Vanja Radišić Biljak

Platelet count, mean platelet volume and smoking status in stable chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD), an increasing global health problem, may be complicated by acute atherothrombotic events. Although systemic inflammation plays the leading role in atherothrombotic processes, platelet activation and increased coagulation together with oxidative stress can significantly exacerbate atherosclerosis in COPD patients. In this study we determined platelet count, mean platelet volume (MPV) and classical markers of systemic inflammation – serum C-reactive protein (CRP), white blood cell (WBC) count and the relative proportion of segmented neutrophils in COPD patients, and compared them to those from the healthy controls. The most important and novel finding of this study was that patients with COPD had a significantly increased platelet count, along with a reduced MPV when compared to healthy controls (286 vs. 260 × 109/l; 9.6 vs. 8.7 fL, respectively). Cigarette smoking had no influence on these results. The presence of systemic inflammation was clearly proved by the increase in classical inflammatory markers (CRP, WBC and segmented neutrophil count).

Glutathione cycle in stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidant/antioxidant imbalance. Glutathione is the most abundant cellular low‐molecular weight thiol and the glutathione redox cycle is the fundamental component of the cellular antioxidant defence system. Concentration of total glutathione and catalytic activities of glutathione peroxidase and glutathione reductase were determined in peripheral blood of patients (n = 109) and healthy subjects (n = 51). Concentration of total glutathione in patients was not changed in comparison to healthy controls. However, we found statistically significant difference between patients with moderate and severe disease stages. Glutathione reductase activity was increased, while glutathione proxidase activity was decreased in the patients with COPD, when compared to healthy controls. We found no significant difference in glutathione peroxidase and glutathione reductase activities between stages. Patients who smoked had lower concentration of total glutathione compared with former smokers and never‐smoking patients. Lung function parameters were inversely associated with glutathione level. Evidence is presented for differential modulation of glutathione peroxidase and glutathione reductase activities in peripheral blood of patients with stable COPD. We suppose that in addition to glutathione biosynthesis, glutathione reductase‐dependent regulation of the glutathione redox state is vital for protection against oxidative stress. Copyright

Laboratory diagnostics of chronic kidney disease in Croatia: state of the art.

Introduction Early identification and management of chronic kidney disease (CKD) is highly cost-effective and can reduce the risk of kidney failure progression and cardiovascular disease. In 2014, the Joint Croatian Working Group (JCWG) for laboratory diagnostic of CKD on the behalf of Croatian society of medical biochemistry and laboratory medicine (CSMBLM) and Croatian chamber of medical biochemists (CCMB) conducted a survey across Croatian medical-biochemistry laboratories to assess the current practice in this area of laboratory medicine. The aim of this study was to present the data collected through the survey and to give insight about laboratory diagnostics of chronic kidney disease in Croatia. Materials and methods An invitation to participate in the survey was sent to all Croatian medical-biochemistry laboratories (N = 196). The questionnaire was designed in a form of questions and statements, with possible multiple answers, comprising 24 questions. Results The response rate was 80/196 (40.8%). 39 answers were from primary medical-biochemistry laboratories. 31/78 (0.40) laboratories measure creatinine with non-standardized method (uncompensated Jaffe method). 58/78 (0.74) of laboratories that measure creatinine do not report eGFR values. Similar number of laboratories (58/80, 0.73) do not measure urine albumin or protein. Conclusions There is a large heterogeneity among Croatian laboratories regarding measuring methods, reporting units and reference intervals (cut-off values), both for creatinine and urine albumin or protein. The two key prerequisites for CKD screening, automatic reporting of eGFR and albuminuria or proteinuria assessment, are not implemented nationwide. There is a need for harmonization in laboratory diagnostics of CKD in Croatia.

Validation of Point-of-Care Glucose Testing for Diagnosis of Type 2 Diabetes

Point-of-care (POC) glucose technology is currently considered to be insufficiently accurate for the diagnosis of diabetes. The objective of this study was to investigate the diagnostic accuracy of an innovative, interference-resistant POC glucose meter (StatStrip glucose hospital meter, Nova Biomedical, USA) in subjects with a previous history of dysglycaemia, undergoing a 75 g diagnostic oral glucose tolerance test (oGTT). Venous and capillary blood sampling for the reference laboratory procedure (RLP) and POC-glucose measurement was carried out at fasting and 2 h oGTT, and categories of glucose tolerance were classified according to 2006 WHO diagnostic criteria for the respective sample type. We found an excellent between-method correlation at fasting (r = 0.9681, P < 0.0001) and 2 h oGTT (r = 0.9768, P < 0.0001) and an almost perfect diagnostic agreement (weighted Kappa = 0.858). Within a total of 237 study subjects, 137 were diagnosed with diabetes with RLP, and only 6 of them were reclassified as having glucose intolerance with POC. The diagnostic performance of POC-fasting glucose in discriminating between the normal and any category of disturbed glucose tolerance did not differ from the RLP (P = 0.081). Results of this study indicate that StatStrip POC glucose meter could serve as a reliable tool for the diabetes diagnosis, particularly in primary healthcare facilities with dispersed blood sampling services.

Validation of a laboratory and hospital information system in a medical laboratory accredited according to ISO 15189

Introduction The aim of the study was to present a protocol for laboratory information system (LIS) and hospital information system (HIS) validation at the Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital, Zagreb, Croatia. Materials and methods: Validity of data traceability was checked by entering all test requests for virtual patient into HIS/LIS and printing corresponding barcoded labels that provided laboratory analyzers with the information on requested tests. The original printouts of the test results from laboratory analyzer(s) were compared with the data obtained from LIS and entered into the provided template. Transfer of data from LIS to HIS was examined by requesting all tests in HIS and creating real data in a finding generated in LIS. Data obtained from LIS and HIS were entered into a corresponding template. The main outcome measure was the accuracy of transfer obtained from laboratory analyzers and results transferred from LIS and HIS expressed as percentage (%). Results: The accuracy of data transfer from laboratory analyzers to LIS was 99.5% and of that from LIS to HIS 100%. Conclusion: We presented our established validation protocol for laboratory information system and demonstrated that a system meets its intended purpose.

Impact of creatinine methodology on glomerular filtration rate estimation in diabetes

AIM To evaluate the influence of creatinine methodology on the performance of chronic kidney disease (CKD)-Epidemiology Collaboration Group-calculated estimated glomerular filtration rate (CKD-EPI-eGFR) for CKD diagnosis/staging in a large cohort of diabetic patients. METHODS Fasting blood samples were taken from diabetic patients attending our clinic for their regular annual examination, including laboratory measurement of serum creatinine and eGFR. RESULTS Our results indicated an overall excellent agreement in CKD staging (kappa = 0.918) between the Jaffé serum creatinine- and enzymatic serum creatinine-based CKD-EPI-eGFR, with 9% of discordant cases. As compared to the enzymatic creatinine, the majority of discordances (8%) were positive, i.e., associated with the more advanced CKD stage re-classification, whereas only 1% of cases were negatively discordant if Jaffé creatinine was used for eGFR calculation. A minor proportion of the discordant cases (3.5%) were re-classified into clinically relevant CKD stage indicating mildly to moderately decreased kidney function (< 60 mL/min per 1.73 m2). Significant acute and chronic hyperglycaemia, assessed as plasma glucose and HbA1c levels far above the recommended glycaemic goals, was associated with positively discordant cases. Due to a very low frequency, positive discordance is not likely to present a great burden for the health-care providers, while intensified medical care may actually be beneficial for the small number of discordant patients. On the other hand, a very low proportion of negatively discordant cases (1%) at the 60 mL/min per 1.73 m2 eGFR level indicate a negligible possibility to miss the CKD diagnosis, which could be the most prominent clinical problem affecting patient care, considering high risk of CKD for adverse patient outcomes. CONCLUSION This study indicate that compensated Jaffé creatinine procedure, in spite of the glucose-dependent bias, is not inferior to enzymatic creatinine in CKD diagnosis/staging and therefore may provide a reliable and cost-effective tool for the renal function assessment in diabetic patients.

Analytical verification and quality assessment of the Tosoh HLC-723GX HbA1c analyzer

Objectives Ion-exchange high-performance liquid chromatography (IE-HPLC) has long been used as a reproducible and versatile analytical tool for HbA1c measurement. In this study, we performed analytical verification and quality assessment of the recently introduced small IE-HPLC Tosoh HLC-723GX HbA1c analyzer, and a comparison of results to immunoassay (IA) and capillary electrophoresis (CE). Design and methods The total imprecision of Tosoh HLC-723GX was verified according to CLSI EP15-A2 protocol using commercial control materials (C-QC) and pooled human whole blood samples (HWB). The Sigma metric was used for the evaluation of quality targets. HbA1c results were compared to automated CE (MiniCap Flex Piercing, Sebia, France) and IA (Tina-quant HbA1c Gen 2, Cobas Integra 400+, Roche Diagnostics, USA) procedures. Results The total imprecision of Tosoh HLC-723GX-HbA1c for IFCC(mmol/mol) and NGSP(%) units was: 1.91/1.25% (HbA1c=31 mmol/mol/5.0%) and 0.51/0.63% (HbA1c=84 mmol/mol/9.8%) for C-QC, and 0.39/0.2% (HbA1c=47 mmol/mol/6.5%) and 0.77/0.46% (HbA1c=94 mmol/mol/10.8%) in HWB samples, respectively. Bland-Altman analysis did not reveal any deviation of the results between Tosoh HLC-723GX and CE: mean difference 0.0% (95%CI: −0.02927 to 0.02653%), while the mean HbA1c difference against IA was −0.07% (95%CI: −0.1039 to −0.02765). At the selected HbA1c clinical decision level (48 mmol/mol/6,5%), six sigma analysis gave σ value of 3.91, within a desirable classification of performance. Conclusion The analytical performance of the Tosoh HLC-723GX complies with the rigorous quality criteria for clinical use of HbA1c, with the results comparable to the CE procedure. Tosoh HLC-723GX provides a plausible analytical choice for reliable HbA1c measurement in low-volume laboratories.

The role of laboratory testing in detection and classification of chronic kidney disease: national recommendations

Chronic kidney disease (CKD) is a common clinical condition with significant adverse consequences for the patient and it is recognized as a significant public health problem. The role of laboratory medicine in diagnosis and management of CKD is of great importance: the diagnosis and staging are based on estimation of glomerular filtration rate (eGFR) and assessment of albuminuria (or proteinuria). Therefore, the joint working group of the Croatian society of medical biochemistry and laboratory medicine and Croatian chamber of medical biochemists for laboratory diagnostics in CKD issued this national recommendation regarding laboratory diagnostics of CKD.
Key factors for laboratories implementing the national guidelines for the diagnosis and management of CKD are:
1. Ensure good communication between laboratory professionals and clinicians, such as nephrologists or specialists in general/family medicine,
2. Ensure all patients are provided with the same availability of laboratory diagnostics,
3. Ensure creatinine assays are traceable to isotope dilution mass spectrometry (IDMS) method and have minimal bias and acceptable imprecision,
4. Select the appropriate GFR estimating formula. Recommended equation is the 2009 Chronic Kidney Disease Epidemiology Collaboration (CKD – EPI) equation,
5. In reporting the key laboratory tests (creatinine, eGFR, urine albumin-to-creatinine ratio, urine protein-to-creatinine ratio) use the appropriate reporting units,
6. Provide adequate information on limitations of creatinine measurement.
The manuscript has been organized to identify critical points in laboratory tests used in basic laboratory diagnostics of CKD and is based on the Kidney Disease: Improving Global Outcomes (KDIGO) 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease.

Serum delipidation but not high-speed centrifugation is effective in clearing lipemia interference in serum lipase activity measurement.

Background: Acute pancreatitis is commonly followed by hypertriglyceridemia. Most current diagnostic guidelines indicate that lipase should be preferred over amylase. However, grossly lipemic samples may interfere with routine laboratory methods for measurement of serum lipase activity. We aimed to evaluate the accuracy of lipase activity measurement by a method with declared lipemia interference in lipemic samples cleared by two different techniques: high speed centrifugation and delipidation using alpha- cyclodextrin (non- ionic natural macrocyclic polymer of glucose). Materials and methods: Thirty-four lipemic serum samples with triglyceride concentration range of 4.27 to 124.6 mmol/L were used in this study. Lipase activity was measured by two measuring principles: Roche Lipase colorimetric method (elective reference laboratory method accurate for lipemic samples), in native lipemic samples on Roche Cobas Integra 400 plus analyzer. Beckman Coulter colorimetric method (lipemia interference >10% above 700 mg/dL Intralipid) in native lipemic samples, and in two serum aliquots cleared of lipemia on Beckman Coulter AU400 analyzer. The results were compared using Bland & Altman plot. Interference of triglycerides on bias was checked by linear regression. All results were analyzed using MedCalc 9.4.2.0 statistical software (MedCalc Software bvba, Mariakerke, Belgium). Results: Bland & Altman plot revealed statistically significant bias between the two compared procedures measured in lipemic samples (differences (%): -36.35, 95% CI = -50.5834 to -22.1214) and aliquots cleared by high speed centrifugation (differences (%): -22.1744, 95% CI = -33.7913 to -10.5575). However, there was no statistically or clinically significant bias between the lipase activity measured in aliquots cleared by serum delipidation (differences (%):-4.1871, 95% CI = -9.7848 to 1.4107) nor it was affected by varying triglyceride levels (R2 = 0.03692, P=0.276). Conclusions: Serum lipase activity can be accurately measured using Beckman Coulter colorimetric method in grossly lipemic plasma samples cleared of lipemia by serum delipidation.

Impact of under-filled blood collection tubes containing K2EDTA and K3EDTA as anticoagulants on automated complete blood count (CBC) testing

*Corresponding author: Vanja Radišić Biljak, Merkur University Hospital, Department of Medical Biochemistry and Laboratory Medicine, Zajčeva 19, Zagreb 10000, Croatia, Phone: +385 1 2334 110, Fax: +385 1 2334 110, E-mail: vanja.radisic@gmail.com Sandra Božičević, Maja Krhač and Marijana Vučić Lovrenčić: Merkur University Hospital, Clinical Department of Medical Biochemistry and Laboratory Medicine, Zagreb, Croatia Letter to the Editor