HISTOPATH Flashcards by marisol encanto

Dolor means

Pain

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Site of collection for hormonal studies

Upper quadrant of vaginal wall

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Prosector of autopsy

Pathologist

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Cells with fried egg appearance

Parabasal cells

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Stain for copper pigments (recall)

Lindquist rhodamine

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Ratio of 10% formalin

1:9 (1 parts of stocksolution: 9 parts of water)

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Most common metallic fixative

Mercuric chloride

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Most rapid fixative

Carnoy’s

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Pathology means

Study of suffering/pain

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Tissues with intercalated disk

Cardiac muscle

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Flat, polyhedral cells with a pyknotic nucleus

Superficial cells

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Removes nicks

Honing

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Scott’s tap water is employed for

Blueing Agent

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Alkaline Fast green is useful as a stain for

Histones

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Fastest method of paraffin impregnation

Vacuum

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Ideal fixative to tissue ratio

20:1

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-Itis means

Inflammation

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Ab class used in IHC

IgG

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Both a nuclear and histochemical fixative

Newcomer’s

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Stain for glomerular basement membrane

PAS (Periodic Acid Schiff)

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Inventor of Sliding microtome

Adams

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Epon is employed as

Plastic embedding medium for Electron microscopy

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Non-appearance of organ

Agenesia

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Clearing agent that becomes milky

Xylene

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Routine fixative for exfoliative cytology

95% ethanol

Recommended fixative for cell detail in tissue photography

Mercuric chloride

Negri bodies are seen in which viral disease

Rabies

Presence of mononuclear cells are associated with which type of inflammation

Chronic inflammation

Most reliable feature of malignancy

Metastasis

Double embedding technique

1st: Celloidin ; 2nd: Paraffin

Tissue examination can be performed in _____ or ______

Fresh or Preserved tissues

Examined in its living state; Allows examination of protoplasmic activities;

Fresh tissues

What are the protoplasmic activities of fresh tissues?

Phagocytosis Pinocytosis Mitosis Motility (PPMM)

Tissues are not permanent: Fresh or Preserved ?

Fresh tissues

4 Methods of FRESH TISSUES examination

Teasing/ Dissociation Squash Preparation/Crushing Smear Preparation Frozen Section

Tissues is immersed in a watch glass with NSS; Dissected or separated using needle

Teasing/ Dissociation

In Teasing, tissues is immersed in a watch glass with _____

NSS

Microscope needed in Teasing method

Phase contrast/ Bright field

Stain used in Teasing method

Methylene Blue

Small tissues are placed in a slide; Tissues are compressed with another slide or coverslip

Squash Preparation/Crushing

In squash method, what is the size of tissue needed to be place in a slide

1mm or less

Stain used in squash method

Supravital stain

Cellular materials are spread lightly over a slide

Smear preparation

Method of fresh tissue examination: Useful for cytology

Smear Preparation

4 types of Smear Preparation

Streaking Spreading Pull- Apart Touch Preparation

Material is added using an applicator stick of loop in a direct or zigzag fashion

Streaking

Material is placed on a clean slide and spread into a moderately thick film by teasing with an applicator stick

Spreading

Advantage of spreading smear

Maintains intercellular relationship

Use of 2 slides in preparing the smear

Pull-Apart

Smear used for thick secretions

Pull-Apart

Touch preparation is A.K.A

Impression smear

Freshly cut piece of tissue in brought into contact and pressed on to the surface of a clean slide

Touch Preparation

Touch preparation is used for

Lymph nodes

The slide of touch preparation should be ____ and _____

sterile and with polished edges

For rapid diagnosis of fresh tissue examination

Frozen section

Time needed for: Rush Frozen Section

5-15 mins

Apparatus used in Frozen section

Cryostat/Cold microtome Freezing Microtome

Method of tissue examination: Used for demonstration of lipids, nervous tissue elements ,enzymes

Frozen section

Preserved tissue processing: Permanent or Not Permanent?

Permanent

Frozen section is done: Intraoperatively or Preoperative or Postoperative

Intraoperatively

First and most important step in histopathology

Numbering (Accessioning)

In numbering, the specimen number is preceded by either: S= A= C=

Surgical Autopsy Cytology

Example: S99-0345, what does it mean?

345th Surgical specimen in the year 1999

The MT will then write down the description for processing at the ________ back of the request or in-front of the request ?

back of the request

Use ___ in writing the description of the tissue specimen

Pencil

Specimen size for processing:

2 x 3 cm 3-5 mm thick

What can be added for small tissues

1% eosin

For electron microscopy, specimen size should be?

1 mm^3

Most crucial step in histopath

Fixation

Process of preserving cells and tissue constituents in a condition identical to that existing during life

Fixation

Fixation prevent ____

Autolysis

Self-destruction of tissues

Autolysis

3 Methods of Fixation

Heat fixation Perfusion Immersion

Fixation via blood flow

Perfusion

2 Mechanism of action in fixation

Additive Non-additive

The fixative becomes part of the tissue by formation of cross links/complexes

Additive

Stabilizes the tissue proteins

Additve

Ex: of additive fixation

Mercury Formalin Osmium tetroxide

Fixative not incorporated into the tissues

Non-additive

Alteration of tissue composition Additive or Non-additive?

Non-Additive

Stabilizes the tissue by removing water Additive or Non-additive?

Non-Additive

Ex of Non-additive fixation

Alcoholic fixative

Ex of mordant/accentuators in fixation

Osmium tetroxide Potassium dichromate

pH of fixation

6-8

Traditional temp for fixation

Room temp

Autotech temp for fixation

40Degree Celsius

EM and Histochem temp for fixation

0-4 Degree Celsius

Rapid fixation temp

60 Degree Celsius

Temp of fixation for tissues with TB

100 Degree Celsius

Thickness of fixation for EM

1mm ^3 or 1-2 mm^2

Thickness of fixation for LM

2x3 cm or 2cm^2

Tissues should not be more than ___ thick

4-5 mm

Thickness of edematous lung tissues

10-20 mm or 1-2cm

Suspended in whole and tie with string

Brain tissues

Brain tissues should be tie at the string of ___ (recall)

Circle of Willis

Fixative for brain tissues

10% buffered formalin

Brain tissues should be fixed for how many?

2-3 weeks

Ex of large solid tissues

Uterus

Large solid tissues should be ____ to allow the fixative to enter the tissue

Cut open

Osmolality of fixation

Slightly hypertonic solution aroung 400-450 mOsm

Ideal for immunolectron microscopy

0.25% glutaraldehyde

Time duration of primary fixation in buffered formalin

2-6 hours

Time duration of EM fixation

3 hours then placed in a holding buffer

Speed of fixation

<1hr or immediately

Why does fixation need to be done immediately

To prevent autolysis and putrefaction

Rate of penetration of formalin

1mm/hr

Volume or ratio of ideal fixation

10-20x of that tissue or 20:1 (fixative:tissue)

Volume for expensive fixative

5-10x reduced

Example of expensive fixative

Osmium tetroxide

Volume for museum preparation

atleast 50x of that tissue

Duration of tissue depends on

tissue structure

Duration of fixation of fibrous tissue

longer fixation time

Duration of fixation of small or loosely textured tissues

shorter fixation time

Duration of fixation can be hastened by:

Heat Agitation Vaccum Microwave "HAV Mi"

If autopsy materials are not anle to be fixed, these should be placed in:

Mortuary refrigeration at 4 Degree Celsius Undergo Arterial Embalming

Organs that need to be grossed after fixation

Brains and eyes

Example of hollow organs

Stomach and intestine

Organs that tend to float in fixation

Hollow organs and Air filled lungs

Remedy for hollow organs in fixation that tend to float

Put cotton inside

Remedy for air filled lungs that tend to float in fixation

Wrap in gauze

Lungs of pneumonia patient tend to float or sink during fixation?

Sink Due to combination of increased fluid content and consolidation in the lungs of pneumonia patients leads to a loss of buoyancy, causing them to sink during fixation.

What considereation should be done in Eyes specimen during fixation ?

Inject with formol alcohol

Example of hard tissues

Cervix,fibroids,hyperkeratotic skin, nails,teeth,bone

Consideration need to be done in hard tissues

Immersed the tissue in a softeners such as Perenyi's or Lendrum's before sectioning

Fixative types according to composition

Simple Compound

Type of fixative that contains 1 fixative

Simple fixative

Type of fixative that contains 2 or more fixative

Compound fixative

Ex. of simple fixative

Heat Aldehyde Metallic Fixative "HAM"

Fixatives according to action

Microanatomical Cytological

Fixatives that permits general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of tissues

Microanatomical

Fixative that preserve a specific part/ element of cells

Cytological

3 Types of cytological fixative

Nuclear Cytoplasmic Histochemical

Fixative that preserve nucleus and chromatin material and contains glacial acetic acid

Nuclear fixative

Purpose of glacial acetic acid

Increase affinity to nuclues

pH of nuclear fixative

4.6 or less

Fixative that preserves cytoplasm and organelles and no glacial acetic acid

Cytoplasmic

pH of cytoplasmic fixative

4.6 or more

Fixative that preserve chemical components

Histochemical

Example of Microanatomical fixative

Bouin's Brasil's 10% Formol Saline 10% Neutral Buffered Formalin Formol sublimate Zenker’s Heidenhain's Susa "BB FNF Z Heiden"

Example of Nuclear Fixative

Flemming's Carnoy's Bouin's Newcomer's Heidenhain's Susa "FC Ba Ni Heiden"

Example of Cytoplasmic Fixative

Flemmings without HOAC(Acetic acid) Formalin with post chroming Orth's Regaud's Kelly's "FFORK"

Example of Histochemical fixative

Absolute ethanol 10% Formol Saline Acetone Newcomer's "A FAN"

10% formol saline is what type of fixative

Microanatomical and histochemical

Heidenhain's Susa is what type of fixative

Microanatomical and Nuclear

Bouin's is what type of fixative

Microanatomical and Nuclear

Newcomer's is what type of fixative

Nuclear and histochemical

Example of aldehyde fixative

fixative with suffix -aldehyde And gendre's/alcoholic formalin/alcoholic bouin

Gas produced from oxidation of methanol

Formalin/Formaldehyde

% of stock/concentrated formalin

37-40%

Routine formalin

10% formalin

Actual concentration of 10% formalin in 37-40% stock solution

Formalin is usually buffered with

PO4 buffer to pH 7 - to prevent formalin pigments

How to retards decomposition to formic acid

add 10% methanol

Formalin added with 10% methanol is no longer suitable with ____

Electron microscopy

Usual fixation time of formalin

12-24 hours

The advantage of this fixative is easy to prepare,cheap,compative with many stains

Formalin

The advantage of this fixative is irritating to nose and eyes and can cause allergic dermatitis

Formalin

Use what type of gloves in preparing formalin to avoid allergic dermatitis

rubber gloves

Prolonged storage of formalin can lead to what formation

White precipitate (Paraformaldehyde)

What is the remedy for white precipitate formation in formalin?

Filtration Add 10% methanol to dissolve

What color of precipitate form in blood contaning tissue when formalin is used

Brown or black crystalline precipitate

Brown or black crystalline precipitate is AKA

Acid Formaldehyde Hematein

Example of blood containing tissues that cause brown or black precipitate

Spleen

What is the reason of formation of brown/black precipitate

Due to the action of formic acid with blood

What is the remedy for removal of formalin pigments

1) Kardasewitsch's method (70% ethanol & 28% ammonium water) 2) Lilie's method (hydrogen peroxide and 28% ammonium water) 3) Picric acid method (saturated alcoholic picric acid) 4) 1% KOH in 80% alcohol

10% formol saline is diluted with

NaCl

Fixation of CNS tissues and General Post Mortem Tissues for Histochemical examination

10% formol saline

Ideal for silver impregnation

10% formol saline

Best fixative for Iron containing pigments and elatic fibers

10% neutral buffered formalin /PO4 buffered formalin

pH of PO4 buffered formalin

Recommended routine fixative but longer to prepare

10% Neutral Buffered Formalin / PO4 Buffered Formalin

Aldehyde fixative that contains Mercuric chloride

Formal Corrosive/ Formol Saline

Excellent for stains like Silver Reticulin Method

Formal Corrosive/ Formol Saline

Aldehyde fixative with no washing out and fixes lipids

Formal Corrosive/ Formol Saline

Has 95% ethyl alcohol with picric acid and glacial acetic acid

Gendre's / Alcoholic formalin/ Alcoholic Bouin

AKA as Alcoholic Bouin

Gendre's

Good for glycogen and microincineration technique

Gendre's

Fixes Sputum

Gendre's

Contains 2 formalin residues linked by 3 carbon chains and acts similarly to formalin

Glutaraldehyde

% of Glutaraldehyde in small and large tissues

2.5% = small tissues 4% = Large tissues

Recommended for enzyme histochem and fixative for EM

Glutaraldehyde

Primary Fixative for TEM (Tranmission Electron Microscopy)

Glutaraldehyde

Better preservation of cellular and fluid proteins Less irritating but expensive

Glutaraldehyde

Glutaraldehyde specimen vial should be ___

refrigerated

Polymer formalin in white powrder forn

Paraformaldehyde

Concentration of Paraformaldehyde

Use for thin and ultrathin sections for plastic embedding and EM

Paraformaldehyde

3 Types of Metallic Fixative

Mercuric chrloride Chromate Lead

Most common metallic fixative

Mercuric fixative

Concentration of metallic fixative

5-7%

What type of fixative according to component is Mercuric chloride ? Simple or Compound

Compound fixative

Fixative that produce black granular deposit

Mercuric chloride

All mercuric chloride produce black granular deposit except

Heidenhain's susa

Removal of black granular deposit can be done by

Dezenkerization (Washing out)

Dezekerization solution contains

Saturated iodine solution of 96% alcohol & 5% sodium thiosulfate

Metallic fixatives that penetrate and harden tissue rapidly

Mercuric chloride

Routine fixative of choice for preservation of cell detail in tissue photography

Mercuric chloride

Fixation of hematophoetic and reticuloendothelial tissues

Mercuric chloride

Example of Mercuric chloride

Zenker's Zenker's formol/ Helly's/Kelly's Heidenhain Susa B5 Fixative "ZZ H5"

Mercuric chloride that contains glacial acetic acid

Zenker's

Recommended for small pieces of liver,spleen,CT fibers and nuclei

Zenker's

Recommended for trichrome staining

Zenker's

Contains Potassium Chromate and formalin

Zenker's formol/Helly's/Kelly's

Excellent for microanatomical fixative of pituitary gland,BM and blood containing organ

Zenker's formol/Helly's/Kelly's

Preserves cytoplasmic granules and produce brown pigments

Zenker's formol/Helly's/Kelly's

what is the remedy of brown pigments due to kelly's

Remove using picric acid or NaOH(sodium hydroxide)

It composed of TCA,Glacial cetic acid and formalin

Heidenhain Susa

Recommended for skin tumor biopsies

Heidenhain Susa

Has anhydrous Na acetate

B5 Fixative

For bone marrow biopsies and lymph nodes

B5 Fixative

3 Example of Chromate Fixatives

Potassium Dichromate Regaud's /Muller's Orth's Fluid "PRO"

Concentration of chromate fixative

1-2% aqueous solution

It is a strong oxidizing agents and should not be combined with reducing agents

Chromate fixative

It preserved carbohydrates

Chromate fixative

It contains 3% aqueous solution

Potassium Dichromate

Preserves lipids and mitochondria at pH 4.5-5.2 cytoplasm,chromatin and chromosomes are fixed

Potassium Dichromate

Regaud's is AKA

Muller's

Fixed chromatin,mitochondria,mitotic figures ,golgi bodies,rbc, and colloid containing tissues

Regaud's

For early degenerative process and tissue necrosis

Orth's fluid

Demonstration of Rickettsia

Orth's fluid

Preserves myelin

Orth's fluid

Contains 4% aqueous solution and recommended for Acid mucopolysaccharide

Lead Fixatives

Fixes mucin also

Lead Fixatives

Usually used in a strong or saturated solutions

Picric acid

Chemically known as 2,4,6 Trinitophenol

Picric acid

Can be used as stain (yellow) and small tissue fragments can be seen

Picric acid

Explosive when dry

Picric acid

Remedy for yellow stain cause by picric acid

Saturated solution of Lithium Carbonate 70% ethanol 5% Sodium Thiosulfate

Excellent for glycogen

Picric acid

Example of Picric Acid

Bouin's Brasil's Hollande's solution "BB Hollande"

Recommended for embryonic and pituitary biopsies

Bouin's

Bouin's are stained using

Masson's trichrome stain

Not for kidney fixation

Bouin's

Has TCA ,better and less messy than Bouin's

Brasil's Alcohic Picroformol fixative

For GI tract samples and endocrine tissues

Hollande's solution

Less lysis than Bouin's

Hollande's solution

Decalcifying property

Hollande's solution

Glacial acetic acid is what type of fixative? Simple or Compound?

Compound

Glacial acetic acid solidies at

17 Degree Celsius

Not for cytoplasmic fixation

Glacial acetic acid

What will happen if Glacial acetic acid is used for cytoplasmic fixation

Cell swelling

Example of Alcoholic Fixatives

Newcomer's Methanol Isopropyl Carnoy's Ethanol "New MICE"

Rapidly denatures and precipitates protein

Alcoholic fixatives

Alcoholic fixatives acts as both

Fixative and Dehydrating agent

Use for glycogen fixation

Gendre's Picric Alcohol fixative

Alcohol fixatives preserves: Nuclear stain or cytoplasmic stain?

Nuclear stain

Alcoholic fixatives dissolves

Fats and Lipids

Methanol is AKA

Wood Alcohol

Concentration of Wood Alcohol

100%

Alcohol fixatives used for blood smear, bone marrow smear, wet and dry smear

Methanol

Isopropyl Alcohol is AKA

Rubbing Alcohol

Concentration of Rubbing Alcohol

95%

Alcohol fixatives that fixes touch preparation and for wright giemsa

Isopropyl Alcohol

Ethanol is AKA

Grain Alcohol

Concentration of Grain alcohol

70-100%

All alcohol fixative is excellent for glycogen fixation except

Ethanol

Fixative useful for PCR

Ethanol

Most Rapid fixative

Carnoy's fixative

Fixation time of Carnoy's fixative

1-3 hours

Fixative that contains Absolute alcohol, Chloroform,Glacial acetic acid

Carnoy's fixative

Fixes Nissl granules and Cytoplasmic granules

Carnoy's fixative

Recommended for mucopolysaccharide and nuclear proteins

Newcomer's

Acts as both nuclear and biochemical fixative

Newcomer's

Osmium Tetroxide is AKA

Osmic Acid

Produce pale yellow powder that dissolves in water (6% at 20 Degree Celsius)

Osmium Tetroxide

Fixes and stains unconjugated fats which appear Black

Osmium Tetroxide

Secondary fixative for EM after aldehydes

Osmium Tetroxide

Preserves mitochondria and Golgi bodies

Osmium Tetroxide

Osmium Tetroxide, Regaud, Potassium Dichromate all fixes

Mitochondria

Regaud's and Osmium tetroxide both fixes

Golgi bodies

It is expensive, cause Corneal blindness and inhibit hematoxylin

Osmium tetroxide

Osmium tetroxide ihibits

Hematoxylin

Fixative that contains Osmium tetroxide

Flemming's

Most common chrome-osmium acid fixative

Flemming's

Excellent fixative for nuclear strctures Flemming's or Flemming's w/o Acetic acid?

Flemming's

For cytoplasmic structures especially mitochondria Flemming's or Flemming's w/o Acetic acid?

Flemming's w/o Acetic acid

Weak Decalcifying agent

TCA

Used at Ice cold temperature from -5 to 4 Degree Celsius

Acetone

Acetone is used at Ice cold temperature from

-5 to 4 Degree Celsius

For diffusible enzymes such as Phosphatases and Lipases

Acetone

For fixing brain tissues:Rabies

Acetone

Thermal coagulation of tissues proteins

Heat Fixation

For frozen tissue section and preparation of bacteriologic smears

Heat Fixation

Physical agent similar to heat(Oven)

Microwave technique

Increases movement of molecules and accelerate fixation,staining,decalcification,EM and immunohistochemistry

Microwave technique

Optimum temp for Microwave technique

45-55 Degree Celsius

Microwave technique can penetrate tissues with thickness of

10-15 mm

4% formalin ,Formol Saline, Acetone,Formalin for cryostat sections are all fixatives for

Enzymes Histochemistry

What are the fixatives for EM

Glutaraldehyde Paraformaldehyde Osmium tetroxide

Pallde's Millonig's, S.collidine are all contains

Osmium tetroxide

Optimum temp for EM fixation

4 Degree Celsius

Best Fixative for Electron Histochemistry and Electron Immunocytochemistry

Karnovsky's

Karnovsky's contains

Paraformaldehyde and Glutaraldehyde

Mixture of methanol and PEG; Cost effective alternative to formalin; Recover RNA,DNA, proteins for molecular analysis

UMFIX

Big size and Thicker Retarded/ Enhaced?

Retarded

Presenced of Mucus Retarded/ Enhaced?

Retarded

Presence of Pseudomucinous cyst of ovary Retarded/ Enhaced?

Retarded

Remedy for the presence of Mucus

Wash with NSS

Presence of Fat Retarded/ Enhaced?

Retarded

Presence of Blood Retarded / Enhaced?

Retarded

Remedy for the presence of blood

Wash with NSS

Cold temperature Retarded / Enhaced?

Retarded

Thinner and small size Retarded / Enhaced?

Enhaced

Agitation Retarded / Enhaced?

Enhaced

Agitation reduced processing time by how many percent?

30%

Heat Retarded/ Enhanced?

Enhanced

Placing an already fixed tissue into another fixative to improve demonstration of substances and ensure further and complete hardening

Secondary Fixation

Secondary fixation that uses 2.5-3% Potassium Dichromate for 1 day

Post-Chromatization

Mordant used in Post-Chromatization for 1 day

2.5-3% Potassium Dichromate

Potassium Dichromate serves as what in post-chromatization

Mordant

Post-Chromatization is AKA

Post-Mordanting

Removal of excess fixative to improve staining and remove artifacts

Washing-out

Tap water can wash out?

Chromates, Formalin,Osmium tetroxide

50-70% alcohol can wash out

Picric Acid

Alcoholic iodine can wash out

Mercuric chloride

Transport medium for unfixed tissues (renal,skin,oral mucosa biopsies); refrigerated; not a fixative

Michel's solution

Removal of calcium or lime salts from bones or calcified tissues following fixation

Decalcification

Decalcification has ________ concentrated acid solutions decalcify more rapidly but may destroy tissue less or more

Ratio and Room of decalcification

20:1 Room temp

Heat and Agitation _____ decalcification hastens/slows

hastens

Suspending the tissue in the decalcifying fluid hastens decalcification using what type of container Metal / Glass

Glass due to acid agents

Duration of decalcification

1-2 days

Types of decalcifying agents

Acids Chelating Agents Ion Exchange Resins Electrophoresis "An ICE"

Widely used for routine decalcification

Acid Decalcifying agents

Types of Acid Decalcifying Agents

Nitric Acid HCl Formic Acid TCA Sulfurous Acid Chromic Acid

Most common and rapid decalcifying agent

Nitric Acid

% of Nitric Acid

5-10%

Nitric Acid can be removed by

70% Alcohol

Remedy for nitric acid causing yellow color

Add 5% sodium thiosulfate / Urea crystals

This decalcifying agent imparts a yellow color due to formation of nitrous acid

Nitric Acid

Contains formalin and less tissue destruction; Rapid for urgent biopsies

Formol Nitric Acid

Decalcifies and softens tissues at the same time

Perenyi's Fluid

No maceration occurs because of chromic acid and alcohol

Perenyi's fluid