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IX.E.001 Paroxysmal Nocturnal Hemoglobinuria (PNH)


Paroxysmal nocturnal hemoglobinuria (PNH). Flow cytometry. In PNH cells, there is a synthetic defect in the glycophosphatidyl inositol (GPI) linkage that attaches a number of cell surface proteins to the external cell membrane. At least two of these proteins, CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis, MIRL), are expressed on red cells of normal individuals, functioning to inactivate the complement attack complex. In patients with PNH, there is partial or complete loss of these proteins, resulting in inappropriate lysis of red cells by activated complement components. Expression of these proteins can be analyzed by flow cytometry on red cells and on leukocytes. Expression is always compared in conjunction with a normal blood cell sample stained in parallel. Shown in this figure are the findings for red cell from normal blood (left column) and from a patient known to have PNH (right column). Top panels show the red cell gates using light scatter on samples stained with single color reagents on unlysed samples. Although red cells will far outnumber leukocytes on such samples, a glycophorin A stained tube is used as a positive control to confirm that the gated region represents red cells. In the lowest two rows of histograms, data are shown for red cell expression of CD55 and CD59. Note that in normal blood, there is a single weakly positive peak for CD55 on red cells and at least a partial loss of this expression in the PNH patient sample (shift of population to the left, against the y axis, shown by the M1 marker). In the case of CD59, the normal sample shows a single peak, indicating that all red cells are CD59 positive, whereas in the PNH sample, there is a bimodal peak, indicating that a subpopulation of red cells demonstrates loss of CD59 expression (lowest right graph, M1 marker). In most affected patients with PNH, not all red cells will be CD55 or CD59 negative and red cell transfusion may also diminish the percentage of affected cells seen by flow cytometry. Leukocytes are often analyzed in addition and the reaction of these cells will not be affected by red cell transfusion. On leukocytes, other GPI-linked proteins such as CD14 and CD16 may also be analyzed as a surrogate assessment of GPI-linked complement regulatory proteins.

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