Aneuploid and Polyploid Cellular DNA Heterogeneity in Insect Cell Material of Diptera Species Analyzed by Flow Cytometry

Abstract Flow cytometric 1-parameter DNA analysis and 2-parameter DNA /protein analysis have been performed with cell material of the diptera species Chironomus thummi, Drosophila melanogaster, Calliphora vicina and Musca domestica using an impulse cytophotometer with a new quartz objective, that was especially manufactured for cytofluorometric investigations. The occurrence of heterogenous cell populations with aneuploid and polyploid DNA content within the cell material of different developmental stages of diptera species have been determined, whereby in larvae polyploid cell populations and in imagos aneuploid cell populations predominate. Partially separation of 2C cells from other cell populations with higher DNA content can be done by Ficoll-Hypaque centrifugation as demonstrated with cell material from Chironomus larvae. For flow cytometric DNA analysis of insect cell material a simple and rapid cell preparation and staining technique is presented by using the DNA-specific fluorochrome DAPI in combination with the protein fluorochrome sulforhodamine 101. Employment of flow cytometry in diptera genetics might be a new tool for cytological and cytogenetic investigations as shown with the classical genetic objects Chironomus and Drosophila

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Flow cytometry, cellular DNA content, and prognosis in human malignancy.

Journal of Clinical Oncology ◽

10.1200/jco.1987.5.10.1690 ◽

1987 ◽

Vol 5 (10) ◽

pp. 1690-1703 ◽

Cited By ~ 192

Author(s):

D E Merkel ◽

L G Dressler ◽

W L McGuire

Keyword(s):

Flow Cytometry ◽

Data Base ◽

Dna Content ◽

Dna Analysis ◽

Clinical Decision Making ◽

Risk Groups ◽

Clinical Decision ◽

Tumor Type ◽

Cellular Dna ◽

Bladder Carcinomas

The use of flow cytometry to analyze the cellular DNA content of human malignancies has become increasingly commonplace. The relationship between abnormalities in DNA content or proliferative characteristics and prognosis is becoming clear for a variety of malignancies in part through new techniques that permit analysis of archival material. High- and low-risk groups of patients with early breast and bladder carcinomas, non-small-cell lung cancer, and colorectal, ovarian, and cervical carcinoma can be distinguished on the basis of abnormal stemline DNA content. In several hematologic and common pediatric malignancies, the prognostic relevance of DNA content flow cytometry has been similarly established. Though the interpretation of tumor cell cycle analyses is less certain, this characteristic may also be prognostically important. However, generalizations cannot be made when applying flow cytometric DNA analysis to clinical decision making. The prognostic importance of an abnormal DNA histogram for an individual patient must be assessed on the basis of the relevant data base for that particular tumor type. The current extent of this data base for various malignancies is reviewed.

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Flow cytometric DNA analysis in conservatively treated renal tumours

Urologia Journal ◽

10.1177/039156039506201s39 ◽

1995 ◽

Vol 62 (1_suppl) ◽

pp. 141-143

Author(s):

P. Beltrami ◽

M. Lazzarotto ◽

G. Giusti ◽

C. Tallarigo ◽

G. Malossini ◽

...

Keyword(s):

Flow Cytometry ◽

Dna Analysis ◽

Tumour Size ◽

Prognostic Significance ◽

Dna Flow Cytometry ◽

Additional Parameter ◽

Flow Cytometric ◽

Renal Tumours ◽

Dna Histograms

— The DNA histograms of 21 conservatively resected renal tumours were studied using DNA flow cytometry. Five patients had an imperative and sixteen an elective indication for conservative resection of the renal tumour. On the basis of DNA histograms twelve aneuploid tumours were pointed out. A mean follow-up of 34.2 months was considered to see whether the ploidy would provide criteria with a prognostic significance, to be useful as an additional parameter. None of the twenty-one patients had local recurrence or distant metastasis: in our series the DNA analysis had no influence on the prognosis of this group of patients. The tumour size seems to be the only selective parameter for choosing renal-conserving surgery.

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Flow Cytometrically Determined DNA Content of Breast Carcinoma and Benign Lesions: Correlations with Histopathological Parameters

Tumori Journal ◽

10.1177/030089168607200209 ◽

1986 ◽

Vol 72 (2) ◽

pp. 171-177 ◽

Cited By ~ 10

Author(s):

Raffaella Uccelli ◽

Alberto Calugi ◽

Donato Forte ◽

Francesco Mauro ◽

Paolo Polonio-Balbi ◽

...

Keyword(s):

Flow Cytometry ◽

Dna Content ◽

Cell Subpopulation ◽

Aneuploid Cell ◽

Breast Carcinomas ◽

Benign Lesions ◽

Dna Index ◽

Tissue Samples ◽

Normal Tissues ◽

Relative Dna Content

The relative DNA content of cellular samples from 54 patients affected by breast carcinomas and 20 affected by benign breast lesions (including 11 fibroadenomas) was measured by flow cytometry. All normal tissue samples and 17/20 (85%) specimens from benign lesions exhibited a cytometrically diploid DNA distribution, 3/20 (15%) benign lesions an abnormal DNA content, and 35/54 (65%) carcinomas at least one aneuploid cell subpopulation. Furthermore, 9/54 (17%) tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results also indicate that flow cytometry can be used to recognize lymph nodes infiltrated by aneuploid cells. Statistically significant correlations were evidenced between the occurrence of aneuploidy or the ploidy level measured as DNA index and the nodal infiltration status. The percentage of S cells can also be extracted from DNA content distribution histograms. Statistically significant differences (p < 0.01) were also observed for the percentage of S cells between normal tissues (6.2±3.2 SD) and benign lesions (11.1±6.6 SD), normal tissues (6.2 ± 3.2 SD) and aneuploid tumors (19.7 ± 10.3 SD), benign lesions (11.1 ± 6.6 SD) and aneuploid tumors (19.7 ± 10.3 SD), and diploid (7.9 ± 4.0 SD) and aneuploid tumors (19.7 ± 10.3 SD).

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Flow cytometric analysis of small DNA content differences in heterogeneous cell populations: Human amniotic fluid cells

Cytometry ◽

10.1002/cyto.990050204 ◽

1984 ◽

Vol 5 (2) ◽

pp. 118-123 ◽

Cited By ~ 15

Author(s):

H. Koch ◽

T. Bettecken ◽

M. Kubbies ◽

D. Salk ◽

J. W. Smith ◽

...

Keyword(s):

Amniotic Fluid ◽

Dna Content ◽

Flow Cytometric Analysis ◽

Cell Populations ◽

Human Amniotic Fluid ◽

Flow Cytometric ◽

Amniotic Fluid Cells

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Biological Characterisation of Superficial Bladder Cancer by Bivariate Cytokeratin 7/DNA Analysis, Flow Cytometric Assessment of MIB-1, and an Immunohistochemical Study

Analytical Cellular Pathology ◽

10.1155/2000/672354 ◽

2000 ◽

Vol 21 (1) ◽

pp. 21-33 ◽

Cited By ~ 3

Author(s):

Elena Leonardi ◽

Paolo Dalla Palma ◽

Alessandro Reich ◽

Orazio Caffo ◽

Lucio Luciani

Keyword(s):

Bladder Cancer ◽

Dna Content ◽

Dna Analysis ◽

Immunohistochemical Study ◽

Statistical Significance ◽

Multiparameter Flow Cytometry ◽

Cytokeratin 7 ◽

Biological Behaviour ◽

Flow Cytometric ◽

Mib 1

A total of 238 cases of bladder carcinoma stages Ta, Tis, T1 were submitted prospectively to multiparameter flow cytometry and immunohistochemical study in order to determine the biological aggressiveness of the tumour. DNA index (DI), S-phase fraction (SPF) obtained by bivariate cytokeratin 7=DNA analyses, and the immunohistochemical evaluation of p53 and MIB-1 were studied in relation to the traditional prognostic factors in bladder cancer (stage and grade). The variance analysis results showed that DNA aneuploidy was significantly associated with high stage (p= 0:0001), high grade (p= 0:0001), high SPF value ≥5.5% (p= 0:0001), MIB-1 positivity ≥31% (p= 0:0001) and high expression of p53 (staining involving >50% of cells,p= 0:0001). Even if there was no statistical significance the hypotetraploid class (1.70<DI<1.89) showed poor prognostic biomarkers more frequently than the other aneuploid classes. Out of 238 cases, 101 were also submitted to flow cytometric measurement ofMIB-1 (fMIB-1) to study the correlation between cell proliferation and DNA content. Data obtained from fresh, 3 : 1 methanol=acetone fixed samples were compared with values obtained from both cell cycle analysis methods and routine application of the MIB-1 immunostaining in histological sections. fMIB-1 values were positively correlated with SPF values (r= 0:801,p< 0:01) and S+G2M fraction (percentage of cells in S and in G2M phases) (r= 0:763,p< 0:01) but no correlation with paraffin sections was found. A fMIB-1 value >7% was strongly associated with aneuploidy (p= 0:0001). The determination of DNA content coupled with the study of the epithelial (cytokeratin 7) and proliferative (MIB-1) markers could be useful in providing important information on the biological behaviour of superficial bladder tumours.

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Diagnosis and risk stratification of Barrett’s dysplasia by flow cytometric DNA analysis of paraffin-embedded tissue

Gut ◽

10.1136/gutjnl-2017-313815 ◽

2017 ◽

Vol 67 (7) ◽

pp. 1229-1238 ◽

Cited By ~ 14

Author(s):

Won-Tak Choi ◽

Jia-Huei Tsai ◽

Peter S Rabinovitch ◽

Thomas Small ◽

Danning Huang ◽

...

Keyword(s):

Flow Cytometry ◽

Risk Stratification ◽

Dna Content ◽

Dna Analysis ◽

Diagnostic Marker ◽

Oesophageal Adenocarcinoma ◽

Dna Flow Cytometry ◽

Low Grade ◽

Area Of Interest ◽

Formalin Fixed Paraffin Embedded

ObjectiveThe diagnosis of dysplasia in Barrett’s oesophagus (BO) can be challenging, and reliable ancillary techniques are not available. This study examines if DNA content abnormality detected by flow cytometry can serve as a diagnostic marker of dysplasia and facilitate risk stratification of low-grade dysplasia (LGD) and indefinite for dysplasia (IND) patients using formalin-fixed paraffin-embedded (FFPE) BO samples with varying degrees of dysplasia.DesignDNA flow cytometry was performed on 80 FFPE BO samples with high-grade dysplasia (HGD), 38 LGD, 21 IND and 14 negative for dysplasia (ND). Three to four 60-micron thick sections were cut from each tissue block, and the area of interest was manually dissected.ResultsDNA content abnormality was identified in 76 HGD (95%), 8 LGD (21.1%), 2 IND (9.5%) and 0 ND samples. As a diagnostic marker of HGD, the estimated sensitivity and specificity of DNA content abnormality were 95% and 85%, respectively. For patients with DNA content abnormality detected at baseline LGD or IND, the univariate HRs for subsequent detection of HGD or oesophageal adenocarcinoma (OAC) were 7.0 and 20.0, respectively (p =<0.001).ConclusionsThis study demonstrates the promise of DNA flow cytometry using FFPE tissue in the diagnosis and risk stratification of dysplasia in BO. The presence of DNA content abnormality correlates with increasing levels of dysplasia, as 95% of HGD samples showed DNA content abnormality. DNA flow cytometry also identifies a subset of patients with LGD and IND who are at higher risk for subsequent detection of HGD or OAC.

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ROR-1 Is a Highly Discriminative Marker in Flow Cytometric Minimal Residual Disease (MRD) Detection in Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v128.22.3197.3197 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3197-3197 ◽

Cited By ~ 2

Author(s):

Michaela Patz ◽

Barbara Pentok ◽

Kathrin Cremer ◽

Stefanie Linnartz ◽

Esther Lilienweiss ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Research Funding ◽

Residual Disease ◽

Lymphocytic Leukemia ◽

Cell Populations ◽

Discrimination Analysis ◽

Flow Cytometric ◽

Correct Discrimination ◽

Minimal Residual

Abstract Introduction:With the advent of new potent therapies for chronic lymphocytic leukemia (CLL) minimal residual disease (MRD) detection becomes increasingly important to assess remission depth. While molecular MRD detection for CLL remains laborious and time consuming flow cytometry is a fast, economic and sensitive method in detecting low frequencies of CLL cells. The usefulness of the antigens CD81, CD5, CD20, CD43 and CD79b has been previously described for this purpose. ROR-1 has recently been identified as a signature gene in CLL and mantle cell lymphoma. The potential utility of ROR-1 in flow cytometric minimal residual cell analysis has not been evaluated yet. Methods: 10 normal samples and 77 remnants of randomly selected samples from diagnosed patients undergoing CLL therapy were analyzed by flow cytometry. A customized dry formulation of an antibody panel was used, comprising antibodies directed against CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 (DuraClone RE CLB). Linearity, repeatability and inter-operator variability of data analysis of the method were examined. B cell populations comprising at least 50 positive events (46 normal B cell populations, 25 CLL populations, paired and unpaired) were analyzed for their expression profile as assessed by respective mean fluorescence intensities of the antibody labels within classified populations. The expression profiles were subject to supervised discrimination analysis (DA). Results: Between124,000 and 2,122,000 (683,000 ± 450,000) CD45+ events were acquired from the 87 samples. The background of cells with a CLL-like phenotype in the normal samples was determined as <0.001% of CD45+ events. Linearity was confirmed in the range from 1% to 0.0025%. The Repeatability analysis and the inter-operator variability showed concordance with typical Poisson distribution characteristics. The 46 populations with a typical normal B cell phenotype ranged from 0.014% to 9.592% with an average of 2.45% ± 2.75 of CD45+ events. The 25 populations with a classical or non-classical CLL phenotype ranged from 0.007% to 5.459% with an average of 1.41% ± 1.65 of CD45+ events. Posterior discrimination analysis revealed 100% correct discrimination for CLL populations and 96% correct discrimination for normal populations when relying on ROR-1 expression alone in CD19+CD45+ B cells. This result was only surpassed by the complete antibody combination (100% / 100%) but not by any other of the markers, neither in single use nor in combination Conclusion: The 8-color dry flow cytometry panel comprising CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 demonstrated sensitive, linear and specific detection of residual CLL cells in a relevant low range of frequency. ROR-1 revealed to be a highly discriminative marker in the analysis of residual CLL cells by flow cytometry. Utilizing this flow cytometry approach, MRD detection showing sensitivity comparable to molecular techniques can be achieved in CLL. Disclosures Hallek: AbbVIe: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria; Glaxo-SmithKline: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria; Roche: Consultancy, Research Funding, Speakers Bureau. Kreuzer:Gilead Sciences: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche Pharma GmbH and Mundipharma GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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