Standardization of a multiplex assay to identify weak D types in a mixed-race Brazilian population.

RH allele variability is caused by several types of variants, resulting in altered RhD and RhCE phenotypes. Most of the weak D phenotypes in European-derived populations are weak D types 1, 2, or 3, which are not involved in alloimmunization episodes. However, the Brazilian population is racially diverse, and the accuracy of molecular and serologic tests developed in recent years has allowed for the identification of other RH variants, that are common in the Brazilian population, such as weak D type 38 or weak partial 11, the latter involved in alloimmunization cases. Furthermore, patients with these two weak D variants must be transfused with D- red blood cell units, as do patients with weak D type 4 or DAR, which are also common D variants in Brazil. Weak D type 38 and weak partial 11 can be serologically misclassified as weak D types 1, 2, or 3 in patients, based on European experience, or as D- in donors. Additionally, pregnant women may unnecessarily be identified as requiring Rh immune globulin. RhCE phenotypes are reliable indicators of RhD variants. For individuals with the Dce phenotype, the preferred approach is to specifically search for RHD*DAR. However, when encountering DCe or DcE phenotypes, we currently lack a developed method that assists us in rapidly identifying and determining the appropriate course of action for the patient or pregnant woman. Two multiplex assays were proposed: one for the identification of RHD*weak partial 11, RHD*weak D type 38, and RHD*weak D type 3 and another for RHD*weak D type 2 and RHD*weak D type 5. The multiplex assays were considered valid if the obtained results were equivalent to those obtained from sequencing. Expected results were obtained for all tested samples. The proposed multiplex allele-specific polymerase chain reaction assays can be used in the molecular investigation of women of childbearing age, patients, and blood donors presenting a weak D phenotype with DCe or DcE haplotypes in a mixed-race population, such as Brazil.

Stability of complete blood count in different storage conditions using the ABX PENTRA 60 analyzer.

INTRODUCTION: Sample stability is essential to obtain reliable results in the clinical laboratory. This study was conducted to investigate the reliability of hematological parameters using ABX Pentra 60 in samples stored for up to 72 hours at different temperatures. METHODS: A total of 651 blood samples were analyzed at different analysis times: 2, 24, 48, and 72 hours and forms and storage: room temperature (25°C) and at 4°C. The imprecision of the results was evaluated by the analytical coefficient of variation (CVa%) obtained by the typical error (TE) and Kruskal-Wallis analysis, to compare the averages. The reliability of the results was evaluated by the CVa (%) within the maximum allowable analytical variation and by the difference of means of the results in relation to the baseline sample (2 hours). RESULTS: Red blood count, hemoglobin, and MCH parameters showed stability up to 72 hours at room temperature and at 4°C. The other complete blood count parameters showed imprecision results emitted by the ABX Pentra 60 from 24 hours of sample storage, independent of the storage temperature. In addition, there were significant oscillations in the mean values, particularly for the samples stored at room temperature, with the exception of platelet parameters that exhibited mean changes also at 4°C. CONCLUSION: The results demonstrate the importance of the clinical analyst's knowledge about the behavior of the CBC parameters over time under different storage conditions, and mainly the imprecision of the hematological equipment used, for the suitable interpretation of the results.

RHD alleles in Brazilian blood donors with weak D or D-negative phenotypes.

The RHD gene is highly polymorphic and the existence of a large number of alleles results in RhD variant phenotypes. RHD genotyping has been used to distinguish normal D antigen from D variants due to limitations of serologic methods. The purpose of this study was to determine the phenotypic frequency of RhD and RhCE antigens and to investigate the RHD alleles present in samples with the weak D or D- phenotypes from Brazilian blood donors. A total of 2007 donors were phenotyped for D, C, c, E and e antigens. Samples phenotyped as D- were genotyped by polymerase chain reaction-sequence specific primers, and exon 10 and intron 4 of the RHD gene were analysed. D- samples containing the RHD gene or samples considered weak D were further characterised using genotyping platform or nucleotide sequencing. Using serologic methods we found that 87.3% of the donors were D+, 11.9% D- and 0.8% weak D. The frequency of RHD gene in D- individuals was 9.2%. Five RHD alleles from phenotypically D- donors were characterised in six molecular backgrounds: RHDΨ, RHD-CE-D(s), RHD-CE-(2-9)-D, RHD/RHDΨ, RHDΨ/RHD-CE-D(s) and RHD-CE(2)-D. The most common weak D antigens types found were 1, 3, 4.0/4.1 and 4.2, whereas the most prevalent weak D type was 4.2 (or DAR). The RHD genotyping proved to be a necessary tool to characterise RHD alleles in donors phenotyped as D- or weak D to increase the transfusion safety in highly racial mixed population.