Flow cytometry is one of the five pillars used to diagnose haematological malignancy. It works by shooting a laser at individual cells in suspension, measuring their physical properties (size, granularity) and — most importantly — which surface and intracellular antigens they express (immunophenotyping). This tells you what lineage the abnormal cells belong to (myeloid vs. B-lymphoid vs. T-lymphoid vs. NK), whether they are clonal (malignant) or reactive, and helps sub-classify the disease. Without flow cytometry, you cannot accurately classify most leukaemias and many lymphomas. ^[1]

1. Principle of diagnosis of haematological malignancy 2. Principle of flow cytometry 3. Application of flow cytometry in diagnosis and management of haematological malignancy

• Clinical practice: When a CBC + blood film shows abnormal white cells or blasts, flow cytometry is the immediate next step to determine lineage — it directly changes treatment (e.g., ALL vs AML regimens are completely different).
• Exam relevance: HKUMed in-house papers directly test CD marker interpretation, the principle of flow cytometry, and the diagnostic workup sequence for leukaemia. The 2024 Fourth Summative MCQ tested CD markers specifically (Questions 5–8). ^[7]

• Morphology first: You see blasts on the film → you suspect acute leukaemia. But blasts can look alike under the microscope — you cannot always distinguish AML from ALL by morphology alone.
• Flow cytometry second: It answers the critical question — is this myeloid or lymphoid? Is it B or T? Is it mature or immature? This determines treatment.
• Cytogenetics and molecular: Refine diagnosis, assign prognosis, guide targeted therapy, and later monitor for residual disease.

Peripheral blood smear findings:

• Genuine thrombocytopenia with occasional giant platelets
• Abnormal lymphoid cells: small in size, high N/C ratio, roundish nuclei, clumped chromatin, scanty basophilic agranular cytoplasm, smooth cytoplasmic border
• Diagnosis: Lymphoproliferative disease → Suggest flow cytometry study

Why this case is illustrative: The morphology tells you "these look like abnormal lymphocytes" but cannot definitively classify the disease. Flow cytometry is needed to confirm lineage, assess clonality (kappa/lambda light chain restriction), and determine exact phenotype.

Flow cytometry is a technique where antibody-bound cells are analysed individually in suspension, allowing large numbers of cells to be analysed very quickly, and enabling detection of co-expression of more than one antigen on one cell. ^[1]

This is the single most important conceptual sentence for the exam: co-expression is key. Unlike immunohistochemistry (IHC), which shows one marker per slide, flow cytometry can show 6–10+ markers simultaneously on each individual cell, creating a "fingerprint" of the cell.

Two fundamental physical measurements ^[1]:

Why this matters: Before you even look at fluorescent antibodies, the forward scatter vs. side scatter plot separates the major WBC populations:

This is shown on the lecture's lysed whole blood scatter plot — three distinct clusters appear. ^[1]

Clinical point: If blasts are present, they typically appear as a distinct cluster (often low side scatter, variable forward scatter) that doesn't fit into the normal lymphocyte/monocyte/granulocyte gates.

• When a cell passes the laser, it generates a voltage pulse ^[1]
• Low signal height = dim staining (few binding sites) → High signal height = bright staining (many binding sites)
• The emitted fluorescence intensity is proportional to the number of binding sites on the cell for that particular antibody ^[1]
• Fluorescence data is typically displayed on a log scale (because expression levels vary over several orders of magnitude)

• Antibodies are conjugated to fluorescent dyes (fluorochromes), e.g., FITC (fluorescein isothiocyanate), PE (phycoerythrin), Cy5, Cy7 ^[1]
• Each fluorochrome emits at a different wavelength when excited by the laser
• Dichroic mirrors and optical filters separate the emitted light by wavelength, directing each color to a different detector ^[1]
• This allows simultaneous detection of multiple markers on a single cell

Gating strategy from the lecture ^[1]:

1. First gate: On the FSC/SSC plot — select the region containing the cells of interest (exclude debris, dead cells)
2. CD45 gating: CD45 (leukocyte common antigen) vs SSC — different WBC populations have characteristic CD45 intensity and SSC patterns. Blasts have WEAK CD45 expression ^[3]
3. Lineage-specific gates: e.g., gate on CD19+ cells (B cells) to further assess kappa/lambda light chain expression

Exam trap: CD5 is a T-cell marker but is aberrantly co-expressed on B cells in CLL/SLL and mantle cell lymphoma. Seeing CD5+/CD19+ on flow = think CLL. ^[3]

Lineage-specific vs lineage-associated markers ^[3]:

• Lineage-specific: MPO (myeloid), CD3 (T-cell), CD79a (B-cell) — these define lineage
• Lineage-associated: CD13, CD33 (myeloid), CD19, CD20 (B-cell) — these support lineage but may be aberrantly expressed

This is one of the most powerful applications of flow cytometry in B-cell lymphoproliferative disorders. ^[1][3]

Why it works:

• Normal B cells express a mixture of kappa and lambda light chains on their surface immunoglobulin (roughly κ:λ ≈ 2:1)
• A clonal (malignant) B-cell population derives from one cell → all express the same light chain (either all kappa OR all lambda)
• Flow cytometry detects this light chain restriction by gating on CD19+ B cells and assessing κ vs λ

From the lecture's flow cytometry plots ^[1]:

• The CD19+ gated population shows kappa-FITC vs lambda-PE
• If > 90% express one light chain → monoclonal = malignant
• In the case shown: 94.1% lambda+, 4.8% kappa+ → lambda light chain restriction → monoclonal B-cell proliferation ^[1]

The lecture walks through a case of suspected lymphoproliferative disease with detailed flow cytometry interpretation ^[1]:

Flow cytometry is not just diagnostic — it is used for:

1. Sub-classification: e.g., distinguishing B-ALL from T-ALL
2. Prognostication: e.g., ZAP70 in CLL (ZAP70+ = worse prognosis) ^[1], CD34 expression in AML
3. Monitoring minimal/measurable residual disease (MRD): After treatment, flow can detect 1 abnormal cell in 10,000 → predicts relapse risk
4. Guiding targeted therapy: e.g., CD20+ → rituximab eligible ^[7]

From GC 060 (High White Cell Count) — highest yield exam source ^[6]:

1. Morphology (APL, AML, ALL) on PB and BM
2. Cytochemistry (MPO and SBB) — AML vs ALL
3. Flow cytometry — for sub-classification lineage specification
4. Cytogenetics (and FISH) — diagnostic and prognostic information
5. Molecular genetics — diagnostic and prognostic information

From the 2024 Fourth Summative MCQ ^[7]:

"What is the role of flow cytometry in haematological malignancy?" → Model answer: "Flow cytometry performs immunophenotyping on fresh cell suspensions to determine the lineage (myeloid, B-cell, T-cell, NK-cell) and maturity of abnormal cells, detect clonality through surface immunoglobulin light chain restriction, sub-classify disease, guide treatment selection, and monitor measurable residual disease."

"Describe the principle of flow cytometry." → Model answer: "Cells in single-cell suspension pass through a laser beam via hydrodynamic focusing. Forward scatter measures cell size; side scatter measures granularity. Fluorochrome-conjugated antibodies bound to cell surface or intracellular antigens emit fluorescence proportional to the number of binding sites, detected by optical filters and detectors, allowing simultaneous multi-parameter analysis of individual cells."