DNA measurement errors with a scanning microdensitometer in cytologic and histologic samples of breast cancers

The influence of various DNA measurement errors using a commercially available scanning microdensitometer was evaluated on Feulgen-stained cytologic and histologic samples prepared from paraffin blocks containing invasive ductal breast cancers. The overall average total measurement error was 5.5% for the cytologic specimens and 10.9% for the 4 micron histologic sections. Components of the error included microscopic adjustment variation and focussing errors (3.5% and 1.1%, respectively, for both cytologic and histologic samples) and background intensity estimation errors (3.0% for the cytologic samples and 10.0% for the histologic samples). Measurements of the integrated optical density had a minimal error of 0.5% and an average error of 1.0%. Limitations due to the histologic architecture and/or heterogeneous cell population gave rise to large differences in the selection of nuclei when differently sized scanning masks were used. To improve the reproducibility, masks used should be based on the individual cell size, and background intensity values should be carefully estimated in the vicinity of the selected cells. Overall, the cytologic tumor samples were preferable to the histologic samples for static DNA measurements. It was easier to select cells suitable for measurement in the cytologic samples, and the cytologic measurements were less time consuming and produced a smaller measurement error.     

Genome size and environmental factors in the genus Pinus

Nuclear 1C DNA content in haploid megagametophyte tissue of 18 North American and one exotic Pinus species was determined using scanning microspectrophotometry. The nuclear DNA content in root meristematic cells of Zea mays L. ssp. mays, inbred line Va35 (4C = 10.31 pg) was used as a standard. DNA content measured by microspectrophotometry was verified using laser flow cytometry with two additional standards, Hordeum vulgare cv. Sultan (2C = 11.12 pg) and P. eldarica (2C = 47.30 pg). DNA values obtained by both methods were significantly correlated (r = 0.987). The 1C nuclear DNA content ranged from 21 pg to 31 pg. The ratio of DNA content in embryo tissue of P. eldarica to that in megagametophyte tissue was 1.72 by scanning microspectrophotometry and 1.74 by laser flow cytometry. To date, this is the most comprehensive data set available for North American Pinus species. Relationships between genome size of 18 North American Pinus species and climatic factors and indices of growth were investigated using regression and correlation analyses. Positive correlations were observed between nuclear DNA content and growth indices, minimum seed-bearing age, and seed dimensions. Strong negative correlations were observed between nuclear DNA content and two climatic factors, the lowest mean annual and monthly precipitation (excluding January) and the highest mean monthly spring air temperature. These correlations suggest that the large genome size and its variation in Pinus are adapted responses to the habitats of these species.     

The nuclear DNA content of vulvar skin using Feulgen microspectrophotometry and "four-quarter" analysis

The nuclear DNA contents in subbasal (nonreplicating) cells of 161 vulvar biopsies from 31 patients were studied by Feulgen staining, integrated scanning microspectrophotometry (densitometry) and subsequent "four-quarter" analysis of the measurements. The results showed a constancy of the nuclear DNA content in the nonreplicating cells, regardless of the patient or the biopsy site.     

DNA cytophotometry of human chromosomes.

The applicability of Feulgen-based parameters to detect variant metaphase chromosomes involved in deletions or translocations, was investigated and algorithms developed to compute such parameters. This report is focused primarily on the magnitude of the errors involved during the prerequisite procedures of photography, measurement and computation. Measurements were performed by stage-scanning of photographic negatives of Feulgen-stained metaphases. In the scanned images the initial chromosome boundaries were obtained by thresholding, while definite chromosomal areas and local background values were obtained by expansion of the initial boundaries. The integrated density profiles and the relative DNA content were computed for the individual chromosomes (straight as well as bent). Total DNA content, DNA arm ratio, as well as length and centromere index can be obtained from the profile. It was shown that under such conditions the experimental errors associated with the measurements are small compared to biologic variations (e.g., differences between homologues) and that the procedures applied allow to detect polymorphisms. In addition to this, mean and standard deviations of both DNA and length parameters are given for metaphases of five subjects. Comparison of the applicability of DNA and length parameters is realized by a classification experiment.     

Dysembryoplastic neuroepithelial tumor. Features distinguishing it from oligodendroglioma on cytologic squash preparations

OBJECTIVE: To evaluate the diagnostic usefulness of intraoperative cytologic preparations from dysembryoplastic neuroepithelial tumor (DNTs). STUDY DESIGN: We reviewed squash preparations from 17 DNTs and compared them to those from 12 oligodendrogliomas and the histology of frozen sections from the same tumors. RESULTS: The floating neurons and extracellular mucin typical of DNT were more easily demonstrated in cytologic preparations than in frozen sections. Cytologically, oligodendroglialike cells of DNT were distinguished from oligodendrogliomas by larger nuclei with frequent nuclear indentation and multiple, small nucleoli, while oligodendrogliomas consistently showed nuclear roundness of tumor cells with 1 or 2 occasional nuclei. The presence of eosinophilic granular bodies in the background was an ancillary sign of DNT. CONCLUSION: The cytologic features of squash preparations of DNT are fairly characteristic and reliable for the correct intraoperative diagnosis of DNT, helping to determine the appropriate neurosurgical procedure.     

Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin-fixed lymphoid tissues

Flow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin-embedded, formalin-fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin-fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the S-phase fraction was higher in paraffin-embedded tissues than in unfixed cells. This difference could be attributed to 4', 6'-diamidino-2-phenylindole dihydrochloride (DAPI), a DNA-binding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin-embedded tissues generally showed a greater S-fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of S-fraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.     

DNA assessment in thin sections of lymphomas by densitometric and stereological methods: comparison with imprint and flow cytometric results

The nuclear DNA content was estimated in 2 microns sections of 18 lymphoma cases by two methods: (1) Feulgen densitometry using QTM 900 with correction by Bins' procedure which allows size-independent DNA distributions; (2) stereological unfolding as proposed by Cruz-Orive giving sphere-size distributions. A general correlation was found between results and DNA measurements obtained by imprint and flow cytometric techniques in the same specimens. When histologic DNA profiles were compared to cytologic histograms, a high correlation was found between the distribution of ploidy classes by correspondence analysis. However two highly proliferating lymphomas were erroneously classified as aneuploid. Conversely, sphere-size distributions allowed the identification of the majority of aneuploid lymphomas but failed to recognize proliferating ones. It appears that when cytologic specimens are not available, densitometric studies on sections may provide valuable information on DNA content, with complementary data obtained from stereological procedures.     

Comparison between slide and flow cytophotometric DNA measurements in breast tumors

Nuclear DNA analysis was performed in 37 human mammary adenocarcinomas in order to elucidate the difficulties and pitfalls connected with the interpretation of DNA histograms obtained using different methodologic approaches. For each tumor, DNA profiles were obtained by means of slide microspectrophotometry on a fine needle aspirate, slide cytophotometry on a 4-micron histologic section and flow cytometry on a suspension prepared from a cube of fresh tissue. When the DNA histograms were interpreted according to criteria usually applied to discriminate low-grade malignant tumors from high-grade malignant tumors, some tumors classified as euploid by one method were classified as aneuploid by another method. The main reasons for this weak correlation seem to be in specimen preparation and in tumor cell representation within the specimen between the methods. Another reason is that slide and flow techniques exhibit different sensitivities for malignancy-associated nuclear DNA changes: minor alterations of the DNA content of the tumor stemlines seem to be more exactly reported by means of the flow technique whereas structural alterations of the nuclear chromatin seem to be more sensitively recorded by means of the slide technique. It is suggested that thorough control of each step of the various DNA analysis procedures and the use of information obtainable by slide and flow techniques taken together may significantly improve the prognostic value of DNA measurements.     

Low-grade cribriform ductal carcinoma in situ of the breast. Fine needle aspiration cytology in three cases.

This paper presents the cytologic features of fine needle aspiration biopsy specimens from three cases of ductal carcinoma in situ characterized by small and uniform tumor cells growing in a predominantly cribriform pattern without comedo necrosis (low-grade cribriform ductal carcinoma in situ). On cytology, most of the tumor cells were clustered in three-dimensional ductal structures. Occasionally in the clusters the tumor cells were seen bordering central lumina, quite similar to the architecture in histology. A few single tumor cells and no myoepithelium were seen. The background was clear or slightly hemorrhagic, without necrosis. The tumor cells were uniform and had a cylindroid shape, with round or oval nuclei. Morphometrically the mean largest nuclear diameter was 1.5-1.6 times that of a red blood cell. The chromatin was finely granular, with a minute nucleolus and slight condensation along the nuclear membrane. In cut sections all three tumors showed strong immunoreactivity for neuron-specific enolase. Unless the cribriform growth pattern is recognized in the smear, the cytologic diagnosis of this entity is difficult.     

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

OBJECTIVE: To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. STUDY DESIGN: Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. RESULTS: Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. CONCLUSION: There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.     

Fine needle aspiration cytologic characteristics of hyalinizing trabecular adenoma of the thyroid

The cytologic characteristics of a histologically proven hyalinizing trabecular adenoma are presented. The solitary thyroid nodule was diagnosed cytologically as benign and histologically as malignant. Cytologically, the fine needle aspirate showed increased cellularity, with mild nuclear atypia and eosinophilic nuclear pseudoinclusions. Collections of an amorphous colloidlike material were noted surrounded by follicular cells. Inflammatory cells in the background suggested the possibility of a thyroiditis. Since the patient's antibodies were negative, a hemithyroidectomy was performed to rule out a neoplasm. The frozen section was interpreted as suggestive of medullary carcinoma since the tumor showed a poorly differentiated pattern of cells with marked nuclear atypia and abundant fibrous stroma (thought to be amyloid) separating the cells. The results of immunopathology and electron microscopy ruled out a medullary carcinoma. Upon review of the histology at a later date, the case was recategorized as a hyalinizing trabecular adenoma, proving the negative cytologic diagnosis to be correct.     

Retinoblastoma cell cycling

Techniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter. The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors. Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material. Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd. BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy. Approximately 14% of viable cells were in the synthesis-phase of the cell cycle. The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48). DNA analysis of these tumors was also performed using flow cytometry. Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak). There was no correlation between abnormal DNA content and the percent of cells in synthesis.     

Evaluation of applying feulgen stain for DNA analysis on destained hematoxylin-eosin-stained cytologic smears

OBJECTIVE: To evaluate the feasibility and reliability of DNA analysis performed on the original hematoxylin-eosin (HE)-stained cytologic specimens by destaining the slides and restaining with the Feulgen method. STUDY DESIGN: Image cytometric analysis of DNA ploidy status was performed in a comparative study on 20 cytologic preparations from 10 nasopharyngeal carcinomas. Ten smears were stained directly by the Feulgen method, and the others were Feulgen stained after HE destaining. RESULTS: There was 90% overall concordance in DNA determination and a good correlation (r = .97, P < .001) between the DNA indices determined by the 2 methods. Discordance was probably due to tumor heterogeneity. CONCLUSION: This study demonstrated that image cytometric DNA analysis on previously routinely HE-stained cytologic preparations is feasible and reliable. This method permits retrospective studies on archival cytologic material from patients with long term follow-up data.     

Chemical and Spectrometric Evaluation of Glycogen After Routine Histological Fixatives

A detailed comparison of fixatives used for the demonstration of glycogen has been based on chemical assay and microspectrophotometry. Rat liver containing known amounts of glycogen was fixed in formol alcohol, Rossman's fluid, 10% neutral formalin, Bouin, Helly, SUSA, and Zenker's fluid at 4 C and 18 C. Chemical assay was carried out before and after fixation and paraffin sections were prepared from the fixed material. Sections were stained with PAS and the silver methenamine method. Visual examination was carried out with a comparison microscope and quantitative estimations on PAS-stained sections were performed by scanning microspectrophotometry. The histochemical methods were compared with the chemical results obtained from the same tissue and a reasonable degree of correlation between the sets of results was observed. Cold formol alcohol and cold Rossman's fluid preserved the most glycogen and Zenker and SUSA fixation preserved the least. Cold formol alcohol was the only fixative that preserved threshold values of glycogen i.e. 0.3% and the silver methenamine method is recommended for the demonstration of these small amounts.     

Fine needle aspiration cytology of invasive micropapillary carcinoma of the breast: review of cases in a three-year period.

OBJECTIVE: To describe the fine needle aspiration cytology findings of invasive micropapillary carcinoma and correlate them with the histologic appearance. STUDY DESIGN: We reviewed the cytologic features of three cases of pure invasive micropapillary carcinoma in the files of Pamela Youde Nethersole Eastern Hospital from 1998 through 2000. Immunohistochemical study for epithelial membrane antigen was performed retrospectively on the cell block sections. Ultrastructural examination was also carried out on one of the cases. RESULTS: Two of the tumors were at pathologic stage II, and the remaining case was at stage III. Ipsilateral axillary lymph node metastases with similar morphology were seen in two of them. Cytologically, the smears were of moderate cellularity and composed of three-dimensional tumor cell balls, abortive and sometimes branching papillae, angulated tumor cell clusters, morules and occasional acini. Some of the tumor cell balls possessed scalloped borders. Focally, the tumor morules clustered together and were separated from each other by small, slitlike spaces. A small number of isolated malignant cells was also present in the background. The cell block sections showed mainly dispersed acini of tumor cells. The "reverse polarity" highlighted in histologic sections by immunohistochemical study for epithelial membrane antigen was not consistently demonstrated in the cell block material. Ultrastructural examination confirmed the focal presence of surface microvilli on the periphery of the tumor cell morules. CONCLUSION: Invasive micropapillary carcinoma of the breast possesses some subtle but distinctive cytologic features. With the help of cell block morphology and ancillary techniques, the preoperative suspicion of this rare subtype of ductal carcinoma, which carries a high propensity for lymphatic permeation, is possible.     

Light-modulation of nitrate reductase activity in leaves and roots of maize

The nuclear DNA content in ray cells from the 1-year-old vascular cambium of white ash ( Fraxinus americana L.) trees was determined at intervals during the annual cycle of cambial activity and dormancy by using Feulgen microspectrophotometry. By 10 September, these cells had entered dormancy in G₁ with a normal DNA distribution and a minimal average DNA content of 2.65 pg. The average amount of DNA increased to 3.51 pg by 30 November, remained at this elevated value until at least 30 March, when the cambium was still dormant, then declined to the minimum level on 1 May and 10 June, when the cells were mitotically active. The springtime decline appeared to occur both before and during cell division. Between 1 May and 10 June, the prophase (4C) and telophase (2C) DNA contents decreased significantly. The amount of nuclear DNA measured by microspectrophotometry was verified by using flow cytometry and image analysis. The results support the view that there is an annual oscillation in the nuclear genome size of shoot meristematic cells in tree species native to the northern temperate zone.     

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.     

Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry

A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry.     

The diagnostic significance of nuclear deoxyribonucleic acid measurement in automated cytology.

Flow cytometric analysis of cytologic samples from four different organs shows that nuclear DNA content of malignant cell populations depends to a large extent on organ of origin of the tumor. This fact must be considered in planning screening systems.