TECHNOLOGIES

PhenoPath Announces New Division of Flow Cytometry
PhenoPath Laboratories is proud to announce a new Division of Flow Cytometry. Find out more about services available, sending specimens, and flow cytometry staff.

IHC and IF | FISH | CISH | Flow Cytometry | PCR

The horizons of surgical pathology have been expanding in recent years with the explosion of information about the cellular and molecular bases of disease and the development of many new technologies that can be applied to tissue analysis. While the hematoxylin and eosin-stained section will likely retain its central importance in pathologic diagnosis in the foreseeable future, ancillary techniques that can be applied to fresh, frozen or formalin-fixed tissue will continue to have a dramatic impact on diagnostic surgical pathology.

These techniques, which include immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH), and DNA- and RNA-based polymerase chain reaction (PCR) techniques, will play an increasingly important role in the treatment of patients with targeted therapies. The pathologists and technologists at PhenoPath Laboratories are at the forefront of applying these technologies to surgical pathology specimens.

Immunohistochemistry (IHC) and immunofluorescence (IF) are immunochemical techniques employing specific antibodies along with highly sensitive detection systems that produce a colored reaction product visible under the light microscope (IHC) or fluorescence microscope (IF). IHC can be applied to either frozen or fixed tissues, whereas IF is largely restricted to frozen tissue. The power of these techniques is the simultaneous retention of tissue morphology, permitting the integration of morphologic and immunophenotypic information. PhenoPath Laboratories maintains a large and ever-expanding library of over 250 antibody reagents, each of which has been validated and optimized for use in diagnostic tissue-based studies.

Antibody List

Fluorescence in situ hybridization (FISH) has become an essential tool in the diagnosis and management of a variety of hematologic malignancies and non-hematopoietic tumors, as well as an aid in the identification of particular genetic disorders. FISH can be applied to fresh, frozen, or fixed tissues. FISH techniques employ DNA probes comprised of specific nucleic acid sequences attached to fluorescent molecules. These probes can be used to look for increased copies of specific genes (such as HER2 amplification in breast cancer) or entire chromosomes (such as trisomy 17 in partial hydatidiform moles), as well as decreased copies of specific genes (such as 1p and 19q in oligodendroglioma) or chromosomes (such as monosomy 7 in a subset of myelodysplastic syndromes). FISH can also identify specific chromosomal translocations, such as the t(14;18) characteristic of follicular lymphoma, and the t(9;22) characteristic of chronic myeloid leukemia. PhenoPath Laboratories has a growing collection of FISH probes suitable for ancillary testing of pathology material, including fresh, frozen, and formalin-fixed specimens. FISH preparations are typically examined with a MetaSystems™ image analysis system that is integrated with a fluorescence microscope, permitting highly accurate and precise quantitative analysis of these genetic alterations.

Probe List

Chromogenic in situ hybridization (CISH) utilizes similar amplification and visualization techniques as immunohistochemistry, but by hybridizing the complementary strand of a nucleotide probe to the sequence of interest, CISH can identify the presence of specific nucleic acid sequences (genes) in their cellular environment. Principal applications of CISH at PhenoPath Laboratories include the detection of viral-specific DNA and RNA sequences, such as the EBER1 mRNA associated with Epstein-Barr virus-infected cells.

Flow cytometry is a technique for evaluating antigen expression in suspensions of dispersed living cells. Various combinations of antibodies conjugated to fluorescent molecules, or fluorochromes, are incubated with the cell suspension according to standard protocols. The mixture of cells and bound antibodies is then introduced into the flow cytometer, and the cell-associated fluorescence in response to illumination with one or more lasers is recorded. Computer-assisted analysis of the patterns of fluorescence helps indicate whether the cells of interest are benign or malignant and, in cases of malignancy, often suggests a specific diagnosis. While flow cytometry has historically been used to characterize hematolymphoid cell populations, this technique can be applied to any monodispersed cell suspension. At PhenoPath Laboratories, cell-associated fluorescence is measured on a state-of-the-art, 3-laser/9-color Becton Dickinson LSRII flow cytometer, utilizing a collection of nearly 100 fluorescently-labeled antibodies.

Flow Cytometry Panels

Polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA or RNA, in order to produce sufficient genetic material to be adequately tested. It can readily identify unique DNA or RNA sequences that are present at even a few copies per cell, and is thus far more sensitive than methods such as CISH and Southern blotting. Leukemias and lymphomas, in particular, are strongly associated with genetic alterations in a number of critical genes. Knowledge of some of these abnormalities can also provide prognostic information that can be used to guide treatment. PCR technology is currently utilized at PhenoPath for identification of clonal immunoglobulin heavy chain gene rearrangements characteristic of B cell lymphomas, and clonal T cell receptor gene rearrangements characteristic of T cell lymphomas. Given that each B cell and T cell expresses a unique immunoglobulin or T cell receptor, respectively, the identification of clonally expanded B cell or T cell populations by PCR may be critical in confirming a neoplastic process, especially when immunophenotyping is not conclusive. PCR techniques are increasingly replacing the Southern blot in the molecular evaluation of B cell and T cell proliferations. It is, however, important to bear in mind that due to variations in the quality of formalin-fixed tissue sections, and issues related to pseudoclonality and oligoclonality, false positive and negative results are an inherent limitation of this assay. Therefore, it is critical to correlate PCR findings with histologic and immunophenotypic information before reaching a final diagnosis.

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