Molecular RH blood group typing of serologically D-/CE+ donors: the use of a polymerase chain
Donatella Londero1, Mauro Fiorino, Valeria Miotti
1Responsible Immunohematology Laboratory, Department of Transfusion Medicine, AOU "S. Maria della Misericordia", Udine, Italy.
The known presence of RHD blood group alleles in apparently D– individuals who are positive for C or E antigens leads to an appropriate investigation for the RHD gene on the red blood cells (RBCs) of D– blood donors, thus preventing their RBCs from immunizing D– recipients. Ready-to-use polymerase chain reaction–sequence-specific primer (PCR-SSP) typing kits are available and allow single-sample results. The need to perform this testing on a large number of donors affiliated with the Transfusion Department of Udine (Northern Italy) led to the use of molecular genetic RH blood group typing with PCR-SSP test kits and DNA samples mixed in pools. From a population of 35,000 blood donors screened for D antigen by serologic typing, a total of 235 samples, distributed in pools of 5 DNA samples, were investigated. Positive results were reevaluated by opening the pools and retesting single samples. Validation of DNA-pool typing with commercial kits was done. Among 235 genotyped samples, 12 were found to be PCR positive (5.1%), exhibiting DEL genotype and RHD-CE-D hybrid alleles. Our data demonstrate that the use of a PCR-SSP commercial test kit with pooled samples is a helpful and valid method to correctly detect RHD alleles. As a consequence, we reclassified our donors as carriers of potentially immunogenic alleles.
The known presence of RHD blood group alleles in apparently D– individuals who are positive for C or E antigens leads to an appropriate investigation for the RHD gene on the red blood cells (RBCs) of D– blood donors, thus preventing their RBCs from immunizing D– recipients. Ready-to-use polymerase chain reaction–sequence-specific primer (PCR-SSP) typing kits are available and allow single-sample results. The need to perform this testing on a large number of donors affiliated with the Transfusion Department of Udine (Northern Italy) led to the use of molecular genetic RH blood group typing with PCR-SSP test kits and DNA samples mixed in pools. From a population of 35,000 blood donors screened for D antigen by serologic typing, a total of 235 samples, distributed in pools of 5 DNA samples, were investigated. Positive results were reevaluated by opening the pools and retesting single samples. Validation of DNA-pool typing with commercial kits was done. Among 235 genotyped samples, 12 were found to be PCR positive (5.1%), exhibiting DEL genotype and RHD-CE-D hybrid alleles. Our data demonstrate that the use of a PCR-SSP commercial test kit with pooled samples is a helpful and valid method to correctly detect RHD alleles. As a consequence, we reclassified our donors as carriers of potentially immunogenic alleles.
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