Int J Gynaecol Obstet 17: 68-72, 1979
Further Experience with a Capillary Tube Pregnancy Test H. Lorrin Lau, Katharine W. Lawrence and Theodore M. King Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
ABSTRACT Lau HL, Lawrence KW, King TM (Dept of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA). Further experience with a capillary tube pregnancy test. Int J Gynaecol Obstet 17: 68-72, 1979 Further evaluation of the nonrefrigerated capillary tube pregnancy test is presented. A 127-mm by 2-mm glass capillary tube containing lyophilized antibody to human chorionic gonadotropin and latex indicator particles is used. Urine is drawn into the tube by capillary action; then, the tube is tilted to allow the urine to dissolve the antibody and to suspend the latex particles. The test results are read after a one-hour incubation period. Test results were evaluated against histopathologic and clinical data. Special efforts were made to stress the false-positive rate of the test by tripling the number of negative cases and doubling the sample size. A 99.82% agreement, a 0.18% false-negative rate and a 0% false-positive rate were obtained. In 52 confirmed pregnancies, the more sensitive capillary test detected human chorionic gonadotropin, whereas the commercial tests did not. Human chorionic gonadotropin was also detected one week earlier than reported in the first series.
INTRODUCTION T h e results of an earlier, initial evaluation of a simple, sensitive, nonrefrigerated capillary tube pregnancy test with a series of 1486 cases were reported previously (11). An agreement of 99.65% between the test results and histopathologic evidence was obtained with a false-positive rate of 0.07% a n d a false-negative rate of 0.28%. Because these false result rates appeared too low, another study with a greater n u m b e r of cases was initiated which screened more nonpregnant women. T h e results of this study are reported here. MATERIALS A N D M E T H O D S T h e method of performing the capillary tube Int J Gynaecol Obstet 17
pregnancy test which has a sensitivity of 0.5 I U of h u m a n chorionic gonadotropin (HCG) per milliliter of urine has been slightly modified. It is currently performed in the following manner. Both the tube and urine container are held as close to a horizontal position as possible. T h e end with the red mark is dipped into urine which ascends by capillary action to the red mark. After the outside of the tube is wiped, the tube is tilted to allow the column of urine to flow into the region containing the antibody. T h e urine is then rocked back-and-forth ten times over the lyophilized antibody to dissolve it. T h e urine is not allowed to enter the latex region during this time. After dissolution of the antibody, the tube is tilted further to move the urine column into the latex region, at which time the tube is again tilted back-and-forth ten times to suspend the latex particles. T h e tube is sealed at both ends by inserting the ends into the same tray of clay as is used for microhematocrit determinations. T h e tube is then placed vertically in a clay tray and read at 60 and 120 minutes. T h e end point is viewed against a black background with a strong light source striking the tube from either the side or back (indirect lighting), but not the front. Holding the tube u p against a strong light source (direct lighting) for reading is undesirable as the dispersion of light by the particles may make every end point appear positive. Although faster readings can be obtained by physically agitating the particles on a mechanical rocker for ten minutes, readings at one hour have been found to be clinically practical by permitting the health personnel to set u p the test, obtain an adequate history and physical examination and then read the end point of the test. A negative end point of large floccules in a clear solution can be obtained as soon as 25 minutes, but a positive end point of a milky white homogeneous suspension should not be recorded as such until the end of 60 minutes. T h e test begins with a "positive" end point and sufficient time must be permitted for flocculation to occur. Unlike some other immunoassays, the capillary test is not affected by vibration.
Capillary lube pregnancy test
The commercially available tests used for comparison were performed in strict accordance with the manufacturer's instructions. The sensitivity of both the capillary tube and commercially available tests was confirmed by titration against the United States Pharmacopeial, the Second International Standard Preparation and commercial standards. Positive and negative controls were run with each batch. Histopathologic results from each case were obtained from the gynecologic pathology division of the Johns Hopkins Hospital. Urine specimens from 2118 patients were tested: 1668 patients were from the Fertility Control Center (FCC) from December 16, 1975, through July 5, 1977, and 450 patients were from the Gynecological Clinic (Gyn Clinic) of the J o h n s Hopkins Hospital from August 13, 1976, through October 31, 1977. When a pregnancy test was required or requested by these clinics, a covalent slide test (4) with a sensitivity of 2.0 IU of H C G / m l of urine was performed in the clinic. A hemagglutination tube test (6) with a sensitivity of 0.9 IU of H C G / m l of urine was also performed in the Gyn Clinic. T h e urine samples were transferred to a research laboratory and tested with the capillary test within two hours after they were obtained. A case was categorized as pregnant if the histopathologic examination showed decidua and villi and if a fetus and placenta were present. A case with a histopathologic report of decidual action, Arias-Stella reaction or "decidua only, no villi" was designated as positive for pregnancy only if it was accompanied by a final diagnosis of incomplete
69
abortion, missed abortion or ectopic pregnancy. A case was designated as nonpregnant if the histopathologic results showed secretory or proliferative endometrium and if two consecutive menstrual periods occurred commencing within six weeks from the last menstrual period and if a confirmatory pelvic examination was obtained within six weeks from the last menstrual period. Cases were eliminated if they lacked any one of the following: histopathologic results, medical records, clinical confirmation of outcome with dates or pelvic examination. Data were entered on cards, verified for errors and analyzed by computer. Study numbers were assigned to urine specimens rather than patients as some patients had two or more pregnancy tests. At no time during the testing did the technician know the results of the histopathology, the history or the pelvic examination.
RESULTS A total number of 2209 tests were performed: 1721 from the F C C a n d 488 from the Gyn Clinic. Eliminated for the reasons described were 36 tests, one from the F C C a n d 35 from the Gyn Clinic. T h e number of tests with confirmatory evidence was 2173: 2162 tests were performed during pregnancy or in the nonpregnant state (Table I) a n d 11 tests were performed after termination of pregnancy. T h e total number of tests which were associated with clinical and histopathologic confirmation of pregnancy was 1759 or 81.4% (Table I, categories
Table I. Categories of pregnancy test results by pathology results. Pregnancy Tests Category No.
a b
Pathology
Capillary Test (0.5 lU/ml)
Tube Test (0.9 lU/ml)
Slide Test (2.0 lU/ml)
P P P P P P P P N P P N N N N
P U P U P N N U N N U N U U P
P P U U N P N N N N N N N U N
P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 Early P 10 Early P 11 N 12 N 13 N 14 N 15 P = positive; early P = positive
Further Experience with a Capillary Tube Pregnancy Test H. Lorrin Lau, Katharine W. Lawrence and Theodore M. King Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
ABSTRACT Lau HL, Lawrence KW, King TM (Dept of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA). Further experience with a capillary tube pregnancy test. Int J Gynaecol Obstet 17: 68-72, 1979 Further evaluation of the nonrefrigerated capillary tube pregnancy test is presented. A 127-mm by 2-mm glass capillary tube containing lyophilized antibody to human chorionic gonadotropin and latex indicator particles is used. Urine is drawn into the tube by capillary action; then, the tube is tilted to allow the urine to dissolve the antibody and to suspend the latex particles. The test results are read after a one-hour incubation period. Test results were evaluated against histopathologic and clinical data. Special efforts were made to stress the false-positive rate of the test by tripling the number of negative cases and doubling the sample size. A 99.82% agreement, a 0.18% false-negative rate and a 0% false-positive rate were obtained. In 52 confirmed pregnancies, the more sensitive capillary test detected human chorionic gonadotropin, whereas the commercial tests did not. Human chorionic gonadotropin was also detected one week earlier than reported in the first series.
INTRODUCTION T h e results of an earlier, initial evaluation of a simple, sensitive, nonrefrigerated capillary tube pregnancy test with a series of 1486 cases were reported previously (11). An agreement of 99.65% between the test results and histopathologic evidence was obtained with a false-positive rate of 0.07% a n d a false-negative rate of 0.28%. Because these false result rates appeared too low, another study with a greater n u m b e r of cases was initiated which screened more nonpregnant women. T h e results of this study are reported here. MATERIALS A N D M E T H O D S T h e method of performing the capillary tube Int J Gynaecol Obstet 17
pregnancy test which has a sensitivity of 0.5 I U of h u m a n chorionic gonadotropin (HCG) per milliliter of urine has been slightly modified. It is currently performed in the following manner. Both the tube and urine container are held as close to a horizontal position as possible. T h e end with the red mark is dipped into urine which ascends by capillary action to the red mark. After the outside of the tube is wiped, the tube is tilted to allow the column of urine to flow into the region containing the antibody. T h e urine is then rocked back-and-forth ten times over the lyophilized antibody to dissolve it. T h e urine is not allowed to enter the latex region during this time. After dissolution of the antibody, the tube is tilted further to move the urine column into the latex region, at which time the tube is again tilted back-and-forth ten times to suspend the latex particles. T h e tube is sealed at both ends by inserting the ends into the same tray of clay as is used for microhematocrit determinations. T h e tube is then placed vertically in a clay tray and read at 60 and 120 minutes. T h e end point is viewed against a black background with a strong light source striking the tube from either the side or back (indirect lighting), but not the front. Holding the tube u p against a strong light source (direct lighting) for reading is undesirable as the dispersion of light by the particles may make every end point appear positive. Although faster readings can be obtained by physically agitating the particles on a mechanical rocker for ten minutes, readings at one hour have been found to be clinically practical by permitting the health personnel to set u p the test, obtain an adequate history and physical examination and then read the end point of the test. A negative end point of large floccules in a clear solution can be obtained as soon as 25 minutes, but a positive end point of a milky white homogeneous suspension should not be recorded as such until the end of 60 minutes. T h e test begins with a "positive" end point and sufficient time must be permitted for flocculation to occur. Unlike some other immunoassays, the capillary test is not affected by vibration.
Capillary lube pregnancy test
The commercially available tests used for comparison were performed in strict accordance with the manufacturer's instructions. The sensitivity of both the capillary tube and commercially available tests was confirmed by titration against the United States Pharmacopeial, the Second International Standard Preparation and commercial standards. Positive and negative controls were run with each batch. Histopathologic results from each case were obtained from the gynecologic pathology division of the Johns Hopkins Hospital. Urine specimens from 2118 patients were tested: 1668 patients were from the Fertility Control Center (FCC) from December 16, 1975, through July 5, 1977, and 450 patients were from the Gynecological Clinic (Gyn Clinic) of the J o h n s Hopkins Hospital from August 13, 1976, through October 31, 1977. When a pregnancy test was required or requested by these clinics, a covalent slide test (4) with a sensitivity of 2.0 IU of H C G / m l of urine was performed in the clinic. A hemagglutination tube test (6) with a sensitivity of 0.9 IU of H C G / m l of urine was also performed in the Gyn Clinic. T h e urine samples were transferred to a research laboratory and tested with the capillary test within two hours after they were obtained. A case was categorized as pregnant if the histopathologic examination showed decidua and villi and if a fetus and placenta were present. A case with a histopathologic report of decidual action, Arias-Stella reaction or "decidua only, no villi" was designated as positive for pregnancy only if it was accompanied by a final diagnosis of incomplete
69
abortion, missed abortion or ectopic pregnancy. A case was designated as nonpregnant if the histopathologic results showed secretory or proliferative endometrium and if two consecutive menstrual periods occurred commencing within six weeks from the last menstrual period and if a confirmatory pelvic examination was obtained within six weeks from the last menstrual period. Cases were eliminated if they lacked any one of the following: histopathologic results, medical records, clinical confirmation of outcome with dates or pelvic examination. Data were entered on cards, verified for errors and analyzed by computer. Study numbers were assigned to urine specimens rather than patients as some patients had two or more pregnancy tests. At no time during the testing did the technician know the results of the histopathology, the history or the pelvic examination.
RESULTS A total number of 2209 tests were performed: 1721 from the F C C a n d 488 from the Gyn Clinic. Eliminated for the reasons described were 36 tests, one from the F C C a n d 35 from the Gyn Clinic. T h e number of tests with confirmatory evidence was 2173: 2162 tests were performed during pregnancy or in the nonpregnant state (Table I) a n d 11 tests were performed after termination of pregnancy. T h e total number of tests which were associated with clinical and histopathologic confirmation of pregnancy was 1759 or 81.4% (Table I, categories
Table I. Categories of pregnancy test results by pathology results. Pregnancy Tests Category No.
a b
Pathology
Capillary Test (0.5 lU/ml)
Tube Test (0.9 lU/ml)
Slide Test (2.0 lU/ml)
P P P P P P P P N P P N N N N
P U P U P N N U N N U N U U P
P P U U N P N N N N N N N U N
P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 Early P 10 Early P 11 N 12 N 13 N 14 N 15 P = positive; early P = positive
