Methodologic aspects of nuclear DNA assessment of gliomas with astrocytic and/or oligodendrocytic differentiation. Correlation of image and flow cytometric studies on paraffin-embedded specimens

A total of 239 samples from paraffin-embedded, formalin-fixed astrocytic and/or oligodendrocytic gliomas from 111 patients were deparaffinized and disaggregated for image cytometric (ICM) and flow cytometric (FCM) DNA assessments. Each measurement technique produced evaluable histograms in about 85% of the samples analyzed. In the 10% that could not be analyzed by FCM, the background counts were too high and the coefficients of variation were too broad for precise evaluation. The failures with ICM were due to a shortage of Feulgen-stained tumor cell nuclei after the deparaffinization and disaggregation procedures. The results obtained were identical in 77% of the samples evaluable by both methods and practically identical (i.e., euploid versus aneuploid) in an additional 18%. The reasons for completely divergent DNA ploidy patterns in 5% of the samples could not be clarified. About 80% of the histopathologically highly malignant gliomas were found to consist of neoplastic cells with an aneuploid or tetraploid nuclear DNA distribution pattern. The results show that cytometric DNA assessments can be reliably performed on paraffin-embedded specimens of gliomas with astrocytic and/or oligodendrocytic differentiation by means of FCM and ICM on deparaffinized and disaggregated specimens.     

Reliability of DNA cytometric S-phase analysis in surgical biopsies: assessment of systematic and sampling errors and comparison between results obtained by image and flow cytometry

Three major parameters in DNA histograms that contribute to the reliability of S-phase analysis were evaluated. These parameters are (1) the extent of background in relation to the amount of S-phase cells (and the validity of its subtraction), (2) the size of the "free" S-phase range (S(free)), and (3) the sampling error of cell counting. Tests in histograms obtained from surgical biopsies by flow cytometry (FCM) showed that the background subtraction is reliable if the found S-phase fraction is higher than the fraction of background events in the histogram range of the cell population. The size of S(free) was determined in computer-generated test histograms as a function of variables such as the coefficient of variation (CV) and the DNA index (DI). To calculate the sampling error of cell counting above background and in S(free), a model was developed that was validated by experimental data. This error can serve as an indicator of the uncertainty in S-phase analysis. The poor correlation found between %S values measured by image cytometry (ICM) and FCM in surgical biopsies was assigned to high uncertainty by low cell numbers in ICM histograms. A method is proposed to estimate quantitatively the reliability of S-phase analysis that can facilitate the interpretation of results.     

Cytophotometric DNA measurements of chondrosarcoma: methodologic aspects of measurements in tissue sections from old paraffin-embedded specimens

The methodologic prerequisites of cytophotometric DNA measurements of normal and tumor cells in tissue sections obtained from paraffin blocks and preserved as archival material were investigated. The optimal time of hydrolysis in 5-N HCl at room temperature was one hour for the different cell types analyzed. Paraffin-embedded tissues, stored for two decades, were still suitable for quantitative cytophotometric DNA determinations of Feulgen-stained nuclei. Different cell types in the Feulgen-stained sections could be identified with accuracy. The 90th percentile of fibroblast (internal control cells) DNA values was used as an upper limit of the diploid DNA content. By determining the number of tumor cells with DNA values exceeding this limit, nondiploid (hyperploid) tumor cell populations could be discriminated from diploid tumor cell populations. Cell population analysis of ploidy level, performed in this way, was found to be accurate in tissue sections of 4 micrometer. The accuracy of this analysis was not improved by increasing the section thickness. Since tissue sections obtained from old paraffin blocks could be used for the determination of hyperploidy, the prognostic significance of this parameter in different tumors can be assessed retrospectively.     

Methodologic aspects of evaluating brush samples from biliary strictures by cytology and DNA flow cytometry

OBJECTIVE: To present a method of increasing the cell yield from brush samples of the biliary tree for measurement of DNA content by flow cytometry (FCM). STUDY DESIGN: One hundred eight cell specimens from 86 patients were studied by FCM for DNA ploidy and cell cycle composition. All specimens were cytologically classified into benign, suspicious for malignancy and malignant. Two methods for preparation of the cell material were compared. RESULTS: Enzymatic treatment of formalin-fixed brushes for release of cell nuclei was superior to mechanical removal of the cells. The fraction of samples not possible to assess was reduced from 27% to 4%, and good quality histograms increased from 21% to 62%. Aneuploidy was detected in 7% of benign and 57% of suspicious malignant samples. Using DNA analysis in addition to cytology as a diagnostic marker for cancer, the sensitivity increased from 12% to 31%. CONCLUSION: FCM of cells from biliary strictures can be used routinely as an adjunct to cytology for DNA analysis.     

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry

Abstract. Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation ( CV ) from 2 to 16%; (ii) constant measurement CV or CV 's increasing from G₁ to G₂ phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time.
The fractions of cells in the G₁, S, and (G₂+ M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10–20%. However, most methods tended to overestimate G₁ fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.     

Multiparametric comparison of mesenchymal stromal cells obtained from trabecular bone by using a novel isolation method with those obtained by iliac crest aspiration from the same subjects

Trabecular bone fragments from femoral heads are sometimes used as bone grafts and have been described as a source of mesenchymal progenitor cells. Nevertheless, mesenchymal stromal cells (MSC) from trabecular bone have not been directly compared with MSC obtained under standard conditions from iliac crest aspiration of the same patients. This is the ideal control to avoid inter-individual variation. We have obtained MSC by a novel method (grinding bone fragments with a bone mill without enzymatic digestion) from the femoral heads of 11 patients undergoing hip replacement surgery and compared them with MSC obtained by standard iliac crest aspiration of bone marrow from the same patients. We have shown that trabecular bone MSC obtained by mechanically fragmented femoral heads fulfil the immunophenotypic and multilineage (adipogenic, osteogenic and chondrogenic) differentiation criteria used to define MSC. We have also differentially compared cellular yields, growth kinetics, cell cycle assessment, and colony-forming unit-fibroblast content of MSC from both sources and conclude that these parameters do not significantly differ. Nevertheless, the finding of slight differences, such as a higher expression of the immature marker CD90, a lower expansion time through the different passages, and a higher percentage of cycling cells in the trabecular bone MSC, warrants further studies with the isolation method proposed here in order to gain further knowledge of the status of MSC in this setting. The present study was partially supported by grant HUS01B07 from the Consejería de Educación and by grant SAN196/SA13/07 from the Consejería de Sanidad, Junta de Castilla y León, Spain.     

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.     

A comparison of algorithms for the identification of specimens using DNA barcodes: examples from gymnosperms

In order to use DNA sequences for specimen identification (e.g., barcoding, fingerprinting) an algorithm to compare query sequences with a reference database is needed. Precision and accuracy of query sequence identification was estimated for hierarchical clustering (parsimony and neighbor joining), similarity methods (BLAST, BLAT and megaBLAST), combined clustering/similarity methods (BLAST/parsimony and BLAST/neighbor joining), diagnostic methods (DNA–BAR and DOME ID), and a new method (ATIM). We offer two novel alignment‐free algorithmic solutions (DOME ID and ATIM) to identify query sequences for the purposes of DNA barcoding. Publicly available gymnosperm nrITS 2 and plastid matK sequences were used as test data sets. On the test data sets, almost all of the methods were able to accurately identify sequences to genus; however, no method was able to accurately identify query sequences to species at a frequency that would be considered useful for routine specimen identification (42–71% unambiguously correct). Clustering methods performed the worst (perhaps due to alignment issues). Similarity methods, ATIM, DNA–BAR, and DOME ID all performed at approximately the same level. Given the relative precision of the algorithms (median = 67% unambiguous), the low accuracy of species‐level identification observed could be ascribed to the lack of correspondence between patterns of allelic similarity and species delimitations. Application of DNA barcoding to sequences of CITES listed cycads (Cycadopsida) provides an example of the potential application of DNA barcoding to enforcement of conservation laws. © The Willi Hennig Society 2006.     

Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the Methyl Green-Pyronin technique with the Gallocyanin chromalum and Feulgen procedures using image cytometry

Summary For simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA.The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen analtion. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm.After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum.We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.     

DNA quantification of squamous cell carcinoma of the oesophagus by flow cytometry and cytophotometric image analysis using formalin fixed paraffin embedded tissue.

This is the first comparative study of DNA quantification of oesophageal squamous cell carcinoma by flow cytometry (FCM) and image cytometry (ICM) using formalin fixed paraffin embedded tissue. The potential advantages of ICM include the identification of a reliable control cell population; avoidance of non-tumour stromal and inflammatory cell nuclei, nuclear fragments, degenerate cell nuclei and doublets, triplets etc., which are not possible with FCM using archival tissue. Twenty-eight cases, all of the same stage (stage 2a) and similar grade (well or moderately differentiated) were analysed. The cases were separated into two groups, those that had succumbed to tumour in less than 18 months (group A) and those that were tumour free at least 18 months post-resection (group B). Using ICM all 28 tumours yielded interpretable histograms by comparison to 25 of 28 using FCM. Aneuploidy was identified in 100% of cases in group A using ICM (in comparison to 73% by FCM) and in 73% of group B using ICM (in comparison to 44% by FCM). Any tumour aneuploid by FCM was also aneuploid by ICM. Nine cases aneuploid by ICM were euploid by FCM. The mean 5C exceeding rate (% of cells whose nuclei contain a DNA mass equivalent to > 5 sets of 23 chromosomes) was 21% in group A and 14% in group B (P < 0.01). Euploidy was confined to tumours of those patients disease free for more than 18 months. The conclusions of this study are that: firstly, ICM is superior in its yield of interpretable histograms to FCM using formalin fixed paraffin embedded tissue; secondly, ICM is more sensitive in the identification of aneuploid stemlines than FCM; and thirdly, euploid tumours (as detected by ICM) appear to have a better prognosis than aneuploid tumours of similar stage and grade.     

Flow cytometric DNA and cytogenetic studies in human tumors: a comparison and discussion of the differences in modal values obtained by the two methods

The numerical chromosome values in 53 human tumors were determined and compared with the modal DNA values as measured by flow cytometry. In tumors with chromosome counts in the diploid and tetraploid range, the modal DNA values were found to correspond to the modal values based on the chromosome counts. In tumors with chromosome counts in the triploid range, however, the modal DNA values were about 15% higher than expected. In order to explain this difference, the ratio between large and small chromosomes in the karyotyped metaphases was assessed. In addition, the DNA content of individual chromosomes, including markers and minutes, was calculated as a reflection of the DNA content of the whole cell. The ratio of large to small chromosomes did not deviate from the normal ratio found in cells with diploid, triploid, and tetraploid chromosome counts. Neither difficulties in karyotyping nor short-comings in the flow cytometric methodology could be used to explain the discrepancy between the expected and empirical modal DNA values. Some of the chromosomes in triploid tumors may, therefore, contain an increased amount of DNA.     

Cytogenetic effects of methotrexate on human cells in vivo: comparison between results obtained by chromosome studies on bone-marrow cells and blood lymphocytes and by the micronucleus test

Cytogenetic studies were performed in 22 patients treated with methotrexate (MTX). In some patients, metaphases from both bone-marrow cells and peripheral blood cells were studied. In the bone-marrow preparations an increased number of structural chromosomal aberrations was present, whereas abnormalities were not observed in the peripheral blood cells. An examination of the bone-marrow chromosomes must therefore be included in the study of the possible chromosome-breaking effect of chemical agents. The results obtained with the micronucleus test and chromosome studies were compared in 10 patients treated with MTX. The micronucleus test was more sensitive than the chromosome analysis as regards the clastogenic effect of MTX.     

Hormonal monitoring of ovarian activity using the Ovarian Monitor,part I. Validation of home and laboratory results obtained during ovulatory cycles by comparison with radioimmunoassay

A study was conducted to determine the accuracy and reliability of the Home Ovarian Monitor for measuring estrone glucuronide (E1G) and pregnanediol glucuronide (PdG) during ovulatory cycles as a means of monitoring ovarian activity. Approximately 60 ovulating women in three centres collected timed specimens of urine (3h or more) for a total of six cycles each. The women measured the E1G and PdG excretion per 24h in their urine specimens using the Monitor. A local laboratory using the Monitor also measured the excretion. Urine specimens from 18 to 19 cycles were sent frozen to the WHO Reference Laboratory in London where they were analysed for E1G and PdG by the Monitor and by radioimmunoassay (RIA). The correlation coefficients between the Monitor and radioimmunoassay results obtained in London were better than 0.84 in 80% of the cycles. A urine bias caused the Monitor E1G results to be higher than those obtained by radioimmunoassay but the daily patterns were the same. In 50% of the cycles, this bias caused a delay of up to 3 days in identifying the beginning of the E1G rise compared with radioimmunoassay. Timing of the preovulatory E1G peak and the postovulatory PdG rise agreed within the experimental errors of the two systems. The study confirmed that women using the Monitor at home obtained results that were as accurate as those obtained by laboratory procedures. Careful supervision was required to maintain laboratory levels of quality control and interpretation of results.     

Differentiation of Indica-Japonica rice revealed by insertion/deletion (InDel) fragments obtained from the comparative genomic study of DNA sequences between 93-11 (Indica) and Nipponbare (Japonica)

DNA polymorphisms from nucleotide insertion/deletions (InDels) in genomic sequences are the basis for developing InDel molecular markers.To validate the InDel primer pairs on the basis of the comparative genomic study on DNA sequences between an Indica rice 93-11 and a Japonica rice Nipponbare for identifying Indica and Japonica rice varieties and studying wild Oryza species,we studied 49 Indica,43 Japonica,and 24 wild rice accessions collected from ten Asian countries using 45 InDel primer pairs.Results indicated that of the 45 InDel primer pairs,41 can accurately identify Indica and Japonica rice varieties with a reliability of over 80%.The scatter plotting data of the principal component analysis (PCA) indicated that:(i) the InDel primer pairs can easily distinguish Indica from Japonica rice varieties,in addition to revealing their genetic differentiation;(ii) the AA-genome wild rice species showed a relatively close genetic relationship with the Indica rice varieties;and (iii)the non-AA genome wild rice species did not show evident differentiation into the Indica and Japonica types.It is concluded from the study that most of the InDel primer pairs obtained from DNA sequences of 93-11 and Nipponbare can be used for identifying lndica and Japonica rice varieties,and for studying genetic relationships of wild rice species,particularly in terms of the Indica-Japonica differentiation.     

Differentiation of Indica-Japonica rice revealed by insertion/deletion (InDel) fragments obtained from the comparative genomic study of DNA sequences between 93-11 ( Indica ) and Nipponbare ( Japonica )

DNA polymorphisms from nucleotide insertion/deletions (InDels) in genomic sequences are the basis for developing InDel molecular markers. To validate the InDel primer pairs on the basis of the comparative genomic study on DNA sequences between an Indica rice 93-11 and a Japonica rice Nipponbare for identifying Indica and Japonica rice varieties and studying wild Oryza species, we studied 49 Indica, 43 Japonica, and 24 wild rice accessions collected from ten Asian countries using 45 InDel primer pairs. Results indicated that of the 45 InDel primer pairs, 41 can accurately identify Indica and Japonica rice varieties with a reliability of over 80%. The scatter plotting data of the principal component analysis (PCA) indicated that: (i) the InDel primer pairs can easily distinguish Indica from Japonica rice varieties, in addition to revealing their genetic differentiation; (ii) the AA-genome wild rice species showed a relatively close genetic relationship with the Indica rice varieties; and (iii) the non-AA genome wild rice species did not show evident differentiation into the Indica and Japonica types. It is concluded from the study that most of the InDel primer pairs obtained from DNA sequences of 93-11 and Nipponbare can be used for identifying Indica and Japonica rice varieties, and for studying genetic relationships of wild rice species, particularly in terms of the Indica-Japonica differentiation. Translated from Journal of Fudan University (Natural Science), 2006, 45(3): 309–315 [译自: 复旦学报(自然科学版)]