Post-transfusion Purpura Research Articles

Human Platelet Antigen Alleles in 998 Taiwanese Blood Donors Determined by Sequence-Specific Primer Polymerase Chain Reaction

Polymorphism of human platelet antigens (HPAs) leads to alloimmunizations and immune-mediated platelet disorders including fetal-neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP), and platelet transfusion refractoriness (PTR). HPA typing and knowledge of antigen frequency in a population are important in particular for the provision of HPA-matched blood components for patients with PTR. We have performed allele genotyping for HPA-1 through -6 and -15 among 998 platelet donors from 6 blood centers in Taiwan using sequence-specific primer polymerase chain reaction. The HPA allele frequency was 99.55, and 0.45% for HPA-1a and -1b; 96.49, and 3.51% for HPA-2a and -2b; 55.81, and 44.19% for HPA-3a and -3b; 99.75, and 0.25% for HPA-4a and -4b; 98.50, and 1.50% for HPA-5a and -5b; 97.75 and 2.25% for HPA-6a and -6b; 53.71 and 46.29% for HPA-15a and -15b. HPA-15b and HPA-3a, may be considered the most important, followed by HPA-2, -6, -1, -5, and -4 systems, as a cause of FNAIT, PTP, and PTR based on allele frequency. HPA-4b and HPA-5b role cannot be excluded based on their immunogenicity. A larger-scale study will now be conducted to confirm these hypotheses and to establish an apheresis donor database for the procurement of HPA-matched apheresis platelets for patients with PTR.

Studies on CD36 deficiency in South China: Two cases demonstrating the clinical impact of anti-CD36 antibodies

CD36 (also known as GPIV) deficiency is known to be responsible for the production of anti-Nak(a) antibodies in different clinical settings such as fetal/neonatal alloimmune thrombocytopenia (FNAIT), platelet transfusion refractoriness (PTR) and post-transfusion purpura (PTP). However, no data regarding the relevance of CD36 immunisation is currently available for China. In this study, healthy blood donors were typed for CD36 deficiency using flow cytometry. Nucleotide sequencing was performed to identify the molecular basis underlying the CD36 deficiency. Anti-Nak(a) antibodies in CD36-deficient individuals were analysed by ELISA and flow cytometry. By analysis of 998 healthy blood donors, 18 individuals failed to express CD36 on their platelets. In 5/12 individuals no CD36 expression was detected both on platelets and monocytes. This result suggested that the frequencies of type I CD36 deficiency (platelets and monocytes) and type II CD36 deficiency (platelets only) are approximately 0.5 and 1.3%, respectively. Nucleotide sequencing analysis of type I CD36 deficient individuals revealed eight different mutations; four of them were not described so far. However, 1228-1239del ATTGTGCCTATT and 329-330delAC appear to be the most common mutations related to type I CD36 deficiency in South Chinese population. Further analysis showed that 1/5 type I CD36 deficient individuals developed anti-Nak(a) antibodies. In addition, anti-Nak(a) antibodies could be identified in two cases of thrombocytopenia associated with FNAIT and PTR. In conclusion, more than 0.5% of CD36 type I-deficient individuals are at risk to be immunised through blood transfusion or pregnancy in China. Testing of anti-Nak(a) antibodies should be considered in FNAIT and PTR suspected cases. A registry of CD36-deficient donors should be established to allow treatment of immune-mediated bleeding disorders caused by anti-Nak(a) antibodies.

Current understanding of allergic transfusion reactions: incidence, pathogenesis, laboratory tests, prevention and treatment

Non-haemolytic transfusion reactions are the most common type of transfusion reaction and include transfusion-related acute lung injury, transfusion-associated circulatory overload, allergic reactions, febrile reactions, post-transfusion purpura and graft-versus- host disease. Although life-threatening anaphylaxis occurs rarely, allergic reactions occur most frequently. If possible, even mild transfusion reactions should be avoided because they add to patients' existing suffering. During the last decade, several new discoveries have been made in the field of allergic diseases and transfusion medicine. First, mast cells are not the only cells that are key players in allergic diseases, particularly in the murine immune system. Second, it has been suggested that immunologically active undigested or digested food allergens in a donor's blood may be transferred to a recipient who is allergic to these antigens, causing anaphylaxis. Third, washed platelets have been shown to be effective for preventing allergic transfusion reactions, although substantial numbers of platelets are lost during washing procedures, and platelet recovery after transfusion may not be equivalent to that with unwashed platelets. This review describes allergic transfusion reactions, including the above-mentioned points, and focusses on their incidence, pathogenesis, laboratory tests, prevention and treatment.

Post-transfusion purpura in an African-American man due to human platelet antigen-5b alloantibody: a case report

IntroductionPost-transfusion purpura is a rare immunohematological disorder characterized by severe thrombocytopenia following transfusion of blood components and induced by an alloantibody against a donor platelet antigen. It occurs primarily in women sensitized by pregnancy and is most commonly caused by anti-human platelet antigen-1a antibodies. Here, we describe what we believe to be the first documented case of an African-American man who developed post-transfusion purpura due to an anti-human platelet antigen-5b alloantibody after receiving multiple blood products.Case presentationA 68-year-old African-American man initially admitted with atrial flutter was started on anticoagulation treatment, which was complicated by severe hematemesis. On days 4 and 5 of hospitalization, he received six units of packed red blood cells, and on days 4, 13 and 14 he received plasma. His platelet count began to drop on day 25 and on day 32 reached a nadir of 7 × 109/L. His platelet count increased after receiving intravenous immune globulin. An antibody with reactivity to human platelet antigen-5b was detected by a solid-phase enzyme-linked immunoassay. Our patient was homozygous for human platelet antigen-5a.ConclusionThis case emphasizes the importance of including post-transfusion purpura in the differential diagnosis for both men and women with acute onset of thrombocytopenia following transfusion of blood products. The prompt recognition of this entity is crucial for initiation of the appropriate management.

Prévalence des anticorps antiplaquettes spécifiques chez les receveurs de concentrés plaquettaires avec effet indésirable transfusionnel

Purpose of the studyIn practice, platelet transfusions are frequently performed in patients with haematologic and/or oncologic diseases. Subsequent to these transfusions, platelet specific antibodies may develop and could induce adverse events, such as platelet transfusion refractoriness or post-transfusion purpura. In order to evaluate the prevalence of platelet specific antibodies in these recipients, adverse events with a request for platelet specific antibodies testing, were studied. Patients and methodsRecipients of platelet units with adverse event, excluding platelet transfusion refractoriness or post-transfusion purpura, were evaluated. ResultsFrom January 1st 2009 to October 31st 2011, 125 adverse events with platelet specific antibodies screening, corresponding to 116 recipients (64 females, 52 males) were included. The main aetiology of the adverse event was a febrile non-haemolytic transfusion reaction in 62 cases (49.6%) and allergy in 40 (32.0%). Most samples tested were post-transfusion solely (101 adverse events, 80.8%). Seven of these samples had free platelet specific antibodies, including four anti-HPA-5b, one anti-HPA-2a and two anti-GPIaIIa. In the cross-match test, platelet specific antibodies were found in two pre-transfusion samples (anti-GP IaIIa) and in five post-transfusion samples (anti-GPIaIIa, three cases; anti-GP IbIX, one case; anti-GP IIbIIIa and -GPIbIX, one case). ConclusionAccording to this study on platelet transfusion related adverse event, few platelet specific antibodies were detected on pre- and post-transfusion samples. The implementation of platelet specific antibodies testing before transfusion would give more accurate data and help prevent adverse events as typed platelets would be given when platelet specific antibodies were found.

Influnce of Pregnancy on Women's Platelet Antibody

Objective To explore the influence of pregnancy on women's platelet (PLT) antibody.Methods Research objects were divided into the observation group and control group.The members in the observation group were 291 pregnant women who received the prenatal examination in the blood group reference laboratory of Liaocheng Blood Center from February 2009 to December 2011.The members in the control group were 4613 healthy female blood donors donated blood in our station during the same period.Blood samples of observation group were collected when the pregnant women were pregnant in the 16th,20th,24th,28th,30th,32nd,34th and 36th week.Blood samples of control group were collected when they donated blood.Apply simplified sensitized erythrocyte platelet serology assay (SEPSA) method and randomly take 5 samples of mixed platelets of type O blood to test platelet antibody.The Yantai Haimo New Generation of Blood Safety and Service System was adopted for data processing and χ2 inspection was adopted for statistical analysis (The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Liaocheng Blood Center). Results In the observation group, PLT antibody positive rate was 20.5％ (45/219).PLT antibody positive rate increased with the increase of the number of pregnancy (χ2=16.409,P＜0.05).During the 16 to 28 weeks of pregnancy,the positive rate increased with the increase of the time of pregnancy (χ2 =16.082,P＜0.05).But during the 28 to 36 weeks of pregnancy,the difference of positive rate was not statistically significant (χ2 =0.054,P＞0.05). Incontrol group,there were 131 positive cases and 4482 negative cases.Positive rate of women who had pregnancy history were higher than who had no pregnancy history (χ2 =134.676,P＜0.05).For women who had pregnancy history,positive rate increased with the increase of the number of pregnancy (χ2 =235.846,P＜0.05),but the positive rate reduced with the increase of the time to the last pregnancy (χ2 =48.387,P＜ 0.05). Conclusions There was closely relationship between pregnancy and women's PLT antibody producing. From the 16th to 28th week of pregnancy,the positive rate of platelet antibody increased with the pregnant time increasing.When a woman with positive platelet antibody was pregnant again,the monitor changes of platelet antibody during the 16th to 28th week pregnancy could prognose the same immune thrombocytopenic purpura.Test the platelet antibody of women who have pregnancy history could effectively prevent the post-transfusion purpura, post-transfusion acute lung injury, platelet transfusion refractoriness,non-hemolytic febrile transfusion reactions and other transfusion reactions. Key words: platelet; antibody; pregnancy

Fiches techniques : aide à l’analyse des effets indésirables receveurs

In order to help the analysis of adverse effects of transfusion, factsheets have been written by working groups of the French agency for the safety of health products ANSM. Each factsheet deals with a blood transfusion side effect and is composed of five parts, including pathophysiological mechanisms, diagnostic criteria, management recommendations, etiologic investigations and rules for filing the notification form to ANSM. Since 2006, 11 factsheets have been published on the French haemovigilance network website. The major characteristics of the two last sheets published “post-transfusion purpura” and “non erythrocyte incompatibility reaction” are presented, followed by the updated card for “allergy”. These factsheets give relevant guidelines allowing better evaluation of recipients’ adverse reactions, particularly their diagnosis, severity and accountability. They also could initiate studies among European and international haemovigilance networks.

Intravenous immunoglobulin-associated renal failure in a patient with post-transfusion purpura.

Intravenous immunoglobulin (IVIG), initially developed for immunodeficiency disorders, has now been used for multiple autoimmune diseases and infections. These are generally well tolerated, with few adverse effects. Acute kidney injury has been described in very rare instances. We report an interesting case of a 59-year-old African American male with a pertinent history of diabetes mellitus, hypertension, endocarditis, and peripheral vascular disease, who was diagnosed with post-transfusion purpura. He was then treated with IVIG and subsequently developed an acute worsening of renal function in a time span of 3 days. The etiology remained elusive even after an extensive workup. Renal biopsy was done finally, which showed findings suggestive of osmotic nephropathy that was traced to the sucrose used as a stabilizing agent in the IVIG. In light of the increasing use of IVIG, it is therefore highly recommended that clinicians are well aware of this side effect of IVIG.

Genetic polymorphisms of human platelet antigens-1 to -6, and -15 in the Malaysian population.

Human platelet antigens (HPA) are determinant in several platelet-specific alloimmune disorders, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura and platelet transfusion refractoriness. The distribution of HPA systems in the Malaysian population is not known. Defining the patterns of HPA systems provides a basis for risk assessment and management of the above complications. The aim of this study was to investigate the distribution of HPA -1 to -6 and -15 in the three major ethnic groups (Malay, Chinese and Indian) in the Malaysian population. A total of 600 random donor samples, 200 from each of the three ethnic groups, were genotyped by means of real time polymerase chain reaction (PCR) with hydrolysis probes and PCR-restriction fragment length polymorphism (PCR-RFLP). The most common genotype observed in this study was HPA-1a/1a-2a/2a-3a/3b-4a/4a-5a/5a-6a/6a-15a/15b (17%) followed by HPA-1a/1a-2a/2a-3a/3a-4a/4a-5a/5a-6a/6a-15a/15b (14.33%). The allele frequencies of HPA in Malays and Chinese were found to be similar those of other East and South-East Asian populations, while those of Indians were comparable to the frequencies found in Europeans. The results of this study have been useful for determining the distribution of HPA polymorphisms in this region and for potential clinical implications.

Human platelet antigen genotypes in Turkish and Caucasian blood donors in Germany

Exposition to allogenic human platelet antigens (HPAs) can lead to antibody formation causing neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura or platelet (PLT) transfusion refractoriness. The frequencies of HPA differ between ethnical groups which could be associated with different potential alloimmunization risk. The Turkish population is the largest ethnic minority group living in Germany. However, no data are available about the HPA frequency among Turkish population. We compared the frequency of HPA between Caucasian and Turkish blood donors. DNA from blood samples of 119 Caucasian and 117 Turkish blood donors was isolated. The genotype of HPA-1, -2, -3 -4, -5 and -15 was determined using a commercialized polymerase chain reaction kit with sequence-specific primers. In Turkish blood donors, the gene frequencies of HPA-1a/1b, -2a/2b, -3a/3b, -4a/4b, -5a/5b and -15a/15b were 0.863/0.137, 0.868/0.133, 0.607/0.393, 0.996/0.004, 0.893/0.107 and 0.474/0.256, respectively. In Caucasians, we observed 0.798/0.202, 0.908/0.092, 0.567/0.432, 1.000/0.000, 0.916/0.084 and 0.517/0.483 for HPA-1a/1b, -2a/2b, -3a/3b, -4a/4b, -5a/5b and -15a/15b, respectively. No statistically significant difference between genotypes in these populations could be observed. Due to the similar distribution of HPA genotypes in both ethnical groups, no increased risk of NAIT for children of mixed couples or of post-transfusion purpura or PLT transfusion refractoriness secondary to antibodies to HPAs for recipients of PLT concentrates from blood donors of the other ethnicity is given.

Bleeding disorders in obstetrics and gynaecological practice.

The objective of the study was to determine the frequency, types, presenting symptoms and management options in patients with bleeding disorders in the local obstetric and gynaecological practice. This was a descriptive study of oneyear duration conducted at the Pakistan Atomic Energy Commission General Hospital, Islamabad. Types of bleeding disorders in decreasing order of frequency were HELLP syndrome in 27.7% (n=5), gestational thrombocytopenia in 22% (n=4), Von Willbrand disease in 16.66% (n=3), Glanzmann's thrombesthenia in 11.11% (n=2), autoimmune haemolytic anaemia in 11.11% (n=2), post-transfusion purpura in 6% (n=1) and factor V deficiency in 6% (n=1). HELLP syndrome and gestational thrombocytopenia were the commonest bleeding disorders in pregnant patients and Von Willebrand disease is the the commonest bleeding disorder in gynaecological patients with menorrhagia.

Comments on: frequency of human platelet antigens in oncohematological patients with thrombocytopenia and the probability of incompatibility to platelet transfusions

Comments on: frequency of human platelet antigens in oncohematological patients with thrombocytopenia and the probability of incompatibility to platelet transfusions

The relevance of HPA-15 antigen expression for anti-HPA-15 antibody detection

The HPA-15 antigen system is characterized by a low antigen expression on platelets. The antibodies against this antigen are implied in fetal/neonatal alloimmune thrombocytopenia (F/NAIT), post-transfusion purpura, and refractoriness to platelet transfusions. Detection of these antibodies appears to be related to the level of HPA-15 expression on the platelets used in the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay. We performed genotyping of 300 healthy blood donors for HPA-15 by TaqMan real-time PCR technology, and the HPA-15 antigen expression was investigated in 13 HPA-15aa and 19 HPA-15bb individuals. We also investigated the relevance of HPA-15 antigen expression on donor platelets used in MAIPA for antibody detection in 223 multitransfused hematological patients and 271 women with suspected F/NAIT. In Polish donors, the HPA-15a allele frequencies were lower than the HPA-15b (0.480 vs. 0.515). We identified three HPA-15 expression groups: high (36.7 ± 8.36 MFI - eight cases), medium (19.5 ± 6.2 MFI - 21 cases), and low (6.5 ± 5.9 MFI - three cases). The HPA-15 expression was stable over time. The HPA-15aa and HPA-15bb platelets with high antigen expression were used for anti-HPA-15 antibody detection; anti-HPA-15 antibodies were detected in 4/223 (1.8%) patients receiving multiple transfusions but in none of the 271 women with suspected F/NAIT. Further examination of the four sera by MAIPA with various platelets revealed the optical density in the assay to be closely related to the level of HPA-15 antigen expression. Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level.

Molecular testing for platelet and granulocyte antigens

Human platelet antigen (HPA) genotyping is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patient sera, complementing the clinical history in the diagnosis of alloimmune platelet disorders such as neonatal alloimmune thrombocytopenia (NAIT), post‐transfusion purpura (PTP) and platelet transfusion refractoriness (PR). Likewise, human neutrophil antigen (HNA) typing also plays a critical role in the investigation and diagnosis of some clinical conditions induced by granulocyte alloantibodies, like neonatal alloimmune neutropenia (NAIN) and transfusion‐related acute lung injury (TRALI).Up to now, 27 HPA alloantigens have been described, including the 6 most clinically relevant biallelic systems and a significant number of low‐frequency or rare HPAs, which continues to increase with new reports. The genetic polymorphisms underlying these antigens have been characterized and in all cases but one, are single nucleotide polymorphisms (SNPs) in the coding sequence of the different membrane proteins where the HPA antigens are expressed. In contrast, the molecular basis of the HNA antigens are a little bit more heterogeneous. Within the 5 recognized HNA systems, the first alloantigens to be described, HNA‐1a and HNA‐1b differ in their coding sequence by 5 nucleotides, which result in 4 aminoacid changes in the protein sequence of the FcγRIIIb. Another alloantigen, the HNA‐2a, has been identified on the CD177 glycoprotein but cannot be genotyped, despite knowing the corresponding coding sequence. The HNA‐2a negative phenotype is due to splicing defects in the mRNA and no mutations have been detected in the gene sequence. The remaining HNA alloantigens result from single nucleotide changes, including the clinically relevant HNA‐3a, which has recently been characterized as the result of a single base change in the choline transporter‐like protein (CTL) 2 gene.Currently, genotyping methods are routinely applied to HPA and HNA testing in all laboratories performing platelet and granulocyte immunohematology studies. Many different techniques have been developed for typing these polymorphisms, including PCR with sequence‐specific primers, which is still the most widely used, 5′‐nuclease assays and high‐throughput SNP typing platforms. These latter ones are based on specific hybridization to oligonucleotide probes immobilized in a solid suport, usually a glass slide or beads. Among these, the newest platform developed for HPA typing, ID‐HPA, is based on the xMAP® technology and the beads are analyzed with the Luminex 100/200™ System, which is a robust and convenient system, already implemented in most HLA laboratories. Interestingly, the same technology can recently be used for the detection of HPA and HNA antibodies, which offers the possibility of multiple applications with a unique platform. These features may foster the introduction of such flexible assay systems in the near future. In any case, already at present, different high‐throughput genotyping platforms analyzing HPA polymorphisms are already being used for large‐scale blood or platelet apheresis donor typing, and further automation will probably lead to their final implementation in centralized National Blood Centers, facilitating the provision of antigen‐negative platelets for alloimmunized patients.Finally, it is worth to remark the novel strategies that have been recently developed for the non‐invasive fetal HPA‐1 typing from the maternal plasma DNA. These highly sensitive approaches may be applied in the future to other clinically significant HPA specificities, and will represent a major improvement in the management of pregnancies at risk of NAIT.

Detection of human platelet antigen‐1a alloantibodies in cases of fetomaternal alloimmune thrombocytopenia using recombinant β3 integrin fragments coupled to fluorescently labeled beads

Testing for alloantibodies against human platelet antigens (HPAs) is essential for the clinical diagnosis of fetomaternal alloimmune thrombocytopenia (FMAIT), posttransfusion purpura, and platelet (PLT) refractoriness. Most of the methods currently used for HPA alloantibody detection rely on the availability of panels of HPA-typed PLTs and some rely on validated monoclonal antibodies (MoAbs) against the PLT glycoproteins. Recombinant β3 integrins displaying the HPA-1a (rHPA-1a) or HPA-1b (rHPA-1b) epitopes have been produced as an alternative source of antigen. The suitability of these integrin fragments was evaluated for the development of an HPA-1a alloantibody screening assay, using Luminex xMAP technology. A 3-plex bead assay was developed by coupling biotinylated rHPA-1a, rHPA-1b, and recombinant glycoprotein VI to LumAvidin microspheres. Forty patient samples referred for FMAIT diagnostic testing, which were previously screened by the MoAb-specific immobilization of PLT antigens (MAIPA) assay, were used to assess the assay. The rHPA-1a- and rHPA-1b-coupled beads were able to detect HPA-1a and HPA-1b alloantibodies in all patient samples tested that were previously confirmed to contain HPA-1-specific antibodies. Furthermore, HLA Class I antibodies did not cross-react with the coupled beads. The 3-plex bead assay can be used to detect HPA-1a antibodies with sufficient specificity and sensitivity for use in the clinical setting of FMAIT. The development of other recombinant integrin fragments with the use of Luminex xMAP technology may assist in providing more rapid HPA antibody detection, enabling prompt diagnosis of alloimmune PLT disorders.

Thrombocytopenia in the Intensive Care Unit Patient

The many comorbidities in the severely ill patient also make thrombocytopenia very common (∼40%) in intensive care unit patients. The risk of bleeding is high with severe thrombocytopenia and is enhanced in intensive care patients with mild or moderately low platelet counts when additional factors are present that interfere with normal hemostatic mechanisms (eg, platelet function defects, hyperfibrinolysis, invasive procedures, or catheters). Even if not associated with bleeding, low platelet counts often influence patient management and may prompt physicians to withhold or delay necessary invasive interventions, reduce the intensity of anticoagulation, order prophylactic platelet transfusion, or change anticoagulants due to fear of heparin-induced thrombocytopenia. One approach to identify potential causes of thrombocytopenia that require specific interventions is to consider the dynamics of platelet count changes. The relative decrease in platelet counts within the first 3 to 4 days after major surgery is informative about the magnitude of the trauma or blood loss, whereas the dynamic of the platelet count course thereafter shows whether or not the physiologic compensatory mechanisms are working. A slow and gradual fall in platelet counts developing over 5 to 7 days is more likely to be caused by consumptive coagulopathy or bone marrow failure, whereas any abrupt decrease (within 1-2 days) in platelet counts manifesting after an initial increase in platelet counts approximately 1 to 2 weeks after surgery strongly suggests immunologic causes, including heparin-induced thrombocytopenia, other drug-induced immune thrombocytopenia, and posttransfusion purpura.

Universal DNA Arrays for High-Throughput SNP Detection of Human Platelet Antigens (HPA)

AbstractAbstract 4403 Background:DNA analysis of human platelet antigens is known to improve the diagnosis and treatment of neonatal alloimmune thrombocytopenia and minimize the risks of post transfusion purpura and refractoriness to random donor platelet therapy. The availability of reliable molecular platforms such as HPA eMAP assays analyzing HPA-1, -2, -3, -4, -5, -6, -7, -8, -9, -11, and -15, are routinely used and eliminate the need for reference sera or large platelet volumes. New generations of HPA assays (HPA eMAP-S Beadchip™ Kit) described here are developed by identification of specific allele in solution and detection of targets by a generic probe array called the Universal BeadChip™ by imaging, HPA-13 was also incorporated in the panel. These assays are easily automatable due to fewer protocol steps. Methods:The BioArray Solutions HPA eMAP-S BeadChip Kit uses the novel Elongation Mediated Multiplexed Analysis of Polymorphisms in Solution (eMAP-S) technology (Lin, X., et al., 2010) to identify the presence or absence of the selected alleles associated with a given phenotype. We amplified 10 DNA fragments covering 12 allele variants which are associated with 12 platelet antigens by using multiplex PCR and HPA gene specific primer pairs. PCR was followed by multiplex allele specific primer extension (ASPE) and Beadchip detection by using the AIS-400 Array Imaging System. To evaluate the overall performance of HPA eMAP-S BeadChip™ Kits, a total 552 clinical samples with known HPA eMAP phenotype and/or serology were analyzed to determine polymorphisms associated with common human platelet antigen (HPA-1, -2, -3, -5, -15) and less common human platelet antigen (HPA-4, -6, -7, -8, -9, -11, -13). For internal study, 176 clinical samples were analyzed at BioArray Solutions (BAS). For the external study, 186 and 190 (total 376) donor samples were tested at two sites and analyzed by BASIS™ software. Phenotype results obtained from the HPA eMAP-S Beadchip™ Kit during the studies were compared to the data generated from the HPA eMAP BeadChip™ Kit and/or serology. Results:For internal and external studies, the concordance between the HPA eMAP-S Beadchip™ kit and HPA eMAP Beadchip™ kit was 99.99%. The discordant call (0.01%) on HPA-1b antigen was investigated. One HPA-1 discordant sample was sent out for sequencing and re-analyzed by using increased amount of DNA from DNA precipitation. The result from sequencing is consistent with the result from HPA eMAP Beadchip™ Kit. The HPA eMAP-S result from higher concentration DNA is also consistent with the result of sequencing. The discordance in the original HPA eMAP-S assay was attributed to less DNA input in the multiplex PCR, which will be corrected in the Package Insert of HPA eMAP-S Beadchip™ Kit. Conclusion:HPA eMAP-S Beadchip™ Kit could be used reliably for the determination of human platelet antigens using DNA analysis. Disclosures:Hashmi:Immucor: Employment.

Molecular insight into human platelet antigens: structural and evolutionary conservation analyses offer new perspective to immunogenic disorders

BACKGROUND:Human platelet antigens (HPAs) are polymorphisms in platelet membrane glycoproteins (GPs) that can stimulate production of alloantibodies once exposed to foreign platelets (PLTs) with different HPAs. These antibodies can cause neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PLT transfusion refractoriness. Most HPAs are localized on the main PLT receptors: 1) integrin αIIbβ3, known as the fibrinogen receptor; 2) the GPIb-IX-V complex that functions as the receptor for von Willebrand factor; and 3) integrin α2β1, which functions as the collagen receptor.STUDY DESIGN AND METHODS:We analyzed the structural location and the evolutionary conservation of the residues associated with the HPAs to characterize the features that induce immunologic responses but do not cause inherited diseases.RESULTS:We found that all HPAs reside in positions located on the protein surface, apart from the ligand-binding site, and are evolutionary variable.CONCLUSION:Disease-causing mutations often reside in highly conserved and buried positions. In contrast, the HPAs affect residues on the protein surface that were not conserved throughout evolution; this explains their naive effect on the protein function. Nonetheless, the HPAs involve substitutions of solvent-exposed positions that lead to altered interfaces on the surface of the protein and might present epitopes foreign to the immune system.

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.

Génotypage en immunologie plaquettaire : quand ? Comment ? Limites

Platelet alloantigens named Human Platelet Antigens (HPA) are involved in immune conflicts such as post-transfusion purpura, platelet transfusion refractoriness and neonatal alloimmune thrombocytopenia. Biological diagnosis relies on: (1) detection of alloantibodies; (2) identification of the alloantigen involved in the immune conflict. Since the development of methods based on molecular biology, platelet genotyping is preferred to phenotyping. Today, most of the Platelet Immunology Units use PCR-RFLP or PCR-SSP, and few use real-time PCR. An increasing amount of commercial kits based on new technologies is now available, for example microarrays, fluorescent or coloured microbeads, or a combination of both technologies. However, an increasing number of polymorphisms have been discovered that are responsible for erroneous platelet genotypings. Consequently, it would be of interest to develop alternative technologies based on antigen/antibody interaction instead of DNA.