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.     

DNA ploidy as a prognostic factor in rhabdomyosarcoma. Analysis of 35 cases with image cytometry

OBJECTIVE: To correlate DNA ploidy in rhabdomyosarcoma (RMS) with other prognostic factors and patient survival and to search for possible reasons for inconsistent conclusions in similar, published studies. STUDY DESIGN: DNA content was measured in archival specimens obtained from 35 patients (23 children and 12 adults) with RMS. Cell suspensions were prepared by the modified Hedley technique, stained by the modified Feulgen-thionin method and analyzed by automated high-resolution image cytometry. DNA ploidy was assessed on the basis of DNA index values. We used the chi 2 test to correlate DNA ploidy with other prognostic factors, Kaplan-Meier procedure to estimate overall survival in terms of individual prognostic factors, log-rank test to calculate differences in survival between groups and Cox multivariate regression analysis to determine the independence of variables in relation to survival. RESULTS: A statistically significant correlation was found only between DNA ploidy and histologic subtype of RMS, patient sex and patient age. A hyperdiploid DNA pattern predominated among patients with embryonal RMS, and a tetraploid pattern dominated among patients with alveolar RMS. The highest 5-year survival rate was seen among patients with hyperdiploid RMS, followed by those with diploid, tetraploid and hypertetraploid RMS. Although DNA ploidy was a significant prognostic factor in univariate analysis, it did not retain its independent prognostic value in multivariate analysis, in which patient age, tumor size and histologic subtype were the only significant factors. We found 12 articles reporting on the association between DNA ploidy and survival of patients with RMS: 6 found a correlation, and 6 did not. The main reasons for the discrepancies seem to be the inclusion of chemotherapy-treated and nontreated patients, low number of patients and differences in grouping DNA histograms. CONCLUSION: The precise prognostic value of DNA ploidy in RMS remains equivocal. Larger, cooperative studies could give statistically more reliable results.     

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.     

DNA ploidy and cell cycle analysis in ear malignant melanoma by flow and image cytometry.

Sixteen ear malignant melanomas (MM) were studied for ploidy and cell cycle analysis by flow and image cytometry. The results were compared with clinical (age, sex, stage), histologic (depth of invasion, level, type) and prognostic (recurrence, death) parameters. Single nuclear suspensions were obtained from fixed, paraffin-embedded tumor and adjacent normal tissue processed separately according to Hedley's technique. These, a "spiked" specimen of normal tissue and tumor, and a spleen diploid control were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, California, U.S.A.). Feulgen-stained Cytocentrifuge preparations of nuclear suspensions of normal, MM and diploid spleen were analyzed with the CAS 200 Image Analyzer (Cell Analysis Systems, Inc., Elmhurst, Illinois, U.S.A.) against commercial calibration rat hepatocytes defined as diploid. Six (37.5%) MM were diploid, and 10 (62.5%) were aneuploid; 8 (90%) were hypodiploid, for a high frequency. There were no statistically significant correlations between clinical, pathologic, prognostic or cell cycle analysis parameters and ploidy, although poor prognostic features tended to be in aneuploid lesions.     

Establishment of a diploid reference value for DNA ploidy analysis by image cytometry in mouse cells.

OBJECTIVE: To establish a diploid reference value for DNA ploidy analysis of mouse cells (Mus musculus) by image cytometry using the CAS 200, an analysis system suitable for DNA content studies in human cells. STUDY DESIGN: To establish this standard, we used spleen imprints from 26 normal animals. A minimum of 150 lymphocytes present in each imprint was counted. The mean DNA content (pg/cell) of the G0/G1 peak and the DNA index observed in all samples were statistically analyzed. Cytospins with peritoneal cells from the same animals were then analyzed with this reference DNA value to confirm the diploid range. RESULTS: The DNA diploid reference value was determined by the mean DNA content of all spleen samples, which was 6.42 +/- 0.234 pg/cell, and the diploid range, defined as the diploid value +/- 10%, was 5.78-7.06 pg/cell. All the peritoneal samples showed a DNA diploid histogram, with a mean value for the G0/G1 peak DNA content of 6.742 +/- 0.15. CONCLUSION: The diploid reference value found in this study differs from those reported for other species, including the human being, and should be used in further studies of mouse pathology.     

Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer.

The DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by ICM. Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.     

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.     

DNA ploidy in soft tissue sarcoma: comparison of flow and image cytometry with clinical follow-up in 93 patients

In soft tissue sarcoma, the prognostic importance of DNA ploidy status is limited. One possible explanation may be technical; small non-diploid stemlines will be diluted in relation to the presence of normal diploid cells and may not be detected by flow cytometry (FCM). We assessed DNA ploidy status in 93 tumors with both FCM and image cytometry (ICM). ICM may permit the exclusion of non-relevant cells. The ability of the two methods to detect non-diploid stemlines was compared, as were the prognostic consequences. The patients (54 males) had a median age of 69 years. Surgical procedures were performed on all patients. None of the patients had received preoperative radiotherapy or chemotherapy. FCM and ICM were performed with standard methods. The prognostic value was assessed with univariate and multivariate analysis. In 82 of the 93 tumors, a concordant ploidy status by FCM and ICM was found. In 5 FCM type 1-2 tumors (diploid), the identification of non-diploid stemlines by ICM did not influence the metastatic rates. Increasing tumor size, histotype other than liposarcoma, increasing malignancy grade, tumor necrosis, and ICM non-diploidy were univariate prognostic factors for metastasis. In a multivariate analysis, only tumor size larger than 9 cm was a prognostic factor. In about 10% of the tumors, a discrepancy between FCM and ICM ploidy status was found, but we could not find a consistent prognostic consequence of this. Neither FCM nor ICM ploidy status was an independent prognostic factor.     

Proliferative characteristics and ploidy of human brain tumors by DNA flow cytometry

We studied nuclear DNA distribution by flow cytometry in 59 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and unicellular suspensions were obtained with a mechanical dissociation technique. Nuclear DNA was stained with propidium iodide. Normal human brain tissue was used as a diploid reference standard. In 86.3% of benign tumors an unimodal DNA distribution with a DNA index usually within the diploid range was found. Among malignant tumors, 64% had un unimodal DNA distribution with diploid or near-diploid modal DNA content. The remaining 36% showed an additional cell peak with a DNA index ranging from 1.15 to 1.92. The percentage of S-phase cells was higher in malignant (median = 3.8) than in benign tumors (median = 1.9) (p less than .001), without correlation to histological tumor subtype.     

DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry

OBJECTIVE: To evaluate a rapid and simple method for DNA content analysis of urinary tract epithelial tumors with laser scanning cytometry (LSC). STUDY DESIGN: The subjects were 25 patients (37 specimens) who underwent surgery for urinary tract epithelial tumors. Tissue specimens of such tumors were frozen immediately after tumor resection and stored at -80 degrees C until used. Touch preparations were made and fixed in ethanol at room temperature. The cell nucleus was stained with propidium iodide solution containing RNase, and DNA ploidy was analyzed by LSC. Nuclear debris and overlapping nuclei were gated out by special statistical filters. In LSC, a normal diploid reference peak was determined by observing lymphocytes morphologically on the computer display of the instrument and/or under the microscope. RESULTS: DNA ploidy could be evaluated in all tumor tissues. The time it took from preparing the tumor specimen to the last measurement was about 40 minutes at the shortest, and measurement of all the specimens was completed within one hour. The coefficient of variation was 2.8-7.8% (mean, 4.4%). All eight specimens (100%) at grade 1 (G1) were DNA diploid, but 20% and 85.7% of the G2 and G3 cells, respectively, were DNA aneuploid. In total, 15 of the 37 specimens were DNA aneuploid. All 17 pTa-pT1 specimens (100%) were DNA diploid, but 100% and 50% of the T2 and T3 tumors, respectively, were DNA aneuploid. CONCLUSION: One can now supplement a morphologic diagnosis with useful information using LSC of touch preparations of tumors obtained at surgery or of imprints of archived, frozen specimens. LSC provides excellent DNA histograms for surgical specimens and has great potential for clinical application in pathology.     

Genome size and ploidy of Paracoccidioides brasiliensis reveals a haploid DNA content: flow cytometry and GP43 sequence analysis

The aim of this study was to evaluate genome size and ploidy of the dimorphic pathogenic fungus Paracoccidioides brasiliensis. The cell cycle analysis of 10 P. brasiliensis isolates by flow cytometry (FCM) revealed a genome size ranging from 26.3+/-0.1Mb (26.9+/-0.1fg) to 35.5+/-0.2Mb (36.3+/-0.2fg) per uninucleated yeast cell. The DNA content of conidia from P. brasiliensis ATCC 60855-30.2+/-0.8Mb (30.9+/-0.8fg) -showed no significant differences with the yeast form, possibly excluding the occurrence of ploidy shift during morphogenesis. The ploidy of several P. brasiliensis isolates was assessed by comparing genome sizing by FCM with the previously described average haploid size obtained from electrophoretic karyotyping. The analysis of intra-individual variability of a highly polymorphic P. brasiliensis gene, GP43, indicated that only one allele seems to be present. Overall, the results showed that all analysed isolates presented a haploid, or at least aneuploid, DNA content and no association was detected between genome size/ploidy and the clinical-epidemiological features of the studied isolates. This work provides new knowledge on P. brasiliensis genetics/genomics, important for future research in basic cellular/molecular mechanisms and for the development/design of molecular techniques in this fungus.     

Fluorescent image cytometry: from qualitative to quantitative measurements

Image analysis is being increasingly used in biology and medicine; however, in order to obtain truly quantitative data and thus avoid errors in interpretation, a certain number of precautions must be taken when the image is digitized, well before any attempt is made to analyse or interpret the data. This is particularly true for image microfluorometry. In this article we will examine an image analysis system for fluorescent images composed of a mercury lamp, a microscope, a high sensitivity video camera and an image analyser and evaluate the principal sources of random and non-random errors, various constraints, and their relative importance. A signal correction protocol is proposed to minimize non-random errors during digitalization. A few examples are given to illustrate its efficiency.     

Problem of size dependence in fluorescence DNA cytometry

The relationship among fluorescence intensity, area, thickness, and DNA concentration of DNA-bearing polyacrylamide film, stained with 50 ng/ml DAPI, was studied using an epi-illumination cytofluorometer. Measurements and theoretical analysis suggest the inevitability of the size-dependent effect on human cell nuclei in which the order of the DNA concentration is approximately 10 mg/ml, and that this effect can be virtually negated if the nuclei are stretched and flattened to less than 0.33 microns in thickness on the smears. Alternative practical ways to minimize this effect are to decrease the dye concentration of a staining solution and to make smears as thin and uniform as possible by some means such as hypotonic treatment and/or using an automatic centrifuge smearing apparatus. The size-dependent effect, nevertheless, should be taken into account particularly when determining the DNA content of different cell types and the DNA aneuploidy of tumor cells, regardless of the technique used for measurement.     

样方大小对苔藓植物生态学指标的影响

为了分析样方大小对苔藓植物生态指标的影响,在环境相对一致的条件下,在各样点以巢式取样法调查苔藓植物盖度,取样的大小分别为20 cm× 20 cm,30 cm× 30 cm,40 cm×40 cm,50 cm× 50 cm,60 cm×60 cm.通过统计发现,随着取样面积的增加,目测法所获得的优势种、总的苔藓植物的盖度...     

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.     

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r²=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that conventional polyploid evolution has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.     

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.     

Influence of macroinvertebrate sample size on bioassessment of streams

In order to standardise biological assessment of surface waters in Europe, a standardised method for sampling, sorting and identification of benthic macroinvertebrates in running waters was developed during the AQEM project. The AQEM method has proved to be relatively time-consuming. Hence, this study explored the consequences of a reduction in sample size on costs and bioassessment results. Macroinvertebrate samples were collected from six different streams: four streams located in the Netherlands and two in Slovakia. In each stream 20 sampling units were collected with a pond net (25×25 cm), over a length of approximately 25 cm per sampling unit, from one or two habitats dominantly present. With the collected data, the effect of increasing sample size on variability and accuracy was examined for six metrics and a multimetric index developed for the assessment of Dutch slow running streams. By collecting samples from separate habitats it was possible to examine whether the coefficient of variation (CV; measure of variability) and the mean relative deviation from the “reference” sample (MRD; measure of accuracy) for different metrics depended only on sample size, or also on the type of habitat sampled. Time spent on sample processing (sorting and identification) was recorded for samples from the Dutch streams to assess the implications of changes in sample size on the costs of sample processing. Accuracy of metric results increased and variability decreased with increasing sample size. Accuracy and variability varied depending on the habitat and the metric, hence sample size should be based on the specific habitats present in a stream and the metric(s) used for bioassessment. The AQEM sampling method prescribes a multihabitat sample of 5 m. Our results suggest that a sample size of less than 5 m is adequate to attain a CV and MRD of ≤ 10% for the metrics ASPT (Average Score per Taxon), Saprobic Index and type Aka+Lit+Psa (%) (the percentage of individuals with a preference for the akal, littoral and psammal). The metrics number of taxa, number of individuals and EPT-taxa (%) required a multihabitat sample size of more than 5 m to attain a CV and MRD of ≤ 10%. For the metrics number of individuals and number of taxa a multihabitat sample size of 5 m is not even adequate to attain a CV and MRD of ≤ 20%. Accuracy of the multimetric index for Dutch slow running streams can be increased from ≤ 20 to ≤ 10% with an increase in labour time of 2 h. Considering this low increase in costs and the possible implications of incorrect assessment results it is recommended to strive for this ≤ 10% accuracy. To achieve an accuracy of ≤ 10% a multihabitat sample of the four habitats studied in the Netherlands would require a sample size of 2.5 m and a labour time of 26 h (excluding identification of Oligochaeta and Diptera) or 38 h (including identification of Oligochaeta and Diptera). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Plant DNA flow cytometry and estimation of nuclear genome size

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.