Introduction

Approximately 40% of clinical tests rely on plasma analysis, however, further biomarker discovery using plasma is stymied due to being comprised of 22 highly abundant proteins that limit the detection of much else. Further, the very processing steps required to isolate plasma, such as chosen anticoagulant, can introduce variability into the results. Whole blood is an attractive alternative to plasma as it can be collected without additional processing required, although it has the same high abundance protein issue with a very wide dynamic range. The increased focus on remotely collected blood samples using dried blood spots and volumetric absorptive microsampling (VAMS) devices has further driven the need to shift focus to developing methods for whole blood analysis.

​

Methods

Blood was collected from healthy volunteers and processed into either whole blood or plasma using a variety of anticoagulant vacutainers. All samples were applied to 30µL Mitra VAMS tips and left to dry for 24 hours. In a test of sample stability, a number of samples were stored for 3 months at room temperature, 4 °C, or -80 °C. All other samples were processed for analysis immediately after drying. To process for analysis, sample-bound tips were incubated then washed in an extraction solution (lithium chloride + Tris) before being digested in tip with trypsin (1 µg/µL in lysis buffer). Peptides were quantified and normalised to 0.2 µg/µg and were analysed by LCMS on HF-X Orbitrap mass spectrometer.

​

Preliminary data

By utilising VAMS to immobilise the sample for washing and digestion, the high abundance protein problem of plasma and whole blood was mitigated which was demonstrated by a significant improvement in the dynamic range of both whole blood and plasma. Immobilisation of plasma into VAMS produced up to 2519 protein identifications for plasma. Whole blood performed even better with up to 3318 protein identifications. In fact, for every metric measured whole blood outperformed plasma.

In a reproducibility test of whole blood and plasma from VAMS, whole blood was found to be superior with a median %CV of 6.1% compared to 10.7% for plasma. In a comparison of the effect of various anticoagulants, the number of protein identifications for plasma changed by 1.9-fold between the highest and lowest results compared to 1.1-fold for whole blood, with mean % CVs of 35% and 11% respectively. It was also found that small deviations in plasma production protocols such as double spinning plasma (compared to single spinning plasma) can result in a loss of 1307 identifications in double spun plasma and 1400 differentially expressed proteins between the processing methods.

Further, in a test of storage stability, plasma was found to be highly affected by storage temperature with a 76% reduction in number of proteins detected after 3 months of storage at room temperature and 42% reduction at 4 °C. Whole blood conversely lost 29% of the IDs after room temperature storage and no detectable loss at 4 °C. Overall, these results demonstrate that when immobilised in VAMS and dried, whole blood is more reproducible, produces more datapoints, and is more resilient to temperature associated changes than plasma.

 

Novel aspect

Novel methods for whole blood analysis enable the production of more datapoints than plasma and analysis of remotely collected samples.