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Hereditary spherocytosis is an inherited haemolytic anaemia due to fragile red cells. This article gives a brief overview of the pathophysiology of this red cell disorder, and presents the key points on the different screening tests.
by Dr May-Jean King
Membrane structure of human red blood cell and associated defects
The human red blood cell (RBC) is discoid or biconcave in shape. It deforms when navigating through blood vessels and capillaries. The integrity and elasticity of RBC are maintained and regulated by a series of interactions between two layers of proteins localised to the outer lipid bilayer and the cytoskeleton on the cytoplasmic side [Figure 1]. The resulting RBC membrane is a 3D structure composed of specific transmembrane proteins (the band 3 macro-complex, and the glycophoein C-protein 4.1R) and a 2D network of skeleton proteins spectrin, actin, protein 4.1R and other minor components [1]. A qualitative or quantitative abnormality in one of these membrane proteins will lead to fragile red cells and haemolytic anaemia. Hereditary spherocytosis is associated with defects in the vertical interaction of the band 3 macrocomplex (i.e., band 3, CD47, and Rh complex) with protein 4.2, and ankyrin to which β-spectrin binds directly [Figure 1]. Hereditary elliptocytosis has abnormalities in protein 4.1R or defective spectrin self-association [2]. A partial deficiency of protein 4.1R can affect its interaction with glycophorin C and P55 in a junctional complex, which is stabilised by a band 3-adducin-spectrin bridge [3]. The mutations located in the self-association site for spectrin αβ heterodimers can affect the formation of tetramers or higher oligomers that enable the extension of the spectrin-based cytoskeleton to cover the cytoplasmic side of the red cell membrane. HS and hereditary elliptocytosis are not single-gene diseases.
Hereditary spherocytosis
Hereditary spherocytosis (HS) is more prevalent among the Northern European populations (about 1 in 2000 to 5000 births) than in other ethnic groups. Where the cytoskeleton fails to attach to band 3 in the membrane via protein 4.2 and ankyrin, that area of membrane becomes detached and is pinched off from the intact RBC. This continuous loss of membrane lipid and integral membrane proteins reduces the RBC volume and transforms it into a spherocyte. Splenic sequestration of spherocytes reduces their lifespan in circulation to <120 days. Therefore a patient with HS presents a haemolytic anaemia with reticulocytosis, jaundice and possibly gallstones and/or splenomegaly [4]. The clinical phenotype of HS is heterogeneous, ranging from asymptomatic, mild, moderate to severe haemolysis requiring blood transfusion. HS is diagnosed in newborn or as late as in the fifth to seventh decade of life. A mild HS condition can be exacerbated by an infection (e.g., Parvovirus B19, CMV, Herpes 6, gastroenteritis), resulting in a severe haemolytic anaemia.
Laboratory testing for HS
Membranopathy is suspected when the cause of haemolytic anaemia remains unknown after the exclusion of enzymopathy, haemoglobinopathy and other extrinsic factors. Finding spherocytes in a blood smear indicates HS, but is not necessarily definitive. Exclusion of immune haemolytic anaemia (AIHA) is important because this condition also presents with spherocytosis [5]. Typical HS is expected to present almost all of the following features: evidence of a haemolytic process (e.g., raised bilirubin and LDH, low or no haptoglobin), low Hb, reduced mean cell volume, elevated mean cell haemoglobin concentration, and raised reticulocyte count [Figure 2]. The raised MCHC and increased % hyperdense RBCs are useful markers [6]. The diagnosis of dominant HS (75% of cases) is straightforward when family history of HS and the results for the red cell indices and blood chemistry are available. In the case of recessive HS, the proband may present a severe haemolytic anaemia with the blood smear showing anisocytosis and occasional cell fragments whereas the parents are apparently asymptomatic.
The majority of subjects with HS can be diagnosed by using a screening test without resorting to further investigation [Figure 2]. Two traditional screening tests for HS are still in use: the osmotic fragility test [7] and the acid glycerol lysis time test [8] [Table 1]. The cryohaemolysis test uses a change in temperature to effect red cell lysis [9]. The ektacytometer gives specific deformability profiles for a range of red cell disorders [Table 1]. However, this technique can give similar profiles for both HS and AIHA. SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins is the confirmatory test because it detects all the membrane proteins known to be associated with HS [Figure 3, panel I]. Molecular analysis of membrane protein genes is usually performed by research laboratories. However, knowing the membrane protein defects and the associated protein gene mutation(s) does not influence the management of HS patients [12]. Unlike the aforementioned HS screening tests, the unusual feature of the EMA (eosin-5’-maleimide) Binding test [13] is the use of a flow cytometer, which analyses individual intact RBC in a sample. Confocal microscopy of EMA-labelled RBCs showed emission of both green and red fluorescence. RBCs of different sizes and shapes are labelled [Figure 3, panels II and III], [14]. The test is robust, only a low volume of patient specimen (5 µL packed RBC) and test reagent is required, and the test gives consistently reproducible results.
Conclusion
There is no screening test that has 100% sensitivity and 100% specificity for the diagnosis of HS. The adoption of the EMA Binding test is because it is easy to use and an abnormal result often indicates a membrane-associated red cell disorder. When this flow method is used in conjunction with the Osmotic Fragility test, differential diagnosis of HS and hereditary stomatocytosis can be made [described in 12].
References
1. Mohandas N & Gallagher PG. Red cell membrane: past, present, and future. Blood 2008; 112: 3939-3948.
2. Gallagher PG. Update on the clinical spectrum and genetics of red blood cell membrane disorders. Current Hematol Reports 2004; 3: 85-91.
3. Anong W et al. Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton, and regulates membrane cohesion. Blood 2009; 114: 1904-1912.
4. Perrotta et al. Hereditary spherocytosis. Lancet 2008; 372:1411-1426.
5. Packman CH. The spherocytic haemolytic anaemias (historical review). Br J Haematol 2001;112: 888-899.
6. Cynober T et al. Red cell abnormalities in hereditary spherocytosis: relevance to diagnosis and understanding of the variable expression of clinical severity. J Lab Clin Med 1996;128:259-269.
7. Parpart AK et al. The osmotic resistance (fragility) of human red cells. J Clin Invest 1947; 26: 636-640.
8. Zanella A et al. Acidified glyceraol lysis test: a screening test for spherocytosis. Br J Haematol 1980; 45:481-486.
9. Streichman S & Gescheidt Y. Cryohemolysis for the detection of hereditary spherocytosis: correlation studies with osmotic fragility and authemolysis. A J Hematol 1998; 58:206-212.
10. Clark MR et al. Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance. Blood 1983; 61: 899-910.
11. Johnson RM & Ravindranath Y. Osmotic scan ektacytometry in clinical diagnosis. J Ped Hematol Oncol 1996; 18: 122-129.
12. Bolton-Maggs et al. Guidelines for the diagnosis and management of hereditary spherocytosis – 2011 update. Br J Haematol 2011; doi:10.1111/j.1365-2141.2011.08921.x
13. King M-J et al. Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia. Br J of Haematol 2000; 111: 924-933.
14. King M-J et al. Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis. Cytometry Part B 2008; 74B: 244-250.
15. wKing M-J et al. Eosin-5-maleimide binding to band 3 and Rh-related proteins forms the basis of a screening test for hereditary spherocytosis. Br J Haematol 2004; 124:106-113.
The author
May-Jean King
Membrane Biochemistry
NHS Blood and Transplant
North Bristol Park
Filton
Bristol BS34 7QH
UK
e-mail: may-jean.king@nhsbt.nhs.uk
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
It is thirty years since the first diagnoses of AIDS were reported, since when, according to the most recent UNAids report, 25 million people have died from HIV-related causes and around 34 million people are currently living with the virus. However, as we enter the fourth decade of this devastating pandemic, there is certainly some light at the end of the tunnel, reflected in the theme that was adopted for this year’s World AIDS Day and until 2015: ‘Getting to Zero’. Although more aspirational than achievable, very real progress has been made.
The good news is that the number of AIDS-related deaths last year was the lowest (1.8 million deaths) since the peak of 2.2 million deaths in 2005, predominantly because of increasing access to antiretroviral treatment (ART) in low- and middle-income countries where the disease burden is heaviest, and where nearly seven million people are now receiving appropriate therapy. The UN-backed Global Fund against AIDS, TB and Malaria has played a significant role in this achievement. The incidence of HIV has also fallen in 33 countries, two thirds of which are in sub-Saharan Africa. Not only has ART reduced transmission, including vertical transmission, of the virus, but education and condom provision, more widespread HIV testing even in low-resource settings and counselling if necessary have all had a large impact. While efforts to introduce an effective HIV vaccine continue to be disappointing, results from trials on the pre-exposure use of antiretrovirals for prophylaxis are encouraging, and more easily tolerated drugs, such as rilpivirine, will improve life for some patient groups.
So what is the bad news? Firstly in some Western countries, where infected people can be diagnosed and treated early and have a near-normal lifespan, the incidence of HIV is actually increasing. And more important globally, there are still around ten million people waiting for treatment, the number of people with new infections remains higher than the number of people starting ART, and the staunch efforts of the Global Fund may now be affected by the financial fraud which was exposed in four recipient countries earlier this year as well as by the global economic crisis. Last month it was revealed that whilst international donors (the principal donors are the US, Germany, France and Japan) have been asked for donations totalling around fifteen billion Euros, the Global Fund has received only eight and a half billion Euros, which is lower than the amount needed to maintain its current programmes for the next three years. It is indeed a tragedy if millions of people continue to suffer from HIV because of the greed and mismanagement of a powerful few.
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
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
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