Commutability Assessment of Candidate Reference Materials for Lipoprotein(a) by Comparison of a MS-based Candidate Reference Measurement Procedure with Immunoassays.

BACKGROUND: Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment. However, the lack of global standardization of current Lp(a) measurement procedures (MPs) leads to inconsistent patient care. The International Federation for Clinical Chemistry and Laboratory Medicine working group on quantitating apolipoproteins by mass spectrometry (MS) aims to develop a next-generation SI (International system of units)-traceable reference measurement system consisting of a MS-based, peptide-calibrated reference measurement procedure (RMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) [apo(a)] content. To reach measurement standardization through this new measurement system, 2 essential requirements need to be fulfilled: a sufficient correlation among the MPs and appropriate commutability of future serum-based RMs. METHODS: The correlation among the candidate RMP (cRMP) and immunoassay-based MPs was assessed by measuring a panel of 39 clinical samples (CS). In addition, the commutability of 14 different candidate RMs was investigated. RESULTS: Results of the immunoassay-based MPs and the cRMPs demonstrated good linear correlations for the CS but some significant sample-specific differences were also observed. The results of the commutability study show that RMs based on unspiked human serum pools can be commutable with CS, whereas human pools spiked with recombinant apo(a) show different behavior compared to CS. CONCLUSIONS: The results of this study show that unspiked human serum pools are the preferred candidate secondary RMs in the future SI-traceable Lp(a) Reference Measurement System.

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.

Assessment of kidney proximal tubular secretion in critical illness.

BACKGROUNDSerum creatinine concentrations (SCrs) are used to determine the presence and severity of acute kidney injury (AKI). SCr is primarily eliminated by glomerular filtration; however, most mechanisms of AKI in critical illness involve kidney proximal tubules, where tubular secretion occurs. Proximal tubular secretory clearance is not currently estimated in the intensive care unit (ICU). Our objective was to estimate the kidney clearance of secretory solutes in critically ill adults.METHODSWe collected matched blood and spot urine samples from 170 ICU patients and from a comparison group of 70 adults with normal kidney function. We measured 7 endogenously produced secretory solutes using liquid chromatography-tandem mass spectrometry. We computed a composite secretion score incorporating all 7 solutes and evaluated associations with 28-day major adverse kidney events (MAKE28), defined as doubling of SCr, dialysis dependence, or death.RESULTSThe urine-to-plasma ratios of 6 of 7 secretory solutes were lower in critically ill patients compared with healthy individuals after adjustment for SCr. The composite secretion score was moderately correlated with SCr and cystatin C (r = -0.51 and r = -0.53, respectively). Each SD higher composite secretion score was associated with a 25% lower risk of MAKE28 (95% CI 9% to 38% lower) independent of severity of illness, SCr, and tubular injury markers. Higher urine-to-plasma ratios of individual secretory solutes isovalerylglycine and tiglylglycine were associated with MAKE28 after accounting for multiple testing.CONCLUSIONAmong critically ill adults, tubular secretory clearance is associated with adverse outcomes, and its measurement could improve assessment of kidney function and dosing of essential ICU medications.FUNDINGGrants from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK/NIH) K23DK116967, the University of Washington Diabetes Research Center P30DK017047, an unrestricted gift to the Kidney Research Institute from the Northwest Kidney Centers, and the Vanderbilt O'Brien Kidney Center (NIDDK 5P30 DK114809-03). The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.

Baseline Assessment of 25-Hydroxyvitamin D Reference Material and Proficiency Testing/External Quality Assurance Material Commutability: A Vitamin D Standardization Program Study.

The Vitamin D Standardization Program (VDSP) coordinated a study in 2012 to assess the commutability of reference materials and proficiency testing/external quality assurance materials for total 25-hydroxyvitamin D [25(OH)D] in human serum, the primary indicator of vitamin D status. A set of 50 single-donor serum samples as well as 17 reference and proficiency testing/external quality assessment materials were analyzed by participating laboratories that used either immunoassay or LC-MS methods for total 25(OH)D. The commutability test materials included National Institute of Standards and Technology Standard Reference Material 972a Vitamin D Metabolites in Human Serum as well as materials from the College of American Pathologists and the Vitamin D External Quality Assessment Scheme. Study protocols and data analysis procedures were in accordance with Clinical and Laboratory Standards Institute guidelines. The majority of the test materials were found to be commutable with the methods used in this commutability study. These results provide guidance for laboratories needing to choose appropriate reference materials and select proficiency or external quality assessment programs and will serve as a foundation for additional VDSP studies.

Baseline Assessment of 25-Hydroxyvitamin D Assay Performance: A Vitamin D Standardization Program (VDSP) Interlaboratory Comparison Study.

The Vitamin D Standardization Program (VDSP) coordinated an interlaboratory study to assess the comparability of measurements of total 25-hydroxyvitamin D [25(OH)D] in human serum, which is the primary marker of vitamin D status. A set of 50 individual donor samples were analyzed by 15 different laboratories representing national nutrition surveys, assay manufacturers, and clinical and/or research laboratories to provide results for total 25(OH)D using both immunoassays (IAs) and LC tandem MS (MS/MS). The results were evaluated relative to bias compared with the target values assigned based on a combination of measurements at Ghent University (Belgium) and the U.S. National Institute of Standards and Technology using reference measurement procedures for the determination of 25(OH)D2 and 25(OH)D3. CV and mean bias for each laboratory and assay platform were assessed and compared with previously established VDSP performance criteria, namely CV ≤ 10% and mean bias ≤ 5%. Nearly all LC-MS/MS results achieved VDSP criteria, whereas only 50% of IAs met the criterion for a ≤10% CV and only three of eight IAs achieved the ≤5% bias. These results establish a benchmark for the evaluation of 25(OH)D assay performance and standardization activities in the future.

Comparison of Two ELISA Methods and Mass Spectrometry for Measurement of Vitamin D-Binding Protein: Implications for the Assessment of Bioavailable Vitamin D Concentrations Across Genotypes.

Studies using vitamin D-binding protein (DBP) concentrations to estimate free and bioavailable vitamin D have increased dramatically in recent years. Combinations of two single-nucleotide polymorphisms (SNPs) produce three major DBP isoforms (Gc1f, Gc1s, and Gc2). A recent study showed that DBP concentrations quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) did not differ by race, whereas a widely used monoclonal enzyme-linked immunosorbent assay (ELISA) quantified DBP differentially by isoform, yielding significantly lower DBP concentrations in black versus white individuals. We compared measurements of serum DBP using a monoclonal ELISA, a polyclonal ELISA, and LC-MS/MS in 125 participants in the Chronic Renal Insufficiency Cohort (CRIC). Serum free and bioavailable 25OHD were calculated based on DBP concentrations from these three assays in homozygous participants, and race differences were compared. We confirmed that the monoclonal ELISA quantifies DBP differentially by isoform and showed that the polyclonal ELISA is not subject to this bias. Whereas ≤9% of the variability in DBP concentrations quantified using either LC-MS/MS or the polyclonal ELISA was explained by genotype, 85% of the variability in the monoclonal ELISA-based measures was explained by genotype. DBP concentrations measured by the monoclonal ELISA were disproportionately lower than LC-MS/MS-based results for Gc1f homozygotes (median difference -67%; interquartile range [IQR] -71%, -64%), 95% of whom were black. In contrast, the polyclonal ELISA yielded consistently and similarly higher measurements of DBP than LC-MS/MS, irrespective of genotype, with a median percent difference of +50% (IQR +33%, +65%). Contrary to findings using the monoclonal ELISA, DBP concentrations did not differ by race, and free and bioavailable 25OHD were significantly lower in black versus white participants based on both the polyclonal ELISA and LC-MS/MS, consistent with their lower total 25OHD. Future studies of DBP and free or bioavailable vitamin D metabolites should employ DBP assays that are not biased by DBP genotype. © 2016 American Society for Bone and Mineral Research.

Significance of serum 24,25-dihydroxyvitamin D in the assessment of vitamin D status: a double-edged sword?

BACKGROUND: 24,25-Dihydroxyvitamin D [24,25(OH)2D] in serum may be both a nuisance and nutritionally valuable. METHODS: We investigated the impact of 24,25(OH)2D3 on the performance of commercially available immunoassays for serum total 25-hydroxyvitamin D [25(OH)D] using (a) serum from a nationally representative sample of adults, (b) serum from a spiking experiment, and (c) data from the UK Vitamin D External Quality Assurance Scheme (DEQAS). We also investigated the utility of the serum ratio of 24,25(OH)2D3 to 25(OH)D as an index of inactivation and of response to vitamin D supplementation using randomized controlled trial (RCT) data. Measurement of 24,25(OH)2D in sera by a LC-MS/MS method allowed for an investigation of its impact on immunoassay-derived serum 25(OH)D values as well as its clinical utility. We report data from a nationally representative sample of adults, a recent vitamin D RCT in older adults, and DEQAS. RESULTS: 24,25(OH)2D3 contributed to the positive bias observed in some immunoassays relative to LC-MS/MS-derived estimates for total 25(OH)D. A spiking experiment showed that the degree of cross-reactivity with 24,25(OH)2D was high and may underpin this positive bias. Adjustment for 24,25(OH)2D3 concentration brought estimates closer to true values. Data from the vitamin D RCT showed that the ratio of 24,25(OH)2D3 to 25(OH)D was associated with serum 25(OH)D3 and with response of serum 25(OH)D to vitamin D supplementation. CONCLUSIONS: Our findings highlight that the effect of 24,25(OH)2D3 in serum is a double-edged sword-an interferent for some immunoassays, yet potentially informative of nutritional status.

Plasma vitamin D is associated with fasting insulin and homeostatic model assessment of insulin resistance in young adult males, but not females, of the Jerusalem Perinatal Study.

OBJECTIVE: To examine cross-sectional relationships between plasma vitamin D and cardiometabolic risk factors in young adults. DESIGN: Data were collected from interviews, physical examinations and biomarker measurements. Total plasma 25-hydroxyvitamin D (25(OH)D) was measured using LC-tandem MS. Associations between 25(OH)D and cardiometabolic risk factors were modelled using weighted linear regression with robust estimates of standard errors. SETTING: Individuals born in Jerusalem during 1974-1976. SUBJECTS: Participants of the Jerusalem Perinatal Study (n 1204) interviewed and examined at age 32 years. Participants were oversampled for low and high birth weight and for maternal pre-pregnancy obesity. RESULTS: Mean total 25(OH)D concentration among participants was 21·7 (sd 8·9) ng/ml. Among males, 25(OH)D was associated with homeostatic model assessment of insulin resistance (natural log-transformed, ß=-0·011, P=0·004) after adjustment for BMI. However, these associations were not present among females (P for sex interaction=0·005). CONCLUSIONS: We found evidence for inverse associations of 25(OH)D with markers of insulin resistance among males, but not females, in a healthy, young adult Caucasian population. Prospective studies and studies conducted on other populations investigating sex-specific effects of vitamin D on cardiometabolic risk factors are warranted.

The impact of inpatient point-of-care blood glucose quality control testing.

Analyze the effectiveness of mandated point-of-care (POC) blood glucose (BG) meter quality control (QC) testing. All POC BG QC tests were analyzed to evaluate operator and strip/meter error rates and institutional cost. POC BG QC test failure (17/103,580 over 24 months) was low and no meters failed subsequent linearity testing. Examining individual QC measures shows that operator error occurs frequently and total error rate is related to QC familiarity (>50 QC tests/month, 2.4%; <50 QC tests/month, 3.8%, p < .001). Even among the most competent operators, strip/meter error (1.2 ± 0.3%) accounted for 50% of total error. Compared with manufacturer-recommended QC testing, Joint Commission mandated POC BG QC testing during 2008/2009 incurred excess costs of approximately US$127,000. POC BG meter failure within current guidelines is rare and does not justify the cost of daily QC testing. Frequent QC testing can identify operators needing retraining in POC testing. Strip/meter QC errors are common, are not prevented by current QC testing standards, and may contribute to clinical errors.