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.

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 2 ligand binding assays - impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of ligand binding assays (Part 2) for the determination of serum total 25-hydroxyvitamin D [25(OH)D]. Fifty single-donor samples were assigned target values for concentrations of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] using isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 2 includes results from 17 laboratories using 32 ligand binding assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 50% of the ligand binding assays achieved the VDSP criterion of mean % bias ≤ |± 5%|. For the 13 unique ligand binding assays evaluated in this study, only 4 assays were consistently within ± 5% mean bias and 4 assays were consistently outside ± 5% mean bias regardless of the laboratory performing the assay. Based on multivariable regression analysis using the concentrations of individual vitamin D metabolites in the 50 single-donor samples, most assays underestimate 25(OH)D2 and several assays (Abbott, bioMérieux, DiaSorin, IDS-EIA, and IDS-iSYS) may have cross-reactivity from 24R,25(OH)2D3. The results of this interlaboratory study represent the most comprehensive comparison of 25(OH)D ligand binding assays published to date and is the only study to assess the impact of 24R,25(OH)2D3 content using results from a reference measurement procedure.

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.

Reference values of 25-hydroxyvitamin D revisited: a position statement from the Brazilian Society of Endocrinology and Metabolism (SBEM) and the Brazilian Society of Clinical Pathology/Laboratory Medicine (SBPC).

Hypovitaminosis D is a common condition with a negative impact on health. This statement, prepared by experts from the Brazilian Society of Endocrinology and Metabolism and the Brazilian Society of Clinical Pathology/Laboratory Medicine, includes methodological aspects and limitations of the measurement of 25-hydroxyvitamin D [25(OH)D] for identification of vitamin D status, and identifies individuals at increased risk for deficiency of this vitamin in whom 25(OH)D measurement is recommended. For the general population, 25(OH)D levels between 20 and 60 ng/mL are considered normal, while individuals with levels below 20 ng/mL are considered to be vitamin D deficient. This statement identifies potential benefits of maintaining 25(OH)D levels > 30 ng/mL in specific conditions, including patients aged > 65 years or pregnant, those with recurrent falls, fragility fractures, osteoporosis, secondary hyperparathyroidism, chronic kidney disease, or cancer, and individuals using drugs with the potential to affect the vitamin D metabolism. This statement also calls attention to the risk of vitamin D intoxication, a life-threatening condition that occurs at 25(OH)D levels above 100 ng/mL.

Reference values of 25-hydroxyvitamin D revisited: a position statement from the Brazilian Society of Endocrinology and Metabolism (SBEM) and the Brazilian Society of Clinical Pathology/Laboratory Medicine (SBPC)

ABSTRACT Hypovitaminosis D is a common condition with a negative impact on health. This statement, prepared by experts from the Brazilian Society of Endocrinology and Metabolism and the Brazilian Society of Clinical Pathology/Laboratory Medicine, includes methodological aspects and limitations of the measurement of 25-hydroxyvitamin D [25(OH)D] for identification of vitamin D status, and identifies individuals at increased risk for deficiency of this vitamin in whom 25(OH)D measurement is recommended. For the general population, 25(OH)D levels between 20 and 60 ng/mL are considered normal, while individuals with levels below 20 ng/mL are considered to be vitamin D deficient. This statement identifies potential benefits of maintaining 25(OH)D levels > 30 ng/mL in specific conditions, including patients aged > 65 years or pregnant, those with recurrent falls, fragility fractures, osteoporosis, secondary hyperparathyroidism, chronic kidney disease, or cancer, and individuals using drugs with the potential to affect the vitamin D metabolism. This statement also calls attention to the risk of vitamin D intoxication, a life-threatening condition that occurs at 25(OH)D levels above 100 ng/mL

Validation of an immunoassay for anti-Müllerian hormone measurements and reference intervals in healthy Brazilian subjects.

BACKGROUND: Anti-Müllerian hormone is marker of ovarian and testicular reserve. The clinical use of this hormone requires proper standardization of reference intervals. The aims of this study were to validate the Anti-Müllerian hormone Gen II immunoassay, to establish Anti-Müllerian hormone reference intervals in healthy subjects, and to evaluate the influence of hormonal contraceptives, smoking, and body mass index on Anti-Müllerian hormone. METHODS: The validation of the Anti-Müllerian hormone Gen II assay (Beckman Coulter Company, TX, USA) was performed using a simplified protocol recommended by Clinical Laboratory Standard Institute. One-hundred and thirty-three healthy females and 120 males were prospectively selected for this study. RESULTS: The analytical and functional sensitivities of the Anti-Müllerian hormone Gen II immunoassay were 0.02 and 0.2 ng/mL, respectively. Intra-assay coefficients ranged from 5.2 to 9.0%, whereas inter-assay precision ranged from 4.6 to 7.8% at different concentrations. In females, Anti-Müllerian hormone showed progressive decline with increasing age (r=-0.4, p<0.001), whereas in males, age showed no influence on Anti-Müllerian hormone concentrations. In females, Anti-Müllerian hormone concentrations did not differ between users and non-users of hormonal contraceptives, smokers, and non-smokers and obese and lean individuals. However, there was a negative and significant correlation between Anti-Müllerian hormone and body mass index in males (r=-0.3, p=0.008). CONCLUSIONS: Anti-Müllerian hormone Gen II assay was reliable for determining serum Anti-Müllerian hormone concentrations. Anti-Müllerian hormone concentrations declined with aging and presented a wide inter-individual variability. The lack of influence of hormonal contraceptives, smoking, and obesity on Anti-Müllerian hormone in both sexes allowed us to refine the normative concentrations for the Brazilian population.

Síndrome de Cushing por secreçäo ectópica de corticotrofina (ACTH) / Cushing's syndrome due to ectopic ACTH secretion

Avaliamos oito pacientes (4F e 4M) com quadro clínico de síndrome de Cushing por secreçäo ectópica de ACTH. A idade cronológica variou de 15 a 45 anos e o tempo de duraçäo da doença de 3 a 48 meses. Todos os pacientes apresentavam aspecto cushingóide e hipertensäo arterial e quatro revelavam hiperpigmentaçäo. Cinco pacientes portavam glicemia de jejum elevada e todos, exceto um caso, apresentavam hipopotassemia; K sérico = 2,2 a 3,9 m Eq/l. O ritmo circadiano do cortisol estava ausente em todos os pacientes e os níveis basais de cortisol estavam elevados em todos os pacientes, exceto em um caso; os níveis de ACTH, em sete pacientes avaliados elevados em seis (29 a 1.050pg/ml MRC). Em seis casos avaliados, näo houve depressäo dos níveis de cortisol sérico e de 17-OH urinário após dois dias de 2 mg de dexametasona; após dois dias de 8 mg de dexametasona, um paciente apresentou depressäo normal e um paciente, depressäo parcial dos níveis de 17-OH urinários. O teste da metopirona (750 mg VO 4/4 horas x 6 doses), realizado em realizado em sete pacientes, mostrou resposta normal de composto S e 17-OH urinário no caso que apresentava depressäo normal após 8 mg de dexametasona. Quatro pacientes eram portadores de carcinóides (três de timo e um de brônquio), dois de tumor de ilhotas do pâncreas, um de feocromocitoma bilateral e carcinoma medual e um carcinoma oat cell do pulmäo e carcinoma medular da tiróide. Seis pacientes faleceram num período de três anos após o diagnóstico. RX simples de tórax identificou lesäo primária em quatro casos, confirmada pela tomografia computadorizada (CT). A CT de abdome identificou feocromocitoma bilateral e revelou aumento difuso das adrenais em todos os casos. Sugerimos que RX de tórax e tomografia computadorizado de crânio, tórax e abdome sejam realizados rotineiramente em todos os casos de síndrome de Cushing, já que os achados clínicos e hormonais podem ser semelhantes na secreçäo ectópica de ACTH e doença de Cushing

Male pseudohermaphroditism due to 5 alpha reductase deficiency associated with gynecomastia

Os autores apresentaram três casos de ambigüidade genital por deficiência de 5 alfa-reductase, diagnosticada pelos níveis elevados da relaçäo T/DHT. Os pacientes mais jovens apresentaram ginecomastia leve (Tanner II), ainda näo descrita nesta síndrome. Concluimos que o desenvolvimento de ginecomastia leve pode ocorrer no pseudo-hermafroditismo por deficiência de 5 alfa-reductase

Defeito de síntese adrenal na etiologia do hirsutismo / Defect of adrenal synthesis in the etiology of hirsutism

Estudaram-se 15 pacientes com hirsutismo e nove pacientes normais após estímulo agudo com ACTH e depressäo com dexametasona. Observou-se a presença de defeito de síntese adrenal em sete casos: quatro pacientes com deficiência da 21-hidroxilase, dois com deficiência da 3ß-hidroxiesteróide deidrogenase e uma paciente com deficiência da 11-hidroxilase. Após o teste com dexametasona, todas as pacientes portadoras de defeito de sintese adrenal apresentaram normalizaçäo dos níveis adrogênicos. Indica-se realizaçäo sistemática do teste de estímulo com ACTH e depressäo com dexa, na investigaçäo do hirsutismo. Quando näo for possível a realizaçäo de ambos os testes, é preferível realizar o teste com dexa, que identifica as pacientes que väo se beneficiar desta terapêutica