Importance of cerebrospinal fluid (CSF) collection protocol for the accurate diagnosis of Alzheimer's disease when using CSF biomarkers

Dear Editor,

The recent clinical use of amyloid‐targeting therapies for Alzheimer's disease (AD) has prompted increased interest in the use of cerebrospinal fluid (CSF) biomarkers of AD. Decreased amyloid β (Aβ1‐42, Aβ42) and increased phosphorylated Tau (pTau) in CSF are surrogate markers for brain amyloid pathology. ¹ Currently there are two FDA‐approved assays for measuring these biomarkers in CSF: Fujirebio Lumipulse G β‐Amyloid Ratio (Aβ42/40) and Roche Elecsys pTau181/Aβ42 ratio. ² , ³ In these assays, a decreased Aβ42/40 ratio or an increased pTau181/Aβ42 ratio in CSF are consistent with the presence of brain amyloid pathology.

Historically, AD CSF biomarker measurements have been challenging as they may be influenced by pre‐analytical sample collection. This is particularly problematic for Aβ42 as the peptide is prone to aggregation and surface binding potentially leading to a falsely low Aβ42 concentration when the CSF is not collected under optimal conditions. ⁴ Current guidelines for the handling of CSF for routine clinical measurements of Aβ42 and tau include the use of polypropylene (PP) low bind (LoB) tubes to mitigate the loss of Aβ42. ⁵ In addition, the CSF fill volume (FV)  to tube surface area differences need to be considered as a low CSF volume versus tube surface area might reduce Aβ concentrations, even when using LoB tubes. ⁶ Our laboratory recommendations for CSF collection include the use of a PP LoB tube with at least a 50% FV. Unfortunately, 14% of all submitted samples for the Aβ42/40 ratio, and 4.1% of samples for the pTau181/Aβ42 ratio assays fall below 50% FV. Given the invasive nature of the CSF collection, we are often pressured to provide results from samples collected outside our predefined collection conditions.

TABLE 1.

Measured CSF AD biomarkers for case 1 and case 2.

		Case 1			Case 2
Analyte	Non‐amyloid pathology cutoff	CSF collection (FV 60%)	CSF collection (FV 4%)	% change	CSF collection (FV 60%)	CSF collection (FV 6%)	% change
Aβ42 (pg/mL)	>834	791	261	−67%	747	295	−61%
pTau181 (pg/mL)	≤21.6	15.2	15.7	3%	95.8	109.0	14%
pTau181/Aβ42	≤0.028	0.019	0.060	216%	0.128	0.369	188%
pTau181/Aβ42 interpretation (AD pathology)		Negative	Positive		Positive	Positive
Aβ40	NA	8453	3570	−58%	24777	11349	−54%
Aβ42	NA	819	220	−73%	796	231	−71%
Aβ42/40	≥0.073	0.097	0.062	−36%	0.032	0.020	−38%
Aβ42/40 interpretation (AD pathology)		Likely positive	Negative		Positive	Positive

Abbreviations: AD, Alzheimer's disease; CSF, cerebrospinal fluid; FV, fill volume (%); NA, not available.

Here, we describe two cases where the improper collection of CSF could have resulted in inaccurate interpretation and diagnosis of AD. Two samples from the same collection with very different relative FVs were received from two patients for determination of the pTau181/Aβ42 and Aβ42/40 ratios. Case 1 submitted samples included a 2.5‐mL PP LoB tube with 1.5 mL of CSF (60% FV), and a simultaneously submitted 15‐mL PP tube with 0.6 mL of CSF (4%FV). Case 2 submitted samples included a 2.5‐mL PP LoB tube with 1.5 mL of CSF (60% FV) and a simultaneously submitted 15‐mL PP tube with 0.9 mL of CSF (6% FV). In both cases the tubes with <10% FV, which were initially submitted for pTau181/Aβ42 ratio testing, were poured over into a 2.5‐mL PP LoB tube to enable instrument analysis. We then comparatively performed both pTau181/Aβ42 and Aβ42/40 ratios, on both specimen tubes for each patient to determine the effects of extremely low FV on each ratio. Table 1 shows the relative % change in measured concentrations of Aβ42, Aβ40, pTau181, and calculated ratios.

While changes in specimen tube FVs did not significantly alter the measured pTau181 concentrations, it resulted in significant differences in the measured Aβ42 and Aβ40 concentrations and in the calculated pTau181/Aβ42 and Aβ42/40 ratios (Table 1). The change in the calculated pTau181/Aβ42 ratio misclassified one of the cases as positive for the presence of the amyloid brain deposition. ⁷ While the Aβ42/40 ratio has been suggested to be more resistant to variations in pre‐analytical procedures due to assumed relatively equimolar adhesion of different amyloid beta isoforms, ⁷ there were observed relative reductions in the Aβ42/40 ratio in the low FV specimens. Although the magnitude of the reduction in Aβ42/40 ratios (≈35%) was 5‐fold less than the corresponding increases in the pTau181/Aβ42 ratio (+200%) when relative FV was decreased, low FV also resulted in misclassification by Aβ42/40 ratio in one case.

These cases provide evidence for the importance of adhering to laboratory specified CSF collection protocols in order to preserve the diagnostic accuracy of CSF AD biomarkers. Low FVs can dramatically increase pTau181/Aβ42 and less dramatically decrease Aβ42/40 ratios leading to potential misclassification/misdiagnosis of patients. Despite the irretrievable nature of collected CSF specimens, these results further demonstrate the need for clinical laboratories to enforce compliance with their optimized CSF collection protocol, which may lead to rejection of specimens with low FVs despite potential dissatisfaction from clinical providers.

CONFLICT OF INTEREST STATEMENT

A.A.S. reports advisory board participation for Fujirebio Diagnostics, Roche Diagnostics, and Siemens Healthineers; honoraria for lectures from Roche Diagnostics. J.A.B. reports consulting fees for Sunbird Bio and honoraria for lectures from Roche Diagnostics. Author disclosures are available in the supporting information.

Supporting information

Supporting Information