December 12, 2022
Sysmex Corporation【Overview presentation】
15th Clinical Trials on Alzheimer’s Disease (CTAD)

Effects of pre-analytical parameters on plasma β-amyloid levelAuthors

Kengo Ishiki1, Kazuto Yamashita1, Shunsuke Watanabe1, Masahiro Miura1, Shigeki
Iwanaga1, and Toshiyuki Sato1

1Central Research Laboratories, Sysmex Corporation

Overview
presentation

Backgrounds
Blood-based biomarkers of Alzheimer’s disease (AD) are expected to be promisingtools for assisting diagnosis in combination with conventional biomarkers such asneuroimaging and cerebrospinal fluid (CSF) biomarkers. In particular, the ratio ofplasma β-amyloid1-42 (Aβ42) to β-amyloid 1-40 (Aβ40) is known to be associated withbrain Aβ pathology. Therefore, it could assist in the selection of the candidates forclinical trials of disease-modifying therapies targeting Aβ. We have developed assaysthat can specifically measure Aβ40 and Aβ42 in plasma using a fully automatedimmunoassay platform, the HISCLTM series. The assay showed high performance forpredicting brain Aβ pathology defined by amyloid positron emission tomography(PET).
However, it is widely recognized that plasma Aβ peptides are unstable molecules,
and thus the measured values are affected by pre-analytical parameter such asexternal factors, measurement equipment and sample handling. Especially, bloodcollection and storage conditions in sample handling affect the quality of plasma Aβlevels, leading to false selection.
Therefore, clarification of the influence of pre-analytical parameters on plasma Aβlevels measured by our assay is required to obtain reliable data in clinical trials.

Objectives
The purpose of this study was to clarify the effect of pre-analytical parameters onplasma Aβ levels measured by HISCL series.
Methods

Whole blood samples were collected in K 2 EDTA tubes from healthy volunteers.
After centrifugation, plasma Aβ40 and Aβ42 levels were immediately measured using
the HISCL series and plasma Aβ42/Aβ40 was calculated.
We compared the impacts of the 11 pre-analytical parameters such as duration
between blood collection and centrifugation at room temperature (RT) or 4°C, theinterval between plasma storage at RT or 4°C before measurement, the number offreeze/thaw (F/T) cycles, the conditions of thawing, the length of time that the plasma
was thawed before measurement, the types of tubes and manufacturers for storingplasma, the tip type using plasma separation, and the number of tube transfers on
plasma Aβ42/Aβ40.

Results
Plasma Aβ42/Aβ40 levels remained unchanged after 2 hours at RT and 6 hours at
4°C after blood collection. After the plasma separation, it is unaltered even after 18
hours of storage at 4°C. Longer sample storage time and higher storage temperature
after plasma separation tended to affect the reduction of the plasma Aβ42/Aβ40 ratio,
but did not have an effect beyond our established criteria. The time for holding thethawed plasma at RT was also investigated, and no difference was detected until 4hours. For the freezing samples, up to three F/T cycles did not significantly change
the Aβ42/Aβ40 ratio compared to the reference value. Thawing at RT and 37°C werealso used to assess the thawing state. The 37°C conditions were verified using awater bath, a dry block incubator, and an air incubator. Furthermore, neither of thethawing conditions affects the Aβ42/Aβ40 values. The Aβ42/Aβ40 values areunaffected by tube manufacturer, tip type, or tube transfer up to one time.
Conclusion
In this study, we evaluated the influences of 11 pre-analytical parameters on plasma
Aβ42/Aβ40 ratio measured by HISCL series. In our evaluation, the sample storage
time and temperature after plasma separation were the most influential factors inplasma Aβ42/Aβ40 ratio. These results were consistent with the previous reports ofpre-analytical handling using another assay platform.
This study will contribute to the determination of pre-analytical handling for plasma
Aβ measurement using HISCL. By establishing pre-analytical handling based on thefindings of this study, the high amyloid PET predictive performance we have shown
so far could be achieved in clinical trials.
SessionPoster, Clinical Trials: Biomarkers including plasma（LP74）