Effects of repeated freeze-thaw cycles on anticardiolipin antibody immunoreactivity

The effect of repeated freeze-thaw cycles on anticardiolipin antibody levels was evaluated using an enzyme-linked immunosorbent assay. Normal human serum was spiked with known quantities of freeze-dried human polyclonal anticardiolipin antibody IgG and IgM (19 samples each) or IgA (11 samples). Each spiked sample was split into four identical aliquots; one aliquot was never frozen, and the remaining three were taken through successive freeze-thaw cycles. All aliquots from each sample were evaluated on the same day using the same plate and reagents. A significant decline in mean anticardiolipin IgG levels occurred between the aliquot which had never been frozen and the one which had been through three freeze-thaw cycles (Student's t-test, P = .04). Although mean IgM and IgA values declined as well, the differences were not significant. When individual samples were evaluated the decline appeared to occur most often between the second and third freeze-thaw cycle. Eight anticardiolipin IgG and three IgM-containing samples which had been positive initially became negative by the third freeze-thaw cycle. These data show that handling and storage of serum used to perform anticardiolipin antibody assays are important potential sources of assay variability.