The highest Duffy (FYX) allele frequency ever reported for Scottish population: A cross-sectional study.

Background and Aim: The Duffy (FY) blood group system has six known antigens among which the Fya and Fyb are known as major antigens. Fyx phenotype forms as a result of two point mutations in the FYB allele leading to instability of Duffy protein and so reduction of Duffy antigen expression in the cells. This study aimed to investigate the FYX allele frequency in the Scottish population. Methods: The Duffy blood group system was serologically and molecularly investigated in 222 samples collected from donors of Aberdeen Regional Blood Transfusion Center (BTC). The haemagglutination and BeadChip microarray chemistry methods were used for phenotyping and genotyping. Confirmatory tests were also used to check the discrepant results. Results: In this study, the frequency of Duffy blood group phenotypes including Fya+, Fya+b+, and Fyb+ were 17.57%, 42.79%, and 39.64%, respectively. Furthermore, the frequency of FYA/FYA, FYA/FYB, and FYB/FYB genotypes was estimated to be 14.41%, 45.95%, and 39.64%, respectively, using the Bioarray method. In the present study, based on Duffy DNA sequencing results, 12 samples (5.41%) had just one FYX allele. Conclusion: The frequency of the FYX allele in this study was estimated to be 0.0270% which is more than the results reported so far.

Direct comparison between two quantitative assays in the measurement of maternal anti-HPA-1a antibody in neonatal alloimmune thrombocytopenia (NAIT).

BACKGROUND: Around 80% of severe neonatal alloimmune thrombocytopenia (NAIT) cases in the Caucasian population are due to anti-HPA-1a antibodies. However, the relationship between anti-HPA-1a antibody quantity and severity of NAIT has been subject to debate, possibly due to discrepant results between studies incorporating different assays (such as ELISA and MAIPA) and the number of samples. The aim of this study was to compare the level of maternal anti-HPA-1a antibodies in the same samples, using two different methods; quantitative ELISA and quantitative MAIPA. At the same time, the relationship between the maternal anti-HPA-1a antibody quantity and the newborn platelet count was examined. MATERIALS AND METHODS: Plasma samples were obtained from HPA-1a negative mothers giving birth to children with different platelet counts (i.e. NAIT vs normal platelet count). The antibody levels were quantified blindly in the respective assays using standards calibrated against the international standard NIBSC 03/152. RESULTS: A linear correlation was observed between quantitative MAIPA and quantitative ELISA results. However, there were many individual variations giving a mean ratio of disagreement between the quantitative ELISA and quantitative MAIPA of 2.84. Furthermore, regression analysis revealed a significant relation between anti-HPA-1a antibody quantity measured by MAIPA, but not ELISA, and the newborn platelet count. CONCLUSION: The results indicate that differences observed between various studies with regard to NAIT severity and anti-HPA-1a quantity could partly be due to different assays used.

The application of a new quantitative assay for the monitoring of integrin-associated protein CD47 on red blood cells during storage and comparison with the expression of CD47 and phosphatidylserine with flow cytometry.

BACKGROUND: After the introduction of universal leukoreduction, the role of factors other than white blood cells in red cell (RBC) storage lesion is attracting increasing attention. These include changes in the levels of CD47 and phosphatidylserine (PS) markers on RBCs during storage. The aim of this study was to monitor these changes with both flow cytometry (FACS) and a newly developed quantitative enzyme-linked immunosorbent assay (ELISA). STUDY DESIGN AND METHODS: A new quantitative ELISA (monoclonal antibody immobilization of RBC antigens [MAIRA]) was developed. The assay yielded consistent linear curves that enabled the measurement of CD47 expression on RBCs. In addition, FACS was used to measure both CD47 expression and PS on RBCs (n = 3 units) during storage (Days 4, 10, 24, and 31). RESULTS: A significant reduction in CD47 expression was observed both by MAIRA assay and by FACS by Days 24 and 31 (p < 0.01), and the correlation between the two assays was significant (p < 0.01). In addition, a significant increase in PS was observed by the same storage days with FACS (p < 0.01). CONCLUSION: The MAIRA assay appears to be suitable for the quantitative measurement of RBC markers during storage. Significant changes in CD47 and PS levels were observed during storage, which may have detrimental immunomodulatory and hemostatic effects on the transfused RBCs.

Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.

BACKGROUND: To assess the value of antenatal screening to detect neonatal alloimmune thrombocytopenia (NAIT) due to anti-HPA-1a, a prospective study was carried out to quantify the potential clinical benefits and determine whether screening would be cost-effective. STUDY DESIGN AND METHODS: An observational prospective controlled study was carried out on 26,506 pregnant women over 2 years. HPA-1a phenotyping was performed in the first trimester and women confirmed HPA-1a-negative were tested for anti-HPA-1a during pregnancy, at delivery, and 10 to 14 days after birth. Babies of HPA-1a-negative women were tested at delivery for thrombocytopenia and examined for signs of bleeding. Economic evaluation was undertaken on the basis of the data collected during the study. RESULTS: Twenty-five of 318 women (7.9%) had anti-HPA-1a detected for the first time. Eight women (43 per 100,000) gave birth to babies with NAIT, and 5 (27 per 100,000) had severe thrombocytopenia. Three babies had mild signs of bleeding, and no cases of intracranial hemorrhage (ICH) or fetal loss were detected. It is estimated that it would cost 60,596 pounds (98,771 US dollars) to detect a case of severe NAIT, where anti-HPA-1a has been identified for the first time, and 1,151,323 pounds (1,876,656 US dollars) to prevent a case of ICH, assuming that detection allowed successful intervention. CONCLUSIONS: Our data suggest that severe HPA-1a NAIT is underdiagnosed in the absence of routine antenatal screening. Serious bleeding complications and ICH, however, occur less frequently in first cases of NAIT than suspected from the literature, and the costs of screening and possible intervention must be balanced against the procedural risks.

The development of a quantitative ELISA for antibodies against human platelet antigen type 1a.

BACKGROUND: Severe neonatal alloimmune thrombocytopenia is often due to antibodies against human platelet antigen type 1a (HPA-1a). The aim of this study was to develop a quantitative ELISA for the measurement of antibodies against HPA-1a. STUDY DESIGN AND METHODS: HPA-1a glycoprotein (GP) IIb-IIIa was immobilized and mixed with recalcified anti-HPA-1a-positive plasma overnight at 4 degrees C. The beads were washed, the antibodies against HPA-1a were eluted, and the eluate pH level was promptly adjusted. The purified antibodies were dialyzed and used for the development of an ELISA incorporating HPA-1a-coated plates. RESULTS: Serial doubling dilutions of the purified antibodies resulted in consistent sigmoid standard curves with a sensitivity of 0.5 microg per mL. To determine the reproducibility of the ELISA, antibodies against HPA-1a in five plasma samples (Samples A-E) were measured at serial doubling dilutions in four separate assays. Three of the samples (Samples A-C) contained antibodies against HPA-1a. The mean amounts in microg per mL (+/- SD, percentage of CV) obtained in the four assays were as follows: Sample A, 133 (9.4, 7.1%); Sample B, 16.5 (1.7, 10%); and Sample C, 8 (0.8, 10%). The amounts in the two antibody-negative controls (Samples D and E) were consistently less than 0.2 microg per mL. CONCLUSION: Using immobilized HPA-1a1a, antibodies against HPA-1a has been purified, and a quick and simple quantitative assay has been developed.