**2. Patients and methods**

#### **2.1. Patients**

13 male patients with co-infection of pulmonary tuberculosis (TB), chronic viral hepatitis C (HCV) and human immunodeficiency virus (HIV) were investigated during the study. All the patients used also the injections of heroin (Table 1).

The patients started their history as a rule from heroin using (mean duration – 9.5 years) and later all of them acquired HCV (mean duration – 7.1 years), HIV (mean duration – 4.7 years) and TB at last (the duration of TB infection of the most part of patients was less than 1 year).

The diagnosis was established after careful examination of the patients: the anamneses of diseases and life, laboratory analyses, virological and morphological studies. Serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was expressed. The upper limit of normal (ULN) of ALT was 41 U/L, AST – 31 U/L.

### **2.2. Histological evaluation**

To refine the diagnosis as well as for detection of the activity of pathological processes in the liver, aspiration biopsy was taken from all the patients. All liver biopsies were performed to the routine medical follow up program, using the standard Menghini procedure [17, 18]. Criteria for adequacy of the biopsy specimens included a core length of 10 mm and at least 5-6 portal tracts. So, only 9 biopsy specimens were used for further histological evaluation. Four biopsy specimens were fragmented and weren't used (Table 1). Samples were formal‐ in-fixed and paraffin-embedded. Serial paraffin sections were cut at 5 mcm. Hematoxylineosin and tolluidine blue stains were used.

Each biopsy for necro-inflammatory activity and fibrosis was assessed by two hepatologists. Knodell Histology Activity Index (HAI) was used to grade histopathological lesions [19]. HAI was graded as minimal (scores 1–3), mild (scores 4–8), moderate (scores 9–12), or severe hepatitis (scores 13–18). METAVIR group scoring system was used for detecting the stage of fibrosis [20]. Fibrosis was staged on the scale from F0 to F4, as follows: F0 = no fibrosis, F1 = portal fibrosis without septa, F2 = few septa, F3 = numerous septa without cirrhosis, F4 = cir‐ rhosis. Only single patient showed any signs of cirrhosis. Fibrosis was also staged by Ishak scoring system[21]. In the Ishak scoring system interface hepatitis (piecemeal necrosis), focal necrosis in the lobule, portal inflammation were scored from 0 to 4, incomplete cirrhosis (bridging necrosis with occasional nodules) and cirrhosis were scored from 5 to 6.

Computer Image Analysis of Liver Biopsy Specimens in Patients with Heroin Abuse and Coinfection ... http://dx.doi.org/10.5772/52971 237


**Table 1.** Characteristics of patients with heroin abuse and co-infection of TB, HCV, HIV. The patients are arranged according to their age.

#### **2.3. Computer digital analysis**

ciency virus (HIV), pulmonary tuberculosis (TB) were studied by the morphological and

13 male patients with co-infection of pulmonary tuberculosis (TB), chronic viral hepatitis C (HCV) and human immunodeficiency virus (HIV) were investigated during the study. All

The patients started their history as a rule from heroin using (mean duration – 9.5 years) and later all of them acquired HCV (mean duration – 7.1 years), HIV (mean duration – 4.7 years) and TB at last (the duration of TB infection of the most part of patients was less than 1 year).

The diagnosis was established after careful examination of the patients: the anamneses of diseases and life, laboratory analyses, virological and morphological studies. Serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was expressed. The

To refine the diagnosis as well as for detection of the activity of pathological processes in the liver, aspiration biopsy was taken from all the patients. All liver biopsies were performed to the routine medical follow up program, using the standard Menghini procedure [17, 18]. Criteria for adequacy of the biopsy specimens included a core length of 10 mm and at least 5-6 portal tracts. So, only 9 biopsy specimens were used for further histological evaluation. Four biopsy specimens were fragmented and weren't used (Table 1). Samples were formal‐ in-fixed and paraffin-embedded. Serial paraffin sections were cut at 5 mcm. Hematoxylin-

Each biopsy for necro-inflammatory activity and fibrosis was assessed by two hepatologists. Knodell Histology Activity Index (HAI) was used to grade histopathological lesions [19]. HAI was graded as minimal (scores 1–3), mild (scores 4–8), moderate (scores 9–12), or severe hepatitis (scores 13–18). METAVIR group scoring system was used for detecting the stage of fibrosis [20]. Fibrosis was staged on the scale from F0 to F4, as follows: F0 = no fibrosis, F1 = portal fibrosis without septa, F2 = few septa, F3 = numerous septa without cirrhosis, F4 = cir‐ rhosis. Only single patient showed any signs of cirrhosis. Fibrosis was also staged by Ishak scoring system[21]. In the Ishak scoring system interface hepatitis (piecemeal necrosis), focal necrosis in the lobule, portal inflammation were scored from 0 to 4, incomplete cirrhosis

(bridging necrosis with occasional nodules) and cirrhosis were scored from 5 to 6.

computer morphometric analysis.

236 Liver Biopsy - Indications, Procedures, Results

**2. Patients and methods**

**2.2. Histological evaluation**

eosin and tolluidine blue stains were used.

the patients used also the injections of heroin (Table 1).

upper limit of normal (ULN) of ALT was 41 U/L, AST – 31 U/L.

**2.1. Patients**

Quantitative morphometric analysis was performed using an image analysis system consist‐ ing of a microscope (Leica DM 2500) with attached digital camera (Leica DFC 320 R2) and a computer. Serial pictures of biopsy slices of patients with co-infection were photographed by light microscope and were saved electronically. Serial microphotographs of biopsies were made by an objective x20. The further process was performed with the computer pro‐ gram Adobe Photoshop CS 5.0. Serial microphotographs were mounted to receive the gener‐ al picture of liver biopsy (Figure 1). The digital image was converted into a binary image. The two-dimensional patterns were measured by direct pixels counting on the binary im‐ ages under simultaneous visual control of the light microscopy.

**Figure 1.** General picture of the liver biopsy composed by computer microscopy (Obj. x20) using Adobe Photoshop CS 5.0. Total area of the biopsy is 11449177 pixels

Three main parameters were used for quantitative evaluation: the total area of portal zones, the total area of intralobular infiltrates and necroses, as well as the total area of hepatic vessels (central and sublobular veins). We considered the total amount of these main param‐ eters as non-parenchymal elements. Liver plates and sinusoids were attributed to the hep‐ atic parenchyma.

The appearance of focal lymphohistiocyte infiltrates and the formation of numerous intra‐ lobular necroses, containing hepatocytes, surrounded by lymphocytes (encircled hepato‐

Computer Image Analysis of Liver Biopsy Specimens in Patients with Heroin Abuse and Coinfection ...

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239

**Figure 3.** Subfigure with two images. Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Variants (a, b) of the development of intralobular necroses containing encircled hepatocytes at the

In the liver parenchyma the narrowing of sinusoids, as without of inflammatory infiltration signs and with the elements of lymphohistiocyte infiltration and chains of lymphocytes in‐

There were features of moderate protein and vacuole dystrophia in all biopsies. In two cases some hepatocytes contained large lipid inclusions (Figure 5). Disturbance of the lobular ar‐

middle part of liver lobule. Hematoxylin-eosin. Obj. x40

side of them, was predominated (Figure 4).

chitecture was observed only in one biopsy (Figure 6).

cytes) were typical to peripheral and middle zones of liver lobules (Figure 3).

The measurement of portions (in percentages) of portal area, foci of intralobular necroses, and vessels was estimated.

### **2.4. Statistical analysis**

Statistical analysis was performed by tabulated processor Microsoft Excel 2003 and STA‐ TISTIKA 9.0.
