Veterinary Dermatology 2003, 14, 147 151

Blackwell Publishing Ltd.

A modified biopsy technique to improve histopathological

evaluation of avian skin
C. S. NETT,* E. C. HODGIN, C. S. FOIL,* S. R. MERCHANT* and T. N. TULLY*
*Department of Clinical Sciences, Louisiana State University, Veterinary Teaching Hospital, Baton Rouge, LA
70803, USA Louisiana Veterinary Medical Diagnostic Laboratories, Louisiana State University, Baton Rouge,
LA 70803, USA
(Received 12 August 2002; accepted 24 January 2003)

Abstract Skin biopsies are a viable diagnostic tool in avian dermatology, however, the thinness of avian skin
makes it difficult to prevent rolling and contraction of skin biopsy specimens during collection and fixation. The
difficulty orienting such rolled samples during processing ultimately interferes with the establishment of a histopathological diagnosis. We describe a modified skin biopsy procedure for obtaining avian skin biopsy specimens. In this technique nontranslucent self-adhesive tape (Scotch tape) was attached to skin biopsy sites before
obtaining skin biopsies using a standard skin biopsy punch instrument. A total of 23 skin biopsy specimens were
obtained: 15 from nonfeathered skin of 12 normal Hispaniolan parrots, 3 from feathered skin of 2 normal birds
and 5 from feathered skin of 3 psittacines presented for pathologic feather-picking. All 23 skin specimens consistently adhered to the tape during the biopsy procedure. The specimens were fixed in 10% neutral phosphatebuffered formalin. During processing, no curling or rolling of specimens occurred, and all specimens could be
easily orientated for correct trimming and subsequent histopathological evaluation. The tape technique did not
produce any appreciable artefacts. Remnants of the tape were microscopically evident above the stratum corneum
assuring that none of the stratum corneum was lost during processing. Obtaining avian skin biopsy specimens
using this modified tape technique is easy and ensures flat fixation of the skin biopsy specimens, which later allows
trimming at right angles, and through the longitudinal diameter of feather follicles for accurate histopathologic
evaluation.
Keywords: avian skin, histopathology, sample orientation, skin biopsy technique, tape

INTRODUCTION
Skin biopsy specimens are commonly used to diagnose
many skin diseases in both people and animals. In
avian dermatology skin biopsy specimens are likewise
used to diagnose a wide variety of integumentary
disorders.13 In birds, thin skin and a lack of subcutaneous fat make it difficult to use mammalian biopsy
procedures because skin specimens often curl and roll
into cylinders during the process of obtaining the
biopsy or during fixation. In 1972, Lucas & Stettenheim
suggested a method for obtaining avian skin biopsy
specimens that addressed the importance of fixing
samples flat.4 This technique allowed for later trimming perpendicular to the surface to obtain a full
cross-section of the epidermis and dermis. These
authors suggested spreading out the skin biopsy specimen, fastening it to a support (parchment paper,
woven wire cloth) and then fixing it in formalin.
Because rolling of avian skin specimens tends to occur

This study was presented as a poster at the annual ISVD meeting in

Nice, France, 25 September 2002.
Correspondence: C. S. Nett, Louisiana State University, Veterinary
Teaching Hospital, Baton Rouge, LA 70803, USA. E-mail: cnett@freesurf.ch
2003 European Society of Veterinary Dermatology

first during the harvesting process (Fig. 1), the fresh

sample has to be stretched immediately. Stretching of
a fresh skin specimen can lead to mechanical damage
resulting in artefacts, particularly at the edges, where
the biopsy specimens are grasped by forceps. These

Figure 1. Standard method of obtaining an avian skin biopsy.

Because of the thinness of the avian skin, the biopsy specimen rolls
during the harvesting process. Subsequent stretching of the sample
is needed prior to fixation to allow for later orientation. Note the
rolled specimen.
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C. S. Nett et al.

Figure 2. Step 1 of new procedure. A 3-cm long, nontranslucent

self-adhesive tape is placed on the skin surface.

artefacts can interfere with dermatopathological evaluation and even lead to misinterpretation.5
The major reason for preventing distortion of the
skin during fixation is to allow correct trimming. The
standard way of trimming a skin biopsy is to use a
scalpel blade to cut the specimen transversely, thereby
creating a flat edge from the epidermis to the subcutis.
In mammalian haired skin specimens, this approach
allows the histopathologist to cut parallel to the long
axis of the hair follicles, so that the location of the hair
bulb in the dermis or subcutis can be evaluated accurately, and the stage of the growth cycle of the hair can
be determined.5
In haired skin, the hair protruding from the skin surface allows the epidermal and dermal surfaces to be
readily identified at trimming. In avian skin, feathers
can similarly direct the pathologist, but because samples roll into cylinders, orientation at the time of trimming can still be difficult. In addition, in nonfeathered
skin, gross distinction between dermal and epidermal
surfaces is frequently impossible. This subsequently
leads to misorientation of specimens during processing
and prevents the pathologist from accurately reading
and interpreting the biopsy specimens. Such poor orientation can occasionally lead to histological sections
in which large portions of the epidermis or dermis are
completely absent due to perpendicular cutting.
Here we describe a simple skin biopsy procedure
that minimizes the rolling or curling of avian skin during collection and fixation, and allows easy orientation
and subsequent correct histopathological processing
and evaluation.

MATERIALS AND METHODS

Twelve healthy Hispaniolan Amazon parrots (Amazona
ventralis), originally obtained from the Department of
Natural and Environmental Resources, Arecibo, Puerto
Rico, and housed at the Louisiana State Department
of Laboratory Animal Medicine facility for the last
two years were used in this study. All animals were
allowed food and water ad libitum and cared for accord-

Figure 3. Step 2: the skin biopsy punch is placed directly over the
tape, the skin is tensed, and gentle pressure is applied as the punch is
used to cut through both the tape and skin.

Figure 4. Step 3: the specimen is collected using forceps. Note that

the skin specimen is adhered to the tape.

ing the guidelines set forth in the NIH Guides for the
Care and Use of Laboratory Animals. All birds were
healthy and evaluated by veterinarians on a regular
basis. None had evidence of any skin disease. Skin
biopsy procedures were performed while the birds were
under general anaesthesia employing a mask and isoflurane as the inhalant anaesthetic agent. Fifteen punch
biopsy sites were selected from nonfeathered skin. The
skin was gently cleaned with a dry cotton ball to
remove natural dander (powder). A 23-cm long,
nontranslucent self-adhesive tape (Scotch tape, 3M,
Minneapolis, MN) was attached to the chosen biopsy
site (Fig. 2). A 6-mm biopsy punch was placed directly
over the taped site and the biopsy procedure was performed through the tape by applying a gentle clockwise
and counter-clockwise twisting force (Fig. 3). Once
the skin biopsy punch penetrated the tape, only minimal
force was applied to the avian skin so as not to traumatize the underlying musculature. The skin biopsy
specimen, adhered to the tape, was collected and placed
in 10% neutral phosphate-buffered formalin for fixation (Fig. 4). Nontranslucent tape was chosen because
a pilot study showed that even though the translucent
tape stuck better to the skin biopsy site, the biopsy
punch did not grip easily, which made exact positioning

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 147 151

A modified avian skin biopsy technique

149

Figure 5. Avian skin. Note the properly orientated and well-aligned

specimen. The arrows indicate remnants of the adhesive tape (H&E
staining).

Figure 7. (a) Avian skin specimen obtained with the tape technique
(H&E, 100 magnification). Note correct alignment and orientation
for easy histopathological evaluation. (b) Avian skin. Skin specimen
obtained with standard punch biopsy technique. Note curling of the
entire specimen with impaired orientation (H&E).

Figure 6. Avian skin. Biopsy specimen obtained with the tape

technique from feathered skin demonstrating correct trimming
along the long axes of a feather follicle (H&E).

of the biopsy punch difficult. Also, the translucent tape

is firmer and therefore more resistant to cutting
increasing the likelihood of bruising and traumatizing
the underlying musculature.
After collection, specimens were fixed for at least 6 h
prior to trimming. All skin biopsy specimens were still
adhered to the tape when removed from the formalin
containers for trimming. None of the specimens
had rolled or balled up. The biopsy specimens were
dissected and placed in tissue cassettes for further

processing without removing the tape. Orientation at

trimming was easy because of the tape sticking to the
epidermal side of the biopsy. No rolling and curling
occurred during fixation or at the time of paraffin
embedding and all specimens were correctly orientated
at histopathological evaluation (Fig. 5).
Because many avian integumentary diseases also
involve the feather follicle and biopsies will most likely
be obtained from feathered skin in clinical cases, it was
felt important to confirm the usefulness of the tape
technique in feathered skin. For this purpose, three
skin biopsies were obtained from feathered skin from
two other avian cases without skin disease. To promote
adherence of the tape, feathers were clipped even with
the surface of the skin carefully avoiding traumatizing
the latter. Punch biopsies were then performed as
described previously.
To verify that this technique was also reproducible
in clinical cases, the tape method was then used in
three pet psittacines presented for pathological featherpicking to the Louisiana State University, Veterinary
Teaching Hospital. A total of five punch biopsies from
feathered skin was obtained.
In skin specimens containing feather follicles (8/23)
it was attempted to trim the biopsy in a right angle to
the skin surface through the longitudinal axis of the
feather follicle (Fig. 6).

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C. S. Nett et al.

RESULTS
All of the skin biopsy specimens from nonfeathered
skin (15/15) adhered tightly to the tape and no curling
or rolling occurred during harvesting or fixation.
In one specimen obtained from feathered skin (1/8),
the margin of the tape came loose resulting in mild
curling at the edge, however, correct orientation for
trimming was still possible. In all other cases (7/8), the
specimens adhered consistently to the tape during
harvesting and fixation despite the presence of clipped
feathers at the biopsy site.
The histological examination of all 23 specimens
(15 nonfeathered and 8 feathered) revealed correct orientation of the dermis and epidermis with no distortion
or rolling. In all but one of the eight biopsies obtained
form feathered skin, the feather follicles were cut
through the longitudinal axis. The biopsy procedure did
not produce appreciable artefact in any of the cases.
Remnants of the tape above the stratum corneum were
evident microscopically in all evaluated specimens but
did not interfere with histological evaluation. Final
specimens were flat and well orientated compared with
specimens obtained without the tape technique optimizing histopathological interpretation (Fig. 7a,b).

a line with an indelible marker on the skin surface in

the direction of the hairs prior to harvesting the skin
biopsy specimen. This line later helped with orientation and correct trimming in the longitudinal axis of
the hairs. Using our technique, this line can be drawn
on the tape over the feather follicle before obtaining the
biopsy, thus providing guidance for easy orientation
when trimming the specimen.
Our modified biopsy technique may even be beneficial in mammalian dermatology when obtaining skin
specimens from extremely thin skin as, for example, in
alopecic, atrophied skin disorders associated with
endocrinopathies or in cats, hamsters and other small
mammals.
It is important that the biopsy punch be sharp in
order to cut through the tape. Because of the thinness
of the skin, caution must be observed when performing
the biopsy as too much force will lead to cutting injuries of the underlying tissues. It may therefore be advisable to practice the tape technique on a dead bird to
experience how much pressure is needed to cut through
the tape without injuring the underlying tissues.
In summary, we believe that the advantage of this
technique lies in its simplicity and ease. The materials
are easily obtainable and the method is readily applicable and offers a solution for optimal orientation and
dermatopathological evaluation of avian skin specimens.

DISCUSSION
This modified biopsy procedure was easy to perform
and resulted in high-quality specimens of normal and
diseased interfollicular and follicular avian skin for histological processing. The main difference between this
technique and those described previously, both for
mammals and avian skin, is in the use of tape to fix
the skin prior to obtaining the biopsy. In previously
described techniques, the biopsy is obtained first and
then placed dermis side down on a piece of cardboard
allowing the serum to dry and stick the skin to the
card (prior to placing it in the fixative) to maintain its
orientation and to prevent rolling.5 However, avian
skin tends to roll and curl at the time it is harvested and
if placed on a card, it has to be teased and stretched to
remove wrinkles.4 This increases the possibility that the
forceps may cause compression or crush artefacts at
the edge of the specimen. This modified technique is
different because it uses adhesive tape on the surface of
the epidermis to maintain flatness of the skin, both at
the time of biopsy and also during fixation. In cases in
which some curling at the edges of the biopsy still
occurs, the biopsy can also be placed on a card to
prevent this. This technique may be of limited use in
extremely crusty, powdery or oily skin because of
inadequate adherence of the tape to the skin.
In feathered skin where it is important to trim specimens through the long dimension of the feather follicle
for accurate evaluation, the line method recently
described by Mauldin et al.6 may be beneficial to use
in combination with the described tape technique for
optimal results. Briefly, these authors suggested drawing

ACKNOWLEDGEMENTS
The authors would like to thank Dr Orlando Diaz for
his technical assistance. This study was approved by
the Animal Care and Use Committee of Louisiana
State University, Baton Rouge, LA, and was partially
supported by a grant from the Department of Veterinary Clinical Sciences Organized Research Fund of
Louisiana State University, School of Veterinary
Medicine, Baton Rouge, LA, USA.
REFERENCES
1. Perry, R. Avian dermatology. In: Burr, E., ed. Companion
Bird Medicine. Iowa State University Press, Ames, 1987:
4050.
2. Perry, R., Gill, J., Cross, G. Disorders of the avian integument. Veterinary Clinics of North America: Small Animal
Practice 1991; 21: 130727.
3. Romagnano, A., Heard, D. Avian dermatology. In:
Olsen, G., Orosz, S., eds. Manual of Avian Medicine. Mosby,
St. Louis, MO, 2000: 95123.
4. Lucas, A., Stettenheim, P. Avian anatomy. Integument.
Agricultural Handbook 362. Agriculture USDo, Washington, DC, 1972.
5. McGavin, M., Fadok, V. Factors limiting the usefulness
of histopathologic examination of skin biopsies in the
diagnosis of large animal dermatoses. Veterinary Clinics of
North America: Large Animal Practice 1984; 6 (1): 20313.
6. Mauldin, E., Castle, S., Davenport, G. et al. A simple
biopsy technique to improve dermatopathologic interpretation. Veterinary Medicine 2002; 4: 2868.

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Rsum Les biopsies cutanes sont un examen intressant en dermatologie aviaire, cependant la finesse de la
peau des oiseaux rend difficile la prvention de lenroulement et de la contraction des prlvements pendant lacte
et la fixation. La difficult orienter des prlvements enrouls pendant le technicage peut interfrer avec le diagnostic. Cet article rapporte une technique modifie de biopsie pour obtenir des prlvements de peau doiseau.
Un morceau de ruban adhsif (Scotch) est attach au site biopsique avant de raliser la biopsie classiquement.
Un total de 23 biopsies cutanes ont t obtenues: 15 partir de la peau sans plume de 12 perroquets normaux,
3 partir de la peau avec plumes de 2 oiseaux sains et 5 partir de la peau avec plumes de 3 psittacids prsents
pour un pica. Tous les prlvements ont correctement adhr la cellophane pendant la biopsie. Les spcimens
ont t fixs dans du formol 10%. Pendant le technicage, aucun enroulement des prlvements nest apparu, et
tous les prlvements ont pu tre facilement orients pour lexamen. Cette technique na pas provoqu dartfact
notable. Des restes de cellophane taient prsents au dessus du stratum corneum, ce qui indique que tout le stratum corneum a pu tre prlev. Lobtention de prlvements cutans de peau doiseau par cette technique est
facile et permet une fixation plat qui autorise une orientation et une interprtation correcte des biopsies
cutanes.
Resumen Las biopsias de piel son una herramienta de diagnstico viable en dermatologa aviar; no obstante,
la fineza de la piel aviar hace que sea difcil evitar el repliego y contraccin de las muestras de piel durante la toma
y la fijacin de las mismas. La dificultad en la orientacin de dichas muestras interfiere en ltima instancia con
el establecimiento de un diagnstico histopatolgico. Este artculo describe un procedimiento modificado para
la obtencin de una muestra de biopsia de piel en aves. En esta tcnica una cinta auta-adhesiva no-transcida
(Scotch tape) fue pegada a la piel a biopsiar antes de obtener las biopsias de piel utilizando un instrumento
estndar tipo punch. Se obtuvieron un total de 23 muestras de biopsia de piel: 15 de piel no-emplumada de 12
loros Hispaniolan normales, 3 de piel emplumada de 2 pjaros normales y 5 de piel emplumada de 3 psitcidas
con alteraciones patolgicas de plumas por picajes. Las 23 muestras de piel se adhirieron constantemente a la
cinta adhesiva durante el procedimiento de la biopsia. Las muestras fueron fijadas en una solucin de formalina
al 10% tamponada con fosfato. Durante el proceso no se produjo el encorvamiento o repliegue de las muestras,
y todos las muestras (23/23) se pudieron orientar fcilmente para el corte correcto y la evaluacin histopatolgica
subsiguiente. La tcnica de la cinta adhesiva no produjo ningn artefacto apreciable. Los restos de la cinta eran
microscpicamente evidentes sobre el estrato crneo y aseguraba que ste no se perdiera durante el proceso. La
obtencin de muestras de piel de aves con esta tcnica modificada de la cinta adhesiva es fcil y asegura la fijacin
plana de las muestras de biopsia de piel, lo cual permite ms adelante el corte en el ngulo perpendicular adecuad,
y a travs del dimetro longitudinal de los folculos de la pluma para la evaluacin histopatolgica correcta.
Zusammenfassung Hautbiopsien sind fr die Diagnosefindung in der Vogeldermatologie wertvoll, es ist jedoch
schwierig, bei der dnnen Vogelhaut Einrollen und Kontraktion der Hautbiopsieproben whrend der Entnahme
und Fixierung zu vermeiden. Schwierigkeiten mit der Orientierung solcher eingerollter Proben whrend der Bearbeitung kann schlussendlich die histopathologische Diagnosefindung erschweren. Dieser Artikel beschreibt eine
modifizierte Hautbiopsieprozedur zur Entnahme von Hautbiopsieproben beim Vogel. Bei dieser Technik wurde
ein undurchsichtiges Selbstklebeband (Scotch tape) auf die zu biopsierenden Stellen aufgeklebt, bevor Hautbiopsieproben mit einer normalen Hautstanze entnommen wurden. Insgesamt wurden 23 Hautbiopsieproben
genommen: 15 von nicht-befiederter Haut 12 normaler Papageien, drei von befiederter Haut zweier normaler
Papageien und fnf von befiederter Haut dreier Psittazine, die auf Grund von Federausrupfen vorgestellt wurden.
Alle 23 Proben blieben whrend der Biopsieentnahme mit dem Klebeband verbunden. Die Proben wurden in
10%-igem, neutralem Phosphat-gepufferten Formalin fixiert. Whrend der Bearbeitung wurde weder Kruseln
noch Einrollen der Proben gesehen und alle Proben (23/23) konnten gut fr korrekte Bearbeitung und nachfolgende histopathologische Bewertung orientiert werden. Die Klebebandtechnik war nicht mit feststellbaren Artefakten verbunden. berbleibsel des Klebebandes waren mikroskopisch ber dem Stratum corneum feststellbar
und Anzeichen dafr, dass vom Stratum corneum whrend der Bearbeitung nichts verlorenging. Die Entnahme
von Hautbiopsien beim Vogel mit dieser Technik ist leicht und stellt die Fixierung der Hautbiopsieproben sicher,
die spter Trimmen im richtigen Winkel und den Lngsdurchmesser der Federfollikel fr akkurate histopathologische Bewertung erlaubt.

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 147151