THE INVISIBLE HUMAN CHORIONIC GONADOTROPIN
P. Bischof, C. Gruffat and A. Campana
Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein (a and ß subunit) produced by trophoblastic tissue and by certain tumors (1). The a-subunit of hCG is composed of 92 amino acids, 5 intrachain disulfide bonds and 2 N linked glycosylation sites (asparagines 52 and 78). This subunit is identical to the a subunit of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH, 2). The ß subunit of hCG is composed of 145 amino acids, 6 intrachain disulfide bonds, 2 N-linked oligosaccharides (asparagines 13 and 30 and 4 O-linked oligosaccharides (serines 121,127,132 and 145, Fig. 1). Although they are obviously different, the ß subunits of hCG, LH, FSH and TSH exhibit considerable homology (2). This is particularly true for the ß subunits of LH and hCG. ßhCG is 23 amino acids longer than ßLH (in the C terminus of the peptide), the rest of the ß chain differs only by 24 residues out of 121 and none of these residues are involved in disulfide bonds (Fig. 1). The biochemical similarity between hCG and LH is thus quite extensive and great difficulties have been encountered to develop assays capable of distinguishing between these molecules. Vaitukaitis et al (1) developed the first radioimmunoassay (RIA) which was capable to distinguish between LH and hCG.
Since this first RIA, many different types of assays have been developed to measure hCG. Several monoclonal antibodies to this glycoprotein hormone became available and led to the development of more specific multiple sites sandwich immunoassays (3,4 among many others).
Multiple site assays are constructed in the following way: antibodies which capture the molecule to be measured are immobilized on the test tubes walls. Antibodies which detect the captured molecule (at a different site then the capture antibodies) are labelled either radioactively (immunoradiometric assays IRMA) or enzymatically (immunoenzymometric assays IEMA). These technologies are now widely available on automates.
The circulating forms of hCG
The biosynthesis of hCG (as for all glycoprotein) is a highly complex process: the human genes for ßhCG and ahCG are located on chromosome 7 and 19 respectively. Transcription of the genetic information into specific RNA is followed by the processing of the RNA into a mature RNA (elimination of non coding region). Mature RNA moves from the nucleus into the cytoplasm of the cell where it is translated into polypeptides. The peptides are then processed further by cleavage of the signal peptide, glycosylation of the subunits, folding of each subunit into its three-dimensional structure, formation of the aß complex, attachment of the O-linked oligosaccharides and further " trimming " of the N-linked oligosaccharides (5). This complicated biosynthetic pathway ends-up by the secretion of biologically active hCG. The patway is not " tight " so that besides the known secretion of holo hCG and free a and ß subunits, abherrent glycosylation products can appear in the circulation. Recently the presence of hCG or free ß subunit with missing peptide linkages (peptide bonds 44-45 or 47-48 of the ß subunit), called nick ßhCG, have also been found in serum (6) and urin (7). Finally, in the urine of pregnant women, ß-core fragment, a post-translational deglycosylated ß subunit, is also detected. Thus, hCG circulates in the maternal blood at least under eight different molecular forms: holo hCG, nicked hCG, holo hCG missing the C terminus, free ßhCG, free ßhCG missing the C terminus, nicked free ßhCG, free ahCG and different glycosylation forms. These different isoforms of hCG are represented schematically in Fig. 2.
According to the physio-pathological conditions of the patient, the relative amount of the different isoforms of hCG change. The concentration of free a subunit increases continuously during pregnancy (from less than 1% in the first trimester to as much as 30% of intact hCG at term). In patients with hydatidiform moles or trophoblastic disease, the concentration of free ahCG is below 1% (8). Conversely, free ß is high during the first trimester in normal pregnancies, in hydatidiform moles and trophoblastic disease (up to 37% of the hCG concentration) and low in term pregnancies (8). Free ß subunits and nicked free ß subunits are also elevated in ectopic pregnancies (9) and in patients carrying a Down syndrome infant (10).
What do we measure?
Quantitative determinations of hCG are usually performed with only one type of assay in routine laboratories. Since the hCG " composition " of the serum to be measured varies with the physio-pathological status of the patients, it is expectable that some isoforms of hCG will not be measured. These are called the invisible hCG. This might be clinically irrelevant most of the time but is important in some cases (Down Syndrome, ectopic pregnancies). The detection of the different isoforms will depend on the antibodies used in the assay and the assay construct. In a recent study, Kardana and Cole (11) tested several antibodies with different specificities towards nicked ß subunits. Important differences appeared: In patients with hydatidiform moles, free ß subunits represented 0.61% of holo hCG when measured with an antibody which does not recognize nicked ß (1E5) or 6.4% when measured with an antibody which cross reacts with nicked ß (B204). If 1E5 can be used to determine hCG in normal pregnancies it is absolutely contraindicated to use it to screen for Down syndromes since under these conditions nicked ß is highly increased. In contrast, using B204 to measure normal pregnancies will not modify the results significantly since free ß is only moderately increased in these conditions but B204 will detect quite a large proportion of Down syndromes. This is illustrated in Fig. 3, where the results of four collaborating laboratories established the median values of hCG in the same serum samples (30 per weeks of pregnancy). The samples measured with the IMX automate (Abbott) using the total hCG kit are significantly higher than the samples measured with other automates (Delphia or Stratus) or with the same automate but with a different kit (IMX hCG). These differences are due to the fact that the Abbott total ßhCG kit overestimates nicked ß by 240% (12, Fig. 4). This kit offers truly an advantage for screening for Down syndromes but overestimates the hCG content of samples which have been stored at room temperature for more than 2 days or at 4°C for more than one week. Interestingly, no variations were observed when the stored samples were measured with the IMX hCG kit (unpublished observations). This seems to indicate that storage of the sample will increase nicking.
Thus according to the pathophysiological condition of the patient, (trophoblastic disease, testicular cancer or normal pregnancy), one type of assay will perform better than the other.
In order for the reader to select the appropriate hCG assay, we have tabulated the results of Cole and Kardana (11), together with the assay construct (Fig. 4).
The Stratus (Baxter) measures hCG by capturing the a subunit and detecting the ß subunit. This assay will thus not measure any free hCG subunit either nicked or not. It will however measure holo hCG and possibly nicked holo hCG. The maia clone assay (Serono) captures hCG by a particular epitope on the intact hCG. Thus this kit also will not detect the free subunits of hCG but will read intact, deglycosylated and C terminus missing hCG. The Hybritech tandem hCG, and the Delphia automate (Pharmacia) measure hCG by capturing the ß subunit and reading the a. Under these conditions, free a and ß subunits are not measured but with the hybritech kit (Pharmacia) nicked hCG is overestimated by 200%. The Organon NML kit or the IMX total ßhCG kit (Abbott) are both based on a ß capture and ß detection. These kits do thus measure the free ß but not the free a subunit. They both overestimate nicked hCG but the Organon kit does not detect the forms of hCG missing the C terminus of the ß subunit.
The large variety of hCG assays based on different constructs and on different monoclonal antibodies recognizing different epitopes has generated a bulk of discordant data. As a rule of thumb for routine laboratories dealing with hCG measurements in normal, abnormal pregnancies, trophoblastic diseases, Down syndrome screening and testicular cancers, assays measuring the free ß subunit and the intact molecule should be chosen. Furthermore, since nicked hCG is clearly increased in Down syndromes, it is advisable to choose a kit which estimates (overestimates?) this molecular form of hCG. Using the IMX automate, we tested the same 2 patients with Down syndrome with the IMX hCG (measuring only holo hCG) and with the IMX total ßhCG (measuring free ß and nicked hCG). In one case hCG went from 1.98 multiple of median (MoM) to 2.54 MoM increasing her risk of Down from 1:350 to 1:160. In the other case the risk went from 1:600 to 1:490. This clearly illustrates the better performances of a kit estimating nicked hCG in Down syndrome screening.
The authors thanks Dr. Wheis (Maternity Hospital, Lausanne), Dr. Stricker (Dianalab Geneva) and Dr. Horak (Laboratoire Riotton) for sharing their hCG medians with us.
Edited by Aldo Campana,