Background
Considerations during study design
Item 1: collection of sociodemographic, lifestyle, and health information
Item 2: sample size consideration
Item 3: selection of analytical platforms
Platform | Technology | Core AD biomarker (Assay range in pg/ml) | Remarks |
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IP-MS-Shim [18] | IP-MALDI-TOF | Aβ40 (40–640)a, Aβ42 (11–180)a | • Sample volume: 250 μL • Also measure other Aβ peptides including Aβ38, Aβ39, Aβ3-40, and APP669-711. |
IP-LC/MS/MS | Aβ40 (11–26,400)b, Aβ42 (2–3,920)b, various p-tau and np-tau variants | • Two clinically approved assays were developed based on this platform for identifying brain amyloid plaques in cognitively impaired patients aged 55 and above: PrecivityAD™ measures Aβ42/40 and ApoE proteotype, while PrecivityAD2™ also includes the p-tau217/np-p-tau217 ratio. • Sample volume: 450 μL for Aβ peptides and 1,000 μL for tau variants. | |
LC-MS-Arc [70] | LC-MS/MS | Aβ40 (50–1,000)a, Aβ42 (10–200)a | • Antibody-free LC-MS • Sample volume: 200 μL |
IP-LC/MS/MS | Aβ40 (50–800)a, Aβ42 (10–2000)a, various p-tau and np-tau species | • Sample volume: 250 μL for Aβ peptides and 1,000 μL for tau | |
Quanterix (HD-X) | Bead-based Simoa | Aβ40 (4.08–280), Aβ42 (1.51–100), p-tau181 (8–1280), p-tau217 (0.007–30), t-tau (0.248–360), NfL (1.38–2000), GFAP (0.248–360) | • Up to 4 plex. • Sample volume: 25 to 38 μL per analysis plus 30 μL dead volume |
Lumipulse G1200 | ECL | Aβ40, Aβ42, p-tau181, p-tau217, NfL | • Singleplex • Sample volumeL 70 to 130 μL per analysis plus 100 μL dead vlolume |
Mesoscale Discovery | ECL | p-tau181 (0.46–990), p-tau217 (5.90–2,400), p-tau231 (15.0–15,000), t-tau (0.12–160), NfL (5.40–3,600), GFAP (0.32–850) | • Singleplex for p-tau; 3-plex for NfL, GFAP and t-tau • V-PLEX Aβ Peptide Panels exhibit sub-pg/ml sensitivity, but they haven’t been validated in human plasma. • Sample volume: 25 μL per analysis. |
Roche Elecsys (Cobas) | ECL | Aβ40, Aβ42, NfL, p-tau181, p-tau217, GFAP | • Comercial assays are validated only in CSF samples, with the exception of NfL. Prototype assays are available for other biomarkers [72]. |
Ella [65] | Microfluidic immunoassay | Aβ40 (24.5–2,400), Aβ42 (6.55–1,600), GFAP (72.1–110,000), NfL (2.7–10,290) | • Can simutenously measure up to 8 plexes • Sample volume: 25 μL per analysis |
Alamar (ARGO HT) [73] | NULISA™ | Aβ40, Aβ42, p-tau181, p-tau217, p-tau231, NfL, GFAP, t-tau | • Both singleplex or multiplex assays are available. • NULISAseq CNS Panel targets ~ 120 biomarkers whcih includes all listed biomarkers and other key proteins associated with neurodegenerative disorders.Use 10/20 μL per analysis. • Exhibits fg/ml sensitivity with large dynamic range (up to 12 logs). |
MagQu [74] | IMR | t-tau (1–100), Aβ40 (1- 200), Aβ42 (1–100), p-tau181 (0.02–200), NfL (0.01 to 100), GFAP (1 to 100) | • Singleplex • Sample volume: 40 to 60 μL per analysis. • In contrast to other immunoplatform assays, MagQu employs only a single antibody as a probe for each biomarker, rather than a pair of capture and detection antibodies. • MagQu assays for Aβ peptides often exhibited different trend comapred to other immunoassay platforms [75]. |
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Amyloid plaques, one of the primary pathological features of AD, consist mainly of amyloid beta peptides [77, 78]. While CSF Aβ42/40 has been used in clinical settings to assess brain Aβ plaques, the association of blood Aβ42/40 with AD pathologies has been controversial [79, 80]. Several immunoassays and MS assays are available to measure blood Aβ peptides [20], but overall, there is low inter-platform reproducibility [75, 81]. MS assays generally exhibit superior predictive power for brain Aβ compared to immunoassays, possibly due to higher specificity obtained through MS assays [81].
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CSF t-tau is a biomarker for neurodegeneration or neuronal injury [82]. However, plasma t-tau shows low correlation with CSF t-tau due to potential contamination with tau from peripheral sources [83, 84]. Improved plasma t-tau assays have been reported recently [85, 86]. In addition, recently developed Simoa assay targeting brain-derived tau showed a better correlation with CSF t-tau and improved biomarker performance [21].
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CSF p-tau is a biomarker for neurofibrillary tangles [15, 87, 88]. Despite their low abundance in the blood, several assays are available to measure p-tau species in the blood [13, 89‐92]. Unlike plasma Aβ assays, p-tau assays exhibited overall strong inter-platform concordance [20, 93‐96]. P-tau181, p-tau217, and p-tau231 are the most widely studied p-tau species. P-tau212 is a new marker recently reported [97]. Different p-tau species might increase at different stages of the AD continuum [91, 98]. Unlike their CSF counterparts, blood p-tau exhibits better association with Aβ plaques rather than neurofibrillary tangles.
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GFAP is a biomarker for reactive astrogliosis [99], a cellular response often associated with brain Aβ plaque pathology in AD [100]. Plasma GFAP positively correlated with Aβ burden and tau pathology in AD [101, 102]. Plasma GFAP level may be impacted by non-AD brain injuries and is an FDA-approved biomarker for detecting intracranial lesions after brain injury [103].
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Neuronal damage/injury leads to elevated secretion of NfL into the extracellular space [104]. Although non-AD specific, NfL is an excellent biomarker for neurodegeneration to monitor the disease progression of AD patients [105, 106]. Head-to-head comparison of Simoa and Ella assays in a multiple sclerosis cohort demonstrated a strong correlation between the platforms [64, 65]. Plasma/serum brain-derived tau showed stronger specificity to AD pathophysiology versus related non-AD disorders.
Item 4: selection of blood specimen
Item 5: blood collection from remote areas, under-resourced settings, or home care
Considerations during blood collection
Item 1: preparation of participants for the blood draw
Item 2: blood draw devices
Item 3: blood collection tubes
Item 4: blood draw order
Item 5: blood collection tube filling height
Item 6: proper mixing of blood samples
Considerations during blood processing
Item 1: serum clotting time
Item 2: pre-centrifugation delay time
Item 3: centrifugation settings, including speed, time, and temperature
Item 4: post-centrifugation storage delay
Item 5: good laboratory practice (GLP)
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All blood samples and associated collection devices should be considered potentially infectious, and proper personal protective equipment (PPE) should always be used to minimize exposure risk.
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To protect the confidentiality of research participants, personal information should not be included on specimen labels. To avoid sample mix-up, all tubes should be clearly labeled, preferably using printed labels or barcodes rather than handwritten ones. This labeling should be done in advance of the participants’ visits for blood collection.
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Good pipetting skills are essential for ensuring high sample quality. When pipetting plasma/serum from the blood collection tubes, gently draw the liquid from the top and gradually move the pipette down with the liquid. It is important to avoid disturbing the buffy coat and the cell layers in the plasma tubes and clots in the serum tubes. If allowed, leave the bottom ~ 10% of plasma/serum behind to prevent cross-layer contamination.
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If plasma/serum samples are to be aliquoted into more than one tube, it is important to transfer them from the blood collection tubes to a second, intermediary tube (such as low protein binding conical tubes) after centrifugation. Before aliquoting, the samples should be mixed by inverting the conical intermediary tube or pipetting up and down multiple times to ensure homogeneity. Direct aliquoting from the blood collection tubes right after centrifugation may lead to heterogeneity among aliquots due to the impact of centrifugation forces.
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Hemolysis significantly deteriorates sample quality and is the primary cause of unusable specimens for clinical assays [140]. Therefore, samples should be inspected for signs of hemolysis which may impact the assay results. We recommend using a quick reference chart (such as the CDC Hemolysis Reference Palette) to record the hemolysis scale during specimen collection and checking the influence of hemolysis during data analysis.
Item 6: general procedures for serum collection
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CRITICAL: If not using the glass red top tubes, phlebotomists should gently invert the blood tubes 5 times immediately after blood draw.
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CRITICAL: Place the filled blood collection tubes upright at room temperature for 30 to 60 min to allow the clot to form.
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CRITICAL: If the blood is not centrifuged immediately after the clotting time (30 to 60 min at room temperature), the tubes should be refrigerated (4 °C) for no longer than 2 h.
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Centrifuge clotted tubes balanced by weight for 10 min at 1500 to 2000 × g at 4 °C.
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Use the disposable transfer pipette to transfer the serum (top layer) to a 15 mL conical tube (or 50 mL conical tube if collecting 30 to 100 mL of blood). Be careful not to disturb the clot containing red blood cells, white blood cells, platelets, etc.
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If more than one tube is collected, combine the serum samples from all tubes into the same conical tube.
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Gently invert the conical tube 8–10 times to mix. Aliquot 250 μl to 1 ml into labeled microtubes or cryovials with O-ring-sealed screw leads. Residual aliquots can be saved and pooled as QC samples for repeated analysis.
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Store all aliquots upright in a specimen box in an -80 °C or colder freezer.
Item 7: general procedures for plasma collection
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CRITICAL: Immediately after blood collection, gently invert/mix (180-degree turns) the EDTA tubes 8–10 times. Place the tubes upright on a rack until centrifugation.
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CRITICAL: It is advisable to store collected blood at 4 °C instead of at room temperature before centrifugation. Blood samples should be centrifuged within 2 h of blood collection to minimize degradation of AD biomarkers.
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CRITICAL: In case of unavoidable prolonged centrifugation delay, place blood samples in the refrigerator for no more than 24 h. Avoid direct contact of blood tubes with ice to minimize cell lysis.
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Centrifuge balanced blood collection tubes for 10 min at 1500 to 2000 × g at 4 °C.
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Use the disposable transfer pipette to transfer the plasma (top layer) to a 15 mL conical tube (or 50 mL conical tube if collecting 30 to 100 mL of blood). Be careful not to disturb the buffy coat layer (the whitish layer in the middle) and the red blood cell layer (the red layer at the bottom).
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If more than one tube is collected, combine the plasma samples from all blood collection tubes into the same conical tube.
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Gently invert the conical tube 8–10 times to mix. Aliquot 250 μl to 1 ml into labeled microtubes or cryovials with O-ring-sealed screw leads. Residual aliquots can be saved and pooled as QC samples for repeated analysis.
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Store all aliquots upright in a specimen box in an -80 °C or colder freezer.
Item 8: general procedures for buffy coat collection
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Note: Prepare the following reagents ahead of time and store them at 4 °C.
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Ammonium chloride solution: 7.72 g/L
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Ammonium bicarbonate solution: 0.79 g/L
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Freezing mixture: TriPotassium Citrate: 17.8 g, Sodium Phosphate, monobasic: 2.4 g, Sodium Phosphate, dibasic: 2.8 g, Glycerin (Glycerol): 400 ml; bring volume to 1 L with distilled water.
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Freshly prepare RBC lysis buffer by combining 45 ml ammonium chloride solution and 5 ml ammonium bicarbonate solution.
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After removing the plasma (top layer) from the EDTA or heparin tubes, use another transfer pipette to draw the buffy coat (the whitish layer on top of the RBC layer) and place into the RBC lysis buffer tube (50 ml).
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Mix by pipetting up and down to separate any leftover cells from within transfer pipette.
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Cap the 50 ml tubes with lysis buffer + buffy coat and gently invert several times to mix.
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Incubate at room temp for at least 20 min.
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Add 10% bleach or Cavicide to the used blood tubes (lower layer with RBC) with leftover blood in them; discard in an appropriate biohazard bag.
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After 20 min incubation, centrifuge 50 ml tubes at 4 °C for 20 min at 2500 rpm
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After centrifuging, a white pellet will be visible at the bottom of the tube.
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If no pellet is visible, centrifuge for an additional 20 min.
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If pellet is visible, pour the red supernatant into a beaker filled with 10% bleach or Cavicide.
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Let pellet dry (approximately 10–20 min).
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Add 1 ml of freezing mixture to pellet. [Freezing Mixture: TriPotassium Citrate: 17.8 g, Sodium Phosphate, monobasic: 2.4 g, Sodium Phosphate, dibasic: 2.8 g, Glycerin (Glycerol): 400 ml; bring volume to 1 L with distilled water].
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Gently mix to break the pellet into single cell suspension.
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Transfer whole (cells + freezing mix) into cryotubes.
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Store at -80 °C for subsequent DNA isolation for genetic studies.
Item 9: detailed step-by-step SOP for blood collection and processing
Considerations for biobanking
Item 1: storage tubes and temperature
Item 2: freeze/thaw cycles
Item 3: transportation
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Include an accurate sample list (including accurate volumes). Ideally, provide a Microsoft Excel sheet (or similar) with columns for sample IDs, their corresponding volumes, and their location in the box. Adding a pictorial illustration of sample arrangements in the boxes is also helpful.
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Inform the receiving laboratory ahead of time so that they will keep a lookout and be able to receive and store them in good time. The receiving lab may need to find freezer space before your samples arrive. Therefore, it is important that they are informed ahead of time.
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Ensure that the package contains an adequate amount of dry ice. For long-distance transportation that spans multiple days, choose a reliable company that can refill the dry ice midway.
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Print labels using a computer, rather than handwriting them, to ensure better legibility.
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Use tubes with caps that do not become loose accidentally.
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Sort the samples in the order you want them analyzed. If you are unsure of the order, there are two main rules:1)If your samples are in groups: randomize them, so that all groups are represented in all analytical runs.2)If you have longitudinal samples, keep all samples from the same participant together and in the order in which they were collected. Ensure your sample coding reflects this ordering.
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Do the final sorting of the samples BEFORE you send them. It might take longer and be too time-consuming for the receiving lab to do it.
Considerations during biomarker measurements
Item 1: preparation of samples for measurement
Item 2: inclusion of calibrators
Item 3: inclusion of QC samples
Item 4: sample measurement order
Item 5: sample blinding
Item 6: assay order
Item 7: the use of bridging samples
Item 8: longitudinal samples
Considerations for result reporting
Item 1: demographic and clinical information
Item 2: full description of methods
Item 3: disclosure of assay performance
Unresolved issues
Certified reference materials
Real world applications
Cutpoints
Conclusion
Pre-analytical | Analytical | Post-analytical |
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• Participant preparation: fasting status, blood draw time • Plasma vs. serum • Blood draw devices • Blood collection tubes • Blood draw order • Blood tube filling height • Blood tube inversion • Serum clotting time • Time from blood draw to centrifugation • Centrifugation parameters, including speed, time, and temperature • Time from centrifugation to storage • Storage temperature • Microtubes for specimen storage • Freeze/thaw cycles • Transportation temperature • Proper tube labeling to minimize error | • Sample thawing and homogenization • Centrifugation for particulate removal • Prompt storage of unused specimen • Inclusion of calibrators • Inclusion of QC samples • Sample measurement order • Sample blinding • Assay orders for multi-assay studies • Inclusion of bridging samples • Order for longitudinal samples • Batches/lots for reagent and consumable • Selection of analytical platforms | • Evaluation of calibrator curves • Evaluation of QC results • Adjustment of potential moderating variables • Full description of methods • Disclosure of assay performance |