Apparent Heparin Interference with Restriction Enzyme
Digestion of Genomic DNA, Dennis M. Todd and Frank
J. Buccini (GenCare Biomedical Research
Sheffield St., Mountainside, NJ 07092)
Corp., 271
The increased use of DNA probes to identify clonality in
lymphoproliferative
malignancies
presents
numerous
problems
associated
with colletio
techniques
and sample
stabilities. While analyzing blod and bone marrow samples submitted for T and B cell gene rearrangement analysis, we noticed that those samples submitted in Vacuminer Tubes”
(Becton Dickinson Corp., E. Rutherford, NJ)
containing
sodium
heparin as the anticoagulant
showed
aberrant restriction fragments
consistently
identifiable
from patient to patient. These patterns were not seen if the
anticoagulants
EDTA or acid-citrate-dextrose
(ACD) were
used in sample collection.
To determine whether indeed these aberrant bands were
caused by the anticoagulant,
we collected whole blood from
a healthy individual (shown through immunophenotyping
studies not to have any lymphoproliferative disorders) into
Vacutainer
Tubes containing
either potassium
EDTA, sodium hepann, or ACD. These anticoagulants
were chosen
because they are commonly used for immunophenotyping
or cytogenetic analysis and represent the anticoagulants
most frequently encountered in our laboratory.
Genomic
DNA was purified according to standard procedures (1) and
digested with a fivefold excess of the enzymes BamHI,
EcoPJ, and Hindffl according to the manufacturer’s
specifications (Promega Corp., Madison, WI). The digested DNA
3.5 kb. In Figure lb, the heparinized sample (lanes D, E,
and F) shows an aberrant fragment at -9 kb for the EcoRJ
digest (lane E), and the Hindffl digest (lane F) again shows
the typical pattern associated with a partial digestion. In
both cases, one can still see the expected
germline fragments. For the EDTA and ACD samples, one can see the
expected
germline banding patterns
for all three enzyme
digestions but, again, no indication of a partial digestion.
Similar patterns, indicating partial digestion of genomic
was split, and individual aliquots were size-fractionated on
0.8% agarose gels, then transferred
to ZetabindTM 0.45-&m
(pore size) nylon membranes (Cuno, Inc., Meriden, CT)
according
to the method of Southern
(2). Each blot was
individually hybridized with a nucleic acid probe, 32Plabeled by the method of Feinberg and Vogelstein (3),
which recognized either the joining region of the immuneglobulin
heavy chain gene or the constant
region of the T
cell receptor beta chain gene.
of the genomic DNA with BamHI, EcoPJ, and
followed by hybridization with the immunoglobulin heavy chain joining region probe, should yield, respecDigestion
Hindu,
tively, 18-, 17-, and 10-kb germline fragments. In Figure
la, the HindIII digest of the heparinized
sample (lane F)
shows a pattern typical of a partial digestion, i.e., numerous high-molecular-mass
fragments as well as a faint
germline band at 10kb. The BamHI digest (lane D) and the
EcoRI digest (lane E) show the expected
germline fragments with no indication of partial digestion. The BamHI
digests (lanes A, D, G, and J) also show a weak-intensity
band at -12 kb, which represents a weakly cross-reacting
fragment having partial homology with the probe. Similar
weakly cross-reacting bands at --3.5 kb are seen in the
Hindffl digests (lanes C, I, and L). In every instance,
the
EDTA and ACD samples gave the germline banding patterns with no evidence of partial digestion.
Upon hybridization
with the T cell receptor beta chain
gene, the BamHI digests should give one germline fragment at 24 kb; the EcoRI digests, two fragments at 11 and
4 kb; and the HindIll digests, three fragments at 8,6.5, and
362
CLINICAL CHEMISTRY, Vol. 39, No. 2, 1993
Fig. 1. (a) Autoradiographic endpoint of the analysis for the joining
region of the immunoglobulin heavy chain gene; (b) the same
samples digested, size-fractionated by electrophoresis in agarose,
transferred, and hybridized with a nucleicacid probe recognizing the
T cell receptor beta chain gene constant
region
MW representsa Lambda DNNHindIII size
marker (PromegaCorp.),which
yieldsfragmentsof 23/130,9416,6557,4361,2322, and2027 bp. Lanes A, 0,
G, and J represent the BsmHl digests of the genomic DNA. Lanes B, E, H.,
and K represent EcoRl digests, and lanes C, F, I,and L representtheHindlll
digests. A, B, C, 0, H. and I: whole-bloodsamples collected
in EDTA; 0, E,
and F:samplescollected
inpotassiumhepann;J, K, and L,samples collected
in ACD. The arrow (b) indicates
the aberrant9-kb band seen In the EcoRl
digest of samples collected with hepann as the anticoagulant
DNA, were obtained with EcoRJ and HindIll when we
probed the heparinized
bloods with the ber probe,
TransProbe-1TM
(Oncogene Science, Inc., Manhasset, NY).
Bgl II digests were unaffected by heparin (data not shown).
Apparently, heparin, when used as an anticoagulant
in
samples submitted for the molecular analysis of genes
associated
with lymphoid malignancies, causes the appearance of DNA fragments that could easily be confused with
rearrangements
and lead to an inappropriate conclusion of
clonality. The appearance and number of these aberrant
bands would obviously depend on the probes and restriction
enzymes used in these studies.
Attempts in our laboratory to reverse the effects of
heparin by increasing the concentrations of the restriction
enzymes, i.e., by using 10- and 20-fold excesses, repeated or
sequential digestions with 5- and 10-fold excesses of enzyme, or predialysis of the sample, have been unsuccessful.
We conclude that the anticoagulants of choice for molecular studies involving restriction enzymes and nucleic acid
hybridizations
are either sodium or potassium EDTA or
ACD. Heparin should be avoided.
References
1. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a
laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory, 1982:458-9.
2. Southern EM. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J Mol Biol 1975;98:
503-17.
3. Feinberg AP, Vogelstein B. A technique forradiolabeling DNA
restriction endonuclease fragments to high specific activity. Anal
Biochem 1984;137:266-7.
A Rapid, Automated
Method for Glycohemoglobin,
Laurence M. Demers (Depts. of Pathol. and Med., M. S.
Hershey Med. Center, The Pennsylvania State
University, Hershey, PA 17033)
The measurement
of glycohemoglobin
has become an
important
part of the care given to diabetic patients (1).
Historically,
affinity chromatography and ion-exchange
methods have been the mainstay of the technology for this
analyte (2). However, these methodologies, although much
improved, still have limitations from a practical point of
view. The batch analysis and manual methods that dominate this technology adversely affect the turnaround
time
for results for both outpatient clinic and hospitalized patients. In response to our diabetologists request that we
provide same-day clinic results for glycohemoglobin,
we
recently implemented an automated, affinity binding assay
(referenced to hemoglobin A1) available with the Abbott
Vision analyzer
(3). The method in pack form is based on
affinity binding of glycohemoglobin
to 3-aminophenylboronic
acid immobilized on a divided agarose support.
We compared results by this method (y) with those by an
affinity-chromatography
method (x; Bio-Rad, Hercules,
CA) in a study of 105 patients (y
0.44 + 1.OOx, standard
error of the estimate, 0.68) (Figure 1). We also compared
=
C
0
a
Blo-Rad
Fig. 1. Comparison between Bio-Rad affInity chromatograpny
methodand Abbott Vision test pack methodfor glycohemoglobinin
105 patients
fingerstick results (y) with results obtained from venipuncture samples (x) obtained from patients at the same time (y
=
0.30 + 0.98x, standard
error of the estimate, 0.27, n =
12). The fingerstick approach is particularly helpful with
pediatric patients when children have an aversion to venipuncture.
We found the Vision method to be more precise
than our chromatography method (4), with an interassay
CV of 3.3% at a mean glycohemoglobin
concentration of
5.3% (n = 10). The mean ± SD value for the Vision results
for the 105 patients was 7.4% ± 2.0%; that for the Bio-Rad
chromatography metho#{224}
was 7.0% ± 1.9%.
We conclude that the Abbott Vision test pack method for
glycohemoglobin
is accurate and precise and is capable of
providing rapid turnaround
of test results. Use of this
approach in our laboratory has allowed provision of glycohemoglobin results within the time frame of patients’ clinic
visits. Patients’ samples are drawn just before the clinic
visit and results are provided to the diabetic nurse practitioner to share with the patient at the time of the clinic
visit. This approach has markedly improved the relations
between clinical laboratory services and the diabetic clinic
in the treatment of diabetic patients.
References
1. Goldstein D, Little R, Wiedmeyer H, England J, McKenzie E.
Glycated hemoglobin: methodologies and clinical applications.
Clin Chem 1986;32(Suppl):B64-70.
2. Kienk DC, Hermanson GT, Krohn RI, Fujimoto EK, Mallia AK,
Smith PK, et al. Determination
of glycosylated
hemoglobin
by
affinity chromatography: comparison with colorimetric and ionexchange methods and effect of common interferences. Clin Chem
1982;28:2088-94.
3. Fiechtner M, Ramp J, England B, Knudson MA, Little RR,
England JD, et al. Affinity binding assay of glycohemoglobin by
two-dimensional centrifugation referenced to hemoglobin A1,,.Clin
Chem 1992;38:2372-9.
4. Wettre 5, Von Schenck H. Batch-to-batch imprecision in the
affinity chromatography assay of glycated hemoglobin. Clin Biochem 1986;19:364-6.
CLINICAL CHEMISTRY, Vol. 39, No. 2, 1993 363