Effect of storage on antibody reactivity after adsorption in the presence of polyethylene glycol

Effect of storage on antibody reactivity after adsorption in the presence of polyethylene glycol In a recent report,1 the pros and cons of using polyethylene glycol (PEG) in adsorption procedures to detect alloantibodies in the presence of autoantibodies were discussed. One of the concerns mentioned was the possibility of losing weak alloantibody reactivity after adsorption in the presence of PEG, as a result of the precipitation of protein. It is well known that PEG precipitates protein.2-4 Leger and Garratty1 noted that after one or two adsorptions in the presence of PEG, the appearance of the plasma and sera samples changed, becoming clearer. It was also noted that, after storage overnight at 4°C, a precipitate formed in the serum–PEG mixtures. Although a loss of antibody reactivity was not demonstrated, we suggested testing the serum– PEG mixture at the time of adsorption (and not after storage) to decrease the likelihood of missing weak antibodies.1 PEG adsorption results from one of our reference laboratory cases prompted us to reinvestigate the possible loss of antibody reactivity after 4°C storage of PEG-adsorbed sera. This case involved a sample from a patient with multiple alloantibodies, as well as antibodies to a high-incidence antigen. PEG adsorption, using allogeneic red cells, was utilized to separate the antibody specificities. When the freshly PEG-adsorbed serum was tested, the reactivity of the antibodies ranged from 1+ to 3+. The serum containing PEG was left at 4°C over the weekend. When work continued (3 days later), a precipitate was noted in the tube of PEGadsorbed serum, so the PEG–serum mixture was centrifuged before testing, and the supernatant was used for testing. The range of reactivity of the antibodies in the adsorbed serum decreased to 0 to 2+, with some specificities no longer detectable. To investigate the frequency of this problem, seven sera containing single antibody specificities (anti-E, -K, -Fya, Jka) were tested after two sham adsorptions with antigennegative red cells in the presence of PEG. The PEGadsorbed sera were divided into two aliquots: one was mixed before subsampling (“mixed”); the precipitate was allowed to settle in the other before sampling of the clear supernatant (“settled”). The PEG-adsorbed sera were tested in parallel with an equivalent mixture of unabsorbed serum plus PEG at the time of adsorption and at 1 and 2, 3, or 4 days after adsorption (after storage at 4°C). Two “settled” adsorbed sera (anti-E and anti-Fya) reacted more weakly (1⁄2 + vs. 1+ and 1+ vs. 2+) than parallel “mixed” sera after storage; the other five stored, “settled,” and “mixed” adsorbed sera reacted to the same degree as the adsorbed sera tested at the time of adsorption. Five of the sera were tested 4 days after adsorption, and only one (anti-Fya) showed weakening. The amount of precipitate formed varied for each sample, but it was visible on the sides as well as on the bottoms of the tubes of PEG-adsorbed sera. Resuspension of the precipitate was easily accomplished by vigorous agitation (vortexing). Once the precipitate was centrifuged, it could not easily be resuspended. Although we did not replicate the results observed in the index case above (i.e., some results became negative), it seems that antibody reactivity can be weakened or lost when PEG-adsorbed sera are stored, especially if precipitate is not remixed into the rest of the sample. While adsorbed sera are generally tested at the time of adsorption, complex cases such as the case above may take several days to resolve. We again caution serologists to test PEGadsorbed serum on the day it is prepared; if the adsorbed serum must be stored before testing is completed, it should be remixed and not centrifuged before sampling.