Flow cytometry (CD4 count and immunophenotyping)

Principle

• Cells in suspension pass single file through a laser beam by hydrodynamic focusing.
• Forward scatter (FSC) relates to cell size; side scatter (SSC) relates to internal complexity and granularity.
• Cells are stained with fluorochrome-labelled monoclonal antibodies (e.g. FITC, PE, PerCP, APC) against CD antigens; emitted light is split by filters and measured by detectors.
• Gating selects populations: e.g. CD45 versus SSC to identify lymphocytes, then CD3+CD4+ helper T cells.

Specimen

• EDTA whole blood for CD4 counts, kept at room temperature (not refrigerated) and processed within the time the method allows (usually 24–48 hours).
• Bone marrow, blood, fluids or tissue suspensions for leukaemia/lymphoma immunophenotyping.

Procedure

• Stain blood with antibody cocktail, lyse red cells (lyse–no-wash for CD4), acquire on the cytometer and analyse.
• Absolute count: single-platform method using counting beads (cells/µL = cell events ÷ bead events × beads per tube ÷ sample volume) is preferred; dual-platform method multiplies CD4% by the haematology analyser lymphocyte count.

Results

• Typical adult CD4 count about 500–1500 cells/µL; local reference ranges apply.
• HIV: CD4 below 200 cells/µL (or below 14%) defines AIDS and increases opportunistic infection risk; percentage is preferred in young children.
• Immunophenotyping: blasts CD34, CD117, HLA-DR; myeloid CD13, CD33, MPO; B-lineage CD19, CD10, CD20, CD22, cytoplasmic CD79a; T-lineage cytoplasmic CD3, CD7, CD5.
• CLL: CD19+, CD5+, CD23+, weak CD20 and weak surface immunoglobulin.
• Kappa/lambda light chain restriction indicates a clonal B-cell population.

Quality control and pitfalls

• Daily instrument setup with fluorescent beads; correct compensation for spectral overlap; use appropriate negative (isotype or FMO) controls.
• Run stabilised whole-blood process controls and participate in EQA.
• Poor gating, clumped cells, old or refrigerated samples and incorrect pipetting of bead tubes cause errors.

Clinical use

• HIV monitoring, diagnosis and classification of leukaemias and lymphomas, minimal residual disease, PNH (CD55, CD59, FLAER), primary immunodeficiency evaluation, stem cell (CD34) enumeration.