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

Abstract

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. BackgroundPre‐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.ObjectivesEvaluating the effect of repeated FTC on anticardiolipin (aCL) IgM/IgG and anti‐beta‐2 glycoprotein 1 (aβ2GPI) IgM/IgG antibody titer.Patient/Methods42 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 ConclusionFor 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.

Stability of serum precipitins to Aspergillus fumigatus for the diagnosis of allergic bronchopulmonary aspergillosis

“…While we do not expect repeated freeze-thaw cycles in routine clinical diagnostic settings, serum specimens that have been frozen as a result of repeated serological testing for different diseases without aliquoting are expected to yield results similar to fresh sera. Our results are consistent with previous studies evaluating the stability of IgM and IgG after repeated freeze-thaw cycles via sensitive enzyme immunoassays [10,11] and adds to the body of evidence that the precipitating characteristics of antibodies (e.g. F c region [5]) against Aspergillus have not been functionally altered.…”

International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing

“…The authors studied PT, APTT, Fibrinogen, D‐Dimer, clot‐based FII/FV/FVII/FVIII/FIX/FXI/FXII, chromogenic FXIII, AT activity, PC activity, PS activity plasminogen, DRVVT and anti‐Xa (LMWH). Five freeze‐thaw cycles did not cause significant changes in anticardiolipin or anti β2 glycoprotein 1 antibody testing 82 . Taken together, these studies indicate that many coagulation tests can be safely performed after 2 cycles of freeze thaw when thawing is done in a water bath for 4‐5 minutes and freezing/refreezing is done at −70°C, provided the interval between thaw and refreeze takes account of sample stability (see Section 6 above).…”

“…Five freeze-thaw cycles did not cause significant changes in anticardiolipin or anti β2 glycoprotein 1 antibody testing. 82 Taken together, these studies indicate that many coagulation tests can be safely performed after 2 cycles of freeze thaw when thawing is done in a water bath for…”

Effect of multiple freeze–thaw cycles on the detection of anti-SARS-CoV-2 IgG antibodies

“…Therefore, it is recommended to store sera in aliquots to reduce sample exposure to multiple F/T cycles [6]. Although several studies have suggested that repeated F/T cycles have a minimal effect on antibody stability against specific pathogens [2,7,8], other researchers are questioning the reliability of the data generated from using such samples [9].…”