Tissue image cytometry or tissue cytometry [1] is a method of digital histopathology and combines classical digital pathology (glass slides scanning and virtual slide generation) and computational pathology (digital analysis) into one integrated approach with solutions for all kinds of diseases, tissue and cell types as well as molecular markers and corresponding staining methods to visualize these markers. Tissue cytometry uses virtual slides as they can be generated by multiple, commercially available slide scanners, as well as dedicated image analysis software – preferentially including machine and deep learning algorithms. [2] [3] [4] Tissue cytometry enables cellular analysis within thick tissues, retaining morphological and contextual information, including spatial information on defined cellular subpopulations. [1] [5] In this process, a tissue sample, either formalin-fixed paraffin-embedded (FFPE) or frozen tissue section, also referred to as “cryocut”, is labelled with either immunohistochemistry [6] (IHC) or immunofluorescent markers, scanned with high-throughput slide scanners and the data gathered from virtual slides is processed and analyzed using software that is able to identify individual cells in tissue context automatically and distinguish between nucleus and cytoplasm for each cell. [2] [4] Additional algorithms can identify cellular membranes, subcellular structures (like cytoskeletal fibers, vacuoles, nucleoli) and/or multicellular tissue structures (glands, glomeruli, epidermis, or tumor foci). [7]
Fluorescence Activated Cell Sorting (FACS) is a method of analysis that measures fluorescence signals on single cells, where the signal comes from antibody-mediated staining techniques and phenotypes detected by flow cytometry. [8] The major limitation of flow cytometry is that it can only be applied – as the name suggest – to cells in solution. Although methods of “solubilizing” solid tissue exist, any such processing irrevocably destroys the tissue architecture and any spatial context. Hence, tissue cytometry complements the use of flow cytometry and fluorescence microscope [9] in basic research, clinical practice, and clinical trials by providing FACS-like analyses on solid tissue sections (as well as adherent cell cultures) in situ. The advantage of tissue cytometry against flow cytometry is that tissue cytometry does not require the cells to be suspended in fluid, aiding in maintaining the integrity of the tissue structure, morphology, and contextual information, further assisting in precise and accurate contextual analysis that are not possible in flow cytometry.
Immunohistochemistry is used in clinical practice, where tissue biopsies from every potential cancer patient are collected, fixed in formalin and embedded on paraffin. These tissue sections are serially cut in a microtome to provide thin sections, representing the diagnostic material for clinical diagnoses. [6] Once stained initially with hematoxylin and eosin stain to detect cancer cells. Multiple marker staining is performed for proliferation, lineage, prognostic and oncogenic targets. Pathologists used optical microscope for the evaluation through the objective lenses and conclude the diagnosis by scoring the staining in percentage or as positive/negative. Visual evaluation provides a subjective diagnosis and plan of treatment. A more robust and automated system was designed to perform flow cytometry-like analyses on immunostained cells in a fixed tissue and termed tissue cytometry. [10] [11] The technique was introduced in the 1990s based on patents by Steiner and Ecker (CEO/founder TissueGnostics), [12] describing a procedure for “Cytometric Analysis of Diverse Cell Populations in Tissue Sections or Cell Culture Visualized Through Fluorescence Dyes and/or Chromogens".
Additional patents were filed in the early 21st century by Hernani et al. to perform virtual flow cytometry on immunostained tissue. [13] The latter's basics were derived from the procedure presented in 1982 by Gillete et al., describing the qualitative analysis of spectral mixtures by using factor analysis in conjunction with a spectral reference library. [14] Following this study, Zhou R et al. published a method to quantify prostate-specific acid phosphatase (PSAP) in histologic sections of prostate tumor with the peroxidase-antiperoxidase (PAP) complex technique using diaminobenzidine (DAB) as a substrate. [15]
Tumor Microenvironment: Tissue cytometry is heavily used in research to characterize the tumor microenvironment including e.g. identification of the immune landscape or tumor-vascularization, within IHC/IF-processed tissue sections. One reason is that by using this technology the complex tissue architecture stays intact and therefore also spatial relationships between cellular phenotypes and/or multicellular structures can be analyzed. [16]
By utilizing tissue cytometry multiple research groups were able to demonstrate the impact of various immune cell subpopulations (CD4, CD68, CD8, CD20, Foxp3, PD1) on patient survival in different cancer types (e.g. breast cancer, colon cancer, gastric cancer, melanoma, non-small cell lung cancer). [16] Since in cancer therapy a novel treatment strategy is targeting immune checkpoints (molecules that inhibit the antitumoral immune reaction), the insights gained by tissue cytometry may help to find new target molecules/biomarkers as well as to determine the best treatment strategy for patients. [16]
Immunology: Immune cell context is important for delineating the etymology of inflammatory diseases, which often result from impaired function of adaptive and/or innate immune cells. Tissue cytometry is useful for detecting and localizing specific cells, especially heterogeneous populations, within their native tissue environment and identifying the cues behind the disease. [17] For example, it was used to investigate IgG4-related diseases: one paper reports about fibrosing mediastinitis being driven by CD4+ CTLs rather than Th2 cells where infiltration of CD4+ CTLs was illustrated by tissue cytometry. [18] Follow-up studies investigated how follicular T cells influence B-cell class-switching events in IgG4-related disease and Kimura disease – researchers found a correlation between AICDA+CD19+ B cells and IgG4 expression using tissue cytometry. [19]
Mesenchymal Stem Cells Characterization: Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity differentiate into several sub-types such as bone, cartilage, muscle, developing teeth and fat tissue which has clinical importance for regenerative medicine. [20] However, although there are defined minimal phenotypic criteria, MSCs due to their heterogeneous nature need to be further characterized regarding their distinct biomarkers. [21] Tissue cytometry promisingly assists to describe the biomarkers of quiescent MCSs and furthermore characterize the effect of hyaluronan on this population. [22] Tissue cytometry can also used to investigate MSCs interaction with glioblastoma: to characterize cell fusion, extracellular vesicle transfer and intercellular communications. [23] Additionally, tissue cytometry is utilized to image the murine hippocampus and visualize M1/M2 microglia in mice with MSCs transplantation as a model for Alzheimer’s disease. [24]
COVID-19: COVID-19 pandemic required various tools to outline the disease progression and severity. Using tissue cytometry, researchers reported about interplay of immune cells and SARS-CoV-2 virus and its effect on disease: for instance, one study showed that CD4+ cytotoxic T cells expanded significantly in the lungs in severe COVID-19. [25] Another finding illustrates loss of germinal centers in lymph nodes and spleens in acute COVID-19, which was shown by multi-color immunofluorescence cytometry. [26]
Neuroscience: Tracking neurodevelopmental processes is an active field of research in neuroscience. Quantitative tissue analysis is widely employed in the field to determine the role of different stimuli in the nervous system. [27] [28] [29] [30] A research group reported about the effect of the magnetic field on neural differentiation of pluripotent stem cells, where the phenotypic effects were observed using tissue cytometry. [27] Another application of tissue cytometry in neuroscience was shown in a study designed to evaluate the effect of stress on hypothalamic neurons. [28]
A lymphocyte is a type of white blood cell (leukocyte) in the immune system of most vertebrates. Lymphocytes include T cells, B cells, and Innate lymphoid cells (ILCs), of which natural killer cells are an important subtype. They are the main type of cell found in lymph, which prompted the name "lymphocyte". Lymphocytes make up between 18% and 42% of circulating white blood cells.
A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist. The process involves extraction of sample cells or tissues for examination to determine the presence or extent of a disease. The tissue is then fixed, dehydrated, embedded, sectioned, stained and mounted before it is generally examined under a microscope by a pathologist; it may also be analyzed chemically. When an entire lump or suspicious area is removed, the procedure is called an excisional biopsy. An incisional biopsy or core biopsy samples a portion of the abnormal tissue without attempting to remove the entire lesion or tumor. When a sample of tissue or fluid is removed with a needle in such a way that cells are removed without preserving the histological architecture of the tissue cells, the procedure is called a needle aspiration biopsy. Biopsies are most commonly performed for insight into possible cancerous or inflammatory conditions.
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods.
Graft-versus-host disease (GvHD) is a syndrome, characterized by inflammation in different organs. GvHD is commonly associated with bone marrow transplants and stem cell transplants.
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo", meaning tissue. Albert Coons conceptualized and first implemented the procedure in 1941.
Hybridoma technology is a method for producing large numbers of identical antibodies. This process starts by injecting a mouse with an antigen that provokes an immune response. A type of white blood cell, the B cell, produces antibodies that bind to the injected antigen. These antibody producing B-cells are then harvested from the mouse and, in turn, fused with immortal myeloma cancer cells, to produce a hybrid cell line called a hybridoma, which has both the antibody-producing ability of the B-cell and the longevity and reproductivity of the myeloma. The hybridomas can be grown in culture, each culture starting with one viable hybridoma cell, producing cultures each of which consists of genetically identical hybridomas which produce one antibody per culture (monoclonal) rather than mixtures of different antibodies (polyclonal). The myeloma cell line that is used in this process is selected for its ability to grow in tissue culture and for an absence of antibody synthesis. In contrast to polyclonal antibodies, which are mixtures of many different antibody molecules, the monoclonal antibodies produced by each hybridoma line are all chemically identical.
The regulatory T cells (Tregs or Treg cells), formerly known as suppressor T cells, are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Treg cells are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Treg cells express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naïve CD4+ cells. Because effector T cells also express CD4 and CD25, Treg cells are very difficult to effectively discern from effector CD4+, making them difficult to study. Research has found that the cytokine transforming growth factor beta (TGF-β) is essential for Treg cells to differentiate from naïve CD4+ cells and is important in maintaining Treg cell homeostasis.
GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans.
FOXP3, also known as scurfin, is a protein involved in immune system responses. A member of the FOX protein family, FOXP3 appears to function as a master regulator of the regulatory pathway in the development and function of regulatory T cells. Regulatory T cells generally turn the immune response down. In cancer, an excess of regulatory T cell activity can prevent the immune system from destroying cancer cells. In autoimmune disease, a deficiency of regulatory T cell activity can allow other autoimmune cells to attack the body's own tissues.
Stromal cells, or mesenchymal stromal cells, are differentiating cells found in abundance within bone marrow but can also be seen all around the body. Stromal cells can become connective tissue cells of any organ, for example in the uterine mucosa (endometrium), prostate, bone marrow, lymph node and the ovary. They are cells that support the function of the parenchymal cells of that organ. The most common stromal cells include fibroblasts and pericytes. The term stromal comes from Latin stromat-, "bed covering", and Ancient Greek στρῶμα, strôma, "bed".
Antibody-dependent cellular cytotoxicity (ADCC), also referred to as antibody-dependent cell-mediated cytotoxicity, is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system kills a target cell, whose membrane-surface antigens have been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response, can act to limit and contain infection.
Memory T cells are a subset of T lymphocytes that might have some of the same functions as memory B cells. Their lineage is unclear.
Exosomes are membrane-bound extracellular vesicles (EVs) that are produced in the endosomal compartment of most eukaryotic cells. In multicellular organisms, exosomes and other EVs are found in biological fluids including saliva, blood, urine and cerebrospinal fluid. EVs have specialized functions in physiological processes, from coagulation and waste management to intercellular communication.
Flow-FISH is a cytogenetic technique to quantify the copy number of RNA or specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols.
Mesenchymal stem cells (MSCs) also known as mesenchymal stromal cells or medicinal signaling cells are multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes and adipocytes.
MHC multimers are oligomeric forms of MHC molecules, designed to identify and isolate T-cells with high affinity to specific antigens amid a large group of unrelated T-cells. Multimers generally range in size from dimers to octamers; however, some companies use even higher quantities of MHC per multimer. Multimers may be used to display class 1 MHC, class 2 MHC, or nonclassical molecules from species such as monkeys, mice, and humans.
Mass cytometry is a mass spectrometry technique based on inductively coupled plasma mass spectrometry and time of flight mass spectrometry used for the determination of the properties of cells (cytometry). In this approach, antibodies are conjugated with isotopically pure elements, and these antibodies are used to label cellular proteins. Cells are nebulized and sent through an argon plasma, which ionizes the metal-conjugated antibodies. The metal signals are then analyzed by a time-of-flight mass spectrometer. The approach overcomes limitations of spectral overlap in flow cytometry by utilizing discrete isotopes as a reporter system instead of traditional fluorophores which have broad emission spectra.
In cell biology, single-cell variability occurs when individual cells in an otherwise similar population differ in shape, size, position in the cell cycle, or molecular-level characteristics. Such differences can be detected using modern single-cell analysis techniques. Investigation of variability within a population of cells contributes to understanding of developmental and pathological processes,
A T memory stem cell (TSCM) is a type of long-lived memory T cell with the ability to reconstitute the full diversity of memory and effector T cell subpopulations as well as to maintain their own pool through self-renewal. TSCM represent an intermediate subset between naïve (Tn) and central memory (Tcm) T cells, expressing both naïve T cells markers, such as CD45RA+, CD45RO-, high levels of CD27, CD28, IL-7Rα (CD127), CD62L, and C-C chemokine receptor 7 (CCR7), as well as markers of memory T cells, such as CD95, CD122 (IL-2Rβ), CXCR3, LFA-1. These cells represent a small fraction of circulating T cells, approximately 2-3%. Like naïve T cells, TSCM cells are found more abundantly in lymph nodes than in the spleen or bone marrow; but in contrast to naïve T cells, TSCM cells are clonally expanded. Similarly to memory T cells, TSCM are able to rapidly proliferate and secrete pro-inflammatory cytokines in response to antigen re-exposure, but show higher proliferation potential compared with Tcm cells; their homeostatic turnover is also dependent on IL-7 and IL-15.