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Les diagrammes suivants illustrent la methode. 1 2 3 4 5 6 MICROCOPY RESOLUTION TEST CHART ANSI ond ISO lESl Chart No 2 1.0 I.I I4i 128 l_ 113 2 L. hi I 2.5 i| 2.2 2.0 1.8 1.25 1.4 1.6 A -AP PL IE D I^A>^GE Inc rjS Rocfester. New fork '460y JSA ■^ "'6) *«»2 - 0300 - Phone mmmmmm^mmmmi^mmm^ -; • ,'^.' ~^ »*• t»'» «.• • •* *' -i^-\q^' I Cat. Lyiiil'lt tiO'l ■ ili'l li\.'i Ml -i.|f[i.Tl |K.r.M fc„ .i!iv f,,..IIh .[fv. !«if ,iL'oL„„ 1„ ,,..cf„„m •,:,„I .HyirMyniA If.f-.lfriol.poraivl,,,) «;,(,. yilM„oiei;Tv> ' '^ .,<':nuv«i.«> itff.,, , ■],,„■/ „^,, ,[^;.„ ,.^.,jj .,, j,_..^.^.^^ ^ j^^ ^_.., 4 . , .f.tl'>8nq-ii>', bfxilfj te..ra.-,io^r,u,», ...„, !.,.-;i .^ob n „to,-( t,„! ^: .,„.-.^fi ol -rr.li.m^ 4- .■..■.( bo.S f,n. not ., ,(,,,,;: „., ^j;„g uiiTlom^d bovhvn r i;;- a:( vlul. .„. THE SPLEEN AND ANAEMIA EXPERIMENTAL AND CLINICAL STUDIES BY RICHARD MILLS PEARCE, M.D., Sc.D. PHOrESSOR OF OESEARCH MEDICINE WITH THE ASSISTANCE OF EDWARD BELL KRUMBH.\AR, M.D., Ph.D. AI8IST1NT rROrSMOR Or RESEARCH MEDICinS AMD CHARLES HARRISON FRAZIER, M.D., Sc.D. PBOFEMOR OF CLINICAL BUBOEBT, DNrVEBaiTT 0» PENHSTLTANIA 16 1LLUSTRATW.\S. COLOR AND BLACK AND WHITE. PHILADELPHIA AND LONDON J. B. LIPPINCOTT COMPANY OPTBIQHT, IriiEi{ than Splenectomy -201 XIV. Tre-vtment: Value of Splenectomy as a Theiiapeutic Prckedurr mia 265 VIII. Test for Resistance of Erythrocytes. Salt Solution fNace) Varying in Strength from 0.3 to 05 Per Cent 267 IX. Reticulated Red Blood-cells. From a Case of Hemolytic Jaundice g^^g X. The Relationsiiip of the Tail of the Pancreas to the Posterior Aspect of the Pedicle 346 XL The Peritoneal Prolongation Between the Spleen and the Splenic Fle.xure of the Colon, Which Must be Divided Before the Organ Can be Completely Mobilized ... 347 XII. Left-side Bevan Incision for Splenectomy 350 XIII. First Step in the Mobilization of the Spleen 352 XIV. To Control Bleeding After Mobilization of the Spleen a Large Tampon of Gauze, Wrung out in Hot Water, is Introduced into the Left Hypochondrium 352 XV. Fractional Ligation After Isolation of the Vessels of the Pedicle by Blunt Removal of Fat and Connective T's'^ue 35S XVI. The Double-clamp Method of Dealing with the Pedicles 35S ix * ILLUSTRATIONS TEXT CUTS 1. Composite Cui^-e of the Red Blood-ceU Count of Seven Dogs After Splenectomy ,„ 2. Composite Curve of the Hemoglobin Estimation of Seven Dogs After Splenectomy j^ 3. Composite Curve of the Leucocyte Counts of Five Dogs After Splenectomy .„ mi^^smi^msm^jm^ma^mmMs^^^^^^ammB^ PARTI EXPERLAIENTAL STUDIES BY R. M. PEARCE ."?£- ■^i^-'>:s'- .f:;^>y/V*- "-. '-fmmm^fm^W^'^: :''rmm?^'''-''^^m^ THE SPLEEN AND ANEMIA CHAPTER I THE HISTORY OF EXTIRPATION OF THE SPLEEN The statement is frequently seen that the ancients practised removal of the spleen in the belief that it improved the wind of runners. It is also stated in the older literature that the swift giraffe is spleenless, an erroneous supposition that may have had something to do with the views of the ancients concerning the spleen and the speed of runners. Aristotle " assumed that the spleen is not necessary for the maintenance of existence, and Erasistratus insisted that it is of no use whatever. Galen more conservatively considered it an organ full of mys- tery (Mysterii pleni organon) and that it probably removed the melancholy of the blood going from the liver to the stomach. The iirst positive statement as to its extirpation (not excision) is made in Pliny's^" Natural History. An old English «" translation runs as follows: " This member (the spleen) hath a propriete by itself e sometimes, to hinder a man's running; whereupon pro- fessed rmmers in the race that bee troubled with the splene, have a devise to burne and waste it with a hot yron. And no marveile; for why? They say that the snlene may be taken out of the body by way of incision, and yet the crea- ture live neverthelesse; but if it be man or woman that is thus cut for the splene, he or she looseth their laughter by the means. For sure it is that intemperate laughers have always great splenes." In connection with the last state- 3 ^ft ^.--iPr-cyy''^-- ■B^ * THE SPLEEN AND AN.EMIA ment it is interesting that the modern German word for hypochondriasis is " Milzsncht." Phny is said to have per- formed experiments involving the removal of the spleen from dogs, hut this is probably merely an incorrect quota- tio.i of the foregoing. The belief that splenectomy im- P'-oved the wind of runners was not lost sight of in the nmldle ages, as is shown by the couplet quoted by Brog- sitter from Murer's Belagerung von Babylon: "Ich han mir Ion diss milz schnydcn, Dass ich mag hinfvn wcgt urul veer." ^^ He also quotes Paracelsus, who considered the spleen des Le.l,ers und Lebens Unkraut," and advised phv- s.cans to excse it whenever possible. Van Ilelmont, /m the other hand, attributed to it important and even vi.al tunctions. The first authentic experimental splenectomv in the cog was performed l)y Zambeccari -^= in ir.80, with appar- ently an uneventful recovery. .Alarcello Malpighi,^^^' the discoverer of the lym].hoid follicles of the spl.en had pre- viously described the effects of ligating the splenic vessels of a dog. It not only quickly recovered from the oi>era- tion, with no noticeable injury to health, but became more voracious and much lazier and fatter. Nothing abnormal was noted in the stools. A second operation on this d.x. performed some time later, showed almost complete dis- appearance of the spleen, but no other clianges except slight enlargement of the liver and engorgement of the n.csentenc vessels. Clarke-^ successfullv extirpated a a„ier, „f Somerset, in an attempted sui- CMde drove li.s butcher's knife into his left side. The spleen part of the onu-ntmn, and the intestines protruded fnun the wound, and his eon.pam.Miv left him for dead Three days later a surgeon re,,laeed ;,e intestines, eut awav the sp een and omentum, and sewed up the wound. " The patient quiekly recovered, was (,uite well a year later, and then nuKratcd to New K,.^dand. where he' lived happilv and m ^ood health for son.e years. The second case wa's of hke character. Xieolaus Mattlna,-' the town sur EXTIRPATION OF THE SPLEEN 9 the spleen. In fact, these reports indicate that an indi- vidual may live to -in advanced a^e aiid exhibit no abnor- mality traceable to the absence of the spleen. The case reported by Ilodenpyl '«' exhibited a ^^eneral lymphoid hyperplasia, and it is probable that, as after splenectomy there is a ^rpadual adajjtation of the l)lood-re.020,(KX)i i4,(XX).(XX)* 4,1!«),(XX) 4,30(),(XK) 3,»)S0,(KX)! 3,8(iO,(KW)| 4,(XX),00()' 4,2()0,(K)(r l,;ilO,(XK) 4,S00,0(K) 5, IS 1,000 5,3i-iO,000 2,8S0,O(K) 3,010.(KX) 3,2(K),(XX)! 5,700,000 3,220,000, 5,3,so,{XK). 5,280,{KKJ 4,9(iO,(HK): 4,580,000, 5,100,000 5,120,(X)0 |5,I25,O0Oi 3,940,000^ 3,810,000; 4,280,(XX)! 4,4-10,0(X)' 4,390,0(X)i 3,150,000 4,140,00) 3,00,(HK) 4.120,000 i4,510,(X)O 4,010,000 4,49(),(KX) 4,570,0(X) 4,9,V(),(,y)0 4,7(X),(XX) 1.340. (XX) 5, 1(M),00() 5,4(K),(XX) (!,2.'-)0.000 5,5.5().(XX) t liI'o,i!f"T> ","' ""' ''"I"'"" '"for to the Picture oMI„.b|,.,„|',,,,;;^'.'j'|; f hi.-ontn'^l^^yj'^Al"^-!"^^ ""''■"-«"">>•, S per cent -1 • 94 per cent . SO per cent. 1* 3 5 7 90 85 SO 80 70 92 70 , 92 70 1 82 70 82 105 95 S3 70 64 48 55 65 65 9 12 15 80 75 70 88 70 70 70 i 72 72 1 02 85 98 77 62 70 18 65 70 ' 00 104 77 70 21 65 58 70 24 05 60 98 71 27 50 55 90 30 30 70 80 oo 57 95 68 55 50 70 42 48 70 52 58 55 55 70 75 61 76 80 76 74 64 60 80 78 82 05 72 84 89 66 90 78 70 75 90 78 72 80 90 70 79 90 80 84 82 75 88 78 94 72 !K> ()6 90 97 88 104 90 62 85 94 124 95 75 80 D5 90 150 210 97 94 100 85 75 68 110 104 105 105 300 102 1 92 81 108 • The fi( sures in the 6rat cfjlumc refer to th e number o days after splenectom y than Ufore splenectomy. This is true of both the red cells and the hiiMiioglobin. Lamson ^"^' has recently shown that strong emotions, such as rage or fear, by stiinulating adrenalin secretion and thus changing the concentration of the blood constituents, can produce considerable changes in the red blood-cell count per cubic millimetre. In the EXPERIMExNTAL STUDIES U animals whose blood counts are here reported, however, various observations indicate that such disturbing factors need not be considered. In the first place, the amount of emotion produced in the process of securing blood counts is slight, temporary, and often almost entirely absent. Fur- thermore, normal animals have been followed over consid- erable periods of time without appreciable change in their blood counts. hemo Qlobin Be]-ore Splenec- After Splenectomy Time in Da,ys i tomy 1 3 5 7 9 12 15 18 21 24 21 30 36 At 46 54 GO 6672 eo 68 96 \M 124 isi MC 100% 95 7, 1 ,A, 90% N. \/' -" 851 ^ V f— ^ > 1 80 i \ / ^ /'^ s/ 75Z \ V 70X V -r v ^ ^ &5% V* •^ J 60Z _j _j _i Fij. 2. — Composite curve of the hasmoiilobin estimation of peven dogo after pplenectomy. The nature of the ana:mia that follows splenectomy will be discussed later (see page 87), after some other factors which are dependent upon the removal of the spleen and have a possible relation to the anaemia have been pre- sented. Here it may be stated, however, that the stage of repair is not characterized by the constant appearance in the peripheral blood of nucleated or other abnormal types of red cells. Careful differential counts of the blood of three dogs at regular intervals for 138 days failed to reveal in two the presence of nucleated red cells, and in the other tliey were demonstrable only five times, the largest number seen in one count being three. In none of 16 THE SPLEEN .\.\D .IN.iiMLV these animals were othe- changes evident in the red cells, except tlie pale stauung corresponding to a low h^nioglo- hm content. In a large number of other animals in which differential counts were made at irregular intervals, changes in the red cells have been found very rarelv, Tims in one dog, hve weeks after splenectomy, f.ve normoblasts and one megaloblast were found (in counting 100 leucocvtes) as was also evidence of poikilcK-ytosis and polvchromato- philia, and, a vyeek latc^-, one normoblast and one megalo- hlast. Ihese hnd.ngs were in the first davs of begin'.in.r repair, the red cells and ba>moglobin having a few dav^ heore reached the lowest level obsened .luring the expeiV nent haemoglobin, 50 per cent.; red cells. 2,970,000. In another dog. two months after splenectomy, again at the stage of b'.'gmmng repair (ha n.ficance only in that they probably indicate the period of begmning repair. ^ The very definite nuclear particles found bv .Arorris ^""' " m- •;"■ '" ^"' • '''''''-'' '■" ''''' ''"^'' ^^"^Pit^ -Teated txannnatK.ns made smce Morris's second publication This The number of skeined or reticulated ervthrocvtes is ery shglitly, ,f a- all. increase,] during the 'earl v 'stal follow,ng removal of the normal dog's spleen. Gates^-' on the other hand, has shown that there is an increa in the number of reticulated cells when the nna-mia is greates" >: i'. EXPERIMENTAL STUDIES 17 that is, just before the period of beginning repair. The blood-phitelets also are only slightly increased after re- moval of the normal spleen. White Ceils. — As our problem was one concerning the red rather than white cells, the latter have not been studied as thoroughly as have the red cells. We have, how- ever, total leucocyte counts of five dogs and differential counts of three dogs, in each instance covering long periods of time,'" ' and on many others for shorter periods. The results of the total leucocyte counts are presented in Table III and Fig. 3, and of the differential counts in Table IV. TABLE III Leucoctte Count After Splenectomy Preliminary count !• 3 5 7 9 12 15 18 21 24 27 30 36 42 48 54 60 66 72 80 88 96 104 i24 240 DOK32 y.ooo 26,000 26,000 28,000 2r,,ryx) 28,000 22,000 25,0(X) 19,(X)0 18,000 15,000 33,000 16,000 11,000 13,000 14,000 13,000 15,000 n g I 1 Dog 44 12,000 33,000 22,500 21,0(K) 23,000 19,000 21,0(X) 18,000 19,000 U,000 15,000 15,000 11,0(X) 12,000 11,000 12,000 12,000 13,000 13,000 13,000 36,000 21, .500 13,000 13,000 14.000 13,000 16,000 18,100 16,400 12,0(X) 14,200 11,200 10,000 11,200 0.000 11,000 12,400 Dog 57 Dog 59 14,400 14,200 38,100 21,100 17,000 22,000 19,200 19,600 18,700 18,400 18,600 10.000 16,900 19,000 19,200 15,000 16,100 17,600 16,400 16,000 18,100 20,100 16,000 16,400 28,700 13,800 11,400 17,900 14,400 17,800 16,800 12,100 11,800 13,400 15,300 12,100 17,400 13,100 11,600 12,200 1.3,600 9,000 K5,000 * The figures in this column refer to the number of days after splrnectomy. 2 18 THE SPLEEN AND ANJ^MIA The leucocyte picture was quite constant. On the day after splenectomy the white cells rose from a normal level of 9000-14,000 to 38,000 per cubic niillin.etre, or even higher, and in a few days fell rapidly to about 20,000, after which there was a more gradual decrease with return to approximately the normal level after a period ranging from one to four months. The initial leucocytosis was due mainly to an increase in the polymorphonuclear neutro- philic leucocytes, and was probably a post-operative effect Fia. 3— Composite curve of the leucocyte count* of five d">rs after splenectomy and not due to the specific absence of the spleen. Lympho- cytosis, although usually described, has been found only to a slight degree in this series. At no time were the lymphocytes recorded as higher than 34 per cent., while the average count in all dogs was about 18 to 26 per cent. In the parallel observations on the results of diverting the splenic blood from the liver without removal of tlie spleen, the early polymorphonuclear leucocytosis and later lym- phocytosis were also noted. The behavior of the eosino- philes has varied; in two dogs (not presented in Table IV) an eosinophilia of 10 to 32 per cent, persisted for 113 days in one, and in the other an eosinophilia of 6 to 11 EXPERIMENTAL STUDIES 19 TABLE IV Differential Cocnts of White Cells Aftek Spu:.\ectomt Dog I Time Leucocytes Polj-nudcars t Lympho- cytes t Kosinophilea t Other forms Ik-fore operation 12,500 8,025 3,000 500 375 •1 4,5,200 37,100 5,SS0 4.50 1,770 3 22,400 17,2,50 ■i;MA) 5t)0 1,2:50 7 2,'), GOO 1S,700 4,340 1,280 1,280 13 2,5,100 19,,500 4,UXJ 1,000 500 21 IS, 000 13,580 4,4(K) 370 190 33 17,100 11,9,50 4,:™" indicate the nun.ber of .lays after operation; n.,n,l,ers in the ottur CO iinm« in|iic,itc ihe nui.ilicr of cells per cubic millimeter. T Calculated on basis of I'lXJ cells counted. 80 TilE SPLKEX AND AN.K.MIA per cent, for 107 days. On the other hand, in three dogs the eosinophiles disappeared entirely ior long periods of time, cor sponding roughly to the third to eleventli week,* but this disapj)earance was followed later hy an increase varying from (J to 20 per cent, of the total white cell count. This disappearance, as may he seen in Tal)le 1\', corre- sponded to the i)oint of severest ana-mia (thirty-third, twenty-first, and tliirteenth (hiys), and the reappearance to the period wlien the blood has returned, or nearly so, to its normal level (eighty-second, ninety-sixth, ninety-sixth day). As to its significance wc have no opinion. In all diffeiential counts especial search has been made for unusual cells, as myelocytes. These have seldom been found. In the counts shown in Table IV, represent- ing three different dogs, basophilic myelocytes were ff)und only four times, and no other atypical leucocytes were seen. In a fourth animal, two months after splenectomv, baso- philic myelocytes (5 to 100 cells) were found once at the period of beginning repair. The literature of experimental splenectomy, while it presents rather widely varying results, is, on the whole, in accord with our experience. Dissimilar results are re- ported by Paton, Gulland, and Fowler,' '" who state that in the dog, cat, and rabbit the removal of the spleen has no influence on the red corpuscles. An examination of their tables shows, however, that in the dog they did obtain a slight anaemia, a decrease of to 18 months after splenectomy. In none was a drop of more than .5 per cent, hivmoglohin or 100,000 red hlood corpuscles note^*«- .:^)^T-''^)m^:'im^^Wl- EXPERIMENT.\L STUDIES 18 m numb'^r of red cells and iunouut of ha'inoglobin occurs; and furtlier, if un I^t the latter circumstancej an iron-rich (llesh) diet is ^iven, the hlood picture (juickly returns to normal; {'2) Kichet's •"* observation that in order to main- tain splenectomizeii dogs at the same weight as normal dogs, a much larger ((uantity of focnl is necessary, and (3) Paton's '-'•' conclusion opposed to that of Richet, that splenectomy in the dog has* no influence upon general metabolism. In regard to Asher and Vogel's contention, we have not found by a direct quantitative study (see page 112) of the elimination of iron that splenectomy seriously influ- ences iron metabolism." Moreover, in our opinion, the time of improvement in the anjemia which these investi- gators descri})e as the result of feeding iron-rich food corresponds to the spontaneous repair of the ana?niia which usually begins about the end of the fourth week. In other words, the improvement was, in our opinion, due in part at least to the normal repair and not to the effect of the iron-rich food. Their conclusions would be more convincing If they iiad prevented entirely, or lessened, the severity of the anaemia by beginning the feeding imme- diately after splenectomy instead of waiting nearly three weeks. As to Richet's point, it may be noted that we have not seen noteworthy changes in the weight of our splenecto- mized animals. p\)r a few days after splenectomy, a slight loss may occur, but in all long time experiments an in- crease beyoFKl the original weight has been observed. The studies of Paton and his associates as to the changes in the blood after splenectomy are the most carefully conducted of any in the literature and for this reason we have been greatly disturbed that our results were so differ- ti THE SIMEN .\M> .LN^KMU ent. Their studies, l.owever, were ]i,„ite,l to two spleneeto nnzed an,n,als and two eontrols and it „,av tTlZt e,,a„ee the o„,,cr eorrespond to the ,„iider aL„ ias whitl « t observed In the.r studies of the blood »" diet is not ment,„„ed hut in the n.etabolisn, '-' work the do 's were lor part of the time at least on a meat (ln>l, irorT dirf whK-h, ,f used in the hlorf work also, nn«ht ha ™ e^ i^ Asher and Vogel are correet, a factor in deel , !;, e ana.n„a. It ,s evident fron, this brief review that d e^n ay be n nnportant factor in detennining the degree of ana" ,?, follownig splenectomy. "ian,iniia Until our special inyestigations of the influence of diet ere undertaken, all anin.als, except those „sed In t le stu,ly of ,r„„ ,„et„bolisn,,== ha.l been kept upon the sa ! f:ir' ''"'r ""''"? "' ""■"'■ ''-»'l. -™ls and c . tables-„, all essentials, the " table scraps " upon wlii-h dogs are usually fed. This was always " '.plied i,al" ance .and each dog received all he wou.d''.t. d a ^ t records show that the splenecto,„i.e,i dogs .luring peri^ of .sever.al n,o„lhs gaine.l in weight on this diet, , e c^n .dered , h.ghly satisfactory. However, we ,lid not k nl ^t wfs ™'""r,?'"' "! «"■' ""-'1 diet and, n.oreover, »s .t wa esscnt,,lly a boiled ,liet, it might possibly be d fic.ent ,„ s„„,e substance essential to the proper fun tion of the h™op„,et,e system. Therefore, in ,, or first gro, chetary expernncnts anin.als we,-e placed on ealoricall ■ ufficent d,ets, the protein bcng furnished in the forn. of beef he.a,t beef spleen, or e.,..,„ercial casein. an,i the fa and carb„l.y7 12-18 105 j2«-40 i 10-60 After splenectomy .5- 7 10-14 ls-23 2ti-33 :iS-4l> 4.J-4N 52-CI 8 8 7,420,000 fl7 .'vO 7,!>10,000 1 7,230,000 !W ;,720,0(K) ().2,'>0,000 1(8 8.6 6,.'i00,000 li,810,00() llf> 8.7 7,270,000 n.8 . ,3(iO,000 KM 8.3 6,S80,(XH1 10 .^) l),f;!»0,OIKI 100 h..! 6,2tiO,0(HI lU.S 0,640. OUO y.j 8.0 6,240,000 lO.'J 103 Itii 101 !)7 <0,(K)() 6,S(K).000 6,000,000, percent. 07 102 HO 90 order to shorten the table, only enough blood counts are given to show the general trend of the blood picture. The figures for iron and of nitrogen in the diet are based on the average of several estimations of the food materials used. These figures with a calculation of the caloric value of the food arc given in Table VIII. By comparing Tables V, VI and VII it is at once evident that in no instance did the general nutrition of the animals suiTer. A slight loss of weight occurred after operation, but this was soon regained. Also it is seen that w THE SPLEEN ANO AX.i:MU constant l.vel „f red cell an.l l„™,„sl»■' ™n here one can iwnll, ,efe, to the condition present as a frank anicnna It .s, however, of sisniHeance that in all instances the val TABLE VI _^ iNFLtE.NCE OF DiET Ca.scin, lard, and broad Day, j j>o<5^2_(^pienectom^ . ^^r^:;;{:;;z:~^ (Weight Red cell ; Hemo-'.^ . ,, I ' I rijunt I globin ."'"«'' t i Cawin, lard, and bread Before splenectomy kilos. 10.7 Days Dog 80 (control) 1 4- ,,7 '2-.- 4()-<)0 Weight '^'''^ ''fll I count 9 1 J*. 3 U.2 ' 7 >*'>0,000 i',- 96 103 t,ons arc nore marked than in the controls and also that they usually occur after ahont four weeks, the period in post.spleneeto„,y ana,nia, usually marked hv the bwesl conn s. On the other hand, the question arises are ,he,e results m .some way due to the diet il,.,i. ■ I 1 character of the die,, o t^^^^" '"""'' of iron' That iron in the die! fsTf o^s Z Z,™:; S^vT, ■«",' '■';* *!'"' *"" "' "- «>- »"" "^785 „ !f ;„ "■; "■'^ '''''■'"• *'"'■"' ""' ">"»» """rked Chan ™ of any ,n the group. Tlcef spleen was selected heca "el EXPERIMENTAL STUDIES 87 — X O CJ X ^ b b r-. pi t.T ^^ j;- *. en O '--1 '— V. V '-I GO j; ^ y: ic in ti 30 p p p p p p p 5 £ 5 2 S jR 2 — — 5 3 5 2 CO 00 00 -» *■ M too g en 00 to 10 i— * Zn ^iZn jLfi >«>. rfi> C5 to =J to 2 o) X ■— p ^i CO 10 p - 1 — oc o O P o o o o g2§25Jg 25p 5 5o O -O X Cr DC »I C; 4* 0< to w* Ci b -I X *. w c to 4» CaS to — ^ tX 01 < " 000 ooo< 01 OJ o o a Z3 2? 2:3 DO 1 a w 2! O H O H g 2« THE SPLEEN AND .LNJiMlA cmtau.s a large anmunt of iron, accord ,> our analvses 2So n.g. per 1 00 g, a.ns, presu,nably in 1 part in or^^an c con.h.nat,on and therefore readily utih.abk. F esiX -rt^and casein, on the other hand, contain onlv 4.6 mg and .2 mg per lOO gnuns respectivelv, and if iron is fn m,portant factor in prever.t.ng an<.,nia after .splenectonu" TABLE VIII K™oo..v ..vn IHO.V CO.VX..S. ..„ C...oa.c V..™ o. D.... ok T.„..s Dog Xo Actual tctal per day Nitrogen em. 79 6.5 83 9.8 81 6.6 82 13.6 84 10.6 80 7.4 8o 4.6 S« 8.3 87 6.8 90 4.6 "ic w„ul,l n„t expect ;,„in,„ls fel ,viti, spleen to .,i„nv the • ."Ke. evKlen. ,„ the ««„,« .iven f„,. D„,, g, ,,„, « uih^r. one «„uhl expect H„n,cs ,,,, in cxpenn.cnl 8T. The .»ng« ,n these llnec ,„,in,als, i„ »|| ,,„,|„,|,intv, re „ese he vanat,,.,, to he expcCe,, h, any «,oup of .,„in,als. TI the a,l„,nn,l,-at,on of al,u,.,lant organic iron in the for,, of hccf .spleen. ,i„l not p,,.vc„t the ana,nia i, i„ accord w.th o„r .,n,lics == of i,,„, n,etaholis„,. in the ah.^e'ce of fc^splccn „„.! o, .,,.,1 ,„ „,e „,neU,sion of Asher an.l _0.lftc_otheH«m^^ ,|,e .,|ijj|„ ,|,„„jj^^ ^,|,,_.,^ * See page 112. ~~ - EXPERIMENTAL STUDIES 20 occurred iii some of the animals, it is impossible to avoid the question as to whether a diet adequate for the nonnal do^ is in some way inadequate for the spleneetomized dog. If the latter could be demonstrated the value of our views concerning the severer types of ana-mia following sple- nectomy, based on our earlier experiments upon dogs fed on a general mixed diet, would depend upon whether or not the inadequacy of diet held for all animals operated upon, or only for animals without a spleen. If anemia occurred in dogs fed on the mixed diet after other operations than sple- nectomy, it would be at once evident that the food, while sufficient for a normal dog, was not sufficient for a con- valescent dog. On the other hand, if the anii?mia could be demonstrated only after splenectomy, there would be cstai)lished a point of importance in regard to the spleen in its relation to metabolism, and our observations on the an.Tmia after splenectomy would not onl_y be substantiated, but would gain an added importance. To settle this point, it was essential, therefore, to study in animals on our rou- tine mixed diet the effect of splenectomy, and as a control some other simple operation involving the removal of an organ. Xephrectomy was selected as an operation quite analogous, from the technical point of view, to splenectomy, and accordingly two healthy dogs were placed upon ordi- nary kennel diet for seventeen days; upon each dog a nephrectomy was then performed and the animals kept on tiie same diet for twenty-three days longer; splenectomy was then performed upon each dog and the animal kept on the same diet for thirty-eight days more. Blood counts were made at frequent inter\\als throughout the experi- ment. Whereas during the seventeen days on the diet before operation and during the twenty-three days follow- 80 THE SPLEJiX .YAD .VS.EM1A animal, after the snlene'to m h ^^' T "''^'""'^^^ '" ^'^'^^^ fall in I.a.n,oo.lol.n aXed I 'n""^ ^^ vvell.narked --•thy. aI.o:tlJt Itlf^^^^^^^ Jtisnote- occurred. ieiat,;clj sJ,g],t change in weigJit July 3 July 10 July 18 July 2V Aug. 4 From these ohservi^;,,nc e factory f„,„, ,•„, an .fj"'""^ ^"■'•l' <'-' - ••. -..V «.at „n II,K .li,.t ™ •'"" rff""' operations; (2) "r;ran other tl, n t^' , ';""''""-' *'"= —"1 "fa. EXPERIMENT.!!. STUDIES si IS c thti (a) because M.nie toxic substance, which operates m the absence of the spleen, is present in this paSar the spleen. In connection with this last cr,nc'asion it a cooked d,et ,t was possible that in the cookin.r there occurred the destruction by heat of some vitan.n-hke sub stance nonnally utilized by the spleen. To "ontrol t's TABLE X The W...CK Upo.v ru. A...m:. Fou.ow.va Sp.....ck.m. o. . R.w ^„ , CooKKD Diet ■*'"' * Dng No. Diet Before Bplenectomy ■s ^ a ■c .»^« fti ii^S fiayx 4S Raw 28 1,^4 i)/ ** 2.5 10.9 63 " 24 8.0 52 41 8.5 62 Cooked 4S li; ,S 6ti''| 1 121 1 1 8.4 per cen/ 99 '.5,4,50,000 100 0,220,0(K) f»!> 6,140,000 104 : 6,910,000 10.5 I 6,760,000 88 0,250,000 Controls Cth- Othl 14 Oi 6th-I2th| 12 4 Mh-12th' 8 9 10th-1.3th' 8 6 "th- 9thi 11 4 Cth-lOth: 7 8 5.590,000 4,499.000 4,920,0fX); 5,551,000 5,1.30,000 4,880,000, Food vaJuea per kilo, of body I weight 4 0.41, 69 0.461 75 0.74: 69 0.15 72 0.40 69 0.40 73 9 Raw 108 11.4 102 6,020,000 (initiiil 12.9 95 period) 6,590,000 (final 56* Cooked 121 8.2 100 f)eriod) 6,4('>O.(M)0 (initial 8.4 88 I>eriod) 6,2.50.000 (final poriod) • Thi. animal wa, n^^Y.st ..-^^.,,1;^^,^;—^^—;^^^-^^^^ i.iiir was splenertnTDiErd 82 THE SPLEEN AND AN/EMIA point (see Ta!)le X) a new series ,.f ol.servatic.ns were luidertaken. Six animals were ])laee(i upon a ealorieallv siiiheient diet, aceurately determined, the onlv difiVrenee I'ein^r that four reeeived raw and two cooked meat. J«:xai i- inations of the blood were made at intervals of not lon-er than seven days. At the same time metabolism studFes the results of whiih are described elsewhere,* were made on some (Xo. 48. .37, .',2, and .-,0) of the animals. The diet m each of these experiments consisted of beef-heart lanl, and su^ar, a small amount of sodium chloride, and sufhcient bone-ash to ensure tirm f;eees. Details of nitro- gen content anmoglobin content of its bloocE ^loreover, this animal was the only one showing a per- sistent loss of weight after s})lenectomy. Definite con- '•lusions cannot be drawn from such a small number of experiments, but the fact that splenectomized animals on cooked bee*" develop an ana?mia of a degree more closely approaching that of animals on the usual kennel diet, essentially a cooked diet, while animals on a raw diet have a less severe ana-mia, suggests that heat brings about some change in the diet, which in the absence of the spleen is a factor in causing anamia. In view, however, of the rela- tively slight diff'erences which we have found, experiments on a large i. ,il)er of animals on diverse diets must be made before a final decision can be reached on this point. Our views at present may be summarized as follows: 1. The anamia which develops after splenectomy is most marked in animals on a mixed " table scrap " diet of meat, bread, cereals, and vegetables, which is essentially a cooked diet. 2. Control studies in which a unilateral nephrectomy precedes splenectomy demonstrates that the anamia is 84 THE SPLEKX AND ANEMIA not due to operation, Iicinoirlia^e, or accidents of conva- lescence, but develops only in the absence of the spleen. 3. The results of studies of tlie intluence of food con- tainin-r a Jar^a- amount of iron in presumably easily utiliza- hle form, as in raw beef spleen, doc-s not support "the view that the ana-miu is due to lack of iron in the focxl. 4. Obsen-ations on the influence of a diet of raw meat as contrasted with cooked meat show a more severe ana-mia in animals on the cooked diet, and sunr^rest the possibility that heat alters some substance which the body cannot utilize in the absence of the spleen. Bi.ooD CiiAXGKs IX Man Aiter Si'i.kxectomy Concerniiimoglohin had returned to 100 per cent, and the red hlood-cells to 4,700,000. Dr. Darling has heen good enough to write us that three years after oi)eration the hlood examination was as follows: Ha>mo- glohin, 100 i)ercent.; red hlood-cells, 5,300,000; and leuco- c>i:es, 10,000, of which polymorphonuclears form 7.5 per cent., small lymphocytes 12 per cent., large mononuclears 5 i)er cent., and eosinophiles 8 per cent. Catellani,"" in a preliminary count hefore extirpation of a movahle spleen, found haemoglobin 70 per cent, and red hlood-cells, 4,435,500. One month after operation the ha>moglohin had dropped to 00 per cent, and the red blood- cells to 3,500,000. After ten weeks the hjcmoglobin had returned to 75 per cent, and the red blood-cells to 6,050,- 000; white cells, 28,310. Meyers ^^ reports a child o'f eleven years .'?plenectomized three days after an injury causing rupture of the spleen. On the evening of operation " THE SPLKKX AM) AX.E.MlA the blootl picture was: Hvd cells, -,,2.50 OOO- ^vhito r.ll 28,000. SVmn tuoty-lVnu. lu.uns the C^- f'd M^' j|,0;«,,000,andatti.ccn,,ortwelve.,aysto;^,^^^^^^ l>^e„.o..loh,n l.^ures (Tall.vist) offer httle of value: Gre J var.a ,o„s ,u the re.l cell count were ohsen-ed for tl ee n.o,.th.s w.th ^n-a,lual in.prove.nent. hut not con.plete re urn to normal in that time. 1 "-it-i<-iurn McCoy ■''■■" reports two cases of rupture, one of a nor mal, the secon.l of a n.alarial spleen. In the firs , on w-h." i seven counts were nwule. the lu^no-Wohin fell fr „. 70 cer.t. before operation to 20 per cent, three davs at't r <.perat.on and steadily rose to over 100 per cent sixteen 3,408 000 before o,,erat,on, fell to 2,300,000, and steadilv rose to o., 00,000. The patient with nudarial spleen si , ha.n.o,dolHn 70 per cent., red blood cells 4,000,000 be , ■! dT.tdv":f't; t ' ''" 1- '■' ''' ^•^"*- ^"^ -^^^« ««^^ "- - •■ 4.-6,000 seven months later. In the case reported by Matthew and Miles ==- a count 3 800,000 red blood-cells and 80 per cent. ha^mo^Wobin, and after two years. 4.800,000 red blood-cells and 85 per ce" hjemo^lobm. '■ Heaton^^= reports a splenectomy for traumatic rupture >n a ch.ld n,ne years of a^e. The operation was done fi" and one-half hours after the injur>^ when serious svm ,! toms of m ernal hemorrhage had developed. Xo prelinnn ar>' or early counts were made-a count seven davs af er operation showed 4,100,000 red cells and h^rmoc^fohin 40 t^blToVn r '^" """'^^ "' "^'^^^"'^^ Perfect^recoverv the blood p,cture was 4.480.000. ha-moglobin 75 per cent. The usual leucocytosis was obser%^ed. -I n EXI'LRIMKNT.A STUDIES Fowler,'-" a lew hours after spleiieetoiny for movable spleen, found the red eelLs to number li,iH)0,(H)0 and the leueoe\ tes :U,-2()0. Daily eounts thereafter for twenty- seven days showed a gradual deerease in the red cells, with a sHo-ht inerease at the end of that time to 4,147,000, the white eells at the same time being 12,880. Unfortunately, no blood examination was made before operation. Musscr's ''" ease of cyst of the spleen illustrates well the degree of ana?mia which may occur and the long duration T.\BLE XI Blood Kxamimatiovs Before and AfTEu Removal or the Spleen for Simple Ciai Hoforo oppration S il:iys after .... l.S i];iy.>i after. .. . !• iiioiitlis after. . lo iiiontlis after. . '22 iiiontlis after. . 'J.-j iiumtlis after. . 2s nionth.'i after. . •10 months after. . lI»mo- Red tilood glubin cells 70 4,3f)0,000 yo 4,042,000 87 3,1(1 ti,(XM) SO 4,400,000 (.■) 4,220,0(X) /o 2,750.000 SO 3,400,000 7S 3,400,000 .SO 3,0itO,O(X)i Leurf>- CJtPS 11,400 11,400 13,9(H) 17,000 14,S0O 12,000 13,200 14,800 17,200 of the .same. The patient was a young woman, twenty-five years of age, with, for seven years, a swelling in the left side of the abdomen. At operation was found a large benign cyst of the spleen measuring 18 cm. in diameter. The spleen with cyst wall (after evacuation) weighed 400 grammes, so that, exclusive of the cyst, the spleen itself was not greatly enlarged. In the detailed statement of the blood examinations, presented in Table XI, it will be seen that a slight anaemia existed before operation. Staehelin^'2<^> has collected from the literature up to 1903 twenty-one cases of splenectomy following injury I Poljmor- ! phonu- j cl»,-ara i Lym- pho- cytes Mono- 7,410 8,0,-,0 8,340 11,950 3,310 1,000 3,340 4,250 and transi- , tiunala I 9,370 7,310 340 1,390 2,220 700 38 THE SPLEEN \SD ANEMIA i V V f ;T; "''"'' """^'^^ "^' '''' ""--1 spleen. In ^e ^ ew of these cases was preli.ninary exanunatLi of he hlooc n,ade, and in only three instances was the hlood ^xannned more than three times, and usually at very ir- regular n.tervals. In all cases there is evid;nce of some ...rade o ana-nna after operation, but the counts were not ^c a, ,ourse o the ana-nua. The figures as to leuco- c\ toMs ^-aIy greatly. from a large nun.her of reports, uost of which, because they present no prelin.inary counts or onlv occasion! of the influence of splenectomy on the blood picture. Such evulence as we have shows very distinctly that in man bo h 1: X '"r^'f '■" ''''''''' ''^ '^'^^- "-- than t e former, after splenecton.y. Some confusion exists beca.se of the .mprovement u, the blood picture that follows opera- t.on for removal of the enlarged spleen of the various types of sp en.c ana.m,a." In these cases, however, an abnor- njal splec. .s ren.oved under abnonnal conditions of t I blood, and no a normal spleen under nonnal conditions and. as we w.ll show later, several factors are to be con- sidered m connection with the changes in the blood follow- 'npT removal of the pathological spleen. 11. INCREASED RESISTANCE OF RED BLOOD CELLS Early in our work with splenectonu/ed anin.als it wis found that they are n.orc resistant to ha>nu>lytic p son than are normal animals. This fact had pre^-iously b en observed m the dog In- Bottazzi.-^ Ranti,- Puglie'se an Luzzatt,.3.u «,,, .Toannovics.- while Domenici ' oundt I EXPERIMENTAL STUDIES SO true also of the rabbit. Evidence of the increased resist- ance was based on the fact that to get the full toxic efFect of a lupniolytic poison it was necessary to give a splenecto- niized dog doses two or three times greater than were re- quired for the normal dog. As a result of such observa- tions the theory was advanced that the spleen was concerned in some way in influencing the normal destruction of aged anrl effete erv-throcytes, and that, as this influence was lost after splenectomy, ha-molytic agents were correspond- ingly less effective. This was the basis of Bottazzi's theory of the spleen as a hcemocatatonistic (i.e., preparing red blood-cells for destruction) organ, which 'vas later adopted by Hanti and supported to some extent bv Joannovics. Pugliese aiul Luzzatti,^«^ on the other hand, denied the influence of the spleen, and pointe' extensive study, found as an average ot hlty-eight determinations that in normal dogs the first trace of haemolysis occurred in 0.42 per cent, salt solution as compared with 0.35 as the average for thirty observa- tions on splenectomized dogs; the average ooncentration at which hemolysis was complete was, for normal .logs 0.30 per cent, salt solution, and for splenectomized dogs' 0.23 per cent. Thus in both series of observations the mcreascd resistance of the splenectomized animals was the equivalent of 0.07 per cent, salt solution. The difference may be expressed in another way: in the forty-eight observations on normal animals hfemolvsis Iiegan in all except one test in solutions of 0.48 to 0.40 per cent., while EXPERIMENTAL STUDIES 41 of thirty obsen-ations on splenectoniized animals, in all I)ut two it began in solutions of 0.38 to 0.30 per cent. The lesistance of the red cells was found to increase gradually and to reach its maximum at the end of about two months ; after further lapse of time up to two years and four months' there was no tendency to return to normal. Pel makes a general statement concerning the influence of the serum, to tlie effect that the serum of a splenectomized dog added to the red cells of a normal dog does not increase the re- sistance of the latter to hypotonic salt solution, and, vice versa, that the addition of normal serum to the red cells of a splenectomized dog does not decrease their resistance. IJlood counts showed a slight decrease in the number of red cells after splenectomy, but not enough, in the opmion of Pel, in view also of only slight changes in the percentage of h.-vmoglobin, to indicate a relation to the increased resistance of the red cells. As for the fac- tors responsible for the increased resistance. Pel offers no explanation. Ver>' recently Gates "' has demonstrated that the red blood-cells of splenectomized animals are more resistant than are those of nonnal animals when submitted to the mechanical damage of long-continued shaking. The increased resistance of splenectomized animals to blood poisons we observed early in our work with hemoly- tic senim,"" and as a result of occasional tests with hypo- tonic solutions of sodium chloride we reached the tentative conclusion that it was due, in part at least, to increased resistance of the red cells. This supposition we confirmed in a special study,'"' in which the cells were tested not only ngainst various strengths of salt solution but by the accii- rate methmls of immunology against specific hsemolytic 4^ THE SPLEEN AND ANAEMIA serum, with also investigation of possible antihaemolj-tic action of the serum and changes in complement content. In these tests six dogs were usetl. One had been splenectomized ten days, a second thirty days, and a third four months before. As the last animal had been given a specific hftmolytic immune serum two months before, thus introducing a new factor, the observations on it were con- trolled by a fourth dog not splenectomized, which had been given ha'molytic serum five weeks l)efore. Two normal dogs were used as general controls. Tests ^Vith Varying Gkadks or IIypoioxic S.vlt Solution Chemically pure sodium chloride was dried for two hours at 170" C. and immediately weighed in amounts necessary to make 500 cubic centimetre volumes of salt solution, ranging from 0,1 to 0.5 per cent, in steps of 0.025 per cent. In order to be sure of approximately the same volume of corpuscles in the anaemic as in the normal bloods, the gently defibrinated blood was centrifuged and the serum drawn off. One-tenth of a cubic centimetre of the corpuscular mass was measured accurately in a graduated pipette and placed in three cubic centimetres of each of the various salt solutions. Standard colorimetric scales for comparison were made by laking red cells with distilled water; thus the hiking of 0.4 of a cubic centimetre of the corpuscular mass in twelve cubic centimetres of distilled water represented a standard of 100 per cent, haemolysis. Dilutions of this solution were made so as to have tubes showing the color values of 80, fiO, 40 and 20 per cent, ha'molysis. Less than 20 per cent, haemolysis was con- sidered as a trace of ha;molvsis. In most instances this EXPERIMENTAL STUDIES 43 > Corpuscles of a o (X) > •a O o B a o 9 a 83 o o at) 17) u o CO 5^ o 00 o to o ~ 2 o" 5 p g 3 ■< g » Si s- B 2; o 3 o n .2 5 3 ns 8 8 8 8 100 100 0.275 i MA 8 1 8 8 1 8 8 0.300 S § 8 1 8 1 8 8 o s 8 8 8 8 ►^ t— • o g 8 s 8 s 8 8 H H ^- O g 1 s i 4^ O 8 to «1 H H o 3 3 o 8 5 3 8 S H H o o o i o S H p o o o o o IJ s 3 S a 44 THE SPLEEN .\ND ANEMIA scale was entirely satisfactory, but occasionally, althouL^h a tube showed 100 per cent. ha:.molvsis colorinietrically tlure was, on shakin- a slight macroscopic sediment of incompletely hivmolyzed corpuscles; this result was indi- cated by a minus si^m after the approximate percentaffc of ha-molysis. Upon adding the corpuscles to the salt solution, a preliminary readin- was made and the mixtures were placed in the refrigerator. The final readings were made at the end of eighteen hours. The results are shown in lable XII, It will be seen that the blood of the normal do^s (.30 and 53) shows haemolysis in fairly hi^h percentages of salt solution, but that the resistance, both maximum and n.m.mum, is increased in all the splenectomized animals. inV-'l r' ^' ?^ f ' ' ■'^P'-^^^^t^n^i^ed animal, shows initial ha>molysrs m the same percentage of salt solution as normal dog 53. but inspection will show that, whereas in the normal dog haemolysis is complete at 0.350 per cent ;t IS not complete in dog 46 until 0.300 per cent, is reached' l^rrr, I" "",^""^* *'"'' '^' '^"^ "f '^"^ 24, the animal which had been longest splenectomize" have found that in experimental ananiia (in the rabbit) due to phenylhydrazine these ceils are no more resistant to h.-em^lysis by hypotonic salt solu- tion than are normal red cells. It has therefore l)een impossible for us to bring forth definite evidence that this increased resistance of the cells after splenectomy is due to the presence of young forms. On the other hand, in view of the fact that this phenomenon is present in ana?niia in animals with intact spleen, we are not prepared to eliminate an.-vmia entirely as a factor. Banti,^" however, denies the influence of anaemia. He argues that (1) the degree of increase in resistance is not proportional to tlic aniemia, (2) it may develop m the absence of anawia, (-i) It may persist for many years, and (4) that red cells in the splenic vein are less resistant than those in the gen- eral circulation. He rJso makes the point (5) that after the mjection of h.-^molytic serum, in spite of the marked 8St THE SPLEEN AND ANEMIA anemia and the many young cells in the circulation, the resistance to this poison is decreased, not increased. Our opinion on these points is as follows: 1. Although we believe that the increased resistance is closely associated with the amernia and may probably be due to some factor accompanying it or the initial regenera- tive processes in the blood, we have not committed our- selves absolutely to this view, as Banti seems to think we have, for the reason that we have never been able to obtain satisfactory proof on this point. We do not, however, consider Banti's objection to be very substantial, for, although it is (juite true that the degree of ana-mia arising in a splenectomized animal is not always proportional to the increase in resistance, there is no reason to assume that there should be a proportional relation. 2. Banti's observations concerning increased resistance in the absence of ana?mia are not conclus"\e. We have never f-iiled to find a decrease of red cells and 'ia«moglobin at some time after splenectomy; this may come early or may be late, but in our experience it never fails, and, though as a rule long continued, may be slight and evan- escent. It may. however, easily be missed if continued counts art not made at frequent intervals for long periods of time. In Banti's experiments ihe four anin.als that faded to develop anaemia, but did show increased resist- ance, Mere examined on (1 ) the second and twenty-fourth (2) the third and thirteenth. (3) the third and fifteenth' and (4) the fifth and twenty-seventh days, respectively Our experience has shown that in dogs on special diets the change m the blood may be long delayed or may be present m slight evanescent form in the interval periods On the other hand in some dogs we have found occasion- EXPERIMENTAL STUDIES 58 ■ all}- - slight increase of resistance before the frank fall in the number of red cells, but, as it coincided with or was (,uickly followed by a decrease in hemoglobin, we could not rule out the influence of tiie factors causing ana?mia. 3. Sc also, the observation concerning increased resist- ance in man eight years after splenectomy is inconclusive, for Banti's blood examination shows 4,950,000 red cells and 70 per cent, hemoglobin, a figure for hemoglobin, of doubtful interpretation. Much more work, both experi- mental i.nd clinical, must be done before we can reach a definite conclusion concerning the points here discussed. 4. The observations concerning the lessened resist- ance of cells in the splenic vein are offered in support of Banti's theory* of the spleen as an organ concerned in hemolysis. While we believe the spleen is concerned in the destruction of rer' cells, our observaMons do i t confirm the experii :ts put forth by Banti to prove the decreased resistance of cells of tl- splenic vein blood. This will be discussed elsewhere,t as will also, in that it brings in a new factor, the matter of (5) decreased resistance of red cells immediately after the injection of a hemolytic serum. Although we offer these statements to support the possibility of an association between anemia and increased resistance, we do so without presenting definite proof Moreover, we do not b-se this support, though Banti seems to think we do, on the presence of young cells in the blood Based on Bottazz. s theory that the spleen has special (h«mo- oataton.sfc) action upon red blood-<-ells as they pass thro, . the organ and as a result of whieh they became less resistant. T absence of he spleen according to this theory, does away with th.s action, and the red cells therefore are mere resistant in splenectomized animals T see page 87. 54 THE SPLEEN AND AN.EMIA The skeined cells we have shown are no more resistant than are the mature cells. We do think, however, that it is some factor intimately associated with the causation or repair of the ana?mia followint^ splenectomy, and not the mere ahsence of the spleen, that is responsible for the in- creased resistance, and that this factor is operative in other ana'mias, in the presence of the spleen. That this factor may be entirely independent of the anaimia we willingly admit. III. LESSENED TENDENCY OF HEMOLYTIC AGENTS TO CAUSE H-ffiMOGLOBINURIA AND JAUNDICE AFTER SPLENECTOMY In the preceding section we have presented the evidence concerning the increased resistance of the erythrocytes as determined by the behavior of these cells to various lytic agents. In the present section we offer the evidence concerning the closely related phenomenon — the lessened tendency to haemoglobinuria and icterus — exhibited by splenectomized dogs. To establish this point we have ex- amined tlie urine for Iitemoglobin and bile-pigment^"' ^'" and determined the changes in the blood as shown by red-cell counts and haemoglobin estimations.''"' The hjemolytic agent used in every instance has been hasmo- lytic immune scrmn.* Female dogs were used almost exclusively in order that the tests for ha-inoglobin and bile might be made on urine obtained by catheterization. All operations were under ether ana-sthesia . as were also the injections of serum. * Rabbits were injeetcd five times with five to ten cubic centimetres of dog's blood nt intervals of five to seven days and bled about one week after the last injection. ^^^0t.c^^;kt^^ .:^^^^^.i^ EXPERIMENTAL STUDIES 55 Tlie injections were either into a small vein of the leg or into the jugular vein. Each experinant on a splenecto- niized animal was controlled hy the injection of a normal animal with the same serum. When the animals were of approximately the same weight they received the same amounts of serum ; but when the weight varied more than half a kilo they received, with a few exceptions, corre- sponding doses per kilo, of body weight. The urine of all animals was examined for coagulable protein, haemoglobin, and bile pigment. For the diagnosis of jaundice, the appearance of bile-pigment in the urine has been deemed sufficient. Table XV shows the effect of a weak lutmolytic serum, administered three days after splenectomy. TABLE XV Effect of H>«molytic Serum Administered Three Dats After Splenec- tomy, With Control Date Dec. 11, 1911 Dr.-. 14, 1911 Dec. 15, 1911 Doc. 10, 1911 Dec. 17, 1911 Doc. 18, 1911 Dec. 19, 1911 Dec. 20, 1911 We. 21-22, 1911 Dec. 23, 1911 Dog U. Weight 10,000 gm. Dog 13. Weight 8,990 gm. Splenectomy Urine normal; 2.5 c.c. lytic serum in vein Urine normal lucmo- Uriiie normal; 4.5 c.c. of same serum in vein Trace of albumin; no bile, no lia-moglobin Trace of albumin; no bile, no hirraoglobin Trace of albumin; no bile, no hiTUioglobin Ligation of common bile duct under ether anaesthesia Hile-pigmenta in urine Bile-pigmentain urine incrra.''inB Rile-pi({ment8 in urine increasing Killed by chlorofonn Control Urine normal; 2.5 c.c. haemo- lytic serum in vein Bile test positive; no hemo- globin, no albumin Bile test positive- no tueroo- globin, no albmnm; 4.5 c.c. of same serum in vein Bile test positive; albumin pres- ent, no Wmoglobin Bile test positive; albumin pres- ent, no Wmoglobin Bile test positive; albumin pres- ent, no Wmoglobin Died, under ether, during oper- tion to remove spleen I 56 THE SPLEEN AND ANEMIA In this experiment the hemolytic serum was not power- ful enough to cause a destruction of blood of sufficient grade to produce ha?moglobinuria, although it did cause in the control animal enough destruction to produce jaun- dice; on the other hand, the splenectomized animal was free from jaundice. The objection might be raised, in connection with this experiment, that the jaundice of the control animal might be due to the fact that, as the smaller of the two animals, it received a relatively larger dose of serum. This objec- tion is not tenable, as is shown by Table XVT. In the experiment here presented, a stronger serum was used and the amount injected was adjusted to the weight of the animals. TABLE XVI Effect of Hemolytic Serum Six Dats After Splenectomy, With Control Date Dec. 14, 1911 D?c. 20, i'JU Dec. 21, 1911 Dec. 22, 1911 Dec. 23, 1911 Dec. 24, 1911 Dec. 25, 1911 Dec. 26-27, 1911 Dec. 2S, 1911 Dec. 29, 1911 Dog 3. Weight n,4(X) gm. Splenectomy Urine nnnnal 0.5 c.c scrum per kilo. ILEmoglobinuria; no bile-pig- ment HiEmog'.ohinuria; no bile-pig- ment Hipmoglobinuria; no bile-pig- ment Trace of hspmoglobinuria; no bile-pigment No hspmoglobinuiia; :o bile- pigment Faint trace of bile-pigment No liile-pigmcnt No bile-pigment Uo(r 7. Weight 10,5.S0 gm. Control Urine normal 0.5 c.c. serum per kilo. Hscmoglobinuria; marked jaun- dice Haenioglobinuria; marked jaun- dice No haemoglobinuria; marked jaundice No hiemoglobinuria; marked jaundice No hemoglobinuria; marked jaundice Much hile-pigment in urine Much bile-pigment in urine Much bile-pigment in urine t?g4,, k'Jim EXPERIMENTAL STUDIES 67 That the same results are obtained after longer periods of time have elapsed is shown in Table XVII, which pre- sents the results obtained sixty-five days after splenectomy. TABLE X\1I Decreased Tendency to Jaundice StxTT-rrvE Days Ajteb Splenectomy Date Dog in. Weight 9,720 gm. Dec. 9, 1911 Fel). 12, 1912 Feb. 13, 1912 Feb. 14, 1912 Feb. 15, 1912 Feb. 16, 1912 Feb. 17, 1912 Fob. 18, 1912 Fel). 19, 1912 Feb. 20-21, 1912 ISplenectoray L'rine normal 0.25 c.c. serum per kilo, into vein Urine free from bienioglobin and biio-pigment 1 c.c. same serum per kilo, into vein No haemoglobin uria; no bile- pigment No hajmoglobinuria; no bile- pigment 2 c.c per kilo, of and !ier scrum into vein Ha:>mogIol)inuria; faint trace of bile-pigment No hsemoglobinuria ; faint trace of bile (?) No hemoglobinuria; faint trace of bile (?) Ojmmon bile-duct ligat«d Large amount of bile-pigment in the urine Chloroformed Dog 22 Weight 6,710 gm. Control Urine normal 0.25 c.c. serum per kilo, into vein No hjjumoglobinuria; bile-pig- ments present 1 c.c. per kilo, of same serum into vein Marked hsemoglobinuria; much bile-pigment No hsemoglobinuria; much bile- pigment No third injection. Spleen ex- cised Well marked bile reaction Well marked bile reaction Well marked bile reaction Well marked bile reaction Chloroformed In all of these experiments the splenectomized dogs sliow less tendency to jaundice and usually to hsemoglo- binuria than do the normal dogs with corresponding doses of the .same hemolytic serum, and this, as will be shown liiftr. we have found to be characteristic of animals that have ])een splenectomized for various periods up to one year. Beyond that period we have made no observations. CHAPTER III CONCERNING THE SUPPOSED REGULATORY INFLUENCE OF THE SPLEEN IN BLOOD DESTRUCTION AND REGENERATION A. IN RELATION TO THE DECREASED TENDENCY TO HEMOGLOBINURIA AND JAUNDICE: (1) THE RELATION OF SPLEEN TO THE LIVER AND THE FORMATION OF BILE FROM H-ffiMOGLOBIN. (2) THE INFLUENCE OF THE COURSE OF THE BLOOD TO THE LIVER. (3) THE INFLUENCE OF ANiEMIA. (4) THE INFLUENCE OF THE INCREASED RESIST- ANCE OF THE RED CELLS. (5) ARE SPLENIC EX- TRACTS H-EMOLYTIC? (6) POSSIBLE INFLUENCE OF FATTY ACIDS AND LIPOIDS. In the preceding chapters have been presented the three most important phenomena — anaemia, increased re- sistance of the erythrocytes, and decreased tendency to jaundice — which follow splenectomy. A discussion of these involves a presentation of the literature and of ex- periments dealing with the supposed regulatory influence of the spleen. As the increased resistance of the red cells has a relation both to the problem of anaemia and to that of the decreased tendency to jaundice, it will not be dis- cussed separately, but in relation to each of these. How does the absence of the spleen influence the oc- currence of hfemoglobinuria and jaundice? (1) One improbable explanation, that the absence of the spleen prevents the secretion of bile by the liver, may be dismissed immediately, for, as shown in Tables XV and XVII, the ligation of the bile-duct in the splenectomized 58 F>^. .A*'-!-.. ^•.:-- *i^'•i<~-■ ■•-ft^^.-ij^^ J REGULATORY INFLUENCE OF THE SPLEEN 59 animal ■'^'' causes the aijpeaiance of bile in the urine within twtnty-four hours. On the other hand (Table XVII), the excision of the spleen in an animal with hemolytic jaundice does not inmiediately lessen the elimination of bile through the urine. Does the spleen take part in the formation of bile- pigment from ha>moglobin ? As the solution of this prob- lem necessitated the study of h{emoglobin?emia in normal animals, we undertook an extensive investigation "'^* under the following heads: (1) The degree of ha?moglobina?mia necessary in order to recognize free htemoglobin in the serum; (2) the degree of hjEmoglobinaemia necessary for the escape of hemoglobin through the kidneys: (3) the percentage of hfemoglobin eliminated by the kidneys; (4) the degree of retention of ha?moglobin necessary to cause jaundice; (5) the influence of the absence of the spleen on the elimination or retention of hjemoglobin and the occur- rence of jaundice. Methods Defibrinated dog blood was hsemolyzed with distilled water, sodium chloride was added to render the solution isotonic with dog blood, the hwmolyzed blood was centrifu- galized to remove the stroma, and the haemoglobin content was then determined with a Fleischl hsmoglobinometer. Definite amounts of the haemoglobin solution, always freshly prepared, were allowed to flow from a burette into a small branch of the femoral vein. The first appearance of haemoglobin in the urine was determined by a catheter in the bladder or by a catheter in one ureter. In order to aid the flow of urine, each dog received 300 cubic centimetres of water by stomach-tube. From time to time the skin was punctured and blood was drawn into capillary 00 THE SPLEEN AND AN^MU -.-* tubes to deteniiine how early free htemoglobin appeared in the serum. The elimination of h.-emoglobin in the urine was esti- mated by rendering the urine acid with hydrochloric acid to about X/10 and comparing this solution of acid ha?ma- tin, suitably diluted, with a 1 per cent, solution made according to the Sahli method from blood containing 100 per cent, of hfemoglobin by the Fleischl scale. A Dubosc colorimeter was used for making the comparison. The quantities of hemoglobin are designated in the table (Table XVIII) in grammes, calculated on the assumption, for the sake of convenience, that blood giving a reading of 100 per cent, by the Fleischl scale contains 14 per cent, of hemoglobin. This figure is, of course, only approximately correct, but, as only relative quantities are of importance in this work, an approximate detertnination of the absolute quantities of hemoglobin is sufficient. In order to determine quantitatively the amount of hemoglobin which must be retained in order to cause jaun- dice, decreasing amounts of hemolyzed blood were injected intravenously into a series of normal dogs; in each case the percentage elimination by the kidney and occurrence or non-occurrence of bile-pigment in the urine were noted. The results of these experiments are shown in Experi- ments I to IX in Table XVIII. It is seen (Experiments II, VT, VII, VIII, IX) that the retention of 0.39 gramme of hemoglobin per kilo, caused marked choluria; of 0.23 gramme, slight choluria for twenty-four hours; and of 0.22 gramme, a very faint choluria for eight hours; the retention of 0.18 and 0.19 gramme per kilo, of body weight failed to cause choluria. The , ercentage of hemoglobin eliminated by the kid- ^m^-:i REGULATORY INFLUENCE OF THE SPLEEN 61 X < •< I-, »-H Nl (— ( X rSf'S >-^ t-^^ t^ ^m^ ^14 ::i < 3 o g •«* It- -: S g 3 3 75 n a S 3 << »•< B 9 a 22:2:22:2; 000000 P§§i§3 — -s ■a t; ? ? to ►-* t— • "— • tv 171 rfi t— oe tc i3> 00 OS o b to io =1 to p o o b 8 ?; 8 £ ;/; 10 o> IJ w o b -J to pppppp toS £So 8 4*0 :;i o *a cb u CO to OS p CO CO CO CO c;i to totoco»-toto ^ S S ig ^ 8 8 JiggSSJS to to o to oc o CO 00 o CO to o CO p o o cj 000000 ag 2 £ S 82SSgg5 D o5 ? O .►- p to S 00 S 9! I<^ p r* p C;i ►-• Cn • to to r^ r* .'"' ►- bl i— o ►- >- to bo en pop 00"-ppp goo ^ cocnb^b^ c-tOCO Cn ~4» tOtOtOOl 01 b «0 H-00Cn»eOH- 38- to to 1— 00 ,» ip o to I& to CO to 01 to >k »-> H' H' tJ 1-1 to to Vi H^ CO oo-^tO*->-^ " 4^ -J en >^ CO £ s li o to o to 0\ o o £5 g p p p pppcoo *(^ Oi >^ to to CO H' H-t CO 4^ 00 00 toco^ocooQ 00 00 05 00*J05Cp<00 H- jS K tOW^OMM to b ►- «otocni»M«e ?-2 2 l- ^ & ^ s. s. !<3 g'£$ 5 S S mmim^mmy^^^M I ^-^ r^t'r M THE SPLEEN /VXD AX/EMU ney appears to be a variable quantity. Thus, in Experi- ment \'l, 3-2.5 per cent, of the hii-nioglobin injeeted was eliminated by the kidney; in Experiment VI 1, 2G.8 per cent.; in ExiKiiment Vlll, 17.1 per cent.; in Experiment IX, 35.9 per cent. In these four experiments the haemoglobin solution was rapidly injected during a period of from four to thir- teen minutes. When the solution was introduced more slowly a much larger amount could apparently l)e cared for in the liver witliout the production of jaundice. Thus, if we refer again to Table XVII I, Experiment I, in which the solution was introduced at intervals throughout a period of «ft\-six minutes, we find that an amount of hemoglobin was retained equal to 0.33 gramme per kilo., without bile-pigments occurring in the urine. These experiments seem definitely to establish the mechanism by which free ha?moglobin is removed from the blood-serum under normal conditions. Our conception of this mechanism is as follows: The kidney does not elim- inate hicmoglobin until its concentration in the blood-serum reaches a certain level. This concentration, we conclude from Experiments I, II, III, is about that produced by the j)resence of 0.06 gramme of free hirnioglobin per kilo, of body weight. As soon as the concentration of the hfpmo- globin in the serum is above this point, the h.-pmoglobm passes through tlie kidneys and we have ha>moglobinuria, but as soon as it falls below this amount, the haMiioglobln- uria ceases. However, other tissues, of which presumably the liver is the most important, appear to take up hemo- globin as soon as mere traces are present in the serum, and continue to remove it from the serum, whether the renal threshold is exceeded or not. Tlierefore, whenever 91^: mM;^'' REGLXATORY INFLUENCE OF THE SPLEEN 63 the kidney is removing hemoglobin from the serum, these other tissues are also removing it. Under the conditions of these experiments the kidneys removed 17 to 36 per tent., and tlie liver (and other tissues r) 04, to 83 per cent. The hicmoglobin which the liver removes is changed into bile-pigment, which, if it is not produced in too large amounts, or if the hicmoglobin is not taken to the liver too rapidly, passes out as bile-pigment in the usual manner through the bile-passages. On the other hand, if the luemo- globin is taken up by the liver in larger quantities, and especially if this occurs rapidly, the bile-pigment is formed faster than the bile capillaries can remove it, and it is re- absorbed into the circulation and appears in the urine. The effect of splenectomy on this process was deter- nimed in part by observations on the same animal before and after splenectomy, and in part on animals splenecto- mized for various lengths of time (Table XVIII. j:xperi- ments X to XV) . These six experiments on splenectomized animals, in all of which bile-pigments appeared in the urine for a short time and in small quantities after the retention of 0.44, 0.31, 0.25, 0.28. 0.26, and 0.22 gm. per kilo., re- spectively, indicate that the threshold for jaundice in splenectomized dogs is approximately 0.22 gramme per kilo., the same as in the experiments (VI to IX) with normal dogs, in which the threshold was found to be be- tween 0.18 and 0.22 gramme per kilo. When we examine the percentage of ha?moglobin elim- inate! by the kidneys in the six splenectomized animals, we find that it runs a trJle lower than the limits deter- mined for normal animals, being 18.8, 16.8. 21.1. 27..5, 7.2 ( ?) , and 1 8.4 per cent, (average, excluding the fifth fi^re^ •*ij; 64 THE SPLEEN AND AN.EMIA 20.5 per cent.), as compared with 32.5, 26.8, 17.1, 35.9, 16, and 26.5 per cent. (Experiments VI to XI), with an average of 25.8 per cent. This difference is, however, so slight that we can conclude that splenectomy has no influence in increasing the elimination of free haemoglobin by the kidneys, nor dots it, as is shown by the occurrence of choluria in each of the experiments, influence the ability of the liver to form bile-pigments from haemoglobin, or interfere with the elimination of these pigments. Thus one of the possible explanations for the failure of jaundice to follow the administration of a ha?molytic serum in sple- nectomized animals is shown to be untenable. (2) The Influence of the Course of the Blood to the i.iaer Although the experiments described in the preceding section led to negative conclusions, there is another possible factor, a purely mechanical one; that is, the relation of the spleen to the blood supply of the liver. Inasmuch as the spleen is undoubtedly a seat of destruction of red cells, the splenic blood must carry to the liver haemoglobin in larger an. )unts, or more concentrated, than does the blood reach- ing the liver through the hepatic artery in the absence of the spleen. This difference in haemoglobin content, under the conditions mentioned, might be suiRcient to explain, in a purely mechanical way, the lessened tendency to jaun- dice after removal of the spleen. This possibility was investigated in two groups of experiments : in one ^^ haemo- globin was injected in the general circulation (femoral vein) and into portal circulation (mesenteric vein), and the influence of these two procedures on the occurrence c' hsemoglobinuria and jaundice was studied. In another '*• m REGULATORY INFLUENCE OF THE SPLEEN 65 set of experiments the blood from the spleen was diverted from the liver by ligation of the splenic vein, as well as by an Eck fistula or an anastomosis of the splenir vein with the vena cava, and the occurrence, under these ' jnditions, of jaundice due to the administration of htemolytic agents was studied. In connection with this problem it may be recalled that Pontick^** applied the term " spodogenous " (anoSoc waste products) to the spleen of hemolysis, swollen in con- st(iuence of the accunmlation of disintegrating erythro- cytes, and that he noted that simultaneously the liver elim- inates a bile very rich in pigment, and suggested that this latter is derived from the lia?moglobin set free in the spleen, carried by the portal circulation to the liver, and removed by this organ. Ponfick further expressed the view, based on experiments not quoted in detail, that the liver could completely remove and transform into bile-pigment liber- ated haemoglobin up to the extent of one-sixtieth of the total haemoglobin of the body, but that hwmoglobin set free in excess of this amount passes through the liver and is eliminated by the kidneys, causing hsemoglobinuria. One-sixtieth of the total haemoglobin in the dog is about 0.1 8 gm. per kilo. In the preceding section we have shown that the injection of 0.14 to 0.85 gm. per kilo, of hsemo- glohin as laked blood will cause the appearance of hemo- globinuria, but that a factor of great importance, appar- ently overlooked by Ponfick, is the rate at which the hemo- globin is liberated in the circulation. The more slowly it is introduced the larger is the quantity that the liver can take up without permitting the concentration in the blood to reach at any time that required for the production of hemoglobinuria. Also, we have shown that, while small '--'':<; ^'-^-iL •» THE SPLEEN AND ANEMIA amounts of injected hsnioglobin are removed by the liver and presmuably excreted as bile-pi^ients in the bile with- out the occurrence of jaundice, if the injected hemoglobin be in excess of 0.30 to 0.40 gm. per kilo, the liver i . unable to eliminate all the bile-pigment formed from the excess of haemoglobin, and some of the bile-pigment is reabsorbed from the liver and under these circumstances appears in the urine. It was noted in this respect, also, that the rate of injection is of greater importance in determining the amount of ha?nioglobin that the liver will tolerate without the appearance of bile-pigments in the urine. Very slow but long-continued acniiinistration of hemoglobin can eventually overtax the hepatic excretory power and lead to the appearance of bile-pigments in the urine, although the hiemoglobin transformation may have been slow enough to jjerniit of its continued adecjuate removal from the circulation by the liver with at no time the development of ha?moglobinuria. Thus the first effect of haemoglobin liberation into the blood is an increased bile pigment con- tent of the bile. This was shown experimentally by T,.r- chanoff'.*-" I f the amount of ha'moglobin be small enough and its liberation slow enough, this is the only efFect. A slightly larger amount, rapidly liberated, wnll produce hemoglobinuria. A still laiger amount, extremely slowly liberated, will produce bile in the urine. An equal amount liberated at an intermediate rate may produce both hemo- globinuria and bile-pigments in the urine. Following Ponfick. miny other workers have attribute.) Mar. 20 Mar. 26 Fel). 2.5 Apr. 15 Jan. 9 Jan. 15 Mar. 2 Mar. 26 Jan. 7 Jan. 15 Feb. 19 Mar. 2 Mar. 20 Feb. 25 .\pr. lo 0.085 0.043 0.024 0.026 0.017 0.025 0.014 Dna 12 (0 3 inn. ner kilo.) 0.043 0.029 naff ^^ (0 3 ma. Der kilo.) Doff 3 (0 3 nn. ner kilo.) None None Splenectomized Trace Door 49 (0 3 irni. Her kilo.) None Three montlis after splenectomy 0.010 The results of the study of the degree and persistence of jaundice (a.s indicated by bile-pigments in the urine) in the dogs after the two types of injection are shown in Table XX. It will be seen that in the six dogs studied, the jaundice was distinctly more persistent after mesenteric than after femoral injection, and this was true regardless of which injection was made first. In Dog 4 two successive injec- tions were made into the femoral vein to determine whether the second injection would give a result notably different from the first. Sucli was not the c^ise, the duration of REGULATORY INFLULNCK OF THE SPLEEN 71 the bile-pigments being the same after each injection when both were made into the femoral vein. In our studies both of hasmoglobinuria and of the per- sistence of jaundice after haemoglobin injections we have employed splenectomized as well as normal dogs, but have found that the mere absence of the spleen has no influ- ence upon the fate of haemoglobin injected into either the general or portal circulation. The place of injection is the important factor. TABLE XX Peusistence of Bile-Pigment in the Urine After Ksmoolop'n Injection AS Determined by Point of Injection D»t« Pcraijtence after injection into Hsmoglobin iDjectiona Femoral vein Mesenteric vein dayg dayt Dog 4 fO.3 gm. per kilo.) Jan. 9 Jan. 16 Dog 5 (0.3 gm. per kilo.) Jan. 9 3 Jan. 15 12 + Dog 25 (0.4 giu. per kilo.) Mar. 21 Mar. 26 7 + Dog 26 (0.4 gni. per kilo.) Mar. 20 Mar. 26 7 + Dog 12 (0.3 gm. per kilo.) Feb. 25 5 Apr. 15 iJog 3 (0.3 gm. per kilo.) Jan 7 Jan. 15 9 Splenectomized Feb. 19 Mar. 2 4 Mar. 20 I>uK 49 (0.3 gm. per kilo.) Feb. 25 4 riirce mouths after splenect rny Apr. 15 6 These experiments indicate, therefore, that when hsemo- globiii is set free in the portal circulation a larger amount i.s held by the liver and converted rapidly into bile )igment than is the case when it is set free in the general circulation, and that under the former condition overloading of the liver with bile-pigment more readily occurs and jaundice is more apt to develop. 7t THE SPLEEN AND AN^MU This mechanicjil iiiHuence must therefore be a factor, though not necessarily the only factor, in the lessened tendency pfter spLnectoniy to the jaundice which follows blood destruction due to hfprnolytic agents, for whether the spleen be an active factor in destroying the erythrocytes or whether it plays merely a passive part as a place for the deposition of the ('.isir»tegrating cells, there can be no question that in this organ a large number of cells imdergo their final disintegration after the action of ha;molytic poisoiis, and that the h:cmoglobin there liberated passes by the portal system directly to the liver. When the spleen is removed this disintegration occurs in other organs, nota- bly in the lymph-nodes and bone-marrow, and the hemo- globin from these organs passes not into the portal but into the general circulation, from which it reaches the liver more gradually and in a more dilute form. Jaundice is therefore less a{)t to occur under such circumstances, when a ha?molytic agent is administered, than is the case in the normal animal. Other experiments giving essentially the same result and supporting the injection experiment described above are those* in which, by ligation of the splenic vein and blood-vessel anastomosis, the splenic blood was diverted from the liver and ha?molytic agents were then adminis- tered.'^"" These showed the same decreased tendency to jaundice that is shown by splenectomized animals. (3) The Influence of AN.aEMiA This we have investigated in some detail.'''"' In our earlier work it was noticed more or less accidentallv that ♦ Sec p.igc 1 .SO. ^4^::<-^^ -i^:^ jm REGULATORY INFLUENCE OF TOE SPLEEN 73 an injection of haemol^'tic' seruin into anaemic (logs, whether splenectoniized or not, did not cause jaundice so readily a^ in dogs with normal red-cell count and normal ha?mo- ^lohin content. This observation suggested that it might not be the mere absence of the spleen, but secondary changes in the blood consequent upon the absence of the spleen, that in addition to the mechanical factor prevented the appearance of jimidice in splenectomized animals. We therefore turned our attention to the condition of the hlood in all splenectomized animals. As we have shown in the first chapter, splenectom\ is followed by a moderate ana?mia in which a decrease of lia'nioglobin is especially prominent. The lowest level of this anaemia corresponds to the third to the sixth week, and it occurred to us that, in this early period, the lessened toiidency to jaundice might be associated in some way with the coexistent anaemia. Obviously it was possible to test this hypothesis by studying the effect of a hicmolytic scrum on dogs rendered ana?mic in some other way than by splenectomy. This was d.me by bleeding, as is shown in an experiment (see Table XXI) in which a normal (log with high hemoglobin content is contrasted with a dog rendered moderately anemic by bleeding. In this experiment the anemic dog, although it received the same amount of serum, proportionately, and, on ac- count of its greater weight, twice as much, actually, as the control, failed to develop hemoglobinuria or jaun(Iice, and this in spite of the fact that the actual fall in hemoglobin and red blood-cells was more rapid and greater than in the control. Anemia would appear, therefore, to be an impor- tant factor in lessening the tendency to jaundice. How the anemia acts to lessen the jaundice Is not indicated by 74 THE SPLEEN AND ANEMIA our experimental data. A possible explanation which may- be offered, however, is the following. VVe have shown that the liver exhibits a saturation point for haemoglobin, so TABLE XXI Effect of H^emolttic Serum on a Dog Rendered Anaemic by Bleeding. NoRUAL Control Dat« AprU 18, 1912 AprU 20, 1912 April 21, 1912 10 A. M.. . . April 21, 1912, 11.10 A. M. 5 P. M. April 22, 1912 April 23, 24, 2.5, 1912 April 24, 1912 April 2o, 1912 Anietnic dog WeiRht, 10,740 em. Urine: free of aTl)umin 179 c.c. of bioo<' tHJven from jugular vein 150 c.c. of blwKl taken from jugular vein Urine: no albumin, no bile-pigmcnt Blood:re(J cells, 4,450 000; ha-moglobinH? [lercent. Fragility: 0.3 + ; 0.4- Date April 16, 1912 11.45 A. M. 2.45 P. 3.15 P. M. M. 6.30 P. M. Control dog Received 0.25 c.c. ha-mo- lytic serum per kilo, in vein Urine: no albumin, iio l)i]e Blood :red cells, 3,400,000; April 17, 1912 lucmoglobin 5-t jx;r cent. Urine: no haemoglobin, no bile ' Blood:red cells, 3,2.50,000;! ha;muglobin49i)ercent. j April 18, 1912 Urine: no bile-pigment Blood : red celk, 3,040,(K)0; hifriioglol nil 42 i)cr cent . Blood : red celLs, 2.910,000; lueuioglobin 42 [x^r cent. April 19, 1912 \y eight, 5,350 gm. Urine: no albumin, no bile. Blood: red cells, 5,390,- 000; hemoglobin 107 per cent. Fragility: 0.4 + ; 0.5-. Received in vein 0.25 c.c. per kilo, of .same -serum as ana-niic dog. r ''h bile in urine. Blood: rod cells, 5,330,- 000; haemoglobin 98 ix;r cent. I rine; contaiiis haemo- globin and much bile Blood: red celLs, 4,660,- 000; hamoglobin 85 jx'r cent. Urine contains haemo- globin and bile-pig- ment Blood, red cells, 4,470,- 000; liujmiigiobin 73 per cent. Urine: no haemoglobin, but trace of bile Urine: no bile, no albu- min Blood: red cells, 4,930,- 000 that, if hicnioglohin he supplied to it in excess of a given amount, jaundice will result, but that, conversely, ha?mo- globin supplied to it in quantities less than this amount will not give rise to jaundice. Now it inav well be that ■^1M w REGLLATORY INFLUENCE OF THE SPLEEN 75 in an animal rendered anemic either by bleeding or by insufficient blood formation the daily blood-cell destruc- tion may be less than in the normal animal and hence the liver be further from its normal saturation point for hemo- globin. In such an animal more hemoglobin could be liber- TABLE XXII Effect op HjuioLrnc Sebum on Sple.nectomized Dooa with Normal Red Ckll Count Diite Ajiril 17, 1912 April 21, 1912 il.25A. M. 5.20 P. M. April 22, 1912 April 23, 1912 April 24, 1912 Four day splenertomy Date W eight, 7,8S0 gm. Splenectomy Uriiie : no albumin, no bile Bloe removed by the liver without exceeding the saturation point of the liver and thus jaundice would not occur. ^t is therefore at once evident that if it is the piosence "f an anaemia, subsequent to the splenectomy, which is of T6 THE SPLEEN AND A\.EML\ importance in preventing the development of jaundice, then ha'moiytie serum administered to an animal soon after splenectomy, before anjemia has developed, or long after splenectomy, when the blood has again returned to normal, should induce the appearance of the bile-pigments in the urine. Both these experiments were performed and the results tabulated in Table XXIT. In both of these splenectomized animals bile-pigments appeared in the urine, and a trifle more abundantly in that animal which at the beginning had the higher bamoglobin and red blood-cell count. Thus anaemia would appear also to be a factor in lessening the tendency to jaundice after administration of a h^emolytic agent. (4) Ini-xuence of thk Ixcrk.\sed Resistance of the Red Cells In neither of the splenectomized animals in Table XXII was the jaundice quite so marked as in the control animal given the same dose of the same serum (see Table XXI), and in neither did the ha^moglobinuria occur that was observed in the control. The rate of fall of ha?moglo- bin and red blood-cells in these animals shows that the rate of blood destruction in the control animal was much greater than in either of the splenectomized animals. In the ani- mal splenectomized four days the eventual blood destruc- tion equalled that of the control, but it occurred nmch more slowly; and in that splenectomized for sixty-six days the amount of blood destruction was relati\-ely slight. This ccmstitute a peculiarity in the reaction of a splenectomized dog to ha?molytic agents which necessitates detailed dis- cussion. The splenectfmiized animal may show an eventual I REGLL.\TORV INFLUENCE OF THE SPLEEN 77 blood destruction, following ha-niolytic agents, less than, LqwA to, or greater than, the controls, but almost always the rate of blood destruction is slower. We believe that at least one factor although not the only one, in this phe- nomenon is the influence of the increased resistance of the red blood-cells. In confirmation of this it may be noted that in the three animals under discussion the rate of blood destruction was proportionate to the fragility of the red blood-cells. Such increased resistance of the red blood- cells has been shown to be characteristic of all splenecto- mized dogs. Consideration of the results obtained upon introduction of free hfemoglobin into the circulation at various rates indicates at once the importance of a slower rate of blood destruction which must lead to a diminished tendency both to hfemoglobinuria and to jaundice. Hence the increased resistance of the red blood-cells, in that it causes a slower rate of blood destruction after administra- tion of hcTmolytic agents, is r third factor to be considered in any attempt to explain the lessened tendency in these animals to hajmoglobinuria and jaundice. Ana-mia is, of course, no longer operative in animals splenectomized for long periods of time in which the blood picture has returned to normal. In such the increased resistance of the red cells, which" persists indefinitely — our longest observation covers twenty months — must be a factor which cooperates with the mechanical factor pre- viously discussed to limit the degree of jaundice. In such animals a faint trace of jaundice — never as much as in the control animal — is not unusual. This persistence of the lessened tendency to jaundice one year after splenectomy is shown in the following table: MICROCOPY RESOLUTION TEST CHART ANSI and ISO TEST CHART No 2 1.0 I.I 150 *== m 1^ I- ||2j> 12.2 zo 1.8 1.25 1.4 -^ -APPLIED INA/IGE Inc =" 1653 Eas' Mam Si'eel p^ Rochester. Ne<» fo'k U609 USA -^ (716) 482 - 0300 - Phone =.■= (716) 288 - 5989 - Fo« 78 THE SPLEEN AND AX.EMIA TABLE XXIIl Effect of Hemolytic Sebum One Year After Splenectomy, With CoNxaoL Time One yeur after aplenectumy (dog 42; Before injei-- Weight, 6,400 gm. Urine ; no albumin, no bile Ist in 'inn Alter iiijtction 1-3 days 3 days 2d injectidii 4 dj\s 6 days 6 davs Received in vein 0.5 c.c. serum i)cr kilo. Urine: trace of albumin ; faint trace of bile Received in vein 1 c.c. of same Fcruin i)er kilo Marked luumoiiloliiiiuria Faint hicmonlDbinuria; doubtful bile te,«t Fiiint h;cinoglol)inuriri; faint but detiiiite bile test Died. No evidence of jaundice at autop.sy Time Before injeo- tion 1st injection .\ft or injection 1 dav 2-3 days 3 days '2d injection 4 (lays o days days ' days Control (dog 43) Weight, 0,61.") gm. Urine: no albiuiiin, no bile Received same dose of same senini as dog 42 Urine: trace of albumin; well marked bile test I'rine: no bile Received ,'iame dose of same senirn a.s dog 42 Marked lixmoglobinuria Moderate ha'moglobin- uria; marked bile test Faint luemoplobinuria; large amount of bile Urine: no hamoglobin; deeply bile-stained Chloroformed. At au- to] )sy general bile staining of tissues AVe may ct)nclude, therefore, that three factors are concerned in the decrea.'^ed tendency' to jaundice when hii'inolytic ajjfents are a(hninistered to splenectoniized ani- mals. The most important is the mechanical factor, the disturbance of the spleen — liver circulation; the second, always present, is the increased resistance of the red cells. These two factor., apjjarently always work together. A third possible factor, not always clearly demonstrable, is that of anivmia. {,')) II.KMOLVTU' POWKR OF Spi.EXIC EXTRACTS The histologic evidence of the destruction of erythro- cytes by phagocytic cells of the spleen has naturally sug- gested the possibility of the liberation by the, e cells of a REGULATORY INFLUENCE OF THE SPLEEN 79 ferment capable of acting extracellularly. If it could be demonstrated that such a free ha?niolysin is present in the si)leen we would have at once an adequate explanation of the decreased tendency to jaundice in the splenectoniized animal, for with the spleen absent fewer red cells would be destfv)yed and less hcCnio% which has recently received support from Banti ="' and his colleague, Fumo."^ In the course of an investigation of hii'inolytic splenomegaly, they studied normal animals and those receiving ha?niolytic serum and came to the con- clusion that free ha?moglol)in can l)e demonstrated in the blood of the splenic vein both in normal animals and in those receiving hfemoiytic serum. Sometimes they found it in the blood of other vessels, but always in less amounts than in the splenic vein. These findings tiiey consider «s evidence of haemolysis in the spleen. Also the red blood-cells of the splenic vein were found to be less resistant to hypotonic salt solution than were those of the general circulation. On the other hand, investigations by Chalier and Charlet"* on the resistance of red cells in the splenic artery and vein gave very different results. Although they found that venous blood in general was slightly less resistant than arterial blood, in the splenic system this was reversed, so that the blood of the splenic vein was more resistant than that of the splenic artery and much more so than the blood of other veins. Hammarsten also, according to Gabbi/«» found that the splenic vein blood was more resistant than the arterial. In the observations of Banti and Fumo, the reference is to free htemoglobin in the serum and not to the increased haemoglobin content of venous or splenic blood described by several investigators. This claim is most surprising, in tliat they state that the dissociated hemoglobin of the 90 THE SPLEEN AND AN.EMIA serum (" emoglobin (iist'iolta dal siero ") is not only always present in the splenic vein of normal animals, but some- times in sufficient quantities to be measured by a Sahli lutmoglobinometer. It is to these observ-ations that we have given especial attention in our work. Methods. — From dogs under ether anjBsthesia blood was obtained directly as it flowed from the splenic artery and the splenic vein. Great care was exercised to disturb the vessels and the organs as little as possible, as it has been shown by Grigorescu ''^ and Pribram ■ '^ that the cell content of the blood may be greatly increased by conges- tion of the spleen. From a nick in the wall of one of the branches of the artery or vein fresh blood was drawn directly into Thoma blood-counting pipettes -md the capil- lary tube of a v. Fleischl hsmoglobinometer. From an- other branch blood was withdrawn by a s\Tinge and imme- diately distributed to tul>es containing different strengths of hypotonic salt solution designed to test the resistance of the red cells. Some of the blood was also set aside for similar tests with washed cells. For the determination of the presence of free haemoglobin in the serum blood was collected in three ways: (1) in a paraffined centrifuge tube, (2) in a tube containing potassium oxalate, and (3) by drawing it directly into tubes through capillary points, which were then sealed. All three samples were centri- fuged and the serum examined for Iwuioglobin by visual inspection and the spectroscope. Smears for diff'erential counts were made at times from the blood flowing directly ^rom the vessel, and at times from a drop from the syringe. Finally, tests for reticulated or skeined (young) red blood- cells \v»re made. This was done by letting a few drops of blood fall into a solution of brilliant cresyl blue, and, after REGULATORY UsFLLENCE OF THE SPLEEN 91 standing fifteen or twenty minutes, the skeined forms in proportion to the unskeined or mature forms were counted in fresh smears. For the purpose of controls, blood from the femoral vein and from the capillary circulation (by puncture of the skin) was occasionally collected. Results. — The figures -^* for the red and white cells, differential counts, and total hemoglobin in a series of five dogs show that, so fai as these estimations are con- cerned, the blood of the splenic vein does not differ greatly from that of the artery. The variations are not uniformly on one side, and are all within the limit of error inherent in the methods of blood examination.* It is of interest that in six of eight animals the red cells of the vein showed more or less marked anisocytosis and * Since these observations were made, Morris '"' has published himilar studies on the cat. He comes to the conclusion that Ihe spV^u plays a definite role in the fomiation of the red blood-cells. This conclusion is based on the counting of the red cells in the splenic artery and vein, the number in the latter being one to four million greater than in the artery; in one animal, for example, 4,400,000 per c.nmi. in the artery as against 9,120,000 in the vein. Morris's technic ,'il)pt'ars to differ from ours onlj' in that he collected his blood from the stagnant stream between two clamps, while we took the free-flowing blood as it passed out of a small nick in the vessel wall. Comparative tests which we have made, since his publication, of stagnant and flow- ing splenic vein blood show that sometimes in the former the count may be one to three million higher than in the latter ; in other instances, it is the same. We are therefore inclined to think that Morris's high counts may be due to mechanical causes, especially as in repetitions of our earlier work, three dogs being used, we found a variation between the artery and vein of never more than 500,000 cells. The liigher count occurred twice in the venous blood and once in the arterial Mood — variations well within the limit of error of blood-counting iiicthods. 9t THE SPLEEN .VND ANAEMIA inequality of staining, which were not seen to the same degree in the blood of the artery. Polychroniatophilia was about equal in blood of the artery and the vein. In two of the eight experiments a few normoblasts were found in the splenic vein blood only. Control smears from the femoral vein of four dogs showed changes in the red cells about equal to that of the splenic vein, indicating that these changes are characteristic of venous blood in general rather than any specific change caused by passage through the spleen. In regard to the presence of free hctmoglobin in the serum, if we had depended on one tube only we would have occasionally found apparent hitmoglobinicmia, both in the general circulation and in the splenic vein; but as in every set of three tul)es, in a series of seven dogs, at least one was free of htemoglobin, we cannot support the view that free h.Tmoglobin in demonstrable amomits is present normally either in the splenic vein or the general circulation of the dog. Our experience forces us to the conclusion that the findings of other investigators are due to hitmolysis after collection or are dependent upon the method of separating the serum.* As regards the resistance of the red cells of the vein * During the prist few years, in connection with investigations on coagulation of blood. Abdirlialden's theory of protective enzymes, Folin's inicrochciniral methods, and the phenomena of ana))hylaxis. nuieh time and attention has l>een given in this laboratory to the collec- tion of plar.ma and serum from the dog and rabbit. In our exi)erience careful collection always yields these fluids free of hemoglobin; dis- colored sera we have always regarded as due to errors in the method of collection. With our experience in mind, we cannot support the statements of Banti and I'urno that free hn-moglobin, in amounts sufficient to be recognized, occurs normally in the serum. REGULATORY INFLUENCE OF THE SPLEEN 93 as compared with the artery, tests were made on eight dogs: in five no difference was found; in the other three the venous corpuscles were shghtly less resistant. Two control tests with cells from the femoral vtin showed these to have the same resistance as cells of the splenic vein blood. The question arises, therefore, as to whether the differ- ences described heretofore are not those of arterial and venous blood in general. In seven comparative tests for skeined or reticulated red corpuscles these were found to be more abundant in five in the splenic vein and in two in the artery; the differ- ences were never very striking. Five controls from the femoral vein corresponded more closely to the splenic artery counts than to those from the splenic vein. As a result of these various observations we conclude that the slight differences tietween the arterial and venous blood of the spleen are within the limits of error inherent in the methods of blood examination, and are not to be explained by a peculiar action of the spleen. In some instances peculiaiities shown by the splenic venous blood are common to the venous blood of the general circulation. Ranti and Furno's observation concerning the presence of free ha?moglobin in the blood of the splenic vein is not confirmed. 2. Influence of Splenic Extract Upon Blood Formation Xearly all investigators grant the spleen a function in the destruction of red cells; and some ascribe to it a part in red-cell formation. This latter view is based largely on the fact that in fetal life red cells are formed in the spleen, and that under pathological conditions myeloid M THE SPLEEN AND ANEMIA metaplasia may occur. We have, however, no satisfactory evidence that this function is continued under normal con- ditions beyond a short period after birth. Some work has been done with splenic extracts, as by Danilewsky,'^ to show that the spleen contains a substance which stinmlates the formation of red cells in the bone-marrow. Danilewsky found a sin-prising increase in ha-moglobin and red blood- cells after a single subcutaneous or intraperitoneal injection of extracts of spleen. This increase reached its height in from three to seven days and continued as long as the ex- periment lasted, usually eight days. In dogs with a dietary anaemia,* splenic extract caused an even greater rise; for example, of 40 per cent, haemoglobin and almost 2,000,000 red cells. This influence of the splenic extract was not destroyed by heating. Danilewsky assumed that his re- sults were due to a stimulation of the bone-marrow. As Danilewsky's work is uncontrolled by injection of other organ extracts, we have repeated his experiments. Silves- tri"*"® records a single observation in which a dog, pre- sumably dying from ana?mia, was apparently saved by the injection of splenic extract. In this connection it must also be noted that the clinical literature of this subject con- tains several reports of the use of extracts of spleen and bone-nuirrow with good results in the treatment of anaemia. Method. — We have tested the effect of splenic extract on four dogs, using as controls extracts of other organs similarly prepared and extracts of erythrocytes. The usual examinations of the blood were made, as were also determinations of the resistance of the erythro- cytes to hypotonic salt solution an d of the p ei- centage o f iTAntemia due to a diet of rice only; red cells fell to 3,980,000. REGUL:\.TORY INFLUENCE OF THE SPLEEN 95 skeined cells. As a rule, two counts were made before injection and daily counts after the injection until the blood picture had returned to normal, usually a period of from three to four days. Extracts were prepared from organs removed aseptically from dogs bled to death under ether ana?sthesia. The finely chopped organ was ground in a sterile mortar to a homogeneous pulp and extracted with double the volume of salt solution for two houx'S in the ice-chest. Ten cubic centimetres of the filtered extract was injected intraperitoneally into dogs of about the same weight. As the splenic extract contained a considerable amount of blood, it was necessary to use as control defib- rinated blood (10 c.c), diluted with normal salt solution (T to 20), in order to determine the possibility of the rise in red cell count being due to the influence of some con- stituent of the red cells. In no case did peritonitis or other infection result from the injection. The result ''^'' in one of these experiments is shown in Table XXVI. TABLE XXVI Effect on the Blood Picture of Injections of Splenic Extract Date (1914) Fel). 6 Feb Feb. Feb. Fob. F'c!). Frb. Fol). Feb, Feb. 19 7 8 9 10 U 12 13 14 ir< 16 HsBtnoglubin Red blood cells 102 5,250,000 101 (10 O.C. splenic extract No. 16 injected) 5,650,000 110 6,500,000 (16 c.c. of same extract injected) 110 7,040,000 105 6,800,000 96 5,330,000 95 6,290,000 (15 c.c. splenic extract No. 88 injected) 101 6,700,000 (10 c.c. of same extract injected) 104 6,880,000 98 6,8r<0,000 % 6,120,000 106 5,540,000 96 THE SPLEEN AND ANiEMIA This experiment shows that intraperitoneal injection of splenic extract causes a sharp rise in hsnioglohin and red cell count, lasting only one or two days. This rise is repeated on reinjection of either the same or another splenic extract. In each of three other experiments with splenic extract an increase in the number of red cells was obtained, but this increase was not always as marked as in the experiment presented; it was nevertheless always greater than that caused by the use of control extracts of liver, kidneys, or blood. The study of the resistance of the red cells in these experiments may be dismissed with the statement that no noteworthy differences were found after injection of any extract. The skeined cells also showed no constant change. We had hoped that, as the latter are supposed to be young forms of erythrocytes, they would be found to be increased after the injection of splenic extract had caused a rise in the red cellcount. Only once, however, in which instance the percentage rose from 0.5 to 2, was this noticed. On the other hand, in two experiments they were not found at all in the blood after injection. Intraperitoneal injection of splenic extract is usually followed by an increase in the total number of leucocytes, consisting chiefly of the polymorphonuclear forms. A similar rise occurred in one of three experiments with liver and kidney, and in one of two with defibrinated blood. Several grades of "transitional cells" appeared in in- creased numbers. Eosinophiles were present in increased numbers in two of the four dogs receivir g splenic extracts, liut were also definitely increased in two of the five con- trols receiving other organ extracts. REG lATORY INFLUENCE OF THE SPLEEN 97 It would appear, therefore, that the intraperitoneal injection of saline extracts of fresh spleen constantly causes a sharp increase in red-cell count and haemoglobin content. The rise is evanescent, lasting but one or two days, and may be followed by an equally evanescent drop below noi-nial. Similarly prepared extracts from other organs fail to give this rise. Xo noteworthy change is found in the resistance of the red blood-cells to hypotonic salt solu- tions or in the number of skeined or reticulated erythro- cytes after the injections of the various organ extracts. A temporary increase of polymorphonuclear and tran- sitional leucocytes usually follows the use of spleen ex- tract, but may occur also, but less frequently, after the injection of liver and kidney. The constant increase of red cells in the peripheral circulation after injection of spleen, in view of the ten- dency to anamia following splenectomy, suggests that the spleen normally may exert a stimulating effect upon the formation of refl cells in the bone-marrow. 3. IXFLUENCF. OF FEEDING SPLEEN TO SpleNECTOMIZED Dogs This study complements that just described in that spleen in large amounts was fed to splenectomized ani- mals. The object was to determine whether through the influence of some necessary substance in the spleen the ana>mia following splenectomy might be prevented. The procedure is, of course, analogous to thyn Id feeding in insufficiency of the thyroid gland, and has an advantage over the injection of extracts in that it may be continued over long periods of time without the possibility of the com- plications occasionally occurring after injection. These 98 THE SPLEEN AND ANAEMIA experiments, it was hoped, would show whether or not the spleen exerts some ett'ect upon the hemopoietic system through peculiar bodies analogous, perhaps, to those of an internal secretion. Thus if the anremia following sple- nectomy depends upon the absence of a stinmlus, fur- nished normally by the spleen, to the hfemopoietic system in general, or to some part of it, as the bone-marrow, the feeding of normal fresh spleen unmodified by heat or chemicals might supply this stimulus and there would then be no ana-mia after the removal of the spleen. Method. — Five dogs were used. Four of these were given a diet consisting of raw hashed beef spleen, lard, and cracker-meal in amounts estimated, according to the weight of each animal, to suit its caloric needs. Of these, three were splenectomize various percentages of hypotonic salt solution. The two experiments given in detail in Tables XXVII and XXVIII represent the longest periods that animals have been obsen-ed after the administration of haemolytic serum. In all other experiments of tliis group, although the animals were carried for shorter periods, the general course of the ana?mia was the same. The two tables are deemed sufficient, therefore, to illustrate the onset and repair in this type of ana?mia. The first experiment (Table XXVII) is of interest chiefly in connection with "sl In lot THE SPLEEN AND AN.EMIA the question of the len^h of time necessary, in the sple- nectoinized as coniparcd with the norma', dog, for a com- plete regeneration of the hlf)od to occur after the destruc- tion caused hv hiemolvtic serum. It will he seen that in tablp: XXVII ErrecT of IIwEmolttic Sebum on V />od Picture or Splbnectomized Dou AND Control * Hsmolytic Immune Herum 61 DoK St. Wriifht s,li;() gm (2t) iaye after bdIp- Dog 53. Weight 7,7S0 (tm 2.5 re. h«pmolytic nectumyjO I'i'.r hipniolylirseruiiiperkjlo in vein Berum per kilo, injeeted lIipmoKluhin, per cent Kc.J bl.Kxi cells liefore HsenioglohiD, per cent. 92 Red blood cellj Before 90 5,230,000 6,200,000 23-2 hours 72 3,800,000 3 hours 78 4,970,000 1 day 00 3,5in,(XX) 1 day 38 2,060,000 3 days 06 3,920,000 2 days 30 2,2,'iO,000 6 days 54 2,730,(XX) 4 days 38 3,100,000 8 days 38 2,310,000 6 days 49 3,420,000 10 dava 55 3,100,000 8 days 42 3,400,000 14 davs 48 2,870,000 10 days 42 3,110,000 17 days 53 3,220,000 12 days 50 3,980,000 22 days 51 3,000,(X)0 17 days 64 3,810,000 28 days 67 3,510,0(K) 24 days 80 4,230,000 34 days M 3,31H).(XX) 31 days 86 4,, 530,000 44 days 76 4,880,000 66 days 81 5,120,000 r)6 days 85 4,210,()(KJ 86 days 92 5,380,000 70 days 63 3,.')10,(KM) 107 days 105 6,510,000 79 days 79 4.120,(K)0 100 davs 86 ■1,01(),(KK) 130 davs 82 4,J0(),(KK) 200 days 110 6,200,000 • The period intcrvrninK after .«pleiiorromy is inclicatod in parenthewa after the number of the uninial. The wDni "■hcfnrf" in thi- tiinL- eolmnn refers to Ihe blood count niude a Mhort time befon* the injection of the hiPinolytir a^enl and not to the blood count before Kplenrctomy. Likewise hours und daya in the ^a^ie column are indicative of the length of time after euch iajectioQ. the normal dog the lowest figures were those of the second day, and that an approach to normal figures was evident after two months, hut that the latter was not actually reached in the case of ha'moglohin until the third month and of the red cells until 107 days; on the other hand, the splenectomized animal cxhihiting a more gradual fall did REGULATORY INFLUENCE OF TIIE SPLEEN lOS TABLE XXVIII RECOVEBT FbOM iNiBCTION OF HEMOLYTIC SeRUM OP SpLENE("rOMIZED DoO A.ND CONTBOL I Time July 24, 1912 May 21, 1013 l3t injection 24 lire, later Ten moDtba ipleQectumy, dug 59| Time CuDtrol. dog 54 2d injection 24 hre. later 2d day. 3d day . 4th day. Splenectomy Weight 10,900 gm. Blood: red cells 5,200,000; ha-moglcjbin 86 [ler cent. Fragility: 0.3+, 0.45- Urine: no bile-pigment; trace of alhuniiu; no casts 0.2 c.c. hemolytic scrum Istinjection {)cr kilo. : May 21, 1913 Weight 12,8a) gm. j Ulood: red cells 5,400,000 ha'inoglohui 104i)er(ent. Fragility: 0.3 + , 0.5- I'rine: not obtained. Blood: red cells 4,820,000; hemoglobin 90 fwr cent.; serum free of hamoglobin Fragility: 0.35 +, O.tj- Urine: trace of albumin; trace of bile-pigment 0.2 c.c. per kilo, of same i scrum i I'fine shows faint trace of ; bile after 10 niin. 24 hrs. later : Blood: red cells 5,050,000; hamoglobin 94 [lercent. ; serum f reeof hu'moglobin Fragility: 0.35 + , 0.6- L'rine; trace of albumin; moderate amount of bile- pigment present Same dose of same serum 2d injection rejieated Blood: red cells 5,620,000; 24 hrs. later haemoglobin 95 fier cent. Trine: no hamoglobm; faint but definite trace of bile-pigment; trace of albumin Blood: red cells 4,030,000; 2d day. haemoglobin 75 per cent. Urine: bile-pigment abun- dant but less than in i dog 54 Bl : 0.325 +, 0.55 - Weiglit 12,450 gm. Blood: re00 Before 68 3,770,(M)0 \:>XM 3 davs i 70 2,:j20.(XK) 10,400 7day,s ; 74 3.1{X).0^ to 13 days after scrum was administered . 20 to 95 days after oc-rum was administered. Per cent, ult Bolution .).2J 0.3 0.35 0.4 0.45 0.5 0.55 + + P P o o + + 1' P p o () + + P P p p p + -1- P P p p p + + P P p o () + + P P o o o + + P P -1- + P P p p p + + P P p p p + + P P p o o p p o o o () p o * + inilicstes complete bsmolysia: I*, partial bcmolyaU, and O, do hamoljEis. i : U i,! )| M 110 THE SPLEEN AND AN.EMIA Here it is seen tluit the erythnxytes of the splenec^to- niized and the normal dog liad tlie same resistance to salt solution before the injection, and likewise the same de- crease in resistance ei<^ht days after the injection of ha^mo- lytic serum. Tlie cells of the normal animal, however, returned to almost the ori<,''inal : esistance after iwenty days, while those of tlie splcnectf)mized animal did not return to the same demia occurring early after splenectomy, and which remains present, although latent for months, rendering any new ha>molytic agent more effective and delaying the recovery from the anaemia which it causes. It would appear, therefore, that of the phenomena as- sociated with the absence of the spleen, two — the increased resistance of the red cells and the decreased tendency to jaundice after the administration of hicmolytic poisons — are correlated, but that the aiicTmia itself is dependent upon some factor, as yet unknown, which operates in the ab- sence of the spleen. After the administration of a ha'mo- lytic agent to a splenectomized animal this unknown factor dependent on the absence of the spleen prolongs the aiuenjia and retards repair, and the animal does not recover as quickly as does the normal animal. This, at the present stage of our knowledge, is the only explanation of the more severe and more prolonged ana?mia occurring in splenecto- mized animals receiving haemolytic a^^-ents. Nevertheless, the increase in the red blood-cell count that we have found to follow the intraperitoneal injection of splenic extract suggests that the normal spleen exerts a stimulating effect on the bone-marrow, which naturally is lost after splenec- I If ! 112 THE SPLEEN AND ANAEMIA toniy. It is probable that this loss may at least contribute to the retardation of repair in splenectomized animals, but the evidence on this point is insufficient to allow definite conclusions. 5. The Txfluekce of the Spleen Upon Iron Metabolis:m This investifration was undertaken to determine whether the tendency to anemia in splenectomized dogs and the delayed regeneration of the blood, after the admin- istration of hicmolytic agents to such dogs, might be due in part to some influence of the spleen upon the iron metab- olism, as has been claimed by Asher and his co-workers. Our present knowledge concerning iron metabolism may be sunmiarizee in intimate organic com- bination, the so-called food iron, incapable of giving the characteristic microchemical reaction, or whether it be in the form of an organic or inorganic salt of iron. Moreover, from the work of Hansermann '''° and of Abderhalden,^ it appears that, though iron salts are absorbed, the body is unable, or but very poorly able, to utilize them for the building of haemoglobin, being dependent for this construc- tive work upon the intimately combined food iron. On the other hand, iron salts are effective stinmlants to the REGLXATORY INFLUENCE OF THE SPLEEN 118 blood-fonning organs and conspicuously increase the utili- zation by them of the food iron. The elimination of iron occurs almost wholly through the intestines, especially the colon, the quantity passing out in the urine constituting less than one per cent, of the total excretion in man and the dog. In the fasting dog t!ie output found by Voit ^''^ was O.GO mgm. per kilo, of liddy weight per day, and on an adequate but iron-poor diet Gottlieb's ''' dog excreted 0.34 mgm. For man the Hgurcs are lower. Cetti and BreithauptJ" while fasting, eliminated about 0.10 to 0.13 mg- i. per kilo, per day, and in various studies on man 0.10 to 0.25 mgm. per kilo. per day have l^een found to be the intake required to maintain iron equihbrium. However, there is ever\' rea- son to believe, as is suggested by the work of Schniidt,^*' who fed mice for months on a diet extremely poor in iron, l)ut obtained no fall in the hemoglobin, that the organism possesses great power of conserving its iron and of reutil- izing it through some form of intermediary metabolism. ^Vhen, however, Schmidt withdrew iron from the diet for several generations, the younger generations were ex- tremely anemic, and this anaem.ia disappeared upon re- storing iron to the diet. As the iron-poor diet led to the disappearance of microchemically demonstrable iron from the liver, but affected to a much slighter degree that of the spleen, Schmidt concluded that the liver is the depot for iron from the food, and that the spleen, on the other hand, is the depot for iron fron tissue and erythrocyte catabolism, and thus an important factor in the interme- diary me'.abolism of iron. If the spleen plays this part in iron metabolism, its absence might well interfere with the reutilization of iron j ,4 1 114 THE SPLEEN AND ANAEMIA by the orguiiisin and lead to an increased iron elimination, and this Asher'^ and his co-workers, Grossenhacher '" a 1 Zinunerniann '' claim to have demonstrated in dogs. X ney studied the iron elimination of four puppies from two litters; one from each litter was splenectomized and one from each kept as a control. The iron estimations were made at intervals of a few weeks, two months, and ten months after splenectomy, and in all their experiments they found an output nmch higher, often double, in the sple- nectomized animals as compared with the controls. Methods. — In our earlier experiments we studied the iron elimmation during four-day periods, but found that such periods led to irregular results. In the work here reported, therefore, we present only observations based on periods of longer duration. The animals were placed in metabolism cages with glass floors, and afler they had been fed for several days on constant weighed amounts of the diet selected the rectum was emptied by the use of morpliine; iron-free charcoal was added to the next feeding and the collection of faeces begun from the appearance of the charcoal; at the close of the period the rectum was again emptied with morphine, carmine added to the next feeding, and the fa?ces coUectetl until carmine appeared in them. In the earlier experi- ments the urine also was analyzed, but as only traces of iron, less than one per cent, of the total elimination, were found the urine was omitted in our later analyses. To avoid the introduction of extraneous iron, the faeces were collected by means of a nickel spatula soon l ' n- being j)asseiO I 9 days Intake • 1.00 1.59 1.71 Output * Time after flplenectomy 1.42 1.89 1.88 Splenectomized 83 9 51 8,400 8.800 10,000 10 days 9 days 9 davs 1.42 1.35 1.32 1.39 1.56 1.42 27-37 dava 9 months 20 months * Figuru'9 represent milligrammes of iron per kilo, per day. Inspection of Tahle XXXIII shows that the iron out- puts of Dogs 88 and 35 was unchanged hy splenectomy, hut tliat Dogs 30, 44 and 79 showed some increase. On the other hand, in Table XXXIV it will be seen that all three splenectomized dogs exhibited an output of iron as com- t f •II ! m 118 THE SPLEEN .VXD AN.EMIA pared with the intake elosely comparable with that of the controls. From these studies it would appear, therefore, that during the tirst two weeks after splenectomy some, but not all. (logs show a slight increase in the output of iron, but that at one month, nine months, and twenty months after splenectomy we lind no indication of such increased iron output. The occasional evanescent and inconstant increase in elimination of iron does not justify the conclu- sion that the spleen exerts an important intluenee on iron metabolism. Our results are obviously different f i-oni those of Asher and his associates, and as a possible explanation of this we would call attention to the extreme shortness of the periods — one to three days — employed by Asher and Grossenbacher, and tc. their failure to mark in any way the stools. In the studies of output ten months after splenec-tomy, as given by Asher and Zimmermann, the splenectomized dog in most of the experiments was much larger than the control, so that if the iron output of their dogs be calculated per kilo, of body weight it will be found that the output of the splenectomized animals approaches ver\' closely that of the normal controls and is in some instances identical. It seems possible that in these studies ten months after splenectomy the apparent increase in iron output of the splenectomize.■) dnys for dog 4H; 13, 41, and 70 diivn for dog 5J; 10 and 86 daya fur dog 5ft, and 3 daya for dog 57. In three of four dogs no Important change in the elimin- ation of iron occurred after splenectomy. In the fourth (Dog 56) there was an increase of 1.0 mg. per day during the ptHod (ten days) immediately after splenectomy, amounting to an increase of 21.6 per cent, over the fore- 4 :i iS 1^20 THE SPLEEN AND .VN^:M1A period. In the final period three months after splenectomy the output showed an increase of 148 per cent, over the fore-period and double that of Period II. The intake of iron was not determined, hut, since the food i take was constant throu^^iiout all the periods, we liave reason to be- lieve that this was a constant factor. Of the four animals, two showed no ana-mia and a third only a slight reduction in hivmo^lobin and red cells. The fourth do^ (No. .56) showed a relatively severe ana-mia (see Table I.I 1 1 in sec- tion devoted to metabolism) . As this last dog- was the only one to show any unusual elimination of iron after splenec- tomy, the question naturally arises: Is the increased elimi- nation of iron due to the an.Tmia or to the absence of the spleen f We incline to the former view and conclude that the spleen exerts no constant and inijiortant influence upon iron metabolism.* * One otluT attempt to elucidate tlie iron proMeiii lias been the feeding of an inorganic salt of iron before and after spleneetoniy. Sucli ex])eriinents 'n the normal animal are, as a rule, of little v iliie, as iron so ade istered is not absorbed to any appreciable extent, but is eliminated . most eomi>leli ly."'' It seemed worth while, however, to see if any change in absorption occurred in the absence of the spleen. 1 Avo dogs on the usual eonstan! diet were, therefore, fed daily .">.") mgm. of iron as ferrous sul|)h.ite during :i ])eriod of one week before and ,i like period after s])leii( etomy. In e.ieh inst.ince the second l)eri()d of analysis began four days .after splcneitomy. No essential or constant difTerence was found in the elimination of the two periods. In one animal, of ;"),'> mgms. of iron gi\en daily, .'iS mgm. were eliminated daily before and .'50..') .after splenectomy: in the other animal the figures wire .'■)1.'2 before and .V,'..j after splenectomy: essentially negative results. l! ! !i CHAPTER V CONTROL EXPERIMENTS: THE DIVERSION OF THE SPLENIC BLOOD FROM THE LIVEB WITHOUT REMOV.\L OF THE SPLEEN (1) BY LIGATION OF THE SPLENIC VEIN, (2) BY TRANSPLANTATION OF THE SPLENIC VEIN INTO THE VENA CAVA, (3) BY ECK FISTULA. IIiTHKUTo in all our discussions of the phenomena f'ollowin^^ splenectomy— the an;emia, the increased resist- ance of the red cells, and the decreased tendency to jaun- dice — we have assumed that, inasmuch as the spleen was ahsent, the changes descrihed are prohahly due to the loss of some function peculiar to that organ". As to the exact nature of the function lost, no definite opinion has thus far been given. Three possibilities, how- ever, readily suggest themselves: (1) The loss of some function of blood destruction or regeneration resident in the spleen itself; (2 ) the loss of an internal secretion acting on the distant luemopoietic tissues, as the bone-marrow! and (3) the obliteration of the venous drainage of the spleen, which, in that it is an important source of the portal blood, may have an essential relation to some peculiar function of the liver. It is obvious that the value of some of these hypotheses might be tested without removal of the spleen by diverting its venous outflow from the liver. We have therefore repeated "'^ many of our experiments, l)ut. instead of removing the spleen, the splenic vein has either been ligated or transplanted in the vena cava, or an Kck fistula has Ix^en established. So far as we know, exactly similar experiments have not previously been at- 121 1! ( ; : >. Ui THE SPLEEN AND AN.EMIA ;l 1 limber, color, and ;i hearing on the tempted, though two ohservalions 1 problem. Xa^s;•ll•'• found that t resistance of the red cells remained unchanged after simple Eck fistula, but, as he gives only one count before and one after the operation, his observations are not conclusive. The other report wliich concerns us is Pribram's'"' arti- ficial constriction of the splenic vein with resultant passive congestion of the spleen. The slight anaemia that this caused is analogous to some of the results we report below. If the aiKcmia following splenectomy is caused by the removal with the spleen of some necessary factor in blood formation or of a hormone essential to the hannopoietic tissues, this factor should not operate in the vein trans- plantation and 'Ick fistula experiments unless it is also essential that such a substance have direct approach to the liver for its proper functioning or activation. If, on the other hand, the aiuemia is largely due to the inter- ference with the supply of sjilenic blood to the liver, it should occur in all the animals. These problems and also that concerning the influence of the experimental pro- cedure on increased resistance of the red cells will be con- sidered first, and then j>roblems such as the decreased tendency to jaundice and the prolonged repair of ana[?mia in the splcnectomized animal when hiPinolytic agents are given. Methods.— Tn the ligation experiments it was found necessary, on account of the immerous branches and the anastomoses of t^e splenic vein with veins from the stomach, to tic all branches of the splenic vein shortly after they left the spleen. In the earlier operations one of the subdivisions of the artery was also ligated. to lessen the supposed danger of rupture. This, later, was abandoned CONTROL EXPERIMENTS 123 vhen it was found to be uiuieccssarv as well as undesirable on account of infarct production. Post-mortem examina- tion of animals subjected to this operation showed in each case that all brandies had been tied, that the vessel beyond the li^ration was much diminished in size or completely obliterated, and that there was usually little attempt at compensatory development of new veins from the adherent omentum. When such new-formed veins were present it was found that seldom did they empty into the portal system and therefore, as a rule, did not complicate the experiment. Yjck fistula was performed in the usual manner, the proximal end of the portal vein being tied off above its new anastomosis with the vena cava just before its entrance into the liver. Transplantation of the splenic ve-'n into the inferior vena cava ])resented considerable difficulty on account of the smallness of the vein and the necessity of stretching it a little to make it reach the vena cava. In only one experiment, however, was it found that the trans- j)lanted vein had been occluded by thrombus. The success of the operation was always determined by examination of the vessels at autopsy, and in some instances injection specimens were prepared and dissected to make doubly sure that no new anastomoses had formed. The particular phenomena studied were (1) the quanti- tative changes in counts of the red blood-cells, leucocvtes, and haemoglobin; (2) the resistance of the red cells to hypotonic salt solution; (3) the general condition of the animals as indicated by their weight and condition of the urine, and (4) the gross and microscopical appearance of the organs at autopsy. The dogs were kept on the usual mixed diet of " table scraps," which has been shown to maintain properly normal ill 124 THE SPLEEN AND ANEMIA dogs as well as those convalescing from operations other than splenectomy without the development of ana'mia."' Our studies have been made on twelve dogs, grouped as follows: Ligation of splenic vein, 4; transplantation of splenic vein, 2; Eck Hstula. 'J: and as controls: Sple- nectomy, 8; transplantation of thr inferior mesenteric vein, 1.' In the case of the last dog it was intended to transplant the splenic vein, and it was not until autopsy that we found that the inferior mesenteric vein had been used by mistake. Changes in the Spleen In those dogs in which all branches of the splenic vein had been ligated, and in \\hich adetjuate new venous chan- nels had failed to develop, the spleen showed considerable change. Before the operation was completed the spleen had increased to almost double its size and taken on a dark- purple color, li' the animal was allowed to survive two or more months the spleen, at a itopsy. was found to be considerably smaller and much firmer than normal. The capsule was slightly thickened and jjuckered, and the organ had a pale-bluish coloi". On section the tissue cut with increased resistance. A few small shrunken infarcts were occasionally founal)le evph,,uation seems to be that the elastic spleen is able not only to accommo- date the increasing pressure without rupture, but. with the aid of the minute capillaries in the adherent omentum, to maintain sufficient degree of nutrition to prevent Jiecro- sis. We have seen, however, that the exchange is not sufficient to prevent atrophy. At the time of the operation for Eck fistula and the splenic vein-vena cava anastomosis there is necessarily a temporary (X'clusion of the large veins. The spleen and intestines I>ecome verv' dark and turgid, hut when the clamps are removed these organs return quickly to normal color. In animals so treated no changes are found at autopsy except adhesions and perisplenitis. Changes in tiie Blood All example of the changes in the blood which occur after ligation of the splenic vein is given in Table XXXVI. It may be seen that *' is animal developed a moderate degree of anaemia lasting several weeks and similar to, but less severe than, that following splenectomy. The ana?mia is accompanied by a temporarv' slight increase in resistance of the red cells to hypotonic salt solution. As in splenec- tomy, there is an immediate leucocytosis, due to polymor- phonuclear and transitional fonns, but prolonged by a more persistent IjTnphocji^osis and eosinophilia. f : 126 THE SPLEEN AND ANEMIA A very slight drop in weight oceurs after operation, hut later there is a distinct increase above the original weight. It has been shown elsewhere,-'^' by the study of control operations such as nephrect(jniy, that the esults here descril)ed are not merely post-operative. Other dogs with ligated veins gave similar results, as, for example, TABI^ XXX\"I Blood Changes Followino LauATio.v of Splenic Veins a ! . HiDmolysia • \ ^ i ..;. 'Z Ued UooA 1 ^S p* « g « JS Dog 47 cells per 1 " s •- fc- *' a •C 1 cmm. = £ »1 1 ^ 5 Begins Com- plete 2» e a o a "5 ^ Eo u S o ■J S 1^ CL, CO "^ i* per crnt. kxlc. Before 102 6,275,000 0.475 0.35 9,S0o! 7,200 700 1,800 100 6.0 1 (lay after ys 6,110,000 19,00015,200 950 2,850 5.7 4 (lays after 92 5,;}5(),(KK) 0.475 0.275 16,500 5.6 1 week after IK) 5,520,OO(J 0.425 0.275 il6,S00 13,600 1,3(X) 1,600 300 5.4 2 weeks after 72 4,9(K),0(M1 0.425 0.3 18,80015,400 1,<.KK) 1..500; 3 weeks after 70 4,tK10,(«X) 0.45 0.3 21,(XX) 16,800 3,100 i 300i300 4 weeks after 78 5,000,(KX) 18,600 6.4 .5 weeks after 74 4,;5SO,(KM)' 15,000 7 weeks after SO 5,aio,ooo 0.475 0.275 12,80010,000 1,700 i,ioo; 7.3 10 weeks after 82 5,0.')0,00() 0.45 0.3 112,400 12 weeks after 95 5,S30,00(» 0.475 0.325 14,800 9,500 3,400 1,500 400 7.5 1,'5 weeks after 92 l),4 10,000 0.475 0.35 14, 9,200 3,600 1,200' 900 14 weeks after 95 1 6,150,0001 15,60012,200 2,500 1 ' 600 100 7.4 * The figures in those coldmns refer to the strongest perrentflceg of salt solution in which hnmoly^ia was drat nuticcj ami in which complete hieniulyais lirat occurred. Dog .51 with a nuixiiniim drop of 14 per cent, of hitmoglo- bin and 1,200,000 red cells, and Dog 74 with a hivmoglobin loss of 24 per cent, and in red blood-cells of 1,800,000. The effect of diverting the splenic venous blood from the liver by transplanting the splenic vein into the vena cava is shown in Table XXXVII. Many of the same changes as after ligation are found CONTROL EXPERIMENTS lil liere. In another dog (No. 71) siniiJarly treated the in- creased resistance was much more marked; before opera- tion, hsemolysis began at 0.50 and was complete at 0.35; soon after operation it began at 0.425 and was complete at 0.25. Anaemia was also present, as shorni bj- a drop of 20 per cent, in ha-moglobin and of 1,800,000 red blood- cells. A third animal (10) lost 35 per cent, hemoglobin TABLE XXXVII Blood Changes Following Anastomosis OF THE Splenic Vein ^ 'iTH THE Vena Cava a Hamolyais • • a 'S s jg Dog 4 ^ Red blood l-e t£ B 12 c r s cmm. Begiua Com- plete e a o a 1 a- ■e o 1 •*' — 0. a tJ K ^ per cent. 1 kilot. Before 97 6,960,000 0.45 0.35 16,100 8,400 4,70011,600 1,400 9.4 1 day after 104 7,(M0,000 0.425 0.3 34,000 29,600 3,100 1,3001 1 week after 75 5,.5.50,000 0.45 0.3 19,300 12,400 3,700 1,900 1,30Q 9.7 2 week; after 68 5,020,000 0.475 0.275 16,900 11,800 2,7-^) 2,200 20f| 9.4 3 weeks after 70 4,530,000 96 4 weeks after SO 4,510,000 0.475 0.3 14,700 9,600 2,800 1,500, son 10.4 5 weeks after 74 5,440,000 0.475 0.325 14,000 9,800 2,800 420| 1,08(1 10.5 6 weeks after 78 5,630,000 0.45 0.3 11,200 6.300 4,000 560' 36(1 < weeks after 82 5,770,000 0.45 0.325 1 11,100 7,400 2,900 200 60(1 12.9 8 weeks after S5 6,060,000 0.45 0.325 '.» weeks after 96 6,500,000 1 1 13.2 * The fiKurcs in these coliimna refer to the utrongent porrentages of anlt solution in which ha^mulysis w:« tJral noticed uuil in which complete haimolyais firat orcurnd. and o> a- 2,000,000 erythrocytes, and a fourth, 22 per cent, hemoglobin and over 1,000,000 en'throcytes. The changes in an Eck fistula experiment are seen in Table XXXVIII. In another Eck fistula dog (Xo. 68) the increased re- sistance of the red cells was more marked (before opera- tion, haemolysis began at 0.45 and was complete at 0.3; soon after operation it began at 0.425 and was complete S'"--! ii:)i I 44, 128 THE SPLEEN AND .\A^MIA at 0.225), and the ani.iial lost 33 per cent, hajmoglobin and 2,000,000 red hlootl-cells in four weeks' time after operation. In a third (Xo. 5) with an even greater drop, the blood picture was complicated by the occurrence of infection, for whicli n-.-ison the figures are not given. That interference with the portal circulation inflow from organs other than the spleen may cause changes in TABLE XXX \ 111 13loi)d Chanue:* Following Kck Fistula •2 j 1 H»moly5iJ* O J3 4 ..a 0, d J5 ^ Red blood &E E iS Dog 31 w celU per C if "" BE c *« g t cmm. Com- = s ?.i — s ffi a J3 * Begins plete Sg. "o 3 86"^ 1 X _>j^ b. w - per cent. hlo$. Before 99 0,500,000 0.45 0.325 13,200 9,800 2,700 500 200 16.1 1 (lay after SS 0,040,(X)0 0.475 0.325 36,0(JO 32,400 2,800 800| t 3 days after 84 6,300,000 0.45 0.325 32,4t)0 27„500 2,tHX) 1,000 400 14.2 5 clays after 72 5,40().(KX) 0.425 0.3 24,0(M)17,;iO<> 3,800 2,400 .500 14.2 1 week after 74 5,500,000 0.425 0.3 18,80013,700 3,400 800 900 13.6 2 weeks after ()8 5,040,(X)0 0.425 0.275 19,60015,500 3,900 000 600 13.5 3 weeks after 73 5,110,0(X) 0.425 0.275 i 1 4 weeks after 67 4,460,000 0.425 0.3 21,00015,500 4,400 000 .500 14.6 5 weeks after 72 4.8.80,000 0.475 0.32.5 18,400 12,900 3,700 6(>0il,200 14.2 6 weeks after 84 5,600,000 0.475 0.325 16,000 10,500 3,800 400:1,300 14.9 7 weeks after 90 6,200,000 0.45 0.3 15,200 10,300 15,80011,400 2,800 t)001,.500; 15.3 8 weeks after 98 6,520,0(X) 0.45 0.3 3,500 1 300 600; 15.4 * The figurt'9 in these columns indirate the pcrrentaKPs of salt solution in which hemolysis was 6rat noticed and in which hEemoljsis first became complete. the blood is shown in another experiment (see Table XXXIX) in which the inferior mesenteric \ein was anas- tomosed with the vena cava. This dog developed an anaemia of mild grade and re- covered from it sooner than did the other dogs of this series. Although little weight can he placed on a single experi- ment such as this, the observation tends to support the theory that the supply of portal blood to the liver is a CONTROL EXPERIMENTS WJ factor in the produc-tiuii of the changes we have obsened to occur after splenectomy. From these ohsen-ations the following conclusions may he drawn: 1. In donrs in which the spknic vein has been ligated or transplanted into the inferior vena cava, or in which an Eck fistula has been made, an ana;mia occurs which re- TAULE XXXiX Blood CHANOEa Folluwino Tkansi-lantation of Inferior Mesenteric Vein. Control Kxpekiment Dog 55 Before 2 days after . 3 days after . 10 days after. 2 weeks after 3 weeks after. 4 weeka after. ■) weeka after, ti weeks after. Hsemu- Ucil Lluod rells p«r cmm. ilOMilO lysis • gtobin Weight Begioa Cumplete per cent. kilo$. 100 5,940,000 0.45 0.3 14.7 76 o,240,(KX) 0.4 0.25 75 5,10(J,(H)0 0.4 0.25 13.9 7(j 4,S.S0,000 0.45 0.275 85 5,400,000 0.425 0.25 84 5,S90,(X)0 0.425 0.25 78 5,600,000 14.7 88 5,720,000 92 5,800,000 0.425 0.275 15.3 -.. fi J,\^ 'i*"T '" '.hesc rnlumn'< indicate the perrpntsup.H of salt solution in whirh hemolysis wag first noticed and in which hjemulysis first became complete. u'oiyi sembles that following splenectomy and shows the same general variations in degree and duration. 2. The resistance of the red cells to hypotonic salt solu- tion is quickly increased, sometimes coincident with and sometimes preceding the anaemia. It gradually returns to normal in about the same length of time as it takes the anfcmia to di.sappear, differing in this particular from the results after splenectomy. 3. There is an initial leucocytosis, involving at first the polymorphonuclear leucoc^-tes and transitional cells. As the total leucocytosis diminishes there is both a relative 1 1 ,^. 130 THE SPLEEN AND ANJiML\ and actual incrcahc of small lymplu>cytes and usually also (.r eosiuophilcs. This may either he temporary or last during the rest of the period of ohservation, and thus differs from the ordiriaiy postoperative leueocytosis. 4. Ligation of the splenic vein is followed hy consid- erahle atrophy of the spleen, hut not hy necrosis or throm- bosis. There is rarely adecjuate new vein fonnation. The other operations cause little or no change in the spleen. .5. \Vhether the disturbances described are due to the loss of a certain volume of blood to the liver, or, as has been previously suggested, to the loss of a splenic 1 ormone acting on the ha-mopoietic tissues, it is impossible to say. If due to the former, the method of production of the ana?mia still remains unexplained. It is evident, also, that the latter theory has no value uidess it is assutned. also, that the supposititious hormone normally is activated by passage through the liver. RESISTANCE TO HEMOLYTIC AGENTS In the previous pages has been discussed the effect upon the blood picture of tliree methods ( transplantation of the splenic vein into the vena cava, Eck fistula, and ligation of the splenic vein) of diverting the splenic blood from the liver. It now remains to present the res\dts of a study of the influence of these procedures on the action of hjrmolytic agents. Do they bring about a decreased tendency to jaundice when a ha?molytic agent is given and retard the repair of an artificially produced anaemia, as is the case in splenectomizcd animals? As ha-mohiiic agents we have employed both toluylene- diamine and ha?molytic immune senmi. The Iwmolytic senun was prepared by injecting, at regular intervals, the CONTROL EXPERIMEN'I^ 131 erythicK.'vt.e.s of the do^ into rabbits. All injections of strum so prepared were made intravenously into the do^. Merck's nieta-toluylenediamine was given by stomach-tube, and in adequate doses never failed to cause anaemia and jaundice. In each experiment the control animal received the same proportionaiie amount of dru^ or serum per kilo, of l)fMly weight as did the test animal. Dogs of approxi- mately the same size were selected, and, on account of the occasional necessity of catheterization, female dogs were used whenever available. Splenectomized as well as nor- mal animals were included in the series tf) further com- parison with our previous results. Daily and, later, weekly examinations of urine and complete blood examinations ( including resistance of erythrocytes to hvpotonic salt solu- tion) were made and records of weight were kept. Each animal was studied as to condition of urine and blood be- fore the experiment. Both cage urine and catheterized TABLE XL Jaundice After Administration- of Toi.uTLENEDiAunN (0.34 Gm. Per Kilo.) D..K 71 Don "9 Dog 51 - Splenic vein trans- Sploneoiomv Do« 75 NorniaT control Time aftpr admiaistratioQ plant <2 mo8.) (I'i moa.) (2 mo« ) 42.') B H- 40 B. H - 0.4.5 B. H.- 42« B H (1 32.5 C. H.* 275C H. 275 C. H. 27.5 C H No ^.nemia No an»mi:i .flight anemia No an»mia Before Urine normal Urine normal t'rine normal I'rine normal 1 day after Bile trace Bile slight Bile moderate Bile marked 3 days after Hile heavy trace Bile slighi Bile moderitr Bile marked 5 days after Bile trace Bile moderate Bile moderate Bile marked 7 da>'s after Bile absent Bile moderate Bile moderate Bile marked 9 da>-8 after Bile absent Bile moderate Bile moderate Bile moderate 1 1 daj-s after Bile absent Bile trace Bile faint trace Bile trace 13 days after Bile aljsent Bile trace Bile absent Bile trace 15 da\'s after Bile absent Bile trace Biln absent Bile trace 17 da>-8 after Bile absent Bile absent Bile absent Bile absent * Thi§ space in this and following tables refers to the condition of the blood before beinnnlng the eiperimenl The finures inriiciite strenrth nf salt solution at which hffmolvsis occurs; B H indicates beirinnine hrmolvsis. C. H. complete hfpmolvsis. Increasing amounts of bile in the unne are expressed in the following terms; faint trace, trace, h»avy trace. Blight, moderate, marked. '$m If '" ^1 i; 132 THE SPLEEN AND AN.EMIA specimens (in doubtful cases) were examined, and the presence of bile-pij^ment in the urine, determined by Gmelin's and Rosenbach's tests, was taken as the surer . evidence of jaundice. At the termination of the experi- ment tlie animals were sacrificed in order to determine the exact anatomical disturbance caused by the operation. As may be seen in Table XL, in all animals receiving toluyknediamine, control as well as experimental, some bile appeared in the urine. The bile was less in amount, how- ever, and lasted for shorter periods in the test animals than in the normal controls. In fact, animals with the vein transplant and Eck fistula showed even less tendency to jaundice than the splenectomized animal. TABLE XLI Jaundice After Administration of Toluylenediamin (0.13 Gm. Per Kilo. ) — ^-;- Don OK DoK ss Dog 77 Eck fintula MpFienterii- vein trans- Nornial control Time after (1 mo.) plant (1 'j mos.) No anffniia administralion 0.4 H. H -<)275C. H. 0.4:^5 B H.-O 27.'-- C. H. 0.425 B. H.-O 35 C. H. Anaruia prcsenl No anten-.ia Before Ifriiie normal I'rine normal Urine normal 1 dav after Hile absent Bile trace Bile trace 2 davs after Hile faint traee Bile trace Bile marked 3 davs after Uile faint trace Bile ni(Hlerat;e Bile moderate 5 days after 7 days after 9 days after Hile absent Bile doubtful Bile moderate Hile absent Bile al)sent Bile trace Bile absent Bile absent Bile absent Another experiment (see Table XLI) in which a smaller single dose was administered gave similar results. It was in this experiment that the animal that was sup- posed to have had the splenic vein transplanted was found at autopsy to have a brancli of the inferior mesenteric vein transplanted ])y mistake, thus unwittingly causing an excel- lent double control for the Eck fistula test (No. 68) . The increased resistance to jaundice of this dog (No. 55) as CONTROL EXPERLMENTS iss compared to the normal (Xo. 77) is still further evidence ol the importance of the mechanical factor of blood supply to the liver. In a third experiment in which three smaller repeated doses of toluylenediaminewere given to animals with splenic vein transplant and splenic vein ligation and to a normal control these results were confirmed. The vein transplant dog, though his red cells at the time were the least resistant of the three, failed to develop jaundice; the other two did. The jaundice in the ligation experiment, however, lasted three days, and that of the control nine days. In the experiments with hemolytic immune serum the same results were obtained. In a i.eliminary experiment TABLE XLII Jaundice After Admini8tration of H^moi ytic Sercm (Sercm No 3 4 c c Per Kilo.) ' Time Kfter , Do(t4 Vein twitch (2 mo8.) Dog 2 npiriiectomy (2ia mos.) ^^ Dog 25 Normal control 0.40 B. H.-0 325C. H. Slight aniemia 0.425 B. H.-0.25 C. H. Slight antemia 0.475 B. H -fl 35 C. H. No ancemia Before 1 day after ~ days after 4 days after () days after Urine normal No bile No bile No bile No bile Urine normal Bile moderate Bile moderate Bile moderate Bile absent Urine normal Bile trace Bile moderate Bile absent with three animals, in which large doses of senim were given, the Eck fistula dog alone survived and developed jaundice; one with vein transplant and also a normal con- trol died in a few hours; the control, ho-vever, had already developetl hamioglobinuria, whereas the vein transplant dog failed to develop either hcTmoglobinuria or jaundice. With a weaker serum (see Table XLII) the greater resistance of the vein transplant animal is shown. A result 11 * 134 THE SPLEEN AND AN.EMIA contrary to our former experienc.-s was obtained in this experiment, in that No. 2 (splenectomized) developed as severe a jaundice as did tlie control. In another experiment (see Table XLIII) '.'le same instructive results were obtained with a serum of less ha^mo- lytic power. TABLE XLIII Jaundke After Admimstkation of H.KMoi.-iTic Serum (Perim No. 3, 0.2 c.c. Per Kilo.) Time altfr admiaidt ration Dog h> Vein transplant ^occluii«'*i vesHcl.*) (1 uio.) Don 1 SpW'nectora> (;j nu)3 1 Dog 51 j5„ 27 Ligation Normal control (t» nios ) OiB H- 2.^1 C H. .\Q»naa pre.sfnt 0.4 B. n - ().i!.5 C H. Slight aneemia 45 B H.- 0.a25 C. H. No anemia O.lj B H - 35 C H No anffmis Before 1 day aftor ? days after 3 days after 5 days after Irine normal No bile No bile No bile No bile Irine normal No bile No bile No bile No bile Trine normal No l)ile Bile marked Bile marked Bile marked (Bile still pres- ent on the 14th day) I'rine normal Bile marked Bile moderate Bile trace Bile absent As the transplanted splenic vein of No. 16 was found at autopsy to have been occluded by a comparatively recent thrombus (probably antedating the administration of serum), this exj)criment must be -egarded as analogous to a ligation experiment. The relatively slight tendency to jaundice in this animal as compared with No. 51 may be largely explained by the greater resistance of the red cells in the former animal. The results of these experiments, while somewhat dis- cordant, indicate that the mechanical factor of the method of the blood's approach to the liver is of importance in de- termining the degree of jaundice after the administration CONTROL EXPERIMENTS 135 of haemolytic agents. Our results in this regard are in accord with those obtained in our comparative studies of the effect of injecting ha?nioglobin into the portal as con- trasted with the general circulation (see page 64). The interpretation of the severity and duration of the anipniia caused in the various test animals is complicated by several factors. In the first place, the original operation iiecessar}' to produce the venous anastomosis has been sliown to cause anaemia. As the test animals were some- times given the hjemolytic agent while more or less ana?mic, we have had to analyze our results with constant reference to this factor. Moreover, we have found that in most cases the severity of the anfcmia largely parallels the resistance of the red cells. In some cases, however, animals with the most fragile cells develop the least anaemia after adminis- tration of ha?molytic agents. Changes in weight in the animals of these experiments seem to liear no definite significant relation to the anaemia. We have previously stated "' that " the anfemia caused in splenectomized animals by h^moh-tic agents is, as shown by direct blood examination, of greater severity, nms a longer course, and has a longer period of repair." By greater severity was meant that the haemoglobin and red- cell count reached lower figures than in the control. The actual blood destruction, however (if determined by esti- niating the change from the condition immediately before administering ha-molytic agents), was usually less in sple- nectomized than in normal dogs. Oiar experiments of this year show that the same statements hold true after trans- plantation of the splenic vein into the vena cava, Eck fistula, and ligation of the splenic veins. The actual blood destruction of the test animals has I i- \-\n 136 THE SPLEEN AND AN.EAUA i?v been constantly less tlian in the normal controls, and in a few instances even the degree of ansemia as expressed by the hicnioglobin and red-cell count was less severe in the test animals. As a constant feature, the greater severity of the anaemia nmst therefore be considered as open to TABLE XLIV DURATKIN AND SEVERITY OF A>f.EMIA AfTER ADMINISTRATIO^f OP ToLUYLENE- DiAMi.v (0.34 Gm. Per Kilo.)* Time after sdministratiou Before 1 day after 3 days afferi 5 days after' 7 days after 9 days after 12 days after 16 days after 22 days after 29 days after, 35 days after 41 days after 49 davs after Time after odminiatration DogTl Splenic vein transplaDt 98 Red blood celL^ 6,785,000 6,940,000 5,400,000 5,340.000 4,820,000 5,070,000 5,8',)O,0OO 6,210,000 6,350,000 6,800,000 Resistance B. H. C. H. 0.45 -0.325 0.5 -0.3 0.475-0.275 0.475-0.325 0.475-0.325 0.5 -0.3 0.5 -0.3 0.5 -0.3 0.5 -0.3 0.475-0,325 Dog 79 Splenectomy Hiemo- globin per cent. 74 84 60 43 62 63 66 69 68 76 88 88 92 Red bIt>od cells Resistance 5,720,000 4,770,000 4,250,000 3,380,000 3,820.000 4,080,000 4,.320,0(X) 4,530,000 4.510.000 5,2,80,000 5,5i)0,000 5,480,000 5,680,000 ' B. H. C. H. i 0.4 -0.275 : 0.5 -0.275 I 0.426-0.275 0.45 -0.25 i 0.45 -0.25 0.425-0.25 0.425-0.25 0.4 0.4 0.4 0.4 0.4 -0.275 -0.25 -0.275 -0.35 -0.25 Before 1 day after 3 days after 5 days after 7 days after 9 dpys after 12 days after 16 days after 22 davs after 29 days after 35 days after 41 days after 49 days after Hsenio- globin Dog 81 Ligation Red blood 1 Dog 75 Normal control pfT rent. 80 90 60 4,020,000 4,.S0O,000 Resistance B. II. c n 0.45 -0.275 0.5 -0.325 3,820,000 0.375-0.25 0.45 -0.3 0.45 -0,275 62 3,3101)00 65 3,9S(),(M)0 72 4,320,000 74 4,6.50,000 7() 4,0.'j(),(>i»<) 84 5,330,000 78 5,340,000 87 5,550,000 0.475-0.35 0.425-0.3 0.45 -0.3 HBmc ' frlobin Red blood cells Resistance per cent. B //, C. II. 85 6,180,000 0.42.5-0.275 96 5,080,000 0.45 -0.35 48 4,590,00*1 0.47.5-0.275 38 2.980,000 0.45 -0.3 45 3,6.30,000 0.475-0.3 55 3,930,000 0.45 -0.3 68 4,800,000 0.47.5-0.3 SO 5,200.000 0.475-0.3 85 6,820,000 0.425-0.3 * TliL- fiiiurf^a in (lie onluinii!! "UewiMtanrf^" indiratp the perreotages of salt sotutioo at which hemolysiB waa tirat noticed and first became complete. CONTROL EXPERLMENTS 137 question, while the actual blood destruction in the test ani- mals is undoubtc-dlj less than in the normal controls. The dui-ation of the anemia of the test animals has been longer than that of the normal controls and paralleled that of the splenectomy controls. The difference, how- oxer, of both test and splenectomy dogs from the normal controls has been less striking than in our previous work, and, like the variable degree of anaemia after splenectomy,' Miust be referred to variations in unknown factors (pos- sibly such as diet, or differences in toxicity of the serum). An experiment with tohiylenediamine showing the char- acter of the blood repair after various forms of experi- mental disturbance of splenic function is seen in Table XLIV. TABLE XLV DnRATION AND SeVERITT OF An.^JHA ApTER AdMINISTOATION of H^MOLTTIC Sercm (0.4 cc. Per Kilo.) Time sfter administration Dob 4 Vein transplant (2 mos.) ring 2 yplenertomy (3;-5 nioa.) Before 1 day after 6 days after 10 days after I () days after 19 days after liO daj's aft«r ■i 1 daj a af t«r Hffmo- dlobin prr cent. 97 83 6,5 62 70 90 96 102 Red blood rella 6,960,000 i 4,840,000 3,940,000 3,750,000 i 4,390,000 i 5,060,000 I 6,010,000 6,400,000 I Hffimo- globin per cent. 85 78 55 67 67 78 82 Ued blood cell* 4,950,t00 4,920,000 3,670,0e raised against its use, and to over- come this objection we attempted to study the marrow of the compact bones. The methods of decalcifying tissues have, however, in our hands failed to yield satisfactory histologic preparations. The alternative, the use of film preparations, obtained successively at intervals over long periods of observations, being impracticable, the study of cover-glass preparations was limited to a single observation CHANGES IN BONE-MARROW 143 at the time of the death of the aiunial. At the same time, however, in many instanees marrow squeezed from the rihs has been obtained in sufficient amount to section and thus to allow a comparison with chancres in the fatty mar- row We have, however, depended lar^^ely upon the study of sections of the marrow of the lon^ bones, and in par- ticular of the femur. We are sati-fied, as the result of our study of the marrow from a lar^e number of normal do^s. that this is, after all. the most rational method of studying compensatory changes, for it is unusual, even in a definitely fatty marrow, not to find numerous centres of blood-form- ing? cells. These may be limited to the periphery' of the marrow or be scattered throughout, but, whatever their position, they afford an excellent starting-point for the study of increased cellular content, as well as of changes in the character of the cells. The fatty marrow is of especial value in the study of the late changes, for in well- fixed and well-stained marrow there can be no doubt about the change from a purely fatty marrow to a red marrow rich in cells. This is so striking as to remove all the doubt which exists when one examines the marrow of compact bone, as of the ribs or vertebne, by either the section or cover-glass method. We have worked exclusively with the marrow of the middle third of the femur, avoiding the marrow at either end, partly on account of its bony nature, but chiefly be- cause of the occasional normal occurrence of more or less red marrow at the ends of the shaft. As onlv adult dors have been used, we feel that the constant use of the middle portion of the marrow gives fairly comparable results. In remo%nng the marrow half the circumference of the bone through the greater part of its length has been chipped 144 THE SPLEEN XSD AN^MLV away, aiid alter separating' the marrow from the bone and cutting it at either end it has been easily removed as a solid cylinder by gently rolling it on to a piece of filter-paper. In carrying these tissues through the process of fixation and imbedding, the filter-paper, which is firmly adherent to the marrow through the coagulation of the attached blood, allows the necessary manipulations without injury to the marrow itself. The routisie procedure has been to fix in Zenker's fluid without previous decalcification, imbed in paraf!ine,and stain with eosin and polychrome methylene blue. Other stains have, however, l)een used whenever necessary •:: bring out certain details. Results. — It may be stated at the outset that we have found no evidence of an early change in the bone-marrow. Splenectomy does not cause, as do successive hemorrhages and hiemolytie poisons, a rapid change of fatty marrow to red marrow. This latter change we have produced read- ily and rapidly in non-splenectomized control animals by the use of specific lutmolytic senmi and by causing hemor- rhage, but we have never seen a frank change from yellow to completely red marrow in the ordinary course of events in the splenectomized animal until many months, usually six or more, had elapsed, and this despite the fact that many of the animals have had, as has been sho>vn in our earlier work, a moderately severe anaemia. This ana?mia has frequently been of as severe degree as that caused by several successive hemorrhages in the normal dog, but changes in the marrow analogous to those caused by hemor- rhage have not been evident in the earlier periods following splenectomy. In this connection it may be recalled that the anamia of splenectomy in the dog follows a gradual downward CHANGES L\ BONE-MARROW 145 course for three to six weeks, the decrease in ha?mogl()bin lici/i>r relatively more marked than the decrease in red cells, and that an equally gradual repair causes the red-cell count and hicmoglohin content to approach normal after three to four months or more. At the same time th^re is a transient initial leucocytosis, due chiefly to polymor- phonuclear leucocytes, and some*^"mes lymphocytosis with a late eosinophilia. Not infrequently the eosinophiles dis- ai)pear from the circulating blood from the third week until the end of the third month. We have, therefore, in the course of our studies at- tempted to determine whether the hyperplasia in the I .le- uiarrow after splenectomy is compensatory in the sense of (1) an overactivity in red-cell formation chiefly, (2) over- activity in the formation of the white cells chiefly, (3) an overactive, orderly reproduction of a new marrow, with involvement of all cells arising within it. The Xormal Marrow ok the Femur of the Dog In our study of the marrow of both normal and sple- nectomized animals we have used as a basis for orientation Bunting's "'' conception of ciythrogenetic and leucogenetic centres, Muir's ^"^ descriptions of erythroblastic arid leuco- blastie reactions, and have received also much aid from Dickson's"'' study of the cytology of marrow. The ar- rangement described by Bunting is by no means a constant and definite one, but in the masses of marrow cells may be seen groups composed mainly of myeloblasts and sur- rounded at times by a nearer zone of myeloc>'tes and an outer zone of leucocytes; in other groups with the same centre the outer zone may be made up of nucleated red cells, with a still more distant zone of normocytes. We 10 «- »=i| I 146 THE SPLEEN AND ANEMIA are not convinced that centres for the production exclu- sively of red cells or of white cells exist, for frequently an intermingling of the two types is seen in one centre, but this conception of definite centres is of great assistance in the interpretation of marrow changes. The study of the marrow of the femur from many normal dogs has led to our recognition of four definite groups of cells: 1. Groups of undifferentiated cells and myelocytes. These lie between fat cells and seem to be in no way con- nected with blood-channels. In all these centres the cells of the connective-tissue reticulum are in evidence. 2. Groups of the character described above, but with a peripheral accumulation of cells in which those of the leu- cocytic series predominate. 3. Groups as in (1 ) , but with a mantle of cells in which those of the erythrocytic series are -rost in evidence; and 4. Groups as in (1), but with an indiscriminate min- gling of cells of red and white series. These groups cannot always be differentiated, for not infrequently an indiscriminate mingling of cells obscures the recognition of centres. Moreover, at times may be seen groups composed purely of white cel^s or of red cells without myeloblastic centres. We have, however, found that search for the groupings described |;ieatly facilitates the study of complex marrow pictures and leads readily to a decision as to whether leucoblastic or erythroblastic activ- ity predominates. In one respect the stiidy of normal marrow has not helped us greatly, ^regakarj-ocytes and polykaryocytes are so infrequent in the normal fatty marrow that wo have no basis, '^^ regard to them, for a comparison with hyper- CHANGES IN BONE-MARROW 147 plastic marrow. The same holds true for the large en- dothelial cells which are phagocytic for red cells and are found so frequently in hyperplastic marrow to contain remnants of red cells and particles of pigment. The Makrow of Splekectomized Animals In Table XL VI the general results of our observations are presented. The terms " yellow " and " red " refer to the gross appearance, not of the surface of the marrow, TABLE XLVI Htperplabia op the Marrow op the Femur After Splenectomt Dog No. 50 23 21 f 81 17 10 39 32 44 41 24 59 57 33 51 Period after •pleaectomy 24 dayu 39 days 40 days GroM appearance 42 60 63 84 da., days daj-B days 7 inoniii monthsi 8 months O'i, rnonths 10 months 1 year m years m vears ' m years u years Yellow Yellow Yellow Yellow Yellow Slieht streaking bellow i{ed Red Yellow Yellow Yellow Red Red Streaked Red Yellow Microscopic change Slight 88 None 88 Slight 65 SL 4ht 96 Slight 92 Slight 76 Slight 6C Complete 78 Complete 68 Slight 97 None 81 None 92 Complete 110 Complete 101 Partial 110 Almost complete 70 Slight I 110 Blood picture ehortly before autopey HiEnioglobin R. B. C. 4,510,000 6,050,000 2,970,000 5,820,000 5,680,000 4,530,000 3,860,000 4,410,000 4,040,000 6,120,000 4,9'. 0,000 4,920,000 6,048,000 5,100,(X)0 .".,206.000 4,480,000 6,200,000 but of the cross-section. " Slight streaking " and " streaked " refer to an intermingling of yellow and red marrow. A marrow is described as " red " only when it is uniformly so. As will be seen by a c '^.parison of gross and microscopic appearances, a man" " yellow " to the naked eye may, microscopically, show evidence of begin- ning hyperplasia. The early changes are indicated by the 148 THE SPLEEN AND ANAEMIA word " slight." The word " complete " indicates that only an occasional fat cell is seen microscopically. " Almost complete " means that fat cells occupy less than one-tenth of the marrow space in the surface area of sections studied. Several purely fatty marrows representing periods be- tween five and twenty-four days after splenectomy are not included in the table. The bone-marrows representing the earlier periods of splenectomy, in that they show praoti(^illy no changes, may l)e dismissed briefly. This is true of a series from animals killed at various intervals from five days to three months. Some of these marrows c^innot be distinguished from those of the normal dog. In others, slight replacement of fatty tissue is seen. Thus one representing the twenty-fourth day shows here and there between the fat cells single rows of blood-forming cells, with now and then clumps of ten to thirty or more. These areas are neither purely erj'thro- genetic nor purely leucogenetic, though in some of the groups with an older type of cells there is a predominance of polynucleated cells. THe endothelial cells of the reticu- lum not infrequently contain large masses of old blood- pigment. Another, representing the fortieth day, presents prac- tically the same appearance, with a tendency, however, to greater erythrogcnesis. On the other Hand, a thirty-nine day dog shows a simple fatty marrow with no evidence of active blood formation. Three other marrows of this period, however, show already the early stages of hyper- plasia; both types of cell groups can occasionally be iso- lated, but usually the groups are mixed. Greater numbers of eosinophile cells, both myelocytic and polymorphonu- clear, are present than have been evident in earlier periods. CHANGES IN BONE-MARROW 149 A number of cells throughout the section correspond to Lon^cope's *''^ small lymphocytes, and a smaller number to Longcope's large lymphwytes. The small lympho- cytes are not, however, in pure groups. Polymorphonu- clear leucocytes are abundant, and the picture, as a whole, is one of leucogenesis rather than of erythrogenesis. Very few giant-cells are seen, and only occasional phagocytes. In another m-.rrow of the sixty-third day a moderate peripheral hyperplasia of mixed type is present. Marked congestion is evident between the fat cells, and hyper- plasia is seen, in places, near the periphefr}-; in some in- stances the erythrocj-tes appear to be outside the vessel, forming distinct areas of hemorrhage. A few phagocytes are present, but giant-cells are rare. Polymorphonuclears are fre(iuent and of mature development. At the periphery erythrogenesis seems to predominate over leucogenesis. Eosinophiles and lymphoid cells are not conspicuous. A marrow of tlie sixtieth day shows less hyperplasia, but leucocytie reaction is more evident, though erj-thro gjmesis is active. Scattered throughout tlie section are many small lymphocytes, but nowhere are these seen in solid clumps. Numerous deposits of pigment are seen. Again, on the eighty-fourth day, an essentially fatty marrow shoMs a narrow cellular strip at the periphery in which erythrogenesis is quite active. Here and. The fact remains, however, that the marrow is actively forming normal red cells. The anemia was, there- fore, not due to abnormally low erj'tlu-ogenesis in thr marrow. Dog SO.— On April 7, 1913, before splenectomv, the red cells numbered 6,528,000, and hjemoglobin was 110 per cent. The lowest point of ana?mia was reached on June 3, the red cells numbering at that time 3,6.50,000; the ha'moglobin was 62 per cent. By July 7 the blood picture had improved (red cells 5,080,000, ha?moglobin 88 per cent.), but on September 11 a late recrudescence of the ancTmia gave red cells 4,040,000 and hfemoglobin 68 per cent. The animal was killed on Xovember 15. The bone- marrow of the femur was of a definite red color. The ana?mia did not affect the general nutrition of the animal, for on April 7 the weight was 12,800 gms.; on November 15 it was 13,950, and the adipose tissue was abundant. This animal, representing practically the same period after splenectomy and the same clianges in the blood as Dog 10, gives very much the same picture in the marrow. Of minor importance is the fact that the marrow is nol so cellular, the proportion of cells to fat being in the ratio of about 3 to 2; also, the myelocytic centres are not so pronounced, but in other respects the marrow is the same. Many giant-cells are present, but lymphoid cells are rare. The formation of red cells and leucocj'tes is perhaps not so rapid (that is, the numbers about any one centre are not so great), bnt, on the other hand, the erj-throgenetic IN M 154 THE SPLEEN AND ANAEMIA activity of the marrow is sufficient to exclude the possi- bility of an inactive bone-marrow being responsible for the late recrudescence of anaemia. Docf 24. — This animal was splenectomized on Febru- ary 10, 1912, and was used for the injection of haemolytic immune serum on March 20 and again on April 7. On June 28 it had recovered from the anaemia then produced (red cells 5,650,000, haemoglobin 89 per cent.), and on July 15 it was treated with sodium oleate. On Septem- ber 26 the red-cell count was 5,780,000 and haemoglobin 90 per cent. On February- 19, 1913, the blood picture had improved (red cells 6,048,000, haemoglobin 110 per cent. ) , and at this time hemolytic serum was again injected. The animal was chloroformed on March 4, 1913. The lapse of time since splenectomy was, therefore, thirteen months. At autopsy a red marrow was found. Owing to the use of various hemolytic poisons, the bone-marrow of this animal may have been influenced by other factors than the absence of the spleen. The his- tological picture, however, is so in accord with the marrow of simple splenectomy that, with this explanation, it is included in the series. Histology. — The marrow is a uniformly solid marrow, with no fat spaces visible in any of the sections examined. It does not, however, appear to be as cellular as the marrow of Dogs 10 and 39. This difference is caused by a greater congestion and distention of the blood-vessels, a slight in- crease in the reticiil iiri, and a lessened tendency of the myelocytic tissue to be grouped in large centres. Ery- throblastic centres are very prominent and very active; leucogenetic centres, on the other hand, are made out with i'iRculty. L>Tnphoid elements are rare. Many cells of ■^^t^m.r/''' T'^m^^m^ ^- ' -^^f^^^^^ :^m CHANGES IN BONE-MARROW 155 the myelocytic type are seen with coarse basic granules and short threads in the nucleus and with little or no protoplasm. In close relation to these are sometimes seen degenerated mitotic cells, but whether all the chromatin masses can be so interpreted is not clear. These degenera- tive changes are doubtless the result of the last injection of haemolytic serum. Dog 59.— On July 24, 1912, this animal was splenec- tortized and used for the study of the progressive anaemia following this procedure. On December 7, 1912, the highest point (red cells 5,250,000, hemoglobin 105 per cent.) of recovery was reached. Continued observation showed a slight decline to 5,2)00,000 red cells and 86 per cent, of hajmoglobin on May 21, 1913, on which date the animal was used in an experiment with sodium oleate. From the moderate ansemia caused at this time the animal recovered, the blood examination on June 9 showing 5,050,- 000 red cells and 86 per cent, haemoglobin, the condition slightly improving as to ha?moglobin content until Novem- ber 18, 1918, when red cells were 5,100,000 and hsemoglobin 101 per cent. The animal was chloroformed on Novem- ber 24. At autopsy the bone-marrow of the femur was soft, succulent, and dark red in color. In connection with the general condition of this animal it is of interest to note that in the last seven months its weight increased from 10,450 grammes to 12,580 grammes, and that adipose tissue was verj' abundant. The administration of sodium oleate introduces a possible disturbing factor, but ns this was given six months before death, and as the anemia which it caused was quickly repaired, it is not considered, in view of our other observations, to have had an important influence on the bone-marrow. t^-tf-Vi*' -fi^^HT^^-'-r 156 THE SPLEEN AND AN.EMLV iss:^: HisToi-(X?v. — The marrow shows some fat cells, the proportion of marrow cells to fat being about 10 to 1. Nothing different from the last four marrows is pre- sented. Leucogentsis and er>-throgenesis proceed at about equal rate, the latter being a little more active. ^Mitotic figures are seen not infrequently, but the type of cell in which they occur is not always evident. Myeloblasts seem to be more abundant than usual. Giant-cells are fairly abundant, but lymphoid cells are rare. Doc/ ,77._On June 23, 1912, the blood of this dog con- tained 5,350,000 red cells per cubic millimetre and 98 per cent, hipmoglobin. On July 2 the spleen was removed. The resulting ana?mia reached its lowest point (red cells 2,970,000, haemoglobin 50 per cent.), on August 5. On October 24, when the bl«)od count showed 5,240,000 red cells and 90 per cent, haemoglobin, the animal received sodium oleate intravenously ; a slight amemia ( fall in hsEmo- globin to 62 per cent., but no marked change in red cells) resulted. In Januan-, 1913, the red cells were 5,206,000, hsemoglobin 110 per cent., and with slight variations this higher level was maintained, accompanied by an increase in body weight, until December 12, 1913, when the animal was chloroformed. At autopsy the animal was found to have a large amount of adipose tissue; the bone-marrow of the femur was definitely reddish in color, with faint yellow- ish streaks. As the sodium oleate given four months after splenectomy and fourteen months before death produced only a slight transient change, we consider that the bone- marrow represents the effect of splenectomy only. Histological Examination. — The relation of the fat to cells is about 1 to 1 ; otherwise nothing new is seen. The marrow is very active, leucogenesis and erythrogenesis CHANGES IN BONE-MARROW 157 being equally prominent. Phagocytic cells and masses of old olood-pignient are quite numerous, as are also giant- cells. More abundant than in other marrows are eosino- philes of the myelocytic type. Lymphoid cells are not conspicuous. Dog ,«.— This animal was splenectomized on May 14, 1012. The blood examination on the previous day showed 4,950,000 red cells and 85 per cent, hicmoglobin. The anicmia following splenectomy reached its lowest point on June 28 (red cells 3,550,000, haMiioglobin 52 per cent.). On September 20 the red cells had risen to 5,490,000 and hicmoglobin to 95 per cent. In November, 1913, the animal passed successfully through pregnancy. In Janu- ary-, 1914, as the animal had developed mange, it was chloroformed. The blood examination on the preceding (lay was red cells 4,480,000, hemoglobin 70 per cent. At autopsy the bone-marrow of the femur was deep red in color. (It should he stated that one and two months before splenectomy the animal had received injections of hfcmolytic serum. From our studies of the effect of hemo- lytic serum in the normal dog, we do not believe that these injections, several weeks before splenectomy and nearly two years before death, are in any way responsible for the hyperplasia of the marrow.) Histological Examixatiox.— This marrow differs in no way from the marrows of Dogs 57 and 59 described above. Dog 51. — The spleen was removed on May 31, 1912, and on June 26 of the same year hemolytic serum was administered. From the anemia thus produced the animal made a slow recovery, but after 200 davs the blood examin- ation showed 6,200,000 red cells and 110 per cent, hemo- I' f 1 ^T«*^i- w*i>^^-»^ . -f U8 THE SPLEEN AND AN.EMIA globin, as compared with (5,210,000 red cells and 100 per cent, ha'nio^lohin before splenectomy. On March 26, 1014, when the animal was chloroformed, its weight was 0750 ^ammes, as compared with 8270 grammes at the time of splenectomy and 8120 grammes when hemolytic serum was administered. The notes made at the autopsy refer to the large amount of adipose tissue, the normal appear- ance of the lymph-nodes, the absence of superimmerary spleens, and the presence in the long bones of a distinctly yellow, fatty marrow. Histological examination of the marrow shows a very slight hyperplasia, with large num- bers of leucocytes and deposits of blood-pigment. Discussion. — It will be remembered that in discussing the anjemia which follows splenectomy it was pointed out that nucleated and other abnormal forms of red cells are rarely found in the circulating blood, and that when they do occur they do not persist for any length of time. Appar- ently their appearance corresponds to the beginning repair and not to the period of hyperplasia of the marrow which we have described. It is difficult, therefore, to bring the changes in the bone-marrow into relation with the changes in the peripheral blood. If the hyperplasia of the bone- marrow is compensatory to increased blood destruction or decreased blood formation, one would expect definite hyperplasia to be present in the earlier period, during the first three months after splenectomy, at a time when the anaemia is evident and repair is taking place, and not after six months to a year or a year and a half, when the blood picture Is normal. It is true that in two of the animals (Dogs 10 and 39) a late recrudescence of anaemia occurred and the marrows of these animals were obtained during this period, but this was not the case in most of the animals C HANGES IN BUNE-MAHROW iffO of the series and is not characteristic of the late periods after splenectomy. It is therefore impossible, on account of the late development of hyperplasia in the man-ow. to explain its occurrence as compensatorv , > the anemia fol- lowing splenectomy. Likewise we cannot accept W^arthin's '«• theory based upon his study of sheep and goats. In these animals Warthm found hyperplasia of the marrow to occur several months after splenectomy and to be associated \ "h evi- dence of increased destruction of red blood-cells in the lymph- and hsmolymph-nodes. This destruction, greater than that in the primitive spleen, is responsible, he be- lieves, for the anajmia following splenectomy, and this is in turn compensated by increased activity 'in the bone- marrow. We have found little to support this theory in our studies of the dog. The lymph-nodes, as well as the endothelial cells of the liver, as we have shown elsewhere,'' ^=' are indeed more active in the phagocytosis and destruction of red cells after splenectomy than in the normal animal, and this is very evident when large numbers of red cells are injured, as by the administration of a hemolytic poi- son ; but in the ordinary course of events, after splenectomy, the IjTnph-nodes present no evidence of excessive blood destniction. An occasional cell containing one or two red cells may be seen, and small amounts of old blood-pigment are occasionally demonstrable, but of excessive hemolysis there is no evidence. Likewise, microchemical tests 'for iron m the lymph-nodes and liver show that little differ- ence exists in this respect between the normal and splenec- tomized animals. For this reason, and because the anaemia is not persistent and progressive, we cannot support the theory that the hyperplasia of the marrow is compen- £l tf^ 'I ti »ii 160 THE SPLEEN AND ANEMIA satory to abnormal blood destruction in the Ijinph-nodes. Another possible explanation is that the bont marrow, in the absence of the spleen, is concerned in the storing and utilization of iron. There is no v^oubt that, in the normal animal, iron set free in the dissolution of red cells is stored in the spleen. After splenectomy a readjust- ment in the storage of iron takes place, and there is some evidence that for a short time after the removal of the spleen iron may be lost from the body. Our investiga- tions "" show, however, that this disturbance of iron utiliza- tion is transient, and that after a few weeks the elimina- tion of iron in the splenectomized animal differs in no way from the process in the normal animal. This suggests naturally that the storage of iron in the absence of the spleen is taken over by other tissues. As microchemical tests for iron showed no definite increase of iron in the lymph-nodL's and liver, it seemed probable that the bon.' marrow might l)e the chief depot of iron storage. Sucii a view was supported by the fact that all hyperplastic bone-marrows contain large amounts of altered blood- pigment, sometimes free, but (w^curring, for the most part, in large phagocytic cells. The activity of these phagocytic cells in transforming the iron of old blood-pigment in order that it may be utilized ff)r red cells might, it was plausible to suppose, stinuilate the other functions of the bone- marrow (that is, the erythrogenetic and leucogenetic func- tions) and cause eventually a replacement of the fatty marrow by a ver\' oellular red marrow. In order to prove this hypothesis it was necessary to obtain some idea of the iron content of these marrows. Direct chemical analysis was nut of the question on account of the small amoimt of material available and the varia- ir^-'r-^:'^'Y\W^ CHANGES IN BONE-xMARROW Ml tions in blood and bone content of different marrows. We therefore made a comparative study based on the use of the microchemical reaction for iron. This demonstrated at once that all red marrows in our series have a large con- tcJil of iron, and that fatty marrows contain very little ()-• no iron. On the other hand, when the marrows of non- splenectomized dogs rendered hyperplastic by anaemia or infection were examined it was found that these also had a large iron content. Thus in a group of seventeen non- splenectomized dogs iron was present in the marrow in large amounts in four, in moderate amounts in two, in siiiall amounts in four, and in seven none was found. On the other hand, in twenty-seven splenectomized dogs, iron was present in large amounts in ten, in modei .te amounts 111 three, small amounts in four, and absent in ten. In both groups the amount of iron was in direct pro- portion to the degree of hyperplasia. These observations pomt, therefore, to the conclusion that a red marrow is always rich in iron, but it is impossible to say whether the cellular hyperplasia or the iron deposition is primary. Tnder the circumstances, it is also impossible to conclude that the late hyperplasia of marrow following splenectomy IS an attempt to conserve iron. Moreover, the irregularity of our results, as shown by the failure of hyperplasia in four animals, representing respectively the eighth, ninth, tenth, and twenty-second months after splenectomy, pre- vents, in the present state of our knowledge, an adequate explanation of the cause of the transformation from yellow to red marrow. However, the tardiness with which hyper- plasia of the bone-marrow appears after splenectomy, de- spite the presence of an aiia?mia of considerable grade, may well bear some causative relation to the slow degree of 11 I I6i THii SPLEEN A%D ANJIMIA i?.. blood regeneration that follows the administration of hsemolytic agents to such animals. The divergent results in this study are characteristic of all phases of experimental work on the spleen, and doubtless are to be explained by the fact that removing the spleen takes away only one organ of a system com- posed of liver, spleen, lymph-nodes, and bone-marrow, and that the interrelations which exist in this system may, or may not, under varying circumstances, bring into play compensations of the greatest importance in determining the degree of blood destruction or regeneration and there- fore the degree of change in the bone-marrow. A search of the literature of splenectomy in man, although it reveals evidence of the occurrence of red mar- row in various forms of splenic ana?mia, offers little of im- portance concerning the changes which occur in the bone- marrow after i moval of the normal spleen. Several refer- ences are made to the occurrence of pain in the long bones after splenectomy, and by some this has been assumed to be evidence of hyperplasia within the rigid bony canal. The only note of the direct examination of the bone-marrow after splenectomy is that of Riegner,"'" who found active proliferation of the marrow of the femur in a man whose leg was amputated for gangrene four weeks after splenC'V tomy for trauma. It is therefore impossible, on account of this paucity of data concerning the changes in man, to bring them into relation with our experimental results. Conclusions. — Splenectomy in the dog causes, as a rule, a transformation of the fatty marrow of the long bones into a richly cellular red marrow. During the early periods, one to three months, the change in the marrow is slight and either focal or pe- CHANGES IN BONE-AL* RROW les ripheral; after six to twenty months the replacement of fat by marrow cells is complete or nearly so. Excep- tions were, however, seen in four animals representing the eighth, ninth, tenth, and twenty-second months respectively. The evidence at hand does not support the theory that this hyperplasia is compensatory either to *he anemia caused by splenectomy or to an increased hemolysis in the lymph-nodes. li is possible that it may be a concomitant of the activity a the bone-marrow in taking over, in the absence of the spleen, the function of storing and elaborat- ing the iron of old blood-pigment for future utilization by new red cells, but our studies do not fully support this view. >'! P m Hi ■..']: CHAPTER VII THE CHANGES IN THE LIVER \SD LYMPH-NODES AFTER SPLENECTOMY DISCUSSION OF THE LITERATURE. CHANGES IN LYMPH-NODES. PHAGOCYTOSIS OF RED CELLS BY ENDOTHELIAL CELLS OF LYMPH-NODES AND LIVER. MICROCHEMICAL TEST FOR IRON IN LYMPH-NODES AND LIVER. Much of the early literature concerning compensatory changes in the lymph-nodes is bound up with the question of the regeneration of the spleen after partial or complete extirpation and more recently witli problems concerning the htemolymph-nodes. As this literature has been col- lected very completely by Warthin,^"' we will give only a general simmiary of it in the li'jfht of his investigation. The earliest detailed investigations are those of Tizzoni and Fileti,*'" who observed in the splenectomized dog an in- crease in size of the retroperitoneal and thoracic lymph- nodes, which were distinctly red in color. In two dogs, splenectomized for fifty-four days and three and a half monllis respectively, they found a new formation of spleen- like nodules in tlie onirntum. These they believed to be formed directly from adipose tissue by an absorption of fat and a transformation of the fat cells into reticulum. I.,eucocytic infiltration followed, as also proliferation of endotlielium, which produced eventually a pulp-like tissue, in the meslies of which were red blood-cells. Around the whole a coimcctive-tissue capsule was formed. Later, in 164 CHANGES IN LIVER AND LYMPH-NODES 165 1882, Tizzoni^^** found somewhat similar bodies in the gastr'osplenic ligament, in connection with indurative sple- nitis in the dog, and after splenectomy numerous newly- formed nodules throughout the subperitoneal fat, over the diaphragm, and in the pelvic, sterno-abdominal and sub- cutaneous fat tissue. Fca,"" in 1883, denied the new formation of such n( des, and explained them as preexist- ing nodes, changed in color by hemorrhage or other patho- logical conditions. In the meantime, however, Wino- gradow'" had described in dogs killed 132, 517, and 760 days after splenectomy changes in existing lymph-nodes similar to those observed by Tizzoni. On account of the presence of red cells and pigmented cells in the sinuses of these nodes, he believed these structures to have a share in blood destruction, and that possibly the anaemia occur- ring after splenectomy could in this way be explained. Zezas "' found that after splenectomy the mesenteric and bronchial lymph-node of the rabbit became swollen, (lark red in color, and firmer in consistency, and quotes Hegar and Simon as finding similar changes in the mesen- teric lymph-nodes of the cat. Tizzoni "« and Ceresole *"» cov'ld not confirm these changes in the rabbit. Mosle- ^^ found in a dog, splenectomized ten months, numerous spleen-like nodules of the size of a pea, scattered through- out the greater and lesser omentum. Microscopically, these were similar in structure to those found by Tizzoni and Winogradow, but Hosier regarded them as neo- plasms — hemorrhagic telangiectatic lynphoma— and not as newly-formed spleen or l>Tnph-nodes. The Ij-mph-nodes generally were not hyperplastic, and the bodies described above were not constantly present; in one dog, for ex- ample, killed after eleven months, no changes in the lym- 166 THE SPLEEN AND AN/EMIA phoid tissue were found. Mosler concludes, nevertheless, that after splenectomy compensatory chanp^es may occur in lymph-glands and bone-marrow. Gibson "° found in splenectoniized dogs enlarged mesenteric lymph-nodes containing both normoblasts and normocj'tes in their sinuses. Of other investigators, Eter- nod '*'^, found in a fox, 161 days after splenectomy, a splenic nodule in the omentum and newly-formed lymph- nodes in the mesentery, the other lymph-nodes being en- larged and of a brownish color; Vulpius*^" found no enlargement of lymph-nodes in dogs dying after a few days, or killed five months after splenectomy; Lauden- bach "" found that hyperplasia of the lymph- nodes was not constant, and that evidence of increased blood forma- tion was present in the bone-marrow only. An examination of Warthin's summary of the litera- ture concerning the lymph-nodes of man after splenectomy shows that no c ""nstant changes have been observed. Tem- porary local enlargement of lymphoid tissue has been ob- served and occasionally general enlargement; on the other hand, in many cases no changes whatever have been seen. In the congenital absence of the spleen, as in the case re- ported by Hodenpyl,'®* general enlargement of all the lymph-nodes of the body is found, as also usually a new formation of lymphoid tissue in the adrenals and liver. Warthin's studies *^^ were upon sheep and goats which normally have nimierous hiemclymph-nodes in the pre- vertebral fat. One and two weeks after splenectomy, lymph- and ha?mol\Tnph-glands presented evidence of an increased nimiber of pigment-bearing phagocytes and eosinophils and a proliferation of Ivmy^hoid tissue gen- CHANGES IN LIVER AND LYMPH-NODES 167 erally. At the end of two months these changes were more marked and, in addition, new formation of haemolymph- nodes in adipose tissue was evident. The changes were progressive, and at the end of five months are described as follows: " Great hyperplasia and new formation of lymph-nodes, new formation of haemolymph-nodes in adi- pose tissue, marked haemolysis, eosinophils in the lym- phoid tissues, pigmentation of the liver, and slight lym- phoid changes in the fatty marrow." Leucocytosis was most marked at the end of two months. From this summary it is evident that two types of changes have been found : ( 1 ) A peculiar new formation of lymph-nodes (Tizzoni, Winogradow and Mosler) , prob- ably identical with the hsemolymph-node described by Warthin, and (2) an inconstant hyperplasia of preexisting lymph-nodes with reddish or reddish-brown discoloration. As to the formation or destruction of red cells by the lymph-nodes after splenectomy, divergent opinions are held. Gibson ^*'' and Laudenbach,'"** among others, sup- port the theory of red-cell formation ; Warthin saw no evi- dence of this. On the other hand, the latter states that his findings indicate that the splenic functions of haemolysis and leucocyte formation are, in the absence of the spleen, taken over by the lymph- and haemolymph-nodes. So far as the function of luemolysis is concerned, this view is supported by Morandi and Sisto,'^* who found in the hfemolymph-nodes of dogs evidence cf increased haemolysis after splenectomy. According to Warthin, " the haemo- lytic function of the hfemolymph-nodes and hyperplastic lymph-glands exceeds that of the primitive spleen, causing an excessive destruction of red cells. The resulting anaemia is later compensated for by an increased activity on the H 168 THE SPLEEN AND ANAEMIA part of the bone-marrow. It would appear, therefore, that the removal of the spleen leads to an increased production or retention of some haemolytic agent usually disposed of by the spleen. The effect of this hremolytic agent is either to stimulate the phagocytes in the htemolymph- nodes to increased activity, or to change the red cells so that they are more easily destroyed by these phagoc\i;es." This view is not shared by Banti,^* who, although he considers the hTiiph-nodes, liver, and bone-marrow to be secondary organs of ha?molysis, denies that they may com- pensate for the hemolytic activity of the spleen. Indeed, it is upon this argument that he bases the beneficial results of extirpation of the spleen in ha?molytic splenomegaly. Changes in thk Lymph-nod ,.;. — All animals used in our studies of the effect of splenectomy have been carefully examined ^^ at autopsy in the hope of finding the ha?mo- lymph-nodes occasionally noted by other investigators. In this we have not been successful, ^ever upon gross ex- amination have we found structures corresponding to Warthin's description, and the occasional doubtful ha'mo- lymph-node has always proved upon histological examina- tion to be a hannorrhagic or otherwise pathologically altered lymph-node. Upon this point we have felt relieved since Dr. Warthin assured us that the dog is a very unsatisfac* ">ry animal for the study of th^^ hsemolymph-node. Still, it has been a matter of surprise to us that we have found in none cf the many dogs we have examined the bodies described by Tizzoni, Winogradow, and Mosser. The second change in the lymph-nodes — a reddish or reddish-brown discoloration — «lescribed by other observers we have frequently seen. More frequently this has been a reddening limited to the centre of the node, at other times a CHANGES IN LIVER AND LYMPH-NODES 169 diffuse reddening. In animals splenectomized ten months or more the red usually gives way to a brownish color, especially in the inguinal and axillary lymph-node. Hyperplasia of the lymph-nodes has been common in animals killed shortly after splenectomy, but in those rep- resenting the longer periods it has been impossible to dis- tinguish any appreciable increase in size and certainly no new formation. On the other hand, the lymph-nodes (as also the liver) in a small group of animals presented changes which appear to be of significance in connection with the general problem of blood destruction in the absence of the spleen. These changes are (1) a proliferation of the endothelial cells, and (2) an increase in the phagocytic power of these cells for red blood-corpuscles. It is not necessary to discuss the voluminous literature concerning the destruction of red cells. This has been well presented up to 1895 by Gabbi "' and up to 1901 by Hunter.'** The more recent literature has added little either in fact or theory that is new. Out of the mass of contradictory statements there is uniformity of opinion on only two points: (1) That large endothelial cells of the spleen (the red blood-corpuscle-carrj'ing cells) have the power to engulf red blood-cells; and (2) that the presence (in anaemia and malaria) of blood-pigment in the cells (KupiFer's cells) of the liver capillaries indicates that these cells play some important part in the destruction of red blood-cells. On the other liand, it is not generally admitted that the endothelial cells of the lymph-nodes likewise have this power. That phagocytosis of red cells, wherever it occurs, leads ultimately to the freeing of haemoglobin, which eventually reaches the liver and is ;^C?ifl^^^^..-^:^.^s-:3' ^^/^? vn THE SPLEEN AND ANAEMIA transformed into bile-pigment, is the opinion of all who support the theory that this mechanism plays a part in the destruction of red blood-cells. There is, however, no uniformity of opinion as to whether the haemoglobin is set free in the liver from red cells carried there by the phago- cytes or whether it is set free by the phagocytes elsewhere and carried to the liver in another way. It is only with one phase of the subject, the role of endothelial cells in engulfing red cells in the absence of the spleen, that we will concern ourselves here. Our hypoth- esis is that in the absence of the spleen the endothelial cells of the lymph-nodes and liver compensate, at times of excessive blood destruction, for the loss of similar cells of the spleen. This possibility was first brought to our attention in the routine examination of tissues from splenectomized dogs which had received specific haemolytic immune serum. So striking were some of the pictures that we undertook, for the sake of control, the study of the liver and lymph- nodes from a number of normal dogs, of normal dogs re- ceiving hsemolj^ic serum, and of dogs which had been splenectomized for various lengths of time, but which had not received hctmolytic serum. The literature of splenectomy offers little aid in determining the histological changes occurring in the lymph-nodes after removal of the spleen. In the litera- ture at our disposal no definite descriptions have been found except those of Warthin, who found in sheep and goats an increase m the phagocytic power of the endo- tiielial cells for red blood-corpuscles. Gabbi, who worked with the guinea-pig, states that a transient increase of the red blood-corpuscle-carrying cells may possibly occur in CHANGES ES' LIVER AND LYMPH-NODES 171 early periods after splenect(Mny, but that after three to six months they are no more abundant than in the normal animal. The IjTiiph-nodes studied have been for the most part the mesenteric, gastrohepatic, prevertebral, and bronchial. In the normal animal these have been examined more particularly for the frequence of mitosis, for the number of endothelial cells in the sinuses, and for the presence of cells containing red blood-corpuscles. Careful study of ncxles from five normal animals showed that mitotic figures are found only after prolonged search and are usually limited to the follicles. The mmiber of endothelial cells varies, but usually is not great, and they never occur in large masses in the sinuses. These cells, however, not infrequently contain old blood-pigment, and occasionally a cell may be seen containing one or two red blood- corpuscles. The IjTTiph-nodes of five animals splenectomized for three, four (two), thirty-nine and eighty- four days and not subjected to the action of haemolytic serum have been studied in the same way. In two animals representing respectively four and eighty-four days the lymph-nodes differed in no way from the normal ; in the other animals mitotic figures were abundant in the follicles, and the endothelial cells in the sinuses were greatly increased in number. Prolonged search, however, failed to demonstrate mitotic figures in the latter cells, and, although they occa- sionally contained one or two red blood-corpuscles, this power of phagocytosis did not appear to be greater than in the non-splenectomized animal. The increase in the number of endothelial cells was, however, verj' striking. The lymph-nodes of five normal dogs, which had received 1 1 '- i Cfv: MICROCOPY RESOLUTION TEST CHART ANSI and ISO TEST CHART No 2 1.0 I.I Si; i||||l£ 1 ^-^ i._ Li 1^ 2.2 1^ IL. Li£ 12.0 1.8 1.25 1.4 1.6 ^ ^PLIED l!VMGE_J =ir- '6-' tost Mo^n Street ~.^ -"'oc'iester. N-w rork U609 USA ' '16) AS2 - 0300 - Phone ^^ .716} 288- 5989 - Fa» 172 THE SPLEEN AND A\/EML\ m^- specific ha>molytic immune serum and had died or been chlorofoi-med, after periods varying: from twenty-four hours to nine days, showed tlie lesions asually described as common to various cytotoxic sera ^" and especially to lymphotoxic sera.' '^ These are (tdema. increase of poly- morphonuclear leucocytes, focal areas of necrosis, abundant TABLE XLVII Changes in the Lymph-nodes Afteb .Splenectomy and the Injection of HiEMOLYTic Serum Experi- period Period after eple- ' after Kffeft of serum HistoloRy neclomy 1 serum I 3 days 36 hours Ha;moglobinuria Extreme phagocytosis of red cells 11 300 (lays 36 hours Severe jaundice Well marked phagocyto- sis of red eel s in 285 days 18 hours No record Well ma-ked i)hagocyto- sis of red cells IV 225 days 48 hours Ha;moglobinuria Moderate phagocytosis of red cells V 15 days 3 days Ha?mogIoliinuria Negative VI 27 days 3 days HKinoglohinuria Proliferation of endothe- lial cells VII 33 days 4 days Jaundice I'roliferation of endothe- lial cells VIII 65 days 8 days HaBmoglobinuria Prohferation of endothe- lial cells IX 6 days 9 davs Hemoglobinuria Negative X 3 days 9 days \o ha>moglobinuria or jaundice Negative XI 103 days 10 days No ha'moglobinuria (spontaneous jaundice) Proliferation of endothe- lial cells XII 25 days 15 days Jaundice Proliferation of endothe- i lial cells mitotic figures in the follicles, and slightly greater fre- quency of large endothelial cells capable of phagocytosis of red cells. Of animals that had been splenectomized and had re- ceived hirmolytic scrum as well, twelve were available for histological examination. Of these, three showed no change in the lymph-nodes and five showed a well-marked in- CHANGES IN LIVER AND LYMPH-NODES 173 crease in the number of endothelial cells in the sinuses, but no increase in power of phagocytosis. In the four remaining animals the sinuses contained a great number of large endothelial cells filled with red blood-corpuscles. The analysis of these findings is somewhat difficult, as three factors must bo considered: (1 ) The length of time after splenectomy; (2) the lapse of time between adminis- tration of hicmolytic serum and the death of the animal; and (3) the degree of red-cell destruction caused by the serum. These factors are brought out in Table XLVII. From this analysis it is seen that the proliferation of the endothelial cells did not occur in the animals (V, IX, and X) splenectomized for periods of from three to fifteen days, but was evident in five (VI, VII, VIII, XI, and XII) in which the time elapsing since splenectomy was 27 to 103 days. On the other hand, the Ivmph-nodes of these animals did not present evidence of increased phao-o- cytosis of red cells. Whether this was due to the perFod which had elapsed (three to fifteen days) since injection of serum could not be determined, but" this was probably tlie case. Certainly it was not due to failure of haunolysis, for at least two of these animals (VI and VIII) presented evidence of extreme blood destruction. That the period of time elapsing may be an important factor is shown by the fact that all animals ( four . in which there was evidence of extensive phagocytosis of red cells represent periods of eighteen to forty-eight hours after injection of the serum. In the absence of exact knowledge of the length of time necessary for the destruction of red cells by pjiago- cytic endothelial cells, it is useless to surmise, but one can- M .J i i r. , I 174 THE SPLEEN .VND ANEMIA ^i^.^ not escape the fact that in this investigation all evidence of active phagocytosis is seen in animals dying within forty- eight hours. It is possible, therefore, that the destruction of red cells by phagocytosis may be completed within forty- eight hours, and this view is supported by the frequency with which pigment is found in the lymph-nodes at later periods. It is also evident that the time elapsing since splenec- tomy bears no relation to the occurrence of phagocytosis of red cells, for the most marked example of the latter was seen in a dog dying three days after splenectomy, while moderate and well-ma: ed phagocytosis occurred likewise after seven and one-half, nine and one-half and ten months. Recently Karsner, Amiral, and Bock ^"* have confirmed these results and have added some interesting observations concerning the time element. Their conclusions are as follows : In cats the same phenomenon of phagocytosis of red blood-corpuscles in the liver and lymph-nodes takes place as in the dog. In the series of splenectomized animals killed three, six, twelve, twenty-four, and forty-eight hours after injection we found at three hours hyaline thrombosis, large numbers of mitotic figures, and in three Ij'mph-nodes two phagocytes containing each one red blood-corpuscle and showing a certain amount of pigment phagocytosis; at six hours there was karyorrhexis in the central parts of the follicles, marked mitosis, phagocytosis of red blood- cells and of pigment, more marked in the central sinuses than in the peripheral siimses. Digestive vacuoles in these phagocytes contained only a few er\'throcytes. At twelve hours there was marked phagoc>i:osis of the red blood-cor- puscles in the central and peripheral sinuses, also phage- CHANGES IN LIVER AND LYMPH-NODES 175 cytosis of pigment granules. At twenty-four hours the necrosis in the follicles did iiot appear to be severe; the phagocytosis of the red blood-corpuscles was seen princi- pally in the peripheral sinuses. At forty-eight hours there was still hyaline thrombosis, very little evidence of necrosis, marked phagocytosis of pigment both in the central and peripheral sinuses. Practically no red blood-corpuscles were within the phagocytes, although simple acidophilic granules, apparently erj-throcytic fraginents, were found. The story of the phagocytosis appears to be that it begins at about three hours after injection of the immune serum, continues progressively, and reaches its height at somewhere between twelve and twenty-four hours after injection, and then the destruction of the corpuscles goes on, so that at forty-eight hours there is nothing left but pigment and corpuscular fragments. Furthermore, with the passage of time the individual phagocytes become more and more filled with erythrocytes until about twelve to twenty-four hours, at which time there is a disappear- ance of the erythrocytes with the substitution of the pig- ment granules. The origin of the phagocytes appears to be particularly the endothelial cells of the" sinuses, but in many of the lymph-nodes it was found that the individual cells of some of the smaller blood-vessels were swollen and also phagocv^ic, and it is possible that this is an important matter in connection with the origin of the phagocytic ceils and also an important way of explaining the presence of red blood-corpuscles within the lymphatic sinuses. Careful examination failed to show anything in the nature of rup- ture of any of the blood-vessels. Another phase of Karsner's work had to do witii the question of h»mopsonins; that is, with the possibility of 176 THE SPLEEN AND AN/EMIA the spleen having some relation to a substance which ren- ders the red cells more susceptible, or otherwise, to phago- cytosis, and the possible increase or decrease of such a substance after splenectomy. He summarizes ^"'* the re- sults of this phase of the investigation, as follows: 1. Splenectomy produces no change in ha'mopsonins of the circulating blood that is clearly demonstrable by in vitro tests. 2. The venous blood returning from the kidney and from the spleen ana the venous blood of the portal vein, of the right auricle, and of the left ventricle showed the same content of ha?mopsonin. 3. PLxtracts of washed spleen, kidney, pancreas, and liver showed no influence over the phagocytic activity of the corpuscle, serum, and exudate mixture used in the opsonic work. 4. Extracts of the lymph-nodes of splenectomized dogs and the extract of the lymph-nodes of normal dogs have no influence over the phagocytic mixture mentioned under heading 3. CiiANCJES IN THE LivEK. — Examination of the stellate endothelial cells (KupfFer's cells) of the liver ^''^ has been rendered difficult on account of the intense congestion and abundant necrosis which occur in the liver after the admin- istration of h.TMiolytic immune serum. For this reason we have not always been able to correlate the evidence of pha- gocytosis in the liver with tlie lesion descril)ed in the lymph- nodes during the early (<^orty-eight hour) period. Definite evidence, however, of phagocytosis has been found in four animals, representing periods of one, two, eight, and nine days after the administration of serum, and representing, respectively, periods of three,'^-' sixty-five, and six days CHANGES IN LIVER AND LYMPH-NODES after splenectoniy. Also in a fif'tli animal, twenty-five dt.ys after splenectomy and fifteen days after the administration of the serum, the cells of the capillaries contained small balls of yellow pigment, apparently representing altered luvmoglobin. As controls we have examined the livers of severaJ nor- mal dogs and of nine splencctomized dogs not receiving serum, hut without finding evidence of phagocytosis on the part of the cells of the liver capillaries, or of prolifera- tion of these cells. The sj)lenectomies in this series repre- sented periods of from three to eighty-four days; rive under ten days and three over twenty days. Likewise we liave examined the livers of nine normal dogs receiving ha-molytic serum. In two of these the endo- thelial cells appeared to be increased somewhat in number, but no undoubted evidence of phagocytosis could be ob- tained. All other livers examined showed no changes in the cells of the capillaries. ]MrcH()CHEMiCAi. Tksts lOR Ihox in Lymph-xodes AND Livj:r Warthin, in his study of the lymph-nodes and Iuppio- lymph-nodes of sheep and goats, found in these organs, several months after splenectomy, evidence of increased blood destruction. This we have observed in the dog, in so far as it is shown by phagocytosis of red cells, only after the administration of a hannolytic poison; in the ordinary course of events, no inci-eased ])hagocytic activity on the part of the endothelial cells of the lymph-nodes of the splenectonu'zed animal is seen. This obsen-ation we have confirmed by examining a numl)er of lymph-nodes and liver by the usual microchemical method ,if demonstrating the presence of iron. The lymph-nodes of fourteen sple- 12 'I I I ii I ^ I SI ' t 11 i 178 THE SPLEEN AND AN.EMU nectoiiiized dogs showed a considerable amount of iron in three, slight amounts in five, and none in six. The animals examined represented periods of eleven days to twenty- two months after splenectomy. In the lymph-nodes of fifteen normal animals similarly examined moderate amounts of iron were found in eight, slight amounts three times, and in four none. It is evident, therefore, that in the dog the iron content of the lymph-nodes after splenec- tomy differs little from normal. The liver likewise shows no increased deposition of iron. Of fourteen livers from splenectomized dogs, four showed slight deposition of iron in Kupffer's cells, while ten showed none. At the same time the livers of six normal dogs were similarly exam- ined; in three slight deposits of iron were found, and in three none. Protocols illustrating the histological changes in the lymph-nodes and liver follow: Dog 32. — Splenectomy was performed under ether anaesthesia on July 19, 1911; on March 8, 1912, specific hasmolytic immune serum was injected intravenously. The red cells dropped within three hours from 6,120,000 to 5,200,000 per cubic millimetre, and the haemoglobin, after twenty hours, to 42 per cent. Haen Dglobinur'a was pres- ent, and death occurred after forty-eight hours. Histology. — The liver-cells are pale, granular, stain poorly, and present here and there small areas of focal necrosis. The capillaries are dilated and contain much granular material and, as seen by the low power, numerous isolated round and oVal clumps of red blood-corpuscles. By higher power of the microscope these clumps of red cells are found to be, in large part, within endothelial cells (Plate I. Fig. 4). Some of the red cells stain well with eosin. others appear as shadows. Other endothelial cells CHANGES IN LIVER AND LYMPH-NODES 179 are seen 'Aliich contain mere fragments of red cells or masses of granular, yellow pigment, or large, yellow hya- line balls of apparently fused red cells. Attempts to dem- onstrate similar phagocj-tic cells in the large vessels of the liver and of other organs failed; they were present, how- ever, in the sinuses of the lymph-nodos. Dog 34. — Splenectomy was performed under ether ansesthesia on March 11, and hemolytic serum adminis- tered intravenously on March 14. Death occurred on March 15, after reduction of red cells to 1,960,000 and hemoglobin to 57 per cent. Haemoglobinuria was marked. Histology. — A mesenteric lymph-node shows hemor- rhage, oedema, and extensive infiltration with polymor- phonuclear leucocytes. The sinuses, both peripheral and central, are closely packed with large, pale, endothelial cells, nearly all of which contain red blood-cells, a single high-power field showing thirty to forty phagocytic cells (Plate I, Figs, l and 2). The number of engulfed red blood-cells varies, but is usually large, ten to twenty not infrequently being found in a single cell. In many en- dochelial cells, on the other hand, the red blood-cells have fused to form large, round, or oval hyaline masses still staining deeply with eosiii. Between the phagocytic cells is much granular, eosin-staining material suggesting dis- integrated red cells, mingled with serum, through which lom irregular threads of fibrin. Here and there in the follicles are small areas of necrosis. Moderate leucocytic infiltration is seen throughout the section. Phagocytic cells cannot be demonstrated in the blood-vessels or in a tangle of lymphatic vessels present at one side of the node. Other IjTnph-nodes (gastrohepatic, prevertebral, and bronchial) present the same lesions. ■:| tl 180 THE SrLEEX AND AN/EMIA The liver of this animal showed widespread necrosis, hut in the non-necrotic areas phagocytic endothelial cells are found in the capillaries (Plate I, Fig. 3). SUM.MAUY In a Inrfre proportion of dogs that have hccn splenec- toniized for periods of two weeks or more one finds a great increase in the nuniher of endothelial cells of the lymph- nodes. In most spleneetdtuized dogs that siiceuml) to an injection of haMiiolytic immune serum within forty-eight hours tliL' sinuses of the lymph-nodes contain large imm- hers of endotlielial cells, pliagocytic for red hlood-cells. Tliis is not seen in normal dogs receiving h;cmolytic serum. I^ikewise a similar power of phagocytosis is seen fre- quently in the stellate colls (KupfFer's) of tlie capillaries of the liver. Both in the lymph-nodes and the liver these cells a})pear to he formed in ffitu: we find no evidence that they have hecn transi)orted to these organs. Such findings suggest the development of a compen- satory funetion on the part of the lymph-nodes and pos- sibly the liver. Xormally the spleen contains cells which have the power to engulf and presumably to destroy the red blood-corpuseles. In certain patliological conditions this function is f]'e(|uently greatly augmented and n)ay sometimes he shared by the lymph-nodes; for example, in tyi>hoid fever, as was first clearly shown by ^Mallory.""" Our observations suggest that in the absence of the s})leen this function of forming red blood-corpuscle {)hagocytic cells, normally a minor activity of the lymph-nodes, be- comes highly developed in the latter organs, and that in times of stress tiicse cells and the stellate cells of the liver tlnis assume, in part at least, the function of destroying red l)lood-corpuscles by phagocytosis. CHAPTER VIII MKTAIUJLISM STUDIKS ON THE DOCi IJEFORE AND AFTER SPLENECTOMY Although early in our work we studied the influence of splenectomy npoii iron metabolism ^see p. 112), it did not at that tinu; set iii advisable, on account of the generally negative results of others, to study the efFect of splenec- tomy upon nitrogen metabolism. Later, however, when interested in the influence of diet in connection with the ana'mia of splenectomy, we became aware of the obsen-a- tions of Kichet.''^ which seemed to iudicate that in order to maintain the weight of a splenectomized dog a much larger amount of food is required than is tlie case with the normal dog. These studies suggested to us a possi1)ie explanation of the contradictory and confusing results obtained in our dietary studies, and. as nitrogen metabolism had never been studied in animals in nitrogen equilibrium, we undertook a detailed investigation in the hope of arriving at some detinite conclusion concerning (1) the influence upon jnetabolism of the absence of the spleen, as contrasted witbi (2) the influence on metabolism of the ana^nia which usually follows splenectomy. Previous Ixvkstioattoxs Paton's "^ investigation included studies of t!;c nitro- gen metabolism and the elimination of salts in a single isi Hi: '. t ,,!■-. 182 THE SPLEEN AND .VN.EMIA dog before and after splenectomy. Obser\'ations were made during fasting and on (1) meat, (2) oatmeal and milk, and (3) rich nuclein diets. The first post-splenec- tomy metabolism study was made twenty-six days and the last four months after the operation. Paton's general conclusion h that under the various conditions of his experi- ments splenectomy causes no essential difference in the course or nature of the metabolism. In Richet's first investigation^"^ nine splenectomized dogs were contrasted with six normal dogs. No metabolism studies and no examinations of the blood were made. Con- clusions were based on records of food taken and the weight of the animals at various inten'als. The increased con- sumption of food by the splenectomized animals is thought by Richet to be due to an increased catabolism in those animals and not to any disturbance of digestion. In a later report ^''* he refers to studies of seventeen splenectomized dogs, of which five were under obser\'ation for about two years, and confirms the conclusions of his earlier report. In this connection it is a matter of importance that the conclusions are based on the averages of two groups of dogs of widely different weights. Richet has not contrasted splenectomized dogs of given weight with normal dogs of the same weight, but if one selects from his tables dogs of the same weight the differences in food consumption are found to be very slight. Only two dogs were studied both before and after splenectomy. Mendel and Jackson,^*^ who investigated the relation of the spleen to purin metabolism, found that in splenec- tomized dogs and cats no changes occurred. Verzar*'*'' has found that extirpation of the spleen in dogs has no appreciable effect upon the respiratory gas METABOLISM SI UDIES ON THE iXA} 183 exchang'e. A siniiiar c(jnclusion was reached by Koren- chevski ^" as regards both gaseous and nitroginous nietab- oHsm. No other experimental studies of the influence of splenectomy are available, except the brief note of Austin and Ringer^' to the effect that in the dog the absence of the spleen does not in any way modify the course of the glycosuria caused by pholornizin. Methods The four dogs used in this study were placed upon a constant diet of beef (usually beef-heart), lard, and sugar, the amounts of each of which constituents varied accord- ing to the caloric needs of each dog. The standard diet con- tained 0.4 gm. of nitrogen per kilo, and 70 calories per kilo, of body weight. A small amount of sodium chloride was given each day, and a suificient amount of bone-ash was added to ensure well-formed faeces. The water intake for each day was constant. To some animals the beef-heart was given raw; in other instances it was boiled. After one or two weeks on the special diet, if the weight of the animal remained constant, a preliminary metabolism study, covering a period of seven days, was made. If the results of this were satisfactory, the animal was then splenecto- mized and the metabolism studies resumed at various inter- vals after the operation. In each experiment the diet after operation was always the same as before, and was con- tinued without change in the intervals between periods of metabolism study. Analyses were made of all foods for fat and total nitrogen. During the periods of study the animals were kept in the usual metabolism cages. They were catheterized at the enl of every twenty-four hours and the fwces marked by cv rmine. i •! i 'i 184 THE SPLEEN AND ANvEMIA In the analysis of the urine the total nitrogen was determined by the Kjeldahl-(iunning method, ammonia by Folin's metliod,'^' creatine and creatinine by Folin's method,'-' and tlie hydrogen ion concentration according to Henderson's techniciue.'"' In the study of faeces the Kjeldahl-Gunning methml was used for total nitrogen, the Folin-Wentwortli niethod for fat,'"- and Neumann's metliod"' for iron. The removal of llic spleen, an essentially bit nlless operation, was done uiu'er ether aiuesthesia. HKsri;rs The details of our studies of nitrogen metabolism are shown in Tables XLVKi. to LI. and of fat metabolism in Tabic L II. Niirogcn MetahoUum, — Table XLVIIl represents the earliest period of metabolic study (three days) after sple- 'leetomy. The animal showed no loss of weight, no ill- effect of the operation, and the conditions were therefore ideal for the detection of any slight early changes in metab- olism which might be due to the absence of the s])leen. Xo variations in nitrogen partition were observed, how- ever, and the nitro;^en e(iiiilil)rium was maintained: an average daily balance before operatitm of 0.4..5 gm. and after operation of 0.40 gm. Table XlilX sliows practically the same results, thir- teen days and eight weeks after splenectomy. The animal was in nitrogen e(iuilibrium before si)Ienectomy, and main- tained that condition after splenectomy. The general metabolism shows entirely normal results. The utilization of nitrogen was in no way interfered with: it was 94 per METABOLISM STUDIES OM HIE DUG 185 cent, before operation aiid 95 and 93 jKir cent, in the post- splenectoniy periods. In Table L, wliich presents observations two, six, ami ten weeks after splenectomy, the results in the third ami fourth periods (sixth and tenth weeks) are similar to tliose Do< 4.s(» in.s: i.sn io.<) 4. so 10.9i4.S0 I 0) d ! to cc. 20.i 17.') •J2-) ■22.-) KH) •2IM) 220 10 29 .'U 37 3S :i7 40 27 i-S cr a y. e^ .s c s: = '" H gm. iVM) .■u.-i < j o AvcraKo. 10.8; 4.80] 219 3o ym. i pm. ijn Arid (i.ltO '.■!.(.-, 0.13 n.2sn0.47S A(icl it)..'>() :!.7()!().1.5 0.2700.3.")7 Aoi.l UVM) :i.4 110.12 0.2S9,0.32(i| Ari.l |i).9() o..'-)7|0.13l0.2,S50.321 Acid |().!K) :!.«.■): 0.1 4 n.28'.10.3:j.-) Ac',<] !(i.'ii) ;f.s(> (1.14 '/..rsiio.^ir)! Aciil |t).!h! 4.02|;<.12 0.2790.3 !o.84|3.fl2!o.l3 0.2S4 0.3(il I am. 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 4. IS +0.02 4.09 +0.71 4.17 +0.(13 4.30 +0.."iO 4.3S +0.42 4. ,59 +0.21 4.7.') I +().(),-) 4.3.5;+0.4o Mar. Mar. M:ir. M.ir. Mar. Mar. Mar. ^lar. Splcncctomj' AvcraKt' 10.S|4.S0 l.-)0 lO.S I.SO 220 10,9J4.S0 2.35 10.9 10.<) 10.9 11.0 I.SO ISO 4. SO I 1,3,5 4.S0J lOTi 4.S0 210 4.5 3.) 37 40 4.-) 37 42 A,kI Acid Acid Acid Acid Acid Acid 10.9!4.S0 IS,-) I 40 ,(5.15 li.so |()..50 3.92 lajtO 3.'.t() ! (1.50 4. OS I .().!.-) 3.<.t5| !().15 3.3() j0.90 3..-)3 ! 0.41 '3.81' O.ISO 0.1.-)0 0.14:0, 0.13 0, 0.1.5 0, 0.130 o.os to, - -i- 0.10 0, 3110 3110 .3320 3020 2S1 .(Mi +0.74 3000.291 0..53 14.34+0.46 * DiiM : rnw h-.-ef, !">() (rrn,; lard, 50 t^m.; eupar, ."0 gm. t KxprcHK(jd a.4 neRutivc lo^nritliniH, shown in Tables XLVIII and XI.IX. In the early period after splenectomy, however, this animal showed a loss of appetite which caused, durinii- the two weeks following? operation, a loss in \veip;ht of 1.4 kilos. This loss of appe- tite was not due to infection or other post-oi^erative dis- i M. 186 THE SPLEEN AND ANAEMIA turbances, but appeared to be due rather to a dislike of the lard ill the diet. When the lard was cut out of the diet the animal ate readily, and later, -Rhen the lard was again added, no trouble was experienced. As may be seen in Table LII, this was the only animal which showed a high DoQ 48. TABLE XLIX NiTROGKN Metabolism Before and Two and Eight Weeks Aiter Splenectomy 1 • Urine z 3 5 03* a c c Dat« a 3 o S a" a "3 o H S a •a 3J a g J as .2 is y. ■< 73 X « *" H < O o U< z"" z ka. ce. 10 gm. gm. gm. gm. gm. Nov. 16 13.4 .5.60 175 56 Acid 0.70 5.35 0.38 0.30010.358 0.34 5.69 -0.09 Nov. 17 i;?4 .5.60 2.50 47 .•Vcid 6.90 5.48 0.37 0.3380.195 0.34 5.82 -0.22 Nov. IS 13.4 5.60 225 60 Acid 6.70 4.91 0.38 9.3.520.480 0.34 5.2.5 +0.35 Nov. 19 1.3.4 5.60 210 43 Acid 6.80 5.33 0.37 0.3680.427 0.34 5.C7 -0.07 Nov. 20 13.4 5.(>0 200 44 Acid 6.90 4.80 34 0.3610.467 0.34 6.14 +0.46 Nov. 21 13.4 5.60 Zm 46 Acid 6.90 4.91 0.31 0.3&80.408 0.34 5.25 +0.35 Nov. 22 13.3| 5.60 265 37 Acid 6.90 4.77 0.27 0.385 0.597 0.34 5.11 +0.49 Average . 13.4,5.00 223 46 6.83 5.08 0.35 0.353[o.419 0.34 5.42 +0.18 Nov. 24 Splenectomy Dec. 7 13.2 5.60 195 55 Acid 7.14 5.19 0.31 0.340 0.575 0.30 5.49 +0.11 Dec. 8 13.1 .5.60 280 35 Acid 6.00 5.36 0.34 0.308 0.513 0.30 5.66 -0.06 Dec. 9 13,1 5.60 290 33 Acid 6.90 4.95 0.29 0.352 0.630 0.30 5.25 +0.35 Dec. 10 13.1 5.t)0 265 39 Acid 6.80 4.65 0.29 0.308 0.513 0.30 4.96 +0.64 Dec. 11 13.1 5.t)0 235 .38 Acid 6.80 4.38 0.29 0.324 0.527 0.30 4.68 +0.92 Dec. 12 13.1 5.60 295 30 Acid 6.80 4.40 0.27 0.329 0.5.58 0.30 4.70 +0.90 Dec. i;i 13.1 5.60 24.- 35 .\cid 6.80 4.52 0.23 D.385 0.672 0.570 0.30 4.82 +0.78 Avemgc 13.1 5.00 256 39 6.75 4.78 0.29 0.3,52 0..30 5.08 +0.51 Jan. 18 13 6 5.70 275 30 Acid 6.80 4.46 0.22 0.3.55 0.398 0.40 4.86 +0.84 Jan. 19 13 6 5.701210 44 Acid 6.80 4.32 0.24 0.346 0.382 0.40 4.72 +0.98 Jan. 20 13.ft 5.70 1 310 20 Acid 6.70 4.58 0.22 0.364 0.361 0.40.4.98 +0.72 Jan. 21 13..T 5.70 300 39 Acid 6.80 4.92 0.21 0.355 0.474 0.40 5.32 +038 Jan. 22 13.'-, 5.70 '2.50 35 Acid 0.70 4.49 0.24 0.311 0.362 0.40 4.89 -1-0.81 Jan. Zi 13 4 5.70 300 39 .A,cid 6.5K) .5.12 0.23 10.337!0. 463 0.40 5.52 +0.18 Jan. 24 13.4 5.70 300 39 Acid 6.90 5.51 0.22 0.326 0.405 0.406 0.40 6.91 -0.21 Average . 13.5 5.70 278 35 6.80 4.77 0.22 0.342 0.40 5.47J+0.53 • Diet: rnw hf-< hc-rt, 2nO nm ; Isrd, fiO «m.; aug»r, fiO gm. t Expressed u negalive loaarithma. 3VIETAB0LISM STUDIES ON THE DOG 187 neutral fat content in the faeces, though what relation there may be between tliis and the dislike of fat is not evident. The practical result of this loss of weight aft«r splenectomy was a moderate retention of nitrogen in the first post- splenectomy metabolism period. However, in the third period, when the animal had returned ^o exactly the same weight as before operation, nitrogen equilibrium was again maintained. It would seem conclusive, therefore, that the loss of weight and nitrogen retention of the earlier periods were due to an influence other than the absence of the spleen. It is of interest that this dog excreted no creatine. In the experiments thus far presented there is no evi- dence that the absence of the spleen influences in any way nitrogen metabolism. In a fourth animal, however, the results were discordant. This animal (Table LI) had serv^ed as a control for the blood counts of the three animals discussed above, and up to the time of our foreperiod had been for ' welve weeks on an adequate constant diet, as was the case in the other animals. Like Dog 52, this animal received boiled meat as a part of the dietarj-. The effect of splenectomy on the nitrogen metabolism, ten days after the operation, was very slight, but a nitrogen equilibrium of +0.48 gm. per day was changed to one of — 0.18 gm., figures not beyond the range of normal variations, but which, in the light of changes to be discussed later, are suggestive of the influence of anemia. At a later period, three months after splenectomy, the animal had not regained the slight loss (0.5 kilo.) in weight, but it appeared to be in excellent condition and the amemia, which had existed for several months, was improving. The plus balance of 1.10 gm. of nitrogen per day (upon a slightly higher nitrogen intake) m i 188 THE SPLEEN ^NJS'D AN/EMLV TABLE L Dog 52. Nitrogen Metabolism Bkkokk and Two, AfTER .Sl'LENEOTOMV Six, and Ten Weeks Date Nov. Xiiv. Nov. Xciv. Nov. Nov. Dee. lO.S ilO.8 lO.S lO.S lO.S ' lo.s lO.S Average Dec. Avcragf Avorag(! Yeh. 10 Fell. 1 1 Feb. 12 Fel). i:i Fell. U 4.77 4.77 4.77 4.77 4.77 4.77 4.77 Urine ! o « 1 'i c a a ! 'A < 1 cc. 170 KiO 225 170 ISO 21,) IGO 10 44 44 33 as 4H 40 43 10.Sj4.77|l83 44 -. 2 I Acid .Vcid Aeid Acid Acid Acid Acid ! I am. :0.-)0 4.17 10..5() !c..")0 0..-)0 ! t)..oi I y. « c a "c t a E K < O :;m. iim. 0.30 o.3r,s' 0.20 ().:',7.V 4.2:i i.4'.J|0.23 0.3t;,S, t.."!") 0.2 J 0.3(JS' 4..") 0.2.J 0.3G.S t.GS 0.2.J|0,3.")N' 4.41 0.2.3 0.351 ym None None None None^ None, None, None tjm 0.3S 0.3S 0.3.S OMS 0,3> 0.3S 0.3S .^ A -\ [ U^' 4.5S -rO.22 4.01 +0.16 4.87 -0.10 4.93 -0.16 4.93: -0.16 5.00,-0.29 4.821-0.05 |0..")0: 1.44,0.25i0.3(i.5| Splenectomy 0.3S 4.,S2 -0.05 9.4 4.70 225 23 9.4 4.70 1.^0 39 9.5 4.70 115 43 1 9.5 t.70 210 35 9.1) 1.70 S5 53 ! 9.7,4.70 275 20 j 9.-; t.70 175 30 1 9.S 4.70! 225 25 .Void |0.90i2.75 0.16 0.213Nonc0.44i3.19l + 1.51 Aci.l ](i..5'.) 3.73 0.25 0.321 None 0.444. 171+0.53 Acul ;ti.s0 2.99 0.15 0.2,sl .None 0.14 3,43i + 1.27 Acid O.SO 2.79 0.15 0.2>9 None 0. 14 3.23l-rl.47 .\cid '(;..'iO 2.4S 0.20 ').21t.-,No!!e 0.4 1 2.92' + 1.7S .\cid r,.70 2.79 0.2.') 0.2in .\one 0.44,3.23 +1.47 Acid (i.70 2.52 0.23 0,2s ,Vcid :().30 3.45 0.22 0.324 None 0.4s ,Vcid 0.30;3..")4 '0.25 0.:i2l .\i.i;c 0.4> ■ ' (1.70 3.3s 0.25 0.311 N ,S,.5 8.4 8.4 8.4 8.4 Urine. 3.43 3.43 3.43 3.43 1 3.43 3.43 3.43 j 100 11. J 140 120 120 200 '.»0 10 39 44 38 42 47 30 Acid Acid Acid Acid Acid Acid Acid S^ ! -e f/m. gm. (jm. urn. I ;;m. t).30 2.03 0.13'o.2210.014i0.35 0.00 2.01 0.1 7 0.2700.0221 0.3.5 (J.13;2.3lt 0.14 0.27<»0.022[0.35 0.30 2.fi:! 0.17ii>.31!M),0r)7 0.;i,-) 0.30, 2,7:i O.li; 0.201 0.1 10 0.3,5 t).,30i2.0I 0.13 0.20.".O.OS4<0.:i.5 (i.l.")'2.(iO 0.1 t 0.2,')3 0.007,0.3.") 8.4 : 3.431 120 i 43 -L .98 .90 .74! .98 i .o,s ,9t) i •hi ] +0.45 +0.47 +0.06 +0.45 + 0.35 +0.47 +0.48 _>j^2 r 2 ilO O.I5 O.207I0.O55; 0.35 i 2.95 Splenectomy +0.48 .\vcranc May 20 May 21 Mav 22 May 23 .MaV 24 8.0 8.0 8.0 S.O 7.9 7.9 7.9 3.37 105 I 3.37! 110' 3.37 i 150! 3.37! 210: 3.37 j 170 1 3.3; 3.3; 8.0 3.3< 130 170 Averui:u. 7.9 13.78 1 130 7.9 |3.7sj 115 7.9 |3.7>;' 130 7.9 ,3.78 1 200 7.9 13.78] 115 47 46 44 40 42 49 38 Acid I Acid .\cid .\cid Acid Acid .\cid 15 3 15:3 50 1 3 .8,5 1 3 15:3 11 0. 00 0. .19 0. 30 0. 32 0. IS 0. 09 0. 13:0.2310 110.2130 14 0.234 0, 10 0.2210, 160.2130. 10 0.2130. 12 0.213 0. .1210.4113, 113 0.41] 3. 0M;0.4i:3. 119 0.41] 3. 144 0.41 1 3. U! 0.4113. 129 0.41:3. 44 0.1 .17 0.14 ,0.220,0.12210. 11 i:i.' 7.9 I3.78i 156 Acid Acid Acid Acid Acid 1.17 !.07 1.24 ;.56; :.47 r .30 0.21.30, 0.21: '), '0.22.-) ().23S0, ii213;o. 029] 0.38 0140.38 010:0.38 01 7! 0.38 014!0.3S 1O.22OO.OI7 0.38 * I)if*t: boilo'J hfQi heart, 75 ( .')(;). This animal showed a loss of wei^-ht, an increased elimination of iron (see p. 119), and a disturbance of creatine metabolism. The fat metabolism was unaltered. The question arises whether these chancres are due to the al)sence of the spleen or to the ana^nia which was present. h Table I.IU are presented the blood examinations of eacli doy at tlie tiiiie of the several metabolism j)eriods. It will I)e seen that Dons 4,8 and .IT showed no aj)precia- able ehanuo in the blood picture after splenectomy, but that I)o,!4s 52 and .jfj did. The blood chancres in l)o; 56 develoi)ed a severe anaemia, already I)rogressive at the time of the first metabolism study, while l)ot?s 48 and 57 were not ana-mic, and Do^ 52 showed only a slight non-progressive deterioration of the blood. The (juestion naturally arises: Is the in^-reased elimination of iron and the disturbance of the creatine metabolism due to the ana?mia and not to an influence on metabolism con- sequent upon the al)sence of the spleen? A few words are necessary concerning Richet's state- ment that the splenectomized dog requires more food to maintain its weight than does the normal dog. In view of our results, Ilichet's eonclrsion is not tenable. Dog 57 (Table XLVIII) maintained its presplenectomy weight without change in diet and with only a slight change in the nitrog-n balance. Dog 48 (Table XLIX) likewise showed only a trifling change during the three weeks after opera- tion and a return to the previous weight after seven to eight weeks. The serious loss of weight in Dog 52 was due to loss of appetite, and that, relatively slight, in Dog 56 was complicated by the coexisting anaemia. Moreover, during the past five years we have fre- quently noticed a tendency for splenectomized dogs to become obese, and this tendency is mentioned also by several investigators who have studied splenectomized ani- mals for long periods of time. This tendency to put on weight is strikingly shown by two of the dogs (48 and 52) of this series. At the close of the metabolism work, pre- sented in Tables XLIX and 1., these animals were not destroyed, on account of the possible necessity of repeating 13 * Is ilH I '*-,'• '■' i 194 THE SPLEExN AND ANEMIA ^^L'' the metabolism studies after longer periods had elapsed. The change from a special to the ordinary kennel d^et ("table scraps") led to a rapid increase of weight in each instance; in tliree months the weight of Dog 48 in- creased from 13.4 to 1.5.8 kilos., while in two months Dog 52 rose from 10.9 to 12.9 kilos. Our results are therefore in accord with those of Paton rather than with those of Kichet, and demonstrate that in the absence of anaemia the removal of the spleen has no influence upon nitrogen or fat metabolism, and in all prob- ability no influence upon iron elimination. Our general results may be summarized as follows : Four dogs have been subjected to metabolism studies before splenectomy and at intervals of three days to three months after splenectomy. In three of the four animals the removal of the spleen was not followed by any dis- turbance of nitrogen metabolism, fat utilization, or iron elimination. Two of these animals showed no anaemia, and the third only a slight reduction in haemoglobin and num- ber of red cells. A fourth animal, studied ten days and three months after splenectomy, develo])ed eventually a definitely pro- gressive ansmia of moderate severity. This animal showed a slight loss of weight, a slight disturbance of nitrogen balance, and of creatine-creatinine partition, with a marked increase in the elimination of iron. We conclude, there- fore, that, under the conditions of our experiments, there is no evidence that the spleen has an influence on metab- olism, and we regard the disturbances occurring in one of our dogs as due to the coexisting anaemia and not to the absence of the spleen. ; CHAPTER IX GENERAL SUMMARY OF EXPERIMENTAL STUDIES After splenectomy in dogs three prominent phe- nomena are observed: 1. An antemia of the secondary type, mild or moderate in character, which usually rvtaches its severest stage after one and a half months and is followed by repair, wh," ^h is well advanced after three or four months but may not be complete for longer periods of time. 2. An increased resistance of the red blood-cells to hypotonic salt solutions, hsemolytic serimi, saponin and cobra venom, and mechanical shaking. 3. A lessened tendency to hfemoglobinuria and jaun- dice, and sometimes an absence of jaundice, after the ad- ministration of hemolytic agents. The ana?mia may be irregular in its onset and severity, as may be also the repair process, but as a rule it has a very definite course. The decrease in hasmoglobin content occurs usually a little later and is often slightly more marked than the decrease in red blood-cells. The former seldom falls below 55 per cent, or the latter below 3,000,- 000. Neither during the period of anaemia nor repair do nucleated or other abnormal types of cell appear in the peripheral blood, except occasionally, at the time of begin- ning repair. The behavior of the white cells is quite con- stant; shortly after operation a marked rise to 26,000 to 38,000 occurs, with a return after a few days to 20,000 and a gradual decrease to normal level after one to four months. The increase in leucocytes is almost entirely an 1« ' t 1- H 196 THE SPLEEN AND .VX.EMIA increase in the polyiiiorphomielear type, the lympliocytes later showing a sh^rJit inerease. The hehaxior of th'j eosinophiles is inconstant. The " blood crisis," so frequently found after removal of the human spleen in splenic disease, is not present after the rem' val of tlie normal do^'s spleen. l)i' ary studies indicate that the ana'niia is not influ- enced by the amount of iron furnished in the food. On the other hand, a small number of experiments furnish some evidence that the ana-mia is apt to l)e mce severe when the animal is fed on cooked food than when it is fed on an uncooked diet. Control experiments, in which other oix?ra- tive procedures, such as unilateral nephrectomy, have pre- ceded splenectomy, prove conclusively that it is the absence of the spleen, and not post-operative accidents or compli- cations, that is the essential factor in the production of ana?mia. A review of the literature of splenectomy in man shows that after removal of the spleen for injury or simple lesion not involving a distui-banee of the function of the spleen, as rupture, twist, and cyst, a mild anjemia of variable course is the rule, and that in the absence of the spleen the repair of antpmia is delayed. The increased resistance of the red blood-cells is a property of the red cell itself and is not due to an anti- hsemolytic power of the serum or to changes in comple- ment content. The exact reason of this increased resist- ance is not evident. Its association with ansemia and the concomitant repair suggests that the presence of young and more resistant cells might explain it. We have not been able, however, to demonstrate an increase in nucleated cells or in the reticulated cells, usually considered as vounsr forms indicative of active blood formation and more resist- SOIMARY OF EXPERIMENTAL STUDIES 197 ant to lytic agents. We believe, however, that the in- creased resistance of the rtd cells is not dependent merely on the absence of the spleen, but is, in part at least, in some way associated with the anc-emiu, or the factor or factors causing it, thuii differing, in our view, from Banti and his associates. In regard to the decreased tendency to hjpmoglobin- uria and jaundice after the administration of hiemolytic agents, we offer experimental evidence to indicate that ( 1 ) absence of the spleen does not prevent the secretion of bile; (2) the spleen does not influence the transformation of free hfemoglobin into bile-pigment, and (3) that fresh splenic extracts have no demonstrable action in vitro. Two im- portant factors appear to be (a) the influence of anremia, and (b) the increased resistance of the red cells, in that each reduces the amount of hemoglobin set free. The most important factor, and a purely mechanical one, concerns the way in which the ha?mogl()bin set free by blood de- struction reaches the liver. Under normal conditions the disintegrating biood-cells accunmlate in the spleen and the liberated hfcmoglobin normally reaches the liver directly and in concentrated form through the portal vein, and, as a result, hemoglobinuria and jaundice are more apt to occur. In the absence of the spleen it reaches the liver through the general circulation (hepatic artery) more slowly and much diluted, and hemoglobinuria is therefore less frequent and jaundice is of less degree or entirely absent. Our experiments in which injections of hemoglo- bin into the mesenteric vein were contrasted with injections of hemoglobin into the femoral vein offer conclusive proof of the correctness of this view, as do also experiments in which, by means of an Eck fistula and by anastomosis of the splenic vein with the vena cava, the splenic blood was il n : I fc a 11 198 THE SPLEEN AND ANAEMIA diverted from the liver without removal of the spleen. Another important mechanical factor connected with the blood supply to the liver is that in splenectomized animals the total volume of the portal blood supply is consider- ably lessened. Further support of this view is found in the ansemia that followed ligation of the splenic veins and in the single experiment where the mesenteric vein was unwittingly transplanted into the vena cava. Another factor which we have discussed in this con- nection is that concerning the influence of fatty acids as brought forth by Joannovics and I'ick. Their views are supported by Eppinger and King, who find, after sple- nectomy in the dog, a decrease in the unsaturated fatty acids and an increase in the cholesterin content of the blood, changes which might well have an influence on the degree and velocity of h£emolysis. Their observations, unfortu- nately, we cannot confirm. In regard to the problem of the ana?mia, detailed com- parison of the arterial and venous blood of the spleen offers no evidence to indicate, by the methods used, that the spleen has an important role in blood formation, or, on the other hand, that it is appreciably active in blood destruction. The injection, however, into normal dogs of fresh spleen extract Joes cause an evanescent but definite in- crease in lijpmoglobin and red-cell content, which is not caused by extracts of other organs. This result would appear to be brought about through a stinmlating action on the l)one-marrow. On the other hand, i"rcsh beef spleen fed liberally to sjilencotomized dogs does not prevent the occurrence of anivmia. If in splenectomized dogs which have recovered fi-om the SUM]MARY OF EXPERIMENTAL STUDIES 199 anaemia following removal of the spleen a second anaemia is produced by administering some haemolytic agent, this anasmia, as shown by direct blood examination, runs a longer course and has a longer period of repair than is the case in the normal dog. Here we have an apparent para- dox, for, as has been pointed out above, the red cells of the splenectomized animal are more resistant to haemo- lytic agents than are those of the normal dog, and theo- retically one would expect a mild a- eemia of shorter course with quick repair. Our explanation of the paradox is that in the spleenless dog some factor is at work, due to the absence of the spleen, which prevents rapid blood regeneration. The normal dog, though suffering as great, or even a greater, blood destruction, has no fault in regeneration, and its blood re- turns quickly to normal. On the other hand, in the splenec- tomized dog the factor delaying regeneration operates after an acute anamia, as it did originally after removal of the spleen, and therefore the anaemia often reaches a lower level and is repaired more slowly. Experiments undertaken to show that this unknown factor might be a function of the spleen concerned in the utilization of iron for the marmfacture of hfemoglobin have given negative results. Occasionally, shortly after splenectomy, a slight increase in elimination of iron was observed, but we have found no evidence of disturbance of iron metabolism one, nine, and twenty months after splenectomy. The most marked disturbance of iron meta- bolism occurred in a dog with a moderately severe anemia, and we believe that an increased elimination of iron is a manifestation of increaseti blood destruction and not di- rectly dependent on the absence of the spleen. «00 THE SPLEEN AND ANJCML\ Control studies, in wliich without removal of the spleen the spk'iic blood was diverted from tlie liver by means of an Lck fistula or an anastomosis of the splenic vein with the vena cava or ligation of the splenic vein, yielded results similar to those following? splenectomy, but varj-ing somewhat in def^ree or duratioii. The ana?mia and the icterus, as also the slow repair of anjcmia, under these circumstances did not differ greatly from the results following splenectomy. If the auivmia is due to the loss of a stimulating action on the bone-marrow, these experi- ments show that access to the liver is necessarj' for the activation of such stimulant. The increased resistance of the red cells, on the other hand, did r- t persist for so long a time as after splenectomy. The lessened tendency to jaundice, on account of the important mechanical factor involved, was quite similar to that following splenectomy. These observations emphasize the importance of the mechanical factor (the disturbance of the direct blood flow to the liver), and suggest, also, some functional relation of the spleen to the liver that is not yet fully understood. Studies of the l)one-marrow after splenectomy show that, as a rule, tlic fatty marrow of the long bone is trans- formed into red marrow. During the early months this change is slight or absent, but after six to twenty months it is fairly constant and complete. There is no evidence that this change is compensatory to the early antemia caused by splenectomy or to an increased hannolysis in the lymph nodes. It may I)e that it is a concomitant of the activity of the bone-marrow in taking over, in the absence of the spleen, the function of storing aiul elaborating th(> iron of old blood-pigment, but our observations do not fully support tliis liypothesis. SUM>L\RY OF EXPERIMENT.VL STUDIES 201 The lymph-nodes, after splenectomy, exhibit, as a rule, a great increase in the number of endothelial cells. In most splenectomized dogs that succumb to a haMiiolytic agent within forty-eight hours the sinuses of the lymph- nodes contain large r.^mbers of endothelial cells filled with red blood-corpuscles. This is seen, also, to a less extent in the case of the stellate cells of the liver capillaries. This phenomenon has not been seen to the same extent in normal dogs receiving hjcmolytic serum. Such findings suggest that in the absence of the spleen the function of forn?ing red-blood-cell-phagocv-tic cells— normally a minor func- tion of the lymph-nodes— becomes highly developed in these structures and is shared also by the liver, and that in times of stress, as after excessive blood destruction, these organs assume, in part at least, the function of destroying red blood-cells by phagocytosis. The small iron content of the lymph-nodes and liver indicates, how- ever, that under noi,nal circumstances, in the splenecto- mized animal, no excessive destioiction of this type occurs. Detailed metabolic studies have demonstrated conclu- sively that the removal of the normal spleen in a noi-mal animal has no effect on nitrogen, fat, or iron metabolism. ^Vhen disturbance of nitrogen and iron metabolism occurs it is due, in all i)robability, to a coexistent antcmia, and not to the mere absence of the spleen. In conclusion, we wish to state frankly that, while the experiiiients here described add to our knowledge of cer- tain i)hases of the relation of the spleen to blood destruc- tion and regeneration, we fire still in doubt about the exact cause of (a) the ana?mia and (h) the increased resistance of the red cells which so constantly follows splenectomy. CHAPTER X METABOLISM STUDIES ON MAN BEFORE AND .UiTER SPLENECTOMY Studies of metabolism after splenectomy in the normal dog, as has been shown in the preceding chapter, are es- sentially negative. In man the situation is entirely differ- ent. Splenectomy, for chronic disease at least, is done in the presence of an anasmia of more or less jeverty, and the alterations in metabolism before operation are largely those dependent on the changes in the blood. The im- provement following splenectomy is to be regarded, there- fore, not as dependent on the removal of a normal fvmction of the spleen, but as dept t on the removal of a ha?mo- lytic or other toxic activil j/ vhich the altered physiology of the spleen is responsible. Metabolism studies in man, under such conditions, thus become valuable adjuvants to the blood examination in determining the type of splenic disease or anjemia in Avhich favorable results from splenec- tomy may be expected. As investigations of this type are comparatively new — to our knowledge only five studies of conditions l)oth before and after splenectomy have been made — we present in con- siderable detail two carried out under our direction: one in cotmection with congenital ha?molytic jaundice"" and the other in connection with pernicious ana^mia.^" Tx CoxGEXiTAi, Hemolytic Jaitxdice The subject of the first of these was a child suffering from congenital ha^molytic jaundice presenting the fol- lowing history: 202 'if METABOLISM STUDIES ON MAN am At birth, at term, the child weighed seven pounds, and is described as lacking the characteristic red color of the newly bom. About twenty-four hours after birth the " alabaster whiteness " of the skin, which the mother de- scribed, changed to a mahogany brown, which lasted three months, gradually fading to a sallow pallor, which has persisted. xVt six months, when the child passed through an attack of pneumonia, it weighed only ten pounds. The general health was poor and gastro-intestinal disturbances frequent. In the fourteenth month the first severe anaemia, accompanied by dark-brown discolorations of the skin and preceded by protracted vomiting and diarrhoea, was ob- served. Two months later a similar attack occurred, with the new feature of marked oedema of the entire body. During these attacks the rectal temperature usually rose to 104 or 105. Periods of recrudescence and exacerbation followed one another imtil the child was two and one-half years old, when an unusually severe attack kept him in bed for five months. Vomiting and diarrhoea were severe, and hemorrhages from the nose and bowel were frequent and difficult to control. During the second month of this period a partial paralysis of the left side developed. At this time an injection of neosalvarsan was given, more for the hsematinic action of the arsenic than with any suspicion of lues. Gradual improvement followed this treatment, and after the paralysis had disappeared, except for a resid- ual spastic palsy of the left leg, tlie child enjoyed fairly good health. The Wassermann reaction, frequently re- peated, has always been negative. During the eight months following this attack salvarsan was given five times by rectum. About the time he was five yerrs old, and again seven months later, he lost the power of speech, was ti f04 THE SPLEEN AND .»u\^ML\ more or less delirious, and eomplainedof pain in his head. At three and one-half and again, at four and one-half years he had an otitis media. His appetite has always heen poor, never normal, and at times he refuses to eat. A tendency to localized oedema, especially of the face and of the hancj, has been constantly noted. He tires easily, and frequently complains of this. Obstinate constipation has heen the rule, and the fa?ces are described as dark or orange in color. From the family history it is found that a sister was jaundiced for ten days and a brother for two days after birth, but these children, now fifteen and thirteen years of age respectively, have otherwise been in good health. The father is said to have had an enlarged spleen and offers a histor\' of exposure to lues, of skin eruption, and chronic abscess of joint and ankle. The mother, three months before the birth of the subject of the present studv, was paralyzed and suffered a separation of the pelvic bones. The deliver}' was under anfpsthesia. Lahoratarji Eoraminatiom. — An examination of the numerous clinical records which have accumulated shows that the urine offers nothing of uimsual interest. The only positive finding is an occasionally slight trace of albumiri. Tests foi- bile have always been negative, and urobilin tests have showTi no increase of this substance. Examina- tions of the f.Tces indicate that food is well digested: tests for occult blood have been negative, and no parasites or ova have heen fomid. Xo records of the early l)lood exam- inations have been preserved, except a brief record that at the time of the first severe attack (when fourteen months old) the h.Tmoglobin fell to 24 per cent, and a diagnosis of pernicious an pounds and he seemed eonsiderahly improved in eolcjr and strength. The appetite was ^ihh\. The eondition of the bowels demanded an oeeasionai eathartie, whieh, however, never eaiised watery stools. Kven before dischartre on February 18, just three weeks after operation, it was evi- dent tliat a marked improvement in the blood pieture had oeeurred, both hiemo^dobin and red blood-cell count being double that obtained on the first admission. After discharge the improvement continued steadily, with greatly increased appetite and strength. The skin lost its sallow hue and became normal in appearance. In the two months since operation the child has enjoyed uninterrupted good health, except for one attack of indi- gestion, the result of overfeeding. Two weeks previous to the last blood count given in the table a rather severe nasal hemorrhage occurred as a result of excoriations. The lowered hipmoglobin in the last count is probably to be explained by this hemorrhage. Pathologic Dksihiption of the Spleen. — The weight is 640 gm.; length, 18.3 cm.; width, 10.8 cm.; thick- ness, 3.8 at one end, increasing to 8.t at opposite end. The organ is of uniform bluish-red color. The capsule is for the most part smooth, with a few fine adhesions at one pole, where there is also a small circumscribed area (1.5 cm. in diameter) of thickening. The vessels of the hilum are normal. Xo supernumerary spleens are seen. On section a large amount of dark fluid blood escapes. The cut surface has a uui' rm smooth, glistening appear- ance of dull-red color. The Malpighian bodies are dis- tinctly visible, but not so large as in the normal spleen. The trabeculfB are not prominent. The consistence is METABOLISM STUDIES ON MAN 209 increased, it being almost impossible to rupture the spleen by pressuie with the thumb. At one end (the larger) of the organ is a distinctly circumscribed, but not encap- sulated, mass (3.5 cm. in diameter), spherical in shape, which shows no Malpighian bodies, but does present a few minute ochre-colored areas. This area is of the same color and consistence as the rest of the spleen. On section it bulges prominently above the cut surface. Tlie weight of the spleen after escape of fluid blood from three longi- tudinal incisions is 435 gm. Gross Diagnosis. — Splenomegaly with area of recent infarction. Microscopic Appearance.— Very slight thickening of capsule with no increase of trabecula;. The sinuses are dilated and congested. The reticulum is increased in amount, and the cells of pulp appear to he decreased in number. The Malpighian bodies show no change except a hyaline thickening of central arteries wliich is evident in arteries elsewhere. ^Macrophages are not numerous and deposition of pigment is not seen. The tumor-like mass described in notes on gross ap- pearance shows intense congestion and hemorrhage without evidence of cell flestruction, and represents, in all proba- bility, the results of occlusion of blood-vesesls at time of operation. Histologic Diagnosis. — Congestion, increase of reti- culum, hyaline degeneration of arteries. ( The rather nega- tive histologic appearance is in general that described for splenomegaly with congenital h?emolytic jaundice.) Methods of Metabolism Study. — The child was kept in a private room of the University Hospital, with a special metabolism nurse in attendance. The complete metab- 14 210 THE SPLEEN AND ANEMIA olisiii study occupied one period ol ten days luid a supple- nientaiy period of five days betore splenectomy, and a period of ten days after splenectomy. The first period extended from December 3 to December 14, after which the child went home for the Christmas holidays. While the child was at home a supplementary period of five days for the study of uric acid elimination extended from Janu- ary 20 to Januarj' 24. The return to the hospital was delayed on account of the desire of the attendant phy- sicians to improve, if possible, the blood picture and the general condition. On January 28, two days after re- admission, the spleen was removed, and on Februarj' 5, after a lapse of eight days, the post-splenectomy metab- olism studies were begun ^'1 continued for ten days. On account of the capricious u^ypetite of the child, it was im- possible to adhere to a constant dietary, such as the Folin diet, and therefore considerable liberty was allowed. The intake was determined by weighing all foods taken and analyzing portions for nitrogen and fat This policy was followed in both of the ten-day periods, but not in the supple;. It I ry fivc-d.:y period when the child was at home. Despite the freedom as to diet, the food intake was quite constant in character from day to day, consisting, in the first period, essentially of milk, eggs, cereals, apple sauce, bread, crackers, potatoes, butter, sugar, rice, and tapioca. During the first five days of this period beef, chicken, or fish was allowed once a day; during the second five days these were entirely eliminated, as they were also in the supplementary period of five days before splenectomy and the ten-day period after splenectomy. Thus, except in the first five-day period, the child was on a practically purin- and creatin-free diet. The calorific value of the diet was METABOLISM STUDIES ON MAN ill adequate. During Periods 1 and II (Table LV) the sub- ject received approximately 1100 calories a day, or about GO calories per kilo, of body weight. During PeritxJs IV^ and V the patient was on a slightly lower calorific intake, but entirely adequate; namely, 960 C!' lories per day, or about jO calories per kilo, of body weight. The nitrogen of tiie food was estimated by the Kjeldahl- Gunning method and the fat by Soxhl'^t extraction. The urine was collected in twenty-four-hour periods, and por- tions passed during that period were preser\'ed under toluene in an ice-chest. The urine was acid to litnms at all times. In the analysis of the urine the total nitrogen was de- termined by the Kjeldahl-Gunning method; ammonia by Folin's '-■'* method; urea by Benedict's *'* method; uric acid by Folin's colorimetric method;'^'' creatin and creatinin by Folin's method; '"' and the hydrogen ion concentration according to Henderson's technique.^" In the study of the fteces the fat content was deter- mined by the Folin-Wentworth method;'^- the iron was estimated by Neumann's method;'''^ nitrogen by the Kjel- liani-Gunning method, and urobilin by a slight modifica- tion of the method recommended by Wilbur and Addis.*'"* Period III (Table LV) was considered a desirable control on account of the high figures for uric acid obtained in the first and second periods. The analyses in this period were therefore limited to those determinations of special interest in this connection. Results of Metabolism Study. — In Table LV are presented the results of the study of nitrogen metabolism. Nitrngen Metaholism. — During Periwls T and 11, be- fore splenectomy, the subject showed a slightly plus nitre- Hi THE SPLEEN AND ANAEMIA c N o I e.s and temper- ature O ■*" -X M f Nilro- bal- ance, gill. d H d - - 1 -r 1 11 rT-r+ 1 ( Total output giu. rt Q cO N X cs 5 1--: 3i q d d ^ d t-' d ^ d CO V d ; d S^ £ fl - - ~ ^ 5J 3 Si i 5 »»«• ic ".T i.-^ xTi 1 ■; dodod d ddooo ^1 cc — c '■■: '-•: CQCOC 1 Hi h- ■* O « t' ccdoc «rc — '-. X c c c d 'd las O) lO -5 o — ^ ?4 « W f) ododd CI O ci C C — |t CJ C-J 01 C-* CM cccco CM CM "Bias o X ci « "_2 ddddd d d d c c d o ~ c r- 1.- — I- t-.- — c CM ?4 C-t CJ .-^ oc:ccc X X X o c ^^ ^ '^ ^ d d c d d X d o-f — q X ^ L-tl L^ ^ t-i d d o d d t c c O C -f O -r (.M -c - ■•r «.■: L- -fl- ;i o o c c c d 2 3 a = - 5 c 5 CO r^ c^ :-i r- ri c. - 1/: =; -r 3J . £5 ■o --r -r ic i.-: d •*• -r t- -r :■) d -r ft -■'•, -r "? ■3 2d = C;«Ci X-r o -?■ r. X tc d lb -r d d X d T. :c ■* -r •^ T .-^ X -f c * ^ a a 'u r o 3 a c ^ d d tb lb d ti3 -id'^d-T q d ■it t- . . . ■ . . . -N : ; ; : -/: i * ■N(NMM(N 11 u O > W ?< OJ :n Ti «c 1"; lo i.-; I/; C — CMirr-r CIC-lCMCMfJ < 1 1 M s MET.VBOLISM SlUDLES ON MAN 21S f-. p ^ ;2 ^ ^ Oi C5 CJl ^ n Oi ■^ ac C C *-. - c^ X si It %: S- C^ + + + ? + o -c >-■: X — P3CS NOl n Ore — -f -*• C X — 70 C-. c< oi :r ci cj coed d oc — c — 1 + I + -i- ».t w c ^ ooco c T -r -r ■* ■-r c d d d c OC' — MO c53d ddcd f' c* c*: >c X c >c c; oooo c ^«0« O CD t" JP O C *o -f ^ «" :c §SSS g d c d d d re O CO r: OOCO c I' X C X deed — «.-■■«? c d d d !•• f re f c*: ce ce X i-e .- >c -r -^ pc' -^ OOOO o q S 'j; c^ ci r,' d 'd -*" ■*' w . ■ ■ 00 : : : C f M?* cj Z4 z> n CCCC COC c c re 'e C-: ;c r: » re le cc n* - - ■ ■ tc ■ • ■ ' hJ »0 »0 ut 1/5 ' ">.*" N ?* C) N N S a .3 u u a = i Ei^^ iU THE SPLEEN AND AN.EMIA geii balance. iVt'ter splenectoiny, (iui-in<^- Period IV, a ^reat retention of iiitrofren oeeurred, altliouersisted. This average (Periods II and III), 0..),30 gm. of uric acid, is very close to the highest average of uric-acid output of an adult on a purin-freo diet. Few figures for normal uric-acid out- put in children are to be found in the literature. Closson,*" METABOLISM STUDIES ON ^LVX 215 ill a cliiUl of about seven years of a^^e, found an average output of 0.23 gni. on a purin-free diet. After splenectomy the average output of uric acid in Periods IV and V decreased 47 per cent, from the average of Periods 11 and 111 before splenectomy. It will be re- membered that the diets of all these periods were purin- free. The appearance of the urine in these later periods was markedly altered, the dark-red color being replaced by a pale yellow, with never a spontaneous precipitate of uric acid. Hydrogen Ion Concentration.— On the same general diet the hydrogen ion concentration of the urine remained constant befor(i and after splenectomy. During Period I. on a mixed diet, an average of .5.87 falls within the normal average (5.94) of Henderson and Palmer.*'" On the purin-free diet yielding a more alkaline ash, the hydrogen concentration before operation is in agreement with that after the operation. It is of interest here to note that the hydrogen ion concentration is not appreciably altered by changes in uric-acid content of urine, although, as shown by Blatherwick,*" the ability of urine to dissolve uric acid' is a function of the hydrogen ion concentration. Ammonia Nitrogen.— The ammonia nitrogen in our experiments shows no variations from the normal. Its close agreement with hydrogen ion concentration may be noted; thus a rise in the hydrogen ion concentration is asso- ciated with a fall in the ammonia output, and vice versa. Creaiinin and Creatin.— The creatinin output showed a great constancy in Periods H and III, before splenec- tomy, on the purin-free diet. During Period I, on a mixed diet] the output is slightly above these periods. During Period IV, after the operation, the creatinin output fell I I I i M . t 'I 216 THE SPLEEN AND i^N^MIA to its lowest lit, a decrease of 25 per cent. fr. in the average of .>t}ier purin-free periods. Wlien the total creatinin (tliat is, preformed creatiiiin and creatin con- sidered as creatinin) output of each period is compared, one readily sees that the total creatinin, in all of th .■ periods on creatin-free diet, shows a remarkable constancy. As will be seen from an inspection of the two tables, the de- crease of creatinin was accompanied by an increase of the creatin amounting to 91 per cent, of the average. Why, in Period IV, the partition of creatinin and creatin changed without an appreciable change of total creatinin is difficult to state. During the last two days of this period the patient suffered from a bronchitis with a rise in temperature, but that these are explanatory factors hardly seems plausible. As regards creatin output, with the exception of the first period, which was not that of a creatin-free diet, the outjjut shows a fair degree of regularity. The increased output in Period IV has already been pointed out. There is a paucity of data on the creatinin and creatin outi)ut of children on controlled diets. The results ob- tained by Folin '-•' on his children offer figures which may serve for comparison. The great constancy of our total creatinin (including preformed creatinin and creatin as creatinin) output leads us to believe that for the purpose of comparison in chil- dren this is the figure to be used rather than the relative or absolute amounts of creatin or creatinin. If we are correct in this view, the total creatinin output afrrees verv well with other published figiires for children, and we therefore believe our creatin nnd creatinin figures to be witliin the range of normal variations. METABOLISM STUDIES ON ALiN 417 }\ts.— The total "ntake of fats and the separation of fats in the fa'ces are shown in Table LVI. In this table Periods I and II represent the pre-splenectomy and Periods IV and V the post-splenectomy studies. Each period represents five days. TABLE LVI Fat Determinations in a Case of Congenital H^molitic Jaundice Before AND After Splenectomv Fuiod Hpfore Splenectomy 1 II After Spleneutomv: IV V Total Total intake. output, gm. gm. 222 8.88 223 7.57 227 13.56 269 13.55 Pnr reut. ul fat utilised Total output fatty acids including Buaps, gm. 96.01 I 96.62 ' 94.(H 94.98 6.8 5.1 !0.1 9.8 Per I rent, 1 fiitly ! ariila 1 in total I fut I output Total output ncutrnl fata Per cent. neutral fata in total fai output 76.8 67.0 2.1 2.5 74.4 I 3.5 72,3 I 3.7 23.2 33.0 25.6 27.7 The metabolism of fats shows no abnormal variations. The fat utilization is good, and well within normal limits. As pointed out by Folin and Wentworth,'-'" as total hi increases more of that fat is put out as fatty acids ( includ- ing soaps). Iron. Table LVIl presents the results of the exam- TABLE LVII Iron EiiMiNxnoN in Vmckh in a Case of Conoenttal H.f:Moi.i-Tir .FArxDiCE Ukfore and After Splenectomy Peiiod Total intake period, nig. (calculated) Total output period, nig. Intake per d:i>', Mli. Output per day, Ulg. Before Splenectomy 37.69 S2.'.)9 3.77 8.29 11 ... After Splenectomy IV V 45.61 41 11 4,56 -^.ll i I 218 THE SPLEEN .VXD .\N.EML\ ination of the faeces for iron. As the iron in human urine seldom exceeds 0.001 mg., the urine is not included. Analyses for iron were made on duplicate samples of dried fa-ces representing periods of ten days, hefore and after splenectomy, respectively. Th.us the first ten days correspond to Periods 1 and II and the second ten days to Periods IV and V. Pericxls II IV, and V represent essentially the same diet. The figures for iron intake were calculated from published records'"^""' of iron content of foods, hence no claim is made for the extreme accuracy of those figiu'cs. They merely serve to show that the iron content of the diet agreed verj' closely and would not account for the large difference of output. Those differences in output before and after splenectomy amounted to about 40 per cent, decrease. That the large output of iron in the period before splenectomy is due to the increased elimination of ii'on consequent on the ex- cessive destruction of red cells seems the most plausible explanation. The decreased elimination after splenec- tomy, with a close agreement of intake and output, shows a cutting off of this loss and presumably a return to normal elimination. Urobilin. — Our interest in the urobilin problem has been limited to the influence of the absence of the spleen on the elimination of this substance. In the urine quali- tative tests for urobilin gave negative results throughout the experiment. At one time, in a concentrated urine, a faintly positive reaction was obtained with Ehrlich's reagent. The large bulk of this constituent was in the fseces. Because of the small bulk of the child's fasces, and the necessity of utilizing considerable portions for other determinations, the use of the wet f'thr<>- l-puk" piT i cytea cytfs lunt. : Nnrl<>ate thrill 5 tl'S I lated I erythro- I cytes, ! per cent 3/28/15 26 4/ S/15 4 15/15 5/ 3/15 6/ 5/15 i 1 6/ 7/15 6/ 8 15 ti/lJ 15 G/I5 15 6 21/15 6,24/15 6,24/15 6 '28/15 7/ 9/15 7/15/15 7/22/15 7/30/ 15 8/ 6/15 8/16/15 8/24/15 8/29/15 8/30/15 1/ 8/16 1,150,000 4,6(U) .\onnobl!vsfs + Mep:ili)l)lii.sts + 25 I 1,620,000, 5,S00 Meg:il<)l)lasts + 20 1,110,000 2,000 20 ! 1,700,000 i 28 1,300,000 6,500, 4,300 N'orni()l>lasts + 2 1 Kcrnarka 40' I 1,810,000 3,800] 37 11,420,000 16,600' 40 1 2,930,000 12,000 Xormobhusta i- + 28 31 35 48 55 69 48 54 70 83' 1,640,(XK» 3,700 .\onnnl)l!i.sts + l.t):;0,(HH)i 6,300, 2.370,(XK); 6,000 XornioMiusts + I MeKul()lilast,s + 2,030,(K)0 S.IOO Xorinoblaf-ts-l- 2,570,()00| 7,100, .Nornioblaala + 2,3(X1,0 ■ ■ ■ -^ 1 »^ «5 i.-^ lit wt X — . C — "") fi Mr: Mi d CO kO »C >-t lit < «C i-t »c ic 10 iC « I- X 3S C -^" < X - i I - MET A HOLISM STUDIES ON >LVN as Cliniad Sotcs.— 'Yhe patient, a luati, aged forty, had coniplaiiifd for twf) years of weakness, dizziness, dyspna'a, and anlenia. These symptoms were stea,0()0 l)eing too slight to permit of signifi- cance l)eing attached to it. Two months after splenec- tomy, however, at a time when the bl(x)d count showed a pronounced and most satisfactory improvement, the uro- bilin output had fallen to one-seventh of its former figure and had reached a low normal elimination. These observations may be summarized briefly as follows : 1. A slight positive nitrogen balance before splenec- tomy was followed by an increased nitrogen retention fourteen days after operation and a return to the pre- operative balance after one month. 2. The output of uric acid, although never exceeding normal limits, showed a decrease of 22 per cent, after operation. 3. The output of iron through the fjtces, although never above normal, showed a decrease of 40 per cent, after operation. 4. The excretion of urobilinogen and urobilin in the faeces before splenectomy was about three times the nor- mal; two months after operation the output was about one-seventh of that before splenectomy. METABOLISM STLUIES ON .NLVN iio Discussion The literature concerning the relation of the spleen to inetaholisni may he considered under five heads: (1) Studies hoth hefore and after splenectomy for disease of the spleen in man; (2) studies in man after splenectomy; (3) studies of eon<^enital hirmolytic jaundice; ( i') studies of anannia; (')) studies of the effect of removal of the normal spleen. 1. In only three instances other than those reported uhove have nietaholic studies heen made hoth hefore and after splenectomy for diseases of the spleen in man. Two of these are T^mher's studies of lianti's disease, and the third Minot's study of pernicious nna'mia. Umher studied two individuals splenectomized for Banti's disease, and Minot one in whom the spleen was removed as a last resort in j)ernieious ana^nia. One of Pml)er's sahjeets''** was a hoy of fifteen with ana'inia and icterus. The post-operative period of study covered twelve days and he^^an twenty- four days after the operation. The diet was pui in-free, and a fully-controlled metaholie study was made. The results showed no pronounced variation in the distrihution of the urinary cynstituents which could he attrlhuted to the ahsence of the spleen. Umher makes a point, how- ever, of the fact that after removal of the spleei it was easier to ohtain n'trogen equilihrium, and attrihuted the pre-operative pathologic destruction of protein to a toxic cause. His figures show also a somewhat greater output of j)urins hefore the operation than after. Tn another case of Banti's disease descrihed in this report *** the " toxic " disturbance of metabolism was not present and splenectomy was not done. In a later study*** Uml^er describes a young man of twentv-one suffering from what 16 226 THE splee:: and anemia he considers as the " toxic " type of Banti's disease. Sple- nectomy led to striking improvement. The metabolism study of this case was limited to a comparison of total nitrogen intake and output before and after splenectomy. The results confirm his former observation, namely, that a persistent negative balance before splenectomy changes to a positive balance after splenectomy. The post-splenectomy study was made three months after operation. Minot's ^^^ patient was a colored woman, aged thirty-five, on whom the second period of metabolic studies were begun fifteen days aft', - splenectomy and blood transfusion. The figures given for five twenty-four-hour periods before and six after splenectomy are not for consecutive days. The examina- tion included total nitrogen in urine and fseces, and urea and ammonia in the urine. The chief results were a change from a slight negative to a slight positive nitrogen balance and an increase in percentage of urea after splenectomy. The uncertaincy of the food intake in the period before splenectomy, the low caloric intake, and the shortness of consecutive periods of observation make these balances of doubtful value. 2. The following studies made after splenectomy have no fore-period for comparison. Lo Monaco "'"^ found in a splenectomized individual no important change in uric- acid elimination. IVfondel and Gibson,^®* in the case of a man with enlarged spleen and secondary ansemia following malaria, studied the metabolism (total nitrogen, urea, uric acid, ammonia, phosphorus, chlorides, and sulphates) after splenectomy, but found no striking variation from the normal distribution of the urinary components. Likewise, Moraczewski,'^"^ who made some stac'es of METABOLISM STUDIES ON MAN 227 -X both nitrogenous and mineral metabolism in a man of tifty-one, seven months after splenectomy for " spleen tumor" (malarial), fomid no important variations. His observations, however, were few in number and were made in the course of an attack of pneumonia which rendered matters of diet and control difficult. 3. The only carefully-conducted and complete study of the metabolism in congenital ha?molytic icterus is, so far as we are a^are, that of McKelvy and Rosenbloom.^®^ The patient, a girl aged eleven, on a Folin diet, was studied for six days. The total nitrogen, fat, and mineral con- stituents of the food were determined and both urine and fa?ces stuu.^d as to nitrogenous and mineral constituents and the fa-ces as to fat. During a period of six days there was a loss of 4.06 gms. of nitrogen, which the authors sug- gest may be due to a toxogenic destruction of protein. The nitrogen partition of the urine was nonnal except in the case of the uric-acid nitrogen, which was increased. This increase, the writers state, might be due to the in- creased, liberation of nucleoproteins through haemolysis of the er>'throcytes. The study of mineral metabolism showed a loss of sulphur, iron, calcium, and magnesium, and a retention of phosphorus. The fat metabolism was normal. No metabolism studies were made after splenectomy. In a woman, aged thirty-nine, with "chronic family javmdice," Tileston and Griffen'^'' studied, for three suc- cessive days md an added odd day, the output of am- monia, urea, creatin, and creatinin on a purin-free and creatin-free diet. They found the elimination of creatinin and urea to be essentially normal, ammonia somewhat high, and uric acid distinctly increased. However, it should be 228 TTiE SPLEEN AND AN.EMIA notetl t?iat only one deterniination of uric acid was made. Ilaal,"" in a case v.f family ha-niolytic jaundice, found an increased excretion of uric acid and of iron. 4. As the changes :i metabolism in various types of anaemia have recently been summarized by Minot, we will not present this literature in detail. The opposing views are represented by Rosenqvist and von Xoorden. Rosen- qvist,'*'** in pernicious ana^nia and bothriwephalus ana-mia, found variations in nitrogen elimination, with periods of alternate increased and decreased excretion. In bothrio- cephalus anamiia a well-marked loss of nitrogen, while the worm was in the l)ody, was followed, after removal of the worm, by a nitrogen retention. Rosenqvist concluded that in both types of ana'inia a pathologic decomposition of protein is present. Von Xoorden ^''' opposes this view, and as a result of his studies concludes that protein de- composition is not increased as the result of ana'mia of the ordinary type. The variations in output, he believes, may be explained by the alimentary and renal disturbances which accompany ana-mia. That an increased output of nitrogen may occur in ana^nia due to parasites is admitted, as is also the possibility in non-parasitic ana^nias of a temporary increase in the output of nitrogen as the result of a sudden destruction of large masses of red cells. As to uric-acid output, von Xoorden refers to Rosen- qvist's high figures and to other observations and concludes that, as a rule, in ana?niia the out])ut is normal, but some- times rises, as in Rosenqvist's work, to twice the normal amount. It is notewortliy that in lK)thriocephalus ana>mia Rosen- qvist found that after removal of the parasite the puriu output increased temporarily and then returned to normal. This temporary increase lie explains as due to the regen- IVIETABOLISM STUDIES ON AL\X 229 eration and incrensed metabolic activity of the blood and somatic cells consequent on the removal of the toxic afjfent. As the cells recovered their normal equilibrium the output of the purins fell to normal level. Halpern/"'' studying one case of pernicious anfpmia and one of splenic ansemia, found normal values for the various urinary constituents. His figures for purin output are in no way abnormal. Samuely,*"' in his studies of metabolism in dogs ren- dered ansemic by poisoning with pyrodin, found no essential changes in protein metabolism. 5. The literature of splenectomy in man for conditions (»ther than chronic antemia, as, for example, gunshot wound, rupture, cyst, etc., shows that no metabolism studies have been made in such conditions. Conclusions concerning the effect on metabolism of removal of the normal spleens in the normal individual must therefore be based on observations on animals. Metabolic studies before and after splenectomy in animals, as we have shown elsewhere (see p. 181), indicate that the removal of the spleen does not influence protein metabolism. As to metabolism studies of substances other than pro- tein and its derivatives, the same paucity of data exists. The literature contains no records of the examination of the fa-ces for fat before and after splenectomy. Tiles- ton and Griffen, in one of their cases of chronic family jaundice, studied, without result, the fats of a single stool. McKelvy and Rosenbloom, in their case of congenital hapmolytic jaundice, report normal fat metabolism. The literature of iron metabolism is, at best, unsatis- factorv', and this is especially true of work on the relation 230 THE SPLEEN AM) .VN^MU of the spleen to iron metabolism. Most of the work is based on Sehmidt's •''■''' conclusions, drawn from the results of the feeding of iron-p(K)r food to normal mice, that the or- ganism possesses great power of consen-ing the iron and of reutilizing it through some form of intermediary metab- olism. In this connection Schmidt regards the liver as the depot for iron from the foml, and the spleen as the depot for iron from tissue and erythrocyte catabolism. The experimental evidence concerning the relation of the spleen to iron metabolism, which we have described in detail elsewhere (see p. 112), is contradictory. Asher and his associates, (Jrossenbacher '" and Zimmermann,''-' claim that the dog after splenectomv eliminates an in- creased amount of iron. Our observations do not support these findings. In our early work we occasionally found a slight increase in the faeces one and two weeks after splenectomy, but in later studies an increase was never found except once in an anaemic animal. We are therefore inclined to view a disturbance of iron elimination in the dog as due to an associated ana-mia rather than to the disturbance of some splenic function. No observations in man, other than our own ( see Table LX) on iron eliminr.tion both before and after splenec- tomy, are at hand. IJayer,''" in the study of iron elimina- tion after si)lei!ectomy for (1) rupture of the spleen and (2) Banti's disease, comjiared his results with those ob- tained in normal individuals. He found an increased molytic jaundice offers no evidence of focal liver ne- crosis, but in view of the attempts of the several investiga- tors we have quoted to show a toxic destruction of tissue in hjemolytic ana?mias, the experimental studies of Jack- son and Pearce are suggestive. The Improvement in functional equilibrium after sple- nectomy, showTi by the studies of protein metabolism, are emphasized by the studies of the exchange of iron. Our METABOLISM STUDIES ON MAN 235 findings in congenital ha-niolytic jaundice and perniciouN anannia can be explaineil, when compared with the direct blood examination, only on the basis of an increased blood destruction before splenectomy and the removal of a hemo- lytic factor by the operation. And this view is tenable despite the fact that the figures for iron elimination iK-fore splenectomy are well within those given for normal indi- viduals (see Table LX). A study of figures for intake and output of iron and of elimination before and after operation leaves no doubt as to the influence on the iron exchange. Our results cannot be brought into relation with other investigations, as in no studies before ours has iron elimination been studied both before and after splenectomy. The results of our study of urobilin are in accord with the older views as to the source of this substance (that is, excessive blood destruction) , and also with the views of Eppinger concerning the decrease of urobilin after sple- nectomy in various diseases of the blood. The study of urobilin elimination has become a matter of considerable importance in prognosis after splenectomy. Its increased elimination is usually associated with other evidences of in- creased hemolysis, as high excretion of iron and uric acid and a discoloration of the skin, and its decrease after sple- nectomy is considered as indicative of the checking of hemolysis and as justifying a favorable prognosis. This is particularly true of congenital hemolytic jaundice, but obser\-ations concerning urobilin elimination in pernicious anemia are somewhat contradictory. Thus Robertson "^ emphasizes the fact that cases which had shown a high uro- bilin excretion before splenectomy and in which, after splenectomy, the urobilin output exhibited only a transient reduction, or none at all, did not show as much improve- THE SPLEEN AND ANEMIA TABLE LX EuMiNATioN or Iron l\ Healthy and Anaemic Ikdividuaub Observer 8ei Von Wendt "• Lehmann, Muel- Male ler, Munk, Male Zuntz »* I Stockman and Male I Greig "« I Male II Female Male 1 2 3 4 5 6 7 8 y Male Male Male Female Shennan «" McKelvy and Rosenbloom*^ Roth '• Male Bayer ' Male Male Male Male Female Female Female > Iron in niKm. Remarks Intake Output per day pe. day 26 i Fasting 7.3 "- I*rofes.sional fastens; 10 21 1 Fasting 7.7 and 6 day i)criodu re- spectively 20 62*' 6.32*< Healthy individuals 5.6 U.46« i5 6.2 8.33 23 1 .•i.5 3.73 11.0 •' 9.0'* Xine periods of observa- . : 6.0 11.0 tion on two healthy m- . 1 10.0 14.0 dividuals . 1 s.o 9.0 . : 17.0 42.0 ■ ; "" 15.0 . f 19.0 24.0 . J 28.0 34.0 . ; 27.0 32.0 . : 5.7 •' 5.5 •« Three healthy individuals . '■ 6.5 8.7 ■ 1 7.1 12.6 1 1 ! 8.8 •' 32.51** Congenital hsemolytic jaundic^e — 5 day fwriod r '6 90.0 •» 6.25** Ha;molytio ana;mia. Sple- 150.0 4.32 nectomized 3 years pre- viously * 7 1 90.0 12.18 Splenectomized one : 200.0 33.07 month previously for trauma of gplocn Two weeks after spleneo- 1 6 240.0 ♦' 9.38 »» 140.0 7.41 tttay for traumatic 130.0 14.54 spleen rupture. Three 80.0 5.92 montlis later 300.0 26.73 1 6 240.0 8.40 Control: Fracture of ! 140.0 7.20 1 tibia 1 6 130.0 8.57 Control: Osteomyelitis; 80.0 3.57 operation 14 days be- 300.0 23.49 fore 1 9 130.0 13.86 Morbus Ranti; 2X years after splenectomy 2 5 130.0 i 10.20 MorbiLs Lanti; }'2 year after splenectomy 2 7 1 60.0 21.46 1 Morbus Basedow; before thymectomy i 60.0 i 32.70 1 Three weeks after METABOLISM STUDIES ON MAN- TABLE LX—("onlinued EUMINATION or Iron in Healthy and Anjuiic iNDiviDnALa 237 1 Iron in mem. Obwrver S^i ' Hemark-* • * 1 1 Intake Output per day per day 60.0 I2.s;j Six weeks after 60.0 19.00 Ten weeks after Male 22 130.0 3.59 Morbus Uanti ; before Hpleuectoiuy Goldschmidt, I'cpiRT and Pearcc "» Male 5 3.77*» 8.29« Congenital hymolytic jaundice. Before sple- nectomy. 10dayi)eriod 4.56 4.11 After .splenectomy, 10 day period Pepper and Aus- tin »« Male 40 16.5 •" 17.0 " PernicioiLS ana-mia. Be- fore solenectomy, 5 day period 16.5 10.0 Two weekf after splenec- tomy, 4 day period •1 Trnn intake dptPrmin**'! by artuftl analysis . . . ■ . ;„ <;-.* « Tw" piM^ods on sTmo .ndivi.lua!; bulk uf f«ce8 in second penod tw.c-e ». great a. m fimt. •• Iron intake estimated from tablin •• Urine and fxcea. •» Feces only. ment in other respects as did those cases in which the urobilin output was permanently reduced. On the other hand, Lee, Vincent, and Robertson "^' state that in some cases' of severe ana-niia which showed marked symptomatic improvement for several months after splenectomy there was in the post-operative period a return to a continued high excretion of urobilin. I^Iore work is necessary before this problem can be con- sidered as settled, and it^is to be hoped that metabolism studies before and after splenectomy will include not only the ansmias, but studies in es^ontially normal individuals, such as those with simple lesions of the spleen, unaccom- panied bv ana?mia. Studies of this latter type would eliminate present doubt as to the Importance of the factor dependent on the absence of the function of the normal 238 THE SPLEEN AM) .VN.EMU spleen and thus offer the essential control, now lacking, for the correct interpretation of the nietaholic disturbances; that is, wliether they are due to ana-mia or the diseased spleen, or both. For the present, however, it seems justifiable to con- elude that splenectomy in the Iwrnolyiic ana?mias is, as a rule, followed by a reduction in the elimination of uric acid, iron, and urobilin, changes indicative of decreased destruction of tissue and blood elements. ^m^ 3 ^ f 1 Part II. CLINICAL OBSERVATIONS BY E. B. KRLTdBH.VAR CHAPTER XI CLASSIFICATION AND ANALYSIS OF TYPES OF SPLENO- MEGALY ACCOMPANIED BY ANEMIA. Enlargement of the spleen is a common accompani- inent of many clinical conditions, but in this chapter will he considered only such splenomegalies as are accompanied by anjemia; as, for example, Banti's disease, splenic ana-mia, Gaucher's disease, hsemolytic jaundice, and per- nicious ana'mia, conditions in which the enlarged spleen seems to have an important relation to the anfemia. The connection between chronic enlargement of the spleen and marked ansemia without leukocytosis, as de- noted in the term " splenic anjemia," was first established fifty years -o by Gretzel,"* in Griesinger's clinic, in Berlin. He describes the case of a child, ten months old, sufTermg from dysentery and severe anjemia, with con- siderable enlargement of the spleen and a lesser degree of enlargeinent of the liver and l\Tnph-nodes. Examina- tion of the blood, by the crude methods then in vogue, showed that the proportion of white to red cells was not increased. Although later authorities have considered this to have been either a case of Hodgkin's or of von Jaksch's disease, it unquestionably served to differentiate a new clinical condition; that is, it was made clear that the disease in question was not leuka-mia. Five yeers later H. C. Womia, h'.i. without leucocytosis. Other case reports of like nature appeared from time to time, but it was not »• 241 fltt THE SPLEEN AND ANEMIA If- until 1900 that Osier's ='='= work familiarized the English- speaking public with the condition now generally known as splenic anaemia. The earlier descriptions, made at a time whtii the pathological anatomy of the spleen was little understood and when the methods of examining the con- dition of the blood were very inexact, constituted a dis- tinct advance, in that they differentiated a new type of disease previously confounded with leukjemia. The term " splenic ana?mia " is now known, however, to include several distinct types, and its use should be restricted, if not, indeed, discarded entirely. The fact that in most cases the etiology or pathogenesis of this group of dis- eases is not yet clearly understood is no more an argument for continuing to group them imder such loose terms as " splenic anaemia " or " splenomegaly with anaemia " than it would have been to continue to group typhoid fever with typhus fever until the discovery of the bacillus typho- sus. Though much still remains to be learned in regard to them, and though probably even their present eponymic and euml)ersome names are only temporary, and will be found to include one or more entities, nevertheless it has already become more profitable to deal with them as inde- pendent affections. The inconvenience resulting from the present use of the term " splenic anosmia " is quickly dem- onstrated to anyone making a critical survey of the litera- ture. Statistical summaries, including valuable detailed information, are thus frequently rendered useless when, on analysis, thoy are found to include several independent types of splenic disease. Cases are occasionally reported under such headings as " simple hypertrophy of the spleen " or " idiopathic splenomegaly," in which not only has annrmia been absent, but histological examination of TYPES OF SPLENOMEGALY 243 the spleen has failed to reveal any peculiar pathological change ( Kidd,'^''* Senator and Krause **•* ) . These reports, however, are so few in number and are based on such slight evidence that the conditions described are not as yet entitled to an independent consideration. In most cases names such as those quoted are used for the want of a more accu- rate designation, a practice that should be discouraged. Certain other diseases, in which the spleen is involved or said to be involved, will not be discussed in this chapter. These are ( 1 ) various ha?molytic anspmias of specific origin ( dibothriocephalus latus and uncinariasis), (2) lesions of tlie spleen associated with thrombosis of the portal vein or artery, and (3) some splenic types of cirrhosis of the liver. It must suffice thereby to indicate such relations. Also, all types of enlarged spleen in which certain features render differentiation easy (as leukaemia, pseudoleuka?mia, and changes secondary' to obstruction or infection, as in heart- disease, typhoid, kala-azar, syphilis, etc.) will be omitted from consideration. This leaves the more definitely dif- ferentiated offsprings of " splenic anfemia " as Gaucher's disease (or large-celled splenomegaly), Banti's disease, v. Jaksch's pseudoleuksemia infantum, the Hayem-Widal or acquired form of ha;molytic jaundice with splenomegaly, and the Chauffard-Minowski or congenital or familial form of the same. Some of the differential points of these dis- eases are indicated in Table LXI. Gaucher's Disease Gaucher's disease, or large-celled splenomegaly, was probably the first variety to be differ'^ntiated on account of its peculiar anatomical picture. Although in reality possessing little in common with the other forms of splenic 244 o o Q THE SPLEEN AND ANEMIA si S3 B a 2 Si a ^ V 11 i * 11 gSCS o o c u + 1 ± a •a g pooiq ai , B ot.^jojn aq | iumi ( j7^ + ~r3~ + ~-3" C O 7: aV 5'" •ti a t % l\^ o • + + + + + + + + + ■c^.S_§ t 0. 5 9 as a> *s *4 >» **, SIT I is O u 11 < Ma s^ » -3 •3 2 S ■s aS£ 9 .0-0 ?.o „> o«« tf ag w U a — t*'3 t 1 — »r- at, 2 9 3 3 a o a o a 3 S? Is 4^ ■■9 o * 9j a M t; a; o w i'l.AH, 11 •' Uu f\',r.*\ / 1 '***'\*?,i .;uv .< **S" •'. »r • .^ »*• .:n i-,«v H*N • I 4 •*•.(.«»■' I'llitr .irranatTin-nt nf h onrentrir nuclei rjir vcsi'Mjlar It TYPES OF SPLENOMEGALY 245 anfemia, it is here considered in some detail, on account of the similarity of the clinical picture and from the fact that it is still included by many writers under that heading. Pathology. — First described by Gaucher/''^ in 1882, as a primitive epithelioma, it was later shown not to possess most of the characteristics of malignancy. Bovaird ''* called it a simple endothelial hypcrjjlasia, and Brill and Mandlebaum '^ showed that the cells e divided into three periods. In the first or pre-ascitic period, usually lasting several years, a grad- ually increasing weakness and pallor is noticed, with diges- tive disturbances and abdominal pain, which may first call attention to the enlarged, smooth, hard spleen. A ten- dency to hemorrhages with a moderate ana?mia of chlo- rotic type is usually present, but may be postponed until the later stages. There is nothing specially characteristic of the anannia, the mcrease of urobilin being the most significant sign of increased blood destruction. The resist- ance of the red cells is unchanged ; signs of a regenerating bone-marrow, as nucleated and reticulated red cells, are slight or absent. After splenectomy, however, an increased resistance of the cells may be noted, and may be marked.* • In a case I recently examined after splenectomy complete ha»nio- lysis did not occur in salt solution as low as 0.25 per cent. No nucleated red cells were found, but reticulated forms were more numerous than before splenectomy. <■* \-^.. TYPES OF SPLENOMEGALY 240 A slight or moderate amount of leukopenia is char- acteristic. The second, or intermediate, stage lasts but a few months, anr' 's characterized by scanty, high-colored urine containing ^ i excess of urobilin, by attacks of dyspepsia and diarrhoea, and by slight increase in the size of the liver. The third stage is ushered in by the symptoms of cir- rhosis, a recurrent, -p -sinless ascites, occasionally slight jaundice, shrunken live., and increasing ana?mia and ema- ciation. After a few years an intercurrent infection or fatal hemorrhage is the terminal event. It is hardly neces- sary to say that such a picture is subject to variation, and that in some cases the three periods cannot be dis- tinguished. The first and second of these periods are usually considered as " splenic anaemia." Etiology and Pathogenesis. — In spite of the great amount of work done on Banti's disease in the past twenty years, not only is its etiolog>' undetermined, but it is still an open question whether it is a disease due to a specific cause or is merely a fairly constant symptom-complex, lianti attempted to demonstrate microorganisms in the blood and viscera of this disease, but failed, as he did also in his various attempts to reproduce the disease in lower animals. He insisted, nevertheless, that the splenic en- largement was primary and due to an unknown infectious agent localized in the spleen. Recently Gibson '*' has reported the finding of a streptothrix in the spleens of certain cases resembling Banti's disease, and Yates, Bunt- ing, and Kristjanson *"' have found diphtheroid organisms in several such spleens. As these findings have not been f 250 THE SPLEEN AND AN^ML\ confirmed, however, and as the great majority of investi- gators have been una'ile to isolate a causative organism ( Senator,* ""^ Sippy,*"* Zancan,*" etc.), the demonstration of an actual ..ifecting agent must be considered as still lacking. Suggestive evidence, however, was recently brought forward by Hollins,'** who by repeated subcutane- ous injections of BaciUus coli was able to produce in the rabbit a distinct splenomegaly with moderately severe anaemia similar to that of Banti's disease. No ha?molytic body or living microorganisms could, however, be demon- strated at autopsy. Banti's later view is that the in- fectious agent is brought to the artery either es a direct toxin or as a substance which is changed by an actual splenic metabolism into a splenotoxin. The earlier changes are therefore to be found in the neighborhood of the fol- licular arteries, and later in the pulp, splenic and portal veins, culminating eventually i tlio liver changes of the third stage. The symptoms, according to Banti, are due to general toxa?mia, and the anasmia to a depression of bone-marrow activity rather than to excessive haemolysis. As the degenerative changes in the spleen are too far advanced to permic conclusions as to such a sequence of events, this theory has never been confirmed by experiment or obser\'!ition. The intimate relationship between the spleen and liver renders intelligible a possible pathologj' of the third stage, especially as Mallory'^^' and, later. Breccia '"' have shown that injurj' to the spleen is followed by focal necroses in the liver. Banti hi.s objected to sug- gested etiological relationship c" intestinal disturbances, despite the many cases in which digestive disturbances are known to precede or usher in the disease. The fact I'LATK IV HiHtol"i»ry oi ■plt-en ul einly B.u.ij'! tiiwJ**-' TYPES OF SPLENOMEGALY 251 that the spleen is involved earlier than the liver would point to a hasmatogenous rather than an enterogenous toxin, and the unquestioned improvement that usually fol- lows splenectomy indicates that the altered spleen is in some way fm important pathogenetic factor. This is still fur- ther emphasized by Umber's*" unique observation. A boy, fifteen years of age, was splenectomized for Banti's disease, and during the operation a small piece of the en- larged liver was excised for histological examination and a distinct peripheral infiltration of the lobules found. Later the liver returned to normal size, a strong indication of the splenogenous origin of the hepatitis, which, if undisturbed, should have progressed to the usual cirrhosis. Trauma to the spleen has been offered as a causative factor in some cases (Armstrong ''*), while another group of authorities consider Banti's disease merely a syndrome, which a great variety of causes are capable of producing (Albu,» Isaac^"'' Luce,"" Neuberg,''" Seiler *"='). There is no question that various conditions, as an atypical cir- rhosis of the liver with early prominence of splenic signs, syphilis, or primary endophleliitis or thrombosis of the splenic or portal vein can produce a picture which cannot be distinguished from that of Banti's disease (Edens,'**^ Goldman *" ) . As endophlebitis or thrombosis is a fre- quent accompaniment of true Banti's disease, it is but natural that the symptomatolog>' of the two conditions should he confounded, Banti himself called attention to the frequency of these changes in his original descriptions, but considered the splenic changes primary. Other authors consider that when the endophlebitis or thrombosis is pri- mary' certain chai acteristic sjTnptoms will often point toward a proper differential diagnosis, Warthin *'^^ in- WhBM 252 THE SPLEEN AND ANzEMlA f sists that an infectious thrombophlebitis of the portal or splenic ve.n is the essential feature of the syinptoni-coni- plex, whereas the upholders of the separate-entity theory claim that such changes are secondary to the changes in the spleen or may be absent altogether (Ziegler "« ) In eer tam cases of syphilis of the liver, also, the splenomegaly, T'TT' ^""^ ^''*°'-' "^ h^matemesis may be so prominent ( Osier -fj'*"'^ ""^ ^^""^'' '^'''^'' '' ^^^y '^""''^y ^^"^^t^ When a consideration of the above featTires allows a differentiation to be attempted it would seem advisable to consider such cases as pseudo-Banti's disease, and to restrict the u.se of the term true Banti's disease for those eases in which no such etiological factor is ajjparent The nature of the splenic enlargement has also ifiven rise to several hypotheses. The view that it is spodoge.ious can be ruled out, on account of the absence of histological evidence to support it, and Barr's '« theon^ of splenio con- gestion due to splanchnic vasomotor paresis needs merely to ne mentioned. The prevailing opinion is that the en- largement is due to a chrunic inflanmiatory process, which m turn results in an increased functional activity (in- creased ha-molysis), with a resultant anaemia. Thi's view IS m accord with Botazzi's ha>mocatatonistic and Banti's ha^molytic theories. Ha'molysis is considered by Harris and ITerzog to be due to an eiythrolytic enzyme elab- orated by the hyperplastic endothelial cells, and by Lint- varew to an increase in the erythropl.agic action of the sp.een-cells. the resultant fibrosis being due to the chronic irritation of the products of red-cell destruction. The absence in the circulating blo^xl of signs of bone-marrow m K' IM \ I K \ I .-:-PM.' +*-i- 5 ;, ■ I I » i - : ;:l ^' • t in TYPES OF SPLENOMEG.\LY 98S activity points, however, to a diminished activity in blood Connation, but against this is the increased urobilin elimina- tion as evidence of increased blood destruction. The probable existence of a splenic hormone to the bone-marrow and its disappearance in splenic disease can be invoked to explain the greater anaemia when the splenic tissue has been largely replaced by fibrosis; but, on the other hand, the improvement in the blood picture that follows splenectomy suggests the removal of a pernicious hasmolytic activity on the part of the spleen. Pathology. — In the pathological histology of the dis- ease there is nothing specially distinctive. The enlarged spleen, as a lule, shows an int -eased amount of fibrous tissue in the capsule and reticulum, usually characterized as " fibroadenie " (that is, iiicreased fibrous tissue, but re- taining an adenoid appearance), and involving both pulp and follicles. The Malpighian follicles, especially in the later stages, are small and scarce; in the earlier stages they may be hyperplastic and the " fibroadonie " be absent. Macrophages, increased amount of pigment, and other evidences of increased blood destruction are usually found. The changes in the liver are those of an ordinary periportal cirrhosis. In summarizing, one might say that, although the eti- ologj' of true Banti's disease is unknown and may well be from several sources, evidence points to the close causative relationship of the spleen. One would not expect the re- moval of a largely fibrotic organ to be attended with marked somatic changes, and it is precisely in the earlier stages of the disease in which splenectomy has proved most beneficial. ■*r' I Von Jaksch's Disease The anaemia infantum pseudoleukaemica of von Jaksch '"*» is in all probability not an independent condi- tion, but represents an atypical response of the infantile ha'mopoietic system to one or other of the primary dis- eases of the blocxl (leuka'mia, pernicious ana-mia, the secondar>' anannia of rickets, syphilis, Banti's disease, or the formerly unrecoprnized types of hjvmolytic jaundice). As a rule, a hitjh-^rade ana-mia, with blood picture some- what resemblinn- pernicious ana-mia, appears in infants of one or two years. Leucwytosis, especially of the small lymphocytes, is a frequent response to any form of aniemia in infancy and childhood. The smooth, hard spleen is con- spicuously lar^e, while the liver, in contrast to the liver of leukaemia in childhood, is very slifrhtly enlarged. Aschen- heim and Benjamin "* have found rickets present in all of a series of such cases examined by them, and suggest the name " Rachitische :Megalosplenie " for this condition. Von Jaksch also associated rickets with this disease. Giffin'^* and others, on the other hand, consider the true v. Jaksch's disease to be nothing more than the infantile form of splenic ana?mia. (Banti's disease.) That von Jaksch's disease is being less and less regarded as a sepa- rate disease is shown by the gradual disappearance of the name from the text-books. H.-EMOLYTic Jaundice The acquired, Hayem-Widal, and congenital or famil- ial, Chaufi'ard-^Minkowski types of ha^molytic jaundice with splenomegaly are frequently grouped by English and American authors under such titles as " ha?molytic jaun- dice " (Thayer '"'), and "chronic family jaundice" TYPES OF SPLENOMEGALY (Tileston*''"). As the two forms possess several rather important and characteristic differences, it is deemed ad- visable to follow the continental custom and consider them a!> independent conditions. Search for their true etiology and pathogenesis (as yet unknown) is more apt to be stinmlated under such an arrangement than if they are grouped together. Furthermore, Widal, Abrami, and IJrule*^" experimenting with toluylenediamine.have offered evidence to indicate, in their opinion, that the two types are of different origin. Symptomatology. — The points of differentiation we will present after outlining the historical development of our general knowledge of ha-molytic jaundice. Although iMurchison,^*** Wilson,^'^ and others had previously de- scribed cases of chronic jaundice (x^curring in several mem- bers of a family (in Murchison's case splenomegaly is not mentioned), it was the more complete description of Hayem,"^ in 1898, that first established the condition as a clinical entity. The clinical picture of the five cases ana- lyzed by him was as follows: All five exhibited a chronic jaundice, with the presence of bile-pigment in the blood- serum, but not in the urine {i.e., acholuric icterus). The other signs of obstructive jaundice, such as itching, brady- cardia, and clay-colored stools, were also lacking. A dis- tinct anaemia, the red-cell count varying from 1,000,000 to 3,000,000, was present in all. Very large, hard spleens were found in each case, and slight enlargement of the liver was also noted. Exacerbations were frequent, and during these the jaundice deepened and bile appeared in the urine. The importance of these exacerbations was emphasized by Widal, who termed them " crises of deglobulization." and considered them highly characteristic of the acquired form. '£V^ • w f..: 'i"?'.': ^1 MICROCOPY RESOIUTION TEST CHART ANSI ond ISO TEST CHART No 2 1.0 I.I iiJ III 2 8 |36 ■ 40 12.5 '- itt 112,2 2.0 1.8 1.25 nil 1.4 ^ .AP PLIED INA^IGE I ^^. 'rbSi East Main Str»pt ~^ Rochester, New Vor* tA609 USA ' i7^6) 482 - 0300 - Phone ^= ('16) 28a - 5989 - Fo» ■B^SiiW ^^ !e56 THE SPLEEN ANT> AN^.MIA .■■a In severe cases the blood count fell below 1,000,000 and haemoglobin appeared in the urine. In all five of Ilayem's cases the family history was negative, and in three the jaun- dice was stated to have appeared first in adult life. Two years later Minkowski ='" described a similar dis- ease occurring during three generations in eight members " one family. This, tlie congenital form, it is now known, is commoner than the acquired form. In addition to tlie symptoms presented by Hayem's cases, an increased amount of urobilin was noted in the urine. Autopsy re- vealed no cirrhosis of the liver or obstruction of the bile- passages. The spleen showed a diffuse hyperplasia and hypera-mia. Pigment deposits were numerous in the kid- neys and in the centres of the liver lobules. The next important contribution to the clinical pic- ture of these diseases was made by ChaufTard," who showed in the congenital type that tlie resistance of the red blood- cells to hypotonic salt solution was much diminished. Increased numbe.- of microcytes and of reticulated red cells by methods of vital staining were found by Chauf- fard "2 in the congenital or familial type, and their presence later confirmed also in the acquired type. Another diagnostic method, the auto-agglutination test, is advocated by Widal, Abrami, and Brule."'* They have found it always positive in the acquired form and always negative in the congenital or familial type. How- ever, in Micheli's^'*^ carefully studied case of the acquired type this test was also negative. Isoh^molysins have occa- sionally been found in both types (Micheli,^" Hopkins '«*) , but are not supposed to possess any pathological signifl- cance. Although both types of hemolytic j „undice usually TYPES OF SPLENOMEG.VLY 257 run a chronic course, Gaisbock "" has shown that an acute uiahgnant form may occur that is fatal in a few montlis. Therefore the cardinal symptoms of the two types of haimolytic jaundice with splenomegaly are found to be a chronic enlargement of the spleen, existing with an acho- luric, non-obstructive jaundice, and anaemia, frequently paroxysmal in character and varj'ing in intensity. In- creased blood destruction is indicated by increased urobilin in the urine, and various characteristic changes are found in the blood. The red cells show diminished resistance to hypotonic salt solution, increased number of reticulated cells with vital staining, and in the acquired form the jihenomenon of auto-agglutination of the red corpuscles. The blood-serum rarely contains auto- or isobemolysins. We have purposely postponed until now a considera- tion of the differentiation of the acquired and familial types and their relation to other conditions, such as those suggested by Gilbert "» and Eanti."' The fact that in the actpiired group the disease is definitely acquired in adult life, whereas in the other there is a family history of the same trouble, is not in itself sufficient to warrant the dis- tinction of independent disease pictures. There are, how- ever, other features which tend to differentiate the two types. In the congenital form the subjects, as Chauffard puts it, "are more icteric than sick." Frequently they come for treatment for other conditions and consider the chronic jaundice as a family idiosjTicrasy not interfering with perfect health. The acquired form, on the other hand, is usually ushered in with a definite attack of illness; the anemia becomes much more grave, sometimes as low as 1 .000.000. and the patient is distinctly more ana'mic than jaundiced. In Decastello's " case, which was greatly im- 17 258 THE SPLEEN AND AN/EMIA :i proved by splenectomy, the red-cell count had previously fallen to 800,000. An analysis of 159 cases of hsemoly tic jaundice in which blood counts are available shows that 55 belong to the acquired type and 104 to the congenital or familial type. Of the latter, only 23 failed to give a positive family his- tory; but pf the 81 remaining cases the disease in 36 de- veloped after birth. The term " familial " would there- fore seem, preferable to that of " congenital," unless a third variety is to be considered. The average red-cell count of the 55 acquired cases is 2,032,000, the counts ranging from 510,000 to 4,500,000. Counts belov 1,000,000 are recorded in ten cases; below 2,000,000 in 27 cases, and over 4,000,- 000 in only four cases. The average count of the 103, congenital and familial, is 3,340,000, the counts ranging from 1,800,000 to 5,700,000.* Xo counts are recorded below 1,000,000 ; eight below 2,000,000, and 25 above 4,000,- 000. If this group is subdivided, the average of the familial cases is 3,281,000; of the congenital, 3,543,000. These figures show that there is a more marked ansmia in the cases of the acquired type than in either the familial or congenital types. Widal and his pupils claiiri that the auto-agglutination test is only positive in the acquired form, and consider this as important evidence that the two diseases have funda- mentally different origins. Attention has already been called to the differential importance of Widal's " crises of deglobulization," but it must be admitted that marked * One familial case reported by v. Krannhals showed 1,000,000 red cells in a single count, but, as the haemoglobin was between 55 and 65 per cent., the accuracy of the count is questionable. T\TES OF SPLENOMEGALY 259 fluctuations in the degree of blood destrnction are present also in the familial type. Numerous reports are at hand of a condition apparently identical with the acquired form, but following attacks of malaria, syphilis, and other infections. In such cases the signs of excessive blood destruction usually disappear when the underlying cause is successfully treated. The familial form, on the other hand, appears more as an inherited dystrophy of the ha^mopoietic system, rendering the red blood-cells more easily destructible. On this basis Chauf- fard at first strongly advised against splenectomy in this type, but subsequently cases have shown such improve- ment after this treatment that it would seem as if the removal of this site of blood destruction was advisable, whether or not it is the primarj-^ seat of the trouble. It nmst be noted, however, that after Kahn's ="" and Roth's '**" successful splenectomies in the familial type the resistance of the red cells failed to return to normal. The congenital type with negative family historj', grouped with the familial type by most authors, offers no definite ground for differ- entiation from the acquired form. The mere fact that the disease has already made its appearance at birth is of it- self not of fundamental importance if there is no history of similar trouble in the family. If the familial form (which, as a matter of fact, is usually, though not always, congenital) were placed in contrast to the acquired form, the time of onset, as indicated by the term congenital, might well be disregarded. The possibility of an early acquisition of the disease is shown in the case reported by Benech and Sabrazes.** With a negative family history, a suckling is su])posed to have accjuired the disease from her wet-nurse, who, together with her two children, had soo THE SPLEEN AND AN/EMLV 1 'i n a chronic hfcniolytic jaundice. Certain authors consider the acquired and congenital types as identical. Hynek,"*' for instance, bases his opinion on two cases observed by him: in one a mother acquired the disease after childbirth, whereas in her child it appeared congenitally. Plehn^'^* reports a case appearing congenitally in father and daugh- ter, but not until the twenty-sixth year in the case of a son. Benjamin and Sluka '- obser\-ed three cases in one family, two appearing congenitally and one in adult life. Many of these cases could undoubtedly be harmonized if the time of onset were disregarded. In favor of the identity of the two types (acquired and congenital) it must be admitted that a series of cases could be selected in which many grades l)etween the two types would be represented. It is obvious that the familial type must at one time or another have originally been acquired. In such an event the difFerence in severity be- tween the acquired and familial still holds. Thus both in Roth's •''**'' and Bychowski's «* cases tiie disease in the parent who acquired it was severe, while in the children who in- herited it it was of the usual mild type. Such facts, how- ever, would not indicate a fundamental difference in the nature of the two diseases. In the small numl)er of cases already accumulated vari- ous atypicalities have l)een reported. Thus, Lommel's "- and Claus and Kalberlah's '" cases of the familial type and Mosse's ^"* and Tixier's *''° cases of the acquired type failed to show any change in resistance of the red ceils (either washed or tlie whole blood), though other evidences of increased blood destruction were present. In a few cases an acholuric j.iundice with splenomegaly has existed to- gether with polychytha'mia instead of anemia. Chauffard TYPES OF SPLENOMEGALY 261 and Vincent"" and Roth ^*' have rece»itly described a form in which hemolysins are present in the blood, and such cases appear to occupy a position midway between htemo- lytic jaundice and paroxysmal ha>nioe dignified with a separate name. As many of their cases were described before the various hasmato- logical methods above described had come into vogue, and are frequently reported in resume, it is difficult to deter- mine conclusively whether or not they should be considered as l)elonging to the types under discussion. Their theory of hepatic origin, thi-ough an infectious angiocholitis, they later abandoned in favor of ChaufFard's idea that the pri- mary change was in the blood. Another occasional variation is in the presence of all symptoms of increased blood destruction but an absence of icterus. This Chauffard describes in a family in which the mother is a typical ha;molytic icteric, while the eighteen- year-old son presents all the signs of the disease except jaundice. When the anaemia is grave the blood may present a picture indistinguishable from pernicious anaemia (v. Stejskal *'") . Chauftard considers that there is an icteric form of pernicious ansemia which, when accompanied by diminished resistance and reticulated red cells, represents the least compensated form cf haemolytic icterus. Widal 262 THE SPLEEN AND ANEMIA :ii a It and Weissenbach *"' have also reported a case of this type. In the usual Biernier type of pernicious ana?mia, icterus, it will he recalled, is absent and the resistance of the red cells increased. Banti ^- has recently proposed the name " haemolytic splenomegaly " for a type of the disease which on analysis is indistinguishable from the acquired form of hemolytic jaundice. Both the cases described by him exhibit a chronic ana'mia, with long-standing splenomegaly, subic- terus, diminished resistance of the red cells, increased nimi- ber of reticulated cells, and urobilinuria. In a later study ^^ of seven cases of this condition he further subdivides " ha?molytic splenomegaly " into regenerative and degen- erative groups, the difference depending chiefly on periods of relapse and remission in the former type, with greater evidences of regeneration in the blood picture. The bene- ficial effects of splenectomy — abolishing the anjcmia and changing the resistance to normal in one case and even changing an aplastic into a reacting bone-marrow — lead him to ascribe a primary role in this disease to the spleen. It must be remembered, of course, that removal of the normal spleen indirectly causes an increase in resistance of the red cells. Banti claims never to have see i Widal's crises of deglobulization in these patients, and emphasizes the presence of a relative lymphoc\i;osis, but this would hardly indicate a different disease. As to the choice of name, Banti's name is open to the same objection as the older one; namely, that splenomegaly may be absent in some cases (Le Gendre,^^'' Pick,''" Gilbert and Lereboul- let,""^ Benjamin and Sluka's third case, and Marchiafava and Nazzari,^") just as jaundice is in others. A name indicating increased blood destruction, such as " ha;mo- TYPES OF SPLENOMEGALY 263 lytic Hypersplenism," without including individual fea- tures, would he less open to objection; but in the mean- time, until hypersplenism can be demonstrated, the name sanctioned by usage is preferable. Pathogenesis. — As I have already indicated in the discussion of the differentiation of the two types, a hepatic or luetic etiology has been largely discarded. Such etiology is, however, still occasionally reported (Bernard, de Beru- iiiann, Gelle), as is also that of malaria (Socquepee) and ankylostomiasis (Darre, Parisot, and Fairise). The two prominent views, however, are ( 1 ) that the primary lesion is in the blood — a dystrophy of the red cells; or (2) either primarily or indirectly in the spleen — an exaggerated hsemolytic a/jtivity. Widal and his school, the extreme supporters of the former view, consider that the congeni- tally weak red blood-cells are destroyed in the circulation and their remains taken up by the spleen, causing a spo- (logenous tmnor, and by the liver, kidney, and bone-mar- row, as shown by the excessive pigment deposits found in these organs at autopsy. This view was supported by Vaquez,*"" von Stejakal, Benjamin and Sluka, Aschen- heim," and Weber and Dorner.^"^ The chief objection to it is that it completely ignores the great improvement fol- lowing splenectomy. A primary increased hemolytic activity of the spleen as the source of the malady was first proposed by Minkowski and supported by von Krannhals and ChaufFard. Its latest adherent, Banti, as I have pre- viously stated, considers that the pathological spleen not only is spodogenous, but actively destroys increased num- l)ers of cells and prepares others for destruction. Though based on incorrect and inadequate experimental evidence, tliis attractive combination of the splenogenous and haemo 264 niE SPLEEN AND AN.EMU catatonistic theories at present seems most plausible. Although it is not possible to demonstrate ha?molysis in the normal spleen, we cannot exclude the poss'bility that it exists and that it is active in some diseased conditions of the organ. Extracts from the spleens of Antonelli's,' Kahn"sr°« and Robertson's ''"'• cases, however, failed to show any ha'molytic activity in vitro. Whatever the source of the increased blood destruction, there results an increased amount of free hemoglobin to be gotten rid of. Most authorities believe that this is changed by the liver into bile in excessive amounts, and that the viscid and highly pigmented bile clogs the bile- capillaries, is reabsorbed into the blood, and thus causes a " pleiochromic icterus." Recent investigations by Whip- ple,^«« however, show that bilirubin can be formed by the action of the endothelium of blood-vessels entirely iso- lated from the hepatic circulation. If this be true, a hematogenous icterus in the narrower sense can be ac- cepted. That the jaundice is not due to gross obstruction is proved by the facts that such obstruction has never been found, that the stools are of normal color, and that the urine does not contain bilirubin. It is interesting to note that in Banti's aplastic or anamopoietic case splenectomy started the formative powers of the bone-marrow, as shown by the appearance of normoblasts in the circulating blood. This would indi- cate that with the spleen was removed a toxin that had inhibited blood formation, and from this it could be argued that the decreased resistance of the red cells of hemolytic jaundice is due, not to a hemocatatonistic action of the spleen, but to an indirect injury to the bone-marrow. Pathological study of the comparatively few cases of - - :..! I'l \ii' \ll 1 |UKi>u-nt. 1; . '» 1 1 ■: :•? T\TES OF SPLENOMEC \T.Y hit hiTHJolytic jaundice thnt have onme [o autopsy or sple- nectomy has yiehled httle in the way of establishing a con stiuit and characteristic pathological picture. I have col- lected descriptions of seven spleens obtained at autopsy and of eight obtained after splenectomy. An analysis of these appears in Table LXII. It will be seen that the chief characteristic of both types of the disease is the marked congestion of the splenic pulp and splenic sinuses, hut this is, of course, found in many other conditions. The Malpighian follicles, the capsule, and trabeculae are frequently left unchanged; and, while pigment deposits and macrophages have usually been found to be increased, in the case reported from this labora- tory this was not found to be true. In all cases in which the bone-marrow was examined at autopsy it was found to be red. Pernicious Anaemia This disease is mentioned briefly here, not as an ex- ample of splenomegaly, but because the recent tendency to treat it by splenectomy brings it into relation with the theory of hypersplenism.* This latter theory has been put forth prominently in recent years by Eppinger*"* and King,^" who consider that the amount of haemolysis in a given case is in definite relation to the amount of unsatu- rated fatty acids and the amount of urobilin in the faeces. They found both these increased not only in Hsemolj'tic jaundice, but also in pernicious anaemia, hypertrophic cir- rhosis, and catarrhal jaundice. For example, the unsatu- rated fatty acids, as may be seen in Table LXIII, rise from a normal (iodine number) of 80 or 90 to 188 to 273 * For iummary of recent literature on this subject see Vogel.*" i{ 266 THE SPLEEN AND ANAEMIA u u o 'A < \< I } o w ^ o o a £ = in C h 2 0^ Ea EC lO S.9 .So . 3 a CO ^ a go a; a ■'a. _^ « « a] « (S « « S 0) S u £• u o ^ - ^J a a £ S^ £ £ S I is 3J 4) * iiii n I ill ii 1 1 a ;3 ^ c o I c a £ * §£ "3 S-2^-^-^ ^ -^ o qS-3-a M £ c o o O O 6. gzz z i: a '■•' a £ f o o u o < ^. u u &» S So a a as CO DO u o _ MO •a -5 s B B a « a; £ s ^ a' a a a I'Ss^.s-go 5^ 1- S *- ? ^- S a o « o Ti *c rrs — *? o c O ^ "^ «■ - C 3 < : a-o e a •3 a ■a -3 -3 -a -a a aa a a Z Z Z E- Z H Z H Z o o ZZ Z Z •a -a s a o o Z Z -<■<;•< ■< o o o a CO a 2. C O < O !2 !5 "3 E 1 1 u. < ■< I1 1 3 3 3 CO c <: u •c.S ^~-a a c*'" a -*^ t I »-. > C «". * * »- • iH M a 2 o a£ "an 03 « ■a o ^1 f ,. t * j k S £ ^ ; - - ■_ :3 z? 5 TYPES OF SPLENOMEGALY 267 in pernicious anaemia. As they had found experimentally that removal of the spleen in the dog caused a great drop in the iodine number and was associated with an increased resistance of the red cells and lessened tendency to haemo- lysis, they favor splenectomy in this type of ansmia. Eppinger's theory of the pathogenesis of pernicious anae- mia is interesting, but not convincing. On histological TABLE LXIII Analysis of Blood Fat in An«mta and Other Conditions • (Modified FROM Table of Eppinoer "^ and Ki.nu"'J Total fat gms. Choles- terin gms. Normal Normal Pernicious autemia Pernicious anaemia Pernicious ansemia H£emolytic jaundice Ha;molytic jaundice Cirrhosis of liver Cirrhosis of liver Cirrhosis of liver Catarrhal jaundice Catarrhal jaundice Catarrhal jaundice Obstructive jaundice Obstructive jaundice Secondary ana!mia, carcinomatous stomach . Secondary antemia, carcinomatous a-sophagus Nephritis Nephritis Polycytha-mia Purpura 5.38 5.90 7.34 8.40 9.37 5.43 6.33 9.01 3.94 7.36 5.70 5.40 5.60 10.63 7.92 6.39 6.97 8.24 15.89 6.93 8.50 0.76 0.86 0.56 0.32 0.14 0.49 y.s5 0.91 0.30 0.56 0.50 0.39 0.48 1.07 0.61 0.71 0.38 0.56 1.26 1.09 0.86 Choles- terin eater gm. 0.52 0.57 0.72 0.01 0.05 0.41 0.31 0.31 0.13 0.12 0.23 0.44 0.67 0.27 0.49 0.52 0.44 0.38 0.07 0.08 0.63 lodin number 90 79 188 213 273 326 258 125 309 82 187 123 122 88 139 36 101 22 69 273 224 • Figures are for 1000 c.c. of blood. examination of the spleen he found thickened walls in the arterioles and intense congestion of the pulp. He assumes, therefore, that the blood seeks the path of less resistance through Weidenreich'sopen capillaries into the pulp, where they are destroyeil, presumably by contact with tlie con- nective tissue. From this rather fantastic point of view, 268 THE SPLEEN AND AN^:MU splenectomy therefore becomes equivalent to tying off a ruptured blood-vessel. Summary. — The several diseases described in this chajiter not only have sufficiently characteristic and con- stant symptom-complexes to permit a differential diagno- sis to be matle, but are also, in all probability, due to dif- ferent causes, or possibly to a common factor operating in different ways. In a strict sense, none of them should be considered as primary anaemias, though in some it is difficult or impossible to find the " causa causorum." From the aspect of the chief lesion found (namely, the changes in the blood), they may be divided into two groups, in one of which increased blocxl destruction and in the other impaired blood formation is characteristic. As will be shown in a later chapter, the relative importance of these features has an important bearing on the results produced by transfusion and splenectomy. From this point of view, the anaMuia of Banti's and Gaucher's diseases is chiefly due to impaired blood formation, while that of the haMiiolytic jaundices is due to impaired blood destruction. Finally, in another disease, pernicious anamia, commonly treated by splenectomy, increased hamolysis predominates, but is usually acx'ompanied by seriously impaired powers of blood formation. CHAPTER XII METHODS OF VALUE IN THE DIAGNOSIS AND PROG- NOSIS OF SPLENIC DISEASE The numerous points of resemblance or of slight dis- similarity in the several clinical conditions analyzed in the previous chapter demonstrate that in a given case careful study must usually be made before a proper diagnosis can l)e reached and such studies continued, if the prognosis and effect of treatment are to be properly gauged. An impor- tant part of these studies is not only the performance of certain special tests to be described in this chapter, but also the proper accomplishment of the usual hlstor>' taking, physical examination, and routine blood examinations. In history taking, emijliasis should be laid on a most thorough inquiiy into the family history- for evidence of disease of a similar nature, either in the present or former generations. Not only should exhaustive search be made into the patient's or parents' past histories for possible underlying or contributory causes, but also the most prob- able time of onset of the disease nmst be carefully investi- gated. Experience has shown that it is in connection with these three points that most defective histories are at fault. The physical examination should always include careful investigation into the presence or absence of jaundice, and the size of the liver, spleen, and lymph-nodes. Evidence of jaundice should be sought not only in the skin and mucous membranes, but by appropriate tests of the urine 269 270 THE SPLEEN AND AN.EMIA and by inspection of the blood se/um.* Frequently re- peated routine blood examinations should be made. The chief fault in connection with such examinations is that they are not repeated often enough, both before diagnosis has been readied and while the effect of treatment is being studied. The special laboratory tests to be described in this chapter are all concerned with attempts to study as nearly quantitatively as may be both the nature and degree of the disease process (estimation of amount of blood destruction, changes in the blood-serum and in the resistance of erythro- cytes), and the ability of the body to compensate therefor (evidences of blood regeneration). It must, however, always be remembered that the constantly changing factors of blood destruction and blood regeneration are being dealt with, so that conditions may be met with in which: (1) Blood destruction is excessive, but powers of regeneration well presen-ed (as in hemolytic jaundice) ; (2) blood de- struction excessive and powers of regeneration insufficient (as in pernicious and aplastic ana?mia) ; (3) blood de- struction not excessive, but powers of regeneration insuf- ficient (as in Banti's and Gaueher's disease), with an in- finite number of intermediate grades. Such considerations are further complicated by the fact that the bone-marrow response may be considerable, but pathological in type, as in remission stages of pernicious anjemia. It must also be rememl)ered that, although fairly accurate indirect methods exist for estimation of the amount of blood destruction, * Blood should be withdrawn from c i- or finger-stab into a pointed glass tube of small calibre, which is then sealed by heat and allowed to stand for several hours. VALUE IN DIAGNOSIS AND PROGNOSIS 471 the study of blood regeneration is still largely qualitative. The tests referred to may be considered under the fol- lowing heads: A. Kesistance of erj'throcytes. B. Evidences of bone-marrow activity (recticu- lated cells; nucleated forms, platelets). C. Agglutinins and ha;niolysins in the blood- serum. D. Urobilin excretion. E. Protein, iu*ic-acid and iron metabolism. A. Resistance of Erythrocytes to Hypotonic Salt Solution The resistance (fragility) of er\'throcytes to various fluids was first studied by Malassez -'^' in 1873, and the mechanism of the destruction of the red blood-cell has been more or less imperfectly understod since the time of Ham- burger's **' investigations on osmosis of body fluids. Although hypotonic salt solution in varying strengths has been the most commonly used in clinical tests, saponin, snake venom, bacterial luemolysins, and specific htemolytic inmiune serums have also been employed either as clinical tests or in attempts to explain the mechanism of the de- struction of the erythrocyte. As a general rule, if the re- sistance of erythrocytes is increased or decreased to one of these agents, it will be so to all, but occasional exceptions have been noted. Thus in obstructive jaundice and in pernicious antemia it has been clai'ned that the resistance of erythrocytes to saponin is diminished, whereas it is in- creased to hypotonic salt solutions. In our own work, on the other hand (see page 42), we have foimd that in animals under various experimental conditions the changes 272 THE SPLEEN AND ANAEMIA '"'if ■■<: in resistance to saponin and hypotonic salt solution were always parallel. On account of such possible divergences, however, it is advisable, for the present at least, to confine routine tests to the hypotonic salt solution method. This method depends on the simple principle that ery- throcytes can remain for some hours in isotonic salt solu- tion without damage, whereas when placed in distilled water they are verj^ quickly ha?molyzed, the hemoglobin being " lakcd out" of the corpuscular stroma. If, then, suitable intem.ediate strengths of solution are arranged, it can be determined in just what strengths of salt solution partial ha'molysis occurs, and at which point complete haemolysis first occurs. The various ways of applying this test have been considered in detail by Ribierre,^'*' the method finally adopted by him being as follows: Glass- ware should be sterilized and the chemically pure sodium chloride should be desiccated before preparation of stock solutions, to get rid of the " water of interposition." In normal cases nine small tubes are arranged in strengths varying as follows: 0.50, 0.46, 0.44, 0.42, 0.40, 0.38, 0.34, 0.32, 0.28 per cent. NaCl. (If it is found that haemolysis occurs at 0.50 per cent., a second test is made with solutions of the strengths 0.60, 0.56, 0.52. ) The finger of the patient is carefully cleansed, pierced in the usual manner, and blood sucked into a pipette to a mark denoting one-fiftieth of the content of the pipette (about 2 c.c). It is then filled with the appropriate strength of salt solution, mixed and emptied into one of another series of small tubes, avoiding as much as possible the admixture of air, and the operation is repeated through the series of tubes. These are then covered with rubber caps, allowed to stand five minutes, centrifuged for one and one-half minutes, and the results observed. Ribierre VALUE IN DIAGNOSIS AND PROGNOSIS 273 has found that, a.'though after twenty-four hours the amount of haemolysis is slightly increased, there is no ap- preciable difference between a five-minute and a three- or four-hour period. Many observers still consider it necessary to defibrinate and wash the erj'throcji;es ; but this not only requires greater quantities of blood and consid- erably increases the difficulty of the examination, but also to a slight extent mechanically injures the cells, so that a slightly lessened resistance is found. To be sure, Widal, Abrami, and Brule showed that in some cases of the ac- (juired form of hfwnoly tic jaundice with apparently normal resistance fragility would be demonstrated if the cells were washed free of plasma. Later work, however, both in this laboratory and elsewhere, has tended to show that any change, when present, is in the cells themselves. In nur work in this laboratory', both on patients and animals, the use of a mixing pipette has been found un- necessary, the measurement of many drops to each tube tedious, and sedimentation for one hour has proved prefer- able to centrifugalization. The test is therefore performed as follows: Stock solutions of sodium chloride are prepared as alK)ve described in strengths varying by 0.02 per cent, from 0.20 to 0.70 per cent. If kept tightly stoppered these may be used for several months, but should be renewed earlier if control tests show any change in concentration of solutions. A series of twelve or more tubes containing 1 c.c. of different strengths of hypotonic salt solution are then arranged, varying by 0.C2 per cent, from 0.25 per cent, to 0.60 per cent, (or even stronger, if diminished resistance 18 174 THE SPLEEN AND ANEMIA K .fe A .■uJ I - Ik It* is suspected) . Into each one drop of whole blood is intro- duced and the tube gently shaken. If the drops have been of differenv size, slightly more blood may occasionally have to be added until the color is the same in all. After stand- ing two hours at room temperature, in the stronger solu- tions in which no hftmolysis has occurred, the unchanged corpuscles at the bottom of the tube will be overlaid with colorless salt solution. In the weakest solutions all cor- puscles will have been hfemolyzed, forming a transparent red solution. In the intermediate tubes can be noted the point at which haemolysis begins and at which it is complete. In normal cases haemolysis begins at al>out 0.45 and is complete at 0.35 per cent. In a case of the familial type recently examined haemolysis began Ps high as 0.7 per cent, and was already complete at 0.475 per cent. In most other anaemias the resistance is nearly always more or less increased, depending on the severity of the anaemia. Hjcmolysis may not begin until solutions as low as 0.36 are reached, or may not be complete before 0.24 or 0.26 per cent. In pernicious ana?mia, while the resistance is usually greatly increased, cases have been reported in which the resistance is normal or even diminished, so that there is a marked resemblance to haemolytic jaundice. B. Evidences of Boxe-Maerow Activity 1 . By Vital Staining. Skeined or Reticulated Cells. Amidst the confusion that surrounds the subject of stain- able granules in the erythrocytes, a few facts are generallv accepted by lijcmatologists. One such is that a basophilic reticulation is demonstrable by the methods of vital stain- VALUE IN DIAGNOSIS AND PROGNOSIS «74 ing in a very small percentage of noraial cells, but in greatly increased numbers in various diseased conditions of tlie blood. This special method of examination was first described by Chaufiard and Fiessinger,'^ in 1907, in connection with their study of a case of congenital ha'mo- lytic jaundice, in which condition the reticulated (granu- lous or skeined) erythrocytes are verj' nmch increased. These authors used Pajjpenheim's pyronine methyl green stain (equal pj'rts of saturated aqueous solutions of pyro- nine and methyl green, prepared at least several days before use, and filtered just before using), but also sug- gest the use of neutral red in isotonic solution, while W'idal recommends polychrome methylene blue, and we have found the best results with brilliant cresyl blue. This simple test is performed as follows : A few grains of the stain are dissolved in a perfectly clean, small test- tube in 1 c.c. of noinial salt solution, together with one or two small cr}-stals of potassium oxalate, to prevent rou- leaux formation.* As the exact strength of the solution is inunaterial, it is sufficient to prepare the stain in this way, aiming to get a strength of solution that is just trnnslucent in a test-tube of 1 cm. diameter. A few drops of ./lood are allowed to flow into this tube, the mixture gently shaken and allowed to stand ten or fifteen minutes. A drop of the sediment is then pipetted off, a fresh cover-slip prepa- ration made and examined under an oil immersion lens. The blue-staining reticulum is easily visible under such conditions, and the percentage of reticulated to non-reticu- lated erythrocytes estimated. The protoplasm of the ery- throcytes is not a ffected by the stain, whereas the various * The solution of sodium oxalate in salt solution may be prepared beforehand and kept on hand, if the test is made frequently. 876 THE SPLEEN AND ANEMIA ■IX- t ' 1 Ik i " '' ■'.■> forms of leucocytes, the platelets, and ha'mokoiiiuin are readily stained and identified. The avera<^e diameter of the reticulated cell, accordinff to Chauff ard and Fiessinger, is 8.1 /*, as compared with an average diameter of (J.3^ for the non-reticulated cell. The reticulum p -rsists longer than the protoplasm of the erythrocyte ur or the conditions of this test, but the majority of both will disappear in the course of a few hours. If an accurate comparative count is to be made, the cover-slip should be prepared and examined between fif*^een and thirty minutes of the time of obtaining the blood. In the blood of normal human subjects, reticulated erythroc}'tes are either entirely absent or constitute but a very small fraction of one per cent. They tend to be slightly increased in any considerable anaemia, but rarely exceed 1 to 4 per cent. Thus Chauff ard and Fiessinger found an average of one cell in .500 and one in 400 in eases of tuberculosis, one in 400 and one in 600 in mitral disease, one in GOO in malaria, one in 500 in plumbism, one in 200 in chronic nephritis, and one in 100 and one in 50 in plumb- ism with nephritis. In a case of tuberculous nephritis with profound amemia, subicterus, and diminished resistance of erythrocytes, the reticulated forms were considerably increased (2 per cent.). In cases of hJEmolytic jaiindice, on the other hand, the percentages of reticulated forms reach as high as 15 to 20 per cent., and in the case reported in the previous section of this book (page 202) the counts were frequently above 5 per cent. The proportion of reticulated cells varies considerably in different species, these cells in the normal dog being even rarer than in man, whereas in the rabbit they constitute about 2 per cent, of the total number. |,-U .i VALUE IN DIAGNOSIS AND PROGNOSIS 277 The nature of the reticulated erythrocytes has not yet been demonstrated, but whether the reticula are nuclear fragments of immature er>'throcytes, indicating a bone- marrow reaction '(as has been generally considered), or are the results of a disease process, their identification is of great diagnostic value, and their diminution or disap- pearance as the result of treatment may be taken as a s'gn of good prognostic significance. 2. Bji Fired Smears (Nucleated forms, Howe -Jolly bodies, etc.). — Although no direct method exists of deter- mining the rate of blood formation, it may be considered as equal to blood destruction (as estimated from urobilin excretion, etc.) so long as the blood counts remain con- stant. Although even this method fails if the count is rising or falling, nevertheless under all conditions some criteria exist for estimating the effort required of the !)lood-form- ing organs (mainly bone-marrow) to maintain the cellular elements of the blood at the level they happen to be. Thus in the stained dried smear of the peripheral blood, stained with any of the Romanousky group of stains, the presence of nucleated forms may l>e taken as evidence that the bone- marrow is so hard pushed that it must allow immature forms to appear in the peripheral blood. If tlie nucleated erythrocytes are megaloblasts. the diagnosis of pernicious aniemia is strongly suggested, but it must not be forgotten that other chronic anaemias, particularly tliose in which small hemorrhages occur over long periods of time, can iilso produce megaloblasts in the peripheral drculation. It must also be remembered that in certain rare condi';ions, such as tumors of the bone-marrow, ervthroblasts mav appear in the peripheral circulation without indicating excessive blood regeneration. 278 THE SPLEEN AND ANEMIA • ■!• .1. HoweU-Jolly bodies and the Cabot ring forms should, like tlie reticulated forms, be taken as evidence of the de- mand on the bone-i.arrow for increased blood formation and of Its ability to respond to the same. The increased number of these and of nucleated forms after blood trans- fusion or splenectomy (so-called " blood crisis ") are simi- larly taken as the response to the bone-marrow stimulation that these operations cause. These so-called "blood crises" will be considered again in the chapter on the results of splenectomy mider the heading of " Pernicious Ans- nua, in which condition they have been most carefdly studied; but it should be noted that thev have also been observed rfter splenectomy in Banti's disease, h^emo'- tic jaui.dice, and other conditions. If absent after splenec- tomy when the blood comit is rising (as in Goldschmidt, 1 epper, and Pearce's case), it would indicate that, with the cessation of increased h.-emolysis, the bone-marrow is no longer forced to put forth immature forms. If on the other hand, both blood crisis and improvement in blood count are wanting, it would indicate that an exhausted bone-marrow is no longer capable of responding to stimu- lation with the production of even these immature forms The blood crisis, which so frequently follows splenec- tomy for splenic disease, has been shown in Chapter II to be practically absent after removal of the normal spleen. This apparent discrepancy is similar to the fluctuations in the red blood-cell count after splenectomy, which falls after removal of the normal spleen, but rises after splenec- tomy m splenic disoase. The most probable explanation of both paradoxes seems to be that in disease the improve- ment IS due to the removal of an agent which both causes excessive haemolysis and depresses Ixine-marrow function. VALUE IN DIAGNOSIS AND PROGNOSIS «70 whereas the anamia following nonnal splenectomy is due to loss of nonnal stimulus to blood formation, and, for the same reason, unusual signs of bone activity are lacking. Polvchromatophilia, like the presence of microcytes and poikilocytes in the blood stream ^Rous)^*'" probably indicates degeneration of the erythrocyte rather than the appearance of immature forms. Macrocytes, like megalo- blasts, are probably signs of perverted bone-marrow activ- ity. The diminution or increase of the number of such forms, mav, however, safely be taken as indicative respect- ively of amelioration or aggravation of the disease process. 3. Blood Platelets and LeiLConjtes.—lf Wright's theorj' l>e accepted, that the blo^jd-platelets are independent elements of the blood formed from the megakaryocytes of the bone-marrow, both leucocytes and platelets*^" enter inu> the problem of blood regeneration. The leukopenia and the diminished platelet counts found in many of the diseases here under consideration should therefore be taken as further evidence of deficient biuod formation. The increase in blooe mad'e with tul)es containing different amounts of the patient's VALUE IN DL\GNOSIS AND PROGNOSIS 281 senun and the results compared with those in similar tests of known normal blood. Wliile positive tests for auto- lysis have, so far as I know, never been reported, Chauf- fard, Widal, and Weissenbach and others have reported cases that at times of exacerbation of the disease ^ive a positive " isolysin " test, indicating the presence in the serum at these times of a free hemolysin. During periods of remission these isolysins were not found. It must, of course, be rememl)ered that Moss^"'"' and others have found that the blood of a certain percentage of individuals is normally isolytic and isoagglutinative to the blood of other individuals of the same species. D. Urobimx Excretion It is now generally recognized that in the estimation of the amount of urobilinogen and urobilin excreted in the urine and stools there exists a fairly accurate index of the amount of blood being destroyed in the body. Emphasis has been laid throughout tliis book on the impor- tance of the study of blood destruction in its relation to the spleen, and it is Eppinger's'"* great serv^ice to have l)rought us to recognize the importance of urobilinogen and urobilin excretion in the stool as the best index of such destruction. Instead of such indefinite criteria as an enlarged spleen, tendency to acholuric jaundice, or the existence of anaemia coincident with active blood formation, Eppinger was led to search for a more definite index, and found it in Charnas's spectroscopic method f.^r the quanti- tative estimation of the amount of urobilinogen and uro- bilin in the stool. Although this has been generally re- placed bv the Wilbur and Addis *''^ method, it still remains jnly roughly quantitative and a rather disagreeable and I «t THE SPLEEN AND ANJiMIA I time-consuming test, so that improved methods are still to be hoped for. Emphasis must be laid, however, on the fact that examination of the stool as well as the urine is essential: for. while urobilin is more apt to be present in the urine when there is an increased amount of blood destruction than in normal conditions, this is in the nature of an overflow through the liver, and may he absent when there are still greatly increased amounts in the stool ; and, furthermore, the amount of urobilinuria may be influenced by a poorly functioning liver. Method.— The method for estimating the amount of urobilinogen and urobilin in the stools is described by Wilbur and Addis as follows: URINE " The method of collection of the twenty-four-hour urine has a considerable effect on the total spectroscopic reading. The vessel in which the urine is collected should be of dark-brown glas.f, and should he kept in darkness. Thymol crystals should be added, for, even in cases in which no obvious fermentation had occurred, we sometimes found a diminution in the total amount if no pres"rvative was present. After measuring the amount of the twenty- four-hour ui-ine, 10 c.c. are mixed with 10 c.c. of a saturated alcoholic solution of zinc acetate and, after a few minutes, filtered. If a number of urines are being examined at the same time, it is convenient to have test-tubes graduated to 10 and 20 c.c. Ten cubic centimetres of the filtrate are taken and 1 c.c, of Ehrlich's solution* is added. It was found that this amount prnduc^d a sufficient concentration * Paradimethylaminolx-nzaldehyde, 20 jftn. ; concentrated hydro- chloric acid, 150 c.c; water, 150 c.c. VALUE IN DIAGNOSIS AND PROGNOSIS 28S of acid in the mixture to give the maximum intensity of the urobilin band and contained enough of the paradi- incthylamidobenzaldehyde for the reaction with urobilino- gen. The development of the urobilinogen band is not instantaneous. We found that, as a rule, it had attained its full intensity in a quarter of an hour. The action can he greatly accelerated by heating, but this is to be avoided. It is better to wai*- for an hour before making the reading, and during this time the solution should be kept in the dark. After three or four hours there is a diminution of the urobilin and urobilinogen in filtrates from urines, so the estimation should be made not later than three hours after adding the Ehrlich solution. We found that Citron's hand spectroscope * was the most convenient instrument to use. The filtrate was washed into a graduate and diluted with tap-water until first one and then the other band of light absorption had disappeared when the full amount of light entered the spectroscope, but were still visible when the light was partly shut off. This gives a fairly definite end-point, and we did not find any great variation in the readings made by different persons, it is important, of course, that the light shall be always of approximately equal intensity. We made the readings in a dark room with a tungsten electric bulb, holding the spectroscope close to the source of light. In highly-colored urines one may be in doubt as to whether or not a trace of urobilin is present, for there may be so much general absorption of hght as to obscure the urobilin band in the undiluted fil- trate. There is no such difficulty with the urobilinogen band, which lies between the red and yellow where there * This can he obtained from Paul Altmann, Luisenstrasse, Berlin, Grrmany. il i It 1 i M 1*1 I i 884 THE SPLEEN AND AN/EML\ *:••> K I ,k Hi ■•''■At S IS no marked Lght absorption. With urines containing bile •f he amount of urobilin is not very large, it is neeessty t^ add some fuller s earth and to leave the mixture s'..-ding Or some t.n.e before filtration, if this is done the urobilin band can usually be read even in the undiluted filtrate. I he dilution required gives the value for 5 e.c. of urine I this figure ,s multiplied by the number of 5 c.c. quan- tities m the twenty-four-hour urine, the number of dilu- tions which would have been necessary if all the urobilino- gen and urob-lm in the twenty-four-hour amount had been concentrates! in a volume of .5 c.c. is obtained. For in- stance ,f in a twenty-four-hour urine measuring lOOO cc a reading of ten dilutions for urobilinogen and of twenty for urobilin were mode, the total un^bilin would '/^^ ^, f ';' 7= ^'«^^- ^^'^ t'-i"! to determine the d [ r ;\\ u" "^ ^"'"'-""^-en and uruoilin absorption hands (Wdbur and Addis n.eth.l), over two per^X Ota hng ten days before splenectomy was 71,250; wheret for two per,o,ls totalling eight days after splenectomy th^ figures were only 7954. ^ Schneider ••'-■has recently shown, by a method of his OH^ devising, that r ,. . titativc- e.tinxate nuy be ma.e of the urobilin, urobilinogen, and bilirubin in the duodenal contents, obtained through a duodenal tube. He eonfi^n^ previous work in finding all the above elements increase in permcious ana-mia and diminished or absent in other conditions simulating pernicious anemia. He considers the pleochromia found to be an expression of the immediate ha.molysis, ui-obilinocholia an expression of the heaped 7fv.'^Ti'"V" *^^ P^'*^^ 'y'''"^' ^"^ *h^ ^'^Sh color-index ot the blood .n pernicious anemia an expression of the ovei^plus of haemoglobin-building material heaped up in the liver. Examination of a single specimen, as practised VALUE L\ DIAGNOSIS AND PROGNOSIS 288 in this nit'thod, iiowever, even though the sul)stance sought for mv- he in greater eoncentration, is so much less de- sirahle than examination of the total collection of several (lays, as in the stool method, that it is doubtful whether tiiis procedure will prove as useful as the Wilbur and Addis test, cumbersome and disagreeable as the latter may be. It has already been pointed out, also, that Wilbur and Addis found extremely wide variations in the urobilin content of bile, and that there is evidence to show that tiie tlaily output of urobilin varies greatly in a given indi- vidual. Although the stool method fails to take acco -nt of the urobilin reabsorbed by the intestine, this disad- vantage is probably more than counterbalanced by weak- nesses in the duodenal method, which ignores variations in the composition of the bile and estimation of the total (juantity of bile secreted, and also is more liable to errors in the collection of material. M 1 I E. Pkoteix, Ukic-acid, axd Ikon Metabolism Expression of the need for proper metabolic studies in the diseases considered in this book, the best methods for studying the same, and the lesults hitherto obtained, both experimentally and clinically, will be found in detail in Chapters VIII and X. In spite of the fact that discor- cordant results are there recorded, both in clinical and ex- perimental work, a few facts of value to the clinician may safely be deduced. In severe anaemias an increased elimi- nation of uric acid and iron is found, and Umber's work demonstrates a pathologically excessive destruction of pro- tein in some cases of Banti's disease. As all these con- ditions of metabolism improve after splenectomy, cases 19 il too THE SPLEEN AND AN.EMIA showing such tlistui-bance of metabolism should probably be considered especially suitable for splenectomy. A final decision on this point, however, cannot be reached until a larger number of metabolic studies are at hand, and for this reason it is desirable that complete studies of this kind should be made whenever conditions are favorable. CIIAPTEIl XIII TREATMENT OF SPLENIC DISEASES BY METHODS OTHER THAN SPLENECTOMY (1) BLOOD TRANSFUSION. (2) MEDICINAL AND HY- GIENIC MEASURES. (3) SURGERY OTHER THAN SPLENECTOMY. Ix tlie treatment of certain phases or of individual eases of splenic disease, splenectomy must either be postponed or be considered as definitely contra-indicated. For in- stance, in very late stages of Banti's disease or in pernicious anifmia, in periods of exacerbation with extreme ana-mia, or in the crises of deglobulization of the hiemolytic jaun- dices recourse to splenectomy may be impossible. At the other extreme of the scale, some cases of familial jaundice, " more icteric than sick," are so little incommoded by the disease that splenectomy may be considered unnecessary. In these, as in other cases where the operation must be postponed, or even in relapses after splenectomy has been performed, certain other procedures have considerable value. 1. Blood Transfusion. — The most important of these is the method of multiple blood transfusions, which, on ac- count of the simpler methods of technique now in use, both for detecting suitable donors and for the actual transfusion, has now come into widespread use. Technique of Tests for Ilwmoli/sis and Agglutina- t'^m. — Lindeman's ^*^ method of testing the suitability of donors is as follows: " The red blood-cells of patient and donor are washed three times with normal saline: v -rying 291 1 m f^] Ih'** 292 THE SPLEEN .VXD AN.EMIA quantities of patient's serum are placed in tliree separate small test-tubes. To each of these are added 0.2.) c.c. of a 2 per cent, suspension of washed red blood-cells of the donor. The same is done with the donur's serum and the patient's cells. Controls are made of donor's serum and donor's cells — patient's serum and patient's cells. Con- trols are also made with donor's cells in normal salt solution and ])atient's cells in normal salt solution. The total vol- ume in each tulx.- is raised with normal saline to 0.5 c.c. of volume. 'J'he test-tubes are incubated in a water-bath for a period of two hours, and readinirs are made. Thev are then set in the ice-box over ni^ht and readiiif^s are made again the following' morning. When a case is urgent, the ice-box test is eliminated. The ice-box test should be elimi- nated only when absolutely necessaiy by the extreme con- dition of the patient, where time is the important factor. When the antount of blood taken from the patient for tests is small, only 0.-2.ji c.c. of serum is used, and controls of patient's serum are eliminated." A Wassermann test of the ' )nor's blood should, whenever j)ossible, Ik? included. This and the subdivision of the donor's blood into the proper a<>glutination groups may readily be done, if the practice is followed of having in reserve a list of pros- pective donois ready to donate blmid on demand. In 1-tG cases in which tests by this method were per- sonally sufjcrvised by Lindeman"not a single case of ha-mo- lysis and not a single death referable to the transfusion occurred. The necessity for careful performance of the test is shown by the fact that chills occurred in only thir- teen instances (0 per cent.), whereas in nine cases in which the tests were not personally supervised chills occurred in five instances {55 per cent.)." TREATMENT OF SPLENIC DISf^ASES 9SS Minot *"- has shown that if one has on hand seruin and corpuscles of subjects belongin/r to Groups II and III (of Moss's four groups), the group to which an adult patient belongs may be detemiined in twenty minutes. If then donors are available that have been previously cata- logued according to groups, transfusion may be performed uitliout danger. The test is performed as follows : A suspension of red cells from the patient is " obtained by collecting one drop of blood in about 1 c.c. of a 1.5 per cent, of citrate solution in 0.9 per cent, salt solution." A drop of this suspension is mixed separately with a drop of each serum (which re- tains its agglutniating power for months), and allowed to stand fifteen or twenty minutes. The presence or absence of agglutinr.tion is then observed microscopically. Similarly ;i drop of the patient's serum is mixed separately with a drop of suspension of corpuscles known to belong to xi roups II and III, and treated as abo e described. ^linot also points out that ha?molysis does not always occur in vivo, when donor and recipient belong to different iso- agglutination groups, because only about 20 per cent, of s(Ta that are agglutinative are ha?molytic. Haemolysis, IiowcA-er, never occurs without being preceded by or asso- ciated with agglutination. " Even when donor and patient Iiolong to the same iso-agglutination group, however, there may wcur. after transfusion, reactions of unknown nature, which are probably of not so severe or serious a nature as lucmolysis." ^Vhile the technique of the various methmis of trans- fusion now in use cannot be considered here, suffice it to say that, with proper precautions, untoward accidents may be practically eliminated and excellent symptomatic results rif 11 r . J- 'i 294 THE SPLEEN AND ANEMIA usually obtained. In all chronic an;pmias under con- sideration, not only is t cnjee of anasniia greatly les- sened after transfusion, l)ut in jjcmicious anaemia especially, a bone-marrow reaction, similar to the '■ blcKid crisis " fol- lowing splenectomy, is apt to occur four to ten days after transfusion. Such a reaction " furnishes the most favor- able time to do a splenectomy in those cases which have been transfused in preparation for the operation" (Vin- cent)*"'' and frequc:>tly initiates in pernicious ana?mia a remission of from three to twelve months. It might then be asked : Why not continue to transfuse instead of having recourse to a major operation such as splenectomy? Un- fortunately, (1 ) the benefit is probably of shorter duration than ■■" the benefit confened by splenectomy; (2) in some cases improvement Ixjcomes less and less after each trans- fusion, and (3) the increased blood destruction, as shown by urobilin excretion, is not lessened as it is by splenectomy. In a study of 212 blood transfusions in 189 cases, Otten- berg and Libman ''-' discuss at length the various condi- tions in wliich transfusion is indicated. Although the best results were foimd in such conditions as simple hemorrhage, liemorrhagic diatheses, and acute poisoning, improvement was also noted in various infections, debilitated conditions, and in the chronic primary anaemias. In twenty-five trans- fused cases of pernicious arifvmia, " fourteen underwent more or less prolf)nged remissions immediately following transfusion, and eleven showed little or no effect. Of these eleven, three were moribund at the time of trans- fusion and died within a few hours or days. The other eight, in spite of marked rise of haemoglobin and temporary symptomatic improvement, showed no interruption in the course of tlic disease, but continued to show blood destruc- TREATMENT OF SPLENIC DISEASES 295 tion of about the same rate as before transfusion. Of the fourteen patients who showed progressive improvement following one or more transfusions, one had a remission lasting approximately three months, three had remissions lasting six or more months, three lasting a year or more, and three lasting over two years." In spite of the remis- sions that are known to occur spontaneously in pernicious aiuemia, a record such as the above leaves but little doubt as to the value of blood transfusion in this condition. For- merly when the technique of whole blood transfusions was difficult single large doses were employed, but with the simpler methods now in vogue repeated transfusions of 400 to 600 c.c. are considered preferable in the chronic ana?mias. Xot only is the desired amount of blood easier to obtain and the discomfort to the donor lessened, but also the danger of hypertransfusion is avoided and the indi- cations about as well met as when larger doses are used. As many as fourteen transfusions have been employed with benefit in chronic conditions (McClure -'") . It must be recognized that in some refractory cases the later trans- fusions in a series have done more good than the earlier ones ; but it is usually true that if the procedure has failed in the first instance, it will probably continue to do so in the future, but is less apt to do so if a different donor is used. From a study of seven cases of pernicious anaemia which had been splenectomized and later transfused, Vogel and Downes gained the impression that the subsequent effects of transfusion were more marked and persisted longer in such cases than in those in which splenectomy had not been perfonned. It would therefore seem that in pern'cious anfemia, if signs of increased ha?molysis are present, transfusions 11 f .1 ..'ii 290 THE SPLEEN ^VXD .VN.EMU ' . i ';? 'f should be eniployed until the patient is in the best condi- tion to submit to splenectomy; and again after splenec- tomy, when the effect of the operation has passed away. If, on the other hand, the case is of the steadily progressive type, unsuitable for splenectomy, transfusions may l)e employed as a palliative iiieasure, according to the state of patient's condition and purse. In the various " indirect methods" (syringe. Erlenmeyer flasks, citrated blood, etc.) the amount ot blood transfused can be accurately measured, but even in the " direct " methods an approxi- mate estimate of the amount of blood received may be obtained by accurately weighing the patient before and after transfusion, 2. Medicinal and Ili/gienir Meamires. — Medicinal and hygienic measures to be employed in the treatment of the disea.ses under discussion are the usual remedies for anjemia, such as iron, arsenic (salvarsan), improved general hygiene, and an ample but sim])le, nutritious diet. Conflicting re- sults have been reported from X-ray treatment of the spleen, but it is possible that properly graded doses may be of distinct value. It is almost sujierfluous to add that in cases of splenomegaly with anaemia, where a causative factor sucii as malaria, lues, uncinariasis, etc., is known to exist, appropriate medication is all-important. Up to a few years ago, l>efore spk-nectomy and blood transfusions came into vogue in the treatment of chronic an;pmia, various medicinal remedies were reported as of value. Organo- therapy (spleen and bone-marrow feeding, Chauffard, Widal) and cholagogues (Chauflard, Cavazza"*), proved of but bttle help, and only with a long-continued course of high iron diet did Widal find any marked improvement. On account of the antiha>molytic properties of arsenic TREATMENT OF SPLENIC DISEASES 897 iCiuiin and Felthaiu "''"^), cholesterin (Chauffard and Gri- <;aut,'^ Parisot and Ileully ^^'*), and calcium chloride ( Iscovesco '■'*), these dru^s have been tried, in some cases uitli considerable improvement. On the whole, however, it nmst be recognized that in the s()-callen, designed to diminish splenic function by squeezing the organ in new-formed connective tissue, is recommended by Schiassi,^"'' under the term " splenocleisis," for cases in which splenectomy could not be performed. The capsule of the organ is scarified, and wrapped with iodoform gauze, which is later gradually witlidrawn. Destruction of the spleen by gradual cauterization, after fixation to the ab- dominal wall, has also been suggested, but the field for such a procedure would seem to be extremely limited and the chances of s';c( .;ss not very great. In similar cases Troell **^ recommends, on the basis of experimental work similar to that done in this laboratory (page 121), ligation of the splenic arteries and veins. Lanz '^' found ligation of the artery beneficial in three cases of wandering spleen, but Roblee '*" states that out of six cases in which ligation was tried by Dr. Skel (discussion of Harris and Ilerzog's paper *"), four died. Although there is no theoretical or H I if 4-. 1 i.ta li ' i\ '. ■mm\ 298 THE SPLEEN AND AN^EMLV L experimental reason for such high mortality, this operation must still be considered to be in the experimental stage. The most promising results from surgical measures other than splenectomy are to be found ' . tiie recent studies of Percy '^^ on tb.e efl'ccts of removing chronic sources of infection in cases of pernicious anaemia in which the spleen is also removed. lie found in pernicious an;emia not only that chronic inflammation may frequently occur in the gall- bladder, appendix, and other organs, as well as in and about the spleen itself, but also that the same strains of bacteria may freciuently be cultivated both from the spleen and the other organ or organs involved. In the hope that a possible cause or contributing factor to pernicious ana-mia may thus be eliminated, he therefore routinely removed gall-bladder, api)endix, tonsils, or carious teeth, or as many as showed signs of chronic infection, either simultaneously with the spleen or as soon thereafter as was practicable. Even the energetic treatment of a complicating pyorrha^ has apparently reinforced the improvement caused by sple- nectomy. Of twenty-four cases treated in this' way, twenty-one showed a marked postoperative improvement,' and fourteen of these have still continued in their improved conditions over periods lasting from eight to thirty-one months. In view of these excellent results and as long as the cause of pernicious auc-emia remains unknown, it there- fore seems highly advisable to supplement splenectomy in this condition by the above procedures in any patient in whom such signs of chronic focal infection can be demonstrated. .. {. CHAPTER XIV TREATMENT: VALUE OF SPLENECTOMY AS A THER- APEUTIC PROCEDURE Splenectomy for rupture or severe injurj- of the spleen is one of the oldest abdominal operations about which we have definite knowledge."''^ It was not until the advent of anaesthesia and the greater surgical skill of the nineteenth century, however, that it was found practicable to remove the chronically diseased organ and thus widen the field of applicability of the operation beyond that of surgical emergencies. Unfortunately, among the chronic diseases of the organ first attacked were the enlargements incident to cirrhosis of the liver and leukemia. The un- favorable results in these two diseases cast discredit upon the operation ; but, nevertheless, in 1 008, Johnston '^"^ was able to collect 708 cases of total extirpation of the spleen with 194 deaths. From 1900 to 1908 there were 355 cases with 66 deaths. If the cases of leukemia and traimia are subtracted, the list is reduced to 235 cases with 27 deaths, or a mortality of 11.5 per cent. Since 1908 our greater knowledge of the physiolog\' and pathologj- of the spleen has resulted in a better selection of cases, so that now the total mortality has been somewhat further reduced (Las- peyres,"^* ^Slichelson,-"" Mayo^*-). In the past three years a more active study of the sur- gical treatment of certain so-called primary ana?mias has led to the much more general use of splenectomy, and it is this application of splenectomy that attracts most attention at present. 209 n| ; 11] i ia ^d I 300 THF SPLEEN AND AN.i':MIA CoxTiLV-iNDicATioNS. — It is iiiost important to know when splenectomy should not be done. We now know that in certain diseases removal of the enlarj^ed spleen as a curative measure is contra-indicated. These include the various forms of Icuk.Tmia, also polycythjtmia, and most cases of malaria, syphilis, and tuberculosis. In certain cases of cirrhosis of the liver (includin/r the hypertrophic form) Eppiri^j^er has recently advcK-ateri splenectomy on account of the evidences of increased blood destruction in this dis- ease when jaimdice is a prominent feature; but in the ordi- nary atrophic forms in the absence of jaundice the desira- bility of splenectomy is questionable. Too much em^ ..jsis cannot be laid on the necessity of ruling out atypical forms of leuka?mia — before the splenectomy is undertaken; but. on account of the fijeat variety of aleukftmic cf)nditions. this is often an extremely difficult task. In no case, how- ever, should splenectomy be advised until the blood picture has been carefully studied over an extended period of time and the presence of leukaemia excluded so far as may be possible. Anything pointing toward a hemorrhagic diathesis should also l)e given careful consideration. Its presence is, as a rule, sufficient to contra-indicate operation, although the repeated hemorrhages from varices or due to other mechanical causes, as in Banti's disease, are more indica- tions for operation than otherwise. In the severer anaemias definite signs of bone-marrow activity should also be forthcoming (nucleated or reticu- lated cells, Jolly bodies, etc.) . If they cannot be provoked by appropriate drugs or by transfusion, it is probable that the marrow is relatively aplastic and splenectomy should not usually be attempted. SPLENECTOMY TREATMENT 301 That the reinoval of tlie iioriiial spleen is followed hy a temporary aiiieinia has heon shown both by cliiiieal observation and animal experimentation; but this should not be eonsidered a eontra-indication to operation. The apparent paradox that, while removal of the normal spleen eauses a temporary anaemia, removal of the spleen in eer- tain bhwHl disease?-, relieves the existing anaeniia, has been commented upon in ancrther section. I.kuk,i:mia. — On account of the almost invariably fatal outcome of splenectomy in early cases of leuka>mia, for some decades the operation has been considered as definitely contra-indicated in this condition. Occasional cases have been reported, however, in which death did not follow sple- nectomy, and, in addition, the success that has attended this operation in recent years has inevitably tended to have the procedure applied to conditions which had previously been considered unsuitable. To help prevent the unwar- ranted inclusion of leukicmia in the scope of splenectomy, some evidence is here furnished as to the result hitherto obtained in this condition. In 1898, Vanverts "' collected twenty-nine cases of splenectomy for leukaemia, to which Fevrier''' adds two more. Of the thirty-one cases, only three survived the operation. Of these three cases there has been considerable doubt as to whether Franzolini's was really a case of leuka^nia, and Banti did not hesitate to call it a case of Banti's disease. Burkhart's case was appar- ently operated upon in an early stage (leucocyte count about 43,000). That the course of the malady was not influenced by the operation is shown by the fact that the patient died eight months later with the typical physical signs and blood picture of leuka?mia. In the third case (Hartmann's) improvement lasted for three years, but ji SI 30-2 THE Sl'LEKN AM) AN.KMIA emaciation and gingival hcniorrhagt-s then became appar- ent. Tlie cause of dcatli in almost every unsuccessful case is hemorrliane, wlietlier from the pedicle of the spleen, from torn adhesions between the spleen and diaphrai^m, viscera, or parietal wall, or from the laparotomy wound. In the single case that I have had opportunity to examine, an aleukcTmic leuka'mia, with a total leucocyte coimt ranging from lO.OUO to l.j.OOO, but with myeloid cells, the spleen was removed apparently without hemorrhage, but the ab- dominal wound showed no tendency to heal. \ entral hernia could not be avoided, and the patient died two weeks after operation from an acute generalized peritonitis. In a few cases, where the great size of the spleen or a complication such as extreme mobility lias been the promi- nent symptom, splenectomy has been practised as a pallia- tive measure. Kuttner - '^ reported one such case of the n)yeh)genous type that continued to improve while under observation, but he recognized that the progress of the dis- ease had not in any way been influenced by the procedure in a curative sense. On account of the high postoperative mor- tality and of the evidence that the course of the disease is not affected by the operation, it is safe to say that splenec- tomy is definitely contra-indicated in the various fom\« of leuka?mia. This is especially true in the acute form, while in the more chronic forms it should only be under- taken when the most urgent indications are present. PoLYCYTH.T.MiA lluBRA. — On account of the fact that polycj-thivmia rubra (erythnemia, Vaquez's disease) is the only primary disorder of the red blood-cell system in which the cell count is increased, and that splenectomy has been attempted unsuccessfully in this condition, it seems advis- able to consider it in some detail at this point, although it SPLENECT«,...IV TREATMENT 303 iWfi cannot, of (.'ourse, be included anion^ the univniias. First described by \'a(iiiez *''' in 181>'J, tliis i)cculiar syndrome was later brou^lit more prominently before the niedical profession by Osier'-' in I'.X).'}. and its various features more exhaustively studied by Cominotti,"'' Ilirsehfeld,''" Senator,"" Abeles," :'nd others. Aceordinmolytic agents has seldom been tested in polycythfemia. In most cases it has been found to be normal, although Guinon, Rist, and Simon ''® reported a case in which the resistance to hypotonic salt solutions ^-^as slightly increased. Pick- ard^^* has very recently described a case of true poly- cythaemia in which hemolysis began at 0.48 per cent. XaCl and was complete at 0.30 per cent. XaCl (essentially normal limits) . An increased resistance to a different kind of ha?mol}'tic agent, on the other hand, was shown by the fact that to antihuman hsemolytic amboceptor the patient's cells showed no ha?molysis when m'xed with twice the amount of amboceptor necessary to hjemolyze normal cor- I gill J '11 Hi S04 THE SPLEEN AND AN^^MIA 1.; ' ,4 i ■'* puscles. That this is not always the case in polycythemia, however, is shown hy the findinjj^s of Freuiul and Rex- ford,' "'•' in whose ease the same test was performed and tlie resistance found to l)e normal. The ctiolo^' is completely unknowTi, though it is proh- able that various toxic or infectious agents may provoke the syndrome, either by direct marrow stimulation or through excessive repair after a primary red blood-cell de- struction. Thus Belonowsky was able to raise both h;vmo- globin and er\thrm'yte c( int by the frecjuently repeated injection of minute doses of luemolytic serum. In true cases it is possible to rule out all mechanical causes, such as occupation, hypertension, heart and lung disease, and adenopathy and other alterations in the blocxl should not be present. The pathogenesis of the disease is probably due to a true ])one-ntarrow hyperplasia, as is shown by the fact that the over-active bone-marrow is but seldom forced to deliver inmiature or nucleated red !)lood-cells to the cir- culation. All the symptoms can be explained as a result of this plethora. The enlarged spleen is largely spodogen- ous; i.e., compensatory attempt to provide for excessiv-e blood destruction, with resultant increased macrophagic action and congestion. On a priori grounds, therefore, it will readily be seen why splenec-tomy should not be undertaken in this disease. If it is correct that the primary trouble is in the hyper- plastic lione-marrow, the overactive spleen should be looked upon as the chief agent to keep the plethora within limits compatible with life. A logical radical treatment woidd he the obliteration of the marrow of one or more long bones, or ligation of nutrient arteries, just as partial excision of the thyroid causes improvement in exophthalmic goitre. SPLENECTOMY TREATMENT 305 mi Sach procedure has not to my knowledge been attempted, and it is doubtful if a sufficient effect could be produced to make it of clinical value. As a matter of fact, sple- nectomy has been but rarely attempted, although Luten- bacher states that " it has been followed in several cases by a rapidly fatal termination from suppuration or hemor- rhage, and in those cases that survived it has caused an evident augmentation in the polyglobulia." A confirma- tion of the protective action of the spleen in this condition is afforded by the increase in polycythsemia in cases where enlarged spleen has been reduced in size by X-ray treat- ment. In some cases, on the con* 'arj' (perhaps where the X-ray dosage to the spleen has been irritative rather than destructive) , this form of treatment has proved beneficial. Destructive X-ray applications to the long bones have not proved of value. Large venesections, repeated at rare intervals and followed by salt-solution injection, have also been of use in some cases, but the consequent improvement is always evanescent and, on the whole, this treatment must be considered unsatisfactory. Diseases in Which Spleneci'omy May Occasionally BE Indicated Cirrhosis of the Liver. — Mention has already been made of the fact that Eppinger had presented an experi- mental basis for extending splenectomy to cases of cirrhosis in which jaundice was a chronic and prominent symptom. On account of the enlargement of the spleen that fre- quently is found at an early stage of cirrhosis of the liver, Kppinger *"* was led to search for evidences of increased blood destniction in this connection also. This was forth- coming, when jaundice was present, not only in the exist- 20 a J i LI ,. J ■: 306 THE SPLEEN A^F AIs^MIA l.'U ' ence of increased aiiEemia, but also in the increased amount of urobilin found in the stool and the high iodine number of the blood. With less justification, but on account of the analog}' that exists between the changes in the liver and spleen in portal cirrhosis and in Banti's disease, other authors have attempted splenectomy in portal cirrhosis even in the absence of jaundice or other signs of excessive blood destruction. Thus Jullien,-*" in 1911, reports seven cases treated in this way, of which two died as a result of the operation, while the others showed considerable im- provement. This even included such important changes as the long-continued disappearance of a chronic ascites, and of the superficial evidences of collateral circulation. Kidd^^' believes that splenectomy should be tried in all cases of cirrhosis showing enlargement of the spleen, and W. J. iVIayo^' has also found splenectomy advisable in cirrhosis of the liver. In addition to one case of Hanot's cirrhosis in which the spleen was removed with " undoubted benefit and possible cure," he has removed the greatly- enlarged spleens from four patients suffering from portal cirrhosis. Although it was too early to know whether or not the end results had justified the operation, three of the four patients showed marked improvement with dis- appearance of the a,scites and anaemia. Although the evi- dence is still meagre on this point, and the evidence at hand insufficient to determine what types of cirrhosis are in- cluded under this term by surgeons, the results are favor- able enough to entitle the procedure to further considera- tion. In estimating their value, however, it should be remembered that the differential diagnosis of portal cir- rhosis from Hanot's cirrhosis and from Banti's disease and other similar conditions is often difficult or impossible SPLENECTOMY TREATMENT 307 to make, and it should also be borne in mind that there is removed with the spleen a reservoir which to some author- ities is of great importance in accommodatinfr the blood which accumulates behind the obstacle of the cirrhotic liver. Whether or not this very accumulation of blowl in the spleen is a factor which may lead to increased blood de- struction is a point for the future to decide. M.vi^vRiA AND Syphilis. — The extreme size attained by the spleen in chronic malaria and the frequency' of malaria in tropical regions early turned the attention of French and Italian surgeons toward the advisability of splenec- tomy in this condition. In spite of almost uniformly fatal results in the early cases, surgeons have persisted in their attempts, on account of the many distressing symptoms (dyspnoea, cyanosis, vomiting, dysiiria, dysmenorrhnea, abdominal pain, etc.) that the tremendously enlarged spleen may cause. The operation has been, however, at- tended with unusual difficulties on account of the friability of the organ and vessels and the great number and density of the adhesions in most cases. This not only promotes the liability to severe hemorrhages, but, on accoinit of the length of time required for the operation, greatly increases the shock of the operation. Of the twelve deaths in the series of forty-seven cases collected by Olgiati,'''^' ten were due to these causes, the other two to peritonitis. Twenty of the thirty-five cases that recovered, on the other hand, had ectopic spleens, which, therefore, did not present ad- hesions to the surrounding organs. As it is precisely this type of spleen that is especially liable to the further com- plication of torsion of the pedicle and rupture, exploratory lap.irotomy would be indicated if the enlarged spleen had failed to respond to medicinal treatment and was causing ]■ 308 THE SPLEEN AND AN/EMIA ' , ) distressing symptoms. It should, however, be recognized that splenectomy was undertaken for the relief of such symptoms ami not as a curative measure. In Roumania splenectomy has been practised in resistant cases of chronic malaria with considerable improvement in many cases, but with the high operative mortality of over twenty per cent. (Racoviceanu '"'"). As all these figures, how- ever, date from more than fifteen years ago, the better tech- nique of the surgery of to-day may eliminate shock and hemorrhage to such a degree that the mortality in malaria may prove to be no higher than in the other diseases. In some cases of long-standing syphilis, also, when the enlarged spleen has proved resistant to specific treatment, it may be advisable to remove the organ that has become the chief cause of the patient's disability. Thus W. J. Mayo^*^ has removed the greatly-enlarged spleen from three patients suffering from chronic syphilis and marked ana?mia. " In one of these specific treatment had been carried out for two years, in another for six months, with- out satisfactory improvement in the general condition of the ana-mia. Following splenectomy, there was marvellous improvement of the anaemia in all of them." The ultimate results of splenectomy in such cases will be awaited with great interest. It may prove that occa- sionally the enlarged spleen, at first protective, eventually assumes a pernicious activity, or that, like the central ner- vous system in some chronic infections, it may become a secluded nidus of infection which cannot be reached by ordinary medical treatment. In either of such contin- gencies its removal, therefore, may become desirable. Nevertheless, for the present at least, enlarged spleens sliould only be routinely removed in those cases where the SPLENECTOMY TREATMENT 309 indications liave been proved to be favorable or else where sudden emergencies require a greater latitude in the em- ployment of this form of treatment. The widespread re- moval of spleens without accurate diagnosis or regard to the suitable indication.'^ will undoubtedly lead not only to many disastrous results but also to the indefinite obscura- tion of the proper field for this important operation. TABLE LXIV Collected Reports of Resulis ok iSPLENECTOMT in Vabiocs Diseases of THE Blood Duease Autho.' Number of caaes Uecov- ered Died Per cent, mortality Gaucher "s Erdman and Moor- head '« Krumbhaar Elliott and Kanavel"" ElUott and Kanavel"" Elliott and Kanavel"" Stillman *'^ 10 183 16 23 9 6 153 8 155 15 22 9 6 123 2 28 1 1 30 20 15.4 6.2 4.3 IJanii's Ha'inolytic icterus ac- quired Hajmoly tic icterus, con- Rcnital and familial Hiemolytic icterus, un- cla.<dmann and Moorhead ""' collected ten cases of large-celled splenomegaly (Gaucher's disease) in which the spleen had been removed, and of these, two died, both within twenty-four hours of operation. While this probably represents too high a mortality, the improvement which followed in the other eight cases cannot always be taken as indicative of eventual cure, for the disease is known to exist independently in the bone-marrow and lymph-nodes. It would therefore seem wiser to restrict splenectomy in this disease to those cases that are unusually handicapped by the results of the disease, but are still good surgical risks, and in such cases to limit the prognosis to improvement and not to promise complete cure. H^i:molytic Jaundice. — The field in which splenec- tomy has been practised with the greatest success is un- doubtedly that of ha?molytic jaundice. Both in the ac- quired form (Hayem-Widal) and the congenital or fa- milial type (ChauflTard- Minkowski), marked improvement and frequently complete cure have resulted from removal of the spleen. In fact, the success obtained in this type of case, where the chief vitium is that of increased blood de- struction, has been a powerful incentive toward extending the operation of splenectomy to the wider range of allied diseases discussed in this chapter. Splenectomy was first tried in this condition by Vaquez and Giroux. As their case, however, died two days after operation, ChaufFard's dictum that hsemolytic jaundice constituted a "Noli me tangere " for the surgeon prevailed for several years. In 1911, however, splenectomy was again tried in hfemolytic SPLENECTOMY TREATMENT S13 jaundice with a very different result from that in Vavquez and Giroux's case. In that year Micheli removed the spleen from a case of the acquired type with the most strik- ing improvement: the blood count, which had been between 980,000 and 2,600,000, quickly rose to almost normal, the acholuric jaundice and urobilinuria disappeared, the fra- gility of the red cells was lessened, and the patient within a few months was apparently cured. Similar beneficial results were obtained by Banti in his two cases of hjemolytie splenomegaly, which we have taken to be identical with the acquired form of haemolytic jaundice. Success was ob- tained in other early operations (Kahn, Roth), and the procedure would undoubtedly have been in more frequent and intelhgent use, in this country as well as Europe, if the xmfortunate grouping of several clinical entities under the cloak of " Splenic anaemia " had not clouded the worth of the procedure. In 1915, Elliott and Kanavel ^'^'- were able to collect forty-eight cases of haemolj-tic jaundice (six- teen acquired, twenty-three familial, and nine unclassified) that had been treated in this way. Of the forty-eight cases, only two died — one shortly after operation, the other from sepsis, six weeks after operation. The other forty-six are reported as "cured," this result being based upon the disappearance of jaundice and exacerbations, and decrease of the amemia and the urobilin excretion. The effect on the resistance of the red cells was not constant; in some instances the resistance returned almost to normal, but in most cases the red cells remained almost as fragile as before operation. In spite of these brilliant results, however, it nmst be remembered that the primary cause of the disease is unknown and is pi'obably not in the spleen. The case ■n \ i V 814 THE SPLEEN AND ANJiMIA i' reported by W'hipham *''' emphasizes this point; although splenectomy was followed by great clinical improvement, return of the red blood-cell count to a level above normal (polyglobulia) and a reduction of their fragility to a nor- mal level, nevertheless, three montlis after operation, jaundice and extreme anaemia returned and the child died in a " crisis of deglobulization." " Whatever the catua causorum, it must be regarded as established that it is throurh the instrumentality of the spleen that pathologi< hiemo ysis is wrought." V. Jaksch's Disease. — The results of splenectomy in V. Jaksch's disease are too meagre to be of value. Only six cases have so far been reported (Stillmann*'^*), and, although all of these were improved by the operation, it must be admitted that ptediatrists claim even greater im- provement or even cure after long-continued medical treat- ment of this condition. It should be noted that the first case reported by Stillmann exhibits certain features more compatible with the diagnosis of ha?molytic jaundice than of v. Jaksch's disease (decreased resistance of erythrocytes, great increase in reticulated en,i:hrocytes, and a practically normal leucocyte coimt) . Pernicious Ax^timia. — The most important disease, from the point of view of its greater frequency and greater severity, to which splenectomy has been applied is per- nicious an.Emia. The striking improvement that has been shown to follow removal of the spleen in such diseases as hsemolytic jaundice and Banti's disease naturally led to an extension of this clinical procedure to allied conditions. Tn 1913 three investigators — Eppinger,*"* Decastello,"' and Klemperer,'^*® — working independently, tried splenec- tomy as a therapeutic measure in pernicious anaemia. It SPLENECTOMY TREATMENT 315 is interesting that Eppinger was led to adopt this pro- cedure by observing after splenectomy a diminished output of urobilin and other evidences of decreased haemolysis. Decastello, on the other hand, had noted the improvement that followed splenectomy in the related conditions, hfcmo- lytic jaundice and Banti's disease; whereas Klemperer was influenced by the clinical observation that splenectomy for such conditions as rupture of the spleen was in some in- stances eventually followed by polycytlia?mia. Such marked improvement was noted in these earlier cases that the procedure was quickly and widely repeated, chiefly in Germany and in this countrj% so that a fairly large group has already become available for study. More prolonged observation, however, has shown a considerable mortality from the operation and postoperative complica- tions, and, moreover, thai very few of the patients con- tinue steadily to improve; in almost all the characteristic blood picture of pernicious anaemia remained, and not a few died from relapses of the disease in the first year or two after operation. The object of this section is to show by a correlation of the published reports, with later informa- tion obtamed by personal communication, just how valu- able splenectomy has thus far proved to be in pernicious anaemia.'^'"* Although it has been impossible to get additional re- ports from some of the German authors, and of some patients who have been lost sight of, nevertheless tiie re- sults obtained in the last two and one-half years are suffi- ciently concordant to give evidence of some value. In drawing conclusions from any such review, however, it must be remembered that certain difficulties are unavoidable. For instance, the dividing line between pernicious anaemia 916 THE SPLEEN ASD AN^.ML\ i::;? i ' «■• and some of the other primary hlood diseases is necessarily such a shadowy one that the possil)ihty of an incorrect diagnosis must always he borne in mind. Then, too, the concept of pernicious ana-mia varies so nuicli witli different authorities that cases included as such by one author miglit easily be rejected by another. Thus one of the earlier cases of splenectomy that was followed by marked and long-contitmetl improvement, when sul)jected to critical analysis, seems to be ratlier a case of acijuired lucmolytic jaundice than of tnie pernicious ana-mia. In another case reported as pernicious anicmia the diagnosis was later changed to ha>molytic jaundice on account of the subse- quent appearance of acholuric jaundice with diminished resistance of the er\i;hrocytes. As such variations in diagnosis are apt to include less serious diseases under the head of " pernicious ana-mia," or diseases in which sple- nectomy is already known to be of benefit, the present sta- tistics will be correspondingly favored by such inclusions. In applying the pn -ent figures to prognosis, however, it is fair to oflt'set the iiu lusiori of such cases with the results that must inevitably follow the l)etter selection of cases and better preparation for operation. A third considera- tion is that the terni " pernicious anicmia " may later be found to include m.oic than one clinical entity. (CoMipare the great variations that occur in the size of the spleen, in the bone-marrow reactions, in the evidences of ha?mol- ysis, and in the duration of the disease.) If this were found to be true, it might well be that some of the appar- ently discordant results that have been observed after sple- nectomy are due to the fact that the operation was of value in one or more types and contra-indicated in the others. The results of these studies are included in Table LXV. ? c H E a S-TS3 =:a^' "s --x (• " ">3 ^ c 5 '•■'•■■ S-;''5s-.-'^p SI s- .TS :?S S^ S.5 # =^:,=:s^-'"'- r-34 •-£ -; o-n 5 ."• 23- ■■ -> 3 — » 3 (p - -) ■a ^ S5-~ ITS* r T3 yO M "^ 3 « *•«».:• =-3 =-cr -1 3- ■-:''< 3 1 C 5 » 22.3 ■^."■_^,S■ ■< -<^< "■ B ■g o - 3 =:• " M ^ < M E;;!!SS3;!2:x C 9 5l- ■ i a.32 =5 ^S io " -• » t;.-' ^ 3 § - - •• '■^■■> •■Bji: ':. •• 6 ._■■ ■■ 'c" " E. 2.3 53 ^£ 3 3 'I ° 2 3 I " S 5.1^5 '^' |3?L^-2^iS.-S' ?.sS>r^i-^ .?23 •Jo- s-£3 3-7-S„ H *♦ ^ H-; to (*-•.• V> M ,2 =S3S;,552--~5.23.f.--£:" ,„,f-=5l=o.a. 5-5 1: 3: o . a . B.; n - o • s: < a- H » ^■X' . -JOiM 1^ < : ■^ ■ 10 *j' |: a ■*■ D ■ 'S • 3: : B: 317 2. 5* CO I"* "8 ' W 1 n ^W>IM MM biV *MtOM^M lOMW-Jt^ > X f >3 13 if *.OWC 00 M "-N- J. — MMW^ ^0«-0*- n3^0 00 M ^*' MOOO»»- OMOC"- w&3W^^ MM o» 00 tx — OOMO ^ow^a ,^ (— W Ot^-MW^-- ^-^»o-*-4 ^•^ < o i? B B 3 2 o 7 > III ' III !■ ': S18 THE SPLEEN AND ANEMIA iK <..■: I'll i Analym of Results.— It will be seen that of the i S8 individuals whose spleens were removed, thirty died within six weeks, presumably from the effects of the operation, a mortality of 19.6 per cent. Of the remaining 123 patients, all but twenty-four showed a distinct improvement, both in general condition and in blood picture. Of the twenty- four individuals that survived the operation but failed to improve, a few were obviously harmed by it. To this group l)elongs Pappenheim's case, splenectomized at a favorable time, when the patient was in tlie stage of a re- mission. The condition, nevertheless, was aggravated by the operation: the blood showed signs of increased destruc- tion and a serious relapse began. The improvement noted in the majority of cases lasted var>-ing periods. Thus at the end of six months, of fifty-three patients who had sur- vived operation for more than six weeks and were still under observation, forty-four ht^a still continued to im- prove and none had died, but nine had already relapsed. TABLE LXVI Late Results Aftek Splenectomy in Pernicious Anemia Number oases known Still improved Relapsed Died subsequently. . After one After two year years 27 6 11 3 7 2 9 1 At the end of the first year after operation there re- mained twenty-seven patients who were still under ob- servation (see Table LXVI ) . Of these, Uecastello's series is the most important, not only because he and Eppinger were che first to try this procedure and therefore the cases oould be followed for a longer time, but because the early SPLENECTOMY TREATMENT 319 successful results have been greatly modified by time. Of six patients at the time of his publication, several months after operation, four showed such great improvement that, except f(-r the microscopic appearance of the blood, they might have almost been considered cured. Two years later, however, two were dead and one was in as poor condition as before operation. Of the other two important early series, Eppinger's and Klemperer's, it has been impos- sible to get additional information. The figires for the whole group of twenty -seven cases, however, show that the initial improvement has been maintained in less than half of the cases. A small but interesting group is formed by six indi- viduals ( see Table LX VI ) that have been knovm to have lived two years or more after operat-on (Descatello [two], iffin, Harpole, Huber, Thayer;. Of these, Giffin's patient haH had the disease for two and one-half years; the anamiia, was not extreme at the time of the operation, and the spleen was much enlarged ( 1640 gm. ) . Improved by the operation, he died three years later from pneumonia. In Decastello's two cases the disease had existed for less than a year; the anaemia was severe and the spleen but slightly increased in size. These patients improved after operation both chnically and as to the blood picture ; but, whereas one in a subsequent report was in poor condition, the other was without symptoms, although the blood picture was still that of pernicious ana?mia. Harpole's patient was known to have had pernicious anir \a. for two years, and at the time of operation the ameo)' was moderately severe and the spleen twice the normal si/e. After splenectomy there fK^curred immediately an active bone-marrow re- action with marked clinical improvement. The patient has w 1 r m ; 1 iW ill t ' ^1 i ■'H 1 Ijillji i ! II 1 1 ;■ l4, i m 1 1 s«o THE SPLEEN AND ANyEMTA ii- , I I ■•« 1 ■» i continued in fair health, with only a slight anaemia, hut with persistence of spinal-oord symptoms. Ruber's patient, who was considered moribund at the time of opera- tion, improved rapidly for seven weeks, relapsed, and later underwent a spontaneous remission. After two and one- half years she was still in good condition and able to do her housework, but still anaemic. Thayer's patient, having had the disease one year, improved after splenectomy, although there was no bono-marrow reaction. After eight- een months the patient relapsed to the same condition as before operation and was last reported in poor condition. Estimation of the value of such a procedure as splenec- tomy in pernicious anaemia must take into consideration not only the actual results obtained, but a comparison, so far as is possible, with the probable results if operation had not been undertaken. Thus, whereas we have seen that splenectomy caused a quick and marked improvement in 64 per cent, of all patients, natural remissions occurred at one time or another in over 80 per cent, of the pa,tiert of Cabot's "^ series treated by the older conservative meth- ods. One cannot maintain from* this that perhaps the im- provement after splenectomy was only a coincidental re- mission, because the onset of improvement was too closely and constantly related to the postoperative period ; but it does offer some basis for the contention that other methods of treatment may yield results as striking as those fol- lowing splenectomy. However, from the aspect of dura- tion of the disease the evidence is more in favor of the sple- nectomized series. Tn Cabot's series, almost half died in the first year of the disease, and of the remainder, one-third died in the next year (compare Table LXVII) . As the dura- tion of the disease in the splenectomized series had already SPLENECTOMY TREATMENT S21 averaged one and one-half years before operation, they should be more properly compared with the remainder of Cabot's group. By tie end of the first year conditions in the splenectomized group were as follows: Of thirty- three patients surviving the operation, twenty-four were still improved, three had failed to show improvement or had relapsed to their pre-operative condition, and six had (lied. If postoperative deaths, however, are included, only about half of those whose fate was known were still alive at the end of the first year. From both these points of view, therefore, there are no clear indications as to the value of splenectomy. TABLE LXVII Results Accordinq to Duration op Disease, Based on Ninett-Fivb Cases - Number of cases Poatoperative reaulta Subse- DuratioQ Deaths "X:^ Improved quently Uievi Undor 6 months .... 17 26 36 16 4 4 3 6 13 3 19 6 27 1 9 Six months to 1 year 3 8 Over 2 years 4 The changes in the blood picture after splenectomy are striking and fairly constant. Forty-seven cases are stat^ to have had a distinct postoperative blood crisis (appear- ance of normoblasts, megaloblasts, reticulated er>i;hrocytes, Jolly bodies, etc., in larger quantities) ; and, as statements that the blood cr' j failed to appear are very rare, it is fair to assume that such a phenomenon is at least a fre- quent occurrence. In most of the patients who recovered the stimulation forms soon grew fewer in number and, coincident wtith thd signs of general improvement, the haemoglobin and red-cell count rose. The csolor-index, how- 21 S II III! IIP I NiB ! 322 THE SPLEEN AND AN.EMIA iK ■ ,1 m 4 • ■',! 1 ■' ^w J .- ".--_■ Im^ .. t' •':•»' ever, usually remained high. As might be expected, also, the nucleated forms became fewer or disappear! d ; and yet statements are almost unanimous that the microscopic features of the blood (tendency to macrocytosis, poikilocy- tosis, etc. ) , even in many cases that showed almost nonnal counts, remained suggestive of pernicious ana?mia. Those patients who died within the first six weeks after operation showed eitlier very slight improvement in the blood picture or an actual deterioration ; whereas, death occurring after that period was in many cases preceded by a distinct im- provement in the blood picture, with the usual signs of a remission. Those few patients who up to the present time have continued well after operation must also be taken into ^n- sideration. By the older methods of treatment, Cabot, in the fir'^t edition of " Modern Medicine," stated that six out of 1200 cases after six years of health might be said to have l)een cured. In the second edition this number was reduced to three. May it not develop that a larger per- centage of such " cures " will follow splenectomy, even though the blood picture does not return absolutely to normal ? Another point brought out by study of the blood picture at the time of operation is that if the operation is undertaken before the blood has reached an extreme degree of deterioration, not only is the operative risk lessened, but the impri ement is greater and more lasting. In any case it is wise to precede it with one or more transfusions. The average age of the patients at the time of operation (see Table LXVIII ) was forty-five ; the average duration of the disease at that time, 1.6 years. If the results are analyzed in groups subdivided according to age, it appears (as one would expect) that less favorable results are ob- SPLENECTOMY TREATMENT 823 tained in patients in the sixth and seventh decades. A similar arrangement on the basis of duration of the disease shows tliat the best results are obtained (after the operation has been successfully passed) in those patients who have TABLE LX\'III Results According to Age of Patient, Based on One Hundred and Twenty-One Cases decade Number cases rustniierative ruaults Subse- quently died Deaths Not improved Improved :id 8 23 43 28 9 2 4 5 4 5 1 2 2 5 2 5 17 36 19 1 4th 2 5th 8 6th 6 7th 1 had the disease for less than one year. Sex has not been found to exert any influence on the results. When arranged according to the degree of anaemia at the time of operation, one fact is patent : that the operative risk is much greater in those cases in which hemoglobin is below 20 (see Table LXIX). This is, of course, for TABLE LXIX Results According to Preoperative Degree of AN.«aiiA, Based on On» Hundred and Fourteen Cases Number of cases 9 29 33 43 Postoperative results Subse- HiBmaglnbin Deaths Not im- proved Improved quently died Rplow 20 7 6 2 7 1 2 2 1 21 29 I5elow 30 3 Below 40 7 7 1 29 5 this disease, a lower level even than that of 1,000,000 erythrocytes. Except for this point, on account of the great fluctua- tions in blood counts incident to the disease and following Sii TIIE SPLEEN AND AN.EMIA I - transfusions, it is difficult to estimate whether or not the previous condition of the blood has any marked influence on the result obtained. The impression is gained, however, that the best results follow splenectomy in those cases that are not extremely ana?mic at the time of operation and that have shown considerable fluctuations in the bhwd picture. Information as to the size of the spleen was secured in eighty-nine cases (see Table T.XX). In twenty-eight eases (31 per cent.) the spleen was either small or approxi- ■^ABLE LXX Results According to Size of jspleen, Based on Eiqhty-.nine Cases Number of casea Postoperative results Subn- Siio of spleen Deaths ^^^ »'"" Improved proved quently died Normal or diniinishwl 28 41 20 9 i 5 4 1 1 1 14 32 18 2 9 Considerably enlurgcil 3 mately normal in size. In forty-one cases (46 per cent.) the spleen was slightly enlarged (between 250 and 500 gm.) ; and in twenty cases (23 per cent.) it was consider- ably enlarged. In other words, although seldom palpable before operation, it was distinctly enlarged in over two- thirds of the cases. If the resul's of splenectomy are subdivided according to the size of the spleen, it will be seen that etter postoperative results were obtained in the cases with enlarged spleens. The third gn up in this arrangement, however, is the only one that could be said to have fared better than another as regards the ultimate outcome of the disease. The EtTKCT Produced by Splenectomy. — Whatever tlie cause of the distinct improvement after splenectomy in those patients who sui-vivc the operation, it is obvious SPLENECTOMY TREATMENT 325 that it is not due to the removal of the sole cause of the disease. If Eppinger's theory of thickened arteriole walls in the spleen, with consequent damming back and destruc- tion of red cells in the splenic pulp, were correct, removal of the spleen should indeed cure the disease ; but our studies show that this is not the case. On Eppinger's theorj', also, the characteristic remissions of pernicious ansmia would be difficult to explain. The postoperative blood crisis discussed above, and the subsequent improvement in the blood picture, decrease in urobilin, etc., indicate both that a stimulus has been applied to the bone-marrow and that a source of blood destruction has been removed. Lee's findings of an increased number of platelets after splenectomy would also support the bone- marrow stimulation theory. Hypotheses to explain these phenomena have been unsatisfactory and the relevant ex- perimental evidence often contradictory. Klemperer be- lieves that the bone-marrow activity is induced by removal with the spleen of an inliibiting hormone, but from ex- pt riments in our laboratory we have found ^*° (in normal animals, to be sure) not only that this bone-marrow activity does not occur until after several months have elapsed, but also that fresh splenic extract stimulates ^'^'^ instead of in- hibiting the bone-marrow. The cause for the blood crisis, therefore, must probably be sought elsewhere : perhaps, as has been suggested, in bone-marrow stimulation from metabolic products or from abnonnal constituents of the erythrocytes that are allowed to remain in the blood by the removal of the spleen (Decastello) . It is also difficult to prove that the lessened hemolysis after splenectomy, as sliown by decreased output of urobilin, is actually due to ii ■i ,: ! - * < ■ L ■ 1- i 1 ^-^ ; , •] 1 i 8M THE SPLEEN AND AN^E^HA t. . ► • -It * the absence of the spleen. Neither normal spleens nor those removed at operation in cases of blood disease (Cole- man, Stewart, Robertson) can be shown to possess demon- strable haemol\i;ic activity, and studies of the blood enter- ing and lea^^ng the spleen have also failed to throw light on the supposed ha?molytic function of this organ (Krumb- haar and Musser ^^* ) . Another factor to be considered is changes in the red blood-cells themselves. In one case that I had an oppor- tunity to study (Stewart's), the resistance of the erythro- cytes was distinctly increased after splenectomy, so that this might constitute one of the factors of improvement after operation. A similar increase in the resistance of the erj'throcj-tcs we have found to be the rule ^"^ after the removal of the spleen in normal animals, and it is present in other blood diseases, but has been denied in some cases of pernicious anaemia (Moffitt). Whatever the cause of the improvement, it is highly probable that the subsequent deterioration is due to other related structures taking over the functions of the spleen (hsemolymph-nodes, accessory spleens, Kupffer cells in the liver, and perhaps ordinary l\Tnph-nodes), although here, also, no positive evidence has as yet been produced. Assum- ing that the cause of the disease has not been removed, the logical sequence would be that when these auxiliary organs have suiticiently developed the interrupted course of the disease would be resumed. As to the few patients who up to the present time have continued well after operation, if the curative acti n of splenectomy is denied, one must assume either that the cau-sative factor has for reasons imknown ceased to op* rate, SPLENECTOMY TREATMENT 327 or that the auxiUarj- organs have failed to develop into pernicious activity. INDICATIOKS FOR SPLENECTOMY IN PERNICIOUS An^- MiA. In what types of pernicious ana?mia, then, should splenectomy be undertaken? One of two lines may be followed, and it is as yet too early to say which, if either, is correct. If splenectomy merely induces a remission— and this is at present the opinion of the majority of ob- servers—it should be logical to undertake it only as a last lesort, when all other measures have proved unavailing, and only with the hope of prolonging life. Even under such limitations, however, the procedure has already proved its value, and in several cases moribund patients have been brought back to life cf comparative well-being for many months. Assuming, on the other hand, that an occasional patient may be, for practical purposes, cured of the disease, and giving due weight to the view that greater and longer continued improvement is obtained if the operation is per- formed before the disease has reached its final stage, it would then be advisable to undertake it as soon as possible. Another factor that may prove to be decisive is whether or not increased haemolysis can be proved. In those cases with clinically enlarged spleens, icteroid appearance, and increased urobilin output, without increased resistance of the er\i:hrocytes, the prognosis is distinctly more favorable than in the opposite types. The condition of the bone- marrow is also important, splenectomy being contra-indi- cated if the bone-marrow is persistently aplastic. It has also been a matter of clinical observation that those indi- viduals in whom spinal-cord symptoms have already de- veloped are less apt to be helped by the operation. ml THE SPLEEN AND .VN^MIA 1 itj • ■i ' 1 Summary of Results of Splenectomy in Pernicious An^.mia 1. Of the 153 patients studied, 19.6 per cent, died within six weeks; a distinct improvement in the clinical condition and in the blood picture occurred in 04.7 per cent., and no improvement in 15.7 per cent. 2. The rather hi^h postoperative mortality (practically 20 per cent.) may be due to poor choice of cases in the early series. As a much greater proportion of the more recent cases has survived the operation, the true postopera- tive mortality is probably much less than 20 per cent. 3. Of the individuals who showed improvement shortly after operation — nearly two-thirds of the total number — a large number have failed to maintain this improvement, or have since died in a relapse or from intercurrent disease. 4. Although a few have continued in good condition during the period of observation (over two years), in no case can it be said that a cure has been effected, and the blood of these individuals continues to show many of the characteristic signs of pernicious anaemia. 5. On account of the improvement that follows splenec- tomy, it would appear to be not only a justifiable, but in many cases an advisable, procedure ; but in no case should a cure be promised or the operation undertaken except under the most favorable conditions. 6. The best results are obtained if the operation is pre- ceded by one or more transfusions, and those patients who relapse after operation may still be greatly helped by trans- fusion. Whether or not transfusions would have produced equally good results in the absence of splenectomy is a question that cannot at present be decided. 7. The most favorable results may be expected in indi- 1^ SPLENECTOMY TREATMENT riuuals who have not passed the fifth dvX^ade, in whom the disease has not progressed for more than a year, and who have a relatively good blood pictm-e (that is, an anaemia that is not of too extreme a degree or of the steady, pro- gressive type). Individuals with enlarged spleens have done better than those in whom the spleen was small or of normal size, as have also those suffering from an an«mia characterized by excessive ha?molysis. 8. The opposite of these conditions should be considered as unfavorable factors, as should also the existence of spinal-cord symptoms or the presence of an aplastic bone- marrow. Choice of Time for Operation.— On general prin- ciples it may be said that splenectomy should be under- taken as soon as the diagnosis is definitely settled. In certain instances, however, exceptions must be made. A " crisis of deglobulization " in haemolytic jaundice or a severe hemorrhage from a mucous membrane in Banti's disease would indicate postponement of the operation until the conditions are more favorable. The existence of a pos- sible contributing factor, as indicated, for example, by the finding of a positive Wassermann reaction or of malarial organisms, would naturally postpone operation until the influence of such factors had been, so far as possible, elimi- nated. When the anaemia is severe— and this applies par- ticularly to pernicious anaemia — a series of blood trans- fusions (^ often four or more) should precede operation. If the patient's blood in this way is temporarily enriched, not only is +he operative risk lessened, but more lasting benefit ensues. Good results have been obtained in per- nicious ansemia by the subcutaneous or intraperitoneal injection of splenic extract,*'' and in cases where the so- ■\ 'm Hi m ml i m M pi 1 3S0 THE SPLEEN AND ANAEMIA *W3f': v.-ft- 'i'^ ''.: ■'] "1, called " blood crisis " fails to materialize after splenectomy, indicating a failure of the bone-marrow to respond, this procedure might be tried. It may be said, in summarizing, that splenectomy has already prove ■•■* '• ) t , > ». it !: HI CHAPTER XV THE SURGICAL TREATMENT OF LESIONS OF THE SPLEEN In the first century Pliny made the observation, of merely historical interest to us, that sometimes the spleen is a peculiar hindrance to runners, so that it is burned awa,y from those who are incommwled by it; also it has been said that the Romans removed the spleen from some of their gladiators to give them greater endurance in contests. It is, however, a far cry from that day to the i neteenth cen- tury, and it was only toward 1860, in the latter part of this century, that splenectomy was admitted to the roll of ac- cepted and approved surgical procedures, while the most important contributions to the surgery of the spleen have been offered during the past five years. The lesions for the treatment of which the surgeon is called upon to operate are varied and numerous. Be- ginning with injuries, such as wounds and rupture, there were added wandering spleen, malarial spleen, abscess of the spleen, Banti's disease, the tAvo types of haemolytic icterus, and, finally, pernicious anaemia. Gtimhot and Stab Wounds.— The spleen enjoys a measure of protection from its position beneath the left costal arch, but occasionally it is injured by bullet and stab wounds. As with all injuries to the spleen, hemorrhage is at once profuse and alarming and, unless surgical relief is immef^ately available, the patient dies of shock or col- lapse. Should the splenic arterj' itself or the vein be punc- tured with or without penetration of the spleen, hemor- 333 !i 334 THE SPLEEN ASB .SJV^MIA i ' I phage will be no profuse that the life is lost before surgical treatment can be applied. The diagnosis can be only conjectural, but suspicion should be aroused by the con- :ititutional signs of profuse hemorrhage, with perhaps dul- TABLE LXXI Results or the Tbe.-tment of 52 Cases of Stab Wodnd bv Vabiocs Methods i^ptsratiun Total csacs 14 24 12 2 52 Recovered Died 9 22 10 1 5 2 2 1 Suture Treatment not siiecified Totals 42 10 19.49 percent. TABLE LXXII Rmultb of the Treatment of 82 Cases of Gunghot Wound bt Vabioub Methods Operation Splenectomy Tamponage Suture Partial excision Ciiuteriz;ition Trratment not specified Diuth previous to opera- tion Wounds not detected at the operation Totals Total caaea ReooTere'i Died 40 25 15 18 10 8 9 5 4 1 1 — 1 — 1 6 1 5 1 — 1 6 — 6 82 42 40 48.2 per cent. ness on percussion in the left flank or when the site or direc- tion of the wound points to the spleen. Immediate opera- tion is imperative, and the treatment of the injured organ will depend somewhat upon the extent and character of the wound. Bullet and stab wounds may be efTectively dealt with by suture, but when there is more or less ex- SURGICAL TREATMENT OF LESIONS 3S5 tensive laceration, when hemorrhage cannot be otherwise controlled, or wlK>n the vessels of the pedicle are involved, splenertomy is clearly indicated. In superficial wounds the insertion of a free epiploon has been used advantage- ously for the control of hemorrhage. Of ninety-nine cases of wounds of the spleen, 50 per cent, of the s'xty-one cases of gunshot wounds and 80 per cent, of the thirty-e\ght stab wounds t^'v vered (Finkelstein ""). Rupture of the Spleen. — Rupture may be designated as traumatic or spontaneous — the former the result of trauma upon a healthy organ, the latter implying an organ already the seat of a pathologic lesion, often of an inflam- matory nature, be it malarial, typhoidal, or tuberculous. Rupture of the spleen follows accidents not unlike those which would cause a rupture of the kidney, but in the ma- jority of instances the kidney will rupture and the spleen escape. The passage over the body of wagon wheels, crushes beneath heavy objects, automobile and railroad accidents, the kick of horse or man, falling upon angular objects, or falls from a distance may be numbered among the possible causer., and together with the ruptured sple-.-', one often finds fracture of one or more ribs. As with gunshot or stab wounds, rupture of the spleen can he surmised only by the character of the injury, by the evidence of an overwhelming hemorrhage with signs of profound shock or collapse, by the presence of dulness in the flank, accompanied in the occasional case by fracture of the ribs in the left side. While there are instances of spontaneous recovery, the majority of cases of rupture of the spleen would be rapidly fatal without surgical inter- ference. Imnip 1 i f te operation should be the rule whenever rupture J tV- .een is suspected, and the measure to be adopted will depend upon the extent and depth of the 'A :j ru .i:l 'ill 336 THE SPLEEN AND ANEMIA i^' (.■: , i ••n»' wound. If the latter be superficial, hamostasis may be effected by simple suture or, as Kirschner*'* suggested, by the envelopment of the organ with fascia. At all events, one should give preference to conservative practice, if not thereby adding to the risk of operation. There may be many instances, particularly after massive hemorrhages, when the more radical operation, splenectomy, may be done more expeditiously than one or the other of the conserva- tive methods and should therefore be the method of choice. Mortality of Rupture of the Spleen table lxxiii I. MiCHELSSON Operation Cases Hecovered Died Splenectomy 254 24 10 10 166 19 8 6 88 5 2 4 '^r;iiTii;M)ii!iirp Total" 298 199 09 33.2 per cent. TABLE LXXIV n. LlTERATUUE OF THE La8T FiVE YeARS Upeiatiun Cases Hecovered 55 8 4 Died 13 6H 11 4 Suture Totals 83 67 16 19.3 JKT rent. Of 208 cases in Michelsson's -"" collection, treated in various ways (Table LXXIII ) , the mortality was 33.2 per cent., while, excluding all but those operated upon from 1910 to 1915, the mortality has fallen to 19.8 per cent., or almost one-half (Table LXXIV.) SURGICAL TREATMENT OF LESIONS 337 The prognosis of spontaneous rupture of the spleen is influenced by the nature of the patholooric process. If, prior to the rupture, the patient's condition have been de- preciated by a more or less virulent and prolonged infec- tion, as of malaria or typhoid fever, the likelihood of siu-- vival after an m-erwhelming hemorrhage is naturally less than when rupture occurs in an other^vise healthy subject or organ. Abscess. — Because of its vascularity and the slowness of its blood current tlie spleen is frequently the site of infec- tion, either by the microorganisms themselves or by infec- tious emboli. Thus we have the secondarj-^ infection of the spleen in the acute fevers, such as typhoid and typhus, or in septicjemia, and the primary involvement of malaria, tuber- culosis, syphilis, and cchinococcus disease. An abscess of the spleen may develop secondarily to any focus of infection and occasionally may result from a contiguous infection, as from perforation of the stomach or subphrenic abscess. Because of the greater frecjuency of malaria, dysenterj', and typhoid fever, in tropical countries, and of the peculiar susceptibility of the spleen to these infections, abscess of the spleen is of greater incidence in the warm and tropical climati , than in the temperate zone. The diagnosis of splenic abscess is frequently not made until the infection has extended beyond the capsular limits. A palpable spleen or one enlarged and tender may be of little significance and the symptoms may vary according to whether the abscess be located in the upper or lower pole. If in the lower pole, an enlarged and painful swelling may be felt; if in the upper pole, the most suggestive signs are those of diaphragmatic invasion or diaphragmatic pleurisy. ^\Tiile there may be theoretical objections to explorntory 22 ] !,t:l,,r fWh I;!; 338 THE SPLEEN AND ANAEMIA aspiration, it seems to have been practiced frequently with- out untoward effects an,. *■ 840 THE SPLEEN AND AN.iiMLV operation should be reserved for those cases in which in- vahdisni is pronounced and where there are no serious changes in liver or kidneys. Not only does improvement follow because of the elimination of an infective focus, but the removal of a weighty organ relieves the patient of dis- comfort, restores to normal the relation of the abdominal organs, and removes the hindrance to their circulation. "Whether splenectomy should be encouraged in the later stages of malaria with ascites, a clinical state not unhke the third stage of Banti's disease, is open to question. Finkelstein,' '" who has had an unusual experience in a malarial chniate, considers v operation advisable when the hanioglobin is not less than thirty or forty per cent., when the red blood-corpuscles are not less than 2,000,000, when there is no (x-dcma of the lower limbs, when there is no parenchymatous lesion of the ' .idneys, when the patient is able to move about. Unfortunately, there are no means of determining serious degeneration of the liver, since the presence of a serious hepatic or renal lesion spells disaster after an operation. In the sur\'iving patients the ascites disappears, and in two of Finkelstein's cases the fluid did not reaccumulate and the condition of the patient left noth- ing to be desired. The operative problems in malaria are virtually those of any disease in which there may be a perisplenitis and consequent adhesions. Tuberculous Spleen.— Tuhercnlosis of the spleen is secondarv to a primary' lesion elsewhere. Up to 1909, Fischer "« had been abl ? to collect but twelve cases, and since that time there have been but few additions. The operation has usually been performetl because of an en- larged and wandering spleen and the presence of tuber- culosis, suspected and not discovered until examination of the sjx imen upon its removal. SURGICAL TREATMENT OF LESIONS 341 Syphilis of the Spleen.— SiAenectomy has been per- formed for both the fruiainatous and non-guinmatous sple- nomegaUes. Giffin '*' speaks of the advantageous removal of the spleen in the non-gmnmatous splenomegaly asso- ciated with syphilis. The spleen has been found to contain spirochete, and after its removal there has been a rapid diminution in the size of a previously enlarged liver. Fur- thermore, whereas before the operation the Wassermann reaction continued positive despite appropriate treatment, after removal the reactions were returned as negative. This relationship of the spleen to the continuation of syphi- lis, the spleen sen-ing as a favorable medium for the propa- gation of the organism, is a recent observation and is preg- nant with possibilities. Wandering Spleen.— The spleen, for various causes, may make wider excursions in the abdominal cavity than any other organ. It has been found in the sac of an ingui- nal hernia and often in the pelvis, where it has been mis- taken for uterine or ovarian tumor. It may change its position, as the patient moves from side to side, when it usually gravitates to the lowest point; it may, however, float upwards on the intestines; it may become fixed by dhesion in an abnormal position, a condition to which is applied the term " dislocated spleen." In most instances the causes of wandering spleen are acquired, although, in exceptional instances, congeni- tal elongation of the mesentery may permit of a wide excursion. The more common causes are accessions in weight, elongation of the pedicle and ligaments, and re- laxation of the abdominal walls often associated with vis- ceroptosis (Glenard's disease). Consequent upon this wider range of motion, which as a matter of fact occurs ami I 4 ■'^■, 342 THE SPLEEN AND ANAEMIA M i •' 'I ;,! ,• J- M. most often in the malarial spleen, certain complications may develop, chief among wliich are engorgement and twists of the pedicle. The former is a gradual process and may add considerably to the weight of the spleen, to the weight of which, under normal circumstances, the blood contributes forty per cent. Twists or rotation of the pedi- cle will, of course, aggravate the engorgement and may in turn lead to hemorrhages, gangrene, and even peritonitis. The rotation may vary from 180 dtgrees to 360 degrees, and upon the suddenness and degree of rotation will de- pend the development of an acute or chronic symptom- complex. Sudden rotation of the splenic pedicle may de- termine an abdominal catastrophe, mistaken often for in- testinal obstruction or peritonitis, marked by tenderness, abdominal rigidity, vomiting, distention, and by the pres- ence of an enlarged, tender, and palpable swelling. In chronic cases there is not only the splenic enlargement, but, by virtue of its weight and displacement, the spleen may disturb the function of other abdominal organs. Epi- gastric distress, nausea and vomiting may result from dragging upon the stomach, and a train of symptoms from pressure upon the uterus and its adnexa. Not only may the stomach be dragged out of position, but the tail of the pancreas may be elongated, and the uterus displaced or even prolapsed. Theoretically, the ideal treatment of wandering spleen is splenopexy. This, however, presumes the presence of a nonr al organ, a condition rarely found. Since in the vast majority of cases wandering of the spleen is primarily due to splenomegaly of one variety or another, often a malarial spleen, splenectomy is the most satisfactory and most logi- cal treatment. ■'*'-(.• 'V'-'-S,- k" '.->jsA^.«, •«5i-. SURGICAL TREATMENT OF LESIONS 343 Tumors and Cysts.— The spleen may be the seat of primary or secondary growths: of the primar>' growths the adenoma, fibroma, and lymphoma have been found, but not on the operating table. The secondary malignant tu- mors of the spleen are not of practical moment, but a limited number of primary sarcomata have been disclosed at exploratory operations and removed. They take their origin from the capsule or trabecula, the lymphoid struc- ture, and the endothelial cells. One may suspect the pres- ence of a sarcoma of the spleen should there be an enlarged and tender organ, of nodular surface and firm consistency, with palpable notches and a rapid increase in size. Provid- ing metastases are not already detected, the organ should be removed. Cysts of the spleen may be large or small, of parasitic or non-parasitic origin. The parasitic or hydatid cyst is quite the most coaimon, and its contents display the fea- tures characteristic of hydatid cysts elsewhere. It is inter- esting to note, hoAvcver, that they usually originate in the upper part of the organ, and, as they increase in size, they may interfere, by pressure upward upon the diaphragm, with the action of both lungs and heart. When feasible, splenectomy is the operation of choice, but when the re- moval of the organ seems prohibitive from the standpoint of safety, the operator must be content with incision and drainage. Non-parasitic cysts of the spleen include the dermoid, serous, blood and lymph cysts. The first mentioned is so rare as not to be of practical moment, while the others are often so small as to pass unrecognized. The blood cyst owes its origin to hemorrhage, either parenchymatous or subcapsular, and is often the result of an injury. A history MICROCOPY RESOLUTION TEST CHART ANSI and ISO TEST CHART No 2 1.0 I.I 1.25 t m ;■ m III 2.5 ||| 2.2 1 2.0 1.8 1.6 ^ -j PPLIED irvt^GE inc ^^ "bbi Lost Mom Stf«t '-S Rochester. New rork 14609 uSA S C^'6) *e? - 0300 - Phone = ('16) 288 - 5989 - Fo» 344 THE SPLEEN AND ANAEMIA . ♦•■• "3' of trauma followed by the sudden appearance of a splenic- enlargement is a significant diagnostic feature. Other diagnostic signs include a tumor of the left hypochondriimi of a cystic nature, pain and tenderness, embarrassment of respiration if the cyst be of large dimensions, and, more specifically, a creaking sensation communicated to the ex- amining hand, as with respiration the roughened surface of the cyst and the abdominal wall come in contact. Splenomegali/. — Of the pathological conditions of the spleen in the treatment of which splenectomy is an accepted method of procedure there remain to be considered the splenomegalies with ana?mia. The classification adapted in this monograph recognizes the following types: 1. Gaucher's disease (large-celled splenomegaly). 2. Banti's disease. 3. von Jaksch's disease (pseudoleukfemia infantum). 4. Chauff ard-Minkowski or congenital form of hitmo- lytic jaundice. 5. Hayem-Widal or acquired form of ha?molji;ic jaundice. 6. Pernicious anosmia. These several types of splenomegaly have been treated in eoctenso in earlier chapters; suffice it, at this juncture, to speak briefly of their surgical aspects. As a general fundamental principle, the best results may be expected from splenectomy in those diseases in which there is evi- dence of increased blood destruction. In this category would fall hfrmolytic jaundice and some cases of pernicious ansv- mia. Good results have also been obtained in Banti's dis- ease and in the few cases of Gaucher's and von Jaksch's disease that have been saidicd. but in accepting these as positive indications for splenectomy the proviso must be SURGICAL TREATMENT OF LESIONS 345 made that promising results can be expected in Banti's disease only when the operation is performed in the early stage. The secondary changes which take place in the liver of the patient with Banti's disease are not materially influ- enced by the removal of the spleen, and when the liver is obviously enlarged the propriety of the operation should be seriously questioned. In the list of doubtful indications for splenectomy I place pernicious ansmia. The surgery of pernicious anne- mia at this wTiting is suh judice. As yet there have been no recoveries following splenectomy, although there have been many instances of prolongation of life, general better- ment of the patient's condition, and in lengthening of the periods of remission. The operation, therefore, is not with- out influence upon the course of the disease and may be practiced in selected cases, with the knowledge, on the part of the patient, that the operation is in the nature of a pal- liative procedure and not in any sense cvu-ative. Further- more, it must be borne in mind that in some cases repeated transfusions may be almost as eff"ective as splenectomy, and that even when the latter is practiced transfusions may be required. The Removal of the Spleen Anatomical Considerations.— The splenic artery, itself a branch of the coeliac axis, divides into seven or more branches which supply the spleen, the pancreas, and the stomach. To the latter is given off the vasa brevia, passing l)etween the layers of the gastrosplenic omentum to be dis- tributed to the great cul-de-sac, and the left gastro-cpiploic, coursing between the layers of the great omentum to the greater curvabire. In the operation of splenectomy the splenic artery is exposed for ligation in the pedicle, but I m .ar iea^id^caKtr: K , * - \.%J 346 THE SPLEEN AND AN.EML\ when ligation of the arter>' is to be substituted for splenec- tomy, tlie artery may be exposed, according to Gerster, through an opening in the lesser omentum just above the lesser cur%'ature of the stomach. The first two inches of the artery lie just beneath the posterior parietal peri- toneum, and may be brought nearer the level of the anterior abdominal incision by placing an Edebohl cushion beneath the patient's back. The splenic vein, or splenic veins— for there are often more than one in the pedicle — unite with the superior mesenteric to form the portal vein, so that a thrombus of the ' 'tter seriously interferes with the splenic circulation. The pedicle of the spleen varies in length. The shorter the pedicle the more difficult are all the stages of splenectomy, including the delivery of the organ and the ligation of its vessels. It is comprised of connective tissue and fat, occasionally of lymphatic nodes, some accessory spleens, and the tail of the pancreas ( Plate X ) , and through the pedicle pass the splenic artery, and one or more veins. The smaller vessels to the stomach lie anterior to the ped- icle. In addition to the splenic artery and veins which enter the spleen at the hilum. there is a leash of vessels which enter the spleen through what Richards calls a sec- ondary hilum near the lower pole. These vessels are car- ried in a fold of peritoneum that is reflected on the surface of the splenic flexure of the colon. The capsule of the spleen is so firmly adherent to the surface that attempts at separation would be Jittended with laceration of the pulp; therefore a subcapsular sple- nectomy is not feasible. A knowledge of the relationship of the spleen to the contiguous structures is of practical moment. The supe- rior pole is in dose relation with the diaphragm and the I'l.All. .\ The ri'lutiuiislnp ■■( the tiul fi Ilu imrn ii;i.-. tu llie l..p^tl'lIur a»iifi-l "t Ihf pttlicle ri Ai i: M i. * "h?". llii- piTitciiiiil ]ir..liii]f:ili.iii l..l\»i'iri 111!' .splicii Mini llii' h|.liliH' lliMiri' i^f tlif i-oliili, wliicli unj!.! Ill' iliMiliil lirliirc I 111' iiiKiiii I nil 111 i"iii|il< 1< l.\ niubilueil. SURGICAL TREATMENT OF LESIONS 347 lower pole through a peritoneal reflexion with the splenic flexures of the colon. In the pancreatic notch behind the hilum lies the tail of the pancreas, and its relation to the structures of the pedicle has already been mentioned. This is a matter of practical consideration, since, unless care be taken to avoid it, a portion of the tail may be amputated in division of the pedicle after ligation. This accident has occurred more than once. So close does the greater curva- ture of the stomach lie to the pedicle of the spleen that a portion of the wall has been inadvertently excised, in one instance with a fatal result. The spleen is maintained in its position under normal conditions by a number of Ugaments, by the gastrosplemc ligament, the lienorenal, by an occasional band to the lower pole derived from the phrenocolic ligament (Plate XI), and by its pedicle. Under abnormal conditions— and it is mostly under these that splenectomy must be performed— the ligaments above mentioned are fortified by adhesions of such density and vascularity that they may make the operation, if not impossible, at least surrounded with many difficulties. The most troublesome adhesions are those from the outer surface and upper pole to the diaphragm, but there may be others to the stomach, large bowel, and parietal peritoneum. W. J. Mayo calls attention to the vascular connections in the deeper portions of the gas- trosplenic omentum, which pass inward and backward to anastomose with vessels along the spine and crux of the diaphragm. These vascular connections must be divided before the spleen can be delivered. Blood Tramfusion.—B\ood transfusion frequently may be called for, either in the preparation for operation or in the after-treatment of the splenectomized patient. Fol- 4 t i }% ? 'i lill'.'ll fir ^^¥^- ; ) -.-■;->/' '-^, m 1 ''^<7^- 3 ' j. ^^v^% 1 >g;^/ 1 • * '*_.^*^v -_''l - J ■ •♦ ■■■ ^^^^^^^H ■■'■i'' WM ,. J.. iPI 348 THE SPLEEN AND AN/EMLV lowing the alarming hemorrhages of wounds and rupture of the spleen, the transfusion of blood, if in the emergency a suitable donor can be found, may be a life-saving remedy. In such situations the transfusion is performed immediately after the operation. When splenectomy is contemplated in the chronic case, be it Banti's disease, ha?molytic icterus, splenic anaemia, or pernicious ana-mia, transfusion plays a verj' important role in the preparation of the patient for operation. Should the ha'moglobin be below thirty or forty per cent., the patient should be transfused forty- eight hours before the time set for the splenectomy and again after the operation, should the latter be attended with much loss of blood. It is, however, not only with reference to the blood picture that inquiry be made into the patient's condition. A careful, systematic examination of the car- diovascular and renal systems should be made to determine whether, irrespective of the anaemia, the patient be a good operative risk. It may well be in certain cases that a patient with 2,000,000 red blood-corpuscles may be a better hazard than one with 3,000,000 corpuscles, but with other handicaps. It is in pernicious anaemia, ho vever, that transfusion plays the most important role, not only in the preparation of the patient for the operation — the phase of the subject with which we are most concerned — but also in preventing relapses and prolonging the peri(Kl of remission. Paren- thetically it may be said that if no improvement follow transfusion little should be expected to follow a splenec- tomy. The effects of one may be said to forecast the effects of the other. Percy, whose large experience compels a hear- ing, has evolved a systematic method of managing the transfusion problem to which I attribute, in part at least, SURGICAL TREATMENT OF LESIONS 349 his low mortality. He has adopted what he styles the step-ladder method of preparing his patients by transfu- sion ; he prefers whole blood to defibrinated blood or blood treated with sodium citrate; the average nuinl)er of trans- fusions for each patient varies from three to five, the aver- age amount of blood is 640 c.c, and the average intei-val between the time of the first transfusion and the splenec- tomy is twenty days. In almost every case improvement follows immediately after the first transfusion, and con- tinues by " step-ladder " progression with successive trans- fusions until the patient is in a state prepared for opera- tion. At the conclusion of the operation, and while still on the table, the patient is transfused again. According to Minot, the most auspicious time for the splenectomy is from four to ten days after the transfusion, when the Howell-JoUy bodies appear in greatest number, together with a rise in the leucocyte count and an increase in the blood-platelets and reticulated cells. The effect of transfusion upon the subject of pernicious ansmia varies somewhat with the stage of the disease. In the early cases transfusion may give a remission of several months and seem almost as beneficial and enduring in its effects as splenectomy. In the late cases the effect is only transitory, a matter, perhaps, of two or three weeks. But, while the transitory effect is fully recognized, transfusion should not be discarded, since it is in itself a humane meas- ure, giving to the patient a sense of bon faisance, increas- ing the appetite and general l>odily comfort, and unques- tionably prolonging life. The obligation of the surgeon in the selection of a suit- able donor need only be mentioned. That vigilance is required to protect the patient from transmissible diseases u :3i S60 THE SPLEEN AND ANAEMIA may be inferred from the transmission of syphilis in one reported case where the donor, denying exposure to infec- tion, had the primary lesion at the time of the transfusion. In the selection of a donor the matter of blood compatibility must be investigated with great care in order to preclude even the remotest possibility of hemolysis or agglutination. Technic of Splenectomtj.—The removal of the spleen under certain conditions is an operation devoid of any peculiar difficulties. When the spleen is not enlarged, or when the adhesions are few and so readily divided that de- livery of the organ is a simple manoeuvre, splenectomy might be compared with an uncomplicated nephrectomy. The problems are somewhat similar: the manner of ap- proach (that is, the incision), the isolation of the organ and its delivery, the management of the pedicle, and the after-care of the wound. But the difficulties of the difficult splenectomy quite exceed the difficulties of the difficult nephrectomy, and the chief point of difference between the two lies in "the presence of dense vascular adhesions with which the spleen may be surrounded. For the preferable line of incision one must choose a midline incision, be- tween an incision at the junction of the middle or outer third of the rectus and an incision in the linea semiluna- ris. Each of these incisions may have its advantages under certain conditions, or perhaps no one should be used to the exclusion of others. Personally, I prefer a left-sided Bevan incision, in the outer rather than the inner half of the rectus (Plate XII), as advocated by Balfour.'' The upper limb of this incision is projected across the rectus an inch below the costal margin, and the flap thus formed, when grasped with a pair of forceps, may serve as a retractor. m ! I'l \il Ml ■il I ~i»| I.cft-Miiied H«-vati im-isidii fur ..plcnectniny. <2 !.»J SURGICAL TREATaIENT OF LESIONS 351 Owen Richards '•' and iVly Bey, whose experience has been chiefly with large and adherent spleens of tlie Egypt- ian splenomegaly, prefer a vertical incision, stretching half-way between the costal angle, the lower margin of the thorax, about two or three inches from the median line, and extending downward a distance of six or eight inches. If the incision be in the midline, access to the outer surface of the spleen is not adequate, and if more external than the incision above described, the costal margin prevents the prolongation of the incision high enough to give access to the vault of the diaphragm. With this left rectus incision Richards has but once had to add a transverse cut in the rectus to give additional room for manipulation. Percy ''"' has quite recently adopted a midline incision and claims for it advantages over those nearer the outer margin of the rectus. No doubt he has been influenced in his selection of a midline incision by his practice of thoroughly exploring the abdomen, particularly the region of the appendix and gall-blatlder, in search for an in- fective focus as having some bearing upon the pathogenesis of pernicious anaemia. However this may be, he has found an associated lesion of the gall-bladder or appendix in many of his cases. In a series of twenty-four splenectomies he had performed twenty-one combined operations, seven- teen splenectomies, appendectomies, and cholecystectomies; three splenectomies and appendectomies, and one splenec- tomy with the removal of carious teeth. After exploring the abdomen with particular attention to the condition of the gall-bladder, biliary passages, and the liver itself, the operator proceeds systematically to prepare the spleen for delivery, directing his atttention first to freeing its upper pole and outer surface. To accom- V M • • i 352 THE SPLEEN AND ANiEAHA i,, * i "i' plish this, the right hand is inserted in the Lf t subdiaphrag- matic space and by blunt dissection with the fingers the adhesions are separated as close to the spleen as possible (Plate XIII). This step of the operation is perhaps the most difficult, because the field is not within view and hemor- rhage may be profuse. It may be necessarj-, should the adliesions be dense, to double-clamp and divide them with scissors. At this stage hemorrhage may be temporari'y con- trolled by the use of a hot pack (Plate XIV), postponing until later ligation of those points still bleeding after the spleen has been removed. The hot pack, a feature of AV. J. JVIayo's technique, serves a two-fold purpose: permanently contn)lling a number of the bleeding points, especially those of venous origin, and, secondly, serving as a support for the subsequent manipulation and mobilization of the spleen. After the adhesions to the upoer and outer sm*- faces are free and the space tamponed, the lower pole is drawn up and the lienorenal ligament with its vessels divided between two ligatures. Before the pedicle can be satisfactorily dealt with there remains to be divided the gastrosplenic omentum, between the layers of which pass the vasa brevia on their way to the stomach. Ihe close relationship of the stomach to the spleen must be borne in mind at this juncture, since in the division and ligation of the gastrosplenic ligament the stomach may be inadvert- ently opened. Freed from all attachments save the pedicle, the spleen is now mobilized cautiously until the pedicle is exposed. To accomplish this an assistant supports the lower pole, and the operator, with his right hand upon the upper pole and his left depressing the outer margin of the abdominal incision, by gentle traction and pressure mandouvres the spleen through the abdominal wound. The i'l.Aii; xiii :i Kir.-,! ^Iii, in tin. irml.iii/Mt,..n .if thr ^plr.i, W.th ill.' ritrhl Iniii.I Hi- .r .'il ..r MlKiriiliB IliP ii.lh.-si.ins 1" tw.Tii thr .■.ii|MTi..r Miif:i.'i- ..( llii- .splci'ii mti.I the .liniilinigni. I'l All; XIV \ ■'" ""■"' l'l''''ll">.' .ift.r nH,l,ili^;,ti,i„ , ,f i1m> ,,,]■.■„ ;, l:,,l,- l:ll, ,,,,,„ ,,f t.„,l,r. „,„I,C ,.lH in lint Will. I. L- nil r... hi. 1.1 11,1.1 I III Irf I In I lii.iiiliHiiii I'l All; \v Tr:\oti"iiiiniiEati<>ii iiftiT is.'I:i1ion -.f tlip vr^^vU nf tlir , r.VuAv V.y bhmt n'tnuvi.l <'f f;it nil. i %M\ ml ii' L 9 I'l Ml. N\ I ; » • « t* "1' Tlii> .Iiiul>li'-cliiii.|i iiiilli.i.; f .li':i)iin! with till- iieilii'Io. SURGICAL TRFATMENT OF LESIONS 353 difficulty in delivery has been said to be in direct pro- portion to the lirnuiess of the adhesions and sometimes in inverse proportion to the size of the spleen. When the spleen is large and weighty sufficient support must be ap- plied to prevent undue traction and laceration of the struc- tures of the pedicle. The most important, though not always the most diffi- cult, step of splenectomy is the treatment of the pedicle. Grave hemorrhage may attend the proce^ ^ of ligation should the ligature slip and the vessel retract, or, after the patient has been returned to bed, should an insecure liga- ture become dislodged. As a general principle, it is a safer plan to ligate the pedicle in sections as the vessels pre- sent themselves (Plate XV), although when the pedicle is short it may be necessary to apply clamps and ligate en masse. By blunt dissection of the peritoneal covering and connective tissue each vessel is isolated for an inch or more and di\ ided between two ligatures, the distal ligature being tied close to the spleen. The ligatures should be tied as far apart as possible and the vessel divided near the spleen so as to leave a long stump centralward. There are usually two or more veins to be dealt with and one or more arteries, according to whether t!^.e artery divide before it enters the hilum. Theoretically the art ry should be tied first in order that the spleen may at least partially empty itself of its blood content. Fractional ligation of the pedicle must be discarded for ligation en masse when the pedicle is too short. Very much as in nephrectomies, the clamp is used when the pedicle is so short that the vessels cannot be easily exposed. Two curved rubber-covered forceps are applied to the ped- icle (Plate XVI), three-quarters of an inch apart, and the 23 I'.i' li.! iliir f \. ' iltv m S54 THE SPLEKN AND ANiEMIA i.:> \-J-. ■ , ) ''|b. c) HiSEl^B'' A- |&j^S|^Bt "H I^^^^^H;. i-i' ■■-'"^Bf l.4i '.^_^i^:^'t . :^^P Ji 1-' fl^ Jl spleen cut away without regard to back bleeding (Mayo), but after the splenic artery has been tied or clamped bleed- ing from the spleen itself is no great loss to the patient, although, as graphically described by Richards, this back bleeding is " mussy and demoralizing." A catgut ligature is thrown about the pedicle as the proximal clamp is loosened and the ligature tied in the compressed area. The distal pair of forceps is used to steady the pedicle while the proximal ligature is tied, and for further security a second ligature is employed. In the application of the clamps by this methotl the operator is again cautioned not to include the wall of the stomach or the tail of the pan- creas. After the pedicle has been ligated and the spleen removed the proximal stump should be given a final in- spection to make sure of the security of the ligature. If there still remain oozing points, these should be taken care of, and should the stump of the pedicle be broad it may be overrun with a fine, continuous catgut suture or covered with omentum. Gerster believes that ligation of the arterial supply at points more accessible than the deeply-situated splenic pedicle will greatly facilitate matters in difficult opera- ations. These points are the splenic artery close to the coeliac axis and the left gastro-epiploic. where it reaches the stomach wall. The coeliac axis is exposed and the sple- nic arter\' ligated through an opening in the lesser omen- tum ; the left gastro-epiploic is ligated just before it begins to send off branches to the arteries and posterior surfaces. With the arterial stre ..ns under control, Gerster feels a greater sense of security in dealing with the vessels of the pedicle should difficulty be encountered or accidents hap- pen. After the vessels of the pedicle have been dealt with, SURGICAL TREATMENT OF LESIOxNS 855 the preliminary' ligatures of the splenic and left gastro- epiploic artery, if only temporary, may be released, so that if any arterial branches in the pedicle have been over- looked they may now be tied. There is no serious objec- tion, according to Cierster, to allowing the ligatures to re- main. While the pancreatic and vasa brevia branches of the splenic artery are shut off from their direct arterial source, the pancreas and stomach wall still have an abun- dant arterial su^^ply through other branches and anasto- mosing relationships. This suggestion of temporary dis- tant ligation of the vessels may prove of sen^ice in excep- tional cases, but so far as I know it has not as yet been put into actual practice. The pedicle divided and the spleen removed, the hot pack is slowly withdrawn and an inspection made for points of hemorrhage from divided adhesions. These are best controlled with mounted ligatures. With htemostasis satisfactorily established, the operation is concluded by closure of the splenic space. This Mayo regards as ex- ceedingly important and introduces what he terms a snaking suture, as follows: " With catgut on a small curved needle, the raw space begimiing at the tied splenic vessels is closed it contains after removal is practically the same m ^'* WhilTthe sources of hemorrhage are many, the im- pression must not be gained that a splenectomy is usually Attended with the loss of a considerable quantity of blood, n h atence of adhesions I have completed the open^^on with the loss of not more than two teaspoonfuls of blood Tnd though the potential sources of hemorrhage are numerous with careful attention to methods of preven- S^n Tnd to methods of control, even in difficult cases, the SURGKWL TREATMENT OF LESIONS 357 have had 'III loss of blood may be within reasonable limits, no experience witli the preoperative administration of adrenalin, which is said to cause marked shrinkage of the organs, but have been content with the effect of normal saline solution, intravenously or with blood transfusion, when the amount of blood lost at the operation might retanl the patient's convalescence. In cases of pernicious ana'mia Percy ^** routinely transfuses the patient while still on the operating table. As complications of splenectomy, apart from hemor- rhage, should be mentioned injuries to adjacent viscera. Mention has been made of the proximity of the stomach and of its occasional injury dm-ing splenectomies and of the intimate relationship of the tail of the pancreas to the pedicle and hilum. Even when a portion of the tiil of the pancreas has been included in the ligature of the pedicle, no seriou. '•onsequences, such as fat necrosis from the pan- creatic secretion, have been reported. The operative sequelae of splenectomy are varied. With the exception of shock and collapse, as in cases of grave hemorrhage, the most frequent complication is bronchitis or pneumonia. The susceptibility of the splenectomized patient to infection is a factor to be reckoned with, but this alone does not explain why the lung should be the site of election for the coincident infection. Nor can the relative frequency of pneumonia be attributed to a transitory para- lysis of the diaphragm, because these pneumonias develop on the right side as well as the left. Whatever may be the cause, the fact remains that pneumonia is a common of m- plication ; and in one of my cases, a splenectomy for Banti's disease, the patient developed after the pneumonia a pneu- mococcic peritonitis, from both of which, however, she I Ml I ,ij,.,i! ) :] ■■■I "^i jjg THE SPLEEN AND AN.EML\ m.de an excellent recovery. Of the causes of death other ^:iXe aUributable to the an.nna, I n.ay -n.on^oc nain hemorrhage, pneumonia, suppression of unne, niesen S' thrombosis, portal thrombosis, and acute ddatat.on of ^''TCoperative risks of splenectomy must of necessity the mortality. As our knowledge of the "'« =" operation has developed as ou. J^^JJ^™ ™ .ta^Xsion atoJ'jaX series of /iaWstei...' who^eo^ * h^^ mortality for 66 cases ro,n «0^^ »/»J»;;^ J ».th a Carstens collected a screes ol J spleneelomies mortality of 27.4 per cent. In » '"''■^ "'J ^^ ^^,^ jeath- i„ malarial suhjeets, ^-^ -f,fXfl; operation ^^'^WnThas'lUntdnld "trially may he inferred can be and has oeen icuu ^^.^^^^p*; with pernicious from the reports of more recent «P r"^«J*/a to 8 per anemia when the -nortahl^rat^haVen educ ^^ ^ P ^ cent. This has heen made poss.me ny -^i^ious standin, of the prohlems of *e o^-^^™;^ ^^t Jhe selec- emplo^-ment of transfusion, and greater tion of cases. BIBLIOGRAPHY il: ^r Nil' . I BIBLIOGRAPHY •Abderhalden, E.: Die Bczieliung dcs Eisene zur Blutbildung. Zeitschr. f. Biol., 1900, xxi, *87. R. A. : Das Vcrhatcn dw Harneiscns oei Hypcrglobuhe. 'Abeles, Zeitschr. f. klin. Med., 1906, lix, 510 Achahd. C, Foix, C, and Salin, H. : Sur le pouvoir humoly- tique dv I'cxtrait dc rate-. Compt. llend. Soc de Biol., 1912, Ixxii, 394. 'Adelmank, J ah iren. G.: Die Wandlungcn der Splenectomie seit .'JO Arch. f. khn. Chir., 1887, xxxvi, 4ii2. „.„., .... Die sogcnannte Bantischc Krankheit. Dcut. mod. VVoch., 1904, XXX, 706. •Ambrose, T. : Splenic Anemia and Splenectoiry. Australas. Med. Claz., 191.'i, xxxiii, 173. 'Anitschkow, N.: Uebcr cxperimentell erzeugtc Ablagerungcn von anisotropen Lipoidsubstanz in der Milz und im Knochen- mark. Beitnige z. path. Anat. u. AUg. Path., 1913, Iv.., 201. •Anschutz: Discussion of Article by Kreuter. Verhandl. d. deutsch. Gcsellsch. f. Chir., 1914, xliii, 232. •Antonelli, G.: Effetti dclla Splenectomia su di una parti- colare forma di ittcro emolitico acquisito con anemia a tipo pcrnicioso. Policlinico (Sez. Med.), 1913, xx, 97. '•Apolant, H.: Ucber die Bezichungen der Milz zur aktwen Gcschwulstimmunitat. Zeitschr. f. Immun., 1913, xvn, 219. "Aristotle: Peri Zoon Morion. Concerning the Parts of Ani- mals. Bk. III. . ^^Vrmstrono, G. E.: Splenectomy and Banti's Disease. Bnt. Med. Jour., 1906, ii, 1273. "AscHENHEiM, E. : Ucbcr familiarcn hamolytischen Ikterus. Munch, med. Woch., 1910, Ivii, 1282. -AscHEKHEiM, E., and Benjamin, E.: Tebor Bezeihungen der Rachitis zu den Hamatoporetischen Organen. I Mitteilung. Die Rachitische Myolosplenie (anemia pseudoleucemia in- fantum). Deut. Arch. klin. Med., 1909, xcviJ, 529. ^ 361 ::i '! i'i I! i ) 1^ ,1 ■ H ^jUkiji • I i <> 1-1;! "'it 362 THE SPLEEN AND AN/EMIA '*AsHF.R, L. : Die Funktion der Milz. Deutsch. med. Woch., 1911, xxxvii, 1-252. ■' AsHF.R, L., and Ejxother: Das Zusammenwirkung von Milz und Leber. Ein Beitrag zur Lehre von der Funktion der Milz. Centralbl. f. Plus., 1915, xx, 61. '"AsiiER, L., and Grossen backer, H. : Beitriige zur Physiologic der Drusen. II Mitteilung. Untersnchungen iibcr dit Funk- tion der Milz. Biochem. Zeitschr., 1909, xvii, 78. "AsHER, L., and Sollberger, H.: Beitriige z. Physiologic d. Drusen. XIX Mitteilung. Fortge>otzte Beitriige zur Lehre von der Funktion der Milz als Organ des Eiweisstoffswechsels. Uber die Konipcnsationsvorgiingc nach Milzexslirpation. Biochem. Zeitschr., 1913, Iv, 13. >« AsHER, L., and Vogel, H. : Beitriige zur Physiologic der Drusen. XVIII Mitteilung. Fortgesteztc Beitriige zur Funktion der Milz als Organ des Eiseiistoffwcch?-^s. Biochem. Zeitschr., 1912, xliii, 386. '"Asher, L., and Zimmermakn, R.: Beitriige zur Physiologic dor Drusen. XII Mitteilung. Fortgesteztc Beitnigc zur Funk- tion der Milz als Organ des EisenstofTwcchsels. Biochem. Zeitschr., 1909, xvii, 297. ^"Assolant: Rccherches sur la Rate. Paris, 1801. (Not avail- able.) =^ Austin, J. H., and Ringer, A. I. : The Influence of Phlorizin on a Splcnectomi^cd Dog. Jour. Biol. Chem., 1913, xiv, 139. "Austin, J. H., and Pearce, R. M. : The Relation of the Spleen to Blood Destruction and Regeneration and to Ha-molytic Jaundice. XL The Influence of the Spleen on Iron Metab- olism. Jour. Exper. Med., 191 1, xx, 122. "Austin, J. IL, and Pepper, O. IL Pehry: Tlie Relation of the Spleen to Blood Destruction and Regeneration and to Haemo- lytic Jaundice. XII. The Importance in the Production of Ha-molytic Jaundice of the Path of Ha-moglobin to the Liver. Jour. Exper. Med. 1915, xxii, 675. BIBLIOGRAPHY 363 "AzzrRiNi, F., and Massart. G. : La morfologia del sangue negli animali smilzati. Arch, di Biol., 1903, Ivii, 802; ibid, 1904, 25 Vol. I, Iviii, 629. Baii.lon : Opera Omnia Med.— Epidem. et Ephemer. Bk. II, p. 183 (de Tournes, Geneva, 1762). -"Baldwin, J. F.: Splenectomy for Pernicious Anaemia, Apparent Recovery, Death. Med. Rec, New York, 1915, Ixxxvii, 230. "Balfoik, D. J.: The Technic of Splenectomy. Surg., Gyn., andObs., 1916, vol. 23, 1. -' Banti, G. : La Milza nelle Itterizie P'ieiocromiche. Gaz. degli Osp.,189.5, xvi, 489 =" Banti, G. : La Splenomogalie hemolytique. Semaine med., 1912, xxxii, 265. Splenomegalie hemolytique anhemopoie- tique, le role de la rate dans I'hemolysc. Semaine med., 1913 ; xxxiii, 313. Splenomcgalia emolitica anemopoieta. Lo Sperimentale, 1913, Ixvii, suppl., 323 =° Banti, G. : La Splenomegalia con cirrosi epatica. Lo Speri- mentale, 1894, Sez. Biol., xlviii, 407. "Banti, G. : La Splenomegalia emolitica, patalogica, 1911, iii, 471 ; La Splenomegalia emolitica anemopoietica, Uffi/io della Milzo nell emolise. Sperimentale, 1913, Ixvii, 323. •'^ Banti, G. : La Splenomcgalia emolitica. Sperimentale, 1912, Ixvi, 91. La Splenomegalia emolitica, anemopoietica. Speri- mentale, 1913, Ixvii, 323. '•'Banti, G.: The Clinical Aspects of Ha-molysis. Tr. Intern. Cong. Med., 1913, Sec. vi. Med., p. 1. ■'* Bardeleben : Note sur des extirpations de la rate et du corps thyroide. Comp. Rend, de I'Acad. de Sciences, Paris, 1844, xviii, 485. ^■' Bari.inc;, G. : Splenectomy for Enlarged Spleens with Ana-mia. Lancet, 1915, i, 220. '"Barr, J.: Three Cases of Banti's Disease. Lancet, 1902, ii, 493. ''Bauer, Joki,, M. : Neuere Arbeiten ueber die Physiologic und Pathologic der Milz. Med. Klin., 191M. ix, 1338. Iii rlfi 'ill!} <..;:■ ,) 4 J84 THE SPLEEN AND ANEMIA '''Bayer F Statistiches uber Splenektomie unci Mitteilung eine FallL von Milzexstirpation wegen idiopathischer Hyper- trophic. Munch.mc-d.\Voch.,190+,li,n6. -Bavkk, R.: Untersuchungen uber den Eiscnstoffwochsel nach dcr Splenektomie. Mittheil. aus d. Grenz.. d. Med. u. Clur 1909, xxi, 335 ; ibid., Weitere Untersuchungen iiber d.e Funk- tionen der MHz, vornehmlich ihre Role im E.senstotfwechsel. mit besondere Berucksichtigung des Morbus Banti. 1J14, -B,^imz.'!u'.: Su di un caso dl morbo di Banti curato colla splenectomia. Riv. Yen. d sci. med., 1915, y.xx>.. 97 ^'Benech, E., and Sabhaze«, J.: Ictere hen^olyf .ue chron.que, avec splenomegalie: Gaz. hebd. sci. mod. d- ordeaux, 1909, XXX, 469. „• , -'Benedict, S. R.: The Estimation of Lrea. Jour. B.ol. Chcm., 1910-11, viii. 405. , • , •. T^ A e,,-v-i F • Feber eine chronisclie nut "Benjamin, E., and Su ka, t.. . i tocr Icterus einhergehen, xxxi, "Blake J B.: Banti's Symptom-complex with Relation to Splenectomy. Ann. Surg., 1915, Ix.i, 315. «B.ANn-SnTTON, S:k J.: Observations on the Surgery of the Spleen. Brit, Jour. Surg., 1913-U, i, 157. BIBLIOGRAPHY 365 -Blatherwick, N. II.: The Specific R61e of Foods in Relation to the Comparison of the Urine. Arch. Int. Med., 1914, xiv, 409. "Blechfr: Subtkutane traumatische Milzzerreissung bei Mor- bus Banti. Splene- omic, Heilung. Munch, med. Woch., 1911, Iviii, 1310. "BoxDi, S.: Morbus Banti mit Splenektomie. Wien. khn. Woch., 1912, XXV, ;J27. " BoTAZZi : La Milza come Organo Emocatatonistico. Lo bperi- mentale. Scz. biol.. 1894, xlviii, 433. - Bov.iRD, D., Jr. : Primary Splenomegaly-Endothehal Hyper- plasia of the Spleen— Two Cases in Children— Autopsy and Morphological Examination in One. Am. Jour. Med. Sci., 19(K), cxx, 3T7. ., „ , r. "BozzoLo: Fol. 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The Resistance of the Bed Blood-colls to Shaking. B. The Effect . I •- 372 THE SPLEEN AND AN^MU of Repeated Bleeding, of Small Amount a year or More after Splenectomy. Read at meetm^' of Am. Soc. for Exp. PatholoRV, New York, Dec. 29, 1916. »»Gaxuv, C. kt BUCX.K. M.: Ictere Hemolytique Congenitale Au- topsie. Bull, et Mem. Soc. Med. des Hop. de Pans. 1909. ". G^^^. : De I'Epithelioma primltif de la rate. TWse de "» 0,0^0"; Tk": The Blood-forming Organs and Blood Forma^ tTon. An Experimental Research. Jour, of Anat. and Phvs., 188.5-86, XX, 100 ; 324. '" G.BSov. A. G. : On Certain Causes of Splenomegaly and Banti » Disease. Proc. Rov. Soc. Med. Sect., 1914, vn, 7. "^GiFFiN- H.Z.: Clinical Observations Concerning Twenty-seven Case; of Splenectomy. Amer. Jour. Med. Sci., 1913, cxlv. 781 "^ GiFFiv, H. Z. : Personal communication to the author. -GiFKiN, H. Z.: Splenectomy for Splenic Anemia in Childhood and for the Splenic Anemia of Infancy, Am. Surg., 1915, -GitnTQ. 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Sci., 1871, Ixii, 373. *"WoLFERTH, C. C: Blood Changes in Albino Rats Following Removal of the Spleen. Arch. Int. Med., 1917, xix, 105. *''"■ Wolff : Discussion of Graff's Communication on Splenectomy in V. Jaksch's Disease. Verb. d. Deut. Ges. f. Chir., 1908, xxxvii, 252. *■* Wright and Kin'nicitt : A New Method of Counting the Blood I'latelets for Clinical Purposes. Jour. Am. Med. Ass'n., 1911 Jvi, 1457. BIBLIOGILVPHY 3911 *"Wi-NTEE, E., and Bland StTroN, Siu J.: Splenectomy for Aclioluric Jaundice. Troc. Roy. Soc. Med., 1913-14, vii, Clin. Sec, 77. ""WvxTEH, W. E., and Bland Suttox, Sir J.: Acholuric Jaun- dice, Splenectomy. Proc. Hoy. Soc. Med., 19U l.j, viii, Clin. Sect. 4. '" Yatks, J. L., BrxTiNG, C. H. and Khistjansox, H. T. : The Etiology of Splenic Ana-mia or Banti's Disease. Jour. Am. Med. Assn., 1914, Ixiii, 2225. ^'^Zambeccaei: Experimente intorno le diverse viscere tagliatc a diversi animal i viventi. Florence, 1680, quoted by Mor- gagni: Animadvers. Anatom., ii, Observ., xxiv. *"Zaxcax, a.: Un caso di morbo di Banti. Spleriectomie e guariqione. Policlinico, 1909, xvi, Sez. Med., 5. *** Zaxda : Sul rapporto f unzionale f ra milza e tiroide. Sperimen- talc, 1893, xlvii, Mem. orig., 14. ^''-Zesas, D. G. : Ueber Extirpation der Milz am Menschen und Thiere. Arch. f. klin, Chir., 1883, xxviii, 157 ; also Beitrag zur Kenntniss der Blutveriindcrungen bei entmilzten Men- schen und Thiren. ; ibid., 815. *''ZiEGLER, K. : Die Bantische Krankheit und ihre nosologische Steilung unter den splenomegalischen Erkrankungen. Ergeb. der Chir. u. Orthop., 1914, viii, 625, i) .1 ,3 J z INDEX li Abscess of splctn, 337 diagnosis, .'i.17 opcrativ uiort.ility, 339 surgical l-crtt.iu-nt of, 337 spltricctoiny, .SS8 splinotomy, 338 Accessory spleens in dog, ^1 Alcoholic icterus, 255 Agglutinins in blood-serum, 280 Ammonia nitrogen in congenital hemolytic jaundice, before and after splenectomy, 215 Anaemia in dogs a factor in in- creased hapmolytic resist- ance, 50 afte- ligation of splenic vein, 195 after splenectomy, 12 artificially produced, 100 methods, 100 results of experiments, 101 repair of. 100 summary of observa- tions, 110 cause of disturbances of metabolism, 201 causal factor unknown. 111 diet, influence of, 22, SO, 34 raw and cooked, 31 experimental conclusions, S3 general summary of ex- perimental studies, 195 hwmorrhagic, 107 blood repair after, 107 26 Anaemia in dogs, after splenec- tomy, iron output in- creased, 119 jaundice, influence of, 72 onset of, 1 2 relation to Iwne-marrow changes, 158 secondary, 1 2 caused by haemolytic agents, 199 sodium oleate type, 105 resistance of red cells in, 110 spleen, absence of. a factor in chronicity .-ind slow repair, 105 spleen feeding, influence of, 97, 198 caused by haemolytic agents. 135 duration of, 137 toluylenediamine test, 136 iron metabolism in, 199 " splenic," 38 extract, injections of, in, other splenettomized animals, 22 in man, after splenectomy, duration of, 37 metabolism, studies of, in, 228 nitrogen balance, 231 pernicious (see pernicious anaemia), 265 icteric form of, 26l "splenic," 241, 242 401 INDEX U '0 i) 1 Ansmia in man, splenomegaly ac- companied by, iitl Antemia infantum pseudoleukffmia, Antihafmolytic })roperty of nor- nnl (lojj serum, l? Auto-af(glutinins in blood-scrum, test for, 280 Autolysins, 280 Bacillus coll. subcutaneous injec- tions of, producing spleno- megaly, 250 Banti's disease, 21'8, 254 a syndrome, 251 blood changes, 252 etiology, 249 infectious agent, 249 trauma of spleen, 251 pathogeneins, 249 pathology, 253 protein destruction excessive in, 290 pseudo- 252 simulated by syphilis of liver, 252 spleen, causative relationship of 253 enlarjT' III. nt of, nature of, 25 V splenectomy in, 344 results of, 330 symptomatology, 248 intermediate stage, 249 pre-ascitic period, 248 third or ascitic stage, 249 treatment of splenectomy, 309 combined with Talma operation, 311 mortality, 310 Hile,test for, after splenectomy, 54 clianpis in, after splenectomy, 68 formation from hsmoglobin, part played by spleen in, 59 in urine of splenectomizcd animals, 76 in absence of spleen, 197 in splenectomizcd animals, 58 Bilirubri in duodenal contents, (lUiintitative estimate, Schnei- der's method, 288 Blood, regeneration of, time of, after destruction by hemo- lytic serum, 102 repair of, in splenectomized dog, after hsemor- rhages, 107 sodium oleate anaemia, 107 of spleen, arterial and venous, comparison of, 87 differential counts, 91 reticulated or skeined red corpnsclefl, 93 total hemoglobin, 91 division of, from liver, re- sults of, 200 splenic extract injections, ef- fect of, on, 198 -cells, red, after splenectomy in dogs (see also ery- throcytes), 12 increased resistance of, 196 white, after wplenectomy in dogs, 17 differential count, 19 changes, after Eck fistula, 127 ligation of splenic vein, 125 INDEX 40S Blood chanjfps. transplantition of inferior mesenteric vein, 128 splenie \(in to vena cava, 126 in man after sj)Ieneetomy, SI crisis, 196. 278, 27!», 291., 321 post-operative, .S2,5 examinations, in pernicious anttinia before and after splenectomy, 221 fat. influence of splenec*'^"iv on, 84 formation, spleen, absence of. unessential to, 198 power of, in, 88 splenic extract, influence of, on, 98 experimental tests, 91 technique, 9t increase of htemoglo- bin content, 97 leucocytes, 96 red cell count, 97 platelets, increase of, after splenectomy, 279 serum, auto-agglutination of, 280 changes in, in splenic disease, 280 hamolvtic power of, test of. 280 autolysis, 280 heterolysis, 280 isolysis, 280 technique, 280 transfusion, 291 and splenectomy, 347 comparative results, 294 in anaemia, effects of, 29* Hlood transfusion, i.. pernicious an»niia, .SJ8 haemolysis and agglutination tests, I.indeman's nietholi-nodes, 177 content of bone-marrow, l60 metabolism after splenectomy, 191 influence of spleen on, 112 storage by bone-marrow after splenectomy, l60 Iso-agglutination of blood-serum, 280 Isolysius, 280 von Jaksch's disease, 241, 251 treatment of, by splenectomy, 314, 344 Jaundice after splenectomy, ansmia a factor, 73 influence of iiicieased red-cell resistance on, 77 htemolytic agents, 197 chronic family, 254 htemolytic, 254 congenital (see Congenital haemolytic jaundice), 202 metal)olisni, 227 influence of ana'mia on, 72 in hrmolytic serum experiments, 133 lessened tendency to, after splenectomy, 72 persistence of, 77 vein ligation and blood vess°I anasto nosis, 72 produced by tohiyicnediaminc experiments, 131 Kupffcr's cells, 176 Leucocyte count after splenectomy in dogs, 1 7 Leucocytosis, after heemolytic serum injections and splen- ectomy, 108 haemolytic agents, 1S8 I^'uka-mia, splenectomy contra- indicated, 10, 300, 301 I.indeman's test for ha-molysis and agglutination in blood trans- fusion, 291 Liver, changes of, after splenec- tomy, 164 and injection of haemolytic serum, 176 phagocyt sis, 176 blood destruction, 169 iron, presence of, micro- chemical bests for, 177 histologic results, 178 compensatory function after splenectomy, 180 diversion of splenic blood from, without splenectomy, 121 saturation point of, for haemo- globin, 65, 74 Lymph-nodes, changes in, after splenectomy, 164, 201 and injections of haemo- Ij'tic serum, 172 blood destruction, l69 discoloration, l68 endothelial cells, prolif- eration of, 171, 173 histological, 170 hyperplasia, 167, 169 INDEX 409 L3rmph-nodes, changes in iron, niicrochemical tests for presence of, 177 histological results, 178 literature, discussion, 15-1 new formation, 167 phagocytosis, 169, 173 course of, 175 time relation of, 173 red-cell formation, l67 compensatory formation after spleneciJiuy, 180 in congenital absence of spleen, 166 haemo-, 168, 177 Lymphocytosis following splenec- tomy in dogs, 18 Malaria, splenectomy in, 307 Malarial spleen, 339 Merck's Saponin Purum, 49 Mesenteric vein, inferior trans- plantation of, blood changes after, 128 Metabolism in man after splenec- tomy, literature on, 226 of normal organ, literature, 229 iron cxchanfTp. 235 before and after splenectomy, literature, 225 urobilin output, 235 studies of, 202 Banti's disease, 225 haemolytic jaundice, 202 history ot case, 203 family, 204 laboratory examina- tions, 201 Metabolism in man, laboratory examina- tions, methods of study, 209 blood, 206 diet, 210 faeces, 211 urine, 2 1 1 operation of splen- ectomy, 207 diagnosis, 209 microscopic ap- pearance o f spleen, 209 pathologic d e - scription, 208 symptoms, 203 a t beginning o f studies, 205 results of, 211 ammonia nitro- gen, 215 creatinin and creatin, 215 fats, 217 hydrogen ion con- centration, 215 iron elimination, 217 nitrogen balance, 211 summary of, 219 uric acid output, 214 urobilin elimina- tion, 218 of pernicious anemia, 220 clinical notes, 223 410 INDEX J' Metabolism in man, pernicious ananuia, results, iron elimination, nitroijen balance, uric acid elimina- tion, urobilin, 22 V summary of, 221' in relation to spleen, ansemia, '^28 changes caused by ha-nio- lytic serum, 2.'?4 Metabolism in man in relation to spleen, congenital ha'molytic jaundice, 227 iron elimination, 230 nitrogen balance, 231 protein destruction, 232 uric acid elimination, 232 in relation to s))lenectomv, ex- perimental studies, 181 blood examinations, 191 body weiglit increase, 193 creatin excretion ab- sent, 187 increased, '89 discussion of observa- tions, 191 earlier investigations. 181 faeces, examination of, 181. fat, 181. fat determina- tions, '90 Metabolism in relation to splenec- tomy, experi- mental studies, greater food re- quirement, 193 influence of an»mia o n , 187, 193, 194 iron elimination, 191 methods, 183 nitrogen determi- nations, 181 results of, \S\ summary, 19 ^ urine, analysis of, 184 literature, 229 iron, influence of spleen on. 112 experimental results. 118 process of, 1 1 2 nitrogen, in congenital jisemo- lytic jaundice before and after splenectomy, 211 not aff'ected by splenectomy, 201 Minot's test for haemolysis and ag- glutination in blood transfusion, 291 Nephrectomy, a control operation for splenectomy in dogs, 29 Nitrogen metabolism in anaemia, literature, 231 pernicious anaemia before and after splenectomy. 223 in relation to splenectomy. 181, 18i Pernicious anaemia, 26,'> blood examinations in case of. before and after splen- ectomy, 221 transfusion in, 348 INDEX 411 I Pernicious aiuemia, hypersplen- ism, 265 iron elimination in case of, before and after splenec- tomy, 2^t metabolism study in case of, before and after splenec- tomy, 2i2() nitrogen balance in case of, before and after splenec- tomy, 2 '2 3 pathogenesis of, 265 Eppinger's theory, 2()7 remission of, after splenec- tomy, 320 splenectomy in, 265, 34i effect on metabolism, clini- cal notes, 223 iron, 222 uric acidj 222 urobilin, 222 treatment of, by blood trans- fusion, 294 and splenectomy, 295 splenectomy, 31-t analysis of results, 318 influence of age, 322 degree of ansmia, 324 sex, 323 size o f spleen, 824. blood changes, 321 crisis after, 321 centra-indications, 327 aplastic bone-marrow, 327, 329 spinal cord symp- toms, 327, 329 effect produced by, 324 blood crisis, 325 Pernicious anaemia, treatment of splenectomy, ef- fect produced by bone-m arrow stimulation, 325 erythrocytes, i n - creased resist- ance of, 326 indications for, 327 mortality, 321 percentage of cures, 321 results of, 317 summary of, 328 time for operation, 329 value of, 315 splenic extract injections, 329 surgical removal of chronic sources of infection, 298 uric acid elimination in case of, before and after splen- ectomy, 224 urobilin elimination in case of, before and after splen- ectomy, 224 Phagocytosis, increase of, after splenectomy and injection of hamolytic serum, 171, 176 Pheiochromic icterus, 264 Pneumonia complicating splenec- tomy, 357 Poikilocytosis after splenectomy in dogs, 16 Polychromatophilia, 279 after splenectomy in dogs, I6 Polycythaemia rubra, 302 etiology, 304 haemolysis in, 303 symptoms, 303 treatment, 304 splenectomy contra indi- cated, .304 X-ray, 305 iU INDEX 1 \'^>';^^M 1 J «'■' -^'t^^^^^l '''^^^^^^^1 ^i^^H y ^ ^x-.^^^i ^'-. ^^^H c yjd^H -^^^H ^ 1. ^^^^^^1 ■■[^^^^H N ^^^^^^1 < ^^^^^1 < Polyglobulia, 314 Protein metabolism, 233 Pseudo-Banti's disease, 252 Pseudoleiikficmia, " splenic vari- ety," 241 Racliitischc mejjalosplcine, 254 Iltd blood-cells increased after in- jections of splenic ex- tract, 97 resistance of, after baemo- lytic serum injections, 109 sodium oleate antemia, 110 in relation to splenec- tomy, 38 test by beemolytic immune serum, 45 hypotonic salt so- lution, 42 saponin, 47 increased after splenec- tomy, 38 cauise of. 50 conclusions of author, 52 influence on jaun- dice. 77 to cobra venom after splenectomy, 50 to httmo lytic agents, in wounds and splenec- tomized dogs, 199 t o hsemolytic agents, summary of observa- tions in normal and splenectomized a n i - mals, 110 Ripper and Schwarzer method, modified, of analysis of fwces for iron, 1 15 Rupture of spleen, 334 Salt solution test for htemolysis, 40 Saponin, resistance of erythrocytes to, 271 Saponin test, 47 Sodium oleate anasmia in splenec- tomized animal, 105 .Spleen a factor in hsemolysis, 72 absence of, a factor in chronicity of antemia and slow re- pair, 105 slow repair of sodium oleate aneemia, 106 congenital, 8 correlation of increased red ' cell resistance and de- creased jaundice after haemolytic poisons, 1 1 1 essential factor in ansmia, 196 influence on haemoglobin and jaundice, 59 nitrogen metabolism, 187 unessential to blood foi-ma- tion, 198 Iisemolysis, 198 arterial and venous blood of, comparison of, 87 difl'"erential counts, 91 reticulated or skeined red corpuscles, 93 total ha-i'ioglobin, 91 changes in, after ligation of splenic vein, 1 ' I dislocated, in mnn^ Sil INDEX 413 Spleen, effect of, on bone-marrow, 111 hsemopoietic system, 98 red cell formation in bone- marrow, 97 enlargement of, 211 in Gaucher's disease, 2 IG experimental studies in animals, 11 extirpation of (see Splenec- tomy), 3 anatomical considera- tions, 345 history of, in animal, 3 in hmnan beings, 6 feeding of, to splenectonii^ed dogs, 97 influence of, 98 method, 98 results, 98 haemolytic function o.', 326 on haemolysis, 39 iron metabolism, 12 experimental results, 118 regulatory, on blood, 58 blood destruction and re- generation, 87 early literature, 87 in relation to ancemia, 87 in Banti's disease, causative re- lationship of, 253 in hasmolytic jaundice, 255 in relation to blood supply of liver, 64 hemoglobinuria and jaundice, 58 haemopsonins, 175 Spleen, in relation to metabolism, 29 in man, literature on sub- ject (see Metabolism), 225 lesions of surgical treatment of, 333 abscess, 337 operative mortality, 339 splenectomy, 338 splenotomy, 338 cysts, non-parasitic, 34S parasitic, 313 gunshot and stab wounds, 333 malarial spleen, 339 rupture, 334 mortality, 336 prognosis, 336 splenomegaly, 344 syphilis of spleen, 341 tuberculous spleen, 340 tumors, 343 wandering spleen, 341 not essential to life, 8 regeneration of, after extripa- tion, 6 simple hypertrophy of, 242 site of disintegration of ery- throcytes, 66 spodogenoHs, 65 trauma of, cause of Banti's dis- ease, 251 venous blood of, more resistant than arterial, litera- ture reports, 89 negative findings, 91 wandering, ligation of arterv in, 297 414 INDEX I'; (.,. Spleen, X-ray trratment, in splt-nic diseases, 2<)fi Spleens, accessory, in dog, 21 Splenectomized animals more re- sistant to lueinolytic poisons, 38 Splenectomy and anieniia, relation of e X p e r i - mental studies, general sum- mary, 195 the anaemia, \<)5 blood crisis, igs diet, influence of, 196 Splenectomy, in dogs, 1 1 accessory spleens, influence of, 21" antemia following, 12, Hi, 158 factor in jaundice. 73 artificially produced, sunmiary of observa- tions, 1 10 blood count emotion, ef- fect of, on, M cosinophiles, 18 erythrocytes, 13 skeined or reticu- lated, 16 haemoglobin content, J 3 lymphocytosis, 18 myelocytes, 20 poikiloeytosis. 1(5 polvchromatophilia, 16 red cells, 13 white cells, 17 character of. 15 Splenectomy in dogs, anamia fol- lowing diet, in- fluence of, on, 22, 24, 30, 34 food, raw and cooked, 31 iron, 26 iron-rich food, 23 experimental c o n e 1 u - sions, .^3 progressive onset of, 12 •tage of beginning re- pair, 16 bile-pigment, changes i n , after, (]S bile test, after, 5't blood changes variable after, 26 blood su})ply of liver ; fter, 197 bone-marrow condition of, after, 147 cholcsterin increase of, after, 84 controlled by previous ne- phrectomy, 29 eff"ects of, 5 mortality, 6 first authentic operations on dogs. 4 hwniojilobin estimation after, n lia;nioglobinuria, after, test for, 54 and jaundice, dec Tased tendency of, after, 54, 197 heemopsonins, change i n , after, 175 history of, 3 INDEX 415 Splenectomy, iron pliininition be- fore and after, 116 experimental results, 118 increase of, after, due to anu>iiiia, 1 19 lessened tendency after, 31 liver changes after ( s c e Liver), Kil, 176 lymph-nodf changes after (see Lymph-nodes), Kil. nature of function lost after, lai in relation to fatty acids and cholcsterin content, 198 red cells, resistance of, to hemolytic agents after, summary of observations, 110 increased after, 38, 196 cause, 196 test by salt solution, 40 shaking, 41 resistance to blood poisons, after, 11 influence of, on blood, 1 1 blood fat, 84 on bone-marrow (see Bone- marrow), 110, 200 cholcsterin, 84 on fertility, 5 general nutrition, 25 lymph-nodes, 201. metabolism (see Metabol- ism), 23, 181 on resistance of red blood- cells, 39 Splenectomy, resistance of red bl.)od cells to hemolytic im- mune serum, 45, 109 to hypotonic salt so- lution, 43 to saponin, 47 on weight, 23 without influence on metabol- ism, 201 Sjilenectomy in various animnls^ antemia after, 22 autopsy findings, 6 Splenectomy in man, 202 anwmia, delayed repair of, in absence of spleen, i'jG duration of, after, 37 anatomical considerations, 345 and blood-transfusion, 347 comparative results, 294 blood changes after, 34 reported cases, 35 blood platelets increased after, 879 supply of portal vein lessened after, 198 choice of time for operation, 329 complications, of hemorrhage, 856 injuries to adjacent viscera, 357 contra-indications, 291, 300 hemorrhagic diathesis, 300 leukaemia, 10, 300, 301 polycythaemia rubra, 302 severe ansemias, 300 first recorded operations, 6 for wandering spleen, 342 history of, 6 416 INDEX SpUncctomy, functional cquilib- riuiii after, 'i'.i i abscess of spleen, 338 ill B'lnti's disease, 341 in cirrhosis of liver, 300 ejsts of spleen, 313 in CJaucher's disease, '2 IT, ^^^ in ha-inolytic jaundice, 3H in V. .laksch's disease, 3 it in niaiarial spleen, 33J) in pernicious anieniia, 3li contra-indications, 327 curative action denied, 32t) indications for, 327 summary of results, 328 in rupture of spleen, 336 in splenic disease, blood crisis following, 278 splenomegaly, 3H in syphiliP of spleen, 341 in tuberculous spleen, 3tO in tumors of spleen, 3i3 in various blood diseases, mor- tality, 309 influence of, 34 on metabolism in pernicious anaemia, 222 on urobilin excretion, 287 iron elimination after, 230 metabolism studies before and after (see Metabolism), 202 mortality, 10. 358 operative risks, 358 positive indic.itions, 309 in Banti's disease, 309 Gaucher's disease, 311 hnemolytic jaundice, 312 V. Jaksch's disease, 314 pernicious antemia, 814 Splenectomy, possible indications, 305 cirrhosis of liver, 305 inalatia, 307 syphilis, 308 sequela' of, 357 bronchitis, 357 pneimioiiia, 357 technique. 350 closure of splenic space, 355 control of hemorrhage, 352 ligation of pedicle, 353 removal of organ, 351 value of, as a therapeutic pr«>- cedure, '299 Splenic blood, diversion of, from liverbyKck fistula, 121 by ligation of splenic vein, 121 by transplantation of splenic vein, 121 without splenectomy, 121 blood crisis following splen- ectomy, '278 diagnostic and prognostic methcds, 269 agglutinins and hsemo- I y s i n s in blood- serum, 280 blood examinations, 270 bone-marrow activity, evidences of, 275 blood platelets and leucocytes, 279 by fixed smears, '277 by vital staining, i7!i INDEX 417 Splenic disease, history taking, 269 hypotonic - It solu- tion, ;i ttciinique, 272 laborntory tests, 270 physical examination, 269 polychroma- tophilia, 279 protein, uric-acid, and iron metabolism, 289 red blood-cell resist- ance, 271 urobilin excretion, 281 urobilinogen excretion, 281 vital staining, 275 treatment of, 291 blood transfusion (see Blood tr(»nsfusion), 291 medicinal and hygienic measures, 296 •urgical, 297 cauterization and d e - struction of spleen, 297 ligation of blood-vessels of spleen, 297 Schiazzi's " splenoclei- sis," 297 extracts, heeniolytic power of, 78 experiments, results of, 82 technique of. 81 literature on subject, 79 influence of, on blood forma- tion, 98 V Splenic extract, influence of, ex- perinient- al ttst, 94 results, g5 technique, 94 leucocytes increased, 96 injections of, in anaemia, 94 stimulating action on blood, 198 on bone-marrow, 19ft vein, ligation of, 121 circulation after adjust- ment of. 125 blood changes after, 125, 200 hasmolytic agents, action of, after, 130 jaundice, lessened tendency to, after, 138 method of experiment, 128 spleen, changes in, 124 transplantation of, into vena cava, 121 blood changes follow- ing, 126 hiemolytic agents, action of, after, 130 jaundice, lessened ten- dency to, after, 138 method of experiment, 123 Splenocleisis, 297 Splenomegaly, 344 heemolytic, 262 " idiopathic," 242 large-celled (see Gaucher 's dis- ease), 243 produced by subci'taneous injec- tions of Bacillus coli, 230 with anemia, 241 418 INDEX Splenomegal}-, classification and types, 2H hepatic cirrliosis (see Banti"- disease), '21-8 Splenopexy in wandering spleen, 342 Splenotomy, in abscess of spleen, 338 Syphilis of spleen, 341 splenectomy in, 307 Test, auto-agglutination in htemo- lytic jaundice, 256, 258 for auto-agglutinins in blood- serums, t.'8() technique, 280 for bile after spltncetomy, 54 for effect of s|)leen feeding to spleneetoini/.ed dogs, 98 for free liaemoglobin in splenic blood-serum, 90 for hemoglobinuria after splen- ectomy, 54 for haemolysis and agglutina- tion in blood transfusion, 291 for haemoiytic power of blood- serum, 280 splenic extracts, 81 resistance of erythrocytes by cobra venom, 50 for influence of splenic extract on blood formation, 94 for iron metabolism in aplenec- Lomized and normal dogs, 1 14 with saponin, 47, 271 for urobilin excretion, Wilbur and Addis method in stools, 284 in urine, 282 I Test, heemolytic serum, 133 I microcheinical, for iron in liver after splenectomy, 177 ! lymph-nodes after splen- I ectomy, 177 I toluylenediamine for jaundice, I 131 leucocytosis caused by, 138 of, resistance of corpuscles, with heemolytic immune serum, 'i5 with hypotonic salt solu- tion, 42 technique, 272 vital staining, 275 technique, 275 Transfusion of blood, 347 in splenic disease, 2pl Tuberculosis of spleen, 340 Tumors of spleer 343 Uric acid elimination before and after splenectomy in congenital haemoiytic jaundice, 214, 23S in pernicious anaemia^ 224 in relation to spleen in anaemia, 232 Urobilin elimination after splenec- tomy, 287 in anaemia, 287 in haemoiytic jaundice, 287 congenital, before and after splenectomy, 218 in pernicious r- -pinia, before and after splenectomy, 224 in relation to splenectomy, 235 in splenic disease, 281 INDEX 419 Urobilin excretion, normal. '2S7 quantitative estimation of, in duodenal contents, Schneider's metJiod, 288 in stools, Wilbur and Addis method, 284 in urine, Wilbur and Addis method, 282 Urobilinogen elimination, 281 quantitative estimate of, in duo- denal contents, Schneider's method, 288 Vaquez's disease (see Polyeyth- aemia rubra), 302 Veins, ligation of splenic, 121 conclusions, 129 transplantation o f , inferior mesenteric, blood changes after, 128 portal (Eck fistula), 121 splenic, 121 conclusions, 129 Vital staining, 275 Wandering spleen, .'JU White blood-cells, influence on, of splenectomy, 108 Widal's "crises of deglobuliza- tion," 2,58 ' Wounds of spleen, 3SS :^m^x^: ":.;^^ .>i._. m\ . mi^i!^^'^jMmM _ :55^i r..'f-;v' % ■'w:-^^* ■?i«J < It