Flow Cytometry, What Is A Flow Cytometry, Analysis, And Results

What Is Flow Cytometry?

Flow cytometry (FCM) is invaluable in the diagnosis and classification of hematolymphoid neoplasms, and in determining prognosis and monitoring response to therapy. Flow cytometry is especially suited for immunophenotypic analysis of blood, fluids  (cerebrospinal fluid [CSF], pleural fluid), and aspirations of bone marrow and lymphoid tissue.

Flow cytometry can characterize surface as well as cytoplasmic protein expression. Furthermore, flow cytometry can provide a highly accurate quantitation of cellular antigens/ molecules.

General Considerations Of Flow Cytometry?

Appropriate samples for Flow Cytometry include blood, bone marrow, lymph node, extranodal tissue biopsies, fine-needle aspirates (FNA), and body fluids. International consensus guidelines on medical indications for Flow Cytometry are available and are based on patient history and presenting symptoms. Timely processing of samples is necessary to maximize cell yield, maintain cell viability and integrity, and prevent loss of abnormal cells of interest.

Flow Cytometry Application?

Lysis is the preferred approach for removing excess erythrocytes (see Stetler-Stevenson et al. for recommendations). In patients with an inaspirable marrow or a “dry tap” submission of several core biopsies for flow, cytometry is appropriate. These cores are disaggregated to release cells into fluid suspension, before flow cytometry.

Principle Of Flow Cytometry?

Intact portions of solid tissue (such as biopsies of bone marrow, lymph nodes, or other tissue masses) must be made into cell suspensions for flow cytometry. Mechanical tissue disaggregation is fairly simple, rapid, leaves the cells relatively unaltered, and is achieved by slicing, mincing, and teasing apart the tissue with commercial devices or manual tools.4 Enzymatic dissociation has been used in processing fibrotic tissue; however, it can alter antigen expression and decrease viability.

Viability Of Flow Cytometry Results?

Decreased viability is noted in solid-tissue samples and aggressive lymphomas. Nonviable cells may nonspecifically bind antibodies and interfere with accurate immunophenotyping. A low-viability sample composed entirely of neoplastic cells can yield meaningful results. 

Flow Cytometry Testing?

No set cutoff exists to dictate specimen rejection for flow cytometry, although, general guidelines suggest rejecting non-irreplaceable samples with less than75% viability. In irreplaceable specimens with poor viability, any abnormal populations should be reported. Failure to identify a neoplastic process in a sample of poor viability should not be viewed as a true negative, as subsequent testing may be informative.

Flow Cytometry Small Specimens?

Diagnosis of lymphoma is frequently based on evaluation of small biopsies, FNA, and body fluids (e.g., CSF, vitreous humor, effusions). Small samples can provide sufficient cells for flow cytometry, even when cell numbers are too low to count by conventional methods. Flow cytometry can be more sensitive than morphology, especially when neoplastic cells are admixed with normal counterparts or associated with a brisk inflammatory response, as in extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) or gastric lymphoma in endoscopic biopsies. Flow cytometry provides increased sensitivity of detection of hematolymphoid neoplasia in FNA. 

What Is A Flow Cytometry?

Flow cytometry is also useful in identifying CNS leukemia and increases the detection rate over cytology alone. Thus flow cytometry is crucial in the evaluation of CSF for hematolymphoid malignancies.16 It should be noted that studies have demonstrated that there is a rapid decline in CSF cell number within the first 30 minutes of sampling, and immediate stabilization with serum-containing media or commercially available stabilizers is vital to preserving the specimen until it reaches the flow cytometry laboratory.

Flow Cytometry In Diagnosis And Classification Of Mature B-Cell Neoplasms?

Flow Cytometry detection of malignant B-cell populations requires extensive knowledge of normal B-cell antigen expression and light scatter characteristics. Markers of B-cell neoplasia include light chain restriction, abnormally large B cells, abnormal levels of antigen expression, absence of normal antigens, and presence of antigens not normally present on mature B cells. Flow-Cytometric Evaluation of Light Chain Expression A B-cell population with monoclonal light chain expression is, with rare exception, considered a B-cell neoplasm.

Flow Cytometry Analysis?

In normal/benign lymphoid tissue, virtually every B cell expresses a single light chain immunoglobulin, and the ratio of kappa expressing to lambda expressing B cells is approximately 60% to 40%.25 Lack of surface immunoglobulin among mature B cells or a deviation from this normal ratio suggests a monoclonal B-cell population. Flow Cytometry is advantageous in that it can recognize monoclonal B cells, even in B-cell lymphopenia, owing to rapid analysis of large numbers of acquired B cells, or in a background of polyclonal B cells, owing to the detection of aberrant antigens on the neoplastic cells. By examining B-cell subsets with differential CD19, CD20, or CD22 expression, an abnormal monoclonal B-cell population may be discovered. 

Detection of a skewed kappa to lambda ratio should prompt a diligent search for an underlying monoclonal population that may be discriminated by CD19, CD20, CD22, or other antigens. For example, peripheral blood with minimal involvement by hairy cell leukemia (HCL) may, at first glance, appear to contain only polyclonal B cells; however, with specific identification of the CD20 bright+, CD22 bright+ B cells, the monoclonal light chain expression of the HCL cells may be revealed.

Flow Cytometry Analysis?

For example, the CD5+ B cells in peripheral blood with involvement by mantle cell lymphoma may be monoclonal, while the CD5 B cells are polyclonal. A simplistic, one-dimensional examination of cells staining with kappa, lambda, and CD5 is clearly ineffective in this case.

The multiparametric analysis is essential in detecting relevant neoplastic populations. The absence of surface immunoglobulin may also indicate a mature B-cell neoplasm, but caution is imperative when interpreting the significance of such a population. In bone marrow aspirates, plasma cells, and most normal immature B cells (hematogenous; benign precursor B cells) also lack surface immunoglobulin.

Technical Considerations In Demonstration of Light Chain Restriction

Technical factors, such as antibody choice and cytophilic antibody artifact can affect a laboratory’s ability to assess the surface light chain. Washing a specimen with phosphate-buffered saline (PBS) before staining and using anti-CD20 or anti-CD19 for B-cell selection prior to flow cytometry analysis is sufficient to eliminate this artifact in most cases. Neoplastic B cells may express light chain epitopes not readily detected by all antibodies. The incorporation of two sets of light chain reagents improves the sensitivity of monoclonal B-cell detection.

Additional Flow-Cytometric Abnormalities in Mature B-Cell Neoplasia

Abnormal B-cell antigen expression can identify malignant B cells. Mature normal B cells express CD19, CD20, and CD22, and, except for plasma cells, failure to express one of these antigens is abnormal. An important caveat is a history of monoclonal antibody therapy (e.g., rituximab, ofatumumab), as the therapeutic antibody may mask detection of the targeted antigen.

The detection of aberrant antigens (not normally expressed on B cells) is also useful in the identification of malignant B cells. Aberrant expression of CD2, CD4, CD7, and CD8 occurs in chronic lymphocytic leukemia (CLL/SLL), hairy cell leukemia, and B-cell non-Hodgkin’s lymphomas.35,36 Demonstration of abnormal levels of expression of various antigens (e.g., abnormally dim or bright staining with antibodies) is also of diagnostic importance and helps in subclassification.

Flow Cytometry Analysis In Diagnosis And Classification Of Mature T-Cell Neoplasms

Flow cytometry immunophenotyping is useful in the diagnosis and may also in the subclassification of mature T-cell neoplasms, though detection of T-cell neoplasia is more intensive and challenging than in B-cell neoplasias. Typically, subset restriction, absent, diminished, or abnormally increased expression of T-cell antigens, presence of aberrant antigens, and expansion of normally rare T-cell populations are indicators of T-cell neoplasia.

T cells fall into two main groups based on TCR expression of either the αβ or γδ chains formed by VDJ gene segments and a constant region. The vast majority of normal and neoplastic T cells express the αβ chain. Commercial antibodies are available against 70% of the human class-specific sequences among the V segments for the TCR β chain (Vβ). T cells in a clonal T-cell population have the same VDJ segment and therefore have identical (“monoclonal”) Vβ protein expression. The distribution (proportion) of Vβ classes in normal CD4+ or CD8+.

Protocol For Flow Cytometry?

T cells are well defined.79 An abnormal expansion of a Vβ-expressing population is consistent with a clonal T-cell population, similar to an expansion of the light chain restricted B cells in a monoclonal B-cell population. Abnormal T-cell populations are detected with a panel of antibodies, and then anti-Vβ antibodies are used to determine the clonality of the immunophenotypically defined abnormal T cells. This approach, known as Vβ repertoire analysis, can be used to establish an initial diagnosis of T-cell neoplasia and to monitor minimal residual disease.

Flow Cytometry In Diagnosis And Classification Of Natural Killer Cell Neoplasms.

Flow cytometry is particularly useful in characterizing blood involvement with NK-cell neoplasms such as aggressive NK-cell leukemia and chronic lymphoproliferative disorders of NK cells (CLPD-NK), also known as NK-cell LGL lymphocytosis, a disease with an indolent clinical course.

Flow cytometry is also helpful in identifying NK cells in extranodal NK/T-cell lymphoma, nasal type, where the tumor often exists in a background of extensive necrosis and inflammation (usually from aspirates or disaggregated tissue samples). Normal NK cells typically express CD45 (characteristically bright, consistent with mature lymphocytes), CD2, and CD7; exhibit a pattern of CD16 and CD56 is slightly heterogeneous in appearance; and are negative for CD3.

Although no specific immunophenotypic markers exist that accurately distinguish reactive from neoplastic NK cells, changes in the pattern of surface antigen and expression may be helpful to identify abnormal NK cells. CD16 may appear unusually homogeneous, and CD56 may be abnormally bright, or uniformly dim, as has been observed in CLPD-NK.91 Diminished expression of CD2, CD7, and CD161; aberrant expression of CD5; and a homogeneous pattern of expression of CD8 may also be helpful features. Additionally, the number and proportion of NK cells and the NK-cell forward scatters properties (presence of large cells) may help to confirm the diagnosis, especially in extranodal NK/T-cell lymphoma, which may have a marked inflammatory background.

Flow Cytometry In Diagnosis And Classification Of Acute Leukemia?

The approach to acute leukemia by flow cytometry often begins with an evaluation of a CD45-versus-SSC plot.  As true myeloid leukemias can aberrantly express lymphoid markers, and vice versa, the use of a comprehensive panel is vital to prevent misdiagnosis.

The WHO classification has incorporated specific genetic alterations and characteristic translocations that carry prognostic and sometimes therapeutic implications into the classification of leukemia. In addition, flow cytometry minimal residual disease detection carries important prognostic implications and may guide further therapeutic options.

Flow Cytometry And The Diagnosis Of Myelodysplastic Syndromes And Myeloproliferative Neoplasms

The utility of flow cytometry in evaluating chronic myeloid stem neoplasms (MSNs), such as myelodysplastic syndromes and myeloproliferative neoplasms, has grown significantly in recent years. Advances in this area have paralleled our increasing understanding of normal patterns of antigen expression on myeloid progenitors and maturing myeloid forms and the routine use of multiparametric flow cytometry in clinical laboratories.

Identifying aberrant antigen expression by flow cytometry can aid in the diagnosis of MSN and, in some cases, flow cytometry may provide additional prognostic data. It should be noted that experience and knowledge of how normal antigenic patterns can shift in various reactive states (such as growth factor administration or with bone marrow regeneration posttherapy) is critical to avoid overinterpretation of the data.

Myelodysplastic Syndromes?

Although bone marrow morphology with concurrent cytogenetic study remains the gold standard for the diagnosis of myelodysplastic syndromes (MDSs), a significant number of patients have blood and bone marrow findings that make diagnosis and classification are difficult.

Flow cytometry in the minimal diagnostic criteria for MDS developed at a 2006 international working conference.128 In addition, the 2008  WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues recognized the utility of flow cytometry in the evaluation of
MDS.

Myeloproliferative Neoplasms?

Virtually little to no role exists for flow cytometry in a patient with CML in a chronic phase with stable white blood cell counts. However, flow cytometry can provide accurate blast characterization and enumeration for patients with increasing white blood cell counts that may be entering an accelerated phase or blast crisis, especially if the blasts are not large, and are difficult to identify by morphology.

Flow-Cytometric Detection of Mature B-Cell Neoplasia?

Blood and bone marrow contain excess serum Ig that binds anti-kappa and anti-lambda antibodies, preventing binding to cells. Serum Ig bound to Fc receptors on cells also stains positive with anti-kappa and anti-lambda, masking monoclonality.

Washing blood or bone marrow specimens with room temperature or 37° C PBS eliminates free serum immunoglobulin and removes serum immunoglobulin bound to the cell surface. When polyclonal B cells are abundant, they obscure the detection of abnormal B cells. Gating on large cells (with increased FSC), cells with abnormal antigen intensity, or cells expressing specific antigens (e.g., CD10) allows detection of monoclonality in abnormal B cells. Malignant B cells are frequently missing a normal antigen (e.g., CD19, CD20, CD22). Monophasic light chain expression (all positive or all negative for anti-kappa and anti-lambda) is abnormal.