IMMUNOHISTOCHEMISTRY (IHC) & DIRECT IMMUNOUORESCENCE (DIF)
IHC and DIF are immunologic-based technologies using speciﬁc antibodies with different detection systems. The reaction product generates a colored reaction product which is evaluated under the standard light microscope (IHC) or ﬂuorescence microscope (DIF). At Vitro, the use of DIF is largely restricted to fresh, skin biopsies for the evaluation of cutaneous-based immunologic conditions. The applications of IHC are innumerable, which makes this assay routinely used for the diagnostic and predictive markers workup in anatomic pathology. As an international anatomic pathology reference laboratory, Vitro has validated hundreds of (over 250) IHC antibodies that are currently being used in diagnostic workups.
FLUORESCENCE IN SITU HYBRIDIZATION (FISH)
While FISH was ﬁrst introduced to the clinical testing arena in the early 90s, it now has become an essential component of the tools employed in the pathology laboratory. FISH applications now include a wide range, either as a diagnostic test helping conﬁrm the diagnosis of a hematolymphoid tumor or a soft-tissue tumor, or as a prognostic/predictive companion diagnostic assay. Most clinical FISH probes include ‘direct labeling’ of speciﬁc nucleic acid sequences (gene speciﬁc, or chromosome speciﬁc) attached to ﬂuorescent molecules. Dr. Yaziji and our pathologists are thought leaders in FISH testing, and have been extensively involved in clinical research on FISH assays since the nineties, and have personally reviewed and reported over ten thousands FISH assays to the pathology and oncology community throughout the United States, Asia and South America. Dr. Yaziji is also the author of a book (Springer, in press) entitled “FISH Testing in The Pathology Laboratory).
CHROMEGENIC IN SITU HYBRIDIZATION (FISH)
Chromogenic in situ hybridization (CISH) CISH utilizes similar detection systems as immunohistochemistry, but by hybridizing the nucleotide probe to the complementary sequence of interest, CISH can identify the presence of speciﬁc genes or chromosome segments in various neoplastic and non- neoplastic tissues. Vitro offers CISH-based testing of EBV mRNA, kappa light chain gene and lambda light chain gene.
Flow cytometry is a technique for detecting and quantitating antigen expression in single cell suspensions. Fluorescein-conjugated antibody ‘panels’ are added to the cell suspension. The mixture of cells and bound antibodies will pass through the ﬂow cytometer, and the cell-associated ﬂuorescence in response to illumination with one or more lasers is recorded. Computer analysis of several patterns of protein expression can identify the immunophenotype of the cells of interest, thus helping to conﬁrm a diagnosis of a speciﬁc hematolymphoid disorder, conﬁrm light-chain restriction (which is a hallmark of neoplastic lymphoproliferative disorder), or detect a small residual population of malignant cells, following treatment. While ﬂow cytometry has historically been used to characterize hematolymphoid cell populations, this technique can be applied to any monodispersed cell suspension. At Vitro 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.
Polymerase chain reaction (PCR) is a technique used to amplify a speciﬁc region of DNA or RNA, which generates sufﬁcient DNA for detection. Most, but not all molecular assays utilize PCR, which is often more sensitive than other molecular methods. Vitro uses PCR-based testing for B- and T-cell clonality testing in lymphoid lesions, and for allele-speciﬁc PCR testing of prognostic / predictive markers in oncology, such as KRAS, BRAF, and EGFR. Example of hematology-based assays offered at Vitro include MPL and JAK2.
ROUTINE DIAGNOSTIC CYTOPATHOLOGY AND HISTOPATHOLOGY
While these technology has been employed over a century ago, some technical advances have been made with regard to cytology monolayer processing and tissue processing. Routine cytopathology and histopathology remains the only gold standard technology used to process patients samples (such as pap smears, cervical biopsies, endometrial biopsies, prostate biopsies, skin biopsies, breast biopsies and GI biopsies) and render pathologic diagnoses on these samples.