Junyan Shi (Presenter)
University of British Columbia
Bio: I am currently a postdoctoral fellow in the laboratory of Dr. Mari DeMarco in the Department of Pathology and Laboratory Medicine, University of British Columbia. I graduated with distinction from the Hebei Medical University in China where I completed my training in Laboratory Medicine. I then earned a MSc in Clinical Microbiology from the Peking Union Medical College focusing on antifungal susceptibility. My study helped launch the first and largest antifungal resistance surveillance network in China. I subsequently earned a PhD in Pathology and Laboratory Medicine from the University of British Columbia, where I expanded my interest to viruses, with specific focus on the protein degradation system. My current research efforts continue to focus on protein chemistry, whereby I am developing quantitative mass spectrometric assay to support diagnostic and clinical research efforts.
Authorship: Junyan Shi (1), Yu Zi Zheng (1), Mari L. DeMarco (1,2)
(1) Department of Pathology and Laboratory Medicine, University of British Columbia; (2) Department of Pathology and Laboratory Medicine, St Paul’s Hospital, Providence Health Care
| Short Abstract We developed a simple and rapid workflow for quantitative analysis of C-reactive protein in plasma. The method uses 10 uL of plasma, and avoids the use of analyte cleanup/enrichment steps and reduction/alkylation steps prior to tryptic digestion, thus facilitating a rapid turnaround time and minimal reagent costs. The new method has a cost-per-test of a few pennies, and requires 15-times less sample volume and has an analytical range 50-times greater than that of a commercial immunometric method. HPLC-MS/MS provides an alternative and cost-effective method for CRP quantification in plasma that is benchmarked against gravimetric targets. |
Long Abstract
Introduction
C-reactive protein (CRP), a positive acute-phase protein that is used as both a diagnostic and prognostic capacity for a variety of diseases states. Due to its broad utility, there is constant demand for measurement of CRP in clinical research studies. Current quantitative analysis of CRP in clinical labs relies on immunoassays requiring upwards of 150 μL of plasma. For many studies, utilizing such a large sample volume is impractical given the scope of analyses desired. Another challenge with immunometric CRP methods is their cost. This is significant issue for studies targeting high-CRP cohorts, where multiple manual dilutions and repeat analyses are required due to the limited analytical measurement range (AMR) of commercial immunoassays. Therefore, we developed an HPLC-MS/MS assay to quantitate CRP that (1) is calibrated against gravimetric standards, (2) has a broad analytical measurement range, (3) utilizes minimal sample volume and (4) has a reagent cost that is a fraction of that of immunometric methods.
Methods
We developed a rapid sample preparation workflow that did not require sample cleanup, analyte enrichment or reduction/alkylation. Briefly, 10 μL of plasma was digested for 20 min at 37°C. A stable-isotope-labeled CRP tryptic peptide was used as an internal standard and quantification was performed by comparison to an external 6-point calibration curve. Samples were analyzed on Shimadzu SIL-20AC HPLC coupled to a SCIEX 5500 triple quadrupole. A method validation was performed following Clinical & Laboratory Standards Institute guidelines including determination of detection capacity, linearity, carryover, precision and stability. A method comparison was performed with the high-sensitivity CRP assay on the Siemens ADVIA 1800. Quality controls (QCs) at low, medium and high concentration were prepared with a mixture of human plasma/serum pools and rat plasma.
Results
The assay used 10 μL of plasma, and only a 20 min digest time without analyte enrichment or cumbersome reduction and alkylation steps. The upper limit of AMR was 100 mg/L, compared to 10 mg/L for the Siemens high-sensitivity immunoassay. At the lowest calibrator concentration, the bias and CV were less than 15 %. No carryover was observed within the AMR. A method comparison to the Siemens ADVIA 1800 (n = 20), yielded the following regression HPLC-MS/MS =0.86×ADVIA+2.20, R2 = 0.92. Evaluation of precision (5 replicates × 5 runs) demonstrated a total CV of 13.7 %, 8.6 %, and 16.6 % for low, medium, and high QCs, respectively. A larger method comparison and long-term (> 6 month) stability of our in-house developed quality controls, along with evaluation of the recently released CRP reference material from the National Institute of Standard and Technology, are currently underway.
Conclusions & Discussion
CRP in plasma can be quantified by HPLC-MS/MS with preliminary data indicating performance comparable to an existing clinical immunoassay. We developed a simple workflow without sample cleanup/enrichment steps such as solid phase extraction and immunoprecipitation and without reduction/alkylation steps, thus facilitating a rapid turnaround time and minimal reagent costs. The new method has a cost-per-test of a few pennies, and requires 15-times less sample volume and has an analytical range 50-times greater than a commercial immunometric method. HPLC-MS/MS provides an alternative and cost-effective method for CRP quantification in plasma that is benchmarked against gravimetric targets.
References & Acknowledgements:
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