Special Techniques in Cytology
Table 31.2 Progressive Staining Method—Cont'd
University o f Chicago section
of cytology laboratory
University of Rochester
Johns Hopkins cytopathology
path #1 coverslip,
1 oz precleaned,
24 x 50 mm
Trior to 1992.
CK Plowden, personal communication, 2007.
Fig. 31.4 A conventional Papanicolaou smear from a patient with a high-
grade squamous intraepithelial lesion. The cell sample was stained with the
progressive method of the University of Chicago (Table 31.2) (x HP).
alcohol is suggested. Som e batches require tw o o r three tim es
m ore h e m a to x y lin th an was p reviously required to produce the
same results. I f the lab o rato ry m akes its o w n dyes, it is advisable
to check the batch n um b er and c olor ind ex (C I) p rinted on the
h e m a to xylin b o ttle and includ e th is in fo rm a tio n in the lab ora-
to ry procedure m anual to ensure the same ingredients are used
to m ake up each batch o f h em ato xylin. Differences in hem a-
to x y lin m ay also be due to differences in th e soil in w h ic h its
source m aterial is grown.
It is im p o rta n t to date h e m a to x y lin w h en it is received in the
lab o rato ry because the dye m ay oxid ize over tim e, especially
in m o is t climates. The lig h te r the crystals, the less oxidized the
h e m a to xylin ; the darker the crystals, the m ore hem atein (o r o xi-
dized h e m a to x y lin ) is present. L illie stated th a t overoxid ation is
one o f the m a in causes o f a p o o r h e m a to xylin stain.
In fo rm a tio n provided b y the m anufacturer m ay n o t indicate
the actual s h e lf life o f dyes. It is best to date the b o ttle w hen
it is received and record the date w h en it is opened. For stock
and w o rk in g solutions, the date w h en it was m ade and the date
w h en it was first used m ay help the lab o rato ry establish the
q u a lity c on trol o f these dyes.
C ertain com ponents in h e m a to x y lin fo rm u la s help transform
h e m a to x y lin in to a usable nuclear dye. These are (1) an o xid iz-
ing agent, (2) a m ord ant, (3 ) a solvent, and (4) a substance used
fo r acidification.
The o xid izing agent is used to begin the rip e n in g process
th a t aids in the tra n sfo rm a tio n o f h e m a to xylin to hem atein, the
active coloring agent. T he o xid izing agents m ost often used are
sod ium iodate and m ercuric oxide. M ercuric oxide is used to
oxid ize and rip en H arris h e m a to xylin , and chloral hydrate is
added as a preservative, as recom m ended b y Mayer. These are
toxic chemicals and sho uld be avoided i f possible.
The m o rd a n t is a substance th a t is responsible fo r the ind uc-
tio n o f c olor in the dye. A lu m in u m sulfate o r a lu m supplies
p o sitively charged ions th a t act as a bridge to chem ically u n ite
the negatively charged hem atein to the negatively charged
phosp horic acid o n the D N A chain.
The solvent in h e m a to xylin is the substance th a t dissolves
hem atein in to s o lu tio n . E thylene glycol is used in certain fo r-
m ulas fo r th is purpose. T he solvent acts as a leveling agent and
helps reduce the rate o f o xid atio n.
A cid ification, the fin a l com ponent, ensures selectivity to
nuclear m aterial and aids in preventing o xid a tio n o f the dye. It
does so b y sta b ilizing the a lu m in u m -h e m a te in com plex. G lacial
acetic acid o r citric acid is used fo r th is purpose.
Soost and coworkers used H arris h e m a to xylin b o th progres-
sively and regressively in a c o n trolle d study.39 T h e ir finding s
showed th a t w ith the progressive m eth od the c hrom a tin was a
fin e ly d istrib uted structure, hyperchrom asia was accentuated,
and there was m oderate sta in in g o f the nuclear envelope. Poly-
chrom asia was accentuated and there was one peak o f nuclear
ab sorp tion at 540 nm .
However, w ith the regressive m ethod , the nuclei were u n i-
fo rm ly d a rkly stained w ith sig n ific an tly hig h er areas o f nuclear
hyperchrom asia. There were textural features as w e ll as con-
trast between nucleus and cytoplasm . There were tw o peaks o f
nuclear ab sorp tion at 530 and 590 nm .
These researchers concluded th a t the progressive m ethod
is better fo r cell m easurem ent and fits the requirem ents fo r