Immunometric assays of ras and c-erbB-2/neu overexpression in breast cancer: a pilot study.

The expression of the ras and c-erbB2 oncoproteins (p21 and p185, respectively), together with estrogen receptor (ER) and progesterone receptor (PgR) determination, has been retrospectively analyzed in 68 primary breast carcinomas and in 19 normal breast tissue samples. The aims of this study were: a) to explore the association between ras and c-erbB2 expression; b) to evaluate the relationship between ras and c-erbB2 expression and both steroid receptor status and the classical clinical and pathological parameters; and c) to compare two different methods for p185 determination. p185 and p21 were measured by enzyme immunoassay (EIA); p185 was also determined by Western blotting (WB); ER and PgR were assayed by radioligand binding assay. The highest value of p185 in benign breast lesions was used as the threshold to distinguish between positive and negative samples. With this threshold the c-erbB2 oncoprotein was overexpressed in 41.2% (with EIA) and in 50% (with WB) of cancer samples. The concordance rate between the two methods was 79.4. No significant association was found between p21 and p185 levels either in cancer or in normal breast tissue samples. Increasing levels of tumor p21 were associated with a shorter time to recurrence and overall survival. Increasing levels of p185 were associated with a significantly shorter time to recurrence (p185 EIA: p = 0.04, p185 WB: p = 0.029) and overall survival (p185 EIA: p = 0.04, p185 WB: p = 0.029).

Cathepsin D versus other prognostic factors in breast cancer. Results and controversies of a multicenter study on 2575 cases.

The aim of this study was the survey of cathepsin D determination in a large group of patients enrolled at several centers, under the coordination of the Italian Committee for Quality Control in the Oncological Laboratory. Cathepsin D was measured with the same methodology, under control of an intra and interlaboratory quality control program, in order to verify the comparability of cathepsin D results from different institutions and to analyze the frequency of cathepsin D positive cases in subgroups of patients stratified according to other prognostic parameters. This retrospective study included 2575 patients with primary breast cancer evaluated in 10 institutions. Cytosol from tumor tissue was the substrate for biochemical cathepsin D, estrogen receptor and progesterone receptor determination, with an interlaboratory quality control survey provided by the E.O.R.T.C. Receptor Group and the Italian Committee for Quality Control in the Oncological Laboratory. The results of the present study can be summarized as follows: 1) Cathepsin D is independent of menopausal status; 2) In spite of standardization of tissue handling and assay methods, different results may be obtained by different institutions. It is therefore essential that each laboratory calculates its own positive/negative cutoff values prior to any routine clinical use of the parameter. This should be a serious consideration when a multicenter study is planned.

Raised level of amniotic endothelin in pregnancies with fetal aneuploidy.

Amniotic fluid endothelin-1 (ET-1) levels were measured in 38 euploid and in 15 aneuploid pregnancies in the 17th gestational week. Varying distribution of the peptide levels was found in the two groups, with higher values in the pathological cases. Should this finding be confirmed in maternal blood, ET-1 could represent a further analyte to be used in prenatal screening for aneuploidy.

Determination of ErbB2 protein in breast cancer tissues by different methods. Relationships with other biological parameters.

Four different methods to measure in parallel the erbB2 protein expression (p185neu) were evaluated in order to: a) compare two enzyme immunoassays with the immunohistochemical assays (IHC) and western blotting (WB) and b) extrapolate eventual relationships between erbB2 and biological parameters. Tissue samples from 248 patients with primary breast cancer were consecutively assayed. We used two different cut-off levels for WB, ELISA, and EIA, defined as follows: 1) the highest level of expression of non malignant tissue was chosen as the discriminant threshold between 'low' and 'elevated' samples: 2) the elevated group was further subdivided into two subgroups: 'intermediate' and 'high', according to their median value. According to the first cut-off, the results were considered 'elevated' in about 52% of cases with the three biochemical methods, while using the second cut-off the percentage lowered to about 26%. Considering this cut-off, the concordance rates between the paired biochemical methods ranged between: 78.4% (WB vs EIA), 93% (ELISA vs EIA), and 82.6% (ELISA vs WB). The comparison between biochemical and immunohistochemical methods gave these concordance rates: 82% (WB vs IHC), 90.5% (ELISA vs IHC), and 85.5% (EIA vs. IHC). According to the first cut off level, 27.5% of tumor samples showed IHC detectable p185 levels, in agreement with other immunohistochemical studies. The relationship between high erbB2 and estrogen and progesterone receptors showed an inverse association. No relationship was found between erbB2 and axillary lymph node positivity or tumor size. In short, the results of the four methods seem generally well correlated; nevertheless, it appears that different methodological approaches of measuring p185neu are not completely equivalent, and there is a need for an authoritative standardization and quality control for clinical applications.

How alternative are immunoassay systems employing non-radioisotopic labels? A comparative appraisal of their main analytical characteristics.

The immunoassay is one of the most sensitive and reliable analytical techniques available in the clinical laboratory. The original label for immunoassays was radioisotopes, and these methods, radioimmunoassay (RIA) and immunoradiometric assay (IRMA) are still the reference methods, because of invulnerability of the radioactive emission with respect to environmental interference. Labels other than radioisotopes have been tested for use in immunoassay to improve the sensitivity and reliability and to avoid some of the disadvantages of radioisotopic techniques. New labels have continued to be developed (Horseradish peroxidase-HPR-, pyrophosphatase, luciferases, pyrodopirazines, europium cryptates, porphirins, phosphors) and new label detection methods have been set up (e.g. chemiluminescence assay, thermometric assay, NADP+ and FADP- based coupled assay). New immunoassay strategies such as simultaneous multianalyte automated test have been developed and the reliability of the assays has in some cases caused division among researchers about the choice between the radioisotopic immunoassay or the non-radioisotopic immunoassay, as considerable effort and investment had been devoted to the search for more sensitive and practicable tests than the classic RIA-IRMA methods. The evolution of immunoassays (Monoclonal Antibodies, non-radioactive tracers, automation) has produced systems which allow a large number of laboratories to determine a great number of analytes with very good practicability. The availability of fully automated systems has generated the opinion that analytical performance of immunoassays can be considered similar to that of many traditional parameters of clinical chemistry. This conclusion seems however too optimistic, in fact data collected from interlaboratory studies demonstrate that problems concerning the analytical reliability of the measurements still remain not completely solved. In the authors' opinion, this opposition between immunological assay based on isotopic or non-isotopic labels is misleading, because each assay (whether it uses isotopic, enzymatic, fluorimetric or luminescent labels) has its own analytical characteristics and performance. For this reason the term "alternative", used to indicate all non-isotopic assays as a unique class of tests, should be abandoned. From a theoretical point of view the choice should not be between isotopic and non isotopic techniques. For each analyte to be tested, it is advisable to use the immunological assay that suits the requirements of the laboratory, irrespective of type of label. From a practical point of view, the choice should be based on the analytical performance and on the characteristics of each assay, on its cost and the type of instrumentation available in the laboratory, and on the experience and the knowledge of the laboratory personnel.

Immunoradiometric versus enzymatic renin assay: results of the Italian Multicenter Comparative Study. Italian Multicenter Study for Standardization of Renin Measurement.

OBJECTIVES: The measurement of plasma renin activity (PRA) is very convenient for estimating the action of the renin system, but its interlaboratory reproducibility is notoriously poor. This multicentre study aimed to examine whether an immunoradiometric assay which quantifies renin directly with monoclonal antibodies can reduce this limitation of the enzymatic assay. The study also aimed to establish the reference values of immunoreactive renin (IrR) in a large sample of normotensive subjects and patients with various pathophysiological conditions. DESIGN AND METHODS: PRA and IrR were measured once in each of the eight participant centres in eight pool plasma samples with a wide range of renin content; in seven centres these measurements were repeated twice more in order to compare the intralaboratory interassay reproducibility of both methods. Finally, PRA and IrR were measured in the supine and standing positions in 503 subjects including normal controls, patients with various forms of hypertension, patients with Cushing's and Bartter's syndromes, patients with hepatic cirrhosis and pregnant women. RESULTS: We found that both the inter- and intralaboratory coefficients of variation for PRA measurements were higher than those for IrR. In plasma samples from normal subjects and from patients, mean +/- SEM supine PRA and IrR ranged, respectively, from 0.08 +/- 0.03 ng/ml per h and 2.6 +/- 0.5 pg/ml in patients with Conn's syndrome to 7.2 +/- 2.5 ng/ml per h and 138 +/- 51 pg/ml in patients with hepatic cirrhosis. PRA and IrR were found to be significantly correlated in all laboratories (mean +/- SEM of correlation coefficients 0.84 +/- 0.03) and for all of the conditions (correlation coefficient ranging from 0.98 in patients with Cushing's syndrome to 0.50 in pregnant women). However, for the pregnant women the slope of the regression line depicting the PRA-IrR relationship was significantly steeper than for all of the other conditions. CONCLUSIONS: In our experience the inter- and intralaboratory reproducibilities of the immunoradiometric assay appear to be greater than can be achieved with the enzymatic assay, the difference being probably due to the greater complexity of the latter. The two methods provide superimposable information on the renin-angiotensin system activity, except in pregnancy, during which the PRA:IrR ratio is much higher than in the other conditions. Therefore, in this and other pathophysiological situations associated with marked angiotensinogen concentration alterations, the enzymatic assay may be still preferable for assessing the activity of the system accurately.

Quality assessment of tumor marker determinations: a proposal for a supraregional scheme.

The use of tumor marker tests has increased progressively in the last decade concomitant with the advent of new monoclonal antibodies and their growing use in clinical oncology for various follow-up programs. External quality assessment (EQA) schemes widely adopted in clinical chemistry, have been extended in the last decade to immunoassays of hormones and tumor markers. EQA results can provide realistic information on the quality of the assays, performed under routine conditions. The goal of this article is to report the main results and discrepancies encountered so far in External Quality Assessment programs on tumor markers.

Comparison between single saturating dose ligand binding assay and enzyme immunoassay for low-salt extractable oestrogen and progesterone receptors in breast cancer: a multicentre study.

An excellent correlation between ligand binding assay (LBA) and enzyme immunoassay (EIA) for both oestrogen (ER) and progesterone (PR) receptors has been reported. Nevertheless, considering that the clinical value of any discrepancy between LBA and EIA probably varies with the receptor level, we undertook a collaborative study in which a single saturating dose (SSD) LBA and EIA were compared in different ER and PR dose ranges. The values of ER measured by EIA were higher in tumours with low or intermediate receptor content, causing a misclassification of ER status in 9% of cases (ER+: 77.5%, EIA, 68.8% SSD). In the case of ER, EIA values tended to be higher than SSD in all centres. For PR, EIA and SSD were generally more comparable (PR+: 66.0% EIA, 72.0% SSD, discordance rate 6%), with EIA showing, however, different trends in different centres. PR concentration was not significantly different in ER SSD-/EIA+ and in ER SSD+/EIA+ cases, suggesting that EIA detects at least in part integer ER. We conclude that although EIA may be a reliable methodological alternative to SSD, the two methods are not interchangeable until effective cut-off levels for clinical decisions are assessed for EIA.

Steroid receptor assays: an Italian quality assessment program.

An Italian Committee for the quality assessment of steroid receptor assay was instituted in 1979; the number of laboratories participating in this program increased from 7 in 1979 to 43 in 1989. The Italian program in collaboration with EORTC (European Organization Research and Treatment of Cancer) initiatives, utilizes as working standards lyophilized samples that tolerate prolonged storage at 4 degrees C. The national representatives agreed, according to EORTC, that all laboratories would perform, for measurement of steroid receptors, the dextran coated charcoal (DCC) method using the same concentration of radioactive ligands (3-H-estradiol, 3-H-ORG-2058) and would determine the non-specific binding with the same compounds (diethylstilbestrol and ORG-2058). The computation method is a potential source of interlaboratory variation; the multipoint Scatchard analysis, though difficult to apply for receptor problems, is the most widely used approach at present. The standardized DCC method is the most common and recognized (Food and Drug Administration) procedure for quantifying hormone receptor in human cancer. Since new technologies are being introduced (receptor enzyme immunoassay, EIA), adequate programs of quality assessment are required; in general, it has demonstrated an excellent correlation between radioligand binding assay (DCC) and immunochemical assay (EIA). Other prognostic factors, such as proteins produced by oncogenes and growth factors of the malignant cells have become more important. Some of these factors, as well as estrogen receptor status, seem the major determinants of recurrence after the first treatment.

Characterization of laboratory working standard for quality control of immunometric and radiometric estrogen receptor assays. Clinical evaluation on breast cancer biopsies. Italian Committee for Hormone Receptor Assays Standardization.

The objective of the study was to characterize a low-cost and reliable working standard material for quality control of estrogen receptor (ER) determination with dextran-coated charcoal (DCC) and enzyme immunoassay (EIA) methods. Human fibromatous uterine lyophilized cytosol demonstrated good characteristics of stability and applicability for this purpose. Eleven laboratories participated in the intralaboratory and interlaboratory quality control study, and they achieved slightly higher coefficients of variation for ER-EIA (interlaboratory, 37.7%; intralaboratory, 22.9%) than for ER-DCC (interlaboratory, 24.2%; intralaboratory, 15.7%). There was an excellent correlation between ER results with ER-EIA and ER-DCC for 268 breast cancer biopsies. Quality assurance for ER assays using DCC techniques and immunometric methods with monoclonal antibodies (ER-EIA) can be set up with this available material of human origin to satisfy the characteristics of both techniques and the species specificity of monoclonal antibodies.

Amiodarone and thyroid status in refractory arrhythmias.

Out of 20 subjects selected for refractory arrhythmias, amiodarone therapy (200 mg/day) was efficacious in 85%. No statistically significant variations in electrocardiographic parameters (QTc) were observed; similarly, there was little evidence of side effects 1 year after initiation of treatment. These results were most likely due to the low daily dosage administered. We observed: 1) a significant increase in rT3 levels; 2) a decrease in TT3; 3) a uniform homeostasis of free fraction (FT3;FT4) These effects are all characteristic patterns of a "Low T3 Syndrome". The dosage of circulating amiodarone in 6 patients with borderline hormonal status (3 hyper- and 3 hypothyroidism) was not found to be an efficacious test for therapeutic monitoring. Identification of a statistically significant linear regression relationship between cumulative dose of amiodarone and rT3 levels may be a useful test in clinical practise for establishing more appropriate therapeutic dosages. Furthermore, it provides a guideline for threshold levels (maximum rT3 = 100-110 ng/dl) which are in close association with several side effects.

[Predictive significance of reticulocyte count (interpretative methodologic approach)]. / Significato predittivo del conteggio dei reticolociti (approccio metodologico interpretativo).

This study concerns the comparison of two methodologies for reticulocyte count on two groups of 100 subjects. The evaluation of the results achieved with the two colorants (brilliant cresyl blue and pre-colored glasses with crystal acetate violet and methylene blue) suggests the application of more reliable parameters (reticulocyte index, reticulocyte production index) for the interpretation of reticulocyte counting. It seems desirable to correct the absolute reticulocyte number with the degree of anemia of the patient and to calculate the medullary production index to identify the effective reticulocytosis.

Testis cytological structure, plasma sex steroids, and gonad cytosol free steroid receptors of heterologous gonadotropin (hCG)-stimulated silver eel, Anguilla anguilla L.

Complete testicular maturation was induced in silver eels, kept at 24 degrees in fresh water, by a single injection of 1000 I.U. of heterologous gonadotropin (hCG). Each week, for 4 weeks, some eels were examined for testis structural pattern, plasma sex steroids, and gonad cytosol steroid receptors. The first effect of the hCG was on the tubular organization of the testis, followed by spermatogenesis. Plasma androgens were not detectable in the untreated eels, whereas a peak was detected a week after in those treated with the injection and afterward a decline. Plasma progesterone and estradiol showed a peak 2 weeks after treatment. Untreated eel gonads showed a high content of cytosolic free estradiol receptors which disappeared in the hCG-treated ones, a peak of free progesterone receptors was found 1 week after injection. The results are discussed in relation to the differentiation and maturation of eels testes.