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Jaleah Hawkins,1 Richard H Aster,2,3 Brian R Curtis2,3 1Versiti, Blood Center of Michigan, Grand Rapids, MI, USA; 2Versiti, Blood Center of Wisconsin, Milwaukee, WI, USA; 3Blood Research Institute, Milwaukee, WI, USACorrespondence: Brian R CurtisVersiti, Blood Center of Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178, USATel +1 414 937 6087Fax +1 414 937 6242Email brcurtis@versiti.orgAbstract: Post transfusion purpura (PTP) is an uncommonly reported post transfusion adverse event that can present with severe thrombocytopenia; sometimes resulting in significant bleeding and hemorrhage. Its diagnosis can be elusive given its substantial symptomatic overlap with other thrombocytopenic syndromes. Underdiagnosis and underreporting make the true incidence of disease difficult to define. While clinical suspicion is key, laboratory evidence of platelet-targeted antibodies and identification of the antigen(s) they recognize are necessary to confirm the diagnosis. A curious aspect of PTP is paradoxical destruction of both transfused and autologous platelets. Although the first case was reported over 50 years ago, this aspect of PTP pathogenesis is still not fully understood and is widely debated. Several theories exist, but conclusive evidence to support most is lacking. Despite limited understanding of disease incidence and etiology, treatment with IVIG (Intravenous Immunoglobulin) has become standard practice and can be highly effective. Although recurrence is rare, precautions should be taken if patients with a history of PTP require transfusions in the future.Keywords: thrombocytopenia, platelet antibodies, transfusion reactions

Background: Blood group A and B antigens are expressed only weakly on platelets (PLTs) of most individuals but are very strongly expressed on PLTs from approximately 1 percent of normal subjects (Type II high expressers). The implications of this trait for transfusion medicine are undefined.Study Design and Methods: A family was studied in which two Group B infants were born with neonatal thrombocytopenia, whereas a third infant whose blood group was A(2) had a normal PLT count at birth.Results: Serologic studies demonstrated a maternal antibody that reacted strongly with PLTs from the father and the two group B children in flow cytometry and with GPIIb/IIIa from their PLTs in solid-phase assays. No PLT-specific antibodies were detected in maternal serum sample, but it contained a high-titer immunoglobulin G antibody specific for blood group B. All PLT-reactive antibody in the mother's serum was removed by absorption with pooled, washed group A and B red cells (RBCs). Studies with monoclonal anti-B and measurement of serum B-glycosyltransferase activity showed that the father and both group B children were Type II high expressers of blood group B.Conclusions: The findings indicate that high-titer blood group antibodies acquired from the mother can cause thrombocytopenia in infants possessing the Type II high-expresser phenotype despite competition for antibody binding by blood group antigens expressed on RBCs and other tissues. [ABSTRACT FROM AUTHOR]

Background: Neonatal alloimmune thrombocytopenia (NATP) is an important cause of morbidity and mortality in the newborn. Optimal management of subsequent pregnancies requires knowledge of the alloantigen that caused maternal immunization, but this is possible only in a minority of cases. This study investigated whether this can be explained in part by maternal immunization against the "rare" alloantigen HPA-9b (Max(a)), implicated previously only in a single NATP case.Study Design and Methods: Archived paternal DNA from unresolved cases of NATP and normal individuals was typed for platelet (PLT)-specific antigens with real-time polymerase chain reaction and direct sequencing. PLT-specific alloantibodies were characterized by flow cytometry and solid-phase enzyme-linked immunosorbent assay. Recombinant GPIIb/IIIa was expressed in stably transfected Chinese hamster ovary cells. Clinical information was obtained directly from attending physicians.Results: Six of 217 fathers were positive for the presence of HPA-9b (Max(a)), an incidence about seven times that in the general population. In each of five cases studied, maternal serum samples reacted with intact paternal PLTs and paternal GPIIb/IIIa. Only one of three serum samples tested recognized recombinant GPIIb/IIIa carrying the HPA-9b (Max(a)) mutation. These seemingly discrepant reactions may reflect different requirements for oligosaccharides linked to residues close to the mutation in GPIIb that determines HPA-9b (Max(a)). NATP in the affected children was severe and was associated with intracranial hemorrhage in three of six infants on whom information was obtained.Conclusions: Maternal immunization against HPA-9b (Max(a)) is an important cause of NATP and should be considered in cases of apparent NATP not resolved on the basis of maternal-fetal incompatibility for "common" PLT antigens. [ABSTRACT FROM AUTHOR]

Agents that react with the platelet glycoprotein (GP) IIb/IIIa complex (alphaIIb/beta3 integrin) to block fibrinogen binding and platelet-platelet aggregation have been proved to be effective in reducing the incidence of complications following coronary angioplasty and are now widely used for this purpose. Acute thrombocytopenia, which is sometimes severe and life-threatening, is a recognized side effect of this class of drugs. In contrast to other types of drug-induced thrombocytopenia, this complication can occur within a few hours of a patient's first exposure to the medication. Accumulating evidence has indicated that drug-dependent antibodies, which can be naturally occurring, are the cause of platelet destruction in such individuals. In this review, we will consider the clinical aspects of thrombocytopenia resulting from sensitivity to GPIIb/IIIa inhibitors and will review evidence that the platelet destruction is antibody-mediated. [ABSTRACT FROM AUTHOR]

Background: Vancomycin has only rarely been implicated as a cause of thrombocytopenia, and there is only limited evidence that this complication is caused by immune mechanisms. We conducted a study to determine whether thrombocytopenia is caused by vancomycin-dependent antibodies in patients being treated with vancomycin.Methods: We identified and characterized vancomycin-dependent, platelet-reactive antibodies in patients who had been referred for testing during a 5-year period because of a clinical suspicion of vancomycin-induced thrombocytopenia. We obtained clinical information about the patients from their referring physicians.Results: Drug-dependent, platelet-reactive antibodies of the IgG class, the IgM class, or both were identified in 34 patients, and clinical follow-up information was obtained from 29 of these patients. The mean nadir platelet count in these patients was 13,600 per cubic millimeter, and severe bleeding occurred in 10 patients (34%). Platelet levels returned to baseline in all 26 surviving patients after vancomycin was stopped. In 15 patients, the drug was continued for 1 to 14 days while other possible causes of thrombocytopenia were investigated. Vancomycin-dependent antibodies were not found in 25 patients who had been given vancomycin and in whom thrombocytopenia did not develop.Conclusions: Severe bleeding can occur in patients with vancomycin-induced immune thrombocytopenia. The detection of vancomycin-dependent antiplatelet antibodies in patients receiving the antibiotic in whom thrombocytopenia develops, and the absence of antibodies in patients given the drug in whom platelet counts remain stable, indicate that these antibodies are the cause of the thrombocytopenia. [ABSTRACT FROM AUTHOR]

Background: We previously described a mouse model in which platelet immunization between selected strains leads to production of alloantibodies and severe autoimmune thrombocytopenia and mimics the human condition posttransfusion purpura (PTP). This report describes studies defining epitopes recognized by these alloantibodies., Study Design: Hybridomas were produced from spleen cells of immunized mice. Glycoprotein (GP) targets of resulting monoclonal antibodies were characterized by immunoprecipitation using platelets from the immunizing strains. Antigens defined by single amino acid (AA) polymorphisms recognized by monoclonal antibodies were identified by mutagenizing target glycoproteins expressed in Chinese hamster ovary cells and observing the effects on antibody binding., Results: Three monoclonal antibodies (417.1, 417.3, 425.1) were produced that recognized GPIIb on immunizing platelets. Monoclonal antibodies 417.1 and 417.3 both required G111 and 425.1 required V37, located on the beta propeller domain of GPIIb, for binding to platelets from the immunizing strains C57 and PWK, respectively. Injection of 417.3 and 425.1 into mice caused platelet destruction only in mice with GPIIb containing the targeted AAs., Conclusions: Findings made provide evidence that alloantibodies produced by mice experiencing thrombocytopenia in a mouse model of PTP are specific for single AA polymorphisms that differ in GPIIb/IIIa integrin of the immunizing and immunized strains and therefore closely resemble the potent alloantibodies found in patients with PTP. The observations show that naturally occurring single AA differences in GPIIb/IIIa integrin of various mouse strains are highly immunogenic in the mouse strains studied and readily induce antibodies comparable to human platelet antigen-specific antibodies found in transfused and pregnant humans., (© 2021 AABB.)

Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14-labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT., (© 2021 by The American Society of Hematology.)

Posttransfusion purpura (PTP) is an uncommon but life-threatening condition characterized by profound thrombocytopenia occurring ∼1 week after a blood transfusion. The hallmark of PTP is a potent immunoglobulin G antibody specific for a transfused platelet-specific alloantigen, usually located on glycoprotein IIb/IIIa (GPIIb/IIIa; αIIb/β3 integrin). It is widely thought that this alloantibody somehow causes the thrombocytopenia, despite absence from host platelets of the alloantigen for which it is specific. In studies described here, we found that cross-strain platelet immunization in mice commonly induces GPIIb/IIIa-specific alloantibodies combined with platelet-specific autoantibodies and varying degrees of thrombocytopenia, and we identified 1 strain combination (129S1Svlm/PWKPhJ) in which 95% of immunized mice made both types of antibody and developed severe thrombocytopenia. There was a strong inverse correlation between autoantibody strength and platelet decline (P < .0001) and plasma from mice that produced autoantibodies caused thrombocytopenia when transfused to syngeneic animals, arguing that autoantibodies were the cause of thrombocytopenia. The findings define a model in which a routine alloimmune response to platelets regularly transitions to an autoimmune reaction capable of causing severe thrombocytopenia and support the hypothesis that PTP is an autoimmune disorder., (© 2020 by The American Society of Hematology.)

Human platelet membrane glycoprotein polymorphisms can be immunogenic in man and are frequently the cause of clinically important immune reactions responsible for disorders such as neonatal alloimmune thrombocytopenia. Platelets from individuals carrying rare polymorphisms are often difficult to obtain, making diagnostic testing and transfusion of matched platelets challenging. In addition, class I HLA antibodies frequently present in maternal sera interfere with the detection of platelet-reactive alloantibodies. Detection of alloantibodies to human platelet antigen 3 (HPA-3) and HPA-9 is especially challenging, in part because of the presence of cell type-specific glycans situated near the polymorphic amino acid that together form the alloepitope. To overcome these limitations, we generated a series of HLA class I-negative blood group O induced pluripotent stem cell (iPSC) lines that were gene edited to sequentially convert their endogenous HPA-3a alloantigenic epitope to HPA-3b, and HPA-9a to HPA-9b. Subjecting these cell lines, upon differentiation into CD41+/CD42b+ human megakaryocytes (MKs), to flow cytometric detection of suspected anti-HPA-3 and HPA-9 alloantisera revealed that the HPA-3a-positive MKs specifically reacted with HPA-3a patient sera, whereas the HPA-3b MKs lost reactivity with HPA-3a patient sera while acquiring reactivity to HPA-3b patient sera. Importantly, HPA-9b-expressing MKs specifically reacted with anti-HPA-9b-suspected patient samples that had been undetectable using conventional techniques. The provision of specialized iPSC-derived human MKs expressing intact homozygous glycoprotein alloantigens on the cell surface that carry the appropriate endogenous carbohydrate moieties should greatly enhance detection of clinically important and rare HPA-specific alloantibodies that, to date, have resisted detection using current methods., (© 2019 by The American Society of Hematology.)

Heparin-induced thrombocytopenia is a relatively common drug-induced immune disorder that can have life-threatening consequences for affected patients. Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive Abs are central to the pathogenesis of heparin-induced thrombocytopenia. Regulatory T (Treg) cells are a subpopulation of CD4 T cells that play a key role in regulating immune responses, but their role in controlling PF4/heparin-specific Ab production is unknown. In the studies described in this article, we found that Foxp3-deficient mice lacking functional Treg cells spontaneously produced PF4/heparin-specific Abs. Following transplantation with bone marrow cells from Foxp3-deficient but not wild-type mice, Rag1-deficient recipients also produced PF4/heparin-specific Abs spontaneously. Adoptively transferred Treg cells prevented spontaneous production of PF4/heparin-specific Abs in Foxp3-deficient mice and inhibited PF4/heparin complex-induced production of PF4/heparin-specific IgGs in wild-type mice. Treg cells suppress immune responses mainly through releasing anti-inflammatory cytokines, such as IL-10. IL-10-deficient mice spontaneously produced PF4/heparin-specific Abs. Moreover, bone marrow chimeric mice with CD4 T cell-specific deletion of IL-10 increased PF4/heparin-specific IgG production upon PF4/heparin complex challenge. Short-term IL-10 administration suppresses PF4/heparin-specific IgG production in wild-type mice. Taken together, these findings demonstrate that Treg cells play an important role in suppressing PF4/heparin-specific Ab production., (Copyright © 2019 by The American Association of Immunologists, Inc.)