Romanowsky staining in cytopathology history advantages and limitations, medycyna, Artykuły, Publikacje

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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.
words:
aspiration
cytology ,
exfoliative
cytology ,
ne
needle
aspiration ,
metachromasia ,
Key
Pap stain , Wright-Giemsa stain
Historical perspective
cancer by smears was presented to the Bucharest
Gynecological Society in 1927 (Babe¸ 1963) and pub-
lished in the prestigious French journal,
Presse Medi-
cale
in 1928 (Babe¸ 1928, 1967, Douglass 1967). Using
this method, Dr. Babe¸ 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 Babe¸ published his
method for diagnosing uterine cancer by cervical
cytology smears, George N. Papanicolaou (1883-
1962) communicated his observations on fortuitously
identi 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, xed the samples in a 50:50 alcohol:ether
mixture, and stained them with a complex mixture of
The diagnostic procedure that we have come to
know as the Pap test, i.e., diagnosis of cervical can-
cer by cytological methods, was 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 rst
used in clinical practice, however, by the Romanian
academic pathologist Dr. Aurel A. Babe¸ (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, xed with methanol and stained with
the Giemsa stain. This method of diagnosis of cervical
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
82
dyes including hematoxylin, orange G, eosin Y, light
green SF and Bismarck brown Y. Ironically, while
the current Pap test stain consists of a modi 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 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 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 lms” prepared from the
sediment of pleural and peritoneal 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 rst researcher to systemati-
cally stain cytological preparations was Paul Ehrlich
(1854-1915). Having previously identi 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 uid (Barcia 2007).
The introduction of paraf 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 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 ne needle aspiration
cytology originated in Europe. Drs. Nils Söderström
(1911-1984) (Söderström 1956, 1958, 1966) and Six-
ten Franzén (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 ne needle aspiration at
the Karolinska Hospital in Stockholm (Linsk 1985,
Schenck 2003, Perez-Guillermo et al. 2005), so as the
technique of 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 xed prepara-
tions usually were employed as well. Currently,
there is wide regional variation in the preferred
type of Romanowsky-type stain. The May-Grün-
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 xation;
Romanowsky-type stains are air dried and Papani-
colaou stains are alcohol 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 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 post xed in methanol before staining with
a Romanowsky-type stain, the longer they stay in
Romanowsky staining in cytopathology
83
this xative, the closer their size approximates that
of wet 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 xed Papanicolaou
stained preparations, ampli 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 xed smears, decreases nuclear size variability,
which makes Romanowsky stained smears prefera-
ble for morphometry (Schulte and Wittekind 1987).
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 ne needle aspirates of the
thyroid and other organs. A common nding in 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 (Lay eld et
al. 2003), but are found across the spectrum of thyroid
pathology and thus are not speci c for any pathol-
ogy. Their presence, however, identi 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 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 dif 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 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
Accentuation of nuclear chromatin
Air drying also in 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 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, 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 de -
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
de nition also enables better appreciation of the
plasmacytoid appearance of certain neoplastic
cells. Such plasmacytoid cells, de 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.
84
Biotechnic & Histochemistry
2011,
86(2):
82–93
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 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, ne needle aspirates of the thyroid
may show follicular epithelial cells with marginal
vacuoles, a phenomenon known as a “ re- are ”
appearance (Das 2006) (Fig. 3). Originally described
as a distinctive feature of hyperthyroidism that could
be identi ed only with Romanowsky-type stains, the
re- are appearance of follicular cells is a nonspe-
ci c nding in both neoplastic and non-neoplastic
disorders (Das et al. 1998). When found in metasta-
sis, however, the re- 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. 3.
Fine needle aspirate of thyroid showing follicular
epithelial cells. Arrows show “ re- are” appearance. Diff-
Quik stain. 400.
While such vacuoles also may be seen in Papanico-
laou and hematoxylin and eosin stained cytological
preparations, they are identi 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 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
Fig. 2.
Cerebrospinal uid with NK/T-cell lymphoma
showing a mitotic gure and a malignant lymphoid cell
with cytoplasmic granules. Wright stain. Original mag-
ni cation
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
1,000.
Romanowsky staining in cytopathology
85
Another characteristic change produced by
the fragility of the cytoplasm is the presence of a
“ frothy ” or “ bubbly ” background in 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 in 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).
Fig. 4.
Fine needle aspirate of metastatic small cell
carcinoma showing paranuclear blue inclusions (arrows).
Diff-Quik stain.
1,000.
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 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 nding is the presence of small frag-
ments of lymphocytic cytoplasm, called lymphoglan-
dular bodies (Söderström 1968), in aspirates from
lymph nodes and lymphoid neoplasms. While not
entirely speci 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).
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 ne needle aspi-
rates of salivary gland tumors with myoepithelial
differentiation (Kapadia et al. 1997). Pleomorphic
adenomas display a 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. 5.
Fine needle aspirate of metastatic seminoma to a
retroperitoneal lymph node showing tigroid background.
Diff-Quik stain.
400.
86
Biotechnic & Histochemistry
2011,
86(2):
82–93
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