Biomedical subjects

Andreas O H Gerstner

Publications and source records attributed to Andreas O H Gerstner.

13 recordsLinked to original sources

PubMed2006

Comparison of immunophenotyping by slide-based cytometry and by flow cytometry.

BACKGROUND: Flow cytometry (FCM) is the gold standard for immunophenotyping of peripheral blood leukocytes (PBLs). Slide-based cytometry (SBC) systems, for example the laser scanning cytometer (LSC(R), CompuCyte), can give additional information (repeated staining and scanning, morphology). In order to adequately judge the clinical usefulness of LSC for immunophenotyping it is obligatory to compare it with FCM. AIM: The aim of this study was to systematically compare immunophenotyping by both FCM and LSC methods and to test the correlation of the results. METHODS: PBLs were stained with directly labeled monoclonal antibodies with the whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in parallel by a FACScan (BD-Biosciences) using standard protocols and by LSC with different triggers (forward scatter, CD45 FITC, or 7-AAD). For 7-AAD measurements by LSC, slides were additionally fixed with acetone before 7-AAD staining. RESULTS: Calculating the percentage distribution of PBLs obtained by LSC and by FCM showed very good correlation with regression coefficients close to 1.0 for the major populations and the lymphocyte sub-populations (neutrophils, monocytes, and lymphocytes; T-helper-, T-cytotoxic-, B-, NK-cells). The best trigger for LSC was 7-AAD. CONCLUSION: LSC can be recommended for immunophenotyping of PBLs especially in cases where only limited sample volumes are available or where additional analysis of the cells' morphology is important. The detection of rare leukocytes or weak antigens is limited; in these cases appropriate amplification steps for immunofluorescence should be engaged.

Dactinomycin↗

PubMed2006

Iterative restaining as a pivotal tool for n-color immunophenotyping by slide-based cytometry.

AIM: Slide-based cytometry (SBC) allows to "ask a cell a second time." We used this tool for detailed immunophenotyping of peripheral blood leukocytes (PBLs). METHODS: PBLs primarily stained for CD-markers and DNA were immobilized on a glass slide and analyzed by laser scanning cytometry. Then, iterative restaining was applied for a second and a third analysis. Based on the cells' fixed location, analyses were merged on a single-cell level. RESULTS: We analyzed six virtual immunostainings by "recycling" the PE-channel for four CD-markers. CONCLUSION: Iterative restaining might prove to be a pivotal tool for n-color immunophenotyping exclusive to SBC concepts.

Antibodies↗

PubMed2006

Sequential photobleaching of fluorochromes for polychromatic slide-based cytometry.

BACKGROUND: Slide-based cytometry is a key technology for polychromatic cytomic investigations. Here we exploit the relocalization and merge feature of Laser Scanning Cytometry for distinguishing fluorochromes of comparable emission spectra but different photostabilities. METHODS: Blood specimens were stained with the fluorochrome pairs: FITC/ALEXA488, PE/ALEXA532, or APC/ALEXA633. Bleaching was performed by repeated laser excitation. RESULTS: Since ALEXA dyes are photostable as compared to the conventional fluorochromes FITC, PE, and APC, a differentiation within one fluorochrome pair is possible. CONCLUSION: The sequential photobleaching method results in an increased information density on a single cell level and represents an important component to perform polychromatic cytometry.

Antigens, CD19↗

PubMed2006

Novel aspects of systems biology and clinical cytomics.

The area of Cytomics and Systems Biology became of great impact during the last years. In some fields of the leading cytometric techniques it represents the cutting edge today. Many different applications/variations of multicolor staining were developed for flow- or slide-based cytometric analysis of suspensions and sections to whole animal analysis. Multispectral optical imaging can be used for studying immunological and tumorigenic processes. New methods resulted in the establishment of lipidomics as the systemic research of lipids and their behavior. All of these development push the systemic approach of the analysis of biological specimens to enhance the outcome in the clinic and in drug discovery programs.

Biomedical Research↗

PubMed2006

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.

Animals↗

PubMed2006

Prediction of upper aerodigestive tract cancer by slide-based cytometry.

AIM: To evaluate slide-based cytometry in screening for and following up of carcinoma of the upper aerodigestive tract using swabs for a minimal-invasive approach. METHODS: Laser scanning cytometry (LSC) was used for multiparametric analysis of cells stained for cytokeratin and DNA to determine the DNA-index (DI) of the tumor cells. Histograms with 0.95 < DI < 1.05 and 1.9 < DI < 2.1 were defined as DNA euploid and any other DI as DNA aneuploid. After subsequent HE-staining, single cells were relocalized in order to document morphology. Conventional cytology was also performed on a subset of the slides. Routine histopathology of parallel biopsies served as gold standard in all cases. RESULTS: 115 swabs from 109 patients were obtained from the entire upper aerodigestive tract. 16 swabs were classified as insufficient for LSC. In the remaining 99 specimens, 1 benign lesion was misclassified as malignant, while 61 of the 75 malignant lesions were correctly identified. This corresponds to predictive values of 98.4% and 62.2% for the detection of malignant and benign samples by LSC. CONCLUSION: This pilot study demonstrates the validity of LSC screening for the identification of tumor malignancy in the upper aerodigestive tract from swab collected cytological material.

Humans↗

PubMed2005

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.

CD3 Complex↗

PubMed2005

Preoperative detection of laryngeal cancer in mucosal swabs by slide-based cytometry.

The aim of our study was to evaluate slide-based cytometry in screening for laryngeal cancer using swabs a minimally invasive approach. Laser scanning cytometry (LSC) was used for the multiparametric analysis of cells stained for cytokeratin and DNA to determine the DNA-index (DI) of the tumour cells. Histograms with DI < 0.95, 1.05 < DI < 1.9, and 2.1 < DI were defined as DNA aneuploid. After subsequent haemotoxylin-eosin (HE)-staining, single cells were re-localised and an analysis by conventional cytology was performed. Additionally, routine histopathology of parallel biopsies was obtained in all cases. Fifty one swabs from 49 lesions were analyzed. Seven and 17 swabs, were classified as insufficient for LSC and cytology, respectively. One and two benign lesions, were misclassified as malignant, respectively. Out of 34 malignant lesions, LSC detected 25 and cytology 14. LSC was superior to cytology in all of the statistical parameters tested. This pilot study demonstrates the validity of LSC for the preoperative detection of malignancy in laryngeal tumours using swabs.

Algorithms↗

PubMed2004

Quantitative histology by multicolor slide-based cytometry.

BACKGROUND: In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings. METHODS: We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed "multiple thresholding," which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA-fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single- (Ar) or dual-laser excitation (Ar-HeNe) in order to analyze single- (FITC) up to four-color (FITC/PE/PECy5/APC) stained specimen. RESULTS: Percentage and cellular density of cell-subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r(2) = 0.96, P < 0.0001). With LSC, semiautomated operator-independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible. CONCLUSIONS: We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions.

Automation↗

PubMed2003

Slide-based cytometry for predicting malignancy in solid salivary gland tumors by fine needle aspirate biopsies.

BACKGROUND: To minimize hospitalization and morbidity for a patient with a solid tumor of a salivary gland, malignancy must be confirmed or excluded as soon as possible. This information cannot be obtained preoperatively by existing standard procedures. Minimal-invasive approaches with adequate diagnostic analysis represent a promising precondition for optimized therapy. METHODS: For fine needle aspirate biopsies (FNABs), laser scanning cytometry (LSC) offers a semi-automated slide-based technology for objective and quantitative analysis. We have established an assay for FNABs from salivary gland tumors. FNAB cells were stained for cytokeratin and DNA followed by LSC analysis. The cells were subsequently HE-stained and were relocalized on the slide. The LSC analysis quantitatively determines the DNA index (DI) of the tumor cells taking leukocytes as internal DNA diploid standard. Histograms with 0.95 < DI < 1.05 and 1.9 2.5 (i.e., 5c exceeding rate, 5cER) was calculated. Samples with DNA aneuploid peaks or with 5cER > 5% were classified as malignant. Routine histopathology was performed as a control. RESULTS: FNABs from 51 solid salivary gland tumors (41 parotid gland, six submandibular, four parapharyngeal) were analyzed with this assay. Eleven of 14 malignant tumors were DNA aneuploid by LSC analysis. All benign tumors showed diploid DNA content. The positive predictive value for malignancy was 1.0, the negative predictive value was 0.93, the correlation with routine histopathology was highly significant (p = 7.6 x 10(-9), Fisher's exact test). The calculated specificity of LSC analysis was 1.0 and the sensitivity was 0.79. CONCLUSIONS: This pilot study demonstrates the validity of slide-based cytometry for the preoperative prediction of malignancy in solid tumors being inaccessible for incision biopsy but suitable for FNABs such as those of the parotid gland.

Adenoma, Pleomorphic↗

PubMed2003

Analysis of ploidy in hypopharyngeal cancer by laser scanning cytometry on fine needle aspirate biopsies.

AIM: To test laser scanning cytometry (LSC) for the analysis of ploidy in squamous cell carcinoma of the hypopharynx (SCCH) and to develop a routine application for minimal samples such as fine needle aspirate biopsies (FNABs). METHODS: From 11 individuals 30 FNABs of primary tumors (n=11) and lymphatic metastases of SCCH (n=11) and non-metastatic lymph nodes (n=8) are analyzed by LSC. This microscope based instrument scans the cells after immobilization on a glass slide and after double staining of cytokeratin and DNA. The location of each cell is stored with the fluorescence data. Therefore the morphology of every cell can be documented by re-staining with H & E; and re-localization on the slide. Additionally, aliquots are Feulgen-stained for image cytometry in 8 specimens. RESULTS: The diploid reference peak is identified taking leukocytes as internal standard. The DNA-index of the carcinoma cells ranges from 0.4 to 3.8. Comparison with image cytometry shows good correlation (r=0.89). CONCLUSION: LSC provides a reliable and objective way to determine the ploidy of SCCH pre-operatively. Colour figures can be viewed on http://www.esacp.org/acp/2003/25-2/gerstner.htm.

Biopsy↗

PubMed2002

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry.

BACKGROUND: To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. METHODS: The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. RESULTS: With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. CONCLUSIONS: With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.

Benzothiazoles↗

PubMed2002

Clinical applications of laser scanning cytometry.

This study reviews existing and potential clinical applications of laser scanning cytometry (LSC) and outlines possible future developments. LSC provides a technology for solid phase cytometry. Fluorochrome-labeled specimens are immobilized on microscopic slides that are placed on a conventional epifluorescence microscope and analyzed by one or two lasers. Data comparable to flow cytometry are generated. In addition, the position of each event is recorded, a feature that allows relocalization and visualization of each measured event. The major advantage of LSC compared with other cytometric methods is the combination of two features: (a) the minimal clinical sample volume needed and (b) the connection of fluorescence data and morphological information for the measured event. Since the introduction of LSC, numerous methods have been established for the analysis of cells, cellular compartments, and tissues. Although most cytometric methods use only two or three colors, the characterization of specimens with up to five fluorochromes is possible. Most clinical applications have been designed to determine ploidy and immunophenotype; other applications include analyses of tissue biopsies and sections, fluorescence in situ hybridization, and the combination of vital and nonvital information on a single-cell basis. With the currently available assays, LSC has proven its wide spectrum of clinical applicability in slide-based cytometry and can be introduced as a standard technology in multiple clinical settings.

Apoptosis↗