Alvarez, Yasel F

Abstract Background Historically, 25-hydroxyvitamin D (25OHD) assays have under- or over-recovered 25-hydroxyvitamin D2 (25OHD2), but assay manufacturers have modified their reagents to address this problem. In this study, we compared the second- and third-generation Roche assays as well as two contemporary offerings from Diasorin and Beckman against liquid chromatography–tandem mass spectrometry (LC-MS/MS). Methods We identified 50 remnant serum samples with 25OHD concentrations from across the analytical range of the second-generation Roche assay. To increase 25OHD2 representation, we identified 25 additional samples from individuals prescribed high-dose vitamin D2 supplements. We tested samples on Roche assays and circulated to laboratories performing Beckman and Diasorin 25OHD assays. We tested samples by LC-MS/MS to obtain concentrations for 25OHD2 and 25-hydroxyvitamin D3. Results Mean overall bias for each assay was 5.1 ng/mL or less against the LC-MS/MS measurement; mean proportional bias was 8.7% to 12.1%. Some individual specimens had much larger bias. 25OHD2 was under-recovered on average, but the bias for the third-generation Roche assay represents a significant improvement over the previous assay, and mean bias for current generation assays was no worse than −3.2 ng/mL. In most cases, clinical classification by automated assay values agreed with clinical classification by LC-MS/MS; where present, disagreements occurred near classification thresholds. Conclusions Automated 25OHD assays continue to improve, and 25OHD2 recovery no longer appears to be a significant concern for the assays evaluated here. All assays evaluated were adequate for clinical classification of vitamin D nutritional status and are suitable for routine use, including in patients prescribed high-dose vitamin D2.

Abstract Background We took advantage of our verification of a new biotin-resistant 25-hydroxyvitamin D (vitamin D) immunoassay formulation to compare analytical performance of the outgoing formulation and assays from Roche, Diasorin and Beckman. Liquid chromatography with tandem mass spectrometry (LCMS) was an independent arbiter of vitamin D concentration and provided quantification of vitamin D2 and vitamin D3. Methods Twenty-five specimens from patients on high-dose vitamin D2 supplementation (50 000 IU/week) and fifty additional specimens spanning our measurable range were identified by the Dartmouth Hitchcock Analytics Institute. Aliquots were tested by the Roche cobas Vitamin D Total II and Vitamin D Total III assays, deidentified, split, and shipped to other study sites for measurement by LCMS, the Diasorin Liaison 25 OH Vitamin D Total assay, and the Beckman Coulter Access 25(OH) Vitamin D Total assay. Estimated vitamin D2 was calculated by subtracting the LCMS-measured vitamin D3 result from the immunoassay total vitamin D result. Bias calculations assume LCMS measurements as the true concentration. Statistical analyses were performed in R. This study was approved by the Dartmouth Hitchcock Medical Center Institutional Review Board. Results No significant differences were observed between platforms in absolute or relative bias in total vitamin D or estimated vitamin D2. Clinical classification was unanimous in 62/75 specimens. For thirteen specimens in which at least one disagreement was observed, immunoassay results for discrepant classification were no more than 10.2 ng/mL from the classification cutoff (median 2 ng/mL). Two specimens with 68 and 70 ng/mL vitamin D were classified as toxic (>100 ng/mL) by one immunoassay each. Conclusion Current vitamin D immunoassays continue to improve over their predecessors and offer good agreement across platforms. Vitamin D2 recovery no longer represents a clinically-concerning obstacle. These immunoassays display positive bias for total vitamin D relative to LCMS but generally result in correct clinical classification.