Inside lab’s experience with assays for Alzheimer’s

Amy Carpenter Aquino

October 2023—Orders for cerebrospinal fluid testing for Alzheimer’s disease have grown at Mayo Clinic since spring 2020, when testing was first offered. When aducanumab was approved in May 2021, test orders jumped. And with lecanemab having received the Food and Drug Administration’s full approval on July 6, “we expect to see another leg up,” said Joshua Bornhorst, PhD, D(ABCC), speaking at the Association for Diagnostics and Laboratory Medicine meeting in July, where he reported Mayo’s experience with the Roche and Fujirebio assays.

“AD biomarkers can be implemented successfully in clinical laboratories,” said Dr. Bornhorst, consultant and assistant professor, Mayo Clinic Department of Laboratory Medicine and Pathology. “There are now FDA-approved options that generally show excellent precision, and there are instruments that are widely accessible.” The strict preanalytical protocols are a downside, and payment is still unresolved, though some insurers have it under consideration, he said.

“One shouldn’t base interpretation results on the ratios alone because comorbidities and other neurological disorders can affect the results of individual components,” said Dr. Bornhorst, who directs Mayo’s metals laboratory and co-directs the clinical immunoassay laboratory with Alicia Algeciras-Schimnich, PhD, professor of laboratory medicine and pathology, who was the scheduled presenter but unable to attend.

A workgroup of the National Institute on Aging and the Alzheimer’s Association presented in July at the Alzheimer’s Association International Conference a draft proposal for new Alzheimer’s disease diagnostic criteria. The criteria are a revision of the 2018 NIA-AA Alzheimer’s research framework and “further elucidate the potential role of biomarkers,” Dr. Bornhorst said. New to the draft revision is the incorporation of blood-based biomarkers.

Aβ42, one of the core CSF biomarkers, is decreased about 50 percent in Alzheimer’s disease due to the brain amyloid deposit.

“It’s thought that as these plaques form, it comes out of the CSF and can be measured in isolation, but more commonly it’s measured as a ratio”—Aβ42/Aβ40—“because Aβ40 is not incorporated as much and is not decreased,” Dr. Bornhorst said. “So it normalizes some of the measurements.”

Phosphorylated-tau represents increased tangle density, and several forms of p-tau are being evaluated, he said. Mayo Clinic uses p-tau181. “There’s more and more evidence that p-tau217 may be as or more effective in some cases, although the literature is mixed,” he said. Total-tau is a nonspecific general marker of neurodegradation and is included in some of Mayo Clinic’s panels. “NfL [neurofilament light chain] is coming on as a nonspecific marker of neurodegradation as well and has been incorporated in some clinical trials in recent studies of Alzheimer’s disease.” Total-tau and p-tau both rise to about 200 percent relative to concentrations found in the CSF of non-AD-affected individuals.

In the NIA-AA’s A/T/N biomarker classification system for Alz­heimer’s disease, A stands for amyloid pathology and is measured by amyloid PET or CSF biomarkers Aβ42 or Aβ42/Aβ40. T is tangle pathology, which is assessed by tau-PET or CSF p-tau. N is neurodegeneration or neuronal injury, detected by 18F-FDG PET, structural MRI, or CSF t-tau. Other nonspecific markers of neuronal injury such as NfL are emerging.

Based on the A/T/N biomarker combinations and whether each marker is negative or positive, there are eight commonly seen biomarker profiles. The four on the Alzheimer’s continuum are as follows: A+T−(N)−: Alzheimer’s pathologic change; A+T+(N)−: Alzheimer’s disease; A+T+(N)+: Alzheimer’s disease; and A+T−(N)+: Alzheimer’s and concomitant suspected non-Alzheimer’s pathologic change. Three profiles in which there may be an elevated N or T marker without a corresponding decrease in amyloid “may be representative of non-Alzheimer’s pathologic change and may point the clinician in a different direction,” Dr. Bornhorst said. Normal Alzheimer’s disease biomarkers are A−T−(N)−.

Mayo Clinic has offered CSF biomarker testing for Alzheimer’s disease evaluation since March 2020, when the laboratory launched its automated Elecsys Gen 1 panel consisting of Aβ42, t-tau, and p-tau181.

Low-bind polypropylene tubes are used to collect CSF and provided by the laboratory. They reduce the amyloid deposition that can occur upon collection (Van Harten AC, et al. Alzheimers Dement. 2022;​18[4]:635–644).

The test is a laboratory-developed procedure, Dr. Bornhorst said. “We based cutoffs on concordance to amyloid PET based on an in-house study. At Mayo, we’re fortunate to have the Mayo [Clinic Study of] Aging cohort, from which we had a large number of samples from people who had been neurologically evaluated and diagnosed with Alz­heimer’s or non-Alzheimer’s.”

Mayo Clinic began last year to also offer the FDA-cleared Fujirebio Lumipulse G β-Amyloid Ratio (1–42/1–40) in vitro diagnostic test.

The p-tau/Aβ42 ratio cutoff point of less than or equal to 0.023 is the important number for the Roche Gen 1 assays, Dr. Bornhorst said. “It provides the optimal balance between negative percent agreement and positive percent agreement when compared to amyloid PET results.” The normal cutoff point for Aβ42 is greater than 1026 pg/mL, for p-tau181 less than or equal to 21.7 pg/mL, and for t-tau less than or equal to 238 pg/mL. “With our population, we’ve got about a 92 percent concordance with both positive and negative with amyloid PET,” he said. Important to note, he cautions, is that these cutoff values can change and have changed with assay modification and restandardization.

The Alzheimer’s Association consensus protocol recommendations call for a lumbar puncture, discarding the first 1 to 2 mL of CSF, and using a gravity-drip method for CSF collection directly into a low-bind polypropylene tube. The syringe-pull method increases collection speed, but the drip method exposes the sample to less manipulation, metal, and plastic and reduces the risk of Aβ42 binding to the plastic of the syringe, which could affect the ratio. The Sarstedt CSF false-bottom tube 63.614.625 (2.5 mL) is preferred.

Internal studies revealed that if the collection tube is at least 50 percent full, there is little volume-to-tube-surface effect, Dr. Bornhorst said. “We put footnotes on tubes that are less than 50 percent full” because low sample volume can affect the overall interpretation.

Compliance with collection tube requirements in 2020 was at about 60 percent. “Any laboratory offering these tests, I believe, will have that compliance challenge,” he said. Footnotes and client education raised compliance to its rate this year of about 90 percent.

In Fig. 1 is a laboratory report. “The important value is the p-tau/Aβ42 value,” Dr. Bornhorst said, noting it is 0.010 and reported as a normal profile, not consistent with the presence of pathological changes associated with Alzhei­mer’s disease. The components are reported quantitatively but without interpretation. An elevated t-tau level may point to other neurological diagnoses.

Dr. Bornhorst reported one year of the laboratory’s experience with the Elecsys Gen 1 assay.

About 25 percent of results were all normal, 25 percent all abnormal, and 40 to 50 percent were mixed ratios. “Because the ratio is diagnostically better and has been established at many points in individual studies, we feel that we make the abnormal/normal call based on the ratio, even if all the markers are not abnormal, and that’s been borne out in our concordance studies to the presence of amyloid PET,” he said.