EFFECT OF AGE AND STORAGE CONDITIONS ON SERUM SAMPLES USED FOR HLA ANTIBODY TESTING.

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EFFECT OF AGE AND STORAGE CONDITIONS ON SERUM SAMPLES USED FOR HLA ANTIBODY TESTING.
Lolita Ardiente, CHT (ABHI), Dennis Arellano, BS, Cydni Boyd, CHS (ABHI), Lynette Brown, CHT (ABHI), Shellie Dieterich, CHS (ABHI), Arlene Duque, CHT (ABHI), Kortney Hamilton, CHS (ABHI), Valeria Petrafeso, CHT (ABHI), Susanne Thornton, CHT (ABHI), Paul Warner, PhD, CHS (ABHI), Danny Youngs, CHS (ABHI) and Karen Nelson, PhD, Dip (ABHI). Seattle WA, USA, Puget Sound Blood Center, 98104, Immunogenetics/HLA.

Factors such as storage conditions and lab transit time may impact the ability to detect and identify HLA antibodies in patient serum samples. To test this, we drew multiple samples of blood from a subject with known HLA antibodies (POS), and from an untranfused male (NEG). The coagulated blood was allowed to sit at room temperature (RT). On days 0, 7, 10, and 14 tubes were centrifuged, and serum was collected and frozen until testing. Individual triplicate samples from each time point were tested for HLA antibodies by solid phase methods (ELISA, flow cytometry with multiple antigen-coated and single antigen-coated beads, and Luminex) and cellular methods utilizing flow cytometric crossmatch and T-cell (AHG) plus B-cell (modified Amos) cytotoxicity. All timepoints tested yielded similar results in the solid phase assays, except for ELISA, which demonstrated slightly lower reactivity of the POS samples after day 7. The flow cytometric crossmatches exhibited a diminished capacity to detect IgG antibodies bound to donor T- and B-cells after day 7, although the decreased channel shift was most noticeable on day 14 (day 0 to day 14 difference = -11 channel shift T-cells, -13 B-cells). In the cytotoxicity assays, there were no differences in any of the time points for AHG-CDC T-cell reactivity. However, B-cell cytotoxicity increased through day 10. NEG samples remained consistently negative in all assays at all time points. In conclusion, serum separated from coagulated blood greater than 7-10 days old may not be suitable for HLA antibody testing, depending upon the methods employed.