Tion of Plasma Volume Changes during Marathon Rapid Real-time Pcr Detection of Hp Del Directly from Diluted Blood Samples Letters to the Editor

Anhaptoglobinemic patients have been reported to experience severe anaphylactic reactions to transfusions due to the production of antihaptoglobin (anti-HP) antibodies (1, 2 ). Anhaptoglobinemia in patients homozygous for HP , which is a deletion of an approximately 28-kb segment of chromosome 16 extending from the promoter region of the HP (haptoglobin) gene to exon 5 of HPR (haptoglobin-related protein), has been adequately characterized only recently (1 ). Use of a simple duplex PCR method has detected the HP del allele in East and Southeast Asian populations at frequencies of 1%–3% but this allele has not been detected in African, West and South Asian, and European populations (1, 3, 4 ). Thus, diagnosing HP del homozygosity prior to blood transfusion or the infusion of blood components into individuals from East and Southeast Asian populations is effective for preventing anaphylactoid shock due to anti-HP antibodies. We have developed a simple method that uses a 5 nuclease real-time PCR assay (TaqMan; Applied Biosystems) to detect the HP del allele without having to isolate genomic DNA. The ethics committee of Kurume University School of Medicine approved this study protocol. To distinguish alleles, we performed real-time PCR assays that detect the 2 regions that encompass the HP del breakpoint and the 5 region of HP exon 1, which is deleted in HP . The 20L PCR reaction contained 200 mol/L deoxynucleoside triphosphates, 1 L of template (diluted blood or genomic DNA), 0.5 U of Ex TaqHS with its buffer (Takara), and the following primers and TaqMan probes (see Fig. 1 legend for sequences): Hp5 -F and -R primers (450 nmol/L); Hp5 –TaqMan probe (125 nmol/L) for detecting the 5 region of HP; Hpdel-F and -R primers (900 nmol/L); and Hpdel– TaqMan probe (250 nmol/L) for detecting HP . The PCR temperature profile was 95 °C for 30 s, followed by 50 cycles of denaturing at 95 °C for 5 s and annealing and extension at 60 °C for 30 s. All oligonucleotides were synthesized by Biosearch Technologies. We monitored amplification progress by monitoring the fluorescence at the end of each cycle with an Mx3000P instrument (Stratagene) with excitation and emission wavelengths of 492 and 516 nm (FAM), and 585 and 610 nm (CAL Fluor Red 610). With genomic DNA (5 ng/ L) as a template, we used dual-color scatter plots to distinguish individuals previously determined to have the HP/HP, HP/HP , and HP /HP del genotypes. Samples with the HP/HP genotype had little FAM fluorescence and plotted along the x-axis, HP /HP samples had little CAL Fluor Red 610 fluorescence and plotted along the y-axis, and HP/HP del samples were located between the homozygote samples in the plot (Fig. 1). To the TaqMan real-time PCR mixture, we directly added 1 L of samples diluted 100-fold with PCR-grade water (previously frozen samples of buffy coat from 47 Indonesians from Surabaya or a freshly drawn blood sample from 1 Japanese individual from Fukuoka). Blood was collected in EDTA-containing tubes (Indonesian and Japanese samples) and in a heparin-containing tube (the Japanese sample). The results from 2 independent experiments showed no discrepancies. In addition, the results obtained with the present TaqMan real-time PCR method were fully concordant with those obtained with a previously described PCR method for the same individuals (i.e., 46 HP/HP individuals and 2 Indonesians with HP/HP ; Fig. 1) (1 ). We previously had collected blood samples from 105 Indonesian individuals from Surabaya and had not found the HP del allele in 58 of these individuals (3 ); however, in the present study we did find 2 HP alleles among the remaining 47 individuals in this population sample. Thus, the HP del allele is also present in Southeast Asian populaLetters to the Editor