Paris: Standardization a Hurdle for Spinal Fluid, Imaging Markers

In the fall of 2009, a large quality control (QC) program started
comparing measurements of cerebrospinal fluid (CSF) biomarkers from labs
across the world (see ARF related news story).
The aim was to come up with standard procedures that would ensure
consistent results. Two years later, the initiative’s main
accomplishment has been to recruit more than 60 labs to participate in
the project, but consistency across labs remains elusive, according to
presentations at the 11th annual Alzheimer’s Association International
Conference (AAIC, formerly ICAD) held 16-21 July 2011, in Paris, France.

Achieving consistency across labs has emerged as a key challenge in
the field, which in the past two decades has amassed strong evidence
that CSF markers can predict AD. Consistency is necessary if this
evidence is ever to be applied to multicenter treatment trials, and
especially in broad clinical settings beyond a few leading academic
medical centers. Brain imaging faces its own standardization challenges.
Compared to CSF biochemistry, this field is at an even earlier stage in
that regard, as large-scale standardization initiatives are only just
beginning to form.

Three times a year, the Alzheimer’s Association Cerebrospinal
Fluid Quality Control Program, headquartered at the Sahlgrenska
University Hospital in Molndal, near Göteborg, Sweden, sends out for
testing three CSF samples to each of the participating labs. Two of the
samples are unique to each round of testing, and serve to gauge the
variability in measurements among labs; the third sample is the same
every time to assess longitudinal stability of measurements. At the same
time, four reference laboratories process several additional copies of
every CSF sample to determine the variability in measurements within a
single lab (see ARF related news story).
The samples are analyzed for the presence of the three most established
biomarkers: amyloid-β42 (Aβ42), total-tau (t-tau) and phosphorylated
tau (p-tau).

At AAIC, Kaj Blennow at Sahlgrenska University Hospital, who heads the QC program, presented the results of the first six rounds of testing (Mattsson et al., 2011).
He showed scatter plot graphs of biomarker measurements, indicating
variation among laboratories in the range of 13 to 36 percent. Variation
of less than 5 to 10 percent is the goal. "You can see quite marked
drift among labs," said Blennow.

Variation was similar irrespective of the assay kit used for the
test (i.e., INNOTEST ELISAs, Luminex xMAP or MSD). Furthermore, the
group did not see differences in variability among rounds of testing, in
other words, variability is not yet going down with repeat testing.
"Variability for tau is a bit less than for Aβ42 but still higher than
we need," said Blennow’s colleague Henrik Zetterberg, also at the Sahlgrenska University Hospital.


left panel shows results from all labs doing the INNOTEST amyloid-β42
(Aβ42) ELISA on one of the unique CSF samples sent to them, with one lab
indicated in color. The panel on the right shows the percent deviation
in measurements obtained by the same lab for the longitudinal sample
over six rounds of testing (two measurements performed for each round).
Both panels show marked variation. Although some of the reference labs
that are part of the QC initiative are able to obtain reproducible
results time after time, most participating laboratories do not yet
produce consistent results. View larger image. Image credit: Kaj Blennow

But despite this seeming lack of progress, Zetterberg said there is
reason to be optimistic. "The reference laboratories that are part of
the initiative are collecting longitudinal data, and those labs do
produce stringent results that are reproducible time after time, except
that we sometimes see changes due to batch variation among kit lots," he
said. "This means that standardization across labs should also be

There are many possible sources of variation in measuring CSF biomarkers, which have been previously discussed on Alzforum (see ARF related news story).
In order to pinpoint the main ones and eliminate them from future
rounds of testing, the Sahlgrenska group has started asking
participating labs to fill out a checklist for each analytical
technique. The checklists include information on assay reagents and
instruments, details on sample handling and storage, such as which kinds
of pipette tips or plates the technician used, as well as questions
about internal control samples, assay conditions, settings for data
analysis, and so on. They will be available on the program’s website.

The QC program has already developed standardized protocols for
participating labs to follow when measuring CSF biomarkers, but they are
"not detailed enough," Zetterberg told ARF. "We are collecting more
detailed information and in the long-run will be able to reduce
variability." The group will conduct hands-on practical courses to train
lab personnel on how to carry out the standardized protocols (see ARF related news story).

Moreover, the program is pushing for better assay kits on the
market. "Biomarkers in other fields of medicine (i.e., troponin-T and
cholesterol) have gone through similar standardization procedures, and
biotech companies producing this type of assay have put in the time,
effort, and money it takes to make highly validated tests. We hope that
the same will come true for these AD CSF biomarkers," wrote Blennow in
an e-mail to ARF.

Zetterberg stressed that the observed variability in CSF
biomarkers measurements among labs does not diminish the value of these
markers in research. "A lab that has established rigorous methods of its
own can reliably monitor relative changes in biomarkers," he said. As
an example, at AAIC, Zetterberg presented a study showing that relative
changes in biomarker levels can be used to predict with high certainty
which patients with mild cognitive impairment (MCI) will go on to
develop AD. His group measured CSF biomarker levels in 137 people with
MCI at the start of the study and then followed these people clinically
for more than nine years. Previous studies have reported that CSF
markers predict AD risk at the MCI stage, but not with follow-up that
long. During those nine years, 54 percent developed Alzheimer’s, and
those people who did had had lower CSF Aβ42 levels and higher t-tau and
p-tau when compared to people who did not. "Aβ is already down nine to
10 years before progression to AD," said Zetterberg. The ratio of
baseline Aβ42/p-tau predicted the development of AD within 9.2 years
with a sensitivity of 88 percent and specificity of 90 percent.

Eventually, researchers would like to have biomarkers that
determine whether someone who has no clinical symptoms of MCI is at risk
for developing AD—somewhat akin to taking a cholesterol measurement to
ascertain one’s risk for cardiovascular disease. At AAIC, Zetterberg
said his group just completed a study of 86 healthy people who were
followed for 13 years. Fourteen of them developed AD during this time.
Preliminary data from his lab indicate that those individuals with CSF
Aβ42 levels lower than 700 pg/mL are at higher risk for developing AD.
"But I do not advocate screening at this stage," said Zetterberg. "These
data are purely for research purposes."

In future, if researchers do find reliable CSF biomarkers for
preclinical AD, having consistent results across labs will be essential
to instituting universal cutoff levels that can be used to determine
risk or establish a diagnosis. Standardization will also be necessary
for comparing results of studies from different labs.

Think Standardizing CSF Is Hard? Try Hippocampal MRI

In the wake of the CSF Quality Control Program, other initiatives are
springing up to make diagnostic markers for the early detection of AD
robust across centers. Giovanni Frisoni of the San Giovanni di Dio Fatebenefratelli Hospital in Brescia, Italy, and Clifford Jack of the Mayo Clinic in Rochester, Minnesota, are heading an initiative formally named A Harmonized Protocol for Hippocampal Volumetry: An EADC-ADNI Effort. (EADC stands for the European Alzheimer’s Disease Consortium, and ADNI is the U.S.-based Alzheimer’s Disease Neuroimaging Initiative).

Hippocampal volumetry has proved its value in aiding AD diagnosis and in tracking disease progression (see ARF related news story on Erickson et al., 2011).
Before it can move into wider clinical use "we have to agree precisely
on what to measure," said Frisoni. "Measurements must be standardized so
that they can be used in all memory clinics all over the world."

As a first step in this process, Frisoni’s group surveyed all
published protocols for assessing hippocampal volume. "If you look at
the hundreds of publications on the subjects, you’ll find tens of
different ways of segmenting the hippocampus. Of these, 12 are the most
popular among scientists," said Frisoni. Segmentation is the way
outlines of brain structures are drawn on magnetic resonance images to
delineate those structures; using different segmentation techniques
leads to different estimates of hippocampal volume.

Frisoni’s group took the 12 most popular protocols and evaluated
the information each one provides and its reliability in measuring
AD-atrophy. These data were then fed to an international task force of
experts who were asked to harmonize currently used segmentation
protocols and come up with a single standard procedure for everyone to
use. The task force comprises principal investigators at EADC and ADNI
centers as well as other imaging centers across the world, comprising
more than 30 groups in all.

The task force is currently developing and testing the harmonized
protocol. Once it has been defined in minute detail, researchers at
different imaging labs participating in this effort will have a chance
to compare it to the segmentation procedures they currently use.
Following this validation step, Frisoni and Jack plan to create a Web
portal for people to obtain certification as an expert Qualified
Hippocampal Tracer, wrote Frisoni in an e-mail to ARF. Tracers are
people in imaging labs with expertise in brain anatomy; for the
certification, they will need to manually trace the boundaries of the
hippocampus slice by slice on a high-resolution computer screen. In
addition, the group plans to develop educational material on how to use
the harmonized protocol and hippocampal probability maps that will
provide a reference for the tracings.

The finalized guidelines should be made available to the research
community in September 2012, said Frisoni. Once available, they will be
open to discussion and validation. More information and updates on the
program can be found on the EADC-ADNI Initiative website.—Laura Bonetta


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