Hematolymphoid/Plasma Cell Neoplasia Assessment (HNA), Immunophenotyping by Flow Cytometry

Whole blood, bone marrow aspirate, body fluids, fresh lymph node, spleen, extranodal solid tissue, biopsy, or needle aspirate

3 mL whole blood or 2 mL bone marrow aspirate, 2 mL body fluid tube. Large volumes of body fluids should be concentrated to <5 mL; 0.5 to 1.0 cm³ fresh tissue.

1 mL whole blood or bone marrow (Note: This volume does not allow for repeat testing.)

Green-top (sodium heparin) tube (preferred), lavender-top (EDTA) tube, or yellow-top (ACD) tube for whole blood or bone marrow (acceptable, not preferred); lavender-top (EDTA body fluids) tube; fresh tissue in lymph node transport bottle containing RPMI

Identify and characterize the following:

• Mature B-Cell lymphoproliferative disorders (chronic lymphocytic leukemia (CLL), mantle cell lymphoma, monoclonal B-cell lymphocytosis, hairy cell leukemia, B-cell lymphomas)

• Mature T-cell and natural killer cell lymphoproliferative disorders (T-cell lymphomas, Sezary syndrome/mycosis fungoides. T-cell and NK cell large granular lymphocyte disorders, adult T-cell leukemia/lymphoma)

• Acute leukemia (acute myeloid leukemia, precursor lymphoid leukemia, acute leukemia of ambiguous lineage)

• Plasma cell neoplasm (multiple myeloma, plasmacytoma, monoclonal Gammopathy)

Immunophenotyping by flow cytometry

In normal or reactive processes, a bimodal distribution of κ- and λ-positive B cells is present in a ratio of approximately 1.5:1. Immunophenotyping using multiparameter analysis (simultaneous staining with a pan B-cell marker and anti-immunoglobulin light chain antibodies) is a rapid and specific method for detecting and confirming the presence of neoplastic B-cell disorders.

Chronic lymphocytic leukemia (CLL) is a clonal lymphoproliferative disorder usually of B-cell origin that has been traditionally diagnosed using clinical and morphologic criteria. Incorporation of immunophenotypic features into the diagnostic criteria is helpful in separating common B-cell CLL from other lymphoproliferative disorders. Detection of cytogenic abnormalities is useful in assessing prognosis. Lymphocytes in B-CLL typically coexpress CD20, CD23, CD5, and a single immunoglobulin light chain, kappa or lambda. CD10 (CALLA) expression is usually absent. Mantle cell lymphoma shows expression of FMC7 with absent or very dim expression of CD23.

Lymphomas include diverse disease entities in the WHO classification, which requires a combination of morphology, immunophenotype, genetics and clinical information for lymphoma diagnosis and classification. Therefore, immunophenotyping by flow cytometry has become a critical component in lymphoma workup. Flow cytometry offers the advantage of rapid multiparameter analysis. Combining light scatter characteristics with patterns of antigen expression provides biological information that is useful in making a diagnosis and assessing prognosis. Various gating strategies can be employed to enhance the detection of minor populations.

T-cell prolymphocytic leukemia is associated with rapid onset, an aggressive clinical course poorly responsive to therapy and decreased survival. Immunophenotyping, in the majority of cases (60% of cases), demonstrates expression of CD3 (a pan T-cell antigen) and CD4 (the helper cell antigen) without CD8 expression (the suppressor/cytotoxic cell antigen). In 25% of cases, the lymphoma cells coexpress CD4 and CD8. The other 15% of cases are CD4 negative and CD8 positive. Genotyping demonstrates a clonal rearrangement of the T-cell receptor gene.

Large granular lymphocyte (LGL) proliferations can be divided into T-cell and natural killer (NK) cell subsets by immunophenotyping. The more common T-cell type expresses CD3, a pan T-cell antigen and CD8, the suppressor/cytotoxic cell antigen. Genotyping demonstrates a clonal rearrangement of the T-cell receptor gene. The NK cell type is relatively rare and expresses CD2 and CD16 and/or CD56. CD3 expression is absent. Genotyping demonstrates a germline configuration of the T-cell receptor gene.

In Sezary syndrome, the neoplastic lymphocytes are T cells with a helper cell phenotype. Expression of CD7, a pan T-cell antigen, is absent and is useful in distinguishing the neoplastic cells from normal T-helper cells. Genotyping demonstrates a clonal rearrangement of the T-cell receptor gene.

In adult T-cell leukemia/lymphoma, the neoplastic lymphocytes are T-cells with a helper cell phenotype. Expression of CD3, CD4, and CD25 is present. Expression of CD7 is absent. Genotyping demonstrates a clonal rearrangement of the T-cell receptor gene.

Detection of a B-cell population coexpressing CD103, CD22, CD11c, and CD25 is useful in establishing a diagnosis of hairy cell leukemia when used in conjunction with morphology and cytochemistry. Immunophenotyping by flow cytometry is a sensitive method for detecting residual or recurrent disease in the peripheral blood of patients with an established diagnosis.

Plasma cells in the blood and bone marrow are detected by expression of CD38 and CD138. Cytoplasmic light chain analysis is used to detect monotypia.

Lineage assignment in acute leukemia is necessary for selecting appropriate therapy and is useful in assessing prognosis. Multiparameter analysis using multi-color immunophenotyping techniques is a rapid and specific method of assigning lineage in acute leukemia.

This profile is also useful in distinguishing lymphoid from myeloid blast crisis in CML and immunophenotyping lymphoblastic lymphoma in blood or bone marrow.

Immunophenotyping and cytogenetic/molecular analysis are increasingly being used to supplement the traditional methods (morphology and cytochemistry) of classifying acute leukemias and to provide prognostic information. Acute lymphoblastic leukemia (ALL) can be classified into T-and B-cell lineages. In ALL of B-cell lineage, expression of CD10 (CALLA) is a favorable prognostic factor. Acute myeloid leukemias (AML) are a heterogeneous group. Immunophenotyping is particularly useful in classifying acute megakaryoblastic leukemia. A combination of characteristic light scattering properties and myeloid phenotype can suggest a diagnosis of acute promyelocytic leukemia. Confirmation of the retinoic acid receptor gene rearrangement by cytogenetic or molecular methods is recommended. See tests listed under Related Information, as well as as well as Fluorescence in situ Hybridization (FISH), Oncology [510669].