Platelet Immunofluorescence Test Research Articles

Detection of alloimmunization in Glanzmann Thrombasthenia and Bernard-Soulier Syndrome: Data from a Brazilian Center

IntroductionThe Glanzmann Thrombasthenia (GT) and Bernard-Soulier Syndrome (BSS) are rare hereditary disorders of platelet function. Their treatment often requires platelet transfusion, which can lead to the development of alloantibodies. ObjectiveIn this study, we aim to develop a strategy for alloantibody detection and to describe the frequency of alloimmunization in a patient population from a single center in southeastern Brazil. MethodsSamples from patients with GT or BSS were tested using the Platelet Immunofluorescence Test (PIFT). If a positive result was obtained, a confirmatory step using the Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) and Luminex bead-based platelet assay (PAKLx) was executed. Main results: Among 11 patients with GT, we detected the presence of alloantibodies in 5 using PIFT, with confirmation through MAIPA and PAKLx in 2 (1 anti-HLA and 1 anti-HPA), resulting in a frequency of 18.1%. Among 4 patients with BSS, PIFT was positive in 3, with confirmation by MAIPA and PAKLx in 1 (anti-HLA), showing a frequency of 25%. The two patients with anti-HLA antibodies exhibited a panel reactive antibody (PRA-HLA) testing greater than 97%. ConclusionOur study highlights the importance of identifying platelet alloimmunization in this patient population. The proposed algorithm for platelet alloantibodies detection allows resource optimization.

Sequestration Pattern in ITP Liver/Spleen Scan Correlates with the Presence of Anti-Glycoprotein V Antibodies in ITP Patients

Introduction Immune thrombocytopenia (ITP) is characterized by low platelet counts due to high clearance and low production. Better insight in the pathophysiology of ITP is needed to personalize management regimes, especially in patients with persisting and chronic ITP. In the Haga Teaching Hospital, we reproduced and validated the ITP liver/spleen scan from St Bartholomew's Hospital in London. With this scan the in vivo sequestration patterns of platelets in either spleen, liver or a combination of both (mixed sequestration) can be assessed, by using Indium-111 labelled autologous platelets . The ITP liver/spleen scan may provide better insights in autoantibody-mediated platelet destruction in ITP patients. Autoantibodies directed against glycoprotein (GP) IIb/IIIa, Ib/IX and V namely are found in a majority of ITP patients and can be detected using specific assays, like platelet immunofluorescence test (PIFT) and Monoclonal Antibody Immobilization of Platelet Antigen (MAIPA). To the best of our knowledge no studies have investigated the possible association between sequestration patterns and glycoproteins. In this study we investigated associations between the sequestration patterns from the ITP liver/spleen scan and the presence of different anti-GP platelet antibodies.Methods All ITP patients receiving an ITP liver/spleen scan between 2014 and 2016 in the Haga Teaching Hospital, a large tertiary referral centre in the Netherlands, are collected from our electronic patient files. We retrospectively collected demographic and clinical characteristics, including duration of ITP, previous management of ITP (corticosteroids, rituximab, thrombopoietin receptor agonists (TPO-RA)), antibody assays (PIFT, direct and indirect MAIPA) and the results from the ITP liver/spleen scan. MAIPA is used as a continuous variable. To investigate the association between anti-GP antibodies and the sequestration pattern from the ITP liver/spleen scan we used linear regression models for the continuous variables and logistic regression models for the binary data. Observational data and scatter plots aid in the interpretation of the possible associations. Both univariate and multivariate regression models including age and sex will be performed.Results 55 ITP patients with a median age of 41 years (IQR 29-57) and 67% females underwent an liver/spleen scan in the study period. The median age of the cohort at diagnosis of ITP was 32 (IQR 22-54) years. Treatment history consisted of corticosteroid therapy in 96% of cases, 44% IVIG, 48% TPO-RA and 21% rituximab. 10% of patients received only 1 treatment line, 23% received 2 lines, 33% received 3 lines, 21% 4 lines, 10% 5 lines and 1 patient received 7 lines (2%). Clinical significant bleedings (WHO bleeding grade ≥2) were reported by 53%, and 8% of the patients received a transfusion in the past. Sequestration pattern of the ITP liver/spleen was in 51% of cases a splenic pattern vs. 49% a mixed/hepatic pattern. A significantly higher prevalence of a splenic pattern was present in patients under 30 years at diagnosis while patients older than 30 years showed a more mixed/hepatic pattern. Anti-glycoprotein antibodies were found in 54% of the patients. Interestingly, linear regression models showed no association between either anti-GPIIb/IIIa or anti-GPIb/IX antibody detection and sequestration pattern. However a significant association was observed between the presence of anti-GPV antibodies and sequestration pattern. A higher anti-GPV antibody level and/or affinity was associated with a higher level of splenic sequestration.Conclusions This study has shown that the presence of anti-GPV antibodies is associated with a splenic sequestration pattern in the ITP liver/spleen scan in patients suffering from persistent and chronic ITP. GPV is usually found as a complex with GPIb/IX in several other bleeding disorders. Surprisingly no association was found in our data between a splenic sequestration pattern and the presence of anti-GPIIb/IIIa and especially anti-GPIb/IX. Further studies are needed to clarify the possible role of anti-GPV antibodies in thrombocyte clearance by the spleen in ITP patients. DisclosuresAmini:AMGEN: Research Funding. Schutgens:Baxalta/Shire: Research Funding; Novo Nordisk: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Pfizer: Research Funding; Uniqure BV: Research Funding. Schipperus:AMGEN: Research Funding; Novartis: Research Funding.

Neonatal alloimmune thrombocytopenia due to a new alloantigen Bl(a) defined by an Asp458Gly substitution in GPIIIa.

Neonatal alloimmune thrombocytopenia (NAIT) commonly arises due to antibodies against a small number of well-defined human platelet antigens (HPAs). A minority of NAIT cases occur due to maternal immunization against low-frequency polymorphisms in platelet glycoprotein that result in new immunogenic epitopes. Antibodies to these novel epitopes can be detected by the incubation of maternal serum with paternal platelets and is usually performed after initial investigation using HPA-typed panel platelets has failed to provide evidence of NAIT. The propositus and the parents from a case of suspected neonatal alloimmune thrombocytopenia (NAIT) were investigated using serologic and molecular techniques to detect and identify relevant platelet-specific antibodies and for HPA typing. Calculations of molecular dynamics were undertaken to explore potential variations in the molecular structure. Maternal antibodies were detected that were reactive only in crossmatch with paternal platelets using the platelet immunofluorescence test (PIFT) and a GPIIb/IIIa monoclonal antibody immobilization of platelet antigen (MAIPA) assay. In the propositus and father, a novel mutation c.1373 A > G was found in exon 10 of ITGB3 resulting in the substitution of an aspartic acid for a glycine (p.Asp458Gly). Recombinant GPIIIa glycoprotein mutated to contain the novel mutation and expressed in HEK293 cells with GPIIb was also specifically recognized by maternal antibodies. Calculations of molecular dynamics identified that the mutation was in a structurally constrained site. This case describes a low-frequency platelet antigen (Asp458Gly) that defines a further alloantigenic target in NAIT. The case emphasizes the role of the platelet crossmatch as the single most useful tool to establish evidence of immunization of low-frequency platelet glycoprotein polymorphisms. A crossmatch should always be performed where there is strong clinical evidence of NAIT but initial laboratory investigations are not confirmatory.

Prediction of Response to Fostamatinib Based on the Presence of Plasma Platelet Autoantibodies in Adult Patients with Immune Thrombocytopenia (ITP) — Exploratory Analyses from Phase 3 Studies

Background: Initial results of an analysis of anti-platelet autoantibodies in patients with ITP from two phase 3 studies showed that patients were more likely to respond to treatment with the SYK inhibitor fostamatinib if they were positive for anti-platelet autoantibodies. Stable platelet responses to fostamatinib were reported in 36% of 28 patients who tested positive for anti-platelet autoantibodies and in 9% of 32 patients who tested negative (p<0.02). This analysis has been expanded to include 85 patients and to evaluate various platelet antigens.Methods: Plasma samples from 85 patients in the phase 3 studies were collected with informed consent at 2 weeks after the first dose of fostamatinib. Samples were analyzed for anti-platelet autoantibodies via Luminex beads-based PakLx assayand FACS-based indirect platelet immunofluorescence test (iPIFT) using platelets from healthy volunteers.Results: Of 85 patient samples, 38 (45%) were positive for one or more anti-platelet autoantibodies and 19 (22%) were from patients with a stable platelet response to fostamatinib. Of 38 patients positive for autoantibodies, 13 (34%) had stable platelet responses. Of 47 patients negative for autoantibodies, 6 (13%) had stable platelet responses.Conclusions: The presence of anti-platelet autoantibodies appeared to be associated with a stable platelet response to fostamatinib. This exploratory analysis provides support for the hypothesis that fostamatinib prevents platelet loss through inhibition of autoantibody-directed platelet destruction in ITP. Further studies are ongoing. DisclosuresMarkovtsov:Rigel: Employment, Equity Ownership. Yi:Rigel: Employment, Equity Ownership. Young:Rigel: Employment, Equity Ownership. Duliege:Rigel: Employment, Equity Ownership. Masuda:Rigel Pharmaceuticals: Employment, Equity Ownership.

Anti-HLA Antibodies in Neonatal Alloimmune Thrombocytopenia - Is There Any Clinical Significance?

Background: Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies raised against paternally inherited alloantigens carried on fetal platelets. Platelets express both HLA class I and specific human platelet antigens (HPA). Although anti-HLA class I antibodies are often detectable in pregnant women, NAIT is considered to be mainly associated with antibodies against HPA. Cases where NAIT has been caused by antibodies against HLA class I are relatively rare and the role of these antibodies in NAIT remains debatable.We hereby describe a sample case of NAIT proved to be caused solely by anti-HLA antibodies and discuss laboratory measures aimed at identification of pregnancies at risk of NAIT related to anti-HLA class I antibodies based on a series of similar cases.Methods: This sample case presents laboratory work-up on a young mother who delivered her first son with a platelet count of 20x109/L, minor petechiae and normal WBC count. Thrombocytopenia in the newborn resolved spontaneously two weeks after birth. Laboratory investigation included platelet immunofluorescence test (PIFT), monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, genotyping of both parents and the newborn for platelet antigens, including rare antigens, and HLA antibody identification using the panel reactive antibodies (PRA) assay (Luminex, USA). A serum sample of this mother, drawn during her second pregnancy, and those of ten other women referred to our laboratory with a similar obstetric history of neonatal thrombocytopenia, were evaluated for the anti-HLA antibody titer using the MAIPA assay.Results: The Rambam Platelet & Neutrophil Immunology Laboratory, as well as 32 other laboratories worldwide, that participated in the 2014 International Workshop organized by the ISBT Platelet Immunobiology Working Party failed to detect anti-HPA antibodies in the mother's serum during her second pregnancy, despite using the most sensitive serological analysis and molecular methods. Only strong anti-HLA antibodies with no single specificity were found in the analyzed samples by all the laboratories. Her second child was born by caesarean section with a platelet count of 50x109/L and maternal anti-HLA antibodies were found in his serum and on his platelets. The anti-HLA antibody titer of the mother, determined by the MAIPA assay, was greater than 1:1024, with antibodies being multi-specific, as demonstrated by PRA. The anti-HLA antibody titer ≥1:16 was found to correlate with low platelet counts in the additional ten cases tested, as opposed to the titer of ≤1:4 in cases with mild and not clinically significant neonatal thrombocytopenia.Conclusions: The presence of anti-HLA class I antibodies should be considered as a potential cause of NAIT, especially in cases with a very high titer of antibodies. The mechanism underlying the effect of these antibodies on fetal platelets needs to be further investigated. DisclosuresNo relevant conflicts of interest to declare.

Detection of clinically relevant platelet antibodies in the Asian population

BackgroundThe detection and identification of the clinically relevant platelet antibodies are important for the diagnosis and management of the affected patients with immune thrombocytopenia.AimsThe aim of this study was to investigate the characteristic of clinically relevant platelet antibodies in the Asian population, and to evaluate the ability of platelet immunology laboratories in Asia to detect and identify these platelet antibodies.MethodsTotal of 635 cases that were diagnosed and studied as NAITP, PTR and PTP by Asian platelet laboratories were reviewed for the clinical and experimental data from the publications.ResultsExcept the anti‐HLA and unknown‐specificity antibody, the most frequently identified platelet antibody was anti‐CD36 (18/635, 18·35%), and then anti‐HPA‐3 and anti‐HPA‐4 (each 5/635, 0·78%), and anti‐HPA‐5 (4/635, 0·62%). Only two cases of anti‐HPA‐1 antibodies (2/635, 0·31%) were reported. In 274 cases, including four cases contained anti‐HLA antibody, the laboratories could not identify the specificity of platelet antibodies. The methods of detection and identification of antiplatelet antibody, such as monoclonal antibody immobilization of platelet antigens (MAIPA), mixed passive hemagglutination assay (MPHA), platelet immunofluorescence test (PIFT), modified antigen‐capture ELISA (MACE) and solid‐phase red‐cell adherence (SPAA), have been used in Asian laboratories.ConclusionWith an increasingly heterogeneous patient population in Asia, diagnostic laboratories should be aware of the ethnic origin of a particular patient to consider the possibility of immunization to antigens. The most important clinical relevant platelet antibodies implicated in immune‐mediated thrombocytopenia among the Asian population are anti‐CD36, then anti‐HPA‐3, anti‐HPA‐4 and others.

Sensitivity of assays for the detection of HPA‐1a antibodies: results of an international workshop demonstrating the impact of cation chelation from integrin αIIbβ3 on three widely used assays

HPA-1a antibodies account for 70-80% of cases of fetal-neonatal alloimmune thrombocytopenia (FNAIT) in Caucasians. However, numerous workshops have demonstrated variability in their detection. We recently showed that exposure of αIIbβ3 to ethylene diamine tetraacetic acid (EDTA) affected binding of many anti-αIIbβ3 monoclonal, and HPA-1a allo-, antibodies; this adversely affected sensitivity of the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay and indirect platelet immunofluorescence test (PIFT). This study presents results from an international workshop studying the impact of cation chelation on HPA-1a antibody detection in routine diagnostic laboratories. Serum and EDTA-anticoagulated plasma samples containing anti-HPA-1a were distributed to 39 laboratories. Participants were asked to detect and identify any HPA antibodies present. 2/39 (5.1%) participants were able to detect and identify anti-HPA-1a in the serum, but not in the plasma sample. EDTA plasma reduced MAIPA assay sensitivity by ≥ 20% in 17/24 (70.8%) laboratories and by ≥ 50% in 9/24 (37.5%) when using HPA-1a1a platelets (mean: 27.7%, range 0-85.1%); when using HPA-1a1b platelets 3/4 (75%), participants reported ≥ 50% loss of sensitivity (mean 65.6%, range 0-96.6%). A small but significant increase in optical densities was observed in antigen capture ELISA assays when using plasma (mean difference: 0.081, P < 0.01). Insufficient PIFT data were returned to draw firm conclusions. Use of EDTA plasma significantly affects the sensitivity of the MAIPA assay and can affect detection of even potent, FNAIT-causing examples of anti-HPA-1a. These data highlight the importance of use of αIIbβ3 in an appropriate conformation for the sensitive detection of anti-HPA-1a.

Platelet antibody detection by flow cytometry: an effective method to evaluate and give transfusional support in platelet refractoriness

BackgroundImmune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80-90% of cases) and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness. ObjectiveThe aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness. MethodsA group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity. ResultsSeventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years) were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% and a negative predictive value of 85.71%. The accuracy of the method was 80.26%. ConclusionThis study shows that the flow cytometry platelet immunofluorescence test has a high correlation with the anti-human leukocyte antigen antibodies. Despite a few limitations, the method seems to be efficient, fast and feasible as the initial screening for platelet antibody detection and a useful tool to crossmatch platelets for the transfusional support of patients with immune platelet refractoriness.

Platelet associated IgG may be related with thrombocytopenia in patients with myelodysplastic syndromes

Thrombocytopenia is common in patients with myelodysplastic syndromes (MDS) and immune destruction of platelets could be an important factor for its occurrence. We prospectively analyzed platelet-associated IgG (PAIgG) through platelet immunofluorescence test (PIFT), mean platelet volume (MPV), platelet size deviation width (PDW) and glycocalicin index (GCI) of 54 patients with MDS, classified according to the International Prognostic Scoring System (IPSS). Thrombocytopenia (platelet count<100×109/L) was correlated with a higher amount of PAIgG, significantly higher MPV and increased GCI. In addition, worse prognosis IPSS groups were associated with a higher positivity of PIFT, which could be indicative of advanced disease.

Identification of platelet refractoriness in oncohematologic patients

OBJECTIVES:To identify the occurrence and the causes of platelet refractoriness in oncohematologic patients.INTRODUCTION:Platelet refractoriness (unsatisfactory post-transfusion platelet increment) is a severe problem that impairs the treatment of oncohematologic patients and is not routinely investigated in most Brazilian services.METHODS:Forty-four episodes of platelet concentrate transfusion were evaluated in 16 patients according to the following parameters: corrected count increment, clinical conditions and detection of anti-platelet antibodies by the platelet immunofluorescence test (PIFT) and panel reactive antibodies against human leukocyte antigen class I (PRA-HLA).RESULTS:Of the 16 patients evaluated (median age: 53 years), nine (56%) were women, seven of them with a history of pregnancy. An unsatisfactory increment was observed in 43% of the transfusion events, being more frequent in transfusions of random platelet concentrates (54%). Platelet refractoriness was confirmed in three patients (19%), who presented immunologic and non-immunologic causes. Alloantibodies were identified in eight patients (50%) by the PIFT and in three (19%) by the PRA-HLA. Among alloimmunized patients, nine (64%) had a history of transfusion, and three as a result of pregnancy (43%). Of the former, two were refractory (29%). No significant differences were observed, probably as a result of the small sample size.CONCLUSION:The high rate of unsatisfactory platelet increment, refractoriness and alloimmunization observed support the need to set up protocols for the investigation of this complication in all chronically transfused patients, a fundamental requirement for the guarantee of adequate management.

Platelet-Associated IgG May Be Associated with Thrombocytopenia In Patients with Myelodysplastic Syndromes

AbstractAbstract 2943 Background.Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by ineffective and dysplastic hematopoiesis, progressive bone marrow failure, cytopenias and a high risk of transformation into acute leukemia. Thrombocytopenia is detected in up to two thirds of patients with MDS and severe thrombocytopenia is present in approximately 10%. Besides ineffective thrombopoiesis, immune destruction of platelets could be an additional factor in the genesis of thrombocytopenia, since immunological abnormalities are also frequent in patients with MDS. The detection of platelet associated IgG (PAIgG) by immunofluorescence (platelet immunofluorescence test or PIFT) is a highly sensitive assay. In addition, some morphological platelet indices (PDW and MPV) are correlated with the occurrence of immune thrombocytopenia. We prospectively analysed platelet-bound IgG and platelet indices (PDW and MPV) in 35 patients with MDS. Methods:Thirty-five patients with MDS (mean age ± SD: 63 ± 19 yo; range 21–89 yo; 15female/20male) were evaluated. According to FAB, 27 patients were classified as RA, 5 as RARS and 3 as RAEB. Clinical manifestations of immunological disorder were not present in this population. Blood samples were analyzed by PIFT, in order to detect platelet associated IgG and results were expressed as a ratio of patient fluorescence/negative control fluorescence (R). Cell-dyn Sapphire blood cell analyzer (Abbott, Illinois, USA) was used to measure platelet count, mean platelet volume (MPV) and platelet size deviation width (PDW). Thrombocytopenia was defined as a platelet count <100 × 109/L. All samples were analyzed on the day of collection. Results.Platelet counts of the entire population ranged from 6.7 to 708 ×109/L, with median of 95.4 × 109/L. Eighteen patients (51.43%) had platelet count <100 × 109/L. A strong association between thrombocytopenia and PAIgG measured by PIFT (R) was demonstrated taking into account three analysis: an inverse correlation between the number of platelets and the fluorescence ratio (p=0.01, r=-0.39, Spearman test), the higher positivity of PIFT in patients with platelet count <100 × 109/L (p=0.007, Wilcoxon rank sum test) and a lower platelet count in patients with positive PIFT (p=0.059, Wilcoxon rank sum test). MPV was significantly higher in patients with platelet count <100 × 109/L (median, min-max: 9.38, 6.9–23.1 vs 8.46, 4.88–12.83; p<0.001, Wilcoxon rank sum test). PDW showed no statistical difference between these groups. Discussion: Immune thrombocytopenia is a relatively frequent hematological disorder of unknown origin and until today depends mainly upon clinical diagnosis. Assays for the detection of glycoprotein-specific antibodies such as MAIPA (monoclonal antibody-specific immobilization of platelet antigens) are highly specific but less sensitive than PIFT. MAIPA is laborious and require a certain amount of platelets, which is not always available in thrombocytopenia Thus, PIFT could be recommended as a screening test to discriminate patients with MDS in whom the hyperdestructive component of thrombocytopenia is important. Our results also showed a higher MPV in patients with platelet count <100 × 109/L, suggesting that this index may be of interest for detection of immune-mediated hyperdestructive thrombocytopenia in MDS. Conclusion:Thus, we propose that a combination of a simple index as MPV and a highly sensitive and easy to perform screening test for PAIgG as PIFT could be applied to select a subset of MDS patients in which we would be able to prevent the overuse of unnecessary platelet transfusions and who could be candidates for an immunosuppressive therapeutic approach. Supported by INCTS, FAPESP, CNPq. Disclosures:No relevant conflicts of interest to declare.

Post‐transfusion purpura caused by anti‐HPA‐3a antibodies that are only detectable using whole platelets in the platelet immunofluorescence test

Post‐transfusion purpura caused by anti‐HPA‐3a antibodies that are only detectable using whole platelets in the platelet immunofluorescence test

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

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

PAKTM-Lx: A New Luminex*®-Based Assay to Detect and Identify Antibodies to Human Platelet Antigens (HPA) in Patient Serum.

Abstract 4187Platelets express a variety of glycoproteins (GPIIb-IIIa, GPIa-IIa, GPIb/IX, GPIV, CD109 and Class I HLA). Human Platelet Antigens (HPA) represent polymorphisms of these platelet surface glycoproteins. While these polymorphisms do not alter the function of the proteins, they can become target of the immune system in mismatched individuals. Alloantibodies against HPA are involved in the pathogenesis of Neonatal Allo-Immune Thrombocytopenia (NAIT), Post-Transfusion Purpura (PTP) and refractoriness to random donor platelets. In addition to generating anti-HPA antibodies, these patients also develop antibodies to Class I Human Leukocyte Antigen (HLA). Most reference laboratories now use a combination of a sensitive screening assay such as the Platelet Immunofluorescence Test (PIFT) along with a solid phase assay for antibody identication, usually either the ELISA and Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) as well as genotyping techniques for at least HPAs 1, 2, 3, and 5. Currently, MAIPA is considered the gold standard in detection of antibodies to HPA. However, all existing methods have significant limitations in terms of sample requirements, time and labor involved. These factors are a major impediment as to how many specificities can be resolved in a rapid, efficient and reproducible manner. Development of microbead-based assays like the xMAP (Luminex®) has provided a technology that can resolve these questions. GTI Diagnostics® and Gen-Probe have developed PAKTM -Lx, a bead-based assay for the Luminex® platform to detect and determine the specificity of anti-HPA antibodies. PAKTM -Lx is capable of resolving the specificities of antibodies to HPA 1, 2, 3, 4 and 5 in addition to detecting antibodies against GPIV and HLA Class-I antigens. Furthermore, it is capable of detecting antibodies to multiple HPA and HLA with only 10 μl of sample. With a total time of less than two hours from addition of samples to end results, PAKTM -Lx is sensitive, rapid and simple to perform. Focused, flexible multiplexing in the range of 1 to 100 analytes provided by Luminex® technology meets the needs to expand the panel to include more HPA combinations, thereby reducing sample requirements, labor and costs. Disclosures:No relevant conflicts of interest to declare.

External quality assessment of platelet serology and human platelet antigen genotyping: a 10‐year review

In this review, the results of an external quality assessment (EQA) over 10 years of platelet (PLT) serology and of human platelet antigen (HPA) polymorphisms genotyping are shown. The detection and identification of PLT antibodies and the distinction between PLT-specific antibodies and HLA Class I antibodies are evaluated. Each year, serum samples from five patients and four donor blood samples for DNA typing were distributed. Laboratories could participate as a screening laboratory (SL; n = 7) or as an identification laboratory (IL; n = 8). SLs scored 57 to 100 percent correct positive and 91 to 100 percent correct negative results in the detection of PLT-specific antibodies. SLs only using a PLT immunofluorescence test (PIFT) scored less well than those using a PLT glycoprotein-based antibody detection method. ILs scored 70 to 100 percent correct positive and 87 to 100 percent correct negative results for, respectively, the detection and identification of PLT-specific antibodies. Both the specificity and the sensitivity for the detection and identification of PLT-specific antibodies were not as good in ILs using solid-phase enzyme-linked immunosorbent assay methods as in those using the monoclonal antibody immobilization of PLT antigens (MAIPA) assay. For HPA-1, -2, -3, and -5 genotyping, incorrect results were observed only twice in 280 genotyping assays. The data underscore the necessity of using the most accurate methods with a high level of knowledge, experience, and technical training. For screening purposes, it is not sufficient to use only the PIFT, whereas for identification of PLT-specific antibodies, the MAIPA assay is the superior assay.

Incidence of transfusion‐induced platelet‐reactive antibodies evaluated by specific assays for the detection of human leucocyte antigen and human platelet antigen antibodies

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). 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(R) and LCT (lymphocytotoxicity test). 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(R) 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 pan-reactive antibodies against glycoprotein IIbIIIa, IaIIa and IbIX. 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.

Platelet and granulocyte alloimmunisation in multitransfused Tunisian patients

We determined the frequency of post-transfusion alloimmunisation against platelet and granulocyte antigens in 51 Tunisian polytransfused patients with haematological diseases. Serum samples were analysed by a standard and an antiglobulin-augmented lymphocytotoxicity technique, a granulocyte agglutination test, a granulocyte immunofluorescence test, a platelet immunofluorescence test and the monoclonal antibody-specific immobilisation of platelet antigens assay. No granulocyte-specific antibodies were detected. HLA antibodies were found in 58.8% of patients. Platelet-specific antibodies were detected in four patients and were directed against human platelet antigen (HPA)-5b, HPA-1b and HPA-3a. The three patients with Glanzmann's thrombasthenia developed anti-GPIIb/IIIa antibodies. This study provides immunogenetic information that could improve the management of transfusion therapy in Tunisia.

Alloimmune neonatal neutropenia and thrombocytopenia associated with maternal anti HNA‐1a, HPA‐3b and HLA antibodies

The incidence of alloimmune neonatal neutropenia combined with neonatal alloimmune thrombocytopenia is very low. We report a case of a neonate who suffered severe neutropenia and thombocytopenia with widespread petechial spots. The presence of alloantibodies in mother's and patient's sera was analyzed by lymphocytotoxicity test, agglutination test, granulocyte indirect immunofluorescence test, platelet immunofluorescence test (PIFT) and solid phase enzyme-linked immunosorbent assay. Human neutrophil antigens (HNA) and human platelet antigen (HPA) genotypes were tested by polymerase chain reaction analyses. The mother's and patient's sera reacted with neutrophils and lymphocytes of the father. PIFT revealed the presence of IgG anti-platelet antibodies in the patient's serum but the test was negative in the maternal serum. Analyses of HNA-1 and HPA genotypes of the family revealed maternal-neonatal HNA-1a and HPA-3b mismatch. The study of the mother's and patient's sera showed the presence of anti HNA1a, HPA-3b and HLA antibodies specific for HLA-A3 and HLA-B38 antigens. These results suggest that the transplacental passage of maternal HNA-1a, HPA-3b and HLA alloantibodies caused neutropenia and thrombocytopenia in this patient.

Evaluation of the Usefulness of Detected Platelet Antibodies for the Prognosis of Treatment Results in Patients with Thrombocytopenic Purpura.

Platelet antibodies are the main cause of platelet destruction in immunological thrombocytopenic purpura. However, the significance of their detection for prognosis of treatment results in ITP patients (idiopathic immune thrombocytopenic purpura) has not yet been determined.The aim of this work was to evaluate the effectiveness of treatment with corticosteroids, splenectomy and immnunosupressive drugs of 409 ITP patients in relation to the detection of platelet antibodies.Platelet antibodies were determined in the ITP patients under observation:in connection with autologous platelets - with platelet immunofluorescence test (PIFT),in serum - with immunoenzimatic test using monoclonal antibodies directed at GPII/IIIa, GPIb, GPIa/IIa (MAIPA - monoclonal antibody immobilization of platelet antigens).In treatment effectiveness prognosis the multifactor statistical analysis was used involving not only platelet antibodies but also other parameters such as age, sex, course of disease, megacariocyte count in bone marrow. In patients after splenectomy -the spleen-liver index and the time lapse between ITP diagnosis and surgical procedure were additionally taken into account.The statistical analysis revealed that platelet antibodies had no significant effect on the treatment results prognosis in ITP patients. However, while analysing groups of patients with different treatment administered, it was found that the effect of platelet antibodies on positive prognosis treatment was significant though not very big effect in the case of patients treated with corticosteroids and immunosuppressive drugs while nonsignificant in the case of patients after splenectomy.Additional factors significantly effecting positive treatment prognosis in all patients were: severe/acute course of disease, increased/normal megacariocyte count, young age, female sex. In splenectomy patients the spleen-liver index (up to 5.7:1) seems to be a significantly beneficial factor as well as the time lapse between diagnosis and procedure (up to 9 years since diagnosis).

The value of crossmatch tests and panel tests as a screening tool to predict the outcome of platelet transfusion in a non‐selected haematological population of patients

Alloantibodies against platelets can be detected by using different laboratory tests. Most of these tests, which use panel cells or antigens as a target, perform poorly in non-selected haematological patients. In relation to these tests, a crossmatch test of transfused platelets and patient's serum may be viewed as the standard and may be superior in predicting donor platelet destruction by alloimmunization. In 95 randomly selected thrombocytopenic patients with haematological malignancies, who were receiving leucodepleted blood products, 184 serum samples were studied in an in vitro crossmatch test by using the technique of the platelet immunofluorescence test (crossmatch-PIFT), in an in vivo crossmatch test detecting in vivo binding of immunoglobulins to transfused platelets according to the PIFT technique (in vivo-PIFT), in the indirect PIFT using five random donors as a target (panel-PIFT) and in an enzyme linked immunosorbent assay using immobilized human leucocyte antigens (HLAs) of 100 standardized donors (ELIHLA). The results of all these methods were related to the recovery at 1 and 16 h post-transfusion. The results of the crossmatch-PIFT were not associated with platelet recovery at 1 and 16 h after transfusion. Even in a subgroup of patients, in whom predefined clinical factors were excluded, no association with platelet recovery was found. The results of the crossmatch-PIFT correlated with those of the in vivo-PIFT (P = 0.02); however, 35 (19%) discrepant results were identified between these tests. The results of the crossmatch-PIFT were not related to the panel-PIFT (P = 0.25), but did relate to those of the ELIHLA (P = 0.02), still revealing 36 (20%) discrepant results. None of the in vivo-PIFT, the panel-PIFT or the ELIHLA was associated with platelet recovery after 1 h, whilst only a positive panel-PIFT was associated with poor platelet recovery at 16 h after transfusion (P = 0.03). In a population at low risk for alloimmunization, the correlation of test outcome and platelet recovery is poor. None of these crossmatch tests or screening tests was identified as superior to any other in this population.