Development and Validation of a Flow Cytometry Protocol for Measuring Tumor Cell Size of B-Cell Non-Hodgkin Lymphomas
Aaron Cotrell, Farzaneh Sayedian, Michelle Huang, Marc Smith, James Huang. Oakland University William Beaumont School of Medicine, Royal Oak, MI
Background: Assessment of the size of neoplastic lymphocytes plays important role in lymphoma classification, grading and prognosis. For B-cell non-Hodgkin lymphomas (B-NHL), low grade lymphomas are predominantly composed of small B-cells while aggressive B-cell lymphomas often have increased large cells. Lymphoma cell size is typically estimated based on microscopic comparison between neoplastic B-cells (NBCs) and normal macrophage or normal lymphocyte on hematoxylin and eosin stain. This method is subjective and poorly reproducible, affecting accuracy and consistency of lymphoma classification and grading. We developed a novel flow cytometry protocol to measure the size of neoplastic lymphocytes in B-NHL.
Design: Cell size was determined by measuring mean channels of forward scatter (FSC). We standardized cell size measurements between lymphoma cases by subtracting the mean FSC of the internal reactive T-cells (IRTCs) from the mean FSC of intact NBCs. We retrospectively analyzed the flow cytometric data of 26 cases of DLBCL, 26 cases of grade 3 follicular lymphoma (FL3), 59 cases of grade 1-2 follicular lymphoma (FL1/2), 22 cases of small non-germinal center B-cell lymphoma (SNGCBL) including small lymphocytic lymphoma (10), marginal zone B-cell lymphoma (8) and mantle cell lymphoma (4). The mean channels of FSC of intact NBCs and IRTCs were collected and compared with a Student's t-test.
Results: The mean FSC of IRTCs within the lymphoma tissue were about the same among different groups of lymphomas (425, 420, 413, and 412 for DLBCL, FL3, FL1/2, SNGCBL, respectively, p>0.05). The mean FSC of NBCs of DLBCL, FL3, FL1/2, and SNGCB were 589, 491, 407 and 412, respectively. The mean differences of FSC between NBCs and IRTCs were 164 for DLBCL, 72 for FL3, 6 for FL1/2, -3 for SNGCBL. No statistical significance (p>0.05) was detected in FSC between IRTCs and NBCs of FL1/2 or SNGCBL. Statistical significance (p<0.01) was observed in FSC of NBCs between DLBCL and FL3, and between FL1/2 and FL3.
Conclusions: First, flow cytometry can identify increased average size of NBCs of B-NHL with increased large cells (DLBCL and FL-3). Second, using our protocol, flow cytometry appears to show distinct differences in lymphoma size among different subtypes of B-cell lymphoma, even within the same grade. Finally, flow cytometric measurement of IRTCs size is highly reproducible and not affected by lymphoma subtype; suggesting that measurement of NRBCs size by flow cytometry may be more reproducible than microscopy with IRTCs as a reference standard.
Tuesday, March 20, 2012 1:00 PM
Poster Session IV # 209, Tuesday Afternoon