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# Flow cytometry and the CD4 count

- Topic

Flow cytometry counts and sorts cells one by one with lasers and fluorescent antibodies. It is how the CD4 count in HIV is measured, for example.

Updated 2026-11-01

## Key points

- Forward scatter = size; side scatter = granularity.
- CD4 below 200 per mm³ = AIDS.
- Absent CD19: Bruton agammaglobulinemia.
- PNH: missing CD55 and CD59.
- CD4 and viral load are read together in HIV.

## What it is

In flow cytometry, cells pass in single file past a laser; the light they scatter reports their size and internal complexity, and monoclonal antibodies tagged with fluorochromes reveal which CD markers they carry. Gating separates populations such as CD4 T cells, CD8 T cells, B cells and NK cells. It is central to following HIV, classifying leukemias and lymphomas and studying immunodeficiencies.

## Step by step

- Blood is stained with fluorescent antibodies against CD45, CD3, CD4, CD8 and others.
- Cells pass one at a time in front of the laser.
- Forward scatter estimates size; side scatter, granularity.
- Detectors record the fluorescence of each marker.
- Lymphocytes are gated (bright CD45, low granularity).
- Subsets are counted and, with reference beads, reported as an absolute count per microlitre.

## Markers

- CD45 (all leukocytes)
- CD3 (T cells)
- CD4 (helper T)
- CD8 (cytotoxic T)
- CD19 and CD20 (B cells)
- CD16 and CD56 (NK)
- CD34 (progenitors)

## Clinical notes

- Immunophenotyping of leukemias and lymphomas: lineage, maturation and clonality.
- Immunodeficiencies: absent CD19 B cells in X-linked agammaglobulinemia; very low T cells in severe combined immunodeficiency.
- Paroxysmal nocturnal hemoglobinuria: loss of CD55 and CD59 on red cells and granulocytes.
- CD34 cell counts for hematopoietic progenitor transplantation.

## In the lab

- Normal adult CD4 count: about 500 to 1200 cells per mm³.
- In HIV, fewer than 200 CD4 cells per mm³ defines AIDS; in young children the CD4 percentage is used.
- CD4 measures immune damage; viral load measures viral replication. They are read together.
- Internal check: T, B and NK cells should add up to close to 100 % of lymphocytes.

## Memory aid

Forward tells how big, sideways tells what is inside: forward light gives size, side light shows the cell's contents.

## Test yourself

Think of the answer, then tap the question to see it.

## What do forward and side scatter tell you in flow cytometry?

Forward scatter relates to cell size and side scatter to granularity or internal complexity.

## Which CD4 count defines AIDS in a person with HIV?

Fewer than 200 cells per mm³ (or an AIDS-defining illness).

## Which cytometry finding suggests X-linked agammaglobulinemia?

Absent or near-absent B cells (CD19 or CD20) in peripheral blood.

## Which markers are lost in paroxysmal nocturnal hemoglobinuria?

CD55 and CD59, GPI-anchored proteins that protect cells from complement.

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## Related

- [ELISA, western blot and rapid tests](https://durgical.com/en/immunology/elisa-western-blot-and-rapid-tests/)
- [Live vaccines: who should not receive them](https://durgical.com/en/immunology/live-vaccines-who-should-not-receive-them/)
- [CD markers: the table to know](https://durgical.com/en/immunology/cd-markers-the-table-to-know/)
- [CD4⁺ helper T cell](https://durgical.com/en/immunology/cd4-helper-t-cell/)
- [B-cell (antibody) deficiencies: XLA, CVID, IgA deficiency, hyper-IgM](https://durgical.com/en/immunology/b-cell-deficiencies-xla-cvid-iga-deficiency-hyper-igm/)
- [Secondary immunodeficiency: HIV, malnutrition, drugs, asplenia](https://durgical.com/en/immunology/secondary-immunodeficiency-hiv-malnutrition-drugs-asplenia/)

## Related conditions

- [HIV infection and AIDS](https://durgical.com/en/conditions/hiv-infection-and-aids/)

## Microorganisms

- [Human immunodeficiency virus](https://durgical.com/en/microbiology/human-immunodeficiency-virus/)

[← All of immunology](https://durgical.com/en/immunology/)

Educational content on the immune system; not a treatment or individual vaccination guide and no substitute for a health professional's advice. No doses are given.

Sources: [Immunophenotyping of Peripheral Blood and Bone Marrow Cells by Flow Cytometry](https://pmc.ncbi.nlm.nih.gov/articles/PMC5954430/) (EJIFCC (PubMed Central), 2006); [CD4 Lymphocyte Count](https://medlineplus.gov/lab-tests/cd4-lymphocyte-count/) (MedlinePlus (U.S. National Library of Medicine), 2024).

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Representative model, for educational purposes only.

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