The impact of repeated freeze‐thaw cycles on antiphospholipid antibody titer

ESSENTIALS: Limited data are available of freeze‐thaw effect on anticardiolipin and anti‐beta2‐glycoprotein I antibodies. Patient samples with antibody titer over a broad range were analyzed in a standardized freezing‐thawing scheme. aCL and aβ2GPI IgG and IgM titers are stable over time and after repeated freeze‐thawing. Repeated use of samples facilitates routine work‐out and use of samples for scientific purposes. BACKGROUND: Pre‐analytical factors, like freeze‐thaw cycles (FTC), can potentially affect results and clinical interpretation. According to the SSC‐ISTH recommendations for antiphospholipid antibodies (aPL) testing, additional FTC should be avoided to maintain the best performance. Patient samples are often analyzed in batch and having one frozen sample aliquot for all aPL tests, that may hamper daily routine work‐out. To use them for study or method validation purpose, sample storage in bio banks is often done in one aliquot also triggering the need for several FTC to be able to use them in different scientific projects. Taking into account the limited scientific literature, the strict guidelines and the potential benefits of repeated FTC we evaluated this pre‐analytical factor. OBJECTIVES: Evaluating the effect of repeated FTC on anticardiolipin (aCL) IgM/IgG and anti‐beta‐2 glycoprotein 1 (aβ2GPI) IgM/IgG antibody titer. PATIENT/METHODS: 42 patient plasmas that were not thawed before, were retrieved from the routine archive (−80°C). All aliquots were analyzed on five consecutive days with an additional, standardized FTC every day. Mann‐Withney tests for statistical differences and a concordance correlation coefficient (CCC) were calculated between the first and following FTC. RESULTS AND CONCLUSION: For all four aPL no statistical difference or degradation from positive to negative was seen, even after five FTC. The CCC between the first and fifth FTC were between 0.98 and 1 for all four aPL. aCL IgM/IgG and aβ2GPI IgM/IgG antibody titer, over a broad titer range, are stable over time and after repeated FTC.