Ultrasonographic visualization of the liver in sites recommended for blind percutaneous liver biopsy in horses Sara C. Sammons, DVM, MS; Tracy E. Norman, VMD; M. Keith Chaffin, DVM, MS; Noah D. Cohen, VMD, MPH, PhD

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valuation of percutaneously obtained liver biopsy specimens is widely regarded as the most sensitive and specific antemortem diagnostic test for suspected hepatopathy in horses.1,2 Evaluation of liver biopsy specimens may establish the presence or absence of liver disease, provide a specific diagnosis, guide treatment, and help determine prognosis in cases of suspected liver disease.3 Multiple techniques for obtaining a biopsy specimen of the liver have been described, including standing laparoscopic surgery,4 blind percutaneous biopsy, and direct and indirect ultrasound-guided biopsy. Ultrasonography is also useful to visualize the echogenicity, size, and location of the liver. The abdomen is scanned for the presence of adequate liver tissue, the biopsy region is marked on the horse, and the area of interest is prepared for sterile biopsy. This technique also allows for a brief abdominal ultrasonographic exFrom the Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843. Supported by a grant from the Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University. Presented in abstract form at the 60th Annual American Association of Equine Practitioners Convention, Salt Lake City, December 2014. Address correspondence to Dr. Sammons ([email protected]. edu).

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Objective—To ascertain the frequency of ultrasonographic identification of liver at sites recommended for blind percutaneous liver biopsy in middle-aged horses and to determine whether the liver is obscured by other organs or too thin for safe sample collection at recommended locations. Design—Prospective case series. Animals—36 healthy middle-aged (between 6 and 18 years old) Quarter Horses or Quarter Horse crosses. Procedures—Blood samples were collected from each horse and submitted for evaluation of liver function. Horses with any indication of liver dysfunction on serum biochemical analysis were excluded. The region just below a line drawn between the dorsal aspect of the tuber coxae and the point of the elbow joint in the right 11th, 12th, 13th, and 14th intercostal spaces (ICSs) was imaged by ultrasonography for the presence of liver. In each ICS, liver thickness and whether there was partial obstruction in viewing the liver caused by other abdominal or thoracic organs were recorded. Results—39% (14/36) of horses had liver imaged on ultrasonographic examination in all of the 11th to 14th ICSs. None of the 36 horses had liver of adequate thickness (ie, liver thickness ≥ 3.5 cm) for biopsy in all of the imaged ICSs. For 22 horses in which the liver was not visible on ultrasonographic examination of an ICS, lung was imaged instead in 12 (55%) horses, intestine in 8 (36%), and both intestine and lung in 2 (9%). Conclusions and Clinical Relevance—On the basis of the results of this study, the practice of blind percutaneous liver biopsy in horses is not recommended because of the risk of serious complications. (J Am Vet Med Assoc 2014;245:939–943)

ABBREVIATIONS CI ICS

Confidence interval Intercostal space

amination following biopsy to ensure that hemorrhage has not occurred as a result of the procedure. On ultrasonographic examination, the equine liver appears as a wedge of weak, homogenously distributed echoes medial to the diaphragm and ventral to the lung margin from the 9th to the 16th ICS.5–7 The liver is most commonly viewed from the right side of the abdomen in a healthy horse, but because of the possibility of atrophy of the right liver lobe in older horses, likely resulting from pressure of the right dorsal colon and cecal base, examination of the liver from the left side may be necessary.7 The left liver lobe can occasionally be imaged from the left 9th to 11th ICS and ventral to the lung margin.1,8 The greatest limitation of ultrasonographic organ localization and biopsy guidance is the inability of ultrasound to transmit through gas-filled structures and bone. These structures act as barriers to sound beam penetration and prevent observation of soft tissue structures deep to them.1 Even with the liver located in a normal anatomic position in a healthy horse, it may be deep to structures that are not able to be penetrated by Scientific Reports

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ultrasound beams. Therefore, gas-filled structures such as lung and intestines positioned between the liver and body wall could inadvertently be penetrated during an attempted liver biopsy. Pneumothorax, excessive blood loss into the abdominal or thoracic cavities, or enterocentesis can occur and require medical intervention.1 Anatomic locations to perform a blind percutaneous liver biopsy are published1,9 and referenced to by many equine veterinarians. The most frequently recommended location for performing a blind percutaneous liver biopsy has been described as the right 12th to 14th ICS at approximately the level of a line drawn between the tuber coxae and the point of the olecranon, directing the needle toward the contralateral elbow joint.9 Another similar location described is the right side of the abdomen between the 10th and 11th or the 11th and 12th ribs just below a line drawn from the point of the tuber coxae to the point of the shoulder, passing the biopsy instrument caudad and ventrad through the intercostal muscles corresponding with expiration to decrease the likelihood of penetrating the lung.1 In 1 study,9 liver biopsy could not be performed in 17% (4/24) of study mares because the liver was not visualized ultrasonographically at the target zone, which is similar to but encompasses a larger area than the recommended locations for blind percutaneous liver biopsy. To the authors’ knowledge, the actual frequency of ultrasonographic identification of the liver in the suggested locations for blind percutaneous liver biopsy in horses has not been reported. Furthermore, we are not aware of any study that has specifically determined the suitability of liver for biopsy at the recommended blind percutaneous liver biopsy sites in a specific population of horses. Therefore, the objectives of the study reported here were to ascertain the frequency of ultrasonographic identification of liver in the recommended location for blind percutaneous liver biopsy in middle-aged horses and to determine on ultrasonographic examination whether the liver is obscured by other organs or too thin for safe sample collection at those locations. We hypothesized that the frequency of identification of liver adequate for biopsy in the recommended locations for blind percutaneous liver biopsy is small and that the lack of sufficient liver tissue in the proposed locations could lead to serious complications resulting from biopsy including hemorrhage, peritonitis, or pneumothorax.

Quarter Horse crosses were included in the study. This sample size was determined on the basis of a binomial model, assuming that the proportion of horses in which liver was not observed within the recommended area was 30% and that we wanted to estimate the proportion with a margin of error of 15% at a confidence level of 95%. A review of records indicates that the mean ± SD age of horses undergoing a liver biopsy at our institution was 12 ± 6.7 years (median, 11 years; range, 2 months to 28 years). On the basis of these findings, horses used for this study were between 6 and 18 years old. Elderly horses (ie, ≥ 20 years) were excluded because liver atrophy has been documented in older horses.7 Only American Quarter Horses or Quarter Horse crosses were used for the study to avoid possible breedassociated anatomic variability. Experimental design—Blood was drawn into plain vacuum tubesa and submitted for serum biochemical analysis to evaluate liver function. Each horse was restrained in stocks, and blood was drawn from a jugular vein to evaluate concentrations of BUN and bile acids and activities of gamma glutamyltransferase, alkaline phosphatase, and aspartate aminotransferase. These specific analytes were chosen because each is associated with hepatocellular or biliary function in horses. Bile acids were included as a specific liver function test. Samples were immediately refrigerated and analyzed within 4 hours after collection. Horses identified by serum biochemical analysis to have any indication of liver dysfunction were excluded from the study. A 3.5- to 8-MHz curvilinear ultrasound transducerb was used to image the region just below a line drawn between the dorsal aspect of the tuber coxae and the point of the elbow joint in the right 11th, 12th, 13th, and 14th ICS for the presence of liver (Figure 1). Clipping of the hair prior to performing ultrasonographic examinations was not necessary because of the short hair of the horses used in the study. The dorsal-most aspect of the probe was situated just ventral to the line and parallel to the ribs within each ICS and directed in an oblique transverse plane. A single

Materials and Methods Study population and animals—This study’s animal use protocol was reviewed and approved by the Texas A&M University Institutional Animal Care and Use Committee. Horses were either privately owned and brought to the hospital for routine farrier appointments or lameness examinations or were part of the university-owned herd of research mares and geldings. Written consent was obtained prior to use of each privately owned horse in the study. Historical information and physical examination findings were used to identify healthy horses for inclusion in the study. Horses with a history of preexisting hepatopathy or those receiving medications for any ailment were excluded. Thirtysix middle-aged healthy American Quarter Horses or 940

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Figure 1—Photograph of a healthy middle-aged Quarter Horse. A line is drawn on the right side of the horse between the dorsal aspect of the tuber coxae and the point of the elbow joint. The 11th, 12th, 13th, and 14th ICSs are designated. The ultrasound probe is positioned just ventral to this line to image the liver. JAVMA, Vol 245, No. 8, October 15, 2014

Figure 2—Photograph of a horse demonstrating ultrasound probe positioning for imaging of the liver between each ICS, parallel to the ribs, directed in an oblique transverse plane. JAVMA, Vol 245, No. 8, October 15, 2014

was performed in the center of the ultrasonographic image in each of the 11th to 14th recommended ICSs. Additionally, the depth from skin surface to abaxial surface of the liver and the total depth from skin surface to axial surface of the liver were recorded. Data analysis—Data were analyzed through both descriptive and inferential statistical methods. For descriptive purposes, proportions were used to summarize categorical data and medians, and ranges were used to summarize continuous data. Confidence limits for proportions were calculated by means of binomial estimation. The upper bound of the 95% CI was estimated according to the formula 1 – α1/n, where α is the significance level (0.05) and n is the number of horses. Because observations were not independent, comparisons between pairs of ICSs were made by methods accounting for pairing. For categorical data, the McNemar test was used, and for continuous data, the Wilcoxon signed rank test was used. For a given variable, the multiplicity of comparisons was adjusted following the Bonferroni method. A significance level of adjusted P < 0.05 was used. Results The study population comprised 36 Quarter Horses or Quarter Horse crosses. The mean ± SD weight of the horses was 497 ± 59.3 kg (1,096 ± 130.7 lb), and all horses had a body condition score of at least 5 of 9. The heights of the horses were not measured, but all had typical Quarter Horse body types. The median age of the population was 11 years (range, 3 to 18 years; mean, 10.9 ± 4.12 years). Of the 36 horses, 17 (47%) were females and 19 (53%) were males (15 geldings [42%] and 4 sexually intact males [11%]). No significant difference was found in the age of males (median, 11 years; range, 4 to 18 years) and females (median, 12 years; range, 3 to 16 years). Of the 36 horses, 14 (39%; 95% CI, 31% to 47%) had liver visible in all of the 11th to 14th ICSs. None of the 36 horses (0%; 95% confidence upper limit, 8%) had liver thickness ≥ 3.5 cm (the thickness deemed appropriate to be sufficient for biopsy collection) in all of the 11th through 14th ICSs. The proportion of horses with visible liver and the median depth from skin surface to abaxial border of the liver, liver thickness, and depth from skin surface to axial border of the liver when liver was visible in each ICS were summarized (Table 1). The proportion of horses with liver visible was higher for the 13th and 14th ICS than for the 12th and 11th ICS, but there were no significant differences among proportions. Also, no significant differences were found among proportions of horses with adequate liver thickness, although the 11th and 14th ICSs had smaller proportions than the 12th and 13th ICSs. No significant differences were found between ICSs for measured depth from skin surface to abaxial surface of liver except for between the 13th and 14th ICS (Table 1). Among the 21 horses that had this depth recorded for both 13th and 14th ICSs, the depth from skin surface to abaxial surface of the liver was significantly (P ≤ 0.001) greater for the 13th ICS than for the 14th, Scientific Reports

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still image was obtained and saved in each ICS (Figure 2). If the liver was identified in a location, the depth from the superficial to the deep surface of the liver was measured by means of postacquisition processing on the ultrasound unit. The presence of other abdominal or thoracic organs that obscured the liver from view was recorded if the liver itself was not visible. Because a standard liver biopsy instrument chamber is 2.5 cm in length, we required that the liver be at least 3.5 cm thick for an adequate sample that could safely be obtained without risk of puncture of deeper structures. All imaging and postimaging evaluation was performed by the same author (SCS). The ability to identify the liver at the designated location (yes vs no) by ultrasonography was recorded. If liver was visible, its thickness was measured at the center of the ultrasound beam to determine whether it was of adequate thickness for biopsy. If liver was not visible or if it was partially obscured such that biopsy could not be performed without penetrating another abdominal or thoracic organ, the other organs at that location were noted. If the liver was not visible in all of the 4 ICSs, the liver was defined as not appropriate for biopsy because the recommended locations for blind percutaneous liver biopsy in horses encompass multiple ICSs. If the liver measured < 3.5 cm in any 1 of the 4 ICSs at the center of the ultrasound beam, it was also defined as inappropriate for biopsy. Therefore, to be defined as appropriate for biopsy, the liver needed to be observable, thick enough for biopsy specimen collection, and accessible if a biopsy

Table 1—Summary of ultrasonographic identification and measurements of the liver at the recommended ICS locations for blind percutaneous liver biopsy in 36 middle-aged Quarter Horses and Quarter Horse crosses. ICS Variable No. of horses with liver visualized (% [95% CI]) Median (range) liver depth (cm) Median (range) liver thickness (cm) No. of horses with adequate liver thickness (% [95% CI]) Median (range) skin to axial border (cm)

11

12

13

14

18/36 (50 [42–58]) 4.5 (1.9–5.8)a,b 2.4 (0.9–6.0) 5/18 (28 [17–39]) 6.8 (4.0–11.3)

20/36 (56 [48–64]) 4.9 (2.4–6.1)a,b 3.3 (1.2–7.9) 8/20 (40 [29–51]) 7.6 (4.3–12.9)

25/36 (69 [61–77]) 4.8 (1.6–6.0)a 2.9 (0.6–5.9) 10/25 (40 [30–50]) 7.5 (4.9–10.2)

25/36 (69 [61–77]) 4.1 (1.1–5.7)b 2.6 (0.7–7.5) 7/25 (28 [18–38]) 6.9 (3.7–11.6)

Within a row, values with different superscript letters are significantly (P < 0.05) different.

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Table 2—Agreement in the ability to image the liver between pairs of ICSs during hepatic ultrasonographic examination in the same horses as in Table 1. ICS 11 vs 12 11 vs 13 11 vs 14 12 vs 13 12 vs 14 13 vs 14

κ

95% CI

Overall agreement

95% CI

0.78 0.50 0.28 0.71 0.36 0.48

0.57 to 0.98 0.21 to 0.79 –0.06 to 0.62 0.48 to 0.94 0.03 to 0.68 0.16 to 0.79

89% (32/36) 75% (27/36) 64% (23/36) 87% (31/36) 69% (25/36) 77% (28/36)

84% to 94% 68% to 82% 56% to 72% 81% to 93% 61% to 77% 70% to 84%

Table 3—Agreement in the presence of adequate liver thickness between pairs of ICSs during hepatic ultrasonographic examination in the same horses as in Table 1. ICS 11 vs 12 11 vs 13 11 vs 14 12 vs 13 12 vs 14 13 vs 14

κ 0.43 0.06 –0.36 0.38 –0.42 0.22

95% CI –0.04 to 0.90 –0.43 to 0.55 –0.59 to –0.13 –0.04 to 0.79 –1.00 to 0.18 –0.24 to 0.69

Overall agreement*

95% CI

77% (13/17) 59% (10/17) 47% (7/15) 70% (14/20) 35% (6/17) 62% (13/21)

66% to 87% 47% to 71% 34% to 60% 59% to 81% 24% to 47% 51% to 72%

*The various denominators reflect the number of horses that had liver visible in both of the listed ICSs.

and the median difference was 0.3 cm (range, –0.2 to 1.1 cm). No significant differences were found in liver thickness among ICSs. Also, no significant difference was found among ICSs in the distance from skin to the axial border of the liver. Agreement with regard to visibility and presence of adequate liver thickness between pairs of ICSs were examined on the basis of κ statistics and overall agreement. The κ statistics, 95% CIs, and overall agreement were tabulated (Tables 2 and 3). In general, there was better agreement between adjacent ICSs than more remote spaces. For 22 horses for which the liver was not visible on ultrasonographic examination of an ICS, lung was imaged instead in 12 (55%) horses, intestine in 8 (36%), and both intestine and lung in 2 (9%). Discussion On the basis of the results of this study, the practice of blind percutaneous liver biopsy in horses is not recommended. Fewer than 40% of horses in this study had liver visible in all recommended ICSs for blind percutaneous liver biopsy, and no horse had thick enough liver in all of the ICSs. The use of ultrasonography prior to liver biopsy is therefore strongly recommended to image the liver and evaluate its thickness to ensure that 942

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an adequate diagnostic sample can be obtained and to avoid puncture of lung or intestines. Our results provide more compelling evidence regarding the necessity to use ultrasonographic assistance for liver biopsy than have previous studies. Pearce et al9 performed a study of liver biopsy procedures in postpartum mares in which 17% of study horses did not have liver imaged at the target biopsy zone, which was a triangle formed by joining the tuber coxae to the point of the olecranon and the scapulohumeral joint between the 12th and 14th ICSs, an area similar in location to but larger in size than the area used for our study. Ultrasonography was used to attempt to guide the precise location for liver biopsy procedures within the target zone and, in all of the 4 cases for which liver was not identifiable, intestine was imaged instead.9 The mares of that study9 were 11, 11, 13, and 18 years old. The mares were reexamined ultrasonographically 2 weeks later, and the anatomic situation was the same. Postpartum mares were used for that study,9 which could cause a slight difference in anatomic configuration of organs within the abdomen, compared with that of geldings. Our study was designed to provide further information about the practice of blind percutaneous liver biopsy, not to estimate the frequency of complications associated with liver biopsy procedures; as such, no liver biopsies were actually performed. Even if ultrasonography is used to plan the best approach to a liver biopsy, there are still risks associated with the procedure. A retrospective study10 evaluating the complications associated with liver biopsy procedures in horses identified 1 case of hemorrhage resulting from diaphragmatic hernia. The authors of that study10 advised that ultrasonographic examination during or directly following liver biopsy could be used to assess the incidence of acute bleeding in horses. If severe hemorrhage is identified ultrasonographically shortly following liver biopsy, proper treatment may be prescribed and further complications prevented. Research regarding the safety of blind percutaneous liver biopsy in humans has shown that ultrasonographic localization of the liver prior to biopsy substantially diminishes the risk of complications in adults.11 In a study12 of percutaneous liver biopsy in children, patients who developed postprocedural bleeding as a complication were all subjected to blind percutaneous liver biopsies, whereas no hemorrhage occurred in patients undergoing ultrasound-guided liver biopsy procedures. Authors of that study10 suspected a decrease in the number of complications following ultrasound-guided biopsy as a result of the greater accuracy in liver localization, ability JAVMA, Vol 245, No. 8, October 15, 2014

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ous liver biopsy procedures in horses. Ultrasound machines are becoming increasingly affordable and portable for equine practitioners. With some training and guidance from an experienced ultrasonographer, a practitioner can use ultrasonographic assistance when performing a percutaneous liver biopsy in horses, thereby reducing the risk of complications that might otherwise occur. a. b.

BD Vacutainer Serum, 10-mL plain tube, BD, Franklin Lakes, NJ. Technos ultrasound unit, Esaote, Genoa, Italy.

References 1.

Modransky PD. Ultrasound-guided renal and hepatic biopsy techniques. Vet Clin North Am Equine Pract 1986;2:115–126. 2. Durham AE, Newton JR, Smith KC, et al. Retrospective analysis of historical, clinical, ultrasonographic, serum biochemical and haematological data in prognostic evaluation of equine liver disease. Equine Vet J 2003;35:542–547. 3. Divers TJ, Bernard WB, Reef VB. Equine liver disease and liver failure—causes, diagnosis, and treatment, in Proceedings. 10th Bain-Fallon Memorial Lecture 1988;35–46. 4. Fischer AT Jr. Standing laparoscopic surgery. Vet Clin North Am Equine Pract 1991;7:641–647. 5. Rantanen NW. Ultrasound appearance of normal lung borders and adjacent viscera in the horse. Vet Radiol 1981;22:217–219. 6. Rantanen NW. Ultrasonic examination of the liver of the horse, in Proceedings. Annu Conv Am Institute Ultrasound Med 1981;81. 7. Rantanen NW, Timon TC. Ultrasound scan techniques for large and small animals. Bellevue, Wash: ATL Inc, 1983;14–23. 8. Dyce KM, Sack WO, Wensing CJG. In: Textbook of veterinary anatomy. Philadelphia: Saunders Co, 1987;503. 9. Pearce SG, Firth EC, Grace ND, et al. Liver biopsy techniques for adult horses and neonatal foals to assess copper status. Aust Vet J 1997;75:194–198. 10. Johns IC, Sweeney RW. Coagulation abnormalities and complications after percutaneous liver biopsy in horses. J Vet Intern Med 2008;22:185–189. 11. Cadranel JF, Rufat P, Degos F. Practices of liver biopsy in France: results of a prospective nationwide survey. Hepatology 2000;32:477–481. 12. Nobili V, Comparcola D, Sartorelli MR, et al. Blind and ultrasound-guided percutaneous liver biopsy in children. Pediatr Radiol 2003;33:772–775.

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to identify and avoid large hepatic vessels and other organs, and the subsequent reduction in number of passes required to obtain an adequate sample with the biopsy instrument.12 Ultrasonography was also found to be useful in detection of postbiopsy hematomas, which are an indication of severe bleeding.12 One limitation of our study was that we evaluated only the right side of horses. The liver is most commonly imaged on the right side of the abdomen in healthy horses, but because of the possibility of atrophy of the right liver lobe in older horses, examination of the liver from the left side can be more productive in some patients. Another limitation of this study is that only horses that were determined to be healthy according to physical examination findings and results of serum biochemical analysis were used in this study, whereas liver biopsy is typically performed in horses with suspected liver disease. Because hepatopathy can result in enlargement of the liver or cirrhosis of the liver, results of a study examining the location and adequacy of liver for biopsy in patients with liver disease may be different. At our institution, a small number of patients are admitted for liver disease; therefore, a multicenter study may be helpful to achieve adequate case numbers in a reasonable period. In this study, because only 1 author performed the ultrasonographic examinations and took measurements at a single time point, no interobserver variability was determined. Because single still images were obtained and measurements were taken once, intraobserver variability could not be calculated; this is considered another limitation to our study. Finally, we used only adult Quarter Horses or Quarter Horse crosses. The location of the liver within the abdomen, the dimensions of the abdomen, and the size of the liver itself may be different depending on the breed of horse evaluated. The results of this study indicated a need to use ultrasonographic assistance when performing percutane-

Ultrasonographic visualization of the liver in sites recommended for blind percutaneous liver biopsy in horses.

To ascertain the frequency of ultrasonographic identification of liver at sites recommended for blind percutaneous liver biopsy in middle-aged horses ...
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