Simultaneous measurement of 25(OH)-vitamin D and 24,25(OH)2-vitamin D to define cut-offs for CYP24A1 mutation and vitamin D deficiency in a population of 1200 young subjects.

Background Simultaneous measurement of 25(OH)D and 24,25(OH)2D is a new tool for predicting vitamin D deficiency and allows evaluating CYP24A1 lack of function. Interpretation of 24,25(OH)2D should be performed according to 25(OH)D levels and a ratio, called the vitamin D metabolite ratio (VMR) has been proposed for such a purpose. Unfortunately, the VMR can be expressed in different ways and cannot be used if 24,25(OH)2D concentrations are undetectable. Here, we propose evaluating the enzyme activity taking into consideration the probability that a normal population presents undetectable 24,25(OH)2D concentrations according to 25(OH)D levels. We thus retrospectively measured 25(OH)D and 24,25(OH)2D in a population of 1200 young subjects to evaluate the 25(OH)D threshold above which the enzyme was induced. Methods Serum samples from 1200 infants, children, adolescent and young adults were used to simultaneously quantify 25(OH)D and 24,25(OH)2D by LCMS/MS. Results Median (interquartile range [IQR]) levels were 20.6 (14.4-27.2) ng/mL for 25(OH)D. 172 subjects (14.3%) presented 24,25(OH)2D values below the LOQ. When 25(OH)D values were <11 ng/mL, 63.1% of subjects presented undetectable 24,25(OH)2D concentrations. Percentage decreased with increasing 25(OH)D values to become 19.7% for 25(OH)D comprised between 12 and 15 ng/mL, 5.1% for 25(OH)D between 16 and 20 and 0.7% for 25(OH)D >21 ng/mL. Conclusions We suggest using a statistical approach to evaluate CYP24A1 function according to 25(OH)D concentrations. Our results also show that vitamin D deficiency, as defined biochemically, could be around 20 ng/mL in infants, children, adolescent and young adults and that vitamin D deficiency could be evaluated on a more individual basis.

Analytical and clinical validation of the new Abbot Architect 25(OH)D assay: fit for purpose?

BACKGROUND: We provide a clinical and analytical evaluation of the reformulated version of the Abbott Architect 25-hydroxyvitamin D assay. We compared this assay with three commercial automated immunoassays and against a VDSP-traceable liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in six different populations. We also supplemented 40 healthy volunteers with either 600,000 IU of vitamin D2 or 100,000 of vitamin D3 to evaluate the performance of the immunoassays vs. the LC-MS/MS. METHODS: Precision and limit of quantification were assessed, 25(OH)D2 and C3-epimer recovery were calculated. Two hundred and forty samples obtained in healthy Caucasians and Africans, osteoporotic, hemodialyzed and intensive care patients and 3rd trimester pregnant women were analyzed by all methods. Correlation was studied using Passing-Bablok and Bland-Altman analysis. Concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and LC-MS/MS. We verified if patients were homogeneously classified with the immunoassays when they took vitamin D2 or vitamin D3 after 1, 7 and 28 days. RESULTS: We observed excellent analytical features and showed a very good correlation to the LC-MS/MS results in the overall population. Compared to the other immunoassays, concordance of the new Abbott assay with the LC-MS/MS was at least similar, and often better in diseased populations. Althought the cross-reactivity with 25(OH)D2 was not of 100%, there was no significant difference in the classifications of the patients, either supplemented with D2 or D3 or after 7 or 28 days. CONCLUSIONS: This modified version of the Abbott Architect assay is clearly improved compared to the previous one and presents a better agreement with the LC-MS/MS.

Analytical and clinical evaluation of the new Fujirebio Lumipulse®G non-competitive assay for 25(OH)-vitamin D and three immunoassays for 25(OH)D in healthy subjects, osteoporotic patients, third trimester pregnant women, healthy African subjects, hemodialyzed and intensive care patients.

BACKGROUND: In this study, we provide a short analytical evaluation of the new Fujirebio Lumipulse®G non-competitive immunoassay for 25(OH)D. Clinical performance was compared with three commercial competitive automated immunoassays against a Vitamin D Standardization Program (VDSP)-traceable liquid chromatography-tandem mass spectrometry (LC-MS/MS) in six different clinically relevant populations. METHODS: Lumipulse®G 25(OH)D precision, measurement uncertainty, recovery, limit of quantification were assessed, as well as 25(OH)D2 and C3-epimer recovery. For method comparison, 250 serum samples obtained in healthy Caucasians and Africans, osteoporotic, hemodialyzed and intensive care patients and 3rd trimester pregnant women were analyzed by all methods. Correlation was studied using Passing-Bablok and Bland-Altman analysis. Concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and the LC-MS/MS. RESULTS: The Lumipulse®G 25(OH)D assay presented interesting analytical features and showed excellent correlation to the LC-MS/MS results (y=1.00×-1.35 ng/mL), as obtained in healthy Caucasian individuals. In the other special populations, Lumipulse®G presented a concordance with LC-MS/MS which was generally higher than competitors, even if all methods significantly under-recovered 25(OH)D in hemodialyzed patients. Intra-assay CV ranged from 12.1% at 9.6 ng/mL to 2.1% at 103.7 ng/mL and inter-assay CV ranged from 16.2 to 3.7% at the same concentrations, respectively. Measurement uncertainty, with a probability of 95%, were respectively 33.1 and 7.6% at these concentrations. LOQ was found to be at 4.6 ng/mL. Mean (95% CI) 25(OH)D2 revovery was 77% (74-81) and no cross-reactivity was observed with C3-epimer. CONCLUSIONS: Fujirebio Lumipulse®G 25-OH Vitamin D Total assay is therefore considered suitable for assessment of vitamin D status in clinical routine.

IDS iSYS automated intact procollagen-1-N-terminus pro-peptide assay: method evaluation and reference intervals in adults and children.

BACKGROUND: We carried out a technical evaluation of the Immunodiagnostic Systems (IDS) automated intact procollagen-I N-terminus propeptide (PINP) assay on the iSYS platform, and established reference intervals for PINP in both adults and children. METHODS: Assay imprecision, recovery and interference were studied. Serum and plasma values were compared, and PINP stability was assessed. Using 828 specimens, IDS iSYS intact PINP and Roche E170 total PINP values were compared. Specimens from 597 adults and 485 children and adolescents were used to establish reference intervals for intact PINP. RESULTS: The method demonstrated good recovery and acceptable imprecision. The assay was unaffected by icterus and lipaemia, but haemolysis decreased measured PINP. Serum and plasma values were comparable. There was a non-linear relation between IDS intact and Roche total PINP values. Pre- and post-menopausal women had comparable PINP values, but there was a difference between women of different age groups. Serum PINP in men showed a decline in young age up to 45 years, but remained steady thereafter. Separate reference intervals were established for four age groups in women and for two age groups in men. Data for children were partitioned into four-year age groups, and these showed PINP to be high with no major gender differences until 12 years of age. Thereafter, values in females decreased in 13-16 years age groups and further in 17-20 years age groups, whereas PINP increased in boys of 13-16 years of age with a subsequent decline at 17-20 years. CONCLUSIONS: The IDS iSYS PINP intact assay appears to be reliable. We have established gender- and age-related reference intervals for children and adults based on a relatively large healthy North European population.

Interpretation of serum PTH concentrations with different kits in dialysis patients according to the KDIGO guidelines: importance of the reference (normal) values.

BACKGROUND: The recommended target range for serum parathyroid hormone (PTH) in dialysis patients has changed from 150 to 300 pg/mL in the KDOQI guidelines to two to nine times the upper normal limit in the KDIGO ones. Although inclusion/exclusion criteria for the reference population are highly important, they are usually not mentioned in the commercial kits. In this study, we used the same reference population of vitamin D-replete normal subjects to establish reference values for 10 commercial PTH kits. We evaluated whether this may improve the classification of dialysis patients according to the KDIGO compared to the use of reference values proposed by the manufacturers. METHODS: We measured serum PTH with 10 different kits in 149 haemodialysis patients, and 240 25-OH-vitamin D-replete (>75 nmol/L) individuals with an estimated glomerular filtration rate >60 mL/min/1.73 m(2). RESULTS: For the 10 kits, our upper normal limit was lower than those of the manufacturers. The difference was, however, variable from one kit to another. The two kits that yielded the lowest and the highest absolute concentrations classified differently 84/149 patients (56.4%) according to the KDOQI and 53/149 (36.2%) according to the KDIGO using the manufacturers' normal values. Using our normal values significantly decreased the discrepancies with 24/149 patients (16.1%) being still classified differently. Taking the measurement uncertainty into consideration, 8% of the patients only remained differently classified by these two kits. CONCLUSIONS: Using the same vitamin-D-replete population to establish the reference range for 10 commercial PTH kits significantly improved the classification of haemodialysis patients according to the KDIGO target range.

Parathormone, bone alkaline phosphatase and 25-hydroxyvitamin D status in a large cohort of 1200 children and teenagers.

Objectives: 25-Hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH) and bone alkaline phosphatase (BALP) are biomarkers of calcium/phosphate metabolism and bone turnover. Although vitamin D deficiency is a well-known cause of secondary hyperparathyroidism, few studies have considered vitamin D status when establishing reference ranges. In this study, we report PTH levels according to the vitamin D status and BALP levels in a large cohort of 1200 children. Additionally, we provide PTH pediatric reference values according to 25(OH)D status as well as BALP pediatric reference ranges.Methods: Serum samples from 1200 children (equally distributed from 5 months to 20 years old) who underwent blood sampling for allergy exploration were used to quantify 25(OH)D, PTH and BALP.Results: The percentage of vitamin D deficient children (<20 ng/ml) progressively increased during childhood starting from 7% in the 0 to 2 year-old subgroup to a mean of at least 50% among teenagers. PTH levels inversely mirrored 25(OH)D concentrations for all age and gender subgroups, and 25(OH)D deficient subgroups presented higher PTH levels than their non-deficient counterparts. In the non-deficient 25(OH)D population, PTH levels were the highest at 11 years old for girls and 14 years old for boys. BALP results were slightly increased during childhood and showed a constant decrease during teenage years starting from 12 years old for girls and 14 years old for boys.Conclusion: Our results highlight the inverse relationship between PTH and 25(OH)D in children and the need for a well characterized 25(OH)D population to establish pediatric reference ranges for PTH.

Analytical validation of the Liaison Calcitonin_II-Gen (DiaSorin).

BACKGROUND: We validated the DiaSorin Liaison Calcitonin_II-Gen, an improved method for calcitonin (CT) measurements, compared this method with the Cisbio_h-CT kit and established the reference range of CT in a normal adult population. METHODS: We determined the precision, functional sensitivity, traceability to the 2nd IS 89/620, linearity and measurement uncertainty, accuracy profile and ß-expectation limits. We evaluated the specificity, the susceptibility to human anti-animal antibodies (HAMA), hook-effect and carry over. To establish a reference range, we selected 267 adults without renal insufficiency presenting with normal thyroid stimulating hormone (TSH), free thyroxin (T4) and calcium concentrations and without anti-thyroglobulin antibodies as our "reference" healthy population. We compared the method with Cisbio on 250 consecutive and 45 samples from a post-pentagastrin stimulation test. RESULTS: Precision (expressed as CV) was < 10% for the measurement range, functional sensitivity: 5.3 ng/L and the method was found linear until to a 1/10 dilution. Uncertainty ranged from 25% to 7.2%, and the risk that one result falls out of the ± 20% acceptance limits was < 5% between 2.9 and 1513 ng/L. The Bland and Altman plot showed no systematic bias between the two methods. The test is still prone to HAMA influence, does not present any hook-effect, although carry over was observed. Ninety-five percent of our adult reference population showed CT concentrations < 7.4 ng/L, with an important gender difference: 95% of the men showed CT values < 9.8 ng/L, whereas 95% of women were < 4.0 ng/L. CONCLUSIONS: The Liaison Calcitonin_II-Gen is an analytically robust method. The important difference in gender observed in our population might lead to re-evaluation of the generally used "10 ng/L" cut-off in a multicentre prospective study.

Analytical validation of serum bone alkaline phosphatase (BAP OSTASE) on Liaison.

BACKGROUND: The goal of this study was to validate the DiaSorin Liaison BAP OSTASE, a new method for measurement of bone alkaline phosphatase (BAP), and to compare this method with the Beckman-Coulter Access Ostase. We also wanted to establish the reference range for BAP in adults and children. METHODS: We determined the precision, functional sensitivity, recovery, linearity and measurement uncertainty, accuracy profile and beta-expectation limits. We defined an adult reference interval using individuals with 25-OH vitamin D >80 nmol/L, parathormone <58 ng/L, and normal calcium, phosphorous and estimated glomerular filtration rate. Each adult subclass (men/non-menopausal women/menopause women) contained 120 individuals. We also determined the 2.5th and 97.5th percentiles from a population of 450 children, stratified according to age and gender. RESULTS: The results of the validation showed: precision <6%, functional sensitivity <0.74 microg/L, mean recovery 98.8+/-4.2% and good linearity. Relative uncertainty ranged from 9.0% to 12.9%, and the risk of one result falling out of the +/-15% acceptance limits was <5% for concentrations between 7 and 94 microg/L. The Bland-Altman plot showed no systematic bias between the two methods. In adults, we did not find any statistical difference between the different subclasses. The upper limit of normality observed in the entire population (n=360) was 21.3 microg/L (90% CI: 18.3-24.2 microg/L). CONCLUSIONS: The Liaison BAP OSTASE is a robust method, and is completely validated between 7 and 93 microg/L: in this range, 95% of the values obtained will be within +/-15% of the true value.

[Epidemiology of urolithiasis in Belgium on the basis of a morpho-constitutional classification]. / Épidémiologie de la lithiase urinaire en Belgique sur base d'une classification morpho-constitutionnelle.

Urolithiasis is a common condition, with a prevalence of ∼10% and a male/female ratio above 1 according to large national series. Various types of urinary stones have been described upon their mineral content and/or their morphology. Hence, a combined morpho-constitutional (M-C) classification has been proposed. In order to detail the prevalence of urolithiasis in general and of each M-C type in particular upon age and gender in Belgium, we retrospectively studied the database of a reference center for urolithiasis analysis. Between 2010 and 2013, 2195 stones were characterized. We excluded 45 non-biological stones and 281 stones, which originated from outside the study zone. Among 1869 stones, 1293 (69.2%) affected men. Prevalence peak of urolithiasis was observed between 50-60 years of age in both genders. The M-C analysis was available for 1854 stones (99.2%): multiple morphological types were concomitantly identified in 49.3%. In the whole population, the main mineral constituent was whewellite (54.4%), mainly organized as type Ia (94%). Weddellite was found in 19.8%, with an equal distribution between types IIa and IIb. Uric acid was the 3rd most frequent constituent in man, with a similar distribution between IIIa and IIIb. Phosphate was uncommon in man (8.2%), but frequent in woman (26.6%) with a type IVa1 organization. Prevalence of M-C types changes with aging, i.e. decrease of weddellite and increase of whewellite and uric acid in both genders. This retrospective analysis of a single-center database of urinary stones helps characterize the M-C epidemiology of urolithiasis in Belgium.

Analytical evaluation of the new Abbott Architect 25-OH vitamin D assay.

OBJECTIVES: Validation of the Architect 25-OH vitamin D assay. DESIGN AND METHODS: Determination of repeatability, reproducibility, accuracy profile and 25(OH)-vitamin D2 recovery on native samples. Comparison with DiaSorin Liaison and RIA. RESULTS AND CONCLUSION: Coefficients of variation: <6% (13.6 ng/mL) and 2.2% (78.1 ng/mL). Functional sensitivity: 5 ng/mL. Accuracy profile shows that the method is validated between 13.6 and 78.1 ng/mL. Recovery of 25(OH)D2: 75,8%( 95% CI: 61.9-89.7%). Good correlation with DiaSorin RIA and Liaison <50 ng/mL; above this threshold a systematic positive bias was observed.