The Use of Monoclonal Antibodies and Flow Cytometry in the Diagnosis of Paroxysmal Nocturnal Hemoglobinuria

  • Sharon B
  • Hall E
  • Rosse W
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Abstract

We have characterized the erythrocytes, granulocytes, and platelets of 54 patients with paroxysmal nocturnal hemoglo-binuria (PNH) with antibodies to glycosylphosphatidylinosi-tol-anchored proteins (anti-CD55, anti-CD59, and anti-CD161 and flow cytometry to establish the usefulness of this tech-nique in the diagnosis of this disorder. All patients demon-strated either completely (PNH 111) or partially (PNH I 11 deficient red cells and granulocytes. Anti-CD59 best demon-strated PNH II red cells, which were present in 50% of the patients. The proportion of abnormal granulocytes was usu-ally greater than the proportion of abnormal red cells; 37% of the patients had >80% abnormal granulocytes. Anti-CD55 did not delineate the erythrocyte populations as well as did anti-CD59. Either anti-CD55 or anti-CD59 could be used P AROXYSMAL nocturnal hemoglobinuria (PNH) is characterized by the deficiency, absolute or partial, of all proteins anchored to the membrane by the glycosylphos-phatidylinositol (GPI) anchor' " ; at least 1 S of these proteins have been shown to be lacking on the abnormal blood cells of patients with this disease. In the past, the diagnosis of PNH has relied on the demonstration of the effect of the absence of two of these proteins, CD59 (membrane inhibitor of reactive lysis [MIRL]? protectin,s,6 membrane attack com-plex inhibiting factor [MACIF],' etc). and CDSS (decay ac-celerating factor [DAF],' which results in the abnormal sen-sitivity of the red cells to the lytic action of complement. " " ' In the present study, we report the results of the use of monoclonal antibodies (MoAbs) against CDSS, CDS9, and CD16 (the Fc,receptor 111) in the analysis by flow cytometry of the red cells, granulocytes, and platelets of 54 consecutive PNH patients. We demonstrate the ability of this methodol-ogy to distinguish the presence of even small populations of blood cells partially or totally deficient in these proteins and to assess accurately the proportion of cells in these popula-tions. By this analysis, we establish for the first time the optimum use of this methodology in the diagnosis of this disease. MATERIALS AND METHODS Patients The blood of S4 patients (23 men, 3 I women) who had or were suspected of having the diagnosis of PNH was tested. The patients ranged in age from 1 1 to 65 years (mean 34 years). Erythrocytes 0006-4971/96/8712-0002$3.00/0 5332 equally well to analyze granulocytes; anti-CD16 did not dem-onstrate cells of partial deficiency. Platelets could not be used for detailed analysis as the normal and abnormal popu-lations were not well distinguished. Flow cytometry of erythrocytes using anti-CD59 or of granulocytes using either anti-CD55 or anti-CD59 provides the most accurate tech-nique for the diagnosis of paroxysmal nocturnal hemoglo-binuria; it is clearly more specific, more quantitative, and more sensitive than the tests for PNH that depend upon hemolysis by complement (the acidified serum lysis [Ham] test, the sucrose lysis test, and the complement lysis sensi-tivity [CLS] test).

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Authors

  • Bv Sharon

  • E Hall

  • Wendell F Rosse

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