Effects of Two Radiocontrast Dyes on the Detection of
Oligoclonal Gamma Globulins by High Resolution
Agarose Gel Electrophoresis
WAYNE R. WECKSLER, PH.D., MOHAMED D. EL-SHATORY, B.S., AND NICK S. HARRIS, PH.D.
The authors examined two radiocontrast dyes (iophendylate
and metrizamide) for their effects on a high resolution agarose
electrophoresis procedure for the detection of oligoclonal
gamma globulins. Each dye was incubated with cerebrospinal
fluid (CSF) and evaluated for effects on sample concentration
and on protein migration. Iophendylate interfered with sample
concentration but could be removed from CSF by centrifugation. Neither dye appeared to alter protein migration. (Key
words: Oligoclonal banding; Radiocontrast dye; Iophendylate;
Metrizamide) Am J Clin Pathol 1983; 79: 607-608
THE PRESENCE of oligoclonal gamma globulins in the
cerebrospinal fluid (CSF) has been taken as confirmatory
evidence in the diagnosis of multiple sclerosis.2"5 Since
workup of these patients frequently includes a myelogram, CSF samples which are contaminated by radiocontrast dyes are received occasionally in the laboratory.
Possible interference with the test for oligoclonal gamma
globulin bands could be caused by an alteration in the
viscosity of the CSF due to the presence of the dye or
by a change in the electrophoretic migration of the CSF
proteins due to dye binding or partial denaturation. We
have evaluated the effects of such contamination by the
two most commonly used dyes—iophendylate and metrizamide.
Bio-Science Laboratories, Van Nuys, California
rose gel (Panagel-16; Worthington Diagnostics, Freehold, NJ) using the application mask provided by the
manufacturer. The agarose gels were electrophoresed
using the Panagel Migration Unit (Worthington Diagnostics) as described by Gerson and colleagues.1 After
electrophoresis, the gels were fixed in picric acid (2.5%
picric acid in 33% acetic acid), dehydrated in denatured
alcohol (95% ethanol-5% methanol), stained with Comassie Blue R250 (0.4% in 45% methanol-10% acetic
acid) and destained in 40% denatured alcohol-10%
acetic acid.
Iophendylate U.S.P. [ethyl lO-(p-iodophenyl) undecanoate] was used as supplied from Lafayette Pharmaceuticals (Lafayette, IN). Metrizamide (2-[3-Acetamido2,4,6-tri-iodo-5(N-methyl-acetamido) benzamido]-2deoxy-D-glucose) was obtained from the Sigma Chemical Company (St. Louis, MO) and reconstituted in normal CSF to 350 mg/mL.
Results
Materials and Methods
Normal human CSF was obtained from Hemodynamics, Inc. (Los Angeles, CA) and stored at - 2 0 ° C .
A calibrator was prepared by diluting serum exhibiting
a fractionation pattern characteristic of a monoclonal
gammapathy to a concentration of 3.5 fig of monoclonal
protein per 10 nL jn deionized water. A control CSF
sample was prepared by spiking normal CSF with three
different sera exhibiting these monoclonal fractionation
patterns.
CSF samples were concentrated 80X by ultrafiltration
(Minicon CS 15, Amicon, Lexington, MA). Concentrated CSF samples (10 ixL) were spotted onto the agaReceived July 26, 1982; received revised manuscript and accepted
for publication September 13, 1982.
Address reprint requests to: Lois Mackey, Librarian, Bio-Science
Laboratories, 7600 Tyrone Avenue. Van Nuys, California 91405.
Two potential sources of interference by sample contamination with radiocontrast dye were considered. The
first was whether or not this contamination presented
technical problems which would hinder sample analysis.
The second was whether or not the dyes bound to CSF
proteins and thereby altered their migration properties
on agarose gels.
When normal CSF was mixed with iophendylate in
various proportions (10%, 25%, and 50% dye, V/V) and
transferred to ultrafiltration cells, concentration of the
mixture proceeded very slowly. In addition, contaminated samples, when applied to the agarose gels, did not
migrate from the application spot. Subsequently, it was
found that centrifugation of all samples (1,500 X g for
10 minutes) and examination for a dye-CSF interface
was a necessary step in sample preparation. This allowed
for easy identification of contaminated specimens and
facilitated separation of the dye from the sample. After
0002-9173/83/0500/0607 $00.90 © American Society of Clinical Pathologists
607
A.J.C.P.-May 1983
WECKSLER, EL-SHATORY, AND HARRIS
608
1
2
3
4
5
6
7
8
9
10
11
12
13
14 15
FIG. 1. Effects of radiocontrast dyes on CSF protein fractionation. Lanes 1, 7, and 12—calibrator. Lanes 2, 8, and 13—normal CSF. Lanes
3, 9, and 14—positive CSF control. lophendylate was overlayered onto normal CSF (lane 4) or positive control CSF (lane 5) and incubated as
described in the Results section prior to centrifugation, concentration, and electrophoresis. Metrizamide was overlayered but not mixed and
incubated with positive control CSF (lane 6). Metrizamide also was mixed with normal CSF (lane 10) and positive control CSF (lane 11) prior
to incubation, centrifugation, concentration, and electrophoresis.
removal of the dye, these CSF samples were found to
concentrate and electrophorese in a normal fashion.
Metrizamide is totally soluble in CSF, and contamination of CSF by metrizamide caused no technical problems in sample concentration or electrophoresis. It did,
however, dilute the specimens.
To test for changes in protein migration, 0.2-mL aliquots of each dye were overlayered with 3.0 mL of
normal CSF or positive control CSF. Some samples were
then mixed by inversion. All samples were incubated for
16-18 hours at 37°C and centrifuged as above. Supernatants from these samples (2.5 mL) were transferred
to concentration wells and processed as described in the
Materials and Methods section. Figure 1 shows the results of this study. There appeared to be no discernable
effect on CSF protein migration as a result of exposure
to the dye at 37°C for 16-18 hours.
In a separate experiment, the positive control was
mixed thoroughly with iophendylate and then incubated
and processed as above. Again, there was no discernable
effect on protein migration.
Discussion
Two radiocontrast dyes were examined as possible
sources of interference in the high resolution agarose
electrophoresis procedure to detect oligoclonal gamma
•globulins. One dye (metrizamide) was totally miscible
with CSF and appeared to cause no problems with sam-
ple preparation or protein migration. lophendylate was
not miscible with CSF and prevented sample concentration by ultrafiltration. However, when CSF samples
contaminated with iophendylate were centrifuged to remove the dye, the CSF samples concentrated and electrophoresed normally. Exposure of CSF to either dye for
16-18 hours at 37°C had no apparent effect on protein
migration.
We have adopted a procedure of centrifugation of all
CSF samples and examination for a dye-CSF interface
in order to identify those CSF samples with iophendylate
contamination. We have encountered no technical problems in utilizing the supernatants from these samples
in our oligoclonal banding procedure.
References
1. Gerson B, Krolikowski FJ, Gerson IM: Two agarose electrophoretic systems for demonstration of oligoclonal bands in cerebrospinal fluid compared. Clin Chem 1980; 26:343-345
2. Gerson B, Orr JM: Oligoclonal bands and quantitation of IgG in
cerebrospinal- fluid as indicators of multiple sclerosis. Am J
Clin Pathol 1980; 73:87-91
3. Johnson K.P, Arrigo SC, Nelson BJ, Ginsberg A: Agarose electrophoresis of cerebrospinal fluid in multiple sclerosis. Neurology
1977; 26:273-277
4. Laterre EC, Callewaert A, Heremans JF, Sfaello Z: Electrophoretic
morphology of gamma globulins in cerebrospinal fluid of multiple sclerosis and other diseases of the nervous system. Neurology 1970; 20:982-990
5. Link H, Moller E, Muller R, et al: Immunoglobulin abnormalities
in spinal fluid in multiple sclerosis. Acta Neurol Scand 1977;
suppl 55:173-191