Image analysis software for automatic DNA ploidy assessment of archival solid tumours.

BACKGROUND: Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS: An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS: DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS: Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.     

Influence of sample size on image cytometry of DNA ploidy measurements

OBJECTIVE: To assess the number of nuclei required for significant image cytometry DNA ploidy measurements on one archival case of breast cancer. STUDY DESIGN: From one case of aneuploid DNA breast cancer, 18 subsets made up of 152-1,524 for the whole population of undamaged nuclei and made up of 74-735 epithelial nuclei had DNA measured. DNA ploidy type and five DNA ploidy indices, allowing DNA ploidy histogram interpretation were evaluated on each population. RESULTS: Three hundred nuclei were always sufficient for DNA typing, whereas reliable results for DNA ploidy indices required at least 750 nuclei. CONCLUSION: To DNA measure the above number of nuclei, fully automated image cytometry DNA ploidy measurements are required.     

利用流式细胞光度术鉴定苹果倍性的研究

利用流式细胞光度术测定了苹果１２个二倍体,５个三倍体细胞ＤＮＡ含量。结果表明：二倍体细胞核ＤＮＡ含量平均为２．２７ｐｇ,三倍体细胞核ＤＮＡ含量平均为３．１３ｐｇ。     

Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obtained by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.     

DNA ploidy assessment method applied to primary breast carcinoma FNA biopsies

The goal of our study was to assess suitability of FNA biopsy material as a source of samples (cell suspension) for DNA ploidy assessment in neoplastic tumors using flow cytometry. DNA ploidy is an established prognostic factor in many types of cancers. Aneuploid breast tumors are characterized by increased aggressiveness which manifests itself through rapid local progression and metastatic spread. Investigated specimens were breast cancer FNA biopsy cell suspensions. Measurements were performed using flow cytometry. Material studied comprised 143 cases analyzed in 1999-2000. We found in this group 101 carcinoma cases with aneuploid type and 42 cases of primary breast carcinoma with diploid type of cell cycle. Immunocytochemical assesssment of estrogen receptor and progesterone receptor status was performed in group of 105 cases. DNA ploidy was compared to receptor status of the investigated cells. DNA aneuploidy correlated with weak or no reaction for the presence of estrogen and progesterone receptors. Our study demonstrates the suitability of DNA ploidy assessment method applied to cytological material from FNA biopsies.     

DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas

DNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P less than 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80-1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 +/- 0.28. The mean DNA index for the three cases of "carcinoma in adenoma" with invasion of the stalk of the adenoma was 1.52 +/- 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.     

The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

Assessment of DNA flow cytometry as a surrogate end point biomarker in a bladder cancer chemoprevention trial

Although conventional cytology represents the most widely performed cytometric analysis of bladder cancer cells, DNA flow cytometry has, over the past decade, been increasingly used to evaluate cell proliferation and DNA ploidy in cells from bladder washings. We have investigated whether DNA flow cytometry and conventional cytology of epithelial cells obtained from bladder washings provide reliable surrogate endpoint biomarkers in clinical chemoprevention trials. We used cytometric and clinical data from a chemoprevention trial of the synthetic retinoid Fenretinide on 99 patients with superficial bladder cancer. A total of 642 bladder washing specimens obtained from the patients at 4 month intervals was analyzed. Intra-individual agreement and correlation of flow cytometric DNA ploidy (diploid vs. aneuploid), DNA Index, Hyper-Diploid-Fraction (proportion of cells with DNA content higher than 2C), and conventional cytologic examination, as assessed by kappa statistics and Spearman's correlation test, were poor from baseline through 24 months. Moreover, no correlation was found between DNA ploidy and cytology at each time point. The same results were obtained when the analyses were stratified by treatment group. In addition, the association between the results of bladder washing (by either DNA flow cytometry or cytology) and concomitant tumor recurrence was significant only for abnormal cytology, while neither biomarker was predictive of tumor recurrence at the subsequent visit. During the time of this study only four patients progressed to muscle-invasive bladder cancer, indicating the "low-risk" features of the patient population. We conclude that DNA flow cytometry and conventional cytology on epithelial cells obtained from bladder washings do not appear to provide suitable surrogate endpoint biomarkers during the early stages of bladder carcinogenesis.     

Vertical heterogeneity of DNA ploidy level assessed by flow cytometry in calli of Passiflora Cincinnata

During in vitro culture conditions, callus tissue is exposed to different intensities of environmental stress, which may induce somaclonal variation. Among the possible resulting abnormalities, callus cells can exhibit distinct DNA ploidy levels, a type of somaclonal variation associated with euploidy and/or aneuploidy. As somaclonal variation has been regarded as both a positive and negative phenomenon, the development of strategies to carefully assess the stability of DNA ploidy level within callus tissue is highly valuable. To this end, the present work aimed to evaluate the presence of intra- and inter-calli heterogeneity in relation to DNA ploidy level and nuclei density by flow cytometry. Calli were induced from cotyledonary leaves of Passiflora cincinnata, a wild passion fruit species. Embryogenic friable calli cultivated for 2, 6, and 9 mo were classified as young, intermediary, and old, respectively. These calli were horizontally sliced from the bottom-up at approximately the same thickness, and a total of 160 layers were evaluated by flow cytometry. Inter- and intra-calli heterogeneities were detected in relation to nuclei density and DNA ploidy level. Additional analysis was performed to identify the most proliferative layer. We conclude that care must be taken when using callus as source material for flow cytometry, since one portion cannot represent the whole cell mass. Moreover, in order to prevent the emergence of undesired ploidies during clonal propagation, callus culture time should not be prolonged.     

基于液氮研磨法的流式细胞术检测马铃薯倍性的研究

染色体组倍性鉴定是马铃薯种质资源评价的重要内容,流式细胞仪能够快速、准确地对细胞核DNA含量进行测定,从而广泛用于检测植物染色体组倍性。建立适于马铃薯倍性鉴定的高通量流式细胞术体系,对马铃薯育种工作提供依据。以20份马铃薯合作88孤雌诱导后代为材料,用液氮研磨法制备叶片细胞核悬液,并将其与传统刀片切碎法制备的细胞核悬液进行比较,对已知四倍体马铃薯合作88和二倍体马铃薯IVP101进行染色体倍性测定,结果发现这两种方法在倍性测定结果之间无明显差异,但是液氮研磨法操作简单、耗时少。基于液氮研磨法的流式细胞术可快速、准确检测其倍性。另外,在液氮研磨法中,对细胞核悬液染色时间的长短(从15 min到12 h)并不会影响倍性测定结果,从而方便研究人员在实际操作中灵活选择染色时间。     

DNA quantitation of distal bile duct carcinoma measured by image and flow cytometry

OBJECTIVE: To evaluate discrepancies between flow cytometry (FCM) and image cytometry (ICM), ploidy incidence and relation between DNA ploidies and survival in distal bile duct carcinomas (DBDCs). STUDY DESIGN: Forty-four archival tumor samples from patients with DBDC who underwent subtotal pancreatoduodenectomy from 1985 to 1996 were examined for DNA ploidy using FCM and ICM. RESULTS: Overall, 59% (26/44) of the tumors were aneuploid by at least one of the two techniques. We detected more cases of aneuploidy with ICM than FCM in formalin-fixed, paraffin-embedded DBDCs, 62% (21/34) versus 33% (13/40), respectively. When results could be compared, moderate strength of agreement (kappa = .45) was demonstrated. No correlation was found between DNA ploidy by FCM, ICM or combined FCM-ICM and survival time (P = .80, P = .35, and P = .54, respectively). CONCLUSION: Approximately 59% of DNA histograms contained aneuploid cell populations. Although ICM, as compared to FCM, is more sensitive in assessing the ploidy status of DBDC, both methods were complementary. Most discrepancies between FCM and ICM were due to the dilution of aneuploid populations by non-neoplastic diploid cells. DNA ploidy assessment in DBDC did not offer the possibility of improving the ability to predict survival.     

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.     

Prognostic significance of DNA cytometry in carcinoma of the uterine cervix FIGO stage IB and II.

OBJECTIVE: To assess the prognostic value of DNA-image cytometry in cervical carcinoma of the uterus and its relation to other established prognostic factors. STUDY DESIGN: The study included 116 cases of cervical carcinoma FIGO stages IB and II which were treated with radical abdominal hysterectomy. The median follow-up was 55 months (range 1-162 months). DNA image cytometry was performed on cytologic specimens prepared by enzymatic cell separation from formalin-fixed, paraffin-embedded tissues. DNA stemline ploidy, DNA stemline aneuploidy, 5c exceeding rate, 9c exceeding rate, 2c deviation index, and DNA malignancy grade were computed. DNA-variables as well as various clinical and histological variables were related to survival rates. RESULTS: In multivariate statistical analysis DNA stemline ploidy using 2.2c as a cut-off value and FIGO stage showed to be statistically significant available presurgery predictors of survival, whereas the postsurgical parameters lymphonodal status, tumor size and parametrial involvement were significantly correlated with survival. The synopsis of all parameters in a multivariate Cox model indicated that - with declining relevance - the number of positive pelvic lymph nodes, DNA stemline ploidy using a cut-off level at a modal value of 2.2c, largest pelvic lymph node, 5c exceeding rate, and ratio of carcinoma area to cervix area, were of predictive value for survival. CONCLUSIONS: Our results suggest that prognostic information deducted from classical staging parameters is successfully complemented by DNA image cytometry which can be applied pretherapeutically.     

Flow and image cytometric DNA ploidy,including 5c exceeding cells,of serous borderline malignant ovarian tumors. Correlation with clinicopathologic characteristics

OBJECTIVE: To analyze DNA ploidy of serous borderline ovarian tumors by flow cytometry (FCM) and image cytometry (ICM), with 5c exceeding cells also analyzed, and to evaluate their correlation with clinicopathologic characteristics of patients and tumors. STUDY DESIGN: Cell suspensions were prepared according to a modified Hedley method from formalin-fixed, paraffin-embedded tissue blocks of 43 tumors. One part of the suspension was used for flow cytometric measurement; from the other part, filter slides were prepared for ICM. RESULTS: FCM and ICM found 2 aneuploid (peridiploid) serous borderline ovarian tumors, and FCM found 1. ICM found 3 tumors with 5c exceeding cells and 2 tumors with octaploid cells. There was no correlation between DNA aneuploidy and presence of 5c exceeding cells with tumor size, International Federation of Gynecology and Obstetrics stage or survival. CONCLUSION: The results confirm a good correlation between FCM and ICM DNA ploidy and the ability of ICM to detect 5c exceeding cells. The prognostic value of DNA ploidy and 5c exceeding cells in serous borderline malignant ovarian tumors warrant further evaluation.     

Prognostic significance of DNA ploidy determined by high-resolution flow cytometry in breast carcinoma

OBJECTIVE: To assess the prognostic value of DNA ploidy in breast carcinoma and its relation to other established prognostic factors. STUDY DESIGN: We evaluated DNA ploidy in 303 breast carcinoma patients with a median follow-up of 63 months. Flow cytometry was performed on frozen tumor material, yielding histograms with narrow peaks (median coefficient of variation of 2.08). DNA ploidy pattern was classified as either diploid versus nondiploid, euploid (diploid and tetraploid) versus aneuploid or diploid/near-diploid (DNA index < 1.2) versus other, and correlated with relapse-free (RFS) and cancer-specific survival (CSS) along with tumor size, histologic grade and type, axillary lymph node involvement, menopausal and steroid receptor status, age and type of treatment. RESULTS: Seventy-one percent of tumors were DNA nondiploid (14% tetraploid and 57% aneuploid). There was a strong association between DNA ploidy and histologic grade. Histologic grade, lymph node status, tumor size and DNA ploidy (regardless of the classification used) were all significantly associated with RFS and CSS in multivariate analysis. CONCLUSION: These results suggest that DNA ploidy, at least when determined from frozen tumor tissue, is an independent prognostic factor in breast carcinoma; however, its prognostic power seems to be inferior to that of histologic grade, with which it strongly correlates.     

Analysis of megakaryocyte ploidy in patients with thrombocytosis

The DNA content of bone marrow megakaryocytes was analyzed in 24 patients with myeloproliferative disorders, 23 patients with secondary thrombocytosis and 15 normal volunteers using 2-color flow cytometry. Compared with normal controls, the majority of patients with secondary thrombocytosis, polycythemia vera and essential thrombocytosis exhibited a relative increase in higher ploidy (greater than 16N) cells. In contrast, patients with chronic myelogenous leukemia exhibited an increase in lower ploidy cells (less than 16N), with a modal DNA content of 8N. Patients with myeloproliferative disorders tended to show a decrease in the 16N megakaryocyte population compared with patients with secondary thrombocytosis. No correlation between ploidy distribution and platelet count was observed.     

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.     

DNA ploidy and chromosomal imbalances in invasive ductal breast cancer. A comparative study of DNA image cytometry and comparative genomic hybridization (CGH).

Chromosomal imbalances were analyzed in 62 breast cancers with different DNA ploidy by CGH. The results of DNA image cytometry and CGH are consistent with peridiploid and aneuploid cases. The peritetraploid tumors harbored a high number of chromosomal imbalances, as a hint for an unfavorable prognosis. The quantitative analysis of imbalances highlighted the role of different physical constituents of the chromosome, and of chromosomal losses in different DNA ploidy groups. The peritetraploid and aneuploid tumors differed from the peridiploid tumors in losses at 8p and 18q. The peritetraploid cancers exhibited more gains at 8q, the aneuploid tumors more losses at 17p than their peridiploid counterparts. The aneuploid cases differed from the peritetraploid tumors in a higher number of losses at 11q and 14q. Combinations of imbalances provide further insights into the genetic background of DNA ploidy. Hypotheses for the progression from peridiploid to nondiploid breast cancers are given.     

DNA ploidy of bladder cancer using bladder biopsy supernate specimens

OBJECTIVE: To perform DNA image cytometry on 119 bladder biopsy supernate (BBS) specimens of transitional cell carcinoma (TCC) bladder to: (1) test the suitability of this cytologic specimen for use in DNA ploidy analysis, and (2) assess the value of DNA ploidy measured on this specimen as to the risk of tumor recurrence and survival. STUDY DESIGN: The histologic grade and cytologic grade were correlated, and the DNA ploidy produced was determined by image analysis of Feulgen-stained nuclei. Kaplan-Meier curves related age, sex, grade and DNA ploidy to recurrence of tumor and survival. Log rank analyses were used to ascertain the difference between the curves for each categorical variable. RESULTS: Urothelial cells derived from the BBS specimen were demonstrated to be representative of the tumor. The tumor recurrence rate was significantly higher (P = .0001) and the survival rate significantly lower (P = .0002) for patients with aneuploid tumors compared to those with diploid tumors. Patients with TCC 2 tumors had a significantly shorter time to recurrence (P = .003), although the relationship between ploidy and survival in this group was of marginal significance. CONCLUSION: The specimen was free of many of the problems associate with the other specimen types used for measuring DNA ploidy. The results show that the BBS specimen is diagnostically useful and suitable for DNA analysis, providing prognostically relevant information.