**3. Results**

#### **3.1. Features of histopathological structure of biopsies**

Morphological analysis of liver biopsies of the patients – heroin addicts with tuberculosis (TB) and virus (HCV, HIV) co-infection showed that the extension of portal zones, the dam‐ age of limiting plates of liver cells and the formation of piecemeal and bridging necroses took place practically in all biopsies (Figure 2).

**Figure 2.** 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 interface hepatitis with piecemeal necrosis at the peripheral zone of portal tract. Hematoxylin-eosin. Obj. x40

The peripheral regions of the portal zones were usually densely infiltrated by lymphocytes and mononuclear histiocytes (Figure 2). Sometimes the lymphoid aggregates adjacent to the damaged bile ducts were formed. Dense connective tissue elements developed more often around the portal vessels (portal veins and hepatic arteries).

The appearance of focal lymphohistiocyte infiltrates and the formation of numerous intra‐ lobular necroses, containing hepatocytes, surrounded by lymphocytes (encircled hepato‐ cytes) were typical to peripheral and middle zones of liver lobules (Figure 3).

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‐

The measurement of portions (in percentages) of portal area, foci of intralobular necroses,

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

Morphological analysis of liver biopsies of the patients – heroin addicts with tuberculosis (TB) and virus (HCV, HIV) co-infection showed that the extension of portal zones, the dam‐ age of limiting plates of liver cells and the formation of piecemeal and bridging necroses

**Figure 2.** 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 interface hepatitis with piecemeal necrosis at the peripheral

The peripheral regions of the portal zones were usually densely infiltrated by lymphocytes and mononuclear histiocytes (Figure 2). Sometimes the lymphoid aggregates adjacent to the damaged bile ducts were formed. Dense connective tissue elements developed more often

atic parenchyma.

and vessels was estimated.

238 Liver Biopsy - Indications, Procedures, Results

**3.1. Features of histopathological structure of biopsies**

took place practically in all biopsies (Figure 2).

zone of portal tract. Hematoxylin-eosin. Obj. x40

around the portal vessels (portal veins and hepatic arteries).

**2.4. Statistical analysis**

TISTIKA 9.0.

**3. Results**

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

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‐ side of them, was predominated (Figure 4).

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

**Figure 6.** Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Strong

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241

**3.2. Quantitative image analysis for evaluation of pathological changes in liver biopsy**

Quantitative computer image morphometric analysis included three indexes. We calculated separately the square (in pixels) occupied by portal zones, the square of intralobular focal infiltrates and necroses and the square of hepatic vessels (central and sublobular veins).

The portal areas were divided into two groups: the portal zones with primary formation of piece-meal necroses and the portal zones with primary formation of bridging necroses. We took into account the calculation of portal zones fragments and septa. We also subdivided the intralobular damages in liver in two groups: the focal lymphohistiocyte infiltrates with‐

As for hepatic vein, we separately considered terminal hepatic veins (central veins) and sub‐ lobular veins. In each case we estimated the relative square of the above-mentioned indexes in pixels and then calculated the specific parts in percents to the total square of biopsy.

We assigned the total sum of a specific part of portal zones, the specific part of intralobular focal infiltrates and necroses and the specific part of the hepatic veins as non-parenchymal elements. Respectively, the hepatic plates and sinusoids were remained in the composition

Then we calculated the parenchyma indexes as the relation of non-parenchymal elements to the parenchyma; these indexes characterized a certain degree of the replacement of the func‐

out hepatocytes and the intralobular piecemeal necroses with encircled hepatocytes.

**structure**

of the parenchyma.

tioning hepatic tissue.

development of bridging fibroses and disturbance of the lobular architecture. Hematoxylin-eosin. Obj. x10

**Figure 4.** Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Severe infiltration of intralobular sinusoids by lymphocytes and histiocytes at the peripheral zone of liver lobule. Hematoxylineosin. Obj. x20

**Figure 5.** Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Expan‐ sion and infiltration of portal areas, presence of intralobular necroses at the middle zone of liver lobules, deposition of lipid droplets were in some hepatocytes. Hematoxylin-eosin. Obj. x10

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**Figure 6.** Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Strong development of bridging fibroses and disturbance of the lobular architecture. Hematoxylin-eosin. Obj. x10

#### **3.2. Quantitative image analysis for evaluation of pathological changes in liver biopsy structure**

**Figure 4.** Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Severe infiltration of intralobular sinusoids by lymphocytes and histiocytes at the peripheral zone of liver lobule. Hematoxylin-

**Figure 5.** Section of the liver biopsy specimen of a patient with co-infection (TB, HCV, HIV) and heroin abuse. Expan‐ sion and infiltration of portal areas, presence of intralobular necroses at the middle zone of liver lobules, deposition of

lipid droplets were in some hepatocytes. Hematoxylin-eosin. Obj. x10

eosin. Obj. x20

240 Liver Biopsy - Indications, Procedures, Results

Quantitative computer image morphometric analysis included three indexes. We calculated separately the square (in pixels) occupied by portal zones, the square of intralobular focal infiltrates and necroses and the square of hepatic vessels (central and sublobular veins).

The portal areas were divided into two groups: the portal zones with primary formation of piece-meal necroses and the portal zones with primary formation of bridging necroses. We took into account the calculation of portal zones fragments and septa. We also subdivided the intralobular damages in liver in two groups: the focal lymphohistiocyte infiltrates with‐ out hepatocytes and the intralobular piecemeal necroses with encircled hepatocytes.

As for hepatic vein, we separately considered terminal hepatic veins (central veins) and sub‐ lobular veins. In each case we estimated the relative square of the above-mentioned indexes in pixels and then calculated the specific parts in percents to the total square of biopsy.

We assigned the total sum of a specific part of portal zones, the specific part of intralobular focal infiltrates and necroses and the specific part of the hepatic veins as non-parenchymal elements. Respectively, the hepatic plates and sinusoids were remained in the composition of the parenchyma.

Then we calculated the parenchyma indexes as the relation of non-parenchymal elements to the parenchyma; these indexes characterized a certain degree of the replacement of the func‐ tioning hepatic tissue.

The data obtained were summarized in the Tables 2, 4, 5, 6 and 7. The samples of biopsies were arranged in sequence of increasing of non-parenchymal elements in bioptats.

> **Biopsy numbe r**

**ALT activity (U/L)**

C by stereometric point morphometry

tive methods of the Ishak score evaluation (Table 4).

**Total area of morphomet ry (points of intersection s)**

**Total number of microsco pic fields(SU) (x400)**

**Parenchy mal elements, %**

**Nonparenchy mal elements , %**

 15, 29450 95 97,84 2,16 0,02 1,79 0,05 0,32 - 20 18910 61 97,54 2,46 0,03 2,00 0,25 0,21 - 57 37690 126 96,49 3,51 0,04 2,30 0,46 0,75 - 14 17980 58 96,40 3,60 0,04 3,18 0,01 0,42 - 26 46190 149 95,70 4,30 0,04 3,15 0,12 1,02 - 104 70060 226 95,37 4,63 0,05 3,25 0,30 1,09 - 15 86800 280 95,30 4,70 0,05 2,91 0,02 1,77 - 42 37820 122 94,94 5,06 0,05 3,81 0,26 1,00 - 35 80290 259 94,82 5,18 0,05 3,93 0,84 0,41 + 441 89900 290 93,36 6,64 0,07 3,29 2,02 1,32 + 214 54560 176 91,33 8,67 0,09 7,24 0,89 0,55 - 187 70680 228 90,54 9,46 0,10 7,57 1,76 0,13 + 333 47720 152 90,32 9,68 0,11 7,51 1,17 1,00 - 107 32860 106 90,29 9,71 0,11 8,32 1,02 0,37 - 38 53514 193 89,44 10,56 0,12 6,23 2,61 1,71 + 122 49600 160 89,11 10,89 0,12 9,07 1,27 0,54 + 596 75330 243 88,24 11,76 0,13 9,26 1,56 0,94 + 162 60760 196 88,07 11,93 0,14 11,49 0,44 0,00 +

**Table 3.** Quantitative characteristics of liver biopsy specimens of the patients with monoinfection of chronic hepatitis

We made the comparative analysis of histopathological changes in liver biopsy structure in the group of the patients – heroin addicts and co-infected using the standard semi quantita‐

We determined the histological activity index HAI according to Knodell [19]. The stages of the fibrosis development were defined by two ways: with the use of the research group French METAVIR [20] recommendations and with Ishak method [21]. Under the METAVIR system we evaluated the fibroses using five indexes where the maximal evaluation was 4

scores (F4 – cirrhosis). It consisted 6 scores (F6 – cirrhosis) according the Ishak system.

**Ratio of nonparenchy mal elements**

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**Total area of portal area, %**

**Total area of spotty infiltrates ,%**

**Total area of hepatic veins, %**

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**Chains of lymphocy tes (absent or present)**

243

The control group included the analysis of cohort of the patients with the monoinfection of chronic virus hepatitis C (Table 3). The morphometric analysis of liver structure of the pa‐ tients belonging to given group was made earlier with the use of the method of the stereo‐ metric point morphometry [8].

#### *3.2.1. The general characterization of morphometric data*

The analysis showed that the specific part of non-parenchymal elements strongly varied in the group of the patients with co-infection and heroin abuse: from 2.65% to 27.43% (Table 2). Mean value of non-parenchymal elements was 12.08±2.38. The specific part of non-paren‐ chymal elements varied in cases of monoinfection of hepatitis C with the different degree of activity from 2.16% до 11.93%, the mean value was 6.94±0.77. Thus, the mean value of nonparenchymal elements of liver biopsy of the patients – heroin addicts with co-infection ex‐ ceeded the mean value of non-parenchymal elements in liver biopsy of the patients with monoinfection of hepatitis C (HСV) in 1.74 times. The maximal value of the specific part of non-parenchymal elements in case of co-infection was in 2.29 times higher than in cases of monoinfection HCV (Tables 2 and 3).


**Table 2.** Quantitative characteristics of liver biopsy specimens of patients with heroin abuse and co-infection of TB, HCV and HIV by computer morphometric analysis


The data obtained were summarized in the Tables 2, 4, 5, 6 and 7. The samples of biopsies

The control group included the analysis of cohort of the patients with the monoinfection of chronic virus hepatitis C (Table 3). The morphometric analysis of liver structure of the pa‐ tients belonging to given group was made earlier with the use of the method of the stereo‐

The analysis showed that the specific part of non-parenchymal elements strongly varied in the group of the patients with co-infection and heroin abuse: from 2.65% to 27.43% (Table 2). Mean value of non-parenchymal elements was 12.08±2.38. The specific part of non-paren‐ chymal elements varied in cases of monoinfection of hepatitis C with the different degree of activity from 2.16% до 11.93%, the mean value was 6.94±0.77. Thus, the mean value of nonparenchymal elements of liver biopsy of the patients – heroin addicts with co-infection ex‐ ceeded the mean value of non-parenchymal elements in liver biopsy of the patients with monoinfection of hepatitis C (HСV) in 1.74 times. The maximal value of the specific part of non-parenchymal elements in case of co-infection was in 2.29 times higher than in cases of

> **Ratio of nonparenchymal elements**

 97,35 2,65 0,03 1,86 0,52 0,27 - 93,39 6,71 0,07 5,80 0,74 0,17 + 93,07 6,93 0,07 6,43 0,21 0,27 - 90,73 9,27 0,10 7,59 1,33 0,35 + 90,52 9,48 0,10 7,93 0,46 1,08 - 88,87 11,13 0,13 10,41 0,37 0,35 + 83,72 16,28 0,19 14,52 1,53 0,53 + 81,19 18,81 0,23 17,99 0,75 0,07 + 72,57 27,43 0,38 27,16 0,31 0 +

**Table 2.** Quantitative characteristics of liver biopsy specimens of patients with heroin abuse and co-infection of TB,

**Total area of portal zones, %**

**Total area of spotty infiltrates ,**

**Total area of hepatic veins, %**

**Chains of lymphocytes (absent or present)**

**%**

were arranged in sequence of increasing of non-parenchymal elements in bioptats.

metric point morphometry [8].

242 Liver Biopsy - Indications, Procedures, Results

monoinfection HCV (Tables 2 and 3).

HCV and HIV by computer morphometric analysis

**Non-parenchymal elements, %**

**Parenchymal elements, %**

**Biopsy number**

*3.2.1. The general characterization of morphometric data*

**Table 3.** Quantitative characteristics of liver biopsy specimens of the patients with monoinfection of chronic hepatitis C by stereometric point morphometry

We made the comparative analysis of histopathological changes in liver biopsy structure in the group of the patients – heroin addicts and co-infected using the standard semi quantita‐ tive methods of the Ishak score evaluation (Table 4).

We determined the histological activity index HAI according to Knodell [19]. The stages of the fibrosis development were defined by two ways: with the use of the research group French METAVIR [20] recommendations and with Ishak method [21]. Under the METAVIR system we evaluated the fibroses using five indexes where the maximal evaluation was 4 scores (F4 – cirrhosis). It consisted 6 scores (F6 – cirrhosis) according the Ishak system.

Standard semi quantitative analysis methods for the most part of biopsies (6 patients from 9) made possible to determine the same fibrosis stage: F3 according to the Ishak system and F2 according to the METAVIR system (Table 4).

*3.2.2. Computer image analysis of portal zones*

dexes were analyzed in details (Table 5).

%

**Total area of**

Number per biopsy

co-infection of TB, HCV and HIV by computer morphometric analysis

nificant, it changed from 1 to 10 (mean value was 6.44±1.01).

changed from 0.12% to 3.37% (mean value was 1.29±0.37).

Total area, % Number per biopsy

**Total area of nonparenchy mal elements, %**

11.08±2.42) (Table 5).

**Biopsy number**

The majority of non-parenchymal elements were situated in portal zones. Therefore these in‐

Minimal size, %

 2,65 1,86 5 0,00 0 0 0 1,86 5 0,14 0,99 6,71 5,80 13 2,98 5 0,10 0,86 2,82 8 0,10 1,23 6,93 6,43 7 0,18 2 0,06 0,12 6,25 5 0,01 3,93 9,27 7,59 9 4,94 4 0,16 2,38 2,65 5 0,14 0,97 9,48 7,93 8 4,89 4 0,90 1,45 3,04 4 0,37 1,35 11,13 10,41 12 0,62 2 0,19 0,42 9,79 10 0,16 2,92 16,28 14,52 12 1,13 2 0,47 0,66 13,39 10 0,12 4,51 18,81 17,99 5 7,92 4 1,17 3,37 10,07 1 0,01 10,07 27,43 27,16 18 11,17 8 0,25 2,39 15,96 10 0,17 4,34

**Table 5.** Quantitative characteristics of portal zones in liver biopsy specimens of the patients with heroin abuse and

The amount of portal zones studied in each biopsy varied from 5 to 18. It depended on the total biopsy volume. The mean value of the portal zones number was 9.8 9±1.34. The amount of portal zones with piecemeal necroses varied from 2 to 8 (mean value was 3.44±0.72). The amount of portal zones with the septa and bridging necroses was more sig‐

In one case (biopsy specimen № 8) the portal zone included several portal tracts forming the extensive confluent bridging necrosis.Thus, the amount of portal zones with bridging ne‐ croses (6.44) exceeded in 1.87 times the amount of portal zones with piecemeal necroses (3.44). The total specific part of portal zones varied from 1.86% to 27.16% (mean value was

The specific part of portal zones with piecemeal necroses varied from 0.18% to 11.17% (mean value was 3.76±1.21). The minimal size of such portal zones characterized mainly its fragment, it changed from 0.06% to 0.9% (mean value is 0.37±0.13). The maximal sizes of such portal zones characterized in general the degree of the portal zone extension, they

**portal zones Portal area with piecemeal necroses Portal area with bridging necroses**

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Maximal size, %

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Maximal size, %

245


**Table 4.** Comparative characteristics of non-parenchymal elements specific parts, grading of histopathological lesions (HAI) and the stages of fibrosis in liver biopsy specimens of the patients with heroin abuse and co-infection of TB, HCV and HIV by computer morphometry and semi quantitative evaluation

At that time the quantitative computer image morphometric analysis showed (Table 2) that among studied biopsies the specific parts of non-parenchymal elements differed significant‐ ly in various biopsies at the same fibrosis stages.

The minimal value of the specific part of non-parenchymal elements was 2.65%. These val‐ ues were 6.71% и 6.93% (two biopsy specimens), 9.27% and 9.48% (two other specimens of biopsy) and 11.13% (one biopsy specimen). Thus, in this case the methods of the semi quanti‐ tative score evaluation reflected only common regularities of the process of the fibrosis devel‐ opment. Meanwhile, the quantitative value of fibrosis was very essential for decision making of the medical treatment tactic and the estimation of the medical treatment effectiveness.

The quantitative value of fibrosis is especially important in the process of repeated studies for the determination of positive or negative dynamics of the fibrosis development. The his‐ tological activity index HAI according to Knodell proved to be more informative. HAI in‐ creased gradually from 8 to 16 points in accordance with the increasing of specific parts of non-parenchymal elements in biopsies.

#### *3.2.2. Computer image analysis of portal zones*

Standard semi quantitative analysis methods for the most part of biopsies (6 patients from 9) made possible to determine the same fibrosis stage: F3 according to the Ishak system and F2

> **Stage of fibrosis by score Ishak**

**Stage of fibrosis by score METAVIR**

**HAI by score Knodell**

 2,65 8 F3 F2 6,71 8 F3 F2 6,93 8 F3 F2 9,27 9 F3 F2 9,48 10 F3 F2 11,13 12 F3 F2 16,28 15 F4 F3 18,81 11 F3 F2 27,43 16 F5 F4

**Table 4.** Comparative characteristics of non-parenchymal elements specific parts, grading of histopathological lesions (HAI) and the stages of fibrosis in liver biopsy specimens of the patients with heroin abuse and co-infection of TB, HCV

At that time the quantitative computer image morphometric analysis showed (Table 2) that among studied biopsies the specific parts of non-parenchymal elements differed significant‐

The minimal value of the specific part of non-parenchymal elements was 2.65%. These val‐ ues were 6.71% и 6.93% (two biopsy specimens), 9.27% and 9.48% (two other specimens of biopsy) and 11.13% (one biopsy specimen). Thus, in this case the methods of the semi quanti‐ tative score evaluation reflected only common regularities of the process of the fibrosis devel‐ opment. Meanwhile, the quantitative value of fibrosis was very essential for decision making of the medical treatment tactic and the estimation of the medical treatment effectiveness.

The quantitative value of fibrosis is especially important in the process of repeated studies for the determination of positive or negative dynamics of the fibrosis development. The his‐ tological activity index HAI according to Knodell proved to be more informative. HAI in‐ creased gradually from 8 to 16 points in accordance with the increasing of specific parts of

according to the METAVIR system (Table 4).

**Nonparenchymal elements, %**

and HIV by computer morphometry and semi quantitative evaluation

ly in various biopsies at the same fibrosis stages.

non-parenchymal elements in biopsies.

**Biopsy number**

244 Liver Biopsy - Indications, Procedures, Results

The majority of non-parenchymal elements were situated in portal zones. Therefore these in‐ dexes were analyzed in details (Table 5).


**Table 5.** Quantitative characteristics of portal zones in liver biopsy specimens of the patients with heroin abuse and co-infection of TB, HCV and HIV by computer morphometric analysis

The amount of portal zones studied in each biopsy varied from 5 to 18. It depended on the total biopsy volume. The mean value of the portal zones number was 9.8 9±1.34. The amount of portal zones with piecemeal necroses varied from 2 to 8 (mean value was 3.44±0.72). The amount of portal zones with the septa and bridging necroses was more sig‐ nificant, it changed from 1 to 10 (mean value was 6.44±1.01).

In one case (biopsy specimen № 8) the portal zone included several portal tracts forming the extensive confluent bridging necrosis.Thus, the amount of portal zones with bridging ne‐ croses (6.44) exceeded in 1.87 times the amount of portal zones with piecemeal necroses (3.44).

The total specific part of portal zones varied from 1.86% to 27.16% (mean value was 11.08±2.42) (Table 5).

The specific part of portal zones with piecemeal necroses varied from 0.18% to 11.17% (mean value was 3.76±1.21). The minimal size of such portal zones characterized mainly its fragment, it changed from 0.06% to 0.9% (mean value is 0.37±0.13). The maximal sizes of such portal zones characterized in general the degree of the portal zone extension, they changed from 0.12% to 3.37% (mean value was 1.29±0.37).

The specific part of the portal zones with bridging necroses (Table 5) changed from1.86% to 15.96% (mean value was 7.31±1.63). Thus, the specific part of portal zones with bridging ne‐ croses was practically in 2 times (1.94) more than the specific part of portal zones with piece‐ meal necroses.

**Biopsy number**

was 0.15±0.02).

**Total area of nonparenc hymal elemen ts, %**

**Total area of intralob ular necrose s, %**

**Total number of intralob ular necrose s per biopsy**

**Total area ,%**

abuse and co-infection of TB, HCV and HIV by computer morphometric analysis

maximal infiltrate was 0.08% (mеan value was 0.02±0.01).

nated, they arranged mainly in the middle zones of lobules.

**Total number per biopsy**

**Focal lymphohistiocyte infiltrates**

**Minimal size, %**

 2,65 0,52 8 0,01 1 0,01 0,01 0,51 7 0,04 0,18 6,71 0,74 22 0,03 6 0 0,01 0,71 16 0,02 0,11 6,93 0,21 6 0,05 2 0,02 0,04 0,16 4 0,02 0,08 9,27 1,33 28 0,09 6 0,01 0,02 1,24 22 0,02 0,25 9,48 0,46 10 0,01 1 0,01 0,01 0,45 9 0,02 0,12 11,13 0,37 16 0,03 3 0,01 0,01 0,34 13 0,01 0,06 16,28 1,53 38 0,10 11 0,01 0,08 1,52 28 0,01 0,36 18,81 0,75 10 0 0 0 0 0,75 10 0,06 0,11 27,43 0,31 9 0,04 3 0,01 0,02 0,28 6 0,03 0,06

**Table 6.** Quantitative characteristics of intralobular necroses in the liver biopsy specimens of the patients with heroin

The total specific part of intralobular necroses varied from 0.21% to 1.53% (mеan value was 0.69±0.14). The specific part of the focal intralobular infiltrates varied from 0.01% to 0.1% (mеan value was 0.04±0.01). The size of the minimal infiltrate was only 0.01%, the size of the

The total specific part of intralobular piecemeal necroses varied from 0.16% to 1.52% (mеan value was 0.66±0.14). The minimal size of the specific part of intralobular piecemeal necroses was 0.01% (mеan value was 0.03±0.01), whereas their maximal size was 0.36 % (mеan value

The analysis of the total biopsy specimen (Figure 1) allowed attributing the topography of the intralobular necroses distribution. Thus, under the middle degree of the parenchyma in‐ jury (HAI according to Knodell system up to 10 points) the small lymphohistiocyte infil‐ trates dominated in periportal zones of lobules. Under the high activity of the process (HAI according to Knodell scoring system exceeded 10 points) the large piecemeal necroses domi‐

Hepatocytes surrounded by lymphocytes were well noticeable in large piecemeal necroses (Figure 3b); it is perhaps connected with hepatocytes death, mediated by lymphocytes.

It is typically that the inflammatory infiltration of sinusoids and the formation "chains" of

lymphocytes in them are mostly expressed in large piecemeal necroses (Figure 4).

**Maximal size, %**

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**Total area, %**

**Intralobular necroses with encircled hepatocytes (piecemeal necroses)**

> **Minimal size, %**

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**Maximal size, %**

247

**Total number per biopsy**

The minimal size of portal zones with bridging necroses characterized in general the septa fragments, it changed from 0.01% to 0.37% (mean value was 0.13±0.03). The maximal size reflected more correctly the specific part of the portal zones with bridging necroses, it changed from 0.97% to 10.07% (mean value was 3.37±0.91).

The quantitative computer image morphometric analysis showed that the significant exten‐ sion of portal zones with the destruction of the limiting plate and the development of piece‐ meal or bridging necroses took place in all bioptats of this patients group. In addition the specific part of portal zones with bridging necroses exceeded considerably (in 1.9 times) the specific part of portal zones with piecemeal necroses.

For comparison: the total specific part of portal zones changed from 1.79% tо 11.49% (mеan value was 5.35±0.68) at chronic hepatitis C monoinfection (Table 3).

Thus, the specific part of portal zones of liver biopsies of the patients – heroine addicts with tuberculosis and virus (HCV, HIV) co-infection was 2.07 times higher than the specific part of portal zones of liver biopsies of the patients with the monoinfection HCV.

Moreover the bridging and piecemeal necroses were absent in the liver of the patients with monoinfection HCV under minimal and low activity. Their appearance was noticed only if

the value of specific parts of non-parenchymal elements exceeded 4.7%.

We have not observed any difference between the amounts of piecemeal and bridging ne‐ croses in biopsy specimens with monoinfection HCV.

#### *3.2.3. Сomputer image analysis of intralobular infiltrates and necroses*

We analyzed the morphometric indexes of intralobular infiltrates and necroses (Table 6).

Intralobular necroses presented in all biopsies, their number varied from 6 to 38 (mean value was 16.33±3.42). The amount of focal intralobular lymphohistiocyte infiltrates was signifi‐ cantly less in comparison with the intralobular necroses containing encircled hepatocytes.

The total number of focal intralobular infiltrates varied in different biopsies from 1 to 11 (mеan value was 3.67±1.09), whereas the total number of intralobular piecemeal necroses varied from 4 to 28 (mеan value was 12.78±2.5).

The relation between piecemeal necroses and focal intralobular infiltrates was especially de‐ monstrative (Table 6). The number of piecemeal necroses in each biopsy was in several times more (up to 10 times) than the number of focal necroses. The total number of intralob‐ ular piecemeal necroses was 115, whereas the number of focal intralobular infiltrates was only 33, i.e. in 3.48 times less.


The specific part of the portal zones with bridging necroses (Table 5) changed from1.86% to 15.96% (mean value was 7.31±1.63). Thus, the specific part of portal zones with bridging ne‐ croses was practically in 2 times (1.94) more than the specific part of portal zones with piece‐

The minimal size of portal zones with bridging necroses characterized in general the septa fragments, it changed from 0.01% to 0.37% (mean value was 0.13±0.03). The maximal size reflected more correctly the specific part of the portal zones with bridging necroses, it

The quantitative computer image morphometric analysis showed that the significant exten‐ sion of portal zones with the destruction of the limiting plate and the development of piece‐ meal or bridging necroses took place in all bioptats of this patients group. In addition the specific part of portal zones with bridging necroses exceeded considerably (in 1.9 times) the

For comparison: the total specific part of portal zones changed from 1.79% tо 11.49% (mеan

Thus, the specific part of portal zones of liver biopsies of the patients – heroine addicts with tuberculosis and virus (HCV, HIV) co-infection was 2.07 times higher than the specific part

Moreover the bridging and piecemeal necroses were absent in the liver of the patients with monoinfection HCV under minimal and low activity. Their appearance was noticed only if

We have not observed any difference between the amounts of piecemeal and bridging ne‐

We analyzed the morphometric indexes of intralobular infiltrates and necroses (Table 6).

Intralobular necroses presented in all biopsies, their number varied from 6 to 38 (mean value was 16.33±3.42). The amount of focal intralobular lymphohistiocyte infiltrates was signifi‐ cantly less in comparison with the intralobular necroses containing encircled hepatocytes.

The total number of focal intralobular infiltrates varied in different biopsies from 1 to 11 (mеan value was 3.67±1.09), whereas the total number of intralobular piecemeal necroses

The relation between piecemeal necroses and focal intralobular infiltrates was especially de‐ monstrative (Table 6). The number of piecemeal necroses in each biopsy was in several times more (up to 10 times) than the number of focal necroses. The total number of intralob‐ ular piecemeal necroses was 115, whereas the number of focal intralobular infiltrates was

changed from 0.97% to 10.07% (mean value was 3.37±0.91).

specific part of portal zones with piecemeal necroses.

croses in biopsy specimens with monoinfection HCV.

varied from 4 to 28 (mеan value was 12.78±2.5).

only 33, i.e. in 3.48 times less.

value was 5.35±0.68) at chronic hepatitis C monoinfection (Table 3).

of portal zones of liver biopsies of the patients with the monoinfection HCV.

the value of specific parts of non-parenchymal elements exceeded 4.7%.

*3.2.3. Сomputer image analysis of intralobular infiltrates and necroses*

meal necroses.

246 Liver Biopsy - Indications, Procedures, Results

**Table 6.** Quantitative characteristics of intralobular necroses in the liver biopsy specimens of the patients with heroin abuse and co-infection of TB, HCV and HIV by computer morphometric analysis

The total specific part of intralobular necroses varied from 0.21% to 1.53% (mеan value was 0.69±0.14). The specific part of the focal intralobular infiltrates varied from 0.01% to 0.1% (mеan value was 0.04±0.01). The size of the minimal infiltrate was only 0.01%, the size of the maximal infiltrate was 0.08% (mеan value was 0.02±0.01).

The total specific part of intralobular piecemeal necroses varied from 0.16% to 1.52% (mеan value was 0.66±0.14). The minimal size of the specific part of intralobular piecemeal necroses was 0.01% (mеan value was 0.03±0.01), whereas their maximal size was 0.36 % (mеan value was 0.15±0.02).

The analysis of the total biopsy specimen (Figure 1) allowed attributing the topography of the intralobular necroses distribution. Thus, under the middle degree of the parenchyma in‐ jury (HAI according to Knodell system up to 10 points) the small lymphohistiocyte infil‐ trates dominated in periportal zones of lobules. Under the high activity of the process (HAI according to Knodell scoring system exceeded 10 points) the large piecemeal necroses domi‐ nated, they arranged mainly in the middle zones of lobules.

Hepatocytes surrounded by lymphocytes were well noticeable in large piecemeal necroses (Figure 3b); it is perhaps connected with hepatocytes death, mediated by lymphocytes.

It is typically that the inflammatory infiltration of sinusoids and the formation "chains" of lymphocytes in them are mostly expressed in large piecemeal necroses (Figure 4).

So, the histological activity index HAI according to Knodell reached 15 points, the total number of intralobular necroses reached 38 (28 from them were referred to piecemeal ne‐ croses) in the biopsy № 7 (Tables 4 and 6). Remarkably that during the cirrhosis develop‐ ment (biopsy № 9, fibrosis stage according to the METAVIR system scale was F4 – cirrhosis) the total number of intralobular necroses considerably reduced (6 piecemeal necroses and 3 focal infiltrates in one large biopsy; see Figure 6).

On the whole it is possible to note the tendencies to the stable extension of vessels and the damage of its internal walls. In addition, the sharp narrowing of intralobular sinusoids adja‐ cent to above mentioned vessels, took part in the contribution of the impairment of the proc‐ esses of the microcirculation inside of liver lobules. Perhaps the worsening of microcirculation lead to the bypass ways of the circulation, this may be one of the reasons of

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The measurement of liver enzyme activities (serum ALT and AST) are important for diagno‐ sis and assessment of liver diseases in clinical practice. However, ALT levels fluctuate in chronic HCV infection and may fall into the normal range [22].The use of many medications have been associated with elevated ALT levels [23]. In chronic hepatocellular injury, ALT in‐ creasing is more typical than AST. However, when the fibrosis progresses, ALT activity typ‐ ically declines, and the ratio of AST to ALT gradually increases [24], especially during the

We observed the increasing of the ALT and AST levels practically among all the patients

**Ratio of AST/ALT**

**ALT (U/L) Activity of AST (U/L)**

**Table 8.** Activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in liver biopsy

So, the ALT level changed from 36 to 162 points (mean value was 89.4±13.45). The AST level varied from 32 to179 points (mean value was 75±14.25). The AST/ALT ratio varied from 0.53

The mostly expressed increase of ALT and AST levels was discovered in the patients with the samples of biopsy having the specific part of non-parenchymal elements up to 10% (Ta‐

specimens of the patients with heroin abuse and co-infection of TB, HCV and HIV

to 1.10 points (mean value was 0.83±0.06).

 90 48 0,53 36 32 0,88 45 42 0,93 140 90 0,64 162 179 1,10 48 39 0,81 90 68 0,75 88 93 1,05 106 84 0,79

**3.3. Investigation of activity of alanin aminotransferase (ALT) and aspartate**

bridging necroses development.

development of cirrhosis [25,26].

**Biopsy number** **Activity of**

(Table 8).

**aminotransferase (AST)**

#### *3.2.4. Computer image analysis of hepatic vessels*

The amount of venous vessels in biopsy samples varied from 2 to 7 (mean value was 3.33±0.63). The central veins with endothelium which are often damaged predominated in all biopsies (Table 7).


**Table 7.** Quantitative characteristics of hepatic vessels in liver biopsy specimens of the patients with heroin abuse and co-infection of TB, HCV and HIV by computer morphometric analysis

Sublobular veins were observed only in two biopsies, perhaps they did not get into biopsies because of large sizes in comparison with central veins.

The total specific part of the hepatic vessels varied from 0.07% to 1.08% (mean value was 0.34±0.1). The specific part of the central veins varied from 0.17% tо 0.35% (mean value was 0.18±0.04). The minimal size of the central vein was 0.01% (mean value was 0.03±0.01), the maximal size was 0.18% (mean value was 0.08±0.02). The specific part of sublobular veins reached 1.48%, maximal size – 0.95%.

On the whole it is possible to note the tendencies to the stable extension of vessels and the

damage of its internal walls. In addition, the sharp narrowing of intralobular sinusoids adja‐ cent to above mentioned vessels, took part in the contribution of the impairment of the proc‐ esses of the microcirculation inside of liver lobules. Perhaps the worsening of microcirculation lead to the bypass ways of the circulation, this may be one of the reasons of bridging necroses development.

#### **3.3. Investigation of activity of alanin aminotransferase (ALT) and aspartate aminotransferase (AST)**

So, the histological activity index HAI according to Knodell reached 15 points, the total number of intralobular necroses reached 38 (28 from them were referred to piecemeal ne‐ croses) in the biopsy № 7 (Tables 4 and 6). Remarkably that during the cirrhosis develop‐ ment (biopsy № 9, fibrosis stage according to the METAVIR system scale was F4 – cirrhosis) the total number of intralobular necroses considerably reduced (6 piecemeal necroses and 3

The amount of venous vessels in biopsy samples varied from 2 to 7 (mean value was 3.33±0.63). The central veins with endothelium which are often damaged predominated in

> **Terminal hepatic veins (central veins)**

> > Minimal size , %

 2,65 0,27 4 0,27 4 0,09 0,11 0 0 0 0 6,71 0,17 2 0,17 2 0,03 0,14 0 0 0 0 6,93 0,27 3 0,27 3 0,03 0,18 0 0 0 0 9,27 0,35 4 0,35 4 0,07 0,11 0 0 0 0 9,48 1,08 5 0,13 4 0,02 0,05 0,95 1 0,95 0,95 11,13 0,35 7 0,35 7 0,01 0,11 0 0 0 0 16,28 0,53 3 0 0 0 0 0,53 3 0,04 0,31 18,81 0,07 2 0,07 2 0,03 0,04 0 0 0 0 27,43 0 0 0 0 0 0 0 0 0 0

**Table 7.** Quantitative characteristics of hepatic vessels in liver biopsy specimens of the patients with heroin abuse and

Sublobular veins were observed only in two biopsies, perhaps they did not get into biopsies

The total specific part of the hepatic vessels varied from 0.07% to 1.08% (mean value was 0.34±0.1). The specific part of the central veins varied from 0.17% tо 0.35% (mean value was 0.18±0.04). The minimal size of the central vein was 0.01% (mean value was 0.03±0.01), the maximal size was 0.18% (mean value was 0.08±0.02). The specific part of sublobular veins

Maximal size, %

Total area, %

Number per biopsy

**Sublobular veins**

Minimal size, %

Maximal size, %

Number per biopsy

focal infiltrates in one large biopsy; see Figure 6).

*3.2.4. Computer image analysis of hepatic vessels*

**Total area of hepatic vessels , %**

**Total number of hepatic vessels per biopsy**

co-infection of TB, HCV and HIV by computer morphometric analysis

because of large sizes in comparison with central veins.

reached 1.48%, maximal size – 0.95%.

Total area ,%

all biopsies (Table 7).

**Total area of nonparenc hymal elemen ts, %**

248 Liver Biopsy - Indications, Procedures, Results

**Biopsy number** The measurement of liver enzyme activities (serum ALT and AST) are important for diagno‐ sis and assessment of liver diseases in clinical practice. However, ALT levels fluctuate in chronic HCV infection and may fall into the normal range [22].The use of many medications have been associated with elevated ALT levels [23]. In chronic hepatocellular injury, ALT in‐ creasing is more typical than AST. However, when the fibrosis progresses, ALT activity typ‐ ically declines, and the ratio of AST to ALT gradually increases [24], especially during the development of cirrhosis [25,26].

We observed the increasing of the ALT and AST levels practically among all the patients (Table 8).


**Table 8.** Activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in liver biopsy specimens of the patients with heroin abuse and co-infection of TB, HCV and HIV

So, the ALT level changed from 36 to 162 points (mean value was 89.4±13.45). The AST level varied from 32 to179 points (mean value was 75±14.25). The AST/ALT ratio varied from 0.53 to 1.10 points (mean value was 0.83±0.06).

The mostly expressed increase of ALT and AST levels was discovered in the patients with the samples of biopsy having the specific part of non-parenchymal elements up to 10% (Ta‐ ble 8, samples of biopsies № 4 and № 5). As a rule the ferment activity rather reduced under the fibrosis intensification. The AST/ALT ratio was increased in 3 patients. In other cases it was closer to the upper border of the normal level.

posed 15 years, HIV – 13 years, TB – 12 years and HCV – 9 years. In accordance with it the cirrhosis developed in the liver of this patient (see Figure 6) and the segment of non-paren‐ chymal elements reached 27.43%. Among them the specific part of portal zones was preva‐

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The other peculiarity was the presence of the same stage of fibrosis (namely fibrosis F2 by

At that time the segment of non-parenchyma elements in liver of these patients varied from 2.65% to 11.13%, and the specific part of the portal zones changed from 1.86% to 10.41%. The detailed information about discussion questions and interpretation of liver biopsy assess‐

The typical changes included the destruction of limiting plate, the expansion of portal areas and the development of interface hepatitis, formation of short septa or bridging necroses. The image analysis allows calculating of portal zones areas and intralobular infiltrates in dif‐ ferent fields of biopsy vision. The expansion of portal zones took place especially during the development of interface hepatitis. As a rule, intensive lymphohistiocyte infiltration pre‐

The region of intralobular infiltrates strongly varies. Our investigation showed that intralob‐ ular infiltrates developed as a result of lymphocyte-mediated death of hepatocytes (apoptosis).

Earlier we studied the apoptosis in liver biopsy specimens of the patients with HCV with the use of the TUNEL method [42, 43]. TUNEL-marked cells looked as small groups similar to intralobular piecemeal necroses. All morphometric parameters were significantly higher

Morphometric image analysis gives a possibility to evaluate quantitative parameters of nec‐ ro-inflammatory and fibrosis changes in liver biopsy of patients with mixed infections and

It is characteristic that the combination of different infections leads to the progression of liv‐ er inflammation and the increasing of the portion of non-parenchymal elements as a total

The investigation showed significant intensification of necroinflammatory lesions. Lympho‐ histiocyte infiltration was typical both for portal zones and intralobular areas. These mor‐ phological indications could be connected with the change of the immune state of patients as a result of combine effect of bacterial, viral infections and heroin abuse. So, numerous fac‐ tors have been associated with an increased risk of fibrosis progression in liver of such type

sum of portal areas, intralobular infiltrates and distended hepatic vessels.

METAVIR scoring) and F3 (by Ishak scoring) in liver of the majority of the patients.

ment by grading and staging systems was presented in recent works [40, 41].

dominates in such a type of portal zones.

in comparison with monoinfection HCV [8].

**5. Conclusion**

heroin abuse.

of patients.

lent (27.16%).

We have not discovered any direct interconnections between the ferment activity levels and the sizes of the specific parts of intralobular necroses. The intralobular piecemeal necroses were dominant in this group of the patients; perhaps, the hepatocytes destruction was caused by the special mechanism of the cell death (apoptosis).
