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Introduction
George Papanicolaou developed what now is known as the “pap stain” in the early 1940’s
(1-3)
during his
studies to detect the action of ovarian hormones on vaginal specimens. Its origins lie in the earlier staining
methods of Shorr and Mallory as reviewed by Marshall
(4)
.
Over the years, the papanicolaou stain has attained a pre-eminent position in the analysis of gynecological
smears and body fluids. The procedure employs hematoxylin as the nuclear stain with Orange G, Eosin
Y and light or fast green SF as counterstains for cytoplasmic elements. Bismark brown was originally
included but was subsequently shown to be superfluous
(4)
.
The papanicolaou stain is designed to meet three staining objectives; good nuclear detail, differential
counterstaining and cytoplasmic transparency.
First, the nucleus is stained with hematoxylin, fulfilling the first objective of good nuclear detail. The
hematoxylin has an affinity for chromatin and RNA-rich cytoplasm. If the cell is quickly and properly fixed
the nucleus will present a fine granular appearance. Depending on the hematoxylin formulation used, either
a regressive or progressive staining method is employed.
The counterstains provide the differential staining of cells to display their maturity and metabolic state.
This differential staining is partially a function of stain penetration, which depends on the size of the stain
molecule and the density of cellular structure
(4,5,6)
.
Orange G, being the smallest of the three (MW = 452), is able to penetrate dense keratinized cells to
provide a yellow-to-orange color. Eosin Y (MW = 692) competes with the Orange G in the superficial
and other cells which it is able to penetrate within the staining period. This results in the superficial cell
generally being stained pink to red.
Light green SF (MW = 793) primarily penetrates the least dense intermediate cells within the staining
period to stain them blue to green. In addition to the dye size in determining cell differentiation, staining
is also dramatically affected by pH
(5,6)
, presumably by altering electrostatic interactions. In addition, a
mordanting effect of phosphotungstic acid for light green has been described
(6,7)
, and other mechanisms
may also be involved
(4)
.
The cytoplasmic transparency is a function of the high ethanol content of the stain. Cytoplasmic
transparency is important in order to view multi-layered cell aggregates. A substantial number of variations
of the papanicolaou stain have been formulated. The most useful probably being the elimination of Bismark
brown which does not appear to stain any component of Pap smears
(8,9)
, and can form precipitates with
PTA
(10,11)
.
A combined OG-EA stain was developed
(12)
, replacement of hematoxylin with thionin was proposed
(13)
and several quick stains have been formulated
(14-17)
. Current commercial practice still focuses on
minor variations of the traditional procedures. Hematoxylin, OG and EA stains of improved stability and
reproducibility are currently offered. Several formulations of the EA stain, i.e. EA-50, EA-65, are offered.
These differ primarily in the concentration of eosin and fast or light green.
Due to the wide range of commercial and private formulations available, the stain is far from standardized.
Formula selection depends primarily on personal or regional preferences. The EA-50 solutions
ELITechGroup provides are filtered through a 0.2 µm filter to provide particle-free staining solutions.
The ELITechGroup Staining System
In adapting the traditional papanicolaou stain to the Aerospray stainer, several benefits have been
incorporated which provide more flexibility in the papanicolaou staining procedure:
1. No Cellular Cross-Contamination
Since only fresh unused stain is applied to the slides, cellular cross-contamination from dislodged cells
in the stain baths is impossible. This benefit eliminates the need to filter stains to rid them of sloughed off
cells. Because each slide is individually stained on a rotating carousel, it is virtually impossible for cells
that may dislodge from slides to deposit on another slide. Varying specimens can be stained in the same
carousel without cellular cross-contamination but are usually separated because of differing staining
Appendix F: Technical Background of Papanicolaou Staining
