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

Abstract

Background: The 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 methods: By 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.

Results: Paroxysmal 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.

Conclusions: These 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.

Figure 1.

Flow cytometry analysis (FCM) of granulocytes with a paroxysmal nocturnal hemoglobinuria (PNH)+ phenotype. Granulocytes were initially identified on the basis of CD45/SSC plot (A-left), further defined by CD15/SSC (A-middle) followed by FSC/SSC (A-right). The granulocytes from the three combined analysis regions (G, Gr and U) were examined for CD55/CD59 (B-left) and CD16/CD66b (B-right) expression. The PNH FCM assay was repeated on the same blood sample after 1 h of incubation with pre-aerolysin at 37°C: PNH⁺ granulocytes were resistant to aerolysin lysis while the non-PNH granulocytes were nearly all lysed (C-left and right).

Figure 2.

Examples of immunophenotypic alterations associated with the underlying bone marrow disease: diagnostic pitfalls and caveats. Case 10-refractory anemia with excess blasts-1: 1.02% CD59 negative (A₁) and 2.03% CD16-negative/CD66b-negative (A₂) cells were detected, which were back-traced to the monocyte region on the CD45/SSC plot (A₃) and showed a low level of CD15 expression (A₄). Case 15-chronic idiopathic myelofibrosis: 12.29% CD55 low/negative cells were detected (B₁) which were CD16 low (B₂) and CD45 low (B₃), corresponding to myeloid cells of intermediate maturation. In all cases illustrated above, the granulocytes were susceptible to aerolysin lysis and were not true PNH⁺ cells (C₁ and C₂).