Hemolytic Anemias😍

• Intrinsic
• Cause is inside → Hb, RBC shape, Membrane, enzyme

• Extrinsic
• Cause is outside → AIHA, MAHA

• IVH
• MAHA () → was sleeping at Night (PNH)
• Murdered inside his room (Intravascular)

• EVH
• HS time → full outside

SKULL XRAY PATTERN APPROACH

NOTE: Different Fanconis

ㅤ	ㅤ
Fanconi disease/syndrome	• Proximal tubular reabsorption problem → Type 2 RTA • Glycosuria, aminoaciduria
Fanconi anemia (Not syndrome)	• Pancytopenia + radial ray
Fanconi Bickel syndrome	• Mutation in GLUT-2   • Bickel → Bi → 2 (GLUT 2) Defect in glucose sensing → ↓ insulin release • Postprandial Hyperglycemia. • Fasting Hypoglycemia • Glycogen accumulation disorder

Stranger things characters

• Dustin (Cleido cranial dysplasia)

• Robin (Cock robin position)

• Ray (Radial Ray) Hopper (Holt Oram ASD)

RBC Shape Abnormalities

Shape	Alias	Seen In	ㅤ
Acanthocytes	Spur cells	• Abetalipoproteinemia	(A for A)
Echinocytes	Burr cells	• Burns, • Renal failure, • Pyruvate kinase deficiency • EDTA changes (BUE)	BURP Burr → Burn → Pyro → PK def
Dacrocytes	Teardrop RBCs	• Myelophthis • Myelofibrosis	ㅤ
Schizocytes	Helmet cells	• MAHA	• Micro: HUS/TTP/DIC/HELLP • Macro: Prosthetic valves
Dog ear projections	ㅤ	• Parvovirus B19	• Leads to PRCA

RBC Inclusions

Inclusion	Composition	Seen in
Howell–Jolly bodies	• DNA remnants	• Post-splenectomy • Megaloblastic • Sickle cell disease (NOT THALASSEMIA) • Dyshematopoiesis • Hemolytic anemia
Reticulocytes	• Residual RNA (bluish network)	• Hemolysis • Blood loss • Response to therapy
Basophilic Stippling	• Ribosomal RNA	• Coarse → Lead poisoning • Fine → Thalassemia, Megaloblastic Anemia • Also seen in 5’ Pyrimidine nucleotidase
Heinz bodies	• Hb denaturation • Stained by Crystal Violet stain • (Supravital stain)	• G6PD
Pappenheimer Bodies	• Iron granules	• Sideroblastic anemia • Post-splenectomy
Cabot Rings	• Arginine-rich • Mitotic spindle remnants • Figure of eight/loop	• Severe anemia • Megaloblastic anemia • Sideroblastic anemia
Hemoglobin H inclusions	• β4 tetramers • (precipitated Hb H)	• α-thalassemia (Hb H disease)
Siderotic Granules	• Iron • In Prussian blue stain	• Sideroblastic states
Target cells	ㅤ	• Thalassemia • Iron deficiency anemia • Liver disease • Post-splenectomy • Hemoglobin C disease
Dohle bodies	• ER remnant	• Bacterial sepsis
Perinuclear hof	• Golgi apparatus	• Plasma cells

Sideroblastic anemia

• Causes
• B6 deficiency
• Alcohol
• ALA synthase (XLR)
• Lead poisoning

Sideroblasts

• Normal immature erythroid precursors in bone marrow

• Present in <30% of marrow cells

• Ringed Sideroblasts
• Bone marrow smear
• Formed when Protoporphyrin is absent
• Iron cannot bind → accumulates as granules around mitochondria

• Iron deposits appear scattered around the nucleus
• ≥5 granules
• ≥1/3rd nucleus

• Detected by:
• Pearl stain/ Prussian Blue Stain

COOMBS TEST

TYPES

• DCT / DAT
• Direct Coombs test /
• Direct antiglobulin test 

• ICT / IAT
• Indirect Coombs test /
• Indirect antiglobulin test 

DIRECT COOMBS TEST (DCT)

• Detects antibodies on the RBC surfaces.
• Mix
• Polyvalent Antihuman Immunoglobulin 
• Antibody-coated RBCs
• ⇒ Agglutination.

• Used to test for autoimmune hemolytic anemia.
• DAT (+) → agglutination.
• DAT (++++) → significant agglutination.

• Alloimmune Hemolysis
• Hemolytic disease of the newborn
• ABO hemolytic disease of the newborn
• Alloimmune hemolytic transfusion reactions

• Autoimmune Hemolysis
• AIHA
• Hereditary spherocytosis
• Cold agglutinin disease
• Infectious mononucleosis

• Drug-induced Immune-Mediated Hemolysis
• Penicillin
• Cephalosporins

INDIRECT COOMBS TEST (ICT)

• Detects alloantibody in the Serum.

• Example: ICT positive (+) → Cold Ab AIHA (as a demonstration).

Major Uses

• Blood Transfusion Preparation
• Cross-matching

• Antenatal antibody screening
• Screens a pregnant patient for IgG antibodies.
• These can cross the placenta.
• Can cause hemolysis in fetal blood.

Comparison: DCT vs. ICT

Autoimmune Hemolytic Anemia (AIHA)

• Warm AIHA (IgG)
• CLL, SLE, Penicillin, Methyl Dopa
• Call, Pen, Sleep, Metha

• Cold (IgM)
• Cold Mosco, Cold Virus
• EBV, Mycoplasma

• Cause: Antibodies against RBCs.

Hemolytic Anemia

• RBC Membrane Proteins:
• Spectrin: Maintains biconcave shape.
• Most abundant: Glycophorin A (never mutated in HS).

Classification by Site of RBC Destruction

Feature	Intravascular Hemolysis	Extravascular Hemolysis
Site	Within blood vessels	Spleen, Liver, Bone Marrow
Examples	• MAHA (eg. HUS) • PNH  • PCH  • Infections • Drugs (some) • Snake bites	• Hereditary Spherocytosis • Sickle Cell Anemia • Thalassemia • G-6-P D deficiency • Warm AIHA • Cold Agglutinin Disease
Common Presentation	Acute > Chronic	Insidious, Rapidly Progressive
LDH	+++ (high)	++ (moderate)
Serum Haptoglobulin	Decreased (Hb bind with Haptoglobulin)	Decreased (↓↓) ????
Investigations	• ↑Hb in plasma & urine • Hemoglobinuria • Hemosiderinuria • ↑↑S. bilirubin	• ↑↑↑↑S. bilirubin (↑ Protoporphyrin) • Splenomegaly • Urobilinogen positive • Tissue iron ↑↑ • S. ferritin ↑↑
Urine Color	High coloured urine	Normal
Both Intra- and Extravascular:	• G6PD Deficiency • Cold AIHA	ㅤ

Drug Induced Hemolysis

Paroxysmal Cold Hemoglobinuria (PCH)

• We drove in Cold 4 degree C → in Landrover (Landsteiner) → in a room We Peed (P antigen) → got syphillis

• Onset:
• Paroxysmal, acute Intravascular hemolysis.

• Antibody Type: 
• Donath-Landsteiner Antibody (Polyclonal IgG)

• Reaction: 
• IgG binds to RBC at 4°C 
• but hemolysis occurs at Room Temperature.

• Antigen:
• Directed against 'p' antigen on RBCs.

• Associated with: 
• Syphilis.

• Coombs Test:
• Usually Positive.

Hereditary Spherocytosis (HS)

• Most common inherited RBC membrane defect

• Characterised by:
• Hemolysis
• Spherocytic RBCs
• Increased osmotic fragility

• >75% cases are autosomal dominant

• Problem: RBC membrane protein defects.

• Mutations:
• Most common (Autosomal Dominant): 
• Ankyrin mutation.
• Severe cases (Autosomal Recessive): 
• Spectrin mutation.
• Band 3 defect:
• Associated with Pincher cells / Mushroom shaped cells.

• Clinical:
• Hemolytic anemia
• Hemolysis Type: 
• Extravascular (spleen breaks down cells).
• Jaundice
• Splenomegaly
• Gallstones
• bilirubin gallstones

Bone marrow:

• Increased reticulocyte count.

Peripheral Smear: 

• Microspherocytes (smaller, round, no central pallor).



Parameter	Finding	Reason
MCV	Low	Smaller cells (spherocytes)
MCHC	High	Hemoglobin concentrated due to water loss from membrane defect
RDW	Increased	Due to presence of microspherocytes and larger reticulocytes

Hemolytic Crisis

• Triggered by viral infections

• Presents with:
• Jaundice
• Anemia
• Abdominal pain
• Tender splenomegaly

• Usually requires only supportive care

Aplastic Crisis:

• ↓ Hb, ↓ Reticulocytes

• Sudden stop in bone marrow production (all cell lines).

• Parvovirus B19 infection causes aplastic crisis in both HS and SCD

• Usually last 10-14 days.

• This can be life-threatening

Splenic sequestration crisis (in SCD):

• ↓ Hb, ↑ Reticulocytes

Screening Test: 

• Osmotic Fragility Test.
• HS cells: high fragility (shift to right)

• Confirmatory (current):
• EMA (Eosin 5 Maleimide) testing by flowcytometry
  (Less EMA dye binds).

Treatment: 

• Splenectomy
• Curative in most
• Eliminates:
• Anemia
• Hyperbilirubinemia
• Reticulocytosis
• Not required in mild cases
• Long-term risks present

Key Notes

• Spherocytes seen in both
• Hereditary Spherocytosis
• Autoimmune Hemolytic Anemia

• Always do Direct Coombs Test to differentiate

Hereditary spherocytosis	Autoimmune hemolytic anemia
• Spherocytes +ve • +ve osmotic fragility test • -ve direct Coomb's test	• Spherocytes +ve • +ve osmotic fragility test  • +ve direct Coomb's test

G6PD Deficiency

• X-linked inheritance.

• Enzyme: Glucose-6-Phosphate Dehydrogenase deficiency.

• Class II Mediterranean type G6PD deficiency: ↓ G6PD.

Mechanism

• Precipitating factors: 
• Fava beans (favism)
• Antimalarial drugs
• Primaquine
• Dapsone
• Sulfa drugs
• Sulfasalazine
• Sulfamethoxazole
• Acetazolamide
• Other drugs
• Nitrofurantoin
• High dose aspirin (>3g/d)
• High dose Vit C (>1g)
• Infections
• E.g., UTIs

Presentation

• Severe Hemolysis

• Jaundice

• Red or Dark Urine

• Back and abdominal pain

• Most patients are asymptomatic
• Until exposed to triggers.

• Gallstones are common.

• Splenomegaly:
• Usually absent (intermittent hemolysis)
• It is an acute intermittent trigger → not chronic → do not cause splenomegaly

Blood Findings:

• Oxidative stress → Heinz bodies (denatured hemoglobin).

Peripheral Smear: 

• Spherical RBCs

• Bite cells (Degmacytes)

• Blister Cells (Hemighosts)

RBC Inclusions

Inclusion	Composition	Seen in
Howell–Jolly bodies	• DNA remnants	• Post-splenectomy • Megaloblastic • Sickle cell disease (NOT THALASSEMIA) • Dyshematopoiesis • Hemolytic anemia
Reticulocytes	• Residual RNA (bluish network)	• Hemolysis • Blood loss • Response to therapy
Basophilic Stippling	• Ribosomal RNA	• Coarse → Lead poisoning • Fine → Thalassemia, Megaloblastic Anemia • Also seen in 5’ Pyrimidine nucleotidase
Heinz bodies	• Hb denaturation • Stained by Crystal Violet stain • (Supravital stain)	• G6PD
Pappenheimer Bodies	• Iron granules	• Sideroblastic anemia • Post-splenectomy
Cabot Rings	• Arginine-rich • Mitotic spindle remnants • Figure of eight/loop	• Severe anemia • Megaloblastic anemia • Sideroblastic anemia
Hemoglobin H inclusions	• β4 tetramers • (precipitated Hb H)	• α-thalassemia (Hb H disease)
Siderotic Granules	• Iron • In Prussian blue stain	• Sideroblastic states
Target cells	ㅤ	• Thalassemia • Iron deficiency anemia • Liver disease • Post-splenectomy • Hemoglobin C disease
Dohle bodies	• ER remnant	• Bacterial sepsis
Perinuclear hof	• Golgi apparatus	• Plasma cells

Tests:

• Screening (older):
• Flurecent Metha
• Fava (Fluorescent spot assay) Malaria (Methaemoglobin reduction test)
• Fluorescent spot assay
• Methaemoglobin reduction test.

• Confirmatory (current):
• Measuring G6PD levels (G6PD assay)
• The definitive test.
• Usually done 6 weeks after the episode.
• If done during hemolytic crisis, results can be equivocal.

• Mnemonic:



• g6PD → Pidich → dog pidich
• Dog (degmetocyte) bite (bite cells) hen (hein) above metha (methamogl)
• Hen passes Malam (malaria)
• Extra (Extravascular) bed vanganam

Clinical Aspects

• Stages (referring to response/progression):
• I: Hypovolemia.
• II: Volume shift (extravascular → intravascular).
• III: EPO ↑, reticulocyte ↑.

• Triad of:
• Acidosis
• Hypothermia
• DIC (Hypercoagulability)

Common Presentations

Treatment

• Stop the precipitating agent.

• Symptomatic Rx.

PNH (Paroxysmal Nocturnal Hemoglobinuria)

• Only acquired RBC defect.

• M/c/c of death → Budd Chiari syndrome

Genetic defect

• PIGA gene mutation.

• Result:
• Failure to form GPI anchors (GlycosylPhosphatydylInositol)
• GPI anchors normally hold CD55 (DAF) and CD59 (MIRL).
• When GPI anchors absent → complement attack the RBC when pH ↓↓
• During sleep (night) → ↓ pH
• MAC attacks RBC membrane
• Leads to hemolysis → hemoglobinuria
• Activates complement proteins (C5–C9)
• → Membrane Attack Complex (MAC)
• Bone marrow goes bizzare
• Pancytopenia
• Leukemia

• Deficiency: CD55 & CD59.

Complications:

• High risk of thrombosis

• Aplastic anemia >>

• Acute Leukemia (least)

Clinical Feature: 

• Nocturnal hemoglobinuria

Hemolysis Type: 

• Intravascular (P for PNH, P for Intravascular).

Tests:

• Screening (older):
• Ham's Acidified Serum test
• Sucrose Lysis test.

• Screening (current):
• Gel card test.
• Normal: CD55/59 positive (line at top).
• PNH: CD55/59 negative (line at bottom).

• Confirmatory:
• Flow cytometry 
• CD55/59 deficiency
• FLAER 
• GPI anchor deficiency
• better, tests direct defect

• Mnemonic:
• 59 (CD59) year old GP (GPI) and his wife 55 (CD55) years was alone at home
• 59 year old → mute/mother (MIRL)
• 55 year old → deaf/dad (DAF)
• a PIG (PIGAa) attacked the house to steal at night (nocturnal)



• MAC (MAC)
• Ham (Ham's Acidified Serum test)
• Sucrose (Sucrose Lysis test)
• Gel (Gel card test)
• Ravile (Ravulizuab) vare kallane Ikkilakki (Eculizumab) → So he Flea (Flaer) away

Treatment: 

• Anti-C5 monoclonal antibody
• Eculizumab
• Ravulizumab (Long acting)

• Hematopoietic stem cell transplant:
• Definitive