Comprehensive Assessment and Standardization of Solid Phase Multiplex-Bead Arrays for the Detection of Antibodies to HLA-Drilling Down on Key Sources of Variation

Authors


To the Editor:

We appreciate the opportunity to respond to the letter by Dr. Maillard and Dr. Mariat [1]. They raise three questions regarding our recent article focusing on inter-laboratory standardization of solid phase multiplex-bead arrays to detect antibodies to HLA within the framework of the Clinical Trials in Organ Transplantation [2]. Our article provides insights into the key sources of variability in commercially available solid phase HLA antibody testing kits. Importantly, our study demonstrates that standardization of reagents and protocols significantly reduces assay variance. Utilization of a global normalization algorithm further reduced median fluorescence intensity (MFI) variations in the protocol, thereby improving comparison of data across laboratories.

Dr. Maillard and Dr. Mariat comment that our study did not address the prozone effect. Although they raise an important point, we do not believe the prozone effect impacted our estimates of inter-laboratory variance, since all participants adopted a standardized protocol, used the same reagents and tested a constant volume of alloantisera. We do agree with Dr. Maillard and Dr. Mariat that adding dithiothreitol, running dilutions or employing other methods to explore potential prozone/interfering factors is worthy of systematic investigation.

Dr. Maillard and Dr. Mariat correctly point out that the %CV decreases within higher MFI strata. Although they indicate this finding is presented in the Bland–Altman plots (2, figure 5), it is actually illustrated in figure 3 of our article, which shows the variation among seven centers across distinct MFI strata. Dr. Maillard and Dr. Mariat suggest that the sudden amelioration in %CV within higher MFI strata is due to saturation of the beads with antibodies. However, as we clearly showed, the decline in %CV begins at 1000 MFI, well below a saturation dosage (<10 000 MFI), which indicates saturation is not the primary reason to explain this result.

Their third point questions the impact of intra-laboratory variability on results and whether the improvement in %CV was due to a reduction in variance within an individual laboratory or between laboratories. Since it is standard of care that clinical laboratories utilize a standard operating procedure for HLA antibody testing, we expect the major cause of assay variance is lot-to-lot differences in test kits. Although we did not specifically address intra-laboratory variability in our report, each data point shown in the Bland–Altman plot (2, figure 5) can be converted into a pseudo “intra-laboratory” %CV [i.e. inline image] representing the variation when a lab repeats the test of same sample and bead across two lots of single antigen kits. The median intra-laboratory %CV was 19%, and boxplots demonstrate a decline with increasing MFI range within each center and overall (Figure 1). On average, the intra-laboratory %CV was less than our reported inter-laboratory %CV (∼25%).

Figure 1.

Intra-laboratory lot-to-lot effects on assay variability. Boxplots of intra-lab %CV in median fluorescence intensity (MFI) distributions within five strata (0–500, 501–1000, 1001–3000, 3001–10 000, 10 001–25 000) grouped by center (AG).

Nonetheless, we acknowledge that other sources of variation in the aspects of the assay can certainly contribute to intra-laboratory variability and that each laboratory needs to address these concerns. We anticipate that both inter- and intra-laboratory variance will decrease with the implementation of standardized testing protocols and the increasing availability of uniform lots of reagents.

Acknowledgments

This research was performed as part of an American Recovery and Reinvestment (ARRA) funded project under Award Number U0163594 (to P. Heeger), from the National Institute of Allergy and Infectious Diseases. The work was carried out by members of the Clinical Trials in Organ Transplantation (CTOT) and Clinical Trials in Organ Transplantation in Children (CTOT-C) consortia. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.

  • E. F. Reed1,*, P. Rao1, Z. Zhang1, H. Gebel2, R. A. Bray2, I. Guleria3, J. Lunz4, T. Mohanakumar5, P. Nickerson6, A. R. Tambur7, A. Zeevi4, P. S. Heeger8 and D. Gjertson1

  • 1 Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA

  • 2 Department of Pathology, Emory University Hospital, Atlanta, GA

  • 3 Transplantation Research Center, Harvard Medical School, Boston, MA

  • 4 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA

  • 5 Department of Surgery, Pathology and Immunology, Washington University School of Medicine, St. Louis, MO

  • 6 Diagnostic Services of Manitoba and University of Manitoba, Winnipeg, MB, Canada

  • 7 Transplant Immunology Laboratory, Northwestern University, Chicago, IL

  • 8 Department of Medicine, Mount Sinai School of Medicine, New York, NY

  • * Corresponding author: Elaine F. Reed, ereed@mednet.ucla.edu

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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