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Published on: May 2, 2013
EDTA Treatment of Serum Unmasks Complement-Mediated Prozone Inhibition in Human Leukocyte Antigen Antibody Testing
Waseem Q Anani1, Adriana Zeevi1, John G Lunz2
1From the Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA.
Insights
Ethylenediaminetetraacetic acid (EDTA) pretreatment of serum samples improves human leukocyte antigen (HLA) antibody detection by mitigating prozone inhibition. This method enhances accurate HLA antibody identification without increasing false negatives.
Area of Science:
- Transplantation immunology
- Clinical chemistry
Background:
- Luminex-based single-antigen bead assays are standard for human leukocyte antigen (HLA) antibody detection in transplant recipients.
- Complement-mediated prozone inhibition can lead to false-negative results in HLA antibody testing.
Purpose of the Study:
- To evaluate the impact of ethylenediaminetetraacetic acid (EDTA) on Luminex-based HLA antibody testing.
- To assess EDTA's effect on assay background reactivity and antibody mean fluorescent intensity.
Main Methods:
- Serum specimens were retrospectively analyzed using Luminex single-antigen beads with and without EDTA treatment.
- Comparisons focused on changes in background reactivity and HLA antibody strength post-EDTA treatment.
Main Results:
- EDTA treatment caused significant fluctuations in weak antibodies (mean fluorescent intensity < 2,000).
- Newly detected HLA antibodies were observed in 16% of class I and 26% of class II beads.
- EDTA did not introduce false-negative reactions compared to untreated samples.
Conclusions:
- EDTA serum pretreatment effectively reduces complement-mediated prozone inhibition.
- This pretreatment enhances the accuracy of HLA antibody detection.
- Assay background reactivity and false-negative rates remained stable.
Objectives:
Luminex-based single-antigen bead human leukocyte antigen (HLA) antibody testing is widely used to define HLA antibodies for transplant compatibility. False-negative results can occur with complement-mediated prozone inhibition. This study assessed the effect of EDTA on the assay background reactivity and fluctuations in antibody mean fluorescent intensity.
Methods:
Serum specimens were retrospectively tested using Luminex-based single-antigen beads with and without EDTA. Treated and untreated serum samples were compared by two measures: changes in background reactivity and changes in HLA antibody strength after EDTA treatment.
Results:
Ten pretransplant and 48 posttransplant specimens were identified: lung (22), heart (10), kidney (21), heart/lung (two), pancreas (one), small bowel (one), and liver (one). After EDTA treatment, weak antibodies (below 2,000 mean florescent intensity) demonstrated the largest fluctuations. Newly identified HLA antibodies were seen in 16% (8/49) of class I and 26% (15/57) of class II beads. EDTA treatment did not result in false-negative reactions compared with untreated serum.
Conclusions:
EDTA serum pretreatment mitigated complement-mediated prozone inhibition and improved accurate HLA antibody detection. The background reactivity and the false-negative rate of the assay appear unchanged.
