Flow Cytometric Immunophenotyping of Systemic Mastocytosis in the Bone Marrow
KJ Jabbar, Y Huh, S Wang, LJ Medeiros, MR Johnson, S Verstovsek, JL Jorgensen. UT MD Anderson Cancer Center, Houston, TX
Background: By WHO criteria, systemic mastocytosis (SM) involves at least one extracutaneous organ. Detection of aberrant expression of CD2 and/or CD25 on mast cells is a minor criterion for diagnosis, and this is commonly achieved through immunohistochemisty (IHC) on bone marrow (BM)biopsies. In this study we assessed flow cytometry (FC) on BM aspirates as an alternative technique for detection of aberrant mast cells, in initial diagnosis and in follow-up after therapy.
Design: We studied 50 adults (23-88 years old) who met WHO criteria for SM, including 20 men and 30 women by flow cytometry. BM aspirates were stained with CD2 and CD25, conjugated to FITC or PE, along with CD45 PerCP-Cy5.5 and CD117 APC for gating. Other antigens included CD11c, CD35, CD59, CD63 and CD69 (all from BD Biosciences, San Diego, CA), and non-specific staining was measured with matched isotype controls. Data were acquired on FACS Calibur cytometers (BD), collecting from 2x105 to 106 events in most cases, and analyzed with CELL Quest (BD). Staining patterns were compared to normal mast cells (in staging marrows negative for non-Hodgkin lymphoma).
Results: By flow cytometry, aberrant over expression could be demonstrated using CD25-PE in 45/50 (90%) SM cases and using CD2-PE in 41/50 (82 %) cases. In cases stained with both CD2 conjugates, 31/40 (77.5 %) cases were positive with CD2-PE while 28/40 (70%) were positive with CD2-FITC. For CD25 staining, cells were brighter with the CD25-PE conjugate, but the same cases were also positive with CD25-FITC. Among cases studied with both FC and IHC there was 100% concordance for CD25 expression, with 25/26 (96%) cases positive. For CD2 staining, FC was more sensitive than IHC, with 14/23 (61%) positive by FC and 9/23 (39%) positive by IHC. The other markers studied by FC were also frequently aberrant, with over expression compared with normal mast cells seen for CD35 in 43/50 (86%) SM cases, CD59 in 46/50 (92%), CD63 in 44/49 (90%), and CD69 in 39/48 (81%). Flow cytometry detected far fewer mast cells (0.002-7%) than estimated by morphology and IHC (2-80%), with 10-fold to 10000-fold lower levels seen by FC.
Conclusions: Flow cytometry is a rapid and sensitive technique for characterizing aberrant mast cells in BM aspirates. Large numbers of cells must be acquired, and this method is not suitable for absolute quantitation. The detection rate is comparable to IHC for CD25 aberrancies, and superior for CD2.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 202, Wednesday Afternoon