W. H. Ouwehand

Management and outcome of 200 cases of fetomaternal alloimmune thrombocytopenia

BACKGROUND: Fetomaternal alloimmune thrombocytopenia (FMAIT) is the commonest cause of severe thrombocytopenia in term neonates but its management remains controversial.

Provision of platelet support for fetuses and neonates affected by severe fetomaternal alloimmune thrombocytopenia

Severe fetomaternal alloimmune thrombocytopenia requires urgent treatment with compatible platelet concentrates. As prompt treatment is sometimes delayed owing to the unavailability of compatible platelets, we established an accredited platelet donor panel to provide effective and timely transfusion support for fetal and neonatal therapy. After a mass screening programme of over 60 000 blood donations, 45 HPA‐1a‐negative donors with no antibodies to HPA, HLA, red cell antigens and granulocytes/lymphocytes, and with low titre anti‐A and/or ‐B were accredited. All accredited donors were fully genotyped for HPA‐1, ‐2, ‐3 and ‐5 by PCR‐SSP. Ninety‐one per cent of the accredited donors were also negative for HPA‐5b.

HPA Genotyping by PCR–SSP: Report of 4 Exercises

Background and Objectives: Polymerase chain reaction with sequence specific primers (PCR–SSP) is widely used for the determination of the alleles encoding the human platelet antigens (HPA)–1 to 5. In order to evaluate and improve performance with this technique, four exercises were organised during 1996–1998. Materials and Methods: Coded DNA samples were distributed from the National Institute for Biological Standards and Control (NIBSC) as follows: exercise one, 18 samples; two, 12 samples; three, 6 samples, and four, 4 samples. Results: Performance improved over the four exercises following the adoption of a consensus protocol and the re–design of one primer. The percentage of incorrect results in each exercise was as follows: exercise one, 9%; two 3.2%, three, 0.8%, and four, 0.3%. Conclusion: The modified PCR–SSP protocol is a reliable method for genotyping HPA–1 to 5 in reference laboratories. DNA–based HPA genotyping has an important role in platelet immunology and further exercises will be included in the bi–annual platelet immunology exercises organised by NIBSC.

Molecular biology for platelet alloantigen typing

SUMMARY. Hitherto, full investigation of patients with alloimmunization to platelet‐specific antigens has been difficult due to the limited availability of both typing reagents and panels of typed platelets. Following recent advances in the understanding of the molecular and genetic basis of platelet alloantigens, it is now possible to genotype individuals for the alleles coding for the epitopes of four platelet antigen systems (HPA‐1–4). This is based on the finding that the two alleles differ by oniy a single base pair substitution, resulting in one amino acid difference in the relevant platelet glycoprotein. The technique involves amplification of the relevant segments of genomic DNA from any nucleated cell by the polymerase chain reaction, followed by restriction fragment length polymorphism analysis. The technique allows investigation of thrombocytopenic individuals and fetuses/neonates, and can be readily applied to large‐scale typing of platelet donors.

Sensitivity of the platelet immunofluorescence test (PIFT) and the MAIPA assay for the detection of platelet-reactive alloantibodies: a report on two U.K. National Platelet Workshop exercises.

SUMMARY. This report presents the results of two National Workshop exercises which were designed to evaluate interlaboratory and interassay variation in sensitivity of techniques used for the detection of platelet‐reactive alloantibodies. Most workshop participants used the platelet immunofluorescence test. Sensitivity of this assay was improved when fluorescence was measured by flow cytometer rather than by microscope. The MAIPA assay was found to be highly sensitive but required considerable technical expertise, and the choice of antiglycoprotein II/IIIa (CD61/41) monoclonal antibody was found to have a significant effect on its ability to detect anti‐HPA‐la.

Platelet Antibody Detection

In the past 40 years a plethora of publications has appeared in the literature, which describe methods to detect platelet antibodies in sera or bound to cells of patients in various clinical conditions. These methods can be broadly subdivided by the principle on which they are based into methods that measure: a. The functional effect that antibodies may have on platelets, such as the induction of aggregation and release or just the opposite, i.e. the inhibition of these processes. b. The immunologic effect that antibodies may have on platelets such as agglutination, complement fixation, complement dependent cytotoxicity or 51Cr-release, cell mediated cytotoxicity and immunophagocytosis. c. The binding of immunoglobulins and/or complement to platelets, either directly or indirectly.