References of "Ammerlaan, Wim"
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See detailMethod optimization for fecal sample collection and fecal DNA extraction.
Mathay, Conny; Hamot, Gael; Henry, Estelle et al

in Biopreservation and biobanking (2015), 13(2), 79-93

BACKGROUND: This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here ... [more ▼]

BACKGROUND: This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here optimization of a stool processing protocol validated for fitness-for-purpose in terms of downstream DNA-based analyses. METHODS: Stool collection was initially optimized in terms of sample input quantity and supernatant volume using canine stool. Three DNA extraction methods (PerkinElmer MSM I(R), Norgen Biotek All-In-One(R), MoBio PowerMag(R)) and six collection container types were evaluated with human stool in terms of DNA quantity and quality, DNA yield, and its reproducibility by spectrophotometry, spectrofluorometry, and quantitative PCR, DNA purity, SPUD assay, and 16S rRNA gene sequence-based taxonomic signatures. RESULTS: The optimal MSM I protocol involves a 0.2 g stool sample and 1000 muL supernatant. The MSM I extraction was superior in terms of DNA quantity and quality when compared to the other two methods tested. Optimal results were obtained with plain Sarstedt tubes (without stabilizer, requiring immediate freezing and storage at -20 degrees C or -80 degrees C) and Genotek tubes (with stabilizer and RT storage) in terms of DNA yields (total, human, bacterial, and double-stranded) according to spectrophotometry and spectrofluorometry, with low yield variability and good DNA purity. No inhibitors were identified at 25 ng/muL. The protocol was reproducible in terms of DNA yield among different stool aliquots. CONCLUSIONS: We validated a stool collection method suitable for downstream DNA metagenomic analysis. DNA extraction with the MSM I method using Genotek tubes was considered optimal, with simple logistics in terms of collection and shipment and offers the possibility of automation. Laboratories and biobanks should ensure protocol conditions are systematically recorded in the scope of accreditation. [less ▲]

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See detailPlatelet mitochondrial membrane potential in Parkinson's disease.
Antony, Paul UL; Boyd, Olga UL; Trefois, Christophe UL et al

in Annals of clinical and translational neurology (2015), 2(1), 67-73

OBJECTIVE: Mitochondrial dysfunction is a hallmark of idiopathic Parkinson's disease (IPD), which has been reported not to be restricted to striatal neurons. However, studies that analyzed mitochondrial ... [more ▼]

OBJECTIVE: Mitochondrial dysfunction is a hallmark of idiopathic Parkinson's disease (IPD), which has been reported not to be restricted to striatal neurons. However, studies that analyzed mitochondrial function at the level of selected enzymatic activities in peripheral tissues have produced conflicting data. We considered the electron transport chain as a complex system with mitochondrial membrane potential as an integrative indicator for mitochondrial fitness. METHODS: Twenty-five IPD patients (nine females; mean disease duration, 6.2 years) and 16 healthy age-matched controls (12 females) were recruited. Live platelets were purified using magnetic-activated cell sorting (MACS) and single-cell data on mitochondrial membrane potential (Deltapsi) were measured by cytometry and challenged with a protonophore agent. RESULTS: Functional mitochondrial membrane potential was detected in all participants. The challenge test reduced the membrane potential in all IPD patients and controls (P < 0.001). However, the response to the challenge was not significantly different between patients and controls. INTERPRETATION: While the reported protonophore challenge assay is a valid marker of overall mitochondrial function in live platelets, intact mitochondrial membrane potential in platelets derived from IPD patients suggests that presumed mitochondrial enzymatic deficiencies are compensable in this cell type. In consequence, mitochondrial membrane potential in platelets cannot be used as a diagnostic biomarker for nonstratified IPD but should be further explored in potential Parkinson's disease subtypes and tissues with higher energy demands. [less ▲]

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See detailMethod validation for preparing urine samples for downstream proteomic and metabolomic applications.
Ammerlaan, Wim; Trezzi, Jean-Pierre UL; Mathay, Conny et al

in Biopreservation and biobanking (2014), 12(5), 351-7

BACKGROUND: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was ... [more ▼]

BACKGROUND: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. METHODS: Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4 degrees C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4 degrees C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. RESULTS: The optimal protocol was a 20-min, 12,000 g centrifugation at 4 degrees C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4 degrees C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. CONCLUSIONS: We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4 degrees C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation. [less ▲]

Detailed reference viewed: 113 (2 UL)