High prevalence of significant liver fibrosis and cirrhosis in chronic hepatitis B patients with normal ALT in central Europe
Abstract
The indication for antiviral treatment of patients with chronic hepatitis B is based on serum HBV DNA levels, transaminases, and histological grade and stage. The relation of liver fibrosis and inflammation to ALT activity in chronic hepatitis B infection was investigated in a non-endemic, European setting. A total of 253 patients with chronic hepatitis B who had undergone liver biopsy at the Clinic of Gastroenterology, Hepatology, and Infectious Diseases, Düsseldorf, Germany over the past 19 years (1990–2009) were evaluated. Thirty-nine patients had persistently normal transaminases, 86 patients had ALT with 1–2 × ULN (upper limit of normal) and 128 patients had ALT >2 × ULN. Liver fibrosis or inflammation was defined as significant for stages or grades ≥ 2 according to the Desmet/Scheuer score. Significant liver fibrosis (F ≥ 2) was found in 36%, cirrhosis in 18%, and significant inflammation (G ≥ 2) in 27% of patients with normal transaminases. There was no difference in the stage of liver fibrosis and the frequency of cirrhosis between patients with normal and elevated transaminases. The most important factor associated with the presence of cirrhosis in multivariate analysis was age ≥40 years (P < 0.003). If concomitant factors like elevated GGT or male sex were furthermore present high prevalences of significant liver disease were found. The data indicate that, in a European setting, patients with chronic hepatitis B infection, and normal transaminases frequently have significant liver fibrosis or cirrhosis. Therefore, liver biopsy or liver stiffness measurement (LSM) should be performed in these patients to determine the stage of liver fibrosis. J. Med. Virol. 83:968–973, 2011. © 2011 Wiley-Liss, Inc.
INTRODUCTION
Chronic hepatitis B infection is one of the leading infectious diseases with an estimated 350–400 million people chronically infected worldwide [European Association for the Study of the Liver, 2009; Sorrell et al., 2009]. The natural history of chronic hepatitis B is variable but may lead to cirrhosis and the development of hepatocellular carcinoma which is currently responsible for about 500,000 deaths per year [Perz et al., 2006]. Recent studies have shown that viral load is an independent risk factor for the development of cirrhosis and hepatocellular carcinoma and that risk increases significantly when HBV DNA is above 2,000 IU/ml (∼10,000 cop/ml) [Chen et al., 2006; Iloeje et al., 2006]. Disease progression is furthermore influenced by factors such as viral coinfections (HCV, HDV, or HIV), other liver comorbidities, grade of inflammation, and probably HBV genotypes [Sagnelli et al., 2004; Liaw et al., 2005; Cornberg et al., 2007]. To prevent patients from developing complications of chronic hepatitis B, antiviral treatment with interferons or nucleos(t)ide analogues is indicated.
Current international guidelines for the management of chronic hepatitis B usually recommend antiviral therapy if transaminases and viral load are elevated [Liaw et al., 2005; Cornberg et al., 2007; Lok and McMahon, 2007; European Association for the Study of the Liver, 2009]. Antiviral treatment is also indicated if significant liver inflammation (G ≥ 2) or fibrosis (F ≥ 2) is detectable irrespective of transaminase level. The current guidelines of the European Association for the Study of the Liver (EASL) recommend initiation of antiviral treatment when transaminases are elevated, whereas the American Association for the Study of Liver Diseases (AASLD) recommends treatment only when ALT levels are two times the upper limit of normal (ULN) [European Association for the Study of the Liver, 2009; Keeffe et al., 2006]. However, studies from non-European countries have shown that a high proportion of patients with chronic hepatitis B infection and normal transaminases have histologically significant disease, which represents an indication for antiviral treatment [Ikeda et al., 2006; Lai et al., 2007; Lin et al., 2007; Kumar et al., 2008; Park et al., 2008]. Individuals with normal transaminases who are frequently found among HBe negative patients are of special interest for the future as the rate of HBe negative patients is increasing worldwide [Hadziyannis and Vassilopoulos, 2001]. In the present study, the liver histology of patients with chronic hepatitis B infection were analyzed in a European setting to determine the prevalence of significant liver fibrosis or inflammation in relation to ALT activity.
PATIENTS AND METHODS
Two hundred fifty three patients with chronic hepatitis B infection who underwent liver biopsy between 1990 and 2009 at the Clinic of Gastroenterology, Hepatology, and Infectious Diseases, Düsseldorf, Germany were analyzed, retrospectively. No patient was receiving antiviral therapy at the time of liver biopsy. Chronic hepatitis was defined as persistence of HBsAg over 6 months. Patients with acute hepatitis B (anti-HBc-IgM positive), coinfection with HCV (anti-HCV positive), HDV (anti-HDV positive), or HIV (anti-HIV positive) were excluded from analysis. Normal transaminases were defined as ALT-levels within the reference range (<23 U/L males, <19 U/L females) at three different time points.
The stage of liver fibrosis was classified according to the Desmet/Scheuer scoring system by two independent pathologists [Desmet et al., 1994], one of them (C.P.) a recognized hepatopathology expert. Discrepancies in staging and grading (<5% of the cases) were discussed using a discussion microscope and a consensus diagnosis was reported. Significant liver fibrosis was defined as a Desmet/Scheuer score ≥ F2. Inflammation was considered to be significant if the grade of inflammatory activity was ≥G2.
HBV DNA was determined by an in-house PCR validated by the Paul Ehrlich Institute or the Cobas Amplicor HBV Monitor Test (Roche Diagnostics, Mannheim, Germany) with a detection limit of 80 IU/ml. HBV genotype was determined by direct sequencing of the HBV surface gene in all patients as has been described previously [Erhardt et al., 2009]. The HBV surface gene sequences of the patients were compared to HBV databank entries of known HBV genotypes E–I with the following GenBank accession numbers: X02763 (for genotype A), D00330 (for genotype B), M12906 (for genotype C), V01460 (for genotype D), X75657 (for genotype E), X69798 (for genotype F), AF 160501 (for genotype G), and AB231908 (for genotype I). Alignments of DNA sequences were carried out with the Lasergene Megalign software (DNAStar Inc., Madison, WI).
Statistical analysis was performed using the SPSS software Vers. 15.0 (SPSS Inc., Munich, Germany). Student's t-test was applied for analysis of continuous variables with normal distribution, the Mann–Whitney U-test and Kruskal–Wallis tests for continuous variables with non-normal distribution and Fisher's exact test and χ2 test for analysis of categorical variables. Logistic regression models were used to examine the association of risk factors with cirrhosis. A P-value of less than 0.05 was considered significant.
This was a retrospective study using data of patients which were collected during routine diagnosis. All patients undergoing liver biopsy had signed a special consent before the procedure. The biopsy samples were delivered to the Department of Pathology for diagnosis, no parts of the samples were used/cut for scientific reasons.
RESULTS
Characteristics of All Patients
The demographic, virological, and histological characteristics of all patients are summarized in Table I. Of the 253 patients, 39 had persistently normal transaminases, 86 had ALT activity within a range of 1–2× ULN and 128 had ALT activity at least twice as high as normal at the time of liver biopsy. One patient with highly elevated transaminases (ALT 2122 U/L) did not have acute hepatitis (anti-HBc IgM negative), but histological signs of the reactivation of chronic hepatitis with cirrhosis. Four patients with normal transaminases at the time of liver biopsy developed spontaneous increase in ALT at subsequent visits and were therefore classified according to their highest ALT activity. Two hundred eight patients (82%) had a Caucasian background; 45 patients (18%) were from Africa or Asia. A significantly higher rate of non-Caucasians was found among patients with normal ALT (Table I) and in HBeAg positive patients compared to HBeAg negative patients.
| ALT ≤ ULN | ALT 1–2 ULN | ALT > 2 ULN | All patients | P-value* | |
|---|---|---|---|---|---|
| N | 39 | 86 | 128 | 253 | |
| Gender | 0.001 | ||||
| Male | 20/39 (51%) | 62/86 (72%) | 102/128 (80%) | 184/253 (73%) | |
| Female | 19/39 (49%) | 24/86 (28%) | 26/128 (20%) | 69/253 (27%) | |
| Age | |||||
| Mean (years) | 39 ± 15 | 41 ± 13 | 40 ± 14 | 40 ± 14 | n.s. |
| Ethnicity | n.s. | ||||
| Caucasian | 28/39 (72%) | 74/86 (86%) | 106/128 (83%) | 208/253 (82%) | (0.064) |
| Non-Caucasian | 11/39 (28%) | 12/86 (14%) | 22/128 (17%) | 45/253 (18%) | |
| Diagnosis of HBV prior to biopsy (years) | 5 ± 6 | 7 ± 9 | 6 ± 8 | 6 ± 8 | n.s. |
| ALT | <0.001 | ||||
| Mean (U/L) | 16 ± 4 | 33 ± 7 | 131 ± 204 | 80 ± 154 | |
| Range (U/L) | 8–22 | 20–45 | 39–2,122 | 8–2,122 | |
| AST | |||||
| Mean ± SD (U/L) | 17 ± 9 | 28 ± 14 | 67 ± 92 | 46 ± 70 | <0.001 |
| GGT | |||||
| Mean ± SD (U/L) | 25 ± 34 | 39 ± 44 | 58 ± 70 | 46 ± 59 | <0.001 |
| HBeAg positive | 16/39 (41%) | 23/86 (27%) | 64/128 (50%) | 103/253 (41%) | n.s. |
| HBV DNA** | |||||
| Mean ± SD (log IU/ml) | 7.16 ± 7.49 | 7.15 ± 7.77 | 7.55 ± 7.25 | 7.39 ± 7.98 | n.s. |
| No viremia (n patients) | 12/39 (31%) | 18/86 (21%) | 16/129 (13%) | 46/253 (18%) | 0.017 |
| HBV genotype | |||||
| A | 4/18 (22%) | 14/51 (27%) | 34/85 (40%) | 52/154 (34%) | n.s. |
| B | 0 | 2/51 (4%) | 3/85 (4%) | 5/154 (3%) | n.s. |
| C | 3/18 (17%) | 5/51 (10%) | 7/85 (8%) | 15/154 (10%) | n.s. |
| D | 11/18 (61%) | 29/51 (57%) | 36/85 (42%) | 76/154 (49%) | n.s. |
| other | 0 | 1/51 (2%) | 5/85 (6%) | 6/154 (4%) | n.s. |
| Histologic stage (mean ± SD) | 1.6 ± 1.5 | 1.9 ± 1.3 | 2.1 ± 1.3 | 1.9 ± 1.3 | n.s. |
| Significant liver fibrosis | 14/39 (36%) | 44/86 (51%) | 76/128 (59%) | 134/253 (53%) | 0.02 |
| Liver cirrhosis | 7/39 (18%) | 17/86 (20%) | 27/128 (21%) | 51/253 (20%) | n.s. |
| Histologic grade (mean ± range) | 1.3 ± 0.7 | 1.6 ± 0.7 | 1.9 ± 0.8 | 1.7 ± 0.7 | 0.002 |
| Significant liver inflammation*** | 8/30 (27%) | 37/73 (51%) | 69/105 (66%) | 114/208 (55%) | 0.001 |
- ALT, alanine aminotransferase, ULN, upper limit of normal, SD, standard deviation, n.s., not significant.
- * Comparison between normal ALT to elevated ALT.
- ** HBV DNA was not available in three patients.
- *** Grade of liver inflammation in liver biopsy only available in 208 patients.
Thirty patients showed elevated ALT despite the absence of viremia. Eleven of these patients presented no histological signs of additional comorbidities apart from alterations which could be attributed to the HBV infection. Sixteen patients (53%) had fatty liver disease confirmed histologically and two patients cholestasis.
Patients with normal ALT were equally distributed in all age groups (data not shown). There was a highly significant difference in histopathological alterations between patients below and above 40 years of age. Above the age of 40 years, a high proportion of significant liver fibrosis, cirrhosis, or significant inflammatory activity was found (Table II).
| <40 years | ≥40 years | P-value* | |
|---|---|---|---|
| Number | 130 | 123 | |
| Histologic stage (mean ± SD) | |||
| All patients | 1.4 ± 1.0 | 2.5 ± 1.4 | 0.001 |
| Elevated ALT | 1.4 ± 1.0 | 2.6 ± 1.3 | <0.001 |
| Normal ALT | 1.5 ± 1.3 | 1.8 ± 1.6 | n.s. |
| Significant liver fibrosis | |||
| All patients | 51/130 (39%) | 83/123 (67%) | <0.001 |
| Elevated ALT | 45/110 (41%) | 75/104 (72%) | <0.001 |
| Normal ALT | 6/20 (30%) | 8/19 (42%) | n.s. |
| Liver cirrhosis | |||
| All patients | 6/130 (5%) | 45/123 (37%) | <0.001 |
| Elevated ALT | 4/110 (4%) | 40/104 (38%) | <0.001 |
| Normal ALT | 2/20 (10%) | 5/19 (26%) | n.s. |
| Significant liver inflammation | |||
| All patients | 52/112 (46%) | 62/96 (65%) | 0.009 |
| Elevated ALT | 49/98 (50%) | 57/80 (71%) | 0.004 |
| Normal ALT | 3/14 (21%) | 5/16 (31%) | n.s. |
| Histologic grade (mean ± SD) | |||
| All patients | 1.5 ± 0.6 | 1.9 ± 0.8 | <0.001 |
| Elevated ALT | 1.6 ± 0.6 | 2.0 ± 0.8 | <0.001 |
| Normal ALT | 1.2 ± 0.4 | 1.4 ± 0.8 | n.s. |
- ALT, alanine aminotransferase, ULN, upper limit of normal, SD, standard deviation, n.s., not significant.
- * Comparison of age <40 years versus ≥40 years.
There was a trend to higher liver fibrosis stages (56% vs. 49%) and lower grades of inflammation (50% vs. 62%) in HBeAg negative patients (data not shown). HBeAg negative patients were older, had lower levels of HBV DNA, and were more likely to be infected with HBV genotype D than HBeAg positive patients.
There was a positive correlation between the level of GGT and stage of liver fibrosis (r = 0.304, P < 0.001) as well as between ferritin and stage of liver fibrosis (r = 0.293, P < 0.001).
Further uni- and multivariate analyses were performed to determine factors associated with cirrhosis (Table III). Univariate analysis included HBeAg status, HBV > 2,000 IU/L vs. HBV < 2,000 IU/L, age ≥ 40 years vs. age < 40 years, GGT (elevated vs. normal), ALT (elevated vs. normal), HBV genotypes, gender, and ferritin levels. Elevated GGT, age ≥ 40 years, elevated ferritin, male gender, and HBV genotype A were associated with a higher risk of cirrhosis. Viral load, HBeAg, and ALT did not show a significant association with cirrhosis in univariate analysis. Factors which were significant in univariate analysis were further studied in multivariate analysis. In multivariate analysis, only age ≥ 40 years remained significantly associated with the presence of cirrhosis (P < 0.003). Patients older than 40 years had an 11-fold higher risk for the presence of cirrhosis than younger patients.
| Parameter | P-value | Odds ratio | 95%CI |
|---|---|---|---|
| Univariate | |||
| GGT elevated versus normal | <0.0001 | 9.5 | 3.9–23.3 |
| Age ≥40 years versus <40 years | <0.0001 | 12 | 4.9–29.4 |
| Ferritin elevated versus normal | 0.03 | 2.3 | 1.1–5.1 |
| Sex: male versus female | 0.018 | 2.8 | 1.2–6.6 |
| HBV Genotype A versus non-A | 0.047 | 2.4 | 1.0–5.8 |
| ALT elevated versus normal | 0.715 | 1.2 | 0.5–2.5 |
| HBeAg negative versus positive | 0.212 | 0.7 | 0.3–1.3 |
| HBV DNA ≥2,000 IU/L versus <2,000 IU/L | 0.093 | 1.7 | 0.9–3.2 |
| Multivariate | |||
| GGT elevated versus normal | 0.066 | 7.5 | 0.9–62.5 |
| Age ≥40 years versus <40 years | 0.003 | 11.2 | 0.7–7.75 |
| Ferritin elevated versus normal | 0.429 | 2.3 | 2.3–55.6 |
| Sex: male versus female | 0.079 | 7 | 0.8–61.3 |
| HBV Genotype A versus non-A | 0.491 | 1.5 | 0.5–4.8 |
- GGT, gamma-glutamyl-transferase; ALT, alanine aminotransferase; CI, confidence interval.
Characteristics of Patients With Persistently Normal ALT
The number of males was lower in patients with normal ALT compared to elevated ALT (P < 0.001). There was no difference in age of patients, HBeAg positivity, viral load, or distribution of HBV genotypes.
Significant liver fibrosis was found in 36% and cirrhosis in 18% of patients with normal transaminases (Table I). The frequency of relevant liver fibrosis increased significantly (P < 0.02) with ALT activity (36% in patients with normal ALT, 51% in patients with ALT 1–2 × >ULN, 59% in patients with ALT > 2 × ULN). The prevalence of cirrhosis was equal in patients with normal ALT in comparison to patients with elevated ALT. Extent and frequency of liver inflammation (P < 0.001) differed significantly between patients with normal and elevated transaminases. Significant inflammation was found in 27% of patients with normal transaminases, in 51% of patients with ALT 1–2× ULN, and in 66% of patients with ALT >2× ULN. Ultrasound of patients with normal transaminases showed chronic liver alterations or signs of liver cirrhosis in 14 patients, 6 patients had signs of fatty liver disease, and 15 patients showed no alterations of the liver. Ultrasound was not available in four patients. In patients with normal ALT and significant liver fibrosis or cirrhosis (n = 14) confirmed histologically, ultrasound diagnosed more severe stages of liver disease correctly in 64% (n = 9) but misclassified 36% (n = 5) of cases.
Analysis of patients with normal or slightly elevated ALT (1–2× ULN) showed high frequencies of significant liver fibrosis or cirrhosis if two or three risk factors were present at the time of liver biopsy (Table IV). Patients ≥40 years with elevated GGT or of male sex had a significantly higher prevalence of liver fibrosis or cirrhosis compared to patients below 40 years. The subgroup of patients ≥40 years, of male sex and with elevated GGT showed the highest rate of significant liver fibrosis (17 of 23 patients) or cirrhosis (13 of 23 patients). A significantly higher frequency of cirrhosis was also found in male patients above 40 years compared to women ≥40 years (43% vs. 13%, P = 0.014).
| Normal GGT | Elevated GGT | Female Sex | Male Sex | Male sex + elevated GGT | |
|---|---|---|---|---|---|
| Age < 40 years | |||||
| Significant liver fibrosis | 18/49 (37%) | 6/16 (38%) | 7/20 (35%) | 17/45 (38%) | 5/13 (38%) |
| Liver cirrhosis | 1/49 (2%) | 4/16 (25%)* | 1/20 (5%) | 4/45 (9%) | 3/13 (23%) |
| Age ≥ 40 years | |||||
| Significant liver fibrosis | 11/28 (39%) | 23/32 (71%)*,** | 9/23 (39%) | 25/37 (67%)**,*** | 17/23 (73%) |
| Liver cirrhosis | 4/28 (14%)** | 15/32 (47%)* | 3/23 (13%) | 16/37 (43%)**,*** | 13/23 (56%) |
- GGT, gamma-glutamyl-transferase; n.s., not significant.
- * P < 0.05 in comparison of normal GGT versus elevated GGT.
- ** P < 0.05 in comparison of age under versus above 40 years.
- *** P < 0.05 in comparison of males versus females.
DISCUSSION
The present study showed a high frequency of significant liver fibrosis or cirrhosis in chronic hepatitis B patients with normal ALT. Among patients with normal ALT activity, 36% had significant liver fibrosis (F ≥ 2) and 18% cirrhosis. Furthermore, 27% of patients with normal ALT showed significant inflammation (G ≥ 2) in liver biopsy. According to the German guidelines for the treatment of HBV infection [Cornberg et al., 2007] initiation of antiviral therapy is indicated in a relevant number of patients with normal transaminases.
A high proportion of significant liver fibrosis in chronic hepatitis B patients with normal ALT was observed in studies including Asian patients [Ikeda et al., 2006; Lai et al., 2007; Kumar et al., 2008; Park et al., 2008]. The frequency of significant liver fibrosis varied in these studies between 13.8% in HBeAg negative patients with normal ALT in India to 60% in South Korean patients with normal or slightly elevated ALT activity. The present study is the first showing a high frequency of significant liver fibrosis in a non-endemic, central European setting with a high prevalence of Caucasians. This study cohort differed from other studies regarding ethnicity (82% Caucasians vs. 18% Asians), route of viral transmission and duration of disease (supposed acquisition in early childhood or later in life vs. vertical transmission), HBV genotypes (A and D vs. B and C), and rate of HBeAg positivity. Nevertheless, consistent with other studies, high frequencies of significant liver disease are found in chronic hepatitis B patients with normal ALT. Given that this study is retrospective and took place in a tertiary centre, severe liver fibrosis may be overrepresented. Comorbidities such as obesity, genetic polymorphisms like Il-28B or high alcohol intake may accelerate chronic liver disease and therefore explain high rates of liver disease despite expected shorter duration of viral infection in Caucasians compared to non-Caucasians [Martinelli et al., 2004; Bondini et al., 2007; Demir et al., 2007; Mota et al., 2010; Thompson et al., 2010].
A recent study from India showed that in patients with normal transaminases ALT flares appear at an annual rate of 4.3% [Kumar et al., 2009]. In the present study, fluctuating transaminases were observed in 9.3% of the patients with normal ALT at initial presentation. If liver biopsy shows no pathological alterations, continuous serological follow-up examinations must be done to identify further patients who may require antiviral treatment.
Despite the absence of viremia at the time of liver biopsy, 30 out of 46 patients had elevated ALT activity. In 60% of these patients, there was a histopathological explanation for the ALT elevation. In the remaining 40%, the ALT elevation has to be considered unclear.
Non-invasive procedures may be helpful for further discrimination of patients with normal ALTs; however most of these tools lack good diagnostic accuracy for the differentiation of lower fibrosis stages. This also holds true for transient elastography which is a reliable tool for the diagnosis of liver cirrhosis but fails to diagnose the lower fibrosis stages which represent an indication for antiviral therapy [Foucher et al., 2006; Marcellin et al., 2009]. Liver biopsy, therefore, is indispensable in the majority of these cases.
Current EASL, AASLD, and APASL guidelines for the treatment of chronic hepatitis B do not give clear recommendations for patients with chronic hepatitis B and normal transaminases. Because of the high proportion of significant liver disease, even in chronic hepatitis B patients with normal transaminases, assessment of liver fibrosis is warranted. Risk factors have to be identified to give definitive recommendations.
In univariate analysis, males are at a higher risk of developing cirrhosis than females (P < 0.018, odds ratio: 2.8, CI: 1.2–6.6) [Kumar et al., 2008; Tsang et al., 2008]. The present study revealed some more risk factors which were associated with an advanced stage of liver fibrosis that could be also identified in patients with normal ALT. According to univariate analysis, these included elevated ferritin, age above 40 years, HBV genotype A and elevated GGT. In chronic hepatitis C, the level of GGT is known to be a negative predictor for a SVR in antiviral treatment, which may be explained by the higher stages of liver fibrosis or steatosis in these patients [Kau et al., 2008; Tahan et al., 2008]. High levels of GGT may be a surrogate marker for liver fibrosis or liver comorbidities (i.e., steatosis).
The frequency of liver fibrosis, cirrhosis, or significant inflammatory activity increased with age. Sixty seven percent of all patients above 40 years of age had significant liver fibrosis and 37% had cirrhosis. Multivariate analysis showed that the most important factor associated with the presence of cirrhosis was age above 40 years (P < 0.003) [Lai et al., 2007]. Therefore, liver biopsy or transient elastography should be considered, especially in older patients with normal ALT.
Table IV demonstrates the prevalence of significant liver fibrosis and cirrhosis in patients with normal or slightly elevated (1–2 × ULN) ALT. Significantly higher frequencies of liver fibrosis (F ≥ 2) were found in subgroups of patients who presented two or three risk factors (elevated GGT, male sex, and age >40 years) at the time of liver biopsy.
In conclusion, this study showed that in a non-endemic area of central Europe, patients with chronic hepatitis B infection and normal transaminases have a high rate of significant liver fibrosis, cirrhosis, or inflammatory activity. In particular, chronic hepatitis B patients aged above 40 years are at risk of having significant liver disease. Concomitant factors like elevated GGT, male sex, elevated ferritin, negative HBeAg, or HBV genotype A are associated with higher stages of liver fibrosis or the presence of cirrhosis. Therefore, biopsy or transient elastography should be performed in these patients to evaluate the status of liver fibrosis and provide an indication for antiviral treatment.
Statement
The present data were collected from patients who were biopsied for grading and staging of their chronic hepatitis B infection as part of the clinical routine. All patients undergoing liver biopsy had signed a special consent before the procedure. The biopsy samples were delivered to the Department of Pathology for diagnosis, none of the samples were used/cut for scientific reasons.
