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The authors evaluated advantages of the glycerol lysis test (AGLT) and its modification the "pink" test in the laboratory diagnosis of hereditary spherocytosis (HS). Both tests were positive in all examined subjects with HS and in some patients with autoimmune haemolytic anaemia (AIHA) and were negative in controls and some other haemolytic diseases. As these tests are easy to perform and quick, they can be recommended as a supplementary or screening examination in hereditary spherocytosis. The "pink" test has, as compared with the ALGT test, the advantage that it is simpler.

Hereditary spherocytosis (HS) is characterised with many deviations of red blood cells properties. We investigated a group of 14 patients with mild HS, 7 of whom were splenectomised. We found changes in the content and/or turnover of polyphosphoinositides and phosphatidic acid accompanied by the higher generation of inositol 1,4,5-trisphosphate. We suggest that the activation of the phosphoinositide signalling system may be crucial for the manifestation of HS.

The authors describe 22 cases of beta-thalassaemia minor in 11 boys and 11 girls from Czech families. The children suffer as a rule mild hypochromic anaemia with marked microcytosis and rather elevated red cell values. The serum ferritin values are normal, serum iron is normal or slightly elevated. In all children the ratio of haemoglobin A2 is raised (to 4-7%) and in 40% the ratio of haemoglobin F is raised (to 1.5 to 5.9%). There are no differences between boys and girls in the investigated parameters. The boy have, as compared with adult men with beta-thalassaemia minor, significantly lower values of red blood cells and serum ferritin. There are no significant differences between girls and adult women suffering from this disease.

Several approaches to the processing of native stroma-free hemoglobin solutions (SFH) were reconsidered regarding present requirements for SFH production and quality. Treatment of outdated red blood cells (RBCs) with chloroform and/or by pasteurization were evaluated for technical ease, speed and efficacy in removing stromata, phospholipids and non-heme proteins from RBC hemolysates. The influence of both procedures upon spontaneous hemiglobin formation in stored, preferably freeze-dried SFH was compared. Among other analytical methods, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS PAGE) and isoelectrofocusing were used for mutual comparison of the purification procedures. Pasteurized samples were significantly better purified, more homogeneous but also more susceptible to spontaneous oxidation, probably due to heat inactivation of enzymic scavengers of oxygen radicals. On the other hand, the chloroform-treated, unheated SFH samples were less purified from non-heme proteins, but were more stable. Fructose and sucrose were equally active in protecting SFH from oxidation during freeze-drying. At present, the easy chloroform treatment and freeze-drying of thus purified SFH with fructose of sucrose seems to offer a plausible technological compromise which merits further investigation.

The effect of mechanical stress on erythrocytes suspended in various media was studied. The ability of the cells to increase their glucose consumption was found to be the major criterion allowing to divide the media into two groups. In plasma, serum or in Ringer's solution supplemented with albumin and glucose the energy consumption by mechanically stressed erythrocytes increased 20 to 50%; no morphological changes of the cells were observed either in suspension or on Giemsa smears. The cells behaved in the same way in Mg2(+)-free medium. The other group included protein-free medium (Ringer's solution supplemented with glucose) and Ca2(+)-free Ringer's solution supplemented with albumin and glucose; under these conditions erythrocytes were unable to raise their energy consumption in response to mechanical stress, and after some period structural impairment of the membrane could be observed on Giemsa smears. No differences in metabolism-associated nucleotide concentrations (ATP, ADP, NAD, NADP) were observed between the samples. Resealed red cell ghosts with high concentrations of intracellular components were prepared as a model of cells with damaged membrane. In these ghosts (with low ATP concentration) mechanical stress produced increased proportions of echinocytes, even in the "native" suspension. These results have confirmed the vital role of the energy-consuming contractile apparatus in the erythrocyte membrane, and supplied a clue to the role of Ca2+ in its activation and to the influence of extracellular proteins on the maintenance of in red cell shape.

Whole red cell deformability was studied by ektacytometry. Reduced shear induced red cell elongation was observed in some hemolytic disorders, chiefly in those where spherocytes occur. With the same technique, red cell membrane deformability and stability were studied following mild proteolytic digestion with trypsin. Reconstitution with spectrin restored in part the membrane elasticity while band 4.1-protein exerted stabilizing effect on the perturbed membrane.

The formation of heterogeneous fibers well observable under a magnifying glass in stirred "stroma-free" hemolysates of outdated banked erythrocytes was found to be predominantly due to random surface denaturation of both hemoglobin and non-heme proteins at the air-liquid interface. Electrophoretic analysis revealed that the precipitated and washed fibers contain residual membrane proteins as well as a certain amount of hemoglobin. The latter, however, largely remains in solution.

Acidified glycerol lysis test (AGLT) was found to be an appropriate technique for detection of sphaerocytes in blood particularly in hereditary sphaerocytosis. The incubation of blood for 5 hours at 37 degrees C prior to measurement exerts positive effect on the reliability of the test and may further suggest the type of haemolytic disorder.

Immunoprecipitation studies showed that the supernatants from the 4-hour mixed cultures of allogeneic mouse lymphoid cells contained a specific component with the alpha2-globulin electrophoretic mobility which can correspond to, or to which a "lymph node activating factor" can be bound. In addition, during allogeneic cell culture serum proteins with the prealbumin mobility were released into the culture medium and in the control culture of cells from only one donor proteins with the albumin mobility predominated. In the supernatants from the 4-hour cultures of human HLA-identical and HLA-diferent lymphocytes beta2-microglobulin was determined by immunodiffusion and radioimmunologically.

Crude spectrin was extracted from the isolated red cell ghosts with low ionic strength buffer at 37 degrees C for 30 min. No significant alterations in crude spectrin extractability in wide range of patients with various hematologic diseases were observed. However, blood samples characterized by elevated reticulocytosis provided crude extracts with increased amount of non-heme membrane skeletal proteins. The presence of ribose in the crude spectrin extracts obtained from reticulocyte-rich blood samples indicates also the presence of nucleic acids which causes the shift of protein peak in the extract from 280 nm towards lower wavelengths. A model experiment with a normal crude spectrin extract mixed with various amounts of RNA allowed us to obtain the correction curve which served for determination of non-heme protein (crude spectrin) extractability.

Factors influencing an increase of glucose consumption in erythrocytes under mechanical stress were studied. Under the shear stress of 2000-5000 s-1 the glucose consumption goes up 20-50%. This effect disappears in protein-free media, in Ca2+-free media and in resealed red ghosts; in all these cases certain morphological deviations were observed in the stressed cells. These results confirm the vital role of energy-consuming contractile apparatus of erythrocyte membrane in restoration of the smooth biconcave shape after deformation.

Low ionic strength extraction (37 degrees C, 30 min) of ghosts from PK-deficient erythrocytes provided crude spectrin extract. No significant differences in the extract composition compared to normal donors were observed. The reticulocyte-dependent spectrin extractability was found among the subjects with PK-deficiency anemia. Likewise ATP-depletion affects spectrin extractability and also leads to the adsorption of cytoplasmic protein MW 50,000 to the reticulocyte membrane. The measurement of membrane fluidity using the fluorescence probe DPH did not reveal significant alterations in the moiety of integral membrane constituents.

The interaction of 99Mo with red cell membrane proteins was found specific for spectrin both in normal red cells and those of hereditary spherocytosis. In addition, no significant quantitative differences were observed in labeling patterns between these two types of red cells, thus indicating no major alterations in the spectrin molecules of hereditary spherocytosis.

Molybdenum in the form of its pentavalent complex binds primarily to spectrin when incubated with erythrocytes. Only the band 1 subunit is involved in this interaction thus indicating some structural differences between spectrin subunits.

The radioactive labeling of spectrin using the pentavalent complex of molybdenum-99 was applied to the study of membrane protein in pyruvate kinase deficient red cells. Compared to the control, the labeling profile of the enzymopathic red cell membrane proteins remained generally unchanged but the molybdenum uptake was found to depend largely on the reticulocyte count. This finding may reflect changes during the cell maturation.

Spectrin oligomers were extracted from the erythrocyte membrane at 37 degrees C with the low ionic strength buffer. The spectrin extractability of HS membranes was enhanced compared with controls in some cases. The major spectrin component in the extract was dimer which was, apart from a tetramer, purified by gel filtration on the Sepharose 4B column. No changes of dimer-tetramer interconversion in the extract from HS membranes were observed. Similarly, the protein composition of the membrane extracts from two types of red cells remained unchanged.

A new simple filtration technique designed for measuring red cell filtrability in the routine laboratory use was developed. The suspension of the whole blood in saline (1:20,000 dilution) was processed on the Sartorius filter membranes, pore size 8 micron. The percentage of passed erythrocytes indicating red cell filtrability was determined. The suitability and perspective applicability of this method for studying various hematological disorders is proposed.