The effect of the immunoassay curve fitting routine on bias in troponin.

OBJECTIVES: Unlike many dose-response curves used in clinical chemistry, the immunoassay curve used to quantitate measurands is often sigmoidal rather than linear. Consequently, a more complex curve fitting model is required. Various models are available, but they can introduce bias, and there can be little awareness of why this error can be introduced. CONTENT: These curve-fitting models include those based on the law of mass-action, empirical models such as splines or linearization models such as the log/logit function. All these models involve assumptions, which can introduce bias as the dose-response curve is 'forced' to fit or minimize the distance between the standard concentration points to the theoretical curve. The most common curve fitting model is the four or five parameter model, which uses four or five parameters to fit a sigmoidal curve to a set of standard points. SUMMARY AND OUTLOOK: Measurement of cardiac troponin is an important element in establishing a diagnosis of acute myocardial infarction. We use troponin, a cardiac biomarker, to demonstrate the potential effect of the bias that the curve fit could introduce. Troponin is used for both rule-in and rule-out decisions at different concentrations and at either end of the dose-response curve. The curve fitting process can cause lot-to-lot reagent (and calibrator) variation in immunoassay. However, laboratory staff need to be aware of this potential source of error and why it occurs. Understanding how the error occurs leads to a greater awareness of the importance of validating new reagent/calibrator assessment using patient samples with concentrations at crucial decision points.

Oral glucose tolerance test to diagnose gestational diabetes mellitus: Impact of variations in specimen handling.

To improve birth outcomes, all pregnant women without known diabetes are recommended for an oral glucose tolerance test (OGTT) to screen for hyperglycaemia in pregnancy (diabetes in pregnancy or gestational diabetes mellitus (GDM)). This narrative review presents contemporary approaches to minimise preanalytical glycolysis in OGTT samples with a focus on GDM diagnosis using criteria derived from the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study. The challenges of implementing each approach across a diverse Australian healthcare setting were explored. Many Australian sites currently collect and transport OGTT samples at ambient temperature in sodium fluoride (NaF) tubes which is likely to lead to missed diagnosis of GDM in a significant proportion of cases. Alternative preanalytical solutions should be pragmatic and tailored to individual settings and as close as possible to the preanalytical conditions of the HAPO study for correct interpretation of OGTT results. Rapid centrifugation of barrier tubes to separate plasma could be suitable in urban settings provided time to centrifugation is strictly controlled. Tubes containing NaF and citrate could be useful for remote or resource poor settings with long delays to analysis but the impact on the interpretation of OGTT results should be carefully considered. Testing venous blood glucose at the point-of-care bypasses the need for glycolytic inhibition but requires careful selection of devices with robust analytical performance. Studies to evaluate the potential error of each solution compared to the HAPO protocol are required to assess the magnitude of misdiagnosis and inform clinicians regarding the potential impact on patient safety and healthcare costs.

Troponins in myocardial infarction and injury.

Troponins are proteins that are integral components of the contractile mechanism of muscle, including cardiac muscle. Cardiac troponins Iand T can be detected in the blood of most people after puberty, at concentrations reflecting cardiac mass, sex and age. Current laboratory assays are approximately 1000 times more sensitive than those used previously. They also have higher sensitivity than point-of-care assays. The measurement of cardiac troponins is used primarily to assist in the diagnosis or exclusion of myocardial injury. Serial tests in acute coronary syndrome are guided by the Universal Definition of Myocardial Infarction.

The intra-individual variation of cardiac troponin I: the effects of sex, age, climatic season, and time between samples.

OBJECTIVES: Knowing the intra-individual variation (CVi), also termed within subject biological variation, of an analyte is essential to properly interpret apparent changes in concentration. While there have been many studies assessing the CVi of cardiac troponin (cTnI), they have been limited in looking at CVi in different settings, and there is no data available on whether CVi might change in different settings. METHODS: We used our large cTnI data bank to look at the CVi of cTnI in Emergency Department (ED) patients who had an acute myocardial infarction event excluded. We looked at the effects of gender, age, climatic season, and time between samples to assess whether CVi changed. To assess the effect of age, after exclusion, we collected two samples from each subject for each study which were used to calculate the CVi between those identified groups. There were 139 males and 98 females aged <65 years and 109 males and 98 females aged ≥65 years. For gender and season, there were 122 males and 94 females in the summer period and 126 males and 102 females in the winter period. To assess long term variation there were 195 males and 153 females who had further admissions after more than 12 months. RESULTS: For the four variables listed, there were no significant differences in within individual variation (CVi), but there was a significant difference in between individual variation (CVg) for men and women with regard to age. The Index of Individuality (II) was <0.20 for all conditions studied. We noted that >90% of subjects had an reference change value (RCV) <9 ng/L. CONCLUSIONS: Because troponin concentration in patients without an identified cardiac condition change so little, delta changes are potentially of great value in assessing patients in the ED. Significant delta changes in troponin can occur without the 99th percentile being exceeded.

Strict Preanalytical Oral Glucose Tolerance Test Blood Sample Handling Is Essential for Diagnosing Gestational Diabetes Mellitus.

OBJECTIVE: Preanalytical processing of blood samples can affect plasma glucose measurement because ongoing glycolysis by cells prior to centrifugation can lower its concentration. In June 2017, ACT Pathology changed the processing of oral glucose tolerance test (OGTT) blood samples for pregnant women from a delayed to an early centrifugation protocol. The effect of this change on the rate of gestational diabetes mellitus (GDM) diagnosis was determined. RESEARCH DESIGN AND METHODS: All pregnant women in the Australian Capital Territory (ACT) are recommended for GDM testing with a 75-g OGTT using the World Health Organization diagnostic criteria. From January 2015 to May 2017, OGTT samples were collected into sodium fluoride (NaF) tubes and kept at room temperature until completion of the test (delayed centrifugation). From June 2017 to October 2018, OGTT samples in NaF tubes were centrifuged within 10 min (early centrifugation). RESULTS: A total of 7,509 women were tested with the delayed centrifugation protocol and 4,808 with the early centrifugation protocol. The mean glucose concentrations for the fasting, 1-h, and 2-h OGTT samples were, respectively, 0.24 mmol/L (5.4%), 0.34 mmol/L (4.9%), and 0.16 mmol/L (2.3%) higher using the early centrifugation protocol (P < 0.0001 for all), increasing the GDM diagnosis rate from 11.6% (n = 869/7,509) to 20.6% (n = 1,007/4,887). CONCLUSIONS: The findings of this study highlight the critical importance of the preanalytical processing protocol of OGTT blood samples used for diagnosing GDM. Delay in centrifuging of blood collected into NaF tubes will result in substantially lower rates of diagnosis than if blood is centrifuged early.

Choice of Statistical Tools for Outlier Removal Causes Substantial Changes in Analyte Reference Intervals in Healthy Populations.

BACKGROUND: Reference intervals are an important aid in medical practice as they provide clinicians a guide as to whether a patient is healthy or diseased.Outlier results in population studies are removed by any of a variety of statistical measures. We have compared several methods of outlier removal and applied them to a large body of analytes from a large population of healthy persons. METHODS: We used the outlier exclusion criteria of Reed-Dixon and Tukey and calculated reference intervals using nonparametric and Harrell-Davis statistical methods and applied them to a total of 36 different analytes. RESULTS: Nine of 36 analytes had a greater than 20% difference in the upper reference limit, and for some the difference was 100% or more. CONCLUSIONS: For some analytes, great importance is attached to the reference interval. We have shown that different statistical methods for outlier removal can cause large changes to reported reference intervals. So that population studies can be readily compared, common statistical methods should be used for outlier removal.

Age-related differences in hs-cTnI concentration in healthy adults.

BACKGROUND: Because the 99th percentile is of such importance in defining myocardial injury and myocardial infarction, it is important to know whether there are real age-related differences in troponin 99th percentiles. METHODS: We went to our database from the Canberra Heart Study where 1062 apparently healthy subjects were extensively screened for occult cardiac disease, and looking at persons aged <65 years and >65 years, for men and women separately, we compared a variety of cutpoints from the 99th percentile down to the 50th percentile. RESULTS: With our rigorous criteria for defining cardiac health, we excluded 67.2% of males aged >65 years and 53.8% of women aged 65 years and older. Even with these rigorous exclusions we found that at every cutpoint examined between the 99th percentile and the 50th percentile, persons aged <65 years had lower troponin I concentrations that persons aged 65 years and older. Similarly, at every cutpoint examined, women had lower troponin I concentrations than did men. For the 4 separate groups examined (men and women, age < 65 years and 65 years and older) after the exclusions of persons with subclinical cardiac disease, the distributions were not significantly different to a Gaussian distribution. CONCLUSIONS: With the rigorous exclusions of persons with subclinical cardiac disease, and the fact that our populations have a Gaussian distribution, our data suggests that age-related hs-cTnI concentrations are real. This has important implications particularly when assessing older persons in the Emergency Department.

Are the changes in diagnostic criteria for gestational diabetes mellitus reflected in perinatal outcomes? A retrospective assessment.

BACKGROUND: Gestational diabetes mellitus (GDM) is glucose intolerance first diagnosed during pregnancy not due to overt diabetes. Recent changes to the diagnostic guidelines have been shown to increase the apparent occurrence of GDM. AIM: The aim of this study was to compare retrospectively the neonatal outcomes between groups defined using the new and old criteria to assess the impact of guideline changes on pregnancy outcomes. METHODS: The study was of singleton babies delivered of 641 women, who had oral glucose tolerance testing and pregnancy care at a single tertiary centre between 2011 and 2015. RESULTS: Compared to the population of women not now considered to have GDM by International Association of Diabetes and Pregnancy Study Groups criteria (two-hour glucose concentration ≤8.4 mmol/L), neonates born to women with the new lower fasting criterion (5.1-5.4 mmol/L) and/or the new 60-min group (glucose ≥10 mmol/L) combined were significantly more likely to have birthweight ≥90th percentile (22% vs 5%, P < 0.0001). In contradistinction, there was a significant excess number of small-for-dates babies (birthweight ≤10th percentile) in all subgroups previously diagnosed and treated for GDM by the Australian Diabetes in Pregnancy Society criteria (17% vs 7%, P = 0.001). Rates for lower uterine segment caesarean section, admission to the neonatal intensive care unit / special care nursery and Apgar scores at one and five minutes were not statistically different across all groups. CONCLUSIONS: Outcomes support the lowering of the fasting criterion to extend management of GDM to limit growth of large birthweight neonates. An unexpected outcome was that in women previously treated for GDM, there were increased numbers of low-birthweight neonates.

The importance of low level QC for high sensitivity troponin assays.

BACKGROUND: With the advent of the new high-sensitivity troponin assays, it is becoming critical to measure troponin accurately to low concentrations. To ensure assay performance is acceptable, appropriate QC must be run. METHODS: In addition to the routine use of commercial QC material, we prepared pools of human QC material with low troponin concentrations close to the limit of quantitation, and ran these regularly on our laboratory analysers. RESULTS: Over 3 years we found no drift or shift in our hs-cTnI assay. We found that only the very low concentration human QC material gave warning of precision problems with the hs-cTnI assay. At the time of the documented poor assay precision, the higher concentration QC material indicated satisfactory performance. CONCLUSIONS: Choice of QC material with an appropriate concentration is important for any assay. For hs-cTn assays, it is of particular importance to use control material with a concentration near to the limit of quantitation.

The investigation of interferences in immunoassay.

Immunoassay procedures have a wide application in clinical medicine and as such are used throughout clinical biochemistry laboratories both for urgent and routine testing. Clinicians and laboratory personnel are often presented with immunoassay results which are inconsistent with clinical findings. Without a high index of suspicion interferences will often not be suspected. Artifactual results can be due to a range of interferences in immunoassays which can include cross reacting substances, heterophile antibodies, autoantibodies and the high dose hook effect. Further, pre-analytical aspects and certain disease states can influence the potential for interference in immunoassays. Practical solutions for investigation of artifactual results in the setting of the routine clinical laboratory are provided.

The relationship of plasma creatinine (as eGFR) and high-sensitivity cardiac troponin and NT-proBNP concentrations in a hospital and community outpatient population.

OBJECTIVES: While persons with overt renal failure have a well-described rise in troponin and NT-proBNP, it is less well described what the relationship is between cardiac markers and persons with impaired renal function, not requiring dialysis. DESIGN & METHODS: We have collected ALL samples referred to our pathology practice over a 24h period and measured hs-cTnI, hs-cTnT, NT-proBNP, calculated the eGFR, and related our measurements to clinical outcomes. RESULTS: For both men and women, for all of hs-cTnI, hs-cTnT and NT-proBNP, there was a graded response, as renal function worsened, the concentration of the cardiac marker increased. CONCLUSIONS: There is a graded inverse relationship between eGFR and the concentrations of hs-cTnI, hs-cTnT and NT-proBNP. For women only there appeared to be an increase in mortality at lowest eGFR.

Statistical considerations for determining high-sensitivity cardiac troponin reference intervals.

The troponin 99th percentile is used as the laboratory decision point in the diagnosis of acute myocardial infarction. A recent publication has shown that the statistical treatment for outlier removal may dramatically change the calculated troponin 99th percentile. We have used our large database from the previously reported Canberra Heart Study to independently assess the effect of various methods for removing outliers on the calculated 99th percentile. We have performed the same exercise using the troponin 97.5th percentile as an exercise to assess how outlier removal may affect calculated upper reference intervals for any analyte which uses this boundary. For healthy males aged <75years the hs-cTnI troponin 99th percentile varied by a factor>3× depending upon the outlier removal method chosen and for the 97.5th percentile the variation was >50%. For women the variation in the hs-cTnI 99th percentile varied by a factor of nearly 2×. Qualitatively similar results were obtained forhs-cTnT. This is not simply a problem for troponin reference intervals. All analyte reference intervals have the potential to be significantly affected by the method chosen for outlier removal. To ensure that studies can be meaningfully compared, guidance on procedures for removing outliers needs to be standardized as a matter of urgency.

Using a thyroid disease-free population to define the reference interval for TSH and free T4 on the Abbott Architect analyser.

OBJECTIVE: Thyroid disease can be subtle in its presentation, and TSH reference intervals may be artefactually increased by including persons with subclinical thyroid disease. We have therefore used a thyroid disease-free population to determine TSH and fT4 reference intervals. DESIGN: Apparently healthy subjects were assessed by health questionnaire, drug history, clinical assessment and measurement of thyroid antibodies. PATIENTS: Healthy subjects in a community setting. MEASUREMENTS: TSH, free T4, antithyroglobulin and anti-TPO were measured on the Abbott Architect analyser. Subjects with clinical abnormalities, consumption of thyroid-active medications or with thyroid antibodies above the manufacturer-quoted reference intervals were excluded. TSH and fT4 data were log-transformed, and the central 95% was used to calculate reference intervals. We assessed whether these data were normally distributed. We compared samples spanning the reference intervals for both TSH and fT4 between different assays looking at biases. RESULTS: From a population of 1,606 subjects, 140 males (18%) and 284 females (34%) were excluded. The central population 95% for TSH was 0·43-3·28 mU/l and for fT4 10·8-16·8 pmol/l. There were no age- or sex-related differences. For both analytes, the distribution was not significantly different to a Gaussian distribution (P > 0·05). For 5 commonly used assays for TSH, the maximum difference in the upper limit of the TSH reference interval was 0·48 mU/l and for fT4 the maximum difference for the upper reference limit was 4·1 pmol/l. CONCLUSIONS: A substantial proportion of apparently healthy persons have subclinical thyroid disease. These subjects must be excluded for any thyroid hormone reference interval studies.

Cross-sectional study of high-sensitivity cardiac troponins T and I in a hospital and community outpatient setting.

OBJECTIVES: Cardiac troponins are specific for the heart, but not for the acute coronary syndrome. We wanted to assess how common elevated cardiac troponin concentrations were, in a population with significant non-cardiac disease. DESIGN & METHODS: We measured both hs-cTnT and hs-cTnI on all samples submitted to the laboratory during one 24h period, and assessed the magnitude of the cTn concentration with the location and severity of disease of the patient. RESULTS: Community patients and patients from the maternity ward had the lowest cTn concentrations with results above the 99th percentile being only 0-2% of the total. As expected, the highest proportion of results >99th percentile came from Coronary Care and Intensive Care. However, substantial numbers of persons on Medical and Surgical wards, without a primary diagnosis of cardiac disease, also had cTn >99th percentile. Particularly for cTnT, there was a highly significant odds ratio predicting mortality when results above and below the 99th percentile were compared. CONCLUSIONS: Significant illnesses apart from the acute coronary syndrome are important causes of a rise in cTn to above the 99th percentile, and appear to reflect the total body burden of disease. Even when the high hs-cTn concentration is not due to the acute coronary syndrome, there is a significant association with all-cause mortality.

Harmonising Reference Intervals for Three Calculated Parameters used in Clinical Chemistry.

For more than a decade there has been a global effort to harmonise all phases of the testing process, with particular emphasis on the most frequently utilised measurands. In addition, it is recognised that calculated parameters derived from these measurands should also be a target for harmonisation. Using data from the Aussie Normals study we report reference intervals for three calculated parameters: serum osmolality, serum anion gap and albumin-adjusted serum calcium. The Aussie Normals study was an a priori study that analysed samples from 1856 healthy volunteers. The nine analytes used for the calculations in this study were measured on Abbott Architect analysers. The data demonstrated normal (Gaussian) distributions for the albumin-adjusted serum calcium, the anion gap (using potassium in the calculation) and the calculated serum osmolality (using both the Bhagat et al. and Smithline and Gardner formulae). To assess the suitability of these reference intervals for use as harmonised reference intervals, we reviewed data from the Royal College of Pathologists of Australasia/Australasian Association of Clinical Biochemists (RCPA/AACB) bias survey. We conclude that the reference intervals for the calculated serum osmolality (using the Smithline and Gardner formulae) may be suitable for use as a common reference interval. Although a common reference interval for albumin-adjusted serum calcium may be possible, further investigations (including a greater range of albumin concentrations) are needed. This is due to the bias between the Bromocresol Green (BCG) and Bromocresol Purple (BCP) methods at lower serum albumin concentrations. Problems with the measurement of Total CO2 in the bias survey meant that we could not use the data for assessing the suitability of a common reference interval for the anion gap. Further study is required.