Rapid Duffy Genotyping in Sickle Cell Disease Patients Using PCR-SSP Is a Valuable Tool for Blood Component Selection.

Abstract

Background: Sickle cell disease (SCD) patients are frequently transfused and, finding adequate and safe blood component for them has a great impact in transfusion services and in patients clinical follow-up. DNA genotyping for RBC antigens is a tool for the management of these patients to overcome the limitations of hemagglutination assays. However, the polymerase chain reaction (PCR) by restriction fragment length polymorphism (RFLP) is time consuming. The aim of this study was to develop a rapid PCR specific sequence primer (SSP) approach for Duffy genotyping in SCD patients.STUDY DESIGN AND METHODS: Blood samples from 91 SCD patients were studied by hemagglutination and by PCR-SSP for Duffy system. All samples typed as Fy(b-) were submitted to adsorption and elution techniques to detect the weak expression of Fyb antigen. DNA was extracted by a commercial DNA extraction kit (QIAamp DNA, QIAGEN, Germany). Detection of FY*A, FY*B, FY*B-33, FY*B-265T and FY*B-298A were performed using specific primers by PCR-SSP. DNA amplification was carried out in a final volume of 20μL with identical reaction mixture and cycling conditions as follows: 0.2μg of genomic DNA, 0.2mM of each dNTP, 5% of glycerol, 1.5 units of platinum Taq polymerase (Invitrogen, Brazil) in the buffer supplied, 5.0mM of MgCl2 and 10.0 pmoles of each primer. PCR fragments were separated for 90 minutes at 100V using 0.5μg.mL-1 ethidium bromide-stained 2.0% agarose gel and visualized in a UV light apparatus (Eagle Eye II, EUA).Results: Table 1 shows the results of Duffy phenotyping, Duffy genotyping and gene frequencies in SCD patients The most common phenotype found in our study was Fy(a–b+), 35.2%. In three samples (3.3%) we detected weak Fyb antigen and identified FY*B-265T alelle. All samples phenotyped as Fy(a–b–) genotyped as (FYB-33/FYB-33).CONCLUSION: PCR-SSP is a method that contributes for easily and accurate Duffy genotyping for SCD patients. In our study, all SCD patients with Fy(a–b–) can be safely transfused with Fy(b+) red blood cell components with no risk for developing anti-Fyb saving rare Fy(a–b–) units.Table 1PhenotypeGenotypenGenotype FrequencyFy(a+b+)FYA/FYB160.176Fy(a–b+)FYB/FYB230.253FYB/FYB-265T90.099Fy(a+b–)FYA/FYA180.198FYA/FYB-3340.044Fy(a–b–)FYB-33/FYB-33210.231TOTAL911.00