Biomedical subjects

D Sgoutas

Publications and source records attributed to D Sgoutas.

At least 19 recordsLinked to original sources

PubMed1993

Analysis of epidermal growth factor receptor gene expression in stained smears and formalin-fixed, paraffin-embedded cell pellets by reverse transcription intron differential polymerase chain reaction.

Previous studies have demonstrated quantitation of epidermal growth factor receptors (EGFR) to be of prognostic significance in breast, bladder, esophageal and other neoplasms. However, the relatively large quantity of unfixed tissue required for epidermal growth factor radioligand binding assays (RLBA) has precluded its application to cytologic specimens and small biopsy specimens. For this reason we evaluated reverse transcription intron differential polymerase chain reaction (RTIDPCR) as an assay of EGFR gene expression. Squamous cell carcinoma (A431 and SiHa), transitional cell carcinoma (HT1376, T24, RT4), mammary (MCF7) and endocervical (HeLa) adenocarcinoma, and leukemia (K562) cell lines were used to compare RTIDPCR and RLBA. RTIDPCR involved reverse transcription of RNA and amplification of cDNA using primers for beta-actin and EGFR. Good agreement was observed between the RLBA and RTIDPCR results. RNA extracted from fresh cells, Diff-Quik-stained smears and formalin-fixed, paraffin-embedded cell pellet sections yielded similar results. These data suggest that RTIDPCR may be useful in evaluating gene expression by cells processed as cytologic specimens.

Adenocarcinoma↗

PubMed1991

Automated immunohistochemical estrogen receptor in fixed embedded breast carcinomas.

The authors immunohistochemically assessed the presence of estrogen receptor (ER) in formalin-fixed, paraffin-embedded tissue sections of 68 breast carcinomas by an automated method using Pronase (CalBiochem, La Jolla, CA) predigestion and alkaline phosphatase detection (Method 1). These results were compared with those obtained by an automated peroxidase-antiperoxidase method with DNAse pretreatment of fixed embedded sections (Method 2), with ER immunostain on frozen sections (Method 3), and with biochemical results (dextran-coated charcoal cytosolic [DCC] assay). Compared with the DCC assay, Methods 1, 2, and 3 gave sensitivities of 54%, 25%, and 89%, respectively. The sensitivity for Method 1 was increased to 74% in those cases with DCC results showing greater than 50 fmol/mg protein. These findings indicate that ER immunohistochemical studies on formalin-fixed paraffin-embedded tissues (as assayed by Method 1) provide useful clinical information when the results are positive. A negative result, especially if surrounding normal elements are not positive, may indicate no receptors, receptor levels less than 50 fmol/mg protein, or improper tissue preservation. In the absence of fresh tissue for ER assay by DCC assay or of frozen sections for immunostaining, and with an understanding of its limitations, this method may be useful.

Automation↗

PubMed1991

Immunohistochemical progesterone receptor assay. Measurement by image analysis.

To determine the efficiency of image analysis in immunohistochemical progesterone receptor (PgR) measurement, 94 primary breast carcinoma tissue samples were evaluated for PgR by biochemical dextran-coated charcoal assay (DCC) and an immunohistochemical method. Frozen sections immunostained for PgR with a monoclonal antibody (Abbott PgR-ICA, Chicago, IL) and the peroxidase-antiperoxidase technique were scored semiquantitatively histologic score by microscopy and quantitatively (percentage nuclear area immunopositivity [PNA] using the CAS 200 image analyzer (Cell Analysis Systems, Elmhurst, IL). There was a positive correlation between dextran-coated charcoal assay and both histologic score (r = 0.82) and PNA (r = 0.69). Selected cutoff points of 60 histologic score and 6.5% PNA based on sensitivity/specificity calculations yielded a predictive value of a negative test of 73% and 80%, respectively, and a positive predictive value of 100% for both; ranges of fmol/mg protein PgR correspond to ranges of histologic score and PNA. The use of an image analyzer to measure PNA in PgR-immunostained sections is a viable alternative to dextran-coated charcoal assay, especially when insufficient fresh tissue is available.

Biochemistry↗

PubMed1991

Immunohistochemical estrogen receptor assay: quantitation by image analysis.

The immunohistochemical estrogen receptor (ER) assay quantitated by image analysis was compared with the biochemical dextran-coated charcoal (DCC) assay in 97 primary breast carcinomas. Frozen sections immunostained for ER with a monoclonal antibody (ER-ICA; Abbott Laboratories, Chicago, IL) and the peroxidase-antiperoxidase technique were scored semiquantitatively by microscopy (HSCORE) and quantitatively [percentage of nuclear area immunopositivity (PNA)] using the CAS 200 image analyzer (Elmhurst, IL). There was a significant and identical correlation between DCC and both HSCORE and PNA. According to sensitivity/specificity calculations, the cut-off points of 10 for HSCORE and 1% for PNA were chosen with positive and negative predictive values of 97.7% and 74.5%, respectively. Low DCC-positive cases between 10 and 50 fmol of ER/mg of protein were immunohistochemically ER positive only in 61.5%. Immunohistochemical results of greater than 150 HSCORE and greater than 15% PNA corresponded to high DCC results of greater than 150 fmol/mg of protein. Immunohistochemical assay quantitated by image analyzer (PNA) is a comparable alternative to biochemical ER, especially when insufficient fresh tissue is available.

Breast Neoplasms↗

PubMed1990

Predictive value of lipoprotein (a) and other serum lipoproteins in the angiographic diagnosis of coronary artery disease.

To determine the relation among lipids in predicting coronary artery disease (CAD), 213 patients undergoing diagnostic angiography for suspected CAD were prospectively studied. Twenty-one patients had normal coronary arteries and 192 had CAD in 1 to 3 arteries at arteriography with measurements obtained with digital calipers. Lipoproteins were measured and lipoprotein (a) [Lp(a)] was also assayed in a subset of 98 patients with CAD. Statistical analysis was performed using uni- and multivariate techniques to test the association among age, gender, systemic hypertension, diabetes mellitus, cigarette smoking, family history, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very low density lipoprotein cholesterol, apolipoproteins (apo) A-I and apo B, ratio of apo A-I to apo B, and ratio of HDL cholesterol to total cholesterol, to Lp(a) and to CAD. All factors except gender, systemic hypertension, diabetes mellitus and cigarette smoking were univariate predictors of CAD. Multivariate predictors were, in decreasing order of significance, family history, age, HDL/total cholesterol ratio and apo B. When Lp(a) was included, multivariate predictors were age, family history, apo B and Lp(a), in that order. Lipid parameters alone showed that the HDL/total cholesterol ratio and that Lp(a) provide the best predictive tests for the detection of CAD in this referral population and may ultimately become important screening tests for CAD.

Coronary Angiography↗

PubMed1990

Estrogen receptor status in malignant melanoma.

Estrogen receptor (ER) positivity demonstrated in malignant melanomas by histochemical and biochemical assays suggested the possibility of hormonal management and improved prognosis as for breast carcinoma patients. We studied the ER status of 5 primary and 28 metastatic malignant melanomas with a commercial immunohistochemical kit (ER-ICA monoclonal), that utilizes monoclonal anti-ER and a peroxidase-antiperoxidase technique, and by a histochemical method using fluorescein-conjugated estradiol (Fluoro-Cep Estrogen assay), on frozen sections. In addition, we conducted a biochemical assay [dextran-coated charcoal cytosolic assay (DCC)] in 16 cases. All 33 cases were ER negative by ER-ICA and Fluoro-Cep: 11 biochemical assays were negative (less than 3 fmol ER/mg protein), four were in the borderline range (3 to 10 fmol ER/mg protein), and one was positive (greater than 10 fmol ER/mg protein) at 11 fmol. The melanomas in 97% of the cases we studied were ER negative by two or three different assays. Low-level estrogen binding of MM tissues may be the result of interactions other than with Type I true ER. The low frequency of ER positivity of malignant melanomas appears to preclude the clinical use of ER status as an indicator for response to hormonal manipulation in patients with malignant melanoma.

Female↗

PubMed1989

Automated immunohistochemical estrogen receptor in fixed embedded breast carcinomas. Comparison with manual immunohistochemistry on frozen tissues.

Estrogen receptor (ER) status of breast carcinomas determines prognosis and treatment. Biochemical ER assays are expensive and time-consuming and require fresh tumor. Immunohistochemical ER was assessed in 68 breast carcinomas, by an automated method using routinely processed formalin-fixed paraffin-embedded tissues, and manually with the use of snap-frozen tissues with a monoclonal anti-ER and peroxidase-antiperoxidase technique. The paraffin sections were digested with DNase to enhance development of signal. Positive nuclear ER was obtained in 9 (13%) fixed tissues and 36 (53%) frozen tissues. The sensitivity, specificity, and predictive value of a positive test result, as compared with the biochemical assay, were 25%, 100%, and 100% for the paraffin section technique, and 89%, 88%, and 89% for the frozen sections. Although it is specific, lack of sensitivity, resulting from loss of ER with fixation and room temperature handling, renders this immunohistochemical technique unacceptable on fixed tissues. However, ER immunostain on frozen tissue is an acceptable alternative to biochemical assay.

Autoanalysis↗

PubMed1989

Reversible impairment of gonadotropin secretion in critical illness. Observations in postmenopausal women.

Gonadotropin levels were measured in 65 postmenopausal women admitted to the intensive care unit. Sixteen patients (25%) were hypogonadotropic as measured by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels of 5 IU/L or less, and only 20 patients (31%) were found to have gonadotropin levels within the expected postmenopausal range (greater than or equal to 30 IU/L). No significant differences in prolactin or free thyroxine levels were found, but the cortisol level was higher and the total thyroxine level lower in the hypogonadotropic group. The patients in the hypogonadotropic group also seemed more seriously ill, with a longer duration of hospitalization, an increased total number of days in the intensive care unit, an increased number of days in the intensive care unit before gonadotropin sampling, a higher incidence of parenteral hyperalimentation, lower serum albumin levels, and higher mortality. Stimulation of the gonadotropin-releasing hormone performed on 10 patients showed a blunted response in patients with baseline LH and FSH levels of 10 IU/L or less. Of 10 patients followed up sequentially, 6 showed a rise in gonadotropin levels that paralleled recovery, 1 died with low gonadotropin levels, and 3 continued to have low gonadotropin levels 17, 34, and 82 days, respectively, after initial screening. Critically ill postmenopausal women may exhibit a marked fall in gonadotropin, LH, and FSH levels. This disease in gonadotropin levels in response to illness is reversible and results from pituitary unresponsiveness to gonadorelin hydrochloride.

Aged↗

PubMed1988

Long-term study of 435 MHz radio-frequency radiation on blood-borne end points in cannulated rats. Part II: methods, results, and summary.

This study investigated the effects of long-term, low-level exposure to radio-frequency radiation (RFR) on various physiological systems in a large rodent population. Two hundred adult male white rats with chronically implanted aortic cannulas were randomly divided into two groups. Animals in the first group were exposed to the low-level RFR environment for approximately 22 hours daily, seven days a week, for six months. Depending on animal orientation within the home cage (all animals singly caged) the estimated whole-body specific absorption rate (SAR) ranged from 0.04 to 0.4 W/kg. The estimated mean whole-body SAR ranged from 0.3 W/kg (medium-sized rats) to 0.35 W/kg (large-sized rats). A second, sham-exposure group was maintained under identical conditions, but were not radiated. Microsamples of blood were withdrawn on a cyclic schedule from the unanesthetized and unrestrained rats. The blood samples were assayed for plasma adrenocorticotropin (ACTH), plasma corticosterone, plasma prolactin, plasma catecholamines (norepinephrine, epinephrine, and dopamine), hematological end points (hematocrit ratio, complete red blood cell count, complete white blood cell count, and a differential count of neutrophils, eosinophils, and monocytes), and cardiovascular end points (heart rate and mean arterial blood pressure). Analysis of the results showed no significant RFR-induced differences in these end points when the RFR-exposed group was compared to the sham-exposed group. Chronic exposure to the low-level, pulsed field resulted in no adverse effects on animal health, as measured by the spectrum of blood-borne end points.

Adrenocorticotropic Hormone↗

PubMed1986

Interaction of cyclosporin A with human lipoproteins.

The ultracentrifugal fractionation of human serum after previous incubation with cyclosporin A showed that, in healthy fasting individuals, 8% of cyclosporin A was found in the very low density lipoproteins (VLDL), 31% in the low density lipoproteins (LDL), 46% in the high density lipoproteins (HDL) and 15% in the non-lipoprotein protein fraction. In non-fasted, healthy and in non-fasted, lipaemic individuals, 7 and 6% of cyclosporin A was found in chylomicrons, 9 and 13% in VLDL, 28 and 30% in LDL, 39 and 37% in HDL, and 12 and 13% in the non-lipoprotein protein fraction, respectively. In patients receiving cyclosporin A the distribution varied from 12 to 19% in VLDL, 21 to 28% in LDL, 33 to 43% in HDL, and 13 to 20% in non-lipoprotein proteins. The interaction between cyclosporin A and isolated normal human lipoproteins was also studied by ultrafiltration. All lipoproteins exhibited a non-saturable, low affinity, high capacity uptake for cyclosporin A. Analysis of the uptake by phospholipid vesicles showed a similar uptake, suggesting that cyclosporin A dissolves in the lipophilic portion of the lipoprotein molecule rather than being associated with specific binding sites.

Cyclosporins↗

PubMed1984

Comparison of phosphotungstate and dextran sulfate-Mg2+ precipitation procedures for determination of high density lipoprotein cholesterol.

Because of the controversy over the best method for assaying high density lipoprotein (HDL) cholesterol in the clinical laboratory, a commonly used phosphotungstate method for precipitating low density and very low density lipoproteins (LDL and VLDL) was compared with a recently recommended dextran sulfate precipitation method. The accuracy and precision of HDL cholesterol determinations were similar for both methods. Either of these procedures would appear to be equally satisfactory for the assay of HDL cholesterol in the clinical laboratory.

Apolipoprotein A-I↗

PubMed1983

A simplified ultrafiltration method for determination of serum free cortisol.

We describe the suitability of the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane for measuring free cortisol in serum. The method combines two independent assays: total cortisol and the ultrafiltrate fraction of added [3H]cortisol. The unbound fraction is determined in 0.25-0.30 ml of ultrafiltrate collected from 0.6 to 1 ml of serum that has been equilibrated with [3H]cortisol at 37 degrees C for 20 min. The assay is rapid (less than 1 h), practical ( no more than 0.6 ml of serum is necessary) and repeatable (CV: 3.8% within-assay and 12.2% in different assays). Error introduced in free cortisol measurement due to dilution effects in dialysis is systematically defined, and the effect of tracer purity on the ultrafiltration method is examined. Dialyzed sera from normal men and women, from patients with Cushing's disease and adrenal insufficiency, and from pregnant women gave ultrafiltration results that accurately duplicated those obtained by previous dialysis.

Adsorption↗

PubMed1983

A simple ultrafiltration method for determining unbound estradiol in serum.

The Amicon MPS-1 centrifugal ultrafiltration device and the YMT membrane were used to measure free estradiol in serum. Two independent assays were required. The first measured total estradiol-17 beta and the second ultrafiltrable fraction. The unbound fraction was determined by counting [3H]estradiol-17 beta in 0.15-0.3 mL ultrafiltrates of 0.5 to 1 mL of serum that had been equilibrated with [3H]estradiol-17 beta at 37 degrees C. The assay is rapid (1 h), practicable (requires 0.6 mL serum) and reproducible (CV 4.6% within assay and 6.5% between assays). Samples from apparently healthy men and women and from pregnant women gave results that agreed with those obtained by equilibrium dialysis. The percentages of free serum estradiol-17 beta were similar during both early and late follicular phases of the menstrual cycle. Samples from women with hirsutism gave values for total and free estradiol-17 beta which were not significantly different from those of normal females when no consideration was given to the phase of the menstrual cycle. The means for both of these female groups were significantly different from those of males.

Adult↗

PubMed1983

Elevated free testosterone levels in women with acne.

Female patients with varying grades of acne were examined to determine whether free testosterone levels were a more sensitive indicator of hyperandrogenism than total testosterone values. Of 24 women with acne studied, four were found to have elevated total testosterone levels, whereas 11 had elevated free testosterone levels. Twenty-four age-matched female control subjects without acne, hirsutism, or irregular menstrual cycles all had normal free and total testosterone values. The mean concentration of free testosterone in the patients with acne was 1.035 ng/dL, while in the control subjects it was 0.54 ng/dL. Other than hirsutism in three patients with acne with elevated free testosterone levels, there were no morphologic clues identified to determine which patients with acne are more likely to have abnormal free testosterone levels. The free testosterone level did not correlate with the type, distribution, or severity of the acne present in the patients.

Acne Vulgaris↗

PubMed1983

Effect of Fluosol-DA on radioimmunoassay results.

Fluosol-DA, an emulsion of perfluorocarbons, is used as a blood substitute. Its presence in samples affects results of certain radioimmunoassays routinely done in our laboratory. In several, added Fluosol-DA caused an upward displacement of the standard curve, the extent of which depended on the structure and quantity of the assayed antigen, the concentration of Fluosol-DA, and the nature of the immunoreagents. Fluosol-DA markedly affected results of assays for triiodothyronine, thyroxin, and digoxin (by one method), but assays for aminoglycosides and protein hormones were unaffected. We determined that Fluosol-DA interacts with unbound labeled (and, presumably, with unlabeled) antigen to form a complex that coprecipitates with the bound antigen-antibody fraction during the separation step. In comparison with the idealized condition we observed an apparent increase in antibody-bound labeled antigen and consequently an apparent underestimation of the measured antigen. Depending on the nature of the immunoreagents, antibodies can extract more or less of their respective antigens from particles of Fluosol-DA emulsion.

Antigen-Antibody Complex↗

PubMed1982

An improved ultrafiltration method for determining free testosterone in serum.

In this method, we use the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane in measuring free testosterone in serum. Two independent assays are combined: total testosterone and the ultrafiltrable fraction of added [3H]testosterone. The unbound fraction is determined in 0.15-0.5 mL ultrafiltrates of 0.6 to 1 mL of variably diluted serum that has been equilibrated with [3H]testosterone at 37 degrees C. The assay is rapid (less than 1 h), practicable (requires 0.6 mL of serum), and reproducible (CV 3.2% within assay, 3.9% between assays). Accuracy was evaluated as the fraction of free testosterone in the ultrafiltrate of dialyzed serum vs that in a prior dialysate; they were the same confirming the validity of the free testosterone measurement. Samples from ostensibly healthy men and women and from hirsute and pregnant women gave results that agreed with those obtained by equilibrium dialysis. Total testosterone concentrations for normal and hirsute women showed considerable overlap, but data on free testosterone concentrations in these populations were better resolved.

Adult↗

PubMed1980

An improved ultrafiltration method for free thyroxine and triiodothyronine in serum.

We describe an ultrafiltration technique for rapidly and directly determining free triiodothyronine or free thyroxine, or both. After equilibrating serum at 37 degrees C with purified tracer of high specific activity, we placed 0.15 mL of serum in 2.8 mL of phosphate buffer (0.1 mol/L, pH 7.4) in the ultrafiltration cell and obtained successive 0.2- and 0.6-mL fractions of protein-free ultrafiltrate. Under our conditions free ligand concentration was independent of flow rate. After purifying the second fraction with protein-coated charcoal, we could determine the proportion of free triiodothyronine or free thyroxine. Samples from normal adult men and women, including women who were taking oral contraceptives or were pregnant, and from hypo- and hyperthyroid patients gave results that agreed with those obtained by equilibrium dialysis. Speed is the main advantage of the method: one technologist can complete the procedure in 2 h and, using a multi-micro-ultrafiltration system, can process many samples in one day. For laboratories where index-type reactions are performed routinely and direct free triiodothyronine or free thyroxine is determined only on selected specimens, this method is superior to dialysis. It is also very convenient for rapidly purifying tracers, to at least 97% radiochemical purity, with 94% recovery and no dilution.

Dialysis↗

PubMed1980

Improved ultrafiltration method for determining unbound cortisol in plasma.

We describe a modified ultrafiltration method for measuring unbound cortisol in diluted or undiluted plasma or serum. After equilibration at 37 degrees C with purified [3H]cortisol, plasma or serum with or without buffer was placed in the ultrafiltration cell and two successive 0.2-mL fractions of protein-free ultrafiltrate were obtained. Under our conditions, free ligand concentration was independent of flow rate. The second fraction (the first is discarded) was used for determining the proportion of unbound cortisol. The assay is rapid (less than 2 h), practical (no more than 1.5 mL of plasma or serum is necessary), and reproducible (CV: 4.5% within assay and 5.2% in different assays). Samples from normal men and women (blood taken at 0800 and 1600 hours), from pregnant women, and from patients with Cushing's disease and adrenal insufficiency gave results that agreed with those obtained by equilibrium dialysis.

Adult↗