Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA

Abstract

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% (R(2) ), 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.

Figure 1. Laboratory variance for single antigen assays using standardized operating procedures versus non-standardized procedures

Comparisons of %CV in MFI distributions (depicted as boxplots) from 5 centers testing SA1 (first three plots) and SA2 (second three plots) kits with CTOT materials processed under SOP (Lot1 and Lot2 ) or with ASHI Proficiency Test materials processed under iHOP (□).

Figure 2. Center effects on assay variation

Left Panel: Histograms of MFI data (log scale) for Centers A to G (9,918 values each) and all centers (Total A–G =7×9918=69,426 values). Average values are marked by the vertical bar within each histogram. Right Panel: Plot shows the relative relationships among seven centers following Multidimensional Scaling (MDS) analysis. The original 7-dimensional array of dissimilarities (i.e., 1–correlation values computed between every pair of centers) was reduced into two major components (Dimensions 1 and 2) that preserved original dissimilarities while explaining ~80% of the variance (see Supplementary Material for details).

Figure 3. Manufacturer and kit effects on assay variation

Boxplots of %CV in MFI distributions grouped according to manufacturer and kit-type in 5 strata (0–500, 501–1000, 1001–3000, 3001–10,000 and 10,000–25,000) and all strata combined (Total) with Lot1 data. The median %CV for each stratum was shown as a thick horizontal line. Boxes were shaded to indicate whether %CV variations were statistically significant: not different from median (□, P>0.05); less than median ( , P<0.001); greater than median ( , P<0.001).

Figure 4. Bead effects on assay variation

Boxplots of %CV in MFI>1000 values grouped according to HLA locus and manufacturer in SA kits. Beads exhibiting significantly more variability than other specificities are boxed with their corresponding plots highlighted ( ). Only the A, C and DQ loci are shown since no significant SA bead anomalies were identified for the B, DR and DP loci.

Figure 5. Lot effects on assay variation

Left Panel: %CV in MFI in Lot1 and Lot 2 are shown by their boxplots grouped by manufacturer (Vendors A and B) and 5 kit types (Scr, PRA1, PRA2, SA1 and SA2). Right Panel: Bland-Altman plots divided into 3 strata based on average bead MFI magnitude (1001–3000 units, 3001–10,000 units and >10,000 units) showing ΔMFI (Lot1 minus Lot2 differences) among matched beads in SA kits. Lines represent the 95% chance boundaries (i.e., mean±1.96SD): 1001–3000 MFI range = 310±1500; 3001–10,000 MFI range = 620±4000; >10,000 MFI range = 1500±5600.

Figure 6. Receiver Operating Characteristic (ROC) analysis of manufacturer agreement

One of the manufacturer’s SA tests and software were used to establish HLA Ab presence (truth). Then, the second manufacturer’s platform was tested for agreement using data from reactions on beads carrying the same antigen specificities. Analyses were performed for each loci nested within class I and II SA kits. Areas under ROC curves (AUC) are listed along with their 95% confidence intervals in parentheses, and the MFI cutoffs (Cut) used to yield maximum correct classification rates (Agreement) are also listed.

Figure 7. Comparison of manufacturer standardization and global normalization methods for reducing assay variation

Boxplots show %CV in MFI distributions using raw, manufacturer’s standardized data and LOESS global normalized data from Lot1’s SA1 and SA2 kits. Values inside boxes represent the median %CV in MFI for a particular grouping. From left to right, the boxes’ lengths represent their interquartile ranges equaled 24%, 28%, 22%, 14%, 17% and 14%, respectively.