American Journal of Obstetrics and Gynecology (2004) 190, 100e5 www.elsevier.com/locate/ajog Accuracy of home pregnancy tests at the time of missed menses Laurence A. Cole, PhD,* Sarah A. Khanlian, Jaime M. Sutton, Suzy Davies, PhD, William F. Rayburn, MD USA hCG Reference Service, Department of Obstetrics and Gynecology, School of Medicine, University of New Mexico, Albuquerque, NM Received for publication May 12, 2003; revised August 7, 2003; accepted August 27, 2003 –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEYWORDS Human chorionic gonadotropin Home pregnancy test Missed menses Objective: The purpose of this study was to investigate the validity of home pregnancy tests (HPTs) around the time of the missed menses. Study design: Levels of human chorionic gonadotropin (hCG) were determined in urine around the time of the missed menses, and 5th and 95th centiles were calculated. In a blind study evaluating 18 brands of HPTs, each was tested six times with ﬁve concentrations of hCG, 0 to 100 mIU/mL. Sensitivity was deﬁned as the concentration at which six of six brands gave positive results, at the suggested reading time or at an extended time. Results: It was estimated that a sensitivity of 12.5 mIU/mL was needed to detect 95% of pregnancies at the time of missing menses. Only 1 of 18 HPTs had this sensitivity. If faintly discernible results considered, 2 of 18 brands gave false-positive or invalid results. Clear positive results were given by only 44% of the brands at 100 mIU/mL hCG. If one accepted faintly discernible and extended reading times, all brands responded at 100 mIU/mL. A test with 100 mIU/mL sensitivity would detect approximately 16% of pregnancies at the time of missed menses. Conclusion: The utility of HPTs is questioned. Clinicians need to be aware of the limitations of current HPT brands. Ó 2004 Elsevier Inc. All rights reserved. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Home pregnancy tests (HPTs) have become a widely accepted ﬁrst step in the detection of early pregnancy. More than 6 million women conceive annually in the United States, and many more wish to know whether a desired or inadvertent conception has occurred.1 Preg- Supported in part by National Institutes of Health grant No. HD35654. * Reprint requests: Laurence A. Cole, PhD, Department of Obstetrics and Gynecology, 2211 Lomas Blvd NE, Albuquerque, NM 87131. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2003.08.043 nancy testing at home offers the advantages of privacy and early detection without the need to go to a clinic. It is no wonder that HPTs are among the most commonly purchased over-the-counter disposable health kits. Since their introduction in 1975, the number of commercially available HPTs has increased from 4 to more than 20 brands. All manufacturers continue to report their HPT as being more than 99% accurate.2 Two major changes have occurred during this period: (1) HPTs now use modern immunometric assay (antibody-dye/ hCG/immobilized antibody) and (2) the tests claim 101 Cole et al use on the ﬁrst day of the missed period or earlier rather than use after 5 weeks’ gestation.2 The objective of this investigation was to determine whether there is validity in the claim advertised by all HPT brands of O99% accuracy and use of the test at the ﬁrst day of the missed period. Material and methods Urine hCG concentrations at missed menses After obtaining approval from our Human Research Review Committee, we ﬁrst determined the urine concentrations of hCG in women during early pregnancy. Urine samples were gathered between 4 weeks 0 days and 4 weeks 3 days from the last menstrual period of 25 women achieving clinical pregnancy while attending an infertility clinic. Samples were random and not selected in any way. All were singleton pregnancies. Four of 25 ended in spontaneous abortion or ectopic pregnancy (16%). None received hCG therapy to induce ovulation. In all cases, early pregnancy was conﬁrmed by measurement of serum hCG. Figure 1 displays a scattergram of hCG concentrations during these 4 days after the missed menses. Individual hCG values are logarithmically distributed.3,4 Values were examined by logarithmic regression using multiple of the median methods. These are established methods for comparing distributions of normal pregnancy hCG concentrations in the ﬁrst3 and second trimesters4 of pregnancy. Results were ranked and centiles calculated as previously described.3,4 By use of these methods, permitted centiles can be calculated for 0, 1, 2, and 3 days after missing the period data from a regression equation established from all 25 patient results. From these data the calculated median result at the time of missed menses was 49 mIU/mL, and the 5th and 95th centiles were calculated as 12.4 and 241 mIU/mL, respectively. An HPT brand, therefore, would need to measure an hCG concentration of 12.4 mIU/mL to detect 95% of intrauterine pregnancies at the time of missed menses. Furthermore, sensitivities of 21, 35, and 58 mIU/mL would be necessary to detect 95% of intrauterine pregnancies at days 1, 2, and 3 after missed menses. Preparation of urine samples Urine samples were pooled from ﬁve healthy nonpregnant women. Currently, the purest and most homogeneous form of hCG available is recombinant hCG (Sigma Chemical Co, St Louis, Mo). This hCG was added to the urine by one of the investigators (L. C.) in amounts calculated to obtain concentrations of approximately 0, 12.5, 25, 50, and 100 mIU/mL. Samples Figure 1 Scattergram displaying 25 urine hCG concentrations during the first 4 days after the missed menses. Logarithmic regression analysis and multiples of the median analysis were used to determine centiles. Lines represent the 95th, 50th, and 5th centiles for the 25 results. of 7 mL (required for each evaluation, at each hCG concentration for each HPT) were blindly coded according to hCG concentrations, then frozen at 80(C. Additional 7-mL urine samples, 13 for each hCG concentration, were thawed and warmed to 37(C multiple times on each day of this study to afﬁrm the constancy of the hCG concentration. No signiﬁcant variations were observed between the calibrated and actual hCG concentrations during the study period, at 0 (all !1.0), 12.5 (12.6 G 0.5), 25 (27 G 2), 50 (52 G 2), and 100 (99 G 3) mIU/mL. HPTs and testing protocol This study evaluated three different lots of 18 brands of HPT, purchased at multiple retail stores. This permitted consideration of lot-to-lot variations and normal shipping and storage variations into our results. All HPT brands work using similar multi-antibody immunometric assay methods with a result window in the HPT to show a positive test and a control window to show that the HPT is functioning properly. Figure 2 displays a ﬂow sheet illustrating the experimental design of the brands to be tested. Each was tested with ﬁve different concentrations of hCG (0, 12.5, 25, 102 Cole et al Figure 2 Flow sheet illustrating how the 18 HPT brands were tested with 5 different urine hCG concentrations by 3 technicians (total, 465 HPT brands evaluated). A random testing protocol was developed for the 3 technicians or readers, for evaluating all HPT brands. Under these protocols, the 3 technicians evaluated 6 samples for each data point. 50, and 100 mIU/mL). Each urine concentration was blindly tested 6 times by the three professionally trained clinical technicians. The readings included three different lots each HPT to consider interlot variation. Five brandsdEckerd One-Step, Rite Aid One-Step, Sav-On Osco One-Step, Target Brand, and Walgreens OneStepdare very similar HPTs produced by one manufacturer and labeled for speciﬁc retailers. Considering the similarity between these brands, they were only tested in triplicate (three HPTs at each hCG concentration, three lots). For validation, the 50 mIU/mL hCG urines were repeated for a total of six valuations at each hCG concentration for each product. Experiments throughout the course of the study were undertaken by use of identical conditions in the same testing laboratory. The study was blinded as to the urine sample hCG concentration and the order in which the kit was tested. It was not possible to blind the individual brands because each had a distinctive shape or label and needed to be maintained in a sealed package until the time of testing. Approximately every 30 minutes the investigator who prepared and coded the urine concentrations (L. C.) thawed a batch of urine samples in a water bath for the three technicians and allowed evaluation only when the samples reached 37(C. The HPT brands were tested strictly according to the package insert instructions. Each HPT was read twice, ﬁrst at the suggested reading time (1 to 5 minutes depending on the kit instructions) and again at an extended time, 10 minutes, to examine maximal results. This was the longest reading time suggested by 14 of 18 HPT instructions and was not considered detrimental by the remaining 4 HPT instructions. Test results that were clearly positive were recorded as ‘‘2,’’ faintly discernible as ‘‘1,’’ and negative as ‘‘0.’’ Sensitivity was deﬁned as the urine concentration at which six of six HPTs of a speciﬁc brand gave a positive result (or 3/3 HPTs in the case of the 5 brands where only 3 HPT brands tested at 12.5, 25, and 100 mIU/mL concen- trations), whether faintly discernible (score = 1) or clearly positive (score = 2). Sensitivity was only calculated for legitimate test results, those giving a valid control line (score = 1 or 2) to show the HPT was functioning properly, as well as a positive test line (score = 1 or 2). Results The Table displays the 18 HPT brands in alphabetic order and the corresponding recommended or claimed earliest time to read the test results. Overall, the brands were unreliable in providing a clearly positive result at any of the four hCG concentrations at the suggested reading time. Brands with a longer suggested reading time (5 minutes) did not have higher sensitivity than those with a shorter suggested reading time (1 minute). Clearly positive results were present in only 44% of the brands at the highest hCG concentration (100 mIU/mL). When a positive result included a faintly discernible line, the sensitivity was improved (83% of brands gave a positive result with the 100 mIU/mL hCG urine) but still inexact. Test sensitivity was improved when HPT brands were reread at an extended reading time of 10 minutes. Despite this, the 18 HPT brands were 100% accurate only when the urine hCG concentration was at the highest level tested (100 mIU/mL), the test was evaluated at the extended time, and when faintly discernible results were included (Table). Certain brands were more sensitive at lower hCG concentrations than others. None provided clearly positive results at the suggested reading time using the 12.5 or 25 mIU/mL concentration. One brand (First Response, Early Results) gave consistent faintly discernible results at 12.5 mIU/mL. Three brands gave some form of a positive result, whether clear or faintly discernible at the suggested reading time using the 25 mIU/mL concentration of hCG (Clear Blue Easy, One Minute; Clear Plan Easy; First Response, Early Results). Three brands 103 Cole et al Table Sensitivity of available HPT brands Results at suggested read time Home pregnancy test Answer Answer, Quick and Simple OneStep Clear Blue Easy, One Minute Clear Choice Clear Plan Easy Confirm CVS E.P.T Eckerd, One-Step Equate First Response, Early Result Inverness Medical, Early Inverness Medical, Early, Cassette K-Mart, American Fare, Easy to Read Rite Aid, One-Step Sav-On, Osco, One-Step Target Brand Walgreens, One-Step Sensitivity, clearly positive onlyy Results at extended read time Suggested read 12.5 25 50 100 12.5 25 50 100 time (min) mIU/mL mIU/mL mIU/mL mIU/mL mIU/mL mIU/mL mIU/mL mIU/mL 2 2 1 5 3 5 3 3 3 1 3 3 5 3 3 3 3 3 Sensitivity, clearly positive or faintly discernibley 4D1 0D0 2D2 0D3 0D1 0D0 0D0 0D2 0D0 0D0 0D6 0D0 0D2 0D0 0D0 0D0 0D0 0D0 0/18 (0%) 1/18 (6%) 3D2 3D2 3D3 1D3 3D3 0D0 0D0 0D5 0 D 0* 0D0 3D3 0D1 2D1 1D3 0 D 0* 0 D 0* 0 D 0* 0 D 0* 0/18 (0%) 3/18 (17%) 6D0 6D0 3D3 2D3 6D0 0D1 0D5 2D4 0D0 0D3 6D0 0D2 5D1 3D2 0D3 1D4 0D6 0D0 4/18 (22%) 8/18 (44%) 6D0 6D0 6D0 3D3 6D0 0D5 5D1 3D3 0 D 3* 0D3 6D0 2D4 6D0 2D4 2 D 1* 1 D 1* 3 D 0* 3 D 0* 8/18 (44%) 15/18 (83%) 3D1 0D0 2D4 0D4 0D6 0D1 0D3 2D0 0 D 0* 4D1 0D6 0D4 0D6 0D4 0 D 1* 0 D 1* 0 D 0* 0 D 3* 0/18 (0%) 5/18 (28%) 6D0 3D2 6D0 0D5 4D2 0D0 2D2 5D1 0 D 2* 6D0 5D1 3D3 3D0 4D2 0 D 3* 2 D 1* 1 D 2* 3 D 0* 4/18 (22%) 12/18 (67%) 6D0 6D0 6D0 4D1 6D0 0D2 6D0 6D0 6D0 6D0 6D0 6D0 6D0 5D1 6D0 5D0 6D0 3D0 13/18 (72%) 14/18 (78%) 6D0 6D0 6D0 4D2 6D0 6D0 6D0 6D0 3 D 0* 6D0 6D0 6D0 6D0 5D1 3 D 0* 3 D 0* 3 D 0* 3 D 0* 16/18 (89%) 18/18 (100%) In each column, the first number represents the clearly positive results, while the second number represents a faintly discernible positive result. The 2 reading times are those suggested by the manufacturer and the extended time of 10 minutes (recommended as maximum read time by 14 of 18 manufacturers). * Eighteen HPT products widely available at US retail stores were blindly evaluated: Answer, Answer Quick and Simple OneStep, and First Response Early Result, produced by Church and Dwight Inc, Princeton, NJ; Clear Blue Easy One Minute and Clear Plan Easy, produced by Unipath Diagnostics, Princeton, NJ; E.P.T., manufactured by Warner Lambert Consumer Healthcare, Morris Plains, NJ; Confirm, distributed by Durex Consumer Products Inc, Norcross, Ga; Clear Choice, manufactured by Phamatech Inc, San Diego, Calif; Equate, produced by LifeCare Medical International, Philadelphia, Pa; Eckerd One-Step, Rite Aid One-Step, Sav-On Osco One-Step, Target Brand, Walgreens One-Step, Inverness Medical Early, and Inverness Medical Early, Cassette, manufactured by Inverness Medical, Waltham, Mass; K-Mart American Fare Easy to Read, manufactured by American Fare, Troy, Mich. y Only 3 HPT brands evaluated at 12.5, 25, and 100 mlU/mL concentrations. z Sensitivity was defined as the urine hCG concentration at which 6 of 6 HPT brands (or 3 of 3 HPT brands, in the case of 5 brands where only 3 HPT brands tested at 12.5, 25, and 100 mlU/mL concentrations) gave a positive result, whether faintly discernible (questionable) or clearly positive. failed to show consistently positive results (6/6 positive results), using both the highest hCG concentration (100 mIU/mL) and the suggested reading time (Conﬁrm, Equate, Sav-On Osco One-Step). It was not until the extended time of 10 minutes that all 18 HPT brands became clearly or faintly positive at the highest hCG concentration (100 mIU/mL). Several brands had indications of technical or design problems. One in six of the Conﬁrm tests gave a clear falsely positive result with the 0 mIU/mL urines at the suggested reading time. This is a false-positive result. Similarly, three of six Clear Choice HPTs tested at 0 mIU/mL yielded false-positive results at the suggested reading time. It was also observed that several tests failed to display a control band, which indicates to users that the HPT is functioning correctly. This occurred at the suggested reading time and at the extended reading time in 1 of 30 evaluations (those at 0, 12.5, 25, 50, and 100 mIU/mL) with the Answer Quick and Simple, 1 of 18 with the Sav-on Osco, 9 of 30 with the Clear Choice, and 10 of 30 evaluations with the Conﬁrm product. Comment Home pregnancy testing was introduced almost 3 decades ago. To appeal to consumers in this competitive market, manufacturers have shortened the time of early diagnosis to the same day of the missed period. One brand (First Response, Early Result) has been reported to be accurate 4 days before the missed menses. Several new brands or new adaptations of existing brands, not evaluated here, now make this earlier use claim. Our research found that in many circumstances HPTs were not sensitive enough to detect the low hCG levels reported 104 at the onset of a missed period, or they provided faintly discernible results at multiple test concentrations. A faintly discernible result is open to misinterpretation. We found in well-dated pregnancies that a wide range of hCG (from 23 to 2438 mIU/mL) is produced by pregnant women by 28 to 30 days after the onset of the last known menstrual period. It was calculated that for a test to detect 95% of pregnancies at the time of missed menses, it would need to consistently detect at least 12.4 mIU/mL hCG. If a HPT brand was used at days 1, 2, or 3 after the onset of the missed menses, then to detect 95% of pregnancies it would need to consistently detect 21, 35, and 58 mIU/mL hCG, respectively. It should be noted that these values are estimates based upon regression lines from only 25 women achieving pregnancy. All 18 HPT brands evaluated use proven multiantibody technology with very similar testing instructions. Tests were credited as being positive regardless of whether the line was clearly positive or only faintly discernible in the result window. A total of 6%, 17%, 44%, and 83% of brands tested at the suggested reading time gave clear or faintly discernible positive results at hCG concentrations of 12.5, 25, 50, and 100 mIU/mL, respectively. In contrast, only 0%, 0%, 22%, and 44% of these brands tested gave clear or indisputably positive results at the suggested reading time at these same concentrations. These results, together with the data indicating hCG concentration around the time of missing menses, clearly indicate that a high percentage of pregnant women will have a negative test result during the ﬁrst few days after the missed period. We found that most tests were consistently accurate only after the urine hCG concentration was at least 100 mIU/mL, or the highest hCG concentration evaluated, allowing for both clearly positive and faintly discernible readings and allowing for readings extended to 10 minutes, rather that at the 1- to 5-minute suggested reading time. The poor ﬁndings reported here may in part be due to consideration of multiple lots of brands purchased from multiple stores and the shipping and storage variations. We estimate that 100 mIU/mL is equivalent to the 84th centile at the time of the missed menses, so that a test with this sensitivity would detect only about 16% of pregnancies. Realizing our ﬁndings, how can manufacturers claim such a high accuracy in very early gestation? The reason for this product claim relates to an arcane Food and Drug Administration (FDA) regulation. The manufacturer needs only to demonstrate that its test results, using a nonpregnant woman’s urine supplemented with commercial hCG, agree with those of an existing test more than 99% of the time. Today’s HPT brands are more sensitive than previous products, so they would be expected to give as many positive results as older products. This FDA evaluation, how- Cole et al ever, has no bearing on the ability to detect early pregnancy. In addition to the inability of these HPT brands to detect the low hCG concentrations associated with very early pregnancies, other reasons may explain a negative result using these brands. The person may have erroneously calculated the time of onset of her missed period, she may have a delayed menses due to an early pregnancy loss5 or delayed ovulation, or implantation may have prolonged the onset of signiﬁcant hCG production.6-8 Although the test instruction sheets were easy to understand, an erroneously performed test or faintly discernible result may also explain a falsely negative result. The purpose of this investigation was not to recommend speciﬁc brands of HPT. Nonetheless, we found that First Response, Early Result was the most sensitive test, consistently detecting 12.5 mIU/mL of hCG (in a supplementary study by Cole et al9 this HPT detected 6.3 mIU/mL hCG), considering the manufacturer’s suggested read time, and both clear and faintly discernible positive results. This is the sensitivity needed to detect 95% of pregnancies at the times of the missed menses (12.4 mIU/mL). It is inferred that this test is the most likely of the 18 brands evaluated to detect a pregnancy at the time of the missed menses. Two of the 18 brands had clear technical or design problems (Clear Choice and Conﬁrm). These tests both gave falsely positive hCG test results with urine containing no hCG and also gave numerous invalid results as indicated by the absence of a conﬁrmation or validity line. Such brands may be unacceptable and may need to be withdrawn and ﬁxed. They should probably be avoided because they may generate false hope and great confusion among users. Costs of the different HPT brands varied from $8 to $16. The more expensive brands contained a second test to be performed later, ‘‘in case the ﬁrst test was negative.’’ This option to purchase a kit with two tests would be reasonable if the patient understands in advance that testing at the day of the missed period or earlier can yield negative or only faintly positive results (even though the user maybe pregnant). Use of two tests is also helpful in determining an early pregnancy loss (ie, test on day of missed menses is positive, whereas test a few days later is negative). Home pregnancy testing continues to be a dominant method for early pregnancy detection despite questions about clarity of positive results, false pregnancy detection as a result of early pregnancy loss, and problems with interpretation of a negative result. Considering the analytic detection limits of HPTs and the wide variability in hCG concentrations, we question the greater than 99% accuracy claims made by the manufacturers. In fairness to the consumer, we suggest that these guidelines be re-examined by the manufacturers. FDA 510(k) regula- Cole et al tions should insist on measurements of clinical sensitivity for detecting pregnancy on the day of the missed menstrual period and at other speciﬁc time points, as support for an accuracy claim. By doing so, a clearer understanding of diagnostic accuracy can be reported on each brand’s package insert rather than the printing boldly ‘‘over 99% accuracy’’ on the outside of the package. All these issues need to be considered to avoid delays in detection of pregnancy, not only for early commencement of prenatal care, but to allow for appropriate changes in medication and behavior consistent with healthy pregnancy (avoidance of alcohol, tobacco, and inappropriate medicines), or to seek earliest pregnancy termination if desired. References 1. Ventura SJ, Mosher WD, Curtin SC, Abma JC, Henshaw S. Highlights of trends in pregnancies and pregnancy rates by outcome: estimates for the United States 1976-1996. Natl Vital Stat Rep 1999;47:1-9. 105 2. Butler SA, Khanlian SA, Cole LA. Detection of early pregnancy forms of human chorionic gonadotropin by home pregnancy test devices. Clin Chem 2001;47:2131-6. 3. Cole LA, Isozaki T, Jones EE. Urine b-core fragment, a potential screening test for ectopic pregnancy and spontaneous abortion. Fetal Diagn Ther 1997;12:336-9. 4. Cole LA, Shahabi S, Oz UA, Bahado-Singh RO, Mahoney MJ. Hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen) immunoassay: a new basis for gestational Down syndrome screening. Clin Chem 1999;45:2109-19. 5. Wilcox AJ, Weinberg CR, O’Connor JF, Baird DD, Schlatterer JP, Canﬁeld RE, et al. Incidence of early loss of pregnancy. N Engl J Med 1988;319:189-94. 6. O’Connor JF, Ellish N, Kakuma T, Schlatterer J, Kovalevskaya G. Differential urinary gonadotrophin proﬁles in early pregnancy and early pregnancy loss. Prenat Diagn 1998;18:1232-40. 7. Bjercke S, Tanbo T, Dale PO, Morkrid L, Abyholm T. Human chorionic gonadotrophin concentrations in early pregnancy after invitro fertilization. Hum Reprod 1999;14:1642-6. 8. Wilcox AJ, Baird DD, Dunson D, McChesney R, Weinberg CR. Natural limits of pregnancy testing in relation to the expected menstrual period. JAMA 2001;286:1759-61. 9. Cole LA, Khanlian SA, Sutton JM, Davies S, Stephens N. Hyperglycosylated hCG (invasive trophoblast antigen, ITA) a key antigen for early pregnancy detection. Clin Biochem 2003;36:647-55.
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