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Stability of Proteins in Dried Blood Spot Biobanks.

Author(s): Björkesten J, Enroth S, Shen Q, Wik L, Hougaard DM, Cohen AS, Sörensen L, Giedraitis V, Ingelsson M, Larsson A, Kamali-Moghaddam M, Landegren U

Publication: Mol Cell Proteomics, 2017, Vol. 16, Page 1286-1296

PubMed ID: 28501802 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared protein levels in matched dried blood and plasma specimens to matched liquid specimens from a single volunteer. Some of the EDTA blood was transferred to a new tube and stored at 4˚C while the remainder was centrifuged for 15 min at 2000 x g to obtain plasma. DBS and dried plasma specimens (DPSs) were prepared in duplicate by allowing a 25 µL droplet of EDTA blood or plasma on the tip of a pipette to absorb onto Whatman DMPK-C sample paper. DBSs and DPSs were left to dry for 3 h at room temperature. Disks were punched from each DBS and DPS using a 1.2 mm Uni-Core micropuncher. The abundance of 92 proteins in DBS, DPS, blood, and plasma were determined using multiplex proximity extension assays (PEA) which uses real-time PCR-based amplification of oligonucleotides conjugated to antibodies to quantify protein levels.

   Summary of Findings:

   Background levels were higher in dried specimens than in matched liquid specimens, but protein levels were very strongly correlated for blood (ρ=0.967-0.972) and plasma (ρ=0.968) regardless of adjustments for the higher background. However, the correlations were higher between technical replicates (ρ=0.999 for wet blood, dried blood, and wet plasma and ρ=0.994 for dried plasma) and between specimens collected from the middle versus edge of the DBS (ρ= 0.992-0.998). Slightly higher levels (<15%, P<0.05) of 10 of the 92 proteins and lower levels of 1 of the 92 proteins were found in the edge compared to the middle of the DBS, but none of the differences remained significant after correcting for multiple testing. As expected, protein levels were only modestly correlated between blood and plasma for both wet (ρ=0.631-0.650) and dried (ρ=0.600-0.604) specimens, reflecting the inclusion of cellular proteins in whole blood.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Other Preservative

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immuno-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Whole blood Plasma
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Dried blood spot sub-sampling (exact positions not specified)
   Biospecimen Preservation	Type of fixation/preservation	Air-dried None (fresh)

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2. Study Purpose

   This study compared levels of 92 proteins in archival neonatal DBS and fresh neonatal DBS. For each storage duration (0.5, 10.5, 20.5, 30.5, and 40.5), five DBSs collected in the month of July of the relevant year were obtained from each of the Swedish and Danish Newborn Screening Programs for Inherited Metabolic Disorders. Swedish specimens stored for 40.5 years were stored at room temperature for six years before transfer to 4˚C. All other specimens in this cohort were stored at 4˚C but humidity control and the type of paper used changed several times. All Danish specimens were kept at -24˚C, but the type of paper used changed over time. For comparisons, five fresh DBSs were prepared in December and stored for a few weeks at 4˚C and five DBSs were prepared in January and stored at -24˚C until assay. Disks were punched from DBSs using a 1.2 mm Uni-Core micropuncher and placed in the reaction solution. The abundance of 92 proteins in DBS, DPS, blood, and plasma were determined using multiplex PEA.

   Summary of Findings:

   During storage at 4˚C, there was a general trend toward decreasing protein levels with increased storage duration, but significance was only evaluated for adjacent storage time intervals (e.g., 0.5 and 20 years versus 10 years but not 20 years versus 0.5 years). DBSs stored at room temperature for six years followed by 34 years at 4˚C had significantly lower levels of protein than those stored at 4˚C for 30 years (P<0.001). DBSs stored for 10 years at 4˚C had lower protein levels than those stored for 0.5 years (P<0.05). Interestingly, freshly obtained DBSs frozen at -24˚C had non-significantly lower average levels of protein than DBSs stored for 0.5 years but then only small decreases were noted with storage at -24˚C until 20 years. Significantly lower average protein levels were noted in DBSs stored at -24˚C for 20 years compared to 10 years (P<0.05) and for 30 years compared to 20 years (P<0.05).

   Further analysis revealed that levels of 61 proteins declined with storage at 4˚C and 22 proteins declined with storage at -24˚C. Of the 22 proteins affected by frozen storage, 20 were also affected by refrigerated storage and the half-life of 19 of these proteins was longer at -24˚C than at 4˚C. Importantly, while protein abundance was higher in specimens stored at -24˚C than 4 ˚C for 10 (P<0.001) or 20 years (P<0.001, and P<0.05, respectively) it was comparable between storage temperatures when stored for 30 years. Among the proteins most affected by storage was TXLNA, for which storage duration accounted for 95% of the variation at 4˚C and 84% of the variation at -24˚C. Overall, storage duration accounted for 37% of the variation in DBS protein levels.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immuno-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	4˚C -24˚C
   Storage	Storage duration	Weeks 0.5 years 10.5 years 20.5 years 30.5 years 40.5 years

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3. Study Purpose

   This study compared levels of 92 proteins in serum and plasma and investigated the effects of storage duration and patient age. Plasma and/or serum samples from six men stored at -70˚C for 45, 22, 16, 13, 7, or 2 years were obtained from the Uppsala Longitudinal Study of Adult Men (ULSAM) Biobank. The specimens may or may not have been thawed for prior studies. To normalize data for patient age and specimen type, an additional five matched plasma and serum specimens were obtained from men in each of the required age groups (50, 60, 70, 80, and 90 ±5 years) and frozen at -70 ˚C. The abundance of 92 proteins in serum and plasma were determined using multiplex PEA.

   Summary of Findings:

   Of the 92 proteins assayed, 58 were significantly affected by donor age and 58 differed between serum and plasma. However, only five proteins (S100A4, TGFα, HGF, ADAM 8, and ANXA1) were shown to be affected by storage at -70˚C after correction for multiple testing. Of these, S100A4, HGF and ADAM8 were also significantly affected by storage of DBS at 4˚C but none were affected by storage at -24˚C. Importantly, only 3.9% of the variation for these specimens was explained by storage duration.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immuno-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	50 ±5 years 60 ±5 years 70 ±5 years 80 ±5 years 90 ±5 years
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Storage	Storage duration	45 years 22 years 16 years 13 years 7 years 2 years

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