Definitions and major prerequisites of direct and indirect approaches for estimating reference limits.

Reference intervals are established either by direct or indirect approaches. Whereas the definition of direct is well established, the definition of indirect is still a matter of debate. In this paper, a general definition that covers all indirect models presently in use is proposed. With the upcoming popularity of indirect models, it has become evident that further partitioning strategies are required to minimize the risk of patients' false classifications. With indirect methods, such partitions are much easier to execute than with direct methods. The authors believe that the future of reference interval estimation belongs to indirect models with big data pools either from one laboratory or combined from several regional centres (if necessary). Independent of the approach applied, the quality assurance of the pre-analytical and analytical phase, considering biological variables and other confounding factors, is essential.

Differing performance of two assays for the measurement of anti-Mullerian hormone in premenopausal women: A cross-sectional study.

OBJECTIVE: To compare the performance of two anti-Mullerian (AMH) assays over a range of concentrations, in samples collected from young women. DESIGN: A cross-sectional method-comparison study of 168 non-healthcare-seeking women. PARTICIPANTS: Included women were aged 18-39 years, not recently pregnant, breast feeding or using systemic hormones. MEASUREMENTS: Serum AMH levels were analysed with the Beckman Coulter Access 2 assay from fresh samples and the Ansh picoAMH assay using samples stored at -80°C, in a parallel setting. Comparisons between the two assays were examined using Bland-Altman plots. RESULTS: Participants had a mean ± SD age of 32.6 ± 5.4 years and body mass index of 28.1 ± 7.9 kg/m2 , and 60.1% were parous. Although the assay results were highly correlated (Spearman correlation .982, P < .001), the relationship between the assays was nonlinear. Serum AMH values below 4 pmol/L were lower with the picoAMH assay compared with the Access AMH assay (mean difference in this range was -0.49 pmol/L), but for samples with a mean value above 10 pmol/L, the picoAMH assay consistently measured higher than the Access AMH assay (mean difference in this range was +8.2 pmol/L). As AMH concentrations increased the absolute discrepancy between the assays also increased. CONCLUSIONS: This study demonstrates that despite the high correlation between two commercially available AMH assays, the assays performed in a discordant manner at high and low concentrations. Hence, the results of these assays are not interchangeable, highlighting the need to establish specific reference limits for individual assays to guide clinical decision-making and the challenge of establishing future universal cut-offs for the application of AMH levels in clinical practice.

Results of the first recorded evaluation of a national gestational diabetes mellitus register: Challenges in screening, registration, and follow-up for diabetes risk.

OBJECTIVE: Gestational Diabetes Mellitus (GDM) increases the risk of type 2 diabetes. A register can be used to follow-up high risk women for early intervention to prevent progression to type 2 diabetes. We evaluate the performance of the world's first national gestational diabetes register. RESEARCH DESIGN AND METHODS: Observational study that used data linkage to merge: (1) pathology data from the Australian states of Victoria (VIC) and South Australia (SA); (2) birth records from the Consultative Council on Obstetric and Paediatric Mortality and Morbidity (CCOPMM, VIC) and the South Australian Perinatal Statistics Collection (SAPSC, SA); (3) GDM and type 2 diabetes register data from the National Gestational Diabetes Register (NGDR). All pregnancies registered on CCOPMM and SAPSC for 2012 and 2013 were included-other data back to 2008 were used to support the analyses. Rates of screening for GDM, rates of registration on the NGDR, and rates of follow-up laboratory screening for type 2 diabetes are reported. RESULTS: Estimated GDM screening rates were 86% in SA and 97% in VIC. Rates of registration on the NGDR ranged from 73% in SA (2013) to 91% in VIC (2013). During the study period rates of screening at six weeks postpartum ranged from 43% in SA (2012) to 58% in VIC (2013). There was little evidence of recall letters resulting in screening 12 months follow-up. CONCLUSIONS: GDM Screening and NGDR registration was effective in Australia. Recall by mail-out to young mothers and their GP's for type 2 diabetes follow-up testing proved ineffective.

Variation in cell-associated unspliced HIV RNA on antiretroviral therapy is associated with the circadian regulator brain-and-muscle-ARNT-like-1.

OBJECTIVE(S): To determine whether variation in cell-associated unspliced (CA-US) HIV RNA in HIV-infected individuals on antiretroviral therapy (ART) has a circadian basis. METHODS: Prospective observational study of HIV-infected individuals on ART. Blood was collected on three occasions and CA-US HIV RNA and mRNA of the circadian-locomotor-output-cycles-kaput (CLOCK)-associated genes quantified by real time PCR. CLOCK-associated proteins were over-expressed in a cell line stably transfected with an HIV long-terminal repeat (LTR) luciferase reporter. RESULTS: Using a mixed effects model, there was a significant increase in log-CA-US RNA at the third visit compared with the first visit (effect size of 0.619 with standard error (SE) of 0.098, P < 0.001) and an independent effect of time of blood draw (effect size 0.051 (SE 0.025), P = 0.040). The CLOCK-associated gene, brain-and-muscle-ARNT-like-1 (BMAL-1) had a significant relationship with log CA-US HIV RNA (effect size 8.508 (SE 3.777), P = 0.028) and also with time (P = 0.045). Over expression of BMAL-1 and CLOCK in a cell line stably transfected with an HIV-LTR luciferase reporter resulted in an increase in luciferase expression and this was reduced following mutation of the second E-box in the HIV-LTR. CONCLUSION: The basal level of HIV transcription on ART can vary significantly and is modulated by the circadian regulator BMAL-1, amongst other factors.

Distinguishing reference intervals and clinical decision limits - A review by the IFCC Committee on Reference Intervals and Decision Limits.

Reference Intervals (RIs) and clinical decision limits (CDLs) are a vital part of the information supplied by laboratories to support the interpretation of numerical clinical pathology results. RIs describe the typical distribution of results seen in a healthy reference population while CDLs are associated with a significantly higher risk of adverse clinical outcomes or are diagnostic for the presence of a specific disease. However, as the two concepts are sometimes confused, there is a need to clarify the differences between these terms and to ensure they are easily distinguished, especially because CDLs have a clinical association with specific diseases and risks, thereby implying that effective clinical interventions are available. It is important to note that, because population-based RIs are derived from the range of values expected in a typical community population, laboratory results that fall outside a RI do not necessarily indicate a disease but rather that additional medical follow-up and/or treatment may be warranted. In contrast, CDLs are associated with a risk of specific adverse outcomes, and are commonly used to interpret laboratory test results, including lipid parameters, glucose, hemoglobin A1c (HbA1c), and tumor markers, to determine risk of disease, to diagnose or to treat. In recent years, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee on Reference Intervals and Decision Limits (C-RIDL) has focused primarily on RIs and has performed multicenter studies to obtain common RIs. However, the broader responsibility of the Committee, from its name, includes "decision limits". C-RIDL now aims to emphasize the importance of the correct use of both RIs and CDLs and to encourage laboratories to specify the appropriate information to clinicians as needed. This review discusses RIs and CDLs in detail, describes the similarities and the differences between these two important tools in laboratory medicine, and clearly explains the processes used to define them. C-RIDL encourages the involvement of laboratory professionals in the establishment of both RIs and CDLs.

Indirect methods for reference interval determination - review and recommendations.

Reference intervals are a vital part of the information supplied by clinical laboratories to support interpretation of numerical pathology results such as are produced in clinical chemistry and hematology laboratories. The traditional method for establishing reference intervals, known as the direct approach, is based on collecting samples from members of a preselected reference population, making the measurements and then determining the intervals. An alternative approach is to perform analysis of results generated as part of routine pathology testing and using appropriate statistical techniques to determine reference intervals. This is known as the indirect approach. This paper from a working group of the International Federation of Clinical Chemistry (IFCC) Committee on Reference Intervals and Decision Limits (C-RIDL) aims to summarize current thinking on indirect approaches to reference intervals. The indirect approach has some major potential advantages compared with direct methods. The processes are faster, cheaper and do not involve patient inconvenience, discomfort or the risks associated with generating new patient health information. Indirect methods also use the same preanalytical and analytical techniques used for patient management and can provide very large numbers for assessment. Limitations to the indirect methods include possible effects of diseased subpopulations on the derived interval. The IFCC C-RIDL aims to encourage the use of indirect methods to establish and verify reference intervals, to promote publication of such intervals with clear explanation of the process used and also to support the development of improved statistical techniques for these studies.

Reporting and ideal testosterone levels in men undergoing androgen deprivation for prostate cancer-time for a rethink?

BACKGROUND: This study aims to review current laboratory reporting strategies across Australia and New Zealand with a view to propose a more useful template for reporting serum testosterone in the context of prostate cancer. MATERIALS AND METHODS: Registered pathology laboratories in Australia and New Zealand were enrolled into the current study. An electronic or a phone survey was utilized to collect data from each participating laboratory. Obtained information included assay utilized, units reported, reference intervals, lowest reported value, and lowest detectable value. To identify recommendations for testosterone testing, a systematic search was performed across Web of Science (including MEDLINE), EMBASE, and Cochrane libraries. RESULTS: Assessment of national pathology laboratories identified significant heterogeneity in the reporting methods. Reports typically used a "normal healthy male of 35 years of age" as a comparator but did not refer to optimal castrate levels, the lowest level that their assay was able to detect, nor did they include appended clinical guidelines relating to the prostate cancer patient cohort. CONCLUSIONS: Across Australia and New Zealand, various methods for testing and reporting serum testosterone exist, while international guidelines remain vague. The fashion in which serum testosterone levels are displayed should be re-evaluated to address the relevant clinical population and reflect an agreed-upon castrate threshold in patients undergoing androgen deprivation therapy.

An update report on the harmonization of adult reference intervals in Australasia.

The Australasian Association of Clinical Biochemists (AACB) has over the past 5 years been actively working to achieve harmonized reference intervals (RIs) for common clinical chemistry analytes using an evidence-based checklist approach where there is sound calibration and metrological traceability. It has now recommended harmonized RIs for 18 common clinical chemistry analytes which are performed in most routine laboratories and these have been endorsed by the Royal College of Pathologists of Australasia (RCPA). In 2017 another group of analytes including urea, albumin and arterial blood gas parameters were considered and suggested harmonized RIs proposed. This report provides an update of those harmonization efforts.

The key incident monitoring and management system - history and role in quality improvement.

BACKGROUND: The determination of reliable, practical Quality Indicators (QIs) from presentation of the patient with a pathology request form through to the clinician receiving the report (the Total Testing Process or TTP) is a key step in identifying areas where improvement is necessary in laboratories. METHODS: The Australasian QIs programme Key Incident Monitoring and Management System (KIMMS) began in 2008. It records incidents (process defects) and episodes (occasions at which incidents may occur) to calculate incident rates. KIMMS also uses the Failure Mode Effects Analysis (FMEA) to assign quantified risk to each incident type. The system defines risk as incident frequency multiplied by both a harm rating (on a 1-10 scale) and detection difficulty score (also a 1-10 scale). RESULTS: Between 2008 and 2016, laboratories participating rose from 22 to 69. Episodes rose from 13.2 to 43.4 million; incidents rose from 114,082 to 756,432. We attribute the rise in incident rate from 0.86% to 1.75% to increased monitoring. Haemolysis shows the highest incidence (22.6% of total incidents) and the highest risk (26.68% of total risk). "Sample is suspected to be from the wrong patient" has the second lowest frequency, but receives the highest harm rating (10/10) and detection difficulty score (10/10), so it is calculated to be the 8th highest risk (2.92%). Similarly, retracted (incorrect) reports QI has the 10th highest frequency (3.9%) but the harm/difficulty calculation confers the second highest risk (11.17%). CONCLUSIONS: TTP incident rates are generally low (less than 2% of observed episodes), however, incident risks, their frequencies multiplied by both ratings of harm and discovery difficulty scores, concentrate improvement attention and resources on the monitored incident types most important to manage.

Depletion of biotin using streptavidin-coated microparticles: a validated solution to the problem of biotin interference in streptavidin-biotin immunoassays.

Background Biotin interference in streptavidin-based immunoassays causes widespread analytical distortions that may lead to clinical confusion, inappropriate patient management and, ultimately, adverse events. Its prevalence has increased recently due to the increased use of high-dose biotin therapy in specific patient groups (notably multiple sclerosis) and possibly the general community. Methods We have developed a method to deplete biotin from samples using the streptavidin-coated magnetic microparticles that are a component of most susceptible assays. Results We show that high concentrations of spiked biotin can be adequately depleted from serum using this approach, and that gross biochemical derangements can be restored to normality. We also show that biotin in samples derived from multiple sclerosis patients receiving 300 mg biotin daily can be adequately depleted to remove associated analytical interference and restore normal results. The method is applicable to competitive and sandwich immunoassays and importantly, because it does not change the volume of the sample, suitable for the measurement of free thyroid hormone assays. Application of the method does not significantly change the precision of measurement, and for the majority of analytes, the accuracy is not substantially altered. Conclusions Adopting this method enables laboratories to confirm biotin interference in the appropriate clinical setting. Moreover, it enables laboratories to remove the interference and report accurate and reliable results, without the need for patients to withhold beneficial therapy prior to blood tests. Until the biotin tolerance of susceptible assays is improved, our method gives laboratories a safe alternative for reporting results using streptavidin-based methods.

Detailed Description of Change in Serum Cholesterol Profile with Incremental Weight Loss After Restrictive Bariatric Surgery.

INTRODUCTION: Dyslipidemia affects up to 75% of morbidly obese individuals and is a key driver of cardiovascular disease. Weight loss is an established strategy to improve metabolic risk, including dyslipidemia. We aimed to determine weight loss goals for resolution of serum lipid abnormalities, by measuring improvements during progressive weight loss in obese individuals. METHODS: We performed a prospective cohort study of obese individuals with the metabolic syndrome undergoing adjustable gastric banding. Lipid levels were monitored monthly for 9 months, then three monthly until 24 months. RESULTS: There were 101 participants included, age 47.4 ± 10.9 years with body mass index 42.6 ± 5.9 kg/m2. At 24 months, total body weight loss (TBWL) was 18.3 ± 7.9%. This was associated with significant improvements in high-density lipoprotein (HDL) (1.18 vs 1.47, p < 0.001), triglyceride (2.0 vs 1.4, p < 0.001), and total cholesterol to HDL ratio (TC:HDL) (4.6 vs 3.6, p < 0.001). Over this time, progressive and linear improvements in HDL, triglycerides, and TC:HDL were seen with incremental weight loss (observed at 2.5% TBWL intervals). Significant improvements occurred after a threshold weight loss of 7.5-12.5% TBWL was achieved, with odds ratio (OR) 1.48-2.50 for normalization. These odds improved significantly with increasing weight loss (OR 18.2-30.4 with > 25% TBWL). Despite significant weight loss, there was no significant change in low-density lipoprotein (LDL). CONCLUSION: Significant improvements in triglycerides, HDL, and TC:HDL occur after 7.5-12.5% TBWL, with ongoing benefit after greater weight loss. LDL needs to be addressed independently, as this was not observed to respond to weight loss alone. TRIAL REGISTRATION NUMBER: Australian Clinical Trials Registry (ACTRN12610000049077).

Characterization of the scope and magnitude of biotin interference in susceptible Roche Elecsys competitive and sandwich immunoassays.

Background Biotin interference is a significant problem to which at-risk laboratories must now be attuned. We sought to systematically characterize the nature of this interference in Roche immunoassays. Methods Known concentrations of biotin were titrated into serum samples and the effects on competitive and sandwich immunoassays were analysed. The maximum and minimum concentrations examined reflect those likely to be achieved in individuals on 5 to 10 mg supplements at the lower end, and 100 to 300 mg biotin at the high end. Results A high variability in biotin tolerance was observed. Some assays, such as troponin T, TSH and antithyroid antibodies, were extremely sensitive to the lower concentrations of biotin (15.6 and 31.3 ng/mL), whereas the majority of assays were relatively resistant. At concentrations ≥500 ng/mL, all assays showed significant interference from biotin but, again, the magnitude of the interference was variable. The more sensitive assays showed profound analytical bias at biotin concentrations that occur with high-dose therapy. Conclusion Our data demonstrate high variability in biotin tolerance across Roche immunoassays. The shape of the dose-response curves provides more detailed information than the single manufacturer-quoted figure for biotin tolerance. Accordingly, these data may be used by laboratories for more accurate risk assessment in predicting the effects of biotin. Our data may also be extrapolated to guide timing of blood tests in patients on high-dose biotin therapy: it demonstrates the number of half-lives required to withhold biotin in order to decrease its concentration to below a given assay tolerance.

Serum 25-Hydroxyvitamin D Insufficiency in Search of a Bone Disease.

Context: Vitamin D "insufficiency" and "deficiency" are defined as serum 25-hydroxyvitamin D [25(OH)D] levels <75 and <30 nmol/L, respectively. We aimed to determine whether these values signal hypocalcemia and hypophosphatemia, secondary hyperparathyroidism, high bone remodeling, low areal bone mineral density (aBMD), microstructural deterioration, or reduced matrix mineralization density (MMD) and so suggest whether bone fragility is present. Methods: Concentrations of 25(OH)D, calcium, phosphate, creatinine, and parathyroid hormone (PTH) were measured in 11,855 participants. Serum C-terminal telopeptide of type 1 collagen, procollagen type 1 N-terminal propeptide (P1NP), aBMD, and distal radius microstructure and MMD were measured in a second subset of 150 participants. Results: A breakpoint for calcium, PTH, and alkaline phosphatase was identified at a threshold 25(OH)D level <30 nmol/L. There was no plateau beyond 75 nmol/L. In the subgroup with measurements of bone morphology, no associations were detectable between serum 25(OH)D concentration, aBMD, trabecular density, cortical porosity, or MMD. Among 1439 participants with serum 25(OH)D <30 nmol/L, 6.1% had low serum calcium, 3.4% had low serum phosphate, 6.1% had high alkaline phosphatase, and 34.2% had elevated PTH. Most participants did not have any abnormalities. Conclusion: At a 25(OH)D threshold of ≤30 nmol/L, abnormalities in biochemical features support the notion of a "deficiency" state predisposing to bone disease. However, no deleterious effects were found in participants within an insufficiency threshold of a 25(OH)D level of 30 to 75 nmol/L, which challenges the rationale justifying vitamin D supplementation in these individuals.

Higher maternal serum prolactin levels are associated with reduced glucose tolerance during pregnancy.

It is unknown if high prolactin levels during pregnancy contribute to the development of gestational diabetes. We hypothesized that higher prolactin levels are associated with reduced glucose tolerance, as determined by higher 2-h glucose level from an oral glucose tolerance test in pregnancy. The 75-g oral glucose tolerance test was carried out at 28 weeks of gestation in 69 participants. A multiple regression analysis was used to determine the relationship between serum prolactin and 2-h glucose levels. Multivariable regression analysis showed an independent and significant relationship between third trimester prolactin and 2-h glucose levels post oral glucose tolerance test. Higher prolactin levels were associated with higher glucose levels independent of age, body mass index, gravidity and parity. Higher prolactin levels associated with reduced glucose tolerance in the third trimester of pregnancy suggests the possible independent role of prolactin in the pathogenesis of gestational diabetes.

Modified thresholds for fibrosis risk scores in nonalcoholic fatty liver disease are necessary in the obese.

BACKGROUND: Obesity and its related comorbidities are significant risk factors for nonalcoholic fatty liver disease (NAFLD). Liver fibrosis is the major determinant of long-term outcomes in NAFLD. A non-invasive tool that accurately identifies obese patients at elevated risk of liver fibrosis would be of significant value. Fibrosis risk scores in patients with NAFLD have been proposed but have not been validated in obese populations. We aimed to validate established simple fibrosis scores in bariatric surgical patients. METHODS: We conducted a prospective study of 107 consecutive high-risk obese patients undergoing primary bariatric surgery. Proposed fibrosis scores (NAFLD fibrosis score; body mass index (BMI), aspartate aminotransferase (AST)/alanine aminotransferase ratio (ALT), and diabetes (BARD); Fibrosis-4 (FIB-4); Forn; and AST to platelet ratio index) were calculated and compared hepatic fibrosis determined by histology of intraoperative liver biopsies. Accuracy was determined, and fibrosis score thresholds were optimized. These modified thresholds were then validated in an independent bariatric surgical population. RESULTS: Liver biopsies were available in 101 patients. Sixty-eight patients had some degree of fibrosis, with 23 patients (23 %) having significant fibrosis (F2-4). The Forn score best predicted significant fibrosis (area under the receiver operator characteristic curve (AUROC) 0.724, p = 0.001). With standard thresholds, the sensitivity for the Forn score for identification of significant fibrosis (F2-4) was 0 %. Using modified thresholds of 3.5, the sensitivity and negative predictive value increased to 85.7 and 94.7 %. This threshold was applied to an independent validation cohort with good accuracy. CONCLUSIONS: Fibrosis risk scores using simple markers have moderate success at delineating obese patients with significant NAFLD-related fibrosis. Thresholds, however, need to be lowered to maximize diagnostic accuracy in this cohort.

Cystatin C estimated glomerular filtration rate and all-cause and cardiovascular disease mortality risk in the general population: AusDiab study.

AIMS: Uncertainties about the role of cystatin C-based estimated glomerular filtration rate (eGFR) in the prediction of cardiovascular disease (CVD) beyond traditional CVD risk factors remain. We assessed contributions of eGFR to CVD and mortality in the general population. METHODS: Using 14 year follow-up data on 9353 adults without a reported history of CVD from the Australian Diabetes, Obesity and Lifestyle study, we assessed the contributions of eGFR (assessed by cystatin C (eGFRcysC ) and serum creatinine (eGFRcr ) and albuminuria (uACR) to total and CVD mortality. RESULTS: After adjusting for age, sex, CVD risk factors and uACR, compared with an eGFRcysC >90 mL/min per 1.73 m2 , eGFRcysC <60 mL/min per 1.73 m2 was associated with 56% and 73% increases in the risks for all-cause and CVD mortality, respectively. The respective changes for the c-statistic when eGFRcysC was added to a risk prediction model were 0.003 (95% confidence interval: 0.001 to 0.005) and 0.002 (95% confidence interval: -0.001 to 0.006). The net proportion of non-events assigned a lower-risk category significantly improved with the addition of eGFR (non-event net reclassification index eGFRcr : 1.0% and eGFRcysC : 1.5%) for all-cause mortality, but for CVD mortality, improvements were only significant when eGFR was combined with uACR. The net proportion of events assigned a higher-risk category was not significantly improved. CONCLUSION: In our community-based cohort, reduced eGFRcysC was associated with all-cause and CVD mortality. The addition of chronic kidney disease measures to risk prediction models improved overall risk stratification among those at low risk as opposed to those at high baseline risk of mortality.

Effects of Bariatric Surgery on Liver Function Tests in Patients with Nonalcoholic Fatty Liver Disease.

OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) affects over 80% of obese patients and is fueled by the metabolic syndrome. Weight loss is strongly advocated as a central treatment for NAFLD and has been shown to induce histological improvement. We aimed to define the patterns of improvement in NAFLD with weight loss and determine target weight goals for NAFLD resolution. METHODS: A prospective study of 84 morbidly obese patients with NAFLD undergoing bariatric surgery was conducted. Intraoperative liver biopsies were taken. Monthly follow-up, including blood tests and measurements, was performed. We monitored improvements in NAFLD by monthly alanine aminotransferase (ALT) and gamma glutamyltransferase (GGT) levels over 1 year. RESULTS: There was rapid improvement in ALT, particularly in the first 6 months following surgery, with statistically significant reduction in ALT at 2 months (35 vs 27 IU/L, p < 0.001). In multivariate analysis, there were significantly increased odds of ALT normalization after a %TBWL of 10-15% (odds ratio 2.49, p = 0.005). The odds of resolution increased with increasing weight loss. Triglyceride levels (odds ratio 0.59, p = 0.021) and baseline NAFLD activity score (odds ratio 0.28, p < 0.001) were also significantly related to ALT normalization. Improvements in ALT occurred prior to metabolic improvement and well before traditional ideal weight goals were reached. CONCLUSION: Improvements in NAFLD occurred rapidly after bariatric surgery and were closely related to weight loss and metabolic factors. A 10-15% reduction in body weight is an appropriate target to achieve substantial improvement in ALT levels. TRIAL REGISTRATION NUMBER: Australian Clinical Trials Registry (ACTRN12610000049077).