Diagnosis and Staging of Alzheimer Disease

Neurobiology of Aging, Vol. 18, No. S4, pp. S33–S42, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0197-4580/97 $17.00 1 .00

PII:S0197-4580(97)00067-5

Diagnosis and Staging of Alzheimer Disease C. DUYCKAERTS1 AND J.-J. HAUW Laboratoire de Neuropathologie R. Escourolle, Hoˆpital de La Salpeˆtrie`re, 75651, Paris, France DUYCKAERTS, C. AND J.-J. HAUW. Diagnosis and staging of Alzheimer disease. NEUROBIOL AGING 18(S4) S33–S42, 1997.—Rather than determining lesions “threshold” between “normal” cases and patients, we prefer to use clinicopathological correlations, assigning a given intellectual deficit to a given amount of lesions with a chosen level of probability. Because large amounts of Ab diffuse deposits may be found in the absence of dementia, we think advisable not to take them into account for the diagnosis. The diffusion of the neurofibrillary tangles in the paralimbic, limbic and isocortical areas (described by Braak and Braak stages or by the number of areas containing tangles) and the density of isocortical senile plaques (Ab focal deposits) as assessed by the CERAD protocol are both correlated with the intellectual status but give complementary information. They should thus be jointly used. We analyzed the variability of the lesions counts, their coefficients of error, and their causes, as a first step toward standardization. We have shown, however, that semiquantitative estimates are presently more reproducible than quantitative measures. © 1997 Elsevier Science Inc. Alzheimer disease Diagnostic criteria Morphometry Standardization

Senile plaques

Neurofibrillary tangles

Ab peptide

Tau protein

accept that a patient with a plaque density of 14.9/mm.2 does not have AD whatsoever. The threshold values, obviously, have to be established by specific studies, the design of which is difficult: how can the ‘normal’ population be defined, among Murphy’s seven meanings for normal (31)? Young people (‘most perfect of its class’)? Old people devoid of intellectual impairment (‘commonly aspired to’)? Old people having a slight intellectual impairment common in aging (‘most representative of its class’)? Finally, the selection of the pathological markers in relation with the clinical signs has the side effect of including cases with AD at onset (i.e., without clinical symptoms or ‘preclinical AD’) in the normal group.

THE very necessity of diagnostic criteria may be questioned. They may, indeed, raise questions, promote research, bring valuable information, and remain open to unforeseen events, but they may also freeze the neuropathological examination in a conventional exercise, forcing the reality into a mold. This is why we would more easily endorse criteria presented as a common vocabulary than as rigid rules of diagnosis. At least two somehow contradictory requirements should then be taken into account: 1) In practice, a definite and clear diagnostic conclusion must be drawn to classify the cases and allow further investigation. Diagnosis should not be obscured by complex and circumstantial neuropathological conclusions. 2) The diagnosis must be documented by an objective (or at least conventional) evaluation of the quantity of lesions in order to permit correlative studies. Similarly, there are two main policies to elaborate criteria for AD as for any disorder lacking a definite marker: 1), The population may be clinically divided into two groups: normal and demented. A histopathological threshold is then fitted to this clinical dividing line (42). 2), Or the severity of the histopathological changes may be assessed and correlated with some estimates of the clinical symptoms, without a clearcut border between normality and disease (6,9). These two points of view fulfill different needs and meet different difficulties.

Correlation, Regression and Staging This approach is based on the hypothesis that the clinical signs are proportional to the number or to the diffusion of lesions. One may apprehend this relationship either by quantifying clinical and histopathological markers (6) or by ‘staging’ the histopathology according to the topography of the lesions (9). This approach makes the definite diagnosis theoretically difficult: should the point where the lesions are in sufficient number, or sufficiently widespread to produce clinical symptoms, be taken as a dividing line or does the disease start as soon as there is one lesion, even if it does not produce symptoms? Only the finding of the direct cause and the mechanism of the disease could probably solve this question. Several correlative clinicopathological studies (6,14,27,54,55) have now been published demonstrating their feasibility: correlation and regression are efficient statistical means in AD because the lesions are not resorbed as quickly as they are produced (in Parkinson’s disease, by contrast, the number of Lewy bodies in the substantia nigra may not be higher in advanced cases). Regression

Threshold When the threshold is made of a mere line between normality and disease, without any gray zone, the diagnostic decision does not take into account the uncertainty due to the threshold itself (we are not sure that the theoretical value is correct in all the cases) and due to the counting procedure (a statistical error is usually present in the data). If, for example, the diagnostic threshold for the count of the senile plaques is 15.0/mm.2, we are personally not ready to

1 To whom requests for reprints should be addressed. Charles Duyckaerts, Laboratoire de Neuropathologie R. Escourolle, Hoˆpital de La Salpeˆtrie`re, 75651, Paris, France.

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between clinical signs and density of lesions permits the observer, to a certain extent, to determine the weight of specific histopathological markers in the final product of the clinical symptoms. In the following, we plan to use the correlative approach utilizing the data that we have accumulated since 1981 concerning a series of 30 cases (Charles Foix Longitudinal study) (14 – 16,23,25–28,39,40,44), all women aged over 75, who had been living in the same institution and had been prospectively assessed by the test score of Blessed et al., BTS: (6). As the study was correlative, the cohort included both normal (the best score was 28) and demented subjects (the worst score was 0) with various degrees of severity without a priori borderline between patients and controls. By chance, and also thanks to the intrinsic properties of the scale devised by Blessed et al., our cases were regularly distributed over the whole range of scores between 0 and 28. We plan to raise the following questions concerning the diagnosis of AD: What? Which lesions should be evaluated? Where? Which region of the brain should be sampled? How? Which are the technical steps to be followed? Judge or count? Should we rely on a quantitative, semiquantitative or qualitative approach? WHAT?

Numerous variables have been assessed in relation with AD dementia. Four of them have been thoroughly studied. Two are positive signs: density of plaques (6,15,27,54) and of tangles (14,54,70); two are negative: loss of neurones (47,53,55,68) and of synapses (13,61,67). In addition, the presence of Lewy bodies (33,36,45,58) has been considered an important covariable, able to alter the course and the clinical presentation of the disease. Plaques and Tangles, Ab- and Tau- Pathologies Immunohistochemistry has modified the understanding of AD pathology. Tangles are only part of a more widespread change of the cytoskeleton (7). Plaques are composite lesions, mixing neuronal processes and extracellular deposits. It may be more convenient to analyze the pathology in terms of immunohistochemical markers (24). Tau immunohistochemistry and Gallyas’ or Bodian’s staining technique reveal the tangles in the cell bodies (NFT), the neuronal processes (neuropile threads) and the crown of the senile plaques (see Table 1). These lesions may be grouped under the label ‘tau pathology’ and the stains used to show them may be described as ‘tau-sensitive’. Ab antibodies, modified Bielschowsky method, thioflavin S, and Congo red may by contrast be named ‘Ab sensitive’ because they show Ab deposits, plaque cores, and amyloid angiopathy. Tau- and Ab- pathologies are two contrasted aspects of AD. They have different distributions, time course, and significance, and they may be considered separately. Tangles have been found in numerous other diseases than AD (72). By contrast, classical plaques with a central core of Ab and a neuritic crown are almost synonymous with AD and aging. However, this contrast between unspecific tangles and specific plaques is somewhat artificial, because both are present together in AD brains, even if sometimes in different cortical areas. We do not think that the alleged unspecificity of tangles is to be considered as a strong fact against their use in the diagnostic procedure. In practice, the tremendous prominence of AD over the other NFT-associated disorders, the special topography of NFT, their preference for middle-sized pyramidal neurons, and their constant association with plaques make them an unmistakable marker of AD. Ab pathology. Ab antibodies label lesions that were previously unknown and have a different status as regards the clinicopatho-

TABLE 1 PROPOSED CLASSIFICATION OF THE LESIONS Ab Immunohistochemistry

Diffuse deposit Focal deposit

tau Immunohistochemistry

Neuritic crown Neuropile threads Neurofibrillary tangle

Ab 1 tau

Senile plaque

Lesions labelled by Ab immunohistochemistry are named “deposits;” two types, diffuse and focal, are distinguished. The neurofibrillary pathology is shown by antitau immunohistochemistry. A senile plaque comprises a core of focal deposit and a crown of tau-positive neurites.

logical correlations (15). In our opinion, the term ‘deposit’ should be preferred when speaking of the lesions shown by Ab immunohistochemistry (Table 1). Deposits may be focal (the core of the senile plaques) or diffuse. The senile plaque, then, comprises a core, made by a focal deposit, and a tau-positive, neuritic crown. Diffuse deposits have convoluted contours and sometimes indistinct margins. They may cover large areas of the neuropile. They may be observed in large amounts in normal aged individuals (16,18). They have been reported in young individuals after brain trauma (60), although this remains controversial (1) and, more frequently, in cases with an epsilon 4 genotype of the apolipoprotein E (56). It should be noticed that the modified Bielschowsky (63) and the silver methenamin methods (73) also reveal some diffuse deposits, which confound the correlation with the intellectual deficit when included in the plaque counts (Fig. 1). Tau pathology. Tau immunohistochemistry and Bodian’s and Gallyas’ techniques reveal the extent of the neurofibrillary pathology. The counts of senile plaques and of tangles after tau immunohistochemistry and after Bodian stain are highly correlated (21). The regression curve between the lesion counts obtained with the two methods has a constant term near zero and a slope close to one, indicating a one-to-one correspondence between the two methods, without threshold. It is hard, in practice, to quantify the density of the neuropile threads (28) but one may readily rely on the tau-sensitive methods to assess the density of neuritic plaques and of tangles. Relation with dementia. The relation between plaques, tangles and dementia is controversial; for some authors, the plaques are correlated with the intellectual score (6) and should be used for the diagnosis (42) because the tangles may be lacking in so-called ‘plaques only’ forms of AD (37,66). For others, the tangles or the neuritic pathology, on the contrary, are the best markers (17,54,70). Correlative studies undertaken in this laboratory as well as in others have indicated that the intellectual status measured by the BTS was proportional to the densities of the plaques and of the tangles (14 –16,21,28). Both lesions accumulate in the course of the disease because they are not, or are only slowly, resorbed. The most significant correlations with the Blessed test score were obtained for the tangles in the hippocampal-parahippocampal region and for the senile plaques in the isocortex. Both relationships appeared to be of low threshold, i.e., present even in mildly affected cases, lower for the tangles in the hippocampal-parahippocampal region, where the only lesions were to be found in some cases identified as ‘transentorhinal’ and ‘limbic’ by Braak and Braak (8,9) and as ‘NFT predominant’ by Bancher and Jellinger (4). The density of tangles in the isocortex was also proportional to the BTS but with a higher threshold; they were indeed present only in advanced cases and then often in high

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FIG. 1. Density of Ab deposits and senile plaques in the superior temporal gyrus as shown by Ab-immunohistochemistry (‘Ab-deposits’), modified Bielschowsky method (‘Bielschowsky’), thioflavin S, Gallyas silver iodide, Bodian technique coupled with luxol fast blue (‘Bodian’), tau (‘tau’), and ubiquitin (‘ubiquitin’) immunohistochemistry in a series of 15 cases (Bielschowsky, thioflavin Gallyas) or of 29 cases (Ab-, tau-, and ubiquitin-immunohistochemistry). The intellectual status had been prospectively assessed by the Blessed test score (‘BTS’). The densities are shown on a log scale. Ab immunohistochemistry shows clearly more deposits than Bielschowsky and thioflavin S methods, themselves showing more plaques than Gallyas, Bodian, and tau- and ubiquitinimmunohistochemistry.

number (20). Below this threshold, the pathology in the isocortex included only Ab or amyloid deposits. The relationship between plaques and intellectual status depended on the type of the lesions which were taken into account; the most significant results were obtained when only the plaques with a crown of neuronal processes were counted. It was much lower when the Ab diffuse deposits were taken into account (15,16), in agreement with other studies (18,54), although a high correlation between clinical status and amyloid load has also been reported (12) without direct comparison with tau markers. In our experience, the counts of the senile plaques obtained with tausensitive methods, although reaching lower numerical values than after Ab immunohistochemistry, are better correlated with the intellectual status and should be recommended for routine work.

be used to avoid this bias (62). A recent (59) and controversial studies (30,41) with this method has indicated that the neuronal loss could be absent or at least of a smaller magnitude than usually thought. However, when specific areas are investigated, the loss may reach 90% (34). Although not diagnostic, the evaluation of the neuronal loss would be a useful complement to understand the heterogeneity of clinicopathological correlations. In some very old patients, a severe loss of neurons is found in the vulnerable sector of the hippocampus (19). The patients had usually been considered demented. This alteration may be associated with Alzheimer lesions and obviously modifies the clinicopathological correlations. This should also be listed among the covariables.

Neuronal Loss

It has been stated that the dementia seen in AD was more directly determined by the loss of synapses, considered by some as the best correlate of the intellectual deficit (48,61,67), although this point remains controversial (17). Unfortunately, the antigen presently used as a marker (synaptophysin) is very sensitive to the post mortem delay and to the duration of fixation. The use of a dot– blot immunoassay could help quantify this loss (2). New markers such as SNAP-25 should be tested (11). Synaptic loss should be considered in the future as an important variable to explain the intellectual deficit and may, as such, be listed among additional data in the diagnostic criteria.

It has been repeatedly stated that the clinicopathological relationship between dementia and neuronal loss was more direct than with the density of plaques and of tangles (53,55,68). However, the neuronal loss seen in AD is far from specific. It is seen in most dementing processes. It is hard to assess (22), because the numerical density of the neurons, i.e., their number per unit area, is measured in a sample where the volume might have been modified by parenchymal atrophy. A measurement of the volume of the cortex has to be simultaneously performed if the total number of neurons is to be correctly evaluated. Moreover, the count of the neuronal profiles in a histological section is biased. The large neurons, being more often cut than the small ones, are overrepresented. When a neuronal population becomes smaller [and this atrophy indeed occurs during aging (65) as well as in AD (64)], their apparent number on the slide decreases [pseudo-loss (29)]. Special procedures, such as the counting of profiles present on a focal plane and absent from the next (optical disector), may

Synaptic Loss

Lewy Bodies The use of ubiquitin immunohistochemistry has made Lewy bodies much easier to detect (46). Double labelling with tau- and ubiquitin- antibodies may facilitate their distinction from ubiquitin positive tangles (5). Their presence in the cerebral cortex in cases with numerous plaques (37), and sometimes spongiosis, raises the

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FIG. 2. Correlation between the density of isocortical senile plaques as evaluated by the CERAD protocol and Braak and Braak staging in the Charles Foix prospective study. The cases were staged by Prof. H. Braak (10).

question of a specific form of AD (33,36,58). The classification of these cases has been much debated; the terms ‘diffuse Lewy body’ (43), ‘Lewy body variant’ (36), and ‘Lewy body type’ (58) of AD have been proposed. The new consensus on the term dementia with Lewy bodies (DLB) (49) greatly simplifies the nomenclature. Discrepancies exist in the evaluation of the incidence of Lewy bodies in AD and this should be more systematically studied in the future. The possibility of geographical heterogeneity should be investigated by international projects. WHERE?

As just seen, two histopathological markers are highly correlated with the clinical symptoms, isocortical plaques (Fig. 1) and hippocampal-parahippocampal tangles. The density of these two markers is actually correlated (Fig. 2). The density of isocortical plaques has been used to determine diagnostic thresholds (42). The distribution of neurofibrillary pathology in allo- and iso- cortices is the basis for staging the cases according to Braak and Braak (9). The density of plaques and tangles, even within a given cytoarchitectural area is heterogeneous; differences between the top, the sides and the sulcus of the gyrus have been repeatedly noticed (50). The isocortical samples should thus systemically cover the whole gyrus including the sulcus. The distribution of both plaques and tangles is layer specific. This is why the evaluation of their density must involve all the layers; in practice, the counting has to be performed in columns of contiguous fields. This technique fulfills the requirement of ‘systematic random sampling’ (35).

FIG. 3. CERAD diagnostic criteria, applied to the cohort of cases of the “Charles Foix Prospective Study.” BTS: Blessed test score. Data presented at the Fourth International Conference on Alzheimer’s Disease. Minneapolis, 1994: C. Duyckaerts, Y. He, D. Seilhean, P. Delae`re, F. Piette, H. Braak, J.-J. Hauw. Diagnosis and stageing of Alzheimer’s disease in a prospective study involving aged individuals.

Samples for the Assessment of Neurofibrillar Pathology The areas where the tangle density has to be evaluated must, on the contrary, be very strictly identified and should include the anterior hippocampus and the adjoining entorhinal cortex (Fig. 4). The samples may include part of the amygdaloid body and should reach laterally the collateral sulcus. Samples of the hippocampus taken more posteriorly at the level of the geniculate body do not contain the entorhinal area but area TF-TH. The density of tangles in that area also appeared well correlated with the intellectual status. Unimodal (e.g., area 22, on the anterior and upper part of the superior temporal gyrus) and multimodal associative cortices (e.g., the supramarginal gyrus) are involved in severely affected cases (the threshold in our series was around BTS 5 10). Primary areas (e.g., the motor cortex or the visual cortex, area 17) are devoid of tangles, except in the most advanced cases. The number of areas containing at least one tangle in a sample of primary and

Samples for the Counting of Plaques In our series of cases, as in the map of the lesions drawn by Arnold et al. (3), the plaques appeared to be rather evenly distributed over the isocortex. Their density is high in associative cortices as well as in primary motor and sensory areas. By contrast, plaques might be relatively sparse in the hippocampal and parahippocampal areas. Because the counts of the isocortical plaques are not sensitive to the topography, we would readily endorse the sampling scheme proposed by the CERAD (52) (Fig. 3). In this protocol, the samples may indistinctly involve large areas of the brain.

FIG. 4. Correlation between the intellectual status evaluated by the Blessed test score (BTS) and Braak & Braak staging in the Charles Foix prospective study. Data from (10).

DIAGNOSIS AND STAGING OF ALZHEIMER DISEASE associative uni- and multi- modal cortices appears to be highly correlated with the clinical status (20). Subcortical Areas The importance of the subcortical lesions has been much debated. Plaques are seen in the mamillary bodies, in the limbic nuclei of the thalamus, as well as in the striatum (63), where they are usually devoid of a neuritic crown. Diffuse deposits are known to be present in the cerebellar cortex. The presence of tangles is well documented in the nucleus basalis of Meynert and in the septal area, in the substantia nigra (mainly the ventral tegmental area), locus coeruleus, nucleus raphe dorsalis, and in numerous other brainstem nuclei. It has been suggested that they were observed mainly in nuclei that had direct projections to the cortex (32). These lesions are probably of little diagnostic significance. They might confuse the diagnosis and erroneously suggest the possibility of progressive supranuclear palsy. The topography of plaques and tangles would justify the systematic sampling of the hippocampal-entorhinal area (mainly for the evaluation of neurofibrillary pathology) and of the isocortex (for the estimation of plaque and tangle density). In this laboratory, we systematically take samples of areas 4 and 17 (primary cortices), 22 (unimodal associative cortex), 40 and 46 (multimodal associative cortices). Tangle and plaque counts may be performed on these samples. The presence of Lewy bodies is evaluated on samples of the SN, cingulate gyrus and insula (43). HOW?

Sampling, Sectioning, and Staining There is a conflict between the constraints of a stereological study, aimed at providing correct quantitative indices [see for example (59)], and those of the neuropathological examination aimed at providing a correct diagnosis. To assess the total number of the lesions (or, for that matter, of the neurons), it is necessary to rely on a specific way of sampling, e.g., ‘systematic random’; the whole structure has to be serially cut from an initial random section, and its volume must be measured. Only a few blocks, systematically picked up in the series, have to be analyzed. The counts have to be performed on thick sections (as thick as possible) to avoid ‘recut’ artifacts, using an unbiased method, such as the disector. The usual neuropathological procedure is quite different. The sampling involves a large number of macroscopically identified structures but is usually sparse, one or two blocks. The microscopical sections are thin (usually 8 mm or less) to ensure the best optical quality. There is no estimation of the volume and when a counting is performed, it is a crude one, simply assessing the number of profiles on the section without taking into account the possible bias due to the shape, the orientation, and the size of the particles to be evaluated. Is there a way to improve this rough quantitative evaluation without losing in image quality and diagnostic efficiency? In this laboratory, several measures have been taken in this direction, which we will now consider. Identification of the area. As previously mentioned, the correct identification of the isocortical areas is important mainly for the assessment of tangles density. We have found it difficult to recognize the areas after the brain has been sectioned even with the use of specific maps. In particular, because atrophy may have affected some cortices and spared other. For this reason, we routinely identify the lobes on the external aspect of the hemisphere (26) with the use of a Brodmann map, and we label them with different colors (eosin, Ponceau red, light green, and cresyl violet). Areas to be sampled are marked with India ink. These

S37 labels are still visible after sections, and they may be identified on the photographic pictures. Systematic sectioning and volume assessment. Our coronal sections are serial. A simple device ensures that the cuts are 1 cm. apart. All the sections are photographed. The photographic slides may be used for the measurement of the area of cut surface of the cortex on sections. The sum of the section areas multiplied by the thickness of the block (1 cm.) gives an estimate of the volume (‘Cavalieri principle’). A volume measurement is useful to correct the effects of atrophy which could artificially increase numerical density. The biological variation of brain volume is of such a magnitude that it is useless to look for extremely precise measures; even a crude estimate is sufficient as long as its coefficient of error does not exceed the biological variation itself. Measurement of the samples before inclusion. Haug (38) found that the shrinkage of the blocks during paraffin inclusion varies with age. It might as well be modified by the amount of gliosis or of neuronal loss. We have systematically measured the length of easily identified and regular borders on each of our blocks to control their shrinkage. Section thickness. The use of thick section, recommended in morphometry, lessens the quality of the microscopic image, but the 7- to 8- mm thick sections that are in current use seriously bias the counts of large structures such as the senile plaques; the biggest ones are overrepresented, the smallest, underestimated. Theoretically then, a lower count could be related to a smaller size of the plaques rather than to a lower density. Practically, two populations of senile plaques different by their size but having the same density would be differently represented on the slide. With a section thickness of 8 mm, plaques of 40 mm in diameter would be 20% more frequent than plaques of 32 mm (29). An estimate of plaque diameters may be used to correct the count and possibly control this bias. The problem is much more difficult for tangles. Their shape is complex and they are probably anisotropically distributed, but we do not know of any means of assessing or controlling this type of error. Staining. The use of different staining methods is one of the major causes of interlaboratory variability, particularly in the assessment of plaque density (23); the plaque is indeed a composite lesion, containing both Ab- and tau-markers in different amounts depending on its maturation stage. Extreme opinions concerning the technique to be used have been expressed; some methods have been said to be mandatory, whereas others have been banned. However, the uses and habits of the neuropathology departments have probably not been much influenced by these debates (71). In the meantime, however, pathology practice has undergone a profound mutation; the routine use of immunohistochemistry has made it a reliable and easily accessible technique. In many laboratories, it is now much easier to obtain an immunolabelling by anti-tau or anti-Ab antibodies than a silver impregnation. No doubt, the diagnostic criteria should assimilate these new ways. The rules to follow in determining the selection of a specific staining method have not been fully elucidated. It is clear, however, that a method which shows the largest number of lesions is not necessarily the best one; sensitivity is not synonymous with specificity. Ab-sensitive techniques usually show many more alterations than the other methods but these changes, including Ab diffuse deposits, are not as well correlated with the intellectual status (16,18). This suggests that the neuritic component of the lesion is more directly responsible for the intellectual dysfunction than the Ab deposition, although the latter may have an important pathogenetic role. To clearly distinguish the two components and to understand their interrelation and evaluate their clinical significance, we would recommend the use of two types of methods; one

S38 should be tau-sensitive (tau immunohistochemistry, Gallyas or Bodian technique) and the other Ab-sensitive (Ab immunohistochemistry, modified Bielschowsky, thioflavin S). We would favor the systematic use of immunohistochemistry, the results of which are easier to analyze, at least theoretically. Judge or Count? We would certainly doubt the results of a biochemist who would measure natremia in 1, 11, or 111. This is, however, the precision which neuropathologists may afford now in the study of the brain, certainly a much more complex system than the serum. This comparison points to the necessity of making our diagnostic tools more objective, more precise, and more reproducible. However, several studies (23,51,71) have shown that neuropathologists are still unable to give a truthful quantitative index that would give comparable results in different laboratories. The apparent precision of lesion counts that could be used as thresholds is thus presently illusory, and semiquantitative estimates are more reliable. We have evaluated some of the factors that impede the quantitation of the lesions (23). ASSESSMENT OF THE MAIN SOURCE OF VARIABILITY IN PLAQUE AND TANGLE COUNTS

A reliable quantification of the plaques and of the tangles should give similar results in different laboratories. This is not presently the case. We have assessed the extent of the variability between laboratories. We have then tried to identify and to study the variables which might determine the scatter of the density values measured in the same microscopic samples. Inter-Laboratory Variation: the Eurage Study (23) This study involved 12 European laboratories. The material which was furnished to them came from six cases selected from the Charles Foix Longitudinal Study. They were chosen on clinicopathologic grounds; two patients were severely affected (Blessed test score 2 and 4), two were moderately affected (Blessed test scores 16 and 19), and two were normal (Blessed Test scores 26 and 28). For each case, eight unstained paraffinembedded sections were furnished from two samples, the hippocampus and the superior temporal gyrus (covering Brodmann’s area 22). Each participant was left free to choose his staining method(s), which was supposed to be the technique(s) he or she routinely used. The observers were requested to examine three cortical regions, two in the hippocampus and one in the isocortex. The examiners did not receive any information concerning the choice of the cases and they were asked the following: 1) to give the density (number of lesions per microscopic field and per square millimeter) of senile plaques and of neurofibrillary tangles; 2) to rank the six cases in increasing order of severity, giving an index of 100 to the most severely affected case and an index of 0 to the least affected one (cohort score); 3) to evaluate the severity of the cases, also on a scale of 100 points, but this time with respect to one’s own experience (general score); 4) to give a final guess of the mental status (demented or normal) of the patient when alive. The mental status of the patient was unknown to each participant, with the exception of the participant from Paris. There was nearly one special technique used per laboratory [for precise references see (23)]. The variability in the counts of the plaques and of the tangles was directly proportional to the density of the lesions, low when they were sparse, high when numerous. The density values given for the senile plaques were highly dependent on the staining method used by the investigator. Values obtained with Absensitive techniques were higher than results obtained with tausensitive methods. Counts of the neurofibrillary tangles were

DUYCKAERTS AND HAUW distributed in only one large cluster but large variations were noted from one laboratory to the other, sometimes reaching a factor ten. The semiquantitative assessments showed some concordance, both for the ranking of the cases in increasing order of severity and for the score evaluating the severity of the cases according to one’s own experience. Most of the observers agreed on the most severely affected cases as well as on the normal ones. Variations were large for the cases of intermediate severity. One might note that the highest correlations with clinical data were obtained with the subjective assessments and the lowest correlations with the senile plaque counts. The neuropathologist was asked to formulate a final guess concerning the premortem mental status of the six cases. There was complete agreement for the two most affected cases. Only one observer disagreed for the least affected case. Concordance was poor for the three cases of intermediate severity. Four Sources of Variation Between Measurements To evaluate some of the determinants explaining the poor inter laboratory reliability, we identified and systematically studied four main sources of variation: interstain, interobserver, intraobserver, and interregion. Interstain variation. Interstain variation was studied by using 15 cases and 8 staining methods. The lesions were counted on serial sections by the same observer in the first temporal gyrus (44). Ten contiguous columns, consisting of fields covering the whole thickness of the cortex from the pial surface to the white matter, were examined with a 3 40 objective, i.e., at a magnification of 3 600. Seven silver methods and thioflavin S were compared. Regression lines were obtained between the test score of Blessed (dependent variable) and the counts of lesions with the various staining methods (independent variable). Data have been published on the same series, including additional cases (30 in all) using the same technique after Ab, tau, and ubiquitin immunohistochemistry (14,15,40). A comparison of these regression lines (Fig. 1) showed that methods sensitive to amyloid substance (such as Bielschowsky’s and thioflavin S) revealed a larger number of senile plaques than methods sensitive to neurofibrillary degeneration (such as Bodian’s or Gallyas’). As far as the neurofibrillary tangles were concerned, the Bielschowsky’s technique, which was often considered as a reference, was indeed very sensitive in the least affected cases, but other techniques, such as Cross’, Bodian’s, and Gallyas’, usually revealed more changes in the more advanced ones. The costs of the stains varied greatly (the least expensive being thioflavin S, the most expensive Bielschowsky’s technique). The time spent per slide was also extremely variable, with thioflavin being probably the easiest technique and immunohistochemistry or Bielschowsky the most complex. Interobserver variation. Interobserver variation was assessed in the following way (unpublished observation). Five observers, four experienced neuropathologists, and a postdoctorate student (Observer 4 in Fig. 5) counted senile plaques and neurofibrillary tangles in two regions (delineated on a microscopic slide) from the first temporal gyrus (case 2812 of the Charles Foix longitudinal study; Blessed score 5 2) and in one section without any recommended region from the first temporal gyrus (case 2825 of the Charles Foix longitudinal study; Blessed score 5 9). The standard error of the densities of senile plaques and neurofibrillary tangles evaluated by each observer was calculated by measuring the standard deviation of the number of lesions per microscopic field (objective 3 40) and dividing the result by the square root of the number of examined microscopic fields. Senile plaques. For the two regions identified on the slide (case 1 area 1; and case 1 area 2 of Fig. 5), all the confidence intervals overlapped. For the slide without any recommended examination

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FIG. 6. Coefficient of error in the evaluation of the density of the senile plaques as a function of the number of examined microscopic field. Notice that the coefficient of error is lower in the case with a higher density of senile plaques. In case 1, 34 fields were found necessary to reach a precision of 20%, 141 fields to reach 10%; the precision of 5% could not be reached even after the scanning of more than 600 fields.

FIG. 5. Density of senile plaques (SP) and of neurofibrillary tangles (NFT) as evaluated by five observers on three regions after Bodian stain. The observers are numbered from 1 to 5. The bars represent two standard errors. (Data presented at: Neuropathology of Alzheimer’s disease and other dementias of old age: A reassessment. NIH, Bethesda, MD, April 2–5, 1990).

region, observers 1 and 2 counted significantly less lesions than observers 3 and 5. These discrepancies could be partly accounted for by regional differences in the density of senile plaques. Neurofibrillary tangles. Observer 4 was considered an outlier (case 1 area 1; and case 1 area 2 of Fig. 5). The performances of the five observers were checked, and conventional means of identifying neurofibrillary tangles were reviewed before the examination of case 2. Further results of observer 4 were then within the common range (case 2, Fig. 5). This high concordance between the counts within a given laboratory has already been mentioned (57). Interregion variation. Interregion variation was evaluated by comparing the counts of neurofibrillary tangles and senile plaques obtained by five observers in two regions located on the opposite sides of the first temporal gyrus (case 1 area 1; and case 1 area 2, mentioned above and in Fig. 5). Density of senile plaques did not vary much from one region to another, whereas there were roughly twice as many tangles in area 1 than in area 2, both areas being located on the same slide. This high regional variation appears to be one of the major difficulties in the estimation of tangles density.

Intraobserver variation. Intraobserver variation was evaluated by having one observer (CD) count the lesions four times in the same region. The surface area which had to be assessed in order to obtain a given precision was then determined. The precision of the count was evaluated by the coefficient of error. Columns of fields were systematically examined. After each new field, standard error was calculated anew. The curve describing the change of the cumulative coefficient of error as a function of the number of evaluated fields (69) was then drawn (Fig. 6). It appeared difficult to lower the coefficient of error below 10%; the precision depended on the density of lesions. When they were numerous, as in case 1, then a small number of microscopic fields was needed to obtain good precision. When they were rarer, as in case 2, the precision had to be improved by increasing the number of fields examined. These results indicate that the precision of the count depends on the number of lesions that has been counted rather than on the surface area that has been examined. One may also conclude that after a certain threshold the gain in precision is costly in terms of time (Fig. 6). Relative weight of the sources of variation. Coefficient of variation (standard deviation divided by the mean) was finally used as an index of variation between the measurements. Concerning the senile plaques, coefficient of variation between the laboratories in the Eurage study reached 93%. We have found in the Charles Foix Prospective Study, that the staining method was a major source of variation because interstain coefficient of variation was 51%, interobserver 5 24%, intraobserver 5 8%, and interregion 5 10%. For the neurofibrillary tangles, coefficient of variation between laboratories was 73%. The regional variations were high, 52%, and the interstain variation was low, 11%. Interobserver variation was 17% and intraobserver 8%. PRACTICALLY

The data that we have presented suggest that the evaluation of a case requires at least 2 estimates. An Estimate of the Topography of the Neurofibrillary Pathology in the Hippocampal-Parahippocampal Region The sample should be taken in the anterior part of the hippocampus to include the entorhinal cortex. Braak and Braak staging is a good way of fulfilling this requirement. Tau immuno-

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histochemistry or Bodian technique could be used instead of the Gallyas method that is recommended by Braak and Braak.

the descriptive diagnosis of ‘AD lesions’ is proposed, qualified by Braak and Braak staging.

An Estimate of the Density of Plaque in the Isocortex

IN THE FUTURE

The semiquantitative assessment proposed by the CERAD protocol (52) is presently useful to fulfill this requirement. The slides may be stained by Ab immunohistochemistry, thioflavin S, or the Bielschowsky technique; only results obtained with the same technique are directly comparable. Severe Cases/Cases at Onset The assessment of the density of tangles in the isocortex is useful in the most advanced stages. By contrast, parahippocampal tangles may be the only signs of AD at onset, and Braak and Braak staging may be the most sensitive way of analyzing these early cases. As the intellectual deficit is then moderate or absent, the formal diagnosis of AD is problematic. Formal Diagnosis of AD We presently rely on the following operational and probability rules. When the case is demented, we determine, on the basis of the correlational studies that we have presented, if the density of the lesions explain the severity of the intellectual deficit. If it does, a diagnosis of AD is considered highly probable. Otherwise, additional factors are sought. In their absence, the diagnosis is considered possible. When the case is not demented but lesions are found, we consider that the distinction between preclinical AD and ‘normal aging’ is impossible, except for special circumstances (trisomy 21; familial AD with a known mutation). In these cases,

As already mentioned, we believe that quantitative indices should be sought. The variability of the counts makes their precision presently illusory. One practical way of standardizing the data would be to rely on the clinico-pathological correlations. A series of samples coming from prospectively assessed cases could be used to ‘calibrate’ the data obtained with the staining and the counting procedures specific to a given laboratory. Instead of providing a count, the neuropathologist would then give a conventional ‘expected deficit’ calculated with the coefficient of the regression curve that he himself, with his own techniques, would have to establish between the density of the lesions and the intellectual status. Such a procedure would imply the collection of unstained reference slides that could be used by several laboratories. They would constitute an indirect way of assessing and improving the quality of the estimation performed in the various laboratories. ACKNOWLEDGEMENTS

Numerous coworkers have contributed to this work. Prof. D. He´nin, Dr. D. Seilhean, and M. Verny have helped us to evaluate the interrater variability. Dr. Y. He, P. Delae`re, C. Lamy, T. Uchihara, Y. Grignon, and M. A. Colle have taken part in many aspects of the study. The clinicopathological correlations would have been impossible without the work of Prof. F. Piette. We thank C. Menninger for reviewing the English text, and the help of the technicians of the Raymond Escourolle laboratory is greatly acknowledged.

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