DETECTION AND FOLLOW UP OF PNH CLONE BY MEASURING CD55 AND CD59 EXPRESSION ON NEUTROPHILS
Abstract
Background. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal haematopoietic stem cell disorder characterised by intravascular haemolysis, bone marrow failure and increased tendency to thrombosis. It is caused by a somatic mutation in the PIG-A gene, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIG-A mutation results in a clone of blood cells with total or partial deficiency of membrane proteins anchored to the cell surface through GPI anchor. For many years, an increased susceptibility of the PNH red cells to complement lysis in acidified serum (Ham’s test) has been essential for diagnosis of the PNH. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of the specific GPI anchor proteins on blood cells. We evaluated a three-colour flow cytometry method for detection and quantification of CD55/59 negative netrophils.
Patients and methods. Nine patients (6 with PNH and 3 with aplastic anaemia) who were previously diagnosed as having PNH or aplastic anaemia were evaluated. In the period of three years the patients were serially evaluated for the extent of PNH clone by quantification of CD55/59 negative neutrophils. Three-colour flow cytometry method was used for quantification of the CD55/CD59 negative neutrophils. We used directly conjugated monoclonal antibodies anti-DC15PC5 for identification of neutrophils and anti-CD59-FITC and anti-CD55-PE for the GPI-linked antigens.
Results. Patients with haemolytic PNH had > 50% CD59/CD55 negative granulocytes. The proportion of the PNH granulocytes was higher in the patient with frequent and serious haemolytic attacks. Over the period of three years slow growth of the PNH clone was seen in two cases. Two patients with PNH diagnosed 19 years ago and in remission at the time of flow cytometric analysis was devoid of the PNH clone. Three patients with aplastic anaemia (hypoplastic PNH) had the proportion of CD59/CD55 negative granulocytes < 40%. In one of them PNH clone increased.
Conclusions. Three colour flow cytometry of granulocytes using combination of anti-CD15/55/59 provides the accurate technique for detection and quantification of the PNH clone. Monitoring the PNH clone size has clinical and prognostic value. The size of the PNH clone is an important determinant for thrombotic risk. Serial studies allow prediction of remission in some cases or progress from hypoplastic to hemolytic PNH in others.
Downloads
References
Rosse WF. Paroxysmal nocturnal hemoglobinuria. In: Hoffman R, Benz EJ, Shattil SJ, Furie B, Cohen HJ, Silberstein LE eds. Hematology: basic principles and practice. 3rd ed. New York: Churchill Livingstone, 2000: 331–42.
Luzzato L. Pathophysiology of paroxysmal nocturnal hemoglobinuria. Haematologica 2000; 84: 203–9.
Schrezenmeier H, Hildebrand A, Rojewski M. Paroxysmal nocturnal hemoglobinuriaa replacement of haemapoetic tissue. Acta Haematol 2000; 103; 41–8.
Richards SJ, Rawstron AC, Hillmen P. Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry 2000; 42: 223–33.
Borowitz MJ. Flow cytometry testing in PNH. How much is enough? Cytometry 2000; 42: 221–2.
Hall SE, Rosse WF. The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood 1996; 87: 5332–40.
Piedras J, Lopez-Karpovitch X. Flow cytometric analysis of glycosylphosphatidyl-inositol-anchored proteins to assess paroxysmal nocturnal hemoglobinuria clone size. Cytometry 2000; 42: 234–8.
Hernandez-Campo PM, Martin-Ayuso M, Almeida J, Lopez A, Orfao A. Comparative analysis of different flow cytometry-based immunophenotypic methods for the analysis of CD59 and CD55 expression on major peripheral blood cell subsets. Cytometry 2002; 50: 191–201.
Brodsky RA, Mukhina GL, Li S, Nelson KL, Chiurazzi PL, Buckley JT, Borowitz MJ. Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 2000; 114: 459–66.
Krauss JS. Laboratory diagnosis of paroxysmal nocturnal hemoglobinuria. Ann Clin Lab Sci 2003; 33: 401–6.
Maciejewski JP, Rivera C, Kook H, Dunn D, Young NS. Relationship between bone marrow failure syndromes and the presence of glycophosphatidyl inositol-anchored protein-deficient clones. Br J Haematol 2001; 115: 1015– 22.
Hillmen P, Richards SJ. Implication of recent insights into the pathophysiology of paroxysmal nocturnal haemoglobinuria. Br J Haematol 2000; 108: 470–9.
Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural history of paroxysmal nocturnal hemoglobinuria. N Engl J Med 1995; 333: 1253–8.
Hall C, Richards S, Hillmen P. Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria (PNH). Blood 2003; 102: 3587–91.
The Author transfers to the Publisher (Zdravniški vestnik/Slovenian Medical Journal) all economic copyrights following form Article 22 of the Slovene Copyright and Related Rights Act (ZASP), including the right of reproduction, the right of distribution, the rental right, the right of public performance, the right of public transmission, the right of public communication by means of phonograms and videograms, the right of public presentation, the right of broadcasting, the right of rebroadcasting, the right of secondary broadcasting, the right of communication to the public, the right of transformation, the right of audiovisual adaptation and all other rights of the author according to ZASP.
The aforementioned rights are transferred non-exclusively, for an unlimited number of editions, for the term of the statutory
The Author can make use of his work himself or transfer subjective rights to others only after 3 months from date of first publishing in the journal Zdravniški vestnik/Slovenian Medical Journal.
The Publisher (Zdravniški vestnik/Slovenian Medical Journal) has the right to transfer the rights, acquired parties without explicit consent of the Author.
The Author consents that the Article be published under the Creative Commons BY-NC 4.0 (attribution-non-commercial) or comparable licence.
