Quantitative histology by multicolor slide‐based cytometry

Abstract

BackgroundIn 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.MethodsWe 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.ResultsPercentage 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 (r2 = 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.ConclusionsWe expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions. © 2004 Wiley‐Liss, Inc.

“Virtual flow cytometry” of immunostained lymphocytes on microscopic tissue slides: iHCFlow™ tissue cytometry

“…The result is highly visual and akin to FC and provides both size and stain density population distribution at a glance. Like FC, which is a standard of clinical hematology practice, TC may supplant the often subjective immunostaining reporting in pathology and may also complement other fluorescence and laser-based technology like laser scanning cytometry (LSC) (1,12,14,15).…”

“…However, LSC has difficultly in analysis of tissue immunofluorescence because of overlapping cells and hence may be difficult to implement as single cell analysis in tissue sections. LSC nevertheless, has been applied to fresh tissue sections to quantitate in low power view, the tissue distribution of the lymphoid population with good results using multicolor dyes (1). A modification using confocal microscopy could obtain size and shape information in low power view (11)(12)(13), but likewise loses the common hematoxylin stained microscopic nuclear details familiar to most pathologists and research scientist.…”

“…We applied a novel, useful, and accurate algorithm that was made feasible after resolving the issues of performing a population or cell-based analysis and contributed to advances in cytomics (1)(2)(3)(4)(5). Using tissue immunohistochemistry (IHC) and iHCFlow TM approach, we produced results similar to FC dot-plot histograms.…”

Laser scanning cytometry in the characterization of the proapoptotic effects of transiently transfected genes in cerebellar granule neurons

“…LSC, a method that has been applied to such diverse research interests as cell cycle analysis (17), oncology (18), gene therapy (19), chemotaxis (20), immunophenotyping of peripheral blood leucocytes (21,22), histological analysis of tissue sections (23,24), and G-protein coupled receptor activation (25), offers the noninvasive capture of multiple fluorescence parameters from adherent cells (13). As such, it is a technology well suited to measuring the morphological correlates of neuronal apoptosis.…”

Merging of data files in laser scanning cytometry—Seeing is believing?

“…Although in flow cytometric analyses the sample is consumed during analysis, the specimen in SBC remains on the slide. This is advantageous not only for relocating measured events of interest, but also to modify the sample or instrument settings and perform additional measurements of the same specimen (5)(6)(7)(8)(9). Such reanalyses yield more information about one and the same sample than it is possible with a single measurement.…”