Takashi Terasawa

Diagnosis of paroxysmal nocturnal haemoglobinuria by phenotypLc analysis of erythrocytes using two-colour flow cytometry with monoclonal antibodies to DAF and CD59/MACIF

Summary We investigated the relationship between the complement lysis sensitivity test and two‐colour flow cyto‐metric analysis using monoclonal antibodies to decay accelerating factor (DAF) and CD59/membrane attack complex inhibitory factor (MACIF) in patients with paroxysmal nocturnal haemoglobinuria (PNH) and other haematological diseases.

Heterogenous expression of decay accelerating factor and CD59/membrane attack complex inhibition factor on paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes.

Summary In order to clarify the characterization of pheno‐types of paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes (E), we analysed the expression of decay accelerating factor (DAF) and CD59/membrane attack complex inhibition factor (MACIF) on the membrane surface of PNH‐E by means of the flow cytometric method using anti‐DAF and/or CD59/MACIF monoclonal antibodies in nine PNH‐patients. In two‐colour analysis, this expression on PNH‐E was classified into three fractions; negative, intermediate and positive according to intensity. The negative fraction was classified into two groups; one group an exclusively negative population, and the other a negative population having slightly DAF‐positive E. The intermediate fraction was recognized on PNH‐E of cases with PNH II‐E and extremely heterogenous. In the positive fraction, this expression was almost the same as on normal E except for case 8. In single‐colour analysis for DAF or CD59/MACIF, three fractions were classified as well as the definition in two‐colour analysis. In single‐colour analysis, the expression on PNH‐E was also heterogenous in each fraction and among PNH‐patients. However, the intermediate fraction for CD59/MACIF was not found on PNH‐E of cases without PNH II‐E, although intermediate fraction for DAF was recognized on PNH‐E of some cases with PNH III‐E in addition to those with PNH II‐E.

Paroxysmal nocturnal haemoglobinuria with coexisting deficiency of the ninth component of complement: lack of massive haemolytic attack

A 47‐year‐old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement‐sensitivity lysis tests, 80% of her erythrocytes were markedly complement‐sensitive (PNH‐III). Laser cytofluorimetry with a monoclonal antibody against decay‐accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF‐negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusions of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patients erythrocytes. When the patients serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.

Complement sensitivity of erythrocytes in a patient with inherited complete deficiency of CD59 or with the Inab phenotype

We investigated the complement sensitivity of erythrocytes from three patients, one with inherited complete deficiency of CD59, one with the Inab phenotype, and one with paroxysmal nocturnal haemoglobinuria (PNH). The complement lysis sensitivity units on the erythrocytes were 11.7, 4.6, and 47.6 for inherited CD59 deficiency, Inab phenotype, and PNH, respectively. Two‐colour flow cytometric analysis showed that the erythrocytes from the three patients consisted of a single population negative for CD59, negative for decay accelerating factor (DAF), and negative for both proteins, respectively. In addition, only the Inab phenotype patient had no haemolysis in vivo. These facts suggest that CD59 deficiency plays a more important role than DAF deficiency in complement‐mediated haemolysis in vitro and in vivo, and that deficiency of both proteins, but not CD59 or DAF alone, causes complement sensitivity corresponding to that of PNH III erythrocytes in vitro.

Decay-accelerating factor (DAF) on the blood cell membranes in patients with paroxysmal nocturnal haemoglobinuria (PNH): measurement by enzyme-linked immunosorbent assay (ELISA)

Summary. We developed a quantitative enzyme‐linked immunosorbent assay (ELISA) for decay‐accelerating factor (DAF) on blood cell membranes using monoclonal anti‐DAF antibodies. DAF is an integral membrane protein of several blood cells. It regulates the C3 and C5 convertase of the classical and alternative pathways of complement activation. It is partially or completely deficient in the membranes of blood cells of patients with paroxysmal nocturnal haemoglobinuria (PNH).

Characterization of the erythropoietic precursors (BFU‐E) in a patient with juvenile chronic myelogenous leukaemia by the analysis of Gγ and Aγ globin chains

Summary. In order to investigate the pathogenesis of juvenile chronic myelogenous leukaemia (J‐CML) we examined the biosynthetic rates of Gγ and Aγ globin chains in the erythropoietic bursts from the bone marrow of a patient with J‐CML. Globin chains were labelled with 14C‐labelled amino acids, separated by isoelectric focusing and quantitated by fluorography. The synthesis of γ‐chains in the erythropoietic bursts comprised 89·0% of the total non‐α‐chains. The Gγ: Aγ ratio was 0·67, which is within the ratios obtained in newborns. Furthermore, individual erythropoietic bursts contained varying ratios of both γ and β chains and all revealed more Gγ than Aγ chain synthesis. The relative proportions of Gγ and total γ chain biosynthesis in 62 separate erythropoietic bursts were 0·69·0·06 and 0·86·0·06, respectively. Cumulative frequency distributions of individual bursts differing in the ratios of γ/(γ+β) and Gγ/(Gγ+Aγ) approached normal frequency distributions. These results suggest that the levels of Hb F in J‐CML are controlled by qualitative changes in a single population of erythropoietic precursors, in which normal switching of the Gγ:Aγ ratio has not occurred, rather than by the abnormal proliferation of an F‐cell clone.

Characterization of the erythropoietic progenitor cells (BFU-E) in paroxysmal nocturnal haemoglobinuria (PNH)

Summary. In order to investigate erythropoiesis in paroxysmal nocturnal haemoglobinuria (PNH), the relative proportions of complement‐mediated lysed erythroblasts in individual bursts of two cases of PNH were analysed by a modified complement lysis sensitivity test using the trypan blue dye exclusion method. All bursts contained both complement‐sensitive and complement‐insensitive erythroblasts and the ratio between them varied from burst to burst. The means ±SD of the haemolytic percentages of individual bursts in cases 1 and 2 were 30.9 ± 10.8% (n= 20, ranging from 13.2% to 52.8%) and 54.8 ± 16.4% (n= 31, ranging from 26.4% to 82.5%), respectively. In addition, the cumulative frequency distributions of the proportions of complement‐mediated lysed erythroblasts in individual bursts approached normal frequency distributions. These results suggest that two red cell populations with different susceptibilities to complement in PNH are derived from a single population of erythropoietic progenitor cells rather than a dual population of normal and PNH erythropoietic progenitor cells.

ERYTHROPOIESIS OF COMPLEMENT‐SENSITIVE CELLS IN A PNH PATIENT WITH IRON DEFICIENCY ANEMIA DURING IRON THERAPY

Paroxysmal nonturnal hemoglobinuria (PNH) is an acquired disorder characterized by increased susceptibility of erythrocytes (E) to complement (C’)-mediated hemolysis. Although patients with PNH may be treated with androgenic hormones, prednisone and iron, these agents are not always effective. Here we report the changes in the proportion of C’-sensitive E, and the C7 sensitivity of erythroblasts observed by the complement lysis sensitivity (CLS) test and erythropoietic cell culture during iron therapy in a PNH patient with iron deficiency anemia (IDA). Case: A 58-yr-old male was admitted to our hospital because of epistaxis and anemia in June, 1979. Both Ham’s and sugar-water tests were positive, and the patient was diagnosed as having PNH. On 3 June, 1987, laboratory findings showed that the serum iron was 50 pg/dl, total iron binding capacity 440 kg/dl, and serum ferritin 8.6 ng/ml. Therefore, he was diagnosed as having IDA and was treated with sodium ferrous citrate 150 mg/d from July 6, 1987. Iron therapy was continued until September 9, 1987. Slight exacerbation of hemoglobinuria occurred on September 1 and 2, 1987, but it was not necessary to stop the iron therapy (Table 1). Material and methods: The CLS test of E in peripheral blood (PB) and erythroblasts from bursts in bone marrow was performed during the clinical course from June 3, 1987 to September 9, 1987. A modification (1) of the CLS test (2) developed by Rosse & Dacie