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.

A new abnormal variant of spectrin in black patients with hereditary elliptocytosis.

Seven black patients with mild hereditary elliptocytosis (HE) from five unrelated families were studied. The erythrocytes of these patients exhibited an abnormal thermal sensitivity (between 45 degrees C and 47 degrees C instead of 49 degrees C). An important defect of spectrin dimer self-association was detected in two ways: (1) the proportions of spectrin dimer (SpD) extracted from membranes at 4 degrees C under low ionic strength conditions were increased between 25% and 56% (normal value 15% +/- 2%); (2) the spectrin dimer----tetramer conversion in solution were defective with an association constant value between 0.

Pathologic and nonpathologic variants of the spectrin molecule in two black families with hereditary elliptocytosis.

Five patients with hereditary elliptocytosis (HE) from two unrelated black families were studied. The patients had prominent elliptocytosis and a decreased erythrocyte resistance to heat treatment. In one infant blood smears showed elliptocytosis and poikilocytosis; his erythrocytes fagmented at a lower temperature than those of his mother and sister, both having typical mild HE.

A variant of erythrocyte membrane skeletal protein band 4.1 associated with hereditary elliptocytosis.

A family comprising three patients (a mother and two children) with mild hereditary elliptocytosis was studied. Each patient had prominent elliptocytosis, reduced red cell deformability, and normal erythrocyte thermal sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the erythrocyte membranes in each patient showed decreased levels of band 4.

Molecular defect of spectrin in the family of a child with congenital hemolytic poikilocytic anemia.

We present the study of a black family in which the proband suffered from a severe neonatal hemolytic anemia with poikilocytosis. Both the parents, sister's, and brother's proband were clinically normal. The presence of poikilocytes in proband led to a search for a red cell membrane skeleton defect.

[Study of erythrocyte deformability using visco-diffractometry (ektacytometry) in hereditary hemolytic anemias].

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.

[Neonatal hemolysis secondary to congenital poikilocytosis].

Hereditary pyropoikilocytosis is a congenital haemolytic anaemia recently described. A new case is reported in which the condition was diagnosed by a study of erythrocyte membrane proteins in the parents. The unusual clinical features of this case lead to a discussion of the relationship between hereditary pyropoikilocytosis and other rare forms of elliptocytosis in children.

[Congenital hemolytic anemia with erythrocyte thermal instability and defect of spectrin tetrameric polymerization. Study of erythrocyte deformability in a new case of hereditary pyropoikilocytosis using diffraction viscosimetry].

Hereditary pyropoikilocytosis (HPP) is a rare congenital hemolytic anaemia observed so far in patients of black extraction. In many cases, the severity of the anaemia has led to early splenectomy, which uniformly improved the hematological conditions. The disease is characterized by extreme anisocytosis and poikilocytosis with erythrocyte fragmentation.

[Effects of 3 varieties of antisickling substances measured by ektacytometry as a function of oxygen pressure].

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.

[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].

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 effect of cell hydration on the deformability of normal and sickle erythrocytes.

The deformability of the erythrocyte (RBC) is greatly influenced by its state of hydration. The purpose of this investigation is to quantitate this relationship by measuring the deformability of an RBC population over a broad range of cell water content. By manipulation of the ion content of the RBC, we performed all of the experiments in media which were isotonic with plasma.

Spectrin beta-chain variant associated with hereditary elliptocytosis.

An electrophoretically fast-moving variant of the spectrin beta-chain was discovered in the erythrocyte membranes of a woman and her father who both exhibited elliptocytosis and mild hemolytic anemia. This abnormal beta'-subunit (Mr = 214,000) co-existed with a decreased normal beta-chain and represented about half of the total beta-chains in the membrane. In contrast to the spectrin beta-chain, the beta'-chain was phosphorylated neither in the membrane by endogenous protein kinases nor in solution by pure membrane casein kinase whether or not the spectrin was dephosphorylated by erythrocyte cytosolic spectrin phosphatase.

[A new test for hemolysis related to tocopherol deficiency: hemolysis caused by sodium azide (NaN3). Results in cholestasis in children].

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.

Echinocytic sensitivity and deformability of human newborn red cells.

Echinocytic red cells are present in the peripheral blood of normal human newborns in small but significant numbers. This is in contrast to normal adults in whom no echinocytes are found in peripheral blood. In addition, red cells from newborns have increased susceptibility to echinocyte transformation induced by naturally occurring phospholipid lysolecithin.

Quantitation of red cell deformability during progressive deoxygenation and oxygenation in sickling disorders (the use of an automated Ektacytometer).

1. The Ektacytometer, which allows quantitation of cell fluidity under known environmental conditions, has been recently modified so that cells can be exposed to any desired O2 tension during shear stress. 2.

The influence of suspension osmolarity and erythrocyte volume on cell deformability.

Erythrocytes were suspended in dextran solutions of phosphate buffered saline with solution osmolarities from 400 to 20 mosM/kg. The dilute suspensions were subjected to linear shear and their deformation determined by laser diffractometry (Ektacytometer). Cell volumes were measured using a Coulter counter following fixation in glutaraldehyde to eliminate the influence of deformability on the volume measurement.

Evidence for imbalanced furosemide-sensitive Na+, K+ cotransport in hereditary stomatocytosis.

The red cells from 5 related patients with hereditary stomatocytosis were investigated. Maximal rate constant of Na+ passive permeability was increased while that of K+ passive permeability was nearly normal. Ouabain-sensitive Na+ efflux was elevated.

Separate mechanisms of deformability loss in ATP-depleted and Ca-loaded erythrocytes.

Membrane rigidity has been widely accepted as the dominant cause of reduced deformability both of ATP-depleted erythrocytes and erythrocytes containing excess calcium (Ca). However, recent studies have shown normal membrane deformability in ATP-depleted erythrocytes. In addition, Ca accumulation causes massive ion and water loss, and it has been shown that extensive dehydration causes an increase in intracellular viscosity with attendant loss of whole cell deformability.

Factors that limit whole cell deformability in erythrocytes after calcium loading and ATP depletion.

Whole cell deformability of ATP-depleted and Ca-loaded red cells has been measured at various osmolalities to determine those cellular factors responsible for the reduce deformability of these cells. For cells depleted of ATP in Ca-free medium, a progressive loss of hypotonic deformability identified membrane loss with reduced surface area-to-volume ratio as the dominant mechanism of deformability loss. For cells treated with Ca and the ionophore A23187 without prior depletion, a rapid loss of isotonic deformability, reversible in hypotonic medium, identified dehydration with increased internal viscosity as the dominant mechanism of deformability loss.