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Diagnosis and tests

Flow cytometry (immunophenotyping)

A microscope shows what a cell looks like, but it cannot always tell two similar cells apart. Flow cytometry “reads” special proteins on the surface and inside the cell and identifies precisely what it is. This topic explains how the test works, how it defines the type of leukemia and what entries such as “CD19+” and “CD34+” on your report mean.

Last updated: ~6 min read
Translucent cells flowing in single file along a thin glowing channel on a dark navy background; a fine blue and green beam of light crosses the channel and lights up one of the cells.
Figure 1.Concept: cells pass one by one in a thin stream through a laser beam.

01Short answer

Flow cytometry is a laboratory method in which cells from blood, bone marrow or another fluid are labelled with antibodies carrying fluorescent dyes and passed one at a time in a stream of fluid through a beam of light (a laser). The instrument records each cell’s size, its internal structure and which markers (antigens) it carries. Most markers are named with a CD number. From this information the laboratory works out which line the leukemia belongs to — myeloid, B-lymphoid or T-lymphoid — how mature it is and how it differs from normal cells; this is called immunophenotyping. Flow cytometry is a key test for telling AML from ALL, determining whether ALL is of B or T type, and confirming CLL and hairy cell leukemia. During treatment it is used to look for leftover leukemia cells that cannot be seen with an ordinary microscope (MRD). Some markers are also targets for immunotherapy. Discuss your results with your hematologist.

02Simple explanation

Every cell carries small protein “flags” on its surface. Different types of cells carry different sets of flags — a kind of “uniform”. Flow cytometry finds and counts these flags to work out who the cell is.

In the laboratory, special antibodies are added to the sample. Each antibody sticks to only one flag and glows in its own colour. The cells then pass one by one in a thin stream in front of a laser beam, and the instrument records which colours light up.

On a report, “CD19+” means the marker is present on the cell, and “CD19−” means it is absent. Sometimes “dim” or “bright” is added — this shows how much of the marker there is.

Flow cytometry is very fast: it measures thousands of cells per second and is often one of the first specialised results to come back. It helps the doctor understand the type of leukemia quickly and plan treatment in good time.

After treatment, the same method can be used to look for a very small number of remaining leukemia cells. This is called MRD, and it shows how deeply treatment has worked.

03Scientific explanation

How the method works: according to the NCI, in flow cytometry the cells in a sample are stained with a fluorescent dye, placed in a fluid and passed one at a time through a beam of light; the test measures the number of cells, the percentage of live cells, their size and shape and the presence of tumour (or other) markers on the surface. Modern instruments measure 8 or more colours at once. The European EuroFlow consortium has developed standardised antibody panels so that results can be compared between laboratories. The ACS notes that the same antibody principle is applied to biopsy tissue under the microscope — this is called immunohistochemistry.

Lineage markers: immature blasts often carry CD34. The B line is characterised by strong CD19 together with CD79a, cytoplasmic CD22 or CD10; the T line by strong cytoplasmic or surface CD3; and the myeloid line by myeloperoxidase (MPO) and markers such as CD13, CD33 and CD117 (NCI). If markers of more than one line are strongly expressed in the same leukemia, it may be a mixed phenotype acute leukemia (MPAL) — a rare situation that needs a special approach.

In chronic leukemias: according to the NCI, in CLL the blood usually contains 5,000/mm³ or more lymphocytes with a characteristic immunophenotype — B cells positive for CD5 and CD23, with low levels of surface immunoglobulin and a single light chain (kappa or lambda). That “single light chain” shows the cells come from one clone — one original cell. If there are fewer than 5 ×10⁹/L B cells and no other signs, the condition is called monoclonal B-cell lymphocytosis (MBL). In hairy cell leukemia, CD11c, CD25, CD103 and CD123 are typical, and the diagnosis can often be made with flow cytometry of the blood.

MRD and treatment targets: according to the 2021 ELN consensus, flow-cytometry MRD in AML is measured by looking for a “leukemia-associated immunophenotype” that differs from normal cells, and a level of 0.1% or more is considered MRD-positive. In ALL, flow cytometry routinely detects 1 leukemia cell in 10,000. Some markers are drug targets (CD19, CD22, CD33, CD20), so flow results can also play a part in choosing treatment.

04What happens in the body?

Where does the sample come from?

Bone marrow aspirate or blood is used most often; sometimes spinal fluid or other tissue is tested. In CLL and hairy cell leukemia the diagnosis can often be made from a blood sample alone. The method needs living cells, so the sample has to reach the laboratory quickly.

Lasers and coloured lights

As antibody-labelled cells pass through the laser beam in a thin stream, each one gives off its own “colour signature”. The instrument collects these signals and a computer groups similar cells together. A specialist analyses these groups to find cells that differ from what would be expected in a normal sample.

A large translucent round cell on a dark navy background; small Y-shaped antibodies in different colours — blue, green, orange — glow softly on its surface.
Figure 2.Coloured antibodies attach to markers on the cell surface.

Sorting cells into “families”

In the end the cells appear on screen as separate “clouds”: normal lymphocytes, monocytes, neutrophils and an unusual group of blasts. Leukemia cells often stand out from healthy cells because of their particular combination of markers. This forms the basis both for the diagnosis and for later MRD tracking.

Small glowing dots on a dark navy background gathered into several separate “clouds”; one of them glows differently, in violet, with no axes or labels.
Figure 3.The instrument groups similar cells and reveals an unusual group.

When will the result be ready?

Flow cytometry often gives a result sooner than other specialised tests — sometimes within a day or two. This matters especially in acute leukemia, when the treatment plan needs to be made quickly. Genetic tests usually take longer.

05How does flow cytometry work?

Four steps — from the sample to the leukemia’s “ID card”.

Sample — Marrow aspirate, blood or another fluid.1SampleMarrow aspirate, blood or another fluid.
Antibodies — Coloured antibodies stick to the markers.2AntibodiesColoured antibodies stick to the markers.
Laser — Cells pass one by one through a beam of light.3LaserCells pass one by one through a beam of light.
Analysis — Cell groups and the immunophenotype are defined.4AnalysisCell groups and the immunophenotype are defined.
LineageMyeloid, B or T.
TargetsMarkers such as CD19, CD20, CD22, CD33.
MRDMeasuring invisible leftover cells.

06Typical immunophenotypes (simplified)

LeukemiaCommonly seen markersNote
AMLCD34, CD117, CD13, CD33, MPOMyeloid line; varies by subtype
B-ALLCD19, CD79a, CD22, CD10, TdT, CD34B-cell precursors
T-ALLCytoplasmic CD3, CD7, CD2, CD5, TdTT-cell precursors
CLLCD19, CD5, CD23; dim CD20 and surface immunoglobulinSingle light chain (kappa or lambda) — a sign of a clone
Hairy cell leukemiaCD11c, CD25, CD103, CD123Diagnosis is often possible from blood
MPALMarkers of more than one lineRare; special treatment approach

* This table shows the general picture; the combination of markers can differ in each leukemia, and the diagnosis is made by weighing all the tests together. Discuss your results with your hematologist.

07Key terms

Flow cytometry
A method that measures cells by passing them in a fluid stream through a laser beam.
Immunophenotyping
Identifying cells by their markers using antibodies.
CD marker
A numbered protein on the cell surface (“cluster of differentiation”).
Antibody
A protein molecule that attaches precisely to a specific protein.
Fluorescent dye
A substance that glows in its own colour under a beam of light.
Lineage
The family a cell belongs to: myeloid, B or T.
Clone
A group of identical cells descended from a single cell.
Light chain
The kappa or lambda part of an immunoglobulin; points to clonality.
MPAL
Mixed phenotype acute leukemia — markers of more than one line.
Immunohistochemistry
Applying antibodies to a tissue section under the microscope.

08Key points

  • Flow cytometry measures the markers on cells.
  • It shows whether a leukemia belongs to the myeloid, B or T line.
  • It is a key test for telling AML from ALL.
  • In CLL and hairy cell leukemia, diagnosis from blood is often possible.
  • “+” means the marker is present, “−” means it is absent.
  • Results are often ready quickly.
  • After treatment it is used for MRD tracking.
  • Some markers are targets for immunotherapy.

09Frequently asked questions

Do I need a separate procedure for flow cytometry?

Usually not. The test is run on the sample taken for blood tests or a bone marrow aspiration. Because the laboratory works with living cells, the sample simply needs to be delivered quickly.

My report lists lots of CD numbers. Do I need to know them all?

No. That list is for specialists. What matters to you is the bottom line: which type of leukemia was found and what it means for treatment. Ask your hematologist to explain it in plain words.

Does flow cytometry replace genetic testing?

No. Flow cytometry shows the cell’s proteins, while genetic tests show changes in its DNA. They complement each other: one refines the type, the others refine the risk group and targeted drug options.

Does “CD20+” affect treatment?

It can. In some leukemias and lymphomas, CD20 is the target of anti-CD20 antibodies. But the choice of treatment depends on many factors — only your hematologist can make that decision.

When is flow cytometry done for MRD?

Usually at particular stages of treatment — for example, at the end of induction — on a bone marrow sample. The schedule depends on the type of leukemia and the protocol.

Can the markers on leukemia cells change?

Yes, sometimes — especially at relapse or after some immunotherapies. That is why, in repeat tests, the laboratory also takes the original immunophenotype into account.

10Questions for your doctor

Take these questions to your appointment. The ones you save are collected on the “My questions” page and stay on this device only.

  1. What type of leukemia did flow cytometry show?

  2. Which immunophenotype markers were found?

  3. Will this result be used for MRD monitoring?

  4. When will we repeat flow cytometry?

My questions

12Sources

  1. National Cancer Institute (NCI). NCI Dictionary of Cancer Terms: flow cytometry
  2. National Cancer Institute (NCI). NCI Dictionary of Cancer Terms: immunophenotyping
  3. National Cancer Institute (NCI). Adult Acute Myeloid Leukemia Treatment (PDQ®) — Patient Version
  4. National Cancer Institute (NCI). Chronic Lymphocytic Leukemia Treatment (PDQ®) — Health Professional Version
  5. National Cancer Institute (NCI). Childhood Acute Lymphoblastic Leukemia Treatment (PDQ®) — Health Professional Version
  6. National Cancer Institute (NCI). Hairy Cell Leukemia Treatment (PDQ®) — Health Professional Version
  7. American Cancer Society. Tests for Acute Myeloid Leukemia (AML)
  8. American Cancer Society. Acute Lymphocytic Leukemia Subtypes and Prognostic Factors
  9. American Cancer Society. Tests for CLL
  10. Testing.com. Immunophenotyping by Flow Cytometry Explained
  11. Blood (American Society of Hematology), 2021. 2021 Update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party
  12. Leukemia, 2012. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes
  13. Leukemia, 2022. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms
  14. Blood (American Society of Hematology), 2018. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL
Medical disclaimerThis information is for educational purposes only and does not replace medical advice.
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