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.

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.

Estrogen and progesterone measurement and its quality control in breast cancer: a reappraisal.

This article illustrates the two main methods for routine measurement of cytoplasmic estrogen receptor status in neoplastic biopsy. The first is the Dextran Coated Charcoal Technique (D.C.C. Assay) which is still the method of choice in the majority of clinical laboratories for its simplicity, reproducibility and low cost. The second is a more advanced technique based on the specific binding, enzymatically displayed, of commercially available antiestrogen monoclonal antibodies (Enzyme Immuno Assay - ABBOTT). The sui generis characteristics of endocrine sensitivity assessment on tumor tissues and the importance of decision-making connected with the assay justify rigorous quality assurance schemes. The quality control design proposed by the Italian Committee concerned the evaluation of several lyophilized preparations with scalar receptor content; this permits the identification through linear regression analysis of systematic and non-systematic errors. The Italian Committee has currently connected 50 labs from most regions of the country.

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.

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.

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).

Quality assurance for steroid receptor assay in human breast cancer: six years experience of the Italian Committee.

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.

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.

Hormone receptors and breast cancer: correlations with clinical and histologic features.

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.

Quality control for estrogen and progesterone receptor assay in human breast cancer: the influence of computation methods on intra and interlaboratory variability.

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.

Results of the Italian interlaboratory quality control program for estradiol receptor assay.

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.

[Clinical and instrumental study program of nodular diseases of the thyroid]. / Programma di studio clinico-strumentale delle affezioni nodulari della tiroide.

Authors have passed from a combined clinical and radioisotopic analysis to an integrated polyinstrumental approach by adding thermography and above all echography. In order to achieve the purpose Authors have confronted in retrospective a "clinico-radioisotopic group" of 103 cases, from 1965 to 1975, and an "integrated polyinstrumental group" of 53 cases, since 1976. Comparing the "clinico-radioisotopic method" with "polyinstrumental approach" the main results could be summarized as follows: the diagnostic specificity improved in benign lesions from 77% to 96%, however in malignant lesions it remained about the same (63%). Considering these results the Authors have subdivided thyroid nodules, mainly based on echography, in two distinct categories with the following orientation: I. Solitary Thyroid Nodule: a) definitely cystic -- demonstrated by echography; b) "cold" -- by conventional scintygraphy; c) "negative" -- by thermography; d) non uptake of tumor seeking radiopharmaceutical. The control, therapy and surveilance of these cases should be limited to needle aspiration of the cystic cavity followed, of course, by citologic examination. This aspirations is both diagnostic and possibly therapeutic. II. Solitary Thyroid Nodule: a) definite solid -- echography; b) "cold" -- convential scintygraphy; c) "positive" --thermography (possibly "negative"); d) non uptake of tumor seeking radiopharmaceutical. Here the therapeutic orientation is clearly surgical.

[Comparative diagnostic value of echography and oral radiologic examination of the gallbladder in relation to surgical findings]. / Valore diagnostico comparato dell'ecografia e dell'esame radiologico per os della colecisti al vaglio chirurgico.

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.

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.

[Value of various hematologic indexes proposed for identifying healthy carriers of the beta-thalassemic trait]. / Considerazioni sul valore di alcuni indici ematologici proposti per identificare il portatore sano del trait beta-talassemico.

The results of a screening for beta-heterozygous thalassemia conducted on 999 school-boys aged from 11 to 13 are the following: 7.5% of the subjects were taker of the thalassemia trait; 74 subjects over 75 with beta-heterozygous thalassemia had "Mean Corpuscolar Volume" (MCV) values below 70 fl. The results of the subjects with globular volume less than or equal to 79 fl (with or without beta-thalassemia) were used to compare the diagnostic accuracy of beta-thalassemia for the hematological indexes of England-Fraser, Mentzer, Shine-Lal and the MCV estimations. This comparison has shown better results in terms of sensibility and specificity for MCV with respect to the other indexes. A more extensive application of the study could be convenient in order to evaluate if the MCV level of 70 fl is effective to discriminate between beta-thalassemia and non thalassemic microcytosis.

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.

[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.

[The significance of antipolysaccharide C antibodies from Streptococcus group A. Their importance in the diagnosis of streptococcal infections]. / Sul significato degli anticorpi antipolisaccaride C dello streptococco di gruppo A. La loro importanza nella diagnosi di infezione streptococcica.

Here is underlined the role of quantitative determination of streptococcus A polysaccharide C antibody for the diagnosis of streptococcal infection. The Authors suggest to associate this determination with antistreptolysin titer and/or bacterial esoenzyme antibody titer, in order to achieve a wider spectrum of streptococcus induced antibodies for therapeutic and prophylactic applications.

[Macro-CK type 1: a methodological study approach and its clinical significance]. / Macro-CK tipo 1: approccio metodologico di indagine e suo significato clinico.

Description of an analytical procedure in order to show the atypical-CK carriers that simulate an abnormal raising of CK-MB circulating levels in dosages for immune inhibition. This procedure was applied to two samples taken from patients of such a type, immune form neoplastic and cardiac pathology. A systematic dosage of a "real" MB isoenzyme is desirable in all the cases of very raised CK-MB activity.