1
NEUROPATHOLOGICAL ASSESSMENTS OF THE PATHOLOGY IN
FRONTOTEMPORAL LOBAR DEGENERATION WITH TDP43 POSITIVE
INCLUSIONS
An Inter-laboratory study by the BrainNet Europe consortium
Irina Alafuzoff, Maria Pikkarainen, Manuela Neumann, Thomas Arzberger, Safa Al-Sarraj, Istvan
Bodi, Nenad Bogdanovic, Orso Bugiani, Isidro Ferrer, Ellen Gelpi, Stephen Gentleman, Giorgio
Giaccone, Manuel B Graeber, Tibor Hortobagyi, Paul G Ince, James W Ironside, Nikolaos
Kavantzas, Andrew King, Penelope Korkolopoulou, Gábor G. Kovács, David Meyronet, Camelia
Monoranu, Tatjana Nilsson, Piero Parchi, Efstratios Patsouris, Tamas Revesz, Wolfgang
Roggendorf, Annemieke Rozemuller, Danielle Seilhean, Nathalie Streichenberger, Dietmar R. Thal,
Steven Wharton and Hans Kretzschmar
Affiliations:
I. Alafuzoff, Section of Clinical Pathology Uppsala University Hospital, Department of
Immunology, Genetics and Pathology, Uppsala University, Uppsala Sweden;
T. Arzberger, H. Kretzschmar, Centre for Neuropathology and Prion Research, München LudwigMaximilians-University, München, Germany;
S. Al-Sarraj, I. Bodi, A. King, Department of Clinical Neuropathology, King’s College Hospital and
MRC London Neurodegenerative Diseases Brain bank, Institute of Psychiatry, London, UK;
N. Bogdanovic, Geriatric Department, Institute for Clinical Medicine, Oslo University, Oslo,
Norway
O. Bugiani, G. Giaccone, IRCSS Foundation Istituto Neurologico Carlo Besta, Division of
Neuropathology and Neurology 5, Milano, Italy;
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I. Ferrer, Institute of Neuropathology, Bellvitge University Hospital, University of Barcelona,
CEBERNED, Barcelona, Spain:
E Gelpi; Neurological Tissue Bank of the Biobanc-Hospital Clínic-IDIBAPS, Barcelona, Spain
S. Gentleman, Neuropathology Unit, Department of Medicine, Imperial College London, London,
United Kingdom;
M.B. Graeber, Brain and Mind Research Institute, Faculty of Medicine and Faculty of Health
Science, The University of Sydney, Sydney Australia;
T. Hortobagyi, Department of Neuropathology, Instutute of Pathology; University of Debrecen,
Debrecen, Hungary;
N. Kavantzas, P. Korkolopoulou, E. Patsouris, Department of Pathology, National and Capodistrian
University of Athens, Athens, Greece;
G.G. Kovács, Institute of Neurology, Medical University of Vienna, Vienna, Austria; PG Ince, SB
Wharton, Sheffield Institute for Translational Neuroscience, University of Sheffield, United
Kingdom;
J. W. Ironside, National CJD Research & Surveillance Unit, University of Edinburgh, Western
General Hospital, Edinburgh, United Kingdom;
D. Meyronet, N. Streichenberger, Hospices Civils de Lyon, centre de Pathologie et de
Neuropathologie Est, Lyon Neuroscience Research Center, Universite Lyon, Lyon, France;
C. Monoranu, W Roggendorf, Abteilung Neuropathologie, Pathologisches Institut der Univeristät
Wurzburg, Wurzburg, Germany;
M. Neumann, Department of Neuropatology, University of Tubingen and DZNE, German Center of
Neurodegenerative Diseases, Tubingen, Germany;
T. Nilsson, Department of Geriatrics, Karolinska Institutet, Huddinge, Sweden;
P. Parchi, Department of Biomedical and Neuromotor Sciences, University of Bologna, IRCCS,
Istituto delle Scienze Neurologiche, Bologna, Italy;
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M. Pikkarainen, Department of Clinical Medicine, University of Eastern Finland, Kuopio, Finland;
T. Revesz, Queen Square Brain Bank, Department of Molecular Neuroscience, UCL Institute of
Neurology, London, UK
A. Rozemuller, VU University Medical Center, Amsterdam, The Netherlands;
D. Seilhean, Laboratoire de Neuropathologie Raymond Escourolle, Université Pierre et Marie Curie
and INSERM, Assistance Publique-hopitaux de Paris, Paris, France;
D.R. Thal, Institute of Pathology - Laboratory of Neuropathology, University of Ulm, Ulm,
Germany.
BrainNet Europe http://www.brainnet-europe.org/.
Source of support: EU Grant FP6, BNEII No LSHM-CT-2004-503039
Correspondence to:
Prof Irina Alafuzoff,
Uppsala University, Department of Immunology, Genetics and Pathology,
Rudbeck’s Laboratory,
751 85 Uppsala, Sweden,
Tel: +46 18 6110235,
e-mail: [email protected]
Justifications: I.Alafuzoff, M.Pikkarainen, M.Neumann, T.Arzberger and H.Kretzschmar / Design
of the study, providing instructions regarding the assessments, primary assessment of slides,
summary of all data and preparation of the manuscript. Each of the participating BNE centres
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carrying out stainings and assessments is represented by 1 to 3 authors. All authors contributed to
manuscript revision.
Keywords: ubiquitin, p62, TDP43, phosphorylated TDP43, FTLD-TDP, BrainNet Europe,
immunohistochemistry, inter-laboratory study, tissue microarray
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ABSTRACT
The BrainNet Europe (BNE) consortium assessed the reproducibility in the assignment of the type
of Frontotemporal lobar degeneration (FTLD) with TAR DNA binding protein (TDP) 43 following
current recommendations. The agreement rates were influenced by the immunohistochemical (IHC)
method and by the classification strategy followed. p62-IHC staining yielded good uniform quality
of stains, but the most reliable results were obtained implementing specific Abs directed against the
hallmark protein TDP43. Both assessment of the type and the extent of lesions were influenced by
the Abs and by the quality of stain. Assessment of the extent of the lesions yielded poor results
repeatedly; thus, the extent of pathology should not be used in diagnostic consensus criteria. While
31 neuropathologists typed 30 FTLD-TDP cases, inter-rater agreement ranged from 19 to 100 per
cent, being highest when applying phosphorylated TDP43/IHC. The agreement was highest when
designating Type C or Type A/B. In contrast, there was a poor agreement when attempting to
separate Type A or Type B FTLD-TDP. In conclusion, we can expect that neuropathologist,
independent of his/her familiarity with FTLD-TDP pathology, can identify a TDP43 positive FTLD
case. The goal should be to state a type (A, B, C, D) or a mixture of types (A/B, A/C or B/C).
Neuropathologists, other clinicians and researchers should be aware of the pitfalls while doing so.
Agreement can be reached in an interlaboratory setting regarding type C cases with thick and long
neurites, whereas the differentiation between types A and B may be more troublesome. Inclusion of
assessment of sequential neuroanatomical distribution of TDP43 pathology as recently
recommended will probably improve the agreement rates and should thus be facilitated.
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INTRODUCTION
The neuropathological entity of frontotemporal lobar degeneration with transactivation responsive
DNA-binding protein (TDP) 43 positive inclusions (FTLD-TDP) was recognised in 2006
(Neumann et al. 2006). The lesions seen in FTLD-TDP were originally visualised with antibodies
(Abs) directed against ubiquitin (Lowe et al. 1988; Jackson et al. 1996) or ubiquitin-binding protein
p62/sequestosome (Arai et al. 2003; Pirici et al. 2006; Kuusisto et al. 2008; Pikkarainen et al. 2008).
Since 2006, Abs directed against TDP43 (Neumann et al. 2006; Arai et al. 2006; Davidson et al.
2007) and phosphorylated TDP43 (pTDP43) (Hasegawa et al. 2008; Neumann et al. 2009) have
been used. Four histopathological subtypes of FTLD-TDP have been described, based on the
predominant type, distribution and density of lesions (Mackenzie et al. 2006; Sampathu et al. 2006;
Cairns et al. 2007). In 2011, a harmonised classification system for FTLD-TDP pathology was
proposed that was based on the previously published classification systems (Mackenzie et al. 2011)
To date, immunohistochemical (IHC) methods applying various Abs, pretreatment strategies and
detection systems have been utilised to visualise the hallmark lesions seen in FTLD-TDP. The type
of lesions, their distribution and density form the basis for typing of the disorder (Mackenzie et al.
2006, 2011; Sampathu et al. 2006; Cairns et al. 2007). IHC methodology is prone to considerable
variability that might cause differing results, as was elegantly reported by Mackenzie (Mackenzie et
al., 2006). Today, it is acknowledged that use of the IHC methods require knowledge and
competence both regarding methodology but also regarding interpretation of the result obtained
(McNicol et al. 1998; Shi et al. 2001; Gelpi et al. 2007; D’Amico et al. 2009; Pikkarainen et al.
2010; Karlsson et al. 2011; Kovacs et al. 2012). In addition, the agreement rate for the designation
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of a specific type of a disease by numerous investigators might be less optimal, as previously
reported by BrainNet Europe (BNE) (Alafuzoff et al. 2008b, 2009a, 2009b).
The objectives of this BNE inter-laboratory study were to evaluate the quality of stains applied,
reproducibility in the recognition of lesions and to assess the level of agreement in assigning
histological types of FTLD-TDP.
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MATERIALS AND METHODS
This study involved 18 BNE centres and a number of experienced neuropathologists. The study
included two separate parts with four separate trials. In the first part of this study (Figure 1 left
column), methodological aspects were assessed including quality of staining and assignment of
lesions, i.e. Trials 1–3. In the second part (Figure 1 right column), agreement in stating the
histopathological type of FTLD-TDP was assessed while applying current recommended
classification strategies, i.e., Mackenzie et al. 2006, Sampathu et al. 2006 or Cairns et al. 2007
(supplement Figure 1). Each part included individual assessments carried out by participating
neuropathologists followed by joint assessment around a multi-headed microscope. Both parts of
the study followed the design previously described by BNE (Alafuzoff et al. 2006, 2008a, b, c,
2009a, b).
Part I. Assessment of methodology
The tissue microarray (TMA) blocks were constructed as previously described from the blocks that
were delivered to the co-ordinating centre (supplement Table 1) (Alafuzoff et al. 2006, 2008a,
2008c; Kauppinen et al. 2006). Prior to the distribution of the TMA sections to the participants, the
uniformity / comparability of the sections, i.e. cores, was assessed by the co-ordinating centre as
previously described (Alafuzoff et al. 2006, 2008a, 2008c). Briefly, the presence of p62
immunoreactive (IR) cytoplasmic inclusions and neurites was assessed in a set of TMA sections
constructed for Trials 1 and 2. To facilitate the comparison of results, all cores with concomitant
pathologies, i.e. hyperphosphorylated tau and/or α-synuclein, were excluded. Furthermore, 15
representative core samples with variable TDP43-IR pathology were selected while summarising the
final assessment results in Trials 1–3 (Supplement Table 1).
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BNE participant efforts
BNE participant efforts in Trials 1 and 2 as well as in Trial 3, i.e. inter-rater assessments, are
summarised in Figure 1, left column. In brief, each BNE centre received one unstained section for
each assessor, the protocol with recommended staining practices for Trial 2, assessment instructions
including figures, and data sheets for recording of the assessments for Trials 1–3, wherein the
assessment instructions and data sheets were the same for all three trials (Figure 2).
In the first trial, the participants were asked to use a staining of their own choice, i.e. Abs directed
against ubiquitin, p62 or TDP43 without strict methodological recommendations. Thereafter,
obtained results were discussed at a joint meeting and subsequently, the Abs to be used in Trial 2
were agreed upon. Trial 3 was carried out after two joint assessments around a multi-headed
microscope and several months after Trial 2. Eight to nine evaluators assessed the same TMA
section of good/acceptable quality stained with ubiquitin, p62, monoclonal TDP43 (mono),
polyclonal TDP43 (poly) or pTDP43-IHC. Briefly, here are the summary protocols with optimal
results applied in Trial 3: ubiquitin MAB1510 Ab (clone Ubi-1, Chemicon, Temecula, CA, dilution
1:50 000, autoclave/120°C 10 min in aqua), p62 lck ligand (clone 3, BD Biosciences, Pharmingen,
San Diego, CA 1:1,000, autoclave/120°C 10 min in citrate buffer pH 6,0), monoTDP43 (clone 2E2D3, Abnova, Taipei, Taiwan, 1:300, autoclave/120°C 10 min in citrate buffer pH 6,0), polyTDP43
(10782-1-AP, Proteintech Group, Chicago, IL, 1:1,000, autoclave/120°C 10 min in aqua) and
pTDP43 (rat mono Ab, clone 1D3 (Neumann et al 2009), 1:50, autoclave/5x3 min in 10 mmol/L
citrate buffer).
Figure 2, panel 1 delineates the ubiquitin-, p62- or TDP43-IR structures that were to be assessed in a
dichotomised manner (i.e. determination of the absence or presence) and semiquantitatively (Figure
2, panel 2) in Trials 1–3.
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Data analysis
The quality of staining in Trials 1 and 2 was evaluated from the whole sections stained by the
participating BNE laboratories on a scale incorporating both staining intensity and background
staining. The stainings were assessed as being of good quality when the staining clearly labelled the
lesions evenly in most cores in a TMA section, as acceptable when lesions were detectable, but the
intensity of staining between the cores varied with regard to the lesions and/or background, and as
poor when the lesions were not detectable in some of the cores.
For dichotomised assessments, the percentage of positive (yes) assessment results for each Ab was
calculated (Trials 1–3). For semiquantitative assessments, the range and the most frequent score
were given (Trials 1–3). In addition, in Trial 3, participants were asked to state the density (Figure
2, panel 2) and predominant lesion in a given core (cytoplasmic inclusions or neurites).
Part II. Designation of a subtype of FTLD-TDP
Thirty cases from four centres were included. Regions selected were frontal cortex and
hippocampus based on the current recommended assessment strategies (Mackenzie et al. 2006;
Sampathu et al. 2006; Cairns et al. 2007) (supplement Figure 1). All included cases displayed
TDP43-IR lesions. A total of eight sets of 7-m-thick sections were produced. There were 15
females (mean age at death 69 years) and 15 males (mean age at death 64 years); the mean age at
death of the whole cohort was 67 ± SE 2 years (standard error of means).
Immunohistochemistry
Abs used included: ubiquitin (one set), p62 (three sets), mono TDP43 (two sets) and pTDP43
(two sets). All sets were manually stained applying IHC method optimised during Trial 3.
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Reference assessment and construction of assessment instructions
The members of the reference group (IA, MN, TA, HK, MP) assessed all 30 cases in ubiquitin,
p62, poly TDP43 and pTDP43 stains first individually and then jointly using a multi-headed
microscope. The reference group assigned a FTLD-TDP subtype to each case (Mackenzie et al.
2006, 2011; Sampathu et al. 2006; Cairns et al. 2007) when possible.
Assessment instructions were written by two members of the reference group (IA, MP). The
instructions included a detailed description of the samples, general instructions regarding
assessment and detailed tabulated guidelines for the subtyping (supplement Figure 1). In
addition, participants were urged to read the original publications (Mackenzie et al. 2006;
Sampathu et al. 2006; Cairns et al. 2007).
Inter-observer assessment
Twenty-six participants from 18 centres assessed and classified each case as instructed. Five
participants assessed the cases that were stained applying ubiquitin, seven applying p62, eight
applying poly TDP43 and six participants assessed the cases stained applying pTDP43. The
results were recorded on the assessment sheets (supplement Figure 2), which were sent to the coordinating centre. These assessment sheets included detailed observations of IR pathology in
addition to the assigned types.
Consensus meeting and joint assessment
Following the phase of individual assessment, the group convened at a meeting to jointly assess
the IHC labelled sections around a multi-headed microscope. The diagnostic features of each
type were discussed. While assessing the stained sections under the multi-headed microscope,
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the actual observations were compared with the data recorded in the assessment sheets.
Inconsistencies in these observations were discussed and pitfalls were sought.
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RESULTS
Part I. Assessment of methodology
Trial 1
The co-ordinating centre received 30 stainings and 32 assessments from 17 BNE centres in Trial 1.
Most of the received TMA sections (16) were stained with ubiquitin (12 stains with polyclonal and 4
with monoclonal Abs), nine were stained with p62 (one stain with polyclonal and 8 stains with
monoclonal Abs) and five were stained with TDP43 (3 stains with polyclonal and 2 stains with
monoclonal Abs). (Supplement Table 2)
The participants had used a variety of IHC methods that yielded variable staining quality from poor
to good (Supplement Table 2). In general, the intensity of IR structures, in particular neurites,
varied notably between the different stainings. The intensity of IR also varied between the core
samples within a TMA section in some of the stainings. When either polyclonal or monoclonal
TDP43 Ab was used, the nuclei of cells were very intensively stained, which hindered the detection
of lesions. Briefly, 56 % of the ubiquitin stained sections (9 out of 16), all p62 stained sections and
none of the TDP43 stained sections were of good/acceptable quality. Unless otherwise stated,
results of Trial 1 given below include only assessments of slides having good/acceptable quality.
Table 1 lists the results of the dichotomous (presence or absence) assessments using the ubiquitin
and p62 Abs, i.e. screening Abs (Supplement Table 3). In all 15 core samples that were assessed,
there was 100 % agreement in the assessment of the presence of pathological ubiquitin-IR when
compared with 93 % while applying p62. The agreement was not as good when assessing
designated lesions, i.e. cytoplasmic inclusions and neurites. The agreement in assignment of thin
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respective thick neurites ranged from 33 to 53 per cent while applying ubiquitin, and from 33 to 73
per cent while applying p62 Abs. The most discrepant results were obtained when assessing
intranuclear inclusions; the agreement was 33 % while applying ubiquitin and 40 % while applying
p62 Ab.
The agreement in semiquantitative assessments of cytoplasmic inclusions or neurites was poor
ranging from 13 to 33 per cent (Table 1, Supplement Table 4).
After a joint meeting during which the obtained staining results were viewed and discussed, a
choice of method was agreed upon. The participants were asked to carry out stains for Trial 2, i.e.
the participants were asked to follow prescribed staining instructions.
Trial 2
Fifteen BNE centres participated in Trial 2. Fourteen TMA sections were stained with p62 (2 stains
with polyclonal and 12 with monoclonal Abs), and 11 were stained with TDP43 (9 stains with
polyclonal and 2 stains with monoclonal Abs); thus, in total the co-ordinating centre received 25
assessments.
Even after harmonisation of the IHC methods, there was some variability in the methodology, most
notably regarding the selection of heating methods and the time used for pre-treatment (Supplement
Table 5). Furthermore, there was great variability in the selection of the detection systems.
However, most of the received stainings were of good/acceptable quality (Supplement Table 5).
Briefly, 71 % of the p62 stained sections (10/14), and 82 % of TDP43 stained sections (9/11) were
of good/acceptable quality.
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Regarding the p62 Ab, the dichotomous and semiquantitative assessment results were comparable
in Trials 1 and 2 (data not shown). Thus, results of the second trial below only include assessments
of TDP43 stained slides of good/acceptable quality (Table 1).
There was good to excellent agreement in the dichotomous assessment of IR with both the poly
TDP43 and mono TDP43 (in 87 % and 73 % of cores, respectively). In line with the previous Trial,
the disagreement increased while assigning the lesions, i.e. cytoplasmic inclusions and neurites
ranging from 20 to 73 per cent while applying polyclonal and from 27 to 67 per cent while applying
mono TDP43 (Table 1, Supplement Table 6).
The agreement in the semiquantitative assessments of cytoplasmic inclusions or neurites was good
in the majority of assessed core samples with the mono TDP43, whereas the agreement was poor
with the poly Ab (Table 1, Supplement Table 7).
During a joint meeting, TMA sections produced by the participants during Trials 1 and 2 were
assessed around a multi-headed microscope. Based on the obtained results, a set of TMA sections
with optimal staining results were chosen for assessment in Trial 3. To disclose the influence of
methodology, all participants were asked to assess the same cores in a TMA section stained with
ubiquitin (8 assessors), p62 (8 assessors), TDP43 monoclonal (9 assessors), TDP43 polyclonal (8
assessors) and a newly produced TMA section stained applying pTDP43 (7 assessors). All stains
were of good/excellent quality and the selected 15 cores represented all patterns of TDP43
pathology.
Trial 3
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In total, the participants carried out 40 assessments. A good to excellent agreement, ranging from 87
to 100 per cent, was achieved in the dichotomous assessment of pathological IR, independent of the
staining applied (Table 2, Supplement Table 8). The Ab directed against the protein of interest
seemed to perform better when compared to ubiquitin and p62, and the best results were obtained
while applying the pTDP43. The separation of thick from thin neurites was still less optimal (73 vs
67 per cent).
The assessment of the density of IR structures is summarised in Table 3. The most even assessments
of the densities were achieved with the pTDP43. In 3 out of 15 cores, all evaluators had given the
same density; moreover, in 10 out of 15 cores, most agreed with the assessment. The agreements in
the densities were quite equal with p62, ubiquitin and polyclonal TDP43. The most variable
assessments of the densities were obtained while assessing monoclonal TDP43 stained TMA.
The results of the predominance of IR structures (none, cytoplasmic inclusions, neurites or both) are
provided in Table 4. All evaluators had given the same assessment in 13 %/ubiquitin-, 13 %/p62-, 33
%/monoclonal TDP43-, 20 %/polyclonal TDP43- and 27 %/pTDP43-stained core samples.
Part II. Designation of subtypes of FTLD-TDP
The members of the reference group (IA, MN, TA, HK, MP) agreed after the session around a
multi-headed microscope that there were four cases that fulfilled the criteria for Type C
(Mackenzie et al. 2011), 11 fulfilled criteria for Type B; however, three were designated as being
atypical, and nine cases fulfilled criteria for Type A. It was impossible to differentiate between
Type A or B in four cases; thus, these were designated as being mixed (A/B). In two cases, the
pathology was not sufficient in order to designate a subtype (Tables 5 and 6). No cases with
Type D pathology were included.
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Inter-observer assessment
Thirty one participants from 18 centres classified the 30 included cases following the
classification recommendations provided by Mackenzie et al. 2006 and Sampathu et al. 2006. In
addition, 26 of the assessors also stated the type following recommendations by Cairns et al.
2007. This statement was given after a joint meeting where classifications results were overall
discussed. The results are summarised in Table 5. The agreement rate between the reference
group and the 31 assessors varied from 19 to 100 per cent. The highest agreement was noted for
the mixed Type (A/B) independent of stain or classification system applied. High agreement was
also noted for Type C cases. Poorest agreement was noted for atypical B cases. Overall
agreement was 62% (ubiquitin-72%, p62-56%, monoclonal TDP43-61% and pTDP43 63%)
when following Mackenzie et al classification system from 2006 and when only the 28 cases
classified by the reference group were included. The overall agreement rate was 38% (ubiquitin56%, p62-49%, monoclonal TDP43-38% and pTDP43-53%) when following Sampathu et al
classification system from 2006.
Consensus meeting and joint assessment
While jointly assessing the cases and discussing the obtained results, it was agreed that
differentiation between Types A and B was difficult, independent of stain or classification systems
used. In contrast, cases of Type C displaying prominent thick neurites were easily recognised and
thus yielded good agreement results.
After the joint meeting 26 participants typed by the reference group classified 28 cases following
the Cairns et al. classification system from 2007 (Table 5). Overall agreement was 66% (ubiquitin66%, p62-61%, monoclonal TDP43-73% and pTDP43 68%).
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DISCUSSION
Here, we assessed the reproducibility in the assignment of a type of FTLD-TDP while following
current recommendations and noted that agreement rates were influenced by the IHC method, i.e.
Abs applied, by the classification strategy followed (Mackenzie et al. 2006; Sampathu et al. 2006),
by the subtype of FTLD-TDP and by training (Cairns et al 2007). The IHC method applied included
the screening Abs directed against ubiquitin, p62 and the specific Abs directed against TDP43
mono/poly and pTDP43. It is noteworthy that in addition to variability in the Abs, a wide range of
pretreatment strategies and detection systems have been utilised. Previous methodological studies
dealing with staining carried out on post-mortem brain material have emphasised that the key factor
for the quality of a staining is not only the Ab but also the antigen retrieval method (Alafuzoff et al.
2006,2008a,b; Croisier et al. 2006). When the participating laboratories followed their own IHC
practices, the variations were remarkable particularly in ubiquitin stains (poor quality in 44 %). We
noted that the same commercial ubiquitin-Ab yielded both good and poor results. For example,
formic acid pretreatment abolished the staining, and a short incubation time at room temperature
was deemed to be of poorer quality when compared with a longer incubation time at +4ºC. When a
poly TDP43 or a mono TDP43 was used, nuclei were intensively stained with a strong stain, which
interfered with the detection of lesions. The quality of TDP43 stains was improved when the
participants received detailed instructions and it was noted that the p62-IHC staining, commonly
used by all participants, yielded quite good uniform quality. Both poor and good results were
produced independent of the mode of stain, i.e. manual vs automatic.
Both assessment of the type and the extent of lesions were influenced by the choice of Ab as well as
by the quality of staining. In addition, we found that while assessing the same TMA core, the
observers did indeed reach an absolute agreement while assessing the existence of pathological
lesions. In contrast, there were some inconsistent results when the assessors were asked to identify
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specific lesions such as cytoplasmic inclusions or neurites. These results were improved in Trial 3
in comparison with Trials 1 and 2. In Trial 3, all investigators assessed the same cores in the same
TMA section; thus, the differences due to staining quality were deleted. Moreover, Trial 3 followed
two joint assessment sessions functioning as training sessions harmonising the interpretation of
obtained IR. A general observation was that the assignment of lesions became more homogenous
after the repeated trials and joint assessments around the multi-headed microscope, emphasising the
need for similar sessions as part of training and not only for less experienced colleagues. The
assessment of extent yielded, however, repeatedly poor results. Based on these observations, it
became obvious that extent of pathology should not be used in diagnostic consensus criteria due to
poor reproducibility in an inter-laboratory setting. This observation is in line with BNE’s previous
results while assessing inter-rater agreement in assessing -synuclein pathology (Alafuzoff et al.
2009a).
The histological typing of FTLD-TDP includes semiquantitative assessments of cytoplasmic
inclusions and/or intranuclear inclusions and neurites ranging from a few to numerous (Mackenzie
et al. 2006, 2011; Sampathu et al. 2006; Cairns et al. 2007). Another significant variable to be taken
into account is the predominant type of lesion (Mackenzie et al. 2006, 2011; Sampathu et al. 2006;
Cairns et al. 2007). Moreover, the evaluation of the distribution (superficial vs transcortical) of
pathology is emphasised in two of the typing schemes (Samptahu et al. 2006; Cairns et al. 2007).
The observed results obtained here in Trials 1 to 3, while assessing methodological aspects
constitute a severe obstacle in the subtyping of FTLD-TDP. All parameters to be assessed are
highly dependent on the staining quality obtained. Of note, the classification strategies, i.e.
Mackenzie et al 2006 and Sampathu et al 2006 were originally designed while applying screenings
antibody Ubq. These instructions have then been applied while using other antibodies such as p62,
mono/poly TDP43 and pTDP43. Each of the antibodies tend to stain different lesions in various
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extent and this might in addition influence the outcome while assigning a type of FTLD-TDP and
using different antibodies.
Here, we noted that while 31 assessors stated the type of FTLD-TDP, the agreement with the
reference group ranged from 19 to 100 per cent. In particular, the highest agreement was obtained
while assessing lesions visualised with specific Abs (TDP43 and pTDP43) when compared with the
screening Abs (Ubq, p62). The significance of the IHC method was emphasised in a recent report
indicating that the use of phosphorylated when compared with non-phosphoryletad TDP43 resulted
in a higher inter-observer variation when five experienced neuropathologist classified14 FTLD
cases into subtypes (Tan el al 2013). Furthermore, it was noted that when the classification strategy
required the distribution of pathology in addition to the assessment of type of lesion, the results
were poorer when compared with classification strategy primarily based on the type of pathology
(Mackenzie et al. 2006 performed better when compared with Sampathu et al. 2006). It was
apparent that classification of a type with clearly identifiable lesions such as long neurites (Type C)
yielded higher agreement when compared with types that were quite similar (Types A and B), i.e.
many/moderate neuronal cytoplasmic inclusions with many/few neurites. The visualisation of the
latter pathology, i.e., neurites is highly dependent on the staining quality. In cases that were not
discernable (mixed Type A/B), the agreement rate while applying specific antibody (TDP43) and
following type oriented classification strategy (Mackenzie et al. 2006, 2011) was also acceptable.
While jointly assessing the cases, it was noted that in most of them the pathology was distributed
throughout the entire thickness of the cortex; subsequently, it was difficult to agree on the
predominant distributional pattern of pathology even around the multi-headed microscope. Thus, it
is impossible to strictly follow the given instructions, including statements such as, infrequent
neurites in deep and superficial cortical layers in Type B, and predominance of small neurites and
inclusions in superficial cortical layer in Type A. This might partly explain the poor agreement
observed here while assigning Type A or Type B cases. What is reported here is probably not
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exceptional but rather the rule, i.e., while classifying cases into Types A and B, the results might
depend on the variability in the staining quality (influencing the extent and type of pathology that
can be seen). Furthermore, one should not rule out significant influence of “experience”, i.e.
assessing one FTLD-TDP case/year vs assessing one FTLD-TDP case/month. Some influence on
agreement results was noted after the joint meeting when the classification systems were discussed
(38% agreement when following Sampathu et al 2006 when compared with 66% agreement when
following Cairns et al. 2007). The difficulty in reaching high agreement rates in the inter-laboratory
setting was also emphasised in previous BNE publications, indicating that even with respect to
common pathologies such as Alzheimer’s disease related lesions or -synuclein pathology, full
agreement is not reached by experienced neuropathologists even though very detailed instructions
are available (Alafuzoff et al. 2008b, 2009a, b).
Results obtained here are in principle in line with the results obtained previously by BNE
consortium. It has previously been reported that choice of Ab and use of methodology is of
significant importance. The community should be aware of the multitude of issues that influence the
staining outcome (fixative, fixation time, storage time of sections) and that this influence varies
from one epitope to another (McNicol et al. 1998; Shi et al. 2001; Gelpi et al. 2007; D’Amico et al.
2009; Pikkarainen et al. 2010; Karlsson et al. 2011; Kovacs et al. 2012). Another issue that the BNE
consortium has become aware of is the need for precise detailed instructions. In all previous trials, it
became obvious that the main factor influencing the outcome of “staging” or “typing” a given
pathology was the neuroanatomical distribution of pathology. Recent reports suggest that in line
with other neurodegenerative diseases a staging of TDP43 pathology based on sequential regional
distribution can be carried out (Brettschneider et al 2013, 2014, Josephs et al 2014). A stepwise
neuroanatomical progression pattern has been identified in behavioural variant of FTLD-TDP with
regions such as orbilat gyrus and amygdala primarily involved, in Amyotrophic Lateral Sclerosis
with agranular motor cortex and medulla oblongata at the level of nerve XII primarily involved and
22
in Alzheimer’s disease with amygdala and entorhinal cortex primarily involved (Brettschneider et al
2013, 2014, Josephs et al 2014). Based on our observations and the recent reports suggesting a
sequential evolvement of pathology, TDP43 pathology should probably initially be assessed in at
least three regions, i.e., amygdala, hippocampus and medulla oblongata as observed or not seen
(TDP43-IR inclusions and neurites), leading to verification of existing FTLD-TDP pathology.
Subsequently in a case of Amyotrophic Lateral Sclerosis, behavioral variant of FTLD-TDP or
Alzheimer’s disease additional neuroanatomical regions should be assessed as recently
recommended (Brettschneider et al 2013, 2014, Josephs et al 2014). Furthermore, regarding FTLDTDP, if many long neurites are seen, then Type C could be designated. Types A and B are not fully
discernable based on our results and thus when not convinced about the type, Type A/B (mixed)
should be assigned. Furthermore, p62 pathology in the cerebellar cortex should be investigated on
all Types of FTLD-TDP cases in order to identify subjects with C9orf72 repeat expansion (Figure
3) (Cairns et al. 2007; Pikkarainen et al. 2008, 2010, 2011; Mahoney et al. 2012). In addition when
the subjects display familial clustering appropriate genetic assessment should be carried out and
thus sampling of frozen tissue should always be done.
In conclusion, in line with previous BNE studies, there are limitations to reaching broad consensus
in inter-laboratory settings. It is possible to achieve staining results of high quality and also some
level of agreement regarding the interpretation of staining results. Currently, we can expect that
neuropathologists, independent of their familiarity with FTLD-TDP pathology, can identify a
TDP43 positive FTLD case. Some agreement can be reached regarding Type C with thick and long
neurites, whereas the differentiation between Types A and B may be troublesome. In general
neuropathologists are urged to state defined types of FTLD (A,B,C.D and mixed A/B, A/C, B/C),
however, one should be aware that the given type by one neuropathologist might not be in
agreement with the type given by another neuropathologist. Recent reports describing a sequential
23
neuroanatomical evolvement of TDP43 pathology in behavioral variant of FTLD-TDP,
Amyotrophic Lateral Sclerosis and Alzheimer’s disease will certainly influence the future
agreement rates (Brettschneider et al 2013, 2014, Josephs et al 2014). It has previously been
reported by BNE that dichotomized assessment of pathology in defined neuroanatomical regions
disregarding the extent of lesions tend to yield good agreement rates in inter-laboratory setting
(Alafuzoff et al 2008b, 2009a). In Figure 3, a flowchart is given summarizing the general handling
of a case with neurodegenerative diseases with respect to TDP43 pathology.
24
Acknowledgments
We thank Tarja Kauppinen and all other laboratory technicians of the BNE members for their skilful
technical assistance and Meena Strömqvist for her critical reading of the manuscript. We
acknowledge the following BNE centres (in alphabetical order of the cities) for contributing the
material with generic data used in this study: Netherlands Brain Bank (Amsterdam), Hospital de
Bellvitge/Universitat de Barcelona (Barcelona), National Institute of Psychiatry and Neurology
(Budapest, OPNI) closed in 2007, Georg-August-University (Göttingen), Karolinska Institutet
(Huddinge), Kuopio University Hospital (Kuopio), MRC London Neurodegenerative Disease Brain
Bank Institute of Psychiatry (London), Hospices Civils de Lyon (Lyon), Istituto Nazionale
Neurologico Carlo Besta (Milan), Ludwig-Maximilians-University of Munich (Munich), Medical
University Vienna (Vienna) and University of Wuerzburg (Wuerzburg).
This study was supported by the European Union grant FP6: BNEII No LSHM-CT-2004-503039.
TA, HK and DRT were also supported by the German ministry for education and research (BMBF;
FTLDc). IA was also supported by the local grants (ALF) from Uppsala University Hospital,
Sweden. This article reflects only the authors' views, and the Community is not liable for any use
that may be made of the information contained herein. The study has been authorised by the Ethics
Committee of Kuopio University Hospital.
Disclosure: DRT received consultant honorary from Simon Kucher and partners, GEHealthcare, and Covance Laboratories, speaker honorary from GE-Healthcare and collaboration with
Novartis Pharma AG.
25
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33
FIGURE LEGENDS
Figure 1. Flowchart summarising the structure of the study. The study is based on the work of 1) a
defined co-ordinating group, 2) a reference group including neuropathologists working individually
and jointly around a multi-headed microscope and finally 3) more than 20 participants working
individually and participating at the joint viewing and discussions.
Figure 2. Summary of constructed assessment instructions and assessment sheets for Trials 1, 2 and
3
Figure 3. Recommended flowchart while handling brains of subjects with a neurodegenerative
disease with emphasis on TDP43 pathology
Supplement Figure 1. Summary of the published subtyping recommendations in tabulated form and
the instructions of assessment of immunoreactive lesions
Supplement Figure 2. Assessments sheet used in part II.