Endocannabinoid analysis in GlucoEXACT plasma: Method validation and sample handling recommendations.

Endocannabinoids (ECs), such as anandamide and 2-arachidonyl glycerol (2-AG), contribute to the pathology of inflammatory, malignant, cardiovascular, metabolic and mental diseases. The reliability of quantitative analyses in biological fluids of ECs and endocannabinoid-like (EC-like) substances depends on pre-analytical conditions such as temperature and "time-to-centrifugation". Standardization of these parameters is critical for valid quantification and implementation in clinical research. In this study, we compared concentrations obtained with GlucoEXACT blood collection tubes versus K3EDTA tubes and employed the optimized procedure to assess ECs profiles in patients with inflammatory skin disease and healthy controls. A UHPLC-MS/MS method was validated for human plasma from GlucoEXACT blood collection tubes according to EMA and FDA guidelines, and pre-analytical conditions were systematically modified to assess analyte stability and optimize the procedures. The results showed significantly lower concentrations of ECs and EC-like substance concentrations with GlucoEXACT tubes compared with K3EDTA tubes, and GlucoEXACT extended the time window of stable concentrations. The strongest method-disagreement occurred for 1/2-AG suggesting that GlucoEXACT delayed ex vivo isomer rearrangement. Hence, GlucoExact tubes were superior in terms of stability and reliability. However, although absolute concentrations obtained with GlucoExact and K3EDTA differed, linear regression studies showed high agreement (except for 1/2-AG), and both methods showed similar EC profiles and similar disease-dependent pro-inflammatory patterns in dermatology patients. Hence, despite the obstacles in EC analyses, implementation of optimized pre-analytical blood collection and sample processing procedures provide reliable insight into peripheral ECs.

Successful harmonization in EpiBioS4Rx biomarker study on post-traumatic epilepsy paves the way towards powered preclinical multicenter studies.

OBJECTIVE: Project 1 of the Preclinical Multicenter Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) consortium aims to identify preclinical biomarkers for antiepileptogenic therapies following traumatic brain injury (TBI). The international participating centers in Finland, Australia, and the United States have made a concerted effort to ensure protocol harmonization. Here, we evaluate the success of harmonization process by assessing the timing, coverage, and performance between the study sites. METHOD: We collected data on animal housing conditions, lateral fluid-percussion injury model production, postoperative care, mortality, post-TBI physiological monitoring, timing of blood sampling and quality, MR imaging timing and protocols, and duration of video-electroencephalography (EEG) follow-up using common data elements. Learning effect in harmonization was assessed by comparing procedural accuracy between the early and late stages of the project. RESULTS: The animal housing conditions were comparable between the study sites but the postoperative care procedures varied. Impact pressure, duration of apnea, righting reflex, and acute mortality differed between the study sites (p < 0.001). The severity of TBI on D2 post TBI assessed using the composite neuroscore test was similar between the sites, but recovery of acute somato-motor deficits varied (p < 0.001). A total of 99% of rats included in the final cohort in UEF, 100% in Monash, and 79% in UCLA had blood samples taken at all time points. The timing of sampling differed on day (D)2 (p < 0.05) but not D9 (p > 0.05). Plasma quality was poor in 4% of the samples in UEF, 1% in Monash and 14% in UCLA. More than 97% of the final cohort were MR imaged at all timepoints in all study sites. The timing of imaging did not differ on D2 and D9 (p > 0.05), but varied at D30, 5 months, and ex vivo timepoints (p < 0.001). The percentage of rats that completed the monthly high-density video-EEG follow-up and the duration of video-EEG recording on the 7th post-injury month used for seizure detection for diagnosis of post-traumatic epilepsy differed between the sites (p < 0.001), yet the prevalence of PTE (UEF 21%, Monash 22%, UCLA 23%) was comparable between the sites (p > 0.05). A decrease in acute mortality and increase in plasma quality across time reflected a learning effect in the TBI production and blood sampling protocols. SIGNIFICANCE: Our study is the first demonstration of the feasibility of protocol harmonization for performing powered preclinical multi-center trials for biomarker and therapy discovery of post-traumatic epilepsy.

Comparative analysis of different nuclear medicine techniques in evaluation of renal function.

INTRODUCTION: Nuclear medicine (NM) methods play an important role in the evaluation of renal function in a wide range of clinical indications. The aim of our study was to evaluate the correlation between measured GFR (mGFR) obtained by the three-plasma sample slope-intercept NM method (TPSM) - reference method vs. estimated GFR (eGFR) using Fleming's single plasma sample method (SPSM) at 120 min, 180 min, and 240 min and correlation of reference method with eGFR with camera-based Gates' protocol. MATERIAL AND METHODS: A total of 82 subjects (33 male/49 female) with a mean age of 54.87 ± 15.65 years were included and mGFR value was obtained by the three-plasma sample slope-intercept NM method and eGFR was obtained with Fleming's single sample method. eGFR was also quantified with the camera-based Gates' protocol after i.v. application of [99mTc]Tc-DTPA. RESULTS: Our study revealed a very strong positive significant correlation between all three SPSMs with the TPSM as the reference method. Between the Gates' method and the TPSM in the group of patients with mGFR ≥ 61-84 mL/min/1.73 m2 and mGFR ≥ 84 mL/min/1.73 m2, a moderate positive statistically significant correlation was obtained. CONCLUSIONS: The SPSM method shows a very strong correlation with the reference and low bias in all three groups of patients and can be routinely used for GFR estimation.

Comparison of Intravenous Microdialysis and Standard Plasma Sampling for Monitoring of Vancomycin and Meropenem Plasma Concentrations-An Experimental Porcine Study.

Microdialysis is a catheter-based method suitable for dynamic sampling of unbound antibiotic concentrations. Intravenous antibiotic concentration sampling by microdialysis has several advantages and may be a superior alternative to standard plasma sampling. We aimed to compare concentrations obtained by continuous intravenous microdialysis sampling and by standard plasma sampling of both vancomycin and meropenem in a porcine model. Eight female pigs received 1 g of both vancomycin and meropenem, simultaneously over 100 and 10 min, respectively. Prior to drug infusion, an intravenous microdialysis catheter was placed in the subclavian vein. Microdialysates were collected for 8 h. From a central venous catheter, plasma samples were collected in the middle of every dialysate sampling interval. A higher area under the concentration/time curve and peak drug concentration were found in standard plasma samples compared to intravenous microdialysis samples, for both vancomycin and meropenem. Both vancomycin and meropenem concentrations obtained with intravenous microdialysis were generally lower than from standard plasma sampling. The differences in key pharmacokinetic parameters between the two sampling techniques underline the importance of further investigations to find the most suitable and reliable method for continuous intravenous antibiotic concentration sampling.

Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.

The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.

A comparative study of different methods of estimating glomerular filtration rate in a subset of Filipinos with normal renal function

@#Kidney function is commonly quantified using the glomerular filtration rate (GFR). However, the gold standard of measuring GFR, inulin clearance, is not practical for daily clinical use. This study compares different methods of GFR estimation based on serum creatinine, plasma levels of 99mTc-diethylenetriaminepentaacetic acid (DTPA), and camera acquisition of 99mTc-DTPA uptake. Seventy-five Filipino adults between ages 20 and 35 presumed to have normal kidneys were recruited. Each subject underwent gamma camera scintigraphy using the Gates and Inoue protocols after receiving a dose of 99mTc-DTPA. Blood samples were subsequently extracted at 1 hour and 3 hours after tracer injection, and GFRs were calculated based on single- and double-plasma sampling methods (SPSM and DPSM, respectively). Serum creatinine was also measured to derive GFR using the CKD-EPI, MDRD, and CockroftGault equations. Each method was correlated with a reference standard (DPSM) based on accuracy, linear regression, bias, and precision. SPSM tends to overestimate GFR unlike the other methods evaluated, but otherwise shows the most favorable diagnostic performance among the six methods when correlated with DPSM. The Inoue method appears modestly better than the routinely utilized Gates protocol, though both methods exhibit lack of precision. The CKD-EPI formula shows similar, if not slightly superior, diagnostic properties to the MDRD and Cockroft-Gault equations, thus confirming its validity for use in this Filipino population subset. Further studies are needed, particularly involving SPSM and CKD-EPI, to determine the applicability of our findings in Filipinos with varying degrees of kidney function. It is hoped that modifications to these methods can be made that are tailor-fit to derive more accurate and population-specific GFR values.

Comparison of Glomerular Filtration Rate Measurement Methods between Radionuclide in vivo Scintigraphic Gates' and Plasma Sampling.

AIM: To correlate between the radionuclide in vitro plasma sampling method (using single and dual blood samples) and Gates' GFR measurement using Tc-99m diethylene triamine penta-acetic acid (Tc-99m DTPA) renal scintigraphy (in vivo method). METHODS: This study included 40 renal donors (group 1) and 40 patients with obstructive uropathy (group 2). Group 1 included 22 males and 18 females with an age range from 22 to 65 years, while group 2 included 24 males and 16 females with age range 27 to 64 years. Both groups subjected to renal Scintigraphy after administration of 5 mCi 99m-Tc DTPA, GFR was calculated using Gates' method (in vivo method), then plasma sampling was acquired at 60 mins and 180 mins post-injection of the tracer, samples were counted in well counter and GFR was calculated using in vitro technique either using single plasma sample (SPSM 60 mins) or dual sample (DPSM 60 & 180 min). Additionally, GFR was measured by estimated equations based on serum creatinine. RESULTS: In group 1, the mean GFR using in vivo Gates' method was 115.7 ± 29 ml/min, while using the SPSM was 100.1 ± 16.1 ml/min, and the DPSM was 100.3 ± 20.1 ml/min. In group 2, mean GFR using in vivo method was 74.1 ± 14.5 ml/min, while using in vitro SPSM it was 77.5 ± 24.9 ml/min and DPSM was 76.8 ± 24.8 ml/min. There was no significant difference between mean GFR values using in vivo and in vitro methods (single or dual samples) in group 1 and 2 (p > 0.05). There is high significant correlation between SPSM and DPSM in groups 1 and 2 (r = 0.90, r = 0.91 respectively), moderate significant correlation was found between in vivo Gates' method and in vitro SPSM in group 1 and 2 (r = 0.46 and 0.57 respectively) and moderate correlation was evident between in vivo and in vitro DPSM in both groups (r = 0.42 and 0.68 respectively). By using the DPSM as the reference standard significant high correlation was found with SPSM and significant-high moderate correlation with in vivo Gates' scintigraphic method. Conclusion: In vitro plasma sampling considered as a reliable, accurate |method for GFR calculation yet it considered relatively complex, both single and dual sample in vitro techniques showed a very high correlation, and hence SPSM can replace DPSM. CONCLUSION: Renal scintigraphy and GFR estimation using Gates' in vivo method is considered inaccurate, yet given its simplicity in performance it can still be used if corrected GFR is standardised for Egyptian population-based on studies with large numbers of patients from multiple centres.

The right blood collection tube for therapeutic drug monitoring and toxicology screening procedures: Standard tubes, gel or mechanical separator?

Stability data of toxics or drugs in gel-based or mechanical separation blood collection tubes are lacking, especially for therapeutic drug monitoring and clinical toxicology procedures. According to ISO 15189 accreditation standard, laboratories need to master the entire preanalytical process including the stability of analytes in a specific tube. Here we explored the impact of BD PST™ II and Barricor™ separator tubes on the stability of 167 therapeutic compounds and common drugs of abuse in plasma samples using LC-MS/MS. Forty drugs were significantly affected by the use of PST™ II tubes, including antidepressants (11/26), neuroleptics (9/13), cardiovascular drugs (5/26), anxiolytics and hypnotics (4/25) and some drugs of abuse (5/26). Six compounds exhibited significant reduction by the mechanical Barricor™ tubes. Ten drugs exhibited low (<85%) but non-significant recoveries due to inter-assay variability. Besides, a logP > 3.3 was determined as a cut-off value to predict a potential lack of stability in PST™ II gel tubes with an 86.4% sensitivity and a 61.4% specificity. As a consequence, determination of drugs with a logP > 3.3 should be carried out with caution in plasma samples withdrawn on PST™ II. The study showed the Barricor™ and non-gel tubes cause less drug interference and are recommended for the drugs studied.

Optimization of plasma sampling depth and aerosol gas flow rates for single particle inductively coupled plasma mass spectrometry analysis.

We performed experiments to assess the separate and also the combined effect of the sampling depth and the aerosol gas flow rates on the signal formation in single particle inductively coupled plasma mass spectrometry (spICP-MS) measurements by using dispersions containing Ag and Au NPs. It was found that the NP signal can significantly be improved by the optimization of the sampling depth. With respect to the "robust" setting, a signal improvement of nearly 100% could be achieved, which translates into a 25-30% improvement in size detection limits. It was also found that the shape of the spICP-MS signal histograms also change with the change of the plasma sampling depth. It was demonstrated that nanoparticle peak separation can also be significantly enhanced by using sampling depth optimization. The effect of the aerosol dilution gas flow, now standard in most ICP-MS instruments, on the spICP-MS signal formation was also studied for the first time in the literature, as this flow was hoped to make spICP-MS measurements more practical and faster via the on-line dilution of the aerosol generated from nano-dispersions. Our experimental results revealed that the dilution gas flow can only be used for a moderate aerosol dilution in spICP-MS measurements, if the gas flow going to the pneumatic nebulizer is proportionally lowered at the same time. This however was found to cause a significant worsening in the operation of the sample introduction system, which gives rise to a strong NP signal loss. Thus it was concluded that the use of the aerosol dilution gas flow, in its present form, can not be suggested for spICP-MS analysis.

Comparison of glomerular filtration rate measured by plasma sample technique, Cockroft Gault method and Gates' method in voluntary kidney donors and renal transplant recipients.

BACKGROUND: There are numerous methods for calculation of glomerular filtration rate (GFR), which is a crucial measurement to identify patients with renal disease. AIMS: The aim of this study is to compare four different methods of GFR calculation. SETTINGS AND DESIGN: Clinical setup, prospective study. MATERIALS AND METHODS: Data was collected from routine renal scans done for voluntary kidney donors (VKD) or renal transplant recipients 6 months after transplantation. Following technetium-99m diethylene triamine penta acetic acid injection, venous blood samples were collected from contralateral arm at 120, 180, and 240 min through an indwelling venous cannula and direct collection by syringe. A total volume of 1 ml of plasma from each sample and standards were counted in an automatic gamma counter for 1 min. Blood samples taken at 120 min and 240 min were used for double plasma sample method (DPSM) and a sample taken at 180 min for single plasma sample method (SPSM). Russell's formulae for SPSM and DPSM were used for GFR estimation. Gates' method GFR was calculated by vendor provided software. Correlation analysis was performed using Pearson's correlation test. RESULTS: SPSM correlated well with DPSM. GFR value in healthy potential kidney donors has a significant role in the selection of donors. The mean GFR ± (standard deviation) in VKD using SPSM, DPSM, camera depth method and Cockroft Gault method was 134.6 (25.9), 137.5 (42.4), 98.6 (15.9), 83.5 (21.1) respectively. Gates' GFR calculation did not correlate well with plasma sampling method. CONCLUSIONS: Calculation of GFR plays a vital role in the management of renal patients, hence it was noted that Gates GFR may not be a reliable method of calculation. SPSM was more reliable. DPSM is reliable but cumbersome. It is difficult to accurately calculate GFR without a gold standard.