When rare becomes common: N2 gene-positive, E gene-negative SARS-CoV-2 PCR results between 2021 and 2022.

Nucleocapsid gene-positive, envelope gene-negative (N2+/E-) SARS-CoV-2 PCR results obtained with the Cepheid Xpert Xpress SARS-CoV-2 assay are an infrequent phenomenon. We assessed the validity of the N2+/E- cases with an indirect approach by analyzing their occurrence in relation to overall positive PCR rates and absolute number of PCR tests (24,909 samples, collected June 2021 to July 2022). Additionally, 3022 samples were analyzed with the Xpert Xpress CoV-2-plus assay in August/September 2022. The incidence of monthly N2+/E- cases closely followed the overall frequency of positive tests (p < 0.001), while there was no correlation with the monthly number of PCR test. The observed distribution of N2+/E- cases implicates, that they are not merely artefacts, but rather represent samples with a very low viral load. This phenomenon will persist with the Xpert Xpress SARS-CoV-2 plus assay, which also produced more than 10% results where only one target gene replicated with a very high Ct value.

Laboratory policies and practices for thyroid function tests in Croatia: survey on behalf of Working Group for Laboratory Endocrinology of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

Introduction: Laboratory plays important part in screening, diagnosis, and management of thyroid disorders. The aim of this study was to estimate current laboratory preanalytical, analytical and postanalytical practices and policies in Croatia. Materials and methods: Working Group for Laboratory Endocrinology of the Croatian Society of Medical Biochemistry and Laboratory Medicine designed a questionnaire with 27 questions and statements regarding practices and protocols in measuring thyroid function tests. The survey was sent to 111 medical biochemistry laboratories participating in external quality assurance scheme for thyroid hormones organized by Croatian Centre for Quality Assessment in Laboratory Medicine. Data is presented as absolute numbers and proportions. Results: Fifty-three participants returned the questionnaire. Response rate varied depending on question, yielding a total survey response rate of 46-48%. All respondents perform thyroid stimulating hormone (TSH). From all other thyroid tests, most performed is free thyroxine (37/53) and least TSH-stimulating immunoglobulin (1/53). Laboratories are using nine different immunoassay methods. One tenth of laboratories is verifying manufacturer's declared limit of quantification for TSH and one third is verifying implemented reference intervals for all performed tests. Most of laboratories (91%) adopt the manufacturer's reference interval for adult population. Reference intervals for TSH are reported with different percentiles (90, 95 or 99 percentiles). Conclusion: This survey showed current practices and policies in Croatian laboratories regarding thyroid testing. The results identified some critical spots and will serve as a foundation in creating national guidelines in order to harmonize laboratory procedures in thyroid testing in Croatia.

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 three different protocols for obtaining hemolysis.

OBJECTIVES: Hemolysis is associated with erroneous or delayed results. Objectives of the study were to compare four different methods for obtaining hemolysis in vitro on three different analyzers. METHODS: Hemolysis was prepared with addition of pure hemoglobin into serum pool, osmotic shock, aspiration through blood collection needle, freezing/thawing of whole blood. Biochemistry parameters were measured in duplicate at Architect c8000 (Abbott, Abbott Park, USA), Beckman Coulter AU680 (Beckman Coulter, Brea, USA) and Cobas 6000 c501 (Roche, Mannheim, Germany), according to manufacturers' declarations. Cut-off value was defined as the highest value of H index with corresponding bias lower than acceptance criteria. RESULTS: We were not able to obtain results with freezing protocol. On all three platforms, lowest number of analytes were sensitive to hemolysis at H=0.5 using method of adding free hemoglobin. When osmotic shock was used, cut-off values for the most analytes were generally met at lower values. Hemolysis significantly interfered with measurement of potassium and lactate dehydrogenase (LD) at H=0.5 on all platforms. The most of the tested analytes had the lowest acceptable H index when aspiration method was used. At the low level of hemolysis (H=0.8) glucose, sodium, potassium, chloride, phosphate, and LD were affected on all analyzers, with some additional analytes depending on the manufacturer. CONCLUSIONS: Hemolysis interference differs on different analyzers and according to protocol for obtaining hemolysis. Aspiration method was generally the most sensitive to hemolysis interference, while addition of free Hb was the most resistant.

Test validation, method comparison and reference range for the measurement of ß-hydroxybutyrate in peripheral blood samples.

INTRODUCTION: The measurement of ß-hydroxybutyrate (ßOHB) concentrations is a corner stone of the diagnosis of diabetic ketoacidosis and other ketonic states. The aim of this study was to perform a validation of a peripheral blood ßOHB assay (Randox) on a Roche cobas c502 analyser and to establish a ßOHB reference range for the validated assay. MATERIALS AND METHODS: Precision, linearity and limit of detection and blank (LoD, LoB) were determined according to Clinical and Laboratory Standards Institute (CLSI) EP05-A3, EP 06-A and EP17-A2 guidelines, using commercial control material and residual patient sample pools. As method comparison, for 190 semi-quantitative measurements of urine ketones we determined the corresponding ßOHB blood concentration. The reference range was based on the CLSI C28-A3 guideline, using 304 randomly selected serum samples from population based German National Cohort (GNC) study. RESULTS: Coefficients of variation for the validated assay ranged from 1.5% for high concentrations (3.1 mmol/L) to 6.5% for low concentrations (0.1 mmol/L). Detection capacity was LoB = 0.011 mmol/L and LoD = 0.037 mmol/L. Linearity of the assay ranged from 0.10 to 3.95 mmol/L. The agreement between the semi-quantitative urine ketone test and the ßOHB blood test was moderate (Kappa = 0.66). The obtained 95% serum reference range was estimated as 0.02 to 0.28 mmol/l ßOHB. CONCLUSIONS: The Ranbut ßOHB assay showed good precision and analytical performance. Our results confirm that ßOHB measurement in peripheral blood is indeed a preferable alternative to the semi-quantitative measurement of urine ketones.

Precision, accuracy, cross reactivity and comparability of serum indices measurement on Abbott Architect c8000, Beckman Coulter AU5800 and Roche Cobas 6000 c501 clinical chemistry analyzers.

BACKGROUND: The aim of our study was to perform verification of serum indices on three clinical chemistry platforms. METHODS: This study was done on three analyzers: Abbott Architect c8000, Beckman Coulter AU5800 (BC) and Roche Cobas 6000 c501. The following analytical specifications were verified: precision (two patient samples), accuracy (sample with the highest concentration of interferent was serially diluted and measured values compared to theoretical values), comparability (120 patients samples) and cross reactivity (samples with increasing concentrations of interferent were divided in two aliquots and remaining interferents were added in each aliquot. Measurements were done before and after adding interferents). RESULTS: Best results for precision were obtained for the H index (0.72%-2.08%). Accuracy for the H index was acceptable for Cobas and BC, while on Architect, deviations in the high concentration range were observed (y=0.02 [0.01-0.07]+1.07 [1.06-1.08]x). All three analyzers showed acceptable results in evaluating accuracy of L index and unacceptable results for I index. The H index was comparable between BC and both, Architect (Cohen's κ [95% CI]=0.795 [0.692-0.898]) and Roche (Cohen's κ [95% CI]=0.825 [0.729-0.922]), while Roche and Architect were not comparable. The I index was not comparable between all analyzer combinations, while the L index was only comparable between Abbott and BC. Cross reactivity analysis mostly showed that serum indices measurement is affected when a combination of interferences is present. CONCLUSIONS: There is heterogeneity between analyzers in the hemolysis, icteria, lipemia (HIL) quality performance. Verification of serum indices in routine work is necessary to establish analytical specifications.

Soluble intracellular adhesion molecule-1 and omentin-1 as potential biomarkers of subclinical atherosclerosis in hemodialysis patients.

PURPOSE: Atherosclerotic cardiovascular complications represent significant cause of mortality in hemodialysis (HD) patients. The aims of this study were to: (a) investigate association of sICAM-1, sVCAM-1, omentin-1 and other non-traditional risk factors with subclinical atherosclerosis; (b) examine the diagnostic value of these specific markers in the early detection of subclinical atherosclerosis; and (c) examine their role as predictors of mortality in group of patients with subclinical atherosclerosis on regular HD. MATERIALS AND METHODS: Starting from November 2011, a cohort of 210 HD patients participated in this 3-year follow-up study. The subjects were divided into three groups according to the presence of atherosclerosis. Atherosclerotic disease was assessed by measuring carotid intima-media thickness (IMT). Samplings were withdrawn at baseline and thereafter every 12 months until the end of follow-up. RESULTS: IMT showed weak correlation with sICAM-1 (r = 0.39, P = 0.001), sVCAM-1 (r = 0.27, P = 0.015) and omentin-1 (r = -0.25, P = 0.020), and also omentin-1 showed good correlation with parameters of systolic and diastolic function (r = 0.52, P = 0.001 and r = 0.51, P = 0.001). Multivariate analysis showed that sICAM-1 and sVCAM-1 concentrations were a strong independent correlate of IMT (P = 0.031 and P = 0.010, respectively). The Cox proportional analysis showed that sICAM-1 and omentin-1 concentrations were strong predictors of cardiovascular death (HR 1.85, CI 1.18-2.32, P = 0.021 and HR 4.14, CI 1.38-12.1, P = 0.004, respectively) and that serial measurements of these markers predict IMT progression (HR 1.98, 95 % CI 1.21-2.38, P < 0.002 and HR 2.91, 95 % CI 1.57-4.72, P < 0.001, respectively). CONCLUSIONS: Our study demonstrated that sICAM-1 and omentin-1 levels are strong predictors of cardiovascular death in HD patients with subclinical atherosclerosis.

Croatian laboratories have a good knowledge of the proper detection and management of hemolyzed, icteric and lipemic samples.

BACKGROUND: Endogenous interferences are an important source of biased laboratory results. Hemolysis, lipemia and icteria are the main source of endogenous interference in laboratory medicine. Accreditation according to ISO 15189 improves the overall quality of the laboratory procedures. The aim of our study was i) to assess the level of knowledge of Croatian medical biochemists about the proper detection and management of hemolysis, lipemia and icteria; and ii) to identify possible differences in the level of knowledge respective to the laboratory accreditation status. METHODS: An on-line self-report survey was carried out by the Working Group for Preanalytical Phase of the Croatian Society of Medical Biochemistry and Laboratory Medicine during April to May 2015. Survey included 14 statements (Q1-Q14) about procedures for samples with interferences and participants were asked to assess the degree of agreement with the statement using a 4-point Likert scale. RESULTS: The lowest level of knowledge was observed for statements Q10 (dealing with icteric sample; 40.9% participants agreed with the correct procedure), Q12 (allowable error for interference; 47.2%) and Q11 (dealing with lipemic sample; 60.1%). Almost all participants (97.4%) agreed that laboratories in Croatia should have a harmonized protocol for management of samples with interferences. Participants from accredited laboratories showed higher knowledge of hemolysis detection (p=0.031), rejection of hemolyzed sample (p<0.001), management of icteric samples (p=0.038) and allowable error for interferences (p=0.040). CONCLUSIONS: Croatian laboratories have a good knowledge of the proper detection and management of hemolyzed, icteric and lipemic samples. Accreditation is associated with higher knowledge about management of samples with interferences.

Serum omentin-1 levels as a possible risk factor of mortality in patients with diabetes on haemodialysis.

AIM: The main cause of mortality in haemodialysis (HD) patients is cardiovascular disease. Serum omentin-1 level was found to be associated with cardio-metabolic disorders. The aim of this study was to examine the role of omentin-1 as a predictor of mortality in a group of diabetes positive HD patients. METHODS: A total of 120 prevalent HD patients were included in the study from December 2012 to May 2014. Patients were divided into two groups according to the presence or absence of diabetes. Venous blood samples were taken at months 0 and 18 following an overnight fast (prior to a midweek HD session). Serum omentin-1 level was assessed by enzyme-linked immunosorbent assay. RESULTS: A total of 84 HD patients were analysed at the end of an 18-month follow-up. Omentin-1 levels of HD patients with diabetes were found to be lower than of HD patients without diabetes (9.1±5.8 ng/mL vs. 11.4±4.1 ng/mL, respectively; P=0.015) at the end of follow-up. Omentin-1 levels of survived patients with diabetes were found to be higher than of nonsurvived patients with diabetes (16.5±10.1 ng/mL vs. 12.9±5.3 ng/mL, respectively; P=0.045). During follow-up, 36 patients (30%) died, of whom 25 had diabetes (34%). CONCLUSIONS: Serum omentin-1 levels were significantly lower in HD patients with diabetes. A decrease in omentin-1 levels could be an independent mortality risk factor in this patient group. Further investigation in a greater number of patients is needed.

How well are pregnant women in Croatia informed about the oral glucose tolerance test?

INTRODUCTION: Preanalytical errors still constitute the largest source of errors in laboratory work. Proper patient preparation and patient's knowledge about a particular procedure affects its accuracy and reliability. We hypothesized that most of pregnant women are not well enough informed about the proper procedure for the OGTT. The aims of this study were to investigate: (i) how well pregnant women are informed about the OGTT; (ii) the most common way to inform pregnant women about OGTT and (iii) whether pregnant women's level of knowledge about the OGTT differ regarding source of information. MATERIALS AND METHODS: The anonymous questionnaire was conducted across the country in 23 Croatian primary and secondary healthcare centres. The questionnaire contained 9 questions on certain demographic data and familiarity with OGTT procedure. All 343 participants filled the questionnaire before the first blood draw. RESULTS: 42% of the participants demonstrated high and 38% adequate level of knowledge about the OGTT procedure. Majority of participants were informed about the procedure by gynaecologist (56%). The level of knowledge differed among participants with different sources of information (P=0.030). Further analysis showed that the level of knowledge was lower in pregnant women having received information from their gynaecologist compared to pregnant women who received information from the laboratory staff. CONCLUSIONS: In general, pregnant women are familiar with OGTT procedure, main source of information about the OGTT procedure is their gynaecologist, but the level of knowledge was higher in women who received information about the OGTT procedure from the laboratory staff.

Evaluation of the BD Vacutainer(®) RST blood collection tube for routine chemistry analytes: clinical significance of differences and stability study.

INTRODUCTION: Preanalytical variables account for most of laboratory errors. There is a wide range of factors that affect the reliability of laboratory report. Most convenient sample type for routine laboratory analysis is serum. BD Vacutainer(®) Rapid Serum Tube (RST) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) blood collection tube provides rapid clotting time allowing fast serum separation. Our aim was to evaluate the comparability of routine chemistry parameters in BD Vacutainer(®) RST blood collection tube in reference with the BD Vacutainer(®) Serum Separating Tubes II Advance Tube (SST) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). MATERIALS AND METHODS: Blood specimens were collected from 90 participants for evaluation on its results, clotting time and stability study of six routine biochemistry parameters: glucose (Glu), aspartate aminotransferase (AST), alanine aminotransferase (ALT), calcium (Ca), lactate dehidrogenase (LD) and potassium (K) measured with Olympus AU2700 analyzer (Beckman Coulter, Tokyo, Japan). The significance of the differences between samples was assessed by paired t-test or Wilcoxon Matched-Pairs Rank test after checking for normality. RESULTS: Clotting process was significantly shorter in the RSTs compared to SSTs (2.49 min vs. 19.47 min, respectively; P < 0.001). There was a statistically significant difference between the RST and SST II tubes for glucose, calcium and LD (P < 0.001). Differences for glucose and LD were also clinically significant. Analyte stability studies showed that all analytes were stable for 24 h at 4 °C. CONCLUSIONS: Most results (except LD and glucose) from RST are comparable with those from SST. In addition, RST tube provides shorter clotting time.