Ann Lab Med.  2019 Nov;39(6):572-576. 10.3343/alm.2019.39.6.572.

EDTA Treatment for Overcoming the Prozone Effect and for Predicting C1q Binding in HLA Antibody Testing

• Kim HS
• Choi AR
• Yang M
• Oh EJ

• KMID: 2450953
• DOI: http://doi.org/10.3343/alm.2019.39.6.572

Abstract

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.

Keyword

Prozone effect; EDTA; Single antigen bead assay; C1q binding; HLA antibody

MeSH Terms

Complement System Proteins
Dithiothreitol
Edetic Acid*
Fluorescence
Humans
Immunoglobulin G
Transplant Recipients
Complement System Proteins
Dithiothreitol
Edetic Acid
Immunoglobulin G

Figure

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  Fig. 1 Comparison of MFI values in naïve and EDTA-treated sera. (A) Class I IgG-SAB results in 19 sera (1,843 beads); (B) Class II IgG-SAB results in 16 sera (1,520 beads).Abbreviations: MFI, mean fluorescence intensity; SAB, single antigen bead.

• 

  Fig. 2 MFI values for naïve samples and samples treated with 1:8 dilution, DTT, and EDTA, and C1q assay using sera from five sensitized patients. (A) MFI values for all samples with various treatments. (B) Correlations between MFI values for naïve, diluted, DTT-treated, and EDTA-treated samples.Abbreviations: MFI, mean fluorescence intensity; DTT, dithiothreitol.

• 

  Fig. 3 Correlation of MFI values from C1q- and IgG-SAB assays using (A) naïve sera and (B) EDTA-treated sera.Abbreviations: MFI, mean fluorescence intensity; SAB, single antigen bead.

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