Markedly high population of affected reticulocytes negative for decay- accelerating factor and CD59 in paroxysmal nocturnal hemoglobinuria

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) blood cells lack glycosylphosphatidylinositol-anchored membrane proteins such as decay-accelerating factor (DAF) and CD59. This lack is of diagnostic value in PNH. Because reticulocytes in PNH are not yet well characterized, we analyzed reticulocytes obtained from 12 patients with PNH and from 5 healthy volunteers by two-color flow cytometry with a membrane-permeable fluorescent dye, thiazole orange, to identify reticulocytes and monoclonal antibodies to DAF and CD59. Healthy individuals had no affected cells. In all patients, the population of affected reticulocytes negative for DAF and CD59 was markedly higher than the population of affected erythrocytes. Moreover, the population of affected erythrocytes became obviously low in patients who received transfusions and suffered from hemolytic precipitation, whereas the population of affected reticulocytes was unchanged. The persistently high population of affected reticulocytes, despite cytolytic exclusion and an inherently short lifetime, might possibly be explained by relative reticulocytosis caused by an anemia-induced feedback stimulation of erythropoiesis in PNH. Thus, affected reticulocytes could be a reliable marker for the diagnosis of PNH and for the evaluation of erythropoiesis by PNH stem cell.

Shortened lifespan of paroxysmal nocturnal haemoglobinuria‐affected RBC estimated from differences in ratios of CD59‐negative populations between reticulocytes and whole RBC

“…As reported by others (Iwamoto et al, 1995;Ware, Rosse & Hall, 1995), the flow cytometric analysis of reticulocytes with the PNH phenotype is a sensitive method for diagnosing PNH. Our method, which enables the detection of a small PNH-affected erythropoiesis is useful for following up PNH patients, especially those such as Patient No.…”

Serum erythropoietin levels in paroxysmal nocturnal haemoglobinuria: implications for therapy

“…In contrast, in PNH the reticulocyte count is a measure of the effectiveness of Epo in causing the erythroid hyperplasia that is often prominent in this disease. Since, in PNH, the majority of reticulocytes belong to the PNH clone, the positive correlation between Epo levels and reticulocytes is further evidence that the PNH clone remains subject to the normal homeostatic controls of haemopoiesis (Iwamoto et al, 1995).…”

Immunophenotypic discrepancies between granulocytic and erythroid lineages in peripheral blood of patients with paroxysmal nocturnal haemoglobinuria

“…Because of the inherently short lifetimes which are unaffected by the cytolytic action of complements, the pattern and proportion of abnormal granulocytes, compared with erythroid cells, should more closely re¯ect current haematopoiesis by mutated PNH stem cells in the bone marrow. Reticulocytes are better than erythrocytes in estimation of size of PNH clone(s) and diagnosis of PNH (31).…”

“…The enumeration of reticulocytes was performed entirely by¯ow cytometry using TO. This method identi®es an almost identical reticulocyte population to conventional microscopic counting using new methylene blue (30,31). The reticulocyte production index (RPI) provides valuable information about the function of erythropoiesis in the diagnosis and monitoring of anaemia.…”