The biological variation of insulin resistance markers: data from the European Biological Variation Study (EuBIVAS).

OBJECTIVES: An insulin resistant state is characteristic of patients with type 2 diabetes, polycystic ovary syndrome, and metabolic syndrome. Identification of insulin resistance (IR) is most readily achievable using formulae combining plasma insulin and glucose results. In this study, we have used data from the European Biological Variation Study (EuBIVAS) to examine the biological variability (BV) of IR using the Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) and the Quantitative Insulin sensitivity Check Index (QUICKI). METHODS: Ninety EuBIVAS non-diabetic subjects (52F, 38M) from five countries had fasting HOMA-IR and QUICKI calculated from plasma glucose and insulin samples collected concurrently on 10 weekly occasions. The within-subject (CVI) and between-subject (CVG) BV estimates with 95 % CIs were obtained by CV-ANOVA after analysis of trends, variance homogeneity and outlier removal. RESULTS: The CVI of HOMA-IR was 26.7 % (95 % CI 25.5-28.3), driven largely by variability in plasma insulin and the CVI for QUICKI was 4.1 % (95 % CI 3.9-4.3), reflecting this formula's logarithmic transformation of glucose and insulin values. No differences in values or BV components were observed between subgroups of men or women below and above 50 years. CONCLUSIONS: The EuBIVAS, by utilising a rigorous experimental protocol, has produced robust BV estimates for two of the most commonly used markers of insulin resistance in non-diabetic subjects. This has shown that HOMA-IR, in particular, is highly variable in the same individual which limits the value of single measurements.

A standard to report biological variation data studies - based on an expert opinion.

There is a need for standards for generation and reporting of Biological Variation (BV) reference data. The absence of standards affects the quality and transportability of BV data, compromising important clinical applications. To address this issue, international expert groups under the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) have developed an online resource (https://tinyurl.com/bvmindmap) in the form of an interactive mind map that serves as a guideline for researchers planning, performing and reporting BV studies. The mind map addresses study design, data analysis, and reporting criteria, providing embedded links to relevant references and resources. It also incorporates a checklist approach, identifying a Minimum Data Set (MDS) to enable the transportability of BV data and incorporates the Biological Variation Data Critical Appraisal Checklist (BIVAC) to assess study quality. The mind map is open to access and is disseminated through the EFLM BV Database website, promoting accessibility and compliance to a reporting standard, thereby providing a tool to be used to ensure data quality, consistency, and comparability of BV data. Thus, comparable to the STARD initiative for diagnostic accuracy studies, the mind map introduces a Standard for Reporting Biological Variation Data Studies (STARBIV), which can enhance the reporting quality of BV studies, foster user confidence, provide better decision support, and be used as a tool for critical appraisal. Ongoing refinement is expected to adapt to emerging methodologies, ensuring a positive trajectory toward improving the validity and applicability of BV data in clinical practice.

Practical recommendations for biochemical and genetic diagnosis of the porphyrias.

The porphyrias are a group of rare inborn errors of metabolism associated with various clinical presentations and long-term complications, making them relevant differential diagnoses to consider for many clinical specialities, especially hepatologists, gastroenterologists and dermatologists. To diagnose a patient with porphyria requires appropriate biochemical investigations, as clinical features alone are not specific enough. Furthermore, it is important to be aware that abnormalities of porphyrin accumulation and excretion occur in many other disorders that are collectively far more common than the porphyrias. In this review, we provide an overview of porphyria-related tests with their strengths and limitations, give recommendations on requesting and diagnostic approaches in non-expert and expert laboratories for different clinical scenarios and discuss the role of genetic testing in the porphyrias. To diagnose porphyria in a currently symptomatic patient requires analysis of biochemical markers to demonstrate typical patterns of haem precursors in urine, faeces and blood. The use of genomic sequencing in diagnostic pathways for porphyrias requires careful consideration, and the demonstration of increased porphyrin-related markers is necessary prior to genomic testing in symptomatic patients. In the acute porphyrias, genomic testing is presently a useful adjunct for genetic counselling of asymptomatic family members and the most common cutaneous porphyria, porphyria cutanea tarda, is usually a sporadic, non-hereditary disease. Getting a correct and timely porphyria diagnosis is essential for delivering appropriate care and ensuring best patient outcome.

A New Concept for Reference Change Values-Regression to the Population Mean.

BACKGROUND: Reference change values (RCV) are used to indicate a change in analyte concentration that is unlikely to be due to random variation in the patient or the measurement. Current theory describes RCV relative to a first measurement result (X1). We investigate an alternative view predicting the starting point for RCV calculations from X1 and its location in the reference interval. METHODS: Data for serum sodium, calcium, and total protein from the European Biological Variation study and from routine clinical collections were analyzed for the effect of the position of X1 within the reference interval on the following result from the same patient. A model to describe the effect was determined, and an equation to predict the RCV for a sample in a population was developed. RESULTS: For all data sets, the midpoints of the RCVs were dependent on the position of X1 in the population. Values for X1 below the population mean were more likely to be followed by a higher result, and X1 results above the mean were more likely to be followed by lower results. A model using population mean, reference interval dispersion, and result diagnostic variation provided a good fit with the data sets, and the derived equation predicted the changes seen. CONCLUSIONS: We have demonstrated that the position of X1 within the reference interval creates an asymmetrical RCV. This can be described as a regression to the population mean. Adding this concept to the theory of RCVs will be an important consideration in many cases.

Biological Variation Data in Triathletes for Metabolism and Growth-Related Biomarkers Included in the Athlete Biological Passport.

BACKGROUND: When using biological variation (BV) data, BV estimates need to be robust and representative. High-endurance athletes represent a population under special physiological conditions, which could influence BV estimates. Our study aimed to estimate BV in athletes for metabolism and growth-related biomarkers involved in the Athlete Biological Passport (ABP), by 2 different statistical models. METHODS: Thirty triathletes were sampled monthly for 11 months. The samples were analyzed for human growth hormone (hGH), insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein 3 (IGFBP-3), insulin, and N-terminal propeptide of type III procollagen (P-III-NP) by immunoassay. Bayesian and ANOVA methods were applied to estimate within-subject (CVI) and between-subject BV. RESULTS: CVI estimates ranged from 7.8% for IGFBP-3 to 27.0% for insulin, when derived by the Bayesian method. The 2 models gave similar results, except for P-III-NP. Data were heterogeneously distributed for P-III-NP for the overall population and in females for IGF-1 and IGFBP-3. BV components were not estimated for hGH due to lack of steady state. The index of individuality was below 0.6 for all measurands, except for insulin. CONCLUSIONS: In an athlete population, to apply a common CVI for insulin would be appropriate, but for IGF-1 and IGFBP-3 gender-specific estimates should be applied. P-III-NP data were heterogeneously distributed and using a mean CVI may not be representative for the population. The high degree of individuality for IGF-1, IGFBP-3, and P-III-NP makes them good candidates to be interpreted through reference change values and the ABP.

Analytical performance specifications based on biological variation data - considerations, strengths and limitations.

Analytical performance specifications (APS) are typically established through one of three models: (i) outcome studies, (ii) biological variation (BV), or (iii) state-of-the-art. Presently, The APS can, for most measurands that have a stable concentration, be based on BV. BV based APS, defined for imprecision, bias, total allowable error and allowable measurement uncertainty, are applied to many different processes in the laboratory. When calculating APS, it is important to consider the different APS formulae, for what setting they are to be applied and if they are suitable for the intended purpose. In this opinion paper, we elucidate the background, limitations, strengths, and potential intended applications of the different BV based APS formulas. When using BV data to set APS, it is important to consider that all formulae are contingent on accurate and relevant BV estimates. During the last decade, efficient procedures have been established to obtain reliable BV estimates that are presented in the EFLM biological variation database. The database publishes detailed BV data for numerous measurands, global BV estimates derived from meta-analysis of quality-assured studies of similar study design and automatic calculation of BV based APS.

The European biological variation study (EuBIVAS): Biological variation data for testosterone, follicle stimulating hormone, prolactin, luteinizing hormone and dehydroepiandrosterone sulfate in men.

BACKGROUND: Knowledge of biological variation (BV) of hormones is essential for interpretation of laboratory tests and for diagnostics of endocrinological and reproductive diseases. There is a lack of robust BV data for many hormones in men. METHODS: We used serum samples collected weekly over 10 weeks from the European Biological Variation Study (EuBIVAS) to determine BV of testosterone, follicle-stimulating hormone (FSH), prolactin, luteinizing hormone (LH) and dehydroepiandrosterone sulfate (DHEA-S) in 38 men. We derived within-subject (CVI) and between-subject (CVG) BV estimates by CV-ANOVA after trend, outlier, and homogeneity analysis and calculated reference change values, index of individuality (II), and analytical performance specifications. RESULTS: The CVI estimates were 10 % for testosterone, 8 % for FSH, 13 % for prolactin, 22 % for LH, and 9 % for DHEA-S, respectively. The IIs ranged between 0.14 for FSH to 0.66 for LH, indicating high individuality. CONCLUSIONS: In this study, we have used samples from the highly powered EuBIVAS study to derive BV estimates for testosterone, FSH, prolactin, LH and DHEA-S in men. Our data confirm previously published BV estimates of testosterone, FSH and LH. For prolactin and DHEA-S BV data for men are reported for the first time.

Biological variation of inflammatory and iron metabolism markers in high-endurance recreational athletes; are these markers useful for athlete monitoring?

OBJECTIVES: To deliver biological variation (BV) data for serum hepcidin, soluble transferrin receptor (sTfR), erythropoietin (EPO) and interleukin 6 (IL-6) in a population of well-characterized high-endurance athletes, and to evaluate the potential influence of exercise and health-related factors on the BV. METHODS: Thirty triathletes (15 females) were sampled monthly (11 months). All samples were analyzed in duplicate and BV estimates were delivered by Bayesian and ANOVA methods. A linear mixed model was applied to study the effect of factors related to exercise, health, and sampling intervals on the BV estimates. RESULTS: Within-subject BV estimates (CVI) were for hepcidin 51.9 % (95 % credibility interval 46.9-58.1), sTfR 10.3 % (8.8-12) and EPO 27.3 % (24.8-30.3). The mean concentrations were significantly different between sex, but CVI estimates were similar and not influenced by exercise, health-related factors, or sampling intervals. The data were homogeneously distributed for EPO but not for hepcidin or sTfR. IL-6 results were mostly below the limit of detection. Factors related to exercise, health, and sampling intervals did not influence the BV estimates. CONCLUSIONS: This study provides, for the first time, BV data for EPO, derived from a cohort of well-characterized endurance athletes and indicates that EPO is a good candidate for athlete follow-up. The application of the Bayesian method to deliver BV data illustrates that for hepcidin and sTfR, BV data are heterogeneously distributed and using a mean BV estimate may not be appropriate when using BV data for laboratory and clinical applications.

Biological variation estimates of Alzheimer's disease plasma biomarkers in healthy individuals.

INTRODUCTION: Blood biomarkers have proven useful in Alzheimer's disease (AD) research. However, little is known about their biological variation (BV), which improves the interpretation of individual-level data. METHODS: We measured plasma amyloid beta (Aß42, Aß40), phosphorylated tau (p-tau181, p-tau217, p-tau231), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL) in plasma samples collected weekly over 10 weeks from 20 participants aged 40 to 60 years from the European Biological Variation Study. We estimated within- (CVI ) and between-subject (CVG ) BV, analytical variation, and reference change values (RCV). RESULTS: Biomarkers presented considerable variability in CVI and CVG . Aß42/Aß40 had the lowest CVI (≈ 3%) and p-tau181 the highest (≈ 16%), while others ranged from 6% to 10%. Most RCVs ranged from 20% to 30% (decrease) and 25% to 40% (increase). DISCUSSION: BV estimates for AD plasma biomarkers can potentially refine their clinical and research interpretation. RCVs might be useful for detecting significant changes between serial measurements when monitoring early disease progression or interventions. Highlights Plasma amyloid beta (Aß42/Aß40) presents the lowest between- and within-subject biological variation, but also changes the least in Alzheimer's disease (AD) patients versus controls. Plasma phosphorylated tau variants significantly vary in their within-subject biological variation, but their substantial fold-changes in AD likely limits the impact of their variability. Plasma neurofilament light chain and glial fibrillary acidic protein demonstrate high between-subject variation, the impact of which will depend on clinical context. Reference change values can potentially be useful in monitoring early disease progression and the safety/efficacy of interventions on an individual level. Serial sampling revealed that unexpectedly high values in heathy individuals can be observed, which urges caution when interpreting AD plasma biomarkers based on a single test result.

One ring closer to a closure: the crystal structure of the ES3 hydroxymethylbilane synthase intermediate.

Hydroxymethylbilane synthase (HMBS), involved in haem biosynthesis, catalyses the head-to-tail coupling of four porphobilinogens (PBGs) via a dipyrromethane (DPM) cofactor. DPM is composed of two PBGs, and a hexapyrrole is built before the tetrapyrrolic 1-hydroxymethylbilane product is released. During this elongation, stable enzyme (E) intermediates are formed from the holoenzyme, with additional PBG substrates (S): ES, ES2 , ES3 and ES4 . Native PAGE and mass spectrometry of the acute intermittent porphyria (AIP)-associated HMBS variant p.Arg167Gln demonstrated an increased amount of ES3 . Kinetic parameters indicated catalytic dysfunction, however, the product release was not entirely prevented. Isolation and crystal structure analysis of the ES3 intermediate (PDB: 8PND) showed that a pentapyrrole was fully retained within the active site, revealing that polypyrrole elongation proceeds within the active site via a third interaction site, intermediate pyrrole site 3 (IPS3). The AIP-associated HMBS variant p.Arg195Cys, located on the opposite side to p.Arg167Gln in the active site, accumulated the ES4 intermediate in the presence of excess PBG, implying that product hydrolysis was obstructed. Arg167 is thus involved in all elongation steps and is a determinant for the rate of enzyme catalysis, whereas Arg195 is important for releasing the product. Moreover, by substituting residues in the vicinity of IPS3, our results indicate that a fully retained hexapyrrole could be hydrolysed in a novel site in proximity of the IPS3.

A survey of total IgE reference intervals reported by Scandinavian and British medical laboratories - a need for harmonisation.

OBJECTIVES: There appears to be marked discrepancies between total IgE reference intervals (RIs) in use by many laboratories and those recommended by published studies. The aim of this study was therefore to review total IgE RIs currently reported by Scandinavian and British laboratories and to compare these to published RIs identified by a literature review. METHODS: Relevant laboratories were identified by test directories provided by the national accreditation bodies in Norway, Sweden, Denmark and the UK. Total IgE RIs and their sources were acquired by accessing laboratory user handbooks or by an electronic survey. In addition a literature review of published total IgE RI studies was performed. RESULTS: From 172 accredited laboratories providing total IgE analysis, data was acquired from 122 laboratories. An adult upper reference limit between 81 to 150 kU/L was reported by 89% of these. Denmark and Sweden reported the most harmonised RIs whilst Norway and the UK exhibited the least degree of harmonisation. Published adult (n = 6) and paediatric (n = 6) RI studies reported markedly higher upper limits than those currently in use by the laboratories included in this study. There were also large variations in the number of age strata in use for paediatric RIs. CONCLUSION: This study demonstrates large variations in currently utilised IgE RIs by Scandinavian and British accredited laboratories and most report markedly lower RIs than those recommended by recent RI publications. Many laboratories likely utilise outdated RIs and should consider critically reviewing and updating their RIs.

Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation.

Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

Personalized and Population-Based Reference Intervals for 48 Common Clinical Chemistry and Hematology Measurands: A Comparative Study.

BACKGROUND: Personalized reference intervals (prRIs) have the potential to improve individual patient follow-up as compared to population-based reference intervals (popRI). In this study, we estimated popRI and prRIs for 48 clinical chemistry and hematology measurands using samples from the same reference individuals and explored the effect of using group-based and individually based biological variation (BV) estimates to derive prRIs. METHODS: 143 individuals (median age 28 years) were included in the study and had fasting blood samples collected once. From this population, 41 randomly selected subjects had samples collected weekly for 5 weeks. PopRIs were estimated according to Clinical Laboratory Standards Institute EP28 and within-subject BV (CVI) were estimated by CV-ANOVA. Data were assessed for trends and outliers prior to calculation of individual prRIs, based on estimates of (a) within-person BV (CVP), (b) CVI derived in this study, and (c) publically available CVI estimates. RESULTS: For most measurands, the individual prRI ranges were smaller than the popRI range, but overall about half the study participants had a prRI wider than the popRI for 5 or more out of 48 measurands. The dispersion of prRIs based on CVP was wider than that of prRIs based on CVI. CONCLUSION: The prRIs derived in our study varied significantly between different individuals, especially if based on CVP. Our results highlight the limitations of popRIs in interpreting test results of individual patients. If sufficient data from a steady-state situation are available, using prRI based on CVP estimates will provide a RI most specific for an individual patient.

Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation.

Defects in hydroxymethylbilane synthase (HMBS) can cause Acute Intermittent Porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ~⅓ of clinical HMBS variants are missense variants, and most clinically-reported HMBS missense variants are designated as "variants of uncertain significance" (VUS). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino-acid substitutions. The resulting variant effect maps generally agreed with biochemical expectation. However, the maps showed variants at the dimerization interface to be unexpectedly well tolerated, and suggested residue roles in active site dynamics that were supported by molecular dynamics simulations. Most importantly, these HMBS variant effect maps can help discriminate pathogenic from benign variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

Long-Term Within- and Between-Subject Biological Variation Data of Hematological Parameters in Recreational Endurance Athletes.

BACKGROUND: Hematological parameters have many applications in athletes, from monitoring health to uncovering blood doping. This study aimed to deliver biological variation (BV) estimates for 9 hematological parameters by a Biological Variation Data Critical Appraisal Checklist (BIVAC) design in a population of recreational endurance athletes and to assess the effect of self-reported exercise and health-related variables on BV. METHODS: Samples were drawn from 30 triathletes monthly for 11 months and measured in duplicate for hematological measurands on an Advia 2120 analyzer (Siemens Healthineers). After outlier and homogeneity analysis, within-subject (CVI) and between-subject (CVG) BV estimates were delivered (CV-ANOVA and log-ANOVA, respectively) and a linear mixed model was applied to analyze the effect of exercise and other related variables on the BV estimates. RESULTS: CVI estimates ranged from 1.3% (95%CI, 1.2-1.4) for mean corpuscular volume to 23.8% (95%CI, 21.6-26.3) for reticulocytes. Sex differences were observed for platelets and OFF-score. The CVI estimates were higher than those reported for the general population based on meta-analysis of eligible studies in the European Biological Variation Database, but 95%CI overlapped, except for reticulocytes, 23.9% (95%CI, 21.6-26.5) and 9.7% (95%CI, 6.4-11.0), respectively. Factors related to exercise and athletes' state of health did not appear to influence the BV estimates. CONCLUSIONS: This is the first BIVAC-compliant study delivering BV estimates that can be applied to athlete populations performing high-level aerobic exercise. CVI estimates of most parameters were similar to the general population and were not influenced by exercise or athletes' state of health.

Systematic review and meta-analysis of within-subject and between-subject biological variation data of coagulation and fibrinolytic measurands.

OBJECTIVES: The diagnosis and monitoring of bleeding and thrombotic disorders depend on correct haemostatic measurements. The availability of high-quality biological variation (BV) data is important in this context. Many studies have reported BV data for these measurands, but results are varied. The present study aims to deliver global within-subject (CVI) and between-subject (CVG) BV estimates for haemostasis measurands by meta-analyses of eligible studies, by assessment with the Biological Variation Data Critical Appraisal Checklist (BIVAC). METHODS: Relevant BV studies were graded by the BIVAC. Weighted estimates for CVI and CVG were obtained via meta-analysis of the BV data derived from BIVAC-compliant studies (graded A-C; whereby A represents optimal study design) performed in healthy adults. RESULTS: In 26 studies BV data were reported for 35 haemostasis measurands. For 9 measurands, only one eligible publication was identified and meta-analysis could not be performed. 74% of the publications were graded as BIVAC C. The CVI and CVG varied extensively between the haemostasis measurands. The highest estimates were observed for PAI-1 antigen (CVI 48.6%; CVG 59.8%) and activity (CVI 34.9%; CVG 90.2%), while the lowest were observed for activated protein C resistance ratio (CVI 1.5%; CVG 4.5%). CONCLUSIONS: This study provides updated BV estimates of CVI and CVG with 95% confidence intervals for a wide range of haemostasis measurands. These estimates can be used to form the basis for analytical performance specifications for haemostasis tests used in the diagnostic work-up required in bleeding- and thrombosis events and for risk assessment.

Biological variation: recent development and future challenges.

Biological variation (BV) data have many applications in laboratory medicine. However, these depend on the availability of relevant and robust BV data fit for purpose. BV data can be obtained through different study designs, both by experimental studies and studies utilizing previously analysed routine results derived from laboratory databases. The different BV applications include using BV data for setting analytical performance specifications, to calculate reference change values, to define the index of individuality and to establish personalized reference intervals. In this review, major achievements in the area of BV from last decade will be presented and discussed. These range from new models and approaches to derive BV data, the delivery of high-quality BV data by the highly powered European Biological Variation Study (EuBIVAS), the Biological Variation Data Critical Appraisal Checklist (BIVAC) and other standards for deriving and reporting BV data, the EFLM Biological Variation Database and new applications of BV data including personalized reference intervals and measurement uncertainty.

Frequency of JAK2V617F, MPL and CALR driver mutations and associated clinical characteristics in a Norwegian patient cohort with myeloproliferative neoplasms.

Myeloproliferative neoplasms are hematological disorders characterized by increased production in one or more myeloid cell lines, associated with driver mutations in JAK2-, MPL- and CALR-genes. The aims of this study were to investigate the prevalence of these driver mutations in a Norwegian patient cohort with myeloproliferative neoplasms, and to assess whether the different mutations were associated with different clinical presentation and natural history.Results from 820 patients in whom analysis for JAK2V617F-, CALR- and MPL had been performed at Haukeland University Hospital in the period 2014-2019 were retrieved and analyzed together with clinical variables related to diagnosis, hematological blood parameters and complications, obtained from patient records.We identified 182 cases of myeloproliferative neoplasms: 78 with JAK2V617F, 28 with CALR-mutations, two with MPL-mutations and 23 cases without a driver mutation. There was a lower prevalence of JAK2V617F mutation than expected in the polycythemia vera group, likely related to overdiagnosis. In patients with essential thrombocytosis, we found significantly higher levels of hemoglobin and erythrocyte volume fraction for JAK2V617F-mutated disease, and significantly higher levels of platelets and lactate dehydrogenase for CALR-mutated disease. Patients with JAK2V617F-mutated primary myelofibrosis had significantly higher levels of hemoglobin, and there was an increased number of smokers or former smokers in this group compared to patients with CALR-mutations.Except for a lower prevalence of JAK2V617F-mutation in polycythemia vera, the mutational distribution in our patient cohort was similar to previous findings in other populations. The novel finding of a higher prevalence of smokers in JAK2V617F-mutated primary myelofibrosis warrants further investigation.