THE PAPANICOLAOU STAIN

THE “PAP” STAIN

 Learning Objectives

1. State the purpose of the Papanicolaou stain.

2. State the three main advantages to the Papanicolaou staining method
3. State the four main tasks involved in the Papanicolaou staining method
4. List and state the purpose of each ingredient found in the main stains used in the
Papanicolaou staining method.
5. Describe hematoxylin and hematein by:
a) Source
b) Formula
c) Structure (chromophore and auxochromes)
d) Classification.
6. Describe the oxidation of hematoxylin by:
a) Chemical agents
b) Natural ripening.
7.Describe the formation of an alum hematoxylin lake.

2
 Learning Objectives

8. Explain why the surface of Harris hematoxylin should be cleared daily.

9. For the Papanicolaou stain, describe each of the following steps including the
reagent(s) used for:
a) Nuclear staining
b) Differentiation
c) Blueing
10. State the purpose of the running tap water following the differentiation and
blueing steps used in the Papanicolaou method.
11. Explain why gradual hydration and gradual dehydration is thought to be
beneficial.
12. Describe Orange G by:
a) Charge
b) Structure
c) Mechanism of staining

3
 Learning Objectives

13. State the purpose of the 95% alcohol after Orange G and Eosin Azure
staining.
14. Explain the difference between E.A. 50 (or 36) and E.A. 65.
15. Describe Eosin Y by:
a) Source
b) Structure
c) Classification
d) Solubility
e) Mechanism of staining
16. Describe light green S.F. Yellowish by:
a) Charge
b) Structure
c) Mechanism of staining
17. List and state the importance of the steps required to complete the Papanicolaou
stain after the E.A. solution.
18. During the process of mounting, explain the importance of choosing the correct
mounting media and coverslip thickness.
4
 Learning Objectives

19. Identify which dye in the main stains is responsible for staining:
a) parabasal cell cytoplasm
b) intermediate cell cytoplasm
c) superficial cell cytoplasm
d) RBC's
e) keratin
f) white cell cytoplasm
g) nuclei
h) mucous

20. Explain why each stain must be filtered before initial use.
21. Explain the importance of filtering dyes daily.

5
 Learning Objectives

22. Identify four potential sources of cross-contamination of specimens.

23. Identify the potential sources for excessively dark nuclear staining.
24. Identify the potential sources for pale nuclear staining.
25. Explain why an entire slide may have a cloudy appearance upon completion of
the Papanicolaou staining method.

6
 Glossary

ACCENTUATOR: A solution that increases the intensity of staining.

ACID DYE: A dye with a negative charge that stains positively charged tissue
components.

AMPHOTERIC DYE: A dye that behaves as an acid (negative charge) dye or a basic
(positive) dye, depending on the pH of the specimen.

AUXOCHROME: Greek meaning “increase colour” is a group of atoms attached to a

chromophore which modifies the ability of that chromophore to absorb light.

BASIC DYE: A dye with a positive charge that stains negatively charged tissue
components.

CLEARING: Replacement of alcohol by the solvent of the mounting medium during

specimen processing. This raises the refractive index to that of glass and makes the
cells even more transparent.
7
 Glossary

CHROMOPHORE: is the part of a molecule responsible for its color.

DECOLORIZATION: The removal of all macroscopically visible stain.

DEHYDRATION: In specimen processing, the replacement of water with alcohol.

DIFFERENTIATION: As applied to staining (rather than to cell maturation), the

microscopically controlled removal of stain from certain components so that
specific cellu­lar components only are demonstrated.

HYDRATION: In specimen processing, the gradual replacement of alcohol with

water.

MORDANT: An intermediate substance which combines with a weak dye to

increase its affinity for tissue structures.

8
 Glossary

OXIDATION: The loss of two hydrogen atoms. (This forms a chromophore or

"colour radical", which is a chemical structure necessary for staining.)

-PHILIC: A suffix meaning "attracted to." Eosinophilic structures stain easily with
eosin, for example, and orangeophilic cytoplasm easily absorbs Orange G stain.

PROGRESSIVE STAINING: A procedure in which different elements are stained in

sequence; overstaining is not done first.

REGRESSIVE STAINING: A staining procedure in which various elements are

overstained, followed by differentiation.

9
 Introduction

The Papanicolaou (or "Pap") stain is the routine staining technique used in most cytopathology laboratories.
The stain is used to study details of cells, with the aid of the compound light microscope.

Dr. George Papanicolaou originally developed the stain

in 1942; he published modifications of this method in
1954 and again in 1960. Since that time many
modifications have been recommended by
others, most
eliminating what they perceive as unnecessary steps in
the procedure.
The procedure followed today by many

laboratories is referred to as the

Modified Papanicolaou Staining Method

10
Purpose of the Pap Stain

The purpose of the Pap stain is to provide::

1) Nuclear detail- DNA and the chromatin pattern in

normal and abnormal cells is clearly demonstrated.
2) Cytoplasmic transparency- enabling overlapping
cellular material to be properly evaluated. Smears
processed through solutions with high alcohol
content also enable one to see through mucus and
debris.
3) Cellular differentiation (reflecting the maturity and
activity level of the cells)- the different staining
properties of cellular material make it possible to
distinguish between superficial, intermediate and
parabasal cells

11
 Pap Stain

Cytological staining can be divided into four main tasks:

1. Fixation of the specimen

2. Nuclear staining
3. Cytoplasmic staining
4. Clearing

Hydration, dehydration and rinsing of the specimen are also tasks that aid in
successful staining.

12
 Staining Overview

Specimens fixed in 1.
0.95 ethyl alcohol FIXATION

Hydration to prepare for

aqueous staining

REGRESSIVE METHOD
PROGRESSIVE METHOD
Overstaining in hematoxylin
Stain to desired intensity in
H2O rinse 2. hematoxylin
NUCLEAR
STAINING
Dilute HCL to remove excess H2O rinse
hematoxylin

Bluing solution to set hematoxylin,

H2O rinse– bluing solution ? H2O then H2O rinse
rinse

Dehydration to prepare for alcohol stain

Orange G 3. Cytoplasmic Staining

Alcohol rinse

EA polychrome

Alcohol rinse

Final dehydration in absolute alcohol to

prepare for clearing step

4.
Clearing in xylene to prepare for mounting
CLEARING
medium

Mount Slides 13
 Step 1) Fixation

• As discussed previously in the cytopreparation unit, once the cells have been
prepared on a slide, they must be immediately fixed.
• Fixation is a crucial step in preparing specimens for microscopic examination. Its
objective is to prevent decay and preserve cells and tissues in a “life-like” state.
• Improper fixation may result in "drying effect" from drying of the cells before
fixation. This artefact causes the cells to appear "blown up"/enlarged and
degenerated; the smear itself may look excessively pink (eosinophilia).
• Most cytology samples are fixed in 95% alcohol.

14
 Step 2) Nuclear Stain
Hematoxylin
• Cell nuclei are stained by hematoxylin.
• In order to fully understand hematoxylin staining, the nature of the dye
itself must be understood.
• Hematoxylin is a natural substance that is obtained from the heartwood
of the Logwood tree Hematoxylin campechianum. The wood is first
chipped and extracted with ether, then evaporated to dryness and the
residue dissolved in water. (“Haematoxylum” derives from the Greek
word for “blood-wood”) The hematoxylin powder is then crystallized out
of solution. This powder is not readily water soluble.
• Hematoxylin does not contain a chromophore and would therefore be
better described as a potential dye rather than a true dye.
• In order to convert the hematoxylin to a true dye, oxidation to hematein is
required. (Fig 2)

15
Hematoxylin to Hematein

oxidation

16
 Nuclear Stain

• This involves the removal of two hydrogen atoms and by doing so a

chromophore is produced.
• Oxidation may be performed using chemicals. Currently most
formulations incorporate a chemical oxidant such as sodium iodate that
rapidly converts hematoxylin to hematein.
• Oxidation may also occur when exposed to air = natural ripening
• The change in structure results in the conversion of the relatively
colorless hematoxylin to the reddish/brown hematein. In addition, the
oxidation to hematein is necessary in order to bind metal ions such as
aluminum

17
 Hematein + Aluminum salts

• The formula of hematein is C16H12O6 (Figure 2B).

• It has a quinonoid chromophore and three hydroxyl auxochromes that
ionize to give a negative charge, therefore it is considered to be a weakly
acid dye.
• Hematein itself has very poor staining properties and in histological
technique it is used in combination with a mordant.
• Mordants are salts, usually sulfates of a heavy metal such as aluminum
or iron. Double sulfates or alums are commonly used.
• The Papanicolaou utilizes either ammonium aluminum sulfate
(ammonium alum) or potassium aluminum sulfate (potassium alum) to
combine with hematein to form a lake.

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 Lake formation

• In order to form a lake, a dye must contain certain chemical groups in

close proximity.
• A hydroxyl group (OH) and a carbonyl group (=O) are most commonly
involved.
• Aluminum ions from the mordant are bound by the two groups, first
ionically, and eventually as a chelation complex.
• The staining solutions are commonly acidified with either acetic or citric
acid to increase the selectivity of hematein for the phosphate groups of
nucleic acids in DNA and reduce background staining

19
Chelation Complex

20
 Binding with DNA

Lake forming groups

Phosphate groups of
nucleic acid

21
 Regressive and Progressive Nuclear Staining
Technique

• In the Papanicolaou stain the solution is an alum hematoxylin.

• There are many different staining formulae; the one commonly used is
Harris' hematoxylin.
• Alum hematoxylin is a compound staining solution that attaches to the
tissue with the help of a mordant. This is known as indirect staining, i.e.
direct staining does not require a mordant. The staining solution is a
basic (cationic) lake, is positively charged and may be used
progressively or regressively.
• A progressive nuclear staining method entails leaving the slides in the
staining solution only until the desired staining intensity is reached. This
is less commonly used.

22
 Regressive and Progressive Nuclear Staining
Technique

• The regressive technique is much more common and involves

overstaining the nuclei and then differentiating (excess stain is removed).
• When alum hematoxylin is applied regressively, salt linkage occurs with
the phosphoryl groups in the nucleic acids of the nucleus and the
ribosomal RNA in the cytoplasm (Figure 4).
• It will also bind to sulfate groups (in mucins) and, to a lesser extent, with
carboxyl groups in both mucins and proteins.
• As with the binding of the mordant to the dye, the binding of lake to
tissue/cells may also be in the form of ionic attraction or chelation
complexes.
• At this step, there is also general surface adsorption to all the cells.
Examination of slides will show an overall blue-purple colour with little
specific detail.

23
 Summary of Ingredients for common nuclear
stains

• Harris’ Hematoxylin – for regressive nuclear staining

• Hematoxylin - potential dye
• Absolute alcohol (ethanol) - solvent of hematoxylin
• Sodium iodate – oxidizer (replaced toxic mercuric oxide)
• Ammonium alum - mordant
• Distilled water - solvent of mordant
• Glacial acetic acid - accentuator.(intensifies the stain)
• A precipitate may form on Harris' hematoxylin and will leave a metallic sheen on its surface. The surface
of Harris’ hematoxylin should be cleaned daily before use (or the stain should be filtered).
• Gill Hematoxylin – for progressive nuclear staining
• Hematoxylin - potential dye
• Ethylene glycol - solvent of hematoxylin
• Sodium iodate - oxidizer
• Aluminum sulfhate - mordant
• Distilled water - solvent of mordant
• Glacial acetic acid – accentuator(intensifies the stain)
•
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 DIFFERENTIATION
(removal of excess nuclear stain)

• After overstaining with alum hematoxylin, differentiation or microscopic

removal of excess stain is carried out using dilute acid solutions. This is
typically hydrochloric acid in 70% alcohol (acid/alcohol) or dilute acid in
water. The alcohol prevents total ionization of the acid, therefore slowing
down the action of the acid.
• Differentiation of a hematein lake using acid is a complex procedure that
can be simplified by stating that the hydrogen ions compete with the
mordant for tissue groups and effectively block the sites with which the
lake would bind. The lake is more easily displaced from some groups
than others, being extracted first from the carboxyl and sulfate and then
from the phosphate groups.
• Differentiation is allowed to proceed until cell cytoplasm is virtually
colourless and nuclear detail is seen, i.e. the chromatin pattern sharply
defined. It is important that the differentiation is stopped at this point or
nuclear detail will be lost.
25
 Bluing the nuclei (again)

• Acid differentiation causes an undesirable change in colour of

aluminum/hematein lakes, from blue to red. This change in colour is
probably due to the dissociation of the lake and it must be reversed back
to a blue colour after differentiation.
• A mildly alkaline solution will effect this reversal and the nuclei will, once
again, be blue.
• Commonly used bluing agents include alkaline tap water, lithium
carbonate, dilute ammonium hydroxide or Scott's tap water substitute
(sodium bicarbonate and magnesium sulfate).
• Care must be taken after the use of a bluing agent to ensure its complete
removal by thorough washing as any traces could adversely affect the
subsequent staining.

26
 DEHYDRATION

• The solvent of the next dye, Orange G or (OG), is 95% alcohol. The
slides must therefore be dehydrated. To remove the water, the slides
can simply be placed in 95% ethanol.
• Some advocate the use of graduated alcohols as this is thought to be
less traumatic to the cells. In practice this is not necessary. It lengthens
the staining time, uses more solutions and the dilutions take time to
prepare.

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 Step 3) CYTOPLASMIC STAINING
or Counter stains

• Cytoplasmic stains or counter stains such as OG-6 (Orange G plus

phosphotungstic acid) or EA, are synthetic dyes.
ORANGE G
• Orange G is a monochromatic dye that stains keratin a bright orange.
Orange G is a strong acid dye, with an azo chromophore and three
auxochromes, two sulphonic and one hydroxyl (Figure 5).
• It has the smallest molecular weight (452.4) of the acid dyes used in
this method. It stains all cytoplasmic elements by ionic bonding, but is
later displaced by the larger dye molecules.
• The orange G remains in keratin, which has a dense protein
meshwork. Keratin is not normally present in cervical epithelium, but is
found in the presence of a keratinizing squamous lesion.
• The presence of bright staining keratin in a cervical smear is a significant
finding.
28
OG-6

29
 OG-6 Summary

• A small amount of phosphotungstic acid is added to the Orange G dye

solution to maintain a solution more acid than pH 3.0. This results in an
overall net increase in the number of positive charges available in the
cell cytoplasm for the Orange G to attach to.

• Summary of Ingredients for: OG-6

• Orange G = acid dye
• Distilled water
• 0.95 alcohol - solvent of Orange G
• Phosphotungstic acid - accentuator

30
 Keratin in a squamous cell

• Keratin is highlighted by OG 6, in this squamous cell in the center of the

image, as a bright “refractile” orange cell. This is an abnormal squamous
cell

31
 95% Alcohol

• After staining with Orange G, the slides are rinsed in 95% alcohol. This
removes excess OG dye and prepares the cells for immersion in the
Eosin Azure dye, which is also dissolved in 95% alcohol.
• Slides should not remain standing in alcohol for prolonged periods,
however, as this will remove cytoplasmic stain from the cells.

32
 EA
the second cytoplasmic stain

• EA is a polychrome cytoplasmic stain containing two acid dyes, (eosin Y,

light green SF yellowish), phosphotungstic acid and sometimes one
basic dye Bismark brown
• The variation in the amount of light green SF yellowish divides EA into
two main groups: EA50 and EA65. EA-50 contains a larger portion of
light green dye, approximately twice as much as EA-65.
• Generally EA50 is used for gynecological specimens and EA65 is used
for all other types of cytology specimens. The lower concentration of light
green S.F. Yellowish in E.A. 65 is preferable for specimens where excess
mucus may mask cellular detail.
• Single ions and dimers of eosin and the larger aggregates of light green
compete with one another for binding sites in the cytoplasm, but do not
displace the smaller molecular weight Orange G from the keratin.

33
 EA

• Phosphotungstic acid (PTA) is added to the E.A. solution, the

concentration having a direct effect on the pH of the solution. It acts as a
dye excluder, optimally at pH 6.5, enabling eosin and light green to stain
• The eosin in the formula will stain superficial squamous cells, nucleoli,
erythrocytes and cilia.
• Light green will stain intermediate squamous cells, parabasal squamous
cells, columnar cells, (such as endocervical cells),and histiocytes among
others.
• Bismark brown in the EA formula has repeatedly shown to link with the
phosphtungstic acid making it too weak for good use. A newer EA
formula has eliminated the Bismark Brown
• EA is neither an abbreviation for Eosin Azure nor Eosin Alcoholic

34
Cytoplasmic Stain EA

• Superficial cell is stained with

Eosin Y and are said to be
“eosinophilic” or pink/red
• Intermediate squamous cell is
stained with Light green SF
yellowish
• Endocervical and metaplastic
cells also stain with the Light
green SF yellowish
• More metabolically active cells
will stain various shades of
blue/green

35
 Cytoplasmic Staining
Eosin Y
• Especially in an alcoholic solution, Eosin Y appears to have a greenish
yellow fluorescence. This acid dye has a quinonoid chromophore and is
classed as a xanthene dye.
• Eosin has a carboxyl and hydroxyl auxochrome that will attach by salt
linkage to amino groups in cytoplasm, muscle, RBC's and structural
proteins (such as collagen). These tissue components are termed
acidophilic as they have an affinity for acid dyes.
• Different intensities of eosin staining are seen due to the varying size of
protein meshworks and the number of dye binding sites. Eosin has a
larger molecular weight than Orange G (but smaller than light green) and
stains superficial cells (mature squamous cells), nucleoli, cilia and red
blood cells.
• Eosin Y therefore, penetrates the cytoplasm of cells faster, but is often
displaced by light green SF yellowish with increased staining times in
EA.
36
Eosin Y

37
 Light green SF Yellowish

• This acid dye has the largest molecular weight (792.8) of the three dyes
used in the Papanicolaou stain. It has a quinonoid benzene
chromophore, three sulphonic auxochromes, and two ethyl modifiers
(Figure 8).
• Light green attaches by salt linkage to positively charged amino groups
in metabolically active cells, e.g. parabasal, intermediate squamous cells
and columnar cells.
• Porosity (physical theory of staining), is also a mechanism of staining.
These components have a less dense protein meshwork than tissue
components stained by the other dyes. Mucus acquires a bluish-green
colour because of staining by the light green.

38
 Light Green SF Yellowish

• As mentioned previously, light green displaces eosin Y from

the cytoplasm.
• One of the most common staining artefacts seen with the
Papanicolaou stain is artificial eosinophilia in the center of
large groups of cells. Longer staining times in EA stain will
allow sufficient time for the light green to displace the eosin Y
and will provide more uniform staining of large groups of
cells.

39
 Light Green SF Yellowish

Figure 8

40
 EA Summary

Summary of Ingredients for Eosin Azure (EA) - cytoplasmic staining

• Light Green SF Yellowish in 0.95 alcohol - acid dye

• Bismarck Brown in 0.95 alcohol - basic dye (recently excluded)
• Eosin Y in 0.95 alcohol - acid dye with yellow fluorescence
• Phosphotungstic acid - accentuator

After staining in the E.A. solution, the slides are rinsed in 95% alcohol to
remove excess dye. Do not allow slides to remain standing in alcohol as this
will remove cytoplasmic stain from the cells.

41
 Final Dehydration and Clearing

• Specimens are gradually dehydrated in absolute alcohol and cleared

with xylol. Clearing in xylene raises the refractive index of the cells and
is miscible with the solvent of the resinous mountant.
• Xylene (also called xylol) is a toxic agent that is difficult to dispose of.
• Some laboratories have opted to use xylene substitutes, which fall into
two categories. There are citrus derived solvents that principally contain
limonene (e.g. Hemo-De, CitiSolv, etc.). Other xylene substitutes are
based on in the aliphatic (paraffinic) hydrocarbon family (e.g. Pro-Par,
Clear-Rite, etc.). Some find the citrus smell of the limonene products
irritating and may develop sensitivity to it. Additionally, making these
products work with the staining and mounting procedures may take
some work. Some types of mountant media are not compatible with
different substitutes and automatic coverslippers work best with slides
wet with xylene.

42
 Modified Pap Stain used at the Michener

METHODOLOGY
1. 95% ethyl alcohol. . . . . . . . . . . . . . . . . . . . . . 8 dips
2. 50% ethyl alcohol. . . . . . . . . . . . . . . . . . . . . 8 dips
3. DISTILLED WATER . . . . . . . . . . . . . . . . . . 8 dips
4. HARRIS' HEMATOXYLIN . . . . .1 minute for unstained slides (30 sec for previously stained)
5. Stand in tap water. . . . . . . . . . . . Until clear
6. 0.5% HCl in 70% Ethyl alcohol . . . . . . . . . . . 8 quick dips
7. Stand in tap water. . . . . . . . . . . . . . . . 8 dips
8. Scott's Tap water substitute. . . . . . . . . . . . . . 1 minute
9. Stand in tap water . . . . . . . . . . . . . . . 5 minutes
10. 50% ethyl alcohol . . . . . . . . . . . . . . . . . . . . . . 8 dips
11. 95% ethyl alcohol . . . . . . . . . . . . . . . . . . . . . 8 dips

12. ORANGE G. . . . . . . . . . . . . . . . . . . . . 1 min

13. 95% ethyl alcohol . . . . . . . . . . . . . . . . . . . . . 8 dips

14. 95% ethyl alcohol . . . . . . . . . . . . . . . . . . . . . 8 dips
43
 Modified Papanicolaou Stain used at the
Michener cont’d

15. EA (50/ 65). . . . . . . . . . . . . 3 min

16. 95% ethyl alcohol . . . . . . . . . . . . . . . . . . . . . 8 dips
17. 95% ethyl alcohol . . . . . . . . . . . . . . . . . . . . . 8 dips
18. 95% ethyl alcohol . . . . . . . . . . . . . . . . . . . . . 8 dips
19. ABSOLUTE ALCOHOL. . . . . . . . . . . . . . . . .. . . . . 1 minute
20. ABSOLUTE ALCOHOL/XYLOL (equal parts). . . . . . . . .. . . 1 minute
21. XYLENE. . . . . . . . . . . . . . . . . . . . . . . . . . . . ………………….2 minutes
22. XYLENE. . . . . . . . . . . . . . . . . . . . . . . . . . . . ………………….1 minute
23. XYLENE. . . . . . . . . . . . . . . . . . . . . . . . . . . . ………………….1 minute
24. XYLENE. . . . . . . . . . . . . . . . . . . . . . . . . . . . ………………….1 minute

44
 Mounting or Coverslipping

Mounting media should match the refractive index of the coverslip as

closely as possible. Two types of mounting media are available: resinous
and aqueous. Examples of these include:
1) Resinous
• Permount- commonly used for routine smears
2) Aqueous
• Saline
• Glycerol
Aqueous types of mounting media are not suitable for permanent slides, i.e.
cytology slides.
A coverslip thickness of No. 1 is recommended for cytologic preparations.
In size, a coverslip should be selected that covers as much of the specimen
as possible.

45
 Coverslipping the slide

• Manual coverslipping can be done in various ways and should be

performed in a well ventilated area. Most large labs now use an
automated coverslipper. However, when the automated unit fails, the
technologist must be prepared to coverslip slides manually.
• The following mounting procedure is an example of one that can be used
for slides.
• Place the required amount of mounting medium on a coverslip (use a
glass rod, if the bottle does not contain an eyedropper). The amount
depends on the volume and thickness of the smear. Avoid using too
much as this will cause a blurred image.
• Remove the slide from the xylene by holding the labeled end with
forceps. Turn the slide over and lower it onto the mounting medium and
coverslip (Figure 9). Clean any excess mounting media and xylene from
the edges and back of the slide with a tissue wipe.

46
 Coverslipping the slide

• Check for air bubbles and remove them while the mounting medium is
still liquid: press against the coverslip with forceps and push the bubbles
towards the edge. Wipe of any excess mounting medium with a tissue
wipe moistened in xylene.
• The slides are allowed to dry before screening to prevent the coverslip
from moving while marking abnormal cells.

47
Coverslipping (Mounting)

48
 Staining Results

• A cytology specimen stained by the Papanicolaou technique gives the

following results:

-Parabasal cell cytoplasm – blue/green

-Intermediate cell cytoplasm - pale blue/green
-Superficial cell cytoplasm - pale pink, pale pink/yellow, or
blue/green (depending upon maturity)
-RBC's - bright red to orange-pink
-Keratin - orange
-Leukocyte cytoplasm - pale blue
-Nuclei - blue to purple with visible chromatin pattern
-Mucus - light green

49
 Decoloring a Slide

Decolorizing or restaining may be required for several reasons:

• Due to fading in an old slide
• Due to a staining error
• When a special stain is necessary (though many special stains can be
done

50
 Procedure to Decolorize

1. Soak the slide in xylene until the coverslip comes off.

2. Soak the slide in clean xylene until the mounting medium is
gone.
3. Soak slides in 95% ethanol to remove the cytoplasmic
stains.
4. Place the specimen in 1% hydrochloric acid in 70% ethanol until the
hematoxylin is removed. This can be checked periodically with a
microscope.
5. Rinse the smear in tap water to remove the acid.
6. If re-staining with the Papanicolaou stain, begin the procedure with
hematoxylin.

51
 Quality Assurance of the Pap
Stain
• There are many aspects of a laboratory’s quality assurance program that
will impact on the Papanicolaou staining.
• For example, laboratories must have specific policies and procedures
that deal with purchasing, storing, labeling, disposing of solutions and
reagents.
• Likewise, any machinery used in the laboratory such as an automatic
coverslipper or staining machine must be regulated by the laboratories
policies and procedures for validation, calibration, routine and preventive
maintenance, etc.
• Automated stainers and coverslippers are now in frequent use in most
laboratories. Be sure to read the operator’s manual in your laboratory
and complete intial training prior to use.
• There are two specific areas addressed by many quality assurance
requirements for cytopathology: daily quality checks of the staining
procedure and prevention of cross contamination.
52
 Automated Laboratory

Automatic Coverslipper Automatic Linear Stainer

53
 Cross Contamination of Specimens

• Cross contamination refers to the problem of cells from one slide floating
off and onto another patient’s slide while staining. This could potentially
lead to a misdiagnosis.
• Many factors can affect the likelihood of cross contamination during the
Papanicolaou staining procedure. The requirements in general terms
state that those involved in processing and reporting of cytopathology
specimens must be aware of the potential problem of cross
contamination and take precautions to prevent it.
• The choice of slide preparation procedures can have a major impact on
the likelihood of floater contamination. Smears from highly cellular
malignant specimens (e.g. positive/malignant body cavity fluids) are
notorious for causing cell floaters. One major advantage of the ThinPrep
processor for non-gynecological cytology samples is that it is extremely
uncommon for the ThinPrep slides to shed cells (even with malignant
effusions).
54
 Cross Contamination of specimens

• Some laboratories develop a list for the order of staining of different

types of samples. For example, positive fluids and respiratory samples
are very cellular and are often stained last. The idea is to stain samples
more likely to produce floaters after those that are less likely to shed
cells. Gynecological specimens must be stained separately from non-
gynecological specimens.
• In addition, all laboratories must have documented policies and
procedures related to the maintenance of solutions used in staining
(filtering and replacing). Stains for example, should be filtered on a daily
basis. Tap water washes used after hematoxylin should be changed after
each use. Alcohol rinses used immediately after cytoplasmic stains are
discarded more frequently. The fact that this maintenance has been
carried out must also be documented.

55
 Quality Control

Laboratories must also check the quality of the staining

procedure microscopically each day and document this check
in a quality control log.

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Symptom
Nuclear stain too pale
Nuclear stain too dark
Nuclei stain
reddish/purplish/brownish
Nuclear stain lacking detail
(smudged or grey)
Nuclear Stain
Trouble Shooting
Cause
Staining time too short in
hematoxylin
Hematoxylin too weak
(exhausted, diluted, etc.)
Differentiation solution too
strong
Too long in differentiation
solution
Differentiation solution not
adequately rinsed
Too long in hematoxylin
Differentiation solution too
weak
Differentiation solution too
weak
Too short time in
differentiation solution
Bluing ineffective
Hematoxylin too weak
(exhausted, diluted, etc.)
Hematoxylin exhausted
Solution
Increase time in
hematoxylin
Replace hematoxylin
Use weaker solution or
decrease time
Decease time in
differentiation solution
Increase time rinses
Decrease time in
hematoxylin
Replace differentiation
solution
Use stronger solution or
increase time
Increase time in
differentiation solution
Increase time or decrease
alkalinity of blueing
solution
Replace hematoxylin
Replace hematoxylin
57
 Cytoplasm Trouble Shooting

Symptom Cause Solution

Cytoplasmic stains too dark Too long in stains Decrease time in stains

Cytoplasmic stains too light Too short time in stains Increase time in stains
Stains exhausted Replace stains
Too long time in alcoholic rinses Decrease time in rinses

Cytoplasm appears blue/grey Too long time in hematoxylin Decrease time in hematoxylin

Groups and clusters of cells stain Too short time in EA Increase time in EA
eosinophilic in center

Cytoplasm lacks transparency Insufficient alcoholic rinses Increase time in rinses

Rinses too dirty (contain too much Replace rinses

stain)

58
 Miscellaneous Trouble
Shooting

Symptom Cause Solution

Dark golden brown pigment on Diffraction from air trapped on Keep slides immersed in xylene
Squamous cells (“cornflake” Squamous cells during coverslipping
artefact)

Purple crystalline granules in Hematoxylin crystals Filter stains daily and before use
smears

Cloudy macroscopic appearance, Insufficient dehydration Change alcohol rinses, maintain

small clear spheres Water contamination proper levels
microscopically

Moisture contaminant in xylene Filter or replace xylols

Moisture contaminant in mountant Replace mountant

59
 References

• Bibbo, M., Wilbur, D., eds. Comprehensive cytopathology, 3rd ed. China: Saunders, Elsevier Inc.;
2008.
• CLSI. Cervicovaginal Cytology Based on the Papanicolaou Technique; Approved Guideline-Third
Edition. CLSI document GP15-A3 [ISBN 1-56238-679-4]. CLSI, 940 West Valley Road, Suite 1400,
Wayne, Pennsylvania 19087-1898 USA, 2008.
• DeMay, R. M. The art and science of cytopathology. 2 nd ed. Chicago: ASCP Press; 2012.
• Keebler CM, ed. The Manual of cytotechnology, 7 th ed. Chicago: American Society of Clinical
Pathologists Press; 1993.
• Koss LG. Diagnostic cytology and its histologic bases, Volume I, 5 th ed. Philadelphia: Lippincott,
Williams & Wilkins; 2006.
• NCCLS. The Papanicolaou Technique (video recording): The Link to Accurate Diagnosis. Wayne, Pa.:
NCCLS Document GP15-A2 1995. (based on NCCLS, Papanicolaou Technique; Approved Guideline.
NCCLS Document GP15-A, Pennsylvania, NCCLS, 1994.)

60