HEMOGLOBINOPATHIES

 Genetic defect that results in abnormal structure of one of

the globin chains of the hemoglobin molecule
 Although the suffix "-pathy" would conjure an image of
"disease," most of the hemoglobinopathies are not
clinically apparent
 Others produce asymptomatic with abnormal hematologic
laboratory findings
 A very few produce serious disease
 The genetic defect may be due to substitution of one
amino acid for another
 Hemoglobin electrophoresis, isoelectric focusing and/ or
DNA (PCR) analysis may be used to confirm the diagnosis
 Changes in RBC deformability and electrophoretic mobility
can occur  Homozygous/ Disease conditions
 Hgb S is the most common - both globin chains affected
 Hgb C is the second most common - more serious
 Hgb E is the third most common  Heterozygous/ Trait conditions
- only one globin affected

SICKLE CELL DISEASE (HGB SS)  CLINICAL FINDINGS

 Cause: when valine replaces glutamic acid at the position 6 on both
beta chains
 Result: a decrease in hemoglobin solubility and function
 Defect is inherited from both parents
 Occurs most commonly in African-America, African, Mediterranean,
and Middle East population
 No Hgb A is produced, Hgb A2 is variable
 Approximately 80% HgbS and 20% HgbF(compensatory) are seen
 An infant does not begin showing symptoms of the disease until
sufficient Hb F has been replaced by Hb S
 Characterized by: lifelong episodes of pain (“crises”)
 A chronic hemolytic anemia with hyperbilirubinemia
 ⬆ susceptibility to infections, usually beginning in early childhood
 The lifetime of an erythrocyte in sickle cell anemia is <20 days,
compared with 120 days for normal RBCs; hence, the anemia
Symptoms:
- Acute chest syndrome
- Stroke
- Splenic and renal dysfunction
- Bone changes due to marrow hyperplasia
Hemoglobin insolubility
- results when deoxyhemoglobin is formed
Hemoglobin crystallizes
- In erythrocytes It is characterized by classic sickled shape of
erythrocytes
 Two types of electrophoresis :
a) Cellulose acetate at pH 8.6
b) Citrate agar at pH 6.2
- Erythrocytes become rigid and trapped in capillaries:
blood flow restriction causes lack of oxygen to the tissues
resulting in tissue necrosis
- All organs are affected, with kidney failure being a
common outcome: hyposplenism and joint swelling also
occur
- Vaso-occlusive crisis occurs with increase bone marrow
response to the hemolytic anemia
- Crisis can be initiated by many physiologic factors,
including surgery, trauma, pregnancy, high altitude, etc
- Apparent immunity to Plasmodium falcifarum
Sickled cells
- frequently block the flow of blood in the narrow capillaries
w/c causes anoxia -> pain & eventually death (infarction)
of cells
- ⬇ ability to deform & ⬆ tendency to adhere to vessel
walls, and so have difficulty moving through small vessels
- ⬇ O2 tension: high altitudes or flying in a nonpressurized
plane
- ⬇ pH, dehydration
- ⬆ p CO2
- ⬆ conc of 2,3-BPG in erythrocytes
 LABORATORY:
- Severe normochromic/ normocytic hemolytic anemia
with polychromasia resulting from premature release
of reticulocyte;
- Bone marrow erythroid hyperplasia(M:E ratio
decreases)
Cells seen in PBS: Sickle cells, Target cells, Nucleated RBCs ,
Pappenheimer bodies, Howell- Jolly bodies
 Increase bilirubin and decrease haptoglobin are characteristic
due to hemolysis
 Positive hemoglobin solubility testing
 Hgb S migrates with Hemoglobin D and G on alkaline
hemoglobin electrophoresis; can differentiate using acid
electrophoresis
 HEMOGLOBINOPATHIES LAB TEST Therapy:
- Adequate hydration
1) Cellulose acetate (pH 86) hemoglobin electrophoresis - Analgesics
- Hgb A migrates the fastest - Aggressive antibiotic therapy if infection is present
- Hgb A2, C, E and O migrate the slowest - Transfusions in Patient at risk for fatal occlusion of blood
vessels
- Intermittent transfusions with packed red cells reduce the
CATHODE (-) ANODE (+) risk of stroke, but the benefits must be weighed against
(X) Origin A2 S F A the complications of transfusion, which include iron
overload (hemosiderosis), bloodborne infections, and
C D
immunologic complications
E G
O

2) Citrate Agar (pH 62) hemoglobin electrophoresis

- Hgb S is differentiated from D and G
- Hgb C is differentiated from Hgb A2, E and O

ANODE (+) CATHODE (-)

C S (X) A F
Origin A2
D
G
E
O

SICKLE CELL TRAIT (HGB AS) Solubility Test for Hemoglobin S (Sickle cell preparation)
- Reagent - sodium dithionite
 Cause: when valine replaces glutamic acid at position 6 on one beta - Hgb S will crystallize and give a turbid appearance of the
chain solution
 Defect is inherited from one parent - The test will not differentiate homozygous from
 One normal beta chain can produce some Hgb A heterozygous conditions containing Hgb S
 Approximately 60% Hgb A and 40% HgbS are produced, with normal - Follow up a positive solubility test with hemoglobin
amounts of HgbA2 and F electrophoresis
 This heterozygous trait is the most common hemoglobinopathies in
US
 Produces no clinical symptoms
 Anemia is rare, but if present, will be normocytic / normochromic
and sickling occur during rare crisis states (same in hemoglobinSS)
 Positive hemoglobin solubility screening test
 Apparent immunity to Plasmodium falciparum

HEMOGLOBIN C DISEASE (HGB CC)  LABORATORY :

 HgbC disease is caused when lysine replace glutamic acid at - Normochromic/ normocytic anemia
position 6 on both beta chains - Target cells present
 Defect is inherited from both parents - Intracellular rod like C crystals
 Occurs in African – American and African populations - Hgb C migrates with hemoglobin A2, E and O on alkaline
 No HgbA is produced; Approximately 90% HgbC, 2% HgbA2 electrophoresis
and 7% HgbF are produced Mild anemia may be present - can differentiate hemoglobins using acid electrophoresis
- Heterozygous Hgb C trait patient is asymptomatic, with no
 Two types of electrophoresis : anemia
a) Cellulose acetate at pH 8.6 - The one normal beta chain is able to produce
b) Citrate agar at pH 6.2 approximately 60% HgbA and 40% HgbC, with normal
amounts of HgbA2 and HgbF
HGB SC
 Hgb Sc disease is double heteroxygous condition where an
abnormal sickle gene from one parent and an abnormal C gene
from the other parent is inherited
 Seen in African, Mediterranean and Middle Eastern
populations
 Symptoms less severe than sickle cell anemia but more severe
than HgbC disease
 No Hgb A is produce, approximately 50% HgbS and 50% HgbC
are produced Compensatory HgbF may be elevated upto 7%
HGB E
 Cause: when lysine replaces glutamic acid at position 26 on
the beta chain
 Found more commonly is Southeast Asian, African and African
– American populations
 Homozygous are asympthomatic
 Hgb E migrates with hemoglobins A2, C and O on alkaline
electrophoresis
 Two types of electrophoresis :
a) Cellulose acetate at pH 8.6
b) Citrate agar at pH 6.2
HGB CONSTANT SPRING
 A variant in which a mutation in the alpha globin gene
produces an abnormally long alpha globin chain
Quantity of hemoglobin in the cells is low for two reasons:
- First - unstable messenger RNA Some is degraded prior to
protein synthesis
- Second - the constant spring alpha chain protein is itself
unstable
- The result is a thalassemic phenotype
 HEMOGLOBINOPATHIES LAB TEST
1) Hemoglobin F (Kleihauer-Betke Method)
 count dense staining Hgb F cells and the number of ghost cells
containing Hgb A to obtain percentage
- It is used to detect the presence of fetal cells in the
maternal circulation during problem pregnancies because Hgb
F in fetal cells resists acid elution
- It differentiates hereditary persistence of fetal hemoglobin
from other conditions associated with high Hgb F cells
- Normal newborns have 70-90% Hgb F levels
 LABORATORY:
- Moderate to severe normocytic/ normochromic anemia
- With target cells
- Characterized by SC crystals
- May see rare sickle cells or C crystals
- Positive hemoglobin solubility test screening
HGB H
 Cause: when glycine replaces glutamic acid at the position
121 on the beta chain
 Found more commonly Middle Eastern and Indian
population
 Both homozygous and heterozygous conditions are
asymptomatic
 Hgb H migrates with HgbS and HgbG during alkaline
electrophoresis
 One type of electrophoresis :
- Cellulose acetate at pH 8.6
2) Solubility Test for hemoglobin S (Sickle cell preparation)
- Hemoglobin S is insoluble when combined with reducing
agent (sodium dithionite)
- Hgb S will crystallize and give a turbid appearance of the
solution
- The test will not differentiate homozygous from
heterozygous conditions containing Hgb S
- Follow up a positive solubility test with hemoglobin
electrophoresis
- Procedure for identification of normal and abnormal
hemoglobins
- Methodology is based on net negative charges, which
cause hemoglobins to migrate from the negative
(cathode) region toward the positive (anode) region The
distance particular hemoglobin molecule migrates is due
to its net electrical charge
- Migration of hemoglobin is dependent on net negative
charge and buffer pH
 Two types of electrophoresis :
a) Cellulose acetate at pH 8.6
b) Citrate agar at pH 6.2