Hemoglobinopathies

a group of genetic hemolytic anemias caused by the hemoglobin molecule being either:

• structurally normal but produced in reduced amounts

• structurally abnormal

what are the hemoglobinopathies?

• 4 globin chains

• one heme molecule that carries one atom of oxygen

what is the structure of hemoglobin?

• 2 alpha chains consisting of 141 amino acids

• 2 non-alpha chains

what comprises the 4 globin chains of hemoglobin?

it helps control the shape of the hemoglobin molecule

why is the sequence of amino acids in the hemoglobin molecule important?

• hemoglobin A

• hemoglobin A₂

• hemoglobin F

what are the 3 normal types of hemoglobin in adults?

2 alpha and 2 beta chains

what is hemoglobin A comprised of?

95%

what percent of hemoglobin is hemoglobin A?

2 alpha and 2 delta chains

what is hemoglobin A₂ comprised of?

3%

what percent of hemoglobin is hemoglobin A₂?

2 alpha and 2 gamma chains

what is hemoglobin F comprised of?

1%

what percent of hemoglobin is hemoglobin F?

a chronic hemolytic anemia resulting from an inherited alteration of the globin chain of hemoglobin

what is sickle cell anemia?

hemoglobin S

what is produced in place of hemoglobin A in sickle cell anemia?

valine

what substitutes glutamic acid on the sixth position of the beta chain in sickle cell anemia?

the hemoglobin has a slightly different shape that causes the cell to from a sickle-shape at low oxygen tensions

• hemoglobin S molecules aggregate to form a “tactoid” structure

why would a single AA substitution cause a problem?

they intertwine with each other becoming insoluble, blocking microcirculation and cause infarction

what happens if the cell cannot get back to the lungs for oxygen?

they are removed by the spleen

what happens to deformed cells?

autosomal recessive

what type of inheritance is sickle cell inheritance?

Africa, Middle East, and USA

where is sickle cell disease found?

malaria

what is sickle cell associated with?

makes cell sickle and spleen will remove

what effect does the malaria parasite have on cells?

normal

what is the sickle cell trait considered?

when O₂ is reduced to less than 40%

when does sickling occur?

• infarcts to bone, eye, abdomen, brain

• anemia develops

• aplastic crisis

• tendency for infection

what are some sickle cell disease crises?

• NC/NC anemia with sickled forms during a crisis

• signs of autosplenectomy

• increased bilirubin, reticulocytes, WBCs

• target cells may be observed when not in a crisis

what are the laboratory findings of sickle cell disease?

solubility tests

what is a good screening tool for sickle cell disease?

deoxygenated Hgb S has decreased solubility when added to a reducing agent

what is the mechanism for solubility tests for sickle cell disease?

• saponin

• sodium dithionnite

what are some reducing agents used in solubility tests?

dissolves membrane lipids, releasing hemoglobin

what is the function of saponin?

reduces ferrous iron to ferric iron

• unable to bind oxygen, converting the molecule to deoxygenated form

what is the function of sodium dithionite?

Hgb S polymerizes and becomes insoluble, becoming turbid

what happens if Hgb S is present in the solubility test for sickle cell disease?

increased levels of Hgb F

what can cause false negative sickle solutbility?

50µL is added to reducing agent

what should be done if the patient is very anemic for sickle solubility?

• too much sample

• hyperlipidemia

what can cause a false positive sickle solubility?

hemoglobin electrophoresis

what is used to diagnose sickle cell disease?

separates the hemoglobin based on globin chains based on movement to the anode pole

what is the mechanism of hemoglobin electrophoresis?

cellulose acetate agar

what agar is used for hemoglobin electrophoresis?

• Hgb A—closest to the anode (accelerate)

• Hgb F— follows (fast)

• Hgb S—slow

• Hgb C—crawl (closest to cathode or (-) pole)

how fast do each type of hemoglobin move in hemoglobin electrophoresis?

citrate agar

what agar is used to verify Hgb S or C from other variants during hemoglobin electrophoresis?

8.4

what is the pH of cellulose acetate agar?

6.0

what is the pH of citrate agar?

position and thickness of bands

what indicates the type of hemoglobin present and the amount of hemoglobin present during hemoglobin electrophoresis?

provide comfort and support 

• control pain

• adequate hydration

• genetic counseling

• treat infection

what is the treatment for sickle cell disease?

prophylactic penicillin

what is given to children for treatment of sickle cell disease?

• bone marrow transplant

• anti-sickling agents

• apheresis to keep Hemoglobin S quantity low

• hypertransfusion

what are some experimental treatments of sickle cell disease?

a mild chronic hemolytic anemia with splenomegaly

what does hemoglobin C produce?

• NC/NC anemia

• numerous target cells

• hemoglobin C crystals

• negative sickle solubility

• diagnose with electrophoresis

• no treatment is usually needed

what are the laboratory findings of hemoglobin C?

inherited S and C from each parent

how is hemoglobin SC disease transmitted?

positive

what sickle solubility result does hemoglobin SC disease have?

hemoglobin electrophoresis

how is hemoglobin SC disease diagnosed?

all the vaso-occlusive complications of SCD, but the damage is less disabling

how does hemoglobin SC disease compare to sickle cell disease?

a splice site, causing decreased transcription of mRNA

what does hemoglobin E cause?

mild chronic hemolytic anemia

what does hemoglobin E produce?

microcytic, hypochromic with increased target cells

what is the RBC morphology of hemoglobin E?

negative

what is the result of Hgb Solubility Test for hemoglobin E?

Southeast Asian descent

who is hemoglobin E commonly found in?

• structural defect

• qualitative defect in the hemoglobin molecule

• caused by an amino acid substitution

• considered NC/NC despite shapes

• Hgb S; Hgb C; Hgb SE

what are the characteristics of hemoglobinopathy?

• reduced amount

• quantitative defect in the hemoglobin production

• due to a gene rearrangement

• produces hypochromic, mirocytic cells

what are the characteristics of thalassemia?

• reduced rate of globin chain

• defective hemoglobin is produced

• the most common single gene disorder in humans

what characterizes thalassemia?

• alpha thalassemia

• beta thalassemia

what are the two types of thalassemia we will focused on?

Mediterranean East through the Middle East and India to Southeast Asia and South through Africa

what is the thalassemia belt?

genetic disorder resulting in abnormal amount or function of mRNA

what is Beta Thalassemia?

mutations in the coding regions, splice sites, or termination codons affect RNA processing and translation

what causes the genetic disorder in Beta Thalassemia?

• reduced Beta Globin Chain

• absent Beta Globin Chain

what does mutations in Beta Thalassemia produce?

major

what is the type of thalassemia inherited as homozygous?

minor, intermedia

what is the type of thalassemia inherited as heterozygous?

heterozygous state with no hematologic abnormalities or clinical symptoms

what is the inheritance of Beta-Thalassema silent carrier?

heterozygous state with mild hemolytic anemia, microcytic/hypochromic RBCs, no clinical symptoms

what is the inheritance of Beta-Thalassemia minor?

homozygous with severe hemolytic anemia, microcytic/hypochromic RBCs, severe clinical symptoms, and transfusion dependent

what is the inheritance of Beta-Thalassemia major?

mild to moderate hemolytic anemia, microcytic/hypochromic RBCs, moderate clinical symptoms, transfusion independence

what is the inheritance of Beta-Thalassemia intermedia?

• no hemoglobin A

• hemoglobin is very low

• marked expansion of marrow spaces

  • due to ineffective erythropoiesis

• marked anisocytosis, poikilocytosis

• microcytosis, hypochromia

• target cells

• nucleated RBCs

• basophilic stippling

what are the lab findings for Beta-Thalassemia major?

• decreased hemoglobin and hematocrit

• RBCs are normal to increase

• indices are decreased

• RDW is increased in major

what are the CBC results of Beta-Thalassemia major?

• little or no hemoglobin A

• increased hemoglobin A₂ and hemoglobin F

what are the hemoglobin electrophoresis results for Beta-Thalassemia major?

• must give blood transfusions throughout lifetime

• hematopoietic stem cell transplant

• possible gene therapy

what is the treatment for Beta-Thalassemia?

• mild anemia

• microcytic, hypochromic cells with target cells and basophilic stippling

• patients have few symptoms except during pregnancy

what are the traits of Beta-Thalassemia?

genetic deletion

what causes alpha thalassemia?

11

what chromosome are the beta, gamma, and delta chains at with alpha thalassemia?

2

how many genes control beta, gamma, and delta chains?

4

how many genes control alpha chains?

16

what chromosome are alpha chains located on in alpha thalassemia?

1, 2, 3 or 4 gene deletion 

what types of alpha thalassemia can humans have?

hydrops fetalis—death in utero

what does 4 gene deletion cause?

hemoglobin A

what is not produced in 4 gene deletion alpha thalassemia?

Asian descent

who is 4 gene deletion Alpha-Thalassemia primarily found on?

hemoglobin H disease

what is another name for 3 gene deletion Alpha-Thalassemia?

• severe hemolytic anemia with tetramers

• non-functional tetramers cause precipitation in cells

  • cells lyse

• found primarily in Asians

what are the characteristics of Hemoglobin H disease?

cis or trans

what are the forms of 2 gene deletion of Alpha-Thalassemia?

microcytic, hypochromic anemia

what does 2 gene deletion Alpha-Thalassemia cause?

Asians

where is the cis form of 2 gene deletion Alpha-Thalassemia found?

Black people

where is the trans form of 2 gene deletion Alpha-Thalassemia found?

no cell abnormalities

what does 1 gene deletion Alpha-Thalassemia cause?

30% African Americans

where is 1 gene deletion Alpha-Thalassemia found?

no switch from fetal hemoglobin to adult hemoglobin, and beta chain is not produced

what is hereditary persistence of fetal hemoglobin?

15-35% hemoglobin F

what do carriers of Hereditary Persistence of Fetal Hemoglobin have?

100% Hemoglobin F

what do homozygous individuals of Hereditary Persistence of Fetal Hemoglobin have?

• peripheral smears are ethanol fixed and immersed in a citrate acid buffer

• adult hemoglobins are eluted from the RBCs, but Hgb F resists acid elution and remains in the cell

• slides are then stained

• true HPFH will have even distribution of Hgb F in al RBCs

what are the steps to the Kleiheihauer Betke Test?

RBCs containing Hgb F will take up the stain, whereas RBCs containing adult hemoglobin will appear as “ghosts”

what happens when the slides are stained during the Kleiheihauer Betke Test?

1. substitution of amino acid near the alpha and beta chain bond

2. substitution of a charged for an uncharged amino acid in the interior of the molecule

3. substitution of a polar for nonpolar amino acid in the hydrophobic heme pocket

4. replacement of an amino acid with proline in the alpha helix section of the chain

what causes unstable hemoglobin variants?

• red cell morphology is variable

• observe heinz bodies on supravital stain

• spleen will remove the cells

• hemolysis is most commonly observed following ingestion of oxidant drugs

what occurs when hemoglobin denatures?

• oxygen dissociation curve shifts to the left

• observed in erythrocytosis because hemoglobin hemoglobin does not release O₂ to the tissues

what occurs in increased O₂ affinity hemoglobins?