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Q1 — Inflammation and Phagocytosis (20 Marks)

Definition of Inflammation

• Acute - rapid in onset, short duration; characterized by exudation of fluid, plasma proteins (edema), and predominance of neutrophils
• Chronic - longer duration; characterized by lymphocytes, macrophages, plasma cells, new vessel formation (angiogenesis), and fibrosis

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Causes of Inflammation

1. Infections - bacterial, viral, fungal, parasitic; microbial products trigger recognition
2. Tissue necrosis - from ischemia, trauma, thermal/chemical injury; necrotic cells release danger signals (sterile inflammation)
3. Foreign bodies - sutures, implants, splinters
4. Immune reactions - autoimmune diseases, hypersensitivity reactions

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Recognition of Offending Agents

• Toll-like receptors (TLRs) - recognize PAMPs (pathogen-associated molecular patterns) such as LPS of gram-negative bacteria, peptidoglycan of gram-positive bacteria, viral RNA, and unmethylated CpG DNA
• NOD-like receptors (NLRs) - cytoplasmic receptors that recognize microbial products and DAMPs; form the inflammasome which activates IL-1β and IL-18
• DAMPs (damage-associated molecular patterns) - molecules released from damaged cells, e.g., uric acid crystals, ATP, HMGB1, DNA - stimulate sterile inflammation

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Process of Phagocytosis

Step 1: Recruitment of Leukocytes (Leukocyte Extravasation)

• P-selectin and E-selectin on endothelium (stored in Weibel-Palade bodies; upregulated by histamine, thrombin, TNF, IL-1)
• L-selectin on leukocytes
• Selectins bind sialylated carbohydrate ligands (e.g., PSGL-1)

• Exogenous: bacterial products (e.g., formyl-methionyl peptides, N-fMLP)
• Endogenous: C5a (complement), leukotriene B4 (LTB4), IL-8 (CXCL8), platelet-activating factor (PAF)

Step 2: Recognition and Attachment (Opsonization)

• Mannose receptors - bind terminal mannose residues of microbial glycoproteins
• Scavenger receptors - recognize oxidized LDL, microbial surfaces

• IgG antibodies (Fc portion binds Fc-γ receptors on phagocytes)
• C3b (complement fragment; binds complement receptors CR1, CR3)
• Plasma lectins (mannose-binding lectin, collectins)

Step 3: Engulfment

Step 4: Killing and Degradation

• Triggered by the assembly of NADPH oxidase (phagocyte oxidase) on the phagosomal membrane
• O₂ + NADPH → superoxide (O₂⁻) → H₂O₂ (hydrogen peroxide)
• MPO (myeloperoxidase) from neutrophil granules: H₂O₂ + Cl⁻ → HOCl (hypochlorous acid) - the most potent microbicidal agent
• Also formed: hydroxyl radical (OH·), peroxynitrite (ONOO⁻)

• iNOS (inducible nitric oxide synthase) in macrophages catalyzes: arginine → nitric oxide (NO)
• NO reacts with ROS to form peroxynitrite (ONOO⁻), which destroys microbes

• Primary (azurophil) granules of neutrophils: myeloperoxidase (MPO), lysozyme, acid hydrolases, elastase, defensins
• Secondary (specific) granules: lactoferrin (iron chelator), collagenase, B12-binding proteins
• Lysozyme cleaves the muramic acid-acetylglucosamine bond in bacterial cell walls
• Defensins punch holes in microbial membranes

Step 5: Neutrophil Extracellular Traps (NETs)

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Clearance of Offending Agents

1. Dead microbes and debris are phagocytosed by macrophages
2. Neutrophils undergo apoptosis (lifespan 1-2 days in tissues) and are cleared by macrophages via efferocytosis - this actively promotes anti-inflammatory signals (release of TGF-β, IL-10)
3. Short-lived inflammatory mediators (prostaglandins, leukotrienes, PAF, histamine) are rapidly degraded or their synthesis ceases
4. Lipoxins, resolvins, and protectins are biosynthesized from arachidonic acid and omega-3 fatty acids; these are pro-resolution mediators that actively terminate the acute inflammatory response
5. The net result is return to homeostasis, followed by tissue repair through regeneration or scarring

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Q2 — Thrombosis: Definition, Pathology, and Fate of Thrombus (20 Marks)

Definition

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Pathology of Thrombosis - Virchow's Triad

1. Endothelial injury
2. Abnormal blood flow (stasis or turbulence)
3. Hypercoagulability of blood

1. Endothelial Injury

• Physical injury: trauma, surgery, catheter placement
• Hemodynamic stress: hypertension, turbulent flow at bifurcations
• Hypercholesterolemia, homocysteinemia
• Inflammatory cytokines (TNF, IL-1)
• Toxins from cigarette smoke
• Infectious agents

• Procoagulant changes: downregulation of thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor (TFPI); upregulation of tissue factor (TF) → activates extrinsic coagulation pathway
• Antifibrinolytic effects: increased secretion of PAI (plasminogen activator inhibitors) → reduced fibrinolysis
• Exposure of subendothelial collagen and von Willebrand factor (vWF) → platelet adhesion and activation

2. Abnormal Blood Flow

• Disrupts laminar flow, causes endothelial injury
• Forms countercurrents and local pockets of stasis

• Prevents dilution of activated clotting factors by fresh blood
• Retards the inflow of clotting factor inhibitors
• Promotes endothelial activation

• Ulcerated atherosclerotic plaques → turbulence
• Aneurysms → local stasis
• Myocardial infarction → dyskinetic wall motion → stasis
• Atrial fibrillation → stasis in atria (classically in the left atrial appendage)
• Venous thrombosis in bed-ridden patients → stasis in leg veins

3. Hypercoagulability

• Factor V Leiden mutation - point mutation renders Factor V resistant to cleavage by activated protein C (most common inherited cause of hypercoagulability)
• Prothrombin G20210A mutation - elevated prothrombin levels
• Antithrombin III deficiency - reduced inhibition of thrombin and Xa
• Protein C or Protein S deficiency - reduced inactivation of Factors Va and VIIIa
• MTHFR mutation → hyperhomocysteinemia → endothelial damage

• Heparin-Induced Thrombocytopenia (HIT): autoantibodies to heparin-PF4 complexes → platelet activation → paradoxical thrombosis despite heparin
• Antiphospholipid antibody syndrome (lupus anticoagulant): antibodies to β₂-glycoprotein I; causes recurrent thromboses, miscarriages, valve vegetations
• Oral contraceptives, pregnancy → increased hepatic coagulation factor synthesis
• Disseminated cancers → release of procoagulant mucins
• Smoking, obesity → unknown mechanisms

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Morphology of Thrombi

• Frequently occlusive
• Platelet-rich, pale/grey ("white thrombus")
• Typically on ruptured atherosclerotic plaques
• Lines of Zahn (alternating layers of fibrin/platelets and red cells) seen in cardiac/aortic thrombi - indicate formation during life

• Almost invariably occlusive
• Red, "stasis thrombus" - rich in enmeshed red cells within fibrin mesh
• Commonly in leg veins (90% of cases)
• Propagate toward the heart as long casts

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Fate of Thrombus

1. Propagation

• The thrombus enlarges through continued accretion of platelets and fibrin
• Increases the risk of vascular occlusion and embolization
• Most clinically dangerous early fate

2. Embolization

• Part or all of the thrombus dislodges and is transported to a distant site
• Venous thrombi → pulmonary emboli (PE) → right heart failure, hypoxia, sudden death
• Arterial/cardiac thrombi → systemic emboli → stroke, limb ischemia, bowel infarction

3. Dissolution (Lysis)

• Activation of fibrinolytic factors (plasmin cleaves fibrin)
• Effective only for newly formed thrombi - older thrombi resist lysis due to extensive fibrin polymerization
• This is the rationale for giving t-PA within hours of acute coronary thrombosis (narrow therapeutic window)

4. Organization and Recanalization

• Organization - ingrowth of endothelial cells, smooth muscle cells, and fibroblasts into the thrombus
• Recanalization - new capillary channels form within the organized thrombus, partially restoring blood flow through the original lumen
• Over time, the thrombus is incorporated into the vessel wall as vascularized connective tissue
• Occasionally the center undergoes enzymatic digestion (release of lysosomal enzymes from entrapped leukocytes) rather than organizing

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Q3 — Laboratory Diagnosis of Cancer (20 Marks)

Introduction

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1. Morphologic Methods

a) Histopathology (Tissue Biopsy)

• Cellular features of malignancy: nuclear pleomorphism, hyperchromasia, prominent nucleoli, increased N:C ratio, atypical mitoses
• Architectural features: glandular pattern (carcinoma), sheets vs. nests vs. cords
• Invasion: beyond basement membrane (cardinal feature of malignancy)
• Grading: based on degree of differentiation; ranges from Grade 1 (well-differentiated) to Grade 3-4 (poorly differentiated/undifferentiated = anaplastic)
• Staging (TNM system): T1-T4 (primary tumor size/invasion), N0-N3 (nodal involvement), M0-M1 (metastases); staging has greater clinical value than grading

• Excision biopsy: complete removal - gold standard
• Incision biopsy / core needle biopsy: representative sample
• Frozen section: rapid intraoperative diagnosis (within minutes) - used for surgical decision-making; excellent accuracy but inferior detail
• Fine-needle aspiration (FNA): minimally invasive; aspiration of cells from palpable masses (breast, thyroid, lymph nodes); rapid but limited by small sample

b) Cytology (Papanicolaou Smear)

• Shed or scraped cells examined for cytological features of malignancy
• Used for: cervical carcinoma (Pap smear), bronchogenic carcinoma (sputum cytology), bladder carcinoma (urinary cytology), effusions (ascitic, pleural, CSF), endometrial sampling
• Malignant cells are less cohesive, shed easily, show anaplastic features
• Most successful public health application: control of cervical cancer by Pap smear

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2. Immunohistochemistry (IHC) and Flow Cytometry

Immunohistochemistry

Flow Cytometry

• Fluorescently labeled antibodies used to phenotype cells
• Used routinely in classification of leukemias and lymphomas (T-cell vs. B-cell, lymphoblastic vs. mature)
• Determines CD marker patterns (e.g., CD10, CD19, CD20 for B-cell; CD3, CD4, CD8 for T-cell)

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3. Tumor Markers (Biochemical Assays)

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4. Molecular/Genetic Methods

a) Cytogenetics and FISH

• Conventional karyotyping: detects chromosomal translocations, deletions, amplifications
• FISH: fluorescent probes detect specific chromosomal loci; detects gene amplification (e.g., HER2), translocations (e.g., BCR-ABL in CML), deletions; can be done on non-dividing (interphase) cells

b) PCR (Polymerase Chain Reaction)

• Detects minimal residual disease (e.g., BCR-ABL transcripts in CML after treatment)
• Detects viral oncogenes (HPV in cervical cancer)
• RT-PCR for gene expression profiling

c) DNA/RNA Sequencing (Next-Generation Sequencing - NGS)

• Somatic mutation profiling: identifies actionable driver mutations (e.g., EGFR, KRAS, BRAF, ALK, ROS1)
• Guides targeted therapy (e.g., EGFR mutation → erlotinib; BRAF V600E → vemurafenib)
• Germline sequencing: identifies hereditary predispositions (BRCA1/2, TP53 in Li-Fraumeni syndrome, APC in FAP, MLH1/MSH2 in Lynch syndrome)

d) Gene Expression Profiling (Microarrays, RNA-Seq)

• Classifies molecularly distinct subtypes (e.g., luminal A/B vs. HER2-enriched vs. basal-like breast cancers)
• OncotypeDX (21-gene signature) and MammaPrint (70-gene signature): predict recurrence risk in breast cancer and guide chemotherapy decisions

e) Methylation Analysis

• Methylation of MGMT promoter in glioblastoma → better response to alkylating agents (temozolomide)
• MLH1 promoter methylation in colorectal cancer → MSI (microsatellite instability)

f) Liquid Biopsy

• Detection of circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), exosomes in blood, stool, sputum, urine
• Non-invasive; can detect minimal residual disease, treatment resistance mutations, early recurrence
• Growing clinical role for monitoring therapy

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5. Electron Microscopy

• Now largely replaced by IHC and molecular methods
• Still used for: classification of undifferentiated tumors, confirming neuroendocrine origin (dense-core secretory granules), Langerhans cell histiocytosis (Birbeck granules)

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Q4 — Short Notes (4 × 10 Marks)

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(a) Fluorescence In Situ Hybridization (FISH)

Principle

Steps in FISH

1. Probe preparation: DNA clones (from BAC or PAC libraries) containing the sequence of interest are labeled with fluorescent dyes (fluorophores) by nick translation or random priming
2. Specimen preparation: chromosome preparations (metaphase spreads) or interphase nuclei on glass slides
3. Denaturation: both probe and target DNA are denatured (double-stranded → single-stranded) by heat or formamide
4. Hybridization: the labeled probe is applied and incubated; it anneals (hybridizes) to its complementary target sequence
5. Washing: unbound probe is washed off
6. Visualization: fluorescence microscope; each fluorescent signal = one copy of the target sequence

Types of FISH Probes

Applications in Pathology

• Oncology:
  • HER2 amplification in breast/gastric cancer (eligibility for trastuzumab) - counted as HER2:CEP17 ratio
  • BCR-ABL fusion (Philadelphia chromosome, t(9;22)) in CML and ALL
  • ALK rearrangement in lung adenocarcinoma (eligibility for crizotinib)
  • N-MYC amplification in neuroblastoma (poor prognosis)
  • EGFR, ROS1, RET rearrangements in lung cancer
• Congenital anomalies:
  • Detection of microdeletions: 22q11.2 (DiGeorge syndrome), 15q11 (Prader-Willi/Angelman), 7q11.23 (Williams syndrome)
• Prenatal diagnosis: on uncultured amniocytes for rapid aneuploidy detection (trisomy 21, 13, 18; X, Y)

Advantages

• Can be performed on non-dividing (interphase) cells - no need for cell culture
• Higher resolution than conventional karyotyping
• Can be used on FFPE (formalin-fixed paraffin-embedded) tissue sections
• Multiple probes with different colors (multicolor FISH) can be used simultaneously

Limitations

• Only detects targeted regions; not genome-wide
• Cannot detect point mutations or low-level mosaicism
• Balanced translocations without gene disruption may be missed

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(b) Transplant Rejection and Graft-Versus-Host Disease (GVHD)

Transplant Rejection

Mechanisms of Allorecognition

1. Direct pathway: recipient T cells recognize intact donor MHC molecules on donor APCs (in graft) - major pathway of acute rejection
2. Indirect pathway: recipient T cells recognize donor-derived peptides (processed from donor MHC) presented by recipient APCs - more important in chronic rejection

Types of Rejection

• Occurs within minutes to hours of transplantation
• Caused by preformed antibodies against donor endothelium (ABO blood group antigens, HLA Class I)
• Diffuse thrombosis, widespread ischemic necrosis of the graft
• Prevented by pre-transplant cross-matching

• Occurs within days to weeks (can occur months later if poorly immunosuppressed)
• Two forms:
  • Acute cellular rejection: CD4 T cells (via cytokines) and CD8 cytotoxic T cells attack donor parenchymal cells; tubulitis in kidney, bile duct destruction in liver
  • Acute antibody-mediated (humoral) rejection: donor-specific antibodies cause endothelialitis, neutrophil infiltration, C4d deposition on capillary endothelium
• Responds to intensified immunosuppression (corticosteroids, anti-thymocyte globulin)

• Occurs over months to years; major cause of long-term graft failure
• Both cellular and humoral mechanisms involved
• Characterized by intimal smooth muscle proliferation (transplant vasculopathy), interstitial fibrosis, tubular atrophy
• Poorly responsive to immunosuppression; may require re-transplantation

Graft-Versus-Host Disease (GVHD)

Setting

• Allogeneic HSCT for leukemias, lymphomas, aplastic anemia, severe thalassemia
• Recipient's marrow must first be destroyed by irradiation/chemotherapy (conditioning)
• Mature donor T cells contaminating the HSC preparation are the effectors

Pathogenesis

• Donor T cells (both CD4 and CD8) recognize recipient HLA alloantigens on recipient dendritic cells in secondary lymphoid organs
• They become activated, expand clonally, and migrate to target tissues
• Target organs (damaged by conditioning): skin (rash, desquamation), intestines (watery/bloody diarrhea), liver (hepatitis, cholestatic jaundice)

Clinical Features

Beneficial Effect - Graft-Versus-Leukemia (GVL) Effect

• The same alloreactive donor T cells that cause GVHD also recognize and kill residual leukemia cells
• This GVL effect is crucial for the therapeutic success of HSCT in leukemia
• Total elimination of GVHD would increase relapse rates; a degree of controlled GVHD is acceptable

Prevention and Treatment

• HLA matching (more critical than solid organ transplantation, especially MHC Class I and II)
• T-cell depletion of the HSC graft (reduces GVHD but also GVL effect)
• Prophylaxis: cyclosporine + methotrexate; tacrolimus
• Treatment of established GVHD: high-dose corticosteroids, ruxolitinib (JAK inhibitor)

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(c) Various Pigments in the Body and Their Special Stains

Exogenous Pigments

Endogenous Pigments

• Derived from hemoglobin breakdown; insoluble iron-storage complex (ferritin aggregates)
• Golden-brown, granular pigment within macrophages
• Local accumulation: hemorrhage, bruising (the yellow-brown color of old bruise)
• Systemic accumulation: hemosiderosis (excess iron deposition without damage), hemochromatosis (organ damage)
• Stain: Prussian blue (Perls' stain) - hemosiderin stains blue

• Artifact from formalin fixation of blood-rich tissues
• Brown-black, granular, birefringent
• Stain: Not specifically; can be removed by alkaline alcohol

• Yellow-green pigment from heme catabolism (non-iron portion of hemoglobin)
• Deposits in jaundice: bile thrombi in hepatic canaliculi, bilirubin crystals in gallbladder
• Inspissated bile in hepatocytes and bile ducts
• Stain: Hall's bile stain (stains bile duct plugs green); van Gieson's

• Yellow-brown, finely granular pigment in cytoplasm of hepatocytes, cardiac myocytes, neurons
• Composed of oxidized lipids and proteins (lipid peroxidation products)
• Accumulates with aging and oxidative stress; not harmful per se
• Causes brown atrophy of organs (heart, liver)
• Stain: PAS positive; Sudan black B; autofluorescent on fluorescence microscopy

• Brown-black pigment produced by melanocytes from tyrosine via tyrosinase
• Normally in skin, hair, eye (retinal pigment epithelium)
• Pathological accumulations: melanoma, Addison disease (increased ACTH → hyperpigmentation), Peutz-Jeghers syndrome (mucosal)
• Stain: Fontana-Masson stain (silver stain, stains melanin black); Schmorl's stain
• IHC: HMB-45, Melan-A, S-100 protein

• Dark brown/black crystalline pigment derived from hemoglobin degradation by Plasmodium
• Found within infected RBCs and phagocytes in malaria
• Stain: Giemsa stain for blood films; appears brown-black

• Dark brown-black homogentisic acid polymer deposited in connective tissue, cartilage, sclera
• Seen in alkaptonuria (homogentisic acid oxidase deficiency)

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(d) Classification of Storage Disorders and Gaucher Disease

Classification of Storage (Lysosomal Storage) Disorders

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Gaucher Disease - A Detailed Note

Etiopathogenesis

• Enzyme deficiency: Deficiency of acid β-glucocerebrosidase (glucocerebrosidase) encoded by the GBA gene on chromosome 1q21
• Inheritance: Autosomal recessive
• Result: Accumulation of glucocerebroside (glucosylceramide) within lysosomes of macrophages throughout the body (Kupffer cells in liver, red pulp macrophages in spleen, bone marrow macrophages, pulmonary macrophages)
• The accumulating glucocerebroside comes primarily from the normal catabolism of senescent red and white blood cell membranes

Types of Gaucher Disease

Morphology

• The hallmark lesion
• Large macrophage (20-100 μm) with abundant pale cytoplasm showing a characteristic "crumpled tissue paper" or "wrinkled tissue paper" appearance
• Cytoplasm: filled with elongated lysosomes packed with glucocerebroside; fibrillary pattern due to tubular storage structures
• Eccentric, small nucleus
• PAS positive (glucocerebroside is a glycolipid)
• Electron microscopy: elongated, curved (spindle-shaped) lysosomes

• Spleen: Most dramatically enlarged (can reach 10 kg); splenomegaly is the cardinal clinical feature; packed with Gaucher cells in the red pulp
• Liver: Hepatomegaly (Gaucher cells in sinusoids); fibrosis in severe/long-standing disease
• Bone marrow: Replacement of hematopoietic marrow → cytopenias (anemia, thrombocytopenia, leukopenia)
• Bone: Cortical thinning, medullary expansion → "Erlenmeyer flask deformity" of distal femur on X-ray; osteonecrosis (avascular necrosis of femoral head); pathological fractures
• Lungs: Gaucher cells in alveolar capillaries → respiratory failure in severe cases
• CNS: In Types 2 and 3

Clinical Features (Type 1)

• Massive splenomegaly (often the presenting feature)
• Hepatomegaly (less prominent than spleen)
• Pancytopenia (due to hypersplenism + marrow infiltration): anemia, thrombocytopenia (bleeding), neutropenia
• Bone pain, pathological fractures, avascular necrosis
• Growth retardation in children
• No neurological involvement (Type 1)

Diagnosis

• Enzyme assay: Decreased β-glucocerebrosidase activity in leukocytes or fibroblasts (definitive)
• Gene mutation analysis: GBA gene sequencing (N370S mutation - most common in Ashkenazi Jews, associated with Type 1; L444P - associated with neuronopathic forms)
• Serum biomarkers: Elevated chitotriosidase and glucosylsphingosine (lyso-Gb1) - useful for monitoring treatment response
• Bone marrow biopsy: Gaucher cells (PAS-positive crumpled cells)
• Imaging: MRI for bone marrow infiltration, Erlenmeyer flask deformity on plain X-ray

Treatment

• Enzyme Replacement Therapy (ERT): Imiglucerase (recombinant glucocerebrosidase), also velaglucerase alfa and taliglucerase alfa; IV infusion every 2 weeks; reduces organomegaly, improves cytopenias, prevents bone complications; does not cross BBB (not effective for Types 2/3 CNS disease)
• Substrate Reduction Therapy (SRT): Miglustat, eliglustat - inhibit glucosylceramide synthase, reducing substrate accumulation; oral drugs
• Splenectomy: historically used; now avoided as worsens bone disease
• Bone marrow transplantation: curative but rarely done due to associated risks

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