Erythrocyte morphological symmetry analysis to detect sublethal trauma in shear flow

The viscoelastic properties of red blood cells (RBC) facilitate flexible shape change in response to extrinsic forces. Their viscoelasticity is intrinsically linked to physical properties of the cytosol, cytoskeleton, and membrane—all of which are highly sensitive to supraphysiological shear exposure. Given the need to minimise blood trauma within artificial organs, we observed RBC in supraphysiological shear through direct visualisation to gain understanding of processes leading to blood damage. Using a custom-built counter-rotating shear generator fit to a microscope, healthy red blood cells (RBC) were directly visualised during exposure to different levels of shear (10–60 Pa). To investigate RBC morphology in shear flow, we developed an image analysis method to quantify (a)symmetry of deforming ellipsoidal cells—following RBC identification and centroid detection, cell radius was determined for each angle around the circumference of the cell, and the resultant bimodal distribution (and thus RBC) was symmetrically compared. While traditional indices of RBC deformability (elongation index) remained unaltered in all shear conditions, following ~100 s of exposure to 60 Pa, the frequency of asymmetrical ellipses and RBC fragments/extracellular vesicles significantly increased. These findings indicate RBC structure is sensitive to shear history, where asymmetrical morphology may indicate sublethal blood damage in real-time shear flow.

Assessment of morphological, biochemical and rheological blood indicators in men after a 24-hour stay in a thermo-climatic chamber -50°C

Purpose: The aim of the study was to evaluate morphological, biochemical, and rheological blood indicators in men staying in a cryochamber at a temperature of -50°C for 24 hours. In 2018, a scientific-survival project ‘Taming the Frost’ was conducted at the Technoclimatic Research and Working Machines Laboratory of the Cracow University of Technology, under the scientific patronage of the Rector of the Cracow University of Technology, Prof. Jan Kazior, PhD, as well as the Rector of the University of Physical Education in Krakow, the late Prof. Aleksander Tyka, PhD, and the Vice-Rector for Science, Prof. Anna Marchewka, PhD. Material and methods: The blood for the tests was collected from an ulnar vein in fasting participants by a qualified nurse, in the morning, before entering the cryochamber and after 24 hours, i.e. on leaving the cryochamber. Morphological, biochemical, and rheological blood indicators were evaluated. The study group of the scientific-survival project ‘Taming the Frost’ involved men (n=6) who stayed in a cryochamber at a temperature of -50°C for 24 hours. For each participant, a 5-ml blood sample was placed in a tube (BD Vacutainer) with EDTA K2 anticoagulant for blood morphology and blood rheological evaluations in the Blood Physiology Laboratory of the Central Research and Development Laboratory, University of Physical Education in Krakow. Another 5-ml blood sample was placed in a Vacuette tube with a clotting activator for the remaining biochemical analyses in the Department of Clinical Biochemistry of the Krakow Branch of Maria Skłodowska-Curie National Research Institute of Oncology. Results: After leaving the cryochamber with a temperature of –50°C, the participants presented statistically significant increases in monocyte count and high-density lipoprotein and creatine kinase values, as well as decreases in IgA, total cholesterol, and triglycerides. In the assessment of blood rheological indicators, statistically significant increases in the elongation index at the shear stress of 0.30, 0.58, 1.13, and 2.19 Pa and decreases in the elongation index at the shear stress of 31.03 and 60.3 Pa were observed. For red cell aggregation indicators, a statistically significant increase in total aggregation time was reported. The other indicators exhibited a significance level of p>0.05. Conclusions: Staying in a cryochamber at a temperature of -50°C for 24 hours did not exert a negative impact on morphological, biochemical, or rheological blood indicators, which implies the subjects’ adaptation to the arranged conditions.

Use of Red Blood Cell Phenotypes for Second Line Therapy Selection in Sickle Cell Disease

INTRODUCTION In sickle cell disease (SCD), abnormal red blood cells (RBCs) sickle upon deoxygenation due to polymerization of hemoglobin S (HbS). Sickle RBCs exhibit poor deformability and increased viscosity, density, and microvascular adhesion. These rheological properties can be measured using existing devices. An oxygen-gradient ektacytometer measures the deformability of sickle RBCs under normoxic (maximum elongation index, or EImax) and hypoxic conditions (minimum elongation index, or EImin), and the pO 2 level at which sickling beings (point of sickling, or PoS). Dense RBCs are measured using a commercially available hematology analyzer. The hematocrit-to-viscosity ratio (HVR), an oxygen delivery index, is calculated based on the viscosity as measured by a cone and plate viscometer. RBC adhesion in the microvasculature can be modeled using a laminin-lined microfluidics device. These rheological biomarkers correlate with clinical complications such as pain events and acute chest syndrome, and are modified by known, clinically effective therapies such as hydroxyurea (HU) and transfusion (TF). HU is the standard of care for most individuals with SCD and positively modifies EImin, EImax, PoS, HVR, adhesion, and %DRBC. Recently, new agents to treat SCD have emerged including voxelotor, crizanlizumab, and pyruvate kinase activators like etavopivat, which have more targeted effects. It is essential to pair the appropriate novel agent to the patient, addressing their most prominent RBC abnormality remaining after HU therapy. We hypothesize that there is significant variability of rheology biomarkers between individuals with SCD on standard of care therapy, and that the most severe aspects of their RBC pathophysiology can be identified and targeted by novel second line therapies for clinical optimization. METHODS We collected peripheral blood in EDTA under an IRB approved protocol from 312 pediatric patients with SCD ranging in age from 2 to 21 years, 70% on HU. Subjects on chronic TF therapy were excluded. We measured whole blood viscosity at 45s -1 shear and calculated the HVR. %DRBC and complete blood counts were obtained using an ADVIA hematology analyzer (Siemens) and EImin, EImax, and PoS obtained using oxygen gradient ektacytometry (Lorrca, RR Mechatronics). RBCs from 17 HbSS subjects were analyzed for adhesion index to a laminin-lined microfluidics device. Values were assembled for each biomarker into histograms to demonstrate distribution, and quartile ranked. Venn diagrams were constructed comparing overlap between top 25% most severe rheology biomarkers to demonstrate effectiveness of a targeted, precision medicine approach to adding second line therapies to individuals with SCD. RESULTS Distribution of biomarkers in a typical pediatric SCD population in a US academic center are shown in Figure 1. High PoS, adhesion index, %DRBC, and low EImin and EImax, low HVR, are associated with disease severity and clinical complications in SCD; biomarker values were stratified from high to low severity association. The most severe quartile subjects from each biomarker were compared, and percentage of overlap noted (Figure 2). CONCLUSION Our rheologic assessment of a large pediatric cohort heavily treated with HU indicates a broad distribution of RBC phenotypes. Even on HU, patients exhibited loss of deformability, sickling, adhesion, or RBC density abnormalities, with little overlap of unrelated biomarkers associated with disease severity in an individual, i.e. the individuals with very high PoS did not have low HVR (16% of highest severity quartile subjects in common), compared to related biomarkers PoS and EImin or EImax (48% of highest severity quartile subjects in common). Only four subjects, all on HU, were in the quartile associated with highest severity for all biomarkers. Given the lack of overlapping pathology between different red cell abnormalities, selection of the appropriate agent should be straightforward. With three new FDA approved therapies for SCD and novel therapies in clinical trials, it is possible to choose the appropriate second agent to be added to HU based on individual patient RBC phenotype according to the principles of precision medicine. Future goals include CLIA certification for novel devices like the oxygen gradient ektacytometer and adhesive microfluidics at major academic SCD centers and use of these biomarkers in routine patient care. Figure 1 Figure 1. Disclosures Rab: Agios Pharmaceuticals: Research Funding; Axcella Health: Research Funding. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sheehan: Beam Therapeutics: Research Funding; Forma Therapeutics: Research Funding; Novartis: Research Funding.

Effects of remote ischemic preconditioning on the deformability and aggregation of red blood cells in a rat endotoxemia model

BACKGROUND: The prevention of rheologic alterations in erythrocytes may be important for reducing sepsis-associated morbidity and mortality. Remote ischemic preconditioning (RIPC) has been shown to prevent tissue damage caused by severe ischemia and mortality resulting from sepsis. However, the effect of RIPC on erythrocytes in sepsis is yet to be determined. OBJECTIVE: To investigate the effect of RIPC on rheologic alterations in erythrocytes in sepsis. METHODS: Thirty male Sprague-Dawley rats were used in this study. An endotoxin-induced sepsis model was established by intraperitoneally injecting 20 mg/kg LPS (LPS group). RIPC was induced in the right hind limb using a tourniquet, with three 10-minute of ischemia and 10 min of reperfusion cycles immediately before the injection of LPS (RIPC/LPS group) or phosphate-buffered saline (RIPC group). The aggregation index (AI), time to half-maximal aggregation (T1/2), and maximal elongation index (EImax) of the erythrocytes were measured 8 h after injection. RESULTS: The AI, T1/2, and EImax values in the LPS and RIPC/LPS groups differed significantly from those in the RIPC group, but there were no differences between the values in the LPS and RIPC/LPS groups. CONCLUSIONS: RIPC did not prevent rheologic alterations in erythrocytes in the rat model of LPS-induced endotoxemia.

ANALYSIS OF ECONOMIC AND SOCIAL INDICATORS IN OPTIMIZING THE PERFORMANCE OF URBAN GREEN SPACE MANAGEMENT (A STUDY OF MASHHAD METROPOLIS)

In recent decades, Mashhad as the second largest city of Iran after Tehran has experienced rapidly physical expansion regardless of the citizens' need for green space. The importance of Mashhad's urban green space is understandable from two dimensions, the first one is the existing population of the area and its increasing trend as the second most populous city of Iran and the other is the religious position of Mashhad, which annually receives millions of domestic and foreign travelers. Therefore, the aim of this paper is the investigation of urban green spaces performance in term of economic and social indicators in the Mashhad metropolis area. This research is an applied study by considering to its goal, and a descriptive analytical study for respect to its nature. It was also predominantly carried out with a quantitative approach. The statistical population for this research included the total population of Mashhad, and 380 peoples were selected as the sample using Cochran's formula. Also, 20 managers of Mashhad green space were interviewed to obtain more accurate data. SPSS software was used in the form of Cronbach's alpha test and the looseness and elongation index. Data and information reliability were also analyzed by statistical tests. SPSS software was used in the form of Cronbach's alpha test and the looseness and elongation index. Also, data and information reliability were analyzed by statistical tests. Data collection tools included a questionnaire and measurement of items is based on the Likert spectrum and 5-choice options. The research questionnaire includes 15 social questions and 5 economic questions that have been tested and analyzed using SPSS22 software. The results showed that managerial performance in the field of urban green spaces in Mashhad metropolis did not have a significant effect. From the view of citizens and managers, the various dimensions of the social index (communication, education, leisure, access and participation) have been evaluated as desirable.

Oral Administration of FT-4202, an Allosteric Activator of Pyruvate Kinase-R, Has Potent Anti-Sickling Effects in a Sickle Cell Anemia (SCA) Mouse Model, Resulting in Improved RBC Survival and Hemoglobin Levels

Introduction: Sickle cell anemia (SCA) results from a mutant β-globin gene that produces abnormal hemoglobin S (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage, leading to vaso-occlusions and hemolysis. Additionally, sickle RBCs contain less ATP and more 2,3-diphosphoglycerate (2,3-DPG) than normal RBCs; 2,3,DPG allosterically reduces hemoglobin (Hb) oxygen (O2)-affinity [i.e. increases P50], promoting faster unloading of O2, which potentiates HbS polymerization and RBC sickling. FT-4202, a selective and orally bioavailable allosteric activator of RBC pyruvate kinase (PKR), decreases 2,3-DPG and increases ATP in normal human RBCs (Blood, 2019, 134, Supplement 1:616). We hypothesized that oral administration of FT-4202 to SCA mice will increase HbS O2-affinity, and thereby decrease RBC sickling and membrane damage. Methods: Berkeley SCA mice were given 500-1000 mg/kg/day FT-4202 in chow (FT-4202 group) or control chow (control group) in 4 cohorts for 2 weeks (total 17-18 mice/group). In all cohorts, the health status, weight, and average chow consumption of each mouse was determined 3 times/week. Three cohorts were injected with sulfo-NHS-biotin 1 week into treatment (10-11 mice/group), and RBC survival assessed over the next week with serial micro-bleeds while on treatment. The 4th cohort was only bled at 2 week time-point to obtain P50 (Hemox Analyzer) and Hb levels (Hemavet). At experiment termination, all cohorts were terminally bled to determine (a) RBC levels of 2,3-DPG and ATP, (c) plasma levels of FT-4202 by LC-MS/MS, (d) the proportion of irreversibly sickled RBC (ISC) on blood smears (Image-J analysis), (e) the kinetics of experimentally-induced sickling (Lorrca®Oxygenscan) and (f) membrane deformability (Lorrca®Ektacytometry). Results: SCA mice on FT-4202 consumed a similar amount of food, and had similar weights and survival, compared to SCA mice on control chow throughout the 2-week period. As hypothesized, HbS O2 affinity increased, reflected by a decrease in P50 from 29.6 ± 0.62 mmHg (mean ± SEM) in the control group to 27.6 ± 0.58 mmHg in the FT-4202 group (p<0.03). Determinations of 2,3-DPG, ATP and FT-4202 are ongoing and will be presented. As expected, this increased HbS O2-affinity in the FT-4202 group reduced RBC sickling and membrane damage. At 2 weeks, the proportion of ISCs on blood smears was reduced in the FT-4202 group to 2.4 ± 0.3% vs. 5.9 ± 1.4% in the control group (p<0.02). The sickle RBC half-life increased to 1.8 ± 0.07 days in FT-4202 group vs. 1.4 ± 0.1 days in the control group, a 28% increase in RBC survival (p<0.01, Figure 1A). Hence, Hb levels in the FT-4202 group increased from 9.1 ± 0.2 g/dL before treatment, to 10.8 ± 0.3 g/dL 2 weeks after treatment (p<0.001), while Hb levels in the control group remained unchanged (Figure 1B). The reticulocytes remained unchanged in both groups before and after treatment. When sickle RBCs were de-oxygenated from an ambient pO2 of ~150 mmHg to a pO2 of 10-15 mmHg, followed by their re-oxygenation to ambient pO2 at a constant shear stress of 30 Pa (Oxygenscan), the point of sickling (PoS; pO2 level when the EI becomes 95% of the EI at ambient O2) decreased on average from 37% pO2 in the control group, to 30% pO2 in the FT-4202 group (p<0.002, Figure 1C), with a significantly improved Elongation Index at the point of minimum pO2 (EImin), (p<0.05). Next, RBC membrane deformability was measured under ambient pO2 (normoxic conditions), but varying shear stress after the de-oxygenation/re-oxygenation cycle on the Oxygenscan. Sickle RBCs from the FT-4202 group were significantly more deformable [i.e. had a higher Elongation Index (EI)] compared to control sickle RBCs (p<0.01, Figure 1D), as shear stress increased to ≥3 Pa, demonstrating that FT-4202 sickle RBCs sustained significantly less membrane damage following sickling and un-sickling. Conclusion: A 2-week oral FT-4202 administration was well tolerated by SCA mice and demonstrated beneficial biological effects: improved RBC membrane deformability and sickling parameters, with a shift in the PoS to lower pO2, and increased RBC survival and Hb levels. A parallel human phase-I study in healthy subjects and sickle cell disease patients to assess the safety and PK/PD of FT-4202 is ongoing (NCT03815695). Overall, our results suggest that FT-4202 can be a potentially useful orally available agent with significant anti-sickling effect. Disclosures Drake: Forma Therapeutics: Other: Shareholder of Forma Therapeutics. Fulzele:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Guichard:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics; AstraZeneca: Other: Shareholder. Malik:Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.

EXPERIMENTAL STUDY ON APPLICATION OF MARBLE WASTE AS CONVENTIONAL AGGREGATE FOR BASE COURSE MATERIALS

Recently, highway and construction industries utilize a substantial quantity of conventional aggregates. The increasing demands for conventional aggregate cause an increase in the cost of construction, reduction of natural resource, and continuing deterioration of the earth's surface. On the other hand, the waste generated from the marble industries during the process of cutting and polishing was increasing day by day all over the world. In Ethiopia, the use of Marble Waste Aggregate materials in road construction as a base course material was not well-known, and it was simply wasted at every place where the marble production was continuing. Therefore, this paper focused on evaluating the possibility of using marble waste aggregate as conventional aggregate in the base course layer of flexible pavements by experimental method. To achieve the objectives of the research, mechanical stabilization and laboratory tests have been carried out at different percentage replacement of Conventional Aggregate by (0%, 20%, 40%, 50%, 60%, 80%, and 100%) of Marble Waste Aggregate weights. Marble wastes materials are collected from Burayyu city, Alisha marble processing industry. The laboratory test results for MWA indicated SG, ACV, AIV, LAA, Flakiness Index , Elongation Index, Plastic Index, Water Absorption, and CBR of 2.74%, 23.63%, 26.21%, 27.03%, 26.48%, 13.29%, Non Plastic, 0.50% and 73.3%, respectively. These test results fulfilled the ERA standard specification for some tests, and it showed marginal quality values to the standard specification for GB2 and GB3 base course materials. CBR and gradation test results shown failure to meet the standard specification. Thus, mechanical stabilization was done to improve the mechanical and physical properties of Marble Waste Aggregate. Blending of 80%MWA with 20% CA results in SG, ACV, AIV, LAA, Flakiness Index, Elongation Index, Plastic Index, Water Absorption, and CBR of 2.83%, 18.2%, 21.52%, 22.58%, 23.79%, 16.29%, Non Plastic, 0.80%,and 82.5% respectively. At this proportion the gradation also observed to fit with the required ERA standard specification of GB2 and GB3 materials. Therefore, the use of marble waste aggregate up to 80% by weight is recommended for road base course layer, when it is found near to the construction site and in places where the materials are abundantly available.

Changes in the Morphological, Rheological, and Biochemical Blood Indicators in Triathletes

The aim of this study was to assess how the influence of intense physical effort changes the morphological, rheological, and biochemical blood indicators in triathletes. The study group comprised 10 triathletes aged 30-45 years, members of the Active Side of Life Association (Kraków, Poland). Venous blood was collected from the study participants twice, before and after the DiablakBeskid Extreme Triathlon 2016 (the Carpathians, Poland), and once from the control group for analysis of the selected blood indicators. Statistically significant changes were observed in the study group before and after the triathlon in morphological blood indicators, in the elongation index at the shear stress of 0.30 and 0.58 Pa, in levels of electrolytes, creatinine (mmol/l), serum protein parameters, and high-sensitivity troponin (ng/l). No such differences were reported for the remaining parameters. In turn, when comparing the study group before the triathlon with the control group, statistically significant differences were recorded in MCHC (g/dl), in the elongation index at the shear stress of 0.30 and 0.58 Pa, and Cl– (mmol/l) levels. No such differences were reported for the remaining parameters. Blood haematological and biochemical indicators in individuals that participate in triathlons characterize the actual range and direction of effort-related changes well and allow for the diagnosis of transient adaptive effects. Rheological parameters, involving the evaluation of erythrocyte deformability and aggregation, are useful for monitoring the particularly undesirable, short- and long-term effects of practicing extreme sports such as triathlons.