Effects of Hypoxia and Inflammation on Hepcidin Concentration in Non-Anaemic COVID-19 Patients.

Background/Objectives: This study aimed to explore the influence of hypoxia, inflammation, and erythropoiesis on hepcidin and other iron status parameters in non-anaemic COVID-19 patients admitted to the emergency unit before the introduction of therapeutic interventions. Methods: Ninety-six COVID-19 patients and 47 healthy subjects were recruited. Patients were subdivided into hypoxic or normoxic groups and, after follow-up, into mild and moderate, severe or critical disease severity groups. Iron, unsaturated iron-binding capacity (UIBC), ferritin, C-reactive protein (CRP), and interleukin 6 (IL-6) were measured on automatic analysers. ELISA kits were used for hepcidin and erythropoietin (EPO) determination. We calculated total iron-binding capacity (TIBC) and ratios of hepcidin with parameters of iron metabolism (ferritin/hepcidin, hepcidin/iron), inflammation (hepcidin/CRP, hepcidin/IL-6), and erythropoietic activity (hepcidin/EPO). Results: Hepcidin, ferritin, EPO, CRP, IL-6, ferritin/hepcidin, and hepcidin/iron were increased, while UIBC, TIBC, hepcidin/CRP, and hepcidin/IL-6 were decreased in hypoxic compared to normoxic patients as well as in patients with severe or critical disease compared to those with mild and moderate COVID-19. Regarding predictive parameters of critical COVID-19 occurrence, in multivariable logistic regression analysis, a combination of EPO and ferritin/hepcidin showed very good diagnostic performances and correctly classified 88% of cases, with an AUC of 0.838 (0.749-0.906). Conclusions: The hypoxic signal in our group of patients was not strong enough to overcome the stimulating effect of inflammation on hepcidin expression. EPO and ferritin/hepcidin might help to identify on-admission COVID-19 patients at risk of developing a critical form of the disease.

Performance comparison of Westergren modified SRS 100/II and alternate iSED® method for erythrocyte sedimentation rate.

INTRODUCTION: The erythrocyte sedimentation rate (ESR) includes three phases, each prone to different interferences. Due to many disadvantages of the reference Westergren method, modified and alternate methods have been introduced. The aim of this study was to compare the modified Westergren method on SRS 100/II analyzer in citrate blood with the alternate method on iSED® analyzer in EDTA sample. Additionally, possible interfering factors and ESR stability during 6 h at room temperature were evaluated. METHODS: A total of 188 samples were included in the method comparison. Additionally, the effects of inflammation, haematocrit and MCV values on ESR were evaluated. To determine ESR stability in different samples, ESR was evaluated at three time points; within 15 min of blood sampling and after 3 and 6 h in different sample types and analyzers (N = 65). RESULTS: Results indicated the constant difference between tested methods with obtained mean bias of 5 mm (95% CI: 3-7). There was higher absolute mean bias in groups with ESR > 40 mm and elevated inflammation markers (p < 0.001). Regarding different MCV and haematocrit groups there was no statistically significant difference in obtained absolute mean biases for MCV (p = 0.087) while there was higher bias in low haematocrit group compared to normal haematocrit (p = 0.004). In addition, there was a significant difference between ESR values at different time points for iSED® (p < 0.001) and no difference for SRS 100/II analyzer (p = 0.406). CONCLUSION: There are differences in ESR values between tested methods. EDTA sample on iSED® should be analysed as soon as possible to avoid falsely increased ESR.

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.

Faecal calprotectin determination: impact of preanalytical sample treatment and stool consistency on within- and between-method variability.

INTRODUCTION: We assessed the differences in faecal calprotectin (FC) concentrations measured by two assays depending on the stool consistency and extraction method. MATERIALS AND METHODS: Stool samples were extracted using the EliA Stool Extraction Kit, Calex® Cap extraction device and respective weighing methods, while FC concentrations were measured using the EliATM Calprotectin and Bühlmann fCAL® Turbo method and checked for within- and between-method variability with regard to extraction method and stool consistency category. Extraction yield was evaluated for impact of different sample incubation time (10 min and 1 h) in extraction buffer for both methods and for impact of different initial sample dilutions (1:50, 1:100, 1:500) for fCAL® Turbo method. RESULTS: Results determined from Calex® Cap extracts were higher compared to weighing method extracts (mean bias 33.3%; P < 0.001), while no significant difference was found between results obtained with EliA Stool Extraction Kit and weighing method (mean bias 0.1%; P = 0.484), in both cases irrespective of stool consistency. Bühlmann fCAL® Turbo results were higher than EliATM Calprotectin results (mean bias 32.3%, P = 0.025 weighing method; and mean bias 53.9%, P < 0.001 extraction devices), the difference is dependent on stool consistency and FC concentration. Significantly higher FC extraction yield was obtained with longer sample incubation time for both methods (P = 0.019 EliATM Calprotectin; P < 0.001 fCAL® Turbo) and with increasing initial sample dilution for fCAL® Turbo method (P < 0.001). CONCLUSION: Preanalytical stool sample handling proved to be a crucial factor contributing to within- and between-FC assay variability. Standardization is urgently needed in order to assure comparable and reliable FC results.

Corrigendum to: Minimum requirements for the estimation of the measurement uncertainty: Recommendations of the joint Working group for uncertainty of measurement of the CSMBLM and CCMB.

[This corrects the article DOI: 10.11613/BM.2017.030502.].

Various glycolysis inhibitor-containing tubes for glucose measurement cannot be used interchangeably due to clinically unacceptable biases between them.

BACKGROUND: The aim of our study was to determine the difference between glucose concentration measured 30 min after venipuncture in ice-chilled heparin plasma sample and all currently available citrate buffer-containing tubes (Greiner Glucomedics, Greiner FC Mix and Sarstedt GlucoEXACT) and still widely used sodium fluoride/potassium oxalate (NaF/Kox) tubes from Greiner. METHODS: Blood was collected from 20 healthy volunteers and 20 patients with diabetes into LiH, NaF/KOx, Glucomedics, FC mix and GlucoEXACT tubes. Glucose was measured within 30 min from blood sampling in duplicate on the Architect c8000 analyzer. Mean biases between all tube types were calculated and compared to the recommended criteria (1.95%). Additionally, glucose concentrations measured in all five tube types were compared using the Friedman test. RESULTS: In the entire studied population, glucose concentrations measured in Glucomedics, FC mix and GlucoEXACT were higher (7.3%, 3.2% and 2.0%, respectively) than in the ice-chilled LiH tubes. When all glycolysis inhibitor-containing tubes were compared, Glucomedics tubes significantly differed from GlucoEXACT and FC mix tubes (biases -4.9% and 4.0%, respectively). In addition, there was a significant difference between the NaF/KOx tube and Glucomedics, as well as FC mix tubes (biases 7.1% and 3.0%, respectively). CONCLUSIONS: Glucose concentrations measured in recommended ice-chilled lithium heparin- and citrate buffer-containing tubes are not comparable. Significant biases exist between various glycolysis inhibitor-containing tubes; therefore, they cannot be used interchangeably.

Minimum requirements for the estimation of measurement uncertainty: Recommendations of the joint Working group for uncertainty of measurement of the CSMBLM and CCMB.

The International vocabulary of metrology - Basic and general concepts and associated terms (VIM3, 2.26 measurement uncertainty, JCGM 200:2012) defines uncertainty of measurement as a non-negative parameter characterizing the dispersion of the quantity values being attributed to a measurand, based on the information obtained from performing the measurement. Clinical Laboratory Standards Institute (CLSI) has published a very detailed guideline with a description of sources contributing to measurement uncertainty as well as different approaches for the calculation (Expression of measurement uncertainty in laboratory medicine; Approved Guideline, CLSI C51-A 2012). Many other national and international recommendations and original scientific papers about measurement uncertainty estimation have been published. In Croatia, the estimation of measurement uncertainty is obligatory for accredited medical laboratories. However, since national recommendations are currently not available, each of these laboratories uses a different approach in measurement uncertainty estimation. The main purpose of this document is to describe the minimal requirements for measurement uncertainty estimation. In such way, it will contribute to the harmonization of measurement uncertainty estimation, evaluation and reporting across laboratories in Croatia. This recommendation is issued by the joint Working group for uncertainty of measurement of the Croatian Society for Medical Biochemistry and Laboratory Medicine and Croatian Chamber of Medical Biochemists. The document is based mainly on the recommendations of Australasian Association of Clinical Biochemists (AACB) Uncertainty of Measurement Working Group and is intended for all medical biochemistry laboratories in Croatia.

The evidence for clinically significant bias in plasma glucose between liquid and lyophilized citrate buffer additive.

OBJECTIVES: Citrate buffer additive has been suggested to be of supreme performance in inhibiting glycolysis. However, there is little evidence in the literature regarding the comparability of glucose concentrations in liquid and lyophilized citrate buffer containing tubes. The aim of this study was to compare glucose concentrations in tubes containing liquid (Glucomedics) and lyophilized citrate buffer (Terumo VENOSAFE™ Glycemia) additive, measured immediately after centrifugation. DESIGN AND METHODS: Blood was collected from forty volunteers into both Glucomedics and Venosafe Glycemia tubes. Blood was centrifuged within 15min from venipuncture and glucose concentration was measured immediately after centrifugation, on the Abbott Architect analyzer. Differences between glucose concentrations in Glucomedics and Terumo tubes were tested using the paired t-test. Mean bias was calculated and compared to recommended quality specification for glucose (i.e. 2.2%). RESULTS: Glucose concentration in Terumo tubes was 3.4% lower than in Glucomedics tubes (P<0.001). The mean bias was clinically significant. CONCLUSIONS: There is a clinically significant difference between glucose concentrations in liquid and lyophilized citrate buffer additive tubes (Glucomedics vs. Terumo tubes) measured immediately after centrifugation. This difference may affect the patient outcome due to the misclassification of diabetes.

Glucose is stable during prolonged storage in un-centrifuged Greiner tubes with liquid citrate buffer, but not in serum and NaF/KOx tubes.

BACKGROUND: Delayed sample processing can affect accurate glucose measurement. Our aim was to investigate the stability of glucose in samples collected in serum, sodium fluoride/potassium oxalate (NaF/KOx) and Glucomedics tubes processed according to different controlled pre-centrifugation delays (up to 180 min after venipuncture) in order to simulate prolonged sample transport between venipuncture and centrifugation. METHODS: Samples were collected from healthy volunteers (n=80) into either serum or NaF/KOx and Glucomedics tubes. Glucose concentration was measured in samples centrifuged immediately after venipuncture and compared with tubes processed with a delay of 60, 120 and 180 min prior to centrifugation. Differences between baseline and respective delayed centrifugation glucose value for each tube type were tested using the paired t-test. Mean bias calculated for each tube type and delay protocol was compared to recommended quality specifications for glucose (2.2%). RESULTS: Glucose concentrations measured in all three delayed tube types were lower in comparison to respective baseline glucose concentrations measured in immediately processed tube (p<0.001). The highest decrease in glucose was observed in serum tubes in all specified time points (p<0.001), while glucose was most stable in Glucomedics tubes (p<0.001). The decrease in glucose observed for serum and NaF/KOx tubes was clinically significant at all specified time points while the bias for Glucomedics tubes did not exceed the criteria even with a centrifugation delay of 180 min. CONCLUSIONS: Glucose stability in un-centrifuged Glucomedics tubes is much superior to serum and NaF/KOx tubes. Glucomedics tubes can be left un-centrifuged for up to 3 h without affecting glucose concentration.

Liquid citrate acidification introduces significant glucose bias and leads to misclassification of patients with diabetes.

BACKGROUND: Glucose measurements are crucial in diabetes diagnosis. We aimed to assess the effectiveness of liquid citrate acidification in preventing glycolysis and investigate glucose stability in serum, sodium fluoride (NaF/KOx) and Glucomedics tubes. METHODS: Samples from 40 participants were collected in serum, lithium-heparin (LiH), sodium fluoride/potassium oxalate (NaF/KOx) and Glucomedics tubes. Glucose was measured within 60 min (baseline), 120 and 180 min from venipuncture. Serum, NaF/KOx and Glucomedics values at baseline were compared to LiH glucose concentration. Additionally, glucose values measured at 120 and 180 min from each tube were compared with the baseline value. Mean absolute bias for each tube and time point was calculated and compared to recommended criteria. The regression equation obtained comparing citrate to NaF/KOx tubes was used to recalculate glucose results retrieved from the laboratory information system. RESULTS: Glucose measured in Glucomedics was higher (9.9%; p<0.001), while glucose in NaF/KOx and serum was lower compared to LiH (2.4%; p<0.001 and 3.2%; p<0.001, respectively). Biases for all tubes were clinically significant. Glucose remained unchanged at room temperature in all tubes for up to 180 min after venipuncture. Observed bias caused by Glucomedics leads to a 10.6% increase in diabetes prevalence (p<0.001). CONCLUSIONS: Inhibition of glycolysis is most effectively achieved using liquid citrate acidification, compared to LiH, NaF/KOx or serum. Due to clinically significant bias relative to reference glucose, the interchangeable use of different tube types for serial glucose measurements is not recommended. The replacement of NaF/KOx with Glucomedics tubes substantially impacts glucose results, giving marked rise in diabetes prevalence.