David A. Rew

Objective quantitative analysis of eosinophils and bronchial epithelial cells in induced sputum by laser scanning cytometry

BACKGROUND Sputum induction is an important non-invasive technique for measuring airway inflammation in asthma. Cell numbers are often too low for flow cytometric analysis. Laser scanning cytometry (LSC) is a novel technique that allows objective multicolour fluorescence analysis of cells on a microscope slide. METHODS LSC was used to determine sputum eosinophil and bronchial epithelial cell counts. We first confirmed that we could measure eosinophil counts accurately in peripheral blood using α-major basic protein (MBP) immunofluorescent staining. Sputum induction was performed according to standard protocols. Sputum samples from eight normal controls and 12 asthmatic patients were analysed by LSC and manual counting by two independent observers. Octospot cytospins were fixed and stained with mouse-α-human-MBP monoclonal antibody or mouse-α-human-cytokeratin antibody and goat-α-mouse Oregon Green conjugated second antibody. RESULTS Sputum induction provided a mean (SE) of 0.99 (0.2) × 106 cells per donor. More than 3000 cells on three cytospins per slide were analysed per cell type. The intraclass correlation coefficient (R) and standard deviation (SD) of differences in eosinophils determined by manual counting and LSC were 0.9 and 2.1, respectively, and for bronchial epithelial cell counts they were 0.7 and 2.0. Selective detection of labelled cells was confirmed visually after relocation. CONCLUSION Eosinophils and bronchial epithelial cells can be accurately and reproducibly counted in an objective manner. LSC is therefore a potentially powerful new method for immunophenotyping leucocytes and epithelial cells objectively in induced sputum in patients with asthma.

Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry

BACKGROUNDnThe evaluation of the safety of drugs and other chemicals is an important aspect of toxicology work. The mouse micronucleus assay is a standard in vivo genotoxicity assay. Chromosomal damage is an indicator of genotoxicity, which manifests in the formation of micronuclei in polychromatic erythrocytes from bone marrow and in peripheral blood erythrocytes. The assay is laborious to perform by manual counting. The laser scanning cytometer allows automated and rapid quantitation of cellular and subcellular fluorescence in monodisperse cell samples on a microscope slide. The object of this study was to evaluate the application of this new technology in the mouse micronucleus genotoxicity assay. Materials and Methods One hundred forty-four mice of various strains were dosed with combinations of carcinogens and antioxidants. Duplicate blood films were prepared 3 days later. One set of slides was stained with acridine orange, and the proportion of micronucleated erythrocytes was counted in 5,000 cells per slide. The duplicates were stained with propidium iodide (40 microg/ml). Five thousand cells per sample were examined using a laser scanning cytometer. The proportion of micronucleated erythrocytes was measured.nnnRESULTSnA coefficient of correlation of 0.96 was found between the data from the two assays. The automation of the assay on the LSC produced a considerable time saving and efficiency gain.nnnCONCLUSIONSnWe conclude that with further development, laser scanning cytometry is likely to become the preferred modality for the performance of standard genotoxicity assays.

Comparison of flow and laser scanning cytometry for the assay of cell proliferation in human solid tumors.

The introduction of the laser scanning cytometer offers new capabilities in cell proliferation research, through its capacity for validation of each and every cell event through direct visualization on the microscope slide. In this study, we report a direct comparison of proliferation data derived from flow and laser scanning cytometry of human tumor nuclei labeled in vivo with bromodeoxyuridine (BrdUrd). Nuclear suspensions from 19 invasive ductal breast carcinomas and 12 gastric adenocarcinomas were prepared and analysed for BrdUrd uptake and DNA content. Specimens were analysed using a FACScan and then prepared on cytocentrifuge preparations for laser scanning cytometry. DNA index, labeling index (LI), duration of S-phase (Ts) and potential doubling time (Tpot) were calculated using standard procedures. There was an excellent correlation between the two techniques in the calculation of DNA index (R = 0.983, P > 0.0001) and LI (R = 0.924, P > 0.0001). The Ts proved more problematical (R = 0.448, P = 0.0115) but the Tpot showed closer agreement (R = 0.851, P > 0.0001) as the LI was the dominant determinant of Tpot. No single parameter could be identified as the major source of variation between the two techniques. We conclude that the laser scanning cytometer produces data equivalent to that obtained by flow cytometry.

Staging the axilla in breast cancer: an audit of lymph-node retrieval in one U.K. regional centre

AIMSnMany surgeons undertake a level 1 axillary dissection in patients with invasive breast cancer. This dissection yields a variable number of lymph nodes for histological study. In this study, we report the consequences of this policy for staging of the axilla.nnnMETHODSnBetween January 1995 and December 1995, 236 patients with a diagnosis of invasive breast cancer underwent axillary surgery.nnnRESULTSnA median of eight nodes was identified (range 0-30). In only 11 patients less than four nodes were identified. An increase in the number of nodes harvested was associated with a higher proportion of node-positive patients and a higher number of metastatic nodes identified.nnnCONCLUSIONSnWe concluded that a standardized approach to axillary dissection consistently yields an adequate sample of lymph nodes for staging purposes. Most importantly, larger node samples yield higher detection rates for metastasis. This has a significant bearing on patient selection for adjuvant chemotherapy when compared with more limited sampling practices, including solitary sentinel node detection and biopsy.

New technology in the analytical cell sciences : the laser scanning cytometer

New technologies are making a major contribution to progress in applied clinical research in surgical oncology. The laser scanning cytometer is a new machine which combines the analytical capabilities of flow cytometry with the ability to inspect and visualize labelled cells and particles. This substantially reduces the uncertainty associated with assays in a wide range of surgical oncology research applications. This article introduces this new technology.

Image analysis enhancement of the laser scanning cytometer

The laser scanning cytometer offers a range of novel applications and the capacity for direct visual validation of experiments through sample analysis on a microscope slide. Linkage of the instrument to an image analysis system through standard connections and software enhances the capabilities of the instrument in image capture and manipulation. In this technical note, we describe a simple linkage between the LSC and the Kontron KS100 Image Analysis System, an example of a standard commercial image processing instrument.

The flow cytometric analysis of total p53 protein content and proliferation indices in colorectal cancer, in relation to clinical outcome

This study was undertaken to assess the value of flow cytometric measurements of total p53 protein content and proliferation indices derived from in vivo halogenated pyrimidine labelling. Two series of colorectal cancer specimens were studied for which clinical outcome data were recorded. A series of 84 archival, ethanol-fixed, bromodeoxyuridine (BrdUrd) labelled colorectal tumours were analysed by flow cytometry for their total and cell cycle phase p53 protein content using the pAb1801 monoclonal antibody. A second series of 33 freshly obtained tumours was used for assay evaluation and for comparison with the archival material. In the archival series (n=84), the median p53-pAb1801 LI was 81.9% (range: 11.1-99.8%). In only three tumours could significant amounts of p53 protein not be detected. The median phase specific p53-pAb1801 LI in G0/G1 was 71.6%, in S was 95.5%, and in G2/M was 98.5%. In the series of fresh tumours (n=33), the median p53-pAb1801 labelling index (LI) was 94.6% (range: 17.9-99.9%). Only two tumours failed to express significant amounts of p53 protein. There was no significant difference in the generally high levels of p53 protein content between the fresh and archival series. Life-table analysis of the patients in the archival series failed to demonstrate a statistical difference in life expectancy in relation to Dukes stage when tumours were stratified by the median total p53 labelling index. In this study, p53 content and proliferative indices measured by flow cytometry do not have independent predictive value over Dukes grading in determining the outcome of colorectal cancer. Flow cytometry is confirmed as a practical tool for multi-parametric and cell cycle analysis of oncoprotein expression in human tumour biopsies.

Cancer—a degenerative disorder?

Cancer is primarily a disease of ageing epithelia, and of ageing individuals. We now possess detailed insights into the changes in cell regulatory genes and DNA repair systems which accumulate with time and which manifest in malignancy. These demonstrate how cancer is frequently characterized by degenerative change in the genotype, from the most subtle base pair mutations to gross aneuploidy, and by deterioration in cell and tissue regulatory control, be it of proliferation, programmed cell death or signalling. Cancer may thus be as much a phenomenon of loss or deterioration of normal genomic control as of the acquisition of new, neoplastic functions. This distinction may be more than semantic, not least because it governs our approach to the search for therapeutic strategies. This essay considers the concept of cancer as a degenerative disease and its implications, and proposes the neologism aldoplasia to describe this phenomenon of cancer biology.

http://www.hbuk.co.uk—the European Journal of Surgical Oncology and the Internet

The popularization of the Internet through the World Wide Web heralds a new era in public and professional communication. The European Journal of Surgical Oncology is now represented on the Web at a site provided, developed and maintained by the publisher. We may expect to see rapid developments in electronic publishing, although the direction and general utility of these changes are not yet clear. This article introduces the EJSO Web site to the readership and considers the ways in which the Internet Revolution may bring benefits to the readership, the publisher, the editorial process and the Journal staff.

The (continued) importance of the hypothesis in surgical oncology research.

There remains much to be learned about the biology and therapy of human solid tumours, despite remarkable progress in science, medicine and technology in the past century. The well-formulated, explicit and testable hypothesis remains the key to progress in scientific medicine. It should precede experiment and provide landmarks by which experimental results may be judged. Primary clinical research takes three common forms) Experimentation in surgical oncology often reports the measurement of a biological moiety in a laboratory model or series of clinical samples, and correlates it with actual or surrogate measures of outcome. Clinical trials in surgical oncology assess the value of an intervention, such as a modification in surgical technique, a new drug, or a new form of adjuvent therapy, on outcome. Surveys measure a parameter in a cohort of patients. Each form of research mandates a hypothesis. The scientific problem which underpins the scientific hypothesis may be a general or global issue, or it may be a subsidiary issue within a field of study. Unfortunately, many studies in surgical oncology lack a clear foundation hypothesis. This devalues much research effort, is scientifically inadequate and leads to a waste of human, clinical, laboratory and fiscal resources. This paper addresses the centrality of the hypothesis in the context of modern research processes and technologies.