P112 : COMBATING CROSS-REACTIVITY IN HLA-B27 DETECTION BY FLOW CYTOMETRY

• Published in: Human immunology Vol. 75; p. 128
• Main Authors: Voit, Laurie, Keso, Crystal, Hallaway, Lisa, Kreuter, Justin D., Gandhi, Manish J.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.10.2014
• ISSN: 0198-8859; 1879-1166
• DOI: 10.1016/j.humimm.2014.08.174

Presence of HLA-B27 (associated with ankylosing spondylitis, juvenile rheumatoid arthritis, and Reiter’s syndrome) on leukocytes in whole blood can be detected with monoclonal antibodies by a rapid qualitative two-color direct immunofluorescence flow cytometric method. In this method cross-reacting glycoproteins can provide a positive result; thus, some samples need to be confirmed by an alternate test method. We present an overview of our reagent quality control as well as observations requiring modification to the laboratory process. Whole blood samples are tested with monoclonal antibody GS145.2 [Becton, Dickinson and Company Biosciences (BD), San Jose] as per the manufacturer’s suggested protocol acquired on FACS Calibur (BD) utilizing the manufacturer provided template. Results are displayed as mean channel shift and reported as HLA-B27 negative or positive sample based on the kit decision marker. The kit package insert references a study in which some cross-reactive samples fell within a ten-channel zone above the decision marker. Results that fall within this zone should be confirmed by an alternate method. It is our protocol to utilize a molecular method to confirm this zone above the decision marker as well as below (±10). For reagent quality control it is our practice to select two negative, two ±10, and two positive from the current lot and then re-test with the new lot. The HLA typing for these samples is confirmed using a molecular method. Sample 4 for both comparisons was false positive in our test system. QC summary.SampleNew lot 1 comparisonNew lot 2 comparisonCurrent lot MCF decision marker 147New lot MCF decision marker 150HLA-B typingCurrent lot MCF decision marker 150New lot MCF decision marker 149HLA-B typing188818.441041247.8293848.5172828.3531431567.715715927.6541531617.371501637.4251681738.271631657.27617918527.2716216327.35 These reagent quality control results caused us to increase the +10 to +15 above the decision marker for a period of time to monitor the findings. Although this change led to more work and expense in the laboratory, it was justified by patient safety. While following the manufacturer’s suggestions are critical, a stringent internal quality control program can identify samples providing discrepant results.