Predictive medicine and clinical cytomics research: résumé of the 15th Annual Meeting of the German Society for Cytometry (Deutsche Gesellschaft für Zytometrie, DGfZ).
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Flow Cytometry↗
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Cytometry of fluorescence resonance energy transfer.
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Algorithms↗
Assessment of DNA content in formalin-fixed, paraffin-embedded tissue of lung cancer by laser scanning cytometer.
A new cytometric device, a laser scanning cytometer was developed to overcome the limitations of flow cytometry (FCM) and image analyses. The purpose of this study was to develop a method that allows laser scanning cytometry (LSC) to be used for measuring the cellular DNA content of paraffin-embedded tissues. Paraffin-embedded lung cancer tissue from 30 patients was analyzed by both FCM (p-FCM) and LSC (p-LSC). In addition, touch preparations from fresh frozen tissues were prepared to provide material for LSC (f-LSC). The limits of agreement for the DNA indices (DI) measured by p-LSC and p-FCM were -0.07 to 0.07, indicating that for a given case, these methods would be expected to differ by no more than 0.07. The limits of agreement for comparisons between the other materials and methods were wider and depended upon the size of the measurements. Agreement between f-LSC and p-FCM was good for small DI values, but poor for large values. Agreement between f-LSC and p-LSC was poor for small and large DI values, but good for moderately sized values. Discordancies in DNA ploidy status between different materials and methods may have been caused by either the heterogeneity within tumors, sampling errors or differences in the interpretation of histograms. This method allows a comparison of the results of DNA analysis with histologic findings from hematoxylineosin-stained sections and the prognosis of the patients.
Aneuploidy↗
[Basic study of automated cytodiagnosis using flow cytometry--a hybrid method with image analysis].
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Carcinoma, Squamous Cell↗
Comparison of image analysis and flow cytometric measurements of DNA content of canine transitional cell carcinomas.
The DNA content of neoplasms is generally determined by flow cytometry, but quantitative microscopic image analysis is an alternative technique for DNA quantification. To compare these two methods, the DNA content of 31 paraffin-embedded canine transitional cell carcinomas was measured by flow cytometry and image analysis. Interpretable data were available for 26 samples by flow cytometry and 27 samples by image analysis. For 23 neoplasms with interpretable data by both methods, a good correlation (r = .80, P < .001) was obtained between the DNA indices determined by flow cytometry and image analysis. Concordance of DNA ploidy classification was reached in 21 samples; however, for 4 samples an aneuploid cell population, usually tetraploid, was identified by image analysis and not by flow cytometry. Two samples also had aneuploid peaks by flow cytometry and not by image analysis. Although a good correlation was seen between the measurement of DNA content of paraffin-embedded canine tissue by image analysis and flow cytometry, this study did demonstrate that aneuploid cell populations can be missed by both methods and that image analysis was better than flow cytometry in the detection of aneuploid cell populations, particularly tetraploid ones, in samples with excessive cellular debris.
Animals↗
Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging.
The intracellular fate of particulate drug delivery systems is determined by the initial mode of internalization and the subsequent intracellular trafficking. Besides clathrin-mediated endocytosis, different cellular uptake mechanisms for internalization are discussed, including caveolae-mediated endocytosis, phagocytosis, macropinocytosis and the badly characterized non-clathrin-non-caveolae-mediated endocytosis. In addition, fusion processes could also be included. For a targeted drug delivery, the mechanism of caveolae uptake seems to be most promising since it does not transport internalized material to endosomes and lysosomes where internalized material might be degraded. The knowledge of the mode of internalization is therefore the first step for optimized drug targeting. The uptake and the intracellular behavior of pH-sensitive liposomes are investigated in two different cell types. For investigation, two different approaches were taken: a) a combination of different inhibitors was used to selectively block different pathways. These effects were evaluated by flow cytometry. b) Fluorescently labeled markers, specific for the different endocytic routes, were incubated with fluorescently labeled liposomes with a subsequent check for co-localization (spectral bio-imaging). COS-7 and HUVEC internalize pH-sensitive liposomes by means of different mechanisms. HUVEC seem to internalize via clathrin-dependent endocytosis, caveolae-dependent endocytosis and macropinocytosis whereas COS-7 cells behave differently: here macropinocytosis is not involved in the uptake. We show that the combination of flow cytometry and spectral bio-imaging offers the possibility to understand the initial mode of internalization and to follow the intracellular fate of liposomes, both of which are a prerequisite for optimizing drug targeting systems.
Animals↗
Visualization of DNA-containing structures in various species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR Green I dye.
We developed an alternative method of staining cell nuclei and chloroplast nucleoids of algal cells using SYBR Green I (the fluorescent dye used commonly for detecting dsDNA in agarose and polyacrylamide gels as an alternative to highly mutagenic ethidium bromide and for DNA staining of viruses and bacteria followed by flow cytometry, digital image analysis or real-time PCR), which enabled routine staining in vivo. Cells do not need to be fixed or treated chemically or physically before staining, thus the shape, size and position of DNA-containing structures are not affected. The fluorescence signal is sharp and reproducible. Examples of application of the method are shown in color microphotographs for representatives of eukaryotic algae from the taxa Chlorophyta, Rhodophyta and the prokaryotic Cyanophyta. The method is also useful for studying progress of the cell cycle in algal cells dividing by multiple fission, as shown by observation of changes in nuclear number during the cell cycle of the green alga Chlamydomonas reinhardtii and Scenedesmus quadricauda. Staining with SYBR Green I can be recommended as a fast, safe and efficient method for the detection of DNA-containing structures in vivo.
Benzothiazoles↗
A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide.
Ten patients with myeloid leukemias were treated in a phase I trial with escalating doses of mouse monoclonal antibody (mAb) M195, reactive with CD33, a glycoprotein found on myeloid leukemia blasts and early hematopoietic progenitor cells but not on normal stem cells. M195 was trace-labeled with iodine-131 (131I) to allow detailed pharmacokinetic and dosimetric studies by serial sampling of blood and bone marrow and whole-body gamma-camera imaging. Total doses up to 76 mg were administered safely without immediate adverse effects. Absorption of M195 onto targets in vivo was demonstrated by biopsy, pharmacology, flow cytometry, and imaging; saturation of available sites occurred at doses greater than or equal to 5 mg/m2. The entire bone marrow was specifically and clearly imaged beginning within hours after injection; optimal imaging occurred at the lowest dose. Bone marrow biopsies demonstrated significant dose-related uptake of M195 as early as 1 hour after infusion in all patients, with the majority of the dose found in the marrow. Tumor regressions were not observed. An estimated 0.33 to 1.0 rad/mCi 131I was delivered to the whole body, 1.1 to 6.1 rad/mCi was delivered to the plasma, and up to 34 rad/mCi was delivered to the red marrow compartment. 131I-M195 was rapidly modulated, with a majority of the bound immunoglobulin G (IgG) being internalized into target cells in vivo. These data indicate that whole bone marrow ablative doses of 131I-M195 can be expected. The rapid, specific, and quantitative delivery to the bone marrow and the efficient internalization of M195 into target cells in vivo also suggest that the delivery of other isotopes such as auger or alpha emitters, toxins, or other biologically important molecules into either leukemia cells or normal hematopoietic progenitor cells may be feasible.
Adult↗
Intracellular alkalinisation in Vero cells parasitised by Trypanosoma cruzi.
We studied the intracellular pH of Vero cells parasitised by Trypanosoma cruzi, using different methods: fluorimetric measurement after labelling the cells with the pH-sensitive intracellular fluorescent dye 2',7',-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; flow cytometry; and image analysis after staining the cells with neutral-red vital stain. The results show that the intracellular pH of the parasitised cells rose in comparison with that of the uninfected control cells. A study of the population of parasitised cells made by flow cytometry allowed us to subdivide the cells from the infected cultures into two populations according to their pH as obtained by fluorimetric measurements. Image analysis showed that the cell cytoplasm was more alkaline in the vicinity of the sites containing parasites. Treatment of the parasitised cells with amiloride, ouabain, or with 4.4'-diisothiocyano-2,2'-stilbene disulphate consistently lowered the pH values of the parasitised cells, but not sufficiently to return to the values of the non-parasitised control cells. When the control cells were subject to similar treatments with the inhibitors, only amiloride acidified the cytoplasm to any extent. The basification undergone by the parasitised cells was independent of the transport systems and may be a consequence of the release of NH4+ by the intracellular amastigotes.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
DNA image cytometric analysis of paraffin-embedded sections of small renal cortical neoplasms.
DNA ploidy analysis of neoplasms is most frequently performed by flow cytometry of single nuclear suspensions or image analysis of Feulgen-stained cytologic specimens. To assess the prognostic significance of ploidy in renal adenomas (less than 3 cm in diameter), nuclear DNA content of 21 renal cortical neoplasms was analyzed by image cytometry (CAS 200, Cell Analysis Systems, Inc., Elmhurst, IL) on Feulgen-stained 5 microns fixed embedded tissue sections. Five (36%) of 14 small neoplasms (so-called renal adenomas; (mean diameter, 7.2 mm; range, 2-24 mm) were in the aneuploid range. Four (57%) of seven carcinomas (mean diameter, 6.3 cm; range, 3.5-10 cm) were aneuploid, a frequency not significantly different from that in the adenomas (P = 0.4). Mean DNA index (1.38) for aneuploid adenomas and carcinomas was identical. Diploid renal adenomas (mean diameter, 5.9 mm) were smaller, although not significantly so (P > 0.05), than the aneuploid ones (mean, 9.7 mm). Size of diploid (mean, 6.7 cm) and aneuploid (mean, 6.1 cm) renal carcinomas was similar. Clinical follow-up studies to evaluate utility of DNA ploidy and proliferative activity in prognosis and management of small renal cortical neoplasms are indicated.
Adenoma↗
The precision and accuracy of six different methods to determine sperm concentration.
The development of new technologies and software that are routinely used in laboratories has now allowed for a more diverse novel range of methods to determine sperm concentrations more rapidly. The aim of this study was to compare 3 such novel methods developed in our laboratory, including a new flow cytometry approach, image analysis, and a fluorescent plate reader, with more conventional methods (hemocytometry, spectrophotometry, and Microcell analysis). Fifteen ejaculates were collected from 13 bulls at an artificial insemination center. The semen samples were analyzed for sperm concentration using a spectrophotometer, hemocytometry, and a novel flow cytometry technique based on counting a fixed volume of fluid. The raw ejaculate was also diluted fivefold in a long-term diluent and sent overnight to another laboratory, where sperm numbers were assessed using Microcells, an image analysis system, and a fluorescent plate reader. Each ejaculate was assessed 5 times using each of the methods described in order to determine the coefficient of variation for each method. Comparisons between methods were determined using correlation and limits of agreement. The flow cytometry results showed the lowest coefficient of variation (2.3%), with the plate reader showing the highest coefficient of variation (20.0%). There was no significant difference between any of the methods used, and none of them consistently over- or underestimated numbers when compared against each other. It is concluded that flow cytometry showed the highest repeatability of results. However, the method employed by each laboratory should be determined based on a range of factors, including cost, convenience, sample size, and number of ejaculates to be assessed.
Animals↗
Analysis and sorting of chromosomes by flow cytometry: new trends.
Flow cytogenetic is widely used since 1975, and essentially contributes to karyotype analysis and chromosome sorting. The principles of experimentation and its possibilities and limitations are now well known. Recently several new technologies have appeared. What attitude should the cytometrist adopt regarding PCR, microdissection of chromosomes, in situ hybridization, slit-scan flow cytometry or image analysis?
Animals↗
Immunocytometric quantitation of foeto-maternal haemorrhage with the Abbott Cell-Dyn CD4000 haematology analyser.
This study evaluated the extended use of a haematology analyser (Abbott Cell-Dyn CD4000) for the immunofluorescent enumeration of foeto-maternal haemorrhage (FMH) with fluorescein isothiocyanate-labelled monoclonal anti-RhD. Method performance was assessed with artificial FMH standards, and a series of 44 clinical samples. Within run precision was <15% (coefficient of variation, CV) for FMH volumes of 3 ml and above, 18.8% at an FMH volume of 2 ml and 31.7% at an FMH volume of 1 ml. Linearity analysis showed excellent agreement (observed FMH% = 0.98x expected FMH% + 0.02), and a close relationship (R(2) = 0.99) between observed and expected FMH percentages. The lower limit of quantification of the CD4000 (SRP-Ret) method with a maximum CV of 15% was 1.6 ml, and the limit of detection was <1 ml. Parallel Kleihauer-Betke test (KBT) assessments of FMH standards showed an overall trend for higher KBT values (observed = 1.25x expected - 0.38). At an FMH level of 4 ml, KBT observer estimates ranged from 0.57 to 11.94 ml with a mean inter-observer CV of 63%. For 44 clinical samples, there was decision point agreement between KBT and SRP-Ret results for 42 samples with an FMH of <2 ml. Analysis in the low FMH range (<1 ml) showed that small volume foetal leaks could be detected with the SRP-Ret method in most of 23 samples with negative KBT results. CD4000 SRP-Ret method performance for FMH determination was similar to that reported for flow cytometry.
Adult↗
Repetitive sequences: cause for variation in genome size and chromosome morphology in the genus Oryza.
Large variation in genome size as determined by the nuclear DNA content and the mitotic chromosome size among diploid rice species is revealed using flow cytometry and image analyses. Both the total chromosomal length (r = 0.939) and the total chromosomal area (r = 0.927) correlated well with the nuclear DNA content. Among all the species examined, Oryza australiensis (E genome) and O. brachyantha (F genome), respectively, were the largest and smallest in genome size. O. sativa (A genome) involving all the cultivated species showed the intermediate genome size between them. The distribution patterns of genome-specific repetitive DNA sequences were physically determined using fluorescence in situ hybridization (FISH). O. brachyantha had limited sites of the repetitive DNA sequences specific to the F genome. O. australiensis showed overall amplification of genome-specific DNA sequences throughout the chromosomes. The amplification of the repetitive DNA sequences causes the variation in the chromosome morphology and thus the genome size among diploid species in the genus Oryza.
Base Sequence↗
A PC-at based video device for flow cytometrically triggered cell imaging in flow.
A personal computer-based system of imaging in flow is described which takes cell images directly from the transducer of a flow cytometer. Imaging is triggered by the pulses of the flow system. The movement of the cells is frozen by ultra-short flashes from pulsed light emitting diodes (LED). The images are taken up by a video camera, transferred to an add-in board of the PC and then stored on disk. A novel method has been developed for capturing the video images which normally arise in asynchronism with the video frame. Images of different cells and particles exposed during flow analysis are shown.
Computer Systems↗
Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.
Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8(+) lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8(+) T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.
Flow Cytometry↗
[Initial results of image cytometric DNA analysis in the evaluation of prognosis in papillary thyroid cancer].
In this study we analysed DNA-ploidy as a potential prognostic parameter in papillary thyroid carcinoma. Paraffin embedded histological material, obtained by resection from 19 patients with a papillary thyroid carcinoma, was selected for analysis. Tumor areas within the paraffin-embedded material were identified by HE-stained reference sections. One 50 microns section was dewaxed, rehydrated and mechanically and enzymatically prepared to form a suspension of 10,000 cells/ml. 1 ml of the suspension, which contained bare nuclei with small rests of cytoplasma, was centrifuged on glass slides. The fixed nuclei were air-dried and stained by Feulgen SITS technique, which allows for the quantitative measurement of DNA. The DNA analysis was carried out with a computer-controlled single-cell cytophotometry. In contrast to using flow cytometry, only the tumor cells were measured by image-cytometry. Overlapping nuclei, dirt and other artifacts as well as inflammatory cells were efficiently eliminated. With DNA image-cytometry, we could differentiate between diploid (n = 13) and aneuploid (n = 6) tumors. Best prognosis with a survival rate of 92% after 103 months had patients with diploid tumors in contrast to patients with aneuploid tumors who did not survive more than 72 months.
Carcinoma, Papillary↗
Cytometric investigations of bladder irrigation/washing and voided urine.
Several studies have shown that cytometry, including DNA analysis, gives valuable information on the grade and stage of bladder cancer when performed on cytological preparations obtained from urine. Although, cytometry should not be used as a screening tool, it has a role in the follow-up of patients with a previous history of superficial bladder cancer. In this group of patients, the combination of cytological examination with cytometric evaluation allows the detection of the majority of recurrent tumors. In patients treated with chemotherapy or immunotherapy, the presence or the appearance of an aneuploid cell population is a good indication of tumor recurrence or progression. A variety of wet laboratory immunoassays, on-slide immunoassays, in situ hybridization procedures and post-nucleic acid extraction molecular techniques have been designed to complement cytology and cytometry and to improve the overall sensitivity and specificity of the detection of recurrent urothelial neoplasia.
Cytological Techniques↗