Veterinary Pathology Online

http://vet.sagepub.com/ A Multistep Approach in the Cytologic Evaluation of Liver Biopsy Samples of Dogs with Hepatic Diseases
C. Stockhaus, T. Van Den Ingh, J. Rothuizen and E. Teske Vet Pathol 2004 41: 461 DOI: 10.1354/vp.41-5-461 The online version of this article can be found at: http://vet.sagepub.com/content/41/5/461

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Vet Pathol 41:461470 (2004)

A Multistep Approach in the Cytologic Evaluation of Liver Biopsy Samples of Dogs with Hepatic Diseases
C. STOCKHAUS, T.
VAN DEN INGH,

J. ROTHUIZEN,

AND

E. TESKE

Department of Small Animals, University of Leipzig, Leipzig, Germany (CS); and Department of Clinical Sciences of Companion Animals (JR, ET) and Department of Veterinary Pathology (TvdI), Utrecht University, Utrecht, The Netherlands Abstract. Cytologic criteria were evaluated for their diagnostic value in liver disease in dogs. Therefore, histopathologic and cytologic examination was performed on liver biopsy samples of 73 dogs with liver diseases and 28 healthy dogs. Logistic regression analysis was used to select the measured parameters to be included in a multistep approach. With the logistic regression method, different characteristic cytologic parameters could be dened for each histopathologic diagnosis. In malignant lymphoma of the liver, the presence of large numbers of lymphoblasts with a minimum of 5% of all cells was found. Clusters of epithelial cells with several cytologic characteristics of malignancy intermixed with normal hepatocytes were indicative of metastatic carcinoma or cholangiocellular carcinoma. Liver cells in hepatocellular carcinoma were characterized by a high nucleus/ cytoplasm ratio, large cell diameters, increased numbers of nucleoli per nuclei, small numbers of cytoplasmic vacuoles, and frequently, small numbers of lymphocytes. Extrahepatic cholestasis was characterized by excessive extracellular bile pigment in the form of biliary casts, an increased number of nucleoli within hepatocytes, decreased hepatic cell size, and low numbers of lymphocytes. In destructive cholangiolitis, increased numbers of neutrophils and a small mean nuclear size within hepatocytes were seen. Acute and nonspecic reactive hepatitis are diagnosed based on the presence of moderate reactive nuclear patterns, including more pronounced chromatin, prominent nucleoli, increased numbers of inammatory cells, excluding lymphocytes, and the absence of increased numbers of bile duct cell clusters. Increased number of mast cells also was indicative of nonspecic reactive hepatitis. Important cytologic criteria for the diagnosis of liver cirrhosis, in addition to chronic hepatitis, are intracellular bile accumulation and increased numbers of bile duct cell clusters. In summary, the stepwise approach based on logistic regression presented in this study might be helpful in the objective cytologic diagnosis of hepatic diseases. Key words: Hepatic diseases; liver cytology; multistep approach.

Cytologic examination of hepatic tissue obtained by ne-needle aspiration biopsy (FNAB) of the liver is being used with increasing frequency in companion animal medicine.4,5,14,19,22,24,26,27,29 However, the gold standard for the diagnosis of most liver diseases is histologic evaluation of a number of wide-bore needle or wedge biopsy samples. Histologic examination is then based on the assessment of a number of liver acini. In contrast to histologic biopsies, the procedure to obtain ne-needle aspirates is minimally invasive and easy to perform without anesthesia or sedation, and smears can be examined immediately. Furthermore, fragments of histologic biopsy samples can be examined cytologically with impression smears. Cytologic criteria for various liver diseases in the dog have not yet been well dened and often are only extrapolated from human literature. In addition, key criteria rarely are separated from secondary criteria. Not only is sound scientic knowledge lacking in the cytologic diagnosis of liver diseases but also is variation present within cytologic specimens from normal

liver aspirates. In healthy humans10,18 and dogs,25 mild cellular variation and a few inammatory cells are considered part of this normal variation. Furthermore, in healthy dogs there is evidence of age-dependent variation of cellular criteria.25 Objective cytologic criteria in dogs with liver diseases have not been evaluated, making an objective and standardized cytologic examination of FNAB difcult. This study analyzes hepatocellular cytologic criteria in dogs with different hepatobiliary diseases and helps develop a stepwise approach for the cytologic diagnosis of liver diseases in the dog. Materials and Methods
Animals Seventy-three dogs were referred to the Utrecht University Clinic of Companion Animals (UUCCA) with suspected liver disease during the years 19931996. Liver disease was suspected in these dogs by either abnormal liver parameters of clinical chemistry results or abnormal appearance of the liver during abdominal ultrasonography. In addition, 28

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Table 1.

Description of cytologic criteria for evaluating liver impression smears in dogs.

Score

Cytologic Parameter

Cell borders Groups in sheets or 3D clusters* Cell size Nuclear size Nucleus/cytoplasm diameter ratio Nuclei per cell Nucleoli per nucleus Maximal number of nucleoli per nucleus Color of nucleoli Form of nucleoli Nuclear membrane Chromatin pattern Color of the cytoplasm Vacuoles in cytoplasm Intracellular bile pigment Extracellular bile pigment Cell necrosis Number of other cell populations Number of groups of bile duct cells per slide
* 3D three dimensional.

0 not distinct; 1 medium; 2 distinct; mean of 100 cells 0 3D cluster; 1 intermediate; 2 2D sheets; mean in 50 cell groups Mean diameter of 50 cells Mean diameter in 50 cells Mean ratio of 50 cells Mean number per cell in 100 cells Mean number per nucleus in 100 nuclei Maximal number in 100 cells 0 gray red; 1 blue; mean of 50 cells 0 round; 1 irregular; mean of 50 cells 0 normal; 1 irregular, thickened; mean of 50 cells Percent cells with regular or irregular chromatin in 100 cells 0 light blue; 1 some dark blue; 2 dark blue; mean of 50 cells 0 none; 1 some; 2 regular; 3 many; mean of 50 cells 0 none; 1 some; 2 regular; 3 many; mean of 50 cells 0 none; 1 some; 2 regular; 3 many; mean of 50 cells 0 none; 1 some; 2 many; mean of 50 cells Percentage of 250 cells counted: neutrophils, lymphocytes, lymphoblasts, mast cells, Kupffer macrophages, mesothelial cells, brocytes, lipocytes, and eosinophils Mean

healthy blood donor dogs owned by the UUCCA also were included. In all dogs, hematology, clinical chemistries, coagulation screening tests (prothrombin time, activated partial thromboplastin time, and brinogen concentration), and liver evaluation using abdominal ultrasound were performed. Diagnosis of liver disease was made by histopathologic examination of at least three 18-gauge-needle liver biopsy samples. Liver diseases identied included malignant lymphoma (n 8), hepatocellular carcinoma (n 12), metastatic carcinoma (n 6), steroid-induced hepatopathy (n 6), lipidosis (n 1), extrahepatic cholestasis (n 6), nonspecic reactive hepatitis (n 10), acute hepatitis (n 6), chronic hepatitis (CH) (n 7), cirrhosis (n 8), and destructive cholangiolitis (n 3). All 28 healthy control dogs had normal liver histology. Liver biopsy samples In dogs with suspected focal liver disease on abdominal ultrasonography, biopsy samples were obtained under ultrasound guidance using an 18-gauge Tru-cut needle (William Cook Co.). Dogs were clipped, disinfected with alcohol (70%), and placed in right lateral recumbency, and the abdominal wall and the skin were locally anesthetized with 3% lidocaine. Under ultrasound guidance, a Tru-cut biopsy needle was placed through a small stab incision in the abdominal wall made with a surgical blade and advanced to the abnormal area for biopsy. Blind percutaneous liver biopsies were performed in right lateral recumbency in dogs with suspected diffuse hepatic disease, identied by ultrasonography, using a technique as rst described by Lettow (1963).15 Preparations and local anesthesia were similar to

those used for Tru-cut biopsies. A small incision was made through the abdominal wall 12 cm caudal to the xyphoid, and an 18-gauge Menghini needle (William Cook Co.) attached to a 5-ml 0.9% saline-lled plastic syringe was inserted into the abdomen until it reached the left lateral lobe. Liver tissue was then aspirated with slight vacuum. From every dog, three biopsy samples, each 23 cm in length, were obtained. One portion of each piece of hepatic tissue was used for cytologic impression smears, which were air-dried and stained with May-Gru nwald-Giemsa. The other portion was xed in 10% neutral buffered formalin. The xed tissue was routinely embedded in parafn, and 4-m-thick sections were stained according to standard techniques with hematoxylin and eosin, Van Gieson stain, and Gordon and Sweet reticulin stain. Liver histology was evaluated by one experienced liver histopathologist (T. van den Ingh), and cytology was performed by one examiner (C. Stockhaus). Cytologic examination Cytologic smears were coded to prevent the observer from knowing the diagnosis. On initial evaluation of the smear, several representative parts of the slides were selected for further evaluation. As already described by Stockhaus et al.,25 slides were analyzed for general cell criteria, nuclear criteria, cytoplasmic criteria of hepatocytes, and the frequency of cell populations other than hepatocytes (Table 1). The number of intracellular vacuoles and bile pigment content were evaluated and scored 03 (0 no vacuoles; 1 small amounts of vacuoles in a minority of cells; 2 variable amounts of vacuoles in a majority of cells; 3 large

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Table 2. Signicant cytologic parameters of liver impression smears between healthy dogs (n 28) and dogs with hepatic disease (n 73).
Healthy Dogs, Mean (95% Condence Interval) Dogs with Liver Diseases, Mean (95% Condence Interval)

Cytologic Criteria

P-Value

Age-corrected cell size deviation (m) Age-corrected number of nuclei/cell deviation (n) Age-corrected nuclear/cytoplasm diameter ratio deviation Hepatocytes with irregular chromatin pattern (%) Color nucleoli (0 gray to 1 blue) Form nucleoli (0 round to 1 irregular) Cell borders (0 not distinct; 2 distinct) Cytoplasmic basophilia (0 light to 2 dark) 3D structure of cell groups (0 3D; 2 2D)* Extracellular bile (0 none to 3 many) Necrosis (0 none to 2 many) Kupffer macrophages (%) Lymphocytes (%) Neutrophils (%) Fibrocytes (%)
* 3D three dimensional.

0 0 0

( 0.58 to 0.58) ( 0.02 to 0.02) ( 0.01 to 0.01)

3.43 ( 4.47 to 2.39) 0.0617 ( 0.09 to 0.03) 0.06 57.4 0.63 0.71 1.07 0.99 0.96 0.49 0.45 0.45 2.68 2.84 0.62 ( 0.04 to 0.07) (48.2 to 66.6)

0.0001 0.0072 0.0001 0.0001 0.053 0.0014 0.0001 0.003 0.0001 0.0023 0.0002 0.028 0.004 0.0001 0.015

8.5 (3.5 to 13.6) 0.31 0 1.89 0 1.87 0 0 0.04 (0 to 0.9) 1 (0.78 to 1.22) 0.86 (0.6 to 1.11) 0

(0.22 to 0.68) (1.98 to 3.39) (2.3 to 3.38) (0.31 to 0.93)

amounts of vacuoles in almost every hepatocyte). For calculating incidence rates, the number of different cell types per 250 hepatocytes was used. Cell and nucleus sizes were measured with an ocular micrometer. In a previous study,25 signicant age inuences were observed for the parameters cell size, number of nuclei per cell, and nucleus/cytoplasm ratio. Therefore, these parameters were corrected for age inuence by taking the individual difference of this parameter with the mean value of a comparable age group in healthy dogs.25 Statistical analysis Statistical analysis was performed by SPSS Windows 9.05 statistical packages.20 Differences between groups were analyzed with the one-way analysis of variance for interval data, the Kruskal-Wallis test for ordinal data, and the chisquare test for bivariate data. Stepwise logistic regression analysis was performed to identify the signicant variables important to diagnose the different liver diseases. For each step, variables with a P-value less than 0.20 on univariate regression score were included in the multiple regression model with forward-step analysis. In such a logistic regression model, Y ln(P/(1 P)) 0 (1X1) (2X2) (nXn), independent variables (X1, X2, . . ., Xn) are combined in a linear equation that is used to estimate the logarithm (Y) of the probability for the occurrence of an event (P) divided by the probability that it does not occur (1 P).2 Negative parameters in the equation have an opposite effect on the probability of the event as to positive parameters.

Based on the values of the independent variables, the estimated logarithm given by a logistic regression equation can be used to calculate a conditional probability (P) of belonging to the group with the event using the formula P (1 exp(Y))1, where exp(Y) is the exponential value of Y.12 For all signicance testing, P-values less than 0.05 were considered to be signicant.

Results Individual cytologic criteria in the different liver diseases and healthy dogs are given in Table 1. Differences in cytologic parameters between the 28 healthy dogs and the 73 dogs with hepatic diseases are listed in Table 2. Steroid-induced hepatopathy, lipidosis, and extramedullary hematopoiesis were not included in the stepwise logistic regression method because they were considered to be secondary diagnoses (n 7). The remaining 94 hepatic biopsy samples were included in the stepwise approach of cytologic evaluation of these smears. In step 1, malignant lymphoma was diagnosed by the presence of large numbers of lymphoblasts ( 5% of total nucleated cells present in the biopsy sample) among the hepatocytes. During this step, mast cell metastasis to the liver also could be diagnosed. High numbers of mast cells or mast cells in sheets as well

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Table 3. Cytologic characteristics for individual diagnosis of liver diseases, excluding lipidosis. For description of parameter see Table 1.
Normal (n 28) Lymphoma (n 8) Hepatocellular Carcinoma (n 12) Metastatic Carcinoma (n 6)

Cytologic Parameter

Age-corrected cell size deviation Age-corrected NC ratio deviation Nuclear size Age-corrected no. nuclei/cell deviation Mean no. nucleoli/nucleus Max no. nucleoli/nucleus Cell borders Three-dimensional structure of cell groups Color cytoplasm Vacuoles in cytoplasm Intracellular bile Extracellular bile Cell necrosis Nuclear membrane Color of nucleoli Form of nucleoli % Kupfers cells % Lymphocytes % Lymphoblasts % Neutrophils % Mast cells % Fibrocytes No. groups of bile duct cells % Cells with regular chromogen pattern % Cells with irregular chromogen pattern

0.04 ( 2.7 to 3.1) 0 9.43 (8.3 to 10.6) 0 1.11 (1 to 1.3) 2.18 (2 to 3) 1.89 (1 to 2) 1.86 (1 to 2) 0 0.71 (0 to 2) 1.05 (0.5 to 1.5) 0 0 0 0.32 (0 to 1) 0 0.04 (0 to 0.5) 1 (0 to 2) 0 0.86 (0 to 2) 0.21 (0 to 1) 0 1.32 (0 to 3) 91.46 (60 to 100) 8.54 (0 to 40)

6.45 ( 8.4 to 4.0) 0.08 (0 to 0.2) 9.24 (7.9 to 11.2) 0.1 ( 2 to 0.1) 1.1 (1 to 1.2) 2 (2 to 2) 1.25 (1 to 2) 1.13 (1 to 2) 0.13 (0 to 1) 0.44 (0 to 1.5) 1.56 (0 to 2.5) 0.13 (0 to 1) 0.5 (0 to 1) 0 (0 to 0) 0.63 (0 to 1) 0 0 0.13 (0 to 1) 0.38 (0 to 3) 52.5 (5 to 90) 2.62 (0 to 6) 0.13 (0 to 1) 0.13 (0 to 1) 0 0 42.5 (0 to 100) 57.5 (0 to 100)

6.05 ( 12.5 to 8.9) 0.15 (0 to 0.3) 10.42 (8.3 to 13.3) 0.12 ( 0.3 to 0.1) 1.22 (1.1 to 1.5) 2.58 (2 to 3) 0.5 (0 to 2) 0.25 (0 to 1) 1 (0 to 2) 0.71 (0 to 2.5) 0.58 (0 to 1.5) 0.08 (0 to 1) 0.96 (0 to 2) 0.25 (0 to 1) 0.83 (0 to 1) 0.58 (0 to 1) 0.25 (0 to 2) 1.58 (0 to 6) 0 0 1.67 (0 to 7) 0 0 1.25 (0 to 5) 0.08 (0 to 1) 13.33 (0 to 100) 69.17 (0 to 100)

6.54 ( 8.3 to 5.2) 0.06 (0 to 0.1) 8.51 (8.1 to 8.8) 0.19 ( 0.3 to 0.1) 1.13 (1.1 to 1.4) 2.33 (2 to 3) 1.33 (0 to 2) 0.83 (0 to 1) 0 (0 to 0) 0 (0 to 0) 1 (0 to 2) 0 (0 to 0) 0.67 (0 to 2) 0 (0 to 0) 0.67 (0 to 1) 0.17 (0 to 1) 0.33 (0 to 2) 3.17 (0 to 6) 0 0 3.67 (3 to 6) 0.58 (0 to 2) 0 0 0.33 (0 to 2) 30 (0 to 100) 66.67 (0 to 100)

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Table 3.

Extended.
Extrahepatic Cholestasis (n 6) Reactive Hepatitis (n 10) Acute Hepatitis (n 6) Cirrhosis CAH (n 8) Destructive Cholangitis (n 3)

SIH (n 6)

CAH (n 7)

1.61 ( 1.1 to 5.2) 0.05 ( 0.1 to 0) 8.54 (8.1 to 9.4) 0.08 ( 0.2 to 0.11) 1.07 (1 to 1.1) 2 (2 to 2) 2 2 2 2 0 (0 to 0) 2.42 (2 to 2.5) 1.17 (0.5 to 1.5) 0 (0 to 0) 0 (0 to 0) 0 (0 to 0) 0.17 (0 to 1) 0 0 0 0 0.83 (0 to 5) 0 0 1 (0 to 3) 0.75 (0 to 3) 0 0 0.33 (0 to 1) 86.67 (70 to 100) 13.33 (0 to 30)

4.2 ( 8.1 to 0.8) 0.06 (0 to 0.1) 9.37 (9.0 to 9.9) 0.01 ( 0.3 to 0.3) 1.15 (1 to 1.3) 2.67 (2 to 3) 1.17 (0 to 2) 1 1 0 (0 to 0) 1.08 (0 to 1.5) 1.33 (1 to 1.5) 2.33 (1.5 to 3.0) 0.33 (0 to 1) 0 (0 to 0) 0.67 (0 to 1) 0.17 (0 to 1) 0.33 (0 to 2) 2.33 (0 to 4) 0 0 3.33 (0 to 7) 0.58 (0 to 2) 0.33 (0 to 2) 1.33 (0 to 7) 45 (0 to 100) 55 (0 to 100)

2.74 ( 6.0 to 1.2) 0.03 (0 to 0.1) 9.25 (8.6 to 9.7) 0.01 ( 0.2 to 0.2) 1.11 (1 to 1.2) 2.2 (2 to 3) 1.1 (0 to 2) 1.1 (0 to 2) 0.2 (0 to 1) 0.95 (0 to 2) 1.4 (1 to 2) 0.4 (0 to 1.5) 0.3 (0 to 1) 0.05 (0 to 0.5) 0.4 (0 to 1) 0.2 (0 to 1) 0.4 (0 to 2) 2.4 (0 to 6) 0 0 3.4 (1 to 6) 1.7 (0 to 5) 0.1 (0 to 1) 0.9 (0 to 5) 46 (0 to 100) 52 (0 to 100)

2.75 ( 6.6 to 3.7) 0.05 (0 to 0.1) 9.74 (9.0 to 10.5) 0.07 ( 0.2 to 0.1) 1.18 (1.1 to 1.3) 2.67 (2 to 3) 0.67 (0 to 2) 1.17 (1 to 2) 0.17 (0 to 1) 1 (0 to 1.5) 1.5 (1 to 2.5) 0.58 (0 to 2.0) 0.42 (0 to 1.5) 0.08 (0 to 0.51) 0.67 (0 to 1) 0.67 (0 to 1) 1 (0 to 2) 4 (0 to 8) 0 0 4.67 (3 to 6) 0.67 (0 to 4) 0.67 (0 to 2) 1.83 (0 to 6) 43.33 (0 to 100) 53.33 (0 to 100)

2.96 ( 5.5 to 1.9) 0.07 (0 to 0.1) 10.07 (9.6 to 10.8) 0.06 ( 0.1 to 0.1) 1.13 (1 to 1.3) 2.14 (2 to 3) 1 (0 to 2) 0.86 (0 to 1) 0.36 (0 to 1) 0.79 (0 to 1.5) 0.79 (0.5 to 1.5) 0.21 (0 to 1.5) 0 (0 to 0) 0 (0 to 0) 0.71 (0 to 1) 0.29 (0 to 1) 0.71 (0 to 3) 6.71 (0 to 10) 0.57 (0 to 2) 2.86 (0 to 7) 0.5 (0 to 2) 1.07 (0 to 3) 0.14 (0 to 1) 40 (0 to 100) 60 (0 to 100)

1.09 ( 4.1 to 6.8) 0.01 (0 to 0.1) 13 (11.7 to 14.3) 0.07 ( 0.2 to 0.1) 1.09 (1 to 1.2) 2.13 (2 to 3) 1.13 (0 to 2) 1 (0 to 2) 0.25 (0 to 1) 1.69 (1.5 to 2.5) 1.13 (0.5 to 1.5) 0.63 (0 to 2.5) 0.5 (0 to 1) 0.06 (0 to 0.5) 0.63 (0 to 1) 0.38 (0 to 1) 0.25 (0 to 2) 2.75 (0 to 10) 0 0 2.06 (0 to 7) 0.5 (0 to 4) 1.88 (0 to 5) 7.63 (1 to 17) 28.75 (80 to 100) 68.75 (20 to 100)

6.39 ( 9.2 to 1.9) 0.08 (0 to 0.1) 12.57 (11.7 to 13.0) 0.01 ( 0.1 to 0.1) 1.18 (1.2 to 1.2) 2.33 (2 to 3) 0.67 (0 to 2) 0.33 (0 to 1) 0.17 (0 to 0.5) 0.5 (0 to 1.5) 0.67 (0 to 2) 2 (2 to 2) 0.67 (0 to 1) 0.17 (0 to 0.5) 1 (1 to 1) 0.33 (0 to 1) 2.67 (0 to 5) 6.33 (4 to 10) 0 0 6.67 (6 to 7) 1.67 (0 to 3) 0 0 8.67 (0 to 18) 0 93.33 (180 to 100)

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as mast cells with variable degrees of granulation are present in mast cell neoplasia.16,19 During step 1, only eight dogs with malignant lymphoma were diagnosed. The remaining biopsy samples were used for step 2. In step 2, clusters of epithelial cells with cytologic characteristics of carcinoma cells, such as anisocytosis, anisokaryosis, multiple or abnormal nucleoli, variable nucleus/cytoplasm ratio, and abnormal nuclear characteristics including clumped chromatin patterns, intermixed with normal hepatocytes, were indicative for metastatic carcinoma or cholangiocellular carcinoma. The remaining biopsy samples were used for step 3. In step 3, a logistic regression was performed to distinguish normal liver biopsy samples from abnormal biopsy samples not diagnosed in step 1 or step 2. Those variables with a P-value less than 0.2 on univariate regression score were included in the model. With the multiple regression model, only parameters cell borders, vacuoles in cytoplasm, chromatin pattern, and presence of neutrophils were considered signicant. The following regression formula was calculated: Y 1 6.1(cell borders) 5.9(cytoplasmic vacuoles) 0.1(chromatin pattern) 2.9(neutrophils). The probability of an abnormal liver biopsy sample was P (1 exp(Y))1. Values more than 0.50 were considered indicative for abnormal liver biopsy samples, and these can be used for step 4. Using a cutoff point of 0.50, 26 of the 28 normal and 50 of the 52 abnormal biopsy samples were correctly classied. All 52 abnormal biopsy samples were introduced in step 4. In step 4, a logistic regression was performed to distinguish hepatocellular carcinoma from other abnormal biopsy samples not diagnosed in steps 1 through 3. Those variables with a P-value less than 0.2 on univariate regression score were included in the model. With the multiple regression model, only parameters age-corrected nucleus/cytoplasm ratio, agecorrected cell size, number of nucleoli per nucleus, intracellular bile pigment content, and number of lymphocytes were considered signicant. The following regression formula was calculated: Y 367.5 1469.7(age-corrected nucleus/cytoplasm ratio) 345.6(nucleoli) 11.9(age-corrected cell size) 64.9(intracellular bile) 24.4(lymphocytes). The probability of hepatocellular carcinoma was P (1 exp(Y))1. Values more than 0.50 were con-

sidered indicative for hepatocellular carcinoma. Using a cutoff point of 0.50, all 12 hepatocellular carcinomas were correctly classied, and 40 other biopsy samples were correctly classied as not being hepatocellular carcinoma. The 40 biopsy samples were used for step 5. In step 5, a logistic regression was performed to distinguish acute or chronic extrahepatic cholestasis from other abnormal biopsy samples, not diagnosed in step 1 through step 4. Those variables with a P-value less than 0.2 on univariate regression score were included in the model. With the multiple regression model, only parameters extracellular bile, maximal number of nucleoli, age-corrected cell size, and presence of lymphocytes were considered signicant. The following regression formula was calculated: Y 329 70.2(extracellular bile) 64(maximal number of nucleoli) 14(age-corrected cell size) 14.4(lymphocytes). The probability of extrahepatic cholestasis is P (1 exp(Y))1. Values more than 0.50 were considered indicative for extrahepatic cholestasis. Using a cutoff point of 0.50, all six extrahepatic cholestasis biopsy samples were correctly classied and the remaining 34 biopsy samples were correctly classied as not having extrahepatic cholestasis. The 34 biopsy samples were used for step 6. In step 6 a logistic regression was performed to distinguish destructive cholangiolitis from other abnormal biopsy samples not diagnosed in step 1 through step 5. Those variables with a P-value less than 0.2 on univariate regression score were included in the model. With the multiple regression model, only parameters numbers of neutrophils and mean nuclear size were considered signicant. The following regression formula was calculated: Y 1021.6 30(neutrophils) 132.7(mean nuclear size). The probability of destructive cholangiolitis is P (1 exp(Y))1. Values more than 0.50 were considered indicative for destructive cholangiolitis. Using a cutoff point of 0.50, all three destructive cholangiolitis biopsy samples were correctly classied and the 31 other types of biopsy samples were correctly classied as not having destructive cholangiolitis. The 31 biopsy samples were used for step 7. In step 7, a logistic regression was performed to distinguish nonspecic reactive hepatitis samples from other abnormal biopsy samples not diagnosed in steps 1 through 6. Those variables with a P-value less than

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0.2 on univariate regression score were included in the model. With the multiple regression model, only parameters age-corrected number of nuclei per cell, presence of mast cells, maximum size of the nucleus, color of the nucleoli, and number of groups of bile duct cells were signicant. The following regression formula was calculated: Y 2110 3728(age-corrected number of nuclei/cell) 160.3(mast cells) 151.9(maximal nucleus size) 455.5(nucleoli color) 17.7(bile duct cells). The probability of nonspecic reactive hepatitis is P (1 exp(Y))1. Values more than 0.50 were considered indicative for nonspecic reactive hepatitis. Using a cutoff point of 0.50, all 10 nonspecic reactive hepatitis samples were correctly classied and the 21 other types of biopsy samples were correctly classied as not having nonspecic reactive hepatitis. The 21 biopsy samples were used for step 8. In step 8, a logistic regression was performed to distinguish acute hepatitis from CH. Because of the decreasing number of cases left, only those variables with a P-value less than 0.15 on univariate regression score were included in the model. With the multiple regression model, only parameters intracellular bile pigment content, maximum number of nucleoli per nucleus, and number of neutrophils were considered signicant. The following regression formula was calculated: Y 17.5 4.5(intracellular bile) 3.3(maximal number of nucleoli) 0.8(neutrophils). The probability of acute hepatitis is P (1 exp(Y))1. Values more than 0.50 were considered indicative for acute hepatitis. Using a cutoff point of 0.50, four of the six cases of acute hepatitis were correctly classied and 14 of the 15 other types of biopsy samples were correctly classied as not having acute hepatitis. The 15 other biopsy samples were used for step 9. In step 9 a logistic regression was performed to decide if cirrhosis was present in addition to CH. Those variables with a P-value less than 0.1 on univariate regression score were included in the model. With the multiple regression model, only parameters intracellular bile pigment content and number of groups of cuboid bile duct cells were considered signicant. The following regression formula was calculated:

Y 131.7 75(intracellular bile) 75(bile duct groups). The probability of the presence of cirrhosis is P (1 exp(Y))1. Values more than 0.50 were considered indicative for the presence of cirrhosis in addition to the CH. Using a cutoff point of 0.50, all eight cases of cirrhosis and the seven cases without cirrhosis were correctly classied. Discussion Although cytologic examination of liver biopsy samples is becoming more popular, objective cytologic criteria in different liver diseases in the dog have not until now been documented. In the literature, diagnostic criteria are based on histologic criteria or cytologic criteria extrapolated from human studies. Often, these studies did not separate key criteria from secondary criteria. Only rarely a statistical approach has been used to identify important cytologic criteria for a certain hepatic disease.6 By using a stepwise approach, including regression analysis, this study was performed to identify the major cytologic criteria to differentiate between normal and abnormal liver tissue and between the different pathologic abnormalities. A signicant age inuence had been observed in a previous study in healthy dogs for the cytologic parameters cell size, number of nuclei per cell, and nucleus/cytoplasm ratio.25 These parameters were therefore age corrected before they were used in the statistical models of this survey. In this study, lipidosis, steroid-induced hepatopathy and extramedullary hematopoiesis were not included in the regression analysis because these diseases can be easily detected cytologically and are frequently a sequel of other diseases.4,5,14,19,24,26,27 The rst step in the multistep approach was to exclude metastatic tumor disease. High numbers of lymphoblasts within the liver specimens support the diagnosis of malignant lymphoma.16 In this study, the mean number of lymphoblasts was 50% of all nucleated cells present within the biopsy sample; however, in a few cases, the number of lymphoblasts was only 5%. This might be caused by partial tumor remission induced by previous prednisolone therapy or an unequal distribution of lymphoblasts within the liver, which can be missed by FNAB. Diagnosis might be further confused because small numbers of lymphoblasts among plasma cells and lymphocytes may sometimes be detected in patients with CH in this study. Excessive numbers of mast cells or mast cells in sheets as well as mast cells with variable degrees of granulation are detectable in mast cell neoplasia.4,16 Care should be taken not to overdiagnose mast cell

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tumors within the liver. In this study, mast cells could be detected in nearly every disease with higher numbers in nonspecic reactive hepatitis and destructive cholangiolitis. However, these mast cells were mostly well differentiated and appeared as individual cells and not in groups. Furthermore, scattered individual mast cells occur in liver aspirates of healthy dogs.25 At the time of this study, histologically conrmed liver biopsy sample of a mast cell tumor was not available; therefore, this diagnosis was not included in the model. In metastatic carcinoma, normal liver cells intermixed with a distinct population of epithelial cells exhibiting several criteria of malignancy can be detected. These tumors are sometimes difcult to distinguish from primary hepatic carcinoma because of tissue necrosis and reactive cell patterns of liver cells, and identication of the origin of metastatic carcinoma cells by cytologic examination is frequently not possible.1,19 Cochand-Priollet et al.9 observed in humans an overall diagnostic accuracy of cytology of 82.6% in the differentiation of primary and secondary hepatic neoplasia. The use of peroxidase-antiperoxidase staining technique and transmission electron microscopy can be further applied to cytologic specimens, rendering a more accurate diagnosis in selected cases.21 In companion animals, diagnostic accuracy of cytology in the detection of epithelial tumor metastases is not known. After exclusion of metastatic disease, the following step is used to decide whether abnormal cell patterns indicative for primary liver disease are present. Hepatic cell morphology and cell types in healthy dogs have already been described.25 In this survey, less well-dened cell borders of the hepatocytes, cytoplasmic vacuolization, and more pronounced irregular chromatin morphology in addition to excessive numbers of neutrophils were signicant criteria for the identication of primary hepatic disease. These cellular alterations and cell types can be detected in primary neoplastic as well as nonneoplastic hepatic diseases. For the inexperienced cytologist, liver cells can appear very aggressive in different forms of hepatitis and can be confused with hepatocellular carcinoma.4,18 Cytologic diagnosis of hepatocellular carcinoma can be very difcult, particularly in well-differentiated cases.19 In this study, liver cells in hepatocellular carcinoma were characterized by a high nucleus/cytoplasm ratio, larger cell diameters, increased numbers of nucleoli per nuclei, small numbers of cytoplasmic vacuoles, and small numbers of lymphocytes intermixed with hepatocytes. Other characteristics, which could occasionally be observed without reaching signicance, were crowding of liver cells, signicant cellular necrosis, and abundance of cells with irregular and frequently condensed chromatin patterns. Morphologic characteristics of pleomorphic liver cells also have

been described in man, but in well-differentiated tumors, these criteria can be totally absent.13 In human medicine, several criteria to distinguish well-differentiated hepatocellular carcinoma from inammatory or regenerative conditions include cellular crowding with increased nucleus/cytoplasm ratio and enlargement of nucleoli,11 thickened nuclear membrane,8 nuclear vacuolization,7 and giant cells containing multiple nuclei or cells with small satellite nuclei.17 Berman and McNeill3 emphasize that atypical cell populations should show uniformity in contrast to dysplastic cell conditions with markedly atypical cells adjacent to normal-appearing cells. An important diagnostic criterion of hepatocellular carcinoma in man may further be the accumulation of intracytoplasmic hyaline bodies13, which were not detected in this study. In this study, signicant parameters for the detection of extrahepatic cholestasis included a high content of extrahepatic bile pigment in the form of biliary casts and an increased number of nucleoli within hepatocytes on the one hand and a decreased cell size of the hepatocytes and low numbers of lymphocytes on the other hand. Although large quantities of extracellular bile accumulations were detectable in extrahepatic cholestasis and destructive cholangiolitis, mild intrahepatic accumulations could be found in other diseases, including acute hepatitis, nonspecic reactive hepatitis, and CH. The association of extracellular bile pigment accumulation with the presence of extrahepatic cholestasis is a nonspecic change that may occur either in extrahepatic bile obstruction or with intrahepatic cholestasis associated with hepatitis.23 Further identication of destructive cholangiolitis is based on high numbers of neutrophils and small mean nuclear size of hepatocytes. Although these criteria also might be detectable in histologic slides,28 it is difcult to determine whether pericholangiolar inammatory reactions can be denitively diagnosed using cytologic criteria alone. Diagnosis of acute forms of hepatitis like purulent and nonspecic reactive hepatitis was based on criteria that document moderate reactive nuclear patterns, increased numbers of inammatory cells, excluding lymphocytes, and absence of increased numbers of bile duct groups. The frequency of inammatory cells might be difcult to interpret if slides contain large amounts of blood.22 In these cases consideration of the peripheral leukogram in conjunction with the cytologic observation may be helpful.21 In this study, the presence of increased numbers of mast cells was indicative of nonspecic reactive hepatitis. According to observations in human medicine in CH without cirrhosis, the appearance of high numbers of lymphocytes was obvious, whereas in CH with cirrhosis, fewer lymphocytes combined with increased

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numbers of cuboid biliary epithelial cells were detectable.10,18,23 Although increased numbers of lymphocytes could be detected in every patient with CH, it is questionable, in the face of the heterogenous distribution of lymphocytes in CH, whether cytologic examination is sensitive enough to detect CH. Lymphocytes are frequently located within dense connective tissue deposits in portal areas inhibiting exfoliation of cells in cytologic biopsy samples.29 In addition, because anatomic relationships are lost in cytologic preparations, zonal inammatory reactions can easily be missed, whereas diffuse distribution of inammatory cells are more likely detected.22 Important cytologic criteria for the diagnosis of liver cirrhosis are intracellular bile accumulation and appearance of increased numbers of bile ductules, probably related to bile duct proliferation in liver cirrhosis. Although bile duct proliferation can occur in patients with liver cirrhosis, it can also occur in extrahepatic cholestasis and inammatory conditions of the bile duct system. More specic morphologic criteria of CH and cirrhosis, including several types of necrosis, brosis, and formation of regenerative nodules, cannot be diagnosed using cytologic examination. It should be emphasized that CH and cirrhosis were the remaining groups in this stepwise logistic regression analysis. Of course, there exists more than the 11 liver diseases represented in this analysis, and in a larger series, it may prove necessary to apply additional criteria to diagnose CH. Another point is that we have used smears of parts of the histologic biopsy samples, which makes comparison between cytology and histology of the same specimen possible. However, in practice, cytologic specimens will usually be obtained by FNAB. It remains to be investigated whether this described multistep approach also is valid for ne-needle aspirates, which may be less representative of the true disease process. In conclusion, this study demonstrated that key cytologic criteria of different hepatobiliary diagnoses can be identied in the dog using a stepwise logistic approach. Based on these results, diagnostic accuracy of cytologic liver examination in the dog by means of a multistep logistic regression method will need to be evaluated in a prospective study. References
1 Alleman AR: Cytologic evaluation of the liver. Proc Am Coll Vet Intern Med Forum 15:46, 1997 2 Armitage P, Berry G: Further analysis of qualitative data. In: Statistical Methods in Medical Research, ed. Armitage P and Berry G, 2nd ed., pp. 371-407. Blackwell Scientic Publications, London, UK, 1987

3 Berman JJ, McNeill RE: Cirrhosis with atypia. A potential pitfall in the interpretation of liver aspirates. Acta Cytol 32:1114, 1986 4 Blue JT, French TW, Meyer DJ: The liver. In: Diagnostic Cytology and Hematology in the Dog and Cat, ed. Cowell RL, Tyler RD, and Meinkoth JH, 2nd ed., pp. 183 194. Mosby, St Louis, MO, 1999 5 Bolliger AP: Cytology of the liver. Proc Eur Soc Vet Intern Med Annu Congr 6:6667, 1996 6 Bottles K, Cohen MB, Holly EA, Chiu SH, Abele JS, Cello JP, Lim RC, Miller TR: A step-wise logistic regression analysis of hepatocellular carcinoma. An aspiration biopsy study. Cancer 62:558563, 1988 7 Brits CJ: Liver aspiration cytology. S Afr Med J 48: 22072214, 1974 8 Carney CN: Clinical cytology of the liver. Acta Cytol 19:244250, 1975 9 Cochand-Priollet B, Chagnon S, Ferrand J, Blery M, Hoang C, Galian A: Comparison of cytologic examination of smears and histologic examination of tissue cores obtained by ne needle aspiration biopsy of the liver. Acta Cytol 31:476480, 1987 10 Dominis M, Cerlek S, Solter D: Cytology of diffuse liver disorders. Acta Cytol 17:205208, 1973 11 Gondos B, Forouhar F: Fine needle aspiration cytology of liver tumors. Ann Clin Lab Sci 14:155158, 1984 12 Jensen AL, Hoier R: Clinical chemical diagnosis of diseases assisted by logistic regression illustrated by diagnosis of canine primary and secondary hepatobilliary diseases. Zentralbl Vetmed A 40:102110, 1993 13 Khanh Nguyen G: Fine-needle aspiration biopsy cytology of hepatic tumors in adults. Pathol Annu 21:321 349, 1986 14 Kristensen AT, Weiss DJ, Klausner JS, Hardy RM: Liver cytology in cases of canine and feline hepatic disease. Compend Cont Educ Pract Vet Small Anim Med 12: 797809, 1990 15 Lettow E: Die blinde Leberpunktion nach Menghini beim Hund. Berl Munch Tierarztl Wochenschr 76:273 277, 1963 16 Lettow E, Saar C: Zytodiagnostik aus Leberpunktaten bei Hunden und Katzen mit maligner Systemerkrankung. Zentralbl Vetmed A 17:613622, 1970 17 Lundquist A: Fine-needle aspiration biopsy for cytodiagnosis of malignant tumour in the liver. Acta Med Scand 188:465470, 1970 18 Lundquist A, Akerman M: Fine-needle aspiration biopsy in acute hepatitis and liver cirrhosis. Ann Clin Res 2: 197203, 1970 19 Meyer DJ, French TW: The liver. In: Diagnostic Cytology of the Dog and Cat, ed. Cowell RL and Tyler RD, pp. 189197. American Veterinary Publications, Goleta, CA, 1989 20 Norusis MJ: SPSS for Windows, Base System Users Guide Release 5.0. SPSS Inc., Chicago, IL, 1992 21 Pinto MM, Avila NA, Heller CI, Criscuolo EM: Fine needle aspiration of the liver. Acta Cytol 32:1521, 1986 22 Roth L: Comparison of liver cytology and biopsy diagnoses in dogs and cats: 56 cases. Vet Clin Pathol 30:35 38, 2001

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23 So derstro m N: The liver. In: Aspiration Biopsy Used as a Direct Adjunct in Clinical Diagnostic Work, ed. So derstro m N, pp. 122136. Grune and Stratton, New York, 1966 24 Stockhaus C, Teske E: Klinische Anwendbarkeit der Leberzytologie bei Hund und Katze. Kleintierpraxis 42: 687701, 1997 25 Stockhaus C, Teske E, van den Ingh T, Rothuizen J: The inuence of age on the cytology of the liver in healthy dogs. Vet Pathol 39: 154158, 2002 26 Taboada J: Fine needle aspiration cytology of the liver:

how do I get it and what does it tell me? ESVC Proc 5860, 1991 27 Teske E: Fine needle aspiration biopsy of the liver, a new tool in the diagnosis of liver disease. Tijdschr Diergeneeskd 117(Suppl 1):1314, 1992 28 Van den Ingh TSGAM, Rothuizen J, Van Zinnicq Bergman HMS: Destructive cholangiolitis in seven dogs. Vet Q 10:240245, 1988 29 Weiss DJ, Blauvelt M, Aird B: Cytologic evaluation of inammation in canine liver aspirates. Vet Clin Pathol 30:193196, 2001

Request reprints from Dr. E. Teske, Department of Clinical Sciences of Companion Animals, Veterinary Faculty, Utrecht University, PO Box 80.154, 3508 TD Utrecht (The Netherlands). E-mail: e.teske@vet.uu.nl.

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