A Modified Biopsy Technique to Improve Histopathological Evaluation of Avian Skin

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© 2003 European Society of Veterinary Dermatology 147

Veterinary Dermatology 2003, 14, 147–151

BlackwellPublishingLtd.

A modified biopsy technique to improve histopathologicalevaluation 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, LA70803, 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 skinmakes it difficult to prevent rolling and contraction of skin biopsy specimens during collection and fixation. Thedifficulty orienting such rolled samples during processing ultimately interferes with the establishment of a his-topathological diagnosis. We describe a modified skin biopsy procedure for obtaining avian skin biopsy speci-mens. In this technique nontranslucent self-adhesive tape (Scotch tape®) was attached to skin biopsy sites beforeobtaining 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 birdsand 5 from feathered skin of 3 psittacines presented for pathologic feather-picking. All 23 skin specimens con-sistently adhered to the tape during the biopsy procedure. The specimens were fixed in 10% neutral phosphate-buffered formalin. During processing, no curling or rolling of specimens occurred, and all specimens could beeasily orientated for correct trimming and subsequent histopathological evaluation. The tape technique did notproduce any appreciable artefacts. Remnants of the tape were microscopically evident above the stratum corneumassuring that none of the stratum corneum was lost during processing. Obtaining avian skin biopsy specimensusing this modified tape technique is easy and ensures flat fixation of the skin biopsy specimens, which later allowstrimming at right angles, and through the longitudinal diameter of feather follicles for accurate histopathologicevaluation.

Keywords: avian skin, histopathology, sample orientation, skin biopsy technique, tape

INTRODUCTION

Skin biopsy specimens are commonly used to diagnosemany skin diseases in both people and animals. Inavian dermatology skin biopsy specimens are likewiseused to diagnose a wide variety of integumentarydisorders.1–3 In birds, thin skin and a lack of subcut-aneous fat make it difficult to use mammalian biopsyprocedures because skin specimens often curl and roll

into cylinders during the process of obtaining thebiopsy or during fixation. In 1972, Lucas & Stettenheimsuggested a method for obtaining avian skin biopsyspecimens that addressed the importance of fixingsamples flat.4 This technique allowed for later trim-ming perpendicular to the surface to obtain a fullcross-section of the epidermis and dermis. Theseauthors suggested spreading out the skin biopsy speci-men, 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

first during the harvesting process (Fig. 1), the freshsample has to be stretched immediately. Stretching of a fresh skin specimen can lead to mechanical damageresulting in artefacts, particularly at the edges, wherethe biopsy specimens are grasped by forceps. These

This study was presented as a poster at the annual ISVD meeting inNice, France, 25 September 2002.Correspondence: C. S. Nett, Louisiana State University, VeterinaryTeaching Hospital, Baton Rouge, LA 70803, USA. E-mail: [email protected]

Figure 1. ‘Standard’ method of obtaining an avian skin biopsy.Because of the thinness of the avian skin, the biopsy specimen rollsduring the harvesting process. Subsequent stretching of the sampleis needed prior to fixation to allow for later orientation. Note therolled specimen.



148 C. S. Nett et al .

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

artefacts can interfere with dermatopathological evalu-ation and even lead to misinterpretation.5

The major reason for preventing distortion of theskin during fixation is to allow correct trimming. Thestandard way of trimming a skin biopsy is to use ascalpel blade to cut the specimen transversely, therebycreating a flat edge from the epidermis to the subcutis.In mammalian haired skin specimens, this approachallows the histopathologist to cut parallel to the longaxis of the hair follicles, so that the location of the hairbulb in the dermis or subcutis can be evaluated accu-rately, and the stage of the growth cycle of the hair canbe determined.5

In haired skin, the hair protruding from the skin sur-

face allows the epidermal and dermal surfaces to bereadily identified at trimming. In avian skin, featherscan similarly direct the pathologist, but because sam-ples roll into cylinders, orientation at the time of trim-ming can still be difficult. In addition, in nonfeatheredskin, gross distinction between dermal and epidermalsurfaces is frequently impossible. This subsequentlyleads to misorientation of specimens during processingand prevents the pathologist from accurately readingand interpreting the biopsy specimens. Such poor ori-entation can occasionally lead to histological sectionsin which large portions of the epidermis or dermis are

completely absent due to perpendicular cutting.Here we describe a simple skin biopsy procedurethat minimizes the rolling or curling of avian skin dur-ing collection and fixation, and allows easy orientationand subsequent correct histopathological processingand 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 Departmentof Laboratory Animal Medicine facility for the lasttwo years were used in this study. All animals wereallowed food and water ad libitum and cared for accord-

ing the guidelines set forth in the NIH Guides for theCare and Use of Laboratory Animals. All birds werehealthy and evaluated by veterinarians on a regularbasis. None had evidence of any skin disease. Skinbiopsy procedures were performed while the birds wereunder general anaesthesia employing a mask and iso-flurane as the inhalant anaesthetic agent. Fifteen punchbiopsy sites were selected from nonfeathered skin. The

skin was gently cleaned with a dry cotton ball toremove natural dander (powder). A 2–3-cm long,nontranslucent self-adhesive tape (Scotch tape®, 3M,Minneapolis, MN) was attached to the chosen biopsysite (Fig. 2). A 6-mm biopsy punch was placed directlyover the taped site and the biopsy procedure was per-formed through the tape by applying a gentle clockwiseand counter-clockwise twisting force (Fig. 3). Oncethe skin biopsy punch penetrated the tape, only minimalforce was applied to the avian skin so as not to trau-matize the underlying musculature. The skin biopsyspecimen, adhered to the tape, was collected and placedin 10% neutral phosphate-buffered formalin for fix-

ation (Fig. 4). Nontranslucent tape was chosen becausea pilot study showed that even though the translucenttape stuck better to the skin biopsy site, the biopsypunch did not grip easily, which made exact positioning

Figure 2. Step 1 of ‘new procedure’. A 3-cm long, nontranslucentself-adhesive tape is placed on the skin surface. 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 isused to cut through both the tape and skin.

Figure 4. Step 3: the specimen is collected using forceps. Note thatthe skin specimen is adhered to the tape.




A modified avian skin biopsy technique 149

of the biopsy punch difficult. Also, the translucent tapeis firmer and therefore more resistant to cuttingincreasing the likelihood of bruising and traumatizingthe underlying musculature.

After collection, specimens were fixed for at least 6 hprior to trimming. All skin biopsy specimens were still

adhered to the tape when removed from the formalincontainers for trimming. None of the specimenshad rolled or balled up. The biopsy specimens weredissected and placed in tissue cassettes for further

processing without removing the tape. Orientation attrimming was easy because of the tape sticking to theepidermal side of the biopsy. No rolling and curlingoccurred during fixation or at the time of paraffinembedding and all specimens were correctly orientatedat histopathological evaluation (Fig. 5).

Because many avian integumentary diseases alsoinvolve the feather follicle and biopsies will most likelybe obtained from feathered skin in clinical cases, it wasfelt important to confirm the usefulness of the tape

technique in feathered skin. For this purpose, threeskin biopsies were obtained from feathered skin fromtwo other avian cases without skin disease. To promoteadherence of the tape, feathers were clipped even withthe surface of the skin carefully avoiding traumatizingthe latter. Punch biopsies were then performed asdescribed previously.

To verify that this technique was also reproduciblein clinical cases, the tape method was then used inthree pet psittacines presented for pathological feather-picking to the Louisiana State University, VeterinaryTeaching Hospital. A total of five punch biopsies fromfeathered skin was obtained.

In skin specimens containing feather follicles (8/23)it was attempted to trim the biopsy in a right angle tothe skin surface through the longitudinal axis of thefeather follicle (Fig. 6).

Figure 5. Avian skin. Note the properly orientated and well-alignedspecimen. The arrows indicate remnants of the adhesive tape (H&Estaining).

Figure 6. Avian skin. Biopsy specimen obtained with the tapetechnique from feathered skin demonstrating correct trimmingalong the long axes of a feather follicle (H&E).

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



150 C. S. Nett et al .


RESULTS

All of the skin biopsy specimens from nonfeatheredskin (15/15) adhered tightly to the tape and no curlingor rolling occurred during harvesting or fixation.

In one specimen obtained from feathered skin (1/8),

the margin of the tape came loose resulting in mildcurling at the edge, however, correct orientation fortrimming was still possible. In all other cases (7/8), thespecimens adhered consistently to the tape duringharvesting 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 ori-entation of the dermis and epidermis with no distortionor rolling. In all but one of the eight biopsies obtainedform feathered skin, the feather follicles were cutthrough the longitudinal axis. The biopsy procedure didnot produce appreciable artefact in any of the cases.Remnants of the tape above the stratum corneum wereevident microscopically in all evaluated specimens butdid not interfere with histological evaluation. Finalspecimens were flat and well orientated compared withspecimens obtained without the tape technique opti-mizing histopathological interpretation (Fig. 7a,b).

DISCUSSION

This modified biopsy procedure was easy to performand resulted in high-quality specimens of normal and

diseased interfollicular and follicular avian skin for his-tological processing. The main difference between thistechnique and those described previously, both formammals and avian skin, is in the use of tape to fixthe skin prior to obtaining the biopsy. In previouslydescribed techniques, the biopsy is obtained first andthen placed dermis side down on a piece of cardboardallowing the serum to dry and ‘stick’ the skin to thecard (prior to placing it in the fixative) to maintain itsorientation and to prevent rolling.5 However, avianskin tends to roll and curl at the time it is harvested andif placed on a card, it has to be teased and stretched to

remove wrinkles.

4

This increases the possibility that theforceps may cause compression or crush artefacts atthe edge of the specimen. This modified technique isdifferent because it uses adhesive tape on the surface of the epidermis to maintain flatness of the skin, both atthe time of biopsy and also during fixation. In cases inwhich some curling at the edges of the biopsy stilloccurs, the biopsy can also be placed on a card toprevent this. This technique may be of limited use inextremely crusty, powdery or oily skin because of inadequate adherence of the tape to the skin.

In feathered skin where it is important to trim speci-mens through the long dimension of the feather follicle

for accurate evaluation, the line method recentlydescribed by Mauldin et al .6 may be beneficial to usein combination with the described tape technique foroptimal results. Briefly, these authors suggested drawing

a line with an indelible marker on the skin surface inthe direction of the hairs prior to harvesting the skinbiopsy specimen. This line later helped with orienta-tion and correct trimming in the longitudinal axis of the hairs. Using our technique, this line can be drawnon the tape over the feather follicle before obtaining the

biopsy, thus providing guidance for easy orientationwhen trimming the specimen.Our modified biopsy technique may even be benefi-

cial in mammalian dermatology when obtaining skinspecimens from extremely thin skin as, for example, inalopecic, atrophied skin disorders associated withendocrinopathies or in cats, hamsters and other smallmammals.

It is important that the biopsy punch be sharp inorder to cut through the tape. Because of the thinnessof the skin, caution must be observed when performingthe biopsy as too much force will lead to cutting inju-ries of the underlying tissues. It may therefore be advis-able to practice the tape technique on a dead bird toexperience how much pressure is needed to cut throughthe tape without injuring the underlying tissues.

In summary, we believe that the advantage of thistechnique lies in its simplicity and ease. The materialsare easily obtainable and the method is readily applica-ble and offers a solution for optimal orientation anddermatopathological evaluation of avian skin specimens.

ACKNOWLEDGEMENTS

The authors would like to thank Dr Orlando Diaz forhis technical assistance. This study was approved bythe Animal Care and Use Committee of LouisianaState University, Baton Rouge, LA, and was partiallysupported by a grant from the Department of Veteri-nary Clinical Sciences Organized Research Fund of Louisiana State University, School of VeterinaryMedicine, Baton Rouge, LA, USA.

REFERENCES

1. Perry, R. Avian dermatology. In: Burr, E., ed. Companion

Bird Medicine. Iowa State University Press, Ames, 1987:40–50.

2. Perry, R., Gill, J., Cross, G. Disorders of the avian integu-ment. Veterinary Clinics of North America: Small Animal

Practice 1991; 21: 1307–27.3. Romagnano, A., Heard, D. Avian dermatology. In:

Olsen, G., Orosz, S., eds.Manual of Avian Medicine. Mosby,St. Louis, MO, 2000: 95–123.

4. Lucas, A., Stettenheim, P. Avian anatomy. Integument.Agricultural Handbook 362. Agriculture USDo, Wash-ington, DC, 1972.

5. McGavin, M., Fadok, V. Factors limiting the usefulnessof histopathologic examination of skin biopsies in thediagnosis of large animal dermatoses.Veterinary Clinics of

North America: Large Animal Practice 1984; 6 (1): 203–13.6. Mauldin, E., Castle, S., Davenport, G. et al. A simple

biopsy technique to improve dermatopathologic interpre-tation. Veterinary Medicine 2002; 4: 286–8.




A modified avian skin biopsy technique 151

Résumé Les biopsies cutanées sont un examen intéressant en dermatologie aviaire, cependant la finesse de lapeau des oiseaux rend difficile la prévention de l’enroulement et de la contraction des prélèvements pendant l’acteet la fixation. La difficulté à orienter des prélèvements enroulés pendant le technicage peut interférer avec le diag-nostic. Cet article rapporte une technique modifiée de biopsie pour obtenir des prélèvements de peau d’oiseau.Un morceau de ruban adhésif (Scotch®) est attaché au site biopsique avant de réaliser la biopsie classiquement.Un total de 23 biopsies cutanées ont été obtenues: 15 à partir de la peau sans plume de 12 perroquets normaux,3 à partir de la peau avec plumes de 2 oiseaux sains et 5 à partir de la peau avec plumes de 3 psittacidés présentés

pour un pica. Tous les prélèvements ont correctement adhéré à la cellophane pendant la biopsie. Les spécimensont été fixés dans du formol à 10%. Pendant le technicage, aucun enroulement des prélèvements n’est apparu, ettous les prélèvements ont pu être facilement orientés pour l’examen. Cette technique n’a pas provoqué d’artéfactnotable. Des restes de cellophane étaient présents au dessus du stratum corneum, ce qui indique que tout le stra-tum corneum a pu être prélevé. L’obtention de prélèvements cutanés de peau d’oiseau par cette technique estfacile et permet une fixation à plat qui autorise une orientation et une interprétation correcte des biopsiescutanées.

Resumen Las biopsias de piel son una herramienta de diagnóstico viable en dermatología aviar; no obstante,la fineza de la piel aviar hace que sea difícil evitar el repliego y contracción de las muestras de piel durante la tomay la fijación de las mismas. La dificultad en la orientación de dichas muestras interfiere en última instancia conel establecimiento de un diagnóstico histopatológico. Este artículo describe un procedimiento modificado parala obtención de una muestra de biopsia de piel en aves. En esta técnica una cinta auta-adhesiva no-transúcida

(Scotch tape®) fue pegada a la piel a biopsiar antes de obtener las biopsias de piel utilizando un instrumentoestándar tipo punch. Se obtuvieron un total de 23 muestras de biopsia de piel: 15 de piel no-emplumada de 12loros Hispaniolan normales, 3 de piel emplumada de 2 pájaros normales y 5 de piel emplumada de 3 psitácidascon alteraciones patológicas de plumas por picajes. Las 23 muestras de piel se adhirieron constantemente a lacinta adhesiva durante el procedimiento de la biopsia. Las muestras fueron fijadas en una solución de formalinaal 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 fácilmente para el corte correcto y la evaluación histopatológicasubsiguiente. La técnica de la cinta adhesiva no produjo ningún artefacto apreciable. Los restos de la cinta eranmicroscópicamente evidentes sobre el estrato córneo y aseguraba que éste no se perdiera durante el proceso. Laobtención de muestras de piel de aves con esta técnica modificada de la cinta adhesiva es fácil y asegura la fijaciónplana de las muestras de biopsia de piel, lo cual permite más adelante el corte en el ángulo perpendicular adecuad,y a través del diámetro longitudinal de los folículos de la pluma para la evaluación histopatológica correcta.

Zusammenfassung Hautbiopsien sind für die Diagnosefindung in der Vogeldermatologie wertvoll, es ist jedochschwierig, bei der dünnen Vogelhaut Einrollen und Kontraktion der Hautbiopsieproben während der Entnahmeund Fixierung zu vermeiden. Schwierigkeiten mit der Orientierung solcher eingerollter Proben während der Bear-beitung kann schlussendlich die histopathologische Diagnosefindung erschweren. Dieser Artikel beschreibt einemodifizierte Hautbiopsieprozedur zur Entnahme von Hautbiopsieproben beim Vogel. Bei dieser Technik wurdeein undurchsichtiges Selbstklebeband (Scotch tape®) auf die zu biopsierenden Stellen aufgeklebt, bevor Haut-biopsieproben mit einer normalen Hautstanze entnommen wurden. Insgesamt wurden 23 Hautbiopsieprobengenommen: 15 von nicht-befiederter Haut 12 normaler Papageien, drei von befiederter Haut zweier normalerPapageien und fünf von befiederter Haut dreier Psittazine, die auf Grund von Federausrupfen vorgestellt wurden.Alle 23 Proben blieben während der Biopsieentnahme mit dem Klebeband verbunden. Die Proben wurden in10%-igem, neutralem Phosphat-gepufferten Formalin fixiert. Während der Bearbeitung wurde weder Kräuselnnoch Einrollen der Proben gesehen und alle Proben (23/23) konnten gut für korrekte Bearbeitung und nachfol-gende histopathologische Bewertung orientiert werden. Die Klebebandtechnik war nicht mit feststellbaren Arte-fakten verbunden. Überbleibsel des Klebebandes waren mikroskopisch über dem Stratum corneum feststellbar

und Anzeichen dafür, dass vom Stratum corneum während der Bearbeitung nichts verlorenging. Die Entnahmevon Hautbiopsien beim Vogel mit dieser Technik ist leicht und stellt die Fixierung der Hautbiopsieproben sicher,die später Trimmen im richtigen Winkel und den Längsdurchmesser der Federfollikel für akkurate histopathol-ogische Bewertung erlaubt.

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A Modified Biopsy Technique to Improve Histopathological Evaluation of Avian Skin