Paroxysmal Nocturnal Hemoglobinuria Cells Research Articles

Leukocyte Antigen-Antibody Reaction and Lysis of Paroxysmal Nocturnal Hemoglobinuria Erythrocytes

Abstract The interaction between leukocyte antigens and specific antibodies can cause lysis of the red cells of paroxysmal nocturnal hemoglobinuria (PNH) in vitro. It is suggested that the activation of complement ensuing from the antigen-antibody interaction is responsible for PNH cell lysis. The clinical implications of this finding are briefly outlined.

Adenosine triphosphatase activity of the red cell membrane in paroxysmal nocturnal haemoglobinuria.

Summary:The activity of the ATPase complex of red cell ghosts as well as the activity of the ATPase components, Mg2+ ATPase and Mg2+, Na+ and K+ ‐dependent ATPase was studied in patients with paroxysmal nocturnal haemoglobinuria (PNH). In ghosts prepared by osmotic haemolysis a significantly higher ATPase activity was found in PNH cells than in normal red cells. Ghosts exposed to the effect of saponin showed a significant increase of ATPase activity both in normal and PNH red cells; however, the increase of activity was markedly higher in PNH ghosts than in ghosts of normal red cells. Both Mg2+‐dependent and Mg2+, Na+ and K+‐dependent ATPase contribute to this increase. The protein content in osmotic ghosts of PNH red cells was higher than normal; but after treating these ghosts with saponin, no marked difference in protein content was found.By comparison with the results of ATPase activity obtained using red cells from non‐splenectomized individuals with hereditary spherocytosis with similar reticulocyte values it is assumed that the increase of ATPase activity is not due to younger cells in the blood but is a further manifestation of the abnormality of the cell membrane in PNH. ATPase activity is present mostly in the inactive form in the red cell membrane and is activated (unmasked) by structural damage of the cell membrane. It is suggested that the increased ATPase activity in the PNH red cell membrane represents a manifestation of abnormal structural arrangement which is more susceptible to damage.The deviations found in the content and metabolism of organic phosphates are probably caused by the younger population of red cells in PNH.

Brief Report: Freeze-Cleaving of Red Cell Membranes in Paroxysmal Nocturnal Hemoglobinuria

Abstract Electron microscopic studies on dried isolated red cell ghosts have been reported to show lesions associated with cell membranes in paroxysmal nocturnal hemoglobinuria (PNH). In this study, carbon-platinum replicas of membranes of freeze-cleaved, partially hydrated PNH red cells and isolated PNH cell ghosts failed to confirm the existence of these abnormalities. This suggests that the previously described lesions are the products of drying artifacts, although they may reflect hidden structural differences between PNH and normal red cell membranes.

Studies of Paroxysmal Nocturnal Hemoglobinuria Erythrocytes: Increased Lysis and Lipid Peroxide Formation by Hydrogen Peroxide*

When paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes were exposed to H(2)O(2) they lysed excessively and formed greater than normal quantities of lipid peroxides when compared to red cells of normal subjects and patients with most types of hematologic disease. It was also shown that lytic sensitivity to acidified serum was related to the enhanced lytic sensitivity to H(2)O(2). If the lipid of PNH cells was first extracted then exposed to ultraviolet radiation more lipid peroxides were formed than in extracts of normal red blood cells. The possible explanations for these findings and their relationship to the PNH hemolytic mechanism are discussed.

Peroxidation of lipid from paroxysmal nocturnal haemoglobinuria-like erythrocytes.

ERYTHROCYTES from patients with paroxysmal nocturnal haemoglobinuria (PNH) are lysed excessively by agents which peroxidize lipid, and their lipid forms more lipid peroxides than that of normal erythrocytes1,2. Recently we noted that incubation of normal red blood cells in reduced glutathione results in cells with all the usual in vitro lytic features of “natural” PNH cells. In the work reported here we have compared lipid extracts from red blood cells of normal subjects and patients with various haemolytic disorders with those of “natural” and “induced” PNH cells, with respect to peroxidation.

Immune lysis of AET-treated normal red cells (PNH-like cells).

NORMAL human red cells treated with the sulphydryl compounds AET (2-aminoethylisothiouronium bromide) or cysteine in suitable conditions develop some of the characters of the red cells of paroxysmal nocturnal haemoglobinuria (PNH)1. In particular, they lyse in slightly acidified fresh compatible normal serum (pH. 6.5) Mengel et al.2 later reported that they had obtained similar results by incubating normal red cells with another sulphydryl compound, reduced glutathione. Lysis in acidified serum (a positive Ham test) has been considered characteristic of PNH, and appears to be an expression of the peculiar sensitivity of such cells to lysis by complement. This has been investigated with a complement lysis sensitivity test based on the use of red cells sensitized with a constant, optimal amount of an anti-I antibody and then treated with graded amounts of fresh compatible normal human serum as a source of complement PNH cells were found to consist of two populations, one markedly sensitive to lysis by C′ (about twenty-five times more sensitive than normal cells) and the other usually only slightly more (about twice) sensitive than normal cells. The very sensitive population of cells and the existence of two populations of cells are distinguishing characteristics of PNH which have not so far been found in health or in any other disease state.

Serum-red cell interactions at low ionic strength: erythrocyte complement coating and hemolysis of paroxysmal nocturnal hemoglobinuria cells.

Complement coating and hemolysis were observed when erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were incubated in isotonic sucrose solution in the presence of small amounts of serum. Normal cells were likewise coated with complement components but did not hemolyze. Both normal and PNH erythrocytes reduced the hemolytic complement activity of the serum used in this reaction. Experience with other simple saccharides and related compounds suggests that the low ionic strength of the sucrose solution is the feature that permitted complement coating of red cells and hemolysis of PNH erythrocytes. Isotonic solutions of other sugars or sugar alcohols that do not readily enter human erythrocytes could be substituted for sucrose. The mechanism for these reactions may possibly relate to the agglutination observed with erythrocytes tested in the serum-sucrose system. Even though PNH hemolytic activity could be removed by prior heating of serum or barium sulfate treatment of plasma, the agglutination phenomenon still persisted. The in vitro conditions necessary for optimal sucrose hemolysis of PNH erythrocytes were described and compared with those of the classical acid hemolysis test. The requirement for less serum in the sucrose hemolysis system than needed in the standard acid hemolysis reaction makes certain experiments, especially those using large amounts of autologous PNH serum, much more feasible. Additional advantages of the sucrose hemolysis test are that it can be carried out at room temperature in the presence of oxalate and citrate and that critical pH control is not essential. To date, the sucrose hemolysis test has been a sensitive and specific one for PNH. A modified test used for screening purposes, the "sugar water" test, is very easy to perform.

Electron-microscope studies of the red cell in paroxysmal nocturnal haemoglobinuria.

It is generally accepted that in paroxysmal nocturnal haemoglobinuria (PNH) there is a fundamental defect in the red cell, but the nature of the defect has not been completely elucidated. The red cells show no particular abnormal morphological features when viewed by the optical microscope but several studies have been carried out on the red‐cell membrane to see if any defect might be revealed by electron microscopy. Matthes, Schubothe and Lindemann (1951) noted an abnormality in the red cell membrane consisting of a patchy appearance with many clefts. This was subsequently confirmed by Braunsteiner, Gisinger and Pakesch (1956) and by Cecchi and Conestabile (1957). However, results have been variable, possible due to the fact that the usual method of preparing red‐cell membranes (‘ghosts’) by drastic osmotic haemolysis in distilled water results in tears and other damage to the membrane (Ponder and Ponder, 1960, 1961; Danon, 1961). Thus, for example, when Douglas and Eaton (1955) used a slight variation of the usual technique they concluded that the membranes of PNH cells were not abnormal, although it should be remarked that electron‐dense ovoid structures can be seen in the illustration of their shadow‐cast preparation.In order to avoid subjecting red cells to a very low ionic strength, Danon, Nevo and Marikovsky (1956) devised a method of dialysis of red‐cell suspensions against hypotonic NaCl, by means of which a gradual decrease in the salt concentration of the medium surrounding the red cells is achieved. By this method ghosts of red cells have been obtained practically free of haemoglobin and relatively undamaged. Electron microscope studies of PNH cells prepared by this method showed a number of abnormalities, which will be described in this paper. It was observed that many of the PNH cell membranes contained electron‐dense material of varying form, size and amount. In order to investigate the nature of this residual material electron microscopy of ultra‐thin sections of the red cells was also carried out.

PH Dependent Hemolytic Systems. I. Their Relationship to Paroxysmal Nocturnal Hemoglobinuria

Abstract 1. Treatment of normal human red cells with various proteolytic enzymes, cholera vibrio filtrate, influenza virus, and periodate ion results in red cells susceptible to acid hemolysis in compatible normal human serum. 2. The kinetics of hemolysis of these artificially altered red cells resembles in many respects those observed in paroxysmal nocturnal hemoglobinuria (PNH). 3. While the observed differences in behavior between these artificially altered cells and PNH cells do not allow direct comparison, it is felt that these models may offer some clues in the understanding of PNH cell hemolysis, and some insight into the nature of the red cell lesion in paroxysmal nocturnal hemoglobinuria.

Hemolysis of PNH erythrocytes in isotonic solutions of choline esters

When the acetylcholinesterase activity of normal cells suspended in isotonic choline ester solutions is inhibited by the addition of physostigmine, these cells are more sensitive to H+ ions (i.e. hemolyze at a relatively higher pH) than the control cells. Such observations have led to the hypothesis that acetylcholinesterase may control membrane pH. Erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) have been demonstrated to be markedly deficient in acetylcholinesterase activity. PNH cells were incubated in isotonic solutions of choline esters and hemolysis and pH changes were followed with time. Such enzyme-deficient cells did not hemolyze at a relatively higher pH, as reported in the case of physostigmine-treated normal cells. The results of these experiments provide no concrete evidence that the erythrocyte acetylcholinesterase enzyme plays any role as an enzyme buffer system. As yet, no simple explanation has been found to correlate the acetylcholinesterase deficiency of PNH erythrocytes with their known susceptibility to hemolysis in acidified serum. Note: (With the Technical Assistance of Dorris E. Webb) Submitted on January 19, 1959