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

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.

Prozone effect on indirect immunofluorescence assay for anti-nuclear antibody testing / 国际检验医学杂志

Objective To explore the influence of prozone effect on anti-nuclear antibodies (ANA) testing by indirect immunofluorescence assay (IIFA).Methods The samples with high titer of ANA (≥1∶1 000) were selected from 880fresh serum samples, and were subsequently diluted in 1∶100, 1∶1 000and 1∶10 000ratio.Prozone effect was defined as fluorescence intensity from 1∶1 000dilution was stronger than that from1∶100dilution.The samples with prozone effect were determined manually or by Sprinter XL and EUROPattern.The samples with prozone effect were further characterized by combinations of fluorescence patterns, fluorescence intensities and autoantibody specificities.Results A total of 880samples were tested.Importantly, 34samples displayed prozone effect (3.86％in total and 29.57％in samples with ANA≥1∶1 000).Interestingly, prozone effect was identified by manual detection as well as by Sprinter XL with similar fluorescence patterns and fluorescence intensities.Notably, EUROPattern can only select the central area for identification.Among all samples with prozone effect, 74.42％samples exhibited fluorescence intensities of≥1∶10 000.Speckled pattern was the most prevalent fluorescence patterns in samples with prozone effect (46.51％).In addition, anti-RNP antibodies (62.79％) were the most popular autoantibodies in samples with prozone effect, followed by anti-dsDNA antibodies (51.16％) and anti-SSA antibodies (51.16％).Conclusion Prozone effect was present in ANA testing, especially in samples with high titers, resulting in underestimating the titers.The study highlighted that special attention should be paid to the prozone effect in clinical practice.

Prozone effects in microscopic agglutination tests for leptospirosis in the sera of mice infected with the pathogenic Leptospira interrogans serovar Canicola

Mice experimentally infected with a pathogenic strain of Leptospira interrogans serovar Canicola produced false negative results (prozone effect) in a microscopic agglutination test (MAT). This prozone effect occurred in several serum samples collected at different post-infection times, but it was more prominent in samples collected from seven-42 days post-infection and for 1:50 and 1:100 sample dilutions. This phenomenon was correlated with increased antibody titres in the early post-infection phase. While prozone effects are often observed in serological agglutination assays for the diagnosis of animal brucellosis and human syphilis, they are not widely reported in leptospirosis MATs.

Comparison of Quantitative Results among Two Automated Rapid Plasma Reagin (RPR) Assays and a Manual RPR Test / 대한진단검사의학회지

BACKGROUND: We compared two automated Rapid Plasma Reagin (RPR) assay kits with a manual RPR assay kit to evaluate the possibility of using the two automated RPR assays as an alternative to the manual RPR assay for a quantitative monitoring. METHODS: One hundred eighty-five samples were analyzed, including 16 sera from patients with primary, secondary, and latent syphilis. Measured RPR unit (R.U.) values of two automated RPR assay kits, Mediace RPR (Sekisui Chemical Co., Ltd, Japan) and HBi Auto RPR (HBI Co., Ltd, Korea), were compared with the RPR titers of Macro-Vue RPR card test (Becton Dickinson BD Microbiology systems, USA). As a confirmatory test, Anti-Treponema pallidum EUROLINE WB (IgG) and Anti-Treponema pallidum EUROLINE WB (IgM) (Euroimmun, Germany) were used. RESULTS: There was a prozone effect with Mediace RPR at RPR titer (card test) of 1:16, but not with HBi Auto RPR. The R.U. values of the two automated RPR assays did not show proportional increase to the RPR titer. Agreement between manual RPR and two automated RPR assay kits, Mediace RPR assay and HBi Auto RPR assay, were 83.8% and 83.2%, respectively. CONCLUSIONS: The two automated RPR assay kits could not be used as an alternative to manual RPR test for quantitative analysis of RPR titer. As Mediace RPR shows a prozone effect at relatively low RPR titer, caution is needed in the interpretation of the measured values.