Flow Cytometry

Flow cytometry can seem complicated to the outsider, but it is a powerful analytical technique used to rapidly measure physical and biological characteristics of individual cells suspended in a fluid stream. By directing cells past a focused light source, typically a laser, the instrument can detect properties such as size, internal complexity, and the presence of specific surface or intracellular markers through fluorescent tagging. This method is widely used in clinical and research settings to identify and quantify distinct cell populations, evaluate immune function, monitor hematologic malignancies, and assess minimal residual disease. Its indications span diagnostic immunophenotyping, transplantation monitoring, evaluation of immune deficiencies, and quality control in cellular therapies, making it an essential tool for precise, high-throughput cellular analysis across modern laboratory medicine.

In summary: Flow cytometry uses fluorescent markers to sort cell populations to find markers for cancer and other conditions.

Flow cytometry: Shining a spotlight on blood cell conditions

What is Flow Cytometry used for?

It will usually be ordered when a physician suspects one of the following conditions:

Leukemias, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL).

Lymphomas, including non-Hodgkin lymphomas, Hodgkin-related immunophenotyping, and assessment of abnormal lymphoid populations.

Plasma cell disorders, such as multiple myeloma and monoclonal gammopathies.

Other uses: Immunodeficiency states, Autoimmune disorders, Transplant monitoring, Minimal residual disease (MRD) assessment, Paroxysmal nocturnal hemoglobinuria (PNH), Allergy and hypersensitivity workups, Functional immune testing

It is able to detect these conditions because certain types of cells produce specific antigens on their surface. All of these conditions are defined by an overproduction of certain kinds of cells presenting their unique types of antigens. So... just attach a readable (fluorescent) marker and run it through a sorting machine.

And that produces a representative graph:

Flow Cytometry for B-Cell Non-Hodgkin and Hodgkin Lymphomas, Gajzer, David C., Fromm, Jonathan R. 2025.

Someone who works in a lab might find this familiar. It looks a lot like a simple histogram from a CBC (complete blood count) differential. And essentially, it's the same process. Whereas Flow Cytometry can sort by antigens, the basic CBC sorts only by size and side-scatter.

Antigens useful for Diagnosis

Condition	Common Flow Cytometry Antigens
Chronic Lymphocytic Leukemia (CLL)	CD19, CD20 (dim), CD5, CD23, CD200, CD79b (dim), CD43, CD45 (dim), FMC7 (negative/weak), Kappa/Lambda light chains
Acute Lymphoblastic Leukemia (ALL) – B-ALL	CD19, CD10, CD20 (variable), CD22, CD79a, TdT, CD34, HLA-DR, CD45 (dim), CD38, CD58
Acute Lymphoblastic Leukemia (ALL) – T-ALL	CD2, CD3 (cytoplasmic or surface), CD5, CD7, CD4/CD8, TdT, CD34 (variable), CD1a (early T-cell), CD45 (dim)
Chronic Myeloid Leukemia (CML) – Blast Phase	CD34, CD117, CD13, CD33, HLA-DR, MPO; For lymphoid blast phase: B-markers (CD19, CD10, CD22, CD79a, TdT) or T-markers (CD3, CD7, CD2)
Acute Myeloid Leukemia (AML)	CD34, CD117, CD13, CD33, HLA-DR, MPO, CD15, CD64, CD14, CD11b, CD36, CD42b, CD61; Aberrant markers may include CD7 or CD19
Multiple Myeloma	CD38 (bright), CD138, CD56, CD45 (dim/negative), CD19 (dim/negative), CD117, CD200, Cytoplasmic light chains (kappa/lambda)
Lymphoma – B-Cell	CD19, CD20, CD22, CD79b, CD5, CD10, CD23, CD43, Kappa/Lambda light chains, CD11c, CD25, CD103 (subtype-specific)
Lymphoma – T-Cell	CD2, CD3, CD4, CD5, CD7, CD8, TCR α/β or γ/δ, plus assessment for loss of normal T-cell antigens

When does a CBC indicate Flow Cytometry?

A complete blood count (CBC) can provide early clues that flow cytometry is warranted, particularly when abnormalities suggest an underlying hematologic malignancy or immune disorder. Significant lymphocytosis—especially when persistent or composed of small, mature-appearing lymphocytes—often raises suspicion for chronic lymphoproliferative processes such as CLL and prompts further immunophenotyping. Conversely, the presence of circulating blasts, severe cytopenias, or unexplained shifts in white blood cell differentials may indicate an acute leukemia or bone marrow dysfunction that requires rapid characterization by flow cytometry. Additional red flags include atypical lymphocyte morphology, unexplained monocytosis, or abnormal platelet patterns that do not fit common reactive conditions. In these situations, flow cytometry offers detailed lineage analysis, assessment of clonality, and identification of abnormal cell populations, making it an essential next step when a CBC detects patterns that could reflect a serious underlying hematologic disease.

Reactive lymphocyte or cancer cell? Run flow cytometry to find out.

Monitoring HIV patients with Flow Cytometry (CD4/CD8)

Flow cytometry plays a central role in monitoring patients with HIV by providing precise, quantitative assessment of immune system status, particularly through measurement of CD4⁺ T-lymphocyte counts. Because HIV targets and depletes these cells over time, their enumeration is critical for determining the stage of disease, evaluating the risk of opportunistic infections, and guiding the initiation or modification of antiretroviral therapy. In addition to CD4 counts, flow cytometry can also measure CD8⁺ T-cells and calculate CD4/CD8 ratios, offering a broader picture of immune recovery or decline. This technology allows clinicians to track subtle changes in immune function, assess response to treatment, and identify treatment failure or nonadherence earlier than clinical symptoms alone. As a high-precision, rapid, and reproducible method, flow cytometry remains an indispensable tool in the long-term management of individuals living with HIV.

The FUTURE: Advances in Flow Cytometry

Advanced techniques in flow cytometry have expanded the field far beyond simple immunophenotyping, allowing for increasingly detailed cellular analysis and functional assessment. One major development is multicolor flow cytometry, which enables simultaneous detection of dozens of antigens on or within a single cell. Improvements in fluorochrome chemistry, laser configurations, and spectral separation now allow researchers and clinicians to evaluate complex cellular subsets with unprecedented precision. This expansion is especially valuable in immunology and oncology, where subtle differences in surface markers can distinguish between malignant and reactive populations or define rare immune subsets involved in disease processes.

Another transformative advancement is spectral flow cytometry, which captures the full emission spectrum of each fluorochrome rather than measuring light at fixed wavelengths. By analyzing complete spectral signatures, this technology reduces compensation challenges and permits the use of fluorochromes with overlapping emission patterns. As a result, spectral flow cytometry significantly increases panel complexity and improves the ability to resolve dim or closely related markers. It also opens the door to flexible dye selection, making it easier to design high-parameter panels tailored to specific diagnostic or research needs.

Flow cytometry has also evolved to incorporate functional assays that measure cellular behavior rather than simply phenotype. These include proliferation assays, calcium flux studies, cytokine detection through intracellular staining, and oxidative burst testing. Such assays help characterize immune responses, evaluate cellular activation, and diagnose disorders like chronic granulomatous disease. Additionally, cell sorting using fluorescence-activated cell sorting (FACS) allows for the physical separation of highly specific subsets for downstream applications, including molecular profiling, culture, or therapeutic development.

More recent innovations, such as mass cytometry (CyTOF) and imaging flow cytometry, have pushed the boundaries even further. Mass cytometry replaces fluorophores with metal isotopes detected by time-of-flight mass spectrometry, enabling panels with more than 40 measurable parameters without spectral overlap. Imaging flow cytometry combines traditional flow techniques with microscopy, producing detailed cellular images that distinguish processes like apoptosis, nuclear translocation, or morphological abnormalities. Together, these advances have transformed flow cytometry into a highly sophisticated platform that not only identifies cell populations but also reveals their function, structure, and molecular complexity with remarkable depth.