Paroxysmal Nocturnal Hemoglobinuria Cells Research Articles

Complement lysis of human erythrocytes. III. Differing effectiveness of human and guinea pig C9 on normal and paroxysmal nocturnal hemoglobinuria cells.

Complement lysis of human erythrocytes. III. Differing effectiveness of human and guinea pig C9 on normal and paroxysmal nocturnal hemoglobinuria cells.

The “Fabulous Fakes”—Laboratory Style

Methods are described for preparing the following “fake” specimens for laboratory studies: red blood cells deficient in glucose-6-phosphate dehydrogenase, red blood cells that react as paroxysmal nocturnal hemoglobinuria cells, and spinal fluid positive for blood cell counts. The specimens are useful as teaching tools for groups of students and as quality control samples.

Complement Lysis of Human Erythrocytes

Although enhanced sensitivity of erythrocytes to complement-mediated lysis is a hallmark of paroxysmal nocturnal hemoglobinuria (PNH), subpopulations of erythrocytes in such patients vary significantly in this respect. One PNH erythrocyte subpopulation (termed type III) comprises exquisitely sensitive cells, whereas type II PNH erythrocytes are intermediate in complement sensitivity between PNH type III and normal human erythrocytes. Differences in the action of the terminal complement components that would account for the differing lytic behavior of types II and III PNH erythrocytes have been proposed but not directly demonstrated. The present studies, making use of carefully selected cases with pure populations of type II or type III erythrocytes, confirm a prior observation that antibody-coated PNH erythrocytes of both types II and III display comparably supranormal C3 binding in whole human serum. However, when lysis was induced by the isolated C5b-9 membrane attack mechanism, bypassing the requirement for C3 binding, only type III PNH cells exhibited greater than normal lysis. This finding suggests that type III PNH erythrocytes have an additional membrane abnormality not present in type II cells. Thus, the differing lytic behavior of these two cell types in whole serum may reflect the additive effects on type III cells of both exaggerated C3 binding and enhanced sensitivity to C5b-9, whereas the more moderate lysis of type II PNH cells may be determined mainly or entirely by the earlier-acting mechanism producing augmented C3 binding. The failure of guinea pig C8 and C9, as opposed to human C8 and C9, to reveal the true lytic sensitivity of PNH-III E in our earlier study is illustrated, and its implications briefly discussed.

Two Populations of Granulocytes in Paroxysmal Nocturnal Hemoglobinuria

AbstractThe granulocytes in paroxysmal nocturnal hemoglobinuria (PNH) are defective, and the defect is similar to that previously described for the PNH erythrocyte. Using anti-l antibody to activate complement and 51 Cr release to detect cell lysis, we found two populations of granulocytes that differed in their susceptibility to lysis by complement in 5 of 6 patients. A proportion of the cells were lysed by one-fifteenth to one-twentieth the amount of complement required to lyse normal cells; the remainder of the granulocytes appeared to be normal in their susceptibility to the lytic action of complement. The binding of the third component of complement (C3) to PNH granulocytes was at least twice that bound to normal cells, even though the binding of antibody was the same for normal and PNH cells. This suggests that the binding of C3 and probably the efficiency of the terminal steps of complement lysis are increased in the abnormal PNH granulocyte. These defects affect only a portion of the granulocytes, thus suggesting that the disorder is a clonal stem cell abnormality.

Two populations of granulocytes in paroxysmal nocturnal hemoglobinuria

The granulocytes in paroxysmal nocturnal hemoglobinuria (PNH) are defective, and the defect is similar to that previously described for the PNH erythrocyte. Using anti-I antibody to activate complement and 51Cr release to detect cell lysis, we found two populations of granulocytes that differed in their susceptibility to lysis by complement in 5 of 6 patients. A proportion of the cells were lysed by one-fifteenth to one-twentieth the amount of complement required to lyse normal cells; the remainder of the granulocytes appeared to be normal in their susceptibility to the lytic action of complement. The binding of the third component of complement (C3) to PNH granulocytes was at least twice that bound to normal cells, even though the binding of antibody was the same for normal and PNH cells. This suggests that the binding of C3 and probably the efficiency of the terminal steps of complement lysis are increased in the abnormal PHN granulocyte. These defects affect only a portion of the granulocytes, thus suggesting that the disorder is a clonal stem cell abnormality.

Susceptibility to Autoxidation of Lipids of Paroxysmal Nocturnal Hemoglobinuria (PNH)-Like Red Cells

The susceptibility to autoxidation of red cell lipids was studied before and after transformation of normal red cells to PNH-like erythrocytes. The transformation was effected by treatment of the red cells with the sulfydryl compounds D-penicillamine (DP) and N-acetyl-L-cysteine (NAC). The autoxidation was induced by incubating the cells with H2O2 and was estimated by measuring the generated malonyl dialdehyde. The susceptibility to autoxidation was significantly higher in DP-treated cells, while the opposite was true for NAC-treated cells. However, both DP- and NAC-treated cells showed a similar sensitivity to lysis by acid serum and about the same degree of acetylcholinesterase (AChE) activity decrease, thus indicating that the susceptibility to autoxidation of lipids is not involved in the determination of complement sensitivity or in the AChE activity decrease of the sulfydryl-treated cells. Finally, since, as evidenced from most of the reported cases in the literature, increased susceptibility to autoxidation is a feature of PNH cells, it seems reasonable to suggest that DP-treated cells should be used in preference to NAC-treated cells as a laboratory substitute for PNH cells.

Extraction of erythrocyte membrane proteins by sulfhydryl inhibitors.

Human red cell membrane proteins were extracted by incubation of the ghost with hypotonic phosphate buffer (pH 7.4), N-ethylmaleimide and p-hydroxy-mercuribenzoate. In paroxysmal nocturnal hemoglobinuria (PNH), hereditary spherocytosis (HS) and hereditary elliptocytosis, the amount of proteins extracted by these procedures was significantly less than the amount extractable from the ghost of normal and aplastic anemia red cells. Polypeptide patterns of red cell membranes in these hematological disorders were essentially similar to those of normal ghosts. Analysis of the supernatant by SDS polyacrylamide gel electrophoresis revealed that this reduction was mainly due to the reduced amount of peripheral proteins extracted. The extraction of peripheral proteins by sulfhydryl reagents was accompanied by shape changes resulting in the formation of membrane vesicles, suggesting an important role of peripheral proteins in the maintenance of ghost shape. It is also suggested that qualitative abnormalities of peripheral proteins such as altered reactivity to sulfhydryl reagents and/or strong binding to the membrane are present in PNH, HS and hereditary elliptocytosis red cells.

Abnormal solubilization by triton X-100 of erythrocyte membranes from patients with paroxysmal nocturnal hemoglobinuria.

SummaryErythrocyte membranes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were found to undergo a deficient degree of solubilization when reacted with the nonionic detergent Triton X-100. Upon exposure to concentrations of Triton X-100 causing partial membrane solubilization in 5 mM phosphate buffer, pH 7.4, cholesterol and hexoses from patients′ membranes dissociated to a lower degree than the membrane constituents from normal controls and patients with hemolytic anemias unrelated to PNH. The degree of solubilization of membrane protein was identical with all types of erythrocytes. The degree of hemolysis caused by Triton X-100 was similar with PNH and normal red cells. Immunoelectrophoresis of normal membranes solubilized with 1-5% Triton X-100 yielded several α-β1 lines and one β2 line. PNH membranes produced similar α-β1 lines but the β2 line was absent or very faint. Since it was known that the β2 component of normal membranes is a glycolipoprotein with high affinity for Triton X-100,...

Renal Failure in Paroxysmal Hemoglobinuria

To the Editor.— A recent Brief Report inThe Journalby Szwed et al (226:315, 1973) notes an association between renal failure and hemodialysis with the onset of hemolysis in a patient who had previously occult paroxysmal nocturnal hemoglobinuria (PNH). The authors go on to suggest that this represented a complication of the administration of blood products. We would like to suggest an alternative explanation. May et al ( N Engl J Med 289:705, 1973) demonstrated the role of magnesium in initiating the alternate pathway mediated lysis of PNH cells. They found that an increase of as little as 0.6 mEq/liter in the magnesium concentration could initiate PNH lysis. The presence of calcium in normal concentrations was shown to have an inhibitory effect. Perhaps, subtle fluctuations in divalent cation concentration were involved in the PNH cell lysis associated with renal failure. Increasing magnesium concentration and falling calcium concentration and pH , all

Mechanisms of Immune Lysis of the Red Cells in Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum Test and Paroxysmal Nocturnal Hemoglobinuria

The red cells of patients with hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS), a form of congenital dyserythropoietic anemia, and the cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) are lysed more readily than normal cells by certain antibodies, notably cold agglutinins (anti-I) and complement. With some but not other examples of anti-I, HEMPAS and PNH cells adsorbed more antibody than normal cells. Equal quantities of adsorbed antibody bound equal quantities of the first component of complement (C1) to normal, PNH, and HEMPAS cells. However, for a given quantity of bound antibody and C1, much more of the fourth component of complement (C4) was bound to HEMPAS cells than to normal cells. This resulted in the binding of proportionately larger quantities of the third component of complement (C3) to these cells. The same amount of bound C3 was found on the membranes of normal and HEMPAS cells for a given degree of lysis. Hence, the marked increase in lysis of HEMPAS cells is due to the increased adsorption of antibody and/or increased binding of C4.PNH cells bound the same amount of C4 per bound C1 as normal cells but bound more C3 than normal cells. However, the mean concentration of C3 on the membrane of PNH cells was one-third to one-fifth that on normal cells for a given degree of lysis. Hence, the increased lysis of PNH cells is due to the increased binding of C3 and increased hemolytic effectiveness of the bound C3.

Studies of the role of red cell membrane peroxidation in paroxysmal nocturnal haemoglobinuria (PNH).

Summary. No evidence was found to support the claimed role of lipid peroxidation in the pathogenesis and haemolysis in paroxysmal nocturnal haemoglobinuria (PNH) including iron‐induced episodes of gross haemoglobinuria. Therapy with the antioxidant vitamin E failed to influence the course of two patients with PNH. Three separate techniques for inducing lipid peroxidation failed to lower AChE of normal and PNH red cells and to induce susceptibility to in vitro PNH haemolytic tests in normal cells whether iron was added to the test system or not. Under peroxidatic stress, PNH cells did not generate abnormally high amounts of lipid peroxides as measured by malonaldehyde formation. Similar results were obtained with human red cells with induced high concentrations of protoporphyrin IX. Such studies do not, however, vitiate the concept that lipid peroxidation may play an important role in biologic ageing and cell destruction.

Complement-mediated lysis in different cationic media of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria.

SummaryA study was performed of the effects of alkali metal cations on the degree of complement (C)-mediated lysis of red blood cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) as well as of artifically prepared, PNH-like red cells. When rabbit antiserum and human C were used, the alkali metal cations enhanced immune lysis of normal and PNH red cells as follows: K ≥ Rb > Li > Cs ≥ Na. In the acidified serum reaction of PNH and PNH-like cells, however, enhancement of lysis occurred with K, Rb, and Cs, but not with Li, as compared to the degree of lysis in Na. The latter pattern of cationic effect was identical with that obtained upon the final stage of immune lysis of normal human red cells by antibody and C.The results indicate that the influence of the alkali metal cations on the final stage of immune lysis after activation of the classical C pathway is similar to that on the sequence of reactions which forms the alternate pathway. A different pattern of influence of the cations was obs...

The membrane abnormality of the red cell in paroxysmal nocturnal haemoglobinuria.

IN paroxysmal nocturnal haemoglobinuria (PNH) a membrane abnormality renders the red blood cell (RBC) extremely sensitive to the lytic action of complement1. The nature of the abnormality is unknown: an alteration of the cell lipids has been postulated on the basis of the results of some lipid determinations2–8 and the greater tendency of lipids to form peroxides after exposure to ultraviolet light or H2O29–11. Alteration of the membrane proteins has also been proposed as the cause of the cell abnormality12,13. It has been found that treatment with some sulphydryl compounds renders normal RBCs similar to those in PNH in many respects14. In particular, they display a sensitivity to complement lysis twenty-fold greater than normal15 and have a very low acetylcholinesterase activity16. Among the sulphydryl compounds so far examined14,17–20 the radio-protector AET (2-amino-ethyl-isothiouronium bromide) has been shown to be the most effective in producing the PNH-like abnormality21. The PNH-like alteration induced in normal cells by sulphydryl compounds has not yet been identified, although it seems to involve the breaking of membrane disulphide bonds21. We have studied PNH and AET RBC membranes in parallel using sodium dodecyl sulphate (SDS) electrophoresis in Polyacrylamide gels. Our experiments show further similarities between PNH and AET cells and indicate that, in both, the alteration (or at least part of it) is located in the protein moiety of the membrane, where an abnormal polypeptide chain is demonstrable.

Mechanisms of Increased Susceptibility to C Lysis of Abnormal Human Red Blood Cells

Abstract The cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) are abnormally susceptible to lysis by C and cold-reactive antibodies, but for different reasons. For PNH cells, the fixation of antibody, C1 and C4 is the same as for normal cells. However, the amount of C3 fixed/C4 is increased 3- to 7-fold. The amount of C3 fixed for a given degree of lysis is about one-third that of normal cells. In a less sensitive variant of PNH cells, the amount of C3 fixed/C4 was about the same as for the markedly abnormal cells, but the amount of C3 fixed for a given amount of lysis was the same as for normal cells. Normal cells treated with sulfhydryl-reactive agents resembled PNH cells in these reactions. HEMPAS cells fix 2 to 3 times as much of some, but not other, examples of anti-I. The amount of C1 fixed/Ab is normal.

Mechanisms of immune lysis of red blood cells in vitro. I. Paroxysmal nocturnal hemoglobinuria cells.

The effect of five different reactions which activate complement (antibody activation, reduction in ionic strength, acidification, cobra venom factor (CoF) activation, and inulin activation) upon normal and PNH cells was investigated, using normal serum and serum devoid of the fourth component of complement (C4) activity from patients with hereditary angioneurotic edema (HANE) as a source of complement. Both normal and HANE serum lysed paroxysmal nocturnal hemoglobinuria (PNH) cells when complement was activated by acidification, CoF and inulin, indicating that the terminal steps of complement were activated in the absence of C4, presumably by the alternate or properdin pathway. Normal but not HANE serum lysed cells coated with anti-I, indicating that complement was activated by the C1-dependent classic pathway. HANE serum partially supported lysis by serum at reduced ionic strength, indicating that the activation of terminal complement components had occurred through both of the pathways of activation. The amount of the third component of human complement (C3) which was bound to the membrane of lysed and unlysed cells by these procedures was determined by anti-C3 absorption and was found to differ for each method of complement activation. In general, more C3 was bound to lysed cells than to unlysed cells. For given conditions, more was bound to PNH cells than to normal cells. However, very much less bound C3 was required for lysis of the PNH cells than for lysis of normal cells. These two phenomena, especially the latter, account for the marked lysis of PNH cells when complement is activated. Normal cells treated with AET (aminoethylisothiouronium bromide) did not bind more C3 than untreated cells and the lysed cells had less bound C3 than lysed PNH cells.

Study of in vitro lysis of paroxysmal nocturnal haemoglobinuria (PNH) and PNH-like red cells.

Summary. In paroxysmal nocturnal haemoglobinuria (PNH) the intensity of haemolysis obtained in the acidified‐serum and sucrose‐haemolysis tests—the two procedures most commonly used for the laboratory diagnosis of the disease—varies according to the red cells, serum and test used. The influence of these three variables has been investigated on both PNH and PNH‐like lysis, that is lysis of PNH cells and of normal red cells treated in vitro with the sulphydryl compound 2‐aminoethylisothiouronium bromide (AET). The conclusions, almost identical for PNH and AET lysis experiments, suggest that for the laboratory diagnosis of PNH: (1) it is advisable to use both tests in parallel; (2) sera to be used in the two tests should be selected; (3) for this selection AET cells can be employed when PNH cells are not available.

Electron microscope study of PNH red cells and AET-treated normal red cells (PNH-like cells).

The morphology of red cells damaged by 2-aminoethylisothiouronium bromide (AET) has been compared to that of PNH cells by transmission and scanning electron microscopy. The main features of the effect of AET as demonstrated by the scanning electron microscope were spherical and deformed cells, with surface craters and relatively large pits. The transmission electron microscope revealed loss of surface structure, an unusual amount of irregularly shaped electrondense material and fine pits in the cell membranes. The paroxysmal nocturnal haemoglobinuria cells showed, by transmission electron microscope, electron-dense material scattered over the cell and fine pitting; by scanning electron microscope, larger craters were seen and swellings protruded from the cell surface. Although the AET changes were not identical with the natural abnormality of paroxysmal nocturnal haemoglobinuria, the two types of cells were sufficiently similar to each other to support the proposition that AET cells have many of the characteristics of paroxysmal nocturnal haemoglobinuria.

The Life-span of Complement-sensitive and -insensitive Red Cells in Paroxysmal Nocturnal Hemoglobinuria

Abstract The survival of the complement-sensitive and complement-insensitive red cells in four patients with paroxysmal nocturnal hemoglobinuria (PNH) have been determined using either a cohort or a random label. The complement-sensitive cells have a very short survival (T ½) of less than 6 days by either technique). The complement-insensitive cells have a longer survival, with a large component of random destruction. The complement-sensitive cells are not changed into complement-insensitive cells. The proportion of complement-sensitive cells produced per day is very much greater than the proportion of these cells in the circulating blood.

Quantitative Studies on the Sensitivity to Complement Lysis of Red Cells Treated With Thiol-reactive Reagents

Abstract The effect of reduced glutathione on the complement sensitivity of normal and paroxysmal nocturnal hemoglobinuria (PNH) red cells was determined quantitatively. The susceptibility to complement lysis was increased in normal and complement-sensitive and complement-insensitive PNH cells; the percentage increase in susceptibility was inversely related to the initial complement-sensitivity. P-chloromercuribenzene sulfonate (PCMBS) likewise increased the sensitivity of normal cells to lysis by complement; the effect with both compounds appeared to be due to reaction with membrane thiol groups. GSH-treated normal cells were similar to PNH cells in that the susceptibility to immune lysis occurred without concomitant increase in fixation of antibody or first component of complement. GSH-treated normal cells differed from PNH cells in that the membrane acetylcholinesterase activity was normal and the cells were not stable during preparation and short-term storage.

Lysis of erythrocytes by complement in the absence of antibody.

A new pathway of complement-mediated hemolysis has been described. It is independent of antibody and does not require binding of the first four complement components to the target-cell surface. The actual attack of the target cell begins with the attachment of C5, C6, and C7. The binding reaction is catalyzed by C4, 2, 3, an enzyme which may be formed in cell-free solution. C4, 2, 3 may effect binding of C5, 6, 7 by acting from the fluid phase or from the surface of another cell to which it is specifically bound (EAC 4, 2, 3). In either case, the resulting product is EC5, 6, 7 which is susceptible to lysis by C8 and C9. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were particularly susceptible to lysis by the above described mechanism. PNH cells, but not normal human erythrocytes, could also be lysed through activation of complement by cobra factor. These observations allow the operational distinction of an activation and an attack mechanism of complement.