Autoimmune thyroid diseases, comprised by Hashimoto thyroiditis, Graves disease, and their variants, are characterised histologically by lymphocytic infiltration of the thyroid gland, biochemically by the presence of well-defined autoantibodies, and clinically by the impairment of thyroid function. The aetiology and mechanisms of the autoimmune damage depend upon genes and environmental factors such as iodine intake and smoking.Three major autoantigens have been identified for autoimmune thyroid disease: thyroglobulin, thyroperoxidase and thyrotropin receptor. Antibodies against these autoantigens are now part of the clinical toolbox. They are not only used to confirm a diagnostic suspicion, but also to predict recurrence of future onset of thyroid autoimmunity.
by Dr Alessandra De Remigis and Dr Patrizio Caturegli
Background on autoimmune thyroid diseases
Autoimmune thyroid diseases (ATDs) comprise two major conditions: Hashimoto thyroiditis with goitre and euthyroidism or hypothyroidism, and Graves disease with goitre, hyperthyroidism and often ophthalmopathy. Hashimoto thyroiditis is named after Dr Hakaru Hashimoto who described in 1912 the thyroid pathological features of four women who had undergone thyroidecomty because of compressive symptoms [1]. Graves disease is named after Dr Robert Graves who reported three patients with hyperthyroid goitre and ocular involvement (Graves ophthalmopathy) [2]. Both conditions are characterised pathologically by infiltration of the thyroid gland with autoreactive T and B lymphocytes and biochemically by the production of thyroid autoantibodies and abnormalities in thyroid function. It is not uncommon to observe transition from one clinical picture to another within the same patient over time, suggesting the existence of common immunological mechanisms [3]. Numerous clinical variants are described for each ATD. For Hashimoto thyroiditis, in addition to the classical goitrous form, fibrous, juvenile, thyrotoxic, post-partum, and IgG4-related forms are described. For Graves disease, beside the classical goitrous form with hyperthyroidism, there is the variant with hyperthyroidism and ophthalmopathy, the one with just ophthalmopathy (called euthyroid Graves disease), and the one with ophthalmopathy and localised myxoedema. Similar to many other autoimmune diseases, ATDs can occur in isolation or associated with other autoimmune diseases, often affecting other endocrine glands. In some patients this association is clinically recognisable and defined as polyglandular autoimmune syndrome.
ATDs are the most common autoimmune diseases with a population prevalence around 2 % in women and 0.2% in men. These estimates are considered to be 10 times higher if ‘subclinical disease’ is taken into consideration [4].
The pathogenesis of ATDs remains to be elucidated but it is believed to rely on the interaction between endogenous genetic factors and exogenous environmental factors. Genes that confer susceptibility to ATDs have been investigated since the 1970s via candidate gene analysis, linkage analysis and genome-wide association studies. Only a handful of genes have been identified and confirmed to increase the risk of ATDs development, but each gene contributes only a very small effect, with odds ratios typically below 3. These genes include the class II region of the major histocompatibility complex, CTLA-4, PTPN22, CD40, CD25, FCRL3, thyroglobulin, and the TSH receptor [5]. Another endogenous factor that has been studied extensively in ATD is pregnancy [6],which is known to ameliorate disease severity. The two major environmental factors implicated in ATDs initiation and progression are iodine and smoking. High iodine intake triggers lymphocytic infiltration of the thyroid in genetically susceptible animals (BB/W rats and NOD.H-2h24 mice); and is associated with increased prevalence and incidence of autoimmune thyroiditis and overt hypothyroidism in humans [7]. Iodine supplementation should thus be kept within the WHO recommended range to prevent from one side iodine deficiency and from the other side autoimmune thyroiditis [8]. Smoking does not have a univocal effect on AITD. It increases the risk of developing Graves disease and aggravates Graves ophtalmopathy. Smoking cessation is associated wth a better response of Graves ophthalmopathy to immunosuppressive treatment [9]. However, smoking seems to have a beneficial effect on Hashimoto thyroiditis and decreases the levels of thyroid autoantibodies [10].
Significant progress has been accomplished on the identification and characterisation of the thyroid autoantigens that are targeted by the immune system in patients with ATDs, so that antibodies against thyroglobulin, thyroperoxidase and the TSH receptor are now well-established tools in the clinical arena.
Thyroid antigens and antibodies Thyroglobulin
Thyroglobulin is a large glycoprotein made of two identical 330-kDa subunits composed of 2,768 amino acids. Each subunit contains 66 tyrosines that when iodinated and processed make up the thyroid hormones (T4 and T3). Thyroglobulin contains numerous immunodominant epitopes for both T and B lymphocytes. Some epitopes are located in the iodine-rich hormonogenic regions and are affected by the iodine content.
Thyroglobulin antibodies (TgAb) recognise predominantly conformational epitopes, tend to favour the IgG2 subclass and do not fix complement. TgAbs were originally detected by tanned red cell haemagglutination, then by quatitative RIAs or ELISAs, and more recently by automated chemiluminescent EIAs. The analytical sensitivity varies depending on the assay method used, and the cut-off value for positivity is typically set at 100 WHO units/mL.
TgAbs are a marker of underlying thyroid autoimmunity but can also be found in non-autoimmune thyroid diseases as well as in healthy controls [11]. Traditionally they are requested together with thyroperoxidase antibodies to corroborate a diagnostic suspicion of ATDs. Currently, however, the greatest utility of TgAb measurement is in the follow-up of patients with differentiated thyroid cancer. These patients undergo thyroidectomy, possibly combined with radioactive iodine administration, and are then followed by measuring thyroglobulin antigen in the serum. Since thyroglobulin is a thyroid-specific antigen, its serum levels should be undetectable after thyroid ablation in the absence of recurrence or metastasis. If the patient had autoimmune thyroiditis in addition to thyroid cancer, TgAbs can persist for years after thyroidectomy and interfere with the thyroglobulin antigen determination. In particular, they can cause falsely low or undetectable levels of thyroglobulin antigen, and therefore a false positive clinical assessement. This realisation has led to the introduction of reflex measurement of TgAbs any time thyroglobulin is measured in thyroid cancer patients. Numerous studies have attempted to distinguish the type of TgAb based on the specific epitopes they recognise. Latrofa and colleagues have recently reported a pattern of TgAb for patients with ATDs and another for patients with multinodular goitre and differentiated thyroid cancer [12]. TgAbs also seem to recognise distinct epitopes in healthy subjects and patients with clinically manifest disease [13], suggesting the potential clinical utility of TgAbs based on specific thyroglobulin epitopes rather than on the entire thyroglobulin molecule.
Thyroperoxidase
Thyroperoxidase is a large membrane-associated glycoprotein (933 amino acids with a molecular weight of approximately 105 kDa) expressed at the apical (follicular) side of the thyroid cell. It separates to the microvillar/microsomal fraction upon ultracentrifugation and for this reason was originally called M antigen. Thyroperoxidase antibodies (TPOAbs) are predominantly IgG, can fix complement and cause damage to the thyroid cell by cell-mediated cytotoxicity [14]. TPOAbs are considered more specific for autoimmune thyroiditis than TgAbs. They correlate directly with the number of autoreactive lymphocytes infiltrating the thyroid gland as well as with the degree of thyroid hypoechogenicity on thyroid ultrasound. Like TgAbs, TPOAbs were originally measured by semiquantitative methods, then by RIAs or ELISAs, and more recently by automated chemiluminescent EIAs. The analytical sensitivity, as for TgAbs, varies according the assay method used; and the cut-off for positivity is 100 WHO units/mL.
In the third National Health and Nutrition Examination Survey the presence of TPOAbs was strongly associated with TSH values greater than 4.5mUI/l and clinical hypothyroidism as well as with TSH values lower than 0.4 mUI/l and clinical hyperthyroidism [15]. These relationships were not observed for TgAbs, suggesting that their diagnostic and prognostic value is lower than that of TPOAbs [Table 1]. TPOAbs can thus be considered the best serological marker we currently have to establish or corroborate a diagnosis of autoimmune thyroiditis. They also have another unique clinical application in the prediction of post-partum thyroiditis. It has been shown that pregnant women who have TPOAbs at the beginning of pregnancy have a significantly greater risk to develop hypothyroidism in the first year after delivery, as well as permanent thyroid dysfunction in the long-term follow-up [16].
TSH Receptor autoantibodies
The thyrotropin receptor (TSHR) is a G protein-coupled glycoprotein composed of 764 amino acids with a molecular weight of approximately 87 kDa. It is composed of two subunits linked by disulphide bonds: a large extracellular A subunit at the N-terminus (residues 1-418) and a B subunit that spans the plasma membrane seven times and ends with a short cytoplasmic tail (residues 419-764). The region between residues 277 and 418 is called the hinge region, which is critical for defining the relationship among the various TSHR domains. After expression on the plasma membrane, the TSHR undergoes intramolecular cleavage so that a fragment of approximately 50 amino acids called C peptide is removed from the hinge region, leaving the A and B subunits linked by the disulphide bonds. After cleavage, some of the A subunits are shed from the cell surface. The TSHR is the master regulator of thyroid function, being involved in thyrocyte differentiation, proliferation and function [17].
Antibodies to the TSHR are found in Graves disease and are key mediators of the pathogenesis. Different categories of TSHR antibodies (TRAbs) have been identified: those with a stimulatory effect on the thyroid gland responsible for hyperthyroidism, those with an inhibitory effect on the receptor responsible for hypothyroidism, and those with neutral activity. TRAbs can be measured by immunoassays, which determine the presence and titre of the antibody but not the activity, and by bioassays. Immunoassays (the most commonly used) use a monoclonal antibody bound to a solid phase that recognises the native human TSHR produced by recombinant DNA technology in mammalian cells. Then bovine TSH labelled with biotin and the patient serum are co-incubated to compete for binding to the immobilised TSHR: the lower the signal the higher the titre of TRAbs in the patient’s serum. Sometimes it is important to establish not only whether TRAb are present but also their biological activity, usually to rule out blocking antibodies. Bioassays use cultured mammalian cells stably transfected with TSHR and then measure the increase (stimulating antibodies) or the decrease (blocking antibodies) in cAMP production, following the addition of the patient’s serum [Figure 1].
TRAbs are used in four main clinical settings: 1) to predict relapse and clinical course of Graves condition. For example, Graves disease patients with high TRAbs six months after diagnosis and medical treatment are more susceptible to relapse [18]; in addition, by combining TPOAbs and TRAbs measurements, the predictive power increases especially in those patients with moderately elevated TRAbs (6-10 IU/l) [19]; 2) to diagnose an autoimmune pathogenesis in patients with isolated Graves ophthalmopathy; 3) to distinguish in the post-partum period a thyrotoxicosis due to destructive thyroiditis from the hyperthyroidism due to Graves disease; and 4) to forecast the development of neonatal Graves disease in infants born to mothers with Graves disease.
Predictive role of thyroid autoantibodies in the natural history of ATD
In recent years there has been a resurgent interest in autoantibodies, for long considered just a mere disease biomarker rather than an important pathogenic player. Longitudinal studies of patients with autoimmune diseases have shown that autoantibodies precede the clinical diagnosis by several years, establishing the field of predictive antibodies. For ATDs, we have recently carried out a study in female US soldiers and shown that TPOAbs and TgAbs precede a clinical diagnosis of ATD by at least seven years in a significant percentage of the subjects [Figure 2], suggesting that when detected in healthy subjects, autoantibodies should not be overlooked because they can predict the onset of future clinically evident dysfunctions [20].
References
1. Hashimoto H. Archiv für Klinische Chirurgie 1912: 219-248.
2. RJ G. Newly observed affection of the thyroid gland in females (clinical lectures). Lond Med Surg J 1835.
3. Tamai H et al. J Clin Endocrinol Metab 1989; 69(1): 49-53
4. Weetman AP. Horm Res 1997; 48 Suppl 4: 51-54
5. Simmonds MJ, Gough SG. Clin Exp Immunol 2004; 136(1): 1-10
6. Landek-Salgado MA et al. Autoimmun Rev 2010; 9(3): 153-157
7. Teng W et al. N Engl J Med 2006; 354(26): 2783-2793
8. Sundick RS, Bagchi N, Brown TR. Autoimmunity 1992; 13(1): 61-68
9. Vestergaard P et al. Thyroid 2002; 12(1): 69-75
10. Belin RM et al. J Clin Endocrinol Metab 2004; 89(12): 6077-6086
11. Spencer CA et al. J Clin Endocrinol Metab 1998; 83(4): 1121-1127
12. Latrofa F et al. J Clin Endocrinol Metab 2008; 93(2): 591-596
13. Prentice L et al. J Clin Endocrinol Metab 1995; 80(3): 977-986
14. McLachlan SM, Rapoport B. Thyroid 2004; 14(7): 510-520
15. Hollowell JG et al. J Clin Endocrinol Metab 2002; 87(2): 489-499
16. Premawardhana LD et al. Thyroid 2004; 14(8): 610-615
17. Ludgate ME, Vassart G. Baillieres Clin Endocrinol Metab 1995; 9(1): 95-113
18. Schott M et al. Horm Metab Res 2005; 37(12): 741-744
19. Schott M et al. Horm Metab Res 2007; 39(1): 56-61
20. Hutfless S et al. J Clin Endocrinol Metab 2011; 96(9): E1466-71
The authors Dr Alessandra De Remigis and Dr Patrizio Caturegli Johns Hopkins University Department of Pathology Baltimore, MD, USA e-mail: pcat@jhmi.edu
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Development of a uniform algorithmic approach to indeterminate thyroid FNAs is important in providing the most appropriate risk stratification and care to patients with thyroid nodules. Although further clinical studies will help refine guidelines for molecular testing of thyroid samples, the currently recommended panel, highlighted in this article, has shown good sensitivity and excellent specificity when used in the appropriate clinical context. Adjunctive molecular testing should have a growing role in the individualised and multidisciplinary care of patients with thyroid nodules, to better enable accurate diagnosis and appropriate therapy.
by Dr Ericka Olgaard and Dr Lewis A. Hassell
Thyroid carcinoma has been increasing in incidence over the past 30 years, in part due to increased detection by fine needle aspiration (FNA) biopsy of small thyroid nodules [1]. FNA biopsy with cytological examination is currently the most accurate tool for diagnosis or triage of thyroid nodules. Still, a significant portion of FNA biopsies are indeterminate and require further work-up [2]. The introduction of the Bethesda System for reporting thyroid cytopathology in 2010 provided a structure for the diagnosis ,reporting and to a degree the management of thyroid nodules [3].The categories of ‘atypia of undetermined significance’ (AUS) and ‘follicular lesion of undetermined significance’ (FLUS) are reserved for FNA smears that show insufficient architectural or cytological, atypia thus precluding assignment to a more definitive Bethesda diagnostic category such as benign, suspicious for malignancy (SFM), or malignant [4]. Currently, following the thyroid nodule of interest with imaging and a repeat FNA is the standard of practice in cases of AUS, FLUS and SFM, of which up to 40% will be malignant [1]. Most patients with indeterminate FNAs undergo surgery for histologic diagnosis of the thyroid lesion. However, the addition of molecular analysis of these lesions may reduce needless surgeries in these patients [5].
Molecular pathology of thyroid cancer
Most thyroid cancers are of follicular cell origin, consisting of papillary thyroid carcinoma (PTC), accounting for 80% of thyroid malignancies, follicular thyroid carcinoma (FTC), which accounts for 15%, poorly-differentiated carcinoma, and anaplastic carcinoma [6]. Advances in molecular pathology have identified several mutations associated with thyroid carcinomas. The majority of these are due to somatic mutations, acquired alterations in cells that are passed on by cell division. These are the focus of this review.
The most common somatic mutations are involved with the mitogen-activated protein kinase (MAPK) pathway, which controls cell proliferation, differentiation and survival [Figure 1], [2]. Included in the pathway are BRAF, RET, and RAS genes. BRAF mutations have been identified in 45% of PTCs, consisting of two main mutations, BRAFV600found in classic and tall cell variants of PTC [Figure 2] and BRAFK601Efound in the follicular variant of PTC (as well as some benign follicular adenomas). BRAF mutations are associated with more aggressive tumors (advanced stage, metastases, and extrathyroidal extension), higher recurrence rates, and often poor response to radioactive iodine therapy [7].
Mutations of the RET gene (RET/PTC) account for 20% – 30% of PTCs, are associated with classic or solid variants, and have been correlated with a history of ionizing radiation, as well as a younger age and smaller lesions (microcarcinomas) [8]. Point mutations in the RAS genes account for approximately 10% of PTCs, usually of follicular variant.
Most FTCs are also associated with somatic mutations involved in the MAPK pathway, most commonly in the RAS genes (40% – 50% of cases) and the gene fusion of PAX8/PPARγ1 (30% – 40% of cases) [2]. FTCs with RAS mutations often show a more indolent course while FTCs with PAX8/PPARγ1 fusion have demonstrated a higher propensity for vascular invasion. Poorly differentiated (insular) and anaplastic thyroid carcinomas may have molecular alterations as described above. Medullary carcinomas, both sporadic and familial, are derived from parafollicular C cells and have been associated with RET gene mutations.
Clinical utility of molecular testing
There are currently no widely used algorithms for molecular testing in cytological analysis of thyroid lesions. Because somatic mutations overlap in different types of thyroid lesions, molecular testing cannot replace cytological or histologic examination in the diagnosis of thyroid lesions. The question becomes ‘when should molecular testing supplement FNA triage and when should it be performed on resected tissue?’
There is 0% – 3% risk of malignancy in FNAs diagnosed as ‘benign’ [3]. Comparably, the risk of malignancy in FNAs categorised as ‘malignant’ is 97% – 99%. In these two groups, it seems additional molecular testing to confirm the diagnosis with the presence or absence of one of the somatic mutations described above would be of little added value.
In contrast, up to 40% of indeterminate FNAs are malignant. The additional cost of molecular testing should be weighed against the potential savings of avoided surgery and associated complications. Samples showing mutations of BRAF or RET/PTC are reasonably specific for PTC. Although RAS mutations may be found rarely in benign thyroid neoplasms, a positive test plus cytological atypia may be more suggestive of malignancy and aid in therapeutic decisions. A panel of BRAF (both V600E and K601E), RET/PTC, RAS (KRAS, NRAS and HRAS), and PAX8/PPARγ1 was recently recommended by the American Thyroid Association for potential use in indeterminate FNA cases and will detect most mutations in thyroid carcinoma [9]. However, upwards of 30% of thyroid malignancies may not demonstrate a detectable mutation, either due to absence of a known mutation, a rare mutation not included in the molecular testing panel, or insufficient sensitivity of current assays [2]. Additionally, clinical studies have demonstrated that 7% of AUS/FLUS lesions with a negative molecular analysis will actually prove malignant [1]. This number in clinical practice may be significantly higher, posing a difficult dilemma for surgeons and patients in choosing between surgery or clinical follow-up.
Molecular studies may also be helpful in therapeutic decisions, including monitoring for therapeutic success and/or recurrence of tumour and in potential directed therapy. This is where testing on resected tumours is a consideration. Treatment for papillary thyroid cancer has long involved surgical excision and radioactive iodine treatment to eradicate residual thyroidal tissue. However, BRAFV600E mutations have been associated with iodine-refractory PTC and these patients could benefit from more tailored therapy, including more extensive initial surgery, higher dose radioactive iodine treatment, and closer follow-up [5,10]. Alternative investigational therapies such as a MAPK kinase inhibitor targeted at BRAF, much like the those available for patients with metastatic melanoma show promise in phase II trials [10]. Hence, analysis of BRAF mutations in patients with PTC may become very valuable.
Qualitative methods of detection of point mutations (BRAF and RAS) are commonly available, mostly involving PCR-based methods with excellent sensitivity, achieved by using pyrosequencing, melting curve analysis, microarrays, fragment analysis, and conventional (Sanger) sequencing [2, 12]. Detection of chromosomal rearrangements of RET/PTC and PAX8/PPARγ1 requires analysis of RNA, which is less stable than DNA. Samples that are fresh or frozen can be used in reverse-transcriptase PCR (RT-PCR), but formalin fixed paraffin-embedded samples are inappropriate for use in RT-PCR and require fluorescence in-situ hybridisation (FISH), a more labour intensive and less sensitive method.
Advances in analysis of microRNA (miRNA), small segments of non-coding RNA that help regulate gene expression, have identified several unique expression profiles in thyroid cancer [11]. Patterns of overexpressed miRNA can distinguish between papillary, follicular, poorly-differentiated and anaplastic thyroid carcinomas. miRNA assays are currently becoming available and show tremendous potential for diagnostic and prognostic use in the future.
Conclusion
The cytopathologist’s role in the diagnosis of thyroid nodules is still vital, but shifting. Development of a uniform algorithmic approach to indeterminate thyroid FNAs is important in providing these patients with the most appropriate risk stratification and care [9]. Although further clinical studies will help refine guidelines for molecular testing of thyroid samples, the currently recommended panel of BRAF, RAS, RET/PTC and PAX8/PPARγ1 has shown good sensitivity and excellent specificity when used in the appropriate clinical context. Adjunctive molecular testing appears ready for a growing role in individualised and multidisciplinary care of patients with thyroid nodules, to better enable accurate diagnosis and most efficacious therapy.
References
1. Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol 2011;7:569-580.
2. Hassell LA, Gillies EM, Dunn ST. Cytologic and Molecular Diagnosis of Thyroid Cancers. Is it time for routine reflex testing? Cancer Cytopathol 2011. DOI: 10.1002/cncy.20186.
3. Cibas ES, Ali SZ. The Bethesda System for reporting thyroid cytopathology. Thyroid 2010;19:1159-1165.
4. Bongiovanni M, Krane JF, Cibas ES, Faquin WC. The atypical thyroid fine-needle aspiration: past, present, and future. Cancer Cytopathol 2011. DOI: 10.1002/cncy.20178.
5. Nikiforov YE, Ohori NP, Hodak SP et al. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab 2011;96:3390-97.
6. Kang G, Cho EY, Shin JH et al. Review of fine-needle aspiration for evaluating thyroid nodule. Cancer Cytopathol 2011. DOI: 10.1002/cncy.20179.
7. Jin L, Sebo JT, Nakamura N et al. BRAF mutation analysis in fine needle aspiration (FNA) cytology of the thyroid. Diagn Mol Pathol 2006;15(3):136-43.
8. Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med 2011;135:569-77.
9. Cooper DS et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009;19(11):1167-214.
10. Hayes DN, Lucas AS, Tanvetyanon T et al. Phase II efficiacy and pharmacogenomics study of selumetinib (AZD6244; ARRY-142886) in iodine131 refractory papillary thyroid carcinoma (IRPTC) with or without follicular elements. Clin Cancer Res 2012. DOI:10.1158/1078-0432.
11. Menon MP, Khan A. Micro-RNAs in thyroid neoplasms: molecular, diagnostic and therapeutic implications. J Clin Pathol 2009;62:978-85.
The authors Ericka Olgaard, D.O. and Lewis A. Hassell, M.D. Department of Pathology 940 Stanton L. Young Blvd., Rm. 451 Oklahoma City, OK 73104 USA Tel+ 1 405 271 4062 e-mail: Lewis-Hassell@ouhsc.edu
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Ed. by Malcolm D. Richardson and David W. Warnock Pub. by Wiley-Blackwell February 2012, 476 pp, €46.50
This book is a concise and up-to-date guide to the clinical manifestations, laboratory diagnosis and management of superficial, subcutaneous and systemic fungal infections. The highly acclaimed title has been extensively revised and updated throughout to ensure all drug and dosage recommendations are accurate and in agreement with current guidelines. A new chapter on infections caused by Pneumocystis jirovecii has been added. The book has been designed to enable rapid information retrieval and to help healthcare workers make informed decisions about diagnosis and patient management. Each chapter concludes with a list of recent key publications which have been carefully selected to facilitate efficient access to further information on specific aspects of fungal infections. Clinical microbiologists, infectious disease specialists, as well as dermatologists, haematologists and oncologists, can depend on this contemporary text for authoritative information and the background necessary to understand fungal infections.
http://eu.wiley.com/WileyCDA/
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Ed. by Kandice Kottke-Marchant and Bruce Davis, Pub. by Wiley-Blackwell April 2012, 776 pp, e235.00
Expertly edited and endorsed by the International Society for Laboratory Haematology, this is the newest international textbook on all aspects of laboratory haematology. Covering both traditional and cutting-edge haematology laboratory technology, this book emphasises international recommendations for testing practices. Illustrative case studies on how technology can be used in patient diagnosis are included. The book is an invaluable resource for all those working in the field.
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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
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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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Autoimmune thyroid diseases: diagnostic and predictive role of thyroid autoantibodies
, /in Featured Articles /by 3wmediaAutoimmune thyroid diseases, comprised by Hashimoto thyroiditis, Graves disease, and their variants, are characterised histologically by lymphocytic infiltration of the thyroid gland, biochemically by the presence of well-defined autoantibodies, and clinically by the impairment of thyroid function. The aetiology and mechanisms of the autoimmune damage depend upon genes and environmental factors such as iodine intake and smoking.Three major autoantigens have been identified for autoimmune thyroid disease: thyroglobulin, thyroperoxidase and thyrotropin receptor. Antibodies against these autoantigens are now part of the clinical toolbox. They are not only used to confirm a diagnostic suspicion, but also to predict recurrence of future onset of thyroid autoimmunity.
by Dr Alessandra De Remigis and Dr Patrizio Caturegli
Background on autoimmune thyroid diseases
Autoimmune thyroid diseases (ATDs) comprise two major conditions: Hashimoto thyroiditis with goitre and euthyroidism or hypothyroidism, and Graves disease with goitre, hyperthyroidism and often ophthalmopathy. Hashimoto thyroiditis is named after Dr Hakaru Hashimoto who described in 1912 the thyroid pathological features of four women who had undergone thyroidecomty because of compressive symptoms [1]. Graves disease is named after Dr Robert Graves who reported three patients with hyperthyroid goitre and ocular involvement (Graves ophthalmopathy) [2]. Both conditions are characterised pathologically by infiltration of the thyroid gland with autoreactive T and B lymphocytes and biochemically by the production of thyroid autoantibodies and abnormalities in thyroid function. It is not uncommon to observe transition from one clinical picture to another within the same patient over time, suggesting the existence of common immunological mechanisms [3]. Numerous clinical variants are described for each ATD. For Hashimoto thyroiditis, in addition to the classical goitrous form, fibrous, juvenile, thyrotoxic, post-partum, and IgG4-related forms are described. For Graves disease, beside the classical goitrous form with hyperthyroidism, there is the variant with hyperthyroidism and ophthalmopathy, the one with just ophthalmopathy (called euthyroid Graves disease), and the one with ophthalmopathy and localised myxoedema. Similar to many other autoimmune diseases, ATDs can occur in isolation or associated with other autoimmune diseases, often affecting other endocrine glands. In some patients this association is clinically recognisable and defined as polyglandular autoimmune syndrome.
ATDs are the most common autoimmune diseases with a population prevalence around 2 % in women and 0.2% in men. These estimates are considered to be 10 times higher if ‘subclinical disease’ is taken into consideration [4].
The pathogenesis of ATDs remains to be elucidated but it is believed to rely on the interaction between endogenous genetic factors and exogenous environmental factors. Genes that confer susceptibility to ATDs have been investigated since the 1970s via candidate gene analysis, linkage analysis and genome-wide association studies. Only a handful of genes have been identified and confirmed to increase the risk of ATDs development, but each gene contributes only a very small effect, with odds ratios typically below 3. These genes include the class II region of the major histocompatibility complex, CTLA-4, PTPN22, CD40, CD25, FCRL3, thyroglobulin, and the TSH receptor [5]. Another endogenous factor that has been studied extensively in ATD is pregnancy [6],which is known to ameliorate disease severity. The two major environmental factors implicated in ATDs initiation and progression are iodine and smoking. High iodine intake triggers lymphocytic infiltration of the thyroid in genetically susceptible animals (BB/W rats and NOD.H-2h24 mice); and is associated with increased prevalence and incidence of autoimmune thyroiditis and overt hypothyroidism in humans [7]. Iodine supplementation should thus be kept within the WHO recommended range to prevent from one side iodine deficiency and from the other side autoimmune thyroiditis [8]. Smoking does not have a univocal effect on AITD. It increases the risk of developing Graves disease and aggravates Graves ophtalmopathy. Smoking cessation is associated wth a better response of Graves ophthalmopathy to immunosuppressive treatment [9]. However, smoking seems to have a beneficial effect on Hashimoto thyroiditis and decreases the levels of thyroid autoantibodies [10].
Significant progress has been accomplished on the identification and characterisation of the thyroid autoantigens that are targeted by the immune system in patients with ATDs, so that antibodies against thyroglobulin, thyroperoxidase and the TSH receptor are now well-established tools in the clinical arena.
Thyroid antigens and antibodies
Thyroglobulin
Thyroglobulin is a large glycoprotein made of two identical 330-kDa subunits composed of 2,768 amino acids. Each subunit contains 66 tyrosines that when iodinated and processed make up the thyroid hormones (T4 and T3). Thyroglobulin contains numerous immunodominant epitopes for both T and B lymphocytes. Some epitopes are located in the iodine-rich hormonogenic regions and are affected by the iodine content.
Thyroglobulin antibodies (TgAb) recognise predominantly conformational epitopes, tend to favour the IgG2 subclass and do not fix complement. TgAbs were originally detected by tanned red cell haemagglutination, then by quatitative RIAs or ELISAs, and more recently by automated chemiluminescent EIAs. The analytical sensitivity varies depending on the assay method used, and the cut-off value for positivity is typically set at 100 WHO units/mL.
TgAbs are a marker of underlying thyroid autoimmunity but can also be found in non-autoimmune thyroid diseases as well as in healthy controls [11]. Traditionally they are requested together with thyroperoxidase antibodies to corroborate a diagnostic suspicion of ATDs. Currently, however, the greatest utility of TgAb measurement is in the follow-up of patients with differentiated thyroid cancer. These patients undergo thyroidectomy, possibly combined with radioactive iodine administration, and are then followed by measuring thyroglobulin antigen in the serum. Since thyroglobulin is a thyroid-specific antigen, its serum levels should be undetectable after thyroid ablation in the absence of recurrence or metastasis. If the patient had autoimmune thyroiditis in addition to thyroid cancer, TgAbs can persist for years after thyroidectomy and interfere with the thyroglobulin antigen determination. In particular, they can cause falsely low or undetectable levels of thyroglobulin antigen, and therefore a false positive clinical assessement. This realisation has led to the introduction of reflex measurement of TgAbs any time thyroglobulin is measured in thyroid cancer patients. Numerous studies have attempted to distinguish the type of TgAb based on the specific epitopes they recognise. Latrofa and colleagues have recently reported a pattern of TgAb for patients with ATDs and another for patients with multinodular goitre and differentiated thyroid cancer [12]. TgAbs also seem to recognise distinct epitopes in healthy subjects and patients with clinically manifest disease [13], suggesting the potential clinical utility of TgAbs based on specific thyroglobulin epitopes rather than on the entire thyroglobulin molecule.
Thyroperoxidase
Thyroperoxidase is a large membrane-associated glycoprotein (933 amino acids with a molecular weight of approximately 105 kDa) expressed at the apical (follicular) side of the thyroid cell. It separates to the microvillar/microsomal fraction upon ultracentrifugation and for this reason was originally called M antigen. Thyroperoxidase antibodies (TPOAbs) are predominantly IgG, can fix complement and cause damage to the thyroid cell by cell-mediated cytotoxicity [14]. TPOAbs are considered more specific for autoimmune thyroiditis than TgAbs. They correlate directly with the number of autoreactive lymphocytes infiltrating the thyroid gland as well as with the degree of thyroid hypoechogenicity on thyroid ultrasound. Like TgAbs, TPOAbs were originally measured by semiquantitative methods, then by RIAs or ELISAs, and more recently by automated chemiluminescent EIAs. The analytical sensitivity, as for TgAbs, varies according the assay method used; and the cut-off for positivity is 100 WHO units/mL.
In the third National Health and Nutrition Examination Survey the presence of TPOAbs was strongly associated with TSH values greater than 4.5mUI/l and clinical hypothyroidism as well as with TSH values lower than 0.4 mUI/l and clinical hyperthyroidism [15]. These relationships were not observed for TgAbs, suggesting that their diagnostic and prognostic value is lower than that of TPOAbs [Table 1]. TPOAbs can thus be considered the best serological marker we currently have to establish or corroborate a diagnosis of autoimmune thyroiditis. They also have another unique clinical application in the prediction of post-partum thyroiditis. It has been shown that pregnant women who have TPOAbs at the beginning of pregnancy have a significantly greater risk to develop hypothyroidism in the first year after delivery, as well as permanent thyroid dysfunction in the long-term follow-up [16].
TSH Receptor autoantibodies
The thyrotropin receptor (TSHR) is a G protein-coupled glycoprotein composed of 764 amino acids with a molecular weight of approximately 87 kDa. It is composed of two subunits linked by disulphide bonds: a large extracellular A subunit at the N-terminus (residues 1-418) and a B subunit that spans the plasma membrane seven times and ends with a short cytoplasmic tail (residues 419-764). The region between residues 277 and 418 is called the hinge region, which is critical for defining the relationship among the various TSHR domains. After expression on the plasma membrane, the TSHR undergoes intramolecular cleavage so that a fragment of approximately 50 amino acids called C peptide is removed from the hinge region, leaving the A and B subunits linked by the disulphide bonds. After cleavage, some of the A subunits are shed from the cell surface. The TSHR is the master regulator of thyroid function, being involved in thyrocyte differentiation, proliferation and function [17].
Antibodies to the TSHR are found in Graves disease and are key mediators of the pathogenesis. Different categories of TSHR antibodies (TRAbs) have been identified: those with a stimulatory effect on the thyroid gland responsible for hyperthyroidism, those with an inhibitory effect on the receptor responsible for hypothyroidism, and those with neutral activity. TRAbs can be measured by immunoassays, which determine the presence and titre of the antibody but not the activity, and by bioassays. Immunoassays (the most commonly used) use a monoclonal antibody bound to a solid phase that recognises the native human TSHR produced by recombinant DNA technology in mammalian cells. Then bovine TSH labelled with biotin and the patient serum are co-incubated to compete for binding to the immobilised TSHR: the lower the signal the higher the titre of TRAbs in the patient’s serum. Sometimes it is important to establish not only whether TRAb are present but also their biological activity, usually to rule out blocking antibodies. Bioassays use cultured mammalian cells stably transfected with TSHR and then measure the increase (stimulating antibodies) or the decrease (blocking antibodies) in cAMP production, following the addition of the patient’s serum [Figure 1].
TRAbs are used in four main clinical settings: 1) to predict relapse and clinical course of Graves condition. For example, Graves disease patients with high TRAbs six months after diagnosis and medical treatment are more susceptible to relapse [18]; in addition, by combining TPOAbs and TRAbs measurements, the predictive power increases especially in those patients with moderately elevated TRAbs (6-10 IU/l) [19]; 2) to diagnose an autoimmune pathogenesis in patients with isolated Graves ophthalmopathy; 3) to distinguish in the post-partum period a thyrotoxicosis due to destructive thyroiditis from the hyperthyroidism due to Graves disease; and 4) to forecast the development of neonatal Graves disease in infants born to mothers with Graves disease.
Predictive role of thyroid autoantibodies in the natural history of ATD
In recent years there has been a resurgent interest in autoantibodies, for long considered just a mere disease biomarker rather than an important pathogenic player. Longitudinal studies of patients with autoimmune diseases have shown that autoantibodies precede the clinical diagnosis by several years, establishing the field of predictive antibodies. For ATDs, we have recently carried out a study in female US soldiers and shown that TPOAbs and TgAbs precede a clinical diagnosis of ATD by at least seven years in a significant percentage of the subjects [Figure 2], suggesting that when detected in healthy subjects, autoantibodies should not be overlooked because they can predict the onset of future clinically evident dysfunctions [20].
References
1. Hashimoto H. Archiv für Klinische Chirurgie 1912: 219-248.
2. RJ G. Newly observed affection of the thyroid gland in females (clinical lectures). Lond Med Surg J 1835.
3. Tamai H et al. J Clin Endocrinol Metab 1989; 69(1): 49-53
4. Weetman AP. Horm Res 1997; 48 Suppl 4: 51-54
5. Simmonds MJ, Gough SG. Clin Exp Immunol 2004; 136(1): 1-10
6. Landek-Salgado MA et al. Autoimmun Rev 2010; 9(3): 153-157
7. Teng W et al. N Engl J Med 2006; 354(26): 2783-2793
8. Sundick RS, Bagchi N, Brown TR. Autoimmunity 1992; 13(1): 61-68
9. Vestergaard P et al. Thyroid 2002; 12(1): 69-75
10. Belin RM et al. J Clin Endocrinol Metab 2004; 89(12): 6077-6086
11. Spencer CA et al. J Clin Endocrinol Metab 1998; 83(4): 1121-1127
12. Latrofa F et al. J Clin Endocrinol Metab 2008; 93(2): 591-596
13. Prentice L et al. J Clin Endocrinol Metab 1995; 80(3): 977-986
14. McLachlan SM, Rapoport B. Thyroid 2004; 14(7): 510-520
15. Hollowell JG et al. J Clin Endocrinol Metab 2002; 87(2): 489-499
16. Premawardhana LD et al. Thyroid 2004; 14(8): 610-615
17. Ludgate ME, Vassart G. Baillieres Clin Endocrinol Metab 1995; 9(1): 95-113
18. Schott M et al. Horm Metab Res 2005; 37(12): 741-744
19. Schott M et al. Horm Metab Res 2007; 39(1): 56-61
20. Hutfless S et al. J Clin Endocrinol Metab 2011; 96(9): E1466-71
The authors
Dr Alessandra De Remigis and Dr Patrizio Caturegli
Johns Hopkins University
Department of Pathology
Baltimore, MD, USA
e-mail: pcat@jhmi.edu
The role of molecular testing in fine needle aspiration of the thyroid
, /in Featured Articles /by 3wmediaDevelopment of a uniform algorithmic approach to indeterminate thyroid FNAs is important in providing the most appropriate risk stratification and care to patients with thyroid nodules. Although further clinical studies will help refine guidelines for molecular testing of thyroid samples, the currently recommended panel, highlighted in this article, has shown good sensitivity and excellent specificity when used in the appropriate clinical context. Adjunctive molecular testing should have a growing role in the individualised and multidisciplinary care of patients with thyroid nodules, to better enable accurate diagnosis and appropriate therapy.
by Dr Ericka Olgaard and Dr Lewis A. Hassell
Thyroid carcinoma has been increasing in incidence over the past 30 years, in part due to increased detection by fine needle aspiration (FNA) biopsy of small thyroid nodules [1]. FNA biopsy with cytological examination is currently the most accurate tool for diagnosis or triage of thyroid nodules. Still, a significant portion of FNA biopsies are indeterminate and require further work-up [2]. The introduction of the Bethesda System for reporting thyroid cytopathology in 2010 provided a structure for the diagnosis ,reporting and to a degree the management of thyroid nodules [3].The categories of ‘atypia of undetermined significance’ (AUS) and ‘follicular lesion of undetermined significance’ (FLUS) are reserved for FNA smears that show insufficient architectural or cytological, atypia thus precluding assignment to a more definitive Bethesda diagnostic category such as benign, suspicious for malignancy (SFM), or malignant [4]. Currently, following the thyroid nodule of interest with imaging and a repeat FNA is the standard of practice in cases of AUS, FLUS and SFM, of which up to 40% will be malignant [1]. Most patients with indeterminate FNAs undergo surgery for histologic diagnosis of the thyroid lesion. However, the addition of molecular analysis of these lesions may reduce needless surgeries in these
patients [5].
Molecular pathology of thyroid cancer
Most thyroid cancers are of follicular cell origin, consisting of papillary thyroid carcinoma (PTC), accounting for 80% of thyroid malignancies, follicular thyroid carcinoma (FTC), which accounts for 15%, poorly-differentiated carcinoma, and anaplastic carcinoma [6]. Advances in molecular pathology have identified several mutations associated with thyroid carcinomas. The majority of these are due to somatic mutations, acquired alterations in cells that are passed on by cell division. These are the focus of this review.
The most common somatic mutations are involved with the mitogen-activated protein kinase (MAPK) pathway, which controls cell proliferation, differentiation and survival [Figure 1], [2]. Included in the pathway are BRAF, RET, and RAS genes. BRAF mutations have been identified in 45% of PTCs, consisting of two main mutations, BRAFV600found in classic and tall cell variants of PTC [Figure 2] and BRAFK601E found in the follicular variant of PTC (as well as some benign follicular adenomas). BRAF mutations are associated with more aggressive tumors (advanced stage, metastases, and extrathyroidal extension), higher recurrence rates, and often poor response to radioactive iodine therapy [7].
Mutations of the RET gene (RET/PTC) account for 20% – 30% of PTCs, are associated with classic or solid variants, and have been correlated with a history of ionizing radiation, as well as a younger age and smaller lesions (microcarcinomas) [8]. Point mutations in the RAS genes account for approximately 10% of PTCs, usually of follicular variant.
Most FTCs are also associated with somatic mutations involved in the MAPK pathway, most commonly in the RAS genes (40% – 50% of cases) and the gene fusion of PAX8/PPARγ1 (30% – 40% of cases) [2]. FTCs with RAS mutations often show a more indolent course while FTCs with PAX8/PPARγ1 fusion have demonstrated a higher propensity for vascular invasion. Poorly differentiated (insular) and anaplastic thyroid carcinomas may have molecular alterations as described above. Medullary carcinomas, both sporadic and familial, are derived from parafollicular C cells and have been associated with RET gene mutations.
Clinical utility of molecular testing
There are currently no widely used algorithms for molecular testing in cytological analysis of thyroid lesions. Because somatic mutations overlap in different types of thyroid lesions, molecular testing cannot replace cytological or histologic examination in the diagnosis of thyroid lesions. The question becomes ‘when should molecular testing supplement FNA triage and when should it be performed on resected tissue?’
There is 0% – 3% risk of malignancy in FNAs diagnosed as ‘benign’ [3]. Comparably, the risk of malignancy in FNAs categorised as ‘malignant’ is 97% – 99%. In these two groups, it seems additional molecular testing to confirm the diagnosis with the presence or absence of one of the somatic mutations described above would be of little added value.
In contrast, up to 40% of indeterminate FNAs are malignant. The additional cost of molecular testing should be weighed against the potential savings of avoided surgery and associated complications. Samples showing mutations of BRAF or RET/PTC are reasonably specific for PTC. Although RAS mutations may be found rarely in benign thyroid neoplasms, a positive test plus cytological atypia may be more suggestive of malignancy and aid in therapeutic decisions. A panel of BRAF (both V600E and K601E), RET/PTC, RAS (KRAS, NRAS and HRAS), and PAX8/PPARγ1 was recently recommended by the American Thyroid Association for potential use in indeterminate FNA cases and will detect most mutations in thyroid carcinoma [9]. However, upwards of 30% of thyroid malignancies may not demonstrate a detectable mutation, either due to absence of a known mutation, a rare mutation not included in the molecular testing panel, or insufficient sensitivity of current assays [2]. Additionally, clinical studies have demonstrated that 7% of AUS/FLUS lesions with a negative molecular analysis will actually prove malignant [1]. This number in clinical practice may be significantly higher, posing a difficult dilemma for surgeons and patients in choosing between surgery or clinical follow-up.
Molecular studies may also be helpful in therapeutic decisions, including monitoring for therapeutic success and/or recurrence of tumour and in potential directed therapy. This is where testing on resected tumours is a consideration. Treatment for papillary thyroid cancer has long involved surgical excision and radioactive iodine treatment to eradicate residual thyroidal tissue. However, BRAFV600E mutations have been associated with iodine-refractory PTC and these patients could benefit from more tailored therapy, including more extensive initial surgery, higher dose radioactive iodine treatment, and closer follow-up [5,10]. Alternative investigational therapies such as a MAPK kinase inhibitor targeted at BRAF, much like the those available for patients with metastatic melanoma show promise in phase II trials [10]. Hence, analysis of BRAF mutations in patients with PTC may become very valuable.
Qualitative methods of detection of point mutations (BRAF and RAS) are commonly available, mostly involving PCR-based methods with excellent sensitivity, achieved by using pyrosequencing, melting curve analysis, microarrays, fragment analysis, and conventional (Sanger) sequencing [2, 12]. Detection of chromosomal rearrangements of RET/PTC and PAX8/PPARγ1 requires analysis of RNA, which is less stable than DNA. Samples that are fresh or frozen can be used in reverse-transcriptase PCR (RT-PCR), but formalin fixed paraffin-embedded samples are inappropriate for use in RT-PCR and require fluorescence in-situ hybridisation (FISH), a more labour intensive and less sensitive method.
Advances in analysis of microRNA (miRNA), small segments of non-coding RNA that help regulate gene expression, have identified several unique expression profiles in thyroid cancer [11]. Patterns of overexpressed miRNA can distinguish between papillary, follicular, poorly-differentiated and anaplastic thyroid carcinomas. miRNA assays are currently becoming available and show tremendous potential for diagnostic and prognostic use in the future.
Conclusion
The cytopathologist’s role in the diagnosis of thyroid nodules is still vital, but shifting. Development of a uniform algorithmic approach to indeterminate thyroid FNAs is important in providing these patients with the most appropriate risk stratification and care [9]. Although further clinical studies will help refine guidelines for molecular testing of thyroid samples, the currently recommended panel of BRAF, RAS, RET/PTC and PAX8/PPARγ1 has shown good sensitivity and excellent specificity when used in the appropriate clinical context. Adjunctive molecular testing appears ready for a growing role in individualised and multidisciplinary care of patients with thyroid nodules, to better enable accurate diagnosis and most efficacious therapy.
References
1. Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol 2011;7:569-580.
2. Hassell LA, Gillies EM, Dunn ST. Cytologic and Molecular Diagnosis of Thyroid Cancers. Is it time for routine reflex testing? Cancer Cytopathol 2011. DOI: 10.1002/cncy.20186.
3. Cibas ES, Ali SZ. The Bethesda System for reporting thyroid cytopathology. Thyroid 2010;19:1159-1165.
4. Bongiovanni M, Krane JF, Cibas ES, Faquin WC. The atypical thyroid fine-needle aspiration: past, present, and future. Cancer Cytopathol 2011. DOI: 10.1002/cncy.20178.
5. Nikiforov YE, Ohori NP, Hodak SP et al. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab 2011;96:3390-97.
6. Kang G, Cho EY, Shin JH et al. Review of fine-needle aspiration for evaluating thyroid nodule. Cancer Cytopathol 2011. DOI: 10.1002/cncy.20179.
7. Jin L, Sebo JT, Nakamura N et al. BRAF mutation analysis in fine needle aspiration (FNA) cytology of the thyroid. Diagn Mol Pathol 2006;15(3):136-43.
8. Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med 2011;135:569-77.
9. Cooper DS et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009;19(11):1167-214.
10. Hayes DN, Lucas AS, Tanvetyanon T et al. Phase II efficiacy and pharmacogenomics study of selumetinib (AZD6244; ARRY-142886) in iodine131 refractory papillary thyroid carcinoma (IRPTC) with or without follicular elements. Clin Cancer Res 2012. DOI:10.1158/1078-0432.
11. Menon MP, Khan A. Micro-RNAs in thyroid neoplasms: molecular, diagnostic and therapeutic implications. J Clin Pathol 2009;62:978-85.
The authors
Ericka Olgaard, D.O. and
Lewis A. Hassell, M.D.
Department of Pathology
940 Stanton L. Young Blvd., Rm. 451
Oklahoma City, OK 73104
USA
Tel+ 1 405 271 4062
e-mail: Lewis-Hassell@ouhsc.edu
Fungal Infection: Diagnosis and Management, 4th Edition
, /in Featured Articles /by 3wmediaEd. by Malcolm D. Richardson and David W. Warnock
Pub. by Wiley-Blackwell February 2012, 476 pp, €46.50
This book is a concise and up-to-date guide to the clinical manifestations, laboratory diagnosis and management of superficial, subcutaneous and systemic fungal infections. The highly acclaimed title has been extensively revised and updated throughout to ensure all drug and dosage recommendations are accurate and in agreement with current guidelines. A new chapter on infections caused by Pneumocystis jirovecii has been added. The book has been designed to enable rapid information retrieval and to help healthcare workers make informed decisions about diagnosis and patient management. Each chapter concludes with a list of recent key publications which have been carefully selected to facilitate efficient access to further information on specific aspects of fungal infections. Clinical microbiologists, infectious disease specialists, as well as dermatologists, haematologists and oncologists, can depend on this contemporary text for authoritative information and the background necessary to understand fungal infections.
http://eu.wiley.com/WileyCDA/Laboratory Hematology Practice
, /in Featured Articles /by 3wmediaEd. by Kandice Kottke-Marchant and Bruce Davis, Pub. by Wiley-Blackwell April 2012, 776 pp, e235.00
Expertly edited and endorsed by the International Society for Laboratory Haematology, this is the newest international textbook on all aspects of laboratory haematology. Covering both traditional and cutting-edge haematology laboratory technology, this book emphasises international recommendations for testing practices. Illustrative case studies on how technology can be used in patient diagnosis are included. The book is an invaluable resource for all those working in the field.
http://eu.wiley.com/WileyCDA/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
No need too small. No demand too big.
, /in Featured Articles /by 3wmedia