Systems biology and clinical cytomics: The 10th Leipziger Workshop and the 3rd International Workshop on Slide-Based Cytometry, Leipzig, Germany, April 2005.

Despite very significant technical and software improvements in flow cytometry (FCM) since the 1980's, the demand for a cytometric technology combining both quantitative cell analysis and morphological documentation in Cytomics became evident. Improvements in microtechnology and computing permit nowadays similar quantitative and stoichiometric single cell-based high-throughput analyses by microscopic instruments, like Slide-Based Cytometry (SBC). SBC and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures during early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance not only as a cytomics technology platform but also because of low quantities of required reagents and biological material. The exact knowledge of the location of each cell on the slide permits repetitive restaining and reanalysis of specimens. Various separate measurements of the same specimen can be ultimately fused to one database increasing the information obtained per cell. Relocation and optical evaluation of cells as typical SBC feature, can be of integral importance for cytometric analysis, since artifacts can be excluded and morphology of measured cells can be documented. Progress in cell analytic: In the SBC, new horizons can be opened by the new techniques of structural and functional analysis with the high resolution from intracellular and membrane (confocal microscopy, nanoscopy, total internal fluorescence microscopy (TIRFM), and tissue level (tissomics), to organ and organism level (in vivo cytometry, optical whole body imaging). Predictive medicine aims at the detection of changes in patient's state prior to the manifestation of the disease or the complication. Such instances concern immune consequences of surgeries or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Preventive anti-infectious or anti-shock therapy as well as curative chemotherapy in combination with stem cell transplantation may provide better survival chances for patient at concomitant cost containment. Predictive medicine-guided optimization of therapy could lead to individualized medicine that gives significant therapeutic effect and may lower or abrogate potential therapeutic side effects. The 10th Leipziger Workshop combined with the 3rd International Workshop on SBC aimed to offer new methods in Image- and Slide-Based Cytometry for solutions in clinical research. It moved towards practical applications in the clinics and the clinical laboratory. This development will be continued in 2006 at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.     

Regenerative and predictive medicine of cardiovascular disease: the 9th Leipziger Workshop and the 2nd International Workshop on slide based cytometry.

Slide-based cytometry (SBC) and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures at very early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance, not only as a cytomics technology platform but also for patients with low blood volume such as neonates. The exact knowledge of the location of each cell on the slide allows restaining and subsequent reanalysis of the specimen. These separate measurements of the same specimen can be fused to one data file (merging), thus increasing the information obtained per cell. Relocalization and optical evaluation of the cells, a feature typical of SBC, can be of integral importance for cytometric analysis. Due to this feature, artifacts can be excluded and morphology of measured cells can be documented. Predictive medicine aims at the detection of changes in patient's state before the manifestation of the disease or its complications. Such instances concern multiorgan failure in sepsis or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high-risk patients undergoing cancer cytostatic therapy. Early anti-infectious or antishock therapy and curative chemotherapy in combination with stem cell transplantation may provide better chances of patients' survival at concomitant cost containment. Predictive medicine that guides early individualized decrease or cessation of therapy may lower or abrogate potential therapeutic side effects (individualized medicine). Regenerative medicine concerns patients who have diseased and injured organs and may be treated with transplanted organs. However, there is a severe shortage of donor organs that is worsening yearly given the aging population. Regenerative medicine and tissue engineering apply the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Neovascularization is promoted by bone marrow-derived endothelial progenitor cells that lead to the formation of entirely new vessels into ischemic tissue. With this knowledge, many therapeutical borders can be skipped. Diseases formerly uncontrolled can be corrected with stem cells to provide causal healing with regeneration processes. The 9th Leipziger Workshop combined with the 2nd International Workshop on SBC aimed to offer new methods in image cytometry and SBC for solutions in clinical research. It moved toward practical applications in clinics and the clinical laboratory. This development will be continued in 2005 at the upcoming Leipziger Workshop and the 3rd International Workshop on SBC.     

Uses of flow cytometry in hematology

Flow Cytometry, an analytical cytology technic which allows several parameters assessment in thousands cells per seconds is frequently used in hematology. The oldest application is the leucocyte count but several news applications have been developed for the analysis of cytomorphological and functional parameters of normal cells and for detection, classification of tumor cells. Most frequent applications are: The phenotyping of cells and the evaluation of relation between antigens and pathology. The cell cycle analysis which can be different in leukemic cells or which can be modified with chemotherapy or other agents. (Radiotherapy ...) Flow Karyotyping and in situ hybridization will extend applications of Flow Cytometry in hematology.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Expression of CD134 (0X-40) on T cells during the first 100 days following allogeneic bone marrow transplantation as a marker for lymphocyte activation and therapy-resistant graft-versus-host disease.

CD134 (OX-40) is an activation-associated antigen which functions as a costimulatory receptor for CD4+ T cells. In order to determine the expression of CD134 during immune recovery following allogeneic bone marrow transplantation (BMT), we measured its expression on T cells and T cell subsets during the first 100 days following BMT in 26 patients. CD4+CD134+ T could be seen approximately 14 days following BMT cells in patients who did not develop GvHD which required therapy (n = 20). The percentage of CD4+CD134+ cells continued to increase up to the fourth week following BMT to a maximum of 40-50% of CD4+ T cells (normal = 1-8%). Two patients who developed Grade I-II GvHD and who responded to treatment with pulsed high-dose methylprednisolone (MPD) showed a decline of approximately 40% in CD4+CD134+ T cells was seen within 48 hours of treatment. Four patients who developed GvHD that was not responsive to MPD and who later developed high IV GvHD showed increasing CD4+CD134+ T cells up to 85% of the CD4+ T cells. Additionally, rapid increases in CD134+ T cells following antibody-based T cell therapy were associated with GvHD recurrence. In no cases was the percentage of CD134+ CD4+ T cells predictive of clinical GvHD. In this exploratory study, we have shown that CD134, although not predictive of the initial onset of GvHD, may be a useful tool for monitoring the response to early GvHD therapy and identification of patients at risk for reemergence of GvHD who may benefit from anti-T cell therapy. Cytometry (Comm. Clin. Cytometry) 38: 238-243, 1999.     

Multiparametric cytometry for exploration of complex cellular dynamics

The development of polychromatic cytometry has contributed to significant progress in the field of human immunology. Although numerous functional studies of rare cell populations have been performed using this technology, here we used polychromatic cytometry to explore the dynamics of complex cellular systems implicated in innate immunity. We used PBMC stimulated with live influenza virus as an experimental model. We studied the time course of activation of PBMC, which contain DC, monocytes, and NK cells, all of which are, in addition to their innate immune properties, susceptible to Flu infection. We developed 12 color panels to investigate intracellular expression of IFN-α, TNF-α, IL-12, IL-6, IFN-γ, CD107, and influenza virus nucleoprotein simultaneously in these cell populations. These panels allowed reproducible determination of activation markers induced in DC after their direct exposure to various stimulations or in NK cells by indirect DC-mediated activation within the complex cellular environment. The ability to use a low number of cells and reduced quantities of reagents permitted us to perform kinetic experiments. The power of polychromatic cytometry associated with bioinformatic tools allowed us to analyze the multiple functional data generated as dynamic clustering maps. These maps present a readily understandable view of activation events induced in different populations of PBMC. In addition, it reveals new information on the coordination of the complex pathways induced and on the cellular interactions that sustained indirect DC-mediated NK cell activation. Our work shows that polychromatic cytometry is a tool for discoveries in unexplored complex cell systems, at the crossroads of immunology and virology. ? 2012 International Society for Advancement of Cytometry.     

Hyperchromatic cytometry principles for cytomics using slide based cytometry.

BACKGROUND: Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high-content and high-throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object. METHODS AND RESULTS: We demonstrate various approaches for hyperchromatic analysis on a SBC instrument, the Laser Scanning Cytometer. The different components demonstrated here include (1) polychromatic cytometry (staining of the specimen with eight or more different fluorochromes simultaneously), (2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), (3) differential photobleaching (differentiating fluorochromes by their different photostability), (4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and (5) photodestruction (destruction of FRET dyes). Based on the ability to relocate cells that are immobilized on a microscope slide with a precision of approximately 1 microm, identical cells can be reanalyzed on the single cell level after manipulation steps. CONCLUSION: With the intelligent combination of several different techniques, the hyperchromatic cytometry approach allows to quantify and analyze all components of relevance on the single cell level. The information gained per specimen is only limited by the number of available antibodies and sterical hindrance.     

High concordance between marker profiles of 22 human leukemia-lymphoma cell lines tested with the same monoclonal antibodies before and during the second international workshop on human differentiation antigens

Summary Our laboratory participated in the Second International Workshop and Conference on Human Leucocyte Differentiation Antigens. In this international study the reactivity profiles of monoclonal antibodies were analyzed on normal and malignant hematopoietic cells. The Workshop was divided into three categories: the T-cell, B-cell and myelomonocytic cell studies. We blindly tested 159 coded monoclonal antibodies of the panel for the T-cell study on 22 permanently established leukemia cell lines. The monoclonal antibodies were provided by the Workshop Committee and their reactivity with the target cells was visualized by standardized indirect immunofluorescence. After decoding it was recognized that 11 monoclonal antibodies had been examined on these cell lines prior to the Workshop. The reactivity of these 11 monoclonal antibodies was analyzed and compared with the earlier results. From a total of 217 paired tests done blindly in the Workshop study and prior to the Workshop, 191 tests (88%) did not show significantly different data. The possible reasons for discrepancies include nonspecific Fc-receptor-binding on some cell lines and a relatively nonspecific reactivity of some monoclonal antibodies.This analysis demonstrates the stability of the antigen expression on human leukemia-lymphoma cell lines grown at consistently optimal conditions, for the tests, using the same monoclonal antibodies as in the Workshop, had been performed 0.5–5 years prior to the Workshop study. On the other hand, nonspecific Fc-binding, wide specificity of monoclonal antibodies and a shift in antigen expression of the cells (due to poor growth conditions, involuntary induction of differentiation and other factors) must be taken into consideration upon immunological analysis.This study was supported in part by Veterans Administration RAG GrantH. G. D. is recipient of a Research Training Fellowship awarded by the Deutsche Forschungsgemeinschaft (Dr 157/1-1)     

In vitro genotoxicity test approaches with better predictivity: summary of an IWGT workshop

Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage.     

Symbiosis instruction: considerations from the education workshop at the 6th ISS Congress

Herein, we summarize presentations on “Symbiosis” instruction given at the Education Workshop held at the 6th International Symbiosis Society Meeting in Madison WI. We convey our teaching experiences and methods in a variety of venues. Information on target audiences, course syllabi, and laboratory skills, utilizing various symbiotic systems are also presented. We hope this review will contribute to further adoption of symbiotic interactions in the classroom as well as the growth of currently developed courses in this field—a specific mission of this and previous International Symbiosis Society meetings.     

HIF1A predicts the efficacy of anti-PD-1 therapy in advanced clear cell renal cell carcinoma

Immunotherapy for cancer has become a revolutionary treatment, with the progress of immunological research on cancer. Cancer patients have also become more diversified in drug selection. Individualized medical care of patients is more important in the era of precision medicine. For advanced clear cell renal cell carcinoma (ccRCC) patients, immunotherapy and targeted therapy are the two most important treatments. The development of biomarkers for predicting the efficacy of immunotherapy or targeted therapy is indispensable for individualized medicine. There is no clear biomarker that can accurately predict the efficacy of immunotherapy for advanced ccRCC patients. Our study found that HIF1A could be used as a biomarker for predicting the anti-PD-1 therapy efficacy of patients with advanced ccRCC, and its prediction accuracy was even stronger than that of PD-1/PD-L1. HIF1A is expected to help patients with advanced ccRCC choose therapeutic drugs.     

FuGEFlow: data model and markup language for flow cytometry

Background  

Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata.     

Validation of a flow cytometry-based method to quantify viable lymphocyte subtypes in fresh and cryopreserved hematopoietic cellular products

Background aimsAdoptive cellular therapy with immune effector cells (IECs) has shown promising efficacy against some neoplastic diseases as well as potential in immune regulation. Both inherent variability in starting material and variations in cell composition produced by the manufacturing process must be thoroughly evaluated with a validated method established to quantify viable lymphocyte subtypes. Currently, commercialized immunophenotyping methods determine cell viability with significant errors in thawed products since they do not include any viability staining. We hereby report on the validation of a flow cytometry-based method for quantifying viable lymphocyte immunophenotypes in fresh and cryopreserved hematopoietic cellular products.MethodsUsing fresh or frozen cellular products and stabilized blood, we report on the validation parameters accuracy, uncertainty, precision, sensitivity, robustness and contamination between samples for quantification of viable CD3+, CD4+ T cells, CD8+ T cells, CD3–CD56+CD16+/– NK cells, CD19+ B cells and CD14+ monocytes of relevance to fresh and cryopreserved hematopoietic cellular products using the Cytomics FC500 cytometer (Beckman Coulter).ResultsThe acceptance criteria set in the validation plan were all met. The method is able to accommodate the variability in absolute numbers of cells in starting materials collected or cryopreserved from patients or healthy donors (uncertainty of ≤20% at three different concentrations), stability over time (compliance over 3 years during regular inter-laboratory comparisons) and confidence in meaningful changes during cell processing and manufacturing (intra-assay and intermediate precision of 10% coefficient of variation). Furthermore, the method can accurately report on the efficacy of cell depletion since the lower limit of quantification was established (CD3+, CD4+ and CD8+ cells at 9, 8 and 8 cells/µL, respectively). The method complies with Foundation for the Accreditation of Cellular Therapy (FACT) standards for IEC, FACT-Joint Accreditation Committee of ISCT-EBMT (JACIE) hematopoietic cell therapy standards, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q2(R1) and International Organization for Standardization 15189 standards. Furthermore, it complies with Ligand Binding Assay Bioanalytical Focus Group/American Association of Pharmaceutical Scientists, International Council for Standardization of Hematology/International Clinical Cytometry Society and European Bioanalysis Forum recommendations for validating such methods.ConclusionsThe implications of this effort include standardization of viable cell immunophenotyping of starting material for cell manufacturing, cell selection and in-process quality controls or dosing of IECs. This method also complies with all relevant standards, particularly FACT-JACIE standards, in terms of enumerating and reporting on the viability of the “clinically relevant cell populations.”     

Characterization of antibodies submitted to the B cell section of the 8th Human Leukocyte Differentiation Antigens Workshop by flow cytometry and immunohistochemistry

The aim of this study was to characterize the reactivity of monoclonal antibodies (mAbs) that had been submitted to the HLDA8 Workshop. The lineage specificity of target molecules was tested by analyzing their expression patterns on blood cells, leukocytes, and lymphocyte subsets. The expression of target molecules during B cell development, ranging from early precursors to plasma cells, was analyzed using a large panel of B cell lines. Our results have permitted us to characterize the expression of 10 new CD molecules: CD316 (HM1.24, BST2), CD268 (BAFF-R, TNFRSF13C), CD269 (BCMA, TNFRF17), CD267 (TACI, TNFRSF13B), CD275 (ICOSL, B7H2), CD254 (TRANCE, TNFSF11), CD252 (OX40L TNFSF4), CD315 (CD9-P), CD316 (EWI-2, PGRL), and CD307 (IRTA-2 or FcRH5). Three of these new CDs, CD267, CD269, and CD307 presented a B cell-restricted expression pattern. MAbs against these novel cell-surface molecules may offer new tools for research, diagnosis, and therapy.     

Report on the fifth international workshop on the CCN family of genes

The Fifth International Workshop on the CCN Family of Genes was held in Toronto October 18–22, 2008. This bi-annual workshop provides a unique opportunity for the presentation and discussion of cutting edge research in the CCN field. The CCN family members have emerged as extracellular matrix associated proteins which play a crucial role in cardiovascular and skeletal development, fibrosis and cancer. Significant progress has been made in the development of model systems to tease apart the CCN signalling pathways in these systems. Results presented at the conference suggest that targeting these pathways now shows real promise as a therapeutic strategy.     

The applications of induced pluripotent stem (iPS) cells in drug development

The introduction of induced pluripotent stem (iPS) cells has been a milestone in the field of regenerative medicine and drug discovery. iPS cells can provide a continuous and individualized source of stem cells and are considered to hold great potential for economically feasible personalized stem cell therapy. Various diseases might potentially be cured by iPS cell-based therapy including Parkinson’s disease, Alzheimer’s disease, Huntington disease, ischemic heart disease, diabetes and so on. Moreover, iPS cells derived from patients suffering from unique incurable diseases can be developed into patient- and disease-specific cell lines. These cells can be used as an effective approach to study the mechanisms of diseases, providing useful tools for drug discovery, development and evaluation. The development of suitable methods for the culture and expansion of iPS cells and their differentiated progenies make feasible modern drug discovery techniques such as high-throughput screening. Furthermore, iPS cells can be applied in the field of toxicological and pharmacokinetics tests. This review focuses on the applications of iPS cells in the field of pharmaceutical industry.     

Bleaching response of Symbiodinium (zooxanthellae): Determination by flow cytometry

Coral bleaching is of increasing concern to reef management and stakeholders. Thus far, quantification of coral bleaching tends to be heavily reliant on the enumeration of zooxanthellae, with less emphasis on assessment of photosynthetic or physiological condition, these being often assessed separately by techniques such as liquid chromatography. Traditional methods of enumeration using microscopy are time consuming, subjected to low precision and great observer error. In this study, we presented a method for the distinction of physoiological condition and rapid enumeration of zooxanthellae using flow cytometry (FCM). Microscopy verified that healthy looking/live versus damaged/dead zooxanthellae could be reliably and objectively distinguished and counted by FCM on the basis of red and green fluorescence and light scatter. Excellent correlations were also determined between FCM and microscopy estimates of cell concentrations of fresh zooxanthellae isolates from Pocillopora damicornis. The relative intensities of chlorophyll and β-carotene fluorescences were shown to be important in understanding the results of increased cell counts in freshly isolated zooxanthellae experimentally exposed to high temperatures (34, 36, and 38°C) over 24 h, with ambient temperature (29°C) used as controls. The ability to simultaneously identify and enumerate subpopulations of different physiological states in the same sample provides an enormous advantage in not just determining bleaching responses, but elucidating adaptive response and mechanisms for tolerance. Therefore, this approach might provide a rapid, convenient, and reproducible methodology for climate change studies and reef management programs. ? 2012 International Society for Advancement of Cytometry.     

Determining clinically relevant features in cytometry data using persistent homology

Cytometry experiments yield high-dimensional point cloud data that is difficult to interpret manually. Boolean gating techniques coupled with comparisons of relative abundances of cellular subsets is the current standard for cytometry data analysis. However, this approach is unable to capture more subtle topological features hidden in data, especially if those features are further masked by data transforms or significant batch effects or donor-to-donor variations in clinical data. We present that persistent homology, a mathematical structure that summarizes the topological features, can distinguish different sources of data, such as from groups of healthy donors or patients, effectively. Analysis of publicly available cytometry data describing non-naïve CD8+ T cells in COVID-19 patients and healthy controls shows that systematic structural differences exist between single cell protein expressions in COVID-19 patients and healthy controls. We identify proteins of interest by a decision-tree based classifier, sample points randomly and compute persistence diagrams from these sampled points. The resulting persistence diagrams identify regions in cytometry datasets of varying density and identify protruded structures such as ‘elbows’. We compute Wasserstein distances between these persistence diagrams for random pairs of healthy controls and COVID-19 patients and find that systematic structural differences exist between COVID-19 patients and healthy controls in the expression data for T-bet, Eomes, and Ki-67. Further analysis shows that expression of T-bet and Eomes are significantly downregulated in COVID-19 patient non-naïve CD8+ T cells compared to healthy controls. This counter-intuitive finding may indicate that canonical effector CD8+ T cells are less prevalent in COVID-19 patients than healthy controls. This method is applicable to any cytometry dataset for discovering novel insights through topological data analysis which may be difficult to ascertain otherwise with a standard gating strategy or existing bioinformatic tools.     

Increase of sensitivity of sputum cytology using high-resolution image cytometry: field study results

Lung cancer remains the leading cause of cancer deaths in the developed world. There is no widely accepted method to screen for this cancer. The most commonly used method remains conventional sputum cytology, but this method is hampered by low sensitivity. We tested the hypothesis that sensitivity of sputum cytology for early lung cancer can be greatly improved by using image analysis of sputum cells, at a modest reduction of specificity.The study was double-blinded and used sputum samples from subjects with well-characterized clinical diagnoses. There were 177 cancers, 98 dysplasias, and 558 normals. The study samples were separated into two independent sets: training set and test set. Sputum samples were collected prospectively from subjects with a high probability of having lung cancer. Seven institutions from five countries participated in the study. All subjects had complete clinical diagnoses which included, as a minimum, negative chest x-rays for all negative cancers, while all cancers had confirmed tissue pathology. Samples were prepared according to the Saccomanno method. For conventional cytology, slides were stained using Papanicolaou stain. For image analysis, slides were stained using a DNA-specific (Feulgen-Thionin) stain. An automated, high-resolution image cytometer was used for measurements.At 90% specificity, sensitivity of 60% can be achieved for adenocarcinoma, compared to only 14% sensitivity of conventional cytology (at 99% specificity). Similarly, 45% sensitivity at 90% specificity can be reached for stages 0 and I lung cancer, compared to only 14% (at 99% specificity) using conventional cytology.Cytometry combined with conventional cytology shows an increase in sensitivity to early-stage cancer and to adenocarcinomas compared to conventional cytology alone. While the results are encouraging, the sensitivity to detect early lung cancer should be further improved to 70-80% at 90-95% specificity before this test can be considered for screening of high-risk individuals for lung cancer. Cytometry (Clin. Cytometry) 50:168-176, 2002.