Red Cell Aggregation Research Articles

Analytical and physiological variations of some hemorheological and hematological blood tests

In order to determine the analytical precision and physiological fluctuation of a battery of blood tests (hematocrit, blood viscosity, plasma viscosity, blood filterability, red cell aggregation and blood cell counts) volunteers were tested at fortnightly intervals for weeks

Second experiment on aggregation of red cells and blood viscosity under zero gravity: on STS 26, Sept/Oct 1988

Second experiment on aggregation of red cells and blood viscosity under zero gravity: on STS 26, Sept/Oct 1988

Red cell membrane fluidity and red cell aggregation in vascular atherosclerotic disease

Red cell membrane fluidity and red cell aggregation in vascular atherosclerotic disease

Influence of cellular properties on red cell aggregation

Influence of cellular properties on red cell aggregation

Red-cell aggregation in insulin-dependent diabetics

Red-cell aggregation in insulin-dependent diabetics

Quantitative study of morphology of aggregation of red cells in patients blood and in the reconstituted suspensions

Quantitative study of morphology of aggregation of red cells in patients blood and in the reconstituted suspensions

Importance of red cell aggregation in venous pathology

Importance of red cell aggregation in venous pathology

Physicochemical basis and clinical implications of red cell aggregation

Physicochemical basis and clinical implications of red cell aggregation

Rivipansel: A Small Pan-Selectin Antagonist Improves Cerebral Perfusion and Inhibits Leukocyte Adhesion and in Murine Sickle Cell Disease Model

▪The pathophysiology of sickle cell disease (SCD) is complex and heterogenous. Tissue hypoxia leads to endothelial activation, and formation of multicellular aggregates of red cells, leucocytes and platelets in the microvasculature. This results in a vaso-occlusion further reducing tissue perfusion. Patients with SCD experience intense pain crisis. Some SCD patients also exhibit elevated cerebral blood flow velocities in transcranial Doppler (TCD) and impaired autoregulation. Higher TCD velocities are associated with imminent risk of ischemic stroke and are an indication for transfusion for SCD patients. Similar to human SCD patients, SCD mice also exhibit an increase in cerebral perfusion (Branch et al, Blood, 2015) and irregularly dilated cerebral microvasculature compared to wild-type (WT) mice (Manci et al, Blood, 2006). Selectins play a critical role in mediating initial tethering and adhesion of hematopoietic cells with endothelial cells, and are attractive targets to treat vaso-occlusion. Rivipansel is a potent small molecule pan-selectin inhibitor with a 100 fold greater inhibitory acitivity for E-selectin over P-selectin. Previous studies have demonstrated the effect of Rivipansel (GMI-1070) in chimeric SCD mice using intravital microscopy (IVM) where bone marrow from Berkeley SCD mice was transplanted in wild type (WT) mice (Chang et al, Blood, 2010). Here, we further investigate the effect of Rivipansel on leukocyte rolling in vitro and in vivo in Townes SCD mice with corresponding changes in inflammatory markers. We also extended these studies to explore the effect of Rivipansel in a cerebral perfusion model in Townes SCD mice.The effect of Rivipansel on cell adhesion in vitro was examined using the microfluidic BioFlux shear flow system. HL-60 cells were continuously flown under physiological conditions over confluent monolayers of Chinese Hamster Ovary cells expressing E-selectin (CHO-E) or P-selectin (CHO-P) treated with Rivipansel, Immunoglobulin G (IgG) control antibody or vehicle. Rivipansel dose-dependently reduced the adhesion of HL-60 to CHO-E monolayers compared to vehicle treated cells, or CHO-P monolayers.In the Townes SCD mice, Rivipansel was evaluated using IVM of the cremaster. Surgical manipulation and the chronic inflammatory conditions in Townes mice, induce an acute vaso-occlusive condition in the post-capillary venules. Rivipansel or vehicle control were dosed at 5, 10 or 20 mgs/kg intravenously prior to surgical preparation and a second dose was administered 70 minutes later. Labeled neutrophil and platelet specific antibodies were injected to quantify neutrophil-platelet aggregates and neutrophils adhered to the vasculature. Neutrophil rolling velocity increased 2.5 fold and there was a dose dependent increase in rolling neutrophils. Rivipansel, dosed at 20 mg/kg resulted in a 69% decrease in neutrophil adhesion to the endothelium and an 85% decrease in neutrophil platelet aggregates (NPA) attached to the vasculature compared to vehicle treated mice. No effect on was observed in mice dosed a 5 mg/kg. Plasma soluble P and E-selectin levels were significantly reduced in mice dosed at 10 and 20 mg/kg compared to vehicle treated mice.Additionally, cultured primary brain endothelial cells stimulated with TNF-α demonstrated a 700 fold increase in E-selectin levels as measured by RNAseq. Based on this observation, we studied the effect of Rivipansel on cerebral perfusion studies in a TNFα induced occlusion model in SCD and WT mice where changes in cerebral microcirculation were measured in real time using laser Doppler tissue perfusion. TNFα treated SCD mice demonstrated a marked reduction in tissue perfusion in contrast to WT mice. This was rapidly reversed by intravenous injection of Rivipansel (20 mg/kg). Brains harvested from TNF treated SCD mice showed increased platelet microthrombi compared to WT mice, and treatment with Rivipansel reduced the platelet aggregates seen in the cerebral microvasculature in SCD mice.In summary, we demonstrated dose dependent decrease in leukocyte adhesion in vitro and in vivo in the Townes mice. In addition administration of Rivipansel is beneficial in improving cerebral perfusion as well improving peripheral microvascular flow in the SCD mice.All experiments were within guidelines and were reviewed and approved by Pfizer institutional animal care and use committee. DisclosuresJasuja:Pfizer: Employment. Suidan:Pfizer: Employment. Hett:Pfizer: Employment. Desai:Pfizer: Employment. Le:Pfizer: Employment. Bell:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Red blood cell microrheological effects of some antitumor chemotherapy drugs: In vitro study

In consequence of limitation of capillary dilatation reserve the role of erythrocyte membrane elasticity and their microrheological properties is very important for the tissue perfusion. The drugs that enter blood flow could be as signaling molecules and their action can lead to alteration of red blood cell aggregation and whole cell deformability. Therefore the subject of this study was to estimate the effect of some antitumor chemotherapy drugs on red blood cell aggregation (RBCA) and deformability (RBCD). It was found that an exposure of red blood cells (RBCs) to cisplatin and epoetin alfa led to significant positive changes in the RBC microrheological properties. It was shown that cisplatin could activate tyrosine protein kinase (TPK). Preincubation of RBCs with the inhibitor of EGF-R and Src kinase lavendustin A was accompanied by a significant decrease of the cisplatin effect. It was found RBC aggregation rise after cell incubation with 5-fluorouracil. This effect was removed with Ca2+ chelation with EGTA and also with pentoxifylline. The drug for the targeted antitumor therapy sunitinib raised RBCA markedly. Whereas RBC deformability index was altered only slightly. The combination of two chemicals: sunitinib and cisplatin led to an elimination of proaggregative effect of sunitinib. Examination of the effects of some antitumor drugs on red blood cell microrheological properties has enabled us to suggest that the observed changes in red cell aggregation and deformability may be partly related to the effects of tyrosine protein kinase activation. It is accompanied by the alteration of red blood cell microrheology and the blood transport efficacy.

The Effects of Weight Loss on Blood Rheology in Obese Patients

capillaries. This blood flow structure is the result of a specifically ordered disposition of the RBCs surrounded by plasma in the microvessel lumen during flow in the capillaries and in the adjacent arterioles and venules up to approximately 15-20 μm wide [12]. The blood flow structure disturbances produce disordered blood fluidity during microcirculation because of increased blood flow resistance under conditions of constant, or even increased, pressure gradient. Based on perennial studies of blood flow disturbances inside the capillary microvessels, several primary factors that produce increased blood flow resistance in the narrowest blood vessels are as follows: enhancement of the RBC aggregation, lowered RBC deformability, increased local hematocrit and increased local blood plasma viscosity. The impact of a hypocaloric diet on the hemorheology of obese patients has been reported in numerous studies [2,13-17]. Several studies concerned with the influence of a hypocaloric diet on RBC aggregation in obese patients reported that weight loss after dieting in obese patients normalized the aggregation of the RBCs, affecting a corresponding improvement in the erythrocyte aggregation index [13-15]. Other authors have showed the absence of improvement in rheological parameters, including RBC aggregation, after weight loss associated with a low-calorie diet [2,16]. Sola et al. (2004) showed that a very low-calorie diet of 458 kcal/day reduced erythrocyte aggregation in one month; however, a prolonged lowcalorie diet (1500 kcal/day) did not provide additional benefit for red cell aggregation, which returned to its basal value after 3 months of follow-up [17]. The impact of a hypocaloric diet on the obese patients’ blood and plasma viscosities are also not conclusive. According to Poggi et al. [14], after three months of a 500-kcal/day diet, there was a reduction in blood viscosity at low shear rates by significant decreases in RBC aggregation and hematocrit levels. Fanari et al. (1993) also demonstrated that weight loss after dieting in obese patients contributed to improving plasma and blood viscosity [13]. Other studies have shown an absence of improvement in blood and plasma viscosity after weight loss associated with a low-calorie diet [14,15]. The results of research on the impact of diet on hemorheology indicated that a beneficial effect of weight loss on rheological profiles depends on the amount of weight lost and its long-term maintenance. Bariatric surgery is recognized to substantially improve body weight, obesity-associated co-morbidities and long-term survival. The influence of weight loss surgery on hemorheological parameters has been investigated in several publications. In our first papers, verticalbanded gastroplasty was shown to induce several beneficial changes Abstract

Are overall adiposity and abdominal adiposity separate or redundant determinants of blood viscosity?

In line with recent literature showing that both general adiposity and abdominal adiposity are independently associated with the risk of death, we recently reported that body mass index (BMI) and waist-to hip ratio (WHR) were independent predictors of blood viscosity, related to different determinants of viscosity (for BMI: plasma viscosity and red cell aggregation; for WHR: hematocrit). Since this report was challenged by a study showing that abdominal adiposity (as measured with waist circumference WC and not WHR) is the only independent determinant of viscosity, we re-assessed on our previous database correlations among viscosity factors, BMI, WHR and WC. Blood viscosity was correlated to BMI (r = 0.155 p = 0.004), WHR (r = 0.364; p = 0.027) and WC (r = 0.094; p = 0.05). Hematocrit was correlated to WHR (r = 0.524) but neither to BMI (r =-0.021) nor waist circumference (r = 0.053). WC was correlated with plasma viscosity (r = 0.154; p = 0.002) while WHR was not (r =-0.0102 NS). A stepwise regression analysis selected two determinants of whole blood viscosity at high shear rate: BMI (p = 0.0167) and WC (p = 0.0003) excluding WHR. Therefore, in this sample, abdominal fatness expressed by WC and whole body adiposity remain independent determinants of blood viscosity. WHR and WC have not the same meaning, WC measuring the size of abdominal fat while WHR measuring the shape of body distribution regardless the degree of fat excess. Interestingly, hematocrit is rather related to shape (even within a normal range of body size) than the extent of abdominal fatness, and is not related to whole body adiposity.

Polystyrene Nanoparticles Activate Erythrocyte Aggregation and Adhesion to Endothelial Cells.

Nanoparticles (NPs) are drawing an increasing clinical interest because of their potential use as drug carriers. Recently, a new strategy for elevation of NPs in vivo circulation time has been proposed, specifically, utilizing red blood cells (RBCs) as a carrier for NPs, that are loaded with a drug, by interaction (in vitro) of human RBCs with NPs (RBCNP). This class of delivery set-up, combines advantages of natural RBCs and synthetic biomaterials. Previous studies demonstrated that NPs initiated hemolysis of RBC and activated cells aggregation. In the present study, we examined the effect of RBCNP on the aggregation of RBC and their adhesion to endothelial cells (EC). Red cells were treated with polystyrene NPs (PS-NP), and following their washing, were added to suspension of untreated cells at various concentrations. We observed that the PS-NP and RBCNP initiated the formation of red cells aggregates and markedly elevated RBC adhesion to EC. These effects were augmented with (a) increasing concentration of NPs or RBCNP, and (b) with decreasing NP size. This implies that RBCNP are cells with a stronger intercellular interaction, and may thereby induce the formation of large and strong aggregates with untreated RBC, as well as strong RBC/EC interaction.

A simple slide test for erythrocyte aggregability as a risk stratification tool in patients admitted with chest pain to the emergency room

Patients presenting to the Emergency Room with chest pain pose a diagnostic challenge because of a possible acute coronary syndrome. This study was done to determine the diagnostic utility of a simple slide test for red cell aggregability in patients with chest pain and as a prognostic indicator in unstable angina. 32 patients of Unstable Angina and 32 persons with non-cardiac chest pain were included in the study. A simple slide was prepared with the peripheral blood of the subjects and stained and assessed by a pathologist and by digital photographs and grid analysis to grade red cell aggregabilty. 19 patients with Unstable Angina had Grade C or D red cell aggregation while only 2 persons with noncardiac chest pain had Grade C aggregation. Estimation of Erythrocyte Percentage from the grid images and calculation of Receiver Operating Characteristic Curve gave a best cutoff value of 60-70 with a lower EP signifying unstable angina. 8 patients with Unstable Angina and an EP <50 developed acute MI or recurrent angina. To conclude, a simple slide test of the peripheral blood helps in the initial evaluation of chest pain patients for possible unstable angina, and, further has prognostic utility in patients with unstable angina.

Hemodynamic biomarkers levels are not dependent on physical exercise in patients with muscle atrophy

Aim: Evaluation of the impact of the physical exercise in patients with muscular atrophy by analyses of the fibrinogen and thiobarbituric acid reacting substances (TBARS). Cross-sectional study. Convenience sampling: control group (C: n=12) and two groups of wheelchair users: non athletes (NA: n=12) and athletes (A: n=13, professional basketball players). Analyzed biomarkers: fibrinogen and TBARs. Differences were assessed by one-way ANOVA and Newman-Keuls post-hoc. Significance P< 0.05 (IC95). Data are expressed as the mean ± S.E.M.Ethical approval: 462.531 /2013. Fibrinogen values were not different between A (3.67 ± 0.44 mg/dL) and NA (3.44 ± 0.38 mg/dL). On the other hand, it was observed difference between fibrinogen from each group of wheelchair users (A and NA) comparing to C (2.27 ± 0.08 mg/dL) or the standard values of fibrinogen (1.8 mg/dL–3.1 mg/dL): P<0.01and P<0.05, respectively). The TBARS values were not different as compared to the wheel chair users A (3.21 ± 0.24) and NA (3.66 ± 0.27), but they were different (P<0.001) from the C (24.11 ± 1.75). Muscle atrophy of the lower limbs impairs venous return in A and NA. In the absence of difference of both biomarkers between the groups of wheelchair users, it is possible to mention that the upper body exercise does not alter the rheological changes in viscosity, the red cell aggregation (fibrinogen levels), the mitochondrial activity and consequently ROS production (TBARS levels). Therefore, these may be not the only biomarkers to be evaluated.

Red cell distribution width as a predictor of left atrial spontaneous echo contrast in echocardiography.

Red cell distribution width (RDW) represents the heterogeneity of red blood cells (anisocytosis). Spontaneous echo contrast (SEC) is thought to be a manifestation of red cell aggregation and it has been linked to the development of thromboemboli. The aim of this study was to evaluate the association between RDW levels and the presence of left atrial SEC (LASEC).One-hundred and 72 patients who underwent transesophageal echocardiography for various indications were enrolled in the study. All patients were categorized into 2 groups according to the presence of LASEC and into 4 groups according to the severity of LASEC. The baseline clinical characteristics, echocardiographic measurements, and laboratory findings, including RDW, were compared between the groups.The RDW (%) level was higher in the LASEC group (14.95 ± 1.32) compared with the non-LASEC group (12.20 ± 1.45; P = 0.0001). When the relationship between RDW and SEC was evaluated according to the increasing grade of SEC, a significant positive correlation was found (r = 0.645, P < 0.0001). In the ROC analysis, an RDW level >13.8% had 70% sensitivity and 89.2% specificity in predicting LASEC (area under the curve = 0.834, P < 0.0001, 95% CI 0.656–0.773). In multivariate analysis, RDW levels >13.8% and the presence of atrial fibrillation were independently associated with LASEC (odds ratio [OR] 1.697; 95% confidence interval [CI] 1.198–2.085; P = 0.001 and OR 1.586; 95% CI 1.195–2.098; P = 0.003, respectively].Elevated RDW value is associated with the presence and the severity of SEC. RDW may be a useful marker and independent predictor for the presence of SEC.

Conjugation of Aurein 2.2 to HPG Yields an Antimicrobial with Better Properties

Aurein 2.2 is an antimicrobial peptide (AMP) whose mechanism of action is quite well-understood and that has good activity against Gram-positive bacteria. It is, however, highly cytotoxic. Poly(ethylene glycol) (PEG) conjugation (PEGylation) of protein and peptide drugs has been used for decades to improve their in vivo efficacy and blood circulation by enhancing the biocompatibility of the protein or peptide in question. However, the relatively large hydrodynamic size, high intrinsic viscosity, the limited number of functional groups available for conjugation, and immunogenicity of high molecular weight PEG limits its use in bioconjugation applications. Recently, hyperbranched polyglycerol (HPG) has been gaining attention as an alternative to PEG due to its excellent biocompatibility. Here, for the first time, we report the synthesis of HPG conjugates of antimicrobial peptides. Aurein 2.2 peptide was conjugated to high molecular weight HPG with a varying number of peptides per polymer, and the biocompatibility and antimicrobial activity of the conjugates were investigated. The antimicrobial activity of the peptide and its conjugates were determined by measuring the minimal inhibitory concentration (MIC) against Staphylococcus aureus and Staphylococcus epidermidis. The interaction of aurein 2.2 peptide and the conjugates with a model bacterial biomembrane was investigated using CD spectroscopy to understand the mode of action of the conjugates. The biocompatibility of the AMP-polymer conjugates was investigated by measuring red cell lysis, platelet activation and aggregation, complement activation, blood coagulation, and cell toxicity. Our results show that the size of the conjugates and the peptide density influence the biocompatibility of the antimicrobial conjugates. These results will help to further define the properties of HPG-AMP conjugates and set the stage for development of better therapeutic agents.

Impact of sleeve gastrectomy on red blood cell aggregation: a 12-month follow-up study

To investigate the effects of weight loss due to laparoscopic sleeve gastrectomy (LSG) on erythrocyte aggregation and the relationship of anthropometric and plasmatic factors, such as plasma viscosity, fibrinogen and lipids, with erythrocyte aggregation. The RBC aggregation and kinetics of the red blood cell aggregation were performed by the Laser-assisted Optical Rotational Cell Analyser (LORCA). Before the LSG and 6 and 12 months after the LSG, we evaluated the aggregation index (AI), amplitude (AMP) and aggregation half-time (t1/2), plasma viscosity, fibrinogen, glucose and lipids patterns in 15 non-diabetic obese subjects. The static and kinetic parameters of aggregation in obese patients at each time point after bariatric weight loss surgery were calculated and significant differences were observed at 12 months after surgery. AI and AMP decreased from 69.81±5.12% and 27.43±2.9 a.u. at baseline to 64.91±5.94% and 22.15±4.3 a.u. 12 months after surgery, respectively. The t1/2 increased from 1.7 (1.32-2.24) s at baseline compared with 2.02 (1.68-2.42) s at 12 months after the surgery. Plasma viscosity and fibrinogen decreased from 1.50±0.093 mPa s and 3.0±0.41 g l(-1) at baseline to 1.407±0.062 mPa s and to 2.66±0.25 g l(-1) 12 months after surgery, respectively. AI correlated positively with BMI (r=0.74, P=0.001), waist circumference (r=0.68, P=0.005), fibrinogen (r=0.52, P=0.045) and plasma viscosity (r=0.76, P=0.001) and negatively with percentages of weight lost after surgery (r=-0.54, P=0.034). Multivariate analyses found that the BMI, fibrinogen and plasma viscosity independently influenced the AI. The study demonstrated that weight loss due to restrictive bariatric surgery can beneficially affect red cell aggregation parameters. The improvement of the RBC aggregation behaviours among obese subjects with weight loss due to LSG was associated with changes in plasmatic factors, especially fibrinogen.

The effect of pulsed IR-light on the rheological parameters of blood in vitro.

In this study we attempted to assess the effect of light of 855 nm wavelength (IR-light) on the rheological parameters of blood in vitro. As an anticoagulant, heparin was used. The source of IR-light was an applicator connected to the special generator--Viofor JPS®. The blood samples were irradiated for 30 min. During the irradiation the energy density was growing at twelve-second intervals starting from 1.06 J/cm2 to 8.46 J/cm2, then the energy density dropped to the initial value; the process was repeated cyclically. The study of blood viscosity was carried out with a Contraves LS40 oscillatory-rotational rheometer, with a decreasing shearing rate from 100 to 0.01 s⁻¹ over 5 min (flow curve) and applying constant frequency oscillations f=0.5 Hz with decreasing shear amplitude ˙γ0 (viscoelasticity measurements). The analysis of the results of rotational measurements was based on the assessment of hematocrit, plasma viscosity, whole blood viscosity at four selected shear rates and on the basis of the numerical values of parameters from Quemada's rheological model: k0 (indicating red cell aggregability), k∞ (indicating red cell rigidity) and ˙γc (the value of the shear rate for which the rouleaux formation begins). In oscillatory experiments we estimated viscous and elastic components of the complex blood viscosity in the same groups of patients. We observed a decrease of the viscous component of complex viscosity (η') at ˙γ0=0.2 s⁻¹, while other rheological parameters, k0, k∞, and relative blood viscosity at selected shear rates showed only a weak tendency towards smaller values after irradiation. The IR-light effect on the rheological properties of blood in vitro turned out to be rather neutral in the studied group of patients.

Description of a Phenomenon Resembling Spontaneous Echocardiographic Contrast in the Venous System

Introduction. —According to Beppu et al., spontaneous echocardiographic contrast (SEC or smoke) is a pattern of “spontaneous swirling echoes originally visualized by transthoracic echocardiography in the enlarged left atrium of patients with mitral stenosis” (J Am Cardiol 1985;6:744–749). It has since been primarily associated with atrial fibrillation and flutter. The clinical significance of SEC is its association with left atrial thrombus, increased thromboembolic complications, and death. The pathogenesis of SEC seems to involve Rouleau formation between red cells and fibrinogen at low shear rates, which is an in vitro equivalent of blood stasis. Black noted that “Red cell aggregation, manifested as SEC, appears to be a precursor to thrombosis. Left atrial thrombi are rich in fibrin and red cells, resembling venous more than arterial thrombi” (Echocardiography 2000;17(4):373–382). Methods. —If left atrial thrombi are associated with SEC and resemble venous thrombi, it was hypothesized that actual venous thrombi in previously undescribed locations, most notably deep vein thrombosis, may equally be associated with SEC. We asked our coauthors (H.T. and M-C.Y.) to search for such a phenomenon among patients referred to the vascular laboratory for suspected venous thrombosis. Case Presentation. —In the following case series, we report an original phenomenon of swirling echogenicity in the venous system that we believe is SEC. The phenomenon was fairly rare (maximum 1.5% of referred patients, whereas deep venous thrombosis itself was 15-20%). The clinical significance of this phenomenon is discussed.