DBS Project Dried blood spot a new method
DBS Project Dried blood spot, a new method for the quantification of clinical analytes using immunoassay, mass spectrometry and high-performance liquid chromatography Biochemistry Laboratory - Regional Clinical Proteomics Platform Elise Casteleyn, June 2015
DBS Project Table of contents 1. Dried Blood Spot 2. Clinical analytes 3. Methods 3. 1 Lumipulse (immunoassay) 3. 2 Mass spectrometry 3. 3 Adams (High performance liquid chromatography) 4. Results 4. 1 Lumipulse 4. 2 Mass spectrometry 4. 3 Adams 5. Conclusion Elise Casteleyn, June 2015
DBS Project 1. Dried Blood Spot • Robert Guthrie in the 1960 s • DBS has a lot of advantages : Less invasive Less painful Lower biohazard risk Lower volume needed Stable samples (one week) No special transport needed • Replacement of classical venipuncture by DBS sampling for the detection of clinical analytes? • Elderly, patients with higher risk, diabetic patients… Elise Casteleyn, June 2015
DBS Project 2. Clinical analytes • Vitamin D: 10 -55 ng/m. L • Vitamin D 2 (diet supplements) and vitamin D 3 (ultraviolet-B rays) • Deficiency of vitamin D: rickets (children) osteomalacia (adults) osteoporosis (adults) • Indications for vitamin D screening: old age dark skin bone diseases obesity chronic kidney disease pregnancy liver failure Elise Casteleyn, June 2015
DBS Project 2. Clinical analytes Albumin: 35 -50 g/L • Most abundant protein • Maintain the osmotic pressure Transthyretin: 0, 10 -0, 40 mg/L • Transports thyroid hormones • Both are malnutrition markers • Prevalence: hospitalized elderly patients elderly who cannot eat and drink unaided elderly suffering dementia or depression Elise Casteleyn, June 2015
DBS Project 2. Clinical analytes Glycohemoglobin: <6, 5% • Glycohemoglobin = Hb. A 1 c • Diabetes marker • Good parameter for long-term glucose monitoring • Binding of glucose to hemoglobin • Membrane of erythrocytes = permeable • Lifespan of erythrocytes = 120 days Elise Casteleyn, June 2015
DBS Project 3. Methods Vitamn D on Lumipulse G 1200 Albumin and transthyretin on LC-MS Elise Casteleyn, June 2015 Hb. A 1 c on Adams™
DBS Project 3. 1 Lumipulse Chemiluminescent enzyme immunoassay based on sandwich ELISA Elise Casteleyn, June 2015
DBS Project 3. 1 Lumipulse Spots extracted in buffer -> 60µL put in the rack for measurement • 3/4 spots were extracted • Different buffers (H 2 O, PBS/triton, ACN) • Different temperatures (22°C, 60°C) • Different times (30 min, 4 h) Elise Casteleyn, June 2015
DBS Project 3. 2 Mass spectrometry Elise Casteleyn, June 2015
DBS Project 3. 3 Adams (HPLC) One spot in 500µL buffer Buffers Extraction times Extraction temperatres Buffer W 5 min Room temperature Dilution buffer 30 min 37°C Buffer H 80 1 h 60°C PBS 1 X 3 h Overnight Elise Casteleyn, June 2015
DBS Project 4. Results 4. 1 Lumipulse • Results = 4 ng/m. L = out of the dynamic range! • Below the LOQ • 4 ng/m. L = noise Problems? • Dilution (3 spots in 100µL buffer) • LOQ too high • Extraction (buffer? ? ) Elise Casteleyn, June 2015
DBS Project 4. 2 Mass spectrometry 135185 – 205190 ng/m. L 40 – 48 ng/m. L DBS Hemo. Void pre-treated samples gave higher concentrations Elise Casteleyn, June 2015
DBS Project 4. 3 Adams Final procedure: • 1 spot in 500µL buffer W • Extraction 1 hour at RT (while shaking) • 300µL put in rack for measurement Elise Casteleyn, June 2015
DBS Project 4. 3 Adams Repeatability measurement Reproducibility Sample concentration Hb. A 1 c (%) 4251 4252 4253 1 5, 2 5, 1 6, 8 2 5, 3 5, 1 7 3 5, 2 5, 1 7 4 5, 2 5 6, 9 5 5, 2 5, 1 6, 9 6 5, 2 5 6, 9 7 5, 3 5, 1 6, 9 8 5, 2 5, 1 6, 9 Mean 5, 2 5, 1 6, 9 SD 0, 05 0, 06 CV 0, 89% 0, 91% 0, 93% Elise Casteleyn, June 2015
DBS Project 4. 3 Adams 150 samples Elise Casteleyn, June 2015
DBS Project 5. Conclusion • Vitamin D on Lumipulse? No ELISA or LC-MS in the future? • Albumin and transthyretin on LC-MRM using Hemo. Void matrix? Yes Processing of more samples? • Glycohemoglobin on Adams? Yes Recovery? Elise Casteleyn, June 2015
DBS Project Dried blood spot, a new method for the quantification of clinical analytes using immunoassay, mass spectrometry and high-performance liquid chromatography Biochemistry Laboratory - Regional Clinical Proteomics Platform Elise Casteleyn, June 2015
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