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Les diagrammes suivants illustrent la methode. 1 2 3 4 5 6 MICROCOPY RESOIUTION TEST CHART 'ANSI and ISO TEST CHART No 2! 1.0 !^»- m I.I 1.25 m ■•■■ 1.4 ||| j.2 112.0 1.8 1.6 jd dgf LIED IfvMGE Ir ~*S ~:":^*^^:e'. New >:rt. !4609 . 'SA *^— ■•6' ..e: 0300 - Ohone ^= ; '161 288 - 5989 - Fo« ^.^■. STUDIES IN PUNCTURE-FLUIDS STUDIES IN PUNCTURE-FLUIDS A CONTRIBUTION TO CLINICAL PATHOLOCJY MKI\(; A THKSIS Al'PROVKO FOR THK DKGRKK OK D()( lOR OF MKDIC INK IN THK ITNIVF.RSITV OF LONDON O. C. GRUNEK, M.D. London clinical Pathologist at the General liifinnarv, l.eeJi ; late Hon. Pathologist to the Leeds Puhlii Dispensary PHILADELPHIA P. BLAKISTON'S SON & CO. I0I2 WALNUT STREET 1908 I'rinted in Engiaiid] ^i .Civ^ 190? TO MY MOTHER 55059 PREFACE It has now l^ecome generally recognised how great is the as- sistance which the detailed chemical and physical examination of morbid material affords the physician or surgeon in arriving at or confirming a diagnosis. In carrying out the research recorded in this book, the requirements of the clinical pathologist have therefore been steadily kept in view. Probably the greatest value of future investigations into the nature and comjwsition of puncture-fluids will be found to lie in the additional light they may be expected to throw ufwn the subject of metabolic processes in diseases. At present the available data are so scanty that it is impossible to treat this subject in detail, but it is hoped that the special index referring to the puncture-fluids met with in different morbid conditions may prove useful. My grateful thanks are due to the members of the Honorary Staff of this Infirmary, who have kindly permitted me to publish the results of my work upon their cases ; to Dr. G. W. Watson and Mr. J. A. Coupland for assistance in tracing the clinical records of the cases studied ; and to the various members of the Infirmary Staff who have preserved material for analysis. Lajtly I must gratefully acknowledge the debt I owe to the writers of the works I have consulted during the course of my researches. O. C. GRUNER. i'athological laboratory, Genrral Infirmary, Leeds, July 1908. vil CONTENTS INTRODUCTION Objects of study— The caution necessary in interpreting results, owing to the existence of important variables — The insight into metabolic pro- cesses afforded by the chemistry of puncture-fluids— The decomposition products of proteid may be either initially present, or artificially produced by analytical processes— The main methods of physico-chemical analysis The scheme of examination of fluids — Cytodiagnosis . . . />. i SECTION I THE CHEMICAL EXAMINATION OF PUNCTURE-FLUIDS Preliminary remarks— Difficulties attached to the analysis of fluids- Precipitation of albumen by mastic, etc.— Adsorption— Scheme for analysis of puncture-fluids. (A) Preliminary processes. (B) Separation of the globulins— Albumen— Globulin— Albumoses and peptones— Monamino- acids— Ammonia— Hydrolysis— Sugars— Purins and urea. (C) Diamino- acids— Residual nitrogen. (D) The glycoproteids ; pseudo-mucin, its properties and reactions ; paramucin ; synovin — Lecithin, its importance, constitution, allies, and symbiotic •substances, and methods of analysis— The diazo-reaction — The a-naphthol reaction — Ehrlich's glucosamine reaction — Tryptophane — Pigment — The inorganic constituents of punc- ture-fluids — Ferments, their detection, their occurrence, and their im- portance p. 10 SECTION II THE PHYSICO-CHEMICAL EXAMINATION OF PUNCTURE-FLUIDS (A) Osmotic pressure — Theoretical considerations on osmotic pressure ; how to calculate the osmotic pressure ; the degree of dissociation — Theo- retical considerations on electroconductivity ; corrections necessary' for vanations in temperature and in amount of prote.ii in the solutions examined ; achloride electrolytes — Osmotic concentration — Theoretical ix X CONTENTS considerations dealing with the effect of mixtures of many substances on the freezing-point depression and on electroconductivity — The relation of freezing-point depression to specific gravity — Methods of determining the freezing-point depression and the electroconductivity — Results of examination in each case — The plasmolytic metho. 3i8 SECTION VI SPECIAL CASES p. 236 APPENDIX Table I. Ready Reckoner for Chlorides . . . . Table II. Ready Reckoner for Molecular Concentration of Sodium Chloride Solutions Table III. Specific Conductivity of Potassium Chloride Table IV. Rate of Migration of Ions Table V. Obach's Table Table VI. Ready Reckoner for Osmotic Concentration Index of Diseases Literature Index of Authors General Index P- 253 *54 256 257 257 260 XVM 263 281 LIST OF FIGURES VAOB 1. Adsorption 2. Device for Burettes 3 3. Velocity of Reaction ' 4. Scheme of Apparatus for determining Electroconduc- TIVITY 5. Hamburger's Pipette "^ 6. The Critical Solution Point ^^^ 7. Diagram to illustrate the Meaning of the term " Acidity " 123 8. Diagram showing the Apparatus for determining the Concentration of Hydrogen Ions in a Puncturk-Fluid . 129 9. The Viscosimeter of Hess ^30 to. From a Cysto-Adenoma Papilliform Ovarii. . . 174 11. From a Multilocular Ovarian Cyst 178 12. From a Cysto-carcinoma Ovarii *78 13. From a Multilocular Ovarian Cyst I79 14. Dead and Dying Cells from a Colloid Carcinoma . .179 xiii LIST OF TABLES RECORDING THE AUTHOR'S RESULTS PAOB 26 TABLE I. Globulin-content of Puncture-Fluids . II. Albumoses in Puncture-Fluids .... III. Urea-content of Puncture-Fluids .... 3° IV. I'uRiNs in Puncture-Fluids 3' V. Mucins in Puncture-Fluids 4^ VI. Lecithin in Body-Fluids 54 VII. The a-NAPHTHOL Reaction of Puncture-Fluids . . 57 VIII. Tryptophane in Puncture-Fluids 59 IX. Chlorides 61 X. Ferments in Puncture-Fluids 80 XI. Autolysis in Puncture-Fluids 85 XII. The Dissociation of a Pleural Fluid .... 98 XIII. Relation of Specific Gravity to Freezing-point De- pression 109 XIV. Viscosity of Puncture-Fluids 141 XV. Constituents met with in some of the Fluids examined 153 XVI. Osmotic Concentration of Pleural and Peritoneal Fluids 154 XVII. Electrolytes in Cerebrospinal Fluid .... 173 XVIII. Osmotic Concentration of Ovarian Cyst-Fluids . . 182 XIX. Electrolytes in Ovarian Cyst-Fluids . . . .183 XX. Pancreatic Cyst-Fluids 185 XXI. Specific Gravity 198 XXII. Percentage of Albumen in Various Fluids . . 202 XXIII. Chloride vetsus Achloride Electrolytes in Exudates AND Transudates 212, 213 r*oa «7 xvi LIST OF TABLES RECORDING AUTHOR'S RESULTS Unnumbered : Adsorption Experiments Degree of Dissociation of Bile iw Electrolytes in (Edema Fluid '45 Composition of Peritoneal Fluid on Successive Tappings 157 Osmotic Concentration of Cerebrospinal Fluid . . 171 Records of Special Cases . . 238, 241. M*. 243. 244. 246 INDEX TO DISEASES REFERRED TO IN THIS WORK \cuto Hhfiniiatisni, l2.^ Akoliolisiii, H)'» 1 7^. Amyloid Disease, 2,< \namia. 82 Hantis Uisoasc, 34, MM Cardiac railiire. Z},. 25. 2<), J". 3'- 34. 4>. 54. 57. 3'J. f"- **"• '4'' ■50. "SJ. '54- '55. i'3' •«37' 247, 2.S'> Adherent I't-ricardiiiin, i'', 59, 141. -4'> Cirrhosis ot Liver, 25. 2<', 3". 3'- 41, 51, 5'», '>!, 80. 14'. '44. i4'». 15". 153. •54. 15''. 157. ■!I3. 243 Monolol)iilar, 54, 2ui, 245, 248-y Syphilitic, i')i Eclampsia, 170 Empyema, 2'>, 30, 31, 59, f)i, 80, 84. 141, 147, 212. 228, 240 Gout, ifi3 Jaundice, 170 Lung, Gangrene of, 80 Malignant Disease — Stomach, 30, 41. 61, 153, 247 Liver, I47 Pancreas, 41 Omentum, 30, 31, 61, 153, 212, 242, 246 Peritoneum, 3. 11, 23, 26, 30, 31. 41. 54. 55. 59, 61, 141, 150, 154, 157. 200, 206, 212, 229. 239 Meningitis, 16;, 108, i;o Tuberculous, lOO, 168, 171, 172, 17K, 230, 232 Pachy-, Hsemo., 168 Multiple Lymphoma, 150 MultipleMyeloma, 150 Nervous Diseases— Apoplexy, 167, 169 Cerebellar Abscess, 172 Cerebral Hx-morrhage, 167 Cerebrospinal Fever, 167, 231 Disseminated Sclerosis, if>g Epilepsy, 169, 172 Herpes, 230 Hydrocephalus, 170, 171 Mental : Acute Amentia, i')8 Amaurotic Idiocy, if>8 Dementia Paralytica, T67, 169,171. 172, 230 Hysteria, i<^'8, ifK) Neurasthenia, 169 Tabes, 168, 169, 172, 229. 230, 231 Tumor Cerebri, 167, 168, 169, 173 (Edema sine Albuminuria, 80 Pericarditis (tuberculous), 162 Peritonitis — Acute, 155 Chronic, 30, 31, 41, 26, 54, 55. 59, 61, 80, 154, 212, 248 Puerperal, 147 Suppurative, 154 i Tuberculous, 23, 25, 30, 41, 54. j 57. 59, 61. 80, 141, 150, 153, I 154. 155' 200, 212 Pleurisy — Postpneumonic, 212, 239 Simple, 25. 26, 30, 31, 41, 57, I 59, 61, 80, 141, 150, 154, 237, ! 238, 242 i Tuberculous, 25, 26, 30, 31, 4', 55- 57. 59. (^i, 80, 83, 147, 153. 154, 201, 212, 237, 239 Pneumonia, 80, 153, 162 Polyorrhomeniiis, 55, 5^^. 59. *^'' 80. 153, 213, 249 will INDEX TO THE DISEASES REFKkKED TO IN THIS WORK Kiiiiil l)isca»ie — Aciitf. I j4 throinc Tuba! 3, 23, jr., 31., 34, 4'. 3<'. 61, 80, 141, 144, ly,. >.'5.<. «54. «37. """. 2ii. i2i3, ii7. 244 Cliroiiic Interstitial, 23, 30, 54, ^'t. fn, 150, jtV) rripinia, i U> Septic Diseases. 82 Subaratlinoid Il.i niorrhage, 167 SvpliiliN 43 Syphilis continutd Brain, 173 Congenital, 155, \f,y \(„, See alsii iindor C'irrhoitis. Tetanus, 24 Thoracic Aneurj-sm^ 85 Thronil)osis of Portal Win, 30, 31, ("■ I5.V 154. IV>. 200, 213, 240, 241 Tubercli', 227 .SVf also under Peritonitis, Pert- cardiiis, and Pleurisy. KRRATA I'.i^e 8, line 17 from top. for " hysico-" read "physico-" I'aj^e so, Table III, for " single " read "simple " I'age 31, line 5 from top, for " Hurians'' read " Hurian's" Page 32, line 2 from bottom, for " togatose'' read " tagatose " I'a^e 44, line 14 from bottom, for " marrows " read " marrow " I'age 51, line 17 from top, omit "in " I'age 55, line 8 from footnote, for " orhistid in " read " or histidin ' I'age 64, line 2 from bottom, for " H + " read " H+ " I'age 68, line 10 from bottom, for " oxidations " read " oxidative ones " Page 104, to formula in middle of page add " atmospheres " I'age 139, line 2, omit " fluid " Page 173, line 6 from top, for 122 read r3i I'age 180, in the Table, for "hydrops, folliante" read "hydrops. folliculi " Pajic 182, line 11 from top for " cetylalcohol " read "cetyl alcohol" Page 185, last line of Table XX, for '• trypisn " read " trypsin " Page 206, line, 1 5 from top for " Wideroe " read " Muller " Page 234, last line, before Fig. 5 insert '" Plate 11 " STUDIES IN PUNCTURE FLUIDS INTRODUCTION CuNTENTs : Objects of study —The caution necessary in interpretmR results. owinK to the existence of important variablci— The insight into metaliolit pi. .cesses attonleil bv the chemistry of puncturc-Huids - The decomposition procbicts of proteiil may be either initially present, or artilicially produced by analytical processes— The main methods of physico-chemical analysis— The scheme of examination of fluids — Cytodiagnosis. The investigation of the characters of puncture fluids may l)e inaubsequent pages that the attempt has been made to comi)el the physico-chemistry to supplement the c'n'inistry of the puncture-fluid. This has been applied, as i> vscii kiuiwn, h\- several renowned scientists, to the examination (if bloini and iiriitc, but so far as can be made out, there has been '%«."}f.' INTRODUCTION 3 no attempt hitherto to apply the principle to the study of the ordinary i^uncture-fluids. It is believed, however, that such an application of the newest lines of study— those of ionic con- stitution— will afford an insight into the metabolic processes of thos' diseases which are associated with the pouring out of fluid. Not only this, but the attempt has been made to compel chemistry and physico-chemistry together to afford an ex- l)lanation of the concordance or discordance which exists between the diagnosis hitherto made by their aid, and the actual nature of the disease ; this has, however, frequently led one into the subject of the pathology of metabolism, a subject which must of necessity be left in the background in the following jiages. Still, it must i)e reasonable to su])pose that the study of the Huids which are poured out must give great aid not only to the pathology of metabolism of a given disease, but must assist in forming a diagnosis of the disease. To illustrate this idea, the deviations from normal metabolism which obtain in disease of the liver may be referred to. They will dej)end to a large ex- tent on the nature of the disease with which the liver is affected, and more than this, these changes will be different according to whether the disease is jirimarily one of the liver, or primarily one of the heart. Each of these is bound to result in a different succession of changes from those due, for instance, to renal k dise;ise, and the chemical composition of the fluid poured out will ibe corresix)ndingly different. The excessive amount of chlorides fpresent in nephritic effusions as compared with back-pressure [effusions instances the correctness of this contention. Then, again, the investigation of the various decomposition products of proteid that occur in an effusion will afford a not mconsiderable ; light on the catabolic changes met with in the disease with which the effusion is associated. To consider for a moment another example of this j)rinciple. The fluid in a case of iKMitoneal carcinomatosis presents, on cytological examination, desquamated carcinoma cells, some of which are living and some necrotic. 1 hcse cells have a s{)ecialised form of metabolism, though the only known fact about them is that their proteid is essentially different in nature from normal cell-proteid.* This difference in ronstitutinn mast invoivo a * Bergell. See also Hottmann, Mihich. mcd. Woch.. 46, 1907. STUDIES IN rUNCTURE-FLUlDS difference in the jiroducts of brealcdown of the proteid, though exactly what the differences in the two series of cases (healthy metabolism, and carcinoma-cell metabolism) may be, and where they are to be located in the configuration of the proteid mole- cules, is quite unknown at the present time. The presence of both living and dead cells too, not only in the fluid, but all over the surface of the jieritoneum, means that these metabolic products occur in the fluid which they have caused to appear, and a chemical study of such a fluid must afford an insight into the metabolic changes of carcinoma celMife. Not only this, but there is the question of ferment action to be considered. There are ferments in the peritoneal fluid of carcinomatosis cases, which may be specific, and are of importance, because they have actuated this particular form of metabolism. We must not forget meanwhile that the whole of the serosa is not one mass of ])rolilerating carcinoma. There are extensive areas in which there is no carcinoma, and the cliemicai composition will de}>end to some extent on the normal processes of life of these cells, and on their permeability-phenomena. That is to say, we shall meet with the resultant of two series of ])rocesses — quite a different matter from having only a single factor to deal with. If reference be made to the possibility of a diseaseii condition of the hitherto intact serosa following the changes produced in the organism as a whole (cachexia, etc.), we shall at once see clearly that there are so many factors that it would be remarkable if all diagnostic rules in the case of carcinomatous puncture-fluids were always infallible. Perhaps it is more important to suggest that herein lies the means of justly appre- ciating the facts on which one can base a diagnosis, for by bearing the variables in mind, one may attempt to secure more accuracy in one's opinion. These considerations have been gone into at such length, because it is felt that there is an undue tendency to expect such investigations as are recorded in the following pages to enable a positive and certain diagnosis to be made as to the nature and origin of an outpouring of fluid. It is not so. We must say, " such and such a substance (of which the tests are given) is more abundant in a certain percentage of cases of this class of effusion than it is in another class of case." Even these limita- tions do not deprive the methods of all utility, for clinical symp- INTRODUCTION 5 toms often help, and just as pros and cons have to be weighed in ordinary clinical diagnosis, so they frequently have to be wciL'hed in clinKul pathological diagnosis. Attention must be directed also to the errors that may arise from the difference in the structure of various proteids. Many proteids contain the same fundamental substance as an mtegral part of their molecule. The following table, which has been •ibrulged for the present purpose, from the valuable work of Gustav Mann (Chemistry of the Proteids, 1907), will illustrate what is meant. We see, for instance, that glycocoll forms a part of the molecule of serum-albumen, of serum-globulin, of hetero-albumose, and of keratin ; that tyrosin occurs m serum- albumen, in protalbumose, in Bence-Jones proteid, and in keratin ; that tryptophane is only present in kerafn (in this tal)le) and so on. Our chemical analysis may re al the pre- sence of all these bodies, and of course it would only be possible bv a careful separation of each of these bodies to say to which the derivative is to be ascribed. If we find tryptophane or tyrosin in a puncture-fluid, for example, we cannot be sure that it was there beforehand, or whether the analysis has resulted in its being split off from a proteid containing it. ^i^ Serum- ' alliuiiit-n. 1 Seruiii- Hlobulin. Hetero- albumose. Prot- albutnuse. Bencc- Jonei proteid. rrot.-imin (Slurinl Honi- keratin. GlvcocoU + 3-52 + 0-34 Alanin + 2-22 Leucin 18-7 + + + J8-3 Phenylalanin + 3-H4 + 30 3-6 Prolin + 2-76 Glutaminic acid + 2-20 + 14 Aspartic acid ■t- 2-54 2"> 6-8 570 4-58 Cystin Serin 1-2 1-51 Tyrosin ■2-7 , , + + Lvsin 3-5 3-5 ^ 12 Histidin . . . . 2-2 2-2 12-9 Arginin 4-9 49 58-2 2-25 Tryptophane Ammonia . . 175 0-8 0-8 1-6 Ami no valerianic acid . . ! ' •• y? Glucosamin 1 The table reveals a further fact, namely, that the amount STUDIES IN PUNCTURE-FLUIDS of each derivative varies according to the different proteid. Thus, if we find seven parts of lysin we shall not know whether this means that it has all been derived from equal parts of hetero-alhumose or protalbumose, or whether it is a proportion characteristic of some other i)roteid not included in this table. We might even say that we cannot be sure that the numbers in this table do really characterise the particular proteid under which it is placed. But assuming the table absolutely correct", and assuming that our analysis is correct, it is evident that by slightly altering the relative proportions between one deiivative and another we shall really be having a different proteid before us. Such slight differences, which involve us in endless per- mutations and combinations, would afford an e.xplanation of the biological distinctions that obtain between one proteid and another, between fish-albumen, and mollusc-albumen ; not to mention the possibility that stereo-isomeric variations might result in still more refined and none the less absolute distinctions. To sj^eak of such possibilities as these is but to touch on a fringe of the difficulties thai arise in interpretation of results of analysis, and would at first sight deter one from ever attempt- ing an opinion in a case. Fortunately, however, there remain many tests of easy application which will help the clinician to form his diagnosis in a case associated with effusion. Even in spite of all the chances of error which have been indicated, these tests preserve their utili y, and the fact that failure may sometimes arise should not be allowed to relegate them into the background ; and we may hope that with time, a better knowledge of the details to which reference has been made will explain the cause of the failure of such tests in some cases, ^nd enable a better inter})retation. with more frequently successful diagnoses, to be made. The statement may be made that a transudate with decided characters may be certainly distinguished from an exudate possessing decided characters. The border-line cases, or the cases in which inflammation sujx^rvenes on transudation, are those which are resjwnsible for the small utility which chemical diagnosis and cytodiagnosis have possessed. But if we re- member that the microscojje diagnosis of innocent tumours from malignant tumours also fails when we come upon the INTRODUCTION 7 border-line cases, we shall see that there is no more justification for condemning the one method of study than the other In pursuit of the studv of puncture-fluids it has therefore been borne in mind that we shall learn far more from the failures ,n diagnosis than from a long succession of successes and the endeavour to find an explanation for the failures will lead us deeper and deeper towards the solution of the ultimate problems of metabolism. . • . v, k«„„ Turning now to the actual methods of study which have been employed m the course of this work, it will be seen that an ordinary chemical examination has been supplemented by a physico-chemical examination of the puncture-fluids, with the object of obtaining an analysis of the grouping of the ions present in anv givtn case. \ consideration of the details of the ionic theory on which so much of biochemical research is based comes more under the domain of theoretical chemistry. A brief account of this lonic theory, as well as of the theories on which cryoscopy is based have however, been included for the sake of complete- ness • ' but it has been felt desirable that the details of the method of i)erformmgboth cryoscopy and electro-conductivity beomitted. The question of the significance of the concentration of the hydrogen ions as affording an understanding of the meaning of the term acid and base has been entered into, in order to give an explanation of much ^yecia.\ work that has been performed, especially in Italy, and to indicate where such conceptions may be erroneous. The fact that hydrogen ions possess practically the same concentration as the hydroxyl ions in puncture-fluids does not really alter the tact that they have more basic pro- perties than acid, that, indeed, they are not really functionally neutral fluids. The mention of the term cryoscopy inevitably calls up visions of a vigorous controversy between exjwnents of the theory that renal disease is diagnosable by its aid and the opponents of the theory. The fact must be admitted that cryoscopy was unduly advoci ted for a puri)ose for which it is un- suitable, since the estimation of the working power of the kidney cannot be based solely on the molecular concentration of the urine, because the osmotic work iKjrformcd by the kidney con- » See Svante .\rrhenius, Theories of Chemistry, 1907. 8 STUDIES IN ITNtTUKIMLUIDS sists of tlio osmotic work ol wattr-sccrction plus that of water absorption plus that of seloctive a< tion on the constituents of the blood,* a (latum which cryoscopy fails to supply. However, the method is useful as affording,' a value for the osmotic con- centration of a ])unctiue-tluid which, by association with a determination of the electro-conductivity, or a determination of the substances impermeable to red cells (Hamburger's method), enables us to chusify the varieties and amounts of the different kinds of ions present. The determination of the relations be- tween the amount of chlorine ions and of the achloridi ions to the substances for which red cells are imjiermeable,* is valuable for deciding on the molecular constitution of various fluids. Such a study demands, of course, considerable apparatus, J unnecessary for the clinician, it is true, but valuable to the biochemical student to exactly the same extent as is the microscope to the histologist, with the difference that much more care and practice are necessary before the hysico-chemical api^aratus can be effectively used. § Tiie scheme of chemical analysis which is submitted is the result not only of practical experiments but also of study of the various methods which have been advocated by eminent chemists. It was, hovvever, felt that a combination of the different methods of separation of the proteids might be advantageous, and an abstraction of the methods described in many numbers of the Zcitschrijt fiir physiologische (hcmie should be found convenient, especially to clinicians who have not the time to turn up the volumes needed. The confirmatory tests which are given are * Pauli, Physical Chemistry in Medicine, 1907. t The dilference I)et\veen the molecular concentration as given l)y the cryoscopic method and that given by Hamburger's blood-corpuscle method gives the concentration of the bodies for which red c ells are permeable, and those very bodies are all the most important products of ptoteid cataholism. X All this special apparatus barely costs as much as one of the better micioscopes. § The physico-chemical methods of examination may be grouped under the following headings : 1. The determination of the total osmotic concentration of a fluid. 2. The determination of the concentration of the chloride electrolytes in the fluid. 3. The doternuudlion ul Ihe acliloiiUt- electrolytes. 4. The determination of the concentration of the non-electrolytes. 5. The determination of the concentration of the hydrogen ions. INTRODUCTION also a compilation from the literature, but it has been felt that the many confirmatory tests desirable Ix-fore stating definitely that a given substance is present should Ix? collected together. The fluids which have come under consideration have been classified as foil -ws, and are dealt with in the same order : 1. The fluiils which occur in the large serous cavities : (<() Pleural. Exutlations, transudations, pus. (/() Peritoneal. The same varieties. 2. The fluids which occur in the small serous cavities ; such as the joint-effusions. The ccrebro-sjtinal fluid. Fluids derived from cysts : (<0 intra-abdominal ; ovarian ; pancreatic ; rare cysts ; (h) in other regions of the Iwdy. Each of these classes of fluids will be studied from their chemical and their ]ihysico-chemical aspects. Finally, a reference will be made to the cytological characters of various puncture-fluids, as the cellular elements which are present in an effusion have some bearing on its chemical com- position, and are probably largely, if not entirely, responsible for the ferments which it may contain. 4- SFXTION I THE CHEMICAL EXAMINATION OF PUNCTURE-FLUIDS t'oNTKNTs : rrfliininary remarks — Ditticultit-s attachfil to the .•.nalyj'is of thiiils — I'recipitatiou of allmmen l>y mastic, etc. — .Vdsorption — Scheme for analysis of puncture-Uuids. (A) I'rehminary processes. (B) Separation of tlie v;h)l)iihns — Allmmen — (llohulin — .Mhumoses and peptone^ ~ Monaminoacids — .\mmonia — Hydrolysis — Sugars — I'lirins and urea. ((') Dianiinoacids — Residual nitrogen. (D) The glycoiiroteids ; p>eudo-nuicm, its jiroperties and reactions ; para- mucin ; svnovin — Lecithin, its importance. ti)n--titution, allies, and symbiotic substances, an constitution of proteid matter by such an eminent organic chemist as Emil Fischer has jierhaps furnished the most striking increase in our know- ledge not only of pathological chemi^try but of pathology as a whole. The application of this knowledge to a study of the pathological effusions in \arious parts of the body seemed a jiromising field of research, esjwcially as these fluids can be made to afford interesting evidence of the jjrocesses of break- down of proteiil in disease. As L'mber * has said, an effusion into the jieritoneum is an accumulation in a sterile reservoir of the intermediate retrograde i)roducts of proteitl metabolism which are preserved intact for a considerable length of time owing to the processes of absorption being e.-cceedingly slow. * 1 he references to the original articles who>e autliors are quoted are collected into a separate bibliography for each section and placed at the end of this book. lO TlIK CHEMICAL EXAMINATION OF PUNCTURE-H-UIDS I [ The first problem which presents itself is that of devising a Mutable mellwd of analysis wh.ch shall enable the various Ixxhes iik.lv to be j.resent in a ^iven puncture-flui.l to be detected, md ' if ix.ssible. estimated quantitatively. There is. however, the ject will call for further comment on a subse- quent page. 13 STUKIES IN PrNCTlRE-KLL'IDS \\V liavi' tlu-n to >tii(ly tin- pioinitics ot our fluids, not from \hv staii(liM)iiit of a w.ittr-basis, Iml from the stan(l]X)int that the proptrtics arc those posM'ssid l)y colloids towards electrolytes ill ((() ( olioidal and (h) in watery solution. We have also to inar m mind the fact that the proteids present in these tluitls are the hearers of electrical charf,'es. in virtue of the fact that electro- lytes lire present,* ami that the process of boiling, or. indeed, any method of removal of proteid from the solution, destroys or alters the electrical charges, and thus interferes with physico- cluiuicid pn)i)erties. hf^ides allowinf,' free ])lay for new associa- tions to take place. Mkthods for Removing Albumen ^h•re mcntinn of the familiar processes of removal of albumen from a Huid is sufficiint, and those which are advocated for the sti'dv of puncture-tiuids are given in Tables A to D. But an important method of removal of albumen, devised by Michaelis and Kona, calls for more careful consideration, since it makes use of the i)henomcnaof adsorjition for separating off the albumen. These authors ])oint out the fact, already laid stress on, that the ordinary methotis of de-albuminisation of a fluid are unsatis- factory for many purposes, since boiling damages the fluid, alcohol is undesirable in subsequent processes, and salting out introiluces a new element altogether. Starting with the fact that colloids of opposite electrical sign will cause one another to separate out, provided the two bodies are present in just sufficient quantity, the two authors mentioned hit upon the use of a watery solution of mastic which should e.\ert this electrical effect. They found (i) that if a small quantity of mastic be added to a large proportion of albuminous solution, the mastic would not be separable, owing to a protective action exerted on it by the albumen ; (i) that if the i)roportions of the mi.xture were re- versed the ojjposite held good. The explanation of the pheno- menon is afforded by the ultra-microscoi^e, which shows the ])articles of albumen in combination with particles of mastic, in such manner that each i)article of mixture consists of mastic, plus albumen. If there \x excess of the latter, it becomes possible • .Mbumen, pseudo-globulin, and euglobulin show no electrical charge in the absence of electrolytes (Pauli). THE CHEMKAL EXAMINATION OF PUNCTlKE-l LLII.S 13 ,,„ ,ach particle of mastic to »>e completely envelo,>e.l by alhu- , Tic surface tens.on will thus otrly have ettect <.n the :„.n ar..! n<.t on the n.ast.c. Tins -"'«;'«•--;!! ,„„,,, n,ast,c has been ad.lecl that .t cannot all be n.askul. ,,„a ,a that iH,int the whole of the albumen w.ll separate out. The methods which they advocate are : . , , , ■ Method I -A lo-per-cent. s<,lution of mast.c m alcohol is suddudv d,h.ted wilh tw.ce its bulk of water, an.l the result.n^ .n.x.ure .s ad.led to the fluid to be de-albumm.sed u, In pro- ,x,rtion of lo parts n.astic mixture to i of l^u.d ; z pa b, o lo.,.'r-cent. acet.c aci.l are then aclder,od of digestion in a tep.d water-bath, the flu.d xv.ll be found to filter easily and leave a completely albumen- free filtrate. In the event of the removal Ixnng .ncomplete, a third addition of mastic is indicated. MclhoJ B -50 cc. of fluid are diluted with 12-15 parts ol Nvater. and as much acetic acid is adde.l as will clear up the tur- bidity To every 100 cc. of fiuid 20 gm. of kaol.n are added in four or five instalments, vigorous shaking Ix-ing carried out during this process. The filtrate will now Ik quite free of albumen. This method, published in July 1907. obviates the necessity .n method A of first getting rid of excess of albumen by a prel.m.nary alcohol piecipitation, wb.ich renders method A applied to fluids rich in albumen of no advantage over former methods of de- albuminisation. The filtrate from these methods contains monammoacids, diaminoacids, and jwlyi^-ptids ; and i' is necessary to remember that there is present in this filtrate a nitrogen-free sulistance of unknown nature, soluble in water, which will come down with phosphotungstic acid unless an excess of tannin lie added to prevent its precipitation by phosphotungstic acid. The precipitate will contain albumen, globulin, and albumose, but no sugar. Pkkardt's Method of Removing Albumen.— 200 cc. of fluid are boiled for three-quarters to an hour ; then add dilute acetic acid, drop by drop, till just acid. Heat three-quarters of an hour in a '4 STII»IES IN lUNCTUkK-KLLins current of stoaiii. lioil tlu' iv^ulm' witli water, iitul sqmrze it out in a han.. Tlit' mixt-d ftltratt-s are hitereil while hot and the residue washi-il with hot water. The filtrate wil! Iw abso- lutely albumen- tree. A climml inclliihl ot estiniatinj,' albumen has been devised by Deyc ke and ll)rahim. J cc. of fluid arc mixed with j cr. of n '5 soda (forms a Miltiblc alkali-.ill>uminate). ami is then made up with soda to loo (c in a tlask. J5 ic. of this diluted solution are placed in a flask "f o cc. capaeity. and 2 jcc. of jjlaiial acetic a(irot...l .loos hoUl so.luun ,.,,,,„„,, ,„ „s ■"'■^'^*^''";'"V' ,' us.s will ..nlv ropn-sent that ,H,ittoiioi tJu- N.Hl^^'»"^'" ,h.. roiirulum. \\V shouhl say. Lv,nK passo.l '"evocahlv ,nt , th. u ^ Urn ^^^^^^^^ , ^.^^ w.th S. M/., tlut sonv of th. ( 1 .> > ";> > „,.. pn.t...l. ami tlu- ^ >s - accuU-ntal. ,„,,.., ,1». very --.mo M"-""" »""'«'' ,^ „ ..hvs.cal coml.t.on ol ttu- ,.,,Ma,K.> m s.rum ur. ....1.1 >n - '"^ ";^ j^,.,; „^ ,„,.„„, ■,., s.,lvo the VV...le B-ivhss acH-s at U-n.-th into the effect of Congo reel on While Bay hssM ^j adsorption, we nUer-pa,K>r -/" "^;^'tu ngs of others,* who ins.t that rece.ve a rebuH f om ^ J? ^ ^ -j-j,^. ^^^„,, author clescr,lx>s .he reaction. sr^lly^^.^u^<- hut in the ex^H-nment. the a.lsorpt.o.. u. . a . > ^ ^^^ ^^^^^ ^^ ^^^^^^^ „arrate.l U cannot Ix. ^aUn as com 11 ^^.^.t,,„„„, ,,, -isorption, and ^^^^^ ^^ , t:t'washing w.ll remove U gelatine ^ -^^ ^^^ ^ ^^^'^^^ ,i,„g ,,„ remove much less if :;;r"I «: ^..J :i '^^. ^-ause the ^.rcentage of salts on ^^ ?Th :: v^^i t vm mu h less loss of salt at the fourth wash, ^"on .r "r!:.^ there is not. at hist sight, any d.f^rence ^ . n the nrocess descrilx-d by Bayliss in washing out the ri/o'^olT^r^ino a,u, onUnary w...ng o„. by C,«-..„n. In oach case the rate cl loss o( salts w.ll be sl.m. vLlmK to V. Ik-mmclen. the factors on which adsor .- ,„, ■ cM.:nds are : the adsorhing substance, the solvent, the » Above atl, o{ Gustav Mann. I6 STUDIES IN I'UNCTURK-FLUIDS sul)stanrc to bo adsorbed, tlu" state of its inolecules, and the tiMiiptratiirf. 'I'ticsc conditions, liowtvcr. aw not snttuicnt, since there must 1) ■ a liinr I'lcnunt to consider. Fortunately, the sul)ject beconie> more intelhgible from H(')l>ers' clear and distinct diction. This author |)oints out three properties which are characteristic ot adsor])ti()n. 1. The e(piilil)rinni l)et\veen the ad>orl)ed >ubstance and the nonadsorbi'd substance varies according,' to whether the s'X'ond is added en masse or by instalments. It is a surface tension [)licnonienon. and if the surface l>e altered by a iHeliminary addition a further addition to th t mu lace will not meet with the sauH- condition: as did the hrst | iH" i. 2. The reaction between aii ii! and >oKent is reversible oniv for a shtjrt time after its ince])'ion. \. The e(iuilii)rium-constant alters wuh tin .' (" old-age "). To use a mathematical expression, we may say that if an albuminous fluid adsorbs chlorides from a solution of chlorides, the amount a.lsorhed will depend on the concentration of the tiuid antl of the solution. Expressed graphically, a curve rei)re- senting the ratio of the adsorbed to the unadsorbed substance {abscissa) will always be concave towards the abscissa, since the power of adsor})tion of the adsorbing substance is greater at the beginning than it is at a later stage in the process. When this is put into a mathematical formula, we have : 'adsorl)ed subst. - k. unadsorbed subst. where k is called the adsorption coef^cient, and /( is a constant greater than i. This fornmla, however, does not necessarily hole! good only for processes of adsorption, i.e. of depression of the surface tension between fluid and Cv)lloid. by the dis.solved substance. It would equally well expres.-> that a soliil solution is formed in wtii( h tile chloride is divided over the fluid and the solid solvent. There may be a question of electrical charges in this asixnn of the conditions. Without pursuing the theoretical side of the (juestion further ve may come to the conclusionthat if the effect of addmg sodium TIIK CHEMICAL EXAMINATION OF PUNCTl'RE-FLUIDb 1/ fliloride to an albuminous solution can be expressed by the formula given above or by this formula : •^^'Cl in alb.= ''- V ^Cl in water. wo may assume that adsorption exists between these two substances. ■ „*. Several tests were applied in order to endeavour to arrive at a conclusion as to the accuracy ot the cM..ride analyses which have been made in puncture-fluids. The variations m con- ductivity shown by different strengths of saline solution in the presence of different percentages of albumen were determined, and in another series of tests, different solutions of egg-white and sodium chloride were made up, and the amount of NaCl estimated in each volumetrically. In a third series of tests a known strength of natural albuminous fluid was treated with a known quantity of NaCl, and different dilutions made the con- ductivity of each of these being tested, and in a final series o exix^riments a known strength of albuminous solution (human) was treated with a known weight of albumen, and the coagulum v.as washed with distilled water on successive days till no more chloride could be detected in the filtrate. The strength of NaCl and the conductivity of each washing was determined. The washing was carried out by taking up the coagulum out of the filtrate, transferring to a small flask, and adding about 80 cc. of water The contents were then well shaken up and allowed to stand'for a time. The mixture was then filtered, and additional water poured on to the precipitate until 100 cc. of filtrate was obtained. .,, , ^ ., ., , Without entering all the results, we may illustrate the method and the results obtained by the following observations. I. By titration, adopting the usual method of de-albummisa- lion, 100 cc. fluid, containing— s : 4 4 2 2 2 2 albumen (egg) + + I % NaCl yielded 0-85 05 025 I -■>5 01 0-85 % NaCl on titration. 0-45 0*25 I. 092 ,. 05 M 025 .. 01 „ IS STrniKS IN I'UNTTrKK-KLUins 2. By successive washing, Kir.-t filtrsf S( cond Third Fourth „ From these experiments albumen is low there is no k26 = 1800 = 1035 404 an n 20 we see that when the percentage of adsorption of chlorides by proteid, while with higher concentrations of albumen, however, there is a de- cided loss, which is greater the more chlorides there are. Putting the results of the last table in the form of a curve, and comparing it with that from an ordinary case of adsorption (tig. i, «), it is at once evident that there is a dif- ference between the two. From our point of view, then, there is no im- portant error likely to arise in the ordinary method of analysis, pro- vided the percentage of proteid he not large. Bugarsky and Liebermann have settled the cjuestion of adsorption ol XaCl by albumen by determining the Ireezing-jioint dejnession of solution, containing varying weights of albumen. Thus : a roprt'Sfiits tlu' vaiyin;,' adxirption occurring in dil- ftTfiit conctntrations of Congo red and cellulose (Bayliss), while /; shows the conditions which obtain in the case ->( siriMii alhiiniin and XaCl (.Author). Amount of Albumen Kreezinff point 100 cc. 1 NaCl DL-prtssion. 20 0183= 04 Olio 0-8 OI()I 1-6 0194 P 0199 6-4 0-2(>3 Freezing-point Depression if no Adsurption, 0183° 0-183 + 0004 = 0-187 0-183 + 0006 = 0-189 0-183 + 0C09 = 0.192 0183 + 0015 = C198 0183 + 0022 = 0205 They conclude that the similarity of the figures in the second THE CHEMICAL EXAMINATION OF PUNCTURE-FLUIDS I9 and third columns indicates that no union has taken place between the salt and the albumen. However, the fact that the observations are made at ordinary temi^erature and at about o"" C. shows that such an experiment does not decide whether adsorption might not occur at 100'' C, a temperature which all the puncture-fluids have to attain in the course of the analysis recommended in this w^ork. Scheme fok Analysis of Fcncture-fliids After these preliminary observations on the difficulties with which one has to cojie, we may proceed to formulate the method of systematic analysis which has been adopted for the chemical examination of puncture-fluids. The following scheme is based on a very considerable litera- ture, and as by its aid more than 200 puncture-fluids of all kinds have been examined, its practical advantages have hizn carefully tested. It may, however, be noted that there is not always a need for following out each table from start to finish ; a judicious selection can be made of those portions of the scheme which are likely to give information in the particular class of fluid under examination. Where any modification of the methods has been found convenient a mention of it will be made in the appropriate places. In order to make this work more useful to those who wish to apply these suggested methods for purposes of diagnosis, the confirmatory tests, especially for the less known substances, are entered along with the description of the particular substance. Inasmuch as there are constantly being devised for many of these bodies, new tests, which the ordinary reader cannot ascertain without a very extensive search through literature, it has been thought of value to gather together these innovations, esjK-cially those new tests which have been found of use in the Leeds General Infirmary. 20 STUDIES IN I'UNCTrkK-KLriDS TABLE A. PRELIMI The fluid is heated in the presence of jnsl oiniii^h acetic acid cess in a water-bath placed on a sand-hath, in order to prevent to boil, a few drops of dilute acetic acid are added until a coaguluni this can be controlled by litmus). Coagulum. — This consists of albumen, globulins, and nucleo- meshes (such as mucoids). J The Filtrate should still be acid, and if an estimation of by adding distilled water to the coagulum in the filter-paper, the remainder is tested in successive jiortions, thus : Add an equal part of methxlateti spirit. Add liiisic lead ncelale. Precipitate IS hftft'o- nlbiinios€, (c<.;i>r. by ; 65^-.) ; s. p. 26 Solution. Test with potass. ferrocyanide, anil acetic acid. PrecipiUM I 'rote ids not required. Soiution. Remove the lead by H..S. Precipi- tate not required. Solution Ptuttilbu- IllOSf. s. p. 26 Precipi- tate not required. Solution Kvap. in a drop of i)ure nitric acid. Dissolve residue in a few drops of soda. Warm in a platinum capsule. If oily drop forms = leiiciii. 3. p. 27 ! Add .Vninio- niacal Silver .Nitrate : Pre- cipitate = piirins. Or evap. to dry- ness with HN03;add one drop Ammonia. If violet forms =purins or uric add. (See p. 31 for confirm- atory tests ; also footnote P- -6.) * Devoto t 'lo detect miclco-protcid (Jolles). Boil the original fluid witl' excess After 24 ho ;rs, the asbestos filter which has been used, and the precipitate, the filtrate with acetic acid, redigest, and finally purify the precipitate by for pliosphori\s as described on p. 40. The filtrate from ammonium molyb- condenser. The fluid, after claritication. polarises light to the right, and with jihenylhydrazin (therefore mucin). To exclude mucin and nucleo-albumen from nucleoproteid, l)oil the filter while hot. Anipioniacal silver nitrate precipit.Ttcs: nucleoproteid X Willanen. § This is a modification of Seliwanoff's test (p. 32), which I believe to be Tin: ClIKMICAL KXAMINATION OF-" I'L'NCTUIiK-FF^L'IDS 21 NARY SEPARATION until coagulation results; i is convenient to perform this pro- -xcessive heating in the lal stages. When the water is about ceases to appear (only some half dozen drops will be needed, but proteid t, apart from substances that may be entangled in its chlorides be desired, it is made up to loo cc. (the original bulk) 10 cc. of the filtrate are then set aside for the determination, and ^.tturatc i\vi> Ihirds with Amnionium .Su!ptiat4 . Precipi- tate K ntcro- iilbii- iiiose a Solution m.t re- quired. Sat ur .tie the neutral i sulutiun with Ani- : monium bulphate. Precipl- Solution Mite not rt- Dciitiro- quired. albu- mose Concentrate another portion Neutralise a of the filtrate in vacuo, at not portion ot' the more than 40° C , or leave filtrate with :; to cci.centratc spontaneously, potash, and ;! I'lace the fluid in a porcelain then conccn- : basin, and add resorcin till a trate on the fairly strong solution results. water-bath. Heat the basin ovtr a liunsm , If a precipi- llame. after the manner of the 1 tate forms on Gunzberg test for free HCI, adding alco- and if a red colour appear, hoi. suspect soluble in alcohol, then /evti/osr the presence is present. i) The confirmatory of succinic ; tests and other details are to acid (con- I be found on p. 32. fi r m a t o r y tests, p. 194). of water, and acidulate with acetic acid. The precipitate is nucleoproteid. are digested in an Erlenmayer flask with 4 per cent. soda. Ueprecipitate washing with alcohol and ether. 'I'he dried substance is incinerated and tested date is hydrolysed for 2 hours with 30 cc. of 4-per-cent. HCI, with a reflux on removing lead will give the orcein reaction, as well as give a glucosazone fluid with 5 per cent. H,SO„ and neutralise with baryta while hot, and and purins. Mucin and nucico-albuiiicu temaia in solution. convenient. 22 STl'DIKS IN I'UNCTrKK-KI.riDS TABLE B. SEPARATION OF GLOBULINS AND THEIR ASSOCIATES I. Add an equal part of thoroughly saturated pure ammonium sulplialf, and leave to stand ov-ernight (or longer). Solution. Contains chielly albumen and secondary albumoscs.* Nut required. Ketldue. I>i;ilysr, dry, i'ud vveipli. Contains plobiilins, nucleoalbumen, piimary allnimosc, peptone, liistoncs, and lecithin ( I I Knse some of the residue with KOH + KNO;, and te;,; lor phosphorus (nucleo- albumen, lecithin). (2) iJissoKr a portion in water and add ] act til- arid (lip to I ). After prolongtd j stardiuf:, PrecipitaCb - sero .ainmin. ' 2. To another portion add ilouhle the hulk of saturated ammonium sulphate to precipitate the eiiglohuhn.t ♦ As tlie liltrate contains alliunieii. it may be boileil as in Table A and the 'eiKlit of albumen ul'imately obtained. The ratio between albiinien and pjlobulin will tlien have been obtained bv the one process. t .\ simjile e.xj-.eriment which was made will illustrate the importance of delay before collectin'j the precipitates obtained by salting out proteuls. toil cc. of a |ileural lluiil were treati^d with .'■ ■■ cc. ui saturated ammonium sulphate itliiis forming a '>'>-per-cent. solution). .\s usual, there was no effect for some tin.e. but next morniiiK abundant llocculi had separated. Tliey weie, however, left for two more days, and then filtered otf. The fluid, which, it may be incidentally mentioned, was turbid, now yitdded a clear liltrate, anil iiendiuK further study of this liltrate the specimen was left in a i)liii;yiMl ICrlenmeyer Mask for ten days, at the end of which time it was noticed that there was a not at all incon-.i(lerable further tlocculation. This of course shows that ■-.iltiiif,' out may take several clays to comiilete (there ciniUl be no coricentratiou of the lluid durinj; this time, otherwise one minht argue that with increasing concentration of ammonium sulphate a ditteient fraction was sejiarating) In studvitig the ditterent globulins, bv weighing, and ^o on, or in col- lecting globulin in order to estimate associateil lecithin, one conse<|ueii ly neeiK to allow a coie-iderable interval to elapse before regarding the salting out as complete. fa the other hand, in a note by Cecil Bosamjuet {Laiii\t. 1907) he states that the globulins alter on standing, so that the relaticms between the eu- and jiseinlo-globulin fractions to albumen will be ditterent in the same liuid according as it maybe examined immediately or after a time. He con- sidered that an enzymatic change occurred, by wliicli albumen mav be con- verted into globulin. The view is |)robably too pessimistic, because if one adds the values given for " ascitic fluid .\ " one tiniis that the immediate estimation gives total glolnilm : albumen --- j-^ : n, and the later estima- tion still gives J ? : im. The other case certainly shows a change from 2-; : !•; to j-.>< : i^ the pseudo-globulin having decideillv increased. The duration of time allowes the most conspicuous. It is hrst necessary to describe the varieties of globulin which are met with, because it is imi)or- tant to distinguish between them in practice. Frtund and Joachim iiave classified globulins as follows : Name. I'rtcipiiant. .Solubility Solubility I Solubility iVecipi Water. "•''*"^^^'l s^h. tatitin Limits. 1. Paraglobiilin or Ovi- miiiiii • ... 2. Ktigliil'iilin 3. I'arap.st'tido- globiilin or dysglobu- lin* 4. I' s e u (I o - globulin 5. Nucleoglo- biilin (con- j tains I'.)... i Cna^ula- ti. n Temi-era- ture. Irclsat.AiiKSO, Insoliibk II Soluble sat. Am^.ISOj Insoluble Soluble Soluble Insoluble Insoluble , 2'ii-yb Soluble Insoluble 36-44 70-77=C 64 70X 74-76'=C 76^ These authors lay stress on the j^hysiological importance of these varieties of globulin bystating that pseudo-globuhn contains the antitoxin for tetanus, paraglobulin has the i)roperty of l)recij)itating egg-white, cuglobulin favours the precipitation of myosin, while pseudo-globulin inhibits the action of euglobulin.f The same authors point nut that a globulin may be (i) soluble in water (pseudo-globulin), (2) soluble in 06 per cent. NaCl, but insoluble in water (euglobulin), (3) insoluble in either 06 per cent. NaCl or in water, but soluble in 0-25 i)er cent. NaaCOa. This will explain the characters of certain kinds of peritoneal exuda- ♦ Of Ohrmeyor and Pick. t Tseudo-globuiin thus plaj-s the part of an " antiferment," as it were. Till. (IIKMICAL KXAMINATION OF I'f NCTURK-FLUIKS 25 tions, since, if the carbonates be present in excess, the third variety of globulin will be in solution, while if there are deficient salts,or if the chloride concentration of the fluid be less than that of the blood, one may exi^ect euglobulin not to remain in solution, and so cause a turbid })erit()neal fluid. V'e shall have to refer to this matter again wlien we come to de d with opalescent and milky jx^ritoneal fluids. As regards the amount of globulin which is present in a fluid, it cannot be said that this shows any constant relation to the nature of the fluid, though, as a rule, there is less globulin jiresent in jileural than in peritoneal fluids. The abundance of globulin in peritoneal fluids is probably an explanation of tlie strikingly more frequently occurring opalescence in them than in pleural fluids. The varying glolnilin-contcnt of fluids is shown by the follow- ing table of some of the analyses from cases in the Leeds General Infirmary. TABLE I Gl.OBUI.IN-KlXTKNT OF I'lNC TIKK-Fl-IIDS TMonolobular Cirrhosis Tuberculous Pciitonitis Peritoneal, Cardiac Cardiac and Renal... lonolobular Cirrhosis /■F.xudalion (simple) Another case Tuberculous Exudate I " Idiopathic " ^' Ca Ca Mc Pleural Cardiac Failure w >> >• Ovarian Simple Unilocular Cyst 4-S«S % o s68 V 0-25 % 0-4 % 0-06 % 0-S8 % 0-84 % 1-8 % 0-87 :; f36 % 077 % o 04 ", 184% 2C79 % The Substanxes Detected by the Filtrate of Table A Albumoses and Peptones.— To confirm the presence of albumoses, a portion of the filtrate from Table A may be (i) saturated with NaCl, (2) saturated with MgSO,, (3) dialysed. The piotalbumose will pass through, while hetero-albumose will remain behind. Protalbumose made alkaline with potash, and treated with 2 per cent. CuSOi will give no precipitate. Hetcro-albumoso is precipitated. 26 STLItll-S IN I'L'NCTI'UK-FLIIDS The differences l)et\veen these two substances may l>e tabu- lated tliiis : lett. I'lotilbuiiiosc. I Secondary Albumoie. NaCI to saturation r acetic ai id satu- rated with salt Biuret (2 : loo) in neutral solution ... Potaj^, ferrocjanide anil aci tic acid Nitric acid Precipitated No No No precipitated in salt- only precipitated in free solution presence of salt Half saturation with ammonium sul- cnmpUtc pncipila- No precipitation phate tion Tlie investigations which liave been made in order to ascertain in wliich fluids albumoses and iK'i)tones were or were not present are shown in Table II. It will be at once noticed that peptone is unitonnlv absent from both pleural and peritoneal fluids, and that juirulent fluids contain both forms of albumose. This is as mi^,'tit be expectetl, for. as we shall see, proteolytic ferments are a conspicuous component of the polvnuclear leucocytes. Umber states that ])rimary albumose is always present in a fresh exudate, and that deutero-albumose is often met with, while true peptone is absent. It will be seen from Table II that wiiile protalbumose is absent in a few cases, the results are other- wise in accordance with L'mber's statement. TAHI.K II Nature of Case. Hrol- albumose. Hetero- albumuse. Peptone. Pleural ... bilateral (single) ... lubcrculous ,, Cardiac Failure idiopathic Chronic Tubercular .Nephritis ... Empyema ... ... . . ^ II Cardiac (back-pies!-ure) ... .Monolobular Cirrhosis ... I'eritoMcil Cancer -Ad!. Pericardi'.ini ^haik-pri'Fsr.rc) ( hri nic Peritonitis (non-tuber- culous) ... Absent Present Absent Present ,, Absent Present '• )i Trace ' Absent Present Absent Present II Uniformly absent. Peritonea! - Absent 11 Trarc Uniformly absent. ( * Tin: ( IIFMICAI. EXAMINATION OF I'UNC TUkl-FI.UlDS 2/ The Monaminoacids (rIvcocoII, leucin, aspartic acid, serin). , . , . • - Traces of these substances have been described by variou, authors as occurring in exudates. es,>ecially in ,)eritoneal thud.. Vmber states that they are always present, but from the e.v nerience of the cases in the Leeds (ieneral Infirmary they are not found at all frequently. It must be admitted, however, that possibly too small a quantity of fluid was utilised for their detec tion • ^oo cc. or more might furnish better pros,x"- «f /i-naphthalen.-sulpho- chloride (Merck) by the aid of a mechanical shaker. \ separating funnel is now used and the watery fluid is shaken with ether, and rendered acid. The ether takes up the fl-naphthalene-sulphaminoacids out of this acid fluid, especially if a little powdered ammonium sulphate lie added hrst. The aminoacids crystallise out and can be recognised from their physical properties and their microscopic appearance. This method is employed by Erben as a quantitative method, but since he admits that sometimes only 577- ^^nd never more than 80 per cent.-varying according to the particular aminoacul -is recoverable, it can hardly be recommended for quantitative purposes. Confirmatory Tests. LECC.N^JJ5>CH.CH..CH.(NH..,).COOH. I. Very dilute copper sulphate gives a blue colour. 2 Very dilute ferric chloride gives a red colour. 3'. Mercuric salts, in the presence of soda, give a white precipUate. ^^^^ ^^^^ ^^^^ _^ ^^.^^^ ^^^^^^^ ^^^ ^^^^^^ ^^^^ ^^ excess), and boil till ammonia ceases to come ott. Filter, wa.h ♦ More if necessary. The fluid must be quite alkaline. 2S STUniKs IN ll'NCTLkK-II.riDS Willi water, aiKl .vapor.itf down (iltrato cm a water-l)atli. If niT(-«s;ti V. (ilt.r aKaiii, and ranfullv .m idify with acetic- acid. A crystalline [.nripitatc will a|)|H'ar. insoliil.le in ctli.'r. Imt s(.lnl>l.' in alcohol and alkalies. Meltimj-point, _'o5 C. It crystallises nut from alcohol in lon^ ne.dles of isol.iitylhydantoic acid. Detects out {,'ni. lein in. 5- Sc/unr's /Vs/. -Kvaporate the substance in a drop of jmrc nitric a( id. 1' --.,.. tli." residu.' in a few droi)s of soda. Warm in a i-latinuni capsule. If l.-ucin I..- present, an oily .hop will ai)i)ear. rolling alM>ut without wetting the |>latinuni. (>. I'he acpieous solution is htvorotatory ,,„ — -(> 1)5 '. 7. It suhlimes on heatinj,', and givesoff an odour of ethylainine. Ci.Ytocoi.i. CR, (NH,.) . COOH. I. A.M K.nr tmi, . ihr «,,«!„ „, ,|„. ,ul.,taiK,. to 1... t.M.-.l <,t alcoholic M.h.t,o„ of ,„cru M.„t. .;iyc,H:oll p.crate w,ll separate out on c,K,l>nK. -Miltllli; point. I.)<) ( . *" .-. T\w trystal, Irom ,■( naplitiialeiu-siilplioclilori.U- nult at 15.,^ .?. C opp.r Milpliatc «ivrs a l)|iu- colour. (C H \o ) („ + n O 4. On .,M.lat,..ti w,tl, 111)., Klyoxyl.cacul an.riormai.l.hv.lc- an- iornu-.i 5. l>rrii ililoridi- gives a (liip rid colour. AsPARTic Acid COOH . CH (\H,) . CH,. . COOH. 1. Copper at. late giv.'s a l.lue crystalline precipitate. 2. Strongly acid solutions are (lextrorotatorv fa, = ji--, ) .1. MeItin«-l.oint. 270 . > ^ " -=3 7 )■ Th.. chief ,ntc.r.st of this particular substance li.s in its association >s.th flu- all.un,,n of carcmon.a tissue. It n.ay const.tute as .nuch as 5 to 10 per cent of the al ..onen ..ol^culc .n tlu-se cases, and should therlre poss.My be nut wuh in the fluids of caicinoinatous serositis. In its detec- tion ,t IS n.cessary to remove the other n.onaminoac.ds first \s,,artic acid IS an essential constituent of the f-Iobulin molecule. Hefore passing on to consider the i)urins it will be convenient to describe two other analytical processes which are of use in the study of i)uncture-fluids, though not available for routine work, owing to the time which is involved, and the absence (so far) of any definite diagnostic or prognostic deductions to be made from the results. The first and most important is Hausmann's method of hydrolysis of pumturctiuids. 10 cc. are boiled with 29 cc of concentrated Htl for five hours on a sand-bath, using a reflux condenser. Saturate the residue with calcined magnesia and TIIK IIIKMICAI- KXAMINATION or I'LXCTUKK-H llltS 2() (li^til ovt-r tlu- ammonia into decinormal sulphuric aciil. It is moil- safe to placo sufticii'nt nia^m'sia into another tlask and add the result of hydrolysis through a funnel. In this way no gas will Ix- lost. This hrst step gives the amid-N. As soon as distillation is complete the resitlue is dissolved in HCI, brought down to a small bulk, and phosphotungstic acid addeil. After 24 hours the jnecipitate is washed with dilute phos- l)hotungstic acid, acidulated with HCI till the fluid ceases to have a yellowish tinge. a. Filtrate. Make up to 500 cc. and Kjeldahlise 100 cc, to get the tnonaminoacid-N . h. Residue. Dissolve in as little alkali ;i.s jKissible, and make up to a definite volume, and filter. Kjeldahlist>, to get tlm dtaminoacid-N. Scblbsing* Method of Estimating Ammonia.* - 10 cc. of normal sulphuric acid is plactd into a sporulatniK ilish in an t'xsiccator. anil lo cc. of till- fluid to bo tcstctl is platt-d in tin- ixsiccator. 50 cc. of milk of lii'u: is added to the fluid and tliu covir rapidly placid in position (vaseline joint). \fter three days the sulphuric acid is titrated with soda, using metlisl 4 orange as indicator (turns yellow). Every centimetre of the sotla which is used in titrating less than \i<) signifies -lo*) gm. ammonia. The Purin Bodies, and Urea.— The purin bodies have lieen the subject of careful study in many quarters, and it is un- necessary to enter into any description of them other than the facts about their occurrence in puncture-fluids. As regards urea, this may Iw tested for in the filtrate from Table A by pouring the filtrate into excess of 95 per cent, alcohol. After several hours, the extract is evaporated over a low flame, re-extracted several times, and the final extract dried, when cold a few drops of nitrate acid are added, and the typical crystals of urea nitrate are searched for 24 hours later, t The addition of hypobromite to a portion of the original fluid will also reveal the presence of urea owing to the efferves- cence which results. As a rule, however, there is so little gas evolved that an estimation of the amount of urea is not jx)ssible. • Uurig gives numerous useful hllle prdCtiCal details in BtJir.cm. Zeit. iv. t Salkowski. .^o STUDIES IN PUNCTUKE-FLUIOS ■Il= TABLE III Uhea-contknt ok I'l'nctl'hf.-Fluids Abfent in. Kaiiit Trace in. ; Trace. Decided Amount in. riirombosis of Carcinomatous I'leiiral 'Single Pleural Effusion. I'oital Vein Cirrhosis o I Liver M o n o 1 o b u I a r Cirrhosis Toxic Ntphritii Sarcoma of Omentum Tuberculous Peritonitis Ascites (2 Eliiision cases) (3 cases) Cardiac Hack-pressure (2 cases). Empyema. Hydronephrosis, Peritoneal Fluids: Cardiac Batk-pressure. Chronic Peritonitis (single). II „ duet J Cirrhosis of Liver. Peritoneal Cancer. Syphilitic Cirrhosis of Liver, I -42% (Poljakoff). From this table it will be seen that urea is much more fre- quently present in cases of pleural than in cases of peritoneal effusion. The presence of urea in purulent fluid such as empyema may perhaps be expected from the fact that pus contains so many extractives, in virtue of the cellular elements in it (see Section III.). The practical deductions which can be made from the pre- sence of urea are, however, scanty. Its occurrence in various l^leural and peritoneal fluids suggests that one cannot be sure that a fluid from the abdomen is from a hydronephrosis just because urea is jiresent in it. Probably we need to know the amount of urea present, in order to be able to say that there is more than ordinary autolysis (rather than renal secretion) could account for. This subject is again referred to under " Renal Cysts " in Section III. The purin bodies are stated by Umber to occur in minimal traces in exudates. They are best separated by precipitating the phosphates with ammoniacal Ludwig's magnesia mixture, and then adding 05 per cent, silver nitrate in 50 })er cent, ammonia to the filtrate. The precipitate is placed on an ash-free filter-paper, and washed till the filtrate is no more alkaline. The residue is boiled in a Kjeldahl flask with water and some magnesia, and is then Kjeldahlised. In this way the nitrogen of the purins is estimated. The uric acid-nitrogen is also determined in another ixjrtion of \i THE CHEMICAL EXAMINATION OF I'UNCTURE-FLUIDS 3 1 fluid, and the ditference between the two values gives the nitrogen of the purin bases. The time involved in separating out the individual punns is not profitably spent in connection with puncture-fluids.* ConfirmatorvTests.^i. Burians Test.-Make the solution alkaline with so- only fallacy to this test is the presence of histidin.) GLYCOPROTEIDS The diagnosis of a puncture-fluid from an ovarian cyst is occasionally necessary, so that it is important to discuss the properties of those substances which lend a distinctive character to the contents of ovarian cysts from a chemical point of view. The most important of these glycoproteids is pseudo-mucin, which was first carefully studied under the name of metalbumen by Hammarsten in 1882. It is so called because it is not precipi- tated by acetic acid, and yet has a mucinous consistence. The presence of allied substances in the peritoneal fluid and in the synovial fluid renders the subject not only one of academic interest, but also of interest to the diagnostician. Glycoproteids are substances which contain a carbohydrate radicle (mostly in the form of glucosamin) attached to the proteid molecule, and it is the glucosamin which is responsible for the similarity of the reactions between the various substances above named. | • The use of this reagent for distingui-shing between tubercular and non-tubercular exudates is referred to in Section IV. t Tke glycoproteids arc fully described in Mann's " Chemistry of the Proteids," but many pomts of mterest m the present connection lind only scanty notice there, so that one feels justified in entering thus fully into it. 3^ STUDIES IN I'LNCTURE-FLUIDS (llucosamin is dextroso with the H atoms of one of the two CH:;()H {,'ioups rephued by an amino group (XH^), so that it forms, as K. Fisher pointetl out, a link between carlH)hydrate and jMoteid. The reactions of this sul)stance are : 1. The MoHscli reaction is positive (alcohohc u-naphthol fol- lowed by strong sulphuric acid, gives a violet colour which is turned yellow by alcohol, ether, or potash). 2. It does not ferment with j-east. J. It gives Trommer's test. 4. It gives a glucosazon melting at 202" C. 5. It gives Ehrlich's glucosainin test after adding an alkali such as baryta, and then warming. (This test consists in a red colour obtained on adding a 2- to 5-per-cent. solution of /)-dime- thylaminobenzaldehyde dissolved in normal hydrochloric acid * till acid.) TABLE D. SEPARATION OF GLYCOPROTEIDS I'o the tluid add three times its bulk of absolute alcohol. Shake occasionally. I'ortioii I Allow to stand ^4 hours. I'lirtion 2 AMdw to stand j to 3 months, then evaporate the alcohol at i 40' C. Portion 3 Kilter at once. Precipitate. Shake with slightly alkaline distilled water. Filter. Solu- tion. not re- quired Precipitate. Shake with slightly alkaline distilled water. Filler. Solu- tion, not re- quired Resi- due. not re- quired Fil- I trate. Test for ' Pseudo- 1 mucin 1 Resi- Fil- due. trate. not re- Test for quired Cholin, Lecithin, Mucin ; i Resi- Fil- due. trate. not re- Add to quired solution, or lest separate- ly for albu- moses Serosamucin (Umber). — This has been found in peritoneal fluids due to inliammatory processes or to new-growth dis- • Normal HCl is made by adding 5 cc. water to i cc. pure hydrochloric r.cid. Precipitate. Shake with distilled water. Filter. Solu- tion. Test for Albu- moses, Mucin. THK CIIEMICAI. EXAMINATION OK rUNCTURE-FLLIDS 37 stinination. The L,uhstance will come out of solution if to the (U'-alhuniinised fluid a small quantity of very dilute acetic acid i> added. The precipitate will have the following reactions and propertic-; : 1. I.t't ono drop of solution drop from a glass rod into a loo-cc. measure ol distilled water to which a couple of drops of glacial acetic acid have been adiled. A cloud will appear if serosamucin be present. The precipitate- ly soluble in motlerate excess of acid, and is reprecipitated by further dilution, while it may be reilissolved by rendering the fluid neutral or -lf.;htly alkaline (Rivalta's test). 2. It contains a minimal amount of reducing substance. V It is precipitated by alkaloidal reagents, such as potassium ferro- cvanide. nitric acid, copper sulphate, ferric chloride, lead acetate. 4. It is precipitated by an equal volume of saturated ammonium sulphate. 5. It gives the biuret reaction. (k It gives Millon's reaction. 7. It gives the xanthoproteic reaction. 8. It gives a strong furfurol reaction (Molisch reaction). 9. It gives .\damkiewitz' reaction. 10. It gives Liebermann's reaction. 11. It is not coagulated when boiled in neutral solution. 12. It is not separated by dialysis. 13. It forms a precipitate when digested with pepsin. 14. It gives an absorption band in orange, and slight darkening to the kit of red when treated with orcein hydrochloric acid, and extracted with amyl alcohol, possibly indicating pentoses or glycuronic acid. The percentage of nitrogen and sulphur has been remarked on by Umber as showing the body to belong to the mucins. The question as to the nature of this substance is still unsettled. Whereas Umber considered it to be a mucin, though not a true mucin, because it contains too much nitrogen, Langstein objected to this view, on the ground that a reducing substance can be obtained from all proteids. Stiihelin considers the body to be related to globulin, since it possesses similar solubilities, and is precipitated by half saturation with magnesium sulphate, and is not separated out by dialysis. A similar discussion has been raised about the mucin of the urine, which was once regarded as a mucin, then as a body re- lated to Bence Jones' proteid, then as a globulin, then as a nucleo- albumen, then as nucleohiston. Stahelin believes the urinary substance to be identical with that of the peritoneum, and with that found in blister fluid. Rivalta and Primavara, more recently, regard it as a mixture of euglobulin and pseudo-globulin. That 38 STUDIES IN PL'NCTURE-KLUIDS it is not a histone is established by the fact of its yielding no histone even after treatment with 08 per cent. HCl for days. Pseudo-mucin. — The reactions of this substance are : 1. On boiling with a small (juantity of dilute sulphuric acid the fluid ac(iuires the power of reducing FihlinK. 2. The opalescent filtrate tjives a turbidity but no precipitate on boiling. 3. Acetic acid gives no precipitate. 4. Acetic acid and potassium ferrocyanide rentier the fluid viscid and impart a yellow coloration to it. 5. Millon's reagmt yives a bluish red colour on boiling. (>. Glyoxylic acid ♦ followed by sulphuric acid gives a violet coloration. I'seudo-mucin has been fully studied by Otori in order to determine what decomposition pro A sulwtance allieil to mucin has been tlescril)Ctl as occurring in the blood by Zanetti, p:ichhol7., and others. Thi- chief difficulty, however. Hes in differentiating mucin troMi mucoids, which inchide colloid sulistance. jweudo-mucin, and paralbumen. However, pseudo mucin is not precipitated by acetic acid, though it comes down readily in the presence of excess of absolute alcohol. The reactions of this body have been given abovr. The paramucin of Mitjukoff is a jelly-like ma^s. and con- sequently hardly calls for consideration, though it is often found in ovarian cyst. It differs from pseudo-mucin in reducing Fehling without previous treatment with acid. Paralbumen is only imjK-rfectly precipitated by acetic acid, and is supposed to be really i^seudo-mucin with albumen as an impurity. The mucin of bile is regarded by Landwehr as a globulin mixed with bile salts, while Paijkull regards it as a nucleo- albumen. TABLE V Mucins in Puncture-Fluids* Absent in Present in (2 Tuberculous Pleurisies 3 Tuberculous Pleurisies Effusion secondary to Ab- dominal Cancer Renal " Idiopathic " effusion Cardiac Failure (4 cases) Empyema (2 cases) Pericardial, Cardiac Failure Cardiac Failure (3 cases) Renal Disease 1 3 cases) Sarcomatosis „ . ,1 Ga^tric Cancer Peritoneal p.^creatic Cancer (not invading peritoneum) Carcinomatosis Cardiac Failure (l case) „ + Renal Disease ( 1 case) Renal Disease Cirrhosis of Liver (2 cases) Simple Chronic Peritonitis ^Tubercular Peritonitis Broad-ligament Cyst Ovaiian Cysts (4 cases) Not necetsarily " »ero»«niucin.' 42 STl.DIKS IN lUNCTURE-FLUIDS LECITHIN • ii ! I. ■ffi ii i The cht'iiiistry of lecithin is gratUially Ix'coming more de- fined, and the fact that there are niany substances p.esent in the \KH\y which can In* f,'rou|)ed under the name of " ii|)oicls " or of " pliosphatids," taking lecithin as a basis, is being realised as a result of several recent iinjmrtant researches. There is, in addition, an increasing knowledge of the physiological and l)athological |)ro|H'rties of this sulwtance. Since this literature is largely inaccessible to the general reader, it may Ix; usefully discussed, with esjK-cial reference to the influence which the pro|H'rties of lecithin have on the nature and constitution of puncture-fluids. The importance of lecithin is shown by the following dis- coveries : It i)lays a somewhat obscure part in the processes of immunity ; thus it is essential to cobra-venom in the hemolytic process characteristic of )X)isoning by snake-bite.* It plays an inijwrtant part in the process of i)roduction of anccsthesia, since it is assumed that lecithin renders the anaesthetic readily i)er- meable into nerve-cells, and calculations have been made by which laws governing the distribution of anjesthetic over nerve- cells could be formulated. t Thirdly, lecithin is presumably the source of cholin (see p. 170), whose presence in cerebrospinal fluid has been found by Halliburton to be pathognomonic of organic nervous disease. Fourthly, lecithin is an essential con- stituent of all growing tissues | and in bone-marrow. § Fifthly, it is frequently associated with cholesterin, which leads one to susi^ect some subtle relation to exist between the functions of these two substances. Their association recalls the phenomena of symbiosis in living organisms, and there are several observa- tions in the literature which show that the two substances may act in combination better than when kept separate. Thus, both bodies may take a part in the hctmolytic action of bile.y Sixthly, the suggestion that pseudo-globulin retards the action of certain lysins may be brought into line with Joachim's discovery that lecithin is mainly attached to the pseudo-globulin fraction in ascitic fluids (and therefore presumably also in the blood-serum). * ilorf;enroth ; Keys. t Researches on the distribution-coefficient by'Overtoi. I Francliini. § Otolski ; Ghkin. || Gustav Bayer. j PI Ml » THE CHKMUAL EXAMINATION OK PI NCTURE-HAIDS 43 Lastly, the imixjrtant discovery l«as recently Jx'en made by Purges that if a o'i jht cent, aqueous susi)ension of lecithin U- ulded in equal volume to the l.l(K>d-serum of a syphilitic subject, there will app-ar atliKCulent precipitate after hve hours' inruba- tion at J7 C. The reaction li.is. so far. never Ix-en obtamtd m the case of a non-syphilitic subject. Some of these ixnnts ilen.and further consideration. Thus, we may refer to the presence of lecithin m growu.g tissues.* among which a developing hen's egg forms a suitable exanq-le, for its yolk contains a large quantity of lecithin, an.l may be said to l« a reixjsitory for that substance. The growing embryo withm this egg contains, of cours.-, a large quantity of nucleic acids in the cells which are so rapuUy \^mg formed, and it becomes of interest to study the formula; of nucleic acid and lecithm in order to ascertain if there l)e any sort of relation between them. If we take a-guanylic acid as an example of a nucleic acid since a graphic formula of this substance has been made out by Bang and Raaschon, we have- Lecithin. OH C H N(CH,),OH-0-I'.-0-C,H»=.(C„H;„-,a), Vjnj.'v-'-ju fatty acid. a-GuANVLic Acid. OH OH c,H.N.-O^P;^0-C.a(OH) . Cj,H.A o C,H,N»-0-l'-0-C,Hs(OH) C.H.Oi o o CjH.N.-0-P-0-C,H5(0H)CjH.Pi 6 C»H,N.-0- P-0-C,Hs( OH )C,H,0» OH OH The symbols printed in thick letters will show at once ♦ It is most likely ihat much of the conf,r-ir.: which ha« ''-;-«y=;.»° the pre^ile constitution of lecithin is due to its having been mamly studied in this material rather than in the various adult tissues. 44 STUDIES IX ri'NCTURE-FLUIDS 41? U i III how the formula of lecithin can he fitted into that of the nucleic acid, or, more correctly, that the two suhstances are built up in a similar manner, so that the nucleic acid might readily give rise to lecithin in the course of metabolic breakdown. It will thus be seen that we have here only another example of what must Ix" going on all over the body during life, producing the redundancies of ])hysiological chemistry, since a few sub- stances of very complex constitution will suffice to allow of the appearances of an endless number of breakdown products (vital as well as lalwratory) which might be looked upon either as post- or pre-formed in the molecule. It is the determination of whether we are dealing with the one or the other which is the most difficult of the problems of physiological chemistry. But enough has been said on this subject in the first few pages. The interest which the decision of the structure of the lecithin molecule har. to us just at present lies in the fact that those two formulc-E allow us to understand (vaguely, it is true) the vital part which lecithin plays. It is remarkable to find that lecithin has a considerable tenacity of its couiplex structure as long as it remains part and parcel of the living cell. An additional fact about the physiology of lecithin is supplied by other recent researches, namely, that it plays a part in the assimilation of food-substances and in the development of cell- ferments. There has been found a marked similarity in the '.nteraction between ferments and lecithin to that between ferments and mastic. The existence of lecithin in bone marrows suggests an explana- tion for the undoubted effect which X rays have in the treatment of Icucocythaemia. In order to arrive at a conception of the chemistry of lecithin it will be convenient to discuss the subject from the historical point of view. There are two periods in the development of the knowledge of this subji-ct,* a prc-Thudichum period, anti t the period started by Thudichum. We may say that although fatty substances containing phosphorus were known to Fourcry, Vauquelin (i793). Couerbe. and Freny(i84o), the name lecithin was not invented till Gobley had made his investigations (1846) on <'gg-y""<' whence he obtained glycerophosphoric acid. Four years later Liebreich had come to consider that a body which he called " protagon " •iiust be the mother-substance of lecithin, a conception which finished in a complete proof as a result of modern research of the fact that there is no Danilewsky. t Michaclis and Rona. THK CHEMICAL EXAMINATION OF PUNCTURE-FLUIDS 45 ontity " lecithin." but that there are a numberof different fatty phosphorus containing boart in j)uiin metabolism, and, inasmuch as two bases are asso- ciated with lecithin, these other bases may at some period of their ionnation come into relation with the same lipoid. The iinp-ort.ince of these considerations lies in the light which they throw on i ertain physiological questions. Thus, the enzymes of the aininal l)ody have been found by Paul Mayer to act entirely differently on de.viro- from htvo-rotatory lecithin. Possibly its action in activating cobra-venom will depend to some t.'vient on its oj)ticaI character. In '.he'apeutics, lecithin may be beneficial. Claude and Za.\y, speaking at Paris in i()oi, showed that lecithin has a favourable inHuence on the nutrition of a tuberculous subject by increasing the energy of metabolism,* though its use did not prevent the advance of the disease. The association of cholesterin with lecithin is a fact of very great interest, and has engaged the attention of Dr. Craven Moore, v.ho considers that cholesterin exists in the cell in a colloidal state, through the agency of lecithin. When the latter under- goes dissolution, the more stable cholesterin becomes unable to maintain its colloidal condition unless fatty acids or their deriva- tives be jiresent, and it then gradually separates out in crystalline form. The observation of Pascucci shows up what I term the sym- biotic relations between cholesterin and lecithin. The addition of cholesterin to a hicmolysin neutralises the activity of the latter, while lecithin has no action, the varying proportion of lecithin and cholesterin thus affording a means of regulating the effect of hscmolytic substances on the cell. If the OH groups of cholesterin l)e rendered inert, this neutralising property is lost. Lecithin and cholesterin together form means by which substances otherwise unable to }x;netrate the cell-envelope become enabled to do so. Methods of Detection and of Extraction of Lecithin. — Otolski made use of the separating power of an excess of (j() per cent, alcohol ; but if we search through his figures, they do not seem to show very constant N : P ratios. Manasse used warm absolute alcohol for extracting the • Glyccrophosphoric acid appears in the urine when lecithin is taken. irx TIIK CIIKMICAL EXAMINATION OF PUNCTURE-FLUIDS 49 l.x-hhin. and cnployed Salkowsk.'s method of .Ictecting phosphorus m the evaporated extract. This method .s smiple and apparently sufficient for clinical purposes. In slveral of mv own specimens I hav^ used the prec.pUate w,th ammonmm sulphate (Table B) a ..pphe.l Salkowski s ,.ethod of incmeration for phosphorus. If known -e.«hts are used this method can be made a quantitative one. although it nuist' be remembered that all the lecithin present is not necessarily ...timated, as not all of it may be in association with the globulin If this method be adopted we shall be able to endeavour to detec the presence of this substance with the same quantity of fluid that was used for globulin-an important point when only moderate amounts of fluid are available for analysis. The absolute proof that a substance .s lecth.n will involve an f}^^^'^^ of th X P ratio, an estimation of the rotatory power, a preparation of the r "'Active cacl,..un, salts, and finally the '^^-^^J^f^^^ °' .i.tection see v i/i). glycerophosphonc acid, and of fatt> acicis. /.a^r"L.U //.:,-t,tc residue fused with 3 parts KNO. and i part sodium carbonate. The white "s u r Lived in a trace of water and nitric acid added. Ammonium moUbdate is added ^avoiding e.xcess) and the precipitate weighed as in ""■''."^^tlll moreaccurate method.s thatof Neumann. 500-. «u id are treated with pure nitric acid and gra.lually poured into a round-bottomed tla^kcontiniSg 30CC. of boiling nitric acid. There should never be more . It is assumed that . gm. lecithin contains 00384 gm. phosphorus. so STUDIES IN PUNCTUKE-KLUIDS .Hi, than irx) cc. of fluid in tin- flask at once. The fluid can then In- rapidly rc. 5.2-5.?) showing Stent an! Tlierfeldct's scheme is given, however, in order to give some idea of the lengthy procedure necessary in order to make accurate re.searches on this subject. The special feature of their methcxl was the carrying out of the metho , (o^ms a slimy mass in the ' '■""^' ^;'t;:r'^''r ti^ -:S l^e^::: .1: abnuy to Lluce alUaline presence «« -^^^^^ j''^ ^ ., prec.pitatal by strong salt solut.ons and by copper ^"'Py-»^' ;''°^"f ;;„ »,^,e the prc-c.p.tate is soluble m excess of ;r r:::r.h^.;::t^ .uh a;^-^- — t-™ stcompouna of s:::f:s.^S=:A::tnbt::::n;;^a.on^-n;.- -^ — tX::.. a mature of n.a^-^ -;; ZT^t::.^n .n very just as act.v.ty of resea ch on K. th.n ^^^^^ ^^ _^ ^^ ^^^ n,any t.ssues^ ^°;^^^T blood, and m the suprarenals. It is worthy are the spleen, the bram. inc considerable amount of "' r Wh ttrSerrtisatrat^d or not has not been made out. U-c.thm. \Vhether ^holesterm is ^ji^ to Jacobsen. Hennques However, it is important to note ha -^^^^J \.^^,,,,,, ,, jecorm. and Kolisch. the glucose of the Woo ..^traction of the although It has to be admitted that '".^'^'-'^f ""^^^^^.j ^^^h the ether, blood w.th ether son. free- glucose -^J^^^Xlt^: otthe extreme care an ^ ^,,4^ It bears out stances are and what - -t prese^^^^^^^^^^^ ^n^^ ^.^.^^ ^^^ ,^ the view that many of the ^^ ^^ f°;;',,.,„it of decomposition of the detected ,n a body flu.d may - 7;,;'>^ '^XU^^ed to definitely settle proteuls which are present. J^^^"' ^^^^'J^ination. and he comes to the th.s problem of the jecorm -"' ^^bv "thm as a solid solufon or conclusion that the glucose is reta ned ^ it j^^,. ^^^^ ^t is a possibly by adsorption. =»'*Jough h^ ad- th IM^^^^^^^ .^ ^^^^^^^, rr^re::::"';, ^t. ;::^n: a„d says they Le essentia, con- ^^'rr:^ that If ^-^-^-sx^^^^^^^ sugar from its combinations it '"■B >t ^^P^^« '^^, ^^i.l experiments ,W »"d.*r™c. Wn« .1... il gl«co-- I. prc«.., *■ l.-moly.,. Ukc. place very much more rapidly. 52 STTMES IN MNelUKK-I-LUinS ft ■s ** c c K u X _'2 '3 s o rs 1 ' s ^ i 1 "^ n c 60 ! <=) a h- (fl c (A g J5 c o u 2 C « e o u T3 3 (- ;^ lii . CI = 1 I e o <^ "a V 2 a 1 s u . 3 ■• li^ Ti c o U B B O u 5 ^ J; T. c -• « S — -i . *^ u cs ^ O o O u gT3 i> .E ic .-"I =.2 » ^ 41 C c C '- O I. li! 54 STUDIES IN I'UNCTUKE-KLUIDS lAHLF. VI I,K IIIIIN IN ItoDV FiflDS Kliiid. Uiaiase. lecithin. I'Uiiral . I'tritontal Ovarian C'v>l Inllanimatory, (AlKlcminal Cancer) ... Chronic Kll'iision (nature?) Kfnal Disease J't fitoneal Cariinoinatobis rnilcibiilar Cirrhosis ... Kcnal Disease . . Cardiac Failure ... Tubercular IVritonitis ... I'nilocii'ar Cyst... Kroad l.inamcnt Cyst ... Trace. Conspicuous I'rescnt. Absent. Absent. Present. Tlie Study of the jircscncc of lecithin in body fluids may be saitl to date from Joachim's investigations in 1903. when he tvas endeavouring to find the cause of the turbidity of certain ascitic tUiids. In one special case studied by him the patient was suffering from cirrhosis of the liver, and the fluid which had been poured out into the abdomen was turbid, though no fat was present. The essence of this study lay in the fact that pseudo- globulin was found to be present in abumlance. and that with this pseudo-globulin there was associated lecithin. That this was the cause of the milkiness was proved by the fact that dialysis gave a clear fluid, and that the residue contained lecithin, while the clear fluid did not. The lecithin was extracted by the use of ether, and the extract tested for jihosphorus. It was found that only the pseudo-globulin fraction contains phosphorus, namely, ^-45 gm. in every litre of ascitic fluid, which corresponds to 035 gm. lecithin.' Jolles records a case in which the peritoneal fluid (disease not stated) contained abundance of lecithin. These observations proved beyond doubt that lecithin may occur in ascitic fluids, and would be the cause of the turbidity. Moreover, it will be noted that this occurred in a case of effusion due to cirrhosis of the liver. It became a matter of no little interest to know if lecithin occurred in other fluids besides ♦ The precipitate resulting on ailding J vol. of sat. .Xm.SO, consisted o! vuslulrtilin, and if the tiltratf !••'■ hM -;aturat«l .og.iin with Am SO^ a ])rctipitatf of pseudo-globulin results which is soluble in sodium chloride and sodium hydrate solutions. TIIK CHEMICAL EXAMINATION OF PUNCTURE- H.UII'S 55 ascitic fluid, ana if it occurred in ascitic fluid under any other conditions. . » i The actual diagnostic in»i>ortance which Js to be ;it a. liea to the occurrence of lecithin in a body fluid can hardly be made out from so few examples, although one .oj^es to have shown that the question may be of use to the clinician. The Diazoreaction.-This reaction, which has been applied to the study of urine under certain febrile conditions, was care- fully studied bv Clemens in 1904. who found that the bodies which are responsible for the reaction are precipitated by basic lead acetate and are insoluble in alcohol. Tyrosin and histidin both give the reaction, and as the former may be present in a puncture-fluid, it was thought that this test might give usefu results in puncture-fluids, esi^cially when the test, as applied for the detection of histidin, is extremely delicate. The results of the experiments showed, however, that it was very rarely present. It was obtained distinctly m a case of ijleural efiasion associated with abdominal sarcoma, and als<) in the fluid from a tuberculous pleurisy. In only two i^ritoneal fluids was it found-namely, one from a case of polyorrhomenitis and another from a case of chronic i>eritonitis associated with an old gastric ulcer. If the investigations of Clemens represent complete knowledge on the subject we should be able to assume that in these four cases either tyrosin orhistid m or both was present ♦ and the fact of one being a case of carcinoma their presence would be explicable because the products of carcinoma metabolism might well api>ear in this as in other flmds of the body However, the reaction was not obtained in any other case of the same kind nor in a definitely carcinomatous effusion. The reaction was uniformly absent in subcutaneous cL-dema fluids. • Other views as to the nature of the reaction are : (i) That it is due to phenols and amines (Ehrlich). (2) That it is due to paired sulphur acids (Dolgow). ^ ,. , , That it is derived from breaking-down leucocytes (Oeissler, Sahev). 4 That it will appear in urine after taking opium, chrysarobin, naphthalin, morphine, while it is prevented by internal admmistrat.on of tannin. /\jr~„\ (O That it is due to the chromogi of urochrome (Wetss). For Utcraiurc and full details see v. Noorder, Handbuck der Path. rf« SMfw. I.. 660. Berlin. 1906. 56 STri>IKS IN I'tSi TruK-KLL!DS l|t> The Molisch Reaction ; the »-Naphthol Reaction.— The MolJM It icai tioii (<)iisi>t-< in olitaitiiti^' a Molit (dlotir with an al( oholit xiltitioii nt i.-naplitlml in the pii-si-m i- ui ptiio siilpliiiiii' a( iti. This test was a|>|>hi-(l to the oii^'inal tliiiil in Miiif t\i iiriosjty in tiie first placf isiiiii- lh«' icai Ikhi ilf|ifii(ls mi tlif piist-iui' of ( arho- hydiatc laihi !t'>^), and witli the hope tliat it ini^ht form a ifady test loi till' piofiK r ot su' h in a thud. It was soon louiid. howt'Vt-r. that there wen- striking; differ- vnres in the efte( t-. in ihtfereiit eases, so that it has sinee tliat time l)een apjilied a^ a routine pro( edure. I p to the present it is hard to lomiul.ite any rules as to (hajjnosis whieh mif^ht l)e made from it-> presini !• or ahsiiin'. Tlie (hfleience in the de^^ree of rea( tion is perhaps the most striking;, hut at the same time it must l)e admitted tliat a violet colour is never obtained. The rem tion whii h one does ohtaiu is the formation of a red rin^ or a red stainiu(,' of the at id. A 5-per-(ent. solution of a-naphthol in (>5 per cent, alcohol is prepared, and an etjual volume is added to the Huid examined. Stronj,' sulphuric at id is allowed to run down the side of the test-tube, and the contents are now oscillated gently. A red line will ajipear at the junction in a well-marked case, almost instantly ; and, with oscillation of the heavy acid, the latter will become uniformly red (carmine). The results which have been obtained are shown in Table VH. From this table it is evident that as a rule pleural fluids give a much more marked reaction than do peritoneal fluids, and that in cases of " idiopathic " effusion the colour-reaction is very decided. On the other hand, peritoneal fluids, esjiecially those dependent on back-pressure (mechanical effusion), show a very scanty reaction. The difference in the tlegree of reaction dejwnds, presumably, on difference in constitution of the proteid or on difference of substitution products. The presence of reducing substance in the proteid of many jiuncture-fluids (some form of carbohydrate radicle) has been specially remarked on by Landolf of Buenos Ay res. The absence of the reaction in such a peritoneal fluid as is derived from a case of jiolyorriiomenitis is suggestive, lor the fluid from such a case has very marked differences from any THE CIIIMI* AL I XAMINATION oF I'UNfTL'KK-KLLIDS 57 .,thiT jmncture-nuia : it is dear, or slightly opalfsct-nt. (luitc watiMy. ami contains only a trat e of ^-lohulin anle In- tiaminationi (1 u h c r - Effmx'o" ciil.MiM Uliiticr- C 11 I o u s cases (. I'e r i to ■ neal F.f- HydatidCysl lusum in m o n o - lobnl ar cirrhosis, ; Chronic i Perito- nitis (simple). Ehrlich's Glucosamine Test.— As in the case of the Molisch reaction, this test has been applied to a considerable number of fluids, and similar marked variations in degree of reaction obtained. The most striking point seen on comparing Table VII with Table VIll is that the latter shows mainly jwritoneal fluids in the marked jwsitive cases, while in the former the pleural fluids gave the more decided reaction. The test is performed thus. To a small jwrtion of fluid an equal quantity of a 5-per-cent. solution of /)-dimethylamino- benzaldehyde in lo per cent, sulphuric acitl is added, and strong sulphuric acid is run down the sloping test-tube. A deep violet colour, varying in intensity, will appear at the line of junction, and gentle oscillation of the acid will induce a very intense coloration in a positive case. The results of these exi)eriments are shown in Table \'III, and the differences in the reaction must find some e.xplanation. The e.xplanation of the test itself has been the subject of nmdi controversy, and the present opinion -^eems tn be that it is the tryptophane radicle which is the cause of the colour le- 58 STUDIES IN I'UN'CTU RE-FLUIDS art ion. Tin- test is called by its name because glucosamine will give the reaction, as well as other osamines. Neubauer con- sidered that uroliilinogen would give the reaction, but it is not generally accepted that this is the ex])ianation of the test when applied to proteids in general. I'ap|)enheim regards it as settled that this reaction is a pyrrhol reaction, though no confirmation of this can he found in the literature available, although if the reaction be looked on as a tryptophane reaction, the two views are readily reco'iciled, for l)oth tryj)tophane and pyrrhol contain the iniido-group in the same position ; indeed, Holland's formula lor tryptophane contains jiyrrhol : CH, Ml, HC CH toon HC CH NH T. _)toi)liaiie (Holland), The thickened letters rei)resent the pyrrhol group. The (juestion of pyrrhol will again come up in discussing the ferments in leucocytes. If we look upon the reaction as indicating the presence of try|)tophane in the fluids uniler question, we .shall be able to obtain a more tangible idea of the significance of the test as api>hed to pi;ncture-ffuids, and the intensity of colour will ])resumably depend on the amount of tryptophane * present. * I'rvplupliaiH' (ii 'ol.Tininopropionic acul) is to some extent an index i>t the amount ol de . iction of proteids that is Roing on. The substance hears some relation to nulanins. It exists m three optical forms a l.evo- rotatorv. a dextrorotatory and a r.ieeniic. The follouinR tests may be enipldved lor its detittion : (1) The ulyoxyhe test (see below). (i) Bromine water in acetic acid nives a rose-red or violet colour (mixed mono- and ili-bronude ol tryptoi)liane according to Leveiie and Koiiiller;. which i,m be taken up by ann I alcohol. I}) I'yrrhol le.tction. A match-chip jiut in strong IICI, and then into the sohition. turns cherry red. (4) /i-diniethvlaniiniibenzaldi hyde ti.st. It IS soluble in cold, readily in hot. water, but only slightly soluble in .di.--()liiti' dcohol. To sei>ar.i,.- it Irmii leiu in anon Simple Inllam- Adherent Empyema mation Pericardium, etc. Empyema Non - tuber- I Simple Inflam- ciilous EtTu- ; matory sion Effusion (2 cases) I (3 cases) Tuberculous ; .• Pleurisy Simple EtTu- j sion Pericardial Fluid P. Cardiac Back- pressure Kenal Cardiac Back- pressure Cardiac Back- Cirrhosis of prissurc Liver (2 cases) Polyorrho- Tuberculosis menitis (2 cases) Atrophic Cir- i rhosis of ' Liver (2) ; ' Monolobular ! Cir^hosi^. j Carcinoma- i tosis I Renal Adherent Peri- cardium, etc. of Cirrhosis Liver Tuberculous Peritonitis Tuberculous Piritonitis (deep crim- son colour) Back-pressure (cardiac) (2 cases) Monolobular Cirrhosis Chronic In- flammation- (Non-tubcr- culous) Tuberculous Peritonitis Unilocular Ovarian Cyst ( Pancreatic Ovarian Cyst Hydatic 1 Cysts Ovarian Pancreatic (crimson j (Hydatid f"^'"""-) ' The fact that the Aclanikiewitz reaction * depends on the presence of tryptophane will also serve as an inliows partial aiisorption of the rif^ht half of the spectrum, and a dark b.ind at F and a paler one between F and (i. Gases. Free leases may ok ur in i)uncture-fluids from the rupture of hollow viscera or from the perforation of a j)ulmonary roi)ortion of electrolytes. The Inorganic Constituents of Puncture-Fluids.— The most important inorganic constituent which we have to deal with is of course sodium chloride. The presence of carbonates, sulphates, j)hosi)hates. is of less importance, and is conveniently considered under the one heading of " .\chlorides " (see Section II.). The subject is more fully discussed in connection with the theoretical consideiation> on electro-conductivity and later, again, m Section I \'. We have already iliscussed the risk of error likely to arise from adsorption ip. 17) and shown that, at anyrate in fluids containing only a small .iiiu)unt of albumen, the volu- metric methoil ot analy>is is accurate enough. In the case of an iiiual (iiianlity ol lo-pir-ctiit I. ,11111111 i]il'\hic. Aftir .1 short ccntri- fusalisation tlic cli-ar tlniil is iKtaiUid. aiiil lo thi' iishIik' .{-inch in a tist-fubo ol till' lollowinu nuxmrL- is addrii : 1 [ni- ..int. lunuiiK HCl in >1t jht ci'iit. alcohol, with Icrric cliloruli' aiklid to tlu' acid in the proportion ol 4 ]HT cent. .MtiT inixiiiK, tlio tliiul is hoilod ami the supernatant ttiiid will turn mitii or lihu-j;rfin, tiirnini; \ iok-t or n-d on adiliii!; nitric acid. THE CHEMICAL EXAMINATION OK I'UNCTURE-FLUIDS 6l I .Si M in 1/1 t^ r^ f^ i^ a H U !fl O 2 o ? Jr| -a ^ -= > i •§. o E g - .h J ;j Q. r- y •c £ 3 c o-O ;v sb vb ■b u p = - = o ■* :o.= S o » £ VI''' i = 2 5i E - ■p V S o X r- C U H < * in u '-J ^ tfi 3 _0 "a I 3 H L in i/^ i.t-e.> O U .t — u ■ .,- .2 = >'. SJ o 0* O s ^ a: 6 1- S r = .= -c sill £ S -^ S c "5 E c o > c o c U b N « i u %n ! = i-S o ,v "5 S 3 : -i-Q- S ft e B I O u i M.i: 1 r" ^-^ * 62 STUDIES IN PUNCTURF.-FLUIDS cxud.ites we have to logai' the quantity of : hlondes estimated as those whicli are " unattarhed. " A parallel i-timation by in- cineration would he ot interest in deciding how nuuh combined < hloride there was present. The aceompanyinf,' table of the amount of ehlorides present in various fluids is of < <)nsiderai)le interest, inasmuch as it shows strikinf^difieremes in chloride-(ontent in different classes of fluid. Thus, in the jileural series there is a hij^h percentage of chlorides in back-i)ressure cases, and the same is the case with renal effusions into the jieritoneal cavity. The subcutaneous fluids in cases of renal disease also often show a relatively high chloride-content. The methods of analysing chlorides. I. Volumetric. The volumetric method which has been adopted in the studies recorded in this work is that of Salkowski's modification of V'olhard's methoil. A solution of 2(yoj^ gnis. per litre of silver nitrate is needed, and lo cc. of this are i)laced in a flask graduated to loo cc. ; 4cc. pure concentrated nitric acid are added, and distilled water is used to fill up to the mark. The mixture is poured into an 8-oz. flask, and 5 cc. of a saturated watery solution of double sulphate of iron and ammonia adtlcd. A solution containing about 8 gms. of ammonium sulphocyanide is now run in from a burette until the red colour i>roduced by interaction with the double salt has become permanent. From this reading it is easy to find out how much water is to be added to the sulphocyanide solution to make 25 cc. of it correspond to 10 cc. of silver nitrate. It is well to test that this is so before using it for actual analyses. The de-albumenised solution is now useil, ic cc. being put into the graduated flask, .]occ. (about) of distilled water, 4CC. of nitric acid and 15 cc. of the silver nitrate. The contents are made up to 100 cc. with water. After thorough shaking, the fluiil is iiltercil through a thick filter-paper (Scheicher and Scliiill, No. 5y8) until the 8o-cc. mark in a perfectly clean and dry loo-cc. moiisure has been reached. This quantity is then poured into the 8-o7.. flask ami 5 cc. of the saturated solution of double salt is added. Titration is then carried out with the suli)hocyanidc as with the control, and the reading on the burette is noted. The 15 cc. of silver nitrate will be found more than sufficient THE CHKMICAL EXAMINATION OF PUNCTURE-KLUIDS 63 to precipitate aU the chlorides present in these fluids, and the ammonium sulphocyanide measures the excess of silver solution. The formula — Parts NaCl pro millc ^-- (37"5 -o'^ K) 04 Kives the number of grammes of chloride per litre. K represents number of cubic centimetres of sulphocyanide used. Since 10 cc. silver corresjiond to 25 cc. of sulphocyanide, 15 re. corresi>ond to 37.V But only 80 cc. (08 per cent of original fluid) have been titrated, hence only 8 of the reading is really required in order to represent the original filtrate The last figure in the formula is due to each centimetre of AgNOa representing 001 gm. XaCl, so that each centimetre of sulphocyanide represents 04 gm. NaCl. To save mathematics, a " ready reckoner " is given m the appendix. A practical hint may be used in these volumetric procedures. The difficulty of reading accurately to a tenth of a cubic centi- metre on the ordinary burette is got over by the foUowing simple procedure which occurred to me : A strip of white paper is cut sufficientlv narrow to prevent it wrapping right round the burette. 'Oown the centre of this strip is ruled a clear thick l^lack line. The strip is now attacheil with elastic bands to the burette, so that the Figures are foremost (Fig. 2). The upper limit of the fluid causes a break in the line as seen through it, which enables a very exact reading to be taken at any level in the burette. With suitable care the slips will last a very long time. 2. The other methods depend on removal of proteitl, thus : (1) 10 cc. fluid are treated with 20 cc. saturated pure ammonium sulphate, and the mixture heated in a closeil flask on the water-bath. (2) 10 cc. of fluid are diluted with 90 cc. of distilled water and placed in a flask closed with a slitted cork. Boil, add a few drops acetic acid till the albumen separates out in big flakes, (j) Dry the fluid, and heat to dryness. Extract the ash with I-U-. 64 STUDIlN IN liN( Tl'KK-lI.l IKS ph(i>pliai mtwt't iff Ixtilin^' small iiortions ol watii. and boil each extraction. Then (lilut. to 70 (1. with watir. and tiltrr. The objections arc that suiphatcs, and i art onatos intcrtfrc, and it the heat 00 i4irat the chlorides may volatilise. . tluit! are treated with e.xccss of alcoholic tannin, hi nd filtered. !i (I - ( ases the ( hlorine is ultimately estimated iiti ,. alh V -liver nitrate and ammonium sulphocyanidc m til vay des( iU"il. THE FERMENTS Ii ili( ( ase ot cii tain ])ii;icture-fluids the presence or absence of di .icteristic ferments serves as a valuable aid to diagnosis. The tiuid contained in pancre.itic cysts affords the best example ol thi> tilth, since the i)resen' •• of trypsin or of lii)olytic ferment, or of liotl is almost consta-' .\ tyi)e of case, auain, in which the di'tectioii of '-rments wo'i e of great value is illustrated by the follow tig : a marked swt jig appeared in the 1 ii'gion attir ;in operation f( colotoiiiy. and vas a fluid 'ilection ■( ibly purulent, for th- te igh. .'. e:;ploi iry puncture produced t^ ni 1 a ci i-idera le quantity of ix'j>sin, ami i(j be !f)rii. ' hy an enormously dilated ston The presence of ferments in a fluid not < nosis to be establislied in certain cases but n as a routine procedure, with a view tn dt tor' of fluid (exudation, transudation, 01 (\ ' hypogastric )ught to lie erature was vliich con- : ing proved iiiri nin: one blei a Uag- >-> l)e stUiiied vhicb iass ex] t to find a given ferment, and in which on< . ex to no ferment. The universal absence of fer; s of inv ke 1 a ]>uncture-f'uid would enable one to exciuae cer^ iir; saHj^noses, Apart from this, however, the |)resence of these Ikk!" or the evidence of their action, has an important bearing o 1. e pro- cesses of autolysis which occur in body-fluids ; and autolysis is a factor that has to be borne in mind in interpreting the results obtained by this section of the chemical analysis. It may be said that the two main peculiarities of ferments are that they are very sjK'cific in their action, and that they are intimately dependent for theii action upon the nature of the substrate. The s}H>ciftcity is shown by the fact that while H + will produce a great variety of chemical changes analogous to THE CHEMICAL EXAMINATION OF PUNCTURE-KLUIPS 6$ ferment action, a proteolytic ferment, for instance, will not act on starch or fat, in spite of each of these Ixidies nndergoing a hydrolytic chanKe wh-'n fermented. The second |X)int is exemplified by the discovery made by E. Fischer, that certain ferments dejK'nd for their action on the stereo-isomensm of the substrate in which the ferment acts. Si»eakinR broadly, then, we may say that ferment action consists in a preliminary anchorin,:;, of molecules oi the ferment to s|Kcitic grou|)s in the substrate (phase i). followed by a catalytic iecmnposition of the same (phase 2). This catalytic decom- jKJsition continues up to a certain {Hjint and no further ; for eventually a state of equilibriiun is reached which does not allow any further change except a reverse change (reversible reaction).* The catalytic agent, the " ferment," simply alters the rate at which the catalytic decomjiosition occurs, so that in order to understand the practical apj)lication of the theories of ferment action it is necessary to refer to the laws l^earing on velocity of reaction. If the velocity of reaction v lx;tween two substa- :es of con- centration C, and C,, be represented by V --= k CXi> where k is the velocity when the concentration of a and of b is unity, the catalysis or ferment action would simply consist in altering k. But as the process involves the production of two new bodies, c and d. in increasing concentration, I'l will = Ai,C,C , and 7' — !'| = A-C.C,, — ^iC.C, until the diminution of QCb is counterbalanced by the increase C,C , , then comes to = ,. ,. • The increase or decrease of C^Ca over C.Ci, must therefore vary with , , and -Incomes a constant K, characteristic for the particular ferment. It is well known that such a metal as " colloidal platinum " f exerts a catalytic action which bears great resemblance to * This wouUl in actual l.ut In- a simultaneous process, t I'ropartd troin tin- metal liy estahlisliiiig an electric arc between platinum electrodes under water. 66 sTLnii^ IN ruM irui;-i i.riiis tcniiriii aitioii. At tii>t >ik1iI tliiic is a maikcd tliffrrfiic iHtwicii till- action oi (olloni.il i)!atini!iii aii'l an oifjanisfi tfrnu'nt.* iH'iausf a stihstanc.' actol on hv the toinior iH-comis .k'coni|M)Sfy the fornunt tlu' i)ro(i>- onlv contiiuir^ np to a certain point, beyonil wlncli it is not jio-^-ihlc to proufd. It is. however, now estah- lislifd tliat tlicic IS no cssintial distinction Ix'twoen the two scries ot processes, tor it the meihiim in which the organised fer- ment is acting Ix- ihhited. tlie reaction will again coinnience, l)ecaiise the concentration ot the products of action i> diminished by the dilution. The state ot equilihrium which was reached was only a laKe eiiuilibriuin. The law of mass action states that the chemical action is ])ro|K)rtional to the active mass of the liodies which enter into the reaction, i.e. is i)roportional to their concentration. This law holds gcKKl in the casi- of ferment action also. Whether this statement is absolutely correct or not has Ix'en disputetatcs that the velocity of cata- lysis shows three periods: (i) A rectilinear jR-riod. where the masses of the substrate con\erted in equal intervals of time are ai)pro.\imately e(iual. The values of the c()n>tant k in the tormula given below steadily increase during the earlier phases ot the reaction, (i) A lo'^arithmic ]H.'riotl. At this stage the law of mass action is followed. The values for k are constant. This period is best seen when the substrate used is in very ililute solution, and the amount of enzyme is very small, (j) An Tin cliitl points ot companion aro : Ciiiioidal riaunum. Kermtnts. ku\ Ihe rcan takes plati acioriling to tlic IVrrmilii ,h Mic vtloiity of rciction imrcasis to a ma.xiimiin on addiiif; molt- Oil ions. ( I 1011 ilimiiii>lie-. the velocity. K.Sd, accili rales ttur velocity, there is a tempi ratine optinuint. II. S anil HC N act as antilirrnenis. lliere is no limit to llie reai tioii. riie formula is Ditto. '■(■ ■ .;) Ditto. Ditto. Ditto. Ihtto. The action ceases when the proiincis of catalysis reacli a certain con- centration. TIIF CHEMKAL EXAMINATION OK PUNCTURK-KLUIDS 67 euie*ittr*ti»» inlmMnrUhmu ihiuxI. .lue to inoditication .)( the logarithmic rours.- ot the reaction with the progressive destruction of the , atalase The k vahies now tall. We >ee. then, that though the net result contonns to the law of nuiss action, it may be that the details show .letinite fluctuations ami deviations therefrom. W'.- have to deal with two cases (i) in which there is only „ne molecule involved in the decomixjsition-monomolecular reaction. (2) in which there are two molecules interacting. The velocity ot a monomolecular reaction l)ecomes progres- sively less iis the reaction proceeds. This is indicated in Fig. 3. where the curve droiw rapidly during the first few seconds, and then falls more gradually until at last it comes to lie almost parallel to the base line. Ex- pressed mathematically we have : _'''' ^k(A-xi, stance, it will also contain h - v f,'ram molecules of the second. When intef^rated, we ha\f : t VA - x' A - x^ Specificity of Ferment Action. —It ha^ been stated above that one of the most striking characters jH>ssessed bv ferments is that their action is si)ecific. The investigations which Jacoby has made on ferment action have led him to discijss whether all ferments do possess this property. While sonu proteolytic ferments are intensely siK'cilic inasmuch as the synthetic jxjly- jK-ptids of Fischer and Aberhalden are not acted on except in the case of those which occur in nature, other ferments do not xhibit this projxrty. Heat-production in Enzymatic Reactions.— Any reaction in which heat is develojxHl externally (whether measurable in quantity or not) is called exothermal, while if heat i absorl)ed ,1 is called endothermal. Passing up from the al)solute zero, we come ujwn a succession of reactions where the development of heat becomes less and less exothermal until we reach a jxiint at which all the reactions are endothermal. At this stage of the series we come uix)n a condition in which the >ul)stances ordinarily split up by the ferment are regenerated. The more nearly endothermal the reaction the more it approaches a reversible reaction. The relation between ferment action and heat production affords an explanation of the pro- cesses of al)sorption in the intestine, for instance, where reversible reactions are constantly going on. In a tabic given by Holx-r, it is shown that the hydrolytic fermentations have a low pro- duction of heat, while the oxidations have a high one. Thus, maltose, cane-sugar, and lactose give a value of j to 7, fermenta- tion of dextrose into lactic acid gives a value 147, while for the conversion of salicylaldehyde into salicylic acid the value is 72-6, so much greater is the heat of reaction. The occurrence of endothermal reactions has some bearing on the question of autolytic decon^josition. The proiludion of heal by fci ment action may provide a means of detecting and of estimating the amount of ferment present in a fluid. Such a method has Uen elaborated by Tangl and his THE CHEMICAL EXAMINATION OF PUNCTURE-FLUIDS 69 |.u|.ils. The chemical energy of the mixture to be (ermentc.l was determined both Nfore and after the action of the ferment 1,V determining the dry substance, the a^h, the nitrogen, and the calorimetric energy. It was thus estabhshed that when the tryi)sin acts on albumen the chemical energy is not turned mto any other form. t)n the other hand, in hydrolytic ferment action th.' energy progressively diminishes as digestion proceeds. Her- /,.g found that oxidising ferments are associated in their action with the pro^hKiion of thermal energy, whereas reductases show ,u) such change, and the ferments, acting on i^lysaccharoses glucosides, fats, and proteids. only show a small production of luat When heat is produced by the action of a ferment within th. body this will be a factor in the maintenance of the Ixxly heat. The effect of temix>rature on the velocity of reaction is given by the following formula of van't Hoff : In* - ^,, + constant, K 1 where k is the equilibrium constant, and q represents the heat devel .ped during the reaction, Dnd T is the absolute temperature. R is a constant. The bearing which all these considerations have on the study of puncture-fluids is that the progress of an enzymatic reaction in a puncture-fluid can Ixj observed by their means. For instance the conversion of cane-sugar into invert-sugar .an be watclu^d by means of polarimetry, and calculated by the use of the formula given, the degree of rotation being proportional to the velocity of reaction. For instance, if A be the initial concentration of the cane-sugar, A must l)e expressed in terms of degree of rota- tion. Let / be 60 (duration of reaction, one hour). The change of rotation, x, divided by A, can be calculated by means of A ., Ostwald's table, which gives the value for log x^~^- "' however, the velocity be compared with that of a standard reaction, the calculation becomes more simple : k -= I total rotation possible. log. total rotation — rotation at the rnd ol time /. Further remarks about this polnrimetric method will \^ found in Section II., under " Ionic Concentration." MICROCOPY RESOLUTION TEST CHART (ANSI and ISO TEST CHART No 21 1.0 I.I 1.25 ;- iiiiM •"- 13 6 I- 1^ I- ■■■ 1.4 2.5 2.2 2.0 1.8 1.6 ^ -■AP PLIED IK/MGE Inc '-^S ,:'^: *o; - 0300 - (^hone ^^ (?'6) ^88 ^98^ - fan 70 STUDIES IN I'UNCTURE-FLLIDS The Ferments which may be met with.— The following is a list of ferments according to the classification by JoUes : I. Hyih"!ytir. — ln>o\nh\r 1io<1k> ari' lomiilUd to take up water and hiconu- convi-rttil into soliililf hotlif^. Duistii^c. iiWittiisi . liiilds,. tiiluilds, turn bK)-.i> (i.anr->UKar, malt- suf^ar, niilU->UKar) into nionoM> !■>• takiUL; up water (=k1ucom', li'Vulost). Ltpiiii- or '-ti'ap-in ). Jimiilsiii break-, up «hico>icle>, i .;'. aniyyilalm. I 'nasf. Protcfilylu ferments, includin!' erep^in.* II. .Ik/.)/}'//!.— Break up bcxlies without lo-s of water, (.;,'. zymase. Tlu^e enzymes are intracellular and l>lay an important part in the iiuta- bolic processes. III. C'>ii!;iihtiiii;.— form jellies irrnnet. tlironilio>e). IV. Oxyctiiscs accelerate oxydative jirocesses (miaiacum test) and play an important part in the lile of the tissues, e.s;. xanthinoxydase.t V. Cdtaliiseis. which decompose Up,. \l. The Aiitihriniiil'i. METHODS OF DETECTING AND ESTIMATING FERMENTS ON rUNCTURE-FLUIDS The fact that these ferments do not all occur in the different puncture-fluids with which we are concernetl renders it un- necessary to discuss the means by which each is identified. We are only concerneil with diastase, invertase, lipase, proteolytic ferments, oxydase, catalase, precipitins, and antiferments. I. Diastase.— Ascoli and Bonfanti's method.— Add 2 cc. of the fluid to be tested to 100 cc. rice starch (i per cent.), and I cc. toluol. Shake, and incubate twenty-four hours at 37" C. The fluid should lemain sterile, and i per cent, sodium chloride is then added, and the fluid boileil in Soxhlet's apparatus. A few drops of dilute acetic acid are added, and the fluid again raised to the boil. Rapidly filter, and estimate the sugar. Potato starch may be used. * Krepsin acts on peptone, deiitero-alliumose, .iiid cluseiu. coiuertins tliem intolcucin. tyrosin, ammonia, arniinn. l\^in, etc., but cannot act on albumens of blooil and ascites, globulins, or Heiice-Jones jiroteid (Sieber and Schumotl-Simonowski). + Xanthinowdase is one of the four lerinents which play the chiel part in nucleiu metabolism; (i) tiiu.'lvlii fcnnciit. met with in kidney, liver, antl muscle; (i) uuileusc. breakmir up nucleic acid and liberatiii!; purin l,ases. met wiih in i!ie liver and. muscles : ! -i) a il-:<:i.">>'li^ii>S uiment. turmn.!; aminopurins into oxypuriiis ; (4) .\\iiithiii>\\ Invertase -This is detected by the formation of reducing .ub^tance after incubation witli a 5-por-cent. solution of cane- sugar, toluol having been added. The glucose n.ay be estima ed V Lipase.-This ferment is more abundant in exudates than in transudates. r »k,.i (a) The neutral fluid is treated with a few drops of eth\l butvrate in a te.t-tulx- and litmus solution added. After twenty- four hours' incubation the litmus is found red if hpase is present, while a control tube is unaltered. Titration with baryta would enable the amount of ferment to be calculated, supposing that the same length of incubation were given in every experiment. Robscn and Cammulge pent out that a trace of -l-»"^ -IJ --[ ^^ aclcUa to the rtuul to l,e teste.l ,n the cus. of pancreat.c c>,t rtuul. a, l.ancnatic fernunt alone will not react. (b) Alternative. Ethyl butyrate may l>e replaced by an ethereal extract of olive oil containing sodium cartonate, the extract being allowed to evaporate. The advantage of this is that one has a ready means of preparing the requisite neutral fat instead of having to prepare or purchase ethyl butyrate Pepsin —The number of methods which have lieen ad%-o- cated for estimating the amount of pepsin in a fluid is remarkable, and may be taken to indicate that no one of them is quite satisfactory. The chief inethoas may be classilied as loUow.-, : , Methods where the anionnt of albunun lelt untouche.I .s notul. e.,. mJasurement ol the column of albumen lett m .Metfs I"'"- z. Methods where the ,.ro.U.rLs of .ligesfon are ..xam.ned (estnnat.on of alteration in acidity ; Volhards metho' -"•- ° coauuiatea e,K-alhumrn in uhich hydrnrhloric acul i^ present. He Kiys a able which he has prepared by takin, known strengths of pepsin sohi- :y STUDIES IN rUXCTL'RE-KLUIDS lions (Armours |h|>mii) i.iid oliMTViiii; tlic time ()ctiii>iicl in dissolving the ('tjy-nllniimn. The nutlioil lias the t;r<:il (.lijntion lli.it on>- i- lulfy/iss uilhout lii\ tahU. liitin Milhuls. Solnis placrs j cc. ol a o- j5-pir-C(nt. solution ol ricin in 50 percent. XaCl in each ol a -< vies of test-tiil.es, and also places o^cc. decinornial hydrochloric acid into each tube. Successive strengths of the jiepsin solution are added to each mlie (i cc. boiled fluid : o<>cc. cf Imilwl fluid + o- 1 cc. of unboiled Hnid diluted too times ; 8 cc. boiled + 1 cc. unlKiihM • liliited fluid ; tcc. iuiileil + -3 cc. unboiled diluted ; locc. unboiled diliitftl fluid I. The corkeil tuUes are incubated for three hours, and it is then noted in wliicli tube the lluid ha> Income clarified. .\ Hiiul ol which 1 cc. in a liundrc.l-lold dilution clears up the turbidity in three hours is a fluio;, Kleniperer there advocating the nse of edestiu in place of ricin (i-per-cent. solution), but Jacoby and Fuld failed to see any advan- tage in such a modihcation. Keicher recommends Jacoby s method. Iiild's .Uf/Ai)(/.— This was de\ised in order that the presence of pepsin could be (leiected with.in a minute. To .:cc of a i-ixr-cent. solution of cJestiii in ,,?,-, normal HCl is adiT of observations : , = _* |7'(2 ) ,,, .vlur.- lis the .legrcr oi v,sco>ity, wlucl. is s„ll capal.K- ol further .linnnu- non; />l th.. restive a. idity o. the pepsin ; t is the tune o. reaction in s.conds. and k and ii are constants. The onlv objection to the method i. the elaborate apparatus that i idv , atc"l Of course one could at intervals abstract a portion of fluid t::;'::: a wUh the Hess instrument ,see Section ..,. ^-P-^. ';:/°- •„ a constant temperature NVithin, at any rate, a decree, llov.v.r, this luld l": Ipt to become tedious, as the instrument would have to be cleaned out so often durinK a short time. ; Estimz^on of Trypsin.- The l^est methocl is that pub- lished by F. Volhanl. The principle of this method is that U a special pre,,aration of . ..ein. to which hydrochloric acid has beeu added, is treated with ix>psin. the acid caseo.es formed cease to be precipitable by sodium sulphate, so that the fiuid is more acid than before When the casein is exposed to trypsin instead of pepsm, in the presence of alkali, and after this is acidified with a certain •unount of hydrochloric acid, and sodium sulphate added, the Increase in aJidity is agam a measure of the amount of ferment action. In the case of FP^i" the increase in acidity is equal to - where / = quantity of i^psin, and / = time of^igestion. In the case of trypsin, increase in acidity — k*.f. The method is a valuable one, and the procedure is as follows : Preparation of the Casein Solution.-ioo grammes of finely granular casein (Chem. Fabrik, Rhenania. Aachen) is dissolved in Ii litres chloroform water, and 80 cc. of nNaOH added. As soon as the casein has dissolved in the water-bath the fluid is heated rapidly to 90X. to kill ferments and bacteria and, on cooling, the bulk is ma.le up to 2 litres, and toluol added. The reagents must be kept in Woulff bottles. Solutions required : Casein s-olution .,/+ NaOH H HCl 20% Na,SO, Chloroform water * In each case Ai is a constant. STUniKs IN I'L'N highly recommended. .\ strus t ini|i(irtant n-scarclics dtaliny with tlu' estimation ol proteolytic Itrnn'iit action has licen made by Sonn^in. He started with the assumption tluit ;)/ llii' iiiiiin prcttolysis is a hydrolysis with the forma- tion of carhoxyl and amino groups The other groups taking part in the decomposition are assumed to he netjliKible. The amino groups can be " ti.xcd." as it vcre, by formol. and the carboxyl groups can then l:e estimated by titration with 11/5 barium hydro.xide against thymol phthale in. The number of centimetres used multiijlied by jS giyes the number of milligrammes of nitrogen, since the formation of each carboxyl corresponds to the formation of an ammo group. The reaction is exemplitied thus : CTI, CH.N!I, + HCOIl + KOH COOII CH, CH, N: CH, + H/) ^ II.O COOK The conditions for the reaction are (l! a suitable strength of formol : (2) a suitable concentration of hydrogen ions; {>,) a suitable indicator, which shall be sensitiee for the particular degree ol concentration of hydrogi'ii ions; 14) a suitable Noluiueol Hind, ll tlu^e conditions be not fultilkd th-- p-;ir};o:i will rry-v~>\ The objections to the method are that it gives inexact results with proliu anil tyrosin and guanin,ate.l an.l acconhn« to the fornunts .-.np ov.-.l. u. mf.n-nce ,n the vaUu. for .nzynu. act.on as nt.asur.a bv lor.uo a on from that as nuasure.l bv pr.c.p.tat.on w,th tann.c ac.l an.l 1 U n^^ .s v.rv «roat .n.h-d. How.v.r, wnportant ,n.provo.n.nt> !:';;;';neS, l.,.. .0'. cone., n.truct.on, as to .ts us.. n,ay W .■xpec... to lollow. 1 » I * 1 ,. 6 Guanase and Nuclease. -r he ferments may i.e .>oiate1 iiui.li aM' li,i> lucii llu- ■.ulijixt of imitli iliscusMon. Sonn- authors consiil. r it to lie the saim- a^ trypsin, Imt il trypsin Ix' adili'd to mil lease the action ol trypsin will tci-e. This (erment CKCurs normally in the th\tniis, pancrias, ami kidiievs in cows. 7- Oxydases. - Sill Mr «i\es the characters possessecl by three varieties ot ox\ilase>. HI I in water Solubility -' in neutral salts I in alcohol and water Saltirit: out ElRct of hi at Amount cf sugar acted on in two days Kate ol inversion of disaccharids Kate of inversion of polysaccharids destroyed easily destroyed slow I immediate rapid I less rapid 4. destroyed 80% slow less rapid Dflcctinii ,111(1 Estimation of Oxydase (Jolles' Metlioil). — '05 cc. of the fluid to be tested is nii.xed with about 30 cc. of oi» per cent. NaCl in a 50 cc. flask and hlled np to the mark with the saline. 10 cc. of this mixture are treatei wash. IKai tlie ,(iecipil.ile with liCi, iiiler oil the silver chloride, and add H_S and again lilter. The filtrate is evaporated and the purins separate out, are puritied, dried, and weighed. TIIF. CUKMICAL KXAMINATION OF rUNCTUUE-l-LUIDS -Jl The following reaction may W ustxl to lUtort tlu' pivsonci- of oxydase in leucocytes : A'rocentlvprcpartHlsolutionol ..-naphtho! in waft ( i iHicent.). containing l inr cent, of s.,.liuni carbonate, is ,Hmvc,l over the tihn and left for one or two nunutes. After a momentary wash in distilled water, a few drops of I l>er cent, a.p.eous dnnethyl- naraphenylenediamine (Marke Sch«charnheim, it is a pyrrhol reaction (see p. ':>»). 8 PerOXydase. — O. v. Furtl. has ,loscril)eoo -^0''"= solution to be calculated. I. is then diluted till it reaches . per cent. The "ithors recommend ix.rmanganate titration rather than the io IN rL'NCTL'KIMI.riUS iiMi il. In u\ < iiiiiiiiti^ .I'M I'll U.lli I .111,1 tlll.'ti- till IlKllllr wuli III M' l^llllilllti -clllltllPlI .I,L.M1II-1 ^l.lli 11 p.i^tt .1-. iniliditiii'. I'..iili' 'lull -|iiinil~ III Jul II III H . II I A Kill, no Il I llil.iti till iIukI III III' li'^ti ij ti P " Ntiw :i-.irn.im i;i muiiiIh r . i!cco:iipi.s»il. Tlir linu 111 i\|iiisiirr II, til, no, ,hoiil,l .ilwiiv. In- tlir --.lire. Tins tcriiifiit is most likrly to Ik- jMi'Sint in ctfusions con- tuininK lilood, since it has \x'vn found by SillHMfjWt and Mosse tluit till- activity o( tlu- catalytic |)()wt'r (U'ln-nds on the varying content ol red cells, whether healthv or diseased. lO. Precipitins. - In order to estimate the amount of these, Hamburger employs the special tubes which he has devi.sed (Fif^. 5). 'Ihe serinn and the antiserum are measured, mi.xcd, and then centrifuged till the precipitate lias a constant volume. The e.xact reading is made with the help of a lens. This jirocedure has not, so far, I' "u applied to puncture-fiuids. EXPERIMENTS ON THE FERMENT-CONTENT OF VARIOUS SEROUS EFFUSIONS The accompanying table will show the lesults which have been obtained by testing jieritoneal and pleural fluids for their ferment -content. The most convenient methoil of applying such tests has been found to consist in using short test-tubes (4 by \ inches) duly sterilised and plugged. A scries of four-ounce flasks were also sterilised, an 1 the following meilia were prepared in them : a thin emulsion of boiled starch, i-per-cent. solutions of glucose, hii tose, mannite, (.lulcit.\ cane-sugar, and ordinary ccn- trifuged milk coloured with litiiius. loo cc. was found sufficient in each case. A number of the sterile tubes were then filled with these media and, as a precaution, further sterilised in the usual wa\-. In the su.:i;ar media Durham's tubes were inserted (preiiared from ordinary glass-tubing), tlie air being expelled during the i)rocess of sterilisation The cotton-wool plugs were dyed with methylene I'ue, methylene green, aurantia, saffranin. TMi; . IIIMKAI. IXAMINATION .'I It Nl TUKK-I' l.LII.S 79 au.l gentian v.ol..t, >.. ;<^ to ^h^uw^'u.h tlw.,.. n.r.h.i wt.uh sure .(il'nuK'ss. In tostm« a f^iv.i. riiiM. mu- ..I r.u li ol tlu-r ti.U-, w.is . lur«nl w,tl, a small - tnl.utc Huid over nuMlnim. and wnv im nbattd at .i7 f<>i nthfi tw.lvc or twfntv-lour hours in .Uttonnt casts. At tlu- rnd of that time Itrnirntation was note.l. if j.irs.-nt. and tlu- ivartion WiTtaincd h\ drop^ ol htnnis solution. + In the caso of star.h. the nu.humwas t.stid tir.t with h.iu-.r iodi, and. sm.ndly. with 1-ihlinK's solution. In th.' caM- of xhv rani-.u«ar Fohhng s t,st was alone applied . In addition to these media, plain te>t-tul.e> of the same size were used to test the lipolytic ferment by the ethyl butyrate method, and for proteolvtic ferments t.y the use of Mett's IuIk-s ,n (<0 acid and {f>) alkaline medium. The tuUs were not used tor (luantitative tests, but were found convenient, iind the egs- albumen was more easily and more economically stored m this The Reducing Power of Puncture-Fluids. -This is met with m inflammatorv exudates, and also in the cerebrospinal fluid. It has been Ix-st studietl in the blood, urine, and bile {Melon. Migliarini). The reducing iwwer of i cc. of blood is equivalent to U5 cc. ol decinormal KMn()4, that of serum to 51. ^nd is found to be increased in pneumonia and diabetes. The chief value o» the observations lies in the fact that the reducing i>ower is constant at all times, but it varies according to the amount of kMn()4 used, according to the action of the light and according to the time occupied by boiling the blood with the KMn()4. Ferments in Leucocytes.-It is to LcIkm in i8c,i that wo owe the discovery that aseptic pus has the power ot digesting proteid matter. He fomul that such pus will iiquety gelatine at 25 C. Not much notic of these observations seems, however, to have Ix^en made till Erlx-n in vpS publisheil an account of ferment action in the blood ot leuc;tmics, shown by the presence oi albumoses. a tact which I^chunnn pointed out in specimens ♦ Q«-intitativ- ;.n;!!y.i-. wa. a-nwl un.ucc^ary .11 tin, ^cn.■s o( (.xiuriinonts. + More rapid than tliu ii>c ot litmus pap'-r. 8o STri>n> IN 1 INiTlKK-KI.UIDS of hlood i.Miuvia po-t iiioitiMii liiMii l.if.iiiiics. Thi- l>t<).).l sfiiiiii coiitaiiifil (U-iitiio-alhiimoM'-'.lN- >iil>-t,inn-;. anil loiuiii ami (viosin. lAiti.r. X NatUK of >|.riiinrii. - 1 - J -z iii «-| o V 0> Iiihi rriilar I'lciu i->y ..■ Siinplr I'lriii.il KlVu-ion l.liopalliic I'leuial KlTu- siiill ... Tiilii H'lil.ir ricurisy I ardi.ic l'.iiUiii Kmpjcina It • • • ■ * ■ Postpncunionii' Caii- tircno (if liini; Back-prpssiire .. riibtriiilous Cardiac Failure II 'I Chronic Peritonitis II I" rhosis of Liver) Cariinoniatosis tra.i' ' o ... " o . .. o o ... o + + + + o o + + + ' o iCir Polyiirrliomcnitis Atrophic Cirrhosis of I.ivtr Cardiac Faihire Renal (Kdema sine Albumin- uria ... + ' ... O o + t ... + i... o o o i + + o + o o o o + o o o o o o o o ... trati . 'raci ... ... (lac. ... traci o o .. . o o . . irai't I) o o o o o o o o + + o o + o . tract ... ■ : + : ••• . ' o i ... o o o o o o o o o o + + + +■ o o cma/ < luid\ CEdcma / Cardiac Failure Flu g I Tanireatic Cyst ... o ^ -, L'liitui-iilar liroad I.ig:: j ; "u mcnt Cvst f t + .£ Unilocular Cyst ... ; tr:ic<; o + o o . . . tr.icf o 'i o J... 1 1 + 1 * 1 + i + t 1 ... o o TIIK < IIFMIi AL KXAMINATION OK IL'NCTLKK-Fl-UIDS 8l To MiilK-r ami Jochinann wi- owv tli.- further discovory that if l-nruU-nt sputum »)<• pl.uv.l on I.otfl.-r's soruin. (UK.-stioii of thf Uttir will take plair . and, nior.-owr. that tlu- hloo I of Wuctinio l-aticnts produces the same solvent effect. The soKvtU action did not occur if the hloo.l had In-en heated, a Jact which j.rove.l that the leucocytes of leucanuc »)lood contain a ferment capa»)le „l it.tinK on servers have corroborated these observations, tli..ui,'h they were not absolutely new. Thus. Opie found that in intlammatorv exudates due to bacterial causes a proteolytic enzyme was present, and he tested it by the eff.-ct on aleuronat, and a N-estimation by Kjeldahlising after coitjulation. Opic also noted the interfering action of the serum on this leucocyte ferment, and that it was thermolabile. Ffeiffer f.jund the pro- tiolytic ferment in the polymorphonuclear neutrophile leucocytes, which explained the autolytic processes noticed in leucicmia. The autolysis was estiinateil by the amount of non-coagulable nitrogen. Stern and EpiK>nstein showed that the ferment can l>e tletected just as easily by gelatin as by blood-serum. The interesting fact was found that this proteolytic fjrmcnt will withstand admixture with formalin ]Keen first established by Salkowski in i8()0. The problem which is most discussed is as to whether autolysis is in any sense a vital i)rocess. Wiener. Poll, Langstein, and Neubauer considered that autolytic pro- cesses could not take place in a living cell ; but in order to establish this it would be necessary to ascertain whether autolysis can occur in an alkaline medium. That this is actually the case has been made out b> Drjewzki (U)o(>). The practical value of the subject of autolysis lies in the fact that in certain flukls no autolytic changes are to be made out. The existence of certain ferment reactions might be accounted for, on the other hand, by assuming autolytic changes to have taken place. Drjewzki's method of study was as follows : The substance to be tested is diluted with water and 75 per cent, chloroform is added. Another mixture is alkalised with sodium carbonate. A third mixture is boiled. The three mixtures are incubated for a given time, then brought to the boiling-point, made up to a litre, and the fluids filterc.l. Next day the filtrates are concen- tratetl to 400 cc, and in the filtrate the following processes are carried out : (a) From 20 cc. estimate the X. (Kjoldahl) = total X. (h) To 50 cc. add 5 cc. dilute HCl and 10 jx'r cent, phosphotungstic acid till a precipi'tatc ceases to fall. The filtrate is Kjeldahlised, to get iiwmimid-N. (c) 50 cc. receive i cc. dilute sulphuric acid. Saturate with zinc sulphate, and leave to stand twenty-four to forty-eight hours. The precipitate (albumose) is Kjeldahlised = albumose-XA (<0 100 cc. are alkalised with a few tlrops of * Wuns. t Tlu' procipitate imi-t W IhoroUKlily tlry before Kjcldalilisinfe else the flask will burbt. THE CHEMICAL EXAMINATION OF rUNCTURE-FLUIUS S3 ammonia, and the phosphates are filtered off. Add 3 i>er cent, ammoniacal silver nitrate to i)recipitate the purins, an'l after twelve hours (darkness) the precipitate is collected, washed, and Kjeldahlised = purin-N. a.—(h + c + d) = nitrogen of diamino-acid, peptone, and ammonia. The following results were obtained in one of his cases : Analvsi!< «fter 72 hours' autolysi*. A without NajCOa H CO with .N».^0, wiih Na-iCOi. co«|fulated at and ferments. ; no ferments. , once. % total N % total N % total N % total N Total nitrogen Monamino acids Albumoses ... Purin bases ... Diatnino acids pef tones ... 6-545 ! 4'022 0-490 o 826 61 -60 748 . 62 and 4ii2q 2-184 0532 0490 ... ! 3-570 5316 ! 172* 1993 i °6oi 11 91 1 0049 1197 183 09065I 1506 1198 3-«5 - 4823 ••• ! ••• «6-83 ; 0756 : 24 00 1-37 i ... 1 ... I ! 33-57 I . . — This shows that autolysis still occurs in an alkaline medium, although it is true that she velocity is much slower than in an acid medium. Column D is a control. Another answer to the question as to ante-mortem existence of an autolytic ferment lies in the following consideration : Why should a ferment exist in a cell during its whole life, and only come into oi^eration after the death of the cell ? The Importance of .4»/o/y,sts.-The following are examples of autolytic decomposition : (i) breaking down of malignant tumours': {2) acute yellow atrophy of the liver ; (3) effusions in process of absorption ; (4) absorption of pneumonic exudate— the proof of this lies in the occurrence tf amino acids in the urine ; (5) relaxation of rigor mortis. Besides these, the pueri-K?ral changes taking place in the uteru.s and the changes of phosphorus poisoning are examples of auto- lysis. It is natural to supjwse that if autolysis occurs in diseases associpted with effusions we should be able to find evidence of it in the effusion itself. Consequently, the detection of albumose in such fluids acquires a considerable imiwrtance, and a list of the cases in which albumose has been found is given in Table II. It is there seen that albumoses frequently occur in tul>ercnlar H STUDIES IN PUNCTURE-KLUinS l)leural cases, and also in empyema. In a purulent effusion one woukl exix-ct autolytic jirocesses to take place in any case, as. from exi>erience of empyemas which have been opened, one has frequently seen a digestive action being exerted on the exposed surfaces. In the case of i>eritoneal fluids albumoses are frequently l^resent, just as jH^ritoneal fluids in general have been found much more complex in comi)osition than are pleural fluids. That the presence of albumoses does not necessarily mean lireceding autolytic processes has been pointed out by Magnus- Levy and by Chvostek, the latter having found albumoses in the urine as a result of ulceration of the intestine.* Again, there is the possibility that the apjiearance of such bodies as deutero-albumose in a fluid may be the result of hetero- lysis (Jacoby), some ferment, say, in the li\er having something to do with the process. The substances which demand search in order to study this ]>oint are : (ii) Leucin. tyrosin, glycocoU, xanthin bases. (h) Annnonia, cystin, pentamethylenediamine, lysin, arginin, tryptophane, asparagic acid, glutaminic acid, histidin. " (c) Thymin, uracil, H2S, succinic acid, j^entoscs. ((/) Peptones and albumoses are merely intermediate pro- ducts. From this formidable list we may refer especially to leucin, tvrrsin, lysin. arginin, tryptophane, as these have been most searched for in jieritoneal fluids.f The results, which will be found tabulated under the various headings, go far to show that either some autolytic changes are going on in exudates and transudates, or that such bodies appear as the result merely of accidental transmission. TheonK way of distinguishing between autolysis and extra- neous causes ol decomposition is to watch the changes which the fluid undergoes on being kept under aseptic conditions. Such observations may be made without going into lengthy analytical * Sucli authorities as AlxlfrliaKlen have denied that albumoses occur in normal blood, while Freund asserts that the methods employed for their detection in blooil have not been sutticiently sensitive. (See btochem. Zeil. vii., p. S4S.) t Tlie special tests lor each c these bodies will be found indicated under their respective headings. THE CHEMICAL EXAMINATION OF PUNCTURE-FLUIDS S5 processes by determining the variations in the electro-conduc- tivity of the fluid from hour to hour (thymol added), since decomposition of proteid would result in diminished inhibition of the conductivity of the electrolytes of the fluid (cf. p. 9)). The following results were obtained: TABLE XI Autolysis is Punctlre-Fi rit s Duration in \ hours. 1 At outset. 22 46 70 92 H7 142 170 189 3Si 'lui i from Ovarian Pleural Fluid Cyst. Ruptured Thoracic Aneurysm. 888-8 at 19-3° C. 843 at 23° C. 818 889 893 ... 95 1 960 981 1062 1073 985 ... 1059 ■ •• The figures represent the changes in conductivity (expressed in terms of io-'>) and it will be seen that in each case there was a steady increase in the ionisation of the fluid. This seems to ]X)int to the existence of an autolytic decomposition, though only of slow develoi^ment and moderate in amount. The initial fall of conductivity in the case of the ovarian cyst is jwculiar. According to Midler and Magnus-Levy, the autolytic decom- position of albumen is accompanied by breaking down of lecithin and of CHO groups into volatile acids, lactic acid, ami succinic acid. There is also a remarkable .imount of ammonia liberated. Ascoli and Izar discovered that autolytic processes are very greatly accelerated by the presence of inorganic colloids such as colloidal silver, platinum, or gold, .\rsenic, on the other hand, may inhibit oxidation (C. Foa). As regards the location of the enzyme there aio two possi- bilities to consider. In the first place, the autolytic ferment may occur as an endo-enzymo, within the cells of the puncture- fluid, just as it occurs in the liver-cell, the spleen-cell, the lung- cell, and the thymus-cell. In this case one would exjxjct that on filtering the fluid the ferment would l^e removed. The other possibility, however, pievenis this from affording any definite evidence. That is to say, the ferment in the fluid, just as STIDIFS IN lUNCTL'RE-KLUIDS 1^ is the case with anv of the other ferments met with m i-uncture- Ihmls mav have beccme hlx-ruted, owing to the breakmg up of cdls ■ remnants of such colls are easily seen in any film prepara- tion " In whichever situation the ferment may be. the hnal result is the same, and it is a uuai.r of no very great imix>rtance m which way exactlv the ferment came to be in the fluid at all. But when we "consider how many ferments there may occur in anv one cell it comes tc be a question by what means they cm ail be accommodated in so small a compass. Pfeffer and Hofmeister considered that the colloidal structure of protoplasm allows the fe-.ments to be within the cell, distributed not only in space but in tune within the cell; and Jacoby, who has contributed many papers on the subject of ferments and anti- ferments has i.ointed out that ferments may remain in a state ol zvmogen for a long time, the active period being relatively only a momentary one. This author compares a ferment to a toxin, and supposes it possible that a ferment may consist of a hapto- phore and a zvmophoie group. The haptophore group would become attached to part of the cell substance and lead to the formation of new compounds, which, being liberated into the serum act the i)art of an antiferment. It is interesting to note that the solubility of ferments is altered by the presence of lecithin. The bearing which this obserx ation has on the study of puncture-fluids is that if lecithin be absent from a puncture-auid its absence may account for the presence oi absence of any given ferment. .,.«.„ Antiferments —The nature of antiferment reaction is difficult of ecially by sulphates. Fuld's experiments show that the appearance of antiferment can also be mimicked by those reactions in which the order of addition of the reagents is of imiwrtance. This is exemplitied by the need for adding potassium ferrocyanide to acetic acid in the test for proteid, and not vice versa. Possibly something similar will prevent or allow a ferment action. The exi^eriments which Hedin made with charcoal and trypsin might be interpreted in favour of the idea that antiferment action does not dei:)end on any specific substance. We see, then, that certain ferment actions can be brought to a standstill or prevented from occurring altogether by the occurrence of certain antagonistic conditions. It is as reason- able to speak of blue light as anticatalase or of chloroform as antitrypsin as it is to give a si^ecitic name to any "anti- ferment." If antiferments really exist one must rely upon biological exiieriments to establish the fact. The most important evidence is certainly furnished by such hemolytic phenomena as have been carried out by Morgenroth and many others. Thus, the injection into a rabbit of a ferment will result in the production of a serum which has the power of rendering the particular ferment inactive. On the other hand, Liebermann found that active rabbit immune serum only prevents hajmolysis if calcium chloride be present. In this connection the observation by * Thi. tart imparts an inestimable importance to the presence of soiiin ican t that Heit/kt' and Neulx>rg describe ti-emulsin a> connected with the globulin an an we have seen fraction, when that lecithin is also associated with tfie globulin fraction. lacobv. in his sununary of his opinions on the nature of ferment action, sav; The fact that rennet antl pepsni can bt separated from hbrin l)y alkalies, whilst rennet is se|)arated frcm antirennin bv acid, is in favour of the view that the union of ferment with substrate is not the same as the imion of ferment with antiferment." The association of antiterments with globulins reminds us that the action of an antiferment may merely be one of alteration of electrical charges in the colloids C(mcerned in the reaction. Out mere consideration. In the case of trypsin it is now well knf)wn that without enterokinase this ferment will not act on i)roteicl. Suppose now that this fact were not known, one would have found that certain solutions would not digest proteid, and have assumed that these solutions contained antiferment (antitryjjsin). The truth is, of course, that the activator of trypsin is merely absent. But suppose, further, that the enterokinase cculd be interfered with by some other body, then we shall be introducing a third factor into the case, and it would be rea,s( n- able to descuDe this third body as antiferment, though its name would be anti-enterokinase. and not antitrypsin. It is. however, out of place to discuss this subject further, although the amount of information which is being ga-hered together liy observers in all countries is increasing by geometrical progression So much is this the case that it becomes almost impossible to fix the state of knowledge on the subject.* * Tlu- vitws ahoM' expressed were written down before the work •• Uecent .Advances in i'hysiology and Biotheniistry " came to notice. The whole siihji-ct ot leriiient action is so lully dealt with there (by Prof. B. .Moore) as liardlv to need supplement. On the other hand, many valu- able contributions have appeared since Its publication, in ihv Biot'.cmual Journal, in the liiochemischc Zcitschrift. etc., as also a work by Schade. •• Diabetes und Katalysc." Many of the references to literature for KJ07 THE CIIKMKAL FAAMINATION OF PUXCTUKK-FI.UIKS 89 Method of Detection— The tact that pus-cells contain a proteolytic ferment which will produce a small roteolytic ferment in a Jjuncture-Huid. One part of the fluid to Ix- tested is treated with from 5 to 20 parts of pus, and a drop of the mixture is added by means of a platinum loop to the serum tulw. or. as Miiller and Jochmann advocate, a plate of serum, which has the advantaf,'e of ease in working, and also the advantage that the same plate will serve tor many experiments. -t If the serum shows liquefaction after incubation, then the proteolytic ferments of the leucocyte have not been interfered with, and therefore there is no antiferment present in the fluid. On the other hand, liquefaction may not occur, in which case one assumes that antiferment is present. The pus nuist be freshly obtained, and must be sterilised by addition of toluol. The pus used must not be tul)erculous. since tuberculous pus contains no proteolytic ferment. J Antiferment may be estimated, according to Ed. Miiller, l)y ascertaining how many times fresh pus has t(j be diluted before the fluid to be tested will prevent it from liquefying the dried Loffler's serum. For blood-serum the normal limit is 75. The method of detection of antiferment § may be applied to Epiwnstein's method for detecting proteolytic ferment, which has the advantage of employing a readily prepared medium. The fluid to be tested is mi.xed with 10 jier cent, gelatine con- taining I jx-r cent. soda, and a similar quantity of 085 jK-r cent, saline is added to another portion of the same gelatine (to furnish and 1908 will be lounil in the Bibhosraphy on page z(<3 of this work, though only those names are included which have been mentioned in the text. Investigations are being made in order to be able to present as full evidence on this problem as possible. • The serum is cow serum, and contains glucose, or 8 per cent. XaCI. t Tlie chief disailvantage lies in its manufacture. * This fa:t may be of use for distinguishing tuberculous from other sources of suppuration. § It must be understood that these methods demonstrate an anti- ferment action without by any means indicating tlie existence of a .specific antibody. It will Ix- remembered that even .an .-mtitnxin is acting in virtue of special colloidal properties without necessarily con- stituting an mtity or a substance capable of being isolateil. 90 STUnil.S IN I'L'NCTL'RE-FHins u control). Botli an- incubated for twelve hours at body heat. ( )n removal, both Petri dishes are set on ice until the saline s.impie (control) has set. If proteolytic ferment be present the gelatine will no longer set. while, if antiferment be present, or if there be no ferment at all, the gelatine will set. To apply this test for detecting antifernients, it is obviously only necessary to add t .hiolated fresh pus to the fluid, and see the effect on the gelatine. It is, however, convenient to use ordinary leucocytes in place of the pus, and a small quantity of blood may theiefore be collected, washed in citrate, and then in saline, exactly after Wright's method for getting leucocytes for opsonic index determinations. For the small quantity of fluid to be tested this will furnish readily, and at any time or place, the necessarv means of carrying out this modified test. SECTION II THE PHYSICO-CHEMICAL EXAMINATION OF PUNCTURE-FLUIDS Contents. (A) Osmotic prt-ssure— Theoretical considerations on osmetic pros-iuro : how to calculate the ounotic (iressure ; the decree of dis- sociation-Theoretical considerations on electroconductivity ; cor- rections necessary for variations in temperature and in amount ol proteid in the solutions examined; achloride electrolytes -Osmotic concentration- -Theoretical considerations dealing with the elfect ol mixtures of many substances on the freezin;j-paint depression and on electroconductivity The relation of Ireezin- point depression to specific Rravitv- Methods of determining the freezinjj-point de- pression an.l the electroconductivity, -Results oi examination in eacli case -The plasmolvtic Tnethoosing its outer world, but we have to deal with pro- cesses of diffusion and of osmosis. Osmosis is si>ecially important in the case of puncture-fluids, as it atcounts for so many of the phenomena that they exhibit. A consideration of the factors on which osmosis dejiends is therefore needed, although those theo- retical considerations which deal with the question of pouring out 9» 92 STl I)li:s IN I'L'Nt TL'KK-H.lins (if thiii atisorption Ironi them. Iiavf loiiu- tr. Ik- so sjH'cial a subjfct that nffu-nce to it does not COIIK within the ()l)jf of thiN woilc. (A) Osmotic Pressure. When a sul)stance is dissolved in wattr the niolecules ot tliat >nl)stance are evenly distrif)Hted tliioiiKlKtut the water, and exert, not only a pressure on each other, hut also on the walls of the rontaininj^ vessel. The pres- sure transmitted to the (onhnes of the fluid in the endeavour of tile molecules to occupy a larger spice is called the osmotic pressure of the tlui wliidi f,'ovcrn tlie osmotic pressure of a solid dissolved in water have i)een found i)y van't Hoff to k' identical with those which olitain wlun a |,ms is dissolved in water. In other words, a solid tlissolved in water behaves like a gas dissolved in water. If thire he several substances in solution, then the total osmotic pressure is ecpial to the sum of the osmotic pressures exerted by each sui)stance taken separately. The osmotic jiressme of a solution varies according to the temi)erat\ue of the solution as well as according to the number of molecules present. .Ml solutions which contain the same number of molecules in the same volume of fluid e.xert the same osmotic in-essure. In other words, all equimolecular solutions exert the same pressure. I'rtssiirc X volume = prrsMire comintralii'ii constant. It is obvious that the only variable will be the weight of the molecules, so that if two substances be dissolved in a given volume of solvent, the osmotic pressure of each sdution will be related to the other according to the molecular weights of the dissolved substances. It is on this fact that one of the methods of determining the molecular weight of a substance deiH>nds,* but it also affords a ♦ Till' tt)ll()\vinK law> have liitn fo\incomo more as tlu- conversion of g^ work, so work is needeil in causing concentrated, that is. to ac«iuire a higher c«motic pressure If we determine the amount of work (osmotic work) wlucJi is ,x>rformed in ju^t causing solvent and olve.; sul)stance (a solution of known concentration) to separate, we can calculate the amount of pressure. If a solution l>e frozen. irature than that at which the solvent alone would freeze. It is this fact which underlies the use of cryoscopy, that is to say, the determination of the fieezing-i)oint of a fluid. ,. , , • SupiK)se that X grams substance are dissolved my grarm solvent, and that the depression of freezing-iK)mt is then .- L., Ihcn I pm. substance dissolved u. y gm. solvent will cause ^ depression. I gm. mol. (M.) ,. y •• ' and I ICO X X Mzv^ luox ■ nioticiilar " ileprcssion. The molecular depression is a constant (k).* For our purposes we wish to know .v, so that the equation become Mzy ICO k. and supposing there were only one substance present m a given solution (usually water), we could readily ascertain how much substance wa, present, o. . in other words, we should be able to * K for water is 18-5. 94 STUDIES IN lUXt TL'KK-KLUIDS iwr t)ii-> nutlKxl tor |nir|M>sistanci's occur tound that every riS5 depression of free/.in^- l)oint indicates the pressure ♦ of one ^ram-molecule of sul)stance in each litre of water. riie tact that the ecjuation ^iven does not holil ^ood for every individual >ul)st nice brings u|) the imiMirtant (juestion of dis- sociiilioH iij Siills when in solution. A solution of salts such as is met with in urine shows a much greater free/.ing-iKiint depression than its concentration would lead us to ex|x'ct. and the same lioltis good for puncture-fluids where sodium chloride — though the most conspicuous— is not the only salt, but occurs associateil witii sulph.iles. phosphates, and carbonates of potassium, calcium, and magnesium. To take the case of NaCl alone, the formula above given would lead us to exjiect that a gram-molecular solu- tion o. sodium chloride (i.e. a solution containing 58-5 gm. — the molecular weight in grains— of sodium chloride in each litre of water) should have an osmotic pressure of 185 x 1205 atmo- spheres, liecausc all gram-molecular solutions in uater freeze at 1-85 {'.. and e.ich degree of depression of freezing-point means .m osmotic pressure of 1205 atmospheres (Kaoult). As a matter of tact, the solution referred to has an osmotic pressure of ^•51 X i_'()5 atmospheres, for its freezing-|K)int is 5-51 ' C. This discrepancv was exi)lained by .\rrlienius, who showed that when a salt is dissolved in water it breaks up into electrically charged ions, without which the resulting tluiil would never be able to conduct an electric current, and without which the fluid would not exert as great an osmotic jiressuic. It is clear that if a solution of NaCl contains not only NaCl but also Xa and CI ions. the resulting number of constituents present is greater than if all the NaCl has remained as Na(T. The conception that the in- • I'ltltiT tound tli.it It .1 Kr'>"i"iol^'^'"l<' oi any substance Ih' dissoiMii in j.;i ^ litris ol wattr. such a solution will i xirt an osvnotic prtssuri' ol one atinospluri' at o""" C. I'llVSICO-CIIKMUAL EXAMINATION 95 .Uvi.Uuil ions a.t )ust its intU'|H'n.Unt inolrciiks ami txtit an oMuotic pressure which forms the |..th of the theory of electro- Ivtie .liss,K-iat.on -explains the tart that the free/.inK-ix»i"t .U- ,,ression of the solution is Kreater than it shouUl hav U-en. As ,.K)n as sodmm chloride is (liss.)lve(l m water some of it .lissociates into Na and CI. each of which adds to the osmotic pressure, an.l ,,Ko enables an electrical current to pass. In this way. then the . lectroconductivity of a flui.l is de,H.ndent .m the same mtluences iis is the free7.inK-l>oint depression.* Ami the mention of this tact in this place will enable us to understand the link binding the electrocomluctivity method of research to the cryos ..pic iiuthod which is dealt with in the present work. To return to .he solution of NaCl. Such a solution contains : fN«(l ■♦ >Na +.v/»s Xa and CI and S()4. an.l PO4, and Ca and K, etc.. etc. If the solution of all these salts Ix- ren.lered more dilute, the .lissociation will increase, and the ratio l>etween un.lissociated and dissociated salt will alter more and more until, theoretically, at infinite dilution, there will only be free ions present. \\ e wi>li to kn.)w how the proportion between molecules and ions can bo ascertained in a given solution. The method of calculating this can be indicated by reverting once more to the simple example of XaCl. . . , ,. It has been state.l that a gram-molecular solution of sodium chloride freezes at -3-51' C. instead of at -rSs^ This solution ♦ N.B. — Ininase in " trtfzing point il.j)n-.-.ion i-ans :i />(// ol t.-m- piraturc. 96 STUDIES IN I'LNCTURK-KI.UIDS therefore l)eli;ivts, not like a gram-molecular solution, but like a solution containing J-5I 1-85 ; 189 grpm-niolecular solution. In otiier words, by dissolving the gram-molecule of XaCl in water we have niatle it have the same osmotic jiressure as if we had made a i'8() gram-molecular solution of undissociateJ substance. In other dilutions the number will be difterent again, for the more a substance is diluted the greater will be the dis- sociation, but Wf can determine the ratio of the true to that of the theoretical (calculated) osmotic pressure in the way indicated. Putting the facts into a formula, we have : dctrre of ilissoci.ition "a" = IrcczinK-point found trt'ezinir-pnint calculated — I iiuinbir ot luns in one moleiulc ol salt — 1. In the above example, for instance, 3 5' 2 - 1 Sq — I - 0-89. That is to say, oSijof the gram-molcule has dissociateu. and only (jii has remained undissociated. I'nfortunately. however, the tluitls under consiileration are not solutions of NaCl only, but of a numlx?r of other substances, an.l it becomes a matt(>r almost of impossibility to give a value for the amount of dissociation when we do not know how much there is of each salt present. Not only this, but there are sub- ^♦.mces present in a puncture-fluid which introduce the further com])lication that they have the power of j)reventing the salts from dissociating as fully as they otherwise would. They inhibit the dissociation of the constituents of the fluid. However, by making use of the term osmotic concentration (C ), which was introtluceil by Hamburger, we can obviate this o little. Of course, if s is very ■^ 1 , nnn little, this fraction is practically negligible. !>?"IHk .. kl 98 STUDIES IN I'UNCTLRE-FLUIDS find ultimately that we come to a iwint at which, no matter what the dilution may he, we do not alter the conductivity any further. The figure remains constant. This means that the sodmm chloride present has been as completely ionised as i^ssible. It the figure representing the conductivity be now 209, we can ascertain the degree of dissociation of the NaCl in the ongmal fluid from the simjile relation : Coiicliirlivitv of ori);inal fluid ^ 185 CoiiilULtivity at inliiiitL dilution 209 = 0-89. Which means that of every gram-molecule present at first, o'Sg part of this molecule was in a dissociated state. It IS obvious that we are arriving at the same result as we were domg with cryoscopy, when we were calculating the lomsed portion of a fluid on page (,0. with the excep..m that bv the conductivity method we arrive at our result in a very short space of time. All that it is necessary to do is to ascertain by experimen the conductivity of the original fluid at a given temperature, and then dilute the fluid, say, four times. Multiply the new con- ductivity figure by 4, and this gives the conductivity of the fluid as it should have been when undiluted. Repeat this for ever- increasing dilutions until the final conductivity remains the same or attains a maximum. The following example will explain this procedure : TABLE Xll TiiE Dissociation of a Pleural Fluid Distribution of I'leural Fluid. Observed Conductivity. Conductivity x times diluted. I'ndiluted. 84.? 2 537 4 296 8 173-3 16 942 32 45-< 64 28-33 128 1443 2S6 901 5>2 4 35« 843 1074 1184 13864 1507-2 1443-2 I829I 18470 22965 2227 7 The last values for observed conductivity, x times diluted, PHYSICO-CHEMICAL EXAMINATION 99 show that the maximum has been attained, so that we have approximately arrived at " infinite " dilution. The degree of dissociation is therefore . = o^S. It is needful here to pause to consider how far the deductions described are really justified. We have taken a sjxicinien of pleural fluid and diluted it with water, and estimated the electro- conductivity of the successive dilutions. Now, pleural fluid often consists largely *— by volume, if not by weight— of albumen, which is a viscid substance, besides being a non-conductor of electricity. To put it in other words, we ha%-e in serum a mixture of electrolytes and non-electrolytes. Xow, a non-electrolyte is a substance which has the power already referred to of inhibiting the dissociation of salts with which it may he associated. The albumen in the fluid will therefore interfere wifi the conductivity, and if the fluid he diluted a littl- reflection wi show that although the non-electrolyte serum-albumen is l>eing diluted, yet it does not dissociate, while the salts (electrolytes) present are dissociating in the water. The presence of the still undissociated molecules will exercise more retardation than ever on the dissociated mole- cules—number for numlier. That is to say, the conductivity will lie ever less than it should be, and the degree of dissociation will work out less than it should be. The figure 038 in the above example would then be much too small. We are able to correct this error, by the aid of researches by Bugarsky and Tangl, who found that for every degree per cent, of albumen the conductivity of the electrolytes was diminished by 25 per cent. If we make the needful correction for the original conductivity and divide that by the conductivity of serum at infinite dilution, we shall get a degree of dissociation of 041. But we have not corrected for the interference of the diluted serum on the diluted electrolytes. The needful fornmla; for this are not to be had, so that in this case we have to content ourselves with a statement that the degree of dissociaticn of pleural fluid lies between 038 and 041. This is unsatisfactory.+ » Depending on the variety of the effusion (whether exudate or trans- udate). t Fortunatelv, however, the value of a knowledge of the degree of dis.sociation of various !>ody fluids r- r,ot appan-nfly of much importance, and probably depends entirely on the comjiosition (relations between salts lOO STUDIKS IN rUNXTURE- FLUIDS Not only is there the interfering influence of non-electrolytes on electrolytes present in the case of serum, but there is also the interference resulting from friction between the ions. The friction is much greater when the serum is undiluted than when it is diluted, because there is so much albumen present. The very dilute serum exhibits no noteworthy degree of ionic friction. However, we may sum up the retarding influence as that due to the albumen as a whole, and by making use of the following formula, original conHiictivitv x ICO Corrected conductivity = ,oo„ ip^^^rccntage ol albumen x i 5). cori> ct for all errors together. But there is one more point to consider in connection with this part of the subject, and that is as regards the actual electro- lytes i^esent. Is it justifiable to express the concentration of electrolytes in terms of NaCl, or should it be expressed in terms of any other body ? This question must be answered in favour of the latter in some cases, and of the former in others. The following figures will illustrate this conclusion : A given pleural fluid contained oo()2 gram-molecule i^r litre of NaCl. Its conductivity * was 1068, which, for the tempera- ture, is equivalent to that of a solution of NaCl of 0123 gram- equivalent per litre. The difference, 0061, gives the concentra- tion of the achlorides in terms of NaCl. The ratio of chlorides to achlorides is then 0062 : 0061 — 1016. This is a simple calculation. and albunun), so that \vc get no further by this method of study than by an onlinarv chemical examination. Thus, tile lollowins values have been obtained in the case of bile: Fluid. Number of Specimen. Degree of Dissociation. Bile I 3 3 4 \ 7 023 034 048 051 054 041 0-25 • Throughout this account " conductivity " means " specific conduc- tivity," and is expressed in terms of lo"*. PlIYSICO-CflEMICAL EXAMINATION lOI The other method gave these results : The chlorides amounted to 0062 gram-molecule per litre. The degree of dissociation of this (calculated by Arrhenius' formula) is oSGi. The number of molecules plus ions (above formula) is 01 153. The conductivity of such a strength of NaCl subtracted from that of the fluid gave a conductivity which is possessed by a solution of NaoCOj containing 0062 gram-molecule \yeT litre, with a degree of dissociation of 0570, and 01342 molecules plus ions per litre. The ratio NaCl : Na.COj is now 116. This calculation is obviously much more prolonged.* In the case of certain transudations, and in the case of urine, the preponderant electrolyte present is undoubtedly sodium chloride, but in the case of exudations sodium chloride does not occupy such a conspicuous position, and sodium carbonate takes its place to a certain extent. In blood-serum the sodium and the chlorine are the twc ost abundant elements, the jiotassium, calcium, phosphates, and carbonates are the least abundant, as shown by the following analysis of pus-serum by Hammarsten : NaCl Na,SO, ... Na.,HPO,... Na.CO, ... Ca;(PO.),... Mg,(PO,), PO, (in excess) 539% 031 0-46 « 13 0-31 0-I2 005 Chlorides 5'39'; Carbonates ... ... 1"I3 Sulphates 031 Total phosphates ... 094 This shows the prejwnderance of carbonates over j)hosphates and sulphates. It must be admitted that this is not invariable, but it has been found that even if the phosphates be in excess and the mode 01 expression of achlorides be altered accordingly, the ratio still remains similar. It is obvious that the only way of avoiding errors at all would be by complete chemical analysis. The present method is an attempt to obtain useful results by the simple method of conductivity determination. The method of study of the particular fluid would therefore consist in the following observations and calculations. In the first place, we a certain the freezing-point depression, which, • An error may lie in these calculations in the values used for the con- ductivity ot NaCl ami Na.CO, solutions, ^vhlch have been r-tirt-.^Ued in watery solution, whereas they occur in alhuminous solution in the body. The degree of dissociation is vastly different. lo: STUnilS IN I'UNCTURF-FLUIDS when divided by i 85, will give the total number of molecules plus ions, the osmotic concentrotion. Secondly, we ascertain the clectroconductivity at a given temperature. Thirdly, we ascertain the amount of albumen present, if any. Thin the value for the conductivity can be corrected by the use of fornuila on p. 100. There are now two courses oix>n— one can ascertain wh.at strength of sodmm chloride has the same con- ductivity by calculation, or one can ascertain the actual concen- tration of the chlorides in this fluid by means of chei lical analysis and ascertain how much of the conductivity (or of the freezing- point depression) is represented by them. We shall then have the concentration of the electrolytes as a whole expressed in terms of yaCl, and the concentration of the chlorides alone, the difteren'c between the two giving us the concentration of the " achloridt; " electrolytes— that is, the electrolytes other than chlorides, expressed, however, as if they were NaCl. Or, again, we can express the concentration of achlorides in l.-rms of carbomiks, by ascertaining what strength of solution of Na.COa has the same conductivity as " total conductivity- conductivity of the given concentration of NaCl." L. order to ascertain the concentration of carbonate according to a given conductivity it is necessary to ascertain the conduc- tivity of a series of solutions of sodium carbonate of known strength. This is best performed in a number of specimens pre- ]iared by oneself, the intermediate values being either obtained by Lagrange's interpolation formula : >■« = n) ( X -a) (X -c ) ■■■ (X -n)^.^ ^ (a~^"br(a -c) ... (a - n)^* (b - a) (b - c ... (b - n) (X - b) (x - c) ... (X where y^ is the lowest value for omdiicthily. v, the ne.xt value (deter- mined by experiment in each case), and y, the value for conductivity at any strength of N'a.CO, desired, a is the concentration of Na.CO^ of conductivity y^. h that of y,. and so on ; n is the concentration of the strongest .solution whose conductivity one has determined, and -v is the strength of the solution whose conductivity one seeks, or by drawing a curve, on which the intermediate values can be read off.* It is important, as will be shown presently, that all the observations be made at one temperature, say 18X. * While the graphic method is the simpler, it is also the less accurate. PHYSICO-CHEMICAL EXAMINATION «03 Not only is it desirable to know the concentration of the carbonates and chlorides in a given solution, but it is desirable to know tlu number of molecules plus iom of each class o/ electrolyte per Hire of fluid. This demands a more lengthy calculation, which will be found to have been adopted m the series of analyses on puncture-fluids described in the section on differential diagnosis (IV.)- The method of calculation is as follows : Starting with the jiercentage of chlorides in the fluid, we ascertain the same value in terms of " concentration " ; i.e. gram-molecules per litre. From this value the degree of disso- ciation is ascertained from the formula j--^ j ^ where A is the equivalent conductivity of the solution of NaCl. and 1,.„ 1,. the rate of migration of the ions Na and CI at infinite dilution. Now K.. + 1. ^ 44-4 + 65-9 = "0-3. The equivalent conduc- tivity can be ascertained for any strength of solution at 18 C. from the tables given in the Apjicndix. Having ascertained the degree of dissociation, its value is put into the formula of Arrhenius : (i -I- «) gram-equiv. The result gives the number of molecules plus ions m the given concentration of NaCl. The following formula will be found to give the result desired without any further trouble : Mols. + ions I + (■'•-je't. Ill" :■"')> ^ 1 10 3 where a = gm.-equiv. solution of NaCl in question Q ^ ,^ „ in Kohlrauscb's table less strong than a. " " ' , ,. itronger than a. -0 ■ t, = Y = equivalent conductivity of solution C,,. r A, = Having subtracted the value for the conductivity of the fluid being examined from that for the given strength of chlorides (estimated by analysis), the difference gives the conductivity of the achloride electrolytes. Reading off either in one's diagram, o- in the list of conductivities which one has prepared from a series of strengths of Na,CO„ we ascertain to what concentration of NajCOj this conductivity corresponds, so that it is now possible I04 STUDIES IN rUNCTURE-FLUIDS to calculate the numl)cr of molecules and ions in this strength of NajjCO:,. Proceeding in the same way we get this formula : Mols. + iosition to discuss the effet I of mixture of electrolytes and non-electrolytes on the freezing-iH)iiit depression and on the conductivity, esix'cially in cases in which van't Hoft's law does not hold. The simple conceptions already detailed do not hoKl for a wide range of dilutions, but the o-imotic pressure will show de- viations from the law as one passes to high or to weak dilutions. A solution of cane sugar will show a more rapid increase of osmotic pressure on dilution than woulil be the case if the simple law held good. In the case of electrolytes the deviation is even more decided. This question is worthy of consideration. The interaction between them may take three forms: (i) inhibition of dissociation of electrolytes ; (2) chemical interaction leading to the formation of a numlx^r of larger and more complex molecules ; (3) polymerisation. It has been found that the latter does not occur. This question has been carefully gone into by Ernst Tezner, and he made up accurately weighed solutions of mixtures of this kind, in order to discover, if iKjssible, whether there were any relation capable of mathematical expression. The results ob- tained showed that when the concentration in watery solution is increased the osmotic pressure and other colloid properties increase much more rapidly than van't Hoff's law would have. This fact is noticeable in e^•'?n very dilute solutions (more than — ) If there are several different substances present in the solution, the total depression of freezing-point is less than the sum of the components. Starting with van't Hoff's formula : * (k + k|) = osmotic concentration of the dissolved substances, W = latent heat of fusion, T = absolute temperature, R = constant. 11 lOS STI'IUKS IN I'l NrliKh-FHIDS where \V ami T loinain constant whether the siiJwtanres are mixed or wlutlur tluy remain separate, Tezner's exiH'riments show that — lsented by th'' formula : Cone. X A = const, (cone. X)', — X and A being dilYerent electrolytes. {2) In a mi.\ture of salts, the IIIVS!C(M HKMH AL KXAMINATIDN 109 '.ilts arc «lisH«• the Name in tlu' two dilutions (i-oliy, '"' the rcs|Mctivc \oluiius 111 the Milutions i-oniliiitivily ofmixliui- « (v + i\) — oc ♦■ o, r, ; (4) till- ' lon btwitn the ions has also to 1h- consiilereil, a correction lieinsj espi.ially necessary in the case of soliiiiim-. alwiM o 111. ami may attain 1 to } )» rcent. even at the; decree of dilution. TaUinK into account this factor, .Srrlunuis suKRests the formula : i-nnftt. (cnnc, of ions X *) fconi-. of inns A") Cone, of XA =■ / , .. », .^ v . ^ \ ^ a "^; T V- total coix. of ions X + + A, '+•... + A + A, +....) X. \. ! following figures from cases in the Leeds Genera) Infirmary will show the incorrectness of such formuUe : TABl-K XIII Specific Gi«vity, Freezing-point observed. Freezing p.iint ulcululed from Ifif ispecilic tiravity. 1024 I 017 1032 I on 1 01 5 1014 roio I 022 1029 - - ■ ,- 1-459 i i-8o roJ5 127 rqio 1 238 aoo6 1 a-47 1061 1126 0990 1047 1 002 0750 0919 mn 135* .625 1775 2161 I 10 STUDII-S IN I'UNCTURK-KI.LIDS -, I In the case of inuictino-fluids the calculated results arc hojx?- lessly incorrect. Wo now come to the mcllwds of hcrjornting cryoscopy and (Icdroanhliictivity. As regaids the former, the widespread knowledge of the methotl rend( rs any exact account superfluous, A;', regards the second, there are several details worthy of con- sideration, tspicially from the standpoint that the method l>lays an important part i:i the examination of puncture-fluids as advocated in tiiis work. 1. Cryoscopy. — S(iik';es of Eukok. -The first possible source of error lies in an incorrectly graduated thermometer. The calibre of the tube mav not be absolutely uniform, so that each ilegree of the scale mav hul luive the same value. This error can be allowed for liy tl < alinary methods of calibration. The construction of liie w •'.ir may be at fatilt : the sides should slope (juite gradually i ;\\..rds the capillary. The 7,ero-point mav have l)een incorrectly tletermined. The best way of ensuring the coirectness of this is to make several determinations at the outset, and take die mean. Subsequent extreme care in hantUing the instrument, with occasional controls of the zero after tlays or weeks, according to the frequency of its use, will sufhce. It is essential to use absolutely pure water (or distilled water which has been boiled to liberate gases) for the j)uri)ose of fixing the zero-point. It is, however, best to use water which has been twice distilled, and once with glacial phos- phoric acid. rel)oiled, and preserved in absolutely scrupulously clean (boiled with acid, etc.) Jena glass flasks. A further control is obtained by estimating the freezing-point of a i-per-cent. solution of pure sodium chloride in distilled water after each observation of a fluid. It is much easier to obtain the freezing-point of a salt solution than of pure water, and this gives a control on the accuracy of the instrument. The next source of error is undercooling, because the more the fluid is frozen the more ice separates out. and the more con- centrated becomes the residual salt solution. If only halt a degree of undercooling is allowed, then only i bo of the bulk of the fluid has separated out as ice, and the resulting error is negligible. The freezing-point may be reached by this time if the mixture be suddenly energetically stirred as soon as so low a temjierature has been reached. PHYSICO-CHEMICAL EXAMINATIO I I I The error which may arise from using too powerful a freezing- mixture is similar, but the moans of avoiding this have alioady been explained. The temperature of the freezing-mixture should not be more than 3 C. below that of the freezing-point of the fluid to be examined. Any error which may arise from the fluid not being cooled uniformly is avoided by careful enclosure of the apparatus in cotton-wool and the use of a mechanical stirring device. Hamburger recommends that at each determination lie reading of the thermometer should be taken for a i-jier-c '•'.. solution of NaCl,* and also foi distilled water. If this be do.io before and after each series of observations one can control the thermometer itself, as it occasionally happx;ns that the reailings vary during the same day, either from variations in barometric pressure or from changes in the glass following exposure to coid. 2. The Determination of Electroconductivity.— The apparatus which is necessary for determining the electrocon- ductivity of a fluid is usually described sufficiently fully in text- books of practical physics. The following account will, however, save reference to such works, and include certain practical details ■which have been found useful. The following diagram will indicate the various parts of the apparatus, t The vessel which receives the fluid to be examined demands special consideration. It consists of a si)ecially shaped tulv, wide above and narrow below. It is very easily cleaned. As it is made of Jena glass there is no risk of interaction between glass and fluid to be examined. Every time it is used it is thoroughly washed out with distilled water, and wiped dry with absorbent non-medicated cotton wool. Every now and then it should be cleaned by soaking in weak nitric acid overnight. If these precautions are followed there is no need for errors to arise. • To prepare a i-per-cent. solution of NaCl, Hamburger reconiinends that 10 grams of pure NaCl which has been strongly heated in a porcelain basin, to drive out HCl and water, Ix.- dissolved in i kilogram of pure distilled water. By weighing the water there is no ri^k ui errors from temperature of the water or of graduations in the measure. The specific gravity of such a solution is rcx);^ at o"" C and there are 9970 grams per litre, or 1704 mol. per litre, and the freezing-point is-o'sS^'C. By adding a ii'Me thymol, this solution will keep a long time. f Ol- I from Fritz Kohler, Iniversitats Mechaniker, Leipzig. 112 STUDIES IN I'UNCTL'Ki;-FLUIDS mercury. This has been found most any spilling of the mercury should The adoption of this form of vessel also avoids any considera- tion of the many varied forms of vessel that different authors have used and de- scribed, for none of them are so readily cleaned and so little liable to break. The electrodes for this vessel are arranged as in the drawing which shows the apparatus. One electrode passes through the other, and they are formed of strong glass tubing ; into the free end of each the electrode is fused. They are kept in position by being fixed into an ebonite disc grooved to allow them to sit firmly within the tube, and be absolutely vertical in it. The tubes are three jiarts filled with mercury, and receive thin copj^er wires, a layer o f hard paraffin being added, so as to completely fill the tube above the useful for preventing the apparatus be PHYSICO-CHEMK AL EXAMINATION "3 accidentally upset, ami the copixr wire is also kept firmly in place.* The Treatment of the Electrodes.— It is neo -;ary to platinise the electrodes thoroughly before they cnn be us( This increases the conducting surface very greatly. The electrodes are soaked in soda solution (placed in the corresjwnding vessel) for a few hours, and then rejieatedly cleaned with water. They are now placed in aqueous platinum tetrachloride (the usual strength jiurchasable), to which has been added lead acetate to the extent of 0025 i^er cent, and a strong current passed through. Four Daniell cells will serve the purpose. The current flows for five minutes, and is then interrupted, and the connections altered so as to reverse the current, which Hows for a further five minutes. This is re}x>ated till both electrodes are found thickly coated with i)latinuni black. The electrodes are now left in distilled water for some hours before use, in order to soak out impurities entangled in the platinum black. The electrodes are to be kept in pure water, and must never come in contact with the skin or be allowed to dry up. In the latter case they would have to be cleaned and reblacked. To remove the excess of water before dipping the electrodes into the fluid to be examined, all that is necessary is to hold a piece of clean filter-pa{)er against thorn, when they become almost dry. They are then rinsed in some of the fluid to be examined, and subsequently placed in the vessel containing this fluid. It will be fovmd essential for rapid work to have several vessels, which should be marked in successive numbers with commercial hydrofluoric acid.t As regards the size of the electrodes it is only necessary to say that the larger the electrode the more accurate the results, because otherwise ]X)larisation occurs, esjjecially with a low resistance. The result is shown by not being able to find a sjxjt on the bridge where the sound is entirely lost in the telephone. The smaller the electrode the more careful must be the i)latinising, • It should be mentioned that in case of breakage it is best to send the electrodes and vessel straight back to the makers, since there is so much ditliculty in obtainmg hard gla.ss and fusible glass of exactly the same meltmg-point as each other and as the platinum. It is, however, wise to have two sets of electrodes in case of accident. t It is also convenient to etch the " capacity " of the vessel (see below) on the glass. 8 114 STUDIES IN rUNCTURE-FLUIDS The UFC of fairly large electrodes is therefore the most simple and the most nccvnite. The vessel is maintained at a definite temiwrature correct to TOO degree by means of a thermostat. This thermostat consists of an enamelled vessel of suitable size covered with felt and raised on a stand sufficient to allow a small gas-jet to stand beneath. It is tilled with water to within an inch or two of the brim. Into the water dii>s a si^ecially accurate ther- mometer registering to bo- C ., and gradua t ed into tenths of a degree. A lens will allow hundredths to be read off. A toluol regulator also dips into the water. This form of regulator is the most sensitive and satisfactory in all ways. A mechanical stirring arrangement is necessary to keep all parts of the water at the same tem^ierature. The ^Iethou of CARRViNe, out the Determinations.— In the first place it is necessary to determine the resistance- capacity (" C ") of the vessel into which the fluid is placed. By this is meant the resistance presented when a conductor of conductivity i is placed in the vessel. The value will vary with the dimensions of the vessel used. The standard solution of known conductivity is decinormal KCl, and the vessel receives enough to well cover the electrodes. The vessel is placed in the thermostat at, say, i8' C, and the connections are made as shown in the figure, the induction-coil started, and a resistance, say loo, is interposed by means of the box. The movable }>oint is moved alwut till there is silence in the telephone. A few controls may be made, and then a higher resistance is interjiosed, and another reading taken. Again, a third resistance is used, so as to bring the reading on the scale as nearly 500 mm. as ixjssible, since most accurate results are obtained near the centre of the bridge. The capacity is equal to the value for the known conductivity multiplied by the mean resistance found necessary in the box. The mean resistance is obtained thus : Suppose at 100 ohms the reading is 49° and at 120 ,, „ '♦SS and at 90 ,, ,. 5*3 Then the resistance of the fluid will be : as .V : 100 : : 498 : "coo - 49^* = 99 20 as .V : 120 : : 453 : '«» - 453 = 99-384 as .r : 90 : : 523 : 1000 - 523 = 98 636 Mean ... 990S6 Capacity = conductivity of n/io KCl at 18= (01119) + 99 08 = >io I'HYSK O-CllKMICAL l.XAMIN ATION I I When this has Ix'en once tleterminci' for a particular vessel the value shoul'' '' noteil, and it is convenient to make a table showing the < tion that will have to be made tor rvery resistance that .ill be iound to be jwssessed by any fluid that will be subsequently examined. This will be found to save a great deal of Calculation. The procedure with the fluid to be tested is exactly the same. Th" fluid to be tested replaces the decinormal KCl in the vessel after the latter has been cleaned in the manner already described. The little piece of aj)paratus is placed in the thermostat at the same tcmjxjrature. ami then the estimations carried out exactly as before, a mean reading Ix-ing taken. We have now the following relations : The resistance to be estimated : resistance in box : : (i : h, where a is the length from zero on scale to movable jwint wfien the telephone is silent, and h is the distance from the latter to the end of the metre. ICXJO A ready reckoner has been provided by Obach for reading off the values of all these fractions without labour (see Appendix). Now, the conductivity of the fluiil is found when the resist- ance-capacity of the vessel is di-,ided by the resistance of the fluid. This is specific conductivity, or the conductivity possessed by a volume of fluid i square cm. in area, and i cm. deep. This is a convenient mode of expressing the conductivity, and will be found to te adopted in most of the pa})ers published on the subject abroad. The letter k is used to express it in brief. Another mode of expressing the conductivity is in terms of the number of gram-equivalents of electrolyte in a vessel whose electrodes are i cm. apart, no matter how large the electrodes. This is the equivalent conductivity, represented by the symbol A. The molecular conductivity is the value when the result is expressed in terms of numl)er of gram molecules of electrolyte in the vessel. It is the same as the fornici with monovaleiit electrolytes. Ii6 STUDII'.S I.N prNcruKK-FLUins i i Tlif I'ciuivali'iit a)mlu(tivity is (•iilculatc'd by dividing the specific (.oiiductivity by tlic iuuiiIht of giam-e(iuivalcnts ix;r cubic ctntiiiKtio. TIic (>l)jcction to this moile of expression is tliat in the fluids undtr consideration we do not know the grani-i(iuivak'nt per ctiitiniftic, and it is not wise to express it in terms of NaCl for reasons already fully dealt with. Resii.ts of 1'^x.\mis.\ti()N of th^ Fi! following,' table will show the application of the method (from Hamburger's pa}ier, Biochem. Zcit. I.). Kluids. Volume of Uepoails of Ktd Cella after CentrifUKaliiing. J hoi .-. !}hou'. .'jhour. |)hour. Uhour. ijoiiD. 1 lomin. 58 5" 55 50 5« 54 59 54 5S 49 55 5' 56 50 54 49 50 46 5« 4b 48 47 46 46 46 47 47 46 46 4b 50 49 49 49 49 52 50 49 49 49 48 47 47 47 47 48 47 47 47 47 47 4t. 45 45 45 47 46 45 45 45 44 43 43 43 43 44 43 43 43 43 Lymph 0-9% Natl '. o-9S%NaCi; I % NaCl : l05%NaCl! The objections mentioned also apply to the method devised by Sir A. E. Wright, although in this case the apparatus has the advantage of extreme simplicity. For comparing the osmotic concentration of blood-serum and urine of a patient it is very useful, l^ecaase so small a quantity of fluid is needed. In this method the familiar pij)ettes are employed, and a mark Ix-ing made on the stem of the capillary, different dilutions of blood, with a standard solution of NaCl (" N "), until the particular dilution is found that just causes haemolysis. Supiwse two volumes of a N'/ 35 NaCl solution cause haemolysis of one volume of blood, i.e. two volumes of a oi67-ix'r-cent. NaCl cause hjemolysis of one volume of blood. The urine is then diluted in a similar manner, using distilled water in this case until it is found that two volumes of the diluted urine when mi.xed .th one volume of blood just cause haemolysis. Supix)se the dilution of urine is twelve-fold. Then the twelve-fold dilution of urine = N, 35 saline = 0167 j^r cent. NaCl. The urine is therefore equivalent to 2004 \yex cent. NaCl. Finally, the serum to be tested is diluted in the same way, till a dilution is found which just causes haemolysis of one volume of blood. The equivalent of the serum in terms of NaCl is again calculated, and may be compared with that of the urine. The clinical utility of the metho 1 it is not possible to over- estimate, but as an exact means of determir-ng osmotic pressure it is unfortunately of no avail, for the same reasons that the I20 SirhlHS IN I'LNCTUUE-FI.UIDS r hitmolytic inctliod ot HamhinK'^'i' l^i'l-^ <>" '^'i' <>thiT h;iiul, the method may 1k' used in place ot HamhuiKer's to amplify the values ascertained by cryoscopy. ami throw li|,'ht on the abun- dance or otherwise of substances in a tKud whit h are iH^rmeable to red (ells. I.inihtck's mdhod consists in placing i cc. of increasing (by o o j \X'\ cent.) (oncentrationsof NaCl intoeach of sixteen small tubes and adding a trace of blixnl to each. After six hours it is noted in which tube haniolysis has occurred. The method takes into account the resistance of the red cells— quite another subject. The (inferential tcnsimticr of Friedenthal has the ailvantage of <'nabling the variations in osmotic jMessure to he uuitchcd. I)ut neci'ssitates the studv of the body-fluid nithotit its ^ascs. since these will W removed by the mercury pumi) In-longing to the apparatus. B. The Critical Solution-point.— Quite recently (February 1908) W. K. (ielston Atkins published a new })hysico-cheniical method of examination of urine, which he advocated in place of cryoscoiiy for the diagnosis of the functional efficiency of the kidney. Without discussing the value which is to lie attached to this mode of diagnosis, one may refer to the method as one likely to Ix' of interest in the study of those puncture-fluids which do not contain much albumen. The method dei)ends on the fact that if phenol and water be shaken together at room-temi)eraturc, they will not completely mix, whereas on raising the temiK-rature a point will i>e found at which the two fluids just become completely misciblc. This temi)erature is the critical solution-temperature, and is a constant for the particular mixture. Any deviation from the critical temiierature causes an opalescence to api)ear (blue by reflection and brown by transmission). A series of mixtures of phenol and water present a series of increasing critical solution-tempera- tures, a maximum temix-raturc (the critical solution-iwint) being reached on the curve so obtained. In some cases, however, it is the opalescence which is the guide to the critical solution-point, and not the maximum temi^K'rature on the curve. The fact that the addition of a third substance to the mixture raises the criticalsolution ti'm]ierature to a degree depending on the concen- tration of the added substance has led to the suggestion of the method for the pur|X)ses indicated above. riSVSKO-CIIEMKAL EXAMINATION 1^1 The pioa-dun- is as follows : some crystallmi- j^honol (in p. 40^ C.) is placfd ill a dry test-tuln- an ^ 4 IN rUN(Tl KK-H.UIDS lis !! i ( . The Concentration of Hydrogen Ions. It is now \ve only 5 or b X ID ** (i.e. 000000005 gm. ix;r litre), which is at first sight so small an entity as to be negligible. The quantity is, nevertheless, of very great importance, as can be readily shown from a reference to the ionic concentra- tion of ji'(j/t'r itself. In the case of water there is very little dissociation present, and there is a definite relation between the amount of H* and OH", which is expressed by: C„+ X Co,,- = 0-64 X 10-" IIIYSKOt MKMICAL tNAMINATlON 12? whoro of)4x 10 " constitutes the " dissoriation-constant "' W 0aslric Jfj^c€ ^ TropttoUn 000 I Tear» , ., ■ , ^---4^Ptritoneal fluid 8.. "" iP^nol Phthqlein. ^'\ Pancreatic '^ Juice i OD Fia. ;.— Diagram to illustrate the mcaninn of the term 'acidity." watpr at the temUratnre i8' C In a neutral solution C„ = C,,,,, or each has the value 8 x lo ^ while m acid solution C„ is greater than C.,„ ; in alkaline sohition less, the equation holding 124 STUDIES IN ILNCTfKE-FLUIDS goo 1 in all casos. The more the C,; pre[K)nderates the less will be the C,,,,. It ioUows, then, that, f,'iven either C,, or C.„,, one can calculate the other. The diagram (F'ig. 7) has been devised to illustrate these consitlerations gra])hically. The curve represents the varying concentration of H ion> as one passes from a highly acid solution at A to one which is alkaline at B. In this curve the ordinates correspond to fractions of normal solution of HCl, anil it will be ol)served that as one passes from tlie neutral point in the direc- tion of A, the concentration of ions in terms of 10 '^ is constantly diminishing, though the curve does not rise nuich from the hori- zontal. Tl'.at is to say. the distance travelled longituflinally becomes greater and greater with each increment in the strength of acid. On the other hand, near the neutral point a slight journey to the left means little change in concentration of ions, but is commensurate with a considerable loss of acidity, because the curve rapidly turns down to the zero-point. At this point When the curve has passed the neutral point and comes to represent an alkaline solution, the reverse takes place, and a small distance to the right means a small change in con- centration of H ions, but a considerable degree of increase of alkalinity, and as one passes towards B one traverses a con- siderable distance before one reaches any increase in the strength of alkali. Put in other words again, the stronger the acid is, the more rapidly does the concentration increase with each increment of strength : the stronger the alkali is, the more effect on concen- tration of OH does a slight increase in strength of alkali produce. The higher the curve rises above the line, the more acid is the fluid, while the lower it falls below the line the more alkalinity does it represent. The reaction of the fluids of the body is all focussed round the " neutral {xjint," with the sole exception of the gastric juice,* which apix\irs well on the left end of the curve, and it is just about the neutral point that the fluids come to be almost com- pletely dissociated. A solution of HCl which is so weak as to I'aucreatic juice will be well on tlic riijht cid ot thf curve. PHYSICO-CHEMICAL EXAMINATION 125 be neirly neutral may be looked uiwn as completely dissociated, so that its ionic concentration is exactly the same as the gram- equivalent of H^. The ixjsition of the various fluids which api^ear in the table on page 133 is indicaten un the curve as far as the size of the drawing jwrmits. and the degree of acidity which is necessary to jiroduce any effect on the common " indicators " has been marked on the curve in order to bring these con- ceptions more into line with the old conception of acidity or alkalinity. We now come to the curve indicated by dotted lines. In this case, jwsition below the horizontal line means "lative diminution of C„„, and when the curve rises above the line it indicates absolute alkalinity of the fluid. It will be at once evident that the further we go towards B, the greater the C,,„ and the less the C,„ since the Co,, curve is constantly rising and the other falling. Just as a slight increase in acidity makes the curve travel a long way horizontally towards A (corresjx)nding great increase in concentration of H ions), so a slight increase in alkalinity is accompanied by a very great decrease in concentration of OH ions, since the curve elongates in exactly the same way in the other direction. When we come to the neutral point the OH curve actually passes above the line, because the solution is definitely alkaline, and with the increase in alkalinity the C,„, curve constantly rises higher and higher towards infinity. The only substance which coincides with the neutral point IS water, and it is at this point that the two curves cross, showing that C„ = C„„. The symmetrical character of the curves shows that the product C„ x C.,„ remains constant, and the dotted lines drawn between the two curves at the various points representing the special body-fluids demonstrate their reaction. Thus the upper limit of acidity of normal urine is indicated by a line which passes from the C„ line to the C,,H line, the point on the former lying as much above " zero " as does the corresponding point on the C ,« curve lie below it. The varying degrees of H+ ion concentration which are necessary before given " indicators " will react, are shown in the following scheme, quoted by Hober from the work of Saleesky and Fels. 126 STUDIES IN rUNCTUKE-KLUIDS I U Indicator. C'i>li>ur-chanKe. Conccntratiiin of H i63 PhenolphlhaUin Ri.l 0174 Curcumin \V ... Red 024 Lacmoid aiue to Rtd 11 /.-nitrophenol ... Yellow 18 f Yellow (Red 590 Methyl Orange 50000 Congo Red Blue I73S0 Methvl Violet ... Violet 4l6<^o This table shows how low a concentration suffices to produce a blue coloration with lacmoid, whereas methyl orange is turned red with a concentration of 3 x 10 '. There are various means by which the numl^er of hydrogen ions i)resent in a given fluid may be ascertained. The following are the most important : Determination of C„ and C,,„ by the Use of an In- dicator Series. — The observation which has l)een made that different indicators vary in their sensitiveness to acids and alkalis, so that the colour change is juoduced only by a definite concen- tration of H, has led to the idea that a scale of indicators such as is given in the table will enable the concentration of H ions in any particular fluid to be estimated. For instance, blood-serum will not redden with phenolphthalein, so that it must contain at least 01 to 03 x 10 'H^, a value which agrees with that found electrometrically. In the case of fluids which are so highly coloured that one cannot use an indicator, it is jwssible to make one's observations by noting the disappearance of absorption bands in the spectrum (Pels). In the method of Sir A. E. Wright the fluid is diluted so many times with a standard solution of acid, until the mixture ceases to redden litmus paper. The Inversion Method. — This is only available for the study of gastric juice. It depends on the inversion of cane-sugar by HCl. The H*^ acts as a catalyst and undergoes no change in the process. The velocity of inversion is proportional to the concentration of the free H ions, and the reaction is a mono- molecular one. The jxjlarimeter is used, and the degree of rotation observed liefoio the action of acid, and after. The ratio of the velocity of the fluid tested to that of a standard solution of Hri is made nut in this way. This method has already been referred to in Section I., Sub-section " Ferments." Ill PHYSICO-CHEMICAL EXAMINATION 127 The Methyl- \cetate Method requires no complicated ap- paratus, and the calculations are much simpler. Here also there is a catalytic process, and is only applicable to such a strongly acid fluidas gastric juice. A given quantity of fluid is incubated with a given quantity of methyl acetate, a similar quantity of n 20HCI being treated in the same way as a control. After four hours each is titrated. The presence of neutral salts interferes with this reaction (chlorides and nitrates accelerate, sulphates depress the velocity of reaction). The digestion of egg-white dej^nds for its velocity on the concentration of the H ions, so that one could estimate this by noting the degree of digestion (weight before and after). Here again free acid is essential to the process. The Dil.^tometer Method.— This method is unfortunately not available for puncture-fluids because of the small number of OH- ions present, and is uncertain in the presence of neutral salts, while the presence of ammonia renders the method useless because it enters into combination with the acetone, and thus inhibits the velocity of reaction. The method dei>ends on the conversion of diacetone alcohol CHo . CO . CH, . C(CH3) . OH mto acetone, a change which is associated with an increase in the volume of the fluid. The Diazoacetic-ether Method.— Bredig has pointed out that this method forms a very convenient one for ascertaining the concentration of the H ions. WTien in contact with acid the following reaction occurs with great rapidity, and nitrogen is liberated (read off) : N, : HC . COAH. + H,p = OH . H.,C . CO,C.,H, + N, SO that the progress of the reaction is very readily watched. Since the velocity-constant of the reaction is proportional to the C„ it becomes a very useful method, and is quite sensitive even at the temi:.erature of the room. This reaction is also monomolecular. Bredig states that it will measure C,, even in ji/nn to irnnsis " dilution with exactitude, that is jaUamxi S™- hydrogen per 1,000 cc. so that it is applicable to puncture-fluids. It is evident that these methods are either inapplicable to puncture-fluids, or that they are liable to be rendered inaccurate by the presence of salt, which certainly occurs-^ometimes in considerable amount— in these very fluids. 128 STUDIES IN rUNCTURE-FLUIDS (i i ill The only method which does not present these difficulties is that in which " gas chains " are employed, and unfortunately labours under the insuperable objection that it is too complicated and lengthy for general use. However, it is worthy of a brief consideration, as some imjwrtant investigations by C. Fo^ have been made on various physiological fluids which have gone far to show that ionically the fluids of the body are universally j)ractically neutral. Thf. C()nckntk.\tion-chaix Method.— Suppose that in each of two small vessels containing zinc sulphate solutions (each of different strength) there is placed a zinc electrode. In each case there will be a tendency for zinc ions to pass into the solution. This hapix'ns with a certain force depending on the concentration of the zinc sulphate solution. If the osmotic pressure of the zinc ions in the solution be less than the force with which ions tend to leave the electrodes, ions will pass from electrode to solution, while, if the pressure in the solutions is greater, the reverse will occur, and if the two forces are equal, nothing will occur. In the first case there are positively charged ions passing into solution, leaving a negative charge in the electrode ; in the second case the reverse holds good. Therefore, if the two electrodes be joined by a wire, and the two vessels be joined by a tube of fluid, a current will pass from the strong solution of zinc sulphate to the weak, until equilibrium is reached. The electro- motive power of this current will depend on the relative strengths of the solutions. In the same way one can use a gas chain where the fluid is, say, strong hydrochloric acid solution, and a " gas-electrode " * (e.g. hydrogen), while the other vessel contains a weak acid and another hydrogen electrode. A current will again pass, whose force dejiends on the difference in the strengths of the two fluids. The ions concerned in this form of apparatus are hydrogen ions instead of zinc ions, so that by this method we can calculate the concentration of the hydrogen ions. Suppose that in one of the vessels there is a slightly acid fluid (body-fluid), and in the other known strength of HCl, we can calculate the acidity of the fluid in terms of hydrogen ions. If we deal with an alkaline fluid • A gas electrode consists of a platinum electrode coated with platinum black, and saturated with a gas, with which the electrode is also surrounded. I'HYSICO-CHEMICAL EXAMINATION 129 such as blood, and use oxygen electrodes,* and a known strength of sodium hydrate, we can calculate the concentration of the hydroxyl ions, and so ascertain the degree of alkalinity. The use of oxygen electrodes is permissible for acid fluids also when we wish to determine CO,, in an acid fluid. * If one uses oxygen electrodes, and soda solution in one vessel, with an acid (luid in the other, we shall also leaiii the concentration of iJic hvdroxyl ions, 9 ! t 130 STUDIES IN rUNCTURE-FLUIDS Fig 8 will explain the way in which the apparatus is set up The Ras chain is seen to he made up of two six^cially shaped ves.eK each furnished with a platinum (or gold-Foa *) electrode, coated with platinum (or palladium) black. The electrode .s either fused into the glass or fits in with a hermetic glass joint The vessels are joined together by dipping into an indifferent fluid or into a fluid of the same strength as the standard (o-oi n HCl). The osed to have been passed in till the electrodes are covered to the extent shown in the figure, and then con- nections are made with an electrometer, a Wheatstone s bridge, and a battery. The electromotive force can then be determined by PoizKendorff's method, and compared with that of a standard cell, B C 1-^ the kev lor making and breaking the circuit at the time of the observat'ion. and A is the movable l^oint on the bndge.f There are many forms of gas chain J available, the modihca- tions being mainlv with the object of enabling the observations to be made at the "temperature of the body. This aim introduces many complications which come to render the method useless for the clinical pathologist. It is therefore wiser to adhere to the room temperature, and ignore the change which temperature will necessarily introduce. At the same time, it must not be for- gotten that because two given fluids show the same C „ at 18-C.. they will not necessarily show the same at 37^ I" other words, it would not be safe to assume that because any ascitic fluid has » Foa .howed that the koW covered uitli palladium black was the ■,„oresenMt>ve; but this type of electrode compared unfavourably with a mm electrodes covered with platmum black, or w.th .nd.ated mdm.n dec rries-the latter bemg found to acquire a constant potenfa much more rapullv than the second, ami the second than the pallad.ated paha- um Butthe latter adsorbs much more H than does plat.nated platinum X -^matter of fact, .f we compare the C„ values as made out by the different ide t^X in the hands of various observers, there is found to be very httle eal dUference. Farkas workn.g w.th platinated platnu.m electrodes found blood to have the san.e reaction as Fnmkel d.d w,th palladmm electrodes. (". I in each case being I x lo"'. t Hamburger recommends the use of two similar re^stance boxes in place of a bridge, as the enormously increased length of the w.re so ob- a.S ensures n.ore delicate observation. The use of these ^^^ J^^ '"» details of the methoens to have, that therefore in the body, and during life, they are identical in reaction. Relative accuracy, as opjx)sed to absolute accuracy, is one to which the reader needs to become accustomed here, as everj'where throughout this work. It is enough to say that if the distinction between the value at 18° and its significance at the temjxrature of the body be remembered, the results become at once legitimate. The most imjwrtant objection to making the observations at 37^ C, however, is that the various factors which enter into the subse- quent calculation of the value C,, depend on measurements which have only been made out at 18^ and 25' C, so that correction formulae (always to be avoided) become essential, and it is far from certain how far these formulae tyay be trusted. The procedure in an actual exjieriment may be outlined as follows : The apparatus being connected up as shown in the figure, and the various parts being prepared for use,* the commu- tator is arranged so that the current will flow through the normal element. The movable jwint A is adjusted until a momentary closure of the circuit at C produces no movement of the column of mercury in the electrometer. The reading on the bridge is then noted, and the ratio DE _ F. M.F. o f batte n-. i>A ~E.M.K. ol normal elemtnt. 1000 X E.M.F. ol" normal clement. Since DE is itxxj min., E.M.F. of battery = DA The commutator is now altered, so that the gas chain is in circuit and the movable jxiint is again adjusted, so that momentary closure of the circuit at C produces no movement of the column of mercury in the electrometer. The ratio again holds good, and DE ^ E.M.F. of battery DA' E.M.K. of gas chain. or, substituting the symbol tt for the E.M.F. of the gas chain, and inserting 1,000 for DE as before, icxx) K E.M.F. of normal element., ^ - — i,x. or we may still more simplify our procedure by inserting value for the E.M.F. of the battery, when DA' the DA X E.M.F. of normal element. which is the same thing as saying thai the E.M.F. of gas * It is this preliminary preparation that makes the method so tedious. «32 STUDIES IN rUNCTUKi:-KLl.-IDS resistance of gas chain : rc- chain: E.M.F. of normal clement sistance of standard cell. The coupling of the gas chain is indicated thus : !I 1 Fluid to be tested " NaCI* 1 "NaCl + ooinHCl S 8 H The symbols beneath indicate the various contact potentials which have to l)e added up (algebraically) in computing the total K.M.F. of the chain. However, it is not necessary to enter into the details of the calculation and discuss the mode of introducing n, tt", tt", into the formula : 5r = o-o575lop. ^-, which will enable the concentration of the H ions in the given fluid to be calculated, provided the observation is made at ibout 17= C t c. is the concentration of the H ions m the solution of ooi hHCI, while C„ represents the desired concentration of the fluid. Or we may state that log. C„ = log.c,-^,i^-= -"--oItS- If desired, the result obtained in this way may be controlled by ascertaining C„„ in the same way, using o.xygen electrodes in an atmosphere of oxygen, and ooi nNaOH in place of acid.., The formula already given, Cii = Con = 0-8 X 10 , will enable one to see if the result for C„ has been arrived at correctly. , ... \ reference to the two formula;-(a) one for estimating ir ; (b) for estimating velocity of ferment-action-is of interest, since the graphic representation of change of reaction (acidity) m Fig. 7 shows a remarkable resemblance to that representing the velocity . The exac. .strength of this can be '^"^"^ed by ascertaining the strength of NaCl. Nvhich has the same comluctivity as the flu.c to be tested, but this procedure greatly lengthens the t»me occupied m the determination "' \" For other temperatures the formula from which the above is derived would need alteration. ,,, .,^;.i:^,. - The electrodes must not he the same as those used for the acuut> determination, since the whole of the O or the H cannot again be removed, and mistakes will occur in the subsequent use of the apparatus. i'llYSKO-CMEMICAL EXAMINATION 133 of ferment-action (Fig. 3)- Comparison of the two formuhe shows that while one has several symbols, representing constants, the other has similar constants expressed in figures. The rate at which a fluid alters its acidity with alteration in concentration follows exactly the same mathematical law as does the chango of reaction produced by the agency of a ferment. This is rather a suggestive fact. The following table shows some of the results obtained by C. Foi. The first column of figures gives the E.M.F. of the gas chain in volts, the second column gives the value obtained for log C„ from this tt, and the third column gives the corresjKjnding value of the concentration of H ions in terms of 10. - The remaining colunms are added ftr comparison of the ionic acidity with that shown by titration (in terms of iwtash), since these values are more familiar. Table showing some of the results that have been obtained by study of the hydrogen concentration of fluids (Fo^). Keactior; Indi- asKiven cator by I uaeil for Titra- I Titra- tion, tion. Water Blood-scrum (dog) Endocellular Fluid Peritoneal (horse) Pericardial (horse) Cerebrospinal (dog) Amniotic Fluid ... Aqueous Humour ( horse) Vitreous Humour (horse) Bile Pancreatic Juice... Urine (human)... •«543 ■I5S3 •1428 •1362 •1328 •1299 •1251 •248 •06 •094 - 742 > 9 -74400 - 7"34 - 7«072 - 7 049 - 6 998 - 69185 9852 3-785 3681 597 7 8<3 893 1-005 I2l8 410,000 n 690,000 n 900,000 KOH n "50 1,000,000 n HCl -90515 I 008882 -35^ KOH - 5 7820 I 16 i _ „- HCl 1 - 6 3733 I 42-3 n 10 litmus dimeihjl imido- azob. •081 — 6 1472 2,500,000 L " i 1.000.000 I " 6tx),ooo " HCl HCl M : SI 134 STIDIKS IN I'UNCTL'KE-FU'IDS The most conspicuous result of this kind of study is that practically all the normal fluids are at the neutral point, except the secretions, which show striking deviations fntm the rule. I'rine, for instance, is moderately acid, gastric juice is markedly acid, and pancreatic juice is markedly alkaline. The fact that in nephritis the urine shows a very much higher degree of ionic acidity is not only of interest, hut may be of use as a means of clinical pathological research. Some theoretical considerations on the reaction of hlootl- serum which have heen entered into by HoIkt deserve notice here in relation to the reaction of puncture-fluids. The electro- lytes which occur in such fluids are either strong acids combined with strong bases, or consist of strong bases combineil with weak acids, or, htstly, of weak bases combined with strong acids. In the first cases, hydrolysis will produce an acid fluid, and in the other cases, a mi.xture of the two kinds of salt in varying projxjrtions would j)roduce a medium which was either alkaline or neutral. The proteids of the body-fluid are also amphoteric electrolytes, and act either as acids or as bases, according to circum- stances. Hober has pointed out that it is a great advantage to the organism to have these various groupings of acid and base, because it secures a }X)ssibility of addition of more acid or more alkali to the medium without any corres}X)nding risk of an actually acid or alkaline mediimi being produced. This he illustrates by the following consideration : su|)jK)se that the fluid contains Na^ CO3 and NH,C1, then, when dissociated, there will be: Na + HCO, H + . OH NH, + CI' HXO, NH.OH H' being equal to OH , Addition of a strong acid will prevent the dissociation of the Ho CO;, and liberate OH " by acting on the anunonia, so that the fluid will remain neutral. The more weak acid there is, and the more free weak base there is, the more rapidly will the addition of acid or alkali be prevented from disturbing the equilibrium. The fact that according to Frieden- thal 70 times as much soda has to be added to blood-serum as to water in order to produce the same colour-change with phenol- phthalcin, and that 327 times as much acid has to be added to produce the same colour-change with methyl orange shows how great the adaptability of the blood-serum is to the variations I'HYSUO-CHEMICAL EXAMINATION 135 in reaction which niifiht \^c pro.h.ca by disorders of metahohsni. and of course the sanu- apphes to transudates and esl>eaatlY lo exudates. , , The reflection in an exudate of the ,,hysi.-..-chemical characters of the hlood-serun, justifies a brief reference to I'taundlcr's work. He showed that in chiUhen the seiuin is practically neutral, while with a.lvancinK age the reaction becomes more and more alkaline ; on the ^ther hand, the alkalinity reaches a high degree if an infant suffer from chronic nuti itive disease. The presence of OH is necessary to many of the phenomena of vital processes ; the deficient OH content of the blood of the infant nnist there- fore have some relation to the inactivity of their ferments. Again, the fact that arterial blood is much richer in OH * throws some light on the function which the trace of free OH plays m normal vital processes The results of Bc>nedicfs study of the OH content in the blood of dialx:tes lead one to think that the coma of dinlK>tes is not due to the presence of acid in the blood so much as to variations in the equilibrium mechanism already mentioned, while, on the other hand, the possibility of acid being masked by this mechanism renders it difficult to draw conclusions. D. Viscosity.— Recent months have witnessed the introduc tionof anumber of new forms of visco,imeter, designed to i^rovidc the clinician with a tyin. of instrument which is nmch more hamly and easy of manipulation than that hitherto available (Ostwald s). Of these new instruments, it is proposed to describe that of Hess as from practical exiK^rience it has lK.>en found to fully answer a the needs of the clinician, being simple, rapidly used, and smp.ll in bulk. This instrument has lx>en employed in the examination of the cases in the Leeds General Infirmary. The viscosimetcr of Hess consists of two capillary tubes, a and c (Fig. 9), of like bore and length, coupled together at one end by a T-tube, e, to which a hard indiarubber ball, g, is attached. This ball can be used for either suction or expulsion of air ; by means of closing the hole at ^' with the finger ; fluid in a (water) can thus be drawn through at the same time as a fluid (the materml to be tested) in c. By observing the distance through which the water has passed during the time that the fluid to be tested passes from • Holder. 136 STUDIKS IN I'l'NCTURE-Kl.lIDS I om- arbitrary mark to anotluT, the rate ol flow of the latter is compared with that of thi" water. It will 1h' seen from the diagram that the ( apillary tulH\ n, leaiU into a wide tuU-, h. which is provided with a tap, /, before it joins the T- piece. In the same way the capillary tul)e, f, is continuous with a wide iuW\ d, -i\ the same diameter as /'. This tube, how- ever, has no tap. It will also 1k' noticed that there is a wide tube, h, joined on to the capillary, a. This is simjily a reservoir for the water which is used for comparison, and is undetachable. On the other hand, the tube, h, which fits on i n a corresponding situation, is readily detachable, and is used to receive the fluids to he examined. A large number of these tulws are supplied with the instrument, so that a new clean one can be used for each new case. The thermometer I'llVSlCO-t IIIMICAL KXAMINATION i}7 serves as a convetiu iit means of reatlitiR the temjHTAture exartlv at the time ot the .»l>sei vation. Mcthoii «f /Vwf(/Mrf.— The ammonia which hes in the system. «rf. is exi)elle(l by the use of the l.ulh. and air is blown thnnigh for a few monx'nts, in oidti to thy the caiuilary. The stoju-ork, /, is now oiKiif'ii" irpr' 8Tv" jrpr* V, V That is to say, the volume of fluid which passes through the tube containing the ascitic fluid is to the volume of water which passes through the tulie as the viscosity-coefficient of the water is to that of the ascitic fluid. In the instrument, it amounts solely to a determination of the volume of water which has passed * The inventor of the instrument has pubhshed an account of the theory in a journal unfortunately inai-ct^iliie to nic. tuil it is not tjiificult to work out the mathematics. PHYSICO-CHEMICAL EXAMINATION 139 through while the fluid to be examined passes from scale mark o to I. Taking water as i, we now know the fluid visco.sity of the fluid as compared with the water. If we refer to the theory of the ordinary viscosimeters which are available, we find that the vL:osity is measured by noting the time which is occupied by the passage of a given quantity of fluid from one mark to the other. In this case the time is a vari- able and the volume a constant. However, it will lie admitted that it is much easier to measure off a volume than it is to measure off so many seconds or fractions of a second— which necessitates the possession of a stop-watch. In this case the formula becomes : 1 = "»! St Mi where r, is the coefficient of friction of the fluid to be tested, s is its specific gravity, rn is the coefficient of friction of the fluid with which it is to be compared, and s, is its specific gravity ; t and t, are the flowing times of the two fluids in seconds. Now, as it stands, this formula demands the determination of the sjiecific gravity of both the fluid to be tested and a standard flu and it requires a knowledge of the coefficient of viscosity of the standard and a correct observation of the times of flow. Freshly distilled aniline has been found to be practically equal in viscosity to that of blood, so that if aniline be used, t . ■ t 1} = I/, -, or Ignoring ij,, t) = - which means that one is always comparing the fluid with anilinp — a very artificial standard. There is only one other {wint to consider, and that is the influence of temperature on viscosity. Water at 0° C. has a coefficient of viscosity of 00178 eg s. units. Obviously one can avoid the necessity for correction for tem- perature by always studying the viscosity at the same tempera- ture, as could be arranged by the use of a water or air bath. But it is more convenient to dispense with this and correct for results afterwards, since the temperature of the room may be maintained correct within small fluctuations. I40 STUDIES IN rUNCTURE-FLUIUS \i On the other hand, water will not be influenced by tempera- ture to the same extent as would the viscosity of a fluid such as a body fluid. Hess, however, made out from a number of cxiieriments that there is not a greater variation than 4 per cent, within 5= above or below 17=" C. Every degree difference from jy^ requires a correction of 08 per cent. From a large number of observations published by Hess, the blood from different cases showed the following variations in viscosity : Normal Blood 5° 'o S'4 Chlorosis ... ... ... .•• 4'4 Carcinoma Uteii 32 Tubercle 4 9i-5"'5-5 4 Pleiirisy ... ... ... •■• S* Peritonitis Traumat 5^ Tubercular Meningitis with Coma 765 The following table (XIV.) shows the results of examination of various puncture-fluids by this method. It has been found that the higher the albumen-content, the higher the value for the \ iscosity. so that in exudates, as a rule, the viscosity is greater than in transudates. As might be expected, ovarian cyst fluids and purulent fluids have a greater viscosity. According to Rossi, the viscosity of blood-serum runs parallel with the conductivity. Ascoli came to think that the variations of conductivity met with in sera of different degrees of alkalinity might be due to variations in the viscosity of the sera, although an assumption of formation of albumen-salt compounds would afford a more satisfactory explanation. Herz found that the viscosity exerts an influence on the velocity of enzymatic reactions, and that the velocity of reaction is an exponential function of the viscosity. An application of the use of viscosity for estimating the amount of pepsin has been referred to in the preceding section. E. Refractometry.— The study of the refractometric charac- ters of various fluids has led to results which are not only inte- resting but of value in the differential diagnosis of the nature of fluids. The appliances necessary for the work are, however, too expensive to enable the method to be widely used. Since the subject is mainly of interest in the differential diagnosis of exudates from transudates, the description of the results obtained has been placed in Section IV. I'lIYSICO-CIIEMICAL EXAMINATION 141 a 5 > * - H z W p < o O *■♦ M M « 'u O 3 o a) n •5 E E u 11 u p > s E E HUM : e : >.» 3 V en o 11 ; 5-1; ? > 01 V u ._ ^ ^ _><: •5 3 3 2 U w« — ^— SB 3 s ;5 ;5 ^ g •2-s-o '■H 3,2 •5 « « ?= '' - •- "3 ^ 5 21- > « u^ I I 8 M •58 ^ T u > !J = 5-° 08.2 S siiv •a e c . ■ ■*• QJS ."" % 001 1 in QO s 3> r^ s 00 QO ? 3 o m Z, •5 > .b . ^ U : ■-. o ■& .2 •5 . I ii ^ 8 u s c Si £ e : < c I - E :2v, :s 10 146 STUDIES IN PUNCTURE-FLUIDS Prolfids i 137 /'= (mostly iiiKlco-albumcn; aUo albutnosc, peptone arid fibrin ferment *) (also pyosin and pyofsenin f) (xanthin bodies; leucin J) Nuckin 34'i6 Insoluble substance 2056 Lecitliin 7'5 Kats and soaps ... 75 Cholcstcrin ... 7'4 Cerrbrin ... ... S'^ Kxtractives ... 44 <;iycogcn in living pus-cells Salts Miesdicr gives : Potassium phosphate Sodium phospliatc Earthy phosphates and iron phosphate NaCl Organic phosphates 61 ,< 42 1-4 • 314 to 203 , parts As bearing on the differential test fully described in Section IV., attention may l>e directed to the salts of pus-cells.§ The ratio of chlorides to achlorides is ^^=-" ^ ■ ^^' ^^^ ^^^ P°^^" slum salts forms by far the larger projwrtion of the achlorides. The exact composition varies, however. In tuberculous abscesses there will be found leucin, tyrosin, free fatty acids, volatile fatty acids (formic, butyric, and valeri- anic), and urea. The serum of pus is similar to that of the blood, though its relative bulk, as compared with the volume occupied by the pus-cells, is small ; the result is that the composition of the latter will have much influence on the total composition of the pus. Hoppe-Seyler gives : Water Solids Organic solids ... Proteids Lecithin Other organic matters... Inorganic salts ... NaCl 9«37 -9056 863 - 94-35 78-57 - 86-58 62-23— 7721 ^NaCl 15 — 056 14-84— 8-8i 2-51— 2-38 5-22— 5"39 Na.SO, NaiHPO, Na.;cO, Ca,(P6,)., Mg,(PO,), 5-22 0-40 0-98 0-49 049 6-19 The amount of lecithin may be noted, in reference to its appearing in certain inflammatory effusions. ♦ Halliburton. •f Kossel and Frcytag. X Friinkel. § I am indebted to Prof. B. Moore for directing my attention to the composition of the inflammatory cells, which will explain the electrolytic character of exudates as compared with transudates (Section IV.). CIIARACTKRS I'OSSESSED I!Y VARIOUS PUNCTURE-FLUIDS I47 chlorides _ ^ . ... The salt ratios may again be referred to : ^^^^^ ^^^^ — i . i 4. carbonates _ chlorides ^ ^ chlorides ~ ^ " ^° 7 achlorides - • • The pigments in pus are separable by ether and are due to micro-organisms. Crystalline pigments are met with, and also a yellow pigment (pyoxanthin).* The osmotic concentration is usually high, being consider- ably above that of the blood. In a case of pyopneumothorax, for instance, I found a freczing-iwint depression of 0867 (osmotic concentration o^bS), and in the accompanying list are observa- tions by Zangemeister, who noticed that bacterial growth in- creases the osmotic concentratioii.t This would satisfactorily explain the high concentration of pus. Sterile pus will be isotonic with the blood, v. Rzentkowski, however, attributed the high freezing- jxiint depression to the presence of the pus- cells. Osmotic Concentration of Pus Nature of Caie. KreeziofC-point Depretiion. ()$molie ConctnlrmtioH.* I'reMure in Atmoapbercr.* Pneumonic Empyema •684^ •369 8-2 Tubercular , ■828 ■447 99 „ (three other cases) •840 ■454 lo-i Psoas Abscess •539 290 •580 ■^'1 t' Puerperal Peritonitis ■642 •346 77 Mastitis •586 ■316 70 „ (reccnfabscess) •709 ■383 ^ 8*5 Empyema (Herzfeld) •47 and -55 •254 and 297 56 and 6 6 • These valuea are my own. A cai,e of empyema + examined gave the following results : specific gravity 1025, albumen 3 jwr cent., chlorides 14 per cent., concentration of electrolytes 092, of achlorides 068. Globulin was abundant. Urea and glucosamin were not found. The ferment-content is recorded in another place. In another case of empyema which was e.\amined.§ the fluid contained 2*45 per cent, chlorides, 8 per cent, proteid, and had a • Fordos and Liicke. t Herzfeld denies that the molecular concentration is usually increased by Bacilli coli. staph vlococci. or Racilli tuberculosis. I Register No. 7334 (Leeds General Iiitirmary). § Reg. No. 8349. 'l If '1 1 !i i i ! I4K STlItllS IN ILNCTUKK-FLLIDS s|HTific Kiavity c»f loj-'. I'm-a wiis present in large amount, but purins could not Ik- found. l>rot;ili)umosc and hetero-albumose were present. Tlu' tryptopluuv and Klurosamin radicles were not identified. PLEURAL AND PERITONEAL FLUIDS The similarity in the physical and chemical characters of these two fluids will make it more convenient to consider them together. Those pro|xrties, which enable the pleural and peri- toneal exudates to be distinguished from the transudates, will be found dealt with fully in Section IV'. These fluids are generally alkaline and of straw-yellow colour. The brown-stained fluids usually owe this character to the presence of blood, but in some cases h.-emoglobin-colouring is met with (familiar in tuberculous and carcinomatous effusions). An intensely blood-staineil fluid was recently met with in a purely cardiac case.* Pleural fluids from cases of new-growths of the pleura are well known to Iw frequently ha;morrhagic, and they may become darker in colour the more frequently they are tapjied— up to a certain point, after which they become paler t (see also the cytological characters of these fluids. Section V.). Probably this phenomenon is due to the relief of tension on the delicate tumour vessels. Sometimes there is a }K'culiar opalescence, or even actual milkiness, a feature which calls for special consideration. The odour and colour of a fluid give some indication as to the bacterial infection in a given case of [x-ritonitis. We may compare the comjiosition of pleural with that of peritoneal fluids (transudates). Pleural (C. Schmidt). t'crituneal (Hoppe-Seyler). Water Solids ^ ... fProteids Organic solids ^g^tractiv, Inorganic solids Sugar Uric acid 966-24 1 952-9^ 3376 ■ 4638 es / 2682 349 , 428 764 7-2 005:: * present OGOI 5%* •0013 • l>ick»rdt. *fo. 1017 3- t V. Starck. -•co78%« CIIAKACTEUS POSSESSKD I!Y VARIOUS P'TNCn'UE-KI.UIDS I49 Anal>-ses which have lieen made by thf same authorities show that the different effusion* met with in .1 tjivcn case ol Bright's disease may show slight (hffereni. -, in chemical lom- |)osition. The most striking jK)int alx)ut the analyses which are obtainable is the absolutely i' itorm salt-content in each fluid (pleural. j)eritoneal, udeina). The proteids are. as one would exjx'ct, uniformly less in the dropsical fluiil, ami the water is always greater in that Huid than in the case of the |)leural anil jKTitoneal effusion. The pleural fluid usually contain- the largest percentage of proteid and the least w.ifer. The varia- tions, then, are mainly in the organic constituents. From analyses of various pli-ural tiiiuls ntonltd hy llallilmrtoii »t is sii'n that acute plt-iirisits havo a specific Rravity of more than taio. while the hytlrothorax ftuids (whether renal or tarJiac) are U.nv, 1020. The total proteid is < to 5 percent, in the pleurisies, and only 1 01 i in the transudates. The proteid-quotimt shows that Kluhulin may 1 more abundant in exudates, while it is practically always less 111 amount than albumen {proteid-ipiotient ii to i •>) in transiulates Globulins.— In three different sj)ecimens of .iscitic fluid, Freund and Joachim found the following globulins : Peritoneal Fluid. EuKlobulin. I'leurio- globulin. I'ara- glohulin. Parupseudo- Itlotiulin, Nuclro- globulin. Cirrhosis of Liver Carcinoma i. Trace Trace Merest trace + + + I Small amount Moderate i Trace amount Associated with the globulin, there may be a considerable quantity of lecithin* as we shall have to refer to tully presently when dealing with the cause of opalescence of puncture-fluids. The presence of globulins in excess in exudates will have a physio- logical value, since the association — in some unknown way — of globulin with antibodies will serve to protect or help to protect a patient from the dangers of bacterial peritonitis, for instance. Aminoacids have been described as occurring even to the extent of 0062 per cent, in cases of jx^ritoneal effusion associated with Banti's disease. Glycocoll was found amongst them. Considering how difficult it is to reach a high degree of accuracy in the estimations when jO [>er cent, or more of ieucin may escape ♦ Jolies, Joachim. See also page 159- i« ,50 STri)IK> IN lUNtTlUK-ILni'S „, „u- ,>HKe>s ot quantmuiv. .nah-sis. w. ,n.v .onclucle that I nua„..tv ..I ain.n...u..ls .nay U- .n.uh ^u-ator than .. c^..;- UunHvl tv-..M„ ..nu. .n ,K.r..on.al tUu.! meases of c,rrh...s „| thf liv.r, Mcnrdma to Hanunarsten. K,.sn>. u N.rK.K.KN. Th.s .s ,..lat,v.ly h.^h m transudates t.277). as Will as W■^n^^ al.solutfly mcirascl. 28.5 g.u AMM..MA.-M".v than ., n^K. i>.-r cent, was met with m a rise of cirrhosis of the liver. + HHSC F-JoNKS PKOTB.n.-The presence ol th.s substance has iH-en desn.lH.1 by HUmKer as occurring m the ascitic tlu.d m a case of multiple myeloma of the bones.J " SvCAH^.-DcMrosc has been .K-scribed as occurrinR in the ascitic fluid in a case of renal disease. Frnclosc § api^ars in ascitic fluid in cases of carcinoma and ,n some cases of granular kidney, if pven by the mouth. It was found m pleural fluid in cases of nuiU.ple lymphoma though Tl vulose L given by the mouth. I'lckardt states that the otal leducmg substance in ascitic fluid -V amount to o... p^^r cent The following table of analyses made by P.ckardt will Illustrate the variations in composition which may be met with . Diieaie %N. ■*> ■> *A J 1 RoUtion Riducinit Albumen. 1 t'ric AciO. ! Substance. 014 087 0C036 046 2S75 trace 022 '■375 ., 059 .V69 00048 1 o<'5 406 0003 0906 5-663 00025 0188 1175 009 0563 OC06 (lextro- ISEVO- 0084 0070 0106 dcxtro- 0029 l.vvo- , dcxtro- It 0050 0113 Cirrhrsis of Liver I Carcinoma o( Peritoneum VrubircuUr Peritonitis ... Pleurisy ... Nephritic CKdc ma Cardiac Failure (,(i:dema) * Friclnchsen, Vages, and Husches. ' ^;oS"tins substanco has been known for nearly s.xty ycars^.ts "r'Lu. .n,[ conlam, mUhcr glycocoll nor pl.o.pl.o. „,. 3. It is not soluble in (rf. per cent, alcohol. § Ncuburger and Strauss. CHARACTERS KkSSESSKD |,Y VARIOUS PUNCTURE-FLUIDS IS" Reference to the nitroRcn an.l albiunen jK-rcentaRc will show the striking (act that .( the ,x.r.toneal cav.ty contain lo htrc. „( fluid at i to 4 IH-r cent, of alUnnen. there w.U then »>e 625 o 1..50 granunes of proteicl-equ.valent to n.ore than z lH,uncl» of meat-within the jicritoneal cavity ! In serous fluuls tl»e dry matter and total nitrogen are. as one would exiK-ct. less than in the blood-serum, but the numlx^r oi extractives is still U-ss than those in the serum.* Ali-ANToin has l)een found in the transudate in a case of cirrhosis of the liver.t ^lj^^^.,ss.-"Serosamucin" occurs in certain exudates. Tht subject is dealt with in Section I. PiGMENTS.-Bile-pigment and urobilin may occur. There may be an undue quantity of uric acid associated with urobilin. A s.>«.al nutho.1 of stu.lv of l.o.ly flui.ls was .lcvis«l by I^mlolf, which is o^rreu-lt though somewhat com,,hcat..l. The .loscr.pt.on of he ItLT* .^- not seen, compUt... Th. total prot.ul h t.rst en .mate.1 bv KT'uiah^sinK, an.l hv-lrolys.s .h th.-n carncM out by means of strong hldSch^ric aci'l . After hvdrolys.s. polar.metr.c obseTvat.ons are ma.le a 1"°; the Uevo-rotat,onof peptone, wh.ch may be present m thef^nal flu°r This author d.scusses (,.) the rat.o of the insoh-ble albumens to fhe leJc' of rotafon of the hy.lrolyse.1 ftu.cl. (/.) the rat.o of the mso uble . \:lX\Z\\e^ after hy.lrolys.s to the total nitrogen of the hydrolyscd r^M tL^Uo ofth rotaton of this fluul to the total mtrogen. (d) S:„tr^.rrtherc;^c;:s'Liel Lt.mat.l ...ect,y, p.. . V^!.„ nf ffie filtrate alter .stimating these nitrogenous boihes. tLc ch e t.su It o tlu-sc laborious researches .s to show that the albumL of puncture-fluids is much more res.stant to hydrochlor.c acid ^'^Tht f^EnTanalyses are selected from Lando.fs paper : Fluid. Albumen % Dry Reiiaue Pleural Abdominal* I 5861 0947 403 217 6896 1832 509 347 Aih. Urea% NaCl % Re<>uc- ing Siib- ■tancc. inaolubl* Albumen other tC. Fat. 0844 0-55 058 0-I02 25:1 0804 0-62 002 1:3 087 ... 056 0-3 ... 084 0-57 oa4 • •• 0-62 090 ■ Opalescent. • Rzentkowski. ^ V. Jaksch. Biochcm. Zeit., vi. 61. 152 STUniKS IN PUNCTURE-FLUIDS m INORGXMC CoNSTiTrENTs.-Chlorine is retained in the peritoneal fluid, not only in nephritis, but also in other conditions, such as hepatic or cardiac l)ack-j>ressurc. While a peritoneal fluid is accumulating the salts will increase in amount : CI. N. and P, come to be retained, showing a deficient ixjwer in the organism of disassimilation. On the other hand, if the fluid is undergoing absorption these elements are excreted in considerable quantity, and there is P- and Cl-loss. If the nitrogen of the food docs not meet the demand, the albumen of the tissues comes to l>e attacked.* Gases. -Transudates contain CO, and traces of nitrogen and oxygin. Tlie tension of the CO, is greater than that of blood in transudates, esi)eciallv if pus is present (Hammarsten). Physico-chemic.m FE.XTfRES.— As compared with the blood, the physico-chemical characters are usually found similar.f though the concentration of the non-electrolytes is hardly ever the same in the two rases. Osmotic Concentration.-This has been studied by various observers, with a view to the detection of the nature of an effusion, and in the hope that prognostic indications might be obtainable. Thus it has been said that the lower the osmotic pressure as compared with the blood, the more likely is the fluid to become absorbed;* but this is disputed by Herzfeld. who holds that absorption does not depend on the osmotic pressure. From personal observations one is inclined to agree with Herzfeld. but it is diflicult to be dogmatic on such a jwint, and it is most likely that the factors influencing absorption are more comi)licated. Rzentko- ,ki thought that tuberculous pleural exudates have generally a much higher osmotic concentration than the blood, e*l)ecialiy if there be pus present. But this observation, which raised a hope that tuberculous pleurisy could be readily diagnosed from the cryoscopic examination, was soon found to be faulty. The value of the determination of the osmotic concentration of an ascitic or pleural fluid has Iwen stated to lie in the deter- if that is increasing, the fluid may be said to lie increasing in amount. mination of the ratio ^.,^^-j of the urim' * Marischlcr and Ozarkiewicz. t BckIou. J Kctlcy and Torday. CHARACTERS POSSESSED BY VARIOUS PUNCTURE-FLUIDS 153 ~ 'J0|q3« 'D |HII|^|:pf;^;f|i?PH?i ilB^f 1 •p»i» 2 -XtiAiianp -U03 o ^ ro r^ o^ t'i ^ O^ %'Mn •JO ds i§2222§ -^^^i -22222222222 2 2_2 2 = ^ M . o • ** ■X eS E E ••2 2 "c .2 ^ .^ t- u !/)H c : : o ■ • E 3 l- S'S.-s £2a: ■s'^ Ui B O "3 o c = u o .2 S *- > X .2 ?.-£ •o.i: i. o ■C IT g ' '^ fc u u V J; .2 c •ss « fi. '^ u- J9 ._ - « I g-2'^1.2.2 gt S"''- ESS § s g s >w^5;« iT-fi. .-:;£; t-:r^s,j;; 5; S- 30 00 i~» JO oe o e 00 ■ V IBjnay „ o f*^ i'^ f*! ' - inaoao t^ao 00 >0 !• r^ l89UO)U3({ 154 STUDIKS IN I'UNCTURE-t'LUIDS m The following table (XVI.) shows the observations which have been made on cases in this hospital : lAHl.K XVI OsMori. CONCFNTRATION OF Pl-FURAL ANI. PERITONEAL KlUIDS Nature of Cise. Kreeiing- point Depression. Osmotic Concen- tration. Pressure in Atmo- spheres. u: Tubirciilons I»critonitis fari-inomatous „ (stomach I Chronic Peritonitis (gastric iilccn... Carciiioiiuitmis Peritonitis lubcrcular Peritonitis Siippiiralive Tuberculous Peri- tonitis Thrombosis of Portal Vein Cardiac Kailure Multilobular Cirrhosis Cardiac Failure and Cirrhosis ol I.ivcr ... Renal Asciti;s Cirrhosis of I, iver Cirrhosis of 1-iver C hronlc nitis . Pcrito- Cariliac Failure Renal Ascites Acute Nephritis V Interstitial Nephritis •569 520 752 064 •569 ■595 •667 •576 •566 .649 574 •750* •667 ■55» •667 •544 ■586 672' •676* •75' Tuberculous Pleurisy Simple Pleurisy lubereulou^ Pleurisy -56.? 488 -549 -56^ -552 •320 Markedly hypei tonic •299 •274 ■406 ■35« ■307 .?«3 360 •310 •298 ■34' •^02 •395 •360 301 ■360 •294 310 361 364 •405 ■303 •257 297 •298 297 683 6-24 90 80 6-8 7«3 80 69 680 778 689 90 80 6-7 80 65 70 80 81 90 6-7» 586 66 6-7 66 6-6 The molecular concentration is thus seen to vary very greatlv and to be. as a rule, much more marked in iieritoneal than m pleural fluids, and. as a rule, greater m transudates than in exudates. . ..• ■ *u« In nephritic transudates the osmotic concentration is the same as or a little less than, that of the blood of the patient. The 'chlorine retention which occurs in transudates will explain this feature in part. The reference which has l>een made to the other constituents in transudates affords additional information. CHARACTERS POSSESSED HY VARIOUS PUNCTURE-FLUIDS I 55 As regards the eledro-conduclmty and the concentration of the electrolytes, both chloride and achloride, reference need no here be made, as the application of this method to diagnostic purposes is gone into at length m the following section. Some experiments of Sasaki may. however, be referred to, m whi h a nerhritis was artificially induced in rabbits, with a resulting decided increase in the electrolyte-content of tbe peritoneal "^"^ Concentration of Hydrogen-Ions.-The concentration of the hydrogen-ions in ascitic fluid has not been frequently determined The reader may be referred back to the table in ^''fermfnts. Antifermests. and Toxins. -The ferment- content" of various fluids has been discussed on a preceding page where the results of one's own observations are tabu- Led These observations may be amplified by a reference to Marshall's work, ^ho found that both pleural and peritoneal fluids have the power of ha.molysing blood other than human nig rat or goose blood, a fact which goes to show that these flui'ds contain complements which can activate various ambu- ''^TlTe content in complement of some fluids for a given lysin varies independently of the content in complement for other lysins. , ^ Fibrin-ferment is frequently present m exudates. The ascitic fluid in carcinomatous cases is hemolytic, just as the extract of these tumours possesses a lu-emolytic power. ANTIFERMENTS.-Muller found that the amount of anti- ferment rises with the albumen-content and vaiiesin amount whTthe degree of destruction of the leucocytes. The following results were obtained: Ca«r. Antiferment-Content. Asiites due to passive cc ngestion Hydrolhorax Hydrocele Pure lubt rcular I'critonitis Acute Peritonitis In excess Lessened Lost Antiferments to congenital syphilis have been found in the peritoneal fluid in three cases by Hans Bab. 156 STLDIKS IN I'UNCTLKE-KLUIDS Autolytic-U'.aents are said not to occur in puncture-fluids.* PoiSONOrS hlFECT OF ExrUAi'^S AND TRANSfDATES WHEN iNjErTED INTO THE Bi.ooD.— In cirrhosis of the liver the transu- date is no more poisonous than the effusion in pleurisy, showing that the jrortal l>lood is not more toxic than the blood of the general circulation. «•. THE EFFECT OF REPEATED TAPPING ON THE COMPOSITION OF PLEURAL AND PERITONEAL FLUIDS That a chalice in comiHisition resulted from repeated tapping was notetl by Halliburton and discussed by him in some detail. He found that the total proteid first rises in amount and tinalh falls, the globulin-content increasing more than the albumen. This increase in amount of jiroteid is very frequently noticed, though it is not an invariable rule, since in an analysis of peri- toneal fluid by Hoppe-Seyler there was a tlecided fall, l)Oth in total solids and in the proteid- and salt-content. The fact that the composition of puncture-fluids does not always necessarily vary during the i>rogrcss of a case has been several times demonstrated in the series studied in the Leeds (ieneral Infirmary. In some cases there was merely a slight fall in the concentration of the electrolytes. Thus, in a case of {peritoneal carcinomatosis secondary to di.sease of the omentum, the only change noticed during three weeks was a fall in the achloride-content from 03 to 01. A {Mjritoneal fluiil due to cirrhosis of the liver showed a fall in the proteid-content during five weeks. The achloride electrolytes remaineil constant, while the chlorides showed a slight rise. The total osmotic concentration remained practi- cally constant throughout. In another class of case the chloride-content remained con- stant during six weeks, while the achloride electrolytes became increased. In this case one may set off the change in concen- tration against the fall in proteid-content which occurred. The fluid was associated with throml)osis of the jwrtal vein. The diminution of proteid-content would naturally allow an increased ionisation of the electrolytes present, and there would be a diminisheil inhibitory effect on the conductivity. Since, • Zock. CMARACTEKS I'OSSESSEI) HV VARIOUS PUNCTLkK-Kl.UIDS 1 57 however, the diagnosis of mcreased aclUor..!. ;'l-»;"'vtos is based on the cryoscop.c result, .t beco.nes evu ent '-t a a>« - ence m albumen of 14 \^^ "'"t. woul bacteria. y> Due to the presence nt gl. !j) Due to a lecitho-globi >\ (4) Due to a mucin. (5) Due to the physical , (6) Due to other proteid^ ha A. The Tkue Chvlois i:FFi~ iN- plain. In this case the tuibidif ^ du of fat, and an examination o? n uns the case at once, or the fact tl her also explains the class of case ti wh cl or An analysis l.y Hopi>e-Seyki gavi Jin. (TS of the proteid. lobulm f mosT -imple to ex- micn -copic particles will clear up he fluid clear to deal. •d si- rei 1 tibrin, albumen, globulin Water <>34-S I fat, lecithin, cholesterin 708 92 FU4 o » int. it».ini»»', *-«iv.*-^»^ --- ^ Solids 952 I latty acids, soaps, and other organic substances lOS Isalts 44 The dry residue of an ethereal extract contained : Cholesterin < ''3 ,5 Lecithin 7'5 % Olein and palmitin . . 81I % There was no peptone or proteose. The content of fat varies in ditlerent ^pecnnens from 09 to 07 per cent. .\ case was rifently reported by llaminertahr, in which the patient CHARACTERS POSSESSED W VARIOUS PUNCTURK-FLUIOS I 59 • ^ » k,ck in thf neck from a horse, with a double chylothorax as TZt i^ he tt "intent was h.«her ,.-.5 P- cent.)^ I.others.n Xtl twonty-three cases, of wh.ch eleven were clue to traun.a. an.l twelve to compression l)y tumour. The fat may be ideHtified by the use of the microsco,>e, when it IS seen in the form of minute granules Uke micrococo wh.ch do not stain and are soluble in potash and ether Sudan III. may be used. Sometimes, however, the fat ,s enclosed m cell., which may be of very large size. The osmotic concentration of chyle is slightly lower than that of the blood, being 0-291. * Contrasting the characters of chyle with those of lymph, we find that the former is much richer in solids, three times a. dch in fats, but much lx>orer m salts. The extractives are high in each case. See also " Lacteal Cysts." B Pseudo-Chylous Ascites. (1) Due to baderia.-Thc presence of enormous numbers of bacteria is occasionally met with even in non-septic effusions. The use of a Chamberland filter will ther procure a clear filtrate Hamburger's Bacillus lymphagogon is c.casionally met with in these cases, and Plate I. Fig. i. shows an example. (2) Due to the presence of g/o^H/m.-Micheli and Mattirolo state that the cause of turbidity in a case which they rejxjr was a molecular alteration of the globulins present The molecular alteration may be really a combination of globuhn "' (3? a'Sw... as one might term it.-Bernert drew attention to the fact that globulin can combine with lecithin and produce a turbid fluid; but a much more complete study of a cLe of this kind was made by Joachim (1903), who discovered that it is the pseudo-globnlin fraction that has an affinity for ecithin. After removal of the pseudo-globuhn the fluid became clear, the action of boiling m the presence of acetic acid having removed the source of the turbidity in the hrst place. He found 0368 part of lecithin pro miUe. This observation was corroborated by Gross, Micheli and Mattirolo. and Christen. The latter author, however, does not think that lecithin is the sole offender in resi^ct of causing a milky effusion. From one s * Strauss and Grossmann. i6o STUD! IS IN I TNCTrKE-FLUins own obsiiviition^ tlii> rxplanation was touiul to hold good in a few (iiMS. till' Miiin.' ot turbulity iR'inf,' shown by its dis- apiH-araiui- cithfi on boiling or on ^ saturation with amnioniiun snlphatf. (4) Diif to a niiictn. -Thi.' presence of excess of mucin in a fluid may cause a milky or opalescent apiH-arance, esjx.'cially if the electrolvte conditions do not allow of its complete solution. (5) Dm- to the t^hysual properties of the proteids.—The pro- gress which has been made in colloid chemistry enables us to form some conception of a change that may possibly take jilace in an effusion so as to render it turbid. It is sometimes very striking that a first tapping brings out a clear, translucent, typically straw-coloured fluid, while a subsequent tapping brings a perfectly milky fluid to light. A striking example of this was met with in a case in which the only iM>st-inortem change was extreme cloudy swelling of the convoluted tubules of the kidneys. What change had taken i>lace between the two tappings ^ When, as in the above case, there is no ex- planation forthcoming that chyle, bacteria, or lecithin can account for the phenomenon, may it not Ix? assumed that a change has taken place in the physical projx?rties of the colloids ? If a change in the electrical charge takes place by which the attraction l)etween colloidal particles and ions is alteretl, then the former may form aggregates of a size suflicieiitly different from those in the first tapping to make the fluid turbid. That is to say, an alteration in the electrical charge of the colloid particles of the effusion, an alteration in the number of particles, and an alteration in the size of the particles is all that is needed to evoke such a striking macroscopic change in the fluid on different occasions. The ultramicroscope would show evidence of this change, but apparently it has not, so far, been impressed for elucidating this particular problem. Raehlmann, however, in IQ03, applied this instrument to clinical medicine, and in hi', account of the apix-arances produced by various albuminous solutions he refers to glycogen, which in dilute solution has a bluish-white opalescence. In very dilute solution extremely minute particles of glycogen, of varying size, can be detected throughout the solvent, and show a characteristic type of very energetic vibratile movements. Wc have, however, to seek the cause of the alteration in CHARACTERS POSSESSED llY VARIOUS I'UNCTURE-KLCIUS l6l electrical charge, but this is not very diflicult. (or it is well established that addition or subtraction of various electrolytes may cornplettly r*"verse or nullify the electrical charge jwssessed by proteid substances. ' In conne. tion with this line of thought, the lu-haviour of globulin solutions when dropjied into distilled water may \^ referred to. As is wi-ll known, this clinical test for globulin (in urine, e.g.) is due to the fact that in the absence of electrolytes globulin cannot remain in solution. The addition of a few- drops of a fluid rich in globulin to a loo-cc. measure of distilled water is sufficient to produce a turbitl. milky fluid of very similar ap|)earance to these pseudo-chylous effusions. A deficiency of salts and an excess of globulin would thus suffice to produce the effect of nature. The case of toxic nephritis referred to might Ik; explained in this way, lx>cause it is a remarkable fact that there was practically no albumen present, but much globulin, and the salt-content was low. Of course these factors produce their effect by changes in the electrical charges referred to. and it is well known that, after tapping, the globulins may increase at the exi)ense of the albumen. The question is one of very great interest, and is bound up with the ex[)lanation of the phenomena of salting-out of proteids.* (6) Due to other proteids than glohiilin.— The observation of Fuld and Levison that when the vegetable proteid edestin is treated with . and also remarks that the fluid did not clear up on shaking with ether. PERICARDIAL FLUID As regards coloui, specific gravity, and similar characters, pericardial fluid falls in line with pleural and i)eritoneal fluids. ♦ See Neisser and Friedemann, ■Stiidien uber Ausflockungserscheinun- gen," Miiiuh. med. Woch.. 1903, u ; Bechhold. Zcitsch. fur physik.Chemie. II 1 62 STUDIKS IN rUNCTUKK-FLl'IDS Thus in a rase ..f pnouinonia intiTcurrcnt in the course of o.inai caries • th.' i-eri. ar.lial ttui.J was (ouml post niortetn tl, he a hrown, turhi.l fiui.l. and the iHr..ardmm showe.t no tra.e ..( uiflanmiatorv - hani^-e. The alhumen amounted to only iH per rent, the ..hlorides were very abundant (o I.J7 ^i'"- e.n.iv ) an.l the .-..n.hutivity was log.j at l8^ C. showmj; that the a.hloride ele. tn.lvtes amounted to ooiq. and the ratio a. hlorides ; .hlorides was onlv 072. as usual in transudates. Tryptophane was present. ■ Accor.lini,' to Hammarsten. the chemical comi)Osition o» jx-ricardial fluid is : VVatt r ... -/'% I libriii globulin I albumen OS 60 22s Solids 40 I proteids ... ... 29 I Null ... 7-« other soluble salts ... f4 insolubli: salts ... 04 fxtrattivts 2'0 The proteid-quotient is thus .574. and the proteid-extractivo ratio 14- V The analyses K'iven b\ Friend and Halliburton. Gorup-Besanez, Wachsmuth, Hop|H-Seyler, and others, show that there is considerable variation in the comi)osition in different sjK'cimens. An analysis in a case of tuberculous pericarditis, made by Bockelman. showed that this fluid, which was h;cmorrhagic. . on- tained a considerable clot, and had a si>ecific gravity of 1024 at 15^ C. Its frcczing-jxtint depression wa> 0-51'' C, indicating an osmotic concentration of o 275 and a pressure of bi atmospheres (at o C .). The chlorides amounted to 7 gms. per litre, which means a concentration of 012, so that the concentration of the achlorides would amount to 0155 (expressed as NaCl). The albumen, estimated by Esbach's instrument, was 41 i^r cent., and the total nitrogen was 0"2b8 gms. ix;r cent. A case of chylo-iwricardium, recorded by Hasebrock, con- tained lo- ^6 ,)er cent, solids, 73 per cent, albumen, 107 ix^r cent, fat, 033 ix?r cent, of cholesterin, 0177 per cent, lecithm, and 0()3 per cent, of salts. Concctiration of Hydrogen /oH.s.-The normal fluid gives for log C„- 7-4400, meaning an alkalinity of ^ ^^.jj potash. • Reg. No. 8072. CHARACTERS POSSESSED BY VARIOUS PUNXTURE-KLl'IDS 163 SYNOVIAL FLUID Tlu . hief jwruliarity of this fluid is the sp^rial variety of mucin which it contains. This mucin does not reduce FehHnR. and behaves hke a nucleo-albumen or nudeo-proteid. It has been analysed by von Hoist, and by Salkowski, who called it synovin. Synovial fluid is alkalin.-. of yellowish colour, and may be turbid even in health. The following analyses form :\ useful comparison : Normal (tUmmar(Un). SynovilU (lloppc-Seylei) Water Mucin ... Proteid Extractives and fat Salts Sodium chloride 948* 2 39 5 3 6 938 7 5« 4 9 Uric acid may occur in synovial fluid, especially in gouty effusions. Antiferments.— Suppuration joint fluid was found by Miiller to contain no antiferment. As regards the clinical value o« examination of synovial fluid, one must admit that the inti rest here lies mainly in the bacteriological characters, and iierhajis no less imi^rtant are the cytological characters. HYDROCELE FLUID The colour varies from straw-colour to a dark brown or greenish colour. The siJecific gravity of hydrocele fluid varies between loib and 1026. An analysis given by Hammarsten gave 938 jier mille water and 61 per mille solids, which con- sisted of fibrin (0-50), globulin (13-25), albumen (35-94). ether extractives (402), salts (926), sodium chloride (619). Lecithin, and traces of reducing substance and of urea are found. Metalbumen \ and paralbumen have been found in hydrocele • For convenk-nce, the nearest whole number is given. I Devillartl. MICROCOPY RESOLUTION TEST CHART I ANSI and ISO TEST C 'ART No 2i 1.0 I.I ;- Ilia I: 1^ 1 2.5 2.2 ZO 1.8 1.25 1.4 1.6 ^ ^^PLIED irvMGE Inc S^S '^j^-iesler, New ' iri. !46C9 USA '"SS ;7^6> ^er - 03OO - Phone == (716) 2B8 - 5989 - Fox 1 64 STUDIES IN PUNCTURE-FLUIDS fluids. Old-standing cases, as with all cysts, may present abundance of cholestcrin. The jiossihility of finding filaria in hydrocele fluid may be just mentioned.* Succinic acid is said to be present in some e.xamples f and occasionally inosite. Ferments and Antiferments. — In syphilitic cases the hydrocele fluid may give a jMsitive reaction with the serum lest (Wassermann's). Aqueous Humour.— Lohnicyer «ives tlu- lollowins analysis : Water 968-87 per mille, solids lyii per mille, consisting of proteids (serum- albumon, and Rlohulin I'lz). extractives o-^i, NaCl f.-8.». other salts 081. There were no cellular elements present. The specific gravity varies between 1003 and i<>o<>. GriinhaRcn found a reducing substance in it, which was not sugar ; he also found urea and sarcolactic acid (" paralaclic acid "). The absolute <((i(//n. determined by C. Foain the case of a horse, gave : •1328 log. C.I — -•049 ^11 X 10 - 8 8 '93 vhich shows it to be practically ionically neutral. AMNIOTIC FLUID Amniotic fluid has been studied very closely in order to decide whether it is to be regarded as a transudate or as a urinary fluid. + The specific gravity is usually low (1008) and the solids only amount to about 11 per cent. • Salm. t Hammarsten. + Hamburger studied the amniotic and allantoic fluids in order to deci*de this (luestion bv determining the freezing-point depression, in association with his blood-corpuscle method (which informs about those substances for which the red cells are not permeable). He found the freezing-point of the two fluids identical, while the allantoic fluid requires less dilution than amniotic fluid in order to cause h;rmolysis ; the allantoic fluid therefore contains bodies which do -lOt exert any osmotic pressure on the red cells (urea). This means that allantoic fluid is a kind of foetal urine. Jacque had an additional argument for his belief, namely, that the ratio between NaCl and soluble salts vanes greatly, whereas if the allantoic fluid were a transudate the ratio would be constant and the same as the blowl. The same arguments hold for amniotic fluid, though the evidence is less convincing in this case. CHARACTERS POSSESSED BY VARIOUS PUNCTURE-FLUIDS 16$ The substances which have Ijeen found in hydramnios fluid • are : Nitrogen-content t ■.. Globulin Albumen .Sugar {not levulosc) J A mucin § o-2737o 1272 L'rca L'ric Acid ... Allantoin ... Ash 1 01, CO,, p.Oj, so, < Na.O, k ,0, CaO, MgO, Fc oim7„ 00635^ present 7-8367» Osmotic Concentration. — The normal freezing-point de- pression is 0510' i! or o-475-,t which corresponds to an osmotic concentration of 0-275 or 0256, or a pressure of 61 or 5-6 atmospheres at o^ C. In cases of hydramnios* it is less, being found to be 0395° C, and in cases of eclampsia it is increased i| (o-6Kr C). Hamburger finds it isotonic with the blood-serum. In cases of ectopic gestation Zangemeister found the osmotic concentration 0-301, and that the longer the time that has elapsed since the death of the foetus, the more concentrated does the fluid become, owing to the salts passing out from the intestine into the sac. The electro-conductivity in a case observed by me was 1070 at 18° C, and the chloride-content 0-507 per cent., so that the achloride electrolytes amounted to 0030, the ratio of achlorides to chlorides being therefore 035. CoNCENTR.\TiON OF HvDROGEN loNS. — Fo4 obtained the following values : IT log. C„ Ch.x.o"* •1362 — 7-1072 7-813 Scipiades and Farkas found for Ch x io"" g.o. The fluid is practically ionically neutral. Ferments. — Bonoli examined the ferment-content in am- niotic fluid and found that trypsin, chymosin, autolytic- ferment and glycolytic-ferment are never present, while diastase, pepsin, fibrin- ferment and lipolytic-ferment are always present. A ferment which can split up salol is also always present. He found a catalytic action. Antiferments. — Bab failed to find antibodies to syphilis in an amniotic fluid in a case of congenital syphilis. • Stozyzowski. t Scipiades and Farkas. I Griibe and Griinbaum. § Schere and Weyl. ii VicarcUi and Cappone. i66 STUDIES IN PUNCTURE-FLUIDS i CEREBROSPINAL FLUID The utility of a study of this fluid has come to be widely recognised, so that lumbar-puncture * is a frequent occurrence, and regarded as of very great value. While much stress has been hitherto laid on the cellular characteristics, it may be safely said that it is chemistry which will afford us full realisation of the diagnostic information which lumbar-puncture can supply. In recent literature tests have been given which are simple to perform, and promise well for clinical diagnosis. It has boon asserted tliat even if the cerebrospinal fluid served no iliaRnostic purpose it wouhl still be ol use to perform lumbar-puncture as a therapeutic measure. v. Bokay has shown that a therapeutic ettect can be expected, owing to the relief of pressure in the cerebrospinal canal, and to tht removal of bacteria or their toxins, so that lumbar-puncture should be performed two or three times in a bad case. The pressure of the fluid may be measured as it comes out of the cannula. It may be found to vary in amount almost from hour to hour. Cerebrospinal fluid is always slightly alkaline, and has a iow specific gravity (1003-8), which disease does not appreciably alter, except in the case of meningitis, where the specific gravity is increased, t Naked-eye Characters. — Colour. — Normally the fluid should be perfectly dear, aUhough, according to Pilcz, it is not invariably turbid in disease, since in a case of subacute tuberculous lepto- meningitis it was clear. As a rule, however, a clear fluid in- dicates that there is no suppurative meningitis. ♦ Ltnnbar-pnnclme. — The requisites are: (i) A trocar three to four inciies long, of the diameter of an antitoxin syringe-needle. (2) Means for sterilising. (3) Sterile test-tubes. (4) Culture media. The puncture is made a third of an inch to right of mi Globulin is always present, antl the amount of it which is present has been utiUscd by Nonnc and Ajielt for diagnostic purix)ses. They employ ammonium sulphate as a precipitant, using the abundance of the precipitate as a measure for the amount of globulin. The fluid is mixed with an equal quantity of saturated ammonium sulphate. If a turbidity apjiears in three minutes, the reaction is positive. (Phase I. reaction.) Thf. Globulin-reaction in Cfrfbrospinal Filid* Nonne'i Cases. |%ofCa»ef| No. of I in which Cases Ki- Lymt-ho- amined. cy'teswere Present. Literature. No. of Cases. %ofCas« in which l.ympho- cytesweie Present Disease. No. of Cases. Caaca ' where Phase I Reaction was Positive. 76 36 r 1/ 2 35 «9 •3 13 >4 5 30 5 97 9S 75 40 100 33 4 15 33 23 40 o o 33 < 95 14 76 >S 18 «7 3! «5 «5 «4 37 6 98 05 80 40 100 44 6 >5 23 24 65 o Dementia Paralytica 22 100 Tabes Dorsal is 17 93 Lues III. «5 92 Lues II. S 20 Lues Congenita 2 100 Healed Lues 18 Alcoholism 12 Idiopathic Epilepsy 10 Apoplexia Sanguines ... Sclerosis Multiplex Tumor Cerebri 3 33 Neurasthenia, Hysteria 12 Health 12 It is convenient to add acetic acid to the filtrate resulting from ammonium sulphate ; boiling will bring down the albumen, and it may be estimated by weighing.f Siemerling uses magnesium sulphate as an indicator of the character of cerebrospinal fluid. If a saturated solution of magnesium sulphate be added to normal cerebrospinal fluid, and the mixture filtered and boiled, the filtrate will remain clear in normal cases, while if from a case of dementia paralytica, it will become turbid. The addition of acetic acid causes a precipitate of serum-globulin. Protalbumose.— Deutero-albumosc has been occasionally met with by Halliburton and by Donath. Variations in Composition on Successive ra/'/)tHg.— Halliburton * From Mlinch. fned. Wocli., 1907, p. 42. t Nissl. r t| f I STUniKS IN rUNCTURE-KLUlDS W lound thf spt'citic gravity to rise and the amount of protcid to rise from 045 per cent, to ofx) per cent, and 072 per cent, in a case of chronic hydrorephaUis which was tapped at mtervals. He found the reducing substance (see fx'low) also to increase in amount under these circumstances, being only present in trace to begin with. (iiMi'osiTioN UK Ckhkhkiisi'INAi Fluid in IIyhkocki-hai.l's Dry bubslance. Ath. Albumen. Author. Kemarki. II' 09* 109 0-97 0S4 07S 078 0'12 .,■31 OlS (Jrc^bcr •» Neumtistir .Mian of 17 specimens Nitdeo-albmnen is found only in tubercular meningitis,* and it is convenient to precipitate it when searching for tubercle bacilli, as they will become entangled in the precipitate. Reducing 5/ear from the fluid when meningitis develops. Galactose was found in hydrocephalus fluid by Langstein. Lactic Acid has been found to l>e always present in cerebro- spinal fluid by Lehndorff and Baumgarten, though it is specially abundant in cases of meningeal inflammation. Carbaminic acid was found in a case of eclampsia. t Bile-pigments and bile-acids have been found in cases of jaundice. I Cholin. — The discovery of cholin in cerebrospinal fluid by Halliburton and Mott in cases of organic nervous disease, as opposed to functional disease, is the most important advance in our knowledge of the chemistry of cerebrospinal fluid. The • Sahli. t ^<- B- Kofmann. * Gilbert and Castaigne. CHARACTERS POSSESSED BY VARIOUS I'l'NCTURE-KLUIDS 17I cholin is derived from the breaking down of nervous tissue (see Section I. under Lecithin). In order to detect it, the fluid is treated with absoUite alcohol, and the extract evajwrated to dryness. Alcohol is again added, and the process rejx'ated several times, the last alcoholic solution l)eing treated with an alcoholic solution of platinum tetrachloride, when a yellow precipitate results. This is washed with alcohol and dissolved in 15 i)er cent, alcohol. On concentration yellow crystals of the double chloride of cholin and plati.nun separate out. To distinguish these microscopic crystals from a similar l)otassium double salt, a strong solution of iodine in jwtassium iodide is added, when dark brown dichroic plates of cholin iwriodide (C.-.HhNOI . 18) result, while the i>- jm salt does noi alter. On standing, the cholin i)eriodid< .eaks up into oil- globules. A simpler method of difterentiat.un is to look at the crystals with the jwlarising microscope, when the cholin salt comes out bright and the jwtassium salt becomes invisible (Rosenheim's recent method of identifying cholin by the use of alloxan is apparently not reliable). The presence of excess of potassium salts in cerebrospinal fluid is, however, regarded by Halliburton and Mott as in itself evidence of nervous-tissue destruction. other Tests.— (i) Make a saline solution of the alcoholic extract anil inject it into an animal. H cholin be present, the arterial pressure will fall, a phenomenon which will be prevented by a preliminary dose of atropine. (2) Dissolve in alcohol and treat with concentrated alcoholic picro- lonic acid. A precipitate of cholin picrolonatc is produced.* Ferments.— A diastatic ferment has been descril)ed by Cavazzani and Grober as present in the iiuid of chronic hydro- cephalus. And ferments have been found in tuberculous meningitis by Muller, while they are absent in cerebrospinal fever. In syphilitic nervous disease Weygandt found that antilx)dies are present — that is to say, certain albuminous substances in the normal spleen interfere with haemolysis, especially when the cerebrospinal fluid of a tabetic is added. According to Meru and Levaditi, 73 per cent, of cases of general paraK-sis of the » Otori. 172 STUMKS IN rUN( TURE-FLUIDS I! insane contain thcso antilxxlies, and W) jwr cent, of cases of tabt's contain ttu'ni. Is()i<(..\M< Cons I rnKNTS. -Sodium chloride, sodium car- bonate, sodium phosphate, and earthy phosphates are met with. The iK)tassium salts are increased in amount in organic nervous (Usease a( cording to HaUiburton. The ratio between Na and K has been made out by various authors to be 1:2-43 (C. Schmidt), I : 2i(> (Yorn). i : 21-5 (F. Miiller), i : Jji (HaUiburton). It must be remembered that this excess of |)otassium salts which has l)een dcsi ribed is only relative ; the sodium salts are abso- lutely in excess in all cases. I'HVSICO-CHEMICAL CHARACTERS OF CEREBROSPINAL FlUID. The Osmotic Concentration.— Oh^eTva.Uom by Fuchs and Rosenthal in different diseases have given the following results : Diseaie. Freeiinf-point ; Oimotic Depression. Concentration.* Pressure in Atmospberes.' Tubercular Meningitis ... Dementia I'aralytica Chronic Alcoholism Epilepsy ... Various Cases 3898 t Tubercular Meningitis .. 3899 t Old Cerebellar Abscess .. 046 054 054 052 053 0337 0524 ■248 '291 •291 ■281 ■286 ■181 ■283 6-5 6-5 6-2 6-3 40 6-3 The Electrolytes.— Observations of cases in the Leeds General Infirmary have given the results shown in the table on next page. These results show that the achloride electrolytes are always very scanty, whether the meninges be affected or no. The amount of chlorides is usually high and the total concentration of electro- lytes is also considerable. The last column shows a very great uniformity in the different cases. The electroconductivity varies with the freezing-point depression, except in cases of meningitis. Ionic Acidity.— Fok records the following values in a case of norma! cerebrospinal fluid : TT log. Cii Cm X .r* •1428 72234 597 or 6-35 Titration against tornasole gave an alkalinity of KOH. I have added these values. ■f Own observations. CIIARACTKKS POSSESSKD UY VARIOUS PUNCTURE-FLUIDS 173 Viscosity— Too few detoi- minations have Ix-en made to allow any ilecUutions to In- made.* In a case in thi-< hospital the viscosity was found to Iw 1-^2, taking water as I. CYSTIC FLUIDS Ovarian Cysts.— At the present day the comiwsition of ovarian cysts is a subject ot comparatively little import- ance. From the theoretical standpoint, however, their colloidal contents possess very great interest. It does some- times happen, nevertheless, that a fluid supjjosed to be a peritoneal effusion is really derived from an ovarian cyst, so that the chemical and other characteristics of this class of fluid demand consideration. Among cystic fluids, many varieties of which call for study, the ovarian cysts are the mo>t frequent and jxjssess the most decided characters. The numerous varieties of ovarian cysts which have lieen described by gynaecologists frequently show }>erfectly dis- tinct varieties of contents. The unilocular cysts, as a rule, contain perfectly clear and watery fluid, while the multi- locular cj^ts may contain • Fuchs and Rosenthal. .a I s a 2 1. ^ , »» •^ 5 ^ r- ? ■J . 3 I ° £y " > = o > OJ a- o 3 61' 2o L.W O O I 5 I- ■H-S.S -U = ■ u VZ, i o B s £> n 1 % N 1-^ try f 0^ f4 1^ 2 f4 ^ ^ 1 ' 5 i «' 2 - * Xi V s 35 as t^ 1 1 :§ S •74 STUDIES IN rUNCTURE-FLUIDS different (onns of »xtr»iiuly imicmoiis material in the tlifferent loruli ; thus, in one cavity there may Ih> a jelly-like mxss, another may contain a mucilaginous clear fluid which will just iK)ur, another may contain a bright green solid jelly, and so on. This is a rather remarkable fact, and a section throuRh a cyst of this kind consequently furnishes a very striking ai)iM-arance. The explanation of such features may Ik- found in tlu' character of the lining cells, for in some cases there is an abundance of goblet-cells in the epithelium, while in others theie is relatively little. In other ca.ses, again, the epithelium is iK-rfectly intact and stains Ix-autifuUy in the histological preparation (Fig. lo), while in other cases the epithelium is practically universally dead, the only indication of the structure of the original cyst bemg the connective-tissue strands which separate the locules. It seems so remarkable that there should lie these alisolute differences in the physical characters of the fluid in different loculi of tlu- same cyst that the question nmst be worth dis- cussing. In order to arrive at any opinion, we shall have to discuss certain projKTties of colloids, and discuss also the histo- logical apiwarances in the cysts. In the first place, the problem may be merely one of a conversion of a sol into a gel (in this instance, of course, a hydrosol into a hydrogel), but if we endeavour to ascertain what explanation there is to be had for the change from sol to gel in general we find that there are abun- dant theories, but that very little is really dcnnitely under- stood. Perhaps the simplest explanatrn is one which takes into account the surface tension. We have already mentioned, when discussing the difficulties of removing albumen com- pletely, that when mastic is gradually added to an albuminous solution, tl\e particles of mastic lx>come distributed over the particles of albumen (hydrosol) without any visible change occurring until a certain point has been reached, when the amount of mastic causes the separation of the proteid, the particles of the latter having become exceeded in numl)er by the former, so that no more distribution of mastic can take place. The same may hold in the colloid of ovarian cysts. The addition of electrolytes, for instance, from the circulating blood may result in the conversion of the hydrosol into the hydrogel, the process being one of modification of the electrical charges origin- e V yf^Wf Fir,, lo. -From a Cysto Adiiioina Papillifoniin Ovarii, I'l-liowaclivi-ly ■'eiriiinuKol.liir>-. II. (I. qiilluli.il KvXU ; /■, -ii]>ii<.rliiiK ti-l.iiii nf tin- -.itimi ; lii'< ill the lavily lif llu- iya-iil i«>rli..ii» i.f llu- i.lK ,A .»' iin- !.• \c. 4. Fici. II. — Fioin a Miiltilocul.ir Ovarian Cyst. The ilrawini! shuw^ the will limiliii« hKiilu* tt (rum lm.uhi-i '), ami mii e-ach vvl 111 aiiilv nf i-c.tilel telU in a -liiiKle rnw, aii.i the latter nf ti iileiieil niliii-.il eell-;, alsieniins by its sharp ennti^iir, ami by the exti.leil ilmp^ i>( snreti.m. Tlu (litTerenee in the ehar.icler nf the seeret..ry eells i>t the Iw.i ey^ts is -IriVini,', and may lie llseU as showillK the ertetts of pri-s*iire Ircun excessive seereti.m in the l.milus h, although it is in juxt.ii>.isilioa with Ititiilns a, where there is ni) evident internal pies-urc. In tjoth cases there were i;cialimiu3 eunteiits To loci pagt 174- fi m CHARACTERS POSSESSED BY VARIOUS PUNCTURE-FLUIDS 1/5 ally possessed by the particles of " colloid," ♦ now become so larRe Ihat they aggregate into a solid substance. In the case of the capillary electrometer, where the electrical charges of surface tension come into play, there is at the junction of mercury with acid a double electrical layer, which is mfluenced bv an incoming electrical current (change of ix)tential) when the instrument is used. A loss of the ix)sitive charge of the mercury meniscus causes its surface to tend to assume a smaller size So with colloids : acco'ding to Bredig, the particles of colloid are surrounded by water, and the junction line possesses an electrical double charge. If the negative charge of the colloid particle be neutralised the surface of the particle will endeavour To shrink from the surrounding water, and, meeting with other particles will tend to associate into complex groups, tiU by a continuation of the change in electrical sign we ultimately arrive at a point at which the aggregations of particles have attamed so large a size that they fall out as a gel. This would merely be part of what seems to be a genera fact, and has much to do with the elucidation of problems of immunity, namely, that a continuous change can be produced in a colloid without the slightest indication to the naked eye, as it were, until a certain ix)int is reached, when the conditions are so strained that coagulation, or precipitation, or what not, takes place.f The fact that electrolytes have something to do with tnis change of sign demands no more detailed consideration in this place because the process is the same as described above, and it is sjieculation to consider whether more chlorides or less, or more phosphates or less, or more or less of any other salts, do at any particular time enter into the contents of loculi. The most evident feature of the walls of these cysts on micro- scopic examination is the goblet-cells to which we have referred already, although they deserve a more careful consideration. There is a decided secretory activity going on in all quarters • It is a pity that the term " colloid " should be applietl to the material in ovarian cysts at the same time as it is applied to matter in a .lehmte ^'t?n tailing egg-white, for instance, one ,s disturbing the electrical charge of ♦he particles. Th.s again illustrates what I contend .s the T^Imv in chemical study of fluids. We do not reahse that even s.mple boiling may be exerting very far-reachmg effects. 1/6 STUDIES IN PUN'CTURE-rLUIDS -cells nearly all laden with secretion, others emptied, others showing active mitotic figures. The secretion is presumably fluid, even though viscid. Can this secretion become solid (gel) as a result ol the processes detailed above, or may there be another cause ^ For instance, there is a iK)ssibil,ty that the secretion mav continue to such an extent within a limited sized cyst that enormous pressure is mechanically exerted by the non-expanding cvst wall on its contents. Such condition could well varv in different loculi. and a difference m viscidi y of adjoining cysts would hinder or allow expansion of the neigh- bouring cysts. In this connection, then, the first problem is that of chscovering whether enormous pressure can alter the state of a colloidal substance, and if so, by what means, and the second is a histological one. namely, as to whether there is anv evidence of pressure in these cysts. In answer to the first question, we may refer to the observa- tion made by Pauli, that the delicate layers of intercellular material which surround cartilage cells may come to exert enormous pressures when they absorb or gradually give off water as is shown in the process of formation of compact bone. Thus, some proteid matter was forced into steel tubes under enormous pressure, and became so ivory-like that it could be worked with tools. Again, it has been computed that in order to remove the merest suspicion of water from gelatine which has taken up 8-4 parts in the loo of water, would require a pressure of more than 200 atmospheres* We have, then, to take the factor of imbibition mto account as a possible one in the production of the jelly-like matter of ovarian cysts. That is to sav, the colloidal matter secreted by the goblet-cells may take up water, and in so doing become so swollen up in a given space that it has become of the con- sistence of a jelly. Under this supposition, the particles of colloid take up water like bibulous paj)er. for instance : each molecule not only becomes increased in size, but there is less water remaining in solution. The laws governing the process of imbibition of water by a colloid such as agar have been expressed in the following mathe- matical formula by Hotmeistei ; Lei \\' be the weight of water • Xagcl. t " i y ~\ % (Z i^^-Tv;*^^v /^ I'lc. 12. -From a Cysto Carcinoma Ovarii. Tin- .Ir iwiiii; sh.iws tUt cpilluliuiii coviritiH imc siilc ..f the lil.roiH lral>.vula, which ^op:ir,iU- uiic locuhis from ani.lhur. Thi- crowlh is not iiapillifirims. Till- luiimt.- cvst a i- cticloscil l.y Kol.l.t-cills h, h. h, in varyins stase> .>f sccrotioii. Sonif ,ir.' iiniily, iilUirs swolkii, aii.l the ,\u.lol sicri titin tan lie s.eii adheriiiR rmiml the st.imata of' the eell-. Ill ^iiiiie <>i tile celU there apjKar to l>e minute ehannoU (rather too .lark in the .Irawini;), Ihrounh whieh the -ecrelion has to pass l,ef..re enteriiij; the ea\ itv a. f, l>as.il eeil-, prolKil.ly alniut to form «oblct-cells ; J. fll>rims tral.eviila snpiHirlini; the epithelium. This trabeeiila eon-ists mainly of short spindle cells with slender nnelei, anil lymph.iytes f. .. <■ (exuded from the walls of the capil- lary tl, intilirate the lilirons tissue, t', medial cells of the >tratitUil epithelitim. *. mitotic tiuures. The -pecimen show* the commeiiciui; formation of a cy-t. which is Hoini; to ex- pand not only l>y excess of secretion, lint al-o l.y the actual growth of the iiathelinm. In tlii~ CISC the ielly-lormation is ilue to a sul.-e<|ueut chau^;e in the oriaiual nmciuous secretion. To /act pat* t77< CHArACTERS POSSESSED liY VARIOUS PUXCTURE-FLUIDS 177 absorbed during a time t, and P be the maximum amount of turgescence jwssible at the given temjierature, and D the thick- ness of material which is taking up the water in miUimetres, then — W - 1 ■(-■-n) where k is a constant. In order to work this formula out for the conditions which exist in the case of ovarian cysts, several experiments would be necessary. We will, however, pass on to consider the second question — whether there is any evidence of pressure within X\\v cysts. In many examples with which one meets, the pressure exerted by the contents makes itself very evident by the spurt which results on making an incision into a cj'st, and it is remarkable how flaccid are the walls in the case of some jellies, while in other cases the material at once swells out on cutting the tumour 0})en. Microscopic examination shows a remarkable richness in goblet cells, which consist of clear cytoplasm, and relatively large triangular nucleus at the lower end of the cell (Fig. 10), while the upi^er end is widely dilated and contains granular matter presenting somewhat the arrangement of a network thickened here and there by granules. Some of the contents can be seen oozing out of the free end of the cell, although in other cases (Fig. 12) there seems to be a cap of striated sub- stance, as if there were numerous minute channels through which the secretion had to pass. Observations on living goblet cells by Merk showed that movements may occur within the goblet, the granules becoming darker or lighter from time to time, while granules come to the surface and explode, as it were, becoming discharged into the surrounding medium, and lost in it, " like smoke from a chimney." This is well seen in Fig. 10, and it indicates that when the secreted substance comes outside the walls of its manufactory, it undergoes imbibition with water, and so alters its physical characters. All these cells are large and conspicuous, though here and there there are smaller cells, apparently empty (Fig. 12), and in other places again (Fig. iie), the cells seem to be devoid of 12 178 STUDIES IN rUXCTURE-FLUIDS secreting activity altogether. The presence of numerous mitotic figures and the full distension of the goblets, however, show that a very striking degree of secretory activity is present throughout the epithelial lining of these cysts. Changes in the nucleus may also be observed, and it has been supposed by Krausc that it is concerned directly with the process of secretion. Thus, in the salivary glands of cephalopoda he finds that the nucleus exerts a ferment action during secretion, the ferment being of an albuminolytic nature. During secretion the nucleus lies near the base of the cell, and becomes increased in size by nearly 20 per cent., besides losing some of its staining power and showing granulations to a much more noticeable extent than is the case in the resting cell. Then, again, the nucleolus may have something to do with the process, being more conspicuous and even multiple or showing a spiral thread- like structure during secretion (Nussbaum). The substance secreted is a mucin, and one of its characters is still seen in the case of jelly-containing cysts, where so much glucosamine is found. We may suppose that the glycoproteid of the colloid matter in the cyst only differs from ordinary proteid in having relatively many more glucosamine radicles per molecule, and if this be so, it is not so difficult to understand how the cytoplasm may give rise to mucin. We may. for instance, say that cytoplasm of goblet-cell = x albumen + y glucosamine, but secretion of goblet-cell =^ (x-z) albumen + (y-z) glucosamine, where x and y are definite but not knowTi quantities, and z is a variable, and not necessarily the same in the case of the albumen as it is in the glucosamine. That pseudo-mucin as met with in ovarian cysts with fluid- contents is exce*»dingly like " paralbumen," or paramucin in chemical compositio.-, was shown by Otori, who found that both })ossessed identical decomposition products. We may illustrate this by the following: Substance. Author. Mucin of saliva 48-84 6-8o I2"32 084 3r2 Hammarsten „ sputum 48'17 6-91 lo-8 1-42 317 ; Muller Pscudo-mucin ol" ovarian cysts 49'8 69 10'27 | i'25 31 78 ] Hamir.arsteB Paraiiuiciii ol ovarian cysta ... 1 5i 7(J , 7/6 16 7 j 1 Oy ■■ 28 69 j M tjukoff The paramucin contains more C and H and less O. M I'lr,. I ?. I'"roiii a Multilociilar ()\,'iiiaii Cyst. The ~)i"i'i'>" -liiiw-i MM-ril of tin- lonili, wliiili nn ul iliiuriiit -i/i<. a, inl.iri litiiiii; t|iiiluliiini i.t ilniilnl iiitiii:il -li.iin- Tlii> rv^l ii.iit.iitu ■nini-lruii^iiariiil iiMiUtiN ; '>, A l.iriiiT lavilv fillcil with ur.iiinl.ir ni.illir (-In.w- Ji''.»n i.f idl-s with liii;li iHiwiri till' liiiim; nii tnlir.iiu' li.i- ili-^ai>iK'ar('i>nliiinitiu hi>niin;riKoii- iiiatirial, ami lUvniil i.f hiiitii; iiutiiliraiu'. iThi- ililail< nl iln' i.riL'iiial ini<.To-|ihiitriiiliuti<>M 1 ,l/.i,' >,i „i,iiii. ^J^ ^ I I'll.. I.). — Dead and Dyin;,' Cells from a Colloid Carcinoma. Tliis illustratis another nutho.! i.f formation of " colloid ■ nialtrial — direct mrta- niorpho^i^ of tlu- neeroscd ti-lU. ri, II remains of the tralieeiihe siip|iorlini; the cells of the urowth ; //, b, cells whose nuclei are still visible, though their shape is lost ; c, r, " shailows" of carcinoma cell> , ij', .;, liwiig >.eilr. (connecli\e tiB>uc cells, and other wandering cells).— /ms Hotnogen Imm. Cic. 4. To tact page 179, CHARACTERS POSSESSED BY VARIOUS PUNCTURE-FLUIDS 179 It must, however, be admitted that the substances which may be present in ovarian cysts cannot be limited in number to the two indicated on this table. It is probable that there are many varieties of " pseudo-mucin," which may be shown by very slight, but nevertheless distinc* ariations in chemical properties. The well-development of these goblet-cells seems to cast doubt on the existence of pressure within the cyst, whose wall they line, but it nevertheless seems to be a fact that even tense cysts may show a perfectly well-formed goblet-cell epithelium. This is contrary to the well-accepted idea that cavities con- taining fluid under pressure have a flattened or cubical lining. This may be so in many cases, but it is certainly not so in all. In Fig. II it will be seen that on one side of a small loculus the cells are large and conspicuous, while on the other side they are low and cubical. In the latter loculus the contents may have been tightly packed, but in both cases there was " colloid " material. (See also Fig. 12.) In a section prepared from a cyst whose contents were en- tirely gelatinous in consistence (Fig. 13), it will be seen that the cells are large and well formed. In another specimen, where the fluid spurted out on opening the cyst, and there was an adenomatous growth (papillary) all over the walls, the goblet- cells are seen to be peculiarly well formed (Fig. 10). We must therefore conclude that the gelatinous material is due perhaps to an excess of mucinous material in the secretion, but, never- theless, to some physical change which the secretion has subse- quently undergone. This change will be either one of imbibition or will be due to a change in the electrical properties of the material, under the influence either of water or of some electro- lyte, as already referred to. The question as to whether the mucin is ever derived from broken-down cells may be considered for a moment. It is cer- tainly the fact that if we search through the material in the microscopic cysts, we shall find many desquamated cells. It may be presumed that in the colliquative process which has taken place (Fig. 14), special mucinous bodies are liberated, which may have something to do with the question. The fact that nucleoproteid such as is met with in the substance of the nucleus may have some chemical- relation to the production i8o MLMl.S IN llNCTlkL-l LLII>S of mucin is ol mliust. tliough a comparison of the formula' of the two siili>tancts shows that unless there are many carho- hyilrate radicles in the nuc leoproteiil m question, there will not he a possibility of its conversion into mucin. The relative abundance of degenerating,' cells, still less of shed cells, as com- pared with those whii li are evidently actively secretin^, is, however, so small, that this factor does not demand much serious notice. The formation ot " colloid " material must be referred to physical changes occurring in the mucinous sccretivn of the goblet-cells so characti ristic of these ovarian cysts. While the nnicinous substance containeil in ovarian cysts is usually a glycoproteid of the ty|)e fully described in Section I., Halliburton has described a case in which true mucin is present. Hanunarsfen says that no mucoids occur in the cyst if it l)e derived from the Wolffian boily. The following analyses by Halliburton will show the ix-rcentage composition of the fhiiils from various ovarian cysts in a con- venient form : £•6 KindofCyiU o-l | Cfl b Sp. itr. '1 >tal Solids. Hrotcidi. balU. Colloid I'apillary Hvdrops. t' liiantc Hv.li'ps. tul ae Fibre cystic 24 2 IOIO-IO38 i 28-75" 1036 ii6'4 IC09 looS 10 I 8 8-108-3% 102-67 12 63-056* O ' + ; 6-8-7% 6-7 The following details about cases which have come under one's own obser\ation will illustrate the characters of different t\-pes of ovarian cyst. A sjiecimen of multilocular papillomatous ovarian cyst + showed the following varieties of fluid in different loculi : (i) a thin watery opalescent fluid having a conductivity of 1233 (at 25- C.) ; (2) a clear straw-coloured fluid having a conductivity of 1-244 ; (3) a viscid, grumous-looking material with a conductivity of I2i(), and (4) a jelly of dark green (fluorescent) colour whose conductivity could not be measured with the apparatus. There • Fibrin 3-58 per cent. ; globulin, albumen, t No. 6O43. rilARACTERS POSSESSED llY VARIOUS I'UXCTURE-KLL'IDS iSl wa-i the merest trace of chlorides in the watery Huid (o i)<) i>er rnilU'). The s|H'ritic gravity of this tyi>e of i yst-Huid is usually .M.'i) un present. A s|)ecimen of parovarian cysl • contained a clear, highly albuminous fluid (29 jxjr cent.) of watery consistence which was moderately rich in chlorides. The conductivity at 18" was I168, corresponding to an electrolyte content of 0-241. The specific gravity is below loio and the solids amount to only I or 2 per cent. This type of fluid contains only a trace of pseudo-mucin. A unilocular hroad ligament cyst which had been known to e.xist for 3.J years t contained a brown muddy fluid remarkably rich in cholesterin. There was only a moderate amount of albumen, and the conductivity was low. The ferment-content was considerable (see Table X.). Leucin was found in this fluid ; a trace of levulose ; the a-naphthol test gave no definite test, while the glucosan.ine test produced a well-marked reaction. The chlorides amounted to yd pro mille. These in»r.-' 'a- mentous cysts usually have a fairly high six;cific gravity 5) and contain 9 or 10 jier cent, of solids. A unilocular cyst with dark brown fluid-contents is referred to under the appropriate headings in Sections I. and II. An ordinary mulliloctilar ovarian cyst J in a patient aged 21, contained a clear watery (slightly mucilaginous) fluid, of specific gravity 1009. Proteids were scu ty in amount, and were mainly albumen, protalbumose and hetero-albumose being absent. There were marked metalbumen reactions. There was no urea, no purin bodies § or lecithin. The osmotic concentration was 0286, the chlorides were abundant (0145 gm. equiv.), and the concentration of the electrolytes o-2()4. No ferments were detected. One or two mononuclear cells were alone seen, and some stellar phosphates were found. A case of papillomatous ovarian cyst with an opalescent fluid contained 2 per cent, of albumen, 6 pro mille of chlorides, and a • No. 8177. t Nc. S364. ♦ No. 7618. § I.e. detectable in ^he quantity of fluid obtained. 183 STrr)IK> IN PUNCTUUE-FLUms conductivity of 1075. so that the eloctrolyte-content was <> .'43 The osmotic coiKintiatior. was ^Vh). The usual glycoprott-iil reactions wvtv olitairud* Lastly, s|Kcial ntiicncf may Ik- made to a case t ol a multi- locular ovarian cyst wliich contained cholesterin, the typical cellular elenunts, ami liad a remarkably small chloride-content (05 pro mille), with a considerable amount of albumen (25 |)er cent.)- The concentration of the electrolytes was 146. The contents of dermoid cysts are well enough known. Among many other substances they contain oleic, stearic, palmitic, and myristic acids, cetylalcohol, ami cholesterm.J Phvsico-Chemic.vl Ch.\r.\cters.— The osmotic concentra- tion is similar to, or most frequently less than, that of normal blootl. The following values will Ih> of interest : TABLE XVIII Osmotic Coscentkation or Ovarian CvsT-FLUin Fluid. iKrwtinK-Poir.t O»inolit PrJ«Ti?ri'in Depre..ion. Concntration. AtioVphere.. Author. Cystoma Ovarii Parovarian Cyst Miiltilocular Cyst Papillomatous Cyht ■54.^ 53' ■546 53' •J93 ■286 •294 298 286 •;o3 67 iZangcmpister O. C. G. The electrolyte-concentration has been studied by me in several cases where the cyst-contents were sufficiently fiuid. The results are given in the table on opposite page. Reference may here be made to a case reported by Nassauer in which an abundant foul-smelling fiuid was discharged per vaginam, having the apjiearance of clotted milk. The fiuid was of a mucoid character and contained tiakes of coagulated material, and stiffened linen. He states that this fiuid was derived from a tubo-ovarian cyst which l«ad ruptured into the tube. Tlieri was no examination of the fiuid itself, beyond the points which have l)een mentioned. J I.udwig and Zeynek. CHARACTERS PObSESSED BY VARIOUS rUNCTURE-FLUIDS 1 83 2 8 2 u. I u z < < > c o G .J W W 1 > 1 3ii|i- fi i - •/n -« tl .S-:i « 7 ^ xf, I i p ■ 8 i ^ 3, X ' ilF " ? ? I I ! I wit a ? • z 5 - S 8 ?• t 8 1 = o I «i = 8 I ? ? P ^ If. •^ :« U 1 « c: = « ^ ^ ■4 n U u u u c s 1 3 n 3 .2 'C z « c 3 e 3 S 2 I 30 3S 184 STUDIl-S IN rUNCTUKE-FLUIDS I tl: I* i-|! Hccnidtmicti'a — Zangeiiicister i'i-coi\ls a case of atresia of the hymen in which a fluiil was obtained having an osmotic concentration identical with that of tlie liiood (o'joi). (iflatinons masses have been descrilwd as occurring in the ptritontum as the rrsult of rupture of an ovarian cyst. An intrrc^tini; tnse ol tliis kinil is rt'corclci! hy HiR'tor as occurring in n male >u!)jict. Tlu' cy^t had arisen Ironi tlio rupture ol an appendix which liad iniderKOiie t xtrenie c\stic distension, and a ipiantity of sterile rnucilayinous matter was lound in the peritoneal cavity. Two other cases have been recorded, one by Fninkel and tlie other liv Merkel. Tlie p^i':ul(i-iny.\on!ii peritonei, when it bursts into the peritoneum, may give a characteristic appearance to the puncture- thiid. either from tlie presence of gelatinous masses, or from the presence of a large amount of mucinous matter. In this case the fluid will be tenacious, homogeneous, yellowish or whitish in colour, and give strong mucin reactions, tieing completely pre- cipitated by acetic acid. Boiling scarcely alters the appearance of the iluid, since iticre is very little albumen present. There is no pseudo-mucin, but paralbumen has been descrilied as abundantly present (Marchand). The presence of very granular cells may also be noted in the material in the peritoneal cavity. This is owing to the liberation of the so-called com- pound granule cells out of the cyst cavity into the peritoneal cavity'. Pancreatic Cysts. — The most recent account of these cysts is that which is given in the work on Diseases of the Pan- creas by A. W. Mayo Robson and P. J. Camniidge. They de- scribe pancreatic cysts as being cither true or false (distension of lesser peritoneal sac, localised collection of fluid in the vicinity of the pancreas). The true cysts are due either to retention of secretion, to hydatid, to new-growth, to hcTemorrhage, or are of congenital origin. The fluid is of varying api)earance and consistence, being sometimes watery and sometimes of mucous or gelatinous con- sistence. The colour may be brownish (as is usual),* or greenish, or the fluid may oven be purulent. The h;emorrhagic fluids probably indicate that the cyst is false. f The odour is stale. * Liiicnsttin, ai.so my own cases. t Schmidt. rHARACTERS POSSESSED BY VARIOUS PfNCTURE-KLUIDS 185 Normal pancreatic juice may contain : Zilwj Scliumm. (.lUstner. Spccitio gravity ... KrtCiiiMH-point ilipression Water Dry matter... Albuincn ... Nilrugen Ash Orcanic matter soluble in alcohol Alkalescence in terms of soda ... o-6i = 98-5 \ '5 0-07 10 0-49: 1009 9^-5% '5 01 ooS O.Sj 056 0-45 , 1007 o'4ii' 9S7 . '3 017 0-1 0-56 0-51 i(X)7 049^ 9S7% 13 013 008 07 042 The liuitl contained in a pancreatic cyst, on the other hand, may have the following composition : Specific (iravity ... Solids A9 4157 dark straw bile- stained 1012 I lO'O 1010 I trypsin trypisn ... i -t- -t- ! ... i -f -l- O o"4oi o Trace The solid material from two specimens was found to consist 1 86 STUDIES IN I'LNCTUKE-FLUinS I of fatty acids or their salts, cholcstcrin, and potassium carbonate. Another specimen, having a specific gravity ol 1012, con- tained a niaxiniviin (piantity of albumen, but neither sugar nor cholestcrin. This fluid coagulated spontaneously. Proteo- lytic ferment was detected. The ferment-content of such fluids is the most characteristic ])rojierty which they possess. The following ferments may be found : Trypsin is generally present, but may be absent, especially in cystic adenoma of the pancreas.* Pepsin has been found (Schmidt). Diaslcisc. — Equal jiarts of starch paste and fluid will give a reaction with filtering in an hop.r (Schumm). This ferment is, however, present in many fluids ^»ee Section I. Table X.) and was not found in a case described by Lillenstein. Lipase has been looked upon as characteristic of this class of cyst, though reference to Table X. will show that it has been met with in other fluids. The method of testing for these ferments has already been gone into sufficiently fully in Section I. Other Constitients. — Chlorides sue scanty ; phosphorus is not present, f Hsemin crystals have been obtained from the deposit in these fluids, and cholesterin may be readily detected on evaporation of an ethereal extract I * Robson and Canimidgo. t Scluimni. I Tests lor Chiile^ten'ii. (i) Cone. H., 50, and a trace of iodine turn the crystals violet, then blue, through green to red. (2) Hesse-Salkowski. — Dissolve in chloroform and let coiic. H SO, n\:\ in. Shake a little. The cldoroform turns blood-red, then cherry-red, and the acid becomes fluorescent (:iioss-j.reen). Four the chloroform into a watch-glass, when the colour passes through blue and green to violet. (3) Deniges. — Dissolve in chloroform and add J vol. cone. H.SO,. Now add a few drops of acetic anhydride to the chloroform layer. A brilliant red turns to a blooil colour in MSO^. (4) Liebermann. — Dissolve in acetic anhydride. .\dd coiic. H SO, drop by drop when cold. The solution becomes rose-red after a time and then turns blue, and finally green. (5) Treat the alcoholic solution with c -methyl furfurol solution and run in cone. HSO,. A rose-red nng appears at the junction line. On CHARACTERS POSSESSED DY VARIOl PUNCTL'RE-FLLIDS 187 Glucose and Gh'coproteids were not present in Schumm's case. Proteids and their Derxvatives.-\\\ix\t albumen and albumoses are present in varying amount, the derivatives leucm and tyrosm are frequently conspicuous * and may be found by simply allow- ing the fluid to evai>orate in a small watch-glass. The variations in composition have been suggested by Lillenstein recently to l>e due to the anatomical structure of the cyst If there be a lining epithelium the contents will be different from those cases in which the fluid has been extravasated (no definite lining membrane). The Osmotic Co«ce»i/r<(/i oh. -Pinkussohn has determined the freezing-point depression of normal pancreatic juice, and found it to vary from 0-58 to oOb" C. It will be seen from Table XX. that the fluid from a pancreatic cv^t owed a depression of 0401° C. corresponding to an osmotic concentration of 02 17. Another fluid examined from the physico- chemical standpoint showed a conductivity of 1430 at 244 t. Thyroid CystS.-A report of Hoppe-Seyler's shows that one may expect to find mucin, 7 per cent, proteid, cholestenn, and calcium oxalate. The dark colour of these fluids may be due to methffimoglobin or even to altered bile-pigment. Abdominal Cysts.-This loose clinical term refers to aU shaking, the fluid turns a Ii:S IN i'UNCTUKE-FLUinS If m these cysts which are met with in tlic abdominal cavity, and whose natuif has nut bicn enteric cysts, though in the absence of post-mortem examinations it is not certain that they were not retroperitoneal. (2) Here may be mentioned a case of cystic Ivmphangioma of the peritoneum which has been recorded, and which is referred to by Hueter as being probably a mistaken diagnosis for pseudo- my.xoma peritonei (see p. 184), with gelatinous masses in the peritoneum. (3) Another class of abdominal cysts is formed by mesenteric cysls, which form the subject of an inaugural dissertation by Theodor Hein (Leipzig, 1907). In one cyst of this kind Zangemeister found an osmotic concentration of 0-299. A case of mesenteric cyst was described in 1907 by Niosi, who jioints out that these cysts are very important, from their occasional simulation of ovarian cysts. There are several possible varieties: ha;morrhagic, chylous, serous, dermoid.f hydatid, and angiosarcoma, + and congenital cysts (from omphalo- mesenteric duct). Other forms have been supjwsed to arise from softening o' the mesenteric glands as a result of such in- fectious tliseases as typhoid, tuberculosis, etc. The very ex- tensive literature is fully quoted by Xiosi. The case which he describes at great length was one of .1 cyst containing a turbid, tenacious, dark chocolate-lnown coloured fluid of specific gravity 1040, and neutral reaction. Its composition was 89 per cent. * \o. 5«W3. t S. Ko--tlivy. * S«' Pfeiiiiii: — iilir rctrot'critoiiCi'J.c D-.rmniJ'V.l: u. " InsM" Pi=- " Bonn, ic)o;. See al~o H. .Max: Zit, Kasmstik der Mesenterialzysten (cau>inL; pirniciou-; vomiting. ■■ Inaui,'. Diss." Leipzig;. I907. 1 i CHAkACTr.RS I'OSSESSED liV VARIOU:^ I'UNCTURL-KLUID.S I Sq water, lo jxt cent, organic substances, and nearly i per cent, salts. Traces of mucus and peptone were found, and (r2i,^ per cent, of fat. the remainder ot the organic matter consisting of aroumen and globulin. There was neither urea nor sugar present. The salts comprised XaCl b-b per cent., traces of S and P, Ca and Fe. The cellular elements were granular corpuscles and fatty ejiithelial cells, and there were crystals of cholesterin. Nio-;i assigns to this cyst a congenital origin, connected with the Wolffian bodies. There was no adrenal tissue present. (4) Another cystic rctropcrih>neal tumour is described by Heyrovsky. The tumour was the size of an aflult head, and contained mucoid material. It had existed for seventeen years, and was found to be a impilliferous cyst contaming goblet- cells which formed mucus and then brokr down into a colloid carcinoma. Retroperitoneal cysts are usually pancreatic or hydatid, but may be due to softening of a sarcoma. (5) Dermoid cysts, arising by implantation from a ruptured ovarian dermoid. (6) Ha-morrhagic cysts, arising after traumatism. (7) Hydatid cysts. (8) Omental cysts, which may be either h.tmorrhagic .ir dermoid. (9) Peripancrcatic cvsts, due to closure of the foramen of W'insiow by jieritonitic adhesions. (10) Urachal and allantoic cysts due to congenital mal- formations — all these belong to the class of abdominal cysts. Ovarian cysts, pancreatic cysts, ami the swelling of a hydro- nephrosis also, strictly speaking, come under this head. The characters of the fluids n-ed not be enlarged upon in this place. Cysts connected with the Liver.— These have been found to contain cholesterin if they are connected in any way with the biliary passages. Bile-pigment may therefore be expected as well. Such characters are also noted when the cyst IS connected with the gall-bladder itself. Probably these cysts are too small to come under the notice of the clinician, and are usually })ost-mortem findings. Other liver cysts are. however, met with, which are due to the breaking down of neu' gnniihs. Hosch records such a case, which he savs was secomtary to a cancer of the stomach. The only case of the kind which I have oliserved was one I90 STUDIES IN ri'NXTrRE-FLUIDS of breaking down angiosarcoma of the liver of enormous size (No. EE4<), Pathological Miu-eimi, University of Leeds). The patient was aged fifty-six, and he had had pain in his left side for four months before admission to the Infirmary, and noticed a swelling in the hepatic region only during three months. An orange-sized tumour could be felt, which fluctuated in the centre. A diagnosis of hydatid cyst was made, but at the ojieration masses of blood-clot were found in the cavity, and these, on microscopic examination, were found to consist of spindle and round cells. It was not till the patient's death, which occurred soon after, that the real nature of the case could be appreciated. Bile. — In this connection it may be interesting to mention that fiilc has an electroconductivity, ranging from no to 1600 10 ~ ' at 35 C, the conductivity having no relation to the viscosity or to the intensity of the coloration. On the whole, however, the golden-colouretl bik-s have a low conductivity, while the dark brown and dark green biles have a high conductivity. The degree of dissociation varies from 02^ too'5i, which shows that no constant rule can be made from a study of this question. The fact that the osmotic and electrolyte concentration of bile may vary with the time of day shows it to be a fluid with very variable characters. Thus, Engelman.i found a steady rise of osmotic concentration from 0308 at 7 a.m. to 0329 at 2 p.m., after which there was a fall to 0-310 at 6 p.m. and again a low rise to 0-319 at 8 p.m. The conductivity was -0131 at 7 a.m., and rose to -013S at 2 p.m. and then fell to -0133 ai ft p.m. Dilute acetic acid does not precipitate any body like the serosamucin of Umber, and the u-najihthol test, which has been applied, has, however, given a positive reaction, though the gliK osamin test of Ehrlich failed to give any reaction. A body giving the reactions of pseudo-mucin has not been found. Renal Cysts.— A few of those which have been examined have shown varying characters. The chief point of note has been the absence of urea or uric acid, which shows tha*^ these substances cannot be relied on for making a diagnosis of renal origin as opposed to cysts of other organs. Indeed, unless the "cyst" be really a distended pelvis, i.e. a hvdf„nephrosis, and unless there is some functioning kidney substance as well, so as to allow much urea to be present, one cannot distinguish a CHARACTERS POSSESSED BY VARIOUS PUNCTURE-FLUIDS IQI renal cyst. In a cyst,* which proved to come from ;i hyper- nephroma, there was found a considerable quantity of albumen, which was probably due to the presence of much blood ; there was an excessive amount of cholesterin, which had app)eared in the urine. The content in chlorides was very small, but there were copious o.xalate crystals, and also cyst in. Both these substances were sufficient to prove the probability of the cyst being connected with the kidney. There was neither urea nor uric acid. There were no cellular elements to form a guide. Another s}iecimen was diagnosed as frc m a cystic kidney f on these characters : sjiecific gravity, loii ; albumen, i to 4 per cent. ; urea, trace ; chlorides, 475 {>er cent. ; cone, elect., 0168 ; cone, achlor., 087. In a case of hydronephrosis ojiened by the surgeon the fluid contained i j)er cent. urea. Reference may be made to a paranephric cyst which was due to rupture of the {x-lvis of the kidney, recorded by Hildebrand. It is impossible to pass in review the chief }X)ints which will enable a fluid to be distinguished as urine. But it is needful to mention that there is occasionally (apart from the cases described above) a necessity for this. Thus, a fluid was drawn from a cystic swelling J in the posterior vaginal wall, and it was only from examination of this fluid that its origin from a dilated ureter that had become malplaced was possible. There was an abundant effervescence, with sodium hyix)bromite, the reaction was acid, the chlorides amounted to 3-9 per cent, and the conductivity at zy C. corresponded to a concentration of electrolytes of 0158. The acid reaction of such a fluid makes the diagnosis practically undoubted. In cases of hydronephrosis the fluid will vary in comjwsition according to the degree of functional activity of the kidney relics. The siiecific gravity is generally less than loio, the amount of albumen may vary from mere presence to 8 jKjr cent., and the same is true of the salts. The presence of creatinin and of urea or uric acid would not necessarily exclude, say, hydatid cyst in the abdomen. § » No. 4484. t No. tV'Ss. C: Record No. 6047. § Cl. No>-. 5S69 and 6987. 192 STLDIF-S IN 1>LX( TUKE-KLLIDS M it In other words, there are none of the characteristic hocUos present which would lead one to identify the fluid as renal. On the otiier hand, it there is some outlet to the flow of urine, and it the kidney is excreting tolerai)ly well, in spite of its functional power heing much diminished, there will Ix- some urea and other urinary constituents present. The ciimnint of urea, however, is of more importance than its mere presence, Ix-cause urea may he found in many other fluids ; and to base a diagnosis of hydronephrosis fluid or urine on the presence of urea might leatl to fallacy. However, the quantity of urea will Mttle the (|ui>tion. if abundant.' A large quantity of urea will not occur excei)t in urine, although it must be rememl)eretl that " no or very littlf urea " does not mean " no renal origin " lor the fluid. A case of congenital cystic kidney which is of interest was described by Freund, at the Medical Congress in Halle (December ii, Kjo?). The case occurred during labour, and It was a question as to wliether the source of obstruction were ascites, distended bladder, or kidney. At a time such as this, the elaborate details which are referred to throughout these pages are ob\iously useless. The author of this case considers the tumour to have been a multilocular cyst of congenital origin, but gives no details as to the nature of the cyst-contents. Spleen Cysts, which are very rare, are described by Las- cialfnra is including (i) hydatid, 2 cases, (2) serous, (3) sero- haniorrhagic — one case of traumatic origin, but he gives no details of the characters of their contents.* Cysts connected with the Lymphatics. — Toyosumi describes a cystic lymphangio-endothelioma papilliferum which was situate in the abdominal wall, but does not supi>ly any information as to the chemical or other characters of the contents. Dencks describes a lymphangioma of the neck, which con- tained a whitish, milky fluid, with a yellowish tinge like lymph. He states that other cysts of similar nature may contain a brown- ish fluid owing to h;eniorrhage mtc them. He gives no other det-iils about these fluids. Albrecht refers to a case of lyniphangif^ctasis, in which numer- ♦ Stf ctIso Zii-glv.nlhur F.. Cici' niHUiplc urosc Zystcn dcr Mil;. " Inaii^. Dis>."' Miinchcn, 1907. CHARACTERS POSSESSED BY VARIOUS PUNCTURE- FLUIDS 193 ous little cysts ap|x;arcd in the skin. They containetl a milky- white fluid, which contained some blooer cent.), and a low osmotic concentration (0104). The salts include chiefly carbonates. Whether sulphocyanide is present in these fluids or no does not seem to have been investigated. The chief feature will be the presence rl diastatic ferment. C]rsts of Bone.— -Rumjjel refers to cysts of bone, which are usually due to the breaking down of tumours, such as sarcomas. In one case described by him he comes to the conclusion that the cyst, which contained clear fluid, was formed from a softened enchondronui. The cellular elements are the chief characters on which he lays stress, though the interest to him is mainly surgical. Such cysts are not, as a rule, likely to come under the notice of the clinical pathologist. But it would be of great interest to have an analysis of such a fluid, since it would afford excellent ideas as to the variety of decomposition products of new-growth protcid, which is different from normal tissue-proteid. A large cyst was found in the cerebrum in a case by Fahr of Hamburg (December 3, 1907). The walls v»re very delicate, the size was that of the left hemisphere. There was no evidence of hydatid. No mention is made of the contents, the sole interest taken in the case apparently having been its clinical • Unfortunately the only specimen which I had the opportunity of examining, cannut now be tract-ii, and the lecoril ol the result Ol exanuaa- tion cannot be found. I have not nad the opportunity of examining the fluid from a ranula. 13 >94 STUDIES IN I'UNCTURE-FLUIDS symptoms. One would have preferred evidence as to the causalion. Cysts in connection with the Cerebellum.— Cysts in this locality can hardlv tver come under the notice of the clinical pathologist, but for tlu' sake of completeness they may In* referred to. Henschen states that the following varieties may occur ; dermoid cysts, simple serous cysts (which have an eiK-ndymal lining and are related to the fourth ventricle), cystic tumours, luemorrhagic cysts (due to h;emorrhage and softening), parasitic cysts, and serous cysts, which are due to developmental anomalies. Spermatocele. This fluid is colourless, thin, and watery. The siwcitic gravity varies lx?tween loof) and loio. Solids, 1-3 j)er cent. It is not sjxjntaneously coagulable. Apart from the presence of sjxjrmatozoa, which is the diagnostic feature, a re- action was devised by Florence for distinguishing sjx'rmatocele from similar fluids. A solution of iodine (1O5) and jwtassium iodide (254) in water (30 cc), added to prostatic secretion, results in dark brown rhombic crystals (plates, fine needles, and rosettes) soluble in e.xcess of water, ether, alcohol, acids, and alkalies. Sjwrmatocele fluid does not give the reaction. Parasitic Cysts. — Hydatid cysts alone call for considera- tion, though their characters are so well known that nothing new can be added. The sjiecitic gravity is low (generally not more than 1015) ; proteids are usually absent, while salts are abundant.* Sugar is occasionally present, and traces of urea may be exjiected, as well as of creatin. Succinic acid \ is characteristically present. :J: Microscopic examination of the dejwsit is, of course, the most conclusive evidence. • In case No. 6897, ^> P'^'' mill»^ of chlorides were found. t To detect succinic acid in hydatid cysts (Salkowski). In Table A. No. 6, the concentrated alkaline fluid is treated with alcohol, and alkaline salts of succinic .icid will separate out. These are dis- solved in water, the solution filtered and squeezetl. They are obtained pure by adding equal parts of alcohol and ether with HCl. Characters. — Four-sided needles, melting at 182" C, soluble in water and alcohol, very soluble in ether. Heated in .. glass tube, it will sublime. Heated on platinum foil, it gives oft irritating vapours. Neutral lead acetate gives a heavy crystalline precipitate ot lead succinate } ANo present in hydrocele fluid (see p. 1O4). CHARACTERS POSSESSED HY VARIOUS I'UNCTURE-FLUIDS I95 Mourson and Schlagdenhauffcn found a iwisonous ptomaine in the fluid of a hydatid cj-st. If in a sus|>ccted hydatid cyst one finds an alkaline reaction, no urea or sugar, and abundant chlorides, this will not decide Ix-tween hydatid and hydronephrosis, unless hooklets are also found. So that the only really relial le sign is the presence of hooklets. It must lie rememlxTed. however, that absence of hooklets does not, i(>so facto, exclude hydatid. ti SECTION IV THE DIFFERENTIAL DIAGNOSIS OF EXUDATES FROM TRANSUDATE'" CoNTF.NTs. Inductions to l)c inailr from : (./) spfcificRravity. (fc) amount ot total protiid, (V) rtfrattonutry. ((/) viscoMty. ie) prost'ncc of sorosa- imicin, (/) Hivalta's t»st, (f) prtv net- of fructost-, i-fc. (A) prcsonce of firimnts, (i) ttfoct of oral ailmini^tration ot rtant place in clinical pathology, and opinions as to their value have varied from time to time according to failures in diagnosis or to improved methods which prevented recurrence of such failures. As has been insisted in foriner pages, it is not possible to experience uninterrupted successes in diagnosis by these means any more ♦'..ir ! "^ car. he. ex{K'cted in any other branch of medical science. The evidence furnished by the methods to be detailed must always be made subservient to either physical signs or to the medical history of the case concerned. The various methods which are to be advocated are not equally well known, ami are deserving of wider iLse, anil from the experience in all but the biological test (reaction with inunune serum) that has been obtained from cases in the Leeds Cieneral Infirmary one is justified in collecting the methods and offering criticisms. The idea of using the electrolyte-determinations has been the result of the discovery that exudates differ essentially from transudates (us a rule) in their achloride-electrolyte-content. The following tests will be considered seriatim : Specific gravity ; amount of total proteid ; jiresence of scrosamucin, and Rivalta's test ; certain special tests (for fer- ments, for acetone, the reaction in tubercular pus, the reaction 196 EXUDATE^ AND TRANSUPATES 197 witli immiim' strum, the fftert of administration of tUx^s) , tlu' n-lation of chloritk-s to a» hloridfs. A practK.il iiDt.' oil tlw coll ■■ tioii of patholoKicil AuuIh may l>c us.ful. Tlxflm.l >tioul.llHtoll.-ct.-.' intoa |Mrl..lly tl.-an Loltlr. strnli^.l li mc.ssarv l.v tliorouKh luatiiiK in an ..vin. Tli.' in-.truriiintH usi-.l lor taiipiiiK imi-.t liavr Inrn Ik)iI<o s.nt to thf liactinoloRist m a small piict of n^Ass yuUmv, whi( h has b.in drawn out at lath end and sealed up aftiT hllinK- ((«) Specific Gravity. -Tlie most wi-U-known characteristic of transudates is that their spot itic gravity is usually nttuh lower than tfiat of exudates. Tims, transudates usually have a specific gravity of less than 1020, while exudatt>s have one of more than 1020. This is shown in the table on the following page. Reference to the cases will show thnt both pleural and jhti- toneal fluids of transudatory origin have a low s|)ecific gravity. The significance of these variations may be attributed mainly to the amount of albumen present in the tiuid. The more albumen the higher will be the sjwcific gravity , and albumen is more abunilant in exudates than in transudates. The relation between amount of albumen and specific gravity ha>; been the subject of a very large amount of study, especially by Reuss and by Bernheim. According to Reuss, the specific gravity may be taken as a rough index of the amount of proteid,* and he devised the follow- ing elaborate formula, which should enable one to estimate the amount of albumen from the specific gravity : Percentage of albumen =^ i (S-iooo)-2-8, S being the S{)ecific gravity. • Reuss made out that specific gravity ot 1018 means more than 4 P'-'^ cent, of proteid. 1015 .. less ,, i'5 loiz .. .. .. I-5-- ■■ loio ,, ,, .- to-1'5" " 1008-8 „ .. .. 0-5-10,, A series of relations which has far more practical utility, even if not strictly accurate, than any elaborate and necessarily artificial formula. 198 STUDIES IN PUNCTURE-FLUIDS 14 ■A o .2 3 3 M rt — 1/1 i,.S ■H •ox H a, V o n •a -5 « 5^ .2 a ■5 La -a s •J a, a 3 " C M M« U *J , = 2 u u O u 3- -J c u -o > rt C3 U r *n u o r - H ■- « 3 V u EXUDATES AND TRANSUDATES 199 This comparativelv simple relation ♦ was controverted, as might be expected, by Bernheim, who had, by the aid of a mathe- matician and very numerous observations of albumen versus specific gravity, been able to issue the formula : Percentage of albumen = 4-944<>i^ + i-^o4<7S'- ^315 ^or exudates, or 27 y 116S - 1 :.i2^(yS^ - 275216 for transudates, a relation which is obv-'i.lv so artiftnal that though the formula meets the observatioi ^ of Bernheim. one cannot exi^ect it to fit in with every cast h;'*. will ever occur. Bernheim came to u ioUiily difterent conclusion, namely, that the specific gravity is no definite index to the nature of a fluid. The presence of several exudates in the column " below 1020 " iA the table will illustrate the truth of Bernheim's contention. At the same time, it m-ist be admitted that in a fair proportion of cases the si)ecific gravity does help in forming an opinion. As a matter of fact, the specific gravity depends on other bodies besides albumen, and the two cannot be brought into relation with each other, even if albumen play the most important part in the question. Thus, the total nitrogen and the residual nitrogen vary with the specific gravity, and so also do the ammonia and the purin nitrogen. The parallelism between specific gravity and the amido-nitrogen and the nitrogen of the uric acid prevents these substances from helping in the diagnosis, only that altera- tion in circulatory conditions such as occurs in transudates from cardiac disease increases the amount of urea and amidoacids.t (6) Amount of Total Proteid.-This has long been re- garded and rightly so, as a safe index to the nature of an effusion into either abdominal or pleural cavity. Here, as everywhere. it is the borderline cases that present difficulty. The explanation of the exceptions is suggested by Stahelin, who found that the amount of albumen varies with the nature of the disease causing the efiusion with the state of nutrition of the individual, as well as other factors which need to be taken into consideration before making any conclusion. • Compare Chnsten's formula for giving the percentage of albumen (a) from the weight of a litre of tluid in grams at 15° C. (p). (a)=4 (P- 1006-8) This formula he asserted to be correct within "47 gm- P^r litre (-04 _ per cent.), t Otori. i^l 200 STUDIES IN PUNCTURE-FLUIDS A most important scries of observations have been made during recent years on the albumen-content of peritoneal effusions by Engliindcr of Vienna. The albumen was estimated by weighing, and not by Esbach's method. This author, whose results were only published in full last year, found that in hydre- mia the amount of albumen varies between 03 and 05 per cent., while in cases of portal stasis it varies between i and 1-5, rising to 3 per cent, in old exudates, or falling to 04 per cent, in cachexia. In the last group of cases, those due to portal stasis, the albumen was not more than 2 per cent, in 50 cases. On the other hand, cases of carcinoma show a high albumen percentage (up to 7 per cent.), and in chronic exudative and tuberculous peritonitis the minimum is 3 per cent. The chief service afforded by this author is his insistence that the amount of albumen must be considered in relation to clinical facts. Thus, in a case of cirrhosis of the liver, if the albumen in the peritoneal fluid is 2, or less than 2 per cent., there is no peritonitis, while if there is much more than 2 per cent, an explanation must be found; and if the general nutrition is not good, or if the ascites has not existed for some time, or if the abdominal walls are not tense, then some inflammatory condition in the peritoneal cavity must be looked for (either carcinomatous, serositic, or syphilitic). Whereas if the albumen-content be much more than 3 per cent., and car- cinoma can be excluded, one may be sure that there is tuberculous peritonitis superadiled to the liver disease. The possibility of excluding tubercle by finding the per- centage of albumen to be less than 3 is an exceedingly useful point. Such conclusions were expressed by Runeberg, though not to the same extent of completeness, when in iSq; he stated that 4 to 6 per cent, of proteid signified an inflammatory exudate (tubercle or carcinoma), i to 3 per cent, signified a transudate from passive congestion, and 01 to 03 (maximum) meant a purely hydremic transudate. The more chronic the condition, the less proteid will there be in the fluid. By bearing such considerations as these in mind, it becomes possible to diagnose the appearance of dissemination of carcinoma over the peritoneum in a case where the primary growth has merely at first caused portal stasis. And further, these considera- tions will serve to emphasise the fact that it is not necessarily by EXUDATES AND TRANSUDATES 20 1 a single exploratory puncture that we shall be able accurately to decide on a diagnosis, but rather is it by seeing what changes occur in the fluid after -n interval of time that we come to be able to form a reliable opinion, if example were necessary one would dates Assoc, with Care. 1 Ovarii, 51 , Kcnal Empyema, 8 Thrombosis Chronic Cardiac, 5-4 i 1 Cardiac of Portal Nephritis Alcoholic ! 1 Ale. Ci-rho- Vein (2 cases) Cirrhosis / Transu. ' sisol'l.iver Ale. Cirrho- Portal t)b- of Liver, 8 Toxic Nc- sis of Liver; struction 1 ''"'^^ - 1 phritis (2 cases) (Runcberg) Syphililic Syph. Cir- | Cirrhosis rhosis of 1 Perito- neal (Hallibur- Liver / ton) (Po1jakoff)| Carcinoma- Carcinoma- Carcinoma- Fuberc. Simple 1 tosis tosis tosis Peritonitis Chronic Peri- (2 cases) i (2 cases) (RunebergI tonitis, 775 I dates Tuberculous^ Polyorrho- Peritonitis menitis 1 Chylous ascites Carcino- matous Pen tonitis, 10 Tubercular, V 1 (McHcy) 67s QCdema Cardiac cases Fluid Pericar- Tuberculous dial 1 Cysts 1 '' Ovarian Pane r t Cyst Cyst in the course of an effusion excited by bacterial agency, they will enter into combination with the globulin, and so affect the proteid quotient.* These changes have been noted chiefly in regard to the composition of the blood, but inasmuch as the proteid quo- tient is constant both for blood and any effusion that occurs in the given individual, tlie same conditions will hold in the case of * Glassner. EXUDATES AND TRANSUDATES 203 tin- effusion. But when we reflect that the urine of cases of nejih- ritis may show a vaiying proteid quotient * from day to day, the value of such a quotient for diagnosis in effusions comes to require substantiation. If the urinary composition varies in this way. a renal ascites may be expected to show similar daily changes. The relation, too, between proteid and molecular concentration cannot be regarded as having any prospects of being of practical importance. The total proteid may also be compared wit'^ the extractive nitrogen, and these two together may be compared with the protcid-extractivc ratio of blood-scrum. Rzentkowski utilised these ratios in order to decide whether a fluid were a transudate or the result of endothelial jiroliferative processes. If the ratio were much less than that of the serum he would argue against a transudatory origin. He states that while they are accumulating the fluid mainly consists of salts, while when the serous membrane becomes active the proteid element enters into the effusion (an autochthonous process). In an exudate we have the following relations : the dry matter is less than that of the blood-serum, the total nitrogen is also less, but the extractives amount to still less than those of the blood-serum. Be these statements correct or no, they cannot be of much assistance in the differential diagnosis, merely from the fact that they involve tedious methods of research which cannot conveni- ently be employed for diagnostic purposes. (c) Refractometry.— The refractometric observations which have been made by Engl and advocated strongly by him as a moans of distinguishing between the two main classes of efiusion are really nothing more than a refined method of albumen- estimations, since the refractive index of a fluid depends very largely upon the amount of albumen which the effusion contains. It will not be necessary to enter into any details of the methods of refractometry, simply because a suitable instrument is very expensive, and cannot reasonably be added to the instrumenta- rium of the clinical pathologist. Still, it cannot be doubted that the method— given the instrument— is a very handy one, but the results are not so uniform that general use of the method is advisable. In the following table, which is adapted from a ♦ V. Noorden, Noel-Paton, Patella, Moram. 204 STUDIKS IN PUNCTURE-FLUins long series of observations by Engl, the numbers represent the refractive coefficients of the tUiids. (Note the thickened figures.) Refractive Coffeicients of Various Fluids (Engl) ii rieura. Abdomen. Peri- cardium. Nephritic TranBiidales Subacute Tubal '3379 •3571 '•3.37« Chronic Tubal Interstitial '■3372 I-3S72 '3397 Average : i •3375 13374 I-339S Cachtctic i I'ernicious Ana-mia •• ■•337S «33^ 1-3408 I'ransudates 1 , Tubcrc. Abscess of Chesi . '337i^ Avtrace : '3365 '•3374 i •3385 13410 '3382 ••3398 I'assive Cardiac I 3405 1-3401 Cirrhosis of Liver ... ' ^s^s _.„ Congestion - Transutlatcs Empliysema Aortic Valve Disease Average : 1-3378 I 3401 '•3390 '3392 '•3398 13*05 Acute Pleurisy '■3436 — 1 Tuberculous Pleurisy '•3474 — — ; Purulent Pleurisy ... 13480 — — Exudates j Tuberc. Peritonitis... Purulent Peritonitis 13443 '•3479 — 1 Carcinomatous Peritonitis. — 13430 — ; Carcinoma Ovarii ... — • •3442 — I Hemorrhagic Pericarditis. Average : Average : — 1-3446 "•3445 '•3*59 fl Chronic Hydrocephalus •• "•334710 — Vaiious ...- 1 1 Cerebrospinal Fluid 1 Hydrocele ... '•3350 ••335° 1 ••3429 — 1 I '335' — i CEdema Fluid Cachectic Nephritis - '3362 I ''3359 ^~" i The advantages of the method are that the refractive coeffi- cient, which is proportional to the albumen-content, (i) is not affected by suspended particles, (2) necessitates the least quantity of fluid, and (3) is much less tedious than a determination of the dry residue accoraing to Runeberg or a Kjeldahl estimation. Besides this, a comparison of the refrartive coefficient of the effusion can be readily made with that of the blood. The only sources of error are that certain substances other than albumen EXUDATES AND TRANSUDATES 205 influence the rocffic icnt and that different forms of proteid cause variations. While i i)er cent, albumen has a coetliicicnt of 0018, I per cent, of globulin is 0020- 0023 (according as it is globulin, pseudo-globulin, or crystallin). ((/) Viscosity. — The investigations which one has made as to the viscosity of the different fluids that have come under observation have shown very strikingly how much lower the value (as compared with water) is in the case of transudates than it is with exudates. Inasmuch ius the viscosity will be influenced by the amount ot albumen present, a natural explanation for the fact is found. Probably there are other factors to be considered, but the full treatment of the subject will be found in Section II., last sub-section. The clinician can hardly be advised to purchase a viscosimeter in order to assist him in such a diagnostic problem as is under consideration, so that more detailed reference to the values found is out of place in this section. {e) Presence of Serosamucin.— The chemistry of this sub- stance, and of the bodies to which it is allied, has already been fully entered into. It is merely necessary to repeat that the presence of serosamucin in a peritoneal fluid is evidence in favour of its being an exudation,* that is, a fluid associated with inflam- matory processes in the peritoneum, t This fact is the basis of — (/) Rivalta's Test, which consists in adding two drops of glacial acetic acid to 100 cc. of distilled water, and then one drop of the fluid to be tested. The result, if the reaction is " jxjsitive," is that a white cloud appears in the trail of the descending drop, the trail being of varying degrees of whiteness, according to the severity of the inflammatory process. Janowski tested this in a number of cases and obtained uniform results. The results obtained in the Leeds Infirmary series have also given precisely concordant results, and it is hardly necessary to tabulate the findings under such uniform circumstances. Only one point would one emphasise, and that is that unless there be a decided white cloud, which increases as the drop descends, the result must not be regarded as definitely positive. Often a faint turbidity • Rivalta. t Stalulin reports fourteen cases in which he examined the fluid for serosamucin and found its presence in those fluids which were of an in- flammatory nature. Eleven cases were pleural fluids ; the others were from' the abdomen. 206 STUDIES IN rUNcTURE-ILUIDS 1 . i ■iri' will l)c produced with trans iidatos, hut it will he found to disappear as the TKANSUDATKS 209 while no reaction oicurred in the t ase of an exudate, presumably owinjitothelatteriontaininK the necessary eonipieiuent, hei ause ol the (ells in the exudate. However, if thickening,' of the serous membrane (such as the pleura) occur, a transudate may fail to ^i\v the reaction. Again, if the exudation be examined in an early stage the reaction may come off owing to the anti-bodies not having had tune to apj>ear. Whereas the reliability of the reaction is likely to be called mto question, it must be admitted that the practical difficulties in the test, involving as they do a vivisection licence, will prevent its use. The distinction betw en an exudate and a transudate is made a very sharp one by such a reaction as this, and it cannot be emphasised too much that the demarcation line where exudate ends and transudate begins does not exist. In well-marked cases the distinction is easy, but there are and always will be many cases which are not strictly either the one or the other. The fact that lecithin plays a part in immunity reactions, and the fact that lecithin is associated with pseudo-globulin or with euglobulin, would throw some light on the observations of Tedeschi, and only brings us back to the original fact that the amount of albumen (including globulin) may form an index as to the variety of the fluid being studied. ik) The Chloride versus the Achloride Electrolytes.— In the course of studies on the electro-conductivity of the various fluids which have been examined, it was first noticed that the chloride-content of these fluids varied very greatly, according to the causation of the effusion. As one would exiH-ct, the renal drojisies contain a relatively large percentageof chlorides, while the exudates due to severe inflammatory change contain but few. This observation led one to endeavour to ascertain whether the other inorganic constituents of the effusion under- went any change. The comparison of the actual conductivity of the fluid with the conductivity which the fluid would have if only chlorides were present to the amount found by quanti- tative analysis formed the basis of the method adopted for in- vestigating this point. The principles involved in this type of " chemical analysis," and the difficulties in the way of a just interpretation, with indications as to how the errors can largely be obviated, have already been dealt with fully (Section I., under the problem of adsorption of chlorine by egg-albumen ; Section 14 210 STUDIES IN I'UN< Tl'kE-H.UIDS II., siil)-M-< tioti 2). It tliercfore lioromes only necessary tc. ^ive an account ol tli.' loult^ ol>t.iine compared with t»,at of ai hlonde electrolyte>, it will W advantageous to call attention (or the moment to the variations in chloride-content alone which obtain m different classes of fluids. Keleienie to Table IX. will show the strikinK fact that in tran-iidates the loncentration of chloritles expressed as grain-equivalent (grain-molecule jht litre) is always, or nearly always, up to o I. while in thuds associated with varying degrees of intlammatorv change, or with disseminateil malignant disease, the figure harelv reaches oo;. In many cases the tUu.l contained in ovarian cysts falls to a still lower chloride-con- centration, as will 1h> >een on reference to Table XIX. 'Ihe suIm iitaneous fluids resulting liom cardiac failure or from renal ina.-n arnvi-.l at by corrcctinK the ron- (liKlivity fimnil Uith fill lfm|HT.itui. (.ill ' :nl at i.H C ) and Km tlu' iHKtiitaK.- ot all.mn.ii/' Hk- roir«-«iHml Na( 1 ol thf saiiif strtn^'th as the fluid is ii\svn in tin- m-xt loluniii, and llif ditftri'mf iMlwron the two f;ives that iMirtion of tli«- total (ondiii tivity whiih is to In; asiiihed to the arhlorides Keganlinn these as Na,C<), we can express the niimlx-rof moU'ules, />//(> ions, iht litre of achlorides in terms of Na.COj The last loitiiiin hut one ^ives the total concentration ot electrolytes, and the la>t column expresses the ratio of achlorides to chlorides. ("omparmti the diffeient classes of fluid, the strikiuK feature will he found to he that m the case of exudations the ratio named is nearly always f^-nater than unity, and, inchrd. is freical exudate and a lyfical transudate. The most conspicuous differ- ence will In? found in tin- albumen-content. The presence of so large an amount of all)umen might interfere with the estimation of the chlorides by adsorption or other circumstances, or the fact that nearly all the tluitl ilisapi^-ars when so highly albu- minous a fluid is boiled might lead one to susjK'Ct an intrinsic error in the estimation. Hoth of these explanations can lie excl'ided, however, the former on the grounds detailed on j)age 18, and the latter on the ground that there is an undoubted difference in the chloride-content in exudates. Another consideration is as io the ^permeability of the in- flamed serous membrane for the different ions. The difference cannot have any relation to the relative tonicity of blood-serum and fluid, since we have to deal with a difference in the content of various salts. On the other hand, just as with questions of absorption of lymph from a serous cavity there are three factors • Th-- notattnr, ^,.^^i been fmnloved uniformlv throueh this work. t The formulx- available for this correction will be found on pp. 100 and 107. 212 STIDIES IN I'UNCTURE-FLUIDS n M ^ ^ O"* ON N — N r< 1- o -t - o - r^ r^. •f **■ i« — — MNOb — b M f^ f* N N N e t^ O - ao o fo ON— o>^^N^^OP*^ 2C~30 30 **■ w^oo r^oo NOT ©N r^ 1^ C^ ^^ ^ ^^ ^^ ^^ ^^ i^O "^ O Q i^O30-rN-t"1OvO NN* I li-l _ N X - - HO r~ O !>• O — >0 N irisO i^^O O •; *f» O ir> O T « t-^-X N O^ O — o -o-ooo--r> l-»N so O I'NCiB t^ N too *0 t^ t^ t^ •; 00 5>M « 90 CT-ao 00 op op CO op 00 O ^ N r^vo M 00 1^ r" ~" ~ ■" r~ "t-OO O O Tf ^O On oo«0'i-'n>« cooooo-op r^oO « ■>t r~ "I- O O O — ? "o 5 B u 6 c o ^o ^o oc sc vO =Z"J 00 00 IBJn.nH 'ie9iio)!J3j •saivanxa EXUDATES AND TRANSUDATES 213 < a in X < K H I > 'Ox "^ 6 b b b 6 b b 5- b b S5 b b j N ■- f* N « N 1 30 - 00 xrt in O vO xrt xrt •t f^ o K d CQ < M 5 t :^ o o I U >,^1' IflS 00- O 3 . u . O tl Zo E CT^ u^ »* — N", - rr * fO 90 »^9C ON — "^1^ " %o o O O « f^ N fn Wi 90 » t^ ro to i#i 00 c^ O^ 0% r^ 90 r^ §; i m O •« "^90 >0 "^ "1 fJ '^ ON — 90 O f^ >- - - M N - t^ Q O O »0 .£ MO 8" X u ■« 5.S • t ; 1- - 90 V r^ 00 oo 90 apoo N ^ 1- CO QO 1 0>o - N 8^ - - r^ 1 .,„ as. o .J ^ o ^^ jj o .2 '" ^ '^ ^rt ^Sc^^Q •=-.= ■=5' S .^ > S--^ i> 3 le t > t > > c «.- — •- — c O I — n fj Tf "1 ") S^ '^ >O90vOi)9000 >« "1 IBjnaid •(Bjuojiiaj sHiva.isxVrfX nm- 214 STUDIES IN PUNCTUKE-FLUIDS to consider-tho endothelial layer, the capillaries, and the con- nective tissue ( anals-so we shall have to consider the influence which each of these factors can have in allowing certain salts to pass through and not others. But more than this, we have to consider the effects of inflammation of the serous membrane, whereby not only arc the jx-rivascular lymph-spaces filled with fluid of abnormal composition and the endothelial cells under- going proliferation, but we have to remember that the endothelial . cells are being cast off into the fluid, and that angiogenic cells as well as histogenic cells are making their way into the fluid in question. In the case of carcinomatosis of the peritoneum, too, there is also the discharge into the fluid of the carcinoma cells with the products of their disintegration or the products of metabolism of those carcinomatous cells which have not been shed. According to experiments made by Hamburger, artificially induced inflammation or injury short of inflammation of a serous membrane does not cause any change in the phenomena of absorption from that cavity, so that we may presume that the problem to be solved is the same whether we are dealing with the ordinary three factors above referred to or with the same factors in a state of disease. Such experiments, however, as Hamburger jxjrformed (injection of strong salt solutions after chemical and thermal injury to the peritoneum) do not affect the probability that the cellular elements which are discharged into the peritoneal cavity when the membrane is inflamed will cause a change in its composition. Hamburger in a sories of experiments on the influence of pres-sure on absorption from the connective-tissues, came to the conchision that increased pressure does not allow proportionately as much NaCl to pass out from the blooe borne in mintl. Incases where a coagulum has formed before the fluid comes to hand for examination, it may suffice to take a small snipping of the coagulum and examine it fresh (no diluting fluid to lie used). If the result is not satisfactory, some of the coagulum may be stirred about in a small quantity of fluid placed in a watch-glass, using a platinum wire as stirrer. The fluid may then be centri- fugaliscil. The risk is. of course, that the dei)osit will not then exactly represent the characters of the deposit, owing to uneven separation of the cells. The unstained snipping may then be used as a contP'l. Preparation of the Film.— For fresh si^ecimens a drop is simply covered with a cover and cxamineil with the Jth. For stained preparations a thin film is spread on a slide,* preferably albumenised, and allowed to dry on a shelf arranged on the paraffin oven. A saturated solution of Jenner's stain (Grubler, e.g.) in anhydrous acetone-free methyl alcohol (Merck) is prepared in a well-fitting stoppered bottle, and a couple of drops are allowed to fall on the film. A Petri dish is convenient as a cover for the ordinary 3-inch slide. After two minutes t the slide is gently washed in distilled water till the pink colour apj^ears, and dried by the application of "' Huffless " blotting-pajier, followed by a momentary warmth in the hollow of the hand over a Bunsen flame. The preparation is now ready for examination with an oil-immersion lens. The staining effects are illustrated on accompanying plates. Though special works on blood-cell technique will mention the precautions needed in the use of Jenner's stain, besides explaining the chemistry of the stain, it may be mentioned (i) that the stopi^er of the bottle must be " turned off " when not in use ; (2) that no time must be lost in applying the Petri dish after pouring on the stain ; (3) that the time of staining must not be ill * Slides are much preferable to cover-glasses (or cytological and bac- tirioloKical work of all kinds. The whole slide is covered with dejiosit, ■save a strip at one end. which is left free for the fingers. One slide will then ha\ e as much on as many coverslips. and a sliding stage enables the work to be done with much less labour than is involved in using several cover- j-lips. t A two-minute sand time-glass is most handy. CYTODIAGNOSIS 221 exceeded ; (4) that the washing must be carefully watched till the colour just apjx^ars ; (5) that no time must be lost in drying. Another method, recommended by Jagri, is to add 2 ix;r cent, formalin to the fluid whose cells are to be examined, centrifuge ofi the deposit after a few hours, and then stain with a freshly jirepared Giemsa solution in glycerin anil methyl alcohol. The cells should be examined while in the stain. The Results afforded by Cytodiagnosis.— The cells of a puncture-fluid may be either enumerated in a counting-chamlier or may l)e subjected to a " differential count." Sometimes it is advisable to jierform both forms of counting. The second count is, however, much more generally useful, especially as by its means we can watch the progressive changes in the cellular constitution of an effusion— changes which occasionally afford valuable prognostic indications. The insight into diagnosis is indicated by the following considerations: If inflammation suj^ervenes during the course of a passive accumulation of fluid, the cytological picture will become completely changed, as will be shown later ; or if recovery is taking place from an inflam- matory effusion another change will be found to take place in the relative proportion of the cells ; or, yet again, if inflamma- tion arises in the neighbourhood of a serous membrane into which there is already an effusion from jmssive causes, the cellular elements will be altered, even though the inflammation has not affected the serous membrane itself. It will be necessary to refer to these points again, but to avoid repetition it will be more satisfactory to discuss each class of cell that may occur in a puncture-fluid, and its relations to disease, than to discuss the diseases, with reference to their cytological characters, or, as it is termed, their " cytological formula." A series of researches made by Heinz in 1900 throw much light on the occurrence of different cytological formulas, although no particular notice seems to have been hitherto taken of his work. This observer studied the effects of artificially induced in- flammation of the peritoneum in rabbits (injections of emulsions of turpentine and of iodine). The result was an accumulation of leucocytes beneath the endothelium at the spots at which the irritation came in contact. Little raised masses consisting of fibrin filaments (due to coagulation of outpoured serum) in- 222 STUniKS IN PUN(TURE-FLUII)S filtrated by thise leucocytes, aj^iieared. and subsequently fibro- blasts replaced the coagulated material, which was still covered by an intact serosa. As the inrtammation proceeded, however, the endothelial cells l)egan to proliferate and assume a more cubical shaiH', their cell-body Ix-came more granular, and their nucleus larger and richer in chromatm, with development ot mitotic figures. Delicate strands of cells and membranous structures made tluir apiH'arance still later, until the tag-like processes of a cor vtllosim were produced, and these tags were also covered by several thicknesses of cubical epithelial cells showing numerous mitotic figures. These young embryonic cells formed part of a new mucoid tissue which was vascularised from the serosa vessels. lie liberation of such cells, as well as a liberation of i)olvnuclear cells, would give a distmctive character to the e.Kudate, varying according to the stage at which the inflammatorv change had reached. The exact course which the inflammatory processes is going to take, the intensity of the inflammation, ami the degree of l)roliferation of the endothelium of the serous membrane will all influence the cytological formula, and the significance of the findings in a given exudate or transudate will dejiend on a due consideration of all these factors. lit THE CELLS WHICH OCCUR IN EXUDATES AND TRANSUDATES I. Lymphocytes.— The preponderance of lymphocytes in an effusion under certain conditions, especially tuberculous conditions, was the first fact about cytodiagnosis to which attention was directed. If we inquire into the possible source from which the lympho- cytes of an effusion are derived, we shall find that they may be classified into angiogenic, endotheliogenic and histiogenic lymphocytes, though it is not possible to decide between them as they are met in an ordinary preparation. The angiogenic Ivmphocvtes are those which have wandered, by their intrinsic }X)werof wandering. from the blood stream or from the lymphatics. The endotheliogenic Ivmphocvtes include those forms which are reallv i»eudo-lvmphocvtes. the result of degeneration of polynuclear or of endothelial cells. The histiogenic lympho- CYTODIAGNOSIS 223 cytes have come from the lymphatic tissues that enclose the blood-vessels supplying the serous membranes. Signorelli sup- l)oses that lymphocytes may he derived from proliferation at the site of effusion. It may be said that lymphocytes in an effusion are a resjionse to a stimulus of low activity, the reaction on the part of the tissues in the immediate locality of a disease which does not seriously implicate the rest of the organism. They are therefore most frequent in effusions of a chronic nature, and if they do occur in the course of acute disease, it is Iwcause there are repara- tive processes going on in the damaged tissues.* A weak stimulus spread over a long space of time constitutes two factors, which, existing as they do in tulierculous disease, serve to explain the frequency with which lymphocytotic effu- sions occur in tubercle.t Where exceptions to this rule occur, the explanation must be sought in some sujieradded acute condition, or in greater toxicity of the micro-organisms, or in the presence of other micro-organisms as well, unless there lie deficient resistance on the part of the individual. The prejwnderance of lymphocytes is a feature not only of pleural tuberculous effusions + (esjiecially at the end of the second week), but also of tubercle of the peritoneum, jomts. tendon sheaths, and any serous cavity. It may be said that if lymphocytic effusion be of acute onset, it is certainly tuberculous, and that the appearance of lymphocytosis after a lX)lynucleosis indicates a favourable prognosis. When jwly- nucleosis gives place to a lymphocytic phase in the course of secondary tubercle of the serous membranes, there wUl be an absolute increase in the number of cells per cubic millimetre. Lymphocytes are also met with in considerable number in transudates of mechanical origin,§ but in nephritic transudates they number less than 20 per cent.|| According to Signorelli, * Signorelli. t This fact has been verifted by many observers. 3 lo^v ..oc uvv.. ,^ - ^t is only necessary to nLnVi^n WidarRavau'tNvolff. Quincke, Ehrlich, Grawitz, Raubitschek. Koster, Vargas-Suarez, Stassewicz. Jacobsohn. Steinbach, v. Ketley and V Torday. The percentage of lymphocytes varies from 70 to 95- t Attention may be drawn to the fact that the connective tissue of tuberculous lesions is often densely infiltrated with histiogenic lymphocytes. § V. Ketley and v. Torday, Bunting. 11 Stassewciz. 224 STUDIES IN rUN( Tl KE-FI.Uins I Li. IvmphcH vt«•^ in excessive ;um)imt may U' exix-cted m effusions associated witli disease o{ the IvmphatJC system. KkKoKS ok Dl.ViXOSIS OF I.YMi'HDCYTE^ I. /V.«. They include Nos. I and 2 of the alwve list, and also new-formed iinbryonic connective tissue cells (young fibroblasts). Fseudodymphocytes are most frequent in those tuberculous exudates which are associated with lymphocytosis (Koniger). The f«)llowinK are distinguishing features : the size is that of a lymi)hocyte. the nucleus is relatively large, rounil. rich m chromatin, and stains deeply. The cytoplasm is moderately abundant and sometimes contains neutrophile granules. Often they have much resemblance to nucleated red cells. 2. Polynucleosis— The appearance of polynuclear cells in an exudate may be looked on as the expression of a general reaction on the part of the individual against a more severe infection. The more virulent the infection the more decided will the polynucleosis be. It is met with in empyema, pyopneu- mothorax, in the initial stage of tubercle, esjHjcially when starting in another organ . and especially in tuberculous pericardial effusions or when tuberculous disease in the lung is undergoing caseation. As a general rule, the number of these cells diminishes as the case progresses towards recovery, and lymphocytosis takes its place. On the other hand, if {xjlynucleosis takes the place of a lymphocytosis in a pleural effusion in a cardiac case, one should susi^ect the development of an infarct. The state of preservation of these cells occasionally gives a clue to the nature of the case. Ekkors of Diagnosis of Polynicle.\r Cells.— They may be simulated by degeucraie cells of other kinds, owing to fragmentation of the nucleus. Careful focussing will assist in distinguishing them. • Patflla. CYTOl>IA(iNOSIS 22$ Pseudo-lymphocvUs have In-en n-fcrrod to (p. 224). PolynticUar cells may thtinstlves unilergo JegeHeralive changes. Tht-ro is then fragmentation of the nucleus, disapjiear- ance of granules, swelling of the cell-lKKly (" hydroi* "). loss of staining iM)wer of the nucleus, and even bursting of the cells. Such ct, they are derived from proliferation of the endothe- lium of the serous membrane, the proliferated cells l)ecoming shed. They are more likely to apj)ear as a result of rei)eated tapping of a serous cavity, presuming that the effusion is purely of mechani- cal origin. In cases of inflammatory effusion there will f)e no change in the variety of the cellular elements, a fact which is of great imjwrtance in differentiatmg tloubtful cases of tulwrcle, for instance. A large number of endothelial cells in an effusion of traumatic origin may lie looked on as a good prognostic sign, esj^cially if any jwlynuclear cells that may co-exist are in a good state of preservation. It must be Iwrne in mind that some cases of Itiherck show even as much as ()0 })er cent, of endothelial cells in the early stages of an effusion, while they disaptK>ar later on. The presence of endothelial cells in a ^xiritoneal fluid in cases of simple ovarian tumour has been noted by some observers. Errors in Di.\gnosis of Endothelial Cells. 1. Leucocytes.— U endothelial cells, in virtue of their phago- cytic lowers, have taken up leucocytes, they may come to be mistaken for the latter. 2. Carcinoma Cells.— It has often l)een said that one cannot distinguish between these two,* but so definite a negation must • Lately, Sawyer. 1908. X5 226 I r STUniES IN I'UNCTURE-FLUIDS The characters of endothehal cells and of carcinoma be disputed. T cells are shown on Plate i, tigs. 2 ana 4, anc. r.a.c .. "b- -.• — useof colour serving to accentuate the features. We may say hat (.0 the cdothehal cell has usually a very large cell-body with a regular oval nucleus (Plate i, tig. 2). which stains but feebly w,h Tenner While the cell-body may show %-acuolation. it has usua ly a perfectly uniform structure. (I» The cells have all a similar appearance. Contrasting carcinoma cells, we find that here there are hardly two alike, (i) the cytoplasm never stains uniformly, and is (ii) often vacuolate and contains fat granules and cell-inclusions C bird's-eye-l.ke " structures-Erben ; c) the nucleus under favourable circumstances stains deeply, and shows mitotic figures, often of heterotype charac er, besides frequently containing several nucleoli (Plate 2 fig 3) • (d) the adhesion of many cells in a bud-l.ke fashion is frequent. Some of the cells are multinucleate^ (.) It is hardly fair to cite, in exemplification of the ability to diagnose cancer-cells, the appearance of the phenomenon depicted m Plate 2. iig. i. for an opportunity of "s kind mist be rare. It is, however, conclusive^ The fluid in tins particular case was turbid, and the turbidity was found o le due to particles formed of clusters of cells of i>erfectly distinctive character even in the unstained preparation. The drawing is prepared from a paraffin section made from a mass of Te deposit that was dehydrated and embedded. The columnar shape of the cells, which are rather degenerate, and the central stroma, are shown. It is of course not certain that this was from a peritoneal fluid rather than from an ovarian cyst, and, unfortu- nately, neither operation nor post-mortem examination was l^r- mitted. (/) The usual type of carcinomatosis consists of a diffuse growth scattered all over the peritoneum and sheddmg.tsce Is into the fluid that its presence has excited. These cells are often de- generate, and cannot be distinguished from similar y degenerate endothelial cells. A comparison of all the cells of the effusion and a search for some better-preserved types may reveal the nature of the case. The difliculties are of course insuperable where there is much endothelial hyperplasia as well as the car- -momatosis. Solid fragments of tissue would settle the diag- nosis but even in their absence the diagnosis is not as hopeless r ome would have us believe. The drawings referred to have CYTODIAGNOSIS !27 been made from s{iecimens obtained in the Leeds General In- firmary, and are from cases verified post mortem, so that there can be no plea that carcinoma cells were not present in, say, Plate 2, fig. 3. There were enormous numbers of these cells, and there was also pleural carcinomatosis absolutely widespread. 3. Large Mononuclear Ce//s.— These will be considered under the next heading. 4. Large Mononuclear Cells.— These cells are best distin- guished in unstained preparations. Changes in the osmotic conditions of the fluid after tapping tend to alter their characters greatly, so that early examination becomes particularly im- portant. They are depicted in f^g. 4 of Plate i. They i^ssess phagocytic properties, and are apparently derived sometimes from connective tissue cells, sometimes from endothelial cells, and sometimes from the blood-stream, while, according to Marchand, they may come from the perivascular lymphatic tissue (leucocy- toid cells). The phagocytic cells are very large in size, and often have included fragments of degenerate cells. If they are increasing in num " they indicate a favourable prognosis, while if they are absent aom the deposit in an inflammatory effusion, and bacteria appear, their absenre indicates approaching suppura- tion. In early tuberculous effusions they may number i per cent, of the total cells (Bunting). They have been met with in the pleural effusion associated with enteric fever. 5. Eosinophile Cells.— Two classes of eosinophilia may be described— a relative and an absolute eosinophilia. The latter exists when the - ells number from 10 up to 74 per cent, of the total count. The significance of so high a count is unknown, though It has been described as occurring in cases gf acute rheu- matism, in nephritis, and in convalescent cases of tubercle. Burnet regards the phenomenon as indicating diminution of the virulence of the organism, while Bibergeil ascribed it to chemo- tactic influence of broken-down endothelial cells. There is generally eosmophilia of the blood at the same time, though this is not met with in infective cases, with the exception of syphilitic cases, as Widal and Ravaut record. Eosinophilia has been described in several cases of carcinoma, thus, by Erben, by v. Starck, by Kroniger. When it appears in 228 STUDIES IN PUNCTURE-FLUIDS an effusion which developed since a growth was noted in another Tart of the body, U may I. assumed that metastases has taken place. In V Starcks case there were also enormous cells which f *"«;! I^^^^^'^^ rh v'-icuolated and contained peripherally situated nuclei. He were much vacuolated an. J ^^^^^^^^ ^^^^^^^ _^ ^^,^^^ ^,,,^ ;:;:atertap;rr up tTa certain point, after wh.ch .t .ecame „«hter .n colour. 6 Mast Cells.-These may often be seen, especially if the fluid' be examined rapidly. They occur in chrome effusions chiefly, and are pathological, since they are never found m the normal fluid bathing the serous membranes. 7 Red Blood Cens.-Apart from accidental contamination importance has always been attached to the presence of blood n an effusion as mdkatmg ehher tubercle, carcmoma, or rena diseL It may occur, however, in metapneumonic empyema * andTn rheumatl cases.f A large amount of blood is met with in those rare cases, of course, where a thoracic aneurism is gradually leaking into the pleural cavlt^^ In such a case the utmost importance will be attached to the finding. 8 Carcinoma or Sarcoma Cells (Plate 2, fig. 3)-The apiL^nce oMhese cells in an exudate may be looked on as of dEostic value to an extent which does not exist m the diag- nS carcinoma of any solid organ. The presence of carcinon.a cells in the sputum, in the stomach contents, or in the f^es or urL must be'looked upon as mamly mythical as it is out of the question to distinguish between malignant cells and the endo- Tlial or epithelial cells of the passages. We can only hope to diinose the condition from the presence of cell-masses But m srous cavities freer conditions obtain, and although fragmen s of tbsue may occur, the individual cells are much more capa^,le o accurate study. The chief features of diagnosis have been gone mto fully on page 2.(.. and need not be agam described. Quincke described a glycogen reaction m carcmoma cells granules visible on staining with iodme-gum being met with . Raubitschek; also a 'recent case in the Leeds General Innrmary. t l-:arl. CYTODIAGNOSIS 229 in such cells ; but little importance can be attached to this from the diagnostic point of view, in the same way as the glycogen reaction of pus cells so often proves misleading. Errors in DiAGNOSis.-{a) Endothelial cells, when en- tangled in fibrin, may simulate carcinoma cells, (b) Vacuolated cells may occur in ascites connected simply with an ovarian cyst or in benign tumours of the ovary. The most serious difficulty, however, is that there may be no free tumour cells at all in the exudate, or that those which were shed have become dissolved. Grenet and Vitry, too, describe two cases in which the peritoneal fluid in a case of carcinomatosis contained only lymphocytes, large mononuclear cells, and red corpuscles. Certain Special Fluids. Hydrocele FLViD.-Flakes of endothelial cells or isolated cells are met with in large numbers in simple hydrocele, and sometimes they are abundant in chronic hydrocele. As a rule, the other cellular elements follow the familiar tyi^es. Lympho- cytes preponderate in tuberculous hydrocele, or in any chronic inflammatory process. Polynucleosis, on the other hand, is generally (not always) associated with acute mflammation— usually gonococcal. The irritation produced by tapping, and especially by injecting such remedies as iodine, will result m the appearance of endothelial cells and lymphocytes (Julhard). Joint Fluids.— In the effusions into joints resultmg from various causes the various tyi^s of cell-elements give similar indications to those of other serous membranes. Thus, polynu- clear cells predominate in gonorrhceal or acute suppurative cases, though, if there be no pyrexia, there may be only lymphocytes present The same polynucleosis occurs in acute rheumatism, and in irritation after tapping. Lymphocytes indicate a chronic affection, much less frequently tuberculous than with other serous membranes, and a small proportion of endothehal cells is frequently met with. Rice-bodies are associated with lymphocytosis. Red cells are likely to be found in fair number in tuberculosis of joints. Tabetic joint effusions contam very few cells.and these are mostly lymphocytes and large mononuclear cells. Cerebrospinal FLUio.-On reviewing the enormous accumu- 230 STUDI?:S IN PUNCTUUE-KLUIDS 1. lation of literature of the cytology of cerebrospinal fluid alone, we strike upon one great fact, namely, that in functional disease the lumbar j)uncture will show few. if any. cellular elements at all. while if there be organic disease of the brain or spinal cord, we may exjiect to find a considerable number of cellular elements, a differential count of which will assist in the diagnosis of details with similar limitations to those that obtain in the case of pleural and jx-ritoneal fluids. Probably in this kind of fluid the real significance of lymphocytosis oi polynucleosis, or of a mixed type, is similar to that which obtains for pleural and jxiritoneal fluids, namely, that it is not so much a question of tubercle or other organisms, as that in the one case the inflammatory change is of a much less active kind, demanding fewer phagocytes. The varieties of cytological formula which may be met with may be groujied under the following headings : {a) Absence of cellular elements, or, fewer cells than two per cubic millimetre. This is the n.'.rmal condition, as shown by many investigators (Schwarz, Bronstcin, V'erzeanu.Devaux, Schlesinger, and others). Cells are absent in such purely mental diseases as mania, paranoia, melancholia, imbecility, acute alcoholism, delirium, senile dementia (Merzbacher). If there be no cellular elements present, or if their number be not greater than two per cubic mm., this is sufficient to exclude any meningeal inflammation, syphilitic meningitis, and such diseases as tabes, superficial gummata, tumours, hypertrophic pachymeningitis. A similar condition is met with in the cerebro- spinal fluid in cases of herpes. (b) Lymphocytosis. — This term denotes either a relative excess of lymphocytes over the other cell-elements or it means that lymphocytes constitute the only cells jiresent. Lymphocytosis occurs, to a moderate extent only, in some cases of dementia paralytica, of chronic alcoholic paralysis and of disseminated sclerosis, while it occasionally occurs in perfectly normal fluids (Schwarz and Bronstein). Sj^aking broadly, lymphocytosis occurs in any meningeal inflammation that is of chronic nature. It is therefore met with in tuberculous meningitis (esjiecially in children), in syphilitic meningitis, whether con- genital * or acquired.t It has Ijeen found in cases of herpes, * Kretschmer. t Raubitschek, Devaux, Donath. CYTODIAGNOSIS 231 of mumps,* and of tabes, as well as in epileptics f (showing that epilejjsy is not purely a " functional " disease). It has been observed that when lymphocytosis occurs in a syphilitic case mercury causes the cells to disappear. The view has been expressed J that lymphocytosis is not characteristic of syphilitic nervous disease, but that it is only present if the meninges are involved. There are one or two additional points to note about this type of cell-predominance : (i) its development during the course of a case indicates improvement. (2) If polynucleosis were present before, the supervention of lymphocytosis may be regarded as a favourable sign. (3) It is not present during the initial stages of tuberculous meningitis. On the other hand, in the later stages of this disease one may e.\i)ect to find as many as 10,000 cells per cubic millimetre. (c) Polynucleosis. — This generally signifies an acute suppura- tive meningitis, or, at any rate, acute meningeal disease.§ It may develop in the initial stages of tuberculous meningitis, and is otten continuously present in the tuberculous meningitis of adults. It is met with in cases of commencing poliomyelitis, and in cases of herpes. In cerebrospinal fever there is an excess of polynuclear cells at the outset of the disease, and at each exacer- bation of the fever. Sawyer gives : polynuclear cells 84 per cent., lymphocytes 15-6 per cent., degenerate cells 04 per cent. It may occur during the congestive attacks of hemiplegia. It is important to note the state of the preservation of these cells, for if transient aseptic disease, such as during syphilitic nervous disease, is present, the cells will be found particularly well preserved. The number of cells per cubic millimetre in a case of suppura- tive meningitis may reach 100,000, of which 53 or more per cent, will be polynuclears. The number diminishes as the case pro- gresses towards recovery. In a case of cerebral abscess || there were 97 jier cent, of polynuclear, 24 per cent, of lymphocytes, 04 per cent, of degenerate cells, and 02 per cent, of endothelial cells. (One would dispute, from one's own experience, whether endothelial cells can be identified in cerebrospinal fluid.) (d) Other Cellular Eletnenls. — Blood may be present in cere- * Raubitschek. f Merzbacher. X Funke. $ V'erzeanu, Devaux, Donath. jj Sawyer. 232 STUDIES IN rUNCTURE-FLUIDS brospinal fluid, apart from accidental contamination, in cases of intracranial h,-emorrhage where the blood has made its way into the meningeal cavities. In a case of this kind, Sabrazes and Muratet found round or oval px)lyhedral isolated cells or masses of such cells, containing fragments of red corpuscles, or haematoidin granules. These they regarded as " macrophages," derived from the endothelium of the subarachnoid spaces. Such cells may contain fatty granules, fragments of myelin, and pigment. The presence of large monontidear cells with basophile proto- plasm, and eccentric nucleus, has l)een described as occurring in tubercular meningitis, but they also occur in tabes and cerebro- spinal syi)hilis. Tumour cells have been described as occurring in cerebrospinal fluid by Sahli. Degenerate cells in cerebrospinal fluid have been explained as due to decomposition having set in ; but it is satisfactory to learn that Pappenheim has offered the view that the degenerate character is evidence of pathological processes, and that it is the result of a toxic action exerted by the diseased cerebrospinal fluid on the ]X)lynuclear cells. This type of cell occurs most frequently in paralytics, and the proof adduced is that warming the fluid to 56° C. causes it to cease to have any deleterious action.* The presence of organisms such as spirochaetes or trypano- somes does not come within the scope of this work. The rate of flow of the fluid from a lumbar puncture has been laid undue stress on by some authors, but to adduce arguments for believing so is out of place here. Ovarian Cysts. — The cellular elements in these cases are almost pathognomonic, and are so well known as hardly to call for more than mere mention. Columnar epithelial cells, ciliated epithelial cells, and squamous epithelial cells may be met with, according to the character of the growth. Cells with fatty granules • Ont' may emphasi.se this idea of Pappenheim 's by caHing attention to the fact that degenerate appearances which are observed are only too readily ascribed to imperfect technique : thus, a liver which becomes almost fluid on removal from the body is often regarded as an example of early post-mortem decomposition. So with the suprarenal gland. As a matter of fact, most livers and suprarenals do not break down readily, even if the necropsy is delayed 48 hours. The explanation of the undue friability must therefore be sought, as there must be a reason for such changes apart from " accident." CYTODIAGNOSIS 235 are also characteristic, especially in multilocular cysts. Colloid masses may appear in the fluid in cases of colloid carcinomata. The presence of cholesterin crystals is a frequent feature in ovarian tumours, and has even been met with in a simple par- ovarian cyst (in this Infirmary), though such cysts have usually perfectly clear watery contents. Special Findings in the Deposit of Puncture-Fluids. — Hamatoidin crystals occur in empyema, subchronic abscess, pulmonary abscess, and esjwcially in old suppurating hydatid cysts. They indicate antecedent haemorrhage. Food particles may be found in the p)eritoneal fluid after perforation in the course of the alimentary canal. Tumour fragments may be met with. Myelin bodies, com})osed of prota- gon, are referred to by Sahli as being abundant in tumour of the lung which has invaded the pleura. Fatty acid crystals occur in putrid collections of pus. Triple phosphate crystals, calcium, carbonate, and phosphates may be seen in i)urulent collections. Hydatid hooklets or scolices may be found, and renal casts will occur in the corresponding cysts. Spontaneous coagulation of a fluid is an indication that fibrin ferment was present. Scheme for Differential Diagnosis in Special Cases. — ^The following scheme has been devised from the data supplied by Koniger, as it may be found useful : ' Ljmphocytosis c o appearing in the course of in f 1am- matory disease. Without associated j' tubercle endothelial des— | quamation \ syphilis Much the com- moner. See if the "formula" alters after repeated tap- ping, endothelial desquamation Ejirly stages of ill. ness, no endothelial* cells present. pure poly- nucleosis! if cells de- generate With associated =: sarcoma. Indicates diminished virulence of bacterial infection. if cells well-preserved = sterile effusion, 'cells shrunken = tubercular pleurisy, secondary to adja- cent lesion, cell body swollen = acute infective pleurisy, polynucleosis with lymphocytes = pleurisy following tubercle of the lungs ; if fatty granules suspect V caseation. Abd ■ 1 i^^ pyrexia present = tubercle. omina I /where previous puncture was performed — «*"^'°"'^ J cirrhosis of liver. ceU type I [ ofmixed-VfnopyrexUpresentJ^^^^ ,^^ cell-formuU cannot be due to previous puncture < tubercle. 234 STLDIKS IN PUNCTURE-FLUIDS I The Chemistry of the Cell-elements present. — This subject docs not come under cytodiagnosis, but reference is made to it in order to draw attention to the fact that upon the com- position of the cells present in an effusion the chemical characters will to a certain extent dejHjnd. The degeneration of the cells will naturally lead to the substances of which they were comjwsed appearing free in the Huid. The exact substances present will be found on reference to the appropriate headings in the preceding sections, but es|)ecial attention may l)e drawn to the facts made out by Miiller and Jochmann, which go to show that the granules in i)olynuclear cells, not to mention the eosinophile granules, are jirobably of the nature of zymogen granules, and that they are intimately connected with the proteolytic ferment which exists in leucocytes. In this manner it will be obvious that the granu- lar appearance of a })olynuclear cell must be placed on a par with the appearance of gland cells (such as salivary gland cells) at different stages in their activity. EXPLANATION OF THE PLATES Plate i Fig. I. The bulk of the cells in this ligure are lymphocytes, many of them having a granular appearance. In the middle is a large mononucleate cell, also very granular, and of endothelial origin. Just below it wdl be seen a large mononuclear cell of angiogenic origin. The small cluster of lymphocytes will be readily distinguished (in an actual specimen) from the clusters of carcinoma or sarcoma cells depicted in Plate 2. lig. 2. Eyepiece 2, objective { in. Fig. 2. This shows the typical endothelial plaques met with in the deposit in cases of back-pressure. A few lymphocytes are shown, with which the size of the endothelial cells can be compiared. A large mononu- clear cell (angiogenic), a cluster of dead cells and a binucleate cell will also be noticed. Oil-immersion lens. Fig. 3. Cells from a case of polyorrhomenitis. These are mainly degenerate cells, some are vacuolated, though finely granular matter can be seen in the vacuoles. The nuclei in the dff eaerate cells are devoid of chromatin. \ group of pseudo-lymphocytes is shown on the right, as well as true lymphocytes. Eyepiece 2, objective J inch. Fig. 4. Dililerent types of cells, (a) degenerated endothelial cell from a case of cirrhosis of the liver ; (fc) large mononuclear cell, with pale cell-body and pale nucleus ; (c) cell with " mast " granules, from a case of tubercular peritonitis ; (d) large ^^-generated endothelial cell — chronic pleural etlusion ; (f ) enormously swollen endothelial tell from the same case ; (/) lym- phocytes. All the cells are drawn to scale. Oil-immersion lens. Fig. 5. Degenerated cells of difierent types : a, b, c, and d ase endothelial / " ■J 'J CJ /■■/ / d« f \ / 1^ ft ^ • c o ^ rV //./ -. //'/ •/ rt • 9 If ^ d / / /■/// «♦ "' ,* ^ •f " O i S-v- / '• / o - • \ r:/ *l / if. / ^/'^ i / /'/ / / i 1- pp. CYTODIAGNOSIS ^35 cells ; c shows nucleus, with nucleolus, granules, and degenerating cyto- plasm ; d is astruct-ivless mass; e large mononuclear cell. The bacilli are Bacillus lympka n (Hamburger). Oil-immersion lens. Plate 2 Fig. I. Fragments of tissue irom the peritoneal fluid in which there was dissemination of carcinoma. Columnar cells are seen on a t>asement membrane. There art indications of structure within the globules of tissue. The spherical appearance was well seen in the fresh specimen. Eyepiece 2, objective J inch. Fig. 2. Cells from a case of sarcomatosis of the peritoneum. (a) Sarcoma cell whose nucleus has two nucleoli, and large granules are seen in the cytoplasm ; (6) typical clusters of cells with deeply staining nuclei. There were extremely large number, of these clusters present in each lilm. (0 large mononuclear cells ; (rf) lymphocytes (the relative sizes of the cells are readily seen from these) ; (e) swollen degenerate cell ; (/) a few sarcoma cells in juxtaposition, showing deeply staining nuclei, and deeply staining cytoplasm. Eyepiece 2. objective I inch. Fig. 3. Endothelial and carcinoma cells, (a) Red cells; (b) lympho- cytes • (c) endothelial cells ; (d) swollen cells containing several nuclei and nucleoli; (e) carcinoma cell showing mitotic figure; (/) excessively swollen endothelial cell. Oil-immersion lens. Fig. 4. Cells from peritoneal carcinomatosis. Shows large vacuolated cells, some large multinucleate cells, some binucleate cells and large deeply staining mononuclear cells. Oil-immersion lens. i SECTION VI SPECIAL CASES Thk application of the facts recorded in previous pages and of the'vanou. methods of diagnosis which have been advocated is lH>st illustrated by the relation of certain special cases which have been examined. Ihe various observations made on the specimens of fluid received for examination are here recorded, and the deductions which have been made as to the nature of the fluid are introduced. The post-mortem diagnosis subsequently obtained is introduced, and affords an opportunity of commenting on the diagnosis made in those cases where one's conclusions proved to be at variance with the actual ^'^The greatest difficulty is undoubtedly attached to the diag- nosis of the effusions in either chest or abdomen, especially in medical cases, and it becomes more useful to refer solely to these two kinds of fluid. , The cases are groui)ed therefore into (a) pleural, and (b) peritoneal, but no other order is observed. Only those which presented features of particular interest or of difficulty are con- sidered in this section. (A) PLEURAL FLUIDS I G aged 40 —The fluid was straw-coloured, with blood- staining' of the deposit. The specific gravity was 1012. There were no endothelial cells in the deix)s.t, which contamed a few lymphocytes and some red cells. , . , • j » The chemical examination showed an abundance of chlorides (8 gm. per litre), and a very small amount of albumen (o'S per cent.). There was no glycoproteid present. 2^6 SPECIAL CASES 237 The concentration of electrolyte'^ was 0-2460, of which 0-0424 consUted of achlorides. The ratio achloride/chloride was 0-21. The post-mortem examination showed cardiac incompetence due to valvular disease (aortic and mitral). There was also ascites, cirrhosis of the liver, infarcts in the lung, spleen, and kidney. The findings in the pleural fluid jwint to a passive accumulation of fluid (transudation), partly because there was an increase in the chloride-content, and partly on account of the arhloride/ chloride ratio. All the facts made out fall in line with the diagnostic indications which were mentioned in Section IV. II. Richard H., aged 26.— The straw-coloured fluid had a siJecific gravity of 1032, and showed a considerable deposit, mainly of lymphocytes. The chemical examination showed a maximal quantity of albumen, and a considerable quantity of chlorides (7-25 iw cent.). The presence of other substances was not investigated. The electroconductivity examination showed the amount of total electrolytes to be 0-2319, rnd the achlorides were as much as -1533 (total concentration in terms of Xa.COj). The achloride chloride ratio was therefore alxjve unity, 2-95. On the strength of these facts, high albumen-content, high " salt " ratio, and the cytological characters, the diagnosis of inflammatory effusion of tul^erculous origin was made. III. Alfred B., aged 15.— A green, fluorescent fluid, having a specific gravity of 1021, contained 2 per cent, albumen. The concentration of the chlorides was 0043 gram-mol., while the total concentration of the electrolytes amounted to 0113. The achloride electrolyte^ therefore amounted to 0070 gram-mol. and the achloride/chloride ratio was o-i6. The omotic concentration waso-28i, so that the non-electro- lytes amounted to o-i68. The chemical examination showed a trace of urea, no purins, a trace of glycoproteid, a trace of invertase, but no diastase. The a-naphthol test was tardy, b.t the glucosamine test instant and intense. The cellular elements consisted mainly of lymphocytes. 238 STUDIES IN I'L'NCTUKE-FLUins The examination on three . + -r + + + -r + + + + IM III) 01 23 00 (3 0042 0070 0070 0074 0053 01 6 17 075 1. Will be seen that only on June 7 did the •' salt " ratio fall m with the idea of an inflammatory effusion. This case was only of •• idionathic " pleural effusion, and was presumably tuberculous. Commail.-TheK is a certain amount of difl^culty in weighing up this case. The chlorides being relatively low indicates an inflammatory process, although the total concentration of electro- lytes was also low, so that the " salt " ratio is not constant. On the whole, the diagnosis is in favour of the pleural effusion being of inflammatory origin, and probably tuberculous. IV. William A., aged 42.— A milky fluid showing no siwn- taneous coagulum or any dei^sit beyond a few granular cells. The total proteid amounted to 4-65 })er cent., and the chlorides to 30 lier cent. The concentration of the achloride electrolytes was almost equal to that of the chlorides. There was a moderate amount of urea. There was no glycoproteid. Cholesteim was present. The tryptophane reaction was positive. As regards ferments, there was a trace of diastatic and of inverting action. Comment.— l\w microscopic characters and the "salt" ratio are in favour of this fluid having a transudatory origin, although the proteid-content is rather high. The diagnosis is that this fluid is a chronic effusion in which there may at first have been mflammatory processes, but that these are now quiescent. V. Mary A., aged 34.— The pleural fluid was examined on two occasions, but with an interval of only a week between them. SPECIAL CASES 239 The turbid greenish fluid (later, blood-stained), with a small amount of coaguluiii, had asj^cific gravity of 1020, and contained 6 per cent, of total proteid (only 058 j)er cent, globulin). The chloride electrolytes showed a concentration of 0035 on one occasion, the total electrolytes amountmg to o J46Q, and the achloride electrolytes to 01830. The achlonde/ chloride ratio was therefore 2S(). The chemical examination showed the presence of a trace of invertase, urea, no protalbumose, no glycojiroteids, no mon- amino-acids. The a-nanhthol test and the glucosamine test were positive but slight. The diazoreaction, on the other hand, gave a |)ositive result. The cellular elements were mainly lymi>hocytes. This fluid was from a case in which there was carcinomatosis of the jx-ritoneum, the primary growth being in the ovary. The pleural effusion was of inflammatory, but not malignant origin The high salt ratio and other fioints will be nntcd. The e.xamination on the two occasions, when compared, showed no appreciable difference ; not enough to justify reproduc- tion of tlie findings in each case. V'l. Alfred K., aged 28. ^An amber-coloured fluid with an abundant coagulum, had a specific gravity of 1022, and contained 8 per cent, of total proteid, of which only a small projHjrtion was globulin. The concentration of chloride electrolytes was 0064 gram-mol., the total electrolytes amounting to 0-261, and the achloride electro- lytes to 0180. The achloride chloride ratio was therefore .2-23. The chemical examination showed the presence of invertase, urea, albumose. but no purins : no serosamacin. The -.-naphthol and the glucosamine reactions were immediate and marked. The cells consisted mainly of lymphocytes and red cells. The fluid was an inflammatory effusion of tuberculous nature. VTI. William L., aged 21.— A clear yellow fluid with a considerable coagulum. The six;cific gravity was 1024 a.ul i per cent, of albumen. The concentration of the chlorides was 0031, while that 01 the total electrolytes was 0-2319 ; the achloride electrolytes amounted to 0-1733 and the achloride/chloride ratio was 2-95. The osmotic concentration was o-2Cjd relatively very small nucleus. Some multi- nUMcteoe- —some having three or four, others several nuclei- were also -^^»<«Tved. „ . , . TY^ Mrmual examination has shown that the fluid is corn- pa, ativ rich in chlorides (oW. iH.r cent), a.vl that there is about o-i i)ei c t. of urea. The albumen is present in considerable but not in excessive amount (1-95 I'er cent.). The osmotic pressure of this fluid is higher than that normal for blood (equivalent to 9 atmospheres instead of 7). The urine secreted at the time of tapping was examined, and found to have as si^cial characters a high spec.hc gravity and an increased quantity of urea. It was practically free from chlorides, while the osmotic pressure was high, and caused largely by non-electrolytes. SPECIAL CASES The results of examination are tabulated below. 241 Urine Periluneal I'rine Keritoneal Swrroted a' Kluld 1 SrcrrUd at Fluid. time of ; weeks t lime of TappiDf. later. 1 TappirR. Specific gravity Reaction 1012 al". rgranular leucocytes, ~) 1 larKo bi - nucleate 1 1 cclli, mononuclear 1 (. ce'.li J 1030 acid toll alk. 1017 acid Deposit red urates urates Urea 00 1 % y2r. i-9?^o Albumen •9S% ^■5'- nil Chlorides 6-5 %o 005°:, 6-35 : 'S^U Freeziiiji: • point de- pression 0591 1950 06C6 \\(,o Osmotic Concentration 3'9 1052 0360 0644 Osmotic pressure in 1 atmospheres -•« 234 79 143 Conductivity (22° C.) 1161 1772 1181 lies Cone of electrolytes 03^47 O-20I 0128 120 Cone, of achlorides ... 00851 196 0020 0095 Cone, of non-eltctro- lytes 0014 0851 0232 05J4 C clecf. C noil ilect. 216 023 0-5S 024 C achior. | 041 097 015 079 C chlor. ( Comment.— From these figures, showing the changes that have taken place during five weeks, it will be seen that while the two specimens show a similar composition as regards the most essential constituents, the urine shows slight changes. The amount of area has diminished, and that of the chlorides has increased. The concentration of electrolytes has also diminished. On this change there can be laid little stress, owing to the fact that urine is so variable The constancy of the composition of the peritoneal fluids is. however, important, as indicative of a stationary condition. The ratio of electrolytes to non-electrolytes and that of achlorides to chlorides, following the rules formulated, indicates that this fluid is transudatory in nature. The question as to whether there was or was not tuberculous peritonitis was raised, and the answer to that was as follows : Against tubercle (no tubercle bacilli found) : less than 3 per cent, albumen, low specific gravity, absence of spontaneous coagula- bility, high achloride/chloride ratio. Alone in favour of tuliercie was the presence of many mononuclear cells in the deposit. The 242 STUDIES IN PUNCTURE-FLUllxS fact that this preponderance was very slight did not influence one"'^ opinion much in favour of tubercle. P J-mortem diagnosis : thrombose of the PO^^al vem _ II ELLEN D aged 50.-A milky fluid of specific gravity 1018-1019-1020. The fluid was not spontaneously coagulable. There is a large amount of albumen present (3 per cent.). The chlorides amount to 8 gm. per litre (0137 gram-moL). The osmotic concentration is 0272. whUe the --e secreted .U this time showed only a concentration of 0^156. T]>^ """^ had a specific gravity of 1005. contained no albumen, and only X per cent, of chlorides ; the urea was 0-25 P«r ""*• The electrolytes consisted almost entirely of chlorides the total electrolyte concentration being only oi39 gram-mol. per ''^' The deposit was considerable, and consisted of numeroi^ very large mononuclear cells (some bi- or tri-nucleate) ; also red celU^ and some polymorphonuclear leucocytes. Some of the cells contained refractile non-fatty granules. , ^ . . • -,>,,, The percentage of albumen indicates that the fluid is either of a chronic exudative nature (associated with either hepatic trll or with peritoneal new growth). While the percent^e oLbumen just comes within the limit for tuberculous effusions, it is so high that this form of peritonitis may be excluded. April as. '9°6- Abdomind. June 11, 1906- Abdominal- June 19, 1906. Pleural. July 19,190^. Abdominal. Specific gravity ... j 1020 3% Albumen ' Cilycoproteid ... 1 3- 1 37 gm.-mol. Chlorides < Freezing-point de- pression 0502 Osmotic Concen- tration 0-272 Cone, of Electro. lytes 0139 Conc.ofAchlorides 0002 Cone, of Non- electrolytes ... o«33 Achloride/chloride ratio O'OI Deposit ? Sarcoma 1020 17 0-113 1018 02 0097 0115 0018 Enormous cells I (sarcoma ?) and red cells 0-34 Endothelial cells and lymphocytes 1019 2 present 0-046 0-679 ■358 •223 •177 •»35 2-62 Numerous cell* and amorphous coagulated proteid SPECIAL CASES 243 The fluid is an exudate, not a transudate. This case was tapped several times, and was found to show fairly constant composition. In the pleural fluid the achloride ratio was low, just as at first in the abdominal fluid. However, the latter subsequently showed the normal ratio of inflammatory conditions. We might assume that the pleural fluid was not of an inflammatory origin, and that the peritoneal had steadUy become inflamed till eventually it was actually invaded by the growth. Post-mortem Diagnosis.— Round-celled sarcoma of ovary with peritoneal dissemination. III. Joseph B., aged 36.— The clear straw-coloured fluid had a specific gravity of 1012. and contained 08 per cent, of albumen. There was a small amount of urea present, and the chlorides amounted to 055 per cent. (0094 gram-equiv.). The cells m the deposit were mainly endothelial. Physico-chemical Examination.— The osmotic concentration was 0-360, and the concentration of the electrolytes 0135. The achloride electrolytes amounted to 0041 gram-mol. per litre, so that the achloride/chloride ratio was 0-435. The urine secreted at the same time as the fluid was tapped was concentrated, and contained a copious deposit of red urates. The fluid was examined again a month later, and found to still have practically the same characters, and the urine also had very simUar characters, as wiU be seen from this table of results. Ubcervation. Specific Gravity Albumen Urea Chlorides Freezing-point Depression Osmotic Concentration Concentration of Electrolytes . . „ Achlorides Non-electrolytes Achloride/chloride ratio Deposit ... May 2, iy-6. June <>, 1906. Fluid. Urine. Fluid. I Urine. 1012 j 1036 1013 o-s°: 1 none 1-0% OOJ% 3-3% i none 5'5%c 3-5%. i ^Vh 0667 2405 0658 0360 1-326 , 0-355 0135 0-266 1 0131 0041 0-207 0-014 0-225 I 060 0-224 0435 35 OCt2 lirfe mono- nuclear cell* urates aameas bcfora and lympho ' cyt«» and endoiheli* ] cell* 1 t 103s none 3-65% 0-7%* 3-400 I -3 16 0-254 0-243 1062 4;54 . aa depoait 244 STUl.ItS IN I'UNCTUKE-FLUIDS From these fads one arrive, at the conclusion that the ''"Thrnt™,^;".^™'::;, a .irrhos»o. ,he Uver i„ an advanced •a fl^^l.i' received foe examinatton at diff.renl fmes. pilf „»,! Mh. mud ». ,„und .0 be ,o/*,->*» ■» "' mc results o( examination are l«st tabulated ; Specific Gravity Albumen Urea i Chlorides Concentration of Electrolytes Concentraticn of Achloridcs ... Achloride/chlo- ride riilio ... 1 °'*9 Deposit ... • Sporulating j bacilli Chemical notes I Excess of glo- ! bulin. No I glycoprotcid The evidence is in favour of a transudate. -"^^ ^^^^f J^^^; content is high and the achlonde-content -ry^^;^ J^^^^ nature of the case is difficult to explain. The following account ofmLroscic examination of three of the organs obtamed at the nprroDsv will sum up the case : S-M.croscop.c examination shows an extreme degree of fatty infiltration and degeneration. In some parts the Uver cells have almost disapju^ared. The parts m which there fno change are few and far between. The capillaries '" p;!:cmis.-There is no increase in the amount of interstitial tissue. The islands are normal, and there .s no evidence of chronic interstitial pancreatitis. SPECIAL CASES M5 Kidney— The only abnormal condition is to Ik- seen in the convoluteil tubules, where the epithelium is extremely cloudy and the nuclei are in most places lost. In other parts the tubules are apparently quite healthy. The glomeruli cannot honestly be said to show any abnormality. There is no increase in the amount of mterstitial tissue, and there is no leucocytic infiltration. The straight tubules are perfectly normal The convoluted tubules are filled with finely granular detritus, and in some places this matter is so abundant as to form what would be hyaline casts. In some parts the epithelium of these tubules is much swollen, while in others it is low. In the former place the nuclei are generally present, while in the latter they are often absent. Hcie and there the capillaries are widely dilated, but this feature is inconspicuoas. In short, an unprejudiced observer would not diagnose any serious renal disease, and the only conclusion to be drawn is that the kidney has been subjected to some serious toxic agent, which has been excreted through the epithelium of the convoluted tubules and damaged it during its passage. , • u j V Harry E., aged 30, was tapi^il several times, and yielded a clear amber-coloured fluid of sj^ecific gravity loio and contam- ing only 15 per cent, albumen. The chloride electrolytes amounted to 0088 gram-equiv.. and the total electrolytes amounted to -2121 gram-mol. per litre, so that the achioride, chloride ratio was 0-26. The acidity, tested by Wright's method, was equivalent to HjSOi. ^he chemical examination showed the presence of much globulin, of serosamucin, of invertase. but no albumoses or neotones, no monamino-acids and no diastase. The deposit contained very little blood,-of white cells mostly lymphocytes; a few endothelial ceUs, none bemg vacuolated. . , The report on this case was that there was proliferation of the cells of the serosa, but no evidence of new growth or tubercle. Most likely it was due to hepatic cirrhosis. The post-mortem showed the case to be one of monolobular cirrhosis of the liver. It 1000 MICROCOPY RESOIUTION TEST CHART (ANSI and ISO TEST CHART No 2i 1.0 I.I m 13.2 I: i^ 2.5 2.2 [2.0 1.8 1.25 1.4 1.6 ^ .APPLIED IIVMGE ■ ■;-. \f..j,r 'j-fer' i^Jl'* -f. New ■.:,ri. 14609 j'^A , ''6 d2 - 0300 - Phone (716! ?8H - 5989 - Fa» 246 STUDIES IN rUNCTURE-FLUIDS The following table will show at a glance the condition of the fluid on different occasions : Obiervxion. Jan. 23. 1907. Feb. a6, 1907. ! April 10, 1907. Specific gravity Albumen Urea Purins Amino acids Albumoscs Glycoproteid ... Ferments 1 a-naphthol test ... •.• Glucosamine test ... ... j Chlorides (gram-equiv.) ... Osmotic Concentration ... 1 Cone, of Electrolytes | Achloride Electro- 1 lytes Achloride/chloride ratio ... | Cone, of non-electrolytes lOIO ' 5/0 trace 1010 1-7% none 0-0021% N none none none + ! none ! trace invertase, ' No diastase, i no diastase 1 trace invertase 0113 o-i68 OC55 047 + + 0-088 0'2I2 0-0426 0-26 lOII s-s% moderate amount trace trace invertase, no diastase + 0-094 0-300 0-206 VI. Isaac S., aged 23.— A turbid straw-coloured fluid of specific gravity 1015 and containing 2 per cent, albumen. The chlorides amounted to o'li gram-mol. per litre, the electrolytes to o-i2, so that the achlorides were 001. The achloride/chloride ratio was therefore 001. There was no globulin present. The ceUular elements con- sisted of a few mononuclear leucocytes. The " salt " ratio is low (exception to rule), and the low percentage of albumen is in favour of a tuberculous effusion. The case was one of tubercular peritonitis. VII. William D., aged 67.— A turbid straw-coloured fluid of specific gravity 1017, containing 8 gms. per litre of albumen. The chlorides amounted to o-io; gram-mol. per Utre, the electrolytes to o-ii8, so that the achlorides were ooii. and the " salt " ratio 1-5. Chemical examination showed no glycoproteid. The cellular characters were of most interest, and are shown in Plate I. The findings indicate an effusion of inflammatory origin (low NaCl-content, high " salt " ratio, high albumen-content). The case proved to be one of sarcoma of the omentum, with peritoneal dissemmutiuii. SPECIAL CASES 247 VIII. John K., aged 46.— The turbid fluid, in which large white particles were floating, contained 2 per cent, of albumen and 0"4 per i nt. of chlorides (o'o68 gram-mol.). The ek irolytes amounted to 0132 gram-mol, so that the concentration of the achloride electrolytes was 0064 gram-mol. ; the " salt " ratio was therefore 074. No globulin or glycoproteid was found, but triple phosphate crystals were present. The cells included numerous large mononuclear cells, with abundant non-granular cytoplasm and also some large multi- nucleate cells. The salt ratio pointed to a transudatory fluid, the low albumen, content likewise pointed against an inflammatory effusion, while the low chloride-content was decidedly against a transudation. On the other hand, the absence of globuUn or of glycoproteid seemed to indicate a transudatory fluid. The necropsy showed colloid carcinoma of the stomach, with extensive peritoneal dissemination ; on the other hand, there had been a tuberculous pleural effusion. IX. George W., aged 52.— A straw-coloured fluid, forming a small coagulum after a considerable length of time, had a specific gravity 1014. The albumen only reached 0-5 per cent. The chloride electrolytes amounted to 0108 gram-mol. (fairly high), and the concentration of electrolytes was Ii2i, so that the achlorides amounted to 0013 gram-mol. The " salt " ratio was therefore 083. The osmotic concentration was 0-311 gram-mol, so that the non-electrolytes amounted to -070 gram-mol. The chemical examination showed abundant glycoproteid (not metalbumen). The cellular characters were insignificant— a few lymphocytes. In this case the low albumen-content and the fairly high chloride-content point to a transudation. The salt ratio also indicates a non-inflammatory collection. The presence of glyco- proteid is peculiar. The case proved to be one of back-pressure from tricuspid regurgitation and stenosis, and the presence of glycoproteid here falls in line with several other back-pressure effusions which have been examined and found to contain a reducing body after hydrolysis. 248 STUDIES IN PUNCTUKE-FLUIDS m i;! I X. William H.. aged 19.— A highly coloured and slightly glistening fluid of specific gravity 1018, and containing a con- siderable amount of albumen. The fluid deposited a thick layer of finely granular pus, with streptococci ; the polynuclear cells were necrotic. No large cells or multinuclear cells were seen. The precipitation limits for ammonium sulphate were 4-5 and 0, the maximum being at 6. There was a trace of hetero- albumose, and some protalbumose. Glycoproteid was found, and some lecithin The a-naphthol and the glucosamin test gave a negative result. Leucin and tyrosin were absent. Physico-chemical tests were not applied. This was a case of ascites due to back-pressure in a case of adherent pericardium and endocarditis. Fat necrosis was found in the abdomen. XI. Walter S., aged 36.— A foul-smelling, reddish-brown fluid, containing much blood and a small amount of coagulum, had a specific gravity of 1013, and contained 775 per cent, albumen. The chloride electrolytes amounted to 0043 gram-mol. per litre, and the total electrolytes to 2675, the achloride electrolytes amounted to 01875 and the arhloride/chloride ratio was 2-34. The osmotic concentration was high, 0406. The chemical examination showed the presence of much urea, purins, of serosamucin, of protalbumose, and a trace of phosphates. The a-naphthol reaction and the glucosamine test were prompt and intense. Diastase and not invertase was found. The cells consisted mostly of lymphocytes. This was evidently a case in which there was some inflamma- tory condition of the peritoneum, and the post-mortem examina- tion showed a chronic adhesive peritonitis with a coli infection. XII. Arthur S., aged 42. — The amber-coloured fluid had a specific gravity of 1016 and contained 25 per cent, albumen. The chloride electrolytes showed a concentration of 0-042, while the total electrolytes were -211 ; the concentration of the achloride electrolytes was therefore 133, and the achloride/chlo- ride ratio i-68. The osmotic concentration was 0-293. The chemical examination showed a trace of urea ; the SPECIAL CASES 249 r i a-naphthol reaction was slight, and the glucosamine tost decided. Metalbumen or serosamucin was not found. There was a trace of invertase and no diastase. The cells were mostly endothelial. This was a case of monolobular cirrhosis of the liver. Note the high salt ratio. XIII. Emma N., aged 35. — The turbid straw-coloured fluid had a specific gravity of 1013, and contained 3 per cent, albumen. The chloride electrolytes amounted to 01 19 per cent, gram- equiv. and the total electrolytes amounted to o'i4q per cent., so that the achlorides were 0'03o per cent, gram-mol. per litre, giving a " salt " ratio of 025 per cent. Chemical examination showed the presence of much globulin, and of purins, and of albumose. No glycoproteid was found. The diazoreaction gave a positive result, as also the tryptophane test. There were large vacuolated endothelial cells and lympho- cytes in the deposit. This was a case of polyorrhomenitis, but the high chloride- content and the low " salt " ratio points to a transudation. Inas- much as there was no direct evidence of inflammation in the serous membranes, one may conclude that this name "-itis " is not strictly accurate. XIV. Alice D., aged 44. — The deeply blood-stained and spontaneously coagulable fluid had a specific gravity of 1021 and contained a considerable amount of albumen (3 per cent.). The chloride electrolytes amounted to 0104 gram-mol. per litre, and the total electrolytes amounted to O'lig, so that the achlorides were 0"0I5 and the " salt " ratio 014. The fluid contained 098 per cent, globulin ; invertase ; prot- albumose, urea, but no glycoproteid. The deposit consisted mainly of lymphocytes. There were mast cells, but no endothelial or carcinoma cells. This was a case of tuberculous peritonitis. XV. John T. I., aged 44. — The deeply yellow-coloured fluid had a specinc gravity of 1016, and the scanty deposit was made up mainly of polygonal cells like endothelial cells. The chemical examination showed 226 p»,r cent, of albumen, 017 per cent, globulin, and 4"63 gm. per litre of chlorides. There was a large amount of urea. Amino acids were not found. Albu- moses Were not found, but a trace of mucin was noted. 250 STUDIES IN PUNCTURE-FLUIOS .if This was from a case of effusions into all the serous cavities, associated with dilated heart and previous pneumonia. The effusion was of transudatory nature, as shown by the specific gravity, the albumen-content, and the considerable amount of chlorides. The other tests were not applied in this case. XVI. Anthony S., aged 28.— A straw-coloured fluid, having a specific gravity of 1020, and containing 475 percent, albumen. The concentration of the chlorides was 0-037 gram-mol. The osmotic concentration was 0-317. The chemical examination showed the presence of urea, but no albumose, or glycoproteid, or ferments. The a-naphthol test and the glucosamine test were positive, but slight. The cellular elements consisted of large mononuclear cells and vacuolated cells. The characters fit well in with the typical signs of an accumulation of fluid due to back-pressure; the case was one of incompensated valvular disease of the heart. XVII. Florence W., aged 23.— A straw-coloured, blood- stained fluid, yielding a bulky coagulum. had a specific gravity of 1022 and contained 675 per cent, albumen. The concentration of the chlorides was 0059 gram-mol., while the total concentration of the electrolytes amounted to 0-128. The achloride electrolytes therefore amounted to -069 gram-mol., and the achloride/chloride ratio was 1-17. The chemical examination showed the presence of stellar phosphates and oxalates ; no monamino-acids ; no ferments. The a-naphthol test and the glucosamine test were positive, but slight. The cellular elements consisted mainly of lymphocytes and red cells. The characters are in accordance with the diagnosis of tuber- culous peritonitis— low chloride-content, high "salt" ratio, absence of ferments. XVIII. Lucy H., aged 23.— A turbid yellowish fluid of specific gravity 1020 and containing 2 1 per cent, albumen. The chlorides were very scanty, amounting only to 0-05 per cent. Metalbumen was found on examining the precipitate after treating the fluid with thrice its bulk of absolute alcohol. The concentration of electrolytes was 0-113 gram-mol. o-*- SPECIAL CASES 251 litre, the achlorides being 0112 gram-moL, so that the electrolytes were almost solely achlorides. (Achloride/chloride ratio =■ 1 120.) Examination of the deposit showed a very large amount of cholesterin crystals, and also a very considerable quantity of fatty acid crystals. There were some granular epithelial celb, but the bulk of the deposit was unorganised. Report. — The fluid is most probably not ascitic, but derived from an ovarian cyst — to judge by the chemical characters* Operation. — Multilocular ovarian cyst. Comment.— l\it facts in favour of the fluid being ovarian considerable albumen-content, very low chloride-content. are ; presence of metalbumen, the achloride/chloride ratio, as as the cholesterin and fatty acid crystals. well APPENDIX TABLE I - For ready reckoning of Chlorides from No. DF CC. OF Ammonium SULPHOCVANIDE SOLUTION USED cc. gm. 1 cc. gm. 1 cc. gm. cc. urn. cc gm iixd. per liUe. used. p«r litre. u»ed. per litre. 1 used. per litre, used. per litre. ' 30 . . O'OO 26*0 . , 2*00 22'0 . . 4"00 180 . . 600 140 . . 800 399 ■ •05 •9 •• 05 •9 .. 05 •9 . . '05 9 • •03 •8 . . -10 •8 .. -lo •8 .. '10 •8 . . -lo -8 . 10 7 • •15 7 •• 15 7 •• '15 7 • . -15 7 • • 'li •6 . . -20 •6 . . -20 1 -6 . . '20 •6 . •20 '6 . •2 •5 • ■25 255 •■ 225 21-3 .. 425 17-5 • . -25 135 • •23 ■4 • ■ -30 •4 . . -30 ! -4 . . -30 •4 ■ . "30 4 . 30 •3 • 35 •3 •• 35 -3 •• '35 •3 • • -35 -3 •35 ■2 • 40 •2 . . -40 1 -2 . . -40 •2 •40 '2 . -40 •I ■ 45 ■I .. -45 1 •' •• "45 •I . -45 -I •45 a9'o • 50 25*0 . . 250 ; 2fo . . 4"5o 170 . 650 13-0 . 8-50 28-9 . ■55 •9 .. 55 "9 •• "55 •9 ■ "55 '9 ■55 •8 •60 •8 .. 60 -8 .. -60 •8 . -60 -8 . 60 •7 • -65 7 ■• '65 7 •• "65 7 • -63 7 . -65 •6 • 70 •6 . . 70 -6 . . 70 6 . 70 -6 . 70 •3 • 75 24-5 •• 75 205 ■• 75 i6-5 • 75 12-5 75 '4 . -80 •4 .. -So -4 .. -So •4 . . -80 -4 •80 •3 • 85 •3 • • -85 ; -3 • • -85 ■3 .. -83 -3 .. -85 •2 .. 90 •2 . . -90 -2 . . "90 •2 •90 '2 . . -90 •I •95 •I .. -95 ■! •• *95 •I .. -95 I • • -95 28-0 .. 100 24'0 . . 3'oo 20'o . . 5*oo l6'o . . 700 120 . . 900 27-9 . . 05 •9 •• '05 9 •• '05 •9 . . 05 9 •05 •8 10 •8 .. -lo -8 .. -lo •8 . . -lo -8 10 •7 •• '15 7 •• "15 7 •• "15 7 .. -15 7 ■15 •6 . . "20 •6 . . "20 -6 . . "20 •6 •20 '6 ■20 •5 •• '25 23-5 •• "25 19-5 •• "25 155 ■ ■ -25 11-5 ■25 •4 • ■ "30 •4 .. -30 4 .. -30 •4 . . -30 '4 .. 30 •3 •• "35 •3 •■ 35 "3 •• 35 •3 • • -35 -3 •35 •2 .. -40 •2 . . 40 "2 . . "40 •2 ■40 -2 .. -40 •I • • "45 •I .. -45 ■! •■ "45 •1 .. -45 -I •45 27'0 .. 1-5" 230 .. 350 190 .. 550 150 .. 7-50 110 .. 950 26-9 • "55 •9 .. 55 ; '9 •• 55 •9 • • ■« "i .. 55 •8 .. -60 •8 . . -60 -8 . . -60 •3 . . -60 -s .. -fto •7 .. -65 7 .. -65 7 •• "65 7 .. -65 7 .. -65 •6 •• 70 •6 . . 70 , -6 . . 70 ■6 . . 70 , -6 .. 70 •5 •■ 75 22-5 . . 75 : i8-5 . . 75 145 • • 75 10 5 .. 75 •4 .. -So •4 . . -So '4 . . "So •4 . . -So ! -4 .. "So •3 ■• -85 ■3 ■ • -85 -3 • • -85 •3 .. -85 -3 .. -85 •2 .. -90 •2 . . "90 i '2 . . '90 •2 ■90 : "2 .. -90 •I •• '95 •I .. -95 ' •! -• ■'>5 ! •! • • -95 •! -- -95 26'0 .. 2'0O 1 22'o . . 4'oo 1 i8'o . . 6-00 ! I4'0 . . 8'oo i lo'o . . 1000 2-53 iri 254 APPENDIX TABLE II APPENDIX 255 TABLE U— continue J Gm. / Gia.-i»ol. Mol«.+ '' knu. Cm. / Gm.'inol iun*. 3' 525 33 3'33 34 543 35 355 3<' 5"5 57 573 5-8 5-85 59 5-95 6'cxj 605 6- 10 615 62 6-25 63 6-35 6-4 6-43 6-3 ''•53 6-6 665 67 ^75 6-8 685 6-9 695 7"oo 7''35 7-10 715 72 7-25 73 735 7'4 745 7"5 753 7-6 77 775 0088 o"o89 0090 0*091 0'092 0-093 0094 0093 0-095 0*096 0097 0*098 0*099 0*100 0*101 0*102 0*103 0*104 0*105 0*105 o*lo6 0*107 0*107 o*lo8 0*109 0*110 0*1 II 0-II2 0*113 0*114 0*115 0*116 0*116 0*117 0*118 0*119 0*120 6121 0*122 0*123 0*124 0*124 0125 0*126 0127 0128 0*129 0*130 0131 1132 o*M3 0*134 845 845 •844 ■844 •843 ! •843 i 842 ! •842 I ■841 I •841 1 •841 I 840 i ■840 •839 ■838 ; •838 i •837 •837 •837 i •837 ' •837 i •836 i ■836 I 836 •833 I •834 •834 •834 •833 •832 •832 ■832 ■831 •831 •831 •831 •830 •830 •830 •830 829 829 *829 *828 •827 •826 •826 *826 •825 •825 •82s *824 1623 , 7-8 1642 7-83 1659 7 9 *i678 7'95 16CJ5 8*00 •J713 1 8*05 •1731 •I •173» •>3 •1748 •2 1767 25 •1785 •3 •1803 •33 1821 •4 *i838 •43 •1856 •5 •1874 •33 •1892 •6 *l9JO 65 *l9i8 7 •1928 75 •1947 *8 *1964 •85 •1965 •9 •1982 •95 2000 900 *2017 05 •2035 1 *2o54 ■13 •2055 'i -2 •2088 ;i -25 *2io6 •3 2113 •35 2123 ■4 2142 •43 *2l6o •5 *2178 ■55 *2200 i -6 *2214 •2232 *2250 *2267 •2267 *2286 •2303 •2320 ■2337 •2338 •2383 •2390 *24i8 •2436 •2410 65 7 75 *8 •85 •9 •95 10*00 10*05 *1 •15 *2 •25 •35 0*134 I 0*135 ; 0*135 ! 0136 I 0137 ! 0*137 I 0*138 0*139 I 0*140 0*141 \ 0*142 0*143 ' 0*143 0*144 0*145 0*146 0*147 0*148 0*149 0*150 0*151 0*151 0*152 0*153 0154 6* 1 55 0155 0*156 01 57 0*158 01 59 o*l6o 0161 0*162 0*162 0*1' 3 0*104 0*165 0*166 0*167 •167 1 0168 0*169 ; 0170 0171 0171 0*172 0173 0174 I 0175 I 0*176 I 0177 i ■824 ■824 •824 •823 •823 •823 •822 1" I ■822 •822 •821 I •821 *821 1 •821 ' ■820 i *82o *820 •819 819 819 819 819 *8io 818 ■818 •818 817 •817 *8i6 *8l6 •816 •815 •815 •813 •815 •814 *8l4 ; -813 I -813 i 812 1 *8l2 ' *8l2 ■812 ! -811 •811 •811 *8lo •810 *809 809 809 •SoS •244*1 *2463 246a •2479 2470 2497 •2514 •253 « •2550 ■2573 •2585 -2604 *2604 ■2622 •2640 •2657 •2675 •2692 *27io •2728 •3746 •2746 •2764 •2781 •2799 *28i7 *28i5 •2834 •2847 •2869 2887 *2904 •2920 •2940 •2940 2956 2974 •2991 j -3009 3027 ■3026 •3044 ■3062 307a •3096 •3096 ! i^ii 3131 •3147 I -3165 ! 3183 •3200 It I 25^ APPENDIX TABLE 111 specie ConauctivUv o. c>-nor.a. and J..n.uanorn.a. so.ut.on, of •^ I'otassium Chlorulc. At i6' it is 001072 for ^ KC» and ooo2294* 0'> •002659 •002712 •002765 ■002819 •002873 Temperature co-efficients ; NaCl 1 ,. 05 gm. equiv. per litre = -0238 j Correct I let ween -. a and 34°C. I o': O'Ol O'OOI •05 •01 •001 •0001 •00005 0238 '°***? \ Correct uciwcvi -"^■^^i 26 and 40^ C. •0254 I Correct between ('..vcn the conductivity at 18 C, * i.e. 229^4 X 10 = •0253.' = -02238 (c) =02255 = -02269 = •02284 = -02273 tt - i8)-]. •oooo795'-C. 848 ., 850 ,. 740 „ 901 „ 114 APPENDIX 257 TABLE IV Ratk or Mic.BATioN or loss. •0003 gn>. cquiv. per litre •001 •ooj .. '• •005 •• " •01 •• " •03 ». •05 .. Na 43' 4i'> 4i4 41'4 405 3»-3 35■.^ Cl f>4-H '•44 ityo fli'O yi>» VS'. 5"S ito. 60 66 60 35 47 3* TABLE V Obach's Table 0000 1 -ooio •OOiO ■0030 •0040 ■0050 •0060 ■0070 •0081 ■cog I 2 3 4 5 6 7 8 9 10 'OIOI 11 -on I 'OI2I ■0132 14 '0142 15 o'S^ •0163 •0173 •0183 12 13 16 «7 18 100 ■nil •1123 •I 136 ■I 148 •I 161 •1173 •1186 1198 -1211 •1223 19 0194 •1236 •1249 ■1261 •1274 •1287 •1299 •1312 •1325 •«337 •1351 20 -0204 21 0215 22 0225 23 0235 24 -0246 25 0256 26 -0267 27 "0277 28 0288 29 -0299 300 aoo ■2500 ■2516 •2531 •2547 •2563 ■2579 •2594 •2610 •2626 ■2642 •1364 •1377 •1390 •J 403 ■1416 ■1429 •1442 •1455 •1468 •1481 •4306 •4327 •4347 •4368 •4388 ■4409 •4430 •445« •447* 400 •66^)7 •f.694 •6722 •6730 •6779 •6807 •6835 •6863 •6892 •69* ' •2638 •2674 •2690 •2706 ■2723 ■2739 •2755 •2771 •2788 •2804 •4493 •4514 •4535 •4556 •4577 •4599 •4620 •4641 •4663 •4684 •2820 •2837 •2853 •2870 •2887 •2903 •2920 •2937 •2953 •2970 soo i-jooo I 0040 j-cx)8o 1-U121 I-Olfjl I'O202 10243 1-0284 10325 10367 600 700 too 00 •6949 ■6978 •7007 •7036 •7065 •7094 •7'23 •7«S3 •7182 •7212 •4706 •4728 •4749 •477> •4793 ■4815 •4837 •4859 ■4871 •4903 •7241 •7271 •730' •7331 •7361 •7391 •7422 •7452 •7483 ■75'3 1-500 1-506 1-513 I-5«9 1-525 1-532 1-338 1545 I-55« 1-558 1-0408 I 0450 1x492 10534 1-0576 1-0619 l-o66l I -0704 1-0747 I -0790 I 564 1-571 1-577 1-584 1-591 1597 1-604 1-611 1618 1-625 1-0833 1-0877 10921 1-0964 1-1008 1-1053 1-1097 11142 11186 1-1231 2333 2-344 2356 2367 2378 ;i-390 2-401 2-413 2425 2-436 2-448 2-460 2472 2484 2-497 2509 2-521 2534 2-546 2 559 4-000 4023 4-051 4-076 4-102 4128 4-155 4-181 4-208 4-236 4-263 4-291 4-319 4348 4376 4*405 4435 4464 4-495 4-525 1632 1-639 1-646 1*653 1-660 1-667 r674 I -68 1 l(.88 1-693 2571 2-584 2-5^7 2-610 2-623 2-636 2-650 2-663 2676 2-690 9'Oo 9-10 9-20 93» 9-42 953 9-64 975 987 9'9'> lo-iF io'24 10-36 10-49 10-63 10-76 10-90 II 05 1 1 '20 11-35 4556 4587 4-618 4650 4-682 4-714 4-747 4-780 4-814 4-848 1: 50 11-66 11-82 11 99 12-l6 .12-33 12-51 12-70 1289 13-08 17 258 APPENDIX TABLE W— continued Obach's ^^al.E-cnulinued !•! 30 '0309 31 -0320 32 -0331 33 '0341 34 '0352 35 •03f'3 36 -0378 37 "0384 38 -031)5 39 '0400 40 -0417 * 41 -0428 • 42 0438 ■ 43 ■°449 44 -0460 45 '0471 46 -0482 47 ■°493 48 -0504 49 '0515 50 -0526 51 -0537 52 -0549 53 °5^° 54 -0571 55 "OSS^ 56 -0593 57 -0604 58 •0616 59 -0627 60 '0638 ■ 61 -0650 • 62 '0661 ■ 63 -0672 64 -0684 65 -0695 66 -0707 67 -0718 68 -0730 6y -0741 70 -0753 71 -0764 72 -0776 73 -0787 74 -0799 75 '0811 7(1 -0823 77 '0834 78 -0840 79 -0858 V ' APPENDIX 259 TABLE V — coniintud Obach's Tkble— continued a 80 "08 70 81 •0881 82 '0893 83 -0905 84 -0917 85 -0929 86 '0941 87 "0953 88 -0965 89 -0977 90 "0989 91 -looi 92 -1013 93 '1025 94 •1038 95 'lojo 96 -1062 97 •i°74 9? -1086 99 "1099 loo •ilii 432 FF <^ to find -^ '*-^— ' . look down the 400 column till 32 of column , ■ is reached, the number 7606 is the one required.] 26o Arri"Ni'i>^ ■».-ij3iiil^'imiV in Jill'-- ^J.l .„.,„..u^M„o %% ? U ^>. :=■■ r- '^-^rr.rr'r- - - - r. - - - ^ .-^ •uol-ojcI.Kl *■?>?- ~ ?■.-.".-.' ." •' •" •"•"•'•'■ • ■ '^ ',. '_ ■-. -aiainUrallV 111 .un^siia niji HUSO Uill>r3a4l-''313II(IfOUl-lV ni iiu^vaia •noi-iri'iua:iuto rmoia*0 uoirsaiilaa boo -■r-,-r^'ro^^5:X5i:J^:r-.r. t:t ^ »f IT", iT; C '^ *^ ™ . ." ^ • • *_ ■ ■- * « * - IT-, = IT' - vn "-. -^^ = l;^ 1 2 5- i^ c c' P- f>-« » 5'^ ^ 5:,3 ? c ^ - ^^i^« '-i ^, ^. ^, ^ ^ ^1- ^ ?^ ?^ ?• ,.r; '^, "", '^, '*^ ■*^' "^^ ^' .'.'.'. ■ ' • ' ' •nonEiin3^uo:> i DIIOUISO uoi-isaadaa ^ ;ri SI =-. _s- S-, _^. ~i ^ :^ r' T' .•.■.•.••■ • ■ :_ z,,^^ • • • - -■ — '^ •t 't "^^ ^ "^^n - "-.0 "~. = ""■ = ^' 2, -• 5 -• i _ si n' S-. -^ 5" 7 ^ \C u-ir. u-iif.C £~ ■uonciias^noo i ^ 'mi0lU5O ■11015531(130 I ■aaiiiaa 1 ! >313H(l50tl»V III 3ill^^^Jc^ •unnrJin3:'Uo:i 'nijouisO •tioissiidsa jiaiM.iiJ i;, ■it, -.n \r. -^ -.n •^, -^ -m .r, .^. m ^. -. .^•^- -'_'^^^:^^ ./-. "-. ; '^. : "T 5^ ^' « ^' ^ - 5 N 2, IJi -iir; c C t^'>xx..j,5.u.^u.jiy-,fju-. m i--r"t;r;t;tT'T^7'^~.' - U-, ""■ •'■• O ^ ->^ 2 :t 3 !5 1 1 T .t .Tf :^ th.zC t^ f^ f^ '^'^ _ _ - - TIIII^IIMSIJ^' r'*? ?^ ^ ^ ^ ' ^ m APPENDIX 261 r. lixx r s s r f ^^ •' ^ ^ '■ ^ ■? '^ '^^ ? ? ? r j; ";:; ':i t:! i:; ij :: ::; "n ':! '-, 'A 'A i: n- J:- ? ;:■ ? ? ? ? ? ::• ^ ^ ? ? ? ? ? ? •t r->. C ''* "*'X — J ; ?'1 ^ ?r r 5 ?i^?r'?r3-5il-^'?f?-'li'i'?S">S"^ .ijf'fi « «"x ? ? ^ ^ ? ? ? ? ^ ?■ :> ? : J :>> ? : £ : ^: E _? ^,s, p^§ ?.i ? i^i^f ^ f s ? ? ^ I r f- ? ?> tl f ?f t>, ^, C" "^ •* '^ I 2ri£5 7^rir-5l??||?ti'il>li^^MiiiH w • -r »oc C f^ t^x - - - w . r* 7 . - 3 .7 . • . >- . ■ . ■ ■ V, „ V oo«acocdoxocoC3C3CXxac £"££■ b_ Ml ^M PJ ^M '^. '^ '^, ^ "^ •t' ^ £ 5 « X * «-«-x X 5^c?o?x « X X X Xxxxxxxxxxxccx - ■?> C C o *< -N *l N - * - o 3^ _ si M S. r^, S- t u". "1 C C f^ f^=C X ? ? mo "no mo mo ""-om; mp m 5 r r -i tj :C' ^p 7 7 IT' IT -J -i '-■"3' N f^, '^ "t m mc C f^op ^ .- !/■, m -^m«- tN'»-, ;vm-< ixmc^m- . --. a> 2 " - N N r^, -r 'f m mc t^ f^sp '>^ ?' 9 ? T T •4 V V V -J v •-!■ 'ij ir ir '^^ ir ir i:- ::■ ^' :? r^ r- ::■ r- r' ::■:?::■ iT' ;r :r ic^j^^ _ _ _ N ^ ^ tN. t>, r>. (N. tN. IN, ~ - - y. n ,u S ,.. ,-1 d »1 «l IN N N N M c^ m ^^ m mc t^ «Nw X - = ?■ r' r ?«■ ?• r rf i* y'-? f f^ ^^ £ -i. in w". !r m m m tr, m m m m m m m _>r, u-, ir, u-. m^ ^ „yyy--^r>'r >• • ■ • t 1- t -t >r. O C m c m _ -r 7 1Q03 264 LITERATURE Freun9 I edin. Zeit. f. physiol. Chem.. 21 Hedin, S. G. Biocheni. Journ.. 1. 4'^4 Henschen. Zeit. f. klin. Med., Ixiii. 1-4 Hobcr. Physik. Che,n. ic)oft Hofmeister. Zeit. f. physiol. Che,n., iv. 2,3. 1880 V Hoist. Ze.t. f. physiol. Chem., 4H Hopkins and Cole. Journ. of Physiol., 2Q. 1003 Hopixs-Scvlcr. Physiol. Chemte. Hosch Deutsche Zeit. f. Chirurg.. xc. 1-3 Moscn. i^cHiiwi ' A„uiiunB z Analyse des Hams. Huppert, Ncubauer, and\ogcl. Anlettung z. .i"'"y 1800 V. Jaksch. Klinische Diagnosttk. 1004 Joachim. Pfliiger's Archiv, 03 Joachim. Miinch med. WocU.. 44- Jollcs. Wien. med. Woch. 1804 Jolles. Munch, med. Woch., 3. 1008. « Aerzte. Dresden, IQ07 Jollel' li. /. P/O'-^-/. Chen,., xxv. 236 ; Z.7. /• hl.n. Med., xxx.v. 53, '89« Kofmann £>'e<'6. rf- J°''.vs., i- 1-28? KrS^r and Schittenhelm. Z..7. /. physioL Che,n., xxxv. .54 Kutsche. Zeit. f^^^y^'-^'-'H'-^X Lecithide der Schlangen- Kyes. Btochem. Zett., n. 09- ^ "^' gifts " Landwehr. Zeit. f. physiol. Chem., v. 375 Lancstein. Jahrb. Kinderh., Km. 925 Levene and Rouiller. B,ochem. Zer., -v. 322. ^oct .1 phangrupiio im Protcin-molekui;' T J • o„.i 7oT-npV Z£i7 /. physiol. Che,n., p. 23/ mtiirlichen Lecithins " Michaelis and Rona. B../... Z«7.. ii. 2,9. iii- 109. iv. :2. v. 36;. Mitiukoff. Arch. f. Gyncikologie, 49 Melon. Gazz-d. Osped. Padua, p. 149. 1904 7g Versamml. Dent. Xaturf. LITERATURE 265 Moore, F. C. Medical Chronicle. December 1007 Morgcnroth. Biochem. Zeit.. iv. 24«. " Ubcr Toxolccithide " Morner. Shand. Arch. f. Phys.. vi. }}2, 1805 Muller. Mitteil. a. d. tned. Klinik zu Wtirzhiirg, \. :.<«5. ^50 Nakavama. Zeit. f. phvsiol. Chem., xxxvi. y,H Nenski and Zaleski. Zeil. /. phvsiol. Che,,,., xxxiii. 2,17. K'Oi Neubauer. SiUber. d. Gesell. /. Morph. u. Physiol. 1,1 Munchen, u>^^^ Neuberg. Biochem. Zeit., vi. zjf^. " Verschiedenes iibcr Trypto- phan " Neuburg and Strauss. Zeit. f. physiol. Che„i.. xxxvi. 2.^ Niosi. Virchow's Archiv, u«) Oerum, Maly's Jahrb.. xiv. 450 Ohrmeycr and Pick. \Vie,\. kliti. Rundschau, p. 15. i'X)2 Otolski. Bioche,n. Zeit., iv. 124. "Das Lecithin des Knochcn- marks " Otori. Zeit. f. physiol. Chetn., 43 Paijkull. Zeit. /. physiol. Chein., xii. 106 Porges. K. K. GeseUsch. d. Aerzte in Wien, 1008 Reben. Zeit. f. physiol. Chenu. xliii. 320 Rcissner. Virchow's Archiv. xxiv. iQi Rivalta. Riforma Medica. 1805 ; il Policlinico, kk)4 Salkowski. Practicum d. physiol. n. pathol. Chen,., 190. Salkowski. Virchow's Archiv, 131 Sarzin. Inaug. Dissert., Berlin, 1894 Schmiediberg. Arch. /. exp. Path., vii. 166 Schulz. Die Crosse des Etweissmolekiils, ick>3 Soldner. Zeit. f. Biol., xxxviii., 237 Stahelin. Miinch. med. Woch., 34. 1902 Tollens and Krober. Zeit. f. angewandle Chem., iW2 Toyosunii. Mtinch. med. Woch., 40, igo" Umber. MUnch. med. Woch., 28, igo2 ; Zat. f. klin. Med., 48 Weisz. Beitr. z. Klinik. d. Tuherk., viii. 2 ; Wien. kltn. Woch., xxxiii 7 Willanen. Biochem. Zeit., i. 108. " tJber das Verhaltens des Ovomukoids im Organisnius " Zangerle. Miinch. med. Woch., 13, iQOO Zanetti. Ann. di chini. e Far,nacologia, xxvi. 12 FERMENTS Ascoli and Izar. Biochem. Zeit., vi. " Beeinfliissu.ig dar Autolyse durch anorganische KoUoide " Ascoli and Bonfanti. Munch, med. Woch., n)02, 1904: Zeit. f. physiol. Chem., 43 Baer. Miinch. med. Woch., 44, 1906 266 LITERATURE " The Dia8» Bainbridgc and Bcddanl. Biochem. Joun,.. ii. «0. tatic Ferment in Tissues in Diabetes Mellitus Bartarelli. Rhisla d'Igiene e Sanild pubblica, ic>04 ^^ Beam and Cramer. Biochem. Journ., ii. 174- " Zymo>^» Beitzke and Xcuberg. I'erh. d. deutsch. pathol. Gesellsch., 1904 filum and Fuld. Brochem. ZeU., iv. 6.. " Die Best.mmung des Fermentcehaltes im menschlichen Magenmhalt Bredi, Biochem. Zeit., vi. 283 ; " Altes und Neues von der Katalvse " Burian. Zeii. /. physiol. Chent., xliii. 509 Chvostek. IVien. klin. Woch., i»()(y V. Dalmady and v. Torday. Bericht aus d. biolog. SUz. am 3. April 1906 Dastre. C. R. Soc. Biol., Iv. 130 Delezcnne. Comptes rendus, 1903 j » * A. V. Drjewzki. Biochem. Zeit., i. 234. " Beemfliissung der Auto- lyse durch Alkali " V Dungern. Die Aniik'drper, igo6 Ehrlich and Morgenroth. Berl. klin. Woch., 1899-1902 Eppenstein. Munch, med. Woch., 45. ">o6 Erben. Zeit. f. Heilk., 24; ^"'- /• ^in. Med., 40: Hofmeister s Evani"' BiL^m. /owrw., ii. i33- "On Catalytic Decomposition of H,0, " O. V. Fiirth. vii. Internat. Physiol. Congress, 1907 Fush. Verein. f. inn. Med. zu Berlin, v-ii., 1907 Hamburger. Fol. haematolog., ii. 1905. » . » *•* _;„ » S. G. Hedin. BiocA.m. Journ., i. 474- " Trypsm and Antitrypsin Hedin Biochem. J omn., '\i. \12 van Itaaie. Soc. de Biol., January 20. 1906. " Verslagen van d, Koninklyke Akad. v. Wetenschappen te Amsterdam mssen- naturkundige afdeeling," p. 540, 1905- ^^ Jacoby. Biochem. Zeit., i. 53. "• i44. ^47, iv. 21, 47 1- i^er- mente und Antifermente " lastrowitz. Biochem. Zeit., ii. 15? JoUes. 76 F^rsamm/. deut. Naturf. a. Aerzte, Breslau. 1904, and Miinch. med. Woch., 47, 1904 Klemperer. Miinch. med. Woch., p. I4S4. I907 Kiittner. Zeit. f. physiol. Chem., 1. 494 Leber Die Entstehung der Entzundung, Leipzig, 1891 Liebmann. Klin. Untersuch. a. d. .-Ibteilung B des Kgl. Fretdrtchs Hasp. Copenhagen, 1907 _ Lockemann. " 79 Versamml. deut. Naturf. u. Aerzte tn Dresden, 1907 Lohlcin. Hofmeister' s Beitra^e, vn. 120 LITERATURE 267 Michaelis. Deut. med. Woch., 1004 Migliarini. Riv. Ven. di Scienxe medicaU, xxi.. 2. 3, i<>04 Molon. Gazz. d. Osped. Padua, 140. ">o4 Morgenroth. Zentralbl. f. Bacteriologie, xxvi. uo Ed. Miiller. Deutsches Arch. f. klin. Med., xci. 13; Munch, med. IVoch., 53, ifX)7 - Miiller and Jochmann. Mtinch. med. Woch., 20, 31. 4i. 43- "»" Nickel. Die Farhenreactionen der Kohlehydrate. Berlin, 19CK vH. Peters) Opie. Journ. of exp. Med., vii., 1905 Oppenhtimer. Carl. Die Fermente u. ihre bwlogische Bedeutung. Mod. Arzt. Bibliothek, vol. 16 Pfeffcr and Hofmeister. Chemische Organisation der Zelle. Braun- schweig, Kyoi Pfeiffer. Wien. klin. Woch., 42, 1906 Prcti. Biochem. Zeit., iv. 6 Reicher. Wien. klin. Woch., 48, 1907 Robson and Cammidge. Surgery of the Pancreas, \m7 _^ Schade. Miinch. med. Woch., 3«, i907- " Diabetes und Katalyse Schittenhelm and Schmid. Zeit. f. exp. Path. u. Ther., iv. 2 Schittenhelm. Zeit. f. physiol. Chem., xliii. 228, 251, 343 Schumm. Hofmeister' s Beitr., iv. c>-n, v. vii. Schumm. Zeit. f. physiol. Chem., xxxvi. 296 Sieber and Schumfi-Simonowski. Zeit. f. physiol. Chem., xxxvi. 244 ; xlix. 50 Sieber. Zeit. f. physiol. Chem., xxxix. 6 SUbergleet and Mosse. Beit. z. klin. Med. Festschrift Senators, 1904 Solms. Zeit. f. klin. Med., i, 2, 1904 Sprig!|S. Zeit. f. physiol. Ckem., xxxv. 480 Volhard. Munch, med. Woch., 49, 1903 ; 9. 1907 Walther. Die Arbeit der Verdauungsdriisen, Wiesbaden, 1903 Wiens. Deut. Arch. f. klin. Med., xci. 5, 6 F Winkler. Fol. haematologica, iv. 3 Wohlgemuth. Biochem. Zeit., iv. 271 : 79 Versamml. deut. Naturf. u. Aerzte in Dresden, 1907 " SECTION II AUaria. Jahrb. f. Kinderh. Ixiii. i Ascoli. Clin. med. Hal., 12, 1904; Vorlesmigen uber Uramte, Jena, 1903 Asher. Biochem. Zeit., iii. 335 Atkins. Brit. Med. Journ. Feb. 1908 Benedict. Biochem. Zeit., iii. ; FfiUger's Archiv, nS Bergell and Richter. Zeit. f. exp. Path. u. Ther., 1905 jM LITKRATURE ;, i'K'4 La Conductibiliti lonen- \iu ktl "Zur \.vhTc von .Icr Uitfah.gkcit des Scrums in L ramie. Deutsche m,d. Woch., 2H, i'^>2 : also Ze,t. f. khn. Mai., xuoz Bilz. hit lifsliniw. (I. M'^lecurareeiiichts, i-xu Bousma. Mt'l. Ivtlsflirifl i.wi (iemeskutuls, li. . Ccconi. Archif. per U Scume mediche, uioi. elettrica del SiiTo umano " Colin. MitUil. a. d. (.nnznih. d. Med. u. Chir., xvn 2 Fok, C. .-irch. di l-'iswl., i., ii. HamburKtr. Hiochen,. /.eit.. i.. n.. ui. ; Fol. hacm. n. Iflirc " HcrzoK Xeit. f. phvsik. Cluii:., y. 43 Hob^r PIns.k. Chen,, d. /elk u. dew., K/.6 ; PftiigerS .-IrfA.r. iHoo V Koranyi. Die wissenschaflltchen Grundtageu d. Kryoskvfte i» ihrer'klin. Amcemluug. Mod. Aerzt. Bihliothek. Belt i Moore and Koaf. Diochem. Journal, ii. " Direct Measurements of the Osmotic Pressure of Solutions of certain Colloids I'faundler Verh. d. 21. Versammlung. der Gesellsch. /. Ktnder- heilkunde. Wiesbaden, 1005: 76. Versamml. deut. Saturf. u. Aenle, Breslau, 1004 Kichter. Deri. klin. Woch., u^)? Robert -Tcssot. Viscosimeter, Fol. hacm.. iv. 4 Rossi. Arch, di Fisiol., i. 4 Sasaki. Virchow's Archiv, clxxxiii. 2 Schonborn. Gefrierpunke u. Leitfdhig. Beslimm. VVi-sbadcn. IQ04 Strauss. Bedeulung d. Kryoskopie f. d. Diagnose u. Therapie von Sierenerkrankungen. Mod. Aerzt. Bibliothek, Hefte 4. 5 Senator. Deutsche med. Woch., 3. looo Tezncr. Zeit. f. physiol. Chem., liv. i , . .^ . n, .. ;„ Wilson. Biochem. Journal, ii. '• The Conductivity of Blood in Coagulation " SECTION III Albrecht. Deut. Zeit. f. Chirurg., Ixxxvi. 2-4 Bab, Hans. Zeit. f. Get. u. Gyn., p. 60. u.oj ; Munch, med. Woch., 46, igo7 H V Bennecke. Miinch. med. Woch., 44. if^)/ Bernert. Arch. f. exp. Path. u. Pharm., 4Q , , • . ^, Bockelman. Sederlandsk Tydschrift von Geneeskunde, 1. 6, 1904 Bodou. PfliiS^r's Archiv, civ. g-12 V. Bokay. Deut. med. Woch., 47, igo7 Bonoli. Centralhl. f. Gyn., 633. kkj^ Cavazzani. Centralhl. f. Phvsiol., x. 145 Christen. Zentralbl. /. inn. Med., ic;u3-7 Dencks. Miinch. nicd. Woch., 37, 1407 Donath. Journ. of Physiol., xxxiii., 1905 UTEKATUKE 269 Zur Chcmie dcr Zerebro- EnRclmann. Mumh. med. Woch., 41. «'>'3 I-ahr. MitHch. mcd. Woch., ;, Hyo8 Foi. C. Arch, ill Phy-uologta, i. 2, ii;)04 Fonlos. CoHiptes remlus, 51, 56 Frcnkel-Hciden. B'ochtm. Zcit., ii., i ^^■ si)inalflus8iRkcit " Freunil. Munch, med. Woch., 7, igo8 Fuchs ami Rosenthal. Wien. med. Presse, 44-47. "XM Ful.l and Lcvison. Biochem. Zcit, si. A". " Die Feiwinbestim- munR mittcls dcr Edestinprobc " Gilbert and Castaigne. Compter rendus, ii. 70. i-4 Grober. Munch, med. Woch.. 8, 1900 Gross. Arch. /. exp. Path., 44 Gruber and Grunbautn. Miinch. mcd. Woch., o. 1004 : Centralbl. f. Phys., XV. 315. I'yos ; Verhdl. d. physik. mcd. Gesellsch. Wurshurg, ,?7. l<'<^'4 Griinbaum. D. Inaug. Dissert. VVurzburg. kaM Gruner. Biochem. Journ., ii. Griinhagen. PflUger's Archiv. 43 , „ , , Halliburton. .-1 Text-book of Chemical Physiology and Pathology, 1891 ; "Oliver Sharpey Lectures," Lancet, May 4. 1907 Hammarsten. Zeit. f. physiol. Chem., vi. 194 Hammerfahr. Munch, med. Woch., ^A, 1907 Hasebroek. Zeit. /. physiol. Chem., 12 Herzfeld. Zeit. f. klin. Med., Ixiv. i, 2 Heyrovsky. Wien. klin. Woch., 6, u,»o8 Hildebrand. Deut. Zeit. f. Chirurg., Ixxxvi. 5. 6 Hoffman. Arch. f. exp. Path. u. Pharm., xvi. 133 Hoppe-Seyler. Lehrbuch der physiol. Chem. Hueter. Ziegler's Beitrage, xli. 3 Jacque. Bull, de I'Acad. royale des Sciences de Belgique, 4, 1902 Joachim. Munch, med. Woch., 44. 1903 Jolles. Wien. med. Woch., 1894 V. Ketley and v. Torday. Deutsche Arch. /. klin Med., Ixxix. 5, 6 Kofnian' Ergeb. d. Physiol., i. i, 285 Kossel and Freitng. Zeit. f. physiol. Chem., xvii. 45^ Kostlivy. Deut. Zeit. f. Chirurgie, xci. 3, 4 Landolf. Biochem. Zeit. vi. 61. "Differential analysen von Mens- chenblut u.s.w." Landwehr. Zeit. f. physiol. Chem., viii. 114, 363 Langstein. Jahrb. Kinderh., Iviii. 925 Lannois and Boulard. Lyons Medical, 21, 1904 Lascialfara. II Policlinico, July 1907 Lehndorf and Baumgarten. -Zeit. f. Path. u. Ther., iv. 2 Liilenstem. Miinch. med. Woch., 34, 1907 IJO MTERATL'RE Lohnmcycr. num.. I m H,ilhhurlon's Text-hook of Chemical Phy- siology, i«'(i Lnthcrsin. H'len. kttn. Rundschau, I'/i; I-iiikc. Arch f. hint. Chnutg., ,? Marchand. Mtil. (iiselluh. :u Leif<:tg, Juno ii, I'xV Mam- and Ixva.liti. A>i>u,les dt llHshtut I'usteur, Fibruar>' IO07 MarischU-r ancJ Ozarkievvicr. Archiv. /. Verdaiiungshraukhetten, Qd v Marshall Jouru. of exp. Med., vi. ,?4". ><'<>5 Michi'h anti Mattirolo. Witn kitv. Woch., Kjckj Mieschcr. Znt. /. Phvsiol Chem.. 4 Mourson ami Schla« Ncumcistcr. Physiol. Chem., \>. 47^ Niosi. Virchow's A'chiv, Kw Nonne and Ajiclt. Arst. Vereni in HavMurg. Oct. 10. 1Q07 ; Arch f. Psych, u. Scrrenheillt.. Ji. 4.^ "'<^7 V. Noorden. Handhuch d. Path. d. Stoffwech.. Bd. i Olmcr and Audibcrt, Revue de .MfdeciMC, uxH Otori. Zeit. f. phystol. Chem., xliii. }ii Pickardt. lierl. klin. W'och., 1807 Pilcz. Wien. klin. Rundschau, nio' Pincussohn. Diochew. Zeit.. iv. 4S4 gung dcs Pankrcassattes " Poljakoff. Berl. klin. H'och., i. looo Quincke. Deut. Arch. f. klin. Med., xxx. 5. ifi Raehlmann. Miinch. med. Woch., p. Jo8(), 1003 Ricken. Arch. f. klin. Med., 22, i8q6 Rosenbach. Berl. klin. W'och., ifKU Robson and Cammidue. Surgery of the Pancreas, 1007 Rumix^. Berliner med. Gesellsch.. July 17. 1^7 ; Munch, med W'och . p. 1507. iry07 Runeberg. Deut. Arch. f. klin. Med., xxiv. V. Rzcntkowski Berl. klin. W'och., 227, ir>04 arzysteva Lekarskiego W'arszawskiego, iii., ,V Rzentkowski. Przsglad Ickarski, IQ04 Salm. Sederl. Tydsch. voor Geneeskunde, i. 16 Sasaki. Virchow's Archiv, ii. 183 Schmidt. Mitnch. med. W'och., 50. IW7 C. Schmidt. Hoppe-Seyler's Lehrbuch Schumm. Zeit. f. phvsiol. Chem.. xxxvi. jc/i Scipiddfs and Farcas. //rf..r'o BcitySge :ur. Geh u- Gyn., ix. 1 Sicard. Soc. med. des hop. de Paris, iqoi ' Die Gefricrpunktserniedri- Pamigtuik Tow- i(/)4 LITERATURE «7i SlcmcrllnR. fieri */i« Woch.. it, 1004 Stnzyiowski. H'l^H med. H'ock , 4^, i«J04 Straus* and GroMman Deutsch* mtd. Woch.,ftfy^, I'lOi ; lyf^. uniy StrauM. H. Du chnmtscken Siertntntxundungtn, p. 4"- Berlin, :■ »t Toyasumi. Miinch. mtd. Woch., 40, i«jo7 Vicarclli and Cepponp. Giornale dtlla R. Accademia di Mtdtcina di Torino, I'a>i WeyRandt. Physihalisch trud. Gesellsch. x. Wiirthurg, IQ07 : A/iiwfA. med. Woch.. p. 155, ifA>7 Zangcmeister. Mtinch. med. Woch., 41, 1004 Zcchuisen. Ceritralhl. f. inn. Med., xl. «<*6, 1018 Zock. Wien. Mm. Woih.. 15. uio^ SECTION IV Bcmhcim. Virchow's Archiv, 131 Bugarsky and TanRl. Pftiiger's Archiv, 72, 1898 Christen. Zentralbl. f. inn. Med., 13, 1005 Citron. Deutsche Archiv f. klin. Med., 4^. " Zur klin. VViirdigung des Eiweissgchalts und des sp. G. patholog. Fliissigkeiten " D'Este Emer>'. Bact. Diagnosis for ^ »iiioners. 1906. Engl. Berl. klin. Woch., 43. Kjo.l Englander. Munch, med. Woch.. i. 1007; Prager Zeitsch. f. Heilk. xxvii., ir^/) Forssner. MUnch. med. Woch.. i IQ05 Gruner. Biochem. Journ., ii. Halliburton. Text-book of Chemical Physiology, iSgi Hoffmann. Virchow's Archiv, 7». " Albumen Content of .\scitic Fluids"; Virchow's Archiv, 44. "Albumen Content ot CEdema Fluids " Janobki. Berl. klin. Woch., 44. i'^7 V. Ketley and v. Torday. Deul. Arch. f. klin. Med.. Ixxix.. 5, 6 Landolfi. Mediz. klinik. Ospedale Incurabili Xtapoli ; Riv. crit. di Clin, med., 4 Lazarus Barlow. Journal of Physiol., 1896 Mammi. ^ La Clin. med. Ital., 3, 1905 M^hu. Etudes sur les liquides. Archiv. gin. de mid., v. 7, 1872] Miram. Maly's Jahrb., iQoo MuUer. Deut. Archiv /. klin. Med., xci. 3, 4 Neuburger and «■ rauss. Zeil. f. physiol. Chem., xxxvi. 232 Neuenkirchen. Vber die Verwandbarkeit des sp. gr. u. des Eiweiss- gehaltes. Diss. Dor pat, 1888 Noel Paton. Brit. Med. Journ., ii., 1890 V. Noorden. Handbuck der Path. d. Stoffivechiels, 1907 27^ LITERATURE ■;-,t I 1 5 I M III Otori. Ziit. f. Hiilkuii(/i\ xxv. 5. 1004 Patella. Ma/y Jber., i,S.S8 Ranke. Vher Piinktionsjiiissi^keiten. Diss., H'iirsbiirg, 1886 Reiss. Arch. f. exf>. Path. it. Phann., 51 ; 76. Versamml. deut. Xatiirf. H. Aerzie, Breslan, igo4 Reuss. lieitrii^. f. klin. Benrtheiluni^\-. Exiuhitenund Transudaten. Deut. Arch. j. kliu. Med., 24, 28 Rivalta. // Policlinico, xi., xii., 1005 Roth. Arch. f. Atiat. 11. Phys., iSgg Rzentkowski. Paiiiie'uik Torwarzystwa Lekarskiego Warszaws- kie^o, Bd. C. Heft iii, igo4 Rzentkowski. Bert. klin. Woch., 0, kx)4 Runeberg. Berl. klin. Woch., 3^, 1807, "Klin. Studien iiber Transudationsprozesse im Organismus," Deut. Archiv. f. klin. Med., 34, 3;- Scnator. Virchow's Archiv, 111. " t'ber Transudation u. iiber den Einfluss dcs Blutdrucks auf die Beschaffenheit der Transudate." Stiihelin. Mihich. mcd. Woch., laoz. 34. " Uber den durch Essigsaure fallbaren Eiweisskorper der Exsudate " Strauss. Deutsche med. Woch., 2, IQ05. " Refractometrv' " Strauss and Chajes. Zeit. f. kliu. Med., W- " Refractometrische Eiwcissbestimmungen am menschlichen Blutserum " StrubcU. Miinch. med. Woch., p. 6i(, 1902 Tedeschi. Mediz. Klinik., Genua. Umber. Miinch. med. Woch., 10, 1Q05, " Zum Stadium der Eiwcisskorper in Exsuclaten " Zeit. f. klin. Med., xlviii., 1903 Wideroe. Xorsk Magasin f. L'dgevidershahen, 1907 SECTION V ! Barjori nd Mazucl. Arch. Gen. d. Med., 40, IQ03 Biberfjeill. Beitr. f. klin. Med. Festschrift Senators, p. 99, 1904. " Ergebnisse Zytologischer I'nter.iuchungen " Brior. Ceutralhl. f. allgcni. Path., xiv. (j^x) Bunting. John Hopkins Hosf>. Bulletin, xiv. 185, 1903 H. C. Earl. Dublin Journ. Med. Science, 1903 Emanuel. Lancet, January 12, 1906 Etlinger. Berl. klin. Woch., 46, itjo" (".Dggia. Gaz:. degli Ospedali ed Cliniche, 13, k>05. "Certain Leucocvtc Forms in Cerebrospinal Fluid " (iramegna. Rif. Medica. 28. IQ04 Cirawitr. Uber geformie Bestandteile in 48 pleiiritischen Exsudaten. Charite-Annalen, xviii. 1893 Grcnet and X'itry. Comptes rendus de la Soc. de biol., 25, 1903. '■ Cytologic dcs -Vscitcs LITERATURE 273 " Studien fiber Entzundung Heinz. Miinch. med. Woch., 7, 1900. seroser Haute " Jacobsohn. Medizinskoje Obosrenife, 12, 1903 ; Miinch. med. Woch., p. 1438, 1903. " Ubcr die Z>-todiagnostik der Exsudate " Jagri IVien. klin. Woch., 40, 1905. •■ Zur Farbung von Exsa- datzellen " Jansen. Kordisk Tidschrift f. Terapi, 9. 1906; Munch, med. yyoch., p. 1483, 1906. " IJber Zytodiagnostik von Pleura- ergiissen " JuUiard. Revue de Chirurgie, 1902. Th6se de Geneve, iwi V. Ketley and Arpad v. Torday. Deut. Arch. f. klin. Med., 190, Ixvii. I, 2 Koniger. Die zytolog. Untersuchungsmethoden, Jena, 1908 Koster. Kordiskt Medicinskt .-Irkiv, ii. 38. 4. 1905. " Die Zytologie der Pleura und Peritonealergusse " Lewkowicz. Przeglad Lekarski, 32-4, 1904; Wien. klin. Woch., X2, Lotti. kiv. critica di Clin, med., 18. 19. 1904. " Contribute alia cytodiagnosi dei vcrsamenti della sierose " Niedner. Gesellsch. d. Charite Aerzte, January 12, 1904 Patella. Deutsche med. Woch. ic^2. " tJber die Zytodiagnose der Ex- und Transudate " Quincke. Deut. Archiv. f. klin. Med., 1875 Raillon. Thise de Paris, 1904. "Lymphocytosis of Cerebro- spinal Fluid " Raubitschek. Zeit. f. d. Grenzgeb. d. Med. u. Chirurg., ix. 2, 6^ 1906. ^' Roscnbach. (See Literatnre to Section III.) Sahli. Lehrbuch der klin. Untersuchungsmethoden, 1905 Samele. La Clin. med. Hal., 2, 1905. " Uber die Zytologie der Pieuraergiisse " Saw>-er, J. Lancet, February i, 1908 Sicard. Soc. med. des hop. de Paris, 1901 Signorelli. // Policlimco {Sez. med. fasc, 10), 1903 ; " La Pleurite Lmfomatosa." La lUforma medica, 6, 7, ic)04. - Contribute alio studio citologico dei vcrsamenti liquidi infiumonatosi dclle diverse cirrose " V. Starck. Med. Gesellsch. in Kiel, July 6, 1907 Stassewicz. Sitzung der Gesellsch. Russischer Aerzte zu S. Peters- burg, October 22, 1903 Steinbach. Inaug. Dissert., Bukarest, 1903 Symes. ^edical Press, October 4, 1905. " Cvtodiagnosis " Vargas-Suarez. Beitr. z. Klinik. d. Tuherculose, ii. 3, 1904 " Uber Lrsprung und Bedeutung der in Pleuraergiissen vorkommenden Zellen ' 18 274 LITERATURE Widal and Ravaut. Compt. rend, de la Soc. de bid de Paris, June 30. October 13. December 22, iqoo : La Prcsse Mid., 1901. etc.. etc. Wolff. Zeit. f. klin. Med., xlii. 1901 CEREBROSPIN.XL FLUID Devaux. Cer^tralhl. f. Nervenhcilk. u. Psych., J^n^J?. 1903 Donath. Lecture XXXII., Meeting of Hungarian Medical Men in Kolozsvar, 1003 Fuchs and Rosenthal. Wien. mcd. Presse, xhv. 7, 1904 Funkc. Arch. f. Dermatol, u. Syph., Ixix. 340, 1904 Krctschmer. Deutsche med. IVoch., 46, 1907 Luticr. These de Paris, 1Q03 Merzbacher. Xeurol. Zentralbl., 12, 1904 Mever Berl. klin. Woch., 5, 1904 Nicdncr and Mamlock. Zeit. f. klin. Med., liv. i, 2 Nonnc Aerzt. Verein in Ho-'hurg, October i, 1Q07 i'appcnhcim. Zett. f. Heilkunde, xxviii. 10, UiOj Pcrcheron. Thdse de Paris, 1903 Preisisch and Flesch. Berl. klin. Woch., 1904. 44-5 Raubitschek. Zeit. f. d. Grerrzgeh. d. Med. u. Chir., ix. 2, 6-9. 1906 Ravaut and Darre. Gazette des kopitaux, August i 5, 1903 Sabrazes and Muratet. Soc. de Biologic., October 31. November 2., 1903 Schlesinger. Berl. klin. Woch., 28, 1904 Schwarz and Bronstein. Berl. klin. Woch., 35. '903 Siemerling. Berl. klin. Woch., 21, 1904 Verzeanu. Inaitg. Dissert. Bukarest, 1903 Voulcoff.-Montpellier. Thesis MM. Univ., No. 6. 1903 4 Hi INDEX OF AUTHORS i Abderhalden, 68, 84 Burian, 31, 76 Albrecht, 192 Burnet, 227 Allaria, 116 - Apelt, 169 Cammidgc, 71, 18). 186 Arrhenius, 7. 94, loi, 108 Cappone, 165 ■> Ascoli, 70, 85 Castaigne, 170 Ashcr, 15, 16 Cavazzani. 171 Atkins, 120 Christen, 159, 199 [ Chvostek, 84 l; Bab, 155 Claude. 48 Babesch. 167 Clemens. 55 Bacr, 81 Cohn. 116 \ Baldi. 45 Couerbe, 44 Bang, 43 Baumgarten, 170 Dalmady, 76 Bayer, 42 Danilewsky, 44 ; Bayliss, 15 De Coppet, 92 Bechhold, 161 Dencks, 192 Beckmann, 92 Deniges, 186 Beitzke, 88 D'Este Emery, 19/ Benimelen, 15 Devaux, 230, 231 Benedict, 135 Devillard, 163 : Bennecke, 167 Devoto, 20 Bergell, 45 Dc Vries, 117 Bernheim, 197, 199 Deycke. 14 Bibergeil, 227 Diakonow. 45 Blagden, 92 Dolgow, 55 Blum, 72 Donath. 169, 230, 231 Bockelman, 162 Drechsfl, 45, 51 Bodou, 152 Drjew^ki, 82 Bokay, 166 Durig, 29 Bonfanti, 70 Bonoli, 165 Earl, 228 Bosanquet, 22 Ehrlich, 55. 57. 223 Boulard, 170 Eichholz, 42 Brand berg, 72 EUingcr, 150 Bredig, 127, 175 Engelmann, 190 Bronstein, 230 Engl, 203, 204 Bugai'sky, tjy. 106, 118 Englinder, 200 Bunting, 223, 227 Eppenstein. 81, 89 2 75 276 INDEX OF AUTHORS 1 1 Erbcn. 27, 79, 227 Eriandscn, 45, 50 Fahr, 193 Parkas, 130, 165 Pels. 125. 126 Pishcr. 3O, 68 Pisher, E., 10, 65 Plorence. 194 Poi, 85, 128, 130, 133, 164, i'>5 Forssncr, 209 Pourcry, 44 Franchini. 42 Prankel, 130, 146, 184 PrenkelHeidon, 167 Frcny, 44 Prcunil, 24. 84, 149, 192 Prey tag, 146 Prietlcmann, i6i Priedenthal, 120, 134 Prk'drichsen, 150 Pricnd, i(i2 Puchs, 172, 173 Puld, 7Z. 87. 161 Punke, 231 Piirbringer, 168 Piirth, 77 Geisslcr, 55 Gilbert, 170 Gliissner, 185, 202 Glikin, 42 Gobley 44 Gorup iesanez, 162 Grawitz, 223 Grenet, 229 Grober, 170, 171 Gross, 159 Grossman, 159 Griibc, 165 Griinbaum, if)5 Griinhagen, 164 Gryns, 215 Gublor, 142 Gumprecht, 167 Halliburton, 42. 143, 14'^'. '49. I5'>. l6z. lO''. 1'"), 170 172. 180. 202 Hamburger, 7, 78, 9'-. 111, 117, 130, 104, 165. 214, 215 Hammarsten, 35, 101, 142, 143. 150, 152. 162, 163. 164, 178-180 Hamnierfahr, 158 Hasebrock, i<>2 Hausman, 28 Hcdin, 87. 215 Hein, 188 Henriqucs, 51 Henschcn, 194 Herz, 140 Herzleld, 147, 152 Hess, 7i, 135, 140 Hesse, 186 Heyrovsky, 189 Hildebrand, 191 Hirschsohn, 187 Hober, 16, 68, 125, 134. US. 215 Hofman, 170 Hofmeister. 86, 176 Holborn. 105 Holland. 58 i Hoist, 163 Hoppc-Seyler, 45, 145, 14O, 148. 156, 15S, 162, 163, 187 Hosch, 189 Huetcr, 184, 188 Husches, 150 Ibrahim, 14 Itallie, 76 Izar, 85 Jacobsen, 51 Jacobsohn, 223 Jacoby, 72, 84. 86, 88 Jac(i Pinkussohn. 187 Poljakotf. ihi Poll, 82 Prima vara, 37 Quevcnnc, 142 Quincke, i^ii, 167, 168, 223, 228 Raaschon, 43 Raehlmann, 160 Raoult. 02, 94 Raubitschek, 223, 228, 230, 231 Ravaut, 218, 223, 227 Reale, 23 Reuss. 197 Rieken, 167. 168 Rivalta, 37, 205 Robson. 71, 184. 186 Rona, 12. 44 Rosenstein, 143 Rosenthal. 172, 173 Ross, 140 Roth, 214 Roiiillcr. 58 Rumpel. 193 Runeberg, 200. 201, 204 Rzentkowski, 147, 151, 152, 203, 206 Schittenhelm, 31, 73 Schlagdenhautten. 19^ Schlesinger. 230 Schlosing, 29 Schmidt, 148, 172. 184 Schulz. 15 Schunim, 79. 185. 186, 187 Schumoff-Simonowski, 70 Schwantke, 39 Schwarz, 230 Scipiades, 165 Seliwanotif, 20, 32 Sicard, 167 Sieber. 70 Siemerling, 167, 1O9 Signorelli. 223 Silbcrgleet. 78 Solms. 72 Sorensen, 74 Spriggs, 72 Stahelin, 37. 199. 2"5 Starck. 148, 227. 228 Starling, 216 Stassewicz, 223 Stecker, 45 Steinbach, 223 Stern. 46, 50. 52, 81 Stozyzowski, 165 Strauss. 34, 150, 159. 2oC) Strauss. H.. 150 Tangl, 68, 99, 106 Tedeschi, 208, 209 Tezner, 107 Thicrfelder, 46, 50. 52 Thudichum. 44. 45, 46 Torday, 76. 152, 206. 223 Sahli. 166. 170. 218 Saleesky. 125. 126 Saliev, 55 Salkowski, 29, 39- 163. 180. 194 Salm. 164 Sasaki, 116. 155 Sawyer. 231 Schade, 88 Schere, 165 Scherer. 28 Schitf, 187 Umber, 10, 26. 27, 30, 36, 37, 190, 207 49, 60. 62, 83. Vages. 150 van't Hort. 69, 92. 94 i V, Bemmelen, 15 I I V. Bennecke, 167 i V. Bokay, 166 V, Dalmady, 76 v. Fiirth, yj V. Hoist. 163 V. ItallJc, 76 INDEX OF AUTHORS 279 V. KctK'y, 152. 206, 223 V. Noorden, 55. 203, 144 V. Starck. 148, 227. 228 V. Torday. 76. 152, 2(y), 223 Vargas-Suarez, 223 Vauquelin. 44 Verzeneau, 230, 231 Vicarelli. 165 Vitry, 229 Volhard. 71. 73, 62 Wachsmuth, 162 Walth r, 71 Weidel, 31 Weiss, 55 Weygandt. 171 Weyl, 165 Widal, 218. 223, 227 Wideroc. 206 Wiener, 82 Willanen, 20 Wohlgemuth, 71. 88 Wolff. 223 Wright, 119, 126, 219 Yorn, 172 Zaky, 48 Zanetti. 41 Zangemeister, 144, 147, 165, i8j, 184, i88 Ziingerle, 38 Zeehuisen, 187 Zeynek, 182 Zieglwallner, 192 Zilwa, 185 Zock, 157 ; INDEX [The thickened figures indicate main references] A Abdominal cysts. 187 Absence of hetcrolytic power as antiferment, 81 Absolute acidity of aqueous hu- mour, i'>4 Absorption of effusions, 83 Acetic anhydride, 186, 187 Acetone in exudates, 20(1 pancreatic cysts, 185 Achloride electrolytes. 60. loi. 162, lf.5, 172 Action of antitoxin, 89 (footnote) Action of blue li^ht on catalysis, 65 Action of lecithin on pancreatic juice, 88 Action of sotlium chloride on catalysis, 87 Action of sodium chloride on peroxydase, 87 Action of sodium chloride on trypsin, 87 Action of sulphates on tryptic digestion, 87 Actual ions, 122 Adamkiewitz reaction, 37, 59 Adsorption, 12, 14-16, 60, 105, 130 Alanin, 5 Albumen, 11, 23, 202 ; estimation of, 14 ; inhibitory effect on conductivity. 99 .\lbumose-N, 82 Albumoscs. 25. 26, 36, 83, 84 Aldehyde test for tyrosin, 35 Aleuronat, 81 Allantoic cysts, 189 Allantoic fluid. 164 Allautoin, 151, 165 Alloxan, 31 a-Ruanylic acid, 43 a-naphthol, 36, 56, 77, 190 a-naphthylisocyanate. 34 a-thymonuclcate of soila, 75 Amid-N, 29. 199 Amidolipoids, 45 Amidomyelin. 45, 53 .\mino-acids. 87. 149 Ammonia, 5. 84. 85, 150, 199, 207 ; in pseudo-niucin, 38 ' Amniotic fluid, 133, U14. Amount of total proteid, 199 An.esthesia. relation of lecithin to, 42 Analysis of puncture-fluids, scheme 19 Angiosarcoma of liver. 190 Antifernients, 24. 66, 70, 81, 82, 86, 88, 89, 149, 155. 163. 164, 165, 171. 207 Antipyrin in exudates. 208 Apomyelin. 53 Apparatus for determining C„, 129; electroconductivity, 112 Aqueous humour. 133. 164 I Argining. 5. 33, 38, 84 ' Arrhenius formula, 103 Asparagic acid, 38. 84 Aspartic acid, 5, 28 Asi;ociation of anti-emulsin with globulin, 88 ; lecithin ditto, 209 Autolysis, (54, 81, 82, 83. 84, 85, I ^ Autolytic ferments, 70, 81 B Bence- Jones proteid, 5, ^y, 150 Benzoic anhydride, 187 Benzoylising pseudomucin, 38, 39 ^-naphthalenesulphaminoacids, 27 281 2H3 INDKX Bil.-aciils. 170 HiU-pigni. nt, lii. !""■ «87. i8.» HiinoK'ciilar p iction, '>; Biologii;al ilivMiictions botwitn pro , viik'tii of antikTimnts, 87 r.iiirit I' iction. 37 Bli)06, 167, 169. 210. 229 Cerebrum, cysts of, 193 Chemical examination of puncture- fluids. 10 Chemistry of cells, 234 ChloricKs. estimation of, (ii — in amniotic fluid, 165 nephritic effusions, 3 ovarian cy=t"i, iSi 3 Chloride i'. achloride electrolytes, 209 Chlorine retention in transudates, «54 Cholesterin. 42. 51, 52. 143- M''- l^H. lf)2. I'M. 182. 185. 186, 187. 188, 189, 191 Cholin. 34. 3'^'. 42. 4'>. >7". '7" Chondroitinsulphuric aciil. 40 Christens formula, 199 Chyle, i,'59 Chylopericardium, 162 Chylous ascites, 158 Cobra-venom and jecorin, 51 Collection of fluids, 197 Colloidal metals, action on auto- lysis. (15. Wi. 85 — characters of puncture-fluids. 1 1 — contents of ovarian cysts. 174 Composition of ovarian cyst con- tents. 180 Concentration-chain nietho*'> Diazoacitic itlur imthotl. ny Diazobeiizcm-sulphonic acul. 31. 35 Diazo reaction. 55 Diilectricity constant. I"S DiHtTc-ntial countiiiK of ci'lls, 221 — iliaKnosisot rxiulatcs from trans- udates, I3, <»5' ''**■ '■^3 Drjewezki's method, 82, 83 Dulcite, 78. 80 Dysglobulin, 24 Edestin, 72, 161 Ettect of amount of albumen on specific gravity, 197 repeated tapping, i j'l temperature on conductivity, 105 - — osmotic pressure. 92 viscosity on velocity of enzy- matic reactions. 140 Eggwhite. electrical charges, 175 Ehrlich's glucosamine test, 3f>. 57. 190 Electrical charge of colloids. 160, 161, 175 Electro-conductivity, 7. I5' ^S' '"■ i').5. 175 Electrodes, treatment of, X13 Electrolytes. 97. 145. •47. ^7^' ^7 Erepsin. 70 Errors in methods of stuily. ,"5. 5". 118. 204. 224. 22y 229 Estimation of albumen. 14, 201 autolysis. 81 working jwwer of kidney. 7 Ethyl butyrate. 71. 79. «" I'.uglobulin. 24. 37 Kuseriimalbumen. 23 Explanation of reaction of Iwxly- 1 Huids. 134 I ditterence in chemical com- I position of l)ody fluids. 21') False pancreatic cysts. 184 Fatty acid crystals, ii^ Ferments, 4. $'■ 64. 8'. '55- '^'4- 165. 171. 185. 20f) — action of. (>4 — classification of. 70 — experiments with. 78-80 — heat-production by. 08 — in fluids. 80 — in leucocytes. 79 — methods of detecting and esti mating. 70 — relation to inorganic — specificity of, 68 Fibroblasts. 224 Filtration-nitrogen. 144 Fish-albumen. Florence reaction, 194 Foa's results. 133 Formalin, action o.n ferments of leucocytes. 8 1 Formic acid. 38 Formula for c.ilculating C». 132 mols + ions. 103-4 ferments. 284 IMtKX FiiriiiiilH {»T corrrctinif (or albunu-n, KM) ._ .-. — — tcmjHTiitiiri'. 107 — intir|Mil.itinK. loj FriTziiiK point iltpn-i^iDn, <(5. i'><>. 1')^, i«s Friction iKtwoi'n mils, nm, locj Friictosf, 150, io*., ^(jH Furoxyluim ti'st, jj ('. Galactose in crrcliro-spinal fliiul. 170 Gas ilectro). 151). iM, i(let-cells, 174 Graphic methods, 102 — representation of acidity, 124 Guanase, 75 Guanidin, 38 Gynesin, 47 H Haematoidin crystals, 233 Haematonu-tra, 184 Haemin crystals. 186 Haemolysis, 42, 51. 155 Haemorrhagic cysts of cerebellum, 194 Hamburger's blood-corpuscle method, 8, lU>, 117 Hausmann's method, 28 Heinz' researches, 221 Hesse-S.ilkowikrh test, 186 Hrleroalbiiii>o-.<', 5, '1. 2" Meterolytit ferment actio.i, 81 Hi-.tidin, 5, 35. 55. 84 Mt^lone. 38 Huniiii Mibst.-inces. 38 ny Hvilroctle fluid, IO3. 2ti) Hydrogen electrol Hydrothorax. 14<> Hydroxyl ions in blooil)itio:i. i-'i Immune seruin reaction, 208 Importance ot early exaimn.ition of Hiiids, 1 1 — NaCl to liody. 87 (footnote) Inilicators. 122. I2^ Inhibition of conductivity l>y al- buiiun, <>•> - dissociation, i) sis, 85 — constituents of puncture-fluids. («> Inosite in hydrocele fluid, if>4 Intercellular substances, permea- bility of. 2 if) Interpolation by graphic method, ; 102 Invertase. 71 Inversion method. 126 Iodides in exudates, 2o3 Ionic concentration, <>7 Ionic theory. 7, 94, 122 Iridium electrodes, 130 Isobutylhydantoic aciil, 28 Jagri's nu'tho4 KohUr 's apparatii>t. 11 1 (footnoti) I Lacteal cysts. ii Lecithin. 3^. 4a. 47. 4'»- 5.«. ^5' *"'■ 14''. '47. '5". '.VJ. "'•'■ "'3' ^'"'^ classification. 45 ; history. 44 ; imiwrtance of. 42, 44 ; methotl ; of analysis. 48 ; optical varieties. 47 ; properties ot. 4') ; relation to cell-ferments. 44. 4''* : '" cholesterin. 42. 48 ; to neunn. 47 ; to nucleic acitls. 43 ; to X-rays. 44 ; tests for, 4') Leucaemia. 79. 81 Leucin, 5. 27. 38. 84. 146, 150, 187, 207 Levulinic aciil, 38 Levulosc. 21, 31. 3a Liebermann's reaction. 37. i8^ Lijiase. 70. 71. 79. i'>5. 18'), 208 Lipoids. 42 Lner cysts, 189 Liver, pathology of metal)olism. 3 Lufflcr's serum, 81. 89 Lumbar puncture, i(y(f Lymph, 142 Lymphatic cysts, 192 Lymphocytes. 82, 222 Lysatinin, 33 Lysin. 5. 6, 33, 34, 38, 84 M Magnesium sulphate, for diagnosis of cerebrospinal ffuul, 1O9 Manasse's method, 50 Mannitc. 78, So Mast cells, 228 .Mastic tor removini? albumen. 12 .Mastites. 147 MeaninK '»f terms atul and base. 7 acidity. 123 Meat extract, new bas s in. 47 Mesenteric cysts. 1H8 Metabolic ,irotlucts of carcinoma colls, 214 Motalbiimon in hydr.Kole flui ' Mode of interaction between elec- trolyses and nonelct ■. ., tos. 107 Molecular concentration of exu- ilates and transudates. 2o'> I — conductivity. 115 ! — depression, 93 Molecules plu:i ions, , 103 Molisch reaction, y>, 37, 5') Mollusc albumen. 6 Monamidophosphatids. 45 Monamid X. 29. 182 Monaminoacids. 13, 20. 27 Monomolecular reaction, <)7 Morner's test tor tyrosin, 35 Mucin, 20, 36. 40, 41, 151. 160, l'>3, 165, 178 Mucoid contents of ovarian cysts, 174 % 2S6 INDEX Nakayania's list, 5') Niosin. 47 Nt'iiinann's method, 4') N'curin. ?4 Neutral iioiiit, 1 J4 NitroKi-n contt-nt of lyniiih, 144 Non-i'kttrolytts. 104 Normal HCl, 3') NovaiiH', 47 Nuck'ar changes in Koblot cells, 1 78 Nuclease, 70, 75 Nucleo glol)Lilin, 24. 141* Nticleoliistone, 40 Nucleoproteid, 20. 170 O Object ,f study ol punctiire-tluids. Objeci to Hausinan's nutluxl. Oblitin, 47 Oedema-Fluid, ('l Omental cysts, 189 Oitalescent fluids, 151. 1.58 Oral administration of diugs, 208 Orcm test, 32 Osmosis, <)i Osmotic concentration, t/'. 108, 144, 147. '5-. >54. 15". "'.V '"-■ 182, 187, 188 — pressure, 92, 104 — work, <»3 Ovarian cysi, 25, 41. 54. ."i'l. '". 80, 85, 141, 173, 180, 232. 251 Ovomucin, 24 Oxalic acid, 38 Oxidation, influence of arsenic on, Oxyaminoacids, 34 Oxydase, 70, 76, 77 Oxygen electrode, 129 1' Pancreatic cysts, 23, 59, 80, 184, 185 — juice, composition of, 133, 185 p-bromphenylhydrazine test, a Para,s;lobulin, 24, I49 Paralactic acid, i')4 Paralbumen, yt. 41, if>3 Paramucin 41, 178 Paranephric cyst. 191 Paranucleins. 40 Parapseud<)j;lobulin, 24 Parasitic cvsts, 194 Parotid cysts, 193 Parovarian cyst. 181 Pathological metabolism in liver, 3 I'atholosy of metabolism, study Pentainethylenediamine, 84 Pentoses. 32. 37, 84 lVl)siii, 71, I'l.i, 180 Peptones. 25, 84 Pericardial fluid, 61, 133, l^)i, i''2 IVripancreatic cysts, 189 Peritoneal fluid, critical solution point. 121, 133, 148 Pernuabihly of carcinoma cells, -=•7 cells in peritone;d fluid, 4 1 to ions, 214 , cnilothelial cells. 215 ■ inflamed serous membranes, 21 1 1 Peroxydase, 77, 87 i Phenolphthalein, 126 Phenylalanin. 5 I'hloroglucin, 33 Phosphatids, 42, .52 Phosphorus, detection of. 49 I Phosphotungstic acid. 13 Pliysical jiroperties of proteids in turbul effusions, 160 Physico-chemical characters of fluids, 152. 182 (see Electrolytes, etc.) examination of puncture-fluids, 91 sqq. Phvsico-chemistry, to supplement chemical study of puncture-fluid. 2 Pickardt's method of removing al- bumen, 13 Picrolonic acid, 171 Pigments. 59. 127 Pinas test for ty rosin, 35 Platinum electrodes. 130 Pleural fluids, 34, 85. ^S. '48. 227 INDEX 287 Poisonous action of txudatcs and transudatfs. 15^) I'olarimttry, bq. 126, 151 Polymerisation in fluids, 107 Polynucleosis. 223, 224 Polypeptids. 13 I'otassium salts in fluids, 14^'- 172 Potential ions, 122 Practical value of autolysis, 82 Precipitins. 78 Preformation of proteid constitu- ents, 44 Preliminary separation of chemical const:* jents, 20, 21 Pressure of contents of ovarian cysts. 1 7'^' Procedure for determining C,,. 131 Prolin, 5 Propionic anhydride, 187 Protagon, 44 I'rotallnimose, 5, 6, 20 I'rotamin, 5 Proteid Extractive Ratio, i'^2. 203 Protcid of carcinoma cells, 3 Proteid quotient, 143, ia9. i<^'2' 201 Proteolytic ferment. 81. 234 Pseudochylous effusions, ifti Pseudoglobulin, 24, 37, A^. 54- 149 Pscudolymphocytes, 222, 224, 225 Pscudomucin, 35, 38. 178 Pseudomyxoma peritonei, 184 Pseudoserumalbumen, 24 Puerperal changes in uterus, 83 Purin N., 83, 199 Purins, 20, 29, 31, 76 Pus, loi, 145 I'jTocatcchin, 170 Pyrrhol, 58, 77 Q. Quinone test for tyrosin. 35 Rate of flow of cerebrospinal fluid, 232 Ratio of chlorides to achloridcs, 8, 100. loi Reducing-powcr of puncture-fluids, 7