College of Health Sciences

Department of Medical Laboratories

Second Year Second Term
Hematology 1
Practice N !"#
Cell$lose %cetate &lectrophoresis at %l'aline pH
Haemoglobin electrophoresis is used to separate and identify the different
haemoglobins by their migration within an electric field. Haemoglobin variants separate
at different rates due to differences in their surface electrical charge as determined by
their amino acid structure. Haemoglobin electrophoresis at pH 8.48.6 using cellulose
acetate membrane is simple, reliable, and rapid. It is satisfactory for the detection of
most common clinically important haemoglobin variants.

Principle
At alaline pH, haemoglobin is a negatively charged protein and when sub!ected to
electrophoresis will migrate toward the anode "#$. %tructural variants that have a change
in the charge on the surface of the molecule at alaline pH will separate from Hb A.
Haemoglobin variants that have an amino acid substitution that is internally sited may
not separate, and those that have an amino acid substitution that has no effect on
overall charge will not separate by electrophoresis.
Equipment

Electrophoresis tank and power pack. Any hori&ontal electrophoresis tan that will
allow a bridge gap of ' cm. A direct current power supply capable of delivering ()*
+ at )* mA is suitable for both cellulose acetate and citrate agar electrophoresis.
Wicks of filter or chromatography paper.
,lotting paper.

Applicators. -hese are available from most manufacturers of electrophoresis
e.uipment, but fine microcapillaries are also satisfactory.

Cellulose acetate membranes. /lastic0baced membranes "'.6 1 6.* cm$ are
recommended for ease of use and storage.
%taining e.uipment.
Reagents
1

Electrophoresis buffer. -ris234-A2borate "-3,$ pH 8.). -ris0
"hydro5ymethyl$aminomethane "-ris$, 6*.7 g, 34-A "disodium salt$, *.6 g, boric
acid, (.7 g, water to 6 litre. -he buffer should be stored at 489 and can be used up
to 6* times without deterioration.

Wetting agent. :or e5ample, ;ip0prep solution "Helena <aboratories$= 6 drop of
;ip0prep in 6** ml water.
Fixative/stain solution. /onceau %, ) g, trichloroacetic acid, '.) g, water to 6 litre.
estaining solution. (> "v2v$ acetic acid, (* ml, water to 6 litre.
!aemoly"ing reagent. *.)> "v2v$ -riton ?06** in 6** mg2l potassium cyanide.
Method

1. 9entrifuge samples at 67** g for ) min. 4ilute 7* @l of the paced red cells with
6)* @l of the haemoly&ing reagent. Ai5 gently and leave for at least ) min. If
purified haemolysates are used, dilute 4* @l of 6* g2dl haemolysate with 6)* @l of
lysing reagent.

2. With the power supply disconnected# prepare the electrophoresis tan by placing
e.ual amounts of -3, buffer in each of the outer buffer compartments. Bet two
chamber wics in the buffer, and place one along each divider2bridge support
ensuring that they mae good contact with the buffer.

3. %oa the cellulose acetate by lowering it slowly into a reservoir of buffer. <eave
the cellulose acetate to soa for at least ) min before use.

4. :ill the sample well plate with ) @l of each diluted sample or control and cover with
a )*0mm coverslip or a CshortD glass slide to prevent evaporation. <oad a second
sample well plate with ;ip0prep solution.

5. 9lean the applicator tips immediately prior to use by loading with ;ip0prep solution
and then applying them to a blotter.

6. Eemove the cellulose acetate strip from the buffer and blot twice between two
layers of clean blotting paper. 4o not allow the cellulose acetate to dry.

7. <oad the applicator by depressing the tips into the sample wells twice, and apply
this first loading onto some clean blotting paper. Eeload the applicator and apply
the samples to the cellulose acetate.

8. /lace the cellulose acetate plates across the bridges, with the plastic side
uppermost. /lace two glass slides across the strip to maintain good contact.
3lectrophorese at ()* + for 7) min.

9. After 7) min electrophoresis, immediately transfer the cellulose acetate to
/onceau % and fi5 and stain for ) min.

10. Eemove e5cess stain by washing for ) min in the first acetic acid reservoir and for
6* min in each of the remaining two. ,lot once, using clean blotting paper, and
leave to dry.
11. <abel the membranes and store in a protective plastic envelope.
Interpretation and Comments
:igure 67.( shows the relative electrophoretic mobilities of some common haemoglobin
variants at pH 8.) on cellulose acetate. %atisfactory separation of Hbs 9, %, :, A, and F
is obtained " :ig. 67.4 $. In general Hbs %, 4, and G migrate closely together as do Hbs
9, 3, and H
Arab
. 4ifferentiation between these haemoglobins can be obtained by using
2
acid agarose gels, citrate agar electrophoresis, H/<9, or I3:. However, there are slight
differences in mobility between Hbs %, <epore, and 4
/un!ab
and also between Hbs 9 and
3I optimi&ation of the techni.ue will facilitate detection of the difference. Generally, the
<epore Hbs and Hb 4
/un!ab
migrate slightly anodal to Hb % "i.e., they are slightly faster
than %$I Hb 9 migrates slightly cathodal to Hb 3 "i.e., it is slightly slower than 3$.
Separation of haemoglobins on Mylar-backed cellulose acetate membrane
(plate) by alkaline electrophoresis.
Courtesy of Helena BioSciences
%chematic representation of relative mobilities of some abnormal haemoglobins.
9ellulose acetate pH 8.).
3

Eelative mobilities of some abnormal haemoglobins. 9ellulose acetate pH 8.).
4
All samples showing a single band in either the % or 9 position should be analysed
further using acid agarose or citrate agar gel electrophoresis, H/<9, or I3: to e5clude
the possibility of a compound hetero&ygote such as %4, %G, 93, or 9H$
Arab
.
-he .uality of separation resulting from this procedure is affected primarily by both the
amount of haemoglobin applied and the positioning of the origin. Also, delays between
application of the sample and commencement of the electrophoresis, delay in staining
after electrophoresis, or inade.uate blotting of the acetate prior to application will cause
poor results. -his techni.ue is sensitive enough to separate Hb : from Hb A and to
detect Hb A
7
variants.
If an abnormal haemoglobin is present, the detection of a Hb A
7
variant band in
con!unction with the abnormal fraction is evidence that the variant is an J chain variant.
Globin electrophoresis at both acid and alaline pH is also useful in elucidating which
globin chain is affected. However, with the more ready availability of H/<9, it is less
often needed.
5
Bhen an abnormal haemoglobin is found, it may be of diagnostic importance to
measure the percentage of the variantI this can be done by the electrophoresis with
elution procedure for Hb A
7
estimation given on page (** . Kuantitation of Hb % is often
clinically useful, both in patients with sicle cell disease who are being treated by
transfusion and for the diagnosis of conditions in which Hb % is coinherited with J and L
thalassaemia, as outlined in -able 67.) . Kuantitation of Hb % can be done with H/<9,
electrophoresis with elution or by microcolumn chromatography.
Results of laboratory investigations in interactions of Hb S an ! or "
t#alassae$ia in aults
%&' ( S ( ) ( )
2
( *
A% M ()(8 676) N(.) N6
%% M 88O( * N(.) )6*
%2L8 thalassaemia < 88O( * P(.) )6*
%2L
#
thalassaemia < )*O( ((* P(.) 66*
%2H/:H M 6)8* * N(.) 7*()
A%2J
#
thalassaemia M2< 78() 67'* N(.) N6
A%2J8 thalassaemia < 7*(* 68'8 N(.) N6
%%2J thalassaemia M2< 88O( * N(.) 66*
A9+, mean cell volumeI M, normalI <, lowI H/:H, hereditary persistence of fetal
haemoglobin.




)garose +el ,lectro-#oresis
Agarose gels are commercially available as substitutes for both alaline and acid
separation systems. -hey are simple to use and particularly useful in laboratories
that process small numbers of samples.
Reagents and Method
-he manufacturerQs method should be followed.
Interpretation
Bith acid agarose systems, the principle of the test is the same as that of citrate
agar electrophoresis at the same pH, but it should be noted that there are
significant differences in mobility of some variant haemoglobins. Bith alaline
systems, in general the same separation patterns are obtained, but where individual

application notes are available these should be used for reference. ,ecause not all
its provide these, laboratories may need to build up their own data on nown
variants.
!