Quantitation of IgG and IgM human anti-mouse antibodies (HAMA) interference in CA 125 measurements using affinity chromatography.

Currently no available immunoassay system offers complete protection against spuriously elevated or lowered results due to interference by Human Anti-Mouse Antibodies (HAMA). Although routine use of chromatography procedures is not an acceptable option because of the extra cost and workload involved, such a procedure would be highly desirable to ensure accurate immunoassay results. The present report describes a relatively simple affinity chromatography procedure using a HiTrap Protein G column to isolate immunoglobulin G (IgG) HAMA, followed by a HiTrap N-hydroxy-succinimide (NHS)-activated column coupled to goat-anti human immunoglobulin M (IgM) to bind IgM HAMA. To examine the usefulness of this purification procedure we determined CA 125 in forty serum samples prior to and following chromatography. Pre- and post-injection samples were obtained from 20 patients injected with 1 mg of 111In-labelled murine OC 125 F(ab')2 fragments in an immunoscintigraphy study. It is shown that this analytical procedure provides a technique to determine the extent and the nature of the existing HAMA interference in samples of patients after in vivo use of monoclonal antibodies for diagnostic or therapeutic purposes. The procedure can also contribute to the clarification of clinically discordant CA 125 results. Finally, the availability of such a procedure in the clinical laboratory provides an opportunity to test the robustness of newly developed immunoassay systems towards HAMA interference.

Comparison of four 'second generation' immunoassay systems to determine CA 125 in serum by using a graphical approach to method comparison analysis.

Clinical management of ovarian cancer patients is facilitated by CA 125 determinations in serum. Presently, several assay systems based on different concepts and different methodologies are available to measure CA 125. Method comparison analysis of such assay systems is usually performed through (linear) regression analysis, which requires assumptions about the distribution of experimental data and its measurement error. The aim of the present study was to compare four newly developed second generation assay systems for quantitation of CA 125 by utilizing an alternative simple approach to method comparison analysis. This alternative comprises the construction of relative difference plots and mountain plots, previously described by Krouwer et al. (Eur J Clin Chem Clin Biochem 1995; 33:525-7). In addition, the diagnostic value of the assays was illustrated through receiver-operating-characteristic (ROC) curves. Sera obtained from 300 women were assayed for CA 125 using the Abbott IMx CA 125 assay (Abbott), the Centocor CA 125 II RIA assay (Centocor), the Berilux Ov testing kit for CA 125 (Behringwerke), and the CA 125 TR-FIA assay (Wallac Oy). Both the relative difference plots and the mountain plots revealed higher serum concentrations with the Centocor RIA II (Median +33%, P2.5 to P97.5: -25% to 161%) and Berilux (Median +28%, P2.5 to P97.5: -17% to 108%) compared to the Abbott IMx system. The TR-FIA assay system showed lower serum concentrations (Median - 17%, P2.5 to P97.5: -74% to 229%). The combination of relative difference plots and mountain plots demonstrated clearly the wide range of differences between CA 125 assays measuring the same analyte. The relative difference plots provided insight into the distribution of the differences over the range of measurement as well as the identification of outliers. A simple quantitative assessment of the median differences could be made from the overlaying mountain plots. The close correspondence observed between the ROC curves illustrated that assay systems for CA 125 differing in design (type of antibodies used) and format can produce similar results on group level. However, the results of the clinical evaluation underline the importance of the application of assay specific cut-off values.

The use of 'ultrasensitive' prostate-specific antigen assays in the detection of biochemical recurrence after radical prostatectomy.

OBJECTIVE: To determine the gain in lead time obtained when using ultrasensitive prostate-specific antigen (PSA) assays in the diagnosis of biochemical progression after radical prostatectomy. PATIENTS AND METHODS: The post-operative PSA serum concentrations of 137 patients who had undergone radical prostatectomy were evaluated retrospectively. From these patients, 12 were selected who showed biochemical recurrence, as measured by the Hybritech Tandem-E Singlepoint PSA assay. Samples of the serum frozen at the time of the initial analysis were thawed and PSA values were remeasured by the Abbott IMx PSA assay and the Tandem-E Multipoint PSA assay. Analytical thresholds (zero-dose + 3 SD) for the Tandem-E Singlepoint, IMx and Tandem-E Multipoint assay were 1.0, 0.04 and 0.04 ng/mL, respectively. The lead time to the detection of a recurrence obtained when using the IMx and the Tandem-E Multipoint PSA assay was compared with that attained using the Tandem-E Singlepoint PSA assay. As a control, PSA values were determined in 58 serum specimens of nine patients having no evidence of recurrence after radical prostatectomy. RESULTS: All 58 control specimens had PSA levels below the analytical thresholds of the three assays, except one which had a PSA serum concentration of 0.08 ng/mL, estimated by the IMx assay. When compared with the lead time obtained with the Tandem-E Singlepoint assay, the 12 patients with a biochemical recurrence had a median gain in lead time of 327 days (range 60-627) with the IMx assay and of 369 days (range 60-639) with the Tandem-E Multipoint assay. CONCLUSION: A PSA value > 0.04 ng/mL after radical prostatectomy heralds further biochemical progression. The use of the ultrasensitive IMx and the Tandem-E Multipoint assays provided more lead time, but there is no clear evidence that this gain is necessarily of benefit to the patient.

Review on the simultaneous determination of total prostate-specific antigen and free prostate-specific antigen.

BACKGROUND: The total prostate-specific antigen (t-PSA) in serum measured by PSA assays represents the sum of free (f-PSA) and PSA complexed with alpha 1-antichymotrypsin. The f-PSA/t-PSA (F/T) ratio in prostate cancer (PCA) patients is lower than in patients suffering from benign prostatic hyperplasia (BPH). This review summarizes the current literature on the clinical relevance of measurement of the F/T PSA ratio. METHODS: Discussed are: physiology of PSA, assays for t-PSA and F/T ratio, factors which bias the F/T PSA ratio, use of F/T PSA ratio in the detection of PCA, correlation with histological features, and pathological stage. RESULTS: Using the F/T ratio in the intermediate t-PSA range, a reduction of approximately 30% in biopsies can be accomplished in the detection of prostate cancer. CONCLUSIONS: The F/T PSA ratio could become a valuable tool in the differentiation of BPH from PCA. To accomplish this goal, an international standardization not only for the t-PSA measurement but also for the F/T PSA ratio must be a priority for manufacturers of PSA assays.

Humoral anti-OV-TL 3 response after the intravenous administration of radiolabelled Fab' or F(ab')2 fragments in ovarian cancer patients.

The human anti-mouse antibody (HAMA) response was determined in the serum of patients suspected of having ovarian cancer who underwent radioimmunoscintigraphy with either 99Tcm-OV-TL 3 Fab' (n = 20) or 111In-DTPA-OV-TL 3 F(ab')2 (n = 73). Blood samples were collected prior to and at several time points post-intravenous injection. The detection of HAMA was performed with an in-house OV-TL 3 F(ab')2-based sandwich-type immunoradiometric assay (IRMA). The homologous IRMA demonstrated that 8 of 20 (40%) patients had developed HAMA responses after injection of Fab' fragments and that 14 of 73 (19%) patients had developed HAMA responses after F(ab')2 administration. The subclass of the measured HAMA was analysed in a limited number of samples, showing IgG or IgM as well as mixed responses. The kinetics of the HAMA responses varied greatly. Our study showed the relevance of the sampling time and frequency: HAMA responses can be easily underestimated with a low sampling frequency. The homologous IRMA described in this study was able to quantify the OV-TL 3-specific HAMA responses. With additional assays, the subclass of the HAMA could be further analysed. Remarkably, the fraction of HAMA responders after injection of OV-TL 3 Fab' fragments was in the same range as the proportion of HAMA responders after F(ab')2 administration.

Analytical and clinical performance of improved Abbott IMx CA 125 assay: comparison with Abbott CA 125 RIA.

We compared the improved Abbott IMx cancer antigen (CA) 125 assay (cat. no. 7A89) with the Abbott CA 125 RIA. Serum specimens were from healthy perimenopausal women (n = 124) and from patients with benign gynecologic and nongynecologic diseases (n = 124), ovarian carcinoma (n = 104), or other malignancies (n = 193). The IMx assay detected as little as 0.193 kAU/L CA 125 (AU = arbitrary Abbott unit), demonstrated up to 29% overestimation upon serum dilution, low within-assay (2.7-5.6%) and between-assay (4.8-8.2%) CVs, and no high-dose hook effect < or = 46,000 kAU/L nor influence from human anti-mouse antibodies in serum of women injected with OC 125 F(ab')2. Values by IMx were 20% lower than by RIA for healthy perimenopausal women (n = 100; IMx = 0.80 RIA - 2.5 kAU/L), and at least 50% higher for those with benign or malignant ovarian disorders at concentrations < 100 kAU/L. Receiver-operating characteristic (ROC) curve analysis of ovarian neoplasma vs perimenopausal controls indicated a gain of specificity and sensitivity with the improved IMx assay over the RIA, but ROC performance was the same with either assay if patients with benign ovarian disorders were used as controls.

Circulating inhibin levels in lactating and nonlactating women.

OBJECTIVE: To investigate the role of inhibin in the human puerperium, by measuring serum levels of immunoreactive inhibin in both lactating and nonlactating women. DESIGN: Prospective, comparative, open study. SETTING: Department of obstetrics and gynecology of a university hospital. PATIENTS: Fourteen healthy women who delivered at term: seven lactating women and seven nonlactating women treated with the dopamine-agonist CV 205-502. MAIN OUTCOME MEASURES: Serum immunoreactive inhibin, PRL, FSH, LH, E2, and P. RESULTS: All women showed a rapid decline of immunoreactive inhibin levels during the first postpartum days. Thereafter the pattern depended on the way of feeding. Nonlactating women, with their rapid return of pituitary and ovarian function, showed increasing immunoreactive inhibin levels to a maximum on day 24 (950 +/- 180 U/L). Lactating women did not show ovarian activity despite high FSH levels, and immunoreactive inhibin stayed on a low level (230 +/- 40 U/L on day 24). There was a significant correlation between immunoreactive inhibin and E2. CONCLUSIONS: The rapid decline of immunoreactive inhibin (elimination of placental hormone) is followed by an increase in nonlactating women (production by the maturing follicle) and by persistently low levels in lactating women. The lack of adequate levels of immunoreactive inhibin in lactating women may be an explanation of the relatively high FSH levels during lactation.

CA-125 levels in cervical mucus during the menstrual cycle.

OBJECTIVE: To determine CA-125 levels in cervical mucus (CM) during the menstrual cycle and their relationship to gonadal steroids and ovulation. DESIGN: Prospective study. SETTING: Two academic tertiary referral centers. PARTICIPANTS: Thirteen women with a normal fertility work-up. INTERVENTIONS: CA-125 and protein concentrations were measured in CM aspirated from the endocervical canal on alternate days in the early follicular and luteal phases and on a daily basis during the periovulatory period. Results were correlated with hormonal determinations, serum CA-125 levels, and ultrasound examination. RESULTS: Twenty ovulatory nonconceptional cycles were analyzed. Although the mean (+/- SD) concentration of CA-125 in CM (173,900 +/- 128,900 arbitrary U/mL) appeared relatively constant along the cycle, a large variation among the different samples was observed, ranging from 9,000 to 830,000 arbitrary U/mL. No clear trend could be detected as related to hormonal changes and ovulation. However, when the mucus CA-125 concentration was multiplied by the total volume of the correspondent sample, a clear periovulatory increase of total CA-125 levels was found. This was further supported by a similar trend showed by the calculated CA-125:protein concentration ratio. CONCLUSIONS: CA-125 is present in CM in high concentrations that vary widely along the cycle. Although no cyclical variation in CA-125 concentration could be determined, there was an apparent increase of total CA-125 levels parallel to the augmented mucus production during the periovulatory period. This further suggests a possible involvement of this glycoprotein in the secretory process of endocervical glands.

The phase of the menstrual cycle has no influence on the disease-free survival of patients with mammary carcinoma.

We evaluated the outcome of treatment for mammary carcinoma in 89 premenopausal women in relation to the phase of the menstrual cycle. The phase of the cycle was determined on the basis of serum concentrations of 17 beta-oestradiol and progesterone. The serum samples were collected 1 day prior to or on the day of operation. After a median follow-up of 4.1 years no significant differences in disease-free survival were found between the preovulatory (proliferative), periovulatory and post-ovulatory (luteal) groups. No differences in survival were found in these subgroups between the N0 and N1 subgroup. On the basis of this study we cannot confirm that the phase of the menstrual cycle during surgery has any effect on the eventual outcome in mammary carcinoma patients. However larger studies of this type are required before definitive conclusions can be reached.

Identification of patients with persistent trophoblastic disease by means of a normal human chorionic gonadotropin regression curve.

OBJECTIVE: Data from the Dutch Central Registry of Hydatidiform Mole were used to establish a reference human chorionic gonadotropin regression curve after molar pregnancy. STUDY DESIGN: A normal serum human chorionic gonadotropin regression corridor was constructed after fitting data from 130 patients with uneventful human chorionic gonadotropin regression after evacuation of a complete hydatidiform mole. Retrospectively, data from 77 patients with persistent trophoblastic disease were analyzed by means of this normal corridor. Measurements were performed with a radioimmunoassay for both native and free human chorionic gonadotropin beta-subunits. RESULTS: Human chorionic gonadotropin disappearance curves showed a biphasic decline with median serum half-lives of 1.8 and 12.8 days. Median time until normalization was 74 days (range 28 to 430). With the 95th percentile line, 71 of 77 patients (92%) with persistent trophoblastic disease could be identified. In > 50% of cases this could be achieved within 6 weeks from evacuation. CONCLUSION: The normal regression corridor allows identification of patients with persistent trophoblastic disease and an expectant attitude within the limits of the corridor.

Comparison of five immunoassay procedures for the ovarian carcinoma-associated antigenic determinant CA 125 in serum.

Interassay variability in CA 125 values was studied in 77 serum samples (covering a range of CA 125 values between 8.9 and 310 arb. U/ml as measured by the original Centocor RIA) using three 125I-labeled RIA kits (Centocor, Byk and Cis) and two enzyme-labeled immunoassays (Abbott and Roche). Taking the Centocor RIA as a reference, orthogonal regression equations resulted in slopes varying between 0.74 and 1.35, with y-axis intercepts varying between -6.5 and +6.2, and correlation coefficients ranging from 0.88 to 0.94. Compared with the results of the Centocor RIA, the EIAs of Abbott and Roche gave overall lower CA 125 values, whereas the Cis and the Byk RIAs gave higher assay results. At the 35 arb. U/ml Centocor cut-off, serum levels with the other assays varied between 23 and 53 arb. U/ml. The 65 arb. U/ml cut-off level corresponded with CA 125 serum levels between 45 and 94 arb. U/ml. When classifying CA 125 values in three clinically relevant categories based on Centocor RIA results, ('normal' < or = 35 arb. U/ml, 'slightly elevated' > 35- < or = 65 arb. U/ml and 'elevated' > 65 arb. U/ml), discordances ranged from 26% with the Cis RIA to 40% utilizing the Byk RIA. The five CA 125 assays tested do not give equal assay results. As a consequence, the interpretation of CA 125 serum concentrations should be done with caution in disease monitoring and in the assessment of ovarian masses, especially when using different serum assays.

Specific and nonspecific immunoassays to detect HAMA after administration of indium-111-labeled OV-TL 3 F(ab')2 monoclonal antibody to patients with ovarian cancer.

The development of human anti-mouse antibodies (HAMA) may cause problems in radioimmunotargeting studies, but may also improve survival of patients. To identify the presence of HAMA in blood samples from patients intravenously injected with 1 mg of 111In-labeled OV-TL3-F(ab')2, we developed three specific OV-TL 3-based HAMA assays and tested these along with two commercially available nonspecific HAMA assays (Sorin and Immunomedics). The specific assays were positive for HAMA with 10 postinjection serum samples from 7 patients. Eight of the 10 samples were also HAMA positive with one or both nonspecific HAMA assays. Conflicting results were observed with half the number of samples. The two nonspecific assays also reacted positively with another 11 serum samples from 5 patients including their preinjection samples. Despite some contradictory results, the nonspecific HAMA assays identify both pre-existent and Mab-induced HAMA, whereas the specific OV-TL3-based HAMA assays identify specific immune-responses occurring after the OV-TL 3 injection.

Comparison of four serum tumour markers in the diagnosis of colorectal carcinoma.

The assessment of the diagnostic power of four serum tumour markers, CEA, CA 19-9, CA 50 and CA 195 for colorectal carcinoma is described, according to recently formulated guidelines. Preoperative serum concentrations of the four markers were determined in 198 colorectal cancer patients and 57 patients with a benign colorectal disorder. The cumulative frequency distributions of the malignant and benign group show strong overlap for all markers, which indicates low diagnostic ability. This is confirmed by the Receiver Operating Characteristic curves, which have areas under the curve of 0.65 (95% confidence interval (CI) 0.58-0.73) for CA 19-9, CA 50 and CA 195 and of 0.70 (95%) CI 0.63-0.77) for CEA. The new tumour markers appear to be of slightly less diagnostic value than CEA for the primary diagnosis of colorectal cancer, although the discrepancy is not statistically significant. The low diagnostic power of CA 19-9, CA 50 and CA 195 may be due to a high proportion of colorectal cancer patients having the Lewis(a-b-) phenotype, who cannot synthesise these markers.

Evaluation of seven tumor markers (CA 50, CA 19-9, CA 19-9 TruQuant, CA 72-4, CA 195, carcinoembryonic antigen, and tissue polypeptide antigen) in the pretreatment sera of patients with gastric carcinoma.

Preoperative serum levels of the tumor markers CA 50, CA 19-9, CA 19-9 TruQuant, CA 72-4, CA 195, carcinoembryonic antigen (CEA), and tissue polypeptide antigen (TPA) were measured in 94 patients with well-staged adenocarcinoma of the stomach and in 15 patients with benign gastric diseases. In all patients with carcinoma, a laparotomy was done. The serum levels were correlated with the stage of disease, the location of the primary tumor, and the resectability and grade of differentiation. The marker CA 50 was the best, with an overall positivity of 59.5%. For CA 19-9, this figure was 34%; for CA 19-9 TruQuant, 22%; for CA 72-4, 34%; for CA 195, 29%; for CEA, 33%; and for TPA, 50%. The best combination of two markers was CA 50 and TPA; this combination gave a positivity of 81%. There was no evident correlation with stage of disease and the percentage of positive serum levels or the median serum levels. The marker CA 50 gave the widest range of elevated serum levels between the cutoff level and the 90th percentile (54%). Patients with carcinoma of the cardia had higher preoperative serum levels than those with a tumor in other parts of the stomach. There was no correlation with the resectability of the tumor and the preoperative serum level. Patients with an undifferentiated tumors did not have significantly lower serum levels than those with more differentiated tumors. Currently, preoperative determination of serum tumor marker levels in patients with gastric carcinoma has no significant in clinical practice.

The effect of chronic oral methadone treatment on monkey chorionic gonadotropin, estradiol, dehydroepiandrosterone sulfate, progesterone, prolactin and cortisol levels during pregnancy in the cynomolgus monkey (Macaca fascicularis).

The effect of chronic methadone treatment upon the serum levels of Estradiol (E2), Progesterone (P), Prolactin (Prl), monkey chorionic gonadotropin (mCG), dehydroepiandrosterone sulfate (DHEAS) and Cortisol (C) in pregnant Cynomolgus monkeys (Macaca fascicularis) is described in comparison with the hormone levels in a control group. Only DHEAS was significantly decreased in late pregnancy in the methadone group. From these data it can not be concluded that methadone treatment compromises (feto)placental function. The observed intra-uterine growth retardation in the methadone treated group might be a result of a direct influence of methadone upon growth.

Time-resolved fluoroimmunoassay for unconjugated estrogen in urine: comparison with a fluorometric assay for total estrogen and application in an in vitro fertilization program.

A time-resolved fluoroimmunoassay (TR-FIA) for unconjugated estrogens in human urine is described. 6-Keto-17 beta-estradiol-6-(O-carboxymethyl)oxime:bovine serum albumin is immobilized onto microtiter strip wells and the coated wells are incubated with 17 beta-estradiol standard preparations or unknowns with a polyclonal antiserum to 17 beta-estradiol-16,17-monosuccinyl:albumin. The antiserum-bound estrogen is detected by incubation with a europium-labeled anti-rabbit IgG that serves as both second antibody and tracer. After the immunoreactions, the bound portion of the labeled antiserum is quantified by dissociating the Eu3+ in a fluorescence-enhancement solution and measuring its fluorescence with a time-resolved fluorometer. The detection limit of the TR-FIA is 24 pmol of 17 beta-estradiol per liter; the analytical range extends to 1.8 nmol/L. This assay is a convenient alternative to radioimmunoassay and to the automated Kober-Ittrich fluorometry of total estrogen. Its advantages include short counting times; use of nonradioactive, stable reagents, all of which are commercially available; and more nearly complete automation. We conclude that this TR-FIA, compared with the Kober-Ittrich fluorometric assay (J Endocrinol 1957; 16:49-56), provides the clinician with equivalent information during follicular development therapy as part of an in vitro fertilization program.

Heterophilic antibodies in human sera causing falsely increased results in the CA 125 immunofluorometric assay.

An in-house OC 125 monoclonal antibody-based "sandwich" immunofluorometric assay (IFMA) described previously (Clin Chem 1987; 33:2191-4) gave higher results for CA 125 in 37 of 123 serum samples than did a commercially available immunoradiometric assay (IRMA). Discordant results between the two assays became concordant when measurements of the samples were repeated with normal mouse serum (100 mL/L) included in the IFMA reagent. The murine immunoglobulins are thought to block the ability of the heterophilic antibodies in the human serum samples to cross-link the labeled antibody with the solid-phase antibody. Using an enzyme immunoassay, we demonstrated human anti-mouse antibodies (HAMA) in most of the discordant samples examined. We tested the ability of nonimmune sera from other animal species to lower the apparent CA 125 concentrations of the spurious samples and observed that rat, goat, and sheep serum were less effective than mouse serum. One serum sample was discovered to give a falsely increased CA 125 result with the IRMA, but this increase could be prevented by adding murine serum to the IRMA reagent. We conclude that falsely increased CA 125 results are best prevented by adding murine serum (or murine antibodies) to the assay buffer.