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.

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.

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.

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.

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.

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.

Serum 25-hydroxyvitamin D deficiency and the 5-year incidence of CKD.

BACKGROUND: Low serum 25-hydroxyvitamin D (25[OH]D) levels have been associated with chronic kidney disease in cross-sectional studies. However, this association has not been studied prospectively in a large general population-based cohort. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 6,180 adults 25 years or older participating in the baseline and 5-year follow-up phases of the Australian Diabetes, Obesity and Lifestyle (AusDiab) Study. PREDICTOR: Serum 25(OH)D levels <15 ng/mL were considered deficient. OUTCOMES & MEASUREMENTS: Incident chronic kidney disease was defined as being negative at baseline but positive after 5 years for (1) reduced estimated glomerular filtration rate (eGFR; <60 mL/min/1.72 m²) or (2) albuminuria (spot urine albumin-creatinine ratio ≥2.5 mg/mmol [≥22.1 mg/g] for men and ≥3.5 mg/mmol [≥30.9 mg/g] for women). RESULTS: 623 (10.9%) participants were vitamin D deficient, 161 developed incident reduced eGFR, and 222 developed incident albuminuria. In participants with and without vitamin D deficiency, annual age-standardized incidences were 0.92% (95% CI, 0.56%-1.30%) and 0.59% (95% CI, 0.51%-0.68%), respectively, for eGFR <60 mL/min/1.72 m² and 1.50% (95% CI, 1.06%-1.95%) and 0.66% (95% CI, 0.56%-0.76%), respectively, for albuminuria. In multivariate regression models, vitamin D deficiency was associated significantly with the 5-year incidence of albuminuria (OR, 1.71; 95% CI, 1.12-2.61; P = 0.01), but not reduced eGFR (OR, 0.93; 95% CI, 0.53-1.66; P = 0.8). LIMITATIONS: The observational nature of the study does not account for unmeasured confounders. Only baseline 25(OH)D level was measured and therefore may not accurately reflect lifetime levels. Differences in baseline characteristics of participants who were included compared with those excluded due to missing data or follow-up may limit the applicability of results to the original AusDiab cohort. CONCLUSIONS: Our prospective cohort study shows that vitamin D deficiency is associated with a higher annual incidence of albuminuria and reduced eGFR and independently predicts the 5-year incidence of albuminuria. These associations warrant further exploration in long-term prospective clinical trials.

Measuring thyroid peroxidase antibodies on the day nulliparous women present for management of miscarriage: a descriptive cohort study.

BACKGROUND: There has been recent evidence suggesting the presence of anti-thyroid peroxidase antibodies (TPOAb) increases the risk of miscarriage, and levothyroxine can rescue miscarriages associated with TPOAb. We propose the most clinically pragmatic cohort to screen for TPOAb are women presenting for management of a missed miscarriage and have never birthed a liveborn. We measured serum TPOAb among nulliparous women presenting for management of miscarriage, and compared levels with women who have had 2 or more livebirths (and never miscarried). Given its potential role in immunomodulation, we also measured Vitamin D levels. METHODS: We performed a prospective descriptive cohort study at a tertiary hospital (Mercy Hospital for Women, Victoria, Australia). We measured TPOAb and Vitamin D levels in serum obtained from 118 nulliparous women presenting for management of miscarriage, and 162 controls with 2 or more livebirths (and no miscarriages). Controls were selected from a serum biobank prospectively collected in the first trimester at the same hospital. RESULTS: Nulliparous women with 1 or more miscarriages had higher thyroid peroxidase antibody (TPOAb) levels than those with 2 or more livebirths; TPOAb in miscarriage group was 0.3 mIU/L (interquartile range [IR]: 0.2-0.7) vs 0.2 mIU/L among controls (IR 0.0-0.5; p < 0.0001). We confirmed TPOAb levels were not correlated with serum human chorionic gonadotrophin (hCG) concentrations in either the miscarriage or control groups. In contrast, thyroid stimulating hormone, fT3 and fT4 levels (thyroid hormones) either trended towards a correlation, or were significantly correlated with serum hCG levels in the two groups. Of the entire cohort that was predominantly caucasian, only 12% were Vitamin D sufficient. Low Vitamin D levels were not associated with miscarriage. CONCLUSIONS: We have confirmed the association between miscarriage and increased TPOAb levels. Furthermore, it appears TPOAb levels in maternal blood are not influenced by serum hCG levels. Therefore, we propose the day nulliparous women present for management for miscarriage is a clinically relevant, and pragmatic time to screen for TPOAb.

Defining acceptable limits for the metrological traceability of specific measurands.

Although manufacturers are compelled by the European IVD Directive, 98/79/EC, to have traceability of the values assigned to their calibrators if suitable higher order reference materials and/or procedures are available, there is still no equivalence of results for many measurands determined in clinical laboratories. The adoption of assays with metrological traceable results will have a significant impact on laboratory medicine in that results will be equivalent across different laboratories and different analytical platforms. The IFCC WG on Allowable Errors for Traceable Results has been formed to define acceptable limits for metrological traceability chains for specific measurands in order to promote the equivalence of patient results. These limits are being developed based on biological variation for the specific measurands. Preliminary investigations have shown that for some measurands, it is possible for manufacturers to assign values to assay calibrators with a measurement uncertainty that allows the laboratory enough combined uncertainty for their routine measurements. However, for other measurands, e.g., plasma sodium, current assays are too imprecise to fulfil limits based on biological variation. Although an alternative approach based on probability theory is being investigated, the most desirable approach would be for industry to improve measurement methods so that they meet clinical requirements.

Prevalence of vitamin D deficiency and its determinants in Australian adults aged 25 years and older: a national, population-based study.

OBJECTIVE: Vitamin D deficiency is recognized as a global public health problem, but the population-based prevalence of deficiency and its determinants in Australian adults is not known. This study evaluated the vitamin D status of Australian adults aged ≥25 years and risk factors associated with vitamin D deficiency in this population. DESIGN AND PATIENTS: We studied a national sample of 11,247 Australian adults enrolled in the 1999/2000 Australian Diabetes, Obesity and Lifestyle (AusDiab) study drawn from 42 randomly selected districts throughout Australia. MEASUREMENTS: Serum concentrations of 25-hydroxyvitamin D [25(OH)D] were measured by immunoassay. Vitamin D deficiency was defined as a concentration <50 nmol/l. Information on demographic and lifestyle factors was derived from interview-administered questionnaires. RESULTS: The mean serum 25(OH)D concentration was 63 nmol/l (95% CI: 59-67 nmol/l). Only 4% of the population had a level <25 nmol/l, but the prevalence of vitamin D deficiency (<50 nmol/l) was 31% (22% men; 39% women); 73% had levels <75 nmol/l. The prevalence of vitamin D deficiency increased significantly with age, was greater in women, in those of non-Europid origin, in the obese and those who were physically inactive and with a higher level of education. Deficiency was also more common during winter and in people residing in southern Australia (latitude >35°S); 42% of women and 27% of men were deficient during summer-autumn, which increased to 58% and 35%, respectively, during winter-spring. CONCLUSION: Vitamin D deficiency is common in Australia affecting nearly one-third of adults aged ≥25 years. This indicates that strategies are needed at the population level to improve vitamin D status of Australians.

Hyponatremia is associated with higher NT-proBNP than normonatremia after prolonged exercise.

OBJECTIVE: To determine the incidence of and risk factors for exercise-associated hyponatremia (EAH) in cyclists completing a long-distance bike ride and to assess whether postexercise serum NT-proBNP concentration (brain natriuretic protein precursor) differed between riders with and without EAH. DESIGN: Observational study. SETTING: "Around the Bay in a Day" cycle event, October 2010. PARTICIPANTS: One hundred thirty-nine cyclists prospectively enrolled, with 90 completing 210 or 250 km. MAIN OUTCOME MEASURES: Body weight change and fluid intake during the event, and postevent serum sodium concentration ([Na+]) and NT-proBNP concentration ([NT-proBNP]). RESULTS: Four riders (4.5%) were hyponatremic ([Na+] < 135 mmol/L). The lowest postride [Na+] was 126 mmol/L. Hyponatremia was associated with a mean weight gain of 3.4 kg (3.9% of total body weight). Significant negative correlations were found between postride [Na+] and change in weight (r = -0.34; P < 0.01) and fluid intake when expressed as total volume (r = -0.35; P < 0.01), mL/kg body weight (r = 0.33; P < 0.01), mL·kg·h (r = -0.27; P < 0.01), or mL/h (r = -0.29; P < 0.01). NT-proBNP concentrations levels in 3 of the 4 hyponatremic subjects were markedly elevated compared with eunatremic subjects matched for age, sex, distance ridden, training, and medical history. CONCLUSIONS: Exercise-associated hyponatremia was found to occur in 4.5% of the study group and was associated with weight gain during a prolonged bike ride. Postride [Na+] varied inversely with weight change and with fluid intake. Three of 4 hyponatremic riders had significant elevations of [NT-proBNP]. These results support the hypothesis that overconsumption of hypotonic fluids in this setting is the most important cause of EAH.

Preanalytical quality improvement: from dream to reality.

Abstract Laboratory diagnostics (i.e., the total testing process) develops conventionally through a virtual loop, originally referred to as "the brain to brain cycle" by George Lundberg. Throughout this complex cycle, there is an inherent possibility that a mistake might occur. According to reliable data, preanalytical errors still account for nearly 60%-70% of all problems occurring in laboratory diagnostics, most of them attributable to mishandling procedures during collection, handling, preparing or storing the specimens. Although most of these would be "intercepted" before inappropriate reactions are taken, in nearly one fifth of the cases they can produce inappropriate investigations and unjustifiable increase in costs, while generating inappropriate clinical decisions and causing some unfortunate circumstances. Several steps have already been undertaken to increase awareness and establish a governance of this frequently overlooked aspect of the total testing process. Standardization and monitoring preanalytical variables is of foremost importance and is associated with the most efficient and well-organized laboratories, resulting in reduced operational costs and increased revenues. As such, this article is aimed at providing readers with significant updates on the total quality management of the preanalytical phase to endeavour further improvement for patient safety throughout this phase of the total testing process.

Thyroid function and autoantibody status among women who spontaneously deliver under 35 weeks of gestation.

OBJECTIVE: Recent studies report an association between maternal thyroid auto-antibodies and preterm birth. None have made the important distinction between spontaneous and iatrogenic preterm birth. We investigated the association between spontaneous preterm birth before 35-week gestation and both thyroid function and auto-antibody status. DESIGN: Retrospective nested case-control study, samples retrieved from a biobank of around 8000 samples. PATIENTS: Forty-six cases of spontaneous preterm birth <35-weeks gestation compared with 124 cases who delivered at 40 weeks gestation, matched for maternal age and duration of serum storage. All resulted in a singleton liveborn. MEASUREMENTS: First trimester serum levels of TSH, free T4, free T3, anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-Tg) antibodies. RESULTS: Women who had a preterm birth delivered at a median gestational age of 33 (+1 day) weeks gestation (controls 40 + 3 weeks) and had significantly lower birthweight infants [mean (range) weight 1965 (581-3117) grams compared with controls 3526 (2685-4760) grams]. Median levels of anti-thyroglobulin and anti-TPO antibodies were similar in the two groups (Anti-Tg: 3 IU/ml (Range 1-316) v 3 IU/ml (1-691); Anti-TPO 8 IU/ml (3-73) v 9 IU/ml (3-499); P >/= 0.18 for both comparisons, compared using three statistical models). In the preterm group, the median TSH level was 0.70 mIU/l (range 0.04-3.84), similar to controls (0.88 (0.01-2.87), P = 0.1). Free T3 and T4 levels were also similar in both groups. CONCLUSIONS: Thyroid function and thyroid autoantibody status among women who have a spontaneous preterm delivery before 35 weeks gestation are not different from women delivering at term.