DNA ploidy in cell nuclei from paraffin-embedded material--comparison of results from two laboratories.

In 49 pairs of contiguous sections from paraffin-embedded prostatic cancer tissue, the DNA indices (DIs) were determined by flow cytometry (FCM) at 2 different laboratories. In 3 of 45 pairs of evaluable nuclear suspensions, DIs of 1.1 (DNA aneuploid) were found at Laboratory 1, whereas all 3 tumours were classified as DNA diploid at Laboratory 2. In the remaining 42 specimens, the correlation between the DIs was excellent, though the application of strictly defined DNA ploidy ranges led to different DNA ploidy allocation in 3 cases. It is concluded that in 85-90% of the cases, reliable DIs can be obtained by FCM done in paraffin-embedded material at different laboratories. Slight technical variations and interpretation differences may lead to different ploidy allocation in 10-15% of the cases.     

Flow cytometric measurement of p53 protein expression and DNA content in paraffin-embedded tissue from bronchial carcinomas

The nuclear protein p53 has been measured in archival lung cancer biopsies. The monoclonal antibody PAb 1801, which recognizes human p53, was used. After immunostaining, the nuclei prepared from paraffin-embedded tissue were stained with propidium iodide for simultaneous measurement of DNA content; 17 of 24 lung cancers were p53 positive. The S-phase fraction in positive tumors was 22.9 +/- 6.4%, as compared to 13.6 +/- 6.1% in negative tumors (P less than 0.02). In ten of the positive tumors (two small cell carcinomas and eight non-small cell carcinomas), the p53 expression varied through cell cycle, whereas in seven tumors (five small cell carcinomas and two non-small cell carcinomas), no such variation of p53 expression was observed. Freezing the nuclear suspensions did not substantially reduce the p53 signals. Control experiments with the SV40-transformed human foreskin fibroblast cell line HSF4-T12 showed that the enzymatic digestion utilized to dissociate paraffin-embedded tissue did not significantly reduce p53 fluorescence. Immunohistochemical staining of biopsy specimens indicated that only cancer cells were overexpressing p53. In conclusion, using the monoclonal antibody PAb 1801, p53 is detectable in cell nuclei prepared from paraffin-embedded bronchial carcinoma biopsies. P53 positive tumors have increased proliferative activity compared to p53 negative tumors. Furthermore, the lack of cell cycle variation of p53 in small cell carcinomas indicates that this pattern may be related to high-grade malignancy.     

Flow cytometric analysis of DNA content for ploidy determination in human solid tumors.

Flow cytometric studies of cellular DNA content were conducted in 26 patients with a variety of neoplasms. Cell dispersal was achieved with pepsin treatment, and a combination of ethidium bromide and mithramycin was used as DNA specific staining procedure. All measurements were conducted with a new sheath flow chamber in a PHYWE ICP 11 pulse cytophotometer. All but one patient with multiple myeloma had unimodal tumor cell DNA distributions. With human granulocytes as reference standard, 24 of 26 tumors were aneuploid; and of these, 23 showed varying degrees of hyperdiploidy. Except for one patient, ploidy abnormalities were stable on repeat examination.     

DNA content in fresh versus paraffin-embedded tissue. Flow cytometric analysis of 100 tumors.

DNA ploidy analysis was determined on 100 consecutive tumors from a wide variety of sites using both fresh and paraffin-embedded tissue on the same specimen. The correlation coefficient (r) value between the methods was 0.85. Aneuploidy was detected by both methods in 51/100 (51%) of the cases. Fresh tissue analysis yielded 10 additional cases (overall 61% aneuploidy) not detected on corresponding paraffin-embedded sections, whereas paraffin-embedded analysis detected 4 additional cases (overall 55% aneuploidy) not revealed by fresh tissue analysis. Fresh tissue analysis produced lower coefficients of variation and resulted in a cleaner preparation with less cellular debris. Fresh tissue analysis was also superior to paraffin for the detection of hypodiploid, near-diploid and multiple peaks. Analysis of paraffin-embedded material allows examination of archival tissue and provides a more rapid means of long-term follow-up and statistical correlations for prognostic studies. Although the overall correlation of both methodologies for DNA analysis showed a minimal variation in results, in our experience fresh tissue analysis has an advantage and is preferable, when available, for ploidy analysis.     

Flow cytometric analysis of isolated rat liver nuclei during growth

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.     

Flow cytometric analysis for ploidy level differentiation of 45 hairy vetch accessions

Determining the ploidy of plant germplasm is a necessary step in breeding or genetic studies in species. The purpose of this research was to determine the presence of ploidy level differentiation of hairy vetch (Vicia villosd) germplasm. Flow cytometry and root tip chromosome squashing methods were employed to assess 45 accessions labeled V. villosa available through the USDA germplasm collection. Flow cytometry determined that 43 of the accessions were 2C, one accession was 4C, and one accession was 6C. Analysis of accessions by root tip chromosome counts indicated that all accessions were diploid. The 2C accession contains 14 chromosomes and their chromosomes were approximately one-half and one-third in size as compared to the chromosomes of the 4C and 6C accessions, respectively. The 4C accession was observed to have 16 chromosomes and the 6C accession was observed to have 14 chromosomes. The large-scale differences in DNA amounts were due to chromosomal size variability as opposed to ploidy differences. This revealed the incidence of species misidentification of these two V. villosa accessions to be Vicia pannonica. All the V. villosa accessions were observed to be diploid and have similar DNA amounts. Flow cytometry proved to be useful in the efficient assessment of these accessions.     

Flow cytometric analysis of mouse hepatocyte ploidy

Summary The development of liver ploidy in mice aged up to 24 months was investigated by flow cytometry in four mouse strains. A mathematical procedure was applied for correction of flow cytometry histograms. In two of the mouse strains, C3H and DBA, both cellular and nuclear ploidy proceed in the same way. The octoploid cell with two tetraploid nuclei is the most numerous cell type in adulthood. On the other hand, strain NZB and the out-bred strain NMRI show at the corresponding age a higher proportion of diploid cells with strikingly low proportions of 4c cells. In addition, high values of 16c cells and nuclei are present in NMRI. In all strains the proportion of binucleate hepatocytes is in the same range (60%). However, the strains differ in ploidy classes of binucleate cells. Development of liver polyploidization does not depend on life span of the specific strain.     

Technical and statistical improvements for flow cytometric DNA analysis of paraffin-embedded tissue

Flow cytometric DNA analysis of paraffin-embedded solid tumors has permitted review of large series of archival tissue in attempts to relate abnormal DNA content to prognosis. Limitations of the technique include: 1) a laborious, time-consuming procedure; 2) variation in technique between laboratories; and 3) lack of an objective method of computing DNA indices. Critical evaluation of our technique has shortened the time involved in dewaxing and rehydration, selectively utilized patient's own normal tissue as the internal standard, proved reproducibility of stored specimens, standardized DNA index computation, and developed a statistical analysis to confirm aneuploidy. These technical improvements and the development of a statistical analysis provide a way to shorten the procedure time and standardize the data generated from flow cytometric DNA analysis so as to improve the quality of retrospective reviews of paraffin-embedded tumors and accelerate the definition of flow cytometry's role as a prognostic indicator.     

Flow cytometric analysis of mouse hepatocyte ploidy

Preparative and mathematical procedures are presented for the investigation of the ploidy pattern of liver cells. The DNA content of enzymatically-isolated liver cells and of nuclei was measured by flow cytometry. The true DNA content could not be measured directly due to super-position of statistical coincidences (demanding "first mode correction") and incomplete separation of the nuclei in binucleate hepatocytes (demanding "second mode correction"). The statistical coincidences (caused by simultaneous measurement of two or more particles or subsequent reaggregation of particles) were corrected by splitting the "unnatural" i.e., aneuploid DNA content, and classifying it with the normal ploidy classes. In addition, the higher normal ploidy classes were reduced by the proportion of the measured coincidences in favour of the lower ones. The second mode correction applied to nuclear distributions only. It is a probability calculation based on counting nuclear pairs on microscope slides, and resulted in a 10% increase of diploid nuclei and a larger standard deviation between the age groups. 8c and 16c values were reduced. The tetraploid values were unchanged.     

Analysis of protein expression in pure cell nuclei populations isolated from human breast cancer tissue by DNA flow cytometric sorting

In this study, cell nuclei from aneuploid breast cancer samples were sorted with respect to DNA content into pure diploid and aneuploid fractions using flow cytometry. The nuclear proteins were then separated by one-dimensional gel electrophoresis (1D-PAGE) and differences in protein expression patterns, between diploid and aneuploid nuclei from the same tumours, were compared. Using a combination of peptide finger printing and peptide identification by MALDI-TOF mass spectrometry, we identified proteins and confirmed that the proteins were of nuclear origins. The results in this study add further information to the knowledge about the breast cancer disease complexity and heterogeneity at molecular level. For some of the tumours studied different nuclei protein patterns were obtained, in the diploid respective aneuploid nuclei populations, whilst other tumours did not show these differences.     

Flow cytometric determination of ploidy and proliferative activity on isolated bone marrow particles and on total bone marrow aspirate

The nuclear DNA content distribution of peripheral blood (PB) and bone marrow (BM) cells was determined by propidium iodide flow cytometry in 33 patients who underwent BM aspiration for diagnostic purposes. Two types of BM samples were taken during every aspiration procedure: whole BM aspirate, composed of BM particles contaminated by PB cells; isolated BM particles. Proliferative activity was calculated as the percentage of cells with DNA content intermediate between the diploid (2n) and the tetraploid (4n) values (2n-4n%). Ploidy was expressed as the ratio between the modal channel of the G0-G1 peak of the probe and that of an internal reference standard (DNA index, DI). The 2n-4n% was very close to zero in all PB samples. It was significantly greater in BM particles (21.2 +/- 6.6%) than in whole BM aspirate (16.6 +/- 5.5%, p less than .0005), with a close correlation (r2 = 66; p less than .0001) between the two values. Aneuploid stem lines were found in BM but not in PB. The DI of BM stem lines were similar in whole BM aspirate and BM particles, but the percentage of aneuploid cells was usually higher in BM particles. The reduced proliferative activity and the lower percent of aneuploid cells found in whole BM aspirates, with respect to BM particles, can be attributed to the contamination of BM tissue by PB, which had a very low proliferative activity and did not show aneuploidy. BM particles are therefore an easily obtained and reliable sample for routine evaluation of proliferative activity and ploidy of BM cells by DNA flow cytometry.     

Comparison of DNA ploidy in routine fine needle aspiration biopsy samples and paraffin-embedded tissue samples

The nuclear DNA content was determined by flow cytometry (FCM) from unfixed fine needle aspiration (FNA) biopsy samples of 31 human tumors, and from the same tumors after their excision, fixation with formalin and embedding in paraffin. The ploidy of the histograms was the same in 29 (94%) of the 31 cases. The disagreement in two cases may be explained by clonal heterogeneity of the tumors. The DNA index of the aneuploid cases was identical in fresh and fixed samples. The coefficient of variation of the diploid peaks (P less than .001) and the mean percentage of S-phase cells (P = .06) were larger in the fixed samples. It is concluded that routine FNA biopsy is a practical and reliable method for collecting cells for FCM DNA ploidy determination.     

Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues.

BACKGROUND: Cellular expression of receptors for the hormones estrogen and progesterone in human mammary tumors is of diagnostic and prognostic value. Ligand binding assays have been replaced by immunohistochemical analysis of receptor expression. However, both of these techniques are slow, and in the ligand-binding assay it is difficult to measure heterogeneity of receptor expression in individual cells. Flow cytometry has been used extensively for monitoring the expression of cellular receptors in hematopoietic tumors but has been of limited value in the analysis of mammary tumors, which are difficult to disaggregate into single cells for flow analysis. Hormone receptors have a predominant nuclear localization, and it is relatively easy to isolate nuclei from paraffin-embedded archival tissues for flow cytometric analysis of receptor expression. METHODS: Thick sections from formalin-fixed paraffin-embedded archival mammary tumors were digested by different enzyme solutions for the isolation of single nuclei. Different fixatives were used to compare the results on subsequent staining of the nuclei for estrogen receptor (ER) expression. Double staining with propidium iodide and fluorescein isothiocyanate labeled secondary antibodies for ER expression was used for multiparametric analysis of ER and DNA content. RESULTS: Digestion of paraffin sections with low concentration of pepsin and detergents was ideal for isolation of single nuclei. Fixation with paraformaldehyde in the presence of Triton X-100 improved staining of the cells. Isolated nuclei had enhanced immunoreactivity compared with the whole cells, and subpopulations differing in reactivity could be identified in the nuclear fractions. Double staining of nuclei for ER expression and DNA content could allow for multiparametric analysis of these two important parameters. CONCLUSIONS: The procedures described can be used for processing of archival paraffin-embedded mammary tumors for monitoring of ER expression and aneuploidy. These two parameters have important diagnostic and prognostic significance in mammary tumors. Laser flow cytometry by providing multiparametric analysis can allow for correlation of these cellular markers with other important cellular and clinical parameters.     

Flow cytometric analysis of paraffin-embedded material in human gastric cancer

Flow cytometric DNa analysis was performed on formalin-fixed, paraffin-embedded samples obtained by gastroscopic biopsy from 9 patients with histologically normal gastric mucosa (36 specimens) and by radical gastrectomy from 42 cases of human gastric cancer (120 specimens). Ploidy patterns and the distribution of cells in the different cell cycle phases were estimated, and the results were correlated with the histologic and clinical features. All samples of normal mucosa showed a diploid modal DNA content whereas DNA aneuploidy was encountered in 71.4% of the gastric tumors. The correlation between aneuploidy and histologic malignancy grading was statistically significant: aneuploidy was found in 36.4% of highly differentiated (grade 1 and grade 2) tumors and in 75.0% of poorly differentiated (grade 3) tumors (P less than .05). The percentage of cells in S-phase in normal gastric mucosa (median: 5.0%) was lower than that in the tumors (median: 11.3%) (P less than .05). There was a trend for grade 3 tumors to have higher median values (median: 13.4%) than grade 1 and 2 tumors (median: 9.3%); however, this was not statistically significant. An aneuploid DNA pattern was associated with a poorer prognosis, both in early and in advanced stages of gastric tumors, while proliferative activity did not correlate with postoperative survival.     

Modified methodology to improve flow cytometric DNA histograms from paraffin-embedded material

Flow Cytometric (FCM) DNA content analysis of paraffin-embedded tissues has become a widely accepted procedure in assessing the biologic course in some tumors from archival material. Difficulty in interpreting histograms is frequently due to high levels of debris, and wide coefficients of variation (CV). These may lead to underestimating near-diploid abnormality. Although the clinical significance of low-degree aneuploidy has yet to be established, the procedure reported here improved our endeavor to detect DNA Indices (DI) of at least 1.1%. Experience has shown that careful technique can result in overall improvement of DNA histograms by lowering levels of debris and % CV, dramatically so in some cases. Archival histograms can be generated that rival in quality fresh tissue results. Archival material is currently employed for diagnostic and research purposes.     

Flow cytometric determination of DNA content in malignant and benign bone tumours

The nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S- and G2M-phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p less than 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.     

Flow cytometric cell division tracking using nuclei

BACKGROUND: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions. METHODS: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. RESULTS: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined. CONCLUSIONS: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle.     

Flow cytometric sorting of non-human primate sperm nuclei

Pre-determination of the sex of offspring has implications for management and conservation of captive wildlife species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort spermatozoa of non-human primate species for use with assisted reproductive technologies. The objectives of this study were to: (i) determine the difference in DNA content between X- and Y-bearing spermatozoa (ii) sort sperm nuclei into X- and Y-enriched samples; and (iii) assess the accuracy of sorting. Spermatozoa were collected from two common marmosets (Callithrix jacchus), seven hamadryas baboons (Papio hamadryas) and two common chimpanzees (Pan troglodytes). Human spermatozoa from one male were used as a control. Sperm nuclei were stained (Hoechst 33342), incubated and analyzed using a high-speed cell sorter. Flow cytometric reanalysis of sorted samples (sort reanalysis, 10,000 events/sample) and fluorescence in situ hybridization (FISH; 500 sperm nuclei/sample) were used to evaluate accuracy of sorting. Based on fluorescence intensity of X- and Y-bearing sperm nuclei, the difference in DNA content between X and Y populations was 4.09 +/- 0.03, 4.20 +/- 0.03, 3.30 +/- 0.01, and 2.97 +/- 0.05%, for marmoset, baboon, chimpanzee and human, respectively. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94 +/- 0.9 and 93 +/- 0.8%, 94 +/- 0.7 and 94 +/- 0.5%, 91 +/- 0.9 and 97 +/- 0.6%, 94 +/- 0.6 and 94 +/- 0.9%, for marmoset, baboon, chimpanzee and human, respectively). These data indicate the potential for high-purity sorting of spermatozoa from non-human primates.     

DNA ploidy measurements in tissue sections

Nuclear DNA ploidy measurements based on tissue sections, although technically tedious and time consuming, can provide useful diagnostic and prognostic information. Methods for minimizing distributional errors and optimizing interpretation of DNA histograms are presented, and the diagnostic and prognostic significance of DNA ploidy measurements in gynecologic cancer and its precursors is reviewed.