Handling and transportation of CSF samples
Cerebrospinal fluid (CSF) can be collected in the lumbar region by an experienced physician. Taking into account the necessary precautions (e.g. sterile environment) and other medical investigations (e.g. brain imaging), the lumbar puncture is a safe procedure.
Adsorption to plastic polymers can alter the biomarker concentrations and the extent of this adsorption differs between tubes. Therefore, always use the same polypropylene reference tube (within one lab).
The CSF sample must be sent to the local laboratory without delay.
- A CSF cell count is usually performed
The CSF sample is centrifuged in the original tube
The CSF sample is aliquoted for basic and specific neurodegenerative disease biomarkers
- CSF should be aliquoted in tubes made of polypropylene
- The volume needed for CSF analyses may vary between laboratories
The volume needed for Alzheimer specific biomarkers is between 0.5 mL and 1.0 mL
Transportation: CSF samples can be sent by ordinary mail, at room temperature if the shipping time is less than two days. If the CSF sample is taken on a Friday, it can be frozen and sent to the central laboratory on dry ice the next week.
Handling of CSF samples before analyses of specific biomarkers
- All CSF samples should be frozen once before analyses. CSF samples sent at room temperature should be frozen, and CSF samples sent frozen should be kept frozen.
- Sample analysis on fresh samples upon arrival is not sufficiently validated. The specific biomarker reference values could be slightly influenced by the ambient temperature. More experiments are necessary.
CSF biochemical pattern interpretation
Alzheimer’s disease (AD) and pre-clinical stages
Alzheimer’s disease (AD) is the most common neurodegenerative disease and demonstrates exponential increase in prevalence with advancing age beyond 60 years. There are three stages usually described: latency when the disease has started but is asymptomatic, prodrome when the disease has progressed and very mild clinical signs and symptoms are present, and clinical when the disease has advanced and the full clinical spectrum is expressed.
It is critical to realize that latent disease cannot be distinguished from absence of disease by clinical examination or neuropsychological testing, but rather requires some ensemble of laboratory-based methods to detect disease initiation in the absence of symptoms.
How fast the patient will progress in the disease depends on risk-enhancing factors, such as age, modifying genes, cognitive reserve, comorbidities, and so forth.
Individuals can now be identified as being in the preclinical state by the in vivo evidence of Alzheimer pathology (AP), by a biological or molecular “signature” of AD.
CSF Aβ42 and amyloid PET are highly concordant when used to dichotomize individuals as amyloid positive or amyloid-negative, showing 80%–90% agreement across studies.
Tau PET ligand is not routinely available, CSF T-tau/P-tau is the easiest tool for tauopathy evidence. Alternatively, topographical markers include volume changes in the brain (hippocampal atrophy, cortical thickness) assessed by MRI and hypometabolism of neocortical regions measured by fluorodeoxyglucose (FDG)—PET are used.
Optimal and reliable blood-based biomarkers are not yet ready for clinical application.
Only the association of both pathologic hallmarks defines AD even in the absence of cognitive symptoms.
Definition of abnormality threshold for CSF biomarkers
The threshold for “abnormality” for CSF biomarkers is difficult to assess, especially in clinically healthy elderly subjects. There are several approaches to define what is abnormal.
- abnormality may be defined based on comparison between Cognitively normal (having a CSF collection for any other causes than NDDs) and AD groups.
- abnormality can be defined based on the distribution of values within a cognitively normal population, where subjects with values exceeding, for example, 2 standard deviations (SD) below or above the mean can be considered “abnormal”.
- abnormality can be defined based on longitudinal observation of clinical progression in a group starting as healthy and declining to AD at follow-up evaluations.
In healthy control subjects, the cortical uptake of Aβ agents is low in comparison with patients suffering from prodromal AD of the hippocampal type/MCI-due-to-AD(*) or fully developed AD dementia. However, a significant proportion of cognitively healthy elderly show increased cortical Aβ binding and decreased CSF Aβ42. This finding is supported by postmortem histopathological data showing Aβ plaques upwards of 30% of the non-demented elderly population above 75 years of age, likely representing preclinical AD. (*) MCI-due-to-AD, Mild cognitive impairment due to Alzheimer etiology.
CSF T-tau levels increased with age and were higher in ApoE ε4 carriers. The apolipoprotein E (ApoE) polymorphism is the most widely accepted genetic factor increasing the risk for sporadic AD. ApoE ε4 carriers might be predisposed to vascular diseases which in turn could contribute to age-related brain damage and therefore to elevated T-tau levels.
In conclusion, a substantial number of healthy subjects over age 60 (25-40%) has at least one CSF biomarker concentration in range that can be considered abnormal. To minimize the age-related risk factor, the “normality” may be defined using results from clinically and cognitively normal individuals below the age of 50.
Remark: Commercial assays for measurement of CSF biomarkers labeled CE-mark for in-vitro diagnostic propose estimated range of normal values.
Combination of CSF biomarkers to be more useful in prediction
CSF T-tau, P-tau, and Aß42 are valuable as biomarkers of AD. At present, their strength relies mostly in supporting neurodegenerative etiology criteria for MCI and AD, and their reasonable capacity to predict the conversion from MCI to AD. A combination of biomarkers seems to be more useful in prediction than a single analyte.
The below table published by Mattson et al (2012) summarizes the specificities and likelihood ratios at cutoffs for 85% sensitivity for AD dementia according age categories. The specificity of CSF biomarkers decreases with age, as an effect of the high AD prevalence in older ages, but the likelihood ratios are improved when CSF biomarkers are combined.
The CSF biomarkers in combination, eg. low CSF Aβ42 peptide with high total tau and phosphorylated tau, are sensitive and specific biomarkers highly predictive of progression to AD dementia in patients with mild cognitive impairment and of presence of AD etiology even in older populations.
CSF markers for AD risk stratification and predictive value
Model 1: AD risk adapted from Hansson et al, Lancet Neurol 2006;5:228-234
Association between CSF biomarkers and incipient AD – Monocentric longitudinal study (Hansson et al, 2006). The follow-up period was extended (5-10 years) published by Buchhave et al (2012).
95.5 % of patients with mild cognitive impairment and abnormal CSF converted to AD.
Model 2: AD risk adapted from Lewczuk et al. J Neural Transm. 2009; J Alzheimers Dis. 2015
The Erlangen score validated using two cohorts of pre-dementia subjects, the German Dementia Competence Network (DCN, n = 190 subjects with MCI) and the US ADNI 1 (ADNI, n = 292 MCI or cognitively normal (CN) subjects. Erlangen score uses a risk graph approach.
The CSF results of a given patient are scored between 0 and 4 points. A CSF result with all biomarkers entirely normal is scored 0 points; a pattern with only marginal alterations in one biomarkers group (either Aβ or Tau, but not both) results in the score of 1; a CSF result with the alterations in either Aβ metabolism (decreased Aβ42 concentration and/or decreased Aβ42/40 ratio) or Tau metabolism (increased concentrations of T-tau and/or P-Tau) but not both is scored 2 points; a result with clear alterations in one biomarkers’ group (either Aβ or Tau) accompanied by marginal alterations in the other group is scored 3 points; clear alterations in both Aβ and T-tau/P-Tau result in 4 points.
Model 3: AD risk adapted from Lehmann et al. Alzheimer's Research & Therapy 2014, 6:38
The scale’s overall predictive value for AD for the different categories (N= 1,273 patients included 646 AD and 627 non-AD) from six independent memory-clinic cohorts.
This simple scale was evaluated in the different cohorts by using as a criterion of AD the presence of two or three pathologic biomarkers can be used to facilitate the interpretation of CSF pattern in routine.
Remark: The two last illustrations of risk scoring are cutoff values independent, meaning each laboratory can easily supplement it with the cutoff values and normal/abnormal ranges according to analytical method used for biomarker measurement.
CSF biochemical pattern interpretation
The combination of CSF biomarkers permits a diagnosis of AD in earlier stages of the disease. Nevertheless, the clinical identification of cognitive impairment and the use of both structural (CT/MRI) and functional (SPECT/PET) brain imaging are necessary for an accurate differential diagnosis with other neurodegenerative diseases. Mixed pathology, especially in elderly subjects, are frequent.
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Cerebrospinal fluid levels of β-amyloid 1-42, but not of tau, are fully changed already 5 to 10 years before the onset of Alzheimer dementia.
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Aβ 42/40 ratio
Potential added value of Aβ 42/40 ratio
Aβ peptide is produced from a transmembrane Aβ precursor protein (APP), sequentially cleaved by β- and Ƴ-secretase. Cleavage of APP by Ƴ-secretase generates a number of Aβ isoforms. Aβ42, a 42 amino acid-long peptide, has the highest propensity for aggregation and appears to be the predominant species in neuritic plaques. Besides Aβ42, several shorter isoforms of Aβ are present in CSF including Aβ40 and Aβ38. Although the concentration of Aβ40, has been reported to be unaltered in AD, the ratio of Aβ42 to Aβ40 has been suggested to be superior to the concentration of Aβ42 alone in discriminating patients with AD (although assay dependency exists).
In a population of normal subjects and AD patients, the distribution of total Aβ (40 and 42) follows a Gaussian distribution for both normal subjects and AD patients, with Aβ1-40 making up about 70% of total Aβ. Further although many cases fall into the middle of the distribution with the majority having normally total Aβ, outliers are still present. Some AD patients will have high total Aβ and some cognitively normal subjects will have low total Aβ.
This means that AD patients with a high total Aβ (“high producers”) will show an incomplete CSF pattern and vice versa, normal subjects with low total Aβ (“low producers”) will be classify as individual with sign of cerebral amyloidosis. In all three cases (normal, low and high total Aβ) the ratio can correctly classify some doubtful CSF pattern. The ratio led to a reduction by half of the number of indeterminate profiles without changing the conclusion when usual biomarkers (Aβ42 and P-tau) were concordant.
There is a consensus to recognize that Aβ42/Aβ40 ratio might be helpful to
- confirm a cerebral amyloidopathy
- decrease preanalytical and analytical sources of variability among centers
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Amyloid beta peptide ratio 42/40 but not A beta 42 correlates with phospho-Tau in patients with low- and high-CSF A beta 40 load.
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