Immobilization Of Platelet Antigens Assay Research Articles

Low Prevalence of HLA-G Antibodies in Lung Transplant Patients Detected using MAIPA-Adapted Protocol.

Lung transplantation is often complicated by acute and/or chronic rejection leading to graft-function loss. In addition to the HLA donor-specific antibodies (HLA-DSA), a few autoantibodies are correlated with the occurrence of these complications. Recently, antibodies directed against non-classical HLA molecules, HLA-G, -E, and -F have been detected in autoimmune diseases, like systemic lupus erythematosus. Non-classical HLA molecules are crucial in the immunological acceptance of the lung graft, and some of their isoforms, like HLA-G*01:04 and -G*01:06, are associated with a negative clinical outcome. The aim of this study is to determine the frequency of detection of HLA-G antibodies in lung transplant recipients (LTRs) and their impact on the occurrence of clinical complications. After incubating the cell lines SPI-801, with and without three different HLA-G isoform expression, with sera from 90 healthy blood donors and 35 LTRs (before and after transplantation), HLA-G reactivity was revealed using reagents from commercial monoclonal antibody immobilization of platelet antigen assay (MAIPA ApDIA®). Only one serum from one blood donor had specific reactivity against the HLA-G transduced lines. Non-specific reactivity in many sera from LTRs was observed with transduced- and wild-type cell lines, which may suggest recognition of an autoantigen expressed by the SPI-801 cell line. In conclusion, this study allowed the development of a specific detection tool for non-denatured HLA-G antibodies. These antibodies seem uncommon, both in healthy subjects and in complicated LTRs. This study should be extended to patients suffering from autoimmune diseases as well as kidney and heart transplant recipients.

Serum from half of patients with immune thrombocytopenia trigger macrophage phagocytosis of platelets.

Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis. Sera that produced a phagocytosis magnitude greater than a normal human serum mean+ 2 standard deviations were considered phagocytosis-positive. Overall, 42% (8/19) of MHC I alloantibody-negative ITP sera were phagocytosis-positive. The indirect monoclonal antibody immobilization of platelet antigens assay was used to detect immunoglobulin G (IgG) autoantibodies to glycoproteins (GP)IIb/IIIa, GPIb/IX, and GPIa/IIa. Autoantibody-positive sera triggered a higher mean magnitude of phagocytosis than autoantibody-negative sera. Phagocytosis correlated inversely with platelet counts among autoantibody-positive patients but not among autoantibody-negative patients. Select phagocytosis-positive sera were separated into IgG-purified and -depleted fractions via protein G and reassessed for phagocytosis. Phagocytosis was largely retained in the purified IgG fractions. In addition, we assessed serum concentrations of C-reactive protein, serum amyloid P, and pentraxin 3 as potential phagocytosis modulators. Pentraxin 3 concentrations correlated inversely with platelet counts among patients positive for autoantibodies. Taken together, sera from approximately half of the patients with ITP studied triggered macrophage phagocytosis of platelets beyond a normal level. An important role for antiplatelet autoantibodies in phagocytosis is supported; a role for pentraxins such as pentraxin 3 may be suggested.

Improvement of Anti-CD36 Antibody Detection via Monoclonal Antibody Immobilization of Platelet Antigens Assay by Using Selected Monoclonal Antibodies.

Antibodies against human CD36 are responsible for several immune-mediated disorders. The detection of anti-CD36 antibodies using the standard monoclonal antibody (mAb) immobilization of platelet antigens (MAIPA) assay is hampered by a high frequency of false-negative results, most likely due to competitive inhibition of the mAb used as the capture antibody. We generated a panel of mouse mAbs against CD36 and seven hybridomas (GZ-3, GZ-13, GZ-70, GZ-143, GZ-413, GZ-507, and GZ-608), which were selected for MAIPA assays, as they reacted with mouse and human CD36. Fourteen anti-CD36 sera were assayed; all of which showed a positive reaction in a PakPlus (Immucor GTI Diagnostics, Inc., Waukesha, WI, USA) ELISA-based screening (optical density: 0.257-2.292). When the reference anti-CD36 mAb FA6-152 was used in the MAIPA assay, only 6/14 (42.9%) sera displayed a positive reaction. In contrast, anti-CD36 antibodies were detected in 13/14 (92.9%) sera when GZ-70 and GZ-608 mAbs were used. This significant improvement resulted in the identification of anti-CD36 antibodies by an antigen capture assay. Since patient's platelets possibly carrying rare native antigens are used, this method will facilitate the identification of new platelet antibodies against CD36 that are involved in immune-mediated thrombocytopenia and other diseases, such as transfusion-related acute lung injury.

A first WHO reference reagent for the detection of anti-human platelet antigen-15b.

Background and ObjectivesAlloantibodies to human platelet antigen‐15b (anti‐HPA‐15b) have been detected in mothers with foetal–neonatal alloimmune thrombocytopenia and in multiply transfused patients. Assays used to detect this antibody, which aids in disease diagnosis, can be unreliable and vary in sensitivity. The objective was to generate a stable, lyophilized anti‐HPA‐15b preparation and evaluate its suitability as a World Health Organization (WHO) reference reagent for use in the quality control of platelet alloantibody detection assays. Results from an international collaborative study to evaluate the preparation were used to assign a minimum potency at which laboratories can be expected to detect the antibody.Materials and MethodsRecalcified plasma containing anti‐HPA‐15b was aliquotted, lyophilized and coded 18/220. Twenty‐five laboratories in 16 countries tested doubling dilutions of the reconstituted material in glycoprotein‐specific assays such as the monoclonal antibody–specific immobilization of platelet antigen assay and reported the last positive (or endpoint) dilution.ResultsTwenty‐four laboratories (96%) detected antibodies with HPA‐15b specificity in preparation 18/220. Reported endpoint dilutions were normally distributed with a modal dilution of 1 in 16 and ranged from 1 in 2 to 1 in 128. Only two laboratories (8%) failed to detect anti‐HPA‐15b at 1 in 8 dilution.ConclusionsWhen diluted 1 in 8, most laboratories detected anti‐HPA‐15b in preparation 18/220 using HPA‐15bb platelets but not with HPA‐15aa platelets. The participants agreed this to be an appropriate dilution for assignment as the minimum potency. In October 2020, the WHO Expert Committee on Biological Standardization approved 18/220 as an International Reference Reagent.

Glycoprotein V is a relevant immune target in patients with immune thrombocytopenia.

Platelet autoantibody-induced platelet clearance represents a major pathomechanism in immune thrombocytopenia (ITP). There is growing evidence for clinical differences between anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib/IX mediated ITP. Glycoprotein V is a well characterized target antigen in Varicella-associated and drug-induced thrombocytopenia. We conducted a systematic study assessing the prevalence and functional capacity of autoantibodies against glycoprotein V. A total of 1140 patients were included. In one-third of patients, platelet-bound autoantibodies against glycoproteins Ib/IX, IIb/IIIa, or V were detected in a monoclonal antibody immobilization of platelet antigen assay; platelet-bound autoantiglycoprotein V was present in the majority of samples (222 out of 343, 64.7%). Investigation of patient sera revealed the presence of free autoantibodies against glycoprotein V in 13.5% of these patients by an indirect monoclonal antibody immobilization of platelet antigen assay, but in 39.6% by surface plasmon resonance technology. These antibodies showed significantly lower avidity (association/dissociation ratio 0.32±0.13 vs. 0.73±0.14; P<0.001). High- and low-avidity antibodies induced comparable amounts of platelet uptake in a phagocytosis assay using CD14+ positively-selected human macrophages [mean phagocytic index, 6.81 (range, 4.75-9.86) vs. 6.01 (range, 5.00-6.98); P=0.954]. In a NOD/SCID mouse model, IgG prepared from both types of anti-glycoprotein V autoantibodies eliminated human platelets with no detectable difference between the groups from the murine circulation [mean platelet survival at 300 minutes, 40% (range, 27-55) vs. 35% (16-46); P=0.025]. Our data establish glycoprotein V as a relevant immune target in immune thrombocytopenia. We would suggest that further studies including glycoprotein V will be required before ITP treatment can be tailored according to platelet autoantibody specificity.

Proteasome Inhibition with Bortezomib Induces Apoptosis of Long-Lived Plasma Cells in Steroid-Resistant or Relapsed Immune Thrombocytopaenia.

Primary immune thrombocytopaenia (ITP) is the most common haemorrhagic disease. Although most patients respond initially to mainstream therapies, such as corticosteroids, immunosuppressants or rituximab, a large proportion of patients fail to respond or relapse. These treatments only affect B lymphocytes or short-lived plasma cells, but not already existing long-lived plasma cells (LLPCs) which persistently secrete antibodies. We hypothesized that LLPCs may play a role in the corticosteroid-resistant or relapsed ITP patients, and bortezomib, a proteasome inhibitor, may act on plasma cells and offer a therapeutic effect. Although a significant difference in the proportion of CD19-CD38hiCD138+ total LLPCs was not observed by flow cytometry, a glycoprotein (GP) IIb/IIIa-specific enzyme-linked immunosorbent spot (ELISpot) assay of sorted CD19-CD138+ LLPCs confirmed the existence of anti-platelet antibody-secreting LLPCs in ITP patients in contrast to healthy controls. Moreover, the LLPCs could be eliminated in the presence of bortezomib by ELISpot assay, which was also confirmed by flow cytometry. Accordingly, a modified monoclonal antibody immobilization of platelet antigen assay of sorted CD19-CD138+ LLPCs revealed that the concentration of anti-platelet antibodies decreased remarkably when cultured with 0.25 ng/mL bortezomib for 5 days. The apoptosis assay demonstrated that bortezomib could induce apoptosis of LLPCs in a concentration-dependent manner. The proteasome activity assay showed that bortezomib significantly reduced the proteasome activity in sorted CD19-CD138+ LLPCs. Furthermore, in active ITP murine models, bortezomib eliminated LLPCs in vivo and alleviated thrombocytopaenia. We conclude that LLPCs participate in the pathogenesis of ITP and bortezomib may have potential as a novel therapeutic regimen.

The Levels of T Lymphocyte Subsets in Immune Thrombocytopenia Associated with Anti-GPIIb/IIIa- and/or Anti-GPIbα-Mediated Responses Are Differentially Sensitive to Dexamethasone

Objective: The aim of this work was to investigate the influence of T lymphocyte subsets and platelet-specific autoantibodies on immune thrombocytopenia (ITP) with dexamethasone therapy. Methods: The samples were obtained from patients before therapy. T lymphocyte subsets were measured by flow cytometry, and platelet-specific autoantibodies were evaluated by modified monoclonal antibody immobilization of platelet antigen assay. Results: A total of 50 ITP patients were involved in the study. Twenty-three were anti-GPIbα antibody positive and were treated with dexamethasone, with a response rate of 47.8%. Twenty-seven cases were anti-GPIbα antibody negative, with a response rate of 77.8%. A significant difference was detected (p < 0.05). The level of CD4+ T lymphocytes in ITP patients was lower compared with the control group (p < 0.05). The level of CD8+ T lymphocytes was higher than that in the normal controls (p < 0.05). Additionally, the patients with a higher level of CD8+ T lymphocytes and lower level of CD4+ T lymphocytes were more likely to respond to dexamethasone treatment. Moreover, we observed that ITP patients associated with anti-GPIIb/IIIa antibodies had lower levels of CD4+ T lymphocytes and higher CD8+ T lymphocyte levels. Conclusions: There was insensitivity to dexamethasone treatment in ITP patients who were anti-GPIbα antibody positive. The detection of T lymphocyte subsets is useful in ITP patients for forecasting the outcome of dexamethasone treatment. There were some relationships between the different antibodies and the levels of T lymphocyte subsets.

Detection of platelet autoantibodies to identify immune thrombocytopenia: state of the art.

Immune Thrombocytopenia (ITP) is diagnosed by exclusion of other causes for thrombocytopenia. Reliable detection of platelet autoantibodies would support the clinical diagnosis of ITP and prevent misdiagnosis. We optimized our diagnostic algorithm for suspected ITP using the direct monoclonal antibody immobilization of platelet antigens assay (MAIPA), which evaluates the presence of platelet autoantibodies on the glycoproteins (GP) IIb/IIIa, Ib/IX and V bound on the patient platelets. The direct MAIPA was shown to be a valuable technique for the detection of platelet autoantibodies and could possibly become a guide for optimizing therapy towards a more personalized treatment of ITP.

Fetal/neonatal alloimmune thrombocytopenia due to anti-CD36 antibodies: antibody evaluations by CD36-transfected cell lines.

Isoantibodies against CD36 (platelet glycoprotein 4), developed in Type I CD36-deficient mothers are frequently reported as the cause of fetal/neonatal alloimmune thrombocytopenia in the Asian population. Therefore, further detailed characterization of anti-CD36-mediated fetal/neonatal alloimmune thrombocytopenia is warranted. Here, we report the characterization of a patient with fetal/neonatal alloimmune thrombocytopenia in a Taiwanese family caused by anti-CD36 isoantibodies using a novel antigen-capture method. Platelets and monocytes were analyzed for CD36 expression by flow cytometry. Sequencing analysis of the CD36 gene was performed to identify the mutation underlying the CD36 deficiency. Stable transfected human embryonic kidney HEK293 cells expressing recombinant CD36 were established. These cells were used for the characterization of anti-CD36 isoantibodies by flow cytometry, immunoprecipitation, and antigen-capture assay. Flow cytometry analysis revealed a total absence of CD36 on both platelets and monocytes of the mother (Type I CD36-deficient) caused by heterozygous deletions of the CD36 gene (332_333delCA and c.1254 + 6_1254 + 11delTATTTG). Analysis of maternal serum with CD36-transfected HEK293 cells by flow cytometry, immunoprecipitation, and antigen-capture assay demonstrated the presence of anti-CD36 isoantibodies in maternal serum. Interestingly, this antibody could not be detected by the monoclonal antibody immobilization of platelet antigens assay when anti-CD36 monoclonal antibody (clone FA6-152) was used as the capture antibody. This case reemphasizes the role of anti-CD36 isoantibodies on the pathomechanism of fetal/neonatal alloimmune thrombocytopenia. The fact that the monoclonal antibody immobilization of platelet antigens assay does not seem to be reliable for the identification of all anti-CD36 antibodies indicates that screening of anti-CD36 isoantibodies by a monoclonal antibody-independent method, as presented here, should be considered.

Development of quantitative monoclonal antibody-specific immobilization of platelet antigens assay for antibodies against human platelet antigen-1a, 3a, and 5b

Human platelet antigens (HPAs) are platelet-specific alloantigens that cause alloimmunization. Alloantibodies induced via pregnancy and transfusion may result in various clinical syndromes. The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay is the gold standard for the detection and identification of HPA antibodies. The present study aimed to develop standardized quantitative MAIPA assays for antibodies against HPA-1a, 3a, and 5b. MAIPA protocol was applied on serial dilutions of standard or reference reagents containing antibodies against HPA-1a, 3a, and 5b. Optimal experimental parameters were determined, and standard curves were constructed with a linear regression. The sensitivity, specificity, and reproducibility of the assays were also evaluated. The optimum reagents and conditions for the MAIPA assays were decided through repeated testing and comparison. The intra-assay coefficients of variation (CVs) are 1.3~3.6%, 2.7~5.2%, and 4.7~5.3%; the inter-assay CVs are 4.0~5.6%, 4.4~5.9%, and 3.2~6.3%, respectively, for HPA-1a, HPA-3a, and HPA-5b antibodies. In specificity tests, some monoclonal antibodies and sera with other antibodies were tested and no obvious positive result was observed. The standardized quantitative MAIPA assays for antibodies against HPA-1a, 3a, and 5b have good sensitivity, reproducibility, and specificity. They have important application value in clinical diagnosis of alloimmunization disease caused by HPA antibodies and safe transfusion of platelets.

Sensitive detection of platelet-specific antibodies with a modified MAIPA using biotinylated antibodies and streptavidin-coated beads

We have developed a modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) with enhanced sensitivity in detecting antibodies against human platelet antigens (HPA), using biotinylated monoclonal antibodies, streptavidin-coated beads and detection by flow cytometry. The beads-MAIPA gave superior signal-to-noise resolution (>10-fold higher) for detection of anti-HPA-1a and anti-HPA-5b compared with the in-house standard MAIPA. Also, efficient and reproducible detection of anti-HPA-15 (CD109) was shown. The enhanced sensitivity was confirmed using WHO International Reference Reagents for anti-HPA-1a, anti-HPA-3a and anti-HPA-5b, which allowed comparison of detection endpoints with other laboratories. Finally, the beads-MAIPA was validated for quantification of anti-HPA-1a. The lower limit of quantification was 0.4IU/mL for beads-MAIPA, compared to 1IU/mL previously reported for standard MAIPA. Based on improved performance against all HPA-antibodies tested, the beads-MAIPA has replaced the standard MAIPA in our laboratory in diagnostics of conditions due to HPA-immunization, such as fetal and neonatal alloimmune thrombocytopenia (FNAIT).

A new bead‐based human platelet antigen antibodies detection assay versus the monoclonal antibody immobilization of platelet antigens assay

The performance of a newly developed Luminex bead-based platelet (PLT) antibody detection method (PAKLx) was compared with the monoclonal antibody immobilization of PLT antigens (MAIPA) assay and the LifeScreen Deluxe Luminex bead-based HLA Class I antibody detection method (LMX). Six sera containing anti-human PLT antigen (HPA)-1a (n=2), HPA-1b, HPA-2b, HPA-3a, or HPA-5b were tested in titration. A total of 194 sera, including HPA-1a, -1b, -2a, -2b, -3a, -5a, and -5b antibodies with or without HLA antibodies (n=63); glycoprotein (GP) IV antibodies (n=1); PLT autoantibodies (n=3); HLA antibodies (n=45); and samples with no PLT-reactive antibodies (n=82), were tested in both assays. Comparable levels of sensitivity were obtained for the MAIPA and PAKLx. The PAKLx showed four (6%) false-negative results in 67 sera with HPA or GP-reactive antibodies: anti-HPA-3a (n=1) or anti-HPA-5b (n=3). The PAKLx showed in 10 of the total 194 samples (5%) the presence of antibodies not detected by the MAIPA. This concerned broadly GP-reactive antibodies (n=7), anti-GPIIb/IIIa combined with anti-HPA-3a (n=1), anti-HPA-1a (borderline, n=1), and anti-GPIV (n=1). Testing 175 sera for anti-HLA Class I antibodies in the PAKLx and LMX showed four discrepant results: PAKLx negative and LMX positive, n=3 and n=1, respectively. For the vast majority of the specimens tested (93%) the results of the PAKLx were in concordance with the MAIPA. The PAKLx is a fast, easy to perform, and sensitive PLT antibody screening method.

Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble β3 integrin

Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems.

Immune‐mediated pancytopenia induced by oxaliplatin: a case report

Drug-induced immune pancytopenia is considered an uncommon disorder. A 76-year-old woman with metastatic gastric adenocarcinoma received 15 cycles of FOLFOX-6 (oxaliplatin/folinic acid/fluorouracil) with a complete response. Upon disease progression, she was restarted on FOLFOX; during the seventh cycle of treatment, 1 hour after completing her oxaliplatin infusion, she presented oral bleeding, petechiae and generalized hematomas. Her platelet (PLT) count decreased from 164 x 10(9)/L to less than 5 x 10(9)/L within a 3-hour period and her white blood cells (WBCs) decreased from 5 x 10(9) to 1.5 x 10(9)/L. One day later she presented a decrease in hemoglobin level (from 11.4 to 10 g/dL, reaching 8.9 g/dL after 5 days). The patient's PLT and lymphocyte count started to recover after 3 days of immunosuppressive treatment. PLT, red blood cell (RBC), and WBC antibody detection tests were performed in the presence and absence of oxaliplatin. PLT-associated antibodies were evaluated by monoclonal antibody immobilization of PLT antigen assay and flow cytometry; WBC antibodies were tested by flow cytometry; and RBC antibodies were evaluated by gel and indirect antiglobulin test tube testing drug-treated RBCs and untreated RBCs in the presence of drug. Positive reactions were obtained only in the presence of the drug (1 mg/mL) for all tests performed (PLTs, RBCs, and WBCs). Our case convincingly demonstrates that oxaliplatin led to the production of drug-dependent PLT, RBC, and WBC antibodies inducing pancytopenia in the patient. The oxaliplatin was discontinued and patient's hematologic values recovered to normal levels.

Neonatal alloimmune thrombocytopenia caused by an antibody specific for a newly identified allele of human platelet antigen‐7

Neonatal alloimmune thrombocytopenia (NAIT) is a neonatal disorder characterized by maternal alloimmunization against fetal platelet (PLT) antigens inherited from the father. A healthy 30-year-old Japanese woman (Hit) gave birth to her second child after an uneventful pregnancy. Nine hours after birth, the infant presented with severe petechiae and a PLT count of 6 x 10(9)/L. To elucidate the maternal cause of NAIT in the infant, serologic and genetic studies, including PLT genotyping and sequence-based analysis, were conducted. Additionally, serologic screening for the new PLT antigen was performed. Serum from the NAIT infant's mother contained antibodies directed against a human PLT antigen (HPA) of the newborn. Using five-cell-lineage flow cytometry, we localized the antigen to a PLT glycoprotein (GP). Subsequent monoclonal antibody immobilization of PLT antigen assay and PLT immunofluorescence inhibition experiments localized the antigen to the GPIIIa subunit of the GPIIb/IIIa complex. GPIIIa localization was confirmed by sequence-based typing studies, which identified a 1297C>T (407proline>serine substitution) mutation on the ninth exon of the GPIIIa gene. This mutation identified the third allele of HPA-7. Anti-Hit(a) reacted with mutated GPIIIa-transfected cells but not with stable transfectants expressing wild-type GPIIIa. Serologic screening for Hit(a) in the Japanese population revealed a phenotypic frequency of approximately 0.0015. We identified a new third allele of HPA-7, which is characterized by a 1297C>T mutation in the GPIIIa gene. This 1297C>T allele was found in 0.15% of the Japanese population. An antibody against this antigen could be the cause of severe NAIT.

Recipient‐derived HPA‐1a antibodies: a cause of prolonged thrombocytopenia after unrelated donor stem cell transplantation

Patients with human platelet antigen (HPA) specific antibodies in cases of neonatal alloimmune thrombocytopenia and platelet (PLT) refractoriness derive clinical benefit from the use of HPA-selected PLTs. This study describes three patients with underlying diagnoses of acute myeloid leukemia, chronic lymphocytic leukemia, and myelodysplasia, respectively, who underwent allogeneic bone marrow transplantation (BMT) with unrelated donors matched at the HLA-A, B, C, Dr, and DQ loci but who failed to achieve an adequate PLT count. Investigation using PLT immunofluorescence test, monoclonal antibody immobilization of PLT antigens assay, and genotyping revealed the presence of recipient-derived HPA-1a antibodies. In two patients, anti-HPA-1a was detected post-BMT and in the third patient, anti-HPA-1a was detected during pre-BMT chemotherapy. Despite apparent 100% engraftment of donor cells, the patients' PLT counts failed to recover 9-10 months posttransplant. The patients remained PLT-transfusion dependent and failed to achieve satisfactory increments following random donor or HLA-matched PLT transfusions. After the identification of HPA-1a antibodies, the patients were supported by HPA-1a(-) PLTs and satisfactory posttransfusion PLT increments were obtained. These cases illustrate that HPA-1a antibodies may remain detectable for 10 months following apparently successful donor engraftment and the disappearance of recipient-derived HLA antibodies. The prolonged persistence of recipient-derived PLT-specific antibodies following BMT has to our knowledge not been described previously. HPA-1a antibodies were associated with protracted PLT-transfusion dependence and significant hemorrhagic complications. Appropriate and timely laboratory investigation for HPA-specific antibodies followed by transfusion support with HPA-selected PLTs provided the cornerstone of the hemostatic management in these cases.

Relative Efficacy of Steroid Therapy in Ameliorating Autoimmune Thrombocytopenia Mediated by Anti-Platelet GPIIbIIIa Versus GPIbα Antibodies.

Abstract 1323Poster Board I-345 BackgroundIdiopathic thrombocytopenic purpura (ITP) is a common autoimmune disease characterized by autoantibody-induced platelet destruction and consequent bleeding disorders. Despite considerable investigation, the pathogenesis of ITP remains incompletely understood, and we currently lack a consensus for therapy. The major targeted platelet autoantigens are located on two distinctive platelet receptors; the GPIIbIIIa and GPIb complexes. It has been reported that thrombocytopenia induced by anti-GPIba antibodies, which may differ from that induced by anti-GPIIbIIIa, can be mediated via an Fc-independent pathway. We further demonstrated in a murine model that anti-GPIba-mediated-ITP is less responsive to IVIG treatment (Blood. 2006; 108(3):943-6), which is consistent with a subsequent retrospective study in human ITP patients. Since IVIG is expensive and steroid therapy is the first line of treatment for most patients, particularly in developing countries, we tested whether gluococorticosteroid therapy has equal efficacy in ITP patients caused by these two different antibodies. Patients and MethodsA total of 107 ITP patients (71 females and 36 males) were recruited for the present study. Patients were first treated with Dexamethasone (DXM, 20mg/day x 3-5 days), then changed to oral administration of Prednisone (1mg/kg/day). The dose of Prednisone was gradually decreased and maintained for = 30 days. The patients were considered as responders to the therapy if no obvious haemorrhage was detected and their platelet counts were raised to = 50 × 109/L or increased by = 30 × 109/L from their original platelet count before treatment. All patients' plasma samples were collected before therapy and specific autoantibodies against GPIIbIIIa and/or the GPIb complex were measured with a monoclonal antibody immobilization of platelet antigen assay (MAIPA). ResultsAmong the 107 patients, 23% (25/107) had antibodies against GPIIbIIIa, 18% (19/107) had antibodies against the GPIb complex, 34% (36/107) had antibodies against both the GPIIbIIIa and GPIb complex, and 25% (27/107) had no detectable antibody to either of these two antigens. We found that a majority of the patients with antibodies against GPIIbIIIa (15/22) were sensitive to the steroid therapy, which is significantly different from those with antibodies against the GPIb complex (5/19) (X2 = 7.15, P <0.01) or those with antibodies against both the GPIb complex and GPIIbIIIa (6/34) (X2 = 14.5, P <0.01). Although no statistically significant difference was observed between patients with anti-GPIb antibodies and those with both anti-GPIb and anti-GPIIbIIIa antibodies (X2 = 0.55, P >0.05), there is a tendency that patients with double positive antibodies were less responsive to steroid therapy therapy. The most sensitive patients for this therapy were those without detectable antibodies to either of these two autoantigens (21/27). ConclusionsOur data suggest that anti-GPIb-mediated ITP may be less responsive to both IVIG and steroid therapies when compared to anti-GPIIbIIIa-mediated ITP. The pathogenesis and treatment of anti-GPIb-mediated ITP merits further investigation. DisclosuresNo relevant conflicts of interest to declare.

A prospective study of maternal anti-HPA 1a antibody level as a potential predictor of alloimmune thrombocytopenia in the newborn

Neonatal alloimmune thrombocytopenia is most commonly due to transplacental passage of maternal anti-HPA 1a antibodies. A prospective study was carried out to evaluate the pattern and quantity of maternal anti-HPA 1a antibodies in order to predict the level of thrombocytopenia in the neonates. A monoclonal antibody immobilization of platelet antigen assay was used to detect antibodies in maternal samples from 1,990 HPA 1bb women. HLA DRB3*0101 typing was performed in all immunized women by sequencing the HLA DRB3 gene when present. Primary immunization more often took place in connection with delivery than during the first pregnancy. There was a strong correlation between maternal antibody levels and the platelet counts in the newborn (R(2) = 0.49, p < 0.001). A maternal antibody level above 3.0 IU/mL measured in gestational week 22 or 34 had a diagnostic sensitivity and specificity of 93% and 63%, respectively, for predicting the grade of neonatal thrombocytopenia. The women who were negative for HLA DRB3*0101 had significantly lower anti-HPA 1a antibody levels than those who were HLA DRB3*0101 positive (p < 0.007). In contrast to primigravida, in whom anti-HPA 1a antibody levels increased during pregnancy, the antibody level decreased in 92 of 147 women who had been pregnant previously (P(92 or more of 147) = 0.003). The anti-HPA 1a antibody level regularly increased after delivery. Maternal anti-HPA 1a antibody levels in weeks 22 and 34 of pregnancy are good predictors of the degree of thrombocytopenia in the newborn both in the first and subsequent pregnancies. Most mothers became immunized at the time of delivery.

A modified rapid monoclonal antibody‐specific immobilization of platelet antigen assay for the detection of human platelet antigen (HPA) antibodies: a multicentre evaluation

The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay is the cornerstone technique for the detection and identification of human platelet antigen (HPA) antibodies. However, the original technique described by Kiefel and colleagues requires approximately 8 h adding to diagnostic delay. Moreover, proficiency exercises indicate that there are substantial variations in the MAIPA protocol, and that these may account for interlaboratory differences in sensitivity and specificity. A review of current MAIPA assay protocols from six laboratories together with performance in quality-assessment schemes identified several key variables potentially affecting the assay results. An optimized protocol was derived and assay time reduced to 5 h. The modified rapid MAIPA (MR-MAIPA) assay was evaluated using 61 samples with a range of HPA antibodies typically encountered in cases of fetomaternal alloimmune thrombocytopenia (n = 22), post-transfusion purpura (n = 8), platelet refractoriness (n = 7) and other platelet immune conditions (n = 24). The sensitivity of the assay was assessed using three international standards and the recombinant HPA-1a antibody CamTran007. The results obtained were compared with the original findings obtained with the local MAIPA assays. In addition, four different glycoprotein IIb/IIIa capture monoclonal antibodies were evaluated for their effect on assay sensitivity. Complete concordance was found between the original MAIPA results and those obtained with the new assay when testing a selected panel of clinical samples. The modified assay had nanogram level sensitivity for the detection of HPA-1a antibodies and titration of HPA-1a and HPA-5b antibody sensitivity standards yielded end-points equal to or greater than the mean recorded in international workshops. The MR-MAIPA assay offers improved turnaround for the detection of HPA antibodies without loss of sensitivity.

A novel antigen‐specific capture assay for the detection of platelet antibodies and HPA‐1a phenotyping

The antigen-specific assays currently used for the laboratory investigation of platelet antibodies and antigens are technically complex and cannot be used in most routine laboratories. Here, we describe a simple antigen-specific capture assay (ASCA) for the detection of serum platelet antibodies and for human platelet antigen-1a (HPA-1a) phenotyping. For the detection of platelet antibodies, platelets from healthy blood donors were incubated with biotinylated monoclonal antibodies to platelet glycoprotein complexes (GP), then solubilized and mixed with superparamagnetic streptavidin particles. Serum samples from patients with autoimmune thrombocytopenia (n = 39), from patients with platelet alloantibodies (6 HPA-1a, 1 HPA-2b, 1 HPA-3a, 6 HPA-5b), and from healthy blood donors (n = 70), were tested. All serum samples from the patients were investigated in parallel by the indirect monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). For HPA-1a phenotyping, superparamagnetic particles were coated with a monoclonal antibody to HPA-1a and mixed with diluted whole blood samples from healthy blood donors (n = 139), who had previously been genotyped for platelet alloantigens. Results The indirect MAIPA detected autoantibodies in 18%, and the direct MAIPA in 50% of patients tested. In contrast, the new ASCA demonstrated positive results in 77% of patients. All tested alloantibodies reacted positive by the ASCA, and all serum samples from healthy blood donors were negative. The results of HPA-1a phenotyping were in concordance with those of genotyping in all cases. In our opinion, the ASCA is easy to perform and much more sensitive than the currently available antigen-specific assays for the detection of platelet antibodies.