Harmonisation of serum dihydrotestosterone analysis: establishment of an external quality assurance program.

BACKGROUND: Serum dihydrotestosterone (DHT) is an important analyte for the clinical assessment of disorders of sex development. It is also reportedly a difficult analyte to measure. Currently, there are significant gaps in the standardisation of this analyte, including no external quality assurance (EQA) program available worldwide to allow for peer review performance of DHT. We therefore proposed to establish a pilot EQA program for serum DHT. METHODS: DHT was assessed in the 2015 Royal College of Pathologists of Australasia Quality Assurance Programs' Endocrine program material. The material's target (i.e. "true") values were established using a measurement procedure based on isotope dilution gas chromatography (GC) tandem mass spectrometry (MS/MS). DHT calibrator values were based on weighed values of pure DHT material (>97.5% purity) from Sigma. The allowable limits of performance (ALP) were established as ±0.1 up to 0.5 nmol/L and ±15% for targets >0.5 nmol/L. RESULTS: Target values for the six levels of RCPAQAP material for DHT ranged from 0.02 to 0.43 nmol/L (0.01-0.12 ng/mL). The material demonstrated linearity across the six levels. There were seven participating laboratories for this pilot study. Results of the liquid chromatography (LC) MS/MS methods were within the ALP; whereas the results from the immunoassay methods were consistently higher than the target values and outside the ALP. CONCLUSIONS: This report provides the first peer comparison of serum DHT measured by mass spectrometry (MS) and immunoassay laboratories. Establishment of this program provides one of the pillars to achieve method harmonisation. This supports accurate clinical decisions where DHT measurement is required.

Sex steroid hormone stability in serum tubes with and without separator gels.

BACKGROUND: A pilot study showing a decrease in androstenedione concentration in serum collected into gel-containing serum tubes (STs) triggered an investigation of the effect of serum collection tube on steroid hormone stability. METHODS: In the main study, two tube types were examined: BD Vacutainer® SST™II Advance and BD Vacutainer® Serum Tube. Forty-seven serum samples from apparently healthy volunteers were collected and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP) (n=20); and oestradiol (n=27). Primary specimens were centrifuged once, maintained at room temperature and extracted within 2 h for day zero (d0) results. To assess stability following refrigeration (2-8 °C), aliquots were taken from the primary tube on day one (d1) and day five (d5) and analysed immediately. Differences in measurand concentration between tubes at d0 and following storage (d1 and d5) were evaluated for statistical significance. RESULTS: There was a progressive and statistically significant decrease in androstenedione concentration from d0 to d5 (p<0.001) in the SST™II tubes. In addition, there was a statistically significant reduction in testosterone, 17-OHP and oestradiol concentrations at d5 (p<0.01). Interestingly, oestradiol and testosterone concentrations increased with time in plain STs (p<0.01). The only change likely to have a clinical impact was that of androstenedione in serum gel tubes. CONCLUSIONS: To optimise conditions and to reduce pre-analytical error we recommend the use of plain serum collection tubes for androstenedione and rapid separation of serum from cells when oestradiol and testosterone are requested.

Serological Responses to Streptococcus pyogenes Vaccine Candidate Antigens Suggests That Streptococcus dysgalactiae Is the Predominant Cause of Lower Limb Cellulitis.

Background: A future Streptococcus pyogenes (Strep A) vaccine will ideally prevent a significant burden of lower limb cellulitis; however, natural immune responses to proposed vaccine antigens following an episode of cellulitis remain uncharacterized. Methods: We enrolled 63 patients with cellulitis and 26 with invasive beta hemolytic streptococci infection, using a multiplexed assay to measure immunoglobulin G against Strep A vaccine candidate antigens, including: streptolysin O (SLO), deoxyribonuclease B (DNB), group A carbohydrate (GAC), C5a peptidase (ScpA), cell envelope proteinase (SpyCEP), and adhesion and division protein (SpyAD). Responses in the invasive cohort were used to predict the infecting etiology in the cellulitis cohort. Results: Of 41 patients with cellulitis and paired serological samples, 68.3% had evidence of beta hemolytic streptococci infection by conventional anti-SLO and/or anti-DNB criteria. A positive serological response to at least 1 of the tested antigens was seen in 78.0% of the cellulitis cohort. Individually, anti-SLO (58.5%), anti-SpyAD (46.3%), and anti-ScpA (39.0%) were the most common. Based on principal component analysis, increases in these 3 antibodies, without responses to DNB, GAC, and SpyCEP characterized Streptococcus dysgalactiae subspecies equisimilis (SDSE) infection. Conclusions: SDSE appears to be the predominant cause of lower limb cellulitis. Effective Strep A vaccines incorporating antigens that provide additional cross protection against SDSE may prevent a significant burden of lower limb cellulitis.

Immunoregulatory effects of testosterone supplementation combined with exercise training in men with Inclusion Body Myositis: a double-blind, placebo-controlled, cross-over trial.

Objectives: Sporadic Inclusion Body Myositis (IBM) is an inflammatory muscle disease affecting individuals over the age of 45, leading to progressive muscle wasting, disability and loss of independence. Histologically, IBM is characterised by immune changes including myofibres expressing major histocompatibility complex molecules and invaded by CD8+ T cells and macrophages, and by degenerative changes including protein aggregates organised in inclusion bodies, rimmed vacuoles and mitochondrial abnormalities. There is currently no cure, and regular exercise is currently the only recognised treatment effective at limiting muscle weakening, atrophy and loss of function. Testosterone exerts anti-inflammatory effects, inhibiting effector T-cell differentiation and pro-inflammatory cytokine production. Methods: We conducted a double-blind, placebo-controlled, cross-over trial in men with IBM, to assess whether a personalised progressive exercise training combined with application of testosterone, reduced the inflammatory immune response associated with this disease over and above exercise alone. To assess intervention efficacy, we immunophenotyped blood immune cells by flow cytometry, and measured serum cytokines and chemokines by Luminex immunoassay. Results: Testosterone supplementation resulted in modest yet significant count reduction in the classical monocyte subset as well as eosinophils. Testosterone-independent immunoregulatory effects attributed to exercise included altered proportions of some monocyte, T- and B-cell subsets, and reduced IL-12, IL-17, TNF-α, MIP-1ß and sICAM-1 in spite of interindividual variability. Conclusion: Overall, our findings indicate anti-inflammatory effects of exercise training in IBM patients, whilst concomitant testosterone supplementation provides some additional changes. Further studies combining testosterone and exercise would be worthwhile in larger cohorts and longer testosterone administration periods.

Achievements and Future Directions of the APFCB Mass Spectrometry Harmonisation Project on Serum Testosterone.

As an outcome of the 2010 Asian Pacific Conference for Chromatography and Mass Spectrometry in Hong Kong, a collaborative working group was formed to promote the harmonisation of mass spectrometry methods. The Mass Spectrometry Harmonisation Working Group resides under the combined auspices of the Asia-Pacific Federation for Clinical Biochemistry and Laboratory Medicine (APFCB) and the Australasian Association of Clinical Biochemists (AACB). A decision was made to initially focus attention on serum steroids due to the common interest of members in this area; with the first steroid to assess being testosterone. In principle, full standardisation with traceability should be achievable for all steroids as they are small compounds with defined molecular weight and structure. In order to achieve this we need certified reference materials, reference methods, reference laboratories, reference intervals and external quality assurance programs; each being an important pillar in the process. When all the pillars are present, such as for serum testosterone, it is feasible to fully standardise the liquid chromatography - tandem mass spectrometry (LC-MS/MS) methods. In a collaborative process with interested stakeholders, we commenced on a pathway to provide ongoing assessment and seek opportunities for improvement in the LC-MS/MS methods for serum steroids. Here we discuss the outcomes to date and major challenges related to the accurate measurement of serum steroids with a focus on serum testosterone.

Prevalence, characteristics, and prognostic significance of HFE gene mutations in type 2 diabetes: the Fremantle Diabetes Study.

OBJECTIVE: To examine the relationship between iron status, hereditary hemochromatosis (HFE) gene mutations, and clinical features and outcomes of type 2 diabetes in a well-characterized representative sample of community-based patients. RESEARCH DESIGN AND METHODS: HFE genotype data were available for 1,245 type 2 diabetic patients from the longitudinal observational Fremantle Diabetes Study (FDS), representing 96.2% of the total FDS type 2 diabetes cohort. Data were collected at recruitment between 1993 and 1996 and annually until the end of June 2001. Hospitalization and mortality data were available until the end of June 2006. The presence of the C282Y HFE mutation was determined in all subjects and H63D in C282Y heterozygotes. Fasting serum iron, transferrin, and ferritin were measured in all C282Y homozygotes and C282Y/H63D heterozygotes and in 286 randomly selected wild-type subjects. Multiple logistic regression analysis was performed to determine independent baseline associates of prevalent complications (myocardial infarction, cerebrovascular disease, retinopathy, neuropathy, and nephropathy), as was Cox proportional hazards modeling to determine predictors of incident complications and mortality. RESULTS: Although there were expected positive associations between HFE gene mutations and serum iron and transferrin saturation, there were no independent positive associations between HFE gene status and either microvascular or macrovascular complications in cross-sectional and longitudinal analyses. HFE gene status did not independently predict cardiac or all-cause mortality. Measures of iron metabolism including serum ferritin were not associated with combined microvascular or macrovascular end points. CONCLUSIONS: Directed screening for iron overload and/or HFE mutations appears unwarranted in patients with type 2 diabetes.