William A. Bartlett

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.

Reflex and reflective testing: efficiency and effectiveness of adding on laboratory tests.

Background Laboratory investigations may be added to existing requests either automatically on the basis of algorithms (reflex testing) or by laboratory professionals (reflective testing). The clinical utility of reflex and reflective testing is not fully established. We studied efficiency (number of tests that needs to be added to make a diagnosis) and effectiveness (number of diagnoses) of reflex and reflective testing in selected biochemical scenarios. Methods Using fixed rules, we prospectively measured efficiency and effectiveness of reflex and reflective testing in the following scenarios (reflex initiators in parentheses): (1) hypovitaminosis D (hypocalcaemia plus elevated alkaline phosphatase activity); (2) hypomagnesaemia (hypokalaemia or hypocalcaemia); (3) hypothyroidism (high thyroid-stimulating hormone [TSH]); (4) hyperthyroidism (low TSH); (5) haemochromatosis (reflex or reflective addition of iron studies, followed by reflective addition of genetic studies). Separately, using a different data-set, we examined the impact of varying TSH thresholds on outcomes in the biochemical diagnosis of hyper- and hypothyroidism. Results In patients aged over 55 y, 25-hydroxy-vitamin D <50 nmol/L could be predicted with ≥90% certainty when albumin-adjusted calcium was ≤2.1 mmol/L plus alkaline phosphatase >150 U/L. Higher numbers of tests were needed to make a diagnosis in other scenarios. In general, more diagnoses were made by reflex testing. Outside the euthyroid TSH range, efficiency of diagnosis of hyper- and hypothyroidism became asymptotic, while effectiveness declined. Conclusions Near-maximal efficiency of reflex testing can be achieved, depending on the reflex and diagnostic thresholds applied. Reflective and reflex testing are complementary activities, the clinical utility of which depends on the initiators used.

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.

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 Estimates Obtained from 91 Healthy Study Participants for 9 Enzymes in Serum

BACKGROUND We sought to develop estimates of biological variation (BV) for 9 enzymes in blood serum as part of the European Biological Variation Study. METHODS Ninety-one healthy study participants (38 male and 53 female, 21-69 years old) were phlebotomized in each of 10 consecutive weeks at 6 European laboratories. The same preanalytical sample-handling protocol was followed at each center before transport to San Raffaele Hospital, Milan, Italy, for analysis. Sera were stored at -80 °C before analysis in duplicate within a single run on an ADVIA 2400 Clinical Chemistry System (Siemens Healthcare) following a protocol designed to minimize analytical imprecision. Assay traceability was established using frozen sera with target values assigned by reference methods. The results were subjected to outlier analysis before CV-ANOVA to deliver valid BV estimates. Results for 9 enzymes were subsequently partitioned for graphical display allowing visual assessment of the effects of country of origin, sex, and age on BV estimates. RESULTS We found no effect of country upon the observed variation, but overall sex-related differences were evident for alanine amino transferase (ALT), γ-glutamyl transferase (GGT), and creatine kinase (CK). The following estimates for within-subject BV (CVI) and between-subject BV (CVG), respectively, were obtained: ALT: 9.3%, 28.2%; aspartate aminotransferase: 9.5%, 20.3%; GGT: 8.9%, 41.7%; alkaline phosphatase : 5.3%, 24.9%; lactate dehydrogenase: 5.2%, 12.6%; CK: 14.5%, 31.5%; amylase: 6.8%, 30.4%; pancreatic α-amylase: 6.3%, 24.9%; and lipase (LIP): 7.7%, 23.8%. CONCLUSIONS All CVI and some CVG estimates were lower than those reported in the online BV 2014 updated database. Analytical performance specifications derived from BV can be applied internationally.

The EuBIVAS Project: Within–and Between-Subject Biological Variation Data for Serum Creatinine Using Enzymatic and Alkaline Picrate Methods and Implications for Monitoring

BACKGROUND The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) European Biological Variation Study (EuBIVAS) has been established to deliver rigorously determined biological variation (BV) indices. EuBIVAS determined BV for serum creatinine using the enzymatic and alkaline picrate measurement methods. METHOD In total, 91 healthy individuals (38 males, 53 females; age range, 21-69 years) were bled for 10 consecutive weeks at 6 European laboratories. An equivalent protocol was followed at each center. Sera were stored at -80 °C before analysis. Analyses for each patient were performed in duplicate within a single run on an ADVIA 2400 system (San Raffaele Hospital, Milan). The data were subjected to outlier and homogeneity analysis before performing CV-ANOVA to determine BV and analytical variation (CVA) estimates with confidence intervals (CI). RESULTS The within-subject BV estimates [CVI (95% CI)] were similar for enzymatic [4.4% (4.2-4.7)] and alkaline picrate [4.7% (4.4-4.9)] methods and lower than the estimate presently available online (CVI = 5.9%). No significant male/female BV differences were found. Significant differences were observed in mean creatinine values between men and women and between Turkish individuals and those of other nationalities. Between-subject BV (CVG) estimates, stratified accordingly, produced CVG values similar to historical BV data. CVA was 1.1% for the enzymatic and 4.4% for alkaline picrate methods, indicating that alkaline picrate methods fail to fulfill analytical performance specifications for imprecision (CVAPS). CONCLUSIONS The serum creatinine CVI obtained by EuBIVAS specifies a more stringent CVAPS than previously identified. The alkaline picrate method failed to meet this CVAPS, raising questions regarding its future use.

Association between the cholesteryl ester transfer protein TaqI-detectable B polymorphism and low high-density lipoprotein cholesterol concentration in Saudis.

Plasma concentrations of HDL (high-density lipoprotein) cholesterol are low in the Saudi Arabian population. A B polymorphism at the CETP (cholesteryl ester protein transfer) locus that is detectable with the restriction enzyme Taq I is a genetic determinant of the plasma HDL cholesterol concentration. We assessed the relationship between the Taq I B CETP polymorphism and lipid and apolipoprotein concentrations in a study sample of 335 Saudi residents. The Taq I B1 and B2 allele frequencies were 0.54 and 0.46 respectively, similar to those in other populations. HDL cholesterol levels in B2B2 homozygotes were significantly higher than in B1B1 homozygotes [1.01 (0.3) compared with 0.92 (0.2) mmol/l; mean (S.D.); P=0.03]. There was also a significant difference between the B2B2 and B1B1 homozygotes with regard to apolipoprotein AI concentration [123.6 (16.4) compared with 113.7 (13.9) mg/dl; P=0.04]. This genetic variation was independent of metabolic risk factors known to influence HDL cholesterol levels. The allele frequency of the Taq I B CETP polymorphism and its relatively modest impact on HDL cholesterol concentrations argue against an important role for this allele, or for strongly linked loci, in determining the low levels of HDL cholesterol seen in the Saudi population.

The Biological Variation Data Critical Appraisal Checklist: A Standard for Evaluating Studies on Biological Variation

BACKGROUND Concern has been raised about the quality of available biological variation (BV) estimates and the effect of their application in clinical practice. A European Federation of Clinical Chemistry and Laboratory Medicine Task and Finish Group has addressed this issue. The aim of this report is to (a) describe the Biological Variation Data Critical Appraisal Checklist (BIVAC), which verifies whether publications have included all essential elements that may impact the veracity of associated BV estimates, (b) use the BIVAC to critically appraise existing BV publications on enzymes, lipids, kidney, and diabetes-related measurands, and (c) apply metaanalysis to deliver a global within-subject BV (CVI) estimate for alanine aminotransferase (ALT). METHODS In the BIVAC, publications were rated as A, B, C, or D, indicating descending compliance for 14 BIVAC quality items, focusing on study design, methodology, and statistical handling. A D grade indicated that associated BV estimates should not be applied in clinical practice. Systematic searches were applied to identify BV studies for 28 different measurands. RESULTS In total, 128 publications were identified, providing 935 different BV estimates. Nine percent achieved D scores. Outlier analysis and variance homogeneity testing were scored as C in >60% of 847 cases. Metaanalysis delivered a CVI estimate for ALT of 15.4%. CONCLUSIONS Application of BIVAC to BV publications identified deficiencies in required study detail and delivery, especially for statistical analysis. Those deficiencies impact the veracity of BV estimates. BV data from BIVAC-compliant studies can be combined to deliver robust global estimates for safe clinical application.

Biological variation: a still evolving facet of laboratory medicine

Numerical data on the components of random biological variation (BV) have been generated for over 40 years. These have been used for a variety of purposes in laboratory medicine, including setting analytical quality specifications, assessing the significance of the difference seen in serial results from an individual and investigating the utility of conventional populationbased reference values. The generation of such data is not easy. However, applications are facilitated through the availability of databases which give single figures for the components of BV, namely, within-subject (CVI) and between-subject (CVG). Generation of a comprehensive database of components of BV was initiated in 1997 by the Analytical Quality Commission (AQC) of the Spanish Society of Clinical Chemistry (SEQC). A scoring system was designed to evaluate the robustness of data, and this has five essential criteria for inclusion: papers must be designed to examine BV, the performance index (PI1⁄4CVA/0.5CVI) must be <2.0, where CVA is the analytical component of variation, the components must be derived by ANOVA or another recommended calculation method, the data must be from apparently healthy individuals and the median estimates of CVI and CVG must be documented. The structure of the on-line database and the criteria used for generation and update have been recently described. The database provides analytical quality specifications for imprecision, bias and total allowable error, at minimum, desirable and optimum levels of quality. It contains data on 358 measurands from 247 papers as single numbers for CVI and CVG. Where multiple studies exist for a single measurand, the incorporated numbers represent median values. The database has been updated every two years and is currently in its eighth edition. Recently, a number of demerits have become apparent. For example, for 202 measurands, the data appear in a single publication, 129 have data in two to nine publications and 27 in 510 publications. Interestingly, 55% of the measurands in the first group had the maximum scores for both the PI and the statistical methods used, which suggests that these estimates of BV are reliable: similar data exist for the other two groups. It is dogma that, in general, estimates of CVI are similar over time span of study, number of samples, health and disease and other factors. However, considerable heterogeneity of the CVI estimates across studies is a major weakness of the database. Carobene et al. showed that published BV data for alanine aminotransferase, aspartate aminotransferase and gamma glutamyl transferase demonstrated a wide range of values derived from inconsistent protocols. The quality of the presentations of the data was variable. These findings raise further concerns around the utility of the data currently available and highlight the need for critical appraisal of publications on BV. The Working Group on BV (WG-BV) of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has presented these concerns at the 1st EFLM Strategic Conference on Defining Analytical Performance Goals. The many insightful presentations are available on the Internet.

Development and validation of diagnostic triage criteria for liver disease from a minimum data set enabling the ‘intelligent LFT’ pathway for the automated assessment of deranged liver enzymes

Background Liver function tests (LFTs) are commonly abnormal; most patients with ‘incidental’ abnormal LFTs are not investigated appropriately and for those who are, current care pathways are geared to find an explanation for the abnormality by a lengthy process of investigation and exclusion, with costs to the patient and to the health service. Objective To validate an intelligent automatable analysis tool (iLFT) for abnormal liver enzymes, which diagnoses common liver conditions, provides fibrosis stage and recommends management Design A retrospective case note review from three tertiary referral liver centres, with application of the iLFT algorithm and comparison with the clinician’s final opinion as gold standard. Results The iLFT algorithm in 91.3% of cases would have correctly recommended referral or management in primary care. In the majority of the rest of the cases, iLFT failed safe and recommended referral even when the final clinical diagnosis could have been managed in primary care. Diagnostic accuracy was achieved in 82.4% of cases, consistent with the fail-safe design of the algorithm. Two cases would have remained in primary care as per the algorithm outcome, however on clinical review had features of advanced fibrosis. Conclusion iLFT analysis of abnormal liver enzymes offers a safe and robust method of risk stratifying patients to the most appropriate care pathway as well as providing reliable diagnostic information based on a single blood draw, without repeated contacts with health services. Offers the possibility of high quality investigation and diagnosis to all patients rather than a tiny minority.