Allogeneic Platelet Research Articles

Substitutes and alternatives to platelet transfusions in thrombocytopenic patients

Over the past decade, there have been many improvements in both the safety profile and quality of liquid-stored allogeneic platelet concentrates. However, significant problems with the clinical use of such products remain. Efforts to overcome some of these have resulted in the development of an array of novel therapeutic strategies for the manufacture of platelet products and platelet substitutes, as well as other approaches using alternatives to platelet concentrates. These various products or procedures are at various stages of clinical development. This review summarizes some recent advancements in the preparation of liquid and frozen stored platelets, as well as approaches used for the pathogen inactivation of platelets. Thus, the status of lyophilized platelets, infusible platelet membranes, red blood cells (RBCs) bearing RGD ligands, fibrinogen-coated albumin microcapsules, and liposome-based agents are discussed. Pre-clinical studies and phase 1-3 clinical trials have been encouraging for several of these; however, to date, very few have been licensed for clinical use. Potential alternatives to allogeneic platelet transfusions including correction of anemia by RBC transfusions, recombinant activated factor VII and HLA-reduced platelets are also reviewed. With the ongoing technical and scientific development of such diverse products, those properties that may be necessary for such agents to have hemostatic efficacy will become apparent. However, safety and efficacy must be demonstrable in preclinical studies and clinical trials, before novel platelet concentrates, platelet substitutes and alternatives to platelets can be used in patients with thrombocytopenia.

Recombinant human macrophage colony-stimulating factor-induced thrombocytopenia in dogs.

To characterize recombinant human macrophage-colony stimulating factor (rhM-CSF)-associated thrombocytopenia (TCP), in vivo studies were performed in dogs, including the biodistributions and recoveries of radiolabelled autologous and allogeneic platelets. rhM-CSF induced a reversible, dose-dependent decrease in platelet counts. The number of megakaryocytes in spleen and marrow of rhM-CSF-treated dogs was increased two to threefold. Recoveries of allogeneic platelets transfused from rhM-CSF-treated donors into tolerized recipients (n = 3) were not significantly different from allogeneic baseline studies (93 +/- 10% of baseline values at 24 h and 90 +/- 1% at 40 h), whereas autologous platelets infused back into rhM-CSF-treated donors had decreased recoveries (45 +/- 2% of baseline values at 24 h, P = 0.03 and 20 +/- 4% at 40 h, P = 0.001). Platelet biodistribution studies showed increased accumulation of radiolabelled platelets over the spleens and livers of rhM-CSF-treated dogs. Histochemistry showed increased levels of platelet-specific antigen (CD41; glycoprotein IIb) associated with Kupffer cells. The sensitivity of platelets from rhM-CSF-treated dogs to activation from thrombin, as measured by expression of P-selectin (CD62P), was not significantly different when compared with baseline studies (P = 0.18; n = 4). These results support the concept that rhM-CSF induces an activation of the monocyte-macrophage system (MMS), which causes a reversible TCP in a dog model.

Postoperative Bleeding After Coronary Artery Bypass Surgery with Cardiopulmonary Bypass

In Response: We thank Dr. Belcher for his comments regarding our study (1). We would like to make only a few minor clarifications in response. Our objective was to determine whether the technique as described was effective in a large and homogenous group presumed at high-risk for bleeding and transfusion. We did not address mechanisms of effect in detail. We agree that platelets would already be concentrated at intravascular sites of vessel wall damage. The topical application of autologous platelet gel to raw surfaces and wound edges was intended as an extravascular mechanical sealant along the lines of “tissue glue” rather than an attempt to correct for a local platelet deficiency. We noted in our introduction that the efficacy of this aspect of the technique had not been reported, but it was part of our established practice at the time (based on reports in conference proceedings (2) and personal communications), and was quick, easy, and safe to do. Recent publications in the orthopedic (3) and cosmetic (4) surgery literature support use of topical autologous platelet gel and fibrin glue to arrest capillary bleeding from bone and under skin flaps. Autologous platelet gel has also been reported to contain platelet-derived growth factor that may enhance wound healing. Dr. Belcher lists several reasons for platelet dysfunction after cardiopulmonary bypass, all of which we recognized and attempted to prevent or minimize by our pheresis protocol. Therapeutic quantities of platelets were sequestered before heparinization and reinfused only after protamine reversal thus avoiding (in the sequestrate) the heparin and bypass induced plasma change(s) referred to by Dr. Belcher. The degree to which these plasma changes persist in the patient (if they do) after bypass and heparin reversal remains unquantified and deserving of further research. Autologous platelets prepared by whole blood centrifugation and stored for several hours before reinfusion exhibit less activation and greater responsiveness than allogeneic platelets (5). Our study of reoperative CABG patients showed only that therapeutic yield plateletpheresis did not improve the two clinical outcomes we measured. Mean chest tube drainage was close to obligate loss volumes in both groups, making further reduction unlikely. Allogeneic PRBC transfusion was also very low, averaging 1.2–1.5 units per patient. Up to 55% of patients in both groups received some allogeneic exposure, but this includes those patients receiving platelets and fresh frozen plasma as treatment for postoperative bleeding. Low rates of preoperative aspirin exposure, routine antifibrinolytic use, and minimal exposure of blood to nonbiologic surfaces are likely to have contributed to our low control group rates, and thus made a treatment difference less likely. We performed only routine measurements of hemostasis (platelet counts, INR, APTT—we did not measure bleeding times) perioperatively, and patients were transfused with platelets only if clinically bleeding with a count < 100 × 109/L. We stand by our conclusion that while the technique was not beneficial in our unit, it may have a place in other institutions where baseline blood loss and transfusion rates are higher than ours. We agree with Dr. Belcher that platelet transfusions should be restricted to bleeding patients with significant thrombocytopenia, and not administered routinely after cardiopulmonary bypass surgery. Paul Wajon, FANZCA John Gibson, FANZCA

Recipient antigen-processing pathways of allogeneic platelet antigens: essential mediators of immunity.

Clinical studies have convincingly demonstrated that WBC reduction of blood components can reduce the incidence of primary HLA antigen alloimmunization at least in patients with acute myeloid leukemia.1 However, despite receiving WBC‐reduced platelets, approximately 19 percent of acute myeloid leukemia patients still became alloimmunized.1 In addition, it is not known whether WBC reduction will prevent HLA antigen alloimmunization among those recipients who have an intact immune system (e.g., general surgery patients). The biologic mechanisms responsible for immune stimulation by blood components are still incompletely understood but are probably related to both donor and recipient factors. Animal models have proven indispensable for elucidating the basic mechanisms of immunity against foreign antigens and several such models have been developed to study various aspects of transfusion immunobiology.2-4 Our laboratory has developed a murine transfusion model that has allowed us to focus on recipient factors affecting the immune response to WBC‐reduced platelets. This article will describe how the murine immune response to platelet transfusion is critically dependent on recipient antigen‐processing and‐presentation pathways and how these pathways can be exploited to modulate immunization against WBC‐reduced platelets. (Less)

Safety and efficacy of transfusions of autologous cryopreserved platelets derived from recombinant human thrombopoietin to support chemotherapy-associated severe thrombocytopenia: a randomised cross-over study

The increasing demand for platelet products, and concern over the transfusion-associated risks of alloimmunisation and infections, have motivated a search for improved methods aimed at keeping exposure to donor antigens to a minimum. Transfusion of thrombopoietin-derived autologous platelets might provide an alternative strategy. We aimed to compare the safety and efficacy of this strategy with that of transfusion with fresh allogeneic platelets in patients with severe chemotherapy-induced thrombocytopenia. 20 patients with gynaecological malignancies were treated with two doses of 1.2 microg/kg recombinant human thrombopoietin. From day 12, we aimed to collect 50 units of platelets from these patients by plateletpheresis. Harvested platelets were cryopreserved in ThromboSol and 2% dimethyl sulfoxide (DMSO) for use in subsequent autologous transfusions. Patients then received carboplatin for up to six cycles. Patients were randomly assigned to group A (n=10), which received allogeneic fresh platelets at the first instance of severe thrombocytopenia (platelet count <15,000/microL) and then autologous cryopreserved platelets at the next, or to group B (n=10), which received first autologous and then allogeneic platelets. In subsequent cycles, all patients received autologous platelets while available. The primary endpoint was platelet count increment corrected for the number of platelets transfused and the patients' body-surface area. Analysis was by intention to treat. Treatment with recombinant human thrombopoietin significantly increased platelet count (median 2.3-fold [range 1.5-3.3], p<0.0001) in all but one patient in group A. The median number of platelets collected per patient was 53 units (14-66) in two collections (one to three). There was no significant difference in the corrected platelet count increments (CCIs) between the 19 paired transfusions of cryopreserved autologous platelets and fresh allogeneic platelets (median 1-h CCI 15.7 vs 19.8, p=0.398; median 24-h CCI 13.0 vs 18.1, p=0.398). 14 of the 19 patients had a good response (1-h CCI >7.5) to their first transfusion of allogeneic platelets. By contrast, all patients had a good response to their first transfusion of autologous platelets (p=0.063). Moreover, no significant decrease in the CCIs (p=0.405) was seen over six cycles after autologous platelet transfusions (n=63). No transfusion reactions or any serious adverse event was recorded during autologous platelet transfusions. Recombinant human thrombopoietin facilitated collection of multiple units of platelets, which could be cryopreserved and reinfused to counteract severe thrombocytopenia during multicycle chemotherapy. Transfusion of autologous cryopreserved platelets derived from recombinant human thrombopoietin can provide a viable strategy to minimise the risks of allogeneic platelet transfusions and provide a long-lasting supply of platelet support.

Allogeneic Platelet Transfusion: Risks and Alternatives

Allogeneic Platelet Transfusion: Risks and Alternatives

Intraoperative use of platelet-plasmapheresis in vascular surgery

Study Objective: To determine, in a pilot study, whether pheresis of plasma and platelets before surgical blood loss, with reinfusion of the autologous plasma and platelets after completion of the aortic reconstruction, will result in decreased bleeding and decreased transfusion of allogenic blood components in patients undergoing elective aortic reconstruction. Design: Randomized study. Setting: University medical center. Interventions: Patients were randomized to perioperative (acute) platelet plasmapheresis (APP group) versus conventional blood component therapy (control group). In the APP group, blood was withdrawn after induction of anesthesia, to sequester approximately 300 mL of platelet rich plasma (PRP); platelet poor plasma (PPP) and red blood cells (RBC) were sequestered as well. An autotransfusion device was used to collect and re-infuse autologous RBC during the course of the operation in both groups. After completion of the aortic reconstruction, autologous PRP and PPP were re-infused in the APP group. Blood loss, volume of blood component transfusions, and preoperative and postoperative hemoglobin (Hb), hematocrit (Hct), platelet, international normalized ratio (INR), and activated partial thromboplastin time (aPTT) were recorded. Measurements and Main Results: There was no difference between groups in demographics, preoperative laboratory values, or surgical procedures, although more patients were treated for aneurysms (73% vs . 60%) and fewer for occlusive disease (20% vs . 40%) in the control versus APP group. Also, there were no differences between the control and APP groups in duration of operation, blood loss, volume of colloid infused, or volume of allogenic RBC and plasma transfused. Patients in the APP group received a greater volume of crystalloid solution (9.1 ± 3.4 L vs. 6.8 ± 3.0 L; p = 0.002), but fewer units of allogenic platelets than the control group (0.7 ± 1.0 units vs . 0.2 ± 0.4 units; p < 0.04). There were no differences in postoperative Hb, Hct, INR, aPTT, or fibrinogen. The platelet count was lower in the APP group than in the control group (123 ± 40 × 10 3 / mm 3 vs. 182 ± 51 × 10 3 / mm 3 ; p = 0.004). Conclusions: Perioperative platelet plasmapheresis led to fewer allogenic platelet transfusions in patients undergoing elective aortic reconstruction. However, there was no decrease in blood loss and no reduction in transfusion of allogenic RBC or plasma. Perioperative platelet plasmapheresis is not recommended for routine use in elective aortic reconstruction.

Identifying allogeneic platelets by resolution of point mutations in mitochondrial DNA using single-stranded conformational polymorphism PCR.

The purpose of this study was to evaluate single-stranded conformational polymorphism (SSCP)-PCR utilizing two different regions of mitochondrial DNA (mtDNA) as a method to discriminate between donor platelets and recipient cells. Twenty-eight mixtures of platelets (1:1 ratio) were prepared from eight randomly selected persons to simulate donor-recipient combinations after allogeneic platelet transfusion. The mtDNA was extracted from each donor and each prepared mixture. Four primer pairs were designed to amplify two regions of mtDNA, hypervariable region (HVR) 1 and 2. An SSCP-PCR method was developed to analyze the four different amplicons. In addition, the amplified DNA samples containing HVR1 and HVR2 mtDNA of the eight persons were sequenced by using dye-terminator cycle sequencing to determine mtDNA polymorphisms. With four different primer pairs and SSCP-PCR, it was possible to discriminate between donor and recipient DNA in all 28 combinations. DNA sequencing confirmed that the suspected differences were localized within the amplicons examined by SSCP-PCR. SSCP-PCR analysis targeting the HVR1 and HVR2 mtDNA is a promising new method to potentially identify donor cells on the basis of mtDNA polymorphisms. The method does not require prior knowledge of sequence differences between donor and recipient and can be optimized to quantify the amount of residual transfused allogeneic platelets.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

Novel platelet products, substitutes and alternatives

Despite the many advances in the safety, processing, and storage of conventional 22 °C liquid-stored allogeneic platelet concentrates, there still are significant drawbacks to the use of such products. Efforts to overcome these shortcomings have resulted in an array of novel platelet products, substitutes, and alternatives; which are currently at various stages of development. This review summarizes the recent developments in the frozen and cold storage of platelets; their pathogen inactivation; as well as the status of lyophilized platelets, infusible platelet membranes (IPMs), red cells bearing arginine–glycine–aspartic acid (RGD) ligands, fibrinogen-coated albumin microcapsules, and liposome-based agents; as potential alternatives to the use of conventional platelet transfusions. Pre-clinical studies have been encouraging for several of these novel products; however, to date, very few have entered human trials. Nonetheless, with the ongoing development of diverse products, those properties that may be necessary for their hemostatic effectiveness will become apparent. However, safety and efficacy must be demonstrated in pre-clinical and phase I to III clinical trials before these novel agents, substitutes and alternatives can be used clinically for patients with thrombocytopenia.

Transfusion Algorithm Reduces Use of Allogeneic Nonerythrocyte Components.

Transfusion Algorithm Reduces Use of Allogeneic Nonerythrocyte Components. Nuttall et al. (page 773)Among 836 enrolled adult patients who had elective cardiac surgery necessitating cardiopulmonary bypass, abnormal bleeding developed in 92 (11%). In a randomized trial, Nuttall et al. compared the impact of different allogeneic transfusion practices in these 92 patients.After the diagnosis of abnormal bleeding was made, patients were assigned randomly to one of two groups. In the control group, decisions for transfusion of blood components (platelets, fresh frozen plasma, or cryoprecipitate) were left to the discretion of the individual anesthesiologists, with or without guidance from laboratory tests. Transfusion therapy in the protocol group was guided by an algorithm based on coagulation tests. Allogeneic platelets were administered if the platelet count was less than 102,000/μl or the maximum amplitude of the thromboelastogram was less than 48 mm. Fresh frozen plasma was transfused if the prothrombin time was greater than 16.6 s or the activated partial thromboplastin time was greater than 57 s. Cryoprecipitate was transfused if fibrinogen was less than 144 mg/dl.Results showed that patients in the transfusion algorithm group received less allogeneic fresh frozen plasma in the operating room after cardiopulmonary bypass than did the patients in the control group. Platelet transfusions were also less frequent in the protocol group, and these patients experienced less blood loss in the intensive care unit. Patients in the control group also had a significantly greater incidence of surgical reoperation of the mediastinum for bleeding (11.8%vs. 0%). Although the authors recommend a multicenter study to address institutional variances in transfusion practices, they believe that use of an algorithm based on similar measurements could improve transfusion decisions.

Polymorphisms in the non-coding region of the human mitochondrial genome in unrelated plateletapheresis donors.

Human mitochondrial DNA polymorphisms are unique targets to discriminate nucleated cells and platelets between donor and recipient in the setting of transplantation or transfusion. We have previously used this approach to discriminate allogeneic platelets from autologous platelets after transfusion. In the present study, we used DNA sequencing to investigate polymorphisms present in two of the hypervariable segments (HVR1 and HVR2) found within the non-coding region of the mitochondrial genome among 100 plateletapheresis donors. Alignments were made with the Cambridge Reference Sequence (CRS) for human mitochondrial DNA (mtDNA). Combining the sequencing information of HVR1 and HVR2 we could demonstrate that, of the 100 investigated mtDNA samples, none was identical to the CRS. We found a total of 2-17 polymorphisms per donor in the investigated regions, most of them were basepair substitutions (563) and insertions (151). No deletions were found. Sixty-six of the 110 detected polymorphisms were detected in more than one sample. Seven polymorphisms are newly described and have not been published in the Mitomap database. Our results demonstrate that polymerase chain reaction analysis of the many polymorphisms found in the hypervariable region of mitochondrial DNA represents a more informative target than previously described mitochondrial polymorphisms for discriminating donor-recipient cells after transfusion or transplantation.

New technologies for the inactivation of infectious pathogens in cellular blood components and the development of platelet substitutes

Despite the increased safety of blood components, achieved through improved donor selection and testing, transfusion recipients remain at risk of transfusion-associated diseases. Transfusion of cellular blood components has been implicated in transmission of viral, bacterial and protozoan diseases.1 While it is commonly recognized that hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), and retroviruses, such as human immunodeficiency virus (HIV) and the human lymphotrophic viruses (HTLV), can be transmitted through cellular components, other pathogens are emerging as potentially significant transfusion-associated infectious agents. For example, transmission of protozoan infections due to trypanosomes2 andBabesia have been reported.5 In addition to viral and protozoan infectious agents, cases of bacterial contamination of platelet and red cell concentrates continue to be reported 6,7 and may be an under-reported transfusion complication.8 More importantly, new infectious agents continue to enter the donor population, and there is an inherent time delay before the new pathogens are definitively identified and new tests implemented in order to maintain consistent safety of the blood supply. The paradigm for this problem is the HIV pandemic.During the past decade a number of methods for inactivating infectious pathogens in platelet concentrates have been investigated as a strategy to improve the safety of platelet transfusion therapy. One method of treating platelet concentrates to inactive pathogens has now reached the advanced clinical trial phase in the United States and Europe. Similar efforts with a new class of compounds are underway for red cell concentrates, and two of these are in early phase trials. In addition to studies with allogeneic platelets and red cells, a number of laboratories have described methods for developing platelet substitutes or modified platelets to avoid the use of traditional platelet concentrates as a means to improve safety.

Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures

Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P‐selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re‐infusion of autologous platelets on platelet function. Autologous platelet‐rich plasma (PRP) contained a significantly lower (P &lt; 0·05) percentage of P‐selectin‐positive and fibrinogen‐positive platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P &lt; 0·05) percentage of responsive platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P‐selectin or binding fibrinogen increased significantly after CPB (P &lt; 0·05), while the percentage of platelets responsive to in vitro agonists was decreased (P &lt; 0·05 in autologous transfusion patients), consistent with platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re‐infusion of autologous PRP. P‐selectin expression had returned to pre‐CPB levels 24 h post‐operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.

Redo without transfusion in a patient with idiopathic thrombocytopenic purpura

Mitral valve replacement was able to be carried out at redo operation requiring neither allogeneic platelet nor blood transfusion in a patient with idiopathic thrombocytopenic purpura, by means of preoperative high-dose intravenous γ-globulin, autologous blood predonation, use of a centrifugal pump, heparin-coated extracorporeal circuits, and simultaneous splenectomy.

Platelet antigens and antiplatelet antibodies

Transfusion of allogenic platelets (TAP) is a potent therapeutic method for patients with impaired bone marrow platelet-producing function caused by endogenous depression ofhemopoiesis or exogenous factors, such as radiation or cytostatic therapy. Transplantation of allogenic bone marrow from close relatives is impossible without TAP. Allogenic platelets are required for recipients in whom thrombopoiesls in the bone marrow ceases because of fractionated irradiation and cytostatic therapy, while transplanted cells do not yet function. TAP is an expensive procedure. It costs at least $500 to attain a hemostatic effect by means of TAP in one patient. That is why it is particularly disappointing when platelet transfusion is ineffective because of immunological reasons which have been neglected by transfusiologist [ 1,2]. Immune conflict in TAP can be due to allosensitization caused by previous pregnancies and transfusion of blood components [2,10,1 l]. The conflict consists in rejection of donor platelets because of immune antibody production in the recipient. It considerably reduces the efficiency of therapy and even annihilate its therapeutic effect. The immune reaction can be associated with fever (nonhemolytic transfusion reaction) and the absence of increment in platelets count (refraction to platelet transfusion. RPT). The main cause of immune complications in TAP are antibodies to HLA class I antigens expressed on T lymphocytes, polymorphonuclear leukocytes, and platelets [8,9]. It is believed that the more blood component transfusions (erythrocyte and platelet transfusions) are received by patients, the higher the incidence of antibody production and RPT [6]. Sensitizing doses for leukocytes and platelets are 15x10 s and

The reduction of HLA class I expression on platelets: A potential approach in the management of HLA-alloimmunized refractory patients

HE FIRST successful attempt to raise the platelet count in thrombocytopenic patients by the transfusion of whole blood was in 1910 by Duke.~ Gradual improvements in the techniques to prepare and store platelets for transfusion contributed to the ready availability of allogeneic platelet transfusions, which supported major advances in the treatment of leukemia, cancer, and aplastic anemia. However, a major obstacle to the provision of long-term platelet support is the development of a state of refractoriness that results in the accelerated destruction of the transfused platelets and may lead to fatal bleeding complications. Refractoriness to platelet transfusions is related to the clinical condition of the patient and the presence of alloantibodies to antigens expressed on the membrane of the transfused platelets.

Extreme Leukoreduction of Major Histocompatibility Complex Class II Positive B Cells Enhances Allogeneic Platelet Immunity

In a murine model of platelet alloimmunization, we examined the definitive role that mononuclear cells (MC) have in modulating platelet immunity by using platelets from severe combined immunodeficient (SCID) mice. CB.17 (H-2(d)) SCID or BALB/c (H-2(d)) mouse platelets were transfused weekly into fully allogeneic CBA (H-2(k)) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 +/- 0.6/microL and SCID mouse platelets could be prepared to have significantly lower (<0. 05/microL) MC numbers. Transfusions with 10(8) BALB/c platelets (containing approximately 100 MC/transfusion) stimulated IgG antidonor antibodies in 100% of the recipients by the fifth transfusion, whereas 10(8) SCID mouse platelets (containing approximately 5 MC/transfusion) stimulated higher-titered IgG alloantibodies by the second transfusion. When titrations of BALB/c peripheral blood MC were added to the SCID mouse platelets, levels approaching 1 MC/microL reduced SCID platelet immunity to levels similar to BALB/c platelets. Characterization of the alloantibodies showed that the low levels of MC significantly influenced the isotype of the antidonor IgG; the presence of 1 MC/microL was associated with induction of noncomplement fixing IgG1 antidonor antibodies, whereas platelet transfusions, devoid of MC (<0. 05/microL), were responsible for complement-fixing IgG2a production. When magnetically sorted defined subpopulations of MC were added to the SCID platelets, major histocompatability complex (MHC) class II positive populations, particularly B cells, were found to be primarily responsible for the reduced SCID mouse platelet immunity. The presence of low numbers of MC within the platelets was also associated with an age-dependent reduction in platelet immunogenicity; this relationship however, was not observed with SCID mouse platelets devoid of MC. The results suggest that a residual number of MHC class II positive B cells within allogeneic platelets are required for maximally reducing alloimmunization.

Extreme Leukoreduction of Major Histocompatibility Complex Class II Positive B Cells Enhances Allogeneic Platelet Immunity

AbstractIn a murine model of platelet alloimmunization, we examined the definitive role that mononuclear cells (MC) have in modulating platelet immunity by using platelets from severe combined immunodeficient (SCID) mice. CB.17 (H-2d) SCID or BALB/c (H-2d) mouse platelets were transfused weekly into fully allogeneic CBA (H-2k) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 ± 0.6/μL and SCID mouse platelets could be prepared to have significantly lower (<0.05/μL) MC numbers. Transfusions with 108 BALB/c platelets (containing ≈100 MC/transfusion) stimulated IgG antidonor antibodies in 100% of the recipients by the fifth transfusion, whereas 108 SCID mouse platelets (containing ≈5 MC/transfusion) stimulated higher-titered IgG alloantibodies by the second transfusion. When titrations of BALB/c peripheral blood MC were added to the SCID mouse platelets, levels approaching 1 MC/μL reduced SCID platelet immunity to levels similar to BALB/c platelets. Characterization of the alloantibodies showed that the low levels of MC significantly influenced the isotype of the antidonor IgG; the presence of 1 MC/μL was associated with induction of noncomplement fixing IgG1 antidonor antibodies, whereas platelet transfusions, devoid of MC (<0.05/μL), were responsible for complement-fixing IgG2a production. When magnetically sorted defined subpopulations of MC were added to the SCID platelets, major histocompatability complex (MHC) class II positive populations, particularly B cells, were found to be primarily responsible for the reduced SCID mouse platelet immunity. The presence of low numbers of MC within the platelets was also associated with an age-dependent reduction in platelet immunogenicity; this relationship however, was not observed with SCID mouse platelets devoid of MC. The results suggest that a residual number of MHC class II positive B cells within allogeneic platelets are required for maximally reducing alloimmunization.

Fibrinogen-coated albumin microcapsules reduce bleeding in severely thrombocytopenic rabbits.

Severe thrombocytopenia frequently occurs in patients receiving chemotherapy and in patients with autoimmune disorders. Thrombocytopenia is associated with bleeding, which may be serious and life threatening. Current treatment strategies for thrombocytopenia may require transfusion of allogeneic platelets, which is associated with serious drawbacks. These include the occurrence of anti-platelet antibodies, which may result in refractoriness to further platelet transfusions, and the potential risk of transfer of blood-borne diseases. Therefore, we have recently developed a platelet substitute product (Synthocytes), which is composed of human albumin microcapsules with fibrinogen immobilized on their surface. Here we show that the intravenous administration of these microcapsules not only corrects the prolonged bleeding time in rabbits rendered thrombocytopenic either by anti-platelet antibodies or by chemotherapy, but also reduces bleeding from surgical wounds inflicted in the abdominal skin and musculature. No potential systemic prothrombotic effect of the microcapsules was observed in a model of rabbit venous thrombosis. As for the mechanism of action, experiments with normal and thrombocytopenic human blood in an endothelial cell matrix-coated perfusion chamber demonstrated an interaction between the fibrinogen-coated albumin microcapsules and native platelets. It was shown that the fibrinogen-coated albumin microcapsules could facilitate platelet adhesion to endothelial cell matrix and correct the impaired formation of platelet aggregates in relatively platelet-poor blood. This study indicates that fibrinogen-coated albumin microcapsules can act to improve primary hemostasis under thrombocytopenic conditions and may eventually be a promising agent for prophylaxis and treatment of bleeding in patients with severe thrombocytopenia.