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 (
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.
Questions and comments to:
G.K.Valet, E-mail: valet@vms.biochem.mpg.de, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany, Tel: +49/89/8578-2518, -2525, Fax: +49/89/8578-2563, INTERNET address: http://www.biochem.mpg.de/valet/dgz.html
Last edit: Oct.16, 1996
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