Clinical and Analytical Comparison of Monoclonal and Polyclonal Immunoassays for Fecal Pancreatic Elastase.

BACKGROUND: Numerous immunoassays have been commercialized to determine pancreatic elastase (PE) in feces in screening for exocrine pancreatic insufficiency (EPI), but how the different assays compare to one another is controversial, especially in the context that all methods use the same cut-off values for interpreting the results obtained on the presence or absence of EPI or the degree of insufficiency if it is present. Our aim was to analytically verify a new method for determining PE, compare the results with a previous method, and verify the declared cut-off values for interpretation of the results. METHODS: PE in the stool was assayed using a previous monoclonal enzyme-linked immunosorbent assay ("ScheBo ELISA") and a new polyclonal particle-enhanced turbidimetric immunoassay ("Bühlmann PETIA"). The direct method comparison of two immunoassays was performed in 40 samples. Clinical comparisons were conducted against each other for the binary determination of "abnormal/normal" elastase levels and the three-way determination of "severe/moderate/no" EPI in 56 samples. The indirect comparison method used external quality assessment (EQA) data to compare the monoclonal and polyclonal immunoassays for PE, and additionally compare the monoclonal ScheBo ELISA to a monoclonal chemiluminescence immunoassay ("DiaSorin CLIA"). RESULTS: Precision in the series and intra-laboratory precision for Bühlmann PETIA met the manufacturer's specifications for the concentration range of limit/lower values and the range of normal values. The Bühlmann PETIA immunoassay on different analytical platforms yielded comparable results and nearly perfect agreement in the case of three-way classification (kappa = 0.89 with 95%CI from 0.79 to 1.00. ScheBo ELISA tends to generate higher values of pancreatic elastase than the Bühlmann PETIA; agreement between the methods was moderate in the case of binary classification (kappa = 0.43; 95% CI 0.25 to 0.62), and substantial in the case of three-way classification (kappa = 0.62; 95% CI 0.50 to 0.75). EQA data analysis showed a statistically significant difference between ScheBo ELISA and Bühlmann PETIA peer groups (p = 0.031), as well as the DiaSorin CLIA and ScheBo ELISA peer groups (p = 0.010). CONCLUSION: The ScheBo ELISA and Bühlmann PETIA do not appear to be commutable in the analytical and clinical context. Our data address a discordance between different mono- and polyclonal immunoassays for pancreatic elastase and the potential of misclassification using its universal cut-off values in screening suspected patients for exocrine pancreatic insufficiency.

Laboratory medicine in arterial hypertension.

In the initial diagnostics of arterial hypertension (AH) laboratory medicine is a cornerstone, along with a blood pressure (BP) measurement and an electrocardiogram. It mainly refers to routine blood and urine tests for diagnosis and monitoring primary hypertension and its associated conditions such as asymptomatic hypertension-mediated organ damage, chronic kidney disease and hypertensive disorders of pregnancy. In addition, long term non-fatal and fatal risks for cardiovascular (CV) events in hypertension are assessed based on clinical and laboratory data. Furthermore, laboratory medicine is involved in the management of hypertension, especially in monitoring the disease progression. However, antihypertensive drugs may interfere with laboratory test results. Diuretics, especially thiazides, can affect blood and urine sodium concentrations, or angiotensin-converting enzyme inhibitors and angiotensin receptor blockers can affect the blood biomarkers of the renin-angiotensin-aldosterone system (RAAS). It's dysfunction plays a critical role in primary aldosteronism (PA), the most common endocrine disorder in secondary hypertension, which accounts for only small proportion of AH in relative terms but substantial proportion of hypertensives in absolute terms, affecting younger population and carrying a higher risk of CV mortality and morbidity. When screening for PA, aldosterone-to-renin ratio still contributes massively to the increased incidence of the disease, despite certain limits. In conclusion, laboratory medicine is involved in the screening, diagnosis, monitoring and prognosis of hypertension. It is of great importance to understand the preanalytical and analytical factors influencing final laboratory result.

Serum bicarbonate stability study at room temperature - influence of time to centrifugation and air exposure on bicarbonate measurement reported according to the CRESS checklist.

OBJECTIVES: The aim was to evaluate the stability of serum bicarbonate at room temperature, depending on time to centrifugation and air exposure. METHODS: Stability study was conducted in the laboratory of Clinical Hospital Centre Rijeka, Croatia in January-February 2022. Nine samples from 10 volunteers were collected in clot activator gel tubes (Greiner Bio-One). Bicarbonate was measured on Beckman Coulter AU480 (Beckman Coulter, Brea, USA). Three tubes were left at room temperature for 30 min, three tubes for 2 h, three tubes for 4 h until centrifugation. First tube from first group (baseline) was measured immediately after centrifugation. Other measurements were expressed as percentage deviation (PD%) from baseline. First tube was remeasured after 1 and 2 h (OT_0h_1h; OT_0h_2h). Second and third tubes were opened 1 and 2 h after centrifugation (C_0h_1h; C_0h_2h). Second group of tubes was processed the same way with 2-hour centrifugation delay (WB_2h; OT_2h_1h; OT_2h_2h; C_2h_1h; C_2h_2h), and third group with 4-hour delay (WB_4h; OT_4h_1h; OT_4h_2h; C_4h_1h; C_4h_2h). PD% was compared to Maximum Permissible Difference (MPD=5.69%). MedCalc statistical software was used (MedCalc, Ostend, Belgium). RESULTS: Bicarbonate baseline mean value (range) was 27.3 (23.4-29.6) mmol/L. Obtained PD% (95%CI) were: C_0h_1h 0.46 (-1.21, 2.12); C_0h_2h 0.18 (-2.22, 2.57); OT_0h_1h -6.46 (-7.57, -5.36); OT_0h_2h -10.67 (-12.13, -9.21); WB_2h -0.15 (-2.04, 1.74); C_2h_1h 0.01 (-1.52, 1.54); C_2h_2h -0.40 (-2.65, 1.85); OT_2h_1h -5.43 (-7.30, -3.55); OT_2h_2h -11.32 (-13.57, -9.07); WB_4h -0.85 (-3.28, 1.58); C_4h_1h -2.52 (-4.93, 0.11); C_4h_2h -3.02 (-5.62, 0.43); OT_4h_1h -7.34 (-9.64, -5.05); OT_4h_2h -11.85 (-14.38, -9.33). CONCLUSIONS: Serum bicarbonate is stable for 4 h in closed uncentrifuged tubes, another 2 h in closed tubes after centrifugation, and is unstable within 1 h in opened tube.

An analysis of the vitamin D overtesting in a tertiary healthcare centre.

Introduction: Vitamin D testing is excessively used in clinical practice, despite of the clinical guidelines statements against population screening for vitamin D deficiency. This study aimed to assess an annual number of performed 25-hydroxy vitamin D (25(OH)D) tests that were unsupported by the national guidelines for prevention, detection and therapy of vitamin D deficiency in adults and to calculate associated financial burden for the publicly funded healthcare. Materials and methods: A representative sample of requested 25(OH)D tests in 2018 (N = 474) was formed after selection and randomisation of data set (N = 5298) collected from the laboratory information system database of the Clinical Department for Laboratory Diagnostics, the Clinical Hospital Centre Rijeka. Records were classified in two groups depending on associated medical condition(s) according to the national guidelines. An annual cost of the total and group specific vitamin D testing was calculated on the base of a single test price reimbursed by the Croatian Healthcare Insurance Fund (CHIF). Results: Medical conditions with high-risk for vitamin D deficiency were detected in 43% (206/474) of vitamin D requests (group 1). Conditions not associated with vitamin D deficiency were detected in 57% (268/474) requests (group 2). A total cost of 25(OH)D testing for the CHIF was 58,729.50 EUR (25,523.79 EUR in the group 1 and 33,205.71 EUR in the group 2). Conclusions: More than half of all 25(OH)D tests performed in the clinical laboratory represent avoidable cost for the public healthcare. Prevention of population screening by vitamin D testing is needed.

Granulysin expression and granulysin-mediated apoptosis in the peripheral blood of osteoarthritis patients.

Osteoarthritis (OA) is a chronic joint disease caused by mechanical damage and metabolic factors that support the development of low-grade inflammation. Increased levels of T helper 1 pro-inflammatory cytokines in the serum of OA patients may support granulysin (GNLY) mediated cytotoxicity, which in-turn may contribute to the pathogenesis of OA. In the present study, GNLY expression and cytotoxic/apoptotic mechanisms mediated by GNLY in the peripheral blood of OA patients were assessed. A total of 40 non-obese women (median age of 64 years old) with knee OA, and 40 controls (median age 62 years old) were enrolled in the study. GNLY, IFN-γ and IL-4 expression levels were investigated in peripheral blood lymphocytes (PBLs) using flow cytometry, immunocytochemistry and/or confocal microscopy. Natural killer (NK) GNLY-mediated apoptosis through NK effectors against K-562 targets was analyzed using the PKH-26 18-h cytotoxicity assay. Serum GNLY levels were assessed using ELISA. The percentage of GNLY+PBLs was higher in the OA patients than that in the controls due to the increase in the proportions of GNLY+ cells in the natural killer (NK), T and natural killer T (NKT) subsets. GNLY localization inside exocytotic lysosomal-associated membrane protein-1+ granules was ~40% in both groups. However, the intensity of GNLY labeling in PBLs was higher in OA patients than in the controls, and it was supported by the increased expression of IFN-γ relative to IL-4 in NK and T cells from OA patients. The serum GNLY concentration was <0.3 ng/ml in both groups. RC8 anti-GNLY mAb by itself was unable to significantly alter early apoptosis, whereas RC8 anti-GNLY mAb combined with anti-perforin mAb significantly reduced NK-mediated early apoptosis of K-562 targets in the OA patients, whilst not exerting a notable effect in the controls. Anti-perforin mAb by itself did not affect apoptosis significantly. These results suggest that in women with knee OA, GNLY expression in the PBL subsets and GNLY-mediated early apoptosis of K-562 targets are increased compared with the controls and accompanied by intracellular dominance of IFN-γ over IL-4 in NK cells.

National Guidelines for the Performance of the Sweat Test in Diagnosis of Cystic Fibrosis on behalf of the Croatian Society of Medical Biochemistry and Laboratory Medicine and the Cystic Fibrosis Centre - Paediatrics and adults, University Hospital Centre Zagreb.

The sweat test (ST) is a cornerstone in the diagnosis of cystic fibrosis (CF), together with newborn screening and genetic testing. However, the performance of the ST can depend on the operator's skill, so several international guidelines have been published to standardise the ST, but inconsistencies remain. The joint Working Group for ST Standardisation (WG STS) of the Croatian Society of Medical Biochemistry and Laboratory Medicine, in association with cistic fybrosis health professional and the Cistic Fibrosis Centre for Paediatrics and Adults, have issued National Guidelines for the Performance of the Sweat Test in order to ensure consistency in ST performance and accuracy of reported results. Many of the standards were taken from the 2nd Edition of the UK Guidelines for Performance of the ST for the Diagnosis of CF, while others were taken from independent consensus statements from the WG STS based on local ST equipment and practices. The standards cover every step of the ST, from the indications for testing to reporting of results and their interpretation, including the analytical phase and quality control. In addition, National Guidelines include appendices with practical examples in order to aid implementation of the recommendations in routine practice.

The Accuracy of Serum Biomarkers in the Diagnosis of Steatosis, Fibrosis, and Inflammation in Patients with Nonalcoholic Fatty Liver Disease in Comparison to a Liver Biopsy.

Background and Objective: This study was conducted to evaluate the diagnostic performance of various biomarkers for steatosis, fibrosis, and inflammation in comparison to a liver biopsy (LB) in patients with nonalcoholic fatty liver disease (NAFLD). Materials and Methods: This was a cross-sectional study that included 135 patients with biopsy-proven NAFLD. Fatty liver index (FLI), hepatic steatosis index (HSI), cell death markers (CK-18 M30 and CK-18 M65), FIB-4 index, NAFLD fibrosis score (NFS), BARD, and AST to platelet ratio index (APRI) were calculated and analysed. Results: FLI, HSI scores, and the cell death biomarkers showed poor diagnostic accuracy for steatosis detection and quantification, with an area under the curve (AUC) of <0.70. The cell death biomarkers likewise did not perform well for the detection of nonalcoholic steatohepatitis (NASH) (AUC < 0.7). As for the fibrosis staging, only APRI and the cell death biomarkers had moderate accuracy (AUC > 0.7) for advanced fibrosis, whereas FIB-4, BARD, and NFS scores demonstrated poor performance (AUC < 0.70). However, a combination of FIB-4 and NFS with the cell death biomarkers had moderate accuracy for advanced (≥F3) fibrosis detection, with an AUC of >0.70. Conclusions: In this first study on Croatian patients with NAFLD, serum biomarkers demonstrated poor diagnostic performance for the noninvasive diagnosis of liver steatosis and NASH. APRI and the cell death biomarkers had only moderate accuracy for diagnosing advanced fibrosis, as did the combination of FIB-4 and NFS with the cell death biomarkers. Further studies regarding serum biomarkers for all NAFLD stages are needed.

Values of vanillylmandelic acid and homovanillic acid in the urine as potential prognostic biomarkers in ischaemic stroke patients.

BACKGROUND: Suitable biomarkers that have prognostic values are one of the key points of interest in ischaemic stroke. Increased sympathetic nervous system activity in ischaemic stroke causes multiple local and systemic effects that can be detrimental to the outcome. The mechanism of action is increased secretion and activity of catecholamines, whose end metabolic products are vanillylmandelic acid and homovanilic acid. Aim of our study was to determine whether these compounds can be used as potential prognostic biomarkers in ischaemic stroke, as a unique insight into the activity of the sympathetic nervous system. METHODS: Urine samples of 96 patients with ischaemic stroke and transitory ischaemic attacks were analysed. Values of vanillylmandelic and homovanillic acids in urine were tested using liquid chromatography on the first and third day post-stroke. Severity of stroke was determined using the NIHSS scale, while functional outcome was determined using the Modified Rankin Scale. RESULTS: Values of vanillylmandelic and homovanillic acids positively correlated with functional outcome of ischaemic stroke. Favorable outcomes correlated with decreased values, on contrary to increased values, which were associated with unfavourable outcomes. CONCLUSION: Determining the values of these compounds in the urine is an easily available prognostic tool for the ischaemic stroke outcome, while also influencing potential therapeutic changes.

Celiac disease: From pathophysiology to treatment.

Celiac disease, also known as "celiac sprue", is a chronic inflammatory disorder of the small intestine, produced by the ingestion of dietary gluten products in susceptible people. It is a multifactorial disease, including genetic and environmental factors. Environmental trigger is represented by gluten while the genetic predisposition has been identified in the major histocompatibility complex region. Celiac disease is not a rare disorder like previously thought, with a global prevalence around 1%. The reason of its under-recognition is mainly referable to the fact that about half of affected people do not have the classic gastrointestinal symptoms, but they present nonspecific manifestations of nutritional deficiency or have no symptoms at all. Here we review the most recent data concerning epidemiology, pathogenesis, clinical presentation, available diagnostic tests and therapeutic management of celiac disease.

Evaluating performance in sweat testing in medical biochemistry laboratories in Croatia.

INTRODUCTION: Sweat test has a diagnostic role in evaluation of cystic fibrosis. Its performance includes sweat stimulation, collection and analysis. All listed may be sources of inconsistencies in everyday practice. The aim of this study was an evaluation of external quality assessment (EQA) of sweat chloride measurement including sweat test performance in medical biochemistry laboratories in Croatia. MATERIALS AND METHODS: EQA for sweat chloride measurement was provided by Croatian Centre for Quality Assessment in Laboratory Medicine (CROQALM) in five consecutive exercises to medical biochemistry laboratories (MBL) that offered sweat testing. A questionnaire regarding all phases of testing was mailed to involved MBL (N = 10). Survey results were compared to current guidelines for sweat test performance. RESULTS: Reported results of EQA in 2015 exercises showed coefficients of variation (CV) from 28.9%, 29.0% to 35.3%, respectively. An introduction of uniform sweat chloride measurement protocol resulted in CV of 15.5% and 14.7% reported in following two exercises in 2016. All MBL included in this study replied to the questionnaire. Results reported by MBL indicated: lack of patient information policy (7/10), use of unacceptable electrodes (6/9), misuse of minimum of acceptable sweat weight (6/9), lack of internal quality assessment (5/9) and recommended reference ranges (5/9 and 4/9). Agreements to guidelines were found in approach to unsuitable patients (9/10) and sweat collection (8/9). CONCLUSION: Presented results indicate major weak points of current practice in sweat test performance in Croatian MBL and stress the need for its standardization on a national level.

Laboratory testing of extravascular body fluids in Croatia: a survey of the Working group for extravascular body fluids of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

INTRODUCTION: We hypothesized that extravascular body fluid (EBF) analysis in Croatia is not harmonized and aimed to investigate preanalytical, analytical and postanalytical procedures used in EBF analysis in order to identify key aspects that should be addressed in future harmonization attempts. MATERIALS AND METHODS: An anonymous online survey created to explore laboratory testing of EBF was sent to secondary, tertiary and private health care Medical Biochemistry Laboratories (MBLs) in Croatia. Statements were designed to address preanalytical, analytical and postanalytical procedures of cerebrospinal, pleural, peritoneal (ascites), pericardial, seminal, synovial, amniotic fluid and sweat. Participants were asked to declare the strength of agreement with proposed statements using a Likert scale. Mean scores for corresponding separate statements divided according to health care setting were calculated and compared. RESULTS: The survey response rate was 0.64 (58 / 90). None of the participating private MBLs declared to analyse EBF. We report a mean score of 3.45 obtained for all statements evaluated. Deviations from desirable procedures were demonstrated in all EBF testing phases. Minor differences in procedures used for EBF analysis comparing secondary and tertiary health care MBLs were found. The lowest scores were obtained for statements regarding quality control procedures in EBF analysis, participation in proficiency testing programmes and provision of interpretative comments on EBF's test reports. CONCLUSIONS: Although good laboratory EBF practice is present in Croatia, procedures for EBF analysis should be further harmonized to improve the quality of EBF testing and patient safety.

Plasma Levels of Monocyte Chemotactic Protein-1 Are Associated with Clinical Features and Angiogenesis in Patients with Multiple Myeloma.

The aim of this pilot study was to determine the plasma levels of monocyte chemotactic protein-1 (MCP-1) and possible associations with angiogenesis and the main clinical features of untreated patients with multiple myeloma (MM). ELISA was used to determine plasma MCP-1 levels in 45 newly diagnosed MM patients and 24 healthy controls. The blood vessels were highlighted by immunohistochemical staining, and computer-assisted image analysis was used for more objective and accurate determination of two parameters of angiogenesis: microvessel density (MVD) and total vascular area (TVA). The plasma levels of MCP-1 were compared to these parameters and the presence of anemia, renal dysfunction, and bone lesions. A significant positive correlation was found between plasma MCP-1 concentrations and TVA (p = 0.02). The MCP-1 levels were significantly higher in MM patients with evident bone lesions (p = 0.01), renal dysfunction (p = 0.02), or anemia (p = 0.04). Therefore, our preliminary results found a positive association between plasma MCP-1 levels, angiogenesis (expressed as TVA), and clinical features in patients with MM. However, additional prospective studies with a respectable number of patients should be performed to authenticate these results and establish MCP-1 as a possible target of active treatment.

Plasma levels of osteopontin and vascular endothelial growth factor in association with clinical features and parameters of tumor burden in patients with multiple myeloma.

The aim of this pilot study was to determine the plasma levels of osteopontin (OPN) and vascular endothelial growth factor (VEGF) and find possible association between them and main clinical features and parameters of tumor burden in patient with multiple myeloma (MM). Plasma levels of OPN and VEGF were determined in 44 newly diagnosed MM patients and 24 healthy persons by ELISA method. These values were compared with the presence of anemia, renal dysfunction, and bone lesions as myeloma related clinical manifestations and with serum beta-2 microglobulin and Durie-Salmon clinical stage as prognosticators related to tumor mass. The value of OPN was significantly higher in MM patients with evident bone lesions (P = 0.03) and there was also a positive correlation with serum beta-2 microglobulin (r = 0.366; P = 0.04). Furthermore, patients with lower Durie-Salmon stage had significantly lower OPN and VEGF levels (P = 0.05; P = 0.04, resp.). Our preliminary results found positive association between plasma level of OPN, tumor burden, and bone destruction. Further analysis should provide information about the possible use of OPN as useful clinical biomarker for monitoring bone disease and tumor mass, as well as a prognostic factor, or a possible target for pharmacological intervention.

[pp65 antigenemia in evaluation of cytomegalovirus infection after kidney transplantation]. / pp65 antigenemija u pracenju citomegalovirusne infekcije nakon transplantacije bubrega.

INTRODUCTION: Cytomegalovirus (CMV) infection is the most common infectious complication after organ transplantation. Serology is useful only for detecting previous CMV infection. Dissatisfied with serologic follow-up after kidney transplantation, three years ago we introduced detection of CMV antigenemia by an immunocytochemical method using a monoclonal antibody specific for the pp65 CMV matrix protein. This test allows for quantification of positive leukocytes. The purpose of this paper is to present our three-year experience. PATIENTS AND METHODS: From May 1999 till May 2002 CMV antigenemia was examined in 76 patients: 55 patients submitted to kidney transplantation during the study period, and 21 patients previously. Antigenemia became positive at 25.68 +/- 15.51 days after transplantation. These 76 patients were divided into three groups according to the number of positive cells per 200,000 leukocytes: < 5 (group I), 6-20 (group II) and > 20 (group III). The groups consisted of 23, 20 and 11 patients, respectively. The percentage of patients treated by ganciclovir was 4.34%, 15% and 100%, respectively. In group I only one patient received ganciclovir because of geographic indication, in group II three patients because of septicemia, thrombopenia and leukopenia and previous miliary tuberculosis. RESULTS: One patient from group III with steroid diabetes died from pneumonia with abscess formation three days from admission. In another two patients, interstitial pneumonia and abscess of the arm developed. Five patients had an acute rejection episode each and were treated by high doses of methylprednisolone. Five patients had elevated temperature, transaminases were elevated in five patients, and neutropenia with or without thrombopenia was found in six patients. One patient had recurrent CMV disease and lymphocele. Two patients had preemptive treatment by ganciclovir based on positive CMV antigenemia. DISCUSSION: Various centers differ according to the approach to treatment of CMV infection, ranging from prophylaxis to deferred treatment for CMV disease. Determination of pp65 CMV antigenemia allowed us a safe follow-up of patients after kidney transplantation. Compared with previous serologic follow-up antigenemia is a considerable progress. We did not use CMV prophylaxis because it is more expensive and can cause resistance to ganciclovir. A promising novel drug valganciclovir will allow for good prophylaxis owing to its better absorption from the gut. Based on our three-year experience, optimal cut-off for antigenemia has been set at 20 positive cells per 200,000 leukocytes. The existence of symptoms or changes in the level of leukocytes, platelets or transaminases goes in favor of treatment decision. CONCLUSION: Cytomegalovirus pp65 antigenemia is a reliable tool in the follow-up of patients after kidney transplantation. Patients with primary CMV infection, those with rejection episode and threshold of 20 positive cells require preemptive treatment with ganciclovir. The measurement of pp65 CMV antigenemia has clinical, analytical and cost-effective advantages. Intensive monitoring for CMV infection allows for quick and specific detection of active CMV infection. This approach avoids resistance to ganciclovir. The method is simple and specific without expensive equipment. Avoidance of unnecessary prophylaxis adds to its cost-effectiveness.