Does multiple freezing and thawing cycles of serum affect the detection of anti-nuclear antibodies and anti-neutrophil cytoplasmic antibodies by indirect immunofluorescent method?

The aim of this study was to determine whether more than once freezing and thawing of serum affects different patterns of anti-nuclear antibodies (ANA) and anti-neutrophil cytoplasmic antibodies (ANCA) as also to determine for how many days can serum samples be reliably refrigerated (2-8oC) for these tests. A total of 20 ANA (6 homogenous, 3 speckled, 3 centromere, 2 nucleolar, 2 cytoplasmic and 4 mixed) and 6 ANCA (3 PR-3 positive cANCA, 2 MPO positive pANCA, one MPO negative pANCA) positive serum samples were evaluated. The ANA and ANCA tests were studied with the indirect immunofluorescent (IIF) method using commercial kits. The effect of freezing and thawing was evaluated by comparing frozen serum samples with fresh samples during 5 freezing and thawing cycles. In addition, we analyzed the effect of storage time in the refrigerator. In none of the ANA positive serum samples, except for one mixed pattern, was negativity found in the HEp-2 and liver cells after the freezing and thawing cycles and the 21 day refrigerated period. In ANCA positive serum samples, positivity similar to initial findings continued in all samples during five cycles in freeze/thaw group and at the end of 21 days, in the refrigerated group. It may be concluded that ANA and ANCA can be reliably analyzed in serum samples with five freezing and thawing cycles and after refrigeration up to 21 days.