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Aim To systematically assess the effectiveness of EDTA in unmasking clinically relevant HLA alloantibodies Methods Serum samples from transplant (txp) patients collected for clinical testing were pre-treated with various concentrations of EDTA for 15 min at room temperature and then tested with Luminex-based solid phase assays using HLA mixed beads (HMB) and Single antigen beads (SAB) for the presence and identification, respectively, of HLA alloantibodies. Results Utility of pretreating sera with heat inactivation, dithiothreitol and/or EDTA to overcome prozone-like inhibition (PZLI) has been demonstrated. However, lack of details vis-a-vis time and concentration used, prompted us to systematically assess the effectiveness of EDTA in unmasking presence of clinically relevant HLA alloantibodies in serum samples with PZLI. Titration analysis using EDTA concentrations ranging from 0–50 mM indicated that 1.25 mM EDTA was ineffective, while 2.5 mM EDTA was moderately effective, in removing PZLI. EDTA concentration =/>5 mM was effective in removing PZLI in all serum samples. Therefore, we selected 5 mM EDTA for all subsequent studies. We observed that 5 mM EDTA did not give rise to high background and/or false positives, i.e., no unmasking of “new” specificities in serum samples with cPRA = 0% or in serum samples that demonstrated presence of limited HLA alloantibody specificities with MFI Conclusions The PZLI phenomenon observed due to presence of activated complement components in sera, challenges the ability of HLA labs to accurately identify HLA alloantibodies. Our study underscores the utility of treating serum with EDTA in identifying pre-formed and de novo DSAs that would be deleterious to the allograft in txp recipients. |
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