8/7- Hereditary Hemolytic Anemias

Question 1

What is shown here?

Answer 1

Left: reticulocyte

• Excess of membrane which forms tuft (looks like pinched wet clay); this membrane is lost over 24 hr period along with mitochondria, left over RNA, and ribosomes

Right: normal discocyte

Question 2

What determines a RBC’s deformability?

Answer 2

Surface area/volume

• Increased in a discocyte
• Decreased in a spherocyte

Membrane pliability

• Unstable in HS and HE
• Oxidation

Increased internal viscosity

• Increased in SS-Hb and xerocytosis

Question 3

Case: Young man with jaundice

• 21 yo referred from GI clinic to hematology after negative workup for jaundice and RUQ pain
• Pts is “lemon yellow”, but no N/V
• GI eval: ceruloplasmin, SPEP, hepatitis serology, HIV, ANA, EBV, Sm Mus Ab, and normal liver biopsy
• Hemoglboin: 11.5, HCT 30 (MCHC 38)… Mild anemia
• Reticulocyte count 7% (RPI 3.1)… slightly elevated retics
• Normal PT, PTT
• Normal Hb electrophoresis
• LFTs total bili 4.5 with direct of 1.2
• ALT 63, AST 33, LDH 177
• Mother and maternal grandmother had gallbladder removed at young ages
• Spleen tip palpable on physical exam

What is this?

Answer 3

Hereditary Spherocytosis (HS)

• Spherocytic hemolytic anemia seen on PBS
• Family hx of gallbladder removal is indicative of stones due to…?

Question 4

What is the most common hemolytic disease in black people? Caucasians?

Answer 4

Black: sickle cell

Caucasian: Hereditary Spherocytosis

Question 5

Mutations leading to hereditary spherocytosis involve what part of the RBC?

Answer 5

Cytoskeleton

• Problem with vertical interactions (?)

Question 6

What is seen here?

Answer 6

Hereditary spherocytosis

• Not completely spherical; this one has a dimple
• These cells have a reduced SA ratio

Question 7

Genetic characteristics of Hereditary Spherocytosis?

• Inheritance
• Prevalence
• Charactersitics

Answer 7

• Autosomal dominant
• Occurs in 1:2000
• 25% of the time, parents are not affected (pt is de novo)
• Variable degrees of anemia
• Reticulocytosis

Question 8

What is the main underlying pathophysiology of HS?

Answer 8

Ankyrin

• Most common
• Gene on 8p; wide deletions with HS, mental retardation, typical facies and hypogonadism

Alpha and Beta Spectrin Mutations

Band 3

• Truncated
• Pincer cells

Protein 4.2 deficiency

• Japanese
• Sphero-elliptocytosis

Question 9

What is this? What causes it?

Answer 9

Acanthyocytic HS

• Certain beta spectrin mutations

Question 10

What is this? What causes it?

Answer 10

“Pincered” HS

• Associated with truncated band 3

Question 11

What is this?

Answer 11

Spleen

• Graveyard of the spherocyte
• Small openings between sinusoids of the spleen and veins which carry blood back to circulation act as traps for non-deformable RBCs
• If stuck, consumed by macrophages nearby
• Excess membrane may be removed when cells in HS go through slits, losing bits of lipid bilayer each time, since it’s not tethered to the cytoskeleton… thus cells become progressively more spherical in “splenic conditioning”

Question 12

Pathophysiology of HS (figure)

Answer 12

Question 13

What is seen in HS in regards to osmolarity?

Answer 13

There is increased osmotic fragility in HS

• Physiologic [NaCl] is about 0.9%
• RBCs in hypotonic solutions swell, and can normally deal with this because of the increased SA ratio of cells
• When cell swells beyond the point that the cytoskeleton can hold the membrane together, hemoglobin will escape (osmotic hemolysis)
• In HS, loss of membrane causes hemolysis to occur at higher concentrations (0.7%)

Question 14

HS Pathophysiology Summarized

Answer 14

• Spectrin Loss (often due to Ankyrin)
• Defects in Vertical Cytoskelton
• Membrane Instability
• Loss of Surface Area
• Spherical RBCs
• Dehydration (increased MCHC)
• Splenic Conditioning and Trapping

Question 15

How may HS be diagnosed?

Answer 15

• Spherocytosis
• Increased MCHC
• Reticulocytosis
• Family History (although parents don’t always have it)
• Splenomegaly
• Negative Coomb’s Test (to rule out autoimmunity)
• Increased Osmotic Fragility
• Eosin-5 maleimide (EMI) binding to RBC

Question 16

What is the clinical course of HS?

Answer 16

• Neonatal Jaundice (need phototherapy)
• Splenomegaly
• Hemolytic Crises During Infection
• Aplastic Crises with Parvovirus B19
• Gallstones
• Jaundice with Indirect Hyperbilirubinemia
• Folate Deficiency
• Rarely: pulmonary Hypertension, leg ulcers

Question 17

Treatment of HS?

Answer 17

Folate Supplementation

Splenectomy

• Not done in children until 8 or 9 yo
• Not done as often because of possible post-splenectomy issues (sepsis with encapsulated organisms, DVT post abdominal surgery, long term risk for DVT, pulmonary HTN, increased risk for CVD and stroke)

Question 18

What is this?

Answer 18

Hereditary Elliptocytosis

• Problem with horizontal interactions
• Shear stress elongates the cell; normally flips back when it gets into wider venous circulation, but here, they don’t go back

Question 19

What is this?

Answer 19

Hereditary Elliptocytosis

• More than 50% of the RBCs should be elliptical
• Some of the cells resemble the ovalocytes you see in B12 deficiency
• The elliptocytes are not typically macrocytic

Question 20

What is this?

Answer 20

Hereditary Elliptocytosis

• When heated, the elliptocyte spectrin is typically unstable
• Spectrin precipitates from the membrane and blebs of lipid form

Question 21

What is this? What causes it? Epidemiology?

Answer 21

A form of HE/Ovalocytosis in which RBC has a transverse ridge (characteristic triangular shape)

• Abnormal band 3 proteins
• Appear to be resistant to malarial invasion (band 3 is a malarial receptor); protective
• Found in SE Asia and the South Pacific (Malaysia, New Guinea, Indonesia, Philippines)

Question 22

Genetic characteristics of HE?

• Inheritance
• Characteristics
• Epidemiology

Answer 22

• Autosomal dominant
• More severe disorders are usually double heterozygotes
• Bizarre blebbing and microspherocytes: HPP (hereditary pyropoikilocytosis); can have incredibly small MCV (like a mouse)
• Southeast Asian ovalocytosis is associated with resistance to malaria

Question 23

What are the membrane protein defects associated with HE?

Answer 23

Spectrin

• Self-association sites (key feature!)
• Spectrin alpha(LELY)

Protein 4.1

Glycophorin C deficiency

• Leach phenotype 7 kb deletion
• GPC deficiency with elliptocytosis

Question 24

Case) A Day Laborer with Anemia

• 25 yo man employed in plumbing supply store on weekends as day laborer
• Spent a day cleaning chemical storage plants down by the ship channel (Saturday)
• 3 days later, doesn’t feel well; mild dyspnea
• Examination unremarkable
• Lab: Hct 37.8, Hb 13.2, WBC 14.8… so slightly anemic with a little WBC elevation
• Next day had fatigue and yellow eyes
• Lab: Hct 25.6, Hb 9.1 MCV 96, WBC 21.3… so more severe anemia and lymphocytosis
• UA: moderate blood, 4+ bili, no red cells
• Total bilirubin 10.6, direct 2.3
• LDH: 2.3
• Coombs D/I both negative
• Reticulocyte count 6.5%… relatively increased
• Haptoglobin 6

Answer 24

Oxidant hemolysis

• All factors of intravascular hemolysis present

- Blister cells: precipitates of hemoglobin that are so dense they won’t take up dye in the Wright’s stain (although can use Heinz body stain)

Question 25

What is this?

Answer 25

Blister cells stained with Heinz body stain

Question 26

Process of oxidant hemolysis (flowchart)?

Answer 26

Question 27

What is this?

Answer 27

Heinz bodies - May occur from hemoglobinopathy or from metabolic disorder (not producing NADP)

Question 28

G6PD Variants?

Answer 28

Question 29

Benefit of G6PD mutation?

Answer 29

Possibly malaria resistant (metabolically defunct cell can't handle the load?)

Question 30

Epidemiology of GP6D?

Answer 30

About 10% of American black men are G6PD deficient

Question 31

What are some compounds associated with oxidant hemolysis in G6PD?

Answer 31

**Antimalarials** - Primaquine, Quinacrine **Sulfonamides, Sulfones** - Sulfamethoxazole, Sulfanilamide - Diaphenylsulfone (Dapsone) **Nitrofurans** - Nitrofurantoin **Misc** - Methylene blue - Naphthalene (Moth balls) - Rasburicase

Question 32

Review of G6P Deficiency

Answer 32

- Hundreds of variants - X-linked inheritance - A- present in 12 % of black American men - Neonatal hyperbilirubinemia - Mediterranean: may hemolyze with fava beans—divicine; may have chronic hemolysis - Blister and bite cells during hemolysis - Heinz bodies Crystal violet supravital stain

Question 33

Pathophysiology of pyruvate kinase deficiency

Answer 33

**- Autosomal recessive**, but most affected are double heterozygotes - Red blood cell enzyme is deficient and there is little generation of ATP - Most of the circulating cells are reticulocytes because they retain a few mitochondria - Before splenectomy: 5-15 %, after splenectomy 70 % reticulocytes - The morphology of the cell is that of a run down energy depleted red blood cell—mostly echinocytosis with extreme polychromatophilia from the resistant population of reticulocytes.

Question 34

What is this?

Answer 34

**PK Deficiency** - In PK deficiency not enough ATP is generated so all of the membrane pumps run down such that the RBC cannot maintain it’s Na K gradient or the integrety of its lipids—echinocytes form. The only cells that are truly happy are very young reticuloyctes they can make ATP with their mitochrondria.