Standardized Procedures for Blood and Cerebrospinal Fluid Collection and Storage in Neurodegenerative Biomarker Research: A Comprehensive Review

Abstract

Blood and cerebrospinal fluid (CSF) biomarkers are essential tools for the rapid diagnosis and monitoring of neurodegenerative diseases like Alzheimer's disease (AD). However, even minor variations in sample collection and storage procedures can significantly impact biomarker measurements, emphasizing the importance of standardized operating procedures. This review discusses the main pre-analytical factors that influence biomarker stability, outlines the best practices for blood and CSF collection and storage, and extensively analyzes recent research findings to ensure optimal reproducibility in biomarker studies. It also discusses the future directions and recommendations for enhancing biomarker research methodologies, including advancements in automated processing and quality control measures.

Keywords: Biomarker; Blood; Cerebrospinal Fluid; Pre-Analytical Phase.

Fig. 1. Recommended blood sample handling standardized operating procedure. Blood should be drawn in the morning (document any other collection times) using a 21-gauge needle (acceptable range, 19–24 G) and EDTA tubes (alternatives permitted by biomarker), with each tube filled to 10–20 mL and gently inverted 5–10 times or placed on a roll mixer to mix. Samples must be centrifuged as soon as possible (within 24 hours of collection); if delayed by up to 3 hours, they can be stored at RT or on ice. If delayed between 3 and 24 hours, they should be stored at 2°C–8°C. Centrifugation should be performed at 1,800 × g for 10 minutes at RT or 4°C. Following centrifugation, plasma should be aliquoted into 250–1,000 µL PP tubes. If immediate freezing is not feasible, aliquots can be stored at 2°C–8°C for up to 24 hours or at –20°C for up to 14 days. However, all samples must eventually be transferred to –80°C for long-term storage. Freeze–thaw cycles should be limited to two or fewer.

EDTA: ethylenediaminetetraacetic acid, g: gravity, RT: room temperature, PP: polypropylene.

Fig. 2. Recommended CSF sample handling standardized operating procedure. Neuroimaging must be performed within one year prior to the lumbar puncture, which should be done in the morning (before noon) with fasting recommended (document any deviations), using an atraumatic needle at L3–L5. The first 2 mL of CSF is always reserved for routine analysis; in the event of a traumatic tap, the sample should be immediately centrifuged at 2,000 × g for 10 minutes at RT, and contamination must be visually confirmed. An additional ≥5 mL of the sample must be collected for research in Sarstedt or other low-binding PP tubes, which should be filled to >90% capacity and stored upright. Samples should be frozen at –80°C as soon as possible; if storage is delayed ≤6 hours, keep at RT or on ice. If the delay extends for ≤2 days, the sample should be stored at 4°C. Centrifugation is not necessary unless contamination is present. Typically, 0.5–1.0 mL of the sample should be aliquoted into each low-binding PP tube, and freeze–thaw cycles must be limited to two or fewer (any additional cycles must be documented).

CSF: cerebrospinal fluid, g: gravity, RT: room temperature, PP: polypropylene.