13. Hemoglobinopathies

Question 1

Hemoglobinopathies

Answer 1

• A genetic defect that results in abnormal structure of one of the globin chains of the hemoglobin molecule - Very few actually produce a “disease” or disorder - Most are asymptomatic abnormal hematologic findings

Question 2

Pathophysiology of Hemoglobinopathies

Answer 2

• increased/ decreased O2 affinity - Methemoglobinemia (can’t bind to oxygen) - Unstable hemoglobin (hemoglobin precipitates) - Sickling and crystallization

Question 3

Increased O2 Affinity

Answer 3

• Hemoglobin eagerly accepts oxygen but does not want to release it. - The kidney responds to the decrease in oxygen by producing more erythropoietin - Erythropoiesis is stimulated resulting in increased RBCs (high hematocrit) - The genetic disorder is rare; condition is more commonly acquired by stress, cigarette smoking, high altitudes, chronic lung disease

Question 4

Decreased O2 Affinity

Answer 4

• Hemoglobin is reluctant to pick up oxygen = deoxygenated hemoglobin - If the level of deoxygenated hemoglobin exceeds 5 g/dL clinical cyanosis results (Blue lips/nails) - The genetic abnormality is rare; cyanosis can also result from pulmonary dysfunction

Question 5

Methemoglobin

Answer 5

• Low O2 affinity hemoglobin variants characterized by the presence of heme that contains iron in the ferric (Fe+++) rather than ferrous (Fe++). - Genetic abnormalities are termed Hgb M - Enzyme deficiencies - Oxidizing drugs and compounds - “Brown” hemoglobin

Question 6

Unstable Hemoglobin

Answer 6

• Genetic abnormalities cause an intrinsically unstable hemoglobin - Denatured hemoglobin forms RBC inclusions (Heinz bodies) -Denaturation due to enzyme deficiency. (ex. G6PD deficiency makes unstable hemoglobin.) ***Heinz bodies are not visible with Wright stain; must be stained with a supravital stain

Question 7

Sickling and Crystallization

Answer 7

Common hemoglobopathies in the United States: - Hemoglobin S - substitution of valine for glutamic acid - Hemoglobin C - substitution of lysine for glutamic acid

Question 8

Laboratory Tools for looking at Hemoglobinopathy

Answer 8

• CBC - Peripheral blood smear - Hemoglobin separation: Electrophoresis, HPLC (High Performance Liquid Chromatography), Isoelectric focusing - Sickle cell screening

Question 9

Hemoglobin Electrophoresis

Answer 9

• Used to isolate, identify, and quantitate hemoglobin bands - When placed in an electrical field, hemoglobins will migrate according to their: pH (charge) and the Media used Typically (alkaline): 0 = origin A= accelerated (fastest so moves furthest down) F= fast S= slow C= crawl (slowest so it barely makes it of the origin/starting line)

Question 10

Sickle Cell Screening

Answer 10

• Solubility Test (6 min) - Hemoglobin Electrophoresis (> 30 min; long)

Question 11

Sickle Solubility Test

Answer 11

• Sickling hemoglobin in a deoxygenated state will precipitate in a concentrated phosphate buffer solution. - will not become soluble so you can’t see the lines through the tube (Turbid = +) - NYS does not recognize this test - Not specific for Hgb S - Cannot resolve trait vs disease - False positives with increased proteins, hyperlipidemia, high WBC, cold reagents (due to condensation) - Sensitivity approx 20-25% (Infants - have a low Hem count and won’t trip the test, Transfusions)

Question 12

Epidemiology and Genetics of Hemoglobin S and Sickle Cell Disease

Answer 12

• Incidence of S trait in African populations is 40% - Incidence of S trait in African-Americans is 8% - Established itself as a protective mechanism against malaria (because Hem S doesn’t carry oxygen efficiently)

Question 13

Pathophysiology of Hemoglobin S and Sickle Cell Disease

Answer 13

• Under low oxygen tension, hemoglobin S will precipitate forming tactoids - RBCs stretch around the tactoids, forming the characteristic sickled cells - The greater the proportion of Hgb S, the greater the propensity to sickle - Heterozygotes do not sickle except under extraordinary conditions

Question 14

Clinical Findings of Sickle Cell Disease

Answer 14

• Chronic hemolysis: anemia, jaundice, cholelithiasis (gallstones), aplastic crisis (stop making RBC), hemolytic crisis - Vaso-occlusion: dactylitis - swollen and painful feet, auto-splenectomy, renal necrosis, infarctive crisis, leg ulcers and infection (due to poor circulation)

Question 15

Laboratory Findings of Sickle Cell Disease

Answer 15

• Severe normocytic, normochromic anemia - Blood smear: target cells, sickle cells (sometimes), nRBC, schistocytes, siderotic granules (iron overload from transfusions), Howell-Jolly bodies - Reticulocytes increased (body trying to compensate because RBCs don’t last 120 days), except in aplastic crisis (retics are low) - Hemoglobin electrophoresis - Hgb S = 80-90%, Hgb F = 1-20%, Hgb A2 = 2-3% (Beta gene making Hgb S instead of Hgb A) Sickle test - positive

Question 16

Laboratory Findings of Sickle Cell Trait

Answer 16

• normal CBC - normal blood smear - Hemoglobin electrophoresis - Hgb A = 55-65%, Hgb S = 35-45%, Hgb A2 = 2-3% - Sickle test - positive

Question 17

Hemoglobin C: Epidemiology and Genetics

Answer 17

• Incidence in West Africa is 20-25% - Incidence in African-Americans is 2-3%

Question 18

Hemoglobin C: Pathophysiology

Answer 18

• Hgb C forms intracellular blunt-ended crystalloids - Usually not seen with normal splenic function (Only seen if spleen is removed or non-functioning) - Decreased RBC survival

Question 19

Hemoglobin C Disease

Answer 19

-hemolysis is not as severe as Hb SS and vaso-occlusive crisis generally does not occur; prognosis is excellent - Mild normo-microcytic, normochromic anemia (smaller end of normal) - numerous target cells - Hgb C crystals - post-splenectomy - Reticulocyte slightly increased - Hgb electrophoresis - Hgb C= 95%, Hgb F= <7%, Hgb A2 = 2-3%

Question 20

Hemoglobin C Trait

Answer 20

-asymptomatic - target cells, mild microcytosis - Hgb Electrophoresis - Hgb A = 55-65%, Hgb C = 35-45%, Hgb A2 = 2-3%

Question 21

Hemoglobin D

Answer 21

• Both homozygous and heterozygous states are asymptomatic - CBC/ Peripheral blood is normal - Hgb D migrates with Hgb S on alkaline electrophoresis and with Hgb A on acid electrophoresis

Question 22

Hemoglobin E

Answer 22

• Occurs frequently in SE Asians - Currently the most frequent hemoglobinopathy in California - Hemoglobin E Disease - mild anemia, target cells, microcytic, hypochromic cells - Hemoglobin E Trait - clinically aymptomatic, microcytosis, target cells, - Hgb electrophoresis - Hgb A = 70%, Hgb E = 30%, Hgb A2 = 2-3%

Question 23

Hemoglobin SC

Answer 23

-double heterozygote - generally less severe than S Disease - peripheral blood shows target cells, folded RBCs, and intracellular crystals - Hgb electrophoresis - Hgb S = 45%, Hgb C = 45%, Hgb F = 2%, Hgb A2 = 2-3%

Question 24

Hemoglobin SD

Answer 24

• double heterozyhote - clinical severity falls between that of S Disease and S Trait - hemoglobins comigrate on alkaline electrophoresis - acid hemoglobin electrophoresis needed to differentiate (because looks like S on alkaline)

Question 25

Answer 25

1. SS Disease
2. S Trait
3. C Disease
4. SC Disease

Question 26

Answer 26

1. AS (Trait)
2. S disease
3. C disease
4. SC disease

Question 27

Answer 27

1. SS disease
2. S trait
3. C disease
4. SC disease

Question 28

Answer 28

1. S trait
2. D trait

* Note that an Acid electrophoresis must be run to destinguish the two.

Question 29

Answer 29

1. EE
2. E trait
3. E / beta-thal