TUGAS KELOMPOK

ILMU BEDAH KHUSUS VETERINER

“TEKNIK BIOPSI HATI”

Oleh:
Ni Made Widy Matalia Astuti 1609511095
Melinda Bellantari 1609511100
Maria Natalia Nini Kewuta 1609511101
Faccettarial Cylon Marchel Marlissa 1609511103
I Dewa Agung Ayu Irma Aristawati 1609511104

FAKULTAS KEDOKTERAN HEWAN

UNIVERSITAS UDAYANA
DENPASAR
2019
 PENDAHULUAN
1.1 Latar Belakang
Biopsi hati adalah prosedur medis di mana sebagian kecil jaringan hati
atau sampel sel dari hati diangkat lewat pembedahan kecil untuk dianalisis di
laboratorium oleh ahli patologi. Biopsi hati dilakukan dengan memasukkan
sebuah jarum di antara dua tulang rusuk paling bawah di bagian kanan tubuh.
Jarum yang sudah ditusuk itu kemudian akan mengambil sampel hati yang
akan dianalisis. Biopsi hati adalah langkah penting dalam evaluasi pasien
dengan penyakit hati dan diperlukan untuk membuat atau merumuskan
diagnosis, terapi langsung, dan memberikan prognosis yang akurat. Namun,
biopsi hati hanya mengevaluasi sebagian kecil dari hati dan mungkin tidak
mewakili keseluruhan hati. Konsekuensinya, hasilnya harus selalu
dikombinasikan dengan informasi klinis, data laboratorium, dan prosedur
pencitraan untuk merumuskan diagnosis.

Diagnosis sebagian besar penyakit hati memerlukan pemeriksaan

histopatologis jaringan hati. Histologi sangat penting untuk penyakit hati
parenkim seperti hepatitis yang terjadi pada anjing dan penyakit radang saluran
empedu yang umum terjadi pada kucing. Dari penyakit peredaran hati,
hipoplasia vena porta (displasia mikrovaskuler) hanya dapat didiagnosis dengan
kombinasi teknik histopatologi dan imaging (misalnya, ultrasonografi).

Neoplasia dan gangguan vakuolar difus (misalnya, lipidosis, steroid

hepatopati) hati sering dapat didiagnosis dengan pemeriksaan sitologis yang
diperoleh dengan menggunakan fine-needle aspiration . Namun, sitologi tidak
menunjukkan perubahan arsitektur hati yang dapat dilihat dengan histopatologi.
Dalam setiap kasus, semua informasi yang tersedia harus dipertimbangkan
sebelum mencapai diagnosis akhir, dan ini menyiratkan memilih teknik yang
tepat untuk mendapatkan sampel jaringan .

Rasio risiko / manfaat dari melakukan biopsi hati harus ditimbang untuk
setiap kasus. Meskipun kemungkinan komplikasi kurang serius untuk kasus
atau prosedur tertentu, selalu ada potensi komplikasi terjadi. Pengalaman
operator juga memiliki pengaruh yang signifikan terhadap tingkat komplikasi.
 Namun, sebagian besar penyakit hati paling baik didefinisikan dan diobati
setelah biopsi hati dengan pemeriksaan histologis.

1.2 Tujuan dan Manfaat

Biopsi hati umumnya aman dan memberikan informasi bermanfaat

tentang hati. Biopsi bertujuan untuk mendeteksi keberadaan sel-sel abnormal
pada hati, seperti jaringan tumor atau kanker.Namun, pentingnya informasi
yang akan diperoleh dari biopsi harus ditimbang terhadap risiko kepada pasien.
Biopsi hati memberikan informasi penting tentang status hati. Hanya setelah
informasi klinis, biopsi hati, dan histopatologi diperoleh, diagnosis dan
prognosis dapat dibuat. Dengan pelatihan yang tepat dan pengalaman operator
yang memadai, biopsi hati merupakan alat diagnostik yang penting. Selain itu,
biopsi membantu dokter mengevaluasi tingkat keberhasilan pengobatan,
seperti dalam sirosis dan hepatitis. Seorang dokter juga akan menjalankan
biopsi jika tes darah atau tes radiologi menunjukkan adanya masalah pada hati.
 II. PREOPERASI DAN ANESTESI

2.1 Tahap Preoperasi

Tahap preoperasi yang dilakukan sebelum operasi biopsi hati, diantaranya:
1. Persiapan alat dan bahan
Persiapan alat dan bahan yang diperlukan yaitu:
• Alat : spuit 1 ml, scalpel, needle, needle holder, tampon, klem, sarung
tangan, lampu operasi, pinset anatomis, gunting bedah, jarum, alat
tambahan untuk monitoring seperti capnograf, elektrokardiograf, monitor
tekanan darah arteri, dan pulse oximeter.

Gambar 1. Peralatan selama prosedur

Sampel jaringan hati khas diperoleh dengan perangkat biopsi yang
berbeda. Atas: jarum Tru-Cut, yang biasanya digerakkan oleh biopsy gun.
Bawah: Jarum Menghini ujung dengan sampel jaringan yang disedot.
Seluruh lumen jarum tersedia untuk mengumpulkan jaringan; sampel
ditangkap oleh aspirasi dengan salin sambil memasukkan jarum ke hati.
Tengah: jarum Vim-Silverman, tidak lagi digunakan
( Sumber gambar : Rothuizen et al 2009)
• Bahan : Sedangkan bahan-bahan yang digunakan adalah anestesi (opiat
kombinasi benzodiazepine atau kombinasi ketamin dan diazepam) cairan
infus dan dukungan onkotik, analgesik serta benang absorbable
monofilament suture
2. Persiapan Ruang Operasi
Ruang operasi harus dalam kondisi yang bersih, penerangan cukup, terdapat
alas kaki khusus dalam ruang operasi, meja operasi bersih, dan beri alas (underpad).
Ruang operasi dibersihkan menggunakan desinfektan, sedagkan meja operasi
didesinfeksi dengan menggunakan alkohol 70%.
3. Persiapan hewan/pasien
Hal pertama yang dilakukan salah pemeriksaan fisik yang meliputi :
signalemen, berat badan, umur, pulsus, frekuensi napas, suhu tubuh dan
pemeriksaan sistem tubuh lainnya (digestivus, respirasi, sirkulasi, saraf,
reproduksi). Semua pasien harus menjalani tes diagnostik pra-bedah (hitung darah
lengkap, hitung trombosit, profil kimia serum, tes koagulasi, urinalisis) untuk
menentukan perawatan perioperatif mana yang harus diberikan. Sebagai contoh,
hewan dengan penyakit hati mungkin mengalami muntah yang dapat menyebabkan
dehidrasi dan kelainan elektrolit. Kekurangan kalium atau magnesium sebelum
operasi dapat menyebabkan ileus, aritmia, dan komplikasi lainnya selama atau
setelah operasi, sehingga mereka paling baik dikoreksi sebelum anestesi.
Sebelum dilakukan operasi, hewan terlebih dahulu dipuasakan yaitu puasa
makan 12 jam dan puasa minum 6 jam sebelum operasi hal ini guna mencegah
vomitting dan kontraksi deflasi terjadi ketika operasi berlangsung. Baringkan
hewan sesuai posisi operasi, untuk operasi biopsi hati dilakukan dengan posisi
dorsal recumbency. Tutup dengan kain drape bagian yang akan dilakukan operasi,
kemudian hewan siap dioperasi
4. Persiapan operator
Operator harus mempunyai kriteria dalam melakukan setiap operasi diantaranya:
o Memahami prosedur operasi
o Siap fisik dan mental
o Personal hygiene yaitu memiliki kondisi sehat serta melakukan
pembersihan diri seperti memcuci tangan dengan sabun antiseptic, memakai
baju operasi, glove, masker, dan penutup kepala.
o Mampu memprediksi hal-hal yang akan terjadi atau dapat menggambarkan
bahaya-bahaya yang mungkin timbul pada waktu melaksanaan operasi.
o Mampu meperkirakan hasil operasi (prognosis)
o Terampil
2.2 Anastesi
Penggunaan premedikasi yang dilakukan sebelum pemberian anastesi
adalah dengan menggunakan campuran opium (morfin) 0,1 mg/kg BB dengan
benzodiazepine pada anjing dan kucing atau campuran antara ketamin dan
diazepam pada kucing. Jika acepromazine yang digunakan sebagai obat sedative,
dosis total yang diberikan tidak melebihi 0,25 mg/kg BB. Penggunaan anastesi yang
diberikan setelah dilakukannya premedikasi adalah induksi propofol secara
intravena (IV) atau dapat melalui inhalasi menggunakan isoflurane (1-2%).
 III. PROSEDUR OPERASI BIOPSI HATI

Biopsi hati adalah prosedur invasif yang berhubungan dengan resiko.

Perdarahan dari tindakan biopsi hati biasanya minimal tetapi dapat menjadi

komplikasi yang berpotensi mengancam jiwa pasien dari semua jenis biopsi hati

(Kemp et al, 2015). Biopsi hati biasanya merupakan tes paling spesifik untuk

menilai sifat dan tingkat keparahan penyakit hati. Selain itu, dapat bermanfaat

dalam memantau keefektifan dari berbagai perawatan (Bravo et al, 2001). Biopsi

harus dilakukan apabila hati terlihat abnormal atau ditemukannya kadar enzim yang

tidak normal sebelum operasi.Perdarahan setelah biopsi hati dapat dikontrol dengan

mudah menggunakan metode jahitan atau elektrokoagulasi. Adapun prosedur

operasi biopsi hati secara laparoskopi (Rothuizen and Twedt, 2009), yaitu:

1. Biopsi hati ambil dengan forsep biopsi oval 5 mm.

2. Biopsi hati diambil dari tepi lobus hati.

Gambar 2. Biopsi hati dengan forsep pada tepi lobus hati

Sumber: Rothuizen and Twedt, 2009
3. Tampilan biopsi yang diambil dari tepi lobus.

Gambar 3. Tampilan setelah dibiopsi pada bagian tepi lobus hati

Sumber: Rothuizen and Twedt, 2009

4. Biopsi hati yang diambil dari permukaan hati.

Gambar 4. Biopsi pada permukaan hati dengan forsep

Sumber: Rothuizen and Twedt, 2009
5. Tampilan biopsi dari permukaan hati.

Gambar 5. Tampilan hati setelah dibiopsi pada permukaan hati

Sumber: Rothuizen and Twedt, 2009
 IV. Hasil dan Pasca Operasi Biopsi Hati
Setelah prosedur, sayatan ditutup dengan jahitan terputus sederhana, dan
diberikan agen antiseptik untuk mencegah terjadi infeksi. Banamine (Flunixin
Meglumine; 1 mg / kg BB) diinjeksikan secara intramuskular 1 jam setelah
prosedur operasi untuk mengurangi ketidaknyamanan pascabedah. Keseluruhan
prosedur biasanya membutuhkan sekitar 20 menit dari waktu injeksi xylazine
hingga waktu penutupan sayatan kulit. Pada anak sapi, sebagaian besarnya dapat
berdiri kembali setelah dua jam biopsi dilakukan (Swanson et al, 2000).
Pendarahan pada tempat dilakukannya sayatan, Tru-Cut, atau clamshell
dapat dikontrol dengan berbagai metode. Diantaranya dengan, busa gelatin yang
adsorbable yang dimasukan ke dalam sayatan operasi, kemudian di jahit dengan
menggunakan bahan ynag dapat diserap yang dijahitkan disekitar luka operasi atau
ditutup dengan omental dan dijahit diatas luka terbuka bekas operasi tersebut. Atau,
dapat dilakukan penekanan pada daerah dilakukannya biopsi, atau dapat pula
dilakukan kauterisasi dengan menggunakan pengaturan rendah. Membasahi
abdomen dengan menggunakan larutan elektrolit yang seimbang pada pasien yang
mengalami kebocoran empedu, perdarahan berlebih, atau lesi terinfeksi atau
nekrotik.

Komplikasi setelah biopsi hati jarang terjadi tetapi memungkinkan

terjadinya peritonitis empedu, perdarahan, dan sepsis. Risiko terjadi komplikasi
lebih besar pada pasien dengan koagulopati dan trombositopenia. Tingkat
komplikasi utama selama biopsi hati telah dilaporkan setinggi 22% dan 50% pada
anjing dan kucing yang trombositopenik. Banyak pasien dengan penyakit hati
dilemahkan dari hipoalbuminemia dan gangguan fungsi hati, meningkatkan risiko
komplikasi potensial dengan anestesi dan pembedahan seperti hipotensi dan
perubahan metabolisme obat anestesi dan analgesik (Carrier et al, 2006). Ada
beberapa potensi kecil yang menginduksi terjadinya hepatitis nekrotik, jika hewan
belum divaksinasi terhadap penyakit clostridial, jadi sebaiknya diberikan suntikan
penisilin atau antibiotik serupa pada saat pengambilan sampel.
 V. KESIMPULAN DAN SARAN
5.1 Kesimpulan
Biopsi hati adalah prosedur medis di mana sebagian kecil jaringan hati atau
sampel sel dari hati diangkat lewat pembedahan kecil untuk dianalisis di
laboratorium oleh ahli patologi. Prosedur ini bermanfaat khususnya bagi pasien
dengan penyakit/kelainan pada organ hati, karena melalui prosedur biopsi hati dapat
dilihat dan menjadi data pelengkap sampai akhirnya diagnose suatu penyakit dapat
diteguhkan. Prosedur biopsi hati relatif aman untuk dilakukan karena proedur yang
dijalankan tidak begitu rumit dan dilengkapi dengan peralatan yang memadai.

5.2 Saran
Ilmu sains khusunya pada bidang kedokteran hewan akan semakin
berkembang. Prosedur biopsy hati ini merupakan salah satu hasil dari
perkembangan teknologi tersebut. Tidak menutup kemungkinan bahwa suatu saat
Teknik biopsy hati akan terbarukan dengan teknologi yang lebih mutakhir dan
efisien untuk pasien. Sehingga, perbaikan dan revisi dari para pembaca mengenai
penulisan atau tambahan materi sangat diperlukan. Supaya kedokteran hewan di
Indonesia semakin maju, berkembang dan menyelamatkan lebih banyak hewan
lagi.
 DAFTAR PUSTAKA

Bravo, Artutoro A., Sunil G. Sheth, and Sanjiv Chopra. 2001. Liver Biopsy. N Engl

J Med, Vol. 344, No. 7. February 15, 2001. The New England Journal of

Medicine.

Carrier, Amie., BS, Diana Burger.,BS, Karen M. Tobias, DVM, MS, DACVS.
2006. How to perform a surgical hepatic biopsy.( Diakses pada :
http://veterinarymedicine.dvm360.com/how-perform-surgical-hepatic-
biopsy)

Kemp, S.D., K.L. Zimmerman, D.L. Panciera, W.E. Monroe, M.S. Leib, and O.I.

Lanz. 2015. A Comparison of Liver Sampling Techniques in Dogs. J Vet

Intern Med 2015;29:51-57.

Rothuizen, Jan. and David C. Twedt. 2009. Liver Biopsy Techniques. Vet Clin

Small Anim (2009) 469-480. Doi:10.1016/j.cvsm.2009.02.006.

Swason, S. Kelly et al. 2000. Technical note: A technique for multiple liver biopsies
in neonatal calves. Journal of Animal Sience. University Illinois, Urbana.

Swanson, K. S., Merchen, N.R., Erdman JR, J.W., Drackley, J.K., Orias, F.,
Douglas, G.N., Huhn, J.C. 2000. Technical note: A technique for multiple
liver biopsies in neonatal calves. University of Illinois, Urbana. J. Anim.
Sci. 2000. 78:2459–2463
J Vet Intern Med 2015;29:51–57

A Comparison of Liver Sampling Techniques in Dogs

S.D. Kemp, K.L. Zimmerman, D.L. Panciera, W.E. Monroe, M.S. Leib, and O.I. Lanz
Background: The liver sampling technique in dogs that consistently provides samples adequate for accurate
histopathologic interpretation is not known.
Hypothesis/Objectives: To compare histopathologic results of liver samples obtained by punch, cup, and 14 gauge
needle to large wedge samples collected at necropsy.
Animals: Seventy dogs undergoing necropsy.
Methods: Prospective study. Liver specimens were obtained from the left lateral liver lobe with an 8 mm punch, a
5 mm cup, and a 14 gauge needle. After sample acquisition, two larger tissue samples were collected near the center of the
left lateral lobe to be used as a histologic standard for comparison. Histopathologic features and numbers of portal triads
in each sample were recorded.
Results: The mean number of portal triads obtained by each sampling method were 2.9 in needle samples, 3.4 in cup
samples, 12 in punch samples, and 30.7 in the necropsy samples. The diagnoses in 66% of needle samples, 60% of cup
samples, and 69% of punch samples were in agreement with the necropsy samples, and these proportions were not signifi-
cantly different from each other. The corresponding kappa coefficients were 0.59 for needle biopsies, 0.52 for cup biopsies,
and 0.62 for punch biopsies.
Conclusion and Clinical Importance: The histopathologic interpretation of a liver sample in the dog is unlikely to vary
if the liver biopsy specimen contains at least 3–12 portal triads. However, in comparison large necropsy samples, the accu-
racy of all tested methods was relatively low.
Key words: Fibrosis; Hepatitis; Laparoscopy; Needle biopsy.

istopathology of the liver provides information
H about the cause, chronicity, and reversibility of
disease.1,2 However, reliable histopathologic results are
dependent upon a liver sample of adequate size and
quality.3,4 In humans, biopsy specimens containing
6–113,4 portal triads are recommended to ensure accu-
rate interpretation. Samples with few portal triads or
those that fracture into multiple pieces are considered
inadequate.3–5 In dogs the minimum number of portal
triads necessary for accurate histopathologic interpre-
tation is unknown.
The World Small Animal Veterinary Association
(WSAVA) Liver Standardization Group guidelines
suggest that needle biopsy is adequate and that surgical
liver biopsy is unnecessarily invasive.6 However, several
studies in dogs have questioned the accuracy of needle
biopsies.7–9 When histopathologic diagnoses obtained
with needle biopsies were compared to those obtained
in necropsy specimens in dogs, there was only 53%
agreement between samples.7 However, the size of the
biopsy instrument was not reported, nor was the qual-
ity of the samples. Another study demonstrated only a
From the Departments of Small Animal Clinical Sciences,
(Kemp, Panciera, Monroe, Leib, Lanz); and the Department of
Biomedical Sciences and Pathobiology, Virginia-Maryland
Regional College of Veterinary Medicine, Virginia Tech,
Blacksburg, VA (Zimmerman).
Data from this study was presented in part as an abstract at the
2013 ACVIM Forum, Seattle, WA.
Corresponding author: D.L. Panciera, Department of Small
Animal Clinical Sciences, Virginia-Maryland Regional College of
Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061;
e-mail: panciera@vt.edu.
Submitted April 22, 2014; Revised September 22, 2014;
Accepted October 22, 2014.
Copyright © 2014 by the American College of Veterinary Internal
Medicine
DOI: 10.1111/jvim.12508
48% agreement in histopathologic diagnosis between
18 gauge needle biopsies and surgical samples taken
from the same animal.8 Finally, other studies have
demonstrated that punch and cup liver biopsies were
shown to routinely produce samples with greater than
6–8 portal triads,9 while 18 gauge and 16 gauge needle
biopsy specimens produced fewer than 6 portal
triads.8,9
Liver biopsy is an invasive procedure that is associ-
ated with risk. Hemorrhage from the biopsy site is
usually minimal but can be a potentially life threaten-
ing complication of any type of liver biopsy.9–12
Because different methods of liver biopsy have dissimi-
lar risks, morbidity, and cost, it is important to iden-
tify the biopsy technique that results in the most
accurate diagnosis with the least potential to harm the
patient.
Currently, the WSAVA Liver Standardization
Group recommends 14 gauge needle samples in most
dogs, with 16 gauge needles reserved for small
patients.6 The adequacy of samples obtained by this
method is unknown as previous studies have evaluated
smaller biopsy needles. Therefore, the primary goal of
this study was to compare postmortem liver samples
collected by 8 mm punch, 5 mm cup, and 14 gauge
needles and to identify the method that most consis-
tently produced samples that represent the histopathol-
ogy of the liver. We hypothesized that liver samples
obtained via punch, cup, and 14 gauge needle would
result in similar histopathologic diagnoses to those
found with large wedge samples of liver obtained at
necropsy.
Materials and Methods
This study was approved by the Institutional Animal Care
and Use Committee of Virginia Tech. This was a prospective
study of dogs presented to the necropsy service between May
52 Kemp et al
2011 and August 2012 at the Virginia-Maryland Regional
College of Veterinary Medicine, Veterinary Teaching Hospital
(VTH). All dogs were patients of the VTH that died or were
euthanized and written consent was obtained from all owners.
All samples were collected within 3 hours of death and by the
same investigator (SDK). For sample collection a midline
abdominal incision was made with a scalpel blade. After the
liver was visualized and exposed samples were collected from
the left lateral liver lobe in order to simulate sample collection
during percutaneous ultrasound-guided needle biopsy.6 All sam-
ple specimens were taken from near the center of the lobe,
within 5 cm of each other. Samples collected from each cadaver
included an 8 mm punch,a a 5 mm cup,b and a 14 gauge nee-
dlec sample. All techniques were performed in a manner that
simulated collection in living dog undergoing liver biopsy as
closely as possible. The punch sample was collected by advanc-
ing the cutting edge of a biopsy puncha at a 90° angle into the
surface of the liver parenchyma near the center of the left lat-
eral lobe. The cup sample was collected by advancing the open
jaws of the cup biopsy forcepsb at a 90° angle into the surface
of the liver parenchyma near the center of the left lateral lobe.
The needle sample was collected with a semiautomatic biopsy
needlec by advancing the needle into the center of the left
lateral liver lobe at a 90° angle to the surface.
Test samples using each technique were collected until a non-
fractured specimen that completely filled the sampling channel of
the instrument was obtained. The number of attempts required
to fill the sampling channel was not recorded. After test sample
acquisition, two deep tissue samples of approximately
2 cm 9 2 cm 9 1 cm were taken from the left lateral lobe. These
large samples (designated “necropsy” samples in this manuscript)
were used as the standard for morphologic diagnosis and com-
parison with each test sample. A histopathologic diagnosis was
determined using the necropsy samples based on the WSAVA
Liver Standardization Group’s classification of hepatic disorders.
If a focal liver lesion was noted (eg, mass or discoloration), the
procedures for obtaining test samples and necropsy samples were
repeated at the lesion site.
Tissue samples were placed in separate cassettes in the same
container and immediately fixed in neutral-buffered 10% forma-
lin at room temperature. After fixation, samples were arranged in
paraffin cassettes for embedding and processing. Five micron
thick sections were prepared and stained with hematoxylin and
eosin (H&E). Two-hundred eighty-four slides from 71 sample
sites were randomized and evaluated by a board certified veteri-
nary pathologist (KZ), who was unaware of their hospital case
identity, for standardized evaluation as described below.
Samples were assigned a score for 16 histologic features8:
hepatocellular atrophy, hepatocellular hypertrophy, biliary hyper-
plasia, ceroid lipofuscin pigment, hemosiderin pigment, canalicu-
lar cholestasis, congestion, extramedullary hematopoiesis,
vacuolar change, fibrosis, tissue inflammation, lobular collapse,
hepatocellular necrosis, neoplasia, thrombosis, and vascular
abnormalities. Scores were on a scale of 0–3 with 0 representing
no change and 3 representing severe change. Neoplasia was
assessed as present or absent.
Hepatocellular atrophy was identified by cords being closer
together, small hepatocytes, increased numbers of portal triads in
a given area, and a wrinkled capsule.13 Hepatocellular hypertro-
phy was defined by the presence of hepatocytes of increased size
and increased cytoplasmic basophilia.13 Biliary hyperplasia was
scored on the basis of increased number of small biliary duct
profiles located within the portal triad areas.13 Ceroid lipofuscin
was defined as a lightly golden-yellow, granular to globular,
hepatocellular cytoplasmic pigment.13 Hemosiderin was defined
as a brown crystalline pigment within both hepatocytes and
Kupffer cells.13 Canalicular cholestasis was scored based on the
identification of green bile plugs within the bile canaliculi.13
Congestion was diagnosed based on distention of hepatic sinu-
soids by erythrocytes.13 Extramedullary hematopoiesis was diag-
nosed when foci of hematopoietic precursors cells were identified
within the biopsy specimen.13 Vacuolar change was identified
based on the presence of swollen hepatocytes with cytoplasmic
vacuoles that were either distinct or indistinct, and, either single
or multiple, as well as those with finely reticulated cytosol.13
Fibrosis was diagnosed by a proliferation of fibroblasts and col-
lagen appreciable by hematoxylin and eosin stain.13 Tissue
inflammation was classified as acute hepatitis, chronic hepatitis,
reactive hepatitis, and cholangiohepatitis. Acute hepatitis was
characterized as a combination of inflammatory cells with neu-
trophils in majority, hepatocellular apoptosis and necrosis, with
or without regeneration.14 Chronic hepatitis was characterized by
a combination of hepatocellular apoptosis or necrosis with vari-
able lymphoplasmacytic infiltration with or without a neutrophil-
ic component, regeneration and fibrosis.14 Reactive hepatitis was
characterized by neutrophilic or mixed inflammation in portal
areas and the hepatic parenchyma without necrosis.14 Cholangio-
hepatitis was characterized by neutrophilic, lymphocytic, or
mixed inflammation involving portal region hepatocytes as well
as bile ducts.14 Hepatocellular apoptosis was characterized by
shrunken hepatocytes, with eosinophilic cytoplasm, and con-
densed nuclei surrounded by an empty halo.14 Lobular collapse
was diagnosed by loss of normal lobular architecture because of
loss of hepatocytes.13 Hepatocellular necrosis was diagnosed by
the presence of shrunken cells, with eosinophilic cytoplasm, and
fragmented or pyknotic nuclei.14 Neoplasia was diagnosed by
identification of atypical, dysplastic hepatic or metastatic cells in
the sample specimen.13 Thrombosis was identified by the presence
of thrombi within hepatic vasculature.13 Vascular abnormalities
were scored based on identification of small or absent portal
veins, arteriolar proliferation, with or without hepatocellular
atrophy.13 Cirrhosis was characterized by bridging fibrosis with
conversion of normal architecture into structurally abnormal
regenerative nodules, and the presence of portal-central vascular
anastomosis as a diffuse change.14 Regeneration was identified
when hyperplasia was present, particularly in a nodular pattern
accompanied by fibrosis. The criteria scores of the two necropsy
samples were averaged and served as the standard to which the
other samples were compared.
Based on the histologic criteria scores, a morphological diag-
nosis was assigned to each of the 4 specimens (three test methods
and necropsy sample) based on the WSAVA Liver Standardiza-
tion Group guidelines.15 Only histologic criteria scores ≥2 were
considered as part of the final morphologic diagnosis. The mor-
phologic diagnosis assigned to the necropsy samples was consid-
ered the definitive diagnosis. If the morphologic diagnoses from
the two necropsy samples from the same liver did not agree, all
specimens from that dog were censored from further analysis.
Finally, the number of portal triads present in each sample was
recorded. The basis for enumerating a portal triad was the identi-
fication of all three triad structures (hepatic artery, portal vein,
and bile duct).
Statistical Analysis
Agreement between definitive morphologic diagnosis and the
morphologic diagnosis of the test specimens were assessed by cal-
culating kappa coefficients. The sensitivity and specificity of each
sample type as compared to the necropsy samples was calculated.
In these calculations, the same predominant histopathologic
abnormality in the test sample and the necropsy sample was con-
sidered a true positive. Comparison between the sensitivity and
specificity for the 3 sampling methods was tested using the
Mantel-Haenszel Chi-square test. The proportions of concordant
 Liver Sampling Techniques in Dogs 53

sample results were compared with logistic generalized estimating
equations (GEE) analysis. The mean number of portal triads
between sample types was compared with a mixed model
ANOVA. The mean score for each of the 16 histologic features
was calculated for all samples of each test sample type and com-
pared using a linear GEE analysis to detect significant differences
in the histologic characteristics between test samples and nec-
ropsy samples. All analyses were performed using commercial
software.d Significance was determined at P < .05.
Results
Seventy dogs and 71 total sample sites (one dog had
a focal lesion) were included in this study. No cases
were censored because of disagreement between the
two necropsy samples. Morphologic diagnoses in the
necropsy samples were: no abnormality (18/71;
25.4%), vacuolar hepatopathy (18/71; 25.4%), neopla-
sia (8/71; 11.3%), primary fibrosis (6/71; 8.45%),
chronic hepatitis (5/71; 7.0%), congestion (5/71;
7.0%), cirrhosis (5/71; 7.0%), necrosis (3/71; 4.2%),
cholangiohepatitis (1/71; 1.4%), reactive hepatitis (1/
71; 1.4%), and cholestasis (1/71; 1.4%).
There were no significant differences (P = .29) in the
proportion of test samples that agreed with the nec-
ropsy sample between test sample types. Cohen’s
kappa coefficient for the needle, cup, and punch sam-
ples were 0.59, 0.52, and 0.62 respectively.
The mean number and 95% confidence intervals of
portal triads in each sampling method was 2.9 (2.6–3.2)
in needle samples, 3.4 (2.7–4.2) in cup samples, 12.0
(10.3–13.7) in punch samples, and 30.7 (27.0–34.5) in
the necropsy samples. Punch samples had significantly
more portal triads than either cup or needle samples
(P < .001) which were not statistically different from
each other (P = .98). The necropsy samples had signifi-
cantly more portal triads than all the test samples
(P < .001). The number of portal triads could not be
determined in 8 needle samples (diagnosis included neo-
plasia [4], cirrhosis [2], fibrosis [1], necrosis [1]), 11 cup
samples (diagnosis included neoplasia [5], cirrhosis [3],
necrosis [2], fibrosis [1]), 12 punch samples (diagnosis
included neoplasia [5], cirrhosis [4], acute hepatitis [1],
fibrosis [1], necrosis [1]), and 13 necropsy samples (diag-
nosis included neoplasia [5], cirrhosis [4], fibrosis [1],
necrosis [1], chronic hepatitis [1], and congestion [1]),
because of loss of normal hepatic architecture.
The sensitivities and 95% confidence intervals of the
test methods as compared to the necropsy samples
were similar, being 60% (46–73%), 55% (41–68%),
and 66% (52–78%) for needle, cup, and punch sam-
pling, respectively. The specificities were also similar
between methods and were higher than the sensitivi-
ties, being 83% (58–96%), 78% (52–93%), and 78%
(52–93%) for needle, cup, and punch sampling respec-
tively. When the sensitivity and specificity of each test
method was calculated for each diagnosis, the highest
sensitivities were found in dogs with vacuolar hepatop-
athy, normal hepatic histopathology, and neoplasia
(Table 1). Within each diagnosis category where sensi-
tivity and specificity were reported, there were no sig-
nificant differences in the sensitivities or specificities
between the test types. Results were not reported for
necrosis, cholangiohepatitis, reactive hepatitis, or cho-
lestasis because of the small number of cases in each
category. Diagnoses in the 8 livers with neoplasia
included histiocytic sarcoma (3), lymphoma (3), undif-
ferentiated round cell sarcoma (1), and spindle cell sar-
coma (1). The sensitivity for diagnosis of neoplasia
was 75% (95% CI: 0.45–1.0) for needle samples; 63%
(95% CI: 0.29–0.96) for cup samples; and 88% (95%
CI: 0.65–1.0) for punch samples. The specificity for
neoplasia was 100% in all three test types. Overall, the
sensitivity for the diagnosis of fibrosis was low, rang-
ing from 16 to 50% (Table 1).
The mean scores for each of the histologic features
were compared amongst the test sample types and sev-
eral significant differences from the necropsy samples
were identified (Table 2). The needle samples identified
significantly less hepatocellular atrophy, biliary hyper-
plasia, hemosiderin, and congestion compared to the
necropsy samples. The cup samples identified signifi-
cantly less biliary hyperplasia, hemosiderin, and con-
gestion when compared to the necropsy samples.
Finally, the punch samples showed significantly less
hepatocellular hypertrophy and hemosiderin than the
necropsy samples.
In the 6 cases with a predominant histopathologic
abnormality of fibrosis, the mean fibrosis score in the
necropsy samples was 2.5, which was significantly
higher than 1.5 in the punch (P = .014), 1.4 in the cup
samples (P = .014), and 0.5 in the needle samples
(P < .001). In the 5 cases of chronic hepatitis the mean

Table 1. Sensitivity, specificity, and 95% confidence intervals for each biopsy type stratified by morphologic
diagnosis in the necropsy samples.
Needle Laparoscopic Cup Punch
Gold Standard
Diagnosis Number Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity
Normal 18 0.83 (0.66–1.0) 0.75 (0.64–0.87) 0.78 (0.59–0.97) 0.68 (0.55–0.80) 0.78 (0.59–0.97) 0.81 (0.71–0.92)
Vacuolar 18 0.72 (0.51–0.92) 0.96 (0.03–0.91) 0.61 (0.39–0.84) 1 (1.0–1.0) 0.83 (0.66–1.0) 0.96 (0.91–1.0)
Neoplasia 8 0.75 (0.45–1.0) 1 (1.0–1.0) 0.63 (0.29–0.96) 1 (1.0–1.0) 0.88 (0.65–1.0) 1 (1.0–1.0)
Primary fibrosis 6 0.16 (0.0–0.64) 0.98 (0.92–1.0) 0.5 (0.12–0.87) 0.97 (0.89–1.0) 0.5 (0.12–0.87) 0.98 (0.92–1.0)
Chronic hepatitis 5 0.6 (0.17–1.0) 0.98 (0.96–1.0) 0.4 (0.0–0.83) 0.98 (0.96–1.0) 0.4 (0.0–0.83) 1 (1.0–1.0)
Congestion 5 0.4 (0.0–0.83) 0.98 (0.96–1.0) 0.2 (0.0–0.55) 0.98 (0.96–1.0) 0.4 (0.0–0.83) 0.97 (0.93–1.0)
Cirrhosis 5 0.6 (0.17–1.0) 1 (1.0–1.0) 0.8 (0.45–1.0) 1 (1.0–1.0) 0.8 (0.45–1.0) 1 (1.0–1.0)

Only morphologic diagnoses with ≥5 cases are shown.

54 Kemp et al

Abnormality
inflammation score in the necropsy samples was 2.6,

SD = 0.32

SD = 0.44

SD = 0.57

SD = 0.46
Vascular

0.078
which was significantly higher than 1.5 in the cup sam-

0.15

0.18

0.14
ples (P = .002), and 1.4 in the needle samples
(P < .001), but not significantly different from 2.1 in
the punch samples (P = .15).
Thrombosis

SD = 0.12

SD = 0.12
0.015

0.014
0

0
Discussion
0.28 SD = 0.59

Results of this study indicate that 14 gauge needle,

SD = 0.62

SD = 0.61

SD = 0.72
Necrosis

5 mm cup, and 8 mm punch samples of the liver have

0.34

0.33

0.41
a similar proportion of samples in agreement to larger
hepatic samples. However, the level of agreement
could be considered insufficient when a single sample
SD = 0.06

SD = 0.53

SD = 0.60

SD = 0.55
Collapse
Lobular

0.13

0.15

0.21

0.24
is taken by any tested technique. The disparity
Table 2. Mean scores and standard deviations for histologic features of each sample type.

between the test samples and the necropsy samples

seemingly occurs as a result of variable distribution of
Inflammation

SD = 0.64

SD = 0.68

SD = 0.79

SD = 0.80

morphologic features within a liver lobe which might

Tissue

0.32

0.37

0.39

0.41

be overcome by obtaining multiple samples, a larger

single sample, or perhaps biopsies from multiple lobes.
The paired necropsy samples from each dog had iden-
SD = 0.80

SD = 0.97

SD = 0.95

SD = 0.12
Fibrosis

tical histopathologic diagnoses, while the smaller sam-

0.43

0.60

0.49

0.64

ples obtained using the three test methods had less

consistent agreement with the large necropsy samples.
SD = 0.95

SD = 0.87

SD = 0.13

Because all the samples were obtained within 5 cm of

SD = 1.1
Vacuolar
Change

0.88

0.76

0.91

1.01

each other, the size of the specimen obtained by the

test methods was most likely the primary factor influ-
encing the histopathologic interpretation.
Extramedullary
Hematopoiesis

SD = 0.052
SD = 0.41

SD = 0.31

SD = 0.57

Smaller test samples had fewer portal triads. The

0.154

0.11

0.18

0.30

number of portal triads in the needle and cup samples

were not different, and both contained fewer than
what is recommended in humans, while the punch
(P = .0021)
Congestion

samples exceeded the minimum recommendations.3,4

SD = 0.50

SD = 0.54

SD = 0.67

SD = 0.73
(P < .001)
0.19

0.25

0.37

0.48

Despite this, the accuracy of the punch samples was

not greater than the other test methods. Therefore,
recommendations for sampling the human liver do not
Cholestasis

SD = 0.79

SD = 0.82

SD = 0.87

SD = 0.90

appear to be applicable to dogs.

(Bile)

0.37

0.43

0.49

0.57

The median and mean number of portal triads of 3

and 2.9, respectively, in needle samples in the present
Hemosiderin

study was lower than previous reports where 18 gauge

(P = .0033)
SD = 0.95

SD = 0.87

SD = 0.82

SD = 0.92
(P = .024)
(P = .02)

needle biopsies had a median of 4 portal triads8 and

0.71

0.75

0.79

0.95

16 gauge needle samples had a mean of 6–7.9 portal

triads.9 This discrepancy may be attributable to the
Lipofuscin

SD = 0.95

SD = 0.92

SD = 0.75

SD = 0.76

strict criteria used for counting portal triads in this

Ceroid

0.87

0.92

0.96
1.0

study, where all 3 structures comprising the portal

triad had to be clearly identified. Other studies that
did not describe their methodology in detail may have
Hyperplasia

SD = 0.67

SD = 0.90

SD = 0.97
(P < .001)

(P < .001)
SD=0.73
Biliary

included portal areas without all three components of

0.27

0.32

0.45

0.58

the triad visible. Despite the punch samples containing

significantly more triads than the other test methods,
Hepatocellular

the overall histopathological agreement with necropsy

Hypertrophy

SD = 0.45

SD = 0.66

SD = 0.45

SD = 0.66
(P = .017)

(P = .039)

samples was not different. Therefore, when the number

0.18

0.34

0.17

0.29

of portal triads in samples ranges from 3 to 12, the

Significant differences are shaded.

final histopathologic interpretation is unlikely to vary.

However, because of the relatively poor agreement
Hepatocellular

SD = 0.43

SD = 0.49

SD = 0.52

SD = 0.45
Atrophy

with the necropsy samples, it is reasonable to assume

0.13

0.16

0.19

0.19

that biopsies larger than those obtained in this study

might enhance the likelihood of a correct diagnosis.
Because techniques used to obtain larger biopsies
Sample Type

might result in increased risk for hemorrhage, multiple

Necropsy
(N = 71)

Laparo-
scopic
Needle

biopsies from different locations of a lobe might be the

Punch
cup

best method to safely acquire adequate tissue.

 Liver Sampling Techniques in Dogs
Portal triads could not be reported in 13 (18%) of
the necropsy samples because of severe distortion in
the hepatic architecture. This raises concern for the use
of portal triad numbers as the only measure of biopsy
specimen quality, as these samples were large but did
not contain recognizable triad structures. However, the
diagnoses in the majority of these cases were neoplasia
or cirrhosis and it is likely that in such severe disease
large samples with many portal triads may not be nec-
essary for diagnosis.
In this group, the sensitivity of needle samples were
similar to a previous report that compared 18 gauge
needle and surgical biopsies.8 While the sensitivity for
detection of hepatic neoplasia was similar to that of
another study (80%) using a smaller needle biopsy, the
specificity was 100% in all sample methods tested in
the present study.8 Because the majority of neoplasms
in our population were systemic, it is unclear if similar
results would be found in dogs with focal, metastatic,
or multifocal neoplasia.16
In cases where fibrosis was the histopathologic diag-
nosis, all three sampling methods had a significantly
lower mean fibrosis score than the necropsy samples. In
these cases the punch and cup samples had a concordant
diagnosis in 3 samples, and only 1 of the 6 livers with
fibrosis had it identified on needle biopsy. These findings
suggest that large tissue samples may be necessary to
accurately describe the degree of fibrosis when severe
disease is present. This is in contrast with previous stud-
ies where needle biopsy specimens showed higher histo-
logic scores for fibrosis.8 However, the results of the
present study mirror those of several human studies in
which fibrosis scores declined with smaller biopsy
size.5,17 The discordance in the fibrosis scoring is likely
caused by variation in severity of fibrosis throughout or
between lobes, which has been documented in humans.
In a report of patients with primary biliary fibrosis,
whole section scanning of the liver at the time of trans-
plantation revealed that only 20% of these livers had
fibrosis throughout the entire organ.18
All test methods were insensitive for diagnosis of
chronic hepatitis, unlike a previous study that reported
needle biopsies had higher scores for inflammation
when compared to wedge biopsies obtained at sur-
gery.8 In the present study, there were no significant
differences in the histologic scores for inflammation
between the sampling methods. However, when the
five cases of chronic hepatitis were analyzed separately,
inflammation scores for both the cup and needle sam-
ples were significantly lower than those of the necropsy
samples. The punch and cup samples both had concor-
dant diagnoses in 2 cases and the needle samples had
concordant diagnoses in 3 cases. Although the number
of cases in the present study was limited, the results
suggest that histopathology of a single sample may un-
derrepresent the severity of disease when chronic
inflammation is present. This finding is similar to a
report in humans which demonstrated that shorter
needle biopsies produced samples with lower inflam-
matory scores in patients with hepatitis C virus infec-
tion.4 However, it is not known if the lower histologic
55
scores for inflammation reported in the small test sam-
ples in the present study would result in a different
clinical diagnosis in dogs.
The high number of discordant samples amongst all
test methods may be attributable to nonuniform
lesions throughout the liver lobe, even in diffuse hepat-
opathies. For example, marked variation in copper
concentration was found when needle biopsy speci-
mens were compared to wedge samples in dogs.19 Con-
clusions of the small number of studies that have
evaluated the diagnostic accuracy of liver biopsy tech-
niques in dogs have been hampered by limitations in
our understanding of canine liver disease. Studies eval-
uating liver biopsy in humans typically focus on
patients with a specific disease such as hepatitis C virus
or nonalcoholic steatohepatitis, and are aimed to
define the best biopsy method for that specific disease,
whether for diagnostic or prognostic purposes.5,20
Because of limited knowledge of the etiology and clini-
cal markers of specific liver diseases in dogs, any
biopsy technique must be able to identify any of the
histologic features that might be present.
One limitation of this study is reliance on a single
pathologist for interpretation of all of the liver samples.
However, use of a single pathologist likely resulted in
more consistent results between cases, compared with
multiple observers.21 The expertise of the pathologist in
evaluating the liver is another important consideration
in humans and likely is important in veterinary medi-
cine as well.22 Dogs enrolled in the study were not
selected because of known hepatic disease, thus were
not representative of the population in which liver
biopsies would be obtained in clinical practice. The
influence that a higher prevalence of hepatic disease
would have had on the results of this study remains
unclear. It is important to note that in a study of liver
biopsy in population of patients where biopsy was
deemed appropriate for clinical reasons, 28% had no
hepatic disease, similar to the 26% in the present
study.8 Sample collection was performed using methods
that mimicked their antemortem use, but differed from
percutaneous and laparoscopic biopsy as the samples
were obtained through a large abdominal incision and
repeated sampling was attempted until a sufficient sam-
ple was retrieved. The number of attempts required to
obtain a sample that filled the biopsy instrument was
not recorded, but the size of antemortem biopsies varies
within a given method and fragmented samples compli-
cate histopathologic interpretation.9 In addition, sam-
ple acquisition can be affected by the underlying liver
disease. For example, in the authors’ experience, needle
or cup sampling of a severely fibrotic liver often results
in smaller biopsies and frequently multiple attempts are
necessary to obtain an adequate sample. Biopsies
obtained in a clinical setting might be of lower quality
and be limited by the potential for complications of
repeated sampling. Thus, it is possible that the accuracy
of nonsurgical biopsies in clinical cases may be lower
than reported here.
Because the WSAVA Liver Standardization Group
recommends two biopsies for histopathologic
56
evaluation, the present study might have been strength-
ened by evaluating more than one sample using each
test method. However, our study was not designed to
determine the minimum number of samples necessary to
ensure accurate histopathologic diagnosis, rather it was
to investigate the ability of commonly used sampling
methods to accurately reflect the histopathologic diag-
nosis. Sample quality in the present study was con-
trolled by using only samples that fully filled the
instrument’s chamber and were not fragmented, result-
ing in uniform comparisons between sampling methods
and avoiding the influence of variation in biopsy size
which has been shown to affect biopsy interpretation in
humans with hepatitis.4 Because no sampling method
had a strong agreement with the gold standard, it seems
clear that multiple samples should be obtained in the
hope that it would improve accuracy. It is likely that the
number of samples necessary for an accurate histopath-
ologic interpretation would vary depending on the dis-
ease and biopsy quality. Histochemical staining was
limited to H&E in the present study, which may have
led to underestimation of fibrosis, limited assessment of
architectural changes in some diseases, and prevented
identification of some intracellular contents and pig-
ments. However, the same stains and analytic criteria
were applied to each sample, so the detrimental effects
of using a single stain would be limited. Future studies
should address these important issues.
Our study design limited the surgical samples to one
technique that allowed for sampling from the center of
the lobe. Other methods of surgical liver biopsy have
been described in the dog,9 and it is possible that an
alternate method of sampling such as obtaining a lar-
ger wedge from the edge of a liver lobe might improve
accuracy.
The results of this study demonstrate substantial
limitations in the accuracy of a single liver sample by
any of the tested techniques. Obtaining multiple sam-
ples from the liver might be of greater importance than
the method of biopsy.
Footnotes
a canine and feline liver 2. Hepatocellular death, hepatitis and cir-
8 mm Biopsy Punch, Miltex, Inc., Plainsboro, NJ
b rhosis. In: Rothuizen J, Bunch SE, Charles JA, Cullen JM,
Eragon 5 mm biopsy forceps, Richard Wolf Medical Instru-
ments Corporation, Vernon Hills, IL
c Winkle TV, Washabau RJ, eds. WSAVA Standards for
SurgiVet VET-Core Biopsy needle 14 ga, 9 cm, Smiths Medical
PM, Inc. Waukesha, WI
d Liver Diseases. Philadelphia, PA: Saunders Elsevier; 2006:
SAS/STAT software version 9.2. (Cary, NC)
Acknowledgments
The authors thank Dr Stephen R. Werre for statisti-
cal assistance. The study was funded by the Virginia
Veterinary Memorial Fund.
Conflict of Interest Declaration: The authors disclose
no conflict of interest.
Kemp et al
Off-label Antimicrobial Declaration: The authors
declare no off-label use of antimicrobials.
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 C URR ENT CONC EP TS
Review Articles
Current Concepts
L IVER B IOPSY
ARTURO A. BRAVO, M.D., SUNIL G. SHETH, M.D.,
AND SANJIV CHOPRA, M.D.
P
AUL Ehrlich is credited with performing the
first percutaneous liver biopsy in 1883 in Ger-
many.1 After Menghini reported a technique for
“one-second needle biopsy of the liver” in 1958, the
procedure became more widely used. The average du-
ration of the intrahepatic phase of previous liver-biop-
sy techniques had been 6 to 15 minutes.2
Liver biopsy is usually the most specific test to as-
sess the nature and severity of liver diseases. In addi-
tion, it can be useful in monitoring the efficacy of var-
ious treatments. There are currently several methods
available for obtaining liver tissue: percutaneous biop-
sy, transjugular biopsy, laparoscopic biopsy, or fine-
needle aspiration guided by ultrasonography or com-
puted tomography (CT). Each of these methods has
advantages and disadvantages.
The size of the biopsy specimen, which varies be-
tween 1 and 3 cm in length and between 1.2 and
2 mm in diameter, represents 1⁄50,000 of the total
mass of the liver.3 Usually, for evaluation of diffuse
liver disease, a specimen of 1.5 cm in length is adequate
for a diagnosis to be made. The number of portal tri-
ads present in the specimen is important; most hepato-
pathologists are satisfied with a biopsy specimen con-
taining at least six to eight portal triads, especially in
cases of chronic liver disease in which the extent of
injury may vary among portal triads. An adequate spec-
imen is usually provided by all the needles currently
used for liver biopsy. Specimens obtained with stand-
ard thin-bore or spring-loaded needles measure be-
tween 1.4 and 1.8 mm in diameter, and those obtained
with Menghini or Tru-cut needles measure up to
2 mm in diameter.4,5
The indications for liver biopsy are outlined in Ta-
ble 1. Even for patients in whom serologic tests point
to a specific liver disease (Fig. 1 and 2), a liver biopsy
can give valuable information regarding staging, prog-
nosis, and management. For example, in patients with
chronic hepatitis C infection, not only is there a poor
correlation between symptoms or levels of serum ala-
From the Liver Center, Division of Gastroenterology, Beth Israel Dea-
coness Medical Center, Harvard Medical School, Boston.
N Engl J Med, Vol. 344, No. 7 · February 15, 2001 · www.nejm.org · 495
Downloaded from www.nejm.org at BOTSFORD GENERAL HOSPITAL LIB on February 1, 2006 .
Copyright © 2001 Massachusetts Medical Society. All rights reserved.
TABLE 1. INDICATIONS FOR LIVER BIOPSY.
Diagnosis, grading, and staging of alcoholic liver disease, nonalcoholic ste-
atohepatitis, or autoimmune hepatitis
Grading and staging of chronic hepatitis C or chronic hepatitis B
Diagnosis of hemochromatosis in index patient and relatives, with quanti-
tative estimation of iron levels
Diagnosis of Wilson’s disease, with quantitative estimation of copper levels
Evaluation of the cholestatic liver diseases primary biliary cirrhosis and pri-
mary sclerosing cholangitis
Evaluation of abnormal results of biochemical tests of the liver in associa-
tion with a serologic workup that is negative or inconclusive
Evaluation of the efficacy or the adverse effects of treatment regimens (e.g.,
methotrexate therapy for psoriasis)
Diagnosis of a liver mass
Evaluation of the status of the liver after transplantation or of the donor
liver before transplantation
Evaluation of fever of unknown origin, with a culture of tissue
nine aminotransferase and histologic features of the
liver, but also patients with completely normal levels
of liver enzymes may be found to have clinically signif-
icant fibrosis or cirrhosis on biopsy (Fig. 3).6 If the
patient has mild disease and is infected with genotype
1a or 1b of the hepatitis C virus, a decision may be
made to defer treatment. If a decision is made to treat
such a patient with a combination of interferon and
ribavirin and there are adverse effects, the treatment
can be stopped. Conversely, if the patient has moder-
ate-to-advanced disease, treatment will most likely be
offered. If the patient has a virologic response and
tolerable side effects with treatment, continued ther-
apy would be strongly encouraged. The finding of
cirrhosis on liver biopsy will determine the need for
further examinations, such as upper endoscopy to rule
out esophageal varices and screening for hepatocellular
carcinoma with serial determinations of serum alpha-
fetoprotein and liver ultrasonography.
In alcoholic liver disease, the severity of the clinical
symptoms and the degree of liver-enzyme elevation
correlate poorly with the extent of liver damage, par-
ticularly in patients who continue to drink alcohol.
The long-term prognosis depends on the severity of
hepatic injury.7 In patients with alcoholic liver disease
as well as nonalcoholic steatohepatitis (Fig. 4), liver
biopsy may reveal fatty infiltration of the liver, bal-
loon-cell degeneration, Mallory’s bodies, and hepa-
tocyte necrosis, with or without clinically significant
fibrosis or cirrhosis. In primary biliary cirrhosis, serial
liver biopsies help one to study the natural history,
monitor the effects of therapy, or identify a recurrence
of the disease after liver transplantation.8-10
Liver biopsy provides an accurate diagnosis in ap-
 The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne

A
A

B
Figure 1. A Liver-Biopsy Specimen from a 32-Year-Old Man Pre-
sumed to Have Acute Hepatitis.
The specimen shows a portal mononuclear infiltrate with prom-
inent plasma cells (arrow in Panel A) and lobular inflammation
with apoptotic hepatocytes (arrow in Panel B), findings consis-
tent with the presence of autoimmune hepatitis (hematoxylin
and eosin, ¬100).
B
Figure 2. Liver-Biopsy Specimens from a 38-Year-Old Woman
with Increased Iron Saturation and Mild Hepatomegaly.
Panel A (hematoxylin and eosin, ¬100) shows periportal depo-
sition of brown pigment (arrow). Panel B (Perls’ stain, ¬10) shows
periportal distribution of iron. The hepatic iron index was 2.0
(normal value, less than 1.0), which is consistent with the pres-
ence of idiopathic genetic hemochromatosis.

proximately 90 percent of patients with unexplained
abnormalities revealed on liver-function tests.11 The
elucidation of various processes that occur in a trans-
planted liver — including immune rejection, systemic
or infectious complications, drug toxicity, and the re-
currence of primary disease — requires a liver biopsy.12
Liver biopsy can also lead to the diagnosis of systemic
disorders that can affect the liver, such as sarcoidosis,
lymphoma, the acquired immunodeficiency syndrome,
and amyloidosis.
PERCUTANEOUS LIVER BIOPSY
Procedures
Needles for percutaneous liver biopsy are broadly
categorized as suction needles (Menghini needle,
Klatskin needle, Jamshidi needle), cutting needles
(Vim–Silverman needle, Tru-cut needle), and spring-
loaded cutting needles that have a triggering mech-
anism. The cutting needles, except for the spring-load-
ed variety, require a longer time in the liver during the
biopsy, which may increase the risk of bleeding.13 A
greater incidence of bleeding after biopsy has some-
times been observed with large-diameter needles.14 If
cirrhosis is suspected on clinical grounds, a cutting
needle is preferred over a suction needle, since fibrotic
tissue tends to fragment with the use of suction nee-
dles.15 We routinely use spring-loaded needles.
Ultrasonography performed before a liver biopsy
identifies mass lesions that are clinically silent and de-
fines the anatomy of the liver and the relative positions

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 C URR ENT CONC EP TS

A A

B
Figure 3. Liver-Biopsy Specimens from a 45-Year-Old Woman
with Chronic Hepatitis C Virus Infection, in Whom There Was
No Clinical Suspicion of Cirrhosis.
Panel A shows a dense portal infiltrate with the formation of
lymphoid aggregates (hematoxylin and eosin, ¬66). Panel B
shows bridging fibrosis with architectural distortion and early
cirrhosis (Masson trichrome, ¬10).
B
Figure 4. Liver-Biopsy Specimens from a 40-Year-Old Man with
Obesity, Diabetes, and Mildly Elevated Liver-Enzyme Levels.
The specimen in Panel A shows moderate-to-marked steatosis
with increased fibrosis (trichrome, ¬10). The specimen in Panel B
shows a single cell (center) containing intracytoplasmic Mallory’s
bodies (hematoxylin and eosin, ¬160). These findings are con-
sistent with the presence of nonalcoholic steatohepatitis.

of the gallbladder, lungs, and kidneys. Most hepatolo-
gists agree that all patients should undergo ultrasonog-
raphy of the liver before a percutaneous biopsy is per-
formed. However, it is debatable whether the routine
use of ultrasonography to guide the biopsy reduces the
rate of complications, provides a higher diagnostic
yield, or is cost effective.16-23 We routinely use ultraso-
nography to mark the site for percutaneous biopsy.
It is now standard practice to perform liver biopsy
on an outpatient basis, provided that various criteria
are met. The Patient Care Committee of the American
Gastroenterological Association has published prac-
tice guidelines for outpatient liver biopsy.24 The patient
must be able to return to the hospital in which the
procedure was performed within 30 minutes after the
onset of any adverse symptoms. Reliable persons must
stay with the patient during the first night after the bi-
opsy to provide care and transportation, if necessary.
The patient should have no serious medical problems
that increase the risk associated with the biopsy. The
facility in which the biopsy is performed should have
an approved laboratory, a blood-banking unit, an eas-
ily accessible inpatient bed, and personnel to monitor
the patient for at least six hours after the biopsy. The
patient should be hospitalized after the biopsy is per-
formed if there is evidence of bleeding, a bile leak,

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 The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne
pneumothorax, or other organ puncture or if the pa-
tient’s pain requires more than one dose of analgesics
in the first four hours after the biopsy.
Contraindications
The contraindications to a percutaneous liver biopsy
are listed in Table 2. Liver biopsy is a safe procedure
when performed by experienced operators. Froehlich
et al.25 noted a lower complication rate for physicians
who performed more than 50 biopsies a year. Prior
ultrasonographic localization of the biopsy site may
decrease the rate of complications for physicians who
perform liver biopsies infrequently. “Blind” liver biop-
sies should be performed by experienced gastroen-
terologists, hepatologists, or transplantation surgeons
and not by general internists.5
Complications of Percutaneous Liver Biopsy
Although the liver has a rich vascular supply, com-
plications associated with percutaneous liver biopsy
are rare. Sixty percent of complications occur within
2 hours and 96 percent within 24 hours after the
procedure.1,14 Approximately 1 to 3 percent of patients
require hospitalization for complications after a liver
biopsy, especially if the procedure was performed with
a Tru-cut biopsy needle. Pain and hypotension are the
predominant complications for which patients are hos-
pitalized.5,26
Minor complications after percutaneous liver biopsy
include transient, localized discomfort at the biopsy
site; pain requiring analgesia; and mild, transient hypo-
tension (due to a vasovagal reaction). Approximately
one fourth of patients have pain in the right upper
quadrant or right shoulder after liver biopsy. The pain
TABLE 2. CONTRAINDICATIONS TO PERCUTANEOUS LIVER BIOPSY.
Absolute contraindications
Uncooperative patient
History of unexplained bleeding
Tendency to bleed*
Prothrombin time »3–5 sec more than control
Platelet count <50,000/mm3
Prolonged bleeding time (»10 min)
Use of a nonsteroidal antiinflammatory drug within previous 7–10 days
Blood for transfusion unavailable
Suspected hemangioma or other vascular tumor
Inability to identify an appropriate site for biopsy by percussion or ultra-
sonography
Suspected echinococcal cysts in the liver
Relative contraindications
Morbid obesity
Ascites
Hemophilia
Infection in the right pleural cavity or below the right hemidiaphragm
*Although these criteria are considered absolute contraindications by
most hepatologists, they can be corrected by transfusions of platelets or
fresh-frozen plasma and are therefore not truly absolute.12
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Copyright © 2001 Massachusetts Medical Society. All rights reserved.
is usually dull, mild, and brief.27 Ongoing, severe pain
in the abdomen should alert the physician to the pos-
sibility of a more serious complication, such as bleed-
ing or peritonitis.
Although very rare, clinically significant intraperi-
toneal hemorrhage is the most serious bleeding com-
plication of percutaneous liver biopsy; it usually be-
comes apparent within the first two to three hours
after the procedure.14,28 Free intraperitoneal blood may
result from laceration caused by deep inspiration dur-
ing the biopsy or may be related to a penetrating in-
jury of a branch of the hepatic artery or portal vein.
Risk factors for hemorrhage after liver biopsy are older
age, more than three passes with the needle during bi-
opsy, and the presence of cirrhosis or liver cancer.14,26
Findings of free intraperitoneal fluid on ultrasonog-
raphy or CT should be correlated with the clinical as-
sessment of the patient.29 If hemorrhage is suspected,
immediate arrangements for blood, platelets, and plas-
ma should be made, and a surgeon and an angiogra-
pher should be alerted. Measures to improve the pa-
tient’s hemodynamic status by the administration of
intravenous fluids, blood products, or both may be
sufficient. If hemodynamic instability persists for a few
hours despite the use of aggressive resuscitative meas-
ures, angiography and embolization or surgical explo-
ration is indicated.
Small intrahepatic or subcapsular hematomas can
be noted after liver biopsy even in asymptomatic pa-
tients.30 Large hematomas may cause pain associated
with tachycardia, hypotension, and a delayed decrease
in the hematocrit.28 Conservative treatment of hemato-
mas is generally sufficient.
The least common of the hemorrhagic complica-
tions is hemobilia, which usually presents with the clas-
sic triad of gastrointestinal bleeding, biliary pain, and
jaundice14 approximately five days after the biopsy.31
Transient bacteremia has been reported in 5.8 to 13.5
percent of patients after liver biopsy,32 and although
such bacteremia is generally inconsequential, septi-
cemia and shock can develop on rare occasions in pa-
tients with biliary obstruction and cholangitis. Cur-
rently, there are no recommendations for the routine
use of prophylactic antibiotics in patients undergoing
liver biopsy, including those with prosthetic valves
or joints.33
Other rare complications of percutaneous liver biop-
sy include biliary ascites, bile pleuritis, bile peritonitis,
pneumothorax, hemothorax, subcutaneous emphyse-
ma, pneumoperitoneum, pneumoscrotum, subphrenic
abscess, carcinoid crisis, anaphylaxis after biopsy of an
echinococcal cyst, pancreatitis due to hemobilia, and
breakage of the biopsy needle.14,28,34
The mortality rate among patients after percutane-
ous liver biopsy is approximately 1 in 10,000 to 1 in
12,000.13,28 Mortality is highest among patients who
undergo biopsies of malignant lesions. Cirrhosis is an-
other risk factor for fatal bleeding after liver biopsy.
 C URR ENT CONC EP TS

TRANSJUGULAR LIVER BIOPSY

TABLE 3. INDICATIONS FOR TRANSJUGULAR
Transjugular catheterization of the hepatic veins in LIVER BIOPSY.
human subjects was first described in 1967 as an ap-
proach to the biliary tract for cholangiography.35 With Severe coagulopathy
transjugular liver biopsy, the liver tissue is obtained Massive ascites
from within the vascular system, which minimizes the Massive obesity
risk of bleeding. The indications for transjugular bi- Suspected vascular tumor or peliosis hepatis
opsy are outlined in Table 3. Need for ancillary vascular procedures (e.g., trans-
jugular intrahepatic portosystemic shunting,
The procedure involves percutaneous puncturing of venography)
the right internal jugular vein, the introduction, with Failure of percutaneous liver biopsy
the use of fluoroscopy, of a catheter into the right he-
patic vein, and a needle biopsy of the liver performed
through the catheter. The duration of the procedure
is between 30 and 60 minutes. Electrocardiographic
monitoring is required to detect arrhythmias induced
by passage of the catheter through the heart.36 Sam- TABLE 4. INDICATIONS FOR
ples are retrieved from a needle passed through the AND CONTRAINDICATIONS TO
catheter into the liver while suction is maintained. The LAPAROSCOPIC LIVER BIOPSY.
samples obtained are small and fragmented, a disad-
vantage of the technique that may be improved with Indications
newer-generation technology.37 Staging of cancer
Ascites of unclear cause
Adequate tissue for histologic diagnosis can be Peritoneal infections
obtained from 80 to 97 percent of patients in cen- Evaluation of an abdominal mass
Unexplained hepatosplenomegaly
ters where a large number of transjugular biopsies are
performed.38 The tissue specimen is usually 0.3 to Contraindications
Absolute
2 cm long, and the procedure generally requires mul- Severe cardiopulmonary failure
tiple passes. A transjugular biopsy can also be per- Intestinal obstruction
formed at the same time as the placement of a trans- Bacterial peritonitis
Relative
jugular intrahepatic portosystemic shunt. Failure to Uncooperative patient
establish a diagnosis may be due to fragmentation of Severe coagulopathy
the tissue specimen. Morbid obesity
Large ventral hernia
In various studies, the rate of complications asso-
ciated with transjugular liver biopsy ranges from 1.3
percent to 20.2 percent, and mortality ranges from
0.1 percent to 0.5 percent.36,38 Complications of trans-
jugular liver biopsy include abdominal pain, neck he-
matoma, transient Horner’s syndrome, transient dys- abdominal wall, vasovagal reaction, prolonged abdom-
phonia, cardiac arrhythmias, pneumothorax, formation inal pain, and seizures.39
of a fistula from the hepatic artery to the portal vein or
the biliary tree, perforation of the liver capsule (espe- FINE-NEEDLE ASPIRATION BIOPSY
cially in small, cirrhotic livers), and death. Lundquist demonstrated that cytologic diagnosis
based on material obtained by fine-needle liver aspi-
LAPAROSCOPIC LIVER BIOPSY ration compared favorably with the final histologic
Diagnostic laparoscopy is especially useful in the diagnosis based on surgical specimens.40 Fine-needle
diagnosis of peritoneal diseases, the evaluation of as- aspiration biopsy of the liver is performed under ul-
cites of unknown origin, and the staging of abdominal trasonographic or CT guidance. Patients with a histo-
cancer. It can be performed safely under local anesthe- ry of cancer and liver lesions are good candidates for
sia with conscious sedation. However, the use of lap- fine-needle aspiration biopsy. The diagnostic accura-
aroscopic liver biopsy by gastroenterologists has de- cy ranges from 80 to 95 percent3 and is substantially
clined in favor of less invasive radiologic procedures, affected by the expertise of the cytopathologist. Cy-
and very few gastroenterology training programs now tologic findings that are negative for cancer do not
provide instruction in the procedure, which is usually rule it out.
performed by surgeons because of their growing ex- Although ultrasound-guided or CT-guided biopsy
perience with laparoscopic surgery. The indications for is usually reserved for focal hepatic lesions, limited data
and contraindications to laparoscopic liver biopsy are suggest that diagnostically useful material can be ob-
outlined in Table 4. The complications include per- tained with automatic spring-loaded biopsy needles
foration of a viscus, bleeding, hemobilia, laceration of guided by ultrasound in over 95 percent of patients,41
the spleen, leakage of ascitic fluid, hematoma in the including those with diffuse liver disease. Fine-needle

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 The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne
aspiration biopsy is associated with a low risk of seed-
ing of the needle tract with malignant cells and is gen-
erally a very safe procedure, even in patients with he-
mangiomas and echinococcal cysts.42,43
We are indebted to Dr. Imad Nasser of the Department of Pathol-
ogy, Beth Israel Deaconess Medical Center, Boston, for providing the
photographs of liver-biopsy specimens.
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1958;35:190-9.
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13. McGill DB, Rakela J, Zinsmeister AR , Ott BJ. A 21-year experience
with major hemorrhage after percutaneous liver biopsy. Gastroenterology
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14. Piccinino F, Sagnelli E, Pasquale G, Giusti G. Complications following
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15. Colombo M, Del Ninno E, de Franchis R , et al. Ultrasound-assisted
percutaneous liver biopsy: superiority of the Tru-Cut over the Menghini
needle for diagnosis of cirrhosis. Gastroenterology 1988;95:487-9.
16. Smith CI, Grau JE. The effect of ultrasonography on the performance
of routine outpatient liver biopsy. Hepatology 1995;22:Suppl:384A. ab-
stract.
17. Riley TR III. How often does ultrasound marking change the liver bi-
opsy site? Am J Gastroenterol 1999;94:3320-2.
18. Vautier G, Scott B, Jenkins D. Liver biopsy: blind or guided? BMJ
1994;309:1455-6.
19. Lindor KD, Bru C, Jorgensen RA, et al. The role of ultrasonography
and automatic-needle biopsy in outpatient percutaneous liver biopsy. Hep-
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20. Caturelli E, Giacobbe A, Facciorusso D, et al. Percutaneous biopsy in
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diffuse liver disease: increasing diagnostic yield and decreasing complica-
tion rate by routine ultrasound assessment of puncture site. Am J Gastro-
enterol 1996;91:1318-21.
21. Younossi ZM, Teran JC, Ganiats TG, Carey WD. Ultrasound-guided
liver biopsy for parenchymal liver disease: an economic analysis. Dig Dis
Sci 1998;43:46-50.
22. Pasha T, Gabriel S, Therneau T, Dickson ER, Lindor KD. Cost-effec-
tiveness of ultrasound-guided liver biopsy. Hepatology 1998;27:1220-6.
23. Cadranel JF, Rufat P, Degos F. Practices of liver biopsy in France: re-
sults of a prospective nationwide survey. Hepatology 2000;32:477-81.
24. Jacobs WH, Goldberg SB. Statement on outpatient percutaneous liver
biopsy. Dig Dis Sci 1989;34:322-3.
25. Froehlich F, Lamy O, Fried M, Gonvers JJ. Practice and complications
of liver biopsy: results of a nationwide survey in Switzerland. Dig Dis Sci
1993;38:1480-4.
26. Janes CH, Lindor KD. Outcome of patients hospitalized for compli-
cations after outpatient liver biopsy. Ann Intern Med 1993;118:96-8.
27. Castera L, Negre I, Samii K, Buffet C. Pain experienced during per-
cutaneous liver biopsy. Hepatology 1999;30:1529-30.
28. Van Thiel DH, Gavaler JS, Wright H, Tzakis A. Liver biopsy: its safety
and complications as seen at a liver transplant center. Transplantation 1993;
55:1087-90.
29. Hederstrom E, Forsberg L, Floren CH, Prytz H. Liver biopsy com-
plications monitored by ultrasound. J Hepatol 1989;8:94-8.
30. Raines DR, Van Heertum RL, Johnson LF. Intrahepatic hematoma:
a complication of percutaneous liver biopsy. Gastroenterology 1974;67:
284-9.
31. Lichtenstein DR , Kim D, Chopra S. Delayed massive hemobilia fol-
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32. Reddy KR , Schiff ER. Complications of liver biopsy. In: Taylor MB,
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33. Reddy KR, Jeffers LJ. Evaluation of the liver: liver biopsy and laparos-
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liver. 8th ed. Vol. 1. Philadelphia: Lippincott–Raven, 1999:245-66.
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management of a patient with an acute abdomen: a case report with review
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35. Hanafee W, Weiner M. Transjugular percutaneous cholangiography.
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Hepatology 1992;15:726-32.
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liver biopsy: an experience based on 1000 hepatic tissue samplings with this
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5-year experience in a hepatology training program. Am J Gastroenterol
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clinical diagnosis and investigation. Acta Med Scand Suppl 1971;520:1-28.
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Published on dvm360.com
Home > How to perform a surgical hepatic biopsy

How to perform a surgical hepatic biopsy

An open surgical method greatly improves your chances of obtaining a diagnostic liver
biopsy sample. The specific biopsy technique you should use depends on the site
from which you are sampling.

May 01, 2006

By Amie Carrier, BS, Diana Burger, BS, Karen M. Tobias, DVM, MS, DACVS
VETERINARY MEDICINE

In patients with known or suspected liver disease, obtaining a biopsy sample is often
indicated. Liver samples may be obtained by a variety of techniques.1-4 Surgical
biopsy allows the whole liver to be visually inspected and palpated, providing an ideal
opportunity to obtain biopsy samples of focal lesions for histologic examination,
culture and antimicrobial sensitivity testing, and metal analysis. Surgical biopsy also
provides a larger tissue sample for a more complete histologic review and allows the
veterinarian to examine the biopsy site for adequate hemostasis.2 This article features
guidance on when and how to perform a surgical hepatic biopsy.

INDICATIONS

The indications for performing a liver biopsy include substantially or persistently

increased liver enzyme activities or serum bile acid concentrations, hepatic
hyperbilirubinemia, generalized changes in hepatic ultrasonographic echogenicity,
unexplained hepatomegaly, or the presence of solitary or multiple focal lesions within
the hepatic parenchyma.1 Liver biopsy is particularly important when the results of
biochemical testing and advanced imaging modalities such as ultrasonography or
scintigraphy are not adequate to establish a diagnosis. Additionally, histologic
information can be combined with an already determined diagnosis to tailor specific
treatment protocols, evaluate the response to therapy, and determine the prognosis.
Specific indications for a surgical biopsy include microhepatia, a hepatic abscess or
cyst, or a lesion that is difficult to localize on ultrasonographic examination.1 In
addition, excisional biopsy of a pedunculated or solitary mass may provide treatment
opportunities.

SELECTING AND COMPARING LIVER BIOPSY METHODS

Liver samples may be obtained percutaneously by blind or ultrasound-guided biopsy or

surgically by laparoscopy or celiotomy.1-4 Selecting the appropriate method is
determined by the size of the liver, the type and size of the lesion, and the patient's size
and overall health.1,2

Biopsy samples from unstable patients or from patients with severe coagulopathy are
best obtained by percutaneous means such as fine-needle aspiration or Tru-Cut
biopsy, particularly if diffuse disease is suspected.1 However, both of these methods
are significantly inferior to wedge biopsy, potentially affecting an accurate diagnosis.5-
8 Cytologic examination of samples obtained by fine-needle aspiration cannot provide
information about tissue architecture and may be nondiagnostic for lesions that
exfoliate poorly. The correlation between cytologic examination of fine-needle liver
aspirates and histologic examination of hepatic biopsy samples is poor; diagnostic
correlation of samples was 30% in dogs and 51% in cats in one study and 29% in
multiple species in another study.5,6 In fact, cytologic and histologic examinations
have a significantly lower correlation in hepatic tissue than all other tissues studied,
including cutaneous, subcutaneous, nasal, osseous, lymphatic, splenic,
gastrointestinal, cerebral, and ocular tissues.6

Sample size and quality are also variable when ultrasound-guided tissue core biopsy
samples are obtained. In one study, only 77% of intended liver biopsies retrieved
hepatic tissue, and 22% of the samples were less than 2 mm long and lacked
appropriate lobular architecture.7 Thirteen percent of the samples were completely
devoid of tissue, and another 10% contained skeletal muscle, blood, or small intestine.
Only 60% of samples obtained by Tru-Cut biopsy were of diagnostic quality.7

In another study, samples obtained with 18-ga spring-triggered biopsy needles using
ultrasound guidance or direct observation during laparotomy or immediately
postmortem had one-third to one-fourth of the median surface area of those obtained
with wedge biopsy.8 Because of this small sample size, a diagnosis based on needle
biopsy correlated with that of wedge biopsy in less than half the patients. Additionally,
the severity of pathology was graded as significantly less in needle biopsy samples for
most morphologic parameters, with the exception of inflammation, which was graded
more severe as compared with wedge samples. Smaller needle biopsy samples were
unable to achieve the necessary number of portal triads per sample to provide an
accurate diagnosis of portal triad diseases such as portal venous hypoplasia or portal
atresia and were subject to misinterpretation by evaluators.8 Laparoscopic biopsy
allows better visualization of the site compared with ultrasonography but may provide
less tissue volume compared with a surgical wedge biopsy.

PERIOPERATIVE CONSIDERATIONS

All patients should undergo presurgical diagnostic tests (complete blood count,
platelet count, serum chemistry profile, coagulation tests, urinalysis) to determine
which perioperative treatments should be administered. For example, animals with
liver disease may present with vomiting that could result in dehydration and electrolyte
abnormalities. Preoperative potassium or magnesium deficiencies may cause ileus,
arrhythmias, and other complications during or after surgery, so they are best
corrected before anesthesia. Animals with severe liver disease or sepsis may have
prolonged clotting times, requiring crossmatching and transfusion before surgery.2,3
Animals suspected of having neoplasia should undergo staging of their disease,
including thoracic radiography and abdominal ultrasonography. Aspirates obtained
during ultrasonography may provide a diagnosis in some patients, obviating the need
for surgery.

If possible, fast patients for 12 hours before surgery. Preoperatively, administer fluids
and analgesics. Give hypoalbuminemic patients colloidal fluids such as hetastarch or
plasma to provide oncotic pressure support. Choose premedicants and dosages with
care since metabolism of some drugs may be delayed when liver dysfunction is
present. We commonly use an opiate combined with a benzodiazepine in dogs and
cats or a combination of ketamine and diazepam in cats. If acepromazine is
administered as a sedative, the total dose should not exceed 0.25 mg. Induction can
be performed with intravenous propofol or by mask induction with isoflurane. Clip
patients undergoing liver biopsy to the midsternal level, and clip patients receiving
feeding tubes farther laterally on the abdomen. All patients should have an
appropriately sized endotracheal tube with an inflated cuff. During anesthesia,
continuous-rate infusions of fentanyl can be administered to reduce gas anesthetic
requirements.9,10 These infusions can be continued postoperatively to provide
analgesia.

Ideally, respiratory and cardiac function and oxygenation should be monitored during
anesthesia with a capnograph, electrocardiograph, arterial blood pressure monitor, and
pulse oximeter. Maintaining intraoperative blood pressure with fluids and oncotic
support is critical in patients with liver disease since reduced hepatic perfusion can
have deleterious effects on postoperative liver function. Forced-air heating blankets
(e.g. Bair Hugger—Arizant Healthcare) can be used to keep patients warm during the
procedure.

Prophylactic antibiotics are usually unnecessary in patients undergoing liver biopsy.

Be prepared to take culture samples during the procedure and to place feeding tubes
in some patients. Cautery should be available intraoperatively for patients with
coagulopathies. Count the sponges and laparotomy pads before the abdomen is
opened and again before it is closed to prevent iatrogenic peritoneal foreign bodies.
Suction all fluid from the abdominal cavity before closure.

Postoperative treatments, complications, and prognosis vary depending on the

underlying disease process and the patient's condition. Most animals continue to
receive fluid support and analgesics after surgery. In patients that are not vomiting,
small amounts of food and water can be offered within eight hours of the procedure.

OPEN SURGICAL BIOPSY TECHNIQUE

Make a ventral midline abdominal incision. The incision should extend cranially to the
level of the xiphoid to improve exposure of the liver, particularly if it is small. Liver
exposure can be improved by removing the falciform fat and by carefully incising the
triangular ligaments or by placing moistened laparotomy pads between the liver and
the diaphragm.2,3 Examine the entire liver visually and by gentle palpation for nodules,
cavitations, and other abnormalities.
 Obtain samples at the junction of normal and diseased tissue to
ensure that both abnormal and normal hepatocellular structures
are included.11 If the liver is diffusely affected, obtain biopsy
samples from the most accessible location, usually the liver
margin. In conditions in which lesions are distributed irregularly,
obtain samples from multiple lobes to increase the likelihood of
obtaining a diagnostic sample. Although affected liver margins
typically suggest parenchymal disease, their greater distance
from hepatic blood supply may predispose them to fibrosis,
obscuring the underlying pathology. Misdiagnosis of hepatic
fibrosis can be avoided by taking larger or multiple samples.2,12
Figures 1,2 Peripheral or diffuse lesions

The guillotine method is used to sample the hepatic margin of

pointed or sharp-edged lobes. Form a loop with 3-0 absorbable
monofilament suture, using a single throw. Drop the suture loop
over the point of the lobe, settling the suture into a natural
fissure or in notches made by Kelly forceps (Figure 1). Tighten
the suture completely so that it crushes all of the tissue within
the loop, leaving the piece of tissue attached only by vessels
and ducts (Figure 2). While tightening, do not pull the suture
outward or upward, as this may sever the vessels and ducts and
accidentally remove the ligature with the sample. A second
throw can be placed to form a knot but is not necessary for
hemostasis and can increase the risk of tearing the vessels.
Transect tissue 2 to 3 mm distal to the suture from the lobe with Figures 3,4
Metzenbaum scissors or a scalpel blade. If you use a blade,
place a finger under the piece of liver to be removed, and press the blade gently and
firmly through the tissue toward the finger (Figures 3 & 4). If you press slowly but firmly
with the flat portion of the cutting edge, you will easily cut through the liver tissue but
will not damage your gloves. Trim the suture ligature short. To avoid iatrogenic
specimen artifacts, do not use tissue forceps to handle the tissue sample.2,3
Excessive bleeding can be controlled with pressure, ligation, or cautery.2,4

For marginal lesions on rounded liver lobes,

place two parallel, full-thickness guillotine
sutures perpendicular to the liver margin
around the proposed site by using absorbable
suture with a swaged needle (Figures 5-8).
Leave the ends of the second suture knot long,
and then pass one end of the suture around
the base of the tissue pedicle and tie it back to
the other end (Figures 9 & 10). This will crush
the tissues across the base of the pedicle,
resulting in hemostasis along all three sides of
the tissue sample. Tighten the sutures
Figures 5,6,7,8 completely so that they cut through the
tissues, and remove the tissue within the
suture box with scissors (Figures 11 & 12).

Central lesions

Samples from nonmarginal liver can be

obtained with a skin biopsy punch, Tru-Cut
biopsy needle, or laparoscopic clamshell
biopsy instrument.12 To avoid nicking the
large, dorsally located hepatic veins, take the
sample, preferably, from the ventral hepatic
surface, and do not let it exceed half the
thickness of the lobe.

When taking biopsy samples of the liver with

clamshell forceps, place the instrument
against the site of interest with the jaws open.
Insert the forceps into the parenchyma to the Figures 9,10,11,12
level of the angle of the jaws, and close the jaws. After a few seconds of crushing,
twist the instrument until a free piece of tissue is removed. Pulling the forceps straight
out of the liver tends to cause more hemorrhage than twisting.12

Hemorrhage from punch, Tru-Cut, or clamshell biopsy sites can be controlled by a

variety of methods. Absorbable gelatin foam (Gelfoam—Pharmacia & Upjohn) can be
inserted into the defect, a mattress suture of 3-0 absorbable material can be placed
gently around the defect, or an omental flap can be sutured over the defect.
Alternatively, pressure can be applied to the site, or the site can be cauterized by using
a low setting.4,13 Thoroughly lavage the abdomen with a balanced electrolyte solution
in patients with bile leakage, excessive hemorrhage, or infected or necrotic lesions.2

COMPLICATIONS

Complications after liver biopsy are uncommon but may include bile peritonitis,
hemorrhage, and sepsis. The risk of complications is greater in patients with
coagulopathies and thrombocytopenia.14,15 Major complication rates during hepatic
biopsies have been reported to be as high as 22% and 50% in dogs and cats that are
thrombocytopenic.14 Many patients with liver disease are debilitated from
hypoalbuminemia and compromised liver function, increasing the risk of potential
complications with anesthesia and surgery such as hypotension and altered
metabolism of anesthetic and analgesic drugs.

Diana Burger, BS
Amie Carrier, BS
Karen M. Tobias, DVM, MS, DACVS
Department of Small Animal Clinical Sciences
College of Veterinary Medicine
The University of Tennessee
Knoxville, TN 37996-4544

REFERENCES

1. Day DG. Indications and techniques for liver biopsy. In: Textbook of veterinary
internal medicine. 5th ed. Philadelphia, Pa: WB Saunders Co, 2000;1294-1298.

2. Martin RA, Lanz OI, Tobias KM. Liver and biliary system. In: Small animal surgery. 3rd
ed. Philadelphia, Pa: WB Saunders Co, 2003;713-717.

3. Fossum TW. Surgery of the liver. In: Small animal surgery. 2nd ed. St. Louis, Mo:
Mosby, 2002;450-457.

4. Harvey CE, Newton CD, Schwartz A. The liver and biliary tract, spleen, and pancreas.
In: Small animal surgery. Philadelphia, Pa: JB Lippincott Co, 1990;407-411.

5. Wang KY, Panciera DL, Al-Rukibat RK, et al. Accuracy of ultrasound-guided fine-
needle aspiration of the liver and cytologic findings in dogs and cats: 97 cases (1990-
2000). J Am Vet Med Assoc 2004;224:75-78.

6. Cohen M, Bohling MW, Wright JC, et al. Evaluation of sensitivity and specificity of
cytologic examination: 269 cases (1999-2000). J Am Vet Med Assoc 2003;222:964-
967.

7. de Rycke LMJH, van Bree HJJ, Simoens PJM. Ultrasound-guided tissue-core biopsy
of liver, spleen and kidney in normal dogs. Vet Radiol Ultrasound 1999;40:294-299.

8. Cole TL, Center SA, Flood SN, et al. Diagnostic comparison of needle and wedge
biopsy specimens of the liver in dogs and cats. J Am Vet Med Assoc 2002;220:1483-
1490.

9. Valverde A, Doherty TJ, Hernandez J, et al. Effect of lidocaine on the minimum

alveolar concentration of isoflurane in dogs. Vet Anaesth Analg 2004;31:264-271.

10. Hellyer PW, Mama KR, Shafford HL, et al. Effects of diazepam and flumazenil on
minimum alveolar concentrations for dogs anesthetized with isoflurane or a
combination of isoflurane and fentanyl. Am J Vet Res 2001;62:555-560.

11. Garner MM, Raymond JT, Toshkov I, et al. Hepatocellular carcinoma in black-tailed
prairie dogs (Cynomys ludivicianus): tumor morphology and immunohistochemistry for
hepadnavirus core and surface antigens. Vet Pathol 2004;41:353-361.

12. Richter KP. Laparoscopy in dogs and cats. Vet Clin North Am Small Anim Pract
2001;31:707-727.

13. Kim EH, Kopecky KK, Cummings OW, et al. Electrocautery of the tract after needle
biopsy of the liver to reduce blood loss. Invest Radiol 1993;28:228-230.

14. Bigge LA, Brown DJ, Penninck DG. Correlation between coagulation profile findings
and bleeding complications after ultrasound-guided biopsies: 434 cases (1993-1996).
J Am Anim Hosp Assoc 2001;37:228-233.

15. Weber JC, Navarra G, Jiao LR, et al. New technique for liver resection using heat
coagulative necrosis. Ann Surg 2002;236:560-563.
© 2019 MultiMedia Animal Care LLC. All rights reserved. Reproduction in whole or in part is prohibited. Please send any technical
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Source URL: http://veterinarymedicine.dvm360.com/how-perform-surgical-hepatic-biopsy

Liver Biopsy Te ch niq ues
Jan Rothuizen, DVM, PhDa,*, David C.Twedt, DVM, PhDb

KEYWORDS
 Liver biopsy  True cut needle  Menghini needle
 Laparoscopy  Wedge biopsy  Fine needle aspiration
 Gall bladder punction  Coagulation

Liver biopsy is an important step in the evaluation of a patient with hepatic disease and
is required to formulate a diagnosis, direct therapy, and provide an accurate prog-
nosis. However, a liver biopsy evaluates only a small percentage of the liver and
may not represent the entire liver. Consequently, the results should always be
combined with the clinical information, laboratory data, and imaging procedures to
formulate a diagnosis.1 There are advantages and disadvantages of each method of
obtaining a liver biopsy. This article presents the indications, technique, and diag-
nostic accuracy of the various biopsy methods, including fine-needle liver aspiration,
needle biopsy, laparoscopic-assisted biopsy, and surgical biopsy. Descriptions of
many of the surgical techniques are beyond the scope of this article; specific details
are available in most surgical texts.
The diagnosis of most liver diseases requires a histopathologic examination of liver
tissue. Histology is especially important for parenchymal liver diseases such as hepa-
titis occurring in dogs and inflammatory biliary tract disease common to the cat. Of the
circulatory diseases of the liver, portal vein hypoplasia (microvascular dysplasia) can
only be diagnosed by a combination of histopathology and imaging (eg, ultrasonog-
raphy) techniques. Diffuse liver diseases may be sampled randomly, but focal lesions
require careful selective sampling using ultrasound-guided needle biopsy, laparo-
scopic guidance, or surgically. Large focal lesions should be sampled in the periphery
of the lesion because a neoplastic mass may have a necrotic center and the malignant
characteristics are best observed in the periphery of the mass.
Neoplasia and diffuse vacuolar disorders (eg, lipidosis, steroid hepatopathy) of the
liver can often be diagnosed by cytologic examination obtained using fine-needle
aspiration. However, cytology does not show the architectural changes of the liver
that can be seen with histopathology. For example, differentiation of liver cell
adenomas and carcinomas often cannot be distinguished without evaluating the histo-
pathology. Needle biopsies also have limitations due to their small sample size; for

a
Department of Clinical Sciences of Companion Animals, University Utrecht, Yalelaan 108, P.O.
Box 80.154, 3508 TD Utrecht, The Netherlands
b
Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences,
Colorado State University, Fort Collins, CO 80523, USA
* Corresponding author.
E-mail address: j.rothuizen@uu.nl (J. Rothuizen).

Vet Clin Small Anim 39 (2009) 469–480

doi:10.1016/j.cvsm.2009.02.006 vetsmall.theclinics.com
0195-5616/09/$ – see front matter ª 2009 Elsevier Inc. All rights reserved.
470 Rothuizen & Twedt

example, with macronodular cirrhosis, needle aspirates may only sample a hyper-
plastic nodule and inflammatory and fibrotic areas may be missed.
In every case, all available information must be considered before reaching the final
diagnosis, and this implies selecting the proper techniques for obtaining tissue
samples (representative and big enough), adhering to the important requirements of
tissue handling, and providing the pathologist with all available essential clinical infor-
mation. The clinician should then consider the information from the history, physical
examination, clinical pathology, and ultrasonography or other imaging procedures
with the histologic results of the liver biopsy before making the diagnosis.

GENERAL CONSIDERATIONS

The risk/benefit ratio of performing a liver biopsy must be weighed for every case.
Although the chance of serious complications is low for a specific case or procedure,
there is always the potential for complications to occur. Operator experience also has
a significant influence on the complication rate. However, most diseases of the liver
are best defined and treated following a liver biopsy with histologic examination.
It is important that the patient fasts for approximately 12 hours to ensure that the
stomach is small. Fasting aids the ultrasonographic examination and is required
before sedation or anesthesia. The stomach covers the caudal visceral surface of
the liver and a distended stomach may prevent the biopsy needle reaching the liver.
Anesthesia is required for surgery and laparoscopy, and possibly for some dogs
and all cats undergoing a needle biopsy. Needle biopsies can be obtained in cooper-
ative dogs using local anesthesia of the skin and abdominal wall. Fine-needle aspira-
tion is usually performed using minimal or no sedation and without local anesthesia.

Coagulation Testing
As the liver produces all the clotting factors except Factor VIII, bleeding from a liver
biopsy is reported to be the most frequent complication. With every liver biopsy there
is always a small amount of blood loss, which can be seen by ultrasonography or
during laparoscopy or surgery. The average amount of blood loss from a liver biopsy
is reported to be around 2 mL in normal dogs. 2,3 Fine-needle aspirates are generally
considered safe and there are few reports of serious bleeding following a needle aspi-
rate.4 If the coagulation tests are normal, bleeding becomes less of a concern.5 The
reserve capacity of the liver in producing clotting factors is so huge that, for most liver
diseases, factor production is rarely decreased to the point whereby it becomes
a limiting factor. Abnormalities in coagulation tests may also not preclude a liver
biopsy but may increase the likelihood of bleeding. The results of routine coagulation
tests have shown the degree of bleeding from a biopsy does not correlate with the pa-
tient’s clotting times.6 However, a liver biopsy should be avoided if there is clinical
evidence of bleeding or marked abnormalities in the coagulation tests. Thrombocyto-
penia (<80,000 platelets) or prolongation of the coagulation times increases the risk of
bleeding following a liver biopsy. The presence of marked abnormalities in coagulation
in a patient with liver disease becomes prognostic and suggests significant hepatic
dysfunction.7
Ideally, before a liver biopsy the coagulation status should be assessed by evalu-
ating the intrinsic pathway (activated partial thromboplastin time [APTT]), the extrinsic
pathway prothrombin time (PT), fibrinogen content, and platelet count. A buccal
mucosal bleeding time (BMBT) is also recommended especially in breeds associated
with von Willebrand’s disease. Patients with von Willebrand’s disease usually have
normal coagulation tests and platelet numbers but have significantly decreased
 Liver Biopsy Techniques 471

platelet function, which increases the BMBT. Disease of the liver can also lead to an
increased consumption of fibrinogen and other clotting factors with a significant effect
on coagulation.8 This situation occurs in diffuse diseases of the liver associated with
pronounced hepatocyte necrosis/apoptosis leading to subclinical diffuse intravas-
cular coagulation (DIC). Associated diseases are active forms of hepatitis and malig-
nant lymphoma in the liver. In such cases, fine-needle aspiration of the liver is still
possible, allowing identification or exclusion of malignant lymphoma. A fibrinogen
concentration less than 50% of the lower reference level can be used as an absolute
contraindication for taking a liver biopsy. The activation of factors II, VII, IX, and X
depends on the availability of vitamin K1. In prolonged and complete obstruction of
the bile flow, the intestinal absorption of fat-soluble vitamins K1 may be severely
impaired due to the lack of bile acids entering the intestine. In these cases, it is useful
to administer vitamin K1 (1–5 mg/kg subcutaneously daily for several days) and then
re-evaluating the effect on the clotting times before biopsy. Some investigators
recommend administering vitamin K1 to all patients if the clotting times are at all
abnormal. However, unless there is vitamin K deficiency, there will likely be little
improvement. With abnormalities in PT, APTT, or BMBT fresh frozen plasma should
be administered 2 hours before the procedure and the patient should be monitored
closely following the biopsy. Suspected bleeding following biopsy should be evalu-
ated with abdominocentesis or ultrasonography, and is evident within 0.5 to 1 hour
after the procedure. Life-threatening bleeding following biopsy is treated with fresh
frozen plasma, fresh whole blood, or, rarely and as a last resort, surgery to stop the
source of the bleeding. Bleeding, with a dropping hematocrit level, is usually evident
within the first 5 hours following the biopsy.6

Ultrasound Examination
It is important to evaluate the structure of the liver, biliary tract, and portal vein in prep-
aration for a liver biopsy. This evaluation is usually done with ultrasonography, but may
also be performed by laparoscopy or surgical inspection. Systematic evaluation
should be performed to determine: (1) the size of the liver; (2) the presence of focal
lesions; (3) the liver architecture and structure; (4) the diameter of the lumen and the
thickness of the wall of the extrahepatic and intrahepatic bile ducts and the gall-
bladder; (5) vascular changes, especially of the portal vein but also the presence of
arteriovenous fistulas; (6) the presence of free abdominal fluid; and (7) echo Doppler
evaluation of the portal blood flow velocity and direction. These evaluations provide
important information for the clinician and the pathologist. Histologic findings become
optimally meaningful when combined with the relevant clinical, clinicopathologic, and
imaging findings.

Risk Factors
Precautions with respect to the coagulation process are not the only factors associ-
ated with complications of a liver biopsy. The experience of the operator must also
be considered. With an experienced operator most techniques are safe and have
a low complication rate. A third complication associated with a needle biopsy is the
induction of vagotonic shock immediately following the procedure.9 Rapid-firing auto-
matic biopsy needles increase the risk for this complication. The automatic spring-
loaded biopsy guns have been reported to produce such a strong impulse to the liver
that it causes a lethal shock reaction in cats but this has not been observed in dogs.
The larger bile ducts and the gallbladder have a dense autonomic innervation and
trauma induced by penetration with a wide core biopsy needle may also induce an
autonomic reaction with bradycardia and deep shock within 30 minutes of the
472 Rothuizen & Twedt

procedure, which may be especially relevant if dilated bile ducts of an extrahepatic

bile duct obstruction (EHBDO) have been hit. Other biopsy techniques are recommen-
ded if an obstruction associated with duct dilation is present. The authors have also
observed biopsy-induced shock following liver biopsy when large liver cell carcinomas
are sampled. A small percentage of these cases may develop severe shock requiring
intensive care treatment, but this is not a reason for avoiding the procedure. The owner
should be informed in advance of a potential higher risk. In these situations, it may be
better to avoid ultrasound-directed needle biopsy.

What Constitutes a Good Liver Biopsy

An ideal liver biopsy should be of proper size and taken from a location that represents
the primary liver pathology. In diffuse liver disease, the biopsy will likely represent the
entire liver but focal or regional disease becomes more problematic. Ultrasound exam-
ination or visual inspection is important to determine the presence of focal disease.
Ideally, at least 2 and preferably 3 biopsies should be obtained from separate liver
lobes.1 If one area seems normal and other areas seem abnormal, representative
samples from each area should be taken. Occasionally the normal looking areas
may actually be abnormal. Because of the smaller sample size obtained from needle
biopsies, there is greater potential for the needle sample not to represent the entire
liver. It is stated that 1 good core needle biopsy represents only a 50,000th of the entire
liver.10 The authors believe that 18G needle biopsies are generally too small, fragment
easily, and do not contain enough portal areas to be of diagnostic value. Good samples
can be obtained with 14G needle diameter for dogs and 16G for small dogs and cats.
Laparoscopic wedge and surgical wedge biopsies generally provide larger samples
but the procedure is more invasive. Samples from laparoscopic biopsy forceps are
usually approximately 5 mm in diameter. Surgical wedge samples should be at least
1 cm, preferably 2 cm deep. Subcapsular tissue contains more fibrous tissue and
may have nonspecific inflammatory changes that could be misleadingly interpreted
as representing the entire liver. With the larger wedge samples, this may be less of
a concern and the pathologist should interpret subcapsular change with caution.
Appropriate tissue handling and histologic interpretation is critical for a correct diag-
nosis. Box 1 provides guidelines for tissue handling of liver samples. An accurate
interpretation of the biopsy requires the presence of a sufficient number of portal
and central areas because different diseases are associated with different sublobular
zones. Many pathologists believe that 6 portal areas are necessary to make an
adequate diagnosis of inflammatory liver disease.11,12 The quality of a clinical diag-
nosis of liver disease depends largely on the histologic description. There is often
confusion about which criteria are essential to diagnose a certain disease, and indi-
vidual pathologists may have different interpretations or even different diagnoses on
the same sample. The World Small Animal Veterinary Association (WSAVA) has initi-
ated standards for unification of diagnostic criteria and nomenclature of liver diseases.
A team of leading specialists, clinicians and pathologists, have reviewed most known
liver diseases of companion animals and have described clear-cut and well-illustrated
criteria in the publication, WSAVA Standards for Clinical and Histologic Diagnosis of
Canine and Feline Liver Diseases in 2006.13 The clinician should expect a detailed
description according to these international guidelines from their pathologist.
In addition to tissue for histopathology, samples may also be procured for culture or
quantitation of copper or other metals. Ideally, approximately 20 to 40 mg of liver (wet
weight) is required for copper quantitation using atomic absorption spectrophotom-
etry methods. This amount equates to a sample of approximately 2.5 mm diameter
or one full 14G (2-cm long) needle biopsy sample. Smaller samples may decrease
 Liver Biopsy Techniques 473

Box 1
Guidelines for proper handling of liver tissue

1. Verify the quality of the tissue; it should be unfragmented and at least 1 cm, preferably 2 cm
in length. Samples obtained surgically should be cut into thin slices <5 mm thick. The center
of thicker pieces will not be fixated adequately in formalin or another fixative.
2. The tissue should be put into the fixative within 5 minutes; 10% neutral buffered formalin is
used routinely.
3. The fixation time should not be too long; after long formalin fixation
immunohistochemistry becomes impossible with many antibodies.
4. Think in advance about the specific requirements. It may be necessary to perform electron
microscopy, specific stains for metabolic diseases, and so forth. In case of doubt, the
pathologist should be contacted about the best way to preserve the tissue.
5. For quantitative measurement of metals in liver such as copper, avoid saline if using neutron
activation analysis for measurement of small amounts in biopsy samples because the analysis
is affected by the presence of sodium. Tissue should be sampled in a metal-free plastic
container and freeze-dried; thereafter closed containers can be stored at room temperature.
6. Fill the container completely with fixative so that the sample is not stirred and broken
during transportation.

the accuracy of the metal analysis. Laparoscopic cup biopsy forceps provide 45 mg of
liver tissue, a 14G Tru-Cut–type biopsy needle provides 15 to 20 mg, whereas an 18G
needle biopsy provides only 3 to 5 mg of liver tissue. Liver tissue taken for culture
should be approximately 5 mg in size and placed in appropriate transport broth or
medium that preserves aerobic and anaerobic bacteria.

FINE-NEEDLE ASPIRATION

Fine-needle aspiration (FNA) with cytologic examination is commonly performed in

small animals with liver disease because it is cheap and easy to do. FNA can be per-
formed at low risk to the patient and usually without the need for sedation or local
anesthesia.4,14 It is best suited for diffuse hepatic disease. If focal lesions are present
ultrasound direction becomes necessary. Bleeding complications from FNA of the
liver are uncommon and there is rarely a need to perform coagulation tests unless
overt hemorrhage is identified before the aspiration. However, there are limitations
to FNA and examination of the liver cytology. These include failure to correctly identify
the primary disease due to the small sample size and the cytology does not reflect the
morphology of the parenchymal architecture. Several studies have compared liver
aspirates and their cytologic interpretation with the histopathologic diagnosis from
a biopsy. In a large study of 97 cases involving dogs and cats, only 30% of the canine
cases and 51% of the feline cases had overall agreement between the histopathologic
diagnosis and the cytologic diagnosis.15 In this study, the diagnosis of vacuolar hep-
atopathy was the category with the highest percentage of agreement, whereas inflam-
matory disease was correctly diagnosed only 25% of the time when dogs and cats
were grouped together. Other similar studies also point out the low correlation of
cytology with histopathology. Although FNA is frequently used by clinicians to support
the diagnosis of hepatic lipidosis in cats, there is a report of 4 cats incorrectly diag-
nosed as having hepatic lipidosis instead of lymphoma.16 A multistep approach to
cytologic evaluation of nonvacuolar diseases may provide useful standardization for
cytologic evaluation and improve the diagnostic accuracy.17
474 Rothuizen & Twedt

The location of the entry site for most FNAs is determined using ultrasound to direct
the needle to focal lesions or to certain areas in the liver. Alternatively a blind method,
in which a random sample of the liver is obtained, may be adequate if a palpable large
liver is present and diffuse disease such as malignant lymphoma or diffuse vacuolar
hepatopathies (lipidosis) of the liver is suspected. If a large palpable liver is present
and if using a blind technique, the usual entry point is caudal to the costal arch. The
liver can also be approached on the right side at the 10th intercostal space at the level
of the rib-cartilage junction. For either approach there is no need for local or general
anesthesia in dogs or cats if using thin needle sampling. Most FNAs are performed
using a 20G to 22G needle. A 3-inch disposable injection needle is usually sufficient,
but longer needles may be required for deep-lying lesions. The tissue is aspirated in an
identical procedure to that used for peripheral structures such as lymph nodes. The
needle attached to a 5 or 12 mL syringe is advanced under ultrasound guidance
into the liver. At the site of aspiration the needle is quickly advanced and withdrawn
0.5 to 1 cm several times. Cells within the needle are then blown on the microscope
slide with the syringe. Others prefer to apply negative pressure on the syringe plunger
while aspirating the liver. Pressure on the plunger is released and the needle with-
drawn. This technique collects more cells but also results in more blood contamina-
tion, which must be considered in the cytologic interpretation. Liver aspirates are
placed on a glass microscope slide, air-dried and routine cytologic staining used. It
is also possible to use specific staining methods such as copper stains (rubeanic
acid or rhodanine stain) for intracellular copper granules, Sudan stain for lipids, and
periodic acid-Schiff stain for the presence of glycogen.

NEEDLE BIOPSY

Several different needles and biopsy techniques are used to obtain liver tissue for
examination, each with advantages and disadvantages. The Menghini technique
involves tissue aspiration (using saline) with a syringe attached to a large bore hollow
needle. The tip of the needle is convex and is sharp around the circumference. Most
needles have a blocking device in the shaft to prevent liver tissue going into the
syringe. The Menghini or blind technique is not suitable for ultrasound guidance and
is almost always performed using the needle tip as a probe to palpate the appropriate
location.1 When the site is determined, suction is applied to the syringe and the needle
is quickly thrust into the liver and then rapidly retrieved. A core of liver is then sucked
into the needle. The samples are larger than those obtained with Tru-Cut needles
because the entire lumen is filled with tissue and the length of the sample can be pre-
determined. The Menghini technique is not suitable for cats.
The Tru-Cut–type needle is generally performed using ultrasound guidance, but
may also be done under visual control during laparoscopy or surgery. This technique
is the most widely used. The Tru-Cut–type needle has an outer cannula and an inner
notched shaft in which a tissue specimen is retained. The notched portion has a 2-cm
long indentation which is first advanced into the liver, so that the tissue can fall in the
indentation. Then, the outer cutting cannula slides over the inner notched shaft so that
the tissue is sliced off. The entire instrument is finally withdrawn and the tissue slice
retrieved. Tru-Cut needles have a sharp tip and should therefore only be used under
visual control such as ultrasound guidance or during surgery. There are 3 types of
Tru-Cut needles: manual, semi-automatic, and those used in a biopsy gun device.18
Manual devices are cheap but they are the most difficult to handle and their use is
not advised other than during surgery under direct visual control. Semi-automatic nee-
dles are the most expensive but easy to use. These needles are recommended for
 Liver Biopsy Techniques 475

cats. Biopsy gun devices require a larger financial investment, but the Tru-Cut needles
used with them are inexpensive. The gun-driven needles are recommended for
centers where biopsies (not only of the liver but also of kidneys and other structures)
are routinely taken under ultrasound guidance.19 An advantage of Tru-Cut guns is that
they operate so quickly that a firm, fibrotic liver or a liver with concurrent ascites tissue
is more easily sampled. In these situations, the liver may be hard to puncture using
conventional needles. As a rule, most semi-automatic and gun-driven Tru-Cut needles
advance 2 cm into the liver so it is important to note the amount of liver tissue available
in front of the needle before advancement so that no structures other than the liver are
hit with the needle.

LAPAROSCOPIC LIVER BIOPSY

Diagnostic laparoscopy is a technique used to view and biopsy the organs in the
abdominal cavity.20 The technique involves distention of the abdominal cavity with
gas followed by placement of a rigid telescope through a portal (cannula) in the
abdominal wall to examine the contents of the peritoneal cavity. Biopsy forceps or
other instruments are then passed into the abdomen through adjacent portals to
perform various procedures. Laparoscopy requires general anesthesia but the limited
degree of invasiveness, diagnostic accuracy, large biopsy sample size, and rapid
patient recovery make laparoscopy a valuable technique for obtaining liver tissue.
The excellent view of the liver with magnification is an advantage but limitations
include the expense of the equipment, adequate operator training, and increased
time over needle biopsy techniques. Laparoscopy is frequently used to obtain biop-
sies of the liver, pancreas, kidney, spleen, lymph node, and intestine. Laparoscopy
may also reveal small (0.5 cm or less) metastatic lesions, peritoneal metastases, or
other organ involvement not easily observed by other techniques. One of the ancillary
diagnostic techniques using laparoscopic guidance also includes gallbladder aspira-
tion (choleocystocentesis).
Laparoscopy is a step between needle liver biopsy and surgical laparotomy for
obtaining tissue for histopathology. Because most liver diseases are nonsurgical,
laparoscopy is often the preferred technique over laparotomy. The advantages of
laparoscopy over conventional surgical laparotomy include improved patient recovery
because of smaller surgical sites, lower postoperative morbidity, and decreased infec-
tion rate, postoperative pain, and hospitalization time. Other less obvious benefits of
laparoscopy are related to fewer stress-mediated factors than are reported to occur
with surgery.
Discussion of basic laparoscopic equipment and a detailed description of the tech-
niques for laparoscopy are beyond the scope of this article and the reader should
consult specific texts or articles on laparoscopy. For most diagnostic laparoscopic
procedures a 5-mm diameter 0 field of view telescope is recommended. The 0 desig-
nation means that the telescope views the visual field directly in front of the telescope
in a 180 circumference. Angled viewing scopes such as the 30 telescope enable the
operator to see into areas with a small field of view. However, angled telescopes make
the orientation more difficult for the inexperienced operator. Five-millimeter diameter
forceps with oval biopsy cups are most often used for liver biopsy.21
The patient should be fasted for at least 12 hours. Under general anesthesia
2 cannula portals are placed through the abdominal wall, one for the telescope
and the other for the biopsy forceps. There are 2 basic entry sites for liver biopsy.
The first uses dorsal recumbency in which the telescope is placed on the midline
behind the umbilicus. This position provides a view of the entire surface of the liver,
476 Rothuizen & Twedt

however the falciform ligament is often a nuisance and the pancreas is more difficult
to identify. The second entry site requires placement of the animal in left lateral
recumbency with the telescope portal in the right mid-abdominal wall. This lateral
approach is preferred by the authors because the falciform ligament is not in the
way, the right limb of the pancreas is easily seen, and the extrahepatic biliary system
can be easily followed to where it enters the duodenum. Using this approach the left
lateral lobe of the liver is difficult to examine completely.
The liver, extrahepatic biliary system, and other abdominal structures are examined
and can be palpated using a specialized palpation probe. The palpation probe is used
to move or elevate lobes of the liver for complete inspection and to aid in selecting
representative areas of the liver to be sampled. Using the oval biopsy cups either
an edge of the liver or the surface of the liver is sampled. To take a biopsy of the
surface of the liver, the forceps are directed at approximately 90 angle to the liver,
opened and pushed into the liver, and then closed. Sample size will vary with the
operator’s technique and depth of penetration. With either sampling technique the
forceps are closed tightly for 15 to 30 seconds and then gently tugged away from
the liver. The biopsy site is then examined for bleeding which usually subsides quickly.
Because of the magnification of the telescope, 1 to 2 milliliters of blood may seem
excessive. Abnormal hemorrhage rarely occurs but if present can be controlled by
several methods. Some prefer using monopolar electrocautery while compressing
the liver tissue with the biopsy forceps. Electrocautery is reported to affect only the
periphery of the biopsy sample.2 A second technique is to place absorbable gelatin
coagulation material in the biopsy site. It is also possible to apply compression over
the bleeding area with forceps or the palpation probe.
Intrahepatic lesions observed using ultrasound may be more difficult to identify.
Deeper samples of the liver can be obtained using laparoscopic direction of a biopsy
needle. Needles are passed directly through the abdominal wall and can be guided to
the area to be sampled without the need for a cannula. Laparoscopic-guided chole-
cystocentesis can also be performed if inflammatory or infectious biliary tract disease
is suspected. A 22G long needle is used to collect bile for culture and cytology. The
needle is directed through the abdominal wall behind the diaphragm into the
gallbladder and the contents aspirated. It is important to remove as much bile as
possible to empty the gallbladder and prevent leakage when the needle is removed.
As previously stated it is important to biopsy areas that seem normal as well as
those that seem abnormal. Some investigators suggest that biopsies taken at the
edge of the liver often do not reflect deeper lesions and that the histopathology at
the subcapsular edge of the liver is usually more reactive.22 Others suggest that the
samples collected by laparoscopic cup biopsy are so large that this should not be
considered a major concern.
The complication rate of laparoscopy is low. In an unpublished review of a series of
cases involving diagnostic laparoscopy, the complication rate was less than 2%.
Serious complications include anesthetic- or cardiovascular-related death, bleeding,
or air embolism. Minor complications are generally operative and are associated
with inexperience or failure to understand the limitations and potential complications.

SURGICAL LIVER BIOPSY

Liver biopsy alone is rarely an indication for a laparotomy. However, there are indica-
tions for surgical procedures such as investigation of an extrahepatic biliary obstruc-
tion or for correction of a vascular anomaly. In these cases, a liver biopsy is always
indicated. Liver biopsies are also often obtained in conjunction with other surgical
 Liver Biopsy Techniques 477

procedures. A biopsy should be performed if the liver looks abnormal or abnormal liver
enzyme levels are discovered before surgery. It is recommended that a surgical liver
biopsy be taken early during the laparotomy because hepatocellular changes can
result from prolonged anesthesia, vascular changes, and manipulation of the
bowel.2,23
There are several techniques for obtaining liver tissue during a laparotomy. The
most commonly used method is the suture fracture technique. With this method,
samples are generally obtained from the tip of a liver lobe. A 5- to 6-mm skin biopsy
punch has also been described to sample focal superficial lesions. Once the core
sample is obtained with the punch, gelatin coagulation material is placed in the biopsy
site. Readers should refer to surgical texts for details of surgical methods of liver
biopsy.
The advantage of surgery is the exposure, ability to manipulate the tissues, and
ability to monitor the biopsy site for bleeding. A review comparing two 18G needle
Tru-Cut biopsy samples with a larger wedge biopsy found poor histologic correlation,
pointing out the advantage of larger surgical or laparoscopic biopsies.20 The extrahe-
patic biliary system can also be completely investigated with surgery and bile is easily
sampled through directed aspiration. The disadvantage of surgical biopsy is the need
for general anesthesia, the large abdominal incision, and the postoperative recovery

Fig. 1. Laparoscopic liver biopsy. Liver biopsies are taken with 5-mm oval biopsy forceps.
(A) View of a liver biopsy taken from the edge of a liver lobe; (B) view showing the liver
following the biopsy; (C) view showing a liver biopsy taken from the surface of the liver;
and (D) view showing the liver following the biopsy.
478 Rothuizen & Twedt

Fig. 2. Local anesthesia given for Menghini needle liver biopsy. The incision is made through
the skin and abdominal wall just 1 to 2 cm caudal to the xyphoid in the midline. The position
of the last rib is indicated.

time. The sample size of a biopsy obtained through surgery is the largest of any of the
methods described, providing more than adequate tissue for histopathology, copper
analysis, and culture. Bleeding can also be controlled easily using focal pressure,
suturing methods, or electrocoagulation.

GALLBLADDER PUNCTURE

Although this is not strictly a liver-sampling procedure, sampling of bile in all cases in
which inflammatory/infectious biliary disease is suspected, is an essential diagnostic
step.24,25 Puncture of the gallbladder can be performed safely using the ultrasound-
guided thin needle technique. There is no need to approach the gallbladder transhe-
patically; any approach is safe. Thin needle sampling does not lead to vagal reactions
and shock. Puncture of the gallbladder should be avoided in cases of extrahepatic bile
duct obstruction because of the risk of inducing rupture or bile leakage. Sampling of
bile for cytology and culture is especially important in cats, in which cholangitis is
a major hepatobiliary disorder.

Fig. 3. Typical liver tissue samples obtained with different biopsy devices. Top: a Tru-Cut
needle, which is usually driven by a biopsy gun. Half of the diameter of the inner needle
is available to collect the biopsy. Bottom: Menghini needle tip with the tissue sample aspi-
rated. The entire lumen of the needle is available to collect tissue; the sample is caught by
aspiration with saline while advancing the needle into the liver. Middle: a Vim-Silverman
needle, no longer in use.
 Liver Biopsy Techniques 479

SUMMARY

A liver biopsy is generally safe and provides useful information about the liver.
However, the importance of the information to be obtained from a biopsy must be
weighed against the risk to the patient. A liver biopsy provides important information
on the status of the liver. Only after the clinical information, liver biopsy, and histopa-
thology are obtained can a diagnosis and prognosis be made. With proper training and
adequate operator experience liver biopsy is an important diagnostic tool (Figs. 1–3).

REFERENCES

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small animal medicine. London: Saunders; 1999. p. 448–97.
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tology. Baillière’s Clinical Gastroenterology, vol 9, nr 4. London: Baillière Tindall;
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a quantitative reference standard. Hepatology 1998;28(2):323–31.
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logical diagnosis of canine and feline liver diseases. Edinburgh: Saunders; 2006.
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needle aspiration of the liver and cytologic findings in dogs and cats: 97 cases
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logic evaluation of liver biopsy samples of dogs with hepatic diseases. Vet Pathol
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ultrasound-guided biopsy techniques. Vet Radiol 1986;27:99–101.
20. Monnet E, Twedt DC. Laparoscopy. Vet Clin North Am Small Anim Pract 2003;33:
1147–63.
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22. Patrelli M, Scheuer PA. Variation in subcapsular liver structure and its significance
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24. Cole TC, Center SA, Flood SN, et al. Diagnostic comparison of needle and wedge
biopsy specimens of the liver in dogs and cats. J Am Anim Hosp Assoc 2002;
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See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/12337718

Technical note: A technique for multiple liver biopsies in neonatal calves

Article  in  Journal of Animal Science · October 2000

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 Technical note: a technique for multiple liver biopsies in neonatal calves
K. S. Swanson, N. R. Merchen, J. W. Erdman, Jr, J. K. Drackley, F. Orias, G. N. Douglas
and J. C. Huhn

J ANIM SCI 2000, 78:2459-2463.

The online version of this article, along with updated information and services, is located on
the World Wide Web at:
http://jas.fass.org/content/78/9/2459

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 Technical note: A technique for multiple liver biopsies in neonatal calves1

K. S. Swanson*, N. R. Merchen*†2, J. W. Erdman, Jr.*, J. K. Drackley*†,

F. Orias†, G. N. Douglas†, and J. C. Huhn‡3

*Division of Nutritional Sciences, †Department of Animal Sciences, and ‡Department of Veterinary Clinical
Medicine, University of Illinois, Urbana 61801

ABSTRACT: Our objective was to develop a rapid
and safe liver biopsy technique that could be repeated
on multiple occasions in individual neonatal calves. A
pilot study was performed to verify the efficacy of seda-
tion and restraint procedures and to evaluate different
biopsy instruments. Following the pilot experiment, a
biopsy trocar was fabricated and an experiment was
conducted using this procedure. Liver biopsies were
performed in neonatal calves on d 4, 9, 15, 21, and 28
of life to evaluate the effect of vitamin A intake on
liver vitamin A concentrations. On these days, a single
injection of ceftiofur sodium was administered i.m. 1 to
2 h prior to the procedure. Calves were lightly sedated
with xylazine and placed on a surgical table in left-
lateral recumbency. The right caudo-thoracic area was
clipped and scrubbed with an iodophor agent. Following
administration of a local anesthetic (lidocaine), a small
incision was made in the skin between the 12th and
13th ribs approximately 15 cm from the dorsal midline.
The biopsy trocar was inserted through the body wall
and peritoneum and introduced into the liver paren-
chyma, and a liver sample was collected. Following the
biopsy, the cutaneous incision was sutured and an anti-
septic agent was applied to prevent infection. An i.m.
injection of an analgesic was administered 1 h following
the procedure to alleviate postsurgical discomfort. Most
calves were able to stand within 2 h after the biopsy.
The entire procedure, which could be performed by a
single individual, usually required about 20 min from
initial sedation until skin closure. Although liver sam-
ples of up to 500 mg were obtained, most samples
weighed 75 to 150 mg (wet weight). A total of 156 liver
biopsies were performed on 33 calves. Complications
due to the biopsy procedure were observed in only two
calves. Therefore, this procedure can be useful for stud-
ies designed to monitor changes in liver composition or
enzyme activities over time.

Key Words: Biopsy, Calves, Liver

2000 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2000. 78:2459–2463

Introduction tration is the best indicator of vitamin A status in most

Liver samples obtained from live animals can be used
to help diagnose diseases or metabolic disorders and can
be used to determine liver concentrations of nutrients,
drugs, or other compounds without killing the animal.
Because the liver is the main storage organ for vitamin
A (approximately 90% of the body stores in vitamin A-
sufficient animals; Basu, 1996), liver vitamin A concen-
1
The authors wish to thank Nancy Bower, Robert Riggs, and Gene
McCoy for assistance in care of the animals in this experiment.
Thanks are also expressed to Amy Waggoner for her assistance with
the liver biopsy procedure.
2
Correspondence: 162 Animal Sciences Lab., 1207 West Gregory
Drive (phone: (217) 333-4189; E-mail: nmerchen@uiuc.edu).
3
Current address: Pfizer Central Research, Eastern Point Road,
P.O. Box 8200-40, Groton, CT 06340.
Received November 5, 1999.
Accepted April 12, 2000.
animals. A rapid and simple liver biopsy technique that
could be repeated in individual animals would be a
useful tool for monitoring vitamin A status over time.
Several techniques for liver biopsy have been published
(Hughes, 1962; Smart and Northcote, 1985; Buckley et
al., 1986), but these procedures were developed for
adult bovines. Our research group was interested in
a biopsy technique that could be used repeatedly on
neonatal calves to monitor vitamin A status over time
in response to differences in vitamin A intake. Biopsy
instruments developed in previous experiments were
too large and were not acceptable for use in small calves.
In addition, many procedures did not use sedatives or
anesthetics or did not report on their use; this was
desired in our study for ethical reasons and to ensure
adequate restraint of the animals. Therefore, the objec-
tive of this study was to develop a rapid and uncompli-
cated liver biopsy technique that could be repeated on
multiple occasions in individual neonatal calves.

2459

Downloaded from jas.fass.org by guest on January 7, 2012

2460 Swanson et al.
Materials and Methods
Selection of a Biopsy Instrument
Liver biopsies performed on adult cattle are usually
done in the standing position between the 11th and
12th ribs (Hughes, 1962; Smart and Northcote, 1985;
Buckley et al., 1986). However, we expected to sedate
and restrain the calves in our work in lateral recum-
bency. It was unknown whether the location of the liver
and surrounding organs would differ depending on posi-
tion. It was also necessary for us to verify that we could
obtain a moderate sample size (100 to 150 mg) of liver.
In addition, gross evaluation of the liver from animals
subjected to repeated liver biopsies was needed to as-
sess whether any instruments tested resulted in seri-
ous damage.
All procedures used in these experiments were de-
scribed in protocols that were submitted to the Univer-
sity of Illinois Laboratory Animal Care Advisory Com-
mittee and approved before the work was initiated. A
pilot experiment to verify the efficacy of our sedation
and restraint procedures and to evaluate different bi-
opsy instruments was conducted. Three Holstein bull
calves (1 to 2 wk of age, 47 ± 6 kg) were used. For
the pilot experiment, calves were transported from the
Dairy Research Farm to the Large Animal Clinic at the
College of Veterinary Medicine before the first biopsy.
Calves were housed together in the Laboratory Animal
Care Facility of the College of ACES between biopsies.
Sedation, anesthesia, presurgical preparation, and
postsurgical care of the calves were identical to proce-
dures described in a following section.
Transcostal ultrasound guidance was used to verify
the location of the liver and liver samples were taken
on three occasions (1-wk intervals) in each of three
calves. Three different biopsy instruments were tested
in each calf at each sampling. After the third biopsy,
one calf was euthanatized by barbiturate overdose and
necropsy was performed. Examining the location of the
liver in the euthanatized calf allowed us to more pre-
cisely define anatomical landmarks that would allow
rapid sampling of the liver with minimal abdominal
complications. Gross postmortem examination of the
liver allowed visual evaluation of the instruments with
regard to procedural trauma inflicted by the different
biopsy instruments.
One of the instruments evaluated was a 14-gauge
Precision Cut biopsy needle (Becton Dickinson Co.,
Rutherford, NJ), which measured 2.1 mm × 15.2 cm.
This instrument was advantageous in that it was easy
to handle, it easily and rapidly penetrated the perito-
neum and liver, and it caused very little visible external
trauma to the liver. However, due to the very small
sample size (10 to 15 mg) that could be obtained with
this instrument, it was not chosen for use in further ex-
periments.
Another instrument tested was an ethmoid rongeurs
(R. Wolfe Medical Instrument Corp., Rosemont, IL)
Downloaded from jas.fass.org by guest on January 7, 2012
commonly used as an arthroscopic surgical instrument.
The instrument consisted of blunt-ended opposing cups
(10 × 4 mm) with cutting edges. The jaws of the instru-
ment were hinged and operated in scissor-like fashion.
With the blunt tip of the instrument held lightly against
the parietal surface of the liver, the operator opened
the jaws and then closed them to obtain a biopsy “bite.”
This procedure also could be performed rapidly, and
the instrument was easily handled. Sample sizes were
approximately 20 to 50 mg. However, distinct disadvan-
tages of this instrument were observed. Because this
tool had a blunt end, an independent incision in the
peritoneum was required for abdominal entry. This in-
creased bleeding and postsurgical recovery time. Other
disadvantages were that it was difficult to detect paren-
chymal penetration with this instrument and it caused
substantial gross damage to the liver and its capsule.
For these reasons, this instrument was not considered
for further use.
The instrument chosen for use in future work was a
biopsy trocar fabricated from the design of Hughes
(1962) that was intended for use in adult cattle. This
instrument was composed of three main parts. A stain-
less steel trocar fit snugly within a stainless steel can-
nula and a neoprene “O” ring, which was located near
the base of the cannula. The cannula had a length of
approximately 31 cm, an o.d. of 9.5 mm, and an i.d. of
8 mm. The trocar had a length of approximately 34 cm
with a diameter that fits snugly into the cannula. The
“O” ring was used to form a seal against the trocar,
which created a vacuum within the cannula when the
trocar was withdrawn. The base of the trocar had a
knurled end for gripping with the fingers while per-
forming the procedure (Hughes, 1962). As with the Pre-
cision Cut instrument, only a skin incision was required
because the trocar readily penetrated the body wall and
peritoneum with minimal resistance.
The operation of the trocar was more unwieldy than
that of either the Precision Cut biopsy needle or the
ethmoid rongeurs. Gross examination of the liver re-
vealed that the biopsy trocar seemed to cause more
damage than the Precision Cut needle but less than
the ethmoid rongeurs. The main advantage of the trocar
was that comparatively large liver samples of approxi-
mately 50 to 300 mg could be obtained. The main disad-
vantage of the trocar was that it was more difficult to
operate and control than the other two instruments
tested. However, this instrument was still relatively
simple to use and was chosen for future experimenta-
tion because of the larger quantity of liver sample ob-
tained and consequent minor trauma to the liver.
Design of the Trocar
Because the biopsy trocar described above was de-
signed for use in adult cattle, a smaller version was
fabricated by personnel in the Machine Lab of the Uni-
versity of Illinois Division of Operations and Mainte-
nance. Both the trocar and cannula were made from
 Multiple liver biopsies in neonatal calves
stainless steel. The cannula was approximately 17 cm
in length with an o.d. of 7 mm and an i.d. of 5 mm. The
trocar was approximately 20 cm long and fit snugly
inside the cannula (Figure 1).
Surgical Procedures and Operation of the Trocar
Following the pilot experiment and fabrication of the
biopsy trocar, an experiment was conducted investigat-
2461
ing the effect of vitamin A intake on liver vitamin A
concentrations in preruminant Holstein bull calves
(Swanson, 1999). As part of this experiment, a total
of 156 liver biopsies were performed in 33 calves. All
surgical instruments were sterilized by autoclaving
(121°C, 15 psi, minimum 20 min) prior to use. A single
injection of 2 mg/kg BW ceftiofur sodium (Fort Dodge
Animal Health, Fort Dodge, IA) was administered i.m.
1 to 2 h prior to the procedure. Calves were placed

Figure 1. The unassembled (a) and assembled (b) biopsy trocar used in the experiment.

Downloaded from jas.fass.org by guest on January 7, 2012

2462
on a surgical table in left-lateral recumbency following
mild xylazine (Bayer Corp., Shawnee Mission, KS) se-
dation (0.01 to 0.05 mg/kg BW i.m.). Once on the table,
the legs of the calf were restrained. The right caudo-
dorsal thoracic area was clipped and scrubbed using an
iodophor detergent (Aaron Medical Ind., St. Petersburg,
FL) followed by an anti-infective agent prior to the bi-
opsy procedure. Local anesthetic (1 mL lidocaine; Ab-
bott Laboratories, N. Chicago, IL) was used to desensi-
tize skin and underlying body wall. A small incision (1
to 2 cm) was made in the skin parallel to the ribs and
approximately 15 cm from the dorsal midline. The inci-
sion was made between the 11th and 12th ribs and was
just large enough to admit the trocar. The trocar and
cannula were inserted through the peritoneum between
the 11th and 12th ribs. Once the trocar point contacted
the visceral surface of the liver, the trocar was retracted
4 to 5 cm, and the cannula was advanced 2 to 3 cm into
the liver tissue using a circular motion. The tip of the
cannula was beveled inward to form a sharp cutting
edge, allowing easy penetration of the liver. Following
the liver penetration, the trocar was retracted an addi-
tional 2 to 3 cm to produce a partial vacuum, allowing
the sample to be drawn into the cannula. The instru-
ment was then withdrawn from the abdominal cavity,
and the sample was deposited in a sterile vial and im-
mediately placed on ice.
Although the 11th intercostal space was used as the
incision site for most biopsies, it was sometimes neces-
sary to penetrate the 10th or 9th spaces as the calves
became older in order to obtain an acceptable sample.
Samples of as much as 500 mg (wet weight) of liver
were obtained from one biopsy, although most samples
weighed 75 to 150 mg (wet weight). If an inadequate
amount of liver was obtained during the first attempt,
another liver sample was obtained immediately after-
ward. As many as five liver samples were taken during
a given session to obtain an adequate amount of tissue.
Following the procedure, the incision was closed with
a simple interrupted suture, and an antiseptic agent
(Red-Kote; H. W. Naylor Co., Morris, NY) was applied
to prevent infection. Banamine (Flunixin Meglumine;
1 mg/kg BW; Schering-Plough Animal Health Corp.,
Kenilworth, NJ) was given by i.m. injection 1 h follow-
ing the procedure to alleviate postsurgical discomfort.
The entire procedure usually required about 20 min
from the time of the xylazine injection to the time of
skin incision closure. Most calves were able to stand
within 2 h after the biopsy.
Results and Discussion
One hundred fifty-six liver biopsies were performed
on 33 neonatal Holstein bull calves to monitor vitamin
A status over time. Liver biopsies were performed on
d 4, 9, 15, 21, and 28 after birth. Because the calves
were sedated, they were very calm and easy to handle
throughout the procedure. This reduced the chance of
accidental puncture of other organs by the biopsy in-
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Swanson et al.
strument, which could occur with struggling animals.
After gaining sufficient experience and developing pro-
ficiency in the technique, a single individual was able
to perform the surgery. A single individual performed
approximately 50% of the biopsies in the trial.
Thirty-five calves were originally allotted to the
study. Calves were housed individually in outdoor
hutches as per normal management of dairy calves.
Calves received two meals in equal amounts per day.
Calves were fed 10% BW/d during the 1st wk of life and
12% BW/d for wk 2 to 4. Milk replacer was reconstituted
with water to contain 12.5% solids. Five calves were
eliminated from the study before they reached d 28
after birth. Two calves were dropped from the study
within 48 h after birth due to development of atresia
coli. Because these calves were excluded from the exper-
iment prior to d 4, the liver biopsy technique was not
performed on these calves.
One calf was removed from the experiment on d 20
and was euthanatized, and a necropsy was performed.
The necropsy report indicated that the calf had a mesen-
teric abscess, an umbilical vein abscess, and diffuse,
severe peritonitis with multiple adhesions localized
near the jejunum. The necropsy report stated that the
cause of the illness of this calf was probably due to
the umbilical vein abscess, which led to the mesenteric
abscess and peritonitis. Thus, the illness of this animal
probably was not a result of complications arising from
the biopsy procedure. The biopsy procedure had been
performed on this calf three times before it became ill.
Another calf was euthanatized on d 12 of life (d 8 of
experiment) due to illness. The necropsy report noted
that the calf was suffering from enteritis, but no specific
cause (viral, bacterial, parasitic) was identified. Immu-
nological tests suggested a failure of passive transfer
of colostral antibodies in this calf. This calf had under-
gone the liver biopsy procedure once before becoming ill.
A fifth calf was euthanatized on d 14 of life (d 10 of
experiment) and a necropsy was performed. The nec-
ropsy report stated that the cause of illness was diffuse,
severe peritonitis, which was possibly induced by the
biopsy procedure. Thus, of five calves that had to be
removed from the study, only one seemed to have com-
plications that were attributable to the biopsy pro-
cedure.
One calf developed a pneumothorax from the biopsy
procedure; however, this condition did not necessitate
removal of the calf from the study. During the second
biopsy on this calf (d 13 of life), the diaphragm was
punctured and ambient air entered the pleural space.
This calf was not treated but was closely monitored for
further complications throughout the study. Although
this calf demonstrated labored respiration throughout
most of the remainder of the experiment, appetite,
growth, and demeanor were not affected. The calf’s res-
piration had normalized by d 28.
There was no positive control (unbiopsied) group of
calves included to compare effects of the biopsy proce-
dure on growth performance. However, the 30 calves
 Multiple liver biopsies in neonatal calves
that completed the experiment from d 4 to 28 seemed
to grow at rates representative of calves fed only this
amount of commercial milk replacers (12% BW/d). Total
weight gains from d 4 to 28 for all 30 calves that com-
pleted the study averaged 11.0 kg or 458 g/d (Swan-
son, 1999).
With any surgical procedure, secondary complica-
tions that lead to illness or death are always a risk.
For example, in the procedure developed by Hughes
(1962), a single liver biopsy was performed on 500 cattle
and three deaths resulted from blood loss caused by
penetration of hepatic portal blood vessels.
Conclusions
The liver biopsy technique described in this report
was successful in allowing multiple liver samples to be
obtained from individual calves between d 4 and 28
after birth. The procedure can be performed in approxi-
mately 20 min by a trained individual and can be per-
formed by a single person. The ease of handling a se-
dated calf during the biopsy procedure and the reduc-
tion in discomfort to the animal justify the use and cost
of drugs. Sedation also reduces the chance of accidental
damage to other organs during the biopsy, which may
occur with struggling animals. More than 150 biopsies
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2463
were performed on 33 animals. Complications related
to the biopsy procedure were observed in only two
animals.
Implications
A biopsy technique was developed to allow for fre-
quent and multiple liver samples to be collected from
individual preruminant calves. This procedure can be
useful for studies designed to monitor changes in liver
concentrations of nutrients or other compounds, gene
expression, or enzyme expression or activity over time.
Literature Cited
Basu, T. K. 1996. Vitamin A. In: T. K. Basu and J. W. T. Dickerson
(ed). Vitamins in Health and Human Disease. pp 148–177. CAB
International, Wallingford, U.K.
Buckley, W. T., G. K. Eigendorf, and W. J. Dorward. 1986. A liver
biopsy instrument for large animals. Can. J. Anim. Sci.
66:1137–1140.
Hughes, J. P. 1962. A simplified instrument for obtaining liver biops-
ies in cattle. Am. J. Vet. Res. 23:1111–1112.
Smart, M. E., and M. J. Northcote. 1985. Liver biopsies in cattle.
Compend. Contin. Educ. Practicing Vet. 7:S327–S332.
Swanson, K. S. 1999. Influence of dietary vitamin A level on serum
and liver vitamin A concentrations in the pre-ruminant calf.
M.S. thesis. Univ. of Illinois, Urbana-Champaign.
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