Fixation + Addtl. Discussion Flashcards by JESSABELLE DECANO

This step includes entering the details of the specimen in a log book

NUMBERING

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Gross description is always done by?

PATHOLOGIST

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Most important and most crucial step in tissue processing. It has to be carried out adequately.

FIXATION

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Step in tissue processing that is define as the killing, penetration and hardening of tissues

FIXATION

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May also be defined as the alteration of tissues by stabilizing protein so that tissues become resistant to further changes

FIXATION

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Primary goal of Fixation:

To preserve the morphological and chemical integrity of the cells and tissues as close to the original as possible

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Secondary goal of Fixation:

To harden tissues;
To facilitate easy cutting;
To protect the tissue from trauma of further handling

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2 Fixation Methods:

PHYSICAL and CHEMICAL

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Types of Physical Method:

Heat Fixation and Microwave Technique
Cryo-preservation (freeze drying)/ freeze substitution

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Mostly done in microbiology to fix bacterial
smears
Not usually carried out in Histopath (rarely used)
For rapid diagnosis
For frozen tissue section

Heat Fixation

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Fixatives under Chromate Fixatives: (CROP)

Chromic acid (1-2%)
Regaud’s/ Moller’s fluid
ORTH’S FLUID
Potassium Dichromate (3%)

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physical technique that is widely used but not a common method.
it increases movement of molecules thereby accelerating fixation, staining, decalcification, immunohistochemistry and EM

Microwave Technique

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Neurochemical substances in brain that can be used by microwave techniue

Acetylcholine

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A method of fixation where the specimen is immersed in chemical fixative

CHEMICAL METHOD

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Reagent used to fix tissue is called?

Fixative

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Factors involved in fixation/ practical considerations

Temperature
Thickness/ Size
pH
Osmolality
Concentration
Time and Duration of Fixation
Volume

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Temp. required for EM and histochemistry

0-4 degree Celsius with Autotechnicon

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Fixation is traditionally carried out at what temperature?

Room temperature

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Required temp. in Autotechnicon?

40 degree Celsius

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TRUE or FALSE. Autotechnicon is done usually in laboratory and it uses constant agitation?

TRUE

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TRUE or FALSE. Formalin @ 60 deg C may be use to fix tissues with TB

FALSE

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TRUE or FALSE. Formalin at 60 deg C is for rapid fixation for urgent biopsy

TRUE

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Temperature for formalin required in order to fix tissues with tuberculosis

Formalin @ 100 deg C

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3 Fixatives for Smear (SME)

Methanol
Ether/ Ethanol
Schaudinns Fluid

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Required thickness of tissue spx for electron microscopy (EM)

1-2 mm^2

Required thickness of tissue spx for light microscopy (LM)

2 cm^2 wide and not more than 4mm thick

TRUE or FALSE. The thickness of tissues spx for LM should be more than 0.5 cm

FALSE; should be no more than 0.4 cm - 0.5 cm (4-5 mm) for LM

TRUE or FALSE. Many laboratories use tissue processors that work at 45 deg C for regular tissue processing?

FALSE; 40 deg C

TRUE or FALSE. Refrigeration is used to speed up decomposition if the tissue needs to be photographed and cannot be fixed immediately

FALSE; refrigeration is used to SLOW DOWN

TRUE or FALSE. Brain cells can deteriorate very quickly

TRUE

TRUE or FALSE. Nucleic acids react with fixatives to any extent at room temperatures and chemical reactions including those involved in fixation are more slower at higher temperatures

FALSE; nucleic acids does not react; rapid at higher temperatures

TRUE or FALSE. An increase in temperature can increase the rate of fixation but can also increase the rate of autolysis

TRUE

Required thickness for processing lung specimen

1-2 cm

TRUE or FALSE. Tissues should not be more than 4-5mm except in processing lung specimen

TRUE

TRUE or FALSE. Uterus and other large solid tissues must be opened/ slice thinly to improve penetration of fixatives

TRUE

TRUE or FALSE. Fecal matter and stomach contents can inhibit the penetration of fixatives and must NOT removed before the fixation process

FALSE; it should be remove before fixation process

Brain is usually suspended whole in what fixative for how many weeks to ensure fixation and some hardening prior to sectioning

10% buffered formalin for 2-3 weeks

TRUE or FALSE. Most tissues can be cut and trimmed without prior fixation such as the brain

FALSE; most tissues EXCEPT brain is generally soft when unfixed so it must be fixed before sectioning

TRUE or FALSE. An incompletely fixed tissue may lead to improper and incomplete clearing and impregnation, and may later prove to be a hindrance to normal sectioning and staining of specimen

TRUE; FIXATION IS MOST IMPORTANT STEP IN TISSUE PROCESSING

TRUE or FALSE. Penetration into a thin section will occur more rapidly than for a thick section

TRUE

TRUE or FALSE. Formalin penetrates tissues slowly so specimens may need to be opened, incised, or sliced and left to fix for an adequate period of time prior to processing

TRUE

TRUE or FALSE. Larger tissues require more fixatives and longer fixation time

TRUE

TRUE or FALSE. To maintain an adequate fixation time of 4- hours, the recommended size of the tissue is 2 cm^2 and no more than 4mm thick

TRUE

Fixation is best carried out close to neutral pH, in what range?

Between pH of 6-8

TRUE or FALSE. Acidity favors formation of formalin-heme pigment that appears black, polarizable deposits in tissue

TRUE

Common buffers includes...

Phosphate Bicarbonate Cacodylate Veronal

Commercial formalin is buffered with phosphate at a pH of...?

pH of 7

What will happen if the cells are fixed in a hypertonic solution?

Cell Shrinkage

Effect of hypotonic solution?

Cells may swell

Preferred osmolality for fixation

Slightly hypertonic (400-450 mOsm)

What component is commonly added to osmium tetroxide fixatives for electron microscopy?

Sucrose

Osmolality classification used in practice and may be use as holding solutions for tissues to be transported to frozen sections or kidney biopsies for special processing

Isotonic solution

TRUE or FALSE. Concentration of fixative should be adjusted down to the lowest level possible

TRUE

Concentration of fixative used for formaldehyde?

10% solution

Concentration of fixative used for glutaraldehyde and used for EM

3% solution

Ideal concentration of fixative for immuno-electron microscopy

0.25% glutaraldehyde

Ideal time to perform fixation

20-30 minutes following interruption blood supply

TRUE or FALSE. In order to maintain tissue morphology, samples SHOULD BE FIXED IMMEDIATELY after removal or death to prevent autolysis or putrefaction.

TRUE

TRUE or FALSE. More cellular organelles will be lost, more nuclear shrinkage and artefactual clumping will occur and tissue spx can be irreversibly damaged when the fixation is delayed

TRUE

It refers to the period the tissue is exposed to formalin

Fixation time

Fixation is retarded by:

Large and thick tissues Presence of mucus Presence of blood Presence of fat Cold temperature (can make the fixation time LONGER/ PROLONG)

TRUE or FALSE. Cold temperature can inactivate enzymes

TRUE

TRUE or FALSE. Cold temperature can inactivate enzymes

TRUE

Fixation is accelerated by:

Smaller and thin tissues Agitation — continuous stirring Heat (37-56 degrees C) (can make fixation time SHORTER/FASTER)

Required temperature for heat in tissues

37-56 deg C only

TRUE or FALSE. Beyond 56 deg C heat is acceptable for tissues spx

FALSE; 37-56 deg C heat only, beyond 56 deg C can be damaging to tissues

Fixative volume for maximum effective fixation

20x the volume of the specimen

Ratio of fixative to tissue

20:1

Formalin diffuses into the tissue at the rate of:

1mm per hour

Fixatives used for EM: (KAPOG)

Karnovsky’s Acrolein Paraformaldehyde Osmium tetroxide Glutaraldehyde

Volume required if osmium tetroxide is used for EM

5-10 times the volume of specimen

Volume required for museum preparations

Should not be less than 50-100 times the volume of the specimen

TRUE or FALSE. Agitation will also enhance fixation of the specimen

TRUE

Most common error in histotechnology is...

Insufficient ratio of tissue volume to fixative

Human brain must undergo in what method in fixation process

INTRAVASCULAR PERFUSION

This is used to wash out blood in human brain during intravascular perfusion

RINGER'S LACTATE

TRUE or FALSE. Eyes should be dissected before they are fixed

FALSE; it SHOULD NOT be dissected; tissues may wrinkle; inject formol alcohol before immersing the organ to fixative

If the autopsy materials is not possible to fix immediately after death, the body must be placed where and at what temperature?

Mortuary ref (4 deg C)

Hard tissues (cervix, uterine, fibroid, etc.) must undergo in what method?

LENDRUM'S METHOD

What method washes with running water overnight then immerse specimen in 4% aqueous phenol for 1-3 days.

Method of Lendrum's Method

Lendrum's method is done before, during or after fixation?

After fixation

Hollow organs (stomach, intestine) should be...

Packed with cotton soak in fixative; The spx can be sliced and it must be completely open before immersing it in adequate fixing solution

What specific organ may float on fixative?

Air filled lungs

How to prevent air filled lungs floating on a fixative?

May cover it on several layers of gauze to keep it at the bottom of the container

Effects of fixatives in general:

Harden tissues Makes cells resistant to damage Increase optical differentiation of cells Acts as mordants or accentuators to facilitate easy staining

(Problems in Fixation) What artefacts that may appear in tissue spx when the washing was incomplete?

Presence of artefacts —> white paraformaldehyde, precipitate artefacts

PROBLEMS IN FIXATION

1. Incomplete washing may lead to: presence of artefacts on tissues 2. Overfixation will render the tissue brittle and hard, shrinkage and swelling 3. Loss of substance soluble in fixing agent, loss or inactivation of enzyme may result from wrong choice of fixative.

Cause of failure to arrest early autolysis of cells

Failure to fix immediately (the tissue was probably allowed to dry before fixing); Insufficient fixative

Cause of removal of substances soluble in fixing agent

Wrong choice of fixative

Presence of artefact pigments on tissue sections

Incomplete washing of fixative

Cause of issues are soft and feather-like in consistency

Incomplete fixation

Cause of loss or inactivation of enzymes needed for study

Wrong choice of fixative

Cause of shrinkage and swelling of cells and tissue structure

Overfixation

Cause of tissues block are brittle and hard

Prolonged fixation

Well-known artifact that may be produced under acid conditions

Formalin pigment

What substance should use to eliminate the pigment or may be used to reduce the fixation of the specimen?

Phenol-formalin

May be found in surgical specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue stained in H&E stained sections

Crush artifact

Can be used to vapor-fix freeze-dried tissues

Paraformaldehyde and Osmium tetroxide

TRUE or FALSE. Fixation does not prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body

FALSE; fixation PREVENTS

TRUE or FALSE. Fixation in 10% buffered formalin will initially cause slight swelling of tissue specimens

TRUE

TRUE or FALSE. During processing, the spx may shrink and lose 20%-30% of its volume

TRUE

TRUE or FALSE. The particular fixative employed will also influence the degree to which individual elements will stain with various histochemical and immuno-histochemical reagents

TRUE

TRUE or FALSE. Leaving a tissue spx in air for a prolonged period of time will cause it to dry out, and will result in distortion of its morphological appearance

TRUE

BENEFITS OF FIXATION

1. Allows thin sectioning of tissue by hardening it 2. Prevents autolysis and inactivates infectious agents (except prion diseases) 3. Improves cell avidity for special stains

Factors to be considered when choosing the right fixative

1. The need for immediate examination: urgency of the case (urgent biopsy = use a rapid fixative in action) 2. Type of specimen to be processed 3. The tissue structure to be studied. 4. Structure technique to be applied (the fixative must be compatible with the stain to be used)

Types of fixatives as to MECHANISM OF ACTION

Additive Non-additive

Fixatives that when used is ABSORBED BY THE TISSUE; stabilizes tissue proteins

Additive Fixatives

Examples of Additive Fixation

Formaldehyde Mercuric Chloride Chromium Trioxide Picric Acid Glutaraldehyde Osmium Tetroxide Zinc Sulfate or Chloride

Fixing agent that is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attach to H-bonds of certain groups within the protein molecule.

Non-additive Fixation

Fixative is not absorbed by the tissue; alteration of tissue components

Non-additive Fixation

Example of Non-additive Fixatives

Alcohol Acetone

Acts by cross-linking proteins

Aldehyde and Oxidizing Agents

Protein-denaturing agents

Alcohol based fixatives

Acts by forming insoluble metallic precipitates

Metallic fixatives

- Cheap - Stable - Safe to handle - Must kill the cell quickly - Must inhibit bacterial decomposition and autolysis - Produce minimum shrinkage of tissue - Permit rapid and even penetration of tissues - Must harden tissues - Must be isotonic (some are hypotonic solutions) - Must make cellular component insoluble to hypotonic solutions and render them insensitive to subsequent processing - Must permit subsequent application of many staining procedures to facilitate easier and more profitable examination

Characteristics of a good fixative

Non-additive fixative that alters the tissue component

Acetone

Types of Fixative According to Action: (MCH)

Cytological Microanatomical Histochemical

Use to preserve PART OF THE CELLS (nucleus or cytoplasm)

Cytological

Preserves parts of cytoplasm; pH greater than 4.6; DO NOT CONTAIN GLACIAL ACETIC ACID because HAc destroys the mitochondria and golgi bodies of the cytoplasm

Cytoplasmic Fixatives

Fixatives that (should) contain glacial acetic acid; pH of < 4.6; Preserves nuclear structures like nuclear chromatin and chromosomes

Nuclear Fixatives

Examples of Nuclear Fixatives: (BF(e)NCH)

Bouin's Fluid Flemming's Fluid Newcomer's Fluid Carnoy's Fluid Heidenhain's susa

Fixatives for Cytoplasmic: (HORFF)

Helly's/ Kelly’s/ Zenker Formol Orth's Fluid Regaud's Fluid (Moller's) Flemming's without HAc Formalin with post-chroming

Fixes Rickettsia org. and other bacteria

Orth's fluid

Fixative that reacts with viruses, and causes the loss of their infective power

Mercuric Chloride

TRUE or FALSE. Glacial acetic acid destroys mitochondria and golgi bodies of the cytoplasm

TRUE

Gives the best quantitative results using frozen tissues as the standard

Ethanol and Acetone

Allows the GENERAL MICROSCOPIC STUDY of tissue structures WITHOUT ALTERING THE STRUCTURAL PATTERN normal intercellular relationship of tissues

Microanatomical Fixatives

Fixatives for Microanatomical:

10% Formol saline 10% Neutral buffered formalin Heidenhain's susa Formol sublimate/ formol corrosive Zenker's formol (helly's) Zenker's solution Bouin's Brasil's

Used to preserve chemical components of tissues like enzymes

Histochemical

Fixatives for Histochemical: (FAcNAe)

10% Formol saline Acetone Newcomer's fluid Absolute ethyl alcohol

Fixative used for the detection of rabies

Acetone

Types of Aldehyde Fixatives: (FGG)

Formaldehyde/ Formalin Glutaraldehyde Glyoxal

Types of Metallic Fixatives: (MCL)

Mercuric Chloride Chromate Fixatives Lead Fixatives

Types of Picric Acid:

Bouin's solution Brasil's alcoholic picformol Holllande's solution

Types of Alcohol Fixatives (MINCER)

Methyl alcohol Isopropyl Newcomer's Carnoy's Ethyl alcohol Rossmann' solution Others: Clarke's solution, Methacarn

Used for routine; m Most common fixative reagent Soluble in water to the extent of 37-40% solution volume AKA 100% formalin

Formaldehyde/ Formalin

Fixative recommended for mailing specimens (tolerant fixative) and for colored tissue photography

Formalin

Diluted form of the concentrated solution; Routine tissue fixative; Most commonly used fixative

10% formalin

Advantages of Formalin:

Cheap Easy to prepare Readily available Can preserve fats Stable

Disadvantages of Formalin:

Fumes are irritating May cause dermatitis on prolonged contact May form brown pigment on blood containing tissues like spleen

Methods in removal of Formalin pigments:

Kardasewitsch method Lillie's method Picric Acid method 1% KOH in 80% alcohol

Component of Kardasewitsch method:

70% ethanol 28% ammonia water

Components of Lillie's method:

Hydrogen Peroxide 28% ammonia water Acetone

Components of Picric Acid method:

Saturated Alcoholic Picric Acid

Most widely used fixative for routine histology buffered to pH 7 with phosphate buffer

10% neutral buffered formalin (phosphate buffer)

It is the considered the fixative of choice for many other procedures that require paraffin embedding, including immunohistochemistry and interphase Fluorescent In-Situ Hybridization (FISH)

10% neutral buffered formalin

Fixative which is for post-mortem tissue and CNS tissues Classified as histochemical fixative

10% formol saline

Remedy for precipitation of white paraformaldehyde

May add 10% methanol or filter it

Prolonged storage of formaldehyde = ?

Precipitation of white paraformaldehyde

TRUE or FALSE. Overnight fixation is generally indicated for 10mm thick slices of tissues

TRUE

TRUE or FALSE. Variations in time and conditions of fixation cause the majority of problems in histochemistry

TRUE

TRUE or FALSE. Formalin cannot preserve fats, mucin, and glycogen

FALSE; Formalin can preserve

TRUE or FALSE. Formalin can preserve proteins but it does not precipitate proteins

TRUE

TRUE or FALSE. Formalin does not make tissues brittle and therefore, it is the recommended for nervous tissue preservation

TRUE

TRUE or FALSE. Formalin is a soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding

TRUE

Prolonged fixation may produce:

1. Bleaching of the specimen and loss of natural tissue colors 2. Dispersal of fat grom the tissue into the fluid 3. Dissolution or loss of glycogen and urate crystals

Recommended for fixing tissues with iron pigments and elastic fibers

10% Neutral Buffered Formalin or Phosphate Buffered Formalin

AKA FORMOL SUBLIMATE

Formol Corrosive

Mercuric chloride + formaldehyde; recommended for lipids, neutral fats, and phospholipids

Formol corrosive/ Formol sublimate

AKA Gendre's solution

Alcoholic Formalin

Composition of Alcoholic Formalin:

95% ETOH Picric Acid Glacial Acetic Acid

Fixative to use to fix sputum specimen and for microincineration techniques

Alcoholic Formalin

When small tissues are burn into ashes to identify mineral elements from the ashes

Microincineration Techniques

Fixative used for enzyme histochemistry and electron microscopy

Glutaraldehyde

Formula of 10% formal-saline:

40% formaldehyde: 100ml Distilled water: 900ml Sodium dihydrogen phosphate monohydrate: 4gm Disodium hydrogen phosphate anhydrous: 6.5gm

Formula of 10% neutral -buffered formalin

Distilled water 40% formaldehyde Sodium dihydrogen phosphate, anhydrous Sodium dihydrogen phosphate

Fixative that considered as alternatives to mercuric chloride formulations; Can give improves results with immunohistochemistry

Zinc Formalin

Fixatives under Glutaraldehyde that are also for EM

Karnovsky's paraformaldehyde-glutaraldehyde and ACROLEIN

Required solution of glutaraldehyde for small tissue fragments

2.5% solution

Required solution of glutaraldehyde for large tissues less than 4mm thick

4% solution

TRUE or FALSE. Glutaraldehyde can cause rapid and irreversible changes but it can fixes quickly and well suited for EM, it fixes well at 4 deg C and gives best overall cytoplasmic and nuclear detail

TRUE

TRUE or FALSE. Glutaraldehyde does not cause dermititis

TRUE

TRUE or FALSE. Glutaraldehyde is more expensive and less stable

TRUE

Supplied as 40% aqueous solution; Commercially available; Suited for urgent biopsies

Glyoxal

Fast acting fixative; Can fix tissues rapidly; Smallest aldehyde fixative; Suited in urgent biopsies

Glyoxal

Surgical specimens are fixed within how many hours using glyoxal fixativesurgical specimens are fixed within how many hours using glyoxal fixative

4-6 hours

Small biopsy specimens are fixed within how many minutes using glyoxal fixative

45 minutes

Most common metallic fixative and may form black mercury deposits

Mercuric Chloride

Remedy for black mercury deposits formed by Mercuric Chloride

Wash tissue with alcoholic iodine (treating the section with 0.5% iodine solution in 70% ethanol for 5-10 minutes)

Mercuric Chloride is excellent for:

Trichrome staining

TRUE or FALSE. Mercuric chloride precipitates all proteins; has greater affinity to acid dyes and preferred in lieu of formaldehyde for cytoplasmic staining.

TRUE

It is recommended for renal tissue, fibrin, connective tissue and muscle

MERCURIC CHLORIDE

Contains mercuric chloride and glacial HAc; Recommended for fixing liver, spleen, connective tissue fibers, and nuclei

Zenker's Fluid

AKA Helly's Fluid

Zenker's formol

Contains potassium dichromate and 40% formaldehyde; Preserves pituitary glands, bone marrow, and other blood containing organs

Zenker's formol

Mercuric deposits may be removed by immersing tissues in ALCOHOLIC IODINE prior to staining, through a process known as ...

De-zenkerization

Done by oxidation with iodine to form mercuric iodide

De-zenkerization (iodine - sodium thiosulfate - water)

Excellent fixative for bone marrow, extramedullary hematopoiesis and intercalated discs of cardiac muscle

Zenker's Formol or HELLEY'S

Fixative combined with anhydrous acetate; For preserving bone marrow

Lillie's B5 fixative

Composition of B-5 Fixative

4% aqueous formaldehyde with 0.22M chloride and 0.22M acetic acid

TRUE or FALSE. Mercuric chloride ensures rapid structural stabilization and also facilitates bright staining by many of the dyes used in microtechnique

TRUE

Estimate fixation time for Lillie's B5 fixative

4-8 hours

Fixatives under Mercuric Chloride: (BHZZ)

B5 Heidenhain's susa Zenker's Fluid Zenker's Formol Others: Schaudinn's Ohlmacher's, Carnoy-Lebrun solution

Used for acid mucopolysaccharides and tissue mucin

Lead Fixatives

Chromate fixative that preserves carbohydrates and precipitates all proteins

Chromic acid (1-2%)

Chromate fixative that preserve lipids and mitochondria

Potassium dichromate (3%)

AKA Muller's fluid

Regaud's

Chromic fixative for mitochondria, mitotic figures, golgi bodies, RBC, and containing colloid tissues

Regaud's (Moller's)

- chromate fixative for rickettsia and other bacteria - also for tissue necrosis, and the early degenerative process.

Orth's fluid

pH value used in potassium dichromate to fix mitochondria

pH 4.5-5.2

Agent can act as a fixative, stain, and decalcifying agent

PICRIC ACID

Excellent for glycogen demonstration and Brilliant staining with trichome method

PICRIC ACID

Major drawback of Picric Acid

Imparts a YELLOW color when used as a fixative

Remedy for drawback (yellow impart) of Picric Acid:

Saturated solution of Lithium carbonate in 70% alcohol then wash with water The tissue is then placed in 70% ethanol followed by sodium thiosulfate and washed with water

TRUE or FALSE. Orth's fluid preserves myelin better than buttered formalin

TRUE

TRUE or FALSE. Regaud's and Orth's fluid can preserve fats

FALSE; Regaud's and Orth's fluid does not preserve fats

Good fixative for connective tissue, preserves glycogen well, and extracts lipids to give superior results in immunostaining of biogenic and polypeptide hormones

Picric Acid Fixatives

TRUE or FALSE. Picric Acid is explosive in dry form

TRUE; should be stored in moist distilled water or saturated alcohol

This stain use combinations of anionic dyes with phosphotungstic or phosphomolybdic acid to impart contrasting colors to cytoplasm, collagen fibers and other components of tissues

Trichrome stains

- Fixative used for the fixation of embryo, pituitary biopsies, and endometrial curetting - Usually an excellent fixative for preserving soft and delicate structures

Bouin's solution

- This fixative is NOT for kidneys, lipids and mucus - It abolishes Feulgen's reaction

Bouin's Fluid

Demonstrates RNAs and DNAs

Feulgen reaction

Excess picric should be washed from tissues prior to staining with...?

70% ethanol

A fixative known to be excellent for glycogen

Brasil's Alcoholic Picformol

Fixative for GIT biopsies and endocrine tissues

Hollande's solution

TRUE or FALSE. One disadvantage of using Bouin's solution is that it causes RBC hemolysis and reduces the amount the demonstrable ferric iron in tissue

TRUE

TRUE or FALSE. Hollande's solution produces LESS lysis than Bouin's solution

TRUE

Fixative that is considered as Picric Acid and Alcohol

Gender's fluid

It is the preferred fixative for tissues to be stained by Masson's trichrome for collagen, elastic or connective tissue; It does not need "washing out"

Gender's fluid

Fixative that is better and less messy than Bouin's solution

Brasil's Alcoholic Picroformol Fixative

The major effects of Acetic Acid are...

Precipitate DNA and for preservation of nuclei

A compound fixative, recommended for nucleoproteins

Glacial Acetic Acid

Glacial acetic acid solidifies at what temperature?

17 deg C

TRUE or FALSE. Acetic acid is always incorporated into other fixatives to form a compound solution, most commonly at a concentration of approximately 5%

TRUE

Can be a fixative or decalcifying agent; Also used for precipitation of proteins and nucleic acids

Trichloroacetic Acid (TCA)

TRUE or FALSE. TCA is a poor penetrating agent, and suitable only for small pieces of tissues or bones

TRUE

- Fixative used at ice cold temperature (-5-4 deg C); - Can also fix and dehydrate tissue at the same time

ACETONE

Fixative recommended for preservation of water-diffusable enzymes (lipases, phosphatases) and for to fix brain tissue for diagnosis of rabies

ACETONE

Type of fixatives that rapidly denatures and precipitates proteins; Can also act both as fixative and dehydrating agents; Ideal for small tissue fragments

Alcohol Fixatives

TRUE or FALSE. Alcohols are protein denaturant and can cause too much brittleness and hardness, hence, not used routinely for tissues

TRUE

TRUE or FALSE. Solid specimens taken from patients with gout are usually fixed with 70% methanol for subsequent histochemical detection of sodium urate crystals

FALSE; 95% ETHANOL

Alcohol fixative that appears to give the most usable DNA fragments for PCR

ETHANOL

TRUE or FALSE. Alcohol can cause tissue shrinkage and not good for EM

TRUE

TRUE or FALSE. Absolute alcohol can be used to fix and preserve glycogen, pigments, blood, tissue films and smears

TRUE

Alcohol fixative for fixing wet and dry smears, blood smears and BM tissues; Fixes and dehydrates at the same time

METHANOL (100%) or Methyl Alcohol

Alcohol fixative that is recommended for touch preparations, for special staining (Wright-Giemsa staining)

ISOPROPYL (95%)

For blood, tissue films and smears, and DNA (alcohol fixative); Preserves nucleoproteins and nucleic acids, used for histochemistry especially for enzyme studies

ETHYL ALCOHOL (70-100%)

Most rapid fixative; Contains alcohol, glacial HAc, and chloroform; Can also used as fixative and can dehydrate at the same time

Carnoy's fluid

Most rapid alcohol fixative; For urgent biopsies (Chromosomes, Lymph glands), brain for diagnosis of Rabies

Carnoy's fluid

- Fixative that is classified both as nuclear and histochemical fixative - Can also preserve mucopolysaccharide and nuclear proteins

NEWCOMER'S FLUID

Alcohol fixative for CT mucins and umbilical cord

ROSSMANN'S

TRUE or FALSE. Lower concentration of ethanol (70-80%) will cause TBC hemolysis and inadequately preserve leukocytes

TRUE

Alcohol fixative that preserves Nissl granules and cytoplasmic granules; Also permits good nuclear staining and differentiation

CARNOY'S

TRUE or FALSE. Carnoy's fluid causes considerable tissue shrinkage and is suitable only for small pieces of tissues due to slow penetration. It also dissolves fat, lipids, and myelin

TRUE

- Alcoholic fixative - Recommended for frozen sections and smears - Can produce fair results after conventional processing if fixation time is kept very short - Preserves nucleic acids but extracts lipids.

Clarke's solution

Fixative for EM that is not commonly used and quite expensive; It is also slow-acting fixative; Excellent stain for lipids in membranous structures and vesicles

OSMIUM TETROXIDE

Tissues fspx of OSMIUM TETROXIDE

Myeline Peripheral nerves Processing neurological tissues

Most common chrome osmium acetic acid fixative, and excellent for nuclear structures

Flemming's

- Known for cytoplasmic fixatives - Preserves cytoplasmic structures particularly the mitochondria - Composed of osmic acid and chromic acid

Flemming's without HAc

Fixation time for Flemming's solution

24-48 hours

Flemming's solution has a tendency to form artifact pigments, what is the remedy of it?

Washing the fixed tissue in running tap water for 24 hours before dehydration

Fixatives for Enzyme Histochemistry:

4% formaldehyde Formal saline

Fixative for electron histochemistry and electron immunocytochemistry

Karnovsky's paraformaldehyde-glutaraldehyde

What substance is used in post chromatization during secondary fixation occurs

2.5-3% potassium dichromate (chromate-containing) - Acts as a mordant for better staining

Placing an already fixed tissue to a second fixative to: - Improve demonstration of particular substance - Ensure complete hardening and - For special staining (not a mandatory step in tissue processing)

Secondary fixation

What fluids or solution that can be used during washing out process in order to remove excess fixative in tissue?

1. Tap Water (often used) - Excess chromates in tissues fixed and in Helly's, Zenker's and Flemming's - To remove excess 2. Alcoholic Iodine 50-70% alcohol

First step of tissue processing wherein the spx will be placed in 10% formalin for the first time?

Primary fixation

Microscopic study of NORMAL tissues

HISTOLOGY

4 Types of Tissues:

Connective tissues Epithelial tissue Muscle tissues Nervous tissue

Microscopic study of ABNORMAL tissues

HISTOPATH

3 Types of Specimen that is processed in Histopathology:

Specimen of cytology Autopsy specimen Biopsy specimen

Tissues specimen obtained from a patient (ALIVE) for the examination in the lab

Biopsy specimen

Most common type of biopsy:

Incisional Excisional Needle biopsy/ Aspiration/ Fine needle aspiration biopsy (FNAB)

Type of biopsy that are not usually carried out:

Core biopsy and Shave biopsy

Type of biopsy that involves the removal of a part of a mass or organ

Incisional biopsy

Type of biopsy that involves removal of ENTIRE mass or organ; Known as the most reliable and will give a greater chance to detect cancer

Excisional biopsy

Type of biopsy that uses needle and syringe to collect tissue spx

FNAB or Needle biopsy

Type of specimen that is obtained from a dead patient; Also called as necropsy

Autopsy specimen

Specimens obtained for studying cell biology; Example is PAP's smear

Cytology specimens

Small fragments are shave from a surface usually skin

Shave biopsy

- Kind of specimen that will not undergo tissue processing - It allows examination of protoplasmic activity (phagocytosis, motility) - Tissues are not permanent it cannot be kept for future purposes

Fresh specimen

- Kind of specimen that usually facilitate or examine in histopath lab - Specimen that are better and more effective - Can keep the specimen on slides for future references - Better and more effective

Processed tissues

- Type of cytology spx - Example is PAPANICOLAU STAIN - Used as a screening procedure for cervical cancer

Gynecologic specimens

- Cytology specimens such as sputum, CSF, gastric lavage, urine - Can be used to detect urothelial malignancies

Non-gynecological specimen collected

4 method of examination are:

Teasing (Dissociation, Squash Preparation (Crushing) Smear Preparation Frozen Section

Steps in Teasing/ dissociation:

1. Get the watch glass, place small pieces of tissues, then add NSS 2. Using a loop or needle aspirator, dissociate or separate. 3. Place the separated tissues on a slide and examine it under the microscope

Stains that can be used in fresh tissue specimen

Supravital dyes/stains

Type of microscope that can be used to detect movement and mitotic division

Phase Contrast Microscope

Steps in squash preparation:

1. On a slide, place the specimen 2. Get another slide 3. Compress the tissue in between two slides (slide-to-slide then compress)

4 types of smear preparation

1. Streaking 2. Spreading 3. Pull-apart 4. T ouch preparation

Most recommended method of examination if dealing with cells

Smear preparation

Manner used in Streaking preparation

Zigzag manner

Advantage of Spreading preparation

Preserve intercellular relationship

Types of smear preparation that is suited for viscous specimens

Pull-apart and Streaking

Smear preparation that also called Impression smear and Abraded cytology

Touch preparation

Recommended glass slides for Touch Preparation A. Frosted Glass Slide B. Polished Ends Glass Slide

B. Polished Ends Glass Slide

Method of examination for rapid diagnosis and done intraoperatively; Method used to demonstrate heat sensitive structures

Frozen section

Smear technique that allows slide to come in contact with freshly cut tissues (lymph node)

Touch Preparation/ Impression Smear/ Abdraded Cytology

Smear technique recommended for fresh sputum and bronchial aspirate, and thick mucoid secretions; It also maintains intercellular components

Spreading

Method used for demonstration of fats/ lipids, nervous tissues elemets, and enzymes

Frozen section

Apparatus used in Frozen Section:

Freezing Microtome and Cryostat/ Cold Microtome

- Rapid diagnosis during surgery - Diagnostic and research enzyme histochemistry - Demonstration of soluble substances like lipids and carbohydrates - Immunofluorescent and immunohistochemical staining - Special staining in neuropathology

Applications of Frozen Sections:

FREEZING AGENTS:

1. Liquid nitrogen 2. Isopentane cooled by liquid nitrogen 3. Carbon dioxide gas 4. Aerosol sprays

BENEFITS OF FIXATION/ EFFECTS OF FIXATION:

- Prevents autolysis, reduce risk of infection - Allows thin sectioning - Acts as mordant thus facilitating staining

Results from frozen section must be released by how many minutes? (TAT)

5-15 minutes

Most common freezing agent used

Carbon dioxide

Average temp of cryostat or comb microtome

-20 deg C

First and most important step; This is where you enter the details of patient in LOGBOOK

Numbering/ Accessioning

TRUE or FALSE. BRAIN tissues must be FIXED BEFORE GROSSING

TRUE

Before Fixation, large solid tissues should be...?

Opened or sliced thinly

Factors that can accelerate fixation:

1. Heat application = 37-56 deg C only 2. Size and thickness of tissue 3. Agitation - continuous mixing —> allows rapid entry of fixative to tissue

Request form must include...

- Name of the patient - Age - Attending doctor - Initial diagnosis - Type of specimen

Not computerized

NUMBERING

Computerized

ACCESSIONING

Size of a tissue cassette

2.5 x 4 cm

Depth of a tissue cassette

5mm

An automatic tissue processor that can do fixation, dehydration, clearing, and infiltration; Consists of 10 1L beakers that arranged in circular position

Autotech

Highest concertation of formalin

37 - 40% Formalin

Factors that can DECREASE the fixation time:

Increased heat Agitation Vacuum Microwave Small and thinner tissues specimen

Factors that ENHANCE fixation process:

Size and thickness Agitation Moderate heat (37 deg C)

TRUE or FALSE. Presence of mucus and blood in a specimen can be flush with NSS if there's too many

TRUE

1. Spx should be transferred to fixative immediately after surgery (< 1 hr.) 2. Fixative to specimen: 20:1 or 10:1 3. Anatomical barriers to fixation (should be removed) - fascia (covering of organ) - bones - feces - thick tissue 4. Large specimens must be sectioned or inflated with fixative or opened and cleaned to allow penetration - Lungs —> inflate/ sectioned - Intestines/gastrointestinal tract —> opened 5. Fixatives diluted and contaminated fixative - should be replaced to ensure effectiveness 6. Pinning of spx to corkboard or inserting a paper or gauze “wick” into tubular structures can improve fixation and reduce tissue distortion

Practical Considerations to Optimize Fixation of Tissue

May be more difficult to reverse and may also result in loss of immunohistochemical antigenicity

Prolonged fixation Increased fixation --> decreased immunohistochemical antigenicity

2 types of COMPOSITION for fixative:

Simple fixative Compound fixative

Composition of Saline:

Distilled water NaCl

Formaldehyde linked by 3C'

Glutaraldehyde

TRUE or FALSE. Methyl alcohol can cause BLINDNESS

TRUE

Microanatomical fixative should never contain ________ because it inhibits hematoxylin (for staining nucleus)

Osmium tetroxide

Preserving or demonstrating chemical substances in the tissue

Histochemical fixatives

Formaldehyde waste ways:

1. Recycle by distillation 2. Drain disposal 3. Disposal by a licensed waste howler 4. Detoxification by a commercial product

Mercurial reagents/ used to dezenkerize

- Releases mercury and must not go through drain

Disposal of mercury is expensive but it can replaced by ____

Zinc formalin or Glyoxal solutions

Fixative agent cannot be used in lipid fixation

ALCOHOL

Agents used in Lipid Fixation: (FB MAP D)

- Formaldehydes - Baker's formol-calcium -> for phospholipids - Mercuric chloride - Aldehydes - Potassium dichromate - lipid for cryostat - Digitonin -> cholesterol for ultra-structural demonstration

Fats (Frozen Section)

- Frozen section - Formalin - Potassium dichromate - Osmic acid - Formol calcium

Agents for Protein Fixation:

- Neutral Buffered Formalin - Formaldehyde Vapor

Agents for Carbohydrate Fixation:

- Alcoholic fixatives (glycogen fixation) - Rossman's fluid - Cold absolute alcohol

BRAIN must suspend WHOLE in ____ for 2-3 weeks to ensure hardening

10% buffered formalin

TRUE or FALSE. Formaldehyde should never be neutralized because it may cause violent explosion

TRUE

REMOVAL OF PARAFORMALDEHYDE

- Use methanol to prevent its decomposition to formic acid - Can filter paraformaldehyde

Color of formalin pigments

Brown or Black

Formalin pigments occur because of...?

Reaction of formic acid and hemoglobin (blood)

Formaldehyde (Formalin) recommended for ___

CNS

It fixes sputum since it coagulates mucus

Alcoholic formalin (Gendre's)

All mercuric chloride fixatives cause black precipitate except...? A. Zenker's fluid B. Zenker's formol C. Heidenhain's Susa D. B5

C. Heidenhain's Susa

TRUE or FALSE. Mercuric chloride is used for tissue photography

TRUE

What to use for removal of black deposits from Mercuric chloride?

Use saturated iodine in 96% alcohol

- Contains glacial acetic acid, mercuric chloride, potassium dichromate, sodium sulfate, and distilled water - Recommended for small pieces of liver, spleen, connective tissue fibers, nucleic components

Zenker's fluid

- Zenker's formol - Contains formaldehyde, mercuric chloride, potassium dichromate - Good for microanatomical fixative - Pituitary gland, bone marrow, blood-containing organs (spleen and liver)

Helly's fluid

- Recommended for tumor biopsies of the skin

Heidenhain's Susa

Fixative used for PRESERVATION OF BONE MARROW BIOPSIES

B5 fixative

Composition of Bouin's solution:

Picric acid saturated aqueous solution 40% Formaldehyde Glacial acetic acid

- Process of removing the excess fixative after fixation - To improve staining - To remove artifacts from the tissue

Washing out

- Less concentrated alcohol fixatives = less lysis - Used in small tissue fragments

Alcoholic fixatives used in concentration of 70-100%

In WASHING OUT: - Excess chromates in tissues fixed and in Helly's, Zenker's and Flemming's - To remove excess formalin and cosmic acid

Tap water (washing out)

In WASHING OUT: - To remove excess picric acid fixatives (Bouin’s solution)

50-70% alcohol (washing out)

In WASHING OUT: - To remove excess mercuric fixatives

Alcoholic iodine (washing out)

- Tissue in a fixated - 450 watts at 55 deg C for 1.5 minutes to 4 minutes - May create fumes

Microwave assisted fixation

ADVANTAGE OF FIXING TISSUES IN MICROWAVE:

- Tissues is heated through the block for a very short time; potentially allowing rapid study of cellular processes. - Can used in neurochemical substances (brain, acetylcholine) - For rapid fixation: routine surgical specimens - Immunohistochemistry and In-Situ hybridization

DISADVANTAGE OF FIXING TISSUES IN MICROWAVE:

- Only penetrates tissues to thickness of 10-15mm - No significant cross-linking of protein molecules, and subsequent chemical fixation may be needed - Viable spores and pathogens may remain in tissues processed with alcohol-based fixatives or microwave alone

Physical method that preserves tissue by rapid exposure of the spx to cold temperatures (-160 to -180 dec C)

Freeze drying/ Freeze substitution

3 types of Specimen

Biopsy Autopsy Cytology

Fixation + Addtl. Discussion Flashcards

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