Lowered reference limits for hCG improve follow-up of patients with hCG-producing tumors.

BACKGROUND: Human Chorionic Gonadotropin (hCG) is produced by germ cell tumors, but can also be elevated in benign conditions such as primary hypogonadism, where hCG is produced by the pituitary gland. In our experience, the reference limits for hCG (Elecsys hCG+ß-assay, Roche Diagnostics), were unnecessarily high and did not reflect levels encountered in clinical practice. We wanted to establish new reference limits to increase the clinical utility of the hCG-assay. METHODS: We analysed hCG in serum samples from a healthy adult population and in a cohort of testicular cancer survivors. The gonadotropins LH and FSH were measured in the cohort and in a selection of the reference population to assess gonadal function. RESULTS: We found low hCG levels for all men and women <45years (97.5 percentiles 0.1 and 0.2IU/L, respectively) from the healthy population (n=795) having normal FSH and LH. Due to assay limitations, we suggest a common reference limit of <0.3IU/L. For the age group ≥45, the 97.5 percentiles in the healthy population were 0.5IU/L for men and 6.0IU/L for women. In all subjects from both the reference population and the cohort (n=732), hCG levels exceeding the reference limit could be fully explained by reduced gonadal function indicated by elevated LH and FSH levels. CONCLUSION: The Elecsys hCG+ß-assay should have lower reference limits than recommended by the manufacturer, with important implications for tumor follow-up. Elevated hCG is rare with intact gonadal function, both in a normal population and among survivors of testicular cancer, and should lead to further investigations when encountered in clinical practice.

The heterogeneity of human chorionic gonadotropin (hCG). II. Characteristics and origins of nicks in hCG reference standards.

hCG, the hormone produced by the trophoblast throughout pregnancy, has peptide bond cleavages, or nicks, in the beta-subunit. We sought to compare the nature of these nicks in standard reference preparations of hCG, to determine the enzymes that may be responsible for generating the peptide bond cleavages, and to devise means of separating nicked from intact hormone. The standard reference preparations of hCG, which are purified from a commercial product made from large pools of pregnancy urine, were found to have varying concentrations of nicked hormone. The preceding report showed that 11 of 13 hCG preparations isolated from individual pregnancy urine samples were nicked at the beta 47-48 bond, with 2 of 13 having a second nick at beta 44-45. As shown here, all of the hCG reference standards are nicked to similar extents at both the beta 47-48 bond and the beta 44-45 bond. The percentage of peptide bond nicking in the various hCG standard preparations ranged from 10-20% and appeared higher in the more recent preparations. We showed that human leukocyte elastase is capable of specifically cleaving the beta 44-45 bond, and in extended digests it can also cleave the beta 48-49 and beta 51-52 peptide bonds. Thus, human leukocyte elastase may be the origin of some of these cleavages in the individual samples and the reference standards. Furthermore, we report that a monoclonal antibody directed to hCG alpha-beta dimer binds preferentially to nonnicked hCG and much less to nicked hCG.

The heterogeneity of human chorionic gonadotropin (hCG). III. The occurrence and biological and immunological activities of nicked hCG.

Nicks, or missing peptide linkages, have been found in hCG beta-subunit between residues 44 and 45 and between residues 47 and 48. We examined the occurrence and biological and immunological activities of nicked hCG. As shown by sequence analysis, CR127 standard hCG is approximately 20% nicked, half at beta 44-45 and half at beta 47-48. Treatment with human leukocyte elastase increased the extent of nicking of CR127 standard hCG. The longer the incubation of CR127 standard with human leukocyte elastase (0, 2, and 21 h), the greater the extent of nicked hCG (20%, 46%, and 89%). As the extent of nicking increased, the receptor-binding ability diminished, as did the ability to stimulate progesterone production by rat corpus luteal cells in vitro (0.9, 0.74, and 0.29 microgram/microgram hCG, respectively). In a regression analysis, a linear relationship was indicated between the extent of nicking and receptor binding values (97% correlation) and between the extent of nicking and steroidogenic activity in vitro (99% correlation). From the intercepts of the regression lines, it was estimated that nicks reduced receptor binding by 11-fold and reduced the steroidogenic activity of hCG by 5-fold. We examined eight individual hCG preparations, three purified from pregnancy urine, three from urine from patients with hydatidiform mole, and two from urine from women with choriocarcinoma. In descending order, the eight individual hCG preparations were 100%, 100%, 85%, 76%, 42%, 41%, 0%, and 0% intact. Although no correlation was observed between the percent intact and the ability of the eight individual samples to displace 50% [125I]hCG in binding CG/LH receptor (r less than 0.5), a close correlation was noted between the percent intact and the steroidogenic activity in vitro (98% correlation). This separated the effects of nicking on receptor binding and steroidogenic activities and indicated that while multiple factors influence receptor binding, only nicking suppresses the steroidogenic activity of bound hCG. We examined the recognition of nicked hCG molecules by different hCG immunoassays. The Hybritech Tandem assay measured total hCG and did not distinguish nicked and intact hCG molecules (in a regression analysis, immunoactivity vs. percent intact hCG, r less than 0.5). In contrast, the immunometric assay using B109 hCG dimer-specific monoclonal antibody and anti-beta-peroxidase only detected the intact component of hCG (in a regression analysis, immunoreactivity vs. percent intact hCG, 98% correlation). We used these assays together to estimate the percentage of intact hCG and to deduce the extent of nicking.(ABSTRACT TRUNCATED AT 400 WORDS)

Beta-core fragments are contaminants of the World Health Organization Reference Preparations of human choriogonadotrophin and its alpha-subunit.

Highly purified preparations of human choriogonadotrophin (hCG), hCG alpha and hCG beta, including those preparations which are being distributed by the World Health Organization as International Standards, cross-reacted in a new radioimmunoassay with increased relative specificity for the beta-core fragment of hCG. A major portion of the beta-core immunoreactivity in the hCG and hCG alpha preparations eluted from Sephadex G-100 in a position (approximate apparent molecular size 15,000-18,000) corresponding to that of purified beta-core fragment prepared from pregnancy urine. However, in the case of hCG beta-subunit preparations, virtually all of the beta-core cross-reacting material eluted from Sephadex G-100 in the same fractions as the native hCG beta-subunit. Quantitatively, the cross-reacting beta-core material accounts for less than 1% (w/w) of the total hCG or subunit immunoreactivity, as measured by conventional radioimmunoassays. The presence of the beta-core fragments as discrete molecular components of the hCG and hCG alpha preparations should be borne in mind when these preparations are used to calibrate new radioimmunoassays for hCG-related molecules.

Pathophysiological importance of various molecular forms of human choriogonadotropin.

Human chorionic gonadotropin (hCG), its subunits and fragments are widely used for diagnostic purposes. In addition to the diagnosis of pregnancy and pregnancy related disorders, hCG determinations are used for diagnosis of trophoblastic and recently also nontrophoblastic tumors. The use for diagnosis of nontrophoblastic tumors requires highly specific and ultrasensitive assays. With these, it is possible to measure the concentrations of both hCG, the free beta-subunits and the so called beta-core fragment in healthy subjects. Therefore it is important to establish reference values for these and also to be aware of the influence of physiological factors on the serum and urine concentrations. Improved standardization of the assay methods is also essential for these novel applications of hCG determinations to become useful.

Use of an immunoradiometric assay and a radioimmunoassay for the detection of free beta-human chorionic gonadotropin.

An immunoradiometric assay and a radioimmunoassay (RIA) were used to quantitate human chorionic gonadotropin (hCG) in the sera of ten pregnant women at term and of six women with gestational trophoblastic neoplasia. The two techniques show good correlation (Pearson correlation coefficient .96) in the assay of pregnancy serum. Because only the RIA, and not the immunoradiometric assay, measures the free beta-subunit of hCG, a comparison of the results obtained by the two immunoassay methods permits a semi-quantitative assessment of the free beta-subunit. The numerical results may not reflect the actual concentration of free beta-subunit in that two different immunoassay methods are used.

Urinary gonadotropins in management and prognosis of testicular tumor.

Ninety-three cases of germinal testicular tumors with urinary gonadotropin (HCG) values, estimated by biologic or radioimmunoassay techniques, were reviewed. Preoperative values of HCG and subsequent postoperative values were related to response to treatment and prognosis. Twenty-five patients (26.9 per cent) comprised the seminoma group, and 68 patients (73.1 per cent) the nonseminomatous groups of germinal neoplasms. High HCG values were observed in the nonseminomatous group pre- and postoperatively by both assay procedures. Most significant was the high HCG values postoperatively (p smaller than 0.05, x2 equals 5.21 and p smaller than 0.05, x2 equals 5.23) for the biologic assay and radioimmunoassay, respectively, with high HCG values being associated with a poor cumulative 24 per cent two-year survival rate. Urinary HCG assay is of prognostic value in the ongoing management of testicular neoplasms. In the future, serum HCG assays may be of additional benefit.

Measurement of inaccuracy and imprecision of HCG methods using dilutions of the WHO 4th IS-HCG standard and a pregnant patient's serum.

INTRODUCTION: Differences in human chorionic gonadotropin (hCG) results obtained by seven different methods were documented by analyzing dilutions of the WHO 4th International Standard (IS) and a pregnant patient's serum. MATERIALS AND METHODS: Biases of +30.9 to -37.5% and +36.8 to -36.1% from the target concentration were found for the WHO 4th IS and patient sample dilutions, respectively. RESULTS: Imprecision was calculated from replicate measurements of hCG on the different sample dilutions. Imprecision ranged from 1.0% to 18.9% and 1.1% to 5.3% for the WHO 4th IS and patient sample dilutions, respectively. Using a maximum allowable error of 12.5% for hCG measurements, we found that two instruments were so biased that their hCG measurements could not be interchanged with hCG values produced by any of the other systems. DISCUSSION: It is ideal to use only one hCG methodology for the serial monitoring of hCG; otherwise, hCG methods should be carefully chosen to minimize inter-method bias.

Serum human chorionic gonadotrophin levels in early pregnancy.

Serum hCG reference intervals for various gestational periods in normal pregnancies were determined using three commercial assays--two standardized against the WHO 2nd IS (Amersham Amerlex-M beta HCG RIA (AMX) and Abbott beta-HCG 15/15 (ABB] and one standardized against the WHO 1 IRP (Hybritech Tandem -E HCG (HYB]. Serial samples from patients with accurately determined gestational periods were analyzed. We correlated these assays to determine the validity of the common practice of interchanging values between assays using the same WHO standard and of converting 1st IRP values to 2nd IS values by a fixed factor. The slope of correlation between the two 2nd IS assays (AMX, ABB) was 1.43, r = 0.960; whereas between the 1 IRP assay (HYB) and the two 2nd IS assays the slopes were 1.67, r = 0.963 and 1.22, r = 0.971 for AMX and ABB, respectively. In a prospective study of 52 patients with normal pregnancies, serum beta-hCG values in 46% of samples taken at 28-35 days gestation fell below the lower limit of the reference curves supplied with the AMX kit. Ninety-two percent of samples were within the newly established intervals. These results indicate that supplier's reference limits may not be accurate; in addition, a common factor should not be used to convert values from one commercial kit to another.

Use of beta-human chorionic gonadotropin in gestational aging.

Quantitative radioimmunoassay (RIA) of the beta chain of human chorionic gonadotropin (B-hCG) in serum has been used to evaluate the gestational status of 99 normal early pregnancies in contrast to 29 ectopic, threatened, aborted and/or terminated cases. Quantitative measurement of serum B-hCG-RIA standardized against the second international standard (2dIS) accurately established age of normal pregnancies in utero up to but not after three weeks postconception and with an accuracy of plus or minus four days between the third and eighth week of gestation. Quantitative urinary hCG-RIA standardized against the 2dIS were not useful for gestational aging. Useful serum hCG-RIA were identically linear and parallel with the 2dIS, had negligible crossreactivity with LH, FSH and/or TSH, and had low nonspecific binding. Of 13 hCG-RIA evaluated, only assays having these latter characteristics were able to detect ectopic pregnancies, spontaneous abortions, and/or threatened pregnancies with up to 90 percent accuracy. However, some assays not standardized to the 2dIS gave over 200 percent error in hCG serum values. Thus, correct choice of quantitative B-hCG reagents is necessary for early pregnancy assessment.

Quantitation of human chorionic gonadotrophin (hCG) by radioreceptor assay.

A sensitive radioreceptor assay was developed for pharmaceutical preparations of human chorionic gonadotrophin with the use of rat testicular membranes as receptor preparation and human 125I-chorionic gonadotrophin as tracer. The addition of unlabelled human chorionic gonadotrophin or luteinizing hormone inhibited the binding of 125I-chorionic gonadotrophin to the receptors in a concentration dependent way. Concentrations of human chorionic gonadotrophin between 30-300 mIU ml(-1) were normally used for a three-dose assay fulfilling pharmacopoeial statistical requirements for assay validity. The relative standard deviation for five assays was 7%. Estimates of potency of commercial preparations of human chorionic gonadotrophin obtained with the radioreceptor assay correlated well with corresponding estimates from in vivo assays. The proposed radioreceptor assay, however, provides a considerable saving in the number of animals required, requires less technical support, and is more precise than the in vivo method.

Modern management of ectopic pregnancy.

The modern management of ectopic pregnancy has been influenced greatly by recent advances in human chorionic gonadotropin determination and ultrasound. Serum progesterone determination holds promise as a means of identifying abnormal gestations. Early diagnosis of tubal pregnancies has prompted conservative surgical treatment and the use of medical therapy in selected cases. Because of the improvement in diagnostic aids and conservative treatment, we are documenting a change in epidemiologic profiles. The incidence of ectopic pregnancy has increased, with a concomitant decrease in mortality. Fertility after conservative surgical procedures appears improved over that with radical treatment. However, women with ectopic pregnancies continue to have reduced fertility potential.

PIP: The number of ectopic pregnancies reported to the Centers for Disease Control quadrupled between 1970 and 1985. Nearly 78,400 cases, or 1 in 66 pregnancies, were reported in 1985. Over the same 15-year interval, there was a sevenfold decline in maternal deaths related to ectopic pregnancies, 33 such deaths reported in 1985. The decreased mortality rate is due to earlier diagnosis of the condition, which allows for earlier surgical intervention. Extensive use of laparoscopy, wide availability of HCG radioimmunoassays, and technical advances in ultrasound have resulted in earlier diagnosis of ectopic pregnancy. Earlier intervention has potentiated the application of conservative surgical procedures, such as salpingotomy, salpingostomy, fimbrial expression, and segmented resection, to treatment of ectopic pregnancy. But despite improvements in surgical techniques, subsequent fertility in patients with ectopic pregnancy remains impaired. Recently medical treatment of ectopic pregnancy, usually performed with methotrexate, has been applied successfully, offering an additional treatment option.

The analytical specificity of human chorionic gonadotropin assays determined using WHO International Reference Reagents.

BACKGROUND: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone with considerable molecular heterogeneity. There is uncertainty regarding which hCG variants are detected by different hCG assays. The analytical specificity of 8 hCG assays was investigated. METHODS: WHO International Reference Reagents for hCG, nicked hCG (hCGn), beta subunit (hCGbeta), nicked beta subunit (hCGbetan), and beta core fragment (hCGbetacf) were individually added to hCG-free human serum. Specimens were analyzed with 8 commercially available hCG assays. Equimolar detection of hCG variants was defined as a recovery of 90-110%. RESULTS: All assays detected hCG and hCGn with mean recoveries of 98.3 and 94.6%, respectively. Seven assays detected hCGbeta (mean recovery 103.8%) but with high variation, and equimolar detection was observed only in four. The mean recovery of hCGbetan was 85.5% but was highly variable with only two assays showing equimolar detection. With a mean recovery of 53.4%, two assays detected hCGbetacf and both underestimated it considerably. Information provided by the assay manufacturer regarding hCG variant analytical specificity was inadequate or unclear in 75% of the assays. CONCLUSIONS: hCG assays vary considerably in their ability to detect different hCG variants. Manufacturers of hCG assays should clearly indicate the hCG variant specificity of their reagent systems.