25OHD Assays Research Articles

B-031 Digital Elisatotal 25-Hydroxyvitamin D

Abstract Evaluation of digital ELISA using a high-resolution computational optical system for measurement of total 25-hydroxyvitamin D Background Biomarkers such as vitamin D occur at very low concentrations in blood, and sensitive detection methods are required in order to measure them. It is difficult to accurately measure low-concentration biomarkers using conventional enzyme-linked immunosorbent assays (ELISA) with fluorescence staining. By developing a digital ELISA (DELISA, Small Machines, Seoul, Korea) platform, a low concentration substance was realized by analyzing the digitalized signal through a high-resolution computational optical system in a microfluidic chip. We evaluated DELISA for measurement of total 25-hydroxyvitamin D (25OHD) in a fluidic chip. Methods The sample with monoclonal antibody bead conjugates mixture was incubated for 20 min at 37°C, then 30 uL sample mixture was applied to inlet of the chip. After additional incubation for 2 min at room temperature and 2 min after sample application, 120 uL of washing buffer was applied onto the washing hole and the chip was incubated for 10 min. 25OHD molecules in the sample bond to magnetic beads coupled with anti-25OHD at the end of the membrane. Chips were inserted into DELISA, and 25OHD results were derived by analysis of the computational method of microscopic imaging, proportional to the concentration of 25OHD. Assay performance characteristics of the limit of detection (LoD), precision and analytical measurement interval (AMI) using Vitamin D Depleted, Low and High Serum (Molecular Depot, San Diego, CA) were determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. 87 Samples for method comparison studies were obtained from routine clinical samples submitted for 2 OHD determination or from apparently healthy normal volunteers. The study was approved by the Institutional Review Board of Dongguk University Ilsan Hospital (202207024). Results LoD was 10.3 ng/mL and within-laboratory precision was 11, 12, and 10% coefficient of variation at 14.1, 56.6, and 113.2 ng/mL, respectively. The best fit polynomial was a cubic equation from 14.1 to 113.2 ng/mL and the difference between the third-order equation and the first-order equation at Level 3 and 4 concentrations was outside the non-linearity limit. Comparison with chemiluminescent assay (Elecsys Vitamin D total II, Roche Diagnostics, Mannheim, Germany) yielded a 0.904 of correlation coefficient and 0.921 slope in the range from 7.0 to 57.9 ng/mL. Conclusions The DELISA 25OHD assay has a suitable LoD and precision, however, it is necessary to improve the AMI at high concentration values and correlation with the automated analyzer.

Analytical variation concerning total 25-hydroxyvitamin D measurement, where are we now? A DEQAS review of current assay performance

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants’ performance of specific methods for 25-hydroxyvitamin D (25OHD) and other vitamin D metabolites. In this review an assessment of the state of the art in the performance of 25OHD methods is presented. This assessment is based on an analysis of data submitted by scheme participants for the 2021/22 distribution cycle, which comprised of four distributions each containing five DEQAS samples. These distributions enabled the assessment of performance across a wide concentration range and included samples containing endogenous 25OHD2. Overall analytical performance continues to improve, but there is still significant method variation and bias in some automated methods. These automated methods continue to be challenged in measuring 25OHD at the extremes of the measuring range and in the presence of 25OHD2. LC-MS/MS methods still show superior performance with regards to bias, but are out-performed by some automated methods in terms of assay variability. Through participating in an accuracy based EQA scheme, such as DEQAS, laboratories are able to assess the accuracy of their methods in comparison to a gold standard reference measurement procedure. It is crucial for all laboratories to be aware of the performance and limitations of their 25OHD assays and to educate their users accordingly in order to ensure reliable assessment of vitamin D status.

Biotin supplementation causes erroneous elevations of results in some commercial serum 25-hydroxyvitamin d (25OHD) assays

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants’ performance of specific methods for 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)2D). DEQAS occasionally circulates samples containing high levels of substances found in certain clinical situations e.g. 25-OHD2, 24,25-(OH)2D3, hypertriglyceridemia. The increased availability and use of health supplements containing biotin has led to case reports of assay interference in methods utilizing a biotin-streptavidin detection system. In October 2018, DEQAS included a serum sample (545) containing exogenous biotin (concentration =586 μg/L) which was analyzed by a total of 683 laboratories using 35 different methods. The same serum sample (544) without exogenous biotin was also included in the 5-sample set. All methods (760 laboratories) performed satisfactorily on sample 544 giving an All-Laboratory Trimmed Mean = 50.2 ± 6.5 nmol/L (±SD, CV = 12.9 %). The target value for this sample 544 (& 555) was 47.4 nmol/L as determined by Centers for Disease Control and Prevention (CDC) Atlanta, Georgia using their LC–MS/MS reference method. In contrast, #545 containing the exogenous biotin was reported by only 683 laboratories and gave an All-Laboratory Trimmed Mean = 66.8 ± 37.6 nmol/L (±SD, CV = 56.3 %). As expected, LC-MS/MS methods (143 labs) reported similar results for both 544 = 48.9 ± 4.4 nmol/L (±SD) and 545 = 48.3 ± 4.5 nmol/L (±SD) showing that assays involving chromatographic steps are unaffected by the presence of biotin. Several of the antibody-based assays including Abbott Architect, DiaSorin Liaison, Beckman Unicel and Siemens Centaur are also unaffected by the addition of biotin. Two assays, IDS-iSYS and Roche Total 25OHD, both of which use biotin-streptavidin, exhibit biotin interference yielding values with a significant positive bias for 545 of 102.6 nmol/L ± 78.7 nmol/L (±SD) and 517.8 nmol/L ± 209.8 nmol/L (±SD) respectively. Interestingly, the failure to report sample 545 data from 77 laboratories is due solely to those running Roche Total 25OHD or Roche Vitamin D Total II assays. Given the prevalence of the adversely affected assays (25 % of DEQAS users) and the high volume of 25OHD testing, clinicians using these assays should, where possible, only measure 25OHD when patients are off biotin.

SAT-514 The Decrease in Serum Parathyroid Hormone on Vitamin D Treatment Is Similar When Comparing Increases in Serum Free or Total 25 Hydroxyvitamin D in White and Black Women

Background: Recent advances in measurement of total serum 25OHD (T25D) led to development of free 25OHD (F25D) assays. The free hormone hypothesis states that only F25D which is about 0.02% of T25D is available for conversion to the active metabolite 1,25(OH)2D, the rest is bound to D binding protein (DBP) and albumin. In a previous paper (J Int Med 2018 Aug 23 online)we did not find a change in BMD or bone markers compared to F25D or T25D. A one month vitamin D study reported F25D was more highly correlated than T25D with serum PTH. If confirmed then F25D measurement would be clinically important. Methods: In two 12 month vitamin D placebo/dosing studies in young, old white and black women, serum T25D, F25D and PTH were measured at baseline and 12 months. The main inclusion criteria was vitamin D insufficiency (Diasorin serum T25D ≤ 20ng/ml). Serum T25D was remeasured by LCMS and F25D was measured by Elisa (Future Diagnostics). Serum PTH was measured by immunoassay (Diasorin). Group means were compared by t-test. Linear regression was used to assess percent change in T25D and F25D on percent change in PTH, accounting for age (young, old), race (Caucasians, African Americans) and interactions. Results: In the older women, there were 109 Caucasians and 79 African Americans with baseline measurements of F25D, T25D and PTH. Mean age (± SD) was 69 (± 7.3) years. In the younger women, there were 81 Caucasians and 31 African Americans, mean age 37 (± 5.6) years. In older women, serum T25D was similar in black compared to white women 18.70(±5.57) and 19.63(±5.19) ng/ml, p=0.24. Serum F25D was lower in older African Americans compared to Caucasians 3.38(±1.21) vs 3.73(±1.39) pg/mL (p=0.075). In younger women T25D was significantly lower in blacks 14.33(±4.13) compared to 22.09 (±5.45)ng/ml in whites, p<0.001. F25D was lower in blacks 3.55(±1.17) versus 4.64(±1.25) pg/ml p<0.001. Baseline mean serum PTH was higher in the older black women 44(±21) compared to whites 38 (±14)pg/ml (p=0.03). In young black women serum PTH was higher 44(±18) than white women 31(±10)(p<0.01). Serum PTH decreased on vitamin D. The decrease in PTH compared to levels of serum F25D was similar in young and old, black and white women. The decrease in PTH was 6% for each 1% increase in F25D. The slope for the regression of PTH on serum T25D was steeper in blacks than whites (p<0.01) but did not differ by age. The percent decrease in PTH was 13% in blacks and 5% in whites for each 1% increase in T25D. Summary: The percent decrease in serum PTH after vitamin D was related to increasing free and total 25D in young and old, black and white women. However, black women showed a significantly larger decrease in PTH with T25D, possibly because of more secondary hyperparathyroidism. Because the parathyroid gland has the megalin and cubilin receptors mechanism for uptake of DBP it may not be unexpected that the correlations between serum PTH and free and total 25(OH)D are so similar.

Circulating 25-hydroxycholecalciferol in Relationship to Central Dual-Energy X-Ray Absorptiometry Assesses. A clinical study

The aim of the research was to realize a clinical study on menopausal patients, focused on 25-hydroxyvitamin D (25OHD) assays versus Dual-Energy X-Ray Absorptiometry (DXA) categories. This transversal, observational, real-life study was effectuated on Caucasian Romanian females. A total of 60 subjects were grouped according to lumbar T-score: normal T-score (N=28), osteopenia (N=22), and osteoporosis (N=10). The lowest average value of 25OHD is found in patients with osteoporosis, which is statistically significant lower than in patients with osteopenia. The average values of PTH were within normal levels for each group. 25OHD did not correlate with PTH or lumbar BMD. Overall the mean values of 25OHD are in deficient ranges regardless osteoporosis, osteopenia or normal DXA.

Verification of Abbott 25-OH-vitamin D assay on the architect system

ObjectivesAnalytical and clinical verification of both old and new generations of the Abbott total 25-hydroxyvitamin D (25OHD) assays, and an examination of reference Intervals. MethodsDetermination of between-run precision, and Deming comparison between patient sample results for 25OHD on the Abbott Architect, DiaSorin Liaison and AB SCIEX API 4000 (LC-MS/MS). Establishment of uncertainty of measurement for 25OHD Architect methods using old and new generations of the reagents, and estimation of reference interval in healthy Irish population. ResultsFor between-run precision the manufacturer claims 2.8% coefficients of variation (CVs) of 2.8% and 4.6% for their high and low controls, respectively. Our instrument showed CVs between 4% and 6.2% for all levels of the controls on both generations of the Abbott reagents. The between-run uncertainties were 0.28 and 0.36, with expanded uncertainties 0.87 and 0.98 for the old and the new generations of reagent, respectively. The difference between all methods used for patients’ samples was within total allowable error, and the instruments produced clinically equivalent results. The results covered the medical decision points of 30, 40, 50 and 125nmol/L. The reference interval for total 25OHD in our healthy Irish subjects was lower than recommended levels (24–111 nmol/L). ConclusionIn a clinical laboratory Abbott 25OHD immunoassays are a useful, rapid and accurate method for measuring total 25OHD. The new generation of the assay was confirmed to be reliable, accurate, and a good indicator for 25OHD measurement. More study is needed to establish reference intervals that correctly represent the healthy population in Ireland.

French law: what about a reasoned reimbursement of serum vitamin D assays?

The number of serum 25-hydroxyvitamin D (25OHD) assays has increased tenfold in France in less than 10 years, sometimes for invalidated reasons. In 2013, the French National Authority for Health (Haute autorité de santé, or HAS) limited the indications for serum 25OHD measurements to rickets/osteomalacia, older adults with recurrent falls, monitoring of kidney transplant in adults, and surgical treatment of obesity in adults. Our aim here was to note that other indications for serum 25OHD measurements are supported by previous literature and by a number of national and international recommendations, in particular the following: any situation of bone fragility, any chronic renal failure <45 mL/min/1.73m2, any situation of malabsorption, clinical signs consistent with vitamin D deficiency or vitamin D overload, and calcium phosphorus evaluation. We suggest that the measurement of serum 25OHD concentration should remain reimbursed as part of these extended indications.

Looking beyond linear regression and Bland-Altman plots: a comparison of the clinical performance of 25-hydroxyvitamin D tests.

The systematic evaluation of the clinical concordance of various 25-hydroxyvitamin D (25OHD) testing methods is presented. The need for this approach is raised by the discrepancies in the analytical performance of the available assays. The analytical and clinical performance of six automated 25OHD assays and an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was investigated. Leftover serum samples (n=162, SA: n=114) were analyzed and all 21 assay combinations were evaluated. The utility of Cohen's κ values was assessed by transforming them into minimum percentage agreement (MPA). McNemar's hypothesis test was employed for testing the symmetry of the disagreeing classification outcomes within each method pair. Depending on the assay method, the ratio of results classified as positive (<20 ng/mL) was 13.5%-40.0%. The percentage agreement (PA) was 74.1%-92.6%. Compared to other methods, significantly more hypovitaminosis cases were delivered by DiaSorin Liaison® 25 OH vitamin D Total (DL) and significantly fewer by IDS-iSYS 25-Hydroxy Vitamin DS (II). The strongest clinical concordance was exerted by II vs. LC-MS/MS. The κ-derived MPA showed close similarity to the PA scores. McNemar's tests confirmed the asymmetry of the disagreement in the classification in 14 method combinations. The presented approach allows the prediction of the clinical consequences of a 25OHD method transfer. Differences in the clinical classification of assay results are likely encountered when transferring to a new method, even between assays standardized according to the Vitamin D Standardization Program (VDSP) Reference Method Procedure (RMP).

Low vitamin D level in pediatric patients with new onset type 1 diabetes is common, especially if in ketoacidosis.

Vitamin D deficiency (VDD) adversely affects bone health. US data on prevalence of VDD or vitamin D insufficiency (VDI) at diagnosis of type 1 diabetes (T1D) is lacking. Low serum 25-hydroxyvitamin D (25OHD) is speculated to increase the risk of developing T1D. Assess the prevalence of pediatric VDD and VDI at diagnosis of T1D, and investigate correlations with demographic and clinical parameters. We performed a retrospective Institutional Review Board (IRB)-approved chart review of all T1D cases diagnosed from January 2011 to August 2012, all having the same 25OHD assay performed at Quest Diagnostics. Definitions for VDD, VDI, and vitamin D sufficiency (VDS) were 25OHD levels (ng/mL) ≤ 20, 21-29, and ≥ 30, respectively. We termed 25OHD <30 ng/mL as Low-D. We identified 185 autoantibody positive T1D subjects (51% female) with 25OHD measured, 51% Caucasian, 25% Hispanic, 4% mixed-Hispanic, 4% African American, and 16% other/mixed race. Mean age 9.8 yr (0.9-18.6). Most had Low-D (58%), 40% VDI, 18% VDD, and 42% VDS. No gender or age differences among the 25OHD groups. Low-D was more common (p < 0.0001) in Hispanics (81%) vs Caucasians (44%), but VDD rates were similar. Low-D subjects were heavier than VDS (p = 0.018). All four with elevated celiac titers were VDS. Diabetic ketoacidosis was present in 33%, but more common (44%) in Low-D vs. VDS (18%) (p < 0.0001), and trended higher in VDD (61%) vs. VDI (36%). Seventy-seven percentage with DKA had Low-D. VDD and insufficiency are common, even in Caucasians, at onset of T1D in pediatrics, worse in those with DKA.

Serum PTH reference values established by an automated third-generation assay in vitamin D-replete subjects with normal renal function: consequences of diagnosing primary hyperparathyroidism and the classification of dialysis patients.

To determine parathyroid hormone (PTH) reference values in French healthy adults, taking into account serum 25-hydroxyvitamin D (25OHD), renal function, age, gender, and BMI. We studied 898 healthy subjects (432 women) aged 18-89 years with a normal BMI and estimated glomerular filtration rate (eGFR), 81 patients with surgically proven primary hyperparathyroidism (PHPT), and 264 dialysis patients. 25OHD and third-generation PTH assays were implemented on the LIAISON XL platform. Median PTH and 25OHD values in the 898 healthy subjects were 18.8 ng/l and 23.6 ng/ml respectively. PTH was lower in subjects with 25OHD ≥30 ng/ml than in those with lower values. Among the 183 subjects with 25OHD ≥30 ng/ml, those aged ≥60 years (n=31) had higher PTH values than younger subjects, independent of 25OHD, BMI, and eGFR (P<0.001). Given the small number of subjects aged ≥60 years, we adopted the 95% CI of PTH values for the entire group of 183 vitamin D-replete subjects (9.4-28.9 ng/l) as our reference values. With 28.9 ng/l as the upper limit of normal (ULN) rather than the manufacturer's ULN of 38.4 ng/l, the percentage of PHPT patients with 'high' PTH values rose to 90.1% from 66.6% (P<0.001), and 18.6% of the dialysis patients were classified differently in view of the KDIGO target range (two to nine times the ULN). When only subjects with 25OHD ≥30 ng/ml were included in the reference population, the PTH ULN fell by 22.4%, diagnostic sensitivity for PHPT improved, and the classification of dialysis patients was modified.

The Association of Vitamin D Status and Pre-operative Physical Activity in Patients with Hip or Knee Osteoarthritis.

Vitamin D is important for musculoskeletal health and may have significant implications for maintaining physical activity in elderly patients. Our goal was to investigate whether serum 25-hydroxyvitamin D (25OHD) levels are associated with pre-operative physical activity in patients who are offered elective knee or hip joint replacement surgery. We performed a single-center, retrospective analysis of patients who had elective knee or hip replacement surgery from 2002 to 2012. To investigate the association of serum 25OHD levels with pre-operative physical activity, as assessed by the University of California, Los Angeles (UCLA) activity scale, we performed a multivariable logistic regression analysis while controlling for age, sex, race, body mass index, American Society of Anesthesiologists physical status score, and season. We identified 182 patients who met inclusion criteria. Mean (± standard deviation) 25OHD level and UCLA activity scale score were 29±13 ng/mL and 4±2, respectively. Patients with 25OHD levels <20 ng/mL were almost three times more likely to have UCLA activity scale scores ≤3 [adjusted odds ratio 2.78; 95% confidence interval 1.72-9.17]. Further adjusting for "type of 25OHD assay" or "type of joint surgery" did not materially change this result. In our cohort of knee or hip joint replacement surgery patients, 25OHD levels were associated with pre-operative physical activity scores. Prospective, randomized, clinical trials are needed to verify whether optimizing pre-operative vitamin D status may improve physical activity and influence clinical decision-making in knee or hip joint replacement surgery candidates.

Serum 25-Hydroxyvitamin D Levels: Variability, Knowledge Gaps, and the Concept of a Desirable Range.

Hypovitaminosis D is prevalent worldwide but proportions vary widely between regions, depending on genetic and lifestyle factors, the threshold to define deficiency, and accuracy of 25-hydroxyvitamin D (25OHD) assays used. Latitude, pollution, concealing clothing, sun exposure, gender, dietary habits, and lack of government regulation account for up to 50% in variations in serum 25OHD levels, whereas genetic polymorphisms in the vitamin D pathway account for less than 5%. Organizations/societies have developed guidelines for recommended desirable 25OHD levels and vitamin D doses to reach them, but their applicability across age groups and populations are still debated. This article and the accompanying online Supporting Information highlight sources of variations in circulating 25OHD levels, uncertainties and knowledge gaps, and analytical problems facing 25OHD assays, while keeping efficacy and safety data as the dominant factors when defining a desirable range for 25OHD levels. We propose a desirable range of 20 to 40 ng/mL (50 to 100 nmol/L), provided precise and accurate assays are used. Although slightly lower levels, 15 to 20 ng/mL, may be sufficient for some infants and adults, higher levels, 40 to 60 ng/mL, may still be safe. This desirable range allows physicians to tailor treatment while taking season, lifestyle, vitamin D intake, and other sources of variation into account. We reserve 25OHD measurements for at-risk patients, defined by disease or lifestyle, and the use of 25OHD assays calibrated against the recommended international standards. Most target groups reach desirable target levels by a daily intake of 400 to 600 IU for children and 800 IU for adults. A total daily allowance of vitamin D of up to 1000 IU in the pediatric age groups, and up to 2000 IU in adults, tailored to an individual patient risk profile, is probably safe over long durations. Additional data are needed to validate the proposed range and vitamin D doses, especially in children, pregnant women, and non-white populations.

Vitamin D and insulin resistance

Vitamin D is a steroid hormone, which in active form binds to the vitamin D receptor. Expression of the vitamin D receptor in diverse cell types (pancreatic islet cells, myocytes, hepatocytes and adipocytes) raises the suspicion that vitamin D may be involved in multiple cellular processes, including the response to insulin. Insulin resistance is a characteristic feature of type 2 DM, and its attenuation may reduce the incidence of type 2 DM and cardiovascular disease. In observational studies, low serum 25-hydroxyvitamin D (25-OHD) concentrations are associated with an increased risk of type 2 DM. It has been suggested that increasing serum 25-OHD concentrations may have beneficial effects on glucose and insulin homeostasis. However, cross-sectional and interventional studies of vitamin D supplementation provide conflicting results and demonstrate no clear beneficial effect of vitamin D on insulin resistance. These studies are complicated by inclusion of different patient cohorts, different 25-OHD assays and different doses and preparations of vitamin D. Any possible association may be confounded by alterations in PTH, 1,25-dihydroxyvitamin D or tissue vitamin D concentrations. We identified 39 studies via MEDLINE and PUBMED. We review the evidence from 10 studies (seven observational and three interventional) examining vitamin D and type 2 DM incidence, and 29 studies (one prospective observational, 12 cross-sectional and 16 interventional trials) examining vitamin D and insulin resistance. Based on this data, it is not possible to state that vitamin D supplementation has any effect on type 2 DM incidence or on insulin resistance. Data from the multiple ongoing randomized controlled trials of vitamin D supplementation due to report over the next few years should help to clarify this area.

Serum 25-Hydroxy Vitamin D Levels in Bulgarian Patients with Prostate Cancer: a Pilot Study

The antiproliferative effect of the active form of vitamin D on cancer cells and its ability to induce cell differentiation and suppression of tumor-induced angiogenesis in the last decade has provoked enormous research for the elucidation of its role in the prevention of different types of cancer and in slowing down the malignancy progression. The aim of the present pilot study was to determine the circulating 25-hydroxy vitamin D (25OHD) levels in Bulgarian prostate cancer (PCa) patients and to investigate their relationship with various determinants associated with the severity and progression of the disease. A total of 53 male patients (mean age 67.0 ± 7.1 years) with clinical suspicion for PCa were enrolled in the study. All patients were subjected to systemic transrectal ultrasound-guided tru-cut prostate biopsies (10 cores at least). Detected tumors were graded using the Gleason grading system. Prostate specific antigen (PSA) serum levels were measured immunochemically. The 25OHD assay was performed by a validated HPLC-UV method. Other covariates (BMI, age, family history of PCa) were collected by interview at the time of hospitalization. One-way ANOVA with Kruskal Wallis statistics was used for comparison of medians of different parameters. The level of significance was set at p < 0.05. Significantly lower 25OHD levels were detected in PCa patients compared to those with benign prostate hyperplasia (BPH) (p < 0.05). Patients with high grade tumors (Gleason score ≥ 7) showed significantly lower 25OHD levels, while those with low grade tumors (Gleason score < 7) revealed better 25OHD status (50.49 vs. 63.17 nmol/L, p < 0.05). A moderate negative correlation between 25OHD levels and the Gleason score was established (Spearman r = -0.46, p < 0.05). Significant seasonal variations in 25OHD levels, both for PCa and BPH patients, were detected (p < 0.01). This preliminary study shows an association between 25OHD status and classical markers characterizing the severity of PCa. The results might suggest a potential beneficial role of vitamin D for PCa patients. Further prospective studies are needed to strengthen the interrelationships between 25OHD levels and variables related with PCa and to test them for causality.

Impact of assay design on test performance: lessons learned from 25-hydroxyvitamin D

Current automated immunoassays vary significantly in many aspects of their design. This study sought to establish if the theoretical advantages and disadvantages associated with different design formats of automated 25-hydroxyvitamin D (25-OHD) assays are translated into variations in assay performance in practice. 25-OHD was measured in 1236 samples using automated assays from Abbott, DiaSorin, Roche and Siemens. A subset of 362 samples had up to three liquid chromatography-tandem mass spectrometry 25-OHD analyses performed. 25-OHD₂ recovery, dilution recovery, human anti-animal antibody (HAAA) interference, 3-epi-25-OHD₃ cross-reactivity and precision of the automated assays were evaluated. The assay that combined release of 25-OHD with analyte capture in a single step showed the most accurate 25-OHD₂ recovery and the best dilution recovery. The use of vitamin D binding protein (DBP) as the capture moiety was associated with 25-OHD₂ under-recovery, a trend consistent with 3-epi-25-OHD₃ cross-reactivity and immunity to HAAA interference. Assays using animal-derived antibodies did not show 3-epi-25-OHD₃ cross-reactivity but were variably susceptible to HAAA interference. Not combining 25-OHD release and capture in one step and use of biotin-streptavidin interaction for solid phase separation were features of the assays with inferior accuracy for diluted samples. The assays that used a backfill assay format showed the best precision at high concentrations but this design did not guarantee precision at low 25-OHD concentrations. Variations in design among automated 25-OHD assays influence their performance characteristics. Consideration of the details of assay design is therefore important when selecting and validating new assays.

Vitamin D Measurements — Facts and Fancies

Clinical laboratories have seen an increased interest in vitamin D measurements as new knowledge about the vitamin's pathobiology escalates. Clinicians now recognise that besides bone health, vitamin D insufficiency may be associated with potential health risks such as coronary artery disease, diabetes, depression, epilepsy, polycystic ovaries, musculoskeletal pain, autoimmune disease, multiple sclerosis, and cancers. While vitamin D testing and supplementation has been touted for these patients, benefit is evident only for the prevention of falls and fractures. The main form of vitamin D measured is 25-hydroxy-vitamin D (25OHD). While 25OHD assays have improved, they remain a work in progress.

Evaluation of two routinely used 25OHD assays and serum variables in patients on dialyses

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Preanalytical evaluation of serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 measurements using LC–MS/MS

BackgroundVitamin D testing is increasing worldwide. Although immunoassays are still widely used in Japan for the measurement of serum 25-hydroxyvitamin D (25OHD) as an indicator of vitamin D status, development of a simple and high-throughput MS-based method is still needed for routine use in clinical laboratories. MethodsWe designed a method using a triple quadrupole mass spectrometer equipped with a two-step separation approach that used the Aria TLX-2 HPLC system in the selected reaction monitoring mode. Analytical performance of the system and effects of various preanalytical factors were tested. ResultsHigh-throughput quantitative analysis of 25OHD3 and D2 at 15samples/h was achieved using 25μl of serum/plasma. Intra- and inter-assay CVs for 25OHD3 were 5% and 7%, respectively. Limit of detection for 25OHD3 was 0.31ng/ml. No significant effects were seen for clotting time, repeated freeze–thaw cycles, anti-coagulants and possible interfering substances. A good correlation (r2=0.947) was found between the present system and the DiaSorin radioimmunoassay. Serum 25OHD3 levels in apparently healthy Japanese subjects were 25.5±9.8ng/ml for men and 20.9±7.1ng/ml for women. ConclusionsThis high-throughput LC–MS/MS 25-OHD assay has the potential to be used as a routine clinical laboratory assay for assessing vitamin D status.

Analytical methods and performance of the immunoassay methods for determination of vitamin d in comparison to mass spectrometry / Analitičke metode i izvođenje imunometrijskih određivanja vitamina d u poređenju sa masenom spektrometrijom

Summary Demand for vitamin D testing has been on a constant rise worldwide, partially due to mounting evidence linking vitamin D status to overall health and well-being. Currently available assays measure 25-hydroxy vitamin D (25-OHD), a major circulating form of vitamin D. Available methodologies include immunoassays and mass spectrometry based methods (LC-MS/MS). Until recently, the only immunoassays available for diagnostic use in the US have been DiaSorin radioimmunoassay (RIA) and an automated immunoassay on a LIAISON® platform. Within the last year, Siemens and Abbott successfully launched immuno - assays for determination of total vitamin D on their respective automated platforms, Centaur® and ARCHITECT®. Development of robust and precise Vitamin D immunoassays has historically been plagued with difficulty. One of the major challenges is development of specific antibodies against such a small antigen. Vitamin D is also highly hy - drophobic molecule predominantly bound to vitamin D binding protein (DBP). It is likely, therefore, that immuno - assays might be affected to varying extent by the DBP concentration. Adoption of LC-MS/MS into clinical laboratories has enabled development of accurate and almost fully automated methods that could handle increasing volume demands, especially in large volume reference laboratories. Smaller to mid-size hospital laboratories as well as physician offices have neither funds nor technical expertise to implement LC-MS/MS based testing. Our laboratory at the University of Chicago Medical Center has also seen in - crease in vitamin D volume and currently performs close to 20,000 25-OHD assays per year. We have recently deve - loped an LC-MS/MS method for quantitation of 25-OHD2 (obtained from plant sources) and 25-OHD3 (endogenous and animal sources). Prior to acquisition of LC-MS/MS instrument, we performed 25-OHD analysis by RIA. Du - ring the transition period, we encountered several challenges, including the necessity to streamline sample preparation as well as the bias introduced by calibration dif ferences. We chose to match our LC-MS/MS method to the RIA method in order to make this transition transparent to the clinician. Most immunoassays available today are acceptable for clinical use and might be method of choice for smaller laboratories. Larger clinical laboratories and aca demic institutions that possess technical expertise, particularly the ones with large pediatric population where assay sensitivity and specificity may be important, might find LC-MS/MS methodology a more suitable choice.

Preanalytical, analytical (DiaSorin LIAISON) and clinical variables potentially affecting the 25-OH Vitamin D estimation

BackgroundVitamin D (25-OHD) physiological functions have been expanded beyond traditional bone health, increasing the importance of its estimation in Laboratory Medicine, which renders validation of available methods mandatory. Aims and methodsWe evaluated some preanalytical and analytical aspects of 25-OHD determination and the effects of potentially confounding clinical variables by using the DiaSorin “LIAISON 25-OH Vitamin D TOTAL”. Results25-OHD samples were extremely stable, at least in the short term, without requiring special transport or storage. Precision intervals (CV%) were: within run (7–11%) and total precision (8–11.5%). Mean (SD) recovery was 96 (2)%. The assay was linear on dilution. Comparison with radioimmunoassay (RIA) yielded acceptable correlation (Inter-rater agreement/kappa coefficient=0.94) and clinical equivalence in the interval from 6 to 55ng/mL.The assay was evaluated on a general population (N=476, age: 60±14years, 65 males). The status of 25‐OHD resulted inversely related to parathyroid hormone levels (r=−0.21, p<0.001), and aging (r=−0.17, p<0.001), but not to sex. Levels of 25-OHD were found to be sufficient (≥30ng/mL) only in 54 samples (12%). Marked seasonal 25-OHD variations were observed in 13 subjects (p<0.05). Moreover, a marked seasonal fluctuation was seen in samples collected during the period of February 2010–October 2011 (p≤0.01).Lower 25-OHD concentration was observed in subjects with diabetes (19±9 vs 14±7ng/mL, p<0.01) and hypertension (20±9 vs 17±9ng/mL, p<0.01). Moreover, 25-OHD inversely correlated with BMI (r=−0.25, p<0.001). Conversely, no difference in 25-OHD levels was observed between subjects due to smoking habits and dyslipidemia.In multiple logistic regression models, aging is the only significant independent risk factor for low 25-OHD levels (Odds ratio, 95% confidence intervals: 3.1, 1.3–7.3; p≤0.01). ConclusionsResults confirm the LIAISON 25-OHD assay as a useful tool for 25-OHD estimation in the clinical practice. Lack of vitamin D is common among Italian adults, and appears associated with several cardiovascular risk factors.