key: cord-0807542-4k2frmz6
authors: Zeeuw van der Laan, Eveline A.N.; van der Velden, Saskia; Porcelijn, Leendert; Semple, John W.; van der Schoot, C. Ellen; Kapur, Rick
title: Evaluation of Platelet Responses in Transfusion-Related Acute Lung Injury (TRALI)
date: 2020-09-16
journal: Transfus Med Rev
DOI: 10.1016/j.tmrv.2020.08.002
sha: 14c6302c393384c76c680e7f696226231711b946
doc_id: 807542
cord_uid: 4k2frmz6

Platelets are versatile cells which are capable of eliciting non-hemostatic immune functions, especially under inflammatory conditions. Depending on the specific setting, platelets may be either protective or pathogenic in acute lung injury and Acute respiratory distress syndrome (ARDS). Their role in Transfusion-related acute lung injury (TRALI) is less well defined, however, it has been hypothesized that recipient platelets and transfused platelets both play a pathogenic role in TRALI. Overall, despite conflicting findings, it appears that recipient platelets may play a pathogenic role in antibody-mediated TRALI, however, their contribution appears to be limited. It is imperative to first validate the involvement of recipient platelets by standardizing the animal models, methods, reagents and read-outs for lung injury and taking the animal housing environment into consideration. For the involvement of transfused platelets in TRALI, it appears that predominantly lipids such as ceramide in stored platelets are able to induce TRALI in animal models. These studies will also need to be validated and moreover, the platelet-derived lipid-mediated mechanisms leading to TRALI will need to be investigated.

Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-associated fatalities and is characterized by the onset of acute respiratory distress within 6 hours of a blood transfusion [1, 2] . The pathogenesis has proven to be challenging to decipher and consequently specific therapies are not yet available [1, 3] . Overall, the pathogenesis of TRALI can be viewed as a 2-hit phenomenon, where both hits are required for induction. The first hit is conveyed by pre-existing clinical risk factors present in the transfused recipient, often signified by a state of inflammation, while the second hit is represented by factors in the transfusion product [1] . These factors may be anti-leukocyte antibodies, such as anti-human leukocyte antigen (HLA) class I/II antibodies or anti-human neutrophil antigen (HNA) antibodies, or biological response modifiers such as accumulated lipids in aged platelets [1, [4] [5] [6] . The role of recipient leukocytes in the pathogenesis of antibody-mediated TRALI has been investigated, with CD4+ T regulatory cells (Tregs) and dendritic cells (DCs) being protective via IL-10 [7] and neutrophils (PMNs), monocytes and macrophages being pathogenic cells [1, [8] [9] [10] . The role of recipient platelets, however, has sparked a debate [11] . In this paper, we will review the evidence for the involvement of recipient platelets, as well as the involvement of transfused platelets in the pathogenesis of TRALI.

Platelets are classically known for their role in hemostasis [12] , however, in the last decade it has become clear that platelets can also function as immune cells [13] [14] [15] . This includes immune-sensing functions for antimicrobial host defense and their ability to communicate with and regulate a variety of immune cells. An example of their antimicrobial defense is illustrated by studies showing that platelets are able to inhibit bacterial growth through encapsulating the bacteria and secreting the anti-microbial peptide β-defensin-1 as well as via J o u r n a l P r e -p r o o f Journal Pre-proof signaling of polymorphonuclear cells (PMNs) to extrude neutrophil extracellular traps (NETs) [16] . Bacterial trapping by platelets has also been described to occur via platelet the adhesion receptor glycoprotein (GP)Ib [17] or during sepsis via platelet toll-like receptor (TLR)4 resulting in NET formation [18] . More recently, migrating platelets were shown to be mechano-scavengers that collect bacteria deposited at the vascular surface [19] . Furthermore, platelets can communicate with other immune cells by releasing CD40L, secreting several cytokines and chemokines, shedding of platelet microparticles or via their MHC-class I molecules [13] [14] [15] . These immunomodulatory functions of platelets are discussed in more detail in a companion article in this issue of Transfusion Medicine Reviews [20] . In the next section of this paper we will first briefly discuss how platelets may play a role in a setting of acute lung injury and acute respiratory distress syndrome (ARDS).

The versatility of the immune functions of platelets is evident from the fact that platelets have the ability to influence both physiological as well as pathological responses in the lungs [21] .

Platelets may enter the pulmonary circulation after being formed in the bone marrow from megakaryocytes, but it has also been described in mice that platelets may be produced by megakaryocytes in the lungs although this needs to be verified in humans [22] . In the lungs, platelets may mediate protective or injurious pathogenic responses depending on the specific setting of ARDS or the type of acute lung injury. One study showed that platelets have an Similarly, in the setting of pneumonia by the gram-positive Streptococcus Pneumoniae, induction of thrombocytopenia in mice by administration of anti-mouse thrombocyte serum also reduced survival, resulted in higher bacterial burden in lungs, spleen and blood but did not affect lung inflammation despite increases in plasma pro-inflammatory cytokine levels [24] . Treatment with the platelet P2Y12 receptor inhibitor clopidogrel prolonged the bleeding time, but had no effect on the bacterial loads [24] . Collectively, these studies indicate that platelets are protective in a setting of gram-negative or gram-positive pneumonia by inhibiting bacterial growth, a function which no longer occurs when the platelets are depleted in vivo. In contrast, a pathogenic role of platelets was found in influenza A-induced pneumonia and acute lung injury [25] . Mice infected with influenza A demonstrated a massive increase of aggregates of activated platelets in the lungs and activation of platelet protease-activated receptor 4 (PAR4), exacerbated acute lung injury and increased mortality in mice. On the other hand, deficiency of platelet receptor GPIIIa, or administration of anti-platelet agents such as the GPIIb/IIIa-antagonist eptifibatide, the PAR4-antagonist MRS 2179 or clopidogrel, protected mice from influenza-virus induced lung injury and mortality [25] . This study underlines the ability of platelets to regulate pathogenic responses in influenza-induced acute lung injury. In a different murine model of acid-induced acute lung injury (modeling acute lung injury due to pulmonary aspiration of gastric content, which may damage the alveolarcapillary membrane), it was found that platelet P-selectin-dependent platelet-PMN interactions occurred in blood and lung vessels [26] . The onset of acid-induced acute lung injury could be prevented by reducing the number of circulating platelets or by blocking Pselectin. Furthermore, activated platelets induced endothelial ICAM-1 expression and increased the adhesion of PMNs to endothelial cells. Targeting the platelet-PMN aggregation, using a specific thromboxane receptor antagonist (platelet-PMN adhesion to untreated human pulmonary microvascular endothelial cells was shown to require endothelial cell thromboxane J o u r n a l P r e -p r o o f Journal Pre-proof receptors), reduced the recruitment of PMNs and prevented endothelial permeability improving survival [26] . This study demonstrates another pathogenic role of platelets which occurs via interaction with PMNs. The multifactorial roles of platelets and their protective or pathogenic aspects in acute lung injury and ARDS appear to be dependent on the specific setting and the readers are referred to a recent comprehensive review [21] . In this paper the focus will be directed more on the involvement of platelets in a setting of blood transfusiontriggered acute lung injury or TRALI.

Animal models of TRALI have significantly contributed to obtaining insights into the pathophysiology of TRALI [1] . This is especially true for the clinically-relevant TRALI model based on injection of the anti-major histocompatibility complex (MHC) class I antibody, 34-1-2S [1, 27] . Like in humans, this model features the 2 hits of TRALI pathophysiology with the first hit being represented by, for example, low levels of IL-10 (IL-10 KO mice or via depletion of CD4+ Tregs or DCs) [7] , infusion of C-reactive protein (CRP) [28] or by priming with low-dose LPS [9, [29] [30] [31] [32] . The second hit is conveyed by the 34-1-2S antibody infusion. Furthermore, this murine TRALI model displays other characteristics observed in human TRALI patients such as, the induction by anti-leukocyte antibodies, the onset of TRALI within 2 hours post-induction, the presence of pulmonary PMN infiltration and the occurrence of pulmonary edema. Also, IL-10 levels have been described to be low [33, 34] and CRP levels have been described to be increased [35, 36] in human TRALI patients.

34-1-2S TRALI models has been extensively utilized to investigate the contribution of recipient platelets in the pathogenesis of TRALI, a notion that seems reasonable based on the capability of platelets to elicit a wide range of immune functions. Strikingly, several J o u r n a l P r e -p r o o f Journal Pre-proof discrepancies have arisen regarding the role of recipient platelets in affecting TRALI responses by 34-1-2S [11] . Some studies have found recipient platelets to be pathogenic [29, 37] , while other studies found recipient platelets to be dispensable for the onset of TRALI [38, 39] or not completely dispensable but with limited pathogenic involvement [30, 31] . An early study by Looney et al found that in LPS-primed mice, in vivo platelet depletion using a rabbit anti-mouse platelet serum administered 2 hours before TRALI induction with 34-1-2s protected against TRALI [29] . In addition, treatment with Aspirin also prevented the onset of TRALI, however, blocking of P-selectin with an anti-P-selectin antibody or inhibition of macrophage-1 (MAC-1)-dependent platelet-PMN interactions had no effect on the prevention of TRALI [29] . Strait et al also used a similar rabbit anti-mouse serum to deplete platelets before TRALI induction and confirmed these findings [38] , however, instead of just antibodyinduced thrombocytopenia, they hypothesized that the anti-platelet antibodies blocked the development of TRALI by either depleting complement or desensitizing effector cells [38] .

When they administered the rabbit anti-mouse serum 2 days and 1 day prior to 34-1-2S infusion, they did not find an effect of platelet depletion on the occurrence of TRALI [38] . In addition, they also depleted platelets in vivo using a non-immune mechanism through treatment with neuraminidase and found that this also did not affect the onset of TRALI [38] .

There were remarkable differences noted between the methodologies used by Looney al) also found an important role for platelets as inducers of NETs in murine TRALI [37] .

Platelet functions were targeted in vivo using Tirofiban and Aspirin and both treatments decreased NET formation and associated platelet sequestration. In the case of Tirofiban, the link with lung injury was also investigated and this was found to protect against TRALI.

Another study, by Thomas et al, also reported the presence of NETs in murine TRALI, however, depletion of platelets with neuraminidase did not prevent NET formation in the lungs [39] . This discrepancy between the studies by Caudrillier et al and Thomas et al is particularly challenging to analyze as the data of the neuraminidase experiment by Thomas et al was not shown and no details were provided. Furthermore, Hechler et al thoroughly investigated the involvement of recipient platelets in LPS-primed and 34-1-2S-induced murine TRALI by using a wide variety of platelet-targeting approaches [30] . In vivo platelet depletion was efficiently achieved by administrating diphtheria toxin (DT) to platelet factor 4 (PF4)-Cre/inducible DT receptor (iDTR) mice. The authors also administered a rat anti-GPVI monoclonal antibody (clone JAQ1) to mice to induce specific depletion of the GPVI-receptor [40] . In addition, the mice were also treated with Aspirin or Clopidogrel or transfused with GPIIb/IIIa-deficient platelets post-DT treatment. They consistently found that all these platelet-targeting strategies did not prevent the occurrence of TRALI suggesting that recipient platelets are dispensable for TRALI induction [30] . The authors did, however, observe that the development of lung hemorrhages was inhibited in the three experimental regimes to deplete platelets; DT treatment of the PF4-Cre/iDTR mice, anti-GPVI treatment or upon GPIIb/IIIa-deficient platelet transfusions [30] . This suggested that recipient platelets may play a limited pathogenic role in the development of TRALI. In addition, Hechler et al [30] was unable to confirm that Aspirin treatment protected against TRALI, in contrast to the studies of Looney et al [29] and Caudrillier et al [37] . LPS priming was performed in both studies, however, the read-outs for lung injury were also different. Hechler et al used blood oxygen J o u r n a l P r e -p r o o f Journal Pre-proof levels, BAL protein levels and lung histology (without using a damage score), while Looney et al used bloodless extravascular lung water, EVPE and lung MPO-activity. It cannot be excluded that the use of different read-outs, at least to some extent, could significantly affect the assessment and conclusion of the occurrence of significant lung injury. A recent study by Cognasse et al also examined the involvement of recipient platelets in 34-1-2S-mediated TRALI [31] . This study depleted platelets in vivo using a rat anti-GPIbα polyclonal Ab or they inhibited platelet activation by pre-treating mice with the PAR4-pathway inhibitor ML354 [31] . Both platelet-targeting interventions did not prevent the onset of TRALI, however, they did reduce the severity of the disease [31] . All these studies investigating the involvement of recipient platelets in 34-1-2S-mediated TRALI are summarized in Table 1 .

Overall, discrepancies are present in these studies, which all utilized 34-1-2S to induce murine TRALI. Not all studies primed mice with LPS and the studies that utilized LPS, did not all use the same type of LPS (Table 1 ). In addition, most studies had a TRALI duration of 2 hours, but there was also a study which only monitored TRALI up to 30 minutes (Table 1) .

Remarkably, there was a very high degree of variation in the read-outs that were used to assess the lung injury (Table 1) . Another important factor which may also be contributing to the heterogeneity may be the gastrointestinal microbiota. It was demonstrated that mice housed in a barrier-free (BF) animal facility were susceptible to 34-1-2S dependent TRALI induction, while mice housed in a more sterile specific-pathogenic free (SPF) animal facility were resistant to antibody-mediated TRALI [32] . This resistance in SPF housed mice could be overcome by priming the mice with LPS or by conducting fecal transfer using fecal matter from susceptible BF housed mice [32] . It is also likely that one SPF-animal facility may TRALI models in certain SPF animal facilities (e.g. [31] ), while in other SPF animal facilities mortality does generally not occur (e.g. [9] ). The nature of the recipient platelet response in TRALI may therefore be dependent on the composition of the gastrointestinal microbiota.

Despite these conflicting findings, it appears conceivable that recipient platelets have a pathogenic involvement in TRALI, but the extent of this involvement may be limited.

An interesting recent study found an important role for platelets in preventing endothelial cell leakage during leukocyte diapedesis [41] . Platelets were found to dock to endothelial von Willebrand factor (VWF) preventing leakage during leukocyte extravasation by stimulating endothelial Tie-2. How this mechanism relates to a TRALI setting will be interesting to investigate.

Regarding TRALI in humans, a mild thrombocytopenia occurs in TRALI patients thus this supports that platelets are at least involved to some extent in the pathogenesis of TRALI [1] . Moreover, it has not been described that a low platelet count in humans is a protective factor for the onset of TRALI, indirectly indicating the recipient platelet involvement in the induction of TRALI may be minor.

Platelet transfusions can elicit fatal TRALI reactions in humans [42] . Several studies have researched the contribution of transfused platelets to the development of TRALI, using in vivo/ex-vivo animal models or by in vitro analysis [6, [43] [44] [45] [46] [47] [48] . The first hit in all these studies was priming with LPS and the second hit was either the transfusion of platelets (stored or treated) or their derived products such as supernatants, lipids or platelet microparticles. An important early study of Silliman et al demonstrated that lipids in stored platelets could induce TRALI in an ex-vivo rat model [43] . In this study, transfusion of 5-day stored human platelet plasma, lipid extracts from human plasma, high-performance liquid chromatography (HPLC) J o u r n a l P r e -p r o o f purified lipids from human platelet plasma or purified lyso-phosphatidylcholines (lyso-PCs) all increased the pulmonary edema index thereby contributing to the development of TRALI.

platelets (stored up to 7 days) and that transfusion of these aged platelets induced TRALI in mice [6] . Mice were also transfused with stored platelets (up to 5 days) which were treated with the acid sphingomyelinase (ASM) inhibitor ARC39 or with stored platelets (up to 7 days) from ASM-deficient mice, which both diminish the formation of ceramide. Compared with the wild-type aged platelets, transfusion of stored ARC39-treated platelets or stored ASM-deficient platelets alleviated the development of TRALI [6] . Using a sheep model of protein levels and damage observed on lung histology) compared to non-irradiated platelets [44] . Remarkably, however, there was no effect on the lung wet to dry (W/D) weight ratios, which is a direct measure for the degree of pulmonary edema. Since BAL protein levels can also be increased in inflammatory settings without significant injury and the fact that the lung histology was not scored for lung damage, a more appropriate conclusion may be that UVBirradiated platelets only have a slight tendency to increase TRALI severity. Chi et al [46] and Caudrillier et al [47] both investigated the effect of Mirasol-treated human platelets transfused into LPS-primed mice. Chi et al found that transfusion of human platelets mediated TRALI, regardless of Mirasol treatment [46] whereas Caudrillier et al concluded that Mirasol-treated human platelets did not induce TRALI [47] . Despite the apparent conflicting outcome of these and sCD40 in the development of TRALI [48] . TRALI was assessed by measuring HMVEC-L viability (damage) and it was found that PMPs and CD40L (which is present in high levels in PMPs) could activate PMN-mediated damage in the LPS-primed HMVEC-L [48] . All these studies investigating the involvement of transfused platelets in TRALI are summarized in Table 2 .

Overall, with respect to transfused platelet involvement in TRALI, it seems that the supernatant of stored platelets, and more specifically, lipids such as ceramide can mediate TRALI in animal models ( Table 2) . UVB-irradiated human platelets may have increased tendency to induce murine TRALI, while Mirasol-treatment of platelets does not directly appear to contribute to murine TRALI (Table 2 ). PMPs and sCD40L can activate PMNmediated damage of pulmonary endothelial cells, but this was not further investigated in vivo (Table 2) .

For the human setting, it is important to keep in mind that transfusion of lipids in stored platelets may not always elicit a TRALI reaction, as this is also dependent on the tightly regulated interplay with recipient first hit factors. Also within the same patient, any change in inflammatory parameters may influence the ability of the accumulated lipids to trigger TRALI.

There are several similarities between TRALI and COVID-19. Hospitalized COVID-19 patients also develop shortness of breath and life-threatening hypoxemic respiratory failure due to pulmonary edema, also presumed to be caused by leaking pulmonary vasculature and damaged endothelium. In addition, it appears that plasma IL-6, IL-8, CRP and NETs are also increased [e.g. [49] [50] [51] , and also complement has been described to be involved in COVID-19

[e.g. 52]. The contribution of platelet transfusions to the occurrence of TRALI in COVID-19 patients has, to best of our knowledge, not been investigated. Notably, however, transfusion of convalescent plasma has been shown to induce cases of TRALI in COVID-19 patients [e.g. 53, 54] . Further research may clarify the similarities between human TRALI and COVID-19, including the contribution of transfused platelets.

Platelets have non-hemostatic immune functions which may translate into a pathogenic or protective effect in acute lung injury or ARDS, depending on the specific triggers and the nature of the inflammatory environment. In TRALI their involvement is less clear, as conflicting outcomes have been reported by studies investigating the contribution of recipient platelets. This heterogeneity could be due to variations in the utilized animal models, methods, reagents, read-outs for acute lung injury or animal housing conditions, despite the fact that all these studies used the antibody 34-1-2S to induce murine TRALI. It seems plausible, however, that platelets are pathogenic in TRALI but that their involvement is minor. In vivo targeting of platelets may not prevent the onset of TRALI, but limit its severity and thus, targeting recipient platelets does not seem to be an attractive approach to search for potential therapies. It may be more important, however, to first further investigate the contribution of recipient platelets in TRALI by standardizing the experimental design, parameters and conditions. Regarding the involvement of transfused platelets in TRALI, J o u r n a l P r e -p r o o f lipids, particularly ceramide, in stored platelets have been shown to contribute to TRALI as demonstrated in animal models. This will also need to be further validated and the mechanisms leading to lung injury will need to be researched. Table 1 : Recipient platelet involvement in TRALI as investigated using 34-1-2S-based mouse models. BAL = bronchoalveolar lavage; Bloodless extravascular lung water = [Wet lung weight / (Dry lung weight, corrected for the dry weight of blood remaining in the lung using hemoglobin levels)]; DT = diphteria toxin; EVPE (extravascular plasma equivalents) = 125 I-albumin radioactivity in the lung homogenate -( 125 I-albumin concentration in plasma sample x Calculated plasma volume in lungs), in 125 I-albumin-instilled mice; MPO = myeloperoxidase; NETs = neutrophil extracellular traps; Pehn = changes in breathing pattern shown by barometric plethysmography as "enhanced pause"; Percentage water weight of lungs = difference between the pre-and post-lyophilization lung weight / pre-lyophillization lung weight x 100%; PF4-cre/iDTR = inducible DT receptor expression in platelet factor 4(PF4)-positive cell populations (megakaryocytes and platelets). Indicated symbols (*,#,) should only be compared within each row. 

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A consensus redefinition of transfusion-related acute lung injury

Targeting Transfusion-Related Acute Lung Injury: The Journey From Basic Science to Novel Therapies

Antibody-mediated transfusion-related acute lung injury; from discovery to prevention

Pathogenesis of non-antibody mediated transfusion-related acute lung injury from bench to bedside

Acid sphingomyelinase mediates murine acute lung injury following transfusion of aged platelets

T regulatory cells and dendritic cells protect against transfusion-related acute lung injury via IL-10

The Pathogenic Involvement of Neutrophils in Acute Respiratory Distress Syndrome and Transfusion-Related Acute Lung Injury

Osteopontin mediates murine transfusion-related acute lung injury via stimulation of pulmonary neutrophil accumulation

M1-polarized alveolar macrophages are crucial in a mouse model of transfusion-related acute lung injury

The contribution of recipient platelets in TRALI: has the jury reached a verdict?

Platelets and hemostasis: a new perspective on an old subject

Nouvelle cuisine: platelets served with inflammation

Platelets as immune-sensing cells

The nonhemostatic immune functions of platelets

Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation

Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance

Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood

Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria

The immune nature of platelets revisited

Amicus or Adversary Revisited: Platelets in Acute Lung Injury and Acute Respiratory Distress Syndrome

The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors

Thrombocytopenia impairs host defense in gram-negative pneumonia-derived sepsis in mice

Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis

Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation

How different animal models help us understand TRALI

C-reactive protein enhances murine antibody-mediated transfusion-related acute lung injury

Platelet depletion and aspirin treatment protect mice in a two-event model of transfusionrelated acute lung injury

Platelets are dispensable for antibody-mediated transfusion-related acute lung injury in the mouse

Platelet depletion limits the severity but does not prevent the occurrence of experimental transfusion-related acute lung injury

Gastrointestinal microbiota contributes to the development of murine transfusionrelated acute lung injury

Low levels of interleukin-10 in patients with transfusion-related acute lung injury

Cytokines and clinical predictors in distinguishing pulmonary transfusion reactions

Elevation of C-reactive protein levels in patients with transfusion-related acute lung injury

Two Episodes of Transfusion-related Acute Lung Injury (TRALI) Occurring within a Short Period: A Case Report

Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury

MHC class Ispecific antibody binding to nonhematopoietic cells drives complement activation to induce transfusion-related acute lung injury in mice

Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice

Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice

Platelets docking to VWF prevent leaks during leukocyte extravasation by stimulating Tie-2

Fatalities Reported to FDA Following Blood Collection and Transfusion Annual Summary for Fiscal Year

Plasma and lipids from stored platelets cause acute lung injury in an animal model

Ultraviolet B light-exposed human platelets mediate acute lung injury in a two-event mouse model of transfusion

A novel in vivo ovine model of transfusion-related acute lung injury (TRALI)

Human platelets pathogen reduced with riboflavin and ultraviolet light do not cause acute lung injury in a two-event SCID mouse model

Transfusion of Human Platelets Treated with Mirasol Pathogen Reduction Technology Does Not Induce Acute Lung Injury in Mice

Platelet-derived microparticles induce polymorphonuclear leukocyte-mediated damage of human pulmonary microvascular endothelial cells

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Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis

Early safety indicators of COVID-19 convalescent plasma in 5000 patients

Mortality reduction in 46 severe Covid-19 patients treated with hyperimmune plasma. A proof of concept single arm multicenter trial

This work was supported by Sanquin (grant PPOC-18-08).

None.J o u r n a l P r e -p r o o f Journal Pre-proof

5 or 10 µM, lung perfusion, 165 min post LPS, analysis after 30 min Gelderman 2011 [44] In vivo  SCID miceGender not specified, 6