FALSE-POSITIVE ELUATE REACTIVITY DUE TO THE LOW-IONIC WASH SOLUTION USED WITH COMMERCIAL ACID-ELUTION KITS

BACKGROUND: During the use of commercial red cell (RBC) acid-elution k its for adsorption and elution (adsorption/elution) studies with anti- D, unexpected reactive eluates (anti-D) were obtained from D-RBCs. Suc h results were not obtained with a parallel xylene method or, historic ally with heat and ether methods. STUDY DESIGN AND METHODS: Single-don or 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 us e of commercial kit wash solution versus phosphate-buffered saline ver sus solutions of various ionic strengths. RESULTS: Anti-D was eluted f rom 20 of 22 samples of D- RBCs after incubation with commercial polyc lonal 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-don or 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-RB C eluates reacted, as compared with 9 of 11 D-RBCs that yielded positi ve (1+-2+) eluates with the commercial wash solution. If the recommend ed initial phosphate-buffered saline wash was omitted before the use o f 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. Antiglobuli n tests on all adsorbing D- were negative. CONCLUSION: Commercial wash solutions used for acid elution are at low ionic strength and commonl y yield superior eluates, but in the presence of high-titer antibodies , false-positive eluates can result. It is our belief that the low-ion ic-strength wash solution caused aggregation of IgG and nonspecific at tachment 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.