Attributes and applications of flow cytometry

Flow cytometry (FCM) permits high speed measurement of fluorescence from individual cells flowing in a liquid medium. Cells stained with fluorescent dyes that bind specifically and proportionally to desoxyribonucleic acid (DNA) emit fluorescence that reflects their DNA content. Since neoplastic cells often contain abnormal amounts of DNA, FCM can be used as a rapid means to detect neoplasia. Flow cytometry can also provide cell kinetic information by quantitating cells in the various phases of the cell cycle. These capabilities make FCM a useful tool in the study of patients with neoplasia.     

General principles of multiparameter flow cytometric analysis: applications of flow cytometry in the diagnostic pathology laboratory

Recent developments in the design of flow cytometric instrumentation and advances in computerized methods of data analysis have produced flow cytometers that are compatible with the clinical laboratory setting. Because flow cytometry is impacting on virtually every area of diagnostic pathology (particularly the subspecialties of hematology, immunology, infectious disease, transplantation monitoring, neoplasia, and genetics), it is essential that pathologists develop a general understanding of flow cytometric techniques. In addition to new developments in instrumentation, simplification of laser design and advances in fluorochrome chemistry have provided several new sophisticated tools to make multiple correlative biological measurements on normal and neoplastic cells in the clinical setting. Flow cytometric analysis thus allows the pathologist to more effectively diagnose, predict prognosis, and monitor disease progression. This report reviews the basic components of flow cytometric instrumentation, the physical and biological parameters that may be assessed using flow cytometric techniques, and the multiparametric data analysis systems available for the clinical laboratory.     

Multicolor chromosome painting in diagnostic and research applications

For many years whole chromosome painting probes have been the work-horses in a large variety of clinical and research molecular cytogenetic applications. In recent years painting probes have been complemented by an increasing number of further region-specific probes, which allow the specific staining of centromeres, subtelomeres or other regions within the genome. This development of new probe sets was greatly facilitated by the Human Genome Project from which well-characterized probes for any region within the genome have emerged. Furthermore, the evolution of different multicolor fluorescence in situ hybridization (FISH) technologies now allows the cohybridization of multiple DNA-probes of different colors. These developments have paved the way for FISH-based automated karyotyping or the simultaneous analysis of multiple defined regions within the genome. Using appropriate instrumentation and image processing, the analysis can be performed two-dimensionally on metaphase spreads or three-dimensionally in intact interphase nuclei. Here we summarize some of the most recent developments and discuss the application of painting probes in different scenarios.     

Applications of immunocytochemistry to diagnostic cytopathology

Immunohistochemical technique is now successfully applied to the cytologic materials. This review article describes details of staining procedures for immunocytochemistry at both light and electron microscopic levels. The clinical application of the technique includes aspiration biopsy cytology for breast lesions, thyroid lesions, lymph nodes, the nervous system, and others. The establishment of carcinoma cells in the body fluid by the demonstration of carcinoembyronic antigen (CEA) and the establishment of specific diagnoses in small-cell or large-cell anaplastic tumors by immunocytochemistry warrants special emphasis.     

DNA flow cytometry in solid tumors: practical aspects and clinical applications

Investigators are currently using techniques of DNA flow cytometry to measure ploidy status (DNA content) and proliferative potential (S phase fraction) in a wide variety of solid tumors. These measurements have shown relevance for diagnosis, prognosis, and treatment for patients with cancer. National cooperative group studies are beginning to evaluate these measurements in controlled clinical trials to further define their clinical utility as well as limitations. The purpose of this report is to discuss both practical aspects of DNA flow cytometry and clinical applications of these measurements in a variety of solid tumors. We describe in detail our methods for sample handling, processing, and interpretation of DNA histograms. Although there are multiple ways of processing samples and interpreting histograms, we present methodology and interpretations that have proven efficient and reproducible. The review of clinical applications of flow cytometry to solid tumors focuses on breast cancer, but includes a discussion of other solid tumors.     

Quantitative analysis of nucleolar margination in diagnostic cytopathology

Summary The diagnostic value of nucleolar margination, defined as the percentage of nucleoli touching the nuclear membrane, was investigated in 359 cytological preparations of benign and malignant lesions of the thyroid, breast, prostate and central nervous system. Premalignant lesions of the uterine cervix and non-invasive papillary carcinomas of the bladder were also examined. It was observed that the percentages in benign lesions were, in general, lower than in the malignant and that the values increased progressively with increasing grade in the cervix and bladder. When the overlap index was calculated, this gave exact information on the usefulness of nucleolar margination in distinguishing benign from malignant lesions, particularly in the prostate and thyroid and, to a lesser extent, in the breast and central nervous system. As for lesions of different grades, the calculation of the index allowed the identification of two subgroups, one corresponding to low grades (mild cervical dysplasia or urothelial papillary carcinoma of grade 1), the other subgroup to high grades (severe cervical dysplasia and carcinoma in situ, or papillary carcinoma of grade 3). Moderate dysplasia cases and grade 2 papillary carcinomas do not appear as separate intermediate categories but rather show values falling into the range of either the higher or lower grades. The margination values obtained from the cytological preparations corresponded well to those in the histological slides obtained from the resected specimens. In conclusion, nucleolar margination appears to be a feature which is easy to evaluate in a reproducible way and useful in cytological diagnosis.     

Flow cytometry of human solid tumours: clinical and research applications

The measurement of DNA content of nuclei isolated from solid human neoplasms was one of the first large-scale applications of flow cytometry. Due to the development of directly labelled monoclonal antibodies mainly directed against cell-surface-associated proteins expressed by haematopoetic or lymphoid cells, flow cytometry rapidly evolved to an indispensable clinical technique. However, progress in multiparameter flow cytometry of solid tumours lagged behind.Recent technical improvements in preparing and staining cell suspensions obtained from solid tumours allows the simultaneous measurement of high-resolution DNA content and tumour-associated protein expression of fresh, frozen and even paraffin-embedded clinical samples. These new developments might give an impulse to clinical multiparameter flow cytometry of human solid neoplasms.     

New disc-based technologies for diagnostic and research applications

The role of genotypic analysis in disease diagnostics and drug response assessment is continually expanding. New genomic discoveries combined with new, novel technologies may provide a greater range of testing capabilities in the near future. We describe the application of nanotechnology, in which DNA microarrays have been placed in a microchannel environment that can be read and analyzed in an optical (CD/DVD) disc drive system. The potential exists to combine molecular and immunological applications together into a rapid, low-cost, high-capacity screening platform. The relevance of this technology is discussed in respect to infectious agent detection, pharmacogenomics, neurogenomics and genetic variations associated with neurologic diseases.     

Inflammatory pseudotumor: a diagnostic dilemma in cytopathology

Inflammatory pseudotumor (IPT) is a rare space-occupying lesion of unknown etiology that can mimic malignancy on clinicoradiological and pathological examination. A review of the cytopathology archives at The Johns Hopkins Hospital identified 12 cases from eight patients with histologically proven IPT (lung, seven patients; liver, five patients). There were six men and two women with an age range of 28-84 yr (mean age, 59 yr). Presenting complaints of IPT of the lung included shortness of breath and hemoptysis, and in cases of IPT of the liver complaints included abdominal pain and elevated liver function tests (LFTs). All cases were found to have mass lesions suspicious for a neoplasm on radiographic examination. Cytological specimens consisted of fine-needle aspiration (FNA; seven specimens) and bronchial brush/wash (five specimens). Diagnostic accuracy of cytology for IPT was low (5/12, 42%). IPT showed hypercellular smears (on FNA) with an admixture of various cell types including inflammatory cells with predominance of plasma cells, fibroblastic proliferation, granulation tissue formation, and atypical-appearing histiocytes with enlarged nuclei and intranuclear inclusions. Fibroblastic proliferation with mitoses may mimic mesenchymal neoplasms. Cytomorphology is nonspecific and IPT usually is a diagnosis of exclusion.