Convolutional neural networks-based red blood cell detection and tracking with automatic data generation

It is crucial in microcirculatory investigation to evaluate red blood cell (RBC) deformability. However, there are almost limited information and indirect method of analyses. The authors tried to measure RBC deformability automatically by a combination of a newly developed algorithm and recording of individual RBC images clearly with an image-intensified high-speed video camera system. The length and the diameter of individual RBCs were measured automatically for RBC velocity measurement. RBC deformability was calculated from the histogram of density at each pixel by the authors' algorithm. To ensure reliability of this system the authors compared actual measurement with automatic analysis for RBC velocity and RBC deformability. Each correlation value (R/sup 2/=0.99) showed the validity of this system. In addition this system can shorten the time needed for measuring. The authors could confirm that the deformability of diabetic RBCs of HbAlc 12.1% was much more decreased compared with that of Hbalc 6.4%. The deformability of diabetic RBCs might depend on hyperglycemic degree of diabetic patients. In addition, the pentoxifylline improved the deformability of diabetic RBCs.

The Role of Red Blood Cells in Microvascular Blood Flow Alterations Caused by Gut Ischemia-Reperfusion Injury

Red blood cell (RBC) deformability is an essential component of microcirculatory function that appears to be enhanced by physiological shear stress, while being negatively affected by supraphysiological shears and/or free radical exposure. Given that blood contains RBCs with non-uniform physical properties, whether all cells equivalently tolerate mechanical and oxidative stresses remains poorly understood. We thus partitioned blood into old and young RBCs which were exposed to phenazine methosulfate (PMS) that generates intracellular superoxide and/or specific mechanical stress. Measured RBC deformability was lower in old compared to young RBCs. PMS increased total free radicals in both sub-populations, and RBC deformability decreased accordingly. Shear exposure did not affect reactive species in the sub-populations but reduced RBC nitric oxide synthase (NOS) activation and intriguingly increased RBC deformability in old RBCs. The co-application of PMS and shear exposure also improved cellular deformability in older cells previously exposed to reactive oxygen species (ROS), but not in younger cells. Outputs of NO generation appeared dependent on cell age; in general, stressors applied to younger RBCs tended to induce S-nitrosylation of RBC cytoskeletal proteins, while older RBCs tended to reflect markers of nitrosative stress. We thus present novel findings pertaining to the interplay of mechanical stress and redox metabolism in circulating RBC sub-populations.

Counting morphologically normal cells in human red blood cells (RBCs) is extremely beneficial in the health care field. We propose a three-dimensional (3-D) classification method of automatically determining the morphologically normal RBCs in the phase image of multiple human RBCs that are obtained by off-axis digital holographic microscopy (DHM). The RBC holograms are first recorded by DHM, and then the phase images of multiple RBCs are reconstructed by a computational numerical algorithm. To design the classifier, the three typical RBC shapes, which are stomatocyte, discocyte, and echinocyte, are used for training and testing. Nonmain or abnormal RBC shapes different from the three normal shapes are defined as the fourth category. Ten features, including projected surface area, average phase value, mean corpuscular hemoglobin, perimeter, mean corpuscular hemoglobin surface density, circularity, mean phase of center part, sphericity coefficient, elongation, and pallor, are extracted from each RBC after segmenting the reconstructed phase images by using a watershed transform algorithm. Moreover, four additional properties, such as projected surface area, perimeter, average phase value, and elongation, are measured from the inner part of each cell, which can give significant information beyond the previous 10 features for the separation of the RBC groups; these are verified in the experiment by the statistical method of Hotelling's T-quare test. We also apply the principal component analysis algorithm to reduce the dimension number of variables and establish the Gaussian mixture densities using the projected data with the first eight principal components. Consequently, the Gaussian mixtures are used to design the discriminant functions based on Bayesian decision theory. To improve the performance of the Bayes classifier and the accuracy of estimation of its error rate, the leaving-one-out technique is applied. Experimental results show that the proposed method can yield good results for calculating the percentage of each typical normal RBC shape in a reconstructed phase image of multiple RBCs that will be favorable to the analysis of RBC-related diseases. In addition, we show that the discrimination performance for the counting of normal shapes of RBCs can be improved by using 3-D features of an RBC.

Preliminary Pharmacodynamic Results from a Multicenter Phase 2/3 Study of Next-Generation HbS Polymerization Inhibitor GBT021601 for the Treatment of Patients with Sickle Cell Disease

Background: The development of abnormalities in red blood cell (RBC) deformability in patients undergoing hemodialysis remains a major problem, because it is related to peripheral microcirculation, oxygen supply, and various complications in such patients. γ-Linolenic acid (GLA; 18:3n-6), one of the polyunsaturated fatty acids and a precursor of prostaglandin E₁, is reported to have a favorable effect on the deformability of circulating blood cells in diabetic patients. Methods: In order to clarify the efficacy of GLA on RBC deformability in 7 patients undergoing maintenance hemodialysis, we examined in a pilot study the changes in the deformability of RBC and the changes in the phospholipid fatty acid composition in both plasma and RBC membrane before and after high-dose oral supplementation with GLA derived from Mucor circinelloides for 12 weeks. Results: Before supplementation, the micropore passage time of RBC suspension, which is an indicator of RBC deformability, in these patients was markedly longer than that in healthy control subjects. After administering GLA, the prolonged passage time of the patients both rapidly and steadily decreased and nearly reached control levels. Light microscopic observations of RBCs using Giemsa stain revealed a decreased number of poikilocytes after supplementation. An analysis of the fatty acid composition before treatment and 8 weeks after starting the treatment showed the dihomo-γ-linolenic acid (DGLA; 20:3n-6) level in the plasma to have increased (p < 0.05), while the arachidonic acid (AA; 20:4n-6) concentration in the RBC membrane decreased (p < 0.05). The level of DGLA in the RBC membrane, the level of GLA, and the ratio of GLA + DGLA/AA in plasma and RBC membrane did not change significantly; however, these all tended to increase. Conclusion: The results of this pilot study indicate that the oral supplementation of GLA extracted from M. circinelloides improves the poor RBC deformability in hemodialysis patients, partly by inducing changes in the composition of fatty acids in plasma and RBC membrane.

Ischemic diseases of people and animals are accompanied with deterioration of microrheologic properties of their blood, in particular, with impairing red blood cells (RBC) deformability. In this work, the analysis of human and rat RBC deformability in norm and ischemia was performed by means of the laser diffractometry - a modern technique allowing for measuring the flexibility of RBC, which determines the blood flow parameters in vessels. Human RBC were obtained from the blood of healthy individuals and from patients suffering from ischemic diseases. Human RBC deformability from both groups of individuals was measured. Rat RBC were obtained from a control group of animals and from a group with experimentally induced ischemia (EII). This animal model is frequently used for studying the response of an organism to ischemia. The effect of <i>Semax</i>, a medication that is frequently used for therapeutic treatments of human brain diseases in clinical practice, on RBC deformability was studied with its application in vitro and in vivo. It is shown that in human ischemic patients, the deformability of RBC was lower than that from healthy individuals. Both in vivo and in vitro applied semax positively influences the impaired deformability properties of RBC of ischemic rats.

Previous work in our laboratory has demonstrated a decrease in red blood cell (RBC) deformability in sepsis. This has not been studied following hemorrhagic shock. We tested the hypotheses that hemorrhagic shock, associated with soft tissue trauma, leads to decreased RBC deformability and that this is related to alterations in the resting shape of the RBC. Elongation index (EI), a measure of RBC deformability, was determined over a range of shear stresses from 0.3 to 30 Pa in 26 male rats before and at various times after 90 min of hemorrhagic shock. RBC resting shape was determined by scanning electron microscopy. The data demonstrate that EI decreased significantly at the end of shock (before resuscitation), and remained below normal throughout the 6-h postshock period. Eight of the 26 animals decompensated during shock, requiring return of a portion of the shed blood to maintain a mean arterial pressure of 30-40 mmHg. Four of eight decompensated animals died before the end of the study period, compared with none of the compensated rats. The decompensated rats had significantly lower EI at 0.3 Pa by the end of the shock period (0.050 +/- 0.009) than the compensated shock group (0.058 +/- 0.006; P < 0.05). RBC shape alterations were first demonstrated at the end of the shock period and persisted throughout the 6-h postshock resuscitation period. These data indicate that trauma and hemorrhagic shock cause RBC shape alterations and a significant decrease in RBC deformability, which becomes manifested during the shock period and persists for at least 6 h postshock. Additionally, a direct relationship appears to exist between the magnitude of the physiologic insult and the degree of RBC damage.

Mesenteric lymph duct ligation decreases red blood cell alterations caused by acute pancreatitis

Use of red cell deformability measurements to assess the impact of pyruvate kinase activators on combined drug and transfusion therapies

Red blood cell (RBC) deformability is a valuable hemorheological biomarker that can be used to assess the clinical status and response to therapy of individuals with sickle cell disease (SCD). RBC deformability has been measured by ektacytometry for decades, which uses shear or osmolar stress. However, ektacytometry is a population based measurement that does not detect small-fractions of abnormal RBCs. A single cell-based, functional RBC deformability assay would complement ektacytometry and provide additional information. Here, we tested the relative merits of the OcclusionChip, which measures RBC deformability by microcapillary occlusion, and ektacytometry. We tested samples containing glutaraldehyde-stiffened RBCs for up to 1% volume fraction; ektacytometry detected no significant change in Elongation Index (EI), while the OcclusionChip showed significant differences in Occlusion Index (OI). OcclusionChip detected a significant increase in OI in RBCs from an individual with sickle cell trait (SCT) and from a subject with SCD who received allogeneic hematopoietic stem cell transplant (HSCT), as the sample was taken from normoxic (pO2:159 mmHg) to physiologic hypoxic (pO2:45 mmHg) conditions. Oxygen gradient ektacytometry detected no difference in EI for SCT or HSCT. These results suggest that the single cell-based OcclusionChip enables detection of sickle hemoglobin (HbS)-related RBC abnormalities in SCT and SCD, particularly when the HbS level is low. We conclude that the OcclusionChip is complementary to the population based ektacytometry assays, and providing additional sensitivity and capacity to detect modest abnormalities in red cell function or small populations of abnormal red cells.

Hypertonicity and/or Acidosis Induce Marked Rheological Changes Under Hypoxic Conditions in Sickle Trait Red Blood Cells

Введение. Микрореологические свойства эритроцитов — их деформируемость и агрегация в значительной степени определяют текучесть цельной крови и ее кислородотранспортный потенциал. При этом эритроциты — это клетки-мишени, на которые действуют сигнальные молекулы. Последние могут регуляторно изменять микромеханические свойства эритроцитов и реологию крови в целом. К сигнальным молекулам относятся и газотрансмиттеры (ГТ) — такие соединения, как оксид азота (NO), сульфид водорода (H2S) и монооксид углерода (CO). Доноры ГТ могут быть основой для разработки лекарственных препаратов для коррекции реологических свойств крови. Цель работы: изучение изменений микрореологических характеристик эритроцитов под влиянием доноров трех газотрансмиттеров — NO, H2S и CO. Материалы и методы. Эритроциты и их восстановленные тени инкубировали с нитропруссидом натрия (НПН, 100 мкмоль) — донором NO, гидросульфидом натрия (NaHS, 100 мкмоль) — донором H2S и трикарбонилхлор (глицинат) рутением (II) (CORM-3, 50 мкмоль) — донором СО. После этого регистрировали деформируемость эритроцитов (ИУЭ), их агрегацию (ПАЭ) и вязкость суспензий (ВС). Результаты. Установлено, что под влиянием всех трех доноров ГТ происходят заметные изменения микрореологических характеристик эритроцитов и их теней (в среднем на 13–16%; p &lt; 0,05). Важно заметить, что сдвиги ИУЭ, ПАЭ и ВС под влиянием доноров газотрансмиттеров были сходными по величине, и суммарная разница их микрореологических эффектов на три разных донора ГТ не превышала 3%. Заключение. На основании полученных данных и их анализа можно заключить, что эритроциты качественно и количественно примерно одинаково отвечают на действие трех разных доноров ГТ положительными изменениями их микрореологических характеристик и особенно агрегации, которая снижалась более чем на 30%. Background. Erythrocytes microrheological properties (deformability and aggregation) largely determine the fluidity of whole blood and its oxygen transport potential. At the same time erythrocytes are target cells for signaling molecules acting. The latter can regulate erythrocytes micromechanical properties and blood rheology in general. Signaling molecules also include gasotransmitters (GT) — compounds such as nitrogen oxide (NO), hydrogen sulfide (H2S) and carbon monoxide (CO). GT donors can form the basis of drugs for blood rheology correction. Objectives: to study the changes of erythrocytes microrheological characteristics under the influence of donors of three gasotransmitters — NO, H2S and CO. Materials/Methods. Erythrocytes and their restored shadows were incubated with sodium nitroprusside (SNP, 100 μmol) — NO donor, sodium hydrosulfide (NaHS, 100 μmol) — H2S donor, and with tricarbonylchlor (glycinate) ruthenium (II) (CORM-3, 50 μmol) — CO donor. Thereafter we registered erythrocytes deformability (ED), their aggregation (EA) and suspensions viscosity (SV). Results. All three GT donors noticeably changed microrheological characteristics of erythrocytes and their shadows (on average by 13–16%; p &lt; 0.05). It’s important that the shifts of ED, EA, and SV under the influence of gasotransmitter donors were similar in magnitude, and the total difference in their microrheological effects on three different GT donors did not exceed 3%. Conclusions. Erythrocytes qualitatively and quantitatively approximately equally responded to the action of three different GT donors by positive changes in their microrheological characteristics and especially by aggregation that decreased by more than 30%.

Capillary blood vessels, the smallest vessels in the body, form an intricate network with constantly bifurcating, merging and winding vessels. Red blood cells (RBCs) must navigate through such complex microvascular networks in order to maintain tissue perfusion and oxygenation. Normal, healthy RBCs are extremely deformable and able to easily flow through narrow vessels. However, RBC deformability is reduced in many pathological conditions and during blood storage. The influence of reduced cell deformability on microvascular hemodynamics is not well established. Here we use a high-fidelity, 3D computational model of blood flow that retains exact geometric details of physiologically realistic microvascular networks, and deformation of every one of nearly a thousand RBCs flowing through the networks. We predict that reduced RBC deformability alters RBC trafficking with significant and heterogeneous changes in hematocrit. We quantify such changes along with RBC partitioning and lingering at vascular bifurcations, perfusion and vascular resistance, and wall shear stress. We elucidate the cellular-scale mechanisms that cause such changes. We show that such changes arise primarily due to the altered RBC dynamics at vascular bifurcations, as well as cross-stream migration. Less deformable cells tend to linger less at majority of bifurcations increasing the fraction of RBCs entering the higher flow branches. Changes in vascular resistance also seen to be heterogeneous and correlate with hematocrit changes. Furthermore, alteration in RBC dynamics is shown to cause localized changes in wall shear stress within vessels and near vascular bifurcations. Such heterogeneous and focal changes in hemodynamics may be the cause of morphological abnormalities in capillary vessel networks as observed in several diseases.

From temperature conditions in blood storage units to those observed in patients with severe thermal burns, it is obvious that the human blood cells are subjected to various temperature ranges and conditions during their lifespan. It is also known that temperature affects the ability of blood cell to transverse thin microcapillaries, although the extent remains unknown. In this study, we employed a three-dimensional (3D) nonlinear multiscale meshfree approach to investigate the effects of freezing and heating temperatures on the deformability of the human red blood cell (RBC). The optical tweezers experiment was numerically simulated in order to quantify the deformability of red blood cells as a function of the relationship between its deformed axial and transverse diameter. We observe that the deformability of red blood cell membrane decreases as temperature increases. It is concluded that increase in temperature leads to increase in membrane rigidity and decrease in overall membrane deformability, which may be due to the denaturation of RBC membrane underlying cytoskeleton protein.