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Objective To investigate the frequency of platelet antibodies in voluntary blood donors and patients in Zhongshan, Guangdong Province, and to study the specificity and cross-matching of platelet antibodies. Methods Platelet antibodies of blood donors and patients were screened by solid-phase immunoadsorption (SPIA), rechecked by flow cytometry (FCM), and antibody specificity was identified by PakPlus enzyme immunoassay, and platelet cross-matching was simulated by SPIA. Results A total of 1 049 blood donor samples and 598 patient samples were tested, with 6 (0.57%) and 49 (8.19%) samples positive for SPIA,respectively(P

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare condition in which maternal alloantibodies to fetal platelets cause fetal thrombocytopenia that may lead to intracranial hemorrhage (ICH). Off-label intravenous immunoglobulin (IVIg) has for 30 years been the standard of care for pregnant women who previously have had a child with FNAIT. The efficacy of this treatment has never been tested in a placebo-controlled clinical trial. Although IVIg treatment may improve the neonatal outcome in women who previously have had a child with FNAIT-associated ICH, the question is whether IVIg is necessary for all immunized pregnant women at risk of having a child with FNAIT. The results from some recent publications suggest that antenatal IVIg treatment is not necessary for women who are (1) HPA-1a-immunized and HLA-DRB3*01:01-negative, (2) HPA-1a-immunized with a previous child with FNAIT but without ICH or (3) HPA-5b-immunized. If IVIg is not used for these categories of pregnant women, the amount of IVIg used in pregnant women with platelet antibodies would be reduced to less than ¼ of today's use. This is important because IVIg is a scarce resource, and the collection of plasma for the treatment of one pregnant woman is not only extremely expensive but also requires tremendous donor efforts. [ABSTRACT FROM AUTHOR]

Objective To analyze the platelet antibody detection results and the influence factors of 937 patients in our hospital，explore the impact of platelet antibodies on clinical transfusion effects，provide basis for the application value of platelet antibody detection，and ensure that platelet transfusion is more effective and safe. Methods From June 2019 to April 2021，samples from 937 patients were randomly collected from inpatients who applied for blood preparation or blood transfusion, and platelet antibodies were detected. The patients’ general clinical data were collected through the electronic medical record system and LIS system. Results 1. The positive rate of platelet antibody in patients with multiple transfusions was 13.23%, and the positive rate of platelet antibody correlated with the number of transfusions with statistically significant differences；2. The positive rate of platelet antibody test was higher in women than in men （15.95% vs 10.57%，P < 0.05)；3. The positive rate of platelet antibody test was higher in medical patient than in surgical patient （16.31% vs 3.88%, P < 0.05)，among which the top three department of medicine with positive platelet antibody test rates were the hematology department，the emergency department，and ICU （19.07% vs 18.42% vs 16.95%)；4. For patients in the hematology department, the number of chemotherapy，the combined use of more than 2 types of chemotherapy drugs，and the number of previous transfusion of platelets were significantly correlated with the detection rate of platelet antibodies （P < 0.05); 5. The positive rate of antibodies in the effective and ineffective groups of platelet transfusion was statistically different （P < 0.05)；6. The detection rate of HLA antibodies among platelet antibodies in non-hemolytic febrile reactions was as high as 67.85% (19/28) . Conclusion 1. The production of platelet antibody is positively correlated with the number of blood transfusion，and the factors affecting the positive rate of platelet antibodies included: the number of platelet transfusions，female maternal history，the number of chemotherapy treatments in chemotherapy patients，and the use of chemotherapy drugs；2. The investigation and study of platelet antibody detection has the enormous application value in the ineffectiveness of platelet transfusion, the assessment of transfusion efficacy in patients with hematologic diseases and chemotherapy, the establishment of transfusion strategy, and the diagnosis of non-hemolytic febrile transfusion adverse reactions; 3. The effect of Platelet transfusion can be significantly improved by coordinating platelets transfusion, and can effectively reduce the occurrence of ineffective platelets transfusion. [ABSTRACT FROM AUTHOR]

Objective To investigate the factors influencing the production of platelet antibody and its effect on clinical platelet transfusion. Methods This is a single-center prospective observational study. The research subjects were patients with hematological diseases in West China Hospital of Sichuan University from October 1, 2018 to September 30, 2019, and their plasma were collected before platelet transfusion to detect platelet antibodies using solid-phase agglutination method. According to the results of platelet antibody screening, the patients were divided into platelet antibody positive group and negative group. The t test and nonparametric Mann-Whitney U test were used to compare the transfusion efficacy of two groups. Patients’ demographic and clinical information, including age, gender, diagnosis, the units of platelets and RBC transfused, were collected via HIS6.2.0 and whole process management system of blood in clinical (version 3.0) to analyze the influence of age, gender and the disease on the positive rate of platelet antibodies, as well as the profile of platelet antibodies in patients with different diseases, the correlation between the positive rate of platelet antibodies and the history of blood/platelets transfusion. In additional, the platelet transfusion process was observed on site. Results A total of 316 patients with hematologic diseases were included in this study, mainly with acute myeloid leukemia(188/316, 59.5%). All patients were transfused 1671 U platelet [1~17(5.3±3.1)U each person] and 1896 U RBC products [0~38(7.8±4.6)U each person] during the treatment. Out of the 316 patients, platelet antibodies were found in 85 (26.9%) of them. No significant differences in the positive rates of platelet antibody after transfusion were notice by genders or ages(P>0.05). The incidence of platelet antibody was related to diseases (P0.05) between two groups. The minimum PLT count(5×109/L vs 9×109/L, P0.05)between two groups. According to transfusion events on site, the incidence of acute adverse reactions to transfusion was 13% (169/1 291). Conclusions The positive rates of platelet antibodies in patients with hematologic diseases were relatively high. In addition, the efficacy of platelet transfusion in positive group were worse than that in the negative group. It is recommended that platelet antibody testing should be routinely performed before transfusion in hematologic disease patients to select crossmatch-compatible platelets in order to improve the effectiveness of platelet transfusion.

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by low platelets and an increased risk of bleeding. Platelet autoantibodies target major platelet glycoproteins and cause Fc-mediated platelet destruction in the spleen and reticuloendothelial systems. As mechanisms of disease, platelet autoantibodies are important therapeutic targets. Neonatal Fc receptor (FcRn) antagonists are a new class of therapeutics that reduce the half-life of immunoglobulin G including pathogenic platelet autoantibodies. Spleen tyrosine kinase (Syk) inhibitors interfere with Fc-mediated platelet clearance. Bruton's tyrosine kinase (BTK) inhibitors and B-cell activating factor (BAFF) inhibitors reduce antibody production. The efficacy of these targeted therapies provides new support for the role of platelet autoantibodies in pathogenesis of ITP even these antibodies can be difficult to detect., Areas Covered: This review includes an in-depth exploration of the pathophysiologic mechanisms of ITP, focusing on autoantibodies. Treatments outlined in this review include a) FcRn antagonists, b) complement inhibitors, c) B-cell directed therapies such as BTK inhibitors, and anti-BAFF agents, d) Syk inhibitors, e) plasma-cell directed therapies, and f) novel cellular therapeutic products., Expert Opinion: Platelet autoantibodies are often elusive in ITP, yet novel treatments targeting this pathway reinforce their role in the pathogenesis of this autoimmune platelet disorder.

BACKGROUND: With the widespread use of platelet transfusion, patients with ineffective platelet transfusion are increasing year by year. OBJECTIVE: To provide a reference for improving the effect of platelet transfusion in patients with ineffective platelet transfusion by detecting and analyzing platelet antibody and cross-matching. METHODS: The platelet antibody test kit (solid phase agglutination method) was used to detect platelet antibody and cross-matching test in 112 patients with ineffective platelet transfusion from April to December 2020. RESULTS AND CONCLUSION: (1) The results of platelet antibody test were positive in 103 of 112 cases. The positive rate was as high as 92%. (2) The results of the platelet cross-matching type showed that 25 patients were incompatible with the donor’s platelets in the cross-matching experiment, and 18 patients were matched with all the donor’s platelets. (3) Relationship between platelet antibody test results and matching rate: The results of platelet antibody test were 84% in patients with negative platelet antibody test, 75.4%, 71.6, 54.6%, and 27.6% in patients with 1+, 2+, 3+, and 4+ platelet antibody test. Differences between groups were statistically significant (P < 0.05). (4) Relationship between the positive rate of platelet antibodies and the patients’ multiple examinations: The positive rate of platelet antibody was 90.2% and 94.2% in the number of platelet transfusions < twice and ≥ twice (P > 0.05). (5) Effect of platelet infusion on the patient’s matching rate: In each group with the same number of inspections, there was no statistically significant difference in the matching rate between the initial inspection and repeated inspections (P > 0.05). In all patients with repeated inspections, the average matching rates of the initial inspection and repeated inspections were 41.2% and 50.6%, respectively, and the difference was not statistically significant (P > 0.05). (6) These results suggest that the higher the positive degree of platelet antibody test, the lower the matching rate was. The positive intensity of platelet antibody did not show an increasing trend after infusion of matched platelets. The patient obtained a stable transfusion effect. Accurate detection of platelet antibody and infusion of matched platelets have an important clinical value for improving the efficacy of platelet transfusion. [ABSTRACT FROM AUTHOR]

Alexander Duncan,1 Angela Kellum,2 Skye Peltier,3 David L Cooper,4 Hossam Saad4 1Department of Pathology & Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA; 2Louisiana Center for Bleeding and Clotting Disorders, Tulane University, New Orleans, LA, USA; 3Center for Bleeding and Clotting Disorders, University of Minnesota Medical Center – Fairview, Minneapolis, MN, USA; 4Novo Nordisk Inc., Plainsboro, NJ, USACorrespondence: Alexander DuncanEmory University School of Medicine, Atlanta, GA, USATel +1 404 712 4637Fax +1 404 712 4632Email aduncan@emory.eduPurpose: Glanzmann’s thrombasthenia (GT) is a rare bleeding disorder caused by a mutation in the αIIbβ 3 integrin essential for optimal platelet function and hemostasis. The aim of this study was to identify the burden of GT on patients and caregivers through better understanding of the management and psychosocial impact of this disorder.Patients and Methods: Participants for this online survey were recruited using a rare disease specialty recruiter from Comprehensive Health Education Services. Data were collected from January 31 through March 12, 2019. The questionnaire was designed to collect information regarding demographics, diagnosis, treatment, and psychosocial impact.Results: Of the 45 respondents (24 patients and 21 caregivers), the majority were female (58%), white (64%), and employed full-time (53%) and had no family history of GT (64%). Many patients reported significant bruising at birth (76%), and the mean age at diagnosis was 2.6 years. About half of the patients experienced 1 bleed per day, and 13% had over 500 bleeds of any severity per year. Most bleeds were skin bruising or mouth bleeds, but patients also reported joint/muscle and gastrointestinal bleeds. Most patients reported receiving a platelet transfusion (82%), and some had developed platelet refractoriness (38%) or antibodies (32%). Common treatments were antifibrinolytics (82%) and recombinant activated factor VII (rFVIIa) (42%), likely due to the presence of antibodies. Many (58%) patients experienced issues with excessive bleeding at school; 38% reported missing school as a result. Female patients struggled to find a gynecologist with knowledge of the management of GT. Most patients were satisfied with the support they receive from their current partner (65%) and their friends (76%).Conclusion: Most patients with GT are diagnosed early. Patients experience considerable psychosocial impact. Patient and physician education concerning treatment alternatives and the support of the GT community are critical.Keywords: bleeding disorder, psychosocial impact, recombinant activated factor VII, platelet transfusion, platelet antibodies

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which autoantibodies and/or autoreactive T cells destroy platelets and megakaryocytes in the spleen and bone marrow, respectively. Thrombopoietin receptor agonists (TPO-RA e.g. Romiplostim and Eltrombopag) have made a substantial contribution to the treatment of patients with ITP, which are refractory to first-line treatments and approximately 30% demonstrate sustained elevated platelet counts after drug tapering. How TPO-RA induce these sustained responses is not known. We analyzed the efficacy of a murine TPO-RA in a well-established murine model of active ITP. Treatment with TPO-RA (10 ug/kg, based on pilot dose escalation experiments) significantly raised the platelet counts in ITP-mice. Immunomodulation was assessed by measuring serum IgG anti-platelet antibody levels; TPO-RA-treated mice had significantly reduced IgG anti-platelet antibodies despite the increasing platelet counts. These results suggest that TPO-RA is not only an efficacious therapy but also reduces anti-platelet humoral immunity in ITP.

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: Platelet refractoriness complicates the provision of platelet transfusions- a critical and essential part in management of thrombocytopenia in Oncology patients. Platelet refractoriness poses challenge due to alloimmunization to HLA (Class I) and human platelet antigens (HPAs) and is associated with adverse clinical outcomes and increases health care costs. Aim: A prospective, observational study was planned in medical oncology patients having thrombocytopenia to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory patients. Methods: Eighty oncology patients having thrombocytopenia in a tertiary care centre; both solid organ and hematological malignancies, requiring platelet transfusion were included in this study. ABO-compatible, leucoreduced, random donor platelets with less than 72 hours storage and platelet cross matched were transfused. In case of platelet refractoriness and presence of platelet incompatibility, platelet antibody screening test was performed using SPRCA technique. A P-value < 0.05 was considered statistically significant. Results: Study population was 18-85 years with maximum number of cases (31.3 %) in 60-70 years age group with equal number of both genders. 80% cases showed platelet cross-match compatibility, while 20% were platelet cross-match incompatible. Amongst incompatible platelet cross matches, 87.5% cases showed presence of platelet alloantibodies and all cases except one showed platelet refractoriness. Platelet yield in compatible platelet cross match was higher than in patients with incompatible platelet cross match (P-value < 0.001). Previous exposure in the form of Pregnancy (61% cases) and history of transfusion (54% cases) played a vital role in platelet refractoriness and development of platelet alloantibodies. Patients treated with chemotherapy (78.8%) had significant risk of platelet refractoriness and platelet alloimmunization. Conclusion: Platelet cross matching along with testing for anti-platelet antibodies using SPRCA method is an effective, useful tool and rapid, first-line approach for selecting compatible platelets from the local inventory as compared to HLA-matched platelets in the treatment of thrombocytopenic cancer patients. Blood services must be aware of the measures to prevent alloimmunization and correct identification of refractoriness to provide adequate transfusion support for oncology patients reducing hospital length of stay and minimizing health care cost.

Summary: Platelet function of immune thrombocytopenia (ITP) has been controversial because of methodological problems associated with low platelet counts. In this study, we evaluated platelet function in 21 patients with chronic ITP (cITP) using the recently developed flow cytometry (FCM)‐based platelet aggregation assay (FCA) along with a PAC1/CD62P assay. Since ITP platelets are larger than controls, whole platelets (whole gating method) and size‐adjusted platelets (size‐adjusted method) were analysed in the PAC1/CD62P via FCM. We found that: (i) aggregation was equivalent [phorbol myristate acetate (PMA) or adenosine diphosphate (ADP)‐induced] or enhanced [protease‐activated receptor 1‐activating peptide (PAR1AP)‐induced] in cITP compared with control by FCA; (ii) PAC1 or CD62P was also equivalent or enhanced in cITP in the whole gating method; and (iii) in sharp contrast, the size‐adjusted method revealed that ADP‐, PAR1AP‐, and collagen synthetic liquid reactive peptide (SRP)‐induced PAC1 and ADP‐induced CD62P were impaired in cITP. These data suggested that an increase in the number of larger‐sized platelets may compensate for the impaired platelet function of cITP, leading to non‐inferiority of overall platelet function in cITP. Furthermore, we revealed that ADP‐induced aggregation was impaired in the patients with thrombopoietin receptor agonists (TPO‐RAs) or platelet‐associated anti‐αIIbβ3 antibodies compared with the control, suggesting that the presence of anti‐αIIbβ3 autoantibodies and/or administration of TPO‐RAs may have a negative impact on platelet function. [ABSTRACT FROM AUTHOR]

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which autoantibodies and/or autoreactive T cells destroy platelets and megakaryocytes in the spleen and bone marrow, respectively. Thrombopoietin receptor agonists (TPO-RA e.g. Romiplostim and Eltrombopag) have made a substantial contribution to the treatment of patients with ITP, which are refractory to first-line treatments and approximately 30% demonstrate sustained elevated platelet counts after drug tapering. How TPO-RA induce these sustained responses is not known. We analyzed the efficacy of a murine TPO-RA in a well-established murine model of active ITP. Treatment with TPO-RA (10 ug/kg, based on pilot dose escalation experiments) significantly raised the platelet counts in ITP-mice. Immunomodulation was assessed by measuring serum IgG anti-platelet antibody levels; TPO-RA-treated mice had significantly reduced IgG anti-platelet antibodies despite the increasing platelet counts. These results suggest that TPO-RA is not only an efficacious therapy but also reduces anti-platelet humoral immunity in ITP. [ABSTRACT FROM AUTHOR]

Glanzmann's thrombasthenia (GT) and Bernard-Soulier's syndrome (BSS) are wellunderstood congenital bleeding disorders, showing defect/deficiency of platelet glycoprotein (GP) IIb/IIIa (integrin aIIbß3) and GPIb-IX-V complexes respectively, with relevant clinical, laboratory, biochemical, and genetic features. Following platelet transfusion, affected patients may develop antiplatelet antibodies (to human leukocyte antigen [HLA], and/or aIIbß3 in GT or GPIb-IX in BSS), which may render future platelet transfusion ineffective. Anti-aIIbß3 and anti-GPIb-IX may also cross the placenta during pregnancy to cause thrombocytopenia and bleeding in the fetus/neonate. This review will focus particularly on the better studied GT to illustrate the natural history and complications of platelet alloimmunization. BSS will be more briefly discussed. Platelet transfusion, if unavoidable, should be given judiciously with good indications. Patients following platelet transfusion, and women during and after pregnancy, should be monitored for the development of platelet antibodies. There is now a collection of data suggesting the safety and effectiveness of recombinant activated factor VII in the management of affected patients with platelet antibodies. [ABSTRACT FROM AUTHOR]

Background and Objectives The aim of the 18th Platelet Immunology Workshop was to characterize platelet-reactive alloantibodies by the use of in-house MAIPA protocol and/or commercial platelet antibody testing kits, to identify whether anti- HPA-1a quantification could be compared between laboratories, to evaluate whether a protease inhibitor could preserve CD109 and the expression of the HPA-15b antigen and to prove HPA genotyping methods. Materials and Methods Workshop materials (plasma samples, DNA and additional reagents) were shipped by the Organizing Laboratory in Stockholm, Sweden. Thirty-two laboratories from 19 countries participated in the 18th International Platelet Workshop. Results A majority of the laboratories agreed on the serological identification of anti- HPA-1a and anti- HPA-5b antibodies. For the quantification of anti- HPA-1a, however, the result differed greatly between the laboratories with high coefficient of variation and the lowest concentration 0·5 IU/ml not detectable. Furthermore, false-positive reactions due to cross-reactivity with other glycoproteins did often interfere with the serological evaluation of anti- HPA antibodies. Detection of anti- HPA-15 antibodies remains challenging; the addition of a protease inhibitor did not seem to preserve HPA-15 expression on platelets. For genotyping, correct results were achieved in four of five samples. A mutation near HPA-3 in one sample caused false interpretation of the typing results from five laboratories. Conclusions This workshop has identified three specific areas in platelet serology with need for improvement: methodology amendment to reduce non-specific binding and cross-reactivity; standardization of antibody quantification (such as anti- HPA-1a); and detection of anti- HPA-15 antibodies. The use of universal monoclonal antibodies for antigen capture assays as well exactly defined references of well-defined specificity and concentration should be envisioned. [ABSTRACT FROM AUTHOR]

Neutrophils are dynamic, motile cells that provide the first line of defence against many pathogens. Therefore, neutrophils are equipped with a number of receptors. Some of these receptors are known polymorphic carrying immunogenic structures. Immunization against these antigenic structures, known as human neutrophil antigen ( HNA), can lead to the production of antibodies. These antibodies are implicated in a number of clinical conditions including immune-mediated neutropenia, refractoriness to granulocyte transfusions and transfusion-related acute lung injury. Based on the development of molecular genotyping, intensive studies on the allelic distribution of HNAs have been conducted all over the world. Significant disparity on the allelic distribution was observed, indicating different clinical relevance and priority of HNAs in different ethnic groups. Currently, except in Caucasians, only limited studies on the occurrence of neutrophil alloantibodies have been reported among other populations. These studies may be hampered by the current approach of neutrophil antibody detection due to the fact that several methods still need to be used to obtain reliable results. In this short review, current state on the distribution of HNAs, occurrence of antibodies against HNA and their clinical relevance among different populations will be illustrated. [ABSTRACT FROM AUTHOR]

Lack of reliable laboratory parameters is the main challenge in the management of fetal and neonatal alloimmune thrombocytopenia ( FNAIT). Despite the long-known association between the HLA- DRB3*01:01 allele and human platelet antigen 1a ( HPA-1a) alloimmunisation, maternal human leucocyte antigen ( HLA) typing has been of little clinical value. Recently, other DRB3 allele variants have been suggested to predict the severity of FNAIT. In this nationwide population-based retrospective cohort study, we performed extensive HLA typing of 96 women, accounting for 87% of our cohort of 110 families with confirmed or possible HPA-1a-immunisation. The HLA type was compared with anti- HPA-1a levels, severity of neonatal disease and responsiveness to maternally administrated intravenous gammaglobulin ( IVIG). HLA haplotypes were constructed to investigate further HLA associations. Despite significantly lower anti- HPA-1a levels in DRB3*01:01-negative women, the carrier status of this particular allele could not be used to confirm or rule out FNAIT in the absence of detectable antibodies. In the haplotype analysis, the DRB3*01:01 allele was the actual factor associated with FNAIT. No other HLA allele was shown to be of additional value as a predictor of severe FNAIT or non-responsiveness to IVIG treatment. Thus, HLA genotyping was not found useful in differentiating high- and low-risk pregnancies or in guiding antenatal treatment in affected families. [ABSTRACT FROM AUTHOR]

Background: The identification of platelet alloantibodies is indispensible for the diagnoses of fetal/neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusions. Methods: We compared the results obtained with the gold-standard assay for the detection and specification of platelet antibodies, the monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA), with those from two bead-based assays, simultaneous analysis of specific platelet antibodies assay (SASPA), and the recently commercialized bead-based assay Pak Lx. Results: For the detection of alloantibodies by SASPA, the sensitivity was 94.6%, the specificity was 99.5% The respective results for Pak Lx were 98% and 99.8%. Some antibody specificities were revealed by SASPA or Pak Lx but not by MAIPA. However, there were also antibodies detected by MAIPA that were not seen by SASPA or Pak Lx. Antibody dilution experiments suggest that SASPA or Pak Lx are more sensitive than MAIPA. Conclusions: All three assays have limitations, but Pak Lx is suited for screening. However, other assays are necessary if results are not conclusive or for evaluations beyond the commercial assay's design. [ABSTRACT FROM AUTHOR]

Background No diagnostic tests reliably distinguish primary immune-mediated thrombocytopenia ( pIMT) from other causes of thrombocytopenia. Objectives The purpose of the study was to evaluate diagnostic sensitivity and specificity using modified direct and indirect platelet-associated immunoglobulin ( PAIg) assays and reticulated platelets ( RP) by flow cytometry for the classification of thrombocytopenic dogs and differentiating pIMT. Methods Platelets were isolated from plasma samples of thrombocytopenic dogs and nonthrombocytopenic healthy and ill dogs. For direct PAIg, they were analyzed by flow cytometry after incubation with anti-human amylase fluorescein isothiocyanate ( FITC, negative control), anti-canine IgG- FITC, anti-canine IgM- FITC, and anti-human CD61-conjugated fluorochrome ( AF647). For indirect PAIg, platelets from normothrombocytic dogs were incubated with thrombocytopenic dog plasma and analyzed similar to direct PAIg. RP percentages were determined based on forward light scatter vs thiazole orange fluorescence. Results Seventy-five thrombocytopenic dogs, 16 nonthrombocytopenic ill dogs, and 24 healthy dogs were evaluated. Diagnostic sensitivity and specificity utilizing direct IgG was 29.4% and 75.9%, respectively; when combining direct/indirect assays (IgG/IgM), it was 76.5% and 65.5%, respectively, for distinguishing pIMT. For RP, no significant difference between pIMT and sIMT was noted. RP > 8% with positive PAIg had a sensitivity of 94% and specificity of 27.6% for distinguishing pIMT. There was a significant difference in platelet concentration and CD61% staining between control and pIMT. Conclusions The combined modified assays resulted in fair diagnostic sensitivity and specificity for the diagnosis of pIMT. The modification of the immunoglobulin assays improved diagnostic accuracy; however, a single panel to accurately classify thrombocytopenia remains elusive. [ABSTRACT FROM AUTHOR]

Background The main aims of the 16th ISBT Platelet Immunology workshop were as follows: (i) to test the ability of the laboratories in the identification of platelet alloantibodies, (ii) to compare the detection of HPA-1a alloantibodies in serum and plasma, (iii) to quantify HPA-1a alloantibodies, (iv) to measure platelet-bound HPA-1a alloantibodies in lysates, (v) to evaluate a new bead-based assay for anti- HPA-1a and anti- HPA-1b detection, (vi) to evaluate the efficiency of a human monoclonal antibody for HPA-5a phenotyping and (vii) to test the ability to genotype for HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6, HPA-9, HPA-12w, HPA-13w and HPA-15. Materials and methods Seven exercises were developed to encompass the aims of this workshop. All materials and procedures were distributed for 34 laboratories from 20 countries. Results The platelet alloantibodies identification exercise presented good results compared with previous workshops. Comparison between serum and plasma in titration study showed comparable results for HPA-1a and HPA-5b alloantibodies. For the quantitative determination of HPA-1a alloantibodies, most of the laboratories could reliably assign a similar concentration. It was also possible to identify bound anti- HPA-1a alloantibodies in cell lysates within 2 weeks. The bead assay demonstrated good reproducibility between laboratories although some anti- HPA-1a samples remain an issue. Phenotyping of HPA-5b showed high false-negative error (6%). DNA typing presented excellent results, demonstrating that DNA tests for HPA are very well established in all laboratories. Conclusions This workshop presented high complexity exercises with very good results achieved in most of the exercises and approximately 100% consensus among the participants. [ABSTRACT FROM AUTHOR]

Background/Aim: The aim of this study was to evaluate the levels of platelet-associated antibodies (PAIgG and PAIgM), activated platelets and serum leptin in children with acute immune thrombocytopenic purpura (ITP). Methods: The study included 40 patients with ITP and 40 healthy age- and sex-matched controls. PAIgG, PAIgM and activated platelet levels were estimated by flow cytometry, and serum leptin levels were estimated by ELISA. Results: Activated platelets and serum leptin were significantly higher in the ITP patients than in the controls. The percentage and mean florescence intensity of PAIgG and PAIgM staining were significantly higher in the patients than in the controls. Serum leptin and activated platelet levels in patients with thrombocytopenia of brief duration were significantly lower than those in patients with thrombocytopenia of prolonged duration. The levels of activated platelets, serum leptin and PAIgG were positively correlated, and the levels of serum leptin, activated platelets and platelet counts were negatively correlated. Conclusion: The increased levels of activated platelets, serum leptin and platelet-associated antibodies in children with acute ITP suggest that these factors could play a role in ITP pathogenesis. Additionally, activated platelets and serum leptin could have prognostic significance in paediatric acute ITP. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background and Objectives The aim of the 15th ISBT Platelet Immunology Workshop was to evaluate the detection of free platelet-reactive autoantibodies from ITP patients by the use of a standardized MAIPA protocol, to compare sensitivity and specificity of antibody detection for anti-HPA-1a and serologically difficult-to-assess antibodies against HPA-3, to identify whether anti-HPA-1a titration results can be compared between laboratories, and to evaluate HPA genotyping methods. Materials and Methods Workshop materials were shipped from the organizing laboratory in Giessen, Germany. Thirty laboratories from 19 countries participated. Results Results for the detection of autoantibodies differed greatly between the laboratories and no consensus was reached for one of the two sera. Detection and titration of antibodies against HPA-1a, in contrast, gave largely congruent results. Serologically difficult-to-assess antibodies recognizing HPA-3a and HPA-3b were not detected by many laboratories. For genotyping, good agreement was achieved. Conclusions Detection of HPA-1a antibodies, titration of anti-HPA-1a, and HPA genotyping are well performed in most participating laboratories. The workshop has identified two specific areas with room and need for improvement: the detection of autoantibodies and the detection of HPA-3 alloantibodies. Recommendations of the Working Party on techniques that can help to overcome these problems are desirable. [ABSTRACT FROM AUTHOR]

Immune thrombocytopenia (ITP) is a common autoimmune disease characterized by low platelet counts and an increased risk of bleeding. Antibody-mediated platelet destruction has been the prevailing hypothesis to explain ITP pathogenesis, supported by the efficacy of B-cell depletion therapy; however, the recent success of thrombopoietin receptor agonists lends support to the notion that platelet production is also insufficient. Best practice for the management of chronic ITP has not yet been established because data from comparative trials are lacking. Despite renewed interest in novel drugs capable of increasing platelet counts, ultimate treatment goals for ITP patients must be kept in mind: to improve patients' health and well-being. In this article, the pathophysiology of ITP is reviewed and key remaining questions about mechanism are explored. A rational approach to the management of ITP in adults is outlined, acknowledging evidence and evidence gaps, and highlighting the need for clinically important endpoints in future clinical trials. [ABSTRACT FROM AUTHOR]

Background and Objectives The aims of the 14th ISBT Platelet Immunology Workshop were to evaluate in-house methods for detection of antibodies to human platelet antigens, to compare the sensitivity and specificity of antibody detection using a panel of monoclonal antibodies and to evaluate genotyping methods and establish procedures for drug-dependent antibody detection. Materials and Methods Forty-two laboratories from 23 countries participated. Samples and reagents provided for the five different exercises. Results The ability of participating laboratories to correctly identify the HPA antibody specificity in the nine samples ranged from 20% to 97%. The greatest difficulty was observed with samples that contained antibodies against HPA-3b and GPIV. The significant differences in optical density values by monoclonal antibody of immobilization of platelet antigens (MAIPA) assay were observed when testing the same platelet-specific antibodies. HPA genotyping of DNA with novel mutations did not significantly affect the results. The overall average discrepancy rate was 2·15% for genotyping of 10 DNA samples from well-characterized Epstein-Barr virus transformed cell lines. For detection of drug-dependent antibodies, excellent results for specificity and sensitivity were obtained by the laboratories using the MAIPA and flow cytometry. Conclusions Most laboratories were able to identify the majority of HPA antibodies; however, significant disparities were observed in proficiency testing. MAIPA assay sensitivity is influenced by the monoclonal antibody clone used. DNA with new mutations and EBV cell lines are valuable samples to ensure accurate genotyping. A sensitive and specific drug-dependent antibody assay performed well in the hands of participants. [ABSTRACT FROM AUTHOR]

Background and Objectives The aim of this work was to study the incidence of transfusion-induced platelet-reactive antibodies in a selective patient population and evaluate different methodologies for platelet antibody screening (PAS). Materials and Methods The patients were retrospectively selected and divided into three separate groups: haematological malignancies (Group 1: n = 33); cardiac and orthopaedic patients (Group 2: n = 31) and a control group (Group 3: n = 23) selected with the same diagnoses of Group 2. PRE- and POST-transfusion samples were tested for PAS by the following tests: PIFT (platelet immunofluorescence test), MAIPA (monoclonal antibody immobilization of platelet antigen), Flow PRA® and LCT (lymphocytotoxicity test). Results There was not a 100% concordance among the methodologies used. PIFT, MAIPA and Flow PRA presented very similar results whereas that of LCT differed from the other methods. A high rate of positive results (32%) was found in the PRE samples followed by an increase of almost 50% after blood transfusion (POST samples: 42·5% of positivity), but there was a statistical difference ( P < 0·05) between the PRE and POST transfusion sample only for the Flow PRA® technique tested on Group 2. Human leucocyte antigen (HLA) class I antibodies were present on 97·4% of POST positive samples, 5·4% presented anti-human platelet antigen (HPA)-1b antibodies and 8·1% presented a mix of panreactive antibodies against glycoprotein IIbIIIa, IaIIa and IbIX. Conclusions Blood transfusion did not increase the rate of alloimmunization in our haematological patients (Group 1); however, the patients were already admitted with a high rate of alloimmunization (12%). Group 2 patients are being immunized and the impact of this procedure remains to be studied as these patients may eventually undergo further hospitalization and receive more blood transfusion. [ABSTRACT FROM AUTHOR]

Objective: There are only few studies on agonist-inducible platelet activation in chronic idiopathic autoimmune thrombocytopenia (cAITP). Materials and methods: We compared agonist (TRAP-6, ADP, Arachidonic acid, Epinephrine, and Ristocetin) -inducible P-selectin expression and PAC-1 binding in 40 patients with cAITP (f/m ratio 23/17) with those in 20 healthy controls. Results were correlated with platelet counts, detectable platelet antibodies, and reticulated platelets. Results: The in vivo activation of platelets determined the in vitro inducible response to agonists. The stronger the in vivo activation the less the number of platelets responding to agonists, as illustrated by the inverse correlation of P-selectin expression ex vivo and the relative increase after the exogenous addition of agonists. The agonist-inducible platelet activation was not associated with the presence of detectable platelet antibodies to GPIb/IX or GPIIb/IIIa. Agonist-inducible platelet activation was also not correlated with counts of reticulated platelets. Conclusion: Agonist-inducible activation of platelets in cAITP is affected mainly by their in vivo activation. [ABSTRACT FROM AUTHOR]

Objective: To investigate the generation of platelet antibodies in hepatitis C virus (HCV)-infected individuals and their relation to the development to thrombocytopenia with the aim of using their detection as a diagnostic aid of immune thrombocytopenia in these patients. Materials and methods: We tested by the monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA) for the presence of platelet antibodies against specific glycoprotein (GP) targets (GPIIb/IIIa, GPIb/IX, GPIa/IIa, GPIIIb, GPV, and FcR γIIa) in 48 HCV-infected individuals of various stages of disease and compared the results with those from 35 patients with alcoholic liver cirrhosis. Results: Thirty-two HCV-infected individuals (66%) had detectable platelet antibodies. The most common target was GPIIb/IIIa, but all other GP were also targets. Results were not different from patients with alcoholic liver cirrhosis. There was no correlation between antibodies and platelet counts, or the stage of disease, or the viral genotype, or a discernible influence of treatment with α-interferon. Conclusion: While platelet autoantibodies are common in individuals with HCV infection, their detection does not assist in the diagnosis of immune thrombocytopenia. [ABSTRACT FROM AUTHOR]

To evaluate the mechanisms underlying prolonged thrombocytopenia after allogeneic hematopoietic stem cell transplantation (SCT), an index for plasma glycocalicin normalized for the individual platelet count (GCI), plasma thrombopoietin (TPO), and circulating B cells producing anti-GPIIb-IIIa antibodies were measured in 50 SCT recipients with or without prolonged thrombocytopenia, 42 patients with idiopathic thrombocytopenic purpura, nine patients with aplastic anemia, and 22 healthy individuals. All three indices were significantly higher in the SCT recipients with thrombocytopenia than in those without (P<0.01 for all comparisons), and were significantly correlated with the platelet count in SCT recipients. Stepwise multiple regression analysis of the samples from the SCT recipients revealed that GCI and TPO independently pointed to specific mechanisms of thrombocytopenia. The GCI and TPO status in SCT recipients with thrombocytopenia had a pattern similar to that seen in aplastic anemia, suggesting a major role for impaired thrombopoiesis. An antiplatelet antibody response was frequently detected in SCT recipients, but the development of thrombocytopenia is likely to depend on additional factors, such as reticuloendothelial function. In summary, post transplant prolonged thrombocytopenia is associated with complex mechanisms, including impaired thrombopoiesis and increased platelet turnover.Bone Marrow Transplantation (2006) 38, 377–384. doi:10.1038/sj.bmt.1705444 [ABSTRACT FROM AUTHOR]

The aims of the 12th International Society of Blood Transfusion (ISBT) Platelet Immunology Workshop were to evaluate the proficiency of molecular human platelet antigen (HPA) genotyping and detection of platelet antibodies of unusual specificity or reactivity, to assess whether quantification of anti-HPA-1a is practicable, and to determine the variability of reagents and components used in the monoclonal antibody immobilization of platelet antigens assay (MAIPA). Forty participants from 23 countries were sent 10 samples for DNA typing, five samples for antibody detection, a freeze-dried anti-HPA-1a standard, three samples for anti-HPA-1a quantification and a MAIPA method questionnaire. The detection and identification of HPA antibodies varied from 2·7 to 95% of participants. The number of HPA genotyping errors per sample ranged from 0 to 3·96% per HPA loci. The majority of laboratories were able to assign an arbitrary number of units/ml of anti-HPA-1a activity to the unknown samples. The MAIPA questionnaire indicated a wide variation among participants, both in method and in reagents used. The results obtained from this workshop highlighted deficiencies in testing regimes and identified a need for internationally available reference materials. [ABSTRACT FROM AUTHOR]

Serological evaluation of maternal sera for platelet antibodies in suspected fetal/neonatal alloimmune thrombocytopenia (FNAITP) discloses in only≈ 30% of individuals a platelet-specific antibody. Transfusion-induced alloimmunization against human platelet antigen-15 (HPA-15) has been reported to be about as common as against HPA-5, the second most common platelet antibody. Thus, anti-HPA-15 may also contribute significantly to yet-unclear cases of FNAITP.In this retrospective analysis, we provide data on maternal platelet antibodies from 309 mothers who delivered an offspring with suspected FNAITP.Genotyping maternal and paternal samples (togethern = 573) revealed a gene frequency of 0·496 for HPA-15a and a gene frequency of 0·504 for HPA-15b. HPA-15 antibodies were detected in 2% of all samples. Anti-HPA-15a and -15b were detected in two and three samples, respectively. One serum reacted equally with HPA-15a and -15b platelets. The most frequent platelet-specific antibodies were anti-HPA-1a (22%), but anti-HPA-5b (8·4%) were more frequent than anti-HPA-15. In addition, panreactive (5·5%) or autoreactive (5·2%) anti-GPIIb/IIIa or anti-GPIb/IX were detectable in maternal samples.These data indicate that HPA-15 alloimmunization needs only to be considered in subjects with suspected FNAITP if no other platelet-specific antibody is detectable. The presence of panreactive or autoreactive antibodies should also be considered in neonatal thrombocytopenia. [ABSTRACT FROM AUTHOR]

Background and Objectives The aims of the Eleventh International Workshop were to evaluate proficiency in platelet genotyping and antibody detection, to equip laboratories to perform Gov antigen system genotyping and antibody detection, and to evaluate the laboratory and clinical approach to cases of neonatal alloimmune thrombocytopenia (NAIT). Materials and Methods There were 34 participating laboratories from 22 countries on five continents. Participating laboratories were provided with 10 DNA samples, 15 unknown sera, and three monoclonal antibodies for titration, as well as primer pairs and a protocol for Gov genotyping and Gov antibody screening. They were also provided with a questionnaire on investigation and clinical management of patients with NAIT. Results Thirty-three participants reported human platelet antigen (HPA)-1, -2, -3 and -5 genotyping results, 25 reported HPA-4 typing results, 17 reported HPA-6 typing results and 24 reported Gov typing results. For HPA-1–6 genotyping, 23 laboratories were concordant with a majority vote for all allotypes tested, five laboratories reported one deviation, three laboratories reported two deviations and one laboratory reported three deviations. For Gov genotyping, six deviations occurred in three of the 24 laboratories reporting results. Antibody detection was 90% concordant for anti-HPA-1a, anti-HPA-5a and anti-HPA-5b detection. Anti-HPA-2b and anti-Gova were detected by 20 and 14 out of 33 laboratories, respectively. Approaches to the clinical management of NAIT vary widely, especially for mothers with a history of a previous infant with mild NAIT. Conclusions The overall error rate for HPA-1–6 genotyping decreased from 2·7% in the tenth workshop to 0·8% in the eleventh workshop. The majority of laboratories were able to perform Gov genotyping, although the error rate was 7·5%. Detection of common clinically significant antibodies was good, although detection of the much rarer HPA-2b was problematic. There was considerable progress in the detection of anti-Gova . The lack of consensus over treatment of NAIT demonstrates uncertainty over optimal management of these patients. [ABSTRACT FROM AUTHOR]

Intracerebral hemorrhage in leptospirosis is a rare event. We report on a fatal case of intracerebral hemorrhage complicating leptospirosis in a 47-year-old sewage drain worker. Since substantial thrombocytopenia was observed during the course of the disease, postmortem autopsy was performed to further elucidate the genesis of platelet destruction. Due to immunohistological findings, immunologically mediated thrombolysis was considered responsible for thrombocytopenia, whereas no signs of disseminated intravasal coagulopathy or deranged platelet production in the bone marrow were detected. [ABSTRACT FROM AUTHOR]

Background and Objectives The aims of the 10th International Platelet Serology and Genotyping Workshop were to evaluate the proficiency of platelet immunology determinations. Materials and Methods There were 40 participants from 25 countries of four continents. Thirty-eight institutions reported results for genotyping, and 38 institutions reported their serological results. For genotyping, EDTA-anticoagulated whole-blood samples were provided (to allow the inclusion of DNA-separation methodology in the analysis) as well as separated DNA of a and b alleles for human platelet antigen (HPA)-1 to -6. For serological evaluations, sera contained allo- and autoantibodies, and for sensitivity testing a standard freeze-dried sample of HPA-5 antibody. Results All participants reported HPA-1, -2, -3 and -5 genotyping results; HPA-4 was determined in 29 laboratories and HPA-6 in 21. Results from 16 laboratories were concordant with the majority vote for all allotypes, eight institutions reported one deviation, five laboratories two, and nine laboratories three or more deviations. Twelve institutions had no deviation from the majority vote for HPA antibodies, nine had one, three had two, and 14 had three or more deviations. Most laboratories reported a reactivity of the standard anti-HPA-5b sample with HPA-5b platelets at a dilution of 1 : 4–1 : 8. Four laboratories detected anti-Gova in one sample. Seventeen laboratories reported no deviation from the majority vote for pan-reactive platelet antibodies, 12 had one deviation, two had two, and seven had three or more deviations. In addition, seven participants reported antibodies against glycoprotein IV (GPIV), three against glycoprotein V (GPV) and three against CD109. These results were discussed at a meeting organized jointly with the International Society of Blood Transfusion (ISBT) 2000 Congress. Conclusion The results for pan-reactive antibodies were heterogeneous with most discrepancies from the... [ABSTRACT FROM AUTHOR]

We performed a prospective study on the incidence of thrombocytopenia (t‐penia) and its immunological origin in unselected 26 275 mothers and 24 101 newborns. Platelet antibodies were examined by the platelet immunofluorescence test (PIFT) and the monoclonal antibody immobilisation of platelet antigens assay (MAIPA). T‐penia (platelet count <100×109/l) was found in 124 (0.5%) mothers (in 0.04% severe, <50×109/l) and in 116 (0.5%) newborns (in 0.15% severe); 90 (72.6%) and 112 (96.6%), respectively, were available for further studies. In both groups non‐immune t‐penia was diagnosed about 4.5 times more often than the immune t‐penia. Among 90 mothers, t‐penia was severe in 11.1%, antibodies were detected in 17.8%; both factors were not prognostic for delivering thrombocytopenic newborns. Among 112 babies, 21 were delivered by thrombocytopenic mothers and 91 by mothers with normal platelet count; among newborns with immune t‐penia the proportion of alloimmune (NAIT) to autoimmune was equal (10 with NAIT, 10 with autoimmune, 4 of them born by mothers with hidden autoimmune t‐penia). In 33% of the neonates t‐penia was severe, most often among NAIT. In conclusion, although t‐penia in mothers as well as in infants is not frequent and severe, and an immune origin not often found, the search for antibodies, in particular alloantibodies, should be done. Even if the serological results are not helpful at the moment, they can be of importance in subsequent pregnancy and for related pregnant women. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that immune platelet destruction observed in an AIDS-free HIV-infected patient was associated with the presence of a cross-reactive antibody recognizing both HIV-glycoprotein (gp)120 and platelet gpIIIa (CD61). We have now investigated the presence of such antibodies in other HIV-infected patients, together with the molecular structure of the cross-reactive epitope. Platelet gpIIb/IIIa antibodies were characterized in sera from HIV-infected patients with immune thrombocytopenic purpura by means of an EI.ISA and a radio-immunoprecipitation procedure (RIP). The platelet antibodies were purified and tested for their ability to recognize HIV-gp. We also tried to characterize the antibody target epitope on HIV-gp120 using recombinant gp and synthetic peptides. IgG with anti-gpIIb/IIIa activity were detected, by means of an ELISA with purified gpIIb/IIIa, in 101/138 (73%) sera from HIV-infected patients with immune thrombocytopenic purpura. The platelet antibodies were purified from 23 sera by absorption/elution on purified immobilized platelet gpIIb/IIIa, and recognition of gpIIIa was confirmed in eight cases with a RIP. Furthermore, the presence of a cross-reactive antibody between HIV-gp120 and platelet gpIIIa was demonstrated in 18/18 patients (including the eight with a confirmed gpIIIa antibody) by the ability of the serum HIV-gp160/120 antibodies to bind to purified gpIIb/IIIa. The cross-reactive epitope was shown to be independent of the carbohydrate moieties of gp120, since deglycosylation of two recombinant (r)-gp120s did not abolish antibody binding. However, the antibody did not recognize synthetic gp120 peptides spanning 355 of the 516 amino acids of gp120, particularly the four regions exhibiting sequences of four or five consecutive amino acids that are identical between r-gp120 and gpIIIa. Our results thus support the hypothesis that the cross-reactive antibody recognizes the conformational structure of gp120. These results strongly suggest that molecular mimicry between HIV-gp120 and platelet gpIIIa may be important in the pathogenesis of immune thrombocytopenia in AIDS-free HIV-infected patients. [ABSTRACT FROM AUTHOR]

Platelets can adhere to surfaces by forming pseudopods. This ability is made use of here to simplify conventional platelet suspension immunofluorescence tests, since steps like serum and FITC incubations, washings and microscopic evaluation are done on platelets adhered to glass surfaces. By eliminating time-consuming manipulations like washing in individual test tubes and mounting platelets to microscopic slides, large scale studies can now be performed (up to 480 tests per day and technician). Antibodies can be detected with fresh as well as with frozen platelets from a well defined cell panel. The PAIFT is 2 to 3 times more sensitive than the NIH-standard-micro-lymphocytotoxicity-test (NIH-LCT), thus allowing earlier recognition of alloimmunization of patients. [ABSTRACT FROM AUTHOR]

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Thirteen cases of posttransfusion purpura (PTP) which were diagnosed in Germany and Austria from 1977-1985 are described. All patients were women with a mean age of 58.6 years (range, 36-77 years). All but one had been pregnant and received blood transfusions 2 to 12 days prior to the onset of PTP. The thrombocytopenic purpura was always severe with a nadir of platelet counts below 10×10/l and lasted between 3 and 60 days. All patients recovered from PTP. Optimal therapy consisted of administration of high-dose IgG. Twelve of the 13 patients had developed platelet-specific Zw antibodies (eight of them together with HLA antibodies), in one Zw positive individual only HLA antibodies were detectable. Five of six HLA-DR typed patients carried DR3 which is considered an immunogenetic risk factor in PTP. Clinical awareness of this rare, but serious iatrogenic transfusion complication is prerequisite for prompt diagnosis and improved therapy. [ABSTRACT FROM AUTHOR]

We have studied target platelet antigens in 22 patients with lupus anticoagulants and a primary antiphospholipid syndrome in order to determine whether any specificities of platelet autoantibodies are correlated with thromboembolism, and if these antibodies cross-react with phospholipids, which would suggest their role in the development of thromboembolic disease. Platelet counts were median 203 x 10(9)/l, range 100-298 x 10(9)/l. Platelet antibodies were found in six thrombocytopenic patients and in further nine patients. All these 15 patients had antibodies against GPIIb/ IIIa, five patients against GPIb/IX, and six patients against GPIV. Anti-GPIb/IX and -GPIV occurred only in combination with anti-GPIIb/IIIa antibodies. There was no correlation between the presence of detectable platelet antibodies or any of their glycoprotein specificity and thrombocytopenia or the history of a thromboembolic disease. Eluates from platelets contained only GPIIb/IIIa reactivities, but neither anti-GPIb/IX nor anti-GPIV. None of the eluates contained lupus anticoagulant activity. In one case, the platelet eluates contained anti-GPIIb/IIIa and anticardiolipin IgG antibodies. These results suggest that in patients with a primary antiphospholipid syndrome the presence of platelet autoantibodies neither indicate a risk for thromboembolic disorder nor have lupus anticoagulant activity. [ABSTRACT FROM AUTHOR]

Autoimmune thrombocytopenic purpura (AITP) is most frequently induced by platelet-specific autoantibodies against epitopes on platelet GP Ib/IX or GP IIb/IIIa. These antibodies are reliably detected on the patients' autologous platelets. So far, studies on the characterization of platelet autoantibodies have been restricted to IgG antibodies. We used the monoclonal antibody immobilization of platelet antigens assay (MAIPA) in a modified version to detect GP-specific IgG, IgM, and IgA antibodies. Platelets of 46.2% of patients carried elevated amounts of IgG antibodies. IgM and IgA antibodies were observed less frequently and showed only weak OD signals in the MAIPA assay. Circulating IgG antibodies in serum were found in 11.5% of patients. Circulating IgM autoantibodies were observed in 8.9% and IgA antibodies in no patient with AITP. Results of direct MAIPA assay were compared with the reactivity of eluates in the platelet adhesion immunofluorescence assay and were found to be highly concordant. Patients with AITP in remission carried high percentages of anti-GP IIb/IIIa. Findings made in this study suggest that autoantibodies of the IgM and IgA classes play only a minor role in the pathogenesis of AITP. [ABSTRACT FROM AUTHOR]

The detection of platelet antigens and platelet antibodies has always been difficult. Recent technical achievements are due to the availability of more specific and sensitive reagents (i.e. F(ab)2 fragments; higher specific activity of labels; monoclonal antibodies directed against platelet membrane constituents etc.) and the application of advanced immunological assays to platelet immunology (i.e. blotting assays, 'capture'-ELISA's and radioimmunoprecipitation in conjunction with SDS polyacrylamide gel electrophoresis). These techniques have permitted the definition and immunochemical characterization of new platelet allo- and autoantigens, have assisted in clinical diagnosis and promoted our understanding of pathogenic mechanisms. Illustrative examples are presented and discussed. [ABSTRACT FROM AUTHOR]

Most platelet-reactive autoantibodies and alloantibodies are not able to fix complement in vitro. However, exceptions have been found. These antibodies are usually characterized by the conventional platelet complement fixation test. A recently developed competitive enzyme immunoassay for quantitation of platelet-associated immunoglobulins and a modification thereof allowed the quantitative study of fixation of C3d and the membrane attack complex (C5b-9) on platelets by HLA antibodies, human platelet autoantibodies, and drug-dependent antibodies (ddab). The highest amounts of both complement products were fixed through ddabs, whereas autoantibodies only showed moderate complement fixation. This enzyme immunoassay is a valuable tool for the characterization of the complement-fixing properties of platelet-reactive antibodies. [ABSTRACT FROM AUTHOR]

A 23 year-old man developed thrombocytopenic purpura at the end of the second week of the clinical evolution of hepatitis A confirmed by viral markers. The bone marrow of this patient showed megakaryocytic hyperplasia. Circulating in his serum immune complexes were demonstrated by solid phase conglutinin enzymo-immunoassay. Platelet-reactive serum factors were also detected by an indirect immunofluorescence test using fresh donor platelets as targets. The evolution of both the hepatitis and the purpura were benign with no therapy other than bedrest. Platelet count normalized within five weeks of the onset of purpura, and IgM antibodies against hepatitis A virus as well as circulating immune complexes dropped to normal levels. It is postulated that the thrombocytopenia of this case was caused by nonspecific deposition of immune complexes at the platelet surface. [ABSTRACT FROM AUTHOR]

Despite recent advances in the understanding and treatment of immune thrombocytopenia (ITP), its diagnosis remains clinical due to the lack of sensitive laboratory tests. The detection of anti-platelet antibodies (APA) in plasma, although highly specific, is notoriously insensitive. Specialised clinical platelet laboratories routinely perform a screening test of only one dilution for indirect APA testing by flow cytometry. We evaluated the presence of APA using several dilutions of plasma from 61 ITP patients. Herein, we report that serial dilutions can improve the diagnostic value of indirect APA assay for ITP. We show that performing just two dilutions (1:2 and 1:25) would capture over 90% of patients with detectable plasma APA. This method enables indirect testing to become a valuable tool to be incorporated into the management algorithm for ITP., (Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Background: Platelet transfusion refractoriness (PTR) remains a difficult problem in patients requiring long-term platelet supportive care. However, there are little data on the frequency of platelet antibodies in multiply transfused Chinese patients. Moreover, the relationship between peripheral regulatory T cells (Tregs) and PTR remains unclear., Methods: We retrospectively studied the frequency of alloimmunization against platelet antigens in patients receiving multiple transfusions between 2013 and 2017. Monoclonal antibody solid-phase platelet antibody test (MASPAT) kits were used to screen for platelet antibodies before each platelet transfusion. Peripheral Tregs and CD4 + CD25 + CD127 - T cells were detected by flow cytometry, while transforming growth factor-beta (TGF-β) and interleukin (IL)-17 cytokines were detected by enzyme-linked immunosorbent assay., Results: A total of 399 patients who met the inclusion criteria were enrolled for the analysis of platelet antibodies and refractoriness. Among these patients, 10 (2.5%) were positive for platelet antibodies before transfusion and 47 (11.8%) became antibody-positive during the study period. The number of alloimmunized patients was significantly higher in patients with hematological disease as compared with other disease groups (p < 0.05). Refractoriness and alloimmunization occurred in 77 (19.3%) and 22 (28.6%) patients, respectively. There were no significant differences in CD4 + , CD8 + , and CD4 + CD25 + CD127 - T cell numbers and plasma levels of TGF-β1 and IL-17 between patients with PTR and the control group., Conclusions: Refractoriness was common in patients undergoing multiple platelet transfusions (19.3%), with alloimmunization observed in 28.6% of patients. However, Tregs in peripheral blood may not play a key role in PTR., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)