The dilemma of screening for antibodies against low‐frequency human platelet antigens (2005)

In this issue, Kroll and colleagues1 describe the immunohematologic investigations of a number of unique cases of neonatal alloimmune thrombocytopenia (NAIT) caused by alloantibodies against low-frequency human platelet (PLT) antigens (HPAs). Why does this warrant an editorial? The discovery of low-frequency PLT-specific alloantigens is not new, and alloantibodies against these antigens are known to cause severe thrombocytopenia in the neonate. The studies reported here, however, are important for the translation of the initial discoveries to better clinical care. The majority of low-frequency HPAs have been reported in the past decade but the PLT immunology community has struggled to translate these discoveries into improved care of families afflicted by this serious condition. The first PLT-specific alloantigen Zwa (PlA1, now HPA-1a) was discovered some 40 years ago. The definition of the molecular basis of HPAs had to await the advent of the polymerase chain reaction, which was used to define the single-nucleotide polymorphisms (SNPs) underlying HPAs.2 For a long time, the PLT immunologist was hampered by the technical complexities of HPA antibody detection (PLTs also express HLA Class I and 25% of multiparous women have HLA Class I antibodies) and of HPA antigen typing. The latter has been resolved now that the genetic basis of 16 of the 18 HPAs has been resolved.3,4 The former was greatly improved by a key discovery from the same German team in 1987 with the description of the monoclonal antibody immobilization of PLT antigens (MAIPA) assay.5 The MAIPA assay is a versatile tool for the sensitive and specific detection of HPA antibodies and was used by several laboratories to define novel HPAs, especially the low-frequency ones. Investigating maternal serum samples for the presence of antibodies against low-frequency HPAs, however, requires paternal PLTs. This is impractical for two reasons. First, ABO group mismatch between parents complicates serology tests in which paternal PLTs are used as a panel cell. Second, paternal PLTs are often not available; that is, the National Blood Service Platelet Immunology laboratories in the United Kingdom receive paternal blood samples in approximately 50 percent of referrals for NAIT. The lack of paternal PLTs prevents completion of the investigation of referred cases of neonatal thrombocytopenia, and many reference laboratories report accordingly, warning clinicians that the investigations are incomplete and that there is a small chance of alloimmunization against a low-frequency alloantigen. This is unsatisfactory, but most laboratories have found it practically impossible to have collections of “rare” cryopreserved panel PLTs, because the allele frequencies are very low. This is where the authors made their next critical contribution. They developed two novel key reagents: 1) B-lymphoblastoid cell lines derived from donors heterozygous for the low-frequency alleles and, more importantly, 2) a panel of Chinese hamster ovary (CHO) cells that have been stably transfected with the rare alleles of the ITGB3 gene, encoding the β3-integrin or PLT glycoprotein (GP) IIIa. By cotransfection with the ITGA2B gene encoding αIIb or PLT GPIIb, cells have been created that faithfully express the rare GPIIIa alleles. The efficacy of this approach has been proven by using these novel cellular reagents as surrogate panel cells in MAIPA with serum samples known to be positive for alloantibodies against rare HPAs. The question then arises when to use these unique CHO reagents. NAIT referrals from Caucasoid cases are generally tested for HPA-1, -2, -3, -5, and -15 alloantibodies, and positive results are obtained in approximately 20 percent of referrals. Where does this leave the PLT immunologist with the testing for antibodies against low-frequency HPAs? It would be wrong to advocate that the 80 percent with a negative HPA antibody test all need screening for “rare” alloantibodies. The decision for further investigations needs to be informed by dialogue with the referring clinician. Was this a typical case of alloimmune thrombocytopenia in a term and otherwise healthy neonate or have other causes, that is, intrauterine infection, intrauterine growth restriction, or prematurity, come to the fore? If the clinical case is typical, then laboratories are advised to pursue additional investigations with the same rigor as our colleagues in red cell immunohematology. But screening large panels of recovered CHO cells expressing the different low-frequency HPAs is not an option. First, DNA of the neonate should be tested for all SNPs known to be at the basis of low-frequency HPAs. This becomes increasingly simple with fourth-generation multiplex PCR technologies combined with SNP detection arrays.6 If a low-frequency SNP is uncovered, then the corresponding CHO cells carrying the cognate alloantigen can be used as panel cells in the MAIPA with the maternal serum. It may be good practice in such cases to request a 4 to 6 weeks’ postdelivery serum sample from the mother because it has been well documented that the rate of false negatives is reduced when postdelivery samples are included. For this to happen, reagents must be freely exchanged between PLT immunology laboratories. This proposal is a logical extension of the highly regarded principle in the world of immunohematology, which respects the principle of altruism and freely donated blood that is at the basis of most of our blood supply organizations.7 The use of reference DNA samples for validation of genotyping is good practice and including the unique panel of CHO cells will benefit the care of families in whom the health of their offspring may be jeopardized by alloimmunization against PLTs. What about the future? All low-frequency alleles should be included, and a CHO cell transfected with an engineered ITGB3 gene carrying all rare SNPs is worth further study. We also need to carefully investigate all clinical cases with a classical presentation of NAIT with a negative HPA antibody screen. In the past, the majority of HPAs have been discovered by this route. With completion of the human genome project8 and planned efforts to define common polymorphisms in all human genes, there will be a new era for the investigative PLT immunologist by applying the principle of “inverted immunology” to the discovery of novel human PLT alloantigens.