Membrane Defects

hemolytic anemias part 1 - hereditary membrane defects and enzyme deficiencies

Hereditary Membrane Defects

• hereditary spherocytosis

• hereditary elliptocytosis

• hereditary stomatocytosis

• acanthocytosis (abetelipoproteinemia)

Acquired Membrane Defects

Paroxysmal Nocturnal Hemoglobinuria (PNH)

Enzyme Defects

• G6PD Deficiency

• Pyruvate Kinase Deficiency

Hemoglobinopathies

• Hb S, Hb C, Hb E (main variants)

• Thalassemias (alpha and beta)

Hereditary Spherocytosis

• hallmark: spherocytes

• defect type: spectrin/ankyrin deficiency

• mechanism of hemolysis: extravascular (splenic removal)

Diagnosing Hereditary Spherocytosis

• clinical manifestations: anemia, jaundice, splenomegaly

• laboratory testing

  • CBC: Hgb decreased, MCHC >36, MCH & MCV normal

  • blood smear: spherocytes on PB smear

  • retic: RPI is increased

  • Osmotic Fragility Test: increased

  • Coombs test: to differentiate from autoimmune hemolytic anemia (also called the direct antiglobulin test or DAT)

Osmotic Fragility Test

• principle confirmatory test for hereditary sphereocytosis

• tests measures RBC resistance to hemolysis

• RBC resistance depends on shape, volume, surface area, and integrity of the cell membrane

• RBCs swell when put into a hypotonic solution by taking in more water to achieve equilibrium

• because of decreased surface: volume ratio of spherocytes, they cannot expand like normal RBCs

• Spherocytes have an increased osmotic fragiltity

Hereditary Elliptocytosis

• Hallmark: elliptocytes

• Defect type: spectrin or protein 4.1 defect

• Mechanism of Hemolysis: mild extravascular

Diagnosing Hereditary Elliptocytosis

• clinical manifestations: anemia (usually none, but can be mild to severe)

• laboratory testing

  • CBC: relatively normal

  • blood smear: elliptocytes on PB smear (could be the only morphologic clue to this disease because patients are asymptomatic). schistocytes may also be present (elliptocytes are likely to fragment)

  • retic: RPI is increased

Hereditary Stomatocytosis

• hallmark: stomatocyte

• defect type: Na+/K+ membrane permeability defect

• mechanism of hemolysis: mild extravascular

Diagnosis Hereditary Stomatocytosis

• clinical manifestations: anemia (usually mild: 8-10g/dL)

• laboratory testing

  • CBC: increased MCV, MCH… decreased MCHC

  • blood smear: stomatocytes (10-50% of cells are stomatocytes)

  • retic: RPI is increased

Acanthocytosis

• Hallmark: Acanthocytes

• due to an abnormal membrane lipid composition

• associated with spur cell anemia and abetalipoproteinemia

Acanthocytosis Spur Cell Anemia

• acquired condition associated with severe liver disease in which lipoproteins increase, leading to an increase in cholesterol

• mechanism of hemolysis: extravascular

Acanthocytosis Abetalipoproteinemia

• rare autosomal recessive disorder characterized by the absence of serum B-lipoprotein, low serum, cholesterol, low triglyceride, low phospholipid, with increased ratio of cholesterol to phospholipids

• minimal hemolysis with little or no anemia

Paroxysmal Nocturnal Hemoglobinuria (PNH)

• a rare, acquired intrinsic disorder of RBC membrane with paroxysmal (sudden) occurrence

• membrane defect makes the cells abnormally sensitive to complement lysis

• results from a stem cell somatic mutation that leads to an abnormal clone of differentiated cells that affect all cell lines (RBC, WBC, Platelet)

• there is a deficiency in the regulation of complement activation - once complement attaches to the RBC membrane, the cell is marked for hemolysis

PNH Regulatory Proteins

• deficiency of membrane glycolipid Glucosyl-phophatidyl inosital (GPI) that normally anchors Decay Accelerating Factor (DAF) and membrane inhibitor of reactive lysis (MIRL)

• DAF expresses CD55 on cell surface

• MIRL expresses CD59 on cell surface

• complement activation remains unregulated when DAF and MIRL cannot bind

Type 1 PNH

little to no hemolysis by complement; nearly normal GPI-linked protein expression

Type 2 PNH

moderately sensitive to complement lysis: intermediate GPI-linked protein expression

Type 3 PNH

highly sensitive to complement lysis; no expression of GPI-linked proteins

Clinical Findings PNH

• four basic disease manifestations

  • hyperhemolysis

  • venous thrombosis

  • infection

  • bone marrow hypoplasia

• patient usually seeks help when they note reddish-brown urine (hemoglobinuria)

• PNH can result in iron or folate deficiency

Laboratory Testing PNH

• CBC: anemia (Hgb 8-10 g/dL)

• retic: increased RPI

• immunophenotyping (CD55 expression is low, CD59 expression is low)

Sucrose Hemolysis Test

for PNH, the patient’s blood is incubated in sucrose solution that promotes binding of complement, because PNH cells are complement-sensitive, they will lyse

Ham’s Test

for PNH, aka acidified serum lysis test - has been replaced by immunophenotyping

Enzyme Deficiencies

mature RBCs have no nucleus, mitochondria, or ribosomes, so they keep metabolically active by secondary pathways

• Hexose monophosphate shunt (HMP)

• Glycolytic pathway (Embden-Meyerhof pathway)

HMP Shunt

• catabolizes 10% of the cells glucose and is necessary to maintain adequate levels of reduced glutathione (GSH)

  • GSH protects the RBC from oxidant damage

  • GSH is maintained by high levels of conversion of NADPH to NADP through glutathione reductase (GR)

  • G6PD enzymes restores the levels of NADPH, thereby restoring levels of GSH

• if G6PD is reduced or missing, HMP shunt is deficient and oxidized hemoglobin accumulates (heinz bodies)

  • as cell goes through the spleen, Heinz bodies are removed and the cell is lysed

G6PD Deficiency

• G6PD is the most common RBC enzyme disorder to cause intravascular hemolysis

  • sex-linked recessive disorder

  • self-limiting form of hemolytic anemia precipitated by antimalarials, sulfanomides, and fava beans (meaning patients are asymptomatic as long as they stay away from these)

  • hemolysis occurs 2-3 days after ingestion of substance

Clinical Presentations G6PD Deficiency

• acute, acquired hemolytic anemia (episodic - including favism)

• hereditary nonspherocytic hemolytic anemia (chronic hemolysis not triggered by drugs or infections, although they may exacerbate the condition)

• neonatal hyperbilirubinemia with jaundice (mechanism unknown, severe jaundice & kernicterus possible)

Favism G6PD Deficiency

• G6PD Mediterranean variant

• severe hemolytic episode triggered by ingestion of fava beans

• the beans have high concentrations of certain glucosides that cause severe damage to RBCs when they are deficient in G6PD

• first signs of favism are: malaise, severe lethargy, nausea, vomiting, abdominal pains, chills, tremor, or fever

• hemoglobinuria occurs a few hours later and jaundice can become intense

Laboratory Testing G6PD Deficiency

• CBC: anemia (sudden drops in hemoglobin to as low as 3g/dL)

• peripheral smear: bite cells

• supravital stain: heinz bodies

• increased bilirubin: hyperbilirubinemia (newborns & kernicterus)

• urine: hemoglobinuria

Qualitative Fluorescent Spot Test

• whole blood is added to a mixture of glucose-6-phosphate, NADP, and saponin

• drop is placed on a piece of filter paper

• examine under UV light for fluorescene

• the G6PD enzyme present in RBC metabolized the G6P, producing NADPH, which fluoresces

• NADP does not fluoresce, so lack of fluorscence indicates a G6PD deficiency

• high false negative rate (around 25%)

Dye Reduction Test

• incubate a hemolysate of patient’s blood with G6P, NADP, and the dye brilliant cresyl blue. If hemolysate contains G6PD, the NADP is reduced to NADPH, which reduced the blue dye to colorless

• normal G6PD color change happens within 30-60 minutes

• may miss moderate deficiencies

• false negatives after hemolytic episodes (G6PD deficient cells destroyed, leaving behind younger cells with higher G6PD)

Pyruvate Kinase Deficiency

• due to a pyruvate kinase enzyme mutation

• 2nd most common RBC enzyme disorder

• PK deficiency compromises the conversion of ADP to ATP which decreases the energy source to maintain the cation pumps, the cell membrane is deformed, leading to splenic sequestration and cell destruction

• to diagnose PK- would need to perform enzyme test to confirm mutations (PKLR gene mutation)

• likely to see echinocytes and polychromasia on the peripheral blood smear