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, in 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. Today, we'll be talking about Alzheimer's CCCSF biomarkers.
These are our disclosures.
The two neuropathologic features found in the brain of patients with AD dementia are
the presence of plaques composed of amyloid beta peptides and intracellular neurofibrillary tangles
containing hyperphosphorylated tau proteins. These two groups of molecules are the most established
biomarkers of the disease using clinical and research practice. In recent years, measurement
of a-beta peptides, especially a-beta 42 and tau proteins, including total and phosphorylated
forms of tau, in CSF are being proposed as an alternative to PET imaging to assess AD pathology.
In CSF, the changes observed include a 50% reduction of A-beta 42 as consequence of
amyloid deposition in the brain, and an increase of p-Tau and total tau.
The increases in both forms of tau are believed to reflect the extent of neurodegeneration,
with phosphorylated tau being a more specific marker for Alzheimer's Disease.
Recently, the use of these biomarkers has been included in the consensus research diagnostic
criteria for AD proposed by the National Institute of Aging and Alzheimer's Association
research framework. This is referred as the A/T/N system,
and it captures the three main neuropathologic findings related to Alzheimer's Disease.
A refers to A-beta pathology, measured by either amyloid PET
or CSF A-beta 42, or the A-beta 42 to A-beta 40 ratio.
T represents tangle pathology, and is assessed by either Tau PET, or CSF phosphorylated tau.
N stands for neurodegeneration, or neuronal injury, detected by
either FDG PET, structural MRI, or CSF total tau.
The normal and abnormal classification of these markers result in eight different ATN biomarker
profiles, as shown in this figure. Based on the definition of Alzheimer's Disease pathological
change, the ATN biomarker system aligns every individual to one of the three biomarker
categories: individuals with normal AD biomarkers, those with the Alzheimer's
continuum, and those with normal amyloid biomarker but an abnormal T, or N, or both.
The Mayo Clinic Laboratory 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 amyloid beta 42, total tau and phosphorylated tau at position
181. All these assays are from Roche Diagnostics. We require that the CSF sample is collected on a
low-bind polypropylene tube, and the report includes the individual biomarker results
in addition to the calculation of the p-Tau 181 to A-beta 42 ratio.
The clinical performance of this assay was validated at Mayo in collaboration with Dr. Ron
Peterson. The study included participants from the Mayo Clinic Study of Aging, and the Mayo Clinic
Alzheimer's Thesis 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-Tau 181 to A-beta
42 ratio, as demonstrated by the higher area under the curve of the receiver operator curve of 0.97.
Using the cut points indicated on this table, the p-Tau 181 to A-beta 42 ratio
shows 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 BioFINDER and Washington University,
using the same assays from Roche Diagnostics reported the use of similar cut points
for the p-Tau/A-beta 42 ratio, as well as similar, positive and negative agreements with amyloid PET.
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-Tau 181 to A-beta 42 ratio, interpretation of the results is only made based
on the ratio. In this example, the p-Tau 181 to A-beta 42 ratio is abnormal, which
is consistent with the presence of pathological changes associated with Alzheimer's Disease.
This next report is an example of a case where the A-beta 42 concentration is above
the measuring millimeter of the assay. In this case, A-beta 42 is reported as greater than
1700 micrograms per mL. This result will not be consistent with pathological changes associated
with Alzheimer's Disease, and there is no need to calculate the p-Tau 181 to A-beta 42 ratio.
We have seen some cases where the p-Tau 181/A-beta 42 ratio is normal in spite pf an
abnormally low A-beta 42 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 phosphorylated tau. Alternatively, this may represent a disorder of CSF dynamics,
such as normal pressure hydrocephalus, where both A-beta 42 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/A-beta 42 ratio is normal,
or close to the cutoff as shown in this case, the recommendation will be to consider
other non-AD causes of cognitive decline, and repeat testing in about 6 to 9 months.
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 A-beta and tau protein
concentrations. Most importantly, these include tube material, and Aliquot, and fill volume.
The absorption of A-beta CSF marker analysis is related to the tube type.
This is more relevant for the A-beta 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 A-beta 42 measurement, and consequently,
produce a falsely high p-Tau over 181 to A-beta 42 ratio. This potentially
increases clinical suspicion of AD. One reason for this is that a decrease in A-beta 42
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 A-beta 42 does.
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 A-beta 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 A-beta peptide it's 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 A-beta stickiness.
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 A-beta 42 concentrations,
potentially then affecting clinical interpretation.
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 to 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 A-beta. Absorption occurs within 30 seconds of the transfer.
Because exposing A-beta to more surface area can reduce the measured concentrations of A-beta,
one should avoid tube transfer prior to sending to Mayo Clinical 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 volume." This
footnote will alert clinicians for the potential of a low sample volume submission on results.
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 to 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 AB beta 42 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.
As both of these phenomenon, 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 A-beta 42 concentration.
This affects subsequent interpretations, as well as the p-Tau to A-beta 42 ratio."
For additional information, both in terms of interpretation of this assay,
appropriate ordering, and specimen collection instructions, one can refer to the Mayo Clinical
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. At this point, I'd like to thank
you for your attention. Feel free to contact Mayo Clinical Laboratories with further questions.