The Cutting Edge. Word from the Chair. Contents. Volume 21, Issue 3

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1 August 2017 Histology Group of South Australia Volume 21, Issue 3 The Cutting Edge Word from the Chair The program of educational sessions has drawn experience from both scientists and pathologists. The most recent talks on Helicobacter pylori complemented each other. Maria Collis provided a comprehensive overview of the organism and its aetiological association with gastric pathologies. She described the pathophysiology of infection with the organism and the mechanisms by which it caused disease. She concluded with current testing methods to detect infection. Dr Sophia Otto used a current medical-legal case to endorse the utilisation of sensitive IHC for the detection of the organism in all gastric biopsies, not just suspected cases of infection. Both sessions have been kindly offered to the committee for distribution. The committee have been planning for the remainder of the calendar with meetings in August and October. Flyers for the August meeting at Clinpath should be up on notice boards soon and is being presented by Clinpath s Dr Georgina England and Mike Rentsch from Australian Biostain, who will boggle us with some of his lifetime s worth of experience and knowledge. Registration for the National Histology Conference in Hobart is available at The program is comprehensive with something for everybody. Numbers are looking excellent at this point and the good news is early bird registration will be extended to September 30 th. The Christmas Dinner is planned for December with details to follow. Contents Educational Session Review #1: Diagnosis of H. pylori 2 Educational Session Review #2: Hell hath no fury like a Helicobacter scorned 6 August Educational Session 9 Upcoming Events 11 Competition 13 Your Committee 15 Contact Us 15 I take this opportunity to thank our trade partners for their commitment in supporting the Histology Group of SA. We hope that our newsletters and social media presence provides the exposure that their companies deserve. Alex Szabo

2 Diagnosis of Helicobacter pylori and the development of chronic inflammation and tumour Maria Collis, SA Pathology Deanna Wallis-Hill - Helicobacter pylori, or H. pylori, is a gram negative, spiral shaped motile bacterium with multiple flagella (figure 1) which colonises the gastric epithelium of approximately half the world s population. Although more than half of the population is in fact infected, less than 2% will develop gastric cancer. Gastric infections caused by H. pylori result in chronic gastritis, gastroduodenal ulcers and are the strongest identified risk factors associated with lymphoid hyperplasia, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinomas (figure 2). The noncardia regions of the stomach are where H. pylori infections occur. This unique and interesting pathogen was discovered in 1982 by Dr. Robin Warren and Barry Marshall. It was first seen in 1875 by German scientist s, and in 1982 Guilio Bizzozoro described and drew what appeared to be spiral shaped bacteria living in his dog s stomach. It was Prof. W Jawroski in 1899 who suggested that it may be a cause of gastric disease. Robin Warren, an expert in Gastric pathology and bacterial staining in pathological specimens, reported findings of an unusual bacteria in biopsies from patients suffering from gastritis in the late 1970 s. His reports were initially met with disbelieve from the general medical community who simply believed that lifestyle, food and stress were the main causes. This changed in 1981 when a young registrar in gastroenterology, Barry Marshall, became interested in Warren s findings and this which sparked the beginning of a Noble prize-winning partnership. J J.R. Warren and B.J. Marshall, 2005 Nobel Prize Winners Nobel Prize winners J.R. Warren and B.J. Marshall In 1982 Warren and Marshall isolated and cultured the bacterium and in 1989 DNA sequencing classified the bacterium as H. pylori. This initiated a review of all patients from who had peptic ulcer disease and they nearly all had H. pylori. To further support their hypothesis, in 1985 Marshall drank a culture of H. pylori and a biopsy taken 8 days later showed H. pylori on the gastric mucous-secreting epithelial cells of his stomach. This rather bold attempt revolutionised the treatment of duodenal and gastric ulcers and they together were awarded the Nobel Prize for Medicine in

3 3 Diagnosis of Helicobacter pylori and the development of chronic inflammation and tumour Maria Collis, SA Pathology Deanna Wallis-Hill - H. pylori is very clever in the way it survives the harsh acidic environment of the human stomach. The spiral shape of the pathogen, plus the flagella hooks (figure 1), allow the pathogen to burrow into the mucosal layer of the stomach, which is less acidic. Helico secretes an enzyme called urease, which reacts with urea to form ammonia and hydrogen bicarbonate. The production of ammonia decreases the acidity of the stomach, making a nice and cozy environment for helico to grow. Immune cells that normally recognize and attack invading bacteria accumulate near sites of the infection, however they can t reach the stomach lining, resulting in gastritis and ulcers. Figure 1. Scanning electron microscope image of H. pylori 16000:1 (at 15x12cm) Figure 2. The clinical outcomes of Helicobacter pylori infections. Although H. Pylori is one of the most common bacterial infections worldwide, not all individuals develop gastric neoplasms. Its clinical outcome depends on both the host and bacterial virulence factors. The pathogenesis begins when H. pylori infects normal gastric mucosa. The individual develops an acute infection followed by a chronic infection. Chronic gastritis, if untreated, can lead to gastro-duodenal ulcers, gastric MALT lymphomas or gastric adenocarcinoma (figure 2). There are about 20 different types of H. pylori species, with many that do not cause disease. Two strains; CagA+ strain which injects toxins into hosts cells and VacA strain, which is a pore forming toxin, both increase the risk of cancer. CagA+ strains are the most damaging of all. The bacterium has a needle like appendage that secretes a toxin into the host s cell, disrupting the cell-cell junction, altering signal transduction and promoting cell proliferation. Recent studies have confirmed that CagA+ functions as a bacterial oncoprotein that inhibits p53 (tumour suppressor) in gastric epithelium. H. pylori-induced deregulation of p53 increase the risk of gastric cancer in infected individuals. Approximately half of the world population is affected by H. pylori with a variable prevalence in different countries attributed to geography, age, and socioeconomic factors. Developing countries have a higher infection rate of H. pylori compared to those living in western countries. This is due to the transmission nature of H. pylori. It is easily transmittable though oral or close contact with an infected person, poor hygiene, and contaminated water or food. There are a variety of symptoms including, heartburn, acid reflux, chest pain, gastritis weight loss, nausea, vomiting and blood in stools.

4 4 Diagnosis of Helicobacter pylori and the development of chronic inflammation and tumour Maria Collis, SA Pathology Deanna Wallis-Hill - Since the discovery of H. pylori, several diagnostic methods have been developed for the aim of accurate detection of this organism. These tests include non-invasive & invasive methods. Non-invasive tests include serology, urea breath test and stool antigen test. The urea breath test is the most sensitive and specific test but false negatives are a possibility. Invasive tests, those which require an endoscopy, are the rapid urease test, culture and histological examination which includes H&E (figure 3), special stains and immunohistochemistry (IHC). The rapid urease test is very quick and inexpensive with a sensitivity of 95% and specificity of 99%. Although there can be a decrease in sensitivity in patients with bleeding ulcers. IHC is the best method regarding sensitivity and specificity and is considered the gold standard. It uses anti-helicobactor Pylori a rabbit monoclonal to detect H. pylori antigens in infected individuals. The special stain of choice would be the Giemsa (figure 4) as they are more sensitive than a H&E. A routine H&E is also examined as can be useful in providing more information about the degree of inflammation and any associated pathology present. Current Therapeutic Guidelines in Australia, recommend PPI-based triple therapy as the first-line measure for eradication which consists of two antibiotics and a proton pump inhibitor. Figure 3. H&E stain of gastritis at 400x magnification. Figure 4. Giemsa stain of gastritis at 400x magnification. Infection by H. pylori remains the most frequent and persistent bacterial infection worldwide; the development of chronic inflammation and gastric cancer is a multistep process. The diagnosis of H. pylori by non-invasive and invasive methods is critical in the treatment, management and prognosis of the patient. Piper Robertson - Cryptic Corner Arctic elections minus the Spanish, but with the capital of Spain Answer on page 15

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6 Hell hath no fury like a Helicobacter scorned Dr. Sophia Otto SA Pathology 6 Dr. Sophia Otto presented a pathologist's worst nightmare... a misdiagnosis, whereby failure to detect the presence of a gastric pathogen in a gastric biopsy years later has developed into a gastric cancer. In a current class action by 20 individuals, one individual had developed gastric adenocarcinoma following a misdiagnosis of a gastric biopsy many years earlier. Re-assessment of the archival biopsy and utilisation of sensitive IHC (figure 5) revealed the presence of the organism Helicobacter pylori. The other claimants suffered a range of neoplastic processes, some with chronic gastritis or dyspepsia, others with Malt lymphoma. IHC verified the presence of H. pylori when their archival tissue samples were tested. Dr. Otto posed the question how does this class action direct the future approach to examination of biopsies from patients with suspected H. pylori infection? Are there better methodologies we could be using to reduce the risk of missing the organism? Helicobacter pylori (H. pylori) affects between 50 to 70% of the world s population and, thus, is one of the most frequent and persistent bacterial infections worldwide. H. pylori is associated with peptic ulcer disease, gastric ulcers, mucosa-associated lymphoid tissue lymphoma and gastric cancer (figure 2). In a histological section, H. pylori appear as a curved or spiral bacillus on the epithelial surface, in the mucus layer, and within gastric glands. Other Helicobacter species, such as Helicobacter heilmanii are also detected in the human stomach. H. heilmanii, a zoonotic infection in humans that can be acquired from cats or dogs and can cause chronic gastritis, is present in about 0.1% of gastric biopsies. H. heilmanii is straight and much longer than H. pylori; thus, the two species can be easily distinguished. Figure 5. IHC stain at 400x magnification

7 7 Hell hath no fury like a Helicobacter scorned Dr. Sophia Otto SA Pathology The standard method to diagnose H. pylori infection is histological examination which provides critical information related to the mucosa (e.g., presence and severity of inflammation, intestinal metaplasia, glandular atrophy, dysplasia, and neoplasia). Dr. Otto performed a small audit of specimens over a two-week period. Her objectives and results of this targeted audit are summarised below: 1. To identify the incidence of H. pylori in biopsies referred to her laboratory. In 29% of the biopsies examined, H. pylori was not identified by HE stain alone. H. pylori not identified by HE were confirmed positive by IHC in 8% of the cases. IHC provided a methodology that HE could not match in terms of cleanliness of background and sensitivity. 2. To determine the advantages of IHC when compared to conventional HE staining, the current protocol being routine HE stain without Giemsa or Diff Quik. H. pylori was identified by IHC and HE in 26% of the biopsies examined. H. pylori was detected by IHC but not observed in the HE sections in 25% of the biopsies examined, validating the sensitivity of IHC compared to routine HE. 3. To correlate the level of inflammation (degree and form) with the presence of H. pylori. Typically, biopsies of the stomach in a setting of H. pylori exhibit active chronic gastritis - active describing the presence of neutrophils in the surface epithelium or penetrating gastric pits and chronic inflammation describing a dense background of lymphocytes and plasma cells. In her study, 88% of biopsies showed acute inflammation, 12% no acute inflammation, 94% showed chronic inflammation and 6% no chronic inflammation. One biopsy showed no inflammation at all, but alarmingly H. pylori was detected by IHC. 4. To correlate the presence or absence of intestinal metaplasia with the presence of H. pylori. This refers to the presence of small intestinal and large intestinal goblet cells which typically don't occur in the stomach. Of the 8 biopsies displaying intestinal metaplasia, 4 revealed H. pylori by IHC. 5. To determine whether the gastroenterologists were abiding with the Sydney system, the gold standard for gastric biopsy collection. The system indicates sampling from five biopsy sites. Two biopsies should be obtained from the gastric antrum, two biopsies from the corpus and one biopsy from the incisura angularis (figure 6). This provides a global picture of the stomach and with more tissue to examine, is more likely to find pathology and H. pylori. It enables the pathologist a more accurate assessment of any histological changes that a single biopsy may not reveal and help distinguish between disease states.

8 Hell hath no fury like a Helicobacter scorned Dr. Sophia Otto SA Pathology 8 Despite the recommendations, this approach for biopsy collection was not reflected in the audit findings, with only 12% of cases being adequately sampled. The risk is that with analysis of fewer biopsy samples than recommended, this can lead to an underestimation of the presence of H. pylori, sampling error, and false negatives. Based on her findings of this small audit (65 biopsies), Dr Otto recommended the use of IHC on all gastric biopsies. Figure 6. The five gastic biopsy sample sites as per the Sydney protocol. It is a relatively cheap and sensitive method for identifying H. pylori in tissue sections. When combined with the rapid urease test on which the CLO test system is based, and the noninvasive Urea Breath Test, the detection rate of H. pylori is also improved. She also talked briefly of advances in genotyping H. pylori to identify a subspecies of the organism that is resistant to the triple and quadruple therapy. Alex Szabo SA Pathology Like, Follow and Share Welcome to the HGSA s Facebook page! Like us and follow us here: View our event photo galleries and feel free to tag yourselves. Don t forget to share! Also check out our website: Here you will also find information on all our upcoming events.

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11 Upcoming Events 11 Check out our website and Facebook page for further details of the following events. Don t worry, you will also get sent an closer to each event, so make sure you are on our mailing list! Educational Session #3 Newsletter Edition #4 Educational Session #4 National Histology Conference HGSA Christmas Dinner Monday 21 st August:, Kent Town Monday 25 th September Monday 16 th October: Royal Adelaide Hospital Friday 17 th Sunday 19 th November: Hobart, Tasmania Monday 4 th December: Le Riad, Adelaide The Histology Group of South Australia, along with the Histology Group of Victoria, Histotechnology Group of Queensland and the Histotechnology Society of New South Wales, are hosting this year s National Meeting in the picturesque city of Hobart, Tasmania. Anyone within the medical science, clinical and research fields, commercial scientists and students are invited to attend. There are a range of workshops and plenary sessions offered, aimed to provide continuing education and professional development. Modern equipment and consumables will also be showcased by trade sponsors. Check out the full program here REGISTRATION: *Early closes 30 th September Full early $450 Full early + dinner $590 Full standard $550 Full standard + dinner $690 Full student (full-time) $150 Saturday early $270 Saturday standard $320 Saturday student (full-time) $100 Sunday early $180 Sunday standard $230 Sunday student (full-time) $ 75 Friday morning and afternoon workshops run concurrently and cost $95 each, which include morning/afternoon tea. MORNING: Tissue Recognition The Basics Pathology of the Surgical Cut-Up: What you need to know before making the cut AFTERNOON: Tissue Recognition The weird, the wonderful and the wacky Perfecting the Gram Stain

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13 13 Competition: WORD SEARCH G D G A C O B I B F A F L G C H E L I C O B A C T E R A I B P F S O B M J I K C Q S L P A Y A P T T P R R I E T I U a C P K T U T O W N N R H R R R T O T X H L E E I I P E B E I E C U Y Y O G R T O F R V A E R S U P W O G I R L A U W S T I O A L C T S E U A L T F E A U D J N U L A X N I B L C Q L M N O B E O E C N E L U R I V N E T T R E S Y A F S C F U V A F P C C M L B I O F I L M S L J I N O I T A T U M J S U M Z M U L C E R S I S P G B L C X Which of the 18 words from the list below is missing from the word search? BACTERIUM GASTRITIS ONCOGENIC ULCERS BARRETTS HELICOBACTER PARIETAL UREASE BIOFILMS MICROAEROPHILIC PATHOGENESIS VIRULENCE CULTURE MUCOID PYLORI ENDOSCOPY MUTATION REFLUX your answer along with your contact and workplace details to kbampton@clinpath.com.au by 28/08/17 for your chance to win a ticket to the HGSA Christmas Dinner!

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15 15 Your Committee Chairperson Secretary/Editor Your Committee Karen Bampton Editor Caroline Loft Treasurer Sharin Prakash SA Pathology, FMC Rod Coombe SA Pathology, RAH Ruth Ninnes SA Pathology, TQEH Rebecca Dyer Melissa Clements Ha Tran SA Pathology, RAH Chairperson Alex Szabo SA Pathology, FMC Sharin Prakash SA Pathology, FMC Contact Secretary Us Editor For feedback, advertising, article Treasurer and all general suggestions/submissions enquiries please contact us:member Committee Alex Szabo 16 Congratulations to our June Edition Word search Karen Vanwinners: Bochove Anne - Australian National University Caroline Loft Oulaivanh - SA Pathology, Lyell McEwin Hospital We hope you enjoy your EventRAH Cinemas movie Rod Coombe SA Pathology, vouchers! Ruth Ninnes Rebecca Dyer Melissa Clements Ha Tran The Histology Group of SA is an organisation representing and educating the histopathology community of South Australia. SA Pathology, FMC SA Pathology, TQEH Spring is almost in the air.. SA Pathology, RAH Find us on the Web: Cryptic Corner Answer Frozen Sections Contact Us Did you get it right??!!! For feedback, advertising, article Cryptic CornerandAnswer suggestions/submissions all general MICROSCOPES enquiries please contact us: Did you get it right??!!! committee@histosa.org.au From Intestinal glands aka Crypts of Lieberkuhn Photo credit: