CRASH COURSE PART 3 CHEMICAL Pathology — Notes OmpathStudy

SECTION 5: Cytology & Cytopathology, Special Techniques, IHC, Vitamins, Trace Elements & PEM (Pages 405–566) --- 1. Cytology vs Cytopathology Cytology: The branch of biology studying cell structure, function, and chemistry in both normal and pathological states. Cytopathology: A subspecialty of pathology that diagnoses disease by examining cells from body fluids or tissue samples — looking for changes in cell morphology that indicate cancer, infection, or inflammation. Key distinction: Cytology is the broader science; cytopathology is the clinical diagnostic application. Cytology Slide Preparation — Steps 1. Sample collection — scraping, brushing, aspiration, or fluid collection 2. Fixation — alcohol or methanol immediately applied to prevent degradation 3. Smearing — thin uniform layer spread on a glass slide 4. Staining — Pap stain (gynaecological/general), Wright's/Giemsa (haematology), H&E (general) 5. Air drying and coverslipping 6. Microscopic examination — low to high magnification 7. Interpretation and reporting — cytopathologist identifies abnormalities, issues a diagnostic report Advantages of Cytopathology Minimally invasive compared to surgical biopsy Cost-effective and relatively quick Used in screening programmes (e.g., cervical cancer via Pap smear) Limitations No tissue architecture — only individual cells are seen May miss disease if sample is non-representative False negatives possible in early or focal disease --- 2. Exfoliative Cytology (Topic VII) Definition: Examination of cells that have been naturally shed from body surfaces or collected by scraping/washing from mucosal surfaces. Techniques and Applications Pap Smear (Papanicolaou Smear): Purpose: Cervical cancer screening Procedure: Spatula or brush scrapes cells from the cervical transformation zone; spread on a slide and stained with Pap stain Detects: Cervical dysplasia (pre-cancer) and invasive cervical cancer Reporting system: Bethesda system — NILM (normal), ASCUS, LSIL, HSIL, carcinoma Sputum Cytology: Purpose: Diagnose lung disease, mainly cancer Procedure: Patient expectorates sputum; cells examined for malignant changes Detects: Squamous cell carcinoma of lung (most amenable to sputum cytology) Urine Cytology: Purpose: Urinary tract evaluation Procedure: Voided urine or catheter specimen processed, concentrated, and stained Detects: Transitional (urothelial) cell carcinoma of the bladder FNA (as an exfoliative technique): Aspirates cells from a mass using a thin needle Used for thyroid, lymph nodes, breast, salivary glands Body Fluid Cytology: Examines cells in pleural, peritoneal, pericardial, CSF, synovial fluids Detects: Malignant effusions, infections, inflammatory conditions Principles of Exfoliative Cytology Cells collected from where they naturally shed or are accessible Fixed, stained, then examined for changes in: cell size, shape, nuclear-to-cytoplasmic ratio, nuclear hyperchromasia, abnormal mitoses, and arrangement --- 3. Immunohistochemistry (IHC) — Detailed Definition: A laboratory technique using antigen-antibody binding to detect specific proteins (antigens) in tissue sections, allowing their localisation to be visualised under a microscope. Core Principle A primary antibody binds specifically to the target antigen in the tissue A secondary antibody (conjugated to an enzyme or fluorophore) binds to the primary antibody to amplify and visualise the signal Detection: Enzyme-based (HRP + DAB substrate → brown precipitate) or fluorescent (fluorophore emits light) Counterstain: Haematoxylin applied to show tissue architecture in blue/purple contrast Full IHC Process (Step by Step) Step Detail --- --- 1. Fixation Formalin (10% NBF) — preserves morphology 2. Embedding Paraffin wax blocks 3. Sectioning Microtome, 3–5 µm sections on glass slides 4. Deparaffinisation Heated + xylene to remove wax; rehydrated through alcohol series 5. Antigen retrieval HIER (heat in buffer — microwave or water bath) or enzymatic retrieval — unmasks antigens masked by fixation 6. Blocking Serum applied to prevent non-specific antibody binding 7. Primary antibody Applied and incubated (1 hour to overnight) 8. Washing Buffer (PBS or TBS) to remove unbound antibody 9. Secondary antibody Conjugated antibody applied; shorter incubation 10. Detection DAB substrate → brown colour where antigen present 11. Counterstaining Haematoxylin → blue nuclei for background 12. Dehydration & mounting Alcohol series, xylene, coverslip applied 13. Microscopy Analysis and image capture Types of IHC By detection method: Chromogenic — enzyme-linked (HRP/AP + substrate); produces coloured precipitate; used in routine diagnostics Fluorescent — fluorophore-conjugated; allows multiplexing (multiple targets simultaneously) By antibody type: Monoclonal — single clone, highly specific, one epitope (e.g., ER, HER2) Polyclonal — multiple clones, higher sensitivity, less specific By target: Tumour markers — Ki-67 (proliferation), p53 (tumour suppressor), HER2 (breast c