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Appendix F: Technical Background of Papanicolaou Staining
requirements. Since a small volume of stain is applied, stain usage is comparable to dip staining.
2. No Dilution
Stain concentration does not vary with time due to evaporation or dilution with carryover alcohol and loss
during daily filtering procedures as in the traditional dip method. The wash alcohol is always fresh and
uncontaminated of stain or cell debris. Alcohol usage is reduced up to 50% compared to the dip method of
staining.
3. Centrifugal Clearing
The Aerospray Cytology employs a centrifugal clearing to remove excess reagents. See Stain Chemistry -
Clearing and Mounting below for more information.
4. Throughput
Through optimization of the staining times required, and employing spray washes, the cycle time for the
Aerospray Cytology is approximately 4.5 to 32 minutes. This is ideal for quick turnaround times. Using the
default settings the instrument is shipped with, the instrument has a capacity of about 80 slides per hour
using the 12-slide carousel, and 120 slides per hour when using the 30-slide carousel.
Stain Chemistry
1. Hematoxylin Stain
Hematoxylin is a natural product extracted from the logwood tree of South America. It is not a dye but
is oxidized to hematein. which is the staining principle when combined with a suitable mordant such as
aluminum to form AL- hematein.
The oxidation can be accomplished with a wide variety of oxidants
oxyhematein is produced. This is considered to be a cause of stain degradation and contributes to poor
nuclear staining. Another mechanism of stain degradation is precipitation. Most hematoxylin stains are
aqueous solutions of aluminum hematein. Since hematein is only sparingly soluble in water, this leads to
precipitation of the active ingredient and hence, a weakened stain concentration.
The Gill formula
is much more soluble in ethylene glycol than water
Baker and Jordan
attractive properties
ELITechGroup offers two concentrations of a Gill type formulation, providing excellent long-term stability. It
is formulated by actual hematoxylin content as measured by the method of Marshall and Horobin
commercial hematoxylins can vary considerably in purity. The Gill hematoxylin is used in a progressive
staining method yielding fine nuclear detail.
2. Bluing Agent
In order to "blue" the hematoxylin, the pH of the cellular environment must be raised to above 5.0.
This can be accomplished with a variety of bluing agents–even distilled water of the correct pH
Aerospray employs a dilute solution of sodium bicarbonate which can also be used for hydrating the cells
prior to hematoxylin staining. Since the molarity of the rinse is low, the acidity of the hematoxylin solution
overpowers the weak buffering capacity of the rinsing agent until the hematoxylin solution is essentially
removed from the slides. In addition, the low molarity avoids the need to remove any excess bluing agent
by a water wash. This unique modification of the ELITechGroup papanicolaou stain avoids the need for a
separate water washing system and shortens the staining time.
A second unique modification to the bluing reagent is the 10% alcohol content. This low percentage of
alcohol helps to reduce the forces exerted on the slide when it is subsequently exposed to 100% alcohol
for the dehydration series prior to the OG stain.
3. Orange G Stain
Orange G is a small, negatively-charged dye important to the papanicolaou stain for visualizing keratinized
tissue. It also enhances the EA stain by acidification of the smear
, is a popular modern commercial stain. It recognizes Baker's observation that hematein
(19)
, and the capabilities of a high-contrast progressive stain as described by Cole
(21)
of a mordant quotient of 8-16 is also provided.
(23)
. If hematoxylin is overoxidized,
(4)
. It employs half oxidized hematoxylin according to
(20)
.
(6)
. The
(22)
, since
(24)
. The
(20)
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