Data Interpretation of Hb

and protein
Electrophoresis

Data Interpretation of Hb and

protein Electrophoresis
z Hemoglobin electrophoresis
z Normal Hb
z Pathological Hb
z Sickle cell Anemia
z -thalassemia
z -thalassemia
z Protein electrophoresis
z Normal
z Pathological
z multiple myeloma
z other pathological profiles

1
 Anemia
z Depletion anemia
z Production defect anemia
z Aplastic anemia/ marrow replacement
z factor deficiency
z Vitamin B12
z Folic acid
z iron deficiency
z hemoglobinopathies

2
 Normal Hb in fetal stage

z Embryonic
z Gower 1 = 22
z Portland 1 = 22
z Gower 2 = 22

z At birth
z Hb A = 22 (25%)
z Hb F = 22 (75%)

3
Normal Hb in adult

z Hb A = 22 (97%)
z Hb F = 22 (<1 %)
z Hb A2 = 22 (2.5%)

4
 Pathological conditions
z Hemoglobinopathies
(a) Structural Hb variants
Substitution, addition, or deletion of one or
more amino acids of the globin
(b) Thalasssemias
Quantitative defect in globin chain production
(c) Combination of (a) and (b)
(d) Hereditary persistence of fetal Hb

Nomenclature of Hb variants
z HbA, HbF, and HbS were first discovered
z Additional variants start from HbC
z Too many variants were found(>500)
z Hb with similar electrophoretic motility
z distinguished by adding the place of discover
z Some new Hb were named by pts family
z New system
z HbS B6 Glu Val (E B6 V)
z HbC B6 Glu Lys (E B6 K)

5
Classification of Hb variants

z Amino acid substitution

z e.g. Sickle cell anemia
z Deletion and insertion
z e.g. thalassemia ( chain deletion)
z Unequal cross over (fusion genes)
z Chain elongation
z Frame shift variants

6
 Clinical consequences of abnormal Hb

z Asymptomatic if they dont interfere with

Hb functions unstable Hb
z Produce disease
z affect the stability, shape, or function of Hb
z Homozygous for abnormal Hb
z HbS
z Heterozygous are mostly mild
z HbC, HbD, HbE but not HbA-HbS

Unstable Hb

z Hemolyticanemia
z Hemichrome formation
(Heme iron form various side chain with globin)
globin)
z Inclusion body formation (Heinz bodies)
z Altered oxygen dissociation
z Altered hemoglobin stability Increase
methemoglobin (Fe++ Fe+++) and
sufhemoglobin
z Altered solubility (Target cells)

7
 Sickle cell anemia
zB chain 6th amino acid E V substitution
results in polymerization of deoxy form
within the red cells
z The sickled-shape cells block
microcirculation
z Stasis hypoxia and ischemic infarction of
liver, kidney, heart, bone, nervous system
z Hemolytic anemia or even DIC

8
 Varying clinical severity of the
different sickle syndrome
z Sickle cell trait
z SA (30-40% HbS) (mild severity)
z SF (70% HbS) (mild severity)
z SC (50% HbS) (+++ severity)
z Sickle cell anemia
z hemolytic anemia
z aplastic crisis
z vaso-occlusive
z Sickle cell-HbC disease

9
 Thalassemia
z Decreased rate of globin chain production
z thalassemia , , embryonic death
z Classification
z Thalassemia major
z thalassemia minor
z thalassemia minima
z Alpha-thalassemia
z Beta-thallasemia
z + or 0-thalassemia

10
11
 Hemoglobin protein has two alpha
subunits and two beta subunits.

z Thetwo chromosomes #11 have one beta

globin gene each (for a total of two
genes).
z Thetwo chromsomes #16 have two alpha
globin genes each (for a total of four
genes).
z Each alpha globin gene produces only
about half the quantity of protein of a
single beta globin gene.

12
 Alpha-thalassemia
z Defective -chain synthesis
z No elevation of HbA2 and HbF
(in -thalassemia)
z Consequence of diminished -chain synthesis
z decrease production of HbA, HbF, HbA2
z Excess -chain and -chain
z Hb Barts (4)
z HbH (4)

13
The offspring that inherits the
double deletion from one
parent and the single from
the other will have
Hemoglobin H disease
(Scenario 1).
The offspring who inherits no
alpha genes from the parents
dies in utero (Scenario 2;
hydrops fetalis).

Beta-thalassemia
z Defective -chain synthesis
z Diminished (+ or ++)or absent (0 ) of -globin
z elevation of HbA2 & HbF in -thalassemia
z Consequence of diminished -chain synthesis
z decrease production of HbA,
z Excess -chain and -chain
z HbF
z HbA2
z No Hb Barts (4)
z No HbH (4)

14
15
 Hb electrophoresis (alkaline)
z Separation on cellulose acetate (pH8.4)
(-) A2 S F A1 (+)
z Most frequently used
z Resolve most of the major Hbs (A1, A2, S, F)
z A2 can not be separated from C
z Can not resolve HbD, G; and HbC, E

16
 Hb electrophoresis (acidic)
z Separation on agarose gel (pH6.0)
(-) F A S C (+)
z Mostly used in confirmation
z E can be separated from C
z HbC, HbS migrate toward the Anode
z HbA, E migrate toward the cathode

17
 Protein electrophoresis
z Multiplemyeloma and Immunoglobulins
z Data of protein electrophoresis
z Acute reaction pattern
z Nephrotic syndrome
z Chronic inflammation
z Cirrhosis of liver
z a1-antitrypsin deficiency (chronic diseases)
z polyclonal gammopathies
z hypogammaglobulinemia
z Multiple myeloma (M spike)

18
 Issues to be discussed
z Structure and function of Immunogloblins
z Clonal deletion and clonal expansion
z Development of lymphocytic lineage
z Multiple myeloma
z Consequences of multiple myeloma
z Normal patterns of protein electrophoresis
z Pathological patterns

19
20
 Multiple myeloma
z Maturation of plasma cells
z Proliferation of plasmacytoid lymphocytes
z Proliferation of plasma cells
z Principle of data interpretation
z Blood
z Hyperviscosity
z Cryoglobulinemia
z M spike
z Bence Jones proteinuria

21
22
 Bone marrow aspirate demonstrating plasma cells of
multiple myeloma. Note the blue cytoplasm, eccentric
nucleus, and perinuclear pale zone (or halo).

multiple punched-out lesions in a patient with multiple myeloma

23
24
 Bence Jones proteins
z The multiple myeloma cell clone produces an
excess of monoclonal (M proteins) and free light
chain proteins.
z The M proteins may be recognized as IgA, IgD,
IgG, IgE or IgM, depending on their heavy chain
class.
z The light chain proteins may be designated as
kappa or lambda. They may precipitate and
deposit, producing organ damage. The organ
most commonly affected is the kidney.
z When these monoclonal light chains appear in the
urine, they are called Bence Jones proteins.

25
 Routine laboratory
z Pancytopenia, abnormal coagulation,
hypercalcemia, azotemia, elevated alkaline
phosphatase and erythrocyte sedimentation rate,
and hypoalbuminemia.
z Proteinuria, hypercalciuria, or both. Urine
dipstick tests may not indicate the presence of
Bence Jones proteinuria.
z All patients with suspected multiple myeloma
require a 24-hour urinalysis by protein
electrophoresis to determine the presence of
Bence Jones proteinuria and kappa or lambda
light chains.

26
 Protein electrophoresis
z Supporting matrix
z Molecular charge
z Agarose,
Agarose, cellulose acetate
z Molecular charge and size
z Starch and polyacrylamide
z pH and ionic strength of buffer
z pH 8.6 most proteins are negative charge
z barbital, Tris-
Tris-barbital, boric acid, Tris and EDTA
z Visualization
z Coomasie brilliant blue, Ponsceus S (albumin>globulin)
z Amido black (agarose
(agarose gel), bromphenol blue,

27
 Data interpretation of protein
electrophoresis
z Protein electrophoresis
z Data interpretation of
z Normal
z Acute reaction pattern (2, 3)
z Nephrotic syndrome (4)
z Chronic inflammation (5)
z Cirrhosis of liver (6, 7, 8)
z a1-antitrypsin deficiency (chronic diseases) (9)
z polyclonal gammopathies (10)
z hypogammaglobulinemia (11)
z Multiple myeloma (M spike) (12)

28


1. Normal 2 3 4
5 6 7 8
9 10 11 12

29