Hemolytic Anemia Membrane and Enzymes Defect

Ahmad A. Al-Qudah

Clinical Hematology I LM-753

Hemolytic Anemia - Introduction to Hemolytic Anemia : - Definition - Evidence of Hemolysis - Signs & Symptoms , Lab Findings - Classifications & differential diagnosis - Hemolysis due to Membrane Defect ( Structure , Permeability )

- Hemolysis due to Enzyme Defect

- What is New In Hemolytic Anemia ?

Hemolytic Anemia - Is a form of anemia due to hemolysis, the abnormal breakdown of red blood cells (RBCs), either in the blood vessels (intravascular hemolysis) or elsewhere in the human body (extravascular) .

- Increased red cell destruction (and increased erythropoiesis)

- Cells are being produced at the same rate as they are hemolyzed; this can develop into anemia if : - Erythrocyte destruction accelerates beyond the compensatory capacity of the marrow -The B.M suddenly stops producing erythrocytes

Hemolytic Anemia Sites of destruction

- Intravascular hemolysis: (within blood vessels)Caused by :-Activation of complement on erythrocyte membrane- Physical or mechanical trauma- Toxic substances

* Hemoglobinemia (free Hb in plasma)* Hemoglobinuria (Hb in urine)* Hemosiderinuria( hemosiderin granules in urine)* Methemoglobinemia

Hemolytic Anemia Sites of destruction

-Extravascular hemolysis :

- Erythrocytes removed by tissue phagocytes - More common than intravascular . - increase bilirubin , urobilinogen . - Decrease haptoglobin . - Spleen(slightly damaged RBC),liver( extensively damaged) - Antibodies against RBCs (immune mediated)

Hemolytic Anemia

Hemolytic Anemia

Evidence of Hemolysis

- Jaundice and hyperbilirubinemia- Reduced plasma haptoglobin

* Increased Hb breakdown:

- haemoglobinuria- methaemalbuminaemia- haemosiderinuria

Hemolytic Anemia

* Damage to red cells: - Spherocytosis & increased fragility - Fragmented RBCs - Heinz bodies - Increased plasma LDH

* Erythroid hyperplasia: - Reticulocytosis - Macrocytosis & polychromasia - BM erythroid hyperplasia

Evidence of Hemolysis

Hemolytic Anemia Signs & Symptoms

• Jaundice Accumulation of Bilirubin

• Gallstones / red urine Increased excretion of bilirubin into the biliary tract

• Splenomegaly Accumulation of damaged RBCs

• Pulmonary hypertension Increased pressure over the pulmonary artery

• Pallor , fatigue , cardiac symptoms General Signs of Anemia

Hemolytic Anemia - Testing and Special Approaches :

- Reticulocyte Count : Young Cells , Contain RNA . Stained by Supravital Stain , 0.5-2% .

RPI = (%Retics /RMT )*(Hct/45)1 --> 45 1.5 --> 35 2 --> 25 2.5 --> 15 RPI > 2 – 2.5 , indicate a hemolytic state .

- COOMBS ( DAT ) :Detection of Auto Antibody Next Lec.

Hemolytic Anemia - Osmotic Fragility :

0

20

40

60

80

100

0.3 0.4 0.5 0.6

NaCl (% of normal saline)

% H

emol

ysis

Normal HS

Hemolytic Anemia Lab Findings

Increased erythrocyte destruction Increased BM production of erythrocytes

AnemiaSpherocytes,schistocytes,poikilocytes+ve DATDecreased haptoglobinDecreased glycosylated HbIncreased urobilinogenIncreased bilirubinHemoglobinemia,hemoglobinuria,hemosiderinuria,methmoglobinemia ( intravascular hemolysis)

Reticulocytosis LeukocytosisNucleated erythrocytes in P.BPolychromasia of erythrocytesNormoblastic erythroid hyperplasia in the B.M

Hemolytic Anemia Complications :

- Hemolytic crisis : due to accelerated hemolysis.

- Aplastic crisis : due to maturation arrest and associated with megaloblastic changes

- Pigmented gallstones : Increased hemolysis of red blood cells leads to increased bilirubin levels, because bilirubin is a breakdown product of heme. The high levels of bilirubin must be excreted into the bile by the liver, which may cause the formation of a pigmented gallstone, which is composed of calcium bilirubinate. Since these stones contain high levels of calcium carbonates and phosphate, they are visible on x-ray.

- Abnormally low hemoglobin A1C levels : the life span of the red blood cells is decreased, providing less time for the non-enzymatic glycosylation of hemoglobin. Thus, even with high overall blood sugar, the A1C will be lower than expected.- Leg ulcer.

- Folate deficiency : caused by increased bone marrow requirement.

Hemolytic Anemia Complications :

Hemolytic Anemia Differential diagnosis

Presence of HemolysisIncrease RBC Production Increase RBC destruction

COOMBS ( DAT ) +ve -ve

IHA PB Smear RBC MorphologyLab Investigation

( LDH , Bili , Retics ..)

Definitive Diagnosis

Hemolytic Anemia Classifications

- Also it can classify into : INTRACORPUSCULAR HEMOLYSIS

-Membrane Abnormalities-Enzyme defects

EXTRACORPUSCULAR HEMOLYSIS-Nonimmune -Immune

- Generally , Hemolytic Anemia Classify depends on the causes of Defect into : - Hereditary - Acquired

Hemolytic Anemia Membrane Defects

- The membrane protein and lipid interactions associated with abnormal erythrocyte membranes can be divided into two categories:

1- Vertical interactions :interactions between the skeletal lattice on the cytoplasmic side and the integral proteins and lipids. Any defect cause uncoupling of the lipid bilayer from the skeletal lattice, selective loss of portions of the lipid bilayer, decrease in the surface area to volume .

Hemolytic Anemia

Hemolytic Anemia

2- Horizontal interactions :Parallel to the plane of the membrane and provide mechanical stability to the membrane .any defect lead to disruption of the skeletal lattice and membrane destabilization which lead to cell fragmentation and formation of poikilocytes .

Membrane Defects

Hemolytic Anemia Hereditary spherocytosis

- Autosomal-dominant, most common disorder of the red cell membrane (1:2000).- Deficiency of spectrin , combined deficiency of spectrin and ankyrin , mutations of ( ankyrin , α or β-spectrin , protein 4.2,band 3)- Influx of Na+ 10 times the normal rate- Increased cytoplasmic viscosity

Gene Locus

ANK1 8p11.2

SPTB 14q22-q23

SPTA 1q21

SLC4A1 17q21-q22

EPB42 15q15

Hemolytic Anemia

Hemolytic Anemia

Hemolytic Anemia

Normal or decreased Hb> 8% Retics60-87 fl MCVnormal MCH> 36g/dl MCHCNormoblastic erythroid hyperplasia, increased iron storage

BM

increased Osmotic fragilityincreased Indirect bilirubinincreased LDdecreased haptoglobin

RBCs small and lack the central pallor PB Smear

Laboratory findings

Hemolytic Anemia Hereditary elliptocytosis

- Autosomal dominant- Defect in the horizontal membran protein interaction:*Decreased association of spectrin dimers to form tetramers*Defect in band 4.1*Abnormalities in glycophorin C, abnormal anion transport(band 3) with increased affinity to ankyrin-The cells are abnormally permeable to Na+

- For Diagnosis : >25% of PB smear --> Elliptocyte

Hemolytic Anemia

Hemolytic Anemia Hereditary Pyropoikilocytosis

- Autosomal recessive , closely related to HE- Presents in infancy as severe HA with extreme poikilocytosis- HPP cell membranes fragment when heated to 45-46 C°

- Two defects :1. Related to a deficiency in α-spectrin 2. The presence of mutant spectrin that prevents association

of heterodimers to tetradimers

Hemolytic Anemia Hereditary Pyropoikilocytosis

PB Smear : - Fragments, Microspherocytes , Elliptocyte .- Decreased MCV 22-55 fl- Autohemolysis increased - Osmotic fragility is abnormal

Hemolytic Anemia

Hemolytic Anemia Hereditary Stomatocytosis Syndromes

- Autosomal dominant HA ,erythrocytes exhibit abnormalities in Na+ and K+ permeability.

- Osmosis leads to the red blood cell having a constant tendency to swell and burst.

- In the hereditary stomatocytosis, the passive leak is increased and the cell becomes swamped with salt and water.

Hemolytic Anemia Hereditary Stomatocytosis Syndromes

OHS : the RBC membrane is abnormally permeable to Na+ , K+ ( the net gain of Na+ > the net loss of K+) ,the capacity of cation pump is exceeded Water enters the cell ------> Stomatocyte

DHS :The net loss of intracellular K+ exceeds the passive Na+ influx decreased water and cation content ----> Xerocyte

* Variants :

Hemolytic Anemia

Hemolytic Anemia

Hemolytic Anemia

Hemolytic Anemia Abnormal lipid composition

Acanthocytosis- Acquired or inherited abnormalities of the membrane lipids- Liver disease, abetalipoproteinemia- lipid of the membrane exchange with plasma lipids- Acquiring excess lipids cause abnormal shapes.- Sequestered in spleen

Hemolytic Anemia Spur cell anemia

- Acquired hemolytic condition associated with severe hepatocellular diseaseIncreased cholesterol and phospholipid leads to :1- Decreased membrane fluidity and deformability2- Membrane fragments are lost during splenic passage( spur cells)*Moderate to severe normocytic normochromic anemiaHb 5-10 g/dLReticulocyte 5-15%Acanthocytes 20-80%Increased bilirubin, liver enzymes.

Hemolytic Anemia

Hemolytic Anemia

Hemolytic Anemia Rare forms

- Lecithin-cholesterol acyl transferase deficiency

- Autosomal recessive affects metabolism of HDL- LACT catalyzes the formation of cholesterol esters from cholesterol.- Low HDL & LDL, high VLDL & lipoprotein X- Mild HA, target cells

Other Forms : - McLeod phenotype with Kx & K antigen deficiency, Acanthocytosis with band 3 abnormalities

Hemolytic Anemia Enzyme deficiencies

- RBCs require constant energy to maintain biconcave disc shape and hemoglobin in reduced form. - Without adequate energy, red cells lyse and/or deform. - Energy from glucose is derived from metabolism- An inherited deficiency in one of the erythrocyte enzymes can compromise the integrity of the cell membrane or Hb and cause hemolysis.

Hemolytic Anemia

Hemolytic Anemia

Hemolytic Anemia

Hemolytic Anemia Hexose monophosphate shunt

Maintain adequate conc. Of GSHHb in the reduced state

The most common enzyme deficiency is G6PD

Heinz bodies

Hemolytic Anemia G6PD deficiency

- The most common erythrocyte enzyme disorder - G6PD deficient cells are more resistant to malarial parasites and/or more readily phagocytosed .- Sex linked carried by gene on the X chromosome.- Heterozygote females have two population of cells ( deficient & normal) - The majority of people have no clinical expression of the deficiency unless they have neonatal jaundice, exposed to oxidative drugs, or have severe infections .

Hemolytic Anemia Pathophysiology The generation of NADPH,GSH is impaired

and cellular oxidants accumulate

erythrocyte injury

Hb is oxidized to metHb (heinz bodies)

increased cell permeability to cations osmotic fragility, cell rigidity

bite and blister cells, spherocytes

Hemolytic Anemia

Hemolytic Anemia Females with G6PD deficiency

- Females heterozygotes for G6PD deficiency contain two populations of cells ( normal & with G6PD deficiency)- All cells in affected males are G6PD deficient- Females may have no clinical expression or may be affected as severely as males- Case reports of homozygous-deficient females

Hemolytic Anemia

- Favism : the sudden severe hemolytic episode that develops in some G6PD deficient individuals after ingestion of fava beans Signs :- malaise, nausia, vomiting, abdominal pain, tremor, fever.- Hemoglobinurea, jaundice- Severe favism affects children between the ages of 2-5 years

Hemolytic Anemia Methemoglobin Reductase pathway

- Maintains iron in the ferrous (Fe++) state.

- In the absence of the enzyme (methemoglobin reductase), the oxygen carrying ferrous ion (Fe2+) of the heme group of the hemoglobin molecule is oxidized to the ferric state (Fe3+).

- Methemoglobin accumulates and it cannot carry oxygen

- Hypoxia occurs due to the decreased oxygen-binding capacity of Methemoglobin, as well as the increased oxygen-binding affinity of other subunits in the same hemoglobin molecule which prevents them from releasing oxygen at normal tissue oxygen levels.

Hemolytic Anemia

Hemolytic Anemia Pyruvate kinase deficiency

- One of The most common enzyme deficiency in glycolytic pathway.- Autosomal recessive , > 180 different mutations in the PK gene (PKLR gene on chromosome 1q21”RBC”)- Clinically significant HA are associated with the homozygous or double heterozygosity for two mutant enzymes .

*Variation in clinical phenotype is related to the genotype and interaction with physiological and environmental factors : - ineffective erythropoiesis , splenic function, epigenetic modifications, polymorphism of other enzymes- Acquired PK deficiency is seen in some leukemias & myelodysplastic disorders- Single heterozygotes are asymptomatic

Hemolytic Anemia Pyruvate kinase deficiency

PathophysiologyEnergy producing reaction is prevented

failure of cation pumps

potassium loss, calcium & sodium gain dehydration(echinocytes)

sequestration in splenic cord and phagocytosis

Hemolytic Anemia

Hemolytic Anemia

Echinocytosis

Hemolytic Anemia Other enzyme deficiencies in the glycolytic pathway :

- Phosphoglyceratekinase deficiency: sex linked, HA and mental retardation in males females have milder form - Phosphofructokinase deficiency: indicated when subunits of the PFK enzyme are found in various tissues. Appear as myopathy or HA or both

- Triosephosphate isomerase deficiency: severe abnormalities in RBCs ,severe hemolysis, death in infancy, abnormalities in striated muscle & CNS

Hemolytic Anemia Other enzyme deficiencies in the glycolytic pathway :

- Glucose phosphate isomerase deficiency : cause hemolytic anemia, all mutants are unstable,show partial response to splenectomy

- Hexokinase deficiency: two types : 1.associated with HA that responds to splenectomy 2.associted with HA & other abnormalities

The deficiency in this enzyme interferes with the production of 2,3-BPG, patients tolerate anemia poorly.

Hemolytic Anemia What is New In Hemolytic Anemia ?

خير ! كل- The most recent and new research's , concentrate on the Autoimmune type of hemolytic anemia. - A lot of aspects in Autoimmunity still unknown .- Most of hereditary Hemolytic Anemia is well defined .

Hemolytic Anemia

Thank You Thank You Thank You Thank You Thank You Thank You Thank You Thank You Thank You Thank You Thank You Thank You