histology group of south australia · before performing the next step in ihc protocol. these are...

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HISTOLOGY GROUP OF

SOUTH AUSTRALIA

The Cutting Edge

September 2012

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CCOONNTTEENNTTSS

Page

A word from the chair 3

Meeting reviews May 21st – WCH, Angela Nuske 4

July 16th

– RAH, Maria Collis 5

July 16th

– RAH, Ruth Davies 6

Forthcoming local events 10

Forthcoming national events 12

Your Committee 13

Editorial Note 13

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FFRROOMM TTHHEE CCHHAAIIRR

The next Education session will be hosted by Peter Holt from Clinpath Laboratories on Monday 17th

September. Sponsors for the evening will be ThermoFisher Scientific. Dr Richard Logan will present

the clinical features and pathology of common oral diseases.

The last meeting for the calendar will be on Monday 5th

November at APP. Details will follow. The

Christmas dinner has been booked for Monday 3rd

December at the Singapore House, Glen Osmond

Road. The Butterfly Room can hold a maximum of 40 people so book early to ensure a seat.

The dates for the National Meeting in 2013 have been set for April 27-29th

April.

We have had excellent attendances at the meetings this year, due mainly to the interesting presenters

and topics, but also due to the revitalised committee. Rebecca Dyer has brought some fresh ideas, and

her flyers are a reflection of them.

We welcome back our seafarer colleague Lisa Mills, back from a long absence overseas and island-

hopping. It’s good to hear she has docked at the Womens & Childrens Hospital. We can’t wait to hear

what trouble she landed in while away and hope she will return to the committee.

Peter Holt has been beaming even more broadly than usual with the news he will be a father in

November – who would have thought!

Alex Szabo

SA Pathology

Flinders Medical Centre

Chairperson Histology Group of South Australia

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Meeting Review

21st May, 2012

Women’s and Children’s Hospital

Chronic Gastrtis and Helicobacter Luke D’Arcy and Alex Szabo

Luke began the session with a run-down of the origins and rates of helicobacter; a gram-negative

bacterium that can be present in the upper gastrointestinal tract. The infection rates of which are as high

as 90% in developing countries, and 50% in developed countries. The symptoms caused by having

these bacteria present include gastric ulcers in humans, and septicaemia, enteritis and even reproductive

diseases in sheep. Luke described how the lipopolysaccharide layer surrounding helicobacter allow this

bacterium to not only survive, but thrive, in the acidic pH of gut linings. It was discovered some years

ago that there are two genes which H. pylori may express; cytotoxin associated gene A (cagA) and

vacuolating cytotoxin gene A (vacA). The proteins produced by these genes have different and

significant pathological effects on the virulence. There are considered to be two strains of cagA H.

pylori; the Western strain and the East Asia strain, both of which increase the likelihood of gastric

cancer in the patient, with the East Asia strain being a higher risk. Immunohistochemical antibodies

have been developed to demonstrate the cagA strain.

Alex Szabo then backed up Luke’s presentation by discussing the histological significance, including

some excellent photographs, of an unusual looking Helicobacter he had seen. This was much larger

than average (10μm instead of approximately 4μm), and rather than a spiral appearance, its shape was

relatively straight. After some research, it was found to be an uncommon strain called Helicobacter

heilmanni. This type is much less common than H. pylori, but may be present with it also. Although the

histological effects may first appear similar to H. pylori, it will be negative for immunohistochemical

stain of H. pylori and will more likely have lymphocyte infiltration in the gastric faveolae.

Lysosomal Storage Disease: research into effective therapy Helen Beard from the Lysosomal Diseases Research Unit at the WCH

Last to present was Helen Beard, who gave an interesting overview of the research she is involved in

regarding effective enzyme therapy in Lysosomal Storage Disease (LSD), using a mouse model. LSD

involves a defect in which unwanted matter accumulates inside the lysosomes of cells. The disease

causes numerous and varying symptoms, including: seizures, development problems and memory loss.

These are generally seen in young subjects and the patients often die at an early age. Helen’s research

included using IHC techniques on sections of mouse brain to study the effects of enzyme uptake during

treatment of LSD. There is currently no cure for LSD, so it is important to research the effects of

treatments to assist these cases.

Angela Nuske- Adelaide Pathology Partners

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Meeting review 16

th July, 2012

Royal Adelaide Hospital

“Diagnosis of Pheochromocytoma Catecholamine-secreting tumours”

Pheochromocytomas are catecholamine-secreting tumours of chromaffin cells of the adrenal medulla or

of extra-adrenal paragangliomas affecting 2 to 8 per 1,000,000 people in United States yearly. Most

pheochromocytomas are sporadic and approximately 90% of pheochromocytomas are benign. These

tumours if untreated, may be fatal, causing a range of metabolic and cardiovascular effects.

Establishing the diagnosis of pheochromocytoma by biochemical, imaging, histological and genetic

testing is critical in the treatment, management and prognosis of the patient.

While treatment for benign pheochromocytoma remains surgical resection, therapy for malignant

disease is unsatisfactory. Further studies and development of pharmaceutical drugs targeting the

enzymes in the biosynthetic pathway of catecholamines may provide useful in treatment, management

and possibly a future cure of this rare catecholamine-secreting tumour.

Maria Collis, Senior Technical Officer, SA Pathology RAH

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Pre-Analytical Factors and Immunohistochemistry (IHC) by Dr Jane Radford

Dr Radford presented the pre-analytical factors that affect IHC results for frozen specimens, cell

preparations and formalin fixed paraffin embedded (FFPE) tissue. Pre-analytical variables include all

steps; from harvesting of tissue until IHC assessment. These variables can affect both diagnostic and

research specimens.

Harvesting of the tissue is the initial step for all specimens. Whether diagnostic or research, the time

taken between warm ischemic time (cross clamp time) clamping of blood vessels and stopping the

blood flow is important. Once blood flow has been haltered, cell autolysis will begin.

In a research setting, standardisation is very important when performing IHC on different specimens

from one animal (e.g. mice). The opening of the animal and the removal of organs such as the brain,

kidney, liver and heart tissue must be in the same sequence so that any loss of antigenicity due to the

retrieval process will be the same between each animal sacrificed.

Another pre-analytical variable is cold ischemia time. This is the time between removing the tissue and

placing it into fixation. In some instances specimens are transported to laboratory, either on gauze or in

saline before being placed into fixative.

Frozen tissue: In both the diagnostic and research setting, specimens are frozen, sections taken and then

followed by IHC. Freezing of the specimen can be performed in different ways, but within each

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laboratory, or for a specific research project this method must be standardised if staff wish to compare

results or perform accurate analysis.

How the specimen is frozen can also cause artefacts. Specimens need to be frozen relatively quickly as

to reduce ice crystals formation which can distort the tissue morphology and also lead to IHC artefact.

Another consideration with frozen sections is the storage of cut sections with -80° C being the optimum

temperature. Length of time and type of fixation used is also an important pre-analytical variable when

assessing IHC staining.

Cell preparation: The volume of solution for smears will affect the cell layer thickness. The drying

time, fixation method, storage and centrifugations are all variables which can lead to variation in

intensity and reproducibility of IHC staining.

FFPE: As mentioned earlier, tissue harvesting and warm ischemia is an important variable along with

tissue handling. Rough handling can lead to tissue distortion which can make it difficult to determine

morphological detail and thereby IHC localisation.

The type of fixation (diagnostic laboratories usually use 10% buffered formalin) plus length of time are

very important for all IHC assessments! With semi-qualitative assays, fixation needs to be monitored

and standardised to ensure accurate IHC results.

An IHC protocol for any given antibody initially requires evaluation, optimisation and then validation.

The established protocol is then reliant on all subsequent specimens treated in the same manner. Using

oestrogen (ER) protocol as an example; if samples used to establish the protocol were fixed for 24-36

hours, a test specimen fixed for an extended length of time; >48 hours (i.e. over the weekend), using

same protocol may give inaccurate results. The extended fixation results in increased cross-linkages,

and to expose the antigen longer antigen retrieval is required. With insufficient antigen retrieval the

patients could show negative staining for ER. A consequence of this would be that the patient didn’t

receive hormone therapy.

Other variables which can affect IHC reproducibility is at the specimen processing stage. Examples

include a lower fixation temperature set on the automated processing machine leading to a slower

penetration rate of the fixative, the overly large specimen squashed into a cassette for processing and

the reduced frequency of changing the processing reagents on automated processing machines. All

these factors can cause under fixed or inadequately processed specimens resulting in possible altered

IHC staining.

Processing schedules are established and are reliant on standardized methodologies, so tissue sample

size, consistent reagents temperature, fresh solutions all help to optimise fixation penetration and tissue

block processing.

Another important consideration is embedding. Tissue antigens can deteriorate if held at high

temperatures for extended periods of time. Therefore, once specimens are removed from the processor

they must be embedded within a reasonable time.

In some instances specimens are required to be reprocessed. The block is placed on the embedding

station to melt away the wax prior to reprocessing and the time taken between melting and starting the

reprocess procedure should be minimal.

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Control sections for FFPE tissue are often pre-cut. The length of time these sections are kept should be

monitored as antigenicity will decline over time. Antigenicity will also be reduced if the cut section is

heated over 70° C.

Antigen retrieval is often required by IHC protocols and this can be in the form of enzyme or heat. The

pH of the solution used in heat induced antigen retrieval (HIER) and the type of heat which may be

water bath, pressure cooker, microwave or on board automation are all variables. If HEIR is employed,

the length of heating is another consideration followed by the time allowed for HEIR solution to cool

before performing the next step in IHC protocol. These are all pre-analytical variables.

As demonstrated above, each step in the process from removing of tissue specimen, fixation,

processing and IHC staining include variables. The take home message was standardisation. For

optimum and reproducible IHC results, all processes must be standardised. However when a variation

from the standard methods occurs, as happens in the real world, it is important that these factors are

noted and recorded. Therefore if there is an unexplained result, the pathologist/ scientist/

technician/researcher can make an informed decision.

Ruth Davies – SA Pathology, QEH

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FFOORRTTHHCCOOMMIINNGG LLOOCCAALL

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Educational meeting, September 17

th 2012 at Clinpath laboratories

Educational meeting, November 5th

2012 at Adelaide pathology Partners

Xmas Dinner: Singapore House, 203 Glen Osmond Road, Frewville

December 3rd

2012

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Histology Group of South Australia

Presents

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pppaaattthhhooolllooogggiiieeesss"""

Location: - Clinpath Pathology,

19 Fullarton Road,

Kent Town

Time: - 17th September,

6 pm for supper and 6:30 pm meeting

Sponsored by

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FFOORRTTHHCCOOMMIINNGG NNAATTIIOONNAALL

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National Histology Meeting April 26-28 2013

Crown Casino Melbourne Provisional program

Workshops Speaker Topic

Dr Thomas Haas (Basic) – Tissue identification for the Histologist

Dr Guy Orchard (Advanced) – MOHS technique Dr Thomas Haas (Advanced) – Stalking the Big Four: New

developments in the diagnosis of breast, prostate, colon and lung carcinomas

Various (Basic) - Basic Immunohistochemistry: Focus on stain identification and recognition of some popular antibody markers

Provisional Program (Confirmed speakers to date)

Dr.Keith Byron

Introduction to Molecular Techniques

Dr. Guy Orchard IHC Diagnosis of Malignant Melanoma

Dr Beena Kumar & Piero Nelva

Technical aspects related to Breast cancer diagnosis

Dr Thomas Haas Sentinal Lymph Nodes: A look at the significance from a histotech’s perspective.

Soeun Kinh Mom Male infertility : Testicular biopsies

Natalie Kvaleheim Abalone virus ISH

David Gan Making the most of your specimen in IHC

Greg Jenkins Histology disasters

Anne Prins & Alana Treasure

Interesting Histology

Andrew Griffin NATA: Aspects of the standard in reference to laboratory accreditation.

Naomi McCallum TBA (Diagnostic Electron microscopy group)

Dr Nina Fotinatos Career Paths/ research opportunities post the Medical Laboratory Science course

Sarah Morabito TBA

Anthony Van Galen TBA

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YOUR COMMITTEE:

Chairperson Alex Szabo

SA Pathology FMC

Secretary

Ruth Ravies

SA Pathology TQEH

Treasurer Sharin Prakash

SA Pathology rah

Editor Peter Holt

Clinpath Laboratories

Committee member Rod Coombe

SA Pathology RAH

Committee member Bec Dyer

Adelaide Pathology Partners

EDITORIAL NOTE:

The Histology Group of South Australia includes advertisements in it’s newsletter at a very reasonable

cost ($70 per A4 page). Advertisements should be sent as original .jpg format where possible, this

enables them to be optimised as required.

Articles and advertisements should be sent directly to:

[email protected]

Peter Holt

Clinpath Laboratories

mailto:[email protected]

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