Rachel,
The following notes may help
Tony

 PAPANICOLAOU  STAINING


The original staining procedure was developed by
George N. Papanicolaou who modified his hormonal stain
in order to visualize cancer cells.

The three main advantages of this staining procedure
are:

 (1)  Good definition of nuclear detail.

 (2)  Cytoplasmic transparency.

 (3)  Indication of cellular differentiation of
squamous epithelium.

It is a polychrome staining method which depends on
degree of  cell maturity and cellular metabolic
activity.

The cell samples are smears and must be well fixed in
95% alcohol or a substitute e.g. 100% methanol, 80%
propanol, 95% alcohol: ether = 1:1.

The haematoxylin nuclear stain demonstrates
chromatinic patterns of normal and abnormal cells. 
The counterstains, Orange-G and E.A.  (eosin-azure)
have a high alcoholic concentration which provides
cytoplasmic transparency.  This enables clear
visualization through areas of overlapping cells,
mucus and debris.

There are four main steps in the staining procedure:

                    (1)  Fixation.

                    (2)  Nuclear staining.

                    (3)  Cytoplasmic staining.

                    (4)  Clearing.

>From fixative (95% alcohol) the cells are hydrated
through a graded series of alcohols to water
preparatory to haematoxylin immersion.  The
haematoxylin is an aqueous solution.  The cells are
then dehydrated prior to immersion in the alcohol
based cytoplasmic counterstains. Grading the alcoholic
solutions in a stepwise manner is  thought to minimise
cellular distortion and reduce cell loss from the
glass slide, due to convection currents in the
solutions.

The haematoxylin nuclear stain is a natural stain
which has been used for over 100 years in histology. 
It has affinity for chromatin, attaching to sulphate
groups on the D.N.A. molecule.  Harris' haematoxylin
is the commonest cytologically and has the advantage
of being usable almost immediately after preparation,
whereas other types need to mature. The regressive
stain method (recommended) gives especially clear
contrast.

1. Overstain components by unacidified alum
haematoxylin. This acts as a basic stain with affinity
for basic  nucleoproteins.  Its pH is 2.5 to 3.
2. Remove excess stain by briefly dipping in dilute
hydrochloric acid.
3. Rinse off hydrochloric acid in water as the acid
action will continue.
4. `Blue' in a suitable solution, e.g. Scotts Blue,
Lithium  carbonate, dilute ammonium hydroxide, running
tap water, for ten minutes.  The pH of these solutions
vary from 8 to 8.5.  This subjects  the haematoxylin
to alkaline conditions and changes its colour from red
to blue.
5. Removal of the bluing solution as its action
continues.
6. Cytoplasmic staining in alcoholic Orange-G 6 and
rinsing off in 95% alcohol.  This a monochromatic
stain which  colours keratin a brilliant orange.  The
dye has small molecules which penetrate the cytoplasm
rapidly.
7. Cytoplasmic staining E.A. 50 and rinsing off in 95%
 alcohol.  This is a polychrome mixture of eosin,
light green and Bismarck brown.

   Eosin gives a pink colour to:

            (a)  cytoplasm of mature squamous cells
            (b)  nucleoli
            (c)  cilia

     Light green stains cytoplasm of metabolically
active cells blue:

            (a)  parabasal squamous cells
            (b)  intermediate squamous cells
            (c)  columnar cells.

     Bismarck brown does not add a characteristic
colour to the cytoplasm.

8.   Immerse cells in absolute alcohol to dehydrate
completely  preparatory to clearing.

9.   Clearing, in xylol results in cellular
transparency and precedes mounting.  Xylol is the
commonest clearing agent  and is miscible with both
alcohol (absolute only) and D.P.X. mountant. Xylol is
colourless, chemically non-reactive and has almost the
same refractive index as glass which is important to
give the best possible transparency of the image.
                 R.I.  xylol  =  1.494
                 R.I.  glass  =  1.515

The presence of water in xylol causes cloudiness due
to water droplets.  Water and xylol are immiscible.

10.  Mounting.  The mountant:
       (a)  acts as a permanent bond between slide and
 coverslip,
       (b)  protects cell material from air drying and
shrinkage
       (c)  acts as a seal against oxidation and
fading of the stain.

Causes of inconsistent staining

 1.   varying thickness of material on slide

 2.   type of fixative used

 3.   inadequate filtering of stain solutions

 4.   age of staining solution

 5.   degree of usage of staining solutions

 6.   use of chlorinated tap water

 7.   pH of water can effect nuclear staining

 8.   temperature of water

 9.   insufficient rinsing after acid

10.   air drying of slides between solutions

11.   improper draining of slides during staining.
 


The PAP stain is designed to meet 3 staining
objectives:

1.  Good nuclear detail,
2.  Differential counterstaining,
3.  Cytoplasmic transparency.

Cytoplasmic transparency is a function of high ethanol
concentration of the stain. This is important in order
to view multilayered cell agregates.

The vital components of the PAP stain are:

1.  Harris's haematoxylin as a nuclear stain,
2.  Orange G,
3.  Eosin Alcohol 50 (EA50 or EA65).

The effects of Orange G are only evident in smear when
keratinised cells are present. However it is likely
that it enhances red blood cell staining and acts as a
mordant to the subsequent EA stain.

One major problem that is frequently encountered in
PAP staining is batch to batch variation of commercial
haematoxylin samples. These differ remarkably in dye
composition and dye content (1).

Disadvantages of haematoxylin (1):

1. Batch to batch variation of commercial samples,
2. Nonspecific staining of nuclear and cytoplasmic
proteins,
3. Blueing and differentiation are critical steps,
4. Staining solution is not stable (haematein, the
active reagent in haematoxylin solutions, is oxidised
to oxyhaematin),
5. The haematein-metal relationship is hard to
control.

Schulte & Wittekind (1) suggest that these
disadvantages can be avoided by the use of such
synthetic dyes as the thiazine dye thionin and the
triarylmethane dye Victoria Blue B, especially when
used in an alcoholic solution.

Both dyes are chemically pure, stable in solution,
donot overstain and therefor do not require
differentiation and can be used for dye fixation.
Disadvantage is they are sensitive to air drying,
staining intensity decreases when material dries. In
the absence of water, the Coulomb forces between dye
and substrate decrease (1).

In the PAP stain, the phosphotungstic acid-Orange G
solution with two sulphonic groups (SO3Na) and the
eosin Y with two auxochromic groups (COONa and NaO)
are acid dyes that demonstrate an attraction to basic
proteins, such as prekeratin. The amphoteric dye,
light green SF with a C2H5N+ reactive group, possesses
an affinity for the ribonucleic acid of the ribosomes.
The process of prekeratinisation to keratinisation of
epithelial cells appears to progress from a green,
glassy appearance (corresponding to abundant free
ribosomes and tonofilaments) or pale orange, glassy
appearance (corresponding to few free ribosomes and
tonofilaments) to an intense, glassy orange colour
(corresponding to an approximate 1:1 ratio of
tonofilament:amorphous matrix). Cytologically,
tonofilament bundles can be recognised with PAP stain
as concentric rings or their fragments throughout
stages of the keratinization process (5).


A change in the relative proportion of the components
of the EA solution results in a change in the staining
of the cytoplasm of the non-keratinised (noncornified)
intermediate cells, ranging from turquoise (EA36) to
blue (EA65). The cytoplasm of keratinised superficial
squames cells stain pink with all modifications (2). 

Drijver & Boon (2) found that Bismark Brown had no
effect on staining results and omission of Orange G
did not affect the accuracy of diagnosis (since
keratin and rbc are also stained 
by eosin). 

Phosphotungstic acid seems to have two functions in EA
solutions (2):

1.  That of an accentuator, in which its action is the
same as HCl.
2.  That of a mordant for the amphoteric dye light
green.

Drijver & Boon (2) suggest following changes to PAP
stain to save time and money:

1. Omit staining with Orange G since it does not add
to the diagnostic value of the stain,
2. Bismark Brown is omitted from EA stain,
3. Since  they prefer the cytoplasm of non-keratinised
cells to be turquoise, they add enough PTA to give a
solution pH of 4.5.
4. Instead of dissolving the dyes in 96% ethanol, they
use 50% ethanol,
5. Use tertiary butanol rather than xylene as clearing
agent. Less unpleasant to use and cheaper.

Akura & Takenaka (6) have presented an EA stain that
contains Eosin Y, Light Green SF, PTA, HCl and Fast
Green FCF. The staining results are claimed to be
excellent and there was good balance between fast
green and the other dyes. Fast green  is more stable
than light green and less susceptible to fading.

There are two basic approaches to the control of
reagents (3):

1.  Physiochemical standardisation of dye properties,
2.  Standardisation of performance of staining
solutions on a representative substrate.

Difficulty of comparing cytology samples
quantitatively is due in part to variations in the
staining process. Dyes vary from batch to batch.

Turner & Collins produced standardised specimens,
derived from biological extracts and composed of
mixtures of proteins and nucleic acids. The
characteristics of such a preparation are:

1. Large number of specimens can be fabricated that
are reproducible to a high degree of precision,
2. Each replicate reacts in the same way with various
staining solutions,
3. Each sample stains uniformly with minimal variation
in light transmission.

Turner & Collins used extracts of bovine liver acetone
powder and nucleoprotamine from salmon testis.

To calibrate stain performance, a standard specimen
must have a spectral response similar to that of the
object or objects being stained.

Schulte (4) studied the effects of air drying on
cytological material. Found air dried nuclei stained
purple, where as immediately alcoholic fixation gave
blue nuclei. Seems that at least the part of the cell
surface that adheres to the slide is already air dried
before being flooded by the fixative (preparatory air
drying).

Air drying is a physiochemical process where there is
more or less complete loss of water from the cells
(especially from the nuclei) connected with structural
alterations of the cell, as spreading of the nucleus
or reduction of the staining reaction after
application of the PAP stain (4).

Alterations caused by airdrying (4):

1. Spreading of cells on the slide surface with a
change of  nuclear area. 3D cell nuclei become flat,
2. Condensation of chromatin. This cannot be fully
restored after reimmersion in water,
3. Favouring/preventing staining reactions. Airdrying
is detrimental to the PAP stain but is essential for
the Romanowsky staining pattern.
The variations of the chromatin texture and
dye-substrate affinity are attributed to alterations
of the tertiary structure of the nuclear proteins (4).


REFERENCES:

1.  Schulte & Wittekind (1990) Analyt Quant Cytol
Histol 12(3):149-160.
2.  Drijver & Boon (1983) Acta Cytolog 27(6):693-698.
3.  Turner & Collins (1987) Acta Cytolog 9(6):521-530.
4.  Schulte (1986) Diagn Cytopathol 2:160-167.
5.  Hess etal (1981) Acta Cytolog 25(2):111-133.
6.  Akura & Takenaka (1991) Diagn Cytopathol 7(3):317.




--- Rachel Stoat  wrote: > Does
anyone out there know how and why the Pap stain
> does what it does?  Any 
> info/references would be much appreciated!
> 
> Thanks,
> Rachel
> 
>
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>  

=====
Tony Henwood JP BAppSc GradDipSysAnaly CT(ASC)
Laboratory Manager
Histopathology Department
The Childrens Hospital at Westmead
Locked Bag 4001 WESTMEAD, 2145 Australia. http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html

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