Andreas W. Machl, Simone Planitzer, Manfred Kubbies
Boehringer Mannheim GmbH, Biotechnology Therapeutics Research Center, 82377 Penzberg, Germany
Retroviral vectors are effective shuttle systems by introducing therapeutically relevant genes stable into the genome of proliferating cells. The majority of vectors applied for research or clinical applications use neomycin for cell selection and identification. To circumvent the time consuming and potential toxic G418 selection process in transduction studies we constructed a novel marker vector using l-NGFR as cell surface marker to identify DNA- repair defective Fanconi Anemia cells complemented with the FAC-gene. The new vector constructed is based on a MoMuLV backbone, a signal peptide deleted l-NGFR receptor gene under control of a LTR-promoter and the therapeutically relevant FAC gene placed downstream of a SV40 promoter. Supernatants containing high titers of amphotrophic viruses from FACS cloned cell cultures were obtained and tested for primary transduction rates, rapid detection of transduced cells within 48 hrs and correction of mitomycin C induced cell cycle G2 phase accumulation in a single assay using multiparameter, dual laser flow cytometry. Primary transduction efficiency detected via
9. ANEUPLOIDY OF HUMAN GRANULOSA CELLS IN FOLLICULAR FLUIDS
OF IN-VITRO FERTILIZATION (IVF) PATIENTS
P. Melsheimer1, K. Grunwald2, K. Feldmann2, K. Klinga2, T. Rabe2, B.
Runnebaum2, H.H. Rummel1
1 Abteilung fur Gynäkologische Morphologie, Universit&aauml;ts-Frauenklinik,
Vossstr. 9, D-69115 Heidelberg,
2 Abteilung fur Gynäkologische Endokrinologie, Universitäts-Frauenklinik
Heidelberg
ABSTRACT:
OBJECTIVES: The aim was to study the proliferational behaviour of granulosa
cells found in follicle fluids of patients after hormone stimulation in the
framework of in vitro-fertilization with gonadotropins.
STUDY-DESIGN: The deoxyribonucleic acid ploidy and the proliferation
indices of granulosa cells in fresh and unfixed follicles (n= 119) of gonadotropin
stimulated patients (n=32) were analysed by flow cytometry, using a
standardised protocol for high-resolution analysis of nuclear DNA content
employing the fluorochrome DAPI. In the follicle fluids the concentrations of
testosteron, DHEAS, estradiol, progesteron, LH, FSH, Prolactin was analysed.
RESULTS:
Aneuploid cells were found in a large number of follicles (65/119)
as well as patients (25/32). A small number of follicles (8/119) and patients
(7/32) even contained multiploid cells. There was no correlation between
proliferation indices and ploidy. Granulosa cells were the predominant part of
cells in the follicle fluids. No malignant cells were found in any case. There was
a significant lower concentration of testosteron, progesteron and DHEAS in the
follicle fluids with aneuploid cells.
CONCLUSION:
This is the first report concerning the high incidence rate of
aneuploidy in ovarian granulosa cells in IVF patients. The clinical relevance of
the phenomena is not clear. There should be further study to find out wheather
there is any link to a previously discussed possible relation between
gonadotropin stimulation in women trying to have children and the occurrence
of ovarian cancer or granulosa-cell tumours. Of further interest might be a
possible relation between ploidy and proliferation indices of stimulated
granulosa cells as well as on side effects of gonadotropin therapy and
biological parameters like maturity, fertilizability of oocytes and rates of
pregnancy.
10. THE INCIDENCE OF ANEUPLOIDIES IN MULTIPLE MYELOMA DOES NOT
CORRELATE WITH STAGE OF DISEASE
R. Nowak, U. Oelschlägel, U. Range and G. Ehninger
Medical Clinic I, Technical University Dresden, 01307 Dresden
The multiple myeloma (MM) is a disease with abundant occurence of
aneuploidy in malignant cell population.
Concerning the relationship between stage of the disease and occurence of
aneuploidies contrictory flow cytometric results have been reported. Even in
monoclonal gammopathy of undetermined significance (MGUS) the
percentage of flow cytometrically detected aneuploidies varied between 0%
and 50%. One cause of these discrepancies is the low percentage of plasma
cells in early stages of this disease. With simultaneous measurement of DNA-
content and CD38 or B-B4 antigen expression aneuploidies could also be
recognized in low numbers of bone marrow plasma cells. Investigating 60
patients with a monoclonal serum component we have found no differences
between the stages of this disease and the incidence of aneuploidies (Tab).
=========== MGUS Multiple Myeloma
=========== Stage I Stage II Stage III
No. of patients 11 15 6 26
Incidence of aneuploidy 5 (45%) 12 (80%) 4 (66,7%) 16 (61%)
Correlation Coefficient 0.886 0.7971 0.7903 0.1630
CD56+/Aneuploidy
p-Value for Correlation <0.001 <0.003 0.210 0.1630
In accordance to other authors, the percentage of aneuploid plasma cells
correlated with the expression of CD56 in plasma cells. But there were great
differences of this correlation between the different stages. Whereas in early
stages the CD56 positivity corresponded with the percentage of aneuploid
plasma cells no correlations could be found in the later stages. The clinical
importance of the subgroup of CD56 negative aneuploid plasmocytomas has
to be tested.
11. NON-SYNGENEIC CELL MODELS FOR SCREENING OF DNA-REPAIR
RELEVANT GENES IN FANCONI'S ANEMIA VIA DIFFERENTIAL DISPLAY
TECHNIQUE
Simone Planitzer, Andreas W. Machl and Manfred Kubbies
Boehringer Mannheim GmbH, Biotechnology Therapeutics Research Center,
82377 Penzberg, Germany
Fanconi anemia (FA) is a DNA-repair deficiency syndrome. In vitro FA cells
show a significant accumulation in G2-phase and only modest, if at all,
alteration of cell activation (G0/G1-delay). One gene (group C) out of at least
five complementation groups has been cloned recently.
More recently a new
molecular approach was developed comparing mRNA expression between the
mutant cell line and a syngeneic control. Using the statistical approach via
differential display technique, within a short period of time almost all mRNA
species can be compared using statistical primers. However, for studying
DNA-repair defective mutant cells from FA, no syngeneic cell model is
available. Theoretically it could be expected that many different bands show up
due to diversity of untranslated regions. After screening of about 13000 c-DNA
bands, only 0,5 % were found to be different expressed between FA and
control cells. The fragment length varied between 350 and 900 bp, and 25
potential FA candidate c-DNA bands have been excised and amplified.
However, to minimize false positive/negative c-DNA bands due to cell cycle
differences, high resolution flow cytometric BrdU/Hoechst quenching technique
was applied for adjustment of cell kinetics of FA and control cells. For rapid
screening of complementation, FA c-DNA candidate genes will be cloned into
a GFP vector (green fluorescence protein), and cell cycle alteration of GFP
transfected cells will be analyzed by dual laser flow cytometry (GFP versus
Hoechst 33342).
12. ALTERATIONS IN CELLULAR MITOCHONDRIAL POTENTIAL UNDER
INFLUENCE OF APOPTOSIS INCUCING DRUGS
Pogrebniak A, Stoetzer OJ, Wilmanns W, Nüssler V
GSF-Research Center for Health and Enviroment, Institute for Clinical
Hematology, Munich
Background:
Mitochondrial membrane potential (MMP) is an indirect indicator
for mitochondrial function (oxygen utilisation). Pertubations in mitochondrial
function are often involved in the process of cellular proliferation and
programmed cell death. Furthermore mitochondrial membranes are the
intracellular sites of oxygen radical generation. Oxidative stress is responsible
in part for programmed cell death after cytotoxic drug treatment.
Methods: MMP determintation was performed using two different stains
namely 5,5',6,6 tetrachloro-1,1',3,3 -tetraethylbenimidazolcarbocyanine iodide
(JC-1) and rhodamin-123. JC-1 and rhodamine staining intensity correlates
with grade of MMP polarisation. Cells were incubated 30 at 37 C and then
analysed with a FACScan using Lysis II software. Photomultipliers were FL1
for rhodamin-123 resp. FL1 and FL2 for JC-1, but only FL2 values were
relevant for estimation of MMP with JC-1. Four cell lines (HL-60, Jurkat,
Carpas, K562) were incubated with etoposide (50-100 umol) for 12hrs.
Forward- and side scatter were used to gate out cellular debris.In one part of
our experiments cell viability was estimated with Propidium Iodide (PI). Peak
channel and mean values were used for determination of fluorescence
intensity (FI).
Results: Dot blot FSC/SSC showed two distinct subpopulations: one with high
FSC (1) and another with lower FSC and/or high SSC (2) dependent to cell
line. Population 1(P1) was entire PI-negative and showed higher fluorescence
intensity than P2. Fluorescence intensitiy in PI-negative cells from P2 was as
high as the FI of dead cells. Therefore we decided to concentrate on Pl. MMP
uncouplers (CCCP) induced a decrease in JC-1 and rhodamine fluorescence,
whereby JC-I decrease was faster and stronger.
After 7hrs of etoposide incubation FI of JC-1 was increased and remained
increased until cell death occured. This phenomenon was observed in all four
cell lines. A slight increase in rhodamine FI after etoposide treatment was seen
only in K562 an Jurkat, whereas in Carpas and HL-60 rhodamine data were
inconsistent. This was probably due to rhodamine induced alterations in
FSC/SSC, which made discrimination between P1 and P2 difficult.
Conclusions: Contrary to other investigators we determined an increased
MMP in cells after lethal etoposide treatment. That might be due to our
improved gating technique, which allowes us to discriminate homogenous
functional active subpopulations instead of observing an entire PI negative cell
population. In our hands JC-1 data appeared to be more reliable than
rhodamine data because of faster and stronger onset of FI alterations.The
alterations might be due to etoposide action because VP-16 is only cytotoxic
for K-562 cells and does not evoke apoptosis.
13. DNA DIPLOIDY - A RARE FEATURE OF OVARIAN CARCINOMA?
Reich O, Pürstner P.
Department of Obstetrics and Gynecology, University of
Graz, Austria
Background: In ovarian carcinoma several studys have suggested that DNA
diploidy is associated with grading I tumors. In contrast. DNA non-diploidy was
found in grading II/III tumors. However, the evaluation of DNA ploidy is directly
depending on the measuring accuracy(1). Our aim was to investigate wether
DNA diploidy is detectable by using high resolution flow cytometry.
Method: Fresh tumor tissue in 33 patients with ovarian carcinoma were
analyzed (26 serous, 3 endometrioid, 2 mucinous, 2 clear cell; 5 grading I, 12
grading II, 16 grading III/IV). 3-8 (mean 5) tumor biopsies per case were frozen
up to 30 minutes after operation. For control, tumor tissue slides were stained
with hematotoxylin and eosin. For DNA analysis, a PAS III cytometer was
used. The cells were stained with DAPI according to Göhde and Otto. The
degree of ploidy was given by the DNA index, defined as the population of
DNA in the G1 cell peak channel compared to the DNA content of known
diploid human lymphocytes. The mean coefficient of variation for all G1 peaks
was smaller than 3%. The following ploidy groups were discerned: diploid (DI:
0,97-<1,03), near diploid (DI: 1.03-<1,05; 0,95-<0.97), near tetraploid (Dl: 1,9-
2,1), hypoploid (Dl: <0,95) low aneuploid (DI: 1,05-<1,1), high aneuploid (DI:
1.1-<1,9; >2,1).
Results: 33 of 33 carcinomas (100%) were DNA non-diploid. 31 of 33
carcinoma (94%) showed high aneuploid, multiploid and hypoploid
measurements. 2 of 33 carcinoma (6%) were near diploid.
Conclusion: Using high resolution flow cytometry, DNA non-diploidy seems to
be a constant sign of ovarian carcinoma. DNA diploidy is not.
1. Göhde W. et al.: Die Bedeutung der Meßgenauigkeit bei der DNS-
Zytophotometrie. Verh.Dtsch.Ges.Zyt. 19: 1995: 156-70
14. DISCRIMINIATION OF OVARIAN TUMORS BY DNA CYTOMETRY
M.Ruhnke, A. Ditzenbach and W. Kühn
Department of Gynecologic Morphology, Academic Medical Center Benjamin
Franklin. Free University of Berlin, Hindenburgdamm 30, D-12200 Berlin
Introduction: Borderline tumors of the ovary are difficult to distinguish from
proliferating adenoma and well differentiated carcinoma. The diagnosis iackss
by the inter- and intraobserver disagreement. While adjuvant multiagent
chemotherapy is an essential part in the treatement of ovarian carcinomas,
borderline tumors only needs surgery. To avoid overtreatement more objective
methods for the differentiation of ovarian tumors are necessary.
Material and methods: The DNA-content of 150 ovarian tumors (24 benign, 53
borderline and 73 malignant tumors) was analysed. All specimen were
reevaluated by an expert pathologist (WK). Paraffin embeded sections were
stained according to Feulgen and measured with a CAS 200 image analysis
system. The statistical analysis (U-test) was performed with the relative
amount of cells with a DNA-content of 2c, > 5c, and >9c. From 24 benign
ovarian tumors 14 were proliferating, 10 nonproliferating. 42 serous and 11
mucinous borderline tumors were investigated. From the ovarian carcinomas
12 were grade 1, 26 grade 11 and 35 grade III.
Results: The median of the relative amount of diploid cells was 78%, 31% and
0% for benigne, borderline, and malignat tumors, respectively. In benign
tumors 3% of the cells had an DNA-content > 5c, compared with 20% in
borderline and 43% in malignant tumors. The median of cells >9c was 6% in
carcinoma an 0 in borderline and benign tumors. There was no significant
difference between proliferating and nonproliferating benign, or between
serous and mucinous borderline tumors. The amount of cells with a DNA-
content of 2c, >5c, and >9c was significantly different between borderline
tumors and well differentiated carcinoma. Based on diploid and aneuploid cells
(>5c), proliferating adenoma and borderline tumors were significantly different.
Conclusions: DNA-cytometry is an additional tool for the differentiation of
benign, borderline and well differentiated malignant tumors of the ovary.
15. ANGIOGENESE IN OVARIALKARZINOMEN: ERSTE ERFAHRUNGEN MIT
BILDANALYTISCHER QUANTIFIZIERUNG DER ENDOTHELIALEN
IMMUNOREAKTIVITAET UND IHRER MOEGLICHEN PROGNOSTISCHEN
BEDEUTUNG
W. Schöll, O. Reich, D. Pieber, F. Gücer
Geburtshilflich-Gynäkologische Univ.-Klinik, Graz
Hintergrund und Fragestellung: Für das Wachstum eines Tumors und seiner
Metastasen ist die Bildung von Blutgefäen essentiell. Das Ausmaß dieser
Angiogenese bestimmt die Oxygenierung und die Anlieferung von Substraten
an die Tumorzellen und wird zu einem determinierenden Faktor für das
Tumorwachstum. Ist die Tumorvaskularisation bildanalytisch quantifizierbar
und ist ihre Zunahme Ausdruck gesteigerter Tumoraggressivität?
Material und Methodik: Patientinnen mit Ovarialkarzinomen desselben
histologischen Typs, Gradings und Stadiums und derselben operativen und
adjuvanten Therapie, jedoch völlig unterschiedlichen Krankheitsverlaufes
(Versterben am Tumor innerhalb von 5 Jahren versus Rezidivfreiheit über 10
Jahre) werden bezüglich ihrer Tumorvaskularisation verglichen.
In paraffiniertem Material werden endotheliale CD34-, CD31- und Faktor VIII
Antigene mit entsprechenden monoklonalen Antikörpern markiert. Die
Dedektion erfolgt durch ein Biotin-Streptavidin amplifiziertes System mit "Fast
Red" als Chromogen. Jene Schnitte, die bildanalytisch vermessen werden,
bleiben zur isolierten Darstellung der Gefäe ohne Gegenfärbung. Mittels CCD
Camera wird ein digitales Graubild im Computer gespeichert. Nach
programmierten Schritten der Bildvorverarbeitung ("dynamische
Diskriminierung", Tiefpaßfilterung mit Subtraktion des Hintergrundes) wird ein
Binärbild erstellt, in dem nur mehr Bildpunkte erfaßt werden, die den
ursprünglich gefärbten Endothelzellen entsprechen. Hintergrund gelangt nicht
zur Darstellung. Der prozentuale Flächenanteil, der von den Endothelien im
eingesehenen Gesichtsfeld eingenommen wird, wird bestimmt. Infolge der
Automatisierung der Methode können bei geringem Zeitaufwand mehrere
Quadratmillimeter am Schnitt vermessen werden.
Ergebnisse:
Karzinome mit schlechter Prognose zeigen im epithelnahen
Tumorstroma Endothelzellflächenanteile bis uber 10 %. Tumore mit klinischer
Rezidivfreiheit haben im Vergleich dazu stromale Endothelflächenanteile unter
5 %. Bei Berücksichtigung der epithelialen Tumorkomponente werden bei
beiden Karzinomformen Werte von 3% nicht überschritten.
Diskussion:
Erste Ergebnisse bestätigen die in der Literatur vorliegenden
Hinweise, daß das Ausmaß der Vaskularisation ein wichtiger prognostischer
Faktor bei Ovarialkarzinomen sein kann. Der Einsatz der Bildanalyse kann die
quantitative Erfassung der Vaskularisation gegenüber einer einfachen
Gefäzählung stark beschleunigen und objektivieren. Der
Endothelzellflächenanteil ist reproduzierbar und unterliegt weniger
Störeinflussen als die Bestimmung des Flächenanteils des Gefälumens oder
eine Gefäzählung.
Literatur:
Hollingsworth H. C. et al.: Tumor angiogenesis in advanced stage ovarian
carcinoma. Comment in Am J Pathol. 1995; 147(1):33-41
Simpson J. F. et al.: Endothelial area as a prognostic indicator for invasive
breast carcinoma. Cancer 1996; 77:2077-85
16. NUMERICAL ABERRATIONS OF CHROMOSOME 17 IN DIPLOID AND
ANEUPLOID TUMORS.
Schnekenbühl S. Polackova J, Nagel E, Hemmer J.
Divison of Tumor Biology, University of Ulm
The flow cytometric DNA ploidy has proved predictive in many human
malignancies. As changes in chromosome numbers substantially contribute to
the expression of aneuploid DNA contents, and since many genes suggested
to be functionally involved in malignancy progression are localized to
chromosome 17, we used the FISH technique to study the significance of #17
numbers in diploid and aneuploid solid tumors. Only one of eight diploid head
and neck tumors showed an abnormal trisomy 17 while numerical aberrations
were expressed in 14 of 17 aneuploid cases. Two diploid and four aneuploid
renal cell carcinomas presented normal disomy 17, three other aneuploid
cases showed abnormal #17 numbers. Recurrent #17 aberrations in aneuploid
carcinomas of the head and neck may signify an association with malignancy
progression in this type of cancer. Numerical #17 abnormalities appear to be
less involved in carcinoma of the kidney.
17. PROLIFERATION KINETIC IN GASTRIC CANCER"IN VIVO"
A. Sendler* , K.-P. Gilbertz , A. Rhein , K. Matzen*, U. Fink*, l. Becker+, D.
van Beuningen
*Chirurgische Klinik und Institut für Pathologie der TU München, Klinikum
rechts der Isar, 81644 München, Institut für Radiobiologie, Akademie des
Sanitäts- und Gesundheitswesen der Bundeswehr, 80901 München, Germany
The clinical value of the potential doubling time Tpot as prognostic factor
and/or predictive parameter in gastric cancer is still controversial. Following
approval by the local Ethical Committee, we administered preoperatively 200
mg Bromodesoxyuridine to 52 patients with histologically proven
adenocarcinoma of the stomach. Resection was done 5 - 7 hours after
injection. BrdU incorporation and DNA content (BrdU/DNA assay) were
measured flowcytometrically (FACS), to obtain DNA-ploidy, S-phase, BrdU
labelling index (L. I.). From this data, duration of S-phase (Ts) and Tpot were
calculated. Furthermore, a reference histology was analysed and classificated
according to Lauren (intestinal vs. non intestinal type).
20 adenokarzinoma of the stomach diploid, 32 aneuploid. According to Lauren
classification, 22 tumors were classificated "intestinal" and 30 "non intestinal"
typ. The detection of BrdU was possible in 82 % (n = 43) of the cases, Ts und
Tpot could be calculated in 71 % (n = 37). In diploid carcinoma, BrdU was
detectable in 75 % of the cases, the calculation of Tpot was possible in 55 %.
Median of BrdU Ll was 16.1 % (3.5 - 39.2 %), Median of Ts was 12.5 hours (6
80 h.). Median of Tpot was 3.7 days (1.4 - 23.1 d.). Taken this median as
borderline between slow and fast proliferating tumors, 51% of the tumors were
fast and 49 % slow proliferating.
In this investigation, Ll BrdU, Ts and Tpot were individual parameter of a
tumor. There was no correlation to epidemiological, clinical or patho-
histological data. Furthermore, there was no correlation to ploidy or S-phase of
the tumors. Before "in vivo" BrdU could used in clinical routine as a prognostic
or predictive parameter, the sensitivity of the method has to be strengthen.
=== Immuneophenotyping and Analysis of Cell Populations ===
18. FLOW CYTOMETRIC ANALYSIS OF LYMPHOMA CELL
IMMUNOPHENOTYPE USING KNOWLEDGE BASED CLUSTER SOFTWARE
S. Barlage, G. Rothe, G. Schmitz
Institute for Clinical Chemistry and Laboratory Medicine, University of
Regensburg, Germany
Flow cytometry has become an extremely helpful method for diagnosing and
classifying hematopoietic malignancies. Multiparametric analysis of antigen
coexpression and expression density allows the detection even of small
abnormal cell populations and their classification according to lineage
derivation and cellular maturity. The complexity of multiparameter analysis
requires standardization of the immunophenotyping technique itself, as well as
standardization of data analysis, in order to achieve inter-sample and
interlaboratory comparability of results. In this study the use of a commercially
available cluster software ("Attractors", Becton Dickinson) for the automated
analysis of data obtained from a three colour immunophenotyping of 45
peripheral blood and bone marrow samples regarding the presence and the
immunophenotype of lymphoma cells was investigated (B-CLL (23), Hairy cell
leukemia (7), Plasmocytoma (5), Immunocytoma / cc/cb-Lymphoma / varia
(10)). In order to obtain a comparable set of data, all samples were processed
according to the same protocol of sample preparation and staining. A panel of
29 different antibodies was analyzed after incubation of whole blood or bone
marrow samples with antibodies, followed by lysis of erythrocytes. Due to the
redundant patterns of expression of multiple antigens, all main physiological
cell populations including meyloid cells could be characterized and separated
automatically from the lymphocyte immunophenotypes. The basic
classification of B-cell NHL was performed in combination with verification of
clonal immunoglobulin light chain expression by the analysis of antigen
expression and expression density of CD19, CD22, FMC7, CD23, CD5,
CD103, CD25, CD11c and membrane immunoglobulins. Plasma cells were
analyzed regarding their cytoplasmic immunglobulin expression. Using cluster
software for data analysis, the automated identification of cell populations and
population subsets was achieved by defining multispace population
boundaries, based on the expectations of the populations location in two
dimensional scatter and fluorescence dot plots. As the cluster software is able
to adapt these boundaries to population-shifts due to e.g. different densities of
antigen expression also abnormal populations are identified. Using hierarchical
structured attractors, B- and T-cell subpopulations as well as characteristic
pathological phenotypes could easily be differentiated. Clonality of B-cells
could be detected down to a detection limit of 1% of lymphocytes. The direct
transfer of results into a data spreadsheat allowed the rapid preparation of a
physician report form. Taken together, the cluster software allows a
standardized analysis of complex flow cytometric data, providing a high inter-
sample and interlaboratory reproducibility of data analysis. The definition of
patient specific attractor sets should further be useful in the monitoring of
therapy and detection of residual cells.
19. FACS-ANALYSIS OF SELECTIVE SKIN INFILTRATING BLOOD CELLS
DUE TO AN ANTIGENIC CHALLENGE IN VIVO
B&oouml;hm I., Bauer R.
Universitäts-Hautklinik Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn
Contact dermatitis, a typical delayed type of hypersensitivity, results from
complex interactions of epidermal cells and blood derived skin infiltrating cells
(SIC). Lmmunocompetent epidermal cells and antigen presenting cells
selectively attract by mediator release antigen-specific T-cells and other
effector cells.
In order to get insight into the qualitative and quantitative composition of SIC,
we analyzed flow cytometrically the cell content of spongiotic vesicles of
positive routine patch test reactions and of non-iatrogenic developed contact
dermatitis. Freshly obtained SIC were stained with a panal of mAbs by using
the two color technique and the results were compared with those of the
peripheral blood.
Our data show that lymphocytes were main SIC in contact dermatitis, but not in
patch test reactions. 70 - 86% of SIC derived from non-iatrogenic contact
dermatitis were CD3+ CD4+, whereas only 36% of the patch test reactions
could be identified as T-helper cells. CD4+ SIC could be characterized as
memory cells with the marker constellation CD45RO+ CD25+ HLA-DR+. CD3+
CD8+ cells were found in equal amounts in the peripheral blood and in the
skin. Only in patch test reactions we found monocytes. They did not express
any special activation marker. Surprisingly, polymorphonuclear neutrophils
were found in 14 - 74% (mean 49%) in the vesicle fluid. These cells expressed
in 46% CD45RA on the cell surface, while in the peripheral blood only 26% did
so. Finally, we detected eosinophils within the vesicle fluid. Generally the
number of eosinophils of the vesicle was only slightly increased in comparison
to the peripheral blood. But one case had 5% eosinophils in the peripheral
blood and 27% in the vesicle fluid.
Quantitative and qualitatitve analyses of SIC comparing non-iatrogenic
developed contact dermatitis and patch test reactions revealed that 1)
lymphocytes dominate in the vesicle fluid of contact dermatitis and, 2) patch
test reactions additionally contain monocytes, neutrophils and eosinophils in
approximately similar percentages like in the peripheral blood. But activation
and proliferation markers are more prominently expressed on SIC as
compared with the peripheral blood.
20. CD69 EXPRESSION AS A MEASURE FOR CHANGES IN PROLIFERATION
OF LYMPHOCYTES IN HIV-AB+ PATIENTS AND PATIENTS WITH CFIDS
Janssen M., Braun P. and Knechten H.
PZB Aachen, Blondelstr. 9, 52062 Aachen, Germany
CD69 is a homodimer glycoprotein, which is constitutively located on
thymocytes and thrombocytes and is expressed on activated T- and B-
lymphocytes, NK-Cells and neutrophiles.
In this study we examined the CD69 expression on CD4+ and CD8+ cells from
HIVAb+ patients (n=78), patients with CFIDS (n=37) and normal donors (n=49)
by three colour flow cytometry.
Whole blood is collected using sodium heparin anticoagulant. Aliquots of
heparinized whole blood were incubated with and without (control samples)
PHA (20 ug/rnl) for 4 hours at 37 C. After this time stimulated and control
samples were stained for 15 min with FastImmune(TM) three colour antibody
conjugate combinations (Becton Dickinson). Samples were lysed and fixed
with Lysing solution for at least 15 min and analyzed by CellQuest in the
FACScan.
The percentage of CD3+CD4+ lymphocytes expressing CD69 after activation
was significant lower in blood of HIV-Ab+ patients than in the samples from the
group of normal donors. The percentage of CD3+CD8+ cells expresing CD69
in the HIV-Ab+ group was lower with and without activation by mitogen in
comparison to the samples of normal donors. There couldn't be found a
significant change of activation of the examined cell populations between the
collective of patients with CFIDS and the group of normal patients, but a trend
of a decreasing activity in both cellpopulations were observed, too.
The results of this study will be presented and discussed at the "9. Zytometrie
Symposium" in Heidelberg.
21. BLOCKING OF THE HIV-1 ENVELOPE GLYCOPROTEIN (GP120)
BINDING TO CD4+ T LYMPHOCYTES BY ANTI-HIV MONOCLONAL
ANTIBODIES
Kröpelin, M., Süsal, C., Daniel, V., and Opelz, G.
University of Heidelberg, Institute of Immunology, 69120 Heidelberg, Germany
Objectives:
Hl-virions, gp120 expressed on infected cells or free glycoprotein
bind mainly to CD4+ T cells leading to dysfunction and/or a reduction in CD4
counts. Our aim was to determine CD4-binding sites (bs) of the envelope and
to block the gp120-CD4 interaction by anti-gp120 monoclonal antibodies
(MAbs).
Methods:
CD4+ Iymphocytes from peripheral blood of healthy volunteers were
isolated by Ficoll-Hypaque gradient centrifugation and after labelling with
CD19-FITC (B cells), CD16-PE (NK cells) and Leu-2a-PE (CD8+ cells) sorted
in a FACStar plus. Negatively selected CD4+ T cells were incubated with
recombinant FlTC-labeled gp120 (IIIB) in the absence or presence of anti-CD4
MAbs Leu-3a, OKT4a or lOT4a, or anti-gp120 human MAbs F105 and/or
murine MAb 87-135/9 (Niedrig, M. et al.). rGp120-FITC binding to CD4+ cells
was determined by FACScan (BD) analysis.
Results:
rGp120(-FITC) bound to CD4+ T cells hindered Leu-3a or OKT4a to
bind, whereas IOT4a binding was less effected. rGp120 has two major
CD4contact sites which were blocked by anti-gp120 MAbs. In contrast to MAb
F105, known to bind to a discontinuous site of viral gp120 including the
constant regions 2, 3, 4 and 5, MAb 87-135/9 recognized an epitope in the
gp120 constant region 1 (C1). 87-135/9 hindered rgp120 to bind to CD4+ cells
dose-dependent and synergistic. The gp120bs of 87-135/9 was localized to the
gp120 amino acids HEDIISLWDQSLK (aa 105-117) by ELISA-testing. The
blocking capacity of this Ab was approximately 50% when compared with
F105.
Conclusions:
A passive immunotherapy using a combination of anti-gp120
CD4bs MAbs or one bispecific antibody could have benefits and/or support an
anti-viral drug treatment of HIV/AIDS patients.
Correspondence to:
marianne.kroepelin@krzmail.krz.uni-heidelberg.de
22. CYTOMETRIC ANALYSIS OF ANTIBODY-SECRETING CELLS
R. A. Manz1, A.Thiel1, S. Miltenyi2 and A. Radbruch1,
1Institute for Genetics, University of Cologne, Germany, 2Miltenyi Biotec GmbH, Bergisch Gladbach,
Germany.
Plasma cells are the effector cells in an humoral immune response. Despite
the important role of these cells in vivo, our knowledge of this cell population is
mostly restricted to data generated from cell lines, because of their low
frequency and lack of exclusive surface markers of normal plasma cells. We
have developed a method for cytometric analysis of live cells according to their
secretion products (Manz et al., PNAS 92, 1921, 1995). Here we describe the
use of this technology to analyse and isolate antibody-secreting cells from a
specific murine humoral immune response to ovalbumin (ova). In spleen and
bone marrow, the ova-specific plasma cells are contained within a fraction of
IgG1-secreting cells which make up less than 0.1% of total cells. When
isolated by FACS and cultured for 3 days in vitro, only these cells but not
control cells secreted ova-specific IgG1 antibodies. After enrichment we
analysed this population by FACS. This method enables us to analyse
antibody secreting cells for their phenotyp and function.
23. An IMPROVED FLOW CYTOMETRIC WHOLE BLOOD ASSAY FOR
RETICULATED PLATELETS
G.B. Matic, G. Rothe, G. Schmitz
Institute of Clinical Chemistry and Laboratory Medicine, University of
Regensburg, Germany
"Reticulated platelets" are characterized through their nucleic acid content
(predominantly RNA). They are proven to be the youngest platelet population
in circulation analogous to the reticulated erythrocyte. Two fluorescent dyes for
flow cytometric analysis of reticulated platelets are currently in use. Thiazole
orange and auramine orange express high quantum yields and fluorescence
enhancement upon binding to RNA. Excitation can be achieved at a
wavelength of 488nm with an argon laser beam in routinely used flow
cytometers.
The dyes readily permeate live cell membranes and non-fixed platelets
continuously accumulate these dyes. (Para)Formaldehyde fixation prior to
staining allows saturation of staining to be achieved after incubation times from
1 to 2 hours. Methods currently available mostly use platelet rich plasma. They
differ in incubation times applied, use of fixed or non-fixed platelets, definition
of threshold levels to discriminate RNA-positive and negative cells,
concentrations of the dyes and lead to reference values in the normal
population that range between 1 and 10% reticulated platelets. Use of platelet
rich plasma is laborious and selection or loss of subpopulations are to be
considered. The published incubation times vary between 3 minutes in unfixed
platelets and 2 hours in fixed platelet systems. The former does not allow
proper manual handling since saturation cannot be reached and the latter is
time-consuming.
We developed a method combining the quick accumulation of thiazole orange
in unfixed platelets with formaldehyde fixation afterwards. Diluted whole blood
was incubated with purified thiazole orange and an additional phycoerythrin-
conjugated "second antibody" to distinguish platelets from debris. Incubation
time in the presence of thiazole orange was reduced to 15 minutes with
stability of fluorescence for at least two hours after addition of the fixative.
With the use of whole blood all drawbacks of platelet rich plasma are omitted.
Threshold was set against samples incubated without thiazole orange and
normal values were in the range of 10-15% reticulated platelets. Considering
an experimentally suggested survival of reticulated platelets from 1 to 2 days
and an overall platelet survival of 10 days in circulation, this value is
appropriate and allows also the diagnosis of diminished reticulated platelets.
24. IN VITRO INVESTIGATIONS OF LYMPHOCYTE HOMING IN
INFLAMMATORY RHEUMATIC DISEASES USING FLOW CYTOMETRY
AND IMAGE ANALYSIS
J. Neumüller (1), J. Menzel (2), A. Dunky (3)
(1) Ludwig Boltzmann Institute for Rheumatology and Balneology, Vienna, (2)
Institute of Immunology, University of Vienna, (3) 5th Department for Internal
Medicine, Wilhelminenspital, Vienna, Austria
Psoriatic arthritis (PA) is characterized by alterations of the synovial
microvasculature. Inflammatory cells adhere to endothelial cells (EC) and
migrate through the vascular wall of postcapillary venules located in the
subintimal layer of the synovial membrane.
The aim of our study was a fourfold one:
firstly, to investigate the phenotype of
Iymphocytes (LC) of PA patients using flow cytometry (FC) with regard to
activation antigens and adhesion molecules; secondly, to study the adhesion
of LC of PA patients on cultivated human umbilical vein endothelial cells
(HUVEC), both resting and activated (with thrombin, IFN-y or LPS) by counting
the Feulgen-stained nuclei of adherent LC and HUVEC using a CCD TV
camera connected to an image analysis system; thirdly, to investigate the
synthesis of IL-6 and IL-8 in both LC and HUVEC 24 h after cell contact. These
cytokines were determined qualitatively by immunofluorescence and
quantitatively at the single cell level by FC as well as in the supernatants of the
cultures using commercial cytokine ELISAs; and fourth, we investigated
whether or not the LC adhesion on HUVEC as well the cytokine production
could be inhibited by MoAbs against LC or EC specific adhesion molecules.
In contrast to controls, PA patients showed an increased surface expression of
CD11 a, b and c as well as of CD44 but a reduced surface expression of
CD49d/CD29, CD49e/CD29 and cell bound fibronectin (FN) on CD3+ LC. The
activation markers CD25 and HLA-DR were found to be slightly enhanced in
PA. Cell adhesion was generally enhanced in PA patients in comparison to the
controls. It could be blocked with MoAbs against CD11a and CD18 and
reduced with a MoAb against CD54 on IFN-y activated HUVEC, but was
enhanced after treatment of HUVEC with MoAbs against FN, CD62E and
CD106. Due to LC adhesion on HUVEC, IL-6 and IL-8 were produced in
significantly higher amounts in PA patients than in the controls. This effect
occurred even in resting HUVEC but was enhanced when the cells were
activated. While IL-6 was mainly produced by HUVEC, but in smaller quantities
also by LC, IL8 was synthesized only by HUVEC and could be modified by
preincubation with MoAbs against LC or EC specific adhesion molecules in
parallel with the cell adhesion.
The experiments show that the main adhesion pathway in LC homing of PA
patients is the interaction of the LC adhesion molecule CD11a/CD18 with
CD54 on EC followed by an enhanced synthesis of proinflammatory and
chemotactic cytokines. These results favour the hypothesis that some of the
pathological alterations of the microvasculature in PA patients are generated
by altered homing processes.
25. DETECTION OF CYTOKERATIN 8-18 AND P53 POSITIVE CELLS BY
FLOW CYTOMETRY IN HUMAN RENAL CARCINOMA
Eugen Plas, Ruth Jilch*, Sonja Thury*, Michael Fischer*, Heinz Pflüger
Dept. of Urology and LBI for Urology and Andrology, Lainz Hospital
*Dept. of Laboratory Medicine, Lainz Hospital Vienna, Austria
DNA-ploidy analysis of tumors of the genitourinary tract are used for additional
information concerning the patients' prognosis. Further, immunohistochemical
detection of epithelial antigens and proliferation markers are gaining
importance in cancer prognosis. We investigated the detection of cytokeratin 8-
18 and pS3 antigen positive cells by flowcytometry in tissue samples of
patients with human renal carcinoma and correlated the different expression of
Cytokeratin 8-18 and p53 in normal and tumorous tissue samples.
In 25 patients, tissue samples of normal renal parenchyma (n-RP), from the
central region of the renal cancer (c-NCA) and the peripheral region of the
tumor (pNCA) were obtained after radical nephrectomy. DNA-ploidy analysis of
each sample was done in all cases by flowcytometry. After performing a single
cell suspension from the tissue samples by the Medimachine(R) (Dako Inc ),
10-6-cells were fixed and permeabilised by paraformaldehyde (1%) and
methanol (70%). Cells were then stained either by using FITC-marked
Anticytokeratin 8-18 antibodies (Becton Dickinson Inc.) or by Anti-p53
antibodies (DO-7; DAKO Inc.). The suspensions were finally stained with
probidiumjodid for DNA-analysis. For detection of Cytokeratin 8-18 or p53
positive cells more than 30000 cells were evaluated per sample and analyzed
by FACSCAN (Becton Dickinson Inc.) and the CELLFIT-program (Becton
Dickinson Inc.).
Ploidy analysis of the n-RP were diploid in all cases. 7/25 patients (28%)
showed a diploid tumor, 18/25 patients (72%) had an aneuploid tumor. A
hypoploid renal cancer was seen in 5/18 cases (27.8%) and a hyperploidy in
13/18 cases (72.2%). All samples stained positive for Cytokeratin 8-18. In n-
RP 43.69%+ 18.92SD of the cells were stained positive, in c-NCA
26.34%+18.16SD and 33.58%+24.6SD of the p-NCA were stained positive for
Cytokeratin 8-18 Antigen. There was a significant difference in the percentage
of Cytokeratin 8-18 Antigen positive cells in n-RP and c-NCA (p<0.05),
however, a significant difference between normal renal parenchyma and p-
NCA was not found (p>0.05). p-53 Antigen positive cells were detected in 75%
of all n-RP, c-NCA and p-NCA samples. The percentage of pS3 positive cells
in normal tissue was 0.78%+0.81SD, 0.91%+1.0SD in c-NCA and
1.44%+1.6SD in p-NCA. A significant difference between pS3 positive cells in
normal renal parenchyma and c-NCA was not found (p>0.05), however, there
was a significant difference of pS3 positive cells between normal tissue and
the peripheral tumor samples (p<0.05).
The flowcytometric detection of Cytokeratin 8-18 Antigen positive cells was
possible in all cases of normal and tumorous tissue, whereas p53 Antigen
positive cells were only seen in 75% of all samples. The increased percentage
of Cytokeratin 8-18 positive cells in peripheral tumors samplss compared to the
central tumorous regicn could be due to a loss of epithelial cells in the central
tumor. The increased percentage of p53 postive cells in the peripheral tumor
samples might be caused by enhanced mutations in the peripheral tumor
region.
26. Institute for Genetics, University of Cologne, Germany, #Miltenyi Biotec
GmbH, Bergisch Gladbach, Germany.
IFN-y AND IL-10 CAN BE DETECTED ON THE SURFACE OF SECRETING
CELLS BY MAGNETOFLUORESCENT LIPOSOMES: SPECIFIC MARKERS
FOR CYTOKINE SECRETION
A. Scheffold&, M. Assenmacher&. J. Schmitz#, S. Miltenyi# and A. Radbruch&
T cells exert an important part of their regulatory functions by the secretion of
specific cytokines. Despite the high clinical relevance of T cell cytokine
patterns there are no available surface markers for identification of
subpopulations of T cells secreting specific cytokines. By using
magnetofluorescent liposomes we have detected IFN-y and IL-10 on the
surface of activated human and murine T cell blasts as well as on the surface
of murine and human cell lines transfected with respective cytokine cDNA. The
cytokines are expressed in low copy numbers, virtually undetectable by
conventional immunofluorescence. Surface expression is correlated to
secretion of the particular cytokine by the same cell. T cells sorted for surface
IFN-y expression from a mixed Th1/Th2 culture secrete almost all IFN-y
detectable in the supernatant and only little IL4 and IL-5. Similarly, surface
cytokine expressing cells are also positive for the respective cytokine in the
cytoplasm. Binding does not involve the known cytokine receptors and the
receptor binding sites of the membrane-bound cytokines are accessible.
27.IMMUNOLOGICAL ALTERATIONS DURING PEDIATRIC CARDIAC AND
VASCULAR SURGERY: THE INFLUENCE OF THE CARDIO-PULMONARY
BYPASS (CPB)
Tarnok A., Hambsch J., Kinzel P., Borte P., Sack U., Schneider P.
Pediatric Cardiology, Heart Center Leipzig - University Hospital - Russenstr.
19, 04275 Leipzig, Germany
Children undergoing major cardiac surgery with CPB can develop a post
operative "capillary-leaksyndrome" (CLS). Children that undergo major
vascular surgery without CPB may develop similar but less pronounced
response. The aim of this study was to determine the immunological response
to CPB. The immunophenotype of peripheral blood leukocytes was determined
for 10 children with and 10 without CPB. Blood samples were taken at eight
pre-, intra- and post-operative time-points, stained and analyzed by flow-
cytometry.
Both groups of children did not significantly differ in the population kinetics of
peripheral blood granulocytes, monocytes or Iymphocytes as well as T-* B-
lymphocytes and NK cells. However! during CPB the following alterations were
found:
1. the fraction of naive T-cells increased 2-3-fold as detected by the
CD45RA+/CD45RO+ T-cell ratio,
2. the helper-cytotoxic T-cell ratio (CD3+4+/CD3+8+) decreased by 30% and,
3. the HLA-DR expression on monocytes decreased by 90%.
These differences indicate a modulating immunosuppressive effect of the CPB.
However, no direct conclusions on the immediate influence of these alterations
on CLS can be drawn.
In an additional approach children undergoing surgery with CPB were divided
into a group with and a group.without post-operative complications. Post-
operative complications were e.g. formation of edema and blood effusion into
the pericard. Children belonging to the complication group had increased
preoperative values of CD4/CD8- and CD45RA/CD45RO-ratio. In this group
during CPB the CD4/CD8ratio decreased (p<0.05) but the CD45RA/CD45RO
ratio did not change significantly. On the other hand in the complication-free
group the CD45RA/CD45RO-ratio increased (p<0.005) but no significant
change was found in the CD4/CD8-ratio. Our results demonstrate pre-
operative differences in the immunstatus of both groups and are potentially
important for the prognosis of the development of CLS.
28. MEASUREMENT OF PROTEIN-PROTEIN-INTERACTION BY FLOW
CYTOMETRY USING FLUORESCENT MICROBEADS
Tarnok A.1, Nöhrenberg U.2, Schuhmacher S.2, Vollmer H.J.2, Rathjen F.2
Pediatric Cardiology, Heart-Center Leipzig - University Hospital - Russenstr. 19,
Leipzig
2 Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany
Certain neuronal adhesion molecules that are expressed during embryogenesis
are essential for the axonal pathfinding and the development of neural
connections. The knowledge of interactions of these molecules is essential for
the understanding of their function in the organism. Adhesion molecules can
interact in different manners:
1. by adhesion with themselves (homophilic
adhesion) or 2. with other molecules (heterophilic adhesion). By these
interactions they support the axonal guidance through the tissue.
We used and further developed a simple and reliable method for the
measurement of these interactions using fluorescent microbeads and flow
cytometry. This method enables for the detection of homo- or heterophilic
interactions. Furthermore, the binding affinity between molecules can be
estimated. It is possible to determine the binding domains of the molecule by
the use of antibodies directed against certain protein domains or protein
fragments.
Neuronal adhesion molecules of the immunoglobuline-superfamily or the
tenascin-family isolated from chick brain were analysed. Immuno-affinity
purified molecules were covalently or non-covalently coupled to microbeads
(Covaspheres oder Dynabeads, Duke Sci.Corp.; 0.5um Dia., CV<5%). For the
investigation of heterophilic interactions different molecules were coupled to
beads of different colours (blue, red or green). Measurements were done on an
EPICS ELITE cell sorter or FACScalibur flow cytometer. From the measured
particle numbers the numbers of beads in homo- and/or heterophilic
aggregates were calculated. From these values the percentage of beads in
aggregates and the aggregate size was determined.
Homophilic interaction was found for the molecules NgCAM, NCAM and
NrCAM, with aggregate sizes NgCAM>NrCAM>NCAM (Neuron, 11: 1113,
1993). Heterophilic interaction was detected in the combinations: NrCAM/F11,
Tenascin-R/F11 (Neuron 10: 711,1993). The binding domain of Tenascin-R
was analyzed in detail. Using fragments of the molecule it was found, that the
third of 8 fibronectin type III domains was responsible for the binding to F11
(J Cell Biol, 130:473, 1995). The flow cytometrically found binding
characteristics could be confirmed in cell culture models of nerve cells. Our
investigations demonstrate that flow cytometric analysis of the adhesion of
fluorescent microbeads is an easy-to-handle and reliable method to determine
protein interactions.
29. FLOW CYTOMETRIC CHARACTERIZATION OF HUMAN TROPHOBLASTS
AFTER ISOLATION FROM FRESH PLACENTAL TISSUE
Thieme B., Koop F., Harbeck N., Kolben M., *Ugele B., Schneider KTM.,
Graeff H. and Schmitt M.
Departments of Obstetrics & Gynecology, Technical University, * Ludwig-
Maximilians-University, Munich, Germany
Proteolytic factors like uPA, uPA-R, matrix metalloproteinases as well as their
respective inhibitors play a very important role in physiology and pathology of
trophoblast invasion into the uterine wall:
Zini et al.(l992); Kolben et al.(l994);
Librach et al.(1994); Cross et al.(l994)
Objectives:
We opted to isolate trophoblasts from term and pre-term
placentas and analyze these single cells flow cytometrically in order to in
insights into proteolytic and regulatory systems involved in trophoblast invasion
at different stages of normal as well as pathological pregnancies.
Methods:
Fresh placenta was mechanically dissected immediately after
delivery, cell aggregates enzymatically separeted (trypsin and DNase) and the
resulting cell suspension then further separated using a percoll gradient
(modified Kliman protocol, 1986 ). Trophoblast containing layers were
collected, the cells washed and fixed in 1 % paraformaldehyde. They were
then analyzed flow cytometrically (FACSCalibur, Becton Dickinson,
Heidelberg) after incubation with several monoclonal antibodies.
Results: A specific reaction of trophoblasts was seen with antibodies against
cytokeratin CK 8,18 ( Medac, Hamburg), 8 HCG and h PL (Dako, Hamburg).
Cells contaminating the trophoblast suspension were identified using
antibodies against CD 3, CD 15 (Dako), CD 45 (Caltag, San Francisco) and
fibroblasts (Sigma, München). Multiparameter fluorescence flowcytometry
confirmed a purity of isolated trophoblasts of about 95 %. Isolated, unfixed
trophoblasts can be taken into short term culture until syncythia are being
formed. Thus, surface antigens that had been damaged by trypsin treatment
can reappear on the cell surface.
Conclusions:
This method enables quantitative analysis in isolated fresh or
cultivated single trophoblasts using flow cytometry. We are currently compaing
the expression of proteolytic factors of single trophoblasts from term
pregnancies, with those of first trimester pregnancies, with those of
complicated pregnancies where pathological trophoblast invasion is present.
Such an analysis may contribute to a better understanding of the basic
processes in physiological and pathological trophoblast cell invasion.
30. CHARACTERIZATION OF A PATIENT WITH REDUCED PLATELET CD36
EXPRESSION AND ENHANCED GLYCOPROTEIN Ib-lX EXPRESSION.
A. Zwick, E. Orso, J. Stöhr, E. Ajzner, S. Barlage, W. Drobnik, G. Rothe, C.
Aslanidis and G. Schmitz.
Institute for Clinical Chemistry and Laboratory Medicine, University of
Regensburg,D-93042 Regensburg, Germany
Inherited disorders of platelets are the cause of primary bleeding problems
such as Glanzmann Thrombasthenia or Bernard-Soulier Syndrome. The
abnormalities in the variant forms of these thrombasthenic diseases may be
due to defects of the glycoprotein expression as well as to functional defects of
ligand binding. In conventional flow cytometric platelet analysis only expression
densities of membrane glycoproteins are detected (GpIIb-lIla, Gplb). To identify
defects in platelet function these assays were optimized with the examination
of activation-dependent ligand binding (e.g. fibrinogen or vWF) beside the
characterization of glycoprotein expression. A further improvement of platelet
assays was platelet in vitro stimulation with different agonists of platelet
activation in order to detect specific defects in platelet stimulation.
Recently we have identified a patient with mild bleeding tendency, moderately
increased mean platelet volume and thrombocytopenia. Using multiparametric
whole blood flow cytometry decreased surface expression of CD36 on platelets
and monocytes and an increased expression of GplX and Gplb on platelets but
no defects in ligand binding or stimulation were detected. Therefore, molecular
techniques were approached to reveal the underlying defect of aberrant
glycoprotein expression and platelet dysfunction.
Neither in GplV nor in Gplba any molecular defects could be identified by DNA
sequence analysis. However, during examination of CD36 many mRNA splice
variants could be detected in thrombocytes of the patient but not in controls.
Even though the patient has also a normal CD36 cDNA sequence, the
aberrant alternative splice pattem strongly indicates that the underlying defect
may be associated with a splicing defect. In conclusion the molecular analysis
revealed that highly diverse patterns of glycoprotein expression may be related
to abnormalities of platelet function. Therefore, platelet function tests, analysis
of different epitopes, and molecular biology may be necessary for the
characterization of platelet dysfunction.
=== Image Analysis and Cell Topology ===
31. Microscope control, image acquisition and visualization in a network
environment: towards "online", long distance telemicroscopy
by Matthias Nagorni, Joachim Bradl, Bernhard Schneider,
Michael Hausmann, Christoph Cremer
Institute of Applied Physics, University of Heidelberg,
Albert-Ueberle-Str. 3-5, D-69120 Heidelberg
An acquisition method for digital CCD cameras with long time integration
capability (Fa. Kappa, Gleichen, Germany) under the Linux operating system
for 3D-image acquisition of cell nuclei or 2D-images of metaphase plates in
fluorescence microscopy has been realized using PC's. For this approach, no
frame grabber has to be used, since the digitization of the analog image signal
is performed in the camera itselŁ The image data are digitally transfered via an
ISA-bus card and are displayed using the X-Window system. With the software
developed so far, the images can be visualized, analyzed and stored on hard
disk. Since all the necessary routines for camera initialization and operation
exist as single programs, automatization of recording whole image sequences
can easily be achieved in combination with e.g. a Unix shell script.
Because visualization is performed using the X-Window system, single images
can be displayed on other workstations which are integrated in a network
running the same graphical display system. This was achieved via the Internet.
As an example, long distance telemicroscopy was realized between
Heidelberg and Braunschweig. Another possibility may be to use ISDN or a
modem together with e.g. the PPP-protocol (Point to Point Protocol). By these
means, it is also possible to run the whole image acquisition from a remote
workstation, if-as in the case of our setup-important parts of the microscope
can be operated by the acquisition computer. This appears to be feasible with
e.g. the new generation of the Zeiss or Leitz microscopes Axiophot or DM RB
respectively. An automatization of the xy-microscope stage and the focus unit
only would already allow a remote operation of the microscope as well as the
data acquisition for instance.
32. Axialtomographical Microscopy for High Resolution Studies of the
Interphase Genome Organization
B.Rinke1, P.Edelmann1, J.Bradl1, A.Esa1, B.Schneider1, M.Hausmann1,
C.Cremer1,2
Institute of Applied Physics, University of Heidelberg 2
Interdisciplinary Centre for Scientific Computing (IWR), University of
Heidelberg
Highly resolving microscopical techniques and digital image analysis can be
used to test the hypothesis and methods concerning the correlation of
genetically activity and inactivity with their spatial configuration to DNA-regions
in chromosome territories or relative to proteins crucial for the replication and
transcription [1]. A principle limitation in conventional and confocal
fluorescence microscopy is that the resolution is depending on the direction.
The resolution in direction of the optical axis is less than half of the lateral one
in the focal plane.
In a practical arrangement, the resolution of FITC labelled regions in
Lymphocyte cell nuclei counterstained with propidium iodide (PI) was estimated
to 430 nm lateral and 860 nm axial [2]. The ratio of axial to lateral resolution is
conserved under practical conditions. Therefore, it is important to arrange the
investigated objects in a rotatable way and thus to compensate the worse z-
axis resolution by means of two perpendicular records [3]. In the new
approach, the objects are attached to the surface of a glass fibre [4]. With
fluorescent beads it has been shown that a physical rotation into the optimal
recording position has advantages even for "simple objects". Beads of 3.15 um
in diametre were adhered to a borosilicate glass fibre and mounted in a
mixture of glycerine/water (9:1 v:v). A standard cover slip (160 1lm) was used.
Three complete confocal data stacks each tilted by 90 degrees were recorded.
In two positions two beads were aligned serially. However, they were resolved
in the zero position as two particles side by side. Without any rotating option
one would have segmented only two objects but not three even in confocal
serial sections. The bead diametre is in the order of an interphase
chromosomal territory painted by fluorescence in situ hybridization [5].
The adaptation of the new tilting device to the LEICA TCS 4D shows the
suitability of the micro axialtomographical setup in the field of confocal laser
scanning fluorescence microscopy. For the quantitative analysis of fluorescent
objects being close together, the possibility to adjust the largest distance in the
projected epifluorescence image minimizes the error in distance
measurements.
[I] T.Cremer et al., Cold Spring Harb. Symp. Quant. Biol. LVIII (1993) 777-792
[2] B.Rinke et al., Fluorescence Microscopy & Fluorescent Probes, ed.
J.Slavik, Plenum Press
(1996) 178-183
[3] J.Bradl et al., J. Microsc. 176 (1994) 211-221
[4] J.Bradl et al., Proc. SPIE 2628 (1996) 140-146
[5] B.Rinke et al., Bioimaging 3 (1995) 1-11
33. A NEW PRINCIPLE IN CONFOCAL MICROSCOPY:
INVARIABLE LIGHTPATH LASER-SCANNING MICROSCOPY
VON STELDERN DU,1, Milicev M., 2
1Biotechnologie CONSULTING, D-69 1 8 1 LEIMEN 2 MG MICROSCOPE
GMBH, D-69245 BAMMENTAL
State-of-the-art scanning microscopy comprises either of scanning the
specimen by moving the stage along the x- and y-axis as well as in z-direction
in a repeated raster pattern or the field of view is scanned by a minute focused
spot of the illumination beam, wheras movement along z-axis is still achieved
by stepping the stage up and down. (Fig. 1 resp. Fig 2). Reconstruction and
display of usable 2-D or 3-D images is only possible by retrieving the stored
data of all the single point measurements and presentation by use of additional
(electronic) display media.
We herewith introduce a confocal scanning microscope system (Fig. 3), based
upon a German patent application dated 1991 (Patent granted: DE 41 13 297),
where all elements within the optical path from illumination light source matrix
to specimen and detection matrix respectively remain in stationary unchanged
positions to each other.
The specimen is illuminated by successive activation of the light source matrix
while detection is achieved through the corresponding pinholes of the detection
matrix.
Comparison of Principles
Fig.1 Fig.2 Fig.3
The following advantages, compared to state-of-the-art instruments, result
from the given
design arrangement:
- 2-D confocal images can primarily be created without any electronic imaging
devices (which still can be done optionally). Images can be observed by eye or
photographed directly.
- Coordinates of the single points within the focal plane can be absolutely
reproduced. Generation of images of higher precision at remarcably higher
speed result in the ability to follow up dynamic processes (3-D time-lapse
imaging).
- For the first time it is possible to combine and to produce precise and fast
confocal images with transmitting illumination. Consequently, the new system
enables upgrading of existing scanning systems to transmitting illumination
scanners (transmitting light option).
34. CONFOCAL 3D-MICROSCOPY: ACQUISITION, DOCUMENTATION AND
PRESENTATION
P.J.,S. Hutzler,
GSF-Forschungszentrum, Oberschleißheim
Confocal Laser Scanning Microscopy (CLMS) provides volume imaging in
microscopy. C'onfocal filtering realizes oplical sectioning, and a sequence of
optical sections results in a voxel data set similar to macroscopic imaging
lechniques like MR or CT. The image signals in most cascs are fluorescence
signals and might range from ultraviolet to infi a-red. On aquisition this range
is subdivided inlo a sequence of discrete spectral channels. Thus CLSM
application in general results in digital 3D multi spectral imagc data. CLSM0
data aquisition producos large data sets. One individual volume, corresponding
to one field of view in the microscope often contains morc than 10 MBytes.
Therefore data archiving requires largc capacitics, and should supply random
access at low price. One praclical solution is the use of recordable CD-ROMs.
Visulisation of volume data in microscopy is a special challenge. Especially
in biomedical applications microscopic objects are semi transparent. This
excludes surface rendering techniqucs and requires transparent modes of
volumc rendering. Different spectral signal channels are generally
visualized in different colors, in analogy to the visual perception when
looking through the eyepiece. However, special care is necessary, not to
confuse the human visual pcrception system with rcspect to space and color.
Various cxamples from biomcdical applications are given.
35. MORPHOMETRIC ANALYSIS OF AgNOR PROTEINS FOR CELL DOUBLING
TIME EVALUATION IN TUMOR PATHOLOGY
Massimo Derenzini
Dipartimento di Patologia Sperimentale, Universitŕ di Bologna Italy
In the past few years a great effort has been made by pathologists in order to
obtain a better biological characterization of cancer lesions. Particular attention
has been paid to prccisely define cell kinetics of human tumors. Indeed, fthe
growth rate of a cancer mass has an important effect on the paticnt clinical
outcome. The tumor growth rate is contitioned by the number of proliferating
cells and the rapidity of their proliferation. The former variable can be
evaluated by using the following methods: Ki67/MIBl immunostaining, [3H]-
thymidine or bromodeoxyuridine (BrdU) incorporation, and DNA flow
cytometry. The rapidity of cell proliferation can be evaluated by measuring the
tumor potential doubling time by in vivo BrdU incorpoxation followed by DNA
flow cytometry, or by the quantitative analysis of AgNOR proteins. The AgNOR
protein parametcr has been recently introduced in tumor pathology for the
evaluation of the rapidity of cell prolifcration. The AgNOR proteins, which are
located in the nucleolus during interphase, are selectively stained by silver in
routinely processed eyto-histological samples and precisely quantified by
image analysis. Nucleolin and protein B 23 are the main AgNOR proteins.
They are involved in ribosomal biogenesis. In cells stimulated to proliferate
thec AgNOR protein quantity progressively increases from the G1-phase,
reaching the maximum value at the ent of the S- phase of the cell cycle.
Therefore, it is not surprising that the AgNOR protein quantity is also related to
the number of cycling cells. On the other hands the importance of the AgNOR
protein parameter for cell kinetics evaluation is due to the fact that, in
continuously proliferating cells, the AgNOR protein quantity is strictly related to
the rapidity of cell proliferation. The evidence of this relationship comes from
the following studies. 1) Using in vitro cultured human cancer cell lines
characterized by different doubling times (DT), it was shown that differences of
only 4 hours in the DT were enough to determine significantive variations of
the AgNOR protein amount (greater the AgNOR protein quantity, faster the
cell proliferation); 2) the DT of 24 human carcinoma xenografts growing in
nude mice was significantly related to the AgNOR protein quantity (r-0.903,
p<0.001), and 3) the DT of 18 human hepatocellular carcinoma nodules
evaluated by measuring the volume variations over a fixed period of time by
"real time" ultasonography was inversely related to the AgNOR protein quantity
measured in liver biopsies at thc time of diagnosis, (r-0.68M p<0.001).
A significant correlation has been found between AgNOR protein expression of
tumor lesions and patient survival. In many cancers the AgNOR protein
variable showed an independent predictive value when entered in multivariate
analysis together with the wellestablished prognostic indexes commonly
considered for each type of cancer.
=== Standardization and Quality Control ===
36. QUALITY ASSESSMENT FOR IMMUNOPHENOTYPING OF LEUKEMIAS
AND LYMPHOMAS
J.W. Gratama1 and J.C. Kluin-Nelemans 2, for the Dutch Cooperative Study
Group on Immunophenotyping of Hematological Malignancies (SIHON)
1 Department of Clinical and Tumor Immunology, Daniel den Hoed Cancer
Center, Rotterdam, and Department of Hematology, University Hospital, Leiden, the Netherlands
A quality assessment scheme for immunophenotyping of leukemias and
Lymphomas must not only address technical aspects of the analysis, but also
the pre-analytical phase (i.e., selection of reagents hased upon a tentative
clinical diagnosis) and the post-analytical phase (i.e., the interpretation of
results and the physician's report). The Dutch Cooperative Study Group on
Immunophenotyping of Hematological Malignancies (SIHON) was initiated in
1985 to set up such a quality assessment scheme. Ever since, biannual send-
outs, each consisting of cryopreserved specimens generally containing >80%
abnormal cells and accompanied by an unstained blood smear or cytospin
preparation, brief clinical data and a tentative clinical diagnosis, were
distributed. The participants were requested to perform immunophenotyping
according to their own protocol and to guidelines formulated by SIHON (see
below), to report all antibody results as % positive cells (i.e., fraction of
mononuclear cell suspension) and to provide an immunological diagnosis to
the coordinating laboratory (University Hospital, Leiden). After data processing
by the coordinating laboratory the results were presented and discussed non-
anonymously by one of us (J.C.K.N.) at biannual plenary meetings. During
these meetings emphasis was laid on the adequate composition of antibody
panels and the appropriate interpretation of results.
To date, 16 send-outs comprising 48 specimens have been organized. The
analysis of the results of these send-outs yielded the following conclusions:
The markers with the highest frequencies of discordant scores (i.e.,
percentages reactivity transformed to either 'positive' or 'negative' scores, and
classified as 'concordant' or 'discordant' with the median score) were
cytoplasmatic (c)CD22, TdT, CDllc, CD13, CD15, CD33, CD4 and CD7.
Technical problems played a major role with cCD22 and TdT assessments.
Discordant scores for the myeloid markers CD13 and CD33 were especially
frequent on ALL specimens. The 'aberrant' expression in such cases is
typically associated with dim fluorescence signals which are a major source of
interlaboratory variability. The same argument goes for the dim expression of
CD4 and CD7 by many AML cases.
The quality of the immunological diagnosis was generally very good for most
mature Bcell malignancies, common ALL and AML specimens, i.e., > 90%
(almost) correct diagnoses. A frequent error, especially in SIHON's early years,
was the assignment of FAB classifications M1 - M6 on the basis of
immunological results. Only FAB classifications M0 and M7 can be made on
immunological marker data. That error was frequently cautioned for at the
plenary meetings.
The immunodiagnosis of T-cell malignancies was associated with significant
problems (i.e., ~60% acceptable diagnoses). A major problem was the
incorrect interpretation of TdT immunofluorescence patterns, or, even worse,
failure to assess TdT at all.
About 6% of immunological diagnoses were based on a too limited panel of
antibody results, e.g., a diagnosis 'ALL' made in the absence of reported CD34
and TdT reactivities.
During its first 10 years, SIHON has evolved trom a working party of 12
participants to a collaborative group of ~ 40, covering all laboratories involved
in immunophenotyping of leukemia and Iymphoma in The Netherlands. Further
improvement in quality is to be achieved by focusing on the pitfalls in
immunodiagnosis as summarize above: by means of the biannual quality
assessment surveys and themed workshops.
37. EXPERIENCES FROM COLLABORATIVE STUDIES ON QUALITY
ASSURANCE OF FLOW CYTOMETRIC DNA MEASUREMENTS
Friedrich J. Otto, Ulrike Westhoff
Fachklinik Hornheide, Abteilung fur Tumorforschung, D-48157 Münster
During the last five years collaborative studies on standardization and quality
assurance of flow cytometric DNA measurements have been performed. Up to
56 laboratories participated in these studies. The laboratories were asked to
stain and measure the ethanol-fixed animal and human cells provided by the
organizers according to their own protocols. Additionally, in the last two studies
standard staining protocols were included to minimize the heterogeneity of the
methods.
In all studies the original data as well as the results calculated from the
histograms exhibited considerable deviations. The highest measuring accuracy
on average as well as the best coefficients of deviation in detail were obtained
using DAPI staining in combination with high-pressure mercury arc lamp
equipped instruments. The subsequent studies over the years demonstrated
progression in measuring accuracy.
The use of standard staining protocols yielded significantly improved results.
The proposed DAPI staining protocol increased the precision and homogeneity
of the original data and reduced the deviations of DNA index and S phase
fraction determinations. A similarly good Pl staining protocol has to be
developed.
In the last study stained and fixed standard cells were used to adjust and
monitor the instrumental performance and stability. The results show that
controlling the instrumental adjustments as well as standardizing the staining
methods efficiently improves the accuracy and reproducibility of the flow
cytometric data.
38. EUROPEAN CONSENSUS PROTOCOL FOR THE FLOW CYTOMETRIC
CHARACTERIZATION OF PLATELET FUNCTION
G. Rothe for the European Working Group on Clinical Cell Analysis - Task
Force on Platelet Analysis (J. Kappelmayer, R. Lenkei, G. Rothe, G. Schmitz)
An increased or disturbed activation and aggregation of platelets plays a major
role in the pathophysiology of thrombosis and hemostasis and is related to
cardiovascular disease processes. In addition to qualitative disturbances of
platelet function also changes in thrombopoiesis or an increased elimination of
platelets e.g. in autoimmune thrombocytopenia are of major clinical relevance.
Flow cytometry is increasingly used for the specific characterization of such
phenotypic alterations of platelets which are related to the cellular activation,
the hemostatic function as well as to maturation of precursor cells. These new
techniques allow to study the in vitro interaction of platelets with platelet-spe-
cific stimuli and the modification thereof under platelet-targeted therapy as well
as the characterization of platelet-specific antibodies. Following the clinical
introduction of thrombopoietin, furthermore, the monitoring of platelet matura-
tion will be of increasing relevance. The increasing sophistication of these flow
cytometric techniques has greatly enhanced the sensitivity and specificity of
the above assays. At the same time, their application by an ever-widening
range of laboratories calis for technological standardization, establishment of
common assay protocols and the implementation of external quality
assessment schemes.
Recently, an initiative has been started by 16 groups active in the field of
clinical flow cytometry in 13 European countries to define the current consen-
sus on flow cytometric methods in the clinical laboratory as a basis for future
quality assurance and control programs. Specific protocols are developed
within specific task forces which bring together members of the group and fur-
ther scientists from the different European regions for 6 areas of technical
standardization as well as 4 areas of diagnostic application.
In a first out of a series of such consensus conferences, a task force group of
35 European scientists on platelet analysis recently met in Milan, Italy, in order
to discuss a protocol on flow cytometric platelet immunophenotyping and
function assays. In this protocol, specific recommendations for the
preanalytical phase, sample preparation and reagents, flow cytometric
measurement as well as data analysis are given based on a description of the
current state of flow cytometric methodology. These recommendations are an
attempt to promote the use of these new techniques and to increase their
interlaboratory reproducibility. Furthermore, in a second step, such consensus
should be of value for the development of study protocols in order to confirm
the clinical utility of these assays for their various diagnostic applications.
39. USE OF ELECTRONIC CELL COUNTER ANALYSER SYSTEMS FOR
QUALITY CONTROL
5W-Rule: Who did what, when, with what and why? (GLP)
Schärfe System GmbH, Emil-Adolff-Straße 14, D-72760 Reutlingen
GLP- Good Laboratory Practice. Everyone is talking about it, yet the number of
practical questions arising indicate that it is not simply defined. One field GLP
does handle with is the inner life of laboratories and their use. This includes
e.g. materials and reagents, or devices which aim is to output data.
The data and results gained in the laboratory have to be " identical and
reproducible". The prerequisite is, that the methods used are also identical and
reproducible.
Which characteristics a Cell Counter Analyser System requires in order to be
considered for being a GLP-conform method will be illustrated.
Practical examples will show how ,,the same" does not necessarily mean
"identical".
40. EXTERNAL QUALITY ASSESSMENT SCHEME "RETICULOCYTES"
(INSTAND E.V.)- A THREE YEARS EXPERIENCE
Stefan Serke,
Virchow-Klinikum der Humboldt-Universität zu Berlin, Abteilung
Innere Medizin und Poliklinik, Hämatologie und Onkologie, Augustenburger
Platz 1, 13353 Berlin, FAX: +49.30.4505.3914
The determination of immature red blood cells (RBC), recently released from
bone marrow, is a routine diagnostical laboratory test in haematology Due to
the fact that these cells, termed reticulocytes in view of the characteristical
staining pattem visible upon staining with DNA-/RNA-polysomes precipitating
dyes, are accounting for only a minor proportion of all RBC, microscopical
("manual") determinations are subject to poor reproducibility. As an altemative
to the manual determination of reticulocytes, flow-cytometric techniques have
been introduced recently. These techniques are based on a number of dyes,
which all bind to DNA or RNA. Reproducibility applying these techniques is
excellent, as several thousands of RBC, typically 30.000, are measured in one
determination.
On behalf of INSTAND e.V. I have organized the send-outs for extemal quality
assessment in reticulocyte-counting, both for the manual and the flow-
cytometric techniques. Identical samples have been shipped for both
techniques. Reviewing the data from four send-outs (1x:1994; 2x:1995;
1x:1996), results in the exspected range have been determined with regard to
the manual technique. With median percentages of 1,5 to 3,5 in the various
send-outs, the coefficient of variation (CV) for all participants (about 350 in
each send-out) was about 40%.
As a finding of particular interest and clinical impact, the CV among all
flowcytometric techniques was identical to that as determined for the manual
technique. Clustering all flow-cytometric data according to the particular
method used, it became evident that the hererogeneity of the flowcytometric
data was due to the method-specific recognition of the counting-event
"reticulocyte". For example, in one of the send-outs the median percentages
with one technique were 1,1 (CV 35%; sample-A) and 1,7 (CV 13%; sample-
B), as with another technique the respective values were 2,5 (CV 55%;
sample-A) and 4,4 (CV 38%; sample-B).
I am concluding that the currently available flow-cytometric techniques for
reticulocyte-counting are characterized by an important heterogeneity. The
general use of this variety of techniques will not result in a more standardized
counting of reticulocytes.
=== Microorganisms and Biotechnology ===
41. FLOW CYTOMETRY WITH PHENOL DEGRADING BACTERIA AFTER IN
SITU HYBRIDIZATION AND DAPI STAINING
C. HERRMANN
Abteilung Biotechnologie, Fakultät für Biowissenschaften, Pharmazie
und Psychologie, Universität Leipzig, Permoserstr. 15, 04318 Leipzig.
Alcaligenes eutrophus JMPl34 and Acinetobacter sp. can utilize acetate and
various aromatic compounds like phenol and benzoate. Their substrate affinity
and growth dynamics were tested after rRNA and DNA staining with flow
cytornetric measurements. For in situ hybridization short, synthetic, fluorescent
monolabeled oligonucleotide probes (15-20 nucleotides) were used which find
their complementary target in the sequence of 16S rRNA and 23S rRNA. A
universal oligonucleotide probe for the domain of bacteria, two group-specific
probes, a genus- and a strain-specific probe were applicated to detect and
identify Acinetobacter sp. and Alcaligenes eutrophus JMP134. Exponential
growing bacteria could be detected using one monolabeled nucleotide probe
for hybridization. Signal amplification was necessary to visualize all phases of
bacterial growth, including lag- and stationary state. Both strains could be
identified with differently labeled probes (dyes: CY3 and fluorescein) in an
epifluorescent microscope. With flow cytometric measurements a separate
detection was achieved by DAPI staining of mixed populaUons. The correlation
between growth rate and rRNA content was measured with cells of
Acinetobacter sp. in transient-state cultivation. A linear trend for growth rates of
0,05 to 0,3 h 1 was found. Cells of Alcaligenes eutrophus JMP134 were
analyzed under stress of phenol and 2,4-dichlorophenoxyacetic acid after
nutristate cultivation experiments. With increasing concentration of an aromatic
substrate growth rate and measured fluorescent signal after in situ
hybridization decreass in same ways.
42. LIGHT SCATTER MEASUREMENTS OF BACTERIA: MODEL
CALCULATIONS AND EXPERIMENTS
Andreas Lösche
Projektgruppe Biosignale, Abteilung Biotechnologie, Fakultät für
Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig,
Permoserstr. 15, D-043 18 Leipzig
With flow cytometric measurements the dynamics of bacterial populations in
biotechnical processes can be investigated. The information of single cells is
obtained from both the single cell fluorescence intensity from specifically
stained cell compartments and the light-scatter behaviour at different scatter
angles. However the light scattering behaviour of cells the size of bacteria
(1....2 um) differs clearly from that of larger
cells like tissue cells (10......20 um).
The angle dependence of the scattered light intensities depends on the
excitation wavelength and the particle size. For very small particles (dimensions less
than 1/10 wavelengths) the intensities of forward and back scattered lights are nearly
equal (Rayleigh scattering). Larger particles (over a range of particle size to tenfold
of wavelength) lead to interferences, which increase the amount of light scattered
in the forward direction, and only the exact MIE theory is applicable. The scatter
behaviour in the forward direction for even larger particles (e.g., yeast or tissue) is also
considered within the framework of geometrical optics. Here the scattering intensity is
approximately the same as the diffraction intensity for the diffraction at a circle
disc, and so it is much easier to comprehend.
Thus, for the model calculation of the scatter behaviour of bacterial cells the
exact MIE theory is necessary. An important parameter here is the refractive index of the
cells. For instance, it depends on the kind of pretreatment of the cells (e.g., fixation) and
thus also on the structure of the cell wall. Moreover, for a larger relative refractive index
there is no reversible unequivocal connection between scatter intensity and cell size.
Nevertheless, the measurement of scattered light as an additional parameter is
useful to get further information about the cells, and requires a close interdisciplinary
collaboration between biochemist and physicist.
43. MICROORGANISMS IN CONCENTRATED FRUIT JUICES
Moustafa M., Wartenberg R. and Sachsse W.
Depart. of cytogenetic Univ. of Mainz
In the last twenty years there has been a dramatic rise in microbial spoilage of
fruit juices. This spoilage is more common in the summer months and is mostly
caused by microorganisms, which are acid tolerant and heat resistant. It has
been proven that the majority of the contamination is a result of unwashed or
poorly washed fruit. It is also very common in fruit which has fallen to the
ground.
Concentrated fruit juices from some countries (Eastern- and Southern Europe,
Africa and Lat. America) were more contaminated than fruit from other
countries. In spite of the usual pasteurisation process during the production of
fruit juice, there are many cases of microbial spoilage which are caused by
acid tolerant bacteria, yeasts and moulds. Reports about heat resistant yeasts
are unknown. For this reason we have examined the concentrated apple juice
from 3 different countries (Southern-ltaly, Turkey and Argentina). The
concentrated juice was incubated for 3h at 60 C and the living yeast cells
were isolated. In comparison to Saccharomyces cerevisiae, the following
examinations were perforrned:
DNA- GC (content).
Growing rate of yeasts.
Alcohol and lactic acid fermentation.
Oleic acid concentration.
From the results of this study we can conclude that:
There is an increase in the GC-content (3,8 +/- 0,2%)
There is a decrease in the growth rate at 35 C
There is a decrease of alcohol fermentation
There is an increase of lactic acid fermentation
There is an increase in oleic acid concentration.
We postulate that the heat resistant yeasts prefer slightly higher growth
temperature than Saccharomyces Cerevisiae. By the usual pasteurisation
process a complete inactivatio can not be expected.
Further research using molecular genetic methods (primer-synthesis and DNA
hybridization) are necessary for the characterization and classification of such
yeasts and their mutants.
44. ASSESSMENT OF BACTERIAL VIABILITY STATUS BY FLOW
CYTOMETRY AND SINGLE CELL SORTING
G. Nebe-von Caron*, P. Stephens and R.A.Badley
Unilever Research, Colworth Laboratory, Sharnbrook, Beds., MK44 1LQ, UK
Rapid bacterial detection and viability measurements have been greatly
enhanced by recent advances in the use of fluorescent stains in cytometry. We
have previously shown that four physiological states can be distinguished:
reproductively viable, metabolically active, intact and permeabilized. Previous
sorting experiments have shown that not all intact cells readily grow, but cells
negative to metabolic activity measurements can grow. To circumvent the
limitations imposed by active dye extrusion or cell dormancy on viability
measurements used to date (e.g. enzyme activity or cell polarization), a fast
triple fluorochrome staining procedure has been developed that takes account
of these problems. This allows further cellular characterisation of intact cells
by: active exclusion of ethidium bromide (metabolically active cells), uptake of
ethidium bromide but exclusion of bis-oxonol (deenergized but with a polarized
cell membrane) and uptake of both dyes (depolarized). Permeabilized cells
were identified by propidium iodide uptake. The method was validated using an
electronically programmable single cell sorter and starved Salmonella
typhimurium cells. Reproductive viability was determined by sorting single cells
according to their staining pattern directly onto agar plates. Most metabolically
active and inactive cells could be recovered as well as a significant fraction of
the depolarised cells, demonstrating that depolarisation is a sensitive measure
of cell damage but a poor indicator of cell death.
45. GERMINATION AND OUTGROWTH OF SPORES CHARACTERIZED BY
FLUORESCENT PROBES AND FLOW CYTOMETRY
G. Nebe-von Caron* and W.A. Anderson,
Unilever Research, Colworth Laboratory. Sharnbrook. Beds., MK44 1LQ United Kingdom
We have now studied the viability of vegetative bacteria using fluorescent
probes and flow cytometry for several years. Because of the advantages of
that approach over growth dependent methods we wanted to test the
feasability of studying bacterial spores. Thus, fluorescent probes for membrane
integrity and membrane potential have been used to characterize the
outgrowth behaviour of spores.
The phase change observed in the microscope has been shown to correlate
with the to uptake of ethidium bromide as a supravital stain. The change to
vegetative cells was accompanied by further increase in ethidium bromide /
nucleic acid staining.and subsequent exclusion of bis-oxonol. Dead spores
showed uptake of propidium iodide as well, but membrane integrity staining
only worked after the initial Phase change or rehydration.
46. FLOW CYTOMETRY OF MESOPHYLL AND BUNDLE SHEATH
CHLOROPLASTS OF VARIOUS PLANTS WITH C4 PHOTOSYNTHESIS
E. Pfündel 1,2 and A. Meister1,
1 Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-
06466 Gatersleben.
2 Universität Leipzig, Institut fur Botanik, Johannisallee 21, D-04103 Leipzig
Previously, we introduced a method for sorting of pure mesophyll (MES) and
bundle sheath (BS) chloroplasts from the C4 species maize (Pfündel and
Meister (1996) Cytometry 23, 97-105). The method uses the chlorophyll
autofluorescence of two spectral windows as the sorting criterion. Here, we
report on the extension of our method to 15 plant species with C4
photosynthesis. The species belong to two different biochemical subtypes of
C4 photosynthesis: the NADP-malic enzyme and the NAD-malic enzyme type.
In the two types of C4 photosynthesis, the chlorophyll fluorescence
characteristics of MES chloroplasts relative to BS chloroplasts differ. By using
flow cytometry in combination with conventional isolation procedures for MES
and BS chloroplasts, we obtained two typical pattern of particle populations
which were specific for the two biochemical groups of C4 plants. Hence, the
biochemical type of photosynthesis of C4 species with unknown biochemistry
can be estimated by using flow cytometry. Flow cytometry also revealed a
large heterogeneity of chlorophyll fluorescence characteristics within the two
biochemical groups of C4 photosynthesis. The analysis of purified MES and
BS chloroplasts by low temperature (77 K) fluorescence confirmed the results
from flow cytometry and indicated that, within the individual biochemical groups
of C4 photosynthesis, different membrane compositions exist in MES and BS
chloroplasts.
47. THREE-LASER FLOW CYTOMETRY FOR SIMULTANEOUS
MEASUREMENT OF PHOTOSYNTHESIS PIGMENTS AND PROTEIN
CONTENT OF PHYTOPLANKTON POPULATIONS IN LAKES AND RIVERS
Schäfer,H.1,2; Bruckmeyer,B.1,2;Steinberg, C.2 and Beisker, W.1
(1): GSF-Durchfluszlig;zytometrie Ingolstädter Landstr 1, D-85764 Neuherberg, Germany
(2): Institut fur Gewässerökologie und Binnenfischerei Müggelseedamm 310,
D-12587Berlin, Germany
Using a FACStarplus flow cytometer we have developed a technique to
simultaneously measure chlorophyll content, chlorophyll pigment groups as
well as protein content of phytoplankton populations. An optical design with
three lasers in a commercially available FACStarplus has been developed.
Chlorophyll pigment ratios can be quantified by registering chlorophyll
fluorescence excited at two different wavelengths: 632nm and 528nm. A third
wavelength (488nm) is used for excitation of FITC to study at the same time
protein content of the algae. As a first thresholding parameter we use the
chlorophyll as well as phycocyanine fluorescence excited by 632nm with a
SOmW Helium-Neon laser. Collinear with this laser beam we use a 15mW air-
cooled Argon-Ion laser for excitation of FITC. The accessory pigment analysis
(carotenoides) is done by excitation of chlorophyll as well as of phycoerythrin
with a third laser beam (an ArgonIon laser at 528nm running at approx.
lOOmW). In order to facilitate the optical design, we use the 528nm laser as
first laser and the 488nm and 632nm as second laser in the flow cytometer.
Before the laser beams passing the focussing lens of the FACStarUS we use a
Dove prism to invert the beams. Therefore at the focus the lasers with 488nm
and 632nm are first whereas the laser with 528nm is second. We have found
that the addition of the chlorophyll fluorescence excited by the 488nm and
632nm laser does not disturb the pigment analysis due to the low power ofthe
488nm beam (15mW) compared to the 632nm beam (50mW). With our
method we can differentiate diatoms, Chrysophycee and dinoflagellates (with
carotinoids) from Chlorophycee and Euglenophycee as well as phycoerythrin
containing Cyanophycee and Cryptophycee.
We present in our report data from river Würnitz and from acidified lignite
mining lakes which demonstrate the applicability and reliability of the
developed method for 'half taxonomical' studies in limnology and aquatic
ecotoxicology.
48. NON-INVASIVE BIOPROCESS MONITORING: POPULATION DYNAMICS
Bernhard Sonnleitner
Institut für Biotechnologie, ETH Zürich
Microbial populations are often treated as statistically distributed or non
segregated. This is plausible because of the large size of these populations.
The mathematical analysis of microbial cultures is simplified by the
assumption of steady state conditions. A true steady state, however, is an
artificial situation and not the rule. Even minute changes in the micro
environmental conditions may cause significant effects on the population
dynamics.
Such aspects are investigated using the example of a bakers yeast,
Saccharomyces cerevisiae. In this context, it is important that the monitoring
techniques do not distrub or influence the dynamics of the cultures; this is why
non-invasive analytical methods are necessary. Cytometry is, therefore, very
useful.
Yeast cultures do have a "memory":
A yeast culture in (pseudo) steady state responds differently to an increase of substrate supply depending on how long
it was allowed to grow under constant conditions before. The intracellular
enzyme pool must be adapted to the new conditions which may result in an
observable overshoot of the residual substrate concentration. At the same
time, population dynamic events can be triggered, for instance the partially
synchronous entry into the S-phase of the cell cycle. Such events are typically
quantified using cytometric methods. Besides classical off-line flow cytometry
we have adopted a non-classifying online variant successfully. 3 examples will
be given to discuss the performance of these methods.
1) A disperse distribution of the population is dominant after transfer of a
culture into fresh medium but, in a later phase, cells aggregate again in typical
classes.
2) Segregation into at least 2 subpopulations is the basis for the persistent
spontaneous oscillations in chemostat culture. Those subpopulations do
resynchronize themselves.
3) Population dynamic events can be triggered by minute changes of the
microenvironment. Among others, a reduction of the generation time can be
forced.
49. OUTGROWTH AND VITALITY DISTRIBUTIONS OF INJURED
LACTOBACILLUS PLANTARUM CELLS ASSESSED BY FLOW CYTOMETRY
Ueckert J.and Ter Steeg,P.F.
Unilever Research Laboratorium P.O. Box 114, 3130 AT Vlaardingen, NL
Lactobacillus plantarum is a spoilage and biotechnology production
microorganism. It has been selected as model Gram-positive bacteria to study
the physiology of survival under extreme conditions and resistance towards
inactivation. Flow cytometry is an ideal tool to study population heterogeneity
of survival of an inactivation process and subsequent outgrowth in a (product)
environment. Aim is to identify the physiological success factors of individual
microbial cells within populations for survival and fast outgrowth, which
physiological "before" characteristics are indicative of the process sensitillsvity
of individual cells and which "after" characteristics determine the outgrowth
distribution. Subsequent manipulation of (pre-) process and environmental
outgrowth conditions can be directed towards increasing process sensitivity
and lowering the outgrowth potential as means to control spoilage. Effects of
mild heat inactivation and nisin (a food-grade bacteriocin) treatments were
assessed on outgrowth and vitality distributions of Lactobacillus plantarum
cells. Combination stains of fluorescent probes for membrane integrity and cell
divisions can identify clusters of growing and non-growing cells and assess the
extent of damage. Vitality (maintenance of internal pH, rate of cell
energization) was monitored by a non-inhibitor covalently bound fluorescent
internal pH-probe. In this way cells can be labelled prior to the inactivation
process and tracked and assessed for vitality afterwards. Fractions of
populations seemed to resist inactivation. Flow cytometry relevated the
presence of clusters after treatment differing in membrane integrity and vitality.
Single cell sorting and subseqent cultivation were employed to establish
whether these clusters could identify vital injured, respectively dead
subpopulations.
=== New Technologies, Procedures and Concepts ===
50. FLUORESCENCE LIFE-TIME MEASUREMENTS IN FLOW CYTOMETRY
PRINCIPLES AND FIRST APPLICATIONS
Beisker, K. and Klocke, A.
GSF-Durchflußzytometrie Ingolstaedter Landstr 1, D-85764 Neuherberg, Germany
A fully digitized signal processing system for fluorescence life-time detection in
flow cytometry using calculation of phase-spectra has been designed.
Modulation of the excitation laser beam is done by an acousto-optical
modulator with modulation frequencies between 5 and 25 Mhz depending on
the exspected life-time. An epiillumination design was used to collect as much
light as possible, with numerical apertures up to 1.25. Data of fluorescence
signals and phase reference signals as well are acquired with a high-speed
digital oscilloscope. Digital signal processing was done by a fast Fourier
transform technique on standard PC's. The recorded fluorescence signals can
be handled just as slit-scanning data with about 2048 or 4096 data points per
cell and channel. Therefore, we could use our DAS data analysis package for
calculating the phase shift, life-time and fluorescence intensities. The final data
are stored as standard list-mode parameters.
Calculations of signal-to-noise ratios for analogue processing and for our new
digital technique are compared with measurements and literature data of
artificial beads and mammalian cells stained with different DNA dyes.
Energy transfer between different molecules can change the fluorescence life-
time as well as the fluorescence depolarization degree. Fluorescence
depolarization measurements are performed with a standard FACStarPlUS
equipped with a quartz cuvette, a polarization rotator and a Glan-Thompsen
prisma as analyzer. Data from the interaction of propidium iodide as well as
ethidium bromide as acceptor and mithramycin as donor are presented.
51. COMPREHENSIVE MONITORING OF CELLS IN CULTURE: THE
PHYSIOCONTROL-MICROSYSTEM
M. Brischwein a*, A. Schwinde a, W. Baumann a, R. Ehret a, P. Seidl b and B.
Wolf a
a AG Medizinische Physik und Elektronenmikroskopie, Institut für
Immunbiologie, Universität Freiburg, Stefan-Meier-Str. 8, 79104 Freiburg;
Germany,
b Physikalisch Technische Studien GmbH, Leinenweberstr. 16,, 79108
Freiburg, Germany
Keywords: cellular biosensor, Physiocontrol-Microsystem, transducer array.
We present an array of differntly constructed transducer elements, housed in
adequately designed culturo- and reaction chambers. These chambers are
suited for all important modes of analytical light microscopy. The system fits
small quantitative of adherent cell types, cultures growing in suspension and
tissue biopsies. Usally the specimen are pre-cultivated and inserted into the
chamber during measurements.
Tile transducer array consists of both microelectrodes and planar sensors
directly contacting the specimen. Currently we include sensors for pH, oxygen
(detecting metabolic rates), electric impedance and semiconductor devices for
ions and bioelectric signals. Since single parameters can be traced by both
optic and electric methods, the system allows to directly compare and evaluate
new analytical approaches.
A modular electronic interface provides data acquisition and control of sensors,
pumps and microvalves. Particulay we pay attention to long-term maintenance
of the different specimen and to the avoidance of manual interferences of transducer
functions.
The parallel detection of multiple kinetic sensor signals in addition to
intracellular analysis on a single-cell level by fluorescent tracer molecules
helps to understand different cellular response modes to physical, chemical or
biological stimuli. On the other hand, the system can also be applied as a
global biosensoric device using specialised target cells for numerous
applications in biomedical and environ,ental research (e.g. toxicology,
chemotherapy, drinking water control).
Besides a description of system components some selected applications will
be presented.
52. MEASUREMENT OF INTRACELLUAR ENZYME ACTIVITY BY THE AID OF
MULTIPARAMETRIC FLOW CYTOMETRY
Rolf Eckhardt,
Coulter Electronics GmbH, Europark Fichtenhain B13, 47807 Krefeld
The CellProbe reagents are a new tool in cell analysis. The product line
consists of 38 synthetic substrates, optimised to measure intracellular enzyme
activity, plus a positive control. The patented formulations allow the substrates
to pass through the cell membrane unhindered, while enzyme specificity and
activity are enhanced through the addition of cofactors and inhibitors. Inside
the cell, enzymatic hydrolysis of the substrate frees a fluorescent dye in
amounts proportional to the concentration or activity of the target enzyme. The
resulting fluorescence can be analysed by flow cytometry, fluorescence
microscopy, laser-scanning microscopy, image analysis and even
spectrofluorometry.
Research applications for CellProbe reagents cover an extensive range of
possibilities - from unraveling cellular processes such as apoptosis, activation,
maturation, differentiation, proliveration and function, to examin the effects of
drug administration on cells and the impact of desease processes on
intracellular enzyme activities.
53. FORMATION OF CELLULAR AGGREGATES DURING ROUTINE
IMMUNOSTAINING: RELEVANCE OF THE FCY RECEPTOR IIA
POLYMORPHISM FOR CLINICAL FLOW CYTOMETRY
J.W. Gratama,1 R. v.d. Linden,l C. de Beukelaer,1 J.G.J. van de Winkel 2 and
R.L.H. Bolhuis1
Department of Clinical and Tumor Immunology, Daniel den Hoed Cancer
Center, Rotterdam
and 2 Department of Immunology, University Hospital, Utrecht, the
Netherlands
Variation of the forward (FSC) and sideward (SSC) light scatter characteristics
of peripheral blood leukocytes as a function of the type of mAb used for
immunophenotyping compromises routinely used flow cytometric analytical
techniques using fixed FSC,SSC selection criteria for Lymphocytes
('Lymphogating') set on the CD45,CD14-stained sample. That situation is
caused by the selective escape of events from the Iymphogate because thev
form aggregates with other cells.
First, we observed significant shifts of events to the right in the FSC and SSC
histograms as a function of mAb cocktail using a panel of 7 double stainings
(all IgGI subclass mAb) on peripheral blood mononuclear cells (PBMC) of 72
healthy donors. These shifts were strongest in the stainings CD2/FITC +
CD3/PE and HLA-DR/FITC + CD3/PE, intermediate in CD4/FITC + CD8/PE
and CD3/FITC + CD16+56/PE, low in CD19/FITC + CD5/PE and absent
relative to unstained cells in CD45/FITC + CD14/PE and the isotype control
staining. A wide but reproducible variation between cell donors was noted.
After simultaneously staining the PBMC with the DNA/RNA stain LDS-751
(detection in FL3), we defmed cellular aggregates as events with FL3width
(FL3w) 22x that of Iymphocytes, i.e., requiring twice the time needed by
Iymphocytes to pass the laser beam. Events with FL3w 22x Iy had significantly
higher FSC and SSC signals than those with FL3w <2x Iy, confirming the
contention that they represented cellular aggregates.
The codominantly expressed Fcy receptor for IgG, type IIa (FcyRIIa) features a
functionally relevant genetic polymorphism, in which histidine as aa 131 codes
for a receptor with low affinity for murine IgG,, whereas arginine on that
position yields a receptor with relatively high affinity. Investigation of a panel of
78 donors, 12 of which were repeatedly (up to 6x) investigated, revealed:
a significant correlation between the frequency of cellular aggregates and
FcyRIIa polymorphism in CD4/FITC + CD8/PE-stained PBMC (low in HH,
intermediate in HR and high in RR, explaining 34% of the variation in
aggregate frequency), whilst this correlation was only weak in HLA-DR/FITC +
CD3/PE;
a strong, and as yet unexplained, effect of cell donor on the frequency of
cellular aggregates in HLA-DR/FITC + CD3/PE, explaining 72% of the variation
in aggregate frequency;
Furthermore, aggregate formation was prevented in single-color stainings by using mAb conjugated with PE (large
molecule) rather than FITC (small molecule), indicating interference by PE of
the interaction between IgG1subclass mAb and FcyRIIa;
reduced proportional to titrating the concentration of the FITC-labeled mAb;
enhanced by extending the time lapse between immunostaining and flow
cytometry, and by not vortexing the cell pellet prior to washing out unbound
mAh or adding fixative (1 % paraformaldehyde in PBS).
These results show that the interaction between mAb reactive with certain
Iymphocyte surface molecules and FcyRIIa polymorphism is an important
determinant for the occurrence of immunostaining artefacts due to formation of
cellular aggregates.
54. LABELING AND ANALYSIS OF RECOMBINANT HIRUDUIN USING FLOW
CYTOMETRY
Malsch, L. Piazolo, W. Lianchun, A. Timmermann and J. Harenberg
I. Medizinische Klinik, Fakultät für klinische Medizin Mannheim der Universität
Heidelberg, Theodor Kutzer Ufer, 68167 Mannheim
Hirudin is a natural anticoagulant, which is isolated from the salivary glands of
the leech hirudo medinalis. Hirudin is a stable and strong thrombin inhibitor
(Km = 0.8 x 10-10 mol/l, pH 7.4. 20 C). The compound is succesfully used for
the prophylaxis and therapy of thrombosis in clincal trials. Recombinant
biotechnology provides sufficient amounts of substances that can be used for
the development of the anticoagulant drug. The binding of hirudin to human
blood cells contributes to the distribution of hirudin. The analysis of r-hirudin
(Knoll AG, Ludwigshafen) can be performed by fitc labeling and by hirudin
antibodies. Here we describe flow cytometry analysis of fluorescent labeled r-
hirudin on human blood cells.
5 mg r- hirudin was labeled with a 10 fold excess of fitc in a 10 mM phosphate
buffer (pH 7.5, 25 C). The antithrombin activity of the compound was 72 U/mg
and was measured using the chromogenic substrate S 2238. The labeled
hirudin-fitc showed a fitc/protein ratio of 0.14.
Fluorescent labeled hirudin was incubated 10 min in the dark with human
whole blood. After a washing step the blood cells were analyzed by FACSCAN
analysis and fluorescence microscopy. Fluorescence microcopy showed a
significant binding to human leukocytes using hirudin-fitc sample (1.2 mg/ml).
For FACSCAN analysis concentrations from 1000 ng to Img/ml hirudin-fitc
were used. The granulocytes showed a binding from 0. lto 1.2 mg/ml. The
monocytes bound from 0.2 to 1.2 mg/ml. The erythrocytes, Iymphocytes and
thrombocytes showed a binding from 0.6 to 1.2 mg/ml. Different concentrations
of r-hirudin 0.03 to 10 mg/ml and thrombin 0.01 to 30 IU were analyzed to
displace hirudin-fitc from the blood cell surface after it was bound. The results
indicate that binding of hirudin to human blood cells is low. Fluorescent labeled
hirudins were used to study the binding of hirudin to human blood cells using
fluorescence microscopy and flow cytometry. Further applications of
fluorescent hirudins are the detection of thrombin on tissues and cellular
surfaces.
Supported by a grant from the Forschungsfond of the Faculty of Clinical
Medicine Mannheim.
55. ANALYSIS OF THE ROLE OF THE FAC-GENE IN THE REGULATION OF
THE CELL CYCLE AND APOPTOSIS WITH A NOVEL FLOW CYTOMETRIC METHOD
Martin Poot, Frank Kruyt2, Hans Joenje2, Victoria L. Singer and Richard P.
Haugland.
Molecular Probes, Inc., Eugene, OR 97402, U.S.A. und Department
of Human Genetics 2, Free University of Amsterdam,1081 BT
Amsterdam, The Netherlands.
Dissection of the pathways toward apoptosis has up to now been hampered by
the fact that many of the parameters relating to apoptosis can only be
assessed after cell fixation. In order to overcome this limitation we screened a
series of viable cell nucleic stains for their ability to distinguish apoptotic and
non-apoptotic cells. Our screen was conducted in cultures of human B-cell
lines immortalized by Epstein-Barr virus transformation. This cell type was
chosen, because these cells can easily be generated from peripheral blood
samples of patients and healthy volunteers. Of the stains screened, SYTO TM
11 gave the best resolution between apoptotic and nonapoptotic cells. By
SYTO 11 /Hoechst 33342 double staining we were able to resolve the cell
cycle distribution of early-apoptotic and non-apoptotic cells. This novel method
for the analysis of apoptosis in viable cells was applied to the analysis of the
mitomycin C (MMC)-hypersensitivity of cells from Fanconi anemia (FA)
patients (Kruyt et al., (1996) Blood 87, 938). This pediatric disorder is
characterized by progressive pancytopenia and skeletal malformations. FA
shows autosomal recessive inheritance and can be resolved into five
complementation groups. The gene for complementation group C has been
isolated; the gene product is a protein localizing to the cytoplasm, where it
forms a complex with three other proteins. From a patient of complementation
group C a cell line has been derived and transfected with the DR2 vector. This
cell line maintained an elevated rate of spontaneous and MMC-induced
apoptosis. In addition, this cell line showed an increased level of early
apoptotic cells in the G1 and G2 phase of the cell cycle after exposure to
MMC. Transfection with, and overexpression of, the wild type FAC-gene led to
a decrease in the number of apoptotic cells and to strong tetraploidization of
the culture. Exposure of a cell line from a healthy volunteer to MMC led to the
formation of early apoptotic cells in the G1 and S phase of the cell cycle. We
conclude that the FAC-gene controls a combined cell cycle/apoptosis
checkpoint.
56. DISSECTION OF APOPTOTIC PATHWAYS IN LIVE CELLS BY
MITOTRACKER RED CM-XROS AND SYTO DYE FLOW CYTOMETRY
Martin Poot, Lisa L. Gibson, Victoria L. Singer and Richard P. Haugland.
Molecular Probes, Inc., Department of Biosciences, Eugene, OR 97402, U.S.A.
Cultures of four human hematological cell types (H9, HL-60, Jurkat and
Lymphoid B-cells) were induced to undergo apoptosis by treatment with
camptothecin. After staining live cells with the SYTO nucleic acid stains, two
signal clusters were detected. Human Iymphoid B-cells showed resolution
between apoptotic and non-apoptotic cells with the SYTO 11, 12, 13, 14 and
16 dyes. The H9, HL-60 and Jurkat cells showed resolution with SYTO 1 1, 13,
14 and 16 only. Treatment of fixed Iymphoid B-cells with RNase A led to
strongly reduced fluorescence after staining with the SYTO 12 dye, while the
other SYTO dyes showed little or no RNase A sensitivity. Thus, the decreased
fluorescence after SYTO 12 staining may reflect breakdown of RNA during
apoptosis. Even after RNAse A digestion, none of the SYTO dyes showed a
fluorescence distribution that was stoichiometric with cellular DNA content. The
decreased fluorescence with SYTO 11, 13, 14 and 16 dyes in apoptotic cells
may be due to an alteration in chromatin structure during apoptosis. In all cell
types tested, clear resolution between apoptotic and non-apoptotic cells was
found with the MitoTracker Red dye CM-XRos. In double staining
experiments, the cells exhibiting reduced SYTO 11 fluorescence were the
same as those showing decreased CM-XRos fluorescence. We conclude that
different SYTO dyes allow resolution of apoptotic and non-apoptotic cells
depending on the cell type. The CM-XRos dye revealed mitochondrial demise
in all cell types analysed, suggesting that this may be a common aspect of
several apoptotic pathways.
57. EXCIMER FLUORESCENCE COMPARED TO DEPOLARIZATION IN THE
FLOW CYTOMETRIC CHARACTERIZATION OF LATERAL MEMBRANE MOBILITY
G. Rothe, B. Schäfer, G. Schmitz
Institute for Clinical Chemistrv. University of Regensburg, Germany
An altered cellular membrane fluidity secondary to changes of cholesterol
metabolism is a potentially important pathomechanism in atherogenesis.
Especially in blood platelets an increased sensitivity for stimulation dependent
aggregation which is a risk factor for vascular thrombosis has been
experimentally linked to abnormal microviscosity in patients with disorders of
lipid and lipoprotein metabolism. The goal of this study was the development of
a new flow cytometric assay for the direct analysis of cellular membrane
microviscosity in correlation to activation associated phenotypic changes of
platelets in vitro.
The analysis of fluorescence polarization following the selective staining of
hydrophobic lipid regions of cell membranes with the fluorescent dye
1,6diphenyl-1,3,5-hexatriene (DPH) is a well established method for the
analysis of membrane fluidity. The extent of fluorescence anisotropy
dependent on the rotational mobility of this fluorochrome is indirectly
proportional to the microviscosity of the stained membrane subcompartment.
In this study, an alternative method based on the diffusion dependent excimer
formation of pyrenedecanoic acid (PDA) which allows a more simple analytical
approach was characterized in comparison to the DPH method as a reference.
Human platelets at room temperature showed a rapid uptake of both DPH and
PDA resulting in the staining primarily of the piasma membrane after up to 30
min of incubation. Staining analyzed by flow cytometry at 351 nm argon laser
excitation resulted in a saturation of the depolarization coefficient of DPH at 20
uM but an increase of the excimer to monomer ratio of PDA with increasing
dye concentration. A "membrane fluidity coefficient" which saturated at 5 uM
PDA was calculated as the excimer fluorescence divided through the square of
monomer fluorescence thereby correcting for the influence of dye
concentration on the frequency of collision which leads to excimer
fluorescence. The temperature dependent changes of membrane viscosity
were further used as a simple model for the comparison of both methods. Cells
analyzed at temperatures between 12 C and 33 C showed a linear increase of
the PDA fluorescence coefficient by a factor of 3.1. The depolarization
coefficient of DPH, in contrast, decreased only 2.2-fold with relatively smaller
changes occurring above 24 C. Cholesterol depletion of platelets using
cholesterol-poor phosphatidyl choline-cholesterol liposomes resulted in
significant increase of the PDA fluorescence coefficient while the change of the
DPH polarization coefficient was to small to allow reproducible analysis. This
higher sensitivity of the PDA method was further confirmed through the
analysis of patient blood samples where the PDA fluorescence coefficient of
platelets showed a negative correlation to serum LDL cholesterol in contrast to
no significant correlation for the DPH method. In conclusion, the analysis of the
excimer fluorescence of PDA is a technically simple, sensitive, and highly
reproducible method for the flow cytometric analysis of an altered membrane
fluidity of platelets.
58. FLOW CYTOMETRY OF ALKALINE AND ACID PHOSPHATASE
ACTIVITIES - COMPARISON OF THE DIAZOIC COUPLING WITH THE
METHYLUMBELLIFERON METHOD
E. Severin und E. Seidler,
Institut fur Strahlenbiologie der Universität Münster,
Robert-Kochstr. 43, D-48149 Münster
Many procedures to demonstrate cellular alkaline and acid phosphatase
activities are published in the histochemical literature. This indicates the
importance of determining these enzymes. However, only few of the published
protocols are suitable for flow cytometry.
The method of coupling naphthole AS with fast red violet LB salt is compared
here with the 4-methylumbelliferone method.
Using the coupling procedure, both the alkaline and the acid phosphatase,
separately, can be demonstrated by choice of alkaline (pH 9) or acid (pH 5)
incubation buffer. The cellular enzymes convert the added substrate
naphthol AS phosphate to a fluorescent but diffusible moiety which must be
coupled with the stable diazonium salt for in situ dye formation yielding an
intracellular red fluorescence, the intensity of which is proportional to the
cellular phosphatase activity. A detailed protocol is presented for bi-parametric
measuring of the phosphatase activities in cultured endothelial cells. The
histograms correlate DAPIDNA content with fluorescence intensity of the diazo
dye i.e. phosphatase activity of single cells.
By comparison, by incubating the cells with 4-methylumbelliferyl phosphate
only the alkaline phosphatase can be demonstrated because the fluorescent
product is not fluorescent at acid pH. Considering this drawback together with
the impaired biparametric histogram resolution of methylumbelliferon/ ethidium
bromide- DNA staining, the diazo method seems to be best suited for
application in flow cytometry to determine cellular phosphatase activity.
Alkaline Phosphatase (Diazo-Method according to Kamalia et al., modified)
1 Mio cells in 0.1M Tris buffer, pH 9
Prefixation: 0.25 % paraformaldehyde, 1 min, wash
Incubation: 2 min, 37 C, cells in 1 ml Tris
0.03 mg naphthole-AS-phosphate (Sigma) in dimethyl formamide (1.5 % final
concentration)
0.1 mg fast red violet LB salt (Sigma) Stop incubation with cold buffer, wash
Postfixation: 0.25 % paraformaldehyde, 1 hour, 4 C
For acid phosphatase use 0.1 M Walpole's acetate buffer, pH 5, instead of
Tris-buffer, pH 9.
59. PHASE-SENSITIVE FLOW CYTOMETRY: LIFETIME-BASED TECHNOLOGY
FOR ANALYZING CELLS\CHROMOSOMES LABELED WITH FLUORESCENT PROBES
John A. Steinkamp
Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM
87545, USA.
A phase-sensitive flow cytometer has been developed that combines flow
cytometry (FCM) and fluorescence lifetime spectroscopy measurement
principles to provide unique capabilities for making frequency-domain
measurements on cells/chromosomes labeled with fluorescent probes (Rev.
Sci. Instrum. 64:3440, 1993). No other instrument can resolve heterogeneous
fluorescence based on differences in lifetimes and quantify lifetimes directly in
real time. Cells are analyzed as they intersect a high-frequency, intensity-
modulated (sine-wave) laser excitation beam. Fluorescence signals are
processed by 1) phase-sensitive detection electronics to resolve
heterogeneous fluorescence based on differences in lifetimes expressed as
phase-shifts, 2) phase shift and amplitude demodulation electronics to quantify
fluorescence lifetime, and 3) low-pass filtering to obtain conventional FCM
signals, and are displayed as frequency distribution histograms and bivariate
contour diagrams. The technology has been characterized with respect to
measurement precision, linearity, sensitivity, and dynamic range. Lifetime
histograms have been recorded on autofluorescent human lung fibroblasts;
murine thymus cells labeled with antibodies conjugated to fluorophores for
studying fluorescence quenching as a function of antibody dilution and F/P
ratio; and cultured cells, nuclei, and chromosomes stained with DNA-binding
fluorochromes. Phase-resolved, fluorescence signal-intensity histograms have
been measured on thymus cells labeled with Red 613 antiThy 1.1 and
propidium iodide (PI positive dead cells) to demonstrate the resolution of
signals from highly overlapping emission spectra. Studies directed at
eliminating background interference caused by cellular autofluorescence in
low-level immunofluorescence measurements are underway. This technology
will increase the number of fluorescent markers usable in multilabeling studies
and lifetimes can be used as spectroscopic probes to study the interaction of
markers with their targets, each other, and the surrounding microenvironment.
(Supported by the U.S. Dept. of Energy and NIH Grants RR013151 and
RR07855).
60. PROGNOSTIC SURVIVAL ESTIMATION OF COLORECTAL CANCER
PATIENTS BY CLASSIF1 ANALYSIS OF MULTIPARAMETER DATA FROM
G6PDH OXYGEN INSENSITIVITY TEST, SOD AND LIPID PEROXIDATION
DETERMINATION ON HISTOPHATOLOGICAL CRYOSECTIONS.
G.Valet1), B.E.M. Van Driel2), H.Lyon3), U.Hansen3), J.Song2), C.J.F. Van
Noorden2)
1) Max-Planck-lnst.Biochemie, 82152 Martinsried, Germany, 2) Lab.Cell
Biol.and Histol, Academic Med.Center, Univ.Amsterdam, NL, 3) Dep.Pathol.,
Kobenhavns Kommunes Hvidovre Hosp., Univ.Copenhagen, Denmark
The activity of glucose-6-phosphate dehydrogenase (G6PDH) as regulatory
enzyme of the pentose phosphate shunt increases at early stages of
malignancy prior to morphological changes in the sequence: normal tissue,
adenoma, carcinoma. Tetrazolium salts as final electron acceptors convert
into water insoluble and coloured formazan. Formazan deposition is
prevented in normal cells but still occurs in malignant colon, stomach, breast
or bronchial cells when neotetrazolium chloride under 100% oxygen is
utilized.
Cryosections from fresh postoperation samples were formazan stained.
Normal and cancer tissue areas were measured in a standardized way with a
Vickers M85a scanning and integrating cytophotometer at 585nm. CuZn-SOD
(superoxide dismutase) and Mn-SOD were quantitated by immuneperoxidase
staining, and lipid peroxidation (LPO) by a FeCI3/ascorbic acid/naphtaic acid
hydrazide (NAH)/Fast Blue B method both following 4% formaldehyde fixation.
29 database columns were available for the unattended self learning
classification containing patient age, tissue differentiation grade, Dukes
staging, local Iymph node affection and tumor invasion into local blood vessels
as well as 10 measured cytochemical values and 2 % values of G6PDH in
tumor (tu) and normal (no) tissue with and without 2 Furthermore 6
differences and 6 % values were calculated between cancer and normal tissue
from the above 10+2 cytochemical values.
The normal and malignant tissues were diagnostically recognized in >95% of
the cases by the CLASSIF1 triple matrix classification program. An important
aspect of the analysis concerned the prognostic data classification for patient
survival and death during the maximal post operation observation period of 56
month. The fatal outcome in 64.3% of the ultimately dead patients (n=42) was
correctly prognosticated and no confusion with surviving patients occured i.e.
100% of the fatal prognostications were correct. Surviving patients (n=22) were
predicted in 100% of the cases but the remaining 35.7% of the dead patients
were also classified as survivors i.e. the CLASSIF1 classification "survivor"
was only correct in 100/135.7=73.6% of the cases. The selected classification
pattern could be narrowed down by the program to 11 of the 29 original
database columns. Patients had a fatal prognosis with low: age, tu-CuZn-
SOD, tu-Mn-SOD, no-%-resid.G6PDH activity under 2 % and difference of
tu/no G6PDH under N2 as well as differences tu/no CuZn-SOD and tu/no LPO
in combination with high: Iymph node infiltration, Dukes stage, % tu/no-%-
resid.G6PDH under O2.
The interesting fact emerges that the standardized triple matrix pattern
classification of image analysis data from cytochemical assays in cryosections
permitted a significantly better single case patient prognosis than traditional
parameters like Dukes staging or histopathological grading.
61. FLOW CYTOMETRIC ASSESSMENT OF SHOCK WAVE INDUCED
ALTERATIONS OF PLASMA MEMBRANE POTENTIAL.
G. Woelfl, J. Bette, E. Endl, F. Hofstädter,
Institute of Pathology, Univeristy of Regensburg, Germany
Extracorporally generated ultrasound shock waves (high energy shock waves,
HESW) have been routinely used to disintegrate urinary calculi and have
recently been applied to several conditions of soft tissue pain. Despite the
definite improvement of the patient's condition, the underlying mechanism is
completely unknown. As a first step we have analysed the influence of HESW
the plasma membrane potential of cells of a neuronal phenotype. To calibrate
the plasma membrane potential we had to modify the already existing
possibilities of potential assessment.
Cell suspensions of the rat pheochromocytoma cell line PC12 were exposed to
HESW in an experimental HESW generator. Cells were stained with the
anionic potential sensitive oxonol dye DiBAC4(3) (Molecular Probes) and
fluorescence was assessed with a FACScan flow cytometer (BD). Dead cells
were excluded by simultaneous staining with propidium iodide. Subpopulations
defined by light scattering were identified by the dye SYTO13 (Molecular
Probes).
Administation of 2000 HESW with a energy densitiy of 0.2 mJ/mm2 resulted in
hyperpolarisation of approximately 8V which is an increase of 30 %. Effects
were dose and time dependent suggesting direct influence of HESW on the
resting membrane potential of cells with neuronal differentiation. We also found
culture conditions changed the plasma membrane potential.
These findings may indicate a basic explanation for the analgesic mode of
action since hyperpolarisation can increase the threshold to start an action
potential.
Further investigations as the influence of the number of applicated HESW and
the establishment of an optimal dose have still to come. Using a different cell
line for example differentiated PC12 cells can help to find out more about the
mode of action.
=== POSTERS ===
62. HIGH SPEED FISH FOR REPETITIVE AND PAINTING PROBES
M. DURM1,3, D. WOLF1, K. ALDINGER1, F.-M.HAAR1,M. HAUSMANN1, C.
CREMER1,2
1;Institute of Applied Physics, University of Heidelberg, Germany
2;Interdisciplinary Centre for Scientific Computing (IWR), University of
Heidelberg, Germany
3;Institute of Physical Chemistry, University of Heidelberg, Germany
Fluorescence in situ hybridization (FISH) has become an important tool in
ytogenetics. Recently we developed a rapid FISH technique (Fast-FISH)
using a buffer not containing any formamide or equivalent chemical denaturing
agents. Following simultaneous denaturation of both, chromosomes and DNA
probes, the hybridization time was reduced down to several minutes. In
addition, only one washing step was used. Due to these conditions the whole
procedure of target labelling was performed within less than two hours with
indirectly labelled probes, and in less than one hour with directly labelled
probes.
This technique has been shown to be suitable for specific centromere labelling
with two repetitive DNA probes (pUC 1.77 and D15Z1) [1;2]. With respect to
the variation of base-sequences of different probes, it was necessary to vary
two parameters (target/probe hybridization-temperature and hybridization-time)
of the basic protocol.
With this method it was possible to optimize the technique for different a-
satellite probes and for different purposes. As an example for three oc-satellite
probes (12 ocsatellite probe D12Z1 / 8 a-satellite probe D8Z2 / X oc-satellite
probe) the optimized conditions are outlined [3].
A slight modification of the procedure allowed fast labelling of the single human
chromosome #11 in a hamster/human hybrid cell line by genomic DNA
extracted from a human cell line (nc37). With this system, FISH in suspension
was performed on isolated chromosomes under the same buffer conditions.
The procedure can be used for model investigations of chromosome aberration
detection after irradiation or chemical exposure in slit-scan flow analysis [4].
We describe a new, extremely fast protocol for chromosome painting (express
chromosome painting, ECP) using a commercially available, directly
fluorescence labelled probe for chromosome #8 [5]. The hybridization
conditions used omit preannealing procedures and denaturing chemical
agents. The renaturation time required for chromosome painting was reduced
to 15 and 30 minutes, respectively. As in previous protocols, the ECP-
procedure also required one washing step only (0.9 % NaCL, 0.2 % Tween 20
at RT). As a consequence, the entire painting prodcedure was fe7ZRihle in
about half an hour and less.
References:
(1): Celeda et al., Cytometry, (1994), Vol. 17, 13-25; (2): Haar et al.;
BioTechniques, (1994), Vol. 17, Nr.2, 346-353; (a): Durm et al.; Z.
Naturforschung, (1996), Vol. 51c, 253-261; G): Hausmann et al.; Z.Med.Physik
(1996), Vol. 6, 59-67; (S): Durm et al.; Z. Naturforschung, (1996), Vol. 51c,
435-439
63. FAST FISH DETECTION AND AUTOMATED DIGITAL IMAGE ANALYSIS OF
NUMERMICAL CHROMOSOMAL ABERRATIONS IN BONE MARROW AND
PERIPHERAL BLOOD INTERPHASE NUCLEI OF HEMATOONCOLOGICAL
PATIENTS
A. Esa1. L. Trakhtenbrot2. M. Hausmann1, J. Ben-Bassat2, C. Cremer1
1 = Institute of Applied Physics, University of Heidelberg, Albert-Ueberle-Str. 3-
5, D-69120 Heidelberg, F.R. Germany
2 = The Institute of Hematology, The Chaim Sheba Medical Center, Tel
Hashomer, Israel
The clonal chromosomal abnormalities of neoplastic cells are viewed as
disease associated markers which can provide diagnostic and prognositic
information and can also be employed for monitoring of remission relapse
status, detecting of minimal residual disease and thus affect the therapeutic
plan. The Fast FISH technique (Celeda et al., 1994; Haar et al., 1994), based
on thermal, formamide-free DNA chromosomal target hybridization, and
automated image analysis of hybridization sites (spots) were used for the
detection of numerical aberrations of chromosomes 8 and 12 in bone marrow
cells and peripheral blood lymphocytes of AML and CLL patients. Optimization
of Fast FISH parameters as well as the computer analysis of fluorescence
images and a comparison beween standard FISH and Fast FISH sensitivity
and resolution has been done.
For Fast FISH, following fixation of cells, it was used a) a joint denturation
procedure for both chromosomal target DNA and probe DNA, in the absence
of any other hybridization reagents except a common buffer; b) an elevated
hybridization temperature; c) a short hybridization time (as low as 15 minutes).
A quantitative analysis of FISH images obtained indicated a contrast almost as
good for the Fast FISH procedure applied as for the standard FISH. The
images were recorded by a one chip true color CCD-camera (Iiappa CF 15
MC) on a Leica fluorescence miroscope. For registration and evaluation, the
commercially available software package OPTIMAS (BioScan, Edmonds, WA,
USA) was running on a PC. In this software package, a program subroutine
was implemented, which was designed for automated spot finding and
evaluation (M. Durm et al., 1996). It was shown that Fast-FISH allowed a rapid
and highly reproducible automatic quantitative evaluation of interphase nuclei
with chromosomal numerical alterations.
Literature
Celeda et al., Cytometry 17: 13-25 (1994); Haar et al., Biotechniques 17: 346 -
353 (1994); Durm et al., Z. Naturforsch. 51c: 253-261 (1996)
64. AUTOMATIC OBSERVATION OF THE DROPLET BREAKOFF POINT
Joachim W. Ellwart. Ingolf Karls*
GSF-National Research Center for Environment and Health, Institute of
Experimental Hematology, Marchioninistr. 25, D-81377 Munich, Germany
*Siemens AG, Munich, Germany
For sorting pure cell populations with a flow-in-air cytometer a stable droplet
breakoff point is nessesary. Usually the position of the breakoff point is
observed visually by the operator. When the position of the droplet breakoff
point changes he interrupts the sorting or readjusts the position by hand.
During the delay time until the operator acts false sorting occurs. So the purity
of the sorted fraction decreases. A further disadvantage of the visual
observation of the droplet breakoff point is the constant attention needed from
the staff. Our equipment for automatic observation of the droplet breakoff point
reliefs the operator from this task. When the breakoff point shifts the sorting is
interrupted nearly without any delay. This guaranties the purity of the sorted
cells.
For the automatic observation of the droplet breakoff point we use a 486-PC
with a monochrome framegrabber for the ISA-bus. The frame grabber digitizes
in real-time the video signal from the camera at the sorter, a FACStar Plus
from Becton Dickinson. An image analysis program continuously calculates the
coordinates of the droplet breakoff point and compares them with the first
coordinates after starting the program. The image analysis program is written
in C und runs under Windows 3.1. It recognizes changes and gives then alarm.
With the aid of a relais-interfacecard sorting is interrupted and acoustic alam is
given. An electro-magnet removes the sample holder from the area of fluid
jets. To alarm the operator we use a siren or a radio installation.
email: ellwart@gsf.de
65. CELL CULTURE TESTING OF BONE REPLACEMENT MATERIALS GLASS
IONOMER CEMENT AND POLYMETHACRYLATE
Dr.Dr. S. Hassfeld, Dr. R. Frombach, Dr.Dr. R. Wiedenmann, PD Dr.Dr. J.
Zöller
Oral and Maxillofacial Surgery, Ruprecht-Karls-University Heidelberg Im
Neuenheimer Feld 400, 69120 Heidelberg, Germany
The biocompatibility of the bone replacement materials glass ionomer cement
und polymethacrylate Refobacin Palacos has been tested in -vitro on 3T3 -
mouse fibroblasts.
Quantitative growth testing of fibroblasts on material
surfaces, toxicity testing (Agardiffusion DIN V 13930) and flow cytometry were
performed.
The ionomer cement did not reduce the growth of 3T3-swiss-
mouse fibroblasts. After one hour of polymerization no toxic effects could be
observed. Flow cytometry (cell cycle distribution) showed no differences to a
fibroblast monolayer growing on a glass surface. Polymethacrylate did
drastically affect cell growth. On polymethacrylate surfaces survival of 3T3-
mouse fibroblasts could be observed for the first time after two hours of
polymerization, still showing severe toxic effects. Flow cytometry exhibited a
shift in cell cycle stages. After passing the cell cycle once cells arrested in G2-
stage. G2-arrest was released after 67 hours of incubation.
Conclusion:
We are expecting a much better clinical performance of glass ionomer cement in
maxillofacial bone reconstruction compared to polymethacrylate. A prospective
randomized clinical study is just being performed.
66. CHANGES OF LYMPHOCYTE SUBSETS AND THEIR CELL HOMING
RECEPTOR CAPACITY IN HUMAN BLOOD AFTER TOTAL SLEEP DEPRIVATION
T.O. Kleine*, W. Schreiber**, J. Albrecht***
*Funktionsbereich Neurochemie,**Klinik fur Psychiatrie, Med. Zentrum fur Nervenheilkunde, Philipps-Universitat
Marburg; ***Becton Dickinson, Heidelberg
Significant circadian changes of T cells, their subsets, and of B cells were
demonstrated by cosinor-rhythmometry in human peripheral blood [1]. The
effect of total sleep deprivation on the circadian chanqes was studied here.
Differentiation of leukocytes and lymphocyte subsets was done by FACScan
analysis of venous EDTA-blood after lysis using Becton Dickinson reagents [2].
During three consecutive days blood was collected on 7.00 h, 13.00 h, and
19.00 h with total sleep deprivation between day 1 and 2 and recovery sleep
between day 2 and 3.
Significant decreases of lymphocyte counts (20% and 31%) were found only at
7.00 h of day 2 and 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 found for CD3+, CD3+44+ and CD3+LFA-1+ cells at days 2 and 3,
whereas only CD3+44- cell counts increased. CD19+, CD19+44+ and
CD19+LFA-1+ B cell counts decreased significantly only at day 3 at 7.00 h,
whereas CD19+44- and CD19+LFA1- B cells increased partially singnificantly
at days 2 and 3. Our results point to distinct alterations of the circadian
lymphocyte pattern of T and B cells in blood after total sleep deprivation with
changes of the homing receptor CD44 expression (to a smaller extent of LFA-1
expression) which indicate distribution changes of peripheral blood
lymphocytes of the cellular immune system.
[1] Kleine TO, et al., J Interdiscipl Cycle Res 1993;24:236-8.br
[2] Kleine TO, Hackler R, Raffael, A. In: Schmitz G,
Rothe G, editors.
Durchflußzytometrie in der klinischen Zelldiagnostik.
Stuttgart, New York- Schattauer. 1994:217-28.
67. TRANSFER OF LYMPHOCYTES THROUGH THE BLOOD/CSF BARRIER OF
HUMANS
T.O. Kleine*, H.J. Werner*, J. Albrecht**
*Med. Zentrum fur Nervenheilkunde, Funktionsbereiech Neurochemie,
Philipps-Universitat Marburg
** Becton Dickinson, Heidelberg
Transfer of hematogenous leukocytes from blood through the bloodbrain-
barrier into the central nervous system (CNS) requires 9 to 12 h in animal
models. Only activated T lymphocytes pass the transendothelial route [1]. In
humans the transfer of lymphocytes through the blood/CSF barrier was studied
by FACScan analysis in venous blood and CSF samples, simultaneously
collected, with Becton Dickinson reagents [2,3]. The ratio was established
between cell counts of venous blood and cell counts of lumbar CSF.
With controls there was no difference between blood/CSF ratio of CD3+HLA-DR+ T
cells and CD3+ T cells. Both ratios were diminished with acute and subacute
meningitis. Our findings do not indicate a facilitated transfer of HLA-DR
activated T cells through the blood/CSF barrier in patients. Indications for an
existence of the LFA-1 : ICAM-2 adhesion pathway and the CD2 : LFA-3
adhesion pathway were obtained in patients [2] where the blood/CSF ratios for
CD3+LFA1+ T cells, respectively CD3+2+ T cells were lower than those of
CD3+LFA- T cells, respectively CD3+2T cells. Similar results were found with
CD19+LFA and CD19+CD2 B cells in the same patients.
Summarizing up, transfer of lymphocytes through the blood/CSF barrier is low with controls:
1900:1 for CD3+ T cells, 10,500:1 for NK cells, and 30,000:1 for B cells;
transfer increased by multiple receptor:counter-receptor interactions with
various CNS inflammations.
[1] Lassmann H, et al.,Brain Pathology, 1991;1:115-23.
[2] Kleine TO, et al., J Lab med 1996;20:164-5.
[3] Kleine TO, et al.. Eur J Clin Chem Clin Biochem
1994;32:45-52.
68. DETECTION OF H202/L-HISTIDINE INDUCED CHROMOSOME
ABERRATIONS BY MEANS OF SLIT-SCAN FLOW FLUOROMETRY
Heiko Lentfer, Dietmar Wolf, Martin Crone, Klaus Aldinger, Michael
Hausmann, Christoph Cremer
Institute of Applied Physics, University of Heidelberg, Albert-Ueberle-Str. 3-5,
D-69120 Heidelberg, F.R. Germany
An application of the Heidelberg Slit-Scan System is presented showing the
analysis of chromosomes and the detection of dicentric chromosomes after
chemical exposure with H202/L-Histine of the cells. Due to DNA double strand
breaks induced by this treatment, similar chromosome aberrations were
expected as after exposure with ionizing radiation. For the analysis, the
chromosomes were isolated, stained with DNAspecific fluorochromes and
injected into the fluid stream of the slit-scan sorter. After hydrodynamic
focusing of the fluid the chromosomes passed a "ribbon" like shaped laser
beam with a velocity of 10 m/s. The laser was focused to less than 2 ,um in the
direction of the fluid stream. This allowed to excite the chromosome
fluorescence in a time resolved way. Perpendicularly to the laser beam
direction and the fluid stream direction, the fluorescence pattern of the
chromosome (relative fluorescence vs. time of flight) was detected. Relative
centromere position as well as centromere number were registered as
additional parameters to chromosome length and relative DNA content. Real
time data analysis allowed fast profile analysis and offered additonal
possibilities for chromosome sorting according to parameters that could be
freely chosen from an appropriate combination of chromosome length,
integrated fluorescence intensity and centromere values. The system was
designed for biological dosimetry, e.g. for the determination of the frequency of
dicentric chromosomes. Typical examples are shown. A preliminary estimate
of the dose correlated frequency of dicentric chromosomes after H202/L-
Histidine treatment is given. For comparison, measurements with quantitative
fluorescence microscopy were performed, too.
Literature: Hausmann et al., Opt. Eng. 31: 1463-1469 (1992); Hausmann et al.,
Microsc. Anal. 7/95: 27-29 (1995); Hausmann et al., Z. Med. Phys. 6: 59-67
(1996)
69. A NEW FLOW CYTOMETRIC ASSAY FOR BASOPHIL DEGRANULATION
Thomas Nebe, Karin Hartmann and Waltraut Pfirrmann
Institut fur Klinische Chemie. Klinikum Mannheim, D-68135 Mannheim
In our attempt to diagnose Type I hypersensivity in patients with allergies
against bee venom and latex, the commercial fluorescence immunoassay for
specific IgE (CAP, Pharmacia, Uppsala, Sweden) partially failed. Clinical
histories were characteristic and skin prick tests often proved type I reaction
while the in vitro test remained negative. We therefore used in addition two
types of in vitro tests for basophil degranulation: i) an immunoassay against
acylated histamin (Immunotech, Marseille, France) and ii) a new flow
cytometric approach to prove for basophil degranulation.
After review of the current literature we tried several dyes specific for
basophilic granules and surface proteins considered to be expressed on
basophils. We compared a flow cytometric test published recently using CD45
and IgE expression density with a new one that uses a Iysosomal protein
(gp55) that becomes expressed on the surface of basophils upon activation.
Using the chemotactic peptide fMLP as a positive control we obtained a good
correlation of the histamin release as measured by immunoassay and the
cytometric antigen expression. Grass pollen allergens as a classical allergen
could be detected by all assays including CAP but latex only with those using
the natural allergen. However, CD45 and IgE expression densities were
unreliable esp. when plotted against titrations of the allergen.
The cytometric gp55 assay was easier and faster to perform while the histamin
release test was more sensitive towards lower allergen concentrations (two
logs). However the test result was the same and correlated well with the
clinical diagnosis. Therefore, flow cytometry will provide an easy access to test
for immediate hypersensitivity in those cases where commercial tests are not
available or insufficiently validated. The new test also reflects the effects of
antihistaminic drugs like the histamin release assay. Combined with the
Iymphocyte activation test measuring the CD69 antigen, eg. hypersensitivities
against drugs could be resolved better in the future.
70. COMPARISON OF DIFFERENT METHODS, TARGETS AND INDUCERS OF APOPTOSIS
Thomas Nebe1, Jutta Wucher1, Monika Bühl2, Gerolf Maier1, Ingrid Brechtel1
and Werner Hirt2
1 Institut fur Klinische Chemie, Klinikum Mannheim, D-68135
Mannheim and 2 ORPEGEN Pharma, D-69115 Heidelberg, Germany
In the last few years apoptosis has gained an enormeous interest in cancer
research, because it offers a new biological and pharmaceutical approach to
treat tumor cells more specifically and with lower side effects.
In order to study the programmed cell death, several biochemical and
cytometric approaches have been published. The ideal method should work in-
vitro and ex-vivo to study new drugs and pharmacological principles. However
there is still a discrepancy between in-vitro studies on established cell lines
and the clinical setting.
Therefore, we have compared and challenged the flow cytometric methods by
using cell lines and clinical samples like peripheral blood and bone marrow.
Not surprisingly several cytostatic drugs currently used in cancer
chemotherapy have shown to be potent inducers of apoptosis. Interestingly,
special combinations of cell types and inducers were more effective than
others. Even classical substances like cortisone showed a remarkable
heterogeneous response on Iymphocytes in different activation states and
patients.
The following methods were compared:
Method A (Pl exclusion) measures the partial loss of membrane barrier
function by analyzing the low propidium iodide uptake in viable apoptotic cells.
Dead cells show the intensive staining of total nuclear DNA.
Method B (Pl sub G) analyzes the fraction of hxed cells that has partially lost
their DNA due to the strand breaks. Cell cyle analysis has been done either by
propidium iodide or 7-AAD.
Method C (tunel assay) measures the incorporation of a fluorescent nucleotide
(dUTP-FITC) after enzymatic amplification in combination with a cell cycle
analysis or surface marker staining. Apoptotic cells show a lower FSC signal
and surface marker density compared to normal Lymphocytes. Several
modifications and improvements have been discribed.
Method D (annexin V) uses the fact that the outer membrane of the lipid bilayer
changes with apoptosis which is a strong inducing signal for phagocytosis (eg.
for removal of apoptotic cells from the circulation or in the thymus).
The results of the comparisons will be discussed in the light of clinical
requirements.
71. COMPARISON OF DIFFERENT TECHNIQUES TO ANALYSE MULTI-DRUG
RESISTANCE
Thomas Nebe1, Susan Kranzpiller1, Verena Zunftmeister1 M. Cianfriglia3, and
Alexandra Dorn-Beineke2
1 Institut für Klinische Chemie, Klinikum Mannheim, D-68135 Mannheim,
2 Labor Prof. Seelig, Kriegstral3e 99, D-76133 Karlsruhe, Germany and 3 Inst.
f. Sanita Superiore di Roma, Italy
In the last decade multi drug resistance (MDR) of tumor cells has been in
question to be responsible for unresponsiveness of tumors to drugs that are
substrates of the mdr transporter (gp170). In order to study the MDR, several
cytometric approaches hav been published. The ideal method should reflect
the clinical behavior of the gp170 protein in-vitro and ex-vivo to study new
drugs and pharmacological principles. However there is still a discrepancy
between in-vitro studies on and the clinical response. The best correlation has
been in patients with acute myloid leukemia using the R123 effflux assay
(Ludescher, Innsbruck). Several methods have been described, which can be
divided into protein expression analysis by immunofluorescence or
immunohistochemistry and functional assays measuring the effflux or influx of
antracyclins or indicator dyes like rhodamin 123 (R123). The so far
disappointing results published in the literature might be due to methodological
problems and discrepancies. For example, as shown by us in a previous study
on CLL, protein expression does not correlate wiith effflux. Therefore, we have
compared and challenged the flow cytometric methods by using cell lines and
clinical samples like peripheral blood and bone marrow. The following methods
were compared:
Method A (immunofluorescence) measures the expression of the MDR1
transporter gp170 on viable cells. Several antibodies against intra- and
extracellular domains of the gp170 have been compared.
Method B (R123 effflux) analyzes the reduction of R123 fluorescence after 15
minutes of loading and 60 min absence of the dye.
Method C (DNR influx) measures the incorporation of a fluorescent anticancer
drug (Daunorubicin, DNR) in comparison with a control run that contains a
inhibitor (verapamil) in combination with surface marker staining. The
fluorescent drug is used in concentrations comparable to plasma levels in vivo.
Immunfluorescence studies on cell lines with a known and graded functional
effflux response gave unsatisfying results when trying to quantitate the
cytoplasmic epitopes. The two monoclonal antibodies against surface epitopes
that we have tested (MRK16 and MM4.17) revealed both the same abnormal
binding characterisitcs i.e. no saturation at 1-10 ,ug per 1 million of cells (with
no parallel increase in staining of gp170 negative control cells). 25-50 p9 per 1
million of cells showed a correlation of efflux and expression density at least in
some cell lines (RT112, RT112-D21). In the functional test, the leaky, dead or
apoptotic cells give false positive results unsing the R123 effflux assay.
Therefore we came up with the DNR influx method. DNR also reflects the true
affinity to the true target DNA inside the cell compared to R123 binding more
loosely to mitochondria. Comparative data will be discussed.
72. PROLIFERATION VERSUS APOPTOSIS: THE BIOLOGICAL ACTIVITY OF
THYMIC PEPTIDES ON LYMPHOCYTES OF HEALTHY DONORS AND HIV PATIENTS
Thomas Nebe1, Gerolf Maier, Karin Hartmann, Ingrid Brechtel and Gerhard
Becker2
1 Institut fur Klinische Chemie, Klinikum Mannheim, D-68135 Mannheim and
2 Sanorell Pharma,D-72270 Baiersbronn, Germany
Recently a new in-vitro assay for Iymphocyte activation was described (VC
Maino et al., Cytometry 20:127-133, commercially available as
FASTIMMUNE(R), Becton Dickinson). It measures the expression of CD69 by
flow cytometry which should appear on T cells as early as 4 hours after
stimulation with mitogen or antigen in-vitro. The authors claimed a strong
correlation between the classical test of thymidine incorporation and CD69
expression. We investigated the in-vitro effects of peptides from calf thymus
(Thymosand(R)) on lymphocytes with and without costimulation with the
mitogen Concanavalin A (Con A) at low doses. Three procedures were
performed in parallel:
Method A (proliferation test) used Ficoll isolated Iymphocytes cultivated for 72
hrs at 37 C in RPMI1640 + 10%FBS w and w/o ConA and 5% C02 in the
presence of bromodesoxyuridine (BrdU) during the last 8 hrs. BrdU
incorporation was analyzed by enzyme immuno assay (Boehringer
Mannheim).
Method B (activation test) used the same culture system w and w/o ConA.
After 24 hrs activation was analyzed by measuring the expression of the early
activation antigen CD69 on CD4 and CD8 lymphocytes respectively by flow
cytometry.
Method C (tunel assay for apoptosis) used similar culture conditions and
harvest after 48 hrs. Cells were hrst stained with CD4-PE and CD8-PE-Cy5,
then fixed and after amplification with the enzyme terminal deoxynucleotidyl
transferase (TdT) the DNA strandbreaks were detected by dUTP-FITC.
Apoptotic T cells show a lower FSC signal and surface marker density
compared to normal lymphocytes.
The results were the following:
1) ConA gives a dose dependent activation and proliferation that is more
pronounced on CD4 than on CD8 T cells in healthy volunteers and vice versa
in HIV patients.
2) HIV patients show a higher rate of spontaneous apoptosis, esp. in CD8 T
cells.
3) Thymosand(R) causes a dose dependent suppression of the ConA activation
and even more of the proliferation in the cultures of healthy and HIV subjects.
4) This suppressive effect is stronger on CD8 than on CD4 Iymphocytes and at
lower concentrations of the stimulating mitogen.
5) The gap between CD69 expression and proliferation was due to an incresed
rate of apoptosis.
Conclusion:
CD69 expression on T cells does not always correlate with
proliferation. The observed suppressive effect of the thymic peptides present in
Thymosand(E) on the clinical course of autoimmune diseases may be
explained by directing the activated T cells towards apoptosis instead of
proliferation.
73. PROLIFERATION, APOPTOSIS OR PHAGOCYTOSIS: THE SUPPRESSIVE
EFFECT OF AUTOANTIBODIES ON LYMPHOCYTES OF HEALTHY DONORS AND HIV PATIENTS
T.Nebe1, G.Maier1, M.Müller1, I.Brechtel1, K.Hartmann1, F.Thienel2,
B.Buchholz3, K.Friese4, A.Willer5 and K.P.Becker6
1Inst für Klinische Chemie, 2I.Med.Klinik, 3Kinderklinik, 4Frauenklinik
5III.Med. Klinik, 6Institut fur Mikrobiologie und Hygiene, Klinikum Mannheim,
D-68135 Mannheim, Germany
In the immunopathogenesis of the HIV infection Iymphocyte proliferation is
considerably diminished in the early phase even before CD4 counts drop. This
suppression facilitates viral and bacterial infections, which in turn, cause CD4
activation and subsequently HIV replication. Therefore, we investigated the
mechanisms which may contribute to this effect. We compared the viral load
with Iymphocyte associated immunoglobulin, with activation and proliferation
after mitogenic costimulation with apoptosis and with phagocytosis. Six
procedures were performed in parallel:
Method 1 (viral load) measures the gp120 binding to Iymphocytes by three
colour immunofluorescence (gp120+Goat-anti-mouse-FITC, blocking with
mouse serum, then CD4-PE + CD3PerCP)
Method 2 (viral load) detects soluble p24 in serum after dissociation of immune
complexes by an enzyme immunoassay (Abbott, Wiesbaden).
Method 3 (Iymphocyte associated immunoglobulin) detects human IgG and
complement C3 with appropriate antisera from rabbit (DAKO, Hamburg) on T
cells counterstained by CD4-PE and CD3-PerCP (Becton Dickinson,
Heidelberg).
Method 4 (activation test) used the whole blood culture system w and w/o
ConA. After 24 hrs activation was analyzed by measuring the expression of the
early activation antigen CD69 on CD4 and CD8 Iymphocytes respectively by
flow cytometry (commercially available as FASTIMMUNE2), Becton
Dickinson). It does not always crrelate with proliferation as shown on by
another study presented by Nebe et al. on this meeting.
Method 5 (tunel assay for apoptosis) used similar culture conditions and
harvest after 48 hrs. Cells were first stained with CD4-PE and CD8-PE-Cy5,
then fixed and after amplification with the enzyme terminal deoxynucleotidyl
transferase (TdT) the DNA strandbreaks were detected by dUTP-FITC
(Boehringer Mannheim). Apoptotic T cells show a lower FSC signal and
surface marker density compared to normal Lymphocytes.
Method 6 (phagocytosis test) looks for phagocytosis of opsonized Iymphocytes
by adherent monocytes. After panoptical staining macrophages containing
Iymphocytes are counted under the microscope.
The results will be discussed in the light of CD4 cell depletion in HIV disease.
74. FLOW CYTOMETRIC DETECTION OF LOW LEVELS OF ANEUPLOID
CELLS IN BONE MARROW OF PATIENTS WITH ACUTE LYMPHOBLASTIC.
LEUKEMIA (ALL)
U. Oelschlägel, R. Nowak, U. Krebs, A. Schaub, C. Köppel, R. Herbst, G.
Stamminger, D. Riemann, P. Hinze, H.-D. Kleine, E. Siegert, G. Ehninger for
the Diagnostic Study Group.
Medical Clinic I, Technical University, 01307 Dresden.
Numerical aberrations from normal DNA content are of prognostic significance
in ALL patients. Beside other methods like conventional cytogenetics and
fluorescence in situ hybridization (FISH) flow cytometry is a relative simple
method to determine DNA aneuploidies. We investigated 57 newly diagnosed
ALL patients concerning their DNA content with simultaneous
immunophenotyping and found 35% to represent at least one aneuploid
leukemia cell clone. The used two-parameter method has some advantages
compared to DNA quantification alone. At first it offers the opportunity to
characterize the residual normal hematopoiesis concerning their DNA content
as internal standard in comparison to the leukemia cells. This results in a
higher sensitivity for detection of small aberrations in DNA content. In 8
patients heterogeneities in DNA content could be detected. With the
biparametric method it was also possible to show that these heterogeneities at
the DNA level can be associated with differences in the antigen expression. In
addition the sensitivity for detecting small percentages of aneuploid cells could
be increased which is especially important in searching for residual aneuploid
leukemia cells after chemotherapy. Follow up with at least one additional DNA
analysis at remission control was performed in 12 of 18 patients with aneuploid
clones. Even in 3 out of 9 patients fullfilling the criteria of a complete remission
aneuploid cells between 0.05% and 1.10% could be detected. Otherwise one
patient with only a partial remission untill day 189 but without aneuploid cells is
now disease free foe 533+ days. This clinical course implicated that the
repeated slightly increased cytomorphological evaluated blast counts were
rather due to regenerating normal hematopoiesis than to residual leukemic
cells. We propose the described flow cytometric method for DNA quantification
in immunophenotyped cells as supplementation to cytogenetics at time of
diagnosing ALL. The importance for detecting minimal residual disease after
chemotherapy is being tested in a prospective multicentric diagnostic study.
This work was supported by Coulter-Immunotech, Becton Dickinson and Dako
Diagnostika.
75. INDUSTRIAL PROCESS ANALYSIS: FLOW CYTOMETRY IN THE BREWING
TECHNOLOGY
Petzold, Louise1; Müller, Susann1, Hutter, Karl-Josef2, Bley, Thomas 3
1 Universität Leipzig, Abteilung Biotechnologie, Permoserstr.15, Leipzig
2 DKFZ Heidelberg, Im Neuenheimer Feld 280, Heidelberg
3 TU Dresden. Institut Lebensmittel- und Bioverfahrenstechnik, Dresden
Today's technological developments in the fermentation control are directed to
the fact, that growing amounts of beer must be produced in shorter times. To
ensure the quality of the product under these circumstances, new test methods
are necessary. One of this is the method of flow cytometry, which can be used
for the on- and off-line analysis of the physiological state of yeast cells in
breewing technology.
There excist some important intracellular parameters, which influence quantity
(the initiation of the proliferation) and quality (fermentation activity) of yeast
cells. To reach information about the cells the DNA content, the content of the
membrane bound 3s-hydroxysterols and neutral lipids were determined.
Parallel to the fermentation process samples were taken from the working ZKG
each day. The samples were stained with both DAPI (DNA) and nystatinA1-
FITC (3s-hydroxysterols) and DAPI (DNA) and nil red (neutral fats), and
measured by flow cytometry.
A high content of membrane bound 3X-hydroxysterols at the beginning of the
fermentation process is the basis for a high biomass yield, because of the
shorter time,needed for the initiation of the proliferation cycle. A high content of
3s-hydroxysterols at the end of the fermentation process produces yeast cells
with an excellent effficiency in their performances. Precondition is, that all cells
are found in the G1-phase of the cell cycle before beeing harvested.lf there
excists a high amount of cells in the G2-phase of the cell cycle at this time the
harvested yeast would have a lower abilrty for fermentation, definitely. That
means a waste of time up to 12 hours.
The synthesis of neutral lipids is an answer to stress situations. Normally, cells
growing under optimal conditions possess a low content of neutral lipids in
contrast to cells that grow under limiting condrtions. Neutral lipids serve as
energy and carbon reserve, and provide a pool of sterylester, important for the
sparking funktion of the sterols with regard to the initiation of proliferation.
By using these investigations proposrtions of the metabolic state of the yeast
cells could be made during the fermentation process and because of that
disturbed metabolic situations can be determined immediatele and can be
removed afterwards.
76. ANALYSIS OF CHROMOSOMAL IMBALANCES IN BIOPSIES FROM ORAL
SQUAMOUS CELL CARCINOMAS AND PRECURSOR LESIONS BY A
COMBINATION OF MICRODISSECTION, UNIVERSAL DNA-
AMPLIFICATION AND COMPARATIVE GENOMIC HYBRIDIZATION (CGH)
Martin Scheer1,2,4; Ruthild Weber 2,4; Christof Hofele 1; Stefan Joos 4; 1.
Antonio Born 3; Peter Lichter 4; Joachim Zöller 1; Thomas Cremer 2,5
1 Department of Oral and Maxillofacial Surgery, 2 Institute of Human Genetics
and 3 Institute of Pathology, University of Heidelberg, 4 German Cancer
Research Center, 69120 Heidelberg, Germany and 5 Institute of Anthropology
and Human Genetics, 80333 München. Germany
Biopsies conducted to examine potentially malignant lesions of the oral cavity
are routinely evaluated histopathologically. The aim of this study was to further
characterize biopsy material classified as oral squamous cell carcinomas,
carcinomas in situ and dysplastic leucoplacias by comparative genomic
hybridization. CGH is a recently developed technique which allows the
detection of chromosomal imbalances on a genomic scale in a single
experiment. Deletions, gains and amplifications in tumor DNA can be identified
at a resolution of 10-20 mega base pairs and localized to normal metaphase
chromosomes. The following experimental procedure was used. Formalin-fixed
paraffin-embedded tissue sections were stained with hematoxilineosin. This
allowed the definite identification of areas consisting of only dysplastic or tumor
cells which were then scratched from the glass slide with a microdissection
device. Prior to CGH, the microdissected material was universally amplified by
DOP-PCR. Using this method, chromosomal copy number changes were
determined in biopsies from oral squamous cell carcinomas and their precursor
lesions.
77. DISTANCE MEASUREMENTS OF SINGLE TARGETS IN INTERPHASE
NUCLEI BY WAVEFIELD MICROSCOPY
Bernhard Schneider 1, Joachim Bradl 1, Ingolf Kirsten 1, Matthias Nagorni 1, Bernd
Rinke 1, Michael Hausmann 1, Christoph Cremer1,2
1, Institut für Angewandte Physik, Universität Heidelberg, Albert-Ueberle-Str. 3-
5, D-69120 Heidelberg
2 Interdisziplinäres Zentrum far wissenschaftliches Rechnen (IWR), Universität
Heidelberg.
key words: distance measurements in fluorescence microscopy, axialtomography
In cytogenetics one of the mostly used technique to localize specific regions in
metaphase chromosomes or in chromatin of interphase nuclei is the labeling
with fluorescence dyes by in situ hybridization followed by the detection in
fluorescence microscope. For the investigation of the functional
compartmentalization of the genome, high resolution 3D-distance
measurements of the labeled objects are required.
To increase the resolution for distance measurements between small objects
in one dimension, interferometric excitation of the dye is preferred (Baily et al.
1993, Lanni et al. 1993). Using a beamsplitter, two light beams are superposed
in the specimen. A variation of the crossangle allows the adjustment of the
fringes. By shifting the wave field different sections of the object are
illuminated. A wave field microscope was build which separates objects in the
orientation of the optical axis. To investigate the influence of the local refraction
index in the specimen, the spacings of the fringes for different refraction
indizes and crossangles were evaluated. Theoretical calculations of the wave
field point spread function were confirmed by experimental measurements with
fluorescent particles (Latexbeads Polysience; 0 140nm; Bex 488nm kem
~520nm). The full width half maximum of the centered maxima were recorded
and determined to 0.14um.
To increase the resolution of distance measurements in a second dimension, a
quartz glass capillary or a glass fibre instead of an object slide can be used for
rotating the object in the same way as it has been reported for in conventional
microscopy (Bradl et al. 1994).
B. Bailey, D.L. Farkas, D.L. Taylor & F. Lanni, Nature, 366, 44-48, 1993
F. Lanni, B. Bailey, D.L. Farkas & D.L. Taylor, Bioimaging, 1, 187-196, 1993
J. Bradl, M. Hausmann, B. Schneider, B. Rinke, C. Cremer, J. Microsc., 176,
211-221, 1994
78. HISTKOM - FIELDTEST IN GERMANY FOR TELEPATHOLOGY AND
TELECYTOLOGY
P. Schwarzmann, B. Binder, J. Burkart, J. Schmid
Institut für Physikalische Elektronik, Universität Stuttgart
In HISTKOM equipment for telepathology and telecytology is developed, and in
a fieldtest evaluated. To cover all expected modes of application of telepa-
thology the telemicroscopy equipment was designed to allow fully remote
diagnosis in the most ambitious application - frozen section diagnosis. To
provide the remote pathologist with the ability to screen a slide, as he normally
does, nearly all microscope functions can be controlled remotely. This holds for
x and y position of the table, focus, magnification, condensor setting, illumi-
nation and an opportunity to get images of the removed organ and an overview
image of the total slide. The microscope station and the remote display station
are connected via the ISDN telephone net. Transmitted is the field of view of
the microscope, voice, signals to control remotely the microscope and the TV-
camera, voice and telepointers within the images. Data compression and
bundiing of several ISDN lines provide an improved online impression to the
operator. Presently 3 stations with different microscope equipment of different
manufacturers are in operation, a fourth system will be ready fall 96. All
microscope and display stations are interoperable.
The user interface is designed to free the operator of as many computer
activities as possible to let him concentrate fully to the investigation of the
slide. All operation parameters like frame rate, resolution, focus, illumination
may also be selected automatically by the system according to his activities.
In the field test about 20 groups participate. Each group gets a system for 4-6
weeks and uses it for a self designed investigation programm. The first larger
test with 118 cases of frozen section preparations of lung diseases began in
fall 95 as a double blinded test and was completed in spring 96. The key
features of the evaluation phase are: false positive and false negative rate as
well as the rate of undiagnosable cases compared with the conventional frozen
section service and with respect to the final diagnosis with embedded material.
Further is recorded the time elapsed until a diagnosis has been made
dependent on the difficulty of the case and the number of fields of view and
magnification changes used during the investigation of a case.
HISTKOM is funded by the German Telekom AG and the Robert Bosch
Foundation and takes part in the European Telepathology project EUROPATH.
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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