Increased plasma decay-accelerating factor levels in paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a complement regulatory membrane protein that is often absent from the cell surface of blood cells in paroxysmal nocturnal hemoglobinuria (PNH). DAF has also recently been found in the body fluids of healthy individuals. However, its precise structure and biological significance are not yet clear. To clarify the clinical and pathological implications of free DAF, we measured plasma DAF levels in PNH patients, using a newly developed quantitative enzyme-linked immunosorbent assay (ELISA), with a measurable range between 0.2-12 ng, for soluble DAF. ELISA assays revealed significantly increased plasma DAF levels in PNH patients (258 +/- 150 ng/ml, mean +/- S.D., n = 9) as compared with healthy controls (80 +/- 41 ng/ml, n = 17) (p less than 0.01). Taken together with the finding that DAF is synthesized in and released extracellularly from affected PNH cells, plasma DAF levels would be useful for clinical diagnosis and for the quantitative evaluation of the clonal expansion of affected cells in PNH.     

Amelioration of lytic abnormalities of paroxysmal nocturnal hemoglobinuria with decay-accelerating factor.

Purified decay-accelerating factor (DAF), from the stroma of normal human erythrocytes, was incorporated into the membranes of erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH), and its effect on the complement sensitivity of the cells was investigated. Reconstitution with exogenous DAF restored the ability of the affected PNH cells to resist assembly of the homologous C3 convertase, C4b2a, on their surfaces, and decreased the susceptibility of the cells to lysis in acidified serum. Conversely, treatment of normal erythrocytes with monoclonal or polyclonal anti-DAF antibodies abrogated the capacity of the normal cells to circumvent C4b2a assembly and rendered the cells sensitive to acid lysis. These findings show that the previously reported association of DAF deficiency with PNH is causally related to the lytic abnormalities of the cells and clarify the molecular basis for restriction of autologous convertase formation on normal human erythrocytes.     

Immunocytochemical staining of decay-accelerating factor (DAF) on erythrocytes: paroxysmal nocturnal hemoglobinuria (PNH)]

Affected erythrocytes in patients with paroxysmal nocturnal hemoglobinuria (PNH) have been detected as complement-sensitive cells by the complement sensitivity assay. Decay-accelerating factor (DAF), a molecular mass of 70 kDa complement-regulatory membrane glycoprotein, has been reported to be deficient on affected PNH blood cells. In the present study, DAF on erythrocytes from 12 patients with PNH were stained by an immunocytochemical method and the ratio of DAF+ erythrocytes was compared with their laboratory data concerning hemolysis, almost all normal human erythrocytes stained positively for DAF. In contrast, various percentages of DAF+ erythrocytes were found in the patients with PNH. The percentages of DAF+ erythrocytes correlated positively with the total amounts of DAF on erythrocytes measured by an enzyme-linked immunosorbent assay (ELISA), negatively with % hemolysis in Ham's test and in sucrose hemolysis assay and with percentages of PNH type III (PNH-III) erythrocytes. In some patients with PNH, subpopulations of erythrocytes weakly-positive for DAF were demonstrated in addition to DAF- erythrocytes. The immunocytochemical method for staining DAF on erythrocytes developed in the present study is useful for the detection of affected PNH erythrocytes and applicable to further studies on membrane defects in PNH.     

Membrane expression of decay-accelerating factor on neutrophils from normal individuals and patients with paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF), a complement-regulating glycoprotein, was found to be a maturational protein for normal neutrophils, and a remarkable correlation was found between the DAF level and alkaline phosphatase activity in neutrophils. We studied the relationship between the amount of DAF on the membrane and cell maturity in total nucleated bone marrow (BM) cells, mature BM and peripheral blood (PB) neutrophils from normal subjects, and patients with paroxysmal nocturnal hemoglobinuria (PNH) using a fluorescence-activated cell sorter with anti-DAF monoclonal antibodies. Percentage distributions of differentiating neutrophils from normal total nucleated BM cells showed that the proportion of immature cells (myeloblasts plus promyelocytes) decreased, while that of mature ones (bands plus segmented forms) increased as the fluorescence intensity increased. For PB neutrophils, no apparent correlation was found between DAF expression and cell maturity. This may have resulted from margination of the fully matured neutrophils with a high amount of DAF. In PNH patients who have low levels of DAF, this study showed that DAF expression in their neutrophils differs from that in normal subjects, and abnormalities occur in PNH cells from a very early stem-cell stage.     

Different distribution of decay-accelerating factor on hematopoietic progenitors from normal individuals and patients with paroxysmal nocturnal hemoglobinuria

Deficiency of decay-accelerating factor (DAF) occurs in blood cells in paroxysmal nocturnal hemoglobinuria (PNH), characterized by an unusual susceptibility to hemolysis by complement activation. This study examined DAF expression on hematopoietic progenitors from normal individuals and PNH patients using a fluorescence-activated cell sorter (FACS) with monoclonal antibodies to DAF. Nonphagocytic mononuclear marrow cells expressing different density distributions of DAF were sorted into DAF-, DAF+/-, DAF+, and DAF++ fractions. The cells from each fraction were cultured in methylcellulose and assayed for CFU-E, BFU-E, CFU-GM, and CFU-Mix. The percentages of distribution of DAF- negative normal progenitors increased in the order of CFU-E, CFU-GM, BFU-E, and CFU-Mix, whereas those of DAF-positive cells inversely decreased in this order. These results indicate that DAF expression may accompany differentiation from CFU-Mix to CFU-E. On the other hand, most progenitors in PNH patients had little, if any, expression of DAF on their cell surfaces. These findings were supported by another approach using a complement-dependent cytotoxicity method with the anti- DAF monoclonal antibodies. Abnormal expression of DAF was found on the progenitors in the bone marrow as well as on mature cells circulating in the blood in PNH.     

The population of paroxysmal nocturnal hemoglobinuria neutrophils deficient in decay-accelerating factor is also deficient in alkaline phosphatase

In patients with paroxysmal nocturnal hemoglobinuria (PNH) the RBCs, neutrophils (PMNs), monocytes, and platelets derived from the abnormal clone are deficient in the complement-regulatory protein decay-accelerating factor (DAF). RBC acetylcholinesterase (AChE) and leukocyte alkaline phosphatase (LAP) activities are also characteristically low. DAF, AChE, and LAP are known to be anchored within cell membranes to glycophospholipid-containing phosphatidylinositol (PI). Because PNH progenitors contain DAF that appears to be lost with maturation, it has been proposed that this disorder results from abnormal tethering of these and possibly other proteins to membrane PI. We were puzzled, therefore, that our two PNH patients consistently had normal LAP levels. Consequently, we studied their isolated PMNs to compare DAF and LAP activities in individual cells. PMNs were separated by flow cytometry into DAF-positive and -negative populations by using rabbit anti-DAF antiserum and fluorescein-conjugated goat antirabbit IgG. In both patients the majority of PMNs were DAF deficient, and these cells contained very little alkaline phosphatase activity. In contrast, the smaller, DAF-positive cell populations were phosphatase replete. This is the first demonstration that abnormalities in DAF and LAP activity occur in the same PNH PMN population and strengthens the hypothesis that defective anchoring of proteins to membrane glycophospholipid underlies the pathophysiology of this disorder.     

阵发性睡眠性血红蛋白尿症致门静脉高压症1例报告

门静脉高压症是指肝静脉压力梯度>10 mm Hg[1]。由于门静脉压力升高将导致腹腔脏器血管压力升高,形成侧支循环,极易发生并发症,严重影响患者预后。门静脉高压症大多由肝硬化引起,少数患者存在肝前性或肝后性因素。现报道1例由肝前性和肝后性因素共同导致的门静脉高压症。     

Dysfunction and deficiency of decay-accelerating factor (DAF) demonstrated in the lymphocytes from a patient with paroxysmal nocturnal hemoglobinuria (PNH)

A defective cell-mediated immunity was seen in a 62-year-old female with paroxysmal nocturnal hemoglobinuria (PNH). We studied the functional defect of the patient's lymphocytes and its relation to the deficiency of decay-accelerating factor (DAF) on the lymphocytes. T cells (CD 5+) and B cells (CD 20+) were obtained by cell-sorting using fluorescence-activated cell sorter (FACS-IV). These two types of cells from the patient were demonstrated to be deficient in DAF by the fluorometric measurement of DAF content using monoclonal anti-DAF antibodies. These cells were shown to be more susceptible to complement-mediated lysis than normal human lymphocytes by a complement-mediated lysis study. It was carried out by treatment of the lymphocytes with either anti-CD 5 or anti-CD 20 antibody plus rabbit complement. The lymphocytes became more susceptible to complement-mediated lysis by an additional treatment with an anti-DAF antibody both in PNH and in normal controls. From these results, we suggest that DAF plays an inhibitory role against complement activation on human lymphocytes. The mononuclear cells of the patient responded poorly to phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). Skin tests both for PPD and for DNCB showed negative. From these findings, we suggest T cell function in the patient is impaired. Causative relations of the deficiency of DAF to the poor responses of the lymphocytes to lectins and to negative skin tests were discussed.     

Expression of decay-accelerating factor and CD59 in lymphocyte subsets of healthy individuals and paroxysmal nocturnal hemoglobinuria patients

The expression of phosphatidylinositol (PI)-anchored complement-regulatory membrane proteins on circulating blood cells has been well clarified; however, the PI proteins on lymphocyte subsets have not been fully analyzed yet. We examined the expression of decay-accelerating factor (DAF) and CD59 on the T lymphocytes (CD2^?, CD3⁺, CD4^?, and CD8^?) and CD20⁺ B lymphocytes in ten healthy volunteers and 12 paroxysmal nocturnal hemoglobinuria (PNH) patients by cytofluorometry. In healthy controls, each subset of lymphocytes showed a small population of cells weakly positive and a large population of cells strongly positive for DAF and CD59, while erythrocytes showed a single population of cells positive for the PI proteins. The two-population expression of DAF was most distinctive in CD8^? T cells among the subsets. In PNH, each subset of lymphocytes showed a moderately higher population of cells weakly positive and a smaller population of cells strongly positive for the membrane proteins compared with those in the healthy controls. Moreover, in some PNH cases, a negative population for the proteins was found in all subsets. Thus the analysis of PI-anchored proteins on lymphocyte subsets (especially CD8^? T cells) was considered to be of diagnostic value in PNH patients who receive blood transfusion after hemolytic attack of affected erythrocytes. Furthermore, the two-population expression of PI proteins in normal lymphocytes suggests that membrane PI protein would be a new subset marker of lymphocytes.     

Altered metabolism of membrane glycosphingolipids in erythrocytes of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is currently accepted to be a stem-cell disorder of a clonal nature with increased susceptibility to autologous complement attack. Consequent hemolytic feature has been partly explained by lack of complement regulatory membrane proteins such as decay-accelerating factor (DAF) or C8-binding protein that anchored to membrane via glycosyl-phosphatidyl inositol (GPI) lipids. Recent reports suggest essential PNH lesion is the synthetic defect of sugar moiety of the PI-anchor. In PNH, the abnormal expression of C3b/C4b receptor (CRI) glycoproteins, or glycophorin-alpha have been also pointed out. These altered expression of glycoproteins and glyceroglycolipids, especially in the carbohydrate structures, prompted us to analyze biochemically the membrane glycosphingolipids as one of major glycoconjugates in PNH. As results, PNH erythrocytes showed altered metabolism of gangliosides in comparison to control erythrocytes from healthy donors. IV6 NeuAc-nLc4 Cer and highly polar gangliosides variably disappeared in PNH erythrocytes, partly due to impaired sialylation of glycolipids. These results suggest metabolic disorder of carbohydrates of membrane glycoconjugates as a new aspect of PNH.     

Acetylcholinesterase and lymphocyte function-associated antigen 3 found on decay-accelerating factor-negative erythrocytes from some patients with paroxysmal nocturnal hemoglobinuria are lost during erythrocyte aging.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria are deficient in decay-accelerating factor (DAF), a factor called C8-binding protein or homologous restriction factor, acetylcholinesterase (AchE), and lymphocyte function-associated antigen 3 (LFA-3). These proteins share a common feature that glycan-inositolphospholipid anchors the protein to the membrane, suggesting that an abnormality related to this glycolipid causes multiple protein deficiencies. The relationship between the DAF, AchE, and LFA-3 defects was studied by fluorescent flow cytometric analysis. In five patients, DAF-negative erythrocytes were also AchE-negative. In three patients, a fraction of DAF-negative erythrocytes expressed subnormal levels of AchE, indicating that AchE was synthesized in these DAF-negative cells. Erythrocytes from the patients having DAF-negative, AchE-positive cells were separated according to density and analyzed for expression of DAF and AchE. Both proteins decreased with increase of cell density, suggesting that DAF-negative, AchE-positive cells become AchE-negative during erythrocyte maturation by losing AchE. A low level of LFA-3 was found on DAF-negative erythrocytes from one patient and decreased with erythrocyte maturation. These results support an idea that complete deficiency of glycan-inositolphospholipid-anchored proteins on erythrocytes could result from abnormally early termination of surface recruitment of these proteins, and subsequent dilution through cell divisions and loss from the surface.     

Treatment of paroxysmal nocturnal hemoglobinuria

Patients with PNH may be treated with a number of known agents. As in all patients with a chronic disease, a regimen tolerable over a long period of time must be selected. Knowledge and anticipation of complications and their proper treatment are essential parts in the treatment. When these principals are used, many patients may live reasonable lives for very long periods of time.     

Pulmonary hypertension in paroxysmal nocturnal hemoglobinuria.

Pulmonary arterial hypertension (PAH) and cor pulmonale were found in a patient with paroxysmal nocturnal hemoglobinuria (PNH). Autopsy revealed widespread thromboses in pulmonary microvasculature. Vascular thromboses attributed to hypercoagulability have been found in PNH in many organs, including the lungs. PAH has not been reported, however. This disease should then be considered a rare cause of PAH.     

Marrow transplantation for paroxysmal nocturnal hemoglobinuria.

Between 1971 and 1990, nine patients ranging in age from 14-38 years received marrow transplants for paroxysmal nocturnal hemoglobinuria (PNH). Six were transplanted for aplastic complications of PNH. Four of these were from HLA-identical siblings, and the patients were conditioned with cyclophosphamide. One graft was form a syngeneic twin without conditioning, and one from a two HLA-antigen nonidentical father after conditioning with cyclophosphamide and total body irradiation. Three of the four recipients of allogeneic marrow developed acute and two chronic graft-versus-host disease (GVHD). Five of six transplanted for severe aplastic anemia are long-term survivors with follow-up ranging from more than 6.2 to more than 19.1 years. The HLA nonidentical transplant recipient experienced graft rejection and died of a pulmonary hemorrhage. Three patients were transplanted for nonaplastic complications of PNH consisting of life threatening recurrent thromboses or refractory hemolysis. Two of these patients received marrow grafts from HLA-identical siblings after conditioning with busulfan and cyclophosphamide. They are surviving with normal hemograms greater than 2.2 and greater than 2.5 years and had mild chronic GVHD which resolved, although one has biochemical evidence of PNH in 15% of the red cells. One received a syngeneic marrow graft without conditioning but reverted to PNH. He is alive greater than 8.6 years after transplantation. Marrow transplantation for aplastic complications of PNH is successful, well tolerated, and compatible with long-term survival when an HLA-identical sibling or a syngeneic donor is available. For patients without aplasia, one must weigh the complications of transplantation with the life threatening nature of thrombotic episodes and hemolysis.     

Danazol for paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal stem-cell disorder in which blood cells lack complement inhibiting membrane proteins, and become susceptible to complement-mediated injury, leading to chronic intravascular hemolysis and pancytopenia. Glucocorticoids have been a mainstay of therapy. For patients refractory to glucocorticoids and requiring blood transfusions, an alternative therapy is needed. We studied danazol therapy in 5 patients refractory to other treatments. Four of the 5 benefited, showing rise in hematocrit and eventual cessation of transfusion requirements. Remissions lasted ≥2 years in 3 and 10 years in 1 patient. Danazol was well-tolerated without serious side effects. Danazol appears to be a good alternative treatment in PNH. Am. J. Hematol. 54:149–154, 1997 © 1997 Wiley-Liss, Inc.     

A closer look at paroxysmal nocturnal hemoglobinuria

Knowledge of the molecular mechanisms leading to the paroxysmal nocturnal hemoglobinuria (PNH) phenotypes has substantially increased in the past two decades. The associated intravascular hemolysis, hypercoagulablilty, and bone marrow failure result in a wide range of clinical sequlae. Although treatment has usually been symptomatic through several modalities and rarely curative through hematopoietic cell transplantation, recent development of the novel targeted therapeutic agent eculizumab has offered new promises for this highly morbid and fatal disease. This review summarizes current knowledge of the pathophysiology, diagnostic modalities, clinical implications, and treatment approaches of patients with PNH.     

The pathophysiology of paroxysmal nocturnal hemoglobinuria

The molecular basis of PNH is known. Somatic mutation of the X-chromosome gene PIGA accounts for deficiency of glycosyl phosphatidylinositol-anchored proteins (GPI-AP) on affected hematopoietic stem cells and their progeny. However, neither mutant PIGA nor the consequent deficiency of GPI-AP provides a direct explanation for the clonal outgrowth of the mutant stem cells. Therefore, PNH differs from malignant myelopathies in which clonal expansion is directly attributable to a specific, monogenetic event (e.g., t(9;22) in CML) that bestows a growth/survival advantage upon the affected cell. Multiple, discrete PIGA mutant clones are present in many patients, suggesting that a selection pressure that favors the PNH phenotype (i.e., GPI-AP deficiency) was applied to the bone marrow. The nature of this putative selection pressure, however, is speculative, as is the basis of clonal expansion. In many patients, the majority of hematopoiesis is derived from PIGA mutant stem cells. Yet clonal expansion is limited (nonmalignant), and the contribution of the mutant clones to hematopoiesis may remain stable for decades. Understanding the basis of clonal selection and expansion will not only delineate further the pathophysiology of PNH but also provide new insights into stem cell biology and suggest novel therapeutic strategies for enhancing marrow function.