Elise Casteleyn, June 2015 DBS Project Biochemistry Laboratory - Regional Clinical Proteomics Platform Dried blood spot, a new method for the quantification of clinical analytes using immunoassay, mass spectrometry and high-performance liquid chromatography

Table of contents Elise Casteleyn, June 2015 DBS Project 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

1. Dried Blood Spot Robert Guthrie in the 1960s 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: ng/mL Vitamin D2 (diet supplements) and vitamin D3 (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: 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 = HbA1c 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 Elise Casteleyn, June 2015 DBS Project Vitamn D on Lumipulse G 1200 Albumin and transthyretin on LC-MS HbA1c on Adams™

3.1 Lumipulse Elise Casteleyn, June 2015 DBS Project Chemiluminescent enzyme immunoassay based on sandwich ELISA

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 (30min, 4h) 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 Elise Casteleyn, June 2015 DBS Project BuffersExtraction timesExtraction temperatres Buffer W5minRoom temperature Dilution buffer30min37°C Buffer H801h60°C PBS 1X3h Overnight

4. Results 4.1 Lumipulse Results = 4ng/mL = out of the dynamic range! Below the LOQ 4ng/mL = noise Problems? Dilution (3 spots in 100µL buffer) LOQ too high Extraction (buffer??) Elise Casteleyn, June 2015 DBS Project

4.2 Mass spectrometry Elise Casteleyn, June 2015 DBS Project – ng/mL 40 – 48ng/mL DBS HemoVoid pre-treated samples gave higher concentrations

4.3 Adams Elise Casteleyn, June 2015 DBS Project Final procedure: 1 spot in 500µL buffer W Extraction 1 hour at RT (while shaking) 300µL put in rack for measurement

4.3 Adams measurement Sample concentration HbA1c (%) ,25,16,8 25,35,17 35,25,17 45,256,9 55,25,16,9 65,256,9 75,35,16,9 85,25,16,9 Mean5,25,16,9 SD0,05 0,06 CV0,89%0,91%0,93% Elise Casteleyn, June 2015 DBS Project Repeatability Reproducibility

4.3 Adams Elise Casteleyn, June 2015 DBS Project 150 samples

5. Conclusion Vitamin D on Lumipulse? No ELISA or LC-MS in the future? Albumin and transthyretin on LC-MRM using HemoVoid matrix? Yes Processing of more samples? Glycohemoglobin on Adams? Yes Recovery? Elise Casteleyn, June 2015 DBS Project

Elise Casteleyn, June 2015 DBS Project Biochemistry Laboratory - Regional Clinical Proteomics Platform Dried blood spot, a new method for the quantification of clinical analytes using immunoassay, mass spectrometry and high-performance liquid chromatography

Process DBS sampling Elise Casteleyn, June 2015 DBS Project PatientDisinfection of the sampling areaPrick in the fingertip with a lancet Deposit five (6mm diameter) spots on filter paper Drying 1 to 3 hours at RT Transport to laboratory in a bag with a dessicant Spot (2-6 mm diameter)Extraction with appropriate bufferAnalyses

Noviplex™ card Elise Casteleyn, June 2015 DBS Project

Adams Elise Casteleyn, June 2015 DBS Project

Hemoglobin Elise Casteleyn, June 2015 DBS Project