Background/Case Studies: Therapeutic plasma exchange (TPE) is a crucial treatment for antibody-mediated rejection (AMR) post-organ transplantation that removes pathogenic donor-specific HLA antibodies (DSA). However, HLA laboratory tests to detect DSA have limitations, including a prozone (PZ)-like effect that mask DSA detection by blocking reporter antibodies. Previous studies have revealed that the PZ-like effect is mediated by complement components and can be mitigated by dilution or EDTA treatment of patient serum. In the case discussed herein, the PZ-like effect led to the incorrect conclusion that the patient was not responding well to TPE, which was corrected by EDTA treatment.
Study
Design/Methods: Patient serum samples were analyzed using the LABScreen Single Antigen HLA Class I and II kit (OneLambda). EDTA was added to the patient serum to overcome the PZ-like effect. Nephelometry was utilized to measure C3 and C4 complement levels (Binding Site).
Results/Findings: A 36-year-old male with orthotopic heart transplant presented with heart failure and reduced ejection fraction. HLA antibody testing revealed multiple class I and II DSA (Figure A). After 5 TPE treatments, an unanticipated increase in DSA was observed raising suspicion of PZ-like effect. This was investigated by repeat testing of pre-TPE patient serum with and without EDTA treatment, which revealed higher DSA levels with EDTA treatment than in the absence of EDTA treatment (Figure A). Interference by complement components in the pre-TPE sample was supported by physiologic levels of complement (C3: 102.2 mg/dL; C4: 26.0 mg/dL) capable of complement fixation. However, in the post-TPE sample the complement level was lowered below reference interval (C3: 40.6 mg/dL; C4: 10.2 mg/dL). We hypothesize the hemodilution effect from plasma replacement with albumin during TPE mitigated the complement-mediated PZ-like effect in the post-TPE sample, much like the EDTA treatment on the pre-TPE sample. Conclusions: The case illustrates that a PZ-like effect can lead to false negative results on Luminex single antigen testing when assessing the outcome of TPE. Hemodilution during TPE likely reduced the PZ-like effect in the post-TPE sample. The lower complement levels that resulted in higher antibody levels post-TPE support the concept that complement components contribute to the PZ-like effect. We recommend the use of EDTA-treated serum samples for testing efficacy of TPE removal of DSA to prevent false negative results.
Importance of research: The case demonstrates the potential for the PZ-like effect to cause misleading HLA antibody results in the setting for TPE. The study highlights the importance of using EDTA-treated serum samples for testing and recognizing the PZ-like effect to prevent misinterpretation of results which, in turn, can lead to misinformed medical management decisions. The findings provide valuable insights into improving the accuracy of HLA antibody testing during TPE for AMR.
