A Dissection of the CD45/Side Scatter (SS) Blast Gate in Non-Acute Myeloid Disorders and Non-Neoplastic Bone Marrows
AM Harrington, H Olteanu, SH Kroft. Medical College of Wisconsin, Milwaukee, WI
Background: CD45/SS gating is widely used for isolating blasts in flow cytometry (FC), with events in this blast gate (BG) often equated with "blasts" in the literature. However, other cells are present in this region, including granulocytes (grans), monocytes (monos), lymphocytes, basophils (basos), erythroid precursors, and hematogones. Thus, CD45/SS gating is imprecise, and may lead to inaccurate blast immunophenotype (IP) determination and enumeration when blasts are few in number or have abnormal CD45 expression. We thus evaluated the contents of the CD45/SS BG in a variety of bone marrows (BMs) with low blast counts.
Design: Diagnostic BMs of 16 myelodysplastic syndromes (MDSs), 12 myeloproliferative neoplasms (MPNs), and 7 chronic myelomonocytic leukemias (CMMLs) were compared to 2 control groups: 20 non-neoplastic cytopenia/cytosis BMs and 20 (-) lymphoma staging BMs. 4-color FC with cluster analysis was used, with tubes designed to delineate all BG populations using: CD10, CD11b, CD13, CD14, CD15, CD16, CD22, CD33, CD34, CD36, CD38, CD45, CD56, CD64, and CD117. BGs were uniformly applied based on conventions derived from standard FC literature.
Results: No differences were found between the control groups. The mean % of events in the BG was higher in CMMLs (7.8%; p<0.001) and MDSs (6.3%; p=0.012), but not in MPNs (2.8%) vs controls (2.7%). There were no significant differences across groups in mean % of BG events represented by blasts (14.9-21.1%), grans (23.9-33.2%), or lymphocytes (2.1-2.9%). Monos were a larger % of the BG in CMMLs (24.2%) vs controls (6.2%), MPNs (2.9%), and MDSs (7.7%). Basos averaged 37.8% of the BG in MPNs, vs 12.6% in controls, 10.9% in MDSs, and 3.1% in CMMLs (all p<0.001). Hematogones averaged 0.5% of the BG in CMMLs (p=0.002), 1.7% in MDSs (p<0.001), and 2.3% in MPNs (p=0.001) vs 10.2% in controls. Erythroid precursors were 11.8% of the BG in MDSs vs 1.0% in CMMLs (p= 0.021) and 4.2% in controls (p=0.058). The % of blasts (defined by cluster analysis) that fell in the BG averaged 90.7% in controls vs 78% in CMMLs (p=0.003), 82.4% in MPNs (p=0.032), and 84.9% in MDSs (p=0.106).
Conclusions: On average, blasts account for ∼20% of events in a traditional BG in normal/reactive BMs, MDSs, MPNs, and CMMLs. Not surprisingly, monos in CMMLs and basos in MPNs heavily contaminate the BG. Notably, ∼20% of blasts fell outside the BG in CMMLs and MPNs. Our data highlight pitfalls in using a traditional BG for blast analysis in non-acute myeloid disorders, and supports application of more robust methods.
Tuesday, March 23, 2010 1:00 PM
Poster Session IV # 180, Tuesday Afternoon