Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and Vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.

Factors influencing cord blood viability assessment before cryopreservation.

BACKGROUND: Cord blood (CB) viability determines product quality and varies with time and temperature of exposure before cryopreservation. Global viability assessment may not reflect viability of white blood cell (WBC) subsets, CD34+ cell viability, or hematopoietic stem/progenitor cells function. STUDY DESIGN AND METHODS: We compared trypan blue (TB) and acridine orange/propidium iodide (AO/PI) staining with flow-cytometric (7-aminoactinomycin D [7-AAD]) viability in total WBCs (Tot-AAD), granulocytes, monocytes, lymphocytes, and CD34+ cells and total nucleated cell, CD34+, and colony-forming cell (CFC) recovery as a function of time and temperature (4, 24, and 37 degrees C) before cryopreservation. RESULTS: TB, AO/PI, and Tot-AAD viability was concordant up to 72 hours (4 degrees C) and 48 hours (24 degrees C) postcollection; however, CD34+ viability was significantly higher due to loss of viable granulocytes. In contrast, at "physiologic" temperature (37 degrees C), the decline in TB, AO/PI, and Tot-AAD viability was significantly lower than the rate of viable CD34+ and CFC loss. At all times and temperatures, CFC recovery correlated best with CD34+ viability and recovery. CONCLUSIONS: CB cell populations exhibit differential time- and temperature-dependent susceptibility to in vitro cell death; consequently, global viability measurements using TB, AO/PI, or 7-AAD (Tot-AAD) significantly underestimate (4-24 degrees C) or overestimate (24-37 degrees C) CD34+ viability and CFC recovery. Our results demonstrate the limitations of global viability assessment with TB, AO/PI, and total AAD; endorse the routine use of CD34+ cell viability measurements; emphasize the importance of temperature control during shipment; and have implications with regard to establishing acceptable "cutoff" values for viability measurements and CB collection through processing time.