CLIN.CHEM. 24/6,936-937 (1978)
Hemoglobir
Electrophoresisat Alkaline pH on Agarose Gels
Cyrus A. Lepp and Barry I. Bluestein
We describe a modified thin agarose-gel system for use
in primary hemoglobin screening by electrophoresis. The
system makes use of a Tris-EDTA-borate-buffered
aga-
rose gel (pH 8.8) and a sodium barbital electrophoresis
buffer (pH 8.6). Separation is effected in 20 mm at a constant potential of 250 V. Eight samples can be simultaneously separated and the patterns made visible in 47 mm.
There are fewer operative steps needed in running and
staining. Normal and the more common abnormal hemoglobins are separated into easily visualized and differentiated bands, with the separation of HbA and F significantly
improved
over that attainable
by present methods.
Additional Keyphrases: screening
pathies
for
hemoglobino-
separation of Hb A and Hb F
Screening for hemoglobinopathies
has become very important within the past five years. Many hospitals now screen
all incoming surgical patients for such abnormalities.
Electrophoresis on cellulose acetate is the primary procedure used
in the Federal Sickle Cell Disease Program (1). Consequently,
cellulose acetate electrophoresis at pH 8.4-9.2 (2) currently
is the most common electrophoretic screening method for
hemoglobinopathies. However, Hicks and Hughes (3) have
found that cellulose acetate, although used as a primary
screening
procedure, is not acceptable
in identifying
FS, FA,
F-fl thalassemia, and S-fl thalassemia
patterns. Thus, in
questionable cases, one must proceed to a secondary procedure such as electrophoresis on citrate agar to obtain a more
definitive diagnosis. Wieme (4) contends that no satisfactory
separations of hemoglobins can be obtained on any medium,
except at acid pH.
A screening system involving agarose gel electrophoresis
is currently marketed by Corning Medical (Corning Glass
Works, Medfield, Mass. 02052), which separates hemoglobins
at pH 8.6 by use of a discontinuous buffer system consisting
of 2-amino-2-methyl-l-propanol.
This method separates Hb
FA and FAS1 with approximately the same resolution as cellulose acetate methods and requires 45 mm of separation
time.
In this report, we describe a modified agarose gel and buffer
system, which can be used with Corning Medical apparatus.
This procedure discretely separates Hb F, A, 5, and C and can
be used as a primary electrophoretic screening method that
takes only 20 mm for adequate resolution.
Materials and Methods
Buffer.
Tris-EDTA-borate
Corning
Glass
Works,
Research
buffer (pH 8.8 at 23 #{176}C,
ionic
& Development
Laboratories,
Corning, N.Y. 14830.
I Nonstandard
abbreviations
used: Tris, tris(hydroxymethyl)methylamine; EDTA, ethylenediaminetetraacetic
acid (or -acetate);
and Hb, hemoglobin(s).
Received Dec. 19, 1977; accepted Mar. 24, 1978.
936
CLINICALCHEMISTRY,Vol. 24, No. 6, 1978
strength 0.78) was prepared by dissolving 6.44 g of Tris base,
0.62 g of EDTA, and 0.4 g of boric acid in 900 ml of de-ionized
glass-distilled
water. The pH was checked, adjusted with
saturated boric acid, and the volume made up to 1 liter. This
buffer was used to prepare the gel.
Agarose.
Gel was prepared by adding 1.0 g of agarose
(Seakem; Marine Colloids, Inc., Rockland, Maine 04841), 5.0
g of sucrose, and 15 mg of polyvinylalcohol
(99% hydrolyzed;
Matheson, Coleman and Bell) to 100 ml of Tris-EDTA-borate
buffer. The mixture was heated, with stirring, in a boiling
water bath for 30 mm after total dissolution of the components
had occurred.
Preparation of gel. Empty cassette molds were obtained
from Corning Medical and used to form the thin gels. The gel
solution was cooled to 65-70 #{176}C
before being injected into the
lower nipple of the mold with a glass syringe to which a piece
of plastic tubing, 3 mm in diameter, was attached. The mold
was held in an upright position during pouring to facilitate
removal of air from the upper nipple. After injection of about
5 ml of gel, the cassette mold was placed on a flat bench top,
and a flat weight of about 500 g was placed on the molds to
expel any excess gel. After the gels set (about 5 mm at room
temperature), they were wrapped in stretch plastic and aluminum foil and stored at 4 #{176}C
for 24 h before use. The gels
were stable for at least six months if kept tightly sealed.
Alternative
gel preparation
method.
A satisfactory alternative method for preparing
an alkaline agarose gel is the
following.
A Corning Medical Agarose Universal
Electrophoresis Film (cat. no. 470102) is pressed between a hard flat
surface and three “Sta-Moist”
papers (cat. no. 470158) for
5-10 mm under a 500-g flat weight. The dehydrated
film is
then soaked for 10 mm in the gel buffer just described. The
gel buffer contains, additionally,
50.0 g of sucrose and 150mg
of polyvinylalcohol
in 1 liter. This rehydrated film is drained
thoroughly of excess buffer. Sample wells are blotted dry with
facial tissue paper.
Electrophoresis.
For the separations we used a Corning
Medical electrophoresis
cell connected
by leads to a Model
3-1155 regulated power supply (Buchler Instruments,
Fort
Lee, N.J. 07024), rather than the fixed-voltage
Corning
Medical power supply. Hemolysates
were prepared
according
to the Corning Medical Electrophoresis
Operations
Procedures Manual.2 One microliter
of hemolysate was applied to
each sample well.
Each well of the cell base was filled with 75 ml of 4#{176}C
running buffer (50 mmol/liter
sodium barbital containing 0.35 g
of disodium EDTA per liter, adjusted to pH 8.6 with 2 mol/
liter HCI). Gels were placed in the gel holder-cover and set into
the cell base so the gel edges were immersed in buffer. A constant potential of 250 V was applied for 20 mm.
Visualization.
Gels were stained after separation by immersion in Amido Black stain for 5 mm. The stain consisted
2
Corning
Medical
Electrophoresis
Corning Glass Works, Medfield,
Operations
Mass.
Procedure
Manual,
Table 1. Comparison of Operational Times for
Other Hemoglobin Separation Systems vs.
Corning Medical (CM) System a
Lenath of tIme (minI
-
A
F
S
-
C
-
-
OperatIon
-
Membrane
Poured
CM
Soaked
CM
Helena
none
20
20
10
2
20
5
20
5
20
10
40
10
60
10
Gelman Beckman
preparation
-
-
Run
Stain
Rinse
ORIGlN
2
Drying
Clearing
Total
2
2
2
2
20
none
20
none
15
3
15
3
15
3
47
67
70
80
92
a Times are taken from operation instructions available from these menu-
facturers.
Fig. 1. Facsimile of separation patterns of different hemoglobin
phenotypes on Tris-EDTA-borate agarose gel
Left to right: AC, FS (trace A), ASC, FASC control (Center for Communicable
Disease), SC, FAS, cord blood FA, A
I
ORIGIN
tt.
distinct advantages
in primary screening proce-
provides
dures. The total process time (Table 1) from preparation
of
the agarose film to final drying is only 47 mm (70 mm if the
U-
-+
Fig.2. Hemoglobins separated by the alternative
gel method
Left to right: normal hemoglobin (tracks 1 and 2), cord blood (tracks 3 and 4),
5 and 6), C trait (tracks 7 and 8)
sickle cell trait (tracks
of Amido Black lOB (Sigma Chemical
Co., St. Louis, Mo.
63178), 2.0 g, dissolved
in dilute acetic acid (50 ml of glacial
acid and 950 ml of water). The gel was then rinsed in the dilute
acetic acid and dried in a Corning Medical drying oven for 20
mm. The dried gel was completely destained in the dilute
acetic acid and rinsed in fresh destaining solution to completely remove background color.
Results and Discussion
The quality of the separations that can be achieved by this
in Figures 1 and 2. Normal hemoglobin
and the main hemoglobin
variants are clearly resolvedespecially F, which is cleanly separated
from A and S in FAS
and FA. Newborn phenotypes
and adult phenotypes,
where
relatively
small amounts
of Hb F are present, can therefore
be run on this gel.
It was necessary to use 250 V to achieve adequate separation
method is illustrated
of Hb F from Hb A. When this gel was run in the Corning
Medical system at 90 V for 1 h, F and A could not be resolved.
The increased ohmic heating caused by the higher voltage does
not appear to damage the gel or hinder separation in any way;
cooling made no significant difference in the separation,
did lengthen separation time.
This improved
methodology
over the use of cellulose
acetate
but
alternative
procedure is used). It requires no presoaking immediately before a run as does cellulose acetate (presoak gels
can be stored for up to one week), nor does it require clearing
and multiple rinse steps afterwards.
Uniform patterns can be
obtained from batch to batch of gel prepared with agarose of
known electroendosmosis
characteristics,
unlike cellulose
acetate, which is notably non-uniform
between batches. In
addition, the patterns obtained are larger than on cellulose
acetate and, therefore, less prone to mistakes in interpretation.
Preparation
of gels is simple and requires only 2 h. Large
batches of gels can be prepared and are stable for at least a
year without deterioration.
By the alternative procedure, only
20 mm is required
to prepare a gel before a separation.
Additionally, these gels can be stored for at least a week in a moist
chamber
before use.
The modified agarose gel system presented here meets the
criteria for an optimal electrophoretic
system stated by LeCrone et al. (5). It clearly differentiates
hemoglobin
variants
with a minimum
of time, effort, and materials.
We are grateful for the assistance of Pam Pasakarnis and Carolyn
Steinberg of the Technical Consultation Center, Corning Medical
Division, Medileld, Mass., in supplying some of the abnormal hemoglobins for our studies and for their thoughts on hemoglobin
electrophoresis.
We are also grateful to Ms. Eve Blake, Chief of the
Biochemistry
Laboratory
of the Georgia Department
of Human Resources, Atlanta,
Ga., and her staff for supplying
us with many abnormal hemoglobin
samples for our studies.
References
1. Schmidt, R. M., Laboratory diagnosis
Am. Med. Assoc. 224, 1276 (1973).
2. Kohn, J., Separation
Pathol.
22, 109 (1969).
of hemoglobins
of hemoglobinopathies.
on cellulose
acetate.
J.
J.
Clin.
3. Hicks, E. J., and Hughes, B. J., Comparison of electrophoresis
on
citrate agar, cellulose acetate, or starch for hemoglobin identification.
Clin. Chem. 21, 1072 (1975).
4. Wieme, R. J.. Agar Gel Electrophoresis,
Elsevier,
Amsterdam,
1965, p 192.
5. LeCrone, C. N., Broszeit, H. K., Jones, J. A., and Detter, J. C.,
Adaptation
trophoresis.
of the acid citrate-agar
gel method
Clin. Chem. 22, 1743 (1976).
for hemoglobin
CLINICAL CHEMISTRY, Vol. 24, No. 6, 1978
elec-
937