A new, more sensitive tube test for pregnancy evaluated with a selected hospital population LOCISE

LANG,

M.I‘.(A.S.C.P.)

ALICE

34. KEISS.

I.IND.4

SMITH

M.4RCIA

PH.D.

KERBER

.4 nezi’ tube test j;~r pregnancy, hazling a sensitivity of 0.5 I. U. oj human chorionic gonadotropin per milliliter, urns evaluated, along with two other commercially available kits, it1 a hospital patient population. Of 586 patients in the study, 302 7wr1~ in nonobstetric servic,r,s and many ?tlere acutely ill. Approximately one fourth of the women ww ago JO or older. The medical records of 66 per cent of the patirnts were retrieved to obtain [email protected] on the drugs administered prior to pregnancy testing. .4 small number of problems did occur in the pregnancy test procedures. Incon&sive results were recor&d for both pregnant and nonpregnant patients and could not be correlated with disease .\tates andior medications. The importance of a reagent control,for tu,be test procedures is discussed. (AM. J. OBSTET. GYNECOL. 126: 693, 1976.)

Mattial

T H E n E v E Lo P M E NT 0 F simple immunologic tests for pregnancy based on the detection of human chorionic gonadotropin (HCG) has provided a practical means by which women in the childbearing age can be screened quickly and reliably. For a period of two years, our laboratory has routinely used the Gravindex* slide test, the UCG-test,? and a tube test under development that is now commercially available as the Gravindex” tube test. The new tube test procedure has a sensitivity of 0.5 1.U. of HCG per milliliter and does not require dilution of the urine specimen prior to testing. A reagent control tube is provided for each specimen to establish that there are no urinary constituents causing interference in the reagent system.

A total of 1,461 urine speciriiens were tested in parallel with the three test kits mentioned above. After study of the first 875 specimens, a change in the buffer system of the Gravindex tube test was made to reduce the number of inconclusive results which had necessitated repeat testing. The findings with the use of the modified buffer system on 586 patients are presented in this paper. The result of each test was recorded, together with appropriate patient identification, age, and clinical diagnosis. Each specimen was tested for specific gravity and protein content. Known nonpregnant patients were selected for the study with the expressed purpose of seeking unsuspected urinary constituents that could interfere with pregnancy test procedures. The patients included women of menopausal age, 40 to .55 years old, and postmenopausal age, over 55 years old, as well as patients with acute myocardial infarct, hepatitis. pancreatitis, acute cholecystitis, carcinoma, diabetes, urinary tract infection, and drug overdose. To study the possible effects of drugs on test accuracy, the hospital charts of 387 of the 586 patients were retrieved after the test results had been tabulated. Notation was made of each drug and the dose level

From Mercy Hospital, Hamilton, and Ortho Research Institute of Medical Sciences, Ortho Diagnostics, Inc., Raritan. Rrcekwd

for puhlicution

.4ccrptd

April

February

20, 1976.

9, 1976.

Rep&t requests: Louise Lang, M.T.(,4.S.C.P.), Hospital, Hamilton, Ohio 45011. *Ortho

Diagnostics,

iWampole

Lab., Div.

Inc., Raritan,

Mercy

New Jersey 08869.

oj’ Denver

Commurca Rd., Stamford,

and methods

Chem. Mfg. Co., 35 Conrwcttiut 06904.

693

694

Table

Lang and Aeiss

I. Results obtained

on 586 patients Test results

Patient

categor)i

No. tested

Positive

( %)

Negative

(%I

Inconclusmr

(75)

Pregnant

Gravindex tube test UCG-test Gravindex slide test Abnormal

231 231 231

224 220 218

(97.0) (95.2j (94.3)

0 (0) 7 (3.0) 13 (5.6)

7 (3.0) 4 (1.7) 0 (0)

fn-egnancks

Gravindex tube test UCG-test Gravindex slide test

53 53 53

39 (73.6) 27 (50.9) 25 (47.2)

12 (22.6) 22 (41.5) 28 (52.8)

2 (3.8) 4 (7.5) 0 (0)

302 301 302

4* (1.3) 4* (1.3) 3* (1.0)

291 (96.4) 295 (98.0) 299 (99.0)

7 (2.3) 2 (0.7) 0 (0)

Nonpregnant

Gravindex tube test UCG-test Gravindex slide test

*One patient, age 68, all tests positive, 3+ albumin, arterial sclerotic heart disease (ASHD); one patient, age 19, alI tests positive, 3+ albumin, acute cystitis (an unrecognized abortion cannot be ruled out with certainty); one patient, age 45, tube tests positive, 1+ albumin, pyelonephritis; one patient, age 61, tube tests positive, 2+ albumin, terminal hepatic coma; one patient, age 23, slide test positive, 1+ albumin, uterine bleeding. administered in the 48 hour period prior to performance of the pregnancy tests. The Gravindex tube test was used according to the directions in the clinical research protocol which corresponds to the direction circular in the commercially available kit. The other two kits were used according to the manufacturers’ directions.

Results The patients studied and test results are shown in Table I. The 231 patients who were categorized as pregnant include those in all trimesters of pregnancy as well as a small number of postpartum women. No false negative reactions were recorded with the Gravindex tube test as compared to 7 false negative results with the UCG-test and 13 with the Gravindex slide test. The seven inconclusive results with the Gravindex tube test and the four with the UCG-test required repeat testing of the same specimen or testing of a new specimen. The 53 abnormal pregnancies include abortions (threatened, incomplete, and missed) and ectopic pregnancies. The increased sensitivity of the Gravindex tube test is readily apparent in this group with wellrecognized low levels of HCG. Twenty-eight of the 53 patients had concentrations of HCG less than 3.5 I.U. per milliliter as determined by the Gravindex slide test. Twenty-two patients had levels less than 3.0 I.U. per milliliter as determined by the UCG-test, and 12 patients, with abnormal pregnancies not detected by the Gravindex tube test, had concentrations of less than 0.5 I.U. of HCG per milliliter of urine. The 302 nonpregnant female patients included many that were acutely ill. The rationale for studying

these patients was that the shortcomings of a pregnancy test would be recognized much more quickly in a hospital patient population. About one fourth of this group consisted of women in the menopausal and postmenopausal age groups. The clinical diagnoses in the cases of the four hospitalized patients yielding false positive pregnancy tests are given at the bottom of Table I. The 6%year-old and 61-year-old patients were moribund at the time of testing. The 19-year-old patient with a diagnosis of cystitis had positive results with all three test procedures. Although clinical follow-up showed the patient to be nonpregnant, an unrecognized abortion cannot be ruled out with certainty. The 45-year-old woman with a diagnosis of pyelonephritis had a false positive result in both tube test procedures but a negative result with the slide test method. Numerous other patients with cystitis and pyelonephritis were tested with accurate results. It is possible that certain bacteria or bacterial or viral products, when present in sufficient quantities, adsorb to the surface of the carrier particles and prevent antigenantibody interaction by steric interference. This can be demonstrated indirectly for some of these problems by washing the HCG-carrier particles after the false positive reaction has occurred. The addition of these washed HCG-carrier particles to reagent anti-HCG and known problem-free nonpregnant urine will show that the HCG-carrier particles still cannot be agglutinated. While such manipulations are not always successful, the results do indicate that false positive reactions should not invariably be interpreted as resulting from pituitary luteinizing hormone (LH). It can be seen that a number of the urine specimens

New tube test for pregnancy

in each category were reported as inconclusive for the tube test procedures. In the absence of the respective reagent controls, there would have been no warning for the laboratory of an inherent problem with the urine specimen. It can also be seen that the interfering factors did not affect the Gravindex slide test reactions. A number of cases of drug overdose established by thin-layer and gas chromatography were included in the study. Urine samples containing hard drugs and methadone were also included. None of these cases showed interference in the respective test systems. These patients are not included in the 387 whose charts were retrieved for the purpose of reviewing the number and type of drugs administered in the 48 hour interval prior to performance of the pregnancy tests. A summary of the number of patients receiving medications within a major category was compiled and is shown in Table II. The number of drugs prescribed was much greater than expected. Medications given averaged three per patient. with a range from zero (patients tested upon admission) to 27 for one patient and 26 for another. Neither of these patients gave false pregnancy test results. Some patients were receiving seven to IO drugs simultaneously. It is not surprising that a recent publication’ suggested routine screening of female subjects admitted to the hospital to identify pregnant patients prior to prescription of drugs and performance of certain diagnostic procedures.

Comment Several significant findings emerged from this study. The increased sensitivity of the Gravindex tube test (0.5 I.U. of HCG per milliliter) did not pose problems that could be ascribed to the increased levels of LH known to be present in women of menopausal and postmenopausal ages. This finding is consistent with that previously reported by Albert’ who showed that the urine of a castrated woman known to excrete an extremely high level of LH was not able to neutralize anti-HCG in the Gravindex slide test even after a tenfold concentration of urine by a tannic acid procedure. In fact, the level of LH after concentration with the tannic acid procedure was only 5 per cent of that required for a false positive result. Cross-reactivity between anti-HCG and LH is well established.3-5 However, the conclusion that false positive results obtained when testing urine samples of older women are attributable to LH is not always justified. The problem of interfering substances being responsible for inconclusive results with the UCG-test was previously reported by Lang.” In a recent publication by Garment and associates,’ inconclusive results were

Table II. Drug study on 387 patitantx: received during pregnancy testing Patients (NO.) 168

162 103 87 87 x0 7” sr, 34 30 17 9 9 100 77

,%4ajor

drug

Medication

c rttc,fpy

Barbiturates Tranquilizers. seciativcs Narcotics Hormones, rnzynw Analgesics, saliryl.ltc,s Antihistamine5 Antibiotics .4ntipruritic5 Laxatives Oxytocic compounds Vitamins Diuretics Anticoagulants Defined miscellaneous* Cndrfined miscellaneoust

*For instance, 27 of’ 100 received antineoplastic tFor

instance,

695

14 of 77 received

gall bladder

agents.

medication.

shown for the Dri-Dot* slide tesl as \$ell as for the Accusphere” tube test procedure. In our experience, the factors causing problems in the interpretation of pregnancy tests (with special reference to tube tests for pregnancy), although increased in older women, nevertheless ma\’ occur at any age and do not appear to be hormone related. Some interfering factors result in the agglutination (to a variable degree) of the sheep red cell-H(:G antigen in the absence of anti-HCG. The problem can be discerned only when a reagent control ~.rtbe, with the identical buffer system present in the test reagent, is an integral part of the testing proc-edurr. 111 the absence of a reagent control. the rube test result would be recorded as negative for pregnancy. ‘l’hese interfering factors are ,just as frequent in apparently healthy pregnant women as in nonpregnant women. Often, the interfering factors are sufficiently labile as to disappear in the time interval required ro I-epcrat the test. When this does not occur, a repeat specimen usually gives clear-cut results. The unstable and elusive nature of the interfering substances has made c-hat-acterization extremely difficult, but the substances are not related to pH, specific gravity, or protein concentration in the urine. The possible interference of’ drugs or drug metabolites cannot be ruled out with absolute certainty in view of the almost infinite number ($1 cotnbinations and dose levels, but the data do indicate that medications need not be a ma-jor concern svit’h regard to the reagents studied. *Organon. Inc.. 375 Mt. Pleasant .4ve.. Mi(.st Orange, New Jersey 070.52.

696

Lang

and

Reiss

REFERENCES

1. Garment, M. B., Ladinsky, J. L., Gottlieb, L. I., and Larson, F. C.: A study on the advisability of routine hospital pregnancy testing. J. Lab. Clin. Med. 84: 920. 1974. 2. Albert, A.: Procedure for concentrating urine for use with an immunologic test for pregnancy, Mayo Clin. Proc. 45: 114, 1970. 3. Paul, W. E., and Ross, G. T.: Immunologic cross-reaction between human chorionic gonadotropin and human pituitary gonadotropin, Endocrinology 75: 352, 1964.

4. Reiss, A. M.. Singher, H. O., Hawk, J. B., et al.: Immunologic cross-reaction between luteinizing hormone and antisera to human chorionic gonadotropin, Int. Arch. Allergy 30: 561, 1966. 5. Wide, L.: An immunological method for the assay ot human chorionic gonadotropin. Acta Endocrinol. 41: 49. 1962. (Suppl. 70.) 6. Lang, L.: Interfering substances of menopausal urine fol red cells coated with human chorionic gonadotropic hormone, Am. J. Med. Technol. 31: 373. 1965.

A new, more sensitive tube test for pregnancy evaluated with a selected hospital population.

A new, more sensitive tube test for pregnancy evaluated with a selected hospital population LOCISE LANG, M.I‘.(A.S.C.P.) ALICE 34. KEISS. I.IND.4...
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