ALKALINE HAEMOGLOBIN ELECTROPHORESIS (pH 8.

6)

(Marengo & Rowe, 1965 with modifications)

This is a technique to separate and identify various haemoglobin variants in an electric field
using their differences in charges in a medium of alkaline pH. The technique can be used for
both qualification and quantification of normal and most abnormal Hb variants.

Principle

At an alkaline pH, haemoglobin has a net negative charge. This makes it migrate from the
cathode (negative pole) to the anode (positive pole) when an electric field is applied.
Different mutations, leads to different amino acid substitutions in the haemoglobin molecule.
The different electrophoretic mobilities’ of abnormal haemoglobins are caused by change in
electrical charge due to different amino acid substitutions.

Any abnormal Hb that arises from mutations without any change in electric charge of the Hb
molecule shall migrate in the position of HbA. Hence, over 70% of abnormal
haemoglobins exhibit similar migration pattern to the same position as HbA.

At alkaline pH, electrophoretic migration of HbC, E, A2 and O is similar. Also, HbS, D, G

and Lapore co-migrate to the same position. These can only be separated by using
additional techniques that allow for their separation.

Alkaline electrophoretic reagents and materials

 Electrophoretic setup (power pack, 

tank)

 Cellulose acetate papers

 Applicator

 Samples haemolysate

 Control specimen

 Filter paper

 Tris-EDTA-Boric acid buffer

(pH 8.6)

 Staining solutions

 Clearing solutions
 Alkaline electrophoretic reagents preparation

Buffer1

Boric acid...................18.6g
Sodium hydroxide......3.4g
D H2O.........................1000ml

Buffer 2

Trishydroxymethylamine .....10.2g

Boric acid..............................3.2g

EDTA....................................0.6g
D H2O..................................1000ml

Working Buffer

Buffer 1.........................................1000ml

Buffer2..........................................400ml

D H2O...........................................500ml

Mix and store at 4°C. It can be used repeatedly without deterioration

Colourants

1. Ponceau
Ponceau Red S.....................................................5g
Trichloroacetic acid (5%)....................................1000ml

 Bleach reagent

5% Acetic acid

2. Amido black

Amido black 10B...................................................4.5g

Methanol................................................................405ml

Glacial acetic acid..................................................90ml

D H2O.....................................................................405ml

 Bleach reagent

Glacial acetic acid...................................................50ml

Methanol.................................................................475ml

D H2O......................................................................475ml

ACID ELECTROPHORESIS

BUFFER PREPARATION (pH 6.2)

Na2HPO4.............................................................................................2.02g
NaH2PO4.............................................................................................7.66g
DH2O...................................................................1000ml
Store in the refrigerator at 4°C

Agar gel phosphate

Agar-agar..............................................................500mg
Phosphate buffer (pH 6.2).....................................25ml

HAEMOLYSATE PREPARATION
 Collect about 5ml of blood in EDTA
 Centrifuge the sample at lower speed and discard the supernatant plasma
 Wash the blood cells 3X with physiological saline and discard supernatant
 Take 2volumes of washed cells and add 1volume of DH2O and mix vigorously to
lyse cells
 Add 1volume of carbon tetrachloride(CCL4), shake vigorously and centrifuge at
3000rpm for 30mins
 Transfer topmost layer of pure Hb into a clean tube and adjust the conc., to 4-6g/dl
with DH2O.
 The pure Hb can then be run on the electrophoretic machine or stored at -20oC for up
to 3months.

ELECTROPHORETIC RUN OF HAEMOLYSATE

 Disconnect the power supply of the equipment and fill the electrophoretic tank with
Tris-EDTA-Borica acid buffer (TEB buffer)
 Soak strips of cellulose acetate in the TEB buffer for 10-15mins
 Blot excess buffer with absorbent paper or filter paper
 Place the cellulose strip across the bridge of the tank with the dull absorbent surface
upwards.
 Using the applicator, apply the haemolysate approx. 1cm from the end of the strip
near the negative pole (cathode).
 Wait for a minute for samples to absorb into the strip
 Connect the power supply and run at 220-320v for 30- 60 mins or until a visible
separation is seen.
 Disconnect the power supply and remove the cellulose strip.

STAINING OF CELLULOSE STRIP

Staining procedure

1. Remove cellulose acetate strip at the end of run and stain in Ponceau S for 3 -5mins.
2. Decolourise in three changes of 5% acetic acid
3. Analyse the fractions and put strip in a clearing solution to remove excess stain.
4. Read test bands along the control samples ensuring to include all haemoglobin
bands no matter how small the bands may be.
For densitometry and prolonged preservation of strip

5. Dehydrate strips in methanol for 1min

6. Transfer to clearing solution for 40 sec.
7. Place strip on a flat glass surface
8. Dry in oven at 60°C for 2mins

Reporting Hb electrophoresis result

 List all Hb bands seen as phenotype, no matter how small, in the order of decreasing
concentration.

Example 1.

Hb Phenotype: FA or FAA2 or AF or AFA2 or AA2 or A.

Interpretation: no abnormal Hb seen. Result is consistent with normal Hb genotype

(A)or Hb disorders such as thalasaemias, in which no abnormal Hb is produced.

Specific genotype cannot be established by this technique alone, more specific studies
are required to confirm thalassaemia.

Example 2

Hb phenotype: FAS or AFS or ASFA2 or ASA2F or ASA2 or AS

 Interpretation: result is consistent with sickle cell trait (AS). The heterozygous
condition for the sickle cell gene.

Note (HbS, D and G co-migrate).

Sickling test must be reported along Hb electrophoresis result for presumptive

identification of HbS since only HbS sickles.

Example3.

Hb Phenotype: FS or SF or SFA2 or SA2F or SA2

Interpretation: result is consistent with sickle cell disease (HbSS) or the following
other genotypes S/beta zero thalassaemia (Sβo), S/delta-beta-zero thalassaemia
[S(δβ)o], or S/HPFH (hereditary persistent of foetal haemoglobin).

Example4.

Hb phenotype: FSA; SFAA2; SAFA2; SAA2F; SAA2

Interpretation: result is consistent with sickle cell disease S/beta plus thalassaemia
(Sβ+ Thal) (HbS,D,G co-migrate, confirm suspected SDG with other method).

 The age of the patient is very important in interpreting electrophoresis result.

 Newborns have large quantities of foetal HbF and small quantities of adult Hb

 Adults have less quantity of foetal HbF (< 2.0%)

 HbA2 is less than 3.4% in normal adults and co-migrate with HbC, E and O and is
seen as a light band on cellulose acetate in alkaline