The Human Protein Atlas Project is carrying out the systematic exploration of the human proteome using antibody-based proteomics, thus providing an invaluable publicly available HPA portal tool for pathology-based biomedical research. As part of the project, the Uppsala-based Science for Life Laboratory tissue profiling group has so far cut more than 200,000 slides from over 1400 tissue microarrays (TMAs). This article describes how the tissue microarrays and slides are made, and how a rotary microtome with different cutting modes and an automated Section Transfer System together ensure that high-quality, reproducible sections are generated.
by Ing-Marie Olsson, Catherine Davidson and Dr Caroline Kampf
A publicly available protein dictionary
Molecular tools developed in the research arena are making a significant contribution in the evolution of tissue-based diagnostics. Immunohistochemistry (IHC) is now well recognised as a means of enhancing morphological analysis, with protein expression patterns considered as effective diagnostic and prognostic indicators for various cancers. For example, within diagnostic pathology, IHC could determine the origin of poorly differentiated tumours and also be used to stratify tumours for optimum treatment regimes.
Consequently, the Human Protein Atlas (HPA) project was initiated in 2003 by the Knut and Alice Wallenberg Foundation to enable the systematic exploration of the human proteome using antibody-based proteomics. Since then, the publicly available HPA portal (www.proteinatlas.org) has amassed a database of millions of high resolution images showing the spatial distribution of proteins in 46 different normal human tissues and 20 different cancer cell types, as well as 47 different human cell lines. As such, the HPA can provide an invaluable tool for pathology-based biomedical research, including protein science and biomarker discovery for disease identification [1].
Tissue profiling
One of the key sites involved in this immense project is the Uppsala-based Science for Life Laboratory (SciLifeLab Uppsala) tissue profiling group [2]. This highly experienced group is focused on histopathology, with special emphasis on tissue microarray (TMA) production, immunohistochemistry and slide scanning. The enormity of profiling the human proteome requires the use of high throughput techniques, prompting the SciLifeLab team to adopt a TMA format to enable them to perform simultaneous multiplex histological analyses.
TMAs are paraffin blocks containing cores of selected tissues or cell preparations assembled together for subsequent sectioning to enable the effective and efficient utilisation of valuable tissue samples, as well as reducing the use of expensive IHC reagents. Multi-tissue blocks were first introduced by Battifora in 1986 with his ‘multitumour (sausage) tissue block’ [3]. Then in 1998, Kononen and collaborators standardised the technology and developed instrumentation which uses a sampling approach to produce tissues of regular size and shape that can be more densely and precisely arrayed [4].
As part of the HPA project the SciLifeLab Uppsala tissue profiling facility has constructed over 1400 TMAs containing over 100,000 tissue cores, in addition to 180 cellular microarrays (CMA) containing over 23,800 cell cores. Over 200,000 slides cut from these arrays have then been stained using immunohistochemical techniques, of which more than 100,000 have been scanned for further analysis. The SciLifeLab team evidently holds a great deal of practical experience in TMA production and, in fact, now offers an external TMA production service [2]. Consequently, its experts handle many different types and combinations of tissues, for which they observe that high quality sectioning is fundamental to TMA production, the primary aim of which is to amplify a scarce resource.
TMA production
The most efficient method of constructing tissue microarrays is by extracting cylinders of donor tissue with a sharp punch and then assembling them into a recipient block that has uniformly sized holes in a grid pattern. Tissue and cell microarrays are made according to a preset standard within the HPA, where paraffin blocks are used in a matrix containing from 72 up to 120 tissue cores. The standard diameter of each core is 1 mm (tissues) and 0.6 mm (cells), with a length of 2-4mm. This is achieved by using a needle to remove relevant tissue from a donor paraffin block which is then inserted into a recipient paraffin block.
Once all tissue cores are in position within the array, it is then ‘baked’ at 42ºC to melt them together into a homogenous paraffin block. This 40 minute baking period ensures that every core is merged with the melted paraffin in the block and, therefore, totally secured for sectioning into 4 µm sections prior to mounting onto glass slides. Thereafter, these multiplex tissue sections are ready for further histological analysis and final slide scanning to transf orm stained glass slides into digital high-resolution images.
Quality sectioning
When sectioning TMAs, the greatest risk of valuable tissue loss or damage can occur during transfer to a water bath. For this reason, the SciLifeLab tissue profiling group uses microtomes with a ‘waterfall’ system (Thermo Scientific HM355S and Thermo Scientific Section Transfer System) to eliminate such risks. A ‘waterfall’ automated Section Transfer System stretches sample ribbons as they are cut, whilst simultaneously transporting them from the blade into the attached circulating laminar flow bath. From this water bath, sections can be extracted and mounted onto a glass slide. Mounting two microarray sections per slide can further reduce IHC reagent usage and enhance workflow within the tissue profiling group.
By using the Section Transfer System the group routinely obtains over 200 quality sections per TMA, depending on the size of donor block and representative tissue within it. Although it is possible to obtain many more sections, for quality assurance (QA) purposes the SciLifeLab team performs a QA after every 50th section, introducing replacement cores where required to ensure that at least 85% of the tissue cores are always present.
The actual composition of a tissue array can also cause complications when sectioning, dependent on whether tissues are homogenous cancer types, or normal tissues where heterogeneity is greater. Furthermore, fatty tissue such as that from brain and breast should not remain within a warm water bath for an extended period due to risk of tissue melting. Conversely, other tissue such as skin and thyroid gland, needs to remain in the water bath for longer in order to ensure that it is sufficiently stretched.
To overcome such issues with tissue composition, SciLifeLab experts group tissues into those with similar texture and hardness when sectioning to make set up easier and improve workflow. For example, the HM355S microtome offers a choice of four mechanised cutting modes that give SciLifeLab greater control over section generation according to varying requirements. Mechanised cutting delivers the slow, smooth, even and controlled action necessary for sectioning harder consistency specimens.
A further sectioning consideration at SciLifeLab Uppsala is the fact that the TMAs are paraffin embedded. Consequently, a peltier-cooled attachment (Thermo Scientific Cool Cut) is used on the group’s microtomes to prolong the cutting period by maintaining a cool block temperature. By using such a cooling tool, 50 TMA sections can be cut consecutively in 50 minutes without the need to remove and re-cool the block on ice, again ensuring effective throughput and efficient laboratory operation.
SciLifeLab tissue profiling services
Tissue Microarrays (TMAs) are coming to the fore as an ideal means of providing multiplex tissue analysis, not only for research based applications, but also for clinical applications: identifying biomarkers for identification of disease, histological grading and detecting disease recurrence [5,6,7]. Some hospital laboratories are also starting to utilise TMAs as controls for diagnostic comparisons.
With over 100 personnel working on the HPA project alone, the SciLifeLab facility provides access to its extensive protein profiling results to laboratories throughout Sweden and beyond. In addition, leveraging their expertise gained in constructing tissue arrays for high throughput protein screening, the SciLifeLab team in Uppsala has also recently extended its capabilities to offer an external TMA production, sectioning and scanning service [2].
Working to a user specified template, the facility can turnaround 120 core duplicate arrays within 24 hours from receipt of the donor tissue blocks. The venture operates as cost neutral, utilising the team’s experience in generating high quality sections at a resolution of 2µm-10µm to provide consistent and reproducible material for downstream analysis. Since its inception, the TMA service has produced more than 100 custom arrays, supporting investigation of clinical models for a wide range of disease states, including cancer, diabetes, heart disease and neurodegenerative disorders.
Establishments utilising the tissue profiling group’s TMA services include university research, hospital and even veterinary laboratories. Such is the experience of this SciLifeLab group, it has been able to produce TMAs on almost any kind of tissue. Although bone and skin can prove difficult, the team can even produce TMAs for these by careful orientation of skin samples and decalcification of bone prior to final preparation.
Advanced technical know-how and state-of-the-art equipment, combined with a broad scientific knowledge, all mean that the SciLifeLab tissue profiling facility is ideally placed to meet high throughput, high quality TMA production needs for the HPA, whilst simultaneously ensuring service excellence for external customers.
References
1. Pontén F et al. The Human Protein Atlas – a tool for pathology. J Pathol 2008; 216(4): 387-93.
2. http://scilifelab.uu.se/technologyplatforms/Proteomic/Tissue_Profiling_Center/?languageId=1
3. Battifora H. The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest 1986; 55(2): 244-8.
4. Kononen J et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nature Medicine 2008; 4: 844-847.
5. Rimm D et al. Cancer and Leukemia Group B Pathology Committee Guidelines for tissue microarray construction representing multicentre prospective clinical trial issues. J Clinical Oncology 2011; 29 (16): 2282-2290.
6. Schmidt L et al. Tissue microarrays are reliable tools for the clinicopathological characterisation of lung cancer tissue. Anticancer Research 2009; 29: 201-210.
7. Smith V et al. Tissue microarrays of human xenografts. Cancer Genomics & Proteomics 2008; 5: 263-274.
The authors Ing-Marie Olsson, Team Leader TMA production, sectioning and scanning, Human Protein Atlas (HPA), Tissue Profiling Centre, Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala, Sweden Tel. +46 18 471 5040 e-mail: ingmarie.olsson@igp.uu.se
Dr. Caroline Kampf, Site Director Human Protein Atlas (HPA), Tissue Profiling Centre, Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala, Sweden Tel. +46 18 471 4879 e-mail: Caroline.Kampf@igp.uu.se
https://clinlabint.com/wp-content/uploads/sites/2/2020/08/C01_Fig1_TMA-example.jpg3253003wmediahttps://clinlabint.com/wp-content/uploads/sites/2/2020/06/clinlab-logo.png3wmedia2020-08-26 09:48:032021-01-08 11:39:39Ensuring sectioning quality in TMA analysis for the Human Protein Atlas project
Newly revised guidelines for the diagnosis of coeliac disease (CD) place greater emphasis on laboratory testing, enabling the number of small-intestinal biopsies performed to be significantly reduced. The detection of antibodies against tissue transglutaminase (anti-tTG) or endomysium (EmA) remains a cornerstone of diagnosis, while further diagnostic procedures have gained new significance. The molecular genetic determination of the human leukocyte antigens (HLA) DQ2 and DQ8 now plays a central role in diagnosis, thanks to a better understanding of the genetic factors underpinning the disease. Moreover, state-of-the-art assays for antibodies against deamidated gliadin peptides (DGP), as oppose to native gliadin, now constitute a highly sensitive and specific analysis to support diagnosis. In the new guidelines, anti-tTG and anti-DGP are recommended as first-line tests in symptomatic individuals, while HLA-DQ2/DQ8 analysis is the initial step for screening asymptomatic persons with a high disease risk.
by Dr Jacqueline Gosink
CD, which is also known as gluten-sensitive enteropathy or non-tropical sprue, is an autoimmune disease caused in genetically predisposed individuals by consumption of gluten-containing cereals. The disease process is triggered by protein components of gluten known as prolamins, of which gliadin is the most common. Partially digested prolamin peptides are chemically modified (deamidated) in the intestine wall by the enzyme tTG. The immune system of genetically predisposed persons reacts with both the deamidated peptides and tTG, causing chronic inflammation of the small-intestinal mucosa, which results in atrophy of the villi and reduced resorption of nutrients. The only effective treatment for CD is observance of a gluten-free diet.
A clinical chameleon
The classic symptoms of CD are fatigue, abdominal pain, diarrhoea, effects of malabsorption such as weight loss, anaemia and growth retardation in children, vomiting, constipation and bone pains. However, CD is now recognised to be a multifaceted condition which can manifest in many ways. Some patients have non-typical symptoms such as osteoporosis, neuropathies, carditis, pregnancy problems or lymphoma. CD patients may also suffer from Duhring’s dermatitis herpetiformis, a recurrent skin disease characterised by subepidermal blisters.
The disease may also present in silent, latent or potential forms [1]. In the silent form, patients are asymptomatic, but nevertheless exhibit CD-specific antibodies, relevant HLA alleles and villous atrophy. Those with latent CD have previously had a gluten-dependent enteropathy, but are now free of enteropathy; they may or may not exhibit antibodies and/or symptoms. In cases of potential CD, individuals have positive antibodies and compatible HLA, but as yet no symptoms; they may or may not go on to develop CD.
While the prevalence of symptomatic CD is around 0.1%, the prevalence of the disease in all its forms is estimated to be as high as 1%. Many experts now speak of the coeliac disease iceberg, in which classic CD represents only the tip.
New ESPGHAN diagnostic criteria
Early in 2012, the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) released a revised version of its 1990 guidelines for the diagnosis of coeliac disease [1], which were compiled by a group of 17 international experts in the field. The new diagnostic criteria are defined by two algorithms: algorithm 1 [Figure 1A] is applied to symptomatic individuals, while algorithm 2 [Figure 1B] is used for asymptomatic individuals with a high disease risk, for example first-degree relatives of CD patients and patients with type 1 diabetes mellitus, Down’s syndrome, autoimmune thyroid or liver disease, Turner’s syndrome, Williams’ syndrome or selective IgA deficiency.
In algorithm 1 the first-line approach is the determination of anti-tTG antibodies of class IgA in patient serum. In order to exclude the possibility of an IgA deficiency, either total IgA or specific IgG (e.g. deamidated gliadin) should be investigated in parallel. If the anti-tTG antibody titre is very high (>10 times upper normal limit), and if this result is reinforced by positive EmA and compatible HLA, it is no longer considered necessary to perform a biopsy. If serological/genetic findings are inconclusive, results must be confirmed by histological examination of duodenal biopsy tissue to demonstrate villous atrophy and crypt hyperplasia. Diagnostic tests should be carried out in individuals on a gluten-containing diet. A gluten challenge is now only performed under exceptional circumstances.
In algorithm 2, the genetic parameters HLA-DQ2/DQ8 are initially determined to establish the genetic susceptibility. If these are negative, the risk of CD is negligible and no further tests are required. If HLA alleles are compatible with CD, specific antibody tests are used to follow up. In this group a duodenal biopsy is a prerequisite for a definite diagnosis of CD.
The individual diagnostic parameters and the technologies used to detect them are reviewed in the following sections.
Antibodies against tissue transglutaminase (anti-tTG, EmA)
Autoantibodies against tTG of immunoglobulin class IgA are the most important serological marker for CD, as they possess a very high sensitivity and specificity for the disease. While they are virtually never found in healthy individuals or patients with other intestinal diseases, their prevalence in untreated CD is near to 100%. Anti-tTG antibodies are alternatively known as EmA, depending on the test method used: EmA are determined using indirect immunofluorescence [Figure 2], while anti-tTG are detected using monospecific test systems such as ELISA [Figure 3].
Detection of EmA using indirect immunofluorescence is considered the reference standard for CD-specific antibodies due to its unsurpassed sensitivity and specificity. However, the microscopic evaluation required is demanding and dependent on the proficiency of laboratory staff. Enzyme immunoassays for detection of anti-tTG antibodies are often preferred due to their simplicity, cost-effectiveness and automatability, combined with their high sensitivity and specificity. Modern ELISAs for determination of anti-tTG antibodies are based on recombinant human tTG. A multitude of clinical studies have confirmed the efficacy of this method, with high-quality tests yielding a sensitivity of 90-100% and a specificity of 95-100% for active CD.
Antibodies against deamidated gliadin peptides (DGP)
Antibodies against DGP have recently assumed a more important diagnostic role, due to the development of highly sensitive and specific test systems to detect them. Conventional assays based on native full-length gliadin, which frequently yield unspecific reactions with sera from healthy persons, are now obsolete.
The advances in test design were precipitated after research revealed that only a tenth of the epitopes of the gliadin molecule are diagnostically relevant, and these must be present in deamidated form [2]. Based on these observations a novel recombinant gliadin-analogous fusion peptide (GAF) consisting of two nonapeptide components expressed in trimeric form (3X) was created [Figure 4]. The remaining 90% of the molecule was omitted, as it serves predominantly as a target for unspecific reactions.
This designer fusion protein is now used as the target antigen in the Anti-Gliadin (GAF-3X) ELISA, which provides vastly superior performance compared to conventional anti-gliadin ELISAs [3, 4]. In a multicentre study using a total of over 900 sera, the new test yielded a sensitivity (at 95% specificity) of 83%/94% (IgA/IgG) compared to 54%/31% for a conventional anti-gliadin ELISA. This represents an increase of 29% for IgA and 63% for IgG, significantly enhancing the relevance of the analysis.
Use of the Anti-Gliadin (GAF-3X) ELISA in combination with the Anti-tTG ELISA significantly increases the serological detection rate for CD and dermatitis herpetiformis [5]. The IgG version of the ELISA is particularly valuable for identifying CD patients with an IgA deficiency [6], which is frequently associated with CD. Determination of antibodies against DGP is also suitable for assessing disease activity and for monitoring a gluten-free diet or a gluten-load test.
HLA-DQ2 and DQ8
HLA-DQ2 and DQ8 are the principle determinants of genetic susceptibility for CD and are found in virtually all patients. The strong genetic background to CD is highlighted by familial prevalences of 10% in first-degree relatives of patients, 70% in identical twins and 11% in non-identical twins. However, the presence of HLA-DQ2/DQ8 is not sufficient by itself to cause CD. Around a third of the healthy population exhibits DQ2/DQ8 alleles.
Although not a particularly specific parameter, HLA-DQ2/DQ8 is a valuable tool for exclusion diagnostics. If neither DQ2 or DQ8 are present, then CD can be virtually ruled out. It is for this reason that DQ2/DQ8 analysis is now recommended as the first-line test for screening asymptomatic persons at high risk of CD, as defined by the presence of an associated disease or family history (algorithm 2). If DQ2/DQ8 is negative no further follow up is necessary. HLA-DQ2/DQ8 also functions as a confirmatory parameter in symptomatic persons (algorithm 1), and it is one of a triad of laboratory parameters that can be employed to diagnose CD without biopsy in these individuals. DQ2/DQ8 analysis is also helpful for clarifying cases in which diagnosis is inconclusive due to ambiguous serological/biopsy results, especially in infants or in patients who are already on a gluten-free diet, and for differentiation of CD from other intestinal diseases.
HLA-DQ2/DQ8 alleles can be determined using microarray test systems such as the EUROArray system. This analysis is simple to perform, requiring no previous knowledge of molecular biology. Disease-associated gene sections are amplified from purified genomic patient DNA samples by the polymerase chain reaction (PCR) [Figure 5]. The fluorescently labelled PCR products are then detected using microarray BIOCHIP slides composed of immobilised complementary probes. The evaluation [Figure 6] and documentation of results is fully automated using specially developed software (EUROArrayScan). In clinical studies employing precharacterised samples, this microarray yielded a sensitivity of 100% and a specificity also of 100% [7], demonstrating its ability to deliver accurate and reliable results in HLA analysis.
Conclusions
The publication of updated ESPGHAN guidelines for the diagnosis of coeliac disease has reinforced the indispensible role of anti-tTG and EMA in diagnosis and propelled further laboratory diagnostic parameters such as HLA-DQ2/DQ8 and anti-DGP into the limelight. In clear-cut cases, a thorough serological and genetic investigation is now considered sufficient to obtain a diagnosis, allowing the costs and patient discomfort associated with biopsy to be avoided. The state-of-the-art diagnostic tools available today are not only a boon for patient diagnosis, but will also help to advance our understanding of this enigmatic and seemingly widely occurring disease.
References
1. Husby S et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition Guidelines for the diagnosis of CD. JPGN 2012; 54: 136–160.
2. Schwerz E et al. Serologic assay based on gliadin-related nonapeptides as a highly sensitive and specific diagnostic aid in celiac disease. Clin Chem 2004; 50: 2370-2375.
3. Prause C et al. Antibodies against deamidated gliadin as new and accurate biomarkers of childhood CD. JPGN 2009; 49: 52-58.
4. Prause C et al. New developments in serodiagnosis of childhood celiac disease. Ann NY Acad Sci 2009; 1173: 28-35.
5. Kasperkiewicz M et al. Novel assay for detecting celiac disease-associated autoantibodies in dermatitis herpetiformis using deamidated gliadin-analogous fusion peptides. J Am Acad Dermatol [Epub ahead of print] (2011).
6. Villalta D et al. IgG Antibodies against deamidated gliadin peptides for diagnosis of celiac disease in patients with IgA deficiency. Clin Chem 2010; 56: 464-468.
7. Pfeiffer T et al. Microarray based analysis of the genetic risk factors HLA-DQ2/DQ8 – a novel test system for the diagnostic exclusion of celiac disease. 44th Annual Meeting of The European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), Italy, May 2011.
The author Dr Jacqueline Gosink EUROIMMUN AG Seekamp 31 23560 Luebeck Germany Tel: +49 451 5855 25881 e-mail: j.gosink@euroimmun.de
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The challenge of providing an effective allergy testing service in the face of the increased prevalence of the condition and a limited number of local allergists is being met by a centralised and specialised South African lab with the help of state-of-the-art equipment and facilities.
by Dr Cathy van Rooyen
Allergy is on the increase, especially in developing countries where industrialisation, lifestyle changes and socioeconomic factors lead to an increase in allergy prevalence. South Africa is no exception, as shown by data from prevalence studies of asthma in rural and urban populations [1]. The challenge in South Africa is to provide an adequate and comprehensive allergy service to a growing allergic population, in a healthcare system that has until recently been ill-prepared to meet allergy demands. South Africa has a dire shortage of trained allergists, as a postgraduate allergy diploma has only recently been introduced by the college of family medicine in South Africa and recognition of Allergy as a subspecialty of Paediatrics and Internal Medicine has only been introduced this year. There is no stand-alone postgraduate specialisation in Allergy or Immunology as seen in European countries.
The diagnostic laboratory not only faces the increased demands for allergy testing from the handful of clinicians with formal allergy training, but also the majority of requests from general practitioners without adequate allergy knowledge and who in conjunction with allergy testing, require in-depth interpretation of results, and advice on additional testing and patient management.
Allergy testing at AMPATH pathologists
AMPATH is one of the largest private pathology providers in South Africa and also provides the most extensive diagnostic service for allergy in South Africa. After consideration was given to the most effective model for the provision of this service, a model of centralisation of in vitro allergy testing was adopted. AMPATH built a National Reference Laboratory (NRL) for centralisation of allergy and other specialised tests, which has been operational since July 2011.
In addition to the centralised in vitro testing services offered, in vivo allergy diagnostics, including skin prick testing, intradermal testing and patch testing is also offered at selected centres. AMPATH also provides a diagnostic referral clinic for Allergy, where patients with complicated allergy can be referred for a detailed workup, which includes history, examination and appropriate testing. Patients are referred back to the clinician with a detailed report on the patient’s allergies and management advice. A separate service for allergen immunotherapy is also available at this clinic.
Centralisation of allergy tests in the AMPATH NRL
The AMPATH NRL is the main testing hub for specialised laboratory tests in AMPATH, South Africa. Centralisation has many advantages, which include cost-savings by increasing and optimising test volumes on a single platform and staff and resource savings, uniform test results, pooling of expertise in test performance and interpretation, etc. However, specimens do take longer to reach a centralised testing centre. The biggest challenge in such an environment is to provide a guaranteed and acceptable turnaround time (TAT), as many specimens are transported from various locations throughout the country.
The major factors that should be addressed to improve TAT are pre-analytical (easy registration of specimens, frequent, reliable and speedy transport, immediate specimen processing and sorting), analytical (24 hour continuous analysis of patient specimens on robust platforms with a large and high-speed processing capacity) and post-analytical (real-time result reporting and interpretation). It is also important to recognise that the perception of TAT for referred work from peripheral sites is largely influenced by intricate overnight transport options to the laboratory with same-night processing, ensuring patient result availability by 6h00 the following morning.
AMPATH rose to this challenge by providing logistical and pre-analytical solutions to transport specimens quickly to the AMPATH NRL using different transportation systems. Once the specimens reach the laboratory, processing is automated and specimens are sorted by high-speed sorters (Beckman automate 2550) [Figure 1] and transported regularly to the Serology Department, where allergy testing is performed. In the Serology department further sorting and aliquotting is performed by another high-speed sorter (Beckman automate 2550) down to test / analyser level. Specimens are processed continuously as soon as they reach their sorting targets.
From a logistic, quality, cost and time-management perspective, AMPATH adopted a policy of automation and consolidation of assays onto automated platforms where possible. A large autoline from Beckman Coulter was installed in the Autolab where various chemistry, immunochemistry and infectious disease testing was consolidated [Figure 2]. Allergy and autoimmune testing was not available on this system and after meticulous research a decision was made not to integrate third-party analysers onto the system. A separate system was therefore required to manage automation of allergy and autoimmune testing.
Previously allergy testing was done on one Phadia 1000 and two Phadia 250 instruments from ImmunoDiagnostics*. These systems were chosen for the quality of their assays, ease of use and reliability. We therefore looked to ImmunoDiagnostics for higher capacity instruments that would meet our needs. We were informed of the new Phadia 2500, which is an integrated and automated system which provides for high volume allergy and autoimmune testing on one platform. This would provide an ideal solution in terms of staff and space saving, easier workflow by consolidating tests on one instrument and increased processing capacity to accommodate increased test volumes due to centralisation. At the same time we would still have the same superior test quality that we require. Unfortunately the Phadia 2500 was not ready for installation when we moved into our new laboratory facility in July 2011, but ImmunoDiagnostics prioritised our request and our Phadia 2500 was installed in October 2011. After a full instrument validation, we have transferred all of our IgE mediated allergy testing as well as a significant portion on our autoimmune testing to the Phadia 2500 [Figure 3].
Performance of the new Phadia 2500 in the AMPATH NRL
We have been extremely happy with the performance of the Phadia 2500 in our laboratory. We have experienced the following advantages after switching to the system:
– Consolidation of allergy and autoimmune diagnostics onto one platform which can be operated by a single technologist, thereby saving one full time equivalent staff member.
– Space saving, as the footprint of the Phadia 2500 is significantly smaller than the three previous allergy and autoimmunity instruments (Phadia 1000 and two Phadia 250s)
– Continuous random access with continuous specimen processing.
– Large processing capacity, which eliminates bottlenecks over peak times.
AMPATH routinely tests for approximately 260 individual allergen-specific IgEs, but there are approximately 700 different allergens available which can be ordered when requested. ImmunoDiagnostics is also constantly adding new allergens and recombinant allergen components to its test menu. We are therefore able to offer the widest range of allergens available for allergy testing in South Africa.
Autoimmune testing in the AMPATH NRL
Patients are screened for connective tissue diseases using immunofluoresence on HEP-2 cells. In patients with positive screening tests, further testing is done by performing a connective tissue disease (CTD) screen for extractable nuclear antigens (ENA) on the Phadia 2500 instrument. This screen contains 16 Individual ENAs. Should the screen be positive, a breakdown of the individual autoimmune components is performed. The new Phadia 2500 streamlines this process through high sample throughput and software solutions which supports our workflow protocols, thereby ensuring optimal TAT.
Range of allergy tests performed in the AMPATH NRL
AMPATH’s main objective is to be the national leader in allergy diagnostics. This is not only beneficial for individual patients, but also leads to a competitive advantage in our local market. We spend a lot of time on research and development in allergy and also on implementing scientifically proven methods or tests in our testing repertoire.
In addition to IgE mediated allergy testing to crude allergen extracts, AMPATH is also placing emphasis on testing to different allergen components. This is either available as singular allergens on the Phadia 2500, or as part of a multiplex microchip array (ISAC or Immuno-Solid Array Chip) from ImmunoDiagnostics. The latest ISAC assay detects specific IgE to 112 recombinant allergens and can provide additional diagnostic insight by the prediction of cross-reactivity, prediction of risk for severe reactions, prediction of whether allergies will be outgrown and additional information on the heat-stability and bio-degradibility of certain allergens.
There has also been an increasing focus on cellular allergy (non-IgE mediated allergy) and AMPATH has expanded its test repertoire by offering Basophil Activation Testing (BAT) by flow-cytometry for foods and food additives, drugs, venoms and inhalants [Figure 4]. Our BAT was developed in-house, and commercially available allergens from Buhlman Laboratories are mostly used, except for drug allergies, where the suspect drug itself is used and tested in different concentrations in comparison to a non-allergic control patient. A similar testing strategy is followed for T-cell mediated allergies, where a modified T-cell proliferation assay (LTT) is used, mainly for drug allergies, metal allergies and occupational allergies. The demand for these services has increased substantially and cellular testing volumes are approaching a third of our total allergy testing requests.
A new philosophy in allergy testing – from bench to bedside
Experience has showed us that in vitro allergy testing only isn’t sufficient in our local environment.AMPATH has therefore instituted in vivo allergy testing at multiple allergy testing depots throughout the country. A range of in vivo allergy testing, such as skin prick testing (SPT), prick-prick testing, intradermal testing and patch testing may be performed at some of these depots.
Although AMPATH’s combined allergy testing services with careful interpretation by allergy consultant pathologists is usually adequate, there is a small minority of complicated patients that still require additional testing or for whom the most appropriate testing protocol cannot be identified. After becoming aware of this void, we decided to take allergy testing from the bench to the bedside by providing a diagnostic referral clinic for allergies and immunology. Clinicians can refer complicated patients to this clinic for an appropriate diagnostic service where the patient’s history and physical examination is considered before testing and management guidelines are given based on test result interpretation. Allergen immunotherapy is also provided where indicated [Figure 5].
Lessons learnt from centralisation and automation
Automation can lead to significant benefits in diagnostic pathology when applied appropriately. The full benefits of automation can only be reached when both pre-analytical as well as analytical steps are automated. Optimal workflow planning is essential to the success of the automation project. Automation works optimally in a simplified workflow environment – too many rules, exceptions and workarounds slow the process. Automation also works optimally if hardware, instruments and software are from the same supplier. Although third party analysers can easily be connected to an automated track, software may often not be fully compatible with middleware and can lead to suboptimal TAT.
The way forward
We want more automation, more integration and more consolidation. We envisage a smaller staff complement of highly trained staff. We are looking at more automation solutions, e.g. slide processing and other manual techniques performed in our serology department. We are also aiming to expand our cellular allergy/immunology department with further research and test development. We are also looking at software solutions to aid with allergy diagnostic interpretation, especially considering unique South African sensitisation patterns and cross-reactive allergens. Through these efforts and by dedication to our patients and clinicians, we aim to embrace the future of allergy diagnostics in South Africa.
References
1. Weinberg EG. Urbanisation and childhood asthma: An African perspective. JACI 2000; 105(2):224-231.
The author Dr Cathy van Rooyen, MBChB, MMed Path, FRC Path Ampath Laboratories, South Africa
*ImmunoDiagnostics (formerly Phadia) is part of Thermo Fisher Scientific
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Opened in 2011, the Institute of Molecular Biology gGmbH (IMB) at Johannes Gutenberg University, Mainz, Germany is housed in a brand new building with state-of-the-art scientific facilities. The architect suggested that ultra-low temperature freezers housed in the Institute’s Core Facilities should be capable of being linked to a central water-cooling system, an integral part of the building’s modern infrastructure.
Centre of excellence
Described as ‘a centre of excellence in the life sciences in the heart of Europe’, IMB has been funded for an initial period of ten years by the Boehringer Ingelheim Foundation, a charity dedicated to promoting outstanding research in medicine, biology, chemistry and pharmaceutical science. As a non-profit entity, which operates like an innovative academic research centre, IMB focuses on key questions in developmental biology, epigenetics and DNA repair. The aim is to transform our understanding of how we develop, adapt to our environment, age and develop diseases such as cancer.
IMB’s core facilities
Researchers at IMB have access to cutting-edge technologies and the latest equipment provided centrally. This arrangement has significant benefits, as Dr Bernhard Korn, Director of IMB Core Facilities, explains: ‘The decision to set up centralised resources comprising cost-intensive instrumentation and high-level expertise enables even small research groups at IMB to run ambitious major projects’.
‘Secondly, it is much more economical for the Institute to run instruments and equipment, such as freezers, at the limit of their capacity by sharing resources among multiple users. In addition, by locating our ULT freezers together in a dedicated area we can make use of a centralised monitoring and alarm system which constantly checks the temperature, power supply and cooling water for all units.’
Selecting ULT freezers
IMB chose to equip its core facilities with 23 SANYO MDF-U74V upright -86ºC freezers and three of the company’s MDF-C2156VAN cryogenic freezers. All are equipped with the water-cooled condenser option, to meet a requirement to utilise the Institute’s water-cooling system. The space-saving -86ºC freezers are used to hold a collection of more than 25,000 different human protein-coding clones, while the -150ºC freezers store a large number of human cell lines, tumour cell lines and tissue samples. Later, patient specimens will also be stored prior to the extraction of nucleic acids and proteins.
According to Bernhard Korn, ‘Installing SANYO freezers was essentially a ‘plug-and-play’ experience for us. The freezers arrived, they were hooked up to the cooling water, switched on and they worked. They provide a very stable, constant environment with no temperature fluctuation – this is what we really love about them. The insulated inner doors on the MDF-U74 model reduce temperature change when the freezer is opened and there is enough space for large boxes. A wide choice of shelves provides the flexibility to accommodate the storage needs of our different research groups. These freezers are very easy to operate and we like the fact that the main power switch is on the side. This means there is no danger of a unit being turned off accidentally – a recognised hazard if this switch is placed on the front panel.’
Advantages of water-cooling
The architect of the IMB building suggested that wherever possible, equipment should be water-cooled, rather than relying on traditional air cooling. So, in addition to ULT freezers, there are centrifuges, laser systems and sterile work benches which are also connected to the building’s central water-cooling system.
As Dr Korn points out, there are various advantages to water-cooling freezers, ‘With less heat dissipated in the freezer room only ventilation is needed, rather than air-conditioning that not only requires energy but wastes heat. In fact with a total of over 100 instruments throughout the Institute hooked up to the water-cooling system, only a very few labs and measurement rooms, around five per cent of the total space, require active cooling.
‘Water is circulated at 18ºC and leaves the freezers at 23–24ºC. However, this heat energy is not wasted as the warmed water is used to contribute to the central heating for the whole building. Therefore the Institute is able to reduce energy costs and benefit the environment.’
SANYO’s water-cooled ULT freezers incorporate a double plate heat exchanger which maximises energy transfer from the refrigerant to a closed water circuit. As water is more efficient than air at removing heat, the compressor efficiency is improved. As a result, not only is energy consumption 15–20 per cent lower than for an equivalent air-cooled model, but temperature recovery after door opening and sample loading is faster, protecting samples.
Further developments at IMB
2012 will see IMB expanding to its full complement of around 12 groups, with the core facilities being extended to support the additional researchers. The success of the current water-cooled ULT freezers is underlined by plans to add five or six more of the same -150ºC freezers and up to ten of the -86ºC models, all with water-cooled condensers.
Dr Korn commented, ‘SANYO is well known not only for the quality and reliability of its ULT freezers, but also for building good customer relationships and providing excellent technical support. Another important factor for IMB is the high level of service and full guarantee provided by EWALD Innovationstechnik GmbH, the German distributor. Although initially more expensive than equivalent air-cooled freezers, choosing the water-cooled condenser option enables the Institute to use less energy and make significant savings in the medium and long term. We believe this is the way to go for the future.’
Institute of Molecular Biology GmbH funded by the Boehringer Ingelheim Foundation Ackermannweg 4 55128 Mainz Tel: +49-6131-39-21501 www.imb-mainz.de
https://clinlabint.com/wp-content/uploads/sites/2/2020/08/C02-Sanyo-Dr-Bernhard-Korn.jpg4503003wmediahttps://clinlabint.com/wp-content/uploads/sites/2/2020/06/clinlab-logo.png3wmedia2020-08-26 09:48:032021-01-08 11:39:38For a dynamic new biomedical research centre, water-cooling is the way to go
AB SCIEX achieves ISO 13485 certification for manufacturing of LC/MS systems, and opens Asia Pacific Application & Training Centre in Singapore’s biomedical hub
AB SCIEX, a global leader in life science analytical technologies, today announced that it has achieved ISO 13485 certification for its quality management system. This certifies an international standard that requires a manufacturer to demonstrate a comprehensive and compliant quality management system suitable for the design and manufacturing of medical devices. Such certification is often considered a first step toward complying with European regulatory requirements for a device to be cleared for use in the clinic. AB SCIEX currently manufactures and sells LC/MS/MS systems for research use only.
‘AB SCIEX is taking the necessary steps to be compliant with regulatory requirements that govern entry into clinical diagnostics,’ said Rainer Blair, President of AB SCIEX. ‘Achieving the ISO 13485 certification is an important measure toward realising the vision of our LC/MS/MS systems to be cleared for use in clinics. The potential impact of mass spectrometry technologies on clinical diagnostics is significant.’
The ISO 13485 certification covers AB SCIEX’s manufacturing facility in Singapore as well as its R&D design center in Toronto, Canada. AB SCIEX is a leader with more than 20 years of innovation and market leadership. Its instrumentation is used in clinical research, forensic toxicology, biomedical research and drug discovery and development. The achievement of ISO certification offers customers and suppliers an additional level of confidence in the quality and reliability of AB SCIEX instruments as well as the company’s commitment to continuous improvement.
AB SCIEX has also announced the opening of its newest APAC Regional Application & Training Centre. Located in Singapore’s biomedical hub at Biopolis, this centre provides comprehensive service, support and application development to enable the scientific community in Singapore and the rest of ASEAN, Australia New Zealand, Japan and Korea to continue its increasing use of mass spectrometry technologies for a broad range of applications. The new Singapore facility complements the regional application support centre that the company opened last year in Shanghai, China.
This Singapore facility serves as a regional hub for the scientific community to learn about the latest innovations in analytical-based laboratory instrumentation. Its primary functions include sample analysis, instrument and workflow demonstrations, comprehensive training programs, region-specific applications development, on-site and remote customer support, and scientific collaborations with research leaders in a variety of life science disciplines. The company has a long history of partnership throughout the life science industry within Singapore and across the Asia Pacific region. It serves a broad range of customers in government agencies, academic research, clinical research and pharmaceutical industries.
‘Leading companies such as AB SCIEX continue to play an important role in the development of Singapore’s biomedical sciences sector by providing the latest tools to advance our efforts in drug discovery research. This new centre is an excellent example of how companies can foster synergies and partnerships with the research community in Biopolis to develop innovative and region-specific solutions for Asia,’ said Mr. Kevin Lai, Deputy Director, Biomedical Sciences, Singapore Economic Development Board.’
‘AB SCIEX continues to be a trusted partner for our customers and collaborators in Singapore and throughout the Asia Pacific region,’ said Johnson Ho, Vice President of Sales, Asia-Pacific. ‘Our new APAC Regional Application & Training Centre represents our commitment to deliver world-class service and support to help our customers address critical issues, such as food safety, environmental contamination and the accuracy of clinical research results.’
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https://clinlabint.com/wp-content/uploads/sites/2/2020/08/25791-CLI-FLC-apr-may-12-FINAL.jpg9927003wmediahttps://clinlabint.com/wp-content/uploads/sites/2/2020/06/clinlab-logo.png3wmedia2020-08-26 09:48:032021-01-08 11:39:37Freelite – Don’t Miss the signs
https://clinlabint.com/wp-content/uploads/sites/2/2020/08/25919-INOVA-NovaView-ad-CLI-FPg.jpg10007003wmediahttps://clinlabint.com/wp-content/uploads/sites/2/2020/06/clinlab-logo.png3wmedia2020-08-26 09:48:032021-01-08 11:39:37See the future of digital IFA. Today.
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Ensuring sectioning quality in TMA analysis for the Human Protein Atlas project
, /in Featured Articles /by 3wmediaThe Human Protein Atlas Project is carrying out the systematic exploration of the human proteome using antibody-based proteomics, thus providing an invaluable publicly available HPA portal tool for pathology-based biomedical research. As part of the project, the Uppsala-based Science for Life Laboratory tissue profiling group has so far cut more than 200,000 slides from over 1400 tissue microarrays (TMAs). This article describes how the tissue microarrays and slides are made, and how a rotary microtome with different cutting modes and an automated Section Transfer System together ensure that high-quality, reproducible sections are generated.
by Ing-Marie Olsson, Catherine Davidson and Dr Caroline Kampf
A publicly available protein dictionary
Molecular tools developed in the research arena are making a significant contribution in the evolution of tissue-based diagnostics. Immunohistochemistry (IHC) is now well recognised as a means of enhancing morphological analysis, with protein expression patterns considered as effective diagnostic and prognostic indicators for various cancers. For example, within diagnostic pathology, IHC could determine the origin of poorly differentiated tumours and also be used to stratify tumours for optimum treatment regimes.
Consequently, the Human Protein Atlas (HPA) project was initiated in 2003 by the Knut and Alice Wallenberg Foundation to enable the systematic exploration of the human proteome using antibody-based proteomics. Since then, the publicly available HPA portal (www.proteinatlas.org) has amassed a database of millions of high resolution images showing the spatial distribution of proteins in 46 different normal human tissues and 20 different cancer cell types, as well as 47 different human cell lines. As such, the HPA can provide an invaluable tool for pathology-based biomedical research, including protein science and biomarker discovery for disease identification [1].
Tissue profiling
One of the key sites involved in this immense project is the Uppsala-based Science for Life Laboratory (SciLifeLab Uppsala) tissue profiling group [2]. This highly experienced group is focused on histopathology, with special emphasis on tissue microarray (TMA) production, immunohistochemistry and slide scanning. The enormity of profiling the human proteome requires the use of high throughput techniques, prompting the SciLifeLab team to adopt a TMA format to enable them to perform simultaneous multiplex histological analyses.
TMAs are paraffin blocks containing cores of selected tissues or cell preparations assembled together for subsequent sectioning to enable the effective and efficient utilisation of valuable tissue samples, as well as reducing the use of expensive IHC reagents. Multi-tissue blocks were first introduced by Battifora in 1986 with his ‘multitumour (sausage) tissue block’ [3]. Then in 1998, Kononen and collaborators standardised the technology and developed instrumentation which uses a sampling approach to produce tissues of regular size and shape that can be more densely and precisely arrayed [4].
As part of the HPA project the SciLifeLab Uppsala tissue profiling facility has constructed over 1400 TMAs containing over 100,000 tissue cores, in addition to 180 cellular microarrays (CMA) containing over 23,800 cell cores. Over 200,000 slides cut from these arrays have then been stained using immunohistochemical techniques, of which more than 100,000 have been scanned for further analysis. The SciLifeLab team evidently holds a great deal of practical experience in TMA production and, in fact, now offers an external TMA production service [2]. Consequently, its experts handle many different types and combinations of tissues, for which they observe that high quality sectioning is fundamental to TMA production, the primary aim of which is to amplify a scarce resource.
TMA production
The most efficient method of constructing tissue microarrays is by extracting cylinders of donor tissue with a sharp punch and then assembling them into a recipient block that has uniformly sized holes in a grid pattern. Tissue and cell microarrays are made according to a preset standard within the HPA, where paraffin blocks are used in a matrix containing from 72 up to 120 tissue cores. The standard diameter of each core is 1 mm (tissues) and 0.6 mm (cells), with a length of 2-4mm. This is achieved by using a needle to remove relevant tissue from a donor paraffin block which is then inserted into a recipient paraffin block.
Once all tissue cores are in position within the array, it is then ‘baked’ at 42ºC to melt them together into a homogenous paraffin block. This 40 minute baking period ensures that every core is merged with the melted paraffin in the block and, therefore, totally secured for sectioning into 4 µm sections prior to mounting onto glass slides. Thereafter, these multiplex tissue sections are ready for further histological analysis and final slide scanning to transf orm stained glass slides into digital high-resolution images.
Quality sectioning
When sectioning TMAs, the greatest risk of valuable tissue loss or damage can occur during transfer to a water bath. For this reason, the SciLifeLab tissue profiling group uses microtomes with a ‘waterfall’ system (Thermo Scientific HM355S and Thermo Scientific Section Transfer System) to eliminate such risks. A ‘waterfall’ automated Section Transfer System stretches sample ribbons as they are cut, whilst simultaneously transporting them from the blade into the attached circulating laminar flow bath. From this water bath, sections can be extracted and mounted onto a glass slide. Mounting two microarray sections per slide can further reduce IHC reagent usage and enhance workflow within the tissue profiling group.
By using the Section Transfer System the group routinely obtains over 200 quality sections per TMA, depending on the size of donor block and representative tissue within it. Although it is possible to obtain many more sections, for quality assurance (QA) purposes the SciLifeLab team performs a QA after every 50th section, introducing replacement cores where required to ensure that at least 85% of the tissue cores are always present.
The actual composition of a tissue array can also cause complications when sectioning, dependent on whether tissues are homogenous cancer types, or normal tissues where heterogeneity is greater. Furthermore, fatty tissue such as that from brain and breast should not remain within a warm water bath for an extended period due to risk of tissue melting. Conversely, other tissue such as skin and thyroid gland, needs to remain in the water bath for longer in order to ensure that it is sufficiently stretched.
To overcome such issues with tissue composition, SciLifeLab experts group tissues into those with similar texture and hardness when sectioning to make set up easier and improve workflow. For example, the HM355S microtome offers a choice of four mechanised cutting modes that give SciLifeLab greater control over section generation according to varying requirements. Mechanised cutting delivers the slow, smooth, even and controlled action necessary for sectioning harder consistency specimens.
A further sectioning consideration at SciLifeLab Uppsala is the fact that the TMAs are paraffin embedded. Consequently, a peltier-cooled attachment (Thermo Scientific Cool Cut) is used on the group’s microtomes to prolong the cutting period by maintaining a cool block temperature. By using such a cooling tool, 50 TMA sections can be cut consecutively in 50 minutes without the need to remove and re-cool the block on ice, again ensuring effective throughput and efficient laboratory operation.
SciLifeLab tissue profiling services
Tissue Microarrays (TMAs) are coming to the fore as an ideal means of providing multiplex tissue analysis, not only for research based applications, but also for clinical applications: identifying biomarkers for identification of disease, histological grading and detecting disease recurrence [5,6,7]. Some hospital laboratories are also starting to utilise TMAs as controls for diagnostic comparisons.
With over 100 personnel working on the HPA project alone, the SciLifeLab facility provides access to its extensive protein profiling results to laboratories throughout Sweden and beyond. In addition, leveraging their expertise gained in constructing tissue arrays for high throughput protein screening, the SciLifeLab team in Uppsala has also recently extended its capabilities to offer an external TMA production, sectioning and scanning service [2].
Working to a user specified template, the facility can turnaround 120 core duplicate arrays within 24 hours from receipt of the donor tissue blocks. The venture operates as cost neutral, utilising the team’s experience in generating high quality sections at a resolution of 2µm-10µm to provide consistent and reproducible material for downstream analysis. Since its inception, the TMA service has produced more than 100 custom arrays, supporting investigation of clinical models for a wide range of disease states, including cancer, diabetes, heart disease and neurodegenerative disorders.
Establishments utilising the tissue profiling group’s TMA services include university research, hospital and even veterinary laboratories. Such is the experience of this SciLifeLab group, it has been able to produce TMAs on almost any kind of tissue. Although bone and skin can prove difficult, the team can even produce TMAs for these by careful orientation of skin samples and decalcification of bone prior to final preparation.
Advanced technical know-how and state-of-the-art equipment, combined with a broad scientific knowledge, all mean that the SciLifeLab tissue profiling facility is ideally placed to meet high throughput, high quality TMA production needs for the HPA, whilst simultaneously ensuring service excellence for external customers.
References
1. Pontén F et al. The Human Protein Atlas – a tool for pathology. J Pathol 2008; 216(4): 387-93.
2. http://scilifelab.uu.se/technologyplatforms/Proteomic/Tissue_Profiling_Center/?languageId=1 3. Battifora H. The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest 1986; 55(2): 244-8.
4. Kononen J et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nature Medicine 2008; 4: 844-847.
5. Rimm D et al. Cancer and Leukemia Group B Pathology Committee Guidelines for tissue microarray construction representing multicentre prospective clinical trial issues. J Clinical Oncology 2011; 29 (16): 2282-2290.
6. Schmidt L et al. Tissue microarrays are reliable tools for the clinicopathological characterisation of lung cancer tissue. Anticancer Research 2009; 29: 201-210.
7. Smith V et al. Tissue microarrays of human xenografts. Cancer Genomics & Proteomics 2008; 5: 263-274.
The authors
Ing-Marie Olsson, Team Leader
TMA production, sectioning and scanning, Human Protein Atlas (HPA), Tissue Profiling Centre, Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala, Sweden
Tel. +46 18 471 5040
e-mail: ingmarie.olsson@igp.uu.se
Catherine Davidson, Sectioning Product Manager
Thermo Fisher Scientific, Anatomical Pathology, Runcorn, UK
Tel. +44 (0) 1928 534122
e-mail: catherine.davidson@thermofisher.com
www.thermoscientific.com/pathology
Dr. Caroline Kampf, Site Director Human Protein Atlas (HPA), Tissue Profiling Centre, Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala, Sweden
Tel. +46 18 471 4879
e-mail: Caroline.Kampf@igp.uu.se
Diagnostic strategy for coeliac disease in line with new ESPGHAN guidelines
, /in Featured Articles /by 3wmediaNewly revised guidelines for the diagnosis of coeliac disease (CD) place greater emphasis on laboratory testing, enabling the number of small-intestinal biopsies performed to be significantly reduced. The detection of antibodies against tissue transglutaminase (anti-tTG) or endomysium (EmA) remains a cornerstone of diagnosis, while further diagnostic procedures have gained new significance. The molecular genetic determination of the human leukocyte antigens (HLA) DQ2 and DQ8 now plays a central role in diagnosis, thanks to a better understanding of the genetic factors underpinning the disease. Moreover, state-of-the-art assays for antibodies against deamidated gliadin peptides (DGP), as oppose to native gliadin, now constitute a highly sensitive and specific analysis to support diagnosis. In the new guidelines, anti-tTG and anti-DGP are recommended as first-line tests in symptomatic individuals, while HLA-DQ2/DQ8 analysis is the initial step for screening asymptomatic persons with a high disease risk.
by Dr Jacqueline Gosink
CD, which is also known as gluten-sensitive enteropathy or non-tropical sprue, is an autoimmune disease caused in genetically predisposed individuals by consumption of gluten-containing cereals. The disease process is triggered by protein components of gluten known as prolamins, of which gliadin is the most common. Partially digested prolamin peptides are chemically modified (deamidated) in the intestine wall by the enzyme tTG. The immune system of genetically predisposed persons reacts with both the deamidated peptides and tTG, causing chronic inflammation of the small-intestinal mucosa, which results in atrophy of the villi and reduced resorption of nutrients. The only effective treatment for CD is observance of a gluten-free diet.
A clinical chameleon
The classic symptoms of CD are fatigue, abdominal pain, diarrhoea, effects of malabsorption such as weight loss, anaemia and growth retardation in children, vomiting, constipation and bone pains. However, CD is now recognised to be a multifaceted condition which can manifest in many ways. Some patients have non-typical symptoms such as osteoporosis, neuropathies, carditis, pregnancy problems or lymphoma. CD patients may also suffer from Duhring’s dermatitis herpetiformis, a recurrent skin disease characterised by subepidermal blisters.
The disease may also present in silent, latent or potential forms [1]. In the silent form, patients are asymptomatic, but nevertheless exhibit CD-specific antibodies, relevant HLA alleles and villous atrophy. Those with latent CD have previously had a gluten-dependent enteropathy, but are now free of enteropathy; they may or may not exhibit antibodies and/or symptoms. In cases of potential CD, individuals have positive antibodies and compatible HLA, but as yet no symptoms; they may or may not go on to develop CD.
While the prevalence of symptomatic CD is around 0.1%, the prevalence of the disease in all its forms is estimated to be as high as 1%. Many experts now speak of the coeliac disease iceberg, in which classic CD represents only the tip.
New ESPGHAN diagnostic criteria
Early in 2012, the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) released a revised version of its 1990 guidelines for the diagnosis of coeliac disease [1], which were compiled by a group of 17 international experts in the field. The new diagnostic criteria are defined by two algorithms: algorithm 1 [Figure 1A] is applied to symptomatic individuals, while algorithm 2 [Figure 1B] is used for asymptomatic individuals with a high disease risk, for example first-degree relatives of CD patients and patients with type 1 diabetes mellitus, Down’s syndrome, autoimmune thyroid or liver disease, Turner’s syndrome, Williams’ syndrome or selective IgA deficiency.
In algorithm 1 the first-line approach is the determination of anti-tTG antibodies of class IgA in patient serum. In order to exclude the possibility of an IgA deficiency, either total IgA or specific IgG (e.g. deamidated gliadin) should be investigated in parallel. If the anti-tTG antibody titre is very high (>10 times upper normal limit), and if this result is reinforced by positive EmA and compatible HLA, it is no longer considered necessary to perform a biopsy. If serological/genetic findings are inconclusive, results must be confirmed by histological examination of duodenal biopsy tissue to demonstrate villous atrophy and crypt hyperplasia. Diagnostic tests should be carried out in individuals on a gluten-containing diet. A gluten challenge is now only performed under exceptional circumstances.
In algorithm 2, the genetic parameters HLA-DQ2/DQ8 are initially determined to establish the genetic susceptibility. If these are negative, the risk of CD is negligible and no further tests are required. If HLA alleles are compatible with CD, specific antibody tests are used to follow up. In this group a duodenal biopsy is a prerequisite for a definite diagnosis of CD.
The individual diagnostic parameters and the technologies used to detect them are reviewed in the following sections.
Antibodies against tissue transglutaminase (anti-tTG, EmA)
Autoantibodies against tTG of immunoglobulin class IgA are the most important serological marker for CD, as they possess a very high sensitivity and specificity for the disease. While they are virtually never found in healthy individuals or patients with other intestinal diseases, their prevalence in untreated CD is near to 100%. Anti-tTG antibodies are alternatively known as EmA, depending on the test method used: EmA are determined using indirect immunofluorescence [Figure 2], while anti-tTG are detected using monospecific test systems such as ELISA [Figure 3].
Detection of EmA using indirect immunofluorescence is considered the reference standard for CD-specific antibodies due to its unsurpassed sensitivity and specificity. However, the microscopic evaluation required is demanding and dependent on the proficiency of laboratory staff. Enzyme immunoassays for detection of anti-tTG antibodies are often preferred due to their simplicity, cost-effectiveness and automatability, combined with their high sensitivity and specificity. Modern ELISAs for determination of anti-tTG antibodies are based on recombinant human tTG. A multitude of clinical studies have confirmed the efficacy of this method, with high-quality tests yielding a sensitivity of 90-100% and a specificity of 95-100% for active CD.
Antibodies against deamidated gliadin peptides (DGP)
Antibodies against DGP have recently assumed a more important diagnostic role, due to the development of highly sensitive and specific test systems to detect them. Conventional assays based on native full-length gliadin, which frequently yield unspecific reactions with sera from healthy persons, are now obsolete.
The advances in test design were precipitated after research revealed that only a tenth of the epitopes of the gliadin molecule are diagnostically relevant, and these must be present in deamidated form [2]. Based on these observations a novel recombinant gliadin-analogous fusion peptide (GAF) consisting of two nonapeptide components expressed in trimeric form (3X) was created [Figure 4]. The remaining 90% of the molecule was omitted, as it serves predominantly as a target for unspecific reactions.
This designer fusion protein is now used as the target antigen in the Anti-Gliadin (GAF-3X) ELISA, which provides vastly superior performance compared to conventional anti-gliadin ELISAs [3, 4]. In a multicentre study using a total of over 900 sera, the new test yielded a sensitivity (at 95% specificity) of 83%/94% (IgA/IgG) compared to 54%/31% for a conventional anti-gliadin ELISA. This represents an increase of 29% for IgA and 63% for IgG, significantly enhancing the relevance of the analysis.
Use of the Anti-Gliadin (GAF-3X) ELISA in combination with the Anti-tTG ELISA significantly increases the serological detection rate for CD and dermatitis herpetiformis [5]. The IgG version of the ELISA is particularly valuable for identifying CD patients with an IgA deficiency [6], which is frequently associated with CD. Determination of antibodies against DGP is also suitable for assessing disease activity and for monitoring a gluten-free diet or a gluten-load test.
HLA-DQ2 and DQ8
HLA-DQ2 and DQ8 are the principle determinants of genetic susceptibility for CD and are found in virtually all patients. The strong genetic background to CD is highlighted by familial prevalences of 10% in first-degree relatives of patients, 70% in identical twins and 11% in non-identical twins. However, the presence of HLA-DQ2/DQ8 is not sufficient by itself to cause CD. Around a third of the healthy population exhibits DQ2/DQ8 alleles.
Although not a particularly specific parameter, HLA-DQ2/DQ8 is a valuable tool for exclusion diagnostics. If neither DQ2 or DQ8 are present, then CD can be virtually ruled out. It is for this reason that DQ2/DQ8 analysis is now recommended as the first-line test for screening asymptomatic persons at high risk of CD, as defined by the presence of an associated disease or family history (algorithm 2). If DQ2/DQ8 is negative no further follow up is necessary. HLA-DQ2/DQ8 also functions as a confirmatory parameter in symptomatic persons (algorithm 1), and it is one of a triad of laboratory parameters that can be employed to diagnose CD without biopsy in these individuals. DQ2/DQ8 analysis is also helpful for clarifying cases in which diagnosis is inconclusive due to ambiguous serological/biopsy results, especially in infants or in patients who are already on a gluten-free diet, and for differentiation of CD from other intestinal diseases.
HLA-DQ2/DQ8 alleles can be determined using microarray test systems such as the EUROArray system. This analysis is simple to perform, requiring no previous knowledge of molecular biology. Disease-associated gene sections are amplified from purified genomic patient DNA samples by the polymerase chain reaction (PCR) [Figure 5]. The fluorescently labelled PCR products are then detected using microarray BIOCHIP slides composed of immobilised complementary probes. The evaluation [Figure 6] and documentation of results is fully automated using specially developed software (EUROArrayScan). In clinical studies employing precharacterised samples, this microarray yielded a sensitivity of 100% and a specificity also of 100% [7], demonstrating its ability to deliver accurate and reliable results in HLA analysis.
Conclusions
The publication of updated ESPGHAN guidelines for the diagnosis of coeliac disease has reinforced the indispensible role of anti-tTG and EMA in diagnosis and propelled further laboratory diagnostic parameters such as HLA-DQ2/DQ8 and anti-DGP into the limelight. In clear-cut cases, a thorough serological and genetic investigation is now considered sufficient to obtain a diagnosis, allowing the costs and patient discomfort associated with biopsy to be avoided. The state-of-the-art diagnostic tools available today are not only a boon for patient diagnosis, but will also help to advance our understanding of this enigmatic and seemingly widely occurring disease.
References
1. Husby S et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition Guidelines for the diagnosis of CD. JPGN 2012; 54: 136–160.
2. Schwerz E et al. Serologic assay based on gliadin-related nonapeptides as a highly sensitive and specific diagnostic aid in celiac disease. Clin Chem 2004; 50: 2370-2375.
3. Prause C et al. Antibodies against deamidated gliadin as new and accurate biomarkers of childhood CD. JPGN 2009; 49: 52-58.
4. Prause C et al. New developments in serodiagnosis of childhood celiac disease. Ann NY Acad Sci 2009; 1173: 28-35.
5. Kasperkiewicz M et al. Novel assay for detecting celiac disease-associated autoantibodies in dermatitis herpetiformis using deamidated gliadin-analogous fusion peptides. J Am Acad Dermatol [Epub ahead of print] (2011).
6. Villalta D et al. IgG Antibodies against deamidated gliadin peptides for diagnosis of celiac disease in patients with IgA deficiency. Clin Chem 2010; 56: 464-468.
7. Pfeiffer T et al. Microarray based analysis of the genetic risk factors HLA-DQ2/DQ8 – a novel test system for the diagnostic exclusion of celiac disease. 44th Annual Meeting of The European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), Italy, May 2011.
The author
Dr Jacqueline Gosink
EUROIMMUN AG
Seekamp 31
23560 Luebeck
Germany
Tel: +49 451 5855 25881
e-mail: j.gosink@euroimmun.de
Automation, consolidation and expansion in allergy diagnosis: embracing the future
, /in Featured Articles /by 3wmediaThe challenge of providing an effective allergy testing service in the face of the increased prevalence of the condition and a limited number of local allergists is being met by a centralised and specialised South African lab with the help of state-of-the-art equipment and facilities.
by Dr Cathy van Rooyen
Allergy is on the increase, especially in developing countries where industrialisation, lifestyle changes and socioeconomic factors lead to an increase in allergy prevalence. South Africa is no exception, as shown by data from prevalence studies of asthma in rural and urban populations [1]. The challenge in South Africa is to provide an adequate and comprehensive allergy service to a growing allergic population, in a healthcare system that has until recently been ill-prepared to meet allergy demands. South Africa has a dire shortage of trained allergists, as a postgraduate allergy diploma has only recently been introduced by the college of family medicine in South Africa and recognition of Allergy as a subspecialty of Paediatrics and Internal Medicine has only been introduced this year. There is no stand-alone postgraduate specialisation in Allergy or Immunology as seen in European countries.
The diagnostic laboratory not only faces the increased demands for allergy testing from the handful of clinicians with formal allergy training, but also the majority of requests from general practitioners without adequate allergy knowledge and who in conjunction with allergy testing, require in-depth interpretation of results, and advice on additional testing and patient management.
Allergy testing at AMPATH pathologists
AMPATH is one of the largest private pathology providers in South Africa and also provides the most extensive diagnostic service for allergy in South Africa. After consideration was given to the most effective model for the provision of this service, a model of centralisation of in vitro allergy testing was adopted. AMPATH built a National Reference Laboratory (NRL) for centralisation of allergy and other specialised tests, which has been operational since July 2011.
In addition to the centralised in vitro testing services offered, in vivo allergy diagnostics, including skin prick testing, intradermal testing and patch testing is also offered at selected centres. AMPATH also provides a diagnostic referral clinic for Allergy, where patients with complicated allergy can be referred for a detailed workup, which includes history, examination and appropriate testing. Patients are referred back to the clinician with a detailed report on the patient’s allergies and management advice. A separate service for allergen immunotherapy is also available at this clinic.
Centralisation of allergy tests in the AMPATH NRL
The AMPATH NRL is the main testing hub for specialised laboratory tests in AMPATH, South Africa. Centralisation has many advantages, which include cost-savings by increasing and optimising test volumes on a single platform and staff and resource savings, uniform test results, pooling of expertise in test performance and interpretation, etc. However, specimens do take longer to reach a centralised testing centre. The biggest challenge in such an environment is to provide a guaranteed and acceptable turnaround time (TAT), as many specimens are transported from various locations throughout the country.
The major factors that should be addressed to improve TAT are pre-analytical (easy registration of specimens, frequent, reliable and speedy transport, immediate specimen processing and sorting), analytical (24 hour continuous analysis of patient specimens on robust platforms with a large and high-speed processing capacity) and post-analytical (real-time result reporting and interpretation). It is also important to recognise that the perception of TAT for referred work from peripheral sites is largely influenced by intricate overnight transport options to the laboratory with same-night processing, ensuring patient result availability by 6h00 the following morning.
AMPATH rose to this challenge by providing logistical and pre-analytical solutions to transport specimens quickly to the AMPATH NRL using different transportation systems. Once the specimens reach the laboratory, processing is automated and specimens are sorted by high-speed sorters (Beckman automate 2550) [Figure 1] and transported regularly to the Serology Department, where allergy testing is performed. In the Serology department further sorting and aliquotting is performed by another high-speed sorter (Beckman automate 2550) down to test / analyser level. Specimens are processed continuously as soon as they reach their sorting targets.
From a logistic, quality, cost and time-management perspective, AMPATH adopted a policy of automation and consolidation of assays onto automated platforms where possible. A large autoline from Beckman Coulter was installed in the Autolab where various chemistry, immunochemistry and infectious disease testing was consolidated [Figure 2]. Allergy and autoimmune testing was not available on this system and after meticulous research a decision was made not to integrate third-party analysers onto the system. A separate system was therefore required to manage automation of allergy and autoimmune testing.
Previously allergy testing was done on one Phadia 1000 and two Phadia 250 instruments from ImmunoDiagnostics*. These systems were chosen for the quality of their assays, ease of use and reliability. We therefore looked to ImmunoDiagnostics for higher capacity instruments that would meet our needs. We were informed of the new Phadia 2500, which is an integrated and automated system which provides for high volume allergy and autoimmune testing on one platform. This would provide an ideal solution in terms of staff and space saving, easier workflow by consolidating tests on one instrument and increased processing capacity to accommodate increased test volumes due to centralisation. At the same time we would still have the same superior test quality that we require. Unfortunately the Phadia 2500 was not ready for installation when we moved into our new laboratory facility in July 2011, but ImmunoDiagnostics prioritised our request and our Phadia 2500 was installed in October 2011. After a full instrument validation, we have transferred all of our IgE mediated allergy testing as well as a significant portion on our autoimmune testing to the Phadia 2500 [Figure 3].
Performance of the new Phadia 2500 in the AMPATH NRL
We have been extremely happy with the performance of the Phadia 2500 in our laboratory. We have experienced the following advantages after switching to the system:
– Consolidation of allergy and autoimmune diagnostics onto one platform which can be operated by a single technologist, thereby saving one full time equivalent staff member.
– Space saving, as the footprint of the Phadia 2500 is significantly smaller than the three previous allergy and autoimmunity instruments (Phadia 1000 and two Phadia 250s)
– Continuous random access with continuous specimen processing.
– Large processing capacity, which eliminates bottlenecks over peak times.
AMPATH routinely tests for approximately 260 individual allergen-specific IgEs, but there are approximately 700 different allergens available which can be ordered when requested. ImmunoDiagnostics is also constantly adding new allergens and recombinant allergen components to its test menu. We are therefore able to offer the widest range of allergens available for allergy testing in South Africa.
Autoimmune testing in the AMPATH NRL
Patients are screened for connective tissue diseases using immunofluoresence on HEP-2 cells. In patients with positive screening tests, further testing is done by performing a connective tissue disease (CTD) screen for extractable nuclear antigens (ENA) on the Phadia 2500 instrument. This screen contains 16 Individual ENAs. Should the screen be positive, a breakdown of the individual autoimmune components is performed. The new Phadia 2500 streamlines this process through high sample throughput and software solutions which supports our workflow protocols, thereby ensuring optimal TAT.
Range of allergy tests performed in the AMPATH NRL
AMPATH’s main objective is to be the national leader in allergy diagnostics. This is not only beneficial for individual patients, but also leads to a competitive advantage in our local market. We spend a lot of time on research and development in allergy and also on implementing scientifically proven methods or tests in our testing repertoire.
In addition to IgE mediated allergy testing to crude allergen extracts, AMPATH is also placing emphasis on testing to different allergen components. This is either available as singular allergens on the Phadia 2500, or as part of a multiplex microchip array (ISAC or Immuno-Solid Array Chip) from ImmunoDiagnostics. The latest ISAC assay detects specific IgE to 112 recombinant allergens and can provide additional diagnostic insight by the prediction of cross-reactivity, prediction of risk for severe reactions, prediction of whether allergies will be outgrown and additional information on the heat-stability and bio-degradibility of certain allergens.
There has also been an increasing focus on cellular allergy (non-IgE mediated allergy) and AMPATH has expanded its test repertoire by offering Basophil Activation Testing (BAT) by flow-cytometry for foods and food additives, drugs, venoms and inhalants [Figure 4]. Our BAT was developed in-house, and commercially available allergens from Buhlman Laboratories are mostly used, except for drug allergies, where the suspect drug itself is used and tested in different concentrations in comparison to a non-allergic control patient. A similar testing strategy is followed for T-cell mediated allergies, where a modified T-cell proliferation assay (LTT) is used, mainly for drug allergies, metal allergies and occupational allergies. The demand for these services has increased substantially and cellular testing volumes are approaching a third of our total allergy testing requests.
A new philosophy in allergy testing – from bench to bedside
Experience has showed us that in vitro allergy testing only isn’t sufficient in our local environment.AMPATH has therefore instituted in vivo allergy testing at multiple allergy testing depots throughout the country. A range of in vivo allergy testing, such as skin prick testing (SPT), prick-prick testing, intradermal testing and patch testing may be performed at some of these depots.
Although AMPATH’s combined allergy testing services with careful interpretation by allergy consultant pathologists is usually adequate, there is a small minority of complicated patients that still require additional testing or for whom the most appropriate testing protocol cannot be identified. After becoming aware of this void, we decided to take allergy testing from the bench to the bedside by providing a diagnostic referral clinic for allergies and immunology. Clinicians can refer complicated patients to this clinic for an appropriate diagnostic service where the patient’s history and physical examination is considered before testing and management guidelines are given based on test result interpretation. Allergen immunotherapy is also provided where indicated [Figure 5].
Lessons learnt from centralisation and automation
Automation can lead to significant benefits in diagnostic pathology when applied appropriately. The full benefits of automation can only be reached when both pre-analytical as well as analytical steps are automated. Optimal workflow planning is essential to the success of the automation project. Automation works optimally in a simplified workflow environment – too many rules, exceptions and workarounds slow the process. Automation also works optimally if hardware, instruments and software are from the same supplier. Although third party analysers can easily be connected to an automated track, software may often not be fully compatible with middleware and can lead to suboptimal TAT.
The way forward
We want more automation, more integration and more consolidation. We envisage a smaller staff complement of highly trained staff. We are looking at more automation solutions, e.g. slide processing and other manual techniques performed in our serology department. We are also aiming to expand our cellular allergy/immunology department with further research and test development. We are also looking at software solutions to aid with allergy diagnostic interpretation, especially considering unique South African sensitisation patterns and cross-reactive allergens. Through these efforts and by dedication to our patients and clinicians, we aim to embrace the future of allergy diagnostics in South Africa.
References
1. Weinberg EG. Urbanisation and childhood asthma: An African perspective. JACI 2000; 105(2):224-231.
The author
Dr Cathy van Rooyen, MBChB, MMed Path, FRC Path
Ampath Laboratories, South Africa
*ImmunoDiagnostics (formerly Phadia) is part of Thermo Fisher Scientific
For a dynamic new biomedical research centre, water-cooling is the way to go
, /in Featured Articles /by 3wmediaOpened in 2011, the Institute of Molecular Biology gGmbH (IMB) at Johannes Gutenberg University, Mainz, Germany is housed in a brand new building with state-of-the-art scientific facilities. The architect suggested that ultra-low temperature freezers housed in the Institute’s Core Facilities should be capable of being linked to a central water-cooling system, an integral part of the building’s modern infrastructure.
Centre of excellence
Described as ‘a centre of excellence in the life sciences in the heart of Europe’, IMB has been funded for an initial period of ten years by the Boehringer Ingelheim Foundation, a charity dedicated to promoting outstanding research in medicine, biology, chemistry and pharmaceutical science. As a non-profit entity, which operates like an innovative academic research centre, IMB focuses on key questions in developmental biology, epigenetics and DNA repair. The aim is to transform our understanding of how we develop, adapt to our environment, age and develop diseases such as cancer.
IMB’s core facilities
Researchers at IMB have access to cutting-edge technologies and the latest equipment provided centrally. This arrangement has significant benefits, as Dr Bernhard Korn, Director of IMB Core Facilities, explains: ‘The decision to set up centralised resources comprising cost-intensive instrumentation and high-level expertise enables even small research groups at IMB to run ambitious major projects’.
‘Secondly, it is much more economical for the Institute to run instruments and equipment, such as freezers, at the limit of their capacity by sharing resources among multiple users. In addition, by locating our ULT freezers together in a dedicated area we can make use of a centralised monitoring and alarm system which constantly checks the temperature, power supply and cooling water for all units.’
Selecting ULT freezers
IMB chose to equip its core facilities with 23 SANYO MDF-U74V upright -86ºC freezers and three of the company’s MDF-C2156VAN cryogenic freezers. All are equipped with the water-cooled condenser option, to meet a requirement to utilise the Institute’s water-cooling system. The space-saving -86ºC freezers are used to hold a collection of more than 25,000 different human protein-coding clones, while the -150ºC freezers store a large number of human cell lines, tumour cell lines and tissue samples. Later, patient specimens will also be stored prior to the extraction of nucleic acids and proteins.
According to Bernhard Korn, ‘Installing SANYO freezers was essentially a ‘plug-and-play’ experience for us. The freezers arrived, they were hooked up to the cooling water, switched on and they worked. They provide a very stable, constant environment with no temperature fluctuation – this is what we really love about them. The insulated inner doors on the MDF-U74 model reduce temperature change when the freezer is opened and there is enough space for large boxes. A wide choice of shelves provides the flexibility to accommodate the storage needs of our different research groups. These freezers are very easy to operate and we like the fact that the main power switch is on the side. This means there is no danger of a unit being turned off accidentally – a recognised hazard if this switch is placed on the front panel.’
Advantages of water-cooling
The architect of the IMB building suggested that wherever possible, equipment should be water-cooled, rather than relying on traditional air cooling. So, in addition to ULT freezers, there are centrifuges, laser systems and sterile work benches which are also connected to the building’s central water-cooling system.
As Dr Korn points out, there are various advantages to water-cooling freezers, ‘With less heat dissipated in the freezer room only ventilation is needed, rather than air-conditioning that not only requires energy but wastes heat. In fact with a total of over 100 instruments throughout the Institute hooked up to the water-cooling system, only a very few labs and measurement rooms, around five per cent of the total space, require active cooling.
‘Water is circulated at 18ºC and leaves the freezers at 23–24ºC. However, this heat energy is not wasted as the warmed water is used to contribute to the central heating for the whole building. Therefore the Institute is able to reduce energy costs and benefit the environment.’
SANYO’s water-cooled ULT freezers incorporate a double plate heat exchanger which maximises energy transfer from the refrigerant to a closed water circuit. As water is more efficient than air at removing heat, the compressor efficiency is improved. As a result, not only is energy consumption 15–20 per cent lower than for an equivalent air-cooled model, but temperature recovery after door opening and sample loading is faster, protecting samples.
Further developments at IMB
2012 will see IMB expanding to its full complement of around 12 groups, with the core facilities being extended to support the additional researchers. The success of the current water-cooled ULT freezers is underlined by plans to add five or six more of the same -150ºC freezers and up to ten of the -86ºC models, all with water-cooled condensers.
Dr Korn commented, ‘SANYO is well known not only for the quality and reliability of its ULT freezers, but also for building good customer relationships and providing excellent technical support. Another important factor for IMB is the high level of service and full guarantee provided by EWALD Innovationstechnik GmbH, the German distributor. Although initially more expensive than equivalent air-cooled freezers, choosing the water-cooled condenser option enables the Institute to use less energy and make significant savings in the medium and long term. We believe this is the way to go for the future.’
Institute of Molecular Biology GmbH
funded by the Boehringer Ingelheim Foundation
Ackermannweg 4
55128 Mainz
Tel: +49-6131-39-21501
www.imb-mainz.de
SANYO E&E Europe BV
Biomedical Division
Tel. +44 (0) 1509 265265 www.eu.sanyo.com/biomedical
Industry news
, /in Featured Articles /by 3wmediaAB SCIEX achieves ISO 13485 certification for manufacturing of LC/MS systems, and opens Asia Pacific Application & Training Centre in Singapore’s biomedical hub
AB SCIEX, a global leader in life science analytical technologies, today announced that it has achieved ISO 13485 certification for its quality management system. This certifies an international standard that requires a manufacturer to demonstrate a comprehensive and compliant quality management system suitable for the design and manufacturing of medical devices. Such certification is often considered a first step toward complying with European regulatory requirements for a device to be cleared for use in the clinic. AB SCIEX currently manufactures and sells LC/MS/MS systems for research use only.
‘AB SCIEX is taking the necessary steps to be compliant with regulatory requirements that govern entry into clinical diagnostics,’ said Rainer Blair, President of AB SCIEX. ‘Achieving the ISO 13485 certification is an important measure toward realising the vision of our LC/MS/MS systems to be cleared for use in clinics. The potential impact of mass spectrometry technologies on clinical diagnostics is significant.’
The ISO 13485 certification covers AB SCIEX’s manufacturing facility in Singapore as well as its R&D design center in Toronto, Canada. AB SCIEX is a leader with more than 20 years of innovation and market leadership. Its instrumentation is used in clinical research, forensic toxicology, biomedical research and drug discovery and development. The achievement of ISO certification offers customers and suppliers an additional level of confidence in the quality and reliability of AB SCIEX instruments as well as the company’s commitment to continuous improvement.
AB SCIEX has also announced the opening of its newest APAC Regional Application & Training Centre. Located in Singapore’s biomedical hub at Biopolis, this centre provides comprehensive service, support and application development to enable the scientific community in Singapore and the rest of ASEAN, Australia New Zealand, Japan and Korea to continue its increasing use of mass spectrometry technologies for a broad range of applications. The new Singapore facility complements the regional application support centre that the company opened last year in Shanghai, China.
This Singapore facility serves as a regional hub for the scientific community to learn about the latest innovations in analytical-based laboratory instrumentation. Its primary functions include sample analysis, instrument and workflow demonstrations, comprehensive training programs, region-specific applications development, on-site and remote customer support, and scientific collaborations with research leaders in a variety of life science disciplines. The company has a long history of partnership throughout the life science industry within Singapore and across the Asia Pacific region. It serves a broad range of customers in government agencies, academic research, clinical research and pharmaceutical industries.
‘Leading companies such as AB SCIEX continue to play an important role in the development of Singapore’s biomedical sciences sector by providing the latest tools to advance our efforts in drug discovery research. This new centre is an excellent example of how companies can foster synergies and partnerships with the research community in Biopolis to develop innovative and region-specific solutions for Asia,’ said Mr. Kevin Lai, Deputy Director, Biomedical Sciences, Singapore Economic Development Board.’
‘AB SCIEX continues to be a trusted partner for our customers and collaborators in Singapore and throughout the Asia Pacific region,’ said Johnson Ho, Vice President of Sales, Asia-Pacific. ‘Our new APAC Regional Application & Training Centre represents our commitment to deliver world-class service and support to help our customers address critical issues, such as food safety, environmental contamination and the accuracy of clinical research results.’
AB SCIEX www.absciex.com
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