Aasne Karine Aarsand

Background: The clinical application of cardiovascular disease (CVD) biomarkers requires robust biological variation (BV) data to enable both interpretation of serial results and definition of appropriate analytical quality. A critical review and meta-analysis of eligible BV studies of natriuretic peptides and other CVD-related biomarkers was therefore undertaken, with a focus on differences observed in different states of health and the impact of sampling intervals on BV. Methods: BV studies of N-terminal pro–B-type natriuretic peptide (NT-proBNP), brain natriuretic peptide, N-terminal pro–A-type natriuretic peptide, galectin, copeptin, soluble suppression of tumorigenicity-2, and growth differentiation factor 15 were identified by a systematic literature review and assessed using the Biological Variation Data Critical Appraisal Checklist. A meta-analysis of eligible studies was used to derive within-subject (CVI) and between-subject BV estimates, which were used to calculate analytical performance specifications, indices of individuality, and reference change values. Results: Twenty-one studies were identified, only 3 of which achieved an A grade on the Biological Variation Data Critical Appraisal Checklist, indicating full checklist compliance. For NT-proBNP, meta-analysis–derived CVI estimates depended on sampling frequencies and study duration: 15 days (34.3%; 95% CI, 25.0–60.0). For the other biomarkers, from one to 5 BV studies were identified, reporting on different states of health and sampling intervals. Conclusions: This systematic review provides quality-assessed BV data for natriuretic peptides and other CVD markers. Our results highlight the influence of sampling frequency on CVI for NT-proBNP, potentially impacting the intended use. Further BV studies applicable to multiple clinical scenarios are required.

Background: There is a lack of robust biological variation (BV) data for porphyria-related biomarkers. Our study aimed to estimate BV of erythrocyte total, metal-free, and zinc protoporphyrin IX in patients with erythropoietic protoporphyria (EPP) and healthy subjects and to explore the clinical implications of these data. Methods: Fourteen patients with EPP and 15 healthy subjects were sampled quarterly for 2 years, and erythrocyte protoporphyrin analyses were performed in duplicate in all samples. A Bayesian method was used to estimate the within-subject (CVI) and personal (CVP(i)) BV. Results: Based on clinical and laboratory assessments, EPP patients were stable during the study, with only 2 data points excluded. CVI in the EPP cohort was estimated as 9.8% (95% credible interval 8.5%–11.5%) for erythrocyte total protoporphyrin, 10.5% (9.0%–12.3%) for metal-free protoporphyrin, and 5.9% (4.3%–8.0%) for zinc protoporphyrin. Baseline metal-free protoporphyrin ranged from 6.9 to 139.8 µmol/L, but the CVP(i)s derived for each patient were similar (20th and 80th percentile of predicted distribution 9.5%–11.5%), and data were homogeneously distributed. Metal-free protoporphyrin was not measurable in the healthy cohort. Data for zinc protoporphyrin were heterogeneously distributed in both study cohorts. Conclusions: The EPP patients had different set points for metal-free protoporphyrin, but the CVP(i) was similar, supporting the use of the same treatment goals when monitoring. This study is the first to use Bayesian analysis to demonstrate that personal BV is similar in patients with stable, chronic disease and different set points.

Background: Conventional population-based reference intervals (popRIs) are established on the ranking of single measurement results from at least 120 reference individuals. In this study, we aimed to explore a new model for popRIs, utilizing biological variation (BV) data to define the reference interval (RI) limits and compared BV-based popRI from different sample sizes with previously published conventional popRIs from the same population. Methods: The model is based on defining the population set point (PSP) from single-measurement results of a group of reference individuals and using the total variation around the PSP, derived from the combination of BV and analytical variation, to define the RI limits. Using data from 143 reference individuals for 48 clinical chemistry and hematology measurands, BV-based popRIs were calculated for different sample sizes (n = 16, n = 30, and n = 120) and considered acceptable if they covered 90% of the population. In addition, simulation studies were performed to estimate the minimum number of required reference individuals. Results: The median ratio of the BV-based to conventional RI ranges was 0.98. The BV-based popRIs calculated from the different samples were similar, and most met the coverage criterion. For 25 measurands ≤16 reference individuals and for 23 measurands >16 reference individuals were required to estimate the PSP. Conclusions: The BV-based popRI model delivered robust RIs for most of the included measurands. This new model requires a smaller group of reference individuals than the conventional popRI model and can be implemented if reliable BV data are available.

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.

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.

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.

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.

ObjectivesThere 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.MethodsRelevant 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.ResultsFrom 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.ConclusionThis 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.