Flow cytometric analysis of glycosylphosphatidyl-inositol-anchored proteins to assess paroxysmal nocturnal hemoglobinuria clone size

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by total or partial deficiency of membrane proteins anchored to the cell surface through a glycosylphosphatidyl‐inositol (GPI) moiety. The relationship between the size of the PNH clone, determined by the expression of GPI‐anchored proteins (AP; CD14, CD48, CD55, CD59, and CD66b) on erythrocytes, lymphocytes, monocytes, and granulocytes using forward and side scatter analysis, and severity of the disease was evaluated in 19 PNH patients. CD55 antigen expression did not delineate abnormal erythrocytes as well as did anti‐CD59.The proportion of monocytes deficient in CD55, CD59, CD48, and CD14 (48–97%) and of granulocytes deficient in CD55, CD59, and CD66b (60–99%) was greater than the proportion of erythrocytes deficient in CD59 (24–95%) and the proportion of lymphocytes deficient in CD55 and CD59 (30–98%). There were no significant correlations among reticulocyte, leukocyte, and platelet counts and GPI‐AP–deficient immunophenotypes in red and white blood cells. However, high coefficients of determination were seen between hemoglobin levels and granulocytes deficient in CD59 (r2 = 0.76), CD55 (r2 = 0.74), and CD66b (r2 = 0.74) antigens and between hemoglobin and monocytes deficient in CD55 (r2 = 0.73), CD59 (r2 = 0.80), and CD14 (r2 = 0.75) antigens. These results are interpreted as indicating that the size of PNH clone is better assessed by immunophenotypic analysis of monocytes and granulocytes rather than of lymphocytes and erythrocytes. Cytometry (Comm. Clin. Cytometry) 42:234–238, 2000. © 2000 Wiley‐Liss, Inc.

Normal patterns of expression of glycosylphosphatidylinositol‐anchored proteins on different subsets of peripheral blood cells: A frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria

“…The increasing interest in both the study of different subsets of PB leukocytes and of other markers, in addition to CD55 and CD59, may be explained to a large extent because of two major observations. First, it has been shown that in PNH patients, the PNH clone frequently represents a greater fraction of the monocytes and neutrophils than of the red cells and platelets, both at diagnosis (7,14,15) and during follow-up (8), which is probably related to the shorter life-time and quicker turnover of these subsets of leukocytes in PB (7,8). Secondly, increasing evidence exists about the potential involvement of the deficit of specific GPI-AP other than CD59 and CD55 in different subsets of PB cells on the development of clinical complications of the disease other than complement-mediated blood cell lysis, such as infection and thromboembolism (1,5).…”

“…At present, consensus exists on the need to demonstrate deficient expression of two of more normally brightly expressed GPI-AP in at least two different populations of PB cells, for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) (8,10,15). For this purpose, detailed knowledge about the exact patterns of expression of GPI-AP on the different subsets of PB cells is required.…”

“…Despite this, analysis of CD16, CD24, and/or CD66b expression on neutrophils and/or of CD14 on monocytes has also been found to be of great help for the diagnosis and monitoring of PNH (10). This relates to their deficiency in these cell populations being more evident than that of CD55 and CD59; this is of particular relevance since the size of the PNH clone in an individual may largely vary among different hematopoietic cell populations, and it has been shown that the proportion of affected cells is frequently lower within the red blood cell and lymphocyte compartments in comparison to that of the neutrophils and monocytes (14,15).…”

“…Accordingly, for diagnosis of PNH it is recommended that the cells demonstrate deficient expression of at least two different GPI-AP and that two or more cell lineages be involved (8,15). Despite this, it should be noted that the pattern of expression of GPI-AP in minor populations of normal hematopoietic cells still remains largely unknown.…”

“…Previous studies have indicated that CD48 could be the most useful marker for the identification of PNH lymphocytes (15,21,24,25). Accordingly, we observed the highest levels of expression for this protein on T cells and, at lower amounts, also on B-and NK cells.…”