CV Range Research Articles

Abstract 6409: A multiparametric flow cytometry assay to monitor regeneration of B-cells following anti B-cell therapy in cancer and autoimmune patients

Abstract Purpose Functional dysregulation of B-cells leads to a variety of autoimmune disorders and blood cancers. Multiple B-cell depleting therapies (e.g., BTK inhibitors, antibodies or CAR-Ts) are currently in active clinical trial investigation. Following therapeutic elimination of pathogenic B-cells, it is critical to monitor the productive regeneration of fully functional B-cells as they mediate innate and adaptive immune responses. To this end, we developed a multiparametric flow cytometry assay for exquisite monitoring of early stages of B cell development, maturation, and antibody production to correlate it with treatment outcomes and determine if continuous depletion of B-cells is necessary to maintain clinical benefits for patients. Study Design We designed a high throughput 12-parameter flow cytometry panel incorporating primary differentiation markers namely, CD45, CD3, CD38, CD10, CD27, CD19, CD20, IgD, Lambda & Kappa Light Chains required for identification of different developmental stages of B-cells in peripheral blood using an in vitro diagnostic grade flow cytometry system (BD FACSLyric). The optimized method underwent robust fit-for-purpose analytical validation per CLSI H62 guidelines with emphasis on establishing assay sensitivity, specificity, and reproducibility across experimental days, operators, and instruments. Results We were able to reliably identify different stages of B-cell development, namely, Naïve (CD19+CD20+IgD+CD27-), Unswitched/Marginal (CD19+CD20+IgD+CD27+), Memory (CD19+CD20+IgD-CD27+), Transitional (CD19+CD20+IgD+CD27-CD10+), Antibody Secreting Cells (CD19+CD20-IgD-CD27+CD38hi) as well as clonality (CD19+CD20+Lambda+ or Kappa+). The lower limit of quantitation was determined to be around 60 events, based upon analysis of 90 data points with good reproducibility (CV range: 0-11.7%) across experimental days, operators and instruments demonstrating sensitive detection and robust performance. Conclusion In summary, we have developed and validated a 12-parameter flow cytometry assay focusing on monitoring the regeneration of different subsets of B-cells in various B-cell malignancies (Non-Hodgkin’s Lymphoma, B CLL) and autoimmune disorders (Multiple Sclerosis, Rheumatoid Arthritis, Systemic Lupus Erythematosus). The high reproducibility/robustness of the assay demonstrates its suitability for use in clinical trials as a valuable tool for safety monitoring and disease evaluation enabling development of novel therapeutic agents. Citation Format: Amit Kumar Mehta, Ruby Tandon, Anil Pahuja, Jane Gao, Kevin Nguyen, Naveen Dakappagari, Zeni Alfonso. A multiparametric flow cytometry assay to monitor regeneration of B-cells following anti B-cell therapy in cancer and autoimmune patients [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 6409.

Assessing the temporal within-day glycemic variability during hospitalization in patients with type 2 diabetes patients using continuous glucose monitoring: a retrospective observational study

AimsFrequent and extensive within-day glycemic variability (GV) in blood glucose levels may increase the risk of hypoglycemia and long-term mortality in hospitalized patients with diabetes. We aimed to assess the amplitude and frequency of within-day GV in inpatients with type 2 diabetes and to explore the factors influencing within-day GV.MethodsWe conducted a single-center, retrospective observational study by analyzing hospital records and 10-day real-time continuous glucose monitoring data. Within-day GV was assessed using the coefficient of variation (%CV). The primary outcome was the amplitude and frequency of within-day GV. The frequency of within-day GV was assessed by the consecutive days (CD) of maintaining within the target %CV range after first reaching it (CD after first reaching the target) and the maximum consecutive days of maintaining within the target %CV range (Max-CD). The target %CV range was less than 24.4%. We evaluated the factors influencing within-day GV using COX regression and Poisson regression models.ResultsA total of 1050 cases were analyzed, of whom 86.57% reduced the amplitude of within-day GV before the sixth day of hospitalization. Of the 1050 hospitalized patients, 66.57% stayed within the target %CV range for less than two days after first reaching the target and 69.71% experienced a Max-CD of fewer than four days. Reducing the average postprandial glucose excursion (hazard ratio [HR]: 0.81, 95% confidence interval [CI]: 0.77–0.85; incidence rate ratios [IRR]: 0.72, 95% CI: 0.69–0.74) and the use of α-glucosidase inhibitors (IRR: 1.1, 95% CI: 1.01–1.18) and glucagon-like peptide-1 agonist (IRR: 1.30, 95% CI: 1.02–1.65) contributed to reducing the amplitude and decreasing the frequency of within-day GV. However, the use of insulin (HR: 0.64, 95% CI: 0.55–0.75; IRR: 0.86, 95% CI: 0.79–0.93) and glinide (HR: 0.47, 95% CI: 0.31–0.73; IRR: 0.84, 95% CI: 0.73–0.97) may lead to an increased frequency of within-day GV.ConclusionsAn increasing frequency of within-day GV was observed during the hospitalization in patients with type 2 diabetes, despite the effective reduction in the amplitude of within-day GV. Using medications designed to lower postprandial blood glucose could contribute to minimize the risk of frequent within-day GV.

The Precision of Lumbar Spine BMD and TBS on Different Vertebral Combinations

OBJECTIVE The objective of this study is to compute the precision and LSC of BMD and TBS at the lumbar spine using dual-energy X-ray absorptiometry in different vertebral combinations. METHODS Thirty female participants (age 58.8 ± 4.0 years, height: 155.0 ± 5.2 cm, weight: 57.4 ± 8.6 kg and BMI: 23.9 ± 3.6) were scanned at the lumbar spine twice in the same day using DXA. The precision and LSC of BMD and TBS were computed in terms of the RMS-SD and %CV using the ISCD Advanced Precision Calculation Tool. RESULTS The precision and LSC of BMD of the 4 vertebrae combinations showed the best precision and also showed a similar trend (0.005 g/cm2 and 0.62%, 0.013 g/cm2 and 1.71%), respectively. For TBS, the precision and LSC followed a similar pattern as BMD but was inferior to those of BMD. The %CV LSC of BMD for all vertebrae combinations did not exceed 5.3% as recommended by ISCD (15). The %CV LSC of TBS for 2-4 vertebrae combinations were within 5.8% with the exception of the individual vertebra which was unacceptable (%CV range 6.17-8.98). CONCLUSIONS All vertebrae combinations had an acceptable level of precision and LSC for BMD monitoring. However, the precision and LSC of TBS were inferior to those of BMD, and individual vertebra were not appropriate for TBS monitoring. KEYWORDS precision, least significant change (LSC), bone mineral density (BMD), trabecular bone score (TBS)

Performance Investigation of Precision Seeding of a Multi-Rows Pneumatic Cylindrical System for Wheat

Highlights By using the seed barrier mechanism, the cylindrical metering device was able to locate seeds through the six rows successfully and precisely. Each row's seed quality index was above 90%, with minor variations within and between rows. An oblong shape was used on the metering cylinder to be comfortable for wheat seeds. During field trials, the coefficient of variation for seed distribution was 3.43%. Abstract. The main objective of this study was to investigate the performance of a pneumatic cylindrical system with six rows for wheat precision seeding. The cylinder was coupled with a plastic barrier to prevent seeds from creeping over the cylinder and permit precise seed distribution. Results disclosed that the optimum negative pressures associated with 15, 20, and 25 rpm were 2.4, 2.7, and 3.00 kPa, respectively. 15 rpm resulted in a quality index of 90.96%, 90.88%, 90.86%, 90.76%, 90.56%, and 90.38%, while 20 rpm attained 91.30%, 91.02%, 91.06%, 90.86%, 91.32%, and 90.82%, and 25 rpm achieved 90.92%, 90.72%, 90.84%, 90.86%, 90.62%, and 90.80% for rows 1, 2, 3, 4, 5, and 6, respectively. The highest seed feed index (91.32%) was obtained by row 4 at a speed of 20 rpm, while the lowest value (90.56%) was achieved by row 6 at 15 rpm. Both the separate rows' stability and the rows' consistent CV resulted in a CV range of less than 1.5 percentage points, with some tendency of 25 rpm to be more consistent than 15 and 20 rpm. Results concluded that the multi-row cylinder and the plastic barrier technique could satisfy the required seed precision without damage and be employed for further investigations. Keywords: Multi-row cylindrical, Pneumatic system, Seed precision, Wheat.

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 < 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.

Investigating the impact of inter-limb asymmetry in hamstring strength on jump, sprint, and strength performance in young athletes: comparing the role of gross force.

The main purpose of this cross-sectional study was to examine the impact of the inter-limb asymmetry of hamstring strength on jump, sprint and strength performance and to compare the effects of inter-limb asymmetry of hamstring strength with gross force (GF) of the hamstring on these physical qualities in youth volleyball athletes. Eighty-one youth volleyball players (age: 16.6 ± 1.9years; training experience: 3.0 ± 0.9years; height: 191.4 ± 7.1cm; body mass: 78.5 ± 12.9kg; lean body mass: 63.5 ± 10.5kg; body fat rate: 18.6% ± 6.1%) performed a mid-season battery of tests consisting of morphological test, depth jump (DJ), counter movement jump (CMJ), squat jump (SJ), 10m sprint, isometric mid-thigh pull (IMTP) and hamstring strength test. All tests reported good to excellent reliability (ICC range = 0.815-0.996) and acceptable variability (CV range = 3.26-7.84%). Results show a significant negative relationship between inter-limb asymmetry of hamstring strength and all physical qualities (r = -0.271 to -0.445; p < 0.05), and a significant positive relationship between GF of hamstring and all physical qualities (r = 0.303 to 0.664; p < 0.05). Additionally, GF of hamstring was more relevant to IMTP-PF (peak force) (r = 0.664) and inter-limb asymmetry of hamstring strength was more relevant to 10m sprint (r = -0.445). The findings from this study indicate that, for youth athletes, the GF of the hamstring is crucial for overall lower limb strength performance, and the importance of inter-limb symmetry of hamstring strength increases with the complexity of the task.

Optimisation and Validation of a conventional ELISA and cut-offs for detecting and quantifying anti-SARS-CoV-2 Spike, RBD, and Nucleoprotein IgG, IgM, and IgA antibodies in Uganda.

There is an urgent need for better immunoassays to measure antibody responses as part of immune-surveillance activities and to profile immunological responses to emerging SARS-CoV-2 variants. We optimised and validated an in-house conventional ELISA to identify and quantify SARS-CoV-2 spike- (S-), receptor binding domain- (RBD-), and nucleoprotein- (N-) directed IgG, IgM, and IgA binding antibodies in the Ugandan population and similar settings. Pre- and post-pandemic specimens were used to compare the utility of mean ± 2SD, mean ± 3SD, 4-fold above blanks, bootstrapping, and receiver operating characteristic (ROC) analyses in determining optimal cut-off optical densities at 450 nm (OD) for discriminating between antibody positives and negatives. "Limits of detection" (LOD) and "limits of quantitation" (LOQ) were validated alongside the assay's uniformity, accuracy, inter-assay and inter-operator precision, and parallelism. With spike-directed sensitivity and specificity of 95.33 and 94.15%, respectively, and nucleoprotein sensitivity and specificity of 82.69 and 79.71%, ROC was chosen as the best method for determining cutoffs. Accuracy measurements were within the expected CV range of 25%. Serum and plasma OD values were highly correlated (r = 0.93, p=0.0001). ROC-derived cut-offs for S-, RBD-, and N-directed IgG, IgM, and IgA were 0.432, 0.356, 0.201 (S), 0.214, 0.350, 0.303 (RBD), and 0.395, 0.229, 0.188 (N). The sensitivity and specificity of the S-IgG cut-off were equivalent to the WHO 20/B770-02 S-IgG reference standard at 100% level. Spike negative IgG, IgM, and IgA ODs corresponded to median antibody concentrations of 1.49, 3.16, and 0 BAU/mL, respectively, consistent with WHO low titre estimates. Anti-spike IgG, IgM, and IgA cut-offs were equivalent to 18.94, 20.06, and 55.08 BAU/mL. For the first time, we provide validated parameters and cut-off criteria for the in-house detection of subclinical SARS-CoV-2 infection and vaccine-elicited binding antibodies in the context of Sub-Saharan Africa and populations with comparable risk factors.

Spatially continuous mapping of hourly ground ozone levels assisted by Himawari-8 short wave radiation products

ABSTRACT Ground-level ozone concentration has demonstrated a noteworthy increasing trend in easternChina over the past 20 years, exerting inescapable influence on atmospheric chemistry, climate change, and air pollution. To support epidemiological research and prevent further environmental pollution, it is imperative to monitor the spatio-temporal distribution of ground-level ozone pollution. Geostationary satellites prove a promising approach to make up for the limitation of ground-based measurement and polar-orbiting observation by acquiring regional atmospheric measurements of high spatio-temporal resolution, yet inevitably exists spatial discontinuity caused by the cloud cover. This study established an effective model to produce spatially continuous estimation of hourly 5 km ground-level ozone (O3) concentrations covered most of China by firstly integrating hourly Himawari-8 SWR product from 1 March 2018 to 28 February 2019. The R2 values of established model of all available samples for direct fitting, 20-fold cluster-based CV, 10-fold site-based CV, and 10-fold sample-based CV reaches 0.95, 0.69, 0.87 and 0.89, respectively, implying superior universality and spatial scalability. The R2 values of seasonal site-based CV range from 0.78 (Winter) to 0.86 (Autumn), and that of hourly site-based CV ranges from 0.73 (0900 BST) to 0.86 (1300 BST). In addition, this study displayed the spatial distribution of the estimated ground-level ozone from the temporal scales of quarter, week, and day. Summer (87.1 ± 28.2 μg/m3) proves to be the most polluted season, and the least polluted season is winter (59.4 ± 16.1 μg/m3). 1400 BST appears to be the most polluted hour (87.4 ± 26.6 μg/m3) and 0900 BST is the least polluted hour (59.1 ± 16.8 μg/m3). Remarkable “weekend effect of ozone” has been detected in northeast Hebei and Sichuan Basin and Yangtze River valley.

Reliability of the Coimbra Reactive Agility Soccer Test (CRAST).

Agility is a fitness-skill-related component that should be a part of the standard physiological testing for soccer players and one of the key performance indicators in soccer. The present study aimed to assess the reliability of the CRAST as a research tool in the study of soccer skills. Twenty-one university soccer players (chronological age: 19.3 ± 1.4 years; body mass: 69.6 ± 8.2 kg; stature: 173.5 ± 6.5 cm; federated training experience: 9.7 ± 3.6 years) volunteered for the testing protocol. The CRAST requires players to complete random courses six times as quickly as possible. In addition, the CRAST requires players to control and dribble the markers (four different colors: green, yellow, blue, and red). The soccer players completed three trials, each separated by one week. The first trial accounted for familiarization; the second and third were considered for analysis. The correlation for overall performance was very strong. The reliability of the CRAST was slightly better for total time than that for the penalty score (0.95 vs. 0.93). The TEM and the associated CV range of 7.04%-7.54% were for the penalty score and the total time, respectively. For both measurements, the ICC values also represent excellent reliability, as both values were over 0.900. The CRAST is a reliable protocol for assessing agility in soccer players.

Intra-unit reliability and movement variability of submission grappling external load as measured by torso mounted accelerometery.

Submission grappling consists of skills and movements used in combat sports to physically control opponents whilst trying to apply choke holds and joint locks. There is currently no accepted method of monitoring external load in grappling-based sports due to the absence of key variables such as distance, velocity or time. The primary aim of this study was to determine whether PlayerLoad is a reliable variable for measuring external load of submission grappling movements, with a secondary aim of determining the between repetition variance of submission grappling movements. 7 experienced submission grapplers were recruited. Each wore a torso mounted Catapult® Optimeye S5 microelectromechanical systems (MEMS) device and completed 5 repetitions of each of the following: 4 submission techniques; 5 transition techniques; 2 guard pass techniques; 2 takedown techniques. Accumulated PlayerLoad (PLdACC) was recorded as a marker of absolute load, with accumulated PlayerLoad per minute (PLdACC∙min-1) representing relative load. Reliability of each was assessed using intraclass correlation coefficient (ICC(3,1)) (≥ 0.70). Between repetition movement variation was assessed via coefficient of variation with 95% confidence intervals (CV, 95%CI) (acceptable ≤ 15%, good ≤ 10%). PLdACC ICC(3,1) range = 0.78-0.98, with CV range = 9-22%. PLdACC∙min-1 ICC(3,1) range = 0.83-0.98, with CV range = 11-19%. Though several variables displayed CV > 15%, all had 95%CI lower boundaries ≤ 15%. Whilst PlayerLoad was found to be a reliable measure for submission grappling, relatively high CVs across most techniques examined suggest PlayerLoad may not be appropriate for measuring changes in external load for individual movements in submission grappling. However, it may prove a useful tool for monitoring the external load of full, grappling-based, training sessions within an individual.

Reliability of biomarkers of physiological stress at rest and post-exertional heat stress.

The purpose of this study was to assess the reliability of blood biomarkers that can signify exercise-induced heat stress in hot conditions. Fourteen males completed two heat stress tests separated by 5-7 days. Venous blood was drawn pre- and post- heat stress for the concentration of normetanephrine, metanephrine, serum osmolality, copeptin, kidney-injury molecule 1 and neutrophil gelatinase-associated lipocalin. No biomarker, except copeptin, displayed systematic trial order bias (p ≥ 0.05). Normetanephrine, copeptin and neutrophil gelatinase-associated lipocalin presented acceptable reliability (CV range: 0.9-14.3%), while greater variability was present in metanephrine, osmolality and kidney-injury molecule 1 (CV range: 28.6-43.2%). Normetanephrine exhibited the largest increase (p <0.001) in response to heat stress (trial 1 = 1048 ± 461 pmol. L-1; trial 2 = 1067 ± 408 pmol. L-1), whilst kidney-injury molecule 1 presented trivial changes (trial 1 = -4 ± 20 ng. L-1; trial 2 = 2 ± 16 ng. L-1, p >0.05). Normetanephrine, copeptin and neutrophil gelatinase-associated lipocalin demonstrated good reliability and sensitivity to an acute bout of heat stress. These biomarkers may be suitable for application in laboratory and field research to understand the efficacy of interventions that can attenuate the risk of thermal injury whilst exercising in the heat.

Load-Velocity Relationship Variables to Assess the Maximal Neuromuscular Capacities During the Back-Squat Exercise.

The relationship between the external load lifted and movement velocity can be modeled by a simple linear regression, and the variables derived from the load-velocity (L-V) relationship were recently used to estimate the maximal neuromuscular capacities during 2 variants of the back-squat exercise. The L-V relationship variables will be highly reliable and will be highly associated with the traditional tests commonly used to evaluate the maximal force and power. Twenty-four male wrestlers performed 5 testing sessions (a 1-repetition maximum [1RM] session, and 4 experimental sessions [2 with the concentric-only back-squat and 2 with the eccentric-concentric back-squat]). Each experimental session consisted of performing 3 repetitions against 5 loads (45%-55%-65%-75%-85% of the 1RM), followed by single 1RM attempts. Level 3. Individual L-V relationships were modeled from the mean velocity collected under all loading conditions from which the following 3 variables were calculated: load-axis intercept (L0), velocity-axis intercept (v0), and area under the line (Aline = L0·v0/2). The back-squat 1RM strength and the maximum power determined as the apex of the power-velocity relationship (Pmax) were also determined as traditional measures of maximal force and power capacities, respectively. The between-session reliability was high for the Aline (coefficient of variation [CV] range = 2.58%-4.37%; intraclass correlation coefficient [ICC] range = 0.98-0.99) and generally acceptable for L0 and v0 (CV range = 5.08%-9.01%; ICC range = 0.45-0.96). Regarding the concurrent validity, the correlations were very large between L0 and the 1RM strength (rrange = 0.87-0.88) and nearly perfect between Aline and Pmax (r = 0.98-0.99). The load-velocity relationship variables can be obtained with a high reliability (L0, v0, and Aline) and validity (L0 and Aline) during the back-squat exercise. The load-velocity relationship modeling represents a quick and simple procedure to estimate the maximal neuromuscular capacities of lower-body muscles.

Test-retest reliability of skeletal muscle oxygenation measurement using near-infrared spectroscopy during exercise in patients with sport-related iliac artery flow limitation.

The ankle‐brachial index is an accurate tool for detecting claudication in atherosclerotic patients. However, this technique fails to identify subtle flow limitations of the iliac arteries (FLIA) in endurance athletes. Near‐infrared spectroscopy (NIRS) is a noninvasive technique that measures skeletal muscle tissue oxygenation status. The aim of the present study is to examine the absolute and relative test–retest reliability of NIRS and evaluate its potential as a diagnostic tool in FLIA. NIRS‐derived exercise variables were analyzed during exercise and recovery in FLIA 17 patients and 19 healthy controls. The relative reliability of absolute variables (such as the maximal value) were slight to yet predominantly substantial (intraclass correlation coefficient [ICC], ICC range: 0.06–0.76) with good to excellent absolute reliability (absolute limits of agreement [ALoA], ALoA range: 0.8 ± 10.2 to 0.7 ± 13.1; coefficient of variation [CV], CV range: 5%–11%). Absolute values encompassing signal amplitudes showed moderate to almost perfect relative reliability (ICC range: 0.51–0.89) and poor to good absolute reliability (ALoA range: −1.3 ± 7.0 to −2.5 ± 15.7; CV range: 15%–32%). Kinetic variables showed moderate to almost perfect relative reliability for most recovery kinetics variables (ICC range: 0.54–0.86) with fair to good absolute reliability (ALoA range: 0.4 ± 12.2 to 3.9 ± 37.9; CV range: 18%–27%). Particularly, kinetic variables showed significant differences between patients and healthy subjects. NIRS is found to be a reliable method for examining muscle tissue oxygenation variables. Given the significant differences in especially recovery kinetics between normal subjects and patients, NIRS may contribute to diagnosing FLIA in endurance athletes.

Multiple-Ascending-Dose Study of the Soluble Guanylate Cyclase Stimulator, IW-1701, in Healthy Subjects

▪Background: Nitric oxide (NO), a key regulator of vascular health, modulates smooth muscle tone and blood pressure, inhibits vascular smooth muscle proliferation, and suppresses inflammation and platelet activation. These protective functions are mediated by soluble guanylate cyclase (sGC), a signaling enzyme that catalyzes the formation of cyclic guanosine monophosphate (cGMP) in response to NO binding. The compromised vascular health characteristic of the hemoglobinopathies may in part be due to deficient NO bioavailability and inadequate sGC-cGMP signaling. IW-1701 is an orally available sGC stimulator that binds to sGC and enhances NO-sGC-cGMP signaling.Methods: A Phase 1b placebo-controlled, randomized, multiple-ascending-dose study was conducted to assess safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (blood pressure, heart rate, platelet function, and plasma cGMP) of IW-1701 in healthy subjects. Five successive cohorts of 11 subjects each (8 active and 3 placebo) were randomized. The first 3 cohorts received 7 days of repeated once-daily (QD) doses of 1-, 2- or 5-mg IW-1701 respectively, and the last 2 cohorts received 7 days of lead-in QD doses of 3 mg followed by 7 days of up-titrated QD doses of 5 or 6 mg, respectively.Results: IW-1701 doses ranging from 1 to 6 mg were tolerated and no safety concerns were identified. There were no serious adverse events (SAEs), severe adverse events (AEs), or withdrawals due to AEs. All AEs were mild or moderate, most resolved on the day of onset. Among the 40 subjects who received IW-1701 for 7 consecutive days, the most common AEs were tachycardia and headache (each reported in 7 subjects), and hypotension and nausea (each reported in 3 subjects). Among the 16 subjects who received an up-titrated dose of IW-1701 for 7 additional days, headache was the only AE reported in >1 subject (2 subjects). IW-1701 was rapidly absorbed, with median Tmax achieved from 2 to 4 hours postdose. Systemic exposure to IW-1701 was less than dose proportional after a single dose (Cmax slope, 0.701 [90% CI, 0.578-0.824] and AUCtau slope, 0.833 [90% CI, 0.726-0.939]) and was dose proportional after 7 days of QD dosing (Cmax slope, 1.01 [90% CI, 0.873-1.14] and AUCtau slope, 1.03 [90% CI, 0.892-1.17]). Intersubject variability in PK was low to moderate (AUCtau %CV range, 10.6-39.1%). The accumulation ratio (based on RAUC) ranged from 1.95 to 2.63 across doses of 1-, 2-, 3-, and 5-mg IW-1701 after 7 days of QD dosing. Mean CL/F (apparent total body clearance) and Vz/F (apparent volume of distribution) ranged from 1080 to 1520 mL/h and 49.4 to 58.9 L, respectively. Mean terminal t1/2 was 30 hours, which is consistent with a QD dosing regimen. After 7 days of QD dosing, least squares mean changes from baseline in 24-hour ambulatory systolic blood pressure (±SE) (in cohort order) were -1.17±1.24 (placebo), -2.98±1.80 (1 mg), -5.37±1.71 (2 mg), -6.67±1.69 (5 mg), -6.55±1.95 (3 mg), and -4.98±1.68 mmHg (3 mg). After 14 days of QD dosing in the 2 cohorts that received up-titrated doses, the least squares mean changes from baseline in 24-hour ambulatory systolic blood pressure (±SE) were -1.51±2.42 (placebo), -6.85±2.03 (5 mg), and -8.64±2.08 mmHg (6 mg). Plasma cGMP levels were highly variable; in general, mean plasma cGMP levels were increased from baseline at dose levels ≥3 mg IW-1701. There were no clinically meaningful changes from baseline in platelet function assessment by PFA-100® at any dose level.Conclusion: IW-1701 was tolerated in healthy subjects with only mild and moderate AEs. Common AEs were consistent with the effects of sGC stimulation. Lead-in dosing with a lower dose (3 mg) appeared to improve the tolerability of higher doses (5 and 6 mg). The PK of repeat IW-1701 dosing was dose proportional with low-to-moderate intersubject variability and half-life consistent with QD dosing. Pharmacodynamic results, including blood pressure and plasma cGMP levels, demonstrated target engagement. Clinical evaluation of IW-1701 in diseases associated with NO deficiency, including sickle cell disease and achalasia, are planned or ongoing.– The authors gratefully acknowledge the early contributions of Bernard J. Lavins. – DisclosuresMittleman:Ironwood Pharmaceuticals: Employment, Equity Ownership. Wilson:Ironwood Pharmaceuticals: Employment, Equity Ownership. Sykes:Ironwood Pharmaceuticals: Employment, Equity Ownership. Mihova:Ironwood Pharmaceuticals: Employment, Equity Ownership. Chickering:Ironwood Pharmaceuticals: Employment, Equity Ownership. Hall:Ironwood Pharmaceuticals: Employment, Equity Ownership. Milne:Ironwood Pharmaceuticals: Employment, Equity Ownership. Currie:Ironwood Pharmaceuticals: Employment, Equity Ownership. Chien:Ironwood Pharmaceuticals: Employment, Equity Ownership.

Measuring tibial hemodynamics and metabolism at rest and after exercise using near-infrared spectroscopy.

The bone vascular system is important, yet evaluation of bone hemodynamics is difficult and expensive. This study evaluated the utility and reliability of near-infrared spectroscopy (NIRS), a portable and relatively inexpensive device, in measuring tibial hemodynamics and metabolic rate. Eleven participants were tested twice using post-occlusive reactive hyperemia technique with the NIRS probes placed on the tibia and the medial gastrocnemius (MG) muscle. Measurements were made at rest and after 2 levels of plantarflexion exercise. The difference between oxygenated and deoxygenated hemoglobin signal could be reliably measured with small coefficients of variation (CV; range 5.7-9.8%) and high intraclass correlation coefficients (ICC; range 0.73-0.91). Deoxygenated hemoglobin rate of change, a potential marker for bone metabolism, also showed good reliability (CV range 7.5-9.8%, ICC range 0.90-0.93). The tibia was characterized with a much slower metabolic rate compared with MG (p< 0.001). While exercise significantly increased MG metabolic rate in a dose-dependent manner (all p< 0.05), no changes were observed for the tibia after exercise compared with rest (all p> 0.05). NIRS is a suitable tool for monitoring hemodynamics and metabolism in the tibia. However, the local muscle exercise protocol utilized in the current study did not influence bone hemodynamics or metabolic rate. Novelty: NIRS can be used to monitor tibial hemodynamics and metabolism with good reliability. Short-duration local muscle exercise increased metabolic rate in muscle but not in bone. High level of loading and exercise volume may be needed to elicit measurable metabolic changes in bone.

Dose Escalating and Bioavailability Phase 1 Studies Assessing Safety and Tolerability and Pharmacokinetics of Tildacerfont, a Small-Molecule CRF1 Receptor Antagonist

Background: Tildacerfont (SPR001; LY2371712), a second generation, potent, selective, nonsteroidal, oral small-molecule antagonist of corticotropin-releasing factor type-1 (CRF1) receptors in the pituitary gland, is in late stage development as a potential treatment for adults with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). We report the results of 3 randomized Phase 1 studies of safety, tolerability, and pharmacokinetics (PK) of tildacerfont in healthy adults and compare the relative bioavailability of 2 tildacerfont formulations. Methods: Two Phase 1 studies were double-blind, placebo-controlled and evaluated escalating doses (2–800 mg QD) as single doses (SAD; n=18) and 50-200mg as multiple doses (MAD; n=47). The studies also included pilot food effect and drug-drug interaction sub studies, respectively. A randomized, balanced, open-label, crossover study (n=42) examined the relative bioavailability (BA) of single oral doses of 2 tildacerfont formulations, 200 mg powder-in-capsule (PIC) compared to 200 mg low-drug load tablet. Results: In the SAD and MAD studies, the most common, non-procedural adverse events were headache and cough. In the BA study, the most common, non-procedural adverse events were constipation and headache. There were no study drug-related serious adverse events (SAEs) in any study. In the SAD and MAD (PIC formulation), tildacerfont concentrations declined in a multi-exponential manner with Cmax occurring moderately late, ~5 hours post dose, and a high degree of variability was observed in AUC and Cmax (CV range: 54–122%). A food effect was observed in the SAD. Fourteen days of dosing achieved near steady state exposure with an AUC accumulation ratio of 2.51 to 3.65 in the MAD study. Midazolam, a sensitive probe substrate for CYP3A4 exhibited ~2-fold increase in AUC and a 1.8 increase in Cmax when co-administered with tildacerfont suggesting moderate CYP3A4 inhibition. The tablet formulation reduced the variability (AUC and Cmax CV range: 69–72%) compared to the PIC formulation resulting in a more predictable pharmacokinetic profile. The half-life of the tablet was ~60 hrs. Bioequivalence in AUC was established with the tablet and PIC formulations (fed state) with Cmax ~20% higher for tablet than PIC due to a reduction in and more consistent Tmax, median [range], from 6 [4.5, 24] hours to 3 [2, 6] hours. Conclusions: These results suggest that tildacerfont is safe and generally well-tolerated in healthy adults and suitable for further study in patients with CAH. The tablet formulation provided a more consistent pharmacokinetic profile with reduced variability in all parameters and is being evaluated in late stage studies of tildacerfont in adult CAH.

Unilateral or Bilateral Standing Broad Jumps: Which Jump Type Provides Inter-Limb Asymmetries with a Higher Reliability?

This study aimed to compare the between-session reliability of performance and asymmetry variables between unilateral and bilateral standing broad jumps (SBJ). Twenty-four amateur basketball players (12 males and females) completed two identical sessions which consisted of four unilateral SBJs (two with each leg) and two bilateral SBJs. Mean and peak values of force, velocity and power, and impulse were obtained separately for each leg using a dual force platform. Inter-limb asymmetries were computed using the standard percentage difference for the unilateral SBJ, and the bilateral asymmetry index-1 for the bilateral SBJ. All performance variables generally presented an acceptable absolute reliability for both SBJs (CV range = 3.65-9.81%) with some exceptions for mean force, mean power, and peak power obtained with both legs (CV range = 10.00-15.46%). Three out of 14 variables were obtained with higher reliability during the unilateral SBJ (CVratio ≥ 1.18), and 5 out of 14 during the bilateral SBJ (CVratio ≥ 1.27). Asymmetry variables always showed unacceptable reliability (ICCrange = -0.40 to 0.58), and slight to fair levels of agreement in their direction (Kappa range = -0.12 to 0.40) except for unilateral SBJ peak velocity [Kappa = 0.52] and bilateral SBJ peak power [Kappa = 0.51]) that showed moderate agreement for both SBJs. These results highlight that single-leg performance variables can be generally obtained with acceptable reliability regardless of the SBJ variant, but the reliability of the inter-limb asymmetries in the conditions examined in the present study is unacceptable to track individual changes in performance.

Reliability of Throwing Velocity during Non-specific and Specific Handball Throwing Tests.

Throwing velocity is one of the most important factors for scoring goals in handball. This study aimed to identify the type of throw and procedure for selecting the final test outcome that provide throwing velocity with the greatest reliability. Fifteen experienced handball players and 33 non-experienced participants were tested in two sessions. Each session consisted of 4 trials of 3 different throwing tests (unspecific, 7-meters, and 3-steps). The maximum value of 4 trials, average value of 4 trials, and average value of the 3 best trials were considered. Throwing velocity was highly reliable (coefficient of variation [CV]≤3.3%, intraclass correlation coefficient≥0.89) with the exception of the unspecific throw for the non-experienced group (CV≥5.9%, intraclass correlation coefficient≤0.56). The 3-steps throw (CV=1.7%) was more reliable than the 7-meters throw (CV=2.1%) (CVratio=1.19) and unspecific throw (CV = 3.8%) (CVratio=2.18), the 3 procedures provided a comparable reliability (CV range=2.4-2.6%; CVratio≤1.07), and the experienced group (CV=1.0%) presented a higher reliability than the non-experienced group (CV=4.0%) (CVratio=3.83). These results support the 3-steps throw to maximise the reliability of throwing velocity performance.

Reliability and Validity of the iLOAD Application for Monitoring the Mean Set Velocity During the Back Squat and Bench Press Exercises Performed Against Different Loads.

Pérez-Castilla, A, Boullosa, D, and García-Ramos, A. Reliability and validity of the iLOAD application for monitoring the mean set velocity during the back squat and bench press exercises performed against different loads. J Strength Cond Res 35(2S): S57-S65, 2021-This study aimed to evaluate the reliability and validity of a smartphone application (iLOAD) for the monitoring of mean concentric velocity (MV) during resistance training sets. Twenty males completed 2 identical sessions consisting of one set of 10 repetitions against 4 loads (25, 40, 55, 70% of the one repetition maximum [1RM]) during the back squat and bench press exercises. The MV of the 5 initial repetitions and for the whole set were determined simultaneously with the iLOAD application and a linear velocity transducer (LVT). Two independent researchers operated the iLOAD application during the experimental sessions to evaluate the interrater agreement for the assessment of MV. An acceptable but generally lower reliability was observed for iLOAD (coefficient of variation [CV] range: 5.61-9.79%) compared to the LVT (CV range: 4.51-8.18%) at 25-40-55% of 1RM, whereas the reliability at 75% of 1RM was acceptable for the LVT during the bench press (CV range: 6.37-8.26%), but it was unacceptable for the iLOAD during both exercises (CV range: 11.3-12.8%) and for the LVT during the back squat (CV range: 11.3-17.4%). Small to moderate differences (ES range: 0.24-1.04) and very high to practically perfect correlations (r range: 0.70-0.90) were observed between the iLOAD and the LVT. A very high agreement was observed between both raters for the recording of MV during the back squat and bench press exercises (r ≥ 0.98). Taken together, these results suggest that the iLOAD application can be confidently used to quantify the MV of training sets during the squat and bench press exercises not performed to failure.

Determination of digitoxin-induced elevated hydrogen peroxide levels in SK-Hep-1 cells using a chemiluminescent method

Drug-induced intracellular hydrogen peroxide overproduction is proposed as an upstream event in apoptosis signaling. It is therefore important to develop a sensitive method to quantify hydrogen peroxide in biological samples undergoing cell death. We report herein a luminol-based chemiluminescent assay with a suitable detection range of best linearity from 1 to 0.1 μM hydrogen peroxide with a CV range of 5.7% to 21.7% for intraday measurement at the detection range. We hypothesized that digitoxin-treated human hepatoma SK-Hep-1 cells produce hydrogen peroxide to cause cytotoxicity, which was verified by measuring the spent media by our chemiluminescent method in a dose-dependent manner. Taken together, our sensitive luminol-based chemiluminescent assay has potential use in the quantification of trace amounts of digitoxin overdose-induced hydrogen peroxide in biological samples.