Comprehensive Assessment and Standardization of Solid Phase Multiplex‐Bead Arrays for the Detection of Antibodies to HLA

• Published in: American journal of transplantation Vol. 13; no. 7; pp. 1859 - 1870
• Main Authors: Reed, E. F., Rao, P., Zhang, Z., Gebel, H., Bray, R. A., Guleria, I., Lunz, J., Mohanakumar, T., Nickerson, P., Tambur, A. R., Zeevi, A., Heeger, P. S., Gjertson, D.
• Format: Journal Article
• Language: English
• Published: Hoboken, NJ Wiley 01.07.2013; Elsevier Limited
• Subjects: Algorithms; Antibodies - blood; Antibodies - immunology; Antibody Specificity - immunology; Arrays; Biological and medical sciences; Clinical trials; desensitization; diagnostic markers; donor‐specific antibodies; Flow Cytometry - methods; Histocompatibility Testing - methods; Histocompatibility Testing - standards; HLA antibodies; HLA Antigens - immunology; Humans; humoral immunity; immune monitoring; immunological markers; Laboratories; Lymphocytes - immunology; Medical sciences; multicenter studies; posttransplant monitoring; quality assurance; quantitative; Reproducibility of Results; ROC Curve; Solids; Standardization; Studies; Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases; transplantation immunology; Transplantation Immunology - immunology; Suboptimal donor; HLA-System; diagnostic markers; Major histocompatibility system; Standardization; Biological marker; Homotransplantation; immunological markers; Quality assurance; Immunology; Immunological investigation; HLA antibodies; Surgery; Graft; Diagnosis; Class I histocompatibility antigen; Humoral immunity; Monitoring; Quantitative analysis; Human; Desensitization; Immune response; donor-specific antibodies; Antibody; multicenter studies; immune monitoring; posttransplant monitoring; transplantation immunology; Surveillance; quantitative; Solid phase; Detection; Arrays
• ISSN: 1600-6135; 1600-6143; 1600-6143
• DOI: 10.1111/ajt.12287

Solid phase multiplex‐bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte‐based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R2), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing. Using a reference set of sera and identical test reagents exchanged among multiple laboratories, this study elucidates the sources and magnitudes of variation present in solid phase multiplex‐bead arrays for the detection and characterization of HLA antibodies and suggests that assay variability can be reduced to near quantitative accuracy with protocol standardization and use of appropriate normalization strategies. See more in the Clinical Trials in Organ Transplantation series, pages 1859–1904.