Real-time amplification of glyceraldehyde-3-phosphate dehydrogenase gene for quality control of leukopoor platelets.

BACKGROUND: Leukoreduction of blood products is crucial to prevent white blood cell (WBC)-associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time-consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest. STUDY DESIGN AND METHODS: Real-time polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was developed for quantifying WBCs in leukopoor platelets (LPPs). After normalization by the cell-free prefiltrated and postfiltrated plasma DNA, the relative copy number of GAPDH gene in the platelet (PLT) concentrate and its corresponding LPPs was calculated according to the equation of 2(-ΔΔCt) of which Ct is defined as the threshold cycle. The percentage and the number of WBCs that remained in LPPs were consequently determined. This method was compared to Nageotte hemocytometry and was validated by using serially diluted PLT concentrate and 10 pairs of PLT concentrate-LPP samples. RESULTS: Consistent with the removal of WBCs after filtration, the Ct values for the LPP samples were increased when compared to their corresponding PLT concentrate. As revealed by real-time PCR of GAPDH gene, there is a correlation between the calculated and theoretical WBC count in the serially diluted PLT concentrate (correlation coefficient, 0.9532). The WBC counts for the 10 LPP samples were comparable between Nageotte and real-time PCR method and were all below 3.3 × 10(6) WBCs/L. CONCLUSION: The real-time PCR method we report in this study is applicable for routine quality assurance during leukoreduction process.

Two prevalent h alleles in para-Bombay haplotypes among 250,000 Taiwanese.

Alpha(1,2)-fucosyltransferase catalyzes the transfer of fucose to the C-2 position of galactose on type II precursor substrate Gal beta1-4GlcNAc beta1-R. It plays an important biological role in the formation of H antigen, a precursor oligosaccharide for both A and B antigens on red blood cells. Aberration of alpha(1,2)-fucosyltransferase activity by gene mutations results in decreased synthesis of H antigen, leading to the para-Bombay phenotype. In this study, we collected about 250,000 blood samples in Taiwan during 5 yr and identified the subjects with para-Bombay phenotype. Then we analyzed the sequence of the alpha(1,2)-fucosyltransferase gene by direct sequencing and gene cloning methods, using the blood samples of 30 para-Bombay individuals and 30 control subjects who were randomly selected. The goals of this study were to search for new h alleles, to determine the h allele frequencies, and to test whether the sporadic theory is applicable in Taiwan. Six different h alleles (ha, 547-548 AG-del; hb, 880-881 TT-del; hc, R220C; hd, R220H; he, F174L; and hf, N327T) were observed. Two h alleles, he and hf, were newly discovered in Taiwan. The he allele has a nucleotide 522C>A point mutation, predicting the amino acid 174 substitution of Phe to Leu; the hf allele has missense mutation of nucleotide 980A>C, predicting the amino acid 327 substitution of Asn to Thr. Frequencies of the 6 alleles are ha 46.67%, hb 38.33%, hc 5.00%, hd 1.67%, he 3.33%, and hf 5.00%, respectively. These findings in the Taiwanese population confirm previous observations in other populations that the Bombay and para-Bombay phenotypes are due to diverse, sporadic, nonfunctional alleles, predominantly ha and hb, leading to H deficiency of red blood cells. In contrast to previous reports of non-prevalent associations of h alleles with para-Bombay phenotype, our results suggest a regional allele preference associated with para-Bombay individuals in Taiwan.