Flow Cytometry in Oncologic Diagnosis
Summary of Key Points
Key Methods
• Fluorescently conjugated antibodies that are bound to cell-surface or intracellular proteins allow enumeration and detailed characterization of subsets of cells in heterogeneous mixtures.
• Fluorescent DNA-binding dyes allow determination of tumor ploidy and can assess cell-cycle characteristics of tumors.
Applications for Lymphoma and Chronic Lymphoproliferative Disorders
• Flow cytometry is suitable for use with cell suspensions of tissue, fine-needle aspirates, fluids, and blood and marrow.
• Clonality of B-cell processes are readily detected by light-chain restriction assay.
• Many lymphoid disorders are defined by phenotypic profiles.
• Minimal residual disease assessment helps in the management of patients with chronic lymphocytic leukemia and myeloma.
1. Flow cytometry differs from other diagnostic techniques because:
A It can identify and enumerate blast populations.
B It can measure properties of individual cells in heterogeneous samples.
C It can determine changes in level of expression of cellular proteins.
2. In which of the following diseases is flow cytometry least useful for diagnosis and/or classification?
A Acute myeloid leukemia (AML)
B Chronic lymphocytic leukemia (CLL)
3. Blasts can be identified in flow cytometric analysis of bone marrow based on:
C The combination of CD45 and right angle scatter.
D The presence of abnormal combinations of stem cell and myeloid antigens.
4. Which of the following statements is true about flow cytometry studies of bone marrow in persons with myeloma?
A They can help to predict progression of disease.
B They play no role because plasma cells are underestimated by flow cytometry.
C Plasma cells are best identified by loss of expression of CD38.
D It is essential to demonstrate that a plasma cell population is clonal.
5. All of the following statements about abnormalities that can be seen by flow cytometry in myelodysplastic syndrome (MDS) are true EXCEPT:
A Blasts can express abnormal combinations of myeloid antigens.
B The pattern of acquisition of myeloid differentiation antigens is disrupted.
C Mature neutrophils have decreased right-angle light scatter (side scatter).
6. Expression of which of the following markers can be used to distinguish normal B-cell precursors from B lymphoblasts?
7. Minimal residual disease (MRD) detection by flow cytometry has been shown to have significant prognostic value in all of the following diseases except:
8. Flow cytometric analysis of DNA content is not used clinically for prognostic assessment in most cases of epithelial malignancies because:
1. Answer: B. Although flow cytometry can identify blasts and they can be counted, the blasts counts by flow do not necessarily reflect those seen by morphology, and classification systems in hematologic malignancies are based on morphologic blast counts. Bulk methods using DNA- or protein-based assays can assess for the presence of clones or for levels of expression of various proteins, but the unique features of flow is that it provides multiparameter information about single cells or groups of cells that can be identified as a discrete subpopulation of an entire sample.
2. Answer: C. Flow cytometry is critical to the diagnosis of AML and CLL and plays an adjunct role in MDS and myeloma. However, in chronic-phase CML, flow cytometry plays no significant role (it is useful in the blast phase). Although the bcr-abl fusion protein can be detected by flow cytometry, in practice better methods are available to do this.
3. Answer: C. Cell size alone does not identify blasts. Whereas most blasts are CD34+, this marker does not define blast cells. Abnormal antigen expression is useful for distinguishing normal and abnormal blasts, but blasts can be seen in normal bone marrow as well. The most universally accepted way to identify blasts is to look at CD45 versus right angle scatter, although one must be aware that some other cell types (for example, basophils) will overlap the “blast” gate and in some cases of acute leukemia, blasts will be found elsewhere.
4. Answer: A. Several studies suggest that the abnormalities in plasma cells can help to determine which patients with monoclonal gammopathy of undetermined significance or smoldering will progress to more aggressive disease, although this is not yet part of routine diagnostics. Although plasma cells are underestimated by flow cytometry, so that one cannot use flow plasma cell counts to apply conventional criteria, the plasma cell abnormalities are still predictive. Plasma cells express very bright CD38, although this brightness may be slightly lower in myeloma than in normal plasma cells. Finally, although cytoplasmic light chain restriction can be demonstrated by flow cytometry, it also can be demonstrated by immunohistochemistry in bone marrow, and of course, serum protein electrophoresis and immunofixation are standard ways of demonstrating plasma cell clonality.
5. Answer: D. Although not every case of MDS will show abnormalities, the types of abnormalities seen include abnormal antigen expression both within the myeloblasts and the maturing myeloid elements. Because side scatter reflects cellular granularity, the hypogranular neutrophils that are typical for MDS will show abnormally low side scatter. B-cell precursors have been reported to be decreased or absent in persons with MDS, but those that are present are phenotypically normal.
6. Answer: F. No leukemia-specific marker exists; all those listed are expressed on both normal and abnormal B-cell precursors, but it is the level of expression of these markers in combination (with each other and with other markers) that is used to determine that the cells are abnormal. (CD19 usually is used as a gating marker and is only occasionally expressed at an aberrant levels.)
7. Answer: D. MRD detection by flow is a very powerful prognostic marker in persons with acute leukemia, and clearance of MRD in persons with CLL has been associated with improved survival. Several studies suggest that MRD in persons with myeloma is prognostic, especially after transplant. Very little data exist about the use of MRD studies in non-Hodgkin lymphoma in general, and diffuse large B cell lymphoma in particular is not suitable for analysis in part because of the low frequency of involvement of marrow and also because of poor recovery of large cells.
8. Answer: A. Although many published studies using robust analytical methods show a correlation between S phase in particular and outcome in many tumor systems, the methods needed to generate high-quality data were difficult to standardize. Because many results were found to be unreliable, and because newer markers became available that were highly predictive, the role of flow cytometry for solid tumor prognostication has become limited at best. It can, however, be performed on fixed paraffin-embedded tumors. Some laboratories still perform this procedure, and some clinicians use the results, but most DNA cytometry is now done for research purposes.
