E&M, Cytologic Techniques, Tissue Processing Flashcards
(from the Greek mikros, meaning “small”, and temnein, meaning “to cut”) is a tool used to cut extremely thin slices of material, known as sections.
Microtome
The rotary microtome is the most used microtome in the Histopathology laboratory.
_____ is an instrument that is used to analyze and process samples by fixing, staining, dehydrating, or decalcifying them. It removes excess water from the tissue for it to be able to coat in melted paraffin wax.
Automatic tissue processor
_____ is designed to ensure that reagent solution spread evenly over the slide, and remain in place during the ensuing incubation period.
Automatic slide staining machine
It is designed for high-precision temperature control of embedding mediums like paraffin wax.
Paraffin oven
_____ is an essential step in the histology process, which allows the paraffin ribbon to smooth out and stick to the glass slide when placed.
Water bath
_____ is designed to assist with the handling of paraffin wax samples in histology and pathology laboratories. It is essentially a hot distilled water floating out bath that allows for the meticulous manipulation and location of sections onto glass slides.
Paraffin embedding wax bath
_____ is a rotor-driven instrument designed to separate liquid samples at high rates of speed.
Centrifuge
_____ is an appliance for removing excess paraffin from tissue cassettes
Paraffin block trimmer
_____ is designed to rapidly dry slides by blowing warm air over the surface of the slides.
Slide dryer
_____ is used to visualize very minute objects such as cells, microorganisms, giving a contrasting image, that is magnified.
Microscope
95% alcohol
-Pap smear staining-
Used in fixation (15-30 min)
80% alcohol
-Pap smear staining-
Used in hydration (2 min)
60% alcohol
-Pap smear staining-
Used in hydration (2 min)
1st Coplin jar of distilled water
-Pap smear staining-
Used in hydration (5 dips)
2nd Coplin jar of distilled water
-Pap smear staining-
Used in hydration (5 dips)
Hematoxylin stain
-Pap smear staining-
1st nuclear stain (3 min)
Running water
-Pap smear staining-
Gentle washing (30 sec)
Scott’s tap water
-Pap smear staining-
Blueing agent, pH=8.02 (3 min)
Distilled water
-Pap smear staining-
Dehydration (3 min)
60% alcohol
-Pap smear staining-
Dehydration (2 min)
80% alcohol
-Pap smear staining-
Dehydration (2 min)
1st Coplin jar of 95% alcohol
-Pap smear staining-
Dehydration (2 min)
Orange G stain
-Pap smear staining-
1st counterstain (3 min)
1st Coplin jar of 95% alcohol
-Pap smear staining-
(2 min)
2nd Coplin jar of 95% alcohol
-Pap smear staining-
(2 min)
Eosin Azure stain
-Pap smear staining-
2nd counterstain (3 min)
1st Coplin jar of 95% alcohol
-Pap smear staining-
(2 min)
2nd Coplin jar of 95% alcohol
-Pap smear staining-
(2 min)
1st Coplin jar of absolute alcohol
-Pap smear staining-
(2 min)
2nd Coplin jar of absolute alcohol
-Pap smear staining-
(2 min)
Step in tissue processing prior to fixation
Obtaining a fresh specimen
_____ is used to preserve cells and tissue components in a “life-like state” or as little alteration as possible to the living tissue.
Fixation
Uses formalin/formaldehyde as a fixative or phosphate-buffered solution
_____ is the process of removing calcium from tissues
Decalcification
_____ is simply the removal of water from aqueous-fixed tissue. This step is necessary to prepare the tissue for embedding in non-aqueous media like paraffin.
Dehydration
Uses increasing concentrations of ethanol solutions to avoid excessive distortion of tissue
The purpose of _____ is to remove dehydrating agents from tissues and to prepare the tissues for impregnation with the embedding agent. It also removes a substantial amount of fat from the tissue.
Clearing
Uses xylene immersions (1 to 3 kinds) for 5 min each
The role of this agent is to remove the clearing agent from the tissue and to completely permeate the tissue with paraffin wax. This step is called?
Infiltration/Impregnation
Uses melted paraffin wax (usually at 60 C, then allowed to cool at 20 C to solidify). These waxes are a mixture of purified paraffin wax and resins such as styrene or polyethylene.
_____ is the process in which the tissues or the specimens are enclosed in a mass of the embedding medium using a mold (cassette).
Embedding
Epoxy resins are the most commonly used embedding media for semi-thin and ultrathin sections.
_____ is cutting a fixed tissue or organ to create a flat surface with the correct orientation.
Trimming
_____ is the technique of making very thin slices of tissue specimens for microscopic examination.
Section cutting/Microtomy
Specimens for H&E routine staining are cut 3-5 micrometers in thickness.
Parafinized ribbons are removed from the microtome using a wooden tongue depressor. Uses albumin and poly-l-lysine additives as a bonding agent.
Mounting
After drying at 60 C, the slide is passed through another series of chemical reagents.
Staining
Uses xylene to remove paraffin, then uses absolute alcohol to remove xylene.
It affects infiltration and subsequent sectioning of tissues.
Tissue density and thickness
It increases the flow of fresh fluids around the tissue.
Agitation
It can speed up fluid penetration and tissue processing protocols
Temperature (37 C-45 C)
Reduced pressure can increase the infiltration rate and decrease the time needed to complete the steps in tissue processing protocols.
Vacuum and Pressure
_____ is a diagnostic technique used to examine cells from body fluids and solid tissues to determine the nature of the disease.
Cytology
_____ is the microscopic study of normal tissues of the body. While _____ is the microscopic study of the tissues affected by the disease.
Histology
Histopathology
Cells shed from bodily surfaces, such as the inside of the mouth, are collected and analyzed.
Exfoliative Cytology (Ex: urine, sputum, CSF, effusions, etc.)
Cells are obtained directly from the surface of the target of interest or from dislodged cells from the body’s surfaces or mucosal linings.
Abrasive Cytology (Ex: Scrapings from the cervix (pap smear), vagina, oral cavity (buccal mucosal smear), and skin lesions, bronchial wash and swabs)
____ is a technique used for obtaining material from organs that do not shed cells on their own. It is also called Fine Needle Aspiration Cytology (FNAC) because it uses a fine needle to obtain samples.
Aspiration Cytology (Ex: subcutaneous soft tissue, tumors, thyroid, lymph nodes, salivary glands, and breast)
It is a polychromatic stain that contains multiple dyes to differentially stain various components of the cell and it is considered the most common stain used in cytopathology.
Papanicolaou stain (PAP smear stain)
This approach is used to distinguish cells in the smear preparation of various gynecological specimens (pap smears) and materials comprising exfoliative cytology smears.
Pap smear staining
This technique is useful in lesions that are easily palpable. For non-palpable, it requires the use of ultrasound or CT guidance.
Aspiration Cytology
_____ are used to differentiate cells for microscopic examination in pathological specimens. Used in FNACs, and fluids
Romanowsky stains
Type of Romanowsky stains
Leishman’s stain - staining blood smears
Wright’s stain - staining peripheral blood smears, urine samples, and bone marrow aspirates
Giemsa’s stain/May Grunwald Giemsa - gold standard staining technique that is used for both thin and thick smears
Jenner’s stain
Field’s stain
_____ stain is also used in FNACs and fluids. Mainly used as histopathology stain.
H&E (Hematoxylin and Eosin)
This is the most common and easiest preparation of the cellular material.
Direct smear
Another method of preparation of the cellular materials are:
Cytospin preparation
Membrane filter preparation
Liquid-based preparation
Cell blocks
Air-dried fixation is done to smears that use what stain?
MGG/May Grunwald Giemsa/Giemsa
Air-dried fixation is later wet-fixed in ______ for 15-20 mins during the staining process
Methyl-alcohol
______ done using cytosprays is employed in many labs as an easier alternative to wet fixation
Spray fixation
The ideal angle for spray fixation is ____
45 degrees
Ideal distance for spray fixation is _____
15-25 cm
The fixative agent that is held too close, causes ____
dislodgement of the cells from the smear
The fixative agent that is held too far, causes ____
insufficient amount of fixative
The fixative agent that is forcefully sprayed, causes _____
creates cellular artifacts by the fracturing of cytoplasm
Father of Cytopathology
George Papanicolaou
_____ stains basic component of cells
Acid dye
_____ stains acid component of cells
Basic dye
Nuclei are stained by _____
hematoxylin solution
This stain is used as a cytoplasmic stain
Orange G stain
A polychromatic solution contains a mixture of _____ that is responsible for the demonstration of differentiation of squamous cells
Eosin Y
Light Green SF
Bismarck Brown Y
It stains the cell nuclei blue.
Harris Hematoxylin (most common) Gill's Hematoxylin and Hematoxylin S (other stains)
It is the first acidic counterstain (cytoplasmic stain) which stains mature and keratinized cells
Orange Green 6 (OG-6)
It is the second counterstain which is a polychrome mixture of Eosin Y, Light Green SF, Bismarck Brown. It gives pink color to the cytoplasm of mature squamous cells, nucleoli, cilia, and red blood cells.
Eosin Azure 50 (EA-50)
It stains blue to cytoplasms of cells like parabasal squamous cells, intermediate squamous cells, and columnar cells.
Light Green SF
It stains nothing instead it stabilizes the staining.
Bismarck Brown Y
Three types of PAP stain:
Progressive
Regressive
Rapid
The nucleus is stained with hematoxylin to an intensity desired
Progressive method of PAP stain
The nucleus is deliberately over-stained with a non-acidified hematoxylin.
Regressive method of PAP stain
Color of stain: Nuclei
Blue
Color of stain: Acidophilic cells
Red
Color of stain: Basophilic cells
Blue-green
Color of stain: Erythrocytes
Orange-red
Color of stain: Keratin
Orange-red
Color of stain: Superficial cells
Pink
Color of stain: Intermediate and Parabasal cells
Blue-green
Color of stain: Eosinophils
Orange-red
List of Precautions:
Immediate fixation of smear is essential
Solutions and other stains are filtered daily after use to keep them free of sediment
Alcohols are hygroscopic
Stains are discarded and replaced as the quality of the stain deteriorates
Place the coverslip on the microslide slowly without trapping air bubbles
Stains keep longer if they are stored in dark-colored stoppered bottles
When alcohol is left open, the concentration _____
decreases
Blueing solution and HCl should be replaced _____
once daily
Water rinses should be changed _____
after use
Alcohol used for the process of dehydration prior to the cytoplasmic stains should be replaced _____
weekly
Accurate patient identification, orientation of samples and adequate fixation rest are responsibilities of _____.
OT staff
Appropriate grossing, sampling are responsibilities of ____.
Junior pathology staff
Adequate processing, appropriate embedding techniques, microtomy, staining, and avoiding unacceptable artifacts are responsibilities of _____.
Histopathology technicians
Inspection of control, immuno-histochemical methods, and reporting of the slides are all responsibilities of _____.
Expert pathologist
Tissues are collected and fixed in _____.
10% formalin
Stain for testicular biopsy.
Bouin’s solution
Test requisition form should contain:
patient demographics detail of patient's history anatomic site of biopsy number of containers sent specimen transportation instruction etc.
Specimens are transported at what temperature?
room temperature
Specimen for transport must be labeled with what?
10% formalin hazard label
Specimen for transport must be placed in what?
well-sealed leak-proof container
Specimen identification is maintained across steps like:
Specimen labeling
Grossing
Block labeling
Slide labeling etc.
Bony or cartilaginous tissue are placed in decalcifying solution for how many days?
1-7 days
The tissue is washed _____ times in distilled water
3-4
Grossing station must be cleaned with _____ daily after grossing.
1% sodium hypochlorite
Formalin is volatile and toxic and causes irritation to what parts of the body?
eyes, mucus membranes, skin
Define OSHA:
Occupational Safety and Health Administration
average formalin exposure?
- 75 ppm / 8hr
2. 0 ppm / 15 minutes (short-term)
Alcohol concentrations in Dehydration:
50% alcohol - 90 min 70% alcohol - 90 min 80% alcohol - 90 min 95% alcohol - 90 min 100% alcohol - 90 min 100% alcohol - 12 hr
Clearing agent used is ____
Xylene
It is the simplest, least invasive test and uses the smallest needle to simply remove cells from the area of abnormality.
Fine Needle Aspiration/Fine Needle Aspiration Cytology
It removes not only cells but also a small portion of the surrounding tissue. This provides additional information to assist in the examination of the lesion.
Core needle biopsy
It takes out even more surrounding tissues. It takes some of the abnormality but not all. This process involves slicing into the lesion but removing only a portion of it.
Incisional biopsy
It generally removes the entire area in question.
Excisional biopsy
It is considered the primary technique for obtaining diagnostic full-thickness skin specimens. This technique involves the use of a circular blade that is rooted down through the epidermis and the dermis, and into the subcutaneous fat, yielding a 3-4 mm cylindrical core of the tissue sample.
Punch biopsy
It is where all small fragments of tissue are shaved from a surface (usually a skin)
Shave biopsy
It is where tissue is scooped or spooned to remove tissue or growths from the body cavity such as the endometrium or cervical canal.
Curettings
It is a process whereby a selected tissue specimen is immersed in isotonic salt solution such as normal saline solution or Ringer’s solution in a petri dish or watch glass, carefully dissected with a needle, and separated by direct or zigzag spread using an applicator stick
Teasing or dissociation
It is a process whereby small pieces of tissue are placed in a microscopic slide and forcibly compressed with another slide or with a cover glass.
Squash preparation/Crushing
With an applicator stick or a platinum loop, the material is rapidly and gently applied in a direct or zigzag line throughout the slide, attempting to obtain a uniform distribution of secretion.
Streaking (Smear preparation)
A selected portion of the material is transferred into the clean slide and gently spread into moderately thick film by teasing the mucus strands with an applicator stick. It is specially recommended for smear preparation of fresh sputum and bronchial aspirate, and also for thick mucoid secretions.
Spreading (Smear preparation)
This is done by placing a drop of secretion or sediment upon one slide and facing it unto another clean slide.
Pull-apart (Smear preparation)
This is a special method of smear preparation whereby the surface of a freshly cut piece of tissue is brought into contact and pressed on the surface of a clean glass slide.
Touch Preparation/Impression smear
Immediate diagnosis is accomplished through the use of ______
Frozen section
Fresh tissue is frozen on a microtome with CO2 or on a ______, which is a cold chamber kept at an atmospheric temperature of -10 to -20 C.
cryostat
It is generally used in histochemistry. It is the most rapid of the commonly available freezing agents.
liquid nitrogen
The main disadvantage of using liquid nitrogen as a freezing agent is:
It produces ice crystals or freezes artifacts on the tissue
A freezing agent that is liquid at room temperature
isopentane
Other freezing agents are:
CO2 gas and aerosol sprays (cryokwik)
It is a refrigerated apparatus used for fresh tissue microtome which maintains the temperature between -5 to -30 C (the average is -20 C)
Cryostat/Cold microtome
It is capable of freezing fresh tissues within 2-3 minutes
Thermostat
It is a special way of preserving tissues by rapid quenching of fresh tissue at -160 C and subsequently removing ice molecules without using any fixative.
Time-consuming and expensive
Freeze drying/Rapid freezing
vacuum at -40 C
Sublimation
It is a process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient temperature dehydration.
More economical and less time consuming
Freeze substitution
Fixative for frozen tissue:
Rossman’s Formula
1% acetone
Reagent for dehydration of tissue:
Absolute alcohol
Reagent change:
Absolute alcohol - alternate day
Xylene - every 3rd day
Wax - weekly
Formalin - every day
A special stone used to sharpen the knife in a microtome is called _____. The process of sharpening is called _____
Hone
Honing
It is the process of polishing an already fairly sharp edge of the knife that may be flexible or rigid. The material is called _____.
Stropping
Strop
Labeling is an important step and must be done using _____
diamond pencil
