ABSTRACTS 2002
BIOCHIPS AS A NEW MOLECULAR DIAGNOSTIC TOOL.
José Remacle, Christophe Van Huffel, Francoise Delongueville, Sandrine Hamels, Nathalie.Zammatteo, Isabelle Alexandre.
Eppendorf Array Technologies, 20 Rue du Séminaire, 5000 Namur, Belgium
AAT@AAT-Array.com
Biochips are a new miniaturization of molecular detection which allow to assay for hundreds of genes or DNA sequence in one test. DNA chips technology is composed of 7 steps which are:
1, the fixation of the DNA capture probes on the support;
2, the type of support used;
3, the addressing of a given capture probe at a specific spot of the surface;
4,the nature and composition of the capture probe;
5,the label used for the DNA;
6, the detection system and
7, the signal treatment. We will review the main technologies now available in the 7 steps mentioned here above for obtaining a full process of DNA chips with the emphasis given on their performances and their limitations. This is essential if we want to understand the type of applications for which they are developed and to evaluate their performance
In EAT, we developed a platform for colorimetric detection of biochips which is well adapted for the research and diagnostic applications; The spots of the microarrays appear as black and white spots and are quantified with a scanner. The method is suitable for glass as well as for plastic.
The platform was adapted for genomic as well as for gene expression chips. The first genome-based chip was the StaphyChips which is able to differentiate between the different species of Staphylococcus and their resistance to antibiotic.
The first gene expression chips was the Hepatochips for the screening of potentially toxic compound. The chips are used in the early screening of new developed drugs in order to avoid toxic or secondary effects. Several other chips linked to medical research applications are in development stage and will be released in the next few months.
The platform can also be adapted for protein chips.
FIRST EXPERIENCES OF THE BELGIAN EXTERNAL QUALITY ASSESSMENT SCHEME FOR IMMUNOPHENOTYPING.
Marjan Van Blerk, Michel Bernier, Xavier Bossuyt, Bernard Chatelain, Jean-Luc D'Hautcourt, Christian Demanet, Luc Kestens, Dirk Van Bockstaele, Jean-Claude Libeer
Department of Clinical Biology, Scientific Institute of Public Health, Brussels, Belgium
EQA Advisory Board, Belgium
The department of Clinical Biology of the Belgian Scientific Institute of Public Health initiated in 2000 a voluntary external quality assessment scheme (EQAS) open to all laboratories routinely performing flow cytometric lymphocyte phenotyping. Until now the EQAS comprised 8 send-outs. Over this period 23 blood samples were sent for evaluation to 38 to 47 laboratories. Specimens consisted of fresh EDTA anticoagulated whole blood and were sent by overnight mail. The specimens had to be tested for white blood cell count, percentage of lymphocytes, and percentages and absolute counts of CD3+, CD4+, CD8+, and CD19+ cells. The laboratories were required to mention the date of receipt, the date of sample analysis, and to provide details of the type of flow cytometer, the sample preparation technique, the source of antibodies, the gating strategy, and the data analysis software used. A non-parametric approach (method of Tukey) was employed for the statistical evaluation of the results of the participants.
Up to seven different flow cytometers were used by the 47 participants. The percentage of participants employing a single-platform approach increased from 8 to 15 % during the programme. All the laboratories applied the whole blood lysis technique. Half of the laboratories reported using 3-color fluorescence. Five laboratories were still using one-color analysis. 43.5 % of the participants used a light-scatter gate to identify lymphocytes, 45.7 % used CD45 fluorescence combined with side scatter and 10.9 % CD45/CD14 backgating.
Median intralaboratory coefficients of variation were 1.0, 1.3, 1.7, and 3.2 % for CD3+, CD4+, CD8+, and CD19+ cell percentages, respectively. Interlaboratory variability was consistently lower than 6.5 % for CD3+ and CD4+CD3+ cell percentages, and lower than 9.5 % for CD8+CD3+ cell percentages. Median coefficients of variation for the absolute values were higher, ranging from 10.4 % for CD4+CD3+ cells to 16.4 % for CD19+ cells. The overall CVs were larger for small subsets such as CD19. Between-laboratory variability remained stable over time. Analysis of individual laboratory performance indicated that some labs markedly improved their results.
The percentage of double positive CD4+CD3+ cells and in particular of double positive CD8+CD3+ cells was for several samples statistically significant lower than the percentage of total CD4+ and CD8+ cells. The between-laboratory variability for the determination of CD4+CD3+ and CD8+CD3+ cells was lower than for the measurement of total CD4+ and CD8+ cells. The number of laboratories measuring total CD4+ and CD8+ cells decreased with 30 % during the programme.
The effects of different gating strategies on the results of the lymphocyte subset assessments were tested and proved not to be significant.
These data provide valuable insights into the state of the art of flow cytometric lymphocyte phenotyping as practiced in the Belgian laboratories, allow us to conclude that these results are comparable with those obtained in similar EQAS of other countries and illustrate the important value of a national EQAS for lymphocyte phenotyping as a tool to identify existing weaknesses and to improve interlaboratory comparability of laboratory results.
A MICROBEAD ASSAY FOR THE ANALYSIS OF ENZYMES AND ENZYME INHIBITORS
Jialiang Hu, Philippe E. Van den Steen and Ghislain Opdenakker
Rega Institute for Medical Research, Katholieke Universiteit Leuven,Minderbroedersstraat 10, B-3000 Leuven
Fluorescence detection by flow cytometry is widely used as a quantitative and qualitative method to analyze immune cells. By using polystyrene beads of approximately 10 µm as a substrate carrier, St.-Pierre et al. (1996) adapted flow cytometry for enzymatic reactions.
Protease substrates, e.g. gelatin or casein, are chemically labeled with fluorescein and the labeled fraction is purified by gel filtration chromatography. Then, the labeled substrate is coupled to the polystyrene beads and the coupling efficiency is analyzed by flow cytometry. Equivalent amounts of the labeled beads (e.g. 20.000 beads per reaction vessel) are mixed with enzyme preparations, which cleave the substrate and hence diminish the fluorescence of the tagged beads. Quantitative analysis by flow cytometry is then used to measure the enzyme activity.
We used this method to quantify gelatinase activity (sensitivity down to 1 nanogram of pure gelatinase B). After standardization the method was used to define gelatinase inhibitors. Classical metalloproteinase inhibitors and metal chelators, such as EDTA and 1,10-o-phenanthroline, were confirmed to be gelatinase B inhibitors in the assay. Furthermore, we found that the active site mimic poly-histidine inhibits gelatinase B. The miniaturized method can thus be used for the high throughput screening of gelatinase B inhibitors.
Another advantage of this method is the observation that it is useful to detect net enzyme activity in crude cell supernatants and biological fluids. As an example of the former, it was found that interferon-beta decreases the net gelatinolytic activity of stimulated human dendritic cells (Bartholomé et al., 2001). The application on biological fluids is exemplified by the observation that synovial fluids of patients with rheumatoid arthritis contain net gelatinolytic activity, whereas in degenerative joint disease (osteoarthritis) no net activity was detected (Van den Steen et al., 2002). Finally, depending on the labeled substrate the principle of this assay can be adapted for the detection of enzymes and inhibitors of many kinds.
References
St. Pierre et al. Cytometry 25, 374-380 (1996)
Bartholomé et al. J. Interferon Cytokine Res. 21, 495-501 (2001)
Van den Steen et al. The FASEB J. 16, 379-389 (2002)
STEM CELL PLASTICITY
A.Gothot
University of Liège
Introduction
In many recent publications, unexpected findings were reported that adult stem cells residing in a given tissue might differentiate into cells belonging to other tissues. It was observed that bone marrow cells may give rise to neural tissue, skeletal and myocardial muscle, hepatocytes, endothelial cells, and skin, gut and lung epithelia1-7. It was also suggested that neural stem cells could generate hematopoietic progenitor cells8. The concept of stem cell « plasticity or transdifferentiation » was coined to describe these events. Following an initial wave of surprise and excitement, stem cell plasticity has become highly controversial since it has been used as much to attract interest to incomplete or preliminary studies as to bring forward truly groundbreaking discoveries. To get a better understanding of this rapidly evolving field the present paper will reiterate the basic properties of stem cells and the scientific standards that have been used for decades in stem cell research. A critical evaluation of some of the data that has been presented as evidence of stem cell plasticity will be presented.
Embryonic and adult stem cells
Both murine9, and more recently, human embryonic stem (ES) cells10 have been isolated from the inner cell mass of pre-implantation blastocysts and can be grown in an unspecialized state for numerous generations. ES cells are pluripotent: they can differentiate into specialized cell types of any tissue derived from all three germ layers of the embryo (endoderm, mesoderm, neuroectoderm). In essence, ES cells are thus plastic.
In the adult, cell turnover and tissue regeneration are dependent on the proliferation of adult, or somatic, stem cells. Adult stem cells have been identified in brain, bone marrow (which harbors distinct stem cell types as will be discussed below), skeletal muscle, liver, pancreas, epithelia of the skin and digestive tract, cornea, retina and dental pulp. Until recently, it was generally considered that adult stem cells could only give rise to specialized cells belonging to the tissue from which they were isolated. The recent cloning of a sheep from the nucleus of an adult tissue led to a renewed interest over the differentiation capability of somatic stem cells11. The current controversy over stem cell plasticity concerns the extent of the differentiation potential of adult stem cells.
Methodology of stem cell research and potential pitfalls of plasticity studies
Hallmark characteristics of adult stem cells include: 1) a self-renewal ability or the capacity to make identical copies of themselves ; 2) through asymmetric divisions, the ability to balance self-renewal with the « robust » generation of vast numbers of differentiated and specialized cells in a flexible and tightly regulated manner ; 3) the capacity to give rise to numerous distinct cell lineages. The latter property is not shared by all stem cell types. Hematopoietic stem cells, for example, are able to differentiate into numerous lineages, while other somatic stem cells, such as skeletal muscle satellite cells, produce only one cell type.
The standard methodology in stem cell research is based on clonal analysis, which is the evaluation of a cell population (or of many cell populations) derived from a single pre-existing cell. Therefore, plasticity studies should first identify and purify from a starting tissue a cell population endowed with standard features of stem cells, i.e. self-renewal and differentiation capacities. Subsequently, it should be described that the same cells may give rise, in a clonal manner, to other specialized cells when placed in a different tissue, while maintaining self-renewal capability. Most importantly, plasticity studies should include a demonstration that the « transdifferentiated cells » integrate in the recipient tissue through defined cell-cell and cell-matrix interactions and function like normal mature cells. To date, there has been few studies which meet these rigorous standards.
In many cases, the starting cells were not purified stem cells, but often crude bone marrow cells, among which many stem or progenitor cell types may be present. In the context of regenerative medicine, the exact nature of the cells responsible for tissue repair in recipient organs may be of secondary concern. However, future research aimed at optimizing cell transition and differentiation will require the knowledge of the exact nature of the starting stem cells.
Another concern frequently encountered in interpretation of plasticity studies is the rare nature of putative transdifferentiation events. In addition to the technical difficulties involved in identifying the exact phenotype of these rare cells, they may also result from genetic or epigenetic aberrations rather than from reproducible plasticity12. Furthermore, recent data indeed indicate that bone marrow cells and neural stem cells may fuse, albeit at a low probability, with ES cells in vitro, generate tetraploid pluripotent cell lines expressing donor markers and thus mimick stem cell plasticity13; 14.
Finally, the function of transdifferentiated cells has rarely been documented. Also, it should not be overlooked that plasticity events occurring after transplantation of bone marrow or hematopoietic stem cells may result from tissue infiltration by macrophages and phagocytosis of recipient cells, with subsequent co-localization of donor and recipient cell markers.
The heterogeneity of stem cell types
Data reported in the papers cited above may be interpreted as evidence that stem cells normally committed to generate specific cell types may be reprogrammed to an alternative differentiation pathway upon relocation to another tissue. Most, if not all, data is consistent with a different interpretation which considers that multiple stem cell types may coexist, notably in the bone marrow, and maintained in an undifferentiated state. When transplanted to other organs or tissues, such cells may respond to local environmental cues and reveal the corresponding differentiation potential. The latter hypothesis is strongly supported by the recent characterization of multipotent adult progenitor cells (MAPC) isolated from bone marrow15. A rigorous clonal analysis has demonstrated that individual MAPCs display broad capabilities and give rise to differentiated cells from all three germ layers when stimulated with specific growth factors in vitro. In vivo, single MAPCs were able to contribute to most somatic cell types when injected into early blastocysts. Therefore, multiple stem/progenitor cell types may reside in the adult bone marrow and possibly derive from even more primitive MAPCs.
Beyond stem cell plasticity
Whether or not stem cell plasticity will be rigorously demonstrated and reproduced, the renewed interest in somatic stem cell biology has provided a wealth of information potentially useful in so-called regenerative medecine. For the basic researcher, a major challenge will be to identify the genetic programs regulating self-renewal and the maintenance of stem cells in an undifferentiated state16; 17. Also of utmost interest will be to delineate microenvironmental components directing self-renewal and differentiation18 since « the most relevant way to view a stem cell is as a biological activity rather than a discrete independent cellular entity »19.
References
1. Brazelton TR, Rossi FM, Keshet GI, Blau HM. Science 2000;290:1775-1779.
2. Gussoni E, Soneoka Y, Strickland CD, et al. Nature 1999;401:390-394.
3. Orlic D, Kajstura J, Chimenti S, et al. Nature 2001;410:701-705.
4. Petersen BE, Bowen WC, Patrene KD, et al. Science 1999;284:1168-1170.
5. Lagasse E, Connors H, Al Dhalimy M, et al. Nat Med 2000;6:1229-1234.
6. Asahara T, Masuda H, Takahashi T, et al. Circ Res 1999;85:221-228.
7. Krause DS, Theise ND, Collector MI, et al. Cell 2001;105:369-377.
8. Bjornson CR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL. Science 1999;283:534-537.
9. Evans MJ, Kaufman MH. Nature 1981;292:154-156.
10. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Science 1998;282:1145-1147.
11. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Nature 1997;385:810-813.
12. Morshead CM, Benveniste P, Iscove NN, van der Kooy D. Nat Med 2002;8:268-273.
13. Terada N, Hamazaki T, Oka M, et al. Nature 2002;416:542-545.
14. Ying QL, Nichols J, Evans EP, Smith AG. Nature 2002;416:545-548.
15. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Nature 2002;418:41-49.
16. Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA. Science 2002;298:597-600.
17. Terskikh AV, Easterday MC, Li L, et al. PNAS 2001;98:7934-7939.
18. Hackney JA, Charbord P, Brunk BP, et al. PNAS 2002;99:13061-13066.
19. Lemischka I. Exp Hematol 2002;30:848-852.
Interested readers may refer to extensive information on stem cells available on the NIH website at www.nih.gov/news/stemcell/index.htm.
János Szöllösi
Applications of FRET
Long wave fluorophores
DETECTION AND LOCALIZATION OF MAC-1 AND L-SELECTIN INTRACELLULAR ANTIGENS IN BOVINE POLYMORPHONUCLEAR NEUTROPHIL LEUKOCYTES BY FLOW CYTOMETRY AND CONFOCAL MICROSCOPY
Araceli Diez-Fraile, Evelyne Meyer, and Christian Burvenich.
Ghent University, Faculty of Veterinary Medicine, Department of Physiology, Biochemistry, and Biometrics, Salisburylaan 133, B9820 Belgium.
Objective: This study aimed at investigating the existence and the localization of Mac-1 (CD11b) and L-selectin (CD62L) intracellular pools in bovine polymorphonuclear neutrophil leukocytes (PMN). Therefore, a flow cytometric technique to detect the surface bound as well as intracellularly stored CD11b and CD62L in bovine PMN was developed. In addition, confocal microscopy was used as a first means of examining the localization of these glycoproteins.
Methods: Immunofluorescence staining of surface antigens on bovine PMN was performed in heparinized blood aliquots of 100 µl incubated for 30 min at 37C with saturating amounts of anti-bovine CD11b or CD62L monoclonal antibody (mAb). After incubation, red blood cells were lysed and fluorescein isotiocyanate (FITC)-labeled secondary antibody was added for 30 min at 4C in the dark. Stabilization of cell membranes with preservation of intracellular antigenicity was achieved with 4% paraformaldehyde. This pretreatment fixes the cells and prevents their destruction. Thereafter, permeabilization was performed with different concentrations of saponin or n-octyl-β-D-glucopyranoside. Cells were further incubated with the primary mAbs and FITC-labeled secondary antibody in order to stain the intracellular antigens. Cellular fluorescence was analyzed with a FACScan flow cytometer. The different leukocyte subsets were identified by forward and side light scattering characteristics and by specific mAb. The PMN population was gated and results were expressed as mean fluorescence intensity (MFI). For examination by confocal microscopy, cell nuclei were additionally stained with propidium iodide and cellular distribution of CD11b and CD62L was evaluated.
Results: Saponin treatment elicited only small changes in PMN fluorescence of control samples and left the PMN intact. On the other hand, n-octyl-β-D-glucopyranoside treatment resulted in an increase of PMN autofluorescence and cellular debris. The optimal concentration of saponin for measuring the total detectable pool of CD11b and CD62L was found to be 1%. To evaluate the precision of the method, ten separate blood samples from one animal were collected and non-permeabilized and saponin-treated samples were analyzed within one day. The average MFI for the extracellular and the total pool of CD11b was 21.7 (CV = 5.8%) and 34.7 (CV = 6.9%), respectively. For CD62L this average was 25.9 (CV = 2.2%) and 34.9 (CV = 4.1%), respectively. The MFI in unstimulated PMN as assessed in 17 cows was found to increase 1.52 and 0.57 fold for CD11b and CD62L, respectively. Additional confocal microscopy of permeabilized PMN indicated that the increased amount of CD11b was distributed throughout the cytoplasm while CD62L was located close to the cell membrane.
Conclusions: Research conducted in our laboratory has lead to the optimization of a method that allows flow cytometric assessment of intracellular antigens in bovine PMN. Detergent permeabilization studies in bovine PMN indicate that CD11b and CD62L are stored in latent pools as detected by flow cytometry. Confocal microscopic studies showed that both molecules exhibit a different pattern of subcellular distribution.
This study was supported by a Marie Curie Research Training Grant from the European Community (no FAIR-BM-974214) and the Exploring European Research Grant from the Ghent University (no 011V0401).
Preferred format: Poster presentation
E-mail: Araceli.Diez@rug.ac.be
MULTIPLE MYELOMA DISEASE PROGRESSION IS A MULTISTAGE AND DYNAMIC PROCESS OF DIFFERENTIATION, PROLIFERATION, INVASION APOPTOSIS AND ANGIOGENESIS.
Kewal Asosingh1, Hendrik De Raeve2, Ivan Van Riet1, Ben Van Camp1, Karin Vanderkerken1.
1Deparment of Hematology and Immunology, Vrije Universiteit Brussel (VUB), 2Department of Pathology, Universitair Instituut Antwerpen. K. Asosingh and K. Vanderkerken are Postdoctoral Fellows of the Fund for Scientific Research-Flanders (Belgium) (F.W.O.-Vlaanderen).
At time of diagnosis, multiple myeloma (MM) is already an well established disease. The processes involved in the earlier stages of the disease are, however, unknown.
We investigated the differentiation, proliferation, invasion and apoptosis of MM cells during disease progression in the 5T2MM murine model. Naive mice were injected with 5T2MM cells and from the onset of the experiment three mice were sacrificed in time intervals of one week until the end stage. Myeloma cells were isolated from the bone marrow and were selected by sequential gating of 5T2 idiotype positive cells by flow cytometry. Microscopic analysis of these sorted 5T2 idiotype positive cells confirmed their identity as true myeloma cells. During the entire development of the disease, there was a good correlation between serum M-component (paraprotein) concentration and tumor load in the bone marrow (R2= 0.987). Based on the paraprotein concentration, three disease stages were distinguished: a quiescent stage of slow tumor progression (increase of paraprotein from 0-0.20 g/dl, in a time interval of 7 weeks after tumor injection), an intermediate stage of moderate tumor progression (increase of paraprotein from 0.30-0.57 g/dl, within a time interval of 3 weeks, (7-10 weeks after tumor injection)) and an end stage of accelerated tumor progression (increase of paraprotein from 0.64-1.2 g/dl, in a time interval of 1 week (10-11 weeks after tumor injection)).
In the quiescent stage, the majority of the myeloma cells had a CD45+CD138-IL-6Rα+ phenotype, corresponding with an immature, invasive and apoptosis resistant nature. In the end stage the majority of the myeloma cells were CD45-CD138+IL6Rα- of phenotype, corresponding with a mature, less invasive and apoptosis sensitive nature. In the intermediate stage a gradual transition from the quiescent phenotype towards the end stage phenotype was observed. In line with these data, functional analysis of sorted 5T2MM cells demonstrated a decrease in Matrigel invasive capacity (5.8 + 1.0% and 2.4 + 1.2%, mean + SD values of quiescent stage and end stage, respectively, p< 0.02) and an increase in apoptosis sensitivity (propidium iodide/Annexin V-FITC staining) (2.4 + 0.5% and 25.6 + 2.7%, mean + SD values of quiescent stage and end stage, respectively, p< 0.0001) of the MM cells during disease progression. Bromodeoxyridine labeling demonstrated an increase in MM cell proliferation (9.2 + 2.3% and 30.9 + 3.7%, mean + SD values of quiescent stage and end stage, respectively, p< 0.001). In the intermediate stage a gradual transition of invasion, apoptosis and proliferation towards the end stage was observed. Microvessel density, assessed by histological analysis of bone marrow samples stained for CD31, demonstrated an increase in angiogenesis during the disease progression (positive correlation with paraprotein and tumorload, R2 = 0.981 and 0.987, respectively). These data suggest that myeloma disease progression is multistage and dynamic process of differentiation, proliferation, invasion, apoptosis and angiogenesis.
ASSESSMENT OF OXIDATIVE STRESS ON SUPEROXIDE ANION PRODUCTION, MEMBRANE PERMEABILITY, SIZE AND GRANULARITY OF FOUR BACTERIAL STRAINS BY FLOW CYTOMETRY.
Baatout S., De Boever P.(•), Derradji H., Marty F., Hendrickx L., Mergeay M.Laboratory of Radiobiology and Laboratory of Microbiology (•), Belgian Nuclear Research Center, SCK-CEN, Mol, Belgium (sbaatout@sckcen.be; tel : +32-14-33.27.29).
Flow cytometry provides a powerful means to measure a wide range of cell characteristics. However, its use in microbiology is still scarse. In order to estimate fine variations associated with oxidative stress, preliminary flow cytometric tests were performed to estimate the maintenance of membrane integrity and the production of superoxide anions in four bacterial strains. These strains were chosen for their potential use in either nuclear bioremediation or life support systems for use in space (Ralstonia metallidurans, Escherichia coli, Shewanella oneidensis and Deinococcus radiodurans).
The maintenance of membrane integrity is commonly measured as an indicator of cell damage or cell death. Propidium iodide (PI) is a fluorescent stain that can be used to estimate membrane integrity. It passively enters stressed or injured cells and intercalates into DNA and RNA. Since the influx of PI can be correlated with the extent of bacterial wall permeability, the mean fluorescence of the bacterial population submitted to the stress will be inversely proportional to the number of viable cells.
The monitoring of reactive oxygen species (ROS) levels in bacteria is critical for both studying the mechanism of damage and evaluating the impact of oxidative stress. The production of superoxide anions (O2•-) was studied with the use of hydroethidine in combination with the above mentioned bacterial strains. Hydroethidine is a nonfluorescent compound that can diffuse through cell membranes, and that is rapidly oxidized in ethidium (measured in red fluorescence) under the action of O2•-.
In this study, the oxidative stress consisted in a 30 minutes incubation with H2O2 at various concentrations (0; 27,5; 55; 110; 220; 440 and 880 mM). Membrane integrity analysis was based on the capacity of the above-mentioned bacterial strains to exclude PI and superoxide anion production was reflected by the production of ethidium. Variations in forward scatter, side scatter and red fluorescence were monitored for each cell on an EPICS XL (Beckman-Coulter) using logarithmic amplification gain. The discriminator was set to trigger on forward scatter. Triplicate samples were analysed for each experiment.
Treatment with H2O2 induced differential effects on the four strains tested. For E. coli and R. metallidurans, treatment with H2O2 induced a dose-dependent increase of membrane permeability and ethidium production. In the case of S. oneidensis and D. radiodurans, a trend for reduced membrane permeability with increased H2O2 concentrations can be observed. However, this phenomenon did not correlate with a dose-response related change of ethidium production. The forward scatter of S. oneidensis and D. radiodurans cells increased with H2O2 dose, whereas the side scatter was reduced. The side scatter was increased for E. coli and R. metallidurans when applying a H2O2 stress.
In conclusion, this study shows that flow cytometry may be used to detect the different sensitivities and/or reactions of bacteria towards oxidative stress. Flow cytometry analytical technique may allow a detail and precision of measurements of cell function and physiological status. Further studies are underway to investigate the differential behaviour of the above mentioned strains and to measure additional viability parameters such as variations in membrane potential, enzymatic activity and intracellular pH. These analyses will be compared with classical culture platings to estimate survivability. This work has been partially supported by ESA/ESTEC (contract number n15680/01/NL/ND).
DETECTION AND QUANTIFICATION OF CBFB-MYH11 FUSION GENE TRANSCRIPTS IN BONE MARROW AND PERIPHERAL BLOOD IN ACUTE MYELOID LEUKAEMIA BY REAL-TIME RQ-PCR AT DIAGNOSIS AND DURING FOLLOW-UP.
N. Boeckx, C. Brusselmans, W. Goossens, X. Bossuyt
Laboratory Medicine, Dept. of Haematology, University Hospital Leuven, Belgium
INTRODUCTION
Real-time quantitative polymerase chain reaction (RQ-PCR) using TaqMan technology allows detection and quantification of leukaemia-specific chromosome aberrations such as inv(16)(p13;q22) or t(16;16) (p13;q22) resulting in a CBFB-MYH11 fusion gene transcript.
MATERIALS AND METHODS
We analysed retro- and prospectively 9 diagnostic (5 bone marrow (BM) and 4 peripheral blood (PB)) samples and 46 follow-up (18 BM and 28 PB) samples taken from 5 de novo acute myeloid leukaemia (AML) patients with a CBFB-MYH11 fusion gene transcript, type A. Patients were followed for a median of 10 months (range: 1-26) after diagnosis.
The RQ-PCR primers and probes used were developed in an international collaborative study (Europe Against Cancer program). All analyses were performed in triplicate. Threshold cycles were determined after 50 cycles. A standard curve was established using serial dilutions of the corresponding plasmids. Copy numbers of fusion genes were normalised to the housekeeping gene b-glucuronidase (GUS).
RESULTS
At diagnosis, all patients displayed the type A CBFB-MYH11 fusion gene transcript in BM and/or PB. At follow-up, 13 out of 18 BM and 12 out of 28 PB samples were positive.
GUS quantification was possible in all samples with amounts of transcript ranging from 9201 to 199366 (median, 36397) in BM and from 76 to 199851 (median, 21356) in PB. The amount of CBFB-MYH11 fusion gene transcripts ranged from 22637 to 390557 in diagnostic BM samples and comparable amounts were found in diagnostic PB (range 17050 to 369707). In follow-up samples ranges from 1 to 19736 (median, 7) in BM and from 1 to 165 (median, 5) in PB were found.
After induction therapy, all patients were in hematological remission, but no patient achieved a molecular remission. The disease reduction following induction course ranged from 390- to 47000-fold in 4 available BM samples. The decrease of leukaemic cells in PB was even more pronounced. A further reduction of minimal residual disease (MRD) levels was seen after first and/or second intensification, or transplantation.
Clinical outcome of the patients: 1 patient died 5 weeks after diagnosis due to respiratory insufficiency and multiple organ failure, 2 patients achieved a molecular remission, 1 patient is still MRD positive after first intensification, and 1 patient remains MRD positive 3 months after completion of consolidation therapy. Moreover, in this patients a significant (>10 fold) increase of MRD was found in her latest samples, which might be an indication of impending clinical relapse.
CONCLUSION
In AML with a CBFB-MYH11 fusion gene transcript, RQ-PCR can be used to monitor the kinetics of the tumor-load reduction during early treatment in BM and PB. RQ-PCR is a powerful method to quantify MRD levels post-chemotherapy and post-transplantation.
PMA INDUCES SIMULTANEOUSLY NEUTROPHIL METABOLIC BURST AND ACCELERATION OF APOPTOSIS.
B. Cantinieaux,F Ntakibirora, C Kails, and V Kerrels.
Laboratory of Hematology. Centre Hospitalier Universitaire Saint-Pierre. Université Libre de Bruxelles. 322, rue Haute 1000. Brussels. Belgium
Background: Oxidative stress has been reported to be one major cause of apoptosis. However, this fact is controversed in neutrophils (PMN). These cells are specialised in the production of Reactive Oxygen Species (ROS) to ultimately kill ingested bacteria and are constitutively programmed to undergo apoptosis.
Aim: The purpose of our study was to objectivate in cytometry the relationship between the production of ROS induced by 3 different stimulants and the apoptotic process. Fmlp activates PMN by membrane receptors, NaF by protein G and PMA by protein kinase C (PKC) stimulation. Effect of PMN priming by PMA on the apoptosis was also assessed.
Methods: Neutrophil suspension from 30 healthy controls was performed thanks to Ficoll-Hypaque. Incubation was done during 2 hours in presence of PMA in priming concentration (10-9M), in stimulating concentration (10(6M), in fmlp (5 10(6M), in NaF (25 mM).
Simultaneous measurements of apoptosis/necrosis and metabolic burst were performed by:
-Annexin V FITC / propidium iodide (PI) exclusion
-DCFH-DA/ CD16-PECy5
In a second time, 3 inhibitors of the metabolic burst were added 15' min before the PMA 10(6M to dissociate the role of PKC as concomitant stimulant of ROS production and of apoptosis: catalase (2000 U/ml), superoxide dismutase (SOD; 2000 U/ml) and staurosporin (10-5M), inhibitor of PKC.
Results: Neutrophil apoptosis was significantly (•••) increased (Annexin-V fixation increase and CD16 decrease ) by incubation in presence of only PMA in stimulating concentration. DCFH-DA mean was significantly increased in presence of fmlp and PMA (•••) and stronger increased in PMA than in fmlp. Necrosis reached 25% in NaF and was no significant in the 2 other medias.
(•): p<0.05; (••): p<0.01; (•••): p<0.001.
| Buffer | fmlp 5*10-6M | PMA 10-6M | |
| Annexin V % | 5 +/- 2.6 | 7 +/- 3.9 | 30 +/- 15.7 • |
| CD16 % | 76 +/- 13.5 | 71 +/- 16.6 | 19 +/- 12.5 ••• |
| DCFH-DA mmn | 24 +/- 18.6 | 36 +/- 25.3 ••• | 144 +/- 113.1 ••• |
| IP % | 2 +/- 3 | 2 +/- 2.9 | 5 +/- 3.1 |
In presence of PMA 10(6M , SOD (•), catalase (••) and staurosporin (•••) decreased the DCFH-DA mean fluorescence intensity. Catalase (•) and staurosporin (••) increased the CD16 %. Staurosporin decreased the DCFH-DA % (••) and normalized the CD16 ( %:•• and mean:•••) but had no effect on the Annexin-V fixation.
A correlation between the CD16 % decrease and the DCFH-DA mean increase in PMA was objectivated: r=0.95; n=8 (•••).
Discussion: Physiological stimulation by fmlp doesn't induce apoptosis allowing PMN to kill eventual ingested bacteria. On the opposite, massive ROS production with PMA induces the apoptotic process.
PKC stimulation seems to be crucial, both for metabolic burst and apoptosis in PMN. ROS appear to be related with the CD16 expression but not with Phosphatidyl-Sérine translocation
The relationship between ROS production and the apoptotic process remains however doubtful. Other mechanisms of caspases stimulation by PMA through PKC could be involved.
TYPING OF PLATELET ANTIGEN HPA1 POLYMORPHISM BY CYTOMETRY AND ELISA.
Hennaux V(1), Delcourt J(2), Larcin S(1), Osselaer JC(2)
(1)Laboratoire d'hématologie Hopitaux St-Joseph,Ste-Thérèse et IMTR 6060 Gilly.(2)BTC Mont-Godinne, Cliniques Universiataires UCL de Mont-Godinne, 5530 Yvoir
INTRODUCTION:
The polymorphism of platelet antigen HPA 1 is carried by the GPIIIa and is responsible for 80 to 90 % of the cases of neonatal allo-immune thrombocytopenia (NAIT) after a maternal alloimmunisation against this platelet antigen.Fetal or neonatal thrombocytopenia, when severe, may result in intracerebral hemorrhage leading to hydrocephalus or death of the fetus or newborn. Neurological impairment may also occur.
It is essential to type the mother's HPA1 system to predict this immunisation. HPA1a negative mothers are at high risk of developping such an immunisation. For this reason, they need an adapted follow up.
AIM OF THE STUDY:
The aim of the study was to type the polymorphism HPA1 (HPA1 a/a or HPA1 a/b or HPA1 b/b) of 100 pregnant women by two methods (cytometry and ELISA), to determine the most accurate and easily adaptable to routine conditions.
The HPA1a negative or the HPA1 b/b and the uncertain results were typed by PCR with specific primers.
METHODS:
Cytometry: the whole blood is incubated simultaneously with 2 monoclonal antibodies. One present a weak affinity for the HPA1 b antigen and the other one binds the GPIIIa independently from the polymorphism of HPA-1.
The quantification of the binding capacity of the two MoAbs allows us to calculate the ratio and then deduce the HPA1 phenotype.
ELISA: an anti-HPA1a conjugated to the peroxydase is added to a microplate coated with an anti-GPIIbIIIa monoclonal antibody.The whole blood is incubated and revealed the presence of absence of HPA1a.
RESULTS:
The frequency obtained by cytometry is:
HPA1 a/a: 68%
HPA1 a/b: 30%
HPA1 b/b: 2%
All the HPA1a positive results with ELISA were also positive with cytometry.
The two negative results (HPA1b/b) with cytometry were confirmed by PCR. One of these samples gave an ambiguous result with ELISA.
CONCLUSION:
Cytometry is an accurate technique for HPA1 platelet antigen typing
It is nevertheless important to notice that the specimen must be treated within 4 days to yield valid results.
FLOW CYTOMETRY AND BIOSAFETY : PROTECTIVE MEASURES
Philippe HERMAN, Florence TARDY, Bernadette VAN VAERENBERGH and William MOENS, Section of Biosafety and Biotechnology, Institute of Public Health, Brussels, Belgium.
INTRODUCTION : Biosafety aspects in a flow cytometry (FCM) routine or research laboratory should be taken into account mainly when working with unfixed and infectious materials. Biohazards can theoretically arise either from sample handling or more specifically from aerosols and droplets generated by the flow itself. Biological specimens can contain documented or not documented pathogens. As a consequence, flow cytometer users are subjected to occupational exposure through accidental inoculation, droplets or aerosols. Such exposure risk is even higher when flow cytometry is organized as a central service, the operator not being necessary aware of all the details concerning the safety status of the handled material. These considerations justify some following recommendations about sample handling, FCM procedures related to aerosols containment and waste management.
1. Sample handling :
Awareness about the origin of the sample, the potential presence of infectious agents or genetically engineered material is a key to protection of the lab scientists. All unfixed materials (blood samples, various body fluids, cultured cells and environmental samples) are a priori considered as biohazardous. The use of a fixative is not always sufficient to eliminate all type of biohazards. The use of appropriate safety cabinet for sample preparation is highly recommended. Gloves and protective clothing should always be worn by flow cytometer operators.
2. FCM procedures related to aerosols containment :
Only experienced and well-trained operators should perform potentially biohazardous cell sorting. Cell-sorters are equipped with a nozzle to form a jet of microdroplets : this experimental step can is likely to generate aerosols. Instrument failures such as clogged sort nozzle or air in the fluidic system can drastically increase aerosol formation.
For this reason biohazardous materials should not be sorted unless supplementary suitable containement measures are applied. For instance a droplet containment module should be installed to reduce the risk of exposure to generated droplets and aerosols.
3. Waste management :
All biohazardous material must be decontaminated in a separate room and placed in leak-proof containers which are sealed before evacuation.
CONCLUSIONS : All scientists in the FCM field must be aware of the potential hazards associated with their discipline. Guidelines on handling and proper disposal of biohazardous materials should be applied in all FCM laboratories. General recommendations approved by the International Society of Analytical Cytology should help to settle a basis for biosafety guidelines in FCM laboratories (Schmid et al., 1997). A biosafety sheet should be filled in and approved by the FCM laboratory team before starting any new set of experiments. The establishment of a local biosafety committee including experts in FCM is strongly recommended in order to control the containement efficacy of the cell sorter.
Reference :
Schmid et al.,(1997) Biosafety Guidelines for Sorting of Unfixed Cells.
Special reports, Cytometry 28 : 99-117.
ORGANOTYPIC CULTURE: AN EFFECTIVE IN VITRO MODEL FOR THE DEVELOPMENT OF NEW IMMUNOTHERAPEUTIC APPROACHES FOR MUCOSAL (PRE) NEOPLASTIC LESIONS
N. Jacobs, I. Renard , P. Hubert, L. Havard, , J. Boniver and P. Delvenne
Dpt of Pathology, University Hospital of Liège, CHU Sart Tilman, 4000 Liège, Belgium
The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous carcinoma which develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (IFNγ and TNFα) and by integrating activated lymphocytes, dendritic cells or lymphocytes armed with bispecific antibodies into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium which develop on a mucosal surface.
DETECTION OF HUMAN INFLAMMATORY CYTOKINE SERUM LEVELS USING THE CBA KIT-I (BD")
H. Jongen(1), E. Bailleul(1), V. Peeters(1), R. Cartuyvels(1), K. Magerman(1), A.Mewis(1), R.Claeys(1), M. Raes(2) and J.L.Rummens(1)
(1) Laboratory of hematology and immunology, (2) Department of pediatrics; Virga Jesse Hospital, Hasselt, Belgium.
Well-known laboratory parameters predicting infection such as leucocyte count, differential count and C-reactive protein (CRP) are either unspecific or not useful for the early diagnosis of serious bacterial infections, especially in infants and young children. More recently the concentration of cytokines such as IL-6 and IL-8 have been reported to offer a better diagnostic accuracy in specific clinical situations such as fever without localising signs.
In this study we evaluated the usefulness of the Cytometric Bead Array (CBA) Kit-I (BD") for the determination of cytokines in serum. The CBA kit allows the simultaneous measurement of six human cytokines (IL-8,IL-1β,IL-6,IL-10,TNF-α and IL-12p70) in a single sample using microparticle-based flowcytometric technology. This method combines the principles of the "sandwich" immunoassay with the capability of flow cytometry for simultaneous measurement of the characteristics of multiple particles. We compared the CBA Kit with the conventional quantitative sandwich enzyme immunoassays (ELISA - PharMingen").
One hundred consecutive drawn samples collected from a random population of 50 children and 50 adults were analysed. On each sample CRP, leucocyte count and differential count were performed. All samples were incubated with a mixture of capture Ab-bead reagent and detector Ab-phycoerythrin (PE) reagent, and analysed using flow cytometry. The fluorescence intensity measured is proportional to the concentration of the cytokines and is quantified from a calibration curve.
Significant differences were found in cytokine levels determined with the CBAKit-I compared with the ELISA assays (p<0.024 to p<0.0001- Mann-Whitney U), except for IL-6 and IL-1β concentrations in children (p=0.676 resp. p=0.977). These results were in contrast with previously reported data obtained in healthy donor's serum.
No correlation could be demonstrated between different levels of CRP, absolute neutrophil and lymphocyte counts and any of the cytokine concentrations. Pearson's product correlation coefficients varied from 0.0006 to 0.0942 in children and from 0.0090 to 0.2545 in adults.
The CBA provides some important advantages : only a small sample volume is needed, a single set of diluted standards is used, the experiment takes less time than a single ELISA and has a wider dynamic range (up to 5000 pg/ml) which leads to less dilution errors than ELISA. These dilution errors could be one of the causes for the lack of correlation between both methods.
We concluded that CBA is an appropiate tool for the evaluation of cytokine concentrations in human serum. The potential for application of cytokine determinations depends on the technique used. Data obtained in this study may indicate that the determination of inflammatory cytokine levels by CBA has to be considered as an independent parameter for the evaluation of human inflammatory diseases. Prospective studies are needed to confirm these results and to define evidence-based recommendations for the application of CBA in different clinical settings.
ISOLATION AND CHARACTERIZATION OF ENDOTHELIAL CELLS DERIVED FROM SAPHENOUS VEIN SEGMENTS WITH THE AIM OF SEEDING THEM ON A BIOMATERIAL.
A.Ooms, M. Humblet #, M.-E. Dumez, P.Motte +, N.Schaaf Lafontaine # and J.-O.Defraigne.
CREDEC Tour de Pathologie, CHU , ULg Liège, # Laboratoire d 'Hématologie , CHU, ULg Liège,+Laboratoire de Biologie cellulaire végétale , Département des Sciences de la Vie ,ULg, Liège
Objectives : In a first step, the isolated cells from saphenous vein segments are cultured in vitro .The growing cells are harvested and selected according to the expression of CD31. Then, the expanded cells are characterized and their growth capacity will be tested on a biomaterial such as Cardiofix (from Sulzer Carbomedics, Austin Texas).
Methods : The endothelial cells from saphenous veins were derived according to an enzymatic procedure ( with a 0,2% collagenase solution).The mixture of cells obtained after collagenase digestion were cultured altogether in a endothelial cells growth medium. After a few days in culture, when the cells reached confluence, they were harvested . An enrichment step was performed with an anti-CD31m Ab, recognizing the surface marker PECAM-1 expressed by endothelial cells, conjugated to magnetic beads. This procedure gives a new cell fraction cultured in the same conditions. The expression of von Willebrand marker, a well-known endothelial marker is evaluated by immunostaining (avidin-biotin method). The level of expression of CD31 and CD34 markers was assessed by flow cytometry analyses.The cells functioning was improved by measurement of HLA-DR and E-Selectin level of expression with or without treatment of endothelial cells respectively in the presence of γIFN or TNFα. After this characterization step, the cells were seeded on Cardiofix membrane.The staining of actin filaments with phalloidin FITC has made possible the visualization with a laser scanning microscope, of the endothelial cells plated on Cardiofix membrane.
Results : After the enrichment step, the majority of cells expressed the von Willebrand factor. Also the CD31 and CD34 positivity was measured. The level of expression of HLA-DR and E-selectin molecules was strongly upregulated when the endothelial cells were activated by respectively γIFN and TNFα.We have preliminary results showing the localization of actin filaments in the cells seeded on cardiofix membrane.
Conclusions : The preliminary data show that we have the capability to derive cells with endothelial phenotype from saphenous vein segments and that is possible to seed them on Cardiofix membrane.
DIAGNOSTIC STRATEGIES IN A CASE OF SEVERE COMBINED IMMUNODEFICIENCY
J. Penders1, H. Vandenbussche1, E. Vandecruys2, J. Verlooy2, F. De Baets3, S. Van Daele3, F. Haerinck3, P. Schelstraete3, J. Philippé1, B. Verhasselt1
1Clinical Biology, 2Pediatrics Hemato-Oncology and 3Pediatrics Pneumology, University Hospital Ghent, Belgium
Introduction: A 5-month old girl from consanguine parents, showing respiratory complaints since 2 months and one episode of viral meningitis, was admitted to our hospital with fever and clinical symptoms of severe sepsis.
Materials and Methods: Flow cytometry was performed using a FACSort (BectonDickinson); serum immunoglobulin analysis on a Behring Nephelometer II analyser; serum total protein on a Modular(r) P System (Roche) and WBC count on a XE2100 System (Sysmex).
Results:
| upon | after transplantation | upon diagnosis | |||
| diagnosis | 20 days | 2 months | |||
| WBC/µL | 4000 | 4000 | 5510 | Protein g/dL | 4.9 |
| Lymphocytes | 560 | 720 | 937 | IgG g/L | 0.5 |
| T cells CD3+ | 39 | 137 | 272 | IgG2 g/L | 0.14 |
| Th cells CD3+4+8- | 39 | 130 | 187 | IgG3 g/L | 0.023 |
| Tc/s cells CD3+4-8+ | 0 | 7 | 84 | IgM g/l | 0.2 |
| B cells CD19+ | 0 | 0 | 0 | IgA g/l | absent |
| NK cells CD56+16+3- | 403 | 497 | 412 | IgE kU/L | <4.4 |
Upon diagnosis, T cells were positive for IL7R, TCR αβ (60%), TCR γδ (40%) and HLA1, a subfraction of the lymphocytes (CD7 +) expressed HLA-DR. Additional tests showed that adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities were present and rag1 and rag2 mutations were absent.
Discussion: Based on the combined clinical, chemical and flow cytometric data, a diagnosis of SCID was obvious. Furthermore, flow cytometry was helpful in excluding several subtypes: e.g. X-linked SCID (girl, NK+), JAK3 (B cells absent), ZAP-70 deficiency (CD4+ absent), Bare lymphocyte (HLA-DR+, CD8 absent) and IL2 deficiency (low CD3+, CD8+ absent). Measuring enzymatic activities could exclude two more subtypes (ADA and PNP deficiency). Genetic analysis is needed for a final diagnosis but has not been conclusive so far.
After transplantation, flow cytometry is one of the best techniques to follow the cellular evolution as illustrated in the table.
Conclusion: In diagnosing immunodeficient individuals, flow cytometry plays a central role in first line strategies because it is an easy-to-use, widely available and fast technique. Combining flow cytometric data with clinical and chemical findings permits a diagnosis of SCID to be made. Moreover, with flow cytometry, classification is possible for most frequent SCID-subtypes.
Enzymatic and genetic analysis are needed for final diagnosis but especially the latter being much more time-consuming and restricted to specialised laboratories.
EVALUATION OF CHEMOKINE RECEPTOR ANTAGONISTS: COMPARING FLIPR AND FLOW CYTOMETRIC TECHNIQUES
K. Princen1, S. Hatse1, K. Vermeire1, E. Fonteyn1, G.J. Bridger2, E. De Clercq1 and D. Schols1.
1 Rega Institute for Medical Research, K.U.Leuven, Belgium; and 2 AnorMED, Langley, Canada.
The bicyclams were identified as potent small-molecule HIV entry inhibitors, interacting specifically with CXCR4, the main coreceptor used by pathogenic T-tropic (CXCR4- using) HIV variants to infect the cells. AMD3100, 1,1'-[1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclo-tetradecane, the prototype compound of the bicyclams, is the most potent and selective CXCR4 antagonist that has been described so far. Here, we evaluated two cell-based fluorescent methods, (i) the 96-well plate Fluorometric Imaging Plate Reader (FLIPR) system, an effective screening tool with high throughput capabilities, and (ii) flow cytometry (FACScan, FACScalibur), to measure the SDF-1-induced intracellular Ca2+ mobilization via CXCR4. In four CXCR4+ T lymphocytic cell lines (Sup-T1, MOLT-4, MT-4 and CEM/0 cells) and one monocytic cell line (THP-1 cells) commonly used inHIV screening laboratories, SDF-1 elicited a transient increase in intracellular Ca2+ concentration. For each cell line, the magnitudeof Ca2+ responsecorrelated closely with the level of CXCR4 expression on the cell membrane, i.e. the cells with the highest CXCR4 level showed the strongest calcium response. AMD3100 dose-dependently inhibited the SDF-1-induced Ca2+ flux in all cell lines examined. The FLIPR was more sensitive than the flow cytometer in detecting minor Ca2+ responses andin evaluating the ability of compounds to blockthe specific chemokine-induced transient intracellular Ca2+ flux. In freshly isolated PBMCs, the Ca2+ signaling was solely due to the stimulation of monocytes and granulocytes, whereas the T lymphocyte population, although clearlyexpressing CXCR4, surprisingly did not respond at all. This phenomenon could be observed with the aid of the flow cytometer and not by the FLIPR. Stimulation of T lymphocytes with PHA and IL-4 for 7 days induced an upregulation of cell surface CXCR4, which resulted in an increase in intracellular Ca2+ concentration upon stimulation with SDF-1. Also, in these activated T cells AMD3100 inhibitedthe SDF-1-induced intracellular Ca2+ mobilization in a dose-dependent manner. In conclusion, combination of resultsobtained by FLIPR and flow cytometry assays can lead to new insights that can help inthe search for novel CXCR4 antagonists with potent anti-HIV activity.
Preferred format: Poster or Oral presentation
DIAGNOSIS AND PROGNOSIS OF ACUTE LEUKEMIA USING CELLULAR DNA CONTENT BY FLOW CYTOMETRY.
V. Schlesser(1), M. Chatelain(1), C.Goffinet(1), B.Chatelain(1), C.Chatelain(1), Y.Cornet(1), V.Deneys(2), A.Dromelet(1)
(1) Laboratoire d'Hématologie et d'Oncologie Expérimentale, Cliniques Universitaires de Mont-Godinne, Yvoir, Belgique,(2) Laboratoire de cytométrie en flux, Cliniques Universitaire St Luc, Clos Chapelle aux Champs, BP 30-52, Woluwé, Belgique
While immunophenotyping is one of the usual techniques for investigating Acute Leukemia (AL), the measurement of the cellular DNA content by flow cytometry is rarely achieved. We report here the case of a 6 year old female referred for aregenerative anemia. The DNA analysis allowed to precise the etiology. In this patient, we found an aneuploid peak by the technique of Vindelov (DNA Index = 1.26), an excess of bone marrow blasts (8 %) and a negative cytogenetics. Four months later, the diagnosis of Acute Lymphoblastic Leukemia (ALL) was set up. The aneuploid peak and DNA index are similar to the initial ones. As reported in the literature, the incidence of the aneuploid peak is weak in our experience (6 aneuploidia on 33 AL). The detection sensitivity of the aneuploid population can be improved by the double staining combining DNA labeling and membrane immunophenotyping. The method of Loken is simple and at least as sensitive as the Vindelov's method (Peak CV : 2.70 % vs 3.24 % respectively).
The DNA analysis on separated nuclei (Vindelov's method) is simple, rapid, accurate and cheap. It is a diagnostic and prognostic tool and an easy way to determine a residual disease of AL and Myelodysplastic Syndrome. The use of double staining technique (DNA labeling and membrane immunophenotyping) improves the detection sensitivity of the aneuploid population.
DETECTION OF RESIDUAL NEUROBLASTOMA CELLS IN BONE MARROW: COMPARISON OF THREE DIFFERENT DETECTION TECHNIQUES
Swerts K.1,2, De Moerloose B.1, Dhooge C.1, Benoit Y.1, Laureys G.1 and Philippé J.2
Department of Pediatrics1, Ghent University Hospital, Ghent, Belgium. Department of Clinical Chemistry, Microbiology and Immunology2, Ghent University Hospital, Ghent, Belgium.
Neuroblastoma, a tumor originating from the sympathetic nervous tissue, is the most frequent extra cranial solid tumor in young children. Approximately 50% of the neuroblastoma patients older than one year present with high-risk, metastatic disease, involving bone marrow (BM) and bone. The presence of marrow disease is associated with relapse and unfavorable outcome. Consequently, the development of sensitive and specific methods to detect rare tumor cells in BM is important for risk assessment at diagnosis and for evaluating response to therapy. Several techniques can be used to detect the residual neuroblastoma cells. In this study, we developed an immunocytochemical assay, a flow cytometric test and a real-time quantitative RT-PCR to evaluate minimal residual disease (MRD) in neuroblastoma patients. The immunocytochemical technique is based on the use of a monoclonal antibody (14G2a) against the GD2 disialoganglioside which is almost ubiquitously expressed by neuroblastomas. The flow cytometric assay is based on the combination of CD9, CD81, CD56 and 14G2a. CD45 is used as a negative marker. Tyrosine hydroxylase, the first enzyme in the catecholamine pathway, is used as a target for the real-time quantitative RT-PCR. The sensitivity of the three assays was evaluated using the cells of a neuroblastoma cell line (CLB-GA or IMR32) spiked in normal peripheral blood. Using immunocytochemistry or real-time RT-PCR, one neuroblastoma cell in 106 mononuclear cells could be detected. The flow cytometric assay reached a sensitivity of 1 neuroblastoma cell in 102-103 mononuclear cells. No positive cells were found in the negative controls.
Between April 2001 and July 2002, 11 BM samples from 7 neuroblastoma patients, taken at diagnosis or during follow-up, were analyzed using immunocytochemistry, flow cytometry and real-time quantitative RT-PCR. In seven BM samples no residual neuroblastoma cells were found. Three patients scored positive for the RT-PCR, but were negative using immunocytochemistry or flow cytometry. In one sample, residual neuroblastoma cells could only be detected by immunocytochemistry.
Both, immunocytochemistry and quantitative real-time RT-PCR can be used to identify contaminating tumor cells in the bone marrow of neuroblastoma patients. However, immunocytochemistry is very labor intensive and the interpretation of the results is often difficult due to background or aspecific staining. The advantages of real-time quantitation are numerous. The technique is very sensitive and accurate and allows good intra-assay and inter-assay reproducibility. In addition, it combines high throughput and speed with the elimination of post-PCR handling steps, preventing carry-over contamination. Although flow cytometry allows the analysis of large numbers of cells and makes it possible to combine various monoclonal antibodies in a multiparameter test, we found this technique not suitable for the measurement of minimal residual disease in the BM of neuroblastoma patients because of the low sensitivity.
In conclusion, for the detection of residual neuroblastoma cells in bone marrow quantitative real-time RT-PCR for tyrosine hydroxylase mRNA is preferred to immunocytochemistry or flow cytometry.
APPLICATION OF DIFFERENTIAL LEUKOCYTE COUNT IN FRACTION-COLLECTED BOVINE MILK
Vangroenweghe F., Duchateau L., Dosogne H., Burvenich C.
Ghent University, Faculty of Veterinary Medicine, Department of Physiology, Biochemistry and Biometrics, Salisburylaan 133, 9820 Merelbeke
Introduction - Inflammation of the bovine mammary gland (mastitis) is an important health problem in animal husbandry. Nowadays, the control of mastitis is mainly monitored by counting the number of cells in milk (somatic cell count, SCC). Recently, two flow cytometric differential leukocyte count methods have been developed (1,2(. Using one of these techniques (1(, a rapid analysis of the variation in differential leukocyte count of bovine milk fractions was performed during milking.
Material and methods - Six healthy Holstein cows in mid-lactation were selected. During milking, five different fractions were collected (3(: (I) foremilk, (II) cisternal milk, (III) main milk, (IV) strippings and (V) residual milk. SCC and fat content were determined. For milk cell isolation, diluted milk (3/5; v/v) was centrifuged (1000 g, 15 min, 20C). Four hundred and ninety (l RPMI was added to the cell pellet, followed by 10 (l diluted SYTO"13 solution (Molecular Probes). This mixture was incubated for 10 min. and samples were analysed flow cytometrically. Flow cytometric differential leukocyte count of milk after SYTO"13 staining was quantified using the SSC - green fluorescence dot plot (FL-1). The percentage of PMN, macrophages, lymphocytes and cells with apoptotic features was calculated [1].
Results - SCC did not differ between fractions. Fat content was significantly different between fractions I-II, III, IV and V (P<0.05), with the highest content in residual milk. The percentage of lymphocytes and macrophages was significantly lower (P<0.05) in fraction V as compared to the other fractions. The percentage of cells with apoptotic features was highest in residual milk (P<0.05). The percentage of PMN was significantly higher (P<0.05) in fraction I-II. Fat content was positively correlated with the percentage of cells with apoptotic features (P<0.001).
Discussion and conclusions - To our knowledge differential leukocyte count has not been performed previously in different fractions during milking. However, the cell pattern of foremilk is in accordance with other flow cytometric data for differential leukocyte count (2,4(. Cells with apoptotic features were not differentiated by a specific marker (such as annexin-V-FITC), but rather on their flow cytometric morphological characteristics (FS-SSC and FL-1). The high percentage of cells with apoptotic features in residual milk is remarkable, and seems contradictory with the high viability observed in the same fraction (3(. However, due to the high fat content in this fraction, phagocytic cells might have taken up fat globules, thus activating the oxidative burst. This increased oxidative burst activity might have enhanced programmed cell death (5(. New experiments should be carried out to elucidate this phenomenon.
References
(1( Dosogne et al. (2002). Journal of Dairy Science, accepted.
(2( Pilai et al. (2001). Journal of Dairy Science, 84, 1413-1420.
(3( Vangroenweghe et al. (2002). The Veterinary Journal, accepted.
(4( Leitner et al. (1997). Journal of Veterinary Medicine B - Infectious Diseases and Veterinary Public Health, 47, 581-589.
(5( Watson et al. (1997). Journal of Immunology, 158, 945-953.
This study has been supported by the Belgian Ministry for Agriculture and Small Enterprises (S/5871 and S/6014) and the National Fund for Scientific Research (grant n 31504200).
EXPRESSION OF CD13, A MYELOID MARKER, ON THE CD19+ NEOPLASTIC CELLS OF WALDENSTRöM'S MACROGLOBULINAEMIA
J. WAWRZYNIAK, J-Y. SERVAIS, N. HOUGARDY, P. GOFFINET
Laboratoire de cytométrie en flux - Cliniques du Sud Luxembourg - Arlon - BELGIUM
Waldenström's macroglobulinaemia is a lymphoproliferative disorder consisting in small lymphocytes presenting, in variable proportions, a plasmacytoid differenciation, associated with a secretion of a large amount of an IgM paraprotein, which can lead to additionnal symptoms in relation with blood hyperviscosity.
CD13, previously described as a marker of myelomonocytic origin, has been already reported to be present in rare cases of lymphoid neoplasms, essentialy in acute lymphoblastic leukemia of B lineage (B-ALL) [1].
In our laboratory practice, we found an aberrant expression of this CD13 marker (CD13+/CD19+ elements) in 3 cases of 5 clinically and cytologicaly proved Waldenström's diseases. 1 case was studied completely, the other ones were studied retrospectively.
Although the positivity of CD13 on human progenitor mast cells (CD34+/CD117+/CD13+), this marker is not expressed on human tissues mature mast cells [2]. Therefore, the expression of CD13 on CD19+ elements can not be associated to the increased presence of these mast cells in Waldenström's macroglobulinaemia.
The immunophenotype of the cell population was CD19+ , CD20+ , CD10- , CD5- , CD22+ , CD79b+ , FMC7- , CD23- , CD103- , CD43+/-, heavy chain µ + , CD34- , CD117- , CD33- , CD13+ .
In all these 3 cases, a difference in the expression of CD33 and CD13 was found on the elements defined by a lymphocytes and blasts gating on the FSC/SSC scattergram. These markers are usually expressed in the same proportion on the myelomonocytic lineage. An index of expression, described as CD13/CD33, can be used to determine this lack of correlation between the two markers. The indexes for the 3 particuliar cases were 18.6 , 10.6 and 8.9 , while the values for normal cases were 2.4 +/- 1.3.
However, no biological or clinical differences between CD13+ and CD13- cases have been noticed. Since Tabernero et al found that BCR/ABL rearrangements can lead to the expression of CD13 on B-ALL neoplastic cells [3], this translocation can be an element for further investigations. The clinical importance (severity, survey, monitoring) has yet to be estimated with a more important population and a longer period of evaluation.
[1] Immunophenotypic and cytogenetic changes in acute leukemia at relapse - M.Hur et al - Clin Lab Haematol 2001 Jun ; 23(3) : 173-9
[2] Demonstration that human mast cells arise from a progenitor cell population that is CD34+ / c-kit+ and expresses aminopeptidase-N (CD13) - A.S.Kirshenbaum et al - Blood, vol 94 No7 (october 1), 1999 : pp 2333-2342
[3] Adult precursor B-ALL with BCR/ABL gene rearrangements displays a unique immunophenotype based on the pattern of CD10 , CD34 , CD13 and CD38 expression - MD Tabernero et al - Leukemia, 2001 oct ; 15 (10) : 1671-2