Filtrer par mots-clé

Filtrer par application

Filtrer par produit/solution

Diagnostics (Basel, Switzerland), 8

Aneuploid CTC and CEC.

Lin, Peter Ping

Conventional circulating tumor cell (CTC) detection technologies are restricted to large tumor cells (> white blood cells (WBCs)), or those unique carcinoma cells with double positive expression of surface epithelial cell adhesion molecule (EpCAM) for isolation, and intracellular structural protein cytokeratins (CKs) for identification. With respect to detecting the full spectrum of highly heterogeneous circulating rare cells (CRCs), including CTCs and circulating endothelial cells (CECs), it is imperative to develop a strategy systematically coordinating all tri-elements of nucleic acids, biomarker proteins, and cellular morphology, to effectively enrich and comprehensively identify CRCs. Accordingly, a novel strategy integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH), independent of cell size variation and free of hypotonic damage as well as anti-EpCAM perturbing, has been demonstrated to enable in situ phenotyping multi-protein expression, karyotyping chromosome aneuploidy, and detecting cytogenetic rearrangements of the gene in non-hematologic CRCs. Symbolic non-synonymous single nucleotide variants (SNVs) of both the gene (P33R) in each single aneuploid CTCs, and the cyclin-dependent kinase inhibitor 2A ( ) tumor suppressor gene in each examined aneuploid CECs, were identified for the first time across patients with diverse carcinomas. Comprehensive co-detecting observable aneuploid CTCs and CECs by SE-iFISH, along with applicable genomic and/or proteomic single cell molecular profiling, are anticipated to facilitate elucidating how those disparate categories of aneuploid CTCs and CECs cross-talk and functionally interplay with tumor angiogenesis, therapeutic drug resistance, tumor progression, and cancer metastasis.

Digital object identifier (DOI): 10.3390/diagnostics8020026

Cancer letters, 412, 99--107

Quantified postsurgical small cell size CTCs and EpCAM, [email protected], circulating tumor stem cells with cytogenetic abnormalities in hepatocellular carcinoma patients determine cancer relapse.

Wang, Liang, Li, Yilin, Xu, Jing, Zhang, Aiqun, Wang, Xuedong, Tang, Rui, Zhang, Xinjing, Yin, Hongfang, Liu, Manting, Wang, Daisy Dandan, Lin, Peter Ping, Shen, Lin, Dong, Jiahong

Detection of hepatocellular carcinoma circulating tumor cells performed with conventional strategies, is significantly limited due to inherently heterogeneous and dynamic expression of EpCAM, as well as degradation of cytokeratins during epithelial-to-mesenchymal transition, which inevitably lead to non-negligible false negative detection of such "uncapturable and invisible" CTCs. A novel SE-iFISH strategy, improved for detection of HCC CTCs in this study, was applied to comprehensively detect, in situ phenotypically and karyotypically characterize hepatocellular and cholangiocarcinoma CTCs (CD45 /CD31 ) in patients subjected to surgical resection. Clinical significance of diverse subtypes of CTC was systematically investigated. Existence of small cell size CTCs (≤5 μm of WBCs) with cytogenetic abnormality of aneuploid chromosome 8, which constituted majority of the detected CTCs in HCC patients, was demonstrated for the first time. The stemness marker EpCAM aneuploid circulating tumor stem cells (CTSCs), and EpCAM small CTCs with trisomy 8, promote tumor growth. Postsurgical quantity of small triploid CTCs (≥5 cells/6 ml blood), multiploid (≥pentasomy 8) CTSCs or CTM (either one ≥ 1) significantly correlated to HCC patients' poor prognosis, indicating that detection of those specific subtypes of CTCs and CTSCs in post-operative patients help predict neoplasm recurrence.

Digital object identifier (DOI): 10.1016/j.canlet.2017.10.004

Biological procedures online, 20, 13

Optimization of Immunofluorescent Detection of Bone Marrow Disseminated Tumor Cells.

Axelrod, Haley D, Pienta, Kenneth J, Valkenburg, Kenneth C

Cancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the blood and bone marrow, respectively. The most promising strategy is fluorescence-based immunostaining using cancer cell-specific markers. However, despite recent efforts to develop robust processing and staining platforms, results from these platforms have been discordant among groups, particularly for DTC detection. While the choice of cancer cell-specific markers is a large factor in this discordance, we have found that marker-independent factors causing false signal are just as critical to consider. Bone marrow is particularly challenging to analyze by immunostaining because endogenous immune cell properties and bone marrow matrix components typically generate false staining. For immunostaining of whole tumor tissue containing ample cancer cells, this background staining can be overcome. Application of fluorescent-based staining for rare cells, however, is easily jeopardized by immune cells and autofluorescence that lead to false signal. We have specifically found two types of background staining in bone marrow samples: autofluorescence of the tissue and non-specific binding of secondary antibodies. We systematically optimized a basic immunofluorescence protocol to eliminate this background using cancer cells spiked into human bone marrow. This enhanced the specificity of automated scanning detection software. Our optimized protocol also outperformed a commercial rare cell detection protocol in detecting candidate DTCs from metastatic patient bone marrow. Robust optimization to increase the signal-to-noise ratio of immunofluorescent staining of bone marrow is required in order to achieve the necessary sensitivity and specificity for rare cell detection. Background immunofluorescent staining in bone marrow causes uncertainty and inconsistency among investigators, which can be overcome by systematically addressing each contributing source. Our optimized assay eliminates sources of background signal, and is adaptable to automated staining platforms for high throughput analysis.

Digital object identifier (DOI): 10.1186/s12575-018-0078-5

Nature Scientific Reports, 7(9789), 2-10

Comprehensive in situ co-detection of aneuploid circulating endothelial and tumor cells

Peter Ping Lin, Olivier Gires, Daisy Dandan Wang, Linda, Li, Hongxia Wang

Conventional circulating tumor cell (CTC) detection strategies rely on cell surface marker EpCAM and intracellular cytokeratins (CKs) for isolation and identification, respectively. Application of such methods is considerably limited by inherent heterogeneous and dynamic expression or absence of EpCAM and/or CKs in CTCs. Here, we report a novel strategy, integrating antigen-independent subtraction enrichment and immunostaining-FISH (SE-iFISH), to detect a variety of aneuploid circulating rare cells (CRCs), including CTCs and circulating tumor endothelial cells (CECs). Enriched CRCs, maintained at high viability and suitable for primary tumor cell culture, are comprehensively characterized by in situ co-examination of chromosome aneuploidy by FISH and immunostaining of multiple biomarkers displayed in diverse fluorescence channels. We described and quantified for the first time the existence of individual aneuploid CD31+ CECs and co-existence of “fusion clusters” of endothelial-epithelial aneuploid tumor cells among enriched non-hematopoietic CRCs. Hence, SE-iFISH is feasible for efficient co-detection and in situ phenotypic and karyotypic characterization as well as quantification of various CRCs, allowing for their classification into diverse subtypes upon biomarker expression and chromosome ploidy. Enhanced SE-iFISH technology, assisted by the Metafer-iFISH automated CRC imaging system, provides a platform for the analysis of potential contributions of each subtype of CRCs to distinct clinical outcome.

PLoS One, 11(8), e0161369

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors.

Brunner, Clemens, Brunner-Herglotz, Bettina, Ziegler, Andrea, Frech, Christian, Amann, Gabriele, Ladenstein, Ruth, Ambros, Inge M., Ambros, Peter F.

Tumor touch imprints (TTIs) are routinely used for the molecular diagnosis of neuroblastomas by interphase fluorescence in-situ hybridization (I-FISH). However, in order to facilitate a comprehensive, up-to-date molecular diagnosis of neuroblastomas and to identify new markers to refine risk and therapy stratification methods, whole genome approaches are needed. We examined the applicability of an ultra-high density SNP array platform that identifies copy number changes of varying sizes down to a few exons for the detection of genomic changes in tumor DNA extracted from TTIs.DNAs were extracted from TTIs of 46 neuroblastoma and 4 other pediatric tumors. The DNAs were analyzed on the Cytoscan HD SNP array platform to evaluate numerical and structural genomic aberrations. The quality of the data obtained from TTIs was compared to that from randomly chosen fresh or fresh frozen solid tumors (n = 212) and I-FISH validation was performed.SNP array profiles were obtained from 48 (out of 50) TTI DNAs of which 47 showed genomic aberrations. The high marker density allowed for single gene analysis, e.g. loss of nine exons in the ATRX gene and the visualization of chromothripsis. Data quality was comparable to fresh or fresh frozen tumor SNP profiles. SNP array results were confirmed by I-FISH.TTIs are an excellent source for SNP array processing with the advantage of simple handling, distribution and storage of tumor tissue on glass slides. The minimal amount of tumor tissue needed to analyze whole genomes makes TTIs an economic surrogate source in the molecular diagnostic work up of tumor samples.

Digital object identifier (DOI): 10.1371/journal.pone.0161369

Prenat Diagn, 34(11), 1066--1072
November, 2014

A new marker set that identifies fetal cells in maternal circulationwith high specificity.

Lotte Hatt, Marie Brinch, Ripudaman Singh, Kristine M{\o}ller, Rune Hoff Lauridsen, Jacob M{\o}rup Schl{\"u}tter, Niels Uldbjerg, Britta Christensen, Steen K{\o}lvraa

Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking.Blood samples from 78 normal pregnancies were used for testing the marker-set CD105 and CD141 for fetal cell enrichment. Fetal cell candidates were subsequently stained by a cocktail of cytokeratin antibodies, and the gender of the fetal cells was explored by fluorescence in situ hybridization (FISH) of the X and Y chromosomes.Fetal cell candidates could be detected in 91\% of the samples, and in 85\% of the samples, it was possible to obtain X and Y chromosomal FISH results for gender determination. The concordance between gender determined by FISH on fetal cells in maternal blood and gender found at birth reached 100\% if three or more fetal cells with FISH signals could be found in a sample.The marker set identifies fetal cells with specificity high enough to make cell-based noninvasive prenatal diagnosis realistic.

Prenat Diagn, 32(8), 742--751
August, 2012

Identification of circulating fetal cell markers by microarray analysis.

Marie Brinch, Lotte Hatt, Ripudaman Singh, Kristine M{\o}ller, Steffen Sommer, Niels Uldbjerg, Britta Christensen, Steen K{\o}lvraa

Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester.Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem cell microarray analysis.Twenty-eight genes were identified as fetal cell marker candidates.Of the 28 fetal marker candidate genes, five coded for proteins, which are located on the outer surface of the cell membrane and not expressed in blood. The protein product of these five genes, MMP14, MCAM, KCNQ4, CLDN6, and F3, may be used as markers for fetal cell enrichment.

Forensic Science International: Genetics Supplement Series, 3(1), e35-e36

Automated scoring of Sperm Hy-Liter™-stained spermatozoa by the MetaSystems Metafer image analysis software system in sexual assault specimens

De Moors, A., Fr{\'e}geau, C.J.

The MetaSystems Metafer image analysis software system was purchased three years ago in the hope of developing a routine approach in the RCMP Forensic Laboratories to automate the scoring of human spermatozoa in sexual assault exhibits. This would enhance case throughput, increase assay sensitivity and standardize the search for spermatozoa. The development of appropriate classifiers was challenging but essential to teach the software system to specifically recognize human spermatozoa fluorescently stained using the Sperm Hy-Liter™ kit (Independent Forensics). Optimized classifiers were tested/validated using a diverse set of slides prepared from mock sexual assault samples containing a limited or a large number of spermatozoa (fecal swabs, vaginal swabs, all mixed with different semen dilutions in addition to urine, blood and yeasts for a subset of those swabs). The performance of Metafer was recorded with respect to false positive counts, false negative counts and time required for the detection of spermatozoa in each sample. Automated spermatozoa counts were further compared to manual spermatozoa scoring in addition to comparing the time spent executing the identification. An excellent concordance was noted between automated and manual counts. The results of this study indicate that automated scoring of fluorescently stained spermatozoa in mock sexual assault exhibits can be carried out reliably and reproducibly using well-developed classifiers for the MetaSystems Metafer image analysis software system. The automated scoring of spermatozoa combining Sperm Hy-Liter™/MetaSystems Metafer will be tested on a large number of sexual assault cases as part of a pilot project within an operational setting.

Int J Legal Med, 124(6), 513--521
November, 2010

Laser capture microdissection in forensic research: a review.

Mado Vandewoestyne, Dieter Deforce

In forensic sciences, short tandem repeat (STR) analysis has become the prime tool for DNA-based identification of the donor(s) of biological stains and/or traces. Many traces, however, contain cells and, hence, DNA, from more than a single individual, giving rise to mixed genotypes and the subsequent difficulties in interpreting the results. An even more challenging situation occurs when cells of a victim are much more abundant than the cells of the perpetrator. Therefore, the forensic community seeks to improve cell-separation methods in order to generate single-donor cell populations from a mixed trace in order to facilitate DNA typing and identification. Laser capture microdissection (LCM) offers a valuable tool for precise separation of specific cells. This review summarises all possible forensic applications of LCM, gives an overview of the staining and detection options, including automated detection and retrieval of cells of interest, and reviews the DNA extraction protocols compatible with LCM of cells from forensic samples.

Pediatr Blood Cancer, 54(1), 158--160
January, 2010

Detection of early precursors of t(12;21) positive pediatric acutelymphoblastic leukemia during follow-up.

Ren{\'a}ta L{\'a}szl{\'o}, Don{\'a}t Alp{\'a}r, B{\'e}la Kajt{\'a}r, Agnes Lacza, G{\'a}bor Ott{\'o}ffy, Csongor Kiss, Katalin Bartyik, K{\'a}lm{\'a}n Nagy, L{\'a}szl{\'o} Pajor

DNA-, RNA-, and cell-based methods provide different biologic information for determining the presence of minimal residual disease (MRD). We monitored the responses of patients with pediatric acute lymphoblastic leukemia (pALL) using DNA markers, TEL/AML1 expression, and scanning fluorescence microscopy (SFM). Using SFM, 36\% of patients exhibited 1.5-3.1 log and 2.9-4.2 log higher MRD levels compared with those based on DNA and RNA markers, respectively. CD10+ ancestor cells with germline antigen receptors, but silent TEL/AML1 expression, may reside in the lymphoid stem cell compartment of treated t(12;21)-positive patients and might act as a potential source of cells for late relapses.

Cancer Genet Cytogenet, 173(1), 23--30
February, 2007

Automated detection of residual leukemic cells by consecutive immunolabelingfor CD10 and fluorescence in situ hybridization for ETV6/RUNX1 rearrangementin childhood acute lymphoblastic leukemia.

Don{\'a}t Alp{\'a}r, B{\'e}la Kajt{\'a}r, M{\'a}ria Kneif, P{\'a}l J{\'a}ks{\'o}, Ren{\'a}ta L{\'a}szl{\'o}, L{\'a}szl{\'o} Kereskai, L{\'a}szl{\'o} Pajor

Among the various methods available for analyzing minimal residual disease, a new procedure for the cell-based approaches using consecutive phenotypic and genotypic analysis as revealed by immunofluorescent labeling and subsequent fluorescent in situ hybridization (FISH) has been developed. We are introducing a fluorescent microscopy-based technique by which not only cellular targets and immunological marker positivity, but also the FISH pattern was identified by automated scanning. For the latter one translocation-specific FISH pattern recognition was accomplished by using an automated scanning mode for the 3D determination of valid distances between FISH signals, to define the cutoff value for the shortest green-red spot distance differentiating positive cells from negative ones. The procedure was tested with CD10(+) acute lymphoblastic leukemia cell line harboring the t(12;21)(p13;q22) resulting in the ETV6/RUNX1 rearrangement (formerly TEL/AML1), as well as peripheral blood lymphocytes of healthy individuals. Using the combined, automated method, a sensitivity of 98.67\% and a specificity of 99.97\% were obtained. The mean false positivity + 2 standard deviations cutoff level (0.09\%) allows detection of leukemic cells with high accuracy, even a bit below the tumor load dilution of 10(-3), a value reported to be critical in clinical decision making.

Fetal Diagnosis and Therapy, 20, 106- 112

Fetal cells in maternal blood: a comparison of methods for cell isolation and identification.

B. Christensen, J. Philip, S. K{\o}lvraa, L. Lykke-Hansen, I. Hromadnikova, D. Gohel, T. L{\"o}rch, A. Plesch, J. Bang, S. Smidt-Jensen, J. Hertz, H. Djursing

OBJECTIVE: A variety of methods have been used to select and identify fetal cells from maternal blood. In this study, a commonly used 3-step selection method is compared with selection directly from whole blood. Identification of fetal origin by XY FISH of male cells was also evaluated. METHODS: Maternal blood was drawn either before invasive chorion villus sampling (pre-CVS) or after (post-CVS) from women carrying a male fetus. Fetal cells were isolated either by density gradient centrifugation succeeded by CD45/CD14 depletion and CD71-positive selection from CD45/CD14-negative cells, or by CD71-positive selection directly from whole blood. The true origin of fetal cells recovered by the two methods was established by two rounds of XY chromosome FISH in reverse colors, in some instances combined with anti-zeta (zeta) or anti-zeta/anti-gamma (gamma) antibody staining. RESULTS: In blood samples taken post-CVS and enriched by CD71 selection directly from whole blood, fetal cells were identified with a frequency that was almost four orders of magnitude higher than in post-CVS samples enriched by the 3-step method. In blood samples taken pre-CVS and enriched by the 3-step procedure, no fetal cells were identified by reverse color FISH in 371 ml of blood. In similar samples enriched by CD71 selection on whole blood, two fetal cells were identified in 27 ml of blood. Rehybridization with X and Y chromosome probes with reverse colors was necessary to exclude false Y chromosome signals. Not all fetal cells identified by the presence of a true Y chromosome signal stained with anti-zeta antibody. CONCLUSIONS: Selection of fetal NRBCs from maternal blood by CD71-positive selection directly from whole blood is superior to density gradient centrifugation succeeded by CD45/CD14 depletion and CD71 selection of CD45/CD14-negative cells. Combining two markers for fetal origin is recommended for unambiguously identifying a cell as fetal.

Clin Cancer Res, 10(9), 3020--3028
May, 2004

Reliable and sensitive identification of occult tumor cells usingthe improved rare event imaging system.

Stine-Kathrein Kraeft, Andras Ladanyi, Kevin Galiger, Anna Herlitz, Andrew C. Sher, Danielle E. Bergsrud, Gaelle Even, Stephanie Brunelle, Lyndsay Harris, Ravi Salgia, Tom Dahl, John Kesterson, Lan Bo Chen

The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors.We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis.The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40\%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile.REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.

Cancer letters, 197, 29- 34

Disseminated tumour cells in the bone marrow - chances and consequences of microscopical detection methods

P.F. Ambros, G. Mehes, I.M. Ambros, R. Ladenstein

The detection of disseminated tumor cells (DTCs) in the hematopoetic system is important for various reasons. It is essential for tumor staging. According to the International Neuroblastoma Staging System (INSS) only the cytomorphological examination of bone marrow smears is accepted despite the fact that an infiltrate below 0.1%, can hardly be detected and even infiltrates of more than 10% are sometimes overlooked. Another important aspect is the monitoring of the disease response to cytotoxic drugs by quantifying DTCs. Moreover, bone marrow aspirates represent an ideal source to determine the genetic and biological make up of DTCs at diagnosis and during follow up. Key issues that can be tested on DTCs are: determination of the proliferation capacity, the apoptotic rate, the drug sensitivity etc. The prerequisite for such a bone-marrow diagnosis, however, is the unequivocal identification of disseminated tumor cells. Thus, in order to avoid false positive and false negative results, which are a risk in bone-marrow diagnostics, a system was developed to distinguish tumor cells from non-neoplastic cells and to facilitate the gain of insights into the biological make-up of tumor cells more easily.

Leukemia, 15, 275- 277

Unequivocal identification of disseminated tumor cells in the bone marrow by combining immunological and genetic approaches--functional and prognostic information.

P.F. Ambros, G. M{\'e}hes, C. Hattinger, I.M. Ambros, A. Luegmayr, R. Ladenstein, H. Gadner

The detection and quantification of disseminated tumor cells (DTC) present in the bone marrow (BM), peripheral blood (PB) and apheresis products (AP) are becoming increasingly significant in the treatment of cancer patients. Three different applications are implemented in the clinical practice of pediatric and adult solid tumor patients: (1) the identification of tumor cells in the BM and PB at diagnosis; (2) the response of occult tumor cells to high-dose chemotherapy; and (3) the presence of tumor cells in the autograft. In solid tumors the clinical significance of DTCs at diagnosis or during the course of the disease, usually termed minimal residual disease (MRD) testing, is still under debate. These indistinct results are mainly due to methodical reasons. Therefore, a fully automated system (RCDetect/metafer) combining the detection of 'tumor-specific' immunological features together with 'tumor-typical' DNA aberrations has been developed allowing the unambiguous visualization of tumor cells in a hematopoietic surrounding.

Medical and Pediatric Oncology, 36, 205- 209

Automatic detection and genetic profiling of disseminated neuroblastoma cells.

G. M{\'e}hes, A. Luegmayr, C.M. Hattinger, T. L{\"o}rch, I.M. Ambros, H. Gadner, P.F. Ambros

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

Medical and Pediatric Oncology, 36, 205- 209

Automatic detection and genetic profiling of disseminated neuroblastoma cells

G. M{\'e}hes, A. Luegmayr, C.M. Hattinger, T. L{\"o}rch, I.M. Ambros, H. Gadner, P.F. Ambros

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.