DEPARTMENT OF PATHOLOGY - SECOND BLOCK INTERNAL ASSESSMENT

Hematology, Blood Banking, Clinical Pathology, Cytology

Complete Model Answers (Distinction Level)

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SECTION A: MULTIPLE CHOICE QUESTIONS (20 Marks)

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Scenario 18 (Q1 & Q2) - 5-year-old boy, weakness/fatigue 3 years, repeated transfusions, Hb 6.2 g/dl, MCV reduced, MCH reduced, MCHC reduced, RDW normal

Q1. The most probable diagnosis:

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Q2. The confirmatory test for Thalassemia Major:

• HbF markedly elevated (>90% in beta-thalassemia major)
• HbA absent or markedly reduced
• HbA2 variable

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Scenario 19 (Q3 & Q4) - 25-year-old postpartum hemorrhage, pale, hypotensive, bleeding from multiple sites

Q3. The probable diagnosis:

1. Bleeding from multiple sites (not just the surgical/delivery site)
2. Hypotension and pallor
3. Obstetric context (amniotic fluid embolism, placental abruption, retained products trigger the extrinsic coagulation pathway)

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Q4. The best lab investigation in DIC:

• Consumption of clotting factors → prolonged PT and aPTT
• Consumption of fibrinogen → decreased fibrinogen (so D is partially correct but the question asks "helpful")
• Secondary fibrinolysis → increased D-dimers and FDPs - this is the hallmark test

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Scenario 20 (Q5 & Q6) - 25-year-old female, menorrhagia, weakness, Hb 6.2 g/dl, MCV 68 fl, MCH 20 pg, MCHC 22 g/dl, RDW 28 (elevated)

Q5. The most likely diagnosis:

• MCV low (68 fl, normal 80-100) = microcytic
• MCH low (20 pg) = hypochromic
• MCHC low (22 g/dl) = hypochromic
• RDW elevated (28, normal <15) = marked anisocytosis
• Menorrhagia = ongoing blood loss (iron depletion)

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Q6. Her peripheral blood smear is likely to be:

• Microcytes (small cells, MCV reduced)
• Hypochromia (increased central pallor, pale zone >1/3 of cell diameter)
• Anisocytosis and poikilocytosis (pencil cells, target cells, elliptocytes)
• RDW elevated confirms the anisocytosis seen on smear

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Scenario 21 (Q7 & Q8) - 9-year-old boy, fever, fatigue, bone pain 2 weeks, generalized lymphadenopathy, CBC shows anemia + thrombocytopenia + leukocytosis with 70% blasts

Q7. The probable diagnosis:

• Age: Child (peak age ALL = 2-10 years)
• 70% blast cells (acute leukemia, not chronic)
• Generalized lymphadenopathy (lymphoid involvement)
• Pancytopenia features (marrow infiltration by blasts)
• Bone pain (marrow expansion)

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Q8. The most useful cytochemical stain for blasts in ALL:

• PAS (Periodic Acid-Schiff) stain - lymphoblasts show block/chunky PAS positivity
• Sudan black B and Myeloperoxidase (MPO) are positive in AML (myeloblasts), negative in ALL
• Alkaline Phosphatase is elevated in neutrophils in leukemoid reactions; Leukocyte Alkaline Phosphatase (LAP) is low in CML

Note: Sudan black B (A) is for myeloid lineage. The correct answer for lymphoblasts is PAS stain (D).

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Scenario 22 (Q9 & Q10) - Healthy 30-year-old donating blood

Q9. Disease routinely screened before blood donation:

• HBsAg (Hepatitis B) - mandatory
• Anti-HCV (Hepatitis C) - mandatory
• HIV 1 & 2 - mandatory
• VDRL/RPR (Syphilis) - mandatory
• Malaria antigen/antibody - in endemic areas

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Q10. Test commonly used to screen Hepatitis B:

• Sensitive and specific
• High throughput for blood bank use
• Cost-effective
• Used for HIV, HCV, HBsAg screening

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Scenario 23 (Q11 & Q12) - 45-year-old, yellowish discoloration of eyes, pale stools, dark urine, urine positive for bile pigments and bile salts

Q11. The most likely diagnosis:

• Pale/clay-colored stools (no stercobilinogen reaching gut - bile duct blocked)
• Dark urine (conjugated bilirubin excreted in urine)
• Urine positive for bile salts AND bile pigments (conjugated bilirubin is water-soluble and appears in urine only in obstructive/hepatocellular jaundice)

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Q12. Test used to detect bile salts in urine:

• Benedict's test = glucose
• Rothera's test = ketone bodies
• Benzidine test = occult blood

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Scenario 24 (Q13 & Q14) - 10-year-old girl, recurrent nosebleeds, easy bruising, mother similar history, platelet count normal, bleeding time prolonged, PT normal, aPTT mildly prolonged

Q13. The most likely diagnosis:

• Family history (autosomal dominant)
• Normal platelet count (platelets present, but cannot adhere - vWF deficient)
• Prolonged bleeding time (platelet adhesion requires vWF → primary hemostasis defect)
• Normal PT (extrinsic pathway intact)
• Mildly prolonged aPTT (vWF carries Factor VIII; deficiency → mild Factor VIII reduction → mild aPTT prolongation)
• Female with mucosal bleeding (epistaxis, menorrhagia typical)

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Q14. Test classically abnormal in vWD:

• Ristocetin fails to agglutinate platelets (absent or reduced vWF)
• RIPA is absent or markedly reduced - this is the hallmark diagnostic test for vWD

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Scenario 25 (Q15 & Q16) - 30-year-old female, severe anemia, needs blood transfusion

Q15. Most appropriate blood product for severe anemia:

• Maximum RBC mass per unit
• Minimal volume overload risk
• Removes plasma (reduces transfusion reactions)

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Q16. Temperature at which PRBCs are stored:

• Shelf life: 35-42 days depending on anticoagulant-preservative solution (CPDA-1 = 35 days; AS-1/SAGM = 42 days)
• -20°C = platelets are NOT stored this way (platelets stored at 20-24°C with agitation)
• FFP stored at -20°C or below
• Cryoprecipitate stored at -20°C

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Scenario 26 (Q17 & Q18) - 32-year-old female, tiredness, recurrent petechiae, Hb 6.5 g/dl, WBC 2000/cmm, platelets 45,000/cmm

Q17. Reduction in all cell lines is called:

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Q18. Bone marrow in aplastic anemia shows:

• Bone marrow is markedly hypocellular (cellularity <25% in severe aplastic anemia)
• Normal hematopoietic cells replaced by adipocytes (fat cells)
• "Empty marrow" on trephine biopsy with fatty spaces
• No blast cells (rules out leukemia)
• Resembles a "dry tap" on aspirate

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Scenario 27 (Q19 & Q20) - 50-year-old male, high fever 5 days, WBC 60,000/cmm, predominantly neutrophils, band forms, metamyelocytes, myelocytes, Hb and platelets normal, LAP score HIGH

Q19. The likely diagnosis:

• Leukemoid reaction: LAP score elevated (reactive process - neutrophils are functionally active)
• CML: LAP score is characteristically low/absent (despite massive leukocytosis, the cells are abnormal)
• The neutrophilia with band forms, metamyelocytes in context of HIGH fever + HIGH LAP = leukemoid reaction (reactive to infection/sepsis)
• Normal Hb and platelets also favor leukemoid reaction over CML

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Q20. Following feature is observed in leukemoid reaction:

• Toxic granulation = coarse dark granules in neutrophil cytoplasm (increased lysosomal granules)
• Dohle bodies = blue cytoplasmic inclusions
• Cytoplasmic vacuolation

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SECTION B: LONG ESSAY (10 Marks)

Q1. A 10-year-old boy - severe pain in both legs, frequent vaso-occlusive pain crises, Hb 6.5 g/dl, reticulocyte count 10%, USG abdomen shows small spleen, Hb electrophoresis confirms diagnosis.

a) What is the probable diagnosis?

• Child with severe pain (vaso-occlusive crisis)
• Frequent episodes = recurrent crises
• Hb 6.5 g/dl (severe hemolytic anemia)
• Reticulocyte count 10% (markedly elevated = compensatory erythropoiesis due to hemolysis)
• Small spleen on USG = autosplenectomy (repeated splenic infarcts from sickling → fibrosis → small/non-functional spleen)
• Hb electrophoresis confirms = HbSS (>90% HbS, absent HbA, elevated HbF)

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b) Etiopathogenesis of Sickle Cell Anemia

• Glutamic acid → Valine substitution at position 6 of the beta-globin chain
• This produces HbS (alpha2 beta2^S) instead of normal HbA (alpha2 beta2)

1. On deoxygenation, HbS molecules undergo polymerization (tactoid formation)
2. HbS polymers form long rod-like fibers that distort the RBC into sickle/crescent shape
3. Sickling is reversible initially (with reoxygenation) but becomes irreversible after repeated cycles
4. Irreversibly sickled cells have damaged membranes, are rigid and dehydrated

• Hypoxia
• Acidosis
• Dehydration
• Cold
• Infection
• High altitude

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c) Laboratory Investigations

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d) Complications of Sickle Cell Anemia

• Most common complication
• Ischemic pain in bones, joints, chest, abdomen
• Precipitated by infection, dehydration, cold, hypoxia

• Fever, chest pain, new pulmonary infiltrates, hypoxia
• Can be fatal; may require exchange transfusion

• Children: ischemic stroke (large vessel occlusion)
• Adults: hemorrhagic stroke

• Repeated splenic infarctions → fibrotic, small, non-functional spleen
• Increased susceptibility to encapsulated organisms: Streptococcus pneumoniae, H. influenzae, Neisseria meningitidis
• Prophylactic penicillin + pneumococcal vaccination mandatory

• Parvovirus B19 infects erythroid precursors → sudden drop in Hb, reticulocytopenia
• Medical emergency

• Massive splenic pooling of blood → sudden drop in Hb + shock (more in children before autosplenectomy)

• Femoral head most common → hip pain, disability

• Papillary necrosis (medullary hypoxia/sickling)
• Isosthenuria (inability to concentrate urine)
• Hematuria

• Gallstones (pigment stones) from chronic hemolysis
• Jaundice (unconjugated hyperbilirubinemia)
• Leg ulcers (chronic ischemia)

• Salmonella osteomyelitis (most common organism for osteomyelitis in SCA)
• Increased susceptibility post-autosplenectomy

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SECTION C: SHORT NOTES (7x5 = 35 Marks)

Q2. Blood Transfusion Reactions

A. Acute Immune Reactions

• Most dangerous acute reaction
• Cause: ABO incompatibility (clerical error - wrong blood given)
• Mechanism: IgM antibodies fix complement → intravascular hemolysis
• Features: Fever, chills, back/flank pain, hemoglobinuria (red/brown urine), hypotension, shock, DIC, renal failure
• Management: STOP transfusion immediately, maintain IV access, fluids, monitor urine output, treat DIC

• Most common reaction
• Cause: Antibodies against donor leukocyte antigens; cytokines in stored blood
• Features: Fever (>1°C rise), chills, rigors within 1-6 hours
• Management: Stop transfusion, paracetamol, use leukodepleted blood for future

• Cause: Antibodies against donor plasma proteins (especially IgA in IgA-deficient recipients)
• Features: Urticaria, itching, flushing
• Management: Antihistamines, resume transfusion slowly if mild

• Rare; IgA-deficient patients with anti-IgA antibodies
• Features: Hypotension, bronchospasm, angioedema - NO fever
• Management: Epinephrine, steroids; use washed RBCs in future

B. Acute Non-Immune Reactions

• Cause: Donor antibodies against recipient neutrophil antigens → neutrophil activation in lungs
• Features: Acute respiratory distress within 6 hours, bilateral pulmonary infiltrates, hypoxia, NON-cardiogenic pulmonary edema
• Management: Oxygen, ventilatory support

• Cause: Rapid transfusion / excess volume in elderly/cardiac patients
• Features: Dyspnea, hypertension, pulmonary edema
• Management: Slow transfusion rate, diuretics

• Cause: Bacterial contamination (most common: Yersinia enterocolitica in RBCs; Staphylococci in platelets)
• Features: High fever, rigors, hypotension, shock
• Management: Stop transfusion, blood cultures, broad-spectrum antibiotics

C. Delayed Reactions

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Q3. Semen Analysis

Collection

• Abstinence: 2-5 days before collection
• Collected by masturbation into sterile container
• Examined within 1 hour of collection
• Temperature maintained at 37°C during transport

Normal Parameters (WHO 2021 Reference Values - 5th edition):

Terminology for Abnormal Results

Steps in Semen Analysis

1. Macroscopic: Volume, color (whitish gray), pH, viscosity, liquefaction
2. Microscopic: Wet preparation - motility assessment (progressive, non-progressive, immotile)
3. Concentration: Hemocytometer (Neubauer chamber) or automated analyzer
4. Morphology: Papanicolaou or Shorr stain - assess head, midpiece, tail defects
5. Vitality: Eosin-nigrosin stain (dead cells take up eosin)
6. MAR test / IBT: For antisperm antibodies if >50% sperm with adherent particles

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Q4. Hereditary Spherocytosis

Genetics

• Autosomal dominant (75% of cases); some autosomal recessive
• Mutations in genes encoding: Ankyrin (most common, 40-65%), Band 3 protein, alpha-spectrin, beta-spectrin, protein 4.2

Pathogenesis

1. Deficiency of membrane skeletal proteins (particularly ankyrin-spectrin-Band 3 complex)
2. Loss of membrane lipid bilayer (membrane vesiculation)
3. RBC surface area decreases relative to volume
4. Cells become spherical (minimum surface area for given volume)
5. Spherocytes are rigid and unable to deform in the spleen
6. Trapped in splenic sinusoids → extravascular hemolysis
7. Repeated cycles of partial hemolysis create "conditioned" spherocytes
8. RBCs become increasingly dehydrated (MCHC elevated)

Clinical Features

• Anemia: Mild to moderate (Hb 8-12 g/dl typically)
• Jaundice: Intermittent, unconjugated hyperbilirubinemia
• Splenomegaly: Due to splenic hyperactivity (red pulp congestion)
• Gallstones: Pigment gallstones from chronic hemolysis
• Aplastic crisis: Triggered by Parvovirus B19

Laboratory Findings

Diagram: Osmotic Fragility Curve in HS

% Hemolysis
100%  |      HS curve (shifted right = increased fragility)
      |     /
      |    /
 50%  |   /    Normal curve
      |  / /
      | / /
   0% |/__/____________________________
      0.8  0.6  0.4  NaCl concentration (%)

Treatment

• Splenectomy: Curative (removes site of hemolysis); post-splenectomy vaccines essential
• Folic acid supplementation (increased demand)
• Cholecystectomy if gallstones symptomatic

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Q5. Pernicious Anemia

Pathogenesis

1. Dietary B12 binds to haptocorrin (R-binder) in saliva
2. In duodenum, pancreatic proteases release B12
3. B12 binds to Intrinsic Factor (IF) (secreted by gastric parietal cells)
4. B12-IF complex absorbed at terminal ileum (specific receptors: cubam complex - cubilin + amnionless)
5. Transported by transcobalamin II in blood

• Autoimmune gastritis: T-cell mediated destruction of parietal cells
• Two types of autoantibodies:
  1. Anti-parietal cell antibodies (APCA): Present in 90% of PA patients (sensitive but not specific)
  2. Anti-intrinsic factor antibodies: Present in 50-60% (Type I = blocking; Type II = binding; highly SPECIFIC for PA)
• Loss of parietal cells → IF deficiency → B12 malabsorption
• B12 deficiency → defective DNA synthesis → megaloblastic changes in rapidly dividing cells

• Large cells (megaloblasts) in bone marrow - arrested in S-phase
• Hypersegmented neutrophils (>5 lobes in >5% neutrophils)
• Pancytopenia in severe cases

Clinical Features

• Macrocytic anemia (MCV >100 fl, often >120 fl)
• Weakness, pallor, mild jaundice (lemon yellow tint)

• Glossitis (sore, smooth, beefy red tongue - Hunter's/Moeller's glossitis)
• Atrophic gastritis (achlorhydria)

• B12 deficiency → defective myelin synthesis (methionine synthesis disrupted)
• Demyelination of dorsal columns (posterior) and lateral corticospinal tracts
• Features: Peripheral neuropathy (tingling/numbness in feet), loss of vibration and position sense, spastic paraparesis, positive Romberg's sign
• SACD does NOT occur in folate deficiency (important distinction)

Laboratory Investigations

Treatment

• Hydroxocobalamin (Vitamin B12) intramuscular injection: 1000 mcg IM daily x 7 days, then weekly x 4, then monthly for life
• Oral B12 NOT effective (no IF for absorption)
• Response: Reticulocytosis in 3-5 days; Hb normalizes in 8 weeks; neurological improvement slow

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Q6. Immune Thrombocytopenic Purpura (ITP)

Classification

• Primary ITP: No identifiable cause
• Secondary ITP: Associated with SLE, HIV, H. pylori, hepatitis C, drugs, CLL
• Acute ITP: Children, self-limiting, often post-viral
• Chronic ITP: Adults (especially women 20-40 years), persistent >12 months

Pathogenesis

1. Autoantibodies (predominantly IgG) produced against platelet surface glycoproteins, mainly GPIIb/IIIa and GPIb/IX
2. Antibody-coated platelets recognized by Fc receptors on splenic macrophages
3. Extravascular hemolysis (destruction) of platelets in the spleen
4. Antibodies may also inhibit megakaryocyte maturation → impaired platelet production
5. T-cell dysfunction contributes (cytotoxic T cells directly kill platelets)

Clinical Features

• Petechiae, purpura (non-palpable, non-blanching)
• Easy bruising (ecchymoses)
• Mucosal bleeding: epistaxis, gingival bleeding, menorrhagia
• GI/GU bleeding in severe cases
• Intracranial hemorrhage - rare but life-threatening
• No splenomegaly (distinguishes from hypersplenism)
• No lymphadenopathy

Laboratory Findings

Treatment

• Corticosteroids: Prednisolone 1-2 mg/kg/day (reduces antibody production and Fc receptor blockade)
• IV Immunoglobulin (IVIG): 1 g/kg/day x 2 days (blocks Fc receptors on macrophages); used for rapid response needed (surgery, severe bleeding)
• Anti-D immunoglobulin: In Rh-positive non-splenectomized patients

• Splenectomy: Removes main site of platelet destruction; 60-70% long-term remission
• Rituximab (anti-CD20): For refractory ITP

• Thrombopoietin receptor agonists (TPO-RA): Romiplostim, Eltrombopag (stimulate platelet production)
• Immunosuppressants: Azathioprine, cyclosporine

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Q7. Beneficence (Bioethics)

1. Beneficence
2. Non-maleficence
3. Autonomy
4. Justice

Key Aspects

Applications in Hematology/Laboratory Medicine

Distinction from Non-maleficence

Conflict with Autonomy

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Q8. Advantages and Disadvantages of Fine Needle Aspiration Cytology (FNAC)

Principle

Advantages

Disadvantages

FNAC vs. Core Needle Biopsy vs. Excision Biopsy

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SECTION D: SHORT NOTES - APPLIED QUESTIONS (4x5 = 20 Marks)

Q9. 6-year-old boy, recurrent painful swelling of knee joint after minor trauma, platelet count and bleeding time normal, aPTT prolonged

a) Likely Diagnosis: HEMOPHILIA A (Factor VIII Deficiency)

b) Pathogenesis and Laboratory Findings

• Genetics: X-linked recessive disorder
  • Gene: F8 gene on X chromosome (Xq28)
  • Males (XY) affected; females are carriers (XX carrier)
  • ~30% are new mutations (no family history)
  • Most common mutation: Inversion of intron 22 (~45% of severe cases)
• Factor VIII Function:
  • Factor VIII is a cofactor in the intrinsic coagulation pathway
  • Acts as cofactor for Factor IXa in the "tenase complex"
  • Tenase complex activates Factor X → starts common pathway → thrombin → fibrin clot
• Effect of Factor VIII deficiency:
  • Intrinsic pathway (contact activation pathway) is impaired
  • Factor X activation is severely reduced
  • Insufficient thrombin generation → inadequate fibrin clot
  • Primary hemostasis (platelet plug) is INTACT (platelets and vWF normal)
  • Secondary hemostasis (fibrin reinforcement) is DEFECTIVE

• Joints (hemarthroses) are highly susceptible because synovial fluid lacks thromboplastin (tissue factor), so the extrinsic pathway cannot compensate
• Repeated hemarthroses → iron deposition → synovitis → cartilage destruction → hemophilic arthropathy

• Factor VIII concentrate (recombinant or plasma-derived) - replacement therapy
• Prophylactic infusions 3x/week in severe hemophilia
• DDAVP (desmopressin) for mild hemophilia (releases vWF-Factor VIII stores)
• Gene therapy: emerging curative option

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Q10. 55-year-old man, weight loss, massive splenomegaly, Hb 9.5 g/dl, WBC 93,000/cmm, platelets 1.7 lakhs/cmm, peripheral smear shows granulocytes at different stages of maturation with basophilia

a) Diagnosis: CHRONIC MYELOID LEUKEMIA (CML)

b) Pathogenesis

• CML is caused by the Philadelphia chromosome (Ph chromosome)
• Translocation t(9;22)(q34;q11): BCR gene (chromosome 22) fuses with ABL1 gene (chromosome 9)
• Creates the BCR-ABL1 fusion gene on the derivative chromosome 22 (Ph chromosome)
• BCR-ABL1 encodes a constitutively active tyrosine kinase (p210 protein)
• This kinase continuously activates proliferative signaling pathways (RAS, JAK/STAT, PI3K/AKT)
• Results in: Uncontrolled myeloid proliferation + inhibition of apoptosis

1. Chronic Phase (3-5 years): Indolent, elevated WBC, manageable with TKI
2. Accelerated Phase: Increasing blasts (10-19%), increasing basophilia, clonal evolution
3. Blast Crisis: >20% blasts (myeloid or lymphoid crisis), resembles acute leukemia

c) Peripheral Smear Findings

1. Leukocytosis: WBC markedly elevated (often 50,000-200,000/cmm)
2. Left shift: Full spectrum of myeloid maturation - myeloblasts, promyelocytes, myelocytes, metamyelocytes, band forms, mature neutrophils ("myelocyte bulge" = peak at myelocyte stage)
3. Basophilia: Absolute basophilia is a hallmark (>1% basophils - distinguishes CML from leukemoid reaction)
4. Eosinophilia: Also present
5. Thrombocytosis: Often present (platelets >400,000/cmm in early CML)
6. Minimal blasts in chronic phase (<5%)

• Hypercellular, myeloid hyperplasia
• All stages of granulopoiesis
• Increased reticulin fibrosis in accelerated/blast phase

• Cytogenetics: Philadelphia chromosome (karyotype)
• FISH: BCR-ABL1 fusion (sensitive)
• RT-PCR: BCR-ABL1 transcript (quantitative - monitors treatment response)
• Bone marrow trephine biopsy: Hypercellular, grade fibrosis

• Imatinib (Gleevec) - first generation TKI (Tyrosine Kinase Inhibitor); transformed CML from fatal to chronic disease
• Second generation: Dasatinib, Nilotinib (for imatinib resistance or T315I mutation)
• Third generation: Ponatinib, Asciminib
• Allogenic stem cell transplantation: For blast crisis or TKI failure

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Q11. 30-year-old man, weakness, fever, night sweats, weight loss 1 year, multiple enlarged, rubbery, discrete lymph nodes in neck, lymph node biopsy shows owl eye cells

a) Diagnosis: HODGKIN'S LYMPHOMA (Classical Hodgkin Disease)

b) Classification, Gross and Microscopy

• Large binucleated or multinucleated cell
• Each nucleus contains a large prominent eosinophilic nucleolus (resembling "owl eyes" or "mirror image")
• Background: Reactive inflammatory cells (lymphocytes, eosinophils, plasma cells, histiocytes)
• RS cells are the neoplastic cells (clonal B-cell origin - CD30+, CD15+, CD20 variable, CD45-)
• RS cells are a minority (<5%) surrounded by reactive inflammatory infiltrate

Classification (WHO Classification of Classical Hodgkin Lymphoma - 4 subtypes):

• Young women, mediastinal involvement (anterior mediastinal mass)
• Lacunar cells (variant RS cells - retraction artifact in formalin fixed tissue)
• Broad collagen bands dividing lymph node into nodules
• EBV association: Low (<10%)
• Prognosis: Good

• Older adults, males > females
• Classic RS cells in mixed inflammatory background (eosinophils, plasma cells, lymphocytes, histiocytes)
• Diffuse effacement of lymph node architecture
• EBV association: High (70%)
• Prognosis: Intermediate

• Background predominantly lymphocytes
• Classic RS cells sparse
• Good prognosis

• Elderly, immunocompromised, HIV patients
• Many RS cells, few lymphocytes
• Diffuse fibrosis or "sarcomatous" variant
• EBV association: Very high
• Worst prognosis

• Separate entity
• Neoplastic cells = "Popcorn cells" (LP cells, previously called L&H cells)
• CD20+, CD30-, CD15- (opposite of classical HL)
• Good prognosis

Gross Appearance

• Enlarged lymph nodes: Rubbery, discrete, non-tender
• Cut section: Gray-white, "fish flesh" appearance
• Nodular sclerosis: White fibrous bands visible on gross
• "Contiguous spread": Hodgkin lymphoma spreads in predictable contiguous fashion (cervical → mediastinal → para-aortic → spleen)

Microscopic Features (Classical HL)

1. Reed-Sternberg cells - pathognomonic (the neoplastic cells)
2. Variant RS cells depending on subtype:
   • Lacunar cells (NS)
   • Mononuclear RS variants (Hodgkin cells)
3. Background inflammatory infiltrate appropriate to subtype

• CD30 positive (membrane + Golgi pattern) - hallmark
• CD15 positive (in most cases)
• CD45 (LCA) negative
• CD20 variable (usually negative)
• PAX5 weakly positive

Staging (Ann Arbor/Lugano Staging):

• Stage I: Single lymph node region
• Stage II: Two or more regions, same side of diaphragm
• Stage III: Regions on both sides of diaphragm
• Stage IV: Disseminated extranodal involvement

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Q12. 62-year-old man, chronic back pain, weakness 3 months, X-ray skull shows multiple punched-out lytic lesions, anemia, hypercalcemia, raised serum creatinine and ESR

a) Probable Diagnosis: MULTIPLE MYELOMA (MM)

b) Pathogenesis and Laboratory Diagnosis

1. Plasma cell transformation: Normal plasma cells undergo malignant transformation, often with cytogenetic abnormalities (t(4;14), t(14;16), del 17p, del 13q)
2. Bone marrow infiltration: Malignant plasma cells proliferate in marrow (>10% in symptomatic MM)
3. Monoclonal protein (M-protein) production: All malignant plasma cells produce identical immunoglobulin (usually IgG > IgA > IgM > IgD > IgE or light chains only = Bence Jones protein)
4. Bone destruction:
   • Malignant plasma cells produce RANKL and inhibit OPG (osteoprotegerin)
   • RANKL activates osteoclasts → osteolysis without osteoblast activation
   • Results in lytic lesions (no bone formation around them - "punched out")
   • Osteolysis → hypercalcemia → polyuria, polydipsia, constipation, confusion
5. Renal failure (Myeloma kidney):
   • Light chain cast nephropathy: Bence Jones proteins (light chains) precipitate in renal tubules → tubular obstruction and inflammation
   • Hypercalcemia, amyloidosis, urate nephropathy also contribute
6. Immunosuppression: Normal immunoglobulin production suppressed ("immune paresis") → increased infection risk
7. Anemia: Marrow infiltration, renal failure (reduced EPO), chronic disease

CRAB Criteria (symptomatic myeloma):

• Calcium elevated (>11.5 mg/dl)
• Renal failure (creatinine >2 mg/dl)
• Anemia (Hb <10 g/dl)
• Bone lesions (lytic lesions, osteopenia, pathological fractures)

Laboratory Diagnosis

• Symptomatic MM: Clonal BM plasma cells ≥10% + any CRAB criterion OR myeloma-defining events (BM plasma cells ≥60%, SFLC ratio ≥100, >1 focal lesion on MRI)

• VRd regimen: Bortezomib (proteasome inhibitor) + Lenalidomide + Dexamethasone
• Autologous stem cell transplantation (ASCT) in eligible patients
• Bisphosphonates (zoledronic acid) for bone disease
• Daratumumab (anti-CD38 monoclonal antibody): newer highly effective option

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SECTION E: REASONING QUESTIONS (5x3 = 15 Marks)

Q13. Role of Anticoagulants Used in Hematology Lab - Name all anticoagulants and their uses

Definition

Why Anticoagulants Are Needed

• Cells to remain unaltered
• Clotting factors preserved (for coagulation tests)
• Cell-plasma ratio maintained (for hematocrit)

Anticoagulants - Complete List and Uses

• Tube color: Purple/lavender
• Mechanism: Chelates (binds) calcium ions → prevents all calcium-dependent coagulation steps
• Uses:
  • Complete Blood Count (CBC) / Full Blood Count
  • Peripheral blood smear
  • Reticulocyte count
  • HbA1c estimation
  • Blood grouping and cross-matching
  • Immunohematology (Coombs test)
  • CD4/CD8 counts (HIV monitoring)
• Advantages: Best preserves cell morphology; prevents platelet clumping
• Special note: K3-EDTA preferred over K2-EDTA; EDTA causes platelet satellitism (pseudo-thrombocytopenia) - repeat with citrate if suspected

• Tube color: Blue
• Mechanism: Chelates calcium (reversible chelation); anticoagulant effect reversed by adding calcium
• Ratio: 9 parts blood : 1 part citrate (critical - must be exact fill)
• Uses:
  • Prothrombin time (PT/INR) - most important use
  • aPTT (activated partial thromboplastin time)
  • Fibrinogen estimation
  • D-dimer assay
  • All coagulation studies
  • ESR (Westergren method): 4:1 ratio (blood:citrate)
• Advantages: Reversible chelation allows addition of calcium to activate clotting in PT/aPTT assays

• Tube color: Green
• Mechanism: Activates antithrombin III → inhibits thrombin (Factor IIa), Factor Xa, IXa, XIa, XIIa
• Uses:
  • Plasma chemistry tests requiring plasma (not serum)
  • Chromosomal studies (cytogenetics): Heparin is anticoagulant of choice for lymphocyte cultures (karyotyping)
  • Plasma osmolality
  • Arterial blood gas (sodium heparin - ABG syringe)
  • Some specialized tests
• NOT used for: CBC (heparin causes clumping/poor staining of cells), coagulation tests

• Tube: Now largely replaced by citrate and EDTA
• Mechanism: Precipitates calcium as calcium oxalate (irreversible)
• Historical uses: Blood glucose (with fluoride), older coagulation tests
• Fluoride-Oxalate combination (Grey tube):
  • Sodium fluoride inhibits glycolysis (preserves glucose for 24+ hours)
  • Oxalate anticoagulates
  • Uses: Blood glucose estimation, GTT (glucose tolerance test)

• Solution A or B
• Mechanism: Citrate chelates calcium + acid preserves cells + dextrose provides energy for RBC metabolism
• Uses:
  • Blood banking: Storage of whole blood and red cell concentrate (extends shelf life)
  • HLA typing
  • DNA studies
  • Paternity testing

• Mechanism: Citrate (anticoagulant + preservative) + Phosphate (maintains ATP) + Dextrose (energy) + Adenine (resynthesizes ATP)
• Uses:
  • Blood banking: Storage of PRBC for 35 days at 2-6°C
  • Most widely used blood bag anticoagulant-preservative

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Q14. Heat Coagulation Test for Protein - Why Only Upper Third Is Heated; Principle; Causes of Proteinuria

Why Only the Upper Third Is Heated

1. The lower two-thirds remain unheated as a control
2. When heat coagulates the upper portion, any turbidity that appears is compared to the clear lower portion
3. This eliminates the possibility of phosphates or carbonates causing turbidity - phosphates precipitate on heating but redissolve on adding dilute acetic acid; protein precipitate does NOT dissolve
4. The lower clear portion confirms the baseline turbidity of the sample
5. If the entire tube was heated, comparison would be impossible

• Protein turbidity persists/increases on adding acetic acid
• Phosphate/carbonate turbidity dissolves on adding acetic acid

Principle of Heat Coagulation Test

1. Protein denaturation: Heat (at ~60-100°C) disrupts hydrogen bonds and hydrophobic interactions that maintain protein secondary/tertiary structure
2. Unfolding: Polypeptide chains unfold, exposing hydrophobic core
3. Aggregation: Exposed hydrophobic regions of multiple unfolded proteins interact and aggregate
4. Precipitation: Aggregated proteins precipitate out of solution → visible turbidity/flocculation
5. At pH near the isoelectric point (pI), proteins have zero net charge → minimum solubility → maximum precipitation
6. Acetic acid adjusts pH toward isoelectric point of albumin, maximizing its precipitation

1. Take clear urine (centrifuge if turbid, filter if deposits)
2. Fill 3/4 of the test tube with urine
3. Hold the tube at 45° and heat only the upper 1/3 over flame
4. Observe for turbidity/precipitate
5. Add 2-3 drops of 1% acetic acid:
   • Phosphates dissolve → not protein
   • Protein turbidity persists → heat coagulation test positive
6. Grade: Faint haze (trace), definite white cloud (1+), heavy flocculation (2-3+), solid coagulum (4+)

Causes of Proteinuria

• Loss of glomerular filtration barrier (podocytes, GBM, fenestrated endothelium)
• Causes: Nephrotic syndrome (minimal change disease, membranous nephropathy, FSGS, diabetic nephropathy), glomerulonephritis
• Predominantly albumin loss (>3.5 g/day in nephrotic syndrome)

• Impaired reabsorption of normally filtered low molecular weight proteins
• Causes: Acute tubular necrosis, Fanconi syndrome, heavy metal poisoning (lead, cadmium), aminoglycoside nephrotoxicity
• Low MW proteins (beta-2 microglobulin, alpha-1 microglobulin)

• Plasma proteins exceed reabsorptive capacity of normal tubules
• Causes:
  • Bence Jones proteinuria (Multiple myeloma): Free immunoglobulin light chains
  • Myoglobinuria (rhabdomyolysis): Myoglobin
  • Hemoglobinuria (hemolysis): Hemoglobin

• Addition of protein to urine below the glomerulus
• Causes: UTI, pyelonephritis, urogenital tumors, Tamm-Horsfall protein (normal renal tubule secretion)

• Transient, benign
• Causes: Orthostatic/postural proteinuria (common in young men, resolves lying down), fever, exercise, emotional stress, cold exposure

• Increased delivery of proteins to glomerulus
• Severe hemolysis, extensive tissue injury (crush syndrome)

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Q15. CSF Forms a Cobweb Clot on Standing in Suspected Meningitis

a) What is the Diagnosis?

• High fibrinogen content in the CSF (elevated protein in TB meningitis)
• The fibrinogen polymerizes on standing to form a delicate cobweb/pellicle

b) Differentiation from Pyogenic Meningitis

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Q16. 6-month-old baby, pallor, increased reticulocyte count

a) Reasoning and Causes for High Reticulocyte Count

• Reticulocytes are immature RBCs that still contain residual RNA (ribosomal RNA and mRNA)
• Normally: 0.5-2.5% of circulating RBCs
• Elevated reticulocyte count = increased erythropoietic drive in the bone marrow (response to anemia)

• At birth, HbF predominates; switch to HbA starts at ~3-6 months
• Physiological nadir of Hb occurs at 2-3 months (physiological anemia of infancy)
• Reticulocytosis at 6 months with pallor → consider: Thalassemia major (HbF switching to HbA reveals the deficit), G6PD deficiency, hereditary spherocytosis, or hemolytic disease

b) Stains Used for Reticulocytes

• Method: Equal volumes blood + NMB solution, incubate 10-15 min at 37°C, make smear
• Staining: Residual RNA precipitates as blue-green granules/filaments (reticulum)
• WHO recommended method for reticulocyte count
• Also precipitates: HbH inclusions (multiple small blue dots throughout cell in HbH disease), Heinz bodies

• Similar to NMB; older method
• Stains RNA as blue reticulum
• Used in some centers

• Binds RNA, visualized under fluorescence microscope
• More sensitive, used in automated analyzers
• Orange fluorescence indicates RNA content

• Uses fluorescent dyes (thiazole orange or oxazine 750) that bind nucleic acids
• Automated reticulocyte count + immature reticulocyte fraction (IRF) - markers of marrow activity

• Adults: 0.5-2.5%
• Neonates: 2.5-6.5% (higher - transitioning from fetal erythropoiesis)
• Absolute reticulocyte count: 25,000-75,000/mcL

CRC = Reticulocyte % × (Patient's Hb / Normal Hb)

RPI = CRC / Maturation factor RPI >3 = adequate regenerative response (hemolysis/blood loss) RPI <2 = hypoproliferative (bone marrow failure, nutritional deficiency)

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Q17. Why is Pap Smear Recommended as a Routine Screening Test? Describe All Normal Cells Seen in Pap Smear

Why Pap Smear is Recommended as Routine Screening

1. Cervical cancer is preventable: Pap smear detects precancerous lesions (CIN - Cervical Intraepithelial Neoplasia) years before they progress to invasive cancer, allowing timely intervention
2. HPV (Human Papillomavirus) as causative agent: Nearly 100% of cervical cancers are caused by high-risk HPV (types 16, 18, 31, 33, 45). Pap smear detects HPV-related cytological changes (koilocytes, dysplastic cells)
3. Long premalignant phase: CIN I → CIN II → CIN III → CIS → Invasive cancer takes 10-15 years, providing a wide window for detection and treatment
4. Simple, inexpensive, non-invasive: Easy to perform in primary care settings; cost-effective for mass screening
5. Proven mortality reduction: Mass Pap screening has reduced cervical cancer mortality by >70% in countries with organized screening programs
6. Detects other conditions: Also detects endometrial cells, vaginal infections (Trichomonas, Candida, bacterial vaginosis), and inflammatory changes

• Start screening at 21 years (or within 3 years of sexual debut)
• Repeat every 3 years (cytology alone)
• Co-testing with HPV DNA every 5 years (age 30-65)
• Stop at age 65 if adequate prior negative screening

Collection

• Ectocervical scraping: Ayre's spatula (samples ectocervix and transformation zone)
• Endocervical sampling: Cytobrush/endocervical brush (samples endocervical canal)
• Liquid-based cytology (LBC): SurePath or ThinPrep - preferred modern method (reduces inadequate samples)
• Conventional smear: Spread directly on glass slide, fix immediately in 95% ethyl alcohol

Staining

1. Harris's Hematoxylin (nuclei)
2. OG-6 (Orange G - stains keratin)
3. EA-50 or EA-65 (Eosin-Azure - cytoplasmic staining)

Normal Cells Seen in Pap Smear

• Columnar, tall cells from endocervix
• Picket-fence / honeycomb arrangement in sheets
• Abundant pale cytoplasm with mucin vacuoles
• Small basal nucleus
• Presence on smear indicates sampling of transformation zone (adequate smear)

• Small, round cells in tight clusters ("two-cell pattern" with stromal cells)
• High nuclear-cytoplasmic ratio
• Normal to see in first 10-12 days of menstrual cycle
• Abnormal if seen after day 12 in postmenopausal women (endometrial pathology)

• From transformation zone (squamocolumnar junction)
• Round/oval with abundant cytoplasm, dense cytoplasm
• Appearance intermediate between squamous and columnar
• "Spider" or "caudate" cells: Metaplastic cells with cytoplasmic projections

• Neutrophils (polymorphs): Small, lobulated nuclei; normal in small numbers
• Histiocytes (macrophages): Bean-shaped/kidney-shaped nucleus; vacuolated cytoplasm; normal as single cells
• Lymphocytes: Rare; increased in follicular cervicitis

• Lactobacilli (Doderlein's bacilli): Normal vaginal flora; large, plump bacilli; indicate healthy acidic vaginal environment (pH 3.8-4.5)

• Most important area to sample
• Where columnar epithelium (endocervix) meets squamous epithelium (ectocervix)
• Most cervical cancers arise here
• Presence of both endocervical cells and metaplastic cells confirms adequate sampling

Reporting (Bethesda System 2014)

• NILM: Negative for Intraepithelial Lesion or Malignancy (normal)
• ASC-US: Atypical squamous cells of undetermined significance
• LSIL: Low-grade squamous intraepithelial lesion (CIN 1, HPV effect)
• HSIL: High-grade squamous intraepithelial lesion (CIN 2, CIN 3, CIS)
• Squamous cell carcinoma
• Glandular abnormalities: AGC, AIS, Adenocarcinoma

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Summary of All Key Answers

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What You Have - Complete Exam Answers

MCQ Section (20 Marks)

Long Essay (10 Marks)

• Diagnosis (HbSS with clinical + electrophoresis findings)
• Full molecular pathogenesis (Glu→Val substitution, polymerization, sickling mechanism)
• Complete lab investigations table
• All 10 major complications with mechanisms

Short Notes (35 Marks)

1. Blood transfusion reactions - acute/delayed, immune/non-immune, full table with management
2. Semen analysis - WHO parameters, terminology (azoospermia, oligospermia, etc.), procedure
3. Hereditary spherocytosis - pathogenesis, osmotic fragility diagram, labs, treatment
4. Pernicious anemia - IF pathway, SACD, Schilling test, labs, B12 treatment
5. ITP - pathogenesis (anti-GPIIb/IIIa), labs (increased megakaryocytes), treatment ladder
6. Beneficence - four principles of bioethics, clinical applications, distinction from non-maleficence
7. FNAC - comprehensive advantages/disadvantages table, comparison with core biopsy

Applied Questions (20 Marks)

1. Hemophilia A - intrinsic pathway defect, Factor VIII assay, severity classification
2. CML - BCR-ABL1, Philadelphia chromosome, peripheral smear (left shift + basophilia), LAP score low, imatinib treatment
3. Hodgkin lymphoma - RS/owl-eye cells, all 4 subtypes (NS/MC/LR/LD) with gross/micro, Ann Arbor staging
4. Multiple myeloma - CRAB criteria, RANKL-mediated lytic lesions, SPEP M-spike, Bence Jones protein, treatment

Reasoning Questions (15 Marks)

1. Anticoagulants - EDTA, citrate, heparin, oxalate, ACD, CPDA-1 with mechanisms and uses
2. Heat coagulation test - why upper third only, principle, all causes of proteinuria
3. CSF cobweb clot - TB meningitis diagnosis, full differentiation table vs. pyogenic meningitis
4. Reticulocytes in infant - causes of high retic count, all stains (new methylene blue, BCB, acridine orange), corrected count formula
5. Pap smear - why recommended (HPV, CIN, 10-15 year window), all normal cells (superficial/intermediate/parabasal/endocervical), Bethesda reporting system

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All Answers Are Above - Navigation Guide

MCQ Answers (Quick Reference)

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Long Essay - Sickle Cell Anemia

• Diagnosis: HbSS (sickle cell anemia) - autosplenectomy, reticulocytosis, Hb electrophoresis confirms
• Pathogenesis: Glu→Val mutation at beta-6, HbS polymerization on deoxygenation, sickling, vascular occlusion
• Labs: Peripheral smear (sickle cells, target cells), elevated reticulocytes, high LDH/bilirubin, Hb electrophoresis
• Complications: Vaso-occlusive crisis, acute chest syndrome, stroke, autosplenectomy, aplastic crisis, AVN, gallstones, renal papillary necrosis

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Short Notes Topics Covered

1. Blood Transfusion Reactions - AHTR, FNHTR, TRALI, TACO, delayed reactions
2. Semen Analysis - WHO parameters, all terminology (oligospermia, azoospermia, teratospermia, etc.)
3. Hereditary Spherocytosis - ankyrin defect, osmotic fragility, MCHC elevated, negative Coombs
4. Pernicious Anemia - autoimmune, anti-IF antibodies, SACD, Schilling test, B12 IM treatment
5. ITP - anti-GPIIb/IIIa antibodies, increased megakaryocytes, steroids/IVIG/splenectomy/TPO-RA
6. Beneficence - "do good," four principles, conflict with autonomy, clinical applications
7. FNAC - advantages (quick, cheap, minimal invasion) vs. disadvantages (no architecture, operator-dependent)

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Applied Questions

• Hemophilia A - X-linked, Factor VIII deficiency, prolonged aPTT only, ristocetin normal (distinguishes from vWD), Factor VIII assay confirms
• CML - t(9;22) BCR-ABL1, LAP score LOW, basophilia, left shift, imatinib (TKI) treatment
• Hodgkin Lymphoma - RS cells (owl eyes), 4 subtypes (NS > MC > LR > LD), ABVD chemotherapy
• Multiple Myeloma - CRAB criteria, punched-out lytic lesions, M-spike on SPEP, Bence Jones protein, VRd + ASCT

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Reasoning Questions

• Anticoagulants - EDTA (CBC), Citrate (PT/aPTT), Heparin (cytogenetics), Fluoride-oxalate (glucose), CPDA-1 (blood banking)
• Heat Coagulation Test - upper 1/3 only so lower 2/3 acts as control; acetic acid differentiates phosphates from protein
• CSF Cobweb Clot - TB meningitis; vs. pyogenic: TB has lymphocytes + high protein + low glucose + ADA elevated; pyogenic has neutrophils + very low glucose + gram positive organisms
• Reticulocytes - high count = regenerative anemia; stains: New Methylene Blue (best), Brilliant Cresyl Blue, Acridine orange
• Pap Smear - detects CIN (10-15 yr window before invasive cancer); normal cells: superficial (pyknotic nucleus, pink), intermediate (vesicular nucleus, blue-green), parabasal, endocervical (honeycomb pattern), metaplastic cells

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HEMATOLOGY INTERNAL ASSESSMENT - COMPLETE ANSWERS

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PART 1: MCQs (20 Marks)

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SCENARIO 18 - 5yr boy, weakness 3 yrs, repeated transfusions, Hb 6.2, MCV low, MCH low, MCHC low, RDW NORMAL

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SCENARIO 19 - 25yr female, postpartum hemorrhage, pale, hypotensive, bleeding from MULTIPLE sites

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SCENARIO 20 - 25yr female, menorrhagia, Hb 6.2, MCV 68, MCH 20, MCHC 22, RDW 28 (HIGH)

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SCENARIO 21 - 9yr boy, fever, fatigue, bone pain, generalized lymphadenopathy, 70% blasts, anemia + thrombocytopenia + leukocytosis

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SCENARIO 22 - Healthy 30yr man donating blood

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SCENARIO 23 - 45yr man, yellowish eyes, PALE STOOLS, DARK URINE, bile pigments + bile salts in urine

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SCENARIO 24 - 10yr girl, recurrent nosebleeds, easy bruising, family history, platelets NORMAL, BT prolonged, PT normal, aPTT mildly prolonged

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SCENARIO 25 - 30yr female, severe anemia, needs transfusion

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SCENARIO 26 - 32yr female, tiredness, petechiae, Hb 6.5, WBC 2000, platelets 45,000

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SCENARIO 27 - 50yr male, high fever, WBC 60,000, neutrophils + bands + metamyelocytes + myelocytes, Hb normal, platelets normal, LAP score HIGH

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PART 2: LONG ESSAY (10 Marks)

A 10yr old boy - severe pain, frequent vaso-occlusive crises, Hb 6.5, reticulocytes 10%, small spleen on USG, Hb electrophoresis confirms diagnosis

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a) PROBABLE DIAGNOSIS: SICKLE CELL ANEMIA (HbSS Disease)

• Recurrent vaso-occlusive pain crises in a child
• Severe hemolytic anemia (Hb 6.5)
• High reticulocyte count (10%) = compensatory bone marrow response to hemolysis
• Small spleen on USG = AUTOSPLENECTOMY (repeated splenic infarctions → fibrosis → small non-functional spleen)
• Hb electrophoresis: >90% HbS, absent HbA, elevated HbF = confirms HbSS

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b) ETIOPATHOGENESIS

• Point mutation in beta-globin gene on chromosome 11
• Glutamic acid → Valine at position 6 of beta-globin chain
• Produces HbS (alpha2-betaS2) instead of normal HbA
• In heterozygotes (HbAS): sickle cell TRAIT - usually asymptomatic
• In homozygotes (HbSS): sickle cell ANEMIA - full disease

1. On deoxygenation, HbS molecules undergo polymerization
2. HbS polymers form long rod-like fibers (tactoids) that distort RBC into sickle/crescent shape
3. Early sickling is reversible (reoxygenation restores shape)
4. After repeated cycles, sickling becomes IRREVERSIBLE
5. Irreversibly sickled cells: rigid, dehydrated, damaged membranes

• Hypoxia / low pO2
• Acidosis
• Dehydration
• Cold temperatures
• Infection
• High altitude

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c) LABORATORY INVESTIGATIONS

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d) COMPLICATIONS

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PART 3: SHORT NOTES (7 x 5 = 35 Marks)

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NOTE 2: BLOOD TRANSFUSION REACTIONS

Classification: Immune vs Non-Immune / Acute (<24hrs) vs Delayed (>24hrs)

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ACUTE IMMUNE REACTIONS

• Most DANGEROUS acute reaction
• Cause: ABO incompatibility (wrong blood given - clerical error)
• Mechanism: Recipient IgM antibodies + donor RBCs → complement activation → INTRAVASCULAR hemolysis
• Features: Fever, chills, back/flank pain, hemoglobinuria (red/brown urine), hypotension, DIC, renal failure
• Management: STOP transfusion immediately. IV fluids. Monitor urine output. Treat DIC. Send blood to lab (repeat cross-match, DAT, urine for Hb).

• MOST COMMON reaction
• Cause: Recipient antibodies against donor leukocyte antigens; cytokines in stored blood
• Features: Fever (>1°C rise), chills, rigors within 1-6 hours of transfusion
• Management: Stop transfusion. Paracetamol. Use leukodepleted blood in future.

• Cause: Antibodies against donor plasma proteins
• Features: Urticaria, itching, flushing - NO fever
• Management: Antihistamine. Can resume transfusion slowly if mild.

• Rare. IgA-deficient recipients with anti-IgA antibodies
• Features: Hypotension, bronchospasm, angioedema - NO fever
• Management: STOP transfusion. Adrenaline/epinephrine. Use washed RBCs in future.

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ACUTE NON-IMMUNE REACTIONS

• Cause: Donor antibodies against recipient neutrophil antigens → neutrophil activation in lungs
• Features: Acute respiratory distress within 6 hours, bilateral pulmonary infiltrates, hypoxia, NON-cardiogenic pulmonary edema
• Management: Stop transfusion. Oxygen. Ventilatory support.

• Cause: Rapid transfusion / excess volume in elderly or cardiac patients
• Features: Dyspnea, hypertension, pulmonary edema, raised JVP
• Management: Diuretics. Slow transfusion rate. Sit patient upright.

• Cause: Bacterial contamination (Yersinia enterocolitica in RBCs; Staphylococci in platelets)
• Features: High fever, rigors, hypotension, shock
• Management: Stop transfusion. Blood cultures (patient + bag). Broad-spectrum antibiotics.

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DELAYED REACTIONS

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NOTE 3: SEMEN ANALYSIS

Collection

• Sexual abstinence for 2-5 days before collection
• Collected by masturbation into sterile wide-mouth container
• Examined within 60 minutes of collection at 37°C

Normal Parameters (WHO 2021 - 6th Edition)

Terminology for Abnormal Results

Steps in Semen Analysis

1. Macroscopic: Volume, color (whitish-grey), pH, viscosity, liquefaction time
2. Microscopic (wet preparation): Motility assessment (progressive, non-progressive, immotile)
3. Concentration: Improved Neubauer hemocytometer
4. Morphology: PAP or Shorr stain - head, midpiece, tail defects assessed
5. Vitality: Eosin-nigrosin stain (dead cells take up eosin = pink; live cells = white)
6. MAR test / IBT: For antisperm antibodies

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NOTE 4: HEREDITARY SPHEROCYTOSIS

Genetics

• Autosomal dominant (75%) or autosomal recessive
• Mutations in: Ankyrin (most common, 40-65%), Band 3, spectrin, protein 4.2

Pathogenesis

1. Deficiency of membrane skeletal proteins (ankyrin-spectrin-Band 3 complex)
2. Membrane lipid is lost by vesiculation
3. Surface area decreases relative to cell volume
4. RBC becomes SPHERICAL (minimum surface area for given volume)
5. Spherocytes are RIGID - cannot deform in splenic sinusoids
6. Trapped and destroyed in spleen = extravascular hemolysis
7. MCHC elevated because cells become dehydrated

Clinical Features

• Anemia (Hb 8-12 g/dl typically)
• Jaundice (intermittent, unconjugated)
• Splenomegaly (splenic hyperactivity)
• Pigment gallstones (from chronic hemolysis)
• Aplastic crisis (Parvovirus B19)

Laboratory Findings

Treatment

• Folic acid supplementation (ongoing)
• Splenectomy = curative (removes site of destruction); give pneumococcal, meningococcal, Hib vaccines before splenectomy
• Cholecystectomy if symptomatic gallstones

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NOTE 5: PERNICIOUS ANEMIA

Normal Vitamin B12 Absorption Pathway

1. Dietary B12 + haptocorrin (salivary R-binder) in stomach
2. Pancreatic proteases release B12 in duodenum
3. B12 binds Intrinsic Factor (IF) (secreted by gastric parietal cells)
4. B12-IF complex absorbed at terminal ileum (cubam receptor)
5. Transported by transcobalamin II in blood

Pathogenesis of Pernicious Anemia

• Autoimmune T-cell mediated destruction of parietal cells
• Type I antibodies (blocking): Against IF binding site for B12 - present in 50-60% (SPECIFIC)
• Type II antibodies (binding): Against IF-B12 complex
• Anti-parietal cell antibodies: Present in 90% (SENSITIVE but not specific)
• Loss of parietal cells → no IF → no B12 absorption
• B12 deficiency → defective DNA synthesis (thymidine synthesis) → megaloblastic changes

Clinical Features

• Macrocytic anemia (MCV often >120 fl)
• Weakness, pallor, mild lemon-yellow jaundice

• Glossitis (Hunter's glossitis) - smooth, beefy red, sore tongue
• Atrophic gastritis, achlorhydria

• B12 deficiency → defective myelin synthesis (methionine pathway disrupted)
• Demyelination of: posterior columns (vibration + position sense loss) + lateral corticospinal tracts (spastic weakness)
• Features: Tingling/numbness in hands and feet, loss of vibration sense, positive Romberg's sign, spastic paraparesis
• SACD does NOT occur in folate deficiency - important exam point

Laboratory Findings

Treatment

• Hydroxocobalamin 1000 mcg IM daily x 7 days → weekly x 4 → monthly for LIFE
• Oral B12 NOT effective (cannot absorb without IF)
• Response: Reticulocytosis in 3-5 days; Hb normalizes in 8 weeks

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NOTE 6: IMMUNE THROMBOCYTOPENIC PURPURA (ITP)

Classification

• Primary ITP: No identifiable cause
• Secondary ITP: Associated with SLE, HIV, HCV, H. pylori, drugs, CLL
• Acute ITP: Children, self-limiting, post-viral
• Chronic ITP: Adults (especially women 20-40 yrs), persists >12 months

Pathogenesis

1. Autoantibodies (IgG) produced against platelet surface glycoproteins - mainly GPIIb/IIIa and GPIb/IX
2. Antibody-coated platelets recognized by Fc receptors on SPLENIC macrophages
3. Extravascular destruction of platelets in the spleen
4. Antibodies may also inhibit megakaryocyte maturation → impaired platelet production
5. T-cell dysregulation: cytotoxic T cells directly kill platelets

Clinical Features

• Petechiae (pinpoint, non-blanching)
• Purpura and ecchymoses (easy bruising)
• Mucosal bleeding: epistaxis, gingival bleeding, menorrhagia
• GI/GU bleeding in severe cases
• Intracranial hemorrhage - rare but life-threatening (platelet count <10,000)
• NO splenomegaly - distinguishes from hypersplenism
• NO lymphadenopathy

Laboratory Findings

Treatment

• Prednisolone 1-2 mg/kg/day (reduces antibody production, blocks Fc receptors)
• IV Immunoglobulin (IVIG) 1 g/kg x 2 days (Fc receptor blockade) - for rapid response needed
• Anti-D immunoglobulin (in Rh-positive, non-splenectomized patients)

• Splenectomy - removes main site of platelet destruction; 60-70% long-term remission
• Rituximab (anti-CD20) - for refractory ITP

• TPO receptor agonists: Romiplostim, Eltrombopag (stimulate platelet production)

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NOTE 7: BENEFICENCE

Four Principles of Biomedical Ethics

1. Beneficence - do good
2. Non-maleficence - do no harm (Primum non nocere)
3. Autonomy - respect patient's right to make decisions
4. Justice - fair distribution of healthcare resources

Components of Beneficence

• Positive beneficence: Actively provide benefits, prevent harm, remove harm
• Utility/proportionality: Balance benefits against risks (risk-benefit analysis)
• Not merely "doing something good" but ensuring benefit OUTWEIGHS the risk

Applications in Hematology/Laboratory Medicine

Beneficence vs Non-maleficence

Conflict with Autonomy

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NOTE 8: ADVANTAGES AND DISADVANTAGES OF FNAC

Advantages

Disadvantages

FNAC vs Core Biopsy vs Excision Biopsy

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PART 4: APPLIED QUESTIONS (4 x 5 = 20 Marks)

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Q9: 6yr old boy, recurrent painful knee swelling after minor trauma, platelet count NORMAL, bleeding time NORMAL, aPTT PROLONGED

a) Likely Diagnosis: HEMOPHILIA A (Factor VIII Deficiency)

b) Pathogenesis and Laboratory Findings

• X-linked recessive (gene: F8, Xq28)
• Males affected; females are carriers
• Most common mutation: inversion of intron 22 (in ~45% of severe hemophilia A)
• About 30% are new mutations (no family history)

XIIa → XIa → IXa + VIIIa (TENASE complex) → Xa → Thrombin → Fibrin

• Factor VIIIa acts as cofactor for Factor IXa in the tenase complex
• This complex activates Factor X → common pathway → thrombin → fibrin clot
• In Hemophilia A: Factor VIII is absent/markedly reduced
• Tenase complex cannot form properly
• Factor X activation is severely impaired
• Insufficient thrombin → inadequate fibrin clot
• Primary hemostasis (platelet plug via vWF) is INTACT
• Secondary hemostasis (fibrin reinforcement) is DEFECTIVE

• Factor VIII concentrate replacement (recombinant preferred)
• Prophylactic infusions 3x/week in severe disease
• DDAVP (desmopressin) for mild hemophilia A (releases stored vWF-FVIII)
• Emicizumab (bispecific antibody) - new preventive treatment

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Q10: 55yr man, weight loss, MASSIVE SPLENOMEGALY, Hb 9.5, WBC 93,000/cmm, platelets 1.7 lakhs, peripheral smear shows granulocytes at ALL stages of maturation WITH BASOPHILIA

a) Diagnosis: CHRONIC MYELOID LEUKEMIA (CML)

b) Pathogenesis

• Reciprocal translocation: t(9;22)(q34;q11)
• BCR gene (chromosome 22) fuses with ABL1 gene (chromosome 9)
• Creates BCR-ABL1 fusion gene on the derivative chromosome 22 (Philadelphia chromosome)
• Encodes p210 BCR-ABL1 protein - a constitutively active tyrosine kinase
• This kinase continuously activates: RAS/MAPK, JAK/STAT, PI3K/AKT pathways
• Results in: Uncontrolled myeloid proliferation + inhibition of apoptosis + impaired differentiation

1. Chronic Phase (years): Indolent; well-controlled with TKI
2. Accelerated Phase: Blasts 10-19%, increasing basophilia, clonal evolution
3. Blast Crisis: >20% blasts (myeloid or lymphoid) - resembles acute leukemia; very poor prognosis

c) Peripheral Smear Findings in CML

1. Marked leukocytosis - WBC typically 50,000-200,000/cmm
2. Full left shift (myeloid spectrum): myeloblasts → promyelocytes → myelocytes → metamyelocytes → band forms → mature neutrophils. "Myelocyte bulge" - peak at myelocyte stage
3. BASOPHILIA - absolute basophilia is the HALLMARK of CML (distinguishes from leukemoid reaction)
4. Eosinophilia - also present
5. Thrombocytosis - platelets often elevated (>400,000) in early CML
6. Minimal blasts (<5%) in chronic phase
7. No toxic granulation or Dohle bodies (unlike leukemoid reaction)

• Cytogenetics: Philadelphia chromosome (karyotype)
• FISH: BCR-ABL1 fusion - sensitive
• RT-PCR (quantitative): BCR-ABL1 transcript levels - monitors treatment response
• Bone marrow trephine: Hypercellular, myeloid hyperplasia, increased reticulin

• Imatinib (1st generation TKI) - transformed CML from fatal disease to chronic manageable condition
• Dasatinib, Nilotinib (2nd gen TKI) for resistance/intolerance
• Ponatinib, Asciminib (3rd gen TKI)
• Allogenic stem cell transplant for blast crisis or TKI failure

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Q11: 30yr man, weakness, fever, NIGHT SWEATS, weight loss 1yr, multiple RUBBERY DISCRETE lymph nodes in neck, biopsy shows OWL EYE cells

a) Diagnosis: HODGKIN'S LYMPHOMA (Classical)

b) Classification, Gross and Microscopy

• Large binucleated or multinucleated cell
• Each nucleus contains a LARGE prominent EOSINOPHILIC NUCLEOLUS resembling an owl's eye
• Seen within a reactive inflammatory background
• RS cells are the NEOPLASTIC cells (clonal B-cell origin)
• CD30 positive, CD15 positive, CD45 negative, CD20 usually negative
• RS cells are a MINORITY (<5%) - surrounded by reactive inflammatory cells

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CLASSIFICATION (WHO - 5 types):

• Young females, mediastinal mass
• Variant RS cells: LACUNAR CELLS (retraction artifact in formalin)
• BROAD COLLAGEN BANDS dividing lymph node into nodules
• EBV association: Low (<10%)
• Good prognosis

• Older adults, males > females
• Classic RS cells in MIXED inflammatory background (eosinophils, plasma cells, lymphocytes, histiocytes)
• Diffuse effacement of architecture
• EBV association: HIGH (70%)
• Intermediate prognosis

• Background predominantly lymphocytes
• Classic RS cells sparse
• Good prognosis

• Elderly, HIV patients
• Many RS cells, few lymphocytes
• EBV association: Very high
• Worst prognosis

• Neoplastic cells = "POPCORN CELLS" (LP cells / L&H cells)
• CD20+, CD30-, CD15- (opposite of classical HL)
• EBV negative
• Best prognosis

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GROSS APPEARANCE

• Lymph nodes: Enlarged, rubbery, discrete, non-tender
• Cut surface: Gray-white, "fish flesh" appearance
• Nodular sclerosis: White fibrous bands visible on gross cut section
• Contiguous spread: HL spreads in PREDICTABLE contiguous pattern (cervical → mediastinal → para-aortic → spleen)

MICROSCOPY

1. Reed-Sternberg cells (pathognomonic)
2. Variant RS cells specific to subtype (lacunar cells in NS; popcorn cells in NLPHL)
3. Background inflammatory infiltrate (varies by subtype)

• CD30 positive (membrane + Golgi pattern)
• CD15 positive
• CD45 (LCA) negative
• PAX5 weakly positive
• CD20 usually negative

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STAGING (Ann Arbor / Lugano)

• Stage I: Single lymph node region or single extralymphatic site
• Stage II: Two or more regions, SAME side of diaphragm
• Stage III: Regions on BOTH sides of diaphragm
• Stage IV: Disseminated extranodal involvement

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Q12: 62yr man, chronic back pain, weakness 3 months, X-ray skull shows MULTIPLE PUNCHED-OUT LYTIC LESIONS, anemia, HYPERCALCEMIA, raised creatinine, raised ESR

a) Probable Diagnosis: MULTIPLE MYELOMA (MM)

b) Pathogenesis and Lab Diagnosis

10% clonal plasma cells in bone marrow (symptomatic MM) → normal hematopoiesis suppressed → anemia, leukopenia, thrombocytopenia.

• Myeloma cells produce RANKL (receptor activator of NF-kB ligand) and inhibit OPG (osteoprotegerin)
• RANKL activates OSTEOCLASTS → lytic bone destruction
• NO compensatory osteoblast activation → pure lytic lesions (no sclerosis)
• "Punched-out" lytic lesions on X-ray (skull, spine, pelvis, ribs)
• Osteolysis → HYPERCALCEMIA

• Free light chains (Bence Jones proteins) filtered by glomeruli, precipitate in tubules
• Cast nephropathy → tubular obstruction and inflammation
• Also: hypercalcemia nephropathy, amyloidosis, contrast nephrotoxicity

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CRAB Criteria (Symptomatic Myeloma):

• C - Calcium elevated (>11.5 mg/dl or >1 mg/dl above upper normal)
• R - Renal failure (creatinine >2 mg/dl)
• A - Anemia (Hb <10 g/dl)
• B - Bone lesions (lytic lesions/osteoporosis/pathological fractures)

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LABORATORY DIAGNOSIS

• VRd: Bortezomib (proteasome inhibitor) + Lenalidomide + Dexamethasone
• Autologous Stem Cell Transplantation (ASCT) in eligible patients (<65-70 yrs)
• Bisphosphonates (zoledronic acid) for bone disease prevention
• Daratumumab (anti-CD38 monoclonal antibody) - highly effective newer agent

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PART 5: REASONING QUESTIONS (5 x 3 = 15 Marks)

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Q13: Role of Anticoagulants in Hematology Lab - Name all anticoagulants and their uses

Why Anticoagulants Are Needed

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1. EDTA (Ethylenediamine Tetraacetic Acid)

• Tube: Purple/lavender top
• Mechanism: Chelates (binds/sequesters) calcium ions irreversibly → blocks all calcium-dependent coagulation steps
• Uses:
  • CBC / Full Blood Count (gold standard anticoagulant for CBC)
  • Peripheral blood smear
  • Reticulocyte count
  • HbA1c
  • Blood grouping and cross-matching
  • Coombs test (DAT/IAT)
  • CD4/CD8 counts (HIV monitoring)
  • DNA studies
• Note: Causes pseudo-thrombocytopenia (platelet clumping in some patients) - if suspected, repeat with citrate tube

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2. Sodium Citrate (3.2%)

• Tube: Blue top
• Mechanism: Chelates calcium REVERSIBLY → anticoagulation can be reversed by adding calcium back in the assay
• Critical ratio: 9 parts blood : 1 part citrate (tube must be filled exactly)
• Uses:
  • PT/INR - most important
  • aPTT
  • All coagulation studies
  • Fibrinogen estimation
  • D-dimer assay
  • ESR (Westergren method): 4 parts blood : 1 part citrate
• Why citrate and not EDTA for coagulation tests? Because citrate chelation is reversible - calcium can be added back to activate clotting in the PT/aPTT assay.

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3. Heparin (Lithium heparin / Sodium heparin)

• Tube: Green top
• Mechanism: Activates Antithrombin III → inhibits thrombin (Factor IIa), Factor Xa, IXa, XIa, XIIa
• Uses:
  • Plasma chemistry tests
  • Chromosomal studies (cytogenetics/karyotyping) - anticoagulant of choice for lymphocyte cultures
  • Arterial blood gas (sodium heparin syringe)
  • Plasma osmolality
• NOT used for CBC - causes cell clumping and poor staining

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4. Fluoride-Oxalate

• Tube: Grey top
• Two components:
  • Sodium fluoride: Inhibits enolase enzyme → blocks glycolysis → preserves glucose for 24+ hours
  • Potassium oxalate: Precipitates calcium as calcium oxalate → anticoagulation
• Uses:
  • Blood glucose estimation
  • Glucose Tolerance Test (GTT)
  • Lactate measurement

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5. ACD (Acid Citrate Dextrose)

• Tube: Yellow top / blood bag (solution A or B)
• Mechanism: Citrate (anticoagulation) + Acid (lowers pH, reduces ATP consumption) + Dextrose (energy for RBC metabolism)
• Uses:
  • Blood banking - storage of whole blood
  • HLA typing
  • DNA studies
  • Paternity testing
  • Lymphocyte preservation

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6. CPDA-1 (Citrate Phosphate Dextrose Adenine)

• Used in: Blood collection bags
• Mechanism: Citrate (anticoagulant + preservative) + Phosphate (maintains 2,3-DPG and ATP) + Dextrose (energy) + Adenine (ATP resynthesis)
• Uses:
  • Blood banking: Storage of PRBCs for 35 days at 2-6°C
  • Most widely used blood bag anticoagulant-preservative solution

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Summary Table

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Q14: Heat Coagulation Test for Protein - Why Only Upper Third Heated; Principle; Causes of Proteinuria

Why ONLY the Upper Third is Heated

1. When the upper third is heated, any turbidity that appears must be compared to the CLEAR lower portion to confirm it is genuine
2. Phosphates and carbonates in urine also precipitate on heating → these can give false positive turbidity
3. To distinguish: 2-3 drops of 1% acetic acid are added to the turbid upper portion
   • Protein turbidity: PERSISTS or INCREASES on adding acetic acid (acetic acid brings pH toward isoelectric point of albumin → maximum precipitation)
   • Phosphate/carbonate turbidity: DISSOLVES on adding acetic acid
4. The unheated lower portion confirms that baseline urine is clear (rules out pre-existing turbidity from phosphates, cells, bacteria)

Principle of Heat Coagulation Test

1. Heat disrupts hydrogen bonds and hydrophobic interactions maintaining protein tertiary structure
2. Protein chains unfold (denaturation) - exposing hydrophobic core regions
3. Multiple unfolded proteins interact via exposed hydrophobic regions → AGGREGATION
4. Aggregated proteins precipitate → visible turbidity
5. Precipitation is maximum at the protein's isoelectric point (zero net charge = minimum solubility)
6. Acetic acid adjusts urine pH toward albumin's isoelectric point (pI ~4.7) → maximizes albumin precipitation

1. Take clear urine (centrifuge if turbid)
2. Fill 3/4 of test tube
3. Hold at 45° and heat ONLY upper 1/3 over flame
4. Observe for turbidity/precipitate
5. Add 2-3 drops 1% acetic acid
6. Interpret and grade:
   • Trace: Faint haze
   • 1+: Definite white cloud
   • 2+: Heavy flocculation
   • 3-4+: Solid coagulum

Causes of Proteinuria

• Loss of glomerular filtration barrier
• Causes: Nephrotic syndrome (minimal change disease, membranous nephropathy, FSGS, diabetic nephropathy), glomerulonephritis
• Predominantly albumin (>3.5 g/day in nephrotic syndrome)

• Impaired tubular reabsorption of low molecular weight proteins
• Causes: Acute tubular necrosis, Fanconi syndrome, heavy metal poisoning, aminoglycoside toxicity
• LMW proteins: beta-2 microglobulin, alpha-1 microglobulin

• Plasma proteins exceed tubular reabsorptive capacity
• Causes: Multiple myeloma (Bence Jones = free light chains), myoglobinuria (rhabdomyolysis), hemoglobinuria (hemolysis)

• Protein added to urine below the glomerulus
• Causes: UTI, pyelonephritis, urogenital tumors

• Orthostatic/postural proteinuria (common in young men, resolves lying down)
• Fever, vigorous exercise, emotional stress, cold exposure

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Q15: CSF forms a COBWEB CLOT on standing from suspected meningitis

a) Diagnosis: TUBERCULOUS MENINGITIS

• TB meningitis causes markedly elevated CSF protein (especially fibrinogen)
• On standing, fibrinogen polymerizes into fibrin
• Forms a delicate cobweb-like clot (pellicle) at the top of the tube
• This pellicle contains MTB bacilli → smear the clot for AFB staining (increases AFB detection)

b) Differentiation: TB Meningitis vs Pyogenic Meningitis

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Q16: 6-month-old baby, pallor, INCREASED reticulocyte count

a) Reasoning and Causes for High Reticulocyte Count

• Reticulocytes are immature RBCs retaining residual ribosomal RNA
• Normal range: 0.5-2.5% in adults; 2.5-6.5% in neonates
• Elevated reticulocyte count = REGENERATIVE anemia = bone marrow is actively producing RBCs in response to increased demand
• Elevated retics + pallor = hemolysis OR blood loss (marrow compensating)

• At birth: predominantly HbF; switch to HbA begins at 3-6 months
• Conditions presenting at this age: Thalassemia major, G6PD deficiency triggered by infection, hereditary spherocytosis, hemolytic disease of newborn (if severe)

b) Stains Used for Reticulocytes

• Equal volumes of blood + NMB solution → incubate 10-15 min at 37°C → smear
• Residual RNA precipitates as BLUE-GREEN granules/filaments (reticulum)
• Also demonstrates: HbH inclusions (multiple small blue dots = golf ball appearance) in HbH disease; Heinz bodies

• Similar principle to NMB; older method still used in some centers
• Stains RNA as blue reticulum

• Binds nucleic acids; visualized under fluorescence microscope
• More sensitive; used in automated analyzers
• Orange fluorescence indicates RNA content

• Fluorescent dyes: Thiazole orange or Oxazine 750
• Provides: Absolute reticulocyte count + Immature Reticulocyte Fraction (IRF) - best marker of bone marrow activity

Corrected Reticulocyte Count (CRC) = Reticulocyte % × (Patient Hb / Normal Hb)

Reticulocyte Production Index (RPI) = CRC / Maturation factor

• RPI >3 = adequate regenerative response (hemolysis/blood loss)
• RPI <2 = hypoproliferative (marrow failure, nutritional deficiency)

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Q17: Why is Pap Smear Recommended as Routine Screening? Describe All Normal Cells Seen

Why Pap Smear is Recommended as Routine Screening

1. Cervical cancer is preventable: Pap smear detects precancerous lesions (CIN - Cervical Intraepithelial Neoplasia) YEARS before they progress to invasive cancer
2. HPV causation: ~100% of cervical cancers caused by high-risk HPV (types 16, 18, 31, 33, 45). Pap smear detects HPV-related cytological changes (koilocytes = pathognomonic of HPV infection)
3. Long premalignant phase (10-15 years):
   • Normal → CIN I → CIN II → CIN III → Carcinoma in situ → Invasive cancer
   • This 10-15 year window allows DETECTION and TREATMENT before malignancy
4. Simple, inexpensive, non-invasive: Performed in OPD without anesthesia; cost-effective for mass screening
5. Proven mortality reduction: Mass Pap screening reduced cervical cancer mortality by >70% in countries with organized programs
6. Detects other conditions: Vaginal infections (Trichomonas, Candida, bacterial vaginosis), endometrial cells (flag for endometrial pathology in postmenopausal women), inflammatory changes

• Start at age 21 (or within 3 years of first sexual activity)
• Cytology alone: every 3 years
• Co-testing with HPV DNA: every 5 years (age 30-65)
• Stop at age 65 with adequate prior negative screening

• Ectocervix: Ayre's spatula (samples transformation zone)
• Endocervix: Cytobrush
• Fix immediately in 95% ethyl alcohol (conventional) OR liquid-based cytology (ThinPrep/SurePath - preferred)

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Normal Cells Seen in Pap Smear

• Origin: Uppermost squamous layer
• Size: Large, flat, polygonal
• Nucleus: PYKNOTIC (small, dark, condensed, <6 microns) - hallmark
• Cytoplasm: EOSINOPHILIC (pink/orange) - keratin
• Seen predominantly in: Proliferative (follicular) phase; high estrogen states
• Karyopyknotic index (KPI) = % superficial cells = estrogen index

• Origin: Intermediate squamous layer
• Size: Large, polygonal (slightly smaller than superficial)
• Nucleus: VESICULAR (open, pale, larger than superficial cell nucleus)
• Cytoplasm: CYANOPHILIC (blue-green); may contain glycogen vacuoles
• Navicular cells = boat-shaped intermediate cells with folded edges (seen in pregnancy and luteal phase)
• Seen predominantly in: Luteal phase, pregnancy, progestogen influence

• Origin: Lower squamous layers
• Size: Smaller, oval/round
• Nucleus: Large relative to cytoplasm, vesicular
• Cytoplasm: Cyanophilic, dense, scant
• Seen predominantly in: Estrogen deficiency (menopause, postpartum, breastfeeding) = atrophic smear
• Normal in postmenopausal women

• Smallest squamous cells
• Large nucleus, scant cytoplasm
• Seen only in severe atrophy

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• Origin: Columnar epithelium lining endocervical canal
• Appearance: Tall columnar cells arranged in HONEYCOMB or PICKET-FENCE pattern in sheets
• Nucleus: Small, round, basal
• Cytoplasm: Pale, foamy, mucin-containing (mucin secretion)
• Single cells appear "goblet cell-like"
• Presence on smear = adequate sampling (transformation zone sampled)

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• Origin: Transformation zone (squamocolumnar junction) - most important area to sample
• Appearance: Round/oval cells, intermediate between squamous and columnar
• Dense cytoplasm, may have cytoplasmic projections ("spider cells" or "caudate cells")
• Immature metaplastic cells have high N/C ratio
• Normal finding - represents physiological replacement of columnar by squamous epithelium

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• Small, round cells in tight clusters
• "Two-cell pattern": Epithelial cells + stromal cells together
• High N/C ratio
• Normal to see in first 10-12 days of menstrual cycle
• Abnormal if seen after day 12 in postmenopausal women (requires endometrial investigation)

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• Neutrophils (polymorphs): Small, multilobed nuclei; normal in small numbers; increased in infection/inflammation
• Histiocytes (macrophages): Bean-shaped/kidney-shaped nucleus; vacuolated cytoplasm; single cells; normal finding
• Lymphocytes: Rare; increased in follicular cervicitis (lymphoid follicles in submucosa)

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• Lactobacilli (Doderlein's bacilli): Large plump bacilli; normal vaginal flora; indicate healthy acidic environment (pH 3.8-4.5); produce lactic acid from glycogen in intermediate cells

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Reporting System - Bethesda System 2014

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END OF COMPLETE ANSWERS