Volume 63, Issue 5 - Highlight Issue: External Quality Assurance (EQA): A Fundamental Tool for Quality in Laboratory Medicine; Issue Editor: Christoph Buchta

This is the first in a series of five papers that detail the role and substantial impact that external quality assessment (EQA) and their providers‘ services play in ensuring in-vitro diagnostic (IVD) performance quality. The aim is to give readers and users of EQA services an insight into the processes in EQA, explain to them what happens before EQA samples are delivered and after examination results are submitted to the provider, how they are assessed, what benefits participants can expect, but also who are stakeholders other than participants and what significance do EQA data and assessment results have for them. This first paper presents the history of EQA, insights into legal, financing and ethical matters, information technology used in EQA, structure and lifecycle of EQA programs, frequency and intensity of challenges, and unique requirements of extra-examination and educational EQA programs.

External quality assessment (EQA) cycles are the smallest complete units within EQA programs that laboratories can use to obtain external assessments of their performance. In each cycle, several samples are distributed to the laboratories registered for participation, and ideally, EQA programs not only cover the examination procedures but also the pre- and post-examination procedures. The properties and concentration range of measurands in individual samples are selected with regard to the intended challenge for the participants so that each sample fulfils its purpose. This aims to ensure the most significant possible information gain in every cycle using the lowest possible number of EQA samples and thus, under economically optimal conditions. Participants examine samples and the results are reported to the EQA provider, who compares them with the target values for individual measurands in every sample. The EQA provider assesses the laboratory performance, and finally communicates the assessment results to the participant. The participants evaluate the outcomes of the assessment of their examination results and can draw conclusions in the case of both failing and passing and, if necessary, define improvement measures. After completion, each cycle is evaluated by the provider so that limitations and weaknesses of the EQA program can be identified and appropriate measures taken, or to confirm its continued suitability and appropriateness.

Providers of external quality assessment (EQA) programs evaluate data or information obtained and reported by participant laboratories using their routine procedures to examine properties or measurands in samples provided for this purpose. EQA samples must offer participants an equal chance to obtain accurate results, while being designed to provide results in clinically relevant ranges. It is the responsibility of the EQA provider to meet the necessary requirements for homogeneity, stability and some other properties of the EQA items in order to offer participants a fair, reliable and technically interesting EQA experience. Thus, the samples are at the heart and in the centre of EQA and its success depends on their quality. This manuscript describes the requirements for EQA samples and the activities of EQA providers to achieve them.

The main stakeholders in external quality assessment (EQA) programs are the participants, in whose interests these challenges are ultimately organised. EQA schemes in the medical field contribute to improving the quality of patient care by evaluating the analytical and diagnostic quality of laboratory and point-of-care tests (POCT) by independent third parties and, if necessary, pointing out erroneous measurement results and analytical or diagnostic improvement potential. Other benefits include the option of using EQA samples for other important laboratory procedures, such as the verification or validation of in vitro diagnostic medical devices (IVD-MDs), a contribution to the estimation of measurement uncertainty, a means of training and educating laboratory staff through educational EQA programmes or samples, or even for independent and documented monitoring of staff competence, such as on samples with unusual or even exceptional characteristics. Participation in an EQA scheme for beneficiaries like medical, microbiological and histo- and molecular pathology laboratories, users of POCT and self-testing systems as well as National Metrology Institutes, calibration laboratories and reference laboratories that are dedicated to specific tasks and have particular expectations of the EQA scheme are presented here.

External quality assessment (EQA) enhances patient safety through the evaluation of the quality of laboratory-based and point of care testing. Regulatory agencies and accreditation organizations utilize the results and the laboratory’s response to them as part of assessing the laboratory’s fitness to practice. In addition, where EQA samples are commutable and the assigned value has been determined using reference measurement procedures (RMPs), EQA data contributes to the verification of metrological traceability of assays as part of the post-market surveillance of in vitro diagnostic (IVD) medical devices (IVD-MDs). More broadly, the scientific and medical communities use EQA data to demonstrate that medical laboratory examination procedures are fit for clinical purposes, to evaluate common reference intervals, and inclusion of data in clinical databases. Scientific groups, the IVD industry, reference laboratories and National Metrology Institutes can work with EQA providers to identify measurands, which should urgently be supported by the development of reference materials or methods. The ability of health systems to respond effectively to fast-evolving medical challenges, such as the Coronavirus Disease-19 (COVID-19) pandemic, is reliant on EQA to demonstrate confidence in the performance of new laboratory methods and testing services. EQA providers are uniquely positioned to assess the performance of IVD-MDs in addition to individual laboratories and testing sites. Although the primary focus of EQA providers remains the improvement of the performance of individual laboratories, there are many stakeholders who benefit from EQA performance data.

Although the concept of bias appears consolidated in laboratory science, some important changes in its definition and management have occurred since the introduction of metrological traceability theory in laboratory medicine. In the traceability era, medical laboratories should rely on manufacturers who must ensure traceability of their in vitro diagnostic medical devices (IVD-MD) to the highest available references, providing bias correction during the trueness transfer process to calibrators before they are marketed. However, sometimes some bias can be observed arising from an insufficient correction during the traceability implementation. This source of bias can be discovered by the IVD-MD surveillance by traceability-based external quality assessment and confirmed by ad-hoc validation experiments. The assessment of significance should be based on its impact on measurement uncertainty (MU) of results. The IVD manufacturer, appropriately warned, is responsible to take an immediate investigation and eventually fix the problem with a corrective action. Even if IVD-MD is correctly aligned in the validation steps and bias components are eliminated, during ordinary use the system may undergo systematic variations such as those caused by recalibrations and lot changes. These sources of randomly occurring bias are incorporated in the estimate of intermediate reproducibility of IVD-MD through internal quality control and can be tolerated until the estimated MU on clinical samples fulfils the predefined specifications. A readjustment of the IVD-MD by the end-user must be undertaken to try to correct the bias becoming significant. If the bias remains, the IVD manufacturer should be requested to rectify the problem.

Important advancements have been made in understanding the pathogenetic mechanisms underlying acute and chronic lung disorders. But although a wide variety of innovative biomarkers have and are being investigated, they are not largely employed to evaluate non-neoplastic lung diseases. The current work aims to examine the use of Krebs von den Lungen-6 (KL-6), a mucin-like glycoprotein predominantly expressed on the surface of type II alveolar epithelial cells (AEC2s), to evaluate the stage, response to treatment, and prognosis in patients with non-neoplastic lung disorders. Data analysis suggests that KL-6 can be utilized as an effective diagnostic and prognostic biomarker in individuals with interstitial lung disease and as a predictor of clinical outcomes in subjects with SARS-CoV-2-related pneumonia. Moreover, KL-6 can be reliably used in routine clinical settings to diagnose and predict the outcome of patients with chronic obstructive pulmonary disease (COPD) exacerbation. The optimal cut-off points within the European population should be defined to improve KL-6’s diagnostic efficacy.

Objectives Preanalytical phase is an elemental part of laboratory diagnostics, but is prone to humane errors. The aim of this study was to evaluate performance in preanalytical phase external quality assessment (EQA) cases. We also suggest preventive actions for risk mitigation. Methods We included 12 EQA rounds (Labquality Ltd.) with three patient cases (36 cases, 54–111 participants, 7–15 countries) published in 2018–2023. We graded performance according to percentage of correct responses in each case as ≥900 % excellent, 70–89 % good, 50–69 % satisfactory, 30–49 % fair and <30 % poor. Performance was simultaneously failed with ≥10 % of responses leading to harmful events. Results Overall performance was excellent in 7, good in 12, satisfactory in 10, fair in 4 and poor in 3 cases. Additionally, 7 cases showed failed performance. Routine requests with incorrect sample tubes or incorrect sample handling were detected with good performance. Lower performance was seen with sudden abnormal results, with rare requests, with false patient identification (never-events) and with incorrect test requests. Information technology (IT) solutions (preanalytical checklists, autoverification rules and patient specific notifications) could have prevented 33 of 36 preanalytical errors. Conclusions While most common errors were detected with good performance, samples with rare requests or those requiring individualised consideration are vulnerable to human misinterpretation. In many instances, samples with preanalytical errors should have been identified and rejected before reaching the laboratory or being directed to analysis. Optimising IT solutions to effectively detect these preanalytical errors allows for focus on infrequent events demanding accessible professional consultation. EQA preanalytical cases may help in education of correct actions in these occasions.

Objectives Emergency department (ED) crowding is a widespread problem that positions patients at risk. The desire to improve the ED throughput requires novel approaches. Point-of-care testing (POCT) has emerged as useful technology that could contribute to create more efficient patient flow and better timeliness in the ED. The main objective of our study is to demonstrate, in a multicenter study, that POCT benefits care timeliness in the ED. Methods We conducted a multicenter and cluster randomized study. A total of 3,200 patients. We randomly assigned patients to a POCT group or Central Laboratory Group. The primary outcome was the ED time to clinical decision. The secondary outcome included the length of stay and the laboratory turnaround time. Readmission within the seven after discharge was also calculated. Results The primary finding of this study is a strategy based on POCT that aims to significantly improve care timeliness in the ED. We found significant reductions in all outcomes regardless of presentation reason, patient disposition or hospital type. Time to clinical decision decreased by 75.2 min (205–129.8), length of stay by 77.5 min (273.1–195.6) and laboratory turnaround time by 56.2 min (82.2–26) in the POCT group. No increase in readmission was found. Conclusions Our strategy represents a good approach to optimize timeliness in the ED. It should be seen as a starting point for further operational research focusing on POCT for improving throughput and reducing crowding in the ED.

Objectives Accurate assessment of calcium levels is crucial for optimal management of regular Haemodialysis (HD) patients. Different calcium adjustment equations and albumin methods; including bromocresol purple (BCP) and bromocresol green (BCG) assays are employed by laboratories, which cause considerable discrepancies between reported results. The aim of this study is to assess the influence of albumin assays on calcium status in stable haemodialysis patients against free calcium (fCa) as a gold standard test. Methods A total of 103 paired serum and fCa samples were collected from a cohort of stable HD patients. Albumin levels were measured by either the BCP or BCG method, and samples were also analysed for the total calcium (T.Ca), phosphate, bicarbonate, and pH levels. The performance of BCG-based and BCP-based adjusted calcium equations was compared using Z-scores scatter plots, intraclass correlation coefficient and Cohen Kappa statistic, with fCa being the reference standard. Results Unadjusted T.Ca achieved a 70 % overall classification agreement with fCa and identified 61 % of the “true” hypocalcaemic samples. Adjusted calcium concentrations, calculated by either BCP- or BCG-based equation, were poor predictors of fCa; with more than 50 % of the hypocalcaemic samples being misclassified as normocalcaemic. Notably, both equations misclassified the calcium status in 5 (4.9 %) patients with severe hypocalcaemia (i.e., potentially requiring calcium infusion) as mild hypocalcaemia. Conclusions Our study showed evidence of hidden hypocalcaemia being missed by the current practice of using adjusted calcium in HD patients. Therefore, we recommend abandoning the adjustment procedure in samples from stable HD patients in favour of fCa measurement.

Objectives Automated immunoassays for 1,25-dihydroxyvitamin D (1,25(OH) 2 D) have increased the use of serum measurements in clinical and research settings, but disagreement with LC-MS/MS methods remains an issue. Methods In this study, we examined this problem using samples obtained from healthy young adults, n=80, mean age 21.7 (18–32) years, and a large cohort of paediatric samples, n=422, mean age 7.3 (0–17) years. We compared serum concentrations of 1,25(OH) 2 D3/D2 produced by the DiaSorin LIAISON ® XL immunoassay against an LC-MS/MS method with immunoaffinity enrichment and DAPTAD derivation. Results Both assays showed intra/inter-assay imprecision of ≤9.4 % across their respective assay range. DEQAS between April 2020 to Jan 2024 (n=80) showed mean bias (SD, 95 %CI) for DiaSorin −0.6 % (6.2, −12.8 to 11.6) and LC-MS/MS of +1.3 % (7.4, −13.3 to 15.8) against their respective method group means. Comparison of measurements in the adult samples showed a strong correlation (r 2 =0.9331) and concordance (CCC=0.959) between the two methods. LC-MS/MS values were lower than DiaSorin by an overall mean (±SD, 95 %CI) of −1.6 (±14.3, −29.6 to 26.5) pmol/L with an increased negative bias at higher concentrations. In the paediatric samples, weaker correlation (r 2 =0.6536) and concordance (CCC=0.782) were observed, with greater bias mean (±SD, 95 %CI) of −9.8 (±23.4, −55.7 to 35.9) pmol/L. The variability in the paediatric samples was not associated with concentration or participant age. There was an increase in the correlation and concordance when 1,25(OH) 2 D2 was included in the analysis. Conclusions It is likely that the metabolites of vitamin D present in the paediatric population contributed to the measurement of 1,25(OH) 2 D. The inconsistent agreement highlights the need for better assay harmonisation and paediatric reference intervals using LC-MS/MS method.

Objectives In the light of a rapidly increasing use of POCT blood gas testing, where tests and interpretation are performed by non-laboratory personnel, the objective was to investigate the knowledge among personnel in the Nordic countries using blood gas analyzers with focus on the interference from hemolysis. Methods Information was obtained from a self-developed, pre-tested online questionnaire. The questions covered demographic information about the respondents and specific questions on handling of and knowledge about blood gas analyses and the impact of hemolysis. The questionnaire was distributed by e-mail to relevant colleagues on behalf of the Nordic preanalytical scientific working group under the Nordic Federation of Clinical Chemistry. Results A total of 117 respondents completed the questionnaire. 62.7 % respondents both used the analyzer and interpreted the results. 59.6 % respondents did not know to which degree the blood gas analyzer can identify hemolysis. 4.4 % answered that all levels or high levels of hemolysis can be detected. 3.9 % considered the result valid despite hemolysis if it is released from the instrument. 73.7 % of all respondents knew that hemolysis alters potassium measurements, while knowledge about the effect on PaO 2 and bicarbonate measurements were more divergent. Conclusions The knowledge about blood gas analyzers with focus on the interference from hemolysis is sparse among non-laboratory personnel using the blood gas analyzers. This emphasizes the need for better education and competence management, which perhaps is even more important for these analyses than for other point-of-care tests.

Objectives To evaluate urinalysis parameters useful for identifying mixed cultures in urine culture using an automated urinary particle analyzer to assess quality indicators (QIs) for urine sample contamination. Methods A retrospective observational cross-sectional study was conducted with 2,527 urine samples from patients of a quaternary hospital in Brazil. Urine samples were processed simultaneously in Sysmex UF-5000 flow cytometry analyzer (urinalysis) and MALDI-TOF (culture). Results For all samples, a cutoff of 123.8 bacteria/µL was established to discriminate culture-negative specimens. ROC curve indicated the following cutoffs for females and males, respectively: 193.65 and 23.55 bacteria/µL, and 21.35 and 5.05 squamous epithelial cells (SEC)/µL, with the latter two related to scenarios of sample contamination/colonization through mixed cultures. Performing univariate logistic regression, we found a 2.78 (CI95 %: 2.12–3.65) times higher chance of probable mixed culture when SEC values were above the cutoffs for each sex, and 6.91(CI95 %: 4.56–10.47) times for bacteria. For multivariate logistic regression, the OR values were 1.62 (CI95 %: 1.21–2.15) and 5.82 (CI95 %: 3.77–8.98), respectively. Conclusions The fluorescent flow cytometry analyzers could efficiently identify urinary bacteria counts associated with contamination/colonization scenarios using the cutoffs of 21.35 SEC/µL for women and 5.05 SEC/µL for men. The cutoffs for bacteria/µL (193.65 for females and 23.55 for males) indicated that the presence of bacteria in male samples may be more associated with urinary tract infections (UTIs), while in female samples, it may be associated with either UTIs or contamination/colonization scenarios. This makes the analyzer a helpful tool as QI of sample contamination in urine cultures.

Objectives Cardiac biomarkers are useful for the diagnostic and prognostic assessment of myocardial injury (MI) and heart failure. By measuring specific proteins released into the bloodstream during heart stress or damage, these biomarkers help clinicians detect the presence and extent of heart injury and tailor appropriate treatment plans. This study aims to provide robust biological variation (BV) data for cardiac biomarkers in athletes, specifically focusing on those applied to detect or exclude MI, such as myoglobin, creatine kinase-myocardial band (CK-MB) and cardiac troponins (cTn), and those related to heart failure and cardiac dysfunction, brain natriuretic peptide (BNP) and N-terminal brain natriuretic pro-peptide (NT-proBNP). Methods Thirty athletes participated, providing monthly fasting blood samples over 11 months. Samples were analyzed using chemiluminescent immunoassays and statistical analyses were conducted using the classical ANOVA method, a linear mixed model and a Bayesian approach. Results The study observed significant gender differences in biomarker concentrations, with higher BNP and NT-proBNP in females and higher myoglobin and CK-MB in males. Physical activity within 24 h before sampling notably affected CK-MB, myoglobin, and hs-cTnI variability. The BV estimates demonstrated high individuality for most biomarkers, suggesting their potential for personalized monitoring. The study also revealed substantial heterogeneity for NT-proBNP and BNP within the population. Conclusions These findings underscore the importance of considering gender-specific reference intervals and the impact of recent physical activity when interpreting cardiac biomarkers in athletes. The study delivers new BV estimates for CK-MB and myoglobin while emphasizing the need for tailored clinical assessments in athlete populations.

Objectives Mild traumatic brain injury (mTBI) remains challenging to diagnose effectively in the emergency department. Abbott has developed the “GFAP/UCH-L1” mTBI test, to guide the clinical decision to perform a computed tomography (CT) head scan by ruling out the presence of mTBI. We evaluated the diagnostic accuracy of the “GFAP/UCH-L1” mTBI test in a Greek cohort and established age-dependent cut-offs. Methods A total of 362 subjects with suspected mTBI and admitted to the Emergency department of the KAT General Hospital of Athens, Greece were recruited for the study. All subjects underwent a CT head scan to establish the diagnosis of mTBI. GFAP and UCH-L1 were measured using Alinity I (Abbott). 163 healthy subjects served as controls. Results Using the manufacturer’s cut-offs (35 ng/L for GFAP and 400 ng/L for UCH-L1), the “GFAP/UCH-L1” mTBI test had a sensitivity of 99.1 % and a specificity of 40.6 %. However, the specificity dropped to 14.9 % in patients older than 65 years old. By defining a new cut-off of 115 ng/L for GFAP and 335 ng/L specifically for patients older than 65 years, specificity was increased up to 30.6 % without changing test sensitivity and the number of CT head scans avoided was doubled in this subgroup. Conclusions The “GFAP/UCH-L1” mTBI test is an efficient “rule-out test” to exclude patients suffering from mTBI. By adjusting the cut-offs in patients older than 65 years old, we could significantly increase the number of CT head scans avoided without affecting the sensitivity. These new cut-offs should be externally validated.

Objectives Urinary steroid profiling after hydrolysis of conjugates is an emerging tool to differentiate aggressive adrenocortical carcinomas (ACC) from benign adrenocortical adenomas (ACA). However, the shortcomings of deconjugation are the lack of standardized and fully validated hydrolysis protocols and the loss of information about the originally conjugated form of the steroids. This study aimed to evaluate the quality of the deconjugation process and investigate novel diagnostic biomarkers in urine without enzymatic hydrolysis. Methods 24 h urine samples from 40 patients with ACC and 40 patients with ACA were analyzed by untargeted metabolomics using liquid chromatography-high-resolution mass spectrometry both unmodified and after hydrolysis with arylsulfatase/glucuronidase from Helix pomatia. Both approaches were compared regarding the differentiation of ACC vs. ACA via ROC analyses and to evaluate the hydrolyzation efficiency of steroid conjugates. Results Steroid glucuronides were fully deconjugated, while some disulfates and all monosulfates were still largely detectable after enzymatic hydrolysis, suggesting incomplete and variable deconjugation. In unhydrolyzed urine, steroid monosulfates showed the best differentiation between ACC and ACA (highest AUC=0.983 for C 21 H 32 O 6 S, followed by its isomer and two isomers with the molecular formula C 21 H 32 O 7 S). Moreover, several disulfates were highly abundant and increased in ACC compared to ACA. Conclusions This work highlights the limitations of hydrolyzing steroid conjugates before analysis and shows a possible superiority of a direct analysis approach compared to a hydrolysis approach from a methodological point of view and regarding diagnostic accuracy. Several steroid conjugates were found as promising diagnostic biomarkers for differentiation between ACC and ACA.

Objectives An analytical protocol based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), which includes a peptide-based calibration strategy, was developed and validated for the determination of cardiac troponin I (cTnI) levels in clinical samples. Additionally, the developed method was compared with a protein-based calibration strategy, using cTnI serving as a model for low-abundant proteins. The aim is to evaluate new approaches for protein quantification in complex matrices, supporting the metrology community in implementing new methods and developing fit-for-purpose SI- traceable peptide or protein primary calibrators. Methods To establish traceability to SI units, peptide impurity correction amino acid analysis (PICAA) was conducted to determine the absolute content of signature peptides in the primary standards. Immunoaffinity enrichment was used to capture cTnI from human serum, with a comparison between microbeads and nanobeads to improve enrichment efficiency. Parallel reaction monitoring was used to monitor two signature peptides specific to cTnI. Various digestion parameters were optimized to achieve complete digestion. Results The analytical method demonstrated selectivity and specificity, allowing the quantification of cTnI within 0.9–22.0 μg/L. The intermediate precision RSD was below 28.9 %, and the repeatability RSD was below 5.8 % at all concentration levels, with recovery rates ranging from 87 % to 121 %. The comparison of calibration strategies showed similar LOQ values, but the peptide-based calibration exhibited significant quantitative bias in recovery rates. The data are available via ProteomeXchange (PXD055104). Conclusions This isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method, based on peptide calibration, successfully quantified cTnI in human serum. Comparing this with protein-based calibration highlighted both the strengths and potential limitations of peptide-based strategies.

Objectives The aim of the study was to evaluate the predictive value of cell population data (CPD) parameters in comparison with procalcitonin (PCT) and C-reactive protein (CRP) for an early diagnosis of sepsis in intensive care unit (ICU). The effect of renal function on CPD, PCT and CRP, in septic and non-septic patients was also investigated. Methods This is a retrospective, observational and single-center study, performed with data collected from patients consecutively admitted to the ICU of the Edoardo Bassini Hospital in Milan. Patients were divided in septic and non-septic according to Sepsis-III criteria. The control group was formed by critically ill patients without sepsis. Patients with sepsis were further divided in patients with sepsis and patients with septic shock. Results A significant difference between septic and non-septic patients was found for neutrophils complexity (NE-SSC), neutrophils fluorescence intensity (NE-SFL), width of dispersion of neutrophils fluorescence (NE-WY), monocytes complexity (MO-X), monocytes fluorescence intensity (MO-Y), PCT and CRP parameters. PCT, neutrophils sixe (NE-FSC), NE-WY, width of dispersion of neutrophils size (NE-WZ) and MO-X discriminated sepsis and septic-shock patients. CPD parameters were not influenced by renal function. CPD, PCT and CRP had a heterogeneous diagnostic performance efficiency in the prediction of sepsis. Overall, NE-SSC, NE-SFL, width of dispersion of neutrophils complexity (NE-WX), MO-X, MO-Y, PCT and CRP displayed the best diagnostic performance for sepsis. Conclusions This study suggested that some CPD parameters (i.e., NE-SFL and MO-X) might provide useful information for diagnosis and management of sepsis.