Peripheral blood erythrocytes morphology in ovarian cancer

Aim. To assess the morphology of circulating red blood cells in patients with stage III ovarian cancer. Methods. The object of the study was the peripheral blood erythrocytes of primary patients with ovarian cancer (n=25) who had stage III according to International Federation of Gynecology and Obstetrics. Patients were examined in the gynecological department of Ulyanovsk Regional Clinical Oncology Center. The control group included 25 somatically healthy women. Morphological studies were performed using light microscopy. The number of red blood cells with an unchanged and altered shape was expressed as a percentage. By the method of atomic force microscopy, the topology and rigidity of red blood cells was studied. Results. A statistically significant decrease in the number of circulating blood erythrocytes was found in patients with ovarian cancer compared to somatically healthy women. At the same time, the number of discocytes is markedly reduced while the number of morphologically altered forms: echinocytes, stomatocytes, spherocytes and erythrocyte rigidity are increased. Conclusion. With the appearance of altered forms of red blood cells and increase of the transformation index and erythrocyte rigidity in patients with stage III ovarian cancer, total number of red blood cells decreases in circulating blood compared to somatically healthy women.

Erythrocyte Rigidity Affects Blood Clot Contraction and Formation of Polyhedrocytes

Blood clot contraction or retraction has been implicated to play a significant role in hemostasis, reduction of thrombus volume, and wound healing. Clot contraction is driven by forces that are generated by platelets and transmitted by fibrin and results in volume shrinkage followed by the compaction of erythrocytes into the core of the blood clot, resulting in their mechanical deformation towards a polyhedral shape, giving rise to the term polyhedrocytes. Despite the fact that erythrocytes are a major component of blood clots, relatively little is known about the influence of the mechanical properties or deformability of erythrocytes on the process of clot contraction. Increased hematocrit reduces extent of clot contraction due to mechanical resilience of erythrocytes and it is likely that in addition to a volume fraction the stiffness of erythrocytes can also affect the extent and rate of clot contraction. Here we tested this assumption by using artificially or naturally stiffened erythrocytes that have pathophysiological implications. The reduced deformability of erythrocytes is associated with a number of pathological conditions, such as hypertension, diabetes mellitus, atherosclerosis and smoking, but perhaps one of the most well-known diseases associated with increased erythrocyte rigidity is sickle cell disease (SCD). Another example of naturally stiff erythrocyte membrane is that of llama or camel that have red blood cells with increased osmotic resistance. To assess the extent of clot contraction, we used an optical tracking methodology that allows for the quantitative tracking for clot size. To assess the influence of erythrocyte rigidity on clot contraction we also used scanning electron microscopy to evaluate deformations of the erythrocytes, including the presence of polyhedrocytes. Centrifugation of citrated blood can be used to mimic the contractile forces generated by platelets and has been shown to cause polyhedrocyte formation. Increasing the erythrocyte rigidity through their treatment with a low concentration of glutaraldehyde resulted in a decrease in polyhedrocyte formation and the requirement of larger centrifugal forces to observe erythrocyte deformation, suggesting that the mechanical properties of erythrocytes could influence the process of clot contraction. As residual glutaraldehyde may have unwanted effects on platelets, clot contraction experiments were completed using naturally stiffer erythrocytes from SCD patients and llama ovalocytes, which are stiffer than human erythrocytes due to the increased amount of the membrane cytoskeletal protein spectrin. SCD patients were only included in this study if they had Sickle Trait, SCD Hb SS, SCD Hb SC and have not received recent transfusions. The blood samples of SCD patients were examined and on average had a 53% decrease (p<0.0001) in the extent of clot contraction compared to healthy subjects. Likewise, addition of llama ovalocytes to human platelet rich plasma resulted in a 28% decrease in extent of clot contraction compared to human erythrocytes and larger centrifugal forces were needed to see red cell deformation. SCD patients contracted 2.4X slower (p<0.001) during linear contraction (Phase 2) and 2.7X slower (p<0.05) during mechanical stabilization (Phase 3) when compared to healthy subjects. Clot contraction was impaired also by erythrocytes treated with antibodies that bind to the Wright b epitope on the erythrocytes and exert a rigidifying effect on the cells. The binding of the antibody to erythrocytes was determined by flow cytometry and the KD was ~50 nM. Increased red blood cell rigidity following exposure to antibodies was confirmed through mechanical and osmotic resistance and compared to unaltered erythrocytes. Collectively, these results demonstrate that erythrocyte mechanical properties can influence the process of clot contraction so that stiffer cells reduce the rate and extent of clot contraction. A better understanding of the role of erythrocyte deformability in the process of clot contraction has the potential to inform the development of more targeted treatments for limiting bleeding and thrombosis in patients who are prone to having altered erythrocyte content and mechanical properties of these highly abundant cells embedded into blood clots and thrombi. Disclosures Weisel: Bayer: Research Funding.

Membrane-associated sickle hemoglobin: a major determinant of sickle erythrocyte rigidity

Micropipette aspiration tests on single erythrocytes have previously shown that the static rigidity (membrane shear modulus) of oxygenated sickle cells increased with increasing hemoglobin concentration, whereas the rigidity of normal cells was independent of hemoglobin concentration. Moreover, it was observed that after mechanical extension, sickle cells exhibited persistent deformation more frequently and to a greater extent than normal cells. To ascertain if differences in association of normal and sickle hemoglobin with the membrane could account for these observations, we measured rheologic properties of normal membranes reconstituted with sickle hemoglobin and sickle membranes reconstituted with normal hemoglobin. The static rigidity of normal ghosts reloaded with sickle hemoglobin was higher than those of either normal ghosts reloaded with normal hemoglobin or native normal cells. On the other hand, the increased rigidity of native sickle cells decreased to near-normal values following reconstitution with normal hemoglobin. Furthermore, we observed that normal ghosts reconstituted with sickle hemoglobin exhibited persistent bumps after mechanical extension, but no bumps formed on normal ghosts reconstituted with normal hemoglobin. Moreover residual bumps were not produced on sickle cells reloaded with normal hemoglobin. Since mechanical characteristics peculiar to sickle cells could be induced in normal cells by incorporation of sickle hemoglobin, and since normal characteristics could be restored to sickle cells by incorporation of normal hemoglobin, we suggest that the interaction of sickle hemoglobin with the cell membrane is responsible for augmented static rigidity of oxygenated sickle erythrocytes.

Membrane-associated sickle hemoglobin: a major determinant of sickle erythrocyte rigidity

Micropipette aspiration tests on single erythrocytes have previously shown that the static rigidity (membrane shear modulus) of oxygenated sickle cells increased with increasing hemoglobin concentration, whereas the rigidity of normal cells was independent of hemoglobin concentration. Moreover, it was observed that after mechanical extension, sickle cells exhibited persistent deformation more frequently and to a greater extent than normal cells. To ascertain if differences in association of normal and sickle hemoglobin with the membrane could account for these observations, we measured rheologic properties of normal membranes reconstituted with sickle hemoglobin and sickle membranes reconstituted with normal hemoglobin. The static rigidity of normal ghosts reloaded with sickle hemoglobin was higher than those of either normal ghosts reloaded with normal hemoglobin or native normal cells. On the other hand, the increased rigidity of native sickle cells decreased to near-normal values following reconstitution with normal hemoglobin. Furthermore, we observed that normal ghosts reconstituted with sickle hemoglobin exhibited persistent bumps after mechanical extension, but no bumps formed on normal ghosts reconstituted with normal hemoglobin. Moreover residual bumps were not produced on sickle cells reloaded with normal hemoglobin. Since mechanical characteristics peculiar to sickle cells could be induced in normal cells by incorporation of sickle hemoglobin, and since normal characteristics could be restored to sickle cells by incorporation of normal hemoglobin, we suggest that the interaction of sickle hemoglobin with the cell membrane is responsible for augmented static rigidity of oxygenated sickle erythrocytes.

Rapidly recovered transient flow resistance: a newly discovered property of blood

Although blood flows in a pulsatile fashion, little consideration has been given in past studies to its instantaneous resistance to motion when onset and cessation of flow occur abruptly. Hemorheological studies have documented three kinds of blood flow properties. 1) Shear thinning is a fall in viscolity as shear rate rises. 2) Viscoelasticity is a transient shear stress variation due to elastic deformation of erythrocytes. Dilatancy is a viscoelasticity-modifying property attributed to high shear rate erythrocyte rigidity; viscoelasticity is prominent only at low shear rate. 3) Thixotropy is an initial extra flow resistance linked to developing orientation and disaggregation of erythrocytes. Thixotropy returns fully to blood over a period longer than 1 min. Measurements utilizing a fast response Couette viscometer have revealed an extra 10% transient flow resistance after a flow cessation shorter than that between heart beats. The rapidly recovered transient flow resistance has a temporal pattern similar to thixotropy. Its peak and duration are directly related to total shear strain (shear rate x time) over the 8-30 s-1 shear rate range studied. Transient behavior was essentially identical in analyses carried out using three different viscometer gaps. Numerical simulation to test the effect of the newly observed transient behavior on sudden onset tube flow shows that the developing pattern of pulsatile arterial flow can be affected by its presence.