[M] Week 8: Gross Room / Surgical Cut- up - Part 1 Flashcards by Geno Alinsub

Tissues from the body taken for diagnosis of disease processes must be processed in the histopathology laboratory to produce ____ ____ that are viewed under the microscope by pathologists

Microscopic slides

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The persons who do the tissue processing and make the glass microscopic slides are

histotechnologists

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In the laboratory safety precautions must observed at all times because we are dealing with a lot of hazards like?

Biological Hazard
Chemicals
Radiation

Safety first and last!

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The interface between hospital staff (or other visitors) and the pathology personnel (histotechnologists and other clerks that may receive the specimen)
To receive samples safely and securely

Specimen Reception

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SPECIMEN HANDLING AND IDENTIFICATION

True/False
1. Specimen labeled properly attached to the cap of the container
2. Request form is needed

False (should be in the body)
True

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SPECIMEN HANDLING AND IDENTIFICATION

What are included in the request form?

patient information
medical history
description of the site of origin

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SPECIMEN HANDLING AND IDENTIFICATION

surgical pathology request form, contains what information needed?

patient info
clinical diagnosis
specimen site or laterality
operation or procedure done
examinations requested (histopathology, cytology, cell block for safekeeping)
pertinent history or diagnostics done on the patient
The requesting physician.

This information is filled out by the operating room personnel (doctors, residents, nurses).

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SPECIMEN HANDLING AND IDENTIFICATION

In the laboratory, we receive the specimen so take note of
the ____ and ____ received and the person who received the specimen, then we will assign the ____

date and time
accession number

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SPECIMEN ACCESSIONING

A complex number or alpha numeric number that will identify each specimen for each patient
Not universal for all institutions. O’thers may have a different way of assigning.

Accession Number

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SPECIMEN ACCESSIONING

What is the format of accession number?

Procedure - Year - Chronology when spx is received

S for surgical
P for Pap’s smear for cytology
CB for cell block

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After receiving a specimen and labelling it, what will you do next?

check if there is a fixative (such as formalin) in the container.

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After fixing for several hours, the specimen will be?

grossed

like yung EWWW

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specimen dissection area, should have?

Good lighting
Good ventilation
Non-absorbent wipe-clean surfaces
Appropriate protective clothing for the laboratory personnel (fluid resistant laboratory gown and masks for protection from fumes)
Gloves
Cutting tools
Other equipment (photography, tissue macerators, bone saw, trash bin)
Integrated dissection desks (Or a kitchen-like tabletop with tiles and a sink )
Enclosed fluid/fixative feeds
Laminar down-draft ventilation (Alternative: Exhaust fan)

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Tissues are examined by a

pathologist, pathology assistant, or pathology resident

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For gross examination, these are the things we have to take note of, what are those?

Describing the specimen
Placing all or just a part of the specimen in a tissue cassette

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GROSS EXAMINATION

Number of tissue/s present in the specimen container, Shape, Color, Consistency, Size of the specimen (3-dimensional: LxWxH in cm)

Describing the specimen

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GROSS EXAMINATION

Tissue inside the cassette should be dissected to?

3-4mm in thickness

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gross only examination is done with what specimens?

Non-tissue
Tissues unlikely to require adetailed diagnosis and may be examined grossly

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When a malignancy is suspected, then the specimen is covered with ink – to mark the margins of the specimen

Inking the Margins

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INKING THE MARGINS

When sections are made and processed, the ink will mark
the ____ ____ on the slide

actual margin

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INKING THE MARGINS

TOF

It is also used to see if the margin is free of malignant cells and also how far the malignant cells are from the margin.

True

The farther the malignant cells are from the margin, the safer.

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TISSUE PROCESSING

After the removal of a tissue sample from the patient, a series of processes must take place to ensure the final microscopic slides are of?

diagnostic quality

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TISSUE PROCESSING

TOF
Producing quality slides for diagnosis is an accident;

requires skills that are developed through continued practice
and experience.

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TISSUE PROCESSING

Tissue processing aims to?

Permanently preserve the tissue
Stain tissue for demonstration of specific structures
Mount tissue on glass slides with coverslips for permanent keeping

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# TISSUE PROCESSING Tissue processing is designed to remove all ____ ____ from the tissue, replacing it with a support medium that provides sufficient rigidity to enable sectioning of the tissue without damage or distortion

Extractable Water

Give men the tissue processing steps

1. Fixation (most critical step) 2. Dehydration 3. Clearing (De-alcoholization) 4. Impregnation 5. Embedding 6. Trimming 7. Section-cutting 8. Staining 9. Mounting 10. Labeling

What is the usual or routine fixative used?

Formaline or Formaldehyde

For dehydration step, what are the routinely used dehydrating agents?

increasing grades of alcohol

- In this step, we remove the alcohol added during dehydration. - After dehydration, the tissue will become transparent or clear. Hence, the other name for this step is clearing.

De-alcoholization

Since we removed all extractable water: holes, gaps, or spaces will be left in the tissue, making it difficult to cut through the tissue into thin sections. It has to have a support medium obtained by filling up the spaces. It is called?

impregnation or infiltration

For impregnation or Infiltration, what is routinely used?

Melted paraffin

The tissue that was impregnated will be placed in a mold, what will be added?

embedding medium

We are now ready to cut into thin sections. Sometimes tissue blocks will be too big for the block holder, so the excess needs to be removed by?

trimming ## Footnote do the coarse cutting cutting the tissue by **10** to **15** microns thickness

When tissue is already exposed, we will adjust the thickness to **4 to 6** microns to cut into tissue or?

serial sections

Then, sections will be placed on a glass slide to be stained using routinely used

H&E

After staining what will be done?

Mounted, using mounting medium | then label after (last step)

For hard tissues (ex. bones, calcified tissues) what extra step are used?

decalcification (after fixation & before dehydration)

# DECALCIFICATION Hard tissues contain? which make them hard

calcium salts or lime salts ## Footnote Removing these salts will make the tissue softer and easier to cut into thin sections

routinely used decalcifying agent

10% nitric acid

yoooo

study ng tissue processing schedule | okay? ## Footnote =

Tissue cassettes are manually transferred from one reagent to another

Manual tissue processing

- Time is usually set with an alarm clock, cassettes are transferred when the time ends to the next reagent. - This is carried out at **room temperature**

Manual tissue processing

automatic tissue processor, temperature is also employed. This model contains reagents in different containers

Technicon

# AUTOMATIC TISSUE PROCESSOR “TECHNICON” used to hold the tissue cassettes

Tissue Basket

# AUTOMATIC TISSUE PROCESSOR “TECHNICON” Time is set digitally at the ____ ____ of the Technicon, inputted through the digital pads, the timing for each step of tissue processing from when it starts to when it ends.

lower portion

# AUTOMATIC TISSUE PROCESSOR “TECHNICON” the tissue processing up to impregnation will take?

6 to 8 hours

# AUTOMATIC TISSUE PROCESSOR “TECHNICON” For this model, tissue cassettes are placed in a ____ and instead of the basket moving from one container to another, the reagent will be flooded in that area.

Tray

# AUTOMATIC TISSUE PROCESSOR “TECHNICON” The baskets for both models employ agitation, by ____ ____ , and rotation, by ____ ____. These oscillations will aid in tissue processing, as well as the heat they create.

1. Vertical Oscillation 2. Rotary oscillation

# AUTOMATIC TISSUE PROCESSOR “TECHNICON” Tissue cassettes contained in baskets through a series of reagents Vertical oscillation or the mechanical ____ and ____ of the tissue and rotary oscillation provide the ____ needed

1. Raising and lowering 2. Agitation

- The tissue will be placed in molds that can be metal, plastic or disposable mold like the paper boat. - After the paraffin solidifies, a paraffin or tissue block will be produced, and later placed in a **microtome**.

Embedding

# Section-Cutting A microtome has different types, what are routinely used in the laboratory?

Minot or rotary microtome | It can be manually, mechanically, or automatically driven.

# Section-Cutting An example of a non-automatic one. Regardless of whether it is manual or automatic, they all have these basic parts, what are those?

- Block holder - Blade holder - Wheel (moves the block holder) - Knob (sets the thickness)

# Section-cutting has a thickness of 4 to 6 microns which is later transferred to a water bath.

Tissue ribbon

# Floatation The water bath is set at **6-10 degrees** lower than the melting point of paraffin. At this point, the paraffin should not melt, it should just be flattened to remove the wrinkles and will be separated into individual sections.

Floatation

Sections are retrieved by ?

**Fishing out** in a near-vertical position close to the section it will attach to.

This is used to melt the paraffin, making the tissue adhere more to the slide, like heat fixation.

Paraffin oven

What is used for the staining of the slides?

hematoxylin and eosin (H&E)

after the staining, what will be done?

Mounting

describe the mounting medium

Syrupy liquia covered with a coverslip | yum

How much mounting medium is dispensed?

1 to 2 drops, enough to cover the entire tissue

# Mounting TOF After it solidifies or dries up, the coverslip will be permanently placed. Sometimes, we place something to seal around the coverslip to prevent the mounting medium from coming out.

True

The slides are labeled according to?

**Types of specimen and staining** But in actual practice, the labels include the accession number and the institution or hospital. No need to write the name of the patient nor the type of specimen, only the institution or hospital and the accession number.

The size & type of specimen in the tissue cassette determine the need for complete fixation and processing, what is the thickness required?

Only **3-4mm thickness** should be placed in the tissue cassette Do not overfill the cassette so that it will not impede the reagents when we do the processing

- The first and most critical step of tissue processing in histotechnology - Involves fixing or preserving fresh tissue for examination - The entire process of tissue processing will depend on this step.

Fixation ## Footnote If the tissue is fixed incompletely, overfixed, or the wrong fixative is used, it will cause autolysis, damaging the tissue.

What is the primary aim of fixation?

To **preserve the morphological and chemical integrity** of the cell as life-like manner as possible

What is the secondary goal of fixation?

To **harden and protect the tissue** from the trauma of further handling, so that it is easier to cut during gross examination

What are the two mechanism involved in fixation?

- Additive Fixation - Non-additive Fixation

Enumerate why fixation is performed

- To preserve the tissue - To prevent the breakdown of cellular elements - To coagulate or precipitate protoplasmic substances that may leak out during subsequent steps of tissue processing - As soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis

- The chemical constituent of the fixative is taken in and becomes part of the tissue by forming crosslinks or molecular complexes and giving stability to the protein - Ex. formalin, mercury, and osmium tetroxide

Additive Fixation

- The fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H-bonds of certain groups within the protein molecule - Ex. alcohol fixatives

Non-additive Fixation

What are the Main factors Involved in fixation

- Hydrogen Concentration - Temperature - Thickness of Section - Osmolality - Concentration - Duration of Fixation

# MAIN FACTORS INVOLVED IN FIXATION pH 6 to 8

Hydrogen Ion Concentration

# MAIN FACTORS INVOLVED IN FIXATION Provide the temperature needed - Tradtional Temp - Tissue processors - Electron microscopy and Histochemistry

- RT - 40 degree C - 0-4 degree C

# MAIN FACTORS INVOLVED IN FIXATION Small or Thin 1. 1 to 2 mm for electron miscroscopy 2. 2cm for light microscopy 3. No more than 0.4cm for light microscopy

1. Small 2. Small 3. Thin

# MAIN FACTORS INVOLVED IN FIXATION What measurement of osmolality will give the best result?

- Slightly hypertonic solution (400 to 450 mOsm) - Isotonic solutions (340 mOsm)

# MAIN FACTORS INVOLVED IN FIXATION What are the percentage used before the fixative

- 10% formaldehyde - 3% glutaraldehyde

# MAIN FACTORS INVOLVED IN FIXATION what is the duration of fixation?

**2 to 6 hours** if prolonged causes shrinkage and hardening of tissue

What are the practical consideration of Fixation

1. Speed 2. Penetration 3. Volume 4. Duration of fixation

# PRACTICAL CONSIDERATION OF FIXATION - Specimen should be placed in fixative as soon as it is removed from the body - Delaying fixation can cause autolysis

Speed

# PRACTICAL CONSIDERATION OF FIXATION - approximately 1mm/hr & slows down as it goes deeper - Cut large or solid organs into sections for the fixative to reach the deeper portions. - For hollow organs that tend to float, a gauze is soaked in fixative to prevent the specimen from floating.

Penetration

# PRACTICAL CONSIDERATION OF FIXATION - **10-25x** the volume of tissue to be fixed - The tissue should be completely submerged in fixative - The size of the container is crucial. (Large specimens should not be put into smaller containers so that they can be filled with enough fixatives to soak them)

Volume

What are the factors influencing the rate of processing?

1. Agitation 2. Heat 3. Viscosity 4. Vacuum

# FACTORS INFLUENCING THE RATE OF PROCESSING - The rate of exchange is dependent upon the exposed surface of the tissue that is in contact with the processing reagent - In automated processors, like Technicon, vertical or rotary oscillation or pressurized removal and replacement of fluids at timed intervals are employed,

Agitation

# FACTORS INFLUENCING THE RATE OF PROCESSING - Increases the rate of penetration & fluid exchange - It must be used sparingly or with caution to reduce the possibility of shrinkage, hardening, and brittleness of the tissue - Temperatures limited to 45˚C can be used effectively; Higher temperature may be deleterious to tissues especially for immunohistochemical staining

Heat

# FACTORS INFLUENCING THE RATE OF PROCESSING Is the property of resistance to the flow of a fluid

Viscosity

# FACTORS INFLUENCING THE RATE OF PROCESSING The smaller the size of the molecules in the solution, the faster the rate of fluid penetration

Low viscosity

# FACTORS INFLUENCING THE RATE OF PROCESSING If the molecular size is larger, the rate of exchange is slower

High viscosity ## Footnote Smaller is faster; Larger is slower

# FACTORS INFLUENCING THE RATE OF PROCESSING - Using **reduced pressure** to increase the rate of infiltration - If used on the automated processor, it should not exceed **15 inches of Hg (mercury)** to prevent damage and deterioration to the tissue

Vacuum

# EFFECTS OF FIXATIVES True/False 1. Soften hard & friable tissues and make handling and cutting sections easier 2. Make cells resistant to damage & distortion 3. Inhibit virus decomposition

1. False (Harden soft) 2. True 3. False (bacterial decomposition)

# EFFECTS OF FIXATIVES True/False 1. Increase optical differentiation of cells and tissue components 2. Act as mordants or accentuators to promote and hasten staining or inhibit certain dyes in favor of another 3. Reduce risk of infection during handling and actual processing of tissues

1. True 2. True 3. True

CHARACTERISTICS OF A GOOD FIXATIVE just familliarize

- It must be cheap - It must be stable. - It must be safe to handle. - It must kill the cell quickly thereby producing minimum distortion of cell constituents. - It must inhibit bacterial decomposition and autolysis. - It must produce minimum shrinkage of tissue. - It must permit rapid and even penetration of tissues. - It must harden tissues thereby making the cutting of sections easier. - It must be isotonic, causing minimal physical and chemical alteration of the cells and their constituents. - It must make cellular components insoluble to hypotonic solutions and render them insensitive to subsequent processing. - It must permit the subsequent application of many staining procedures to facilitate easier and more profitable examination.

What are the types of fixatives according to composition

- Simple Fixatives - Compound Fixative

# TYPES OF FIXATIVES - made up of only **one component substance** - Aldehydes: formaldehyde, glutaraldehyde - Metallic fixatives: mercuric chloride, chromate fixatives, picric acid, acetic acid, acetone, alcohol, osmium tetroxide

Simple Fixative

# TYPES OF FIXATIVES made up of **two or more fixatives** which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents

Compound Fixatives

What are the types of fixatives according to action

- Microanatomical Fixatives - Cytological Fixatives (Nuclear Fixatives, Cytoplasmic Fixatives) - Histochemical Fixatives

# Fixative according to action - Permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question - 10% Formal saline, 10% Neutral buffered formalin, Heidenhain’s Susa, Formal sublimate (formol corrosive), Zenker’s solution, Zenker-formal (Kelly’s solution), Bouin’s solution, Brasil’s solution

Microanotmical Fixative

# Fixative according to action - preserve specific parts and particular microscopic elements of the cell itself: nuclear or cytoplasmic components

CYTOLOGICAL FIXATIVES

# Fixative according to action Cytological Fixative - preserve the nuclear structures (e.g., chromosomes) - usually contain glacial acetic acid - Flemming’s fluid w/ acetic acid, Carnoy’s fluid, Bouin’s fluid, Newcomer’s fluid, Heidenhain’s Susa

Nuclear Fixatives

# Fixative according to action Cytological Fixative - preserve cytoplasmic structures - Flemming’s fluid without acetic acid, Kelly’s fluid, Formalin with “post-chroming”, Regaud’s fluid (Muller’s fluid), Orth’s fluid

Cytoplasmic Fixative

# Fixative according to action - preserve the chemical constituents of cells and tissues - 10% Formal saline, Absolute ethyl alcohol, Acetone, Newcomer’s fluid

Histochemical Fixatives

# Fixative according to action what are the other fixative under histochemical fixatives?

- Lipid Fixation - Carbohydrate Fixation - Protein Fixation - Glycogen Fixation

# HISTOCHEMICAL FIXATIVES Cryostat or frozen section followed by general lipid stains Mercuric chloride & Potassium dichromate

Lipid Fixation

# HISTOCHEMICAL FIXATIVES Carbohydrate fixation

Alcohol fixatives

# HISTOCHEMICAL FIXATIVES Neutral buffered formalin, formol saline or formaldehyde

Protein fixation

# HISTOCHEMICAL FIXATIVES Alcohol-based, such as Rossman’s fluid or cold absolute alcohol

Glycogen fixation

[M] Week 8: Gross Room / Surgical Cut- up - Part 1 Flashcards

(108 cards)