Deutsche Gesellschaft für Zytometrie
(DGfZ)

DGfZ Abstracts

Abstract Titels

The abstract are printed in the: Proceedings of the DGZ Heidelberg Meetings, DKFZ, Heidelberg 1996, ISSN 0949 - 5347

=== ORAL PRESENTATIONS ===

=== Tumor Biology and Tumor Diagnostics ===

2. IMPROVED SINGLE LASER MEASUREMENT OF TWO CELLULAR ANTIGENS AND DNA-PLOIDY BY THE COMBINED USE OF PROPIDIUM IODIDE AND TO-PRO-3 IODIDE
Willem E. Corver, Gert Jan Fleuren and Cees J. Cornelisse
Department of Pathology, Faculty of Medicine, Leiden University, Leiden, the Netherlands
Recently, it has been demonstrated that TO-PRO-3 iodide (TP3) can be excited indirectly by a 488 nm laser line through energy transfer when added to a solution containing propidium iodide (Pl). We have evaluated this combined use of Pl and TP3 in order to overcome spectral cross talk problems observed using fluorescein isothiocyanate (FITC), R-phycoerythrin (PE) and Pl for the simultaneous detection of two cellular antigens and DNA-ploidy using single laser excitation.
A range of TP3 concentrations (0.001 to 16 uM) was evaluated using a mixture of unlabelled, keratin 8/18-FITC and keratin 8/18-PE labelled MCF-7 breast carcinoma cells of which the DNA was stained with Pl (100 uM). Correction for spectral cross talk of PE/PI into the green fluorescence detector (FL1) could entirely be compensated for and required only 1.3% (FL1 - %FL2) compensation at a TP3 concentration of 2.0 uM in the presence of Pl (100 uM). The Pl spectral cross talk into the orange fluorescence detector (FL2) was reduced from 76% to 38% using the same PMT settings. The CV of GoG1, peak of the DNA histogram was decreased from 3.70% to 2.96%, respectively. TP3 concentrations over 4.0 uM gave less satisfactorily results. The ratio between p53 stained COV362.cI4 ovarian carcinoma cells and unstained COV362.cI4 cells was reduced from 13.3 +/- 0.13 to 10.3 +/- 0.59 after staining the DNA with Pl and even more after staining with Pl/TP3 (7.56 +/- 0.32). However, when triple staining was performed using keratin 8/18-PE as an additional marker, the signal to noise ratio between unstained and p53 stained carcinoma cells was lower when DNA was stained with Pl only (4.84), compared to DNA stained with Pl/TP3 (6.61).
We conclude that the addition of TP3 to Pl improves the combined measurement of DNA ploidy and antigen expression in the epithelial fraction in heterogenous samples by single laser excitation.

3. GRADE OF ANEUPLOIDY (5c- AND 9c-EXCEEDING RATE) AS A PROGNOSTIC MARKER FOR BREAST CANCER
Coumbos, A., Ruhnke, M., Yildirim, S. and Kühn, W.
Dept. of Obstetrics and Gynecology, Klinikum Benjamin Franklin, Free University of Berlin,Hindenburgdamm 30, 12200 Berlin, Germany
Introduction: DNA-image analysis represents an established method in order to classify biologically breast cancer. AUER introduced the method and classified the malignant breast tumors in four groups AUER I-IV according to the amount of diploid, polyploid, triploid and aneuploid cells. As high risk tumors of the groups AUER III and IV are seen frequently (in our study more than 80%) the prognostic value of the method becomes doubtful. We examined in our study the prognostic significance of the number of aneuploid cells in a tumor population (5c- and 9c-exceeding rate). Materials and Methods: Imprint smears of 252 malignant breast tumors were stained by the FEULGEN method and measured by CAS 200 DNA-image analysis system. The patients were observed after surgery in the outdoor department of our clinic. The grade of aneuploidy was correlated to the course of the disease. Results: The median observation interval was 22 months. 215 women (85%) were healthy and disease free. 18 patients (7%) suffered from recurrency, 19 women (8%) died because of breast cancer. As shown in Table 1, healthy women were classified into the group AUER IV in 63%, whereas women who died due to disease were classified into this group in 84%. Table 2 shows the median amount of aneuploid cells in living, healthy women and patients who died from breast cancer. The Wilcoxon Test for the cells >5c and >9c is p<0,0277 and p<0,0026 respectively. There was no significant difference in the number of aneuploid cells in the groups of living patients with or without recurrency. Patients who had died from breast cancer had in 63% more than 4 cells >9c. Living healthy women showed only in 31% more than 4 cells >9c. A combination of more than 10 cells >5c and more than 2 cells >9c was observed in 58 % of the dead women and in 31% of the living heatlthy patients. Summary: The amount of cells >5c and especially >9c represents a prognostic marker for the survival of breast cancer patients. The probability of metastasis can not yet be predicted by DNA-image analysis, as the follow-up interval is too short. A longer followup should allow an answer in the near future.
Table 1: Distribution of patients in AUER groups
===== AUER I AUER II AUER III AUER IV
healthy 3,3% 12,5% 21,4% 62,8%
(n=215)
recurrency 6,3% 16,7% 11,0% 66%
(n=18)
death 0,0% 5,0% 11,0% 84,0%
(n=19)
Table 2: Median amount of aneuploid cells in living or died patients
============= >5c >9c
living healthy pat: 12 2 cells
dead patients: 21 5 cells

.4 THE VALUE OF FLOW CYTOMETRY IN STUDYING MALIGNANT GLIOMAS
V. Ehemann, B. Eifert*, K. Munkel*, A. Lange, H. F. Otto
Institute of Pathology, and Department of Neurosurgery*, University of Heidelberg, Germany
Cell cycle analysis and DNA-index give information about the grade of ploidy and the growth rate characteristics of tumors. We studied malignant gliomas using flow cytometry and primary cell culture. The results were related to tumor type, tumor cell grading and to cytogenetic characteristics of the tumors.
Thirty percent of gliomas were analysed as diploid, seventy percent showed aneuploid tumor cell populations. The DNA-index was heterogeneous ranging from 1,0 to 2,3. The S-phase analysis showed proliferation activity from a very lowrange of 0,7%upto17,0%.In general, diploid gliomas exhibited a lower S- phase activity than aneuploid gliomas. Twenty percent of aneuploid gliomas showed a peridiploid pattern with a DNA-index of 1,1 (CV-range 1,4 to 1,9). Remarkably, in these peridiploid tumors a trisomy of chromosome # 1 could be detected by fluorecence in situ hybridisation (FISH).
We conclude that in malignant gliomas there is a close correlation between DNA-index, proliferative activity, tumor cell grading and cytogenetic tumor characteristics. Therefore, DNA flow cytometry is an additional objective parameter in the biological evaluation of gliomas.

5. STANDARDIZATION OF FLOW-CYTOMETRIC DUAL PARAMETER-DNA MEASUREMENTS ON HUMAN COLON CARCINOMA
Ruth Knüchel, H.Zirngibl*, F.Hofstädter
Institute of Pathology and Dept. of Surgery, University of Regensburg, Germany
Since dual-parmeter-flow cytometry is suggested increasingly as a method for tumor-selective ploidy and proliferation assessment, quality criteria have to be established as a basis for supplementary diagnostic use.
Thus samples from colon carcinomas and related normal tissue were gained from 100 fresh partial colectomies and rectum resections, and cut in half for reference histology and standardized tissue dissociation. Methanol fixed single cell suspensions were stained with an indirect immunofluorescence technique, using a cytokeratin antibody (CK18, Immunotech) and RNAse treatment preceding propidium iodide staining. On a Facscan flow cyometer (Becton Dickinson) a minimum of 20000 events were gathered.
Diploid cytokeratin(CK)-negative cells did not differ significantly in peak position and coefficient of variation from diploid CK-positive cells, indicating negative cells as useful internal controls. Precision of ploidy determination was dependent on cell number, requiring a minimum of 3000 CK-positive cells for reliable determination. CK-gating resulted in the detection of 9 additional non- diploid tumors. S-phase values within the different ploidy groups were found in a more narrow range after CKgating, provided that sufficient cell numbers are maintained in the gated population.

6. FLOW CYTOMETRIC ANALYSIS OF MITOCHONDRIAL FUNCTION OF ONCOGENE-TRANSFORMED FIBROBLASTS ISOLATED FROM 2D- AND 3D-CULTURE
L.A. Kunz-Schughart1,2, R.C. Habbersett1, and J.P. Freyer1
1Los Alamos Natl. Lab., Life Sciences Division, Los Alamos, NM 87545, USA and 2Institute of Pathology, University of Regensburg, 93042 Regensburg, Germany
Rat embryo fibroblasts transformed to different extents by oncogene- transfection represent an excellent model for studying pathophysiological alterations accompanying tumorigenic conversion. Investigations have been carried out in monolayer and threedimensional short and long-term culture using two differently immortalized fibroblast cell lines (Rat1 and M1) and their T24Ha-ras-transfected, highly tumorigenic descendants Rat1-T1 and MR1. We have previously demonstrated that these cell lines significantly differ in their growth behavior and proliferation kinetics in 3D culture. Also, it has been documented that cellular respiration was rather influenced by proliferation characteristics than by degree of tumorigenicity in spheroids while cell line specific modifications were shown in monolayer culture.
In the present study we have stained exponentially grown and confluent monolayer cells as well as cells isolated from small (< 200 ,um), medium size (500 - 700 um) and large (> 1500 um) aggregates with the two mitochondrial- specific fluorochromes 10Nnonyl-acridine orange (NAO) and rhodamine 123 (Rh123) in order to analyze mitochondrial mass and activity. Cells were simultaneously stained with the DNA dye Hoechst 33342. Flow cytometric measurements were carried out on the Los Alamos multilaser/ multiparameter flow cytometer with integrated Coulter volume sensing. Offline data analysis was done using a locally-developed flow data analysis program based on the IDL language. Cell cycle distributions were calculated from the DNA histograms using the MultiCycle AV program (Phoenix Flow Systems).
Our results indicate that differences in mitochondrial activity per cell are mainly due to modifications in cell volume. However, the Rh123 fluorescence/cell to some extent mirrors differences in cellular oxygen uptake of non- versus highly tumorigenic cell lines. Also it could be shown, that the ratio of mitochondrial activity:mitochondrial mass decreases as a function of time in monolayer culture and as a function of the spheroid size which might be due to enhanced cell quiescence. This is in accordance with previous studies showing a fundamental decrease in the uptake of rhodamine 123 from the periphery to inner regions of Ratl -T 1 and MR1 spheroids, but no reduction in the total mitochondrial mass per unit cell volume.
This work was supported by DFG grants Ku 917/1-1/2, NIH grant CA51150, and the National Flow Cytometry Resource RR-01315.

7. CHARACTERISATION OF APOPTOSIS IN LUTEAL CEllS FROM EARLY OVARIAN CYCLE STAGES BY FLOW CYTOMETRY
Bertold Löhrke, Ralf Pöhland, Torsten Viergutz
Research Institute of Animal Biology, Dummerstorf-Rostock
Introduction:
Ovarian follicle cells differentiate to produce pregnancymaintaining steroids ( progestins, i.e., progesterone and related steroids) after ovulation, generating the corpus luteum. The corpus luteum is regressed when fertilisation of the released oocytes did not take place. The regression is attributed to be an ovarian key event, dictating ovarian cyclically. The regression is characterised by a loss of luteal cells and progression of the cycle by shifting the portion of small and large cells, indicating regulated decrease of the portion of certain cells via physiologic cell death ( apoptosis ). However, whether apoptosis or necrosis is responsible for this process is not clear cut and may dependent on the cycle stage. In a number of experimental cell systems the early stage of the apoptosis, i.e. the stage which precedes chromatinolysis is characterised by the breakdown of the mitrochondrial transmembrane potential and changes in plasma membrane structure without disintegration of the plasma membrane.
Methods:
Thus, bovine luteal cells from different ovarian cycle stages were flowcytometricly examined to produce double strand DNA ( dsDNA ) fragments, to uptake propidium iodide (Pl, indicator-for changed plasma membranes) and the negative charged oxonol dye DiBaC4(3) (indicator-for changed transmembrane potentials) as well as to oxidate uncharged dihydrorhodamine (DHR) to charged fluorescent rhodamine 123 (R123) which binds to mitochondria dependent on the inner mitochondrial transmembrane potential (IMTP).
Results and Conclusions:
The findings were that with progression of the cycle stage ( day 5 to day 8 ) the portion of large cells with dsDNA breaks increased whereas R123 fluorescence decreased, i.e. breakdown of the IMTP or/and the oxidative activity occurred. Dissipation of IMTP was accompanied with plasma membrane depolarisation and enhanced Pl uptake. Incubation with PGF2a (16h) did not affect dsDNA fragmentation and increased the portion of DHR oxidating cells. Plasma membrane integrity was maintained as the cells responded to a2-adrenergic stimulation. In conclusion, these data are compatible with the hypothesis that large luteal cells underwent apoptosis characterised by disruption of the mitochondrial potential ( linked to the oxidative activity of mitochondria ) and by an increase in plasma membrane permeability leading to depolarisation and increasing Pl uptake followed by nuclear disintegration.

8. A NOVEL, MEMBRANE RECEPTOR BASED RETROVIRAL VECTOR FOR FANCONI ANEMIA GROUP C GENE THERAPY
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 antibody was between 5 % and 30 % with Fanconi cell lines, 5 /0 with CD34+ cells and 15 % with PBL's. MMC induced G2 phase cell cycle disturbances were fully complemented in Fanconi Anemia B-cell lines of complementation group C but not in B cell lines of another FA complementation group (D). In addition to the normalization of the G2 phase arrest, induction of cell death in the FAC cell line was also decreased 3 to 10 fold at different MMC concentrations.

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.

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