Salivary testosterone changes during oral glucose tolerance tests in overweight and obese men - postprandial or circadian variation?

BACKGROUND: Serum total testosterone (T) decreases postprandially. Postprandial salivary testosterone (SalT) responses, however, have not been studied. We report on the effect of glucose ingestion on fasting SalT concentrations. OBJECTIVE: To investigate the effect of oral glucose ingestion on fasting SalT. METHODS: Salivary and blood samples were collected between 09.00 and 09.30 and two hours after a 75g oral glucose load in 32 men with mean (standard deviation) age of 52 (5.7) years and body mass index of 32.6 (5.56) kg/m2. Free T and bioavailable testosterone (BAT) were calculated using the Vermeulen equation. RESULTS: Two hours following oral glucose, there was a decrease in fasting mean (standard deviation) SalT [178.2 (56.6) vs 146.0 (42.2) pmol/L; p = 0.0003], serum cortisol [332 (105.0) vs 239 (75.3) nmol/L; p = <0.0001], prolactin [193 (75.0) vs 127 (55.9) mIU/L; p = <0.0001] and TSH [1.60 (0.801) vs 1.16 (0.584) mIU/L; p = <0.0001]. Plasma glucose increased [6.2 (0.72) vs 8.1 (3.71) mmol/L; p = 0.0029]. Serum total T, SHBG, albumin, Free T, BAT, gonadotrophins and FT4 remained unchanged. CONCLUSIONS: SalT decreased postprandially. A concomitant decrease in serum cortisol, prolactin and TSH reflecting diurnal variation offers an alternative explanation for the decrease in SalT independent of food consumption. Further studies are required to determine whether morning temporal changes in SalT are related to food consumption or circadian rhythm or both.

Age-specific Reference Intervals of Abbott Intact PTH-Potential Impacts on Clinical Care.

Background: PTH assays are not standardized; therefore, method-specific PTH reference intervals are required for interpretation of results. PTH increases with age in adults but age-related reference intervals for the Abbott intact PTH (iPTH) assay are not available. Methods: Deidentified serum PTH results from September 2015 to November 2022 were retrieved from the laboratory information system of a laboratory serving a cosmopolitan population in central-west England for individuals aged 18 years and older if the estimated glomerular filtration rate was ≥60 mL/min, serum 25-hydroxyvitamin D was >50 nmol/L, and serum albumin-adjusted calcium and serum phosphate were within reference intervals. Age-specific reference intervals for Abbott iPTH were derived by an indirect method using the refineR algorithm. Results: PTH increased with age and correlated with age when controlled for 25-hydroxyvitamin D, estimated glomerular filtration rate, and adjusted calcium (r = 0.093, P < .001). The iPTH age-specific reference intervals for 4 age partitions of 18 to 45 years, 46 to 60 years, 61 to 80 years, and 81 to 95 years were 1.6 to 8.6 pmol/L, 1.8 to 9.5 pmol/L, 2.0 to 11.3 pmol/L, and 2.3 to 12.3 pmol/L, respectively. PTH was higher in women compared with men (P < .001). Sex-specific age-related reference intervals could not be derived because of the limited sample size. Conclusion: Age-specific Abbott iPTH reference intervals were derived. Application of age-specific reference intervals will impact the diagnosis and management of normocalcemic hyperparathyroidism, based on current definitions, and secondary hyperparathyroidism. Additional studies are required to clarify the effect of sex and ethnicity on PTH.

Impact of Variation between Assays and Reference Intervals in the Diagnosis of Endocrine Disorders.

Method-related variations in the measurement of hormones and the reference intervals used in the clinical laboratory can have a significant, but often under-appreciated, impact on the diagnosis and management of endocrine disorders. This variation in laboratory practice has the potential to lead to an errant approach to patient care and thus could cause harm. It may also be the source of confusion or result in excessive or inadequate investigation. It is important that laboratory professionals and clinicians know about these impacts, their sources, and how to detect and mitigate them when they do arise. In this review article, we describe the historical and scientific context from which inconsistency in the clinical laboratory arises. Examples from the published literature of the impact of the method, reference interval, and clinical decision threshold-related discordances on the assessment and monitoring of various endocrine disorders are discussed to illustrate the sources, causes, and effects of this variability. Its potential impact on the evaluation of growth hormone deficiency and excess, thyroid and parathyroid disorders, hyperandrogenism, hypogonadism, glucocorticoid excess and deficiency, and diabetes mellitus is elaborated. Strategies for assessment and mitigation of the discordance are discussed. The clinical laboratory has a responsibility to recognise and address these issues, and although a lot has been accomplished in this area already, there remains more to be done.

Impact of hyperthyroidism and its treatment on the outcome of mental health, occupational functioning, and quality of life: A naturalistic, prospective study.

Context Background: Hyperthyroidism has a major impact on mental health. Aims: We intended to determine the magnitude of the unmet need for mental health support in patients with hyperthyroidism attending an endocrinology clinic. Settings and Design: General Hospital Endocrine Department, Prospective Study. Methods and Material: In a naturalistic, prospective study, consecutive (n = 176) hyperthyroid patients were assessed for anxiety (generalized anxiety disorder, GAD-7), depression (Patient Health Questionnaire PHQ-9), functional impairment (work and social adjustment scale, WSAS), and quality of life (EuroQol ED5D) using standardized instruments. Statistical Analysis Used: Percentages, mean and standard deviation (SD), Chi-square, Student's t-test, ANOVA, Spearman's correlation. Results: A considerable proportion (40.5%) of patients had moderate and severe anxiety, around half (50.6%) had moderate or severe depression, and 20.8% had severe functional impairment at presentation. Mean EQ5D score was 59.6 ± 23.5. The scores of anxiety, depression, and functional impairment were significantly correlated with each other and negatively correlated with QOL. Following treatment of hyperthyroidism, improvement in psychiatric symptoms was noted which could be correlated with the lowering of T4. However, a significant proportion of patients continued to have psychiatric symptoms and functional impairment despite achieving euthyroidism. There was no correlation between the severity of hyperthyroidism to persistence of mental health parameters. Conclusions: Our findings of high prevalence and persistence of impairment of mental health and functional status of patients with hyperthyroidism have highlighted the unmet needs for these patients.

Thyroid autoantibodies.

Thyroid-stimulating hormone (TSH) receptor antibody (TSH-R-Ab or TRAb) testing plays a pivotal role in arriving at the aetiological diagnosis in patients with thyrotoxicosis. A positive test establishes the diagnosis of Graves' disease (GD) while a negative result in conjunction with imaging studies supports other possible aetiologies. In patients with GD, TRAb levels at diagnosis and at the time of withdrawal of antithyroid drugs can identify patients who are unlikely to achieve remission and guide clinical management decisions. We provide an algorithm that incorporates TRAb in the decision-making process for the management of thyrotoxicosis. The utility of TRAb in predicting the risk of fetal and neonatal thyroid dysfunction is established and widely accepted in guidelines. TRAb may also help in the diagnosis of Graves' orbitopathy, especially in euthyroid or hypothyroid patients and its role in guiding its management is evolving as a useful adjunct to the clinical parameters used in making therapeutic decisions.Anti-thyroid peroxidase antibodies (TPOAb) and anti-thyroglobulin antibodies (TgAb) indicate thyroid autoimmunity. The most common use of TPOAb is to identify patients at a higher risk of progression to treatment-requiring hypothyroidism. They also aid the diagnosis of immune thyroiditis and Hashimoto's encephalopathy. Thyroglobulin measurement is used to help guide differentiated thyroid cancer treatment. TgAb is used as an accompanying test with thyroglobulin measurement as its presence can interfere with the thyroglobulin assay. A negative TgAb result reduces the likelihood of, but does not exclude, interference with thyroglobulin assay.

Managing ethylenediaminetetraacetic acid (EDTA) interference in EDTA contaminated samples - selectivity in reporting analytes.

BACKGROUND: The widely accepted practice of not reporting any results from ethylenediaminetetraacetic acid (EDTA) contaminated samples necessitates repeat phlebotomy and could delay clinical management decisions. EDTA, however, interferes variably or not at all in assays. EDTA concentration in contaminated samples, like serum indices, therefore, could be used to selectively report the result of analytes not affected at measured EDTA concentration. METHODS: A serum pool, level 1 and level 3 internal quality control materials were spiked with tripotassium-EDTA to create samples with EDTA concentration up to 6.0 mmol/L. EDTA and 45 common and critically important analytes were measured on Abbott Architect to identify EDTA concentrations for analytes where the change in concentration exceeded their respective reference change value (RCV) for unidirectional change at 95% probability. RESULTS: Serum potassium increased and calcium decreased exceeding RCV at 0.17 mmol/L EDTA. Alkaline phosphatase (ALP) decreased exceeding RCV at EDTA >1.86 mmol/L. The decrease in iron did not exceed a wide RCV of 61.9% until maximum spiked EDTA but exceeded the desirable specification for allowable total error (30.7%) at EDTA >1.86 mmol/L. The small decrease in magnesium did not exceed RCV. EDTA up to the concentration in blood collection tubes did not affect the results of any other measured analyte. CONCLUSIONS: Only serum potassium, calcium, ALP and iron studies, of the 45 analytes studied, should not be reported in EDTA contaminated samples. EDTA concentration cut-offs for selective reporting would further facilitate reporting of these analytes in EDTA contaminated samples.

Effect of automated feedback laboratory comments promoting good phlebotomy practice on the frequency of ethylenediaminetetraacetic acid (EDTA) contamination of serum samples.

BACKGROUND: Ethylenediaminetetraacetic acid (EDTA) contamination of serum samples is common but under-recognized unless EDTA is measured. Incorrect order of draw with closed phlebotomy (vacutainer) does not cause EDTA contamination. EDTA contamination occurs largely or solely during open phlebotomy due to syringe tip or needle-tip contamination when delivering blood into EDTA sample tubes before other sample tubes or direct transfer of blood from EDTA containing tubes to other tubes. Therefore, preference for closed phlebotomy or following the order of tube fill when open phlebotomy is used may reduce EDTA contamination. METHODS: The laboratory's comments for EDTA-contaminated serum samples were amended to encourage closed phlebotomy and with open phlebotomy filling of serum tubes before EDTA and fluoride-EDTA tubes. The weekly frequency of EDTA sample contamination, normalized for weekly urea and electrolyte (U&E) requests, was studied 52 weeks before and 43 weeks after amending the comments. RESULTS: Median (IQR) frequency of EDTA-contaminated samples per week per 10,000 U&Es decreased by 58% [5.6 (3.1-9.2) versus 2.3 (1.1-4.4); P < 0.001] after the introduction of the new comment. CONCLUSION: Explicit automated laboratory feedback comments promoting closed phlebotomy and order of tube fill with open phlebotomy were associated with a 58% reduction in EDTA-contaminated samples and thus may play a role in improving phlebotomy practise.

The Diagnosis of Normocalcaemic Hyperparathyroidism is Strikingly Dissimilar Using Different Commercial Laboratory Assays.

We assessed the impact of intact parathyroid hormone (iPTH) and adjusted calcium analyses on Abbott, Roche and Siemens analytical platforms in the diagnosis of normocalcaemic primary hyperparathyroidism (NCPHPT). These assays are used by over 85% of clinical laboratories in the UK. Over five months, consecutive serum samples from outpatients with NCPHPT in the laboratory with Abbott assays were identified, aliquoted and stored at -80°C. Frozen aliquots were transported monthly to the other two laboratories. After thawing, samples were mixed and analysed immediately for calcium, albumin and iPTH in the laboratories with Abbott, Roche and Siemens analytical platforms. Adjusted calcium was calculated using the equation used in the respective laboratory. Diagnostic concordance of iPTH and adjusted calcium were assessed using manufacturer-provided assay-specific reference intervals and the pathology harmony reference interval respectively. Fifty-five patients with NCPHPT were identified using Abbott assays. Of these, 16 (29.1%) and 11 (20.0%) had NCPHPT, 9 (16.4%) and 13 (23.6%) had hypercalcaemic primary hyperparathyroidism, and 30 (54.6%) and 31 (56.4%) patients had normal results when analysed in laboratories with Roche and Siemens assays, respectively. The diagnosis of NCPHPT was strikingly different depending on the commercial assay used. There is a pressing need for iPTH assay harmonisation and robust reference intervals. Reference intervals may become invalid if an assay drifts, as exemplified by adjusted calcium in this study.

Clinical concordance assessment should be an integral component of laboratory method comparison studies: A regression transference of routine clinical data approach.

Manufacturer-provided assay-specific reference intervals may not compensate for between assay differences leading to method related clinical discordance. Not all laboratories, however, assess clinical concordance as part of method comparison studies. We assessed the utility of method comparison data combined with routine clinical data to assess method related clinical concordance in the diagnosis and management of subclinical hypothyroidism (SCH). Passing-Bablok method comparison regression analysis was performed for both thyroid stimulating hormone (TSH) and free thyroxine (fT4) from 100 samples analysed by Abbott and Roche methods. Primary care samples indicative of SCH were identified from the laboratory information system (LIMS) of two laboratories (one with Roche methods and one with Abbott) over four months. For Roche and Abbott TSH and fT4 results, the Passing-Bablok regression equations were used to predict Abbott and Roche results respectively. The predicted results were interpreted using manufacturer-provided assay-specific reference intervals and compared to those of the previous SCH sample exchange study. On laboratory method comparison, Roche TSH and fT4 results were 30 ± 13% and 16 ± 7% higher compared to Abbott assays respectively. Of those with results indicative of SCH by Roche assays, 76.8% would have had normal thyroid function using predicted Abbott assay results and 46.9% of those with results indicative of SCH by Abbott assays would have had a biochemical indication for levothyroxine replacement using predicted Roche assay results. The results from the regression-based approach were comparable to the previous SCH sample exchange study. A regression-based approach using routine method comparison data and real-world clinical data identifies potential clinical discordance. We suggest that clinical concordance assessment be an integral component of laboratory method comparison studies.

Acid freezes uricolysis in addition to glycolysis: Utility of fluoride-EDTA-citrate tube in the measurement of uric acid for patients administered rasburicase.

BACKGROUND: In samples from patients administered rasburicase, ex vivo uricolysis leads to spuriously low uric acid results. The manufacturer's recommendation of storing the sample in ice-water until analysis, however, does not fully arrest uricolysis. Since uricase activity is affected by pH and metal chelators, we assessed uricolysis inhibition in sodium fluoride-ethylenediaminetetraacetic acid (EDTA)-citrate sample tube (FC Mix tube, Greiner) used primarily for plasma glucose. METHOD: A serum pool was spiked with rasburicase and uric acid measured at 15, 45, 90, 150, 240 and 1080 min in a lithium heparin tube in ice-water, plain tube at room temperature (RT), EDTA tube at RT, FC Mix tube in ice-water, FC Mix tube at RT and FC Mix tube at RT prepared by dissolving FC Mix in serum. RESULTS: The rate of urate decay was lowest in the FC Mix tube independent of temperature, then lithium heparin tube in ice-water, then EDTA tube at RT and highest in the plain tube at RT. Uric acid concentrations in the prepared FC Mix tube at RT and heparin tube in ice-water were, respectively, 98.2% and 93.8% of control values at 90 min, 97.1% and 89.3% of control values at 4 h, and remained higher in the prepared FC Mix tube at all time points. CONCLUSION: NaF-EDTA-citrate mixture largely arrested rasburicase mediated ex vivo uricolysis without the need for sample cooling. We propose that sample tubes containing NaF-EDTA-citrate be used for the measurement of uric acid in patients administered rasburicase.

Biochemical assessment of adequate levothyroxine replacement in primary hypothyroidism differs with different TSH assays: potential clinical implications.

AIM: Thyroid stimulating hormone (TSH) assays provided by Abbott Laboratories and Roche Diagnostics are used by approximately 75% of laboratories in the UK. We assessed the potential impact of Abbott and Roche TSH assay differences on the biochemical assessment of levothyroxine replacement in primary hypothyroidism. METHOD: Samples from 100 consecutive primary care patients (83 women, median age 64 years, IQR 51-73 years) with primary hypothyroidism on adequate levothyroxine based on an Abbott Architect TSH in the reference range were analysed for TSH on Roche cobas within 24 hours. The Abbott and Roche TSH results were compared. Over 1 year, TSH results from patients in primary care from the laboratories with Abbott and Roche methods were compared. RESULTS: The median (IQR) Roche TSH (2.5 (1.3-3.6) mIU/L) was 30%±10% higher (p<0.001) than Abbott TSH (1.9 (1.1-2.6) mIU/L). Although all Abbott TSH results were in the Abbott specific reference range, 14 patients (14%) had Roche TSH results above the Roche specific reference range. In the 1 year gather, Roche TSH (1.9 (1.3-2.9) mIU/L, n=103 932) results were higher (p<0.001) than Abbott TSH (1.5 (1.0-2.2) mIU/L, n=1 10 544) results. The TSH results were above their assay-specific upper reference limit in 10.7% of Roche results and 4.2% of Abbott results. CONCLUSION: Biochemical assessment of levothyroxine replacement may be dependent on the type of TSH assay. Laboratorians and clinicians should be aware that the lack of harmonisation between TSH methods and their assay-specific reference ranges may potentially lead to different patient management decisions. We suggest lot verification in laboratories should include processes to identify cumulative drift in assay performance.

Conflicting effects of haemolysis on plasma sodium and chloride are due to different haemolysis study protocols: A case for standardisation.

BACKGROUND: Haemolysis has been reported as having a positive, negative or no effect on plasma sodium (PNa) and chloride (PCl). We investigated the haemoltytic effect of different haemolysis protocols on PNa and PCl using modelling and laboratory experiments. METHODS: In a modelling experiment, percentage change and recovery due to dilution in routinely (in vitro) haemolysed samples were compared against shear stress haemolysis and samples spiked with haemolysate from whole blood freeze-thaw, packed cells freeze-thaw and osmotic shock protocols. The results were compared against a control base pool. Additionally, for the osmotic shock method, results were compared against saline- and deionised water (DIW)-spiked controls. In a laboratory experiment, percentage change and recovery were similarly compared using haemolysate from whole blood freeze-thaw and osmotic shock protocols. PNa, PCl and H-index were measured on the Abbott Architect and haemoglobin on the Sysmex XN-9000. RESULTS: In the modelling experiment, the percentage decrease in PNa and PCl was similar in in vitro haemolysis, shear stress haemolysis, whole blood freeze-thaw haemolysis and packed cells freeze-thaw haemolysis and this was lower compared to the osmotic shock method. In the laboratory experiment, the change in PNa compared to the base pool was less (p < 0.001) per unit increase in H-index in the freeze-thaw method (-0.33 mmol, 95% CI -0.35 to -0.31) compared to the osmotic shock method (-0.65 mmol, 95% CI -0.66 to -0.64). PCl did not change with haemolysis in the freeze-thaw method and changed by -0.21 ± 0.01 mmol per unit increase in the H-index in the osmotic shock method. Recovery of PNa and PCl increased with increasing H-index in both methods. CONCLUSION: The osmotic shock protocol is inappropriate for haemolysis studies because of dilution with DIW used for cell lysis. Recovery calculations may incorrectly compensate for genuine dilution caused by haemolysis.

Samples spiked with pituitary-derived thyroid-stimulating hormone may disguise the extent of differences between thyroid-stimulating hormone assays.

BACKGROUND: A large discordance in the diagnosis and potential management of hypothyroidism using Abbott and Roche thyroid assays has been reported recently. The difference in Abbott and Roche thyroid-stimulating hormone (TSH) results in these studies was larger than anticipated from the external quality assessment (EQA) reports. METHODS: Abbott and Roche TSH method means in UK NEQAS for thyroid hormones distributions 430 to 454 were compared against the amount of TSH spiked. A TSH deplete serum pool was spiked with various concentrations of pooled high TSH serum and 3rd WHO International Standard for TSH (WHO-IS). Four serum pools with TSH close to clinical decision limits were spiked with two concentrations of WHO-IS. RESULTS: On review of EQA data, median (IQR) Roche: Abbott TSH ratio was lower (p < 0.001) in 48 pools spiked with TSH (1.11 (1.07-1.16)) compared to 41 pools not spiked (1.29 (1.25-1.31)) and the decrease was proportionate to the contribution of spiked TSH to total TSH in the samples (ρ=-0.908, p < 0.001). In spiking experiments, the relationship of Roche and Abbott TSH was different in TSH deplete pool spiked with WHO-IS (RocheTSH=1.13*AbbottTSH-0.52) and high TSH serum (RocheTSH=1.43*AbbottTSH-0.50), respectively. The Roche: Abbott TSH ratio decreased and the method agreement improved on spiking serum pools with WHO-IS. CONCLUSION: Abbott and Roche TSH assays are not in harmony in human serum samples but the agreement was better in samples spiked with WHO-IS which contains pituitary-derived TSH. Use of pituitary-derived TSH spiked samples, such as provided by EQA schemes, may mask clinically significant between-assay differences.