Online Preconcentration in Capillaries by Multiple Large-Volume Sample Stacking: An Alternative to Immunoassays for Quantification of Amyloid Beta Peptides Biomarkers in Cerebrospinal Fluid

A novel electrokinetic preconcentration approach, so-called multiple pressure-assisted large-volume sample stacking with an electroosmotic flow pump (M-PA-LVSEP), was developed to allow in-capillary enrichment and separation of analytes from unlimited sample volumes. With this approach, the inherent limitation of in-capillary electrokinetic preconcentrations to the separation capillary volume can be overcome. The M-PA-LVSEP protocol relies on repeated cycles of pressure-assisted electroosmotic pumping and injection of extremely large sample volumes for analyte stacking and sample matrix removal. This technique was developed to address the challenge of sensitive and simultaneous determination of several amyloid β (Aβ) peptides, which are biomarkers for the molecular diagnosis of Alzheimer’s disease (AD). For the first time, reliable quantification of different species of fluorescently derivatized Aβ peptides, that is, Aβ 1–42, Aβ 1–40, and Aβ 1–38 down to subnanomolar ranges in cerebrospinal fluids (CSF) from AD and non-demented patients (healthy controls) was made possible without recourse to immunoassay, immunoprecipitation, or mass spectrometry approaches. Based on the stacking from a sample plug representing up to 400% of the total capillary volume, sensitive enhancement factors up to 170 could be achieved with this “antibody free” approach. Quantification limits for these Aβ peptides down to 0.05 nM with capillary electrophoresis coupled with laser-induced fluorescent detection could be obtained. Excellent agreement between results from M-PA-LVSEP and the gold standard ELISA method was achieved for measurements of Aβ 1–42 in CSF, with a determination correlation (<i>r</i>²) better than 0.993

KFLC in CSF and serum.

<p>Immunoglobulin kappa free light chain (KFLC) in CSF and serum of patients with MS, CIS, pathogen-related diseases and NIND (for abbreviations see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088680#pone-0088680-t001" target="_blank">table 1</a>). Q KFLC: CSF- serum ratio of KFLC. Data are shown as the median and IQR.</p

Sensitivity, specificity, positive and negative predictive value for elevated KFLC, MRI parameters and OCB regarding conversion of clinically isolate syndrome to definite multiple sclerosis.

<p>Data are shown as percent and 95% confidence interval. Q KFLC = CSF-serum ratio of KFLC above the approximately upper reference value described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088680#pone-0088680-g002" target="_blank">figure 2</a>. OCB = cerebrospinal fluid oligoclonal bands of IgG class not detectable in serum. Intrathecal IgG- Synthesis according to Reiber quotients diagrams. IgG- Index = CSF/serum IgG:CSF/serum albumin >0.7. Barkhof = 3 of 4 Barkhof criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088680#pone.0088680-Barkhof1" target="_blank">[21]</a> fulfilled.</p

CSF-serum ratio of KFLC (Q KFLC) among different oligoclonal IgG band patterns.

<p>There are five classic patterns of oligoclonal bands <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088680#pone.0088680-Andersson1" target="_blank">[48]</a> (type 1, no bands in CSF and serum; type 2, oligoclonal IgG bands in CSF but not in serum, indicative of intrathecal IgG synthesis; type 3, oligoclonal bands in CSF plus identical oligoclonal bands in serum and CSF, indicative of intrathecal IgG synthesis; type 4, identical pattern of oligoclonal bands in CSF and serum. There was no patient with type 5 (identical patterns of monoclonal bands in CSF and serum) in the study. Horizontal solid line indicates median, Kruskal-Wallis test among groups revealed a significant difference (p<0.001), significant P-values for pairwise comparisons (Mann-Whitney U test) are displayed.</p

Cerebrospinal fluid – serum ratio of kappa free light chain (Q KFLC) is plotted against CSF-serum ratio of albumin.

<p>Dashed lines indicate the 99% confidence interval of the linear regression line (straight line). The upper 99% confidence interval (dark dashed line) indicate the approximately upper reference value of Q KFLC based on a control group of 77 non-inflammatory neurologic diseases and a range of Q Albumin from 1.9 to 28.2.</p

Demographic data and basic cerebrospinal fluid findings.

<p>Data are shown as the median and IQR. Abbreviations: B-CNS-I bacterial central nervous system infections, CIS clinically isolated syndrome, CSF cerebrospinal fluid, MS multiple sclerosis, NB neuroborreliosis, NIND non-inflammatory neurological diseases, OCB oligoclonal IgG bands, V-CNS-I viral central nervous system infections.</p

CSF-serum ratio of KFLC (Q KFLC) was elevated in patients with positive oligoclonal IgG (OCB). Q KFLC of 77 CIS patients are shown.

<p>Dashed line indicates the approximately upper reference value of Q KFLC, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088680#pone-0088680-g002" target="_blank">figure 2</a>. Black rhomb indicate positive OCBs, white rhomb indicate negative OCBs.</p

CSF NfH in ALS and controls.

<p>Box and dot plots show CSF NfH^SMI35 in ALS, Parkinson's disease (PD), and controls (CTRL). ALS fast  =  patients with rapid progression of disease over follow-up of 6 months, ALS slow  =  patients with slow progression of disease over follow-up. The box represents the 25^th to 75^th quartile, the whiskers represent the range, and the horizontal line in the box represents the median. Difference between the groups was significant (p<0.001, Kruskal-Wallis Analysis of Variance on Ranks), with post-hoc analysis (Dunn's method) showing patients with ALS to have significantly higher CSF concentrations as compared to patients with PD and controls (p<0.05 each).</p

Sensitivity, specificity, positive (PPV) and negative (NPV) predictive value in percent (exact 95% confidence interval in brackets) for CSF and MRI parameters regarding conversion of clinically isolated syndrome to definite multiple sclerosis.

<p>MRZR  =  AI for measles, rubella, zoster, two or more AI ≥1.5, MRZS  =  MRZ score >10, OCB  =  oligoclonal bands in cerebrospinal fluid, MRI  =  two or more lesions in T2-weighted magnetic resonance imaging of the brain.</p

CSF and Serum sAPPα, sAPPß, NfH^SMI35, and Progranulin (PRGN) in patients with ALS, Parkinson's disease (PD), and controls (CTRL).

‡<p>Comparison across all groups, Kruskal-Wallis Analysis of Variance on Ranks.</p><p>*Comparison of ALS fast vs. ALS slow, Mann-Whitney Rank Sum Test.</p><p>Fast  =  ALS patients with fast progression of disease over follow-up, slow  =  ALS patients with slow progression of disease over follow-up, S  =  statistical significance.</p