The Action of Two Sulfhydryl Compounds on Normal Human Red Cells: Relationship to Red Cells of Paroxysmal Nocturnal Hemoglobinuria

Treatment of normal human red cells with AET and cysteine, under suitableexperimental conditions, modifies them in such a way that their behavior inin vitro hemolysis tests becomes similar to that of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

It is felt that alteration of the red cells is due to the -SH groups possessedby both substances. A possible mechanism of action is hypothesized.

Submitted on April 7, 1964 Accepted on June 17, 1964     

Paroxysmal Nocturnal Hemoglobinuria: Evidence for Monoclonal Origin of Abnormal Red Cells

Paroxysmal nocturnal hemoglobinuria(PNH) was diagnosed in a 26-year-oldNigerian woman who subsequently diedof amebic colitis. The patient’s red cellsexhibited mosaicism with respect to glucose 6-phosphate dehydrogenase, in thatsome of them had the A, and some ofthem had the B variant of this enzyme(as expected in female subjects heterozygous at this sex-linked locus). The redcells bearing the PNH abnormality onlyhad the B variant, suggesting that theyall belonged to a single abnormal clone.

Submitted on October 27, 1969 Accepted on January 12, 1970     

Observations on Certain Enzyme Activities of Normal Human Red Cells Treated with Sulfhydryl Compounds

Some enzyme activities of normal human red cells treated with AET andcysteine have been investigated. Treated red cells showed low acetylcholinesterase activity and low O₂ uptake in the presence of methylene blue; on thecontrary, glucose-6-phosphate-dehydrogenase activity and lactic acid production in the presence of glucose were found to be normal. Results are brieflydiscussed.

Submitted on May 24, 1965 Accepted on November 13, 1965     

Paroxysmal Nocturnal Hemoglobinuria: From Bench to Bed

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia with highly variable clinical symptoms making the diagnosis and prediction of its outcome difficult. It is caused by the expansion of a hematopoietic progenitor cell that has acquired a mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene that results in deficiency of the glycosylphosphatidylinositol anchor structure responsible for fixing a wide spectrum of proteins particularly CD55 and CD59. The clinical features of this disease arise as a result of complement-mediated hemolysis in unprotected red cells, leukocytes, and platelets as well as the release of free hemoglobin. Patients may present with a variety of clinical manifestations, such as anemia, thrombosis, kidney disease, smooth muscle dystonias, abdominal pain, dyspnea, and extreme fatigue. PNH is an outstanding example of how an increased understanding of pathophysiology may directly improve clinical symptoms and treat disease-associated complications when we inhibit the terminal complement cascade. This topic will discuss PNH overview to assist specialists looking after PNH patients.     

Diagnosis of Paroxysmal Nocturnal Hemoglobinuria: Recent Advances

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder with its protean clinical manifestations. This is due to partial or complete absence of ‘glycophosphatidyl-inositol-anchor proteins’ (GPI-AP). The main aim of this review is to highlight various diagnostic modalities available, basic principle of each test and recent advances in the diagnosis of PNH. Recently among various tests available, the flow cytometry has become ‘the gold standard’ for PNH testing. In order to overcome the difficulties encountered by the testing and research laboratories throughout the world, International Clinical Cytometry Society has come up with guidelines regarding the indications for testing, protocol for sample collection, processing, panel of antibodies as well as gating strategies to be used, how to interpret the test and reporting format to be used. It is essential to test at least two GPI-linked markers on at least two different lineages particularly on red cells and granulocytes/monocytes. The fluorescent aerolysin combined with other monoclonal antibodies in multicolour flow cytometry offered an improved assay not only for diagnosis but also for monitoring of PNH clones. It is equally important to diagnose this rare entity with high index of suspicion.     

The Effect of Iron Therapy in Paroxysmal Nocturnal Hemoglobinuria

Two patients with paroxysmal nocturnalhemoglobinuria who were iron deficientwere treated with parenteral iron compounds. In both instances, a marked increase in hemoglobinuria followed,beginning 4 days after the first administration of the iron compound. The risein hemolysis paralleled the rise in reticulocyte count. When erythropoiesis wassuppressed with transfusion, the administration of iron did not bring about anincrease in hemolysis. This indicates thatthe effect of iron in bringing abouthemolysis in patients with paroxysmalnocturnal hemoglobinuria is due to anincrease in erythropoiesis, resulting in theproduction of excessive numbers of complement-sensitive cells.

Submitted on March 20, 1970 Revised on May 8, 1970 Accepted on May 28, 1970     

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

1. Treatment of normal human red cells with various proteolytic enzymes,cholera vibrio filtrate, influenza virus, and periodate ion results in red cellssusceptible to acid hemolysis in compatible normal human serum.

2. The kinetics of hemolysis of these artificially altered red cells resemblesin many respects those observed in paroxysmal nocturnal hemoglobinuria(PNH).

3. While the observed differences in behavior between these artificiallyaltered cells and PNH cells do not allow direct comparison, it is felt thatthese 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.

Submitted on July 14, 1960     

Paroxysmal Nocturnal Hemoglobinuria: The Role of Antibodies in the Diagnostic Thrombin Test

1. The thrombin test for paroxysmal nocturnal hemoglobinuria (PNH) consists in the demonstration that PNH hemolysis in vitro is increased by theaddition of a small amount of thrombin. This ability of thrombin is not substantially changed by preliminary removal of the heterophil antibodies whichare present in commercial preparations of bovine thrombin, provided the testis performed in the way it was originally described.

2. Dilution of the serum with saline and the addition of imidazole bufferabolish the phenomenon of thrombin enhancement of PNH hemolysis.

3. Certain commercial thrombin preparations contain additives which inactivate the PNH hemolytic system. These materials are unsuitable for usein the thrombin test.

Submitted on July 13, 1959 Accepted on August 10, 1959     

阵发性睡眠性血红蛋白尿患者骨髓粒单系祖细胞的体外培养研究

本文采用造血祖细胞体外培养技术，研究了阵发性睡眠性血红蛋白尿（ＰＮＨ）病人骨髓粒单系祖细胞（ＣＦＵ－ＧＭ）的增殖能力；骨髓细胞经酸化ＡＢ型血清处理后ＣＦＵ－ＧＭ的增殖能力；和ＣＦＵ－ＧＭ对粒单系集落刺激因子（ＧＭ－ＣＳＦ）的反应能力，发现ＰＮＨ病人骨髓ＣＦＵ－ＧＭ集落数明显低于正常；骨髓细胞经新鲜酸化ＡＢ型血清处理后培养的ＣＦＵ－ＧＭ集落数明显低于经热灭活酸化ＡＢ型血清处理后培养的集落数；ＣＦＵ－ＧＭ对ＧＭ－ＣＳＦ的剂量反应曲线低平。因此认为ＰＮＨ病人骨髓粒单系祖细胞膜缺陷导致其在酸性条件下对补体的敏感性增高而致损伤或溶解和导致其对粒单系集落刺激因子的敏感性降低。     

Brief Report: The Exacerbation of Hemolysis in Paroxysmal Nocturnal Hemoglobinuria by Strenuous Exercise

Muscular exercise in patients with paroxysmal nocturnal hemoglobinuria hasbeen shown to result in systemic acidosis with lacticacidemia and exacerbationof in vivo hemolysis. It is suggested on the basis of this study that patientswith PNH be advised to avoid severe muscular exercise.

The acid hemolysis test should be performed in all patients that aresuspected of having March hemoglobinuria.

Submitted on February 24, 1967 Accepted on April 17, 1967     

Clinical Manifestations of Paroxysmal Nocturnal Hemoglobinuria: Present State and Future Problems

The clinical pathology of paroxysmal nocturnal hemoglobinuria (PNH) involves 3 complications: hemolytic anemia, thrombosis, and hematopoietic deficiency. The first 2 are clearly the result of the cellular defect in PNH, the lack of proteins anchored to the membrane by the glycosylphosphatidylinositol anchor. The hemolytic anemia results in syndromes primarily related to the fact that the hemolysis is extracellular. Thrombosis is most significant in veins within the abdomen, although a number of other thrombotic syndromes have been described. The hematopoietic deficiency may be the same as that in aplastic anemia, a closely related disorder, and may not be due to the primary biochemical defect. The relationship to aplastic anemia suggests a nomenclature that emphasizes the predominant clinical manifestations in a patient. This relationship does not explain cases that appear to be related to myelodysplastic syndromes or the transition of some cases of PNH to leukemia. Treatment, except for bone marrow transplantation, remains noncurative and in need of improvement.     

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.     

Paroxysmal Nocturnal Hemoglobinuria: Mechanism of the Enhancement of Hemolysis by Bovine Thrombin

1. Crude bovine thrombin contains most, if not all, the proteins of bovineserum.

2. The effect of crude bovine thrombin in enhancing the lysis of paroxysmalnocturnal hemoglobinuria erythrocytes has been shown to be an effect on theerythrocytes themselves.

3. By enzyme inactivation, absorption technics and Sephadex chromatography the thrombic clotting activity has been separated from the PNH enhancing activity nullifying any role of thrombic clotting activity in the enhancement of PNH hemolysis by crude bovine thrombin.

Submitted on October 20, 1966 Accepted on February 21, 1967     

Production of Paroxysmal Nocturnal Haemoglobinuria-like Red Cells by Reducing and Oxidizing Agents

S ummary . Normal red cells incubated with high concentrations of compounds containing sulphydryl groups are known to develop several characteristics of red cells obtained from patients with paroxysmal nocturnal haemoglobinuria (PNH). These include positive acid and sucrose haemolysis tests and a decline in acetylcholinesterase (AChE) activity. The present studies demonstrate that incubation of normal red cells with the non-sulphydryl reducing agent sodium borohydride also results in the production of PNH-like characteristics. Evaluation of this phenomenon utilizing the sulphydryl agents glutathione (GSH) and dithiothreitol (DTT) reveals that the transformation is not dependent upon the presence of oxygen, is not associated with a significant decline in red cell vitamin E levels, and is not observed when GSH is rapidly oxidized by azoester. It appears that the most likely mechanism for the formation of PNH-like red cells under these conditions is through the reduction of cell membrane disulphide groups.
Production of a positive sucrose and acid haemolysis test as well as a decline in AChE activity was also observed in two experimental systems which produce oxidative free radical damage to the red cell membrane: gassing of normal red cells with ozone, and irradiation of red cells from patients with erythropoietic protoporphyria with 400 nm light.