Hemolysis In Blood Samples Research Articles

Prevalence of and factors associated with Na + /K + imbalances in a population of children hospitalized with febrile urinary tract infection

We aimed to assess the prevalence of and factors associated with Na + /K + imbalances in children hospitalized for febrile urinary tract infection (fUTI). This retrospective Italian multicenter study included children aged 18 years or younger (median age = 0.5 years) who were discharged with a primary diagnosis of fUTI. Na + /K + imbalances were classified as hyponatremia (sodium < 135 mEq/L), hypernatremia (sodium > 145 mEq/L), hypokalemia (potassium < 3.5 mEq/L), hyperkalemia (potassium > 5.5 mEq/L), and concurrent hyponatremia and hyperkalemia, in the absence of evidence of hemolyzed blood samples. Among the 849 enrolled children, 23% had hyponatremia, 6.4% had hyperkalemia, 2.9% had concurrent hyponatremia and hyperkalemia, 0.7% had hypokalemia, and 0.4% had hypernatremia. In the multiple logistic regression analysis, after applying the Bonferroni correction, only C-reactive protein (C-RP) levels were significantly associated with hyponatremia (OR = 1.04; 95% CI: 1.02–1.06; p < 0.001), only age was significantly associated with hyperkalemia (OR = 1.7; 95% CI: 1.1–2.7; p = 0.01), and only CAKUT was significantly associated with concurrent hyponatremia and hyperkalemia (OR = 4.3; 95% CI: 1.7–10.8; p = 0.002). Even after adjusting for the presence of kidney hypoplasia, abnormal renal echogenicity, pelvi-caliceal dilation, ureteral dilation, uroepithelial thickening of the renal pelvis, bladder abnormalities, pathogen other than E. coli, concurrent hyponatremia and hyperkalemia persisted significantly associated with CAKUT (OR = 3.6; 95% CI: 1.2–10.9; p = 0.02).Conclusion: Hyponatremia was the most common Na + /K + imbalance in children hospitalized for fUTI, followed by hyperkalemia and concurrent hyponatremia and hyperkalemia. C-RP levels were most strongly associated with hyponatremia, age with hyperkalemia, and CAKUT with concurrent hyponatremia and hyperkalemia (suggestive of transient secondary pseudo-hypoaldosteronism). Therefore, in children who develop concurrent hyponatremia and hyperkalemia during the course of a fUTI, an underlying CAKUT could be suspected.What is known:• Na+ and K+ abnormalities can occur in patients hospitalized for febrile urinary tract infection (fUTI).• Concurrent hyponatremia and hyperkalemia during fUTI may suggest transient secondary pseudo-hypoaldosteronism (TPHA), for which limited data on prevalence are available.What is new:• The most common Na+/K+ imbalance in children hospitalized with fUTI was hyponatremia (23%), followed by hyperkalemia (6.4%), concurrent hyponatremia and hyperkalemia (2.9%), hypokalemia (0.7%), and hypernatremia (0.4%).• Concurrent hyponatremia and hyperkalemia were mainly associated with CAKUT, while hyponatremia alone correlated with high C-reactive protein and hyperkalemia alone with younger age. In cases of concurrent hyponatremia and hyperkalemia during fUTI, an underlying CAKUT should be suspected.

Novel In-Line Hemolysis Detection on a Blood Gas Analyzer and Impact on Whole Blood Potassium Results.

Preanalytical error due to hemolyzed blood samples is a common challenge in laboratory and point-of-care (POC) settings. Whole blood potassium (K+) measurements routinely measured on blood gas analyzers are particularly susceptible to hemolysis, which poses a risk for incorrect K+ results. The GEM Premier 7000 with IQM3 (GEM 7000) blood gas analyzer provides novel integrated hemolysis detection within the sample measurement process. Therefore, the GEM 7000 can detect and flag hemolyzed whole blood samples at the POC, warning the operator of potentially erroneous results. Heparinized venous or arterial whole blood samples were used for K+ interference studies and assessed for hemolysis agreement utilizing either a traditional volumetric method or chemistry analyzer serum index measurements with the Roche cobas c311 or Abbott Alinity c. Hemolysis interference studies performed at 2 different K+ concentrations (3.8 and 5.3 mmol/L) identified that a plasma free hemoglobin ≥116 mg/dL can impact K+ results on the GEM 7000. Hemolysis agreement studies demonstrated an excellent agreement of >99% with the volumetric method, 98.8% with cobas H index, and 96.4% with Alinity H index. GEM 7000 K+ results were correctly flagged for both native and spiked samples. GEM 7000 hemolysis detection provides a novel technology to detect hemolysis in whole blood samples. Moreover, the GEM 7000 demonstrates excellent agreement with traditional laboratory hemolysis detection methods and offers an integrated technological solution for assuring the quality of whole blood K+ results in POC settings.

Hemolysis in blood samples

Hemolysis is one of the interfering factors that can limit the results of various biochemical tests, and this study aimed to assess the interference of hemolysis in the reliability of biochemical tests conducted in clinical-surgical procedures in the veterinary hospital routine. The methodology was retrospective, based on the analysis of records of animals treated at the Veterinary Hospital Unit (SUHVU) of the Federal University of Fronteira, Campus Realeza (Paraná) from January of 2020 to December of 2022. The tests conducted in the Veterinary Clinical Analysis laboratory included serum determination of total proteins, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), creatinine and urea. Based on the biochemical results, animals were divided into three groups according to the degree of hemolysis observed in the serum: mild hemolysis (HD); moderate hemolysis (HM) and intense hemolysis (HI). The control group (GC) was determined based on reference values specific to the species. Data were tabulated in spreadsheets and analyzed using Pearson's correlation test (p). Mild hemolysis was predominant and, in dogs, did not affect the analyzed biochemical values in comparison to the reference averages for the species, regardless of the degree. Isolated, most measurements increased. In cats, changes were more significant concerning the group average, with ALP showing the greatest variation, not consistent with the elevation of hemolysis. None of the parameters had a strong correlation of interference between hemolyses, only moderate negative and positive correlations in ALP and urea.

Biological sample collection and handling methods for fat-soluble vitamin and trace mineral analysis

Diagnostic reports of biological samples submitted from farms are essential to correctly identify any underlying issues in a herd, including disease and improper nutrition. Proper sample collection, handling, and storage are critical to most accurately diagnose health complications or nutritional status. When possible, sample pigs before they eat, keep tissue samples frozen, avoid hemolyzed blood samples, and minimize transport time to the diagnostic laboratory. Concerns regarding sample collection and storage can be addressed with a veterinary diagnostic laboratory.

Influence of biological sample pre-analytical manipulation for fat-soluble vitamin analysis

Objective: Determine how sample handling affects nutrient analysis of fat-soluble vitamins and minerals. Materials and methods: In experiment 1, blood was collected in either plasma or serum blood tubes and exposed to 4 hours of light or wrapped in aluminum foil to protect from light. In experiment 2, blood was collected at hours 0, 1, 2, 3, 4, 6, and 12 after the consumption of feed. In experiment 3, vitamins and minerals were assessed in varying degrees of hemolyzed blood samples. Experiment 4 evaluated liver samples exposed to various temperatures for up to 12 hours. In experiment 5, serum and liver samples were processed the day of, 1 day after, or 2 days after collection and subsequent placement into coolers with icepacks. Results: There was a significant difference (P &lt; .05) for the interaction of tube type and light exposure for vitamin D (25-hydroxyvitamin D3) and a tendency (P &lt; .10) for a tube type and light exposure interaction for vitamin A (retinol). Experiment 2 found serum vitamin concentrations changed post feed consumption both linearly and quadratically. Alpha-tocopherol peaked at 4 hours post meal consumption, whereas retinol peaked at 6 hours. In experiment 3, the degree of hemolysis affected (P &lt; .05) nutrient concentration. Experiment 4 and 5 showed no differences (P &gt; .05) in degradation of retinol and alpha-tocopherol. Implication: As many pre-analytical factors can affect laboratory results, care must be taken when collecting, handling, and storing samples for diagnostic analysis of vitamins and minerals.

Reducing blood sample hemolysis in the emergency department using S-Monovette® in aspiration mode

BackgroundBlood sample hemolysis continues to be a significant problem in clinical practice. In vitro hemolysis rates up to 77% have been reported in literature. The use of manual aspiration techniques for blood sampling has previously been shown to reduce the burden of erythrocyte injury in the pre-analytical phase compared to the vacuum collection technique. This study compares the hemolysis rates between two blood sampling methods: 5.0 ml BD Vacutainer® SST™ (BDV) and 4.9 ml S-Monovette® serum gel tubes in aspiration mode (SMA). MethodsThis was a prospective randomised controlled study conducted in an Emergency department (ED). A convenience sample of 191 adult patients, aged 18–90 years old, presenting at the ED and requiring blood samples for serum electrolyte was included in the study. Paired blood samples were obtained through an intravenous cannula from each patient with randomised order of blood draw using SMA or BDV. Patient data was obtained and hemolysis index (HI), serum lactate dehydrogenase (LDH), and serum potassium (K) levels measured. ResultsThe adjusted mean HI (35.2 vs 21.5 mg/dL, p < 0.001), serum K (4.38 vs 4.16 mmol/L, p < 0.001) and LDH levels (259.6 vs 228.4 U/L, p < 0.001) were significantly higher in blood samples taken using BDV compared to SMA. The frequency of severely hemolyzed (>150 mg/dL) samples was also higher in blood collected using BDV (16.2%) compared to SMA (0%). ConclusionsThe burden of hemolysis in blood samples taken from IV cannulae can be effectively reduced with the use of manual aspiration using the S-Monovette® blood collection system as compared to BD-Vacutainer.

C-peptide level concomitant with hypoglycemia gives better performances than insulin for the diagnosis of endogenous hyperinsulinism: a single-center study of 159 fasting trials.

Diagnosis of endogenous hyperinsulinism relies on the occurrence of a hypoglycemia, concomitant with inadequate high insulin and C-peptide levels. However, diagnostic cutoffs are not consensual among the different learned societies. The objective of this work was to propose optimized cutoffs for these three parameters for the diagnosis of endogenous hyperinsulinism. All the patients having performed a fasting trial in Cochin Hospital Endocrinology Department between February 2012 and August 2022 were included. The results of glycemia, insulin and C-peptide levels during fasting trial were collected and analyzed. One hundred and fifty-nine patients were included: 26 with endogenous hyperinsulinism and 133 without endogenous hyperinsulinism. ROC analysis of glycemia nadir during fasting trial identified the value of 2.3 mmol/L as the optimal cutoff, ensuring a sensitivity of 100% associated with a specificity of 81%. ROC analysis of insulin and C-peptide levels concomitant with hypoglycemia <2.3 mmol/L showed very good diagnostic performances of both parameters with respective cutoffs of 3.1 mUI/L (=21.5 pmol/L; sensitivity = 96%; specificity = 92%) and 0.30 nmol/L (sensitivity = 96%; specificity = 100%). Insulin to glycemia ratio as well as C-peptide to glycemia ratio (in pmol/mmol) at the time of glycemia nadir did not show better diagnostic performances than C-peptide alone. A C-peptide level 0.3 nmol/L concomitant with a hypoglycemia <2.3 mmol/L appears as the best criterion to make the diagnosis of endogenous hyperinsulinism. Insulin level can be underestimated on hemolyzed blood samples, frequently observed in fasting trial, and thus shows lower diagnostic performances.

A Single-Center Prospective Study of the Effects of Different Methods of Phlebotomy in the Emergency Department on Blood Sample Hemolysis Rates

IntroductionHemolysis is more commonly seen in the emergency department and causes delays in diagnosis, hospitalization, discharge, and treatment of patients. The aim of this study was to determine the most appropriate phlebotomy method and device to reduce blood sample hemolysis in the emergency department. MethodsThis prospective, comparative descriptive study involved patients who presented to the emergency department with any medical condition and required blood sampling. Patients were divided into 6 groups according to the method of phlebotomy and the device used for phlebotomy. Data were analyzed with logistic regression. ResultsA total of 715 patients participated in the study. The blood sample hemolysis rate in the emergency department was 25.7%. When the hemolysis rates were compared with a steel straight needle or intravenous catheter, it was found that the use of steel straight needle significantly reduced hemolysis. Blood drawing through a 20 G intravenous catheter with Luer-Lock access device reduces the risk of hemolysis. Male sex and difficult blood collection also have been shown to increase the risk of hemolysis. DiscussionBlood should be drawn with a steel straight needle instead of an intravenous catheter. However, when that is not possible, we recommend the use of a 20 G intravenous catheter with Luer-Lock access device if a blood sample is to be drawn from intravenous line.

Massive intravascular hemolysis is an important factor in Clostridium perfringens-induced bacteremia.

Clostridium perfringens bacteremia is rare but often fatal. In particular, once bacteremia with massive intravascular hemolysis (MIH) occurs, the mortality rate is extremely high. However, because of its rarity, the detailed pathophysiology of this fulminant form of bacteremia is unclear. To elucidate the detailed pathogenesis of MIH, we retrospectively reviewed the data of all patients with C. perfringens bacteremia from two university hospitals from 2000 to 2014. The medical records and laboratory data of 60 patients with bacteremia, including 6 patients with MIH and 54 patients without MIH, were analyzed. Patients with MIH had higher rates of intense pain at onset, impaired consciousness, shock at presentation, hematuria, metabolic acidosis, and gas formation than patients without MIH. The antibiotic susceptibility of the clinical isolates was not significantly different between the two groups. All patients with MIH, although treated with appropriate antimicrobial agents, died within 26h of admission due to rapidly progressive acute lung injury or acute respiratory distress syndrome, and the median time from arrival at the hospital to death was only 4h and 20min. When clinicians observe intravascular hemolysis in blood samples from patients with characteristic symptoms of MIH, they should prepare for a severe disease outcome. The underlying pathophysiology of fulminant cases must be investigated.

Co-Relation of HBA1C Level with Diabetic Retinopathy on Fundoscopy in Patients Suffering from Type-II DM for more than 10 years Duration

Defects in insulin secretion or its metabolic functionality results in increased levels of glucose causing metabolic disorders. Aims: To check the Co- relation of HbA1c level with diabetic retinopathy on fundoscopy in patients suffering from Type II DM for more than 10 years duration. Study Design: Cross sectional study. Methodology: Patients (n=61) who were diagnosed cases of diabetes mellitus type II according to American diabetes association guidelines for the past 10 years were included into the study. HbA1c was assessed on autoanalyzer from Roche and NGSP certified using turbidimetric inhibition immunoassay (TINIA) method of hemolyzed blood samples. Eye examination of patients were carried out by expert ophthalmologist after proper pupillary dialatation was carried out. After proper fundoscopy patients were classified into four stages of diabetic retinopathy based on ETDRS levels. Statistical analysis: Data was analyzed using SPSS version 26. Results were presented as frequency and percentage. Age was presented as mean± SD. Results: Out of 61 patients 38 (62.3%) were male and 23 (37.7%) were female. HbA1c levels and diabetic retinopathy showed a statistically significant association when assessed using chi square test (p=0.001). Comparison of HbA1c levels among various groups based on diabetic retinopathy grading revealed a statistically significant difference (p=0.001). Conclusion: It was concluded that HbA1c levels and diabetic retinopathy were significantly associated. Higher levels of HbA1c which reflected the poor glycemic control that can be used as a predictor for the severity of developing retinopathy. Keywords: Diabetes Mellitus, Diabetic Retinopathy, Fundoscopy, Microvascular Complication and Type-II Diabetes Mellitus

Effectiveness of food concentrate phenolic compounds of apples in experimental membrane pathologies

Apple fruits are an available source of phenolic compounds that exhibit a wide range of biological activities (antioxidant, anti-inflammatory, membrane stabilizing, etc.). The antioxidant properties of food concentrate phenolic compounds of apples (Concentrate) were studied in vitro in models of spontaneous and ascorbate induced lipid peroxidation (LPO) in rat liver homogenate, and acute carbon tetrachloromethane hepatitis was chosen as in vivo model in rats. Membrane stabilizing activity was evaluated by the degree of hemolysis in blood samples from the tail vein. The effect of Concentrate on vascular permeability was studied considering the time of animal skin papules staining at the site of injection of phlogogenic substances. Hepatoprotective activity in the model of acute carbon tetrachloride hepatitis was assessed by changes in prooxidant-antioxidant status in liver homogenate and liver enzymes activity in serum. Significant antioxidant effect of Concentrate was fixed in models of spontaneous and ascorbate induced LPO (TBA reactants’ content was 3.12 times and 2.25 times lower than control for spontaneous LPO and ascorbate induced LPO, respectively) and under tetrachloride hepatitis (Concentrate antioxidant activity was 47.8%). The membrane-protective activity of the studied Concentrate was also high and reached 50.1%. Also, Concentrate demonstrated capillary-strengthening properties, reducing the permeability of the vascular wall, which was caused by three different chlorogens, most notably by zymosan (Concentrate significantly delayed the stain utilization from the bloodstream by 2.14 times compared to control). Newly developed concentrate showed complex hepatoprotective activity, improving the indices of antioxidant-prooxidant status and activity of liver cytolysis enzymes in rats with tetrachloromethane hepatitis. The transparent corrective effects of Concentrate are the result of synergism and additivity of its multiple components and indicate the prospects of its further research in order to develop medications for the prophylaxis and treatment of diseases associated with membrane damage.

Pattern of C-peptide response to oral glucose tolerance test: Interest and cut-off values

ObjectiveThe oral glucose tolerance test (OGTT) classifies subjects as normal, glucose intolerant or diabetic depending on glycemia at 120 min (T120) post-test. Five insulin profiles associated with different incidences of diabetes over 10 years’ follow-up were previously described following OGTT. However, insulin measurement is sensitive to hemolysis, and can be replaced by C-peptide assay on hemolyzed samples. However, little is known about patterns of C-peptide response to OGTT. Design and methodsIn total, 128 patients were included, to establish preliminary baseline C-peptide values and to evaluate C-peptide response to OGTT in comparison to insulin response, using the Liaison XL immunoanalyzer. ResultsHundred patients had a normal glycemic response, 19 were classified as glucose intolerant and 9 as diabetic. In normal subjects, median C-peptide values (nmol/L, with 5–95 percentiles) were 0.53 (0.23–1.37) at baseline, peaking at 2.36 (0.94–1.83) at T60, and decreasing to 2.09 (1.13–4.36) at T120. The C-peptide response pattern was similar but flatter than the insulin pattern because of different catabolism pathways. Nevertheless, C-peptide and insulin response profiles were discordant in only 9.4% of cases. Profile 3 (C-peptide peaking at T60) was the most prevalent in normal patients whereas profile 4 (peak at 120 min and lower level at T30 than at T60) was the most prevalent in glucose intolerant and diabetic patients. ConclusionsIn OGTT, C-peptide could replace insulin determination on hemolyzed blood samples to predict the risk of type 2 diabetes.

Hemolyzed Blood Samples in the Emergency Department - Finding Our “Why”

When patients present to the emergency department, they expect strong communication from the hospital staff and short wait times and to be treated with empathy and compassion.1,2 Patients commonly present during a time of high stress and uncertainty, so addressing these areas of patient care as well as other areas that invoke patient harm have remained a focus for ED leaders: one area for improvement of ED care that has not received as much attention as its potential warrants is the hemolysis of blood samples.

The Hidden Cost of Hemolyzed Blood Samples in the Emergency Department.

On average, patients with hemolyzed potassium samples spend about 1 h longer in the emergency department (ED), regardless of acuity level or disposition. We aimed to quantify the direct expenses associated with poor-quality preanalytic blood samples collected in the ED. We created a simple table with a range of direct expenses (i.e., costs) and rates of hemolyzed sample draws, allowing for identification of potential high-level cost-of-care impact analysis. We included a range of costs informed by review of literature on the topic. Those costs range from $600 to $3000 per bed-hour. This amount was inflation adjusted from 1996 to 2020 (1.68 × [direct cost per visit] × [100000 visits per year/365 days/24 h]). We provided a range of hemolysis incidence based on previously reported data. We showed that for an ED with 100000 annual visits, a 40% draw rate for routine chemistries (including potassium), and a 10% hemolysis incidence, the direct cost impact of hemolysis waste is approximately $4 million/year as a result of the 1 h of added length of stay on average for a patient with a hemolyzed blood sample. This amount represents an annualized estimated cost of caring for a patient in the ED with an avoidable extended length of stay. The financial burden of poor-quality blood samples can be estimated using cost per bed-hour and rate of sample failure. Similar methodology may identify additional QC issues with previously invisible financial implications.

Emergency Nurses’ Knowledge, Attitudes, and Practices Related to Blood Sample Hemolysis Prevention: An Exploratory Descriptive Study

IntroductionThe aim of the study was to identify emergency nurses’ knowledge, attitudes, and practices related to blood sample hemolysis prevention and explore associations between these factors and demographic characteristics. The current state is unknown. Understanding baseline knowledge, attitudes, and practices addresses a gap in the literature. MethodAn exploratory, descriptive design with cross-sectional survey methodology employing a study-specific instrument was used. ResultsRequest for participation email was sent to a random sample of 5000 Emergency Nurses Association members, and 427 usable surveys were returned (response rate = 8.5%). Mean years in nursing was 13.85 (standard deviation = 10.78), and 226 (52.9%) were certified emergency nurses. Only 85 participants (19.9%) answered all 3 knowledge questions correctly. Answering the 3 knowledge questions correctly was significantly associated with being a certified emergency nurse (χ2 = 7.15, P < .01). Participant responses to attitude items about the sequelae of blood sample hemolysis were skewed toward agreement, and most attitude items were associated with whom participants reported as being primarily responsible for phlebotomy. Emergency nurses remain primarily responsible for phlebotomy as well as addressing hemolyzed samples, but few reported that blood sample hemolysis was addressed at a departmental level. DiscussionFindings suggest that emergency nurses lack some knowledge related to blood sample hemolysis prevention best practices. Attitudes toward phlebotomy practices may be 1 reason practice has not changed. Every effort should be made to prevent hemolyzed blood samples to decrease delays and costs in emergency care.

Benefits of the application of heat and pressure on peripheral venous cannulation in adults: A randomized controlled trial.

To evaluate the effectiveness of the application of topical heat, high pressure or a combination of both on antebrachial venous cannulation. A cross-over clinical trial blinded for haemolysis analysis. This cross-over clinical trial with two periods was performed in the Clinical Trial Unit of Hospital Universitario de La Princesa (Madrid) during June-July of 2017 in 59 healthy adults who were randomly allocated to one of three interventions: (1) Using dry topical heat for 7min produced by two hot seed bags (N=21), (2) Applying controlled pressure from a sphygmomanometer inflated to 100mmHg (N=18) and (3) combining heat and pressure (N=20) in one period out of two. All interventions were contrasted to standard clinical practice in the other period. The comparator involved a standard tourniquet around the upper arm to restrict venous blood flow. The primary outcome was effectiveness measured as vein cannulation at first attempt. Secondary outcomes were vein perception, pain, haemolysis in blood samples and adverse events. All the interventions were more effective than comparator. Vein perception was optimized in about all individuals. Moreover, pain relief was significantly higher when high pressure was applied. Haemolysis was not affected in any of the three interventions. In addition, no serious adverse events appeared. High pressure is determined to be the most effective in vein catheterization, pain relief, vein perception and quality of blood sample inalterability. Moreover, it is safe considering that only one adverse event appeared. Vein cannulation is a very common invasive technique, where repeated failures have been registered. Thus, we consider it relevant to develop interventions to achieve venous catheterization at first attempt to alleviate the pain and anxiety associated with this technique. We advocate using high pressure intervention for emergency, due to swiftest method and feasible in case of lacking resources, such as sphygmomanometers in the ambulance. Interventions can be extrapolated to healthy young adults, adults and patients who have healthy vein status perception. Pressure intervention could be an alternative to heat intervention when performing vein cannulation due to its lower risk of transient paresthesia for older people who often suffer from arterial hypertension.

Corrigendum to Seeing Red: Blood Sample Hemolysis Is Associated with Prolonged Emergency Department Throughput.

Corrigendum to Seeing Red: Blood Sample Hemolysis Is Associated with Prolonged Emergency Department Throughput.

Simple and fast UPLC-MS method for quantifying the anti-inflammatory candidate 5′-methoxynobiletin in rat plasma: Validation and application in a preliminary pharmacokinetic study

This study presents the development and validation of a fast and simple bioanalytical ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method intended for quantifying the anti-inflammatory candidate 5′-methoxynobiletin (5′-MeONB) in rat plasma. Standard of 5′-MeONB was purified from A. conyzoides extract by using preparative HPLC. After a pretreatment of plasma samples with acetonitrile, chromatographic separations were efficiently achieved with a C18 column using a 9 min gradient system of 0.1% aqueous formic acid and acetonitrile as eluent. Drug candidate 5′-MeONB and chrysin (internal standard, IS) detection were carried out using ESI+ through the extracted ion chromatograms approach, monitored at m/z 433.1494 (for 5′-MeONB, tR:1.78 min) and m/z 255.0657 (for IS, tR:1.57 min). Method was validated according to US FDA guidelines, presenting linearity (R2 > 0.999) over concentration range of 30–750 ng/mL. Relative standard deviation (RSD) of repeatability and intermediary precision respectively ranged between 1.93–3.65% and 2.16–7.54%, considering lower limit of quantitation (30 ng/mL) and quality control (90, 360 and 600 ng/mL) samples, while accuracy was between 82.51 and 109.44%. Moreover, no interference from plasma endogenous substances, no carryover effect, and no influence of extraction method even in hemolyzed blood samples were observed. Sample stability in auto-sampler and long-term −80 °C storage, as well as matrix effect were within acceptable limits. For the first time, using the validated UPLC-MS bioanalytical method, the plasma pharmacokinetics of 5′-MeONB following 2 mg/kg intravenous bolus dosing to Wistar rats was characterized allowing the determination of the parameters describing drug distribution and elimination.

A Reference chart for clinical biochemical tests of hemolyzed serum samples.

BackgroundAlthough the effect of hemolysis has been extensively evaluated on clinical biochemical tests, a practical guidance for laboratory staff to rapidly determine whether a hemolyzed blood sample is acceptable and how to interpret the results is lacking. Here, we introduce a chart as a convenient reference for dealing with such samples.MethodsSerum samples with 0.1%, 0.3%, 1%, 3%, and 10% hemolysis were prepared from sonicated endogenous red blood cells and received 35 wet and 22 dry clinical biochemical tests, respectively. The contributing part in the biochemical test result at each hemolysis condition was derived by subtracting the original test result of this sample with no hemolysis. The net results were used for analyses and preparation of the reference chart.ResultsThe reference chart displayed the analytically calculated hemolysis interference and related statistical analyses. The chart also provided the color appearance of serum samples at each hemolysis condition for clinical staffs to determine whether a hemolyzed sample could be accepted.ConclusionIn clinical laboratories, preparation of such a reference chart is extremely useful in dealing with hemolyzed blood samples for clinical biochemical tests.

Adenosine deaminase 2 activity negatively correlates with age during childhood

BackgroundHuman adenosine deaminase 2 (ADA2) is an extracellular enzyme that negatively regulates adenosine-mediated cell signaling by converting adenosine to inosine. Altered ADA2 enzyme activity has been associated with some viral infections and rheumatic diseases. The potential utility of ADA2 as a biomarker is, however, limited by the absence of established ranges of ADA2 concentration and enzyme activity in the healthy population. It is known that ADA2 enzyme activity is lower in adults, but when (and why) this decline happens is not known. The purpose of this study was to establish normative ranges of ADA2 enzyme activity and protein concentration in the healthy pediatric population.MethodsWe modified a commercially available ADA2 enzyme activity assay to enable higher throughput analysis of fresh, frozen and hemolyzed blood samples. With this assay and ADA2 protein immunoblotting, we analyzed ADA2 enzyme activity and protein concentration in blood plasma from a cohort of children and adolescents (n = 94) aged 5 months to 18 years. One-way ANOVA with subsequent Tukey multiple comparison test was used to analyze group differences. Reference intervals were generated using the central 95% of the population (2–97.5 percentiles).ResultsADA2 enzyme activity was consistent in fresh, frozen, and hemolyzed sera and plasma as measured by our modified assay. Analysis of plasma samples from the healthy pediatric cohort revealed that ADA2 enzyme activity is significantly lower in older children than in younger children (p < 0.0001). In contrast, there was no significant correlation between ADA2 protein concentration and either age or ADA2 enzyme activity.ConclusionWe observed that ADA2 enzyme activity, but not ADA2 protein concentration, negatively correlates with age in a cohort of children and adolescents. Our findings stress the importance of appropriate age-matched controls for assessing ADA2 enzyme activity in the clinical setting.