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PubMed1985

An improved method of preparing nuclei for absorption cytophotometry.

An improved method of isolating nuclei from tissue for Feulgen-DNA measurements has been developed. The optimal nuclear isolation medium (NIM) was found to be a solution of 2% polyethylene glycol (PEG) and 0.6% NP40 in phosphate-buffered saline. The disaggregation procedure consists of gentle mechanical disruption of the tissues, followed by a 10 min incubation in the NIM at room temperature. The mixture is then syringed four times through a 27-gauge needle, and the nuclei are placed onto slides with a cytocentrifuge. Nuclei prepared in NIM without PEG had obvious DNA leakage and tended to form clumps. Addition of PEG to the NIM led to separation of nuclei without any DNA leakage, thus greatly increasing the accuracy of the DNA cytophotometry results. G0/G1 nuclei at the appropriate ploidy levels were found for non-transformed and transformed tissues prepared with this technique. In addition, S-phase liver nuclei prepared in this manner showed the expected incorporation of (3H) thymidine after a 1/2 hr pulse in vivo.

Animals↗
PubMed1979

Feulgen cytophotometry of pine nuclei. II. Effect of pectinase used in cell separation.

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.

Cell Separation↗
PubMed1988

A rapid screening technique for detection of diosgenin through in situ cytophotometry.

Costus speciosus (Koenig) Sm. contains diosgenin, an important drug in family planning programs in India and underdeveloped countries. A simple, rapid method has been developed for in situ quantitation of diosgenin in this plant. The method is based on the formation of a suitable colored product of diosgenin in frozen sections of fresh material followed by determination of its optical density by in situ cytophotometry. The staining reagent is a combination of anisaldehyde, sulfuric acid and acetic acid. A positive correlation has been observed between cytophotometric diosgenin estimates and those derived from thin layer chromatography of extracts. The method is convenient for routine screening of plants for steroidal sapogenins such as diosgenin.

Acetates↗
PubMedDate not supplied

DNA cytophotometry in renal cell carcinoma: a significant prognostic factor?

We report on a prospective DNA cytophotometric study of 66 patients with renal tumors, 61 of whom had renal cell carcinoma (RCC) (pT1-pT4, G1-G3). 16 of the patients had a metastasis at the time of diagnosis. Cell material from 1-5 specimens of each tumor was analyzed for intratumoral heterogeneity. The aim of the study was to evaluate the prognostic value of the following DNA parameters: DNA ploidy, DNA grade of malignancy (DNA MG), mean DNA, DNA index, 2c deviation index (2cDI), and 5c exceeding rate (5cER). In this study 21% of the tumors were non-aneuploid, 79% were aneuploid; however, it proved possible to diagnose 38% of the total collective as aneuploid only by analyzing several tumor areas. In five of 61 RCC patients who died during an observation period of 42 months, at least one area of the primary tumor was aneuploid. Aneuploid primary tumors also accompanied the development of metastases and recurrent tumors in four of the 61 RCC patients. Only DNA 2cDI was found to have a significantly positive clinical correlation with metastasis (r = 0.261) during the clinical course. This was not true, however, for the histopathologic parameters. Significantly positive correlations were found between the tumor stages and the following DNA parameters: mean DNA, DNA index, and 5cER. Histopathologic tumor grading showed a significantly positive correlation with DNA MG, mean DNA, and 5cER. Statistically, the mean values of all evaluated parameters were significantly higher in metastasizing and recurrent RCCs than in non-metastasizing carcinomas (p < 0.05; t-test). DNA cytophotometry cannot substitute histopathologic prognosis. However, the analysis of various DNA parameters helps considerably in evaluating both the malignant potential of kidney tumors and the benign parenchyma of tumor-bearing kidneys.

Adult↗
PubMedDate not supplied

Adjuvant interferon alpha therapy in renal cell carcinoma (RCC): prognostic value of DNA cytophotometry.

BACKGROUND: Adjuvant immunotherapy with interferon alpha in non-metastatic RCC is still controversial. It was the aim of the present study to investigate whether tumor ploidy can help to isolate a group of patients profitting from this treatment. MATERIALS AND METHODS: Survival data of 119 patients undergoing tumor nephrectomy because of a non-metastatic RCC were analyzed retrospectively. Ploidy was measured in every tumor by means of DNA-cytophotometry. 24 patients received an adjuvant therapy with interferon alpha for one year. Statistical evaluation was performed by the Kaplan-Meier-Method with the logrank-test. RESULTS: Ploidy could be measured in all tumors. 56 (47%) tumors showed a diploid, 63 (53%) an aneuploid DNA-distribution. Ploidy was seen to be a good prognostic factor in RCC with significantly better survival in patients with diploid than those with aneuploid tumors. The adjuvant interferon therapy provided a marginal however not significant better survival in diploid tumors. Patients with aneuploid RCC had no benefit from this treatment. CONCLUSIONS: The data show that an interferon treatment in non-metastatic RCC might improve survival in patients with the prognostic better diploid tumors. However further investigations are necessary to give a general recommendation for this therapy in this group of patients. Additional molecular tumor factors like S-phase analysis and others might also improve patient selection.

Aneuploidy↗
PubMedDate not supplied

Immunohistochemical examinations (Ki67, p53, nm23) and DNA cytophotometry in bladder cancer.

Bladder cancer is clinically characterized by a high recurrence rate for superficial tumours up to 70% and by the invasiveness of advanced bladder cancer. To learn more about the biological behaviour of an individual bladder cancer different tumour markers have been investigated. The aim of our study was to compare the potential of aggression of both superficial and invasive bladder tumours by means of the proliferation marker Ki67, the tumour suppressor gene p53, the non metastasizing protein nm23 and the evaluation of DNA ploidy. We examined 36 patients, 28 with a bladder tumour (Ta-T4) and 8 without as a control group. For immunohistochemistry (Ki67, p53, nm23) we took paraffin sections and scored semiquantitatively under a microscope. The DNA cytophotometry was done on bladder washings by evaluating the DNA ploidy of single cells. The results showed that benign tissues were negative for Ki67 and p53 but positive for nm23. The DNA diagnosis was diploid for all benign samples. The superficial bladder cancer (Ta, T1) showed, in comparison to the invasive tumours, significantly lower numbers of aneuploid cells and a higher rate of p53 mutations. On the other hand the invasive tumours (T2-4) were correlated to significantly higher proliferation rates and higher potencies for metastasizing. The combination of the investigated tumour markers allowed a graduation of the biological behavior of an individual bladder cancer. Especially a high p53 mutation rate and a non aneuploid DNA diagnosis were indicators for the recurrence of superficial bladder tumours. Invasive growth of bladder cancer was characterized by high Ki67 proliferation and low nm23 protein binding.

Adult↗
PubMed2005

[A study of DNA depolyploidization and depolytenization of the heterochromatized gonosomal chromatin bodies in the secondary giant trophoblast cells of the field vole Microtus rossiaemeridionalis using cytophotometry].

A study was made of the distribution of the heterochromatized gonosomal chromatin bodies (GCB) material in the course of nuclear fragmentation of secondary giant trophoblast cells resulting in polykaryocyte formation at the late stage of their differentiation. A simultaneous DNA cytophotometry in GCBs and nuclear fragments showed a progressive GCB DNA content decrease proportional to that of DNA content in nuclear fragments. DNA contents in the nuclear fragments corresponded to 2c, 4c and 8c. In most cases 1-2 GCBs were found in the nuclear fragments of different ploidy levels. Both the total DNA content in GCBs and the DNA content in separate GCBs well correlated with the ploidy levels of fragments. The data obtained demonstrate a regular, whole-genome distribution of chromosomal materials into the nuclear fragments exemplified by sex chromosome distribution in compliance with the ploidy of nuclear fragments. We discuss a possible mechanism of nuclear fragmentation that may ensure substantially a balanced genome of nuclear fragments without leading to mitotic cycle renewal in the giant trophoblast cell population.

Animals↗
PubMed1990

[Microscopic DNA cytophotometry of axillary lymph node metastasis in mammary carcinomas].

65 primary mammary carcinomas and their corresponding axillary lymphnode metastases were studied. DNA-histograms were measured in imprints using microscopical Feulgen-cytophotometry (CAS 100). Comparison between DNA histograms according to AUER-typing showed a close consent in all except five cases; with a shifting from euploidy to aneuploidy (one case), and from aneuploidy to euploidy (four cases). The clinical value of these findings should be proved.

Breast Neoplasms↗
PubMed1990

[Histology, immunocytochemistry and DNA cytophotometry of adrenocortical tumors--a clinicomorphological study of 72 tumors].

Surgical specimens of 72 adrenocortical tumours were investigated by conventional histology, immunocytochemistry and DNA-cytophotometry. Histologically, 57 tumours were classified as adenomas and 15 as carcinomas. Nine adenomas weighed more, 2 carcinomas less than 50g. Only in 9 of the latter cases were distant metastases and/or lethal outcome of disease recorded, while the clinical course of the remaining patients was uneventful. No significant differences in DNA content were found between adenomas and carcinomas or between carcinomas with aggressive and indolent behaviour. Neither could immunocytochemistry discriminate between these conditions. Immunostaining with the monoclonal antibody D 11 proved to be the only effective means to definitely type adrenocortical neoplasia. Thirty-one cases exhibited positivity upon immunostaining with a polyclonal antiserum against synaptophysin. This phenomenon has so far not been encountered in non-neuroendocrine neoplasia.

Adenoma↗
PubMed1990

[DNA determination by cytophotometry in bowenoid papules in comparison with condylomata acuminata and Bowen's disease].

The relation between bowenoid papules and Bowen's disease is characterized by largely corresponding histological pictures and diverging clinical-epidemiological features. In bowenoid papules and Bowen's disease, 5 cases each, compared with 5 cases of common condylomata acuminata, we determined the DNA by means of Feulgen's cytophotometry in visible light after Feulgen's staining. The following results were obtained: The average DNA content in bowenoid papules equals that in Bowen's disease, but it is significantly higher than in condylomata acuminata. What is different between the two groups "bowenoid papules" and "Bowen's disease" is the distribution of DNA: In Bowen's disease, DNA spreads into higher ranges of polyploidy. If we take recent molecular biological/virological findings into account, this result can be interpreted as a transition towards carcinoma in situ.

Adult↗
PubMed1987

Effects of chemistry, manufacturing and concentration of the dye basic fuchsin regarding its use in quantitative cytophotometry.

Four separately manufactured preparations of basic fuchsin were compared to determine the effects of concentration, chemistry, and the manufacturing process for their quantitative value in the nuclear Feulgen reaction. In order to make the necessary comparisons, the two wavelength method of quantitative cytophotometry was employed to analyse each stain application regarding DNA measurements. Chicken erythrocytes, and myxamoebae and plasmodia of Didymium iridis were employed as experimental tissues. Results indicated that all four preparations of the stains yielded acceptable and valid quantitative data in relative DNA values. Differing manufacturers and dye concentrations had no appreciable effect on relative quantitative measurements. However, the maximum staining intensity was affected by differences both in the chemical structure of the individual stains and by the products from various manufacturers. The maximum dye intensity and accurate quantitative absolute values for DNA measurement were best obtained by the use of basic fuchsin having the same colour index (Cl) 42510 as that manufactured by Fisher Scientific Incorporated.

Amoeba↗
PubMed1977

Quantification by DNA-based cytophotometry of the 9q+/22q-chromosomal translocation associated with chronic myelogenous leukemia.

DNA-based cytophotometry was used to analyze metaphase chromosomes in four patients with chronic myelogenous leukemia. In three of these patients, both Philadelphia chromosome (Ph1)-positive and Ph1-negative cells were measured. On the basis of these three patients, the characteristic 9q+/22q- translocation of chronic myelogenous leukemia involves the net transfer of 0.325% of the autosomal genome; there is no evidence of net gain or loss of DNA (apart from duplication of the Ph1 chromosome in one patient), and no significant difference is found in the amount of DNA transferred in different patients. Significant differences are found among patients in the derived Chromosomes 9 and the Ph1 chromosomes and are ascribed to preexisting variations in the Ph1-negative cells of these patients. There is no evidence in these patients of any further cytogenetic lesion associated with chronic myelogenous leukemia.

Adolescent↗
PubMed1989

[Results of impulse cytophotometry in malignant tumors of the head and neck. A prognostic parameter?].

Tumour biopsies of 93 patients with head and neck malignancy were analysed by cytophotometry. Ploidy and the proportion of cells in S-phase were measured. Tumours were classified as proliferative if the S-phase fraction was greater than 8%; the rest were classified as being less proliferative. Proliferative tumours produce local and distant metastases earlier and more frequently, so that the proportion of cells in S-phase is of prognostic importance. The response to chemotherapy of irradiation was no different in either group.

Carcinoma, Squamous Cell↗
PubMed1979

[Nonhomogeneity of the distribution of the absorbing substance in cytophotometry].

Results of two-wavelengths cytophotometry may be used to calculate m--the quantity of light absorbing substance, and gamma--the degree of non-homogeneity of distribution of the substance on a preparation. Formula (1) is almost free from any distributional error. Optical density limits of applicability of formula (3) for gamma are shown on fig. 2. An algorithm for calculation of gamma at higher optical densities is proposed.

Absorption↗
PubMed1985

Some practical considerations in quantitative absorbance microspectrophotometry. Preparation techniques in DNA cytophotometry.

An experimental review of the Feulgen and gallocyanine-chrome-alum stains for quantitative cytophotometry of DNA in tissue sections yielded information on the preparation and staining of tissue for quantitative absorbance microspectrophotometry. (1) Tissues routinely fixed in formalin are suitable for either stain. Specimens fixed with glutaraldehyde-containing fixatives are not satisfactory for Feulgen staining, nor are ethanol-fixed specimens, unless they are post-fixed in formalin. (2) The pararosaniline dyes, used in the Feulgen stain, are sufficiently pure to use if a solution of the dye in ethanol shows an absorbance peak at 543 to 546 nm. (3) The Feulgen stain provides good reproducibility when the staining solution is adjusted to pH 1.5. (4) Gallocyanine is the best stain to use on Bouin-fixed or glutaraldehyde-fixed tissues. (5) Where fixation is an option, Carnoy and methanol-formalin-glacial acetic acid are excellent fixatives that can be followed by either stain. (6) Selection of the thickness of a tissue section involves a compromise. Requirements of minimum nuclear overlap and sharp focusing favor a section thickness of 4 micron to 6 micron. On the other hand, the requirement for full nuclear thickness, as judged by absorbance equivalent to that of a touch preparation, demands sections as thick as 8 micron in the case of mouse liver. Within this range, the optimum thickness, therefore, is determined by the particular tissue, the range of its nuclear sizes and its packing density. (7) The refractive index of the mounting medium should be closely matched to that of the background structure of the tissue sections. For animal tissues, we found that media with refractive indices of 1.54 to 1.56 are suitable.

Animals↗
PubMed1985

Cytophotometry of breast carcinoma. Acridine-orange DNA microfluorimetry with Giemsa counterstain.

Investigations have suggested that a correlation exists between DNA ploidy levels and prognosis in human breast carcinoma. Nuclear DNA content can be studied by flow cytometry or cytophotometric analysis. While both methods yield comparable results for DNA distribution, cytophotometry has the advantage of permitting both quantitative cell measurements and cytomorphologic identification of tumor cells. Microfluorimetric analysis of nuclear DNA content was carried out on acridine-orange-stained imprint smears of malignant breast tumors, with the DNA values plotted as a histogram distribution. Quantitative fluorescence measurements of breast carcinoma cells using the acridine-orange stain appeared to be a fairly rapid and simple method for DNA determination as compared to Feulgen DNA analysis. Following cytometric measurements, imprint smears were counterstained by the Giemsa stain and examined by cytomorphologic criteria. With the Giemsa counterstain, the same cytologic preparation could be studied both by quantitative cell measurements and by conventional cytomorphologic criteria. Results are illustrated, and possible implications of the use of this method in the study of tumor behavior and the diagnosis by cytologic methods are discussed.

Acridine Orange↗
PubMed1979

Chromosomal DNA cytophotometry in 20q- nonspecific myeloid disorders.

DNA cytophotometry was used to quantify the chromosomal alterations in the bone marrow and blood of three patients with nonspecific myeloid disorders. All patients possessed a population of cells with a morphologically abnormal chromosome 20, del(20)(qll). In two of the patients, the abnormal chromosome 20 showed nearly identical DNA measurements with a net loss of 0.37% of the total autosomal DNA in one patient and 0.38% in the second. The third patient had a net loss of only 0.25% of the autosomal DNA. Analysis of the DNA content of the long arm and short arm of the abnormal No. 20 indicated that all three cases had chromosomal material added to the short arm (0.10 to 0.14% of the autosomal DNA). About 0.50% of the autosomal DNA was deleted from the long arm in two of the patients; only 0.35% of the autosomal DNA was deleted from the long arm in the third case. Within the limit of resolution, there is no evidence that the material lost has been translocated intact to another chromosome. The origin of the 20q- chromosome as the result of an incomplete pericentric inversion is suggested.

Aged↗