Applications of Flow Cytometry to Diagnostic Hematophathology

Vol. 15 •Issue 3 • Page 19
Applications of Flow Cytometry to Diagnostic Hematophathology

Flow cytometric immunophenotyping is a useful diagnostic tool.

Flow cytometric immunophenotyping (FCI) is a useful tool in diagnostic hematopathology. Types of specimens suitable for FCI include peripheral blood, bone marrow aspirates and core biopsies,1 fine needle aspirates (FNA),2 fresh tissue biopsies and all types of body fluids.3

Advantages of FCI include:

• Distinct cell populations are defined by their size (forward light scatter) and granularity (side light scatter).

• Dead cells may be gated out of the analysis.

• Weakly expressed surface antigens may be detected.

• Multi-color (2-, 3-, 4-) analysis may be performed, allowing for the definition of the surface antigen profile of specific cells.

• Two simultaneous hematologic malignancies may be detected in the same tissue site.

• Tissue biopsy may be obviated by the relatively noninvasive diagnostic evaluation of body fluids and FNA specimens.

Disadvantages of FCI include:

• Sclerotic bone marrow may yield too few cells for adequate analysis.

• Sclerotic tissue may be difficult to suspend for individual cellular analysis.

• A loss of architectural relationships occurs.

• Monoclonal B-cells may not be detected in a T-cell-rich (TCR) or lymphohistiocytic-rich (LHR) B-cell lymphoma (BCL).

• T-cell lymphomas that do not have an aberrant immunophenotype may not be detected.

• Partial tissue involvement by lymphoma with sampling differences or poor tumor preservation may result in falsely “negative” flow cytometry results.4

Table 1 lists the various applications of FCI to diagnostic hematopathology, which will be discussed.

FCI has been reported to be successful in evaluating sites for lymphomatous involvement in 75-90 percent of cases. False negatives may result from tumoral necrosis or sclerosis, partial tissue involvement, T-cell non-Hodgkin lymphoma (NHL) without an aberrant immunophenotype, or a TCRBCL or LHRBCL without detectable monoclonal B-cells.

Accurate subclassification of NHL by FNA and FCI has been reported to be attainable in 71-77 percent of positive cases. Further, evaluation of cell size by combining FCI and cytomorphology (CM) has recently been studied; large cell lymphoma/transformation may be diagnosed reliably if greater than 40 percent large cells are present.5

The following are situations requiring biopsy, based on FNA and FCI results:

• NHL of follicle center cell (FCC) origin with a mixed cellular composition,

• indeterminate results,

• necrosis and polymorphonuclear cells in evaluation of recurrent NHL,

• fewer than 10 percent neoplastic cells detected by FCI,

• a predominance of small cells detected by CM or FCI with clinical signs of transformation, and

• evaluating for an initial diagnostic possibility of or recurrent Hodgkin lymphoma (HL), due to the possibility of a composite lymphoma or a subsequent NHL.

A Particularly Useful Application

FCI is particularly useful in subtyping BCLs/leukemias composed predominantly of small cells (Table 2). Small lymphocytic lymphoma (SLL)/chronic lymphocytic leukemia (CLL) may be reliably differentiated from mantle cell lymphoma/leukemia (MCL) if CD23 is negative; however, dimly positive CD23 expression may be seen in SLL/CLL and MCL and, thus, molecular analysis for cyclin D1 may be necessary in selected cases.6 Atypical CLL, characterized by at least 10 percent lymphocytes with clefted and folded nuclei in the peripheral blood, demonstrates significantly higher expression of CD23, and these patients generally have higher white blood cell counts and probability of disease progression.7

CD5-negative small B-cell leukemias are unlikely to represent CLL and are classified more appropriately as NHL in the leukemic phase.8 CD10 expression is encountered in approximately 80 percent of FCC lymphomas but also may rarely be seen in MCL and hairy cell leukemia; thus, the FCI data must always be correlated with the morphologic features of each case.

Lymphoplasmacytic lymphoma (LPL) may be differentiated from other types of BCL by applying the characteristic FCI findings outlined in Table 2 and by identifying the presence of monoclonal plasma cells by immunohistochemistry (IH). Likewise, LPL may be differentiated from plasma cell dyscrasias (PCD) by the finding of a prominent population of monoclonal B-cells by FCI in LPL.

FCI also has been useful in immunophenotyping large BCLs and differentiating them from anaplastic CD30+ large cell lymphoma (LCL) and from anaplastic PCD. Expression of CD10 is seen in approximately 80 percent of FCC lymphomas and is characteristically strongly expressed in Burkitt’s lymphoma. The blastic and pleomorphic variants of MCL have the same immunophenotype as MCL; these variants are important to recognize, because these patients have a significantly worse prognosis. The majority of anaplastic CD30+ LCLs are of T-cell origin and they strongly express CD30 by FCI. Anaplastic PCD characteristically does not express CD45, B-cell antigens or surface immunoglobulin by FCI, but does express CD138 and variably expresses CD56.

Identifying Peripheral BCL

The lack of surface immunoglobulin (sIg) light chain expression by FCI helps identify peripheral BCL. In a recent study by Shiyong et al9 cases with greater than 25 percent B-cells lacking sIg light chain expression all represented lymphoma. By FCI, the identified sIg light chain-negative population was distinctly separated from the normal polytypic B-cells; in 90 percent of cases, the identified population was larger by forward angle light scatter than the reactive T-cells and polytypic B-cells. In their review of reactive cases, no reactive case revealed greater than 17 percent sIg-negative B-cells.

Prolymphocytic (PLL) and B-lineage acute lymphoblastic leukemias (ALL) may be immunophenotyped by FCI. B-cell PLL may be divided into CD5+ PLL (arising in CLL) and CD5- PLL (de novo PLL). CD5+ PLL has a longer median survival than CD5- PLL and, thus, it is important to distinguish between these two types of B-PLL. Likewise, FCI is extremely helpful in immunophenotyping and subtyping B-lineage ALL. FCI allows for the detection of aberrant myeloid antigen expression in B-lineage ALL, which in adults is associated with a significantly lower complete remission rate and shorter survival. Likewise, FCI allows for the detection of CD15+ early precursor B-ALL, which may occur in infants younger than 1 year of age and in adults, and is associated with t(4;11). This group of ALL is associated with a rearrangement of MLL and a poor prognosis.

Additionally, FCI is able to distinguish bone marrow hematogones from leukemic B lymphoblasts, which is often critical in a post-therapy ALL situation. Hematogones always exhibit a typical complex spectrum of antigen expression that defines the normal antigenic evolution of B-cell precursors and lacks aberrant expression. In contrast, lymphoblasts in precursor B-ALL show maturation arrest and exhibit varying numbers of immunophenotypic aberrancies (e.g., expression of CD13 or CD33).10

When a monoclonal or aberrant B-cell population or an aberrant T-cell population, characterized by a loss of a T-cell antigen, is identified, HL may be excluded only after correlation with the histology, to exclude the possibility of a composite lymphoma. In cases where FCI data is diagnostic, microscopic observations may provide additional information, not only due to sampling, but also due to patterns of involvement and the cytological features of the malignant cells. If no FCI abnormalities are detected, IH may need to be performed in selected cases.

Evaluating T-cell ALL Immunophenotypes

The immunophenotypes of T-cell ALL are outlined in Table 3 and are best evaluated by FCI. T-cell lymphoblastic lymphoma (T-LL) most often has an immunophenotype that corresponds to the common thymocyte stage of ALL; the immunophenotype of thymoma is identical to this stage. However, FCI allows for the distinction between thymoma and T-LL. FCI features characteristic of thymoma include a smear pattern of CD4/CD8 co-expression, a smear pattern of CD3 and TdT expression and lack of T-cell antigen deletion (with the exception of partial CD3). T-LL shows much more variability in expression patterns and is characterized by a tight pattern of CD4/CD8 expression, significant T-cell antigen deletion and absence of the CD3 or TdT smear pattern.11 In addition, distinguishing between thymoma and T-LL must also always rely on correlation of the FCI data with the morphology.

Mature T-cell lymphomas may have variable immunophenotypes by FCI. There may be variable loss of a pan-T-cell antigen (e.g., CD2, CD3, CD5, CD7). Most cases are CD4+; some are CD8+, CD4-/CD8-, or CD4+/CD8+. The characteristic immunophenotype of mycosis fungoides (MF) is CD4+/CD8- with CD7 commonly lost and variable expression of CD2; CD25 is negative. Adult T-cell leukemia/lymphoma (ATLL) has a similar immunophenotype to MF; however, CD25 is characteristically expressed in ATLL. T-cell CLL/PLL expresses pan-T-cell antigens (CD2, CD3, CD5, CD7) with CD4+/CD8- > CD4+/CD8+ > CD4-/CD8-. T-CLL is distinguished from the small cell variant of T-PLL by electron microscopy.

Lymphoproliferative Disorders

FCI is useful in dividing lymphoproliferative disorders of large granular lymphocytes into those of natural killer cell origin (CD2+, CD3-, CD8-, CD16+/-, CD57+/-, CD56+/-) and those of T-cell origin (CD2+, CD3+, CD8+, CD57+, CD16+/-, CD56-/+). Clonality is defined by the detection of a single band for the joined termini of the EBV genome in those of NK cell origin and by the detection of a T-cell gene rearrangement in those of T-cell origin.

FCI also may be useful in identifying a clonal process in post-transplant lymphoproliferative disorders (PTLD), even those with negative results by genotypic studies. FCI and genotypic studies should be routinely performed in PTLD to detect a clonal process, because the detection of clonality is important to categorize the process and for treatment management.12

Distinction of malignant lymphoma from a granulocytic (GS) or monocytic sarcoma (MS) is greatly aided by the use of FCI, because the malignant cells will variably express myelomonocytic markers in GS and MS. Likewise, FCI is useful in differentiating AML from ALL, detecting bilineage and biphenotypic acute leukemias, and defining a blast immunophenotype, which may be most useful in evaluating relapse/residual disease.

In immunophenotyping AML, FCI defines AML, M0, detects CD19+ AML characteristically associated with t(8;21), differentiates hypogranular acute promyelocytic leukemia from acute monocytic leukemia, and defines acute megakaryocytic leukemia. In addition, FCI is useful in immunophenotyping MDS because it allows for the detection of an accurate percentage of myeloblasts; microblasts are characteristic of MDS and often difficult to morphologically differentiate from lymphocytes. FCI also allows for the detection of an accurate percentage of monocytic cells by analyzing CD14 in establishing a diagnosis of chronic myelomonocytic leukemia.


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Dr. Dunphy is associate professor and director of Hematopathology in the Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill.

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