NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Old J, Harteveld CL, Traeger-Synodinos J, et al. Prevention of Thalassaemias and Other Haemoglobin Disorders: Volume 2:
Laboratory Protocols [Internet]. 2nd edition. Nicosia, Cyprus: Thalassaemia International Federation; 2012.

Chapter 3 HAEMOGLOBIN PATTERN ANALYSIS

3.1. CHROMATOGRAPHIC METHODS (FOR HbA2 DETERMINATION)

Quantitative HbA2 determination is the most valuable test for β-thalassaemia carrier identification.
Several methods have been set up, but only a few are now recommended for their accuracy. It should be
pointed out that the precision and accuracy of HbA2 determination using densitometry scanning after
cellulose acetate electrophoresis is unsatisfactory and its use has to be avoided (1). Isoelectric focusing
(IEF) has an excellent resolution allowing for an accurate quantification, but it is cumbersome and time-
consuming. Capillary electrophoresis (CE) is being used more and more in a clinical diagnostic setting
for diagnosis of haemoglobinopathies. Several studies showed an excellent correlation between
capillary electrophoresis and high performance cation-exchange chromatography (HPLC) for the
qualitative and quantitative haemoglobin analysis (2-4).

The most used methods for HbA2 measurement are CE, HPLC and less frequently the
microchromatography on DE-52 (5-8). These methods are very accurate, fast and simple. In addition CE
and HPLC identify and measure many variant haemoglobins, including the commonly encountered
variants HbS, HbC, HbE and Hb D-Punjab. Figure 3.1 shows examples of HPLC chromatograms from 4
different analysers (Variant II, HA 8160, G7 and Ultra 2) in comparison to CE.

3.1.1. HPLC FOR HAEMOGLOBINOPATHIES SCREENING

Cation: exchange high performance liquid chromatography (HPLC) has emerged as the method of
choice for quantification of HbA2, HbF and for detection and quantitation of the Hb variants,
particularly those which may interact with β-thalassaemia such as HbS, E, C, O-Arab, D and Lepore.

Principle: In this method phosphate buffers at different concentrations (mobile phase), pass under
pressure through an ionic exchange column (stationary phase). The stationary phase consists of a
temperature controlled analytical cartridge containing a resin of anionic or cationic particles (3-5 μm).
The chromatographic station delivers a programmed buffer gradient of increasing ionic strength and
pH to the cartridge by two dual-piston pumps, and the haemoglobins are separated according to their
ionic interaction with the stationary phase.

The separated haemoglobins then pass through the flow cell of the filter photometer, where changes in
the absorbance (415 nm) are measured; background variations are corrected by an additional filter at
690 nm. Each haemoglobin is characterised by a specific retention time, which is the elapsed time from
the sample injection to the apex of a haemoglobin peak.

The calibration factors for HbA2, F, A1C are automatically calculated by processing a calibration sample
at the beginning of each run. Specific software turns the raw data collected from each analysis into a
report showing the chromatogram, with all the haemoglobin fractions eluted, the retention times, the
areas of the peaks and the values (%) of the different haemoglobin components. The report presents the
percentages of haemoglobins F, A1C, A and A2 and provides qualitative and quantitative determination
of abnormal haemoglobins.

Sample: Venous blood in any anticoagulant.

Method: A commonly used apparatus is the Variant (Bio-Rad Laboratories). However, reliable results
have been recently obtained also with other instruments, such as HA816 (Menarini Arkay) and G7
(Tosoh- Bioscience) (4).
Results and interpretation: The expected normal range for HbA2 is between 1.7% and 3.2% in normal
subjects, while in β-thalassaemia carriers when it is between 4.0% and 7%. HbA2 values are considered
borderline when between 3.2% and 3.8%. Samples with these levels need further investigation for
possible normal HbA2 thalassaemia (see Table 1.1). The normal range for HbF is usually less than 1.5%
of total haemoglobin.

HPLC machines have analyte identification windows that help in the interpretation of normal and
abnormal haemoglobins detected in the blood sample (9). The windows are defined retention time
intervals in which the common haemoglobin variants are eluted (eg HbS, C and D). However, it should
be pointed out that since other Hb variants may have a similar retention time to the common variants,
(Tables 3.1a and 3.1b), the Hb variant identification is only presumptive (Figure 3.1) and DNA or globin
amino acid analysis is necessary for definitive Hb variant identification (10). HPLC is also being used for
neonatal haemoglobinopathy screening programmes (11).

Note: Different elution patterns are obtained with different instruments.

Limitations of the procedure: Since Hb Lepore and HbE are co-eluted with HbA2, their presence in the
sample gives a falsely high percentage (>10%) of HbA2. This amount of HbA2 is almost never present in
β-thalassaemia carriers (6). Therefore samples found to have a level of HbA2 greater than 10% should
be further tested for the possible presence of a haemoglobin variant running with the HbA2 peak. The
increase of HbA2 levels above 3.5% in HbS carriers is due to co-elution of minor components with HbA2
(possible post-translational modifications of HbS). This may also occur with haemoglobin variants
eluting after HbA2. HbH and Hb Bart’s can be detected in the chromatogram but not quantified because
they are eluted prior to the start of integration.

3.2. ELECTROPHORETIC METHODS

Electrophoresis is a separation technique based on the mobility of ions in an electric field. It is the
classical method of identifying and quantifying the haemoglobin proteins.

The haemoglobin molecules (HbA, HbA2, HbF and variants) in solution are electrically charged at any
given pH. They can have a positive charge or a negative one according to the ionisable groups (acidic or
basic side chain) that they have. Total haemoglobin, which is a mixture of these molecules, has a net
negative charge. When an electrical potential difference is applied, particles will migrate either to the
cathode or the anode depending on their net charge, and molecules with different overall charges will
begin to separate.

3.2.1. CLASSIFICATION OF ELECTROPHORETIC METHODS

There are several electrophoretic methods, mostly classified according to the supportive media. The
broad classification is into “free electrophoresis”, in which the molecules are made to migrate in liquid
and “zone electrophoresis”, in which the molecules, dissolved in buffer, are made to migrate on a more
solid medium.

The supportive media are described as:

a. Liquid - the only “free” method still used to separate haemoglobin molecules is capillary
electrophoresis.

b. Solid - this includes paper, which is no longer used and cellulose acetate, which is one of the most
commonly used media.

c. Gel - such as starch (which is also no longer in use), agar, agarose, and polyacrylamide.

Some of these media effect separation of molecules by utilizing differences in size (mass) of the
molecules, shape of the molecule and the frictional effects of the medium. Therefore the choice of
medium will affect the quality of separation. Likewise the rate of migration and thus the resolution
depends on:

a. The strength of the electric field

b. The molecular mass

c. Whether the molecule is hydrophobic

d. The ionic strength of the buffer

e. The temperature of the buffer.

For example high voltage means faster separation but excessive current causes heat which will distort
the bands by causing evaporation, siphoning of electrolytes and denaturation of molecules. The choice
of buffer is also important since it determines the pH, which influences the rate and direction of
movement of the protein, and the ionic strength, which influences the rate of separation. The
composition of the buffer may interact with a protein causing a change in charge density.

In practice the choice of the electrophoretic method will be influenced by all of the considerations
mentioned above and at least two different methods are recommended to positively identify the
haemoglobins because some haemoglobins have identical migration rates on one medium but separate
on the other. For example HbS, HbD and Hb G-Philadelphia migrate together on alkaline
electrophoresis, as do HbE and C, while their mobility differs on citrate agar. In a reference laboratory
other methods, such as Isoelectric Focusing (IEF), Capillary Electrophoresis (CE) and /or High Pressure
Liquid Chromatography (HPLC), should also be available to resolve diagnostic problems.

In this chapter, the most commonly used electrophoretic methods in thalassaemia population screening
programmes or for diagnosis of homozygote states and identification of variants will be described.
These are Capillary Electrophoresis, cellulose acetate electrophoresis, citrate agar electrophoresis and
isoelectric focusing.

Samples:

a. 2-3 ml of whole blood in EDTA is the usual sample taken for electrophoresis. Other anticoagulants
may also be used.

b. The red cells are lysed in distilled water and carbon tetrachloride and then centrifuged to give a
clear haemolysate that can be used for electrophoresis. The haemolysate is better utilised fresh
although if stored at –80ºC it can be used later. In automated systems, like for example the
Capillarys of Sebia, haemolysing buffer is delivered by the manufacturer.

3.2.2. CAPILLARY ELECTROPHORESIS FOR HAEMOGLOBINOPATHIES SCREENING

Capillary Electrophoresis is an emerging diagnostic tool in many clinical chemistry labs to separate Hb
fractions and calculate the percentage of each fraction. A commercially available apparatus, used in
several European labs, is for example the Capillarys from Sebia (Lisses, France).

Principle: Capillary Electrophoresis (CE) is the technique of performing electrophoresis in buffer-filled

narrow capillaries, 25-100 μm in diameter. The separation relies on differences in the speed of
migration (migration Velocity) of ions or solutions, but the vitally important feature of CE is the bulk
flow of liquid through the capillary, which is called Electric Osmotic Flow (EOF).

The inside surface of the capillary has ionisable silanol groups, which readily dissociate giving a
negative charge to the capillary wall. The negative charge attracts the positive charged ions from the
buffer, creating an electrical double layer and therewith a potential difference close to the capillary
wall. When a voltage is applied across the capillary, cations in the diffuse layer are free to migrate
towards the cathode, carrying the bulk solution with hem. The result is an Electro Osmotic Flow and
separation of the differently charged Hb fractions. These fractions are detected directly at an
absorbance wavelength of 415 nm, which is optimal to haemoglobins, in the following order from
cathode to anode: HbC, A2, E, S, D, F, A, Bart’s, J and H.

In contrast with the pressure driven flow in HPLC, the flow profile of EOF is distributed uniformly along
the capillary. No pressure drops are encountered and the flow velocity is uniform across the capillary,
which leads to higher separation efficiencies (12).

Equipment: The capillary electrophoresis device used is the Capillarys from Sebia (Lisses, France).
Consumables are delivered by the manufacturer and include:

a. Dilution segments green and transparent.

b. Sample rack 0: For use of normal and pathogen control, with tube and barcode.

c. Sample rack 100: The system recognizes this rack as a maintenance rack and the option: “capi.
cleaning “is available.

d. Sample rack Sebia: Used with the green dilution segments. The system recognizes automatically
that dilution preparing is not necessary. For use of patient’s samples and AFSC control (control
containing mix of HbA, HbF, HbS and HbC).

e. Sample rack with a number: The system makes dilutions automatically from the tubes placed into
the sample rack.

Sample preparation: The anticoagulant is EDTA, but the use of citrate or heparin is also acceptable.

Method:

1. Switch on the Capillarys with the button in the rear.

2. Turn on the computer and log on into the system.

3. Then double click the Phoresis icon on the desktop screen and log in to the system with
appropriate ID and password.

4. Check and /or change the reagents by opening the lid on the right front side of the apparatus. Click
OK in the window appearing when performing this procedure.

5. Enter the new Lot numbers and expiration dates and click “OK “.

6. Check the reagents levels visually and move the cursor buttons to adjust if necessary.

7. The status window will show the message “Busy” and changes to “ready”: the apparatus is ready
for use.

Migration control and control cycle:

1. The Control samples are available commercially and should be dissolved in distilled water
according to the manufacturer’s instructions. This control vial can be stored at –20ºC and used 15
times. The control should be placed in position 1 in the sample rack “0”. Pour 4 ml of the
haemolysing solution in a tube and place it in position 8. Place a new green dilution segment on
the rack. (For manual dilution: mix 90 μl of the haemolysing solution with 18 μl of the control in a
green segment and put it on the “0” rack with the appropriate tube. The controls made in this way
can be frozen and thawed three times.)

2. When the status window changes from “busy” into “ready”, the rack “0“ containing the normal
control, is inserted into the Capillarys. Place this rack in the apparatus and push the rack until you
hear a beep. The green light of the feeder belt indicator will switch to red.
 3. The “Select a control” window will appear: Select QC medium in the drop down menu, enter the lot
number, select the right dilution: manual when a diluted green segment is used or automatic when
the Capillarys has to make the new dilution and click “OK” (A second normal control run has to be
done in the following circumstances:

a. After changing the analysis buffer

b. After capillary cleaning or capillary activation

c. After a software upgrade)

d. When the run is completed and the controls are in range, all eight capillaries are calibrated.

4. Insert rack “0“ again with the pathological control and on the first position a tube with the
appropriate barcode. A window appears in which the lot number should be entered, select manual
or automatic dilution and click “OK “.

5. Collect the patient blood for making the dilution. Use the red blood cells after centrifugation. Mix
the red cells carefully and prepare the dilutions by pipetting 90 μl haemolysing solution (Sebia)
with 18 μl red cells, use the green dilution segments.

6. Insert the rack when the status window shows “Ready”.

7. If migration is completed the curves are shown by clicking the video screen-icon (red arrow).

Results and interpretation: When the run has ended, prepare a Sebia worklist by clicking the worklist
icon. The results of the controls appear automatically on the worklist (NORMAL CTL, PATHO CTL). Open
the video screen-icon to analyze the curves:

To open the curves there are two options:

1. Click on a curve to open its review window

2. Click on the icon underneath the blue marker (the third icon from the left) and it shows the first
curve of the run. Use the select buttons to bring up the next curve, the previous curve and the first
or last curve. To return to the profiles of the current analysis: click the ‘save and exit’ button (with
the red arrow).

The Capillarys recognizes automatically the A0, F, C and A2 peaks. Other peaks have a specific position
(zones) for instance HbS, HbD, HbE, HbH and HbJ but they are all labelled as HbX. It is possible to add a
comment with the most probable name of the abnormal Hb variant.

3.2.3. CELLULOSE ACETATE ELECTROPHORESIS

This is an acetate salt of cellulose produced by treating cotton with acetic acid using sulphuric acid as a
catalyst. Migration takes place on the buffer film on the surface of the cellulose acetate plate or
membrane. Separation of the proteins is primarily by charge. Cellulose acetate electrophoresis may be
used for qualitative identification of variants, but also with elution for quantitation of the
haemoglobins, A2, A, S, D, Lepore, α-chain variants, HbH and Hb Bart’s. The positions of various
haemoglobin fractions on cellulose acetate electrophoresis are shown in Figure 3.2.

Reagents and materials:

a. Tris-EDTA Boric Acid (TEB) buffer, pH 8.4:

i. Tris hydroxymethyl amino methane (TRIS): 10.2 g

ii. Ethylene diamine tetracetic acid (EDTA): 0.6 g

iii. Boric Acid: 3.2 g

 Make up to 1 litre with distilled water.

b. Whatman No. 3 chromatography paper.

c. Cellulose acetate membranes are supplied Scheicher and Schuell, 40 x 300 mm. Alternatives are
made by Sartorius and by Shandon.

d. HbA2 control, as supplied by the National Institute for Biological Standards and Control (NIBSC).
The control has been produced by freeze drying a solution of haemoglobin prepared from human
cells and made stable by the addition of sucrose (200 mM), potassium cyanide (6 mM) and
chloramphenicol (1 mg/dl). This control was established by the World Health Organization in 1994
as the international Reference Reagent with an assigned value of 5.3% of total haemoglobins
present.

Equipment required:

a. Power supply capable of delivering a constant current, 0-80 mA and up to 400 volts.

b. An horizontal electrophoresis tank with adjustable bridge gaps and a polarity indicator (eg.
Shandon), as shown in Figure 3.2.1.

c. Roller mixer.

d. A single beam SP6-200 - Spectrophotometer Pye Unicam.

Method:

a. Haemolysate is prepared from whole blood (in K2EDTA) as previously described.

b. The electrophoresis tank is prepared by filling the tank with 900ml approximately of TEB buffer
wicks are cut from Grade No. 3 chromatography paper and were placed along the 22 cm long
bridges in the tank.

c. The cellulose acetate membranes are cut in 40 x l00 mm each and soaked (shiny side down) in TEB
buffer for 5 minutes. Five strips are plotted and placed on the electrophoresis tanks.

d. Voltage current is applied at 250 V for 5 minutes to the membranes to equilibrate the membranes
with the buffer.

e. The current is turned off and 8-10 μl haemolysate (10 g/μl) is applied on each membrane at the
cathodal end using a capillary tube.

f. Then the voltage is set at 250-300 V working at constant current of 2 mA for each strip.

g. The electrophoresis is run for approximately 45 minutes to one hour until there is a clear area
between the bands.

h. The current is then turned off and the separated HbA2 on the cellulose acetate membrane is cut
and immersed in a tube containing 4 ml of distilled water and the HbA in a tube containing 16 ml
distilled water. If a haemoglobin variant is present then this is cut separately into 4 or 16 ml
distilled water depending on the quantity of the variant present. Note that a blank is prepared
from the same run by cutting a piece of clear cellulose acetate strip that was immersed in 4 ml
distilled water.

i. The tubes are then placed on a roller mixer for 30 minutes for the haemoglobin elution.

j. The strips are removed and the tubes are then centrifuged for 10 minutes at 3000 rpm.

k. The absorbance of each haemoglobin is read at 413 nm against the blank on a spectrophotometer.

Interpretation:
The results are calculated as follows:

Absorbance of HbA2 × 100

% of HbA2 =
Absorbance of Total  (HbA × 4) + absorbance of HbA2

The haemoglobins migrate on the cellulose acetate membrane from cathode to anode in the following
order: HbA2, HbE, HbC, HbD, HbS, Hb Lepore, HbF, HbA and the fast moving haemoglobins Bart’s and
HbH (see Figure 3.2). The normal range for HbA2 is 2.4% to 3.2%. A typical example of cellulose acetate
electrophoresis is shown in Figure 3.2.2.

Factors affecting the result are: correct pH, correct concentration of the buffer and the temperature of
the buffer, which may be influenced by the voltage or the environmental temperature. It is advisable,
especially in hot climates to keep electrophoresis tanks with buffer refrigerated at 4ºC. Ideally the
method should be conducted at an environmental temperature below 23ºC. It is necessary, therefore, in
warm climates to have air conditioning in the laboratory. The quality of the carrier membrane must be
good and poor quality should be recognised and discarded. The membrane should be kept moist.

3.2.4. AGAR GEL ELECTROPHORESIS

Agar is a gelatinous substance derived from the cell wall of red marine algae. It is composed of a matrix
of cross-linked molecules with spaces between them. In an electric field the haemoglobin molecules will
move through the matrix so that the migration rates depend on the size and shape of the molecules as
well as the charge. This means that smaller, linear molecules with high electric charge will move
through the gel at a faster rate.

Agar gel electrophoresis is not a satisfactory screening technique because it cannot distinguish many
abnormal haemoglobins from HbA. However it can separate the C group into three fractions: HbC, O-
Arab, and HbE plus HbA2. The method can also distinguish HbS from HbD, HbF from HbA, Hbs Little
Rock, Rainier and Bethesda from HbA, and HbH from HbI.

Reagents:

a. Stock buffer - Citrate buffer, pH 5.9:

Dissolve 73.5 g tri-sodium citrate (Na3C6H5O7.2H2O) in approximately 700 ml distilled water.

Adjust the pH to 5.9 using 0.5 M citric acid (10.5 g per 100 ml). Approximately 34 ml will be
required. Make the solution up to 1 litre with distilled water, and store at 4ºC.

b. Working buffer. Dilute stock buffer 1 in 5 with distilled water. The pH will be 6.

c. Gel: Pre-prepared made Titan IV Citrate Agar plates, provided by Helena Laboratories U.K.

d. Bromophenol blue - for staining of the gel.

Dissolve 20 mg bromophenol blue in 200 ml distilled water containing 2ml glacial acetic buffer.

Equipment:

a. Power supply capable of delivering 30-40 mA is necessary (Vokan SAE 2761, Vokan 400).

b. A southern horizontal electrophoresis tank.

c. Gel slot, a device for making sample application wells.

Method:
 1. Using the gel slot, 5 slots are made on the gel, mid-way between anode and cathode.

2. Using a capillary tube enough haemolysate is delivered to fit each slot on the gel.

3. 700 ml of the working buffer is poured into the electrophoresis tank.

4. Wicks made from Whatman No.3 chromatographic paper, are placed along the bridges of the
electrophoresis tank. The gel is placed along the wicks and is held in position using extra wicks
made of Whatman No.3 chromatographic paper in order to ensure contact with the buffer.

5. A current 30 mA approximately (40 V) is applied across the gel.

6. Electrophoresis is run at 4ºC.

7. After approximately 3 hours, separation is achieved.

8. The agar plate is stained for 20 minutes with the bromophenol blue stain and then rinsed with
distilled water.

Interpretation: Typical results of agar gel electrophoresis are shown in Figure 3.3.

3.2.5. ISOELECTRIC FOCUSING

Isoelectric focusing is the electrophoretic method that separates proteins according to the isoelectric
points. The net charge of a protein is the sum of all negative and positive charges of its amino-acid
chains and their ionisable groups (amino and carboxyl termini). The isoelectric point (pI) is the specific
pH at which the net charge on the molecule is zero (proteins are positively charged at pH values below
their pI and negatively changed at pH values above their pI) (13, 14).

A pH gradient is necessary so that under the influence of an electrical field, a protein will move to the
position where its net charge is zero. A protein with a positive net charge will migrate towards the
cathode becoming progressively less positively charged as it moves through the pH gradient until it
reaches a point that corresponds to its pI value.

Isoelectric focusing requires solid support such as agarose gel and polyacrylamide gel. Polyacrylamide
is a polymer with small interstices - approximately the size of proteins so that apart from surface
charge, separation depends on the size of the molecules. Isoelectric focusing gels contain synthetic
buffers called ampholytes that smooth the pH gradients.

Isoelectric focusing needs high voltage (1000 V or more). It gives good separation with a high resolution
compared to any other method. Resolution depends on:

i. The pH gradient,

ii. The thickness of the gel,

iii. Time of electrophoresis,

iv. The applied voltage,

v. Diffusion of the protein into the gel.

Method 1: Pharmacia PhastSystemTM

The preferred isoelectric focusing electrophoresis method in the Cyprus laboratory is the
PhastSystemTM with dry polyacrylamide gels soaked prior to use in a narrow pH gradient (6.7-7.7). The
pH gradient is made by Pharmalyte® which generates stable, linear pH gradients in the gels during the
run. The haemoglobins migrate under an electric field to a point in the pH gradient that corresponds to
their pI (isoelectric point). The separated Hbs on the stained gel are evaluated by visual inspection.
Protein patterns from known haemoglobin variants are used as references to identify the protein bands
from unknown Hb samples.
Reagents:

a. Phast-Gel Dry IEF plates *

b. Pharmalyte pH 6.7-7.7*

c. Kerosene

d. 20% trichloroacetic acid (TCA) fixative

e. Staining solution stock: one tablet of Phast Gel Blue R tablet * (1 tablet + 80 ml distilled H2O + 120
ml methanol). The solution was stirred for 2 minutes and filtered twice

f. Methanol

g. Destaining solution containing 300 ml methanol + 100 ml acetic acid + 600 ml distilled water
(3:1:6). CuSO4 (5H2O)

h. Working solution for staining was made by mixing 30 ml stock staining solution with 270 ml of
3:1:6 destaining solution and 0.45 g CuSO4 (5H2O). The solution is filtered and is prepared fresh
before use.

i. 0.1% Triton

Note: *These items are provided by the Amersham-Pharmacia company.

Equipment:

Pharmacia LKB - Phast System. The system consists of:

a. a separation unit

b. a developing unit for staining procedures.

c. Phast Gel sample - applicator 8/1

d. Phast Gel sample – well stamp

e. Scanning Densitometer LKB Pharmacia

All functions are controlled by a microprocessor.

Gel preparation: Rehydration of dry gels: dry gels are rehydrated using a 1:16 solution of Pharmalyte
6.7-7.7 (100 μl Pharmalyte + 1500 μl distilled H2O). The dry plate is placed with the gel side down on the
drop of the solution that has been pipetted on a clean plastic surface, for 1 hr checking at intervals that
the plate did not stick to the surface.

After rehydration excess of fluid is removed from the surface of the gel by wiping it gently with the edge
of a piece of filter paper.

Sample preparation: Samples are prepared by diluting 5μl of whole blood in 200 μl 0.1% Triton. The
mix is allowed to stand for 5 minutes and then mixed on a vortex mixer. 5μl is used for the sample
application.

Sample application: To load the sample applicator, depressions are formed on a strip of Parafilm
(using the PhastGel sample well stamp). 5 μl of each sample is placed on the depressions. The PhastGel
sample applicator 8/1 is dipped in the 5 μl droplets on each sample.

Method:

1. The plates are placed on the cooling bed of the Phast System, on which two drops of kerosene are
placed on each plate.
 2. After application of the electrodes on the gel the pre-focusing stage is performed.

3. After pre-focusing, the samples are applied anodally to the gels using the 1 μl applicator.

4. For each type of plate a different separation program on the Phast System is used through the
microprocessor of the Phast unit.

5. For running Phast Gel Dry IEF 6.7-7.7 the following separation program is used:

6. End of separation program:

Sample applicator down at 1.2 vh

Sample applicator up at 1.3 vh

7. The method called separation method 1 was programmed on the Phast System previously and
contains three separating steps, Sep 1.1, Sep 1.2 and Sep 1.3. A final step, Sep 1.4, was added to
finalise the program.

8. The sample applications will be lowered onto the gels at after 75 vh during Step 1. During this 75
vh period, the sample applicators are loaded. An alarm sounded at 73 vh as a warning that the
sample application will occur in 2 vh. The sample applicators are automatically raised at 100 vh of
Step 3 and separation proceeds as indicated in the above program.

Staining: After the focusing steps have been performed, the gels are placed in the staining chamber.
The plates are fixed with 20% TCA for 5 minutes at 20ºC, and washed with distilled water for 2 minutes.
Subsequently they are stained for 10 min with 0.2% Coomassie Blue (1:10 solution, of stock solution:
destain solution), containing 0.15 w/v % CuSO4 (5H2O) at 50ºC.

The plates are destained in the destain solution for 10 min at 50ºC and allowed to dry. Plates are
scanned on a laser scanning densitometer for determination of pI values. Samples containing known
haemoglobins (HbF, HbS, HbA2, HbA, Hb Lepore, HbD) as well as the sample containing the unknown
haemoglobin are run on the same plate. The method does not distinguish between Hb G-Philadelphia,
Hb Lepore, HbE and Hb O-Arab.

Interpretation: The identification of the unknown haemoglobin is achieved by measuring the pI value
of the unknown haemoglobin in a laser densitometer. The pI values of known haemoglobins, separated
on the same plate, are also measured and are used as controls. Despite excellent resolution achieved by
isoelectric focusing, accurate quantification is not possible.

A Phast Gel plate pH 6.7-7.7 showing the separation of various haemoglobins is illustrated in Figure 3.4

Method 2: PerkinElmer RESOLVE® System

The preferred isoelectric focusing electrophoresis method in the Oxford laboratory is the RESOLVE®
Systems K Haemoglobin Kit, used to detect haemoglobin variants in whole blood (EDTA) for antenatal
screening or blood spot samples from Guthrie cards for neonatal screening.

Reagents:

a. RESOLVE® FR-9120 Haemoglobin Kit – contains 5 IEF gels, an anode and cathode solution, Hb
elution solution, electrode wicks, blotting paper and strips and sample application templates.

b. JB-2 Staining System - 2x Gel Stain Concentrate, 2x Stain Buffer and 1x Stain Activator.

c. 10% Trichloroacetic acid -To make up to 500 ml 10% TCA, add 50 ml 6.1N TCA to 450 ml distilled
water.

d. Vnbs Retention Time markers (FASE and FACD) are used as control samples. These are made up
with 1ml de-ionised water, aliquotted into microvials, and frozen at -70ºC until required. Those in
 use are stored at 4ºC. Use FASE twice, one at the beginning and end of the run. Use FADC in the
middle of the run (as well as FASE if space allows).

Equipment:

PerkinElmer RESOLVE® System consists of:

a. Multiphor II electrophoresis unit

b. A circulating water bath to provide constant temperature control to the electrophoresis unit

c. Programmable Power Supply

Sample preparation from whole blood:

1. Pipette 5 μl of whole blood sample into a labelled Eppendorf tube (try not to mix the blood and use
the packed cell portion).

2. Add 50 μl of Hb Elution solution provided with the IEF gel kit.

3. Vortex the tubes and leave to lyse for approximately 10 minutes.

Sample preparation from blood spots on Guthrie cards:

1. Allocate a well number for each specimen. Using a manual puncher, punch 3 mm holes from the
Guthrie cards and place in the corresponding well of the microtitre plate. Use two spots for
samples with HbA levels of less than 3% and abnormal variant levels of less than 2% on the Vnbs.

2. Add 80 μl of Hb Elution solution to each well.

3. Cover the microtitre plate and allow to stand for 30 min at room temperature. Mix the blood spots
using a sonic bath for 5 min.

Electrophoresis:

1. The precast gels and solutions (Hb elution solution, cathode solution and anode solution) are kept
2-8ºC and should be taken out of the fridge approximately 30 minutes prior to use, to bring up to
room temperature.

2. Start up the water bath by turning on the Power Switch and the Refrigeration Switch. The
temperature at temperature controller (Walton SD) will start going down. After a few minutes,
check the ‘outlet’ and ‘inlet’ tubing of the bath and the cooling plate in the electrophoresis unit to
see if they are cooled down. If they are not cool, the bath needs to be switched off and switched on
again observing for bubbles in the lines which may cause airlocks.

3. Pipette water onto the centre of the cooling plate. Take out the gel and using both hands hold the
edges of the gel. Position the gel in the centre of the cooling plate between lanes 4 and 14 and
bending gently, roll from side to side to ensure water spreads evenly under the gel. Be careful not
to trap air bubbles between the gel and cooling plate.

4. Remove the topmost sheet on the gel and blot gently. Any excess water from the periphery of the
gel should be removed with paper towel.

5. Evenly saturate one wick (rough side up) with the cathode solution on several paper towels and
blot to remove excess water. Place the wick between positions 3-4 of the cooling plate. Remove
gloves and repeat with another wick using the anode solution. Place this wick between positions
13-14. Change gloves again.

6. Place the sample template strip carefully onto the gel, next to the cathode wick. Run your finger
gently along the sample template strip to remove air bubbles.
 7. Load controls and specimens according to the worksheet (5 μl for control and neonatal samples,
3.5 μl for adult specimen).

8. Place the electrode holder over the gel with the cathode electrode on left (black) and centre the
electrodes over the wicks. Connect the electrode leads into the connectors on the inside of the tank.
Place the cover over the electrophoresis unit.

9. Switch on the power supply. Press “run” at each prompt until the voltage readout shows increasing
voltage (In some cases the power does not reach the 1500 V but that usually does not cause a
problem).

10. After 20 minutes, press ‘standby’ to pause the program. Remove the sample template strip and blot
the gel. Press ‘Run’ to continue the program. Repeat blotting after 20 minutes (optional).

11. The program takes 1 hour and 30 minutes to run. However, if the bands are separated and clearly
visible on the gel (after approximately 1 hour and 15 minutes), press ‘stop’ to terminate the run.
Switch off both the cooler unit and the power supply. Remove the cover, disconnect the electrodes
and remove the electrode holder.

12. Make sure that the cooling plate and electrodes are wiped down with deionised water and a paper
towel after use. When cleaning the electrodes, do so gently so as to avoid bending and damaging
the electrodes.

Fixing and Staining Gel:

1. Place the gel in a staining tray. Take it to the fume cupboard and add 100 ml of 10% TCA or
sufficient to cover the gel, place the lid on the staining tray and mix it gently on the mixer for 10
minutes.

2. Pour off the TCA down the sink and run plenty of water afterwards. Add deionised water in the
staining tray and mix it gently for a couple of minutes. Leave it to stand for 10 minutes whilst the
gel stain is prepared. Always make the stain fresh.

3. Take the box with the stains into the fume cupboard. Add 15 ml of stain buffer and 27 ml of gel
stain concentrate to the designated 500 ml measuring cylinder. Make up to 150 ml with deionised
water. Add 3 ml of stain activator. Place parafilm over the cylinder and gently mix.

4. Pour off the water on the gel and add the stain. Place the lid on the tray and place it on the mixer.
Leave it to agitate gently for 10-15 minutes or until the HbA band in the neonatal samples is clear.

5. Pour off the stain down the sink with copious amounts of water. Submerge the gel in deionised
water and place it back on mixer for at least 1 hour, changing the water at least once.

6. Air-dry the gel on a paper towel overnight. Write the gel number on the paper towel.

7. Next day, number the lanes and write the gel number on the gel using a marker pen.

Calculation of Results:

1. Measure the gel distance, in mm, from HbA to HbS, HbA to HbC, HbA to HbD and HbA to HbE (use
bands of the control samples: FASE and FADC).

2. Calculate the multiplication factor by dividing the reference distance by the gel distance. For
example, if the gel distance from HbA to HbS is 8 mm, the multiplication factor for any band
positioned between Hb A and Hb S will be 8.5 mm / 8.0 mm.

3. To determine the relative distance of an unknown band on the gel, measure the gel distance
between HbA and the unknown band and multiply it with multiplication factor. The relative
 distance will then be obtained. Thus a band running a 6.5 mm below HbA is recalculated to a
position of 6.9mm.

4. Some bands may appear above the HbA band - these are noted with a negative measurement.

5. The Hb variant may now be given a presumed identification by comparing its relative distance to
those of known variants observed on previous gels, or against the positions marked on the
published isoelectric focusing map of human haemoglobins (13, 14).

6. Additional genetic investigations should be done to confirm the presumptive diagnosis when it is
needed.

Interpretation: An example of isoelectric focusing electrophoresis is shown in Figure 3.5. As each gel
run is slightly different, the band positions for each run should be standardised using the reference
band distances from the published isoelectric focusing map as described above. The reference distances
are: HbA to HbF 3 mm; HbA to HbS: 8.5 mm; HbA to HbC: 16.0 mm. Refer to the isoelectric focusing
maps of human haemoglobins (13, 14) for variants that are out of the AFSC range. Electrophoresis of old
samples may yield “aging bands”. This is due to the degradation or oxidisation of the haemoglobin
molecule.

REFERENCES
1. Lafferty J. College of America Pathologists haemoglobinopathy survey HG-B. Chicago, IL; College of
American Pathologists; 1999.
2. Cotton F., Lin C., Fontaine B., Gulbis B., Janssens J., Varteongen F. Evaluation of a capillary
electrophoresis method for routine determination of haemoglobins A2 and F. Clinical Chemistry.
1999;45:237–243. [PubMed: 9931046]
3. Mario N., Baudin B., Aussel C., Giboudeau J. Capillary isoelectric focusing and high performance
cation-exchange chromatography compared for the qualitative and quantitative analysis of
haemoglobin variants. Clinical Chemistry. 1997;43:2137–2142. [PubMed: 9365399]
4. Delft van P., Lenters E., Bakker-Verweij M., de Korte M., Baylan U., Harteveld CL, Giordano PC.
Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in Multi-ethnic
populations. International Journal of Laboratory Hematology. 2009;31(5):484–495. [PubMed:
19486364]
5. Efremov C.D., Huisman T.H., Bowman K., Wrightstone R.N., Shroeder W.A. Microchromatography of
Haemoglobins. II. A Rapid method for the determination of haemoglobin A2. J. Lab. Clin.
1974;83:657–664. [PubMed: 4817785]
6. Galanello R., Melis M.A., Muroni P., Cao A. Quantitation of Hb A2 with DE-52 microchromatography
in whole blood as screening test for beta-thalassaemia heterozygotes. Acta Haematologica.
1977;57(1):32–36. [PubMed: 402762]
7. Galanello R., Barella S., Gasperini D., Perseu L., Paglietti E., Sollaino C., Paderi L., Pirroni M.G.,
Maccioni L., Mosca A. Evalution of an automatic HPLC analyser for thalassaemia and haemoglobin
variants screening. Journal of Automatic Chemistry. 1995;17(2):73–76. [PMC free article:
PMC2548064] [PubMed: 18925016]
8. Clarke M.C., Higgins T.N. Laboratory investigation of haemoglobinopathies and thalassaemias:
review and update. Clinical Chemistry. 2000;46(8):1284–1290. [PubMed: 10926923]
9. Clarke GM, Higgins TN. Laboratory Investigation of Haemoglobinopathies and Thalassaemias:
review and update. Clinical Chemistry. 2000;46:1284–1290. [PubMed: 10926923]
10. Lorey F, Cunningham G, Vichinsky EP, Lubin BH, Witkowska HE, Matsunaga A, Azimi M, Sherwin J,
Eastman J, Farina F, Waye JS, Chui DH. Universal newborn screening for Hb H disease in California.
Genet. Test. 2001;5:93–100. [PubMed: 11551109]
11. Galanello R, Barella S, Gasperini D, Perseu L, Paglietti E, Sollaino C, Paderi L, Pirroni MG, Maccioni
L, Mosca A. Evaluation of an automatic HPLC analyser for thalassaemia and haemoglobin variants
 screening. Journal of Automatic Chemistry. 1995;17:73–76. [PMC free article: PMC2548064]
[PubMed: 18925016]
12. Frazier RA, Ames JM, Nursten HE. Cambridge, U.K.: 2000. Capillary Electrophoresis for Food
Analysis Method Development. Published by The Royal Society of Chemistry. ISBN 0-85404-492-2.
13. Basset P, Beuzard Y, Garel MC, Rosa J. Isoelectric focusing of human haemoglobins; its application to
screening, to characterisation of 70 variants and to study of modified fractions of normal
haemoglobins. Blood. 1978;51:971–982. [PubMed: 638255]
14. Bain BJ. 2001. In: Haemoglobinopathy Diagnosis Bain Barbara J., editor. Blackwell Scientific;
Oxford: p 25 27 .
Figures
FIG. 3.1 Examples of HPLC chromatograms in comparison to CE (adapted from van Delft et al. 2009 ref
4). From left to right: Capillarys CE, Variant II, HA 8160, G7 and Ultra 2. a = normal; b = HbA/S; c = HbA/C; d
= HbA/E; e = HbA/D; f = High HbA2 Heterozygous β-thalassaemia.
FIG. 3.2 Position of fractions on cellulose acetate electrophoresis.
FIG. 3.2.1 Cellulose acetate electrophoresis equipment.
FIG. 3.2.2 Example of cellulose acetate electrophoresis.
FIG. 3.3 Example of agar gel electrophoresis
1. Lanes 1,4: normal adult
2. HbS/β+-thalassaemia
3. HbS/β0-thalassaemia
FIG. 3.4 A Phast Gel plate pH 6.7-7.7 showing the separation of various Hbs by IEF. Lanes 1, 2, 6, 7, 8 are
cord blood samples lane 3 is Hb Lepore trait Lane 4 is HbD trait Lane 5 is HbS trait
FIG. 3.5 Isoelectric focusing gel showing adult and neonatal samples using the RESOLVE system.
Tables

Table 3.1a Elution times of some haemoglobin variants observed with the BioRad Variant-II HPLC system.

Hb F A1C A0 A2 D S C
Window
Retention 1.05-1.10 1.60-1.70 2.3-2.7 3.6-3.7 4.1-4.2 4.4-4.5 4.7-4.8 5.1-5.2
Time
J- Le Athens- E D-Punjab S Hasharon C
Sardinia Lamentin GA Bari G-Norfolk Winnipeg O-Padova C-
J-Iran N- Tyne Lepore G-Phil. Memphis O-Arab Rothschild
Dagestan Baltimore Creve G- G-St Jose Manioba 2 Shelby G-Siriraj
S. J- Couer Copenhagen Stanleyville Yakima Q-Iran S-Oman
Florida Baltimore New York D-Ibadan 2 Savaria Q-India
J-Broussais Hounslow Belfast Dallas G- Setif
J-Paris 1 Ty Gard Matera Kempsey Waimanalo M-
J-Norfolk Koln Zurigo Tak G-Pest Milwaukee-
J-Pontoise Fort Worth Tarrant Russ 2
Luton Kenya Matsue-Oki Richmond Ta-Li
Andrew Mi D-Iran San St Lukes M-Iwate
Harbin Deer Lodge Antonio Summer Titusville
Dublin G-Tapei Radcliffe Hill
Interlaken Gainsville- Woodville
I-High GA West One
Wycombe
Bexiers
J-
Wenchang-
Wuming
Table 3.1 b (part 1) Elution zones of some haemoglobin variants observed with the Sebia Capillarys Capillary
Electrophoresis system: Zones 1-7.

Zone 1 2 3 4 5 6 7
Hb HbA2 HbF
Window
Potential Santa Ana C O-Arab E S Stanleyville II Q-Thailand
Hb F-Hull Constant Chad Köln Dhofar Osu Alabama
variants T- Spring E- Agenogi Arya Christansborg Chapel Hill
Cambodia F-Texas Saskatoon G-Siriraj Hasharon Leiden Bassett
A2 C-Harlem Santa Ana Handsworth Muravera Barcelone
HbA2 HbA2 A2- Ottawa Matsue-Oki Geldrop
variants of: variants Babinga S-Antilles Muskegen Sant Anna
“Hasharon” of: M- Fort de Summer Hill Swan River
“Winnipeg” “Setif” Saskatoon France D-Ibadan Presbyterian
“Q- “Bassett” Denat. C Hamadan D-Bushman Burke
Thailand” “Swan HbA2 Montgomery D-Punjab Manitoba II
“Q-India” River” variants Denat. O- D-Ouled Richmond
“G-Norfolk” “Manitoba of: Arab Rabah G-San-José
“G-Pest” I” “M-Iwate” HbA2 D-Iran Porto Alegre
“Inkster” “Manitoba variants of: Lepore denatured S
“Memphis” II” “Lombard” Korle-Bu HbA2
“Chapel “Cemenelum” Köln variants of:
Hill” “Jackson” Fort Worth “J-Paris-I”
“Arya” G-Norfolk
“Fort de G-
France” Philadelphia
G-Coushatta
G-Taipei
G-Siriraj
G-Pest
Inkster
Memphis
P-Nilotic
Q-India
Q-Iran
Willamette
Winnipeg
Setif
denatured E
HbA2
variants of:
“J-Toronto”
“J-Rajappen”
“J-Anatolia”
“J-Oxford”
“J-Broussais”
“Mexico”
“J-Habana”
“J-Rovigo”
Table 3.1 b (part 2) Elution zones of some haemoglobin variants observed with the Sebia Capillarys Capillary
Electrophoresis system: Zones 8-15.

Zone 8 9 10 11 12 13 14 15
Hb Window Hb F HbA Denatured
acetylated HbA zone
Potential Atlanta Toulon Hope Kaohsiung Hb Bart’s J-Rogivo N- Hb
Hb variants Alberta Okayama M- Providence Cemenelum N- Seattle I-
Hinsdale Fontainebleau Iwate K-Woolwich Providence Baltimore Texas
Kempsey Camperdown Camden Lombard J-Toronto J-Norfolk
Athens-GA Gorwihl Fannin J-Mexico J-
Phnom Penh Lubbock J-Baltimore Kaohsiung
Silver Springs Andrew J-Calabria
La Coruna Minneapolis J-Rajappen
Bougardirey- Jackson Grady
Mali Himeji J-Anatolia
Austin HbA2 J-Broussais
Buenos Aires variants of: J-Chicago
Chicago “I-Texas” J-Oxford
Raleigh J-Meinung
Hekinan Ube-2
Mosella Mexico
Dallas J-Habana
Aztec J-Paris-I
Little Rock
Frankfurt
Bethesda
M-Boston
Brisbane
Mizuho
Grange
Blanche
San Diego
M-Saskatoon
Malmö
Minneapolis
Laos
Syracuse
Sep. 1.1 1000 V 2.0 mA 2.0 W 15 t 75 vh
Sep. 1.2 300 V 1.0 mA 1.0 W 15 t 25 vh
Sep. 1.3 1500 V 5.0 mA 3.5 W 15 t 550 vh
Sep. 1.4 0V 0 mA 0W 0t 0 vh

© 2012 Thalassaemia International Federation.

All rights reserved. The publication contains the collective views of an international group of experts and does not necessarily represent the decisions
or the stated policy of the Thalassaemia International Federation.

Bookshelf ID: NBK190579