Laser-assisted Optical Rotational Cell Analyzer Research Articles

Investigating the blood rheology in the first trimester pregnancies with high risk for preeclampsia.

Pregnancy is a dynamic process associated with changes in vascular and rheological resistance. Maternal maladaptation to these changes is the leading cause of pregnancy complications such as preeclampsia. This study aimed to assess the hemorheological alterations in pregnancies with a high risk for preeclampsia in the first trimester. Ninety-two pregnant women were allocated into the high preeclampsia risk group (37 cases) and control groups (55 cases). Plasma and whole blood viscosity and red blood cell morphodynamic properties, including deformability and aggregation were assessed by Brookfield viscometer and laser-assisted optical rotational cell analyzer (LORRCA) at 11-14 gestational weeks. Whole blood viscosity was significantly higher in the high-risk group at all shear rates. Plasma viscosity and hematologic factors showed no differences between the groups. Hematocrit levels positively correlated with high blood viscosity only in the high-risk group. There were no significant changes in the other deformability and aggregation parameters. Changes in the whole blood viscosity of pregnant women with high preeclampsia risk refer to impaired microcirculation beginning from the early weeks of gestation. We suggest that the whole blood viscosity is consistent with the preeclampsia risk assessment in the first trimester, and its measurement might be promising for identifying high-preeclampsia-risk pregnancies.

Blood-based protein biomarkers and red blood cell aggregation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant with a low 5-year survival rate. Blood biomarkers may be of value for the noninvasive diagnosis of pancreatic cancer. This study assessed blood-based biomarkers and disturbances in red blood cell aggregation associated with pancreatic cancer. We studied 61 patients who underwent pancreatic resection. Of these 61 patients, 46 patients had PDAC, and 15 patients had inflammatory tumours. Serum VEGF, hypoxia-inducible factor (HIF-1α), elastin-derived peptides (EDPs), total sialic acid (TSA) and resistin levels were measured. Red blood cell aggregation was assessed by a laser-assisted optical rotational cell analyser. VEGF (p < 0.000001), HIF-1α (p = 0.000002), resistin (p = 0.000349), EDP (p = 0.000089) and TSA (p = 0.000013) levels were significantly higher in the PDAC group than in the inflammatory tumour group. The aggregation index (AI), syllectogram amplitude (AMP) and threshold shear rate (γthr) were significantly higher in the PDAC group, whereas the aggregation half-time (t1/2) was lower than in the inflammatory tumour group. Multivariate analyses revealed that VEGF, TSA and EDP levels were variables that predicted PDAC. VEGF levels were the most powerful predictor of PDAC independent of CA 19-9 levels. The cut-off points for VEGF, TSA and EDP levels were 134.56 pg/ml, 109.11 mg/dl and 36.4 ng/ml, respectively, with sensitivities of 97.8%, 87% and 69.6%, respectively, and specificities of 86.7%, 86.7% and 93.3%, respectively. This study indicated that there are significant differences in blood-based biomarkers for differentiating between PDAC and inflammatory tumours of the pancreas. We also confirmed that PDAC is associated with the excessive aggregation of RBCs.

Zinc improved erythrocyte deformability and aggregation in patients with beta-thalassemia: An in vitro study.

Thalassemia patients have reduced red cell deformability and decreased plasma zinc levels in their blood. This study aimed to evaluate the effects of zinc (Zn) on the hemorheological parameters and antioxidant enzyme activities in β-thalassemia major (TM) and healthy volunteers (HV). Hemorheological parameters were measured using LORCA (laser-assisted optical rotational cell analyzer) after adjusting the hematocrit to 40%. Zinc sulfate (ZnSO4.7H2O) was used for Zn incubation with a concentration of 0.5μg/dl. Oxidative stress and antioxidant status were determined using commercial kits. Data showed that after Zn incubation, EImax, the area under the EI-osmolarity curve (Area), and Omax decreased in TM. However, no significant difference was observed in the osmotic deformability parameters of HV. The increased elongation index was obtained at different shear stresses for TM and HV, and SS1/2 decreased in both groups. The AMP and aggregation index (AI) decreased in TM, and the required time for half of the maximum aggregation (t1/2) increased in HV. However, Zn did not affect oxidative parameters in both groups. This study showed that Zn incubation increased deformability and decreased aggregation in thalassemic erythrocytes. It means that Zn supplementation will contribute to microcirculation in thalassemia patients.

L-Arginine-eNOS-NO Functional System in Brain Damage and Cognitive Impairments in Cerebral Small Vessel Disease.

Cerebral small vessel disease (CSVD) is a significant cause of cognitive impairment (CI), disability, and mortality. The insufficient effectiveness of antihypertensive therapy in curbing the disease justifies the search for potential targets for modifying therapy and indicators supporting its use. Using a laser-assisted optical rotational cell analyzer (LORRCA, Mechatronics, The Netherlands), the rheological properties and deformability of erythrocytes before and after incubation with 10 μmol/L of L-arginine, the nitric oxide (NO) donor, blood-brain barrier (BBB) permeability assessed by dynamic contrast-enhanced MRI, clinical, and MRI signs were studied in 73 patients with CSVD (48 women, mean age 60.1 ± 6.5 years). The control group consisted of 19 volunteers (14 women (73.7%), mean age 56.9 ± 6.4 years). The erythrocyte disaggregation rate (y-dis) after incubation with L-arginine showed better performance than other rheological characteristics in differentiating patients with reduced NO bioavailability/NO deficiency by its threshold values. Patients with y-dis > 113 s-1 had more severe CI, arterial hypertension, white matter lesions, and increased BBB permeability in grey matter and normal-appearing white matter (NAWM). A test to assess changes in the erythrocyte disaggregation rate after incubation with L-arginine can be used to identify patients with impaired NO bioavailability. L-arginine may be part of a therapeutic strategy for CSVD with CI.

Hyperbaric oxygenation improve red blood cell deformability in patients with acute or chronic inflammation.

Red blood cells (RBC) are one of the key elements of the microcirculation. Their ability to pass through capillaries and to deliver oxygen to cells is due to their large degree of deformability linked to the characteristics of the RBC membrane. Alterations in RBC deformability as a result of membrane damage, linked in part to increased synthesis of reactive oxygen species (ROS), can be observed in several diseases, such as sepsis, and may contribute to the altered microcirculation observed in these pathologies. Hyperbaric oxygen therapy (HBOT), with inhalation of 100% oxygen, has been proposed in several acute or chronic pathologies, including carbon monoxide poisoning. We investigated the effects of HBOT on oxidative stress from ROS produced by myeloperoxidase (MPO) and on RBC deformability in patients with acute or chronic inflammation (n=10), in patients with acute carbon monoxide poisoning (n=10), and in healthy volunteers (n=10). RBC deformability was evaluated before and after HBOT in the various populations using the ektacytometry technique (Laser-assisted Optical Rotational Red Cell Analyzer - LORRCA). Deformability was determined by the elongation index (EI) in relation to the shear stress (SS) over a range of 0.3 to 50Pa. Oxidative stress was estimated through changes in proteins (chlorotyrosine and homocitrulline) induced by MPO activity measured by liquid chromatography-tandem mass spectrometry analysis. Before HBOT, EI was significantly lower in patients with acute or chronic inflammation than in healthy volunteers and patients with acute carbon monoxide poisoning for the majority of SS values studied. After one session of HBOT, the EI was significantly higher than before HBOT for SS values of 1.93Pa or higher in patients with acute or chronic inflammation. This effect remains constant after 10 sessions. There were no differences before and after HBOT in protein or amino acid oxidation due to ROS generation mediated by MPO in the three populations. Our results confirm altered RBC deformability in patients with acute and chronic conditions associated with an underlying inflammatory process. HBOT improves deformability only after one session and therefore may improve microcirculation in this population. According to our results, this improvement does not seem mediated by the ROS pathway via MPO. These results need to be confirmed in a larger population.

5610325 VARIABILITY OF OXYGENSCAN PARAMETERS ACCORDING TO THE TYPE OF THERAPY IN CHILDREN WITH SICKLE CELL DISEASE: PRELIMINARY DATA FROM A FEASIBILITY STUDY

Background: Sickle Cell Disease (SCD) is characterized by significant phenotypic variability even when the same causative mutation is present (1). Validated biomarkers for routine use in SCD, to evaluate phenotypic severity and predict the evolution of organ damage and response to different therapies, are currently lacking. Oxygenscan (function of the instrument Laser-assisted Optical Rotational Red Cell Analyser) is a novel physiological assay of red blood cells’ deformability at different oxygen tensions; it allows the measurement of parameters such as maximum/minimum elongation index e point of sickling (2,3). Aims: This feasibility study aims at preliminarily evaluating the variability of the point of sickling, as a possible biomarker in SCD, with respect to different clinical conditions and types of treatment. Methods: We’ve prospectively gathered peripheral blood samples of pediatric patients with SCD followed in the Veneto Region Pediatric Sickle Cell Disease Reference Center of Padua University Hospital. The Oxygenscan test has been repeated twice for each sample. Results: We’ve analysed 67 blood samples of 48 pediatric patients with SCD (36 HbSS, 5 HbSbeta0, 7 HbSC). Patients were not receiving a disease-modifying therapy (n.11) or were on hydroxyurea (HU) (n.32), on HU and chronic transfusion regimen (n.2), HU and crizanlizumab (n.2), or after HSCT (n.2, one of them had been evaluated also before HSCT). Parameters obtained by Oxygenscan seem to vary according to the type of treatment received by patients (Figure 1).Conclusion: A higher number of samples and further analyses will be necessary to investigate a possible relationship between the point of sickling and clinical data such as the evolution of organ damage and response to different types of treatment.

The Changes of Elongation Index of erythrocytes caused by storage of blood at low temperature

Abstract Introduction: An important parameter characterizing the ability of erythrocytes to deform depending on the blood flow conditions is the Elongation Index (EI), and it is a parameter defined by the shape of the erythrocyte obtained as a diffraction pattern of erythrocytes at different values of shear stresses. Material and methods: EI measurements at different shear stress were performed by Laser-assisted Optical Rotational Cell Analyzer (LORRCA) for erythrocytes derived from Tissue Bank in Katowice. Measurements were performed immediately after receiving them from Tissue Bank and after 2, 9, and 28 days of storage of samples at the temperature of 4°C in solution with the anticoagulant. Results: An increase in the erythrocytes Elongation Index in the first 9 days of storing samples at low temperatures was observed in the entire range of applied shear stresses. This indicates an increase in the elasticity of erythrocytes during short-term storage at 4°C. In turn, on the 28th day of erythrocyte storage, a significant decrease in the Elongation Index for shear stresses greater than 1 Pa was observed, which indicates the stiffening of the erythrocyte membrane structure, reducing their elasticity. The relative decrease in the Elongation Index of erythrocytes stored for 28 days compared to erythrocytes measured at the beginning was similar and slightly greater than 30% for shear stresses greater than 3 Pa. For shear stresses lower than 3 Pa, the relative change in elongation index was smaller than for shear stresses greater than 3 Pa and increased with the increase in shear stress. Conclusions: The elongation index of erythrocytes stored in the anticoagulant solution at 4°C, initially increases in the entire range of applied shear stresses in the first few days from the moment of blood collection and preparation at the Tissue Bank, and then decreases, but on the ninth day of storage the elongation index is still higher than for blood immediately after collection.

Evaluation of Red Blood Cell Metabolism in Patients with Low-Risk Myelodysplastic Syndrome (LR-MDS): A Proof-of-Concept Study

Low risk myelodysplastic syndromes (MDS) show high clinical heterogeneity ranging from mild anemia with nearly normal performance status to life-threatening cytopenias, frequent hospitalization, and disability. Anemia occurs in 80%-85% of patients and therapeutic strategies are still very limited mainly consisting of of erythropoiesis stimulating agents and red blood cell (RBC) transfusion support. Starting from the working hypothesis that the defective maturation of hematopoietic precursors may dampen the "functionality" of MDS-RBCs, we aimed at evaluating RBC metabolic pathway in anemic patients with LR-MDS. We focused on the glycolytic pathway, which is the prominent metabolic cascade in RBCs and is currently targeted with allosteric stimulators of pyruvate kinase (PK) enzyme in clinical trials of PK deficiency, thalassemia, and sickle cell diseases, which are also marked by ineffective erythropoiesis like MDS. Sixty-six consecutive anemic patients (i.e. Hb<11 g/dL) diagnosed with MDS according to WHO 2016 classification that met IPSS-R criteria for very low, low, or intermediate-risk MDS were enrolled in the study. All patients were sampled for peripheral blood at least 4 weeks after the last RBC unit to reduce possible blood donor contamination. The determination of activities of the glycolytic RBC enzymes pyruvate kinase (PK) and hexokinase (HK) (the terminal and initial steps of the glycolytic pathway), and of ATP levels was performed by standard spectrophotometric method according to Beutler et al, 1984. 2,3 DPG determination was tested by AssayGenie Kit in a subgroup of patients. Ektacytometric analysis by LoRRca MaxSis (Laser-Assisted Optical Rotational Cell Analyzer, Mechatronics, NL) was also performed in a subgroup of patients. As shown in table 1, patients were mainly elderly males, and mostly belonged to IPSS low (78%) and IPSS-R very low/low groups (85%). Median levels of PK activity were 13.7 IU/gHb, with a wide range (7.5-35.9) and were reduced in 23 cases (35%; normal reference range 11.9-16.7 IU/gHb); median values of HK activity were 2.3 (0.8-8.1), increased in 60 patients (91%; normal reference range 0.78-1.32 IU/gHb). This resulted in an abnormally reduced PK/HK ratio (median 5.45, range 2-15) in 63 subjects (95%; (normal reference range calculated on 167 normal subjects: 11.55 - 17.65). To asses if impaired glycolysis may result in altered RBC deformability or in lack of energy, ATP levels, 2,3 DPG, and ektacytometric curve were evaluated. Median ATP levels were 3.4 mcM/gHb (1.3-6.5), reduced in 33/56 tested patients (59%; normal reference range 3.6-4.9 mcM/gHb), of whom 32 had simultaneous reduction of PK/HK activity ratio (figure 1), and 11 decreased PK activity. Concerning 2,3 DPG levels preliminarily analysed in a subgroup of patients, they were close to the upper limit of the normal range (median, 389 nM/gHb, 318-639). Osmoscan, performed in 10 patients did not show altered RBC deformability. Preliminary data show decreased glycolytic activity in RBCs from patients with LR-MDS highlighted by reduced PK activity in more than 1/3 of cases, and increased HK activity in almost all subjects. This results in reduced RBC ATP content in up to 2/3 of patients (possibly due to reduced production or to increased consumption) indicating an overall lack of energy in MDS-RBCs. The latter may be an interesting target for allosteric PK stimulators and warrants further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Red Blood Cell Morphodynamics in Patients with Polycythemia Vera and Stroke.

Polycythemia vera (PV) is a Ph-negative myeloproliferative neoplasm (MPN) which is characterized by erythrocytosis and a high incidence of thrombotic complications, including stroke. The study aimed to evaluate red blood cell (RBC) morphodynamic properties in PV patients and their possible association with stroke. We enrolled 48 patients with PV in this cross-sectional study, 13 of which have a history of ischemic stroke. The control group consisted of 90 healthy subjects. RBC deformability and aggregation analysis were performed using a laser-assisted optical rotational red cell analyzer. The following parameters were calculated: aggregation amplitude (Amp), RBC rouleaux formation time constant (Tf), time of formation of three-dimensional aggregates (Ts), aggregation index (AI), rate of complete disaggregation (y-dis), and the maximal elongation of RBC (EImax). Statistical analysis was performed with the R programming language. There were significant differences in RBCs morphodynamics features between patients with PV and the control group. Lower EImax (0.47 (0.44; 0.51) vs. 0.51 (0.47; 0.54), p < 0.001) and γ-dis (100 (100; 140) vs. 140 (106; 188) s−1, p < 0.001) along with higher amplitude (10.1 (8.6; 12.2) vs. 7.7 (6.6; 9.2), p < 0.001) was seen in patients with PV compared with control. A statistically significant difference between PV patients with and without stroke in aggregation amplitude was found (p = 0.03). A logistic regression model for stroke was built based on RBC morphodynamics which performed reasonably well (p = 0.01). RBC alterations may be associated with overt cerebrovascular disease in PV, suggesting a possible link between erythrocyte morphodynamics and increased risk of stroke.

Calcium/protein kinase C signaling mechanisms in shear-induced mechanical responses of red blood cells

Red blood cell (RBC) deformability has vital importance for microcirculation in the body, as RBCs travel in narrow capillaries under shear stress. Deformability can be defined as a remarkable cell ability to change shape in response to an external force which allows the cell to pass through the narrowest blood capillaries. Previous studies showed that RBC deformability could be regulated by Ca2+/protein kinase C (PKC) signaling mechanisms due to the phosphorylative changes in RBC membrane proteins by kinases and phosphatases. We investigated the roles of Ca2+/PKC signaling pathway on RBC mechanical responses and impaired RBC deformability under continuous shear stress (SS). A protein kinase C inhibitor Chelerythrine, a tyrosine phosphatase inhibitor Calpeptin, and a calcium channel blocker Verapamil were applied into human blood samples in 1 micromolar concentration. Samples with drugs were treated with or without 3 mM Ca2+. A shear stress at 5 Pa level was applied to each sample continuously for 300 s. RBC deformability was measured by a laser-assisted optical rotational cell analyzer (LORRCA) and was calculated as the change in elongation index (EI) of RBC upon a range of shear stress (SS, 0.3–50 Pa). RBC mechanical stress responses were evaluated before and after continuous SS through the parameterization of EI-SS curves. The drug administrations did not produce any significant alterations in RBC mechanical responses when they were applied alone. However, the application of the drugs together with Ca2+ substantially increased RBC deformability compared to calcium alone. Verapamil significantly improved Ca2+-induced impairments of deformability both before and after 5 Pa SS exposure (p < 0.0001). Calpeptin and Chelerythrine significantly ameliorated impaired deformability only after continuous SS (p < 0.05). Shear-induced improvements of deformability were conserved by the drug administrations although shear-induced deformability was impaired when the drugs were applied with calcium. The blocking of Ca2+ channel by Verapamil improved impaired RBC mechanical responses independent of the SS effect. The inhibition of tyrosine phosphatase and protein kinase C by Calpeptin and Chelerythrine, respectively, exhibited ameliorating effects on calcium-impaired deformability with the contribution of shear stress. The modulation of Ca2+/PKC signaling pathway could regulate the mechanical stress responses of RBCs when cells are under continuous SS exposure. Shear-induced improvements in the mechanical properties of RBCs by this signaling mechanism could facilitate RBC flow in the microcirculation of pathophysiological disorders, wherein Ca2+ homeostasis is disturbed and RBC deformability is reduced.

Changes in Red Blood Cell Properties and Platelet Function during Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) is associated with frequent hemorrhagic and thromboembolic complications. The multiple effects of ECMO include inflammatory response on contact with the circuit; hemolysis acquired von Willebrand syndrome likely affects the function of red blood cells (RBC) and platelets. The aim of this prospective observational study was to analyze RBC aggregation and elongation (deformability) and platelet aggregation in the first week of ECMO. Sixteen patients were included. Blood samples were taken prior to initiation of ECMO and on days 1, 2, 3, 5, and 7. RBC aggregation and elongation were analyzed using the laser-assisted optical rotational red cell analyzer (Lorrca). Upstroke, top, and amplitude as indices of aggregation showed significant time effects. RBC elongation was not affected at low shear stress. At high shear stress there was an increase in the elongation index at day 2 (p = 0.004), followed by a decrease. Platelet function was analyzed using multiple electrode aggregometry (Multiplate®). In pairwise comparison in the days 1–7 to the value prior to ECMO there was no significant difference in platelet aggregation by any of the three agonists (ADP p = 0.61; TRAP p = 0.77; Ristocetin p = 0.25). This implies that the rheology of RBCs seemed to be more affected by ECMO than platelets. Especially the red blood cell deformability continues to decline at higher shear stress.

PS1198 MOLECULAR DIAGNOSIS OF RARE CONGENITAL ANEMIAS USING TARGETED NEXT-GENERATION SEQUENCING IN INDIAN PATIENTS

Background: Hereditary spherocytosis (HS) is one of the most frequent hereditary hemolytic disorders in Caucasian populations. With the emergence of the eosin-5’-maleimide (EMA) binding test, osmotic gradient ektacytometry, and targeted next-generation sequencing (tNGS), the role of single and combined diagnostic analyses remains unclear. Aims: To improve accuracy, robustness, and cost-effectiveness of the laboratory diagnosis of HS by the EMA binding test and osmotic gradient ektacytometry, individually and in combination, using the occurrence of diagnostic cytoskeleton protein gene mutations determined by tNGS as confirmation of HS. Methods: We included samples from all individuals referred to our laboratory with suspected HS between May 1st, 2017 and July 1st, 2018. In addition to a modified EMA-binding test using commercial Rainbow Beads as reference, we performed osmotic gradient ektacytometry using the Osmoscan module of the Laser-assisted Optical Rotational Deformability Cell Analyser (LoRRca MaxSis, RR Mechatronics, The Netherlands), as well as a comprehensive tNGS membranopathy panel (including α-spectrin, β-spectrin, ankyrin, band 3, protein 4.1, and protein 4.2). HS was defined as the occurrence of diagnostic cytoskeleton protein gene mutations. Results: A total of 99 individuals were included in the study. Of these, 41 individuals had no proven mutations in red blood cell cytoskeleton protein genes, 34 had one or more diagnostic mutations, and 24 had one or more variants of uncertain significance (VUS). We identified a total 78 different diagnostic mutations and VUS, including 42 novel mutations. The table depicts the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the EMA binding test as well as the two ektacytometry-derived parameters Omin (osmotic resistance) and the maximal elongantion index (EImax). Rated individually, the ektacytometry-derived parameter EImax (<0.579) gave the highest accuracy (91%), when comparing results from individuals with diagnostic mutations and individuals with no mutations. When combining Omin >166 mOsm/kg and EImax <0.6, the highest sensitivity (100%) and accuracy (94.7 %) was reached. Twenty-seven of 34 individuals with diagnostic mutations had EMA ≥40, which was the optimal threshold in receiver operating characteristic (ROC) analysis. Five of 7 individuals with diagnostic mutations and EMA values <40 had mutations only in the SPTA1 gene.Summary/Conclusion: Individually, osmotic gradient ektacytometry was superior to the EMA binding test in the laboratory diagnosis of HS. In individuals with EMA ≥40 combined with Omin and EImax values of ≥166 mOsm/kg and <0.6, respectively, tNGS provided no additional value in the laboratory diagnosis of HS. In cases of discrepancy between results from the EMA binding test and selected Omin and EImax values, DNA testing may help explain this divergence.

The relationship between the rheological behavior of RBCs and angiogenesis in the morbidly obese.

The aim of this study was to investigate the relationship between red blood cell (RBC) aggregation and deformability and angiogenesis parameters in obese patients. We studied 35 obese subjects and 20 non-obese people as a control group. Angiogenesis was detected using Bio-Plex Pro Human Angiogenesis Multiplex Assays. The RBC aggregation and deformability of the red blood cell aggregation were performed by the Laser-assisted Optical Rotational Cell Analyser - LORCA. The aggregation index and the syllectogram's amplitude were significantly higher in the obese patients, whereas the aggregation half-time (t1/2) was lower compared with the control group. The deformability of RBC expressed as EI was significantly lower in the obese group than it was in the control group. All angiogenesis parameters were higher in obese individuals than they were in the control group. Significant differences were observed in angiopoietin 2 (p = 0.048), folistin (p = 0.0017), G-CSF (p = 0.042), HGF (p = 0.016), and PECAM-1 (p = 0.014). The VEGF tended to be higher in the obese patients than in the control group (p = 0.09); nevertheless, the concentration of PDGF-BB was similar in both groups. EI at shear stresses of 18.49 Pa and 30.2 Pa was strongly correlated with all angiogenesis parameters. No correlations were found between the studied RBC aggregation indices and angiogenesis parameters. Multivariate analyses indicated that only HGF was an independent predictor of RBC deformability at 18.49 Pa (β-0.83, P < 0.000005) and at 30.2 Pa (β-0.83, P < 0.00005). The study found that there are relationships between enhanced RBC rigidity and angiogenesis status in obese subjects. Because this correlation between angiogenesis and RBC deformability is presented for the first time, the physiological importance of the relationship requires further research.

Effects of Ankaferd Hemostat on red blood cell aggregation: a hemorheological study

Background/aim Ankaferd hemostat (ABS; Ankaferd blood stopper, İstanbul, Turkey) is a prohemostatic agent affecting erythrocytes. The hemostatic action of ABS depends upon fibrinogen gamma chain, prothrombin, and red blood cells. The aim of this study was to assess the effects of ABS on erythrocyte aggregation via hemorheological analyses.Materials and methods To measure erythrocyte aggregation, blood samples were obtained from healthy, nonsmoker volunteers who had not taken any medication in the previous 10 days. One mL of blood was placed into the laser-assisted optical rotational cell analyzer (LORCA), into the chamber formed by the gap between two concentric glass cylinders. The solution prepared with ABS and saline was added to blood in incremental amounts of 10 µL, 20 µL, 30 µL, 40 µL, 50 µL, 60 µL, 70 µL, and 100 µL. Erythrocyte aggregation was determined by laser-assisted optical rotational cell analyzer at 37 °C. Results AMP was found to be 17.7 ± 2.1 au in the blood without ABS, whereas it was lower in the blood with ABS. AMP was 16.0 ± 3.3 in the ABS-added blood group. RBC aggregates did not form faster when cells contacted ABS. The t t½ value was 4.6 ± 2.6 in the ABS-added blood group and 1.9 ± 0.20 in the control group. Aggregation was faster in the control group (P = 0.03). AI, which is a combination of AMP and t½, was lowered in the ABS group (48.7 ± 12.3) compared to the control group (65.8 ± 1.6) (P = 0.02). It was notable that the γIsc max (sec-1) value of the control was higher (200 ± 106) than the ABS-added blood group (141 ± 51.0). Conclusion ABS has antierythroid aggregation effect. ABS inhibits pathological aggregation of red blood cells. Antithrombotic clinical effects of ABS may be ascribed to the antierythroid aggregan actions of the drug.

Use of Laser Assisted Optical Rotational Cell Analyzer (LoRRca MaxSis) in the Diagnosis of RBC Membrane Disorders, Enzyme Defects, and Congenital Dyserythropoietic Anemias: A Monocentric Study on 202 Patients.

Chronic hemolytic anemias are a group of heterogeneous diseases mainly due to abnormalities of red cell (RBC) membrane and metabolism. The more common RBC membrane disorders, classified on the basis of blood smear morphology, are hereditary spherocytosis (HS), elliptocytosis, and hereditary stomatocytoses (HSt). Among RBC enzymopathies, the most frequent is pyruvate kinase (PK) deficiency, followed by glucose-6-phosphate isomerase, pyrimidine 5′ nucleotidase P5′N, and other rare enzymes defects. Because of the rarity and heterogeneity of these diseases, diagnosis may be often challenging despite the availability of a variety of laboratory tests. The ektacytometer laser-assisted optical rotational cell analyser (LoRRca MaxSis), able to assess the RBC deformability in osmotic gradient conditions (Osmoscan analysis), is a useful diagnostic tool for RBC membrane disorders and in particular for the identification of hereditary stomatocytosis. Few data are so far available in other hemolytic anemias. We evaluated the diagnostic power of LoRRca MaxSis in a large series of 140 patients affected by RBC membrane disorders, 37 by enzymopathies, and 16 by congenital diserythropoietic anemia type II. Moreover, nine patients with paroxysmal nocturnal hemoglobinuria (PNH) were also investigated. All the hereditary spherocytoses, regardless the biochemical defect, showed altered Osmoscan curves, with a decreased Elongation Index (EI) max and right shifted Omin; hereditary elliptocytosis (HE) displayed a trapezoidal curve and decreased EImax. Dehydrated hereditary stomatocytosis (DHSt) caused by PIEZO1 mutations was characterized by left-shifted curve, whereas KCNN4 mutations were associated with a normal curve. Congenital diserythropoietic anemia type II and RBC enzymopathies had Osmoscan curve within the normal range except for glucosephosphate isomerase (GPI) deficient cases who displayed an enlarged curve associated with significantly increased Ohyper, offering a new diagnostic tool for this rare enzyme defect. The Osmoscan analysis performed by LoRRca MaxSis represents a useful and feasible first step screening test for specialized centers involved in the diagnosis of hemolytic anemias. However, the results should be interpreted by caution because different factors (i.e., splenectomy or coexistent diseases) may interfere with the analysis; additional tests or molecular investigations are therefore needed to confirm the diagnosis.

Impact of systemic cryotherapy on the rheological properties of the blood in women with rheumatoid arthritis

Study aim: The aim of this study was to assess the effects of systemic cryotherapy on the rheological properties of the blood in women with rheumatoid arthritis. Study group: The study group consisted of 10 women with rheumatoid arthritis, aged 57.2 ± 9.4, who underwent systemic cryotherapy treatments (3 min treatment time, -120 °C chamber temperature, 10 treatment sessions - 5 times a week). Their average body height was 165.5 ± 4.6 cm, weight 68.5 ± 4.9 kg and BMI 24.8 ± 2.2 kg/m2. In order to analyze morphological and rheological parameters of the blood, venous blood samples were drawn from the participants of the study twice. The first study was held on the day of beginning treatments and the second test was conducted after a series of 10 treatments. Methodology: The morphological blood test - measurements were taken using the ABX MICROS 60 (USA) hematology analyser. Erythrocyte deformability and aggregation were tested using the LORCA analyser (Laser-assisted Optical Rotational Cell Analyser RR Mechatronics, The Netherlands). The results were obtained as the index of elongation and aggregation according to the Hardeman method (2001). Results: Analysing the average values of morphological and rheological parameters of the blood in women with rheumatoid arthritis in the study group, the mean values of RBC, Hct and AI following the series of 10 treatments were significantly higher after cryotherapy in comparison to the measurements taken before treatments. Analysing the mean concentrations of T½, there was statistically significant reduction after the series of 10 treatments. Conclusions: Regular usage of cryotherapy treatments may affect the levels of morphological and rheological parameters of the blood in women with rheumatoid arthritis - RBC, Hct and AI (increase) and T½ (reduction) in the blood. null

Comparison of the simple red blood cell adhesiveness/aggregation test with the laser-assisted optical rotational cell analyzer: Red blood cell aggregation in patients with coronary artery disease and a healthy control group.

Red blood cell (RBC) aggregation influences blood flow properties, impacts blood microcirculation and consequently oxygen delivery. Different methods are established to determine RBC aggregation: under static conditions (i.e. the RBC adhesiveness/aggregation test (EAAT)) or under shear conditions (i.e. the laser-assisted optical rotational cell analyzer (LORCA)). Comparison of these two different methods in detecting the RBC aggregation of patients with coronary artery disease (CAD) and of healthy controls. RBC aggregation was quantified in peripheral venous blood of patients with CAD and healthy controls using EAAT and LORCA. Both methods detected an increased RBC aggregation in patients with CAD compared to the healthy control group: the ratio of clot-free area to whole area (rCFA) detected with EAAT (15.65 vs. 11.30%), and aggregation index (66.33 vs. 53.90%), shear rate of disaggregation (SDA) (105.59 vs. 69.21 s-1), and upstroke/ttop (0.03 vs. 0.02 au/s) detected with LORCA device were increased, aggregation half time (detected with LORCA) was decreased (2.11 vs. 3.60 s). rCFA (EAAT) correlated with SDA (LORCA). Both methods determine an increased RBC aggregation in patients with CAD. However, only one measurement parameter of the LORCA seems to reflect the same RBC aggregation properties as the EAAT.

Comparison of endothelin and nitric oxide synthase blockers on hemorheological parameters in endotoxemic rats.

Septic shock is an important health problem that vastly alters cardiovascular and hemodynamic status. Increased production of nitric oxide (NO) and endothelin is a counterpart of this endotoxemic state. This study was conducted to test the hypothesis that nonselective NO synthesis blocker (L-NAME), inducible NO synthesis blocker (L-canavanine), or endothelin receptor antagonist (bosentan) will reverse the effects of sepsis on hemorheological parameters. Forty-eight male Sprague-Dawley rats were used in 8 groups: saline (control), endotoxin, bosentan, L-NAME, L-canavanine, endotoxin + bosentan, endotoxin + L-NAME, and endotoxin + L-canavanine. Blood was withdrawn at the 4th hour of endotoxemic state. Erythrocyte deformability and erythrocyte aggregation were determined by laser-assisted optical rotational cell analyzer at 37 °C. Plasma viscosity (mPa.s) was measured by a cone-plate viscometer with 0.5 mL of plasma. Endotoxin administration significantly increased aggregation half-time and lowered erythrocyte aggregation amplitude and aggregation index compared to the control, indicating a slower and weaker aggregation pattern. L-NAME and L-canavanine alleviated the effects of endotoxin on erythrocyte aggregation without altering the values in the control animals. However, bosentan did not perform such a restoration. This finding suggests that these restoration effects of the blockers occur via their modulation of nitric oxide synthesis rather than through the endothelin pathway.

Men with Sickle Cell Anemia and Priapism Exhibit Increased Hemolytic Rate, Decreased Red Blood Cell Deformability and Increased Red Blood Cell Aggregate Strength.

ObjectivesTo investigate the association between priapism in men with sickle cell anemia (SCA) and hemorheological and hemolytical parameters.Materials and MethodsFifty-eight men with SCA (median age: 38 years) were included; 28 who had experienced priapism at least once during their life (priapism group) and 30 who never experienced this complication (control group). Twenty-two patients were treated with hydroxycarbamide, 11 in each group. All patients were at steady state at the time of inclusion. Hematological and biochemical parameters were obtained through routine procedures. The Laser-assisted Optical Rotational Cell Analyzer was used to measure red blood cell (RBC) deformability at 30 Pa (ektacytometry) and RBC aggregation properties (laser backscatter versus time). Blood viscosity was measured at a shear rate of 225 s-1 using a cone/plate viscometer. A principal component analysis was performed on 4 hemolytic markers (i.e., lactate dehydrogenase (LDH), aspartate aminotransferase (ASAT), total bilirubin (BIL) levels and reticulocyte (RET) percentage) to calculate a hemolytic index.ResultsCompared to the control group, patients with priapism exhibited higher ASAT (p = 0.01), LDH (p = 0.03), RET (p = 0.03) levels and hemolytic indices (p = 0.02). Higher RBC aggregates strength (p = 0.01) and lower RBC deformability (p = 0.005) were observed in patients with priapism compared to controls. After removing the hydroxycarbamide-treated patients, RBC deformability (p = 0.01) and RBC aggregate strength (p = 0.03) were still different between the two groups, and patients with priapism exhibited significantly higher hemolytic indices (p = 0.01) than controls.ConclusionOur results confirm that priapism in SCA is associated with higher hemolytic rates and show for the first time that this complication is also associated with higher RBC aggregate strength and lower RBC deformability.

Acute and long-term effects of hyperbaric oxygen therapy on hemorheological parameters in patients with various disorders.

Inhalation of 100% oxygen in a hyperbaric chamber has been accepted as a useful treatment for patients with various pathologies who suffer from hypoxia. The oxidative effects of hyperbaric oxygen (HBO) on RBCs have been investigated in animals but there is not enough data on hemorheological parameters in patients following HBO treatment (HBOT).In this study, we investigated the effect of HBO on hemorheological and haematological parameters during treatment. Red blood cell (RBC) deformability and aggregation, blood and plasma viscosity and superoxide dismutase activity were investigated in patients who underwent HBOT. Hematological parameters were determined by an electronic hematology analyzer. A Laser-assisted Optical Rotational Cell Analyzer (LORCA) was used to measure RBC deformability. RBC aggregation was measured for cells in autologous plasma and for cells resuspended in PBS containing Dextran70 (3% ) by using a Myrenne Aggregometer. A Wells-Brookfield cone/plate rotational viscometer was used for viscosity measurements. According to our results, a significant decrement of the hematocrit and the RBC count was observed after the 20th session of HBOT compared to the baseline, but none of the hemorheological parameters changed significantly. Our results showed that HBOT did not cause any significant changes in hemorheological parameters, thereby not representing any problems for the patients.