Verification policies in Croatian medical biochemistry laboratories: a survey of the practice.

Introduction: The aim of this study was to screen practices used in verification procedures for methods/analysers among medical biochemistry laboratories (MBLs) in Croatia. We hypothesized that these procedures differ widely from laboratory to laboratory and wanted to gather specific data on steps used in the verification workflow. Materials and methods: In order to obtain data, an online survey was conducted. The survey, divided in two sections, contained 29 questions and statements addressing general characteristics and specific steps of the verification workflow of each individual MBL. The survey was disseminated among managers of all MBLs in Croatia. Results: A total of 108/196 (55%) laboratories participated in the survey. Forty nine MBLs were excluded from the second part of the survey: 14 have not implemented verification procedures, and 35 MBLs due to the absence of answers. The most relevant results of the second part of the survey showed that: 18/59 (0.31) of the responding MBLs have difficulties when defining acceptance criteria, 27/59 (0.46) used the Clinical and Laboratory Standards Institute protocol for precision estimation; the majority of MBLs used a median of 20 samples for method/analyser comparisons and estimated bias using internal quality control samples; reference intervals provided by external sources are mainly adopted; 60% of MBLs do not include linearity verification in their protocol and do not use the national document for the estimation of measurement uncertainty. Conclusions: Heterogeneous verification protocols are routinely utilized across Croatian MBLs which clearly confirms that a national document might help in the harmonization of verification procedures.

Comparison of diagnostic accuracy for eight SARS-CoV-2 serological assays.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests have been suggested as an additional diagnostic tool in highly suspected cases with a negative molecular test and determination of seroprevalence in population. We compared the diagnostic performance of eight commercial serological assays for IgA, IgM, and IgG antibodies to the SARS-CoV-2 virus. MATERIALS AND METHODS: The comparison study was performed on a total of 76 serum samples: 30 SARS-CoV-2 polymerase chain reaction (PCR)-negative and 46 SARS-CoV-2 PCR-positive patients with asymptomatic to severe disease and symptoms duration from 3-30 days. The study included: three rapid lateral flow immunochromatographic assays (LFIC), two enzyme-linked immunosorbent assays (ELISA), and three chemiluminescence immunoassays (CLIA). RESULTS: Agreement between IgM assays were minimal to moderate (kappa 0.26 to 0.63) and for IgG moderate to excellent (kappa 0.72 to 0.92). Sensitivities improved with > 10 days of symptoms and were: 30% to 89% for IgM; 89% to 100% for IgG; 96% for IgA; 100% for IgA/IgM combination; 96% for total antibodies. Overall specificities were: 90% to 100% for IgM; 85% to 100% for IgG; 90% for IgA; 70% for IgA/IgM combination; 100% for total antibodies. Diagnostic accuracy for IgG ELISA and CIA assays were excellent (AUC ≥ 0.90), without significant difference. IgA showed significantly better diagnostic accuracy than IgM (P < 0.001). CONCLUSION: There is high variability between IgM assays independently of the assay format, while IgG assays showed moderate to perfect agreement. The appropriate time for testing is crucial for the proper immunity investigation.

Validation and verification of examination procedures in medical laboratories: opinion of the EFLM Working Group Accreditation and ISO/CEN standards (WG-A/ISO) on dealing with ISO 15189:2012 demands for method verification and validation.

This paper reflects the opinion of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO). It aims to provide guidance for drawing up local/national documents about validation and verification of laboratory methods. We demonstrate how risk evaluation can be used to optimize laboratory policies to meet intended use requirements as well as requirements of standards. This is translated in a number of recommendations on how to introduce risk evaluation in various stages of the implementation of new methods ultimately covering the whole process cycle.

Documenting metrological traceability as intended by ISO 15189:2012: A consensus statement about the practice of the implementation and auditing of this norm element.

ISO15189:2012 requires medical laboratories to document metrological traceability of their results. While the ISO17511:2003 standard on metrological traceability in laboratory medicine requires the use of the highest available level in the traceability chain, it recognizes that for many measurands there is no reference above the manufacturer's selected measurement procedure and the manufacturer's working calibrator. Some immunoassays, although they intend to measure the same quantity and may even refer to the same reference material, unfortunately produce different results because of differences in analytical selectivity as manufacturers select different epitopes and antibodies for the same analyte. In other cases, the cause is the use of reference materials, which are not commutable. The uncertainty associated with the result is another important aspect in metrological traceability implementation. As the measurement uncertainty on the clinical samples is influenced by the uncertainty of all steps higher in the traceability chain, laboratories should be provided with adequate and appropriate information on the uncertainty of the value assignment to the commercial calibrators that they use. Although the between-lot variation in value assignment will manifest itself as part of the long-term imprecision as estimated by the end-user, information on worst-case to be expected lot-lot variation has to be communicated to the end-user by the IVD provider. When laboratories use ancillary equipment that potentially could have a critical contribution to the reported results, such equipment needs verification of its proper calibration and criticality to the result uncertainty could be assessed by an approach based on risk analysis, which is a key element of ISO15189:2012 anyway. This paper discusses how the requirement for metrological traceability as stated in ISO15189 should be met by the medical laboratory and how this should be assessed by accreditation bodies.

Relevance of KFLC quantification to differentiate clinically isolated syndrome from multiple sclerosis at clinical onset.

OBJECTIVE: The objective of the research was to evaluate diagnostic and predictive value for determination of KFLC in cerebrospinal fluid (CSF) compared to the qualitative procedure of OCB determination in patients with CIS who converted to MS during a two-year period. PATIENTS AND METHODS: KFLC, total immunoglobulin G (IgG), serum albumin and CSF albumin were determined with an immunonephelometric method in 151 patients with suspected MS who were admitted to the Neurology Clinic while CSF/serum quotients (QKFLC, QIgG and QAlb) and indexes were calculated with regards to albumin (QCSF/Qserum). Presence of OCBs was determined by isoelectric focusing with immunofixation. Based on their clinical, OCB and magnetic resonance imaging (MRI) findings, 50 patients were classified as other neurological disorder patients (OND), and 101 patients were classified as CIS, 50 of which converted to MS during the two-year period. ROC analysis, ROC curve comparisons and comparison of median KFLC parameters were used to find optimal cut-off with regards to CIS diagnosis and conversion to MS. RESULTS: CSF KFLC median was 2,01 mg/L in MS group contrary to 0,68 mg/L and 0,17 mg/L in CIS and OND group, and KFLC index was 33,52 mg/L contrary to 9,68 mg/L and 3,71 mg/L (p < 0,0001). ROC analysis for accuracy of detection of intrathecal synthesis for QKFLC and KFLC index showed an AUC of 0,891 and 0,839 and the cut-off of 0,027 and 8,82, respectively (sensitivity 73,2% and 71,3%; specificity 96,0% and 98,0%; +PV 97,4% and 98,6%). The diagnostic accuracy of KFLC index for conversion from CIS to MS showed AUC of 0,840 and a cut-off of 9,092 (sensitivity 90,0%; specificity 73,3%; -PV 93,7%). Life age correlates significantly with serum KFLC (r = 0,34; p < 0,0001) and through aging process lower KFLC indexes can be expected, i.e. likelihood of false negative diagnoses. CONCLUSION: KFLC index showed diagnostic value, although it is not more specific and more sensitive than OCB. Application of KFLC might serve as a screening method while OCB could be used in uninterpretted cases only. Patients who converted to MS have significantly higher KFLC which can contribute to an early diagnosis and prompt therapy with its predictive role.

Comparison of Enzymatic Assay for HBA1C Measurement (Abbott Architect) With Capillary Electrophoresis (Sebia Minicap Flex Piercing Analyser).

OBJECTIVE: To compare the analytical performances of the enzymatic method (EM) and capillary electrophoresis (CE) for hemoglobin A1c (HbA1c) measurement. METHODS: Imprecision, carryover, stability, linearity, method comparison, and interferences were evaluated for HbA1c via EM (Abbott Laboratories, Inc) and CE (Sebia). RESULTS: Both methods have shown overall within-laboratory imprecision of less than 3% for International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) units (<2% National Glycohemoglobin Standardization Program [NGSP] units). Carryover effects were within acceptable criteria. The linearity of both methods has proven to be excellent (R2 = 0.999). Significant proportional and constant difference were found for EM, compared with CE, but were not clinically relevant (<5 mmol/mol; NGSP <0.5%). At the clinically relevant HbA1c concentration, stability observed with both methods was acceptable (bias, <3%). Triglyceride levels of 8.11 mmol per L or greater showed to interfere with EM and fetal hemoglobin (HbF) of 10.6% or greater with CE. CONCLUSION: The enzymatic method proved to be comparable to the CE method in analytical performances; however, certain interferences can influence the measurements of each method.

Corrigendum to: Minimum requirements for the estimation of the measurement uncertainty: Recommendations of the joint Working group for uncertainty of measurement of the CSMBLM and CCMB.

[This corrects the article DOI: 10.11613/BM.2017.030502.].

Minimum requirements for the estimation of measurement uncertainty: Recommendations of the joint Working group for uncertainty of measurement of the CSMBLM and CCMB.

The International vocabulary of metrology - Basic and general concepts and associated terms (VIM3, 2.26 measurement uncertainty, JCGM 200:2012) defines uncertainty of measurement as a non-negative parameter characterizing the dispersion of the quantity values being attributed to a measurand, based on the information obtained from performing the measurement. Clinical Laboratory Standards Institute (CLSI) has published a very detailed guideline with a description of sources contributing to measurement uncertainty as well as different approaches for the calculation (Expression of measurement uncertainty in laboratory medicine; Approved Guideline, CLSI C51-A 2012). Many other national and international recommendations and original scientific papers about measurement uncertainty estimation have been published. In Croatia, the estimation of measurement uncertainty is obligatory for accredited medical laboratories. However, since national recommendations are currently not available, each of these laboratories uses a different approach in measurement uncertainty estimation. The main purpose of this document is to describe the minimal requirements for measurement uncertainty estimation. In such way, it will contribute to the harmonization of measurement uncertainty estimation, evaluation and reporting across laboratories in Croatia. This recommendation is issued by the joint Working group for uncertainty of measurement of the Croatian Society for Medical Biochemistry and Laboratory Medicine and Croatian Chamber of Medical Biochemists. The document is based mainly on the recommendations of Australasian Association of Clinical Biochemists (AACB) Uncertainty of Measurement Working Group and is intended for all medical biochemistry laboratories in Croatia.

Recommendation for the review of biological reference intervals in medical laboratories.

This document is based on the original recommendation of the Expert Panel on the Theory of Reference Values of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), updated guidelines were recently published under the auspices of the IFCC and the Clinical and Laboratory Standards Institute (CLSI). This document summarizes proposals for recommendations on: (i) The terminology, which is often confusing, noticeably concerning the terms of reference limits and decision limits. (ii) The method for the determination of reference limits according to the original procedure and the conditions, which should be used. (iii) A simple procedure allowing the medical laboratories to fulfill the requirements of the regulation and standards. The updated document proposes to verify that published reference limits are applicable to the laboratory involved. Finally, the strengths and limits of the revised recommendations (especially the selection of the reference population, the maintenance of the analytical quality, the choice of the statistical method used…) will be briefly discussed.

Interference of M-protein on prothrombin time test - case report.

The aim of this report was to present a case of interference on prothrombin time (PT) test that directed further laboratory diagnostics and resulted with final detection of monoclonal gammopathy in an 88-year old man. Routine coagulation testing during medical examination at Emergency Department revealed unmeasurable PT (< 7% activity) and activated partial thromboplastin time (aPTT) within reference range. After repeated sampling for coagulation testing, PT was unmeasurable again, as well as fibrinogen level (< 0.8 g/L), thrombin time (TT) was significantly prolonged (107 seconds) and aPTT was within reference range. In both plasma samples refrigerated at 4 ˚C overnight, white gelatinous precipitate was visible between the cell and plasma layers and the presence of monoclonal protein (M-protein) was suggested in our patient. Further laboratory diagnostics revealed total serum proteins at concentration of 123 g/L and the presence of M-protein IgG lambda (λ) at concentration of 47.1 g/L. These results suggested monoclonal gammopathy as an underlying pathophysiological condition in our patient. Activities of coagulation factors II, V, VII and X were within reference ranges or increased. These results and correction of unmeasurable PT result to 67% in mixing test with commercial normal plasma suggest in vitro rather than in vivo interference of M-protein on PT result. In contrast, significantly prolonged TT results in all analysed samples suggest impact of M-protein on this global coagulation test due to possible effect on fibrin polymerization.

Accreditation process in European countries - an EFLM survey.

BACKGROUND: Accreditation is a valuable resource for medical laboratories. The development of quality systems based on ISO 15189 has taken place in many laboratories in the European countries but data about accreditation remain scarce. The EFLM Working Group "Accreditation and ISO/CEN standards" conducted a survey that reviews the current state of the accreditation process in European countries. METHODS: An on-line questionnaire was addressed to delegates of 39 EFLM scientific societies in March 2014. One answer by country was taken into account. The survey was dealing with mandatory status, number of accredited medical laboratories in each country, possibility of flexible scope and concerned medical fields. The status of point-of-care testing (POCT) in each country was also studied. RESULTS: Twenty-nine responses (74%) were registered. All the assessed countries (100%) have begun an accreditation process in various ways. All the national accreditation bodies (NAB) offer or are working to offer an ISO 15189 accreditation. The accreditation process most often concerns all phases of the examination and various medical fields. Medical laboratories are responsible for POCT in 20 (69%) countries. The accreditation process for POCT, according to ISO 15189 and ISO 22870, is also developing. CONCLUSIONS: While there are several variations in the approaches to accreditation of medical laboratories in the European countries, the ISO 15189 accreditation project has been widely accepted. The use of a unique standard and the cooperation among countries due to scientific societies, EFLM, accreditation bodies and EA enable laboratory professionals to move toward uniform implementation of the accreditation concept.

Flexible scope for ISO 15189 accreditation: a guidance prepared by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO).

The recent revision of ISO15189 has further strengthened its position as the standard for accreditation for medical laboratories. Both for laboratories and their customers it is important that the scope of such accreditation is clear. Therefore the European co-operation for accreditation (EA) demands that the national bodies responsible for accreditation describe the scope of every laboratory accreditation in a way that leaves no room for doubt about the range of competence of the particular laboratories. According to EA recommendations scopes may be fixed, mentioning every single test that is part of the accreditation, or flexible, mentioning all combinations of medical field, examination type and materials for which the laboratory is competent. Up to now national accreditation bodies perpetuate use of fixed scopes, partly by inertia, partly out of fear that a too flexible scope may lead to over-valuation of the competence of laboratories, most countries only use fixed scopes. The EA however promotes use of flexible scopes, since this allows for more readily innovation, which contributes to quality in laboratory medicine. In this position paper, the Working Group Accreditation and ISO/CEN Standards belonging to the Quality and Regulation Committee of the EFLM recommends using an approach that has led to successful introduction of the flexible scope for ISO15189 accreditation as intended in EA-4/17 in The Netherlands. The approach is risk-based, discipline and competence-based, and focuses on defining a uniform terminology transferable across the borders of scientific disciplines, laboratories and countries.

The role of classic risk factors and prothrombotic factor gene mutations in ischemic stroke risk development in young and middle-aged individuals.

BACKGROUND: In young individuals, a genetically predisposing hypercoagulability and classic modifying risk factors can act synergistically on the ischemic stroke risk development. The aim of the study was to compare the prevalence of classic vascular risk factors and polymorphisms of the G20210A coagulation factor II (prothrombin), Arg506Glu coagulation factor V Leiden, C677T methylenetetrahydrofolate reductase (MTHFR), and 4G/5G plasminogen activator inhibitor-1 (PAI-1) and the impact of these gene mutations and classic vascular risk factors on the overall stroke risk in individuals aged 55 years or younger. METHODS: The study included 155 stroke patients aged 55 years or younger and 150 control subjects. Stroke prevalence and odds ratio (OR) were assessed for the following parameters: G20210A prothrombin, Arg506Glu factor V Leiden, C677T MTHFR, and 4G/5G PAI-1 polymorphisms; total number of study polymorphisms in a particular subject (genetic sum); and classic vascular risk factors of hypertension, obesity, diabetes mellitus, cigarette smoking, hypercholesterolemia, hypertriglyceridemia, and elevated levels of low-density lipoprotein (LDL) cholesterol and very low-density lipoprotein cholesterol. RESULTS: The prevalence of hypertension (P < .001), smoking (P < .001), decreased HDL cholesterol levels (P < .001), obesity (P = .001), elevated LDL cholesterol (P = .036), C677T MTHFR polymorphism (P < .001), and genetic sum was significantly higher in the group of stroke patients. The following parameters were found to act as independent risk factors for ischemic stroke: decreased HDL cholesterol level (P < .001; OR 4.618; 95% confidence interval [CI] 2.381-8.957); hypertension (P = .001; OR 2.839; 95% CI 1.519-5.305); obesity (P = .040; OR 2.148; 95% CI 1.036-4.457); smoking (P = .001; OR 2.502; 95% CI 1.436-4.359); and genetic sum as a continuous variable (P < .01; OR 2.307; 95% CI 1.638-3.250). CONCLUSIONS: Gene mutations of the procoagulable and proatherosclerotic factors investigated exerted a synergistic action in the development of overall risk of ischemic stroke in young and middle-aged individuals.

The prevalence of preanalytical errors in a Croatian ISO 15189 accredited laboratory.

BACKGROUND: The preanalytical phase is the most common source of laboratory errors. The goal of this descriptive study was to analyze the prevalence and type of preanalytical errors in relation to the site of sample collection (inpatient vs. outpatient) and the type of laboratory unit (hematology and coagulation vs. biochemistry). For the biochemistry unit, the data were also analyzed relative to the type of the analysis (stat vs. routine). METHODS: We retrospectively analyzed the sample and test request form error rate for a 1-year period, from January to December 2008. RESULTS: The frequency of the sample errors differed significantly between the emergency and routine biochemistry unit (0.69% vs. 2.14%; p<0.0001), and between inpatients and outpatients (1.12% vs. 1.36%; p=0.0006). Hemolysis was the most frequent sample error, accounting for 65% of all unsuitable specimens in the emergency biochemistry unit. The total sample error rate did not differ between hematology and coagulation vs. the biochemistry unit. The frequency of test request form errors differed significantly with respect to the sample collection site (p<0.0001), laboratory unit (p<0.0001) and type of the analysis (p<0.0001). Errors in the test request form were least frequent in the outpatient unit (2.98%) and most frequent in the routine biochemistry unit (65.94%). CONCLUSIONS: Sample and test request form errors in our laboratory are occurring with a frequency comparable to that reported by others. Continuous educational action is needed for all stakeholders involved in laboratory testing to improve the quality of the preanalytical phase of the total testing process.

Antibodies targeting mutated citrullinated vimentin in patients with psoriatic arthritis.

Antibodies against mutated citrullinated vimentin (anti-MCV) are of a comparable diagnostic value in rheumatoid arthritis (RA) as antibodies targeting citrullinated peptides (anti-CCP). Anti-CCP are present in up to 15% of psoriatic arthritis (PsA) patients, while the prevalence of anti-MCV in PsA patients has been poorly investigated. The aim of the present study was to assess the prevalence and relevance of anti-MCV antibodies in PsA patients. The study included 56 PsA patients. Clinical features, disease activity, and functional ability were noted by an experienced rheumatologist. Serum samples of all patients were analyzed for anti-MCV and anti-CCP antibodies using enzyme-linked immunosorbent assay. Data on 92 patients with RA, 44 patients with other inflammatory rheumatic diseases, and 107 healthy controls from a previous study were used to compare the prevalence of anti-MCV antibodies in PsA patients. Anti-MCV antibodies were positive in only two out of 56 (3.6%) PsA patients, which was significantly lower compared to RA patients (63%). The anti-MCV level was moderately positive and borderline in one patient each. Both patients had asymmetric polyarthritis, dactylitis, moderate to high disease activity, and were anti-CCP and rheumatoid factor (RF) negative. There was no significant difference in anti-MCV levels according to clinical subtypes of PsA and no correlation of anti-MCV levels with anti-CCP, RF, disease activity variables, and functional ability indices. According to study results, anti-MCV antibodies can be detected in a very small proportion of PsA patients with polyarthritic disease and are primarily related to the polyarthritic pattern rather than the specific diagnosis of RA.

Matrix metalloproteinases and their inhibitors in different acute stroke subtypes.

The aim of the study was to determine serum levels of selected matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs) in the acute phase of different stroke types subdivided according to the Oxfordshire Community Stroke Project (OCSP) classification and the possibility of discriminating stroke types according to their levels. The study included 126 patients with acute stroke within the first 24 h of symptom onset, and 124 healthy volunteers. The stroke group had lower MMP-2 concentrations and MMP-2/TIMP-2 ratios (p<0.001) but higher TIMP-2 (p<0.001) than controls. The level of MMP-9 and the MMP-9/TIMP-1 ratio were higher in patients with total anterior circulation infarct (TACI) than in patients with other stroke subtypes according to OCSP classification (p=0.0019, p=0.0065, respectively) or in controls (p<0.0001, p=0.0024, respectively). A negative correlation of MMP-2 levels with MMP-9 and MMP-9/TIMP-1 ratio was recorded in all stroke subtypes except for TACI. Receiver operating characteristic analysis showed similar discriminating power for MMP-9 levels and Barthel index in the differential diagnosis of TACI. High MMP-9/TIMP-1 ratio (odds ratio 3.263) was associated with TACI. Our results demonstrate that the MMP-9/TIMP-1 ratio may provide information to help in assessing stroke patients in the future as a baseline biomarker of infarct extent.