25OHD Assays Research Articles

The prevalence of vitamin deficiency in clinical practice is assay-dependent

Vitamin D deficiency is an important concern in clinical settings and recently, international agencies have recognised the importance of 25-OHD assays in defining vitamin D status. Thus, our aim was to assess the consistency of different vitamin D assays in clinical practice. 25-OH-vitamin D was measured in 332 patients by ultra-fast liquid chromatography (UHPLC) and two immunoassays (Liaison Total 25(OH) and ADVIA Centaur Vitamin D Total Assay). Samples from the Vitamin D External Quality Survey (DEQAS) and the Standard Reference Material SRM 972 were used for analytical quality control. All methods displayed an acceptable performance with DEQAS samples but immunoassays showed a significant bias against certified materials. Compared to UHPLC, differences were significant for both immunoassays in the deficiency interval but the systematic bias was higher for the ADVIA assay throughout the whole range of concentrations. The prevalence of vitamin D deficiency in clinical practice is assay-dependent and physicians should be aware of the uncertainty associated with vitamin D assessment.

Les dosages des hormones calciotropes : PTH, métabolites de la vitamine D, FGF 23

PTH and 25OHD assays must now be considered routine assays that are available in numerous laboratories. Even if improvements in these measurements are still necessary, they are currently acceptable for the routine practice. Reference values for serum PTH levels need however to be revisited for many PTH kits in order to fit with the official recommendations. Indeed, they should be established in subjects with a normal vitamin D status and a normal kidney function. Calcitriol and FGF 23 measurement are not first-line measurements and should only performed in highly specialized laboratories. Whereas a 25OHD measurement may be prescribed without calcemia, phosphatemia or PTH to evaluate the vitamin D status, a PTH, calcitriol or FGF 23 concentration needs to be interpreted by comparison with other parameters of calcium/phosphate metabolism measured on the same blood sample. Specially, a serum PTH level needs, in any case, to be interpreted with the concomitant calcemia at least, phosphatemia, calciuria, and 25OHD being also frequently necessary.

Current 25-hydroxyvitamin D assays: Do they pass the test?

BackgroundVitamin D testing is becoming increasingly important with recent research demonstrating a correlation between vitamin D insufficiency and metabolic diseases, immunodeficiencies and other diseases. However, existing 25-hydroxyvitamin D (25OHD) assays lack comparability to the candidate reference method, causing difficulties in diagnosis and monitoring of vitamin D deficiency. MethodsWe looked at the accuracy of 3 automated assays (Roche Diagnostics Elecsys® Total 25OHD assay, Abbott Architect® Total vitamin D assay, Advia Centaur® vitamin D Total assay) and Diasorin® Radioimmunoassay (RIA) compared to a routine laboratory Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS). ResultsThe correlation based on Passing Bablok regression was good with the slopes between 0.95 and 1.31 and the intercepts between −3.24 and 3.68. However, a significant positive bias was observed using the Abbott Architect and the Diasorin RIA. Using published analytical goals of coefficient of variation (CV) <10% and bias <5%, most methods did not meet these criteria. Using measurement of uncertainty of 9%, most methods were able to meet criteria using quality control materials but not patient samples. ConclusionInadequacies of these assay performances are contributed by differences in method of extraction of vitamin D from vitamin D binding protein, cross-reactivities to 25OHD2, 25OHD3 and other vitamin D metabolites, matrix interferences and a lack of standardization.

Evaluation of 25-hydroxy vitamin D assay on the immunodiagnostic systems iSYS analyser

We evaluated the recently released chemiluminescence assay for 25-hydroxy vitamin D (25-OHD) on the Immunodiagnostic Systems iSYS (IDS-iSYS) automated analyser. The IDS-iSYS comparison was performed using patient samples previously measured for 25-OHD by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (n = 119) and an IDS enzyme immunoassy (IDS-EIA) method (n = 64). Limit of detection and limit of quantification were determined from a precision profile. Imprecision was assessed using quality control material and pooled serum. External QAP material (Vitamin D External Quality Assessment Scheme, UK) was analysed to establish inaccuracy. Linearity was assessed by two dilution studies. Cross-reactivity was determined by three serial dilution studies of patient samples with known 25-OHD(2) concentrations. The IDS-iSYS correlated well with both established methods (iSYS = 1.03LC-MS/MS - 6.53, R(2) = 0.82 and iSYS = 1.07IDS-EIA - 1.61, R(2) = 0.86). Imprecision of the iSYS assay for IDS control material was 13.4% at 32 nmol/L, 10% at 78 nmol/L, 9.4% at 161 nmol/L, and for the pooled material 9.3% at 72 nmol/L and 5.6% at 158 nmol/L. The evaluation found the assay to be highly accurate (IDS-iSYS = 0.93ALTM + 3.79, R(2) = 0.94) and linear (obs(1) = 0.93exp(1) - 5.05, R(2) = 0.99 (P = 0.256); and obs(2) = 0.97exp(2) + 6.07, R(2) = 0.97 (P = 0.654); ALTM, all-laboratory trimmed mean). Cross-reactivity studies demonstrated no significant difference to the calculated total 25-OHD as measured by LC-MS/MS. Even though the imprecision of the iSYS was found to be greater than that of the LC-MS/MS and EIA methods, the performance characteristics of the IDS-iSYS 25-OHD assay are suitable for routine diagnostic purposes on a high throughput automated analyser.

25-Hydroxyvitamin D Assay Variations and Impact on Clinical Decision Making

Laboratories are increasingly shifting to new automated 25-hydroxyvitamin D (25-OHD) assays, with subsequent variability in results. We describe the experience at our center with such a shift and illustrate its clinical implications. 25-OHD levels were measured in 494 patients using Immunodiagnostic Systems RIA (IDS-RIA) and DiaSorin Liaison assays. Sources of variability between the assays were investigated in a subset of 83 samples, retested in the reference laboratory in the United States, and by reviewing the performance reports issued by the International Vitamin D External Quality Assessment Scheme, DEQAS. 25-OHD cut-points for target levels were used to compare the two assays. 25-OHD concentrations were significantly lower when measured with Liaison as compared to IDS-RIA: mean bias was -5 ng/ml, range was -38.1 to 18.7 ng/ml, P<0.001; the absolute bias was independent of 25-OHD value. Interassay variability was also detected in values obtained in the reference laboratory and in DEQAS reports. Using 20 ng/ml as the target 25-OHD level, 52% of patients required treatment when tested by Liaison, as opposed to 36% by IDS-RIA (P<0.001). Using 30 ng/ml as the desirable level, the proportions were 79 and 64%, respectively (P<0.001). The two assays agreed in only 41-68% of subjects, proportions that depended on criteria used to define agreement. A change in 25-OHD assays has a significant impact on results, patient classification, and treatment recommendations. Such variability cannot be ignored when deriving and applying vitamin D guidelines. It also renders universal assay standardization a pressing call.

Accuracy of 25-Hydroxyvitamin D Assays: Confronting the Issues

Measurement of 25-hydroxyvitamin D (25-OHD) is widely used for assessing vitamin D status. There has been a dramatic increase in 25-OHD requests over recent years prompting many laboratories to consider the use of automated immunoassays. To achieve higher throughput, these methods have abandoned the traditional solvent extraction of samples and are therefore more prone to non-specific interference. The Vitamin D External Quality Assessment Scheme (DEQAS) has revealed method-related differences in 25-OHD results, raising concerns about the comparability and accuracy of different assays. This paper highlights some of the pre-analytical, analytical and post-analytical issues which may influence the accuracy of 25-OHD assays and interpretation of results. Recent attention has focused on reconciling the relatively high results given by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to those of the DiaSorin radioimmunoassay (RIA) on which clinical decision points have previously been based. Data is presented on 20 DEQAS samples which were analysed by an LC-MS/MS assay developed as a candidate reference measurement procedure by the US National Institute of Standards and Technology (NIST). The NIST results were on average 11.2% lower than those given by routine LC-MS/MS methods. If confirmed, these results suggest that most routine LC-MS/MS assays are perhaps overestimating 25-OHD by failing to resolve a molecule having the same mass as 25-OHD(3) and a similar fragmentation pattern. All 25-OHD assays should be monitored by a proficiency testing scheme and the results made available to clinicians and editors of scientific journals.

UK Food Standards Agency Workshop Consensus Report: the choice of method for measuring 25-hydroxyvitamin D to estimate vitamin D status for the UK National Diet and Nutrition Survey

The consensus workshop, organised on behalf of the Food Standards Agency, was convened to recommend the most appropriate and secure method for measuring vitamin D status in the UK. Workshop participants (the Expert Panel) were invited on the basis of expertise in current 25-hydroxyvitamin D (25OHD) assays, or expertise in vitamin D nutrition and metabolism or detailed knowledge and experience in the National Diet and Nutrition Survey (NDNS). A decision support matrix, which set out the particular criteria by which the different options were scored and evaluated, was used to structure the discussion. The Expert Panel agreed that five methods for measuring 25OHD should be evaluated according to eleven criteria, selected on the basis of their relevance to the NDNS. All three of the evaluating subgroups of the Expert Panel produced similar total scores over the eleven criteria for the different methods; they scored LC-MS/MS and HPLC-UV similarly highly, while the scores for the immunoassay methods were lower. The Expert Panel recommended that an LC-MS/MS method should be the preferred method for the NDNS. A detailed specification for the method will be required to ensure comparability between NDNS and the National Health and Nutrition Examination Survey in the US facilitating future comparisons. The Expert Panel also recommended that the method should be carried out in a laboratory with appropriate expertise, competency and history of records of good performance. The method should be standardised against the National Institute of Standards and Technology SRM 972. If the recommended LC-MS/MS is adopted, the Expert Panel indicated that the method should be able to discriminate the C-3 epimer of 25OHD(3), especially if used to measure 25OHD in young infants in the forthcoming Diet and Nutrition Survey of Infants and Young Children, who are known to have high circulating concentrations of the C-3 epimer.

Proficiency testing of 25-Hydroxyvitamin D (25-OHD) assays

The Vitamin D External Quality Assessment Scheme (DEQAS) has been monitoring 25-OHD assay performance since 1989. The scheme has expanded rapidly in recent years and has 670 participants in 35 countries (July 2009). Five samples of human serum are distributed quarterly and the results analyzed to give an All-Laboratory Trimmed Mean (ALTM) and SD. Each participant has internet access to a preliminary report after submission of results and, following the results deadline, a final report is e-mailed to designated staff in each laboratory. The last 15 years has seen an improvement in mean inter-laboratory imprecision (CV), from 32.0% (1994) to 15.3% (2009) and most major methods are now giving results within plus or minus 7.4% of the ALTM (2009). DEQAS has regularly conducted and reported on a number of investigations into the performance of 25-OHD methods. A gas chromatography–mass spectrometry (GC–MS) reference method for 25-OHD is under development and will be used to assess whether the ALTM remains the most appropriate target for DEQAS samples.

Multiple micronutrient deficiencies persist during early childhood in Mongolia.

Data on the prevalence of micronutrient deficiencies in children in Mongolia is limited. We therefore determined the prevalence of anaemia, iron deficiency anaemia (IDA), and deficiencies of iron, folate, vitamin A, zinc, selenium, and vitamin D among young Mongolian children. Anthropometry and non-fasting morning blood samples were collected from 243 children aged 6-36 months from 4 districts in Ulaanbaatar and 4 rural capitols for haemoglobin (Hb), serum ferritin, folate, retinol, zinc, selenium, and 25-hydroxyvitamin D (25-OHD) assays. Children with alpha-1-glycoprotein >1.2mg/L (n=27) indicative of chronic infection were excluded, except for folate, selenium, and 25-hydroxyvitamin D assays. Of the children 14.5% were stunted and none were wasted. Zn deficiency (serum Zn <9.9 micromol/L) had the highest prevalence (74%), followed by vitamin D deficiency 61% (serum 25-OHD<25 nmol/L). The prevalence of anaemia (24%) and iron deficiency anaemia (IDA) (16%) was lower, with the oldest children (24-36 mos) at lowest risk. Twenty one percent of the children had low iron stores, and 33% had vitamin A deficiencies (serum retinol < 0.70 micromol/L), even though two thirds had received vitamin A supplements. Serum selenium values were low, perhaps associated with low soil selenium concentrations. In contrast, no children in Ulaanbaatar and only 4% in the provincial capitols had low serum folate values (<6.8 nmol/L). Regional differences (p<0.05) existed for anaemia, deficiencies of vitamin A, folate, and selenium, but not for zinc or IDA. Of the children, 78% were at risk of > or = two coexisting micronutrient deficiencies emphasizing the need for multimicronutrient interventions in Mongolia.

Assay-Specific Decision Limits for Two New Automated Parathyroid Hormone and 25-Hydroxyvitamin D Assays

The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P <0.001). Regression and Bland-Altman analyses suggested that the LIAISON 25OHD assay reads lower than the DiaSorin RIA at low concentrations but higher at high concentrations. However, the cutoff (50 nmol/L) used in our laboratories to define vitamin D insufficiency with the DiaSorin RIA is applicable to the LIAISON 25OHD assay. In 927 healthy individuals, the 3rd-97th percentile intervals were 3-80 ng/L and 13-151 nmol/L for the LIAISON PTH and 25OHD concentrations, respectively. However, 506 individuals (54.6%) were vitamin D-insufficient; we therefore considered only the 421 individuals with a LIAISON 25OHD >50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.

Serum 25-Hydroxyvitamin D Is Unaffected by Multiple Freeze-Thaw Cycles

Vitamin D deficiency is a common disorder believed to affect as many as 57% of hospitalized patients (1) and 9–50% of outpatients, depending on the characteristics of the study population (2)(3)(4)(5)(6). Vitamin D deficiency can cause secondary hyperparathyroidism and, if severe, osteomalacia. Both of these conditions are associated with loss of bone density, and both are reversible by repletion of vitamin D (7)(8)(9)(10). Recently, the association of vitamin D deficiency with an increased risk for a broader range of diseases, such as prostate cancer (11)(12) and colon cancer(13), has come to attention. Dietary vitamin D supplementation has even been implicated in decreasing the risk of developing type 1 diabetes mellitus (14), and ultraviolet irradiation has been shown to decrease systolic blood pressure in patients with mild untreated hypertension (15). Determining whether vitamin D deficiency is present usually involves assessing the stores of vitamin D by measuring serum 25-hydroxyvitamin D (25OHD) concentrations, in combination with an assessment of parathyroid hormone concentrations. Currently, the preferred clinical 25OHD assay is a two-step procedure that involves a rapid extraction step of 25OHD from serum or plasma, followed by an equilibrium RIA procedure. The …

How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme.

A recent report highlighted the interlaboratory variability in serum 25-hydroxyvitamin D (25OHD) results (1). Concerns have also been raised about the apparent inability of some 25OHD assays to reliably measure 25-hydroxyvitamin D2 (25OHD2) (2)(3)(4). The accurate measurement of this metabolite is essential for the monitoring of vitamin-D-deficient patients receiving ergocalciferol, which is the only supplement used in the United States (5) and widely used elsewhere. The international Vitamin D Quality Assessment Scheme (DEQAS) has been monitoring the performance of 25OHD assays since 1989 and now has >100 registered participants in 18 countries. In essence, DEQAS is an ongoing, multicenter trial of the methods used by its participants and provides a unique opportunity to assess the accuracy and specificity of 25OHD methods as well as the analytical performance of a large number of their users. The organization of DEQAS has been described elsewhere (6), and details are available on the DEQAS website (www.deqas.org). In brief, five samples of normal human sera are sent out at 3-month intervals. Participants are asked to measure the total 25OHD concentration in each and return their results within 6 weeks. After statistical analysis of results (7), participants receive a report giving an All-Laboratory Trimmed Mean (ALTM) and Standard Deviation (SD) for each sample. A small study conducted in 1997 (8) showed that the ALTM was a good surrogate for the “true” (target) value produced by gas chromatography–mass spectrometry. The accuracy of each result is defined by its percentage bias from the ALTM. Results for each sample are also grouped by method, and a method mean (MM) is produced. The overall accuracy of each method can be assessed from the percentage bias of the method mean from the ALTM: {[(MM − ALTM)/ALTM] × 100}. The …

Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation*1

The International Quality Assessment Scheme for Vitamin D metabolites (DEQAS) was introduced in 1989. Initially, the aim was to improve the reliability of 25-hydroxyvitamin D (25-OHD) assays but the scheme was extended in 1997 to include 1,25-dihydroxyvitamin D (1,25(OH) 2 D). DEQAS has 95 members in 18 countries (January 2003). Five serum samples are distributed quarterly and participants are given up to 6 weeks to return their results for statistical analysis. The majority of participants use commercial kits for both analytes. A performance target was set by an advisory panel in 1997 and, at present, requires participants to get 80% or more of their results within ±30% of the All-Laboratory Trimmed Mean (ALTM). The performance targets are under continual review. In 2003, 59% of participants met the target (cf. 52% in 2000). A questionnaire, distributed in January 2003, requested information on methods and the interpretation of results. Reference ranges varied but there was reasonable agreement on the 25-OHD concentrations below which Vitamin D supplementation was advised. A minority (22%) of respondents was unsure whether Vitamin D 3 or Vitamin D 2 was used to treat patients in their locality. The majority (52%) of assays for 1,25(OH) 2 D were done ’on demand’ and others for apparently spurious reasons. Most respondents thought participation in DEQAS extremely important and the planned introduction of on-line reporting should enhance its value.

Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation

The International Quality Assessment Scheme for Vitamin D metabolites (DEQAS) was introduced in 1989. Initially, the aim was to improve the reliability of 25-hydroxyvitamin D (25-OHD) assays but the scheme was extended in 1997 to include 1,25-dihydroxyvitamin D (1,25(OH) 2D). DEQAS has 95 members in 18 countries (January 2003). Five serum samples are distributed quarterly and participants are given up to 6 weeks to return their results for statistical analysis. The majority of participants use commercial kits for both analytes. A performance target was set by an advisory panel in 1997 and, at present, requires participants to get 80% or more of their results within ±30% of the All-Laboratory Trimmed Mean (ALTM). The performance targets are under continual review. In 2003, 59% of participants met the target (cf. 52% in 2000). A questionnaire, distributed in January 2003, requested information on methods and the interpretation of results. Reference ranges varied but there was reasonable agreement on the 25-OHD concentrations below which Vitamin D supplementation was advised. A minority (22%) of respondents was unsure whether Vitamin D 3 or Vitamin D 2 was used to treat patients in their locality. The majority (52%) of assays for 1,25(OH) 2D were done ’on demand’ and others for apparently spurious reasons. Most respondents thought participation in DEQAS extremely important and the planned introduction of on-line reporting should enhance its value.

Update on the Diagnosis and Treatment of Disorders of Calcium Regulation

The availability of PTH, iCa, PTHrP, and 25OHD assays for evaluation of calcium abnormalities in companion animals has been well received [table: see text] by clinicians and diagnosticians. Use of these assays has heightened awareness that some of these disorders are more common than originally thought. Also, there is added insight of alterations of calcium homeostasis as a consequence of other illness or environmental factors such as diet. Animal counterparts of other disorders of calcium metabolism in people are likely to be identified, and use of these assays should play a significant role. As already emphasized, the foundation of using [table: see text] these assays is first assessing whether the calcium abnormality is of a parathyroid-dependent or parathyroid-independent classification.

The value of serum 25-hydroxyvitamin D measurements in hypoparathyroid and pseudohypoparathyroid patients treated with calciferol

In 23 patients with hypoparathyroidism or pseudohypoparathyroidism treated with vitamin D, and in whom the dosage was adjusted downward or upward in response to hypercalcemia or hypocalcemia respectively, assays of serum 25-hydroxyvitamin D (25-OHD) were carried out in addition to the usual serum calcium assays. In 120 assays there was a significant correlation between serum 25-OHD levels and serum calcium levels (corrected for serum albumin). There was, however, no clear distinction between the 25-OHD levels of patients who were hypocalcemic, normocalcemic or hypercalcemic. The highest serum 25-OHD level found in a hypocalcemic patient was 1193 nmol/L and the lowest serum 25-OHD level found in a hypercalcemic patient was 605 nmol/L. It was not possible to predict subsequent episodes of hypocalcemia or hypercalcemia from the serum 25-OHD levels. The 25-OHD assay was found to be useful only in checking compliance. We conclude that the assay of serum 25-OHD is of no more value than serum calcium alone in the management of compliant patients.

Vitamin D and femoral neck fractures in elderly South Australian women.

The vitamin D status of women with femoral neck fractures and of male and female nursing-home residents in South Australia was assessed and compared with that of ambulant normal elderly women and young male and female controls. Serum 25-hydroxyvitamin D (25OHD) was assayed with a competitive protein-binding method including a chromatography step. The mean serum 25OHD levels in the patients with femoral neck fractures (39.2 [SE 2.6] nmol/L) and in the nursing-home residents (28.9 [SE 1.3] nmol/L) were significantly lower than those in the ambulant elderly women (67.6 [SE 3.9] nmol/L) and young controls (76.9 [SE 3.7] nmol/L) (P less than 0.001 in each case). The serum 25OHD levels were unrelated to age in the normal ambulant subjects. There was a significant seasonal variation in the results of routine 25OHD assays performed in our laboratory, but no such variation was seen in the nursing-home residents. We conclude that vitamin D deficiency is common in housebound subjects, and that femoral neck fractures occur mainly in subjects who were housebound before the fracture.

14 Phenytoin determination by the substrate-labeled fluorescent immunoassay (SLFIA): R. C. Wong and J. F. Burd, Miles Labs., Inc., Elkhart, IN 46515, U.S.A.

Vitamin 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.We 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”.25-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 55 ng/mL.The assay was evaluated on a general population (N = 476, age: 60 ± 14 years, 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 (≥ 30 ng/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 ± 7 ng/mL, p < 0.01) and hypertension (20 ± 9 vs 17 ± 9 ng/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).Results 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.

16 Biochemical evaluation of the nutritional and metabolic status of geriatric subjects: D. Shapcott, S. Hontela, J. Vobecky, Dép. de Pédiatrie and Dép. de Santé communautaire, Centre hospitalier universitaire de Sherbrooke, Québec, J1H 5N4 and Douglas Hospital Centre, Montréal, Québec, H4H 1R3

Vitamin 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.We 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”.25-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 55 ng/mL.The assay was evaluated on a general population (N = 476, age: 60 ± 14 years, 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 (≥ 30 ng/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 ± 7 ng/mL, p < 0.01) and hypertension (20 ± 9 vs 17 ± 9 ng/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).Results 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.

Competitive protein-binding radioassay of 24,25-dihydroxyvitamin D in sera from normal and anephric subjects

A competitive protein-binding radioassay for 24,25-dihydroxyvitamin D [24,25-(OH) 2D] in human serum has been developed. Whereas small amounts of [ 3H]24,25-(OH) 2D must be biosynthesized in order to trace the efficiency of the extraction and chromatographic procedures, tritiated 25-hydroxyvitamin D 3 ([ 3H]25-OHD 3) can be used as the assay tracer. Since 25-OHD 3 and 24,25-(OH) 2D 3 are equipotent in their competitive displacement of [ 3H]25-OHD 3 from rat serum, 25-OHD 3 can be used as the assay standard. Liquid-gel partition chromatography on small columns of Sephadex LH-20 can reliably isolate 24,25-(OH) 2D by batch elution. The purity of biosynthesized [ 3H]24,25-(OH) 2D 3 and the 24,25-(OH) 2D fraction isolated from serum was confirmed by high-pressure chromatography on 0.2 × 50 cm columns of 10-μm silica. Serum 24,25-(OH) 2D levels averaged 16% of the serum 25-OHD concentrations in normal subjects. Since chronic hemodialysis patients, without kidneys, had normal serum 24,25-(OH) 2D levels, significant extrarenal 25-hydroxycalciferol 24-hydroxylase activity occurs in these subjects. Since the present assay represents a reasonably simple extension of 25-OHD assay methodology, it should prove to be a useful technique in the analysis of clinical disorders of vitamin D metabolism.