Background/Purpose: Serological positivity defined by presence of anti-nuclear antibodies (ANA) is frequently used in interventional lupus trials as an inclusion criterion and longitudinal biomarker. As such, consistent and sensitive quantification of ANA during conduct of clinical trials is important. In this study we compared ANA testing results between 3 different commercial laboratories vendors using shared samples to assess variability in analysis.

Methods: Serum samples obtained from commercial sources from 19 anonymous SLE donors meeting ACR criteria were banked and aliquotted. Triplicate samples from each donor were randomized and shipped to 3 different commercial labs for ANA testing with staining patterns (HEp-2.) Reports from each laboratory were unblinded to group each sample replicate for both ANA titer and staining pattern. Inter-sample and inter-laboratory variability were then analyzed descriptively.

Results: Identical SLE patient sera samples analyzed by three laboratories providing ANA testing revealed differences in detection, titer level and staining pattern identification. While high titer ANA were readily detected by all three laboratories, there was significant variability in the assessment of staining patterns by some labs, and patterns present at different titers were often missed. In general, titers within an individual vendor between samples were reported within one fold. Lower titer ANAs <1:160 were more consistently scored as equivocal or absent by some labs, indicating a difference in sensitivity limit utilizing the samples provided.  Table 1. ANA identification by testing laboratories

*Of note, staining patterns from Vendor 1 had 2/19 samples that missed patterns in one triplicate assessment, Vendor 2 had 6/19 missed patterns in one or more triplicate assessments, and Vendor 3 had 3/19 missed patterns in one or more triplicate assessments.

Conclusion: This study suggests that commercial laboratories exhibited variability in terms of ANA titers and staining patterns using identical samples, especially in those of low titer. This occurred in a format consistent with collection in a clinical study and provision to a central laboratory for analysis. Variability was present between sample replicates and also the level of detection between different commercial labs performing ANA testing. This variability may potentially impact patient enrollment in clinical studies, and also the ability to accurately assess the impact of therapeutic interventions using these serological readouts. A key limitation to this study is that samples were accrued and banked to be sent to the analytical laboratory, and some vendor laboratory methodology may be more prone to latency of assessment than others.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/comparative-analysis-of-anti-nuclear-antibody-testing-using-blinded-replicate-samples-reveals-variability-between-commercial-testing-laboratories/