Myelosclerosis and Paroxysmal Nocturnal Haemoglobinuria

3 patients with myelosclerosis have subsequently developed paroxysmal nocturnal haemoglobinuria, with positive in-vitro lysis tests (acidified-serum lysis, cold-antibody lysis and sucrose lysis), characteristic of PNH. These cases provide further evidence of a relationship between PNH and myeloproliferative disorders; they support the proposition that PNH is an acquired defect of erythropoiesis due to an abnormal clone. In two of the patients PNH has been severe with marked intravascular haemolysis, so that in these cases the clinical course and management of the original disease have been influenced by the PNH. In the third patient the PNH defect has, so far, been essentially only a phenomenon of in-vitro tests and apparently without clinical effect.     

Red-Cell Density in Paroxysmal Nocturnal Haemoglobinuria

S ummary . Density distribution of red cells has been measured in twelve patients with PNH by centrifuging blood with phthalate-ester mixtures of varied specific gravity. Results have been compared with findings in other blood diseases and in normal subjects. The PNH red cells have an abnormally low density but normal osmotic fragility. The reason has not been established.
The lightest cells had in general a greater phospholipid content than the heavier cells from the same blood sample; but not more so than in normal blood, and there did not appear to be significant variation in the phosphatide composition of the excessively light PNH cells.
By the technique described it is possible to separate a limited fraction containing only the lightest 10 per cent of the cell population. This fraction contains a concentration of cells which are most susceptible to lysis in vituo; it provides a means for increasing the sensitivity of laboratory tests on PNH blood, and it has enabled a diagnosis to be made in an obscure case when the standard in-vitro lysis tests on whole blood have been negative.     

Paroxysmal Nocturnal Haemoglobinuria in Thailand with Special Reference to an Association with Aplastic Anaemia

Eighty-five cases of PNH in Thailand were analysed, with emphasis on the comparison with European series and on an association with aplastic anaemia. Compared with European series, two points emerge: (1) the disease in Thai affects individuals of younger age group with a tendency to male preponderance; (2) the incidence of arterial and venous thrombosis is relatively rare. In association with aplastic anaemia, two main categories of PNH patients, are described, which differ in their clinical and laboratory citeria.     

Surface Charge of Erythrocytes in Paroxysmal Nocturnal Haemoglobinuria

The electrophoretic mobility of erythrocytes from 3 patients with PNH was measured by standard techniques. Erythrocyte mobility is a measure of the net negative surface charge of these cells. In each of the 3 patients studied the mobility was significantly increased over normal cells indicating that there is an increase in negative surface charge on PNH erythrocytes. Following the Ham's and sucrose lysis tests, surviving cells showed mean mobilities that were not statistically different from normal cells. This suggests that these cells with higher surface charge are vulnerable to complement lysis.     

Variations in the Red Cells in Paroxysmal Nocturnal Haemoglobinuria

S ummary . The red cells of 22 patients with paroxysmal nocturnal haemoglobinuria have been evaluated by the complement lysis sensitivity test of Rosse & Dacie. Four patterns of abnormal susceptibility to complement lysis were seen. In Group 1 (10 patients), markedly abnormal complement-sensitive cells were present and cells of a second population were not distinguishable from normal cells. In Group 2 (four patients), markedly abnormal complement-sensitive cells were present and the second population of cells was clearly distinguishable, but not markedly different from normal. In Group 3 (five patients), three populations of cells were present: a markedly abnormal complement-sensitive population, a population of moderately abnormal cells and a population of normal-appearing cells. In Group 4 (three patients), no cells with marked complement sensitivity were seen; two populations were present, one was apparently normal and the other moderately abnormal (3–4 times as sensitive as normal). During the period of observation two patients in Group 4 acquired a markedly abnormal complement-sensitive population.
The proportion of cells in each population varied during the course of the illness. In three patients the proportion of complement-sensitive cells increased during the onset of the illness, and in two patients the proportion decreased gradually during the resolution of the disease. Increase in the total production of red cells resulted in an increased proportion of complement-sensitive cells, as did decrease in haemolytic rate. Increase in the haemolytic rate resulted in a decreased proportion of complement-sensitive cells.     

The Aplastic Anaemia–Paroxysmal Nocturnal Haemoglobinuria Syndrome

Seven patients are described who suffered from the aplastic anaemia-paroxysmal nocturnal haemoglobinuria syndrome. All had been first diagnosed as having aplastic anaemia. In one case the bone marrow became repopulated but the patient developed severe intravascular haemolysis and died of liver failure due to intrahepatic venous thrombosis. Two patients died of marrow aplasia; haemolysis was minimal and was only a laboratory phenomenon. Another patient also died of marrow aplasia but in his case haemolysis had contributed significantly to his illness. In three patients the bone marrow became repopulated completely or partially and the patients have recovered clinically despite the persistence of a small proportion of PNH red cells.
The relationship between aplastic anaemia and PNH is discussed. It seems likely that the link between the diseases is the development of an abnormal clone of haemopoietic cells in a regenerating, previously aplastic, marrow.
The effect of the superimposition of the PNH defect on the prognosis in aplastic anaemia is discussed. In five cases haemolysis had little or no influence on the course of the disease; but in two patients intravascular haemolysis was severe and was undoubtedly harmful.
The influence of splenectomy on the course of the disease was studied. It was performed in four of the patients, but in only one did it seem to have a beneficial effect.     

Erythrocyte and Leucocyte Enzymes in a Case of Paroxysmal Nocturnal Haemoglobinuria

In a patient with paroxysmal nocturnal haemoglobinuria (PNH) enzymatic activities of erythrocytes and leucocytes were studied. Studies of autohaemolysis were also performed. The following erythrocytary enzymes were measured: Glucose-6-phosphate dehydro-genase (G-6-PD), pyruvate kinase (PK), glutathione reductase (GR), and acetyl-cholinesterase (AcChE). The following enzymes were measured in leucocytes: Adenosine deaminase, purine nucleoside phosphorylase, adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and adenosine kinase. Normal activity of G-6-PD, GR and PK in erythrocytes was found. In leucocytes and lymphocytes activity of purine nucleoside phosphorylase was reduced. Autohaemolysis in vitro was increased, which could not be compensated by addition of glucose or ATP.     

Effects of Sulphydryl Inhibition on the Erythrocyte in Paroxysmal Nocturnal Haemoglobinuria

S ummary . Normal erythrocytes exposed to high concentrations of sulphydryl-containing compounds such as reduced glutathione, cysteine, and 2-aminoethyliso-thiouronium bromide (AET) acquire some in-vitro properties of the PNH erythro-cyte with increased sensitivity to acidified serum and cold-antibody lysis. These findings suggested the possibility that an abnormality of membrane sulphydryl metabolism is a feature of PNH. Investigations were undertaken in five patients with PNH and a group of normals. Quantitative determination of measurable SH groups in haemoglobin-free erythrocyte membranes failed to reveal a difference between PNH and normal. Normal erythrocytes exposed to AET had a marked reduction in measurable SH groups. The functional status of the membrane SH groups was studied by the in-vitro effect of sulphydryl-binding compounds, p -chloromercuribenzoate (PMB) and p -chloromercuribenzene sulphonate (PMBS).
In all PNH cases an increased sensitivity to the haemolytic effects of SH blockade as compared to normal erythrocytes was noted. Studies with the inhibitor, PMBS, which acts only on the outermost surface of the membrane, revealed two types of erythrocytes in PNH, one highly vulnerable and the other behaving similarly to normal. Haemolysis occurred without inhibition of glycolytic activity as measured by lactate production or interference with the intracellular SH status. It appears that the PNH membrane defect is associated with an abnormality in the functional activity of membrane SH groups.     

A Case of Paroxysmal Nocturnal Haemoglobinuria Terminating in a Myeloproliferative Syndrome

Summary: This report discusses the case of a 60-year-old man who presented in 1969 with thrombocyto-penia and mild marrow hypoplasia. A diagnosis of paroxysmal nocturnal haemoglobinuria (PNH), was established. The subsequent course included episodes of overt intra-vascular haemolysis. Thrombocytopenia reverted on several occasions during Oxymetholone therapy. The terminal phase of the illness was marked by the development of a leukocytosis and densely hypercellular bone marrow with splenomegaly. The features were those of a myeloproliferative disorder, although frank leukaemia did not develop.     

Study of Two in Vitro Diagnostic Tests for Paroxysmal Nocturnal Haemoglobinuria

S ummary . The variability with time of the serum haemolytic activity of normal subjects in the acidified-serum and sucrose-haemolysis tests towards the red cells of paroxysmal nocturnal haemoglobinuria (PNH) and PNH-like cells (i.e. normal red cells treated in vitro with the sulfhydryl compound AET) was investigated. A wide variability was observed in samples of serum obtained from individual donors at intervals of months, days, and even hours, without evidence of a circadian rhythm. In spite of this variability each donor of serum, considered in respect to other donors, maintained unchanged with time his rank of serum haemolytic activity towards both PNH and AET cells in the acidified-serum test, but not in the sucrosehaemolysis test. From the practical point of view it seems convenient for the laboratory diagnosis of PNH to carry out acidified-serum tests with the serum of one selected normal subject (in addition to the patient's own serum) and sucrosehaemolysis tests with a few random normal sera.     

Paroxysmal Nocturnal Haemoglobinuria and Refractory Marrow Failure Treated by Marrow Transplantation

S ummary . A patient with pancytopenia and paroxysmal nocturnal haemoglobinuria (PNH) following exposure to insecticide spray developed complete marrow failure after inhalation of vapours containing benzol. There was no sign of spontaneous recovery after more than 6 mth of conventional and supportive therapy. The patient was treated with the immunosuppressive agent cyclophosphamide, 50 mg/kg on each of four days, followed in 36 hr by transplantation of marrow from a sibling compatible at the major human histocompatibility locus (HL-A). Intermittent methotrexate therapy was given for 102 days after grafting to prevent graft-versushost disease. The patient showed prompt haemopoietic engraftment indicated by restoration of marrow cellularity and a rise in peripheral blood cell counts beginning on day 11 after the graft. The patient is alive and well with normal haemopoietic function and continued absence of PNH more than 1 yr and 4 mth after transplantation.     

PIG-A gene mutations in Four Taiwanese Patients with Paroxysmal Nocturnal Haemoglobinuria Following Aplastic Anaemia

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic disorder caused by deficient biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor in haemopoietic stem cells. PIG-A , an X-linked gene that participates in the first step of GPI-anchor synthesis, is responsible for PNH. Various abnormalities of the PIG-A gene have been demonstrated in all patients with PNH so far examined. In this study we characterized the somatic mutations in PIG-A gene in four Taiwanese patients with PNH. We identified five novel mutations in the PIG-A gene, three single nucleotide substitution mutations (−342, C → G, codon 335, GGT → AGT and codon 405, GCT → GTT) and two frameshift mutations (codon 22, GGA → G-A and codon 356, TGT → TGTT) in the PIG-A gene. The −342 mutation was judged to be a polymorphism. Furthermore, three patients had previous clinicopathologic evidence which suggested aplastic anaemia (AA), before the development of PNH. One of these was found to have thrombocytopenia during follow-up. We suggest that the somatic PIG-A gene mutations highlight a subgroup of AA having a pathogenetic link with PNH.     

Adenosine Triphosphatase Activity of the Red Cell Membrane in Paroxysmal Nocturnal Haemoglobinuria

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.     

The Lysis of Paroxysmal Nocturnal Haemoglobinuria Red Cells by Serum and Cobra Factor

S ummary . The complement-sensitive red cells of patients with paroxysmal nocturnal haemoglobinuria (PNH) are haemolysed in the presence of serum and a protein factor present in cobra venom (CoF). The reaction of CoF and a serum factor, C₃ proactivator, requires Mg²⁺. Once this interaction has taken place, the activation of C₃ and the terminal components of complement can occur in the presence of EDTA and result in the lysis of PNH red cells.
The concentration of reactants may be adjusted so that all of the complementsensitive red cells of PNH patients and the red cells of no other patients are lysed. A simple but specific and precise test for the presence of the complement-sensitive red cells of PNH was devised. Normal human red cells treated with trypsin and neuraminidase are not lysed, whereas these cells treated with glutathione or 2-aminoethylisothiouronium bromide (AET) are lysed.