Multi-Dimensional Flow Cytometry

Immunophenotyping of Peripheral Blood and Bone Marrow Aspirate Specimens

Abnormal cell populations of suspected hematopoietic malignancies (acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogeneous leukemia, myelodysplasia, chronic lymphocytic leukemia, hairy cell leukemia, non-Hodgkin’s B and T cell lymphoma, plasma cell myeloma), and some instances of non-hematopoietic malignancies can be identified in peripheral blood and bone marrow aspirate specimens using High Resolution Flow Cytometry™. The diagnostic and prognostic information obtained by flow cytometry supplements clinical, morphological, and cytogenetic parameters.

We have spent several years characterizing the expression of cell antigens on normal hematopoietic cells. Neoplastic cells are distinguished from their normal counterparts based on the abnormal expression of cellular antigens. In a reflexive approach, three-color panels of monoclonal antibodies are applied to characterize the neoplastic or reactive process. This multidimensional flow cytometric (MDF) approach to detection of hematological abnormalities based on differences from normal allows identification of aberrant populations even when they constitute a minor proportion of cells (0.5% or less of total nucleated cells). MDF can be used not only for characterization of the cells at diagnosis, but it can be used to detect residual leukemia post therapy or to identify lymphoma present in blood or bone marrow aspirates.

Immunophenotyping of Tissue Specimens

Cells for flow cytometric analysis can also be obtained from lymph node biopsies, fine needle aspirates of lymph nodes or tissue masses, or from body fluids (e.g., CSF or pleural effusions). Detection of neoplastic cells of B lineage origin is based on the cellular expression (surface or cytoplasmic) of immunoglobulin restricted to either kappa or lambda light chain. T lineage neoplasms are identified by the aberrant expression of T lymphoid antigens.

It is crucial to distinguish viable from dead cells in these specimens it since poor cell viability may give false positive or uninterpretable results. We use MDF in combination with a viability dye to assess only the intact live cells with two-color immunofluorescence to accurately phenotype the cells in these tissues.

The combination of cell size by light scatter and immunophenotype correlates well with established histological criteria for determining type and grade of non-Hodgkin’s lymphoma while providing sensitive detection techniques for minimal residual disease.

Clinical Cell Sorting: Tumor Cell Confirmation, Lineage Specific Chimerism, Monoclonality Profiling

When abnormal cells are present at very low levels, flow cytometry, cytogenetics or morphology  often cannot adequately confirm relapse. Therefore, it is useful to  concentrate the abnormal cells via cell sorting for futher analysis and confirmation . We routinely separate cells at frequencies down to 0.2% for analysis by fluorescence in situ hybridization (FISH) or clonality profiling by polymerase chain reaction analysis (PCR), thereby providing an independent assessment of the tumor cells detected at low levels. When appropriate, we will contact the requesting laboratory as to the necessity to sort the cells for genotypic confirmation of relapse.

In a non-myeloablative hematopoietic stem cell transplant situation, it is necessary to assess the chimerism of the T cells and myeloid cells separately. Peripheral blood specimens can be separated into T cell and neutrophil components using fluorescence activated cell sorting. Populations of cells isolated in this manner are >98% pure and can then be assayed for donor/host proportions using either short tandem repeats (STR) analysis or FISH (for opposite sex transplants).

Quantitative Progenitor Cell (CD34) Analysis

The identification of stem cells by MDF permits the detection of these cells as an alternative to time consuming culture systems. In addition to stem cell detection, the enumeration of these cells can be used to define the appropriate timing for harvesting of progenitor cells from peripheral blood following growth factor mobilization. The test quantifies the total percentage of stem cells present by detecting CD34 positive cells while ensuring the exclusion of cell debris, aggregates, and dead cells.


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