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Nuances of the Papanicolaou stain
*Corresponding author: Dr. Meherbano M. Kamal, Professor, Department of Pathology, Government Medical College, Nagpur, Maharashtra, India. dr.mmkamal@gmail.com, prajakta.sathawane20@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Sathawane P, Kamal MM, Deotale PR, Mankar H. Nuances of the Papanicolaou stain. CytoJournal 2022;19:43.
Abstract
The impressive list of achievements of Dr. G. N. Papanicolaou and his tedious journey from normal to abnormal human cell includes the importance of wet fixation of cells and the development of the unique polychromatic Pap stain. The 5-dye Pap stain method evolved through 2 salient phases. The first being the development of wet fixation using alcohol-ether to enhance cellular transparency and the second phase saw the introduction of various cytoplasmic counterstaining methods using orange G and EA (light green, Bismarck brown, eosin) and phosphotungstic acid, facilitating the distinction of cell types. The specific characteristics of the staining method is, the cellular transparency combined with crisp nuclear staining, achieved through tailored cellular fixation and cytoplasmic staining using variable dye and pH combinations. With little modifications if any the Pap stain continues to be applied uniformly globally. However, institutional supply of dyes and chemicals from different companies make minor modifications, that remain consistent, an essential part of the staining protocol. This chapter describes the preparation and principles of various components of the stain that are being currently used in our department.
Keywords
Papanicolaou stain
Pap stain
Wet fixation
Fixatives
Gill’s modified OG-6
Gill’s modified EA
Rehydration of Pap smears
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.
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
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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
- Manual staining procedure for Papanicolaou stain.
| 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 |
EA: Eosin azure
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 |
EA: Eosin azure
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
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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.
ACKNOWLEDGMENTS
None.
LIST OF ABBREVIATIONS (In alphabetic order)
DNA - Deoxyribonucleic acid
DPX - dibutylphthalate polystyrenexylene
EA - Eosin Azure
GMC - Government Medical College, Nagpur
HIMEDIA - company name
LOBA - company name MERCK - company name
OG6 - Orange G 6 Pap – Papanicolaou
RNA - Ribonucleic acid
STWS - Scott’s tap water substitute
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