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False-positive eluate reactivity due to the low-ionic wash solution used with commercial acid-elution kits
R M Leger1, P A Arndt, D J Ciesielski
1American Red Cross Blood Services, Southern California Region, Los Angeles, USA.
Background:
During the use of commercial red cell (RBC) acid-elution kits for adsorption and elution (adsorption/elution) studies with anti-D, unexpected reactive eluates (anti-D) were obtained from D- RBCs. Such results were not obtained with a parallel xylene method or, historically, with heat and ether methods.
Study Design And Methods:
Single-donor and commercial polyclonal anti-D samples were incubated with D+ and D- RBCs. Acid eluates were prepared by the manufacturers' directions. Variations in the wash step of the eluate preparation included the use of commercial kit wash solution versus phosphate-buffered saline versus solutions of various ionic strengths.
Results:
Anti-D was eluted from 20 of 22 samples of D- RBCs after incubation with commercial polyclonal anti-D (titer 512) and from 2 of 3 samples of D- RBCs incubated with single-donor anti-D (titer 256). With a low-titer (16) single-donor anti-D, 0 of 4 eluates from D- RBCs reacted. When phosphate-buffered saline was substituted for the commercial wash solution, 0 of 11 D- RBC eluates reacted, as compared with 9 of 11 D- RBCs that yielded positive 1+(-)2+ eluates with the commercial wash solution. If the recommended initial phosphate-buffered saline wash was omitted before the use of the commercial wash solution, the eluate reactivity was stronger (2+(-)3+). When low-ionic-strength (< 0.03 M) saline was substituted, anti-D was eluted from D- RBCs. All last washes were nonreactive. Antiglobulin tests on all adsorbing D- were negative.
Conclusion:
Commercial wash solutions used for acid elution are at low ionic strength and commonly yield superior eluates, but in the presence of high-titer antibodies, false-positive eluates can result. It is our belief that the low-ionic-strength wash solution caused aggregation of IgG and nonspecific attachment of IgG on RBCs. Aggregates will contain IgG serum antibodies in proportion to the titer of the antibody. It is this nonspecifically bound antibody that is eluted from antigen-negative RBCs.