Flow cytometry is a powerful laboratory method used in hematology to rapidly analyze blood and bone marrow samples. This technique measures multiple characteristics of thousands of individual cells suspended in a fluid stream as they pass through a laser beam. Different cell types carry unique protein signatures on their surface, known as markers. Identifying these specific markers allows clinicians to accurately distinguish between normal and diseased cell populations.
Understanding Myeloid Cells and Surface Antigens
The myeloid lineage is a major branch of blood cell development, giving rise to mature cells including granulocytes, monocytes, macrophages, red blood cells, and platelets. These cells originate from hematopoietic stem cells in the bone marrow, passing through a common myeloid progenitor stage.
As a myeloid cell matures, it expresses a changing pattern of surface proteins, categorized using the Cluster of Differentiation (CD) system. Immature progenitor cells typically express CD34 and CD117, markers progressively lost as the cell differentiates.
The specific combination of CD markers determines a cell’s identity, stage, and function. The systematic study of these markers is called immunophenotyping. In healthy individuals, predictable expression patterns allow specialists to define normal cell populations.
Monocytes are identified by expressing CD14 and CD64, while mature granulocytes are characterized by markers like CD15. Deviation from this expected pattern—such as retaining an immature marker or acquiring one from a different lineage—signals an abnormality. Flow cytometry detects these changes in surface protein expression.
Principles of Flow Cytometry
The flow cytometer analyzes thousands of cells per second. The fluidics system uses a stream of sheath fluid to hydrodynamically focus the sample, forcing cells into a single-file line to pass through the laser interrogation point.
The optics system measures two main types of light signals. Light scatter provides information about the cell’s physical properties. Forward Scatter (FSC) correlates with cell size, while Side Scatter (SSC) relates to internal complexity or granularity. Plotting FSC against SSC allows major cell populations, such as lymphocytes and monocytes, to be initially separated into distinct clusters.
The second signal is fluorescence, which reveals specific surface antigens. Before analysis, cells are mixed with antibodies linked to fluorescent dyes (fluorochromes). These antibodies bind only to matching CD markers on the cell surface.
When the laser hits the bound fluorochromes, they emit light at specific wavelengths. Detectors capture this light, and electronic systems convert the signal into data points representing marker intensity. Using multiple antibodies tagged with different colors simultaneously generates a detailed, multi-color immunophenotype for every cell.
Interpreting Key Myeloid Marker Panels
Flow cytometry interpretation relies on analyzing the expression pattern of a myeloid panel. The first step uses pan-leukocyte markers, like CD45, to identify all white blood cells and exclude debris, followed by lineage-specific markers.
General myeloid markers like CD13 and CD33 are commonly included as they are expressed across many myeloid cells. Progenitor markers, specifically CD34 and CD117, identify very immature cells (blasts), which are normally found only at low levels.
Monocyte identification is confirmed by CD14 and CD64 expression. The combination of CD14 and CD16 allows subdivision into classical, intermediate, and non-classical subsets. Granulocytes are recognized by high Side Scatter (granularity) and expression of CD15 and CD66b.
The panel’s power lies in detecting abnormalities, such as asynchronous expression (markers from early and late stages simultaneously) or cross-lineage expression. A myeloid cell aberrantly expressing a lymphoid marker suggests malignant transformation. This detailed analysis creates a definitive immunophenotype for the cell population.
Using Marker Data for Disease Classification
Immunophenotypic data is applied to diagnose and classify hematologic malignancies, including Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS). AML diagnosis requires identifying an abnormally high percentage of myeloid blasts, characterized by an immature marker profile (CD34+, CD117+).
Flow cytometry counts these abnormal blasts and classifies the AML subtype based on marker combinations. Specific patterns of CD13, CD33, and HLA-DR expression help categorize the leukemia according to the World Health Organization (WHO) system. Detecting these aberrant phenotypes is a rapid and specific method for confirming malignancy.
In Myelodysplastic Syndromes (MDS), characterized by ineffective blood cell production, flow cytometry detects subtle signs of abnormal maturation (dysplasia). This involves identifying quantitative changes, such as reduced expression or unusual marker intensity. Multiple phenotypic abnormalities across myeloid lineages strongly suggest MDS, even without obvious morphological changes.
Flow cytometry is also used for monitoring treatment and detecting minimal residual disease (MRD). MRD refers to remaining cancer cells after therapy that can lead to relapse. Because flow cytometry is highly sensitive, it detects these tiny populations of malignant cells, allowing clinicians to make timely decisions about further treatment.