Evaluation of covalent antisickling compounds by PO2 scan ektacytometry.

The ektacytometer, a device to measure erythrocyte flexibility, has been used to evaluate antisickling agents that covalently modify hemoglobin S (HbS). The instrument has been adapted to produce a continuous gradient of oxygen pressure in the measuring cuvette, which permitted the rapid determination of sickle cell rigidity over the complete oxygenation range. Inspection of curves allows classification of the compounds according to their mode of action: altering oxygen affinity or increasing deoxy-HbS solubility. Reagents that modify amino groups, thiols, and histidine, as well as a crosslinking agent, were examined. The method directly evaluates deoxygenated cell deformability rather than cell shape. Many of the compounds that are effective in preventing the morphological sickling of deoxygenated sickle cells do not necessarily restore cell deformability. The method also readily detects membrane damage brought about by covalent agents that nonspecifically derivatize membrane proteins. Cystamine and pyridoxal appear to improve deformability in deoxygenated SS cells at concentrations that do not damage the membrane. This method, which examines the intact cell, fills a gap in the available experimental techniques for drug evaluation, between studies of isolated hemoglobin and in vivo studies.

The Technicon Ektacytometer: automated exploration of erythrocyte function.

The Ektacytometer is a visco-diffractometric method to preferentially examine the functional properties of erythrocytes. The realization of this method in the form of an automated laboratory instrument, the Technicon Ektacytometer, is described. A suspension of RBC is submitted to a uniform shear stress. The resulting deformation of the cells is seen as an elongation of the diffraction image and expressed as the Elongation Index or EI. EI may be traced on an X-Y recorder as a function of stress, time and of suspending medium osmolality. The osmolality is varied automatically in the viscometer and automatically measured via its conductivity, resulting in an "Osmoscan". The Osmoscan is highly reproducible and requires only 150 microliter of blood and 5 minutes per sample. Its high sensitivity to small changes in erythrocyte properties permits the separation of factors affecting RBC deformability, and provides a measure of the osmotic fragility which correlates very well with the traditional test. Other applications are described, including mechanical fragility assays on whole cells, relaxation times of ghosts and the automatic variation of PO2. Possible future applications are suggested.

[Effects of 3 varieties of antisickling substances measured by ektacytometry as a function of oxygen pressure]. / Action de trois variétés de substances antidrépanocytaires mesurée par ektacytométrie en fonction de la pression d'oxygène.

The ektacytometer measures with precision the deformability of erythrocytes during a continuous change in oxygen tension. Automation of the instrument renders easily feasible the evaluation of proposed antisickling compounds. Drugs representative of the three modes of action presently recognized have been selected: those which increase oxygen affinity (potassium cyanate); those which inhibit hemoglobin polymerization (butylurea); membrane-active compounds. An anionic phenothiazine (metiazinic acid) had an interesting activity on deformability.

[A new technic for the study of the physiology of erythrocytes: measurement of their deformability as a function of osmolarity. Results obtained by an automatized Ektacytometer in normal blood and in various hemolytic anemias]. / Une nouvelle technique d'étude de la physiologie des globules rouges: la mesure de leur déformabilité en fonction de l'osmolarité. Résultats obtenus par un Ektacytomètre automatisé sur du sang normal et dans différentes anémies hémolytiques.

A system has been added to the Ektacytometer to allow continuous variation and measurement of the osmolarity of the suspension medium from 50 to 500 mosm kg-1 permitting the analysis of erythrocyte deformability over a range varying from hypo- to hypertonicity. The curves are obtained automatically in 7 min. from a blood sample of 150 microliters. The "minimum index" observed in hypotonicity indicates that the cells are non-deformable at this point just preceding hemolysis. The osmolarity at which this index occurs provides a measure of the osmotic fragility of the sample. In hypertonicity there is a zero index, corresponding to non-deformable cells, which occurs at an osmolarity dependent on the original internal viscosity of the cells. This "null index" introduces the notion of erythrocyte resistance to deformation in hypertonicity. This new technique permits the evaluation of the erythrocyte response to various factors affecting their shape, internal viscosity or membrane flexibility. We present and discuss examples of curves obtained with normal blood before and after fractionation by density and after treatment with cystamine, as well as pathological bloods from two hemolytic anemias (hereditary spherocytosis and sickle cell anemia).