---

Hemolytic Anemia - Complete MD Pathology Review

Definition & General Pathogenesis

---

Classification Framework

By Location of Defect (Pathologic Basis)

By Site of Hemolysis (Clinical Basis - Most Important)

Extravascular Hemolysis

• Hyperbilirubinemia and jaundice (unconjugated bilirubin from macrophage heme degradation)
• Splenomegaly ("work hyperplasia" of splenic macrophages)
• Pigment (bilirubin-rich) gallstones if long-standing - cholelithiasis in 40-50% of hereditary spherocytosis patients
• Iron is efficiently recycled by phagocytes → no iron deficiency

Intravascular Hemolysis

• Hemoglobinemia (free Hb in plasma - pink plasma)
• Hemoglobinuria (Hb in urine - red/brown urine)
• Hemosiderinuria (chronic episodes - renal tubular cells process Hb → hemosiderin → shed in urine)
• Iron deficiency with persistent hemolysis (iron lost in urine)

---

Pathogenesis of Key Entities (Differential Diagnosis)

1. Hereditary Spherocytosis (HS)

• Inherited mutations in membrane skeleton proteins: spectrin, ankyrin, band 3, band 4.1, band 4.2
• These mutations weaken the connection between the membrane skeleton and the overlying lipid bilayer
• Result: RBCs shed membrane vesicles → surface area decreases progressively → cells become spherical (↓ surface area-to-volume ratio)
• Spherocytes are non-deformable → trapped in splenic cords → phagocytosed by splenic macrophages
• Extravascular hemolysis exclusively

• Dark red spherocytes lacking central pallor on peripheral smear
• Splenomegaly (500-1000 g vs. normal 150-200 g)
• Erythroid hyperplasia in marrow
• Cholelithiasis (40-50% of patients)

---

2. Sickle Cell Anemia

• Deoxygenated HbS polymerizes into long rigid rods → distort and damage the RBC
• Initially reversibly sickled → repeated cycles → irreversibly sickled cells
• Two consequences:
  • Extravascular hemolysis - irreversibly sickled cells phagocytosed by macrophages
  • Microvascular occlusion - sickled cells adhere to endothelium → vascular stasis → ischemia and infarction
• Factors that promote sickling: hypoxia, acidosis, dehydration, cold, infection

• Moderate to severe anemia (Hct 18-30%)
• Hyperbilirubinemia + compensatory reticulocytosis
• Vasoocclusive crises:
  • Hand-foot syndrome (most common in children)
  • Acute chest syndrome (leading cause of death)
  • Stroke
  • Proliferative retinopathy
  • Priapism
• Autosplenectomy by adulthood (splenic infarction) → increased susceptibility to encapsulated bacteria (Streptococcus pneumoniae, Haemophilus influenzae)
• "Crewcut" skull on X-ray (marrow hyperplasia)

---

3. Thalassemia

• β-Thalassemia: Reduced/absent β-globin → excess α-chains precipitate as insoluble inclusions → damage RBC membrane → ineffective erythropoiesis + hemolysis
  • Major (β⁰/β⁰): Severe transfusion-dependent anemia; secondary hemochromatosis; erythroferrone-mediated hepcidin suppression → excess iron absorption
  • Minor (β/β⁰): Mild microcytic anemia; elevated HbA₂
• α-Thalassemia: Deletion of α-globin genes → excess β-chains form HbH (β₄) or HbBart (γ₄) - unstable tetramers

---

4. Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency

• G6PD catalyzes the first step of the hexose monophosphate shunt → generates NADPH → maintains reduced glutathione (GSH) → neutralizes oxidants
• Deficient G6PD → inadequate GSH → oxidants attack globin chains
• Oxidized hemoglobin denatures → precipitates as Heinz bodies (intracellular inclusions)
• Heinz bodies damage membrane → intravascular hemolysis
• Splenic macrophages "pluck out" Heinz bodies → bite cells (degmacytes)
• Hemolysis is episodic - triggered by oxidant stress

• Drugs: Primaquine, dapsone, sulfonamides, nitrofurantoin, aspirin (large doses), vitamin K derivatives
• Foods: Fava beans
• Infections (most common trigger - phagocyte-generated oxidants)

---

5. Autoimmune Hemolytic Anemia (AIHA)

---

6. Microangiopathic Hemolytic Anemia (MAHA)

• TTP (Thrombotic thrombocytopenic purpura) - ADAMTS13 deficiency → ultra-large vWF multimers → platelet microthrombi
• HUS (Hemolytic uremic syndrome) - Shiga toxin (E. coli O157:H7) → endothelial injury → microthrombi; predominantly renal
• DIC - Fibrin-platelet clots in microvasculature
• Malignant hypertension
• Prosthetic heart valves (macroangiopathic)
• Metastatic carcinoma / marrow infiltration

---

7. Paroxysmal Nocturnal Hemoglobinuria (PNH)

---

8. Others

• Hereditary Elliptocytosis: Mutations in spectrin or band 4.1 → elliptical/oval cells; mild hemolysis
• Malaria: Intracellular parasite; Falciparum most severe (P. falciparum = P. falciparum)
• Clostridial septicemia: Phospholipases destroy RBC membranes → intravascular hemolysis
• Transfusion reactions: Alloantibody against transfused RBCs (ABO incompatibility → intravascular; minor incompatibility → extravascular)

---

Clinical Signs and Symptoms

Symptoms

• Fatigue, weakness, exertional dyspnea (anemia)
• Jaundice (unconjugated hyperbilirubinemia) - scleral icterus often prominent
• Dark urine - either hemoglobinuria (intravascular) or bilirubinuria
• Pallor
• Abdominal pain (splenic infarction, gallstone colic)
• Back/flank pain (intravascular hemolysis - acute)
• Symptoms of the underlying disease

Signs

---

Diagnostic Approach

Step 1: Confirm Hemolysis

Step 2: Determine Type of Hemolysis

Step 3: Directed Tests

Step 4: Bone Marrow (when needed)

• Erythroid hyperplasia (M:E ratio reversed, approaching 1:1)
• Megaloblastic change (if folate depleted by chronic hemolysis)

---

Key Laboratory Summary Table

---

Complications of Chronic Hemolysis

1. Pigment (bilirubin) gallstones - cholelithiasis
2. Aplastic crisis - parvovirus B19 infection silences erythropoiesis → sudden severe anemia
3. Megaloblastic crisis - folate depletion (high demand from chronic compensatory erythropoiesis)
4. Iron overload (transfusion-dependent thalassemia)
5. Leg ulcers (sickle cell)
6. Pulmonary hypertension (chronic hemolysis - free Hb scavenges NO)
7. Extramedullary hematopoiesis - thalassemia facies, hepatosplenomegaly

---

Quick Summary for Exam Recall: "5 I's of Hemolysis"

---

• Read and extract content from them
• Answer your haematology questions directly from those textbooks
• Cross-reference with other sources I already have (Robbins, Henry's, etc.)
• Give you exam-focused answers tailored to those specific books

---

HEMOLYTIC ANEMIA - Seminar Notes

---

PART 1: CLASSIFICATION

A. By Site of the Defect

---

B. By Site of Hemolysis (Most Clinically Important Classification)

HEMOLYTIC ANEMIA
│
├── EXTRAVASCULAR HEMOLYSIS
│   (RBCs destroyed by splenic/hepatic macrophages)
│   Examples: HS, AIHA (warm), Thalassemia, Sickle cell
│
└── INTRAVASCULAR HEMOLYSIS
    (RBCs burst within blood vessels)
    Examples: PNH, G6PD deficiency (acute), MAHA, ABO mismatch

---

C. Etiological Classification

---

PART 2: MECHANISM OF HEMOLYSIS

Extravascular Hemolysis - How it Happens

1. RBCs have reduced deformability (spherocytes, sickled cells, antibody-coated cells, thalassemic cells)
2. These RBCs cannot negotiate the narrow splenic sinusoids (normal RBC diameter = 8 µm; sinusoidal gap = 3 µm)
3. They are trapped in the splenic cords → recognized and phagocytosed by splenic macrophages
4. Hemoglobin is degraded within macrophages → globin → amino acids; heme → biliverdin → unconjugated bilirubin (indirect bilirubin) released into blood → conjugated by liver → excreted in bile

Intravascular Hemolysis - How it Happens

1. Severe membrane injury causes RBCs to burst inside blood vessels
2. Free hemoglobin is released directly into plasma
3. Free Hb rapidly binds haptoglobin → Hb-haptoglobin complex → cleared by macrophages → haptoglobin depleted
4. Excess free Hb in plasma = hemoglobinemia → filtered by kidney = hemoglobinuria
5. Renal tubular cells reabsorb some Hb → process it into hemosiderin → shed into urine = hemosiderinuria (chronic marker)
6. Iron is lost in urine → can lead to iron deficiency

---

PART 3: CONSEQUENCES (What Hemolysis Produces)

Consequences of Extravascular Hemolysis

Consequences of Intravascular Hemolysis

Consequences Common to Both

---

PART 4: SALIENT FEATURES OF SPECIFIC TYPES

Hereditary Spherocytosis

• Gene defects: Spectrin, Ankyrin, Band 3, Band 4.1, Band 4.2 → membrane vesicle shedding → spherocytes
• Type: Extravascular (spleen destroys spherocytes)
• Triad: Anemia + Splenomegaly + Jaundice + Cholelithiasis (40-50%)
• Morphology: Dark spherocytes, no central pallor; splenomegaly 500-1000 g
• Splenectomy corrects anemia (spherocytes persist but spleen removed)

Sickle Cell Anemia

• Mutation: β-globin GAG→GTG; Glu→Val at position 6
• Trigger for sickling: Hypoxia, acidosis, dehydration, cold, infection
• Two consequences: Hemolytic anemia + Microvascular occlusion
• Crises: Vasoocclusive (hand-foot, acute chest, stroke), Aplastic (parvovirus B19), Sequestration
• Autosplenectomy by adulthood → susceptibility to encapsulated bacteria

Thalassemia

• β-Thal major: Absent/reduced β-chains → excess α-chains precipitate → ineffective erythropoiesis + hemolysis → transfusion-dependent; iron overload
• Peripheral smear: Microcytic hypochromic, target cells, nucleated RBCs, basophilic stippling
• Erythroferrone suppresses hepcidin → excess iron absorption

G6PD Deficiency

• X-linked; episodic hemolysis triggered by oxidant stress (drugs, fava beans, infection)
• Heinz bodies (denatured Hb inclusions) → membrane damage → bite cells (degmacytes)
• Intravascular hemolysis predominantly
• Test after episode resolves (G6PD-deficient cells selectively destroyed during crisis)

Autoimmune Hemolytic Anemia

---

PART 5: KEY LABORATORY TESTS

Universal Markers of Hemolysis

Markers Specific to Intravascular Hemolysis

Peripheral Smear Morphology → Diagnosis

Directed Tests

---

PART 6: PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) - DETAILED

Definition

"The only hemolytic anemia caused by an acquired genetic defect" - Robbins Pathologic Basis of Disease

---

Epidemiology

• Prevalence: ~1 per million population; incidence 2-5 per million/year
• Age: Any age; most common 10-50 years (mean ~34 years)
• Sex: No sex preference (M:F = 1:1) - unusual because PIGA is X-linked
• Median survival: ~20 years with current therapy
• No family history (purely acquired/somatic)

---

Molecular Pathogenesis - Step by Step

• During sleep, respiratory rate decreases → CO₂ accumulates → blood pH decreases (respiratory acidosis)
• Lower pH activates complement (especially the alternative pathway amplification loop)
• → More MAC formation → more hemolysis during and just after sleep
• Patient passes dark brown urine in the morning (first morning void)
• Important: Classical nocturnal pattern occurs in only ~25% of cases; most patients have chronic, low-grade intravascular hemolysis without dramatic hemoglobinuria

---

PNH Red Cell Types

---

Why Does the PNH Clone Expand?

• Immune escape hypothesis: The PNH clone lacks GPI-linked antigens that are targeted by autoreactive T-cells in aplastic anemia-like settings. The normal HSCs are destroyed by autoimmunity but the PIGA-mutant clone resists immune attack → relative expansion
• This explains the strong association between PNH and aplastic anemia (AA precedes PNH in ~30% of cases; PNH can evolve into AA)

---

Clinical Manifestations

1. Hemolysis (Cardinal Feature)

• Morning hemoglobinuria - dark/cola-colored first morning urine (classic but only ~25% of patients)
• Chronic hemosiderinuria - almost constantly present; more reliable sign
• Chronic intravascular hemolysis → anemia (often normocytic, but can be microcytic due to iron deficiency)
• Iron deficiency from chronic urinary iron loss - paradox: hemolytic anemia with microcytosis

2. Thrombosis (Leading Cause of Death - 40% of patients)

• Unusual venous sites are characteristic:
  • Hepatic vein → Budd-Chiari syndrome (hepatomegaly, ascites, jaundice)
  • Portal vein → portal hypertension
  • Cerebral veins → venous sinus thrombosis
  • Mesenteric, splenic veins
• Arterial thrombosis is rare
• Mechanisms of thrombosis:
  • MAC on platelets → phosphatidylserine externalization → prothrombinase complex assembly
  • Free Hb scavenges NO → platelet activation and endothelial damage
  • Lack of CD59 on platelets

3. Smooth Muscle Dystonias (from NO Scavenging)

• Dysphagia (esophageal spasm)
• Abdominal pain / cramping (intestinal smooth muscle spasm)
• Erectile dysfunction
• Pulmonary hypertension (chronic NO depletion)

4. Cytopenias

• Neutropenia (~60% of patients at some point)
• Thrombocytopenia (~66% of patients) → pancytopenia is common
• DAT is negative (no antibody coating)

5. Associated Conditions

• Aplastic anemia - ~30% of PNH cases evolve into or arise from AA
• Myelodysplastic syndrome (MDS) - PNH clones found in 15-20% of MDS
• Acute myeloid leukemia (AML) - rare (~5% of PNH patients)

---

Diagnosis

Classic Presentation Clue:

DAT-negative intravascular hemolytic anemia + iron deficiency = suspect PNH

Old Tests (Historical - no longer recommended)

• Ham's Acid Hemolysis Test (Acidified Serum Test): Acidified serum activates complement → lysis of PNH RBCs. Sensitive but not specific. Replaced by flow cytometry.
• Sugar Water (Sucrose Hemolysis) Test: Screening test. Low ionic strength activates complement. Less specific.

Gold Standard: Flow Cytometry

• Demonstrates deficiency of GPI-anchored proteins on two or more cell lineages (RBCs + granulocytes/monocytes)
• Markers tested:
  • RBCs: CD55 and CD59
  • Granulocytes/Monocytes: CD24, CD16a (on neutrophils), CD14 (on monocytes)
• FLAER (Fluorescent Aerolysin variant): Binds directly to the GPI anchor itself → most sensitive and reliable reagent for leukocytes; preferred for diagnosing PNH in granulocytes
• Reports clone size (% PNH cells) which correlates with degree of hemolysis

Laboratory Findings in PNH

---

PNH Clinical Categories

---

Complications

1. Thrombosis - leading cause of death; occurs in ~40% of patients
2. Budd-Chiari syndrome - hepatic vein thrombosis
3. Iron deficiency anemia (from urinary iron loss)
4. Aplastic anemia (immune-mediated marrow failure)
5. AML / MDS transformation (~5%)
6. Pulmonary hypertension (chronic NO scavenging)
7. Renal impairment (chronic hemoglobinuria, microthrombi)

---

Treatment

Supportive

• Blood transfusions (washed RBCs preferred)
• Iron and folate supplementation
• Anticoagulation for thrombosis

Complement Inhibitors (Disease-Modifying)

• Abolishes intravascular hemolysis and hemoglobinuria
• Reduces thrombosis risk by up to 90%
• PNH RBCs, now protected from MAC, still bind C3b on their surface → become opsonized → DAT becomes positive → iatrogenic extravascular hemolysis persists (limitation of C5 inhibition)
• Mandatory meningococcal vaccination before starting (risk of Neisseria meningitidis infection due to complement blockade)
• Oral penicillin prophylaxis often recommended long-term

Curative

• Hematopoietic stem cell transplantation (HSCT) - only cure; reserved for:
  • Intercurrent aplastic anemia not responding to complement therapy
  • AML/MDS transformation
  • Patients unresponsive to medical therapy

---

Summary Mnemonic for PNH: "PNH = PIGA = No GPI = No CD55/59 = MAC = Hemolysis"

PIGA mutation (X-linked, somatic)
  ↓
No GPI anchors
  ↓
No CD55 (no C3 convertase inhibition) + No CD59 (no C9 polymerization inhibition)
  ↓
Uncontrolled complement activation → MAC on RBC membrane
  ↓
INTRAVASCULAR HEMOLYSIS
  ↓
Free Hb → Scavenges NO → Smooth muscle spasm + Thrombosis
  ↓
Morning hemoglobinuria / chronic hemosiderinuria / iron deficiency

---