A n n a l s o f C l i n i c a l a n d L a b o r a t o r y S c i e n c e , Vol. 4, No. 4 Copyright © 1974, Institute for Clinical Science

Quantitation o f A2 Hem oglobin by Polyacrylam ide Gel D isc Electrophoresis: A Method with Individual Specim en Standardization

HENRY G. SCHRIEVER, M.D. AND DANUTE M. LEVECKIS, B.A.

Department of Pathology, John F. Kennedy Community Hospital,Edison, NJ 08817

ABSTRACT

A polyacrylamide gel electrophoresis technic for the determination of hemo­globin A2 is presented. Rapid separation is an advantage. The use of a diluted hemolysate as a 4 percent standard avoids overestimation of the A2 fraction by densitometry and also provides a reference for visual comparison. Normal range of A2 hemoglobin by this method is 1.12 to 4.13 g per dkg. Coefficient of variation was 5.84 percent.

In tro d u ctio n

The diagnosis of beta thalassemia minor can be established in three steps. The first step is measuring red cell indices to see if they are in the thalassemic range. The sec­ond is checking the blood smear for the red cell targeting and basophilic stippling which are typical of the condition. The third and final step is measurement of the A2 hemo­globin fraction.12’13 Elevation of A2 hemo­globin is specific for beta thalassemia minor, with the possible exception of pernicious anemia14 which is easily excluded by mor­phology and red cell indices.

Methods for the determination of A2 hemoglobin must provide for both separa­tion and quantitation. Electrophoresis on starch gel,19 starch block,15 polyacrylamide gel,3’5 cellulose acetate,2’8’9’16’17’18 paper4’11 and agar gel20 have been used for separa­tion. Column chromatography has also been used.1’7’10 Quantitative comparison of the two fractions is usually accomplished by

either direct densitometry or elution and colorimetry, with A2 hemoglobin being re­ported as a percent of total hemoglobin. Many of the methods are either time-con­suming or inaccurate or both.

M ethod

The method given here has been used for three years in the diagnosis of more than 100 cases of thalassemia minor. It has two main advantages. The separation by poly­acrylamide gel disc electrophoresis is rapid and distinct. A 4 percent dilution of each original hemolysate is used as a standard for comparison with the A2 band. This avoids the densitometric comparison of a tiny A2 band with a huge Ax fraction, which tends to give false high values for hemo­globin A2. A third advantage is that with the use of the 4 percent “standard”, ele­vated A2 fractions can usually be spotted easily by inspection if densitometry is not available.

H B QUANTITATION B Y PO LYA CRYLA M ID E G E L DISC ELECTRO PH O RESIS 2 5 1

P r i n c i p l e

The normal hemoglobin fractions Ax and A2 are separated by electrophoresis in poly­acrylamide gel, in about 20 minutes. Once clearly separated, the A2 fraction is quanti­tated by densitometry. Two gel columns are electrophoresed for each A2 determina­tion. The extra column carries a 4 percent dilution of the original hemolysate which serves as a standard for densitometric com­parison to the A2 peak.

R e a g e n t s 6

Temed solution. To 0.12 ml of NNN’N’ tetramethylethylene-diamine ( Canalco #204) are added 18.1 g of 2-amino-2- hy- droxymethyl-3-propandiol ( tris) ( Canalco #210), 24 ml of 1 N HC1 and enough dis­tilled water to make 100 ml. The pH range is 8.8 to 9.0. The solution is stabile in­definitely at 4°.

Acrylamide solution. To 28.0 g of acryla- mide monomer ( Canalco #201) are added0.735 g of N,N’methylenebisacrylamide (b is) (Canalco #202) and enough distilled water to make 100 ml. The solution is stable indefinitely at 4° in an amber bottle.

Catalyst solution. To 0.14 g of ammonium persulfate (Canalco #209) are added enough distilled water to make 100 ml. Store solution at 4 ° and make fresh monthly.

Upper gel component 1. To 48 ml 1 of N HC1 are added 5.98 g tris, 0.46 ml temed and enough distilled water to make 100 ml. Adjust the pH to 6.6 to 6.8. The solution is stabile indefinitely at 4°.

Upper gel component 2. To 20.0 g of acrylamide are added 5.0 g bis and enough distilled water to make 100 ml. The solu­tion is stabile indefinitely at 4°.

Riboflavin solution. To 0.004 g of ribo­flavin (Canalco #208) are added enough distilled water to make 100 ml. Store solu­tion at 4°.

Sucrose solution. To 40 g of sucrose are

added enough distilled water to make 100 ml. Solution is good for one month at 4°.

Upper gel solution. To one part compo­nent 1 are added one part component 2, one part riboflavin solution, four parts su­crose solution and one part distilled water. The solution is stabile indefinitely at 4°.

Glycine buffer. To 6.0 g of tris are added 28.8 g of glycine (Canalco #203) and enough distilled water to make one liter. The solution is stabile for approximately six months at 4 °.

Saponin solution. Zap-Isoton (Coulter Electronics, Hialeah F L ).

Chloroform. AR grade.

S p e c i a l A p p a r a t u s

The necessary equipment includes:(1 ) Disc electrophoresis cell with stacking

buffer chambers;*(2 ) Power supply capable of supplying 60

ma current;(3) Densitometer and integrator capable

of scanning gel columns;!(4 ) Glass Columns; 8 X 60 mm, o.d. cut

from glass tubing;(5) Polymerization rack,! or equivalent

constructed from 7 ml Vacutainer stop­pers glued to a solid base;

(6 ) Disposable plastic syringe, 10 cc, fitted with curved #18 blunt ended lumbar puncture needle and

(7) Micropipet to deliver 0.025 ml.§

P rocedureM a k i n g t h e H e m o l y s a t e

Two to seven ml anticoagulated whole blood (EDTA, heparin or citrate) are cen­trifuged at 2500 rpm for five minutes; the plasma and buffy coat are aspirated and discarded. The red cells are washed three

* Model 1200 bath with #1807 Safety interlock adapter, Canalco, Rockville, MD 20852.

f Densicord and Integraph, Photovolt Corpora­tion, New York, NY 10010.

t Canalco #1812.§ Unimetrics, Anaheim, CA 92801.

SCH RIEV ER AND LEV ECKIS

times with normal saline solution, centrifug­ing and discarding the washes each time. One drop of Zap-Isoton is added to the packed cells and the mixture is shaken vigorously for one minute.

One-half ml of chloroform is added to the mixture which is shaken vigorously for one minute and then centrifuged at 2500 rpm for 15 minutes. The supernatant hemo- lysate is then aspirated and transferred to a clean test tube. If not sparkling clear, the procedure is repeated. The hemoglobin con­centration should be 18 to 25 gm per dl. A4 percent “standard” hemolysate solution is prepared by adding 0.1 ml of the original hemolysate to 2.4 ml 40 percent sucrose solution in a test tube.

M a k i n g t h e G e l C o l u m n s 5

One part Temed solution, one part acry- lamide solution and two parts catalyst solu­tion are drawn into a 10 ml syringe through the curved needle. The solutions are mixed, handwarmed and degassed by pulling a vacuum in the syringe with a finger over the outlet. Two glass columns for each specimen are placed in the polymerization rack and one ml of gel solution is added.

Distilled water is carefully layered over the gel mixture using a disposable Pasteur pipet. No mixing of gel and water should

occur. The columns are allowed to stand for 20 to 30 minutes for gelling to occur. Columns may now be capped with poly­ethylene film and stored at 4°. When the columns are to be used, the water layer is shaken off, 0.15 ml upper gel solution is added and layered with water. The columns are placed in ultraviolet, fluorescent or sun­light for polymerization, which takes about20 minutes.

E l e c t r o p h o r e s i s

The water layer on the columns is shaken off and the columns are filled with glycine buffer. With the long-tipped micropipet0.015 ml hemolysate is carefully layered on top of the upper gel so that it forms a thin band. The same is done for the 4 percent “standard” hemolysate on a separate col­umn (figure 1). The columns are inserted in the disc electrophoresis cell, the cham­bers filled with glycine buffer and electro- phoresed at 5 ma per column until the Ax and the slower moving A2 fractions are separated by two mm (figure 2) which takes aproximately 20 minutes.

D e n s i t o m e t r y

The columns are removed and wiped clean with tissue. The densitometer is set for log-linear response (L5 on the Densi-

F i g u e e 1. Gel columns ready for electrophoresis. Hemolysate has been lay­ered under the glycine buffer on top of the upper gel. The greater density of the hemolysate retards diffusion and keeps it a thin band.

H B QUANTITATION B Y POLYACRYLAM IDE G E L DISC ELECTRO PH O RESIS 253

F i g u r e 2. Full strength hemolysate and diluted 4 percent “standard” on left after 20 minute elec­trophoresis. The hemoglobin A» band on right is much more intense than the band of the standard, indicating thalassemia minor.

cord). One tracing is run for the 4 percent “standard.” Two tracings are run for each full strength hemolysate, one with zero set on clear gel and one with zero set between the Ai and A2 peaks. This is to correct for trailing of the Ax fraction.5

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F ig u r e 3. Densitometric tracings of the A» peak (1 ) , the Aa peak with zero set in the valley be­tween Ai and A= bands (2 ) , and the 4 percent “standard” (3 ) . Calculation consists of averaging the tooth count for 1 and 2 and comparing with 3. Result of 2.3 percent is in normal range.

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F i g u r e 4. Densitometric tracings in thalassemia minor. Total tooth count of As band (1 ) and A2 band with zero set in Ai-A2 valley (2 ) is 95, the average of which is 47.5, giving a 5.0 g per dkg A2 hemoglobin result when compared to the total tooth count of 38 for the 4 percent “stan­dard” (3 ) .

C a l c u l a t i o n

The integrator marks (teeth) under the4 percent “standard”, and each of the two tracings of the fullstrength A2 fraction are counted. The average tooth count of the two tracings of the A2 fraction is deter­mined and compared with the 4 percent standard ( figure 3 and 4).

average teeth standard teeth

X 4Percent A2 =

Sources o f Error

B a d H e m o l y s a t e

Contamination with red cell stroma will cause a fuzzy separation. Re-extraction, however, with chloroform will correct this. Hemolysates are generally stabile at 4 ° for months, but they may deteriorate to a brownish color and give poor separation.

D e l a y i n E l e c t r o p h o r e s i s

After adding the glycine buffer, the hemolysates should be layered immediately and electrophoresed without delay. If the

2 5 4 SCH RIEV ER AND LEV ECK IS

buffer is permitted to diffuse into the gel, fuzzy separation will result.

F a in t B ands

The standard and A2 bands should be dense enough to give a fairly high tooth count on the integrator (30 to 90) to avoid the inaccuracy inherent in low numbers. Amount of hemolysate added to the col­umns may be adjusted.

N orm al V alues

In 72 normal patients, the mean value of A2 hemoglobin was 2.62 g per dkg ± 0.75 giving a 95 percent range of 1.12 to 4.13. In 86 patients with thalassemia minor, the mean was 6.2 g per dkg and the A2 hemo­globin had a range of 4.2 to 11.5.

P recision

In 19 replicate determinations, the mean was 3.84 g per dkg ± 0.225, giving a coeffi­cient of variation of 5.84 percent.

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18. S h e e n a , A. H., Fox, F . A., B a y h a , M., a n d S t e v e n s , K. M.: A simple microtechnic for screening abnormal hemoglobins and for quantitation of A2 hemoglobin by electro­phoresis on cellulose acetate. Amer. J. Clin. Path. 5 0 :142-145, 1968.

19. S u n d e r m a n , F. W. J r . : Electrophoretic identi­fication of hemoglobins. Hemoglobin, Its Pre­cursors and Metabolites. Sunderman, F. William and Sunderman, F. William, Jr., eds., Lippincott, Philadelphia, pp. 94-108, 1964.

20. Y a k u l i s , V. J., H e l l e r , P., J o s e p h s o n , A. M., a n d S i n g e r , L .: Rapid demonstration of A2

hemoglobin by means of agar gel electro­phoresis. Amer. J. Clin. Path. 34:28-34, 1960.