Effect of exogenous lipids contamination on blood gas analysis.

Objectives: The purpose of this study was to investigate the effects of contamination of venous blood with a lipid-containing solution on parameters measured by a modern blood gas analyzer. Methods: We collected venous blood from 17 healthcare workers (46 ± 11 years; 53 % women) into three blood gas syringes containing 0 , 5 and 10 % lipid-containing solution. Blood gas analysis was performed within 15 min from sample collection on GEM Premier 5000, while triglycerides and serum indices were assays on Roche COBAS C702. Results: Triglycerides concentration increased from 1.0 ± 0.3 mmol/L in the uncontaminated blood gas syringe, to 39.4 ± 7.8 and 65.3 ± 14.4 mmol/L (both p<0.001) in syringes with 5 and 10 % final lipid contamination. The lipemic and hemolysis indices increased accordingly. Statistically significant variation was noted for all analytes except hematocrit and COHb in the syringe with 5 % lipids, while only COHb did not vary in the syringe with 10 % lipids. Significant increases were observed from 5 % lipid contamination for pO2, SO2 and lactate, while the values of pH, pCO2, sodium, potassium, chloride, ionized calcium, glucose, hematocrit (10 % contamination), hemoglobin and MetHB decreased. All these changes except lactate and CoHb exceeded their relative performance specifications. Conclusions: Artifactual hyperlipidemia caused by contamination with exogenous lipids can have a clinically significant impact on blood gas analysis. Manufacturers of blood gas analyzers must be persuaded to develop new instruments equipped with serum indices.

Effect of syringe underfilling on the quality of venous blood gas analysis.

OBJECTIVES: There is limited information on the influence of collecting small amounts of blood on the quality of blood gas analysis. Therefore, the purpose of this study was to investigate the effects of different degrees of underfilling of syringes on test results of venous blood gas analysis. METHODS: Venous blood was collected by venipuncture from 19 healthcare workers in three 1.0 mL syringes for blood gas analysis, by manually aspirating different volumes of blood (i.e., 1.0, 0.5 and 0.25 mL). Routine blood gas analysis was then immediately performed with GEM Premier 5,000. The results of the two underfilled syringes were compared with those of the reference syringe filled with appropriate blood volume. RESULTS: The values of most assayed parameters did not differ significantly in the two underfilled syringes. Statistically significant variations were found for lactate, hematocrit and total hemoglobin, the values of which gradually increased as the fill volume diminished, as well as for sodium concentration, which decreased in both insufficiently filled blood gas syringes. The bias was clinically meaningful for lactate in syringe filled with 0.25 mL of blood, and for hematocrit, total hemoglobin and sodium in both syringes containing 0.5 and 0.25 mL of blood. CONCLUSIONS: Collection of smaller volumes of venous blood than the specified filling volume in blood gas syringes may have an effect on the quality of some test results, namely lactate, hematocrit, total hemoglobin and sodium. Specific indications must be given for standardizing the volume of blood to be collected within these syringes.

Effects of Recombinant SARS-CoV-2 Spike Protein Variants on Platelet Morphology and Activation.

Platelets are central elements of hemostasis and also play a pivotal role in the pathogenesis of thrombosis in coronavirus disease 2019. This study was planned to investigate the effects of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant spike protein variants on platelet morphology and activation. Citrated whole blood collected from ostensibly healthy subjects was challenged with saline (control sample) and with 2 and 20 ng/mL final concentration of SARS-CoV-2 recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Platelet count was found to be decreased with all SARS-CoV-2 recombinant spike protein variants and concentrations tested, achieving the lowest values with 20 ng/mL Delta recombinant spike protein. The mean platelet volume increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, but especially using Delta and Alpha recombinant spike proteins. The values of both platelet function analyzer-200 collagen-adenosine diphosphate and collagen-epinephrine increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, and thus reflecting platelet exhaustion, and displaying again higher increases with Delta and Alpha recombinant spike proteins. Most samples where SARS-CoV-2 recombinant spike proteins were added were flagged as containing platelet clumps. Morphological analysis revealed the presence of a considerable number of activated platelets, platelet clumps, platelet-monocyte, and platelet-neutrophils aggregates, especially in samples spiked with Alpha and Delta recombinant spike proteins at 20 ng/mL. These results provide support to the evidence that SARS-CoV-2 is capable of activating platelets through its spike protein, though such effect varies depending on different spike protein variants.

Plasma Bile Acid Profiling and Modulation of Secreted Mucin 5AC in Cholangiocarcinoma.

Studies investigating the potential role of circulating bile acids (BAs) as diagnostic biomarkers for cholangiocarcinoma (CCA) are sparse and existing data do not adjust for confounding variables. Furthermore, the mechanism by which BAs affect the expression of the oncogenic mucin 5AC (MUC5AC) has never been investigated. We performed a case-control study to characterise the profile of circulating BAs in patients with CCA (n = 68) and benign biliary disease (BBD, n = 48) with a validated liquid chromatography-tandem mass spectrometry technique. Odd ratios (OR) for CCA associations were calculated with multivariable logistic regression models based on a directed acyclic graph structure learning algorithm. The most promising BAs were then tested in an in vitro study to investigate their interplay in modulating MUC5AC expression. The total concentration of BAs was markedly higher in patients with CCA compared with BBD controls and accompanied by a shift in BAs profile toward a higher proportion of primary conjugated BAs (OR = 1.50, CI: 1.14 to 1.96, p = 0.003), especially taurochenodeoxycholic acid (TCDCA, OR = 42.29, CI: 3.54 to 504.63, p = 0.003) after multiple adjustments. Western blot analysis of secreted MUC5AC in human primary cholangiocytes treated with primary conjugated BAs or with TCDCA alone allowed us to identify a novel 230 kDa isoform, possibly representing a post-translationally modified MUC5AC specie.

Machine Learning Model Comparison in the Screening of Cholangiocarcinoma Using Plasma Bile Acids Profiles.

Bile acids (BAs) assessments are garnering increasing interest for their potential involvement in development and progression of cholangiocarcinoma (CCA). Since machine learning (ML) algorithms are increasingly used for exploring metabolomic profiles, we evaluated performance of some ML models for dissecting patients with CCA or benign biliary diseases according to their plasma BAs profiles. We used ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for assessing plasma BAs profile in 112 patients (70 CCA, 42 benign biliary diseases). Twelve normalisation procedures were applied, and performance of six ML algorithms were evaluated (logistic regression, k-nearest neighbors, naïve bayes, RBF SVM, random forest, extreme gradient boosting). Naïve bayes, using direct bilirubin concentration for normalisation of BAs, was the ML model displaying better performance in the holdout set, with an Area Under Curve (AUC) of 0.95, 0.79 sensitivity, 1.00 specificity. This model, also characterised by 1.00 positive predictive value and 0.73 negative predictive value, displayed a globally excellent accuracy (86.4%). The accuracy of the other five models was lower, and AUCs ranged 0.75-0.95. Preliminary results of this study show that application of ML to BAs profile analysis can provide a valuable contribution for characterising bile duct diseases and identifying patients with higher likelihood of having malignant pathologies.

Bile Acids Quantification by Liquid Chromatography-Tandem Mass Spectrometry: Method Validation, Reference Range, and Interference Study.

Bile acids (BA) play a pivotal role in cholesterol metabolism. Their blood concentration has also been proposed as new prognostic and diagnostic indicator of hepatobiliary, intestinal, and cardiovascular disease. Liquid chromatography tandem mass spectrometry (LC-MS/MS) currently represents the gold standard for analysis of BA profile in biological samples. We report here development and validation of a LC-MS/MS technique for simultaneously quantifying 15 BA species in serum samples. We also established a reference range for adult healthy subjects (n = 130) and performed a preliminary evaluation of in vitro and in vivo interference. The method displayed good linearity, with high regression coefficients (>0.99) over a range of 5 ng/mL (lower limit of quantification, LLOQ) and 5000 ng/mL for all analytes tested. The accuracies were between 85-115%. Both intra- and inter-assay imprecision was <10%. The recoveries ranged between 92-110%. Each of the tested BA species (assessed on three concentrations) were stable for 15 days at room temperature, 4 °C, and -20 °C. The in vitro study did not reveal any interference from triglycerides, bilirubin, or cell-free hemoglobin. The in vivo interference study showed that pools obtained from hyper-cholesterolemic patients and hyper-bilirubinemic patients due to post-hepatic jaundice for benign cholestasis, cholangiocarcinoma and pancreatic head tumors had clearly distinct patterns of BA concentrations compared with a pool obtained from samples of healthy subjects. In conclusion, this study proposes a new suitable candidate method for identification and quantitation of BA in biological samples and provides new insight into a number of variables that should be taken into account when investigating pathophysiological changes of BA in human diseases.

Stability of refrigerated whole blood samples for osmotic fragility test

ABSTRACT Background: The osmotic fragility test (OFT), conventionally used for assisting the diagnosis of many erythrocyte disorders, is a manual and time-consuming analysis not daily performed in many medical laboratories. This study was aimed at defining the stability of whole blood samples used for assessing erythrocyte osmotic resistance. Methods: Twenty-one consecutive routine whole blood samples collected into 5.4 mg K2EDTA were tested immediately after collection (day 0) and at different time intervals afterward (day 1, 2, 3, 4, 7, 10 and 14) after storage at 4 °C. The OFT was performed with the Osmored Monotest (1.3% glycerol; Eurospital, Trieste, Italy). Results at the different time points were compared with those obtained at day 0 and with the reference change value (i.e., 33%). Results: The median value of both hyperosmolar and hyposmolar resistance increased from baseline, reaching statistical significance at day 7 for hyperosmolar resistance and at day 1 for hyposmolar resistance, respectively. The median relative increase of hemolysis percentage values become greater than the reference change value at day 3 for hyposmolar resistance, while this limit was never overcome for hyperosmolar resistance. A significant inverse association was found between the mean increase in hyperosmolar resistance and the baseline value of hyperosmolar resistance (r = −0.92), mean corpuscular volume (MCV; r = −0.46) or mean corpuscular hemoglobin (MCH; r = −0.44), as well as between the mean increase in hyposmolar resistance and the baseline value of hyposmolar resistance (r = −0.86), or patient age (r = −0.56). Conclusions: The sample stability seems critical for the OFT. Whole blood specimens should not be stored refrigerated at 4 °C for >2 days before testing.

Serum Androgens Are Independent Predictors of Insulin Clearance but Not of Insulin Secretion in Women With PCOS.

CONTEXT/OBJECTIVE: In insulin-resistant individuals, hyperinsulinemia is a key compensatory mechanism, aimed at maintaining glucose homeostasis. Increased secretion and reduced clearance of insulin may both potentially contribute to this phenomenon. Insulin resistance and hyperinsulinemia are common findings in women with polycystic ovary syndrome (PCOS). While there is some information on insulin secretion, very few studies have investigated metabolic clearance rate of insulin (MCRI) in these women. Moreover, there is paucity of data on the relationships between MCRI and the pathophysiological characteristics of PCOS. The aim of the study was to explore these issues. PATIENTS: One hundred ninety women with PCOS, diagnosed according to the Rotterdam criteria, with normal glucose tolerance. DESIGN: Assessment of MCRI and clinical, hormonal, and metabolic characteristics of subjects. MCRI and insulin sensitivity were measured by the hyperinsulinemic euglycemic clamp. Serum androgens were assessed by liquid chromatography-mass spectrometry and equilibrium dialysis. A historical sample of healthy women was used to define the corresponding reference intervals. RESULTS: MCRI was impaired in about two-thirds of women with PCOS. Subjects with low MCRI differed from those with normal MCRI for a number of anthropometric, metabolic, and endocrine features. In multivariate analysis, the degree of adiposity, estimates of insulin secretion, and serum androgen concentrations were independent predictors of MCRI. Conversely, age, adiposity, MCRI, and insulin sensitivity, but not serum androgens, were independent predictors of insulin secretion. CONCLUSIONS: In women with PCOS, metabolic clearance of insulin is reduced, contributing to generating hyperinsulinemia. Serum androgens are independent predictors of this phenomenon.

Two-center comparison of 10 fully-automated commercial procalcitonin (PCT) immunoassays.

Background This two-center study was designed to verify comparability of procalcitonin (PCT) values among 10 different commercial immunoassays. Methods A total number of 176 routine lithium-heparin plasma samples were divided in identical aliquots and simultaneously analyzed with 10 different PCT immunoassays, including Kryptor BRAHMS PCT sensitive, Abbott Architect BRAHMS PCT, Beckman Coulter Access PCT (on Access and DXI), BioMérieux Vidas BRAHMS PCT, Diasorin Liaison BRAHMS PCT, Fujirebio Lumipulse G BRAHMS PCT, Roche BRAHMS PCT (on Cobas E801), Diazyme PCT (on Roche Cobas C702) and SNIBE Maglumi PCT. Results Highly significant correlation was always found across multiple comparisons, with correlation coefficients comprised between 0.918 and 0.997 (all p < 0.001). Bland and Altman plots analysis revealed highly variable bias among immunoassays, ranging between ±0.2% and ±38.6%. Diazyme PCT on Roche Cobas C702 and SNIBE Maglumi PCT displayed the larger overestimation, whilst PCT values were underestimated by Cobas BRAHAMS PCT. The agreement was always >80% (all p < 0.001), but varied largely across multiple comparisons, ranging between 90%-99% at 0.1 µg/L, 81%-99% at 0.25 µg/L, 83%-100% at 0.5 µg/L, 94%-100% at 2.0 µg/L and 90%-99% at 10 µg/L, respectively. The larger disagreement was observed comparing Diazyme PCT and Maglumi PCT with the other methods. Conclusions Although we found acceptable correlation among 10 commercial PCT immunoassays, the limited agreement at clinical decision thresholds remains a major issue, especially at lower end of PCT concentration, thus potentially contributing to jeopardize the clinical value of this biomarker.

Impact of low volume citrate tubes on results of first-line hemostasis testing.

INTRODUCTION: Pediatric tubes are increasingly used for drawing blood for hemostasis testing. This study has investigated the potential impact of low volume citrate tubes on results of first-line hemostasis testing. METHODS: The study population comprised 34 patients on warfarin therapy and 17 ostensibly healthy volunteers. Blood was collected into five different evacuated blood tubes from each subject. On right arm, blood was drawn directly into two standard evacuated blood tubes (3-mL Vacuette and 2-mL Vacutest) and one evacuated low volume blood tube (1-mL Vacuette) by straight needle venipuncture. On left arm, blood was drawn using a 5-mL syringe and then transferred within two nonevacuated microtubes (0.5 mL MiniCollect and 0.5 mL Micro Test). Prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen were assayed on ACL TOP 700. RESULTS: Spearman's correlation of PT, APTT, and fibrinogen values obtained using different tubes was always satisfactory (ie, ≥0.93). A statistically significant bias was frequently found by comparing values obtained in different tubes. Nevertheless, the minimum quality specifications for bias were exceeded only by comparing data of Vacuette 1 mL with those of all other blood tubes for PT, by comparing data of Micro Test 0.5 mL with those of all other blood tubes for APTT, and by comparing data of Micro Test 0.5-mL blood tubes with those of Vacuette 3 mL and Vacuette 1. CONCLUSION: First-line hemostasis testing using low volume citrate tubes may display differences sometimes exceeding the minimum quality specifications.

A paradigmatic case of haemolysis and pseudohyperkalemia in blood gas analysis.

A 51-year old male patient was admitted to the hospital with acute dyspnea and history of chronic asthma. Venous blood was drawn into a 3.0 mL heparinized syringe and delivered to the laboratory for blood gas analysis (GEM Premier 4000, Instrumentation Laboratory), which revealed high potassium value (5.2 mmol/L; reference range on whole blood, 3.5-4.5 mmol/L). This result was unexpected, so that a second venous blood sample was immediately drawn by direct venipuncture into a 3.5 mL lithium-heparin blood tube, and delivered to the laboratory for repeating potassium testing on Cobas 8000 (Roche Diagnostics). The analysis revealed normal plasma potassium (4.6 mmol/L; reference range in plasma, 3.5-5.0 mmol/L) and haemolysis index (5; 0.05 g/L). Due to suspicion of spurious haemolysis, heparinized blood was transferred from syringe into a plastic tube and centrifuged. Potassium and haemolysis index were then measured in this heparinized plasma, confirming high haemolysis index (50; 0.5 g/L) and pseudohyperkalemia (5.5 mmol/L). Investigation of this case revealed that spurious haemolysis was attributable to syringe delivery in direct ice contact for ~15 min. This case emphasizes the importance of avoiding sample transportation in ice and the need of developing point of care analysers equipped with interference indices assessment.

Dark chocolate modulates platelet function with a mechanism mediated by flavan-3-ol metabolites.

Cocoa is a rich source bioactive compounds, i.e., flavan-3-ols, and its consumption has been associated with several beneficial effects, such as the positive modulation of the hemostasis targeted by the platelet function. However, these phenolic compounds have a very low bioavailability and extensively undergo phase I and II metabolism, with the appearing into the bloodstream of (epi)catechin conjugates and phenyl-γ-valerolactones and their conjugates, at different times.The aims of this study were to explore the effect of dark chocolate on platelet function and to investigate the relationship between this interplay and flavan-3-ol derived metabolites.Eighteen healthy male volunteers ingested 50 g of 90% cocoa chocolate within 5 minutes. Blood samples were collected immediately before chocolate ingestion (T0) and 4 hours afterwards (T1). Platelet function analyzer (PFA)-100 closure time was assessed using collagen/adenosine-5'-diphosphate (COL/ADP) and collagen/epinephrine (COL/EPI) cartridges. Plasma flavan-3-ol metabolites were identified and quantified by means of liquid chromatography coupled to a triple quadrupole mass spectrometer (UHPLC-ESI-MS/MS).Results evidenced a significant increase of COL/ADP-induced PFA-100 closure time, but not COL/EPI, 4 hours after ingestion of dark chocolate. Total plasma structurally-related (epi)catechin metabolite (SREM) concentration significantly increased at T1, together with 4 out of the 6 detected metabolites. Total phenyl-γ-valerolactone concentrations remained unchanged. Spearman correlations evidenced a strong correlation between COL/ADP closure time and SREMs, mainly led by (epi)catechin-sulfate isomers.These data confirm that the potential beneficial effect of dark chocolate on primary hemostasis may be mediated by flavan-3-ol circulating metabolites.

Middle-distance running acutely influences the concentration and composition of serum bile acids: Potential implications for cancer risk?

BACKGROUND: This study was aimed to investigate the acute effect of medium-distance running on bile acids concentration and composition, in order to verify whether the positive impact of physical exercise on cancer risk may also be mediated by variation of bile acids concentration and composition in serum. METHODS: The concentration and composition of serum bile acids was analyzed in 30 middle-aged and healthy recreational athletes with a reference liquid chromatography-mass spectrometry technique, immediately before and shortly after the end of the running trial. The concentration of bile acids after the run was adjusted for plasma volume change. RESULTS: All athletes successfully completed the trial. After correction of values for the individual plasma volume change calculated after the run, the serum concentration of total bile acids was found to be significantly reduced by approximately 46%. A statistically significant decrease was observed for cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, glycoursodeoxycholic and hyodeoxycholic acids, whereas the concentration of the remaining compounds remained unvaried after the run. A considerable variation of bile acids profile was also observed. No significant association was found between running performance and variation of bile acids concentrations. CONCLUSION: These results show that middle distance running acutely decreases the concentration of total bile acids in serum, especially that of the more mutagenic and carcinogenic compounds, so providing an intriguing support to the favorable effects of physical exercise for lowering the risk of many gastrointestinal cancers.

Mobile phone radiofrequency exposure has no effect on DNA double strand breaks (DSB) in human lymphocytes.

BACKGROUND: The use of mobile phones has been associated with an increased risk of developing certain type of cancer, especially in long term users. Therefore, this study was aimed to investigate the potential genotoxic effect of mobile phone radiofrequency exposure on human peripheral blood mononuclear cells in vitro. METHODS: The study population consisted in 14 healthy volunteers. After collection of two whole blood samples, the former was placed in a plastic rack, 1 cm from the chassis of a commercial mobile phone (900 MHz carrier frequency), which was activated by a 30-min call. The second blood sample was instead maintained far from mobile phones or other RF sources. The influence of mobile phone RF on DNA integrity was assessed by analyzing γ-H2AX foci in lymphocytes using immunofluorescence staining kit on AKLIDES. RESULTS: No measure of γ-H2AX foci was significantly influenced by mobile phone RF exposure, nor mobile phone exposure was associated with significant risk of genetic damages in vitro (odds ratio comprised between 0.27 and 1.00). CONCLUSIONS: The results of this experimental study demonstrate that exposure of human lymphocytes to a conventional 900 MHz RF emitted by a commercial mobile phone for 30 min does not significantly impact DNA integrity.

Patient posture for blood collection by venipuncture: recall for standardization after 28 years

ABSTRACT Background: Although data about the effect of posture on routine hematological testing were published 28 years ago, this pre-analytical issue has not been standardized so far. This study was planned to evaluate whether postural changes influence the results of hematology testing. Methods: A complete blood count was performed in 19 healthy volunteers after 25 min in the supine position, 20 min in a sitting position and 20 min stationary standing in an upright position. Results: The change from supine to sitting position caused clinically significant increases in the hemoglobin, hematocrit and red blood cell count. Furthermore, the change from supine to standing caused clinically significant increases in the hemoglobin, hematocrit, red blood cell, leukocyte, neutrophil, lymphocyte, basophil and platelet counts, and mean platelet volume, and that from sitting to standing caused clinically significant increases in hemoglobin, hematocrit, and red blood cell, leukocyte, neutrophil and lymphocyte counts. Conclusion: The results of this investigation provide further support to the notion that effort should be made to achieve widespread standardization in the practice of phlebotomy, including patient posture.