The reactivity of paired plasma and serum samples are comparable in the anticardiolipin and anti‐β2‐glycoprotein‐1 ELISAs. Reply to a rebuttal

Abstract

We thank Wong and Favaloro for suggesting an alternate method for displaying and analyzing our data concerning the reactivity of paired plasma and serum samples in the anticardiolipin (ACL) and anti‐β2‐glycoprotein‐1 (anti‐β2GP1) ELISAs [1Wong R.C. Favaloro E.J. The reactivity of paired plasma and serum samples are comparable in the anticardiolipin and anti‐β2‐glycoprotein‐1 ELISAs: a rebuttal.J Thromb Haemost. 2006; 4: 1434-5Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar]. The ELISA data from our original report have now been analyzed using Bland–Altman difference plots (Fig. 1A–D) [2Lewis D.A. Pound M.L. Ortel T.L. The reactivity of paired plasma and serum samples are comparable in the anticardiolipin and anti‐β2‐glycoprotein‐1 ELISAs.J Thromb Haemost. 2006; 4: 265-7Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 3Bland J.M. Altman D.G. Statistical methods for assessing agreement between two methods of clinical measurement.Lancet. 1986; 1: 307-10Abstract PubMed Scopus (39517) Google Scholar]. The mean difference of the antibody levels in the two pairs of sample types (value detected in plasma sample minus value detected in serum sample) and 95% limits of agreement for these values are shown as dashed lines on the graphs and are listed in the figure legend. The mean differences for both sample pairs in both ELISAs are relatively small (−0.91–1.89 GPL units) and vary in a random manner, indicating that the sample pairs behave similarly in each of the ELISAs. The absolute values of 95% limits of agreement for the samples pairs are within 18 GPL units for the ACL ELISA and 30 anti‐β2GP1 units for the anti‐β2GP1 ELISA, indicating that the differences we found in the antibody values determined using the different sample types are not clinically relevant. As expected, the mean differences between the sample types in both ELISAs were greater at higher antibody levels. If antibody levels above 100 GPL or 100 anti‐β2GP1 units are excluded from the analysis, the absolute values of 95% limits of agreement are < 12 GPL or 12 anti‐β2GP1 units for both sample pairs in both assays (data not shown). Wong and Favaloro also raised concerns about dilution artifacts in plasma samples. In our original letter the antibody levels determined using citrated plasma samples were corrected for dilution of samples (collection of blood using ethylenediamine‐tetraacetic acid (EDTA) as an anticoagulant does not result in dilution of the sample). We do note, however, that the mean differences in antibody levels and the 95% limits of agreement for the two sets of sample pairs for both ELISAs are greater for citrated plasma and serum pairs compared with the EDTA plasma and serum pairs (Fig. 1A–D). It is unlikely that this difference reflects only a dilution artifact as the results vary in a random manner. Analysis of our data using different plots supports our original conclusion that the reactivity of plasma and serum is comparable in the ACL and anti‐β2GP1 ELISAs developed in our laboratory. These data would suggest that it is valid to compare antigen levels in datasets containing a combination of citrated plasma, EDTA plasma and serum samples. Our interest in this comparison was to confirm the ability to identify ACL and anti‐β2GP1 antibodies in different plasma and serum samples for future research studies. For clinical laboratory testing we would agree with adhering to the recommended laboratory practices and not mixing sample preparations. This work was supported by a cooperative agreement (U18DD00014) with the Hematologic Diseases Branch, Centers for Disease Control and Prevention (T. L. Ortel), and grant (U54‐HL077878) from the National Institutes of Health (T. L. Ortel).