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Deutsche Gesellschaft für Zytometrie (DGfZ)



Abstract Text 1995


8th Heidelberg Symposium
Oct.18-20, 1995



The abstract are printed in the: Proceedings of the DGfZ Heidelberg Meetings, DKFZ, Heidelberg 1995, ISSN 0949-5347
=== ORAL PRESENTATIONS ===
1. CELL KINETIC EFFECTS INDUCED BY INTERFERON GAMMA AND TUMOR NECROSIS FACTOR ALPHA IN HUMAN TUMOR CELLS, INVOLVEMENT OF CYCLINS
H. Baisch
Institute of Biophysics and Radiobiology, University of Hamburg, Martinistrasse 52, D-20246 Hamburg
Human tumor cell lines were treated with TNFa and IFNg. The cell kinetic effects were measured using the bromodeoxyuridine method with flow cytometry. TNFa down regulated the labelling index (LI) while Ts remained constant in MCF-7 cells (breast carcinoma), and only cell loss was induced. In contrast, treated Me 180 cells (cervix carcinoma) had the same LI, Ts and Tpot as control cells, but many cells were killed by TNFa. The cell line BC5637 (bladder carcinoma) was TNFa-resistant. IFNg, on the other hand, induced decreasing LI, prolonged Ts and considerable cell loss. Ts was also prolonged in Me 180 up to a complete stop of DNA synthesis by IFNg, while MCF-7 cells proliferated like controls. The TNFa effects were reversible in the 2 sensitive lines, in contrast, IFNg effects were reversible only in MCF-7, while Me180 and BC5637 cells did not resume proliferation after replacement of IFNg with fresh medium.
All tumor cell lines showed unscheduled expression of cyclins. Treatment with IFNg for more than 2 days induced a general decrease of cyclin per cell rather than a specific effect in one of the phases of the cell cycle. Cytokines seem to influence cyclins in a more indirect way.
2. ROLE OF PROTEIN KINASE ACTIVITY IN REGULATION OF CELL CYCLE PROGRESSION
H. Crissmann, J. Valdez, S. Rose, S. Minter
Life Sciences Division Los Alamos National Laboratory, USA
Evidence from a large number of studies suggests that protein phosphorylation plays a central role in the regulation of cell cycle proliferation, growth and differentiation. Control of cell proliferation involves temporal activation of a series of interrelated primary and secondary kinases that phosphorylate an array of essential cycle regulating proteins. Cell cycle studies indicate that basic cellular processes such as the commitment to DNA replication and the initiation of mitosis are regulated by kinase mediated mechanisms. Recent studies have shown that phosphorylations of cellular proteins, important for cycle traverse, are brought about by complexing of cycle-specific cyclins with cyclin-dependent kinases. Levels of expression of the various cyclins can be observed to modulate at different times in the cell cycle. In order to obtain information on the role of kinase-mediated mechanisms in commitment to mammalian DNA replication, we initiated a series of studies using the general protein kinase inhibitor, staurosporine. Previous studies showed an inhibitory effect of continous low levels of staurosporine (2-20 nM) on progression of nontransformed but not transformed cells through G1 phase of the cell cycle. More recent studies showed that similar effects could be obtained after 1h exposures of asynchronous cultures of cells to relatively high levels of staurosporine (150 nM). Supported by the U. S. Department of Energy and the Los Alamos National Flow Cytometry Resource (NIH Grant P41RRO1315).
3. STROMA ORIGINATING FROM SORTED CD13-POSITIVE BONE MARROW CELLS
J.W. Ellwart, K. Nispel, R.A.J. Oostendorp, G. Ledderose*, P. Dörmer
GSF-lnstitut für Experimentelle Hämatologie, München, *Med. Klinik III, Klinikum Großhadern, Universität München
To study the interactions between stroma cells and hemopoietic progenitor cells we want to enrich pure subpopulations of stroma forming cells. For this purpose anti-CD13-labeled mononuclear cells from cryo-conserved bone marrow were isolated with a FACStar plus and then kept under long-term conditions. After 3 weeks of culture we could observe a stromal layer originating from the CD13-positive cells. 98% of the CD13-positive cells were low positive. They had light scatter properties like monocytes/macrophages and after a few days in culture the adherent cells of this fraction had the appearence of macrophages. They did not form a confluent stromal layer during the next weeks unless mixed with the CD13-high-positive cells. About 100 of CD13-high-positive cells were required to form a fibroblast-like confluent stroma containing adipocytes after replating. The stroma remained vital in culture for about two months and could be replated about 6 times. CD13-negative sorted cells never formed a stroma under our culture conditions. We use anti-CD13 (Aminopeptidase N) instead of STRO-1 to label the stromal cells because this antibody is better defined.
Besides the study of normal interactions between stroma cells and hemopoietic precursors the flow cytometric isolation of different types of stroma forming cells may be important for the improvement of bone marrow long term culture and for the diagnosis and understanding of the pathophysiology of hematopoietic diseases with stroma involvement.
4. CELL CYCLE KINETICS OF UROTHELIAL CELL LINES AS MEASURED BY FLOW CYTOMETRY USING BRDU/HOECHST TECHNIQUES AND IMMUNHISTOCHEMICAL STAINING FOR PROLIFERATION ASSOCIATED ANTIGENS
E. Endl, G. Fauser, P. Steinbach, R. Knüchel and F. Hofstädter Institute of Pathology, Franz-Josef-Strauss Allee 11, D-93053 Regensburg
The incorporation of modified nucleotides (i.e. 3H thymidine or Bromodeoxyuridine (BRDU)) during DNA replication is a widely accepted method for the analysis of proliferative characteristics of cells. Incorporated BRDU can be detected using a combination of an intercalating DNA stain (propidium iodide or 7-Aminoactinomycin D) with Hoechst 33258, whose fluorescence is quenched in the presence of BRDU.
The aim of our study was to establish this method for asynchronously growing tumour cell lines which have been studied less extensively by the BRDU/Hoechst method. Transit times through the different phases of the cell cycle, changes of these transit times and the simultaneous expression of proliferation associated antigens were analysed for in vitro growing cell cultures derived from bladder carcinomas (RT4, J82).
The results reveal differences between the growth characteristics obtained by the BRDU/Hoechst technique and growth curves. The BRDU/Hoechst technique considers the transit times between the different cell cycle phases at a certain time point in culture, whereas population doubling times obtained by growth curves are calculated as an average of cell cycle kinetics over a certain time period. Thus growth curves represent the proliferation characteristics of a cell population as a whole and do not reflect changes of the growth kinetics of individual cells or at individual time points. An additional advantage of the BRDU/Hoechst technique is the possibility of simultaneous analysis of proliferation associated antigens and their relation to certain time points within the populations of cycling and non cycling cells.Cells in the plateau phase of in vitro growth were analysed for the expression of proliferation associated antigens (MIB1, P120) and compared to the results obtained by BRDU/Hoechst staining.
5. DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE MYELOGENOUS LEUKEMIA AND MYELODYSPLASTIC SYNDROMES IN CLINICAL REMISSION BY FACS/FISH
H. Engel1, A. Gooodacre, A. Keyhani, S. Jiang and M. Andreeff
Department of Hematology, University of Texas M. D. Anderson Cancer Center, Houston, USA and 1 Institut für klinische Biochemie der Universität Bonn, Germany
The majority of patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS), especially those with unfavorable cytogenetics, relapse. The mechanism of survival of residual leukemic cells in the bone marrow (BM) is unknown and sensitive detection methods are necessary to monitor their levels. This study was designed to investigate the presence and frequency of minimal residual disease (MRD) in 35 patients with AML or MDS and abnormalities of chromosomes 6, 7, 8, 9, 10, 17 and 18 in clinical remission. BM samples were labeled with bromodeoxyuridine (BUdR), sorted for their leukemia associated immunophenotype by fluorescence activated cell sorting (FACS) and analyzed by interphase fluorescence in-situ hybridization (FISH). The technique allows to detect as few as 3 leukemic in 100,000 normal cells. MRD was detected in 33/35 patients, 15 of them relapsed (8/11 with monosomy7, 4/17 with trisomy 8 and 4/7 others). Levels of MRD (p="0".007) and proliferation index (p="0".011) were significantly higher in patients with monosomy 7 than in patients with trisomy 8 or other cytogenetic abnormalities. Cox-regression analysis showed that S-phase, level of abnormal cells and cytogenetic class were related to time to relapse (p="0".001) with S-phase being the single most important prognostic factor (p="0".0001). We conclude, that the combination of FACS/FISH/BUdR, which determines number, phenotype and proliferation of very rare leukemic cells in patients with AML or MDS in clinical remission, identifies patients with high and low risk of relapse.
6. ANALYSIS OF PARTICLE-SIZE FOR TESTING ON CYTOTOXICITY
D. Fieblinger, K. Grünheid, D. Labes
Pharmakologische Forschungsgesellschaft BIOPHARM GmbH Berlin, Alfred-Kowallke-Strasse 4,10315 Berlin
Toxicity testing on substances are necessary for the evaluation of potential endangering of humans. For this reason the search for possibilities of using other test systems than laboratory animals, which are used in general for this purpose, has particular importance. The use of in vitro systems for investigation of toxicity is important because of the strict correlation between acute toxicity (LD50, oral) and cytotoxicity (IC50).
Well characterised and established cell strains (e. g. V 79 - cells) are suitable for testing of test substances on cytotoxicity as well as on Mutagenicity. By the aid of modern measuring systems like the Coulter Multisizer II, values for evaluation of the vitality of cells, e. g. cell number and changes of size, and consequently toxic proporties of a test substance can well be registered, represented and evaluated. The criterious used for assessment of the toxicity exceed the simple judgement of the change of proliferation. The analysis of the particle-size provides additional qualitative parameters which are applied to the calculation of the IC50 values.
Subtances for instance as 4-Acetamidophenol and Ethylmethanesulfonate influence in different way the growth of a cell culture. This is clearly presented and compared together by a graphic interpretation of the particle-size distribution.
7. ANGULAR RESOLVED LIGHT SCATTERING OF SINGLE PARTICLES AND PARTICLE AGGLOMERATES IN A FLOW CYTOMETER
C.Gohlke, J.Neukammer, and H.Rinneberg
Physikalisch-Technische Bundesanstalt, Berlin-Charlottenburg, Abbestrasse 2-12, D-10587 Berlin, Germany
We have equipped a laser based flow cytometer with an intensified CCD camera (ICCD) for angular resolved detection of light scattered by particles and biological cells. Angular resolved observation of light scattered by blood cells was performed (i) to improve their differentiation, (ii) to distinguish single cells from agglomerates, and (iii) to identify rare cells using integral light scattering for preselection. In addition, knowledge of the angular distribution of light scattered by blood cells might serve to determine the real and imaginary part of their index of refraction.
We have studied angular resolved light scattering of single polystyrene microspheres with diameters ranging from 0.37 um to 10 um and single blood cells at wavelengths of 413.1 nm, 488.0 nm, and 632.8 nm. The angle of observation (630 -1170) was determined by the aperture of the 32x/0.6 microscope objective used. The trigger signal necessary to open the intensifier of the CCD camera was derived from a second probing laser beam located 20 um above the first one. Exposure time was variied between 0.1 us and 2 us. To select a certain particle the integral light scattering caused by its interaction with the probing laser beam was analysed by means of window discriminators. This allows all particles to be imaged with integral light scattering falling into a selected region of interest of a scattering diagram.
Besides angular distribution of light scattered by single polystyrene microspheres we have measured the angular distribution of light scattered by clusters (agglomerates) consisting of two or more polystyrene microspheres. In contrast to light scattering of single spheres, for a particle cluster Mietheory cannot be applied to describe the observed pattern. First results on the angular resolved light scattering of erythrocytes, leukocytes, and thrombocytes will be presented.
8. FLOW CYTOMETRIC S-PHASE DETERMINATION IS A STRONGER PROGNOSTIC FACTOR IN NODE-NEGATIVE BREAST CANCER THAN IMMMUNOCYTOCHEMICAL MIB1 (Ki-67) STAINING
N. Harbeck, P. Dettmar1, C. Thomssen, L. Pache, M. Schmitt, F. Jänicke, W. Nathrath1, and H. Graeff
Frauenklinik and 1 Institut für Pathologie, Technische Universität, D-81675 München, Germany
The proliferation markers, S-phase fraction (SPF) and MIB1-proliferation rate (MIB1-PR) were retrospectively determined on adjacent paraffin sections in primary tumors of 90 patients with node-negative breast cancer. For flow cytometric SPF determination we applied an improved Hedley's technique for release of pure nuclei from formalin-fixed, paraffin-embedded tissue sections. SPF was calculated using ModFit (Verity, Maine, USA). MIB1 (Ki-67) immunostaining was performed using APAAP. MIB1-PR was calculated as the percentage of stained nuclei per 500 randomly chosen tumor cells. Using isotonic regression, optimized cutoff values were determined for SPF (8 %) and MIB1-PR (25 %). Thus, 61 (68 %) tumors had low ( 8 %) SPF; 75 (83 %) tumors had low (< 25 %) and 15 (17 %) high (> 25 %) MIB1-PR. Median follow-up time was 34 months (9-72 months). SPF was significantly correlated to tumor size (p="0".041) and steroid hormone receptor status (p="0".03), MIB1-PR to grading (p="0".018) and medullary histological type (p<0.05). A significant correlation between MIB1-PR and SPF was only found in aneuploid tumors (p="0".025). In univariate analysis, both SPF (p="0".0028) and MIB1-PR (p="0".0224) were statistically significant prognostic factors for disease-free survival (DFS). In multivariate analysis however, SPF was the only significant predictor of DFS (p="0".0073), stronger than MIB 1-PR or established prognostic factors (tumor size, menopausal or steroid hormone receptor status, grading, lymph vessel invasion, tumor necrosis). We conclude that both proliferation markers, flow cytometrically determined SPF and immunocytochemically evaluated MIB1-PR, provide additional prognostic information in node-negative breast cancer. Yet, in our group of patients SPF was of higher prognostic strength and may therefore be better suited for clinical application than MIB 1 (Ki-67).
9. MODEL SYSTEM FOR ISOLATION OF COMPETENT OVARIAN CARCINOMA CELLS FROM FRESH TUMOR TISSUE BY A MAGNETIC SEPARATION SYSTEM (MACS)
N. Harbeck, A. Abdulsalam, S. Schwarze, E. Schüren, P. Dettmar1, W.Kuhn, H. Graeff, and M. Schmitt
Frauenklinik and 1 Institut für Pathologie, Technische Universität, München, Germany
Flow cytometric analysis of living ovarian carcinoma cells from fresh tumor tissue is hampered by cell heterogenity in the tumor and its stroma. We have established a model system for isolation of competent ovarian carcinoma cells from fresh tumor tissue. Fresh ovarian carcinoma tissue was subjected to mechanical disintegration and mild enzymatic treatment (0.005 % collagenase D) to obtain single cells with intact surface antigens. To overcome the lack of tumor-cell specific antibodies, we used "negative tumor cell separation": Non-malignant cells were labeled with monoclonal antibodies against cell surface antigens: CD3 (T-cells), CD14 (monocytes), CD15 (granulocytes), CD45R (T-/B-cells), and 5B5 (fibroblasts). Rat-anti-mouse-IgG coupled to ferrit microbeads (Miltenyi, Bergisch-Gladbach, Germany) was then added. Cells reacting with the microbeads were magnetically retained in a column filled with steel wool matrix (MACS, Miltenyi). Unlabeled tumor cells were washed through the column and recovered in the effluent. This method enables fast and simple isolation of single, competent tumor cells from fresh ovarian carcinoma tissue, ascitic or pleuritic effusions. In a model system consisting of cultured ovarian carcinoma cells and human leukocytes, tumor cell purity was 93 %, and 97 % after a second separation (recovery 75 and 50 %). These still unlabeled tumor cells can be analyzed by flow cytometry or confocal laser scan microscopy for the presence of various surface antigens including receptors for proteases or growth factors. Analysis of cellular constituents such as RNA, DNA, and cell metabolites is also possible. After detergent treatment or fixation, flow cytometric multiparameter analysis such as simultaneous labeling of intracellular and surface antigens, as well as nuclear DNA staining (ploidy, S-phase) becomes possible.
10. INFLUENCE OF FLUORESCENT LABELING OF GLYCOSAMINOGLYCANS (GAGs) FOR BINDING TO LEUKOCYTES
J. Harenberg. R. Malsch. L. Piazolo. G. Huhle. and D.L. Heene
1st Department of Medicine, Faculty for Clinical Medicine Mannheim of the University Heidelberg, Theodor-Kutzer-Ufer, D-68167 Mannheim
Binding of GAGs to leukocytes has been analyzed using a fluorescent labeled derivative (Cytometry, in press). Here we analyze in greater detail the binding of fluorescent labeled GAGs to granulocytes, monocytes and lymphocytes using different labeling techniques and molecular masses of heparin. Five Fitc-labeled GAGs were used: low molecular mass heparin-tyramine-Fitc (LMMH-tyr-Fitc) with molecular weight of 3,700 dalton and 8,100 dalton, where Fitc was tagged by endpoint attachment; three GAG preparations with Fitc bound to the carboxylic groups, i.e. a LMMH-Fitc, a LMM-dermatansulfate-Fitc (LMM-DES-Fitc), and a combination of DES with LMMH labeled with Fitc (sulodexide-Fitc).
Results: 1) The dose-dependent binding of the 5 GAGs were demonstrated for all heparin preparations except for LMMDES-Fitc. 2) The endpoint attached LMMH preparations bound to a higher extent compared to the other 2 heparin preparations produced by binding of Fitc through carboxylic groups. 3) On molar basis similar amounts of both LMMHtyr-Fitc preparations bound to granulocytes, monocytes and lymphocytes. Conclusions: The binding of Fitc to the saccharide backbone of GAGs reduces the affinity to leukocytes. Binding depends on the degree of sulfation rather than on the chain length of heparins. All 5 GAG preparations can be used for affinity studies of GAGs on cellular basis.
11. SERIAL FLOW CYTOMETRY DURING CHEMOTHERAPY: IMPACTS ON THE DEVELOPMENT OF INDIVIDUALIZED TREATMENT STRATEGIES
J. Hemmer
University of Ulm, Germany, Division of Tumor Biology
In oral squamous carcinoma, nearly exclusively aneuploid tumor cells are capable of metastasis as well as of local recurence development. A close association between the expression of high grade malignancy and aneuploidy formation is also reflected by an excellent 90% 5 year survival rate in patients with diploid carcinomas while only 36% of the aneuploid group were long-term survivors. In order to evaluate whether a selective elimination of aneuploid tumor cells might result in a better outcome of the respective patients, tumor biopsies were taken before, during and after induction chemotherapy. A clear reduction of the aneuploid cell numbers during treatment was seen in all 53 cases. Although response to chemotherapy was significantly better in tumors in which the aneuploid cells disappeared completely during treatment than in cases with persisting aneuploid cells, the 5year survival rates were nearly identical in both groups. Accumulation of tumor cells in late S-phase together with a drop of bromodeoxyuridine labeled cells suggested a complete interruption of the proliferative activity while continuous cellular proliferation was reflected by the presence of BrdU-positive cells during chemotherapy. A functional link between the cytotoxic and the cytostatic effect of the treatment was suggested by a complete disappearence of aneuploid cells exclusively in tumors in which BrdU positive cells could not be assessed at the end of treatment. Hence, serial flow cytometry during treatment represents a promising advancement which not only provides fundamental information on instantaneous therapeutic effects of anticancer drugs in vivo, but may also contribute to develop a rational basis for establishing individualized treatment procedures.
12. DIFFERENT APPROACHES TO STUDY THE NUCLEOLAR TERRITORY DURING THE CELL CYCLE
D. Hernandez-Verdun, C. Masson and R. Junera
Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France
In the nucleoli, the ribosomal genes (rDNA) are present as tandem repeats and are the most frequently transcribed genes in cycling cells. The repetetiveness of the rDNA is an advantage that should facilitate the analysis of the 3-D organization during interphase. this makes it possible to investigate the in situ organization of transcribed and non transcribed genes of the same locus and also in situ organization during gene replication within the structural context of the cell.
Identification of the interphase stages was performed in single cells using DNA quantifiacation by cytometry for the G1 and G2 phases while the S-phase was identified by immunolabeling of the proliferating cell nuclear antigen (PCNA). The 3-D organization of the rDNA in the nucleolus was analyzed by fluorescence in situ hybridization using confocal microscopy. The rDNA was heterogeneously distributed in each nucleolus during G1, S and G2 with alternate sites of clustered genes (spots) and of genes in more extended configurations. During mid-S phase, rDNA replicatin occures inside nucleoli and at different sites of the same locus simultaniously. In electron microscopy, the fine 3-D structure of G1 and G2 nucleoli was reconstructed after specific contrast of DNA and RNA, digitization of the serial section images and computer-assisted 3-D architecture. Fibrillar centers (FCs) formed discrete structures (about 10 G1 and 20 in G2) connected by a network of the dense fibrillar component. The 3-D arrangement of the FCs in G1 and G2 are similar to the rDNA spots. In conclusion, the architecture of the nucleoli during interphase reflects the distribution of the rDNA that is characterized by alternation of clustered and extended genes.
13. QUANTITATIVE ANALYSIS AND QUALITY ASSURANCE FOR FLOW CYTOMETRY
H.-G. Höffkes und G. Schmidtke
Abteilung für Hämatologie, Zentrum fur Innere Medizin, Universität Essen, Hufelandstrasse 55, 45122 Essen
In the past, quantification of antigen expression has depended upon the use of techniques other than flow cytometry (RIA, ELISA). Now, with the development of a indirect immunofluorescence assay (quantitative indirect immunofluorescence assay (Qifikit, DAKO Diagnostika, Hamburg), an external calibrator is available which enables flow cytometry to be used in the measurement of antigen density. Both the number of positive cells and the number of antigenic sites per cell may be measured on a single sample. The major advantage is that it is used to calibrate indirect immunofluorescence. The results, therefore, are not affected by differential binding of fluorochrome to individual primary antibodies. The disadvantage of this test system is related to the fact that low density antigens needed a high amount of antibodies to establish a saturated solution, thus, the investigation of these antigens is expansive. Furthermore, only antibodies of the IgG-isotype are considered to be evaluated with indirect immunofluorescence. Overall, the standardized calibration and quantification in combination with a special software analysis program (TallyCall, DAKO) offers new insights in quantitative immunophenotyping for both clinical and scientific applications.
14. ISOLATION AND CHARACTERIZATION OF PERIPHERAL BLOOD DENDRITIC CELLS BY HIGH-GRADIENT MAGNETIC CELL SORTING
Alexander Horst*, Andreas Radbruch#, Stefan Miltenyi* and Jürgen Schmitz*
* Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany and #Institute for Genetics, University of Cologne, Cologne, Germany
We have developed a quick method for efficient isolation of functionaly intact, highly purified dendritic cells (DC) from human peripheral blood (PB) by high gradient magnetic cells sorting (MACS). DC are enriched from PB mononuclear cells (PBMC) by immunomagnetic depletion of CD3, CD14, CD16 and CD19 expressing cells and subsequent enrichment of either CD4 or HLA-DR positive cells. Using this method, DC, present at a frequency of about 1% among PBMC, could be enriched to a frequency of up to 99.5%. Fresh PB-DC can be morphologically devided in two subpopulations: round cells with round or slightly indented nuclei and less round cells with lobulated nuclei. Upon culture PB-DC develop the typical dendritic morphology, they enlarge and exhibit cell processes. Surface immunophenotypic analysis of freshly purified PB-DC by triple-color immunofluorescence revealed expression of the c-kit receptor (CD117) as well as the high affinity FcE-receptor (FcERI). As detected by intracellular cytokine immunofluorencence stimulation of PB-DC with a soluble form of the CD40 ligand results in production of IL-12.
15. COMBINATION OF B-CELL PURGING AND CD34+ SELECTION FOR LYMPHOMA CELL REMOVAL FROM BLOOD STEM CELL AUTOGRAFTS
P. Hundsdörfer1, S. Frühauf2, R. Höft1, R. Haas2, W. J. Zeller1
1 German Cancer Research Center, Research Program Diagnostics and Experimental Therapy, Heidelberg, Germany, 2 Department of Internal Medicine V, University of Heidelberg
Peripheral blood stem cells are about to replace bone marrow for autografing after high dose conditioning therapy in hematological malignancies. This is partly due to a more rapid hematological reconstitution than following autologous bone marrow transplantation. One limitation of PBSC transplantation is a possible tumor cell contamination, which may give rise to a tumor relapse. In malignant Lymphoma, tumor cells can be depleted by direct targeting or by CD34+ selection. With the aim to reach a more effcient tumor cell depletion, we combined direct tumor cell targeting with two different CD34+ selection techniques, immunomagnetic CD34+ selection (IMB) or biotin-avidin CD34+ selection (BAC), and compared the results to CD34+ selection alone. Follicular lymphoma cells (line K422) stained with the fluorescent dye PKH26 were added in different proportions (2.33 +-1013%, n=24) to samples of leukapheresis products containing 018 - 81% CD34+ cells. After B-cell targeting with antibodies against CD19, CD20, CD22, CD23 and CD37 combined with BAC selection (n=3), a mean purity of 76.95 % +-17.72 % CD34+ cells (mean + SEM) in the enriched fraction, a mean recovery of the initial CD34+ cell number of 35.3 % +-12.06 % and a mean purging effciency of log 4.32+- log 0.21 were observed. After tumor cell targeting combined with IMB (n=6), the purity was 40.75% +-30.19 % CD34+ cells, the recovery 47.4 % +- 10.95 % and the purging efficiency log 3.68 + log 0.42 B-cell depletion before IMB selection did not improve the tumor cell removal when compared to IMB selection alone (log 33 +- log 0.86) In comparison, a significant (p < 0.01) higher purging efficiency could be achieved by a combination of B-cell targeting and BAC selection than by BAC selection alone (log 3.02 +- log 0.67). Possibly B-cell-bead complexes or antibody-loaded B-cells were carried over to the CD34+ selection step and were retained during the immunomagnetic bead CD34+ selection, while binding sides for B-cells were not available in the biotin-avidin CD34+ selection system.
Our results argue for the combination of different purging modalities to achieve a maximal tumor cell depletion.
16. A NEW 3-COLOUR LYSE-NO-WASH (LNW), ABSOLUTE COUNT SYSTEM FOR IMMUNOPHENOTYPING AND HIV MONITORING
Luc Ketele
Becton Dickinson Immuncytometry Systems Europe, Erembodegem, Belgium
The presentation will focus on New horizons in Immunophenotyping and HIV monitoring.
We will address a new concept of identifying Lymphocyte subsets, using TriTEST TM Reagents with fluorescece triggering and gating. Unlike traditional methods of light-scatter gating, where lymphocyte gate purity and recovery are concems, TriTEST reagents allow you to directly gate the CD45-positive lymphocyte population or the CD3-positive T lymphocyte population, providing unambiguous identification. In addition, we will focus on important features of the new 3-Colour, Lyse-No-Wash, Absolute Count Capability using TriTEST Reagents, Automated Sample Introduction and Enhanced Data Analysis.
17. "TERBIUM-NUCLEOTIDES" FOR LUMINESCENT LABELLING AND SENSITIVE TIME-RESOLVED DETECTION OF NUCLEIC ACIDS
S. Klingel, M. Collasius and G. Valet
Arbeitsgnuppe Zellbiochemie, Max-Planck-lnstitut für Biochemie, D-82152 Martinsried
Sensitivity of flow cytometric analysis of cells stained by fluorescence in situ hybridization (FISH) is limited due to the background of scattered excitation light and cellular autofluorescence. This makes detection of specific nucleic acids other than ribosomal RNAs and high copy mRNAs with fluorecent dye labels like FITC very difficult. The use of luminescent metal chelates attached to hybridization probes could greatly improve the signal-noise-ratio. Excited with a short light flash such molecules emit photons over much a longer time (10-3sec) than cellular compounds (10-7 sec). After a delay of about 100 microseconds there is no fluorescent background from unlabelled cellular material interfering with the specific luminescence signal. We have developed new deoxynucleoside triphosphates coupled to stable luminescent terbium chelates ("terbium nucleotides") allowing direct enzymatic labelling of nucleic acid probes. These dUTP-derivatives are easily incorporated in DNA by terminal deoxynucleotidyl transferase, nick-translation, randompriming and PCR substituting for dTTP in the reactions. Use of analogous terbium ribonucleotides allowed the efficient labelling of RNA probes by in vitro transcription with phage T3 and T7 RNA polymerases. Detection of the luminescent probes in agarose gels was more sensitive than unspecific staining with ethidium bromide in most cases although photon densities during excitation with flash lamps are clearly suboptimal. Luminescence excitation with pulsed UV-laser light, in contrast, will allow the measurement of picogram amounts of nucleic acids which so far is only possible with radioactivity or enzymatic signal amplification.
Luminescent probes in combination with time-resolved fluorescence detection in a flow cytometer, microskope or other solid support systems should be very useful for sensitive in situ hybridizations but also for in situ nucleic acid labelling, e.g. the detection of DNA nicks generated in the process of apoptosis.
18. IMPACTS OF THE DNA PLOIDY STATUS OF THE PRIMARY TUMOR OKKULT METASTASIS IN ORAL SQUAMOUS CELL CARCINOMA
K. Kraft*, A Schouba#, J.Hemmer#
*Dept of Pathology, Military Hospital Ulm, Germany,# Division of Tumor Biology. University of Ulm, Germany
A study on 485 patients with oral squamous cell carcinoma showed lymph node involvement on admission in only 14% of those with diploid primary tumors but in 53% of those with aneuploid lesions. Delayed metastasis has been observed in 8% of the diploid NO group but in 23% of the aneuploid NO cases. In a 3-year pilot study, also patients without evidence of lymph node involvement from CT and ultrasound scans were therefore submitted for radical neck dissection if their primary tumors were aneuploid by flow cytometry. All cervical lymph nodes were histologically examined in 100u equidistances. Metastasis was verified microscopically in only one of 15 patients with diploid tumors (7%) who underwent lymph node surgery because of clinical evidence of involvement. But metastasis was proved by histology in 9 of 38 patients with aneuploid primary tumors (23%) who would have remained untreated because of lack of clinical evidence of nodal invasion. These histological findings were completely identical with the clincal results of the above mentioned follow up study. The present study confirms again that oral carcinoma patients with aneuploid primary tumors, independent of tumor stage or histological grade, carry a dramatically higher risk of metastasis than those with diploid tumors. Lymph node treatment in the diploid group should be considered only if metastasis is unequivocally assessed by clinical examination. Delayed metastasis can largely be avoided by neck dissection also in the aneuploid NO group, but overtreatment has to be accepted in the majority of these cases.
19. DIFFERENTTIATION OF OVARIAN TUMORS WITH IMAGE ANALYSIS
W. Kuhn, M. Ruhnke, A. Ditzenbach, H. Weitzel
Department Gynecol. Obstet., Klinikum Benjamin Franklin Free University of Berlin, Germany
The differentiation of ovarian tumors by conventional histology is difficult. Especially the diagnosis of lesions of borderline malignancy is connected with problems for the pathologist an clinician. The aim of the present study is to differentiate ovarian tumors with image analysis.
55 ovarian tumors were analysed with a CAS 200 image analysis system. On Feulgen stained nuclei integrated optical density (IOD), nuclear size, shape, entropy, blobness, and ploidy were measured. The mean values of the estimated parameters are given in Tab. 1:
param. | benign | borderl.| malign. |
IOD |102.672 | 127.74 | 271.78 |
SIZE | 54.83 | 60.94 | 129.41 |
SHAPE | 17.37 | 17.86 | 17.38 |
ENTROPY | 1.4735 | 1.4493 | 1.4688 |
BLOBNESS| 0.3973 | 0.4114 | 0.3892 |

In benign lesions no cells with a DNA-content >9c were measured. In borderline lesions 0.22% and in malignant ovarian tumors 11.3% of the measured cells were > 9c.
While differences between benign and borderline lesions are small, borderline lesions can easily be distinguished from ovarian cancer by DNA-content and nuclear size.
20. NEW DEVELOPMENTS IN CONFOCAL LASER SCANNING MICROSCOPY
J.Kukulies
Carl Zeiss Jena GmbH, Unternehmensbereich Mikroskopie, Systemvertrieb Deutschland
In molecular cell research, confocal Laser scanning microscopy (CLSM) has gained increasing importance over the last years: spatially intact cell and tissue preparations can non-invasively be cut in optical sections. These display very high sharpness and resolution in XYZ-direction and are suitable for computer aided 3D-reconstruction using appropriate software-packages. Results are spatial fine-localizations of fluorescently labeled molecular probes (i.e antibodies, DNA-probes) down to the subcellular level. Multiple labeled molecular probes can be visualized at the same time for simultaneous localization of different parameters in living and fixed specimens.
For fluorescence excitation CLSMs for the following laser-lines are available: 351, 364, 457, 488, 514, 543, 568, 633, 647 nm. HeNe-lasers are integrated (543, 633 nm), and air-cooled Ar-Lasers can be cascaded via optical fibres or "optical bench system". On the emission side, up to 4 parallel confocal detection paths (PMT`s) can be used simultaneously. The software of CLSM's in the first place controls the instrument functions (i.e. laser-line, attenuation, scanning speed, zoom factor, confocality-pinhole diameter, series of z-sections etc.). Further sofware provides 3D reconstructions and evaluation of data/images from living cells along the time axis.
The presentation illustrates applications of CLSM in molecular cell research and discusses trends in this modem technique.
21. DETECTION OF MDR-1 mRNA BY RT-PCR AND FLOW CYTOMETRY
D. Lassner, J. Milde, M. Ladusch*, K. Drössler* and H. Remke
Institute of Clinical Chemistry and Pathobiochemistry, *Institute of Zoology, University Leipzig, Liebigstrasse 16, D-04103 Leipzig
The MDR1 gene, responsible for "multi drug resistance"(mdr) in human cells, encodes a broad specificity efflux pump (P-glycoprotein, P-170) (1). Overexpression of mRNA for P-glycoprotein has been shown in vitro to mediate multidrug resistance, and the expression has been found in human tumors and normal tissues.
Detection of mdr1-mRNA in white blood cells of patients suffering from leukemia is a good marker for mdr-phenotype of these cells and is mainly induced by treatment with cytostatics. Expression of P-170 on bone marrow cells is indicated as second stem cell marker, except of CD34 (2). Amplification of mdr-1 mRNA is performed by PCR following reverse transcription of mRNA to cDNA (RT-PCR) which can be detected by conventional methods (3).
We have designed the in-cell PCR (4) for mdr-1 mRNA. This method combines the amplification of mRNA within formalin-fixed cells by RT-PCR using fluoresceinated primers and subsequent analysis of treated cells by flow cytometry. Achieved results correspond to detection of mdr-1 mRNA by conventional methods and in cell-PCR has a potential use in diagnostics of leukemia on cellular level. References:
(1) Roninson,I., Biochem. Pharmacol. 1992, 43:95-103
(2) Chaudhary PM, Roninson I, Cell 1991; 66:85-94
(3) Koehler T, Lassner D, Remke H., Eur.J.Clin.Chem.Biochem. 1993,4:254
(4) Embleton MJ, Gorochov G, Jones PT, Winter G. Nucl.Acids Res. 1992; 20:3831-38
22. COMPARISON OF BrdU INCORPORATION AND PCNA AND Ki67 EXPRESSION IN HL-60 CELLS AFTER INDUCTION AND IRRADIATION
M. Lindinger*, A. Sendler**, K.-P. Gilbertz*, D. van Beuningen*
*Institut für Radiobiologie, Akademie des Sanitäts- und Gesundheitswesen der Bundeswehr, D-80901 München, **Chirurgische Klinik und Poliklinik der TU München, Klinikum "rechts der Isar", D-81644 München, Germany
In oncology, tumor proliferation plays an important role. For estimation of growth fraction, proliferation associated antigens (PCNA, Ki-67) or BrdU (Bromodesoxuridine) incorporation are used. The results are somewhat contradictionary. Using the leukemia cell line HL-60, which proliferation can be modulated with various compounds, these proliferation markers are analysed and compared. Investigations were done by flow cytometry.
Noninduced HL-60 cells proliferate exponentially during the first 4 days and reach the plateau phase on day 5. Up to day 7, PCNA expression is about 90%. After seeding, 70% of cells express Ki-67. From day 1 - 5 Ki-67 expression is over 90% and decreases to 70% at day 7. During the first 5 days BrdU - labelling index (Ll) is 50% and decreases to 10% at day 7. After modulation of proliferation, Ki-67 expression correlates better with the growth curve and BrdU - Ll, than PCNA. After irradiation, Ki-67 and PCNA expression increases. Noninduced cells show a X-ray induced dose dependent increase of PCNA expression in the G2-Phase of the cell cycle.
In this model, Ki-67 seems to be a more exact parameter for evaluation of the growth fraction than PCNA.
23. COMPARISON OF FLOW CYTOMETRIC METHODS FOR THE ANALYSIS OF APOPTOTIC CELLS
Kristin Marx, Michael Nüsse
GSF-Forschungszentrum für Umwelt und Gcsundheit, AG Durchflußzytometrie, D-85758 Oberschleißheim, Germany
The applicability of a method to identify and quantify apoptotic cells depends on the cell system, nature of the inducer of cell death, the particular information that is being sought and the technical restrictions. Three different published flow cytometric methods to reveal dose- and time- dependent effects were compared. In addition, a new method to detect apoptotic bodies, one of the characteristic morphological features of apoptotic cells, was used. The cell system of choice was the human myeloid leukemia cell line HL60, as an inducer the chemotherapeutic agent camptothecin, an Topoisomerase-II-inhibitor was applied. The first method is based on a double staining of ethanol fixed cells with HOECHST 33342 and propidium iodide (PI). After UV excitation, apoptotic cells show a decreased blue fluorescence. The two other methods, namely the in situ nick translation (ISNT) and the sub-G,-peak method, deal with the typical apoptotic DNA strand breaks. In the ISNT assay the DNA is kept inside the cell and strand breaks are labeled directly with fluorescein. In the sub-G,-peak method the DNA fragments are washed out and the DNA is stained with PI to identify apoptotic cells by their lower DNA content. All three methods show a similar time course with little variation in the number of apoptotic cells after addition of different concentrations of camptothecin. Similar time and concentration dependencies were also obtained using the new technique for flow cytometric measurement of apoptotic bodies, although these results were quantitatively different due to the the fact that cell membranes were destroyed to obtain apoptotic bodies.
24. FLOW SORTING OF PRINS LABELLED PLANT CHROMOSOMES
Armin Meister, Uta Pich and Ingo Schubert
Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben
Flow sorting enables the isolation ot single chromosome types, which is an essential condition for construction of chromosome specific DNA libraries and gene mapping. Contrary to animals, only for a few plant species chromosomes are successfully sorted. The reasons are the existence of a cell wall in plants, which causes preparative problems, and the similarity in size and therefore in the DNA content of the majority of plant chromosomes. Therefore the separation on base of DNAspecific dye fluorescence is possible in a few cases only. In the field bean ( Vicia faba) only one out of six chromosome pairs is clearly different from the other ones and can be isolated with a flow cytometer on the base of DNA content. The use of reconstructed karyotypes with clearly different chromosomes extends the possibilities for sorting chromosomes of this species.
The FITC labelling of specific DNA sequences of single chromosomes in suspension by the PRINS (primed in situ) technique opens new possibilities for chromosome sorting. By combination of the DNA-specific propidium iodide signal with the sequence-specific FITC signal, separation of all six chromosome types of karyotype ACB is possible with high purity (on average 95 %).
25. PROGNOSTIC RELEVANCE OF PLOIDY AND S-PHASE IN RESECTED CARCINOMA OF THE PANCREAS
H. Nekarda, J.D. Roder, K. Becker*, A. Rossmann, J.R. Siewert
Department of Surgery and *Institut for Pathology, Technische Universität München, Klinikum rechts der Isar
The prognosis of patients with ductal carcinoma of the pancreas compares unfavorably to other Gl tumors. Analysis of prognostic factors is therefore essential to identify subgroups of patients who might benefit from adjuvant therapy.
The data of 69 patients who had a resection of a ductal carcinoma of the pancreas between 1982 and 1990 were documented prospectively. 32/69 (46 %) patients were totally resected (R0-UICC). Ploidy and S-Phase of the tumor were investigated in nuclear preparations of paraffin embedded tissue by flow cytometry and computed by "Multicycle analysis". Median survival time of all patients was 13 months with a 5year survival rate of 10 %.
The aneuploidy rate was 68 % for all patients and 57 % for totally resected patients (R0-UICC). By multivariat analysis (Cox model) an independent prognostic impact was demonstrated for S-phase (RR: 6-fold) and tumor grading (RR: 2.3fold) compared to all prognostic staging (pTNM) and morphological factors. Median survival was significantly longer in the 24 patients with a S-phase below 8 % as compared to those patients with a S-phase above 8 % (17.5 vs. 6.5 months). Median survival in the 18 patients with well or moderately differentiated tumors was 23 months as compared to 7.5 in the 12 patients with poorly differentiated tumors.
In conclusion only tumor grading and S-phase independently influenced the prognosis of R0-resected patients with carcinoma of the pancreas. These parameters may be helpful to identify those patients who benefit from adjuvant therapy.
26. DEMONSTRATION OF THE NUMBER OF RESIDUAL LEUCOCYTES OR PLATELETS IN FILTRATED RED BLOOD CELL CONCENTRATES AND PLASMA PREPARATIONS
J. Neumüller, *D.W.M. Schwartz, *W.R. Mayr
Ludwig Boltzmann Institute for Rheumatology and Balneology, Vienna, Austria *Blood Donation Center, Austrian Red Cross, Vienna, Austria
The development of new flat polyester filters (Pall RD1746) provides a significant depletion of leucocyte (LC) and platelets (PL) in filtrated red blood cell concentrates (FRCC) and in filtrated plasma preparations (FPP). A depletion of LC to at most 5 x 106/unit and a reduction in PL to at most 5 x 109/unit prevents above all febrile transfusion reactions and reduces the risk of alloimmunization in recipients with LC or PL antigens. The present study shows that the enumeration of residual LC can be performed accurately by FC.
FC was performed on a FACSort (Becton & Dickinson, B&D) using the Lysys-II or the CELLQuest softwares for acquisition and the Lysys-II or the Attractor softwares for analysis. The absolute number of cells was calculated by the determination of the acquired volume using a definite number of reference beads (Standard Brites, uniform FITC and PE cross-linked microspheres, Coulter). LC in FRCC and FPP were determined by FC using a combination of monoclonal antibodies (MAB) against CD45 (FITC labelled) and CD45 (PE labelled, Simultest, B&D). 500 ul of FRCC or FPP were incubated with 50 ul of the MAB mixture for 15'. Red blood cells (RBC) were eliminated by incubation with 10 ml FACS Lysing solution (B&D) for 10' (Iysing of RBC was performed only in FRCC). The remaining cells were washed twice with PBS and resuspended in 450 ul CellFix (B&D) and mixed with 50 ul of Standard Brites. The residual LC were gated in the FSC x SSC dot plot and defined more precisely by gating in the FL1 x FL2 dot plot.
Determination of residual PL: 100 ul of FRCC or FPP were incubated with 10 ul of a FITC conjugated MAB against CD41 (Immunotech) for 15', and diluted 1:10 with 0.15 M citrate buffer, pH 7.4. 100 ul of this solution were mixed with 800 ul of citrate buffer and 100 ul of Standard Brites. PL were demonstrated in a FL1 single histogram after gating in the FSC x SSC dot plot.
With these methods it could be documented that the filtration using the Pall RD1746 filters provide a reduction in LC to 2.9 +- 2.3 x 10/5 and in PL to 3.8 +- 4.1 x 10/7 in FRCC and a reduction in LC to 7.9 +- 2.5 x 10/1 and in PL to 1.3 x 10/8 +- 3.3 x 107 in FPP. These values are under the critical limits that are thought to be responsible for the adverse side effects mentioned just before.
27. ANALYSIS OF MICRONUCLEI BY FLOW SORTING AND FISH
M. Nüsse1, A. Slavotinek2, B.M. Miller3
1 GSF-AG Durchflußzytometrie, D-85758 Oberschleißheim 2 Dept. Clinical Genetics, Churchill Hospital, Oxford 3PRPT. Preclinical Division, Hoffmann La-Roche AG, Basel
The chromosomal composition of radiation-induced MN was studied in mouse 3T3 cells and in various human cell lines with different radiation sensitivity using FISH with centromeric DNA probes and with chromosome-specific painting probes. Most MN in 3T3 cells were found to contain acentric fragments, with increasing DNA content, however, the frequencies of MN containing 1, 2 or 3 hybridization signals increased demonstrating the presence of one or several chromosomes in larger MN. These data agreed with results obtained on fixed cells using a combination of telomeric and centromeric DNA probes. The DNA distribution of radiationinduced MN measured by flow cytometry could be simulated by a random breakage model of chromosomes using these results.
In situ hybridization with whole chromosome painting probes to paint radiation-induced MN was used to further investigate the nature of radiation-induced cytogenetic damage. The results obtained for four chromosomes (1, 7, 11, 14) in three human cell lines with differing radiosensitivity showed that there was a significant deviation of the numbers of signal positive MN from that expected on the basis of DNA proportionality. The results were dependent mainly on chromosome 7 which was underrepresented in the numbers of signal positive MN in the group of chromosomes studied. If it is assumed that the chromosomal content of the MN is an accurate reflection of the radiation-induced damage, then these results support a non-random model of radiation-induced cytogenetic damage.
28. RESULTS OF THE COLLABORATIVE STUDY 1995 ON STANDARDIZATION AND QUALITY ASSESSMENT OF DNA FLOW CYTOMETRY
Friedrich J. Otto
Fachklinik Hornheide, D-48157 Münster
The fourth trial of a collaborative quality assessment study on flow cytometric DNA content measurements supported by the Deutsche Gesellschaft fuer Zytometrie was carried out in January 1995. Test samples consisting of ethanolfixed animal and human cells had been distributed to 53 laboratories. The participants were asked to process, stain, and to measure the samples according to their own protocols. In addition two standard staining protocols were proposed in order to reduce the heterogeneity of methods. Also in this trial the results revealed a considerable interinstitutional variability of the measured data and the issues derived from the histograms. Measuring accuracy as specified by the CVs of the peaks varied from laboratory to laboratory. The highest accuracy on average as well as the best CVs were obtained while using DAPI staining and HBO 100 mercury arc lamp equipped instruments. Comparing the results of the subsequent trials, a tendency towards higher accuracy became recognizable. Exactness of DNA index determination, however, remained still insufficient.
Also the S phase fractions calculated from the histograms exhibited considerable deviations.
The use of standard protocols and reagents supplied by the organizers significantly improved the results. The standard DAPI protocol increased the accuracy and homogeneity of the data. It reduced the variability of DNA index and S phase determinations. The standard Pl protocol diminished slightly the deviations of DNA indices and S phase fractions.
These results confirm that standardization of staining methods is an efficient way to increase accuracy, reliability and reproducibility of the data. Our efforts to assess and improve the quality of flow cytometric DNA measurements will continue with testing standard staining protocols and controlling instrument performance.
29. CYTOMETRIC ANALYSIS AND ISOLATION OF TRANSFECTED CELLS BY SURFACE EXPRESSED SELECTION MARKERS
K. Petry, R. Christine, G. Siebenkotten and A. Radbruch
Institute for Genetics ,University of Cologne, Weyertal 121, D-50931 Cologne, Germany
Genes or their regulatory elements are often investigated in transfected cells to study their function and expression. To assess the transfection efficiency, conventionally transfectants are analysed using enzymes and their activity as an indicator. Only a few methods permit the quantitative proof of single, living cells. We developed expression vectors which code for surface expression of CD4 (human) and H-2Kk (murine). Both vectors, separate or combined, allow the cytometric measurement of transfection efficiency, quantity of expression activity and expression kinetics. Transfected cells of interest, even if they are very rare, can be further investigated after using cell separation techniques like FACS or MACS. Toxic biochemical selection is not necessary.
We used these vectors to analyse recombination in B lymphocytes. Recombination of the vectors leads to deletion of a transcription-stop-signal upstream of the membrane exon of the CD4 respectively H-2Kk gene, which allows indicator expression on the cell surface. First results indicate an increased recombination frequency in activated cells of a B cell line compared to non activated cells. At the moment we analyse the sequences involved in the recombination event.
30. SUBCELLULAR LOCALIZATION STUDIES WITH NOVEL MITOCHONDRIAL STAINS
Martin Poot, Ananda G. Lugade, Jennifer Krämer, K. Sam Wells, Laurie J. Jones, Lisa Gibson, Stephen T. Yue, David Hanzel$, Victoria L. Singer and Richard P. Haugland
Molecular Probes, Inc., P.O. Box 22010 Eugene, OR 97402, and $Molecular Dynamics, Sunnyvale, CA 94086, U.S.A.
Implicit to using fluorescent stains to characterize the structure and function of cell organelles is the assumption that these stains localize specifically to the organelle under investigation. We followed two different approaches to ascertain whether this assumption holds for a variety of mitochondrial stains. First, we analysed co-localization of stains with different emission spectra ("green" and "red" stains) by regular and confocal fluorescence microscopy. Rhodamine 123, nonyl acridine orange, MitoTrackerTM Green, and MitoTrackerTM CMXRos showed perfect co-localization as evidenced by "blending" of the fluorescence from pairs of stains (eg. "green" plus "red" fluorescence becomes "yellow"). No isolated "green" or "red" signals were observed. Second, we performed costaining experiments and analysed potential interaction between the stains by flow cytometry. Rhodamine 123 and nonyl acridine orange showed no fluorescence interaction. Strong fluorescence energy transfer occurred between nonyl acridine orange and CMXRos, but not between nonyl acridine orange and the MitoTracker Green dye. We found rhodamine 123 and CMXRos to be sensitive to alterations in the mitochondrial membrane potential, while nonyl acridine orange was not. We conclude that, while rhodamine 123, nonyl acridine orange, CMXRos and the Mitotracker Green dye appeared to stain the same organelle, only nonyl acridine orange and CMXRos occupy sites in close proximity to each other. These results suggest that combinations of mitochondrial stains can be used to map the submicroscopic structure of the mitochondrion.
31. CALIBRATING A FLOW CYTOMETER WITH FLUORESCENT LATEX BEADS
Martin Poot, Sharon M. Swan, and K Sam Wells
Molecular Probes, Inc., Eugene, OR 97402, U.S.A.
In quantitative flow cytometry daily calibration of the instrument is paramount. First, flow cytometrists need a well defined standard particle to optimally adjust the Instrument. This can be achieved with a sample of strongly fluorescent latex beads. These beads have to be very homogenous regarding their size and fluorescence intensity (CV values of less than 2%). The higher the fluorescence intensity of the particle the lower the CV value will be, since the contribution by the electronic noise from the signal amplifier to the CV value will be lower at lesser signal amplification. In multiparameter flow cytometry one needs either different beads for every wavelength region, or a bead with strong fluorescence in every wavelength range. Such beads became available recently. To compare fluorescence intensities obtained on different days, one has to compensate for different instrument adjustments. Generally a single particle is used as a standard. With this approach the investigator cannot assess differences in the degree of electron saturation of the dynodes of the photomultiplier tubes. These differences manifest themselves as variation in the rabo in fluorescence intensity between particles as a function of the voltage over the photomultiplier tube. To assess this parameter we have recently devised a set of latex beads with different fluorescence intensities. The range of fluorescence intensities of these beads covers two decades. With this novel product it became possible for the first time to calibrate a flow cytometer even at strongly different degrees of signal amplification.
32. EXTERNAL QUALITY CONTROL SURVEY OF IMMUNOPHENOTYPING OF THE CENTRAL REFERENCE INSTITUTION IN GERMANY ISSUES OF SPECIMENS AND INTERLABORATORY VARIABILITY
W. Prohaska, R. Kruse, W.-J. Geilenkeuser, T. Nebe, A. Radbruch, H. Diem, A. von Rücker
Working group of quality assessment in flow cytometry of the German Society of Clinical Chemistry
Since 1993 five quality control surveys in flow cytometry immunophenotyping have been performed with the markers CD3+, CD4+/CD3+, CD8+/CD3+, CD19+, CD1 6,56+/CD3- and CD3+/HLA-DR+. In the first two surveys washed and purified buffy coat, afterwards CDP (Citrate/ Phosphate/ Dextrose) whole blood was used. The buffy coat was magnetically immunodepleted (Milteniy Biotec immuno magnetic beads) to gain pathologically decreased subpopulations. All specimens were shipped on the day of drawing and preparation.
On the average, 109 laboratories participated in the surveys. After changing from immunodepleted buffy coat to CPD whole blood, there was a distinct decrease of the coefficients of variation (CV) in the determination of percentages of Iymphocyte subpopulations: CV's improved from 4,5 - 12 % to 3 - 7,5 % (CD3+ cells) and CV's of CD4+/CD3+ cells moved from 5,8 -14,4 % to 3,2 - 6,6 %. The CV's are lower than those reported in the College of American Pathologists Flow Cytometry Survey (1 ) and other surveys from other countries.
Clinically, the absolute cell count is more important than the percentages which show relatively small CV's. For the absolute cell counts we calculated CV's of 14 22,4 % and 14,5 - 21 % for CD3+ cells and CD4+/CD3+ cells, respectively. These are considerably worse than those for the corresponding percentage counts.
As the leucocyte and lymphocyte count is the basis for calculating of the absolute cell counts of subpopulations the calculation of these entities is important and can raise CV's two- to threefold. Subpopulations with low percentages and/ or inhomogenic expression of marker epitopes (for instance HLA-DR) also yielded high CV's in relative counting.
In summary, fresh CPD whole blood is a natural, sufficiently stable and easy to handle specimen material for quality surveys in immunophenotyping. Aims to improve the results of quantitative immunophenotyping should address white cell and Iymphocyte counting. Further standardisation of flow cytometry methods should improve quantitative immunophenotyping.
1. Homburger, H A., Rosenstock W. Paxton,H. Paton, M L., Landay,A. L; Assessment of Interlaboratory Variability of Immunophenotyping; Ann N.Y. Acad. Sci 677 4349 (1993)
33. ANALYSIS AND ISOLATION OF RARE CELLS
Diether Recktenwald1, Hans Joachim Gross2, Stefan Miltenyi3
1 AmCell Corp., Sunnyvale, Ca, USA 2 Labor MittererstraBe, München 3 Miltenyi Biotec, Bergisch Gladbach
The quantitative detection and analysis of rare cells has gained importance with the recent progress in immunology, cell biology and molecular biology. This is examplified with stem cells and rare tumor cells in blood, fetal cells from maternal blood and antigen-specific lymphocytes. Based on a commercial flow cytometer we have developed a method to detect one cell within more than ten million other cells by immunofluorescence. The method is based on exclusion staining combined with data filtering. In addition, the contamination of probes with cells from previous probes has to be controlled. A genetic algorithm was used for automated data analysis. The improved data analysis cannot yet be used for cell sorting since the algorithms do not run in "real time". Analysis and isolation of rare cells can be improved considerably, however, by pecific pre-enrichment techniques. Highgradient magnetic cell sorting with colloidal magnetic antibody-conjugates is one such method. It has been used for the detection of fetal cells from matenal blood tumor cells from blood and subpopulations of CD34 cells. Effective pre-enrichment will also improve the sensitivity of manual and automated microscopic techniques for the detection of extremely rare cells.
34. HIGH RESOLUTION FLOW CYTOMETRY AND P53 IMMUNOREACTIVITY IN STAGE I BORDERLINE EPITHELIAL OVARIAN TUMORS
Reich O., Pürstner P., Schöll W.
Department of Obstetrics and Gynecology, University of Graz, Auenbruggerplatz 14, 8010 Graz, Austria
Several studys have suggested that DNA aneuploidy and detection of p53 gene mutation is associated with tumor progession in borderline epithelial tumors of the ovary (BOT). 14 stage I BOT (8 serous, 4 mucinous, 1 endometrioid, 1 clear cell mean age of patients: 50 years (20-82); mean size of tumors: 10 cm ( 4cm-18cm)) were studied for DNA-content and for mutant p53 protein overexpression. On 4 - 8 specimens per case, DNA analysis by high resolution flow cytometry (Goehde's technique, Partec PAS III, Multicycle software) and evaluation of mutant p53 protein by immunohistochemistry (DO 7, monoclonal, BioGenex AM 239-5M) were performed. 8 of 14 cases (57%) showed DNA aneuploidy. 6 women (43%) had DNA-diploid tumors. The DNA-indices of aneuploid tumors ranged between 1,06 and 1,90 (1,06; 1,09; 1,10; 1,70; 1,90). 3 cases showed near diploid measurments. Immunohistochemical staining for p53 gene product was not seen in any of the specimens. The study suggests that high resolution flow cytometric offers the opportunity for DNA grading of malignancy in BOT (diploid, near diploid, aneuploid (2c+<10%), aneuploid (2c+>10%)). In contrast mutant p53 protein overexpression seems not to be commonly associated in stage I BOT.
35. QUANTIFICATION OF THE TOTAL CELLULAR CONTENT OF THE MYELOMONOCYTIC PROTEINS MRP8 AND MRP14 AND OF THEIR HETERODIMER RECOGNISED BY THE ANTIBODY 27E10 IN HUMAN MONOSYTES AND GRANULOCYTES USING DUAL LABEL FLOW CYTOMETRY
O. Röhrig, C.L. Klein, T. van Kooten C.J. Kirkpatrick
Institute of Pathology, University Hospital of Mainz, Germany
The myelomonocytoid proteins MRP-8 and MRP-14, also known as calprotectin or L1 protein are important cytoplasmic proteins of cells of the monocyte-macrophage system and of granulocytes. Ca++ dependant formation of heterodimers of MRP8 and -14, and subsequent translocation to cellular membranes and the cytoskeleton have been shown to be associated with the processes of activation and extravasation of monocytes from the circulation. Consequently macrophages stained by the monocylonal antibody 27E10, which recognises a conformational epitope on the heterodimer of MRP8/14 have been termed "inflammatory macrophages". Since the cellular distribution of monomeric MRP8 and -14 molecules and heterodimers (27E10) in circulating monocytes has not yet been determined quantitatively it is still debated wether circulating monocytes expressing 27E10 constitute an "inflammatory subpopulation". Therefore we developped a new method to quantify the total cellular content of MRP8, -14 and the dimer 27E10 in human monocytes and granulocytes directly isolated from human blood. Detection of intracellular antigens was performed using the readily available reagent Fix and Perm. Pre-fixation membrane labelling of CD14 and CD66b(CD67) was performed to discriminate monocytes and granulocytes without need for light scatter based gating. Flourescence intensities were transformed into molecules per cell by quantification using QSC-beads. Direct and indirect (ab-biotin, avidin-fitc) staining of the intracellular molecules was applied and calculations of molecule numbers using both methods were compared. The combined use of pre fixation membrane labelling, intracellular staining using Fix and Perm, and calibration using QSC-beads turned out to be a straight forward method for the quantification of the cellular content of intracellular antigens both, in terms of molecule numbers per cell and percentage of cells expressing the molecules at the quantified level. The results concerning the intracellular distribution of MRP8 and -14 as well as their heterodimer MRP8/14 (27E10) will be presented and their implications on the notion of "inflammatory monocytes/ macrophages" will be discussed.
36. CHARACTERIZATION OF PERIPHERAL BLOOD MONONUCLEAR PHAGOCYTE SUBPOPULATIONS
G. Rothe, J. Stöhr, G. Schmitz
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg
Mononuclear phagocytes (MNP) play a major role in atherogenesis. The goal of our study was the characterization of blood monocyte subpopulations as potential cellular markers of immunological abnormalities in hypercholesterolemia. In normal probands 61.3 + 6.0 % of monocytes showed bright expression of the LPS-receptor (CD14) and Fcy-R-I (CD64) in the absence of Fcg-R-III (CD16) expression. In addition, a subset of these "regular" MNP, 2.1 t 0.8 %, showed expression of NCAM (CD56).Further subsets of monocytes were characterized by simultaneous expression of both Fcg-receptors (25.6 + 5.0 %), isolated expression of Fcg-R-III (9.4 + 1.7 %), or high expression of CD33 (3.7 + 1.1 %) with only dim expression of CD14, respectively. All five subpopulations showed staining for CD68 and myeloperoxidase, phagocytosis, and esterase activity but significant differences in their expression of antigens related to antigen presentation and adhesion.An analysis of peripheral MNP in hypercholesterolemic patients and normal probands revealed an increase of the more mature phenotype of MNP with an isolated expression of Fcg-R-III in association to the apolipoprotein E3/E4 and E4/E4 phenotype. Furthermore, an increased expression of the activation antigen CD45RA in correlation to plasma levels of LDL and Lp(a) suggests systemic abnormalities of MNP in disorders of lipid and lipoprotein metabolism.
37. A NEW FLOW CYTOMETRIC ASSAY FOR THE ANALYSIS OF THE HUMAN LYSOSOMAL ACID LIPASE
G. Schindler, G. Rothe, G. Schmitz
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany
Lysosomal acid lipase (LAL, EC 3.1.1.13)is a hydrolase essential for the degradation of cholesterylesters and triglycerides derived from uptake of plasma lipoproteins. An impaired LAL activity is associated with two genetic disorders, Cholesteryl ester storage disease (CESD) and Wolman disease (WD), and beyond that is considered to be relevant in the pathogenesis of arteriosclerosis. The aim of this study was the characterization of the in situ catabolic activity of the LAL in intact living cells.
LAL activity was investigated in situ by means of fluoresceindilaurate as a fluorogenic probe. Probe internalization and targeting to Iysosomes were the primary concems. Intenalization and targeting were achieved by the use of low density lipoprotein (LDL). FLDL incorporated into the core of LDL (FLDL-LDL) was taken up by fibroblasts specifically through the apolipoprotein-B/Ereceptor mediated pathway as suggested by competition of uptake through non-labeled LDL. In order to quantify enzyme hydrolysis independently from differential rates of FLDL-LDL uptake the LDL-particle was simultaneously labeled with 3,3'-dioctadecylindocarbocyanine (Dil).
LAL activtiy as detemmined with Dil-FLDL-LDL in cultured fibroblast monolayers from normal individuals and from patients affected with LAL deficiency showed significant differences. These results correlate well with experiments using 3H-Cholesteryl-linoleate incorporated into LDL as a substrate in a reference assay.
38. QUALITY CONTROL AND STANDARDIZATION OF FLOW CYTOMETRIC PLATELET ANALYSIS
G. Schmitz, S. Barlage, G. Rothe
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany
Flow cytometric analysis of platelet activation is an important aspect in quality control of platelet concentrates and in the diagnosis of platelet hyper- or hyporeagibility. Although altered surface densities of platelet specific antigens such as GpIlb/lIla (CD41a), GMP-140 (CD62), Gp53 (CD63) and Gplb (CD42b) can serve as markers of platelet activation, the flow cytometric results highly depend on methodological aspects such as preparation techniques, selection of antibodies and data analysis. In order to achieve a better standardization of platelet activation analysis, our laboratory organized two external quality assessment trials in 1994, based on the non-fixative stabilization of test samples, a standardized protocol for sample preparation and calibration of fluorescence intensities using calibrated goat-anti-mouse beads.
The results showed comparable relative fluorescence values for the bright CD42b antigen for most participants. A significant number of participants, however, reported difficulties in analysis of the weakly expressed CD62 and CD63 antigens, and in the antibody specific calibration of fluorescence intensities. These results confirmed the demand for detailed consensus protocols on methodological aspects of platelet analysis.
39. COMPARISON OF FLOWCYTOMETRIC DNA-MEASUREMENT WITH MORPHOMETRIC IMAGE ANALYSIS PARAMETER IN SELECTED OVARIAN CARCINOMA
Scholl W., Reich O., Pürstner P., Taucher A.
Department of Obstetrics and Gynecology, Univ. of Graz, Austria
Objectives: Flowcytometric DNA-measurement from several tumor areas of the same ovarian carcinoma point to an often heterogen structure of cytogenetically different tumor clones, which usually remain hidden in histological assessment. Is it possible to differentiate such cell populations by means of morphometric, only with image analysis systems determinable parameters?
Materials & methods: On several spots in an ovarian carcinoma fresh material for flow cytometric and histologic investigation is taken. The quantitative DNA-determination is done in accordance with a standardised record according to Goehde on the PAS III flow cytometer, the pathohistologic investigation that is strictly correlated with it, is done on an HE cut. Those cases that according to flowcytometry show heterogen cell populations which, however histologically do not reveal distinguishable typing and grading, are being investigated on the Zeiss Ibas 2.0 Image analysis system. Interactively about 150 malignant cell nuclei are investigated, regarding their nucleus area, form factor, maximal diameter, elongation, and nucleus alignment. The measurement results of the various areas are examined for possible differences by means of the Kruskall-Wallis test.
Results: Specimens with corresponding flow cytometrically heterogen cell populations are highly significantly (p < 0,0001) different in nucleus area and form factor from a preparation that is histologically not different and consists flow cytometrically just of a single cell population. Tumor areas with heterogen cell clones with higher aneuploidia show in median larger nucleus areas (e.g.73 um2 vs 0,38 um2 ) as well as smaller nucleus form factors (e.g. 0,65 vs 0,74).
Conclusion: The flowcytometric proof of tumor heterogenity can be confirmed by means of image analysis.
40. QUALITY CONTROL OF IMMUNEPHENOTYPING IN AUSTRIA
E. Schönwald*, R. Jilch**
** Central Laboratory ( Univ.Prof. Dr. M. Fischer ) and * Department of Dermatology ( Univ. Prof. Dr. F. Gschnait ) Municipal Hospital Lainz, Vienna, Austria
Since 1993 an external quality control for flow cytometric immunephenotyping of peripheral blood lymphocytes is performed regularly in Austria. Based upon pilot trials a regular procedure was established. In periodical meetings the achieved results are discussed by the participants and suggestions are made to be included for the following trials. Current status: for 22 laboratories in Austria and for 1 lab in Hungary ( University clinics, general hospitals and private labs ) two whole blood samples are delivered four times a year. The following marker combinations are chosen: CD45/14, CD3/19, CD3/16+56, CD4/8, CD3/4, CD3/8 and CD3/HLA-DR. The leucocytes counts are given by the coordinator of the quality control programme. The participants have to measure the lymphocyte and subpopulation percentages as well as the absolut counts. In addition to this basis programme within every quality control additional antibody combinations ( e.g. CD3/25, CD3/HLA-DR ) of different suppliers are sent for comparison. The working guidelines are following international consensus protocols for routine diagnosis ( whole blood lysing and gating ). First of all the evaluation of mean, standard deviation and the coefficient of variation was performed. At present the evaluation and assessment is provided with a special software programme by OEQUASTA ( Austrian society for quality assurance and standardization of medical diagnostic examination ). The coordinator of the quality control is providing the reference range and the individual weights of the single subpopulations. An interesting expansion to perform additional quality control may be the use of beads for the evaluation of density of receptors and linearity. The final goal of our quality control programme remains, especially for clinical reasons and routine diagnosis, to establish comparable results of all the participants.
41. SOURCES OF ERRORS IN QUANTITAVE IMAGE ACQUISITION IN CYTOMETRY
Schwarzmann P., Mähner G.
Inst. für Physikallshe Elektronik, Univ. Stuttgart,FRG
The quantitative evaluation of images in cytometry requires improved image aquistion techniques and standards, which may differ from that of image quality for mere visual observation. The contribution concentrates on features of imaging technologies Influenncing geometry, densitometry and color representatlon of images.
Evaluation of geometrical and morphometrical features: In pathology minor geomnetrical distortions influencing measurements of length or area can be neglected, Of much more importance is the ability to locate at all borderlines of cells, cell nuclei and tissue limits. This ability is influenced by the optical resolution of the microscope, the aperture of the sensor elements of the CCD camera and naturally by the optical properties of the object borders. Evaluation of density features: Errors in the evaluation of these features are of high interest as this measure is related to the amount of a chemical compound which may be of diagnostic importance. The most prominent example is the DNA histogramm in diagnosis of tumors. Effects influencing the quantitation of a staining component are mainly: non-Lambert-Beer transmission of light through the object, glare of microscope, local and time dependent shading of illumination, diffraction of optics and the object itself and finally the dynamic properties of the sensor elements. Evaluation of color Information for the separation of stained biological objects: Errors introduced here are mainly due to bad registration of the spectral images, systematic color shadings, different resolutions of the color channels and the principal differences of the color TV technology in comparison to the true spectral responce of the object.
42. FLOW CYTOMETRIC DETECTION OF INTRACELLULAR PLATELET ANTIGENS
Peter Sedlmayr1, Barbara Grosshaupt1, 2, Wolfgang Muntean2, Astrid Blaschitz3, Gottfried Dohr3
Departments of Internal Medicine1 and Pediatrics2, and Institute for Histology and Embryology3, Karl-Franzens-University Graz, Austria
Resting platelets contain intracellular structures such as a-granules, lysosomes, and dense bodies. Activation with thrombin leads to exocytosis. As their content is released, the granule membrane fuses with the cell surface membrane, and integral granule membrane molecules like P-selectin (CD62P, for a-granules) or pS3 (CD63, for lysosomes and dense bodies) become detectable on the cell surface. Thrombospondin, an a-granule protein, is secreted following platelet activation and binds to the thrombospondin receptors present on the platelets cell surface. Flow cytometric detection of in vitro activated platelets by the use of antibodies against surface membrane activation markers is a well established technique. We investigated the detectability by flow cytometry of these markers preformed in the cytoplasm of resting platelets. This was possible by permeabilization of the cell surface membrane by either methanol or using the Fix&Perm-kit (An der Grub). In addition we show that interleukin (IL)-1 a, but less so IL-1beta, are detectable inside but not on the cell surface of resting platelets after cell membrane permeabilization. Treatment by methanol, but not using Fix&Perm, permitted the detection of intracellular IL-1. This leads to the question whether the latter treatment causes leakage of (mainly cytosolic) IL-1 from the platelets.
Our data demonstrate the feasibility of flow cytometry as a tool for the study of platelet activation markers preformed inside resting platelets. This technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilizaton procedure does not lead to antigen leakage. For instance, preliminary data suggest that in comparison to adults, platelets from newborns contain the same amount of CD63 but show low expression of CD63 on the surface after thrombin stimulation, suggesting that this hyporeactivity to thrombin of platelets from neonates is not caused by preactivation of platelets during birth to depletion of intracellular stores.
43. CYTOMETRIC EVALUATION OF RUSH BEE-VENOM IMMUNOTHERAPY
J.A. Segura, J. Irsch*, A. Löhndorf*, N. Hünzelmann* and A. Radbruch Institute for Genetic. University of Cologne. Weyertal 121. 50931 Cologne, Germany.*Clinic of Dermatology of the University of Cologne. Joseph-Stelzmann Str. 9.50931 Cologne, Germany
The mechanisms of action of rush bee-venom desensitisation are largely unknown. Here we use flow cytometry to analyse how this therapy affects control of immune responsiveness. Blood was drawn from allergic patients receiving desensitisation treatment, and the cells stimulated with PMA and Ionomycin for 5 hours. Percentages of responding T cells were analysed for cytokine production by intracellular staining and multi parameter flow cytometry. The frequencies of cells expressing the key regulatory cytokines IL-4 and IFN-gamma decrease during the course of the immunotherapy, reaching a minimum at the 5th day of treatment. IL-2 production doesn't change significantly . The percentage of cells expressing the early activation marker CD69 is also reduced during the treatment. In healthy donors cytokines are produced mainly by the CD45RO+ (preactivated) cells. On the contrary, in allergic individuals the frequency of CD45RO+ T cells secreting IL-4 and IFN-gamma is drastically reduced, and the "native" CD45 RO- population now contains a higher number of cytokine secreting T cells.
Thus, the mechanism of rush desensitisation seems to be mediated by a systemic depression of the immune system selectively affecting the activated CD45RO+ T cells.
44. 5-AMINOLEVULINIC ACID UPTAKE, SYNTHESIS OF PROTOPORPHYRIN IX, AND EFFLUX OF PORPHYRINS IN TWO UROTHELIAL CARCINOMA CELL LINES
P. Steinbach1, H. Weingandt1, R. Baumgartner2, M. Kriegmair2, F. Hofstädter1, R. Knüchel1
Institute of Pathology, University of Regensburg and 2Dept. of Urology, University of Munich, Germany
In order to employ 5-aminolevulinic acid (ALA) in photodynamic therapy (PDT) it is necessary to obtain an adequate intracellular concentration of the photosensitizer protoporphyrin IX (PPIX). At present it is not yet completely understood why various cell types differ in the accumulation of PPIX. Therefore we studied differences of porphyrin biosynthesis in two urothelial cell lines derived from malignancies of the bladder. After 4h of incubation in ALA containing medium RT4 cells displayed a threefold fluorescence intensity value as J82 cells. These differences in fluorescence could in part be attributed to a different uptake of ALA, which was photospectrometrically quantified. Following the administration of ALA, for both cell lines a significant increase of fluorescence intensity was at the earliest detectable 6 min after administration. Efflux kinetic of porphyrins was initially faster for RT4 cells as for J82 cells. The differences of ALA induced fluorescence could be correlated with differences of the mitochondrial potential as measured with rhodamine 123, a probe for monitoring mitochondrial membrane potential. Dissipation of the mitochondrial transmembrane potential by the potassium ionophore valinomycin led to no detectable ALA induced fluorescence for both cell lines. The same result was achieved by incubating cells in potassium enriched medium, suggesting that the uptake of ALA is driven by the plasma membrane potential. These data demonstrate that the final ALA induced fluorescence might be attributed to differences in mitochondrial membrane potential (synthesis of PPIX), efflux rates, and ALA uptake, which itself might be dependent on plasma membrane potential.
45. NON-RADIOACTIVE LIFE SPAN MEASUREMENTS OF RED BLOOD CELLS BY FLOW CYTOMETRY AND ITS COMPARISON WITH RADIOACTIVE LABELING
Trastl. C.1; Beisker. W.2; Berg D.3; Hoffmann-Fezer, G.1
(1): GSF - Institut für Immunologie, (2): GSF - Arbeitsgruppe Durchflußzytometrie, (3): GSF - Institut für Strahlenbiologie, Ingolstädter Landstr. 1, D-85764 Oberschleißheim, Germany
The in vivo life span of canine erythrocytes was monitored for a time period of 120 days by flow cytometry as well as by radioactive tracers (51Cr). A proportion of 2% of the peripheral blood of Beagle dogs was biotinylated using Biotin-X-N-hydroxy-succimide ester (B-X-NHS) and reinjected afterewards. Peripheral blood samples were taken weekly and stained with fluoresceinisothiocyanate (FITC) labeled avidin. Biotin-labeled red blood cells could easily be dctected by single laser (488nm excitation) flow cytometny, using forward and/or side scatter for gating red blood cells and green fluorescence (530nm emission pathway). Labeled red blood cells showed an approx. 10 fold higher fluorescence intensity compared to the unlabeled cells. The percentage of labeled red blood cells decreascd during the monitoring period down to 0.2 % . During the first 30 days the biotin labeling technique was comparable to the standard radioisotopic method (International Committee for Standardization in Haematology, 1980). Radioisotopic labeling with 51Cr was comparable after data reduction for radioactive decay of 51Cr as well as for chromium elution during the 120 days The FITC fluorescence intensity per cell remained constant over the full period, which clearly demonstrates the advantage of the method.
46. SENSITIVITY OF MAMMALIAN SPERMATOGENIC CELLS TO IONIZING IRRADIATION
I. Tatchen, W. Göhde
Institut für Strahlenbiologie, Westfälische Wilhelms-Universität, Münster, Germany
Biological indicators are the only way for the determination of irradiation doses individuals have been exposed to accidentially. The conventional cytogenetic methods of 'biological dosimetry' are time-consuming and comparatively unsensitive.
Spermatogenic cells are known to be highly sensitive to treatment with ionizing irradiation; the LD50 for spermatogonia can be as low as 0.25 Gy. Further methodical improvements for the detection of small doses have been made. After irradiation with 60Co-Gamma radiation doses (0.05 - 2.0 Gy) NMRI mice have been been treated with BrDU (1h) in vivo. After removing the testes the S-phase cell frequency has been determined by using a monoclonal antibody against BrDU. The flow cytometric determined Sphase cell frequencies (Partec PAS lil) have been used to produce dose response curves. The maximal decrease of the S-phase cell frequency has been observed between the 2nd and 4th day after irradiation. The dose response curves 2, 4, 7 and 9 days after irradiation show that 0.05 Gy cause a decrease of Sphase cells.
This biological indicator is more sensitive than all known mammalian biological dosimeters. Procedures are discussed in order to receive dose response curves for human spermatogenic cells, too.
47. COMPUTER CLASSIFICATION OF FLOW CYTOMETRIC THREE COLOUR IMMUNEPHENOTYPES FROM BLOOD LEUKOCYTES AND BONE MARROW OF LOW AND HIGHGRADE NON-HODGKIN-LYMPHOMA AND CHRONIC LYMPHATIC LEUKEMIA PATIENTS
G.Valet1, G.Schmidtke2, U.Schmücker2, G.Brittinger2, H.G.Höffkes2
1) Max-Planck-lnst.f.Biochemie, 82152 Martinsried, 2) Abt.Hämatologie, Medizinische Univ.Klinik, 45122 Essen, Germany
The analysis of three colour immunephenotypes provides usually 3x4=12 values for the % freqency of lymphocyte populations in the quadrants of the FITC/PE, PE/CY5 and FITC/CY5 histograms. The majority of the information consisting of total antibody fluorescence, relative antibody surface density, fluorescence ratios, forward (FSC) and sideward light scatter (SSC) as well as SSC/FSC ratios of lympho-, mono- and granulocytes is lost in this way. Diagnosis is made visually and quantitatively by qualified experts. This is tedious, time consuming and subject to interobserver variability due to non standardized evaluation procedures.
The goal of this study was to automatically computer classify CD45/14/20, CD8/4/3, K/CD19/5, g/CD19/5, CD10/23/19 list mode files (BD-FACScan) of blood leukocytes and bone marrow samples of chronic lymphatic (CLL, n=14) and hairy cell leukemia (n=8), centrocytic-centroblastic (n=2), centrocytic (n=1), centroblastic (n=2), low grade (n=1) and B-cell (n=1) lymphoma, immunocytoma (LP-IC, n=9), normal (n=15) and leukocytotic (n=12) persons with the CLASSIF1 program (Ann.NY Acad.Sci 677,233-251(1993), Partec, Muenster).
The CLASSIF1 program extracts 222 values per three parameter immunophenotype and has the capacity to establish instrument and laboratory independent classifiers. 84-100% of patient samples were correctly classified. The clinically difficult distinction between CLL and LP-IC was achieved in 100% of the cases. In addition, 9 out of 10 clinically unassignable patients were assigned to either CLL or LP-IC.
48. EXCIMER FLUORESCENCE COMPARED TO DEPOLARIZATION IN THE ANALYSIS OF HYDROPHOBIC MEMBRANE DOMAINS
M. Wimmer, G. Rothe, G. Schmitz
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany
An altered cellular membrane fluidity secondary to changes of cellular cholesterol metabolism is a potentially important mechanism in the cellular pathogenesis of atherosclerosis. The established method for the determination of membrane fluidity in flow cytometry is the measurement of fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), excited with an argon ion laser at 364 nm. An altemative technique that does not depend on hardware modifications is based on the excimer formation of pyrenedecanoic acid (PDA).As an experimental model the cholesterol content of human Iymphocytes was reduced by incubation with phosphatidylcholine liposomes. They were further labelled with either PDA or DPH. Excimer (478 nm) and monomer (395 nm) fluorescence intensity of PDA or the two polarization directions of the DPH emission were measured by flow cytometry.The measurements showed, that the depolarization ratio of DPH decreases and the excimer monomer ratio of PDA increases with a diminished cholesterol content of the surface membrane. The PDA method in comparison to DPH proved to be the more sensitive technique for the detection of changes of the membrane fluidity.
The technically simpler PDA excimer assay should be of interest for the analysis of disturbed cellular membrane fluidity.
=== Poster Presentations ===
49. CGH IMAGING BY A ONE CHIP TRUE COLOR CCD CAMERA
Harald Bornfleth1, Klaus Aldinger1, Michael Hausmann1, Anna Jauch2 and Christoph Cremer1,3
1=Institute of Applied Physics, 2=Institute of Human Genetics and Anthropology, 3=Interdisciplinary Centre of Scientific Computing (IWR), University of Heidelberg, Heidelberg, F.R. Germany
In fluorescence microscopy, the quantitative measurement of fluorescence intensity ratios on metaphase chromosomes is the basic method for cytogenetic analysis in comparative genomic hybridization (CGH). Usually, the microscope images are acquired by high resolution, highly sensitive black & white (B&W) CCD cameras. This requires subsequent recording of the different color images using appropriate filter combinations for excitation and emission. Here, we show an alternative approach using the one chip true color CCD camera Kappa CF 15 MC and an Omega triple band pass filter for simultaneous registration of the three dyes Texas Red, FITC, and DAPI. An examination of the imaging properties of the system was performed. The camera response in the three color planes R, G, B was evaluated and calibration factors for image correction were calculated. A complete computer program for image analysis of CGH experiments running on a PC 80486 was developed using the commercially available software package OPTIMAS as the basis for image recording. Examples are shown that contirm the suitability of the system to ratio imaging in CGH of tumor cells. The results were compared to results obtained from the same microscope slides by an established setup consisting of a B&W CCD camera (Photometrics) and a software program based on the TCL software-package running on a Macintosh Quadra 950. A good agreement of both evaluations was found for several examples tested.
Literature: Bornfleth H, Aldinger K, Hausmann M, Jauch A, Cremer C: CGH imaging by the one chip true color CCD camera Kappa CF 15 MC. Cytometry 1995 (submitted)
50. SIMULTANEOUS REGISTRATION OF CALCIUM TRANSIENTS AND REACTIVE OXYGEN INTERMEDIATES OF HUMAN POLYMORPHONUCLEAR GRANULOCYTES IN RESPONSE TO ENVOIRONMENTAL AGENTS
W. Grundler1, P. Dirscherl2, K Marx1, I. Beck-Speier2, W. Beisker1, K. Maier2, and A. Stampfl3
GSF-Forschungszentrum für Umwelt und Gesundheit, AG Durchflußzytometrie, Institut für Inhalationsbiologie2 und Institut für Toxikologie3, D-85758 Oberschleißheim
Polymorphonuclear granulocytes (PMNs) play a pivotal role in host defense against pathogens. Besides migratorial. phagocytotic and secretoric capabilities is the stimulus dependent generation of reactive oxygen intermediates (ROI), termed respiratory burst (RB), one of the fundamental functions of these cells. The normally beneficial effects of RB could lead to possible pathological consequences when dysregulation occurs. Incubation of PMNs with sulfite a hazardous substance in certain air pollutants, generates RB alone and modulates the stimulatory effects of PMA and FMLP. We stablished a flow cytometric assay for simultaneous registration of intracellular Ca2+ ([Ca2+]i) and H2O2 ([H2O2]i) to determine interactions of sulfite with signal transduction events. Flow cytometric detection of [Ca2+]i with Indo-l and of [H22]i with DHR123 is combined with a novel evaluation of data provided with the data analysis system (DAS). This allows us to estimate kinetics of [Ca2 l]i transients and [H2O2]i generation in different populations of viable cells. Future experiments will additionally deal with simultaneous measurements of these parameters on single cell level, applying laser scanning microscopy to achieve enhanced spatial and temporal resolution of these events.
51. THE HEIDELBERG SLIT-SCAN FLOW FLUOROMETER
Michael Hausmann1, Alexander Rohrbach1, Dietmar Wolf1, Klaus Aldinger1, Michael Schurwanz1, Jürgen Dölle2, Arnulf Keese3, Martin Crone1, Christoph Cremer1
1=Institute of Applied Physics, University of Heidelberg, D-69120 Heidelberg, Germany (present addresses: 2=lBM Development Laboratory, D-71032 Böblingen, FRG; 3= Bertelsmann Development, D-33311 Gütersloh)
Slit-scan flow fluorometry is a laser-technological approach offering perspectives to further accelerated aberration scoring of isolated metaphase chromosomes in suspension. Details of the Heidelberg system are presented. The fluorescence labelled chromosomes rapidly pass one by another a "ribbon shaped" laser beam. The resolution of the system can be estimated by computer simulation. For this purpose a simulation program has been developed describing wave propagation effects of the focusing elements in the system. Since the chromosomes are orientated perpendicularly to the laser beam by hydrodynamic focusing of the flow stream, the fluorescence intensity along the chromosome axis can be measured time (= spatially) resolved. Different intensity profiles of normal, dicentric, and translocation chromosomes (after FISH in suspension) can be obtained. Time optimized parallel computing allows signal processing of profiles in less than 600,us. According to relevant profile parameters that can be freely chosen, a fast sorting module can be controlled by the computer system. In the order of hundred chromosomes per second can be analyzed and sorted out. This offers new perspectives in cases of rare events as for instance in biological dosimetry where the technique may be useful to sort out the chromosomes classified by "aberrant" fluorescence profiles for further cytogenetic analysis.
Literature: Hausmann M, Doelle J, Schurwanz M, Cremer C: Slit-scan flow fluorometry and sorting of chromosomes: A fast preanalysis system for microscopy. Microscopy and Analysis 7/95 (1995) 27-29
52. FLOW-CYTOMETRIC CHARACTERIZATION OF BATCH CULTIVATED BACTERIA WITH FLUORESCENT, rRNA-TARGETED OLOGONUCLEOTID PROBES
Carsten Herrmann, Andreas Lösche, Thomas Bley
Projektgruppe Biosignale, Abteilung Biotechnologie, Fakultät für Biowissenschaften,Pharmazie und Psychologie, Universität Leipzig, Permoserstr. 15, 04138 Leipzig
The combination of ftow-cytometry and in situ hybridization of bacteria with fluorescent, rRNA-targeted oligonudeotide probes is used to examine the afffinity to substrates and the growth dynamics of Alcaligenes eutrophus JMP 134 and Acinetobacter sp which were cultivated under batch conditions. Both bacterial strains can utilize aromatic compounds like phenole and benzoate. In first batch-experiments both strains were cultivated together or in pure culture using acetate as energy and carbon source. Short, synthetic oligonucleotide probes (15-20 nucleotides) were used for in situ hybridization which find their complemetary target in the sequence of 16S rRNA and 23S rRNA (overview by Amann, 1995) Molecules of rRNA are well suited for phylogenetic studies because of their partly single stranded and amplificated character Furtherwise, different conserved regions in the sequence of rRNA allow the classification at different taxonomic levels (Woese 1987) In this work a universal oligonucleotide probe for the domain of bacteria and two group-specihc probes (for beta and gamma-group of proteobacteria), a genus-specific and a strain-specific probe were used to identify Acinetobacter sp and Alcaligenes eutrophus JMP 134.
The content of rRNA or the number of ribosoms in bacteria is correlated with the rate of growth. Thus, the measured signals of hybridization (signal of fluorescence during flow cytometric analysis) give some information about the physiological activity of cells (Wallner 1993) Several samples of batch cultivated Acinetobacter sp were hybridized with fluorescein-labeled oligonucleotide probes Only exponential growing bacteria could be detected by using one nucleotide probe for hybridization. However all phases of bacterial growth (even lag- and stationary state) could be detected in the case of hybridization with up to three different nucleotide probes.
Another possibility of signal amplification was achieved by using alternativ dyes as CY3 (an indocarbocyanin dye) with increasing quantum yield Bacteria hybridized with the strain-specific oligonucleotid probe labeled with CY3 showed by exitation with 514 nm equal brightness of fluorescence as the three fluorescein-labeled probes did.
A separate detection of the mixed population with the genus and strain-specific nucleotide probes labeled wilh the dyes fluorescein (green) the other with CY3 (red-orange) was not possible wilh flow cytometry (spectral overlapp of dyes) but with epifluorescence microscopy.
Amann, R. I., Ludwig, W, Schleifer, K.-H. 1995 Phytogenetic identification and in situ detection of individual microbial cells without cuitivation. Microbiol Rev 59: 134-169
Wallner, G.,Amann R., Beisker, W. 1993. Optimizing fluorescent in situ hybrydization with rRNA-targeted oligo nucleotide probes for flow cytometrie identification of microorganisms. Cytometry. 14: 136-143
Woese,C.R. 1987, Bacterial evolution Microbiol.Rev.51:221-271
53. ANTI-P53 AUTOANTIBODIES IN SERA OF PATIENTS WITH ORAL SQUAMOUS CELL CARCINOMA
Ch. Hofele*, M. Volkmann+, M. Schmitt*, W. Fiehn+, J. Zöller*. J. Mühling*
(*Dept. of Oral and Maxillofacial Surgery, University of Heidelberg, INF 400, D-69120 Heidelberg, Germany, +Central Laboratory, Medical University Hospital, Bergheimer Str. 58, D-69115 Heidelberg, Germany)
P53 gene aberrations are the most common genetic changes in human carcinogenesis. Frequently, single point mutations are detected, resulting in increased levels of an aberrant p53 protein in tumor cells. This cellular protein appears to become immunogenic during tumor development, inducing the production of circulating antibodies against p53. In our study we investigated so far the sera of 69 patients with oral squamous cell carcinoma before treatment. 79 sera from patients without known or suspected neoplasm served as control. We used an ELISA (Dianova, Hamburg) to detect p53 autoantibodies. In 25% of the patients with primary oral cancer evidence for p53 autoantibodies was found (38% in patients with recurrent or second carcinomas). None of the control sera was anti-p53-positive. The anti-p53-positive patients were younger then the negative. Female patients had a higher rate of anti-p53 positivity. There was no correlation between the p53 specific autoantibody-response and the TN-stage. A correlation of the presence of anti-p53 could be revealed with high histological grading, sonographic tumor volume, and in recurrent or secondary carcinomas, respectively. Our results indicate a possible value of the anti-p53 status in the diagnosis of oral cancer, however, further studies are required to fully assess the clinical usefulness of anti-p53 and whether it can be used in the early diagnosis of preclinical, recurrent or secondary cancer.
54. CELL CYCLE AND CELL SIZE ANALYSIS OF DIFFERENT SACCAROMYCES YEASTS
K.-J. Hutter1, J.Schärfe2
1 DKFZ Heidelberg, Im Neuenheimer Feld 280, R.Süssmuth, Institut f. Mikrobiologie, Univ. Hohenheim, 2 Schärfe System, Reutlingen
Cell growth and cell cycle Or microorganisms are influenced directly by their chemical and physical environment. The observation of these variations during cultivation ean perrormed by electronic cell size analysis and flow cytometric measurements. Yeast eells exhibit different sizes under different growth conditions. After mitosis the smaller daughter cell requires a eritical size before s particular cell cycle can be eompleted. The larger mother cell can start immediately with a new round of cell cycle under optimal nutrient and growth conditions. The dirferent cell sizes of individual yeasts are eorresponding to the age Or the cells in a population. Cell size analysis (CASY) is compared to flow cytometric data of different yeast strains. Ref.: Carter, B.L.A., Jagadish, M.N.: Exptl. Cell Res. 112, 15-24 (1978).
55. TIME SHIFTING OF CIRCADIAN PATTERNS OF LYMPHOCYTE SUBSETS AND EXPRESSION OF CD2, CD11a AND CD44 IN HUMAN PERIPHERAL BLOOD
T.O.Kleine1, R. Hackler1, P. Z&oouml;fel2, J. Albrecht3
1 Med. Zentrum für Nervenheilkunde, Funktionsbereich Neuro- chemie, Universität Marburg a.d. Lahn, 2 Hochschulrechenzen- trum der Universität Marburg, 3 Becton Dickinson, Heidelberg
To study prolonged changes of significant circadian variations of human lymphocyte subsets (1) cytometric measurements using monoclonal antibody reagents were done in EDTA blood from male volunteers during a 48 h sampling period in one-year interval; they were evaluated by cosinor-rhythmometry (cf. 1). Peak times for T lymphocytes and their CD4+3+ or CD8+3+ subsets shifted from 23.00 24.00 h to 14.00-l5.00 h; and to a smaller extent with CDl9+ B cells. The findings were not influenced by daily fluctuations of red cell parameters. Similar peak time shiftings during the one-year interval were found with the expression of CD2 (LFA-2), CDlla (LFA-1a) and CD44 (Pgp-l) on T cells and their subsets which was almost complete. B cells showed some differences in CD44 and /or CDlla staining exhibiting similar circadian variations of positively and negatively stained cells. Our data indicate substantial changes of homing capacities of T cells, their subsets, and B cells besides changes of circadian lymphocyte activation with unknown effects on the immune system. 1. T.O. Kleine, R.Hackler, K Ehlenz, P. Zofel, J. Albrecht. J. interdiscipl. Cycle Res. 24 (1993) 236.
56. INFLUENCE OF TOTAL SLEEP DEPRIVATION ON CIRCADIAN VARIATIONS OF LYMPHOCYTE SUBSETS IN HUMAN PERIPHERAL BLOOD FROM HEALTHY MALE VOLUNTEERS
T.O. Kleine1, W. Schreiber2, R. Hackler1, P. Zöfel2, J.Albrecht3
1 Med. Zentrum für Nervenheilkunde, Funktionsbereich Neurochemie, Universität Marburg a.d. Lahn; 2 Klinik für Psychartrie im Med. Zentrum für Nervenheilkunde, Universität Marburg a.d. Lahn; 2 Hochschulrechenzentrum der Universität Marburg, 3 Becton Dickinson, Heidelberg
Significant circadian variations of T cells, their subsets, and of B cells have been demonstrated by cosinor-rhythmometry in human peripheral blood (1). We studied the influence of total sleep deprivation on these circadian variations in healthy male volunteers. Cytometric measurements using monoclonal antibody reagents (cf. 1) were done in venous EDTA blood during three consecutive days at 7.00 h, 13.00 h and 19.00 h with total sleep deprivation between day 1 and day 2 as well as recovery sleep between day 2 and day 3. Significant decreases of lymphocyte counts (20% and 31 %) were found only at 7.00 h of day 2 and (day 3 compared with day 1 (paired t-test); differences of the 13.00 h or 19.00 h cell counts were not significant. Significant drops at 7.00 h were also obtained for CD3+, CD4 + 3 +, and CD8 +3 + counts, resp., in parallel with NK counts at 2nd and 3rd day. HLADR+ CD3+ and CDl9+ cell counts decreased at 7.00 h of the 3rd day only. Our data indicate significant alterations of the cellular immune system, especially an impairment of circadian lymphocyte patterns induced by total sleep deprivation in normal humans. 1. T.O. Kleine, R.Hackler, K Ehlenz, P. Zoefel, J. Albrecht. J. interdiscipl. Cycle Res. 24 ( 1993) 236.
57. DNA IMAGE CYTOMETRY ON SECTIONS: MEASUREMENT OF ENTIRE OR CUT NUCLEI?
T.KIapperstück and W. Wohlrab
Department of Dermatology, Martin Luther University Halle-Wittenberg, Germany
Cytometric DNA quantifications of tumours are performed by means of flow cytometry and image analysis. The latter allows morphological criteria to be included in selecting the nuclei under investigation. This is an advantage, when the tumour population is small and cannot be detected by flow cytometry. Smears and imprints, as well as isolated nuclei obtained from paraffin embedded tissue were mostly used for image cytometry. The identification of different tissues is even better on sections. However, one must take into account the presence of cut nuclei and nuclear overlap. The aim of the present study was to examine the reliability of measurements on sections.
Smears and the corresponding sections of 25 meianomas were investigated using the digital image analysis system CYDOK (Carl H. Hilgers, Konigswinter, Germany). To obtain the smears, fresh resected tumour material were processed by a citric acid-Tween 20 procedure. At first, 25 smears with known DNA histogram (5 diploid, 20 aneuploid) were selected. The DNA indices of the GO/G1 peaks were in the range of 0.98 to 2.5. These results obtained from entire nuclei were considered as a reference. In order to find out an optimal section thickness, 10 sections cut at 2, 4, 6 and 8 pm were measured. Eight pm sections showed the best histograms and were therefore used for comparing the data. We attempted to exclude cut nuclei from the measurement by careful focusing in either direction. The results of smears and sections were compared in relation to the DNA index of the GO/G1 peaks (Dl), the 5c exceeding rate (5cER), and the proliferation index (Pl) by carrying out the Spearman rank order correlation. Stem lines with a Dl of 0.9 to 1.1 were classified as DNA diploid (reference: spindle-shaped nuclei or nuclei of fibroblasts, respectively).
The histograms of smears showed a better quality as compared with those of sections. However, this did not affect the assessment of DNA ploidy. A high correlation was observed between the Dls (rs="0".95). Both the 5cER and the Pl values showed no such a good concordance (rs="0".84 and 0.77). Nevertheless, the 5cER appears to be useful in sections too, because the presence of nuclei with a Dl>2.5 is probably more important than the exact percentage. It seems to be possible to reduce the number of cut nuclei measured by careful selection and by using a suitable section thickness.
58. FAST KINETICS MEASUREMENT OF INTRACELLULAR Ca2+ ON THE FACScan
Ladusch, M., Jüngel, A., Drössler, K.
University Leipzig, Institute of Zoology, Talstrasse 33, D-04301 Leipzig
Some agonists like fMLP as well as ionophores induce a very rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in leukocytes. Normally these changes occur in a few seconds or even in the region of milliseconds and are therefore not to be followed with the usual flow cytometers. In most cases using these stimulators/ ionophores one can only state that the [Ca2+]i is already increased. The aim of our work was to evaluate the chance to monitor the [Ca2+]i within the first seconds after addition of the stimulator by the FACScanTM flow cytometer.
We have established a simple, low-cost set-up to inject the stimulator into the sample tube in place during the running data aquisition. The dead time between injection and getting first signals was determined with 2.8 s and cannot be considerably shortened without a complete modification of the sample tube and/ or the cuvette and/ or the pressure regulation.
The time point of appearance of cells having already contact to the stimulator were indicated by small (2,65_m), brightly fluorescing latices previously admixed to the stimulator/ ionophore solution in a tested relation to cell density. Dose-response experiments with FMLP on neutrophil granulocytes in a large range of concentrations (2 10-6 to 2 10-13 M) revealed that not the intensity but the time to the appearance of the response depends on the dose. The neutrophils reached the maximum fluorescence in the lowest dose (2-10-l3 M) after 5.6 s and within the dead time (2.8s) in concentrations above 2-10-7 M.
Our measuring set-up allows the flow cytometric demonstration of [Ca2+]i oscillations in human peripheral blood neutrophils. The application of fMLP in concentrations between 10-11 and 10-l2 M leads in a majority of these cells to partially synchronized [Ca2+]i changes with a phase duration of 20 to 23 s. After one oscillation the synchronicity is no longer detectable.
59. FLOWCYTOMETRIC TYPING OF PREADIPOCYTES IN MUSCLE TISSUE
Bertold Löhrke, Torsten Viergutz, Jochen Wegner, Klaus Ender
Research Institute of Animal Biology, Dummerstorf-Rostock
Introduction: In the muscle tissue of growing individuals there is a small portion of fibroblastoid cells which can develope both adipogenic and chondroblastic programs. Little is known about the factors engaged in differentiation. Methods: Bovine muscle specimens were digested, centrifuged at 200g and 2000g and the cells were analysed by receptor pattern, proliferative signal transduction and functional state before and after culture with Insulin (1), cortisol (C), and noradrenaline (NA). In flowcytometry the cells were gated by > 15um high-granulated(I), 10-14 um granulated (II), 5-9 um granulated (111), 5-9 um low granulated (IV). Results Cells without cultivation. 1) The highest cell portion (18% of 4% of the gated cells) with lipid vesicles was in gate (1V) -2000g and in gates (1+11)- 200g, the lowest % in gate (IV)- 200g. 2) High enrichment of macrophages was observed according to the functional tests and casein receptor expression in gates (I) and (11) 200g and 2000g although 30 % ( 1- 200g) and 50 % ( 1-2000g) of the cells expressed Ras. 3) 29-36 % of gate (1)-2000g cells ( 25% of (11)-200g, 20% of (11)-2000g and 15% of (111)-2000g cells) expressed protein S-100. 4) In (1V)200g and 2000g neither Ras nor protein S-100 were detected. Cultured cells. Gate (1V)-200g cells were induced to produce Ras by 1. The Ras expression was inhibited by C or NA and protein S-100 expression by NA. In gate (11)-200g cells Ras expression was stimulated by (I + C) and protein S-100 by 1. Gate (11)-20009 cells responded to (I + NA) or NA with increased Ras and protein S-100 expression (both were inhibited by C). Functional tests revealed considerable phenotypic alteration by 1, C, and NA in macrophages. Conclusion: Gate (IV)-200g cells without detectable lipid vesicles as well as Ras and protein S-100 expression can be induced to produce proliferative signal transduction ( Ras expression ) by I and adipocytes marker protein S-100 by NA. Together with the altered expression of these proteins by I and NA the cells developed an adipocyte-like transmembrane potential-,beta- adrenergic and opioidergic receptor response, suggesting the cell population contained stem cells, beeing adipogenically programable, possibly in successive steps, with proliferative and differentiating effects of I and NA occuring.
60. FLOW CYTOMETRY ANALYSIS USING ACTIVATED MICROBEADS
Reinhard Malsch, Lukas Piazolo, Daniel Melui and Job Harenberg
First Dept. of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Theodor Kutzer Ufer, 68167 Mannheim, Germany.
Activated carboxy-, hydroxy-,aminogroups or tosyl groups can be used to bind ligands to non- and ferromagnetic microbeads. Different activated microbeads of different size were used to bind glycosa ninoglycans and protamine. Using carboxy-activated beads heparin (M = 11000 Da) was covalently bound by the catalysis of cyclocarbodiimide (26 mM) to the surface. For the synthesis of heparin-microbeads activated hydroxy- and aminogroups were successfully used. Parallel reactions demonstrated that the functional groups required for anticoagulation remained intact. The heparin microparticles forrned complexes with antithrombin III and heparin cofactor II by fluorescence microscopy and flow cytometry. Protamine ( M = 6000 Da) was synthesized to using carboxyand tosyl-activated microbeads. Between 0.65 and 0.75 U/ml heparin in solution was neutralized by 0.1,ug protaminemicrospheres. The protamine-microparticles bound dose dependent fluorescent labeled low molecular mass heparin (LMMH-tyr-fitc) in saline solution and in plasma. The relative fluorescence intensity (RFI) was quantified by means of flow cytometry analysis using a direct and an indirect binding assay Qualitative and quantitative measurement of heparin can be performed by flow cytometry using these heparin and protamine-coated microbeads. Supported by Deutsche Forschungsgemeinschaft Ha 1164/3-2.
61. STANDARDIZATION OF PROTAMINE-COATED MICROBEADS USING FLOW CYTOMETRY FOR HEPARIN QUANTITATION
Daniel Melui, Reinhard Malsch, Lukas Piazolo and Job Harenberg
First Dept. of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer, 68167 Mannheim, Germany
For determining the concentration of heparin in plasna and whole blood a microassay was developed using protamine coated microbeads detected by flow cytometry (Piazolo et al: Semin Thromb Haemostas 20: 227-235, 1994. Here we report on the optimization of protamine-microbeads of different procedures.
The following microbeads of different sizes were used: Estapor-Microbeads M1-070/60 (0.8 um) and M1-180/12 (1.6 um) and Dynabeads M450, (4 um) were compared: The influence of different pH (7.0, 7.5, 8.0, 8.5) of the buffer system used for coating the microbeads on their relative size after the coating reaction could not be ascertained.
The binding of Protamine-Estapor-Microbeads and Protamine-Dynabeads to fluorescent heparin (LMMH-tyr-fitc) was studied: After incubation of protamine-microbeads with LMMH-tyr-fitc (14.3 ng/ml to 28.6 ug/ml) the relative fluorescence intensity (RFI) of bound LMMH-tyr-fitcmolecules was quantified by means of flow cytometry analysis. The higher binding rate and sensitivity of the Protamine-Estapor-Microbeads M1180/12 could not be matched by the more homogenous Protamine Dynabeads, whereas the Protamine-Estapor-Microbeads M1-070/60 were equivalent to the Protamine-Dynabeads. Using a competitive binding assay the Protamine-Dynabeads were more favorable. They showed a higher sensitivity and a linear calibration curve as compared to the Protamine-Estapor-Microbeads .
In conclusion the Protamine-Dynabeads should be prefered to the Protamine-Estapor-Microbeads because they can be also used for measuring the concentration of heparin.
Supported by Deutsche Forschungsgerneinschaft, grant Ha 1164/3-2.
62. DETERMINATION OF BACTERIAL VIABILITY WITH A NOVEL 488 NM-EXCITABLE NUCLEIC ACID STAIN
Paul J. Millard, Bruce Roth, Stephen T. Yue, Laurie J. Jones, and Martin Poot
Molecular Probes, Inc., Eugene, Oregon 97402, U.S.A.
SYTOXTM Green dye is a novel fluorescent nucleic acid stain, which because of its positive charge is cell-impermeant. Upon binding, this compound undergoes a 1500-fold enhancement in fluorescence at 523 nm and exhibits a quantum efficiency of 0.64. While the excitation of SYTOX Green dye complexed with DNA is maximal at 502 nm, its fluorescence is readily excited by the argon-ion laser at 488 nm. The SYTOX Green stain is completely excluded from live eukaryotic and prokaryotic cells, whereas all cells with compromised plasma membranes are labelled with bright green fluorescence. Since SYTOX Green dye fluorescence in membrane-compromised bacteria is typically very bright, about 10- 100fold greater than in intact organisms, discrimination of live and dead cells by flow cytometry was possible. The effect of several beta-lactam antibiotics on the viability of Escherichia coli can be assessed by flow cytometry. In addition, fluorescence microscopy of bacteria stained with a combination of SYTOX Green dye and either AMCA-X or Texas Rede conjugates, such as wheat gemm agglutinin or antibodies, was shown to be an effective method for identifying bacteria and simultaneously assessing their viability. These studies suggest that the SYTOX Green nucleic acid stain wiil provide a powerful altemative to propidium iodide for measuring cell viability.
63. FLOW CYTOMETRIC DETECTION OF APOPTOSIS IN RAT T CELL LINES
E.Mix1, U.K.Zettl1,2, J.Zielasek2, M.Stangel3, K.V.Toyka2, H.-P.Hartung2, R.Gold2
1Dept. of Neurology, Hosp. Nerv. Dis.,Universität Rostock, 2Dept. of Neurology, Universität Würzburg, 3Dept. of Neurology, Freie Universität Berlin, Germany
Apoptosis is a major mechanism of T cell elimination in autoimmunity, during ontogeny and tolerance induction. To assess the possible involvement of reactive oxygen and nitrogen species in T cell apoptosis we investigated the effects of H202 and NO* in vitro on activated autoreactive CD4+ T cell lines capable of transfering experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN). For detection and quantitation of apoptotic cells, DNA fragmentation was assessed by insitu tailing with fluorescein-ddUTP and subsequent flow cytometric analysis. H202 applied directly to the cell cultures for 6h to 18h at concentrations of 1OuM to 300uM and active oxygen intermediates released by hypoxanthine/xanthine oxidase (HX/XO) caused apoptosis in a dose-dependent manner in 13-33% of neuritogenic and encephalitogenic T cell lines. NO' released by the penicillamine-derivative SNAP induced high rates of apoptosis in two encephalitogenic (38% resp. 23%) and one neuritogenic (26%) T cell line, whereas the apoptotic effect on ovalbumin-reactive control T cells was somewhat lower (9%). The findings suggest a role of macrophage-derived reactive oxygen and nitrogen species for disease limitation in inflammatory demyelination by elimination of autoreactive and bystander T cells via apoptosis.
64. DNA DISTRIBUTIONS OF SOME BACTERIA: INFLUENCE OF GROWTH PHASE, GROWTH RATE AND NUTRIENTS
Susann Müller, Andreas Lösche and Thomas Bley
Projektgruppe Biosignale, Abteilung Biotechnologie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, Permoserstr.15, D-04318 Leipzig
Microbial state distributions and the investigation of state - dependent performances are not a main objective of biotechnological research to date. Clearly there are different mechanisms in the regulation of the cell cycle for various bacteria at various media but an understanding of the whole process of differentiation of a population into subpopulations with distinctly separate performances regarding the overproduction of metabolites requires detailed flow cytometric monitoring of the DNA content and based on this knowledge on the cell-cycle regulation. The bacterial strains grow at different media at distinct generation times, respectively. Only in the exponential phase of growth (the time without any limitations) the cells synthesize DNA according to the model of Cooper and HelmsteHer ( Cooper, S. and Helmstetter, C.E. (1968) Chromosome replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31, 519 550.). Afterwards, the growth rate 11 is much more lower than in the exponential growth phase. In dependence on the limitation conditions the cells shifl into the C 1 or C 2 content of DNA. In the bacterium Methylobacterium rhodesianum which is able to produce poly-3-hydroxybutyrate (PHB), all cells contain the double content of DNA, if they grow on methanol followed by Nlimitation (Muller, S. et al. (1995) A flow cytometric approach for characterization and differentiation of bacteria during microbial processes. Appl. Microbiol. Biotechnol. 43, 93 101 and Ackermann et al. (1995) Methylobacterium rhodesianum cells tend to double the DNA content under growth limitations and accumulate PHB. J. Biotechnol. 39, 9-20.) In the case of lower growth rates (growth on fructose) only a part of the cells synthesize PHB and only these cells seem to master of the double content of DNA. The distributions of the DNA content of Acinetobacter calcoaceticus V69 show the typical appearance of cells which grow in the beginning of batch cultivation like the model of Cooper and Helmstetter (1968). Already two hours later limitation conditions occur. At the time of stationary phase growth there exist both cells with one and double content of DNA. In Escherichia coli decoupling DNA synthesis is not measurable by flow cytometry. Indeed, the application of rifampicin to E coli cells (according a method to Skarstad, K. and Boye, E. (1993) Degradation of individual chromosomes in recA mutants of Escherichia coli. J. Bacteriol. 175, 5505 5509) shows that exponential growth occurs in the first two hours, only. At this time the DNA content of the cells is at least two- or fourfold. If ampicillin is added the DNA content is fourand eighffold. In the stationary phase all the cells possess of the C1 and C2 content of DNA. Only in the case of E.coli B335 most of the cells are in the C1state if two different C-sources were applicated and nonexponential growth occured until the 12th hour.
65. FLOW CYTOMETRIC ANALYSIS OF THE PHYSICAL ASSOCIATION BETWEEN SURFACE RECEPTOR AND CYTOSKELETON
B. Nebe, J. Rychly
Klinik für Innere Medizin, Universität Rostock
Physical association between surface receptors and the cytoskeleton can be detected with antibodies against the receptor after extraction of cells using triton X-100 containing buffer. Such interaction with the cytoskeleton may be an important mechanism in signal transduction. Using microscopic techniques we found that the association between integrin receptors and the cytoskeleton can be induced in a hepatocyte cell line by receptor cross linking. To obtain more quantitative data we developed a flow cytometric technique to analyze receptor-cytoskeleton interaction. Cells in suspension were incubated with the monoclonal antibody against the integrin receptor and cross linking was performed with the second antibody. After extraction of the cells, incubation was performed with a third FITC labeled antibody. Extracted cells were then analyzed with a FACScan flow cytometer. They revealed a distinct population in the dot plot with a low forward and sideward scatter. We obtained quantitative differences in the receptor quantity associated at the cytoskeleton comparing different integrin receptors. Moreover, differences were observed when cross linking was performed at 37o C and 4o C. We conclude that flow cytometric analysis of receptor-cytoskeleton association is suitable to obtain relative quantitative data.
66. EVALUATION OF FOUR MONOCLONAL ANTIBODIES AGAINST HLA-B27 FOR THEIR RELIABILITY IN HLA-B27 TYPING WITH FLOW CYTOMETRY (FC). COMPARISON WITH THE CLASSIC MICROLYMPHOCYTOTOXIC TEST (MLCT)
J. Neumüller, D.W.M. Schwartz**, E. Dauber**, W.R. Mayr**
Ludwig Boltzmann Institute for Rheumatology and Balneology, Vienna, Austria, **Clinical Department for Blood Group Serology, University of Vienna, Vienna, Austria
Typing for HLA-B27 is usually performed by MLCT but can also be carried out by FC Although there is a controversial view about the value of the determination of this antigen in diagnosis of ankylosing spondylitis, this investigation is frequently requested by rheumatologists. The presence of HLA-B27 in other seronegative spondarthritides such as reactive arthritis or psoriatic arthritis is not of diagnostic value but could influence the course of these disorders. The aim of the present study was to check the accuracy and reliability of this method using different MAB against HLA-B27. A comparative determination of HLA-B27 and cross-reacting specificities was performed by MLCT.
Typing of HLA-B27 by FC was performed using a special software package which requires corresponding calibration beads in order to perform a standardized setup of the flow cytometer. MAB were used from the following producers: Becton & Dickinson (BD), Behringwerke (BE), One Lambda (OL) and Immunotech (IT). The software provides a critical limit of fluorescence intensity (Fl) only for the MAB of BD. For the other MAB a cut off for HLA-B27 positivity was calculated as follows: mean of Fl of HLA-B27+ patients minus twice the standard deviation. The Iymphocytes were prepared according to a standard protocol including incubation of whole EDTA-anticoagulated blood for 15' with a mixture of an anti-HLA-B27 MAB-FITC with an anti-CD3 MAB-PE and a subsequent Iysis of erythrocytes. The binding of the MAB against HLA-B27 was determined in the FL1 single histogram after automatic gating of CD3+ cells in the FSC x FL2 dot plot.
A good discrimination between HLA-B27+ and HLA-B27- patients was obtained with all of the MAB. Cross-reactions with HLA-B7+ patients occurred with all MAB with the exception of OL. False-negative results were found with MAB from OL in 2.4 % and false-positive results if MAB from BD, BE and IT were used. Such errors in the interpretation of the FC analysis might be avoided if more than one MAB (including the non cross-reacting with HLA-B7 from OL) are used.
67. DETECTION OF RED FLUORESCENT MICROPHERES USING AN ALL- SOLID-STATE FLOW CYTOMETER
Stefan Niehren1, Wolfgang Kinzelbach1, Stefan Seeger2, Jürgen Wolfrum2
1 Institute of Environmental Physics, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, 2 Institute of Physical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg
Fluorescent particles with excitation and emission in red light are of interest if high selectivity against unlabeled particles is required We use such particles as tracers in the investigation of fractured media. Because their transport behavior differs from that of solute tracers they supply complementary information on fracture space. This information is crucial in the risk assessment of underground hazardous waste storage sites. To allow quick and on-line continuous monitoring of single particles, a flow cytometer has been developed in the present work. The cytometer is built from solid-state optical components, resulting in a robust and transportable system suitable for field use. To maximize the fluorescence quantum efficiency, a new dye JA22 was used. Use of an avalanche diode allowed considerable increase in the efficiency for photon detection compared to photomultipliers. For complete digital data acquisition and analysis only a personal computer is required The detection efficiency of the cytometer is greater than 25%, the probe flow is 20ml/h and the present accuracy is <160 particles/ml.
Recently a derivate (MR200) of the dye JA22 was succesfully used as label in biodiagnostics and MR200 has the same optical properties than JA22. Hence, d seems to be possible to introduce a new parameter in flow cytometry with the All-Solid-State technique.
68. FLOW CYTOMETRIC ANALYSIS OF T CELL REPERTOIRE: A RAPID METHOD FOR PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE HUMAN a/beta T CELL RECEPTOR
C. Otto, F. Struck, K. Littmann-JanGen, J. Collins, A. Lingnau, K.E.J. Dittmar
Gesellschaft für Biotechnologische Forschung (GBF), Dept. of Applied Genetics, D-38124 Braunschweig, FRG
Flow cytometric analysis of a/beta T cell receptors by specific monoclonal antibodies (mAbs) is an alternative to RT-PCR analysis. With the Iymph node immunization mAbs were produced by hybridoma technology within eight weeks. These mAbs are useful for flow cytometric analysis.
Chimeric protein (human mouse T cell receptor) was used as immunogen for local Iymph node immunization. After six subcutaneous injections the Iymph nodes were removed at day 17 and the B-cells were fused to P3X63.Ag8.653 myeloma cells. Supernatants of B-cell hybridomas were screened by flow cytometric analysis with specific surface transfectants after 8-10 days. Positive hybridomas were then cloned under limiting dilution.
The monoclonal antibody 1 OD6 recognizes 3-4% of peripheral CD3 positive cells from healthy donors and the antibody 1C7 recognizes 0,1-0,5%. The specificity was always confirmed by RT-PCR analysis of TCR mRNA from mAb sorted blood lymphocytes. We will determine the binding constants for these antibodies by BlAcore (Pharmacia Biosensor) and will investigate their application for histopathology.
69. BINDING TO LEUKOCYTES AND INTACT BIOLOGICAL ACTIVITY OF A FLUORESCENT LABELED LMM-HEPARIN
L. Piazolo, J. Harenberg, R. Malsch and D. L. Heene
1st. Department of Medicine, Medical University Clinic, Theodor-Kutzer Ufer, 68167 Mannheim, Germany
The pharmacokinetic and pharmacodynamic properties of an endpoint-attached N'alkylamin derivative of low molecular mass heparin was investigated ex vivo. In our study we labeled LMMHeparin-Tyramine with fluorescein-5-isothiocyanat (Fitc). This LMM-Heparin-Tyramine-Fitc has an antithrombin activity of 5 U/mg and an antifactor Xa activity of 70 U/mg. By flow cytometry analysis we were able to show an in vitro dose dependent binding of LMM-Heparin-Tyramine-Fitc to leukocytes. The fluorescent labeled LMM-Heparin bound in increasing amounts to leukocytes in following order: lymphocytes < monocytes < granulocytes. After intravenous bolus injection of the LMM-Heparin-Tyramine-Fitc to SpragueDawly rats (n = 8) blood samples were taken. The in vivo binding of LMM-Heparin-Tyramine-Fitc was detected by flow cytometry analysis. Using this data and the in vitro standard curves we were able to calculate the blood concentration (ug/ml) of the injected LMM-Heparin-Tyramine-Fitc. Beside this we measured the biological activities in plasma. We used the chromogenic substrate assays S 2238 and S 2222 to detect the antithrombin and antifactor Xa activities.
The pharmacokinetic of the blood concentration was comparable with the pharmacodynamic of the plasma antifactor Xa activities of LMM-Heparin-Tyramine-Fitc.
This work has been supported by the Deutschen Forschungsgemeinschaft, grant Ha 1164/3-2
70. RESOLUTION EFFECTS IN "PRACTICAL" LIGHT MICROSCOPY AND THE APPLICATION OF A 2pi TILTING DEVICE
Bernd Rinke, Joachim Bradl, Bernhard Schneider, Michael Hausmann, Christoph Cremer
Institute of Applied Physics, University of Heidelberg, D-69120 Heidelberg, Germany
The spatial resolution of a conventional light microscope or a confocal laser scanning microscope can be detemmined by calculating the point spread function. Usually, the values are obtained for ideal optical conditions. These conditions are often not fulfilled in biological applications especially in those cases where biochemical requirements (e.g. buffer conditions) influence the specimen preparation on the microscope slide ("practical" light microscopy). The application of a quartz glass capillary for object rotation instead of a standard slide has an additional influence on the resolution. However, such a 2pi-tilting device has advantages in 3D-microscopy. It allows, for instance, to find the optimal perspective for object visualization. Rotating by 90_ can be used to overcome the problem of a reduced z-resolution compared to the lateral resolution. To estimate resolution effects, point spread functions were measured under different modified (non-ideal) conditions using fluorescent microspheres of subwavelength dimensions. Results obtained from standard slide setups were compared to capillary setups in several types of microscopes (conventional, confocal). The results indicate, that in practice the capillary setup leads to an overall 3D-resolution improvement. Effects of refractive index matching and light propagation are discussed using computer simulation.
71. CELL CYCLE OR PROLIFERATION DEPENDANCY OF DIFFERENTIAL EXPRESSION OF CD34 BY HUMAN ENDOTHELIAL CELLS?
O. Röhrig, C.L. Klein, T. van Kooten, F. Bittinger, C.J.Kirkpatrick
Institute of Pathology, University of Mainz, Germany
CD34 is an important, highly glycosylated membrane protein of hemopoietic stem cells and is also present on the luminal surface of human endothelial cells CD34 has been shown to bind selectins and, tnerefore, may act as an important leukocyte-endothelial a&esion molecule. In contrast to in vivo conditions, where CD34 is expressed by almost all endothelial cells, it is downregulated very rapidly dunng in vitro culture of endothelial cells, resulting in only 10 - 30% CD34 positive cells. Since the current knowledge about leukocyte-endothelial interaction is mainly based on m vitro studies using cultured endothelial cells, the reliability of these m vitro models is crucial. Resting and stimulated expression of intercellular adhesion molecules like ICAM-I, VCAM and Eselectin in vitro, have been shown to be in accordance to in vivo observations. So the difference in CD34 expression remains the major phenotypic observed difference between in vivo and in vitro. Based these observations a flow cytometric study was designed to study the relation of cell cycle, proliferation and the expression of CD34 by cultured human umbilical vein endothelial cells (HUVEC).The expression of CD34 by freshly isolated and cultured HUVEC was examined in correlation to DNA-content, cell-cycle and cell proliferation by double and tripple labelling of CD34 and DNA using the BrdU-PI-method. The purity of endothelial cell preparations was confirmed by intracellular staining of clotting factor VIII. In addition, cultured in vitro-CD34-positive endothelial cells were purified by MACS-immunomagnetic separation and the kmetic of CD34 expression was compared to originally ex vivo CD34 positive endothelial cells. The results presented will contribute to the understanding of the differential regulation of CD34 by endothelial cells and, thereby may help to adjust culture conditions to reach in vivo levels of CD34 expression during in vitro culture of human endothelial cells. Thus in vitro models of leukocyteendothelium interaction will be even more reliable.
72. FLOW CYTOMETRIC DNA ANALYSIS OF PROSTATE TUMORS
K. Romanakis1, B. Wullich2, K. Becker3, B. Kopper3, and H. Zankl1
1University of Kaiserslautern, Dept. Human Biology and Human Genetics, 67663 Kaiserslautern, 2University of the saar, Dept. of Urology, 68141 Homburg/Saar, 3Universitv Hospital of Kaiserslautern, 67655 Kaiserslautern
After total resection seventy-six prostate tumors were examined for DNA analysis by flow cytometry. In 50 cases we have been able to examine biopsies from the tumor area as well as from tumor free area, based on histological and macroscopic evaluation. Of 26 tumors studied, 24 biopsies from the tumor area and 1-3 tumor free biopsies could be analysed. The aim of this work was to observe flow cytometrically the intratumorai heterogeneity within one tumor. Of further interest was to observe the rate of abnormal cells in tumor free defined tissues.
According to the slight modificated method described by Otto (Methods in Cell Biology, 1990, Vol.33, 105-110) cell suspensions of the biopsies were prepared, stained with DAPI and analyzed with a PAS lIl flow cytometer (Partec, Munster, Germany). The multi cycle program of cell cycle analysis (Flow Systems, Phoenix) was used for generation of histograms. The CV's showed an average of 1.86 +- 0.56%.
Of the 76 cases studied, 28 showed aneuploidy by FCM. These 28 could be separated into 6 hypodiploid, 8 hyperdiploid, 3 triploid, 1 hypertriploid, 8 tetraploid and 2 hypertetraploid tumors. The biopsies of the tumor free areas were diploid. By observing the heterogeneity we found additional aneuploidy in different biopsies in 8 of the 26 examined cases. Two cases showed next to the aneuploidy of the tumor area, in 2 of 3, i.e. 3 of 4 biopsies diploidy. These results point out the necessity to examine several biopsies from different areas of a tumor to ensure the histological diagnosis, and by this flow cytometric DNA analysis.
The average of the S-phase fraction of the aneuploid cell population (consisting of S- + G2/M-phase) was about 16.6%. 80% of the diploid biopsies of the tumor area showed an increased S-phase fraction (9.25%) compared to the biopsies of the tumor free prostate areas (5.9%). This distinction indicates a clearly increased rate of proliferation, of the diploid tumor cell population too.
Beside the observation of the heterogeneity we examined the effect of collagenase treatment during the preparation of single cell suspensions. Celi suspensions of 22 tumor biopsies were prepared in parallel (8-16h) with and without collagenase. Here we detected a preferential lost, e.g. a quantitative reduction of the aneuploid cell population in the suspensions prepared with collagenase. Because studies with other tumors showed similar effects, collagenase treatment should be avoided in general.
73. DNA-CYTOMETRY AND KARYOMETRIE OF CERVICAL INTRAEPITHELIAL NEOPLASIAS
M. Ruhnke, A. Coumbos W. Kühn
Department Gynecol. Obstet., Klinikum Benjamin Franklin, Free University of Berlin, Germany
DNA-cytometry and karyometry with image analysis are able to distinguish between high- and low-risk cervical smears. In the present investigation a comparison between cytological und histological specimen was done. 400 cervical smears and 78 biopsies of the uterine cervix were analysed with a CAS 200 image analysis system. On Feulgen stained nuclei integrated optical density (IOD), nuclear size, shape, blobness, entropy, and ploidy were measured. The mean values of the estimated parameters are given in Tab. 1:
sample| JOD | Size | Shape | Blob.|Entrop|
noCIN | 94.97 | 58.48 | 16.27 | 0.38 | 1.50 |
CIN 1 |109.55 | 55.60 | 16.35 | 0.38 | 1.49 |
CIN 2 |124.89 | 71.31 | 17.20 | 0.39 | 1.49 |
CIN 3 |182.92 | 82.19 | 18.56 | 0.41 | 1.48 |
Biological and prognostical differences became obvious regarding the relative number of cells with a DNA-content > 9c. In CIN 1 0.067% and CIN 2 0.16% of the measured cells are > 9c, in CIN 3 3.33% were > 9c.
This reflects the unfavourable prognosis of CIN 3 lesions and raises the question whether CIN 2 is a high-grade lesion or not.
74. EXPRESSION OF ADHESION MOLECULES OF THE IMMUNOGLOBULIN SUPERFAMILY ON HUMAN RENAL CARCINOMA CELL LINES
Schardt, C., Gerharz, CD., Gabbert, HE.
Institute of Pathology, Düsseldorf, Germany
23 human renal carcinoma (RCC) cell lines of the clear cell (n=19) and of the chromophilic (n=4) type were examined forthe expression of adhesion molecules including ICAM-1 (CD54), NCAM (CD56), MUC18 (melanoma progession associated antigen) and the HNK-1 epitope (CD57) by flowcytometric analysis and western blotting. All of these adhesion molecules belong to the immunoglobulin superfamily and share a various degree of homology on DNA and protein level. ICAM-1, widely expressed on normal and malignant epithelial cells, was found on 1 7 out of 23 RCCs with molecular weights ranging from 80-97kDa. NCAM, first described in embryonic and neuroendocrine tissue, was detected in 16 out of 23 cell lines. Surprisingly, only the 140 kDa isoform and not the often polysialated (embryonic) 180 kDa isoformwasfound. MUC18 is ubiquitously expressed on vascular smooth muscle cells and was first detected as a melanoma progression antigen. MUC18 was present in 9 out of 23 cell lines with a molecular weight of 1 35 kDa which is slightly above that found in malignant melanomas (113 kDa). The HNK-1 opitope described on NCAM and MUC 1 8 was missing on all cell lines. Clear cell RCCs were positive for ICAM-1 and NCAM in a higher percentage and with a stronger expression than chromophilic RCCs. 2 out of 4 chromophilic RCCs compared to 7 out of 19 clear cell RCCs stained positive for MUC18. Although RCCs are tumours of non-neuroendocrine origin, we could demonstrate de novo expression of the neuroendocrine marker NCAM. MUC18 expression which is restricted to vascular smooth muscle cells and melanomas. was demonstrated in RCCs for the first time.
75. MORPHOMETRIC AND DENSITOMETRIC IMAGE ANALYSIS ON BLADDER CARCINOMA CELLS STAINED WITH FLUORESCENT DYES
Wolfgang Schildbach, Elmar Endl, Pia Steinbach, Gabriele Fauser, Josef R.Aschoff and Ferdinand Hofstädter
Institute of Pathology, University of Regensburg,D-93042 Regensburg, Germany
The nucleolar organizer region (NOR-) prolein p120 is expressed during G1-, S- and G2-phase of the cell cycle. The expression seems to be associated with "hyperactivity" of the nucleolus and is discussed as an indicator of malignancy or proliferative activity. It therefore was the aim of our study to quantify the spatial distribution of p120 in different cell cycle phases and in several cell lines in vitro to find conelations to proliferative activity. The quantification was done by confocal laser scanning microscopy (CLSM) and computerized image analysis.
We developed a library of C++ classes which permits us to make a quantitative assessment of three-dimensional fluorescence patterns. Implemented are image analysis operations such as morphologic operators, morphometric and densitometric measurements, connected-components-algorithms and routines for convolution and deconvolution by fast fourier transform (FFT). For each application short user programs have to be created. These typically load the image data, segment the data and call connected-components-analysis. Finally for each separate object dfferent parameters such as area, form parameters, integrated fluorescence or properties of enclosed objects are measured.
For the present work, the three bladder carcinoma cells MGH-U1, RT4 and J82 of different malignancy grading were stained for p120 presence with fluorescein isothiocyanat (FITC). The nucleus was counter-stained with 7-aminoactinomycin D (7MD). We sampled fluorescence in two channels on the CLSM, using a 535 nm bandpass filter for FlTC-fluorescence and a 665 nm longpass filter for 7MD-fluorescence. Single nuclei were segmented using thresholding; werlapping p120 dots could be separated by preprocessing the FITC channel with a Marr-Hildreth-type convolution.
Applications linked with this library are capable of reliable automated analysis of CLSM images with separation and identification of subnuclear objects, e.g. p120 stained nucleoli.
Our data have been compared with parallel measurements conducted by use of multi-parameter flow-cytometry and cell sorting. We found the data obtained by analvzinq pre-sorted cells compatible with the underlying sort parameters.
76. ENDOMITOTIC CHROMOSOMES IN THE NURSE CELLS OF CHRYSOMYA RUFIFACIES
Michael Schöttke
Zoologisches Institut der Universität Kiel, Olshausenstr. 40, D-24098 Kiel
During oogenesis of the blowfly Chrysomya rufifacies (Diptera, Calliphoridae) the nurse cells multiply their DNAcontents by endoreplication. In stage 2 the nurse cell nuclei go through two endomitotic cycles which differ in the structure ofthe endochromosomes and in their DNAcontents. In the first endomitotic cycle "paired" chromosomes are visible which are characterized by a close association ofthe homologous chromatids. The "paired" chromosomes show a progressive condensation behaviour, leading into an endometaphase. Without an endoanaphase the chromosomes decondense, take on a reticular interphase structure and go through the next Endo-Sphase. Subsequently the second endomitotic cycle starts, in which the homologous chromatids are only loosely associated forming "bundeled" chromosomes. At the end of this second endomitotic cycle the "bundeled" chromosomes fall apart into the single chromatides and again take on a reticular interphase structure. Normally these two endornitotic cycles take place at the 32 C and 64 C level, but in some cases it occurs one endocycle earlier (16 C, 32 C) or later (64 C, 128 C). Together with the measurements of the whole nuclei it was possible to carry out single chromosome measurements. A comparison of the relative DNA-content of the six chromosome pairs between mitotic chromosomes (follicle cell metaphases and neuroblast metaphases) and the endometaphases gave no hint for a differential replication behaviour of the endochromosomes at this early stage of oogenesis.
77. TELEMICROSCOPY - A TOOL FOR THE REMOTE EVALUATION OF CYTOLOGICAL MATERIAL
Schwarzmann P., Schmid J., Schnörr C., Binder B.
Inst. für Physikalische Elektronik, University Stuttgart,FRG
Telemicroscopy allows the local separation of microscope equipment and user resp. an image evaluation facility. The technique is suitable to share expensive mircroscope equip- ment beetween different user group and to make available at the microscope site the expertise of remote specialists what is important for instance for telepathology. Dependent of the allowable costs all types of telecomnunication channels may be used beginning at single ISDN-telephone lines, bundled ISDN Iines, Ethernet, FDDI until to broadband ISDN-connections (ATM-nets) with realtime capabilities. In telemicroscopy a high impression of online and realtime capability, is favourable to create an impression of telepresence. This holds not only for image transmission but also for a perfect handling of the microscope functions like exchange of magnification, focusing, movements of the object on the scanning stage, illumination, camera operation etc. At the authors site systems for broadband and narrow band technology have been realized. The equipment is evaluated now in scientific projects (ultraviolett miroscopy) and in a clinical field test in telepathology. The tool allows it to add to the two already present partners - the surgeon and ths pathologist - facilities for quantitative image evaluation without the necessity that all services are available physically at the same hospital. The project is funded by the Robert Bosch Foundation and the Deutsche Telekom AG.
78. FLOW CYTOMETRIC ANALYSIS OF CELL CYCLE PERTURBATIONS AND MlCRONUCLEI AFTER UV B- AND gamma-IRRADIATION
Weller E.M.1, Hain J.1, Jung T.1, Kinder R.2, Köfferlein M.3, Burkart W.1 and Nüsse M.2
1BfS-lnst. f. Strahlenhygiene, 2GSF-AG Durchflusszytometrie, 3 GSF Inst. f. Biochemische Pflanzenpathologie, 85762 Oberschleiss- heim, Germany
Perturbations of the cell cycle after UV B- and gammairradiation of asynchronous human SCL-2 keratinocytes were analysed over two cell cycles using BrdU/Hoechst flow cytometry. Exponentially growing keratinocytes exposed to UV B radiation (lambda> 295nm) of 800 J/m2 were temporarily blocked in S and G2 phase of the first cell cycle. Subsequently, progression through the second cell cycle was delayed. The cell kinetic perturbations increased with the UV B dose. Irradiation with wavelengths X > 335 nm (UV-A) did not have any detectable effect on cell cycle progression. In contrast, 137Cs gammairradiation (4 Gy) caused a temporary G2 block only. SCL-2 cells did not show a Gl arrest after both types of irradiation due to lack of wilde-type p53 (Hain et al., submitted). Micronucleus frequency in gamma-irradiated cells increased as soon as the cells started to divide and reached a plateau when most of the cells had divided. Continuous treatment with caffeine (1 mM) starting directly after 137Cs irradiation prevented accumulation of cells in G2 phase, but did not alter the frequency of micronuclei. In UV B-irradiated keratinocytes, however, the damage-induced S and G2 blocks were merely reduced by caffeine, but not eliminated. Compared to gamma-irradiation a moderate increase of micronuclei was observed in UV B exposed cells. Caffeine, however, potentiated the in- duction of micronuclei by UV B. These different effects on cell cycle kinetics and micronucleus induction indicate different mechanisms of DNA damage caused by UV B and ionizing radiation that may be repaired at least partially through different signal transduction pathways.
79. FLOW SORTING OF BACTERIA FOR PCR-ANALYSIS
G. Wallner1, S. Spring2, B. Fuchs2, W. Beisker1, and R. Amann2
1 GSF-Forschungszentrum, AG Durchflußzytometrie, D-85764 Oberschleißheim, 2 TU München. Lehrstuhl fur Mikrobiologie, D-80290 München
We sorted bacteria from complex mixed populations in natural samples for the amplification of rDNA (coding for ribosomal RNA) by PCR and subsequent analysis by DNA sequencing or filter hybridisation.
Very large magnetotactic bacteria (> 10 um) in magnetic enrichments from lake sediment were separated from smaller cells and inhibitory sediment components by flow sorting, making use of their strong forward and side scatter signals. The sorted cells were used directly for PCR amplification and cloning of 16S rRNA genes. Comparative sequence analysis rcvealed their phylogenetic position even though they could not be cultivated.
In more complex populations fluorescence in situ hybridisation with rRNA-targeted probes can be used to characterise specific subpopulations. For example, we could identify certain phylogenetic groups of bacteria in activated sludge by rRNA-targeted probes and sort them for PCR and dot blot hybridisations.
The combination of flow sorting and PCR allows the molecular analysis of subpopulations with selected properties in complex microbial communities without the need for cultivation.
80. OXIDATIVE STRESS AND NO* INDUCE APOPTOSIS IN RAT MYOGENIC CELLS
U.K.Zettl1,2, E.Mix1, J.Zielasek2, M.Stangel3, H.Meyer- Rienecker1, K.V.Toyka2, H.-P.Hartung2, R.Gold2
1Dept. of Neurology, Hosp. Nerv. Dis., Universität Rostock, 2Dept. of Neurology, Universitat Würzburg, 3Dept. of Neurology, Freie Universität Berlin, Germany
Reactive oxygen species (ROS) and nitric oxide (NO*) have a pivotal role for tissue damage, tissue regeneration and defense against infections. In this study we investigated, whether exposure to ROS and NO*causes apoptosis of myogenic cells. Skeletal myoblasts obtained from adult Lewis rats were cultured and allowed to fuse to myotubes. Cells were exposed to the ROS H202, the ROS generating system hypoxanthine/xanthine oxidase (HX/XO) and the penicillamine-derived NO* liberator SNAP. Apoptosis was assessed by morphological criteria and DNA gel electrophoresis, and estimated quantitatively by molecular labeling, i.e. by tailing and FACS analysis (for myoblasts) and in-situ nick translation (for myotubes). After 16h exposure to H202 (10-5- 3x10-4M), HX (10-4 -5x10-4M) and SNAP (10-3 - 5x10-3M) a dose-dependent increase of the number of apoptotic myoblasts was observed with a maximum of 40% for the highest concentrations of H202 and SNAP. Apoptosis was specifically inhibited by catalase and hemoglobin, scavengers of H202 and NO', respectively. Apoptosis of myotubes was obselved at 3x1 0-4M H202. It is concluded that exposure to ROS and NO' causes apoptosis of myogenic cells, which may contribute to muscle cell damage in inflammatory and degenerative myopathies.


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1965-2006: Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany
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