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.

[Study of erythrocyte deformability using visco-diffractometry (ektacytometry) in hereditary hemolytic anemias]. / Etude de la déformabilité érythrocytaire par visco-diffractométrie (ektacytomètre) au cours des anémies hémolytiques constitutionnelles.

Deformability is a basic characteristic of red blood cells; its regulating factors are presently well known (surface volume ratio, internal viscosity, viscoelastic properties of the membrane). The contribution of each of these factors in the pathogeny of hemolytic anemias has not been fully established. We report 64 hereditary hemolytic anemias in which we studied the deformability, using a visco-diffractometric method (ektacytometer) on whole blood and various subpopulations separated by density. Comparison of the response of the cells submitted to isotonic and hypotonic media allows the determination of the respective contribution of viscosity and surface volume ratio. Deformability is always decreased in red blood cell membrane diseases and in homozygous or double heterozygous hemoglobinopathies; on the other hand, deformability is normal in all the enzymopathies studied. This decrease could be due either to one regulation factor (increase of internal viscosity easily demonstrated by this method in hereditary xerocytosis and hemoglobin CC) or to several parameters: increase of internal viscosity and decrease of surface volume ratio in hereditary spherocytosis.

[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).

[A new test for hemolysis related to tocopherol deficiency: hemolysis caused by sodium azide (NaN3). Results in cholestasis in children]. / Un nouveau test d'hémolyse lié à la carence en tocophérol: l'hémolyse par l'azoture de sodium (NaN3). Résultats dans les choléstases de l'enfant.

In patients suffering from intra or extrahepatic biliary obstruction defective absorption of liposoluble vitamins results in tocopherol deficiency. Erythrocyte membranes thus have an increased susceptibility to various oxydants. Erythrocytes from 28 infants and children with biliary atresia have been studied. The usual peroxide hemolysis test (H2O2) was compared with a new method using sodium azide (NaN3), an inhibitor of intraerythrocytic catalase, as the hemolyzing agent. A much more significant correlation was found between plasma tocopherol levels and NaN3--induced hemolysis (R. coefficient = -0.872; P = 99.9%) than tocopherol levels and peroxyde hemolysis (RC = -0.477; P = 95-99%). In severely tocopherol-deficient patients (plasma levels below 0.15 mg/dl compared to normal of 0.5 mg/dl) sodium azide hemolysis is completed by the 5th hour of incubation at 37 degrees C. After the 5th hour, hemolysis begin to appear in the control media and when measured at 20 hrs is almost complete especially in the isotonic saline solution (NaCl 0.9 g/l). When abnormal red cells are incubated in their own tocopherol-supplemented plasma a complete normalization of hemolysis due to NaN3, H2O and control media is obtained. The sodium azide hemolysis test is proposed as a new and simple means of measuring tocopherol deficiency in erythrocyte membranes.

[1st instance of the absence of an erythrocyte membrane protein (band 4(1)) in a case of familial elliptocytic anemia]. / Première observation de l'absence d'une protéine de la membrane érythrocytaire (bande 4(1)) dans un cas d'anémie elliptocytaire familiale.

In four members of a family presenting hereditary elliptocytosis erythrocytes were studied, membranes were extracted and proteins were analysed by gel electrophoresis in a polyacrylamide gradient. In one of the patients suffering from severe haemolytic anaemia successfully treated by splenectomy, an almost complete deficiency in band 4(1) was discovered. Endogenous protein kinase activities revealed the absence of radioactivity of band 4(1) in the proband, a result not modified by cAMP. The kinase activity was normal in the parents, which confirms the almost complete absence of protein 4(1) seen in the stained gel of the proband. A moderate increase in the phosphorylation of band 3 was observed in all the members of this family. Deformability was measured by a visco-diffractometric method (Ektacytometry) in a medium of low viscosity (11 cp at 22 degrees C), allowing the recording of curves characteristic of elliptocytosis and revealing a markedly reduced deformability index (DI). At low shear stress, elliptocytes were oriented perpendicular to the flow (a result common to all elliptocytosis). In the proband several years after splenectomy, the DI was extremely reduced at high shear stress, which can be explained by the simultaneous presence of elliptocytes, schizocytes and spherocytes. A more detailed comparative study of the proteins of the various members of the family could lead to more precise information on the possible role of band 4(1) as a linkage protein maintaining the erythrocyte membrane stability.

Discocyte--echinocyte reversibility in blood stored in CPD over a period of 56 days.

Blood collected with CPD and stored was examined with optical (LM) and scanning electron microscopy (SEM) before and after reversal of echinocytes into discocytes. Reversal was achieved by incubation of the red blood cells at 37 C in an adenosine containing medium. The transformation of discocytes into echinocytes occurred rapidly during the first three weeks of storage. A shape/density relationship was observed in the various fractions. The denser cells were found to have the more advanced echinocytic changes. After incubation with adenosine, most of the cells reversed into discocytes and early stages of stomatocytes. When spheroechinocytes I and II were present, they reversed into spherostomatocytes. No echinocytogenic property was found in plasma during 5 weeks of storage at 4 C and no immediate reversion of echinocytes was obtained in fresh plasma, therefore the initial discocyte--echinocyte transformation was explained by intracellular changes. The data from the various fractions showed that the more dense cells were more spherocytic. We suggest that removal of this part of the population of stored red blood cells might improve the survival of transfused cells.

Observation of echinocytosis in eight patients: a phase contrast and SEM study.

We report eight cases of echinocytosis discovered after screening of stained smears. The presence of echinocytes was exceptional in adults and children but was more frequent in neonates. In all cases we confirmed the presence of abnormal red cells by careful examination of the blood in wet preparations observed in phase contrast and after glutaraldehyde fixation and processing for SEM. No discernible common denominator was found to explain the presence of echinocytes in the blood of our patients. No echinocytogenic factor could be demonstrated in the fresh plasma. Echinocytes appeared quickly in patients who received exchange transfusion or transfusion of normal red cells. By contrast, in vitro incubation in the presence of substrates allowing constant ATP level never resulted in the formation of echinocytes when normal red cells were suspended in patient's plasma.