Patient and External Quality Assessment Samples Demonstrate Similar Bias Between Manufacturers in Titer of Antibodies to Nuclear Antigens: Implications for Commutability Open Access

To the Editor.—The presence of antibodies to nuclear antigens (ANAs) above a threshold titer is a defining criterion for classification of systemic lupus erythematosus (SLE) and participation in clinical trials, and the probability of diagnosis of many autoimmune rheumatic diseases varies with the ANA titer.^1,2  Consistent ANA titer results are therefore necessary for clinical and research purposes. Indirect fluorescent antibody (IFA) assays using HEp-2 cell substrates are widely considered the reference method for ANA detection. However, there is significant variability in titer results between laboratories performing IFA, challenging the concept of a universal threshold titer.³ 

Variability in ANA titer could result from reagents, HEp-2 substrates, microscopy conditions, and subjective visual interpretation. We recently analyzed results reported for 11 years from the College of American Pathologists (CAP) ANA proficiency testing survey and found that reported ANA titer was strongly influenced by reagent manufacturer.³  We found remarkable consistency in the relative rank order of ANA titer for each kit over time among all participating laboratories, including specimens producing a variety of different ANA patterns. Although this finding supports the potential for harmonization of ANA titer between laboratories using different kits, it remains unclear whether the consistent difference between manufacturers is also present for clinical specimens. Thus, we sought to determine whether the manufacturer bias that we observed in proficiency testing specimens is consistent in clinical specimens.

We used data reported by Copple et al⁴  who evaluated commercially available ANA IFA assays with serum samples from patients with autoimmune rheumatic diseases. This study evaluated 8 serum samples from patients with SLE and 11 serum samples from patients with scleroderma, each on HEp-2 assays from 5 manufacturers. Consistent with our observations using proficiency testing specimens, some assays typically produced higher titers and others produced lower titers for each patient sample. We used the reported titer from each assay for each specimen to determine the manufacturer rank order. We then compared this manufacturer rank order for clinical samples versus the rank order for proficiency testing samples that we previously reported.³  The kit rankings for proficiency testing samples were nearly identical to the kit rankings for patient specimens (Figure). Linear regression demonstrated strong correlation of kit rankings for proficiency testing specimens and samples from patients with both SLE (r = 0.90, P < .001) and scleroderma (r = 1.0, P < .001).

These results demonstrate that differences in ANA titer between kits are consistent between CAP proficiency testing materials and specimens from patients with autoimmune rheumatic diseases, supporting commutability of proficiency testing material for relative ANA titer, where commutability is defined as a property of materials having the same interassay relationships as clinical samples.⁵  Together with our previous study, these data support the potential to harmonize IFA-based ANA quantitation. Harmonization could be achieved by collaboration between the clinical laboratory community, manufacturers, and organizations including proficiency test providers. Standardization using reference materials to align titer results between manufacturers could improve consistency between clinical laboratories, thus promoting diagnostic accuracy for patients with autoimmune diseases.

The authors gratefully acknowledge Christine Bashleben, MT(ASCP), from the College of American Pathologists Laboratory Improvement Programs.