| 1. |
- Bagwell, C B, et al.
(författare)
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Optimizing flow cytometric DNA ploidy and S-phase fraction as independent prognostic markers for node-negative breast cancer specimens
- 2001
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 46:3, s. 121-135
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Tidskriftsartikel (refereegranskat)abstract
- Developing a reliable and quantitative assessment of the potential virulence of a malignancy has been a long-standing goal in clinical cytometry. DNA histogram analysis provides valuable information on the cycling activity of a tumor population through S-phase estimates; it also identifies nondiploid populations, a possible indicator of genetic instability and subsequent predisposition to metastasis. Because of conflicting studies in the literature, the clinical relevance of both of these potential prognostic markers has been questioned for the management of breast cancer patients. The purposes of this study are to present a set of 10 adjustments derived from a single large study that optimizes the prognostic strength of both DNA ploidy and S-phase and to test the validity of this approach on two other large multicenter studies. Ten adjustments to both DNA ploidy and S-phase were developed from a single node-negative breast cancer database from Baylor College (n = 961 cases). Seven of the adjustments were used to reclassify histograms into low-risk and high-risk ploidy patterns based on aneuploid fraction and DNA index optimum thresholds resulting in prognostic P values changing from little (P < 0.02) or no significance to P < 0.000005. Other databases from Sweden (n = 210 cases) and France (n = 220 cases) demonstrated similar improvement of DNA ploidy prognostic significance, P < 0.02 to P < 0.0009 and P < 0.12 to P < 0.002, respectively. Three other adjustments were applied to diploid and aneuploid S-phases. These adjustments eliminated a spurious correlation between DNA ploidy and S-phase and enabled them to combine independently into a powerful prognostic model capable of stratifying patients into low, intermediate, and high-risk groups (P < 0.000005). When the Baylor prognostic model was applied to the Sweden and French databases, similar significant patient stratifications were observed (P < 0.0003 and P < 0.00001, respectively). The successful transference of the Baylor prognostic model to other studies suggests that the proposed adjustments may play an important role in standardizing this test and provide valuable prognostic information to those involved in the management of breast cancer patients.
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| 2. |
- Baldetorp, Bo, et al.
(författare)
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Different calculation methods for flow cytometric S-phase fraction: prognostic implications in breast cancer? The Swedish Society of Cancer Study Group
- 1998
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Ingår i: Cytometry. - 0196-4763. ; 33:4, s. 385-393
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Tidskriftsartikel (refereegranskat)abstract
- S-phase fraction (SPF), estimated in the flow cytometric DNA histogram, is a prognostic factor in breast cancer. There are, however, some inherent difficulties in the estimation of SPF, such as the influence of debris, aggregates, and normal cells. Most of the available SPF calculation principles try to consider these difficulties, but so far no consensus has been reached with regard to which principle is to be recommended. The aim of the present study was to investigate the prognostic impact of SPF when estimated with different calculation methods in frozen breast cancer samples from 350 patients. Two nonparametric (Rman, Rmin/both rectangle) and three parametric (ACAS/DNA-base, ModFit, and MultiCycle) calculation methods, with and without correction for debris and aggregates, were used. The mean values for SPF varied from 4.3% (ACAS/DNA-base with correction for debris and aggregates) to 9.4% (MultiCycle without any correction for background). The pairwise correlation between methods varied considerably (R = 0.72-0.98). After categorization of SPF values into low SPF (lower two tertiles) and high SPF (upper tertile), all methods yielded statistically significant Pvalues for recurrence-free survival (median follow-up time 67 months), both univariately (0.0004-< 0.0001) and multivariately (0.048-0.0004), after adjusting for nodal status, tumor size, and estrogen receptor status. SPF with background correction did not yield lower P values than SPF without. Regardless of which method was used, SPF showed similar correlations with lymph node involvement, tumor size, and estrogen receptor content. In conclusion, as the mean value of SPF for different calculation methods varies, each laboratory must be restricted to use only one method. Background correction does not seem to improve the prognostic impact of SPF in DNA histograms. Based on the experiences obtained in the present study, S-phase calculation methods without background correction may therefore be the most suitable for routine evaluation of DNA histograms of fresh frozen breast cancer material (ModFit, MultiCycle, and Rman [the latter only for experienced operators]). The nonparametric Rmin, with an automatic setting of the region used for SPF calculation, may be an alternative, but suffers from the disadvantage of not being commercially available yet.
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| 3. |
- Baldetorp, Bo, et al.
(författare)
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Image cytometric DNA analysis in human breast cancer analysis may add prognostic information in diploid cases with low S-phase fraction by flow cytometry
- 1992
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 13:6, s. 577-585
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Tidskriftsartikel (refereegranskat)abstract
- Measurements of DNA ploidy can be performed either with image cytometry (ICM) or flow cytometry (FCM); both methods provide independent prognostic information in primary breast cancer. The aim of the present investigation was to compare the two methods and to relate the findings to prognosis (median follow-up 42 months). Concordance in ploidy status (diploid, tetraploid, aneuploid) was obtained in 76% of the samples (168/222). When the fraction of S-phase cells (SPF) from FCM analysis was also taken into consideration, four different groups of samples were obtained (Flow I-IV), which were considered to correspond to the Auer classification (Auer I-IV) of DNA histograms obtained from image cytometry. Complete concordance between the two techniques now was 70% (155/222). Samples classified as Flow I (diploid or near-diploid with low SPF) and Auer I had a distant metastasis rate of 3/60 (5%), as compared to 62/154 (40%) for all other combinations of the Flow and Auer classifications taken together. Thus, the only findings of prognostic importance were that some samples were Flow I but not Auer I, or vice versa. These two groups represent 17 (7.7%) and 14 (6.3%), respectively, of the total number of samples, and had frequencies of distant metastasis similar to those of the other high-risk groups, namely, 7/17 and 5/14, respectively. In a multivariate analysis, flow cytometric S-phase value was a stronger prognostic factor than either the Flow and Auer classification. We conclude that when routine FCM DNA analysis is used, diploid or near-diploid samples with a low S-phase value should be reanalyzed with ICM.
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| 4. |
- Baldetorp, Bo, et al.
(författare)
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Improved DNA flow cytometric, DNA ploidy, and S-phase reproducibility between 15 laboratories in analysis of breast cancer using generalized guidelines
- 2003
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 56A:1, s. 1-7
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundLack of generalized guidelines for DNA flow cytometric analysis (FCM) may be the main reason for its limited use in the clinical management of breast cancer.MethodsAfter an initial interlaboratory reproducibility study (Round I), we concluded that it was the evaluation of the DNA histograms rather than the technical performance of the analysis that was the main reason for discordant results between laboratories. Guidelines for the interpretation of DNA histograms were therefore drawn up. We present here data from a new reproducibility study (Round II) using these guidelines.ResultsFor 10 laboratories also participating in Round I, use of the guidelines increased the concordance in DNA ploidy status from 89% to 100% for the 46 samples used in both rounds. The concordance rate for SPF also increased; mean rs-value increased from 0.81 to 0.88, and mean kappa value (lower two-thirds versus upper third versus not reported) increased from 0.55 to 0.71. Five new laboratories, participating only in Round II, also agreed with the 10 original laboratories regarding DNA ploidy status. With the inclusion of all 15 laboratories, we obtained a mean rs-value of 0.81 and a mean kappa value of 0.72 for SPF.ConclusionsGeneralized guidelines for DNA FCM increase interlaboratory agreement, which is highly important in clinical routines and in multicenter studies. Furthermore, inexperienced FCM laboratories using generalized guidelines can produce and interpret DNA FCM data equally as well as experienced laboratories.
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| 5. |
- Baldetorp, Bo, et al.
(författare)
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Reproducibility in DNA flow cytometric analysis of breast cancer: comparison of 12 laboratories' results for 67 sample homogenates
- 1995
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 22:2, s. 115-127
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Tidskriftsartikel (refereegranskat)abstract
- Flow cytometric (FCM) DNA analysis yields information on ploidy status and the S-phase fraction (SPF), variables of prognostic importance in breast cancer. The clinical value of the SPF is currently being evaluated in prospective randomized trials. The widespread use of FCM DNA analysis emphasizes the importance of reproducibility (both intra- and interlaboratory). In this study, 67 nuclear suspensions of breast cancer samples were analyzed by 12 laboratories routinely performing FCM DNA analysis in breast cancer. No general guidelines were imposed; each laboratory used its own standard protocols. For DNA ploidy status (diploid vs. non-diploid), agreement was complete for 79% (53/67) of the samples, compared with 64% (43/67) of samples when tetraploidy was considered [i.e., euploid (diploid+tetraploid) vs. aneuploid (the remaining non-diploid)]. For the SPF, pairwise comparison of the results of all 12 laboratories yielded a mean Spearman's rank correlation of 0.78 (range: 0.54-0.93). For those 39 samples being categorized in low or high SPF by all laboratories, all agreed in 14 samples (36%). Similar patterns were obtained with kappa measures, agreement being good for ploidy status (diploid vs. non-diploid; overall kappa = 0.87 and 0.74 for euploid vs. aneuploid), but moderate for the SPF [overall kappa = 0.47 (for low SPF vs. high SPF vs. "no SPF reported")]. Discrepancies were chiefly attributable to differences in the categorization of the S-phase values, rather than in FCM procedures, other critical differences being in the detection and interpretation of near-diploid and small non-diploid cell populations, the definition of tetraploidy, and the choice and execution of the method used for S-phase estimation. Based on the observations of this study, detailed guidelines for FCM analysis and interpretation of data are proposed in the Appendix. Some issues remain, however, e.g., to standardize a method for S-phase calculation and tetraploid definition.
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| 6. |
- Baldetorp, Bo, et al.
(författare)
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Statistical evaluation of cell kinetic data from DNA flow cytometry (FCM) by the EM algorithm
- 1989
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 10:6, s. 695-705
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Tidskriftsartikel (refereegranskat)abstract
- Flow cytometric DNA measurements yield the amount of DNA for each of a large number of cells. A DNA histogram normally consists of a mixture of one or more constellations of G0/G1-, S-, G2/M-phase cells, together with internal standards, debris, background noise, and one or more populations of clumped cells. We have modelled typical DNA histograms as a mixed distribution with Gaussian densities for the G0/G1 and G2/M phases, an S-phase density, assumed to be uniform between the G0/G1 and G2/M peaks, observed with a Gaussian error, and with Gaussian densities for standards of chicken and trout red blood cells. The debris is modelled as a truncated exponential distribution, and we also have included a uniform background noise distribution over the whole observation interval. We have explored a new approach for maximum-likelihood analyses of complex DNA histograms by the application of the EM algorithm. This algorithm was used for four observed DNA histograms of varying complexity. Our results show that the algorithm works very well, and it converges to reasonable values for all parameters. In simulations from the estimated models, we have investigated bias, variance, and correlations of the estimates.
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| 7. |
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| 8. |
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| 9. |
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| 10. |
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| 11. |
- Dreinhofer, KE, et al.
(författare)
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DNA ploidy in soft tissue sarcoma: Comparison of flow and image cytometry with clinical follow-up in 93 patients : comparison of flow and image cytometry with clinical follow-up in 93 patients
- 2002
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Ingår i: Cytometry. - : Wiley. - 0196-4763. ; 50:1, s. 19-24
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Tidskriftsartikel (refereegranskat)abstract
- In soft tissue sarcoma, the prognostic importance of DNA ploidy status is limited. One possible explanation may be technical; small nondiploid stemlines will he 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 nonrelevant cells. The ability of the two methods to detect nondiploid 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 nondiploid stemlines by ICM did not influence the metastatic rates. Increasing tumor size, histotype other than liposarcoma, increasing malignancy grade, tumor necrosis, and ICM nondiploidy 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. (C) 2002 Wiley-Liss, Inc.
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| 12. |
- Fernö, Mårten, et al.
(författare)
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One or multiple samplings for flow cytometric DNA analyses in breast cancer-prognostic implications?
- 1992
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 13:3, s. 241-249
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Tidskriftsartikel (refereegranskat)abstract
- Flow cytometric assessments of DNA ploidy status and the S-phase fraction (SPF) have been shown to yield prognostic information in breast cancer. The aim of the present investigation was to elucidate the reproducibility of results with regard to tumor DNA heterogeneity, and to ascertain whether the prognostic value of DNA measurements might be enhanced by analyzing two pieces of a tumor instead of one. Agreement with regard to ploidy status (diploid versus non-diploid) was obtained in 90% of cases (71/79) when two adjacent sections of the tumor were investigated, and in 77% of cases (10/13) when four biopsies from different quadrants of the tumor specimen were investigated. The corresponding figures for agreement in SPF (divided into three categories, less than 7.0%, 7.0-11.9%, and greater than or equal to 12%) were 75% (59/79; 2-sample series) and 55% (7/13; 4-biopsy series). The main reason for variance in ploidy results was the difficulties in distinguishing near diploid cell populations. Discrepancies in SPF categories could be explained by minor fluctuations in SPF values near the cut-off levels, or by variance in ploidy status, the fraction of non-diploid nuclei, and background noise due to cell debris. There was a stepwise increase in recurrence rate (RR) among patients with increasing SPF category (RR: 20%, 41%, and 53%). Patients whose SPF categories varied, from low or intermediate in one part of the tumor to high in another, seemed to have a poor prognosis (RR = 57%).(ABSTRACT TRUNCATED AT 250 WORDS)
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| 13. |
- Hedenfalk, Ingrid, et al.
(författare)
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Activated cell cycle checkpoints in epirubicin-treated breast cancer cells studied by BrdUrd-flow cytometry
- 1997
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Ingår i: Cytometry. - 0196-4763. ; 29:4, s. 321-327
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Tidskriftsartikel (refereegranskat)abstract
- Genetic alterations, such as p53 mutations, may affect a tumour's response to chemotherapy. We have treated two human breast cancer cell lines that differ in p53 status with epirubicin in order to study if there are differences in cell cycle kinetic response. MCF-7 cells express wild-type p53, while SK-BR-3 cells express only a mutated form of p53. The transition of cells from one cell cycle stage to another was studied by a bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) method. MCF-7 cells showed a block in the G1 phase after treatment with 50 nM epirubicin for 24 hours, in agreement with the actions of p53 at the G1 checkpoint. SK-BR-3 cells, on the other hand, progressed through the G1 checkpoint and were blocked in late S and G2 phases, presumably due to the activation of a later checkpoint. In addition, studies of the mRNA levels of p53 and its effector gene p21 revealed that although both cell lines expressed p53 mRNA, a marked difference in the mRNA levels of p21 was seen. A dramatic increase in the level of p21 mRNA was seen in epirubicin-treated MCF-7 cells, while no such increase was seen in SK-BR-3 cells.
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| 14. |
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| 15. |
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| 16. |
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| 17. |
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| 18. |
- Johansson, Maria C, et al.
(författare)
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Comparison of different labelling index formulae used on bromodeoxyuridine-flow cytometry data
- 1998
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Ingår i: Cytometry. - 0196-4763. ; 32:3, s. 233-240
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Tidskriftsartikel (refereegranskat)abstract
- The essence of the bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) technique is that cells are labelled with the thymidine analogue BrdUrd. They are then allowed to progress through the cell cycle in a BrdUrd-free environment during the postlabelling time period. At a postlabelling time shorter than the length of the S phase (Ts), cells are fixed and prepared for FCM-mediated analysis of BrdUrd and DNA contents. From FCM-derived data, cell cycle kinetic parameters such as labelling index (LI), Ts, and potential doubling time (Tpot) can be calculated. Tpot is believed to be important in the evaluation of tumor aggressiveness and therapy response. Since LI is most commonly used together with Ts to calculate Tpot, it is important that both LI and Ts are independent of the time when cells are sampled. Several formulae to calculate LI and Ts have been presented. In the present paper, we deal with various formulae to calculate LI. These formulae differ in how they take into account unlabelled and BrdUrd-labelled cells in various fractions of the cell cycle. We present a new formula, which takes into consideration cells in the different fractions and thus makes LI theoretically independent of postlabelling time. Our results show that different LI values are obtained when different formulae are used to calculate LI. In addition, we show that the BrdUrd labelling period should be kept as short as possible.
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| 19. |
- Kostova, NN, et al.
(författare)
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Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions
- 2004
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 58A:2, s. 132-139
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Tidskriftsartikel (refereegranskat)abstract
- Background: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. Methods: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. Results: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. Conclusions: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition. (C) 2004 Wiley-Liss, Inc.
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| 20. |
- Lahdetie, J, et al.
(författare)
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Flow cytometric analysis of micronucleus induction in rat bone marrow polychromatic erythrocytes by 1,2;3,4-diepoxybutane, 3,4-epoxy-1-butene, and 1,2-epoxybutane-3,4-diol
- 1997
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Ingår i: CYTOMETRY. - : WILEY-LISS. - 0196-4763. ; 28:3, s. 228-235
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Automation of the analysis of micronucleus induction with flow cytometry was developed by using mouse bone marrow or peripheral blood, In the present study, we report the use of flow cytometry for the identification and quantification of micronuclei (MN)
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| 21. |
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| 22. |
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| 23. |
- Lindblad, Joakim, et al.
(författare)
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Image Analysis for Automatic Segmentation of Cytoplasms and Classification of Rac1 Activation
- 2004
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 57A:1, s. 22-23
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:Rac1 is a GTP-binding molecule involved in a wide range of cellular processes. Using digital image analysis, agonist-induced translocation of green fluorescent protein (GFP) Rac1 to the cellular membrane can be estimated quantitatively for individual cells.METHODS:A fully automatic image analysis method for cell segmentation, feature extraction, and classification of cells according to their activation, i.e., GFP-Rac1 translocation and ruffle formation at stimuli, is described. Based on training data produced by visual annotation of four image series, a statistical classifier was created.RESULTS:The results of the automatic classification were compared with results from visual inspection of the same time sequences. The automatic classification differed from the visual classification at about the same level as visual classifications performed by two different skilled professionals differed from each other. Classification of a second image set, consisting of seven image series with different concentrations of agonist, showed that the classifier could detect an increased proportion of activated cells at increased agonist concentration.CONCLUSIONS:Intracellular activities, such as ruffle formation, can be quantified by fully automatic image analysis, with an accuracy comparable to that achieved by visual inspection. This analysis can be done at a speed of hundreds of cells per second and without the subjectivity introduced by manual judgments.
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| 24. |
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| 25. |
- Loborg, Helena, 1963-, et al.
(författare)
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Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe
- 2000
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Ingår i: Cytometry. - 0196-4763 .- 1097-0320. ; 40:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- Background: It is generally assumed that linker histones contribute to condensation of chromatin and to the repression of genetic activity by binding to chromatin with increased affinity. The aim of this study is to investigate a possible correlation between linker histone binding to chromatin in situ and chromatin condensation using a cytochemical approach that largely preserves nuclear structure.Methods: Ionically bound H1 was extracted with increasing concentrations of sodium chloride. Thereafter, the cells were fixed in formaldehyde and stained with 50 nM 4',6-diamidino-2- phenylindole (DAPI) and fluorescence intensity was measured by image cytofluorometry.Results: The association between linker histones and chromatin was stronger in cultured human fibroblasts and rat smooth muscle cells than in both human T lymphocytes and granulocytes, and in particular, frog erythrocytes, which exhibited highly condensed chromatin. Our data indicate a lower affinity of linker histones for metaphase chromosomes than for interphase chromatin, and we observed a reduction in linker histone affinity during terminal differentiation of frog erythrocytes in vivo.Conclusions: Taken together, our findings imply that the affinity of linker histones for chromatin in situ is unrelated or inversely related to chromatin condensation.
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| 26. |
- Loborg, Helena, et al.
(författare)
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DNA Binding Fluorochromes as Probes for Histone HI-Chromatin Interactions In Situ
- 1997
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Ingår i: Cytometry. - 0196-4763 .- 1097-0320. ; 28:3, s. 212-219
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated using the DNA binding fluorochromes 7-aminoactinomycin (7-AAMD) and 4′,6-diamidino-2-phenylindole (DAPI) as cytochemical probes for linker histone (H1)–chromatin interactions in situ. Human lymphocytes, permeabilized with digitonin, were exposed to increasing concentrations of sodium chloride to remove ionically bound H1 from the nuclei. The cells were stained to equilibrium with 1 μM 7-AAMD or 50 nM DAPI. Lymphocytes stained with 7-AAMD showed a gradual increase from 11% to 36% of HCl treated cell fluorescence intensity when the salt concentration was increased from 0.15 to 0.7 M. The corresponding increase for DAPI was 53–68%. The 7-AAMD obviously showed higher sensitivity for H1–chromatin interactions that DAPI but had disadvantages such as high background fluorescence and an affinity that was dependent on the preparation procedure. DAPI had negligible background fluorescence, and its fluorescence intensity resembles the number of available high-affinity dye-binding sites when used at 50 nM. We conclude that both fluorochromes can be used as probes for H1–chromatin interactions in situ and that our method has a potential to provide new information on such interactions.
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| 27. |
- Loborg, Helena, et al.
(författare)
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High Affinity Binding of 7-Aminoactinomycin D and 4' ,6-Diamidino-2-Phenylindole to Human Neutrophilic Granulocytes and Lymphocytes
- 1995
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 20:4, s. 296-306
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Tidskriftsartikel (refereegranskat)abstract
- The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4′,6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction methods were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid extracted cells, supposedly containing nucleosome-free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ.
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| 28. |
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| 29. |
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| 30. |
- Menghi-Sartorio, Samantha, et al.
(författare)
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DNA copy number amplifications in sarcomas with homogeneously staining regions and double minutes
- 2001
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 46:2, s. 79-84
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Tidskriftsartikel (refereegranskat)abstract
- To identify DNA amplifications in sarcomas, comparative genomic hybridization was performed on 27 cases that were likely to display high-level DNA copy number gains. In all cases, chromosome banding analysis had revealed homogeneously staining regions or double minutes, i.e., cytogenetic signs of gene amplification. In most cases, gains predominated over losses. Low-level amplifications (ratio 1.3:1.5) were seen in 20 cases. High-level amplifications (ratio >1.5) exceeded the frequencies seen in published, unselected sarcomas of similar histotypes and were detected in 16 tumors: 4/4 osteosarcomas, 5/8 malignant fibrous histiocytomas, 3/7 leiomyosarcomas, 1/2 myosarcomas, 0/1 liposarcoma, 0/1 rhabdomyosarcoma, 1/1 pleomorphic sarcoma, 0/1 myxofibrosarcoma, 1/1 malignant mesenchymona, and 1/1 malignant schwannoma, with two to four chromosomal regions involved in nine tumors. Recurrent amplifications involved 1p33-p32, 5p15-p14, 7pter-p12, 7q21-qter, 8q21.3-qter, 11q22-q23, 16p13.2-p12, 19q12-q13.1, 20q11.2-qter, and 22q12-q13. Most of the recurrent gains/amplifications we detected have been reported in sarcomas previously. A novel gain/amplification was seen at 2q14.3-q21 in five cases of four sarcoma types. The disparate pattern of amplified sequences, the poor correspondence between the localization of low- and high-level amplifications, and the chromosomal position of homogeneously staining regions suggest the involvement of many genes in the amplifications and that the genes rarely maintain their native position in these tumors.
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| 31. |
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| 33. |
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| 34. |
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| 35. |
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| 36. |
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| 37. |
- Stal, Olle, et al.
(författare)
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S-phase fraction assessed by a variant of the rectangular model adapted to the flow-cytometric DNA histogram profile
- 1998
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Ingår i: Cytometry. - 0196-4763. ; 33:4, s. 487-491
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Tidskriftsartikel (refereegranskat)abstract
- Measurement of S-phase fraction by DNA flow cytometry is widely used in the analysis of human tumors. The calculation of fraction of S-phase cells is performed either by means of curve-fitting techniques or by more simple planimetric methods. Estimates by the latter often suffer from subjectivity or poor adaptation to complex DNA histograms. We describe a variant which is based on the rectangular model. The height of the rectangle is automatically determined from the lowest density value in the S-phase interval. The variant was tested on 141 DNA histograms, and the results were compared with those obtained with other variants based on the rectangular model. The new variant is thought to circumvent some of the problems concerning subjectivity and disturbances caused by aggregate peaks.
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| 38. |
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| 39. |
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| 40. |
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| 41. |
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| 42. |
- Wählby, Carolina, et al.
(författare)
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Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei
- 2002
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Ingår i: Cytometry. - 0196-4763. ; 47:1, s. 32-41
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundVisualization of more than one antigen by multicolor immunostaining is often desirable or even necessary to explore spatial and temporal relationships of functional significance. Previously presented staining protocols have been limited to the visualization of three or four antigens.MethodsImmunofluorescence staining was performed both on slices of formalin-fixed tissue and on cells microscopy. The primary and secondary antibodies, as well as the fluorophores, were thereafter removed using a combination of denaturation and elution techniques. After removal of the fluorescence stain, a new immunofluorescence staining was performed, visualizing a new set of antigens. The procedure was repeated up to three times. A method for image registration combined with segmentation, extraction of data, and cell classification was developed for efficient and objective analysis of the image data.ResultsThe results show that immunofluorescence stains in many cases can be repeatedly removed without major effects on the antigenicity of the sample.ConclusionsThe concentration of at least six different antigens in each cell can thus be measured semiquantitatively using sequential immunofluorescence staining and the described image analysis techniques. The number of antigens that can be visualized in a single sample is considerably increased by the presented protocol.
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- Zätterström, Ulf K, et al.
(författare)
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Comparison of BrdUrd and [3H]TdR incorporation to estimate cell proliferation, cell loss, and potential doubling time in tumor xenografts
- 1992
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Ingår i: Cytometry. - : Wiley. - 0196-4763 .- 1097-0320. ; 13:8, s. 872-879
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Tidskriftsartikel (refereegranskat)abstract
- In this study, two different methods of estimating cell proliferation were compared: cell loss and potential growth rate of xenografted head and neck cancer grown in nude mice based on the detection of DNA incorporation of bromodeoxyuridine (BrdUrd) in one method, and [3H]thymidine ([3H]TdR) in the other. The 21-d-old xenografts were labelled in vivo, either with BrdUrd or [3H]TdR and excised at intervals during 65.5 h. In tumors containing BrdUrd, the percent labelling was measured in mid-S and mid-G1 phase windows of cytograms from bivariate DNA flow cytometry (FCM). In [3H]TdR-labelled tumors, the percent labelled mitoses (PLM) was determined by light microscopy evaluation of autoradiographs. With a computer program based on a theoretical model, the percent labelling versus time after injection was used to analyze cell cycle time, cell loss, tumor growth fraction, and potential doubling time. The values calculated from DNA incorporation with BrdUrd agreed well with those obtained from labelling with [3H]TdR, i.e., cell cycle time 2.3 vs. 2.4 d, and growth fraction 67 vs. 70%. The estimated potential doubling time was 3.1 d and cell loss factor 40% by both methods. Flow cytometry analysis of BrdUrd-labelling is considerably faster than the evaluation of [3H]TdR-labelling, and the present results provide further support for the BrdUrd labelling method as a promising alternative to the PLM method in cell cycle studies designed to evaluate the relevance of cell proliferative properties in relation to biological behavior in xenografted head and neck cancer.
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