Romanowsky staining in cytopathology: history,
advantages and limitations

KP Krafts

1

, SE Pambuccian

2

1

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

1035 University Drive, Duluth, and

2

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: [e-mail]
© 2011 The Biological Stain Commission
Biotechnic & Histochemistry 2011, 86(2): 82–93.

DOI:10.3109/10520295.2010.515492

82

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

ü

n-

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

é

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

ö

derstr

ö

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

”

or

“

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.

References

Abati A, Skarulis MC, Shawker T, Solomon D (1995)
Ultrasound-guided fi ne-needle aspiration of parathy-
roid lesions: a morphological and immunocytochemical
approach. Hum. Pathol. 26: 338–343.
Abdul-Karim FW, Wasman JK, Pitlik D (1993) Needle
aspiration cytology of chondrosarcomas. Acta Cytol. 37:
655–660.
Aida Y, Takeuchi E, Shinagawa T, Tadokoro M, Inoue S,
Omata Y, Noro M (1993) Fine needle aspiration cytology
of lipid-secreting carcinoma of the breast. A case report.
Acta Cytol. 37: 547–551.
Ang GA, Janda WM, Novak RM, Gerardo L (1993) Nega-
tive images of mycobacteria in aspiration biopsy smears
from the lymph node of a patient with acquired immuno-
defi ciency syndrome (AIDS): report of a case and a review
of the literature. Diagn. Cytopathol. 9: 325–328.

clusions are more distinct in wet fi xed preparations
and unusual chromatin patterns, e.g., the “ salt and
pepper ” chromatin in neuroendocrine neoplasms,
are appreciated more easily. Many of these nuclear
features, such as nuclear grooves in papillary carci-
nomas of the thyroid (Bhambhani et al. 1990) and
nuclear inclusions in both thyroid tumors and mela-
noma, also can be demonstrated in Romanowsky
stained preparations, but they may be more diffi cult
to identify (Fig. 9).

Another limitation of Romanowsky-type stains

is their relative inability to highlight cytoplasmic
keratinization. Because the Papanicolaou stain was
developed to document keratinization (the orange
G dye component binds avidly to keratin), it is supe-
rior to Romanowsky-type stains for this purpose.
Identifi cation of cytoplasmic keratinization may be
essential for diagnosing squamous cell carcinomas.

A further constraint is that Romanowsky-type

stains, including the Diff-Quik stain commonly used
in cytopathology, were created and intended for use
with very thin, evenly smeared preparations. Typical
fi ne needle aspirates commonly contain thicker tis-
sue fragments and a thin smear may be impossible to
make. Thicker tissue fragments, which may consist
of three-dimensional cell groups or partially necrotic
tissue fragments, frequently are poorly stained and
diffi cult to visualize. In such cases, Papanicolaou or
hematoxylin and eosin stains may reveal the cyto-
logical features of the individual cells within the
three-dimensional cell clusters and may show well
defi ned, individual, intact, non-necrotic cells.

The technique for making good air dried smears

from fi ne needle aspiration material is important.
The optimal smearing technique usually requires

Fig. 9. Fine needle aspirate of papillary thyroid carcinoma
showing nuclear size and shape variation and occasional
grooves. A cytoplasmic nuclear pseudoinclusion is
indicated by an arrow. Diff-Quik stain.

1,000.

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

Argyle JC, Schumann GB, Kjeldsberg CR, Athens JW
(1983) Identifi cation of a toxoplasma cyst by fi ne-needle
aspiration. Am. J. Clin. Pathol. 80: 256–258.

Arora VK, Singh N, Bhatia A ( 1997) Charcot-Leyden
crystals in fi ne needle aspiration cytology. Acta Cytol. 41:
409–412.
Atkins KA, Powers CN (2002) The cytopathology of
infectious diseases. Adv. Anat. Pathol. 9: 52–64.
Babe s¸ A (1928) Diagnostic du cancer du col ut é rin par les
frottis. Presse M é d. 29: 451–454.
Babe

s¸

A (1967) Diagnosis of cancer of the uterine cervix

by smears. Acta Cytol. 11: 217–224.
Babe

s¸

LM (1963) Les premieres communications de Dan-

iel et Babe s¸ , en 1927, sur le diagnostic du cancer du col
uterin par les frottis. (Methode roumaine). Gynecol. Prat.
14: 267–269.
Baig MA, Fathallah L, Feng J, Husain M, Grignon DG,
Al-Abbadi MA (2006) Fast drying of fi ne needle aspira-
tion slides using a hand held fan: impact on turn around
time and staining quality. Cytojournal 3: 12.
Barcia JJ (2007) The Giemsa stain: its history and applica-
tions. Int. J. Surg. Pathol. 15: 292–296.
Bardin RL, Barnes CE, Stanton CA, Geisinger KR (2004)
Soft tissue amyloidoma of the extremities: a case report
and review of the literature. Arch. Pathol. Lab. Med. 128:
1270–1273.
Beale LS (1860) Results of the chemical and microscopical
examination of solid organs and secretions. Examination
of sputum from a case of cancer of the pharynx and adja-
cent parts. Arch. Med. 2: 44.
Beale LS (1878)

The Microscope in Medicine . Churchill,

London. p. 237.
Bedrossian CW, Mason MR, Gupta PK (1989) Rapid cyto-
logic diagnosis of Pneumocystis : a comparison of effective
techniques. Semin. Diagn. Pathol. 6: 245–261.
Bedrossian CW (2007) Cytopathology: a global perspec-
tive. Diagn. Cytopathol. 35: 745–748.
Bezabih M, Mariam DW, Selassie SG (2002) Fine needle
aspiration cytology of suspected tuberculous lymphad-
enitis. Cytopathology 13: 284–290.

Bhambhani S, Kashyap V, Das DK

(1990) Nuclear

grooves. Valuable diagnostic feature in May-Grunwald-
Giemsa-stained fi ne needle aspirates of papillary carci-
noma of the thyroid. Acta Cytol. 34: 809–812.
Boon ME, Tabbers-Bouwmeester ML (1980)

Gynaeco-

logical Cytology: a Textbook and Atlas . Macmillan, London.
p. 172.
Boon ME, Drijver JS (1986) Routine Cytological Staining
Techniques. Theoretical Background and Practice. Elsevier,
New York. pp. 16–32, 51–59, 118.
Broders AC (1932) Carcinoma in situ contrasted with
benign penetrating epithelium. JAMA 99: 1670–1674.
Caraway NP, Fanning CV, Amato RJ, Sneige N (1995)
Fine-needle aspiration cytology of seminoma: a review
of 16 cases. Diagn. Cytopathol. 12: 327–333.
Chang F, Vu C, Chandra A, Meenan J, Herbert A (2006)
Endoscopic ultrasound-guided fi ne needle aspiration
cytology of pancreatic neuroendocrine tumours: cyto-
morphological and immunocytochemical evaluation.
Cytopathology 17: 10–17.

Chatzipantelis P, Salla C, Konstantinou P, Karoumpalis
I, Sakellariou S, Doumani I

(2008) Endoscopic ultra-

sound-guided fi ne-needle aspiration cytology of pancre-
atic neuroendocrine tumors: a study of 48 cases. Cancer
114: 255–262.
Chhieng DC, Paulino AF (2002) Cytology of myoepithe-
lial carcinoma of the salivary gland. Cancer 96: 32–36.

Das DK, Gupta SK, Sehgal S

(1986) Extramedullary

plasma cell tumors: diagnosis by fi ne-needle aspiration
cytology. Diagn. Cytopathol. 2: 248–251.
Das DK, Gupta SK, Pathak IC, Sharma SC, Datta BN
(1987) Burkitt-type lymphoma. Diagnosis by fi ne needle
aspiration cytology. Acta Cytol. 31: 1–7.

Das DK, Jain S, Tripathi RP, Parkash S, Khan IU,
Rajwanshi A, Gupta S (1998) Marginal vacuoles in thy-
roid aspirates. Acta Cytol. 42: 1121–1128.
Das DK ( 2006) Marginal vacuoles (fi re-fl are appearance)
in fi ne needle aspiration smears of thyroid lesions: does it
represent diffusing out of thyroid hormones at the base of
follicular cells? Diagn. Cytopathol. 34: 277–283.
De Las Casas LE, Gokden M, Mukunyadzi P, White P,
Baker SJ, Hermonat PL, You H, Korourian S, Malak SF,
Miranda RN (2004) A morphologic and statistical com-
parative study of small-cell carcinoma and non-Hodgkin’s
lymphoma in fi ne-needle aspiration biopsy material from
lymph nodes. Diagn. Cytopathol. 31: 229–234.
DiPalma S, Alasio L, Pilotti S (1996) Fine needle aspira-
tion (FNA) appearances of malignant myoepithelioma of
the parotid gland. Cytopathology 7: 357–365.
Dodd LG, Sneige N, Reece GP, Fornage B (1993) Fine-
needle aspiration cytology of silicone granulomas in the
augmented breast. Diagn. Cytopathol. 9: 498–502.
Douglass LE (1967) A further comment on the contribu-
tions of Aurel Babe

s¸

to cytology and pathology.

Acta

Cytol. 11: 217.

Dundore PA, Aisner SC, Templeton PA, Krasna MJ,
White CS, Seidman JD (1993) Nodular pulmonary amy-
loidosis: diagnosis by fi ne-needle aspiration cytology and
a review of the literature. Diagn. Cytopathol. 9: 562–564.
Dusenbery D (1997) Aspiration cytology of glycogen-rich
squamous cell carcinoma: signifi cance of a tigroid smear
background. Acta Cytol. 41: 941–942.
Finley JL, Silverman JF, Dabbs DJ, West RL, Dickens
A, Feldman PS, Frable WJ (1986) Chordoma: diagnosis
by fi ne-needle aspiration biopsy with histologic, immu-
nocytochemical, and ultrastructural confi rmation. Diagn.
Cytopathol. 2: 330–337.

Flanders E, Kornstein MJ, Wakely PE, Jr., Kardos TF,
Frable WJ (1993) Lymphoglandular bodies in fi ne-needle
aspiration cytology smears. Am. J. Clin. Pathol. 99: 566–569.
Fleury-Feith J, Bellot-Besnard J (1989) Criteria for aspi-
ration cytology for the diagnosis of seminoma.

Diagn.

Cytopathol. 5: 392–395.
Francis IM, Das DK, al-Rubah NA, Gupta SK (1994)
Lymphoglandular bodies in lymphoid lesions and
non-lymphoid round cell tumours: a quantitative assess-
ment. Diagn. Cytopathol. 11: 23–27.
Franzen S, Giertz G, Zajicek J (1960) Cytological diagno-
sis of prostatic tumours by transrectal aspiration biopsy:
a preliminary report. Br. J. Urol. 32: 193–196.

Romanowsky staining in cytopathology 91

of the islet cell tumor of pancreas: a comparison between
computerized axial tomography and endoscopic ultra-
sound-guided fi ne needle aspiration biopsy. Ann. Diagn.
Pathol. 6: 106–112.
Jhala N, Siegal GP, Jhala D (2008) Large, clear cytoplas-
mic vacuolation: an under-recognized cytologic clue to
distinguish solid pseudopapillary neoplasms of the pan-
creas from pancreatic endocrine neoplasms on fi ne-needle
aspiration. Cancer 114: 249–254.
Kapadia SB, Dusenbery D, Dekker A (1997) Fine nee-
dle aspiration of pleomorphic adenoma and adenoid
cystic carcinoma of salivary gland origin. Acta Cytol. 41:
487–492.
Kay PA, Nascimento AG, Unni KK, Salomao DR (2003)
Chordoma. Cytomorphologic fi ndings in 14 cases diag-
nosed by fi ne needle aspiration. Acta Cytol. 47: 202–208.
Khalbuss WE, Fischer G, Ahmad M, Villas B (2006) Syn-
chronous presentation of breast carcinoma with plasma-
cytoid cytomorphology and multiple myeloma. Breast J.
12: 165–167.

Khunamornpong S, Thorner PS, Suprasert P,
Siriaunkgul S (2005) Clear-cell adenocarcinoma of the
female genital tract: presence of hyaline stroma and
tigroid background in various types of cytologic speci-
mens. Diagn. Cytopathol. 32: 336–340.

Klijanienko J, Caillaud JM, Orbach D, Brisse H,
Lagace R, Sastre-Gareau X (2007) Cyto-histological corre-
lations in primary, recurrent, and metastatic bone and soft
tissue osteosarcoma. Institut Curie’s experience. Diagn.
Cytopathol. 35: 270–275.
Koprowska I (1985) Concurrent discoveries of the value
of vaginal smears for diagnosis of uterine cancer. Diagn.
Cytopathol. 1: 245–248.
Koss LG (2003) Aurel Babe s¸ . Int. J. Gynecol. Pathol. 22:
101–102.
Kumar PV, Hambarsoomina B, Vaezzadeh K (1987) Fine
needle aspiration cytology of localized Leishmania lym-
phadenitis. Acta Cytol. 31: 14–16.
Kumar PV, Hodjati H, Monabati A, Talei A (2000) Med-
ullary thyroid carcinoma. Rare cytologic fi ndings. Acta
Cytol. 44: 181–184.

Kumar PV, Sobhani SA, Monabati A, Hashemi SB,
Eghtadari F, Hamidi SA (2004) Myoepithelioma of the
salivary glands. Fine needle aspiration biopsy fi ndings.
Acta Cytol. 48: 302–308.
Kumar PV, Monabati A, Tabei SZ, Ramzy M, Husseini
SV, Khajeh F (2005) Metastatic follicular thyroid carci-
noma diagnosed by fi ne needle aspiration cytology: a
report of 3 cases. Acta Cytol. 49: 177–180.
Kuwabara H, Kohno K, Kishida F, Uda H, Saito K, Shiba-
nushi T (1997) Fine needle aspiration cytology of predom-
inant plasmacytoid myoepithelial cells in pleomorphic
adenoma of the palate. Acta Cytol. 41: 1637–1640.
Layfi eld LJ, Wax T, Jones C (2003) Cytologic distinction of
goiterous nodules from morphologically normal thyroid:
analyses of cytomorphologic features. Cancer 99: 217–222.
Leiman G (2007) My approach to pancreatic fi ne needle
aspiration. J. Clin. Pathol. 60: 43–49.

Lemos LB, Baliga M, Brister T, Cason Z

(1997)

Cytomorphology of tyrosine-rich crystalloids in fi ne

Garbyal RS, Agarwal N, Kumar P

(2006) Leishman-

Giemsa cocktail: an effective Romanowsky stain for air-
dried cytologic smears. Acta Cytol. 50: 403–406.
Giemsa G (1904) Eine Vereinfachung und Vervollkom-
mnung meiner Methylenazur-Methylenblau-Eosin-F ä rbe-
methode zur Erzielung der Romanowsky-Nochtschen
Chromatinf ä rbung. Zentralbl. Bakteriol. 37: 308–311.
Grenko RT, Lee KP, Lee KR (1990) Fine needle aspira-
tion cytology of lactating adenoma of the breast. A com-
parative light microscopic and morphometric study. Acta
Cytol. 34: 21–26.
Grohs HK (1988) The interventional cytopathologist. A
new clinician/pathologist hybrid. Am. J. Clin. Pathol. 90:
351–354.

Guiter GE, Gamboni MM, Zakowski MF

(1999) The

cytology of extraskeletal Ewing sarcoma. Cancer 87: 141–
148.

Gupta R, Mathur SR, Arora VK, Sharma SG

(2008)

Cytologic features of extragonadal germ cell tumors: a
study of 88 cases with aspiration cytology. Cancer 114:
504–511.

Gupta SK, Rajwanshi AK, Das DK

(1985) Fine nee-

dle aspiration cytology smear patterns of malignant
melanoma. Acta Cytol. 29: 983–988.
Gupta SK, Francis IM, Sheikh ZA, al-Rubah NA, Das
DK (1994) Intranuclear Reinke’s crystals in a testicular
Leydig cell tumor diagnosed by aspiration cytology. A
case report. Acta Cytol. 38: 252–256.
Halliday BE, Silverman JF, Finley JL (1998) Fine-needle
aspiration cytology of amyloid associated with non-
neoplastic and malignant lesions. Diagn. Cytopathol. 18:
270–275.
Hasteh F, Pu R, Michael CW (2007) A metastatic renal
carcinoid tumor presenting as breast mass: a diagnostic
dilemma. Diagn. Cytopathol. 35: 306–310.
Henry MJ, Burton LG, Stanley MW, Horwitz CA (1987)
Application of a modifi ed Diff-Quik stain to fi ne needle
aspiration smears: rapid staining with improved cyto-
logic detail. Acta Cytol. 31: 954–955.

Hirschowitz SL, Mandell D, Nieberg RK, Carson K

(1994) The alcohol-fi xed Diff-Quik stain. A novel rapid
stain for the immediate interpretation of fi ne needle aspi-
ration specimens. Acta Cytol. 38: 499–501.
Hughes JH, Volk EE, Wilbur DC (2005) Pitfalls in sali-
vary gland fi ne-needle aspiration cytology: lessons from
the College of American Pathologists Interlaboratory
Comparison Program in Nongynecologic Cytology. Arch.
Pathol. Lab. Med. 129: 26–31.
Hughes JH, Young NA, Wilbur DC, Renshaw AA, Mody
DR (2005) Fine-needle aspiration of pulmonary hamar-
toma: a common source of false-positive diagnoses in
the College of American Pathologists Interlaboratory
Comparison Program in Nongynecologic Cytology. Arch.
Pathol. Lab. Med. 129: 19–22.
Jannotta FS, Sidawy MK (1989) The recognition of myco-
bacterial infections by intraoperative cytology in patients
with acquired immunodefi ciency syndrome. Arch. Pathol.
Lab. Med. 113: 1120–1123.
Jhala D, Eloubeidi M, Chhieng DC, Frost A, Eltoum IA,
Roberson J, Jhala N (2002) Fine needle aspiration biopsy

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

Novotny DB, Maygarden SJ, Shermer RW, Frable WJ

(1991) Fine needle aspiration of benign and malignant
breast masses associated with pregnancy. Acta Cytol. 35:
676–686.
Papanicolaou GN (1925 ) The diagnosis of early human
pregnancy by the vaginal smear method. Proc. Soc. Exp.
Biol. Med. 22: 436–437.
Papanicolaou GN (1928)

New Cancer Diagnosis . The

Race Betterment Foundation, Battle Creek, MI. pp.
528–534.
Papanicolaou GN (1933) The sexual cycle in the human
female as revealed by vaginal smears. Am. J. Anat. 52:
519–637.
Papanicolaou GN, Traut HG (1941) The diagnostic value
of vaginal smears in carcinoma of the uterus. Am. J. Obstet.
Gynecol. 42: 193–206.
Papanicolaou GN (1942) A new procedure for staining
vaginal smears. Science 95: 438–439.
Pathan SK, Francis IM, Das DK, Mallik MK, Sheikh ZA,
Hira PR (2003) Fine needle aspiration cytologic diagnosis
of toxoplasma lymphadenitis. A case report with detec-
tion of a Toxoplasma bradycyst in a Papanicolaou-stained
smear. Acta Cytol. 47: 299–303.
Perez-Guillermo M, Acosta-Ortega J, Garcia-Solano J
(2005) The continuing role of fi ne-needle aspiration of the
prostate gland into the 21st century: a tribute to Torsten
Lowhagen. Diagn. Cytopathol. 32: 315–320.
Powers CN (1998) Diagnosis of infectious diseases:
a cytopathologist’s perspective. Clin. Microbiol. Rev. 11:
341–365.
Quincke H (1881-1882) Ueber die geformten Bestandtheile
von Transsudaten.

Deut. Arch. Klin. Med. (Leipzig) 30:

580–588.
Rau AR, Kini H, Verghese R (2006) Tigroid background
in fi ne-needle aspiration cytology of clear cell sarcoma.
Diagn. Cytopathol. 34: 355–357.
Renshaw AA, Hughes JH, Wang E, Haja J, Wilbur D,
Henry MR, Moriarty AT

(2006) Leukemia/lymphoma

in cerebrospinal fl uid: distinguishing between cases that
performed well and poorly in the College of American
Pathologists Interlaboratory Comparison Program in
Non-gynecologic Cytology. Arch. Pathol. Lab. Med. 130:
1762–1765.
Reyes CV, Thompson KS, Jensen J (1998) Multivesicu-
lated macrophages: their implication in fi ne-needle aspi-
ration cytology of lung mass lesions. Diagn. Cytopathol.
19: 98–101.
Safo AO, Li RW, Vickers SM, Schmechel SC, Pambuc-
cian SE (2009) Endoscopic ultrasound-guided fi ne-needle
aspiration diagnosis of clear-cell pancreatic endocrine
neoplasm in a patient with von Hippel-Lindau disease:
a case report. Diagn. Cytopathol. 37: 365–372.
Sah SP, Prasad R, Raj GA (2005) Fine needle aspiration
of lymphadenopathy in visceral leishmaniasis. Acta Cytol.
49: 286–290.
Schenck U (2003) Cytopathology in Germany.

Diagn.

Cytopathol. 28: 112–113.
Schreiner AM, Yang GC (2009) Medullary thyroid carci-
noma presenting as rectangular cell type on fi ne-needle
aspiration. Diagn. Cytopathol. 37: 213–216.

needle aspirates of salivary gland adenomas. Acta Cytol.
41: 1709–1713.
Lew W, Seymour AE (1985) Primary amyloid tumor of
the breast. Case report and literature review. Acta Cytol.
29: 7–11.
Linsk JA (1985) Aspiration cytology in Sweden: The
Karolinska Group. Diagn. Cytopathol. 1: 332–335.
Liu K, Madden JF, Olatidoye BA, Dodd LG (1999) Fea-
tures of benign granular cell tumor on fi ne needle aspira-
tion. Acta Cytol. 43: 552–557.
Ljungberg O (1972) Cytologic diagnosis of medullary
carcinoma of the thyroid gland with special regard to the
demonstration of amyloid in smears of fi ne needle aspi-
rates. Acta Cytol. 16: 253–255.
Long SR, Cohen MB (1993) Classics in cytology. VI.
The early cytologic discoveries of Lionel S. Beale. Diagn.
Cytopathol. 9: 595–598.
Long SR, Cohen MB (1996) Classics in Cytology VII.
Kun, Lebert, and early efforts at fi ne-needle aspiration
biopsy. Diagn. Cytopathol. 14: 182–183.
Lopes Cardozo P (1950) Het herkennen van gezwelcel-
len in volgens May-Gr ü nwald-Giemsa gekleurde uitstri-
jkpraeparaten. Ned. Tijdschr. Geneeskd. 94: 1635–1643.
Lopes Cardozo P, Posthuma J (1954) Clinical Cytology,
Using the May-Gr

ü

nwald-Giemsa Stained Smear, with a

Chapter on Prostatic Cytology . 2 volumes, Stafl eu, Leyden.
pp. 128 (text) and 190 (atlas).
Lopes Cardozo P (1960) Needle-aspiration cytology of
the palpable swelling. Acta Unio Int. Contra Cancrum. 16:
382–384.
Martin HE, Ellis EB (1930) Biopsy by needle puncture
and aspiration. Ann. Surg. 92: 169–181.
Maygarden SJ, Flanders EL (1989) Mycobacteria can
be seen as “negative images” in cytology smears from
patients with acquired immunodefi ciency syndrome.
Mod. Pathol. 2: 239–243.
Michael CW, Naylor B (1999) Amyloid in cytologic speci-
mens. Differential diagnosis and diagnostic pitfalls. Acta
Cytol. 43: 746–755.
Mullins RK, Thompson SK, Coogan PS, Shurbaji MS
(1994) Paranuclear blue inclusions: an aid in the cytopatho-
logic diagnosis of primary and metastatic pulmonary
small-cell carcinoma. Diagn. Cytopathol. 10: 332–335.
Nayar R, De Frias DV, Bourtsos EP, Sutton V, Bedrossian
C (2001) Cytologic differential diagnosis of papillary pat-
tern in breast aspirates: correlation with histology. Ann.
Diagn. Pathol. 5: 34–42.
Nayar R, Frost AR (2001) Thyroid aspiration cytology: a
“cell pattern” approach to interpretation. Semin. Diagn.
Pathol. 18: 81–98.
Naylor B, Tasca L, Bartziota E, Schneider V (2002) In
Romania it’s the Méthode Babes¸-Papanicolaou.

Acta

Cytol. 46: 1–12.
Nguyen GK (1995) Cytopathology of pulmonary carci-
noid tumors in sputum and bronchial brushings. Acta
Cytol. 39: 1152–1160.
Nicol KK, Ward WG, Pike EJ, Geisinger KR, Cappellari
JO, Kilpatrick SE (1997) Fine-needle aspiration biopsy
of gouty tophi: lessons in cost-effective patient manage-
ment. Diagn. Cytopathol. 17: 30–35.

Romanowsky staining in cytopathology 93

from other malignant neoplasms in fi ne-needle aspiration
biopsy specimens. Arch. Pathol. Lab. Med. 129: 1017–1021.
Tabatowski K, Elson CE, Johnston WW (1990) Silicone
lymphadenopathy in a patient with a mammary prosthe-
sis. Fine needle aspiration cytology, histology and analyti-
cal electron microscopy. Acta Cytol. 34: 10–14.
Tasca L, Ostor AG, Babe

s¸

V (2002) XII. Aurel Babe

s¸

. Int.

J. Gynecol. Pathol. 21: 198–202.
Torlakovic E, Ames ED, Manivel JC, Stanley MW (1993)
Benign and malignant neoplasms of myoepithelial cells:
cytologic fi ndings. Diagn. Cytopathol. 9: 655–660.
Tsou MH, Lin HH, Ko JS (1997) Riu’s stain and the cyto-
logic diagnosis of thyroid fi ne-needle aspiration: a single
cancer center experience. Diagn. Cytopathol. 16: 543–547.
Tsou MH, Lin YM, Lin KJ, Ko JS, Wu ML (1998) Fine
needle aspiration cytodiagnosis of liver tumors. Results
obtained with Riu’s stain. Acta Cytol. 42: 1359–1364.
Us-Krasovec M, Auersperg M, Bergant D, Golouh R,
Kloboves-Prevodnik V (1998) Medullary carcinoma of
the thyroid glad: diagnostic cytopathological character-
istics. Pathologica 90: 5–13.
Ustun MO, Ekinci N, Payzin B (2001) Extramedullary
plasmacytoma of the parotid gland. Report of a case with
extensive amyloid deposition masking the cytologic and
histopathologic picture. Acta Cytol. 45: 449–453.
Viana O (1928) La diagnosi precoce del cancro uterino
mediante lo striscio. Clin. Ostetr. 30: 781–793.
Virtej P, Vasiliu C (2003) Cytodiagnosis in cervical neo-
plasia: from the Babe

s¸

/Papanicolaou smear to the actual

Bethesda System. Clin. Exp. Obstet. Gynecol. 30: 173–177.
Walker WP, Wittchow RJ, Bottles K, Layfi eld LJ, Hir-
schowitz S, Cohen MB (1994) Paranuclear blue inclusions
in small cell undifferentiated carcinoma: a diagnostically
useful fi nding demonstrated in fi ne-needle aspiration
biopsy smears. Diagn. Cytopathol. 10: 212–215.
Wied GL (1964) Editorial. Pap-test or Babe

s¸

method? Acta

Cytol. 8: 173–174.

Wittchow R, Laszewski M, Walker W, Dick F

(1992)

Paranuclear blue inclusions in metastatic undifferentiated
small cell carcinoma in the bone marrow. Mod. Pathol. 5:
555–558.
Wu HH, Cramer HM, Kho J, Elsheikh TM (1998) Fine
needle aspiration cytology of benign adrenal cortical nod-
ules. A comparison of cytologic fi ndings with those of
primary and metastatic adrenal malignancies. Acta Cytol.
42: 1352–1358.
Yang GC (1994) The mathematical basis for the increased
sensitivity in cancer detection in air-dried cytoprepara-
tions. Mod. Pathol. 7: 681–684.
Yang GC (1995) Ultrafast Papanicolaou stain is not lim-
ited to rapid assessments: application to permanent fi ne-
needle aspiration smears. Diagn. Cytopathol. 13: 160–162.
Yang GC, Alvarez II (1995) Ultrafast Papanicolaou stain.
An alternative preparation for fi ne needle aspiration
cytology. Acta Cytol. 39: 55–60.
Zaharopoulos P (2000) Demonstration of parasites in Tox-
oplasma lymphadenitis by fi ne-needle aspiration cytology:
report of two cases. Diagn. Cytopathol. 22: 11–15.

Schulte E (1986) Air drying as a preparatory factor in
cytology: investigation of its infl uence on dye uptake and
dye binding. Diagn. Cytopathol. 2: 160–167.
Schulte E, Wittekind C (1987) The infl uence of the
wet-fi xed Papanicolaou and the air-dried Giemsa tech-
niques on nuclear parameters in breast cancer cytol-
ogy: a cytomorphometric study. Diagn. Cytopathol. 3:
256–261.
Shaw JA, Vance RP, Geisinger KR, Marshall RB (1990)
Islet cell neoplasms. A fi ne-needle aspiration cytology
study with immunocytochemical correlations. Am. J. Clin.
Pathol. 94: 142–149.
Shinde PB, Pandit AA (2006) Application of modifi ed
ultrafast Papanicolaou stain in cytology of various organs.
Diagn. Cytopathol. 34: 135–139.
Sidawy MK, Costa M (1989) The signifi cance of para-
vacuolar granules of the thyroid. A histologic, cytologic
and ultrastructural study. Acta Cytol. 33: 929–933.
Siddaraju N, Yaranal PJ, Mishra MM, Soundararaghavan
J (2007) Fine needle aspiration cytology in recurrent amelan-
otic melanoma: a case report. Acta Cytol. 51: 829–832.
Silverman JF, Frable WJ (1990) The use of the Diff-Quik
stain in the immediate interpretation of fi ne-needle aspi-
ration biopsies. Diagn. Cytopathol. 6: 366–369.
Singh N, Arora VK, Ramam M (1994) Nodular leproma-
tous leprosy: report of a case diagnosed by FNA. Diagn.
Cytopathol. 11: 373–375.
S ö derstr ö m N (1956) Cytologisk punktionsdiagnostik i
praktisk-kuliniskt arbete. Nord. Med. 56: 1645–1649.
S ö derstr ö m N (1958) Identifi cation of normal tissues and
tumors by cytologic aspiration biopsy. Acta Soc. Med. Ups.
63: 53–87.
S ö derstr ö m N ( 1968) The free cytoplasmic fragments of
lymphoglandular tissue (lymphoglandular bodies). A
preliminary presentation. Scand. J. Haematol. 5: 138–152.
S ö derstr ö m N (1966) Fine-Needle Aspiration Biopsy Used
as a Direct Adjunct in Clinical Diagnostic Work . Grune &
Stratton, New York.
Stanley MW, Horwitz CA, Burton LG, Weisser JA (1990)
Negative images of bacilli and mycobacterial infection: a
study of fi ne-needle aspiration smears from lymph nodes
in patients with AIDS. Diagn. Cytopathol. 6: 118–121.
Stelow EB, Stanley MW, Bardales RH, Mallery S, Lai R,
Linzie BM, Pambuccian SE (2003) Intraductal papillary-
mucinous neoplasm of the pancreas. The fi ndings and
limitations of cytologic samples obtained by endoscopic
ultrasound-guided fi ne-needle aspiration.

Am. J. Clin.

Pathol. 120: 398–404.
Stewart FW (1933) The diagnosis of tumors by aspiration.
Am. J. Pathol. 9: 801–812.
Stockard CR, Papanicolaou GN (1917) A rhythmical
“heat period” in the guinea pig. Science 46: 42–44.
Stockard CR, Papanicolaou GN (1917 ) The existence of
a typical oestrous cycle in the guinea pig: with a study
of its histological and physiological changes. Am. J. Anat.
22: 225–283.
Tabatabai ZL, Staerkel GA (2005) Distinguishing pri-
mary and metastatic conventional renal cell carcinoma

Copyright of Biotechnic & Histochemistry is the property of Taylor & Francis Ltd and its content may not be

copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written

permission. However, users may print, download, or email articles for individual use.