Discrimination of ischemic and hemorrhagic strokes using a multiplexed, mass spectrometry-based assay for serum apolipoproteins coupled to multi-marker ROC algorithm

Abstract

Purpose: Typically, apolipoproteins are individually measured in blood by immunoassay. In this report, we describe the development of a multiplexed selected reaction monitoring (SRM) based assay for a panel of apolipoproteins and its application to a clinical cohort of samples derived from acute stroke patients.

Experimental design: An SRM assay for a panel of nine apolipoproteins was developed on a triple quadrupole mass spectrometer. Quantitative data for each apolipoprotein were analyzed to determine expression ratio and receiver operating characteristic (ROC) values for ischemic versus hemorrhagic stroke.

Results: The optimized SRM assay was used to interrogate a small cohort of well-characterized plasma samples obtained from patients with acute ischemic and hemorrhagic strokes. The ROC analyses demonstrated good classification power for several single apolipoproteins, most notably apoC-III and apoC-I. When a novel multi-marker ROC algorithm was applied, the ischemic versus hemorrhagic groups were best differentiated by a combination of apoC-III and apoA-I with an area under the curve (AUC) value of 0.92.

Conclusions and clinical relevance: This proof-of-concept study provides interesting and provocative data for distinguishing ischemic versus hemorrhage within first week of symptom onset. However, the observations are based on one cohort of patient samples and further confirmation will be required.

Figure 1

Workflow for automated development and optimization of SRM assays.

Figure 2

Pinpoint-generated calibration curves for apolipoprotein heavy peptides in a background of 30 µg raw serum digest. The 8-point curves measured concentrations from 250 attomoles to 500 femtomoles on column, in triplicate. LLODs were estimated at 250–500 attomoles and LOQs were calculated between 1 and 5 femtomoles for each peptide. The linear correlation coefficients ranged from 0.93 to 0.99 and the CVs for points above the LOQ ranged from 0 to 20%. (A) apoA-I; (B) apoA-II; (C) apoB; (D) apoC-I; (E) apoC-II; (F) apoC-III; (G) apoD; (H) apoE; (I) apoH.

Figure 2

Pinpoint-generated calibration curves for apolipoprotein heavy peptides in a background of 30 µg raw serum digest. The 8-point curves measured concentrations from 250 attomoles to 500 femtomoles on column, in triplicate. LLODs were estimated at 250–500 attomoles and LOQs were calculated between 1 and 5 femtomoles for each peptide. The linear correlation coefficients ranged from 0.93 to 0.99 and the CVs for points above the LOQ ranged from 0 to 20%. (A) apoA-I; (B) apoA-II; (C) apoB; (D) apoC-I; (E) apoC-II; (F) apoC-III; (G) apoD; (H) apoE; (I) apoH.

Figure 3

(A) Graphical representation of the multi-marker ROC algorithm. The different thresholds are varied for the two different marker populations. The highest TP rate for a given FP rate generates the final ROC curve. (B) Overlaid ROC plots of the single markers and marker pair with the highest AUC for differentiating ischemic from hemorrhagic samples (see Table 3).