TBIH_A_515492.indd

Romanowsky staining in cytopathology: history,
advantages and limitations

KP Krafts

, SE Pambuccian

Department of Anatomy, Microbiology and Pathology, University of Minnesota School of Medicine, Duluth,

1035 University Drive, Duluth, and

Department of Laboratory Medicine and Pathology, University of Minnesota School

of Medicine, 420 Delaware Street SE, MMC 76 Mayo, Minneapolis, Minnesota

Abstract

If the entire discipline of diagnostic cytopathology could be distilled into a single theme, it
would be the Papanicolaou stain. Yet it was the Romanowsky stain upon which the discipline
of cytopathology was founded. Both stains are used today in the cytopathology laboratory,
each for a different and complementary purpose. We trace the history of cytopathological
stains and discuss the advantages and limitations of Romanowsky-type stains for cytological
evaluation. We also provide suggestions for the advantageous use of Romanowsky-type stains
in cytopathology.

Key words:

aspiration cytology , exfoliative cytology , fi ne needle aspiration , metachromasia ,

Pap stain , Wright-Giemsa stain

Historical perspective

The diagnostic procedure that we have come to
know as the Pap test, i.e., diagnosis of cervical can-
cer by cytological methods, was fi rst suggested by
Lionel S. Beale (1828-1906), who stated that “ In cases
of cancer of the uterus, we should expect to meet
with cancer cells in the discharge, when this condi-
tion is suspected, the discharge and also the urine
should be subjected to very careful and repeated
microscopical examination ” (Beale 1878). It was fi rst
used in clinical practice, however, by the Romanian
academic pathologist Dr. Aurel A. Babes¸ (1886-1961)
(Wied 1964, Koprowska 1985, Tasca et al. 2002) with
the help of his colleague, the gynecologist, Con-
stantin Daniel, who procured the samples. Smears
were made from samples collected directly from the
cervix using a platinum loop similar to that used in
bacteriology, fi xed with methanol and stained with
the Giemsa stain. This method of diagnosis of cervical

cancer by smears was presented to the Bucharest
Gynecological Society in 1927 (Babes¸      1963)  and  pub-
lished in the prestigious French journal, Presse Medi-
cale  in 1928 (Babes¸      1928,  1967,  Douglass  1967).  Using
this method, Dr. Babes¸      diagnosed  18  of  20  cervical
cancers, some of them “ incipient, ”  and speculated
that the method would be valuable for diagnosing
precursor lesions (noninvasive cervical carcinoma)
years before this concept generally was accepted
(Broders 1932, Koss 2003). This method of cervical
cancer diagnosis was applied successfully in Italy
(Viana 1928), but soon disappeared from the inter-
national stage, surviving for some time only in its
founder ’ s homeland, Romania (Naylor et al. 2002,
Virtej and Vasiliu 2003).

In 1928, the same year that Babes¸ published his

method for diagnosing uterine cancer by cervical
cytology smears, George N. Papanicolaou (1883-
1962) communicated his observations on fortuitously
identifi ed cancer cells (Papanicolaou 1928) while
studying hormonal changes in vaginal secretions of
women (Papanicolaou 1925, 1933) using a technique
that he had developed for studying the estrous cycle
in guinea pigs (Stockard and Papanicolaou 1917a,b).
He used a small glass pipette to collect vaginal pool
samples, fi xed the samples in a 50:50 alcohol:ether
mixture, and stained them with a complex mixture of

Correspondence: Kristine Krafts, M.D. Assistant Professor
Department of Anatomy, Microbiology and Pathology University
of Minnesota School of Medicine, Duluth 1035 University Drive
Duluth, Minnesota 55812. E-mail: kkrafts@d.umn.edu
© 2011 The Biological Stain Commission
Biotechnic & Histochemistry 2011, 86(2): 82–93.

DOI:10.3109/10520295.2010.515492

Romanowsky staining in cytopathology 83

technique of fi ne needle aspiration spread around
the world, Romanowsky-type stains continued to
be preferred for air dried smears. Papanicolaou or
hematoxylin and eosin stained wet fi xed prepara-
tions usually were employed as well. Currently,
there is wide regional variation in the preferred
type of Romanowsky-type stain. The May-Gr

wald-Giemsa stain is preferred in Europe, while the
Leishman-Giemsa (Garbyal et al. 2006) and the Riu
stain (Tsou et al 1997, 1998) occasionally are favored
in Asia, and the Wright-Giemsa or Diff-Quik (Henry
et al. 1987, Silverman and Frable 1990) stains usu-
ally are used in North America.

Advantages of Romanowsky-type
stains in routine cytological practice

Common practice dictates the use of both a
Romanowsky-type and the Papanicolaou (or hema-
toxylin and eosin) stain to evaluate most cytological
specimens. Because the information provided by each
stain is unique and complementary, both types are
essential for accurate cytological diagnosis. Although
Papanicolaou and hematoxylin and eosin stains show
better nuclear detail and generally perform better on
thick or extensively necrotic smears, Romanowsky-
type stains allow better estimation of relative cell and
nuclear sizes, and superior visualization of cytoplas-
mic details, smear background elements and intercel-
lular matrix components. We discuss each group of
advantages of Romanowsky-type staining below.

Apparent enlargement of cells and nuclei

A major difference between Romanowsky-type and
Papanicolaou stains lies in the method of fi xation;
Romanowsky-type stains are air dried and Papani-
colaou stains are alcohol fi xed. Air drying has a
striking effect on the apparent size and shape of cell
nuclei. Spreading cells onto the glass slide leads to
an increase in their apparent size proportional to
the volume of the nuclei. The degree of perceived
nuclear and cytoplasmic size increase due to air
drying depends on the type of cell; it is smallest in
mature squamous cells. Air dried Giemsa stained
urothelial cells, for example, show an apparent 50%
increase in nuclear area and a 30% increase in cyto-
plasmic area. By contrast, wet fi xed Papanicolaou
stained urothelial cells show a decrease in appar-
ent cytoplasmic area of 15-55% and nuclear area of
10-30% (Boon and Tabbers-Bouwmeester 1980, Boon
and Drijver 1986). Because air dried smears usu-
ally are postfi xed in methanol before staining with
a Romanowsky-type stain, the longer they stay in

dyes including hematoxylin, orange G, eosin Y, light
green SF and Bismarck brown Y. Ironically, while
the current Pap test stain consists of a modifi cation
of the original Papanicolaou stain, the current Pap
test technique most closely resembles that of Aurel
Babes¸ (Koss 2003) by using direct sampling of the
cervix and ethanol or methanol fi xation.

Diagnostic exfoliative cytology in general was

practiced well before Papanicolaou ’ s groundbreak-
ing studies on cervicovaginal smears (Papanicolaou
1942, Papanicolaou and Traut 1941). The fi rst descrip-
tion of malignant cells in a cytological preparation
was published by Lionel S. Beale using unstained
preparations of postmortem pharyngeal aspirates
(Beale 1860-1861, Long and Cohen 1993). He was fol-
lowed in 1881 by Heinrich Quincke (1842-1922), who
studied unstained

“

cell fi lms

”

prepared from the

sediment of pleural and peritoneal fl uid (Quincke
1881-1882). Because unstained preparations did not
allow good differentiation of malignant cells from
“ endothelial ” (mesothelial) cells, Quincke also used
iodine staining. The fi rst researcher to systemati-
cally stain cytological preparations was Paul Ehrlich
(1854-1915). Having previously identifi ed mast cells
and eosinophils using his novel compound blood
stain, he employed in 1882 the same methylene blue-
eosin stain to describe adenocarcinoma cells in pleu-
ral and peritoneal fl uid (Barcia 2007).

The introduction of paraffi n embedding in about

1880 and the cell block technique around the turn of
the 20th century did not supplant cytological meth-
ods, such as smears, which continued to be used for
body fl uids and sputum examination. Variations of
Ehrlich ’ s staining methodology were used for cyto-
logical specimens and hematoxylin and eosin stains
were used on tissue blocks.

Attempts at aspiration biopsy using needle

puncture of tumors had been made during the 19th
century (Long and Cohen 1996) and the beginning
of the 20th century (Martin and Ellis 1930, Stewart
1933). The current practice of fi ne needle aspiration
cytology originated in Europe. Drs. Nils S ö derstr ö m
(1911-1984) (S ö derstr ö m 1956, 1958, 1966) and Six-
ten Franz

n (1919-2008) (Franzen et al. 1960) in

Sweden and Dr. Paul Lopes-Cardozo (1913-2002)
(Cardozo 1950, 1960, Lopes Cardozo and Posthuma
1954) in the Netherlands developed the technique
and employed it successfully in thousands of cases
during the 1950s and 1960s. Because all three sci-
entists were clinicians specializing in hematology,
they used Romanowksy-type stains for the diag-
nosis of aspirates. Many pathologists from the US
and other countries learned fi ne needle aspiration at
the Karolinska Hospital in Stockholm (Linsk 1985,
Schenck 2003, Perez-Guillermo et al. 2005), so as the

84 Biotechnic & Histochemistry 2011, 86(2): 82–93

myoepithelial-rich salivary gland tumors (Torlak-
ovic et al. 1993, DiPalma et al. 1996, Kuwabara et
al. 1997, Chhieng and Paulino 2002, Kumar et al.
2004), melanomas (Siddaraju et al. 2007) and some
carcinomas of the breast (Khalbuss et al. 2006).

Improved visualization of cytoplasmic granules

may be valuable for fi ne needle aspirates of the
thyroid and other organs. A common fi nding in fi ne
needle aspirates of the thyroid is the presence of
paravacuolar granules, which represent lysosomes
containing hemosiderin or lipofuscin pigments
(Sidawy and Costa 1989) (Fig. 1). Paravacuolar gran-
ules are more common in samples from normal thy-
roid than in those from colloid nodules (Layfi eld et
al. 2003), but are found across the spectrum of thyroid
pathology and thus are not specifi c for any pathol-
ogy. Their presence, however, identifi es the aspirate
as coming from the thyroid and helps exclude aspi-
rates of similar appearing cells from the parathyroids
(Abati et al. 1995). Other types of granules potentially
encountered in thyroid fi ne needle aspirates include
the red cytoplasmic granules occasionally seen in
neoplastic cells of medullary thyroid carcinoma
(Us-Krasovec et al. 1998, Kumar et al. 2000). These
granules can be helpful for cytological diagnosis of
this otherwise diffi cult to diagnose malignancy.

The cytoplasmic granules of granular cell tumors

also are demonstrated well by Romanowsky-type
stains, although they can be seen also in Papanico-
laou stained smears. Both Romanowsky-type and
Papanicolaou stained preparations may show fi nely
granular material in the background (Liu et al. 1999)
resulting from the smearing of the fragile cytoplasm
of the neoplastic cells.

Granules are important for recognition of

many hematolymphoid cells and their neoplastic

this fi xative, the closer their size approximates that
of wet fi xed cells and nuclei (Boon and Drijver 1986).
The apparent enlargement of cells and nuclei in air
dried Romanowsky-type preparations, compared to
the cell and nuclear sizes in wet fi xed Papanicolaou
stained preparations, amplifi es the cell and nuclear
size differences within the specimen and permits
more accurate evaluation of relative cell size as well
as nuclear size and shape . In addition, uniform air
drying of well prepared, evenly smeared cytological
preparations stained with Romanowsky-type stains,
as opposed to the unintentional focal air drying of
wet fi xed smears, decreases nuclear size variability,
which makes Romanowsky stained smears prefera-
ble for morphometry (Schulte and Wittekind 1987).

Accentuation of nuclear chromatin

Air drying also infl uences the structure of the nuclear
chromatin, which becomes condensed and thus more
hyperchromatic (Schulte 1986). The accentuation of
size and chromaticity differences between normal
and malignant nuclei produced by Romanowsky-
type stains is a useful feature for evaluating fi ne
needle aspirates, especially when dealing with well
differentiated malignancies (Yang 1994).

Accurate exfoliative cytological diagnosis is

dependent on high power evaluation of nuclear
chromatin changes. By contrast, because they usu-
ally are very cellular and show many small true
tissue fragments, fi ne needle aspirates frequently
can be diagnosed under low power and nuclear
chromatin detail is less important for diagnosis.
Romanowsky-type stains are essential for low
power pattern-based diagnosis (Nayar and Frost
2001, Nayar et al. 2001) owing to their better defi -
nition of cell cytoplasm and their accentuation of
enlarged tumor cell nuclei.

Enhanced visibility of cytoplasmic detail

Romanowsky-type stains enhance cytoplasmic
detail, a useful feature for determining differen-
tiation of neoplastic cells. Clearer cytoplasmic
defi nition also enables better appreciation of the
plasmacytoid appearance of certain neoplastic
cells. Such plasmacytoid cells, defi ned by their
eccentrically placed nuclei, are characteristic not
only of plasma cell tumors (Das et al. 1986), but
also of neuroendocrine tumors of the pancreas
(Shaw et al. 1990, Jhala et al. 2002, Chang et al.
2006, Chatzipantelis et al. 2008), carcinoid tumors
(Nguyen 1995, Hasteh et al. 2007), medullary car-
cinoma of the thyroid (Schreiner and Yang 2009),

Fig. 1. Fine needle aspirate of thyroid showing follicular
epithelial cells with paravacuolar granules in colloid goiter.
Diff-Quik stain.

1,000.

Romanowsky staining in cytopathology 85

While such vacuoles also may be seen in Papanico-
laou and hematoxylin and eosin stained cytological
preparations, they are identifi ed much more easily
and consistently by Romanowsky-type stains.

Cytoplasmic vacuoles and other changes also

may be seen in macrophages. In certain lung lesions,
such as those associated with amiodarone toxicity
and with Rhodococcus equi infection in patients with
AIDS, macrophages may appear multivesiculated
or foamy owing to ingested material (Reyes et al.
1998). In other lesions, such as silicone lymphade-
nopathy and the silicone reaction around implants,
macrophages may display large clear vacuoles con-
taining unstained foreign material (Tabatowski et
al. 1990, Dodd et al. 1993). Large clear vacuoles also
may be found in solid pseudopapillary tumors of
the pancreas (Jhala et al. 2008); their presence is
useful for differentiating these tumors from pan-
creatic neuroendocrine neoplasms with which they
demonstrate an extensive morphological overlap.
All of these cytological changes are seen better in
Romanowsky stained smears.

Another cytoplasmic fi nding of diagnostic value

unique to Romanowsky stained preparations is the
presence of paranuclear blue inclusions (Wittchow
et al. 1992, Mullins et al. 1994, Walker et al. 1994).
These inclusions favor a diagnosis of small cell
carcinoma rather than non-small cell carcinoma or
lymphoma (De Las Casas et al. 2004) (Fig. 4).

Enhanced visibility of smear background
elements

The fragility of tumor cell cytoplasm can give rise to
characteristic patterns that are more visible, or solely
visible, in Romanowsky stained smears. One of

counterparts: eosinophils, mast cells, myeloid pre-
cursors and large granular lymphocytes all have
distinctive granules that are seen poorly or not at all
in Papanicolaou and hematoxylin and eosin stained
smears (Fig. 2). Their recognition in fi ne needle aspi-
rate smears and other cytological preparations fre-
quently is important diagnostically, whether they are
the neoplastic cells or part of the reactive background.
While Romanowsky-type stains frequently show
excellent granule detail, care should be taken with the
Diff-Quik stain, which is an aqueous rather than alco-
hol based Romanowsky-type stain, because granule
contents may be washed away during staining.

Occasionally, fi ne needle aspirates of the thyroid

may show follicular epithelial cells with marginal
vacuoles, a phenomenon known as a

“

fi re-fl are ”

appearance (Das 2006) (Fig. 3). Originally described
as a distinctive feature of hyperthyroidism that could
be identifi ed only with Romanowsky-type stains, the
fi re-fl are appearance of follicular cells is a nonspe-
cifi c fi nding in both neoplastic and non-neoplastic
disorders (Das et al. 1998). When found in metasta-
sis, however, the fi re-fl are appearance is helpful for
identifying the primary tumor, which usually is a fol-
licular carcinoma of the thyroid (Kumar et al. 2005).

Cytoplasmic vacuoles containing lipid can play an

important role in the diagnosis of primary and meta-
static renal cell carcinomas (Tabatabai and Staerkel
2005), aggressive lymphomas such as Burkitt lym-
phoma (Das et al. 1987), pancreatic endocrine neo-
plasms associated with Von Hippel-Lindau disease
(Safo et al. 2009) and secretory breast carcinoma (Aida
et al. 1993). Cytoplasmic vacuoles containing glyco-
gen are seen in seminomas (Fleury-Feith et al.1989)
and tumors of the Ewing family (Guiter et al. 1999).

Fig. 2.

Cerebrospinal fl

uid with NK/T-cell lymphoma

showing a mitotic fi gure and a malignant lymphoid cell
with cytoplasmic granules. Wright stain. Original mag-
nifi cation

1,000.

    Fig. 3.      Fine needle aspirate of thyroid showing follicular
epithelial cells. Arrows show  “ fi re-fl are ”  appearance. Diff-
Quik stain.

400.

86 Biotechnic & Histochemistry 2011, 86(2): 82–93

these patterns is the so-called tigroid background,
which consists of lacy, vacuolated, foamy mate-
rial in a band-like arrangement. Initially described
as characteristic of seminomas and germinomas
(Caraway et al. 1995, Gupta et al. 2008), this fi nding
also has been described in other high grade tumors
with abundant, glycogen-rich cytoplasm (Dusenbery
1997, Khunamornpong et al. 2005, Rau et al. 2006)
(Fig. 5). A similar fi nding is the presence of small frag-
ments of lymphocytic cytoplasm, called lymphoglan-
dular bodies (S

derstr

m 1968), in aspirates from

lymph nodes and lymphoid neoplasms. While not
entirely specifi c for lymphoid tissues and neoplasms
(Flanders et al. 1993), the presence of malignant cells
accompanied by numerous lymphoglandular bodies
favors the diagnosis of lymphoma over other small
round cell tumors (Francis et al. 1994).

Another characteristic change produced by

the fragility of the cytoplasm is the presence of a
“ frothy ” or “ bubbly ” background in fi ne needle
aspirates from normal tissues and tumors with
abundant cytoplasmic lipid such as adrenal tissue,
benign adrenal tumors, lactating breast tissue and
lactating adenomas (Grenko et al. 1990). Recognition
of this background is important for differentiating
benign adrenal lesions from metastatic tumors (Wu
et al. 1998) and for avoiding the diagnostic pitfalls
posed by the marked atypia that accompanies lacta-
tional changes of the breast (Novotny et al. 1991).

Other diagnostically useful background elements

that are readily visible in Romanowsky stained prep-
arations include the presence of caseous necrosis in
necrotizing granulomatous infl ammations (Bezabih
et al. 2002), the presence of abundant thick mucus in
mucinous neoplasms such as pancreatic intraductal
papillary mucinous neoplasms (Stelow et al. 2003),
and the presence of melanin pigment in the aspirate
smears of metastatic melanoma (Gupta et al. 1985).

Superior demonstration of intercellular material

The presence of metachromatic chondroid matrix
material is characteristic for cartilage tumors
(Abdul-Karim et al. 1993) and chordoma (Finley et
al. 1986, Kay et al. 2003), as well as for chondroid
hamartomas of the lung (Hughes et al. 2005). Such
chondroid matrix material usually is spectacularly
visible after Romanowsky-type staining. When only
Papanicolaou stained preparations from pulmonary
hamartomas are examined, this cartilaginous matrix
may not be recognized, which can lead to a false
diagnosis of carcinoid, adenocarcinoma or even
small cell carcinoma (Hughes et al. 2005). Osteoid
also is more clearly visible after Romanowsky-type
stains; it usually appears strongly eosinophilic
(Klijanienko et al. 2007) as opposed to chondroid
matrix, which usually appears magenta.

Recognition of the intercellular matrix also is

important for interpretation of fi ne needle aspi-
rates of salivary gland tumors with myoepithelial
differentiation (Kapadia et al. 1997). Pleomorphic
adenomas display a fi brillary matrix with embed-
ded tumor cells, while adenoid cystic carcinomas
show large, homogeneous, round matrix structures
surrounded by tumor cells (Fig. 6). A small amount
of matrix material may be impossible to identify
in Papanicolaou or hematoxylin and eosin stained
smears, but it is seen easily after Romanowsky-type
stains owing to its metachromasia. Because the pres-
ence and type of matrix material are critically impor-
tant diagnostic criteria, it is important to use both
a Romanowsky-type stain and a Papanicolaou (or

Fig. 4.

Fine needle aspirate of metastatic small cell

carcinoma showing paranuclear blue inclusions (arrows).
Diff-Quik stain.

1,000.

Fig. 5. Fine needle aspirate of metastatic seminoma to a
retroperitoneal lymph node showing tigroid background.
Diff-Quik stain.

400.

Romanowsky staining in cytopathology 87

ratio of colloid to cells is one of the most important
diagnostic criteria for differentiating between col-
loid nodules and potentially malignant thyroid neo-
plasms. While thick colloid can be identifi ed easily
on both Romanowsky-type and Papanicolaou stains,
thin colloid may be extremely diffi cult to visualize
in Papanicolaou stained smears. In Romanowsky-
type stains, thin colloid is recognized easily owing
to its characteristic folding and cracking patterns,
which confer a

“

cracked glass

”

“

crazy pave-

ment ” appearance (Fig. 8).

Crystalloids and crystals also can be identifi ed by

Romanowsky-type stains and may be of diagnostic
importance for several different lesions. Longstand-
ing cysts may contain negatively stained cholesterol
crystals, conditions with abundant eosinophilic infi l-
trates may show Charcot-Leyden crystals (Arora et
al. 1997), Leydig cell tumors may display Reinke ’ s
crystalloids (Gupta et al. 1994), and salivary gland
adenomas may exhibit tyrosine-rich crystalloids
(Lemos et al. 1997). Recognition of the slender, needle
shaped crystals of uric acid in fi ne needle aspirates is
diagnostic of gouty tophi (Nicol et al. 1997).

Advantages of Romanowsky-type
stains in special situations

In certain diagnostic scenarios, wet fi xed prepara-
tions are of little use. In these settings, Romanowsky-
type stains provide the required information.

Detection of microorganisms

Because Romanowsky-type stains originally were
devised to identify better the parasite responsible
for malaria (Giemsa 1904), it is not surprising

hematoxylin and eosin) stain when examining fi ne
needle aspirate material from the salivary glands to
avoid potential pitfalls (Hughes et al. 2005).

After Romanowsky-type staining, amyloid stains

deep blue with possible focal metachromasia (Hal-
liday et al. 1998, Michael and Naylor 1999) (Fig. 7).
Amyloid detection may be important for diagnosing
medullary carcinomas of the thyroid (Ljungberg 1972)
and localized amyloid depositions with or without
plasma cell proliferations ( “ amyloid tumors ” ) of the
parotid gland (Ustun et al. 2001), lungs (Dundore et
al. 1993), breast (Lew and Seymour 1985) and other
soft tissues (Bardin et al. 2004).

Another noncellular element whose identifi ca-

tion is of paramount diagnostic importance is col-
loid. Both the amount of colloid and its quality
are important in thyroid fi ne needle aspirates; the

Fig. 6.

Fine needle aspirate of pleomorphic adenoma

showing fi brillary magenta colored matrix material intimately
admixed with tumor cells. Diff-Quik stain.

400.

Fig. 7. Fine needle aspirate of medullary carcinoma of the
thyroid showing amyloid (center) surrounded by spindled
neoplastic cells. Diff-Quik stain.

400.

Fig. 8. Fine needle aspirate of thyroid showing colloid with
crazy pavement appearance in a colloid nodule. Diff-Quik
stain.

200.

88 Biotechnic & Histochemistry 2011, 86(2): 82–93

ment in the effi ciency of fi ne needle aspiration and
core biopsy procedures. Pathologists, radiologists
and clinicians frequently use various types of imag-
ing guidance, such as computerized tomography
and ultrasound, to obtain fi ne needle aspirate and
core biopsy specimens from deep lesions. In the
past, such specimens were sent to the pathology
laboratory for time-consuming cytological process-
ing and evaluation. Now these specimens can be
evaluated on site using Romanowsky-type stains
that take 30 sec or less to perform. Other rapid
stains occasionally are used including the ultrafast
Papanicolaou stain (Yang 1995, Yang and Alvarez
1995, Shinde and Pandit 2006) and the toluidine
blue stain (Leiman 2007), but none has achieved
the widespread acceptance of the Diff-Quik stain.

Rapid on site evaluation of specimens has

reduced signifi cantly the number of unsatisfactory
procedures, which has decreased the need for the
patient to undergo a repeat biopsy. Further, because
of the pathologist ’ s ability to carry out all of the
duties involved in this process, from performing the
procedure, to evaluating its adequacy, to triaging
material for ancillary studies such as cultures and
fl ow cytometry, a new specialist, the “ interventional
cytopathologist, ” has been forged (Grohs 1988). The
practice of cytopathologists performing their own
fi ne needle aspiration biopsies in turn has given
an enormous boost to the status of cytopathology
(Bedrossian 2007).

Limitations of Romanowsky-type stains
in cytological practice

Most cytopathologists believe that nuclear chroma-
tin is better defi ned in wet fi xed than in air dried
preparations, an opinion that hematopathologists
may not share. Because the contrast between nuclear
chromatin and nucleoli is variable, nucleoli are not
always demonstrated well in Romanowsky-type
stains. Wet fi xed variations of Romanowsky-type
stains have been recommended to overcome this
shortcoming (Hirschowitz et al. 1994); these offer
nuclear chromatin and nucleolar detail similar to
wet fi xed Papanicolaou or hematoxylin and eosin
stained preparations. As expected, however, the
advantages of increased nuclear size and accentua-
tion of nuclear size differences brought about by air
drying are lost.

In addition to the differences in the nuclear

chromatin detail, wet fi xed stains exhibit sharper
nuclear outlines, a feature that is particularly useful
for highlighting the nuclear contour abnormalities
of neoplastic cells. Nuclear grooves and pseudo-in-

that the stain is useful for detecting microorgan-
isms in fi ne needle aspirates, and exfoliative and
abrasive cytological specimens (Powers 1998,
Atkins and Powers 2002). Currently, the most
important diagnostic applications are for identi-
fi cation of Leishmania  (Kumar et al. 1987, Sah et
al. 2005), Toxoplasma  (Argyle et al. 1983, Zaharo-
poulos 2000, Pathan et al. 2003) and Pneumocystis
(Bedrossian et al. 1989). In addition, despite the
fact that Romanowsky-type stains do not stain
Mycobacteria , they outline the organisms, which
makes them stand out as unstained rods, nega-
tive images or “ ghost bacilli, ”  against a blue back-
ground. These negative images of Mycobacterium
kansasii  (Jannotta and Sidawy 1989), Mycobacte-
rium avium-intracellulare

(Maygarden and Flan-

ders 1989, Stanley et al. 1990, Ang et al. 1993) and
Mycobacterium leprae (Singh et al. 1994) have been
described within cells or in the background in a
variety of cytological samples, particularly when
the organisms are numerous.

Diagnosis of hematolymphoid
neoplasms

Because most pathologists and technologists are
familiar with the Romanowsky stained appear-
ance of hematolymphoid cells, Romanowsky-type
stains are superior to wet fi xed preparations for
diagnosing hematolymphoid neoplasms, particu-
larly in body fl uid specimens. Certain characteris-
tic features of hematolymphoid cells either are not
seen or are more diffi cult to recognize in Papani-
colaou or hematoxylin and eosin stains. In a recent
College of American Pathologists Interlaboratory
Comparison Program in Nongynecologic Cytol-
ogy, the rate of individuals misclassifying cerebro-
spinal fl uid slides with leukemia or lymphoma as
benign was signifi cantly greater for Papanicolaou
stained slides than for Romanowsky stained slides
(Renshaw et al. 2006). Although Romanowsky-
type stains are mandatory for accurate interpre-
tation of fi ne needle aspirates of primary lymph
node conditions, many cytopathologists also use
Papanicolaou stained smears for subtyping of
lymphomas, because the better nuclear chromatin
defi nition afforded by this stain aids in differenti-
ating centroblasts from centrocytes.

Rapid on-site evaluation of diagnostic
specimens

The advent of rapid Romanowsky-type stains, such
as the Diff-Quik stain, heralded a marked improve-

Romanowsky staining in cytopathology 89

some practice. Smears should be air dried relatively
quickly (within 5 min) to avoid artifacts. Because
waiting even a few minutes is impractical in the
course of rapid on site evaluations, hand-held fans
can be used successfully without noticeable artifact
formation to shorten the median air drying time
from about 3 min to about 1 min per smear (Baig
et al. 2006).

Finally, cytotechnologists and pathologists

trained only in exfoliative cytology may not
receive training in the use and interpretation of
Romanowsky-type stains. This limitation can be
overcome by including instruction in the interpreta-
tion of Romanowsky-type stains in all current cyto-
technology school curricula, pathology residency
and cytopathology fellowship programs.

Conclusion

Romanowsky-type stains provide several advan-
tages for evaluation of routine cytological speci-
mens including accentuation of cell and nuclear
size differences, accentuation of nuclear chromatin,
enhanced visibility of cytoplasmic detail, enhanced
visibility of smear background elements, and supe-
rior demonstration of intercellular material. In
addition, in certain diagnostic settings in which
Papanicolaou stains are of little benefi t, the use of
Romanowsky-type stains is critical. These settings
include detection of microorganisms, diagnosis of
hematolymphoid neoplasms, and rapid on site eval-
uation of diagnostic specimens.

Although the Papanicolaou stain may be the

best known stain in the practice of cytopathology,
the Romanowsky-type stain remains an indispens-
able tool in the cytopathologist ’ s armamentarium.

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