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Introduction 

Hello. My name is Alicia Algeciras. I’m the co-director of the Clinical Immunoassay Laboratory and Professor of Laboratory Medicine and Pathology in the Division of Clinical Biochemistry and Immunology at Mayo Clinic, Rochester, Minnesota. 

My co-presenter is Dr. Joshua Bornhorst, also director of the Clinical Immunoassay Laboratory and Assistant Professor of Laboratory Medicine and Pathology in the Division of Clinical Biochemistry and Immunology at Mayo Clinic, Rochester, Minnesota.

And today, we will be talking about Alzheimer’s disease CSF biomarkers. 

Disclosures

These are our disclosures.

Alzheimer’s Disease (AD) CSF Biomarkers

The two neuropathologic features found in the brain of patients with AD dementia are the presence of plaques composed of amyloid beta (Ab) peptides and intracellular neurofibrillary tangles containing hyperphosphorylated Tau proteins. These two groups of molecules are the most established biomarkers of the disease used in clinical and research practice. In recent years, measurement of Ab peptides, specifically Ab42, and Tau proteins, including total and phosphorylated forms of Tau (p-Tau), in cerebrospinal fluid (CSF) are being proposed as an alternative to PET imaging to assess AD pathology. In CSF, the changes observed include a 50% reduction of Ab42 as consequence of amyloid deposition in the brain and an increase of p-Tau and total Tau (t-Tau). The increases in both forms of Tau are believed to reflect the extent to neurodegeneration with p-Tau being a more specific marker for Alzheimer’s disease. 

Biological definition of Alzheimer’s disease¹

Recently the use of these biomarkers has been included in the consensus research diagnostic criteria for AD, proposed by the National Institute on Aging and Alzheimer's Association (NIA-AA) Research Framework. This is referred to as the “A/T/N’’ system, and it captures the three main neuropathologic findings related to Alzheimer’s disease. 

• "A" refers to the Ab pathology, measured by either amyloid PET, or CSF Ab42 or Aβ42/Aβ40.
• "T" represents tangle pathology and is assessed by either Tau PET or CSF phosphorylated tau (p-Tau).
• "N" stands for neurodegeneration, or neuronal injury, detected by either FDG PET, structural MRI, or CSF t-Tau. 

The normal and abnormal classification for each of these markers results in eight different A/T/N biomarker profiles as shown in this figure. Based on the definitions of Alzheimer’s disease pathological change, the A/T/N biomarker system aligns every individual to one of three biomarker categories:

• Individuals with normal AD biomarkers.
• Those with the Alzheimer’s continuum.
• Those with a normal amyloid biomarker but an abnormal T, N, or both.

Mayo AD CSF biomarkers assay

The Mayo Clinic Laboratories AD biomarker assay for CSF can be found under the names of Alzheimer’s Disease Evaluation and the Biogen Alzheimer’s Disease Evaluation. The assay includes tests for Aβ42, t-Tau, and p-Tau181. All these assays are from Roche Diagnostics. We require that the CSF sample is collected on a low bind polypropylene tube. The report includes the individual biomarker results in addition to the calculation p-Tau181/Aβ42 ratio.

Mayo AD CSF biomarkers assay: Clinical performance²

The clinical performance of this assay was validated at Mayo Clinic in collaboration with Dr. Ron Peterson. The study included participants from the Mayo Clinic Study of Aging and the Mayo Clinic Alzheimer’s Disease Research Center. It included cognitively normal individuals, individuals with mild cognitive impairment, and individuals with AD dementia. The performance of the assays was compared to amyloid PET. The agreement with amyloid PET was higher for the p-tau181/Aβ42 ratio, as demonstrated by the higher area under the curve (AUC) of the receiver operator curve (ROC) curve of 0.97. Using the cutpoints indicated on this table, the p-Tau181/Aβ42 ratio showed superior positive, negative, and overall percent agreement with amyloid PET when compared to the individual biomarkers. The agreement with amyloid PET when used in the ratio was 92% for both the positive and negative classifications.

These findings are in alignment with other published work. Studies from various cohorts, including ADNI, BIOFINDER, and Washington University, using the same assays from Roche Diagnostics, reported the use of similar cutpoints for the p-Tau/Aβ42 ratio as well as similar positive and negative agreements with amyloid PET. 

Mayo AD assay report: NORMAL AD profile

This is a sample report of the Mayo assay with the normal biomarker profile. For each patient, the individual assays along with the reference value are provided. Given the highest clinical performance of the p-Tau181/Aβ42 ratio, interpretation of the results is only made based on the ratio. 

Mayo AD assay report: ABNORMAL AD profile

In this example, the p-Tau181/Aβ42 ratio is abnormal, which is consistent with the presence of pathological changes associated with Alzheimer's disease.

Mayo AD assay report: UNABLE to calculate ratio

This next report is an example of a case where the Ab42 concentration is above the measuring limit of the assay. In this case, Aβ42 is reported as greater than 1,700 mg/mL. This result would not be consistent with pathological changes associated with Alzheimer's disease and there is no need to calculate the p-Tau181/Aβ42 ratio.

Mayo AD assay sample report: Normal ratio but abnormal Ab42

We have seen some cases where the p-Tau181/Aβ42 ratio is normal despite often abnormally low Aβ42 concentration. In these situations, we need to consider various scenarios. It is possible that this represents an early stage of an AD process, hence the lack of elevation of p-Tau. Alternatively, this might represent a disorder of CSF dynamics, such as normal pressure hydrocephalus, where both Aβ42 and p-Tau will be decreased. In these situations, the normal ratio will be suggestive of either one of these processes.

If there is a high suspicion of Alzheimer’s disease but the p-Tau/Aβ42 ratio is normal or close to the cut-off, as shown in this case, the recommendation will be to consider other non-AD causes of cognitive decline and repeat testing in about 6-9 months. 

AD CSF biomarkers: Pre-analytical variables⁶

Hi, I'm Josh Bornhorst, and I’m going to talk today about pre-analytical variables related to Alzheimer's disease CSF markers.

Alzheimer's disease testing components are relatively stable once they are on an appropriate collection sample container. As can be shown on this screen, once they are frozen, they are stable for at least 30 days. At ambient temperature, they're remarkably stable: up to 12 hours. Refrigerated, they're stable for 14 days.

We do request samples be sent in frozen, and one reason is that there are multiple steps in the pre-analytical process that may, potentially and differentially, affect the measured Aβ and Tau protein concentrations. Most importantly, these include tube material, and aliquot, and fill volume.

The absorption of Aβ CSF marker analysis is related to the tube type. This is more relevant for the Aβ than for the Tau proteins, and that's because the amyloid beta peptides, including the ones used in this assay, are hydrophobic and potentially sticky. They're exceptionally sticky, so there's great potential for absorption of these to the tube surface. When this occurs, this can lead to a falsely low Aβ42 measurement, and consequently, produce a falsely high p-Tau181/Aβ42 ratio. This potentially increases clinical suspicion of AD. One reason for this is that a decrease in Aβ42 upon absorption is not mirrored by a decrease in p-Tau; p-Tau is not as sticky and does not adhere to surfaces nearly as much as the Aβ42 does.

AD CSF biomarker analysis: Sample tube effect

To further explain which tubes are susceptible to the absorption effect, I'm going to talk more about different sample tube types. It has been shown that Aβ peptides showed major variations in concentration, measured concentrations, depending on the tube type they were stored in, with the best recovery found in polypropylene tubes.

This shows two different families of tubes. The polypropylene tubes, designated as PP, are somewhat cloudy, usually, and have better recovery compared to polystyrene tubes in studies that have been published. Because of this, what we ask for is, we ask for the tubes to be collected in polypropylene due to the higher recovery of amyloid beta peptides, as compared to polystyrene tubes. In fact, there's evidence that Aβ peptides may decrease as much as 30% if polystyrene tubes are used. I will note that p-Tau recovery in both tube types is approximately 100%, so this is related to the Aβ stickiness.

AD CSF biomarker analysis: Low bind tubes⁸

In the course of Alzheimer's disease CSF biomarker analysis, there's an additional tube type that we need to talk about. That is the low-bind polypropylene tubes that are available from various manufacturers. They have been shown to be somewhat better than polypropylene tubes that are not designated as low-bind. Because of this, we have a preferred tube, just a Sarstedt Polypropylene low-bind tube, which has a fill volume of 2.5 mL, which is available upon request to clients ordering this test. However, we recognize that there are other acceptable CSF polypropylene tubes out there, and other acceptable tubes that we list are the two Sarstedt tubes shown below.

One thing that we do need to emphasize is that polystyrene collection tubes are not acceptable and will be rejected. This is because exposure of CSF to polystyrene tubes may result in significantly falsely low Aβ42 concentrations, potentially affecting clinical interpretation. 

Polypropylene tube: Fill volume^8-10

Another important pre-analytical variable related to collection tubes is the fill volume of CSF used to fill that tube. The lower the relative fill volume, the lower the recovery due to a relatively larger surface area versus the volume contained in that tube. Thus, in all tubes, a 75% reduction in volume can result in as much as a 20%-40% reduction in measured concentration.

Some things to remember about this is that a lower volume-to-sample tube size ratio results in more absorption of Aβ. Absorption occurs within 30 seconds of the transfer. Because exposing Ab to more surface area can reduce the measured concentrations of Aβ, one should avoid tube transfer prior to sending to Mayo Clinic Laboratories.

As a reminder, if we do get a sample with a short volume, we do recommend that the tubes be as much as 80% or greater full. We will add a footnote that says, "The sample volume (in this example, it was 1.0 mL) was received in a 2.5 mL collection tube. That was less than the 50% required fill line." This footnote will alert clinicians for the potential of a low sample volume submission on results.

Collection recommendations^6,8

To summarize, we will go over the overall collection recommendations for this assay. The Alzheimer's Association has a consensus protocol recommendation, which is performing a lumbar puncture and discarding the first 1-2 milliliters of CSF; that helps mitigate the effects of potential blood contamination and collecting the CSF directly into a low-bind collection tube in the course of Alzheimer's disease analysis by this method.

One thing to note is that while we don't require this, the use of the drip method for CSF collection directly into a low-bind polypropylene tube is preferred over the syringe pull method. While the syringe pull method increases the collection speed, the drip method reduces the risk of Aβ42 binding to the plastic of the syringe used. Also, related to studies and literature, the Mayo recommendation is to fill the sample tube until at least 80% full, and would like to remind clients to use a polypropylene tube; preferably, a low-bind polypropylene tube.

Sample report

As both of these phenomena, the tube collection type selection and the volume submitted, can affect clinical interpretation on all results, we include a warning, or a disclaimer, which is in the red box here. "Failure to adhere to sample collection instructions in the Lab Test Catalog may result in a falsely low Aβ42 concentration. This affects subsequent interpretations, as well as the p-Tau/Aβ42 ratio."

Additional information

For additional information, both in terms of interpretation of this assay, appropriate ordering, and specimen collection instructions, one can refer to the Mayo Clinic Laboratories Test Catalog, just shown at this web address here. Additionally, Mayo Clinical Laboratories has an Alzheimer's disease overview page, and the front door of that is shown on this website here. 

Thank you

At this point, I'd like to thank you for your attention. Feel free to contact Mayo Clinic Laboratories with further questions.

 

References

1. Jack CR Jr, Bennett D, Blennow K, et al: NIA-AA research framework: Toward a biological definition of Alzheimer’s disease. Alzheimer’s Dement. 2018 Apr;14(4):535-562.
2. Van Harten A, Wiste H, Weigand S, et al: Detection of Alzheimer’s disease amyloid beta 1-42, p-tau, and t-tau assays. Alzheimer’s Associate. Open Access. July 2021.
3. Hansson O, Seibyl J, Stomrud E, et al: CSF biomarkers of Alzheimer’s disease concord with myloid-β PETand predict clinical progression: A study of fully automated immunoassays in BioFINDER and ADNI cohorts. Alzheimers Dement. 2018 Nov;14(11):1470-1481.
4. Schindler S, Gray J, Gordon B, et al: Cerebrospinal fluid biomarkers measured by Elecsys assays compared to amyloid imaging. Alzheimers Dement. 2018 Nov;14(11):1460-1469.
5. Willemse E, van Maurik I, Tijms B, et al: Diagnostic performance of Elecsys immunoassays for cerebrospinal fluid Alzheimer’s disease biomarkers in a nonacademic, multicenter memory clinic cohort: The ABIDE project. Alzheimers Dement (Amst). 2018 Sept 12;10:563-572.
6. Hansson O, Batrla R, Brix B, et al: The Alzheimer’s Association international guidelines for handling of cerebrospinal fluid for routine clinical measurements of amyloid β and tau. Alzheimers Dement. March 2021; 17:1575-1582.
7. Lewczuk P, Beck G, Esselmann H, et al: Effect of sample collection tubes on cerebrospinal fluid concentrations of tau proteins and amyloid beta peptides. Clin Chem. Feb 2006:52(2):332-4.
8. Vanderstichele H, Janelidze S, Demeryer L, et al: Optimized standard operating procedures for the Analysis of Cerebrospinal Fluid Aβ42 and the Ratios of Aβ Isoforms Using Low Protein Binding Tubes. J Alzheimers Dis. May 2016;53(3):1121-1132.
9. Willemse E, van Uffelen K, Brix B, et al: How to handle adsorption of cerebrospinal fluid  amyloid b (1–42) in laboratory practice? Identifying problematic handlings and resolving the issue by use of the Ab42/Ab40 ratio. Alzheimers Dement. 2017;885-892.
10. Toombs J, Foiani MS, Wellington H, et al. Amyloid β peptides are differentially vulnerable to preanalytical surface exposure, an effect incompletely mitigated by the use of ratios. Alzheimers Dement (Amst). 2018;10:311-321. 

Questions?

Contact us: mcleducation@mayo.edu

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