Jérôme Vialaret

Current and future use of "dried blood spot" analyses in clinical chemistry.

Abstract The analysis of blood spotted and dried on a matrix (i.e., “dried blood spot” or DBS) has been used since the 1960s in clinical chemistry; mostly for neonatal screening. Since then, many clinical analytes, including nucleic acids, small molecules and lipids, have been successfully measured using DBS. Although this pre-analytical approach represents an interesting alternative to classical venous blood sampling, its routine use is limited. Here, we review the application of DBS technology in clinical chemistry, and evaluate its future role supported by new analytical methods such as mass spectrometry.

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application.

Abstract Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in ‘functional’ studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

Tau Protein Quantification in Human Cerebrospinal Fluid by Targeted Mass Spectrometry at High Sequence Coverage Provides Insights into Its Primary Structure Heterogeneity

Tau protein plays a major role in neurodegenerative disorders, appears to be a central biomarker of neuronal injury in cerebrospinal fluid (CSF), and is a promising target for Alzheimers disease immunotherapies. To quantify tau at high sensitivity and gain insights into its naturally occurring structural variations in human CSF, we coupled absolute quantification using protein standard with the multiplex detection capability of targeted high-resolution mass spectrometry (MS) on a Quadrupole-Orbitrap instrument. Using recombinant tau we developed a step-by-step workflow optimization including an extraction protocol that avoided affinity reagents and achieved the monitoring of 22 tau peptides uniformly distributed along the tau sequence. The lower limits of quantification ranged (LLOQ) from 150 to 1500 pg/mL depending on the peptide. Applied to endogenous CSF tau, up to 19 peptides were detected. Interestingly, there were significant differences in the abundance of peptides depending on their position in the sequence, with peptides from the tau mid-domain appearing significantly more abundant than peptides from the N- and C-terminus domains. This MS-based strategy provided results complementary to those of previous ELISA or Western Blot studies of CSF tau and could be applied to tau monitoring in human CSF cohorts.

Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer's Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies.

Microtubule-associated Tau proteins are major actors in neurological disorders, the so-called tauopathies. In some of them, and specifically in Alzheimers disease (AD), hyperphosphorylated forms of Tau aggregate into neurofibrillary tangles. Following and understanding the complexity of Taus molecular profile with its multiple isoforms and post-translational modifications represent an important issue, and a major analytical challenge. Immunodetection methods are, in fact, limited by the number, specificity, sensitivity, and capturing property of the available antibodies. Mass spectrometry (MS) has recently allowed protein quantification in complex biological fluids using isotope-labeled recombinant standard for absolute quantification (PSAQ). To study Tau proteins, which are found at very low concentrations within the cerebrospinal fluid (CSF), we relied on an innovative two-step pre-fractionation strategy, which was not dependent on immuno-enrichment. We then developed a sensitive multiplex peptide detection capability using targeted high-resolution MS to quantify Tau-specific peptides covering its entire sequence. This approach was used on a clinical cohort of patients with AD, progressive supranuclear palsy (PSP), and dementia with Lewy body (DLB) and with control non-neurodegenerative disorders. We uncovered a common CSF Tau molecular profile characterized by a predominance of central core expression and 1N/3R isoform detection. While PSP and DLB tau profiles showed minimal changes, AD was characterized by a unique pattern with specific modifications of peptide distribution. Taken together these results provide important information on Tau biology for future therapeutic interventions, and improved molecular diagnosis of tauopathies.

Quantitative detection of amyloid-β peptides by mass spectrometry: state of the art and clinical applications

Abstract Alzheimer’s disease (AD) is the most common form of dementia in humans, and a major public health concern with 35 million of patients worldwide. Cerebrospinal fluid (CSF) biomarkers being early diagnostic indicators of AD, it is essential to use the most efficient analytical methods to detect and quantify them accurately. These biomarkers, and more specifically amyloid-β (Aβ) peptides, are measured in routine clinical practice using immunoassays. However, there are several limits to this immunodetection in terms of specificity and multiplexing of the multiple isoforms of the Aβ peptides. To overcome these issues, the quantification of these analytes by mass spectrometry (MS) represents an interesting alternative, and several assays have been described over the past years. This article reviews the different Aβ peptides quantitative MS-based approaches published so far, compares their pre-analytical phase, and the different quantitative strategies implemented that might be suitable for clinical applications.

Clinical Mass spectrometry proteomics (cMSP) for medical laboratory: What does the future hold?

BACKGROUND Mass spectrometry (MS) methods are being widely used these days in medical laboratories for quantifying many small molecular analytes as well as for microbiological purposes. METHODS Little use has been made so far, however, of MS for analyzing peptides and proteins in clinical laboratory (an approach known as clinical MS proteomics (cMSP)). The explanation for this situation may be that cMSP assays are more complex to implement than conventional assays, require large investments in terms of equipment and training, and have not yet been sufficiently validated for clinical applications. In addition, the protein analysis assays currently used in medical laboratories mostly meet both laboratory and clinical requirements in terms of analytical performances, ease of use, and turn-around-time. RESULTS With the spread of MS methods in laboratories, increasing interest seems to be focusing on the development of MS for quantifying new analytes. MALDI-TOF MS methods have already been replacing classical methods of bacterial classification in clinical laboratories, for example, and this can be said to be an important step in this direction. CONCLUSIONS In this paper, the literature available on the topic of clinical MS proteomics is reviewed and the pre-analytical, analytical, and post-analytical challenges which will have to be met in connection with this approach are discussed.

Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry

Tau protein concentration in cerebrospinal fluid (CSF) is currently used as a sensitive and specific biomarker for Alzheimers disease. Its detection currently relies on ELISA but the perspective of using mass spectrometry (MS) to detect its different proteoforms represents an interesting alternative. This is however an analytical challenge because of its low concentration in the CSF, a biological fluid collected in small volume by lumbar puncture, and with a high structural heterogeneity. To overcome these issues, instead of using immunocapture as previously done, we rather relied on an original two steps pre-fractionation technique of CSF: perchloric acid (PCA) followed by micro solid phase extraction (μSPE). We could then measure seven tau trypsic peptides by Multiple Reaction Monitoring (MRM) on a triple quadrupole mass spectrometer. Quantification was performed using isotopically labeled 15N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs). Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides. This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

Cerebrospinal fluid levels of orexin-A and histamine, and sleep profile within the Alzheimer process

To better understand how sleep/wake dysregulation affects Alzheimers disease (AD), we compared the cerebrospinal fluid (CSF) orexin and histamine/tele-methylhistamine (HA/t-MHA) levels of 82 patients (41 probable-AD-high level of evidence, 41 mild cognitive impairment MCI-due-to-AD), 24 other neurologic disorders (OND) and 24 controls. We determined the relationships between these biomarkers, the CSF AD biomarkers concentrations, and the clinical sleep profile. CSF orexin-A but not HA/t-MHA levels were higher in MCI and AD than OND and controls. CSF orexin-A is correlated to CSF amyloid-β42in MCI and AD, independently of age, gender, MMSE, total-tau/phosphorylated-tau, HA or sleep parameters. Nighttime sleep duration was longer in MCI and AD patients than controls. In MCI, nighttime sleep duration negatively correlated with CSF amyloid-β42 and MMSE. To conclude, CSF orexin-A but not HA/t-HMA was upregulated in AD and correlated with amyloid-β42 level. Our data suggested a change in the sleep-wake pattern at an early stage of the disease that needs further investigation to deeply explain the mechanistic interplay between sleep and Alzheimer.

Absolute quantification of 35 plasma biomarkers in human saliva using targeted MS.

BACKGROUND Although the use of human saliva for diagnosing disease has been known to be of great clinical potential, few attempts have been made so far to develop its use. In this work, we developed an MRM-MS approach for 35 plasma biomarkers using human saliva in a clinical environment. METHODS & RESULTS A 30-min micro LC-MS/MS run in MRM mode was conducted in order to quantify the 35 plasma proteins in human saliva. Sample preparation procedures were performed in quadruplicate and analyzed in duplicate. Results show that 32 of the 35 plasma proteins were quantified in human saliva using calibration curves in the 2- log10 dynamic ranges with excellent linearity. DISCUSSION/CONCLUSION Our MRM method is compatible with routine measurements in daily clinical practice.

Development and validation of dried matrix spot sampling for the quantitative determination of amyloid β peptides in cerebrospinal fluid.

Abstract Background: The use of dried blood spots on filter paper is well documented as an affordable and practical alternative to classical venous sampling for various clinical needs. This technique has indeed many advantages in terms of collection, biological safety, storage, and shipment. Amyloid β (Aβ) peptides are useful cerebrospinal fluid (CSF) biomarkers for Alzheimer disease diagnosis. However, Aβ determination is hindered by preanalytical difficulties in terms of sample collection and stability in tubes. Methods: We compared the quantification of Aβ peptides (1–40, 1–42, and 1–38) by simplex and multiplex ELISA, following either a standard operator method (liquid direct quantification) or after spotting CSF onto dried matrix paper card. Results: The use of dried matrix spot (DMS) overcame preanalytical problems and allowed the determination of Aβ concentrations that were highly commutable (Bland-Altman) with those obtained using CSF in classical tubes. Moreover, we found a positive and significant correlation (r2=0.83, Pearson coefficient p=0.0329) between the two approaches. Conclusions: This new DMS method for CSF represents an interesting alternative that increases the quality and efficiency in preanalytics. This should enable the better exploitation of Aβ analytes for Alzheimer’s diagnosis.