Prof. Moustafa RizkFever Hospital Labs Conference

28-4-2016

Moving to a Capillary Electrophoresis

Platform: Assessing the role in

hemoglobinopathies

10/11/2017 4:19 AM 1

agenda…..

THEORY OF ELECTROPHORESIS.

CAPILLARY ELECTROPHORESIS

PRINCIPLE.

HEMOGLOBINOPATHIES

ASSASSEMENT.

10/11/2017 4:19 AM 2

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

10/11/2017 4:19 AM 3

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.

10/11/2017 4:19 AM 4

10/11/2017 4:19 AM 5

THEORY OF ELECTROPHORESIS

THEORY OF ELECTROPHORESIS

The rate of migration is dependant :1-Net electrical charge of the molecule.2-Electric field strength3-properties of the supporting medium.

10/11/2017 4:19 AM 6

THEORY OF ELECTROPHORESIS

4-Size and shape of the molecule.5-Temperature of the operation6-Buffers and its ionic strength

10/11/2017 4:19 AM 7

THEORY OF ELECTROPHORESIS

10/11/2017 4:19 AM 8

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.

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 10/11/2017 4:19 AM 9

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.10/11/2017 4:19 AM 10

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

10/11/2017 4:19 AM 11

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.

Thermic

bridge

Temperature

Controlled by

Peltier device

Anode +Cathode -

Injection

Silica Capillary in

thermo-conductive

resin (25µm diameter)

CAPILLARYSTM TECHNOLOGY

MigrationUp to 10.000 volts

Deuterium lamp

Detector

-+

1- Electrophoresis (electrophoretic migration)

Principle:CE has two types of driving forces:

10/11/2017 4:19 AM 14

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

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

10/11/2017 4:19 AM 16

-+EOF

Electrophoresis + Electroosmosis

0tm (min)

10/11/2017 4:19 AM 17

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

The combination of

Small capillary internal diameter

High voltage

Tight temperature control

Direct proteins measurement

at a specific wavelengthAllows

Fast analysis time

High throughput

Excellent resolution and reproducibility

Optimal prediction with high sensitivity and specificity

CAPILLARYSTM TECHNOLOGY

Ooooo!

It

MOVES

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

10/11/2017 4:19 AM 21

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.

Normal profile

Hb A

Hb A2

10/11/2017 4:19 AM 23

A

A2

Alk.

Anh.

Car.

HbA

HbF

HbS

HbA2

HbC

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

Geographic distribution of Hemoglobinopathies

Thalassemias

10/11/2017 4:19 AM 26

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.

10/11/2017 4:19 AM 27

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

10/11/2017 4:19 AM 29

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)10/11/2017 4:19 AM 30

Heterozygous A/S on Minicap/Capillarys

Hb S

Hb A2

Hb A

10/11/2017 4:19 AM 31

N

A

A2

Anh.

Car.

Alk.

SF

Heterozygous S/C

S

N

C

Alc.

Anh.

Car.

AFSC control

Hb CHb S

Hb F

Hb A2

10/11/2017 4:19 AM 32

10/11/2017 4:19 AM 33

Homozygous S with foetal hemoglobinAFSC control overlaying

Hb A2

Hb S

Hb F

10/11/2017 4:19 AM 34

N

F

A2

Alk.

S

Anh.

Car.

10/11/2017 4:19 AM 35

10/11/2017 4:19 AM 36

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

10/11/2017 4:19 AM 37

N

Anh.

Car.

A

C+A2

Alk.

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

10/11/2017 4:19 AM 38

Heterozygous A/C

Hb A2

Hb A Hb C

10/11/2017 4:19 AM 39

10/11/2017 4:19 AM 40Am J Clin Pathol 2008;130:824-831

10/11/2017 4:19 AM 41HbA2 peak shows slight considerable overlap with HbC

3- HEMOGLOBIN EAbnormality 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 %

10/11/2017 4:19 AM 42

Anh.

Car.

A

E+A2

Alk.

N

Anh.

Car.

Alk.

N

E+A2

A

Heterozygous A/E

Hb A

Hb A2

Hb E

10/11/2017 4:19 AM 43

Homozygous Hb EAFSC control

Hb E

Hb A2

10/11/2017 4:19 AM 44

10/11/2017 4:19 AM 45

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

10/11/2017 4:19 AM 46

4- HEMOGLOBIN DAbnormality 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 %

10/11/2017 4:19 AM 47

Anh.

Car.

A

A2

Alk.

N

D

`AFSC control overlaying

Hb AHb D

Hb A2

10/11/2017 4:19 AM 48

10/11/2017 4:19 AM 49

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

10/11/2017 4:19 AM 50

Decreased synthesis of the alpha chain by gene deletiona a

b bHb A

b ba 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)

10/11/2017 4:19 AM 51

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

Completely asymptomatic

- One gene deleted:

silent a thalassemiaHb A and A2 fractions are normal, (presence of Bart Hb at birth)

- Two genes deleted:minor a thalassemia (mild anemia)

Hb A fraction normalHb A2 fraction slightly decreased or normalHb Bart fraction detected at birth (5 – 10 %),

together with fetal Hb

10/11/2017 4:19 AM 52

Hb Bart

(Baby’s blood)

Hb F

Hb A

Hb Bart’s

10/11/2017 4:19 AM 53

10/11/2017 4:19 AM 54

Alpha thalassemia with Hb H

Hb A

Hb A2Hb H

10/11/2017 4:19 AM 55

10/11/2017 4:19 AM 56

10/11/2017 4:19 AM 57

Alk.

N

A

Anh.

Car.

H

A2

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….

10/11/2017 4:19 AM 58

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 %)

10/11/2017 4:19 AM 59

Beta Thalassemia

Hb A

Hb A2

10/11/2017 4:19 AM 60

10/11/2017 4:19 AM 61

Anh.

Car.

A0+A2

A1

Alk. Ac.

N N

A2

A

10/11/2017 4:19 AM 62

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% )

10/11/2017 4:19 AM 63

Major b thalassemia

10/11/2017 4:19 AM 64

10/11/2017 4:19 AM 65

10/11/2017 4:19 AM 66

10/11/2017 4:19 AM 67

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)

10/11/2017 4:19 AM 68

Hemoglobin variants

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

10/11/2017 4:19 AM 69

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

10/11/2017 4:19 AM 70

From 6/11/2010 to 1-5- 2013

2064 cases were done

- 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

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

10/11/2017 4:19 AM 73

Thank you for

listening.

Any Questions?

Prof. Moustafa Rizk