Human Reproduction vol.7 no.5 pp.701-710, 1992

REVIEW

Pregnancy tests: a review

T.Chard

Materials which might form the basis of a pregnancy test

Department of Reproductive Physiology, and Obstetrics and Gynaecology, St Bartholomew's Hospital Medical College, and the London Hospital Medical College, London, UK

All current pregnancy tests involve die qualitative determination of die concentration of human chorionic gonadotrophin (HCG) by an immunoassay. Usually, diis is applied to a urine sample. However, die odier 'specific' fetoplacental products might dieoretically be suitable provided diat diey appear at concentrations which can be readily detected widiin die first 4 weeks followingfertilization.In practice, die only materials which have been evaluated in this context are Schwangerschaftsprotein 1 (SP1), placental protein 14 (PP14) and 'early pregnancy factor' (EPF) (Canfield and O'Connor, 1991). Schwangerschaftsprotein 1 (SP1) is a glycoprotein of die same family as carcino-embryonic antigen (CEA). Substantial amounts are produced by me early trophoblast. Using a sensitive radioimmunoassay, SP1 can be detected in maternal blood from as early as 9 days following fertilization, and 2 days later in maternal urine (Grudzinskas et al., 1978; Ho et at., 1988). However, altiiough measurement of SP1 might be equivalent to that of HCG, it is in no way superior. The interesting possibility diat it might be a useful test in women receiving exogenous HCG has not been exploited in clinical practice. Placental protein 14 (PP14) was originally described by Hans Bohn as one of a group of proteins which could be isolated from placental extracts (Bohn et al., 1982). Subsequently, it was shown to be secreted by die maternal endometrium and decidua ratiier tiian die placenta (Bell et al., 1985) and to be a member of die beta-lactoglobulin family (Julkunen et al., 1988). Small amounts are present in non-pregnant females, die highest levels being found at die time of menstruation. In early pregnancy, tiiere is a very rapid increase to a peak at 8 — 10 weeks, witfi a fall diereafter. This pattern is virtually identical to diat of HCG and suggests that PPM could be used as a pregnancy test. As witii SP1, however, there is no evidence diat it is superior to HCG in diis respect. 'Early pregnancy factor' (EPF) is a poorly characterized molecule which can be measured by a complex immunological test (Morton et al., 1977). The great interest in diis compound results from the claim that it can be detected in maternal blood widiin hours of fertilization. If true, and if convenient mediodology were to be developed, dien estimation of EPF would rapidly become the most popular of all pregnancy tests. Unfortunately, die test technology has proven difficult to reproduce, and a number of audiors have even doubted the actual existence of EPF (Chard and Grudzinskas, 1987). A rather more convincing 'early pregnancy factor' has been demonstrated in the mouse (O'Neill et al., 1989). This is a phospholipid which, because of its main biological activity,

Pregnancy tests are widely used both by the public and by healthcare professionals. All tests depend on the measurement of human chorionk gonadotrophin (HCG) in urine. Other pregnancy-specific materials have been proposed as pregnancy tests but none can better the sensitivity and convenience offered by immunoassay of HCG. Ultrasound detection is also not as sensitive as HCG measurement. The current generation of tests is based on monoclonal antibodies to the beta-subunit of HCG; these virtually eliminate the possibility of cross-reaction with pituitary hiteinizing hormone (LH) and it is this feature which permits the high sensitivity. However, it is important to recognize that the 'beta-subunit' antibody reacts with both intact HCG, which is the major component in pregnancy serum, and with fragments of the beta-subunit (beta-core), which are the major form hi urine. Both the blood and urine of non-pregnant subjects contain small amounts of HCG. HCG from the implanting blastocyst first appears in maternal blood around 6—8 days following fertilization; the levels rise rapidly to reach a peak at 7 - 1 0 weeks. With most current pregnancy test kits (sensitivity 25 units per litre) urine may reveal positive results 3 - 4 days after implantation; by 7 days (the time of the expected period) 98% will be positive. A negative result 1 week after the missed period virtually guarantees that the woman is not pregnant. With the present generation of test kits, false positive results due to interfering materials are extremely unlikely. Pregnancy tests have now reached a level of sensitivity and specificity which is unlikely to be surpassed either by better tests or alternative technology. Key words: HCG/pregnancy tests/ELISA

A pregnancy test is among the most widely used and valuable of tests in the whole field of clinical biochemistry. Furthermore, it is unique in that it is also used by non-professionals. One-third or more of all women buy and use a pregnancy test during their reproductive lives (Jeng et al., 1991). Unlike the majority of laboratory tests, the result may be die cause of immediate joy or anguish on die part of the consumer. For the purposes of die present discussion a 'pregnancy test' is defined as a procedure intended simply to reveal die presence or absence of a pregnancy. © Oxford University Press

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is known as 'platelet activating factor' (PAF). A similar phenomenon (a reduction in platelet count within a few days of fertilization) has also been noted in the human but, once again, the evidence for this is far from complete. It is worth remembering that the human embryo is approximately the same size as the mouse embryo, but that its products will be diluted into a far larger maternal pool. This, together with the lack of any direct contact between the early blastocyst and the maternal bloodstream, makes it inherently unlikely that there will ever be a reliable test of pregnancy in the human prior to the time of implantation (i.e before 6 - 7 days post-fertilization). Development of HCG measurement as a pregnancy test The development of the first biological assay for HCG, and hence pregnancy tests, can be attributed to Ascheim and Zondek (1927). A number of variations on their procedure were used for the next 30 years (Table I). In the classic study by Tietz (1965), the frog test showed 12% of positive results in the first week after the missed period, 58% during the second week and 93% in the third week. Corresponding results for the more sensitive rabbit test were 77%, 90% and 96%. Subsequent developments in bioassays for gonadotrophk activity (Rees et al., 1973) suggest that it would not be difficult to devise tests with a higher level of accuracy in the first week after the missed period. However, the real disadvantage of the bioassays as clinical tests was that the procedures were cumbersome and expensive, and that it might be several days before results were available. As so often in practical medicine, convenience and cost determine the applicability of a procedure and it is not surprising that the bioassays were rapidly overtaken in the 1960s when immunoassays were introduced (Wide and Gemzell, 1960). Biochemistry of HCG Human chorionic gonadotrophin (HCG) is one of a family of glycoprotein hormones, the other members being luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyrotrophin-stimulating hormone (TSH). Each of these consists of two subunits: an alpha-subunit (92 amino acids) which is virtually identical in all four; and a beta-subunit which is characteristic of the individual hormone (Bahl et al., 1972; Morgan et al., 1975; Pierce and Parsons, 1981). The beta-subunit of HCG is a single chain of 145 amino acids. The first 121 Nterminal amino acids share 80% of the sequence with beta-LH; the C terminus of beta-HCG has a 24 amino acid extension which is not present in beta-LH. Both subunits of the molecule are needed for biological activity but the beta-subunit determines the

specificity of the action (Strickland and Puett, 1981). The alphasubunit is coded by a single gene on chromosome 6 and the betasubunit by a family of seven genes on chromosome 19; only two of the latter genes appear to be active (Fiddes and Talmadge, 1984). In the circulation, the bulk of HCG is in the form of the intact hormone (a-/3 dimer) with only small amounts of free alpha- and beta-subunit. In urine, by contrast, a large fraction of the material which reacts in immunoassays for HCG is a metabolic fragment of the hormone commonly known as 'beta-core' (Wehmann et al., 1989). This consists of two polypeptide chains derived from the /3-subunit of HCG: the amino acid sequences 6 - 4 0 and 55-92 are joined by disulphide bridges (Birken et al., 1988). The fragment lacks the unique 30 amino acid carboxy-terminal peptide but retains conformational immunological determinants also found in intact and free /3-subunit of HCG. Although large quantities are found in the urine of pregnant women and some patients with cancer, it is virtually undetectable in serum. Some workers believe that this fragment is secreted directly into the circulation by the placenta and then rapidly excreted by the kidneys (Kardana et al., 1988); others consider that the major source is intracellular degradation of the intact and /3-subunit of HCG in the renal parenchyma. Functions of HCG Chorionic gonadotrophin has biological activities identical with those of pituitary luteinizing hormone. The trophoblast surrounding the blastocyst begins secretion of HCG into the maternal circulation at the time of implantation, i.e. ~ 7 days post-conception. The rapidly rising levels of HCG in the mother provide a stimulus to the corpus luteum (luteotrophic effect). In a normal, non-pregnant menstrual cycle the corpus luteum would begin to involute at this stage with a fall in secretion of oestradiol and progesterone leading eventually to shedding of the endometrium. However, under the influence of HCG, the corpus luteum continues to grow and secrete increasing quantities of steroids. Thus, HCG is the principal signal from an early pregnancy leading to 'rescue' of the corpus luteum, delay of menses and thus maintenance of the pregnancy. Methods of measurement The biological assays for HCG are now of only historical interest. For a time, considerable use was made of an assay using receptors extracted from bovine corpora lutea (Saxena et al., 1974). However, since the 1960s virtually all assays have been immunoassays.

TaWe I. Biological tests for early pregnancy* Name of test (researcher)

Test animal

Endpoint

Time taken for test

Ascheim — Zondek Friedman Ovarian hyperaemia (Beck et al.) Frog test (Wiltberger, Miller) Toad test (Galli, Mainini, Shapiro)

Mice or rats Rabbits Rats Female frog Male toad

Corpus luteum Corpus luteum Hyperaemia Extrusion of eggs Extrusion of spermatozoa

5 days 48 h 12 — 18 h 24 h

'Modified from Pritchard et al. (1985).

702

2-5 h

Pregnancy tests

Several different forms of immunoassay have been used for qualitative pregnancy tests. The major variants are in the type of label employed and in the specificity of the antibody. The original immunoassays used particles as the label—agglutination assays (Figure 1). Agglutination techniques are still widely used because of their familiarity and relatively low cost. The most recent generations of slide tests have a sensitivity of 500 IU/1 and the tube tests of 200 IU/1. The later ELISA procedures (Figure 2) occur in a variety of different sorts of which 'dipsticks' (Norman etal., 1985) offer great user convenience and a sensitivity between 25 and 50 IU/1. From the point of view of the consumer, professional or non-professional, it is difficult to envisage any procedure which could offer a significant advance over some of these devices. For example, Clearview (Unipath, UK) requires only the addition of five drops of urine to the 'sample window' followed by the appearance of a line in the 'result window' if the test is positive. Similar procedures have been used for quantitative measurements in hospital laboratories. In the latter, extensive use is also made of radioimmunoassay with an isotope of iodine [125I] as the label. The earlier assays used antibodies raised against the whole HCG molecule. Most of these antisera reacted equally well with LH and HCG. The use of antibodies raised against the purified beta-subunit was an important development; such antibodies would react with HCG but not to a significant extent with LH (Vaitukaitis et al., 1972). The resulting ability to measure low levels of HCG in the presence of LH explains the great sensitivity of current pregnancy tests. A further development was the use of monoclonal antibodies. These have two important advantages: first, because of the method of selection it is possible to choose only those antibody-producing clones which have the desired specificity; second, they can be produced in large quantities

3.

(Pj-hCG

with total consistency from one batch to the next (unlike traditional polyclonal antibodies which vary with each individual antibody-producing animal).

What is measured by an assay for HCG? The complex structure of HCG and its subunits and fragments has led to much confusion in both terminology and quantitation. For example, it is very common amongst clinicians to refer to 'beta-subunit assay' or 'beta-subunit' levels. These terms may correctly refer to the specificity of the antibody. However, in blood the vast majority of the materials measured by a betasubunit antibody is intact HCG (i.e. the assay is detecting the a-/3 dimer). The amount of free beta-subunit is very small, — 1 % or less of the total (Ozturk et al., 1987). In urine the bulk of HCG-like material is beta-core (Kato and Braunstein, 1988), which again cannot properly be described as beta-subunit. It is also commonly assumed that antibodies to the (3-subunit are directed to the unique C-terminal peptide. However, although highly specific HCG assays have been developed using antibodies to this peptide (Wehmann et al., 1981; Birken et al., 1982), such antibodies are frequently of low affinity. In reality, many of the specific beta-subunit antibodies used in pregnancy tests are directed to other sites. Although quantitation is not directly relevant to qualitative pregnancy testing, it may have an important bearing on the levels of 'sensitivity' quoted for different assays. The problem is that the International Reference Preparation (IRP) on which the unitage is based is a preparation of highly purified HCG. The most widely used standard at the present time is the First International Reference Preparation (IRP-HCG 75/537) provided by the World Health Organization (Stoning et al., 1980). This highly purified preparation still contains small quantities of subunits (Wehmann etal., 1988) and succeeded the Second International Standard (2nd IS-HCG) which consisted of a mixture of HCG and subunits. One unit of the 2nd IS is approximately

Parties coated with hCG Antibody to hCG with two combining sites Sowphaze anl)-hCG

(?)-hCG

Unne with hCG

Enzyme-label led anti-hCG

(p)-hCG Agglutination

c.

©©-

hCG hCG

urbgwHh

( p ^

h C G

hCG ~

(^Pj-hCG No agglutination

Fig. 1. The principle of the agglutination inhibition assay: (a) the test reagents are particles (red cells or latex) coated with HCG, and an antibody to HCG; (b) when these are mixed with urine containing no HCG, the antibody binds adjacent particles thus causing agglutination; (c) when the reagents are mixed with urine containing HCG, the latter combines with the antibody and thus prevents it from binding and agglutinating the particles. The antibody is usually directed to the beta-subunit. The test can be carried out on slides or in tubes; in the latter a positive result is indicated by deposition of a ring at the bottom of the tube.

Fig. 2. The principle of the enzyme immunoassay [enzyme-linked immunosorbent assay (ELISA); also sometimes known as 'sandwich' or 'immunometric' assays]. HCG in urine is captured by a solid-phase antibody directed to one she on the HCG molecule. The bound HCG is then identified by a second antibody to a different site on the HCG molecule. This second antibody carries an enzyme (®) as label: addition of an appropriate substrate yields a colour reaction. There are many variations on this procedure, especially in the solid-phase support and the method of developing the colour reaction: commercial pregnancy tests involve a range of dipsticks, pots, tubes, etc. The use of two antibodies confers sensitivity because the capture antibody concentrates the HCG on the surface of the solid phase. It also confers specificity because the two antibodies can be directed to sites which will only be found together in HCG. Thus in the classical Hybritech format, the capture antibody is anti-a-subunit and the detection antibody is anti-/3-subunit. Other combinations can be used, and the antibody may be monoclonal or polyclonal.

703

T.Chard

equivalent to 2 units of the 1st IRP, though this varies according to the assay used (Ooi et al., 1989). In this review, levels are usually stated in terms of the 1st IRP. Some authors report values as weight-by-volume; it is generally agreed that 1 ng of the highly purified preparation CR 119 is equivalent to 56 mTU of IRP. To understand the potential problems of basing the assay on a purified HCG preparation, consider two assays, one of which is specific to free beta-subunit and reacts only slightly with intact HCG (assay 1), while the other shows the reverse (assay 2). The IRP will show relatively little activity in assay 1. However, a urine sample from a pregnant woman, which contains predominantly beta-core, will show high levels in assay 1 and low levels in assay 2. This illustrates why different assays can give quite different results on the same clinical specimen. It also indicates why the sensitivity (minimum detection limit) of an assay in respect of the IRP could be different from that in respect of pregnancy urine. HCG levels in normal pregnancy Normal non-pregnant subjects have low circulating levels of HCG: in males and pre-menopausal females the range is 0.02 — 0.8 IU/1; in post-menopausal females it may sometimes range much higher than this (Body et al., 1981; Borkowski et al., 1984; Armstrong et al., 1984; Lee et al., 1991). Cultured blastocysts secrete HCG into the medium from 7 days post-fertilization onwards (Fishel et al., 1984; Hay and Lopata, 1988). Messenger RNA for HCG can also be detected in the trophoblast at this time (Ohlsson et al., 1989). The earliest point at which HCG can be detected in the maternal circulation is — 6—8 days post-conception, i.e. around the time of implantation (Braunstein et al, 1976; Lenton et al., 1981, 1982). It is likely that the appearance of HCG in the mother is a combination of commencement of secretion by the trophoblast, together with the establishment of direct contact between the trophoblast and the maternal circulation. Thereafter, the levels rise rapidly with a doubling time which has been variously estimated at between 1.3 days (Lenton et al., 1982) and 2.3 days (Batzer et al., 1981; Lenton et al., 1981) (Figure 3). The increase is more rapid in the earlier days of the rise (Pittaway et al., 1985; Daya, 1987; Fritz and Guo, 1987). Lenton and colleagues (1991) have shown that the exponential rise can be resolved into two distinct phases: an initial faster component which they attribute to the actual process of implantation (up to 11 days post-LH surge); and a slower component, as trophoblast HCG and maternal circulating HCG come into equilibrium. The rate of rising HCG is slightly reduced in pregnancies which subsequently abort (clinical or sub-clinical 'biochemical' pregnancies) and in pregnancies resulting from in-vitro conception (Smitz et al., 1988; Lenton etal., 1981). The levels of HCG reach a peak at 7 - 1 0 weeks of pregnancy. At this time, the mean concentration is 50 000 U/l with a range from 20 000 to 200 000 U/l; the secretion rate is over half a million units (26 mg) per day (Rizkallah et al., 1969). The levels of HCG in relation to various events in early pregnancy are shown in Table II. Somewhat surprisingly, the levels of HCG in urine are very similar to those in blood (Marshall et al., 1968; Naryshkin et al., 1985; Norman et al., 1987; Lenton et al., 1991). The two fluids 704

are therefore virtually equivalent from the point of view of pregnancy testing. Traditionally HCG is measured in the first specimen of urine passed in the morning. However, specific experiment has failed to show any consistent variation of urinary HCG concentrations over a 24-h period (Kent et al., 1991). Detection of early pregnancy by a pregnancy test For the user there are three main questions about a pregnancy test. What is the earliest time after conception when the test becomes positive? How certain can a woman be that she is not pregnant on the basis of a negative result? How certain can a woman be that she is pregnant on the basis of a positive result? How early does the test become positive? Chorionic gonadotrophin first appears in maternal blood at 6—7 days post-conception. Using a research assay (sensitivity 0.1 - 0 . 3 IU/1), Lenton and colleagues (1982) found circulating HCG in 5.3% of subjects by day 8 following the LH peak, 15.8% by day 9, 53.2% by day 10 and 100% by day 11. Most state-of-the-art pregnancy tests have a sensitivity of 25 IU/1. At this level it appears that some women will yield positive results 3 or 4 days after implantation (Figure 4). Thereafter, the rate of positive results increases rapidly, reaching an estimated

10,000 r

1.000 hCG levels (lu/l)

100

10

10

12

14

16

I

I

18

20

22

Days from LH peak

Fig. 3. The levels of HCG in early pregnancy based on the blood HCG levels reported by Lenton and colleagues (1982) and Ooi et al. (1989). Surprisingly, there is no adequate quantitative information on urinary HCG levels in the first 6 weeks of pregnancy. However, the concentrations of HCG in blood and urine are very similar. The vertical lines show the means ( • ) and range of values reported by Ooi et al. (1989). The means are very similar to those of Lenton and colleagues; the ranges are larger because the published results were aggregated into 4-day periods.

Pregnancy tests

98% by 7 days after implantation, i.e. at around the time of the expected period. Clearly, the earliest time of detection is directly related to the sensitivity of the test. The bioassays and earlier immunoassays with a sensitivity of 2000-2500 IU/1, only became positive at 6 weeks post-LMP. Sometimes published results on sensitivity can be confusing. For example, in a group of tests all of which had an estimated sensitivity of 200 IU/1, Asch and colleagues (1988) reported positive values at the time of the missed menses of 70%, 88% and 95%. A possible reason for this type of discrepancy is shown in Figure 5; relatively small differences in sensitivity can make large differences in positivity rates on a given day of pregnancy. How certain is a negative result? It is generally recognized that pregnancy tests are unreliable (i.e. quite likely to give negative results) before the time of the first missed period (Jovanovic et al., 1987). However, given a state-of-the-art test with a sensitivity of 25—50 IU/1, a negative result > 1 week from the expected time of the missed period can virtually guarantee that the woman is not pregnant. There must still remain a small chance that the woman has levels at the extreme lower end of the normal range (and thus might be undetectable), or that conception took place later than expected on the basis of the menstrual history. This residual uncertainty can be excluded by repeating the test 1 week later. Several current test kits provide a second test for this purpose. How certain is a positive result? With current technology, a positive result is unlikely in the absence of a pregnancy. However, 'false-positive' results can occur, notably in post-menopausal women. There is little doubt that these findings are attributable to low circulating levels of authentic HCG in non-pregnant subjects (Armstrong et al., 1984). In a study using five ELISA kits, there was a 1 - 2 % false-positive rate with three of the kits, while the remaining two yielded much

higher rates (10% and 25%) (Bandi et al., 1987a). Many of these false-positive results appear to have been due to readings between 5 and 25 IU/1; levels above the latter almost always proved to be correct (Bandi et al., 1987b). Problems can also arise with a classical radioimmunoassay, even using an apparently highly specific antibody. Emancipator and Cadoff (1988) showed that 8 out of 9 of women yielding a result between 10 and 30 IU/1 in a radioimmunoassay were not pregnant on follow-up. In the light of this information, the following general rules can be made (Bandi et al., 1989). A level of < 5 IU/1 can be confidently stated to be negative. A level > 25 IU/1 can equally confidently be stated as positive. Between these two is a 'doubtful' zone. At a practical level, a test result of this type should be repeated a minimum of 2 days later. A wide variety of factors have at various times been described as possible interfering agents in a pregnancy test (Table HI). Few if any of these are significant with the current generation of test kits; there is no drug or physiological state which can interfere with a well-designed ELISA carried out on urine. An exception is the woman who has received HCG therapeutically, usually for induction of ovulation. The serum half-life of injected HCG follows a multiphase curve with initial rapid components of 5 h and 24 h (Rizkallah et al., 1969) followed by a further slowing to 2.3 days (Damewood et al., 1989). Exogenous HCG may still appear in the urine up to 14 days after the last dose (Damewood et al., 1989). Levels of HCG are higher following intramuscular than subcutaneous injection (Saal et al., 1991). It has been proposed that other tests may be useful under these circumstances (SP1; Grudzinskas et al., 1978) but the indication is so unusual that it would hardly justify the resources needed to make the technology available. Following abortion in the first trimester, it may take as long as 60 days for HCG levels to return to zero from the very high values present at 7 - 1 0 weeks (Steier et al., 1984). Another and exceedingly rare possibility is that the woman has an HCG-secreting tumour: gestational trophoblastic disease (hydatidiform mole and choriocarcinoma); a gonadal teratoma;

Table n. Events in early pregnancy observed by ultrasound and the corresponding mean HCG levels. Note that most of these studies assayed serum rather than urine, but the levels in these two fluids are very similar. Daya et al (1991a,b) have published a very detailed table of HCG levels versus gestational sac size Author

Event

Days

HCG (IU/1)*

Cacciatore et al (1990)

Gestational sac 1—3 mm Yolk sac apparent Heart action seen Gestational sac 1 cm Gestational sac seen Gestational sac seen: 20% of cases 80% of cases 100% of cases Gestational sac seen Fetal pole seen Fetal heart seen Yolk sac first seen Yolk sac always seen Heart action first seen Heart action always seen

31 36 41

730 (467-935) 4103 (1120-7280) 12 050 (5280-22 950) >6000 >600

Goldstein el al. (1988) Bemaschek et al. (1988) Nyberg et al. (1988)

Fossum et al. (1988)

Daya et al. (I991a,b)

1000 1398 ± 155 5113 ± 298 17 208 ± 3772 1900 5800 9200 24 000

"International Reference Preparation (IRP), if necessary converted by multiplying 2nd International Standard results by two.

705

T.Chard 10,000

10,000 r

1.000

1.000 hCG levels (lu/l)

hCG levels (lu/l)

100

100 -

10

10 10

12

14

16

18

20

22

I 12 14 16 18 20 Days from LH peak

22

Days from LH peak

Fig. 4. Levels of circulating HCG in early pregnancy (see Figure 3), showing that an assay with a sensitivity of 25 IU/l would become positive in some subjects between 10 and 11 days after the LH peak and in most subjects 12 — 13 days after the peak.

or a carcinoma with ectopic secretion of HCG (lies et al., 1990). It is important to emphasize that a pregnancy and a positive test do not guarantee a baby 9 months later. The total loss of pregnancies between conception and term is substantial. Failure of the pregnancy is more common with the new than with the older tests: the earlier the pregnancy is diagnosed, the more likely it is to abort. Positive tests may even occur in the luteal phase of an apparently normal cycle; the pregnancy implants but aborts before the term of the expected period. This is the so-called 'occult' or 'biochemical' pregnancy (reviewed by Chard, 1991). Dating of pregnancy Since HCG levels are related to the stage of gestation, it is logical that a quantitative measurement of HCG in an individual woman might be used to estimate the gestational age. Several authors have shown that this prediction is possible (Lagrew et al., 1983; Westergaard et al., 1985). However, the error of the prediction in relation to the 'gold standard' of an unambiguous menstrual history is as high as ± 2 weeks. This is definitely inferior to dating by ultrasound (gestational sac diameter or crown-rump length) with an error of only 3 - 4 days (Johal et al., 1992). It has also been suggested that a qualitative pregnancy test may be used for dating. Thus, if serial tests are done on an individual woman, the time at which they become positive defines the stage of gestation. However, this approach is fraught with possible errors and is entirely replaced by ultrasound dating. 706

Fig. 5. Why different tests with the same quoted sensitivity may give apparently very different results (Asch et al., 1988). Take three tests with a sensitivity of '200 IU\ It is well-recognized that there may be substantial errors in the estimation of minimum detection limits, such that the three kits might actually have sensitivities of 100, 200 and 400 IU/l. At 16 days of gestation the kit with the 100 IU limit will be positive in all cases; the 200 IU kit will be 80—90% positive; and the 400 IU kit will be positive in only some 2% of cases.

Table m . Materials which have been described as interfering with pregnancy tests on urine. Most of these were described in connection with first generation assays and probably do not apply to current tests. Other factors, such as circulating antibodies to mouse immunoglobulins, rheumatoid factors, proteases and hyperlipidaemia can interfere with HCG assays on serum (Norman, 1991) Acetylsalicylic acid (aspirin) Carbamazepine Methadone Blood Protein High pH Seminal fluid

Other ways of detecting early pregnancy The only significant current competitor to HCG as an early pregnancy test is ultrasound. There are descriptions of changes associated with the earliest stages of implantation and embryonic development, especially using transvaginal ultrasound (Table II). Clearly, the type of equipment and user skills needed to make these observations are of a high order. The minimum level of HCG below which a gestational sac cannot be visualized by ultrasound is ~ 1000 IU/l (Fossum et al., 1988; Daya et al., 1991a,b). For practical purposes, however, routine clinical detection of an early pregnancy by ultrasound is not possible

Pregnancy tests

before 6 weeks of gestation. Measurement of HCG will continue to be the standard method of detecting an early pregnancy for the foreseeable future. Complications of pregnancy Most pregnancy tests are performed simply to satisfy the curiosity of the woman and her relatives. There are also important medical indications. These include situations in which the woman should consider changing her lifestyle to avoid possible risks to the fetus: examples include smoking, drugs and excess alcohol. In addition, pregnancy might indicate the need for specific medical action, as in the mother with diabetes or heart disease. It has also been recommended that a pregnancy test should be performed on all women undergoing endometrial biopsy in the late luteal phase of the cycle (Herbert et al., 1990). An important medical indication is investigation of the woman with abdominal symptoms which might or might not be associated with a pregnancy (Seppala et al., 1980). A positive pregnancy test ( > 25 IU/1) is found in 95 % or more of women with ectopic pregnancy; false-negatives are rare (Taylor et al., 1988; Uribe and Dunn, 1990), although the rate becomes 11.9% if an assay with a sensitivity of 200 IU/1 is used (Romero et al., 1985). Even if the history seems unambiguously to exclude pregnancy, —10% of women may be pregnant (Ramoska et al., 1989). In one study of females admitted as accident victims, random pregnancy tests revealed that 2% were pregnant (Lippman et al., 1988). If the test is negative, it can be safely assumed that pregnancy is not a feature of the case; if the test is positive then laparoscopy should be performed to confirm or exclude an ectopic pregnancy. This policy has led to a great improvement in the accuracy of diagnosis of ectopic pregnancy, with a substantial reduction in unnecessary laparoscopies and other special investigations. The diagnostic approach has been made possible by the widespread availability of simple and sensitive tests. Evaluation of a pregnancy test The main criteria for evaluating and comparing different pregnancy tests are specificity, sensitivity, convenience and cost (Delfert etal., 1987; Lee and Hart, 1990). Specificity refers to the cross-reaction with LH which should be < 1 %. As already noted, specificity is vital to the achievement of a high level of sensitivity. A manufacturer would be most unlikely to market a kit which performed less well than this. The criteria for assessment of specificity have been well described by Porter et al. (1988). Testing should include substantial numbers of samples from men and from non-pregnant women. Sensitivity should be 50 IU/1 or better. Test kits with sensitivity levels of > 100 IU are still on the market but it is difficult to see any argument for their retention. They should definitely not be used for specific clinical indications (abdominal pain), a situation in which a negative result from a poor test could be clinically misleading. The precision of the test at the detection limit should be examined by replicate determination on samples containing HCG levels 50% above and 50% below the limits (Delfert etal., 1987).

Convenience refers to simplicity such that any member of the general public can perform the test without difficulty. Most members of the medical profession can be considered in the same category since they are not usually trained in lab techniques. Simplicity of both procedure and instructions is especially important in the light of the fact that most 'over the counter' pregnancy tests are purchased on the basis of advertisements rather than professional advice from a pharmacist (Coons, 1989). Another aspect of convenience is speed. It should be possible to complete the test within 15 min, including any waiting period for delayed positive results. Cost does not appear to be an issue in the 'over the counter' market where all tests are currently priced at around £8 — 10 ($14-18). Bulk purchases for hospital practice tend to be very competitive and prices of around £1.50 ($2.50) may be expected. AH these prices are very cost-effective in terms of the demand and the value achieved. The ultimate evaluation of the performance of a pregnancy test should be a study on substantial numbers of very early pregnancies (3 weeks post-LMP onwards) with a follow-up to identify all false-positive and false-negative results. Ideal studies of this type are few and far between. Where they have been performed, the results are sometimes disturbing. For example, Doshi (1986) found that only 56% of pregnancies were identified by kits with a claimed accuracy of 98-99%. This study referred to a previous generation of products, but it is still the case that many tests are judged solely on the basis of their specificity and sensitivity in the laboratory, with an extrapolation to their likely performance in clinic. Conclusions The current generation of immunoassay technologies for measurement of HCG can detect or exclude a pregnancy at the time of the missed period. Their simplicity and convenience is such that the same procedures are applied in the hospital or the home. Pregnancy tests have reached a level of sensitivity and specificity which is unlikely to be surpassed either by better tests or alternative technology. Pregnancy testing is remarkable in that it fulfils a strongly felt demand both from the public and the healthcare professions. References Armstrong.G., Ehrlich.P.H., Birken.S., Schlatterer.J.P., Siris.E., Hembree,C. and Canfield.R.E. (1984) Use of a highly sensitive and specific immunoradiometric assay for detection of human chorionic gonadotropin in urine of normal, nonpregnant, and pregnant individuals. J. Clin. Endocrinol. Metab., 59, 867-874. Asch.R.H., Asch.G., Asch,M., Bray.R. and Rojas.F.J. (1988) Performance and sensitivity of modem home pregnancy tests. Int. J. Fertil, 33, 157-161. Ascheim.S. and Zondek.B. (1927) Hypophysenvorderlappenhormon und ovarialhormone im ham von schwangeren. Klin. Wochenschr., 6, 1322. Bahl.O.P., Carlson.R.B., Bellisario.R. and Swaminathan.N. (1972) Human chorionic gonadotropin amino acid sequence of the alpha and beta subunits. Biochem. Biophys. Res. Commun., 48, 416-422. Bandi.S.L., Schoen.I. and DeLara.M. (1987a) Enzyme-linked

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