Effects of Poloxamer 188 on Red Blood Cells Membrane Properties in Sickle Cell Disease

Abstract Introduction: Sickle cell anemia (SCA) is a severe monogenic hereditary disorder characterized by chronic hemolytic anemia and the occurrence of frequent painful vaso-occlusive crises (VOC). SCA classical physiological scheme involves hemoglobin S polymerization under hypoxic conditions, which triggers red blood cells (RBCs) sickling. Recent studies demonstrated that the degree of hemorheological alterations, such as blood hyper-viscosity, determines the risk for VOC. Moreover, sickle RBCs abnormally adhere to the vascular endothelium, triggering microvascular occlusions. Despite extensive research very few drugs are available to efficiently treat VOCs or VOC-like events. A first clinical trial was performed to test the efficacy of poloxamer 188 (P188) in a large sickle cell cohort of adults and children (Orringer et al., 2001). This study demonstrated a significant reduction of pain duration in the children treated with P188. Recently, a new phase III multi-center trial has been started to test the efficacy of this drug during acute VOC in children (Humphries et al., 2015). We conducted in vitro experiments using the commercial formulation named Kolliphor P188 (Sigma-Aldrich) to test the effects of this drug on RBCs biophysical properties of SCA patients. Methods: To measure deformability and mechanical properties of RBCs, we used ektacytometry and microfluidic device mimicking the diameters of the micro vessels. RBCs adhesion assays were performed on HMEC-1 (Human Microvascular Endothelial Cell line) using dynamic flow adhesion platform. RBCs from healthy (AA) and SCA individuals were used for the different experiments. Results: While P188 did not significantly affect blood viscosity in AA, P188 treatment decreased blood viscosity at the lowest shear rates in SCA (Fig 1A). When measured in plasma, RBC aggregation decreased with P188 in SCA patients but not in AA (Fig 1B). RBC deformability assessed by both ektacytometry (not shown) and microfluidic device (Fig 2) was not affected by P188. This is in agreement with the mode of action of P188 suggesting that it binds to hydrophobic surfaces and lowers surface tension without any changes in the organization of the cytoskeleton. We examined the effect of P188 treatment on SCA-RBCs adhesion to the endothelial HMEC-1 cell line. We observed a mean adhesion of 40 cells/ mm2 for the untreated SCA-RBCs versus 20 cells/mm2 in the case of P188 treated RBCs, i.e. a 50% decrease upon P188 treatment (Fig 3). As for RBC aggregation, our findings suggest that the binding of P188 to SCA-RBCs membranes prevent the interaction with endothelial cells. This is of particular importance in the context of SCA since increased RBC adhesiveness has been demonstrated to trigger VOC. Conclusion: In parallel of the phase III clinical trial studying the profit of P188 for sickle cell patients during VOCs, our results bring clarifications regarding its mode of action on RBCs. We show that P188 directly reduces blood viscosity, RBC aggregation and adhesiveness to endothelial cells, making this drug as a potential beneficial therapy in SCA. References: Orringer, E.P., et al; (2001). JAMA, 286, 2099-2106. Humphries, J.D., et al; (2015). Trends Cell Biol, 25, 388-397. Figure 1. P188 treatment decreases blood viscosity (A) and RBC aggregation (B) in SS patients but not in AA controls. Figure 1. P188 treatment decreases blood viscosity (A) and RBC aggregation (B) in SS patients but not in AA controls. Figure 2. P188 treatment does not change SCA-RBC deformability. (A) Design of the microfluidic chip containing eight filtering units organized in parallel. Each filtering unit has a height of 5 µm and pillars are organized to allow an escape route of 20 µm around the unit to avoid occlusion. The surrounding pillars line has 5 µm slits. (B) Retention percentage of untreated and P188-treated SCA-RBC in 5 µm slits. Histograms represent mean of 5 µm slits from the 8 filtering units in one chip expressed in percent of total trapped RBCs for three patients. Figure 2. P188 treatment does not change SCA-RBC deformability. (A) Design of the microfluidic chip containing eight filtering units organized in parallel. Each filtering unit has a height of 5 µm and pillars are organized to allow an escape route of 20 µm around the unit to avoid occlusion. The surrounding pillars line has 5 µm slits. (B) Retention percentage of untreated and P188-treated SCA-RBC in 5 µm slits. Histograms represent mean of 5 µm slits from the 8 filtering units in one chip expressed in percent of total trapped RBCs for three patients. Figure 3. Graph representing adherent cells per mm2 at a flow rate of 1 dyne/mm2. The mean of the 5 patients is expressed as the average number of adherent cells/mm2 ± SD. Paired t test, P < 0.05. Figure 3. Graph representing adherent cells per mm2 at a flow rate of 1 dyne/mm2. The mean of the 5 patients is expressed as the average number of adherent cells/mm2 ± SD. Paired t test, P < 0.05. Disclosures No relevant conflicts of interest to declare.

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Variation of Red Blood Cell Rheology and Adhesiveness during Vaso-Occlusive Crises in Sickle Cell Patients

Blood ◽

10.1182/blood.v128.22.1311.1311 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1311-1311

Author(s):

Regine Hierso ◽

Claudine Lapoumeroulie ◽

Philippe Connes ◽

Sara El Hoss ◽

Catia Oliveira ◽

...

Keyword(s):

Emergency Department ◽

Steady State ◽

Blood Cell ◽

Sickle Cell ◽

Red Blood Cell ◽

Blood Viscosity ◽

Rheological Parameters ◽

Positive Correlation ◽

Rbc Aggregation ◽

Rbc Deformability

Abstract Introduction: Impaired red blood cell (RBC) rheology, increased RBC adhesiveness to the vascular wall, enhanced inflammation and blunted vascular reactivity are involved in the pathophysiology of sickle cell anemia (SCA). Painful vaso-occlusive crisis (VOC) is the most frequent complication encountered by SCA patients. While several studies compared several biomarkers of severity between patients at steady state and others during VOC, very few works compared the same patients in the two conditions. It is therefore difficult to know what happens during VOC. The present study was devoted to compare several hematological, biochemical, and rheological parameters, as well as RBC adhesiveness at steady state and during VOC. Altogether, 36 SCA patients were studied. Methods: This prospective monocentric study was performed at the University Hospital of Pointe-a-Pitre (Guadeloupe, French West Indies), in accordance with the guidelines set by the declaration of Helsinki and was approved by the Regional Ethics Committee (CPP Sud/Ouest Outre Mer III, Bordeaux, France, registration number: 2012-A00701-42). After admission to the emergency department for a VOC episode, patients were informed about the purpose and procedures of the study and gave their written consent. Blood was sampled at the arrival of the patients at the emergency department before they received any medications. A visit to the Sickle Cell Center was then scheduled at least 3 months after the emergency department admission to collect routine blood samples at steady state. Steady state condition was defined as a period free of blood transfusion in the previous three months and without any acute SCA complications in the previous two months. RBC deformability was determined at 3 and 30 Pa by ektacytometry (LORRCA, Mechatronics), RBC aggregation properties (RBC aggregation and RBC disaggregation threshold) by syllectometry (LORRCA), blood viscosity by cone-plate viscosimetry (Brookfield, DVII+ model with CPE 40 spindle) at 225 s-1. Irreversible sickle cells (ISCs) were measured on an Imagestream ISX MkII flow cytometer (Amnis Corp, EMD Millipore). Lu/BCAM, ICAM-4/Lw and the alpha4-beta1 integrin were measured by flow cytometry at the RBC surface (FACSCanto II, BD Biosciences). RBC adhesion to monolayers of transformed human bone marrow endothelial cells (TrHBMECs) was studied in continuous flow conditions in Vena8 Endothelial+ Biochips (Cellix Ltd). Other hematological and biochemical parameters were measured by standard techniques. Results: Compared to steady state values, white blood cell (9.2 [6.7-10.6] versus 12.3 [10.1-16.2] 109/L, p < 0.001) and C-Reactive Protein (3.7 [3.3-6.0] versus 7.1 [3.3-17.5] mg/L, p < 0.05) levels increased during VOC (table 1). Lactate dehydrogenase level slightly increased during VOC (418 [351-564] versus 437 [370-727] IU/L, p < 0.03) but no change was observed for hemoglobin. RBC deformability slightly decreased during VOC (0.34 [0.26-0.44]) compared to steady state (0.38 [0.31-0.46], p < 0.02). RBC aggregation increased during VOC (55 [46-60 %] compared to steady state (51 [46-54] %, p < 0.05). No difference was detected for blood viscosity, RBC surface proteins, RBC adhesion, and RBC disaggregation threshold between the two conditions. During VOC, the percentage of ISCs was inversely correlated with deformability (p < 0.002 and p < 0.006 at 3 and 30 Pa, respectively), but positively correlated with RBC disaggregation threshold (p < 0.002) and with RBC adhesion to TrHBMECs (p < 0.008) (figure 1). At steady state, it was significantly correlated only with the disaggregation threshold (positive correlation, p < 0.03). Discussion: The most striking observation of this study is the positive correlation between ISCs (rigid cells) and RBC adhesion properties during overt VOC. This is in contrast with the general observation that the most deformable RBCs are those exhibiting the strongest adhesiveness and possibly involved in VOC initiation. Strengthened RBC aggregates may also disturb the blood flow into the microcirculation, hence participating to VOC progression and sustention. Drugs targeting RBC deformability (ISCs) and RBC aggregates might be helpful during established VOC in SCA. Disclosures No relevant conflicts of interest to declare.

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Rheological Effects of L-Glutamine in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood-2019-128891 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3567-3567

Author(s):

Celeste K. Kanne ◽

Varun Reddy ◽

Vivien A. Sheehan

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Blood Viscosity ◽

Whole Blood ◽

Blood Cells ◽

Cell Disease ◽

Phase 3 ◽

Whole Blood Viscosity

Background: ENDARITM (oral pharmaceutical L-glutamine powder) received FDA approval in 2017 as a treatment for sickle cell disease (SCD). A pivotal phase 3 clinical study conducted by Emmaus Medical, Inc. showed that L-glutamine resulted in a lower incidence of vaso-occlusive crises (VOC) as well as a lower rate of hospitalizations and shorter hospital stays. No changes in standard clinical laboratory values were noted. The clinical improvements associated with sickle cell complications are believed to be due to an increase in the proportion of the reduced form of nicotinamide adenine dinucleotides in the red blood cells (RBC) of patients with SCD, reducing the oxidative stress. While the endpoints in the phase 3 study are clinically important, it is essential that we identify biomarkers or measurable laboratory changes that can serve as endpoints for future clinical trials assessing dose optimization and the efficacy and safety of L-glutamine in SCD individuals, including those with hepatic and renal dysfunction. RBC rheology is markedly abnormal in SCD; blood is more viscous for a given hematocrit than normal individuals, dense red blood cells (DRBC) are packed with HbS, potentiating sickling, and RBCs are less deformable than those of HbAA or HbAS individuals. High whole blood viscosity, high DRBCs, and poor RBC deformability are associated with higher rates of VOC. Given the demonstrated reduction in pain events, we hypothesized that L-glutamine might improve RBC rheology and sought to test this in vitro and in vivo using a battery of rheological tests. Methods: For the in vitro study, 6 mL of whole blood was drawn into an EDTA vacutainer from ten pediatric patients with sickle cell anemia (HbSS or HbSβ0) during routine clinical checkups under an IRB approved protocol. The cohort included 3 female and 7 male patients, ages 2-19 years old. All patients were on a steady dose of hydroxyurea and did not receive a transfusion within the 3 months prior to sample collection. A 200 mM stock solution of L-glutamine and water was mixed and filtered under light-protected conditions. Aliquots were stored at -20°C to avoid multiple freeze/thaw cycles. L-glutamine was added to 3 mL of whole blood for a final concentration of 1 mM (average in vivo L-glutamine plasma concentration in patients with SCD treated with L-glutamine); 3 mL of the same patient sample with water added served as a control. After a 24-hour incubation period at 4°C, whole blood viscosity was measured using a cone and plate viscometer at 37°C (DV3T Rheometer, AMETEK Brookfield, USA), %DRBCs were measured on an ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Inc., USA), and deformability measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca®) (RR Mechatronics, the Netherlands) with the Oxygenscan module. The Oxygenscan measures RBC deformability at normoxia (Elmax), deformability upon deoxygenation (EImin), and point of sickling (PoS), the oxygen tension at which deformability begins to decline, reflecting the patient-specific pO2 at which sickling begins. Paired samples (with and without added L-glutamine) were analyzed using Student's t-test. For the in vivo study, rheological tests were performed on peripheral blood from one patient (18-year-old male on hydroxyurea) at baseline and treated with L-glutamine as part of his routine clinical care. Results and conclusions: Addition of L-glutamine in vitro significantly reduced the PoS, meaning RBCs incubated with L-glutamine could tolerate a lower pO2 before sickling compared to the control. RBCs incubated with L-glutamine also had significantly higher EImin, meaning deoxygenated RBCs were more flexible and deformable. Whole blood viscosity at 45s-1 and 225s-1 did not change significantly following incubation with L-glutamine; %DRBCs also did not change significantly (Table 1). The in vivo patient sample tested exhibited a similar improvement in PoS and EImin (Figure 1). We therefore propose to further test the performance of the PoS and EImin as possible biomarkers of response to L-glutamine in vivo. If validated, these biomarkers may also help further elucidate the mechanisms of action of L-glutamine in SCD. Disclosures No relevant conflicts of interest to declare.

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Identification of Biomarkers That Are Associated with Clinical Complications of Hemoglobin SC Disease and Sickle Cell Anemia

Blood ◽

10.1182/blood-2021-145376 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 962-962

Author(s):

Minke A.E. Rab ◽

Celeste K. Kanne ◽

Mitchel C. Berrevoets ◽

Jennifer Bos ◽

Brigitte A. van Oirschot ◽

...

Keyword(s):

Board Of Directors ◽

Sickle Cell ◽

Blood Viscosity ◽

Whole Blood ◽

Blood Cells ◽

Research Funding ◽

Advisory Committees ◽

Whole Blood Viscosity ◽

Clinical Complications ◽

Hemoglobin Sc Disease

Abstract Introduction: Sickle cell disease (SCD) is an umbrella term used to describe inherited anemias that originate from a mutation in HBB, the gene coding for β-globin, that causes the formation of the abnormal hemoglobin S (HbS). The two most common genotypes are HbSS, termed sickle cell anemia (SCA) and HbSC, termed hemoglobin SC disease. Due to the higher prevalence and more severe clinical symptoms in SCA, attention is focused on this latter group of patients. Patients with HbSC disease who report significant SCD related symptoms are treated largely the same as SCA patients, despite differences in disease pathophysiology. Objective: To evaluate rheological differences in red blood cells (RBC) and whole blood from individuals with SCA and HbSC, and examine for associations with clinical complications. Methods: We analyzed an adult (n=30) and pediatric cohort (n=226) of SCA and HbSC individuals. Blood samples were evaluated for percent dense red blood cells (% DRBC), whole blood viscosity, and several ektacytometry-derived parameters, such as the cell membrane stability test, osmotic gradient ektacytometry and Point of Sickling (PoS): an oxygen gradient ektacytometry-derived biomarker that depicts the oxygen tension at which sickling is initiated. Subsequently, we assessed in the pediatric cohort (189 SCA and 37 HbSC individuals) if PoS, dense RBCs or blood viscosity are associated with clinical complications. Results: In particular (micro)dense RBCs (Figure 1A and B), blood viscosity (Figure 1C) and point of sickling (PoS, Figure 1D-F), are notably different in HbSC disease compared to SCA. In SCA, PoS was associated with occurrence of acute chest syndrome (ACS, for every 10mmHg increase OR 1.84, p<0.0001, adjusted OR 1.67, p=0.002), but no such association was found in children with HbSC. In contrast, we found an association of blood viscosity and ACS (OR 9.3, p=0.012; adjusted OR 13.5, p=0.015) in HbSC children, while we found no such association in SCA. No significant association of micro dense RBCs was found in SCA or HbSC children. Conclusion: We found that associations between PoS and ACS found in SCA were not found in HbSC. Instead, in HbSC ACS was associated with higher whole blood viscosity. We therefore conclude that sickling is a key factor in the pathophysiology of SCA, whereas in HbSC disease blood viscosity might play a crucial role in development of certain complications like ACS. This study suggests that complications in SCA and HbSC disease are driven by different aspects of blood abnormalities, and may warrant a distinct treatment approach. Figure 1 Figure 1. Disclosures Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Schutgens: Shire/Takeda: Research Funding; Pfizer: Research Funding; OctaPharma: Research Funding; Novo Nordisk: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding. Wijk: Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Beers: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; RR Mechatronics: Research Funding; Pfizer: Research Funding. Sheehan: Beam Therapeutics: Research Funding; Novartis: Research Funding; Forma Therapeutics: Research Funding.

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The Effect of Rigid Cells on Blood Viscosity: Linking Rheology and Sickle Cell Anemia

Soft Matter ◽

10.1039/d1sm01299a ◽

2022 ◽

Author(s):

Antonio Perazzo ◽

Zhangli Peng ◽

Yuan-Nan Young ◽

Zhe Feng ◽

David Wood ◽

...

Keyword(s):

Red Blood Cells ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Blood Viscosity ◽

Blood Cells ◽

Deformable Objects ◽

Blood Capillaries

ickle cell anemia (SCA) is a disease that affects red blood cells (RBCs). Healthy RBCs are highly deformable objects that under flow can penetrate blood capillaries smaller than their typical...

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TCD in Infants: A Report from the BABY HUG Trial.

Blood ◽

10.1182/blood.v106.11.952.952 ◽

2005 ◽

Vol 106 (11) ◽

pp. 952-952 ◽

Cited By ~ 2

Author(s):

Robert J. Adams ◽

Julio Barredo ◽

Duane R. Bonds ◽

Clark Brown ◽

James Casella ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Maximum Flow ◽

Organ Damage ◽

Sample Volume ◽

Phase Iii ◽

Mean Velocity ◽

Increased Risk ◽

The Mean ◽

The Right

Abstract Transcranial Doppler (TCD) is useful in children with sickle cell anemia (SCA) to detect increased risk of stroke. The use of TCD in infants less than 2 years of age is less well established, but has previously been shown to be feasible. As a secondary endpoint in the BABY HUG Trial, TCD is expected to provide useful information on the possible effects of hydroxyurea (HU) in babies with SCA. BABY HUG is an NHLBI-NICHD sponsored phase III clinical trial to compare hydroxyurea to placebo to ascertain effectiveness in preventing end organ damage of the spleen and kidney. Eligible subjects underwent a baseline TCD using the Nicolet Companion (EME) 2-MHz pulsed Doppler. All infants were 8–18 months of age at enrollment, had no history of stroke and were not receiving chronic blood transfusions. Blood flow velocities were recorded using the Stroke Prevention Trial in Sickle Cell Anemia (STOP) protocol with the exception of reducing the standard sample volume to 4 mm. No sedation was used. The time averaged maximum mean was measured to determine the highest velocity on either side to categorize the study as normal (all recordings <170 cm/sec), conditional (170–199cm/sec) or abnormal (≥200 cm/sec). Recordings of the middle cerebral artery (MCA) and internal carotid artery (ICA) bifurcation defined an adequate TCD. TCD was not required for study entry but subjects with an abnormal exam were not eligible for randomization and treatment. TCD exams were read by blinded reviewers at the Medical College of Georgia. TCD results were transmitted to Clinical Trials & Surveys Corporation (C-TASC) for statistical analysis. As of June 24, 2005, 70 TCD exams had been attempted. Two exams were unsuccessful (no data) because of the children’s irritability and 1 was interpreted as inadequate. Of the remaining 67 TCD exams, 66 were normal and one baby had a high conditional TCD (190 cm/sec). No subjects were found ineligible for the study due to TCD results. The mean velocity of the left MCA was 117 cm/sec ±22.9 and that of the right MCA was 114 cm/sec ±24.9. Regression analyses were performed to examine the relationship of maximum flow velocity (VMAX) to age and total hemoglobin (Hb). VMAX was inversely correlated to Hb (left p=<0.0001, right=<0.0014) and directly associated with age (left p=<.0005, right p=<0.0022). When the mean MCA velocity was regressed against age and Hb, both age (p=0.0285) and Hb (p=.0024) were significant. Adequate baseline TCD evaluation was obtained on 67 out of 70 babies. As expected, baseline TCD velocities varied inversely with the degree of anemia and directly with age; all but one was normal by childhood sickle cell disease standards. These studies provide valuable normative data for infants with SCA, and for further assessment of the effect of HU on TCD in infants with SCA as the study progresses.

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Abnormal Red Cell Deformability and Aggregation in Sickle Cell Trait

Blood ◽

10.1182/blood.v120.21.1001.1001 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1001-1001

Author(s):

Jon Detterich ◽

Adam M Bush ◽

Roberta Miyeko Kato ◽

Rose Wenby ◽

Thomas D. Coates ◽

...

Keyword(s):

Blood Flow ◽

Blood Viscosity ◽

Whole Blood ◽

Red Cell ◽

Cell Deformability ◽

Autologous Plasma ◽

Red Cell Deformability ◽

Whole Blood Viscosity ◽

Rbc Aggregation ◽

Rbc Deformability

Abstract Abstract 1001 Introduction: SCT occurs in 8% of African Americans and is not commonly associated with clinical disease. Nonetheless, the United States Armed Forces has reported that SCT conveys a 30-fold risk of sudden cardiac arrest and a 200-fold risk from exertional rhabdomyolysis. In fact, rhabdomyolysis in athletes with SCT has been the principal cause of death in NCAA football players in the last decade, leading to recently mandated SCT testing in all Division-1 players. In SCT, RBC sickle only under extreme conditions and with slow kinetics. Therefore, rhabdomyolysis most likely occurs in SCT when a “perfect storm” of factors converges to critically imbalance oxygen supply and demand in muscles. We hypothesize that in SCT subjects, abnormal RBC rheology, particularly aggregation and deformability, play an important role in abnormal muscle blood flow supply and distribution to exercising muscle. To test this hypothesis, we examined whole blood viscosity, RBC aggregation, and RBC deformability in 11 SCT and 10 control subjects prior to and following maximum handgrip exercise. Methods: Maximum voluntary contraction (MVC) was assessed by handgrip dynamometer in the dominant arm. Baseline blood was collected for CBC, whole blood viscosity, RBC aggregation, and RBC deformability. Patients then maintained 60% MVC exercise until exhaustion. Following 8 minutes of recovery, a venous blood gas and blood for repeat viscosity assessments was collected from the antecubital fossa of the exercising limb. Whole blood viscosity over a shear rate range of 1–1, 000 1/s was determined by an automated tube viscometer, RBC deformability from 0.5–50 Pa via laser ektacytometry (LORCA) and RBC aggregation in both autologous plasma and 3% dextran 70 kDa using an automated cone-place aggregometer (Myrenne). Aggregation measurements included extent at stasis (M), strength of aggregation (GT min) and kinetics (T ½). Results: Baseline CBC and aggregation values are summarized in Table 1. Both static RBC aggregation in plasma and RBC aggregation in dextran (aggregability) were significantly increased in SCT (Table 1). The rate of aggregation formation trended higher in SCT but the strength of aggregation was not different between the two groups. In SCT subjects, red cell deformability was impaired at low shear stress but greater than controls at higher shear stress (Figure 1). Red cell deformability was completely independent of oxygenation status states in both SCT and control subjects. Whole blood viscosity did not different between the two groups whether oxygenated or deoxygenated and prior to or following handgrip exercise. Discussion: Three important hemorheological differences were observed for SCT subjects versus controls: a) RBC deformability was below control at low stress levels yet greater than control at higher stress; b) The extent of RBC aggregation in autologous plasma was about 40% greater; c) The extent of RBC aggregation for washed RBC re-suspended in an aggregating medium (i.e., 3% dextran 70 kDa) was about 30% higher. RBC deformability is a major determinant of in vivo blood flow dynamics, especially in the microcirculation; decreased deformability adversely affects tissue perfusion. RBC aggregation is also an important determinant since it affects both resistance to blood flow and RBC distribution in a vascular bed (e.g., plasma skimming). The finding of greater aggregability (i.e., higher aggregation in the defined dextran medium) indicates that RBC in SCT have an altered membrane surface in which the penetration of this polymer into the glycocalyx is abnormal. The combined effects of these three rheological parameters is likely to impair in vivo blood flow in SCT, perhaps to a degree resulting in pathophysiological changes of the cardiovascular system. Disclosures: Coates: Novartis: Speakers Bureau; Apopharma: Consultancy. Wood:Ferrokin Biosciences: Consultancy; Shire: Consultancy; Apotex: Consultancy, Honoraria; Novartis: Honoraria, Research Funding.

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Haemorheology of the eastern grey kangaroo and the Tasmanian devil

Australian Journal of Zoology ◽

10.1071/zo10083 ◽

2011 ◽

Vol 59 (1) ◽

pp. 26 ◽

Cited By ~ 2

Author(s):

Michael J. Simmonds ◽

Oguz K. Baskurt ◽

Herbert J. Meiselman ◽

Michael Pyne ◽

Michael Kakanis ◽

...

Keyword(s):

Surface Charge ◽

Carrying Capacity ◽

Blood Viscosity ◽

Haemoglobin Concentration ◽

Scanning Electron Micrographs ◽

Tasmanian Devil ◽

Grey Kangaroo ◽

Rbc Aggregation ◽

Oxygen Carrying Capacity ◽

Rbc Deformability

The blood of two Australian marsupials, the eastern grey kangaroo (Macropus giganteus) and the Tasmanian devil (Sarcophilus harrisii), has been reported to have greater oxygen-carrying capacity (i.e. haemoglobin content) when compared with that of placental mammals. We investigated whether alterations of blood rheological properties are associated with the increased oxygen-carrying capacity of these marsupials. Eastern grey kangaroos (n = 6) and Tasmanian devils (n = 4) were anaesthetised for blood sampling; human blood (n = 6) was also sampled for comparison. Laboratory measurements included blood and plasma viscosity, red blood cell (RBC) deformability, RBC aggregation and the intrinsic tendency of RBC to aggregate, RBC surface charge and haematological parameters. Scanning electron micrographs of RBC from each species provided morphological information. High-shear blood viscosity at native haematocrit was highest for the Tasmanian devil. When haematocrit was adjusted to 0.4 L L–1, lower-shear blood viscosity was highest for the eastern grey kangaroo. RBC deformability was greatly reduced for the Tasmanian devil. Eastern grey kangaroo blood had the highest RBC aggregation, whereas Tasmanian devil RBC did not aggregate. The surface charge of RBC for marsupials was ~15% lower than that of humans. The dependence of oxygen-delivery effectiveness on haemoglobin concentration (i.e. oxygen content) and blood viscosity was quantitated by calculating the haematocrit to blood viscosity ratio and was 15–25% lower for marsupials compared with humans. Our results suggest that environmental pressures since the marsupial–monotreme divergence have influenced the development of vastly different strategies to maintain a match between oxygen demand and delivery.

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In Vitro Red Blood Cell Segregation in Sickle Cell Anemia

Frontiers in Physics ◽

10.3389/fphy.2021.737739 ◽

2021 ◽

Vol 9 ◽

Author(s):

Viviana Clavería ◽

Philippe Connes ◽

Luca Lanotte ◽

Céline Renoux ◽

Philippe Joly ◽

...

Keyword(s):

Red Blood Cells ◽

Flow Velocity ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Blood Cells ◽

Vascular Wall ◽

Mean Flow ◽

The Mean ◽

The Impact ◽

Mean Flow Velocity

Red blood cells in sickle cell anemia (sRBC) are more heterogeneous in their physical properties than healthy red blood cells, spanning adhesiveness, rigidity, density, size, and shape. sRBC with increased adhesiveness to the vascular wall would trigger vaso-occlusive like complications, a hallmark of sickle cell anemia. We investigated whether segregation occurs among sRBC flowing in micron-sized channels and tested the impact of aggregation on segregation. Two populations of sRBC of different densities were separated, labeled, and mixed again. The mixed suspension was flowed within glass capillary tubes at different pressure-drops, hematocrit, and suspending media that promoted or not cell aggregation. Observations were made at a fixed channel position. The mean flow velocity was obtained by using the cells as tracking particles, and the cell depleted layer (CDL) by measuring the distance from the cell core border to the channel wall. The labeled sRBC were identified by stopping the flow and scanning the cells within the channel section. The tube hematocrit was estimated from the number of fluorescence cells identified in the field of view. In non-aggregating media, our results showed a heterogeneous distribution of sRBC according to their density: low-density sRBC population remained closer to the center of the channel, while the densest cells segregated towards the walls. There was no impact of the mean flow velocity and little impact of hematocrit. This segregation heterogeneity could influence the ability of sRBC to adhere to the vascular wall and slow down blood flow. However, promoting aggregation inhibited segregation while CDL thickness was enhanced by aggregation, highlighting a potential protective role against vaso-occlusion in patients with sickle cell anemia.

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Characteristics of Children with Abnormal TCD in the Modern Era: Results from the Displace Consortium

Blood ◽

10.1182/blood-2019-129951 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2270-2270

Author(s):

Julie Kanter ◽

Mary Dooley ◽

Logan P Sirline ◽

Martina Mueller ◽

Shannon Phillips ◽

...

Keyword(s):

Board Of Directors ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Stroke Prevention ◽

High Risk Group ◽

Phase Iii ◽

Dissemination And Implementation ◽

Advisory Committees ◽

Transfusion Therapy ◽

The Mean

Background: Stroke is one of the most devastating complications of sickle cell anemia (SCA). In 1998, the Stroke Prevention Trial in Sickle Cell Anemia (STOP), demonstrated that a high-risk group of children with SCA could be identified using Transcranial Doppler ultrasound(TCD) and that chronic red cell transfusion therapy (CRCT) could reduce the risk of first ischemic stroke in this group by over 90% (Adams, et al NEJM 1998).At STOP studyenrollment, 9.7% of children with SCA were identified as having an abnormal TCD. Baby HUG, (NCT00006400), an NHLBI supported phase III trial showed that severe anemia in SCA was associated with elevated white blood cell (WBC) and higher TCD velocities (Lebensburger, et al Blood 2010 ). It is unclear if lower hemoglobin (Hb) and/or higher WBC are causative of elevated TCD velocities or correlative biomarkers. As new disease therapies become available, it is important to know the current rate of abnormal TCD and characteristics of those patients. The DISPLACE (Dissemination and Implementation Looking at the Care Environment) project is a multicenter, NHLBI-funded study whose primary purpose is to identify barriers to implementation of stroke screening in SCA and test novel methods for improving outcomes. DISPLACE is a 3-part study: retrospective assessment of current practice, qualitative review of barriers and facilitators to screening and a cluster-randomized intervention implementation project to improve stroke screening. Part 1 of the study showedthat TCD screening rates varied widely among institutions ranging from 30-75.2% (mean 48.4%, median 47%). We are now reporting on the rate of abnormal TCD and the characteristics and outcomes of patients with abnormal TCD. Methods: DISPLACE is a consortium of 28 US centers. Each site performed a rigorous retrospective chart review of children with SCA aged 2-16 years from 2012-2016. To be eligible for inclusion, children must have been seen at their institution at least 2x during the study period and have confirmation of SCA. A custom electronic data capture (EDC) system facilitated entry of de-identified data including demographics, TCD and MRI results, medications, transfusions, and laboratory values. For children with SCA who had TCD or central nervous system imaging prior to 2012, these results were also entered into the EDC. TCD results were recorded in the EDC as normal, conditional or abnormal based on their institutional interpretation. Labs and vitals were entered for each patient in closest proximity to each TCD. Confirmation and adjudication of each abnormal TCD and associated outcomes were performed. Stroke status was also recorded as well as presence or absence of CRCT. Results: In total, 5247 children with SCA are included in the database of whom 5225 should have received a TCD. Of this cohort, 4210 children (80.6%) had at least one TCD recorded in the database. Within this group, 207 (4.9%) of children had an abnormal TCD and 816 (19.4%) had a conditional TCD. For those children who underwent TCD during the study (2012-2016) period, there were 105 (2.9%) abnormal TCD and 501 (13.6%) conditional TCD (Table 1). The mean age of children at the time of abnormal TCD was 6.6 years (range 2-16 yr). The majority of children were <10 yr at first abnormal TCD. Over 30% of patients with an abnormal TCD were identified as receiving hydroxyurea.The mean Hb associated with an abnormal TCD was 7.8 +/- 1.1g/dl (range 5.7-10.6) and the mean WBC was 13 x109/L+/- 3.6 (range 3.9-23.4) (Table 2). Of the 105 patients with abn TCD during the study period, 18% had a stroke. Of the total 5247 patients in the database, 3093 (59%) had been prescribed hydroxyurea (HU) and 999 (19%) were prescribed CRCT. CRCT was prescribed most often for abnormal TCD (37%) or secondary stroke prevention (31%). Discussion: DISPLACE is the largest contemporary cohort of children with SCA. The incidence of abnormal TCD in the DISPLACE cohort is significantly lower than at randomization in the STOP study. The number of children receiving CRCT is higher than expected which may partly account for the decrease in frequency of abnormal TCD. Many patients with abnormal TCD were receiving HU when their TCD was abnormal and were started on CRCT. Additionally, while the outcomes of children with conditional TCD are still being evaluated, many of those children reverted to normal TCD without intervention. These data may also help us redefine the use and interventions needed for abnormal TCD. Disclosures Kanter: NHLBI: Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy; SCDAA: Membership on an entity's Board of Directors or advisory committees; Guidepoint Global: Consultancy; GLG: Consultancy; Sangamo: Consultancy, Honoraria; Modus: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Imara: Consultancy; Cowen: Consultancy; Jeffries: Consultancy; Medscape: Honoraria; Rockpointe: Honoraria; Peerview: Honoraria. Adams:GBT: Consultancy, Other: consultancy to companies GBT and Blueburd Bio; Bluebird: Consultancy.

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GTx011, a Novel Agent That Improves Rheological Properties Of Sickle Cell Blood By Increasing Oxygen Affinity For Hemoglobin

Blood ◽

10.1182/blood.v122.21.2207.2207 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2207-2207 ◽

Cited By ~ 1

Author(s):

Mira Patel ◽

Donna Oksenberg ◽

Abel Silva ◽

Andreas Betz ◽

Brian Metcalf ◽

...

Keyword(s):

Red Blood Cells ◽

Rheological Properties ◽

Sickle Cell ◽

Blood Viscosity ◽

Whole Blood ◽

Blood Cells ◽

Plasma Proteins ◽

Oxygen Affinity ◽

Employment Equity ◽

Equity Ownership

Abstract Sickle cell disorder (SCD) is characterized by the presence of non-deformable red blood cells. Literature reports indicate that the T-state structure of hemoglobin (Hb) (highly correlated with the deoxy form) is responsible for the formation of HbS polymers that lead to rigid cells. We hypothesized that the likelihood of polymer formation will be reduced if sufficient HbS remains in the R-state (oxy) conformation. We have designed and synthesized a novel series of compounds that increase the O2 affinity of HbS and improve the rheological properties of SCD blood. In a novel 96-well format oxygen dissociation assay (ODA), compounds including GTx006, GTx007 and GTx011 were all more potent than 5-hydroxy furfural (5HMF), an agent being tested in clinical trials in SCD patients. After two hours of passive deoxygenation, GTx011, at an equimolar concentration to Hb, increases the O2 affinity by six-fold and drastically delays polymerization of HbS. Even at substoichiometric concentrations (GTx011:Hb= 1:3) GTx011 elicits a two-fold improvement in O2 affinity for Hb that translated to 16% more oxy-Hb relative to control (Table 1). We then analyzed the agents in a TCS Hemox analyzer using purified Hb at 25µM. At a GTx011:Hb ratio of 1:3 the oxygen affinity was improved by 15%, while at stoichiometric concentrations, the oxygen affinity was increased by 70% compared to Hb control. These biochemical assays indicated that the GTx agents were altering Hb O2 affinity and should therefore assist in maintaining the oxy conformation of Hb and prevent the formation of polymers.Table 1AssayHb in ODAWashed RBC OECWhole Blood OECViscosityunit(Δoxy state)(%Δp50)(%Δp50)(ΔcP)[Hb]3 µM1 mM1 mM1.5 mM[cmp]1 µM3 µM1 mM3 mM1 mM3 mM1.6 mM8 mM5-HMF<1<1306510470.042.4GTx006210597849711.7>2.5GTx007623697863>802.1>2.5GTx0111656768380>802.5>2.5 To test the agents in a more physiological system, oxygen equilibrium curves (OECs) were measured in washed red blood cells (RBCs) and in whole blood at 20% hematocrit (∼1 mM Hb). In washed RBCs, 5HMF, GTx006, GTx007 and GTx011 at a concentration of 1mM produced partial O2 pressures (p50) of 20, 12, 9 and 7 mm Hg, respectively (control RBCs = 30 mm Hg). To determine the effects of plasma proteins, OECs were measured in whole blood from SCD patients, giving p50s of 27, 18, 11 and 6 mm Hg compared to the control blood p50 of 30 mm Hg (agent concentration of 1 mM). Table 1 shows that 5HMF and GTx006 activities were affected by the presence of plasma proteins but GTx007 and GTx011 activities were not altered. SCD patients develop anemia due to hemolysis, which partially compensates for an increase in blood viscosity caused by the non-deformable RBCs (ssRBCs). We monitored the effect of our agents on SCD patient blood rheology, ex vivo, to determine if they were capable of decreasing the viscosity of SS blood under hypoxic conditions. We incubated whole blood from SCD patients (30% hematocrit, ∼1.5 mM Hb) with 5HMF, GTx006, GTx007 or GTx011 for 30 mins and then subjected the blood to 2 hours of hypoxia (2.4% O2). Blood viscosity was then measured in a cone-plate viscometer at shear rates ranging from 60 s-1 to 415 s-1. Of the four compounds, GTx011 showed the most pronounced improvement in rheologic measures (see Table 1), changing the viscosity from 6.46 cP (no GTx011) to 4.00 cP (equimolar GTx011). Normoxic SCD blood had a viscosity of 4.28 cP. A similar improvement in blood viscosity under physiologic conditions may be predicted to decrease the residence time of ssRBCs in hypoxic tissue, and allow for a lower level of polymerization in individual red blood cells. In addition, GTx011 has also been shown to delay polymerization and delay sickling. Thus, GTx011 has the potential to elicit a decrease in HbS polymer concentration, reducing the likelihood of forming the rigid cells that cause vaso-occlusion in SCD patients. Disclosures: Patel: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Silva:Global Blood Therapeutics: Employment, Equity Ownership. Betz:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Employment, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.

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