Clark Henderson (Presenter)
University of Washington
Bio: I am currently a research scientist developing novel proteomic and small molecule assays in Andy Hoofnagle’s laboratory in the Department of Laboratory Medicine at the University of Washington Medical Center in Seattle, Washington. I received my PhD in Biophysics from the University of California Davis, where I studied the relationship between membrane lipid composition and ethanol tolerance in Saccharomyces cerevisiae.
Authorship: Clark M. Henderson, James G. Bollinger, Jessica O. Becker, Jennifer M. Wallace, Thomas J. Laha, Michael J. MacCoss, and Andrew N. Hoofnagle
University of Washington, Seattle, WA, 98195
| Short Abstract Blood samples collected during clinical and epidemiological investigations are often archived using dried blood spots for future retrospective investigations of novel biomarkers of human disease. Several recent studies have investigated protein quantification using dried bloods spots, however few studies have determined their performance compared to matched plasma or serum samples measured using validated clinical assays. We developed a precise and linear bottom-up proteomic method for quantification of proteins in dried blood spots that demonstrated poorer accuracy than anticipated with matched plasma samples analyzed using clinically validated immunoassays. |
Long Abstract
Introduction
Blood samples collected during clinical and epidemiological investigations are often archived using dried blood spots for future retrospective investigations of novel biomarkers of human disease. There have been several recent studies reporting compelling evidence that dried blood spot quantification of proteins in whole blood by multiplexed LC-MS/MS are a potential substitute for traditional venous blood draws and clinical immunoassays. We hypothesized that measurements of proteins in dried capillary blood spots using liquid chromatography-tandem mass spectrometry would be linear, precise, and accurate enough to replicate the serum/plasma protein concentrations in samples from clinical or epidemiological investigations.
Methods
Using a Skyline-based empirical workflow, we developed a trypsin digestion-nanoflow liquid chromatography-tandem mass spectrometric method to quantify apolipoprotein A-I and apolipoprotein B in serum/plasma and dried blood spots. The assay was characterized for precision, linearity, interferences, and stability. To evaluate accuracy, method comparison was carried out vs. clinical nephelometric immunoassays in plasma using samples from 36 normal healthy controls.
Results
The method was relatively precise (total variability derived from 5x5 experiments was 10-11%CV) and linear. Measured protein concentrations in blood spots were affected by storage at 37°C and spiked artificially high concentrations of human globular proteins. Peptides in digests were stable for 48 h at autosampler temperature (7°C). Measurements in capillary derived blood spots were significantly different than for EDTA whole blood spotted onto filter paper. Importantly, when we compared clinical assay measurements of each protein with measurements using our new method, the correlation was poorer than anticipated (r~0.8).
Conclusions & Discussion
Dried blood spots are commonly used to archive blood samples from population studies for later analysis. They are thought to be a promising sampling method for retrospective novel biomarker investigations or discovery proteomic studies. Given the significant differences that we observed between plasma and dried blood spots for commonly measured proteins, it appears that devices or methods that enable the collection of precise amounts of blood and approaches to accurately determine the amount of plasma in each blood spot may be important for useful protein measurements in dried blood spots.
References & Acknowledgements:
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