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.

Compliance to specifications in an external quality assurance program: did new biological variation estimates of the European Federation of Laboratory Medicine (EFLM) affect the quality of laboratory results?

Objectives: The results of external quality assurance schemes are evaluated against specifications generally based on biological variation (BV) data. This study was carried out to determine whether new BV values affected the level of compliance to specifications. Our secondary objective was to identify the conditions that would be compromised as a result of poor analytical performance in disease associated markers. Methods: This study was based on the results of the SEQCML External Quality Assurance scheme for the 2015-2022 period. Deviation of the individual result from the target value was estimated. Additionally, we calculated the percentage of results that met the pre-established specification. Results: In 97 of the 133 analytes, the level of compliance was maintained in 80-90 % of the results obtained in the two study periods. In 23 analytes, the level of compliance ranged from 51 to 79 % in the two study periods. In ALT, AST and sodium, the level of compliance was ≤50 % of the results obtained in the first study period, with sodium being the only analyte that maintained this poor level of compliance in the second study period. Conclusions: The level of compliance to specifications remained independent from the specification used (SEQCML or EFLM) for the majority of the analytes. The results for sodium ion were below the target value, which may lead to misdiagnosis of hyponatremia. Non-compensated alkaline picrate methods overestimate creatinine, which may produce false information suggestive of kidney failure.

Impact of COVID-19 pandemic on HbA1c management and results in pediatric and adult outpatients with diabetes.

Objectives: Diabetes mellitus intensify the risks and complications related to COVID-19 infection. A major effect of the pandemic has been a drastic reduction of in-person visits. The aim of this study was to evaluate the impact of the COVID-19 pandemic on HbA1c management and results among pediatric and adult outpatients with diabetes, considering the laboratory and point-of-care testing (POCT) HbA1c measurements. Methods: Observational retrospective study including patients from pediatric and adult diabetes units was conducted. HbA1c results obtained in the laboratory and POCT over 3 years (2019-2021) were collected from the laboratory information system. Results: After the lockdown, the number of HbA1c plummeted. Children returned soon to routine clinical practice. The number of HbA1c increased gradually in adults, especially in POCT. Globally, HbA1c results were lower in children compared with adults (p<0.001). HbA1c values in children (p<0.001) and adults (p=0.002) decreased between pre-pandemic and post-pandemic periods, though lower than the HbA1c reference change value. The percentage of HbA1c results above 8% remained stable during the study period. Conclusions: Continuous glucose monitoring and a telemedicine have been crucial, even allowing for improvements in HbA1c results. During the lockdown, patients with better metabolic control were managed in the laboratory whereas patients with poorer control or a severe clinical situation were attended in diabetes units by POCT. Adults returned to pre-pandemic management slowly because they were more susceptible to morbidity and mortality due to COVID-19. Coordination among all health professionals has been essential to offering the best management, especially in difficult scenarios such as the COVID-19 pandemic.

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.

Laboratory interpretative comments and guidance: clinical and operative outcomes on moderate to severe hyponatraemia patient management.

AIMS: Hyponatraemia is the most common body fluid disorders but often goes unnoticed. Our laboratory incorporated a standardised procedure to help clinicians detect moderate/severe hyponatraemia. The study aims were to evaluate the outcomes on patient care and clinicians' satisfaction. METHODS: The study, observational and retrospective, included 1839 cases, adult and paediatric patients, with sodium concentration <130 mmol/L. The procedure consisted of interpretative comments in the emergency and core laboratories report and the point-of-care testing blood gas network report. We evaluated hyponatraemia length in two equal periods: before and after the implementation. We conducted a survey addressed to the staff of the clinical settings involved to know their satisfaction. RESULTS: The median hyponatraemia length decreased significantly from 4.95 hours (2.08-16.57) in the first period to 2.17 hours (1.06-5.39) in the second period. The lack of hyponatraemia patients follow-up was significantly less after the procedure implementation. The survey was answered by 92 (60 senior specialists and 32 residents) out of 110 clinicians surveyed. Ninety of them (98%) answered positively. CONCLUSIONS: We have demonstrated the reduction in the time for diagnosing and management by physicians, the higher uniformity in the time required to solve hyponatraemia episodes following our laboratory procedure and the clinicians' satisfaction.

From big data to better patient outcomes.

Among medical specialties, laboratory medicine is the largest producer of structured data and must play a crucial role for the efficient and safe implementation of big data and artificial intelligence in healthcare. The area of personalized therapies and precision medicine has now arrived, with huge data sets not only used for experimental and research approaches, but also in the "real world". Analysis of real world data requires development of legal, procedural and technical infrastructure. The integration of all clinical data sets for any given patient is important and necessary in order to develop a patient-centered treatment approach. Data-driven research comes with its own challenges and solutions. The Findability, Accessibility, Interoperability, and Reusability (FAIR) Guiding Principles provide guidelines to make data findable, accessible, interoperable and reusable to the research community. Federated learning, standards and ontologies are useful to improve robustness of artificial intelligence algorithms working on big data and to increase trust in these algorithms. When dealing with big data, the univariate statistical approach changes to multivariate statistical methods significantly shifting the potential of big data. Combining multiple omics gives previously unsuspected information and provides understanding of scientific questions, an approach which is also called the systems biology approach. Big data and artificial intelligence also offer opportunities for laboratories and the In Vitro Diagnostic industry to optimize the productivity of the laboratory, the quality of laboratory results and ultimately patient outcomes, through tools such as predictive maintenance and "moving average" based on the aggregate of patient results.

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.

Within-subject biological variation estimates using an indirect data mining strategy. Spanish multicenter pilot study (BiVaBiDa).

OBJECTIVES: The estimates of biological variation (BV) have traditionally been determined using direct methods, which present limitations. In response to this issue, two papers have been published addressing these limitations by employing indirect methods. Here, we present a new procedure, based on indirect methods that analyses data collected within a multicenter pilot study. Using this method, we obtain CVI estimates and calculate confidence intervals (CI), using the EFLM-BVD CVI estimates as gold standard for comparison. METHODS: Data were collected over a 18-month period for 7 measurands, from 3 Spanish hospitals; inclusion criteria: patients 18-75 years with more than two determinations. For each measurand, four different strategies were carried out based on the coefficient of variation ratio (rCoeV) and based on the use of the bootstrap method (OS1, RS2 and RS3). RS2 and RS3 use symmetry reference change value (RCV) to clean database. RESULTS: RS2 and RS3 had the best correlation for the CVI estimates with respect to EFLM-BVD. RS2 used the symmetric RCV value without eliminating outliers, while RS3 combined RCV and outliers. When using the rCoeV and OS1 strategies, an overestimation of the CVI value was obtained. CONCLUSIONS: Our study presents a new strategy for obtaining robust CVI estimates using an indirect method together with the value of symmetric RCV to select the target population. The CVI estimates obtained show a good correlation with those published in the EFLM-BVD database. Furthermore, our strategy can resolve some of the limitations encountered when using direct methods such as calculating confidence intervals.

Impact of ultra-low temperature long-term storage on the preanalytical variability of twenty-one common biochemical analytes.

OBJECTIVES: Retrospective studies frequently assume analytes long-term stability at ultra-low temperatures. However, these storage conditions, common among biobanks and research, may increase the preanalytical variability, adding a potential uncertainty to the measurements. This study is aimed to evaluate long-term storage stability of different analytes at <-70 °C and to assess its impact on the reference change value formula. METHODS: Twenty-one analytes commonly measured in clinical laboratories were quantified in 60 serum samples. Samples were immediately aliquoted and frozen at <-70 °C, and reanalyzed after 11 ± 3.9 years of storage. A change in concentration after storage was considered relevant if the percent deviation from the baseline measurement was significant and higher than the analytical performance specifications. RESULTS: Preanalytical variability (CVP) due to storage, determined by the percentage deviation, showed a noticeable dispersion. Changes were relevant for alanine aminotransferase, creatinine, glucose, magnesium, potassium, sodium, total bilirubin and urate. No significant differences were found in aspartate aminotransferase, calcium, carcinoembryonic antigen, cholesterol, C-reactive protein, direct bilirubin, free thryroxine, gamma-glutamyltransferase, lactate dehydrogenase, prostate-specific antigen, triglycerides, thyrotropin, and urea. As nonnegligible, CVP must remain included in reference change value formula, which was modified to consider whether one or two samples were frozen. CONCLUSIONS: After long-term storage at ultra-low temperatures, there was a significant variation in some analytes that should be considered. We propose that reference change value formula should include the CVP when analyzing samples stored in these conditions.

Critical review and meta-analysis of biological variation estimates for tumor markers.

OBJECTIVES: Biological variation data (BV) can be used for different applications, but this depends on the availability of robust and relevant BV data. In this study, we aimed to summarize and appraise BV studies for tumor markers, to examine the influence of study population characteristics and concentrations on BV estimates and to discuss the applicability of BV data for tumor markers in clinical practice. METHODS: Studies reporting BV data for tumor markers related to gastrointestinal, prostate, breast, ovarian, haematological, lung, and dermatological cancers were identified by a systematic literature search. Relevant studies were evaluated by the Biological Variation Data Critical Appraisal Checklist (BIVAC) and meta-analyses were performed for BIVAC compliant studies to deliver global estimates of within-subject (CVI) and between-subject (CVG) BV with 95% CI. RESULTS: The systematic review identified 49 studies delivering results for 22 tumor markers; four papers received BIVAC grade A, 3 B, 27 C and 15 D. Out of these, 29 CVI and 29 CVG estimates met the criteria to be included in the meta-analysis. Robust data are lacking to conclude on the relationship between BV and different disease states and tumor marker concentrations. CONCLUSIONS: This review identifies a lack of high-quality BV studies for many tumor markers and a need for delivery of BIVAC compliant studies, including in different disease states and tumor marker concentrations. As of yet, the state-of-the-art may still be the most appropriate model to establish analytical performance specifications for the majority of tumor markers.

Internal quality control - past, present and future trends.

Objectives: This paper offers an historical view, through a summary of the internal quality control (IQC) models used from second half of twentyth century to those performed today and wants to give a projection on how the future should be addressed. Methods: The material used in this work study are all papers collected referring IQC procedures. The method used is the critical analysis of the different IQC models with a discussion on the weak and the strong points of each model. Results: First models were based on testing control materials and using multiples of the analytical procedure standard deviation as control limits. Later, these limits were substituted by values related with the intended use of test, mainly derived from biological variation. For measurands with no available control material methods based on replicate analysis of patient' samples were developed and have been improved recently; also, the sigma metrics that relates the quality desired with the laboratory performance has resulted in a highly efficient quality control model. Present tendency is to modulate IQC considering the workload and the impact of analytical failure in the patent harm. Conclusions: This paper remarks the strong points of IQC models, indicates the weak points that should be eliminated from practice and gives a future projection on how to promote patient safety through laboratory examinations.

External quality control in laboratory medicine. Progresses and future.

Objectives: An external quality control program distributes same control samples to various laboratories and evaluates results obtained with a common criterion. The aim of this work is to summarize the evolution of various types of external programs, to point out the progresses ant to preclude practical consequences of the participant laboratories. Content: The material consists on a brief revision of the different types of external programs that have been used for the last forty years. The method is the critical analysis of the strong and weak points of each program model, from the light of our experience. External quality assessment (EQA) programs were initiated at half the XX century, evidencing big discrepancies among laboratory results. EQA were developed in various countries and some mechanisms to harmonize them were proposed: to establish common performance specifications derived from biological variation, to use EQS as educational tool. Since the 2000 important advances were seen: to focus EQA to assure the adequate clinical use of laboratory tests, to use commutable controls, to harmonize the different EQA models, to promote a forum for co-operation and exchange of knowledge on quality-related matters for EQA organizers. Summary and Outlook: To participate in an EQA with commutable-reference method assigned values controls allows to know the real inaccuracy of results and their impact on patient' samples. To participate in a EQA with non commutable controls allows to know whether the individual laboratory performance agrees with that from other laboratories using same analytical method.

Biological variation estimates of thyroid related measurands - meta-analysis of BIVAC compliant studies.

OBJECTIVES: Testing for thyroid disease constitutes a high proportion of the workloads of clinical laboratories worldwide. The setting of analytical performance specifications (APS) for testing methods and aiding clinical interpretation of test results requires biological variation (BV) data. A critical review of published BV studies of thyroid disease related measurands has therefore been undertaken and meta-analysis applied to deliver robust BV estimates. METHODS: A systematic literature search was conducted for BV studies of thyroid related analytes. BV data from studies compliant with the Biological Variation Data Critical Appraisal Checklist (BIVAC) were subjected to meta-analysis. Global estimates of within subject variation (CVI) enabled determination of APS (imprecision and bias), indices of individuality, and indicative estimates of reference change values. RESULTS: The systematic review identified 17 relevant BV studies. Only one study (EuBIVAS) achieved a BIVAC grade of A. Methodological and statistical issues were the reason for B and C scores. The meta-analysis derived CVI generally delivered lower APS for imprecision than the mean CVA of the studies included in this systematic review. CONCLUSIONS: Systematic review and meta-analysis of studies of BV of thyroid disease biomarkers have enabled delivery of well characterized estimates of BV for some, but not all measurands. The newly derived APS for imprecision for both free thyroxine and triiodothyronine may be considered challenging. The high degree of individuality identified for thyroid related measurands reinforces the importance of RCVs. Generation of BV data applicable to multiple scenarios may require definition using "big data" instead of the demanding experimental approach.

Within- and between-subject biological variation data for serum zinc, copper and selenium obtained from 68 apparently healthy Turkish subjects.

OBJECTIVES: Trace elements (TrEL) are nutritionally essential components in maintaining health and preventing diseases. There is a lack of reliable biological variation (BV) data for TrELs, required for the diagnosis and monitoring of TrEL disturbances. In this study, we aimed to provide updated within- and between-subject BV estimates for zinc (Zn), copper (Cu) and selenium (Se). METHODS: Weekly serum samples were drawn from 68 healthy subjects (36 females and 32 males) for 10 weeks and stored at -80 °C prior to analysis. Serum Zn, Cu and Se levels were measured using inductively-coupled plasma mass spectrometry (ICP-MS). Outlier and variance homogeneity analyses were performed followed by CV-ANOVA (Røraas method) to determine BV and analytical variation estimates with 95% CI and the associated reference change values (RCV) for all subjects, males and females. RESULTS: Significant differences in mean concentrations between males and females were observed, with absolute and relative (%) differences for Zn at 0.5 µmol/L (3.5%), Cu 2.0 µmol/L (14.1%) and Se 0.06 µmol/L (6.0%). The within-subject BV (CVI [95% CI]) estimates were 8.8% (8.2-9.3), 7.8% (7.3-8.3) and 7.7% (7.2-8.2) for Zn, Cu and Se, respectively. Within-subject biological variation (CVI) estimates derived for male and female subgroups were similar for all three TrELs. Marked individuality was observed for Cu and Se. CONCLUSIONS: The data of this study provides updated BV estimates for serum Zn, Cu and Se derived from a stringent protocol and state of the art methodologies. Furthermore, Cu and Se display marked individuality, highlighting that population based reference limits should not be used in the monitoring of patients.