Ibrahim Unsal

Sample collections from healthy volunteers for biological variation estimates' update: a new project undertaken by the Working Group on Biological Variation established by the European Federation of Clinical Chemistry and Laboratory Medicine.

Abstract Background: Biological variation (BV) data have many fundamental applications in laboratory medicine. At the 1st Strategic Conference of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) the reliability and limitations of current BV data were discussed. The EFLM Working Group on Biological Variation is working to increase the quality of BV data by developing a European project to establish a biobank of samples from healthy subjects to be used to produce high quality BV data. Methods: The project involved six European laboratories (Milan, Italy; Bergen, Norway; Madrid, Spain; Padua, Italy; Istanbul, Turkey; Assen, The Netherlands). Blood samples were collected from 97 volunteers (44 men, aged 20–60 years; 43 women, aged 20–50 years; 10 women, aged 55–69 years). Initial subject inclusion required that participants completed an enrolment questionnaire to verify their health status. The volunteers provided blood specimens once per week for 10 weeks. A short questionnaire was completed and some laboratory tests were performed at each sampling consisting of blood collected under controlled conditions to provide serum, K2EDTA-plasma and citrated-plasma samples. Results: Samples from six out of the 97 enroled subjects were discarded as a consequence of abnormal laboratory measurements. A biobank of 18,000 aliquots was established consisting of 120 aliquots of serum, 40 of EDTA-plasma, and 40 of citrated-plasma from each subject. The samples were stored at –80 °C. Conclusions: A biobank of well-characterised samples collected under controlled conditions has been established delivering a European resource to enable production of contemporary BV data.

Biological Variation Estimates Obtained from 91 Healthy Study Participants for 9 Enzymes in Serum

BACKGROUND We sought to develop estimates of biological variation (BV) for 9 enzymes in blood serum as part of the European Biological Variation Study. METHODS Ninety-one healthy study participants (38 male and 53 female, 21-69 years old) were phlebotomized in each of 10 consecutive weeks at 6 European laboratories. The same preanalytical sample-handling protocol was followed at each center before transport to San Raffaele Hospital, Milan, Italy, for analysis. Sera were stored at -80 °C before analysis in duplicate within a single run on an ADVIA 2400 Clinical Chemistry System (Siemens Healthcare) following a protocol designed to minimize analytical imprecision. Assay traceability was established using frozen sera with target values assigned by reference methods. The results were subjected to outlier analysis before CV-ANOVA to deliver valid BV estimates. Results for 9 enzymes were subsequently partitioned for graphical display allowing visual assessment of the effects of country of origin, sex, and age on BV estimates. RESULTS We found no effect of country upon the observed variation, but overall sex-related differences were evident for alanine amino transferase (ALT), γ-glutamyl transferase (GGT), and creatine kinase (CK). The following estimates for within-subject BV (CVI) and between-subject BV (CVG), respectively, were obtained: ALT: 9.3%, 28.2%; aspartate aminotransferase: 9.5%, 20.3%; GGT: 8.9%, 41.7%; alkaline phosphatase : 5.3%, 24.9%; lactate dehydrogenase: 5.2%, 12.6%; CK: 14.5%, 31.5%; amylase: 6.8%, 30.4%; pancreatic α-amylase: 6.3%, 24.9%; and lipase (LIP): 7.7%, 23.8%. CONCLUSIONS All CVI and some CVG estimates were lower than those reported in the online BV 2014 updated database. Analytical performance specifications derived from BV can be applied internationally.

The EuBIVAS Project: Within–and Between-Subject Biological Variation Data for Serum Creatinine Using Enzymatic and Alkaline Picrate Methods and Implications for Monitoring

BACKGROUND The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) European Biological Variation Study (EuBIVAS) has been established to deliver rigorously determined biological variation (BV) indices. EuBIVAS determined BV for serum creatinine using the enzymatic and alkaline picrate measurement methods. METHOD In total, 91 healthy individuals (38 males, 53 females; age range, 21-69 years) were bled for 10 consecutive weeks at 6 European laboratories. An equivalent protocol was followed at each center. Sera were stored at -80 °C before analysis. Analyses for each patient were performed in duplicate within a single run on an ADVIA 2400 system (San Raffaele Hospital, Milan). The data were subjected to outlier and homogeneity analysis before performing CV-ANOVA to determine BV and analytical variation (CVA) estimates with confidence intervals (CI). RESULTS The within-subject BV estimates [CVI (95% CI)] were similar for enzymatic [4.4% (4.2-4.7)] and alkaline picrate [4.7% (4.4-4.9)] methods and lower than the estimate presently available online (CVI = 5.9%). No significant male/female BV differences were found. Significant differences were observed in mean creatinine values between men and women and between Turkish individuals and those of other nationalities. Between-subject BV (CVG) estimates, stratified accordingly, produced CVG values similar to historical BV data. CVA was 1.1% for the enzymatic and 4.4% for alkaline picrate methods, indicating that alkaline picrate methods fail to fulfill analytical performance specifications for imprecision (CVAPS). CONCLUSIONS The serum creatinine CVI obtained by EuBIVAS specifies a more stringent CVAPS than previously identified. The alkaline picrate method failed to meet this CVAPS, raising questions regarding its future use.

Iodine status in Turkish populations and exposure to iodide uptake inhibitors.

Perchlorate, nitrate, and thiocyanate are competitive inhibitors of the sodium iodide symporter of the thyroid membrane. These inhibitors can decrease iodine uptake by the symporter into the thyroid gland and may disrupt thyroid function. This study assesses iodine status and exposure to iodide uptake inhibitors of non-pregnant and non-lactating adult women living in three different cities in Turkey (Istanbul, Isparta and Kayseri). We measured iodine and iodide uptake inhibitors in 24-hr urines collected from study participants (N = 255). All three study populations were mildly iodine deficient, with median urinary iodine (UI) levels of 77.5 µg/L in Istanbul, 58.8 µg/L in Isparta, and 69.8 µg/L in Kayseri. Perchlorate doses were higher in the study population (median 0.13 µg/kg/day), compared with a reference population (median 0.059 µg/kg/day), but lower than the U.S. EPA reference dose (0.7 µg/kg/day). Urinary thiocyanate levels increased with increasing exposure to tobacco smoke, with non-smokers (268 µg/L) significantly lower than light smokers (1110 µg/L), who were significantly lower than heavy smokers (2410 µg/L). This pilot study provides novel data indicating that study participants were moderately iodine deficient and had higher intakes of the iodide uptake inhibitor perchlorate compared with a reference population. Further investigation is needed to characterize the thyroid impact resulting from iodine deficiency coupled with exposure to iodide uptake inhibitors such as perchlorate, thiocyanate and nitrate.

A new approach to calculating the Sigma Metric in clinical laboratories

In clinical laboratories, the performance of a process as Sigma Metric (SM) is calculated by the equations derived by Westgard. In the present study, we found that the Westgard equations do not reflect the real performance of the process and that the SM calculated using these equations is lower than the real SM. We measured the substance concentration of ten analytes (glucose, urea, creatinine, cholesterol, calcium, magnesium, phosphorus, LDH, sodium, and potassium) in serum and calculated the SM for each using the Westgard equations and z transformations. The SM values for the same measurand using the Westgard equations based on either the absolute or the relative (percentage) results were not equal to each other, and those related to calcium and sodium were even lower than 0. The SM obtained from the z transformation was higher than that from the Westgard equations, and none were lower than 0. We concluded that the equations suggested by Westgard to calculate the SM do not cover all of the data produced by the process and do not reflect reality. From our research, the z transformation was the optimum method of calculating the actual SM for the process.

Post‐translational modifications of transthyretin affect the triiodonine‐binding potential

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys10), which is frequently affected by oxidative post‐translational modifications. As Cys10 is part of the thyroid hormone‐binding channel within the TTR molecule, PTM of Cys10 may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys10 modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype‐ and site‐specific manner with S‐glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function‐specific patterns of TTR with a substantial decrease in S‐sulphonated, S‐cysteinylglycinated and S‐glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys10 seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.

Six Sigma as a Quality Management Tool: Evaluation of Performance in Laboratory Medicine

In medical school, the first concept expressed to students is a Latin phrase, primum non nocere, meaning “first, do no harm.” This phrase is well known among health workers and dates back to Hipocrates. However, in reality, the situation is slightly different. According to the report of the Institute of Medicine, each year in the USA, approximately 98,000 people die from medical errors (Kohn et al., 2000). Unfortunately, more people have died each year during mid-1990s from medical errors than from AIDS or breast cancer (Kohn et al., 2000). Despite this situation, we cannot say that adequate attention has been paid to the application of high standards in the healthcare sector to effectively prevent medical errors. Yet in industry, for more than a century, modern quality control has been applied to prevent errors and produce high quality goods. The result of these long-term efforts is that in many companies, the rate of errors approaches a negligible level. Regrettably, we cannot say the same thing for medical services, because the components that produce errors or defects in medical services are many more than those involved in any industrial or business sector. Despite these facts, it is clear that the quality of medical services is more important than the quality of most other goods. Consequently, healthcare professionals must pay more attention to quality than any industrial professionals do. Among healthcare services, clinical laboratories are particularly important because physicians make their decisions mostly in accordance with laboratory results (Forsman, 1996). In this context, accurate test results are crucial for physicians and their patients. First, the laboratory must be able to produce an accurate test result before any other dimension of quality becomes important. From this point of view, the evaluation of laboratory performance is critical to maintaining accurate laboratory results (Coskun, 2007). In clinical laboratories, we traditionally divide the total testing processes into three phases: pre-analytical, analytical, and post-analytical phases. However, the selection and interpretation of tests are also prone to errors and must be considered in the total testing process. For this reason, in laboratory medicine, we analyze the total testing process in five phases: pre-preanalytical, pre-analytical, analytical, post-analytical, and post-post-analytical phases 13

Sigma metric or defects per million opportunities (DPMO): the performance of clinical laboratories should be evaluated by the Sigma metrics at decimal level with DPMOs.

*Corresponding author: Abdurrahman Coskun, MD, School of Medicine, Department of Medical Biochemistry, Acibadem University, Istanbul, Turkey, E-mail: Coskun2002@gmail.com Wytze P. Oosterhuis: Zuyderland Medical Center, Department of Clinical Chemistry and Hematology, Heerlen, The Netherlands Mustafa Serteser and Ibrahim Unsal: School of Medicine, Department of Medical Biochemistry, Acibadem University, Istanbul, Turkey Letter to the Editor

Analysis of Changes in Parathyroid Hormone and 25 (OH) Vitamin D Levels with Respect to Age, Gender and Season: A Data Mining Study

Summary Background: 25 (OH) vitamin D3 (25(OH)D) and parathyroid hormone (PTH) are important regulators of calcium homeostasis. The aim of this study was to retrospectively determine the cut–off for sufficient 25(OH)D in a four-season region and the influence of age, seasons, and gender on serum 25(OH)D and PTH levels. Methods: Laboratory results of 9890 female and 2723 male individuals aged 38.8±22.1 years who had simultaneous measurements of 25(OH)D and PTH were retrospectively analyzed by statistical softwares. Serum 25(OH)D and PTH levels were measured by a mass spectrometry method and by an electrochemiluminescence immunoassay, respectively. Results: Mean serum 25(OH)D levels showed a sinusoidal fluctuation throughout the year and were significantly (p<0.01) higher in summer and autumn. On the other hand, PTH levels were significantly higher (p<0.01) in women and showed an opposite response to seasonal effects relative to 25(OH)D. Lowest levels of 25(OH)D were detected in people aged between 20 and 40 years whereas PTH hormone levels were gradually increasing in response to aging. The significant exponential inverse relationship that was found between PTH and 25(OH)D (PTH=exp(4.12–0.064*sqrt(25(OH)D)) (r=−0.325, R–squared=0.105, p<0.001)) suggested that the cut–off for sufficient 25(OH)D should be 75 nmol/L. Conclusions: Our retrospective study based on large data set supports the suitability of the currently accepted clinical cut–off of 75 nmol/L for sufficient 25(OH)D. However, the issue of assessing Vitamin D deficiency remains difficult due to seasonal variations in serum 25(OH)D. Therefore, PTH measurements should complement 25(OH)D results for diagnosing Vitamin D deficiency. It is imperative that seasonally different criteria should be considered in future.

Inhibition of Cholesterol Biosynthesis in Hypercholesterolemia – Is It the Right Choice? / Inhibicija Biosinteze Holesterola u Hiperholesterolemiji – Da Li Je Pravi Izbor?

Summary Cholesterol biosynthesis is a complex pathway comprising more than 20 biochemical reactions. Although the final product created in the pathway is cholesterol, the intermediate products, such as ubiquinone and dolichol, also provide vital metabolic functions. Statins are HGM-CoA reductase inhibitors that stop the production of cholesterol by directly inhibiting the mevalonate production. Mevalonate is a precursor of two additional vital molecules, squalene and ubiquinone (coenzyme Q10). We hypothesized that inhibiting the cholesterol biosynthesis with statins for an extended duration may potentiate the oxidative stress, neurodegenerative disease and cancer. Our recommendation was to measure muscle enzymes, antioxidant capacity, and ubiquinone to monitor patients receiving the statins for prolonged periods of time. Kratak sadržaj Biosinteza holesterola je kompleksan metabolički put koji obuhvata više od 20 biohemijskih reakcija, lako je konačan proizvod koji nastaje holesterol, intermedijerni proizvodi, kao što su ubihinon i dolihol, takođe obezbeđuju vitalne metaboličke funkcije. Statini su inhibitori HMG-KoA reduktaze koji zaustavljaju produkciju holesterola direktnom inhibicijom produkcije mevalonata. Mevalonat je prekursor dva dodatna vitalna molekula, skvalena i ubihinona (koenzim Q10). Postavili smo hipotezu da produženo trajanje inhibicije biosinteze holesterola statinima može da potencira oksidativni stres, neurodegenerativne bolesti i kancer. Takođe preporučujemo određivanje mišićnih enzima, antioksidativnog kapaciteta i ubihinona u praćenju pacijenata koji primaju statine u dužem vremenskom periodu.