New Applications of Flow Cytometry
Flow cytometric immunophenotyping is used to evaluate the antigenic characteristics of leukemic cells.
Traditional laboratory evaluation of leukemia is based on microscopic examination of neoplastic cells in peripheral blood or bone marrow and special cytochemical stains. Although these morphologic studies are essential, they don’t consistently predict the immunologic origin of the leukemic process.
Flow cytometric immunophenotyping is used to accurately classify and determine cell lineage in pediatric and adult leukemias. When combined with morphologic studies, immunophenotyping improves diagnostic accuracy and determines optimal chemotherapeutic regimens for leukemia patients. Advanced, sensitive flow cytometric techniques are under investigation and may be used to evaluate bone marrow for residual leukemia after chemotherapy or prior to bone marrow transplantation.
Leukemias are hematopoietic disorders defined as an unregulated proliferation of malignant cells (blasts) originating in bone marrow. The neoplastic cells may circulate in peripheral blood, invade other body organs and replace normal bone marrow cells.
The original French-American-British (FAB) leukemia classification system described morphologic features of bone marrow blasts and cytochemical studies. Unfortunately, immunologic features of leukemic cells were not included in this schemata.
Morphologic evaluation of leukemia and interpretation of cytochemical studies may also be subject to interobserver variation, even among experienced pathologists. Immunophenotyping refers to the antigenic analysis of leukemia using a flow cytometric technique and monoclonal antibodies. These immunologic studies evaluate cell surface, cytoplasmic and nuclear characteristics of neoplastic cells to appropriately classify leukemia, predict clinical outcome, assess remission status and determine optimal chemotherapeutic regimens for individual patients.
Flow Cytometry Instrumentation
A flow cytometer is an automated instrument that measures physical or antigenic characteristics of single cells using a laser light source.
A laser beam provides a constant light source for cellular analysis. Laser light is focused and intensified within the instrument through a series of mirrors and is emitted as a narrow beam of a specific wavelength (monochromatic light). A sample containing a single cell suspension of hematopoietic cells is aspirated into a continuously flowing enclosed stream of isotonic fluid (sheath) through an aspiration nozzle. This pressurized, moving column of fluid draws cells single file past the laser beam.
Light scattered or emitted from a cell is then collected by a series of sensitive light detectors called photomultiplier tubes (PMTs) and converted to electronic signals. A computer system analyzes emitted light and results in a visual and quantitative record of cellular analysis.
Different cellular parameters may be studied using a flow cytometer. Light collected from cells that have not been treated with chemicals or dyes measure intrinsic or native cellular characteristics.
Intrinsic cellular features include forward angle light scatter (FALS) and side scatter (SS). FALS refers to light collected along the axis of the laser beam and relates to cell size. Light deflected from cells at a 90 degree angle to the laser beam is termed SS and may be displayed as a scattergram, where each dot represents a cell of a certain size and density. By combining these two native cellular characteristics of unstained cells, it’s possible to identify different cellular populations.
Extrinsic cellular characteristics are studied using exogenous chemicals such as dyes or fluorochromes. Light of a specific wavelength is emitted from a fluorescent dye excited by the laser beam. The computer system then converts the light signal to a histogram, which plots emitted cellular fluorescence vs. cell count (Fig. 1). This visual display may be further analyzed to determine the relative percentage of cells that stain with a specific probe. Commercially prepared monoclonal antibodies are frequently conjugated to different color dyes and can be used to study several antigens on the surface of a cellular population.
Some research facilities have the capability to perform cell sorting, allowing physical separation of specific cells of interest during cytometric analysis. Cells with predesignated antigenic features are isolated in individual liquid droplets from the sample column. The cytometer computer then identifies and isolates the cell of interest in a charged liquid droplet and deflects the appropriate cell into a collection tube. These specifically selected cells may be used for further analysis or research purposes, including drug sensitivity or culture studies.
Different manufacturers are marketing instruments with various laser light sources and sophisticated computer analysis systems. Many cytometers now have the capability to store raw data (listmode data) obtained from leukemia analyses on computer disks. The operator may then “replay” a prior analysis on a computer system or manipulate the information obtained without physically reanalyzing the specimen.
Current immunologic studies suggest leukemic cells fail to complete a normal maturational sequence and display markers usually seen early in cell ontogeny or aberrant, inappropriate antigenic patterns. Cellular antigens expressed on hematopoietic cells are studied using a flow cytometric technique in combination with commercially prepared monoclonal antibodies conjugated to different dyes.
The antibodies recognize lineage-associated antigens and frequently are given numbers in a cluster designation (CD) system developed by an International Workshop Group (Table 1). The cellular antigens and corresponding monoclonal antibodies are given the same cluster designation number.
Antibodies may be directed to myeloid, lymphoid or other surface, nuclear or cytoplasmic antigens. Since it’s not possible to consistently predict cell lineage and classify acute leukemias based on morphologic features alone, these hematopoietic disorders are typically analyzed with a preselected panel of monoclonal antibodies directed to numerous antigens. If more than 20 percent of cells of interest react with a specific antibody, they are considered positive for the corresponding antigen.
Newly diagnosed leukemias are always assessed morphologically utilizing FAB criteria. The FAB system recognizes three morphologic subgroups of lymphoid leukemia (L1, L2, L3) and eight morphologic types of myeloid leukemia (M0-M7) (Table 2).
Bone marrow or peripheral blood is submitted for flow cytometric analysis and stained with a broad panel of monoclonal antibodies directed to lymphoid, myeloid, monocytic or other cell antigens. The flow cytometric data is then integrated with morphologic findings to confirm the FAB subgroup and establish the cellular origin of the leukemia.
The two major immunologic subtypes of leukemia are lymphoid leukemia (ALL) and myeloid leukemia (AML). ALL may originate from B or T lymphoid cells. Blasts of B-lymphoid origin are characterized by bright nuclear TDT expression with the surface markers HLADR, CD10 (CALLA), CD19 or CD22, occasionally in combination with the immature stem cell antigen CD34.
This phenotypic pattern is termed precursor B phenotype and is one of the more frequently observed patterns of B-cell ALL. It’s the most common form of childhood ALL; low-risk precursor B ALL is treated with a short course of chemotherapy and results in an 85 percent five-year survival rate.
Lymphoblasts that express the heavy chain of the IgM molecule (cytoplasmic M) but lack surface immunoglobulin or nuclear TDT expression are termed pre-B ALL. This immunologic form of leukemia is associated with a higher risk for relapse than the precursor-B type and may be associated with a t(1;19) translocation. No specific FAB subtype of ALL is associated with precursor-B or pre-B immunologic profiles.
Mature B ALL usually displays surface immunoglobulin in combination with B-cell markers such as CD19 and CD20; nuclear TDT is often negative and surface CALLA (CD10) expression is variable. In contrast to the other subtypes previously described, mature B ALL is associated with morphologic features of FAB L3 leukemia (large leukemic cells with dark blue bubbly cytoplasm). Mature B ALL requires aggressive chemotherapy and is associated with a poor prognosis.
Lymphoid leukemias of T-cell origin are less common than those derived from B-cell lines. Again, no specific FAB morphologic appearance can be associated with T-cell ALL. T leukemias often express nuclear TDT as well as T-cell markers such as CD2, CD3, CD5, CD7, CD4 and CD8. The loss of an expected T antigen, co-expression of CD4 and CD8 epitopes on the same cell, or bright nuclear TDT with surface CD3 are considered presumptive evidence of a clonal T-cell process.
T ALL is clinically associated with greatly elevated numbers of leukemic cells in peripheral blood and a mediastinal mass. Specialized chemotherapeutic regimens are required for T leukemias and may result in a 45 percent five-year survival rate. The overall prognosis for T ALL is worse than the more common precursor B ALL.
Immunologic features of myeloid leukemias are not as clearly understood as those of lymphoid leukemias. Several significant associations have been noted, however, between some myeloid FAB subtypes and patterns of antigen expression. Blasts of myeloid origin characteristically display surface HLADR, CD33 and CD13 antigens. Differentiated myeloid leukemias such as FAB M1 or M2 subtypes often express CD15 in addition to CD33 and CD13. Leukemias with a monocytic component (FAB M4 or M5) may express the monocyte-specific surface marker CD14.
FAB M3 (acute promyelocytic leukemia) is a distinctive disease that differs from other myeloid leukemias. It’s characterized by large, granular leukemic blasts that may trigger life-threatening bleeding disorders.
Flow cytometric analysis of M3 leukemia usually reveals bright expression of CD33 and CD13; surface HLADR is absent. This immunophenotypic profile is strongly suggestive of promyelocytic leukemia and may be a helpful diagnostic clue for early recognition of this disorder. Many M3 leukemias have a high remission rate if treated with a retinoic acid chemotherapeutic regimen. Prompt diagnosis of this disorder, however, is essential to prevent complications or death from the bleeding that may be associated with promyelocytic leukemia.
FAB M0 leukemia is characterized by primitive blasts that may be interpreted morphologically as myeloid or lymphoid in origin; cytochemical stains are negative. Immunophenotyping of undifferentiated M0 leukemia often reveals the presence of the stem cell surface antigen CD34 in combination with myeloid markers such as CD13 or CD33.
Weak nuclear TDT activity may also be observed in M0 leukemia. Immunophenotyping is critical and can prevent misdiagnosis of the disorder as a lymphoid leukemia.
Megakaryocytic leukemia (M7) may also have variable morphologic features and negative cytochemical studies. Immunophenotyping may demonstrate the presence of megakaryocytic cell markers such as CD41, CD42 or CD61. These findings, again, should preclude confusion of megakaryocytic/myeloid leukemia with lymphoid leukemia and result in treatment with the appropriate myeloid therapeutic regimen.
Occasional leukemias may co-express surface antigens of both myeloid and lymphoid cell lineages (mixed lineage leukemia). These are a heterogeneous group of leukemias that don’t fit into a defined FAB subgroup. When reasonable, a predominant cell lineage is identified and the patient is treated according to that lineage. Some of these leukemias may be associated with high peripheral blood white counts, cytogenetic anomalies and a poor response to chemotherapy.
Research techniques are being developed using dual laser systems and multiple monoclonal antibodies. These studies have been used to examine bone marrow of leukemia patients treated with chemotherapy in preparation for bone marrow transplantation. Investigators are able, in some instances, to immunologically identify leukemic cells in the absence of microscopic evidence of leukemia (minimal residual disease, MRD).
Dr. Kotylo is an associate professor and director of the flow cytometery laboratory, Pathology and Laboratory Medicine, Indiana University Medical Center, Indianapolis.