Progress and development of platelet antibody detection.

Human platelet antibody (HPA) detection is necessary for the diagnosis and therapeutic decisions for refractoriness to platelet transfusions, post transfusion purpura and fetal and neonatal alloimmune thrombocytopenia. In the last four to five decades many new developments, both in knowledge and methods, have increased the quality of platelet serology. However, the quest for the optimal antibody detection method(s) encountered, sometimes unexpected, difficulties. In this review the various aspects concerning platelet antibody test methods and detection of platelet antibodies both for the diagnostic and screening setting are discussed.

Post-transfusion purpura in a woman with acute myeloid leukemia.

Post-transfusion purpura (PTP) is a rare, but severe transfusion reaction in which both donor and autologous platelets are sequestered due to immunization against HPA-1a antigens in HPA-1a negative recipients (HPA: human platelet antigens). We describe a patient who developed PTP during induction therapy for acute myeloid leukaemia. The pitfalls, delays in diagnosing and therapy options of this serious transfusion reaction are discussed.

Underdiagnosing of antibody-mediated transfusion-related acute lung injury: evaluation of cellular-based versus bead-based techniques.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. To support the diagnosis of antibody-mediated TRALI, HLA and HNA antibodies are tested in involved blood donors. Identification of antibody positive donors is important as exclusion of these donors is part of preventative strategies against TRALI. We compared cellular-based versus bead-based techniques for diagnosis of antibody-mediated TRALI. MATERIALS AND METHODS: All reported TRALI cases in the Netherlands during a 5-year period were evaluated. Donors were screened for the presence of HLA class I and class II antibodies using both cellular-based and bead-based techniques. RESULTS: In total, 100 TRALI cases were reported of which 91 were fully tested. In 113 donors, HLA antibodies were detected of which 84 were only detected by bead-based techniques, 12 only by cellular-based tests and 17 by both assays. Antibody-mediated TRALI was diagnosed in 44 of 91 reported cases. Twenty-one (48%) of these cases would not have been identified using only cellular-based assays. CONCLUSION: Bead-based techniques show a higher sensitivity for detecting incompatible donors in TRALI cases than cellular-based assays. These results suggest that the use of bead-based assays will result in a significant reduction of future TRALI reactions as more antibody positive donors will be excluded from future donations.

External quality assessment of human neutrophil antigen (HNA)-specific antibody detection and HNA genotyping from 2000 to 2012.

Since 2000, Quality Assurance (QA) exercises for the detection and identification of granulocyte antibodies and DNA typing for human neutrophil antigens (HNA) have been distributed within the International Granulocyte Immunobiology Workshops, which are linked to International Society of Blood Transfusion. The exercises were standardised at the outset to enable laboratory performance to be monitored. Between 2000 and 2012, nine exercises were distributed to 20 laboratories. Overall, 45 examples of 42 unique samples containing defined granulocyte reactive antibodies were distributed for serological analysis together with 20 samples for HNA genotyping. The level of satisfactory serological performance was initially set at 50% and later increased to 70%, while the 'cut-off' for HNA genotyping was set at 100% after 2008. Failure to achieve the minimum score in the QA exercises in consecutive years resulted in temporary exclusion. In 2000, the 15 participating laboratories had a mean score of 56.1% for serological analysis and 13 laboratories attempted HNA-1a and -1b genotyping, while 11 attempted HNA-1c typing. Steady improvements in proficiency for serological testing and HNA typing occurred in subsequent exercises. In 2012, the mean score for serology was 88.5% and 12/13 laboratories scored 100% for HNA-1a, -1b, -1c, -3a, -3b, -4a, -4bw, -5a and -5bw genotyping. These QA exercises have provided an invaluable tool to monitor and improve the standard of granulocyte immunology investigations for participating laboratories, thereby enhancing performance for both clinical investigations and donor screening programmes to reduce the incidence of TRALI.

Pseudothrombocytopenia in a neonate due to mother?

UNLABELLED: Ethylenediaminetetraacetic acid (EDTA)-dependent pseudothrombocytopenia (PTCP) is the phenomenon of a false low platelet count reported by an automated haematology analyzer due to in vitro aggregation of platelets. This aggregation is due to the interaction between antibodies and EDTA-dependent crypt antigens on platelets. We observed a new born child whose mother was diagnosed with transient PTCP due to transplacental transmission of maternal immunoglobulin G antibodies during pregnancy. CONCLUSION: Although maternal-neonatal PTCP is rare, it is important to consider this phenomenon as a cause of trombocytopenia, as it can result in unnecessary diagnostic workup and treatment.

Delayed diagnosis of fetal and neonatal alloimmune thrombocytopenia: a cause of perinatal mortality and morbidity.

OBJECTIVE: To evaluate the rate and consequences of a late or missed diagnosis of fetal and neonatal alloimmune thrombocytopenia (FNAIT). DESIGN: Retrospective analysis of prospectively collected data of a national cohort. SETTING: National referral centre for fetal therapy in the Netherlands. POPULATION: Twenty-six women with pregnancies complicated by FNAIT and at least one previous pregnancy with a thrombocytopenic child. METHODS: Retrospective analysis of data from our electronic FNAIT database. In a consecutive cohort managed between July 2008 and July 2010, timing of first diagnosis of FNAIT was correlated to severity and outcome in the subsequent pregnancies. MAIN OUTCOME MEASURES: Occurrence of delayed diagnosis of FNAIT, and possibly associated intracranial haemorrhage (ICH). RESULTS: In four of 26 pregnancies, timely diagnostic testing for FNAIT was not performed despite fetal or neonatal thrombocytopenia or ICH. Down syndrome, dysmaturity and birth trauma were perceived to be the cause of the thrombocytopenia/ICH. In two of these four subsequent, untreated pregnancies, severe fetal ICH occurred. The other 22 women were treated for FNAIT using intravenous immunoglobulin, all children are alive and well. CONCLUSIONS: All neonates with thrombocytopenia at birth should be evaluated for FNAIT. Missing this diagnosis can have severe consequences for subsequent pregnancies.

Allo-exposure status and leucocyte antibody positivity of blood donors show a similar relation with TRALI.

INTRODUCTION: The fraction of transfusion-related acute lung injury (TRALI) cases preventable by deferral of allo-exposed donors has previously been estimated, under the assumption this indirectly estimated the contribution of leucocyte antibodies to the occurrence of TRALI. Our aim was to estimate the fraction preventable by deferral of leucocyte antibody positive donors and to investigate the validity of allo-exposure as a marker for leucocyte antibodies. METHODS: All donors involved in a series of previously published TRALI patients were tested for leucocyte antibodies. The observed number of antibody positive donors was compared to the expected number. From this comparison we estimated the contribution of leucocyte antibodies to the occurrence of TRALI and compared this to the previously reported estimate for allo-exposed donors. RESULTS: Sixty-one TRALI patients were included. Of 288 involved donors 43 were expected and 67 were observed to be leucocyte antibody positive. The observed percentage of positive donors was 8.3% (95% confidence interval (CI): 5.1-11.5%) in excess of the expected. Overall 59% (95% CI: 34-85%) of TRALI cases was estimated to be preventable by the exclusion of all leucocyte antibody positive donors. For plasma-poor products this was 16% (95% CI: -5.0 to 36%). CONCLUSIONS: These estimates were similar to those previously published for allo-exposed donors. This suggests allo-exposure status can effectively be used in donor deferral strategies.

Prevalence of leucocyte antibodies in the Dutch donor population.

INTRODUCTION: Donor leucocyte antibodies have been associated with transfusion-related acute lung injury (TRALI) and can be present in allo-exposed donors. Donor deferral policies aiming at excluding allo-exposed donors are increasingly implemented worldwide. We aimed at assessing the prevalence of leucocyte antibodies in different subgroups of allo-exposed donors in the Dutch donor population. METHODS: Consecutive donors were enrolled during routine whole blood donation. Donors filled out a questionnaire on allo-exposure history. Blood samples were tested for human leucocyte antigens (HLA) (LifeScreen Deluxe and the Lifecodes LSA I/II assays) and granulocyte-reactive (GIFT, GAT, and MAIGA) antibodies. RESULTS: Six thousand and thirty-four consecutive donors (60% men) were included. A total of 2.5% reported a history of blood transfusions, and 51% (of female donors) reported a history of pregnancy. In never allo-exposed donors, the prevalence of granulocyte-reactive antibodies was 2.0% (95% CI: 1.6-2.4), and for HLA antibodies, it was 7.0% (95% CI: 6.3-7.8). In previously pregnant donors, the prevalence of granulocyte-reactive antibodies was increased to 3.0% (95% CI: 2.0-4.0), and for HLA antibodies, it was increased to 33% (95% CI: 30-36). Prevalence of leucocyte antibodies of all types depended on transfusion history, number of pregnancies, time since last pregnancy, and pregnancy outcome. CONCLUSIONS: Fourteen percent of Dutch blood donors are allo-immunized against HLA or granulocyte antigens. Deferral of all self-reported allo-exposed donors will decrease this prevalence to 9%. Deferral of all female donors and transfused male donors will result in a similar prevalence among remaining donors but approximately twice as many deferrals.

Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a potentially devastating disease, which may lead to intracranial haemorrhage (ICH), with neurological damage as a consequence. In the absence of screening, FNAIT is only diagnosed after bleeding symptoms, with preventive options limited to a next pregnancy. OBJECTIVES: To estimate the population incidence of FNAIT and its consequences to prepare for study design of a screening programme. SEARCH STRATEGY: An electronic literature search using MEDLINE, EMBASE and Cochrane database, and references of retrieved articles. No language restrictions were applied. SELECTION CRITERIA: Prospective studies on screening for human platelet antigen 1a (HPA-1a) alloimmunisation in low-risk pregnant women. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed studies for inclusion and extracted data. Main outcome data were prevalence of HPA-1a negativity, HPA-1a immunisation, platelet count at birth and perinatal ICH. We aimed to compare outcome with and without intervention. MAIN RESULTS: HPA-1a alloimmunisation occurred in 294/3028 (9.7%) pregnancies at risk. Severe FNAIT occurred in 71/227 (31%) immunised pregnancies, with perinatal ICH in 7/71 (10%). True natural history data were not found because interventions were performed in most screen-positive women. AUTHORS' CONCLUSIONS: Screening for HPA-1a alloimmunisation detects about two cases in 1000 pregnancies. The calculated risk for perinatal ICH of 10% in pregnancies with severe FNAIT is an underestimation because studies without interventions were lacking. Screening of all pregnancies together with effective antenatal treatment such as intravenous immunoglobulin may reduce the mortality and morbidity associated with FNAIT.

Recommendations of the ISBT Working Party on Granulocyte Immunobiology for leucocyte antibody screening in the investigation and prevention of antibody-mediated transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is currently one of the most common causes of transfusion-related major morbidity and death. Among the many TRALI mediators, leucocyte antibodies have been identified as important triggers of severe TRALI. STUDY DESIGN AND METHODS: These recommendations were compiled by experts of the ISBT Working Party on Granulocyte Immunobiology, based on the results obtained in eight international granulocyte immunology workshops, their personal experiences and on published study results. RESULTS: Leucocyte antibody screening has to include the detection of human leucocyte antigen (HLA) class I, class II and human neutrophil alloantigen antibodies using established and validated techniques. HLA class I antibody detection should be restricted to antibodies clinically relevant for TRALI. To avoid unnecessary workload, TRALI diagnosis should be assessed by consultation with the reporting clinician and thorough exclusion of transfusion-associated circulatory overload/cardiac insufficiency. In patients diagnosed with TRALI having donors with detectable leucocyte antibodies, evidence of leucocyte incompatibility should be provided by either cross-matching or typing of patient for cognate antigen. CONCLUSION: Leucocyte antibody screening for the immunological clarification of TRALI cases as well as for identification of potentially alloimmunized blood donors is feasible and can be performed in a reasonable and quality assured manner. This practice can contribute to the prevention of antibody-mediated TRALI.

[Transfusion-related acute lung injury (TRALI) in the Netherlands in 2002-2005]. / Transfusiegerelateerde acute longbeschadiging (TRALI) in Nederland in 2002-2005.

OBJECTIVE: To determine the number of reported cases of transfusion-related acute lung injury (TRALI) in the Netherlands in 2002-2005 and to determine how many cases were associated with incompatibility between leukocyte-reactive antibodies in the donor plasma and leukocytes or antigens in the recipient. DESIGN: Retrospective national case review. METHOD: Cases of TRALI reported in 2002-2005 were assessed according to the national clinical definition of TRALI, and the relationship between TRALI and transfusion was assessed. Additional clinical details were requested from the treating hospital as necessary. The results of leukocyte serological tests from donors and recipients were linked to clinical cases. For cases with positive leukocyte serological tests, the relevant blood components and the sex of the donor were recorded. RESULTS: Of the 46 cases reported, 6 had insufficient information. 8 cases did not meet the definition or had another more likely diagnosis. There was a trend toward an increase in the number of reports: 12 cases were reported in 2005, corresponding with 1:60,000 blood components. Of the 40 evaluable cases, 32 (80%) met the definition of TRALI and were deemed to be definitely (n = 16), probably (n = 5) or possibly (n = 11) related to transfusion. Severity ranged from moderate to life-threatening, and there was one TRALI-related death. Leukocyte serology was fully investigated in 18 cases: 13 (72%) had leukocyte incompatibility and in 5 cases exclusively fresh frozen plasma from a female donor was implicated.

Fetal thrombocytopenia.

Fetal thrombocytopenia is most often caused by maternal alloantibodies against fetal platelets crossing the placenta and resulting in platelet destruction. This condition, known as fetal and neonatal alloimmune thrombocytopenia, is usually detected after the birth of a symptomatic child who shows signs of bleeding in the skin or in the brain. In the most severe cases, intracranial hemorrhage leads to severe handicap or death. The challenge for the clinician is to provide preventive treatment in the next pregnancy. The current cornerstone of this treatment is maternal intravenous administration of immunoglobulins during the second half of pregnancy.

Fetal/neonatal allo-immune thrombocytopenia (FNAIT): past, present, and future.

UNLABELLED: We reviewed the English, American, and German literature for articles describing the prevalence, clinical presentation, outcome, therapeutic options, and screening possibilities for fetal/neonatal allo-immune thrombocytopenia (FNAIT), published between January 1950 and March 2007. The reported prevalence of FNAIT in human platelet antigen (HPA)-1a-negative women varies between 1/600 to 1/5000 live births among various populations. The typical picture is that of a neonate presenting with purpura minutes to hours after birth, born to a healthy mother with no history of infection or abnormal bleeding, after an uneventful pregnancy with a normal maternal platelet count. Thrombocytopenia in FNAIT can be severe, with intracranial hemorrhage occurring in 10% to 30% of severe FNAIT cases. Several types of neonatal treatment have been proposed, of which transfusion of HPA-compatible platelets is most effective. Antenatal management of FNAIT consists of weekly maternal intravenous immunoglobulin (IVIG) infusions, with or without oral steroid therapy. Serial fetal platelet transfusions can be provided in cases of failure of IVIG therapy, but the multiple cordocenteses that would be required to administer the platelets entail substantial risk. The possibilities for antenatal screening of first pregnancies are limited. Postnatal screening does not prevent neonatal morbidity and mortality. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians. LEARNING OBJECTIVES: After completion of this article, the reader should be able to summarize the many and varied causes of neonatal thrombocytopenia, explain that fetal/neonatal allo-immune thrombocytopenia (FNAIT) is a rare but devastating cause with potential high risk of recurrence, and recall the treatment options for FNAIT as well as their potential side effects.

Acquired Glanzmann's thrombasthenia caused by glycoprotein IIb/IIIa autoantibodies of the immunoglobulin G1 (IgG1), IgG2 or IgG4 subclass: a study in six cases.

BACKGROUND: Acquired Glanzmann's thrombasthenia (GT) is an uncommon bleeding disorder caused by glycoprotein (GP) IIb/IIIa-specific autoantibodies. Covering of the fibrinogen binding site of GPIIb/IIIa results in a moderate-to-severe bleeding tendency. MATERIALS AND METHODS: We performed a diagnostic evaluation and evaluated the underlying risk factors in six patients with a bleeding tendency caused by acquired GT. RESULTS: One patient, with GPIIb/IIIa autoantibodies of the immunoglobulin G2 (IgG2) subclass, used diclophenac and recovered after discontinuation of this drug. A second patient was primarily diagnosed with multiple angiodysplastic lesions. In this patient, the acquired GT was caused by GPIIb/IIIa autoantibodies of the IgG4 subclass that was treated with DDAVP and platelet transfusions. A third patient with Hodgkin's lymphoma and anti-GPIIb/IIIa of the IgG2 subclass was treated for haemorrhagic diathesis with corticosteroids and azathioprin. A fourth patient, with IgG2 anti-GPIIb/IIIa autoantibodies, diagnosed with mantle cell lymphoma, responded well to treatment of an axillary mass with local radiotherapy. The fifth and sixth patients, with IgG1 anti-GPIIb/IIIa autoantibodies, appeared to have GT after splenectomy because of autoimmune thrombocytopenia. They were treated with corticosteroids, intravenous immunoglobulin and Rituximab. CONCLUSION: Although it might be a rare event, one should be aware of acquired GT as a cause of an unexpected primary disorder of haemostasis in patients with lymphoma or autoimmune disease. The lack of platelet destruction in these cases of acquired GT can be explained, either by the subclass of the autoantibodies (i.e. IgG2 or IgG4) or by the arrested platelet destruction by IgG1 (or IgG3) autoantibodies after splenectomy.

Thrombocytopenia in hydropic fetuses with parvovirus B19 infection: incidence, treatment and correlation with fetal B19 viral load.

OBJECTIVE: To examine (1) the incidence of fetal thrombocytopenia in hydropic fetuses with congenital B19 virus infection, (2) the effect of intrauterine platelet transfusions and (3) the correlation between fetal B19 viral load and severity of thrombocytopenia. DESIGN: Retrospective analysis of data from prospectively collected fetal blood samples. SETTING: Leiden University Medical Centre, the national centre for management of intrauterine fetal disease in the Netherlands. POPULATION: Thirty hydropic fetuses treated with intrauterine red blood cell and platelet transfusions for human B19 virus-induced severe fetal anaemia and thrombocytopenia over a 10-year period. METHODS: Fetal blood samples (n= 30) taken before and after intrauterine transfusion were investigated. No cases were excluded, and there was no loss to follow up. MAIN OUTCOME MEASURES: Parameters recorded were gestational age, experienced fetal movements, gravidity and parity, severity of fetal hydrops, severity of fetal anaemia and thrombocytopenia and megakaryocyte and reticulocyte counts. Survival and procedure-associated complications were documented. Quantitative B19 viral load measurements were performed on all fetal samples. RESULTS: Forty-six percent of all hydropic fetuses showed severe thrombocytopenia. No antenatal intracerebral haemorrhage or procedure-associated bleeding occurred. Overall, survival was 77%. Platelet counts increased following platelet transfusion and decreased significantly following red blood cell transfusion alone. No correlation was found between fetal viral loads and platelet counts. CONCLUSION: Thrombocytopenia was frequently encountered in fetal B19V infection, but fetal bleeding complications were not noted. Absence of a direct relationship between fetal B19 viral load and platelet counts suggests a temporal dissociation between these findings. Dilutional thrombocytopenia is frequently seen in the fetus following red blood cell transfusion alone. The clinical significance of this phenomenon is unclear. The risk of fluid overload by fetal platelet transfusion in a severely hydropic fetus should be weighed against the low incidence of fetal bleeding complications.

Collaborative studies to establish the first World Health Organization International Standard for detection of human antibody against human platelet antigen-3a.

BACKGROUND AND OBJECTIVES: The platelet-specific antibody anti-human platelet antigen-3a (anti-HPA-3a) is involved in neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet refractoriness. However, HPA-3a antibodies are often difficult to detect, probably because the antigen is labile. This report describes the production of a freeze-dried preparation of pooled human plasma, coded 03/190, containing IgG antibodies against the HPA-3a. The material is intended for use as a minimum sensitivity reagent in glycoprotein-specific assays currently used for anti-HPA-3a detection. Laboratories can use it to assess the sensitivity of their 'in-house' assays for anti-HPA-3a and to calibrate local controls for routine use in each batch of tests. MATERIALS AND METHODS: Plasma containing anti-HPA-3a was obtained from a mother of two babies both born with severe thrombocytopenia, and following dilution it was freeze dried in glass ampoules. RESULTS: Two collaborative studies demonstrated that the candidate material contained anti-HPA-3a and human leucocyte antigen (HLA) class I antibodies, but no other HPA antibodies that might confuse the detection of the anti-HPA-3a. The minimum dilution that should give a positive result was determined to be 1 : 8 by two further international collaborative studies involving a total of 49 laboratories in 23 countries. CONCLUSION: The material also contains HLA antibodies and is suitable for use only in techniques that are glycoprotein specific (i.e. monoclonal antibody immobilization of platelet antigens and enzyme-linked immunosorbent assay) where only HPA antibodies will be detected. This standard will allow laboratories to measure their sensitivity of detection of anti-HPA-3a and will also allow those laboratories with relatively insensitive techniques to monitor their performance as they improve their methodology.

Low-dose fondaparinux in suspected heparin-induced thrombocytopenia in the critically ill.

BACKGROUND: In critically ill patients, heparin-induced thrombocytopenia (HIT) is estimated to account for approximately 1 to 10% of all causes of thrombocytopenia. HIT exerts a strong procoagulant state. In case of suspected HIT, it is an important clinical decision to stop heparin and start treatment with alternative nonheparin anticoagulation, awaiting the results of laboratory testing for the final diagnosis of HIT (bridging therapy). Fondaparinux acts by factor Xa inhibition and expresses no cross-reactivity with HIT antibodies. Excretion of fondaparinux is mainly renal. We describe our early experience with fixed low-dose fondaparinux bridging therapy and monitoring of anticoagulant activity for safety reasons. METHODS: This retrospective cohort study was conducted in a closed format general intensive care unit in a teaching hospital. Consecutive critically ill patients suspected of HIT were treated with fondaparinux after discontinuation of unfractionated heparin or nadroparin. Anti-Xa levels were determined afterwards. RESULTS: Seven patients were treated with fondaparinux 2.5 mg/day for 1.8 to 6.5 days. Anti-Xa levels varied from 0.1 to 0.6 U/ml. A negative correlation was found between creatinine clearance and mean and maximum anti-Xa levels. No thromboembolic complications occurred. Bleeding complications were only minor during fondaparinux treatment. Transfusion requirements did not differ significantly between treatment episodes with fondaparinux or with heparin anticoagulants. CONCLUSION: In this small sample of critically ill patients suspected of HIT, bridging therapy with fixed low-dose fondaparinux resulted in prophylactic and therapeutic anti-Xa levels. Monitoring of anticoagulant activity is advised in patients with renal insufficiency.

Fetal and neonatal thrombopoietin levels in alloimmune thrombocytopenia.

Thrombopoietin (Tpo) is the main hematopoietic growth factor for platelet production. Plasma Tpo levels in autoimmune thrombocytopenic patients are normal or slightly elevated. Although thrombocytopenia exists, Tpo levels are not increased because the produced megakaryocytes and platelets can bind circulating Tpo, thereby normalizing Tpo levels. In this study, plasma samples from fetuses and neonates with neonatal alloimmune thrombocytopenia (NAIT), a different form of immune thrombocytopenia, were measured. Umbilical cord samples from 50 fetuses before treatment because of severe thrombocytopenia and 51 fetuses after treatment, and peripheral blood samples of 21 untreated newborns with NAIT were analyzed. As controls, plasma Tpo levels were determined in 21 umbilical cord samples of 14 nonthrombocytopenic fetuses with hemolytic disease resulting from red blood cell alloimmunization and in umbilical cord samples of 51 healthy newborns. The values were also compared with the plasma Tpo levels in 193 healthy adults. Mean Tpo levels from the groups of fetuses and neonates, including both NAIT and control plasma, were slightly but significantly elevated compared with levels in healthy adults. Tpo levels in NAIT samples were not significantly different from the levels in hemolytic disease samples or in samples from healthy newborns. Thus, as in autoimmune thrombocytopenic patients, normal Tpo levels are present in NAIT patients.