Percent Coefficient Of Variation Research Articles

Abstract 93: Validation of an impedance-based in vitro potency assay for repeatability and precision

Abstract Cell-based immunotherapies, such as CAR T cells, are emerging as a promising approach to therapeutic intervention against cancer. Developing cell and gene therapy products requires reliable and reproducible methods of quantifying critical quality attributes, such as potency, to ensure product strength and consistency. A variety of assays can be used to assess potency; however, it is crucial that these tests are qualified for assay performance to ensure intra- and inter-assay precision. Although inherently variable, biological assays are often the best methods to investigate a product’s mechanism of action and predict clinical outcomes. Previous work has shown that impedance-based potency assays provide a non-invasive, real-time measure of effector cell-mediated cytotoxicity, thus reducing cell manipulation that impacts data reliability. Here, we describe an in vitro potency assay that demonstrates low variability and high precision across replicates, analysts, and days. Effector cell-mediated cytotoxicity was performed using a CD19-positive liquid tumor cell line (Raji) tethered to the well surface using an anti-CD40 antibody. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of E: T ratios, and cytolysis was monitored for three days. Real-time measurement of cell-mediated cytotoxicity also allowed for the calculation of kill time 50 (KT50), defined as the time required for 50% cytolysis of the target cells. Repeatability was calculated across three replicates per E:T ratio. The co-culture experiment was performed in duplicate by a second analyst and on two subsequent days for a comparison of f three plates as well as two operators to evaluate inter-assay precision. In addition to this, CAR T-mediated cell killing in a multiplate impedance system was also investigated for plate-to-plate variability. The percent coefficient of variation (% CV), a statistical measure that describes the precision and repeatability of an assay, was calculated to assess variability between assay replicates, operators, and plate replicates. In all conditions tested, the percent CV for all E:T ratios was below 20%, illustrating the repeatability and precision of the assay. These data support the use of an impedance-based potency assay for evaluating and characterizing immunotherapy products. Citation Format: Danielle Califano, Jianjie Jiang, Dirk Windgassen, Caitlyn De La Flor, Stacie Chvatal, Denise Sullivan, Daniel Millard. Validation of an impedance-based in vitro potency assay for repeatability and precision [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 93.

Intra-Day and Inter-Day Reliability and Usefulness of Performance, Kinetic and Kinematic Variables during Drop Jumping in Hurling Players

The aim of this study was to estimate the intra-day and inter-day reliability and usefulness of performance (Jump height (JH), ground contact time (GCT) and reactive strength index (RSI)), kinetic (force, power, eccentric rate of force development [E-RFD] and leg stiffness [LS]) and kinematic (velocity) variables during drop jumping (DJ) in hurling players. Seventeen (n = 17; mean ± SD; age = 23.35 ± 5.78 years, height = 178.35 ± 6.30 cm, body mass = 78.62 ± 8.06 kg) male club-level hurling players completed two maximal DJs from 0.20, 0.30, 0.40, 0.50 and 0.60 m drop heights on three testing days separated by 5–9 days of rest. Reliability was assessed using the coefficient of variation percentage (CV% ≤ 15%) and intraclass correlation coefficient (ICC > 0.70). For intra-day reliability, GCT (0.40 m, 0.50 m and 0.60 m), peak force (absolute and relative) (0.40 m and 0.50 m) and leg stiffness (0.40 m and 0.50 m) were found to be unreliable (ICC = 0.32–0.68 and CV% = 3.67–11.83%) from those specific drop heights. All other variables were found to be reliable (ICC = 0.72–0.98 and CV% = 1.07–14.02%) intra-day. All variables were found to be reliable (ICC = 0.72–0.96 and CV% = 2.57–14.68%) inter-day except for relative peak force and absolute and relative eccentric RFD (0.30 m and 0.40 m) (ICC = 0.68–0.90 and CV% = 7.76–16.47%). Practitioners have multiple reliable DJ performance, kinetic and kinematic variables for performance testing and training purposes.

Study of Glycemic Variability in Well-controlled Type 2 Diabetic Patients Using Continuous Glucose Monitoring System.

The world has changed tremendously for patients suffering from diabetes mellitus with the development of cutting-edge technologies like continuous glucose monitoring and flash glucose monitoring systems. Now, the details of constant fluctuations of glucose in their blood can be monitored not only by medical professionals but also by patients, and this is called glycemic variability (GV). Traditional metrics of glycemic control measurement, such as glycated hemoglobin (HbA1c), fail to reflect various short-term glycemic changes like postprandial hyperglycemia and hypoglycemic episodes, paving the way to the occurrence of various diabetic complications even in asymptomatic, well-controlled diabetic patients. This need for advanced management of diabetes and effective monitoring of these swings in blood glucose can be met by using a continuous glucose monitoring system (CGMS). To evaluate the extent of GV in well-controlled type 2 diabetes mellitus (T2DM) patients using a flash CGMS and to assess the correlation between GV and HbA1c. A hospital-based prospective observational study was carried out from May 2020 to Oct 2021 at the Department of Medicine, SMS Hospital, Jaipur, Rajasthan (India), after approval from the Ethics Committee of the institution. A total of 30 patients with well-controlled T2DM (HbA1c was ≥6.5, but ≤7.5) were included in the study using simple random techniques after written informed consent from patients. Patients were studied for glycemic excursions over a period of 7 days by using FreeStyle® Libre Pro™, which is a flash glucose monitoring system. The CGM sensor was attached to the left upper arm of the patient on day 0 and removed on day 7. The data recorded in the sensor was then retrieved using pre-installed computer software and analyzed using standard CGM metrics like standard deviation (SD), percentage coefficient of variation (%CV), time above range (TAR), time below range (TBR), and time in range (TIR), out of which %CV was used to quantify GV. %CV has been used to cluster patients into four cohorts from best to worst, namely: best/low CV ≤ 10%, intermediate CV from 10 to 20%, high CV from 20 to 30%, and very high CV of >30%. Scatterplots are used to establish correlations between various parameters. Data from a total of 30 patients were analyzed using CGMS and thus used for calculating standard CGM metrics; glucose readings every 15 minutes were recorded consecutively for 7-day periods, making it a total of 672 readings for each patient. Interpreting the CGM data of all 30 patients, the following results were found: the mean blood glucose of all cases is 134.925 ± 22.323 mg/dL, the mean SD of blood glucose of all cases is 35.348 ± 9.388 mg/dL, the mean of %CV of all cases is 26.376 ± 6.193%. CGM parameters of time are used in the form of percentages, and the following results were found: the mean of TAR, TBR, and TIR is 14.425 ± 13.211, 5.771 ± 6.808, and 82.594 ± 12.888%, respectively. Clustering the patients into cohorts, the proportion of patients exhibiting best/low %CV (10%) is 0, intermediate %CV (10-20%) is 16.67% (five out of 30 patients), high %CV (20-30%) is 50% (15 out of 30 patients) and very high %CV (>30%) is 33.33% (10 out of 30 patients). Also, there is no significant correlation found between HbA1c and %CV (ρ = 0.076, p-value = 0.690); a significant negative correlation was found between %CV and TIR (ρ = -0.604, p < 0.001S); a positive correlation of %CV with TAR and TBR is significant (ρ = 0.816, p-value of <0.001). Using a flash CGMS device and considering %CV as the parameter and primary measure of GV, the study demonstrated the overall instability of a person's glycemic control, making note of unrecognized events of hypoglycemia and hyperglycemia in asymptomatic well-controlled T2DM patients, revealing the overall volatile glycemic control. The most important finding of this study is that even those diabetics who are considered well-controlled experience a great degree of GV as assessed by CGM-derived metrics. This study also demonstrated that there is no significant correlation between HbA1c and GV, suggesting that patients may not have optimal control of their diabetes despite having "normal HbA1c" values; hence, GV can be considered an HbA1c-independent danger factor, having more harmful effects than sustained hyperglycemia in the growth of diabetic complications. So, by using CGM-derived metrics, the measurement of GV has the potential to complement HbA1c data. In this manner, a more comprehensive assessment of glycemic excursions can be provided for better treatment decisions, thereby facilitating optimal glycemic control, which is essential for reducing overall complications and promoting good quality of life.

Assessment of the variation of the pulmonary gas exchange model in the critical care setting

Background and Aims: In intensive care units (ICUs), clinicians use various mathematical models to assess and monitor respiratory illness. However, evidence suggests that some pulmonary internal and external physiolog¬ical factors are not integrated in the calculation of these models. This research aimed to demonstrate the variation in these models within a short period in critical care settings. Methods: A retrospective cohort study was conducted from 2017 to 2019 with adults admitted to an ICU and mechanically ventilated for more than 24 hours. Two different arterial blood gas samples were selected within a mean of 120 minutes. The coefficient of variation percent [(CV%)=SD/Mean] was calculated. Results: In total, 211 intubated ICU patients were included. The mean age was 57 (SD +/- 19.62) years, and 33% were female. The degree of variation of the average CV% for all the mathematical models was 14%-30%. The mean CV% for the arterial oxygen tension to inspired oxygen fraction (PaO2/FiO2) and arterial/alveolar oxygen tension ratio (PaO2/ PAO2) was 14%, which is a low variation compared to the respiratory index (RI) for which the mean CV% was 30%. Conclusions: The findings of this study indicate that most of the existing oxygen mathematical models used in clinical settings lack physiological realism, necessitating a new mathematical model that integrate both assess illness severity and enhance other models’ accuracy.

Comparison of central corneal thickness and endothelial cell count among diabetic retinopathy and non-diabetic cases

: Diabetes is associated with alterations in the structure and functions of corneal endothelial cells. Current research has been carried out to compare the central corneal thickness and endothelial cell density in patients with diabetic retinopathy (DR) and non-diabetic (ND) individuals. : A Cross Sectional comparative research was carried out in a tertiary eye care hospital, over the period of two years from April 2021 to March 2023. Among 160 study participants, 80 cases with diabetic retinopathy (DR) and 80 non-diabetic (ND) cases as age matched control have been selected. Thorough Ophthalmic assessment was done. We have included only retinopathy cases as they have longer duration of diabetis and poorer metabolic control, for better comparison of the endothelium parameters with non-diabetics. Specular microscopy has been done in all cases for endothelial cell count evaluation and thickness of cornea has been estimated by Pachymeter. Statistical analysis was carried out by students‘t’ test by comparing the variables between two groups. : P-value was not significant for the mean age and sex distribution in the two groups. Mean endothelial cell density was lesser (2512.12±260.23cells/mm2) in DR than in ND group (2699.10±95.68cells/mm2). Mean central corneal thickness was greater (522.65±36.56μm) in DR than in ND group (486.50±18.67μm) (P&amp;#60;0.05). Also, the Co-efficient of variation percentage was more whereas the percentage of hexagonality was found to be statistically less in DR than ND group. Among DR patients, endothelial cell density was significantly reduced and central corneal thickness considerably raised when compared with ND. Our results also suggest that poor metabolic control and advanced Diabetic Retinopathy are risk factors for developing keratopathy.

IMPROVING QUALITY AND EFFICIENCY PERFORMANCE OF ANALYTICAL TESTING PROCESS USING SIGMA METRICS IN EMERGENCY LABORATORY OF KING FAHD ARMED FORCES HOSPITAL, JEDDAH, SAUDI ARABIA

Background: Six Sigma is a popular quality management system tool used for process improvement. Using that, the clinical technologist can directly intervene to improve the quality of test reporting during the analytical phase of the total testing process in the medical laboratory. The present study aimed to assess and continuously improve the performance of individual biochemical and hematological parameters on a Sigma Scale by calculating the Sigma metrics for individual parameters redesigning and customizing the internal quality control (IQC). A sigma metric is a simple measurement of assay quality that compares an assays precision and bias performance to a total allowable error (TEa) goal. This analysis uses the Alinity-ci system, Alinityhq, and Stag for 47 assays from the Emergency Department of King Fahd Armed Forces Hospital. Methods:The present study is retrospective-prospective conducted in a clinical Emergency laboratory of King Fahd Armed Forces Hospital (KFAFH) Medical Pathology, Jeddah, Saudi Arabia, from May 2021 to September 2022. A retrospective secondary data analysis of eight months duration was carried out in an ED laboratory with a follow-up prospective study for more than six months. During this period, 47 analyses were tabulated to analyze the Internal Quality Control (IQC) coefficient of variation percentage and external Quality Control (CAP). Bias %) and total error allowable for the same analytic were obtained monthly, and the sigma metrics were calculated for each analytic. Standardized QC sigma charts were established with these parameters.Root cause analysis (RCA) was used to discover potential problems for the analytes.For analytes with a sigma value &lt;4, appropriate measures were taken to improve the quality of laboratory investigations. Results: At critical decision levels, all data analyzed those parameters and identified the assays that were four Sigma or better. Those assays which meet these criteria are now considered to be verified. The method decision chart showed that out of 47 analyses, 57 % demonstrated a world-class performance of 6 sigma level, whereas 2 % showed an Excellence of 5 σ performance, and 12.0 % showed a good performance of 4 sigma level. In contrast, 30 % showed poor performance of less than four sigma at the QC levels. From root cause analysis, the source of error was detected and corrected. However, for all analyses of less than four sigma levels, indicating the area requiring improvement. In contrast, the SQC control rules have been redesigned for the improvement. Conclusions:For the analyses listed in this report, under the circumstances detailed in the report, Westgard QC, Inc. is proud to re‐verify that the Sigma performance of KFAFH, ED laboratory is achieving the appropriate goals of analytical quality performance. For QC procedure, sigma metric analysis is helpful to evaluate the performance and optimize the protocol for improvement and cost-effectiveness.

Analysis of biochemical analytes using six sigma metrics with two analyzers at an Indian lab setting.

A zero defects goal was implemented in the clinical laboratory settings using a six-sigma model. Daily Internal Quality Control (IQC) and external quality control data from April-September 2023 was extracted to calculate the sigma metrics of 21 biochemical analytes based on Total Error Allowable (TEa), % bias and co-efficient of variation percent (CV%). A retrospective comparative study was conducted in the department of Clinical Biochemistry at Kanva Diagnostic Services Pvt. Ltd, Bengaluru, India. The analytical performance of the 21 biochemical analytes was tested on Cobas 6000 and C311 analyzers. Quality Goal Index (QGI) and root cause analysis was calculated to infer the reason for the deviation of six sigma. Method decision charts were plotted to show the comparison of the problem analytes on both the analyzers. On Cobas 6000 at level 1 IQC, out of 21 analytes, 10 analytes showed σ>6 and 10 analytes showed σ 3-6 and on C311, 15 analytes which showed σ>6 and 6 analytes that showed σ 3-6. On Cobas 6000 at level 2 IQC, out of 21 analytes, 12 analytes showed σ>6 and 8 analytes showed σ 3-6 and on C311 17 analytes showed σ>6 and 4 analytes showed σ 3-6. Creatinine failed to meet minimal sigma performance at both levels of IQC on Cobas 6000.

Pharmacokinetics, pharmacodynamics and efficacy of pemigatinib (a selective inhibitor of fibroblast growth factor receptor 1–3) monotherapy in Chinese patients with advanced solid tumors: a phase i clinical trial

Pemigatinib is a selective fibroblast growth factor receptor (FGFR)1–3 inhibitor and has demonstrated acceptable tolerability and clinical activity in advanced solid tumors in Western population. This phase I trial evaluated pharmacokinetics/pharmacodynamics (PK/PD) characteristics, preliminary safety and efficacy of pemigatinib in Chinese patients with advanced, solid tumors. Patients with unresectable advanced or metastatic solid tumors bearing FGF/FGFR1-3 alterations received oral pemigatinib at 13.5 mg once daily (QD) on a 2-weeks-on/1-week-off schedule. The primary endpoint was PK/PD characteristics; secondary endpoints were safety and efficacy. Twelve patients were enrolled (median age: 61 years, 58.3% males). PK data demonstrated pemigatinib (13.5 mg QD) was rapidly absorbed with a geometric mean elimination half-life of 11.3 h. The geometric mean values of maximum serum concentration and area under the plasma concentration–time curve from 0 to 24 h at steady state were 215.1 nmol/L and 2636.9 h·nmol/L, respectively. The mean clearance adjusted by bioavailability at steady state was low (11.8 L/h), and the apparent oral volume of distribution was moderate (170.5 L). The PD marker, serum phosphate level, increased on days 8 and 15 of cycle 1 (mean: 2.25 mg/dL, CV% [percent coefficient of variation]: 31.3%) and decreased to baseline post 1 week off. Three (25.0%) patients experienced grade ≥ 3 treatment-emergent adverse events. Partial response was confirmed in one patient with FGFR1-mutant esophageal carcinoma and one with FGFR2-mutant cholagiocarcinoma. Pemigatinib had similar PK/PD characteristics to Western population and demonstrated an acceptable safety profile and potential anti-cancer benefit in Chinese patients with FGF/FGFR1-3 altered, advanced, solid tumor. (ClinicalTrials.gov: NCT04258527 [prospectively registered February 6, 2020]).

Population Pharmacodynamic Modelling of the CD19+ Suppression Effects of Rituximab in Paediatric Patients with Neurological and Autoimmune Diseases.

Limited pharmacotherapy and the failure of conventional treatments in complex pathologies in children lead to increased off-label use of rituximab. We aimed to characterize the time course of CD19+ B lymphocytes (CD19+) under treatment with intravenous rituximab in children with neurologic and autoimmune diseases and to evaluate the impact of covariates (i.e., demographics, diagnosis and substitution between innovator and biosimilar product) on rituximab pharmacodynamics and disease activity. Pre- and post-drug infusion CD19+ in peripheral blood were prospectively registered. A population pharmacodynamic model describing the time course of CD19+ was developed with NONMEM v7.4. Simulations of three different rituximab regimens were performed to assess the impact on CD19+. Logistic regression analysis was performed to identify predictors of clinical response recorded through disease activity scores. 281 measurements of CD19+ lymphocyte counts obtained from 63 children with neurologic (n = 36) and autoimmune (n = 27) diseases were available. The time course of CD19+ was described with a turn-over model in which the balance between synthesis and degradation rates is disrupted by rituximab, increasing the latter process. The model predicts half-lives (percent coefficient of variation, CV(%)) of rituximab and CD19+ of 11.6 days (17%) and 173.3 days (22%), respectively. No statistically significant effect was found between any of the studied covariates and model parameters (p > 0.05). Simulations of different regimens showed no clinically significant differences in terms of CD19+ repopulation times. A trend towards a lack of clinical response was observed in patients with lower CD19+ repopulation times and higher areas under the CD19+ versus time curve. Rituximab pharmacodynamics was described in a real-world setting in children suffering from autoimmune and neurologic diseases. Diagnosis, substitution between innovator rituximab and its biosimilars or type of regimen did not affect rituximab-induced depletion of CD19+ nor the clinical response in this cohort of patients. According to this study, rituximab frequency and dosage may be chosen based on clinical convenience or safety reasons without affecting CD19+ repopulation times. Further studies in larger populations are required to confirm these results.

A-156 White Blood Cell Measurands, Flagging and Scatter Plot Patterns in Chronic Lymphocytic Leukemia

Abstract Background In this study, we use Alinity hq hematology analyzer to investigate the differences between white blood cell measurands of a healthy control population vs Chronic Lymphocytic Leukemia (CLL). A repeatability study was also conducted to determine the precision of the white blood cell parameter results on the Alinity hq. In addition, we used Alinity hq to identify unique flagging and patterns in the optical channels for CLL. Methods One hundred and fifteen peripheral blood samples of CLL were compared to a population of four hundred and fourteen healthy controls on the Alinity hq analyzer. Statistical differences in Alinity hq’s white blood cell measurands compared between the control population and CLL. For the repeatability study, five CLL samples (with sufficient volume) will be tested in 10 replicates for WBC measurands. These samples can be part of the 115 samples above or can be different samples. Comparison of scatterplots were also analyzed between CLL and control samples. Lastly, a peripheral smear was performed on all CLL samples. Results Statistical analysis of the means of the white and red cell parameters showed that in comparison to the means of the control population there was a significant statistical (P &amp;lt; 0.05) and clinical difference with CLL for the following parameters: absolute white blood cells (WBC) X 103/µL (43.6 CLL vs 7.41 control), percent neutrophils (%N) (18.25 CLL vs 53.71 control), percent lymphocytes (%L) (77.72 CLL vs 33.47 control), and percent monocytes (%M) (2.71 CLL vs 8.75 control). The WBC repeatability study results showed good concordance on the Alinity hq CBC + Diff + Retic test mode with the following percent coefficient of variance (%CV) results: absolute WBC X 103/µL (0.64, 5.31), percent neutrophils (%N) (1.32, 5.57), percent lymphocytes (%L) (0.20, 2.49), and percent monocytes (%M) (3.99, 26.39). As expected, the %CV range for monocytes is wide due to lymphocyte/variant lymphocyte population extending into the monocyte cluster. Evaluation of flagging showed 67 of the 115 (58.26%) CLL samples had a blast flag and 88 out of the 115 (76.52%) CLL samples had a variant lymphocyte flag. Review of the raw data from the optical channels shows a dominant and elongated lymphocyte cluster on the IAS, FL1 and PSS channels vs ALL for the CLL sample group compared to the control population. In the IAS and PSS vs ALL scatter plots in the CLL samples the lymphocyte cluster extends into the monocyte population triggering the blast flag. Peripheral smear review confirmed the presence of CLL (i.e., numerous smudge cells, mature-looking lymphocytes with characteristic condensed “soccer ball” pattern chromatin and scant cytoplasm) in the experimental group. Conclusion CLL is a cancer of the blood and bone marrow that predominately affects the quantity, and morphology of lymphocytes. Patients present with an abnormally high relative and variant lymphocyte populations. As such, there are a number of signs found in WBC measurands, flagging, and scatter plots on the Alinity hq that can be used as an early screening tool for CLL.

B-021 Analytical Performance of Thyroid Function Test on DxI800 System

Abstract Background Thyroid function tests (TFT) which usually include thyroid stimulating hormone (TSH), total triiodothyronine (TT3), and free thyroxine (FT4) are widely applied in clinical practice for screening, diagnosis and monitoring of thyroid disease. We evaluated analytical performance of TSH, TT3, and FT4 using DxI800 (Beckman Coulter, USA) including comparison to Alinity I (Abbott, USA). Methods Precision was determined by percent coefficient of variation (%CV) measuring low, medium, and high levels of quality control material for 5 days with one run per day, 5 replicates per run. Linearity was evaluated using 5 levels of patient serum samples and concentrations were determined by the mean value of two replicates of each level. Method comparison was performed by Passing-Bablok regression using 40 patient serum samples. Results The within-run imprecision at low, medium and high level were 1.50%, 1.77%, and 2.59% for TSH; 4.10%, 2.45%, and 2.84% for TT3; and 3.23%, 2.76%, and 2.19% for FT4. The within-laboratory imprecision at low, medium, and high level were 1.61%, 1.91%, and 2.87% for TSH; 4.33%, 3.73%, and 3.70% for TT3; and 3.36%, 2.76%, and 2.20% for FT4. Linearity of TFT on DxI800 was demonstrated in the range of 0.01–44.68 µIU/mL, 0.09–7.40 ng/mL, and 0.18–5.52 ng/dL for TSH, TT3, and FT4, respectively. Method comparison between DxI800 and Alinity I showed a slope of 1.21 and intercept of 0.05 (correlation coefficient R = 0.994); a slope of 0.95 and intercept of 0.13 (correlation coefficient R = 0.971); and a slope of 1.81 and intercept of −0.88 (correlation coefficient R = 0.969) for TSH, TT3, and FT4, respectively. Conclusion DxI800 showed acceptable performance in terms of precision and linearity for TFT assay including TSH, TT3, and FT4. This study demonstrated that TFT on DxI800 are suitable in clinical laboratory.

Deep learning reconstruction for brain diffusion-weighted imaging: efficacy for image quality improvement, apparent diffusion coefficient assessment, and intravoxel incoherent motion evaluation in in vitro and in vivo studies

Deep learning reconstruction (DLR) to improve imaging quality has already been introduced, but no studies have evaluated the effect of DLR on diffusion-weighted imaging (DWI) or intravoxel incoherent motion (IVIM) in in vitro or in vivo studies. The purpose of this study was to determine the effect of DLR for magnetic resonance imaging (MRI) in terms of image quality improvement, apparent diffusion coefficient (ADC) assessment, and IVIM index evaluation on DWI through in vitro and in vivo studies. For the in vitro study, a phantom recommended by the Quantitative Imaging Biomarkers Alliance was scanned and reconstructed with and without DLR, and 15 patients with brain tumors with normal-appearing gray and white matter examined using IVIM and reconstructed with and without DLR were included in the in vivo study. The ADCs of all phantoms for DWI with and without DLR, as well as the coefficient of variation percentage (CV%), and ADCs and IVIM indexes for each participant, were evaluated based on DWI with and without DLR by means of region-of-interest measurements. For the in vitro study, using the mean ADCs for all phantoms, a t-test was adopted to compare DWI with and without DLR. For the in vivo study, a Wilcoxon signed-rank test was used to compare the CV% between the two types of DWI. In addition, the Wilcoxon signed-rank test was used to compare the ADC, true diffusion coefficient (D), pseudodiffusion coefficient (D*), and percentage of water molecules in micro perfusion within 1 voxel (f) with and without DLR; the limits of agreement of each parameter were determined through a Bland-Altman analysis. The in vitro study identified no significant differences between the ADC values for DWI with and without DLR (P > 0.05), and the CV% was significantly different for DWI with and without DLR (P < 0.05) when b values ≥250 s/mm2 were used. The in vivo study revealed that D* and f with and without DLR were significantly different (P < 0.001). The limits of agreement of the ADC, D, and D* values for DWI with and without DLR were determined as 0.00 ± 0.51 × 10-3, 0.00 ± 0.06 × 10-3, and 1.13 ± 4.04 × 10-3 mm2/s, respectively. The limits of agreement of the f values for DWI with and without DLR were determined as -0.01 ± 0.07. Deep learning reconstruction for MRI has the potential to significantly improve DWI quality at higher b values. It has some effect on D* and f values in the IVIM index evaluation, but ADC and D values are less affected by DLR.

Simultaneous Estimation of Berberine and Piperine in a Novel Nanoformulation for Epilepsy Control via HPLC.

Berberine (Brb) and piperine (Pip) are salient examples of bioactive nutraceuticals possessing a promising role in controlling epilepsy. However, during the development of novel nanoformulation that augments their effects, an adequate determination of each one separately was a challenge since they have nearly the same detection wavelength and diverse solubility profiles. Consequently, a tailored high-performance liquid chromatography technique was developed for their simultaneous detection in routine analyses. The chromatographic separation was achieved using a C18 column. The linear gradient flow of acetonitrile: 0.1%v/v aqueous phosphoric acid was altered from 55:45 to 80:20v/v over 3min at a 1.2mL/min flow rate until the end of the run. Brb and Pip were eluted at 1.6 and 3.4min, respectively. The linearity of the standard curves was found to be ≥0.999, and the mean % recovery for Brb and Pip lay within the accepted limit. Moreover, the percentage coefficient of variation was <2% for intra- and inter-day precision. Consequently, the developed assay was successfully applied for the quantification of both drugs rapidly with high resolution and minimum interference from each other during the different steps conducted during the nanoformulation development.

Reference ranges for body composition indices by dual energy X-ray absorptiometry from the Bone Mineral Density in Childhood Study Cohort

BackgroundBody composition assessment aids evaluation of energy stores and the impact of diseases and interventions on child growth. Current United States pediatric reference ranges from the National Health and Nutrition Examination Survey (NHANES) include 20% of children with obesity, body mass index of ≥95th percentile. ObjectivesThis study aimed to develop dual energy X-ray absorptiometry (DXA) based reference ranges in a diverse cohort with low-obesity prevalence from the Bone Mineral Density in Childhood Study (BMDCS). MethodsThis is a secondary analysis of a longitudinal, prospective, observational cohort. Healthy children (height and BMI within 3rd to 97th percentiles, ages 5–19 y at enrollment), from 5 United States centers were measured annually for ≤7 visits. Whole body scans were acquired using Hologic scanners. A subsample underwent repeat measurements to determine precision. We generated reference ranges for appendicular and total lean soft tissue mass index (LSTM Index), fat mass index (FMI), and other body composition measures. Resulting curves were compared to NHANES and across subgroups. Sex and age-specific equations were developed to adjust body composition Z-scores for height Z score. ResultsWe obtained 9846 scans of 2011 participants (51% female, 22% Black, 17% Hispanic, 48% White, 7% Asian/Pacific Islander, and 6% with obesity). Precision (percent coefficient of variation) ranged from 0.7% to 1.96%. Median and–2 standard deviation curves for BMDCS and NHANES were similar, but NHANES +2 standard deviation LSTM Index and FMI curves were distinctly greater than the respective BMDCS curves. Subgroup differences were more extreme for appendicular LSTM Index-Z (mean ± SD: Asian −0.52 ± 0.93 compared with Black 0.77 ± 0.87) than for FMI-Z (Hispanic 0.29 ± 0.98 compared with Black −0.14 ± 1.1) and were smaller for Z-scores adjusted for height Z-score. ConclusionsThese reference ranges add to sparse normative data regarding body composition in children and adolescents and are based on a cohort with an obesity prevalence similar to current BMI charts. Awareness of subgroup differences aids in interpreting results.

Protocol Standardization May Improve Precision Error of InBody 720 Body Composition Analysis.

Bioelectrical impedance analysis (BIA) is a popular technique which can be used to track longitudinal changes in body composition. However, precision of the technique has been questioned, especially among athletic populations where small but meaningful changes are often observed. Guidelines exist which attempt to optimize precision of the technique but fail to account for potentially important variables. Standardization of dietary intake and physical activity in the 24hr prior to assessment has been proposed as an approach to minimizing the error of impedance-derived estimates of body composition. Eighteen recreational athletes, male (n = 10) and female (n = 8), underwent two consecutive BIA tests to quantify within-day error, and a third test (the day before or after) to quantify between-day error. All food and fluid intake plus physical activity from the 24hr prior to the first BIA scan was replicated during the following 24hr. Precision error was calculated as the root mean square standard deviation, percentage coefficient of variation, and least significant change. There were no significant differences in precision error of within- and between-day fat-free mass, fat mass, and total body water. Differences in precision error of fat-free mass and total body water, but not fat mass, were less than the smallest effect size of interest. The 24-hr standardization of dietary intake and physical activity may be an effective approach to minimizing precision error associated with BIA. However, further research to confirm the validity of this protocol compared to nonstandardized or randomized intake is warranted.

Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin.

Luminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally certified reference standards. Therefore, there is an urgent need to characterize existing reference standards for the standardization of multiplex immunoassays (MIAs). Here, we report the development and validation of an MIA for the simultaneous estimation of levels of human serum immunoglobulin G (IgG) antibodies for pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), and tetanus toxoid (TT). The MIA was assessed using a panel of human serum samples and WHO reference standards. The WHO reference standards were also studied for suitability in the MIA. Purified antigens (PT, FHA, PRN, DT, and TT) were coupled to the spectrally unique magnetic carboxylated microspheres. The method was validated in accordance with the United States Food and Drug Administration (US FDA), European Medicines Agency (EMA), and the International Committee of Harmonization Multidisciplinary (ICH M10) guidelines, and parameters such as precision, accuracy, dilutional linearity, assay range, robustness, and stability were assessed. Method agreements with commercially available IgG enzyme-linked immunosorbent assay (ELISA) assays were also evaluated. In addition, the study assessed the level of correlation between the IgG levels estimated by the MIA and the cell-based neutralizing antibody assays for PT and DT. We identified that an equimix of WHO international standards (i.e., 06/142, 10/262, and TE-3) afforded the best dynamic range for all the antigens in the MIA. For all five antigens, we observed that the back-fitted recoveries using the four-parameter logistic (4-PL) regression fits ranged between 80% and 120% for all calibration levels, and the percentage coefficient of variation (% CV) was < 20%. In addition, the difference in mean fluorescence intensity (MFI) between the monoplex and multiplex format was < 10% for each antigen, indicating no crosstalk among the beads. The MIA also showed good agreement with conventional and commercially available assays, and a positive correlation (> 0.75) with toxin neutralization assays for PT and DT was observed. The MIA that was calibrated in accordance with WHO reference standards demonstrated increased sensitivity, reproducibility, and high throughput capabilities, allowing for the design of robust studies that evaluate both natural and vaccine-induced immunity.

A computer vision approach to the assessment of dried blood spot size and quality in newborn screening

BackgroundDried blood spot (DBS) size and quality affect newborn screening (NBS) test results. Visual assessment of DBS quality is subjective. MethodsWe developed and validated a computer vision (CV) algorithm to measure DBS diameter and identify incorrectly applied blood in images from the Panthera DBS puncher. We used CV to assess historical trends in DBS quality and correlate DBS diameter to NBS analyte concentrations in 130,620 specimens. ResultsCV estimates of DBS diameter were precise (percentage coefficient of variation < 1.3%) and demonstrated excellent agreement with digital calipers with a mean (standard deviation) difference of 0.23 mm (0.18 mm). An optimised logistic regression model showed a sensitivity of 94.3% and specificity of 96.8% for detecting incorrectly applied blood. In a validation set of images (n = 40), CV agreed with an expert panel in all acceptable specimens and identified all specimens rejected by the expert panel due to incorrect blood application or DBS diameter > 14 mm. CV identified a reduction in unsuitable NBS specimens from 25.5% in 2015 to 2% in 2021. Each mm decrease in DBS diameter decreased analyte concentrations by up to 4.3%. ConclusionsCV can aid assessment of DBS size and quality to harmonize specimen rejection both within and between laboratories.

Micro-titer plate assay for measurement of total phenolic and total flavonoid contents in medicinal plant extracts

Total phenolic content (TPC) measured by using the Folin-Ciocalteu method and Total flavonoid content (TFC) based on a complex formation with AlCl3 assay are widely applied as part of herbal extract quality control. Conducting the assay measurements in microtiter plates may save resources and time. The objective of this study was to evaluate the validity of the micro-titer plates assay to determine the TFC and TPC in several medicinal plants’ extracts. To compare the developed TPC and TFC methods to the conventional assays, the confidence intervals of linear regression were used with a significance set as p < 0.05. Both of the standards showed good linearity in the range of 10–60 mg L−1 of gallic acid and 40–200 mg L−1 of quercetin, (R2 > 0.999). Based on the micro-titer plate assay, the limits of detection (LODs), and quantification (LOQs) for TPC ranged between 1.19 mg L−1 and 3.98 mg L−1 whereas for TFC, they were 1.47 mg L−1 and 4.90 mg L−1. The percent coefficient of variation (%CV) of the intra-day and inter-day assays were lower than 1.46% and 1.91%, respectively, showing adequate precision. The recovery ranged between 101.25% and 105.93% for the TPC assay and between 96.78% and 101.28% for the TFC assay. Twelve samples of medicinal extracts were analyzed by validated microplate assay for TPC and TFC and then compared with the conventional method. Notably, there were no significant differences in the TPC and TFC content detected between the microplate and conventional methods for all samples (p > 0.05). In conclusion, the microplate assay can be potentially used for TPC and TFC determinations.

Impact of bone-equivalent solution density in a thoracic spine phantom on bone single-photon emission computed tomography image quality and quantification.

This study aimed to evaluate the effects of dipotassium hydrogen phosphate (K2HPO4) solution density on single-photon emission computed tomography (SPECT) image quality and quantification. We used a JSP phantom containing six cylinders filled with K2HPO4 solutions of varying densities. Computed tomography (CT) was performed, and CT values and linear attenuation coefficients were measured. Subsequently, SPECT images of an SIM2 bone phantom filled with 99mTc with/without K2HPO4 solution were acquired using a SPECT/CT camera. The full width at half maximum (FWHM), percentage coefficient of variation (%CV), recovery coefficient, and standardized uptake value (SUV) were evaluated to investigate the impact of the K2HPO4 solution density. The CT values and linear attenuation coefficients increased with the K2HPO4 solution density. The CT values for cancellous and cortical bones were reflected by K2HPO4 solution densities of 0.15-0.20 and 1.50-1.70g/cm3, respectively. FWHM values were significantly lower with the K2HPO4 solution than those with water alone (18.0 ± 0.9mm with water alone, 15.6 ± 0.2mm with 0.15g/cm3 K2HPO4, and 16.1 ± 0.3mm with 1.49g/cm3 K2HPO4). Although the %CVs showed no significant differences, the recovery coefficients obtained with water alone tended to be slightly lower than those obtained with the K2HPO4 solution. The SUV obtained using the standard density of the K2HPO4 solution differed from that obtained using the optimized density. In conclusion, SPECT image quality and quantification depends on the presence and concentration of the bone-equivalent solution. The optimal bone-equivalent solution density should be used to evaluate the bone image phantoms.

Design and Construction of Vertical Livestock Feed Mixer

The development of a feed mixer plays a vital role in optimizing the feed production process in various agricultural and livestock industries. Thus, for better nutrition, improved feed efficiency, cost, and waste reduction, locally designed and fabricated feed mixers using locally available materials are essential and highly required. The volume of the mixing chamber, shaft diameter, belt selection, pulley diameter, and required power to drive the shaft were well designed for. The developed feed mixer comprises the mixing chamber, hopper, auger, bearings, electric motor, and pulley, which are major components of the machine. Angle Iron bars and 2mm sheet metal made of mild steel were used in the fabrication of the frame and the mixing chamber, respectively. The degree of mixing of the sample that was collected at the end of each test and the coefficient of variation percentage were used to evaluate the effectiveness of the machine mixing operation. At a mixing shaft speed of 560 rpm and a mixing time of 4.5 minutes, the optimum values of coefficient of variation (12.01%) and degree of mixing (88.21%) were achieved. However, the fabricated feed mixing machine was evaluated to be 92% efficient.