Rapid and quality controlled screening assay for the HLA-B57 antigen by flow cytometry

Abstract

Aim The presence of the genotype HLA-B∗57:01 is highly predictive of Abacavir hypersensitivity. The need to identify the presence of this antigen in the HIV population is pertinent to treatment. Traditional molecular methods of HLA-B∗57:01 with high specificity come often with long turn-around times and high costs. Our laboratory developed a flow cytometric screening method to identify the HLA-B∗57 antigen for an expedited negative result. Methods 59 random patients were tested for the HLA-B∗57 antigen utilizing a two-step flow cytometry staining protocol. Whole blood was incubated with CD3-APC and a biotinylated B17 monoclonal antibody. The monoclonal HLA-B∗17 antibody detects the presence of the HLA- B∗57 or B∗58 antigens on the cell surface. To enable detection of the B57 antibody, streptavidin-PE was added. An internal positive/negative control was developed to confirm assay performance. 10 μL of Flow PRA 1 screening beads (One Lambda) were added to the patient sample. Based on SSC and FSC, beads are easily distinguishable from patient cells. All samples were confirmed by molecular HLA typing using real time PCR or SSOP. Results Of the 59 patient samples, 12 were considered positive with a Mean MFI of 8622 ± 3962. 2 of the 12 positive screens were positive for HLA-B∗57:01 by molecular typing. Based on the data, a cutoff was set with a mean MFI of 500. None of the negative screens had either HLA-B∗57 or B∗58 present by molecular typing. No cross-reactivity was observed from the 5 CREG group. Thus there is a lower likelihood of detecting antigens outside of HLA-B∗57 and HLA-B∗58. Conclusions The screening of patients for the presence of HLA-B∗57:01 is important prior to initiation of Abacavir therapy. An estimated 0.1–2.5% of the US population is positive for HLA-B∗57:01, so the majority of patients will be negative. This rapid flow cytometry assay allows for faster turn-around time for B∗57:01 negative patients. The introduction of an internal positive control into the assay provides quality control that previous HLA-B∗57 screening assays failed to provide.