ABSTRACT

Objectives: Biopsy quality is an essential factor for successful diagnosis of canine liver disease.

Several minimally invasive methods to obtain diagnostic liver biopsies have been described. The aim

of this study was to compare weight and volume of the samples, surgical time required, possible

complications and histological quality between liver biopsies obtained laparoscopically with a pre-tied

ligating loop (PLL) and cup biopsy forceps (CBF).

Materials and Methods: Fifteen client owned dogs underwent laparoscopic liver biopsies for

diagnosis of liver disease. Biopsies were obtained from the same liver lobe using a PLL and CBF. The

resulting biopsies were evaluated for; weight, volume, histological value and the surgical time

required and compared. Any surgical complications were recorded.

Results

Samples obtained with the PLL were significantly heavier and larger in volume than those obtained

with CBF. Samples obtained with the PLL contained significantly more portal tracts and less crush

and fragmentation artefact than the ones obtained with CBF. The duration required to obtain a liver

biopsy with the PLL was approximately double of that of the CBF.

Clinical significance

The use of a PLL is a good alternative technique to the CBF when performing laparoscopic liver

biopsies in dogs.

KEYWORDS: Soft Tissue-General, Laparoscopy, Pre-tied ligating loop, Liver biopsy, Hepatic

biopsy.

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INTRODUCTION

Liver biopsy is integral in the diagnosis, treatment and assessment of prognosis of hepatic disease

(Rockey et al. 2009, Rothuizen and Twedt 2009). However, each available liver biopsy method

presents with specific limitations and risks. The main methods available are fine needle aspiration,

ultrasound guided core needle biopsies, laparoscopy, and open abdominal surgery.

Fine needle aspiration and core needle biopsy are the less invasive and inexpensive options available.

Fine needle aspiration can be diagnostic in some neoplastic processes and vacuolar disorders,

however, cytology does not allow appreciation of structural changes in the liver (Rothuizen and

Twedt 2009). Furthermore, agreement between histopathological diagnosis obtained with needle

biopsy and cytological diagnosis was only found in 30.3% of canine cases and 51.2% of feline cases

in one study (Wang et al. 2004).

Ultrasound guided core needle biopsies are minimally invasive and are usually performed without the

need of general anaesthesia or specialised equipment other than the needle. A study comparing needle

biopsies with surgical wedge biopsies identified agreement in morphological diagnosis in 56-67% of

the paired samples. This study concluded that needle biopsy specimens of the liver of dogs and cats

must be interpreted with caution (Cole et al. 2002). Transjugular liver biopsy is a technique

commonly used in human patients but only one cadaveric study has been published in dogs with a

high complications rates reported (Levien et al. 2014).

Laparoscopy is a minimally invasive method which allows direct visualisation of the liver whilst

obtaining biopsies and resulting in less postoperative pain (Devitt et al. 2005) and a faster return to

normal activity (Culp et al. 2009) in comparison to an open approach. The traditional sampling

instrument used in laparoscopic liver biopsies are the cup biopsy forceps (CBF), which provide a

sample of a maximum volume depending on the size of the forceps. The size of the collected sample

is important and recommendations in the human literature state that it should contain a representative

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amount of parenchyma including more than 11 portal tracts (Rockey et al. 2009). Furthermore, a

study in human patients showed a correlation between fragment size and number of portal tracts and

found that advanced fibrosis was linked to the representation of less than 11 portal tracts (Coral et al.

2016).

Biopsy quality is an essential factor for successful diagnosis of liver biopsies. In the veterinary

literature, two previous studies looking at the quality of canine hepatic biopsies reported only 8-13

portal tracts in one study (Petre et al. 2012) and a mean of 3.4 portal tracts in another (Kemp et al.

2015b) in biopsies obtained with CBF. The latter study also compared methods and found no

statistical significant difference in the number of portal tracts between samples taken with the CBF

and with core needle biopsy (Kemp et al. 2015b). The size of the sample has also shown to affect the

concentration of metal detected and being statistically significantly lower in the core needle biopsy

method in comparison to wedge biopsy specimens in canine livers due to the smaller size of core

needle biopsies (Johnston et al. 2009). Therefore, there are limitations with all the techniques

available and there is scope for better techniques.

A pre-tied ligating loop (PLL) has been previously used to perform liver lobectomies and liver

biopsies in open surgery with low perioperative complications (Cuddy et al. 2013, Goodman and

Casale 2014). This technique allows collection of a biopsy sample of variable size depending on the

amount of tissue incorporated in the loop. PLLs are available for use in laparoscopy and have been

previously used for laparoscopic ovariectomy in horses (Boure et al. 1997) and sheep (Barros et al.

2015), orchiectomy in abdominal cryptorchidism in dogs (Oviedo Peñata and Hernandez Lopez 2013)

and horses (Fischer and Vachon 1998) and also for thoracoscopic and thoracoscopic-assisted lung

lobectomy in dogs (Faunt et al. 1998, Pramatwinai et al. 2006) and horses (Relave et al. 2008).

However, the use of a PLL to obtain laparoscopic liver biopsies has never been reported in dogs.

In the authors’ experience, the sample obtained with CBF can contain less than 11 portal tracts and

show marked crush and fragmentation artefact, making histological diagnosis challenging.

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Furthermore, the amount of tissue obtained in one cup is often insufficient for copper analysis and

extra biopsies need to be obtained for this purpose.

We hypothesise that the PLL is a method as safe as the CBF to obtain laparoscopic liver biopsies in

dogs with similar complication rates than previously reported. In addition, we hypothesise the volume

and weight of the samples collected with the PLL will be statistically significantly larger and of better

histological quality (i.e. higher number of portal tracts and reduced frequency of sampling artefacts),

than biopsies obtained with CBF from the same liver lobeThe aim of this study was to compare the

surgical time, weight and volume of the biopsies, possible complications and histological quality of

the samples obtained between laparoscopic PLL and CBF.

MATERIALS AND METHODS

Animals and inclusion criteria

Based on a power of 0.80 and an alpha error of 0.05 to detect an estimated difference of 25% in tissue

sample volume, the target sample size was a minimum of 14 cases. Seventeen dogs with suspected

diffuse hepatopathy were originally recruited in the study; however, two dogs were excluded because

a large solitary liver mass was identified on laparoscopy and the procedure was converted to an open

coeliotomy and liver lobectomy. Finally, fifteen privately owned dogs undergoing laparoscopic liver

biopsies for suspected diffuse liver disease between January 2016 and December 2016 were enrolled

in the study. Informed consent was obtained from the owners and the project was accepted by the

Veterinary Ethical Research Committee (VERC 68/15). Seven of the 15 cases underwent gallbladder

bile aspiration as an additional procedure at the time of surgery.

Previous investigations

All cases underwent diagnostic tests prior to the procedure including haematology, serum

biochemistry, coagulation panel, endocrine disease screening tests, abdominal ultrasound and fine

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needle aspiration. All dogs had a platelet count, prothrombin time (PT) and partial thromboplastin

time (PTT) performed within two weeks prior to the procedure.

Anaesthetic protocol

Dogs were premedicated according to the anaesthetist preference. Induction was performed with

either propofol (Propoflo Plus, Zoetis) or alfaxolone (Alfaxan, Jurox) to effect. General anaesthesia

was maintained with isoflurane (IsoFlo, Abbott) or sevoflurane (SevoFlo, Abbott) in oxygen. Intra-

operative analgesia was selected according to the preferences of the attending anaesthetist.

Perioperative intravenous antibiotics were administered at induction or after obtaining a biopsy for

bacterial culture using cefuroxime (Zinacef, GlaxoSmithKline) (20 mg/kg).

Surgical technique

Dogs were placed in dorsal recumbency and the ventral abdominal hair was clipped from the xiphoid

to the pubis. The area was aseptically prepared and draped as for a ventral midline coeliotomy. In all

cases, the surgical procedure was performed by the main author as the primary surgeon. Surgical

assistance was provided by either a board-certified surgeon (n=3), another surgical resident (n=10) or

an undergraduate veterinary student (n=2).

A modified Hasson technique (Humphrey and Najmaldin 1994) was used to introduce a 6-mm trocar-

cannula (Storz) assembly 1-1.5cm caudal to the umbilicus. If ascites was present, a Poole suction tip

was introduced through the incision and the peritoneal fluid was suctioned prior to insufflation. The

abdomen was insufflated to 8 to 12 mm Hg with carbon dioxide by means of a pressure regulating

mechanical insufflator (Storz). A 5 mm 0 or 30 telescope (Storz) was introduced. A second 6 mm

blunt trocar-cannula assembly was placed under direct visualisation through an incision, also

performed with an 11 scalpel blade, in the left or right cranial abdominal quadrant. A third, 11 mm

blunt trocar-cannula assembly was placed on the contralateral side under direct visualisation through

an incision performed with an 11 scalpel blade. The abdomen and liver were explored with the aid of

a blunt probe and any abnormalities were recorded. Selection of the liver lobes from which biopsies

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were collected was based upon; the presence of local or diffuse macroscopic abnormalities, the

available access and the potential risks associated with the localisation of the lesion within the liver. A

cup biopsy sample and a wedge biopsy, performed with the PLL, were obtained from the same liver

lobe as detailed below. The order in which the biopsies were obtained was randomised using a toss of

a coin online programme (https://www.random.org/coins/).

After the biopsies were obtained, the biopsy sites were immediately observed for haemorrhage.

Haemorrhage associated with each procedure was recorded as none, mild, moderate or severe. Mild

haemorrhage was defined as not needing any intervention, moderate haemorrhage was recorded when

a collagen haemostatic agent (Lyostypt, B. Braun Medical Ltd) or a second PLL was needed and

severe haemorrhage was recorded when the procedure was converted to an open coeliotomy or when

blood products were administered as a result of haemorrhage.

The abdomen was actively deflated prior to closure. All wounds were closed in three layers using

monofilament absorbable suture material (PDS, Ethicon). The skin was sutured with an intradermal

pattern using monofilament absorbable suture (Monocryl, Ethicon).

The total surgery time was recorded, as well as specific times for: time to establish the two 6 mm

ports, time to establish the 11 mm port, time for observation of the abdomen, time to obtain the

gallbladder bile aspirate (n=7), time to harvest biopsies using CBF, time to harvest a wedge biopsy

with the PLL, time to observe for haemorrhage, time for closure of the two 6 mm wounds and time for

closure of the 11 mm wound.

Cup biopsy technique (CBF)

Five mm CBF (Storz) were used to obtain biopsies from different liver lobes. The number of biopsies

depended on the estimated tissue required for diagnosis. The biopsy forceps were introduced into the

liver with the jaws in the open position. The liver lobe was lifted ventrally, away from the other

abdominal organs with the bottom jaw. The forceps were advanced towards the liver and closed to

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grasp the biopsy. The forceps were held in this locked position for approximately 15 seconds (Twedt

and Monnet, 2003) before the laparoscopic cannula was advanced over the forceps until contacting

the liver. At this point, the forceps were withdrawn, pulling the tissue sample from the liver. This

technique prevented tearing of the liver tissue adjacent to the biopsy site.

All the biopsies obtained were weighed and measured in three dimensions (length, width and height)

in a sterile manner by the main author (NF) with the same scales and ruler. One of the samples from

the same liver lobe as the PLL sample was always submitted for histological analysis. Any remaining

cup biopsies were submitted for bacterial culture or added for mineral analysis depending on the

analytical requirements of the case.

Pre-tied Ligating Loop wedge biopsy technique (PLL)

A 3-0 polydiaxonone pre-tied suture loop (Endoloop, Ethicon) was introduced through the lateral

6mm cannula. A pair of endoscopic Babcock (Storz) forceps were introduced from the contralateral

side (11mm cannula) and passed through the pre-tied loop (Figure 1). The liver lobe was grasped and

lifted ventrally with the endoscopic Babcock forceps. The pre-tied loop was passed around the end of

the liver lobe and the suture was pulled tight whilst advancing the plastic applicator to create a wedge

biopsy using the suture fraction or ‘guillotine’ technique. The suture end was then cut with

endoscopic scissors through the same cannula by which it was introduced and then the scissors were

used to cut the liver parenchyma approximately 5 mm distal to the suture to allow a decent cuff of

tissue to prevent suture slipping. The biopsy was followed with the camera to ensure it could be safely

retrieved through the 11mm cannula.

All the biopsies obtained were weighed and measured in the same way as described for the CBF

biopsies. The wedge obtained with the PLL was divided with a scalpel blade in two portions

macroscopically similar in size and the two portions were weighed and measured again. One of the

portions was submitted for histological analysis and the other for mineral analysis.

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Histological preparation for analysis

The samples for histopathology were fixed in 10% neutral buffered formalin for at least 24 hours. All

samples were trimmed in the same manner (i.e. longitudinally to a slice thickness of 2-3mm), and then

processed for histology and haematoxylin and eosin staining using standard protocols. The sections

were then assessed for total cut area of tissue available, number of portal tracts, degree of crush and

fragmentation artefacts, subjective diagnostic value and histological diagnosis. The score used for

both crushing and fragmentation artefacts had four values, ranging from 0-absent to 3-severe (Figure

2).

Postoperative period and follow-up

Postoperative analgesia was provided with intravenous methadone (Comfortan, Eurovet Animal

Health) boluses (0.1-0.2 mg/kg) every 4 hours for the first 8h and intravenous buprenorphine

(Buprecare, Animalcare) boluses (20 µg/kg) q 8 hours until discharge.

All dogs were hospitalised for a minimum of 12 hours and the hospitalisation time was recorded.

Dogs discharged from the hospital were prescribed a five to seven-day course of oral tramadol

(Bristol Laboratories Ltd) (2-3 mg/kg, p.o., q 8-12h). Dogs requiring a change in the analgesia plan

were recorded. An Elizabethan collar was placed at time of recovery from anaesthesia, and all dogs

were discharged with it.

Dogs that survived to hospital discharge were examined 10-14 days postoperatively. The wounds

were examined and a questionnaire was completed by the consulting veterinarian (ECVS resident)

(appendix 1). The questionnaire used was a modification from the questionnaire used for surveillance

of surgical site infections (SSI) in people, provided by the Centre of Disease Control and Prevention

(CDC) (www.cdc.gov).

Further follow-up was performed four to six weeks after the procedure by routine rechecks or by

telephone contact of the owners or referring veterinarian.

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Complications during the follow-up period were recorded and classified as minor or major.

Complications treated with medical management or dressings were considered minor and

complications requiring a second intervention were considered major. Survival time after surgery was

also recorded for dogs that died or were euthanized in the study period.

Statistical analysis

Descriptive statistics were calculated for all recorded variables (categorical and continuous).

Statistical differences of continuous variables between groups were assessed with a paired t-test for

normally distributed variables and a 1-sample Wilcoxon Signed Rank test for ordinal variables. The

significance threshold was set at p<0.05. All the data was collated and described in Excel (2006,

Microsoft, US), and statistical analysis were conducted in Minitab Express statistical software

(version 1.5.0, Minitab).

RESULTS

Animals

The mean body weight of the dogs in the study was 21.5 (±11.3SD) Kg, and the breeds included

Mixed-breed dogs (n=4), Tibetan terriers (n=2), Labradors (n=2), and one of each Shih Tzu, Border

Collie, German Shepherd, West Highland White Terrier, Bichon Frisé, Dalmatian and Staffordshire

Bull Terrier. There were six males and nine female dogs and the mean age was 6.7 (±3.2) years.

Five (33.3%) of 15 dogs had a low platelet number (<200 x 109/l) with a mean platelet count of 142.0

(±33.7) x 109/l. Two different analysers were used to measure prothrombin time (PT) and activated

partial thromboplastin time (aPTT). Only one dog showed a mildly increased PT at 16.9 s (ref. 5-12 s)

and this dog received an intraoperative fresh frozen plasma transfusion.

Laparoscopy

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The mean time to obtain 2-3 biopsies with CBF was 8.3 (±3.7) minutes, whereas the mean time to

obtain a biopsy with a PLL was 22.4 (±13.3) minutes. The time to obtain one biopsy with a PLL was

significantly longer than the time to obtain 2-3 biopsies with the CBF (Table 1). The mean total

surgery time was 78 (±24.1) minutes. Placement and closure of the first and second ports (6 mm) took

a mean time of 11.3 (±5.8) and 11.6 (±3.56) minutes respectively and placement and closure of the

third port (10mm) took a mean time of 2.8 (±1.6) and 5.2 (±1.57) minutes respectively. A mean time

of 5.4 (±2.3) minutes was used for initial observation of the abdomen and a mean time of 4.33 (±4.81)

minutes was used to observe for haemorrhage once the biopsies were obtained. Finally, gallbladder

bile aspiration was performed in a mean time of 14.1 (±10) minutes (n=7).

There were no major intraoperative or postoperative complications, and biopsies were successfully

obtained with the PLL and the CBF in all cases. The biopsies submitted for histopathology were

obtained from the left lateral liver lobe (n=6), left medial lobe (n=5), right medial lobe (n=2), right

lateral (n=1) and quadrate lobe (n=1). Three cases required a second PLL; in one case the ligature was

cut with scissors inadvertently whilst obtaining the biopsy and a second loop was used to control the

haemorrhage and in the two other cases the first loop was tightened before placing it around the liver

lobe and could not be re-used.

Only one of 15 dogs presented with ascites and the minimum amount of fluid needed was aspirated to

allow adequate visualisation. Mild haemorrhage was recorded in all biopsies obtained with CBF. No

haemorrhage was recorded in 14 of 15 biopsies obtained with the PLL, and moderate haemorrhage

was recorded in the case in which the PLL was cut inadvertently. No cases required conversion to

open approach.

The weight and volume of the biopsies obtained with the PLL were on average more than three times

higher than those obtained with CBF, irrespectively if considered total sample or sample submitted

for histology (p<0.001, Table 1).

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A total of 13 of 15 dogs survived to hospital discharge. The mean hospitalisation time for the dogs

that survived was 1.2 (±0.4) days. Only one dog required rescue analgesia two days after surgery and

the frequency of administration of tramadol was increased to three times daily and subcutaneous

maropitant (Cerenia, Pfizer) (1mg/kg) was administered in case the clinical signs were secondary to

nausea rather than pain.

Biopsy evaluation

The PLL method resulted in a greater area available for histological analysis compared to the CBF

(median 108 vs 26 mm2, p=0.0004, Table 1) with a greater number of portal tracts obtained (median

69 vs 19, p <0.001, Table 1). Four of 15 (26.7%) of the samples obtained with the CBF contained ≤11

portal tracts and all the samples obtained with the PLL contained >11 portal tracts. In addition, there

was reduced crush and fragmentation artefact with PLL samples compared to CBF (p<0.038, Table

1).

All the samples, independent of the method used, were diagnostic. The diagnoses included chronic

hepatitis with fibrosis (n=6), chronic hepatitis (n=2), steroid hepatopathy (n=2), subacute hepatitis

(n=1), fibrosis and biliary hyperplasia (n=1), chronic cholestasis and biliary hyperplasia (n=1),

hepatocellular swelling (n=1) and normal liver (n=1).

Complications and follow-up

Eight dogs were presented to the scheduled recheck appointment 10-14 days after surgery. Three of

the eight dogs had minor wound complications. Two cases showed dehiscence of the 6 mm side

wound and one case showed dehiscence of the wounds on both sides (11 mm and 6 mm wounds).

None of the dogs that developed wound complications wore the Elizabethan collar given to the owner

at discharge. Bacterial culture was performed in two of the three cases which developed wound

complications. One yielded a mixed growth of E. coli and Pasteurella sp and no growth was reported

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in the second one. Both cases received oral co-amoxiclav (15-20 mg/kg, q 12h) for 10 days. All three

cases were also managed with wound dressings.

Of the five remaining dogs, which were not presented to the scheduled recheck appointment, no

wound complication was reported by the owners on follow-up by telephone contact or on routine

recheck appointment with the Internal Medicine Service.

In total, four dogs (26.6%) were euthanased during the study period due to disease progression.

Follow-up time for the rest of the dogs was 26-254 days.

DISCUSSION

The results of the present study confirm that using a PLL is a feasible and safe method to obtain

laparoscopic liver biopsies. In addition, the hepatic sample obtained is significantly larger and

contains significantly more portal tracts and less artefacts than those obtained with CBF from the

same liver lobe.

The size of the sample obtained by traditional open coeliotomy is the largest of any of the methods

described in the literature, providing more than adequate tissue for histopathology, copper analysis

and culture (Rothuizen and Twedt 2009). The sample obtained with the PLL via laparoscopy shows

similar characteristics to biopsies obtained in open surgery with the guillotine technique with a sample

significantly larger than that obtained with CBF and enough tissue to perform histopathology,

bacterial culture and copper analysis for comprehensive evaluation of liver disease. Conversely, the

total amount of tissue obtained with CBF in this study would have not been enough to perform all the

tests required (i.e. quantitative copper analysis). However, for ethical reasons, a smaller number of

hepatic biopsies were obtained with CBF as the aim was not to remove more liver tissue than required

between the two methods.

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The PLL technique for laparoscopic hepatic biopsies has been described previously in a surgical

textbook and in a review on diagnostic laparoscopic techniques in cats (Robertson et al. 2014a,

Robertson et al. 2014b). In both, its use is recommended for animals with increased risk of

haemorrhage such as advance hepatic failure, focal or highly vascular lesions and coagulopathies.

None of the dogs in the current study had a clinically significant coagulopathy and comparing the

degree of haemorrhage between the two methods in dogs with and without coagulopathy in detail was

beyond the scope of the study. However, less haemorrhage was noted when using the PLL in

comparison to the CBF.

In this study, both techniques were used in dogs of a wide range of body sizes and different

histological diagnoses. The PLL was easier to use in dogs with fibrosis as the tissues were more

resilient to handling. On the contrary, the technique was slightly more challenging in dogs with a

friable liver or those with rounded liver edges.

A recent study concluded that the likelihood of obtaining a sample that represents the predominant

histological diagnosis is increased when multiple liver lobes were biopsied. (Kemp et al. 2015a). They

reported that when one liver lobe was sampled, the sample would reflect the prevalent histologic

diagnosis in 92% of the cases, whereas when two liver lobes were sampled, the percentage increased

to 98%. The previous study was published after all the data collection had completed for the current

study with only one liver lobe sampled with the two different techniques. Therefore, we have not

included this circumstance in our experimental design.

Despite all samples being considered of diagnostic value, 33% of the biopsies obtained with the cup

forceps contained less than eleven portal tracts as recommended in the human literature (Rockey et al.

2009). Conversely, all the biopsies obtained with the PLL had more than 18 portal tracts and showed a

significantly less crush and fragmentation artefact making the PLL method a superior method to the

CBF in terms of histologic sample quality. Furthermore, the PLL method provided a total sample 3.23

times heavier and 4.33 times larger in volume than those obtained with CBF. Both weight and volume

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were measured as different hepatopathies may affect weight and volume independently and they may

not increase or decrease together in a proportional fashion.

PLL sampling consistently required more surgical time than cup forceps biopsy. The PLL technique

requires more advanced laparoscopic skills when compared to the CBF and the learning curve is

probably steeper. All the procedures were performed by a resident in training as the primary surgeon

who had previous experience using CBF but had minimal experience with the PLL. This study is

likely to represent part of the learning curve for this procedure and this surgeon. All these reasons

may explain the longer duration required to obtain the necessary biopsies. Additionally, the PLL

method required a third port being placed for triangulation which also added a mean time of 2.8

minutes to the total surgical time.

An assistant was required in all the procedures included in this study. When an assistant is used, the

primary surgeon and the assistant should be able to coordinate their movements and therefore, the

assistant should also have previous laparoscopic experience. Following the study, the procedure has

been performed without a trained assistant, with them only stabilising the camera whilst the surgeon

was positioned at the caudal aspect of the dog between the hind limbs rather than on the side of the

dog.

In the case in which the PLL was cut inadvertently, a collagen sponge was used first to stop the

haemorrhage. However, this was not enough to control the haemorrhage and a second PLL was placed

around the liver parenchyma which immediately controlled the haemorrhage. This could provide an

additional use of the PLL in controlling haemorrhage in laparoscopic surgery.

None of the cases in this study required conversion to an open approach during surgery due to

uncontrolled haemorrhage or laceration of abdominal viscera. In a previous retrospective study

including 80 cases undergoing laparoscopic liver biopsies, the spleen was lacerated in one (1,3%) of

the cases and necessitated conversion to an open approach (Petre et al. 2012). In human laparoscopy,

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the incidence of iatrogenic trauma to abdominal organs and vessels by trocars or Veress needle has

been reported to be as low as 0.18% (Schafer et al. 2001).

A total of 13 of 15 (86.6%) survived to hospital discharge. The two dogs that died or were euthanased

during the study period had a histologic diagnosis of chronic hepatitis with cirrhosis. Cirrhosis is a

poor prognosis indicator in canine liver disease associated to a median survival time of 1.3 months

(Favier et al. 2013).

Only four cases developed postoperative complications. One dog needed rescue analgesia 24 hours

after discharge and three dogs had wound related complications. Tramadol is considered an

unpredictable analgesic and oral tramadol alone provided an inadequate level of analgesia in a

significant number of dogs undergoing an orthopaedic procedure (Davila et al. 2013). Postoperative

wound complications was reported in 16% in a study on dogs undergoing laparoscopic ovariectomy

(Pope and Knowles 2014). In that study, they could not find a statistically significant difference in

wound complications between the wound of 6 mm ports and 11 mm ports. In the present study, three

(20%) cases developed wound related complications which is a similar to previously reported and

only one of the three showed a wound complication in the 10 mm wound.

The goal of this study was to describe the use of a PLL to perform laparoscopic liver biopsies and

compare it to the traditional cup biopsy forceps. The limitations of the present study include the

limited number of cases, the use of single liver lobe for histological diagnosis and the experience of

the primary surgeon. The number of cases that developed complications was too low to allow

meaningful comparison with previous studies. The mean total surgery time was 78 minutes and this

included cases in which gallbladder bile aspiration was performed. Given the steep learning curve for

performing laparoscopy, it is possible that the mean surgery time would have been shorter if the

procedures were performed by a more experienced surgeon.

Conclusion

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The PLL is a successful and safe method to obtain laparoscopic liver biopsies. The resulting samples

were on average >3 times as heavy and larger in volume compared to samples obtained with cup

biopsy forceps. Although both methods resulted in biopsies of diagnostic value, the samples obtained

with the PLL showed superior quality in terms of number of portal tracts, crushing and fragmentation

artefacts.

We propose that obtaining laparoscopic hepatic biopsies with a PLL method can be considered a good

alternative method to the cup biopsy forceps method in dogs.

Conflict of interest

No conflicts of interest have been declared.

References

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Figure 1. Laparoscopic Babcock forceps holding the liver lobe through the pre-tied ligating loop ready

to position the loop around the hepatic tissue (left). Cuff of liver tissue and visible ends of the PLL

(white asterisk) and biopsy sites from the CBF (white arrows) (right).

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Figure 2. Examples showing the different grades (0-3) of crush and fragmentation artefact in the

population of the study. 0 = none, 1 = mild, 2 = moderate, 3 = severe). Please note that grade 3

fragmentation was absent in this dataset. Stain: Haematoxylin and eosin. Bar: 1mm for fragmentation,

500um for crush.

Cup biopsy forceps

(CBF)

Pre-tied ligating loop

(PLL)

Statistical test and

significance

1. T bx (min) Mdn=7 (IQR = 4) Mdn=18 (IQR = 17) 1-s W, WS= 115, p

=0.0020

2. Total W (mg) M=184 (±56.9SD) M=595.8 (±313.2) P-t (>0), t=5.37,

p<0.001

3. W Histo (mg) M=62.87 (±15.57) M=340.00 (±144,98) P-t (>0), t=7.33,

p<0.0001

4. Total V (cm3) M=0.29 (±0.10) M=1.30 (±0.60) P-t (>0), t=6.82,

0.5mm

1mm

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p<0.0001

5. V Histo (cm3) M=0.09 (±0.03) M=0.73 (±0.41) P-t (>0), t=6.03,

p<0.0001

6. A Histo (mm2) Mdn=26.30

(IQR=18.70)

Mdn=107.91

(IQR=137.40)

1-s W(>0), WS=120,

p=0.0004

7. PT (number) Mdn=19 (IQR=24) Mdn=69 (IQR=75) 1-s W(>0), WS=119,

p=0.0004

8. C artefact (0-3) Mdn=1 (IQR=2) Mdn=0 (IQR=1) 1-s W(<0),

WS=13.50,

p=0.0249

9. F artefact (0-3) Mdn=1 (IQR=1) Mdn=0 (IQR=1) 1-s W(<0),

WS=7.00, p=0.0378

Table 1. Summary of the differences between the CBF and the PLL techniques. 1. Time to obtain the

biopsies (minutes). 2. Total weight of the biopsy obtained at surgery. 3. Weight of the sample submitted

for histology. 4. Total volume of the biopsy obtained at surgery. 5. Volume of the sample submitted for

histology. 6. Total area of tissue available for histology. 7. Number of portal tracts. 8. Crush artefact (0-3).

9. Fragmentation artefact (0-3). †, mean (±SD). §, median (IQR). t, paired t-test. 1-s W, 1-sample

Wilcoxon test. Statistical significance <0.05.

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