Validation of a single specimen pneumatic tube system in the clinical laboratory.

The single-specimen pneumatic tube (PTS) is a commonly used rapid specimen delivery system in modern clinical laboratories. However, its impact on sample integrity and laboratory test results remains controversial. The installation and configuration of single-specimen PTS are unique to their institution. We sought to validate our single-specimen PTS by comparing routine chemistry, immunology, and hematology results with a repeat sample integrity index for manual transport. In 2023, 30 employees were randomly selected from the company medical examination, and three tubes of procoagulant serum samples and three tubes of EDTA anticoagulant blood samples were collected from each of them. Group A uses a single specimen PTS at 8 m/s, Group B uses a single specimen PTS at 15 m/s, and Group C uses manual transfer. Specimens from all three groups were simultaneously analysed for ALT, AST, TG, TC, LDL, K, NA, CI, TSH, hs-cTnT, NSE, Cyfra21-1 and haematological analysis. The differences between the three groups of NSE and Cyfra21-1 were statistically significant (P < 0.05). The differences of the rest of the items were not statistically significant. The difference in NSE was not statistically significant between groups A and B (P = 0.401), B and C, and C and A (P < 0.05). The difference in Cyfra21-1 was not statistically significant between groups A and B (P = 0.897), B and C (P = 0.052), and C and A (P = 0.145). Individual sample PTS should be validated for testing prior to use to ensure the results' accuracy.

Impact of regression modeling on the assessment and harmonization of a point-of-care analyzer and commercial laboratory analyzer for feline plasma biochemistry testing.

BACKGROUND: Regression describes the relationship of results from two analyzers, and the generated equation can be used to harmonize results. Point-of-care (POC) analyzers cannot be calibrated by the end user, so regression offers an opportunity for calculated harmonization. Harmonization (uniformity) of laboratory results facilitates the use of common reference intervals and medical decision thresholds. OBJECTIVE: Our aims were to characterize the relationship of results for multiple biochemistry analytes on a POC and a commercial laboratory analyzer (CL) with three regression techniques and to use regression equations to harmonize the POC results with those of the CL. Harmonized results were assessed by recognized quality goals. We used harmonized results to assess the regression techniques. METHODS: After analyzer imprecision assessments, paired clinical samples were assessed with one dataset to calculate regression parameters that were applied to a second dataset. Three regression techniques were performed, and each was used to harmonize the POC results with those from the CL. POC results were assessed for bias and the number of results reaching quality goals before and after harmonization. RESULTS: All regression techniques could be used to harmonize most analytes so that 95% of results were within ASVCP TEa guidelines. Harmonization could be further improved with alternate regression techniques or exclusions. CONCLUSIONS: Regression offers a means to harmonize POC and CL analyzers. Further work is needed to assess how few samples can reliably be used and to assess likely species differences. No regression technique reliably describes the relationship between methods when correlation is poor.

An HPLC-UV Method to Assess Human Plasma 25(OH)D3.

The aim of this study was to validate an HPLC-UV method to assess vitamin D status by determining the linearity and precision of the 25-hydroxyvitamin D3 (25(OH)D3) calibration curve, the limits of detection, quantitation and robustness of the method, and its accuracy. A second stock solution of 25(OH)D3 was prepared (500 ng/mL), and working dilutions (5, 10, 20, 30, 40, and 50 ng/mL) were prepared for a calibration curve. The HPLC equipment had a UV-Vis diode-array detector and utilized an AcclaimTM 120 C18 column (5 µm, 4.6 × 250 mm) with a flow rate of 1.2 mL/min, a column temperature of 30 °C, and the standards and samples were maintained at 4 °C, with an injection volume of 100 µL. Detection of 25(OH)D3 was determined at 265 nm, with a retention time of 4.0 min. The validation was conducted according to the FDA Validation of Analytical Procedures: Guidance for Industry. Vitamin D was extracted from plasma samples using acetonitrile (ACN)-0.1% formic acid (2:1 v/v), and the percentage of recovery was calculated. The proposed method conditions gave excellent linearity (R2 = 0.9989) and the linearity coefficient was R2 > 0.99 for 25(OH)D3. The detection and quantification limits were 1.1703 ng/mL and 3.5462 ng/mL, respectively. Decreasing or increasing the reading temperature by 1 °C decreased the response units (AU) of vitamin D, 25(OH)D3. When the current flow rate decreased by 0.2 mL/min (1.0 mL/min), the retention time increased to 4.913 min, whereas an increase of 0.2 mL/min of the proposed flow rate (1.4 mL/min) decreased the retention time to 3.500 min. The percentage of recovery varied from 92.2% to 97.1%. The proposed method to quantify a vitamin D metabolite (25(OH)D3) in human plasma samples was reliable and validated.

Accounting for differences between serum and plasma: An indirect approach to derive reference intervals for total protein, albumin, and globulin.

BACKGROUND: Observable quantitative variations exist between plasma and serum in routine protein measurements, often not reflected in standard reference intervals. In this study, we describe an indirect approach for estimating a combined reference interval (RI) (i.e., serum and plasma), for commonly ordered protein measurands: total protein, albumin, and globulin. METHODS: We applied an indirect reference interval estimation for protein measurements in serum and plasma using data from July 2018 to February 2024. The data were divided into three Epochs based on a period of plasma separator tube shortage during the COVID-19 pandemic. Bootstrap resampling was used to calculate RIs and corresponding 95% confidence intervals for each month. RESULTS: Our results demonstrate notable changes in RI limits for total protein, albumin, and globulin between Epochs, reflecting the influence of changing sample matrix. A combined RI was identified for all components and verified using plasma and serum samples from 20 healthy individuals and retrospective analysis of flagging rates on our outpatient population using new and historical RIs. CONCLUSION: The study demonstrates notable differences in the RIs for total protein, albumin, and globulin when container type changes. In addition, the results demonstrate the effectiveness of big data analytics in deriving RIs and highlights the necessity of continuous RI assessment and adjustment based on the patient population and acceptable specimen types.

Limitations of glycated albumin standardization when applied to the assessment of diabetes patients.

OBJECTIVES: Glycated albumin (GA) has potential value in the management of people with diabetes; however, to draw meaningful conclusions between clinical studies it is important that the GA values are comparable. This study investigates the standardization of the Norudia Glycated Albumin and Lucica Glycated Albumin-L methods. METHODS: The manufacturer reported imprecision was verified by performing CLSI-EP15-A3 protocol using manufacturer produced controls. The Japanese Clinical Chemistry Reference Material (JCCRM)611-1 was measured 20 times to evaluate the accuracy of both methods. GA was also measured in 1,167 patient samples and results were compared between the methods in mmol/mol and %. RESULTS: Maximum CV for Lucica was ≤0.6 % and for Norudia ≤1.8 % for control material. Results in mmol/mol and % of the JCCRM611-1 were within the uncertainty of the assigned values for both methods. In patient samples the relative difference in mmol/mol between the two methods ranged from -10.4 % at a GA value of 183 mmol/mol to +8.7 % at a GA value of 538 mmol/mol. However, the relative difference expressed in percentage units ranged from of 0 % at a GA value of 9.9 % to +1.7 % at a GA value of 30 %. CONCLUSIONS: The results in mmol/mol between the two methods for the patient samples were significantly different compared to the results in %. It is not clear why patient samples behave differently compared to JCCRM611-1 material. Valuable lessons can be learnt from comparing the standardization process of GA with that of HbA1c.

A Conjugated Polymer-Based Portable Smartphone Platform for Sensitive and Point-Of-Care Detection of Monoamine Neurotransmitter.

The precise and effective detection of neurotransmitters (NTs) is crucial for clinical investigation of neuronal processes, and timely monitoring of NT-related chronic diseases. However, sensitive detection of specific NT with unprecedented selectivity is highly challenging due to similarities in chemical and electronic structures of various interfering neurochemicals. Herein, an anionic conjugated polyelectrolyte Poly[(9,9-bis(4'-sulfonatobutyl)fluorene-co-alt-1,4-phenylene) sodium], PFPS was rationally designed and synthesized for amplified detection and point-of-care (PoC) determination of monoamine neurotransmitter, serotonin (5-Hydroxy tryptamine or 5-HT, also diagnostic biomarker of carcinoid tumor) in human blood plasma. The PFPS displayed a remarkable sensing response with an exceptionally high fluorescence quenching constant of 1.14×105 M-1 and an ultralow detection limit of 0.67 µM or 0.142 ppm, much below the clinical range. Furthermore, a smartphone-enabled portable platform was constructed for real-time onsite detection of 5-HT by quantification of visual fluorescence response of PFPS into RGB values using a color recognizer android application. The smartphone platform could be readily applied for convenient, non-invasive PoC testing of 5-HT levels in complex biological fluids accurately and is expected to revolutionize clinical diagnosis and personalized health care devices.

Analytical validation of the Mindray CL1200i analyzer high sensitivity cardiac troponin I assay: MERITnI study.

OBJECTIVES: This study performed an analytical validation study of the Mindray high-sensitivity cardiac troponin I (hs-cTnI) assay addressing limit of blank (LoB), limit of detection (LoD), precision, linearity, analytical specificity and sex-specific 99th percentile upper reference limits. METHODS: LoB, LoD, precision, linearity and analytical specificity were studied according to Clinical and Laboratory Standards Institute. We used one reagent lot and one CL1200i analyzer. Skeletal troponin I and T, cardiac troponin T, troponin C, actin, tropomyosin, myosin light chain, myoglobin and creatine kinase (CK-MB) were studied for cross-reactivity. Interference with biotin was examined. Lithium heparin samples (one freeze thaw cycle) from healthy males and females were measured to determine the 99th percentiles by using the non-parametric method. Analyses were performed before and after excluding subjects with clinical conditions and/or increased surrogate biomarkers. RESULTS: The Mindray hs-cTnI assay met criteria to be considered as a hs-cTn assay. LoB and LoD was <0.1 ng/L and 0.1 ng/L, respectively. Repeatability had a coefficient of variation 1.2-3.8 %, and within-laboratory imprecision 1.7-5.0 %. The measuring interval ranged from 1.1 to 28,180 ng/L. The analytical specificity was clinically acceptable for the interferents studied. After exclusions, the 99th percentile URLs obtained were 10 ng/L overall, 5 ng/L for females and 12 ng/L for males. CONCLUSIONS: Analytical observations of the Mindray hs-cTnI assay demonstrated excellent LoB, LoD, precision, linearity and analytical specificity, that were in alignment with the manufacturer's claims and regulatory guidelines for hs-cTnI. The assay is suitable for clinical investigation for patient-oriented studies.

End-user verification results of two serum separator tubes for clinical chemistry analytes according to CLSI GP34-A and CLSI GP41-A6.

Tube manufacturers use different composition of gels and blood clot activator formulations in serum tube production. Our aim was to investigate the within-tube (repeatability) and between-tube variation, concordance between comparison results of BD and VacuSEL tubes. Blood samples were collected from control subjects (n = 20) and patients (n = 30) in accordance with the CLSI GP41-A6 and CLSI GP34-A guidelines. Twenty-three clinical chemistry parameters were analysed via Roche Cobas C702 Chemistry Analyzer on T0 (0 hour) and T24 (24 hour). Mean differences % were compared with Wilcoxon matched pair test. Clinical significance was evaluated based on desirable bias according to total allowable error (TEa). VacuSEL tubes demonstrated acceptable performance for the results of 20 parameters with regards to desirable bias % limits. Lactate dehydrogenase (LD) [mean difference % (%95 confidence intervals (CI) values of BD and VacuSEL tubes at T0 [6.41% (4.80-8.01%)]; sodium (Na) and total protein (TP) at T24 [-0.27% (-0.46 to -0.07%) and -1.39% (-1.87 to -0.91), respectively] were over the desirable bias limits (LD: 4.3%, Na: 0.23% and TP: 1.36%, respectively) but not exceeding total biological variation CV % [Na: 0.5 (0.0-1.0) % and TP: 2.6 (2.3-2.7) %). %95 confidence intervals (CI) of T0 LD values overlap with within-subject biological variation % (CI) limits (LD: 5.2 (4.9-5.4) %). The differences between two tubes were not medically significant and necessarily conclusive. VacuSEL serum tubes presented comparable performance with BD serum tubes.

Insight into the status of plasma renin and aldosterone measurement: findings from 526 clinical laboratories in China.

OBJECTIVES: Accurate measurements of renin and aldosterone levels play an important role in primary aldosteronism screening, which is of great importance in the management and categorization of hypertension. The objective of this study is to investigate the current status of plasma renin and aldosterone measurements in China, which is achieved by analyzing the results of 526 clinical laboratories nationwide for three pooled fresh plasma samples derived from more than 2,000 patients. METHODS: Renin and aldosterone in three pooled plasma samples were measured four times in 526 laboratories employing various measurement systems. The inter- and intra-laboratory %CV were calculated and compared. To determine the source of the substantial inter-laboratory %CV, laboratories were categorized according to the measurement systems they are using, and both the inter- and intra-measurement-system %CV were calculated and compared. RESULTS: Regarding renin, the majority of laboratories use four primary commercial immunoassays. However, for aldosterone, in addition to commercial immunoassays, laboratory-developed liquid chromatography-tandem mass spectrometry (LC-MS) methods are also used by laboratories. The median values of intra-laboratory %CVs, intra-measurement-system %CVs, inter-laboratory %CVs, and inter-measurement systems %CVs varied between 1.6 and 2.6 %, 4.6 and 14.9 %, 8.3 and 25.7 %, and 10.0 and 34.4 % for renin, respectively. For aldosterone, these values ranged from 1.4 to 2.2 %, 2.5-14.7 %, 9.9-31.0 %, and 10.0-35.5 %, respectively. CONCLUSIONS: The precision within laboratories and measurement systems for plasma renin and aldosterone measurements is satisfactory. However, the comparability between laboratories using different measurement systems remains lacking, indicating the long way to achieve standardization and harmonization for these two analytes.

Verifying the nonreporting hemolysis index for potassium, phosphate, magnesium, AST, LDH, iron, CA 19-9, and vitamin D, using Beckman Coulter AU5800 and DxI800 automated analyzers.

BACKGROUND: Hemolysis is a common reason for nonreporting results in biochemistry and is measured using the hemolysis index (HI), with nonreporting limits set for analytes by manufacturers. OBJECTIVE: To verify the nonreporting HI limit for potassium, phosphate, magnesium, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), iron, CA19-9, and vitamin D on the Beckman Coulter AU5800/DxI800 analyzers. METHOD: Hemolysate was created from EDTA-lined tubes of whole blood using an osmotic shock procedure. The hemolysate underwent serial dilutions with saline and was spiked in paired serum. The delta changes in HI and analyte concentration were measured, assessed using regression analysis, and compared against calculated reference change values. RESULTS: A linear relationship between increasing HI and increasing analyte concentration (R2 > 0.9) was observed for potassium (y = 0.8864x), phosphate (y = 0.1079x), magnesium (y = 0.0678x), AST (y = 29.035x), and LDH (y = 350x). Increasing HI values did not have a linear effect on iron (y = -0.2544x), CA19-9 (y = 2.7019x), or vitamin D (y = 8.036x) concentrations. CONCLUSION: The results from this experiment support increasing the HI nonreporting limit to 100 mg/dL for potassium; 200 mg/dL for magnesium; and 300 mg/dL for phosphate, CA19-9, and vitamin D. The iron assay is not affected by hemolysis as high as 500 mg/dL. The current HI nonreporting limit of 50 mg/dL is appropriate for LDH.

CLSI-based verification and de novo establishment of reference intervals for common biochemical assays in Croatian newborns.

Introduction: This study aimed to examine whether the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) reference intervals for 19 commonly used biochemical assays (potassium, sodium, chloride, calcium, magnesium, inorganic phosphorous, glucose, urea, creatinine, direct and total bilirubin, C-reactive protein (CRP), total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and lactate dehydrogenase (LD)) could be applied to the newborn population of one Croatian clinical hospital. Materials and methods: Reference interval verification was performed according to the CLSI EP28-A3c guidelines. Samples of healthy newborns were selected using the direct a posteriori sampling method and analyzed on the Beckman Coulter AU680 biochemical analyzer. If verification wasn't satisfactory, further procedure included de novo determination of own reference intervals by analyzing 120 samples of healthy newborns. Results: After the first set of measurements, 14/19 tested reference intervals were adopted for use: calcium, inorganic phosphorous, glucose, urea, creatinine, total bilirubin, CRP, total protein, albumin, AST, ALT, GGT, ALP and LD. A second set of samples was tested for 5 analytes: potassium, sodium, chloride, magnesium and direct bilirubin. The verification results of the additional samples for sodium and chloride were satisfactory, while the results for potassium, magnesium and direct bilirubin remained unsatisfactory and new reference intervals were determined. Conclusions: The CALIPER reference intervals can be implemented into routine laboratory and clinical practice for the tested newborn population for most of the analyzed assays, while own reference intervals for potassium, magnesium and direct bilirubin have been determined.

Quality assurance of add-on testing in plasma samples: stability limit for 29 biochemical analytes.

Introduction: Clinical laboratories should guarantee sample stability in specific storage conditions for further analysis. The aim of this study is to evaluate the stability of plasma samples under refrigeration for 29 common biochemical analytes usually ordered within an emergency context, in order to determine the maximum allowable period for conducting add-on testing. Materials and methods: A total of 20 patient samples were collected in lithium heparin tubes without gel separator. All analyses were performed using Alinity systems (Abbott Laboratories, Abbott Park, USA) and samples were stored at 2-8 °C. Measurements were conducted in primary plasma tubes at specific time points up to 48 hours, with an additional stability study in plasma aliquots extending the time storage up to 96 hours. The stability limit was estimated considering the total limit of change criteria. Results: Of the 29 studied parameters, 24 demonstrated stabilities within a 48-hour storage period in primary plasma tubes. However, five analytes: aspartate aminotransferase, glucose, lactate dehydrogenase, inorganic phosphate and potassium evidenced instability at different time points (7.9 hours, 2.7 hours, 2.9 hours, 6.2 hours and 4.7 hours, respectively). The stability study in plasma aliquots showed that all parameters remained stable for 96 hours, except lactate dehydrogenase, with a stability limit of 63 hours. Conclusions: A reduced stability of primary plasma samples was observed for five common biochemical analytes ordered in an emergency context. To ensure the quality of add-on testing for these samples, plasma aliquots provide stability for a longer period.

Time-of-day dependence of running averages for some components of metabolic panels at our hospital: Relation to strategy for patient-based quality control.

BACKGROUND AND OBJECTIVES: We assessed properties of running averages for our hospital's most common chemistry analytes, for use in real-time patient-based quality control (PBQC). We determined whether there was dependence of any running averages on 24-h clock time (time-of-day, TOD). MATERIALS AND METHODS: We analyzed 3-months' data for measurements of 13 metabolic panel components. Running averages for 20 consecutive results (20-mers) were computed for data restricted to results within reference intervals. This produced an overall mean (X) and standard-deviation (SD) of 20-mers for each analyte. We then computed the average 20-mer result (Y) reported within 1-h bins across 24-hour clock time (t). Y(t) was regarded as having TOD-dependence if either nadir or apex values for |Y-X| exceeded 0.5 SD, occurring within a contiguous series of at least 4 Y(t) values on one side of the mean. RESULTS: Seven analytes (albumin, aspartate aminotransferase, calcium, chloride, CO2, potassium, total protein) demonstrated TOD-dependence of running means for 20-mers. CONCLUSIONS: At our hospital, TOD-dependence of running means was identified for 7 of 13 metabolic panel analytes. TOD-dependence is likely to be hospital-specific. Utilization of TOD-dependent targets for PBQC, rather than fixed targets, would be appropriate in these cases.

The stability of 65 biochemistry analytes in plasma, serum, and whole blood.

OBJECTIVES: The pre-analytical stability of various biochemical analytes requires careful consideration, as it can lead to the release of erroneous laboratory results. There is currently significant variability in the literature regarding the pre-analytical stability of various analytes. The aim of this study was to determine the pre-analytical stability of 65 analytes in whole blood, serum and plasma using a standardized approach. METHODS: Blood samples were collected from 30 healthy volunteers (10 volunteers per analyte) into five vacutainers; either SST, Li-heparin, K2-EDTA, or Na-fluoride/K-oxalate. Several conditions were tested, including delayed centrifugation with storage of whole blood at room temperature (RT) for 8 h, delayed centrifugation with storage of whole blood at RT or 4 °C for 24 h, and immediate centrifugation with storage of plasma or serum at RT for 24 h. Percent deviation (% PD) from baseline was calculated for each analyte and compared to the maximum permissible instability (MPI) derived from intra- and inter-individual biological variation. RESULTS: The majority of the analytes evaluated remained stable across all vacutainer types, temperatures, and timepoints tested. Glucose, potassium, and aspartate aminotransferase, among others, were significantly impacted by delayed centrifugation, having been found to be unstable in whole blood specimens stored at room temperature for 8 h. CONCLUSIONS: The data presented provides insight into the pre-analytical variables that impact the stability of routine biochemical analytes. This study may help to reduce the frequency of erroneous laboratory results released due to exceeded stability and reduce unnecessary repeat phlebotomy for analytes that remain stable despite delayed processing.

Analytical performance of feline plasma on current Heska and IDEXX point-of-care biochemistry analyzers compared with a commercial laboratory analyzer.

BACKGROUND: Point-of-care (POC) biochemistry analyzers are widely used in small animal clinical practice but infrequently independently assessed for performance. OBJECTIVE: To assess the performance of two current model point-of-care biochemistry analyzers (Heska Element DC and IDEXX Catalyst) compared with a commercial laboratory analyzer (Cobas 8000). METHODS: One hundred twenty-one cats from a feline hospital population were sampled with plasma results from a single lithium heparin tube assessed on all three analyzers. Plasma biochemistry results from each POC analyzer were compared with the commercial laboratory analyzer using Bland-Altman difference plots and by determining whether the limits of agreement (LOAs) (95% of differences) fell within various quality goals after correcting for inherent bias. RESULTS: Only 7 of 14 analytes on the Heska analyzer and 2 analytes on the IDEXX analyzer attained the most stringent LOA quality goal, which was being within desirable total error based on biologic variation (TEdes). The number of analytes achieving quality goals increased with less stringent standards such as American Society of Veterinary Clinical Pathologists allowable total error (ASVCP TEA) guidelines or if <95% of clinical comparisons reaching these quality goals is considered acceptable. Widespread bias was found between both POC analyzers and the commercial laboratory analyzer. CONCLUSIONS: The performance of both POC biochemistry analyzers was variable compared with a commercial laboratory analyzer. Performance goals were only able to be attained after the bias for each analyzer was accounted for by offsetting the LOAs and quality goals set by the mean bias for each analyte on each analyzer.

Evaluation of Sensitive Analytes to Hemolysis Interference on an Automated Chemistry Analyzer.

BACKGROUND: Hemolysis is a common reason for specimen rejection in the laboratory. Our experience suggested that hemolysis (H) flag limits are too strict for some analytes leading to unnecessary specimen rejections. This study summarizes H flags for commonly rejected analytes on the Beckman Coulter DxC 700 AU analyzer. METHODS: We evaluated analytes with low-limit H flags and high rejection rates. These included: aspartate aminotransferase (AST), alanine aminotransferase (ALT), iron (IRN), potassium (K), direct bilirubin (DBIL), magnesium (Mg), amylase (AMY), sodium (Na), gamma-glutamyltransferase (GGT), phosphorus (PHOS), albumin (ALB), alkaline phosphatase (ALKP), and lactate dehydrogenase (LDH). Five patient plasma pools without hemolysis were made from 50 patient specimens. Neat pools were analyzed to establish baseline analyte concentrations. A hemolysate was created by diluting whole blood with distilled water. Each analyte was tested after spiking each pool with the hemolysate to specific hemoglobin concentrations corresponding to manufacturer's H flags. Percent differences were calculated between baseline pool means and each flag's pool mean. Acceptance limits were based upon the average of the 2019 CLIA and the method precision limits. Calculated percent differences greater than the acceptance limits were considered significant. RESULTS: Manufacturer-defined hemolysis flags can be updated to greater than 1+ for Na, K, and AST, greater than 3+ for ALKP, and greater than 4+ for AMY and Mg. No changes were noted for the remaining analytes. CONCLUSIONS: The hemolysis criteria set for ALKP, AMY, AST, Mg, K, and Na were updated in the Remisol Advance middleware, which led to a 56% reduction in rejected hemolyzed specimens.

Development of reference intervals for serum biochemistry and haematology of juvenile green sea turtles (Chelonia mydas) in a Thai rehabilitation centre.

No reference intervals for serum biochemistry and haematology of sea turtles in Thailand exists to assist veterinarians who are responsible for sea turtle health management and treatment. This study determined serum biochemistry and basic haematology of healthy juvenile green sea turtles (n = 92) in captivity in Thailand following the American Society for Veterinary Clinical Pathology (ASVCP), Quality Assurance and Laboratory Standards Committee (QALS) guidelines for the determination of reference intervals in veterinary species. Biochemistry tests, including blood urea nitrogen, creatinine, uric acid, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were analysed using an IDEXX VetTest Chemistry Analyzer. Haematology parameters were measured manually using a microhaematocrit for packed cell volume (PCV), Neubauer counting chamber for red blood cell count and cyanmethemoglobin method for haemoglobin concentration. mean corpuscular volume and mean corpuscular haemoglobin concentration were calculated using the PCV, red blood cell count and haemoglobin. Turtles in this study were found to have higher mean values for PCV (28.70%), haemoglobin (92.13 g/L), mean corpuscular haemoglobin concentration (327.03 g/L), uric acid (247.15 µmol/L), alanine aminotransferase (16.53 IU/L), aspartate aminotransferase (209.44 IU/L), and alkaline phosphatase (245.08 IU/L) compared to sea turtles in Brazil. The reference intervals established using high numbers of healthy turtles in this study will assist veterinarians with diagnostic and treatment decisions when evaluating laboratory results for juvenile green sea turtles.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma.

OBJECTIVES: Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. METHODS: A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 µg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from -3.0 to -0.7 % for native serum levels, and from -2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. CONCLUSIONS: A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.

OBJECTIVES: To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 µg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively. CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

In-house standards derived from doping peptides: Enzymatic and serum stability and degradation profile of GHRP and GHRH-related peptides.

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.