Introduction to Diagnostic Methods Pre-Reading Flashcards
what are the distinct areas of a pathology laboratory?
histology, cytology, and the clinical laboratory
what is the first step for specimens sent for histologic examination?
what does this involve?
gross examination-meticulously detailing the tissue/organ description and dissecting the tissue/organ.
all gross examination descriptions should contain color, size ( in 3 dimensions), and texture of the tissue.
needs to be detailed!
for resection specimens in cancer cases, what information from the gross description are the surgeon and pathologist most interested in getting from the sample?
largest dimension of the tumor, where the tumor originates from, what tissues the tumor invades, and the distance between the tumor and the surgical margins
what does histologic examination entail? and what information can it give us?
tissues are sectioned, fixed in formalin and embedded in paraffin, cut into sections and stained. All tissue submitted histologically will be routinely stained with hematoxylin and eosin (H&E stain). In cancer cases, this microscopic examination determines the histologic types of cancer and grade, extent of tissue involvement, and margin involvement.
what do H&E stains show?
Hematoxylin natural dye which stains the cell’s nucleus blue/purple. Eosin is the counterstain which stains the intra- and extra-cellular proteins pink/red/orange.
what is a frozen section and what is the purpose of a frozen section?
A frozen section is when a representative piece of tissue is rapidly processed by freezing, staining, and then examination under a microscope. Frozen sections are used during surgery when a surgeon needs an immediate answer about a tissue sample.
what are the indications for a surgeon to order a frozen specimen?
Surgical margin assessment, sentinel lymph node involvement, and assessment of tissue adequacy.
what is immunohistochemistry?
immunohistochemistry is a technique which uses antibody reagents to detect specific antigens on the surface or in the cytoplasm of cells. These antibodies are harvested from rabbits, goats, or mice and are conjugated to detector molecules such as enzyme, which give off colored products in the presence of a substrate.
how do pathologists utilize immunohistochemistry tests?
to detail the expression of tumor antigens on the cell surface, cytoplasm, or nucleus and for the purpose of diagnosis, prognosis, and treatment options.
For example, in the differential diagnosis of a spindle cell tumor of the GI tract, C117 expression (receptor-linked tyrosine kinase) defines a GI stromal tumor (GIST) and provides valuable therapeutic information, suggesting use of a tyrosine kinase inhibitor.
what is cytology? and how is it different from histology? what is an example of a sample used for cytology testing?
as opposed to histology (where cells are examined in their tissue architecture), cytology is the study of cells in a fluid suspension without architecture.
An example of a cytology specimen is a scraping of the cervical transformation zone submitted for Pap smears.
what type of stain do most cytology samples use?
Papanicolaou stain
what do cytologists look for in their samples?
examine nuclear and cytoplasmic features of cells to assess the type of cells and if the cells are benign, dysplastic, or malignant.
why would a cytology specimen not be diagnostic and what is recommended when this happens?
sometimes cytology specimens are not diagnostic due to issues such as low cellularity. In these instances, biopsy procedures are often recommended for more definitive diagnosis. Also, cytology specimens revealing high grade lesions will require biopsy/resection to evaluate for invasion, histologic type/grade, and metastasis.
what type of genetic alterations can lead to tumor formation?
mutations, translocations, deletions, and amplification/overexpression.
How are genetic alterations in tumors identified for diagnostic, prognostic, or therapeutic implications?
several techniques utilized in routine practice to elicit this key molecular data:
conventional cytogenetic/karyotype analysis
gluorescence in situ hybridization studies (FISH)
polymerase chain reaction (PCR)
what is karyotype analysis? what can it reveal?
examination of the chromosomes. Karyotype analysis can reveal genetic alterations when sufficient genetic material is exchanged, lost, or amplified in translocations, deletions,, or amplifications.
what is the most routine analysis performed in a cytogenetics laboratory?
chromosomal analysis
in what kind of sample can chromosomal analysis be performed? what is analysis dependent upon?
any dividing cell preparation, such as blood, bone marrow, or tissue from a tumor.
analysis is dependent upon isolation of chromosomes in metaphase, following stimulation with a mitogen. chromosomes are stained and arranged in pairs, then examined for any deviations from each other and normal.
how many metaphase cycles are usually examined during routine chromosomal analysis?
20 metaphases are examined.
when tumor cells cannot be stimulated to grow in vitro, karyotypic analysis cannot be performed. What can be done in these cases?
flourescence in situ hybridization (FISH) studies may be employed to target non-dividing cells as well as a larger percentage of tumor cells.
how is FISH utilized? How long does FISH analysis take?
FISH analysis uses specific nucleic acid probes labeled with a fluorescent dye. The probes are constructed against areas of interest in the genome.
FISH can be performed in a shorter period of time (24hrs) compared to karyotypic analysis, which may take several days.
FISH can be used for diagnostic purposes as well as for prognostication.
Certain genetic alterations, such as mutations in growth regulatory genes (protooncogenes) are only detected with very sensitive techniques. What techniques can detect these changes?
PCR and gene sequencing.
How does PCR work?
complementary DNA primers are designed against a genomic area of interest. These primers stimulate replication of the complementary DNA strand, which is repeated over numerous cycles of primer annealing, extending, and strand separation to generate millions of DNA copies. Various methods for detection of PCR products are available including fluorescent-labeled primers, florescent-labeled product probes, melting curve analysis, and restriction enzyme fragment analysis.
what can be detected with PCR?
PCR can detect a single base pair mutation, perhaps responsible for an oncogene’s unregulated growth signal.
