Cellular Immunology & Diagnostic Flow Cytometry

Blood Cell Types

• Lymphocytes: T, B, NK
• Granulocytes: neutrophils (most abundant), eosinophils, basophils
• Monocytes / macrophages

Flow Cytometry Fundamentals

• Single-cell stream intersects laser → light scatter + fluorescence
• Forward scatter ↔ cell size; side scatter ↔ granularity
• Fluorochrome-tagged monoclonal antibodies bind cell-surface markers; laser excitation → emitted light measured
• Multiparameter detection if emission spectra do not overlap

Core Lymphocyte Markers (TBNK Panel)

• \text{CD45}: all leukocytes
• \text{CD3}: all T cells
• \text{CD4}: helper T
• \text{CD8}: cytotoxic T
• \text{CD19}: B cells
• \text{CD16/56}: NK cells

Basic TBNK Assay (LAS1)

1. 20 µL antibody mix + 50 µL whole blood (TruCount tube)
2. Incubate 15 min (RT)
3. Lyse RBC (450 µL), 15 min
4. Acquire on analyser; beads provide absolute counts

Result Interpretation

• Report % and absolute counts (cells \times 10^9/\text{L})
• Low/absent populations or abnormal CD4:CD8 ratio guide diagnoses

Quality & Troubleshooting

• Always confirm unexpected "no-cell" plots: sample volume, reagent addition, clogs, laser/detector faults
• Perform QC, review instrument messages before authorising

Primary Immunodeficiencies Detected by Phenotyping

• Severe Combined Immunodeficiency (SCID): absent T, B, NK; multiple genetic subtypes (X-linked \gamma_c, ADA, PNP, RAG, MHC II)
• DiGeorge (22q11 deletion): thymic aplasia → low T cells; cardiac/facial defects, cleft palate, hypocalcaemia
• Hyper-IgM Syndrome: defective class switching (missing \text{CD40} or \text{CD40L}); high IgM, low IgG/A/E
• Common Variable Immunodeficiency (CVID): variable B-cell/Ig defects; poor vaccine response

Acquired / Therapy-Related Immunodeficiency

• HIV: infects \text{CD4}^+ T cells → low CD4, compensatory high CD8
• Rituximab (anti-CD20): B-cell depletion → very low \text{CD19} counts

Extended Phenotyping Panels

• TCR \alpha/\beta vs \gamma/\delta if CD3 ≠ CD4 + CD8
• \text{CD45RA} (naïve) vs \text{CD45RO} (memory)
• \text{HLA-DR} expression: activation / bare lymphocyte syndrome

Neutrophil Respiratory Burst Assay (DHR Test)

• Principle: PMA stimulation → \text{NADPH oxidase} generates O2^- and H2O_2; oxidises dihydrorhodamine 123 → fluorescent rhodamine
• Procedure: whole blood (EDTA) + DHR, lyse RBC, split into unstimulated vs PMA-stimulated tubes; analyse within 24 h with control sample

Chronic Granulomatous Disease (CGD)

• Defective burst (NADPH oxidase subunits gp91^{phox}, p47^{phox}, p67^{phox})
• X-linked or autosomal-recessive
• Flow: stimulated sample resembles unstimulated (no fluorescence)
• Carrier females show dual peaks
• Management: prophylactic antimicrobials; cure = bone-marrow or emerging gene therapy

Leukocyte Adhesion Deficiency (LAD)

• Absent integrins (e.g., \text{CD18}) → neutrophils stay in blood, cannot reach infection sites
• Detected by phenotyping CD11/18 family markers

Key Takeaways

• Flow cytometry rapidly profiles immune cells by size, complexity, and surface antigens
• TBNK counts guide diagnosis/monitoring of primary, acquired, and therapy-induced immunodeficiencies
• Functional assays (DHR) assess neutrophil oxidative burst; absent activity suggests CGD
• Always validate surprising results; technical errors can mimic severe pathology