Mohd Farid Laporan Latihan Industri Sarjana Muda Sains Bioperubatan UKM Tahun 3 Sesi 2007/2008-Hospital Selayang Part X-Histopathology And Cytology Unit

Histopathology and Cytology Unit

HISTPATHOLOGY LABORATORY

In the histopathology laboratory, the surgical or biopsy specimen (organ or tissue) and request vouchers are given to the pathologist or Medical Laboratory Technologist (MLT) for processing and diagnosing. Specimens should routinely be submitted in 10% buffered formalin. The specimen must not be allowed to autolysis or decompose. Handling of tissue specimen in histopathology gives a guideline for tissue histology examination. Tissue is prepared by performing the following outlined steps 

• Grossing
• Decalcification
• Processing tissue
• Embedding/ Blocking
• Slide mounting
• Sectioning with microtome
• Staining
• Cover slipping

PINCIPLE OF TEST METHOD

No	Test method	Principle of test method	Equipment used
1	Grossing	To obtain the tissue specimen in small piece for tissue histology examination	Shandon gross lab senior
2	Decalcification	Bone and other calcified materials must be fixed before placing them in TBD solution
3	Processing tissue	To embed the tissue in solid media firm enough to support the tissue and give it sufficient rigidity to enable this section to be cut and yet soft enough not to damage knife or tissue.	Shandon PC 1172E9803
4	Blocking (embedding)	Block of paraffin with the tissue in the center are prepared to facilitate subsequent sectioning on a microtome	LEICA EG1160
5	Sectioning	The paraffin embedded tissue in sectioned on rotary microtome to get a thin slice and attached to the slide so that microscopic examination can be taken place	Microtome model: LEICA RM2135
6	Slide mounting	The mounting of tissue sectioned is facilitated by placing the ribbon (2-3 proper sections) on the surface of the water heated to approximately 40-45oC. The ribbon is drawn across the water’s surface in such a manner as to cause stretching of the ribbon. This action will remove all folds or wrinkles that have been caused by sectioning.	Waterbath Electrothermal (10506322-3)
7	H&E Staining	To make the tissue structure visible when viewed through microscope	H&E Stainer- Shandon Varistain XY (XY1048E9803)
8	Frozen Section	The purpose of frozen section is to get a rapid section for urgent diagnosis during operation or to examine the sections for a substance or structure that will be destroyed by preparing sections in the routine way	Cryostst (Freezing Microtome) Model-LEICA CM1850

Processing Tissue

Table below shows Routine Processing tissue method for routine H&E biopsy and autopsy specimen.

No	Reagent	process	Routine H&E (Hour)	Autopsy specimen (Hour)
1	80% Alcohol	Dehydration	0.5	1
2	95% Alcohol	Dehydration	1	23
3	95% Alcohol	Dehydration	1	8
4	Absolute alcohol I	Dehydration	1	8
5	Absolute alcohol II	Dehydration	1	3
6	Absolute alcohol III	Dehydration	2	4
7	Xylene Substitute I	Clearing	1	2
8	Xylene Substitute II	Clearing	1.5	1.3
9	Xylene Substitute III	Clearing	1.45	1.3
10	Xylene Substitute IV	Clearing	1.45	2
11	Paraffin wax I	Infiltration	0.5	3
12	Paraffin wax II	Infiltration	1	3
13	Paraffin wax III	Infiltration	1	3
14	Paraffin wax IV	Infiltration	0.5	3

Embedding

The process whereby tissue is encapsulated or included in a paraffin mold to facilitate sectioning on a microtome is called embedding. The tissue are fixed require some medium to furnish stability and hold cellular structures in proper relation to each other before they can sectioned.

The mold or ‘boat’ is filled with ho liquid paraffin and the infiltrated tissue is placed so that it is at the bottom of the mold touching the copper plate. The molds may be of several varieties: two L-shaped lead with copper plate, paper boats, or plastic boats. The latter two generally are preferred because they are faster and more easily handled in a busy laboratory. Wax impregnated tissue is processed to paraffin blocks using tissue embedding system. The system is complete and it includes paraffin tank, hot plate, warming tank forceps warmer and cold plate.

Method

The tissue is blocked in the following manner:

• The mold is filled with melted paraffin.
• The tissue is transferred from the infiltration bath of melted paraffin to the mold with forceps.
• Plastic cassette is dropped into mold and cool by placing the mold on the cold plate

When the blocks have hardened, they are trimmed and placed in labeled boxes. The blocks are kept in the refrigerator until sectioned.

Sectioning

Tissue embedded in paraffin is sectioned on the rotary microtome. Since paraffin is relatively soft material, a plastic cassette holder is attached to it. The plastic cassette is then inserted into the tissue holding vise of the microtome and is secured in place.

The microtome knife is inserted into the knife-holding vise and is brought as close to the tissue block as possible. It is usual to trim the block of paraffin as close to the tissue as possible prior to sectioning. The sectioning should be done at different part of edge from the trimming. This procedure removes excess paraffin and will keep the knife sharp longer.

The indicator for depth should be adjusted accordingly. The beginner will find that sectioning at 10µ is easier than 4µ. With experience, small depth may be obtained. It must be aware of the danger caused by the exposed microtome knife. The knife or the tissue block must never be touched by the fingers. Serious injuries will occur if individuals become absent-minded or forget this basic rule.

When the tissue block and microtome knife are parallel and are close to each other, the microtome handle is turned until the knife shaves the excess paraffin. Be continued until the knife comes in contact with the tissue. If the knife is sharp and angled correctly, a ribbon will form. This ribbon represents the consecutive sections of the tissue in the paraffin block. The size of the tissue is observed within each paraffin frame. When they are the size desired (usually the same size as the overall tissue), this tissue section is ready for mounting on a slide.

Method

• For routine examination, the sections are cut with a rotary or sliding microtome at 5-7 micron thickness
• The sections are floated on the surface of distilled water (about 38oC). Bubbles are avoided and the sections are stretched to  remove wrinkles.

Problems

Section fail to form ribbon

Caused by

• Paraffin is too hard- re-embed in softer paraffin
• Knife is dull
• Knife is too cold
• Section should be made thinner

Compressed ribbon

Caused by

• Paraffin is too warm
• Knife is too warm
• Knife angle is wrong – readjust
• Knife is dull
• Recheck all screws and retighten

Cracked or torn ribbon

Caused by

• Knife blade has nicks
• Knife is dirty
• Paraffin or tissue has dirt in it
• Crumbling tissue or tissue falls out of block
• Embedding paraffin is too hot
• Dehydration, clearing and/or infiltration was incomplete

Mounting Tissue

Precleaned microslides are coated thinly with Mayer’s albumin. The mounting of tissue sectioned is facilitated by placing the ribbon (2-3 proper sections) on the surface of the water heated to approximately 40 to 45oC. The ribbon is drawn across the water’s surface in such a manner as to cause stretching of the ribbon. This action will remove all folds or wrinkles that have been caused by sectioning. Then, using the blunt part of a teasing needle, each section is cut away from the others.

The albumin-coated microslide is placed in the water directly underneath one of the sections. The microslide then gently rose to lift that portion above the water’s surface. The microslide with its section of tissue is drained to remove all water by allowing it to stand at room temperature in vertical position.

When the tissue section appears dry, it is best to press it gently to the slide. This force out all water or air that has collected between the tissue and the slide. The slide is placed in a dry air incubator set at temperature just below the melting point of the paraffin (40-45oC) for 45 minutes to 3 hours.

The heat causes the albumin to coagulate thereby forming an adhesive bond between the tissue and the microslide. The temperature of the incubator should not cause the paraffin to melt; a temperature that high destroys many tissues.

Staining

After processing, tissues do not retain enough of their natural color to make their structures visible when viewed through a microscope. If therefore becomes expedient to add colors to them by staining them with proper dye.

A stain defined as any substance or solution which will cause tissue differentiation. A stain commonly is thought of as dye-solution that imparts color. The dyes imparting color to tissue are used most commonly in routine laboratories and are divided into two groups; natural dyes and synthetic dyes.

Natural dyes derived from living organic material. Examples of such dyes are hematoxylin, an extract of the logwood tree, Hematoxylon campechianum. Hematoxylin is itself, an unsuitable dye but when oxidized it is converted into the active staining substance hematein. Nuclear structures have a great affinity to hematein, since the nucleus is acid and hematein is basic, thereby making hematein an excellent nuclear differentiating dye. Hematein stains nuclear material in variable shades of blue.

Synthetic dyes. The most commonly used synthetic dye is eosin Y.Eosin Y has an affinity for the cytoplasm of the cell and stains these structure in  variety of red hues (pink to deep red)

Method

No	Reagent	Appropriate time
1	Xylene Substitute I	4 min
2	Xylene Substitute II	5 min
3	Absolute alcohol 100%	1 min
4	Absolute alcohol 100%	1 min
5	95% alcohol	1 min
6	70% alcohol	1 min
7	Wash in running water	1 min
8	Hematoxylene	8 min
9	Wash in running water	30 sec
10	1% alcohol acid	10 sec
11	Wash in running water	1 min
12	Bluing reagent	1 min
13	Wash in running water	1 min
14	Eosin	5 sec
15	Wash in running water	50 sec
16	95% alcohol	1 min
17	70% alcohol	1 min
18	Absolute alcohol I,100%	1 min
19	Absolute alcohol II, 100%	1 min
20	Absolute alcohol III, 100%	1 min
21	Xylene substitude I	2 min
22	Xylene substitude II	2 min
23	Xylene substitude III	2 min

Result Of Attaining

• The nuclear structures retain the basic dye of hematoxylin and remain blue.
• The cytoplasm stains red with the eosin
• Connective tissue fibers stain red

Coverslipping

Stained tissue must be preserved so that it can be studied microscopically and filed for future reference. Coverslipping with DPX is used to accomplish this preservation.

The coverslip used depend upon the size of the tissue. Routinely, coverslips measuring 22 by 22mm and 22 by 45mm may be employed

Method

The tissue must not be allowed to dry prior to coverslipping. Once tissue dries,it becomes cloudy. This cloudiness prevents differentiation, and thus the tissue is of no values.

Work should be done in an area free from lint and dust. All coverslips are precleaned before mounting.the tissue-coated slide to be coverslipped is removed and wiped clean with a lint-free cloth. Rinsed repeatedly with xylene to stop dehydration. It is placed back in xylene until ready to coverslip. Enough DPX is placed on the edge of a cover slip so that it runs parallel to the edge. The precleaned tissue slide is removed;excess xylene is removed and it is positioned so that the long axis is parallel to the edge of coverslip containing the DPX. The slide is gently lowered until it touches the DPX. Capillary attraction will cause the DPX to flow upward carrying the coverslip long with it.

The tissue slide is placed with its coverslip on edge and allowed it to drain. The slide is examined macroscopically and microscopically for the presence of the air bubbles. If they are present, they must be removed. If they cannot be removed by gentle pressing, the coverslip must be removed and the coverslipping process repeated. The slide is then labeled for identification and for microscopic diagnosis.

SPECIAL STAIN IN HISTOPATHOLOGY

NO	SPECIAL STAIN	PURPOSE/PRINCIPLE/RESULTS
1.	PAS [PERIODIC-ACID SCHIFF]	-Carbohydrate -Result Glycogen-Magenta Nuclei-Blue
2.	PAS-D[PERIODIC-ACID SCHIFF-DIASTASES]	-Saliva contains enzyme diastases -Glycogen present -Result Glycogen-Magenta Nuclei-Blue
3.	ZN [ZIEHL NEELSEN]	-Acid Fast Bacili (AFB)/TB/Leprae -Results Acid Fast Bacili-Red Background-Pale Blue
4.	MT [MASSON TRICHROME]	-Mussle/Collagen -Results Cytoplasm Neuroglia Fibre & Musle-Red Nuclei-Blue to Black Collagen & Mucus-Blue/Green
5.	ORCEIN SHIKATA	-To stain liver cell that have Hepatitis B -Results Hepatitis B Positive-Brown to Black Background-Pale Brown
6.	MUCICARMINE	-The aluminium salts in the solution from chelate compound with carmine, thus conferring a next positive charge on a molecule consequent binding to the tissue polyanions -Results Mucins-Red Nuclei-Blue Background-Yellow
7.	RETICULUM [SILVER IMPREGNATION]	-Reticulum -Results Reticular & Nervous Fibre-Black Connective Tissue-Tobacco Brown Collagen-Gold Yellow
8.	PERL’S PRUSSIAN BLUE	-Ferric Iron -Results Ferric Iron-Blue Nuclei-Red
9.	WADE-FITE	-Acid Fast Bacili (AFB)/TB/Leprae -Results Acid Fast Bacili-Red Background-Pale Blue
10.	CONGO RED	-Amyloid (Glycoprotein) -Results Amyloid-Pale Pink to Red Nuclei-Blue
11.	OIL RED O	-Lipid which is fat soluble die -Results Fat-Orange to Bright Red Nuclei-Blue
12.	COPPER-RHODANINE [RUBEANIC ACID]	-To detect Cuprum (Copper) -Results Copper deposits-Brigt Red to Orange Nuclei-Blue
13.	GMS [GROCOTT GOMORI METHANAMINE SILVER]	-Fungi -Results Fungal Hyphae & Yeast Body-Black Bacground-Pale Green
14.	GIEMSA	-Protozoans/Microoganisms (Helicobacter) -Results Protozoans-Blue Background-Pink to Pale Blue
15.	PTAH [PHOSPHOTUNGSTIC ACID OF HEMATOXYLIN]	-Musle -Results Musle striations, Neugrolial Fibre, Fibrin, Amoeba-Dark Blue Nuclei, Red Blood Cells, Cilia-Blue Myelin-Lighter Blue Collagen, Osteoid, Cartilage, Elastic Fibres-Deep Brownish Colour Cytoplasms-Pale Pinkish Brown
16.	AB/PAS [ALCIAN BLUE-PERIODIC ACID SCHIFF]	-Acid Mucopolysaccharides -Results PAS Positive substances(Neutral Polysaccharides)-Magenta Acid Mucopolysaccharides-Blue Nuclei-Blue
17.	MF [MASSON FONTANA]	-Melanin -Results Melanin, Argentaffin, Chromatin, and some Lipofuscin-Black Nuclei-Red
18.	VAN GIESON	-Collagen -Results Collagen Fibres-Red Muscle-Yellow Cornified Epithelium-Yellow Nuclei-Blue Black Cytoplasm-Yellow
19.	VERHOEFF’S ELASTIC STAIN	-Elastic tissues -Results Elastic fibres-Blue Black to Black Nuclei-Blue to Black Collagen-Red Other Tissue Elements-Yellow
20.	GRIMELIUS	-Argirophil Islet Cells -Results Argirophil Islet Cells(+ve)-Brown to Black Argirophil Islet Cells(-ve)

LIVER SPECIAL STAIN

• PAS
• PAS-D
• RETICULUM
• GIEMSA
• MUCICARMINE
• ZN [ZIEHL NEELSEN]
• ORCEIN SHIKATA
• PERL’S PRUSSIAN BLUE
• MF [MASSON FONTANA]

EXAMPLE OF SPECIAL STAIN PRINCIPLE

IRON - PRUSSIAN BLUE REACTION - MALLORY'S METHOD

PURPOSE: To demonstrate ferric iron in tissue sections. Small amounts of iron are found normally in spleen and bone marrow. Excessive amounts are present in hemochromatosis, with deposits found in the liver and pancreas, hemosiderosis, with deposits in the liver, spleen, and lymph nodes.

PRINCIPLE: The reaction occurs with the treatment of sections in acid solutions of ferrocyanides. Any ferric ion (+3) in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called 'Prussian blue" or ferric ferrocyanide.

CONTROL: A known positive control tissue.

FIXATIVE: 10% formalin

TECHNIQUE: Cut paraffin sections 4μ.

EQUIPMENT: Microwave oven, acid-cleaned glassware, non-metalic forceps.

REAGENTS:

5% Potassium Ferrocyanide:

Potassium ferrocyanide 25.0 gm

Distilled water 500.0 ml

Mix well, pour into an acid-cleaned

brown bottle. Stable for 6 months.

CAUTION: Low toxicity if not heated.

Nuclear-fast Red:

5% Hydrochloric Acid:

Hydrochloric acid, conc. 25.0 ml

Distilled water 475.0 ml

Mix well, pour into brown bottle, stable for 6 months.

CAUTION: Corrosive, avoid contact and

inhalation.

Working Solution:

5% potassium ferrocyanide 25.0 ml

5% hydrochloric acid 25.0 ml

Make fresh, discard after use.

CAUTION: Avoid contact and inhalation.

SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation. Potassium ferrocyanide; Low toxicity as long as it is not heated, it will release cyanide gas. Hydrochloric acid; target organ effects on reproductive system and fetal tissue. Irritant to skin eyes and respiratory sytem.

PROCEDURE:

• Deparaffinize and hydrate to distilled water.
• *Working solution, * microwave, 30 seconds. Allow slides to stand in
• solution for 5 minutes, in the fume hood.
• Rinse in distilled water.
• Nuclear-fast red, 5 minutes.
• Wash in tap water.
• Dehydrate, clear, and coverslip.
• *Conventional method: room temperature for 30 minutes.

RESULTS:

Iron (hemosiderin) blue

Nuclei red

Background pink

PAS - McMANNUS' PERIODIC ACID SCHIFF'S – GLYCOGEN

PURPOSE: Glycogen is present in skin, liver, parathyroid glands and skeletal and cardiac muscle. The PAS stain is used for demonstration of basement membranes, fungus secreting adenocarcinoma from undifferentiated squamous cell carcinoma, and mucosubstances secreted from the epithelia of various organs. A routine stain for liver and kidney biopsies.

PRINCIPLE: The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid which form the magenta color.

CONTROL: For staining fungus; use a known positive such as those used for the GMS. Use skin, aorta or normal liver for positive PAS staining.

FIXATIVE: Any well fixed tissue.

TECHNIQUE: Cut paraffin sections 4-5 m( 3m for kidney biopsies).

EQUIPMENT: Rinse glassware in DI water. Coplin jar, microwave oven.

REAGENTS:

0.5% Periodic Acid:

Periodic acid 0.5 gm

Distilled water 100.0 ml

Mix well, label with date and

initial. Stable for 1 year.

Caution: Avoid contact and inhalation.

Hematoxylin, GILL-3 Purchased

through Baxter.

PAS STAIN

Lillie's Cold Schiff's Reagent:

Basic fuchsin 10.0 gm

Sodium metabisulfite 18.0 gm

Distilled water 1000 0 ml

Hydrochloric acid 10.0 ml

Stir solution for 2 hours, set in a

dark cool place overnight. The

solution is now a clear light brown

to yellow color. Add:

Activated charcoal 500.0 gm

(or two heaping spoons)

Stir. Filter through Whatman #2

filter paper into a 1000 ml

graduated cylinder. Change the

filter paper often. Restore volume

to 1000 ml with distilled water.

Store in Refrigerator, solution is

stable for 6 months.

CAUTION: Carcinogen, corrosive.

SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation. Hydrochloric acid: strong irritant to skin, eyes and respiratory system. Target organ effects via inhalation on skin, respiratory, reproductive and fetal systems. Corrosive.

Schiff’s Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is a known carcinogen. Wear gloves, goggles, particle mask and lab coat, while preparing solution. Work under the hood, keep hot, uncapped, solutions under the hood.

PAS STAIN

PROCEDURE:

• Deparaffinize and hydrate to distilled water.
• Place slides into 0.5% Periodic acid for 5 minutes.
• Rinse in distilled water.
• *Schiff's Reagent, microwave HIGH power, for 45 - 60 seconds, until deep magenta.
• Wash in running tap water for 5 minutes.
• Counterstain in hematoxylin for 3 minutes.
• Wash in tap water, blue hematoxylin, rinse in distilled water.
• Dehydrate in alcohol, clear, and coverslip.
• *Conventional method: Schiff's Reagent, room temperature for 30 minutes.

RESULTS:

Glycogen, fungus: magenta

Nuclei : blue

NOTES:

• To check stain, pour 10 ml of formaldehyde in cylinder, add a few drops of Schiff's, it should turn red-purple immediately.
• Discard Schiff's if it turns pink while sitting in the refrigerator.
• The white precipitate at the bottom of the Schiff's can be redissolved if desired by gently warming and stirring the solution.

RETIC - GORDON AND SWEET'S METHOD - RETICULAR FIBERS

PURPOSE: A silver impregnation technique that demonstrates reticular fibers. Reticulum is a support function of the body and is abundant in liver, spleen, and kidney. In a normal liver the fibers are will defined strands, but necrotic and cirrhotic liver show discontinuous patterns. Reticulum also forms characteristic patterns in relationship to certain tumor cells.

PRINCIPLE: The tissue is oxidized, then sensitized with the iron alum, which is replaced with silver. The silver is reduced with formalin to its visible metallic state.

CONTROL: Normal liver.

FIXATIVE: 10% formalin

TECHNIQUE: Cut paraffin sections at 4m to 5m.

EQUIPMENT: Acid cleaned glassware, pipettes.

REAGENTS:

3% Sulfuric Acid:

Distilled water 100.0 ml

Sulfuric acid 3.0 ml

Mix well, label with initial and

date. Solution is stable for 1 year.

CAUTION: Corrosive, avoid contact and

inhalation.

Potassium Permanganate:

Potassium permanganate 0.5 gm

Distilled water 47.5 ml

3% Sulfuric acid 2.5 ml

Make fresh, discard after use.

CAUTION: Corrosive, avoid contact and

inhalation.

1% Oxalic Acid:

Oxalic acid 1.0 gm

Distilled water 100.0 ml

Mix well, label with initial and

date. Solution is stable for 1 year.

CAUTION: Corrosive, avoid contact and

inhalation.

2% Iron Alum

Ferric ammonium sulfate 2.0 gm

Distilled water 100.0 ml

Mix well, label with date and

initial. Solution is stable for 6

months.

RETIC Page: 2 of 4

10% Silver Nitrate Stock

Silver nitrate 10.0 gm

Distilled water 100.0 ml

Acid clean all glassware and rinse

well. Store in a brown bottle, keep

in the refrigerator. Label with

initial and date. Stable for 6

months.

CAUTION: Corrosive, possible

carcinogen.

3% Sodium Hydroxide Stock:

Sodium hydroxide 1.5 gm

Distilled water 50.0 ml

Mix well, label with date and

initials. Store in the refrigerator,

stable for 3 months.

CAUTION: Corrosive, avoid contact and

inhalation.

Ammoniacal Silver

Working Solution:

10% silver nitrate 5.0 ml

Add ammonium hydroxide drop by

drop until clear again.

3% sodium hydroxide 5.0 ml

Add ammonium hydroxide drop by

drop until clear again.

Distilled water 40.0 ml

Using acid clean glassware, agitate

solution continually while adding

ammonium hydroxide. Make right

before use, discard.

CAUTION: Corrosive, avoid contact and

inhalation.

10% Formaldehyde:

Formaldehyde 5.0 ml

Distilled water 45.0 ml

Make fresh, discard after use.

CAUTION: Carcinogen.

0.5% Gold Chloride:

Gold chloride 1.0 gm

Distilled water 200.0 ml

Use acid clean glassware, label

with date and initials. Store in the

refrigerator. Stable for 1 year.

CAUTION: Avoid contact and inhalation.

5% Hypo

See Stock Solution

Nuclear-Fast Red (Kernechtrot)

Aluminum sulfate 25.0 gm

Distilled water 500.0 ml

Nuclear-fast red 0.5 gm

Dissolve the aluminum sulfate in

distilled water, then the nuclearfast

red, using heat. Cool, filter,

and add a few grains of thymol as a

preservative. Label with date and

initials. Stable for 1 year.

CAUTION: Irritant, avoid contact and

inhalation.

SAFETY/PPE: Wear nitrile gloves, goggles and lab coat, avoid contact and inhalation. Potassium permanganate: Skin and eye irritant, ingestion will lead to severe gastrointestinal distress. Strong oxidant. Sulfuric acid: Strong irritant to skin, eyes and respiratory system. Inhalation can produce target organ effects on skin, respiratory, reproductive and fetal systems. Corrosive. can cause severe burns of the eyes, skin or mucous membranes. Toxic by inhalation and ingestion. Target organ effects on kidneys and cardiovascular system, repeated exposure can cause dermatitis. Corrosive. Silver nitrate: severe skin and eye irritant. Oxidizer. Ingestion will produce violent gastrointestinal discomfort. Possible carcinogen: equivocal tumorigenic agent. Ammoniacal silver solution can be explosive, discard down the drain followed by copious amounts of running water. Ammonium hydroxide: severe eye irritant, irritating to respiratory system. Target organ effects on respiratory system. Corrosive. Work under

hood. Sodium hydroxide: severe skin and eye irritant. Corrosive. Formaldehyde: known carcinogen. Sodium thiosulfate: Toxic on ingestion. Can irritate the stomach. Irritant to skin, eyes and respiratory tract.

PROCEDURE:

• Deparaffinize and hydrate to distilled water.
• Potassium permanganate solution, 5 minutes.
• Wash in water.
• 5% oxalic acid until clear.
• Wash in distilled water.
• Iron alum solution, 10 minutes.
• Wash in running tap water, rinse in distilled, 3 changes.
• Silver solution, 7 dips, shake excess solution off slides.
• Distilled water, 2 changes, 3 quick dips each.
• 10% formaldehyde solution until gray black, 30 seconds.
• Wash in distilled water.
• 0.5% Gold chloride, 1 minute.
• Rinse in distilled water.
• 5% hypo, 1 minute.
• Wash in tap water.
• Nuclear-fast red solution, 5 minutes.
• Wash in running tap water.
• Dehydrate, clear, and coverslip.

RESULTS:

Reticular fibers black

Nuclei red

NOTES:

• Use acid clean glassware, or rinse 5x with distilled water.
• When making working silver solution, if over 30 drops of ammonium hydroxide are used to turn the solution, then the ammonium hydroxide is too old. Start over with fresh ammonium hydroxide.
• When adding the 3% sodium hydroxide solution to the silver solution it should turn black, if not make fresh sodium hydroxide.
• Because of the alkalinity of the solution, it may cause some tissues to fall off the slides, celloidinize (see Stock Solutions).
• Change distilled water after every slide , step '9'.

FERROUS IRON - TURNBULL’S BLUE

PURPOSE: To detect ferrous (Fe2+) iron in tissues.

PRINCIPLE: Tissue sections are treated with an acidic solution of

potassium ferricyanide, any ferrous iron present will react to form an

insoluble bright blue pigment called Turnbull’s blue (ferrous ferricyanide).

CONTROL: Routine iron control, which includes an negative.

TECHNIQUE: Cut paraffin section 4μ.

EQUIPMENT: Acid clean glassware, non-metallic forceps.

REAGENTS:

0.006N Hydrochloric Acid

Hydrochloric acid 2.5 ml

Distilled water 497.5 ml

Mix well. Solution is stable for 1

year.

CAUTION: Corrosive acid.

Potassium Ferricyanide

Staining Solution:

Potassium ferricyanide 0.4 gm

Hydrochloric acid, 0.006N 40.0 ml

Prepare fresh, just before use.

CAUTION: Avoid contact and inhalation.

1% Acetic Acid

Acetic acid, glacial 1.0 ml

Distilled water 100.0 ml

Mix well. Solution is stable for 1

year.

CAUTION: Avoid contact and inhalation.

Nuclear-Fast Red:

SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation. Hydrochloric acid; target organ effects on reproductive system and fetal tissue. Irritant to skin eyes and respiratory system. Potassium ferricyanide; Low toxicity as long as it is not heated, it will release cyanide gas.

TURNBULL’S FERROUS IRON

Acetic acid: Irritating to respiratory system. Target organ effects on

respiratory system by inhalation. Corrosive.

PROCEDURE:

• Deparaffinize and hydrate to distilled water.
• Place slides in Potassium ferricyanide staining solution for 1 hour.
• Wash slides in 1% acetic acid.
• Counterstain slides in nuclear-fast red for 5 minutes.
• Rinse well in distilled water.
• Dehydrate, clear and coverslip.

RESULTS:

• Ferrous iron blue
• Background pink red

STAIN FOR ACID FAST BACILLI

BACKGROUND

Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are [fl]-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species

Basic fuchsin binds to negatively charged groups in bacteria. The mycolic acid (and other cell wall lipids) present a barrier to dye entry as well as elution (washing out with solvent) and this is partly overcome by adding a lipophylic agent to a concentrated aqueous solution of basic fuchsin and partly by heating

This method does not use phenol and was developed by R. Ellis, Principal Hospital Scientist, Histopathology, The Queen Elizabeth Hospital, Woodville, South Australia.

METHOD

SECTIONS -Tissue fixed in 10% neutral buffered formalin. 3 - 5 μm paraffin section

CONTROL-known positive material

REAGENTS-Staining solutions

Solution A

• Basic fuchsin 1 g
• Absolute ethyl alcohol 10 ml

Solution B

• L.O.C. High Suds (Amway) 0.6 ml
• Distilled water 100 ml

Note: Other pure, liquid organic cleaners work just as well as the Amway product. The two solutions can be kept as stock solution and mixed immediately before use.

3% hydrochloric acid in 95% ethyl alcohol

• Absolute ethyl alcohol 95ml
• Distilled wate 2 ml
• Concentrated hydrochloric acid 3 ml
• Make up the alcohol solution then add the concentrated acid

0.25% methylene blue in 1% acetic acid

• Methylene blue 0.25 g
• Distilled water 99 ml
• Acetic acid 1 ml

METHOD

• Place the staining solution in a Coplin jar and pre-heat to 60oC for 10 min
• Deparaffinise sections, bring to water and stain in the pre-heated solution for 15 min
• Place the Coplin jar containing the slides into running cold tap water for 2 min before removing the slides from the Coplin jar.
• Remove the slides from the Coplin jar and wash in running water for 1 min
• Differentiate in 3% hydrochloric acid in 95% ethyl alcohol until no more colour runs from the slide
• Wash briefly in water to remove the acid alcohol
• Counterstain with 0.25% methylene blue in 1% acetic acid for 15 to 30 secs
• Wash in water. Dehydrate, clear.
• Mount sections in Cytoseal XYL

RESULT

• Acid fast bacilli-Red
• Nuclei-Blue
• Other tissue constituents-Blue

IMMUNOHISTOCHEMISTRY

Introduction

Immunohistochemistry is the localization of antigens in tissue sections by the use of labeled antibody as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold.

Albert H. Coons and his colleagues (Coons et al. 1941, 1955; Coons and Kaplan 1950) were the first to label antibodies with a fluorescent dye, and use it to identify antigens in tissue sections. With the expansion and development of immunohistochemistry technique, enzyme labels have been introduced such as peroxidase (Nakane and Pierce 1966; Avrameas and Uriel 1966) and alkaline phosphatase (Mason and Sammons 1978). Colloidal gold (Faulk and Taylor 1971) label has also been discovered and used to identify immunohistochemical reactions at both light and electron microscopy level. Other labels include radioactive elements, and the immunoreaction can be visualized by autoradiography.

Since immunohistochemistry involves specific antigen-antibody reaction, it has apparent advantage over traditionally used special enzyme staining techniques that identify only a limited number of proteins, enzymes and tissue structures. Therefore, immunohistochemistry has become a crucial technique and widely used in many medical research laboratories as well as clinical diagnostics.

There are numerous immunohistochemistry methods that may be used to localize antigens. The selection of a suitable method should be based on parameters such as the type of specimen under investigation and the degree of sensitivity required.

Tissue Preparation

Fixation

Tissue preparation is the cornerstone of immunohistochemistry. To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or prolonged fixation may significantly diminish the antibody binding capability.

There is no one universal fixative that is ideal for the demonstration of all antigens. However, in general, many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. The discover and development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories. 

For best results, vertebrate tissues (especially neuronal tissues) usually require fixation by transcranial perfusion for optimal tissue preservation. The most common fixatives used for immunohistochemistry are the followings:

• 4% paraformaldehyde in 0.1M phosphate buffer
• 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer
• PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer
• 4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistry)

Some antigens will not survive even moderate amounts of aldehyde fixation. Under this condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat without infiltrating with sucrose. The sections should be kept frozen at -20 C or lower until fixation with cold acetone or alcohol. After fixation, the sections can be processed using standard immunohistochemical staining protocols

Sectioning:

Since its introduction, paraffin wax has remained the most widely used embedding medium for diagnostic histopathology in routine histological laboratories. Accordingly, the largest proportion of material for immunohistochemistry is formalin-fixed, paraffin-embedded. Paraffin sections produce satisfactory results for the demonstration of majority of tissue antigens with the use of antigen retrieval techniques. 

Certain cell antigens do not survive routine fixation and paraffin embedding. So the use of frozen sections still remains essential for the demonstration of many antigens. However, the disadvantage of frozen sections includes poor morphology, poor resolution at higher magnifications, special storage needed, limited retrospective studies and cutting difficulty over paraffin sections.

Vibratome sections have some advantages when doing immunohistochemistry since the tissue is not processed through organic solvents or high heat, which can destroy the antigenicity. In addition, the morphology of tissue sections is not disrupted due to no freezing and thawing needed. Vibratome sections are often used for floating immunostaining, especially for pre-embedding EM immunohistochemistry. The disadvantage of vibratome sections is that the sectioning process is slow and difficult with soft and poorly fixed tissues. In addiction, the chatter marks or vibratome lines are often appeared in the sections.

Whole Mount Preparation:

Small blocks of tissue (less than 5 mm thick) can be processed as whole mounts. The advantage of whole mount preparations is that the results provide three dimensional information about the location of antigens without the need for reconstruction from sections. However, the major limitation of using whole mounts is antibody penetration may not be complete in the tissue, resulting in uneven staining or false negative staining. So Triton X-100 or saponin treatment are used routinely for whole mount immunohistochemistry to enhance penetration of the antibody.

Antigen Retrieval

The demonstration of many antigens can be significantly improved by the pretreatment with the antigen retrieval reagent that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. The techniques involved the application of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in an aqueous solution (commonly referred to as the retrieval solution). This is called "Heat Induced Epitope Retrieval (HIER)". Another method uses enzyme digestion and is called "Proteolytic Induced Epitope Retrieval (PIER)". 

Microwave Oven, Pressure Cooker and Steamer are the most commonly used heating devices. Other devices also include the use of autoclave and water bath. The heating length of 20 minutes appears to be the most satisfactory and the cooling usually takes about 20 minutes. Citrate buffer of pH6.0 is the most popularly used retrieval solution and is suitable for most of antibody applications. The TRIS-EDTA of pH9.0 and EDTA of pH8.0 are second most used retrieval solutions. Proteinase K is effective enzyme digestion reagent for membrane antigens such as Integrins, CD31, vWF, etc.

PIER methods (such as proteinase k, trypsin, chymotrypsin, pepsin, pronase and various other proteases) has also been reported for restoring immunoreactivity to tissue antigens with different degrees of success. However, the use of enzyme digestion method may destroy some epitopes and tissue morphology. Therefore the optimal enzyme concentration and incubation time need to be tested.  

Combination of Heat Mediated and Proteolytic Enzyme Method is an alternative approach to unmask antigens if other methods did not work. It is especially useful when performing double or triple labeling of two or more antigens simultaneously.

Improving antibody penetration is also important for immunohistochemical staining of frozen and vibratome sections. Triton X-100 is by far the most popular detergent for improving antibody penetration for immunohistochemistry. However, it is not appropriate for the use of membrane antigens since triton X-100 destroy membranes. Some researchers prefer the freeze and thaw method for the improvement of antibody penetration. Sodium borohydride (1% in phosphate buffer) treatment is also widely used to unmask antigens, particularly in glutaraldehyde fixed tissue to reduce the glutaraldehyde linkages.

IHC Methods

Blocking

Background staining may be specific or non-specific. Inadequate or delayed fixation may give rise to false positive results due to the passive uptake of serum protein and diffusion of the antigen. Such false positives are common in the center of large tissue blocks or throughout tissues in which fixation was delayed. 

Antibodies, specially polycolonal antibodies, are sometimes contaminated with other antibodies due to impure antigen used to immunize the host animal.

 

The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. This form of background staining is usually uniform and can be reduced by blocking those sites with normal serum.

Endogenous peroxidase activity is found in many tissues and can be detected by reacting fixed tissue sections with DAB substrate. The solution for eliminating endogenous peroxidase activity is by the pretreatment of the tissue section with hydrogen peroxide prior to incubation of primary antibody.

Many tissues also contain endogenous alkaline phosphatase (AP) activity and should be blocked by the pretreatment of the tissue section with levamisole if using AP as a label.

Some tissues such as liver and kidney have endogenous biotin. To avoid unwanted avidin binding to endogenous biotin if using biotin-avidin detection system, a step is necessary for these tissues by the pretreatment of unconjugated avidin which is then saturated with biotin.

Autofluorescence or natural fluorescence exists in some tissues and can cause background problems when fluorescent dyes are used in the experiments. The simplest test is to view the tissue sections with a fluorescence microscope before any antibody incubation. If autofluorescence is detected in the tissue sections, the best solution is to avoid use of fluorescent method but choose enzyme or other labeling methods.

Controls

Special controls must be run in order to test the protocol and for the specificity of the antibody being used.

Positive control is to test a protocol or procedure and make sure it works. It will be ideal to use the tissue of known positive as a control. If the positive control tissue showed negative staining, the protocol or procedure needs to be checked until a good positive staining is obtained.

Negative control is to test for the specificity of an antibody involved. First, no staining must be shown when omitting primary antibody or replacing an specific primary antibody with normal serum (must be the same species as primary antibody). This control is easy to achieve and can be used routinely in immunohistochemical staining.

Second, the staining must be inhibited by adsorption of a primary antibody with the purified antigen prior to its use, but not by adsorption with other related or unrelated antigens. This type of negative control is ideal and necessary in the characterization and evaluation of new antibodies, but it is sometimes difficult to obtain the purified antigen, therefore it is rarely used routinely in immunohistochemical staining.

Direct Method

Direct method is one step staining method and involves a labeled antibody (i.e. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is short and quick. However, it is insensitive due to little signal amplification and rarely used since the introduction of indirect method.

Indirect Method

Indirect method involves an unlabeled primary antibody (first layer) which react with tissue antigen, and a labeled secondary antibody (second layer) react with primary antibody (Note: The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised). This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. In addition, it is also economy since one labeled second layer antibody can be used with many first layer antibodies (raised from the same animal species) to different antigens. 

The second layer antibody can be labeled with a fluorescent dye such as FITC, rhodamine or Texas red, and this is called indirect immunofluorescence method. The second layer antibody may be labeled with an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase, and this is called indirect immunoenzyme method.

PAP Method (peroxidase anti-peroxidase method)  

PAP method is a further development of the indirect technique and it involves a third layer which is a rabbit antibody to peroxidase, coupled with peroxidase to make a very stable peroxidase anti-peroxidase complex. The complex, composed of rabbit gaba-globulin and peroxidase, acts as a third layer antigen and becomes bound to the unconjugated goat  anti-rabbit gaba-globulin of the second layer. The sensitivity is about 100 to 1000 times higher since the peroxidase molecule is not chemically conjugated to the anti IgG but immunologically bound, and loses none of its enzyme activity. It also allows for much higher dilution of the primary antibody, thus eliminating many of the unwanted antibodies and reducing non-specific background staining.

Avidin-Biotin Complex (ABC) Method

ABC method is standard IHC method and one of widely used technique for immunhistochemical staining. Avidin, a large glycoprotein, can be labeled with peroxidase or fluorescein and has a very high affinity for biotin. Biotin, a low molecular weight vitamin, can be conjugated to a variety of biological molecules such as antibodies. 

The technique involves three layers. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is a complex of avidin-biotin peroxidase. The peroxidase is then developed by the DAB or other substrate to produce different colorimetric end products.

Labeled StreptAvidin Biotin (LSAB) Method

Streptavidin, derived from streptococcus avidini, is a recent innovation for substitution of avidin. The streptavidin molecule is uncharged relative to animal tissue, unlike avidin which has an isoelectric point of 10, and therefore electrostatic binding to tissue is eliminated. In addition, streptavidin does not contain carbohydrate groups which might bind to tissue lectins, resulting in some background staining. 

LSAB is technically similar to standard ABC method. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is Enzyme-Streptavidin conjugates (HRP-Streptavidin or AP-Streptavidin) to replace the complex of avidin-biotin peroxidase. The enzyme is then visualized by application of the substrate chromogen solutions to produce different colorimetric end products. The third layer can also be Fluorescent dye-Streptavidin such as FITC-Streptavidin if fluorescence labeling is preferred.

A recent report suggests that LSAB method is about 5 to 10 times more sensitive than standard ABC method.

Polymeric Methods

EnVision Systems are based on dextran polymer technology. This unique chemistry permits binding of a large number of enzyme molecules (horseradish peroxidase or alkaline phosphatase) to a secondary antibody via the dextran backbone. The benefits are many, including increased sensitivity, minimized non-specific background staining and a reduction in the total number of assay steps as compared to conventional techniques. The simple protocol is i) Application of primary antibody; ii) Application of enzyme labeled polymer; iii) Application of the substrate chromogen. EnVision+ was developed after EnVision to provide increased sensitivity.

ImmPRESS polymerized reporter enzyme staining system is based on a new method of polymerizing enzymes and attaching these polymers to antibodies. The novel approach employed to form enzyme "micropolymers" avoids the intrinsic shortcomings of using large dextrans or other macromolecules as backbones. Attaching a unique "micropolyer with a high density of very active enzyme to a secondary antibody generates a reagent that overcomes steric interference and provides enhanced accessibility to its target. The result is outstanding sensitivity, signal intensity, low background staining, and reduced non-specific binding. The simple protocol is i) Application of primary antibody; ii) Application of enzyme labeled polymer; iii) Application of the substrate chromogen.

CSA Methods From Dako

CSA Systems use Tyramide Signal Amplification. It is ideal for the following applications: i) Detecting small quantities of antigen; ii) Enhancing performance of low affinity mouse and rabbit antibodies; iii) Enabling compatibility of certain "tough" mouse and rabbit antibodies with paraffin embedded tissue sections. The simple protocol is as follows: 

• Application of primary antibody.
• Application of biotinylated linking antibody.
• Application of the Tyramide Amplification Reagent.
• Application of Streptavidin-HRP.
• Application of the substrate chromogen

CSA II - Biotin-free Tyramide Signal Amplification System is a highly sensitive immunohistochemical (IHC) staining procedure incorporating a signal amplification method based on the peroxidase-catalyzed deposition of a fluorescein-labelled phenolic compound, followed by a secondary reaction with a peroxidase-conjugated anti-fluorescein. In the procedure, a mouse primary antibody is first detected with a peroxidase-conjugated secondary antibody. The next step utilizes the bound peroxidase to catalyze oxidation of a fluorescein-conjugated phenol (fluorescyl-tyramide) which then precipitates onto the specimen. The procedure is continued with detection of the bound fluorescein by a peroxidase-conjugated anti-fluorescein. Staining is completed using diaminobenzidine/hydrogen peroxide as chromogen/substrate, and can be observed with a light microscope. In comparison to standard immunohistochemical methods, such as labelled streptavidin biotin (LSAB) or avidin-biotin complexes (ABC), tyramide amplification methods have been reported to be many fold more sensitive. The CSA II System is a simplified version of the extremely sensitive Catalyzed Signal Amplification System (code K1500) that utilizes biotinyl-tyramide. The highly sensitive CSA II System allows for the detection of very small quantities of target protein, as well as for the use of low affinity antibodies. This reagent system utilizes fluorescyl-tyramide, rather than biotinyl-tyramide, and does not contain avidin/biotin reagents, thus eliminating potential background staining due to reactivity with endogenous biotin.

Principles of Procedure: The specimens are first incubated with Peroxidase Block for five minutes to quench endogenous peroxidase activity. The specimens are then incubated for five minutes with a protein block to suppress nonspecific binding of subsequent reagents, followed by a 15-minute incubation with an appropriately characterized and diluted mouse primary antibody or negative control reagent (user provided). This is followed by sequential 15-minute incubations with anti-mouse immunoglobulins-HRP, fluorescyl-tyramide hydrogen peroxide (amplification reagent) and anti-fluorescein-HRP. Staining is completed by a five-minute incubation with 3,3' diaminobenzidine tetrahydrochloride (DAB)/hydrogen peroxide, which results in a brown precipitate at the antigen site.

Multiple Labeling

It is often useful to be able to stain for two or more antigens in one common tissue section. This can be achieved by immunofluorescence method using different fluorescent dyes. Multiple staining can also be done with peroxidase conjugated antibodies developed with different chromogen substrates to produce the end products of different colors. There are three basic approaches in planning multiple staining: parallel, sequential and adjacent. In addition, the antibody dilution and condition are also important factors to be considered.  Finally, appropriate color combination is also crucial since improper color combination may produce poor result and fail to demonstrate multiple antigens in the same section. For best result, the careful design and test of multiple staining protocols are necessary.

NO	IMMUNOHISTOCHEMISTRY	CONTROL TISSUE
1.	CD3 (T cell)	Tonsil
2.	CD15	No Control Tissue
3.	CD20 (B cell)	Tonsil
4.	CD30	Tonsil
5.	CD34	Tonsil
6.	CD45 Ro (T cell)	Tonsil
7.	CD68	Tonsil
8.	Cerb B2	Cerb B2/Breast
9.	Estrogen Reseptor	Cerb B2/Breast
10.	Progesteron Reseptor	Cerb B2/Breast
11.	S100	Colon
12.	VIM	Colon
13.	Cytokeratin (AE1/AE3)	Colon
14.	Chromogranin A	Pancreas
15.	NSE, Glukagon	Pancreas
16.	Insulin	Pancreas
17.	EMA	Appendix
18.	HMWCK	Prostate
19.	PSA	Prostate
20.	Congo Red	Congo Red
21.	CK	Colon
22.	ST	Synap
23.	DES	DES
24.	LCA	Tonsil
25.	HBC Ag	HBS Ag
26.	HBS Ag	HBS Ag
27.	HSA	HBS Ag
28.	HMB 45	HMB

CYTOLOGY

That fact that we as humans are made up of millions of tiny cells, and that other lifeforms around us are similarly constituted, now barely needs explanation. However, the concept of the cell is relatively new. The scientific community did not accept the idea of the existence of cells until the late 18th century. Cytology became, in the 19th century, a way to describe and identify cells, and also to diagnose certain medical diseases.

Cytology, more commonly known as cell biology, studies cell structure, cell composition, and the interaction of cells with other cells and the larger environment in which they exist. Cytology can also refer to cytopathology, which analyzes cell structure to diagnose disease. Microscopic and molecular studies of cells can focus on either multi-celled or single-celled organisms.

Recognizing the similarities and differences of cells is of the utmost importance in cytology. Microscopic examination can help identify different types of cells. Looking at the molecules which form a cell, sometimes called molecular biology, helps in further description and identification. All fields of biology depend on the understanding of cellular structure. The field of genetics exists because we understand cell structure and components.

Another important aspect in the discipline of cytology is examining cell interaction. By studying how cells relate to other cells or to the environment, cytologists can predict problems or examine environmental dangers to cells, such as toxic or cancer-causing substances. In humans and other multi-cellular structures, cytology can examine the presence of too many of one kind of cell, or the lack of enough of a certain kind of cell. In a simple test like a complete blood count, a laboratory can look at white blood cells and identify the presence of an infection, or it may examine a low level of certain types of red blood cells and diagnose anemia.

Certain autoimmune disorders can be diagnosed by abnormal cell reactions. Hashimoto's thyroiditis, for example, is an autoimmune condition caused by abnormal cell reaction. Instead of white blood cells recognizing the presence of normal thyroid cells, these antibodies attack them, causing low thyroid. If untreated, this condition can result in retardation, extreme fatigue, obesity, and ultimately death. Through cytology, the abnormal reactions of these antibodies can be recognized, and treatment can be undertaken long before this condition creates irreversible problems.

Cytopathology has similar aims but tends to look for cells that should not be present in an organism. Urinalysis and blood tests, for example, can scan for the presence of parasites or bacteria which can cause illness and death. Hence, in cytology, understanding single-celled organisms like many forms of bacteria is as important as understanding multi-cellular structures.

Cytopathology is also one of the main diagnostic tools for detecting cancer. A woman's yearly gynecological exam almost always involves a pap smear, a collection of tissues that are analyzed at the cellular structure to detect early formations of cancer cells. Early detection can lead to greater survival rates. Similarly, needle biopsies of lumps in the breast or elsewhere can detect cancer cells and provide an excellent means for diagnosis.

The recognition and the study of cells represent huge improvements in medical care and diagnostics. Cytology, by studying cell interaction, helps us to understand ways in which we can care for humans, animals and plants. Though biology precedes cytology in its development, cytologists are responsible for our modern view of biology and all other life sciences.

A pap smear or smear test is a diagnostic screening for cervical cancer, a serious cancer which kills thousands of women around the world every year. The screening contributes to early cancer detection, increasing the likelihood that a woman will catch her cancer early, thus improving her prognosis. Pap smears are routinely performed as part of an annual exam, which is recommended for all women from their teens to the end stages of their lives.

To perform a pap smear, a physician takes a small sample of cells from the cervix, the lower part of the uterus. These cells are stained and studied under a microscope to look for abnormalities which characterize the development of cancerous growth. Abnormalities indicate that some sort of action needs to be taken, such as removing a pre-cancerous region of the cervix to prevent the onset of a full-blown cancer.

A pap smear can be uncomfortable, as can pelvic exams in general. However, the discomfort is outweighed by the benefits of routine cancer screening and early detection. In regions of the world where women receive annual exams on a regular basis, women's health in general tends to be better, with a longer life expectancy for women thanks to a focus on preventative care. Women should be aware that pap smears only screen for cervical cancers, and that screening for other cancers requires different diagnostic tests.

The test is named for George Papanicolaou, an American doctor who published a paper in 1941 discussing his early work on the exam. By the 1980s, the American College of Obstetricians and Gynecologists was recommending annual pap smears for all women. Along with other routine medical screening, a pap smear ensures that women maintain their fertility and general health. A pap smear may identify other underlying medical issues which are cause for concern in addition to cervical cancer.

Some people may be at greater risk for cervical cancer than others, such as women who have been infected with the human papilloma virus. It is an excellent idea to discuss conditions such as cancer with your doctor, as he or she may identify risk markers such as genetic factors which suggest that you should be more carefully monitored than other patients. You may also want to take advantage of your annual exam as an opportunity to discuss other women's health issues with your doctor, such as family planning and Sexually Transmitted Infections (STIs).

Spesimen

• Coloscopy specimen (Cervix and Vagina)
• Fluid (Peritoneal, Pleural, Sputum, Urin)

Types Of Stain

• PAP Stain
• May Grunwalds Giemsa
• Tzank