Source Journal of Veterinary Science
Review Article
Open Access
A New Technique to Obtain Cell Blocks Based On Snap Freezing
Hugo Enrique Orsini Beserra1*, Fabrizio Grandi2 and Diogo Sousa Zanoni1
1
Laboratory of Investigative and Comparative Pathology, School of Veterinary Medicine and Animal Science,
Universidade Estadual Paulista – UNESP, Botucatu, Brazil
2
Department of Pathology, Botucatu Medical School, Univ. Estadual Paulista – UNESP, Botucatu, Brazil
*
Corresponding author: Hugo Enrique Orsini Beserra, Laboratory of Investigative and Comparative Pathology, School of
Veterinary Medicine and Animal Science, Universidade Estadual Paulista – UNESP, Botucatu, Brazil, Tel: +55 14
38116293, E-mail: enriqueorsini@gmail.com
Abstract
Cytology is a widely used diagnostic test in both human and veterinary medicine. Several new methods have
constantly been described in literature aiming to minimize drawbacks associated with conventional cell block
techniques. In this context, this technical note describes a new method to obtain cell blocks based on snap freezing.
Keywords: cytology, cell block, freeze, cryo, snap, pathology
Received: January 15 2015; Accepted: March 25 2015; Published: April 1 2015
© 2015 Hugo Enrique Orsini Beserra et al; licensee Source Journals
This is an open access article is properly cited and distributed under the terms and conditions of creative commons
attribution license which permits unrestricted use, distribution and reproduction in any medium.
Editor: Prof. Filazi Ayhan
Introduction
The traditional cytological smear provides
important information regarding morphological and
functional status of specific cell populations. However,
in general, smearing techniques are insufficient to
preserve optimal tissue architecture, which poses a
significant limitation, mainly due to essentiality of cell
arrangement for diagnosis [1,2]. Moreover, thick smears
or smears containing a high amount of blood might
difficult visualization of cell features [3-6].
Aiming to minimize drawbacks related to
conventional cytology, researchers developed several
alternative techniques including cell block cytology
[5,7]. Cytology samples obtained by conventional fineneedle aspiration are fixed in 70% ethanol (water-based)
in Eppendorf tubes, and centrifuged. After that, the
supernatant is removed and 2% liquid agarose gel agar
is added to the sample. Finally, samples are centrifuged
to obtain a pellet to be embedded in paraffin, and
routinely processed for subsequent morphological,
cytochemical, immunocytochemical, genomic and/or
proteomic analysis [6,8,9]. Despite numerous
advantages, processing samples for cell block is time-
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consuming, placing important limitations in a routine
setting [10].
Frozen sections are rapid and relatively easy to
prepare, providing good morphological details and
superior preservation of antigenicity. The technique
helps to save time since dehydration steps typically seen
in other methods is not involved. Furthermore,
sectioning, labeling, and visualization of frozen samples
can be usually carried out in one day [11,12].
The objective of this technical note is to describe
a new technique by combining conventional
cryosectioning and cell block method.
Cell block Cryosectioning Method
Ten canine mammary tumors, from 1.6 to 8.7 cm
in diameter, localized in different mammary glands were
used. The cytological samples were obtained by fine
needle aspiration cytology (FNAC) using a 22G
hypodermic needle attached to a 10 ml syringe.
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© 2015 Hugo Enrique Orsini Beserra et al; licensee Source Journals. This is an open access article is
properly cited and distributed under the terms and conditions of creative commons attribution license which
permits unrestricted use, distribution and reproduction in any medium.
Aspirated cells were placed into Tissue Tek®
OCT compound and then subjected to snap freezing at 22°C. Eight-micrometer cryosections (Figure 1) were
obtained onto histological slides and fixed in 95º ethanol
solution for 2 minutes. Following this, samples were
rehydrated in decreasing alcoholic solutions for 1
minute each and, finally, in tap water for 3 minutes. All
slides were immersed in Hematoxylin solution for 1
minute, washed in tap water for 2 minutes, dehydrated
in absolute ethyl alcohol, and stained with Eosin Y for
40 seconds. Next, slides were dehydrated in absolute
ethyl alcohol, mounted with Fisher Chemical
Permount® Mounting Medium, and submitted to
analysis by an optical microscopy [13].
Cellular arrangements was assessed in cell block
samples, and classified according to Masserdoti C [1].
After
cytological
specimen
collection,
excisional biopsies from all tumors were compiled.
Representative samples for cell block analysis from
previously collected areas were immersed in Tissue
Tek® OCT compound, and then submitted to snap
freezing at -22°C. Eight-micrometer cryosections onto
histological slides were obtained, and routinely
processed for H&E staining for further microscopic
analysis by an experienced pathologist. Tumor
histotypes were classified according to published data
[13,14].
Figure 1: A) Deposition of cytological sample into
Tissue Tek® OCT compound. B) Cell block
cryosectioning. C) Ductal/tubular cellular arrangement.
Canine mammary gland showing cell clusters with a
tubular arrangement. The epithelial cells are arranged
in double-layers (arrow), H&E stain, 200x D) Ductal
carcinoma, canine mammary gland. Tissue cryosection
obtained from biopsy sample depicted in C. Note
morphological and structural similarities between both
samples. Double-layer (arrow), H&E stain, 200x.
Cell block
Histopathology
Ductal
carcinomas,
Tubular/ductal
malignancygrade I (n=3) and II
(n=6)
(n=3)
Simple
papillary
carcinomas,
Papillary (n=4)
malignancy grade I (n=4)
Table 1: Cellular arrangement on cell block samples
and histopathological diagnosis
Cell blocks obtained by snap-freezing technique
have demonstrated satisfactory results regarding cellular
architecture. Architectural arrangement is the
morphologic and functional expression of the cell
organization in a specific tissue, reflecting biological
behavior of a cell population [1]. Based on this
assumption, correct classification of cell arrangement,
according to published data for cytology specimens, was
possible.
A papillary arrangement is composed of a
stromal or vascular axis, and a mono or multi-layered
epithelial layer. Ductal patterns can be found in several
types of tumors, including those originated from ductal
portions of glandular tissues. Both types of cellular
arrangement may be seen in several histotypes of canine
mammary tumors [1]. Although other cellular
components commonly present in such neoplasia, even
tumor grade, were not evaluated in this study, its authors
consider this technique suitable for this type of analysis.
Moreover, other types of neoplastic and non-neoplastic
lesions in veterinary, even in human medicine, may be
also assessed.
In addition to above mentioned possibilities,
combination of two cell block method with snap
freezing permits a quick release of cytological reports
when compared to conventional cell block, with the
advantage of obtaining a high quality material.
Moreover, frozen methods are recognized as better
preservers of molecular cell quality, which opens
opportunities for future analysis in a molecular setting.
Finally, a need for further studies concerning
applicability of additional techniques, including
immunocytochemistry and molecular analysis, still
remains in order to validate the value of cell block
cryosectioning technique in a routine and research
setting.
Results and Discussion
Conflict of interests
Matching between cellular arrangement in cell
blocks obtained by snap freezing and histopathology
was 100% (Table 1) [14].
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No conflicts of interest have been declared
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© 2015 Hugo Enrique Orsini Beserra et al; licensee Source Journals. This is an open access article is
properly cited and distributed under the terms and conditions of creative commons attribution license which
permits unrestricted use, distribution and reproduction in any medium.
References
1. Masserdotti C (2006) Architectural patterns in
cytology: correlation with histology. Vet Clin Pathol 35:
388-396.
13. Mikel UV (1994) Advanced Laboratory Methods in
Histology and Pathology, 1st ed. American Registry of
Pathology.
2. Cassali GD, Gobbi H, Malm C, Schmitt FC (2007) E
valuation of accuracy of fine needleaspiration cytology
for diagnosis of canine mammary tumors: comparative
features with human tumors. Cytopathology 18: 191196.
14. Cassali GD, Lavalle GE, Ferreira E, Estrela-Lima A,
De Nardi AB (2014) Consensus for the diagnosis,
prognosis and treatment of canine mammary tumors.
Braz J Vet Pathol 7: 38-69.
3. Sontas BH, Yüzbaşıoğlu Öztürk G, Toydemir TF,
Arun SS, Ekici H (2012) Fine-needle aspiration biopsy
of canine mammary gland tumors: comparison between
cytology and histopathology. Reprod Domest Anim 47:
125-130.
4. Sangha S, Singh A, Sood NK, Gupta K (2011)
Specificity and sensitivity of cytological techniques for
rapid diagnosis of neoplastic and non-neoplastic lesions
of canine mammary gland. Braz J Vet Pathol 4: 13-22.
5. Kulkarni MB, Prabhudesai NM, Desai SB, Borges
AM (2000) Scrape cell block technique for fine needle
aspiration cytology smears. Cytopathology 11:179-184.
6. Sanchez N, Selvaggi SM (2006) Utility of cell blocks
in the diagnosis of thyroid aspirates. Diagn Cytopathol
34: 89-92.
7. Shivakumarswamy U, Arakeri SU, Karigowdar MH,
Yelikar BR (2012) Diagnostic utility of the cell block
method versus the conventional smear study in pleural
fluid cytology. J Cytol 29: 11-15.
8. Zanoni DS, Grandi F, Cagnini DQ, Bosco SMG,
Rocha NS (2012) Agarose cell block technique as a
complementary method in the diagnosis of fungal
osteomyelitis in a dog. Open Veterinary Journal 2:19-22.
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9. Zanoni DS, Grandi F, Rocha N (2011) Use of the
agarose cell block technique in veterinary diagnostic
cytopathology: an “old and forgotten” method. Vet Clin
Pathology 41: 307-308.
10. Nathan NA, Narayan E, Smith MM, Horn MJ (2000)
Cell block cytology. Improved preparation and its
efficacy in diagnostic cytology. Am J Clin Pathol 114:
599-606.
11. Fisher AH, Jacobson KA, Rose J, Zeller R (2008)
Cryosectioning tissues. CSH Protocols 3: 1-2.
12. Silva RDP, Souto LRM, Matsushita GM, Matsushita
MM (2011) Precisão diagnóstica das doenças cirúrgicas
nos exames por Congelação. Col Bras Cir 38: 149-54.
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