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1
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Harris MR, Canterbury A, Worthington JE, Lowe MP, Hampson ME, Poulton KV. Comparison of HISTO SPOT HLA AB With Cross-Match Results. Int J Immunogenet 2025; 52:135-140. [PMID: 40095438 DOI: 10.1111/iji.12710] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2024] [Revised: 02/24/2025] [Accepted: 03/06/2025] [Indexed: 03/19/2025]
Abstract
Single antigen bead assays have revolutionised the identification and definition of HLA-specific antibodies and HLA-specific antibody profiles present in patients awaiting transplantation are routinely characterised to inform organ allocation. For highly sensitised patients with a lower likelihood of finding a compatible donor, de-listing of unacceptable antigens is an option to release organ offers. In this study, 164 serum samples from 106 potential renal transplant recipients were tested using HISTO SPOT HLA AB in parallel with testing by LABScreen Single Antigen (One Lambda) and cross-matching by both CDC and flow cytometry. The results were analysed to assess the ability of HISTO SPOT HLA AB to predict a cross-match result and to understand the relative sensitivity of this test compared with other available assays. 136 samples analysed were positive for donor-specific antibodies (DSAs) using HISTO SPOT HLA AB. Of these, 17 (12.5%) were CDC positive, and 82 (60.3%) were positive by flow cytometry. A total of 28 sera which were negative for DSAs using HISTO SPOT HLA AB were negative by CDC and 25 (89.3%) were also flow cytometry cross-match negative. In this early study, HISTO SPOT HLA AB has a 100% negative predictive value for CDC and 89.3% for flow cytometry cross-matching. HISTO SPOT may therefore prove a useful additional tool to inform de-listing strategies and to facilitate transplantation in highly sensitised patients.
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Affiliation(s)
- Madeleine R Harris
- Transplantation Laboratory, Manchester Royal Infirmary, Manchester University NHS Foundation Trust, Manchester, UK
- Faculty of Biology, Medicine and Health, Division of Medical Education, School of Medical Sciences, University of Manchester, Manchester, UK
| | | | - Judith E Worthington
- Transplantation Laboratory, Manchester Royal Infirmary, Manchester University NHS Foundation Trust, Manchester, UK
| | - Marcus P Lowe
- Transplantation Laboratory, Manchester Royal Infirmary, Manchester University NHS Foundation Trust, Manchester, UK
| | - Marie E Hampson
- Transplantation Laboratory, Manchester Royal Infirmary, Manchester University NHS Foundation Trust, Manchester, UK
| | - Kay V Poulton
- Transplantation Laboratory, Manchester Royal Infirmary, Manchester University NHS Foundation Trust, Manchester, UK
- Faculty of Biology, Medicine and Health, Division of Medical Education, School of Medical Sciences, University of Manchester, Manchester, UK
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2
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Abraha J, Rao P, Morris GP. Modes of assay interference and the effectiveness of serum pretreatment approaches in detection of anti-HLA antibodies. J Clin Pathol 2024; 77:284-290. [PMID: 36600574 DOI: 10.1136/jcp-2022-208371] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2022] [Accepted: 11/30/2022] [Indexed: 12/12/2022]
Abstract
AIMS Several modes of assay interference common to immunoassays affect solid-phase single-antigen bead-based immunoassays (SAB) used to detect antibodies against human leucocyte antigens (HLA). Best practice recommendations include methods to address assay interference, though the clinical impact and optimal approaches are undefined. We sought to evaluate assay interference in HLA SAB to identify an efficient approach for avoiding erroneous results. METHODS Retrospective analysis of 14 059 patient samples tested for anti-HLA antibodies was performed. This included 4685 samples tested prior to implementation of serum pretreatment with EDTA and 4982 samples tested with routine EDTA treatment using the same testing algorithm. An algorithm for efficiently identifying and processing samples with suspected interference was evaluated in a separate cohort of 4392 EDTA-treated samples. RESULTS EDTA serum pretreatment reduced assay interference, but did not eliminate all modes of interference. A protocol for identification and testing of samples with suspected interference facilitated efficient detection of interference while reducing the amount of additional testing required. CONCLUSIONS Our data indicate that a single-method approach is insufficient to address all sources of interference in HLA SAB. A multimodal approach with a proactive screening is a more effective way to minimise risk of erroneous results.
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Affiliation(s)
- Joseph Abraha
- Department of Pathology, University of California San Diego, La Jolla, California, USA
| | - Ping Rao
- Aurora Health Care, Milwaukee, Wisconsin, USA
| | - Gerald P Morris
- Department of Pathology, University of California San Diego, La Jolla, California, USA
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3
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Pedini P, Hubert L, Baudey JB, Etienne JM, Basire A, Vey N, Chiaroni J, Chabrières C, Ladaique P, Picard C. Comparison of HLA antibody identification methods for the selection of platelet products for HLA-mediated platelet refractory patients. HLA 2024; 103:e15276. [PMID: 37947374 DOI: 10.1111/tan.15276] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2023] [Revised: 09/20/2023] [Accepted: 10/19/2023] [Indexed: 11/12/2023]
Abstract
In an ineffective transfusion context, solid-phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA-compatible platelet products. A new HLA antibody identification method, the HISTO SPOT® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT®. The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti-HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center. In total, 1414 class I specificities, 433 HLA-A and 981 HLA-B, were detected by the OL SAB test. The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens. The HISTO SPOT® HLA AB and C1q SAB tests identified 85% and 79% of OL SA-detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB-detected antigens, highlighting the lower sensitivity of these techniques. Interestingly, the donor-specific antibodies (DSAs) identified by the HISTO SPOT® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection. Although the HISTO SPOT® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA-compatible platelet products.
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Affiliation(s)
- Pascal Pedini
- Immunogenetic and Histocompatibility Laboratory, EFS PACC, Marseille, France
- ADES UMR, Aix Marseille Univ, Marseille, France
| | - Lucas Hubert
- Immunogenetic and Histocompatibility Laboratory, EFS PACC, Marseille, France
| | | | - Jean-Michel Etienne
- Immunohematology Laboratory, Institut Paoli-calmettes, EFS PACC, Marseille, France
| | - Agnes Basire
- Immunogenetic and Histocompatibility Laboratory, EFS PACC, Marseille, France
| | - Norbert Vey
- Onco-Hématology Department, Institut Paoli-calmettes, Marseille, France
| | | | - Corinne Chabrières
- Immunohematology Laboratory, Institut Paoli-calmettes, EFS PACC, Marseille, France
| | - Patrick Ladaique
- Onco-Hématology Department, Institut Paoli-calmettes, Marseille, France
| | - Christophe Picard
- Immunogenetic and Histocompatibility Laboratory, EFS PACC, Marseille, France
- ADES UMR, Aix Marseille Univ, Marseille, France
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4
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Gnanaraj J, Doss SA, Stephen S, Pratheeba M, Daniel D. Fallacies of a purely virtual platform: Virtual plus reality versus virtual reality - A case study. Transpl Immunol 2023; 81:101956. [PMID: 37952899 DOI: 10.1016/j.trim.2023.101956] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2023] [Accepted: 11/01/2023] [Indexed: 11/14/2023]
Abstract
Pretransplant immunological assessment of a transplant donor has evolved significantly over the last few decades with the advent of testing platforms with enhanced sensitivity and varying formats. The single antigen bead assay (SAB) assay, a virtual crossmatch (vXM) is used extensively and considered the gold standard for defining donor-specific antibodies (DSA) in many parts of the World. A country like India, is however challenged by the lack of adequate representation of locally frequent HLA alleles and hence in our institution, we continue to perform a physical crossmatch (pXM) on the Complement Dependent Cytotoxicity and flow cytometry platforms alongside the SAB. We report here a case report where the discrepancy between platforms of testing have raised certain pertinent questions in our interpretation of the vXM.
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Affiliation(s)
- John Gnanaraj
- Department of Transfusion medicine & Immunohaematology, Christian Medical College, Vellore, Tamil Nadu, India
| | - Sam Arul Doss
- Department of Transfusion medicine & Immunohaematology, Christian Medical College, Vellore, Tamil Nadu, India
| | - S Stephen
- Department of Transfusion medicine & Immunohaematology, Christian Medical College, Vellore, Tamil Nadu, India
| | - M Pratheeba
- Department of Transfusion medicine & Immunohaematology, Christian Medical College, Vellore, Tamil Nadu, India
| | - Dolly Daniel
- Department of Transfusion medicine & Immunohaematology, Christian Medical College, Vellore, Tamil Nadu, India.
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Ravindranath MH, Ravindranath NM, Selvan SR, Filippone EJ, Amato-Menker CJ, El Hilali F. Four Faces of Cell-Surface HLA Class-I: Their Antigenic and Immunogenic Divergence Generating Novel Targets for Vaccines. Vaccines (Basel) 2022; 10:vaccines10020339. [PMID: 35214796 PMCID: PMC8878457 DOI: 10.3390/vaccines10020339] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 02/07/2022] [Accepted: 02/17/2022] [Indexed: 12/19/2022] Open
Abstract
Leukocyte cell-surface HLA-I molecules, involved in antigen presentation of peptides to CD8+ T-cells, consist of a heavy chain (HC) non-covalently linked to β2-microglobulin (β2m) (Face-1). The HC amino acid composition varies across all six isoforms of HLA-I, while that of β2m remains the same. Each HLA-allele differs in one or more amino acid sequences on the HC α1 and α2 helices, while several sequences among the three helices are conserved. HCs without β2m (Face-2) are also observed on human cells activated by malignancy, viral transformation, and cytokine or chemokine-mediated inflammation. In the absence of β2m, the monomeric Face-2 exposes immunogenic cryptic sequences on these cells as confirmed by HLA-I monoclonal antibodies (LA45, L31, TFL-006, and TFL-007). Furthermore, such exposure enables dimerization between two Face-2 molecules by SH-linkage, salt linkage, H-bonding, and van der Waal forces. In HLA-B27, the linkage between two heavy chains with cysteines at position of 67 of the amino acid residues was documented. Similarly, several alleles of HLA-A, B, C, E, F and G express cysteine at 67, 101, and 164, and additionally, HLA-G expresses cysteine at position 42. Thus, the monomeric HC (Face-2) can dimerize with another HC of its own allele, as homodimers (Face-3), or with a different HC-allele, as heterodimers (Face-4). The presence of Face-4 is well documented in HLA-F. The post-translational HLA-variants devoid of β2m may expose several cryptic linear and non-linear conformationally altered sequences to generate novel epitopes. The objective of this review, while unequivocally confirming the post-translational variants of HLA-I, is to highlight the scientific and clinical importance of the four faces of HLA and to prompt further research to elucidate their functions and their interaction with non-HLA molecules during inflammation, infection, malignancy and transplantation. Indeed, these HLA faces may constitute novel targets for passive and active specific immunotherapy and vaccines.
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Affiliation(s)
- Mepur H. Ravindranath
- Department of Hematology and Oncology, Children’s Hospital, Los Angeles, CA 90027, USA
- Emeritus Research Scientist at Terasaki Foundation Laboratory, Santa Monica, CA 90064, USA
- Correspondence:
| | - Narendranath M. Ravindranath
- Norris Dental Science Center, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA 90089, USA;
| | | | - Edward J. Filippone
- Division of Nephrology, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19145, USA;
| | - Carly J. Amato-Menker
- Department of Microbiology, Immunology and Cell Biology, School of Medicine, West Virginia University, Morgantown, WV 26506, USA;
| | - Fatiha El Hilali
- The Faculty of Medicine and Pharmacy of Laayoune, Ibn Zohr University, Agadir 70000, Morocco;
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Ravindranath MH, Filippone EJ, Amato-Menker CJ, Arosa FA, Das B, Ou Y, Norin AJ. Antibodies to cryptic epitopes on HLA class I and class II heavy chains bound to single antigen beads: Clinically relevant? Transpl Immunol 2021; 69:101482. [PMID: 34656784 DOI: 10.1016/j.trim.2021.101482] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2021] [Revised: 10/08/2021] [Accepted: 10/08/2021] [Indexed: 10/20/2022]
Abstract
Cell surface HLA class I consists of trimers, i.e., alpha - heavy chain, beta - 2 - microglobulin, and a peptide, termed closed conformers (CC) on non-activated lymphocytes. HLA class I and class II may also exist, respectively, as alpha-chain only or alpha and beta - chain only on activated cells termed open conformers (OC). We extend previous studies using an OC-specific monoclonal antibody that demonstrate LABScreen HLA class I and II single antigen beads (SABs) contain a mixture of open and closed conformers. LIFECODES SABs have bound CC only. More HLA class I and class II LABScreen SABs were reactive than LIFECODES SABs due to the presence of OC on LABScreen SABs. We hypothesized that antibody against OC on HLA B antigens would not be detected in cell based cross matches (XMs) with typical lymphocyte targets since anti-HLA OC antibodies would not react with native HLA CC on the cell surface. To test this hypothesis, we performed flow cytometry XM (FCXM) assays with sera of sufficient strength that most laboratories would likely predict positive FCXMs. Sera that reacted strongly with LABScreen SABs (>13,000 MFI) but weakly or not at all with LIFECODES SABs (<1000 MFI) gave negative T and B cell FCXMs. In contrast, sera that reacted with LIFECODES SABs (>13,000 MFI) but weakly with LABScreen SABs (<2100 MFI) exhibited positive FCXMs. Detection of antibodies directed against OC in SAB assays, may lead to inappropriate listing of unacceptable antigens, a decision not to XM or pre-or post - transplant desensitization procedures.
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Affiliation(s)
- Mepur H Ravindranath
- Department. of Hematology and Oncology, Children's Hospital, Los Angeles, CA 90027, United States of America
| | - Edward J Filippone
- Division of Nephrology, Dept. of Medicine, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA 19145, United States of America
| | - Carly J Amato-Menker
- Department of Immunology and Microbial Pathogenesis, West Virginia University, School of Medicine, Morgantown, WV 26506, United States of America
| | - Fernando A Arosa
- Health Sciences Research Center (CICS-UBI) & Department of Medical Sciences, University of Beira Interior, Covilhã 6200-506, Portugal.
| | - Ballabh Das
- Department of Pathology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States of America.
| | - Yijun Ou
- Department of Pathology, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States of America.
| | - Allen J Norin
- Department of Medicine and Cell Biology, Transplant Immunology and Immunogenetics, SUNY Downstate Health Sciences University, Brooklyn, NY 11203, United States of America.
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Launhardt K, Lefevre V, Souplet V, Prantl L, Marget M, Hovoricova B, Wenda S, Olivier C. Concordance with established tests and reproducibility of results obtained with a new single antigen chip array for HLA antibody detection (HISTO SPOT® HLA AB). J Immunol Methods 2021; 491:112971. [PMID: 33549571 DOI: 10.1016/j.jim.2021.112971] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 12/30/2020] [Accepted: 01/21/2021] [Indexed: 11/18/2022]
Abstract
The purpose of this study was to analyze performance of a new single antigen chip array system (HISTO SPOT® HLA AB) developed for HLA antibody detection and compare with results obtained using single antigen Luminex-based systems and serum samples from the Eurotransplant external proficiency testing scheme. Results were analysed from 11 independent Eurotransplant laboratories using HISTO SPOT® HLA AB utilising the Eurotransplant external proficiency testing (EPT) sera and these were compared to published results from 67 labs using the Luminex-based technologies. In addition, QC results from different batches of the test were analysed. Generally, concordance of results with the results from the Luminex technique was good. With the Luminex tests more consensus results and more questionable results were found than with the HISTO SPOT® HLA AB test. Within the HISTO SPOT® HLA AB testing group we found a discrepancy rate from the consensus of 2.9% for the EPT sera which is far below the 25% allowed to pass the quality test and only slightly higher than for the Luminex single antigen tests with 1.2%. The average global coefficient of variation (CV) of the mean signal (raw data) for the HISTO SPOT® HLA AB test was 13% which is lower than the values reported for Luminex tests in the literature. The average global CV for the signal/background ratio was higher with 28%. In the present study, the mean signal is the best parameter to compare results between labs and the new HISTO SPOT® HLA AB test is at least as good in terms of signal reproducibility as the Luminex tests. In conclusion, the HISTO SPOT® HLA AB test is a good alternative to be used in addition or instead of the Luminex tests in clinical labs.
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Affiliation(s)
| | | | - Vianney Souplet
- Innobiochips, 70, rue du Docteur Yersin, 59 120 Loos, France
| | - Livia Prantl
- Central Institute for Blood Transfusion and Immunology, University Hospital, Anichstr. 35, 6020 Innsbruck, Austria
| | - Matthias Marget
- Institute for Transfusion Medicine, University Hospital Hamburg Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
| | - Blanka Hovoricova
- University Hospital F. D. Roosevelta Banska Bystrica, Department of Laboratory Hematology - HLA Laboratory, Namestie L. Svobodu 1, 975 17, Banska Bystrica, Slovakia
| | - Sabine Wenda
- Medical University Vienna, University Hospital for Blood Group Serology and Transfusion Medicine, Währinger Gürtel 18-20 / Ebene 4i, 1090 Vienna, Austria
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8
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DSA-FXM: Accelerated Donor-specific Flow Crossmatch Discriminating Class I and II Antibody Specifically and Only to Donor HLA for Determining True Incompatibility. Transplantation 2019; 104:813-822. [PMID: 31385929 DOI: 10.1097/tp.0000000000002900] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Worldwide, a final crossmatch is the gold standard for determining compatibility between patient and donor before solid organ transplantation and preventing hyperacute rejection. In the absence of autoantibodies, an incompatible crossmatch in a sensitized patient is attributed to mismatched donor HLA. However, current physical crossmatch methods cannot distinguish reactivity to HLA from other clinically irrelevant cell surface targets nor the class of HLA if it is the target. Result interpretation is difficult or impossible when autoantibodies, alloantibodies, or therapeutic antibodies coexist. METHODS Herein, we describe a unique donor-specific flow crossmatch (DSA-FXM) that distinguishes HLA class I or II donor-specific antibody bound to HLA antigens on the donor cell surface in their native conformation that is not impacted by rituximab, anti-thymocyte globulin (after absorption), or autoantibodies. It is HLA specific. RESULTS We compared the results of single-antigen antibody testing, autoreactive and alloreactive flow cytometry crossmatches (FXM) using traditional FXM and our DSA-FXM method from 94 patients (enriched for auto+/allo+ pairs; n = 64) against 110 donors (338 tests) and show that, in our cohort, positive traditional FXM results are not directed to donor HLA 60.25% of the time and negative traditional FXM results are missing HLA donor-specific antibody 36.2% of the time based on the DSA-FXM. CONCLUSIONS We demonstrate that the DSA-FXM is able to define categorically distinct and clinically important HLA antibody profiles in half the time required for the standard FXM, potentially shortening cold ischemia time and providing clinicians with unambiguous essential information regarding HLA compatibility when time is critical.
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9
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Huang Y, Dinh A, Heron S, Gasiewski A, Kneib C, Mehler H, Mignogno MT, Morlen R, Slavich L, Kentzel E, Frackelton EC, Duke JL, Ferriola D, Mosbruger T, Timofeeva OA, Geier SS, Monos D. Assessing the utilization of high-resolution 2-field HLA typing in solid organ transplantation. Am J Transplant 2019; 19:1955-1963. [PMID: 30623581 DOI: 10.1111/ajt.15258] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2018] [Revised: 12/18/2018] [Accepted: 01/03/2019] [Indexed: 01/25/2023]
Abstract
HLA typing in solid organ transplantation (SOT) is necessary for determining HLA-matching status between donor-recipient pairs and assessing patients' anti-HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serologic equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next-generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high-resolution 2-field HLA typing (HR-2F) in SOT by retrospectively evaluating NGS-typed pre- and post-SOT cases. HR-2F typing was highly instructive or necessary in 41% (156/385) of the cases. Several pre- and posttransplant scenarios were identified as being better served by HR-2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of the cases required HR-2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti-HLA antibody issues.
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Affiliation(s)
- Yanping Huang
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Anh Dinh
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Steven Heron
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Allison Gasiewski
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Carolina Kneib
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Hilary Mehler
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Michael T Mignogno
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Ryan Morlen
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Larissa Slavich
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Ethan Kentzel
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Edward C Frackelton
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Jamie L Duke
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Deborah Ferriola
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Timothy Mosbruger
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
| | - Olga A Timofeeva
- Department of Pathology and Laboratory Medicine, Katz Medical School, Temple University, Philadelphia, Pennsylvania
| | - Steven S Geier
- Department of Pathology and Laboratory Medicine, Katz Medical School, Temple University, Philadelphia, Pennsylvania
| | - Dimitri Monos
- Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.,Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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10
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Ravindranath MH, Filippone EJ, Mahowald G, Callender C, Babu A, Saidman S, Ferrone S. Significance of the intraindividual variability of HLA IgG antibodies in renal disease patients observed with different beadsets monitored with two different secondary antibodies on a Luminex platform. Immunol Res 2019; 66:584-604. [PMID: 30324227 PMCID: PMC6244961 DOI: 10.1007/s12026-018-9027-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The accurate measurement of anti-HLA alloantibodies in transplant candidates is required for determining the degree of sensitization and for the listing of unacceptable antigens for organ allocation. Both the configuration of the HLA molecules coated on the beads and the nature of detection antibodies may impede assessment of the presence and strength of anti-HLA IgG- with the Luminex single-antigen-bead assay. Sera antibodies of the end-stage renal disease patients were compared using LIFECODES (LC) and LABScreen (LS) beadsets monitored with polyclonal-Fab (IgHPolyFab) and monoclonal-IgG (FcMonoIgG) second antibodies. Positive results at mean fluorescence intensity (MFI) > 500 (at serum dilution 1/10) were used to calculate panel reactive antibody (cPRA) levels. LS-beadsets are coated with monomeric variants in addition to intact HLA antigens with or without peptides, while LC-beadsets are devoid of monomeric variants and with lesser levels of peptide-free heterodimers. Consequently, IgG antibodies against both classes of HLA were reactive to more antigens with LS than with LC-beadsets. For both classes, MFIs were also frequently higher with LS than with LC. For HLA-I, MFIs were higher with IgHPolyFab than with FcMonoIgG with the exception of sera with MFIs > 5000 where they were comparable. For HLA-II, the reverse occurred, with significantly higher levels with FcMonoIgG regardless of the beadsets. The intraindividual variability observed between beadsets with two detection antibodies elucidates that antigens found as acceptable with one beadset may end up unacceptable with the other beadsets, with the possibility of denying potentially compatible transplants to candidates.
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Affiliation(s)
| | - Edward J Filippone
- Division of Nephrology, Department of Medicine, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA
| | - Grace Mahowald
- Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | | | - Adarsh Babu
- CSRL, University Hospitals Coventry and Warwickshire, Clifford Bridge Road, Coventry, UK
| | - Susan Saidman
- Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Soldano Ferrone
- Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
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11
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Passamonti SM, Cannavò A, Espadas de Arias A, Innocente A, Ramondetta M, Regalia A, Messa P, Ferraresso M, Cardillo M. Pretransplant Single Antigen Bead-Detected HLA Antibodies in Kidney Transplant Long-term Outcome: A Single-Center Cohort Experience. Transplant Proc 2019; 51:707-714. [PMID: 30979454 DOI: 10.1016/j.transproceed.2019.01.063] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2018] [Revised: 12/14/2018] [Accepted: 01/17/2019] [Indexed: 11/17/2022]
Abstract
Single-antigen bead (SAB) platform permits the identification of antibodies not detectable by complement-dependent lymphocytotoxicity test, but their clinical significance is not completely understood. The aim of this study was to evaluate whether the presence of pretransplant SAB-detected antibodies is associated with the development of allograft failure. This is a single-center cohort study with 10-year follow-up in which 573 kidney recipients with negative pretransplant complement-dependent lymphocytotoxicity crossmatch who received transplants at the Kidney Transplant Center of Policlinico, Milan, from deceased donors between 1996 and 2005 were evaluated. Pretransplant plasma samples were retrospectively analyzed by SAB assay. Survival analyses were performed to assess the risk of allograft failures by SAB-detected antibodies. Pretransplant antibodies were found in 160 (28.0%) recipients, of whom 42 subsequently developed an allograft failure for a survival rate of 70.9% (95% confidence interval [CI), 63.5-78.4). Among those without antibodies, 58 (14.0%) returned to dialysis with a survival rate of 84.7% (95% CI, 81.0-88.4). In Cox regression analyses, patients with SAB-positivity had 2-fold higher risk of allograft failure than those who were SAB-negative (hazard ratio, 2.07; 95% CI, 1.39-2.79). Results did not change after adjustment for putative confounders. In conclusion, in this single-center cohort, 10-year allograft survival rate was significantly influenced by the presence of SAB-detected antibodies.
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Affiliation(s)
- S M Passamonti
- North Italy Transplant program (NITp), UOC Coordinamento Trapianti, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy.
| | - A Cannavò
- North Italy Transplant program (NITp), UOC Coordinamento Trapianti, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy
| | - A Espadas de Arias
- North Italy Transplant program (NITp), UOC Coordinamento Trapianti, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy
| | - A Innocente
- North Italy Transplant program (NITp), UOC Coordinamento Trapianti, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy
| | - M Ramondetta
- North Italy Transplant program (NITp), UOC Coordinamento Trapianti, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy
| | - A Regalia
- Unit of Nephrology, Dialysis, and Renal Transplant, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy and University of Milan
| | - P Messa
- Unit of Nephrology, Dialysis, and Renal Transplant, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy and University of Milan
| | - M Ferraresso
- Kidney Transplant Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy and University of Milan
| | - M Cardillo
- North Italy Transplant program (NITp), UOC Coordinamento Trapianti, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy
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Comparison of Two Luminex Single-antigen Bead Flow Cytometry Assays for Detection of Donor-specific Antibodies After Renal Transplantation. Transplantation 2019; 103:597-603. [DOI: 10.1097/tp.0000000000002351] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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13
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Timofeeva OA. Donor-Specific HLA Antibodies as Biomarkers of Transplant Rejection. Clin Lab Med 2019; 39:45-60. [DOI: 10.1016/j.cll.2018.10.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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14
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15
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Alzahrani M, Qahtani Z, Harbi H, Kebasi S, Essa O, Al Attas R. Virtual Crossmatch: Reality of Perception. Transplant Proc 2019; 51:488-491. [PMID: 30879574 DOI: 10.1016/j.transproceed.2019.01.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Results of 773 actual flow crossmatches (aFXMs) and virtual flow crossmatches (vFXMs) performed for living and deceased donor kidney transplantation in our center were analyzed retrospectively and evaluated for their concordance. Prediction of vFXMs was based on antibody identification using single antigen bead assay and locally established mean fluorescence intensity cutoff point compared with donor HLA antigens. The vast majority of aFXMs were in concordance with vFXMs with an overall concordance of 97%. Twenty-three predicted to be negative showed positive aFXMs; 12 of them had 0% calculated panel-reactive antibody, and 11 were found in patients with multiple non-donor-specific HLA antibodies. Three predicted positive vFXMs yielded negative aFXMs; 2 of them had allele-specific antibodies. CONCLUSIONS: vFXMs based on precise characterization of antibody specificities detected by single antigen bead assay using our cutoff point accurately predicted FXMs in the majority of patients and can be used safely to allocate kidney offers without performing physical crossmatches in selected patients.
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Affiliation(s)
- M Alzahrani
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Z Qahtani
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - H Harbi
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - S Kebasi
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - O Essa
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - R Al Attas
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia.
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16
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Al Attas R, Alzahrani M, Al-Otaibi AS, Lopez R, Liacini A, Alzahrani S, Ajlan K, Abduladheem D, Kebasi S, Harbi H. Discrepant Antibody Testing Results: Which One to Believe? Transplant Proc 2019; 51:497-503. [PMID: 30879576 DOI: 10.1016/j.transproceed.2019.01.009] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The impact of solid-phase immunoassay for HLA antibody detection on the field of transplantation has been extremely significant by providing the most sensitive and precise method for characterization of HLA antibodies. However, despite all the benefits, technical limitations and inherent artifacts represent significant challenges, particularly with Luminex-based single-antigen bead (SAB) assay. Discordant results between antibody detection (screening assay) and identification (SAB) is not uncommon. Positive SAB assay in the context of negative screening testing is well documented and attributed to altered tertiary structure of HLA molecules exposing new epitopes or detection of naturally occurring antibodies. However, there are few reports that addressed the opposite scenario when negative SAB appeared in the context of positive screening assay. In such discrepant results, unmissed HLA antibody has to be excluded with certainty by other tests; however, with the availability of variable assays it may be difficult to choose the best combinations that clarify discrepancy without adding more confusion. Here we describe the results of correlation between 2 antibody screening solid-phase immunoassays (LABScreen Mixed using Luminex and FlowPRA Screen) on conventional flow cytometry and compare their outcomes with SAB and crossmatch results.
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Affiliation(s)
- Rabab Al Attas
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia.
| | - Mariam Alzahrani
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Ahmed S Al-Otaibi
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Ricardo Lopez
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Abdelhamid Liacini
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Saber Alzahrani
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Kenana Ajlan
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Dalal Abduladheem
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Shaima Kebasi
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
| | - Hassan Harbi
- Histocompatibility and Immunogenetic Laboratory, Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital-Dammam, Dammam, Saudi Arabia
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17
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Koefoed-Nielsen P, Møller BK. Donor-specific anti-HLA antibodies by solid phase immunoassays: advantages and technical concerns. Int Rev Immunol 2018; 38:95-105. [DOI: 10.1080/08830185.2018.1525367] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Affiliation(s)
| | - Bjarne Kuno Møller
- Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark
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18
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Katalinić N, Starčević A, Mavrinac M, Balen S. Complement-dependent cytotoxicity and Luminex technology for human leucocyte antigen antibody detection in kidney transplant candidates exposed to different sensitizing events. Clin Kidney J 2017; 10:852-858. [PMID: 29225816 PMCID: PMC5716092 DOI: 10.1093/ckj/sfx050] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Accepted: 04/27/2017] [Indexed: 01/02/2023] Open
Abstract
Background The aim of this study was to determine the frequency of exposure to different sensitizing events (SEs) and to assess their effects on human leucocyte antigen (HLA) alloimmunization in transplant candidates using two different HLA antibody screening techniques: complement-dependent cytotoxicity (CDC) and Luminex. Methods This retrospective study included HLA antibody screening results for 163 patients on the kidney transplant waiting list (WL) tested from March 2012 until the end of December 2015 at the Tissue Typing Laboratory, Rijeka, Croatia. All sera samples were tested using the CDC and Luminex techniques in parallel. Results Two-thirds of the patients [114 (70%)] on the WL were exposed to transfusions, pregnancies and/or kidney transplant. The pre-transplant sera of 104 (63.80%) patients were negative for antibodies. In the sera of 23 (14.11%) patients, HLA antibodies were detected by CDC and Luminex and in the sera of 36 (22.09%) patients by Luminex only. Conclusion In patients on kidney WL, previous organ transplantation represents the strongest immunogenic stimulus, followed by blood transfusions (the most frequent SE) and pregnancies. Although Luminex is more sensitive than CDC in HLA antibody detection, the decision on unacceptable HLA antigens in WL patients has to be based on the results of both assays and the patient's immunization history.
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Affiliation(s)
- Nataša Katalinić
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Centre Rijeka, Croatia.,Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Croatia
| | - Alma Starčević
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Centre Rijeka, Croatia
| | - Martina Mavrinac
- Department of Medical Informatics, Faculty of Medicine, University of Rijeka, Croatia
| | - Sanja Balen
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Centre Rijeka, Croatia.,Department of Clinical Laboratory Diagnostics, Faculty of Medicine, University of Rijeka, Croatia
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19
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Sullivan HC, Gebel HM, Bray RA. Understanding solid-phase HLA antibody assays and the value of MFI. Hum Immunol 2017; 78:471-480. [DOI: 10.1016/j.humimm.2017.05.007] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2017] [Revised: 05/26/2017] [Accepted: 05/29/2017] [Indexed: 01/10/2023]
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20
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Akgul SU, Ciftci HS, Temurhan S, Caliskan Y, Bayraktar A, Tefik T, Kaya IA, Canitez IO, Demir E, Yazici H, Bakkaloglu H, Aydin AE, Turkmen A, Nane I, Aydin F, Oguz FS. Association Between HLA Antibodies and Different Sensitization Events in Renal Transplant Candidates. Transplant Proc 2017; 49:425-429. [PMID: 28340805 DOI: 10.1016/j.transproceed.2017.02.004] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
BACKGROUND Human leukocyte antigen (HLA) allo-immunization is caused by various events such as blood transfusions, pregnancies, or organ transplantations, which can lead to sensitization. In this retrospective study, we evaluated different sensitization models and their effects on panel-reactive antibody (PRA) profiles of renal transplantation candidates. METHODS Anti-HLA class I/II antibody screening tests were performed in 906 renal transplantation candidates with the use of a microbead-based assay (Luminex). RESULTS Two hundred ninety-seven (32.8%) of the patients were determined as positive in terms of PRA, and 609 (67.2%) were negative. Sensitized and non-sensitized patients were compared separately in terms of each sensitization type. The anti-HLA class I, II, and I+II positivity rates in patients sensitized only by blood transfusion were 13.1%, 6.3%, and 14.1%, the rates with pregnancy sensitization were 35.5%, 29%, and 45.2%, and rates with previous transplantation sensitization were 15.6%, 34.4%, and 38.9%, respectively. Prevalence of PRA positivity was significantly higher in patients with previous pregnancy than with transplantation and transfusion (odds ratio, 1.003; 95% confidence interval, 0.441-2.281; P = .031). The risk of developing HLA class I antibodies was higher in pregnancies (P < .001), and the risk of developing anti-HLA class II antibodies was higher in patients who had undergone a previous transplantation (P < .001). The rate of developing HLA-B antibodies in patients sensitized by pregnancy were significantly higher compared with sensitization after transfusion (P = .015), as was the rate of developing HLA-DQ antibodies in patients sensitized by previous transplantation compared with sensitization through pregnancy (P = .042). CONCLUSIONS In patients who are waiting for kidney transplantation, sensitization by pregnancy and transplantation have a significant impact on development of HLA class I and class II antibodies.
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Affiliation(s)
- S U Akgul
- Department of Medical Biology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey.
| | - H S Ciftci
- Department of Medical Biology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - S Temurhan
- Department of Medical Biology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - Y Caliskan
- Department of Nephrology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - A Bayraktar
- Department of General Surgery, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - T Tefik
- Department of Urology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - I A Kaya
- Department of Medical Biology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - I O Canitez
- Department of Medical Biology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - E Demir
- Department of Nephrology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - H Yazici
- Department of Nephrology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - H Bakkaloglu
- Department of General Surgery, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - A E Aydin
- Department of General Surgery, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - A Turkmen
- Department of Nephrology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - I Nane
- Department of Urology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - F Aydin
- Department of Medical Biology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
| | - F S Oguz
- Department of Medical Biology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey
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21
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Zerrouki A, Ouadghiri S, Benseffaj N, Razine R, Essakalli M. Reason and Resolution of High Negative Control Beads in Solid-Phase Immunoassay. EXP CLIN TRANSPLANT 2017; 16:38-43. [PMID: 28540842 DOI: 10.6002/ect.2016.0239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
OBJECTIVES The Luminex technology is the most sensitive diagnostic method for HLA antibody detection and identification. However, the interpretation of immunoassays is commonly affected by the artifact, and non-specific background. Sera from some patients show high negative control bead (NC) value, which makes assessing and interpretation of HLA antibodies difficult. In this study, we evaluated the effect of Adsorb Out reagent, dithiothreitol (DTT), and Ethylenediaminetetraacetic acid (EDTA) on the NC median fluorescence intensity value by comparing treated versus untreated patient sera. In addition, we wanted to identify whether kidney disease and administered medication influenced high NC median fluorescence intensity values by comparing patient versus control results. MATERIALS AND METHODS HLA antibody screening was performed on 3500 serum samples. Sera were analyzed using the standard protocol for Luminex antibody screening. Sera with high NC values were preincubated with Adsorb Out, DTT, and EDTA. Screening of these sera was then performed. RESULTS We found that 4% of samples showed high NC values. Adsorb Out, DTT, and EDTA decreased the NC values at 723.5 (299.25-1443) versus 85 (34-218; P < .001), at 723.5 (299.25-1443) versus 184 (106-597; P < .001), and at 723.5 (299.25-1443) versus 455 (131-1177; P = .004). These succeeded in bringing back NC values to normal range in 69.2%, 43%, and 30% of treated sera, respectively. In addition, the differences of corticoids, immunosuppressive, and heparin drugs between patients and controls were statistically significant (P < .001, < .001, and = .043). However, presence of kidney disease was not significant between these groups. CONCLUSIONS All pretreatments had an important effect in decreasing negative control values, with Adsorb Out having highest efficiency. Serum-specific components could contribute to high negative control bead median fluorescence intensity values. Further studies are needed to determine the adequate pretreatment of patient sera.
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Affiliation(s)
- Asmae Zerrouki
- From the UPR d'immunologie, Faculté de médecine et de pharmacie de Rabat, Université Mohamed V, Rabat, Morocco
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22
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Conformational Variants of the Individual HLA-I Antigens on Luminex Single Antigen Beads Used in Monitoring HLA Antibodies: Problems and Solutions. Transplantation 2017; 101:764-777. [PMID: 27495776 DOI: 10.1097/tp.0000000000001420] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Single antigen beads (SAB) are used for monitoring HLA antibodies in pretransplant and posttransplant patients despite the discrepancy between virtual and actual crossmatch results and transplant outcomes. This discrepancy can be attributed to the presence of conformational variants of HLA-I on SAB, assessment of which would increase the concordance between SAB and flow cytometry crossmatch (FCXM) results, thus enabling improved organ accessibility for the waiting list patients and a better prediction of antibody-mediated rejection. METHODS The conformational variants were examined on HLA-I beads, iBeads, acid-/alkali-treated beads, and T cells using HLA-I monoclonal antibodies (W6/32, TFL-006, and heavy chain (HC)-10). RESULTS The affinity of the monoclonal antibodies against HLA-I beads confirmed the presence and heterogeneous density of peptide-associated β2-microglobulin-associated HLA HC (pepA-β2aHC), peptide-free-β2aHC (pepF-β2aHC), and β2-free HC (β2fHC) on every single antigen-coated bead. In contrast, iBeads harbor a high density of pepA-β2aHC, low density of pepF-β2aHC, and are lacking β2fHC. The FCXM analyses confirmed the prevalence of pepA-β2aHC, but not pepF-β2aHC or β2fHC on resting T cells. CONCLUSIONS The strength of a donor-specific antibody should be assessed with a bead-specific mean fluorescence intensity cutoff based on TFL-006 reactivity against HLA-I beads, and HC-10 against iBeads, where the β2fHC or pepF-β2aHC normalized donor-specific antibody level would reveal the true anti-pepA-β2aHC reactivity associated with positive FCXM.
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23
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Fasano RM, Sullivan HC, Bray RA, Gebel HM, Meyer EK, Winkler AM, Josephson CD, Stowell SR, Sandy Duncan A, Roback JD. Genotyping Applications for Transplantation and Transfusion Management: The Emory Experience. Arch Pathol Lab Med 2017; 141:329-340. [PMID: 28234571 DOI: 10.5858/arpa.2016-0277-sa] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Current genotyping methodologies for transplantation and transfusion management employ multiplex systems that allow for simultaneous detection of multiple HLA antigens, human platelet antigens, and red blood cell (RBC) antigens. The development of high-resolution, molecular HLA typing has led to improved outcomes in unrelated hematopoietic stem cell transplants by better identifying compatible alleles of the HLA-A, B, C, DRB1, and DQB1 antigens. In solid organ transplantation, the combination of high-resolution HLA typing with solid-phase antibody identification has proven of value for highly sensitized patients and has significantly reduced incompatible crossmatches at the time of organ allocation. This database-driven, combined HLA antigen/antibody testing has enabled routine implementation of "virtual crossmatching" and may even obviate the need for physical crossmatching. In addition, DNA-based testing for RBC antigens provides an alternative typing method that mitigates many of the limitations of hemagglutination-based phenotyping. Although RBC genotyping has utility in various transfusion settings, it has arguably been most useful for minimizing alloimmunization in the management of transfusion-dependent patients with sickle cell disease or thalassemia. The availability of high-throughput RBC genotyping for both individuals and large populations of donors, along with coordinated informatics systems to compare patients' antigen profiles with available antigen-negative and/or rare blood-typed donors, holds promise for improving the efficiency, reliability, and extent of RBC matching for this population.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | - John D Roback
- From the Center for Transfusion and Cellular Therapies (Drs Fasano, Sullivan, Meyer, Winkler, Josephson, Stowell, Duncan, and Roback) and the Department of Pathology and Laboratory Medicine (Drs Fasano, Sullivan, Bray, Gebel, Meyer, Winkler, Josephson, Stowell, Duncan, and Roback), Emory University School of Medicine, Atlanta, Georgia; and the Department of Transfusion, Tissue, and Apheresis, Children's Healthcare of Atlanta, Atlanta (Drs Fasano, Meyer, and Josephson). Dr Meyer is now with the Department of Pathology, Nationwide Children's Hospital, Ohio State University College of Medicine, Columbus
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24
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Nakamura J, Nakajima F, Kamada H, Tadokoro K, Nagai T, Satake M. Males without apparent alloimmunization could have HLA antibodies that recognize target HLA specificities expressed on cells. HLA 2017; 89:285-292. [DOI: 10.1111/tan.13000] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2016] [Revised: 01/18/2017] [Accepted: 02/10/2017] [Indexed: 11/26/2022]
Affiliation(s)
- J. Nakamura
- Central Blood Institute; Japanese Red Cross Society; Tokyo Japan
| | - F. Nakajima
- Central Blood Institute; Japanese Red Cross Society; Tokyo Japan
| | - H. Kamada
- Central Blood Institute; Japanese Red Cross Society; Tokyo Japan
| | - K. Tadokoro
- Blood Service Headquarters; Japanese Red Cross Society; Tokyo Japan
| | - T. Nagai
- Central Blood Institute; Japanese Red Cross Society; Tokyo Japan
| | - M. Satake
- Central Blood Institute; Japanese Red Cross Society; Tokyo Japan
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25
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Jani V, Ingulli E, Mekeel K, Morris GP. Root cause analysis of limitations of virtual crossmatch for kidney allocation to highly-sensitized patients. Hum Immunol 2017; 78:72-79. [DOI: 10.1016/j.humimm.2016.11.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2016] [Revised: 11/11/2016] [Accepted: 11/14/2016] [Indexed: 10/20/2022]
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26
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Katalinić N, Fućak M, Crnić T, Ćurković M, Starčević A, Balen S. Pretransplantation monitoring of HLA antibodies by complement dependent cytotoxicity and Luminex-based assays. Wien Klin Wochenschr 2016; 129:33-37. [PMID: 27743177 DOI: 10.1007/s00508-016-1094-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Accepted: 09/14/2016] [Indexed: 11/25/2022]
Abstract
BACKGROUND The introduction of more sensitive techniques, such as Luminex® for HLA antibody screening of patients awaiting organ transplantation has resulted in a better understanding of transplantation immunology and improvements in clinical practice. OBJECTIVE The interpretation of the results obtained only by Luminex® can lead to inaccurate evaluation of a patient's antibody status and unjustified rejection of a potential organ donor. The aim of this study was to demonstrate the benefits of performing HLA antibody screening in the sera of patients on the waiting list for organ transplantation by two different assays, complement dependent cytotoxicity (CDC) and Luminex®. METHODS A retrospective analysis was performed on 563 pretransplant serum samples from 141 patients on the kidney transplantation waiting list in Rijeka, tested from March 2012 until March 2015. All samples were tested in parallel by the CDC assay and the Luminex®-based assay. RESULTS Out of the 563 samples tested 302 (53.7%) tested negative for HLA antibodies and 88 (15.6%) positive by both assays. From 173 (30.7%) samples with discordant results 149 (26.5%) were CDC negative and Luminex® positive, while 24 (4.3%) were CDC positive and Luminex® negative. Among the Luminex positive patients seven did not experience any immunizing events. CONCLUSION Evaluation of the HLA antibody status in patients on a waiting list for organ transplantation should be based on the results of the both CDC and Luminex® (or other sensitive) assays in accordance to information about patient's clinical status and exposure to immunizing events.
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Affiliation(s)
- Nataša Katalinić
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Center Rijeka, Tome Strižića 3, 51 000, Rijeka, Croatia.
| | - Marina Fućak
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Center Rijeka, Tome Strižića 3, 51 000, Rijeka, Croatia
| | - Tajana Crnić
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Center Rijeka, Tome Strižića 3, 51 000, Rijeka, Croatia
| | - Milena Ćurković
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Center Rijeka, Tome Strižića 3, 51 000, Rijeka, Croatia
| | - Alma Starčević
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Center Rijeka, Tome Strižića 3, 51 000, Rijeka, Croatia
| | - Sanja Balen
- Tissue Typing Laboratory, Clinical Institute for Transfusion Medicine, Clinical Hospital Center Rijeka, Tome Strižića 3, 51 000, Rijeka, Croatia
- Faculty of Medicine, University of Rijeka, Rijeka, Croatia
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27
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Schinstock CA, Gandhi MJ, Stegall MD. Interpreting Anti-HLA Antibody Testing Data: A Practical Guide for Physicians. Transplantation 2016; 100:1619-28. [PMID: 27140516 PMCID: PMC4961613 DOI: 10.1097/tp.0000000000001203] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
The development of sensitive methods for alloantibody detection has been a significant advance in clinical transplantation. However, the complexity of the data from solid phase and crossmatch assays has led to potential confusion about how to use the results for clinical decision making. The goal of this review is to provide a practical guide for transplant physicians for the interpretation of antibody data to supplement consultation with local tissue typing experts. Sources of variability in both the solid phase and crossmatch assay are discussed as are recent data regarding C1q binding antibodies and IgG subclass testing. Although definitive approaches to alloantibody testing are not possible with our current knowledge, we outline a pragmatic approach that we hope will enhance clinical management in this area.
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Affiliation(s)
- Carrie A Schinstock
- 1 William J. von Liebig Transplant Center, Mayo Clinic, Rochester, MN.2 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN
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Focosi D. Advances in Pretransplant Donor-Specific Antibody Testing in Solid Organ Transplantation: From Bench to Bedside. Int Rev Immunol 2016; 35:351-368. [PMID: 27120091 DOI: 10.3109/08830185.2016.1154051] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Immunological risk stratification has a central role in determining both the feasibility of solid organ transplantation and the type (and amount) of induction and maintenance immunosuppressive therapy. Currently there is poor consensus on how to exactly estimate the global immunological risk, and most transplant centers adopt complicated internal guidelines for risk stratification. Here we systematically review published evidences that should drive appropriateness in risk stratification, focusing on donor-specific antibodies against HLA and other antigens.
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Affiliation(s)
- Daniele Focosi
- a Department of Translational Research , University of Pisa , Pisa , Italy
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29
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Carey BS, Boswijk K, Mabrok M, Rowe PA, Connor A, Saif I, Poles A. A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect. Transpl Immunol 2016; 37:23-27. [PMID: 27109036 DOI: 10.1016/j.trim.2016.04.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2016] [Revised: 04/10/2016] [Accepted: 04/15/2016] [Indexed: 11/28/2022]
Abstract
Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines) [1]. Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect. Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer.
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Affiliation(s)
- B Sean Carey
- H&I, Combined Labs, Derriford Hospital, Plymouth, United Kingdom.
| | - Kim Boswijk
- H&I, Combined Labs, Derriford Hospital, Plymouth, United Kingdom
| | - Mazen Mabrok
- H&I, Combined Labs, Derriford Hospital, Plymouth, United Kingdom
| | - Peter A Rowe
- South West Transplant Centre, Derriford Hospital, Plymouth, United Kingdom
| | - Andrew Connor
- South West Transplant Centre, Derriford Hospital, Plymouth, United Kingdom
| | - Imran Saif
- South West Transplant Centre, Derriford Hospital, Plymouth, United Kingdom
| | - Anthony Poles
- H&I, Combined Labs, Derriford Hospital, Plymouth, United Kingdom; NHS-BT, Filton, United Kingdom
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30
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Michel K, Santella R, Steers J, Sahajpal A, Downey FX, Thohan V, Oaks M. Many de novo donor-specific antibodies recognize β2 -microglobulin-free, but not intact HLA heterodimers. HLA 2016; 87:356-66. [PMID: 27060279 PMCID: PMC5071754 DOI: 10.1111/tan.12775] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Revised: 02/16/2016] [Accepted: 02/24/2016] [Indexed: 11/27/2022]
Abstract
Solid‐phase single antigen bead (SAB) assays are standard of care for detection and identification of donor‐specific antibody (DSA) in patients who receive solid organ transplantation (SOT). While several studies have documented the reproducibility and sensitivity of SAB testing for DSA, there are little data available concerning its specificity. This study describes the identification of antibodies to β2‐microglobulin‐free human leukocyte antigen (β2‐m‐fHLA) heavy chains on SAB arrays and provides a reassessment of the clinical relevance of DSA testing by this platform. Post‐transplant sera from 55 patients who were positive for de novo donor‐specific antibodies on a SAB solid‐phase immunoassay were tested under denaturing conditions in order to identify antibodies reactive with β2‐m‐fHLA or native HLA (nHLA). Antibodies to β2‐m‐fHLA were present in nearly half of patients being monitored in the post‐transplant period. The frequency of antibodies to β2‐m‐fHLA was similar among DSA and HLA antigens that were irrelevant to the transplant (non‐DSA). Among the seven patients with clinical or pathologic antibody‐mediated rejection (AMR), none had antibodies to β2‐m‐fHLA exclusively; thus, the clinical relevance of β2‐m‐fHLA is unclear. Our data suggests that SAB testing produces false positive reactions due to the presence of β2‐m‐fHLA and these can lead to inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA in the post‐transplant period.
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Affiliation(s)
- K Michel
- Transplant Program, Aurora St. Luke's Medical Center, Milwaukee, WI, USA
| | - R Santella
- Transplant Institute, Avera McKennan Hospital and University System, Sioux Falls, SD, USA
| | - J Steers
- Transplant Institute, Avera McKennan Hospital and University System, Sioux Falls, SD, USA
| | - A Sahajpal
- Transplant Program, Aurora St. Luke's Medical Center, Milwaukee, WI, USA
| | - F X Downey
- Transplant Program, Aurora St. Luke's Medical Center, Milwaukee, WI, USA
| | - V Thohan
- Transplant Program, Aurora St. Luke's Medical Center, Milwaukee, WI, USA
| | - M Oaks
- Transplant Program, Aurora St. Luke's Medical Center, Milwaukee, WI, USA
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31
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Chacko MP, Augustin A, David VG, Valson AT, Daniel D. Nonspecific positivity on the Luminex crossmatch assay for anti-human leukocyte antigen antibodies due to antibodies directed against the antibody coated beads. Indian J Nephrol 2016; 26:134-7. [PMID: 27051139 PMCID: PMC4795430 DOI: 10.4103/0971-4065.159305] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
Two cases are described of previously unreported false positivity on the Luminex crossmatch assay due to non HLA specific antibodies directed against the beads. In both cases the Luminex crossmatch indicated the presence of donor specific antibodies to class II HLA antigens, which was not substantiated by the clinical scenario or other assays. We could demonstrate the non specificity of these antibodies through using the same assay in a modified form where beads were unexposed to cell lysate and therefore did not carry HLA antigens at all. These cases further serve to emphasize the absolute necessity of correlating positive results with the priming history, and confirming their relevance using other platforms.
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Affiliation(s)
- M. P. Chacko
- Department of Transfusion Medicine and Immunohematology, Christian Medical College, Vellore, Tamil Nadu, India
| | - A. Augustin
- Department of Transfusion Medicine and Immunohematology, Christian Medical College, Vellore, Tamil Nadu, India
| | - V. G. David
- Department of Nephrology, Christian Medical College, Vellore, Tamil Nadu, India
| | - A. T. Valson
- Department of Nephrology, Christian Medical College, Vellore, Tamil Nadu, India
| | - D. Daniel
- Department of Transfusion Medicine and Immunohematology, Christian Medical College, Vellore, Tamil Nadu, India
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32
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Lopes D, Barra T, Malheiro J, Tafulo S, Martins L, Almeida M, Pedroso S, Dias L, Castro Henriques A, Cabrita A. Effect of Different Sensitization Events on HLA Alloimmunization in Kidney Transplantation Candidates. Transplant Proc 2016; 47:894-7. [PMID: 26036480 DOI: 10.1016/j.transproceed.2015.03.014] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
BACKGROUND HLA alloimmunization is caused by sensitization events (SEs), such as transfusion, pregnancy, or previous organ transplantation, and the effects of particular SEs have not been thoroughly studied. Our aim was to evaluate how each SE affected HLA alloimmunization by considering Luminex assays. METHODS Sera from 722 kidney transplantation candidates were screened per protocol by means of Luminex assays to determine the presence of anti-HLA class I/II antibodies; positive sera underwent single-antigen assay to determine the presence of specific antibodies against HLA A, B, C, DR, DQ, DP loci (positivity if median fluorescence intensity values were >1,000). The effect of each SE was analyzed considering only patients exposed to 1 kind of sensitization. RESULTS In the 453 candidates with ≥1 SE, anti-HLA class I positivity rates were significantly higher in patients with previous transfusion (18.9%; P = .014), pregnancy (38.3%; P < .001) or transplant (75%; P < .001) compared with those with no SE (similar results for class II). The strength (median fluorescence intensity) of specific antibodies was significantly higher in patients with previous transplantation than in those with previous transfusion for HLA-A (8,017 vs 2,302; P = .02), HLA-B (7,765 vs 2,901; P = .018), and HLA-DR (9,835 vs 2,060; P = .003). Other anti-HLA antibody strengths were similar between patients with previous pregnancy or transplantation. CONCLUSIONS Presence of any SE analyzed was associated with a higher prevalence of anti-HLA antibodies for class I ± II compared with nonsensitized patients. Transplantation had the strongest immunization effect on both classes, followed by pregnancy and then transfusion.
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Affiliation(s)
- D Lopes
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal.
| | - T Barra
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
| | - J Malheiro
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
| | - S Tafulo
- Centro do Sangue e Transplantação do Porto, Porto, Portugal
| | - L Martins
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
| | - M Almeida
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
| | - S Pedroso
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
| | - L Dias
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
| | - A Castro Henriques
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
| | - A Cabrita
- Nephrology and Kidney Transplantation Department, Centro Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
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33
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Badders JL, Jones JA, Jeresano ME, Schillinger KP, Jackson AM. Variable HLA expression on deceased donor lymphocytes: Not all crossmatches are created equal. Hum Immunol 2015; 76:795-800. [DOI: 10.1016/j.humimm.2015.09.029] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2014] [Revised: 02/21/2015] [Accepted: 09/28/2015] [Indexed: 12/22/2022]
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34
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Lee H, Oh EJ. Luminex-based Immunoassay for Organ Transplantation. KOREAN JOURNAL OF TRANSPLANTATION 2015. [DOI: 10.4285/jkstn.2015.29.2.54] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Affiliation(s)
- Hyeyoung Lee
- Department of Laboratory Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
| | - Eun-Jee Oh
- Department of Laboratory Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
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35
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Böhmig GA, Kikic Z, Wahrmann M, Eskandary F, Aliabadi AZ, Zlabinger GJ, Regele H, Feucht HE. Detection of alloantibody-mediated complement activation: A diagnostic advance in monitoring kidney transplant rejection? Clin Biochem 2015; 49:394-403. [PMID: 26118475 DOI: 10.1016/j.clinbiochem.2015.05.024] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2015] [Revised: 05/23/2015] [Accepted: 05/28/2015] [Indexed: 11/29/2022]
Abstract
OBJECTIVE Antibody-mediated rejection (ABMR) is an important cause of kidney allograft injury. In the last two decades, detection of complement split product C4d along transplant capillaries, a footprint of antibody-mediated classical complement activation, has evolved as a useful diagnostic marker of ABMR. While it was recognized that ABMR may occur also in the absence of C4d, numerous studies have shown that C4d deposition may indicate a more severe rejection phenotype associated with poor graft survival. Such studies suggest a possible diagnostic benefit of ex vivo monitoring the complement-activating capability of circulating alloantibodies. DESIGN AND METHODS We reviewed the literature between 1993 and 2015, focusing on in vivo (biopsy work-up) and in vitro detection (modified bead array technology) of HLA antibody-triggered classical complement activation in kidney transplantation. RESULTS Precise HLA antibody detection methods, in particular Luminex-based single antigen bead (SAB) assays, have provided a valuable basis for the design of techniques for in vitro detection of HLA antibody-triggered complement activation reflected by C1q, C4 or C3 split product deposition to the bead surface. Establishing such assays it was recognized that deposition of complement products to SAB, which critically depends on antibody binding strength, may be a cardinal trigger of the prozone effect, a troublesome in vitro artifact caused by a steric interference with IgG detection reagents. False-low IgG results, especially on SAB with extensive antibody binding, have to be considered when interpreting studies analyzing the diagnostic value of complement in relation to standard IgG detection. Levels of complement-fixing donor-specific antibodies (DSA) were shown to correlate with the results of standard crossmatch tests, suggesting potential application for crossmatch prediction. Moreover, while the utility of pre-transplant complement detection, at least in crossmatch-negative transplant recipients, is controversially discussed, a series of studies have shown that the appearance of post-transplant complement-fixing DSA may be associated with C4d deposition in transplant capillaries and a particular risk of graft failure. CONCLUSIONS The independent value of modified single antigen bead assays, as compared to a careful analysis of standard IgG detection, which may be affected considerably by complement dependent artifacts, needs to be clarified. Whether they have the potential to improve the predictive accuracy of our current diagnostic repertoire warrants further study.
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Affiliation(s)
- Georg A Böhmig
- Division of Nephrology and Dialysis, Department of Medicine III, Medical University Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
| | - Zeljko Kikic
- Division of Nephrology and Dialysis, Department of Medicine III, Medical University Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
| | - Markus Wahrmann
- Division of Nephrology and Dialysis, Department of Medicine III, Medical University Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
| | - Farsad Eskandary
- Division of Nephrology and Dialysis, Department of Medicine III, Medical University Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
| | - Arezu Z Aliabadi
- Department of Cardiac Surgery, Medical University Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
| | - Gerhard J Zlabinger
- Institute of Immunology, Medical University Vienna, Lazarettgasse 19, A-1090 Vienna, Austria
| | - Heinz Regele
- Clinical Institute of Pathology, Medical University Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
| | - Helmut E Feucht
- Department of Organ Transplantation/Nephrology, Fachklinik Bad Heilbrunn, Wörnerweg 30, 83670 Bad Heilbrunn, Germany
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36
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The dilemma of DQ HLA-antibodies. Hum Immunol 2015; 76:324-8. [DOI: 10.1016/j.humimm.2015.03.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2014] [Revised: 03/03/2015] [Accepted: 03/11/2015] [Indexed: 11/21/2022]
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Visentin J, Guidicelli G, Moreau JF, Lee JH, Taupin JL. Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation. Eur J Immunol 2015; 45:2111-21. [PMID: 25824860 DOI: 10.1002/eji.201445340] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2014] [Revised: 03/06/2015] [Accepted: 03/27/2015] [Indexed: 11/07/2022]
Abstract
Anti-HLA donor-specific antibodies are deleterious for organ transplant survival. Class I HLA donor-specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti-denatured HLA antibodies (anti-dHLAs). Anti-dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti-dHLAs to be discriminated from anti-nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid-treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti-dHLAs. However, some anti-dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti-nHLAs conserved significant reactivity toward acid-treated LSAB. After depleting serum anti-nHLA reactivity with HLA-typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid-treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti-nHLAs and anti-dHLAs, or anti-nHLAs recognizing acid-resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti-HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti-HLA antibodies in human serum.
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Affiliation(s)
- Jonathan Visentin
- Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France.,UMR 5164 CNRS, Université de Bordeaux, Talence, France
| | - Gwendaline Guidicelli
- Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France
| | - Jean-François Moreau
- Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France.,UMR 5164 CNRS, Université de Bordeaux, Talence, France
| | | | - Jean-Luc Taupin
- Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France.,UMR 5164 CNRS, Université de Bordeaux, Talence, France
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38
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Abstract
Human leukocyte antigen (HLA) sensitisation occurs after transfusion of blood products and transplantation. It can also happen spontaneously through cross-sensitisation from infection and pro-inflammatory events. Patients who are highly sensitised face longer waiting times on organ allocation programmes, more graft rejection and therefore more side effects of immunosuppression, and poorer graft outcomes. In this review, we discuss these issues, along with the limitations of modern HLA detection methods, and potential ways of decreasing HLA antibody development. We do not discuss the removal of antibodies after they have developed.
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Affiliation(s)
- Lesley Rees
- Department of Paediatric Nephrology, Great Ormond Street Hospital for Children NHS Foundation Trust, Great Ormond Street, London, WC1N 3JH, UK,
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Konvalinka A, Tinckam K. Utility of HLA Antibody Testing in Kidney Transplantation. J Am Soc Nephrol 2015; 26:1489-502. [PMID: 25804279 DOI: 10.1681/asn.2014080837] [Citation(s) in RCA: 147] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor-specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes.
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Affiliation(s)
| | - Kathryn Tinckam
- Department of Medicine, Division of Nephrology and Laboratory Medicine Program, HLA Laboratory, University Health Network, University of Toronto, Toronto, Ontario, Canada
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40
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Visentin J, Marroc M, Guidicelli G, Bachelet T, Nong T, Moreau JF, Lee JH, Merville P, Couzi L, Taupin JL. Clinical impact of preformed donor-specific denatured class I HLA antibodies after kidney transplantation. Clin Transplant 2015; 29:393-402. [PMID: 25683727 DOI: 10.1111/ctr.12529] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/09/2015] [Indexed: 01/06/2023]
Abstract
Class I single-antigen flow beads (SAFB) carry native and denatured human leukocyte antigen (HLA) molecules. Using a cohort of 179 class I HLA-sensitized kidney recipients, we described incidence and clinical relevance of preformed denatured HLA donor-specific antibodies (DSA) using two different assays: an acid-treated SAFB assay (anti-dHLA DSA) and the iBeads assays (SAFB+/iBeads- DSA). Eighty-five class I DSA were found in 67 patients (median mean fluorescence intensity [MFI] of 1729 [range 520-13 882]). Anti-dHLA and SAFB+/iBeads- DSA represented 11% and 18% of class I DSA and were mainly low MFI DSA (500-1000 MFI). Concordance between these two assays was good (90%). None of the patients with only class I anti-dHLA DSA or only SAFB+/iBeads- DSA developed acute clinical antibody-mediated rejection in the first-year post-transplantation, and their five-yr death-censored graft survival was similar to that of patients without DSA. Moreover, all these patients displayed a negative current T-cell flow cytometry cross-match. Therefore, both anti-dHLA DSA and SAFB+/iBeads- DSA appear irrelevant, which could explain the good outcome observed in some patients with preformed class I DSA.
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Affiliation(s)
- Jonathan Visentin
- Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France; UMR CNRS 5164, Université de Bordeaux, Bordeaux, France
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41
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Lights and shadows of anti-HLA antibodies detected by solid-phase assay. Immunol Lett 2014; 162:181-7. [DOI: 10.1016/j.imlet.2014.08.014] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2014] [Revised: 08/09/2014] [Accepted: 08/21/2014] [Indexed: 11/21/2022]
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Washing red cells after leucodepletion does not decrease human leukocyte antigen sensitization risk in patients with chronic kidney disease. Pediatr Nephrol 2014; 29:2005-11. [PMID: 24777534 DOI: 10.1007/s00467-014-2823-6] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/21/2014] [Revised: 03/11/2014] [Accepted: 04/02/2014] [Indexed: 10/25/2022]
Abstract
BACKGROUND Standard leucodepleted blood transfusions can induce the production of human leukocyte antigen (HLA)-specific antibodies, which are associated with longer transplant waiting times and poorer graft outcomes. We hypothesized that additional washing of leucodepleted red cells might reduce antigenic stimulus by removal of residual leukocytes and soluble HLA. METHODS A retrospective review of HLA antibodies in children with chronic kidney disease stage 4-5 who had ≥ two HLA antibody screens between 2000 and 2009, pre- and post-transfusion, and were HLA antibody-negative at first testing. Patients were divided according to whether they received standard leucodepleted blood or "washed cells". To assess the efficacy of washing methods, total leukocytes were enumerated pre- and post- manual and automated washing of standard leucodepleted red cells that had been supplemented with whole blood to achieve measurable leukocyte levels pre-washing. RESULTS A total of 106 children were included: 23 received no blood transfusions (group 1), six had washed cells only (group 2), 59 had standard transfusions only (group 3), and 18 had both standard and washed cells (group 4). Sensitization rates were 26, 17, 44, and 44 % in groups 1-4 (p = 0.32). Patients in groups 3 and 4 had more transfusions with red cells, platelets, and plasma products. There was no difference in HLA sensitization risk with washed or standard red cells on analysis of co-variance controlling for platelets and plasma transfusions. The red cell washing study showed no significant reduction in leukocytes using manual methods. Although there was a statistically significant reduction (33 %) from baseline pre-washing using the automated method, from 6.54 ± 0.84 × 10(6) to 4.36 ± 0.67 × 10(6) leukocytes per unit, the majority of leukocytes still remained. CONCLUSIONS There was no evidence that using washed leucodepleted red cells reduced patient HLA sensitization rates. Washing leucodepleted red cells is unlikely to reduce the risk of HLA sensitization due to the limited effect on residual leukocytes.
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43
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Lobashevsky AL. Methodological aspects of anti-human leukocyte antigen antibody analysis in solid organ transplantation. World J Transplant 2014; 4:153-67. [PMID: 25346888 PMCID: PMC4208078 DOI: 10.5500/wjt.v4.i3.153] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/21/2014] [Revised: 06/16/2014] [Accepted: 07/25/2014] [Indexed: 02/05/2023] Open
Abstract
Donor human leukocyte antigen (HLA)-specific antibodies (DSA) play an important role in solid organ transplantation. Preexisting IgG isotype DSA are considered a risk factor for antibody mediated rejection, graft failure or graft loss. The post-transplant development of DSA depends on multiple factors including immunogenicity of mismatched antigens, HLA class II typing of the recipient, cytokine gene polymorphisms, and cellular immunoregulatory mechanisms. De novo developed antibodies require special attention because not all DSA have equal clinical significance. Therefore, it is important for transplant clinicians and transplant immunologists to accurately characterize DSA. In this review, the contemporary immunological techniques for detection and characterization of anti-HLA antibodies and their pitfalls are described.
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Gebel HM, Bray RA. HLA antibody detection with solid phase assays: great expectations or expectations too great? Am J Transplant 2014; 14:1964-75. [PMID: 25088978 DOI: 10.1111/ajt.12807] [Citation(s) in RCA: 115] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2014] [Revised: 04/22/2014] [Accepted: 04/24/2014] [Indexed: 01/25/2023]
Abstract
Alloantibodies directed against HLA antigens, are a barrier to long-term solid organ allograft survival. The clinical impact of preformed, donor-directed HLA alloantibodies range from acceptable risk to unequivocal contraindication for organ transplantation. HLA antibodies are key factors that limit patient access to donor organs. Serological methods were once the only approach to identify HLA antigens and antibodies. Limitations in these technologies led to the development of solid phase approaches. In the early 1990s, the development of the polymerase chain reaction enabled DNA-based HLA antigen testing to be performed. By the mid-1990s, microparticle-based technology that utilized flow cytometry for analysis was developed to detect both classes I and II HLA antibodies. These methodologies revolutionized clinical histocompatibility testing. The strengths and weaknesses of these assays are described in detail in this review.
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Affiliation(s)
- H M Gebel
- Department of Pathology, Emory University, Atlanta, GA
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In JW, Rho EY, Shin S, Park KU, Song EY. False-positive reactions against HLA class II molecules detected in Luminex single-antigen bead assays. Ann Lab Med 2014; 34:408-10. [PMID: 25187899 PMCID: PMC4151015 DOI: 10.3343/alm.2014.34.5.408] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2014] [Revised: 04/14/2014] [Accepted: 07/23/2014] [Indexed: 11/19/2022] Open
Affiliation(s)
- Ji Won In
- Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea
| | - Eun Youn Rho
- Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea
| | - Sue Shin
- Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea
| | - Kyoung Un Park
- Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea
| | - Eun Young Song
- Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea
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Oaks M, Michel K, Sulemanjee NZ, Thohan V, Downey FX. Practical value of identifying antibodies to cryptic HLA epitopes in cardiac transplantation. J Heart Lung Transplant 2014; 33:713-20. [DOI: 10.1016/j.healun.2014.02.013] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2013] [Revised: 12/12/2013] [Accepted: 02/07/2014] [Indexed: 10/25/2022] Open
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Alhamid N, Alterky H, Almouslem A, Al-Rayess H, Othman MI. Successful kidney transplant in a patient with IgG anti HLA Class-I auto-antibodies: a case report. Hum Immunol 2014; 75:597-601. [PMID: 24859192 DOI: 10.1016/j.humimm.2014.05.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2013] [Revised: 05/06/2014] [Accepted: 05/06/2014] [Indexed: 11/18/2022]
Abstract
Donor specific antibodies (DSA) play a significant role in graft rejection. Many laboratory methods, varied in sensitivity and specificity, are used to detect them. We report a case of a 38-year-old man presented with end stage renal disease considered for kidney transplantation. He had no history of blood transfusions nor transplantation procedures. Dilemma rose when he got multiple positive crossmatches with matching donors and a positive autologous crossmatch due to IgG anti HLA auto-antibodies, which are at the same time against matched donors. Since positive crossmatch is a contraindication for transplant, we couldn't perform transplant from any matched donor. Therefore, we considered a total mismatched donor then transplantation was performed. Observation after surgery showed normalization of creatinine, blood pressure and a good function of the planted allograft for two years of follow up.
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Affiliation(s)
- Naji Alhamid
- Damascus University, College of Medicine, Syrian Arab Republic.
| | - Hani Alterky
- Damascus University, College of Medicine, Syrian Arab Republic
| | | | - Heba Al-Rayess
- Damascus University, College of Medicine, Syrian Arab Republic
| | - Mohammad Imad Othman
- Head of Nephrology Department and Kidney Transplant Unit AUH, Damascus University, College of Medicine, Syrian Arab Republic
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Schlaf G, Pollok-Kopp B, Altermann WW. Sensitive solid-phase detection of donor-specific antibodies as an aid highly relevant to improving allograft outcomes. Mol Diagn Ther 2013; 18:185-201. [PMID: 24170304 DOI: 10.1007/s40291-013-0063-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Transplant recipients who have had sensitizing events such as pregnancies, blood transfusions and previous transplants often develop antibodies directed against human leukocyte antigen (HLA)-molecules of the donor tissue. These pre-formed donor-specific antibodies (DSA) represent a high risk of organ failure as a consequence of antibody-mediated hyper-acute or acute allograft rejection. As a first assay to detect DSA, the complement-dependent lymphocytotoxicity assay (CDC) was established more than 40 years ago. However, this assay is characterized by several drawbacks such as a low sensitivity and a high susceptibility to various artificial factors generally not leading to valid and reliable outcomes under several circumstances that are reviewed in this article. Furthermore, only those antibodies that exert complement-fixing activity are detected. As a consequence, novel procedures that act independently of the complement system and that do not represent functional assays were generated in the format of solid phase assays (SPAs) (bead- or ELISA-based). In this article, we review the pros and cons of these sensitive SPA in comparison with the detection of DSA through the use of the traditional methods such as CDC and flow cytometric analyses. Potential drawbacks of the alternative methodological approaches comprising high background reactivity, susceptibility to environmental factors and the possible influence of subjective operators' errors concerning the interpretation of the results are summarized and critically discussed for each method. We provide a forecast on the future role of SPAs reliably excluding highly deleterious DSA, thus leading to an improved graft survival.
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Affiliation(s)
- Gerald Schlaf
- Tissue Typing Laboratory, University Hospital Halle/Saale, Martin-Luther University of Halle-Wittenberg, Magdeburger Strasse 16, 06112, Halle (Saale), Germany,
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Otten HG, Verhaar MC, Borst HPE, van Eck M, van Ginkel WGJ, Hené RJ, van Zuilen AD. The significance of pretransplant donor-specific antibodies reactive with intact or denatured human leucocyte antigen in kidney transplantation. Clin Exp Immunol 2013; 173:536-43. [PMID: 23627692 DOI: 10.1111/cei.12127] [Citation(s) in RCA: 72] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/23/2013] [Indexed: 11/30/2022] Open
Abstract
Antibodies recognizing denatured human leucocyte antigen (HLA) can co-react with epitopes on intact HLA or recognize cryptic epitopes which are normally unaccessible to HLA antibodies. Their specificity cannot be distinguished by single antigen beads (SAB) alone, as they carry a mixture of intact and denatured HLA. In this study, we selected pretransplant sera containing donor-specific HLA class I antibodies (DSA) according to regular SAB analysis from 156 kidney transplant recipients. These sera were analysed using a SAB preparation (iBeads) which is largely devoid of denatured HLA class I, and SAB coated with denatured HLA class I antigens. A total of 241 class I DSA were found by regular SAB analysis, of which 152 (63%) were also found by iBeads, whereas 28 (11%) were caused by reactivity with denatured DNA. Patients with DSA defined either by regular SAB or iBeads showed a significantly lower graft survival rate (P = 0·007) compared to those without HLA class I DSA, whereas reactivity to exclusively denatured HLA was not associated with decreased graft survival. In addition, DSA defined by reactivity to class I SAB or class I iBeads occurred more frequently in female patients and in patients with historic HLA sensitization, whereas reactivity to denatured HLA class I was not associated with any of these parameters. Our data suggest that pretransplant donor-specific antibodies against denatured HLA are clinically irrelevant in patients already sensitized against intact HLA.
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Affiliation(s)
- H G Otten
- Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, the Netherlands.
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Ellis TM. Interpretation of HLA single antigen bead assays. Transplant Rev (Orlando) 2013; 27:108-11. [DOI: 10.1016/j.trre.2013.07.001] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2013] [Revised: 07/01/2013] [Accepted: 07/02/2013] [Indexed: 10/26/2022]
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