Red Cell Rheology Research Articles

Red Cell Rheology and Blood Viscosity in Pediatric Individuals Having Received Allogenic Hematopoietic Stem Cell Transplantation or Ex Vivo Autologous Gene Therapy for Sickle Cell Disease

Background: Allogenic hematopoietic stem cell transplantation (HCT) and ex vivo autologous gene therapies (GT) are potentially curative treatments for sickle cell disease (SCD). However, there is debate around how to define cure including what degree of donor chimerism or level of functional hemoglobin constitutes a cure. Information on red cell function post-HCT/GT and how it relates to donor chimerism and resolution of SCD complications is scarce. Red cell function tests are not currently part of follow-up care post-HCT but may provide helpful information on the degree of red cell correction and risk of SCD complications. To address this knowledge gap, we report rheology and whole blood viscosity data on HbSS patients post-HCT or GT. Methods: Blood samples were collected under an IRB-approved protocol at Baylor College of Medicine and Emory University School of Medicine. We analyzed elongation index maximum and minimum (EImax, EImin), point of sickling (PoS), hematocrit-viscosity ratio at shear rates of 45 and 225 s-1(HVR45 & HVR225), and dense red blood cell % (DRBC%). EImax, EImin, and PoS were measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics, The Netherlands). Chimerism was measured by short tandem repeat testing (STR) after cell density sorting and DRBC% was measured on ADVIA cell counter (Siemens, Germany). HCT patients were compared with controls matched to the donor's genotype, and patients post-GT were compared with HbAS controls (HCT: HbAA donor =8, HbAS donor=19, GT=2, HbAA controls=42, and HbAS controls=15). Stata 18.0 (College Station, Texas, USA) was used to perform statistical analyses. Median values and ranges were used to describe the data. Wilcoxon rank sum test was used to compare groups and a mixed model was for the longitudinal data. A p-value <0.05 was considered statistically significant. Results: There were 29 patients with a median age of 6.6 years (range: 2.0-16.3) at HCT/GT and a median follow-up of 2.4 years (range: 0.1-7). A total of forty-three samples were analyzed. Median donor myeloid chimerism was 94% (33-100). Overall, the HbS% ranged from 28.9 - 45.2% with HbAS donors and 0% with HbAA donors. Two HbAS HCT patients had myeloid chimerism ≤ 50% with a follow-up period of 3.3 and 1.9 years, respectively. The myeloid donor% and HbS% were 33% and 50%, and 45.2% and 41.6%, respectively. At the first post-HCT/GT time-point (median 1 year, range: 0.1-6.9), DRBC%, EImax, and EImin were lower in HbAA donor HCT patients compared to HbAA controls (0.593 vs 0.604, p=0.008; 0.581 vs 0.604, <0.001; and 0.75 vs 0.20, p=0.02, respectively). RBC function improved with a longer follow-up from HCT/GT (median 2.9 years, range 1.3-7) with EImax showing a trend of statistical significance (p=0.07). Patients with HbAS donors had significantly higher point of sickling (PoS) values compared to the patients with HbAA donors (p=0.02). There was a trend for a lower HVR225 in patients with HbAS donors compared to the patients with HbAA donors (p=0.08). Seven patients (24%) had one or more red cell function test values outside the genotype-matched control range despite having myeloid donor chimerism of > 25-30% - a value commonly used in clinical practice to define cure (Table & Figure). Conclusion: A substantial number of patients had abnormal red cell function tests following HCT/GT despite achieving donor engraftment. Donor myeloid chimerism ≥ 25% has been reported protective from SCD complications while a chimerism ≥50% normalized tests for hemolysis. Higher odds of SCD-related complications have been reported with higher PoS and lower EImax/EImin. Thus, we propose that red cell function should be included in follow-up care of HCT/GT patients until values normalize and clinical relevance is further assessed.

Pluronic F-68 Reduces the Endothelial Adherence and Improves the Rheology of Liganded Sickle Erythrocytes

A perfluorochemical blood substitute emulsified with Pluronic F-68 has been shown to improve the filterability of deoxygenated sickle red cells. We questioned whether some of the effect was independent of oxygen loading and studied the influence of Fluosol DA (Green Cross, Osaka, Japan) and Pluronic on the rheology and adhesion of sickle red cells saturated with oxygen and carbon monoxide. A 5-vol/vol% concentration of Fluosol or equivalent concentration of Pluronic was equally effective at improving the filtration of washed sickle cells through 5-micron-diameter pores at wall shear stresses approximating 1,000 dyne/cm2. The same concentration of Pluronic reduced the extensional static rigidity of irreversibly sickled cells (ISC) by 25% and also abolished the adherence of gravity-sedimented sickle cells to endothelial monolayers in the presence of saline or plasma. The inhibition of adherence was not reversible by washing and was accomplished with equal ease by isolated treatment of sickle cells or endothelium. Pluronic had no effect on the rheology or adhesion of normal adult red cells. Neither Fluosol nor Pluronic changed sickle or normal cell shape, mean cell volume, mean cell density, or cell density distribution. A lubricating effect of Pluronic on cell surfaces could explain all of the rheological observations and offers another direction of inquiry in the search for therapy for sickle cell disease.

Lethal and Sub-Lethal Irradiation of Sprague-Dawley Rats Causes Development of Dense Red Blood Cells

Background: In vitro irradiation of blood produces morphologic and rheologic changes in the red blood cells. These changes may have clinicla significance, and if the changes are prolonged beyond hematologic recovery, serve as an indication of radiation exposure. However, no rheological studies have been performed on blood from irradiated animals.Objective: Determine the effect of lethal and sub lethal radiation treatment (RT) on whole blood rheology in a rodent model.Methods: 3 SD rats were subjected to lethal doses of irradiation (7.5 Gy), and 3 untreated; ten days following RT, the animals were sacrificed, and their blood analyzed by an ADVIA hematology analyzer, a cone and plate viscometer, and a RheoScan ektacytometer.Next, following baseline CBC and dense cell measurements, 6 rats were subjected to whole body RT at 6 Gy; 6 were subjected to 7.5 Gy with one leg shielded, and 2 were not irradiated. Percent dense red blood cells (%DRBC), CBC, reticulocyte count, and red cell deformability were measured 4 hours, 5 days and then every 10 days post RT.11 week old Sprague Dawley (SD) rats were used in all experiments. Peripheral blood smears were prepared and analyzed from each blood draw to assess RBC morphology.Results: SD rats who received lethal irradiation at 7.5 Gy exhibited a marked rise in %DRBC; 17.1% ±10.5% compared to 0.5%±0.3% in untreated SD rats (p=0.025). The hematocrit to viscosity ratio (HVR), a measure of oxygen carrying capacity, was calculated at two shear rates, 45 s-1 and 225 s-1, to mimic venous and arterial circulation. There was no significant difference in HVR at 45 s-1, but the HVR at 225s-1 was significantly lower in the RT group, p=0.018, suggesting impairment of oxygen carrying capacity due to intrinsic red cell defects caused by lethal RT. Deformability was reduced in the RT group as well (p=0.039).In longitudinally followed SD rats subjected to sub lethal RT, %DRBC rose 4 hours after irradiation, peaked at 5 days post-irradiation, and remained elevated 31 days after RT. At 31 days post-RT, hematologic recovery was observed in both the sub lethal and shielded groups, but %DRBC remained elevated. RBC morphology revealed anisocytosis in the RT groups, but few spherocytes were noted.Conclusions: In vivo RT impairs red cell rheology in SD rats; lethal doses cause a marked rise in %DRBC, reduction in red cell deformability, and reduction in oxygen carrying capacity as measured by HVR. Longitudinal examination of sub lethal RT reveals a rapid rise in %DRBC, without spherocytosis, suggesting that this change may be due to a KCl channel leak rather than spherocytosis from splenic fibrosis. Obese SD rats have higher %DRBC at baseline, and show a significantly higher increase in %DRBC in response to RT, compared to normal weight SD rats. This suggests that obesity may exacerbate the rheological complications of RT.The persistence of %DRBC after hematologic recovery is a significant observation, suggesting that radiation exposure after the window of hematologic recovery has passed could be detected by a point of care test for %DRBC, already available, as a rapid field test. DisclosuresNo relevant conflicts of interest to declare.

Rheological Impact of GBT1118 Cessation in a Sickle Mouse Model

Background: In sickle cell disease (SCD), elevated blood viscosity is a risk factor for vaso-occlusive crisis (VOC). Blood viscosity is influenced by hemoglobin (Hb) levels, red blood cell (RBC) deformability, density, and aggregation. Voxelotor is an HbS polymerization inhibitor that increases the Hb affinity for oxygen and reduces sickling. In human subjects, voxelotor significantly increases Hb levels. A rise in Hb due to voxelotor does not increase blood viscosity the way a similar increase in unmodified HbS would. However, voxelotor transiently modifies HbS, raising concern that if voxelotor was stopped suddenly, the drug induced extra HbS would revert to its unmodified state, causing a significant rise in blood viscosity and increasing the risk of VOC. This effect could continue until the drug induced Hb is eliminated by hemolysis. To investigate this, we treated a sickle mouse model with GBT1118, a voxelotor analog. We then stopped the treatment and determined the effect on whole blood viscosity, the hematocrit-to-viscosity ratio (HVR), a measure of oxygen carrying capacity, and RBC deformability. As GBT1118 has a half-life of 14 hours in SCD mouse, we measured the post-drug effect 48 hours after drug withdrawal. Methods: Ten Townes HbSS mice were fed control chow, 10 were fed chow containing GBT1118 for 7 days, 10 were fed GBT1118 chow for 7 days, and then returned to control chow for 48 hours. RBC deformability was measured by oxygen gradient ektacytometry (Lorrca with Oxygenscan, RR Mechatronics). Blood viscosity was measured with a cone and plate viscometer (Brookfield DVII+). Hb was measured via ADVIA (Siemens). Results: Treatment with GBT1118 significantly increased Hb without increasing blood viscosity. HVR was increased at both 45 s-1 and 225 s-1 shear rates (Figure 1). GBT1118 treatment also improved RBC deformability under both normoxic (EImax) and hypoxic conditions (EImin). Point of sickling (PoS) was reduced, indicating that treated RBC tolerated lower oxygen concentrations before sickling (Figure 2). When the drug was withdrawn for 48 hours, compared to control animals, 1) Hb declined rapidly (7.1 g/dL in control feed, 13.1 g/dL in treated mice compared to 9.0 g/dL after 48 hours off treatment, P<0.001); 2) viscosity did not increase compared to control feed (at shear rate 45 s-1, 12.8 cP after treatment compared to 11.4 cP in control cohort; at shear rate 225 s-1,10.8 cP after treatment compared to 10.3 cP in controls); 3) HVR returned to baseline (at shear rate 45 s-1, 4.5 after treatment compared to 3.6 in controls; at shear rate 225 s-1, 6.6 after treatment compared to 4 cP in controls), 4) EImin remained elevated (0.16 in controls compared to 0.32 after 48 hours off treatment, P<0.001); 5) Elmax and PoS were not significantly different. Conclusions: The GBT1118-treated sickle mouse had a significant rise in Hb and improvement in PoS and deformability. Forty-eight hours after removing the analog compound, Hb dropped markedly, and the PoS was not different from control animals, suggesting a return to native oxygen affinity and clearance of the drug. Interestingly, deformability (EImin) did not return to baseline, suggesting some residual rheological improvement occurred in the red cell population that did not dissipate when the drug was cleared. These data suggest that potential concerns regarding viscosity rising above pre-treatment levels upon sudden cessation of voxelotor are not warranted. This may be due to retained improvement in red cell rheology after voxelotor is stopped combined with rapid return to baseline Hb levels. Further study in patients treated with voxelotor may be needed to determine if voxelotor produces additional benefits, such as reduction in oxidative stress, that outlive its presence in the red cell. Disclosures Pochron: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Oksenberg:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sheehan:Global Blood Therapeutics: Research Funding; Emmaus: Research Funding; Novartis: Research Funding.

Red Cell Rheology Biomarkers to Assess Cure in Gene-Based Therapies

Introduction Red cell rheology is abnormal in sickle cell disease (SCD); red blood cells (RBC) are rigid, dense, and the sickle hemoglobin (HbS) polymerizes with deoxygenation. There are several devices commercially available and under development to assess RBC rheology. One is an oxygen gradient ektacytometer (Lorrca with Oxygenscan, RR Mechatronics) which measures RBC deformability under oxygenated conditions (EImax) and deoxygenated conditions (EImin), and the oxygen concentration at which deformability begins to worsen, point of sickling (PoS). A commercially available hematology analyzer, the ADVIA (Siemens), measures hemoglobin (Hb) by flow and colorimetric methods, permitting automated calculation of the percent dense red blood cells (%DRBC). Allogeneic hematopoietic stem cell transplant (alloHSCT) can provide a cure for SCD, and viable gene-based therapy options are under investigation to serve the many patients without a matched related donor. However, the level of HbS correction or functional Hb induction necessary to achieve a cure is still unknown. Clinical endpoints such as prevention of pain events are important, but it is possible to be pain free for several years while still experiencing organ damage. As gene-based therapy clinical trials move forward, we must assess the level of functional improvement beyond Hb profile and conventional clinical labs. We propose that the goal of any gene-based SCD therapy should be to normalize blood rheology to the level of an individual with sickle cell trait (HbAS), and that EImax, EImin, PoS, and %DRBC may be used to distinguish between HbAS and HbSS/Sβ0 genotypes. Methods Subjects: Blood samples were collected from 257 unique patients (17 HbAS, and 240 HbSS/Sβ0) under IRB-approved protocols at Texas Children's Hospital and University Medical Center Utrecht. Patients were 56% male, ages 9 months to 22 years. Some HbSS/Sβ0 subjects were on transfusion and hydroxyurea (HU) (Table 1). Fetal hemoglobin (HbF) levels ranged from 0-41%. Oxygen gradient ektacytometry: Blood collected in EDTA and standardized to a fixed RBC count was suspended in 5 mL polyvinylpyrrolidone at room temperature. 1.5 mL of the sample solution was injected into test cup. Each sample was run in duplicate. ADVIA: 250µL of blood collected in EDTA at room temperature was aspirated to measure the %DRBC, defined as the percentage of RBCs with a Hb concentration >1.11 mg/mL. Analysis: Patient characteristics were summarized using median with 25th and 75th percentiles, and frequency with percentage. Characteristics and labs were compared by group with t-test, Wilcoxon rank sum test, or Fisher's exact test. Receiver operating characteristics (ROC) analyses were performed to identify HbAS versus HbSS/Sβ0 for each biomarker. All analyses were performed using Stata 15. Results The EImin, EImax, PoS, and %DRBC differed significantly between the HbAS and HbSS/Sβ0 groups, despite including transfused, very young, and HU-treated samples in the HbSS/Sβ0 cohort (p<0.001, p=0.002, p<0.001, and p<0.001, respectively; Table 1). ROC analysis showed that a threshold Elmin greater than or equal to 0.453 identified HbAS with 94.12% sensitivity and 95.43% specificity. EImax greater or equal to 0.569 identified HbAS with 94.12% sensitivity and 81.57% specificity. PoS less than 21.38 mmHg identified HbAS with 93.87% sensitivity and 93.33% specificity. %DRBC less than 1.6% identified HbAS with 88.13% sensitivity and 88.00% specificity (Table 2). Conclusion Conventional laboratory testing may not be able to establish if gene-based therapy has achieved a cure. We propose to define a cure as achievement of an HbAS level of RBC quality in a gene-based therapy edited RBC population. We show that rheological biomarkers EImin, EImax, PoS, and %DRBC differ significantly between individuals with HbAS and HbSS/Sβ0 of all spectrums of severity and treatment regimens. Not only are the rheological biomarker values significantly different, but there is little to no overlap in ranges of values obtained from the different genotypes, even in heavily transfused HbSS patients, with HbS as low as 31%. EImin, EImax, PoS, and %DRBC functionally identify genotype with high sensitivity and specificity; we propose that they be used not to diagnose SCD, but to determine if a HbSS or HbSS/Sβ0 individual who has undergone successful gene-based therapy has achieved the RBC functionality of a HbAS individual. Disclosures van Beers: Novartis: Research Funding; Pfizer: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR mechatronics: Research Funding. Wijk:Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding. Rab:RR Mechatronics: Research Funding. Sheehan:Global Blood Therapeutics: Research Funding; Novartis: Research Funding; Emmaus: Research Funding.

Drug Treatment of Raynaud's Phenomenon of the Nipple.

Drug Treatment of Raynaud's Phenomenon of the Nipple.

Improvement in Red Blood Cell Physiology in Children with Sickle Cell Anemia Receiving Voxelotor

Background: Sickle hemoglobin (HbS) under conditions of deoxygenation polymerizes to cause sickling of red blood cells (RBCs) and other rheological abnormalities. Voxelotor has been previously shown in a preclinical model of sickle cell disease (SCD) to increase HbS affinity to oxygen, thus reducing its polymerization and sickling with subsequent increase in the half-life of RBCs. We hypothesized that given this mechanism of action, we would observe improvements in RBC physiology in patients receiving voxelotor. In the Phase 3 GBT HOPE trial, the use of voxelotor in patients with SCD caused a significant reduction in markers of hemolysis and anemia. Ektacytometry is considered the gold standard to study deformability of RBCs with membrane protein disorders. The deformability of RBCs can be assessed using a defined value of shear stress with an increasing osmotic gradient (osmoscan) as well as with a newer technology to subject these cells to gradual deoxygenation (oxygenscan). Both assays can be measured using the Laser Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics, NL). In this pilot study, we analyzed samples from patients with SCD receiving voxelotor, before and 12 weeks after starting therapy to assess the benefits of voxelotor on RBC physiology. Methods: Our pilot study obtained whole blood from children ages 4-11 years with SCD, who were enrolled in the IRB approved GBT 440-007 clinical trial (NCT02850406; a study evaluating multiple doses of voxelotor at 1500 mg/day equivalent exposure to adults based on body weight) at Emory University/Children's Healthcare of Atlanta. All participants in this cohort continued their stable, optimal hydroxyurea dose during treatment with voxelotor. The below measurements were performed on the pre-dose and Week 12 visit samples. Deformability of RBCs was performed at a shear stress of 30 Pa and varying osmolality gradients (0-600 mOsm/Kg) for Osmoscan. Omin corresponds to the value of the hypotonic osmolality, where 50% of the cells hemolyze in an osmotic fragility assay and provides information on the initial surface area:volume ratio. Maximal deformability or Elongation Index (EImax) near isotonic osmolality informs us of the RBC cytoskeleton mechanics and Ohyper, the osmolality corresponding to 50% of the Elmax, provides information regarding the cytoplasmic viscosity. Oxygenscan was performed but under controlled deoxygenation using nitrogen. Point of Sickling (POS) is a point on the curve during deoxygenation when sickling begins, and EImin corresponds when sickle RBCs can least elongate. Oxygen dissociation curves were obtained using a HemOx Analyzer (TCS Scientific). Complete blood count parameters were determined on a clinical laboratory hematology analyzer (ADVIA, Siemens). Data was analyzed with Prism using a paired T-test. Results: Both pre-dose and Week 12 visit samples were available for 10 participants. Mean hemoglobin at baseline was 9.0 g/dL (7.6-10.0) and at 12 weeks, 10.3 g/dL (8.2-12.3). Six out of 10 participants had a hemoglobin response at Week 12 (defined as an increase in Hb from baseline by >1 g/dL), of which 5 had hemoglobin over 10 g/dL. Mean % change in percentage of reticulocytes was -17.0%. Significant improvement in EImax on osmoscan was noted at Week 12 (p=0.0147), suggesting RBCs were more deformable with improved cytoskeleton mechanics. In addition, oxygenscan curves shifted upwards towards normal with a significant increase in EImax (p=0.0347) and EImin (p=0.0079). These findings combined with a decrease in POS (p=0.0001) during deoxygenation suggests that at low oxygen tension, voxelotor treated RBCs were more deformable possibly from reduced HbS polymer inside these cells. Significant reductions in P50 (p=0.0011) and P20 (p=0.0001) with a left shift of the oxygen dissociation curve further demonstrates the effect of voxelotor on RBCs. Discussion: Voxelotor therapy in children with HbSS is associated with reductions in anemia and reticulocyte response, and recovery in RBC health as early as 12 weeks of treatment. Voxelotor's ability to inhibit HbS polymerization and RBC sickling is associated with specific modulation in red cell rheology at normoxic and deoxygenating conditions. Left shifted oxygen dissociation curves confirm voxelotor's ability to increase oxygen affinity. These findings suggest that voxelotor improves RBC deformability and anemia and delays the initiation of RBC sickling. Figure Disclosures Chonat: Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Baratz:Prolong Pharmacuticals: Honoraria; Global Blood Therapeutics: Research Funding. Pochron:Global Blood Therapeutics: Employment, Equity Ownership. Dixon:Global Blood Therapeutics: Employment, Equity Ownership. Tonda:Global Blood Therapeutics: Employment, Equity Ownership. Lehrer-Graiwer:Global Blood Therapeutics: Employment, Equity Ownership. Brown:Pfizer: Research Funding; Novartis, Inc: Research Funding; Imara, Inc: Consultancy, Research Funding; Global Blood Therapeutics, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Archer:AstraZeneca: Research Funding; Prolong Pharmaceuticals: Consultancy; Global Blood Therapeutics: Consultancy, Research Funding.

Rheological Assessments of Sickle Cell Patients Post Allogeneic Hematopoietic Cell Transplant

Introduction: Mixed donor chimerism (MDC) occurs in nearly 35% of patients with sickle cell disease (SCD) post allogeneic hematopoietic cell transplant (alloHCT). Donor myeloid engraftment of >20% is considered necessary to support disease resolution. However, in a disease known for its clinical variability, we hypothesize that patients (pts) with a severe pre-HCT phenotype may have abnormal RBC rheology at a level of chimerism and %HbS believed to be consistent with a cure. RBC rheology is markedly abnormal in SCD; these abnormalities are associated with SCD related clinical complications. Even fully oxygenated, sickle RBC are less deformable than those of HbAA or HbAS individuals; upon deoxygenation, deformability further declines. Sickle RBC morphology reflects the tendency to polymerize, with characteristic sickle forms. Sickle RBC are more adherent to the endothelial cell lining of the vasculature than HbAA or HbAS RBCs. The goal of any cell-based therapy of curative intent should be normalize the red cell rheology. To determine if the rheology of blood from SCD pts post-HCT with varying degrees of donor chimerism fall into the HbSS range of values, we propose to functionally assess the peripheral blood from a series of 6 post alloHCT SCD patients using a battery of rheological tests measuring deformability, sickling, adherence, percent dense cells (%DRBC), and RBC morphology. Methods: Peripheral blood samples (EDTA) from 6 SCD pts post alloHCT with a matched sibling donor were collected and immediately analyzed using oxygenscan (Lorrca), artificial microvascular network (AMVN), microfluidic image acquisition, and an ADVIA hematology analyzer. The Lorrca with oxygenscan measures RBC deformability (elongation index EI), under a range of pO2 (150-0 mmHg). EImax is the deformability of the oxygenated sickle RBC; EImin is the deformability of deoxygenated RBCs. The point of sickling (PoS) is the pO2 at which RBC deformability rapidly declines. RBC adhesiveness is measured by the difference in rate of perfusion of RBC through the AVMN coated with adhesive proteins (adherent AVMN) and a non-adherent, uncoated AVMN. %DRBC was measured by an ADVIA hematology analyzer. Microscopic RBC images were acquired and classified to determine the fraction of sickled RBCs in well-oxygenated samples. Additional clinical data including Hb profile, donor chimerism, and symptoms were obtained via chart review. Reference ranges were generated as described above using n=45 HbSS samples ages 2-21, on hydroxyurea, chronic transfusion, or untreated; n=14 HbAS, and n=43 HbAA. Results: In Figure 1, we show the measurements obtained by the Lorrca with oxygenscan on 5 pts post-alloHCT with a range of donor chimerism, as well as typical values for HbAA, HbAS, and HbSS patients. Patient 1 and 3 exhibit normal rheology; both were transplanted with a HbAA donor, with high chimerism and no detectable HbS. Patient 2 has 40% whole blood chimerism, well above the 20% threshold described with myeloid chimerism, and a HbS less than 50%, yet their plot resembles that of an untransplanted HbSS pt; the donor to Patient 2 exhibits the expected HbAA deformability. Patients 5 and 6 exhibit rheology in the HbAS range; they have intermediate chimerism from a HbAS donor. Clinical details and biomarker panel values for all 6 pts analyzed are in Table 1, as well as ranges of values for HbSS and HbAS subjects generated in the Sheehan and Shevkoplyas labs. Patient 2 is the only subject with values in the HbSS range, and the only subject with SCD symptoms. Conclusions: Our rheological tests identified a pt with values consistent with a HbSS pt of moderate severity or treated with hydroxyurea despite variable donor chimerism above the 20% threshold and HbS <50% thought to be sufficient for cure. The pt reported pain events beginning two years prior, indicating a clinical correlation supporting the validity of the rheological tests we propose to use to distinguish cure from persistent SCD. Current post-HCT evaluation depends on chimerism and hemoglobin profiles, and would not detect the significant functional abnormalities visible by Lorrca with oxygenscan biomarkers that indicate that Patient 2 is at risk for SCD related complications. Our results suggest that this functional analysis may help with management of post alloHCT SCD pts, and may be even more essential to assessing new gene-based therapy approaches to curing patients with SCD. Disclosures Rab: RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. Shevkoplyas:SSS: Research Funding; New Health Sciences: Consultancy.

Leg electrical resistance predicts venous blood viscosity and hematocrit.

We previously reported that whole body bioelectrical impedance analysis (BIA) measurements are correlated to some hemorheologic factors, suggesting a relationship between viscosity factors and electric properties of flowing blood not only in vitro but also in vivo. Recently we reported that with segmental BIA (analyzing the body considered as composed of 5 cylinders) predictive equations for various determinants of blood viscosity were closer than for the wole body. Another widely used BIA technique uses leg-to-leg impedance measurements so that two cylinders (the two legs) are analyzed. We investigated whether impedance measured with this technique (Tanita TBF-300) is also a predictor of blood viscosity factors. From viscometric measurements performed on venous blood drawn in recreative athletes over the range of shear rates 1 to 6000 s-1 (RHEOMETRE Anton Paar CP 50-1), we found a correlation between leg-leg resistance at 50 kHz (Rx[50 kHz]) and blood viscosity at 1000 s-1 (η1000= 0.0051 Rx[50 kHz] + 1.3265; r = 0.521 p = 0.028 yielding a prediction of η1000 (Bland Altman plot: bias 0.05 [RANGE - 0.24; 0.34]. Neither plasma viscosity nor the red cell rheology index «k» of Quemada's model are correlated with Rx[50 kHz], but hematocrit (Hct) does (Hct (%) = 0.0217 Rx[50 kHz] + 33.783; r = 0.480 p = 0.044) yielding a prediction of Hct (Bland Altman plot: bias - 0.11, [range - 1.67; 1.45]. The discrepancy between actual and predicted Hct is also correlated with resistance at 50 kHz (r = 0.575 p = 0.031) as does the discrepancy between actual and predicted Hct/viscosity ratio (r = -0.651 p = 0.006). Therefore, as other previously studied methods, leg to leg BIA predicts viscosity, suggesting that blood rheology may influence the passage of an electric current in the legs.

Surgical Splenectomy Alters Red Cell Rheology in Patients with Sickle Cell Disease

Background: Many patients with sickle cell disease (SCD) require a surgical splenectomy for repeat splenic sequestration or hypersplenism, resulting in worsening anemia and/or thrombocytopenia, or abdominal discomfort. Higher rates of thrombosis, pain crises and acute chest syndrome (ACS) have been reported following surgical splenectomy, although the reasons for this are not known. We hypothesize that this clinical worsening post-splenectomy is due to hemorheological changes; studies of the effects of surgical splenectomy on hemorheology in non-SCD animal models found significant reductions in red cell deformability and increase in whole blood viscosity, or blood thickness, following splenectomy. Understanding the impact of surgical splenectomy on blood rheology is especially relevant for patients with SCD, who have many clinical complications as a result of their high whole blood viscosity for their given hemoglobin levels, and low hematocrit-to-viscosity ratio (HVR), a measure of oxygen carrying capacity. Another important measure of SCD rheology is percent dense red blood cells (%DRBC), red cells with a density>1.11 mg/mL; they are typically the result of cellular dehydration, and are less deformable and more likely to sickle. We therefore sought to use our existing longitudinal rheology data, including measures of viscosity and %DRBC, to evaluate the impact of surgical splenectomy on our pediatric patients with SCD.Methods: We identified seven pediatric patients with multiple measurements of whole blood viscosity and %DRBC, collected before and after surgical splenectomy between November 2013 and April 2018 from SCD patients at Texas Children's Hospital on an IRB approved protocol. The cohort included 4 female and 3 male patients, ages 3-12 years old. Whole blood viscosity was measured using a cone and plate viscometer (DV3T Rheometer, AMETEK Brookfield, Middleboro, MA, USA) at 37 degrees Celsius within 4 hours of sample collection in an EDTA vacutainer tube. CBC data including %DRBC was measured on an ADVIA 120 Hematology System (Siemens Medical Solutions USA, Inc., Malvern, PA, USA). Samples collected 1 month before or after an emergency department visit or within 3 months of a packed red blood cell transfusion were omitted from analysis.Results: We found a significant rise in %DRBC following splenectomy (p=0.01). There was a significant increase in whole blood viscosity at 45 s-1 and 225 s-1 (p=0.006 and p=0.004, respectively) and a decline in hematocrit-to-viscosity ratio (HVR) at 45 s-1 and 225 s-1 (p=0.03 and p=0.03, respectively) (Table 1). Hemoglobin and hematocrit did not significantly change after splenectomy (p=0.6 and p=0.5, respectively), suggesting that the rise in viscosity was due to intrinsic changes in red cell rheology. Platelets increased markedly (p<0.00002), a side effect commonly seen following splenectomy, known to contribute to thrombophilia.Conclusion: Overall, the changes in %DRBC, viscosity, and HVR show a worsening of blood rheology following surgical splenectomy with no evidence of a return to baseline 800 days after splenectomy. The increase in viscosity and reduction in HVR in the setting of a rise in %DRBC suggests that the spleen may have played a role in removing these dense or irreversibly sickled cells. Further studies with a larger cohort and long term observation are needed to further elucidate the relationship between worsening rheology and SCD-related complications reported in the literature post-splenectomy. These rheological changes should be considered as part of the decision making for elective splenectomy, monitored post-splenectomy, and addressed therapeutically where possible. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New insights into red cell rheology and adhesion in patients with sickle cell anaemia during vaso-occlusive crises.

Painful vaso-occlusive crisis (VOC) represents the most frequent complication encountered by SCA patients. In animal models, increased red blood cell (RBC) adhesion to the endothelium has been demonstrated to initiate VOC by increasing the transit time of RBC through the microcirculation, predisposing them to sickle and occlude the vessels (Kaul et al.,

NMR-Based Lipidomic Analysis of Red Blood Cells Membranes in Type 2 Diabetes

Changes in the composition of red blood cells membranes may lead to in an altered blood rheological pattern. Experimental studies suggest that hyperglycemia affects red cell rheology via direct effects on the membrane, including alterations of the lipid membrane bilayer composition and microviscosity and changes in membrane Na+/K+ ATP-ase function. We aimed to investigatate the 1H NMR-based lipid profile of RBC membranes in type 2 diabetic patients and compare it to that of nondiabetic subjects. Blood samples from 25 nondiabetic and 15 diabetic individuals were collected after an overnight fast. The groups were matched for age, sex and conventional lipid parameters. RBC were lysed and their lipid content was extracted according to a standard procedure. The lipid profile of membranes was recorded on Bruker DRX-500 NMR Spectrometer and analyzed by multivariate data techniques. The NMR-based lipidomic analysis showed that diabetic patients presented a significantly different lipid profile in RBC membranes compared to that recorded in nondiabetic individuals. RBC membranes from patients with diabetes were characterized by altered fatty acid pattern e.g., higher saturated and lower unsaturated fatty acid levels and lower concentrations of omega-3 fatty acids, mainly docosahexaenoic acid. In addition, lower levels of phosphatidylcholine and phosphatidylethanolamine, and higher levels of sphingomyelin and cholesterol were observed in diabetic patients compared to nondiabetic individuals. In conclusion, the lipid composition of RBC membranes in the diabetic state is significantly altered. These disturbances may be involved in the pathogenesis of the microvascular complications of diabetes through the disruption of tissue delivery of oxygen. Additionally, the increased cholesterol content of red cell membranes that reach the lipid core of the atherosclerotic plaques through the vasa vasorum may lead to core expansion, decrease their stability and make them prone to rupture. Disclosure C. Kostara: None. E. Bairaktari: None. M. Elisaf: None. V. Tsimihodimos: None.

EFFECT OF PROPOFOL AND MEMANTINE ON ERYTHROCYTE DEFORMABILITY IN DIABETIC RAT MODEL

OBJECTIVES: In this study we aimed to investigate the effect of diabetes on erythrocyte deformability (ED) and whether it can be changed by an NMDA antagonist propofol or an NMDA agonist, memantine BACKGROUND: Several clinical studies showed that ED decreases in diabetes. Recent studies showed that erythrocytes have N-methyl-D-aspartate (NMDA) receptors (NMDA Rec) on their membrane. METHODS: Thirty rats were allocated to five groups containing 6 rats each. Memantine was given for 30 days to diabetic rats in one group (group DM) single dose propofol injection is added to this regimen in another group (group DP+ memantine) another group received propofol only (group DP). The remaining groups were controls and diabetic controls. ED was measured in each group and compared RESULTS: The deformability index was significantly increased in the diabetic rats ( p <0.0001). However, it was similar in Group DC and DP ( p =0.551), Group DC and DM ( p =1.000), Group DC and DPM ( p =0.176). CONCLUSION: Neither NMDA antagonist propofol nor NMDA agonist memantine affected the altered red cell rheology in diabetic rat model.

Thalassaemia intermedia: the role of erythroexchange in the treatment of an indolent wound.

Thalassaemia intermedia is a haemoglobinopathy in which, by definition, the patient maintains satisfactory levels of haemoglobin without needing transfusions. Indeed, the condition is also called non-transfusion-dependent thalassaemia. Nevertheless, at some time during the patient’s lifetime, transfusion therapy may become necessary to guarantee a level of haemoglobin adequate for the activities of normal life. Thalassaemia intermedia is most commonly associated with a homozygous or compound heterozygous state for two beta-thalassaemia alleles but the severity of the clinical picture is related to the patient’s genotype and the consequent degree of globin chain imbalance1. The level of foetal haemoglobin (HbF) is usually variably elevated. In addition, patients with thalassaemia intermedia can suffer from an atypical form of pseudoxanthoma elasticum (PXE), a multisystem disorder affecting, among others, the elastic tissues of the arteries and leading to degeneration and calcification of the elastic lamina of the arterial wall2. At the level of the ankle, low haemoglobin concentrations associated with abnormal red cell rheology and increased haemoglobin F cause tissue hypoxia that promotes thinning of the skin and subcutaneous fragility. As a consequence, trophic ulcers are a common finding in adult patients3. We report here the difficult healing of a surgical wound in a patient with thalassaemia intermedia and the beneficial effect obtained with erythroexchange that, decreasing the level of HbF in favour of HbA, improved the oxygen availability in the area and promoted healing.

Hematological and Genetic Predictors of Daytime Hemoglobin Saturation in Tanzanian Children with and without Sickle Cell Anemia

Low hemoglobin oxygen saturation (SpO2) is common in Sickle Cell Anemia (SCA) and associated with complications including stroke, although determinants remain unknown. We investigated potential hematological, genetic, and nutritional predictors of daytime SpO2 in Tanzanian children with SCA and compared them with non-SCA controls. Steady-state resting pulse oximetry, full blood count, transferrin saturation, and clinical chemistry were measured. Median daytime SpO2 was 97% (IQ range 94–99%) in SCA (N = 458), lower (P < 0.0001) than non-SCA (median 99%, IQ range 98–100%; N = 394). Within SCA, associations with SpO2 were observed for hematological variables, transferrin saturation, body-mass-index z-score, hemoglobin F (HbF%), genotypes, and hemolytic markers; mean cell hemoglobin (MCH) explained most variability (P < 0.001, Adj r 2 = 0.09). In non-SCA only age correlated with SpO2. α-thalassemia 3.7 deletion highly correlated with decreased MCH (Pearson correlation coefficient −0.60, P < 0.0001). In multivariable models, lower SpO2 correlated with higher MCH (β-coefficient −0.32, P < 0.001) or with decreased copies of α-thalassemia 3.7 deletion (β-coefficient 1.1, P < 0.001), and independently in both models with lower HbF% (β-coefficient 0.15, P < 0.001) and Glucose-6-Phosphate Dehydrogenase genotype (β-coefficient −1.12, P = 0.012). This study provides evidence to support the hypothesis that effects on red cell rheology are important in determining SpO2 in children with SCA. Potential mechanisms and implications are discussed.

Is increased perfusion pressure really necessary during cardiopulmonary bypass?

We read with great interest the work by Siepe et al .[ 1] investigating the effect of increased systemic perfusion pressure during cardiopulmonary bypass on cognitive dysfunction and delirium. In this revolutionary study questioning cerebral autoregulation, they found that maintaining mean perfusion pressure during normothermic cardiopulmonary bypass (CPB) at 80–90 mmHg is associated with less early cognitive dysfunction and delirium after coronary artery bypass grafting (CABG), and elevated perfusion pressure is not associated with increased morbidity and mortality. This finding may lead to severe changes in the routine practice of cardiac surgeons and anaesthesiologists including us. However, we believe that there are some issues that need to be addressed. This study lacks a power and sample analysis and seems to have been conducted in a very small group of patients. For this reason, postoperative complications such as atrial fibrillation (19 vs 36%), bleeding requiring reoperation (2 vs 7%), use of blood products (50 vs 70), cessation of catecholamine support (26 vs 14 h), superficial wound infection (4 vs 7%) and pleural effusion (33 vs 48%) which may be statistically significant in a larger group of patients appears to be insignificant. As the low perfusion group needed longer catecholamine support, it is easy to assume that they were in either low cardiac output (LCO) or vasoplegic state. The incidence of preoperative left ventricular dysfunction, which is a predictor of LCO, is not reported and should be included in the statistical analysis. Furthermore, it is well known that preoperative medication by inhibitors of angiotensin-converting enzyme in coronary artery patients is associated with vasoplegic shock early after CABG. This issue also needs to be addressed. If low perfusion pressure during CPB is to be blamed, then the postoperative period should be comparable in both groups. However, as the LP group suffered from LCO postoperatively, measurement of neurochemical markers (S-100 beta and neuronspecific enolase) immediately after CPB might be appropriate. It is clear that patients with delirium had longer CPB, ventilation and intensive care unit (ICU) stay times, and were still in LCO (probably still in ICU and under major depression) at the time of Mini-Mental-State examination. All of these factors have been shown to be associated with postoperative delirium [2, 3]. Cerebral circulation during CPB may be affected by partial arterial oxygen (hyperoxaemia by impairing red cell rheology and microcirculation) and carbon dioxide pressure (by changing cerebral arteriolar reactivity) [4]. Thus, they should be monitored closely (more frequently than every 20 min) and kept similar in both groups. Spot measurements of transcutaneous cerebral oxygen saturation as measured by near-infrared spectroscopy seem to be similar in both groups. However, it is reported that rSo2 desaturation score, which is calculated by multiplying rSo2 below 50% by time (s), is a more sensitive method of predicting early postoperative cognitive decline than spot measurements [5]. Nitric oxide (NO) formed via endothelial NO synthase and neuronal NO synthase plays crucial roles in the regulation of blood flow through vasodilatation and decreased vascular resistance. Endothelial and autonomic nitrergic nerve functions are impaired in patients with diabetes mellitus, resulting in regional blood flow decrease [6]. As the duration of the diabetes mellitus and insulin dependence may change from patient to patient, it seems necessary to exclude these patients or at least compensate for the severity of their disease.

Effect of Nitric Oxide Exposure on the Red Cell Rheology

Effect of Nitric Oxide Exposure on the Red Cell Rheology

Blood rheology and body composition as determinants of exercise performance in female rugby players

Athletes involved in rugby are characterized by a very specific pattern of body composition with an unusually important muscle mass. In a preceding study about rugbymen we evidenced that they exhibit a correlation between red blood cell aggregability and the amount of body fat although it remains within a normal range, and that red cell rigidity was correlated to isometric adductor strength. We had the opportunity of studying the relationships among exercise performance, body composition and hemorheology in 19 female rugby players (age 19-26, mean: 24.47 ± 0.67 yr) practising 4 - 10 hr/wk (mean 7.15 ± 0.3) since 1-12 yr (mean 4,05 ± 0,694). VO2max was not related by its own to blood rheology, either hematocrit (r = -0.0717 p = 0.7706) or plasma viscosity (r = 0.0144; p = 0.9533), but other markers of performance exhivited a correlation with red cell rheology. Relationships between fitness and body composition were evidenced. Isometric handgrip strength was negatively correlated to red blood cell aggregability (Myrenne M, r = -0.57839; p = 0.00948 M1 r = -0.58910; p = 0.00795). Adductor isometric strength was negatively correlated to red blood cell aggregability Myrenne M (r = -0.5033; p = 0.0280) but not to M1 (r = -0.4227; p = 0.0714). Fat mass is a major determinant of the maximal oxygen consumption VO2max either measured by a field test (r = -0.766; p = 0.00013) or exercise test (r = -0.575; p = 0.00994) and was also negatively correlated to both handgrip (r = -0.4918; p = 0.0325) and RBC aggregability M (r = -0.57839; p = 0.00948 and M1 r = -0.5891; p = 0.00795). Independently of fat mass, FFM appears to be a determinant of blood viscosity (r = 0.4622; p = 0.0463) due to its correlation with RBC rigidity (r = 0.4781; p = 0.0384). Thus, trained young women exercising 4-10 hr/wk and thus exhibiting a low percentage of body fat exhibit clear relationships between body composition and hemorheology, but fat mass being low, the parameter correlated with blood rheology is in this case fat-free mass, consistent with recent findings indicating that high fat mass in women is sometimes correlated with parameters of the metabolic syndrome such as insulin resistance or inflammation. In addition, parameters quantifying fatness even within such a physiological range are in this sample negatively related with exercise performance.

Thyroid hormone: the modulator of erectile function in the rabbit.

The possible role of thyroid hormones in the Nitric Oxide (NO)-mediated response to sexual stimulation, and on prostaglandin E1 (PGE1) and Sildenafil in the treatment of erectile dysfunction was investigated using the corpus cavernosum of the New Zealand rabbit animal model. The parameters studied were penile erection monitored as contractile force of the erectile tissue, sperm count and motility; in parallel with the haematocrit, red cell count or rheology, Heart Rate (HR), Mean Arterial Pressure (MAP), Thyroid Stimulating Hormones (TSH) and Thyroxine levels. Hypothyroidism or thyroidectomy was found to cause depletion of Endothelium Derived Relaxant Factor (EDRF) thereby causing very feeble contraction of the cavernosum muscle, in both prostaglandin E1 (PGE1) and sildenafil, oligospermia and less than 45 % motile sperms. Thyroxine treatment produced contraction proportionate to the concentrations of PGE1 and Sildenafil; providing evidence that the erectogenic actions of both PGE1 and Sildenafil are possible only in the presence of adequate thyroid hormone level.

Nonneuronal Cholinergic System in Human Erythrocytes: Biological Role and Clinical Relevance

Acetylcholine is well known in the medical setting as one of the most exemplary neurotransmitters. Its ubiquity in nature otherwise suggests a theoretically diverse spectrum of action and an extremely early appearance in the evolutionary process. In humans, acetylcholine and its synthesizing enzyme, choline acetyltransferase, have been found in various nonneural tissues such as the epithelium, mesothelium, endothelium, muscle, immune cells and blood cells. The widespread expression of nonneuronal acetylcholine is accompanied by the ubiquitous presence of acetylcholinesterase and nicotinic/muscarinic receptors. Structural and functional dissimilarities are evident between the nonneuronal and neuronal cholinergic systems. An increasing body of evidence throughout the last few years has placed acetylcholine as a major cellular signaling molecule in many pathways. Furthermore, numerous erythrocyte physiological events in the microcirculation are strongly regulated by acetylcholine. Thus, it is time to revise our understanding of the role of vascular acetylcholine in humans. Its biological and pathobiological roles must be evaluated in more detail to eventually achieve novel therapeutical targets. The present article reviews recent findings about nonneuronal acetylcholine in red blood cells, with special regard to (1) red cell rheology, (2) plasma ion concentrations, (3) nitric oxide intracellular translocation and metabolism and (4) band 3 protein phosphorylation.