Paul Metcalfe

Nomenclature of human platelet antigens

1 Division of Haematology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK 2 Department of Haematology, University of Cambridge and National Blood Service Cambridge, Cambridge, UK 3 Platelet Unit, INTS, Paris, France 4 Blood Research Institute, the Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin, USA 5 Finnish Red Cross Blood Transfusion Service, Helsinki, Finland 6 Sanquin Research at CLB, Amsterdam, the Netherlands 7 Saitama Red Cross Blood Center, Hidaka-Shi, Saitama, Japan 8 McMaster University Medical Centre, Hamilton, Ontario, Canada 9 Department of Transfusion Medicine, University of Rostock, Rostock, Germany 10 Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany * Chairman of the ISBT platelet working party

Activation during preparation of therapeutic platelets affects deterioration during storage : a comparative flow cytometric study of different production methods

Three different separation methods, all using centrifugation, are routinely used to prepare therapeutic platelet concentrates from human donor blood. Platelet concentrates derived from platelet‐rich plasma (PRP‐PC), buffy coat (BC‐PC) and apheresis (AP‐PC) were investigated at the end of production, and over an 8 d storage period. Change in platelet surface markers were measured by flow cytometry, using fluorescein‐conjugated antibodies to fibrinogen, P‐selectin (CD62P), GPIIb–IIIa (CD41), GPIbα (CD42b) and GPV (CD42d), and fluorescein‐conjugated Annexin V was used to measure expression of anionic phospholipid.

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Detection of Gov system antibodies by MAIPA reveals an immunogenicity similar to the HPA-5 alloantigens

The glycosylphosphatidylinositol‐linked platelet protein CD109 carries the biallelic alloantigen system Gov. There is limited information on the incidence of Gov alloantibodies in neonatal alloimmune thrombocytopenia (NAITP), post‐transfusion purpura (PTP) and platelet refractoriness. We adapted the monoclonal antibody‐specific immobilization of platelet antigens (MAIPA) assay to the detection of Gov antibodies and determined their incidence in 605 archived samples (112 with HPA antibodies) referred for the aforementioned conditions. Here, we show that CD109 expression was reduced upon platelet storage in saline or by cryopreservation, but was stable when stored as whole blood or therapeutic platelet concentrate. Fourteen of the 605 samples contained Gov alloantibodies (anti‐Gova, n = 10; anti‐Govb, n = 4), with the majority in platelet refractoriness (n = 9) and, of the remaining five, four in NAITP and one in PTP. In seven cases, no other HPA antibodies were detected, three being NAITP cases. The incidence of Gov antibodies was significantly lower than HPA‐1 system antibodies (n = 87), but equalled the number of HPA‐5 system antibodies (n = 14) and outnumbered HPA‐2 and ‐3 system antibodies (10 altogether).

Genotyping for platelet-specific antigens: techniques for the detection of single nucleotide polymorphisms

Accurate typing of patients for platelet‐specific (human platelet) antigens (HPA) is required in several different clinical situations, and blood services need to maintain panels of HPA‐typed apheresis platelet donors and whole‐blood donors to support HPA alloimmunized patients. Six clinically relevant HPA alloantigen systems have been described and, in addition, a significant number of HPA alloantigens with a highly skewed allele frequency or of very low immunogenicity have been reported. Certain well‐characterized biallelic systems such as Gov have not as yet been included in the HPA nomenclature but are included in this review. Biochemical studies have identified the platelet membrane proteins on which the HPA antigens are localized. Cloning of the genes encoding these proteins and the realization that there is adequate mRNA in fresh platelets has led to identification of the molecular basis of HPA antigens over the last decade. All but one of the biallelic platelet‐specific alloantigen systems are based on a single nucleotide polymorphism in the DNA sequence, corresponding to a single amino acid substitution in the encoded primary protein sequence. The discovery of the genetic basis of the alloantigens has allowed the development of polymerase chain reaction‐based techniques for HPA genotyping using genomic DNA. The genetic basis of the HPA alloantigens, the most commonly used genome typing techniques and their pitfalls, and future developments, are discussed in this review.

Microarray-based genotyping for blood groups: comparison of gene array and 5'-nuclease assay techniques with human platelet antigen as a model.

BACKGROUND: Most blood group alloantigens specific for red cells and platelets (PLTs) are based on single‐nucleotide polymorphisms (SNPs) in genes encoding relevant membrane proteins.

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

Background  The monoclonal antibody‐specific immobilization of platelet antigens (MAIPA) assay is the cornerstone technique for the detection and identification of human platelet antigen (HPA) antibodies. However, the original technique described by Kiefel and colleagues requires approximately 8 h adding to diagnostic delay. Moreover, proficiency exercises indicate that there are substantial variations in the MAIPA protocol, and that these may account for interlaboratory differences in sensitivity and specificity.

Interlaboratory variation in the detection of clinically significant alloantibodies against human platelet alloantigens

This report describes the results of eight workshop exercises which were designed to test the proficiency of laboratories in the detection of antibodies to human platelet antigens (HPA). Detection of the most clinically significant alloantibody, anti‐HPA‐1a, is adequate. However, despite improvements in consistency of test results between laboratories over the last 3 years, there is still a high probability that clinically significant antibodies against other HPA alloantigens will not be detected.

HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia

BACKGROUND: The antenatal management of fetomaternal alloimmune thrombocytopenia (FMAIT) due to HPA‐1a antibodies remains controversial, and a test identifying pregnancies that do not require therapy would be of clinical value.

Platelet antigens and antibody detection

Platelets are anucleate blood cells, which play an importantpart in haemostasis and maintenance of the integrity ofblood-vessel endothelium. Interaction of platelet glycopro-teins (GPs) with extracellular matrix proteins is critical tothis function.The original impetus for investigating the alloantigensfound on the GPs of the platelet membrane came from clinicalobservations of pathological conditions involving antibodiesagainst platelet-specific antigens. Since then, dramatic pro-gress has been made in elucidating the biochemical nature,function and molecular biology of platelet membrane GPs.The structure of many of the genes controlling platelet GPshave been determined and further characterization of plateletpolymorphisms has been possible because of advances inmolecular biology and monoclonal antibody techniques.These techniques have also resulted in a significant increasein the reliability of detection and identification of plateletantibodies and antigens in clinical laboratories.