THE PAPANICOLAOU (PAP) STAIN – STAIN PREPARATION AND PROCEDURE IN DETAILS

Among the major achievements in the history of cytopathology, the eponymous Pap stain method formalized by Dr. George N. Papanicolaou in 1942 was foundational.[1,2,3] The PAP stain has been used all over the world since half a century for the staining of cervicovaginal smear. Many modifications have been published after George Papanicolaou described the original staining technique in 1942. The staining procedure varies with various staining protocols and methodologies used in different laboratories. The PAP stain is a polychromatic counterstaining method consisting of stains such as Orange G 6 (OG6) and modified eosin azure (EA). The strength of the Pap stain is such that it results in:

Well-stained nuclear chromatin

Differential polychromatic counterstaining of cytoplasm

Cytoplasmic transparency.

To achieve this, proper fixation of the smear is one of the most important prerequisite factors.

FIXATION AND FIXATIVES

The purpose of cytological fixatives is to maintain the cytomorphologic characteristics and diagnostically essential elements of the cell. Ethyl alcohol is the fixative specifically recommended for cytological preparations. Fixation coarsens the cell structures and sharpens nuclear chromatin pattern and its details. Commonly used cytological fixatives include wet fixatives and dry fixatives.

WET FIXATIVES

95% ethyl alcohol and ether in equal volume. Nowadays, it is not used as it is inflammable in nature and has a pungent odor

95% ethanol: Fixation for routine cytological smear

100% methanol

95% denatured alcohol

80% isopropanol

80% propanol.

The cytology laboratory of G.M.C, Nagpur, uses 95% ethanol as the fixative for routine cytological smears for both cervicovaginal and fine-needle aspiration cytology smears. The slides should be fixed immediately in the fixative solution, as even a slightest air drying of the smear can alter cytomorphological features causing diagnostic problems.[1] The smear should be fixed at least for 20–30 min to assure adequate fixation. However, prolonged fixation for several days or even weeks will not alter cellular features. Fixative solution along with the smear may be refrigerated in such situations to minimize the evaporation.

DRY OR COATING FIXATIVES

Dry or coating fixatives are major substitutes for wet fixatives in special situations like cancer detection camps or when transportation of smears from distant collection centers is required. These fixatives are available commercially and are either aerosol (applied by spraying) or liquid based (dropped over a smear). They are composed of an alcohol base, which fixes the cells and a wax-like substance that forms a thin protective coating over the smear. Any ordinary hair spray can be substituted as coating fixative.

Method

The Pap smear is prepared in the usual way and immediately fixed with “dry fix” spray; or a few drops of liquid based fixative solution are put on the smear

While applying the spray, the bottle must be held at least 10–12 inches away from glass slide, which will prevent layering and hole formation

The slide is then placed on a flat surface for a few minutes to allow the “dry fix” to dry

The coating fixatives must be removed from the smear before staining. For this, slides are kept in 80% ethyl alcohol for 1–2 h. Sometimes, two changes of 80% alcohol may be required to remove the coating fixative completely.

If the coating fixative is not removed entirely before staining, it gives a “bubbling” effect to the smear which interferes with the diagnosis.[2]

STAIN PREPARATION Nuclear stain hematoxylin

Hematoxylin is the most widely used nuclear stain. It is commercially available as an amorphous brown-colored powder form, which is extracted from heartwood of Central America Logwood – “Haematoxylum campechianum.”

Hematoxylin itself is not a dye, it has to be oxidized to “hematin” which is actual staining component

The natural oxidation process or ripening of hematoxylin in aqueous solution takes place gradually over a period of time by keeping the stain bottle in the sunlight area at least for a month

The instant ripening can be achieved chemically by the addition of oxidizing agents such as sodium iodate or mercuric oxide

Hematoxylin stains the DNA and RNA of the cell

This stain can be used either progressively (Box 1) (without the use of acid alcohol differentiation) or regressively (Box 2)

In the bluing step (Box 3), the absorbance peak of hematoxylin is shifted visually resulting in the change of color from red to blue

Harris, Gill’s, and Mayer’s reagents are the most commonly used alum hematoxylin (Box 4) in cytology laboratory.

The cytology laboratory of G.M.C.H, Nagpur, uses the regressive method of staining using Harris and Gill’s hematoxylin followed by differentiation with 0.5% acid alcohol and bluing in the running tap water.

Box 1: Progressive staining

Box 2: Regressive staining

Indirect staining method

Tissue is overstained

Excess stain is selectively removed from all except the structures to be demonstrated.

Excess stain removed by acid alcohol to achieve desired staining

This process of selectively removal of excess stain is called Differentiation

Box 3: Bluing

Alum hematoxylin stain red color to the nuclei, which is converted to blue black when section is washed in weak alkali solution like tap water/ Scott’s tap water/ saturated lithium carbonate/ 0.05% ammonia in distilled water / etc.

Box 4: Alum Hematoxylin

Alum is a chemical compound containing double sulfate salts of metals like aluminum (most common), iron or chromium.

Common alum used in laboratories are: Ammonium aluminum sulfate (i.e., ammonium alum) and potassium aluminum sulfate (i.e., potassium alum).

Alum is a Mordant (Box 5) used for hematoxylin.

Different alum hematoxylin are Harris Hematoxylin, Mayer’s Hematoxylin, Gill’s Hematoxylin, Ehrlich’s Hematoxylin, etc.

Box 5: Mordants

The oxidation product of hematoxylin is Hematin.

Hematin is a natural dye having color properties.

However, this Hematin has poor affinity for tissues and does not stain nuclei when used alone.

This affinity of Hematin to tissues is improved by use of Mordants.

Mordant is a substance or a metal which chemically binds with the dye and acts as a link between dye and tissue.

Most commonly used mordants for Hematoxylin are salts of Aluminum (Alum), Iron, Tungsten, Lead, etc.

Harris hematoxylin: (For 1 L)

Hematoxylin powder 5 gm

(Yucca diagnostics, MERCK)

Absolute methanol/ethanol (solvent) 50 ml

Mercuric oxide (oxidizing agent) 2.5 gm

Aluminum ammonium sulfate (mordant) 100 gm

Glacial acetic acid* 40 ml

Distilled water 1000 ml

Note: *Glacial acetic acid is used as a stabilizer and slows down the oxidation process.

Preparation[3]

Dissolve hematoxylin in alcohol

Dissolve alum in water and bring to boil

Add dissolved hematoxylin to alum solution and again bring to boil

Remove the flask from heat and immediately add mercuric oxide

Stir the solution until a dark purple color appears

Cool the flask in water bath

Filter and store in dark bottle in refrigerator.

Principle

Being a regressive stain, Harris hematoxylin over stains the nucleus and the excess stain is removed with the help of running tap water followed by differentiation using 0.5% acid alcohol

The decolorizing acid is then removed by keeping the slides in running tap water for bluing

Timing in acid alcohol is essential for the final appearance of the nuclear chromatin

If acid bath is inadequate, the contrast between the chromatin and the parachromatin is less and uptake of the counterstain is also lessened.

Gill’s hematoxylin[3]

Used for the procedure of PAP stain. For the preparation of 1 L of Gill’s hematoxylin, the following chemicals are combined in large conical flask in the given order. Stir the mixture for an hour manually or preferably on a magnetic stirrer at room temperature.

Preparation

Distilled water 730 ml

Ethylene glycol 250 ml

Hematoxylin 2 g

Sodium iodate (oxidizing agent) 0.2gm

Aluminum sulfate (mordant) 17.6 gm

Glacial acetic acid* 20 ml

Note: *Glacial acetic acid stabilizes aluminum-hematin complex and slows down the oxidation of dye.

Gill’s hematoxylin can be prepared easily and rapidly

The unoxidized hematoxylin forms a metallic scum on the surface of the stain, which can interfere with the staining and precipitate on the slides

Although no surface or bottom precipitate is recovered, it is a good practice to filter the stain, before staining

Fresh hematoxylin should be added when the level in staining jar drops

Being a progressive stain, Gill’s hematoxylin stains the nucleus to the desired intensity and this is followed by bluing

The optimum time for nuclear staining is 1–1½ min

If Gill’s hematoxylin is used as a regressive stain, then the time of nuclear staining is increased up to 10–15 min followed by differentiation using acid alcohol (0.5 %) and then bluing in running tap water or in lithium carbonate bath.

OG-6 modified (Gill’s modified OG-6)

It is a cytoplasmic counterstain

In OG-6, OG signifies Orange G (“G” is an abbreviation for German word “gelb” which means yellow) and 6 denotes the concentration of phosphotungstic acid added

OG is an acidic protein dye, which can be combined with other protein dyes

Other variants of OG are OG-5 and OG-8

Modified OG is a Gill’s modification and is a combination of OG and phosphotungstic acid. It minimizes precipitation and reduces filtering and staining time

Gill’s modified OG is stable in solution and gives high-quality staining results.

Preparation[4]

Orange G stain Powder* 5 g (HIMEDIA)

Phosphotungstic acid** 1.5 gm

Distilled water 50 ml

Absolute alcohol 950 ml

Glacial acetic acid*** 10 ml.

Dissolve OG and phosphotungstic acid in distilled water, warm if necessary

Add ethanol and glacial acetic acid and mix

Store in a dark bottle, filter before use.

Note: *OG-6 stains mainly the keratin in the cytoplasm as brilliant orange, yellow, or brown (parakeratotic squamous carcinoma cells, erythrocytes, hemosiderin crystals, and asbestos bodies). *OG modified is stored in amber colored bottle. It is a supersaturated solution and hence a thick precipitate may be collected at the bottom of the staining jar which should not be filtered but gently stirred with glass rod before the slides are introduced in the staining jar. After staining about 2000 slides, the used stain is discarded and replaced with fresh stock solution. **Phosphotungstic acid maintains the pH of the stain to acidic level. It is a non-volatile acid which acidifies the solution and increases the uptake of OG. ***Acetic acid plays an important role in the uptake of the dye by the cytoplasm.[4]

EA modified (Gill’s modified EA)

It is also a cytoplasmic counterstain

EA-65 is a polychromatic stain and contains 3 dyes: Eosin Y, Light Green SF yellowish, and Bismarck Brown Y

The number of light green and eosin molecules must be adjusted relatively to one another to ensure the balanced staining

Eosin is purely an acidic dye and binds mainly to protein

EA is followed by a number which denotes proportion of the dyes. There are different formulations of this product (EA36, EA50, and EA65). EA-65 contains higher percentage of Green stain component, also referred to as “PAP EA65 enhanced green.”

In EA modified formula, Bismarck brown has been eliminated.[4] Although not numbered, Gill’s modified EA is similar to EA-36 and EA-50, but offers improved staining performance.

The main ingredients are

Light Green* 2 g

Eosin** (LOBA) 2 g

Phosphotungstic acid*** 1 g

Distill water 480 ml

95% Ethanol 500 ml

Glacial acetic acid**** 20 ml

Dissolve light green, eosin, and phosphotungstic acid in distilled water separately. Then, mix all the three solutions together and add 95% ethanol and glacial acetic acid. Mix them thoroughly.

EA modified contains light green and eosin only

In EA modified formula, Bismarck brown has been deleted as it is considered to have no distinguishable color effect. Combination of Bismarck brown and phosphotungstic acid create precipitation, alters color overtime and reduces shelf life

*Light green stains metabolically active cells, that is, parabasal and intermediate cells, histiocytes, columnar cells, and malignant cells. **Eosin stains the mature squamous cells, erythrocytes, nuclei, and cilia. Red cells stain blue green if eosin is exhausted. ***Phosphotungstic acid is added to adjust the pH of the stains and helps optimize the color intensity. It selectively excludes eosin from cytoplasm of certain cell types and permits it to be stained by Light Green or Fast Green. Those sites remaining unstained by hematoxylin, OG, and Light Green are then stained by eosin.

****Glacial acetic acid is included in formula as it gives better cytoplasmic color differentiation and less background staining. It maintains required pH for optimal staining.

Acid alcohol

0.5% acid alcohol.

Preparation

Conc. HCL 5 ml

70% ethanol or rectified spirit 995 ml.

Bluing agent

Scott’s tap water substitute (STWS), dilute aqueous solution of ammonium hydroxide, and lithium carbonate are the most commonly used bluing agents. Tap water may serve as a bluing agent if the water pH is higher than 8.

STWS - pH 8.02

Distilled water 1000 ml

Magnesium sulfate MgSO4 10 gm

Sodium bicarbonate NaHCO3 2 gm.

(20 g of MgSO47H2O (Epsom salt) can be used instead of 10 g of magnesium sulfate). Mix the ingredients in water and stir them well. STWS should be discarded after each round of staining.

Other bluing agents

3 ml liquid ammonia is added to 97 ml of 70% ethanol

Dissolve 1.5 g of lithium carbonate in 100 ml of distilled water (stock solution) and add 30 drops of this solution to 1000 ml of distilled water.

Stain maintenance

Stain maintenance is necessary to remove contaminants, floaters and to restore the activity of the stain.

Filter the stain daily: It is recommended because washed off cells may be a source of contamination. Hematoxylin forms a scum and OG may precipitate into crystals

Change the staining solutions:

Hematoxylin: It has a long shelf life. Replace with fresh stain after 1500 slides are stained

OG modified: Replace after 2000 slides have been stained. This too has a long shelf life

EA modified: It has a short shelf life and so replace it after 1500 slides have been stained

Xylene: Filter through filter paper to remove contaminant cells

Alcohol: One jar should be replaced daily.

Staining procedure: [Manual method followed in GMC, Nagpur. Figure 1]

From the fixative, the slides are passed through the following solutions

Figure 1:: Manual staining procedure for Papanicolaou stain.

Export to PPT

Title Time Running tap water 2–3 min Gills hematoxylin 10–15 min Running tap water 2–3 min 0.5% acid alcohol 1 Dip Running tap water 3–5 min 95% ethanol 10 Dips Orange G modified 2–3 Dips 95% ethanol 2–3 Dips EA modified 3–5 Dips 95% ethanol 10 Dips Absolute alcohol 10 Dips Dry the slides Xylene 3–5 min

Mount the slides in dibutylphthalate polystyrene xylene (DPX).

Automated stainer (Method followed in GMC, Nagpur)

From the fixative, the slides are taken out and fixed in the slide carrier of the automatic robotic arm of the automatic stainer. The following program is followed.

Running tap water 1 min Gills hematoxylin 15 min Running tap water 5 min 0.5% acid alcohol 1 Dip Running tap water 5 min 95% ethanol 1 min Orange G modified 1 Dip 95% ethanol 1 Dip/1 min EA modified 5 min 95% ethanol 1 Dip/1 min Absolute alcohol 1 min Xylene 2 min Xylene 1 min Results

Nuclear chromatin is stained as classical blue to purple in color

RNA associated with DNA is delicately stained so that nucleoli display their characteristic eosinophilia

Bar bodies are stained conspicuously

Mucus and cell debris are minimally stained, thereby exhibiting an unobscuring background

The first counterstain, OG-6 modified stains keratin a brilliant orange, yellow, or brown

The second counter-stain EA-36stains the cytolasm of superficial cells (cornified) various shades of pink

Cytoplasm of the intermediate cells (non-cornified) stains pale blue or green

Cytoplasm of parabasal cells stains deep green

Candida stains red

Trichomonas vaginalis stains grey green.

Important factors for optimum staining

Excellence of the PAP’s staining depends largely on the delicate tints and transparency of the stained cells. The quality of the stained slides is dependent on the staining time, solubility and percentage of the dye concentration that is used in making nuclear and cytoplasmic stains. The following factors are useful in maintaining this desired quality.

The smears should not be allowed to dry at any time/ between any steps

Prolonged standings of the stained smears in the final grades of alcohols should be avoided as it leads to destaining of the cytoplasm

The stains should be kept well covered as water absorption causes a dense opaque stain. Evaporation of the alcohol changes the color balance and causes precipitation of the stain

Daily filtration of alcohol through a tight pad of cotton wool is recommended. This removes “free floating” cells or “floaters” thus minimizing cross-contamination

After staining approximately 800 slides, alcohol should be filtered with Whatman filter paper 1. This can be done at the weekends

Hematoxylin remains relatively constant in staining. Fresh stains should be added daily to replace stain loss due to evaporation

Daily filtration of xylene should be done through Whatman filter paper 1. This removes the moisture and floaters

The absolute alcohol and xylene of the last few steps must be fresh. All alcohol and xylene should be replaced by fresh if the following is noted

A pale murky staining color

Loss of sharp contrast and staining reaction of nuclear chromatin and cytoplasm

Microscopic water droplets on the smear

When manual staining is done, it is a good practice to blot the slides carrier on a thick pile of blotting paper for a few seconds. This will minimize transfer of stain and alcohol from one jar to another and also save the solution

Shelf life of the stains may be increased by storing them in dark (amber) colored bottles when not in use and keeping the staining jar covered

Purchasing readymade stain is time saving exercise but they are not cost effective. Furthermore, the composition and shelf life are unknown and hence the staining results are not optimal.

DESTAINING AND RESTAINING PROCEDURE

Occasionally, it is desirable to destain and restain poorly stained slides or when special stains are required. They can be destained and restained after the removal of coverslip as follows.[2]

The coverslips are removed by soaking the slides in xylene till the coverslip falls off

The slides are transferred into a jar containing absolute alcohol for a sufficiently long time (½ of h)

The slides are then placed in 1% acid alcohol solution till the smear is completely colorless. It may require a few dips to 1 min or even longer, depending on the thickness of the smear

The slides are gently and thoroughly washed in running tap water so that all the traces of acid is removed

Then the slides are stained by routine PAP staining technique or any other desired stain.

REHYDRATION TECHNIQUE FOR AIR-DRIED SMEARS

The rehydration procedure may be used for air dried smears stored upto 72 hours before staining with HE and Pap.[6] It must, however, be noted that squamous cells may be restored to a considerable extent after rehydration. Whereas cells of the secretory type often suffer irreparable damage. The simplest technique of rehydration is as follows:

Place the air-dried smears in 50% aqueous solution of glycerol for 3 min

Rinse the slides in two changes of 95% alcohol and then stain the slides by routine PAP stain.[3]

METHOD OF COVERSLIPPING

Place coverslips on clean blotting paper

Remove the smear from xylol, drain off the excess xylol

Place one or two drops of mounting medium DPX on the smear, invert the coverslip on the smear

Apply gentle pressure, so that the mounting medium will spread uniformly and to remove air bubbles squeeze out excess mounting medium

Using xylene moistened soft absorbent paper, wipe the edges of the slide clean

Lay the slide flat till dry.

COVERSLIP REMOVAL METHODS

Xylene at room temperature: Most commonly used solvent for coverslip removal. It dissolves DPX. Its turnaround time is more and takes 72–94 h for coverslip removal

Xylene at 56 degree Celsius: Slides are kept in glass jar containing xylene and immersed inside water bath maintained at 56°C.[5] Heat fastens the melting of DPX

Freezing: Fastest method of coverslip removal. Slides are kept in freezing chamber of domestic refrigerator at 0–4°C with coverslip facing downwards

Petrol: Dissolves DPX. Plastic airtight jar with cap is used

Diesel: Dissolves DPX. Plastic airtight jar with cap is used

Liquid nitrogen: Not easily available at the setup

Ultrasonic vibrations: May cause damage to the tissue section

Scratching coverslip along with application of ice block: Causes damage to smear/tissue section on the slide.