Dr Anthony Rudd
email: anthony.rudd@kcl.ac.uk

Alex Hoffman

Robert Grant

James T Campbell

Derek Lowe

Rudd Ag Frcp

MSc Stroke Programme Manager Hoffman A Lcst

Bsc Dip

Stat Statistician

Llb

Msc C Stat

England, Wales and Northern Ireland. How much do we do and how much do we need?

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Stroke Thrombolysis in England, Wales and Northern

Ireland. How much do we do and how much do we need?

Findings: All relevant hospitals participated, submitting data on 11,262 acute stroke patients. 2118 patients arrived within 2 hours and 2596 within 3 hours of the onset of symptoms and 587 people were already in hospital. Therefore 28% (3183) were potentially eligible for thrombolysis based on a 3 hour time criterion. Of these 1914 were under 80 years and 2632 had infarction with 14% (1605) meeting all three NINDS study criteria and so being potentially eligible for thrombolysis. If the time window is increased to 4.5 hours then only another 2% became eligible. If the age limit was removed for treatment the percentage potentially appropriate for tPA increased to 23% within 3 hours and 26% within 4.5 hours.

Overall 1.4% (160) of patients were thrombolysed.

Introduction

The evidence showing that thrombolysis is beneficial within three hours 1 and possibly 4.5 hours 2 of the onset of ischaemic stroke symptoms is strong with significant reduction in long term disability. The earlier treatment is given the greater the chance of a good outcome.

Thrombolysis for stroke is however a treatment with a high risk of adverse effects, particularly intracerebral haemorrhage with 8.7% incidence of symptomatic intracranial bleeding (haemorrhage causing death or clinical deterioration) [START_REF] Wardlaw | Thrombolysis for acute ischaemic stroke[END_REF] Data from the SITS-MOST register [START_REF] Wahlgren | Thrombolysis with alteplase for acute ischaemic stroke in the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST): an observational study[END_REF] suggests a much lower rate of bleeding (1.7%) but it is probable that there is selection bias in this dataset and more weight should be given to data from rigorous randomised controlled trials. Evidence from centres reporting the introduction of thrombolysis into routine clinical care has highlighted the importance of skilled clinical management in minimising the risk of complications. Cleveland, Ohio reported a 15% symptomatic haemorrhage rate in the first year of practice [START_REF] Katzan | Use of tissue-type plasminogen activator for acute ischaemic stroke: the Cleveland area experience[END_REF] , which resulted from widespread failure to adhere to inclusion and exclusion criteria for treatment. The UK has been slow to adopt thrombolysis compared to some other countries [START_REF] Heuschmann | Frequency of thrombolytic therapy in patients with acute ischaemic stroke and the risk of in-hospital mortality[END_REF][START_REF] Bray | Thrombolysis therapy for acute ischaemic stroke: successful implementation in an Australian tertiary hospital[END_REF][START_REF] Steiner | for the participants in the Austrian Stroke Registry for Acute Stroke Units. The quality of acute stroke units on a nation-wide level: the Austrian Stroke Registry for acute stroke units[END_REF] with less than 0.2% of patients reported to have received the treatment in the 2006 National Sentinel Audit of Stroke 8 . A few centres have developed effective acute care and are thrombolysing a significant proportion of their patients 9. The Department of Health National Stroke Strategy in England 10 has been published which identifies, amongst many other aspects of stroke care, the need to improve public awareness of stroke and the need to develop services that deliver high quality acute care including thrombolysis when appropriate. National Institute for Health and Clinical Excellence (NICE) guidance was published on acute stroke care in 2008 in England [START_REF]NICE Guideline on Diagnosis and Initial Management of Acute Stroke and TIA[END_REF] , following on from a Technology Appraisal in 2007 12 , and it recommends the use of thrombolysis.

It is important that services, currently in the process of major restructuring to deliver effective acute stroke care, know the size of the problem they will have to deal with. This study is one of the first to report national level data covering all hospitals and describing potential requirements for stroke thrombolysis. The aims of this study were:

• To use national audit data to establish the proportion of patients receiving thrombolysis and compare this to the proportion that could benefit.

• To assess the proportion of patients that might be eligible for thrombolysis if treatment was extended from three hours to 4.5 hours and if the treatment was routinely given to patients over 80 years • To assess the effect of the time of day of hospital admittance on the availability of thrombolysis • To establish whether patients receiving thrombolysis are admitted to an adequately equipped acute stroke unit

Methods

The National Sentinel Audit of Stroke has been conducted biannually since 1998 with participation currently from all hospitals treating acute stroke patients in England, Wales and Northern Ireland. The audit tool uses standards based upon best available evidence 13 , and was developed by the Intercollegiate Stroke Working party that includes a wide range of experts from specialties involved in delivering stroke care, as well as user representatives. All hospitals within England, Wales, Northern Ireland and Channel Islands were invited to take part in the sixth round of the National Sentinel Audit of Stroke. The minimum requirements were that they had inpatient services for stroke patients and had admitted at least 20 patients with a primary diagnosis of stroke (ICD10 I61, I63 and I64) between 1 st April and 30 th June 2008. Details of the audit questionnaire are available on www.rcplondon.ac.uk/stroke. The clinical proforma was completed on up to 60 consecutive patients (median 59 per hospital) 1 st April and 30 th June 2008 to assess compliance with evidence-based standards, to define case mix and outcome. Using a web-based tool with in-built validation, very high levels of data completeness and internal consistency were achieved. An inter-rater reliability study was conducted on the first 5 patients, to estimate the agreement in data as collected by a second auditor with access to the same information and this demonstrated good to excellent levels of agreement (median kappa 0.77, IQR 0.67-0.86, n=77 categorical questions).

The data collected covered the whole in-patient stroke pathway but did not cover all the inclusion and exclusion criteria as defined by the NINDS study [START_REF]The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, Tissue plasminogen activator for acute ischaemic stroke[END_REF] (i.e. uncontrolled hypertension, profound premorbid dependency or current use of anticoagulants). However data from the 2006 audit which did record premorbid disability and use of anticoagulation prior to admission suggest the number of patients excluded for reasons other than time since stroke onset (more than 3 and more than 4.5 hours), age over 80 years and haemorrhage on brain imaging is likely to be relatively small. We did not collect the data in the 2008 audit for the number of patients admitted on anticoagulants nor on Barthel prior to admission. Data were cleaned and analysed at the Royal College of Physicians' Clinical Effectiveness and Evaluation Unit, using SPSS version 15 software. Times of admission and thrombolysis were collected to the minute, but many participants rounded times to wider intervals, including the nearest hour in some cases, so we present time to thrombolysis to the nearest hour. This will have only a small impact on overall eligibility for reasons discussed below under "relaxing the NINDS criteria". All data were submitted by individual hospitals without any patient identifiers. Where admission times were not known we assumed that these patients would not have been eligible for tPA. Ethical approval was not required for this audit and individual patient consent was not obtained.

Results

All eligible hospitals (216) treating acute stroke patients participated in the audit and they submitted data on 11,369 patients. Of these 107 were excluded for this paper as they received rehabilitation care only. This left 215 hospitals (England 183, Wales 18, Northern Ireland 11 and Islands 3) and 11262 patients. Some 47% (5306) of patients were male, mean age 72 years, with 53% (5956) female, mean age 79 years. Motor deficits were recorded in 77% (8668), dysphasia in 39% (4425) and dysarthria in 39% (4384). Of the 96% (10849) receiving a brain scan, 87% (9431) had infarction on scan and 13% (1418) haemorrhage. At some point in the first week after the stroke, 13% (1476/11262) of patients were recorded as being unconscious. This was higher for haemorrhagic strokes (30%, 423/1418) than infarctions (8%, 797/9431). 78% (8495/10859) were independent in everyday activities before the stroke.

Eligibility for and receipt of thrombolysis.

The date of stroke was known for 77% (8670) and was estimated by hospitals in 23% (2587), data missing for 5 cases. The time of stroke during the day was known for 58% (5031/8670), while 30% (1075/3639) of those with unknown times occurred during sleep. The time of stroke and the time of admission to hospital were both known for 4980, with 262 of these being inpatients at the time of onset. Stroke and admission times were subject to a certain degree of rounding, particularly to nearest half and full hours, and to accommodate this the time elapsing between stroke and admission was rounded to the nearest hour and was computable for 4600 of the 4718 patients admitted after onset of symptoms. Of these 46% (2118) arrived at the hospital within 2 hours of onset, 56% (2596) within 3 hours, 64% (2944) within 4 hours, and 70% (3223) within 5 hours. There were 587 in-patients at time of stroke and so overall 28% (2596+587=3183/11262) were potentially eligible for thrombolysis based on the 3 hour time criterion assuming that the majority of patients in a hospital bed should expect to have acute stroke recognised within three hours of the onset of symptoms although some would not be eligible for reasons such as recent surgery or co-morbidities. Of these, 1914 (17%) were younger than 80 years and 2632 (23%) had infarction. In all, 14% (1605/11262) met all three criteria and were therefore potentially eligible. Only 10% (160/1605) of eligible patients were thrombolysed and half (80) of these were from just 14 hospitals. Almost all hospitals (209) had at least one eligible patient and 65 of these gave thrombolysis to some eligible patients. Of the 9657 patients who were not strictly eligible under the NINDS criteria, 44 (0.5%) received thrombolysis and 24 of these were aged over 80 years, 10 were admitted within 4.5 hours and none were inpatients at the time of stroke.

Hospital variation in eligibility for and receipt of thrombolysis

The percentage of patients available for thrombolysis within 3 hours of symptoms varied between hospitals, with median 28% and IQR 21-35%. The percentage of patients meeting all three NINDS criteria varied with median 13% and IQR 9-18%. Only 31% (65/209) of hospitals with eligible patients used thrombolysis, and these 65 hospitals treated a median of 20%, IQR 14-33% of eligible patients. Hospitals with larger numbers of eligible patients were more likely to treat (Spearman r=0.28, p<0.001) (Table 1).

Relaxing the NINDS criteria

Overall 32% (3558) were admitted within a 4.5 hour window and 35% (3960) within 6 hours, including inpatients at onset. Removing the age restriction increased potential eligibility for patients with infarction from 14% to 23% (2632) for the 3 hour window, to 26% (2951) within 4.5 hours and 29% (3294) within 6 hours. Maintaining the age restriction but extending the time window to 4.5 hours increased eligibility from 14% to 16% (1811).

Time of admission to hospital

Time of admission was known for 10538 (94%) stroke patients who were admitted to hospital. Two-thirds of these, 67% (7070/10538), were admitted between 8am and 6pm of whom 13% (903) were eligible under NINDS, while 33% (3468/10538) were admitted between 7pm -7am of whom 13% (446) were eligible. Significantly more eligible patients received thrombolysis during 8am-6pm than outside these hours (14% (126/903) Vs 5% (24/446), p<0.001). Of 587 inpatients at the time of stroke 44% (256) were eligible, of whom 3.9% (10) were treated. Figure 1 shows the variation in time of day for the 10538 admitted patients and within this the variation in numbers eligible for and treated by thrombolysis can be seen.

Gender

Whereas 18% (937/5306) of men were eligible using NINDS criteria, only 11% (668/5956) of women were eligible. Female patients were however older than male patients and if the NINDS age restriction is relaxed then 24% (1277/5306) of men would be eligible and 23% (1355/5956) of women. Rates overall for males and females entering hospital within 3 hours of onset, including existing inpatients, were similar at 29% (1525/5306) and 28% (1658/5956) respectively and rates for infarction on brain scanning were also very similar at 87% (4914/5667) for women and 87% (4517/5182) for men. A lower proportion of eligible women (9%, 61/668) received thrombolysis than eligible men (11%, 99/937) but this was not statistically significant (p=0.35)

Case mix and admission characteristics

Variables applying at onset or at admission or soon after are summarised in Table 2.

Eligibility rates did not display much variation between patient subgroups apart from existing inpatients (44%). Eligibility variation overall is explainable by the associations between these variables and the factors (admission <3 hours, brain scan infarction, age 80 years or under) determining the NINDS criteria. The overall treatment rate for eligible patients was 10% and there was notable variation (Table 1) in relation to age (under 65's 13% Vs 65-80's 9%), in being capable of independent living before the stroke (Yes 11% Vs No 4%), prior comorbidities (None 18% Vs one or more 8%), use of lipid-lowering treatment prior to stroke (Yes 8%, No 12%), having an ambulance patient record on file and FAST test available (Yes 21% Vs No 7%), being admitted during working hours (8am-6pm 14% Vs 7pm-7am 5% Vs existing inpatient 4%), and having dysphasia, dysarthria and motor deficits during the first 24 hours (None 1% Vs One 4% Vs Two/three 14%). The worst level of consciousness at time of maximum severity in the first week after stroke did not predict thrombolysis use, nor did current smoking, nor alcohol excess.

Acute stroke units and ambulance records

Within 4 hours of admission to hospital 69% (111/160) of eligible and thrombolysed patients were admitted to an acute or combined stroke unit. This compares with 19% (275/1445) of those eligible but not thrombolysed. Five characteristics were used in the audit to identify quality of care in acute or combined stroke units. These were continuous physiological monitoring (ECG, oximetry, blood pressure), access to scanning within 3 hours of admission, direct admission from A&E, specialist ward rounds at least 5 times a week and presence of acute stroke protocols/guidelines. Some 111 of 191 sites with acute or combined stroke units possessed only 4 or fewer of these characteristics and 17 of these 111 carried out thrombolysis on eligible patients in this audit. These 17 sites had 145 eligible patients and thrombolysed 21% (31) of them. Therefore 19% (i.e. 31/160) of all eligible and thrombolysed patients were thrombolysed in centres without fully equipped acute specialist stroke beds.

Outcomes

Inpatient mortality was 12% (19/160) for eligible treated patients and 14% (208/1445) for those not treated (14%, 208/1445). Thirty-day mortality was 12% (19/156) Vs 12% (169/1386) respectively excluding unknown outcomes. New institutionalisation rates for eligible and discharged patients were 4% (6/137) and 7% (81/1196) respectively. Length of stay was known for 1595 eligible patients and median (IQR) stays for treated and untreated patients were 9 (4-24) days and 12 (5-31) days respectively. The numbers are too small to meaningfully stratify by further organisational or clinical features.

Discussion

A number of centres have reported their local stroke thrombolysis rates, ranging from 1.6% to 18% [START_REF] Reed | Treatment with tissue plasminogen activator and inpatient mortality rates for patients with ischaemic stroke treated in community hospitals[END_REF][START_REF] Johnston | Utilization of intravenous tissue-type plasminogen activator for ischaemic stroke at academic medical centres; the influence of ethnicity[END_REF][START_REF] Barber | Why are stroke patients excluded from TPA therapy? An analysis of patient eligibility[END_REF][START_REF] Demaerschalk | Development of a metropolitan matrix of primary stroke centres: The Phoenix experience[END_REF] . Several authors have attempted to model the number of potential patients suitable for thrombolysis from hospital and population based registers, again with large differences between studies ranging from 0.4% [START_REF] Jørgensen | Predicted impact of intravenous thrombolysis on prognosis of general population of stroke patients: simulation model[END_REF] of patients benefiting from treatment to, 8% in Cincinnati [START_REF] Kleindorfer | Eligibility for recombinant tissue plasminogen activator in acute ischaemic stroke: a population based study[END_REF] and 24% [START_REF] Boode | Estimating the number of stroke patients eligible for thrombolytic therapy if delay could be avoided[END_REF] in the Netherlands suitable to be treated.

This study however provides one of the first estimates at a national level of the provision of and need for thrombolysis services. It shows that about 14% of patients admitted to hospital with acute stroke are likely to be eligible for thrombolysis, and that only 10% of these patients actually receive it (1.4% of total stroke population). Provision of a daytime service alone without access to night-time acute stroke care will result in a significant number of patients being denied best quality care. 24 hour services are therefore essential. These data show that a major step change in stroke care is feasible by improved delivery of care.

Provision of high quality acute stroke services, accessible to the whole population rather than the current patchy delivery of care, should be able to increase the number of patients thrombolysed ten-fold. The most important actions, within current European licensing, to increase the proportion of patients eligible for treatment, will be to reduce the delay between onset of symptoms and arrival in hospital and to ensure that all hospitals treating acute stroke patients whould have the skills and resources to offer thrombolysis at all times. This will require both increased public awareness and better professional management in the prehospital phase. It has been shown that where the Face, Arm Speech Test (FAST) [START_REF] Ford | Agreement between ambulance paramedic-and physician-recorded neurological signs with Face Arm Speech Test (FAST) in acute stroke patients[END_REF] is being used patients are more likely to be thrombolysed, but this may simply reflect services organising themselves to deliver care effectively. An Australian randomised controlled trial has shown that a pre-hospital protocol increases access to treatment [START_REF] Quain | Improving access to acute stroke therapies: a controlled trial of organised pre-hospital and emergency care[END_REF] and this has also been demonstrated in the US [START_REF] Wojner-Alexandrov | Houston paramedic and emergency stroke treatment and outcomes study (HoPSTO)[END_REF] . Improving public awareness of the symptoms of stroke and how to respond when they happen is being addressed in England with a major advertising campaign funded by the Department of Health and ongoing awareness raising by the Stroke Association, though the efficacy of such campaigns remains unproven. A recent study reported a significant increase in the proportion of patients thrombolysed admitted to academic medical centres in the US between 2001 and 2004 from 14% of those admitted within two hours of symptom onset to 37.5% but with no significant increase in the proportion arriving within 2 hours (37 to 38%) [START_REF] Lichtman | Hospital Arrival Time and Intravenous t-PA Use in US Academic Medical Centers[END_REF] .

Offering tPA up to 4.5 hours is only going to marginally increase the rates of thrombolysis and because of the high number needed to treat (NNT) between 3 and 4.5 hours 2 this change in practice will have a small impact on outcomes at a population level. The message that treatment is still available up to 4.5 hours may actually make outcomes worse if there is a mistaken belief that the patients and professionals can afford to take their time and therefore not treat each case as an emergency. Around 40% of patients who are in hospital within 3 hours of stroke are excluded from thrombolysis when delivered according to the European license because they are over the age of 80 years. The trial currently being funded by the MRC (IST 3) evaluating among other things whether the drug can be given safely and effectively to older patients is therefore of major importance and if shown would have the biggest impact on the proportion of patients eligible for thrombolysis. If the ongoing trials of treating patients with mismatch on diffusion/perfusion scanning show positive results then again a significant increase in the number of patients eligible for treatment would be expected as some of the patients with 'wake up' strokes and those with unknown time of onset could be treated. A recent review of these trials shows that reperfusion is increased. However, delayed thrombolysis in mismatch patients was not confirmed to improve clinical outcome, although a useful clinical benefit remains possible. However the authors conclude that until a further phase 3 study is completed, 'delayed treatment, even according to mismatch selection, cannot be recommended as part of routine care' [START_REF] Mishra | Mismatch-based delayed thrombolysis: a meta-analysis[END_REF] .

These data show marked differences between centres in the proportion of patients meeting the NINDS eligibility criteria but this is consistent with random variation between hospitals with relatively small sample sizes. That these data do not show significant differences in outcome between those treated and not treated with thrombolysis is likely to reflect the very small numbers of patients that were treated. This is observational data not a randomised controlled trial and differences may also reflect any local level selection biases for thrombolysis in eligible patients due to their clinical condition . The Cochrane review [START_REF] Wardlaw | Thrombolysis for acute ischaemic stroke[END_REF] shows that treatment is worthwhile and the SITS-MOST audit [START_REF] Wahlgren | Thrombolysis with alteplase for acute ischaemic stroke in the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST): an observational study[END_REF] suggests that the treatment can be delivered safely in routine clinical practice although it must be acknowledged that this database may not represent a complete record of all thrombolysis treatments given and may therefore have some bias. What we have shown that is very concerning is that some centres appear to be delivering thrombolysis without the highest quality stroke service in place to support the treatment. Thrombolysis is only one relatively small component of acute stroke care and should only be provided when a high quality environment is established. NICE guidance [START_REF]NICE Guideline on Diagnosis and Initial Management of Acute Stroke and TIA[END_REF]12 states that Alteplase should only be administered within a well organised stroke service with staff trained in delivering thrombolysis and in monitoring for any associated complications, care up to level 1 and level 2 nursing staff trained in acute stroke and thrombolysis immediate access to imaging. Additionally many centres providing thrombolysis are only treating very small numbers of patients. Data from the German Stroke Registers Study Group [START_REF] Heuschmann | Predictors of in-hospital mortality in patients with acute ischaemic stroke treated with thrombolytic therapy[END_REF] have shown that outcomes are better from units performing at least 15 procedures per year. This would suggest that reorganisation of care should concentrate acute stroke care in a smaller number of larger specialist centres. The Stroke Organisational audit in 2009 [START_REF]Intercollegiate Stroke Working Party. National Sentinel Audit of Stroke Organisation of Stroke Care in England[END_REF] showed that, of 210 sites providing acute care, 138 (66%) claimed to have thrombolysis available on site, but only 47 (22% of all acute sites) had administered the treatment to 15 or more patients in the previous year. These numbers are something of an improvement on the organisational audit in 2008 [START_REF]Intercollegiate Stroke Working Party. National Sentinel Audit of Stroke Organisation of Stroke Care in England[END_REF] which showed that, of 213 sites providing acute care, 91 (43%) claimed to have thrombolysis available on site, but only 22 (10% of all acute sites) had administered treatment to 15 or more patients in the previous year. One third of patients 446/1605=28% eligible for thrombolysis were admitted outside normal working hours (8am through 6 pm) and these data show that 24 hour, seven day a week acute stroke care is essential.

The strengths of this study are that it involves all acute stroke providers in England, Wales and Northern Ireland and uses a well established and accepted methodology [START_REF] Rudd | Stroke Units: Research and Reality. Results from the National Sentinel Audit of Stroke[END_REF] that has been used by local hospitals, strategic health authorities and government to develop services. The potential weaknesses are that the number of patients included from each centre is relatively small and the total number of patients treated with Alteplase within the sample is very small. Data are self reported and may therefore be open to bias, both through patient selection and inaccuracy in reporting. We do not believe that this is a major factor particularly for the data items used in this analysis given the good inter-rater reliability. Data were not collected that enabled complete certainty about the proportion of patients that were eligible for thrombolysis but the number in clinical practice that are excluded because of these factors such as hypertension, recent bleeding and use of anticoagulants are relatively small. In the 2006 audit, 7% of patients were admitted on warfarin, 78% were independent prior to admission (Barthel 19 or 20 or a Rankin of <3) with only 7% (from the 2001-2002 audit) being so severely impaired prior to the stroke as to preclude the use of thrombolysis with a Barthel of less than 10. There is also some uncertainty about the time from onset to admission due to rounding of times to the nearest hour, and there are some patients for whom the admission times were not known. Thrombolysis is an important component of stroke care that is currently applicable to about 14% of the stroke population. In England, Wales and Northern Ireland only 10% of eligible patients are currently receiving the treatment.

Extending the thrombolysis time window from 3 to 4.5 hours will have a minimal effect on the number of patients treated. The major factors that might increase the number will be if it can be shown that the drug can be given safely to patients over the age of 80 years and public awareness increases so that a greater proportion of patients arrive within the hospital in time to be treated.

Figure 1 .

 1 Figure 1. Time of day patients admitted to hospital for whole cohort divided by eligibility for, and provision of, thrombolysis (n=10538 admitted)

Table 1 .

 1 Receipt of thrombolysis according to the number of eligible patients

		800		Eligible for / given thrombolysis
				Not eligible
		700		Eligible but not given
				Eligible and given
		600
	Number of patients	500 400 300
		200
		100
		0
		0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23

Table 2 :

 2 Case-mix and admission characteristics in relation to eligibility for, and if eligible the provision of, thrombolysis (n=11262 unless stated otherwise) Fisher's exact test was used, except for where Mann-Whitney test was used with age in years and where chi-squared test was used for worst level of consciousness, N of co-morbidiy groups and N of known symptoms limiting physical function.

		Eligible		Treated	P-value for
	%	Patients	%	Patients	treatment*

Acknowledgements

We are grateful to the Healthcare Commission and HQIP for funding the National Sentinel Audit of Stroke and to all the people who completed the data in all the hospitals in England, Wales and Northern Ireland.


Toni T Seppã¤l㤠

Sanna-Kaisa Herukka

Tuomo Hã¤nninen

MPsych Susanna Tervo

MD, PhD Merja Hallikainen

MD Hilkka Soininen

Tuula Pirttil㤠

Seppälä Tt

Professor Tuula Pirttilä
email: tuula.pirttila@kuh.fi

Toni Seppälä

BM, MSc, PhD B M Sanna-Kaisa Herukka

MPsych, PhD Tuomo Hänninen

Plasma Aβ42 and Aβ40 as markers of cognitive change in follow-up -a prospective, longitudinal, population-based cohort study

Keywords: Alzheimer, amyloid, plasma, dementia, mild cognitive impairment

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Alzheimer's disease (AD) is the most common cause of dementia. At present, its diagnosis is based on clinical criteria and the exclusion of other causes. However, objective biomarkers for the early diagnosis and monitoring of the disease process are clearly needed because symptomatic treatments are available and disease-modifying drugs are already in phase III trials. The presence of amyloid β deposition in senile plaques is one pathological hallmark of Alzheimer's disease (AD) together with neurofibrillary tangles. [START_REF] Selkoe | Alzheimer's disease: a central role for amyloid[END_REF] The amyloid β is a peptide secreted by neurons [START_REF] Selkoe | Alzheimer's disease: a central role for amyloid[END_REF] and platelets [START_REF] Chen | Platelets are the primary source of amyloid beta-peptide in human blood[END_REF], derived from amyloid precursor protein APP via the activity of proteases β and γ secretase. [START_REF] Selkoe | Alzheimer's disease: genes, proteins, and therapy[END_REF] Most of the Aβ deposited in the brain is constituted of 42 amino acids (Aβ42) form. [START_REF] Gravina | Amyloid beta Protein (Abeta) in Alzheimer's Disease Brain[END_REF] Aβ42 has also been shown to be first amyloid form to accumulate with Aβ40 being deposited later in the process of AD pathogenesis. [START_REF] Iwatsubo | Amyloid beta protein (A beta) deposition: A beta 42(43) precedes A beta 40 in Down syndrome[END_REF] The level of Aβ42 in cerebrospinal fluid (CSF) is reduced in patients with mild cognitive impairment (MCI) and AD [START_REF] Blennow | CSF markers for incipient Alzheimer's disease[END_REF][START_REF] Herukka | CSF Abeta42, Tau and phosphorylated Tau, APOE epsilon 4 allele and MCI type in progressive MCI[END_REF] and a combination test of CSF Aβ42 and tau or phospho-tau has been claimed to be helpful in the early diagnosis of AD. [START_REF] Dubois | Research criteria for the diagnosis of Alzheimer's disease: revising the NINCDS-ADRDA criteria[END_REF] Aβ is present in plasma but it is still unknown whether it originates from peripheral sources or from the brain. In Tg2576 transgenic mouse, plasma Aβ levels decline in parallel with their increasing accumulation in the brain. [START_REF] Kawarabayashi | Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease[END_REF] Since Aβ can be transported bidirectionally across the blood-brain-barrier, it has been hypothesized that there may be an equilibrium between CSF and plasma pools of Aβ. [START_REF] Ghersi-Egea | Fate of cerebrospinal fluid-borne amyloid beta-peptide: rapid clearance into blood and appreciable accumulation by cerebral arteries[END_REF][START_REF] Shibata | Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier[END_REF] Seeing that it is well established that CSF Aβ42 levels decrease in conjunction with the cognitive decline, it has been postulated that plasma Aβ42 may decrease similarly. [START_REF] Graff-Radford | Association of Low Plasma Abeta42/Abeta40 Ratios With Increased Imminent Risk for Mild Cognitive Impairment and Alzheimer Disease[END_REF] If so, plasma Aβ would offer a straightforward, non-invasive and economical biomarker for AD. However, patients with known mutations in chromosome 21 causing early-onset familial AD as well as patients with trisomy 21 have increased plasma Aβ42 levels which are detectable before the onset of the symptoms of dementia. [START_REF] Scheuner | Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease[END_REF][START_REF] Schupf | Elevated plasma beta-amyloid peptide Abeta(42) levels, incident dementia, and mortality in Down syndrome[END_REF] Also the first-degree relatives of late-onset AD patients exhibit elevated Aβ levels measured in plasma. [START_REF] Ertekin-Taner | Plasma amyloid B protein is elevated in late-onset Alzheimer disease families[END_REF] Previous studies have suggested that the levels of plasma Aβ40 are increased before the onset of sporadic AD. [START_REF] Mehta | Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease[END_REF][START_REF] Van Oijen | Plasma Aβ1-40 and Aβ1-42 and the risk of dementia: a prospective case-cohort study[END_REF] However, one recent study concluded that low plasma Aβ40 level predicted AD in elderly men. [START_REF] Sundelof | Plasma beta amyloid and the risk of Alzheimer disease and dementia in elderly men: a prospective, population-based cohort study[END_REF] Other studies have found elevated Aβ42 levels in patients who later develop dementia [START_REF] Mayeux | Plasma A[beta]40 and A[beta]42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF], particularly in MCI amnestic type. (aMCI) [START_REF] Sobow | Plasma levels of alpha beta peptides are altered in amnestic mild cognitive impairment but not in sporadic Alzheimer's disease[END_REF] Finally, several studies have not been able to detect any significant difference of Aβ levels between AD converters and cognitively stable controls. [START_REF] Tamaoka | Amyloid beta protein in plasma from patients with sporadic Alzheimer's disease[END_REF][START_REF] Vanderstichele | Standardization of measurement of beta-amyloid(1-42) in cerebrospinal fluid and plasma[END_REF] It seems that a single measurement of plasma Aβ is not useful whereas the change in plasma Aβ levels observed in repeated testing may be of help in the prediction and evaluation of progression of incipient AD or MCI. [START_REF] Irizarry | Biomarkers of Alzheimer disease in plasma[END_REF] However, only a limited number of longitudinal studies have been performed. [START_REF] Graff-Radford | Analysis of plasma Aβ42 as a premorbid biomarker for late onset Alzheimer's disease[END_REF][START_REF] Blasko | Conversion from cognitive health to mild cognitive impairment and Alzheimer's disease: prediction by plasma amyloid beta 42, medial temporal lobe atrophy and homocysteine[END_REF][START_REF] Schupf | Peripheral Abeta subspecies as risk biomarkers of Alzheimer's disease[END_REF] Our aim was to examine whether the change in plasma Aβ levels during follow-up would be more predictive of cognitive decline than straightforward baseline plasma Aβ levels in a population-based cohort of MCI and cognitively intact controls.

SUBJECTS AND METHODS

Subjects

Study subjects were participants in the population-based study (n=806, aged 60-76 years) examining the risk factors and predictors of dementia in the elderly [START_REF] Hanninen | Prevalence of mild cognitive impairment: a population-based study in elderly subjects[END_REF][START_REF] Tervo | Incidence and risk factors for mild cognitive impairment: a population-based three-year follow-up study of cognitively healthy elderly subjects[END_REF] (Table 1). At baseline (years 1997-1998), 52 subjects were cognitively impaired (CDR 0.5 n=51 and CDR 1 n=1). Group 1 of this study included all of the cognitively impaired subjects from the original cohort who provided a plasma sample (n=52). For each of them, we randomly selected 4 -5 cognitively unimpaired (n=217) age-and sex-matched controls from the same cohort. These subjects were clinically re-evaluated after three (n=197) and six years (n=60).

The longitudinal marker group (group 2) included 70 subjects of the original cohort of 269 subjects who provided a 3 year follow-up plasma sample and 43 of them a 6 year followup sample. The group 2 included 11 cognitively impaired (at baseline), non-demented subjects (CDR 0.5) who provided at least one follow-up plasma sample and 59 cognitively intact (at baseline) age-and sex-matched controls with at least one follow-up plasma sample (Figure 1). Drop-outs occurred mainly due to refusal of the participants to continue the study. Written informed consent was obtained from all the subjects and the study was approved by the local Ethical Committee.

Table 1

The baseline demographic information of the subjects.

Population

Clinical evaluation

The evaluation included a structured detailed interview including demographic information, medical history, medication, smoking habits and alcohol consumption, and a subjective assessment of memory disturbances and depression. The evaluation also included clinical examination as well as an assessment of cognitive impairment by applying the Clinical Dementia rating (CDR) and using a battery of neuropsychological tests. Memory: Visual Reproduction Test from Wechsler Memory Scale [START_REF] Russel | A multiple scoring method for the assessment of complex memory functions[END_REF], Word List Recall from the CERAD Neuropsychological Assessment Battery [START_REF] Morris | The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part I. Clinical and neuropsychological assessment of Alzheimer's disease[END_REF], Logical Memory Test from Wechsler Memory Scale-Revised [START_REF] Wechsler | Wechsler Memory scale -Revised manual[END_REF], NYU Paragraph Recall [START_REF] Kluger | Neuropsychological prediction of decline to dementia in nondemented elderly[END_REF], Delayed Recall of the Constructional Praxis from CERAD [START_REF] Morris | The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part I. Clinical and neuropsychological assessment of Alzheimer's disease[END_REF]; Language: vocabulary subtest from the Wechsler Adult Intelligence Scale-Revised [START_REF] Wechsler | Wechsler Adult Intelligence Scale -Revise[END_REF], Abbreviated (15 items) Boston Naming Test [START_REF] Kaplan | The Boston naming test[END_REF];

Attention and executive function: Trail Making Test [START_REF] Reitan | Validity of the Trail Making test as an indicator of organic brain damage[END_REF] parts A and B, Verbal Fluency Test [START_REF] Borkowski | Word fluency and brain damage[END_REF][START_REF] Butters | Episodic and semantic memory: a comparison of amnesic and demented patients[END_REF]; Visuospatial skills: Block Design from the WAIS-R [START_REF] Wechsler | Wechsler Adult Intelligence Scale -Revise[END_REF], Constructional Praxis from CERAD [START_REF] Morris | The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part I. Clinical and neuropsychological assessment of Alzheimer's disease[END_REF]; Global functioning: Mini-Mental State Examination [START_REF] Folstein | Mini-mental state". A practical method for grading the cognitive state of patients for the clinician[END_REF] (MMSE) Clock Drawing Test. [START_REF] Morris | The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part I. Clinical and neuropsychological assessment of Alzheimer's disease[END_REF].

Cognitive decline was defined by the CDR change from 0 to 0.5 or 0.5 to 1.

Measurement of Aβ40 and Aβ42

The A venous blood sample was obtained into heparine tubes and plasma was separated using the standard methods. The samples were aliquotted and stored in polypropylene tubes at -70°C until analyses. Aβ40 was measured by the ELISA method modified from a well established method. [START_REF] Mehta | Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease[END_REF] The capture antibody was 6E10 (Sigma, Saint Louis, MO) and the detection antibody was a biotin labelled G2-10 antibody (The Genetics Company, Schlieren, Switzerland). The synthetic Aβ 1-40 peptide (Bachem, Bubendorf, Switzerland) was used as the standard. Before the analyses, 0.05% Tween 20 -0.5% BSA was added to the samples. Aβ42 was measured by a high sensitivity method of a commercially available ELISA (Innogenetics, Gent, Belgium) which we modified to be suitable for the measurements of more than 7 pg/ml concentrations. Before the analyses, 0.5M guanidine chloride was added to the standards and the samples. The detergents were used to avoid coagulation of samples and to release Aβ peptides from plasma proteins.

In longitudinal analysis, baseline and follow-up samples from one individual were placed on the same plate to prevent inter-assay variation. Thus, we measured baseline samples The inter-assay variation for Aβ40 assay was 23.8 % and for Aβ42 assay 19.1 %. The inter-CVs were measured by using reference samples of medium concentration (∼250 pg/ml for Aβ40 and ∼400 pg/ml for Aβ42). The intra-assay variations for Aβ40 were 0,71 % for high (∼ 1200 pg/ml), 0,95 % for medium and 5,9 % for low concentrations (∼150 pg/ml).

The intra-assay CVs (median) for Aβ42 were 1,6 % (∼1000 pg/ml), 2,5 % and 9,8 % (∼15 pg/ml), respectively.

APOE genotyping

The APOE allele genotyping was done by a PCR based method. [START_REF] Tsukamoto | Determination by PCR-RFLP of apo E genotype in a Japanese population[END_REF] The subjects were subdivided into the APOE ε4 negative and APOE ε4 positive subjects.

Statistics

The statistical analyses were conducted by the SPSS for Windows release 14.0. Excel to analyse the alteration trend of Aβ levels.

RESULTS

Table 1 presents the demographic information about the subjects. The baseline Aβ40 and

Aβ42 levels of 269 individuals were generally low although some subjects exhibited extremely high Aβ42 levels. The limit for the 90 th percentile was 101 pg/ml but the highest measured level was 1341 pg/ml. The Aβ40 levels showed a weak correlation to age (r = 0.186, p = 0.002) but this was not the case with the Aβ42 levels. There were no differences in Aβ40 and Aβ42 levels between the sexes or between the APOE ε4 carriers and non-carriers. Aβ42 levels did not correlate with Aβ40 concentrations.

Baseline Aβ levels and cognitive decline during the follow-up

No significant differences were found in plasma Aβ40, Aβ42 or the Aβ42/Aβ40 ratio between cognitively impaired (n=52) and cognitively intact subjects (n=217) at baseline.

However, 197 of these subjects were clinically assessed after three years and 60 were clinically assessed after six years. The baseline Aβ42 levels were significantly lower in the subjects who showed cognitive decline after three years of follow-up (cognitively stable, n=147: 19 pg/ml (0-1341), cognitive decline, n= 50: 12 pg/ml (0-276), p = 0.001). Baseline Aβ42 levels were also lower in subjects who had declined cognitively after six years (10 pg/ml, n=36) compared to those who remained cognitively stable (18 pg/ml, n=24), p=0.013.. Subjects who had baseline Aβ42 levels in the lowest quartile displayed an OR of 3.12 (95% CI 1.25 -7.79, p=0.015) for cognitive decline after three years and 4.77 (95% CI

Relationship between changing plasma Aβ levels and cognitive decline

The follow-up plasma samples were available from 70 subjects after three years and 43 subjects after six years of follow-up. The median levels of Aβ42 did not change or decreased in subjects with cognitive decline (n=27 after three years and n=14 after six years) whereas they increased in those who remained cognitively stable (Table 2). No statistically significant changes were found in Aβ40 levels. The Aβ42/Aβ40 ratio decreased significantly in the subjects who experienced a cognitive decline.

A trend analysis was undertaken by calculating a slope for each subject and this was used to assess the change of Aβ42 between cognitively stable and cognitively declining subjects. During the follow-up of 3 to 6 years, the cognition of 28 out of the total 70 subjects had declined and there was a decreasing trend of the level of Aβ42 in 24 subjects during the follow-up. The Aβ42 level remained the same in the declining subjects and increased in the cognitively stable subjects (0.0 (4.9) pg/ml/year and 2.1 (21) pg/ml/year; p=0.009 for slope difference). The corresponding changes of Aβ42/Aβ40 ratio were also significant (0.0 (0.029) per year and 0.0036 (0.055) per year; p=0.02 for slope difference).

Plasma Aβ and general health

Table 3 shows the relationship between medication as well as certain diseases on the plasma Aβ levels. The Aβ40 levels were not associated with the use of lipid lowering drugs or NSAIDs at baseline. Hormone replacement therapy was not related to Aβ values in women. The Aβ40 levels were higher in those subjects using ASA (n = 62, p = 0.004) or dipyridamole (n = 12, p = 0.017). The Aβ40 values were lowest in subjects using neither of the drugs, intermediate in subjects using either ASA or dipyridamole and highest in the subjects taking both drugs. The Aβ40 levels were also higher in subjects using insulin alone (n = 5, p = 0.009) or insulin in combination with oral antidiabetic drugs (n = 13, p = 0.003). The Aβ42 levels were not associated with use of any of the drugs. CHD was associated with a high plasma Aβ40 level (p=0.035). There was no statistically significant difference in medication use between the cognitively stable and the cognitive decliners.

DISCUSSION

Our results indicated that low or decreasing plasma Aβ42 levels and Aβ42/Aβ40 ratio were related with cognitive decline during the follow-up. Moreover, possible age-related increase in plasma Aβ42 levels in serial measurements was associated with stable cognitive performance.

We found no differences in plasma Aβ levels between cognitively intact and impaired subjects in the cross-sectional analysis at baseline. Previous cross-sectional studies comparing plasma Aβ levels in patients with sporadic AD and controls have reported contradictory results. Elevations of plasma Aβ40 [START_REF] Mehta | Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease[END_REF] or Aβ42 [START_REF] Mayeux | Plasma A[beta]40 and A[beta]42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF] have been described

whereas other studies have found no differences between AD and controls. [START_REF] Tamaoka | Amyloid beta protein in plasma from patients with sporadic Alzheimer's disease[END_REF] One recent study found increased levels of plasma Aβ42 in women with amnestic MCI compared to healthy controls or affected men. [START_REF] Assini | Plasma levels of amyloid beta-protein 42 are increased in women with mild cognitive impairment[END_REF] However, low Aβ42 levels at baseline were associated with a cognitive decline occurring during the follow-up. In line with our results, a recent study detected an association between a low Aβ42/Aβ40 ratio and cognitive decline. [START_REF] Graff-Radford | Association of Low Plasma Abeta42/Abeta40 Ratios With Increased Imminent Risk for Mild Cognitive Impairment and Alzheimer Disease[END_REF] Respectively, a prospective three-city study of 257 dementia patients found an association of high Aβ42/Aβ40 ratio with a lower risk of dementia in follow-up. [START_REF] Lambert | Association of plasma amyloid beta with risk of dementia: the prospective Three-City Study[END_REF] Another population-based case-cohort study claimed that individuals with a combination of low Aβ42 and high Aβ40 measured from plasma at baseline had an over 10-fold risk of dementia, but found no association between the Aβ42 or Aβ40 levels alone with cognitive decline. [START_REF] Van Oijen | Plasma Aβ1-40 and Aβ1-42 and the risk of dementia: a prospective case-cohort study[END_REF] Other studies have reported different results. The VITA study found no association between baseline Aβ levels and cognitive decline during the followup. Other studies have found elevated concentrations of Aβ42 at the baseline in subjects who developed AD during the follow-up [START_REF] Mayeux | Plasma A[beta]40 and A[beta]42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF][START_REF] Lopez | Plasma amyloid levels and the risk of AD in normal subjects in the Cardiovascular Health Study[END_REF] although plasma Aβ levels were not associated with AD in the fully adjusted multivariate model. [START_REF] Lopez | Plasma amyloid levels and the risk of AD in normal subjects in the Cardiovascular Health Study[END_REF] Differences in study cohorts, for example timing with respect to cognitive decline, assessment of cognitive functioning (different tests) and presence of confounding factors such as medication and other diseases, are all factors that can influence the results. The outcome in some studies has been conversion to dementia [START_REF] Van Oijen | Plasma Aβ1-40 and Aβ1-42 and the risk of dementia: a prospective case-cohort study[END_REF][START_REF] Mayeux | Plasma A[beta]40 and A[beta]42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF] whereas the outcome in our study as well as in some other studies [START_REF] Graff-Radford | Association of Low Plasma Abeta42/Abeta40 Ratios With Increased Imminent Risk for Mild Cognitive Impairment and Alzheimer Disease[END_REF] was cognitive decline. The difference in the selected outcome and the timing of the sample collection may partially explain differences in the results.

Single measurement of plasma Aβ may not be a suitable marker for AD due to many confounding factors. The timing of the Aβ measurement in terms of the natural history of AD may be critical. Experimental studies on transgenic animals suggest that plasma Aβ levels decrease at the time when accumulation of Aβ begins in the brains. [START_REF] Kawarabayashi | Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease[END_REF] It is possible that the increased plasma Aβ concentration is related to the development of AD as suggested by the findings of elevated plasma Aβ levels in AD gene mutation carriers [START_REF] Citron | Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice[END_REF] and in the first degree relatives of the patients with late onset AD. [START_REF] Ertekin-Taner | Plasma amyloid B protein is elevated in late-onset Alzheimer disease families[END_REF] However, since amyloid pathology in the brain begins years before the appearance of the first symptoms, the possible increase of plasma Aβ may not be detected in the symptomatic individuals.

This hypothesis can only be addressed in longitudinal studies. In line with our results, previous studies have suggested that decreasing Aβ levels are associated with cognitive decline [START_REF] Mayeux | Plasma A[beta]40 and A[beta]42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF][START_REF] Graff-Radford | Analysis of plasma Aβ42 as a premorbid biomarker for late onset Alzheimer's disease[END_REF] and Aβ42 levels were lower in patients diagnosed having AD than in those with MCI. [START_REF] Pesaresi | Plasma levels of beta-amyloid (1-42) in Alzheimer's disease and mild cognitive impairment[END_REF] Many studies have shown that plasma Aβ levels increase with age, as was found in the cognitively intact subjects in our study. [START_REF] Mayeux | Plasma A[beta]40 and A[beta]42 and Alzheimer's disease: relation to age, mortality, and risk[END_REF][START_REF] Blasko | Conversion from cognitive health to mild cognitive impairment and Alzheimer's disease: prediction by plasma amyloid beta 42, medial temporal lobe atrophy and homocysteine[END_REF][START_REF] Lopez | Plasma amyloid levels and the risk of AD in normal subjects in the Cardiovascular Health Study[END_REF] In one study, the age-related increase was smallest in those subjects who converted to AD from MCI [START_REF] Blasko | Conversion from cognitive health to mild cognitive impairment and Alzheimer's disease: prediction by plasma amyloid beta 42, medial temporal lobe atrophy and homocysteine[END_REF]. It is possible that age-related changes of plasma levels of Aβ40 and Aβ42 differ in subjects with AD. In this respect, the Aβ42/Aβ40 ratio may be a better predictor for AD than the single markers.

Differences in study cohorts make the comparison between different studies difficult. There are many confounding factors that may influence plasma Aβ levels. Renal dysfunction may increase plasma since plasma Aβ is excreted through the kidneys [START_REF] Arvanitakis | Serum creatinine levels correlate with plasma amyloid Beta protein[END_REF]. Many studies have suggested an association between vascular disease and plasma Aβ levels. [START_REF] Van Dijk | Plasma amyloid beta, apolipoprotein E, lacunar infarcts, and white matter lesions[END_REF][START_REF] Lee | Circulating beta amyloid protein is elevated in patients with acute ischemic stroke[END_REF][START_REF] Gurol | Plasma {beta}-amyloid and white matter lesions in AD, MCI, and cerebral amyloid angiopathy[END_REF] The levels of plasma homocysteine, a possible marker for vascular disease, correlate positively with plasma Aβ40 and Aβ42 levels. [START_REF] Irizarry | Association of homocysteine with plasma amyloid beta protein in aging and neurodegenerative disease[END_REF] Previous studies have also suggested that certain drugs may influence plasma Aβ. [START_REF] Blasko | Effects of medications on plasma amyloid beta (Abeta) 42: Longitudinal data from the VITA cohort[END_REF] We found elevated levels of Aβ40 in the subjects who were using ASA and dipyridamole, i.e. drugs that directly influence platelet function and activation, and subjects who used anticoagulation and antidiabetic drugs, whereas there was no association between the use of NSAIDs or lipid lowering agents and plasma Aβ levels. The association between ASA and plasma Aβ40 was seen also in subjects without cardiovascular diseases. In line with previous studies [START_REF] Blasko | Effects of medications on plasma amyloid beta (Abeta) 42: Longitudinal data from the VITA cohort[END_REF][START_REF] Tokuda | Plasma levels of amyloid beta proteins did not differ between subjects taking statins and those not taking statins[END_REF], no relationship was found between Aβ levels and lipid lowering agents has been found in previous studies.

Differences in methodology and experimental conditions may also influence results.

Erythrocytes and plasma proteins, for example albumin and lipoproteins, bind Aβ and denaturating conditions liberate Aβ into the free pool of plasma. [START_REF] Kuo | Amyloid-beta peptides interact with plasma proteins and erythrocytes: implications for their quantitation in plasma[END_REF] Also, the different antibodies used in the immunological assays may detect different fractions of Aβ. Previous studies suggested that the absolute levels of Aβ vary across different ELISA batches [START_REF] Golde | Biochemical detection of Abeta isoforms: implications for pathogenesis, diagnosis, and treatment of Alzheimer's disease[END_REF].

We also noticed a difference between absolute levels in 70 samples that were measured twice four years apart. Due to these methodological difficulties, the diagnostic value of a single measurement is limited.

The significance of the standardization of the conditions in storing and handling the samples is reported in a work by [START_REF] Vanderstichele | Standardization of measurement of beta-amyloid(1-42) in cerebrospinal fluid and plasma[END_REF]. To better utilize these analyses, the assays used should be commercially available, well standardized and thoroughly validated.

We conclude that plasma Aβ is not a diagnostic marker for AD but the decreasing levels of

  269 baseline samples (group 1) were measured in year 2002-2003. After completing the six year follow-up, we re-analysed the baseline samples of 70 subjects (group 2) together with their follow-up samples in year 2006-2007 (group 2).

  from 70 subjects twice (4 years apart). The absolute concentrations differed between these two measurements; median level for Aβ40 174,5 (year 2003) and 198 pg/ml (74) (year 2007), and Aβ42 17 (year 2003) and 49 pg/ml (110) (year 2007). However, there was a moderately good correlation between these measurements (Aβ40 r=0.674, p<0.001 and Aβ42 r=0.824, p<0.001). The correlation figure is presented in the supplemental data (available at www.jnnp.com).

Fig. 1 .

 1 Fig. 1. Formation of the study population.

  1. (SPSS Inc., Chicago, IL, USA). Due to the non-normal distribution of data, Kruskal Wallis, Mann Whitney and Spearman's correlation tests were used. The categorical data was analysed by χ 2 test. The odds ratios (OR) for cognitive decline of patients in different groups were calculated by logistic regression analysis. We fitted a linear regression slope by Microsoft

Table 2 .

 2 Cognition and changes of Aβ between baseline and follow-ups in subjects.

	Baseline	Cognitively healthy	Cognitively impaired
	N =	59		11
	Aβ40	195 (88)		201 (60)
	Aβ42	50 (121)		46 (92)
	Aβ42/Aβ40	0.236 (0.464)	0.291 (0.487)
		Cognitively stable or
		improved	Cognitive decline
	Follow-up	3 years	6 years	3 years	6 years
	N =	43 †	29 ‡	27	14
	change of Aβ40	18 (55)	33 (45)	21 (24)	33 (75)
	change of Aβ42	3.7 (29)	12 (54)	0.0 (14)*	-2.4(50)*
	change of	0.0093	0.0166	-0.0027	-0.0486
	Aβ42/Aβ40	(0.169)	(0.451)	(0.084)*	(0.251)*

The data is given as medians (interquartile range). The values of p reflect the significance against the cognitively stable subgroup. *p < 0.05 against "stable or improved" † Aβ data of one subject is from a plasma sample of 4 years of follow-up. ‡ Cognition data of two subjects is from the previous year

Table 3 .

 3 Relation of Aβ40 level to medication at baseline.

		Group 1	Aβ40		pg/ml	pg/ml
		n			Users	Non-users
	Sex, female	148	-
	Smoking	21	-
	CHD	61	↑	p=0.035	188 (105 -360)	176 (0 -780)
	Diabetes	22	-
	Anticoagulants	16	↑	p=0.038	198 (144 -360)	176 (0 -780 )
	NSAIDs	22	-
	ASA	62	↑	p=0.004	194 (0 -278)	175 (0 -780)
	Dipyridamole	12	↑	p=0.016	208 (156-278)	176 (0 -780)
	Antidiabetics*	13	↑	p=0.003	219 (161 -426)	179 (0 -780)
	Lipid lowering	24	-
	agents
	*insulin and oral antidiabetics

ACKNOWLEGEMENTS

This study was supported by Health Research Council of the Academy of Finland, EVO grants from the Kuopio University Hospital 5883, 5772720, 5772722 and 5772725, Nordic Center of Excellence in Neurodegeneration and the Finnish Cultural Foundation´s North Savo Regional fund and North Kymenlaakso Regional fund.

The authors have no conflicts of interests. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in the Journal of Neurology, Neurosurgery & Psychiatry editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://jnnp .bmjjournals.com/ifora/licence.pdf).


Declan T Chard
email: d.chard@ion.ucl.ac.uk

Catherine Mary Dalton

Josephine K Swanton

Leonora K Fisniku

Miszkiel A Katherine

Alan J Thompson

Gordon T Plant

David H Miller

K A Miszkiel

MRI-only conversion to MS following a clinically isolated syndrome

Keywords: multiple sclerosis, clinically isolated syndromes, clinical outcomes, brain lesions, benign. Disclosures

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

For most patients who develop multiple sclerosis (MS), the initial clinical event is a clinically isolated syndrome (CIS) suggestive of demyelination. About two thirds of young adults presenting with a CIS exhibit clinically silent lesions on magnetic resonance imaging (MRI) that suggest demyelination. Long term (15-20 year) follow up studies have revealed that subsequent further clinical relapses leading to a diagnosis of clinically definite (CD) MS [START_REF] Poser | New diagnostic criteria for multiple sclerosis: guidelines for research protocols[END_REF] occur in 70-80% with an abnormal scan and 20-25% with normal imaging [START_REF] Fisniku | Disability and T2 MRI lesions: a 20-year follow-up of patients with relapse onset of multiple sclerosis[END_REF][START_REF] Neuritis | Multiple sclerosis risk after optic neuritis: final optic neuritis treatment trial follow-up[END_REF] ; the majority of those who convert do so within the first 5 years [START_REF] Morrissey | The significance of brain magnetic resonance imaging abnormalities at presentation with clinically isolated syndromes suggestive of multiple sclerosis. A 5-year follow-up study[END_REF][START_REF] Tintoré | Baseline MRI predicts future attacks and disability in clinically isolated syndromes[END_REF] .

Since 2001, diagnostic criteria for MS allow for MRI evidence of dissemination in space and time alone to diagnose MS following a CIS [START_REF] Mcdonald | Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis[END_REF][START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] , and follow up studies have shown that MRI conversion is often associated with developing CDMS [START_REF] Dalton | Application of the new McDonald criteria to patients with clinically isolated syndromes suggestive of multiple sclerosis[END_REF][START_REF] Tintoré | New diagnostic criteria for multiple sclerosis: application in first demyelinating episode[END_REF][START_REF] Swanton | MRI criteria for multiple sclerosis in patients presenting with clinically isolated syndromes: a multicentre retrospective study[END_REF] . Although it is known that people with CIS develop clinically silent new lesions during follow up [START_REF] Brex | A longitudinal study of abnormalities on MRI and disability from multiple sclerosis[END_REF][START_REF] Neuritis | Long-term brain magnetic resonance imaging changes after optic neuritis in patients without clinically definite multiple sclerosis[END_REF] , the frequency with which patients develop MS on MRI criteria only after prolonged follow-up has not been studied.

The work we report here sought to address the question: in the longer-term, how many people could be diagnosed as having MS based on the current MRI criteria only [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] ?

Methods

Participants

We retrospectively interrogated data from two cohorts who underwent serial

Magnetic resonance imaging

The 20-year cohort had baseline MRI and 5-year follow-up scanning undertaken using a Picker (Cleveland, USA) 0.5T system, and all other scanning performed using a General Electric (Milwaukee, USA) Signa 1.5T system. Contiguous 5mm axial slices were obtained at all time-points, except for a minority of baseline images that were acquired with a slice thickness of 10mm. At baseline and 5 years spin echo scans (repetition time (TR)=2000ms, echo time (TE)=60ms) were acquired; at 10 years conventional spin echo scans were obtained (TR=2000ms, TE=30/90ms); at 14 years fast spin echo (FSE) sequences was acquired (TR=2000ms, TE=14/98ms); and at 20 years an FSE sequence was obtained (TR=2000ms, TE=17/102ms).

For the 6-year cohort all scanning was undertaken using a General Electric Signa 1.5T system. Contiguous 3mm axial slices were obtained at all timepoints. An FSE sequences (TR=3200ms, TE=15/90ms) was acquired during each scanning session, and at baseline a T1-weighted (TR=600ms, TE=17ms) post gadolinium (Gd) (0.1mmol/kg body weight) image was also obtained.

Brain T2-weighted lesion counts and volumes were determined from baseline images in both the 20-and 6-year cohorts [START_REF] Grimaud | Quantification of MRI lesion load in multiple sclerosis: a comparison of three computerassisted techniques[END_REF] . Baseline brain Gd-enhancing lesion counts were obtained in the 6-year cohort.

Clinical assessment

Patients were classified into four subgroups at last follow up: Expanded disability status scale (EDSS) [START_REF] Kurtzke | Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)[END_REF] scores are reported from the baseline and 6-year follow up visits for the 6-year cohort and from the 5-and 20-year follow-up visits for the 20-year cohort (a baseline EDSS was not obtained in this group). Disease modifying treatments were prescribed only to patients with CDMS who had experienced two or more relapses in the previous two years.

Statistical analyses

Fisher's exact tests were used to assess group-wide differences in gender ratios, all other measures were compared using Mann-Whitney U tests.

Analyses were performed using SPSS 11 (SPSS Inc, Chicago, USA).

Results

Six-year CIS group (Table 1)

The 6-year CIS group were first reviewed an average of 47 days after their first clinical event (median 45, range 2 to 106). They were followed-up for a mean of 6.3 years (median 5.6, range 4.7 to 10.2 years), at which time ~51% had CDMS, ~15% RDMS, ~12% ECIS and ~20% SCIS. Comparing baseline observations between CDMS and RDMS, except for a greater proportion of males in the RDMS group (p=0.005), and a trend towards the RDMS group being older at the time of their first episode (p=0.068), no significant differences were detected (baseline T2 lesion volumes and numbers, Gdenhancing lesion counts, and EDSS scores). Comparing CDMS with the ECIS and SCIS groups, there were significant differences in T2 lesion volume (p<0.001), T2 lesion number (p<0.001) and Gd-enhancing lesion number (CDMS with ECIS p=0.026 and with SCIS p<0.001).

Twenty-year CIS group (Table 2)

The 20-year CIS group were first reviewed an average of 63.3 days after their first clinical event (median 26, range 1 to 361). They were followed-up for a mean of 19.9 years (median 20, range 18.0 to 23.1 years), at which time ~61% had CDMS, ~11% had RDMS, ~11% ECIS and ~16% SCIS.

Comparing baseline observations between CDMS and RDMS, no significant differences were detected (gender, age at first episode, baseline T2 lesion numbers, and 5-year EDSS scores), although a slight trend to a lower baseline T2 lesion volume was noted (p=0.073, with caveat that T2 lesion volumes were only available in a subset of patients, as per Table 2).

Comparing CDMS with ECIS and SCIS, baseline T2 lesion counts were lower (p=0.001 and p<0.001 respectively), as were T2 lesion volumes (p=0.002 and <0.001 respectively); 5-year EDSS scores were lower in the SCIS compared with CDMS group (p=0.048).

Discussion

The main finding of our study was that some CIS patients (11-15%) developed MS using current imaging but not clinical criteria after prolonged follow up (6 to 20 years), so identifying a group of patients who exhibit a clinically silent disease course over a long period.

Although the length of follow up of the two cohorts is very different, conversion to CDMS following a CIS occurs most commonly during the first 5 years. Our findings are consistent with this, showing similar proportions with CDMS in the 6-year and 20-year cohorts (51% and 61% respectively). They also suggest that those classified as having RDMS at 5 years are more likely than not to remain so in the longer term (with 15% and 11% classified as having RDMS in the 6-year and 20-year cohorts respectively), although some will still convert to CDMS.

With the available data, we also investigated whether there were MRI features at presentation that could help distinguish a RDMS from CDMS evolution. However, like CDMS most RDMS subjects had abnormal brain MRI at presentation with similar T2 lesion loads and, in the 6-year cohort, a similar number of gadolinium enhancing lesions. We found that the 6-year RDMS group were more often male and had a slightly older age at CIS presentation compared to the CDMS group. These clinical features tend to be overrepresented in progressive forms of MS [START_REF] Miller | Primary-progressive multiple sclerosis[END_REF][START_REF] Cottrell | The natural history of multiple sclerosis: a geographically based study. 5. The clinical features and natural history of primary progressive multiple sclerosis[END_REF] , and their presence in the 6-year but not 20-year RDMS group might suggest that the former cohort includes subjects who in the longer term will evolve to a secondary progressive form of disease.

It is well established that the presence of brain MRI lesions in CIS patients is associated with a relatively high likelihood of conversion to CDMS and disease modifying treatments beta interferon and glatiramer acetate delay the time to developing CDMS [START_REF] Jacobs | Intramuscular interferon beta-1a therapy initiated during a first demyelinating event in multiple sclerosis. CHAMPS Study Group[END_REF][START_REF] Comi | Effect of early interferon treatment on conversion to definite multiple sclerosis: a randomised study[END_REF][START_REF] Kappos | Treatment with interferon beta-1b delays conversion to clinically definite and McDonald MS in patients with clinically isolated syndromes[END_REF][START_REF] Comi | Effect of glatiramer acetate on conversion to clinically definite multiple sclerosis in patients with clinically isolated syndrome (PreCISe study): a randomised, doubleblind, placebo-controlled trial[END_REF] and are sometimes initiated in CIS patients with abnormal MRI. Our present study nevertheless highlights that conversion to CDMS is not invariable and awareness of the possibility of a long-term RDMS course may be useful in counselling and managing CIS patients.

In addition to the RDMS group, there was a group who accrued new lesions but did not fulfil current diagnostic criteria (ECIS). This group is likely to include subjects with a mild form of MS and some with other causes of white matter lesions, and raises the issue of the specificity of MRI findings. In this regard the present McDonald imaging criteria for MS may beneficially reduce the chance of a false positive diagnosis of MS, albeit at the expense of diagnostic sensitivity. It has been noted that some patients who develop CDMS do not fulfil the current MRI criteria for MS, and recent work supports expanding the imaging criteria for MS in patients with a typical CIS [START_REF] Swanton | MRI criteria for multiple sclerosis in patients presenting with clinically isolated syndromes: a multicentre retrospective study[END_REF][START_REF] Rovira | A single, early magnetic resonance imaging study in the diagnosis of multiple sclerosis[END_REF][START_REF] Montalban | MRI criteria for MS in patients with clinically isolated syndromes[END_REF] .

Further research to improve prediction of outcomes following a CIS is needed, and could include newer genetic, immunological and imaging measures.

There are several limitations to the study. First, cognitive testing was not included in the 20-year follow up cohort and the possibility that some of the RDMS cases developed cognitive impairment cannot be excluded.

Neuropsychological testing was performed at follow up of the 6-year cohort [START_REF] Summers | Cognitive impairment in multiple sclerosis can be predicted by imaging early in the disease[END_REF] and cognitive impairment when present was mild with no clear differences between those who developed MS and those who did not. Secondly, it is possible that some MRI lesions were not due to demyelination; particularly in the older 20-year follow up cohort, some of the new white matter lesions may represent vascular disease. However, the proportion with RDMS in this group was not higher (it was actually slightly lower) than in the younger 6-year cohort, suggesting that the contribution from superadded vascular disease is probably small. Thirdly, some subjects originally recruited could not be followed up with the required clinical and MRI evaluation at the final study time-point; this was more often the case for the 20-year cohort in whom it was also not possible to scan some more disabled MS patients. Finally, there were differences between the CIS cohorts with respect to imaging protocols, frequency of scanning, type of scanner and type of CIS. Notwithstanding these factors, the two CIS cohorts exhibited many similarities -e.g., age, gender and frequency of MRI abnormalities at presentation and of evolution to clinically definite MS -and the baseline clinical and imaging features of both groups are typical for CIS.

Overall, the similarity of findings in both CIS cohorts confirms that a long term RDMS course occurs, albeit less commonly than CDMS. Awareness of a RDMS course may be relevant when counselling and treating CIS patients.

Further prospective follow up studies are warranted to better characterise and understand this evolution of MS.

  clinical and MRI studies following a CIS. The first cohort was recruited between May 1984 and July 1987, and had clinical and MRI follow-up at (means) 1, 5, 10, 14 and 20 years 2 . The second cohort was recruited between June 1995 and November 2002, and had clinical and MRI follow-up at 3 months, and 1, 3 and 6 years 13 . Clinical and MRI data were missing at some time-points in some patients; to be included in the present study, subjects must have had both a clinical and MRI follow up study at the final scheduled 6-year or 20-year time-point. These studies were approved by the National Hospital for Neurology and Neurosurgery and UCL Institute of Neurology Joint Research Ethics Committee. All participants gave written informed consent.

1 .

 1 Clinically definite (CD) MS: those people who had at least one new clinical relapse with documented new symptoms and signs, or who developed progressive neurological deficits 1 ; 2. Radiologically defined (RD) MS: fulfilling the McDonald MRI 7 criteria for dissemination in space and time, but not the CDMS criteria; 3. Evolving (E) CIS: not fulfilling CDMS or McDonald MRI criteria for MS while still accruing new MRI lesions; 4. Stable (S) CIS: with neither clinical nor MRI evidence for new disease.

1

  Mean, standard deviation. 2 Median and range. N=105, except for baseline EDSS, where N=103 (N=53 for CDMS and N=21 for SCIS), and 6-year EDSS scores, where N=104 (N=15 for RDMS). CDMS=clinically definite multiple sclerosis; ECIS=evolving clinically isolated syndrome; EDSS=expanded disability status scale; RDMS=radiologically defined multiple sclerosis; SCIS=stable clinically isolated syndrome.

Table 1 : clinical and MRI baseline observations, and last review EDSS scores, in the cohort followed-up over 6-years from their index clinical event.

 1

					Subtype
			CDMS	RDMS	ECIS	SCIS
	Further events	clinical	Yes	No	No	No
	New MRI lesions Not required Yes	Yes	No
	Gender (F/M)	42/12	6/10	8/5	15/7
	Age at first event (years) 1	33.0, 7.7	36.6, 6.3	34.3, 7.5	32.3, 7.5
	T2 lesion volume
	(ml) 2

Table 2 : clinical and MRI baseline observations, and last review EDSS scores, in the cohort followed-up over 20-years from their index clinical event.

 2 Mean, standard deviation.[START_REF] Fisniku | Disability and T2 MRI lesions: a 20-year follow-up of patients with relapse onset of multiple sclerosis[END_REF] Median and range. N=70, except for baseline T2 lesion volume, where N=53 (N=33 for CDMS, N=4 for RDMS, N=6 for ECIS and N=10 for SCIS), and five year EDSS, where N=55 (N=37 for CDMS, N=5 for RDMS, N=6 for E-CIS and N=7 for SCIS). CDMS=clinically definite

	multiple	sclerosis;	ECIS=evolving	clinically	isolated	syndrome;
	EDSS=expanded disability status scale; RDMS=radiologically defined multiple
	sclerosis; SCIS=stable clinically isolated syndrome.

[START_REF] Poser | New diagnostic criteria for multiple sclerosis: guidelines for research protocols[END_REF]

Acknowledgements

We thank Drs Peter Brex, Kryshani Fernando, Olga Ciccarelli, Sean Morrissey, Jonathan O'Riordan, Michael Sailer for their assistance with data collection and analysis of imaging data, and the people who took part in this study. The NMR Research Unit is supported by the MS Society Great Britain and Northern Ireland, and Department of Health's NIHR Comprehensive Biomedical Research Centre at UCLH.

C.M. Dalton received funding from Novartis, through a grant held by the UCL Institute of Neurology, to perform MRI analysis. A.J. Thompson has received honoraria for consulting services, speaking, and serving on a scientific advisory boards from Novartis, Eisai, Weleda/Society for Clinical Research, Hoffman La Roche, UCB Pharma, Serono Foundation, Sanofi-Aventis, and the MS Society of Great Britain and Northern Ireland. He is Editor-in-Chief of Multiple Sclerosis for which he receives an honorarium from Sage Publications. D.H. Miller has received honoraria from UCB Pharma, Schering, Biogen Idec, GSK, and Wyeth for consulting services, speaking, and serving on a scientific advisory boards. He has received reimbursement for work as co-chief Editor of Journal of Neurology, and research grant support from the MS Society of Great Britain and Northern Ireland, Wellcome Trust, Medical Research Council UK, Biogen Idec, Novartis, GlaxoSmithKline, and Schering. G.T.

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