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 Mario Roederer ( Detlev Loppow
( 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, |
|
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 Christine Dahl (Dept Paediatrics,
AUH): Culture of functional human Mast cells. Immunopenotypic
Analysis of Differentiating Cord Blood Derived Cultured Human Mast Cells |
|
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 |
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17.30 –
? |
|
Wine for the speakers and chairpersons is sponsored by RAMCON.
Train connections: Intercitylyn 23 at
7.44 from
Intercity
121 at 7.00 from
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
Intercitylyn 54 at
17.30 from
Århus to
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,
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
*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,
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,
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,
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‡,
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,
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
1Finsen Laboratory,
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
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.
