Molecular typing for blood group antigens within 40 min by direct polymerase chain reaction from plasma or serum

Abstract

Determining blood group antigens by serological methods may be unreliable in certain situations, such as in patients after chronic or massive transfusion. Red cell genotyping offers a complementary approach, but current methods may take much longer than conventional serological typing, limiting their utility in urgent situations. To narrow this gap, we devised a rapid method using direct polymerase chain reaction (PCR) amplification while avoiding the DNA extraction step. DNA was amplified by PCR directly from plasma or serum of blood donors followed by a melting curve analysis in a capillary rapid-cycle PCR assay. We evaluated the single nucleotide polymorphisms underlying the clinically relevant Fya, Fyb, Jka and Jkb antigens, with our analysis being completed within 40 min of receiving a plasma or serum sample. The positive predictive value was 100% and the negative predictive value at least 84%. Direct PCR with melting point analysis allowed faster red cell genotyping to predict blood group antigens than any previous molecular method. Our assay may be used as a screening tool with subsequent confirmatory testing, within the limitations of the false-negative rate. With fast turnaround times, the rapid-cycle PCR assay may eventually be developed and applied to red cell genotyping in the hospital setting.

Serological weak D phenotypes: a review and guidance for interpreting the RhD blood type using the RHD genotype

“…For patients requiring chronic transfusions, for example, sickle cell disease, thalassaemia and myelodysplastic syndrome, the results of once-in-a-lifetime RHD genotyping may not be available in time for the current transfusion, but would be available for future transfusions (Fasano & Chou, 2016; Chou et al , 2013; ). New methods for blood group genotyping, for example, direct polymerase chain reaction (PCR) amplification without DNA extraction, offer the promise of reducing the time for RHD genotyping to minutes, making RHD genotyping feasible for real-time application in the hospital (Wagner et al , 2017). Patients with sickle cell disease benefit from RHD genotyping, not only because most have a requirement for chronic transfusion, but also because they are at increased risk of alloimmunization to certain Rh and other blood group antigens because of the differences that they inherit from their African ancestry compared to those inherited by the predominately Caucasian donors in Western countries (Vichinsky et al , 1990).…”

“…Currently, in the United States, most RHD genotyping is performed in reference laboratories and, therefore, the turnaround time is more than 1 day, excluding the procedure for patients requiring an urgent transfusion. For patients requiring chronic transfusions, for example, sickle cell disease, thalassaemia and myelodysplastic syndrome, the results of once-in-a-lifetime RHD genotyping may not be available in time for the current transfusion, but would be available for future transfusions (Chou et al, 2013;Fasano & Chou, 2016 to minutes, making RHD genotyping feasible for real-time application in the hospital (Wagner et al, 2017). Patients with sickle cell disease benefit from RHD genotyping, not only because most have a requirement for chronic transfusion, but also because they are at increased risk of alloimmunization to certain Rh and other blood group antigens because of the differences that they inherit from their African ancestry compared to those inherited by the predominately Caucasian donors in Western countries (Vichinsky et al, 1990).…”

Microchip technology applications for blood group analysis

“…Many methodologies are also available to genotype blood groups from laboratory-developed assays to commercially available platforms [61] . For example, direct PCR from plasma or serum with melting point analysis has been developed for faster red cell genotyping to identify blood group antigens within 40 min [62] . A real-time based blood-group genotyping approach was also introduced with automation potential and rapid cycling time [63] .…”

Basic aspects, application and platforms currently available for molecular blood grouping of donor and patient population

“…Single‐specific primer‐PCR (SSP‐PCR), allele‐specific oligonucleotide‐PCR (ASO‐PCR) and restriction fragment length polymorphism‐PCR (RFLP‐PCR) are some of the techniques that have been employed either as singleplex or multiplex for red cell genotyping . The advantage of these methods is that they can be readily introduced into a general molecular laboratory . However, visualization of the PCR product is usually by gel electrophoreses, which precludes up‐scaling and interrogation of multiple alleles in multiple samples, is not amenable for automation and introduces reading and recording errors.…”