Immense random colocalization, revealed by automated high content image cytometry, seriously questions FISH as gold standard for detecting EML4-ALK fusion.

EML4-ALK gene fusion (inv2(p21p23)) of non-small cell lung cancer (NSCLC) predisposes to tyrosine kinase inhibitor treatment. One of the gold standard diagnostics is the dual color (DC) break-apart (BA) FISH technique, however, the unusual closeness of the involved genes has been suggested to raise likelihood of random co-localization (RCL) of signals. Although this is suspected to decrease sensitivity (often to as low as 40-70%), the exact level and effect of RCL has not been revealed thus far. Signal distances were analyzed to the 0.1 µm precision in more than 25,000 nuclei, via automated high content-image cytometry. Negative and positive controls were created using conventional DC BA-, and inv2(p21p23) mimicking probe-sets, respectively. Average distance between red and green signals was 9.72 pixels (px) (±5.14px) and 3.28px (±2.44px), in positives and negatives, respectively; overlap in distribution being 41%. Specificity and sensitivity of correctly determining ALK status was 97% and 29%, respectively. When investigating inv2(p21p23) with DC BA FISH, specificity is high, but seven out of ten aberrant nuclei are inevitably falsely classified as negative, due to the extreme level of RCL. Together with genetic heterogeneity and dilution effect of non-tumor cells in NSCLC, this immense analytical false negativity is the primary cause behind the often described low diagnostic sensitivity. These results convincingly suggest that if FISH is to remain a gold standard for detecting the therapy relevant inv(2), either a modified evaluation protocol, or a more reliable probe-design should be considered than the current DC BA one. © 2018 International Society for Advancement of Cytometry.

Biomarkers for screening of lung cancer and pre-neoplastic lesions in a high risk Chilean population.

BACKGROUND: The mortality of lung cancer (LC), increases each year in the world, in spite of any advances, in development of new drugs to advance stages of LC. The high incidence of LC has been associated with smoking habit, genetic diversity and environmental pollution. Antofagasta region has been reported to have the highest LC mortality rate in Chile and its inhabitants were exposed to arsenic in their drinking water in concentrations as high as 870 µg/L. Non-invasive techniques such as biomarkers (Automatic Quantitative Cytometry: AQC and DR70) and Auto Fluorescence Bronchoscopy (AFB) might be potentially useful as a supplementary diagnostic approach and early detection. Early detection is one of the most important factors to intervene and prevent cancer progression in LC. This is a work of an ongoing prospective bimodality cancer surveillance study in high risk LC volunteers. Enrolment was done in subjects from Antofagasta and Metropolitan regions. In addition, we enrolled subjects who were suspected of having lung cancer. AQC, DR70 and AFB were used as tools in the detection of pre-neoplastic (PNL) and neoplastic lesions (NL). RESULTS: Half of the samples, classified as suspicious by AFB, were confirmed as metaplasia or dysplasia by histopathology. For LC, DR70 showed a higher sensitivity (95.8%) and specificity (91.9%) than AQC. However, for PNL AQC showed a higher sensitivity (91.9%) than DR70 (27.3%), although both with low PPV values. As a pre screener, both biomarkers might be employed as complementary tools to detect LC, especially as serially combined tests, with a sensitivity of 60% and a PPV of 65.2%. Additionally, the use of parallel combined tests might support the detection of PNL (sensitivity 91.2%; PPV 49.1%). CONCLUSION: This work adds information on cellular and molecular biomarkers to complement imaging techniques for early detection of LC in Latin America that might contribute to formulate policies concerning screening of LC. Supported by INNOVA-CORFO, Chile.

Biomarkers for screening of lung cancer and pre-neoplastic lesions in a high risk Chilean population

BACKGROUND: The mortality of lung cancer (LC), increases each year in the world, in spite of any advances, in development of new drugs to advance stages of LC. The high incidence of LC has been associated with smoking habit, genetic diversity and environmental pollution. Antofagasta region has been reported to have the highest LC mortality rate in Chile and its inhabitants were exposed to arsenic in their drinking water in concentrations as high as 870 µg/L. Non-invasive techniques such as biomarkers (Automatic Quantitative Cytometry: AQC and DR70) and Auto Fluorescence Bronchoscopy (AFB) might be potentially useful as a supplementary diagnostic approach and early detection. Early detection is one of the most important factors to intervene and prevent cancer progression in LC. This is a work of an ongoing prospective bimodality cancer surveillance study in high risk LC volunteers. Enrolment was done in subjects from Antofagasta and Metropolitan regions. In addition, we enrolled subjects who were suspected of having lung cancer. AQC, DR70 and AFB were used as tools in the detection of pre-neoplastic (PNL) and neoplastic lesions (NL). RESULTS: Half of the samples, classified as suspicious by AFB, were confirmed as metaplasia or dysplasia by histopathology. For LC, DR70 showed a higher sensitivity (95.8%) and specificity (91.9%) than AQC. However, for PNL AQC showed a higher sensitivity (91.9%) than DR70 (27.3%), although both with low PPV values. As a pre screener, both biomarkers might be employed as complementary tools to detect LC, especially as serially combined tests, with a sensitivity of 60% and a PPV of 65.2%. Additionally, the use of parallel combined tests might support the detection of PNL (sensitivity 91.2%; PPV 49.1%). CONCLUSION: This work adds information on cellular and molecular biomarkers to complement imaging techniques for early detection of LC in Latin America that might contribute to formulate policies concerning screening of LC. Supported by INNOVA-CORFO, Chile.

A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high-content high-resolution multiparameter image-cytometry.

Dissection of complex molecular-networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high-resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image-analysis of data from confocal or widefield robotized microscopes. We have applied this image-cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell-cycle profile and active DNA-replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (γH2AX) and 53BP1, as markers of DNA-damage response (DDR); and (iii) p53 and p21, as checkpoint-activation markers. Our data suggest the existence of cell-cycle modulated mechanisms involving different functions of γH2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell-cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance.

A computational platform for robotized fluorescence microscopy (I): high-content image-based cell-cycle analysis.

Hardware automation and software development have allowed a dramatic increase of throughput in both acquisition and analysis of images by associating an optimized statistical significance with fluorescence microscopy. Despite the numerous common points between fluorescence microscopy and flow cytometry (FCM), the enormous amount of applications developed for the latter have found relatively low space among the modern high-resolution imaging techniques. With the aim to fulfill this gap, we developed a novel computational platform named A.M.I.CO. (Automated Microscopy for Image-Cytometry) for the quantitative analysis of images from widefield and confocal robotized microscopes. Thanks to the setting up of both staining protocols and analysis procedures, we were able to recapitulate many FCM assays. In particular, we focused on the measurement of DNA content and the reconstruction of cell-cycle profiles with optimal parameters. Standard automated microscopes were employed at the highest optical resolution (200 nm), and white-light sources made it possible to perform an efficient multiparameter analysis. DNA- and protein-content measurements were complemented with image-derived information on their intracellular spatial distribution. Notably, the developed tools create a direct link between image-analysis and acquisition. It is therefore possible to isolate target populations according to a definite quantitative profile, and to relocate physically them for diffraction-limited data acquisition. Thanks to its flexibility and analysis-driven acquisition, A.M.I.CO. can integrate flow, image-stream and laser-scanning cytometry analysis, providing high-resolution intracellular analysis with a previously unreached statistical relevance.

Nottingham-defined mitotic score: comparison with visual and image cytometric phosphohistone H3 labeling indices and correlation with Oncotype DX recurrence score.

Prognosis of breast cancer patients has been determined traditionally by lymph node status, tumor size, and histologic grade. In recent years the Oncotype DX recurrence score (RS) assay has emerged as an expensive adjunct prognostic tool. Markers of proliferation play a large role in determination of RS, and we have shown previously that immunohistochemical expression of proliferation markers Ki-67 and phosphohistone H3 (PPH3) correlates with RS. Our current goal is comparison of the hematoxylin and eosin (H&E) mitotic score, defined by the Nottingham grading system, with anti-PPH3 mitotic figure labeling assessed by both visual and automated image analysis and correlation of mitotic score results with RS. Estrogen receptor-positive breast carcinomas from 137 patients with Oncotype DX testing were selected. A representative H&E-stained tumor section was evaluated. Mitoses were counted per 10 high-power fields and tumors graded using the Nottingham criteria by 1 pathologist in accordance with College of American Pathologists-recommended mitotic count cutoffs for a field diameter of 0.55 mm. An additional section was immunostained with PPH3 antibody. PPH3 mitotic scores were determined visually and by automated imaging system. Statistical analysis was performed using univariate tests and Spearman coefficient. There was a statistically significant positive correlation among the 3 methods of mitotic score assessment. Specifically, correlation of tumor grades obtained using visual and automated methods of assessment of mitotic activity with PPH3 stain was the strongest and most statistically significant (weighted κ value 0.84, P<0.001; Spearman coefficient 0.89, P<0.001). There was a statistically significant positive correlation between H&E mitosis score and RS (P<0.001, Spearman coefficient 0.30) and between visual PPH3 mitotic score and RS (P<0.001, Spearman coefficient 0.28). In conclusion, mitotic score by any of the 3 methods studied may be useful in assessing tumor grade, proliferation, and prognosis.

Image cytometric HER2 in gastric carcinoma: is a new algorithm needed?

BACKGROUND: Algorithms for quantitation of HER2 immunohistochemistry were developed for breast carcinoma, where the membranous stain must be entirely around the cell membrane. For gastric carcinoma, although assessment of intensity of immunostain (0 to 3) is similar, the site and percentage of stain differs by lacking the requirement of entire cell membrane positivity (complete, basolateral, or lateral membranous reactivity is sufficient for a positive result). We quantitated HER2 in gastric cancer specimens visually and by image cytometry, comparing results, and where available, with fluorescence in situ hybridization (FISH). The goal was to assess whether lack of concordance among results, might suggest a requirement for changing the image cytometric algorithm. DESIGN: All gastric carcinoma biopsies, resections, and cell blocks studied for HER2 expression/amplification in the past 2 years were included. Immunostain intensity, percentage, and score 0 to 3+ (0, 1+ negative, 2+ equivocal, 3+ positive), were evaluated visually, and by image cytometry with the ACIS score 0 to 3 (0, 1 negative, 2 equivocal, 3 positive). FISH (<1.8 negative, 1.8 to 2.2 equivocal, >2.2 amplified) was performed on all specimens with scores 2 and 3 by image cytometry. Results were compared. RESULTS: Sixty-eight specimens were studied, including 43 (63.2%) biopsies, 17 (25%) resections, and 8 (11.8%) cell blocks. Forty-seven (69.1%) were primary gastric, esophageal, or gastroesophageal junction adenocarcinoma; 19 (27.9%) were metastatic; 3 (4.4%) were well, 14 (20.6%) moderately (17, 25% tubular), and 51 (75%) poorly differentiated (poorly cohesive). Fourteen (20.6%) of cases were HER2 IHC positive with no significant difference in frequency based on type of specimen, site of carcinoma, or differentiation. Of the 14 visually HER2 IHC positive, 13 were positive by image cytometry (93% concordance), all 13 were amplified by HER2 FISH (100% concordance). Of the 3 cases equivocal both visually and by image cytometry, only 1 was FISH amplified. Fifty-one were negative by IHC visually and 52 by image cytometry (98% concordance). None of the 5 HER2 IHC negative were amplified by FISH. CONCLUSIONS: Despite different recommendations for interpretation of HER2 in gastric versus breast cancer, equivocal and positive/amplified results visually, and by image cytometry, and where FISH was performed, are similar. This concordance is noted for biopsy, resection, and cell block specimens, for primary versus metastatic, and for moderately versus poorly differentiated carcinoma; HER2 positivity/amplification is most frequent with poor differentiation, but not significantly so. There seems to be no need for the HER2 image cytometric algorithm used for breast cancer, to be changed when used for assessment of gastric cancers.

A simple reproducible and time saving method of semi-automatic dendrite spine density estimation compared to manual spine counting.

Estimation of spine number and spine density by manual counting under the assumption that all dendrite protrusions equal spines are often used in studies on neuroplasticity occurring during health, brain diseases, and different experimental paradigms. Manual spine counting is, however, time consuming and biased by inter-observer variation. We present accordingly a quick, reproducible and simple non-stereological semi-automatic spine density estimation method based on the irregularity of the dendrite surface. Using the freeware ImageJ program, microphotographs of Golgi impregnated hippocampal dendrites derived from a previously performed study on the impact of chronic restrained stress were binarized, skeletonized, and the skeleton endings assumed to represent spine positions were counted and the spine densities calculated. The results based on 754 dendrite fragments were compared to manual spine counting of the same dendrite fragments using the Bland-Altman method. The results from both methods were correlated (r=0.79, p<0.0001), The semi-automatic counting method gave a statistically higher (approx. 4%) spine density number, but both counting methods showed similar significant differences between the groups in the CA1 area, and no differences between the groups in the CA3 area. In conclusion, the presented semi-automatic spine density estimation method yields consistently a higher spine density number than manual counting resulting in similar significance between groups. The proposed method may therefore be a reproducible time saving and useful non-stereological approach to spine counting in neuroplasticity studies requiring analysis of hundreds of dendrites.

Tumores mamarios caninos: estudio de ADN mediante análisis de citometría estática / Canine mammary tumours: A quantitative DNA study using static cytometry

Un análisis del contenido de ADN por citometría estática fue realizado en muestras fijadas en formalina e incluidas en parafina de 56 neoplasias mamarias caninas (23 benignas y 33 malignas). El contenido de ADN se ha correlacionado con el aspecto histológico, la edad y con marcadores inmunohistoquímicos de las neoplasias. Diez tumores benignos (43,47%) y dieciséis malignos (48,48%) fueron aneuploides. La aneuploidía no estuvo relacionada con la edad o con el carácter maligno de los tumores (P<0,05). La expresión de los marcadores receptor de progesterona, MIB-1, CD-31, p53, c-erbB2, y ciclina D1 no mostró diferencias significativas entre los tumores diploides y aneuploides (P<0,05). Los componentes epiteliales y mesenquimales de los tumores mixtos benignos, adenomas complejos y carcinomas en tumores mixtos tienen un contenido de ADN idéntico en 74,0% de los casos lo que sugiere un origen celular común de ambos componentes(AU)

Static cytometric analysis of DNA content was performed on formalin-fixed paraffin wax-embedded samples of 56 canine mammary neoplasms (23 benign and 33 malignant) and histology, patient age and immunohistochemical markers were compared between diploid and aneuploid tumours. 10 benign tumours (43.47%) and 16 malignant (48.48%) were aneuploid. No association was found between age and ploidy (P>0.05). Aneuploidy was not related to age or tumour malignancy (P<0.05). The expression of the following markers: progesterone receptor, MIB-1, CD-31, p53, c-erbB2, and cyclin D1 did not show significant differences between diploid and aneuploid tumours (P<0.05). Epithelial and mesenchymal components of benign mixed tumours, complex adenomas and carcinomas in mixed tumours had an identical DNA content in 74.0% of cases suggesting a common cell origin of both components(AU)

Automated reconstruction of neuronal morphology: an overview.

Digital reconstruction of neuronal morphology is a powerful technique for investigating the nervous system. This process consists of tracing the axonal and dendritic arbors of neurons imaged by optical microscopy into a geometrical format suitable for quantitative analysis and computational modeling. Algorithmic automation of neuronal tracing promises to increase the speed, accuracy, and reproducibility of morphological reconstructions. Together with recent breakthroughs in cellular imaging and accelerating progress in optical microscopy, automated reconstruction of neuronal morphology will play a central role in the development of high throughput screening and the acquisition of connectomic data. Yet, despite continuous advances in image processing algorithms, to date manual tracing remains the overwhelming choice for digitizing neuronal morphology. We summarize the issues involved in automated reconstruction, overview the available techniques, and provide a realistic assessment of future perspectives.

Image cytometry accurately detects DNA ploidy abnormalities and predicts late relapse to high-grade dysplasia and adenocarcinoma in Barrett's oesophagus following photodynamic therapy.

BACKGROUND AND AIMS: DNA ploidy abnormalities (aneuploidy/tetraploidy) measured by flow cytometry (FC) are strong predictors of future cancer development in untreated Barrett's oesophagus, independent of histology grade. Image cytometric DNA analysis (ICDA) is an optical technique allowing visualisation of abnormal nuclei that may be undertaken on archival tissue. Our aim was to determine the accuracy of ICDA vs FC, and evaluate DNA ploidy as a prognostic biomarker after histologically successful treatment with photodynamic therapy (PDT). METHODS: Nuclei were extracted from 40 mum sections of paraffin-embedded biopsies and processed for ICDA at UCL and FC at UW using standardised protocols. Subsequently, DNA ploidy was evaluated by ICDA on a cohort of 30 patients clear of dysplasia 1 year after aminolaevulinic acid PDT for high-grade dysplasia (HGD). The results were correlated with long-term outcome. RESULTS: In the comparative study, 93% (41 out of 44) of cases were classified identically. Errors occurred in the near-diploid region by ICDA and the tetraploid region by FC. In the cohort study, there were 13 cases of late relapse (7 cancer, 6 HGD) and 17 patients who remained free of dysplasia after a mean follow-up of 44 months. Aneuploidy post-PDT was highly predictive for recurrent HGD or cancer with a hazard ratio of 8.2 (1.8-37.8) (log-rank P=0.001). CONCLUSIONS: ICDA is accurate for the detection of DNA ploidy abnormalities when compared with FC. After histologically successful PDT, patients with residual aneuploidy are significantly more likely to develop HGD or cancer than those who become diploid. DNA ploidy by ICDA is a valuable prognostic biomarker after ablative therapy.

Image cytometric validation of breast carcinoma markers (ER, HER2 and MIB-1) using tissue microarrays: rabbit monoclonal vs. FDA-approved antibodies.

OBJECTIVE: To use the ACIS III (Dako, Carpinteria, California, U.S.A.) with tissue microarrays (TMAs) to compare rabbit monoclonal antibodies (RMab) for ER, HER2, and MIB-1 with FDA-approved monoclonal (FMab) and polyclonal (FPab) antibodies. STUDY DESIGN: TMAs of 43 breast cancers were used. Immunohistochemistry was performed using RMab (LabVision, Fremont, California, U.S.A.): ER (SP1; 1/100), HER2 (SP3; 1/100), and MIB-1 (SP6; 1/200). FMPab (Dako) used: ER (1D5; 1/50), HercepTest kit and MIB-1 (MIB-1; 1/160). The stained TMAs were quantitated visually and by image cytometry (ACIS III). RESULTS: The overall agreement between RMab and FMab for ER using visual (98.45%) and image analysis (97.56%) was excellent, with a kappa level of 0.89 and 0.94, respectively. For HER2, the overall agreement between RMab and FPab was fair for visual (67.44%) and substantial (87.50%) for image analysis, with a kappa level of 0.32 and 0.72, respectively. For MIB-1, there was fair (64.29%) to poor (43.33%) agreement between MIB-1 RMab and FMab using visual and image analysis, with a kappa level of 0.47 and 0.16, respectively. CONCLUSION: RMabs for ER (SP1) and HER2 (SP3) are almost comparable to their counterpart, FDA antibodies; however, MIB-1 RMab (SP6) shows poor concordance with FMab in TMA. Image analysis shows a better concordance than visual quantitation assessment specifically for ER and HER2.

Morphometric analysis of the inferior olivary complex in infants and adults.

In the literature, few studies detailed morphometric parameters of the inferior olivary complex, and mainly applying biased methods based on counting in a two-dimensional plane. In the present work, the unbiased quantitative method of the optic disector was applied in order to analyse neuronal densities, nuclear volumes and total neuron numbers of the principal (PION), medial (MION) and dorsal (DION) nuclei of the inferior olivary complex in adults (16 male, 6 female; mean age: 37 years) and infants (5 male, 5 female; mean age: 5 months). In both adult and infant series, statistically significant differences were not found in neuronal densities between the various inferior olivary nuclei. All the nuclei showed higher volumes and lower neuronal densities in adults than infants, without statistically significant differences in total neuron numbers, thus suggesting postnatal development of the neuropil.

[Implementation of cytology images classification--the Bethesda 2001 System--in a group of screened women from Podlaskie region--effect evaluation]. / Ocena efektów wdrozenia klasyfikacji obrazów cytologicznych wg systemu Bethesda 2001, w populacji kobiet województwa podlaskiego poddanych badaniom profilaktycznym.

UNLABELLED: Increasing knowledge concerning carcinogenesis within cervical epithelium has forced us to make continues modifications of cytology classification of the cervical smears. Eventually, new descriptions of the submicroscopic cytomorphological abnormalities have enabled the implementation of Bethesda System which was meant to take place of the former Papanicolaou classification although temporarily both are sometimes used simultaneously. AIM: The aim of this study was to compare results of these two classification systems in the aspect of diagnostic accuracy verified by further tests of the diagnostic algorithm for the cervical lesion evaluation. MATERIALS AND METHODS: The study was conducted in the group of women selected from general population, the criteria being the place of living and cervical cancer age risk group, in the consecutive periods of mass screening in Podlaski region. The performed diagnostic tests have been based on the commonly used algorithm, as well as identical laboratory and methodological conditions. RESULTS: Performed assessment revealed comparable diagnostic accuracy of both analyzing classifications, verified by histological examination, although with marked higher specificity for dysplastic lesions with decreased number of HSIL results and increased diagnosis of LSILs. Higher number of performed colposcopies and biopsies were an additional consequence of TBS classification. Results based on Bethesda System made it possible to find the sources and reasons of abnormalities with much greater precision, which enabled causing agent treatment. CONCLUSION: Two evaluated cytology classification systems, although not much different, depicted higher potential of TBS and better, more effective communication between cytology laboratory and gynecologist, making reasonable implementation of The Bethesda System in the daily cytology screening work.

Personal cytometers: slow flow or no flow?

BACKGROUND: Although some manufacturers have optimistically described instruments with prices in the 40,000 US dollars range as "personal cytometers", analogy with the personal computer suggests that the target price for a true "personal" cytometer should be under 5,000 US dollars. Since such an apparatus could find a wide range of applications in cytomics in both developing and developed countries, it seemed desirable to consider its technical and economic feasibility. METHODS: Using resolution targets and a variety of fluorescent bead standards immobilized on filters and/or slides, we evaluated high-intensity LEDs as fluorescence excitation sources, relatively inexpensive CCD cameras as detectors, and 35 mm camera lenses and plastic low-power microscope optics for light collection in a simple, inexpensive low-resolution imaging cytometer. RESULTS: The components tested could be combined toproduce an instrument capable of detecting fewer than 10,000 molecules of cell-associated fluorescent label, and thus applicable to a broad range of cytometric tasks. CONCLUSIONS: Given the requirements for light sources, detectors, optics, mechanics, electronics and data analysis hardware and software, and the components presently available, it should be easier to reach the desired 5,000 US dollars price point with an image cytometer than with a flow cytometer.

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.