The Clinical Use of Cerebrospinal Fluid Biomarkers for Alzheimer’s Disease Diagnosis: The Italian Selfie

Abstract

Although the use of cerebrospinal fluid (CSF) amyloid β_1-42 (Aβ₄₂), tau (T-tau), and phosphorylated tau (p-tau₁₈₁) gives added diagnostic and prognostic values, the diffusion is still limited in clinical practice and only a restricted number of patients receive an integrated clinico-biological diagnosis. By a survey, we aimed to do a “selfie” of the use and diffusion of CSF biomarkers of dementia in Italy, the standardization of pre-analytical procedures, the harmonization of ranges, and the participation to Quality Control programs. An online questionnaire was sent to the members of SIBioC and SINdem-ITALPLANED and to main neurological clinics all over Italy. In Italy, 25 laboratories provide biomarkers analysis in addition to a network of 15 neighboring hospitals. In sum, 40 neurological centers require CSF analyses. 7/20 regions (35%) lack CSF laboratories. Standardization of pre-analytical procedures is present in 62.02% of the laboratories; only 56.00% of the laboratories participate in International Quality Control. There is no harmonization of cut-offs. In Italy, the use of CSF biomarkers is still limited in clinical practice. Standardization and harmonization of normal ranges are needed. To optimize and expand the use of CSF biomarkers, a cost–benefit analysis should be promoted by scientific societies and national health services.

Keywords

Alzheimer’s disease diagnosis cerebrospinal fluid neurodegenerative biomarkers survey

INTRODUCTION

There is numerous evidence of the utility of the cerebrospinal fluid (CSF) biomarkers in the diagnostic setting of Alzheimer’s disease (AD) [1]. Indeed, AD patients typically display low concentrations of amyloid Aβ₄₂ (Aβ₄₂), and high concentration of total tau (T-tau) and phosphorylated tau (p-tau₁₈₁), a pattern commonly referred to as the “AD signature” [2 –8]. In particular, combining CSF Aβ₄₂, T-tau, and p-tau₁₈₁ adds diagnostic accuracy, both for identification of AD and to differentiate AD from non-AD dementia, or to predict the progression to AD in patients with mild cognitive impairment [9 –14]. Importantly, the CSF biomarkers have a high predictive value to exclude the AD diagnosis, especially in older individuals [15].

The use of CSF biomarkers, together with the topographical markers (PiB, MRI), has been included in the recommendation for AD diagnosis by both the International Working Group (IWG) and the US National Institute on Aging–Alzheimer’s Association [16, 17]. The new criteria (IGW-2) move from the old concept of AD dementia to a new perspective which points to early diagnosis and discrimination from other forms of dementia, and represent a shift from the clinico-pathological paradigm, based on dementia and autopsy confirmation, to a clinico-biological paradigm, which focuses on measurable in vivo evidence of AD pathology [16, 17]. Moreover, the CSF biomarker analysis has been accepted and adopted with limitations in clinical routine around the world, with many differences between countries [1]; the European Federation of Neurological Societies (EFNS) guidelines recommend the use of CSF analysis in the differential diagnosis of typical and atypical AD [18, 19].

We do not know, however, the actual diffusion of CSF biomarkers in clinical practice, or whether their use is still mainly limited in a research setting. By a survey, we aimed at performing a “selfie” of the use of CSF biomarkers in the clinical practice for the diagnosis of AD and other form of dementia in Italy. Moreover, we investigated the adherence to pre-analytical procedures, the participation in quality control programs, and the methods used in each CSF laboratory, in order to understand the standardization of the analytical phase and the variations between laboratories. Finally, we investigated whether CSF biomarker tests are approved and reimbursed in each Italian region by the Regional Health Services.

METHODS

Joint questionnaires were designed and approved by the Italian Society of Clinical Biochemistry (SIBioC, the Clinical Biochemistry of Biological Fluids excluding the Blood Working group), and by the Italian platform for neurodegenerative disease, part of the Italian Society of Neurology for Dementia (SINdem-ITALPLANED). Anonymous questionnaires (n = 1,815) were sent by e-mail to all members of SIBioC and ITALPLANED, distributed in all the regions of Italy.

Surveys of the compiled questionnaires were performed using Survey Monkey. Data were collected and analyzed, after exclusion of questionnaires coming from the same laboratory. Data are reported as percentages of the collected responses. The levels of CSF biomarkers are expressed in ng/L; range of normality and cut-offs are reported as range (min-max) or mean±standard deviation.

RESULTS

The distribution and activity of CSF laboratories

Based on the data we collected, we drew a map of the laboratories performing CSF biomarker analysis for neurodegenerative diseases in Italy. We received 99 full questionnaires (out of 1,815 sent); questionnaires from the same centers were considered only once, analyzing 97 questionnaires in total.

We found that in Italy CSF analysis for AD biomarkers is currently performed in 25 laboratories, which can be distinguished as “internal laboratories” performing analysis just for their own hospital (10/25), and “centralized laboratories” (15/25), which provide biomarkers analysis for their own and for a network of neighboring hospitals. Indeed, 15 hospitals lack the service of an internal laboratory and send CSF samples to external or centralized laboratories, situated in the same or near city. Therefore, we can conclude that on the whole internal and centralized laboratories can provide biomarker analysis for a sum of 40 hospitals (25 internal plus 15 external).

The number of CSF samples analyzed per month in each laboratory is generally low, i.e., less than 10 (41.66% of the responses) and less than 20 (45.83% of the responses). Only in a few laboratories the amount of CSF assays is more than 20 per month (12.50%) (Table 1).

We tried to verify whether we collected the responses from all the laboratories in Italy performing AD biomarker’ analysis. To do this, we choose an indirect method and asked Fujirebio-Innogenetics and Euroimmun, two major European companies, which confirmed that there are no more than 25 laboratories in Italy, buying the kits widely used to measure CSF biomarkers.

The 25 laboratories performing CSF analysis have a patchy distribution across Italy, with major presence in the North, intermediate in the Center, and minor in the South and the islands (Fig. 1). Notably, centralized CSF laboratories (n = 15) are distributed in 11 regions. Surprisingly, 7 regions (35%) in Italy lack laboratories performing CSF biomarkers: three regions are in the North, one in the Center, and three in the South. It is worth noting that all 7 regions lacking CSF biomarkers laboratories are at the same time devoid of a Regional Section of SINDem, enclosing specialized members interested in the diagnosis of dementia. However, only in 4/7 regions there is a very low number of SINDem members (0–4/per region in the year 2015) that can explain the lack of CSF laboratories. This is not the case for 3/7 regions (Abruzzo, Campania, Calabria) that instead have many SINDem members (13–26 per region in the year 2015), suggesting other causes for their lack of interest in CSF biomarkers (see Supplementary Table 1).

CSF biomarkers laboratories and memory regional centers

Thereafter, we tried to evaluate the relationship between the number of CSF laboratories and the number of neurologic clinics in Italy. There are 333 neurological institutes/centers in Italy as counted by the Società Italiana di Neurologia (see Supplementary Table 1). Therefore, CSF biomarker analysis is requested for a diagnosis of dementia by only 12.0% (40/333) of the neurology centers in Italy.

We also considered the relationship between the presence of Memory and Dementia Regional Centers (Dem-RC) and CSF laboratories. Interestingly, a Dem-RC is present only in 32.5% of the hospitals using CSF biomarkers analysis (n = 13), showing that CSF biomarkers are not used exclusively by the centers dedicated to the study of cognitive impairment (Table 1). Notably, almost all Dem-RC participating in the survey use CSF biomarkers and have an internal laboratory; one hospital with Dem-RC sends CSF sample to a centralized laboratory, whereas only one Dem-RC does not use the CSF biomarkers (no sending and no internal CSF laboratory).

CSF pre-analytical procedures and quality control

Correct pre-analytical procedures are crucial for obtaining reliable results of CSF biomarkers. We refer to standard operating procedures for CSF collection and analysis, including monitoring temperature during sampling collection, use of the correct polypropylene tubes, centrifuging CSF before storing samples, etc. [20]. The data we collected show that standardization of pre-analytical procedures is present in 62.02% of the laboratories participating to the survey. Moreover, only about half of the laboratories (56%) participate in International Quality Control Programs (e.g., Alzheimer’s Association Quality control, JPND, or others); the other laboratories lack of an external control for the inter-assay variability (Table 1).

CSF biomarker ranges of normality

As expected, there is a wide heterogeneity in the definition of normal range for the three biomarkers of neurodegeneration. Only a low number of those interviewed responded to the question about the normal cut-offs (19 laboratories for Aβ₄₂, 14 for T-tau, 17 for p-tau₁₈₁). Normal values used in almost all CSF laboratories are reported in Table 2.

In two laboratories, the normal Aβ₄₂ level is higher than 450 ng/L; in the majority of the laboratories, the Aβ₄₂ normal level in the adult population (52–70 year) is considered higher than 500 or 550/562 ng/L; in one laboratory, normal Aβ₄₂ levels is considered higher than 600 ng/L; finally, in one center normal values are included between 790±228 ng/L. In most of the laboratories, a level of T-tau lower than 400 or 450 is considered normal in adult population (52–70 year); however, in some laboratories, normal T-tau levels are considered lower than 300 or 350, while in one center normal T-tau values are lower than 243±127 ng/L. Lastly, in the majority of the laboratories the cut-off for p-tau181 in normal adult patients is generally <61–67 ng/L, while some centers considered the normal level lower than 52 or 35 ng/L.

Notably, all the centers participating in the survey use the same ELISA assays for the analysis of the three biomarkers (Innotest, Fujirebio), with one exception (Euroimmun).

CSF biomarker reimbursement by Regional Health Services

Currently, CSF biomarker tests are not approved nor reimbursed by the Regional Health Servicesthroughout Italy, with the exception of theUmbria.

Additional analysis for diagnosis of neurodegenerative diseases

We asked if CSF is used in laboratory medicine for diagnostic investigations of other neurological diseases. Lumbar puncture is a routine procedure for diagnostic purpose, and the cyto-morpho-chemical analysis of CSF is widespread in neurologic clinics. Oligoclonal bands, which are used for the diagnosis of multiple sclerosis, are evaluated in 52.78% of the laboratories. Oppositely, the analysis of other parameters that are involved in the study and the diagnosis of neurodegenerative diseases are much more restricted. CSF Aβ₄₀, which is used in combination with Aβ₄₂ to improve the diagnostic accuracy, is employed in 12.19% of the centers. Similarly, CSF alpha-synuclein, a marker for synucleinopathy, is used only in 7.31% of the centers, while the CSF 14-3-3 protein, a marker usually measured for Creutzfeldt-Jakob disease diagnosis, is used in 3.65% of the laboratories.

Moreover, the analysis of blood biomarkers for the diagnosis of neurodegenerative disease is limited: the ApoE polymorphism, a major genetic risk factor for AD, is performed in 18.29% of the laboratories; serum progranulin, a marker of some genetic forms of frontotemporal dementia, in 8.5% (Table 3).

DISCUSSION

Most of the studies on CSF biomarkers focus on the diagnostic utility [1 , 21], or on analytical and pre-analytical factors hampering their use [20 , 22–24], but we lack studies evaluating how frequently and where AD biomarkers are really used. To the best of our knowledge, this report is the first one systematically assessing the use of CSF biomarkers in a large multicenter context, focusing on critical issues in the clinical use of CSF biomarkers.

The data of our “selfie” show that 25 laboratories perform CSF biomarker analyses in Italy, on samples coming from 40 different centers located in 13 out of the 20 Italian regions (65%). Then we could consider “the glass half full”, because in most of the regions there is at least one laboratory for CSF biomarkers. Oppositely, the glass could be considered “half empty”, because despite the apparent high number of centralized laboratories (15/25), the number of samples received from external hospitals is relatively small, and in the majority of the laboratories the number of assays is less than 10 or 20 per month. However, the low number of CSF tests per center could be proportional to the number of new patients with mild to moderate cognitive impairment requiring an etiological diagnosis, according to a recent survey on the use of CSF biomarkers in Europe [25].

We have verified that the laboratories answering our questionnaire are the same buying kits for biomarkers’ analyses in Italy, confirming in this way the accuracy of the selfie regarding the number of laboratories and the amount of CSF biomarker tests per month in the different regions in Italy. One limit of this study is, however, the possible under estimation of the clinical centers requesting CSF biomarker analysis to a centralized laboratory, in case they did not receive the questionnaire or did not give answers to the questionnaire. Therefore, the number of clinical sites using CSF biomarkers could be larger than the 40, we estimated. In conclusion, the 40 clinical centers using CSF biomarker analysis in the diagnostic workflow of dementia in Italy represent approximately 12.0% of all 333 official Neurology centers in Italy.

The map of CSF biomarker laboratories in Italy show that they are grouped in some regions and absent in others, suggesting that the use of CSF analysis in diagnosis of dementia is divided between regions “marker-phile” and “marker-skeptic”. In some “marker-phile” regions, indeed, there are clusters of CSF laboratories and of hospital sending CSF to centralized laboratories. Interestingly, the use of CSF biomarkers in “marker-phile” regions is not limited to highly specialized hospitals, since only 32.50% of the laboratories performing CSF analyses have an internal Memory Center. On the other hand, in many regions (7/20) CSF biomarkers are not included in diagnostic setting of AD, even though in some of them many specialists are adherent of SINDem, suggesting that in such regions the specialists are “marker-skeptics”.

Globally, these data suggest that CSF biomarkers are not considered routine test in clinical practice [26], and their analysis is limited to services that gather all the clinical, psychological, and laboratory specialists dedicated to the diagnosis and treatment of dementia. The reasons for the limitations in the use of CSF biomarkers in different regions in Italy could be multifactorial, in part due to economic reasons linked to the lack of reimbursement of the CSF analysis by the Regional Health Service, and in part due to the lack of standardization in the clinical indications coupled with a lack of consensus on range of normality [27]. Accordingly, a national survey has evaluated the use of CSF biomarkers in clinical practice in France, the reasons for prescriptions and clinician’s expectations, finding that results from clinical research do not really seem applicable to routine clinical practice [28]. Another obstacle to the diffusion of CSF biomarkers in clinical practice can be the lumbar puncture, still considered as an invasive procedure by many physicians and patients. However, several studies demonstrate lumbar puncture to be both “safe and acceptable” in memory clinics, with low incidence of patients needing medical intervention (11 on a total of, 3868; 0.3%) or a blood patch [24, 29]. Indeed, the LP is a frequent routine procedure for diagnostic purposes in other neurologic diseases to the cyto-morpho-chemical analysis and the search of oligoclonal bands in CSF (evaluated in 75.61 and 52.78% of the laboratories, respectively).

An important issue emerging from our survey is the low number of laboratories following standard pre-analytical and analytical procedures. Pre-analytical variables that may influence the final AD biomarker detection could include, for example, the tube type, repeated freeze/thaw cycles, or the length of frozen storage. Therefore, a consensus of the members of the Alzheimer’s Biomarkers Standardization Initiative (ABSI) has been proposed [20], to minimize the differences due to pre-analytical procedures, followed by a consensus on the clinical use of CSF biomarkers [21]. This is a crucial point to allow harmonization between laboratories and standardization of samples sent to centralized laboratories.

Moreover, we found a wide variability in the cut-off values used by the laboratories for the three biomarkers. This is not surprising as even in laboratories using similar assay kits, variations of about 20% to 30% were found [20, 23]. These differences in normal and pathologic CSF biomarker reference values between centers lead to different cut-off values [18]. On the other hand, when a laboratory uses a cut-off established in another laboratory, the clinical diagnostic accuracy could be similar, but sensitivity and specificity are markedly different between the two laboratories [22].

Laboratories can ensure the accuracy of their results by carrying out internal quality control and calculating coefficients of variation, as well as by participating in external quality control programs. Half of our interviewed laboratories do not adhere to an external control program for the inter assay variability. Therefore, there is a need of normalization of cut-offs among the Italian regions to improve the comparability and allow multicenter studies. Actually, differences between centers in the cut-offs of AD CSF biomarkers represent a substantial difficulty in view of the routine clinical use [23].

We do not know how our findings are comparable, because to our knowledge, the real distribution of CSF biomarkers in clinical setting of other countries has never been evaluated. However, the use of CSF biomarkers is widespread in Western countries. For example, in the US, CSF biomarkers are rarely used for clinical diagnosis, but are more commonly required in research and for inclusion of patients in clinical trials. In France, the use of CSF biomarkers is authorized by the government agency to diagnose AD at the dementia stages in young patients and in atypical clinical presentations [30]. In Sweden, CSF biomarkers are largely used for clinical diagnosis of AD since 1995 [31]. Actually, the use of CSF biomarkers is not homogeneous throughout Europe with a consequent difference in the diagnostic accuracy, as observed in Italy [25].

In conclusion, our data demonstrate that the use of CSF biomarkers is still limited in clinical practice and only a restricted number of patients receive an integrated clinico-biological diagnosis in Italy. Obstacles to the diffusion of CSF biomarkers in clinical practice can be represented by: 1) difficulties and prejudices to perform lumbar puncture in patients with dementia; 2) lack of standardization of pre-analytical and analytical procedures; and 3) variability of cut-offs among different centers.

A more accurate clinico-biological AD diagnosis can support the use of appropriate therapy and reduce unnecessary treatment, extending the relevance of CSF biomarkers not only in research trials but also in clinical practice. Additional research is needed to demonstrate the link between potentially inappropriate treatment and adverse health outcomes in misdiagnosed AD patients [32].

To optimize and expand the use of biomarkers in clinical practice, a cost–benefit analysis should be promoted by neurological scientific societies together with national health services.

Footnotes

ACKNOWLEDGMENTS

Thanks to the members of the SIBioC and SINdem-ITALPLANED that participated in the survey. Thanks also to Federica Vergani and Claudia Agosta for their support in the survey development.

The study has been supported by the Italian Ministry of Healthy GR-2011-02349822.

Authors’ disclosures available online ().

Appendix

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