P079High throughput HLA-B27 immunophenotyping; conversion to a 96 well plate

Aim Spondyloarthropathies are more commonly diagnosed in patients that are positive for HLA B27. The Immunology lab in Hamilton Regional Laboratory Medicine Program (HRLMP) currently performs 200 samples/week of HLA B27 Immunophenotyping test by conventional manual tube (5 ml) based flow cytometry method using the Becton Dickinson (BD) HLA-B27 kit. Testing is transferring to the Histocompatibility lab to utilize existing flow cytometry equipment. The aim of our study was to modify the BD HLA-B27 test for processing on a 96 well plate to improve testing efficiency and maintain cost effectiveness. Methods Phenotype testing-BD HLA-B27 Kit (Catalog No 340183). PCR-SSP/RSSO One Lambda Micro SSP and/or Labtype HD or XR kits. Immunophenotyping acquired and analysed by BD HLA-B27 Clinical software on the FACSCanto II. Phase 1-Test Modifications-96 wells plate was used and the lyse solution step was modified from a single 10 min incubation in 2 ml of lysing solution to two 5 min incubations in 120 μl and 200 μl of lysing solution. Wash volume of Phosphate Buffered Saline (PBS) was reduced from 2 ml to 200 μl. All other aspects of the test were as per package insert. Phase 2-Side-by-Side Testing-performed on 55 samples (sample age 1-3 days). Samples were from the HLA testing population with confirmed PCR-SSP/RSSO B locus typings. Phase 3-Blind Comparison Study−20 samples tested by the tube method in the Immunology lab were compared to the modified plate method done in the HLA Lab. Discrepant results confirmed by PCR-SSP. Results There was good correlation between the standard tube method and the Hamilton plate method. Paired T test does show statistical difference between the methods, however, the homoscedastic T Test does not indicate statistical difference in their variances. Despite the mean statistical difference, as per the package insert recommended interpretation there were no false positive or false negative results in the side-by-side study as verified by PCR. Conclusions The 96 well plate method produced reliable results using package insert recommended cut-off guidelines. The modified method appeared to be a very useful and a high throughput cost effective method for HLA B27 testing.