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Investigation
Journal of Veterinary Diagnostic
http://vdi.sagepub.com/content/24/4/710
The online version of this article can be found at:
DOI: 10.1177/1040638712445763
2012 24: 710 originally published online 14 May 2012J VET Diagn Invest
Kellye S. Joiner and Elizabeth A. Spangler
-embedded specimens for use in veterinary diagnostic pathologyEvaluation of HistoGel
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Journal of Veterinary Diagnostic Investigation
24(4) 710 –715
© 2012 The Author(s)
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DOI: 10.1177/1040638712445763
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Brief Research Reports
Processing small or delicate specimens and exudates can be
one of the many challenges in diagnostic veterinary pathol-
ogy. HistoGel™a is an inert aqueous processing gel that can
be used to encapsulate a variety of cytologic specimens and
unfixed or formalin-fixed tissues in a solidified agar-like
medium prior to processing.
Evaluation of cytologic specimens is routinely performed
on aspirates, direct smears, and touch prep applications using
prototypical staining techniques, such as Romanowsky
stains. However, there are relatively few special staining
techniques available for routine cytology, thus limiting the
phenotypic characterization of cytologic preparations. For
example, lymphoma is routinely diagnosed via cytology;
however, immunocytochemistry or surgical pathology with
subsequent immunohistochemistry is necessary for immuno-
phenotypic characterization of the neoplastic cell population.
HistoGel is a readily available media that can be used for
double embedding techniques to incorporate cytologic speci-
mens into a solid medium. In smaller or hypocellular sam-
ples, cell blocks can be readily prepared from specimens
with singly scattered loose cells using conventional centrifu-
gation.3 Double embedding provides improved tissue sup-
port such that specimens can be processed as a routine
histologic sample for further histochemical and immunohis-
tochemical evaluation. HistoGel double embedding method-
ology and immunohistochemistry have been previously used
on peripheral blood samples in people with chronic lympho-
cytic leukemia (CCL).1,3 In a previous study,1 HistoGel cell
blocks were used to evaluate MUM1/IRF4 expression in
peripheral blood CCL cells. While MUM1/IRF4 expression
was variable, the study demonstrated that surface antigens
expressed on circulating leukemic cell lines could be identi-
fied in HistoGel-prepared specimens.
The present study describes the use of HistoGel for encap-
sulating a variety of fresh and formalin-fixed tissues, as well
as highly cellular cytologic, fluid samples. This strategy
helps to maintain the integrity of friable sample and orienta-
tion of small tissues during processing. Cytologic specimens
can retain elements of tissue architecture that are often lost in
standard cytologic preparations and are amenable to standard
methods for immunostaining. The intent of the current study
was to support the use and explore potential applications of
HistoGel in veterinary diagnostic pathology.
Multiple tissues were collected from a 2-year-old, male
Domestic Shorthair cat that presented for postmortem exam-
ination following postoperative anesthetic-related death.
Collected tissues included fragments of nasal turbinates, sec-
tions of gastrointestinal tract, and cerebellum. These repre-
sentative tissues are frequently encountered during routine
surgical biopsy service and may be small or extremely fria-
ble and therefore more labor intensive to process and section.
Five sections of each tissue were collected using endoscopic
biopsy forceps to simulate samples that are often encoun-
tered in surgical pathology submissions. Two sections of
each tissue were fixed by immersion in 10% neutral buffered
445763JVDXXX10.1177/1040638712445763Join
er, SpanglerHistoGel™ in veterinary diagnostic pathology
From Auburn University, Auburn, AL (Joiner, Spangler)
1Corresponding Author: Kellye S. Joiner, Department of Pathobiology,
College of Veterinary Medicine, Auburn University, 166 Greene Hall, AL
36849-5519. bessiks@auburn.edu
Evaluation of HistoGel™-embedded
specimens for use in veterinary
diagnostic pathology
Kellye S. Joiner,1 Elizabeth A. Spangler
Abstract. HistoGel™ is an aqueous specimen-processing gel that encapsulates and suspends histologic and cytologic
specimens in a solidified medium. HistoGel-embedded specimens can then be processed and evaluated by routine histologic
and immunohistochemical methods. This methodology has been used in human diagnostic pathology and is especially
useful for small, friable, or viscous tissue samples that are difficult to process. In addition, special histochemical stains or
immunohistochemistry can be performed on HistoGel-embedded cytologic specimens using standardized methods developed
for histopathology. The current report describes several applications for HistoGel, including use with cytologic specimens,
bone marrow aspirates, retention of tissue orientation for endoscopic biopsy specimens, and evaluation of friable tissues.
Samples were encapsulated in HistoGel, fixed in 10% neutral buffered formalin, routinely processed, paraffin embedded, and
sectioned for histochemical and immunohistochemical evaluation. The results of this study support the use of HistoGel in
veterinary diagnostic pathology.
Key words: Cytology; diagnostic pathology; HistoGel; immunohistochemistry.
HistoGel™ in veterinary diagnostic pathology 711
formalin for 24 hr prior to encapsulating the tissues into
HistoGel. Two sections of each tissue were directly embed-
ded into the gel before fixation, and the fifth section of each
tissue was routinely processed without the addition of
HistoGel. HistoGel was prepared following the manufactur-
er’s recommendations. Briefly, HistoGel is a solid at room
temperature and must be liquefied for use by heating to 50ºC
in a water bath. The liquefied HistoGel was placed in a ster-
ile, flat, 12 mm in diameter polyethylene centrifuge tube cap
and allowed to cool to a semi-solid state at room tempera-
ture. All tissues were embedded in the gel with appropriate
orientation, and additional liquefied HistoGel was added to
completely encapsulate the tissue. The samples were then
refrigerated at 4°C for 5 min to completely solidify the
embedded sample. HistoGel-encapsulated samples were
transferred to a processing cassette. Unfixed tissues were fixed
by immersion in 10% neutral buffered formalin for 24 hr
prior to embedding in HistoGel. Following fixation, HistoGel-
encapsulated tissues were routinely processed, paraffin
embedded, sectioned, and stained with hematoxylin and
eosin for evaluation.
The cytologic specimen evaluated in the present study
was fluid from traumatic catheterization of a dog with tran-
sitional cell carcinoma. Half of the fluid was prepared as a
cytologic direct smear on a glass slide, allowed to air dry,
and stained with modified Wright stain. The remaining por-
tion of the fluid from the traumatic catheterization was
embedded in HistoGel as follows: 1.5 ml of sediment pre-
pared from the fluid was combined with an equal volume of
liquefied HistoGel. Samples were gently mixed to distrib-
ute cells evenly within the gel matrix prior to solidification.
HistoGel-encapsulated samples were transferred to a tissue
processing cassette and fixed by immersion in 10% neutral
buffered formalin for 24 hr. Following fixation, the
sample was routinely processed and evaluated as described
previously.
To demonstrate the retention of immunoreactivity in
HistoGel-embedded specimens, sections of nasal turbinate,
gastrointestinal tract, and cerebellum were examined im-
munohistochemically with a polymer detection kitb using
an autostainerc and commercially available antisera for
cytokeratin,d glial fibrillary acid protein,e muscle specific
actin,f and vimenting with appropriate control tissues for
each antibody (Table 1). HistoGelembedded sections of the
fluid sediment were immunolabeled with commercially
available antisera for cytokeratin and vimentin. Table 1 sum-
marizes the immunohistochemical antibodies and antigen
retrieval technique.
Both fresh and formalin fixed sections of nasal turbinates,
gastrointestinal tract, and cerebellum were found to be easily
embedded into HistoGel with preservation of tissue architec-
ture and proper orientation. All tissues were fixed in forma-
lin for 24 hr prior to processing; however, fixation time may
vary with tissue type and should be evaluated on an individ-
ual basis. In all instances, tissues were easily sectioned, and
there was excellent retention of cell morphology and immu-
noreactivity with several commonly used immunohistochem-
ical antibodies. These results are consistent with previous
studies that evaluated bone marrow aspirates in dogs with
hematopoietic tumors (presented at the 29th annual meeting
of the Veterinary Cancer Society, Austin, Texas, 2009).
During this previous study, bone marrow aspirates from 21
dogs with hematopoietic tumors were evaluated for clinical
staging. Results demonstrated that bone marrow aspirates
could be readily embedded into HistoGel and routinely pro-
cessed as a histologic specimen. Phenotypes of neoplastic
cell lines were further characterized by immunohistochemis-
try using the same methodology applied to staining of tissue
sections.
There were no histocytologic differences in tissues placed
in HistoGel prior to fixation or following fixation when com-
pared to tissues that were not placed in HistoGel. However,
embedding fresh tissues in HistoGel prior to fixation
increased preservation of friable specimens, and proper ana-
tomic orientation was easier. In addition, while there were no
differences in tissue processing techniques including pro-
cessing times, histochemical staining, or immunohistochem-
ical reactivity, double embedding friable and small tissues in
HistoGel as compared to non-embedded samples provided
improved tissue support and sectioning qualities. In some
instances (i.e., sections of cerebrum and gastrointestinal
tract), specimens that were embedded in HistoGel were eas-
ier to section compared to standard, non-HistoGel–embedded
tissue.
Tissues with high lipid content or those that are heavily
myelinated (i.e., brain and spinal cord) are extremely friable
and often fragment with tissue handling, processing, and sec-
tioning. In the present study, sections of cerebellum were
collected and placed in HistoGel prior to fixation in order to
evaluate preservation of tissue integrity in friable specimens.
Table 1. Antigens and immunohistochemical protocols used in the current study.*
Antigen Dilution Antigen retrieval Control tissue
Cytokeratin 1:1,200 Proteinase K Tonsil
Glial fibrillary acidic protein Prediluted Proteinase K Cerebrum
Muscle specific actin 1:100 HIER Skeletal muscle
Vimentin 1:400 HIER Tonsil
* HIER = heat-induced epitope retrieval. Source of antigens and HIER: Dako North America Inc., Carpentaria, CA.
712 Joiner, Spangler
The encapsulated sections of cerebellum were completely
fixed in formalin within 24 hr and remained intact during
processing, embedding, and sectioning. Histologic sections
of embedded cerebellum were well preserved with excellent
tinctorial contrast when embedded in HistoGel before or
after fixation in 10% neutral buffered formalin as compared
to non-HistoGel–embedded sections (Fig. 1). The non-
HistoGel–embedded sample required 10 additional serial
sections to achieve nonfragmented sections; however, non-
fragmented sections were readily obtained with a single sec-
tion of those samples embedded in HistoGel.
Thin, delicate tissues, such as nasal turbinates, may be
difficult to orient when embedding directly into paraffin and
tend to readily fragment during sectioning. Incorporating the
tissue into HistoGel prior to processing allows proper orien-
tation and provides improved tissue support for sectioning.
Sections of fresh and formalin-fixed nasal turbinates were
easily embedded into HistoGel, preserving tissue orientation
and maintaining mucosal integrity, thus validating the use of
HistoGel in delicate specimens (Fig. 2). Ciliated mucosal
epithelial cells were distinct, and strong immunoreactivity
with cytokeratin was maintained in the mucosal epithelium.
Endoscopic biopsies of the gastrointestinal tract are com-
monly encountered in diagnostic surgical pathology.
Anatomic orientation can be difficult when dealing with
small endoscopic-guided biopsy samples (i.e., gastrointesti-
nal or respiratory tract). Proper orientation is necessary to
evaluate the villus-to-crypt ratio, and the mucosal epithelium
lining the villus tips is extremely delicate. Full-thickness
sections of unfixed intestine were directly embedded into
HistoGel following collection to demonstrate a potential
intraoperative use for maintaining tissue orientation and
preservation of tissue architecture. Proper orientation of the
unfixed sections of gastrointestinal tract was readily achieved
and maintained in HistoGel (Fig. 3). The villus-to-crypt ratio
could be assessed, and the mucosal integrity was intact.
Perhaps the most striking potential for HistoGel in veteri-
nary diagnostic pathology involves a multitude of cytologic
applications. While routine cytology provides tremendous
insight into etiologies and disease mechanisms, limitations
Figure 1. Cerebellum; cat. There is excellent retention of cerebellar architecture with preservation of cell morphology and tinctorial
properties in all treatments. Note that fragmented samples of cerebellum were retained in HistoGel™ encapsulation matrix and not lost
upon sectioning. Additional sectioning of the samples that were not embedded in HistoGel was required for comparable results. a, sections
of cerebellum were encapsulated in HistoGel prior to fixation. Hematoxylin and eosin (HE). Bar = 100 µm. b, sections of cerebellum were
encapsulated in HistoGel following fixation. HE. Bar = 100 µm. c, sections of cerebellum were not embedded in HistoGel. HE. Bar = 100 µm.
Figure 2. Nasal turbinates; cat. Thin, delicate nasal turbinates were easily oriented into HistoGel™, preserving tissue orientation and
maintaining mucosal integrity that can be lost during processing of small, friable tissues. Formalin-fixed sections of nasal turbinates that
were not embedded in HistoGel appeared similar histologically, but were more difficult to orient and embed during processing. a, sections
of respiratory tract were encapsulated in HistoGel following fixation. Hematoxylin and eosin (HE). Bar = 100 µm. b, sections of respiratory
tract were encapsulated in HistoGel prior to fixation. HE. Bar = 100 µm. c, sections of respiratory tract were not embedded in HistoGel. HE.
Bar = 100 µm.
HistoGel™ in veterinary diagnostic pathology 713
exist with regard to the amount of diagnostic information
that can be deduced from a cytologic specimen. Preparation
of the cytologic samples embedded in HistoGel was rapid,
and no special equipment was required. Once embedded into
HistoGel, samples were fixed in formalin and processed
exactly as for tissue biopsies, and no special handling of the
sections was required for immunostaining. The use of
HistoGel for cytologic samples may require additional sam-
ple preparation compared to strategies described for immu-
nocytochemistry.2 However, a principal advantage of
HistoGel-encapsulated cytologic specimens over routine
cytology is the ability to further evaluate and phenotypically
classify cell types using an extended array of histochemical
and immunohistochemical techniques. Moreover, additional
sections can readily be obtained from cytologic samples in
HistoGel. With the continuing evolution of clinical treat-
ments directed toward specific cellular phenotypes (i.e., neo-
plastic cell lineages), immunohistochemical categorization
of cell types is critical. HistoGel-encapsulated cytologic
specimens address this dilemma by functionally transform-
ing a cytologic sample into a solid media that can be pro-
cessed as a routine histologic sample. Special histochemical
stains and immunohistochemical antibodies can then be
applied to the sample using routine procedures that have
been developed and standardized for histopathology.
An additional consideration is retained stromal architec-
ture in HistoGel-embedded cytologic specimens. It was
found that in contrast to the conventional cytologic sample,
sections of the fluid sample that were encapsulated in
HistoGel maintained some stromal integrity, as large sheets
of neoplastic transitional epithelium were organized around
a fibrovascular stroma (Fig. 4). This allows the pathologist
to assess the interaction between the neoplastic cell popula-
tion and the neighboring tissue, which is very important
when considering localized and vascular invasiveness that
is frequently associated with several neoplasms. Because
collection of cytologic samples is relatively noninvasive,
HistoGel-encapsulated cytologic specimens may provide
the diagnostician with additional information with mini-
mal patient discomfort as compared to surgical biopsy
procedures.
The findings also illustrate the use of immunohistochemi-
cal antibodies in embedded cytologic specimens. Neoplastic
transitional epithelium was strongly reactive with cytokera-
tin, and the vascular endothelial cells were strongly immuno-
reactive with vimentin (Fig. 4). In addition, encapsulated
sections of cerebellum showed strong immunoreactivity with
glial fibrillary acid protein whether fixed in formalin prior to
or after embedding in HistoGel (Fig. 5). Similar immunoreac-
tivity to cytokeratin and muscle-specific actin was present in
processed sections of nasal turbinates and gastrointestinal
tract, respectively (data not shown). Optimized techniques
were identical to those used for standard histologic speci-
mens. Because some laboratories are limited to immunohisto-
chemistry and do not perform immunocytochemistry, this
technique provides an additional diagnostic tool.
In summary, the current study demonstrates the success-
ful application of HistoGel to encapsulate a variety of
tissues, preserving fluid samples that are traditionally
amendable only to cytology, maintaining the integrity of fri-
able samples and appropriate anatomic orientation of small
tissues. The results validate the utility of HistoGel in routine
veterinary diagnostic cytology and histopathology. Both
fresh and formalin-fixed tissues can be embedded into
HistoGel with no detectable differences in histologic appear-
ance. This suggests that HistoGel tissue encapsulation can be
performed by the surgeon prior to submission such that ana-
tomic orientation of tissue margins is maintained and pathol-
ogist reporting of margins correlates to the surgeon’s
reference point. Due to the additional labor embedding sam-
ples in HistoGel, it is only recommended in cases in which
orientation of small samples is critical or for cytologic proce-
dures in which multiple tests (e.g., immunocytochemistry,
histochemical stains) will be performed.
Figure 3. Gastrointestinal tract; cat. Proper orientation of small unfixed sections of gastrointestinal tract was readily achieved and
maintained in HistoGel™. a, sections of gastrointestinal tract were oriented and encapsulated in HistoGel prior to fixation. Hematoxylin and
eosin (HE). Bar = 100 µm. b, sections of gastrointestinal tract were oriented and encapsulated in HistoGel following fixation. HE. Bar =
100 µm. c, sections of gastrointestinal tract were not embedded in HistoGel. HE. Bar = 100 µm.
714 Joiner, Spangler
Figure 4. Sediment made of fluid from traumatic catheterization of the urinary bladder; dog. a, smear made from the sediment is
abundantly cellular and contains many large sheets of epithelial cells that show moderate anisocytosis and anisokaryosis. Binucleated
cells and mitotic figures are occasionally seen. Modified Wright stain. Bar = 100 µm. b, sections made of the fluid sediment encapsulated
in HistoGel™ show many sheets of neoplastic epithelial cells that are organized around a fibrovascular stroma. Hematoxylin and eosin.
Bar = 100 µm. c, neoplastic cells are strongly immunoreactive with cytokeratin. Bar = 100 µm. d, vascular endothelial cells are strongly
immunoreactive with vimentin. Dual link system. Hematoxylin counterstain. Bar = 100 µm.
Figure 5. Cerebellum; dog. Samples show strong positive immunoreactivity with glial fibrillary acidic protein. a, sample embedded in
HistoGel™ prior to fixation. Bar = 100 µm. b, sample embedded in HistoGel following fixation. Bar = 100 µm. c, sections of cerebellum
were not embedded in HistoGel. Bar = 100 µm. d, negative control. Dual link system. Hematoxylin counterstain. Bar = 100 µm.
HistoGel™ in veterinary diagnostic pathology 715
Acknowledgements
Part of this study was presented as a poster at the 60th annual meeting
of the American College of Veterinary Pathologists in Monterey,
California, December 2009. The authors thank the Auburn University
College of Veterinary Medicine Diagnostic Histopathology Laboratory
and Clinical Pathology Laboratory staff for technical assistance.
Sources and manufacturers
a. HistoGel™, Richard-Allan Scientific, Kalamazoo, MI.
b. Dual Link System-HRP Envision+ (catalog no. K4061), Dako
North America Inc., Carpentaria, CA.
c. Dako Universal autostaining system (model no. LV-1), Dako
North America Inc., Carpentaria, CA.
d. Cytokeratin (catalog no. IR053), Dako North America Inc.,
Carpentaria, CA.
e. Glial fibrillary acidic protein (catalog no. SK200), Dako North
America Inc., Carpentaria, CA.
f. Muscle specific actin (catalog no. IR700), Dako North America
Inc., Carpentaria, CA.
g. Vimentin (catalog no. IR630), Dako North America Inc.,
Carpentaria, CA.
h. Heat induced epitope retrieval, target retrieval solution, Pascal
S2800, Dako North America Inc., Carpentaria, CA.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
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2. Valli V, Peters E, Williams C, et al.: 2009, Optimizing methods
in immunocytochemistry: one laboratory’s experience. Vet Clin
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