[Proposal for a standardized protocol for the assay of nuclear estradiol receptors].
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Biomedical subjects
Publications and source records attributed to A Piffanelli.
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A new enzyme immunoassay (Abbott ER-EIA Monoclonal) for the determination of estrogen receptor in cytosols from breast tumor specimens has been developed by Abbott Laboratories. To establish the correlation of the results from this new technique with currently existing steroid binding methods, a multicenter study was conducted in eight European laboratories. All participants followed the same protocol consisting of a familiarization phase, a proficiency evaluation, and a comparison of existing steroid binding methods with the new immunoassay using panel samples and clinical specimens. ER-EIA was compared with the multipoint dextran coated charcoal assay in six laboratories, four of which followed the EORTC protocol; of the remaining two laboratories, one used a single saturating dose assay, the other an isoelectric focusing assay. The results show no significant difference between reducing agents when used in the ER-EIA. Reproducibility for the immunoassay (interassay coefficient of variation, 6%, interlaboratory coefficient of variation, 11-19%) was somewhat better than that for the steroid binding methods (interlaboratory coefficient of variation, 12-32%). The correlation between the methods was dependent on the origin of the lyophilized specimens. In breast tumor samples, an excellent correlation, (not statistically different from 1) was found between the ER-EIA and the steroid binding method in six laboratories. One laboratory showed a slope of 1.1 for the correlation line; the laboratory using isoelectric focusing showed a slope of 1.9. The mean value determined by the enzyme immunoassay in premenopausal women was 74 fmol/mg cytosol protein, and in postmenopausal women it was 187 fmol/mg cytosol protein with no significant difference in the slope of the correlation line. Results suggest the usefulness of the new standardized enzyme immunoassay for routine use in the clinical laboratory.
Since 1979 the quality control design proposed by the Italian ad hoc Committee has evaluated several lyophilized preparations with scalar receptor content; this permits the identification by linear regression analysis of systematic and non systematic errors. At present 41 laboratories from most of the national regions have joined the Italian Committee. The overall results of five years application of quality assurance in Italy show that there was a different pattern of imprecision with satisfactory indexes for intralaboratory performances but major variations in interlaboratory controls. There was also a remarkable difference of variability indices between the so-called "expert" and "new" laboratories; this problem can be reduced with practical seminars for new centers. On the basis of the results and experience achieved the Committee is starting another program of quality assurance for different new methodologies to provide guidelines for international working reference standards.
The importance of evaluating receptors for estrogen and progestin in human breast cancer has been pointed out by many authors. In the absence of a reference standard, receptor assays must be controlled by intra and interlaboratory quality control programs. Much interlaboratory variability exists due to non-uniform analytical protocols, non-uniform ligands, intrinsic errors and also errors in computation methods. The goals of our Italian Quality Control Program on Multicenter Trials are to standardize the analytical procedures and computation methods. Twenty Italian laboratories participated in the Quality Control Program. Each specimen was assayed for steroid receptor content according to the standardized dextran-coated-charcoal method. Data were subjected to computerized analyses by 5 different methods of calculation (Scatchard plot, direct plot, Lineweaver-Burk method, Brunauer-Emmet-Teller analysis, single-point approach). The results were than evaluated to identify intra- and inter-assay variation coefficients and to define other statistical parameters. The authors suggest different calculation methods depending on the specific experimental and/or physiopathological conditions.
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During the period September 1980-June 1984, abdominal US was performed by the authors on 103 patients with confirmed malignant lymphomas. The results indicate US has accuracy comparable to that of lymphography for lymph node enlargement; furthermore, US has the added advantages of being able to demonstrate mesenteric, renal hilar and other abdominal lymph nodes that are not commonly filled with contrast medium during lymphography. For these reasons, US is now frequently used in the diagnosis and staging of malignant lymphomas as well as for following up the results of therapy.(US = ultrasonography)
Administration of four different contrast media exerted slight but significant and prolonged effects on the hormonal thyroid status, in 53 euthyroid subjects (four subgroups), without any clinical expression of dysthyroidism. Changes in serum T3, rT3 and T4 demonstrated different trends for each iodinated compound, probably depending on the regulatory thyroid mechanisms, and CM pharmacokinetics. Plasma profiles of free fractions (FT3, FT4) slightly reflected consensual changes with circulating iodothyronines, at various time periods of study, demonstrating high correlation indexes with total fractions. The five hormones profiles correlation study suggest that different mechanisms could be responsible for some features encountered of "low T3 syndrome". These effects may be not only equivalent to giving various sources of iodide, but suggest more complex mechanisms. It is apparent that man has a remarkable and possibly unique system of homeostatic thyroid hormone regulation.
The possible relationships between hormone receptor status and several clinical (age, gynecologic history, clinical stage) and morphologic aspects (histologic grade, vascular invasion, lymphocytic infiltration, necrosis, fibrosis, elastosis and lymph node metastasis) were evaluated. A highly significant correlation between estrogen receptor levels, patient age, menses regularity and postmenopausal status was found. The histologic features most significantly related to tumor receptor status were histologic grade, lymphocytic infiltrate, necrosis and elastosis. Since these same histologic aspects appear to influence prognosis in breast cancer, the prognostic significance attributed to tumor receptor levels is substantiated. Therefore the importance of this assay is confirmed, not only for its diagnostic and therapeutic purposes, but also for its prognostic value.
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For nearly 60 years, since it was introduced by Graham and Cole (1924), oral cholecystography (OCG) has been used as the imaging technique of choice in investigation of gallbladder disorders. Recently, the future of OCG has come into doubt, principally as a result of ultrasonography, with the advent of high-resolution real-time scanning. Stones are seen as echoes within the lumen of the gallbladder and are associated with an acoustic shadow, findings which are highly specific. On this basis, to evaluate the accuracy of ultrasonography in detecting cholelithiasis, the A. have performed by real-time cholecystosonography 60 patients with not diagnostic OCG in a group of 546 patients affected by gallbladder diseases. Our results have confirmed that sonography revealed 4 normal gallbladders, 51 gallstones and 1 primary gallbladder cancer, with 5 cases of false positive. The true false negative rate has been difficult to determine, as surgery is usually not performed after a negative study. The accuracy of cholecystosonography for gallstone diagnosis was found to be 88,1% for all three Crade's categories.
Diagnostic results obtained by echography and lymphography in the study of abdominal lymphoglandular involvement in patients with lymphomas are compared. The data indicate that while lymphography is more revealing, the two examinations should be used together in staging. Echography, on the other hand, should be used for long-term surveillance, once the lymphographic contrast medium has been absorbed.
Plasma and red cell folate mean contents have been found to be significantly lower in 41 symptom-free beta-thalassaemia heterozygotes than in 21 controls. Such decreases must be considered as effects of an increased folate utilization caused by the enhanced total, both effective and ineffective, erythropoiesis. Since no close correlation has been found between packed cell volume and plasma or red blood cell folate levels, it seems that in 'healthy' beta-thalassaemia subjects, the degree of anaemia is not influenced by folate body reserves. However, the frequent finding of reduced red cell folate contents suggests that further folate imbalance might lead to clinically significant degrees of folate deficiency.
100 breast symptomatic patients have been examined with ultrasound, using an automated water-path scanner (Senomatic 3D CGR), and commercial B-mode or real-time scanners. All the patients were also examined with xeromammography. Normal breast patterns, as well as benign and malignant breast lesions, are presented.
The Coomassie-Blue method for protein determination is the EORTC uniformed technique for steroid receptor assay in tumor tissues. Many quality control trials have shown unacceptable interlaboratory variation coefficients for protein determination in steroid receptor assay. The objective of this study is to check the critical parameters of the method. The authors propose some suggestions on stability of dye-protein complex, ionic strength and temperature influence to improve the accuracy of the method.
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This article presents results of the first Italian quality control program for determining the estradiol receptor on lypophilized guinea pig and calf uteri. Despite considerable variability in quantitative terms, the results concur in ability to define samples as positive or negative for receptor content. One of the parameters that most strongly influences accuracy of determination of receptor concentration is protein assay. The evaluation of several lyophilized preparations at scalar concentrations permitted identification, by linear regression, for each laboratory of the systematic and non-systematic variables. More comparable results will be forthcoming when a standardized methodology program has been fully adopted.
In the Ferrara province of Italy, a screening programme for the detection of breast cancer was initiated in 1965; by 1976, 21,120 women had been examined. The biologic history of the women participating in the program is routinely collected at registration. Several biological variables can be extracted from the history. We used 15 biologic variables in order to construct discriminant functions between women affected by breast cancer and a group of control women. It was found that four variables suffice to separate the group of the women with breast cancer from the control group, although with some degree of uncertainty. The theory was advanced that the individual values of a discriminant function may be associated with the risk of healthy women for developing breast cancer. Data collected after the construction of the discriminant function hint at such an association. We have attempted to calculate a discriminant value for healthy women participating in screening programs, so that those having a discriminant value below a given threshold may be considered at higher risk than those above the threshold. The women in the high-risk group may then be submitted to an appropriate examination schedule.
Estradiol binding in preparations of female rat thymi is demonstrated by three different approaches (dextran-coated charcoal method, immunofluorescence technique and thin-layer gel filtration). Scatchard's analysis of [3H]-estradiol proves consistent with the existence of a single class of receptor sites, having a dissociation constant (Kd) of about 2 x 10(-10) M. Steroid binding specificity in the thymus is similar to that found in the uterus. On thin-layer gel filtration the binding appears in the 8S region similar to that observed for the uterus. With the immunofluorescence sandwich technique, estradiol proves to bind to a specific cytoplasmic component that subsequently migrates into the nucleus.