Introduction to Diagnostic Methods Pre-Reading

Question 1

what are the distinct areas of a pathology laboratory?

Answer 1

histology, cytology, and the clinical laboratory

Question 2

what is the first step for specimens sent for histologic examination?
what does this involve?

Answer 2

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!

Question 3

for resection specimens in cancer cases, what information from the gross description are the surgeon and pathologist most interested in getting from the sample?

Answer 3

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

Question 4

what does histologic examination entail? and what information can it give us?

Answer 4

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.

Question 5

what do H&E stains show?

Answer 5

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.

Question 6

what is a frozen section and what is the purpose of a frozen section?

Answer 6

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.

Question 7

what are the indications for a surgeon to order a frozen specimen?

Answer 7

Surgical margin assessment, sentinel lymph node involvement, and assessment of tissue adequacy.

Question 8

what is immunohistochemistry?

Answer 8

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.

Question 9

how do pathologists utilize immunohistochemistry tests?

Answer 9

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.

Question 10

what is cytology? and how is it different from histology? what is an example of a sample used for cytology testing?

Answer 10

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.

Question 11

what type of stain do most cytology samples use?

Answer 11

Papanicolaou stain

Question 12

what do cytologists look for in their samples?

Answer 12

examine nuclear and cytoplasmic features of cells to assess the type of cells and if the cells are benign, dysplastic, or malignant.

Question 13

why would a cytology specimen not be diagnostic and what is recommended when this happens?

Answer 13

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.

Question 14

what type of genetic alterations can lead to tumor formation?

Answer 14

mutations, translocations, deletions, and amplification/overexpression.

Question 15

How are genetic alterations in tumors identified for diagnostic, prognostic, or therapeutic implications?

Answer 15

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)

Question 16

what is karyotype analysis? what can it reveal?

Answer 16

examination of the chromosomes. Karyotype analysis can reveal genetic alterations when sufficient genetic material is exchanged, lost, or amplified in translocations, deletions,, or amplifications.

Question 17

what is the most routine analysis performed in a cytogenetics laboratory?

Answer 17

chromosomal analysis

Question 18

in what kind of sample can chromosomal analysis be performed? what is analysis dependent upon?

Answer 18

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.

Question 19

how many metaphase cycles are usually examined during routine chromosomal analysis?

Answer 19

20 metaphases are examined.

Question 20

when tumor cells cannot be stimulated to grow in vitro, karyotypic analysis cannot be performed. What can be done in these cases?

Answer 20

flourescence in situ hybridization (FISH) studies may be employed to target non-dividing cells as well as a larger percentage of tumor cells.

Question 21

how is FISH utilized? How long does FISH analysis take?

Answer 21

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.

Question 22

Certain genetic alterations, such as mutations in growth regulatory genes (protooncogenes) are only detected with very sensitive techniques. What techniques can detect these changes?

Answer 22

PCR and gene sequencing.

Question 23

How does PCR work?

Answer 23

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.

Question 24

what can be detected with PCR?

Answer 24

PCR can detect a single base pair mutation, perhaps responsible for an oncogene’s unregulated growth signal.

Question 25

what types of specimens can PCR be performed on?

Answer 25

PCR can be performed on many different types of specimens, including tissue sections of tumors. Mutation analysis for K-RAS in colon, pancreas, and lung adenocarcinomas, EGFR in lung adenocarcinomas, MSI (microsattelite instability) and mismatch repair enzymes in inherited colon cancer syndromes, and c-KIT in certain hematolymphoid malignancies may be assessed using PCR and has become routine clinical practice for tumor prognostication and employment of targeted therapeutic options.

Question 26

what technique is used for gene expression profiling? what is it used for?

Answer 26

microarray technology.
used in the field of carcinogenesis.
microarrays can be experimentally produced or commercially bought and serve as a nucleic acid template on which complementary nucleic acids (from tumor or normal tissue) can hybridize.

Question 27

what information can gene expression levels give us? what is this technology used for clinically?

Answer 27

gene expression levels can provide valuable insight into mechanisms of tumorigenesis. Currently, the clinical utility of microarray technology is limited to the Oncotype DX breast cancer assay. This assay may be used in a specific group of breast cancer patients to predict recurrence and identify those requiring specific therapeutic regimens.