Specimen Handling Flashcards
When does the pre-analytical stage of tissue handling begin?
Once the tissue leaves the body.
What is autolysis?
The process of degeneration caused by enzymes within the cell, basically self-digestion. This process begins immediately and is accelerated by increased temperatures.
What is the “Cold Ischemic Time”?
The time between when tissue is taken out of the body and when it’s placed in a fixative. The goal is to have this time be as little as possible and has to be less than one hour.
T/F: Epitope damage due to ischemia can be recovered using Ag retrieval techniques.
False, this type of epitope damage is irreversible.
How does formalin fix tissue?
By forming cross-linking methylene bridges between the reactive amino groups in proteins.
What is the minimum time that non HER2 tissue can be formalin fixed?
24 hours.
What is the formalin fixation window for HER2 tissue?
6-72 hours.
How does alcohol fix tissue?
Alcohol fixed by coagulating and precipitating proteins which tends to extract tissue elements such as LMW carbohydrates. Alcohol also tends to dehydrate the tissue causing shrinking and hardening.
What is a main advantage of alcohol fixation over formalin?
Alcohol fixation eliminates the need for Ag retrieval, it initially penetrates more easily than formalin and it is much more useful for molecular work.
What kind of slides are made more effective by formalin fixattion?
Charged slides because formalin fixation makes tissue more acidic (negative) to attract the positively charged slides.
Why are nylon bopisy bags recommended above sponges during grosing?
Because the sponges can soak up excess reagent and transfer reagent between solution containers during processing.
What is one practical application for frozen fixed tissue over FFPE tissue?
Frozen tissue is necessary for DIF (Direct Immunofluorescence).
What is DIF used for?
To demonstrate the Immunoglobulins and complement in skin biopsies with a much shorter turnaround time than with FFPE tissue.
What is a TMA?
A Tissue Microarray (TMA) is a method of control preparation in which multiple types of tumors and normal tissue are combined into a single block. Think: GU control at work.
When should validation be performed?
Anytime there is a modification or the addition of a new: fixative, processing protocol, staining protocol, Ab, ISH protocol, decalcifying agent or control tissue type.
When should validation be reassessed or revisited?
- When new Ab lots are put into use (cross-lots)
- When aberrant or unexpected staining results occur
- When several different staining platforms are being used for the same IHC or ISH tests.
What are the two major categories of fixation?
Fixation by drying and fixation by chemical means.
What is the ideal fixation protocol for frozen tissue?
The usage of coagulant (chemical) fixatives that do not form an additive compound such as Acetone, Zenker and Bouin. Methanol-Carnoy is also works.
What is the fixation window for breast tissue prior to HER2 testing?
6-48 hours in room temperature formalin.
T/F: There is a negative correlation between extended cold ischemic time and loss of Ag stability.
False, there is a positive correlation (the longer it is left out, the worse it will stain).
What is the most common 10% NBF alternative used in routine processing.
Zinc formalin (we use this in lab).
What IHC can be ordered to test for overfixation?
The Vimentin stain.
How does Vimentin test for overfixation?
If the test is negative, then all IHCs for that day should be regarded with hesitation. If the Vimentin is unevenly positive, all parallel sections should only be read in the “positive” sections of the corresponding Vimentin stain.
How does reprocessing affect IHC?
Reprocessing tissue may alter epitope sites which causes irreversible damage and can lead to false negatives.
In the peroxidase-antiperoxidase (PAP) method of Ab detection, the PAP complex must be the same species as what?
The primary Ab in order for the secondary Ab to link them together.
Polyclonal antibody serums often have non-specific antibodies in them. This problem can be greatly alleviated by the manufacturer by:
Affinity chromatography of the polyclonal serum
In reference to antibodies, the terms “light” and “heavy” chains refer to the:
Molecular weight of the polypeptide chains
In immunohistochemical staining, a limitation of polyclonal antisera as opposed to monoclonal antisera is:
The greater cross-reactivity of polyclonals with similar antigens
When staining for neuroendocrine tumors the most beneficial markers of identification are:
A. s-100 and cytokeratin
B. chromogranin and synaptophysin
C. neurofilament and neuron specific enolase
D. neuron specific enolase
B. Chromogranin and synaptophysin.
What does synaptophysin test for?
Synaptophysin stains for synaptic vesicles of the neuroendocrine system.
In immunohistochemical staining of formalin-fixed tissue, proteolytic enzyme pretreatment of a tissue section:
A. enhances background staining
B. unmasks antigen epitopes
C. is needed to demonstrate all antigens
D. has precise end-points
B. Unmasks Ag epitopes.
In peroxidase staining, the colored end product is formed following:
Reduction of hydrogen peroxide and oxidation of a chromogenic substrate
Polyclonal antisera are those:
A. whose antibodies have a polygonal structure
B. that are produced by two or more distinct B-cell clones
C. that are more specific than monoclonals
D. which are more difficult to use than monoclonals
B. That are produced by two or more distinct B-cell clones.
DAB may be enhanced by the use of:
Cobalt, nickel, silver or gold
The peroxidase staining procedure in which reagents are linked exclusively by antigen-antibody reactions without any conjugation steps is:
Peroxidase-anti-peroxidase
Describe the indirect immunohistochemistry staining method:
The test slide is first treated with unlabeled antiserum. A secondary antibody, conjugated to any of several enzymes is then applied. Binding sites are made visible following reaction with a chromogen substrate.
What is a chromogen used in peroxidase histochemistry that generates a product that is soluble in alcohol or xylene but not in water?
9-aminoethylcarbazole (AEC)
In immuno-alkaline phosphatase staining, which control is useful to determine the level of endogenous alkaline phosphatase?
Incubate in chromogen only
What is the most effective method of determining endogenous alkaline phosphatase in a tissue?
To omit all steps except the chromogen. Extensive endogenous alkaline phosphatase will stain with the chromogen alone, thereby allowing you to judge the effectiveness of your primary antibody staining.
How is a negative control used in IHC?
Determine what steps contribute to any non-specific staining. If there is significant staining without the primary antibody used it indicates a problem with some of the other steps.
Of the following, the preferred fixative for demonstration of intermediate filaments contains
A. alcohol
B. formalin
C. mercury
D. dichromate
A. Alcohol or an alcohol based fixative.
As a chromogenic substrate when would amino-ethylcarbazole (AEC) would be preferred to diaminobenzidine (DAB)?
When staining pigmented lesions. In such cases, the brown reaction product of DAB might be confused with melanin pigment. AEC, however is not permanent, nor is it less carcinogenic than DAB.
Polyclonal antibody serums often have non-specific antibodies in them. This problem can be greatly alleviated by the manufacturer by:
Affinity chromatography of the polyclonal serum. Affinity chromatography, which involves trapping of antibodies specific to the antigen of interest, will eliminate most of the non-specific antibodies in a polyclonal serum.
In reference to antibodies, the terms “light” and “heavy” chains refer to the:
Molecular weight of the polypeptide chains. An immunoglobulin molecule consists of two light and two heavy polypeptide chains containing specific sequences of amino acids.
In immunohistochemical staining, a limitation of polyclonal antisera as opposed to monoclonal antisera is:
The greater cross-reactivity of polyclonals with similar antigens. Consequently, polyclonal antibodies may cross-react with similar antigens, resulting in staining that may not be as specific as with monoclonals.
When staining for neuroendocrine tumors the most beneficial markers of identification are:
Chromogranin and synaptophysin. Chromagranin is specific for neurosecretory granules. Synaptophysin stains for synaptic vesicles of the neuroendocrine system.
In immunohistochemical staining of formalin-fixed tissue, what is the function of a proteolytic enzyme pretreatment of a tissue section?
Unmasks antigen epitopes.
In peroxidase staining, the colored end product is formed following:
The reduction of hydrogen peroxide and oxidation of a chromogenic substrate.
What is the primary function of horseradish peroxidase?
Horseradish peroxidase catalyzes the release of oxygen from its specific substrate, hydrogen peroxide (reduction of hydrogen peroxide). When the reaction is coupled with the oxidation of a chromogenic substrate, a colored polymer is formed that is visible by light microscopy.
Polyclonal antisera are those:
A. whose antibodies have a polygonal structure
B. that are produced by two or more distinct B-cell clones
C. that are more specific than monoclonals
D. which are more difficult to use than monoclonals
B. That are produced by two or more distinct B-cell clones. Polyclonal refers to the number of clones of B-cells contributing to the antiserum in the preparation. An antiserum will be polyclonal if two or more B-cells contribute to it.
What is “warm ischemic time”?
The time that blood supply is cut off during surgical procedures
What is the “gold standard” for IHCs?
10% NBF.
When would epitope retrieval not be needed?
Using alcohol in place of formalin fixatives often eliminates the need for epitope retrieval.
How can fixation be enhanced?
Enhancing of fixation can be done using microwave or ultrasound technology.
What are the main drawbacks of microwave fixation?
- May induce uneven fixation depending on the type/size of specimen
- Microwaving causes protein coagulation & may lead to hard/over-cooked
tissues
How does the type of wax effect IHC?
Waxes with lower melting points have been shown to produce
better IHC staining results than those with higher melting points - especially
for T-Lymphocyte markers (CD3, CD4, CD8, etc.)
What type of specimen is “best” for IF?
It is best to perform immunofluorescence staining on fresh specimens rather than formalin fixed specimens, because those that are fixed often demonstrate autofluorescence.
What kind of fixative is recommended for IF?
Holding solutions such as Michel’s transport medium or a PBS solution containing 10% sucrose may be used to hold specimens for transport prior to immunofluorescence staining.
How thick should frozen sections be cut?
Between 4-6 microns.
Is epitope retrieval necessary for frozen sections?
No. Due to exclusion of the antigen masking effects of formalin when using fresh tissue, little or no epitope retrieval may be needed.
