SQ #5
Question 1: Define hemoglobinopathy.
Condition produced by hemoglobin variant. Structural defects in globin chains due to altered amino acid
sequences
Question 2: Differentiate between a hemoglobinopathy and a thalassemia.
Hemoglobinopathy – Hemoglobin’s differ in sequence of amino acids composing globin chain. Qualitative
disorder.
Thalassemia – Characterized by decreased production of hemoglobin resulting from decreased synthesis of one
particular globin chain. Qualitative disorder.
Question 3: Explain how the scientific nomenclature system identifies a specific hemoglobinopathy.
Description of variant can include chains and substitution. Example, homozygous Hb S is a2b2S or a2b26Val or
a2b26Glu-Val
Currently some abnormal hemoglobin’s are assigned a common designation and a scientific designation. The
common name is selected by the discoverer and usually represents the geographic area where the hemoglobin
was identified. A single capital letter is used to indicate a special characteristic of the hemoglobin variants, such
as hemoglobin’s demonstrating identical electrophoretic mobility but containing different amino acid
substitutions, as in Hb G-Philadelphia, Hb G-Copenhagen, and Hb C-Harlem. The variant description also can
involve scientific designations that indicate the variant chain, the sequential and the helical number of the
abnormal amino acid, and the nature of the substitution. The designation [b6 (A3) Glu -> Val] for the Hb S
mutation indicates the substitution of valine for glutamic acid in the A helix in the b chain at position 6.
Hb S: a2b26Val (severe hemolytic anemia; sickling)
Hb C: a2b26Lys (mild hemolytic anemia)
Hb D: a2b2 121Gln (no anemia)
Hb E: a2b226Lys (mild microcytic anemia).
Question 4: Discuss how hemoglobinopathies are classified.
Broad Classification System – Qualitative or Quantitative
Better Classification System has 5 categories: No clinical significance, Aggregating Hgb, unbalance synthesis of
Hgb, Unstable Hgb, and Hgb with abnormal Heme Function).
Question 5: List the four classes of hemoglobinopathies based on the functional abnormality present.
Homozygous - Hb Polymorphisms, Heterozygous - Hemoglobin Associated with Methemoglobinemia and
Cyanosis, Heterozygous - Hemoglobins Associated with Altered Oxygen Affinity; Heterozygous - Unstable
hemoblibins
Question 6: Discuss the demographics and genetics of sickle cell anemia.
1 of 375 African American live births have hemoglobin SS (Sickle Cell Disease); Have 85%
chance to live to age 20.
8-10% of American blacks carry the Hb S trait (heterozygous).
Affects more than 50,000 Americans.
Question 7: Describe what occurs during a sickle cell crisis.
A sickle cell crisis occurs when sickle-shaped red blood cells clump together and block small blood vessels that
carry blood to certain organs, muscles, and bones.
Question 8: Explain the pathophysiology of sickle cell anemia. Include a discussion of how the various organs of the
body are affected.
Sickle cell disease (SCD) is an inherited blood disorder. Hb S is defined by the structural formula a2b26Glu->Val, which
indicates that on the b chain at position 6, glutamic acid is replaced by valine. Hb S molecules within the RBCs
become less soluble, forming tactoids or liquid crystals of Hb S polymers that grow in length beyond the diameter
of the RBC, causing sickling.
All major organs are affected by sickle cell disease. The liver, heart, kidneys, gallbladder, eyes, bones, and joints
can suffer damage from the abnormal function of the sickle cells and their inability to flow through the small
blood vessels correctly.
Hands and feet swell (dactylitis), fingers grow at different rates.
Joint pain in arms and legs first. May affectlungs. Have chest pain and abdominal pain.
Spleen enlarges – cells become trapped. Have decreased blood volume (hypovolemia), and shock.
Repeated splenic infarcts cause splenic dysfunction, increasing susceptibility to infection.
Organs Affected:
Liver: Enlarges, malfunctions, jaundice, hyperbilirubinemia.
Heart: Cardiomegaly, iron deposits.
Spleen: Enlarges leading to infarction and fibrosis. Eventually shrivels and becomes nonfunctional.
Skin: Develop ulcers.
Kidney: Hematuria and eventual failure.
Lungs: Infarction.
Brain: Strokes.
Question 9: List the clinical laboratory findings in sickle cell anemia.
Classified morphologically as normocytic, normochromic.
PBS with Wright-stained: poikilocytosis, anisocytosis with normal RBCs, sickle cells, target cells, nucleated RBCs,
spherocytes, basophilic stippling, Pappenheimer bodies, and Howell-Jolly bodies. The presence of sickle cells
and target cells is the hallmark of SCD. Moderate to marked polychromasia with a reticulocyte count between
10% and 25%, corresponding with the hemolytic state and the resultant bone marrow response.
RBC distribution width (RDW) is increased as a result of moderate anisocytosis.
Hb 6-8 g/dL – severe anemia
Increased MCV
An aplastic crisis can be heralded by a decreased reticulocyte count.
Moderate leukocytosis is usually present (sometimes 40 to 50 3 109 WBC/L) with neutrophilia and a mild shift
toward immature granulocytes.
The leukocyte alkaline phosphatase score is not elevated when neutrophilia is caused by sickle cell crisis alone
when no underlying infection is present.
Thrombocytosis is usually present.
The bone marrow shows erythroid hyperplasia, reflecting an attempt to compensate for the anemia, which
results in polychromasia and an increase in reticulocytes and nucleated RBCs in the peripheral blood.
Levels of immunoglobulins, particularly immunoglobulin A, are elevated in all forms of SCD.
Serum ferritin levels are normal in young patients but tend to be elevated later in life.
Chronic hemolysis is evidenced by elevated levels of indirect and total bilirubin with the accompanying jaundice.
Haptoglobin decreased
Electrophoresis – Hb S present.
Sickling Test – Positive.
Osmotic Fragility – Decreased.
Sed Rate – Decreased
Question 10: Discuss the treatment of sickle cell anemia and predict the prognosis of a patient with sickle cell anemia.
Prevent crisis.
If crisis – keep hydrated. Alleviate pain.
Treat infections with antibiotics. Keep warm. Blood transfusion if needed.
Prophylactic penicillin treatment prevents most deaths due to pneumococcal infections.
RBC exchanges are beneficial.
Developing anti-sickling agents (hydroxyurea) which increases amount of Hemoglobin F in red cells. Decreases
amount of sickling that occurs during crisis.
Bone marrow transplants in children show promise.
Future treatment may include gene therapy.
Question 11: Define sickle cell trait. Describe the clinical laboratory findings for a patient with sickle cell trait.
Heterozygous AS with more HbA than HbS, so condition is compensated for.
Normal CBC – Few target cells or sickle cells may be present.
Sickle solubility test – positive.
Electrophoresis – Both A and S present
Question 12: Define Hb C disease. Describe the clinical laboratory findings.
Amino acid substitution of lysine for glutamic acid at sixth position of Beta chain (a2b2 6Glu-Lys).
HbC crystals on peripheral blood.
Mild to moderate anemia (8-12 g/dL), splenomegaly and abdominal discomfort.
Numerous target cells, few microspherocytes, schistocytes, and folded cells.
May see hexagonal or rod-shaped crystals ("bar of gold”). Usually, intracellular. Are elongated with blunt ends
and parallel sides.
Retic count 4-8% (slightly increased).
Electrophoresis: Most hemoglobin is HbC; no HbA present; may or may not have increase in Hb F.
Question 13: List the clinical laboratory findings associated with Hb D.
Question 14: Describe the clinical laboratory findings associated with Hb E
Migrates with HbS upon electrophoresis, but does not cause sickling of RBCs.
Mild, microcytic, hypochromic hemolytic anemia.
Many target cells.
Electrophoresis shows E band. Normal Hb F, no Hb A.
May protect against malaria.
Question 15: Describe the laboratory findings in Hemoglobin SC disease.
Sickle cell symptoms including splenomegaly.
Positive for anemia. Mild if present.
See target cells, folded, pocket-book cells, and rare sickle cells; “Washington monument" crystals (fingerlike
projections) may be found.
Positive sickle solubility test.
Electrophoresis shows equal amounts of HbC and HbS, no HbA. Hb F normal or elevated.
Question 16: Give examples for each of the following types of abnormal hemoglobins:
a. Multiple amino acid substitutions – Hemoglobin SC,
b. Amino acid deletions - Alpha thalassemia
c. Elongation of polypeptide chain - hemoglobin Tak, hemoglobin Cranston and Hb Pakse.
Question 17: List the clinical laboratory findings associated with hemoglobins with an increased oxygen affinity; with a
decreased oxygen affinity.
Increased Oxygen affinity - Decreased delivery of oxygen to tissues. Hb values from normal to 20 g/dL.
Leukocytes and platelets normal. Normal life span. Often results in polycythemia
Decreased Oxygen Affinity - Increased release of oxygen to tissues. Patient may become anemic.
Question 18: Describe the clinical laboratory findings associated with unstable hemoglobin disease.
Isopropanol Precipitation - Unstable Hemoglobins precipitate out within 5 minutes. Normal Hemoglobins do
not precipitate.
Heat Denaturation Test - Washed red cells hemolyzed with H20. Incubate. Will denature hemoglobin. See
precipitation. Normal Hemoglobin’s do not precipitate.
Heinz Body Staining - Supravital staining. Brilliant Cresyl Blue Stain. Heinz bodies stain pale blue and are
refractile. Eccentrically located.
Question 19: Describe the clinical laboratory findings associated with Hb M.
Blood is chocolate brown.
Mild hemolytic anemia.
Heinz bodies.
M band on electrophoresis.
Question 20: Explain the principle the isopropanol precipitation test based upon. Explain the principle the heat
denaturation test is based upon.
The Isopropanol Precipitation Test - based on the principle that an isopropanol solution at 37° C weakens the
bonding forces of the hemoglobin molecule. If unstable hemoglobins are present, rapid precipitation occurs in
5 minutes and heavy flocculation occurs after 20 minutes. Normal hemoglobin does not begin to precipitate
until after approximately 40 minutes.
The Heat Denaturation Test - When incubated at 50° C for 1 hour, heat-sensitive unstable hemoglobins show
a flocculent precipitation, whereas normal hemoglobin shows little or no precipitation. Significant numbers of
Heinz bodies appear after splenectomy, but even in individuals with intact spleens, with longer incubation and
the addition of an oxidative substance such as acetylphenylhydrazine, unstable hemoglobins form more Heinz
bodies than does the blood from individuals with normal hemoglobins.
Question 21: Discuss the principle for the Heinz body staining technique.
Heinz body inclusions can be either round or irregularly shaped and are composed of denatured hemoglobin.
Observation of these inclusions is made possible by supravital stains such as brilliant cresyl blue or crystal violet. They
are not observed on Wright’s stain.
Question 22: For each of the following tests, discuss the principle of the test, the specimen requirements, interpretation
of results, and sources of error:
1) dithionite screening test
• Principle: (Hemoglobin solubility test) is a screening test for Hb S. Capitalizes on the decreased solubility of
deoxygenated HbS in solution, producing turbidity. Blood is added to a buffered salt solution containing a
reducing agent, such as sodium hydrosulfite (dithionite), and a detergent-based lysing agent (saponin).
Saponin dissolves membrane lipids, causing release of hemoglobin from RBCs, and dithionite reduces iron
from the ferrous to the ferric oxidation state. Ferric iron is unable to bind oxygen, converting hemoglobin to
the deoxygenated form. Deoxygenated Hb S polymerizes in solution, which renders it turbid, whereas
solutions containing non-sickling hemoglobin remains clear.
•
Specimen Requirements: Blood
•
Interpretation: Hemoglobin Solubility Test for the Presence of Hemoglobin S. In a negative test result (left),
the solution is clear and the lines behind the tube are visible. In a positive test result (right), the solution is
turbid because of the polymerization of hemoglobin (Hb) S and the lines are not visible.
•
Source of Error: False-positive results for Hb S can occur with hyperlipidemia, in a few rare
hemoglobinopathies, and when too much blood is added to the test solution; false negative results can
occur in infants younger than 6 months and in those with low hematocrits. Other hemoglobins that give a
positive result on the solubility test include Hb C-Harlem (Georgetown), Hb C-Ziguinchor, Hb S-Memphis, Hb
S-Travis, Hb S-Antilles, Hb S-Providence, Hb S-Oman, Hb Alexander, and Hb Porte-Alegre.
2) cellulose agar hemoglobin electrophoresis
•
Principle: Electrophoresis is based on the separation of hemoglobin molecules in an electric field primarily
because of differences in total molecular charge
•
Specimen Requirements: Blood
•
Interpretation: In alkaline electrophoresis hemoglobin molecules assume a negative charge and migrate
toward the anode (positive pole). Historically, alkaline hemoglobin electrophoresis was performed on
cellulose acetate medium, but it is being replaced by agarose medium. Nonetheless, because some
hemoglobin have the same charge and therefore the same electrophoretic mobility patterns, hemoglobin
that exhibit an abnormal electrophoretic pattern at an alkaline pH may be subjected to electrophoresis at
an acid pH for definitive separation.
3) citrate agar hemoglobin electrophoresis
•
Principle: Electrophoresis is based on the separation of hemoglobin molecules in an electric field primarily
because of differences in total molecular charge
•
Specimen Requirements: Blood
•
Interpretation: In an acid pH some hemoglobins assume a negative charge and migrate toward the anode,
whereas others are positively charged and migrate toward the cathode (negative pole). For example, Hb S
migrates with Hb D and Hb G on alkaline electrophoresis but separates from Hb D and Hb G on acid
electrophoresis. Hb D and Hb G are further differentiated from Hb S in that they produce a negative result
on the hemoglobin solubility test. Similarly, Hb C migrates with Hb E and Hb O on alkaline electrophoresis
but separates on acid electrophoresis.
4) alkali denaturation test
•
Principle: The classic alkali denaturation test is accurate and precise to quantify Hb F in the 0.2% to 50%
range. Most human hemoglobins are denatured on exposure to a strong alkali, but Hb F is not. The Hb F
can be separated and its concentration compared with that of other hemoglobins. Consistent methodology
is required to ensure accurate results.
•
Specimen: Whole Blood
•
Interpretation: Hemoglobin F less than 1% after 1 year of age
Question 23: Define thalassemia.
Hereditary hemolytic diseases caused by faulty hemoglobin synthesis,
Question 24: Explain how thalassemias are inherited.
May be homozygous defect of heterozygous defect. In general, thalassemia is inherited in an autosomal
recessive manner; however, the inheritance can be quite complex as multiple genes can influence the
production of hemoglobin. Most people affected by beta thalassemia have mutations in both copies of the HBB
gene in each cell .
Question 25: Define Beta Thalassemia. Draw a chart demonstrating how beta thalassemias are classified.
Beta (b) - Caused by defect in rate of synthesis in beta chains. Usually caused by mutation. Microcytic,
hypochromic anemia of varying sensitivity.
Mutation
Minor gene
mutation
Type
Silent Carrier State
Minor Point
Mutation
Beta Thal Minor:
B+ Heterozygous
Two
mutations
Beta Thalassemia
Intermedia
Heterozygous or
Homozygous
Severe Gene
Mutation
Beta Thalassemia
Major (Cooley’s
Anemia)
Genotype Clinical Severity
B/B+
Hb A: Normal
Hb A2: Normal
Hb F: Normal
Small decrease in production of beta chain and No
hematological abnormalities
B/B+
Hgb 10 – 13 g/dL
Hb A: Decreased
Hb A2: Normal/Increased
Hb F: Normal/Increased
Bilirubin: Increased
Mild asymptomatic Hypochromic, microcytic, hemolytic
anemia (mimics IDA).
Target and Elliptocytes
Stress increases symptoms – Pregnancy, infection, or F9
deficiency
B/B+ or
Hgb:  7 g/dL
B+/B+
Hb A: Decreased
Hb A2: Normal/Increased
Hb F: Increased
Severity lies between the minor and major
Hypochromic, microcytic, hemolytic anemia
Target and Elliptocytes
Stress increases symptoms – Pregnancy, infection, or F9
deficiency
May become transfusion dependent (Fe Oveload)
B+/B+
Hgb: 4 – 8 g/dL
Hb A: Decreased
Hb A2: Increased
Hb F: Increased
Little or no beta chain is synthesized; consequently, no
(or very little) Hgb A is synthesized
No Beta Chains to combine with Alpha chains. Alpha
chains percipitatie in RBC and < life span (7-22 days)
Severe Hypochromic, microcytic, hemolytic anemia
causes marked bone changes due to expansion of
marrow space for increased erythropoiesis.
Detected in Childhood.
PBS: extreme poikilocytosis, target cells, teardrops,
elliptocytes, basophilic stippling and numerous nRBCs.
Homozygous disorder resulting in severe transfusion
dependent hemolytic anemia.
Question 26: For each of the following types of beta thalassemias, discuss the demographics and clinical laboratory
findings:
Thalassemia major
Thalassemia intermedia
Thalassemia minor
Carrier State
Demographics: The thalassemic gene is ubiquitous, yet it has a particular penetration in Mediterranean areas and in
Middle Eastern, Northern African, Indian, Asian, and Caribbean populations bordering the seas.
Clinical Laboratory: RDW, Serum Iron, TIBC, Serum Ferritin, FEP are Normal for Thalassemia.
Type
Beta Thalassemia
Major (Cooley’s
Anemia)
Beta Thalassemia
Intermedia
Heterozygous or
Homozygous
Beta Thal Minor:
B+ Heterozygous
Clinical Laboratory Findings
Hgb: 4 – 8 g/dL
MCV: 50 – 60fL
Retic: Decreased
Alpha chains precipitate in RBC and < life span (7-22
days)
Iron Overload associated w/transfusions– hemosiderosis
w/o chelation therapy
Severe Hypochromic, microcytic, hemolytic anemia
PBS: extreme poikilocytosis, target cells, teardrops,
elliptocytes, basophilic stippling and numerous nRBCs.
Hb A: Decreased
Hb A2: Increased
Hb F: Increased (represents most of the hemoglobin
present)
Hgb:  7 g/dL
Bilirubin: Increased
Hb A: Decreased
Hb A2: Normal/Increased
Hb F: Increased
Hypochromic, microcytic, hemolytic anemia
Target and Elliptocytes
Iron Overload
Stress increases symptoms – Pregnancy, infection, or F9
deficiency
Hgb 10 – 13 g/dL
RBC: Normal/Slight Elevation
Hb A: Decreased
Silent Carrier State
Hb A2: Normal/Increased (3.5-8.0%)
Hb F: Normal/Increased
Bilirubin: Increased
Mild asymptomatic Hypochromic, microcytic, hemolytic
anemia (mimics IDA).
Target, Elliptocytes, Basophilic stippling
Stress increases symptoms – Pregnancy, infection, or F9
deficiency
Hb A: Normal
Hb A2: Normal
Hb F: Normal
Small decrease in production of beta chain and No
hematological abnormalities
Question 27: Describe how alpha thalassemias are named.
a-thalassemia is divided clinically into a silent carrier state, a-thalassemia minor, Hb H disease, and Hb Bart
hydrops fetalis syndrome.
Question 28: Discuss the demographics, pathophysiology, and laboratory findings of alpha thalassemias.
Question 29: Define Bart's Hydrops Fetalis. Include the demographics and pathophysiology in your discussion. List the
laboratory tests that are used for the diagnosis of thalassemia.
Bart’s Hydrops fetalis is the most severe form for Alpha-Thalassemia and is incompatible with life. There are NO
functional alpha chain genes (- -/- -).
Demographics: Northern Europe and North America
Pathophysiology: Hb Bart has a high Oxygen affinity so it cannot carry oxygenBaby born with hydrops fetalis,
which is edema and ascites caused by accumulation serous fluid in fetal tissues as result of severe anemia. Also
see hepatosplenomegaly and cardiomegaly.
Laboratory Tests for DX: Severe hypochromic, microcytic anemia with numerous nRBCs.Predominant hemoglobin
is Hemoglobin Bart, along with Hemoglobin Portland and traces of Hemoglobin H.
Question 30: Discuss Hemoglobin H Disease. Include the demographics, pathophysiology, and clinical laboratory
findings of the disease.
Hemoglobin H Disease - a moderate to severe form of alpha-thalassemia. There is one functional alpha chain
gene (- -/- a)
Demographics: predominantly seen in Southeast Asia, the Middle East and the Mediterranean.
Pathophysiology: HbH disease is usually caused by inactivation of three alpha-globin alleles leading to
underproduction of alpha-globin chains of Hb, with the formation of beta-4 tetramers (HbH). HbH tetramers have
a high affinity for oxygen, and are highly unstable, precipitating as toxic Heinz bodies. The formation of Heinz
bodies predominates in mature red blood cells, leading to premature hemolysis rather than ineffective
erythropoiesis.
Laboratory Tests for DX: mild-to-moderate microcytic hypochromic hemolytic anemia and hepatosplenomegaly.
Marked poikilocytosis and numerous target cells. Heinz bodies can be detected on blood smears after cresyl blue
staining. Hb biochemical analysis reveals the presence of HbH (5-30%). Diagnosis is confirmed by genetic testing.
Question 31: Explain what is meant by hereditary persistence of fetal hemoglobin. Differentiate between pancellular
HPFH and heterocellular HPFH.
Hereditary Persistence of Fetal Hemoglobin (HPFH) is an unusual condition in which red blood cells contain greater
than normal amounts of hemoglobin F (fetal hemoglobin). About one in a thousand African-Americans have the
HPFH carrier (trait) condition, compared with about 1 in 12 who have sickle cell trait.
HPFH is Classified into two groups according to distribution of Hb F among red cells: Pancellular HPFH Hemoglobin F uniformly distributed throughout red cells. Heterocellular HPFH - Hemoglobin F found in only small
number of cells
Question 32: Discuss the clinical significance of Delta-Beta thalassemias.
Group of disorders due either to a gene deletion that removes or inactivates only delta and beta genes so that
only alpha and gamma chains produced. Similar to beta thalassemia minor. Growth and development nearly
normal. Splenomegaly modest. Peripheral blood picture resembles beta thalassemia.
Question 33: Explain how iron deficiency anemia and thalassemia are differentiated.
RDW
IDA
Inc
Thalassemia Normal
Serum Iron TIBC
Dec
Inc
Normal
Normal
Serum Ferritin
Dec
Normal
FEP
Inc
Normal
Question 34: List the tests that are useful in the diagnosis of thalassemia.
CBC with Diff, Osmotic Fragility, Brilliant Cresyl Blue Stain, Acid Elution Stain, Hemoglobin Electrophoresis, ,
Hemoglobin Quantitation, Routine Chemistry, globin chain testin, DNA analysis
Question 35: Discuss the principle for each of the following tests:
brilliant cresyl blue stain for Hb H
Incubation with brilliant cresyl blue stain causes Hemoglobin H to precipitate. Results in characteristic
appearance of multiple discrete inclusions-golf ball appearance of RBCs. Inclusions smaller than Heinz bodies and
are evenly distributed throughout cell.
acid elution test for Hb F
Based on Kleihauer-Betke procedure. Acid pH will dissolve Hemoglobin A from red cells. Hemoglobin F is resistant
to denaturation and remains in cell. Stain slide with eosin. Normal adult cells appear as "ghost" cells while cells
with Hb F stain varying shades of pink. Useful way to differentiate between pancellular HPFH and heterocellular
HPFH
Question 36: Discuss the thalassemias associated with hemoglobinopathies.
Beta Thalassemia with Hb S - Inherit gene for Hb S from one parent and gene for Hb A with beta thalassemia from
second parent. Great variety in clinical severity. Usually depend upon severity of thalassemia inherited.
Production of Hb A ranges from none produced to varying amounts. If no Hb A produced, see true sickle cell
symptoms. If some Hb A produced, have lessening of sickle cell anemia symptoms.
Beta Thalassemia with Hb C – Shows great variability in clinical and hematologic symptoms. Symptoms directly
related to which type thalassemia inherited. Usually asymptomatic anemia.
Beta Thalassemia with Hb E – Is unusual because results in more severe disorder than homozygous E disease..
Very severe anemia developing in childhood. Transfusion therapy required.