Cerebrospinal Fluid Biomarkers for Neurological Diseases

Because cerebrospinal fluid (CSF) is adjacent to and interacts with the brain’s parenchyma, it yields brain-specific molecules reflective of the pathophysiology of neurological diseases and brain-based disorders. These include bacterial meningitis, multiple sclerosis and Alzheimer’s disease. For this reason, CSF is particularly valuable for analysis, including biomarker discovery. The identification of novel CSF biomarkers could allow for early diagnosis, prognostic prediction, disease monitoring and the development of neuroprotective treatments.

Since the techniques for obtaining even lumbar CSF samples are invasive, samples from healthy controls are rare, and few large CSF biobanks exist. This means that scientific collaboration for access to this resource is at a premium, requiring standardized sampling and storage protocols. In a recent review, Willemse and Teunissen (2015) indicate that enhanced industry understanding of the value of CSF biobanking and strategies to address long-term storage quality control challenges could reap scientific rewards.¹

According to the authors, pre-analytical errors make up 60% of total laboratory errors and represent a significant issue for CSF biomarker analysis. This preanalytical variation includes factors related to the patient/donor as well as differences in collection techniques and assay performance. Additionally, the specific cellular and biochemical composition of CSF as compared with blood (e.g., lower protein concentration and protease activity) can impact protein stability. Since approximately 85% of CSF proteins derive from blood, contamination with blood can also affect biobanked samples.

Pre-Analytical Variation Factors¹

Patient-derived	Laboratory processing	Assay performance
Diet	Transport time and temperature	Buffer composition
Exercise	Time delay to spinning	Machine settings
Diurnal rhythm	Spinning conditions	Compliance with protocol
Clinical history documentation	Time to freezing	Lack of certified reference materials
Sample labeling	Freezing temperature
	Duration of freezing
	Type of laboratory plastics

When it comes to patient-derived variations, the authors recommend careful documentation for long-term storage and future research purposes. This includes fasting status, caffeine intake, smoking status, alcohol use and exercise, as well as specific time of sample collection. These variabilities can also be addressed through the use of electronic patient records linked with research databases. The authors indicate that 2-D bar coding is the gold standard, facilitating automated sample picking, human error reduction and pseudonymization.

For sampling-derived variations, Willemse and Teunissen offer the following best practices for consistent collection and preparation, drawn from the consensus guideline of the BioMS-consortium:²

Collection Procedures¹

Procedure	Ideal Protocol for CSF
Time of withdrawal and storage	Record collection date and time
Preferred volume	At least 12 ml: 1 to 2 ml for CSF assessment and 10 ml for biobanking Record volume taken and fraction banked
Location	Intervertebral space L3-L5 (S1)
If blood contamination occurred	Do not process further Criteria for blood contimination: >500 red blood cells/μL Record number of blood cells in diagnostic samples
Type of needle	Atraumatic
Type of collection tube	Polypropylene tubes with screw cap, >10 ml volume
Other body fluids collected simultaneously	Serum
	Plasma (EDTA over citrate)

 

Processing Procedures¹

Procedure	Ideal Protocol for CSF
Storage temperature until freezing	Room temperature before, during and after centrifugation
Centrifugation conditions	2,000 g (1,800–220), 10 minutes at room temperature
Time delay between withdrawal, processing and freezing	30 to 60 minutes, maximum 2 hours After centrifugation, aliquot and freeze samples immediately. Maximum delay 2 hours.
Tubes for aliquoting	Small polypropylene tubes (2 ml for routine diagnostics, 1 ml for biobanking) with screw caps. Record manufacturer
Aliquoting	Minimum of 2; the recommended research sample volume should provide >10 aliquots
Volume of aliquots	0.1 ml minimum, depending on total tube volume (0.2, 0.5 and 1 ml) Tubes filled up to 75% of volume
Coding	Use unique codes with freezing-proof labels.
Freezing temperature	−80 °C

So far, researchers believe that CSF biomarkers and neurofilament proteins should be stable during long-term storage. However, there are few studies available and some indication that freezing and thawing could produce structural changes that impact CSF samples. Increasing CSF sample stores could produce experimental data useful in answering this question.

The authors also indicate that, even when facilities use the same commercial kits, they may employ different biomarker outcome levels and cut-off levels. This intra- and inter-laboratory variation has been shown to alter diagnosis in 26% and 12% of cases, respectively, specifically for the Aβ42 biomarker.³ The authors suggest that, in addition to standardizing protocols, establishing an independent quality marker for CSF would mitigate this variation. This would require the identification of a panel of sentinel molecules that are each unstable due to a specific preanalytical step and could thus highlight the source of decreased quality. Because CSF proteins are less abundant and even below the limits of detection in some cases, the identification of these markers would be challenging.

Willemse and Teunissen suggest that collaborative efforts with regard to CSF banking and research have ushered in a new era for the diagnosis and treatment of neurodegenerative diseases, particularly via biomarkers. This, combined with standardization of sampling and processing protocols, could improve biomarker research and directly impact individuals with neurodegenerative diseases.

 

References

1. Willemse, E. A. J. & Teunissen, C. E. (2015) “Biobanking of cerebrospinal fluid for biomarker analysis in neurological diseases,”  Biobanking in the 21st Century: Advances in Experimental Medicine and Biology, 864. doi: 10.1007/978-3-319-20579-3_7

2. Teunissen, C.E., et al. (2009) “A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking,” Neurology, 73 (pp. 1914–1922). doi:10.1212/WNL.0b013e3181c47cc2

3. Vos, S.J.B., et al. (2014) “Variability of CSF Alzheimer’s disease biomarkers: Implications for clinical practice,” PLoS One, 9:e100784. doi:10.1371/ journal.pone.0100784.