Effect of methadone on human pregnancy tests

Abstract

The results of studies on 309 samples of urine from 103 patients taking methadone as tested by 4 commercially available pregnancy tests are reported. Urine specimens were subjected to 2 hemagglutination-inhibition tube bests (UCG and Pregnosticon) and 2 latex agglutination-inhibition slide tests (Cravindex and Pregnosticon). Thin-layer chromatography was done on Silpate-22 plates and various narcotics and other drugs were identified by standard procedures. Most of the problem specimens were from patients on high doses of methadone 100 mg or more a day. False positive or inconclusive test results were recorded with the Gravindex slide test on 18% of specimens and with the Pregnosticon slide test on 3.6% of specimens. Using the tube hemagglutination tests only a single false positive test was obtained from 309 specimens. Results suggest that methadone or its metabolites could be resonsible for the inhibition of latex particle agglutination giving the false positive results. Associated proteinuria in 20% of urine specimens may also have been a factor. Since completion of this study in 1971 a new Gravindex test has become available It may eliminate the false positive tests in most cases. The accuracy was 99.7% for the Pregnosticon and UCG tube tests 96.4% for the Pregnosticon slide test and 81.2% for the Gravindex slide test.

Full text

EFFECT
OF
METHADONE
ON
HUMAN
PREGNANCY
TESTS
C.
A.
HORWITZ,
R.
MASLANSKY,
R.
WALDINGER,
H.
CABRERA
and
P.
C.
J.
WARD
Mount
Sinai
Hospital,
Minneapolis,
Minnesota
55404,
and
Mount
Carmel
Hospital,
Columbus,
Ohio,
U.S.A.
{Received
27th
May
1972)
Summary.
A
prospective
study
of
four
commercially
available
preg-
nancy
tests
was
performed
on
309
urine
specimens
specifically
selected
from
patients
in
a
methadone
study
group.
The
accuracy
of
the
various
tests,
based
on
the
total
number
of
correct
test
results,
was
99\m=.\7%
for
the
Pregnosticon
\s=r\
and
UCG\s=r\
tube
tests,
96\m=.\4%
for
the
Pregnosticon
\s=r\
slide
test
and
81\m=.\2%
for
the
Gravindex\s=r\
slide
test.
Analysis
of
the
test
data
revealed
that
most
of
the
false
positive
test
results
were
recorded
on
specimens
received
from
patients
on
100
mg
methadone,
or
more,
per
day.
Semiquantitative
thin-layer
chromatography
suggested
that
methadone
metabolites
could,
at
least
in
part,
be
responsible
for
the
inhibition
of
latex
particle
agglutination
and
incorrect
test
results.
INTRODUCTION
In
a
recently
completed
comparative
study
of
pregnancy
tests,
only
one
false
positive
test
result
was
recorded
with
a
latex
agglutination-inhibition
slide
test
on
unselected
specimens
without
Labstix
©-detectable
blood
or
protein
(Horwitz,
Garmezy,
Lyon,
Hensley
&
Burke,
1972).
This
specimen
was
received
from
a
22-year-old
female
heroin
addict
in
a
methadone
therapy
group
taking
130
mg
methadone
per
day.
It
was
also
noted
in
testing
some
specimens
from
non-pregnant
patients
on
methadone
that
there
was
a
decrease
in
the
usual
boldness
of
latex
particle
agglutination.
The
apparent
interference
with
the
test
endpoint
raised
questions
regarding
the
effect
of
drugs
in
general,
and
of
methadone
in
particular,
on
the
various
slide
agglutination-inhibition
pregnancy
tests.
In
order
to
gain
more
information
about
these
findings,
we
undertook
a
study
on
samples
specifically
selected
from
a
methadone
study
group.
It
became
apparent
that
false
positive
or
inconclusive
test
results
could,
at
least
in
part,
be
related
to
the
presence
of
methadone,
or
methadone
metabolites,
in
the
urine.
*
Presented
at
the
Fourth
National
Methadone
Conference,
9th
January
1972,
San
Francisco,
California,
U.S.A.
Address
for
reprints
:
C.
A.
Horwitz,
m.d.,
Mount
Sinai
Hospital,
2215
Park
Avenue,
Minneapolis,
Minnesota
55404,
U.S.A.
489

490
C.
A.
Horwitz
et
al.
MATERIALS
AND
METHODS
The
test
material
consisted
of
309
urine
specimens
obtained
from
103
different
patients
(seventy-seven
males
and
twenty-six
non-pregnant
women)
in
a
methadone
study
group.
Three
specimens
were
collected
from
each
patient.
The
patients
were
reportedly
taking
from
10
to
240
mg
methadone
per
day.
No
specimen
was
excluded
from
this
study
because
of
proteinuria,
haematuria
or
hyposthenuria.
The
urine
specimens
were
subjected
to
two
haemagglutination-
inhibition
tube
tests
(UCG®
and
Pregnosticon®)
and
two
latex
agglutination-
inhibition
slide
tests
(Gravindex®
and
Pregnosticon®).
All
procedures
were
carried
out
as
directed
by
the
manufacturers.
Thin-layer
chromatography
was
performed
on
Silpate-22
plates
(Brinkman
Instruments
Incorporated,
Westbury,
New
York),
and
various
narcotics
and
other
drugs
were
identified
by
a
standard
procedure
(Davidow,
Li
Petri
&
Quame,
1968).
Iodoplatinate
and
Dragendorff
reagents
were
used
to
achieve
colour
development
for
identification
of
methadone
and/or
methadone
by¬
products.
A
methadone
spot
was
recorded
if
a
visible
spot
with
an
RF
value
and
colour
corresponding
to
that
of
a
methadone
control
was
identified
on
the
chromatogram.
RESULTS
In
Table
1,
false
positive
and
inconclusive
test
results
are
recorded
in
relation
to
four
patient
groups
(A
to
D)
delineated
by
the
size
of
the
daily
dose
of
methadone.
Accuracy
(%)
is
expressed
in
terms
of
correct
negative
test
results
(Table
1).
At
the
conclusion
of
the
above
study,
thirty-five
coded
problem
Table
1.
False
positive
and
inconclusive
test
results
obtained
on
309
selected
specimens
from
103
patients
receiving
methadone
Division
of
specimens
by
daily
methadone
dosage
No.
of
specimens
Gravindex9
slide
test*
FP
IC
Accuracy^
Pregnosticon'1'
slide
test*
FP
IC
Accuracy
Tube
tests*
(UCG®
and
Pregnosticon9)
FP
IC
Accuracy
Group
A
:
200
to
240
mg
methadone/day
(eighteen
patients)
Group
:
150
to
190
mg
methadone/day
(twenty-five
patients)
Group
C:
100
to
140
mg
methadone/day
(thirty
patients)
Group
D
:
<
100
mg
metha¬
done/day
(thirty
patients)
54
75
90
90
64-8%
74-7%
83-3%
94-4%
90-7%
94-7%
98-9%
98-9%
100-0%
100-0%
98-9%
100-0%
309
53
81-2%
10
1
96-4%
1 1
99-7%
Abbreviations:
FP
(false
positive)
and
IG
(inconclusive)
test
results.
*
Gravindex®
(Ortho
Diagnostics,
Raritan,
New
Jersey),
Prognisticon®
slide
and
tube
tests
(Organon
Inc.,
West
Orange,
New
Jersey),
UCG®
(Wampole
Laboratories,
Stamford,
Connecticut),
f
Accuracy—based
on
the
percentage
of
correct
'negative'
test
results.

Methadone
and
human
pregnancy
tests
491
specimens
were
sent
for
independent
appraisal
to
Columbus,
Ohio,
where
the
immunoassays
were
repeated
by
one
of
us
(H.C.).
There
was
agreement
on
32/35
specimens
when
test
results
were
compared
2
to
3
weeks
later.
Thin-layer
chromatography
performed
on
'undiluted'
urines
from
the
Methadone
Clinic
revealed
the
presence
of
drugs
other
than
methadone
in
some
specimens.
As
compiled
from
413
consecutive
specimens
submitted
from
the
Methadone
Clinic
between
1st
May
1971
and
1st
August
1971,
the
drugs
detected
in
this
patient
population
included
Librium,
15-5%
(64/413);
amphetamine,
5-8%
(24/413)
;
morphine,
3-4%
(14/413);
pentabarbital,
3-1%
(13/413);
phénobarbital,
1-7%
(7/413);
codeine,
1-2%
(5/413);
quinine,
1-2%
(5/413);
and
other
unidentified
drugs,
4-8%
(20/413).
Additional
specimens
for
use
in
semiquantitative
thin-layer
chromatography
studies
were
collected
from
patients
being
treated
with
methadone.
After
testing
with
the
various
immunoassays,
a
total
of
200
specimens
were
selected
on
the
basis
of
their
recorded
latex
agglutination-inhibition
test
results.
Of
these,
112
specimens
showed
both
negative
Gravindex®
and
Pregnosticon®
slide
test
results.
False
positive
or
inconclusive
test
results
were
recorded
on
all
of
the
Table
2.
Detection
of
methadone
or
metha¬
done
metabolites
by
thin-layer
chromatography
following
1:20
dilution
of
test
specimens
Division
of
200
test
specimens
by
slide-test
results
False
positive
or
inconclusive
88
Negative
112
Methadone
spot*
detectable
76
7
*
'Methadone
spot'—as
used
in
this
paper—refers
to
the
presence
of
a
spot
on
the
chromatogram
having
an
jRF
value
and
colour
corresponding
to
that
of
a
metha¬
done
standard.
Colour
development
was
achieved
by
spraying
with
Dragendorff
and
Iodoplatinate
solutions.
No
other
drug
or
drug
metabolites
appeared
on
the
chromotograms
following
1:
20
dilution
of
urine
samples.
remaining
eighty-eight
specimens
with
the
Gravindex®
slide
test
and
on
15/88
specimens
with
the
Pregnosticon®
slide
test.
The
200
specimens
were
then
diluted
1:20
and
screened
for
drugs
using
thin-layer
chromatography
as
out¬
lined
above.
While
76/88
specimens
with
false
positive
or
inconclusive
latex
agglutination-inhibition
slide
tests
showed
a
methadone
spot
(Table
2),
only
7/112
specimens
with
initially
recorded
'negative'
latex
agglutination-inhibition
test
results
showed
a
spot.
No
other
drugs
were
detected
on
the
chromatogram
following
the
1:20
dilution
of
the
test
specimen.
DISCUSSION
The
effects
of
drugs
on
the
various
immunological
pregnancy
tests
are
not
widely
appreciated
and
most
reports
deal
with
the
effects
of
psychotropic
drugs
(Marks
&
Shackcloth,
1966;
Ravel,
Riekers
&
Goldstein,
1969;
Kerber,

492
C.
A.
Horwitz
et
al.
Inclan,
Fowler,
Davis
&
Fish,
1970)
including
promethazine
(Phenergan®)
(Tait,
1971)
or
oral
contraceptives
(Horwitz,
Polesky,
Odenbrett,
Gronli,
Horowitz,
Diamond
&
Ward,
1971).
The
present
study
is
based
on
test
results
from
urine
samples
received
from
patients
on
methadone.
False
positive
or
inconclusive
test
results
were
recorded
with
the
Gravindex®
slide
test
on
58/309
(18-8%)
specimens
and
with
the
Pregnosticon®
slide
test
on
11/309
(3-6%)
specimens.
In
this
highly
selected
material,
most
of
the
problem
specimens
were
received
from
patients
on
high
doses
of
methadone
(i.e.
100
mg
or
more
per
day).
In
contrast
to
the
latex
agglutination-inhibition
slide
tests,
there
appeared
to
be
no
significant
inter¬
ference
with
the
test
accuracy
using
the
tube
haemagglutination
tests
(i.e.
only
a
single
false
positive
test
result
was
obtained
from
309
tested
specimens).
The
data
in
Table
2
shows
that
76/88
specimens
with
false
positive
or
inconclusive
latex
agglutination-inhibition
test
results
contained
methadone
or
methadone
metabolites
which
could
be
detected
by
semi-quantitative
thin-layer
chromatography.
By
contrast,
only
7/112
specimens
from
patients
with
clearly
negative
test
results
had
detectable
'methadone
spots'.
These
studies
suggested
that
methadone
or
methadone
metabolites
present
in
the
test
sample
could,
at
least
in
part,
be
responsible
for
the
inhibition
of
latex
particle
agglutination
with
false
positive
test
results.
Because
of
the
presence
of
associated
proteinuria
on
specimens
from
patients
on
high
doses
of
methadone
and
other
drug
metabo¬
lites
in
up
to
20
%
of
urine
specimens
from
this
group
of
patients,
one
cannot
with
certainty
ascribe
false
positive
test
results
to
the
presence
of
methadone
metabolites
alone.
Possible
explanations
for
the
lack
of
agglutination
and
false
positive
or
inconclusive
test
results
with
the
latex
agglutination-inhibition
slide
tests
include
blocking
of
antibody
receptor
sites
or
antigen
sites
on
the
HCG
mole¬
cules
by
drug
metabolites.
This
mechanism
for
inhibition
of
agglutination
is
unlikely
in
view
of
the
fact
that
other
tests
based
on
the
same
principle
(tube
haemagglutination-inhibition
tests)
were
not
similarly
affected.
Adherence
of
drug
metabolites
through
ionic
bonding
to
latex
particles,
however,
could
result
in
steric
hindrance
of
the
antigen-antibody
interaction.
This
bonding
might
also
result
in
an
increase
in
the
surface
charge
density
with
resultant
increase
of
zeta
potential
above
the
critical
value
at
which
agglutination
can
occur.
Pollack
&
Hager
(1965),
Pollack,
Hager,
Reckel,
Toren
&
Singher
(1965)
and
Pollack
&
Reckel
(1970)
have
studied
in
detail
the
zeta
potential
in
relation
to
the
forces
involved
in
haemagglutination.
In
their
studies,
the
dielectric
properties
of
albumin
as
well
as
of
three
synthetic
polymers
enabled
them
to
lower
the
zeta
potential
with
resultant
haemagglutination.
The
importance
of
zeta
potential
rather
than
surface
charge
was
emphasized
as
being
of
paramount
importance
in
determining
the
stability
of
red
cell
suspen¬
sions.
Semi-colloidal
suspensions
of
hydrophobic
polystyrene
particles
(latex)
are
similarly
influenced
by
the
zeta
potential
of
the
particles
and
the
dielectric
properties
of
the
surrounding
media.
Drug
metabolites
might
thus
possibly
increase
the
zeta
potential
with
inhibition
of
agglutination.
The
exact
mechan¬
ism
of
interference
with
the
latex
slide
tests,
however,
remains
to
be
determined.
Since
the
completion
of
this
study
in
August
1971,
a
revised
Gravindex®

Methadone
and
human
pregnancy
tests
493
test
has
become
available
(February
1972).
Preliminary
evaluation
of
test
results
with
the
new
Gravindex
®
showed
promise
in
elimination
of
most
of
the
false
positive
test
results
in
this
selected
study
group.
ACKNOWLEDGMENTS
We
wish
to
thank
Dr
John
Sciarra,
Chairman,
Department
of
Obstetrics
and
Gynecology,
University
of
Minnesota
Medical
School
and
Dr
Milton
Trapold,
Department
of
Psychology,
University
of
Minnesota,
for
their
helpful
sugges¬
tions,
manuscript
review
and
assistance
in
statistical
analysis.
The
technical
assistance
with
thin-layer
chromatography
by
Miss
Dzintra
Dombrovskis,
Miss
Jennifer
Powell
and
Miss
Sharon
Collins
is
gratefully
acknowledged.
The
skilled
technical
assistance
of
Miss
Margaret
Gronli
and
Miss
Donna
Rae
Faro,
Medical
Librarian,
is
likewise
acknowledged.
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