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Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays

BACKGROUND: Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very cruci...

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Autores principales: Singh, Parvind, Tiwari, Aseem Kumar, Mishra, Vikash Chandra, Deshpande, Trupti Vimlakar, Dorwal, Pranav, Bhardwaj, Amit Kr, Kumari, Sneha, Raina, Vimarsh
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Wolters Kluwer - Medknow 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9855212/
https://www.ncbi.nlm.nih.gov/pubmed/36687549
http://dx.doi.org/10.4103/ajts.ajts_145_20
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author Singh, Parvind
Tiwari, Aseem Kumar
Mishra, Vikash Chandra
Deshpande, Trupti Vimlakar
Dorwal, Pranav
Bhardwaj, Amit Kr
Kumari, Sneha
Raina, Vimarsh
author_facet Singh, Parvind
Tiwari, Aseem Kumar
Mishra, Vikash Chandra
Deshpande, Trupti Vimlakar
Dorwal, Pranav
Bhardwaj, Amit Kr
Kumari, Sneha
Raina, Vimarsh
author_sort Singh, Parvind
collection PubMed
description BACKGROUND: Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long-term graft survival. For identification of DSA, usually, either cell-based or HLA bead-based assay is being used in laboratories. However, both cell-based and bead-based assays have certain limitations. One such common limitation is “prozone effect,” which can give false-negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell-based and HLA bead-based assays and its utility in histocompatibility testing. MATERIALS AND METHODS: In a series of four experiments, cell-based assay, flow cytometric cross-match (FCXM), and HLA bead-based flow cytometric panel reactive antibodies (PRAs) were performed. Single-antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti-HLA antibody-antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called “positive concoction” in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. RESULTS: Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. CONCLUSION: In our experimental findings, it was clear that cell-based assay (FCXM) was more severely affected by the prozone, whereas solid-phase (flow PRA) assay remained resistant to prozone.
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spelling pubmed-98552122023-01-21 Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays Singh, Parvind Tiwari, Aseem Kumar Mishra, Vikash Chandra Deshpande, Trupti Vimlakar Dorwal, Pranav Bhardwaj, Amit Kr Kumari, Sneha Raina, Vimarsh Asian J Transfus Sci Original Article BACKGROUND: Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long-term graft survival. For identification of DSA, usually, either cell-based or HLA bead-based assay is being used in laboratories. However, both cell-based and bead-based assays have certain limitations. One such common limitation is “prozone effect,” which can give false-negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell-based and HLA bead-based assays and its utility in histocompatibility testing. MATERIALS AND METHODS: In a series of four experiments, cell-based assay, flow cytometric cross-match (FCXM), and HLA bead-based flow cytometric panel reactive antibodies (PRAs) were performed. Single-antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti-HLA antibody-antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called “positive concoction” in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. RESULTS: Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. CONCLUSION: In our experimental findings, it was clear that cell-based assay (FCXM) was more severely affected by the prozone, whereas solid-phase (flow PRA) assay remained resistant to prozone. Wolters Kluwer - Medknow 2022 2022-07-27 /pmc/articles/PMC9855212/ /pubmed/36687549 http://dx.doi.org/10.4103/ajts.ajts_145_20 Text en Copyright: © 2022 Asian Journal of Transfusion Science https://creativecommons.org/licenses/by-nc-sa/4.0/This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
spellingShingle Original Article
Singh, Parvind
Tiwari, Aseem Kumar
Mishra, Vikash Chandra
Deshpande, Trupti Vimlakar
Dorwal, Pranav
Bhardwaj, Amit Kr
Kumari, Sneha
Raina, Vimarsh
Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays
title Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays
title_full Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays
title_fullStr Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays
title_full_unstemmed Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays
title_short Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays
title_sort prozone phenomenon in pretransplant testing: an interesting conundrum involving solid-phase and cell-based assays
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9855212/
https://www.ncbi.nlm.nih.gov/pubmed/36687549
http://dx.doi.org/10.4103/ajts.ajts_145_20
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