Hemolysis Curve Research Articles

Fragility of erythrocytes in animals affected by lantana poisoning.

Clinical observations on four male cow calves and three other lantana poisoned animals under field conditions have been made along with hematological studies, including observations on plasma bilirubin content and osmotic fragility of erythrocytes. Four healthy male cow calves served as control. Five clinically advanced cases succumbed within 1 to 10 days of observation. Hematocrit values in four of the seven affected animals were abnormally high. The total plasma bilirubin content in the fatal cases was of the order of 5 to 50 times the normal value while, in calves that recovered, it was not more than 3 times. The osmotic fragility of erythrocytes was measured by the rate of hemolysis in decreasing concentrations of saline. The erythrocytes of normal animals resisted hemolysis till the saline concentration was lowered to 0.60%, and it was complete at the low saline concentration of 0.30%. However, in lantana poisoned animals, hemolysis started even in 0.72% saline and was complete at a saline concentration of 0.44%. The hemolysis curves of animals that died were far removed from the normal, but those of the calves that recovered and the one that died but had the lowest bilirubin content closely approached the normal curve.

Alterations of Saponin Hemolysis during Storage of ACD Blood

A dynamic hemolysis curve was examined on stored ACD blood by a new instrument (mean cell volume or MCV continuous analyzer) during treatment with either saponin or with hypotonic medium. Several changes of the dynamic hemolysis curves were recorded during storage. With saponin hemolysis, these changes appeared earlier and were more marked than those of hypotonic hemolysis.

Shear-induced hemolysis with commercial and glow discharge silicones.

The hemolytic characteristics of 14 different polydimethyl-siloxane materials were studied, using a rotating disk device to shear whole human blood for 6000 sec. Disk surfaces included commercial Silastic products with and without silica filler, coatings formed from hexa-methyl and tetra-methyl disiloxane monomer vapors by glow discharge, and heparinized silicones. Results are presented in the form of kinetic hemolysis curves (KHC)--plots of plasma hemoglobin vs. time--and analyzed in terms of the slopes of these curves in the 1500-6000 sec region where the KHC is nearly linear. It was found that most silicone products are low-hemolysis materials, comparable to polyethylene (PE) but often about 20% less ehmolytic; one product was superior by about 40%. The only consistent exceptions to this were the glow discharge surfaces from HEXA-methyl disiloxane (15-80% poorer than PE) and heparinized surfaces (70-280% more hemolytic than PE).

Hemolysis of Sheep Erythrocytes in Guinea Pig Serum Deficient in the Fourth Component of Complement

Abstract The hemolysis of sensitized sheep erythrocytes in serum from guinea pigs with a total genetically controlled deficiency of C4 was studied with purified IgG and IgM anti-Forssman antibodies. It was found that an approximately 20-fold increase in hemolytic antibody concentration was necessary to initiate lysis in C4D serum as compared with normal serum. The kinetic curves of hemolysis in concentrated C4-deficient serum reached a plateau in 30 to 60 min. Lysis in the deficient serum could only be achieved with concentrated serum; hemolytic activity was rapidly lost on dilution. The lytic capacity of C4-deficient serum from individual animals was shown to vary markedly. The cytolytic reaction was specific in that only sensitized sheep erythrocytes were lysed, and there was no lysis of “innocent bystander” cells.

Studies on the biological properties of coelomic fluid of sea urchin 1. Naturally occurring hemolysin in sea urchin

1. 1. Biological properties, especially hemolytic activity, of coelomic fluid of sea urchin were investigated. 2. 2. Dialyzed coelomic fluid preparation obtained from Anthocidaris crassispina was found to have hemolytic activity against rabbit, mouse and human erythrocytes, but not against erythrocytes of other species of animals so far examined, i.e. rat, guinea pig, goat, sheep, dog, pig, hen, snake and frog. A similar hemolysis spectrum was obtained with a coelomic fluid preparation from Pseudocentrotus depressus, while coelomic fluid preparation from Hemicentrotus pulcherrimus showed a capacity to lyse erythrocytes of rat and guinea pig, besides those of rabbit, mouse and man. 3. 3. The hemolytic activity of coelomic fluid preparation was greatly enhanced by the addition of Ca 2+ to the medium, but not by Mg 2+. Addition of EDTA, even in the presence of Ca 2+, resulted in a loss of potency. 4. 4. The degree of hemolysis induced by coelomic fluid preparation was dependent on concentration and temperature of incubation. The kinetic analysis as a function of concentration indicated that the hemolysis curve resembles those of saponin and streptolysin O. 5. 5. The hemolytic factor in coelomic fluid preparation was so labile that complete inactivation was effected after 30 min of incubation at 56°C, and after storage for 24 h at pH 11.3. 6. 6. The active factor may be not a saponin but a protein or a protein-like substance, since its activity was abolished irreversibly by treatment with trypsin or 2-mercaptoethanol.

Use of a Logistic Function in Expressing Kinetic Hemolysis Data

A method of rectifying sigmoidal kinetic hemolysis curves is described. The curves are described, within limits, by the equation t = K(l/1 - l)(1/n) where t is time, K is a constant, l expresses the degree of lysis, and l/1 - l is the ratio of lysed to unlysed cells. This description holds over a wide range of red cell and lysin concentrations, provided the conditions are not substrate limited and the lysin preparation is reasonably stable. Graphical solution of the logarithmic transformation of the equation yields straight lines, and the intercepts of these lines with time ordinates may be used to define a time of initial lysis or, under suitable conditions, a time of 50% lysis. The time of initial lysis is inversely related to the lysin concentration, and thus its reciprocal may be used to precisely define units of lytic activity.

Effect of pH on the haemolysis of rabbit erythrocytes by staphylococcus alpha--toxin.

Summary The effect of toxin concentration on the kinetics of haemolysis of rabbit erythrocytes has been studied at pH values differing by about 0·5 pH units over the range 5·5 to 8·5 for both crude and partially purified toxin at concentrations of 2·5, 5·0, 10, 20, 30, 40, 60 MHD per ml and at 80 and 120 MHD per ml for the partially purified toxin. Increased concentration of toxin caused a decrease in the prelytic lag time (·) of the sigmoid haemolysis curves, a decrease in the time to reach 50 per cent. haemolysis (t1/2), and an increase in the maximum rate of haemolysis (Rm). Plateaux in Rm-toxin concentration, ·-toxin concentration and t1/2-toxin concentration curves were found at all pH values: these were displaced to higher concentrations in plots drawn for the partially purified toxin. The effect of pH on haemolysis is very marked, and pH profiles for the maximum rate of haemolysis were constructed: for the crude toxin, the shape of the profile changed as the toxin concentration was increased, while for the partially purified toxin the shape was independent of toxin concentration. The pattern of the profiles indicates that the crude toxin contains a small concentration of impurity that is strongly lytic at pH 5·5 and less markedly so at 8·5; the impurity has no significant effect at intermediate pH values. The reaction is first order with respect to toxin over a very small range of concentrations only, and the maximum rate of haemolysis is independent of toxin concentration at concentrations greater than 120 MHD per ml. The reaction is first order with respect to erythrocytes at all pH values, and over all measurable concentrations.

Inhibition of staphylococcal alpha-toxin. A kinetic evaluation of aromatic polysulphonic acids as inhibitors of haemolysis.

1. The effect of a number of aromatic polysulphonic acids on the kinetics of haemolysis of rabbit erythrocyte suspensions by crude staphylococcal alpha-toxin was studied at pH8.6 and 6.8. 2. All of the inhibitory compounds caused an increase in the prelytic lag time (tau) of the sigmoid haemolysis curves, an increase in the time to reach 50% haemolysis (t((1/2))) and a decrease in the maximum rate of haemolysis (R(max.)). The most inhibitory compounds caused a 50% decrease in R(max.) at concentrations between 0.1 and 0.2mm. 3. The effect of pH varied considerably: compounds (I) and (II) were almost equally inhibitory at both pH values, compounds (IV) and (IX) were more inhibitory at pH6.8 than at pH8.6, and compounds (VII), (VIII), (X), (XI) and (XII) were more inhibitory at pH8.6. 4. Increased time of premixing alpha-toxin with compound (I) caused increased inhibition. 5. An attempt was made, where possible, to relate the inhibitory activity to the structure of the test compound.

Combined effects of freezing rates and of various protective agents on the preservation of human erythrocytes

Summaryo1.Fifty cubic millimeters of citrated human blood containing various concentrations of glyeerol, DMSO, dextrose, sucrose, or PVP were frozen in capillary tubes which had an outside diameter of 1.5 mm and a wall thickness of 0.3 mm, by abrupt immersion into isopentane baths at temperatures ranging from −5 to −130°C.2.The frozen specimens were thawed rapidly by abrupt immersion of the capillary into a well stirred water bath at 35°C, and the extent of injury to the erythrocytes was measured by a colorimetric determination of the amount of hemoglobin released from the damaged cells.3.The results obtained indicate that the 5 protective agents investigated may be classified into three types: the type glycerol, the type dextrose and the type PVP.4.Protective agents of the type glycerol, which includes DMSO, become increasingly efficient in preventing lysis with increased concentration (up to a limit) at low cooling velocities and increasingly injurious at high cooling velocities.5.At low cooling velocities, at which hemolysis is minimum with protective agents of the type glycerol, the agents of the type dextrose, including sucrose, exert only a slight protection. At high cooling velocities, they become increasingly effective in reducing hemolysis.6.Protective agents of the type PVP give results intermediate between those of the type glycerol and those of the type dextrose. At low cooling velocities they give less protection than the additives of the type glycerol, but more than those of the type dextrose. Conversely, at high cooling velocities, they do not provide the protection afforded by sugars, nor do they exert the marked injurious effects of glycerol.7.The general shape of the hemolysis curves obtained with human blood frozen at various rates with different protective agents is very similar to those observed with bovine blood frozen under the same conditions. Fifty cubic millimeters of citrated human blood containing various concentrations of glyeerol, DMSO, dextrose, sucrose, or PVP were frozen in capillary tubes which had an outside diameter of 1.5 mm and a wall thickness of 0.3 mm, by abrupt immersion into isopentane baths at temperatures ranging from −5 to −130°C. The frozen specimens were thawed rapidly by abrupt immersion of the capillary into a well stirred water bath at 35°C, and the extent of injury to the erythrocytes was measured by a colorimetric determination of the amount of hemoglobin released from the damaged cells. The results obtained indicate that the 5 protective agents investigated may be classified into three types: the type glycerol, the type dextrose and the type PVP. Protective agents of the type glycerol, which includes DMSO, become increasingly efficient in preventing lysis with increased concentration (up to a limit) at low cooling velocities and increasingly injurious at high cooling velocities. At low cooling velocities, at which hemolysis is minimum with protective agents of the type glycerol, the agents of the type dextrose, including sucrose, exert only a slight protection. At high cooling velocities, they become increasingly effective in reducing hemolysis. Protective agents of the type PVP give results intermediate between those of the type glycerol and those of the type dextrose. At low cooling velocities they give less protection than the additives of the type glycerol, but more than those of the type dextrose. Conversely, at high cooling velocities, they do not provide the protection afforded by sugars, nor do they exert the marked injurious effects of glycerol. The general shape of the hemolysis curves obtained with human blood frozen at various rates with different protective agents is very similar to those observed with bovine blood frozen under the same conditions.

魚類血液の生化学的研究-VI

It was found that the red corpuscles of the teleosts studied increased their size, when the salt concentration of the hypotonic solution spproached the threshold concentration where the hemolysis of the corpuscle begins. In this case, the oval form of the natural red corpusele was deformed into the round one. In hypertonic (5%) NaCl solution, however, the dwindled red corpuscle appeared (Table 1). In contrast to other fishes; shark red corpuscle was found to become larger than the normal corpuscle even in the hypertonic solution. In the case of the sea water, fishes, the hemolysis of the red corpuscle took place at the higher concentrations compared to the case of the fresh water fishes. By estimating the dissolved hemoglobin in terms of the optical density, the hemolysis curve was easily obtained (Figs. 1 & 2).

The Use of Equations of the n-th Order to Describe the Action of Simple Haemolysins

In all recent work concerned with the fitting of formulae to curves obtained for the action of the simple haemolysins it has been assumed that the “fundamental reaction” between the cells and the lysin is one in which the latter combines with some component (probably protein) in the membrane of the former, thus forming a new compound as the result of the formation of which the integrity of the cell is destroyed. Thus, the quantity of the cell component, S, destroyed, is proportional to the quantity of lysin, x, used up in the system, and the velocity of the reaction is given by where c is the initial quantity of lysin (in milligrams), where t is the time required to produce lysis of an arbitrary number of red cells, and where S is large compared to c. Since it is assumed that the complete lysis of n cells corresponds to the utilization of a constant quantity of lysin, we obtain, by putting × = const., and varying c in (2), a relation between the time for complete lysis of n cells and c, the initial concentration of lysin; when plotted, this relation gives the “time-dilution curve” for any particular lysin. If we are concerned with the number of cells, N, haemolysed from moment to moment by a particular concentration of lysin, from the beginning of the reaction until its completion, we solve (2) simultaneously with For certain haemolysins under certain conditions, these expressions describe the experimental results excellently. Recently, however, we have examined the action of several simple lysins over very much longer periods than previously, observing the time-dilution and percentage haemolysis curves over periods as long as 300 minutes and as short as 6 seconds.

HEMOLYSIS OF CHICKEN BLOOD

1. The time-dilution curves are given for the hemolytic action of saponin, sodium taurocholate, and sodium oleate on nucleated chicken erythrocytes. 2. Saponin and sodium taurocholate cause hemolysis but leave the nuclei and ghosts in suspension, thereby making the end-point of hemolysis more arbitrary than the clear end-point for non-nucleated cell hemolysis. 3. The curves of hemolysis by saponin and taurocholate are shown to be of the same nature as are found in the hemolysis of non-nucleated cells. 4. Sodium oleate causes first hemolysis and then, in the stronger solutions, causes karyolysis. Two pairs of values for kappa and c = infinity are thus obtainable for the same reaction, one pair for the destruction of corpuscular membrane, the other pair for the destruction of the nucleus. 5. Viscosity changes are found in the lysin-cell system with strong concentrations of sodium taurocholate and sodium oleate. Time-viscosity curves are given for these changes. 6. Microscopically, the action of these lysins on the nucleated chicken red cell appears to be similar to their action on the non-nucleated erythrocytes.

Light Transmission Through Blood Suspensions as Recorded by the Photo-electric Cell.

In an attempt to find a method of securing accurate time curves of red blood corpuscle hemolysis by various hemolytic agents, an apparatus was devised in which a constant beam of light was passed through a dilute blood suspension contained in a revolving glass chamber. The emergent beam was allowed to fall on a potassium hydride photo-electric cell, and the current so set up in the cell was suitably measured.1 Using, under standard conditions, supposedly unchanging blood suspensions in various isotonic fluids including saline, Brinkman's solution,2 several modifications of it, a special phosphate solution, and a modified Locke's solution,3 marked spontaneous variations in the emergent beam were noted during the first one to three hours after the preparation of the suspensions. Usually these variations consisted of a gradual progressive diminution in light intensity; sometimes an increase occurred.Studies of the pH of the diluting fluids before and after the experiments revealed no relationship to the ligh...