InTechOpen uses cookies to offer you the best online experience. By continuing to use our site, you agree to our Privacy Policy.

Medicine » "Trends in Helicobacter pylori Infection", book edited by Bruna Maria Roesler, ISBN 978-953-51-1239-6, Published: April 3, 2014 under CC BY 3.0 license. © The Author(s).

Chapter 8

Helicobacter pylori and Liver – Detection of Bacteria in Liver Tissue from Patients with Hepatocellular Carcinoma Using Laser Capture Microdissection Technique (LCM)

By Elizabeth Maria Afonso Rabelo-Gonçalves, Bruna Maria Röesler and José Murilo Robilotta Zeitune
DOI: 10.5772/57080

Article top


Images of LCM technology used in the study. In A is represented the complete PALM MicroBeam system. In B is illustrated the path of the laser beam passing through the objective lens to reach the tissue slice and the catapulting process for capturing the cells of interest into the Eppendorf cap. Source of the images:
Figure 1. Images of LCM technology used in the study. In A is represented the complete PALM MicroBeam system. In B is illustrated the path of the laser beam passing through the objective lens to reach the tissue slice and the catapulting process for capturing the cells of interest into the Eppendorf cap. Source of the images:
Results of amplification of H. pylori 16S rRNA gene. 1, 2 and 5 are positive samples; 3 and 4 are negative samples. MM: molecular marker, PC: positive control and NC: negative control.
Figure 2. Results of amplification of H. pylori 16S rRNA gene. 1, 2 and 5 are positive samples; 3 and 4 are negative samples. MM: molecular marker, PC: positive control and NC: negative control.
Optical microscopy of FFPE liver fragments of HCC patients with PCR-positive H. pylori 16S rRNA and stained with carbol fuchsin. In (A) and (C) bacteria are represented within sinusoid (arrows) before microdissection (magnification: 610X). In (B) and (D) the same samples are represented after bacterial microdissection (magnification: 610X).
Figure 3. Optical microscopy of FFPE liver fragments of HCC patients with PCR-positive H. pylori 16S rRNA and stained with carbol fuchsin. In (A) and (C) bacteria are represented within sinusoid (arrows) before microdissection (magnification: 610X). In (B) and (D) the same samples are represented after bacterial microdissection (magnification: 610X).
Electropherogram sequence of H. pylori 16S rRNA gene and alignment results of the BLAST databases.
Figure 4. Electropherogram sequence of H. pylori 16S rRNA gene and alignment results of the BLAST databases.

Helicobacter pylori and Liver – Detection of Bacteria in Liver Tissue from Patients with Hepatocellular Carcinoma Using Laser Capture Microdissection Technique (LCM)

Elizabeth Maria Afonso Rabelo-Gonçalves1, Bruna Maria Röesler1 and José Murilo Robilotta Zeitune1

1. Introduction

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver in humans [1]. It is the fifth most common cancer in men (523,000 cases per year, 7.9% of all cancers) and the seventh among women (226,000 cases per year, 3.7% of all cancers) [2], with over a half of million new cases diagnosed annually. It is the second leading cause of the cancer related mortality in the world [3,4,5] and its prevalence differs according to geographic location, gender, age and ethnicity [6].

In general, the distribution of HCC cases presents great geographic variation, with higher incidence in Easter Asia and sub-Saharan Africa where infection with hepatitis B virus (HBV) is endemic with rates of over 20 per 100,000 individuals. Mediterranean countries such as Italy, Spain and Greece have intermediate rates of 10 to 20 per 100,000 individuals. The North and South America have a relatively low incidence (< 5 per 100,000 individuals) [4], although a rising incidence was observed in the USA, probably associated with the rise in hepatitis C virus (HCV) infection. Recent decreases in the incidence of HCC were reported among Chinese populations in Hong Kong, Shangai and Singapore; the incidence in Japan is also decreasing [3].

In Brazil, there are few reports on the prevalence of HCC [7]. It was suggested that the prevalence was considered low according to epidemiologic and retrospective studies [8]. In 1997, Brazilian researchers showed that HVB was the most common cause of liver disease in patients with HCC [9]. After that, another survey demonstrated that in Southeastern and Southern Brazil, HCV accounted for over 55% of cases. In the Northeast and North, HCV accounted for less than 50% and HBV accounted for 22-25% of cases; hepatitis B was more prevalent in the Northern than in the Southern regions [10].

The development of HCC has been attributed to several risk factors. In general, chronic viral infection with HBV and HCV is considered the major cause of HCC in 75-80% of cases [1], although HBV is globally considered the leading risk factor responsible for 50% of cases [11]. Furthermore, cirrhosis [12], exposure to the carcinogenic fungal aflatoxin B1 [13], inherited diseases [14], Wilson’s disease [15] and heavy alcohol consumption [16] are also risk factors attributed to its development. Recently, upcoming risk factors for HCC include obesity, diabetes and related nonalcoholic fatty liver disease [3].

In 1994, researchers described the infectious agent Helicobacter (H.) hepaticus and its role in causing active hepatitis and associated liver tumors in mice [17]. Since then, several studies related to H. hepaticus experimental infection have demonstrated that this bacterium may induce a strong inflammatory change in the liver leading to HCC. Considering that H. pylori was classified as a class I carcinogen [18] and Helicobacter spp. DNA was detected in hepatic tissue from patients with different hepatobiliary diseases, it has been proposed that in humans, as in animals, Helicobacter spp. may also colonize and induce chronic hepatic diseases mainly HCC.

In fact, studies related to the possible association between H. pylori and hepatobiliary diseases have been developed since 1998, when Helicobacter DNA was identified in Chilean patients with chronic cholecystitis [19]. After that, a variety of researches have been conducted to verify the role of H. pylori in the development of HCC [20, 21, 22, 23, 24, 25, 26 e 27]. Considering the role of chronic inflammation and infection in the development of cancer, in the case of HCC, future studies should be performed to verify the role of Helicobacter infection in the liver pathophisiology [28]. However, whether this bacterium causes liver tumor or acts as a cofactor in the process of carcinogenesis needs to be confirmed.

The mechanism by which H. pylori colonizes the human liver is not totally enlightened. The H. pylori DNA detected in the liver tissue may result from bacterial translocation from the stomach into the blood through the portal system, especially in the later stages of chronic liver disease when portal hypertension occurs [1, 29, 30]. In addition, the bacteria may reach the liver by phagocytes and macrophages or circulating retrograde transfer from the duodenum [31]. However, the studies involving the growth of H. pylori from the HCC liver reinforce the bacterial colonization ruling out the possibility of retrograde contamination [32, 33]. Additionally, no other bacteria from the digestive tract are associated with human hepatocarcinogenesis [23, 34].

Several researchers have suggested that H. pylori may damage hepatocytes in vitro by a cytopathic effect in a liver and HCC cell lines [35, 36]. Furthermore, it was demonstrated in a HCC cell line (Huh7) that an inoculum of H. pylori was able to adhere and internalize into hepatocytes and this result was also dependent on virulence factors of bacteria [37]. Proteomic analysis of human hepatic cell line (HepG2) co-cultured with H. pylori have revealed that bacteria may exert the pathological effect on HepG2 cells by up-regulating the expression of some proteins enrolled in transcription regulation, signal transduction and metabolism [38].

In most pathology laboratories, archives of formalin-fixed paraffin-embedded (FFPE) tissues represent the only tissue specimens available for routine diagnostics. A major advantage of such archives is that long-term clinical data is often available [39]. Furthermore, another benefit of using FFPE tissues is that they are the easiest to store and transport [40]. Because of this, FFPE tissues have been used in PCR-based studies related to cancer research, genetics, infectious diseases and molecular epidemiology [41]. Additionally, the use of FFPE tissue also allows employing modern transcriptomic and epigenomic methods with nucleic acids [39].

However, isolating high-quality genomic DNA from FFPE sections remains a challenge for researchers. Formalin is the most commonly used tissue fixative worldwide because it offers the best compromise between cost, practicality and morphological fixative properties [39, 42]. However, the fixation of tissue in formalin leads to extensive protein-DNA cross-linking of all tissue components and nucleic acids isolated from these specimens are highly fragmented [43]. This is particularly troublesome when long DNA regions are amplified, old paraffin blocks are used or fixation time is over three days [41, 44]. Because of this, FFPE tissue requires special protocols in order to extract small amounts of DNA suitable for amplification [45]. Nevertheless, methods of DNA extraction from FFPE tissue are generally laborious and time consuming.

Although studies on the role of the H. pylori in the development of HCC were more frequent in the last decade [46], most of them presents a prospective nature. This probably occurs because the retrospective studies frequently employ FFPE liver tissue and DNA extraction is a limiting factor in this type of sample. However, several researchers have detected H. pylori and its virulence factors (vacA genes and 26 kDa) in paraffin embedded liver samples [21, 27, 34, 47, 48].

Laser capture microdissection (LCM) is a technique that has recently become available for isolation of individual or groups of cells from a heterogeneous tissue sections by microscopic visualization. The technique was first described in 1996 by researchers of the National Institutes of Health (NIH) in Bethesda, MD [49] and allows the isolation of cells reducing the interference from nontarget cell population. The method allows selection of unmixed starting material for DNA, RNA or protein extraction for further downstream analyses [50].

The LCM system is based on an inverted light microscope fitted with a laser device to facilitate the visualization and procurement of cells [51]. The PALM MicroBeam System (Carl Zeiss, MicroImaging GmbH, Göottingen, Germany) was used in this study and it is based on the Laser Microdissection and Pressure Catapulting technology. This system consists of an inverted microscope with a motorized stage and a pulsed “cold” nitrogen ultra-violet (UV) laser. The laser is focused through the objective lenses to a micron-sized spot diameter. The narrow laser focal spot allows the ablation of the material while the surrounding tissue remains fully intact. The microscope stage and UV laser are controlled by a PC, and a video camera allows for tissue sections to be displayed on the PC screen. Cells or regions of interest are then identified and manually delineated on the computer screen using the software program. The microscope is then instructed to collect delineated regions. The noncontact capture of homogeneous tissue samples or individual cells is achieved by means of catapulting using PALM’s patented Laser Pressure Catapulting technology. With the same laser, the separated cells, or the selected tissue area, can be directly catapulted into the eppendorf cap containing a depressed lid [52, 53]. The Figure 1 shows the LCM technology employed in this study.

LCM has been used in a wide variety of applications, including pathology [54, 55], organ transplantation [56, 57], gene expression [52, 58] and molecular characterization of cancer cells [59, 60, 61]. LCM is compatible with most stains and tissue preservation techniques including frozen sections, FFPE tissues, cytology preparations and cultured cells [52, 62]. Because of its high precision and accuracy LCM has been successfully employed to isolation of bacterial cells in FFPE tissues including H. pylori [48, 61, 62, 64, 65].


Figure 1.

Images of LCM technology used in the study. In A is represented the complete PALM MicroBeam system. In B is illustrated the path of the laser beam passing through the objective lens to reach the tissue slice and the catapulting process for capturing the cells of interest into the Eppendorf cap. Source of the images:

2. Clinical samples, methods and results

2.1. Clinical samples

This study was carried out utilizing six cases of FFPE liver tissue from patients with HCC from Department of Anatomic Pathology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. The mean ages of patients was 56.0 years, with 4 male cases (66,6%) and 2 female cases (33,4%). All the fragments of liver were obtained during hepatic surgery (either transplantation or partial hepatectomy). These samples were H. pylori positive previously detected by polymerase chain reaction (PCR) with H. pylori specific 16S rRNA primers [27]. The selection criteria for the paraffin blocks included specimens archived for 5 years (2008 to 2012). The present study was approved by the Ethics Committee of the Faculty of Medical Sciences, UNICAMP (CEP 616/2009).

2.2. Methods

2.2.1. Tissue preparation for LCM

Six H. pylori positive samples were cut into 10µm-thick sections and mounted on 0.17mm PEN membrane-covered slides (Carl Zeiss, MicroImaging GmbH, Göttingen, Germany). After slicing, the sections were placed at 60°C for 30 minutes, then deparaffinized in 3 xylene baths (3x1min), rehydrated in decreasing alcohols (100%, 95% e 70%, each for 30 seconds) and washed for 30 seconds in tap water. Further, the routine staining with carbol fuchsin was performed [66]. At this step the sections have remained in the dye for at most 15 seconds. The stained sections were observed under the microscope for the identification of bacteria.

2.2.2. Bacterial microdissection

Stained bacteria were microdissected using a PALM MicroBeam system (Carl Zeiss, MicroImaging GmbH, Göttingen, Germany) (Figure 1A). The areas with target bacteria were traced around and microdissected together with the pieces of thin membrane by laser microbeam and then ejected into the Eppendorf tube cap by a single laser shot (Figure 1B). The tube was stored at -80°C until DNA extraction.

2.2.3. DNA extraction

After microdissection, the cap was inserted into an Eppendorf tube containing 100µl digestion buffer, prepared with 10mM Tris-HCl (pH 8.0), 1mM EDTA, 1% Tween 20 and 0.3% proteinase K. After that, samples remained in a water bath at 56°C for 3 hours and the tube was heated to 95°C for 5 min to inactivate proteinase K. The crude lysate was directly employed as template for PCR. All of these procedures were previously described with minor modifications [48].

2.2.4. PCR amplification

The samples were further amplified by PCR using H. pylori 16S rRNA primers. The sequence of the sense primer (JW21) was 5’-GCGACCTGCTGGAACATTAC-3’(position 691-710) and the antisense primer (JW22) was 5’-CGTTAGCTCCATTACTGGAGA-3’ (position 829-809) and they amplified a product of approximately 129bp [27]. Briefly, 1µl of DNA extracted was added to 25µl PCR mix containing deoxynucleoside triphosphates (dNTPs) at concentrations of 200 µM each, 2.0µl of 25mM MgCl2, 0.25 µl of GoTaq Hot Start Polymerase (Promega Corp., Madison, WI, USA), 4.0 µl of 5X GoTaq Flexi Buffer (supplied with the enzyme) and 20 pmol each primer (Life Technologies, Carlsbed, CA, USA). Amplification reactions included an initial 2-minute denaturation step at 94ºC, followed by 40 cycles of 30 seconds at 94ºC, 30 seconds at 55ºC and 45 seconds at 72ºC. A final extension step for 7 minutes at 72ºC was performed. The DNA extracted from H. pylori from FFPE gastric tissue was used as positive control and distilled water in place of the DNA samples was used as negative control for PCR assays.

2.2.5. Detection of PCR products

For analysis of the amplified products, 5µl of the amplicons were put on 1,5% agarose gels containing 1µg of ethidium bromide per ml. The amplicons were visualized by UV transillumination.

2.2.6. Sequence analysis

The 16S rRNA amplicons were further identified by sequence analysis using ABI Prism Dye Terminator sequencing kit with AmpliTaq DNA polymerase and the ABI 3500xL Sequencer (Applied Biosystems, Foster City, CA, USA). Sequence comparison was then carried out using the Blast program and GenBank databases.

2.3. Results

Analyzing the tissue sections stained with carbol fuchsin, we visualized microorganisms resembling H. pylori mainly in hepatic sinus from HCC samples. The number of cocci was greater than of bacilli (Figure 3).

Our PCR results showed that all six microdissected samples were positive for 16S rRNA gene (Figure 2) and showed 98% similarity to H. pylori 16S rRNA gene by sequence analysis (GeneBank accession number CP003419.1) (Figure 4).


Figure 2.

Results of amplification of H. pylori 16S rRNA gene. 1, 2 and 5 are positive samples; 3 and 4 are negative samples. MM: molecular marker, PC: positive control and NC: negative control.


Figure 3.

Optical microscopy of FFPE liver fragments of HCC patients with PCR-positive H. pylori 16S rRNA and stained with carbol fuchsin. In (A) and (C) bacteria are represented within sinusoid (arrows) before microdissection (magnification: 610X). In (B) and (D) the same samples are represented after bacterial microdissection (magnification: 610X).


Figure 4.

Electropherogram sequence of H. pylori 16S rRNA gene and alignment results of the BLAST databases.

3. Conclusion

The results described here confirm the identification of H. pylori in FFPE liver tissue from patients with HCC. Although specific primers were used for amplification of the H. pylori 16S rRNA gene, we cannot exclude the possibility of cross-reaction of these primers with other Helicobacter spp [65].

Considering the difficulty of DNA extraction from paraffin embedded samples, the use of LCM simplified the achievement of specific DNA because the DNA extraction process was reduced to a single digestion step of bacterial cells without further purification. Consequently, the crude lysate was used directly as template for PCR amplification. This is particularly advantageous when compared to traditional methods of DNA extraction that are generally laborious, toxic and time consuming [67].

Another advantage of using LCM is that it allowed the exact location of H. pylori in the liver, since bacteria were mainly found in the peritumoral tissue. Considering that our samples presented tumoral and peritumoral tissue in the same paraffin block, the technique was highly effective for obtaining a target bacterial population within a selected area in the HCC tissue. This is very important when we consider that the necrotic state and nuclease content of tissues may influence in recovering intact DNA specially when performing traditional methods for DNA extraction [68].

Furthermore, LCM was useful to reduce the interference from nontarget cell population considering that bacteria were found in small quantities in the liver tissue (Figure 3). In relation to nontarget cell population, it is important to consider that the major obstacle in the analysis of tumoral tissue is that it is composed by different cell types including stroma and inflammatory cells [69, 70, 71] and there is a potential dilution effect of the larger quantities of nontarget DNA found in whole tissue sections [64]. Thus, the employment of LCM was very efficient in isolating H. pylori despite of the reduced bacterial quantity in the HCC tissue.

In summary, we suggest that LCM can be extensively applied for identification of H. pylori in FFPE liver tissue. Further studies will be performed in order to amplify virulence genes of bacteria as well as to isolate H. pylori from other tissues using LCM technique.


This work was supported by grants from FAPESP (2009/09889-5) and FAEPEX 101/2011.


1 - Tu QV, Okoli AS, Kovach Z, Mendz GL. Hepatocellular carcinoma: prevalence and molecular pathogenesis of Helicobacter spp. Future Microbiology 2009;4(10) 1283-1301.
2 - GLOBOCAN. International Agency for Research on Cancer (IARC). 2008. (accessed may 2013).
3 - El-Seragh HB. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology 2012;42(6) 1264-1273.
4 - Mittal S & El-Seragh HB. Epidemiology of hepatocellular carcinoma. Consider the population. Journal of Clinical Gastroenterology 2013;47 Suppl S2-6.
5 - World Health Organization. Mortality Database. WHO Statistical Information System. 2008. (accessed may 2013).
6 - Hall AJ & Wild CP. Liver cancer in low and middle income countries. British Medical Journal 2003;326(7397) 994-995.
7 - Carvalho FM, Pereira TA, Gonçalves PL, Jarske RD, Pereira FEL, Louro ID. Hepatocellular carcinoma and liver cirrhosis TP53 mutation analysis reflects a moderate dietary exposure to aflatoxins in Espírito Santo State, Brazil. Molecular Biology Reports 2013; 40(8) 4883-4887.
8 - Bosch FX, Ribes J, Cléries R, Diaz M. Epidemiology of hepatocellular carcinoma. Clinical Liver Diseases 2005;9(2) 191-211.
9 - Gonçalves S, Pereira FF, Gayotto LC. Hepatocellular carcinoma in Brazil: report of a national survey (Florianopolis, SC, 1995). Revista do Instituto de Medicina Tropical de São Paulo 1997;39(3) 165-170.
10 - Carrilho FJ, Kikuchi L, Branco F, Gonçalves CS, Mattos AA. Brazilian HCC Study Group. Clinics 2010;65(12) 1285-1290.
11 - Parkin DM. The global health burden of infection-associated cancers in the year of 2002. International Journal of Cancer 2002;118(12) 3030-3044.
12 - Gao J, Xie L, Yang W, Zhang W, Gao S, Wang J, Xiang Y. Risk factors of hepatocellular carcinoma - current status and perspectives. Asian Pacific Journal of Cancer Prevention 2012;13(3) 743-750.
13 - Soman NR & Wogan GN. Activation of the c-Kis-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturating gradient gel electrophoresis. Proceeding of the National Academy of Sciences 1993;90(5) 2045-2049.
14 - Taddei T, Mistry P, Schilsky ML. Inherited metabolic disease of the liver. Current Opinion in Gastroenterology 2008; 24(3) 278-286.
15 - Kumagi T, Horiike N, Abe M, Kurose K, Iuchi H, Masumoto T, Joko K, Akbar SF, Michitaka K, Onji M. Small hepatocellular carcinoma associated with Wilson’s disease. Internal Medicine 2005;44(5) 493-443.
16 - La Vecchia C. Alcohol and liver cancer. European Journal of Cancer Prevention 2007;16(6) 497-495.
17 - Ward JM, Fox JG, Anver MR, Haines DC, George CV, Collins MJ Jr, Gorelick PL, Nagashima K, Gonda MA, Gilden RV. Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. Journal of the National Cancer Institute 1994;86(16) 1222-1227.
18 - IARC: Working group on the evaluation of carcinogenic risks to human Helicobacter pylori. IARC Monography Evaluation Carcinogenic Risks Human 1994;61 177-240.
19 - Fox JG, Dewhirst FE, Shen Z, Feng Y, Taylor NS, Paster BJ, Ericson RL, Lau CN, Correa P, Araya JC, Roa I. Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis. Gastroenterology 1998;114(4) 755-763.
20 - Ponzetto A, Pellicano R, Leone N, Cutufia MA, Turrini F, Grigioni WF, D'Errico A, Mortimer P, Rizzetto M, Silengo L. Helicobacter infection and cirrhosis in hepatitis C virus carriage: is it an innocent bystander or a troublemaker? Medical Hypotheses 2000;54(2) 275-277.
21 - Dore MP, Realdi G, Mura D, Graham DY, Sepulveda AR. Helicobacter infection in patients with HCV-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Digestive Diseases and Sciences. 2002;47(7) 1638-1643.
22 - Fan XG, Peng XN, Huang Y, Yakoob J, Wang ZM, Chen YP. Helicobacter species ribosomal DNA recovered from the liver tissue of Chinese patients with primary hepatocellular carcinoma. Clinical and Infectious Diseases 2002;35(12) 1555-1557.
23 - Verhoef C, Pot RG, de Man RA, Zondervan PE, Kuipers EJ, IJzermans JN, Kusters JG. Detection of identical Helicobacter DNA in the stomach and in the non-cirrhotic liver of patients with hepatocellular carcinoma. European Journal of Gastroenterology and Hepatology 2003;15(11) 1171-1174.
24 - Pellicano R, Mazzaferro V, Grigioni WF, Cutufia MA, Fagoonee S, Silengo L, Rizzetto M, Ponzetto A. Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma. World Journal of Gastroenterology 2004;10(4) 598-601.
25 - Ito K, Nakamura M, Toda G, Negishi M, Torii A, Ohno T. Potential role of Helicobacter pylori in hepatocarcinogenesis. International Journal of Molecular Medicine 2004;13(2) 221-227.
26 - Rocha M, Avenaud P, Ménard A, Le Bail B, Balabaud C, Bioulac-Sage P, de Magalhães Queiroz DM, Mégraud F. Association of Helicobacter species with hepatitis C cirrhosis with or without hepatocellular carcinoma. Gut 2005;54(3) 396-401.
27 - Pirouz T, Zounubi L, Keivani H, Rakhshani N, Hormazdi M. Detection of Helicobacter pylori in paraffin-embedded specimens from patients with chronic liver diseases, using the amplification method. Digestive Diseases and Sciences 2009;54(7) 1456-1459.
28 - Gonciarz M, Wloch M, Gonciarz Z. Helicobacter pylori in liver diseases. Journal of Physiology and Pharmacology 2006;57(3) 155-161.
29 - Casafont F, Martin L, Pons-Romero F. Bacterial overgrowth in the small intestine in chronic liver disease. In: Blum HE, Bode JC, Bode Ch, Sartor RB(ed). Gut and the liver. London: Kluwer Academic Publishers; 1998. p 332-337.
30 - Tsunneyama K, Harada K, Kono N, Hiramatsu K, Zen Y, Sudo Y, Gershwin ME, Ikemoto M, Arai Y, Nakanuma Y. Scavenger cells with Gram-positive bacterial lipoteichoic acid infiltrate around the damaged interlobular bile ductus of primary biliary cirrhosis. Journal of Hepatology 2001;35(2) 156-163.
31 - Queiroz DMM & Santos A. Isolation of a Helicobacter strain from the human liver. Gastroenterology 2001;121(4) 1023-1024.
32 - Xuan SY, Qiang X, Zhou RR, Shi YX, Jiang WJ. Helicobacter infection in hepatocellular carcinoma tissue. World Journal of Gastroenterology 2006; 12(15) 2335-2340.
33 - Leelawat K, Suksumek N, Leelawat S, Lek-Uthai U. Detection of VacA gene specific for Helicobacter pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients. Southeast Asian Journal of Tropical Medicine and Public Health 2007;38(5) 881-885.
34 - Al-Soud AW, Stenram U, Ljungh A, Tranberg KG, Nilsson HO, Wadstrom T. DNA of Helicobacter spp. and common gut bacteria in primary liver carcinoma. Digestive and Liver Diseases 2008;40(2) 126-131.
35 - Taylor NS, Fox JG, Yan L. In vitro hepatotoxic factor in Helicobacter hepaticus. H. pylori and other Helicobacter species. Journal of Medical Microbiology 1995;42(1) 48-42.
36 - Chen R, Fan XG, Huang Y, Li N, Chen CH. In vitro cytotoxicity of Helicobacter pylori on hepatocarcinoma HepG2 cells. Al Zheng 2004; 23(1) 44-49.
37 - Ito K, Yamaoka Y, Ota H, El-Zimaity H, Graham DY. Adherence, internalization and persistence of Helicobacter pylori in hepatocytes. Digestive Diseases and Sciences 2008; 53(9) 2541-2549.
38 - Zhang Y, Fan XG, Chen R, Xiao ZQ, Feng XP, Tian XF, Chen ZH. Comparative proteome analysis of untreated and Helicobacter pylori-treated HepG2. World Journal of Gastroenterology 2005;11(22) 3485-3489.
39 - Frankel A. Formalin fixation in the ‘-omics’ era: a primer for the surgeon-scientist. ANZ Journal of Surgery 2012;82(6) 395-402.
40 - Rivero ERC, Neves AC, Silva-Valenzuela MG, Sousa SOM, Nunes FD. Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues. Pathology Research and Practice 2006;202(7) 523-529.
41 - Greer CE, Wheeler CM, Manos MM. Sample preparation and PCR amplification form paraffin-embedded tissues. PCR Methods and Application Journal 1994;3(6) S113-S122.
42 - Klopfleisch R, Weiss AT, Gruber AD. Excavation of a buried treasure - DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues. Histology and Histopathology 2011;26(6) 797-810.
43 - Wu L, Pattern N, Yamashiro CT, Chui B. Extraction and amplification of DNA from formalin-fixed, paraffin-embedded tissues. Applied Immunohistochemistry and Molecular Morphology 2002; 10(3) 269-274.
44 - Karlsen F, Kalantari M, Chitemerere M, Johansson B, Hagmar B. Modifications of human and viral deoxyribonucleic acid by formaldehyde fixation. Laboratory Investigation 1994;71(4) 604-11.
45 - Muñoz-Cadavid C, Rudd S, Zaki SR, Patel M, Moser SA, Brandt ME, Gómez BL. Improving Molecular Detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using parafungal PCR. Journal of Clinical Microbiology 2010;48(6) 2147-2153.
46 - Bulajic M, Panic N, Stimec B, Isaksson B, Jesenofsky R, Schneider-Brachert W, Löhr JM. PCR in Helicobacter spp. diagnostic in extragastric malignancies of digestive system. European Journal of Gastroenterology and Hepatology 2012; 24(2) 117-125.
47 - Nilsson HO, Mulchandani R, Tranberg KG, Stenram U, Wadström T. Helicobacter species identified in liver from patients with cholangiocarcinoma and hepatocellular carcinoma. Gastroenterology 2001;120(1) 323-324.
48 - Tian XF, Fan XG, Huang Y, Dai H, Ying RS. Procuration and identification of bacteria in paraffin-embedded liver tissues of hepatocellular carcinoma by laser-assisted microdissection technique. Acta Pathologica, Microbiologica et Imunologica Scandinavica 2008;116(1) 10-15.
49 - Emmer-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA. Laser capture microdissection. Science 1996; 274(1274) 998-1001.
50 - Aaltonen KE, Ebbesson A, Wigerup C, Hedenfalk I. Laser capture microdissection (LCM) and whole genome amplification (WGA) of DNA from normal breast tissue - optimization for genome wide array analyses. BMC Research Notes 2011;4(3) 1-7.
51 - Liu A. Laser capture microdissection in the tissue biorepository. Journal of Biomolecular Techniques 2010; 21(3) 120-125.
52 - Esposito G. Complimentary techniques: laser capture microdissection - increasing specificity of gene expression profiling of cancer specimens. Advances in Experimental Medicine and Biology 2007; 593: 54-65.
53 - Sluka P, O’Donnell L, McLachlan R, Stanton PG. Application of laser-capture microdissection to analysis of gene expression in the testis. Progress in Histochemistry and Cytochemistry 2007; 42(2008) 173-201.
54 - Agar NS, Halliday GM, Barnetson RS, Jones A.M. A novel technique for the examination of skin biopsies by laser capture microdissection. Journal of Cutaneous Pathology 2003; 30(4) 265-270.
55 - Okuduku AF, Hahne JC, Von Deimling A, Wernert N. Laser-assisted microdissection, techniques and applications in pathology. International Journal of Molecular Medicine 2005; 15(5) 763-769.
56 - Kleheergher W, Rothämel T, Glöckner S, Lehmann U, Kreipe H. Laser-assisted microdisscetion and short tandem repeat PCR for the investigation of graft chimerism after solid organ transplantation. Pathobiology 2000;68(4-5)196-201.
57 - Lehmann U, Versmold A, Kreipe H. Combined laser-assisted microdissection and short tandem repeat analysis for detection of in situ microchimerism after solid organ transplantation. Methods in Molecular Biology 2005; 293: 113-123.
58 - Wang L, Zhu JS, Song MQ, Chen GQ, Chen JL. Comparison of gene expression profiles between primary tumor and metastatic lesions in gastric cancer patients using laser microdissection and cDNA microarray. World Journal of Gastroenterology 2006;12(43) 6949-6954.
59 - Jensen LH, Cruger DG, Lindebjerg J, Byriel L, Bruun-Petersen G, Jakobsen A. Laser microdissection and microsatellite analysis of colorectal adenocarcinomas. Anticancer Research 2006;26(3A) 2069-2074.
60 - Pai CY, Hsieh LL, Tsai CW, Chiou FS, Yang CH, Hsu BD. Allelic alterations at the STR markers in the buccal tissue cells of oral cancer patients and the oral epithelial cells of healthy betel quid-chewers: an evaluation of forensic applicability. Forensic Science International 2002;129(3) 158-67.
61 - Huang C, Yang L, Li Z, Yang J, Zhao J, Dehui X, Liu L, Wang Q, Song T. Detection of CCND1 amplification using laser capture microdissection coupled with real-time polymerase chain reaction in human esophageal squamous cell carcinoma. Cancer Genetics and Cytogenetics 2007;175(1) 19-25.
62 - Cheng L, Zhang S, MacLennan GT, Williamson SR, Davidson DD, Wang M, Jones TD, Lopez-Beltran A, Montironi R. Laser-assisted microdissection in translational research: theory, technical considerations, and future applications. Applied Immunohistochemistry and Molecular Morphology 2013;21(1) 31-47.
63 - Ryan P, Kelly RG, Lee G, Collins JK, O’Sullivan GC, O’Conell J, Shanahan F. Bacterial DNA within granulomas of patients with Crohn’s disease: detection by laser capture microdissection and PCR. American Journal of Gastroenterology 2004;99(8) 1539-1543.
64 - Klitgaard K, Molbak L, Jensen TK, Lindboe CF, Boye M. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization. BioTechniques 2005;39(6) 864-868.
65 - Rabelo-Gonçalves EM, Sgardioli IC, Lopes-Cendes I, Escanhoela CA, Almeida JR, Zeitune JM. Improved detection of Helicobacter pylori DNA in formalin-fixed paraffin-embedded (FFPE) tissue of patients with hepatocellular carcinoma using laser capture microdissection (LCM). Helicobacter 2013;18(3) 244-245.
66 - Rocha GA, Queiroz DMM, Mendes EN, Lage AP, Barbosa A. Simple carbolfuchsin staining for showing C. pylori and other spiral bacteria in gastric mucosa. Journal of Clinical Pathology 1989;42(9) 1004-1005.
67 - Steinau M, Patel SS, Unger ER. Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues. Jounal of Molecular Diagnostic 2011;13(4) 377-381.
68 - Goelz SE, Hamilton SR, Vogelstein B. Purification of DNA from formaldeyde fixed and paraffin-embedded tissues. Biochemical and Biophysical Research Communications 1985;130(1) 118-126.
69 - Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100(1) 57-70.
70 - Cheng L, Zhang S, Wang M, Davidson DD, Morton MJ, Huang J, Zheng S, Jones TD, Beck SD, Foster RS. Molecular genetic evidence supporting the neoplastic nature of stromal cells in 'fibrosis' after chemotherapy for testicular germ cell tumours. Journal of Pathology 2007;213(1) 65-71.
71 - Hanahan D, Weinberg R.A. Hallmarks of cancer: the next generation. Cell 2011; 144(5) 646-674.