MSACL 2017 EU Abstract

Quantifying Proteins in Dried Blood Spots

Andy Hoofnagle (Presenter)
University of Washington

Bio: Dr. Hoofnagle's laboratory focuses on the precise quantification of recognized protein biomarkers in human plasma using LC-MRM/MS. In addition, they have worked to develop novel assays for the quantification of small molecules in clinical and research settings. His laboratory also studies the roles that the lipid metabolism and systemic inflammation play in the pathophysiology of obesity, diabetes, chronic kidney disease, and cardiovascular disease.

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

Short Abstract To improve access to laboratory testing in remote sites and to facilitate more frequent measurements in longitudinal risk assessment, therapeutic monitoring, and clinical research studies, there is great interest in the use of dried blood spots in specimen collection. While there have been several studies investigating the feasibility of protein analysis in dried blood spots, comparisons with current clinically-used assays in serum and plasma are scarce. We used a precise and linear bottom-up proteomics assay to demonstrate that the measurement of apolipoproteins in dried capillary blood spots is not as accurate as needed for facile translation to clinical care.

Long Abstract

Introduction

To improve access to laboratory testing in remote sites and to facilitate more frequent measurements in longitudinal risk assessment, therapeutic monitoring, and clinical research studies, there is great interest in the use of dried blood spots for specimen collection. Several interesting studies demonstrating the feasibility of protein analysis in dried blood spots have been published. 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 effectively substitute for the current venous measurements that are used clinically.

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 serum 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 for each protein. 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 an attractive blood collection method in a variety of settings. It is generally accepted that minimal training is required for adequate specimen quality, although for many patients, this is not the case. 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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