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Paroxysmal nocturnal hemoglobinuria (PNH) develops in patients who have had a somatic mutation in the X-linked PIG-A gene in a hematopoietic stem cell; as a result, a proportion of blood cells are deficient in all glycosyl phosphatidylinositol (GPI)-anchored proteins. Although the PIG-A mutation explains the phenotype of PNH cells, the mechanism enabling the PNH stem cell to expand is not clear. To examine this growth behavior, and to investigate the role of GPI-linked proteins in hematopoietic differentiation, we have inactivated the pig-a gene by homologous recombination in mouse embryonic stem (ES) cells. In mouse chimeras, pig-a- ES cells were able to contribute to hematopoiesis and to differentiate into mature red cells, granulocytes, and lymphocytes with the PNH phenotype. The proportion of PNH red cells was substantial in the fetus, but decreased rapidly after birth. Likewise, PNH granulocytes could only be demonstrated in the young mouse. In contrast, the percentage of lymphocytes deficient in GPI-linked proteins was more stable. In vitro, pig-a- ES cells were able to form pig-a- embryoid bodies and to undergo hematopoietic (erythroid and myeloid) differentiation. The number and the percentage of pig-a- embryoid bodies with hematopoietic differentiation, however, were significantly lower when compared with wild-type embryoid bodies. Our findings demonstrate that murine ES cells with a nonfunctional pig-a gene are competent for hematopoiesis, and give rise to blood cells with the PNH phenotype. pig-a inactivation on its own, however, does not confer a proliferative advantage to the hematopoietic stem cell. This provides direct evidence for the notion that some additional factor(s) are needed for the expansion of the mutant clone in patients with PNH.

A large fraction of the hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) are deficient in membrane expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Current evidence suggests that this deficiency is sufficient to account for the hemolytic and thrombotic manifestations of this disease but not for its frequent association with aplastic anemia, an autoimmune disorder in which the patients' own hematopoietic progenitor cells are the target. Mutations in X-linked gene PIG-A, encoding one of several enzymes required for the biosynthesis of the glycophosphatidylinositol anchor, have been found in all PNH patients studied to date. Recent experiments with murine Pig-a knock-out embryonic stem cells show that although embryogenesis is critically dependent on normal GPI-AP expression, Pig-a-deficient cells can undergo apparently normal hematopoietic differentiation if they develop in a GPI-AP-replete environment. Thus, in an in vitro mouse model of PNH, Pig-a mutations confer no gross proliferative or differentiative advantage or disadvantage, suggesting an unidentified process selecting for these mutations in the bone marrow of patients with the PNH-aplastic anemia syndrome. The rescue of hematopoiesis observed in chimeric cultures of knock-out and normal cells was accompanied by intercellular transfer of GPI-AP, suggesting exciting new possibilities for future therapeutic manipulations in PNH patients.

Myelodysplasia (MDS) and aplastic anaemia-paroxysmal nocturnal haemoglobinuria (AA/PNH) syndrome developed in a severe aplastic anaemia (AA) patient after treatment with immunosuppressive (IS) therapy. Glycosylphosphatidyl inositol (GPI)-linked proteins were determined, and during the AA/PNH phase, a high proportion of neutrophils were found to be negative, without clinical evidence of haemolysis. However, MDS developed with cytogenetic abnormalities of monosomy 7,9q- and a rearranged chromosome 6; the GPI-linked protein negative cells were completely replaced by positively expressing cells. This represents the emergence of a GPI-linked protein positive myelodysplasia clone arising separately from an AA/PNH clone.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease with distinct and rather peculiar characteristics that have puzzled hematologists for more than a century. PNH cells are deficient in a set of membrane proteins that have in common a glycolipid anchor. We refer to this combination of deficiencies as the PNH abnormality or the PNH phenotype. Biochemical analysis has recently made it possible to pinpoint the metabolic block in PNH cells to an early step in the biosynthesis of the glycolipid anchor. This block is due in turn to the deficiency of a protein, called PIG-A, which is encoded by an X-linked gene. Expression cloning of the PIG-A gene has been followed by the identification in patients with PNH of somatic mutations in this gene that inactivate or impair the function of the PIG-A protein. These findings explain in full the molecular basis of the PNH abnormality, but they do not explain how the PNH clone, which is biochemically defective, can expand to the extent of contributing a substantial proportion of the patient's hematopoiesis. Thus a second factor is required to explain the pathogenesis of PNH. This is most likely the coexistence of an element of bone marrow failure that produces, paradoxically, a survival or growth advantage for the PNH clone. The notion that the injury causing failure of normal stem cells spares selectively cells with the PNH phenotype is supported by a number of observations, including the finding of multiple independently arisen PNH clones in patients with PNH.

Treatment of severe aplastic anemia with antithymocyte globulin (ATG) and cyclosporin leads to clinical remission in a large proportion of patients. As many as 10% to 57% of these patients, however, develop paroxysmal nocturnal hemoglobinuria (PNH). We and others have observed that this secondary PNH appears to be more indolent than classical PNH, which results from an acquired mutation in the PIG-A gene. In the present study, we compared PIG-A mRNA transcripts in affected cells from patients with secondary PNH and patients with classical PNH. All four of our aplastic patients who developed PNH had a negative Ham test at diagnosis. Two of the four showed a positive Ham test within 3 months after ATG/cyclosporin administration, one developed a positive test at 6 months, and another at 18 months after immunosuppressive therapy. All four patients remain transfusion-independent with no thrombotic episodes after a mean follow-up of 30 months (range, 6 to 63 months). Reverse transcription-polymerase chain reaction (RT-PCR) of PIG-A transcripts in DAF-/CD59- neutrophils or lymphocyte lines of the four patients showed PIG-A abnormalities in all cases. Transition of C163 to T was found in one, a 14-bp deletion (positions 1141 to 1154) was found in the second, deletion of C39 was found in the third, and two mutations, transition of C55 to T and transversion of T762 to A, were found in the fourth. These abnormalities compared with findings of abnormal RNA splicing causing a 133-bp deletion, a 4-bp insertion (between positions 578 and 579), loss of A767, and loss of C575 in four patients with primary PNH. We conclude that secondary PNH that evolves out of aplastic anemia, like classical PNH, is associated with mutations in the PIG-A gene. The apparent indolent nature of this disease probably reflects early detection.

Bone marrow from 65 patients with aplastic anaemia (AA) was tested for stroma growth in short term cultures (2 weeks) and for colony formation by haemopoietic precursor cells during the course of their disease. In 18 untreated patients, mean stroma growth was 30% of normal and colony formation was virtually absent. After treatment with immunosuppression (IS), as estimated from 90 examinations in 54 patients, stroma growth was approximately 50% and colony growth approximately 10% of normal. Growth impairment of stroma and haemopoietic precursors persisted for 10 and more years after IS. Results of 2-week stroma cultures were compared with results of long term bone marrow cultures in 10 AA patients and 4 controls. At 2 weeks, growth of aplastic marrow was delayed compared to normal, but this difference became less evident with prolonged incubation time. In vitro growth abnormalities were compared with the clinical evolution after IS. The development of late haematological complications (paroxysmal nocturnal haemoglobinuria (PNH)) and myelodysplastic syndrome (MDS), did not correlate with the degree of stroma growth impairment. However, relapse of aplasia was associated with poor stroma growth: 8/29 patients with stroma confluence of < or = 30% during haematological remission versus 1/25 with stroma confluence of > 30% relapsed. We conclude that (i) the haematopoietic microenvironment is frequently coinvolved in the disease process of AA, (ii) a defect is detected in short term rather than in long term stroma cultures and, (iii) relapse is more frequent in patients with poor stroma growth.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia in which abnormal red blood cells with enhanced susceptibility to complement undergo intravascular hemolysis when complement is activated in vivo, resulting in hemoglobinuria. This enhancement of complement susceptibility appears to involve multipotent stem cells and, in this respect, is thought to closely resemble myelodysplastic syndrome. Recently, it has been reported that the increased susceptibility of PNH cells to complement-mediated lysis is related to a deficiency of complement regulatory membrane proteins, especially CD55 and CD59. Both proteins are glycosylphosphatidylinositol (GPI)-anchored membrane proteins. It was found that a deficiency of GPI-anchored membrane proteins in PNH cells is due to the faulty synthesis of the GPI-anchor which may occur during early synthesis. Moreover, it is suggested that an abnormality of the PIG-A (phosphatidylinositol glycan-class A) gene, which is related to the early stage of GPI-anchor synthesis, is responsible for the pathogenesis of PNH. In conclusion, the clinical expression of PNH is dependent on two factors, complement susceptibility of PNH blood cells and changes in bone marrow function. The search for the ideal treatment of this disease may be aided by resolving the relationship between the two.

Immunosuppressive therapy can produce hematologic improvement in a large proportion of patients with severe aplastic anemia. Antithymocyte globulin (ATG) is the current treatment of choice for patients who do not have histocompatible sibling donors or who are otherwise inegligible for allogeneic bone marrow transplantation. About 50% of patients respond to an initial course of ATG, and many nonresponders can be salvaged by subsequent treatment with cyclosporine (CsA). To determine whether simultaneous administration of these agents could further improve response rates, we enrolled 55 patients in a therapeutic trial of 4 days of ATG and 6 months of CsA. Among the 51 patients who had not received previous courses of ATG or CsA, 67% had responded by 3 months, and 78% had responded by 1 year (response was defined as an increase in peripheral blood counts sufficient that a patient no longer met the criteria for severe disease). There was a high incidence of relapse (36% actuarial risk at 2 years), but most relapsed patients responded to additional courses of immunosuppression, and relapse was not associated with a significant survival disadvantage. Evolution to myelodysplastic syndromes and acute leukemia was rare (1 of 51 patients), but the later appearance of paroxysmal nocturnal hemoglobinuria was more common (5 of 51 patients). Actuarial survival was 86% at 1 year and 72% at 2 years. These data support the use of a combination immunosuppressive regimen containing both ATG and CsA as first-line therapy for severe aplastic anemia.

The protein encoded by the yeast gene SPT14 shows high sequence similarity to the human protein, PIG-A, whose loss of activity is at the origin of the disease paroxysmal nocturnal hemoglobinuria. The symptoms of this disease are apparently due to a loss of cell surface, glycosylphosphatidylinositol (GPI)-anchored proteins. Like PIG-A mutant cells, spt14 mutant cells are defective in GPI anchoring due to a defect in the synthesis of GlcNAc-PI, the first step of GPI synthesis. The spt14 mutant causes several other abnormalities including transcriptional defects and a downregulation of inositolphosphoceramide synthesis. We suggest that these defects are indirect results of the loss of GPI anchoring.

The association of paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA) raises the yet unresolved questions as to whether these two disorders are different forms of the same disease. We compared two groups of patients with respect to cytogenetic features, glycosylphosphatidylinositol (GPI)-linked protein expression, protein C/protein S/thrombomodulin/antithrombin III activity, and PIG-A gene expression. The first group consisted of eight patients with PNH (defined as positive Ham and sucrose tests at diagnosis), and the second, 37 patients with AA. Twelve patients with AA later developed a PNH clone. Monoclonal antibodies used to study GPI-linked protein expression (CD14 [on monocytes], CD16 [on neutrophils], CD48 [on lymphocytes and monocytes], CD67 [on neutrophils and eosinophils], and, more recently, CD55, CD58, and CD59 [on erythrocytes]) were also tested on a cohort of 20 normal subjects and five patients with constitutional AA. Ham and sucrose tests were performed on the same day as flow-cytometric analysis. Six of 12 patients with AA, who secondarily developed a PNH clone, had clinical symptoms, while all eight patients with PNH had pancytopenia and/or thrombosis and/or hemolytic anemia. Cytogenetic features were normal in all but two patients. Proteins C and S, thrombomodulin, and antithrombin III levels were within the normal range in patients with PNH and in those with AA (with or without a PNH clone). In patients with PNH, CD16 and CD67 expression were deficient in 78% to 98% of the cells and CD14 in 76% to 100%. By comparison, a GPI-linked defect was detected in 13 patients with AA, affecting a mean of 32% and 33% of CD16/CD67 and CD14 cell populations, respectively. Two of three tested patients with PNH and 1 of 12 patients with AA had a defect in the CD48 lymphocyte population. In a follow-up study of our patient cohort, we used the GPI-linked molecules on granulocytes and monocytes investigated earlier and added the study of CD55, CD58, and CD59 on erythrocytes. Two patients with PNH and 14 with AA were studied for 6 to 13 months after the initial study. Among patients with AA, four in whom no GPI-anchoring defect was detected in the first study had no defect in follow-up studies of all blood-cell subsets (including erythrocytes). Analysis of granulocytes, monocytes, and erythrocytes was performed in 7 of 13 AA patients in whom affected monocytes and granulocytes were previously detected. A GPI-anchoring defect was detected on erythrocytes in five of six.(ABSTRACT TRUNCATED AT 400 WORDS)

Recently a defective glycosylation of glycoconjugates has been implicated in the pathogenesis of a number of heritable or acquired diseases of humans. Herein I discuss them under the name of diseases of aberrant glycosylation. These are: congenital dyserythropoietic anemia type II, carbohydrate-deficient glycoprotein syndrome, I-cell disease, galactosemia in subjects on galactose-free diet, variants of leukocyte adhesion deficiency, and of Ehlers-Danlos syndrome, paroxysmal nocturnal hemoglobinuria, and Tn syndrome. Regarding the present views on the function of glycoconjugates it is probably significant that in most instances defective or missing glycoproteins (or proteoglycans) but not glycosphingolipids, are probably involved in the pathogenesis of these diseases.

The clinical interrelationship between paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA) promoted a search for a pathogenetic link. Since the molecular defect in PNH is a failure to express phosphatidylinositol glycan-anchored proteins (PIG-AP), we investigated whether this defect could also be demonstrated on peripheral blood cells of patients with typical AA. Quantification of the expression of PIG-AP was performed by flow cytometry using the monoclonal antibodies (MAbs) CD16 and CD66b for granulocytes, CD14 and CD48 for monocytes, CD48 and CD52 for lymphocytes, and CD55 and CD59 for erythrocytes. We analyzed cells from 52 patients with acquired AA. A PIG-AP-defective population was identified in 27 of 52 patients (52%) in at least one cell lineage. Granulocytes were involved in 25 of 27, monocytes in 18 of 25, lymphocytes in seven of 27, and erythrocytes in seven of 27 AA patients who were affected by a PIG-AP deficiency. The response rate to standard immunosuppressive therapy was significantly higher in the group of patients without a PIG-AP-deficient population than in patients with a PIG-AP-deficient population in at least one cell lineage (85.7 vs. 30.4%; p < 0.0003). Our results demonstrate that on the basis of PIG-AP expression, the proportion of AA patients who show features of typical AA along with a PNH phenotype is substantially higher than previously recognized. The pattern of PIG-AP expression might identify subgroups of AA patients who differ in the underlying mechanism as well as in the course of their disease.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder that affects both sexes equally. The biochemical defect in PNH resides in the incomplete enzymatic assembly of glycosylphosphatidylinositol (GPI) anchors used for surface protein attachment. In all PNH patients tested to date, the biosynthetic defect occurs at the addition of N-acetyl-glucosamine to the phosphatidylinositol molecule (class A defect). A human cDNA, Piga, that repairs cell lines with the class A GPI-anchor biosynthetic defect has been recently cloned. Mapping of Piga to the X chromosome suggests that a single acquired mutation within Piga could alter GPI-anchor synthesis and result in PNH. However, this finding does not explain why all PNH patients have the class A defect. In the current study, the chromosomal assignment of Piga, as well as of Pigf and Pigh, two additional genes involved in GPI-anchor biosynthesis, has been established using a mouse interspecific backcross mapping technique. In contrast to Piga, both human and mouse Pigf and Pigh genes map to autosomes. The location of Pigf and Pigh suggests that mutations on both alleles of these autosomal genes would be necessary to produce PNH. This helps to explain the predominant class A defect in PNH.

Of 61 patients with aplastic anaemia (AA) diagnosed in our hospitals, 37 survived more than 2 years; actuarial survival of this latter group was 58%, with a median follow-up of living patients of 10.2 years. Laboratory and clinical data pertaining to these long-term survivors was scrutinized to determine the incidence of clonal disorders, which was identified in 43%. Morphological evidence of the myelodysplastic syndrome (MDS) was found in 13 (35%), including four cases of RAEB; four (11%) developed PNH. Of 23 patients studied, four showed karyotypic abnormalities, but these did not always coincide with morphological features of MDS. Although four patients now have completely normal blood and marrow morphology, and another had normal blood and marrow morphology at the time of death due to unrelated disease, the study confirms the high incidence of cytopenia and morphological abnormality, sufficient to justify a diagnosis of MDS, in patients with a history of AA. No definite survival plateau was identified. However, the natural history of MDS secondary to AA seems to be different to that of MDS arising de novo; the clinical course is relatively indolent, possibly implying a different biology.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disorder that resembles in several aspects the Tn-syndrome, in which bone marrow-derived cells are deficient in mucin-type beta 1,3 galactosyltransferase (beta 1,3Gal-T) due to the persistent repression of an intact allele. In the present study, we have investigated phytohemagglutinin (PHA)-activated T cells from the peripheral blood of an individual with the well-established clinical diagnosis of PNH. Only 10% of T cells were deficient in surface expression of the glycosylphosphatidylinositol (GPI)-linked CD48 antigen; in contrast, 95% of the patient's polymorphonuclear leukocytes harbored the defect. The cell-surface density of CD48 on unaffected T cells from this patient was two- to three-fold higher when compared to PHA-activated normal donor T cells. CD48-negative T cells were cloned and shown to belong to the CD4+ or the CD8+ antigenic subset. To test for the possibility that in PNH, as in Tn syndrome, gene repression may be responsible for the deficiency, CD48- T cell clones were treated with 5-azacytidine (5-azaC) and sodium n-butyrate (NaB). While such treatment reproducibly led to reexpression of a sialylated CD43 epitope on affected Tn-syndrome T cell clones, these drugs failed to induce reexpression of CD48 on affected PNH T cell clones. Our data suggest that despite many similarities, different pathogenetic mechanisms are responsible for PNH and Tn-syndrome. These CD48- T cell clones are the first to be described and may be useful to define the PNH lesion at a biochemical and genetic level.

Cold-reactive antibodies may cause peripheral ischaemia, occasionally proceeding to gangrene on exposure to cold. A 46-year-old male with syphilis, presented with an unusual combination of both paroxysmal cold haemoglobinuria and cold agglutinin disease, resulting in peripheral gangrene and requiring bilateral below knee amputations.

This case report concerns a woman thought to have a low blood count myelodysplastic syndrome who responded poorly to cytosine arabinoside. She was treated with Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and had an adverse reaction resembling a capillary leak syndrome. Later on, an episode of post-transfusion hemoglobinuria led to the correct diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). This case report suggests an adverse effect of GM-CSF on PNH.

Water intoxication is considered to be one of the possible causes of haemoglobinuria in calves. As to predisposing factors, prognosis and particularly differential diagnosis, uncertainty is rather frequent. Therefore we tried to provoke this state in an experiment, in which the disiony-induced changes of the acid-base balance (pH, pCO2, HCO3, pO2, BE, SAT of venous blood) were determined in addition to clinical observations (general behaviour, respiratory and pulse rates, body temperatures) and blood analysis (red and white blood cells, PCV and haemoglobin). Experimental paroxysmal haemoglobinuria was induced in eight 2-months-old male calves of the Black Pied breed weighing 55-70 kg that were held on a green diet. After the animals had been given cold water (12-14 degrees C) at an amount of 12% of their body weight, increased volume of the abdominal cavity, muscle tremor, and a stooping posture could be seen, and in 45-60 min. following water administration the first spontaneous haemoglobinuria occurred. Except the first 20 minutes following water gavage, when pronounced tachycardia accompanied by arythmia (100-130 per min.) was recorded, a tendency towards hypothermy, mild bradycardia and bradypnoe was observed; correction of these values occurred within 24 hours. Haematological indices revealed a hydraemic trend that was most pronounced and long-lasting in haemoglobin. Twenty-four hours after water gavage haemoglobin, red blood cells and PCV values were still below their starting levels; the tendency toward leucopoenia changed into leucocytosis. The acid-base balance revealed a slight acidemic trend with decreasing pH, actual bicarbonate and BE levels in the first hour; later, equilibration and increase within the physiological range occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Human umbilical vein endothelial cells (HUVEC) were found by Western blot analysis to express three membrane-bound C regulatory proteins, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. DAF was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 70-kDa molecule under nonreducing conditions in 2% deoxycholate extracts of HUVEC, MCP as a 63-kDa protein and CD59 as a 20-kDa molecule. Northern blot analysis revealed the presence of two species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for DAF, with sizes of about 2.0 kb and 2.7 kb. MCP mRNA was detected at 4.2 kb and a CD59 cDNA probe hybridized with three mRNA species with sizes of about 800, 1400 and 2000 bp. DAF and CD59 were released from the surface of HUVEC by phosphatidylinositol-phospholipase C, demonstrating that both are attached to the cell membrane by means of a glycolipid anchor. The relative contribution of DAF, MCP and CD59 in regulating the sensitivity to lysis of HUVEC by autologous complement was determined by incubation of sensitized endothelial cells with F(ab')2 fragments of polyclonal antibodies raised against these proteins. The susceptibility of sensitized cells to lysis by homologous complement was markedly increased in the presence of F(ab')2 anti-CD59 and to a lesser, but significant, extent in the presence of F(ab')2 anti-DAF. F(ab')2 anti-MCP did not significantly alter the susceptibility of HUVEC to complement-mediated lysis.

We have studied three glycosylphosphatidylinositol (GPI) linked proteins on the erythrocytes of 14 patients with paroxysmal nocturnal haemoglobinuria (PNH). The pattern observed was bimodal in 12 of the patients and trimodal in two. Ten patients had a red cell population with normal CD59 antigen (membrane inhibitor of reactive lysis, MIRL), decay accelerating factor (DAF or CD55) and lymphocyte function-associated antigen (LFA-3 or CD58) and a second abnormal PNH population with absent CD59 antigen, DAF and LFA-3. The other two patients with a bimodal pattern had a red cell population with normal CD59 antigen, DAF and LFA-3 and an abnormal population with reduced, but not absent, CD59 antigen and DAF. The LFA-3 on the abnormal red cells in these two patients appeared to be only slightly reduced. The two patients with a trimodal pattern had a normal population, a population with reduced, not absent, CD59 antigen and DAF, and a population with complete absence of CD59 antigen, DAF and LFA-3. The accuracy of the Ham test in estimating the proportion of red cells with the PNH defect in the two types of PNH was assessed. The case of one patient who appeared to be 'rescued' from severe aplastic anaemia by the development of PNH is described.

A 78-year-old female suffered from idiopathic paroxysmal cold hemoglobinuria (PCH). Her symptom occurred immediately after she worked outside in the cold early morning. This case is characterized by the eldest case with non-syphilitic PCH reported in Japan and by the most advanced anemia (Hb 4.2 g/dl) possibly due to prolonged hemolysis after cold exposure. Analysis of the serum revealed positive Donath-Landsteiner antibody of IgG type, which could react to hemolysis not only below 15 degrees C but also at 15-20 degrees C in vitro.

Although it was common in the preantibiotic era, paroxysmal cold hemoglobinuria (PCH) is now an exceptional condition. A 34-year-old female with anemia and episodic hemoglobinuria provoked by cold is reported. The laboratory studies suggested intravascular hemolytic anemia, and the Donath-Landsteiner test showed a biphasic IgG hemolysin with anti-P specificity. This finding and the evidence of latent syphilis were diagnostic of PCH. Penicillin therapy suppressed the hemolytic episodes, although the Donath-Landsteiner test is still positive. It is concluded that PCH is still an exisiting disease which should be considered in the differential diagnosis of episodic hemoglobinuria.

In the presence of aplastic anemia (AA), therapeutic choices should be determined while taking into account not only changes for immediate improvement, but also both the risks for late-occurring complications and the following quality of life. We report here data concerning a long-term clinical survey (5 to 18 years with a median of 12 years) including 156 nongrafted patients receiving androgen therapy; all patients were alive more than 5 years after diagnosis (40% of patients included at time of diagnosis in our multicentric analysis). Between the 5th and the 13th year follow-up, 21 patients died of various causes either related to AA or to its treatment: 12 of infection or hemorrhage secondary to pancytopenia (6 relapses and 6 that had never been improved; 2 with paroxysmal nocturnal hemoglobinuria [PNH]); 5 of leukemia; 1 of a non-Hodgkin's lymphoma; 2 of late side effects following transfusion (1 acquired immunodeficiency syndrome and 1 chronic B hepatitis); and a single case of myocardial infarction (the latter could possibly result of androgen therapy). Thirteen patients in total developed PNH (among which 10 had clinical symptoms including 2 deaths, and 3 exhibited only biologic abnormalities). Few long-term side effects of androgens could be noticed. Adult height was normal in patients treated during childhood and so was young women's fertility. No malignant hepatoma occurred. This survey allows the recording of late spontaneous hematologic improvement (between 5 and 10 years of evolution). This occurred in 50% of patients that had remained cytopenic 5 years after diagnosis. Although bone marrow stem cell concentration remained abnormal after 10 years of evolution. 85% of patients had a normal red blood cell count, 80% a normal polymorphonuclear count, and 66% a normal platelet count. All patients who did not show late complications had an excellent quality of life.

A young woman who developed a cerebral venous thrombosis shortly after commencement of an oral contraceptive preparation was subsequently found to have paroxysmal nocturnal haemoglobinuria. The aetiology of thrombotic complications in this condition is discussed, with particular reference to the additional risk factor of the contraceptive pill in this case.

A case of severe but transient haemolytic disease occurring after a febrile episode is described. The thermal amplitude of the haemolysin was high during the acute phase, since the autoantibody fixed complement at 31 degrees C. After 4 months complement fixation could exclusively be demonstrated at 4 degrees C. The patient was treated in a room heated to 30-32 degrees C. The treatment consisted of prednisone and azathioprine and during the acute phase plasmapheresis was attempted in order to reduce the antibody concentration. However, the haemolysis decreased when the thermal amplitude of the antibody diminished. 1 year after termination of therapy, she developed sarcoidosis.