Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats

Abstract

BackgroundThe presence of paroxysmal nocturnal hemoglobinuria clones in the setting of aplastic anemia or myelodysplastic syndrome has been shown to have prognostic and therapeutic implications. However, the status of paroxysmal nocturnal hemoglobinuria clones in various categories of myelodysplastic syndrome and in other bone marrow disorders is not well-studied. Design and MethodsBy using multiparameter flow cytometry immunophenotypic analysis with antibodies specific for four glycosylphosphatidylinositol-anchored proteins (CD55, CD59, CD16, CD66b) and performing an aerolysin lysis confirmatory test in representative cases, we assessed the paroxysmal nocturnal hemoglobinuria-phenotype granulocytes in 110 patients with myelodysplastic syndrome, 15 with myelodysplastic/myeloproliferative disease, 5 with idiopathic myelofibrosis and 6 with acute myeloid leukemia. ResultsParoxysmal nocturnal hemoglobinuria-phenotype granulocytes were detected in nine patients with low grade myelodysplastic syndrome who showed clinicopathological features of bone marrow failure, similar to aplastic anemia. All paroxysmal nocturnal hemoglobinuria-positive cases demonstrated loss of the four glycosylphosphatidylinositol-anchored proteins, with CD16 - CD66b-clones being larger than those of CD55 -CD59 -(p<0.05). Altered glycosylphosphatidylinositol-anchored protein expression secondary to granulocytic hypogranulation, immaturity, and/or immunophenotypic abnormalities was present in a substantial number of cases and diagnostically challenging. ConclusionsThese results show that routine screening for paroxysmal nocturnal hemoglobinuria clones in patients with an intrinsic bone marrow disease who show no clinical evidence of hemolysis has an appreciable yield in patients with low grade myelodysplastic syndromes. The recognition of diagnostic caveats and pitfalls associated with the underlying intrinsic bone marrow disease is essential in interpreting paroxysmal nocturnal hemoglobinuria testing correctly. In our experience, the CD16/CD66b antibody combination is superior to CD55/CD59 in screening for subclinical paroxysmal nocturnal hemoglobinuria because it detects a large clone size and is less subject to analytical interference.

Management of Paroxysmal Nocturnal Hemoglobinuria in the Era of Complement Inhibitory Therapy

“…19,20,22,23 The association between PNH and MDS appears to be confined to low-risk categories of MDS, particularly the refractory anemia (RA) variant. [18][19][20]22 Using high-sensitivity flow cytometry in which Ն 0.003% of GPI-AP-deficient RBCs or peripheral mononuclear cells was classified as abnormal, Wang et al reported that 21 of 119 (18%) patients with RA MDS had a population of PNH cells, whereas GPI-AP-deficient cells were not detected in patients with RA with ringed sideroblasts, RA with excess of blasts, or RA with excess of blasts in transformation.…”

“…19,20,22,23 The association between PNH and MDS appears to be confined to low-risk categories of MDS, particularly the refractory anemia (RA) variant. [18][19][20]22 Using high-sensitivity flow cytometry in which Ն 0.003% of GPI-AP-deficient RBCs or peripheral mononuclear cells was classified as abnormal, Wang et al reported that 21 of 119 (18%) patients with RA MDS had a population of PNH cells, whereas GPI-AP-deficient cells were not detected in patients with RA with ringed sideroblasts, RA with excess of blasts, or RA with excess of blasts in transformation. 20 Compared with patients with RA without a population of PNH cells, RA patients with a population of PNH cells had a distinct clinical profile characterized by the following features: (1) less pronounced morphological abnormalities of the blood cells, (2) more severe thrombocytopenia, (3) a lower rate of karyotypic abnormalities, (4) a higher incidence of HLA-DR15, (5) a lower rate of progression to acute leukemia, and (6) a higher probability of response to cyclosporine therapy.…”

“…More recently, the findings by Wang et al that a population of PNH cells was associated only with low-risk MDS variants in Japanese patients were confirmed in a North-American study of 137 patients classified by World Health Organization criteria. 22 When combined with evidence of polyclonal hematopoiesis (based on the pattern of X-chromosome inactivation in female patients), the presence of a population of PNH cells in patients with MDS predicts a relatively benign clinical course and high probability of response to immunosuppressive therapy. 18 A relatively good response to immunosuppressive therapy for patients with MDS and aplastic anemia was also predicted by expression of HLA-DR15 in studies of both North American and Japanese patients.…”

A systematic analysis of bone marrow cells by flow cytometry defines a specific phenotypic profile beyond GPI deficiency in paroxysmal nocturnal hemoglobinuria

“…PNH clones can be detected in the setting of MDS, and their presence may not be related to the pre-leukemic nature of MDS but rather linked to BM failure, similar to aplastic anemia [22]. Furthermore, PNH can represent the clinical evolution of another BM disorder, and especially of MDS cases.…”

Issue Highlights—January 2013

“…It is caused by a mutation in the phosphatidyl inositol glycan-class A gene leading to partial or complete loss of the cell membrane molecule called glycosyl phosphatidyl inositol (GPI) (15,16 (14), data analysis has not been standardized. The majority of current analytical methods use manual gating to enumerate PNH cells (14)(15)(16)(17)(18)(19) although new automated analysis techniques that improve the objectivity of operator input have also been recently developed (19). Gating strategies imply the recognition of the various cell types (monocytes, neutrophil granulocytes, and red blood cells) based on the expression of various phenotypic markers.…”