Danish Society for Flow Cytometry

Dansk Selskab for Flowcytometri

The 26th meeting of the Danish Association for Flow Cytometry will be held with the Immunological Society in Auditorium 2, Søauditoriet, Aarhus University ( see map ), on Thursday, 6th September 2001:

”Flow Cytometry at the dawn of the third Millenium

Please register for attendance by sending an e-mail to dsfcm@dadlnet.dk with "26 meeting" in the title line, or by phone (+45 89492107) by 31.August 2001 as we would like to order lunch and coffee for you.

 

11.10 – 12.10

 

12.10 – 12.40

12.40 – 13.00

13.00 – 13.20

Unravelling the TH1/TH2 paradigm

Hans Jürgen Hoffmann (morning chairperson): Welcome

Mario Roederer (Vaccine Research Center, NIAID, NIH): The rich heterogeneity of the peripheral immune system: Identifying basic components of T cell immunity (Sponsored by Serotec)

Detlev Loppow (Krankenhaus Grosshansdorff, Hamburg): Flow cytometric analysis of induced sputum – from cell differentials to intracellular cytokine profiles. (Sponsored by BD)

Katja Adolf (Dept Respiratory Medicine, AUH): T-cell phenotyping and surface marker expression in a prospective study of sensitization to flour of a cohort of baker apprentices

Jan Christensen (Panum Institute, University of Copenhagen): IL-2 expression as a marker of in vivo CD8+ T cell differentiation during viral infection

13.20 to 14.00

Lunch (sponsored by BD) and poster/exhibition viewing

 

14.00 – 14.20

14.20 – 14.40

 

14.40 – 15.00

15.00 – 15.20

Free Communications

Chairperson: Jørgen K. Larsen

Peter Hokland (Dept Hematology, AUH): Flow cytometric identification of myelopoiesis in health and disease employing CD13, CD14 and CD66 monoclonal antibodies

Christine Dahl (Dept Paediatrics, AUH): Culture of functional human Mast cells. Immunopenotypic Analysis of Differentiating Cord Blood Derived Cultured Human Mast Cells

Carolin Post (Finsen Lab, NUH, Copenhagen): Flow cytometric bivariate analysis of DNA and cytokeratin in colorectal cancer.

Jacob Larsen (Finsen Lab, NUH, Copenhagen): Cytogenetic analysis of DNA aneuploid subclones in mammary carcinomas using fluorescence activated cell sorting and comparative genomic hybridization (sponsored by RAMCON)

15.20 – 15.50

Coffee Break (sponsored by DAKO)

 

15.50 – 16.10

16.10 – 16.30

16.30 – 16.50

16.50 – 17.10

Hardware of the Future

Chairperson: Jørgen K. Larsen

Frans Nauwelaers (Beckton Dickinson): New Development in Cytometry

Anny Thews/Martin Adelman (Beckman Coulter): Visions in Beckman Coulter

Anders Pedersen/Matt Ottenberg, (DAKO/Cytomation): Next Generation Flow Cytometry Systems

Jeff Harvey/Bill Staffopoulos (Guava Technologies): Introducing the Guava Personal Cytometer

17.30 ?

Aarhus Festival Week

Wine for the speakers and chairpersons is sponsored by RAMCON.

Train connections:         Intercitylyn 23   at 7.44 from Copenhagen arrives in Århus at 10.44

                                    Intercity 121     at 7.00 from Copenhagen arrives in Århus at 10.08

The train ticket is valid on bus 14 (10.53) or 3 (11.01) from the Station to the University (Kommunehospital). The Auditorium complex is the new yellow brick building across the road from the bus stop.

                                    Intercities         at 18.02 … 22.02                       from Århus to Copenhagen

                                    Intercitylyn 54   at 17.30                                    from Århus to Copenhagen

Instead of rushing home, visit the Århus Festival, see www.aarhusfestuge.dk for a programme

 

 

ABSTRACTS

 

Unravelling the TH1/TH2 paradigm

 

1. The rich heterogeneity of the peripheral immune system: Identifying basic components of T cell immunity.

Mario Roederer, Vaccine Research Center, NIAID, NIH.

Over the past 7 years, we advanced multicolor flow cytometric technology to simultaneously measure 14 different parameters from every cell (12-color FACS). Using this technology, we can now identify at least 6 distinct lineages of T cells in blood as well as from 4 to 12 distinct differentiation stages for each lineage. Each of these 50+ phenotypically-distinct subsets has a unique functional profile, such as proliferative capacity, cytokine pro-file, cytolytic activity, and apoptotic potential. We are trying to identify the complete functional repertoire of these subsets.

This technology has already proven invaluable in under-standing the immunopathogenesis of disease. For example, we identified CD4 memory subsets with polarized cytokine profiles that are significantly elevated or reduced in classical Th1 (tuberculoid leprosy) or Th2 (lepromatous leprosy or atopy) diseases. We concluded that the functional polarization of the peripheral immune system in these diseases is a consequence of homeostatic or differentiation processes, rather than a change in the functions of individual T cells. We are extending our studies to determine the detailed cytokine profile of these subsets to understand the potential impact on immune function of changes in representation during pathogenesis.

We also identified what may be extrathymically-derived T cells becoming prevalent in the blood of subjects with distinct pathologies (e.g., BMT, chemotherapy, or HIV disease). The origin and function of these cells are poorly understood. However, it may be crucial to immune reconstitution in immunodeficiency, where the normal T cell compartment has been ravaged. Importantly, we identified antigen-specific reactivities within this extrathymic compartment; it remains to be determined whether the functional response is protective, or, as for anti-tumor responses, anergic.

It is clear that the immune system is extremely complex, comprising of at least sixty functionally and phenotypically distinct lymphocyte subsets. Paradoxically, while it seems that the ability to identify so many cell types is a significant complication, the unique identification of specific subsets clarifies immunopathogenesis significantly. The technology allows us to focus on only the relevant subsets, while ignoring the inter-subject variation in the vast majority of cells from other subsets–a variation that only adds significant noise to bulk measurements made by the typical 3- or 4-color flow cytometers.

 

2. Flow cytometric analysis of induced sputum - from cell differentials to intracellular cytokine profiles

D. Loppow1-3, G. Gercken3, H. Magnussen1, R.A. Jörres1,3

1) Krankenhaus Großhansdorf, Zentrum für Pneumologie und Thoraxchirurgie, D-22927 Großhansdorf, Germany; 2) Gemeinschaftspraxis für Laboratoriumsmedizin Dr. Kramer und Kollegen*, Lauenburger Str. 67, D-21502 Geesthacht; 3) Universität Hamburg, Institut f. Biochemie u. Lebensmittelchemie, Abt. f. Biochemie u. Molekularbiologie, D-20146 Hamburg

Sputum induction by inhalation of ultrasonically nebulised saline is a noninvasive method to obtain cellular and biochemical components from the airways and the lung. Consequently, it is increasingly used as an alternative to the invasive bronchoalveolar lavage (BAL). As quality and quantity of recovered cells decrease from blood to BAL fluid to sputum, flow cytometric analysis of induced sputum (iSP) poses a special methodological challenge.

As a first step we established a protocol for leukocyte cell differentiation that was feasible within a routine laboratory setting. iSP of 49 patients was analysed by flow cytometry (CD45 vs. SSC) and compared to microscopic data from May-Grünwald-Giemsa stained cytospin preparations. The detection of eosinophils turned out to be the major difficulty. Therefore, several approaches for assessing eosinophils were evaluated in comparison to microscopical results. The measurement of depolarised sideward scatter yielded the best results and was superior to the combination of CD49d and CD16 expression or autofluorescence. However, more work seems to be needed to improve the reliability within the low range of eosinophil percentages (1-3 %).

Since lymphocytes are key players in the immune system, we subsequently focused on lymphocyte subtyping as usual in BAL fluid. This was performed in iSP from 37 patients. Percentages of T, B, and NK cells as well as check sums were similar to BAL data, both from our own laboratory and literature. Therefore, subtyping of lymphocytes appears to be a valid and reliable method in induced sputum.

In addition to cell counts, cell function is considered to be an important determinant of disease processes. For this purpose, we established the determination of intracellular cytokine profiles within sputum T lymphocytes after stimulation with PMA and Ionomycin. Preliminary results on IFN-g , IL-2, IL-4, and IL-5 showed unexpected distributions of TH1- and TH2 cytokines produced by CD4- as well as CD8-positive cells. The opportunity to assess these profiles within the blood, the BAL fluid, and the induced sputum offers new perspectives for the regional assessment of immune function.

Supported by LVA Hamburg and Christiane Herzog Stiftung

*LADR Laborärztliche Arbeitsgemeinschaft für Diagnostik und Rationalisierung e.V.

 

3. T-cell phenotyping and surface marker expression in a prospective study of sensitization to flour of a cohort of baker apprentices

Katja Adolf, Department of Respiratory Diseases, Århus University Hospital, Århus C, Denmark

Baker’s asthma is used as a model of sensitization and type I allergy.

The expression of 22 surface markers, mainly on CD4+ T-cells, is studied by 4-colour flow cytometry on 268 blood samples obtained from 163 persons over a period of 2 years during their first occupational exposure to flour. The aim of the study is to describe T-cells in the process of sensitization and find possible differences in the development of allergic symptoms like asthma and rhinitis dependent on different distributions of subpopulations of Th-cells and intrinsic properties of the immune system between healthy controls, persons heredetary predisposed for allergy and persons with allergic/non-allergic symptoms from lungs, nose and eyes. The suitability of a range of markers to distinguish Th1-/Th2-/regulatory Th-subsets is tested.

PBMC are stained freshly isolated and after priming for Th0/Th1/Th2-type in whole PBMC cultures.

Results: Usage of CD11a, CD62L, CD45RA and CD4 for Th1/Th2 typing1 has proven useful. CD26, CD195 (CCR5), CD223 (LAG-3), CD183 (CXCR3) are associated with a Th1 type, while CCR3 is associated with a Th2 type. Data analysis of the expression of IL18R, CD152, CD184 (CXCR4), CD213a, CDw137, CD154, ST2L, CD57 and CD134 is currently in process. Also, allergenspecific proliferation and cytokine production of PBMC cultures after unspecific stimulation is assessed in other experiments and provides additional information to determine the activation state of the immune system. Clinical, genetical, environmental and microbiological data of the participants are recorded in collaboration and correlation to the immunological results is investigated.

1) Mitra D.K., De Rosa S.C., Luke A., Balamurugan A., Khaitan B.K., Tung J., Mehra N.K., Terr A.I., O'Garra A., Herzenberg L.A., & Roederer M. (1999) Int.Immunol. 11, 1801-1810.

 

4. IL-2 expression as a marker of in vivo CD8+ T cell differentiation during viral infection

Jan Pravsgaard Christensen, Nanna Ny Kristensen, and Allan Randrup Thomsen. (J.Pravsgaard@immi.ku.dk). Institute of Medical Microbiology and Immunology, University of Copenhagen, DK-2200 Copenhagen N, Denmark

Using infection with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus as model systems, we have investigated the ability of antigen-primed CD8+ T cells generated in the context of viral infections to produce IL-2. Our results indicate that acute immunizing infection normally leads to generation of high numbers of antigen-specific CD8+ T cells with the capacity to produce IL-2. By costaining for IL-2 and interferon (IFN)-g intracellularly, we find that IL-2 producing cells predominantly constitute a subset of cells also producing IFN-g. Comparison of the kinetics of generation reveals that IL-2 producing cells appear slightly delayed compared to the majority of IFN-g producing cells. However, the IL-2 producing subset is preferentially maintained with transition into the memory phase. In contrast to acute immunizing infection, few IL-2 producing cells are generated during chronic LCMV infection. Furthermore, in MHC class II deficient mice which only transiently control LCMV infection, IL-2 producing CD8+ T cells are initially generated, but with time this subset disappears. Eventually also the capacity to produce IFN-g becomes impaired while cell numbers are maintained at a level similar to that in wildtype mice controlling the infection. Taken together these findings indicate that phenotyping of T cells based on their capacity to produce cytokines and especially IL-2, can provide important information as to the functional status of the analyzed cell subset. Moreover, combined analysis of the capacity to produce IL-2 and IFN-g can be used to define distinct stages in the development of anergy vs. memory.

 

Free communications

 

5. Flow cytometric identification of myelopoiesis in health and disease employing CD13, CD14 and CD66 monoclonal antibodies

P Hokland & K Meyer. Department of Medicine and Hematology, Aarhus University Hospital, DK-8000 Aarhus C, Denmark.

Immunophenotyping employing monoclonal antibodies against leukocyte differentiation antigens constitutes a valuable tool for identifying immature hematopoietic cells. On the other hand, the definition of various stages of myelopoiesis by this methodology is less well documented. We have designed a flow cytomeric assay in which the CD13 antigen is using for detection of immature cells and the CD66 antign for mature. In addition, CD14 is used for monocyte identification. Using a multiparameter flow cytometry assay enumerating cells positive for CD13, CD14 and CD66 antigens we determined the asynchronous CD14/CD66 co-expression in unselected bone marrow and peripheral blood samples with suspected malignant blood disorders. CD14/CD66 co-expression > 5% were found in 131/691 bone marrow samples. Only 55 of these exhibited an identifiable population in two-parameter flow cytometry histograms. Of the 55 samples 43 (78%) came from patients with myeloid disorders; e.g. 11 with myelodysplastic syndromes, 15 with chronic myeloproliferative disorders and 17 with acute myeloid leukemia. Only one of these 17 cases was a de novo case, while 8 were secondary to another malignant hematological disease and 8 were from the period after cytoreductive therapy. Notably, CD14/CD66 co-expression patterns were related to disease categories; e.g. in chronic myelomonocytic leukemia and acute myeloid leukemia following a dysplastic phase the co-expression displayed two subsets in peripheral blood, low-avidity CD14 and low-avidity CD66, respectively. The latter disease category also exhibited these two subsets in bone marrow. In all other cases, the CD14/CD66 co-expression in bone marrow was heterogeneous. In conclusion, abnormal CD14/CD66 co-expression might be a valuable parameter in defining asynchronous myelopoiesis in malignant myeloid disorders, especially myeloproliferative disorders and secondary acute myeloid leukemias.

 

6. Culture of functional human mast cells: Immunophenotypic analyses of differentiating cord blood derived cultured human mast cells.

C Dahl*, HJ Hoffmann†, HV Nielsen*, H Saito‡, PO Schiøtz*, *Dep. of Pediatrics, Aarhus University Hospital, Denmark, †Dep. of , Aarhus University Hospital, Denmark, ‡National Childrens Hospital, Tokyo

Background: To study factors affecting the development of human mast cells and mast cell function different methods for culturing human mast cells have been published. Here we present a method for culturing large numbers of pure and functionally mature human mast cells using a serum deprived culturing system.

Materials and methods: Human umbilical cord blood samples were obtained into heparinized syringes from normal full term deliveries following informed consent from the mothers. CD34+ cells were separated from the mononuclear cell layer using a magnetic cell separation system (MACS-system). The CD34+ cells were cultured in serum-free media (StemSpan, Stem Cell Technologies, Canada) for eight weeks. Media were supplemented with adequate concentrations of stem cell factor, IL-6, IL-3 (first two weeks only), 1% penicillin-streptomycin and 5 x 10-5 M mercaptoethanol. From 8 weeks cells cultured in serum-free media were passed into a serum containing media. Monoclonal antibodies used for mast cell immunophenotypic analyses were CD34(BIRMA-K3), CD117(104D2), CD33(WM53), FcεRI-α(CRA-1) and CD13(WM47). Negative controls were done using isotype matched IgG antibodies. Cells were analysed using a FACSCalibur flow cytometer. IgE-dependent histamine release was performed.

Results: Using the serum-deprived culture system up to eight weeks and there after passing the cells on to FCS containing medium 100% pure and functionally mature human mast cells developed. After ten weeks of culture 100% of the cells stained positive with Alcian blue (n=5). They released 45 % of their histamine upon anti-IgE stimulation (10 μg/ml). Mast cell CD34+ precursors coexpressed the pan myeloide antigen CD13 (81,96%+/-8,79 SD) and were positive for the c-kit receptor CD117 (88,2%+/- 8,7 SD), (n=3). After 10 weeks of culture the cells were positive for CD117 (96%+/-3 SD), the myeloid cell marker CD33 (94,78+/-4,0 SD) and cells were found positive for the high affinity IgE receptor FcεRI (28,6%+/ 3,47 SD).

Discussion: Serum is an extremely complex solution and the quantity and quality of this solution is subject to significant
biological variation. Our serum free procedure gives a high yield of human mast cells applicable for various functional assays and using the serum-deprived culture conditions the reproducibility of cell growth improves. We thereby create well-defined culture conditions providing a constant high yield of functional human mast cells and a source for studies of the human mast cell.

 

7. Flow cytometric bivariate analysis of DNA and cytokeratin in colorectal cancer

Carolin Post1, Ib Jarle Christensen1, Henrik Flyger2, Jette Christiansen1 and Jørgen K. Larsen1

1Finsen Laboratory, Finsen Center, Rigshospitalet, Copenhagen, and 2Department of Surgery, Hillerød Hospital, Hillerød, Denmark

There is some debate whether flow cytometric estimates of DNA aneuploidy and/or S-phase fraction (SPF) are useful as supplementary prognostic markers in colorectal cancer. The different conclusions are to some degree associated with the methodologies applied. Using flow cytometric univariate DNA analysis, we have previously investigated the DNA ploidy in colorectal cancer, its heterogeneity within and between tumors and its relation to survival (Flyger et al. 1999, Cytometry 38:293). In order to improve the detection of DNA aneuploid subpopulations and particularly the estimation of their SPFs, we then investigated a method for flow cytometric bivariate analysis of cytokeratin and DNA content. Bivariate DNA/cytokeratin histograms were obtained from fine-needle aspirates of 728 frozen biopsies from 157 colorectal tumors. The aspirates were stained with propidium iodide and FITC-conjugated anti-cytokeratin antibody in a buffer with 0.3% saponin for cell permeabilization. Good quality DNA histograms with low CV, debris and a sufficient number of counted nuclei were selected. The SPF was adjusted to minimize the influence of debris. There were no substantial difference in the estimated DNA ploidy patterns between univariate and bivariate measurements (concordance 92-95 %). The SPFs of cytokeratin-positive histograms were significantly higher than those of ungated histograms, also when DNA aneuploid subpopulations were considered (p < 0.0001). We were not able to demonstrate a prognostic value of SPF i colorectal cancer.

 

8. Cytogenetic analysis of DNA aneuploid subclones in mammary carcinomas using fluorescence activated cell sorting and comparative genomic hybridization

Jacob Larsen1, Claes Lundsteen2, and Jørgen K. Larsen1, 1Finsen Laboratory, Finsen Center, and 2Dept. of Clinical Genetics, Juliane Marie Center, Rigshospitalet, Copenhagen, Denmark

New insights in carcinogenesis may be generated by cytogenetic analysis of subpopulations separated from heterogeneous tumor tissue. As a strategy for cytogenetic analysis of the clonal heterogeneity in tumors we combined the following methodologies: 1) fluorescence activated cell sorting (FACS) of cell subpopulations according to flow cytometric DNA ploidy distribution, 2) whole genome DNA amplification (DOP-PCR) on sorted nuclei, and 3) high resolution comparative genomic hybridization (HR-CGH) for the detection of chromosomal regions with copy number imbalances. This strategy was applied to a small series of mammary carcinomas.