May 27, 1972 - nancy tests was performed on 309 urine specimens specifically .... (Tait, 1971) or oral contraceptives (Horwitz, Polesky, Odenbrett, Gronli,.
EFFECT
OF METHADONE ON HUMAN PREGNANCY TESTS
C. A.
HORWITZ, R. MASLANSKY, R. WALDINGER, H. CABRERA
Mount Sinai
and
P. C. J. WARD
Hospital, Minneapolis, Minnesota 55404, Columbus, Ohio, U.S.A.
and
Mount Carmel Hospital,
{Received 27th May 1972)
prospective study of four commercially available pregwas 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. Summary.
A
nancy tests
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, U.S.A. Address for reprints : C. A. Horwitz, m.d., Mount Sinai Hospital, 2215 Park Avenue, Minneapolis, Minnesota 55404, U.S.A.
California,
489
C. A. Horwitz
490
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 haemagglutinationinhibition tube tests (UCG® and Pregnosticon®) and two latex agglutinationinhibition 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 Incorporated, Westbury, New York), and various narcotics and 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.
Instruments other drugs
RESULTS
In Table 1, false positive and inconclusive test results are recorded in relation 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 to
Table 1. False
positive and inconclusive test results obtained on specimens from 103 patients receiving methadone
of specimens by daily methadone dosage
Division
Group A
:
200
to
FP
IC
Accuracy^
Tube tests*
slide test*
FP
IC
Accuracy
(UCG® and Pregnosticon9) FP
IC
Accuracy
240 mg
methadone/day (eighteen patients)
54
64-8%
90-7%
100-0%
75
74-7%
94-7%
100-0%
90
83-3%
98-9%
98-9%
90
94-4%
98-9%
100-0%
150 to 190 mg
Group methadone/day (twenty-five patients) Group C: 100 to 140 mg methadone/day (thirty patients) Group D : < 100 mg metha¬ done/day (thirty patients) :
of
specimens
Pregnosticon'1'
Gravindex9 slide test*
No.
309 selected
309
Abbreviations: FP
53
81-2%
10
1
96-4%
1
1
99-7%
(false positive) and IG (inconclusive) test results. (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. *
Gravindex®
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 88 112 Negative
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. REFERENCES
Davidow, B., Petri, N. & Quame, B. (1968) A thin layer Chromatographie screening procedure for detecting drug abuse. Am. J. clin. Path. 50, 714. Horwitz, C. ., Carmezy, L., Lyon, F., Hensley, M. & Burke, D. (1972) A comparative study of five immunologie pregnancy tests : an analysis of 1,863 cases. Am. J. din. Path. 58, 305. Horwitz, C. ., Polesky, ., Odenbrett, P., Gronli, M., Horowitz, ., Diamond, R. & Ward, P. G. J. (1971) Clinical and immunologie study of a direct agglutination test for pregnancy (mechanisms of interference including prozoning and circulating macroglobulins). Am. J. Obstet. Gynec. Ill, 808. Kerber, I. J., Inclan, A. P., Fowler, . ., Davis, K. & Fish, S. A. (1970) Immunologie tests for pregnancy. A comparison. Obstet. Gynec, N.T. 36, 37. Marks, V. & Shackcloth, P. (1966) Diagnostic pregnancy tests in patients treated with tranquillizers. Br. med. J. i, 517. Pollack, W. & Hager, . J. (1965) A physico-chemical study of hemagglutination. Biblthca haemat. Li
23, 802. Pollack, W., Hager,
. J.,
Reckel, R., Toren, D. A. & Singher, H. O. (1965) A study of the forces
involved in the second stage of hemagglutination. Transfusion, 5, 158. Pollack, W. & Reckel, R. P. (1970) The zeta potential and hemagglutination with Rh antibodies. A physiochemical explanation. Int. Archs Allergy appi. Immun. 38, 482. Ravel, R., Riekers, H. G. & Goldstein, B. J. (1969) Effects of certain psychotropic drugs on immuno¬ logie pregnancy tests. Am. J. Obstet. Gynec. 105, 1222. Tait, . (1971) Interference in immunological methods of pregnancy testing by promethazine. Med. J. Aust. ii, 126.
