Clinical Applications

Molecular genotyping is useful to determine the fetal risk for neonatal alloimmune thrombocytopenic purpura (NATP). In particular, molecular genotyping alleviates the need for platelets when the thrombocytopenia is so severe that it is not possible to obtain sufficient platelets for pheno-typing.

5.2.1 Identification of a Fetus at Risk for Alloimmune Thrombocytopenia

Analogous to the management of haemolytic disease of the newborn, platelet genotyping is used to determine the paternal antigen inherited by the fetus at risk for NATP. This analysis is relevant when the father is heterozygous for the corresponding allele. Investigators have used chorionic villus, or amniotic fluid-derived DNA to determine the fetal platelet genotype (McFarland et al 1991). These sources avoid the need for fetal blood sampling, which is associated with a higher risk of morbidity or mortality than amniocentesis alone. This risk has been reported to be as high as 10.2% in NATP (Kaplan et al 1994).

We recommend that the decision to perform molecular analysis for clinical use be based on the following:

• The mother has a history of delivery of an infant with NATP.

• The father is heterozygous for the allele or is unknown.

• The genotype is performed on parents and fetus.

• The parental serological phenotypes are determined.

• The ethnicity of the parents is obtained. This can help focus the test approach because some variants are restricted to certain populations (e.g. HPA-4b has a higher incidence in Japanese).

• For the compatible fetus, the genotype is repeated using short-term cultured amniocytes.

• The newborn's platelet antigen genotype is confirmed at birth (cord blood DNA analysis).

Antibodies to antigens in the HPA-1, -3 and -5 systems account for >98% of all confirmed cases of NATP (Warkentin and Smith 1997). However, it is important to recognize that often a population frequency for any given allele is derived from a relatively small number of individuals. The frequency has the potential to represent a bias if obtained from one ethnic group or a geographically isolated area. Therefore, rare alleles may be over-represented or may represent a 'founder allele'.

5.2.2 Genotyping When Appropriate Antisera are not Available

Antibodies to many platelet antigens are not easy to obtain. Furthermore, the platelet surface membrane has a considerable amount of IgG

(2,000-3,000 molecules of IgG/platelet), which complicates the use of serological techniques to determine the phenotype. Thus, serological analysis requires high-titred, well-characterized antisera and the use of 'third generation' techniques (Kiefel et al 1987). In 1989 (Newman et al 1989), the nucleotide polymorphism associated with HPA-1a/-1b was characterized using platelet mRNA and reverse transcription followed by direct PCR sequencing. Since that time, the nucleotide polymorphisms responsible for a number of other antigens expressed on various platelet membrane glycoproteins have been determined. Therefore, molecular techniques have provided the means to type for platelet antigens in the absence of antisera.

5.2.3 Determination of Genotypes in Thrombocytopenic Patients

Molecular genotyping complements serological investigations in the process of antibody identification. This is useful in cases of posttransfusion purpura because it is hard to obtain sufficient platelets for phenotyping. Anti-HPA-1a is the most common antibody associated with the disorder but other antigen systems have been implicated (Waters 1989).

5.2.4 Platelet Panels for Antibody Investigations

Molecular genotyping for platelet antigens can be used to select platelets for panels used in antibody identification. This is possible since inherited defects that cause platelet genotype/phenotype discrepancies are very rare (Skogen and Wang 1996).

5.2.5 Screening for Antigen-negative Platelet Donors

Blood centres should have HPA-1 a-negative donors available for treatment of post-transfusion purpura or for NATP. Microtitre assays have been used to identify these donors (Denomme et al 1996; Bessos et al 1996). It is advisable to confirm the phenotype by molecular genotyping.

Furthermore, the DNA can be archived to genotype for other antigens as the need arises; the donor does not need to be recalled for additional testing.

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