In the development of this assay, we encountered insufficient extraction efficiency and irreproducible recovery.
2. Method Information
• Spot a Whatman 903 DBS card with 50 ul of whole blood, dry for at least 2 hours
• Cut a 6mm disc using a standard hole puncher
• Add DBS disc into a 15 ml conical tube
• Optimize extraction:
o Add 5 ml of extraction buffer into 15 ml conical tube; use extraction buffer with increasing EDTA concentrations, +/- additional compounds such as nitric acid
o Vortex for 1, 5, 15 and 30 minutes at various rpms
o Incubate for 30, 60 and 90 minutes
o Centrifuge for 5 minutes
• Run samples by ICP-MS, injection volume of 2.5 ml
• Interpret and analyze results for accuracy and imprecision
3. Troubleshooting steps
We hypothesized that there were five potential contributors for the insufficient lead extraction from filter paper: extraction buffer, vortexing speed, incubation time, the volume assumption of the punch DBS and the calibration curve. We systematically addressed each parameter to optimize the extraction conditions of lead from the DBS.
The extraction method was optimized. The optimized method for a 6 mm disc from the filter paper card consists of using an extraction buffer containing 5mM EDTA and 0.05% triton, vortex speed of 1 minute at 2,000 RPM, and an assumption of 10 µl of blood from the DBS disc. When adding nitric acid to the extraction buffer we observed no dramatic improvement. Additionally, when increasing the vortexing time at 2,000 rpm we started to observe disintegration of the DBS disc. Importantly we found that using a DBS calibrator is necessary for accurate and reproducible recovery, relative to the use of a liquid calibrator in extraction buffer matrix. The DBS calibrator likely accounts for any matrix effect from the DBS card and corrects for loses during the extraction process. An incubation time of 30 minutes post vortexing is sufficient for maximum extraction with no apparent extraction increase after 90 minutes.