Effect of Refrigeration on Alzheimer’s Disease Biomarker Measurements in Plasma

Abstract Background Mobile phlebotomy is an attractive option for the study of Alzheimer’s disease (AD) in rural and underserved populations, but logistics of collecting, and shipping these samples outside clinical settings can introduce additional sources of variability. Knowledge is limited on the impact of field collection on blood‐based Alzheimer’s disease biomarkers, especially protein stability when dry ice is not available during shipping. Our study looks into the effects of holding plasma samples under refrigeration prior to freezing on AD biomarker concentrations compared to samples that were frozen within one hour of collection as is standard practice. Method We used plasma samples from 30 participants (58‐93 yo, mean age = 72.23, SD = 7.31; 18 females, 12 males) from the Wisconsin Registry for Alzheimer’s Prevention and the Wisconsin Alzheimer’s Disease Center, with aliquots from each of them undergoing three different treatments: a reference condition frozen immediately at ‐80°C, and two treatments involving refrigeration at 2‐8°C for either 48 or 72 hours before freezing. We measured the concentration of five AD blood biomarkers (ALZpath pTau 217 , GFAP, NfL, Aβ 40 , and Aβ 42 ) using the Quanterix HD‐X platform. Finally, we evaluated biomarker stability by comparing refrigeration treatments to the aliquots frozen immediately, via repeated measures ANOVAs and ICC analysis. Result We found consistent concentrations of pTau 217 [F(1.78, 44.49) = 0.16, p = .829; ICC(3,3) = 0.987, p < 0.001], NfL [F(1.45, 39.14) = 2.84, p = .086; ICC(3,3) = 0.992, p < .001], and GFAP [F(1.63, 43.89) = 1.66, p = .205; ICC(3,3) = 0.996, p < 0.001] for up to 72 hours of refrigeration. However, both Aβ 42 and Aβ 40 levels in plasma significantly declined after refrigeration [Aβ 40 : F(1.22, 33.07) = 27.38, p < 0.001; ICC(3,3) = 0.537, p < .001; Aβ 42 : F(1.16, 31.34) = 15.35, p < .001; ICC(3,3) = 0.713, p < .001]. Conclusion Our results highlight the sensitivity of plasma Ab to the storage conditions, and show pTau 217 , GFAP, NfL as candidates to study when ideal handling conditions for plasma samples are not easily accessible. Their resilience under refrigeration can provide flexibility to access more remote communities and increase representation in AD research.