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Obtaining Quality Biopsies of the Liver and Kidney

2004; American Animal Hospital Association; Volume: 40; Issue: 5 Linguagem: Inglês

10.5326/0400352

1547-3317

Clarence A. Rawlings, Elizabeth W. Howerth,

Hepatocellular Carcinoma Treatment and Prognosis

Biopsies of the liver are important to direct treatment and define prognosis based upon a histological diagnosis. The decision to obtain a biopsy is based on the benefits of knowing the disease process versus the cost of the biopsy and risks to the patient. Characterization of liver disease depends on cytological and histological findings in tissues obtained via ultrasound-guided fine-needle aspiration, ultrasound-guided needle core biopsy, laparoscopic cup forceps biopsy, or biopsy by laparotomy. The biopsy technique selected is determined by the likelihood of the test sample being accurate, the appearance of the liver on imaging, the presumptive diagnosis, the results of coagulation tests, the risks for anesthesia and surgery, and the likelihood of postoperative complications. Ultrasound-guided biopsy and fine-needle aspiration have limited roles in diagnosing liver disease because of the high probability of inaccuracies, even in animals with normal livers.Biopsies of the kidney are indicated less frequently than liver biopsies and are most often obtained in cases of acute renal failure or unexplained proteinuria. Biopsies of the kidney can be obtained with ultrasound guidance, with laparoscopy, or during laparotomy.Cytological and histological samples of the liver and kidney are desirable when a combination of clinical signs and abnormal laboratory results require a definitive answer as to the cause of the abnormal findings.1 The purpose of this paper is to present the indications, techniques, and assessments of test accuracy for biopsies of the liver and kidneys in dogs and cats.The liver is an organ with many indications for biopsy. The liver metabolizes food products, stores fat-soluble vitamins and nutrients, transforms toxins, produces bile, aids in fighting infections, and manufactures many of the essential substrates of the body, such as coagulation factors. Many local and systemic diseases produce liver dysfunction.Initial screening tests for liver disease often include a complete blood count, chemistry profile, radiography, and ultrasonography. Biopsy is indicated when abnormal activities of liver enzymes or abnormal hepatic function tests persist for >30 days or when hepatomegaly is present without a known cause. Other indications for liver biopsy are the staging of cancer, determination of liver involvement in systemic disease, and evaluation of the hepatic response to treatment.2 Biopsies are also useful to differentiate acute from chronic disease and to determine prognosis.34A complete evaluation of the liver is used to select the most appropriate biopsy technique. Technique selection considers liver size, the presence of any coagulopathy and/or ascites, whether the disease is diffuse or focal, the need for possible surgical resection or treatment of the liver lesion, anesthesia risks, and the capability of the veterinarian in performing the procedure.13 For example, the diseases most likely to be diagnosed with percutaneous techniques are those that are diffuse throughout the liver.34It is important to note that biopsy sample size and test accuracy vary with the biopsy technique. Figure 1 shows biopsy samples obtained with an 18-gauge, spring-fired needle; a 14-gauge, spring-fired needle; a 5-mm laparoscopic biopsy forceps; and a suture fracture technique. Histological sections obtained with each technique are visible in the two right-hand columns. The 18-gauge needle biopsy sample is fragmented, and generally only portions of lobules (about 12) are present. Zonal patterns of liver disease (e.g., periportal necrosis) and bridging fibrosis are difficult to recognize with this technique, and random foci (e.g., multifocal microabscesses) may be missed. In the 14-gauge needle biopsy specimen, only portions of about 20 liver lobules are present. Problems with interpretation of this size of sample are similar but less severe than those of 18-gauge biopsies. The cup biopsy contains at least eight to 10 complete lobules, and their architecture is well defined, which drastically improves the pathologist's ability to discern zonal patterns and bridging fibrosis. The likelihood of sampling randomly distributed liver lesions is greater with the cup biopsy technique than with needle biopsies. The largest biopsy sample is obtained with the suture fracture technique, although the one in this photograph [Figure 1] is larger than what is usually obtained. The suture fracture technique sample contains numerous, well-defined lobules, which greatly facilitates interpretation for the pathologist. The greater size of the biopsy also increases the likelihood of detecting randomly distributed lesions.The primary contraindication for a liver biopsy is coagulopathy with a tendency to bleed from the biopsy site.12 Coagulation studies (e.g., coagulation times, mucosal bleeding time) and platelet counts are performed before any biopsy procedure, as both the liver and kidney are highly vascular and hemorrhage can occur, especially with percutaneous techniques. Coagulation defects that produced complications following ultrasound-guided percutaneous biopsies were reviewed retrospectively in 434 dogs and cats.6 The presence of thrombocytopenia predicted the likelihood of complications and was considered to be a contraindication for percutaneous biopsy.6 Complications were also more likely when the one-stage prothrombin time was prolonged in dogs and the activated partial thromboplastin time was prolonged in cats.6 Hemorrhage can be visually monitored and controlled during laparoscopy and laparotomy, and laparoscopy decreases the chances of trauma and the likelihood of bleeding.The first historical technique developed for liver biopsy was the blind percutaneous placement of either a fine-needle (20- to 22-gauge) or a core biopsy needle.578 With the advent of ultrasonography and laparoscopy, there is now little justification for the use of the blind liver biopsy technique. Previous indications for blind percutaneous biopsies included a large liver with diffuse disease.23 A blind aspirate was most likely to be diagnostic in diseases such as hepatic lipidosis, masses that exfoliated well (e.g., malignant lymphoma and myeloproliferative cancers), or in diffuse infectious diseases such as histoplasmosis.3Ultrasound guidance is preferred for obtaining a percutaneous biopsy. Ultrasonography identifies normal and diseased liver tissue, the presence of cystic or large vascular structures, and any dilatation in the biliary structures. Contraindications for an ultrasound-guided fine-needle or core biopsy procedure include microhepatica, coagulation defects, large-volume ascites, a suspected hepatic cyst or abscess, vascular tumor, or a lesion that is adjacent to large bile ducts or hepatic vessels.1 The experienced ultrasonographer is often able to identify and selectively sample focal lesions and avoid structures adjacent to the liver.3Usually an 18- or 16-gauge biopsy core needle is used for liver biopsy. With larger needles there is more concern about hemorrhaging, especially with passage of the needle deep into the liver. Biopsy needles also vary from approximately 12 mm to 2 cm in length. The use of a spring-fired instrument to power the biopsy core needle is preferred to manual launching. The spring firing provides more control over the biopsy technique and insures the best cutting of tissue. The spring-fired instrument can be a single spring device that fires the outer biopsy cutting cannula, which is preferred in some cases because the inner stylet can be manually placed within a focal lesion before activating the biopsy cutting cannula. In contrast, the double spring-fired devices launch the inner stylet first and then releases the cutting cannula in a very rapid sequence. Double spring-fired biopsy devices are placed against the biopsy site and then discharged. The sequential actions are so rapid that the human eye cannot detect the two motions.The major limitation of this biopsy technique is the small size of the sample retrieved. For example, a 16-gauge biopsy core needle typically contains only four or five portal triads, without complete hepatic lobules or acini. Histological interpretation can be very difficult with such a small sample.1 Small-diameter biopsy samples have a tendency to become distorted and fragment, even when care is taken to gently remove the sample from the needle and place it into the formalin container. Fragmented and distorted biopsy samples further complicate histological interpretation.Reasons for the increasing popularity of ultrasound-guided percutaneous biopsy techniques are that they are minimally invasive, they do not require surgery, and they can usually be performed without general anesthesia. Another possible advantage is the reported ability to monitor the biopsy site for massive hemorrhage. When combined with prudent avoidance of fluid-filled structures and use of biopsy needles smaller than 16-gauge, postoperative complications appear to be low in dogs and cats with normal coagulation function.Laparoscopy provides excellent visual control and the ability to select samples from multiple sites in the liver [Figure 2]. It is the technique of choice when a nonsurgical liver disease is suspected.8 Large liver masses can be examined during laparoscopy, biopsies can be obtained, and decisions can be made about whether the tumor is resectable [Figures 3A-3C]. If it appears that the tumor can be removed, then the procedure may be converted to an open laparotomy. Laparoscopy is often the preferred biopsy technique in animals with either coagulation or wound healing deficiencies. Animals with coagulation defects often require pretreatment with therapies such as desmopressin acetate, plasma transfusion, and vitamin K.2Animals with ascites can effectively have their liver examined and biopsied by laparoscopy [Figures 4A, 4B]. Liver lobes float on the ascitic fluid. The use of two small trocar holes to obtain biopsies, followed by tight closure of the trocar sites, avoids the necessity of removal of the ascitic fluid and the accompanying protein loss that is inherent with a traditional laparotomy. Although biopsy samples taken from just beneath the liver capsule via laparoscopy are usually representative of generalized parenchymal changes, there is some concern that the sub-capsular location (in relation to hepatic blood supply) might predispose to erroneous diagnoses of fibrosis.9 Large-margin biopsies may overcome this artifact.8Laparoscopy requires general anesthesia and two trocar openings 5 mm in size (one for the viewing port; one for the biopsy cup instrument). Most clinicians find a 0° viewing scope easier to use, but a 30° scope allows the operator to see around the edges of the liver, which is especially helpful when lifting lobes to search for masses and for selectively sampling focal lesions from remote sites within the liver. The trocar for the viewing port may be placed in two different locations. With the animal lying on its back, the trocar can be inserted through the midline using a Hasson technique.10 This positioning provides an excellent view of all of the liver lobes. Another option is to position the animal in left lateral recumbency and insufflate the abdomen using a Verres needlea before blindly placing a sharp-tipped trocar into the right side of the abdominal wall.10 This latter technique can reduce the need for general anesthesia, but it restricts examination of the left side of the liver.If the liver appears different in various areas, then 5-mm biopsy samples are usually taken from each representative site. If possible, the biopsy forceps are directed at a 90° angle to the surface of the liver and then gently thrust deeply into the liver parenchyma. Sample size varies with the operator's technique. It is possible to select biopsy sites that are within millimeters of the biliary structures and major vessels, as these can usually be seen well through the viewing port. Two-dimensional viewing of the abdominal organs is much superior to that of traditional laparotomy, as images are magnified by the laparoscope.In addition to directly watching the sampling site for hemorrhage following biopsy, hemorrhage can also be reduced by using one of several different procedures. The authors prefer to use monopolar electrocautery applied while compressing liver tissue within the biopsy forceps cup,b before extraction of the tissue from the liver. Electrocautery apparently affects only the perimeter of the biopsy sample. Another hemostatic technique is to place a piece of absorbable gelatin foamc next to the biopsy site before obtaining the sample and then moving the foam into the excavation site immediately following the biopsy. Compression of adjacent liver with the biopsy forceps or another manipulating instrument is also routinely applied to the biopsy site after sampling, and any evidence of hemorrhage is directly observed through the laparoscope.Intrahepatic lesions may be more difficult to identify during either laparoscopy or laparotomy as compared to ultrasonography, yet the location of many such lesions can be appreciated during the first two procedures. During laparoscopy, samples can be taken for cytology, histology, or culture from intrahepatic lesions using double spring-fired biopsy needles or via fine-needle aspirates using spinal needles. Although it is possible during laparoscopy to aspirate normal bile from the gallbladder (by passing a needle through the quadrate liver lobe), bile is frequently too viscous to aspirate through the needle in animals with biliary disease.Liver biopsy during laparotomy is preferred in animals with diseases that can be treated with different surgical procedures, such as extrahepatic biliary obstruction or portosystemic shunts. Many animals undergoing laparotomy for other abdominal diseases also have abnormalities identified within the liver during surgery.2 Liver biopsies performed during laparotomy for other procedures are usually quick and seldom produce complications. The authors recommend that liver biopsies be performed early during an exploratory laparotomy in order to reduce the potential for migration of sequestered leukocytes from the intestinal tract to the liver after intestinal surgery.Similar to laparoscopy, exposure and viewing of the liver during laparotomy are excellent. Multiple areas can be sampled, biopsy sites can be examined for hemorrhage following sampling, and other areas of the abdomen can be explored. Another advantage of laparotomy is that most clinicians are more familiar with the liver biopsy techniques that can be performed during laparotomy than they are with the laparoscopy procedure. Disadvantages of laparotomy include the need for general anesthesia, the creation of a large abdominal wall incision for maximal exposure, and the possible need for postoperative analgesics. The resultant biopsy samples, however, are the largest obtained with any technique and have historically served as the "gold standard" for diagnosing liver disease.11When the hepatic disease is generalized, biopsies can be taken from the tip of a liver lobe using a suture fracture technique with a single crushing ligature or a transfixation ligature [Figures 5A, 5B]. The liver parenchyma should be crushed sufficiently so that the vessels, biliary structures, and connective tissue are compressed, preventing the leakage of blood and bile. Rarely, absorbable gelatin foam or electrocautery are also required to maintain hemostasis. When the hepatic lesions are deep cystic structures and have no ultrasound evidence of blood flow, they can be sampled via needle aspiration. A biopsy core needle is usually used on other deep structures. If hemorrhage occurs after deep core biopsies, the placement of mattress sutures around the needle puncture site often controls the bleeding.During laparotomy, the gallbladder may be aspirated by passing a large bore needle through the quadrate lobe or through direct puncture of the gallbladder. The latter procedure is performed by encircling the needle site with a purse-string suture of 5-0 synthetic absorbable suture material.All biopsy samples must be gently manipulated from the cup forceps and core needles using a small-gauge injection needle or by gently flushing with 10% formalin solution. Needle core biopsies tend to fracture, so it is best to place them onto a sponge inserted in a tissue-processing cassette prior to placing the sample in fixation media. Heavy metal analysis (e.g., copper) is typically done on fresh or formalin-fixed tissue, although some laboratories can analyze paraffin-embedded tissues. The amount of tissue needed for heavy metal analysis varies from 2 mg (i.e., equivalent to a 0.5-cm long, 14-gauge biopsy or a 1-cm long, 18-gauge biopsy) to 5 g, depending on the laboratory. Special fixatives are required for electron microscopy (EM), and the tissues submitted for EM are usually minced into 1- to 2-mm cubes.Test accuracy is vital, as an inaccurate diagnosis may result in inappropriate treatment and prognosis. In fact, the greatest cost of a test can be inaccurate interpretation, which may exceed equipment costs, professional fees, and risks to the animal. In a prospective study from the Cornell University Veterinary Hospital, needle core biopsies were compared to biopsies obtained at laparotomy or necropsy in 124 dogs and cats.11 Two ultrasound-guided biopsies were obtained with an 18-gauge needle (22 mm-long and 1 mm-wide biopsy chamber) from each animal, and subsequent wedge biopsies were also obtained via laparotomy or necropsy. Three examiners reviewed each set of needle and wedge biopsies. A wide range of histological diagnoses was made, with test accuracy varying with each disease.11 For individual examiners, agreement ranged from 56% to 67% when needle biopsies were compared to wedge biopsies. In 10 of 33 normal animals, liver disease was falsely diagnosed based on needle biopsy.11 Agreement occurred between the two biopsy specimens in 36 (40%) of the 91 animals with liver disease and in 59 (48%) of all 124 animals. Poor agreement between the biopsy specimens was a reflection of the small size and quality of the 18-gauge needle biopsy samples.11 The study concluded that needle biopsy specimens of the liver from dogs and cats should be interpreted with caution.11A more recent retrospective study compared ultrasound-guided fine-needle aspirates of the liver to biopsy samples taken within 7 days of the needle aspirate.12 Needle gauge was not reported, and the histological diagnosis was based on samples taken by percutaneous needle core biopsy, laparoscopy, laparotomy, or necropsy.12 Overall agreement between needle cytology and biopsy histopathology occurred in 17 (30.3%) of 56 dogs and in 21 (51.2%) of 41 cats.12 This low level of agreement should also serve as a caution to clinicians considering a fine-needle aspirate to be a definitive diagnostic tool.12Although the general guidelines for renal biopsy are the same as for hepatic biopsy, the specific indications and frequency of renal biopsy are much lower. Renal biopsies are indicated when unexplained disorders such as proteinuria (especially if amyloidosis is the primary differential diagnosis) and acute renal failure are present, when acute renal disease from ethylene glycol toxicity is suspected and unconfirmed by other modalities, etc.13 Because many clinical conditions require the examination of glomeruli to confirm a diagnosis, samples are usually taken from the renal cortex. To reduce hemorrhage, the large arcuate vessels in the medulla should be avoided. To avoid extravasation of urine, the renal pelvis should also be avoided.Ultrasound guidance is preferred for percutaneous biopsies. Ultrasonography identifies cortical tissue and large renal vessels and allows monitoring for postbiopsy hemorrhage. The experienced ultrasonographer may also be able to identify and selectively sample focal lesions, such as tumors.14 As with hepatic biopsies, most renal samples are obtained with either an 18- or 16-gauge, single spring-fired biopsy core needle. In the authors' experience, 18-gauge, ultrasound-guided biopsies have contained an average of 10.9 glomeruli, but the quality of the samples was often poor (e.g., 70% were crushed or fragmented, and 20% contained skeletal muscle).15Laparoscopy provides excellent visual control for positioning of the biopsy needle, provides for the application of pressure at the biopsy site, and allows monitoring for post-biopsy hemorrhaging. Laparoscopy for renal biopsy requires general anesthesia and the creation of two 5-mm trocar openings. In addition, a third small penetration is made for a 14-gauge core biopsy needle. To insure that the biopsy needle is sharp, it should be new, and the skin should be incised with a no. 15 Bard-Parker surgical blade. A manipulating instrument is used to position and secure the kidney, while a double spring-fired biopsy needle is placed against the renal capsule and directed tangentially prior to discharge [Figures 6A, 6B]. After biopsy, the needle is withdrawn and pressure is applied with the manipulating instrument.The biopsy sample is gently manipulated from the biopsy needle and placed into 10% formalin solution for light microscopy, into glutaraldehyde for electron microscopy, or it may be frozen for immunofluorescence studies. Because hemorrhage after renal puncture is unlikely, a second biopsy may be obtained from the same kidney or the contralateral kidney. In the authors' experience, biopsies obtained with the 14-gauge needle contained an average of 32.6 glomeruli, with the lowest number being 19 glomeruli in a single sample.15 In addition, none of the biopsies contained muscle, and all were of excellent quality.15Kidney biopsy performed as an additional procedure during exploratory laparotomy is usually quick and has minimal complications. The use of laparotomy to obtain a renal biopsy is usually restricted to those animals requiring laparotomy for other reasons. The major disadvantages of laparotomy are the need for general anesthesia, the creation of a large abdominal wall incision for maximal exposure, and the possible need for postoperative analgesics. Most bleeding from a 14-gauge biopsy needle puncture is controlled by digital compression. Only rarely has hemorrhaging after a biopsy required placement of a mattress suture at the puncture site.The authors are unaware of any published prospective comparison of these three biopsy techniques in small animal medicine. The authors' study of normal dogs that had renal biopsies under ultrasound guidance or laparoscopy found that laparoscopic novices using a 14-gauge needle consistently obtained large numbers of glomeruli.15 Renal biopsy samples obtained with 18-gauge needles had few glomeruli and were usually crushed and fragmented, thereby increasing the difficulty of reaching a diagnosis.15 Ultrasound-guided biopsies frequently included samples of muscle, in contrast to renal biopsies obtained during laparoscopy.15aVeress Pneumoperitoneum Needle (62120J); Karl Storz Veterinary Endoscopy-American, Inc. Goleta, CA 93117-3084bClickline Biopsy Forceps with Teeth; Karl Storz Veterinary Endoscopy-American, Inc. Goleta, CA 93117-3084cGelfoam; Pharmacia & Upjohn, Kalamazoo, MI 49001