Flow Cytometry

Related Subjects: | Oncological Emergencies | Acute Myeloid Leukaemia (AML) | Acute Lymphoblastic Leukaemia (ALL) | Chronic Lymphocytic Leukaemia (CLL) | Chronic Myeloid Leukaemia (CML) | Immune Thrombocytopenic Purpura (ITP) | Multiple Myeloma | Graft-versus-Host Disease (GVHD) | Cytomegalovirus (CMV) Infections |Flow Cytometry |Haematology Laboratory Values |Indications for Irradiated Blood Products

Flow cytometry is a rapid, cell-by-cell technique that measures physical properties (size and internal complexity) and protein expression (immunophenotype) using fluorescently-labelled antibodies. It is especially powerful in haematology and immunology because it can identify and quantify mixed cell populations (e.g., B cells vs T cells, blasts vs mature cells) within minutes, and detect very small abnormal populations (e.g., minimal residual disease) when panels are designed appropriately.

🔬 What flow cytometry measures

• Forward scatter (FSC): correlates with cell size (e.g., lymphocytes smaller than monocytes).
• Side scatter (SSC): correlates with granularity/internal complexity (e.g., granulocytes have higher SSC due to granules).
• Fluorescence intensity: amount of a marker on/in the cell (e.g., CD3, CD19, CD34, CD45, light chains).

⚙️ Core process (step-by-step)

• Sample preparation: blood, bone marrow, lymph node aspirate, CSF, etc. Red cells may be lysed (depending on assay).
• Antibody staining: cells incubated with monoclonal antibodies targeting surface and/or intracellular proteins.
  • Antibodies are conjugated to distinct fluorochromes (multi-colour panels allow many markers at once).
  • For intracellular targets (e.g., TdT, cytoplasmic CD3), cells must be fixed and permeabilised.
• Hydrodynamic focusing: cells are aligned so they pass single-file through the interrogation point.
• Laser interrogation: each cell crosses one or more lasers; detectors capture:
  • scatter (FSC/SSC)
  • emitted fluorescence at different wavelengths
• Data processing: software applies gating (selecting cell populations) and compensation (correcting spectral overlap between fluorochromes).

📊 How results are displayed

• Histogram (one parameter): frequency vs fluorescence intensity of a single marker (e.g., CD10 expression).
• Dot/scatter plot (two parameters): marker A vs marker B (e.g., CD3 vs CD8).
• Density/contour plots: useful when many events overlap (common in marrow samples).

🧭 Understanding quadrants (CD3 vs CD8 example)

In a two-colour plot, axes represent fluorescence intensity for each marker. By setting thresholds, the plot is divided into quadrants to describe positivity/negativity.

• Lower left (CD3− / CD8−): double-negative cells (may include B cells, NK cells, or unstained debris depending on gating).
• Lower right (CD3− / CD8+): CD8-positive but CD3-negative population (often small; can include some NK subsets depending on markers used).
• Upper left (CD3+ / CD8−): T cells that are not CD8 (commonly CD4+ T cells, but confirm with CD4 marker).
• Upper right (CD3+ / CD8+): CD8 T cells (cytotoxic T cells).

🧩 The critical concept: gating

• Interpretation depends on first selecting the relevant population (e.g., CD45 vs SSC to separate lymphocytes/monocytes/granulocytes/blasts).
• Then refine with lineage markers (e.g., CD3 for T cells, CD19/CD20 for B cells, CD16/CD56 for NK cells).
• In malignancy workups, additional markers define maturation and aberrancy (e.g., CD34, CD117, TdT; light chain restriction; abnormal co-expression).

🩺 Clinical uses (high-yield)

Flow cytometry is used to classify cell populations, detect abnormal clones, and quantify immune subsets. Panels are tailored to the clinical question.

• Haematological malignancy diagnosis and classification:
  • Acute leukaemias (blast identification; lineage assignment: myeloid vs B-ALL vs T-ALL).
  • CLL, lymphomas in blood/marrow, plasma cell disorders (including light chain restriction).
  • Minimal residual disease (MRD) monitoring after treatment (high sensitivity when validated).
• Paroxysmal nocturnal haemoglobinuria (PNH): detects deficiency of GPI-anchored proteins (e.g., CD55/CD59 or FLAER-based assays) on granulocytes/monocytes.
• Immunodeficiency assessment: lymphocyte subset enumeration (CD3, CD4, CD8, CD19, CD16/56), naïve/memory phenotypes, and activation markers when needed.
• HIV monitoring: CD4 count and CD4:CD8 ratio as markers of immune status.
• Fetomaternal haemorrhage: classically by flow cytometry (anti-HbF) to quantify fetal RBCs and guide anti-D dosing (local lab practice varies).

⚠️ Common pitfalls (worth knowing)

• Pre-analytical issues: delayed processing, clotted samples, haemolysis, low cell viability can distort results.
• Non-specific binding: especially with Fc receptors (monocytes) — mitigated by controls/blocking.
• Spectral overlap: fluorochromes spill into other detectors → requires correct compensation.
• Interpretation is clinical-context dependent: “positivity” thresholds and abnormal patterns depend on gating strategy, antibody panel, and reference ranges.