College of Health Sciences

Department of Medical Laboratories

Second Year – Second Term

Hematology – 1

Practice NO ( 8 )

Cellulose Acetate Electrophoresis at Alkaline 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 8.4–8.6 using cellulose acetate membrane is simple, reliable, and rapid. It is satisfactory for the detection of most common clinically important haemoglobin variants.

Principle

At alkaline pH, haemoglobin is a negatively charged protein and when subjected to electrophoresis will migrate toward the anode (+). Structural variants that have a change in the charge on the surface of the molecule at alkaline 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 horizontal electrophoresis tank that will allow a bridge gap of 7 cm. A direct current power supply capable of delivering 350 V at 50 mA is suitable for both cellulose acetate and citrate agar electrophoresis.

   Wicks of filter or chromatography paper.    Blotting paper.

  Applicators. These are available from most manufacturers of electrophoresis equipment, but fine microcapillaries are also satisfactory.

  Cellulose acetate membranes. Plastic-backed membranes (7.6 × 6.0 cm) are recommended for ease of use and storage.

   Staining equipment.Reagents

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Electrophoresis buffer. Tris/EDTA/borate (TEB) pH 8.5. Tris-(hydroxymethyl)aminomethane (Tris), 10.2 g, EDTA (disodium salt), 0.6 g, boric acid, 3.2 g, water to 1 litre. The buffer should be stored at 4°C and can be used up to 10 times without deterioration.

  Wetting agent. For example, Zip-prep solution (Helena Laboratories): 1 drop of Zip-prep in 100 ml water.

   Fixative/stain solution. Ponceau S, 5 g, trichloroacetic acid, 7.5 g, water to 1 litre.   Destaining solution. 3% (v/v) acetic acid, 30 ml, water to 1 litre.   Haemolyzing reagent. 0.5% (v/v) Triton X-100 in 100 mg/l potassium cyanide.Method

  

1.    Centrifuge samples at 1200 g for 5 min. Dilute 20 μl of the packed red cells with 150 μl of the haemolyzing reagent. Mix gently and leave for at least 5 min. If purified haemolysates are used, dilute 40 μl of 10 g/dl haemolysate with 150 μl of lysing reagent.

  

2.    With the power supply disconnected, prepare the electrophoresis tank by placing equal amounts of TEB buffer in each of the outer buffer compartments. Wet two chamber wicks in the buffer, and place one along each divider/bridge support ensuring that they make good contact with the buffer.

  3.    Soak the cellulose acetate by lowering it slowly into a reservoir of buffer. Leave

the cellulose acetate to soak for at least 5 min before use.

  4.    Fill the sample well plate with 5 μl of each diluted sample or control and cover

with a 50-mm coverslip or a “short” glass slide to prevent evaporation. Load a second sample well plate with Zip-prep solution.

  5.    Clean the applicator tips immediately prior to use by loading with Zip-prep

solution and then applying them to a blotter.

  6.    Remove the cellulose acetate strip from the buffer and blot twice between two

layers of clean blotting paper. Do not allow the cellulose acetate to dry.

  7.    Load the applicator by depressing the tips into the sample wells twice, and apply

this first loading onto some clean blotting paper. Reload the applicator and apply the samples to the cellulose acetate.

  8.    Place the cellulose acetate plates across the bridges, with the plastic side

uppermost. Place two glass slides across the strip to maintain good contact. Electrophorese at 350 V for 25 min.

  9.    After 25 min electrophoresis, immediately transfer the cellulose acetate to

Ponceau S and fix and stain for 5 min.

  10.  Remove excess stain by washing for 5 min in the first acetic acid reservoir and

for 10 min in each of the remaining two. Blot once, using clean blotting paper, and leave to dry.

   11.  Label the membranes and store in a protective plastic envelope.Interpretation and Comments

Figure 12.3 shows the relative electrophoretic mobilities of some common haemoglobin variants at pH 8.5 on cellulose acetate. Satisfactory separation of Hbs C, S, F, A, and J is obtained ( Fig. 12.4 ). In general Hbs S, D, and G migrate closely together as do Hbs C, E, and OArab. Differentiation between these haemoglobins can be obtained by using

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acid agarose gels, citrate agar electrophoresis, HPLC, or IEF. However, there are slight differences in mobility between Hbs S, Lepore, and DPunjab and also between Hbs C and E; optimization of the technique will facilitate detection of the difference. Generally, the Lepore Hbs and Hb DPunjab migrate slightly anodal to Hb S (i.e., they are slightly faster than S); Hb C migrates slightly cathodal to Hb E (i.e., it is slightly slower than E).

Separation of haemoglobins on Mylar-backed cellulose acetate membrane (plate) by alkaline electrophoresis.Courtesy of Helena BioSciences

  Schematic representation of relative mobilities of some abnormal haemoglobins. Cellulose acetate pH 8.5.

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Relative mobilities of some abnormal haemoglobins. Cellulose acetate pH 8.5.

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All samples showing a single band in either the S or C position should be analysed further using acid agarose or citrate agar gel electrophoresis, HPLC, or IEF to exclude the possibility of a compound heterozygote such as SD, SG, CE, or CO)Arab.

The quality 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 inadequate blotting of the acetate prior to application will cause poor results. This technique is sensitive enough to separate Hb F from Hb A and to detect Hb A2 variants.

If an abnormal haemoglobin is present, the detection of a Hb A2 variant band in conjunction with the abnormal fraction is evidence that the variant is an α chain variant. Globin electrophoresis at both acid and alkaline pH is also useful in elucidating which globin chain is affected. However, with the more ready availability of HPLC, it is less often needed.

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When an abnormal haemoglobin is found, it may be of diagnostic importance to measure the percentage of the variant; this can be done by the electrophoresis with elution procedure for Hb A2 estimation given on page 300 . Quantitation of Hb S is often clinically useful, both in patients with sickle cell disease who are being treated by transfusion and for the diagnosis of conditions in which Hb S is coinherited with α and β thalassaemia, as outlined in Table 12.5 . Quantitation of Hb S can be done with HPLC, electrophoresis with elution or by microcolumn chromatography.

   Results of laboratory investigations in interactions of Hb S and α or β thalassaemia in adults

  MCV % S % A % A2 % F

AS N 35–38 62–65 <3.5 <1

SS N 88–93 0 <3.5 5–10

S/β° thalassaemia L 88–93 0 >3.5 5–10

S/β+ thalassaemia L 50–93 3–30 >3.5 1–10

S/HPFH N 65–80 0 <3.5 20–35

AS/α+ thalassaemia N/L 28–35 62–70 <3.5 <1

AS/α° thalassaemia L 20–30 68–78 <3.5 <1

SS/α thalassaemia N/L 88–93 0 <3.5 1–10

MCV, mean cell volume; N, normal; L, low; HPFH, hereditary persistence of fetal haemoglobin.

        

Agarose Gel Electrophoresis

Agarose gels are commercially available as substitutes for both alkaline and acid separation systems. They are simple to use and particularly useful in laboratories that process small numbers of samples.

Reagents and Method

The manufacturer's method should be followed.

Interpretation With 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. With alkaline systems, in general the same separation patterns are obtained, but where individual

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application notes are available these should be used for reference. Because not all kits provide these, laboratories may need to build up their own data on known variants.

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