Moving to a capillary electrophoresis platform assessing the role in hemoglobinopathies

1. Prof. Moustafa Rizk
Fever Hospital Labs Conference
28-4-2016
Moving to a Capillary Electrophoresis
Platform: Assessing the role in
hemoglobinopathies
10/11/2017 4:19 AM 1

2. agenda…..
 THEORY OF ELECTROPHORESIS.
 CAPILLARY ELECTROPHORESIS
PRINCIPLE.
 HEMOGLOBINOPATHIES
ASSASSEMENT.
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3. Electrophoresis is a comprehensive term that refers to
the migration of charged solutes or particles of any
size in a liquid medium under the influence of an electric
field
The first electrophoresis method used to study proteins
was the free solution or moving boundary method
devised by Tiselius in 1937
Transactions of the Faraday Society 1937;33:524
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4. From World War Two until the late 1960s the
dominant separation techniques were:
- Paper chromatography: for most uncharged analytes
- Paper electrophoresis: for charged analytes such as amino acids
Tiselius was able to separate the 5 most
abundant proteins that occur in human serum
with relative ease and quantify their levels in
both normal and some disease states
Tiselius was awarded a Nobel Prize in 1948
for his work on protein separation by
electrophoresis.
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THEORY OF ELECTROPHORESIS

6. THEORY OF ELECTROPHORESIS
The rate of migration is
dependant :
1-Net electrical
charge of the molecule.
2-Electric field strength
3-properties of the
supporting medium.
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7. THEORY OF ELECTROPHORESIS
4-Size and shape of the
molecule.
5-Temperature of the
operation
6-Buffers and its ionic
strength
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8. THEORY OF
ELECTROPHORESIS
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CAE was popular in the clinical
laboratory because of its
simplicity, reproducibility,
reliability, and for its relative low
cost.
Because of the need for
presoaking and clearing, CAE
was largely replaced by agarose
gel (AGE) in most clinical
applications
The relatively poor resolution of most
commercially available cellulose
acetate membranes limited the
sensitivity of the technique.

9. Firstly adapted for the routine use by Laurell in 1962
Separation is based only on the charge-to-mass ratio of the
protein
In 1972, Laurell reported some clinical advantages of using
high-resolution agarose gel electrophoresis (HR-AGE) systems
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10. Since the mid nineties, CZE has been developed for use
in clinical labs and it was extended quickly in the routine,
being a very sensitive and fully-automated method .
This technique is
a liquid-based
system that
bears some
similarities to the
early Tiselius
system.
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11. Capillary tube:
• The cross-section of which (typically 50 m)
• The length of the capillary differs in different
applications, but is normally in the region of
20 to 50 cm,
• The capillary is filled with running buffer
• Fused silica is by far the most frequently
used material, due to its intrinsic properties,
which include transparency over a wide
range of the electromagnetic spectrum and a
high thermal conductance.
• The inner surface of the capillaries can be
untreated or coated .
-+
High voltage
power supply
The ends of a capillary are placed in
separate buffer reservoirs, each containing
an electrode connected to a high-voltage
power supply capable of delivering up to
30 kV.
• The sample is introduced by
placing one end into the sample
and applying an electric field
(electrokinetic injection) or by
applying external pressure for
a few seconds (Hydrodynamic
injection)
• An electric potential is applied
across the capillary and the
separation is performed
Detector
Computer
•detection of separated analytes
achieved directly through the capillary
wall near the opposite end normally
near the cathode
•The output of the detector is sent to a
data output and handling device such as
an integrator or computer.
•The data is then displayed as an
electropherogram
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12. CAPILLARY ELECTROPHORESIS PRINCIPLE
Detector
BUFFER BUFFER
Migration
High Voltage
TEMPERATURE CONTROLLED BY PELTIER DEVICE
Separation of proteins in a very narrow capillary tube filled with electrolyte solution.
Proteins are detected directly at a specific wavelength providing a high degree of
precision and accuracy.

13. Thermic
bridge
Temperature
Controlled by
Peltier device
Anode +Cathode -
Injection
Silica Capillary in
thermo-conductive
resin (25µm diameter)
CAPILLARYSTM TECHNOLOGY
Migration
Up to 10.000 volts
Deuterium
lamp
Detector

14. -+
1- Electrophoresis (electrophoretic migration)
Principle:
CE has two types of driving forces:
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15. 2- Electroosmotic Flow
EOF
•The inner surface of a fused silica capillary is covered with
silanol groups (Si-OH), which are ionized to SiO– at pH > 2.
•When silanol groups come in contact with the buffer during
CE, They readily dissociate, giving the capillary wall a negative
charge.
•The negatively-charged wall attracts positively-
charged ions from the buffer, creating an electrical
double layer and a potential difference (zeta
potential);
When a voltage is applied across the
capillary, the positive ions in the diffuse part
of the double layer migrate towards the
cathode; carrying water with them.
+ -
INJECTION OF
SERUM
DETECTION OF
PROTEINS
The result is a net flow of buffer
solution in the direction of the
negative electrode, which results in
electroosmotic flow10/11/2017 4:19 AM 15

16. At neutral to alkaline pH
The EOF is sufficiently stronger (silanols are completely ionized)
than the electrophoretic migration and Despite the peptide’s
(negatively charged) electrophoretic migration towards the
positive electrode (anode) the EOF is overwhelming, and the
peptide migrates towards the negative electrode (cathode).
+ _
EOF Electrophoretic
migration
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17. -+
EOF
Electrophoresis + Electroosmosis
0
tm (min)
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18. Protein migration
Electric Field force
EOF Force
+
_
Positive charges
of the buffer solution
Negative charges
of the capillary wall
The Electro Osmotic Flow (EOF) is a
stronger force than the Electrical Field.
As a result, all proteins are carried
towards the cathodic end of the
capillary.
PROTEINS SEPARATION

19. The combination of
 Small capillary internal diameter
 High voltage
 Tight temperature control
 Direct proteins measurement
at a specific wavelength
Allows
 Fast analysis time
 High throughput
 Excellent resolution and reproducibility
 Optimal prediction with high sensitivity and specificity
CAPILLARYSTM TECHNOLOGY

20. Ooooo!
It
MOVES

21. HEMOGLOBIN STRUCTURE
HEME
- Association of a porphyric structure and a divalent iron atom.
GLOBIN
- Protein composed of polypeptidic chains differenciated by their
amino acids sequence
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22. 10/11/2017 4:19 AM 22
Relative
migration times
of the
hemoglobin that
past the detector
in zones from Z1
through Z15,
Based on
standardizing on
the location of
HbA.

23. Normal profile
Hb A
Hb A2
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A
A2
Alk.
Anh.
Car.

24. HbA
HbF
HbS
HbA2
HbC

25. Interpreting Capillarys curve is like having the mountains in front of you!

26. Geographic distribution of Hemoglobinopathies
Thalassemias
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27. Capillary Electrophoresis Hb allows screening of the
main hemoglobin abnormalities of clinical interest.
Identification
Hb S, Hb C, Hb D, Hb E, excess Hb F, Hb H, Hb Bart
(in thalassemia) are the most common abnormal
hemoglobins.
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29. 1- HEMOGLOBIN “S”
Sickle Cells disease
Frequency – localization:
Mediterranean countries, inter tropical African population (40%),
American black population (10%), west Indies.
Characterization:
Mutation b6 Glu (negative) →Val (neutral)
Alkaline buffer: decreases of the total charge → Migration slowed down
on alkaline gel. Migration
faster on Capillarys
Biological signs:
deformation of RBCs giving a sickle aspect, decreased oxygen
affinity for the Hb , hyper hemolysis and anaemia
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30. Clinical signs:
variable according to the form.
Heterozygous form:
Sickle cells trait (generally
asymptomatic)
Hb A fraction: 50 – 70 %
Hb S fraction: 35 – 50 %
Hb A2 fraction: normal
Homozygous form:
Severe hemolytic syndrome
Hb A fraction: absence
Hb S fraction: 80 – 100 %
Hb A2 fraction: normal or
increased
Hb F fraction may be present 0 – 20%
Association with Hb S:
+ Thalassemia
+ Hb C (or other variants)
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31. Heterozygous A/S on Minicap/Capillarys
Hb S
Hb A2
Hb A
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N
A
A2
Anh.
Car.
Alk.
S
F

32. Heterozygous S/C
S
N
C
Alc.
Anh.
Car.
AFSC control
Hb C
Hb S
Hb F
Hb A2
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34. Homozygous S with foetal hemoglobin
AFSC control overlaying
Hb A2
Hb S
Hb F
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N
F
A2
Alk.
S
Anh.
Car.

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37. 2- HEMOGLOBIN C
Frequency – localization:
Abnormality most common in the black population, west and north
Africa, south Italy.
Characterization:
Mutation b6 Glu (negative) →Lys (positive)
Alk. Buffer : Decreases of the total charge →
Migration considerably slowed down , at same level as
Hb A2 on agarose gel , more cathodic to A2 on Capillarys
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N
Anh.
Car.
A
C+A2
Alk.

38. Clinical and biological signs:
Decreased solubility leads to target RBCs
Heterozygous form:
(asymptomatic) or slight anemia
Hb A fraction: 60 – 70 %
Hb C fraction: 35 – 40 %
Homozygous form:
moderate hemolytic anemia
Hb A fraction: absent
Hb C fraction: 95 – 100 %
Association with Hb S:
same pathology as homozygous sickle cell disease
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39. Heterozygous A/C
Hb A2
Hb A Hb C
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40. 10/11/2017 4:19 AM 40Am J Clin Pathol 2008;130:824-831

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HbA2 peak shows slight considerable overlap with HbC

42. 3- HEMOGLOBIN E
Abnormality most commonly found in south East Asia
Characterization:
Mutation b26 Glu (negative) →Lys (positive)
Alk. buffer: Decreases of the total charge → Migration considerably slowed
down like Hb C on agarose gel, more anodic to C on Capillarys
Clinical and biological signs:
Heterozygous form:
(asymptomatic)
Hb A fraction: 65 – 70 %
Hb E fraction: 30 – 35 %
Homozygous form:
(Discrete anemia)
Hb A fraction: absence
Hb E fraction: 100 %
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Anh.
Car.
A
E+A2
Alk.
N
Anh.
Car.
Alk.
N
E+A2
A

43. Heterozygous A/E
Hb A
Hb A2
Hb E
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44. Homozygous Hb E
AFSC control
Hb E
Hb A2
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HbA2 is not completely separated from HbE by HPLC , and the 2
hemoglobins will be measured together. CZE pattern on the same
sample showes that HbA2 does completely separate from HbE.
Am J Clin Pathol 2008;130:824-831

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47. 4- HEMOGLOBIN D
Abnormality most commonly found in India.
Characterization:
Mutation b121Glu (negative) →Gln (neutral) (D-los Angeles = D-Punjab)
(more than 7 different Hb D according to the position of the mutation)
Alk. buffer: Decreases of the total charge → Migration slowed down
like Hb S, more anodic to S on capillarys
Clinical and biological signs:
Heterozygous form:
(asymptomatic, electrophoresis
allows diagnosis)
Hb A fraction: 65 – 70 %
Hb D fraction: 30 – 35 %
Homozygous form:
Very light anemia
Hb A fraction: absence
Hb D fraction: 100 %
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Anh.
Car.
A
A2
Alk.
N
D

48. `AFSC control overlaying
Hb A
Hb D
Hb A2
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50. 5- THALASSEMIA
Genetic hemoglobinopathy regulation.
Absence or reduced synthesis of one or
several hemoglobin chains.
Balancing increased synthesis of the other
chains.
Thalassemias are distributed around the
Mediterranean region, Middle East, Indian
subcontinent, southeast Asia
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51. Decreased synthesis of the alpha chain by gene deletion
a a
b b
Hb A
b b
a variant (major Peak 1)
a a
d d
Hb A2
am am
d d
am am
a a Hb F (if g expressed)
g g
a
g g
am am
am a variant (Peak 2)
a variant (Peak 3)
(if g expressed)
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Decreased synthesis of the alpha chain by gene deletion
In consequence the synthesis of the 3 physiologic hemoglobin,
Hb A , Hb A2 and Hb F is affected
ALPHA THALASSEMIA

52. Completely asymptomatic
- One gene deleted:
silent a thalassemia
Hb A and A2 fractions are normal,
(presence of Bart Hb at birth)
- Two genes deleted:
minor a thalassemia (mild anemia)
Hb A fraction normal
Hb A2 fraction slightly decreased or normal
Hb Bart fraction detected at birth (5 – 10 %),
together with fetal Hb
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53. Hb Bart
(Baby’s blood)
Hb F
Hb A
Hb Bart’s
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55. Alpha thalassemia with Hb H
Hb A
Hb A2
Hb H
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Alk.
N
A
Anh.
Car.
H
A2

58. BETA THALASSEMIA
Hb A affected by a decreased synthesis of the b chain by gene
mutation
Compensation by increased g and d chains synthesis
Hb A2 fraction increased (< 9 %)
Hb F fraction present but not constant
Frequency – localization
The most frequent thalassemia, found around the
Mediterranean countries, in Greece, Italy, north Africa,
Asia….
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59. BETA THALASSEMIA
Minor heterozygous b thalassemia
Clinic: moderate microcytic anemia,moderate
splenomegaly.
Hb electrophoresis: Hb A 85-95%
Hb F N or slightly increased
(1-10%)
Hb A2 > 3,5% (but < 9 %)
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60. Beta Thalassemia
Hb A
Hb A2
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Anh.
Car.
A0+A2
A1
Alk. Ac.
N N
A2
A

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63. BETA THALASSEMIA
Clinic: Anemia associated with frequent
complication:splenomegaly, hemochromatosis,
growth delay
Hb electrophoresis: Hb A Decreased or absence
Hb F increased (major Hb peak)
Hb A2 Normal or increased (> 3,5% )
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Major b thalassemia

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68. More than 750 -International Center on Hemoglobins - (1997)
a chain (141 aa) variants: 217 mutations
b chain (146 aa) variants: 362 mutations
g chain variants: 70 mutations
d chain variants: 32 mutations
others: 69 mutations (double mutation,
deletion, insertion, hybrids)
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Hemoglobin variants

69. Database of hemoglobin variants
 Known hemoglobin variants: > 1000
 A database with all reported hemoglobin mutations is available on
http://globin.cse.psu.edu/
 The type of globin chain and the mutation position on the gene
 % for heterozygotes and homozygotes
 Affected population
 Information about the migration on agarose gel
 Only a genotyping can define the true identity for a rare variant
observed on a capillary electrophoretic profile
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70.  243 case with HB F either increased or just
detected
 192 case with increased HB F
 90 case with increased HB A2
 53 case with HB S
 6 case with HB C
 2 case with HB D
 2 case with HB H
 1 case with HB Bart’s
 1 case with HB O Arab
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From 6/11/2010 to 1-5- 2013
2064 cases were done

71. - A very good Hb A2 focalization allowing
an automatic identification and
quantification of this fraction.
- A direct identification of the main
hemoglobin variants.
The Hb Capillarys allows:
CONCLUSION

72. CONCLUSION
- A separation of hemoglobin C and E from the
A2 hemoglobin
- A slight difference of migration between S
and D hemoglobin allowing an easy
identification by overlaying with a memorized
reference curve
- A perfect individualization and focalization
of F hemoglobin between A and S allowing a
precise quantification

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Thank you for
listening.
Any Questions?
Prof. Moustafa Rizk