How to assess the quality of your analytical method?

Laboratory medicine is amongst the fastest growing fields in medicine, crucial in diagnosis, support of prevention and in the monitoring of disease for individual patients and for the evaluation of treatment for populations of patients. Therefore, high quality and safety in laboratory testing has a prominent role in high-quality healthcare. Applied knowledge and competencies of professionals in laboratory medicine increases the clinical value of laboratory results by decreasing laboratory errors, increasing appropriate utilization of tests, and increasing cost effectiveness. This collective paper provides insights into how to validate the laboratory assays and assess the quality of methods. It is a synopsis of the lectures at the 15th European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Continuing Postgraduate Course in Clinical Chemistry and Laboratory Medicine entitled "How to assess the quality of your method?" (Zagreb, Croatia, 24-25 October 2015). The leading topics to be discussed include who, what and when to do in validation/verification of methods, verification of imprecision and bias, verification of reference intervals, verification of qualitative test procedures, verification of blood collection systems, comparability of results among methods and analytical systems, limit of detection, limit of quantification and limit of decision, how to assess the measurement uncertainty, the optimal use of Internal Quality Control and External Quality Assessment data, Six Sigma metrics, performance specifications, as well as biological variation. This article, which continues the annual tradition of collective papers from the EFLM continuing postgraduate courses in clinical chemistry and laboratory medicine, aims to provide further contributions by discussing the quality of laboratory methods and measurements and, at the same time, to offer continuing professional development to the attendees.

Continuing professional development crediting system for specialists in laboratory medicine within 28 EFLM national societies.

INTRODUCTION: Continuing professional development (CPD) with corresponding crediting system is recognized as essential for the laboratory medicine specialists to provide optimal service for the patients. Article presents results of the survey evaluating current CPD crediting practice among members of European Federation of Clinical Chemistry and Laboratory Medicine (EFLM). MATERIALS AND METHODS: A questionnaire had been forwarded to presidents/national representatives of all EFLM members, with invitation to provide information about CPD programmes and crediting policies, as well as feedback on individual CPD categories, through scoring their relevance. RESULTS: Complete or partial answers were received from 28 of 38 members. In 23 countries, CPD programmes exist and earn credits, with 19 of them offering access to non-medical scientists. CPD activities are evaluated in all participating countries, regardless to the existence of an official CPD programme. Among participating members with mandatory specialists' licensing (22/28), CPD is a prerequisite for relicensing in 13 countries. Main categories recognized as CPD are: continuing education (24 countries), article/book (17/14 countries) authorship and distance learning (14 countries). The highest median score of relevance (20) is allocated to professional training, editor/authorship and official activities in professional organizations, with the first category showing the least variation among scores. CONCLUSIONS: Majority of EFLM members have developed CPD programmes, regularly evaluated and accompanied by crediting systems. Programmes differ in accessibility for non-medical scientists and impact on relicensing eligibility. Continuing education, authorship and e-learning are mainly recognized as CPD activities, although the professional training is appreciated as the most important individual CPD category.

Position paper of the EPMA and EFLM: a global vision of the consolidated promotion of an integrative medical approach to advance health care.

The authors consider acute problems in the quality and management of medical services challenging health care systems worldwide. This actuality has motivated the representatives of the European Association for Predictive, Preventive and Personalised Medicine and European Federation of Clinical Chemistry and Laboratory Medicine to consider the efforts in promoting an integrative approach based on multidisciplinary expertise to advance health care. The current paper provides a global overview of the problems related to medical services: pandemic scenario in the progression of common chronic diseases, delayed interventional approaches of reactive medicine, poor economy of health care systems, lack of specialised educational programmes, problematic ethical aspects of treatments as well as inadequate communication among professional groups and policymakers. Further, in the form of individual paragraphs, the article presents a consolidated position of the represented European organisations. This position is focused on the patients' needs, expert recommendations for the relevant medical fields and plausible solutions which have a potential to advance health care services if the long-term strategies were to be effectively implemented as proposed here.

A position paper of the EFLM Committee on Education and Training and Working Group on Distance Education Programmes/E-Learning: developing an e-learning platform for the education of stakeholders in laboratory medicine.

The progress of information and communication technologies has strongly influenced changes in healthcare and laboratory medicine. E-learning, the learning or teaching through electronic means, contributes to the effective knowledge translation in medicine and healthcare, which is an essential element of a modern healthcare system and for the improvement of patient care. E-learning also represents a great vector for the transfer knowledge into laboratory practice, stimulate multidisciplinary interactions, enhance continuing professional development and promote laboratory medicine. The European Federation of Laboratory Medicine (EFLM) has initiated a distance learning program and the development of a collaborative network for e-learning. The EFLM dedicated working group encourages the organization of distance education programs and e-learning courses as well as critically evaluate information from courses, lectures and documents including electronic learning tools. The objectives of the present paper are to provide some specifications for distance learning and be compatible with laboratory medicine practices.

Clinical chemistry and laboratory medicine in Croatia: regulation of the profession.

Heterogeneity exists across Europe in the definition of the profession of clinical chemistry and laboratory medicine and also in academic background of specialists in this discipline. This article provides an overview of the standards of education and training of laboratory professionals and quality regulations in Croatia. Clinical chemistry in Croatia is almost exclusively practiced by medical biochemists. Although term Medical biochemist often relates to medical doctors in other European countries, in Croatia medical biochemists are not medical doctors, but university degree professionals who are qualified scientifically. Practicing the medical biochemistry is regulated by The Health Care Law, The Law of the Medical Biochemistry Profession and The Law of the State and Private Health Insurance. According to the law, only medical biochemists are entitled to run and work in the medical biochemistry laboratory. University degree is earned after the 5 years of the studies. Register for medical biochemists is kept by the Croatian Chamber of Medical Biochemists. Licensing is mandatory, valid for 6 years and regulated by the government (Law on the Health Care, 1993). Vocational training for medical biochemists lasts 44 months and is regulated by the national regulatory document issued by the Ministry of Health. Accreditation is not mandatory and is provided by an independent, non-commercial national accreditation body. The profession has interdisciplinary character and a level of required competence and skills comparable to other European countries.

Efficiency of test report delivery to the requesting physician in an outpatient setting: an observational study.

BACKGROUND: Clinical laboratories accredited according to ISO 15189 quality standards are obliged to implement and continuously monitor quality indicators for evaluation of the laboratory's contribution to patient care. Reporting laboratory results to the requesting physician is one important phase of the clinical laboratory testing process. Failure to report results may indicate the ineffectiveness of the laboratory service. We aimed to analyze the proportion and type of laboratory reports for outpatients that were not delivered to the requesting physician. METHODS: This retrospective observational study was conducted during an 11-month period from January to December 2007 at our outpatient biochemistry laboratory unit. Data on demographic characteristics, request types and laboratory findings for all uncollected reports were retrieved from the laboratory information system and compared with one random 2-week representative period. RESULTS: During the study period our laboratory issued 22,445 patient reports with more than 150,000 biochemistry analyses. Of these, 464 (2.1%) were uncollected laboratory reports. When compared to the representative period, patients who never collected their laboratory reports were younger (p<0.001) or suffering from some chronic disease. Routine biochemistry tests were the most prevalent (>50%). The majority of routine biochemistry tests were almost equally represented during the study and representative period, while molecular diagnostic tests were several times more frequently uncollected (p<0.001). Reports with electrolytes, metabolites and glucose were the least likely to be uncollected (p<0.001). The total cost for those tests was 30% of the average monthly laboratory budget. CONCLUSIONS: A significant amount of the laboratory budget is wasted for tests that never reach the requesting physician. Such misutilization of the laboratory reveals the substantial lack of medical necessity for test requests. Further studies are needed to explore the possible efficiency of the various interventions in reducing the volume of unnecessary and erroneous testing.

Comparison of visual vs. automated detection of lipemic, icteric and hemolyzed specimens: can we rely on a human eye?

BACKGROUND: Results from hemolyzed, icteric, and lipemic samples may be inaccurate and can lead to medical errors. These preanalytical interferences may be detected using visual or automated assessment. Visual inspection is time consuming, highly subjective and not standardized. Our aim was to assess the comparability of automated spectrophotometric detection and visual inspection of lipemic, icteric and hemolyzed samples. METHODS: This study was performed on 1727 routine biochemistry serum samples. Automated detection was performed using the Olympus AU2700 analyzer. We assessed: 1) comparability of visual and automated detection of lipemic, icteric and hemolyzed samples, 2) precision of automated detection, and 3) inter-observer variability for visual inspection. RESULTS: Weighted kappa coefficients for comparability of visual and automated detection were: 0.555, 0.529 and 0.638, for lipemic, icteric and hemolyzed samples, respectively. The precision for automated detection was high for all interferences, with the exception of samples being only slightly lipemic. The best overall agreement between observers was present in assessing lipemia (mean weighted kappa=0.698), whereas the lowest degree of agreement was observed in assessing icterus (mean weighted kappa=0.476). CONCLUSIONS: Visual inspection of lipemic, icteric and hemolyzed samples is highly unreliable and should be replaced by automated systems that report serum indices.

Metabolic control in type 2 diabetes is associated with sulfonylurea receptor-1 (SUR-1) but not with KCNJ11 polymorphisms.

BACKGROUND AND AIMS: Sulfonylureas are hypoglycemic agents used for promotion of insulin secretion in type 2 diabetics (T2D). They bind to sulfonylurea receptor-1 (SUR-1), which is a functional subunit of the ATP-sensitive potassium channel (K(ATP)). The other component of the potassium channel is Kir6.2, encoded by gene KCNJ11. Polymorphisms in these genes may lead to modulated response to sulfonylurea therapy. The aim of this study was to determine a relationship between SUR-1 [exon 16 (-3C/T), exon 31 (Arg1273Arg; AGG-->AGA) and exon 33 (S1369A)] and KCNJ11 (E23K) polymorphisms and the following parameters of metabolic control in T2D: fasting plasma glucose (FPG), postprandial glucose (PPG) and HbA1c in Caucasian T2D of European origin. METHODS: A total of 228 unrelated patients with T2D on sulfonylurea therapy were included in the study. Genotyping of all polymorphisms was performed by PCR-RFLP method. Biochemical parameters were determined using standard laboratory methods. RESULTS: There was no difference in FPG and PPG concentration in any of the genotype subgroups. However, diabetics with wild-type C/C genotype of the SUR-1 exon 16 polymorphism had significantly lower HbA1c concentration compared to the patients with variant T/T genotype [6.9 (6.2-7.7) mmol/L vs. 8.1 (6.7-8.8) mmol/L; p=0.009]. Also, patients with wild-type G/G genotype of the SUR-1 exon 31 polymorphism had significantly higher HbA1c concentration compared to the patients with variant A/A genotype [7.8 (6.9-8.8) mmol/L vs. 6.3 (5.7-6.8) mmol/L; p<0.001]. CONCLUSIONS: SUR-1 exon 16 and exon 31 polymorphisms are significantly associated with HbA1c concentration.

Methodological issues in genetic association studies of inherited thrombophilia: original report of recent practice.

The aims of this article are to evaluate the methodological quality of genetic association studies on the inherited thrombophilia published during 2003 to 2005, to identify the most common mistakes made by authors of those studies, and to examine if overall quality of the article correlates with the quality of the journal. Articles were evaluated by 2 independent reviewers using the checklist of 16 items. A total of 58 eligible studies were identified. Average total score was 7.59 +/- 1.96. Total article score did not correlate with the journal impact factor (r = 0.3971; 95% confidence interval [CI], 0.1547-0.5944, P = .002). Total score did not differ across years (P = .624). Finally, it is concluded that methodological quality of genetic association studies is not optimal, and it does not depend on the quality of the journal. Journals should adopt methodological criteria for reporting the genetic association studies, and editors should encourage authors to strictly adhere to those criteria.

Association of CYP2C9 gene polymorphism with bleeding as a complication of warfarin therapy.

The aim of this study was to determine the association of bleeding as a complication of warfarin therapy with polymorphism of CYP2C9 gene (alleles 1, 2 and 3). The CYP2C9 is the main enzyme for warfarin metabolism. Study included 181 patients receiving warfarin for at least one month. Allele 1 of CYP2C9 gene (in 94.5%) and genotype *1/*1 (57.5%) prevailed. Allele 3 was found in 12.7% patients. Bleeding side-effects occurred in 18 patients (10%). Patients with allele *1 needed significantly higher maintenance warfarin dose (p=0.011). Those with allele *3 had significantly lower maintenance warfarin dose (p=0.005) and higher prothrombin time (PT) at induction (p=0.034). Bleeding occurred significantly more often in those with lower maintenance warfarin dose (p=0.017). Patients with allele *3 had increased risk of bleeding, with marginal significance (p=0.05). Polymorphism of CYP2C9 could determine dose of warfarin therapy and thus it could be related to the risk of bleeding complications. Allele *3 carriers need lower warfarin dose. Therefore, initially reduced warfarin induction dose in allele *3 carriers could avoid more prolonged PT and decrease the risk of bleeding complication.

Pro-inflammatory and anti-inflammatory cytokines in acute ischemic stroke and their relation to early neurological deficit and stroke outcome.

OBJECTIVES: Our aim was to explore (i) the difference in concentration of IL-6, TNF-alpha and IL-10 between acute ischemic stroke patients and control individuals; (ii) the association of plasma cytokine concentration with stroke severity at admission assessed by NIHSS and stroke outcome in 90 days assessed by Barthel index (BI) and modified Rankin scale (mRS). MATERIALS AND METHODS: Study included 68 stroke patients admitted within 12 h of symptoms onset and 71 controls. RESULTS: IL-6 was increased in patients relative to controls (P=0.035) and this increase was associated with severe stroke (P=0.007) and worse outcome (P=0.030 and 0.019; assessed by BI and mRS, respectively), whereas IL-10 was decreased (P=0.044) and associated with better outcome (P=0.043). TNF-alpha did not differ between studied groups (P=0.302). CONCLUSIONS: Increased IL-6 and reduced IL-10 concentrations are present in early stroke period and are associated with a degree of neurological deficit and/or stroke outcome.

Are serum lipids measured on stroke admission prognostic?

BACKGROUND: It has been hypothesized that serum lipids measured in the early period of stroke are predictive of stroke severity and outcome. The optimal time for lipid measurement is not established. We explored whether lipid profile assessed within the first 24 h after stroke onset: (i) differs from that in stroke-free individuals; (ii) differs between stroke subtypes; and (iii) is predictive of stroke severity and outcome. METHODS: We prospectively enrolled 70 acute ischemic stroke patients who presented to the Stroke Unit within 24 h of the onset of stroke symptoms, and 68 stroke-free control subjects. RESULTS: Triglycerides (p<0.001) and high-density lipoprotein (HDL) cholesterol (p<0.001) were significantly lower in patients than in controls. HDL cholesterol was different across stroke subtypes classified according to the Trial of ORG 10172 in Acute Stroke Treatment scale (p=0.035). Patients with more severe stroke had higher serum triglycerides (odds ratio 2.755; p=0.030). CONCLUSIONS: Serum triglycerides might serve as a prognostic marker in acute stroke patients.

Rare TA repeats in promoter TATA box of the UDP glucuronosyltranferase (UGT1A1) gene in Croatian subjects.

BACKGROUND: Gilbert's syndrome is a chronic or recurrent mild unconjugated hyperbilirubinemia caused by decreased activity of UDP glucuronosyltranferase (UGT1A1). The most common cause of Gilbert's syndrome in Caucasians is homozygous variant of the A(TA)7TAA promoter polymorphism. Alleles with five or eight TA repeats have also been described, but they are very rare in Caucasian populations. METHODS: Over a 6-year period (2001-2006), 1109 subjects with suspected Gilbert's syndrome were included in this study. Genotyping of (TA)6 and (TA)7 alleles was performed using high-resolution electrophoretic separation of amplified PCR products on Spreadex EL300 gels. In seven subjects, aberrant electrophoretic patterns were observed and additionally sequenced on an ABI Prism 310 Genetic Analyzer. RESULTS: Genotype distributions for 1102 subjects with (TA)6 or (TA)7 alleles were as follows: 54.10%, 26.33% and 18.94% for the (TA)7/(TA)7, (TA)6/(TA)7 and (TA)6/(TA)6, respectively. Sequencing of seven samples that could not be identified as one of these alleles identified four subjects with the (TA)5/(TA)7, two with the (TA)7/(TA)8 and one with the (TA)6/(TA)8 genotype. CONCLUSION: Genotyping of TA repeats in the promoter region of the UGT1A1 gene revealed the presence of rare alleles with five or eight TA repeats, with a very high frequency of the (TA)7 allele in subjects suspected of having Gilbert's syndrome.

The methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and cancer risk: the Croatian case-control study.

OBJECTIVES: Methylation abnormalities appear to be important for the pathogenesis of many cancer types. Since methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the methylation process catalyzing reduction of 5,10-methylenetetrahydrofolate to 5-methyl-tetrahydrofolate, C677T polymorphism, which decreases enzyme activity, may be associated with cancer susceptibility. The aim of this work was to investigate the distribution of MTHFR C677T polymorphism between various types of cancer and cancer-free controls and to assess if there is a difference in frequency. MATERIALS AND METHODS: 269 Cancer cases (95 prostate cancer, PC; 81 head and neck, HN; and 93 breast cancers, BC) and 102 healthy controls, free of cancer, were genotyped for C677T MTHFR polymorphism using the PCR-RFLP method. RESULTS: There was no overall difference in C677T genotype distribution between total cancer cohort and controls (p=0.064). However, a significant difference and protective OR was found for the C/T genotype (OR=0.574, 95% CI=0.352-0.935). In a comparison of different cancer types and respective controls, genotype frequencies were significantly different between head and neck carcinoma and controls (p=0.004), again with protective role of C/T genotype (OR=0.356, 95% CI=0.189-0.671). Moderate overrepresentation of C/T was found in respective male controls when compared with prostate cancer patients (p value was 0.074 for C/T vs. C/C comparison). The OR for heterozygous C/T genotype in prostate cancer group was 0.404, pointing to its putative protective role. Genotype and allelic frequencies did not differ significantly between 93 breast cancer patients and their 65 age-matched female controls. CONCLUSION: Our data indicate that the C677T MTHFR polymorphism does not significantly contribute to the inherited genetic susceptibility to breast and prostate cancer, while we show some evidence for possible genetic contribution of this polymorphism to the development of head and neck carcinoma.

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.

[Issues of research in medicine]. / Osobitosti istrazivanja u medicini.

Research in medicine is liable to all rules and standards that apply to research in other natural sciences, since medicine as a science and service fully meets the general definition of science: it is a common, integrated, organized and systematized knowledge of mankind, whereby physician--being more or less aware of doing so-- in his daily activities applies scientific thinking and scientific methods. The procedure of problem solving in scientific work and in medical practice is characterized by many similarities as well as variation. In scientific research, the observation of some phenomenon that cannot be explained by the known facts and theories is followed by making a hypothesis, planning and carrying out experimental investigation resulting in some data. Interpretation of these data then provides evidence to confirm or reject the hypothesis. In medical practice, quite a similar procedure is followed; the initial examination of a patient, when his condition cannot be explained by the data thus obtained, is identical to the observation of a phenomenon which cannot be explained by the known facts; working diagnosis would correspond to making the hypothesis; and experimental investigation would compare to laboratory and other diagnostic studies. The working diagnosis is accepted or rejected depending on these results. Of course, there also are differences in the problem solving procedure between scientific research and daily medical practice. For example, in research a single hypothesis is posed, a single experiment with successive testing and/or repeats is performed, whereas in medical practice several hypotheses are made, multiple studies are concurrently performed to reject current hypotheses and to make new ones. Scientific investigation produces an abundance of systematic data, whereas in medical practice target data are being generated, yet not systematically. Definitive decision making also differs greatly, as in scientific research it only ensues from conclusive evidence, whereas in medical practice definitive decision is made and therapeutic procedures are performed even before reaching final evidence. The general strategy of work and research in medicine can be briefly described by four principles, i.e. good knowledge of one's own work; continuing upgrading of one's own work in collaboration with respective institutions (laboratories, university, and research institutes); implementation of standard, up-to-date and scientific methods most of the time; and publishing work results on a regular basis. This strategy ensures constant progress and treatment quality improvement while allowing due validation and evaluation of the work by the society. Scientific research is based on the pre-existing knowledge of the problem under study, and should be supervised, systematic and planned. Research produces data that may represent some new concepts, or such concepts are developed by further data processing. In research, scientific procedure includes a number of steps that have to be made to reach a new scientific result. This procedure includes (a) thinking about a scientific issue; (b) making a scientific hypothesis, i.e. the main objective of the study; (c) research ethics; (d) determination of sources and mode of data collection; (e) research performance; (f) collection and analysis of all research data; (g) interpretation of results and evidence; and (h) publications. The next section of this chapter brings an example of scientific research in the field of medicine, where the procedures carried out during the research are briefly described; other chapters of this supplement deal with statistical methodology used on processing the data obtained in the study, which is most frequently employed in scientific work in the field of medicine.