Thomas Røraas

Confidence Intervals and Power Calculations for Within-Person Biological Variation: Effect of Analytical Imprecision, Number of Replicates, Number of Samples, and Number of Individuals

BACKGROUND Reliable estimates of within-person biological variation and reference change value are of great importance when interpreting test results, monitoring patients, and setting quality specifications. Little information has been published regarding what experimental design is optimal to achieve the best estimates of within-person biological variation. METHOD Expected CIs were calculated for different balanced designs for a 2-level nested variance analysis model with varying analytical imprecision. We also simulated data sets based on the model to calculate the power of different study designs for detection of within-person biological variation. RESULTS The reliability of an estimate for biological variation and a studys power is very much influenced by the study design and by the ratio between analytical imprecision and within-person biological variation. For a fixed number of measurements, it is preferable to have a high number of samples from each individual. Shortcomings in analytical imprecision can be controlled by increasing the number of replicates. CONCLUSIONS The design of an experiment to estimate biological variation should take into account the analytical imprecision of the method and focus on obtaining the highest possible reliability. Estimates of biological variation should always be reported with CIs.

A checklist for critical appraisal of studies of biological variation

Abstract Data on biological variation are used for many purposes in laboratory medicine but concern exists over the validity of the data reported in some studies. A critical appraisal checklist has been produced by a working group established by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) to enable standardised assessment of existing and future publications of biological variation data. The checklist identifies key elements to be reported in studies to enable safe accurate and effective transport of biological variation data sets across healthcare systems. The checklist is mapped to the domains of a minimum data set required to enable this process.

A systematic review of data on biological variation for alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase

Abstract Background: Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT) are enzymes measured in serum or plasma to investigate liver disease. The aim of this work is to assess the validity of published biological variation (BV) data currently available for these enzymes. Methods: Publications containing BV data for ALT, AST and GGT were identified by searching PubMed using the following keywords: biological varia*, RCV, CVw, CVi, CVb, and CVg. The 95% confidence intervals for the within- and between-subject coefficients of variation were calculated using the analytical imprecision, the number of subjects, samples and replicates. Results: The searches identified 10 publications with ALT, 14 with AST and nine with GGT data. The protocols presented in those publications as used were varied. The ranges of within-subject variation reported were: ALT: 11.1%–58.1%, AST: 3.0%–32.3% and for GGT: 3.9%–14.5%. The median values (ALT: 18.0%, AST: 11.9% and GGT: 13.8%) were similar to those listed in a BV database commonly used as a reference source. Conclusions: Published BV data for ALT, AST and GGT demonstrate a wide range of values derived from inconsistent protocols. The quality of the presentations of the data is variable. These findings raise concerns around the utility of the data currently available and highlight the need for critical appraisal of such publications. The working group on BV of the European Federation of Clinical Chemistry and Laboratory Medicine is undertaking work to develop a critical appraisal checklist for the production and publication of reliable BV data.

Weekly and 90-Minute Biological Variations in Cardiac Troponin T and Cardiac Troponin I in Hemodialysis Patients and Healthy Controls

BACKGROUND Myocardial infarction (MI) is diagnosed by the finding of a single cardiac troponin value above the 99th percentile and a significant time-dependent change in cardiac troponin concentration. The aim of this study was to determine the 90-min and weekly biological variations, the reference change value (RCV), and the index of individuality (II) of high-sensitivity cardiac troponin T (hs-cTnT) (Roche Diagnostics) and hs-cTnI (Abbott Diagnostics) in patients receiving hemodialysis (HD) and in healthy individuals. METHOD Blood samples were collected from 19 HD patients (on an HD-free day) and 20 healthy individuals at 90-min intervals over a 6-h period (between 08:30 and 14:30) and before the midweek HD treatment for 10 weeks. The within-person variation (CVi), between-person variation, RCV, and II were calculated. RESULTS During the 6-h sampling period, the concentrations of hs-cTnT (both groups) and hs-cTnI (HD patients only) decreased on average by 0.8% to 1.7% per hour, respectively. These declining trends were included in the calculation of a 90-min asymmetric RCV: -8%/+5% in HD patients (hs-cTnT), -18%/+21% in HD patients (hs-cTnI), -27%/+29% in healthy individuals (hs-cTnT), and -39%/+64% in healthy individuals (hs-cTnI). The II was low in both groups for both assays. The weekly CVi values were approximately 8% (hs-cTnT) and 15% (hs-cTnI) in both groups. CONCLUSIONS When using a cardiac troponin change of 20%-50% to diagnose an MI, the false-positive rate is likely to be lower for the hs-cTnT assay than for the hs-cTnI assay. The low II suggests that use of a diagnostic cutoff value can be omitted.

Diagnosing Diabetes Mellitus: Performance of Hemoglobin A1c Point-of-Care Instruments in General Practice Offices

BACKGROUND Hemoglobin A1c (Hb A1c) measurement by hospital laboratory instruments, but not by point-of-care (POC) instruments, has been recommended for use to diagnose diabetes mellitus. We evaluated results from 13 Hb A1c external quality assurance (EQA) surveys over a 6-year period in Norway, from both POC instruments used in general practice (GP) offices and instruments in hospital laboratories, against the analytical quality specifications recommended for use of Hb A1c to diagnose diabetes mellitus. METHODS All GP offices (n = 1288) and hospital laboratories (n = 52) measuring Hb A1c in Norway participated in the EQA survey. The percentage of participants that performed measurements within the quality specifications was calculated. Pooled within-laboratory CVs were estimated for the Afinion, DCA 2000, DCA 2000+, DCA Vantage(TM), and Nycocard Hb A1c Reader instruments and for hospital laboratory instruments. RESULTS Between 60% to 90% of Afinion and DCA users and hospital laboratories performed Hb A1c measurements within the quality specifications for both trueness (6.0%) and imprecision (CV ≤2.0%) at 2 levels in each EQA survey. The pooled within-laboratory CVs for the Afinion and DCA instruments and hospital laboratories were below the recommended limit of 2.0% for most of the surveys. CONCLUSIONS A large proportion of GP offices using Afinion and DCA POC instruments to measure Hb A1c fulfill the analytical quality specifications for diagnosing diabetes mellitus, and these instruments demonstrate analytical quality comparable to that of hospital laboratory instruments. When GP offices participate in a stringent quality assurance program and generate Hb A1c measurements that meet analytical quality specifications, these measurements can be recommended for use to diagnose diabetes mellitus.

Biological Variation: The Effect of Different Distributions on Estimated Within-Person Variation and Reference Change Values

BACKGROUND Good estimates of within-person biological variation, CVI, are essential for diagnosing and monitoring patients and for setting analytical performance specifications. The aim of the present study was to use computer simulations to evaluate the impact of various measurement distributions on different methods for estimating CVI and reference change value (RCV). METHOD Data were simulated on the basis of 3 models for distributions of the within-person effect. We evaluated 3 different methods for estimating CVI: standard ANOVA, ln-ANOVA, and CV-ANOVA, and 3 different methods for calculating RCV: classic, ln-RCV, and a nonparametric method. We estimated CVI and RCV with the different methods and compared the results with the true values. RESULTS The performance of the methods varied, depending on both the size of the CVI and the type of distributions. The CV-ANOVA model performed well for the estimation of CVI with all simulated data. The ln-RCV method performed best if data were ln-normal distributed or CVI was less than approximately 12%. The nonparametric RCV method performed well for all simulated data but was less precise. CONCLUSIONS The CV-ANOVA model is recommended for both calculation of CVI and the step-by-step approach of checking for outliers and homogeneity in replicates and samples. The standard method for calculation of RCV should not be used when using CVs.

Biological variation - reliable data is essential

Biological variation (BV) data is a cornerstone in the interpretation of laboratory test results, being the basis for many of the decisions we make every day both in the laboratory and in clinical practise. Among the many applications is its use in diagnosis and monitoring of disease. Most typically this occurs when comparing a person’s level of the analyte of interest against a reference interval, based on the between-subject variation, CV G , or when comparing a change against the reference change value, based on the within-subject variation, CV I . Furthermore, BV data is probably the most commonly used approach for setting analytical quality specifications for bias, imprecision and total error for many laboratory constituents. General assumptions for the uses of BV data are that estimates are reliable, i.e., adequately collected and calculated, and that the estimates are representative for the specific population and setting for which they will be applied.

Sample collections from healthy volunteers for biological variation estimates' update: a new project undertaken by the Working Group on Biological Variation established by the European Federation of Clinical Chemistry and Laboratory Medicine.

Abstract Background: Biological variation (BV) data have many fundamental applications in laboratory medicine. At the 1st Strategic Conference of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) the reliability and limitations of current BV data were discussed. The EFLM Working Group on Biological Variation is working to increase the quality of BV data by developing a European project to establish a biobank of samples from healthy subjects to be used to produce high quality BV data. Methods: The project involved six European laboratories (Milan, Italy; Bergen, Norway; Madrid, Spain; Padua, Italy; Istanbul, Turkey; Assen, The Netherlands). Blood samples were collected from 97 volunteers (44 men, aged 20–60 years; 43 women, aged 20–50 years; 10 women, aged 55–69 years). Initial subject inclusion required that participants completed an enrolment questionnaire to verify their health status. The volunteers provided blood specimens once per week for 10 weeks. A short questionnaire was completed and some laboratory tests were performed at each sampling consisting of blood collected under controlled conditions to provide serum, K2EDTA-plasma and citrated-plasma samples. Results: Samples from six out of the 97 enroled subjects were discarded as a consequence of abnormal laboratory measurements. A biobank of 18,000 aliquots was established consisting of 120 aliquots of serum, 40 of EDTA-plasma, and 40 of citrated-plasma from each subject. The samples were stored at –80 °C. Conclusions: A biobank of well-characterised samples collected under controlled conditions has been established delivering a European resource to enable production of contemporary BV data.

Biological Variation of Hemoglobin A1c: Consequences for Diagnosing Diabetes Mellitus

To the Editor: For optimal monitoring and diagnosing of patients with diabetes by use of glycated hemoglobin (Hb A1c)1 measurements, the analytical CV (CVa) of the Hb A1c assay and the within-person biological variation (CVwp) are of relevance. CVwp refers to an inherent biological variation around an individual patients set point of a biological parameter. Several studies have been published assessing the CVwp of Hb A1c (1–3). However, none of those studies used >1 Hb A1c method to determine the CVwp. The aim of this study was to apply 4 different IFCC and National Glycohemoglobin Standardization Program (NGSP) certified secondary reference measurement procedures (SRMPs) using different assay principles and calibrated in SI units (mmol/mol) and Diabetes Control and Complications Trial (DCCT) units (% Hb A1c) to see whether there were differences in the CVwp obtained. In addition, we addressed the consequences for diagnosing diabetes mellitus of the CVwp found. We recruited 21 presumed healthy hospital employees to participate in the study (11 men and 10 women). Five K2EDTA-treated whole blood samples were collected from each individual every 2 weeks for 2 months. On collection, aliquots of each sample were immediately stored at −80 °C. Full analysis was performed at the end of the 2-month collection period. The samples were analyzed in a single run in duplicate using the following 4 SRMPs:

Calcium, Magnesium, Albumin, and Total Protein Measurement in Serum as Assessed with 20 Fresh-Frozen Single-Donation Sera

To the Editor: The scope of external quality assessment (EQA)1 in laboratory medicine has evolved considerably (1). With the increasing worldwide interest in the use of common reference intervals and/or medical-decision limits, modern EQA schemes need to be better at assessing the standardization status of commercial in vitro diagnostic tests. This need has led to new challenges in the design of EQA surveys. We report the outcomes of a Norwegian pilot study that investigated the use of commutable, fresh-frozen single donations to assess the current standardization status as part of an initiative toward producing common reference intervals (2). The study covered measurements of calcium, magnesium, albumin, and total protein in serum from 20 single-donation blood samples obtained from Solomon Park Research Laboratories. Serum was generated according to CLSI protocol C37-A, with 2 U human thrombin (Sigma-Aldrich) added per milliliter of plasma to facilitate clotting (3); filtration was not used. Aliquots of these samples were sent to laboratories that used the same test systems (instrument, reagent, and calibrator from the same source). Five peer groups (n ≥ 6 laboratories each; N = 47) were considered: Abbott Diagnostics ARCHITECT, Ortho Clinical Diagnostics VITROS, …