Multiplexed micronutrient, inflammation, and malarial antigenemia assessment using a plasma fractionation device

Abstract

Processing and storing blood samples for future analysis of biomarkers can be challenging in resource limited environments. The preparation of dried blood spots (DBS) from finger-stick collection of whole blood is a widely used and established method as DBS are biosafe, and allow simpler field processing, storage, and transport protocols than serum or plasma. Therefore, DBS are commonly used in population surveys to assess infectious disease and/or micronutrient status. Recently, we reported that DBS can be used with the Q-plex™ Human Micronutrient 7-plex Array (MN 7-plex), a multiplexed immunoassay. This tool can simultaneously quantify seven protein biomarkers related to micronutrient deficiencies (iodine, iron and vitamin A), inflammation, and malarial antigenemia using plasma or serum. Serum ferritin, an iron biomarker, cannot be measured from DBS due to red blood cell (RBC) ferritin content confounding the results. In this study, we assess a simple blood fractionation tool that passively separates plasma from other blood components via diffusion through a membrane into a plasma collection disc (PCD). We evaluated the concordance of MN 7-plex analyte concentrations from matched panels of eighty-eight samples of PCD, DBS, and wet plasma prepared from anticoagulated venous whole blood. The results showed good correlations of >0.93 between the eluates from PCD and DBS for each analyte except ferritin; while correlations seen for plasma/PCD were weaker. However, the recovery rate of the analytes from the PCD were better than those from DBS. The serum ferritin measures from the PCD were highly correlated to wet plasma samples (0.85). This suggests that surveillance for iron status in low resource settings can be improved over the current methods restricted to only measuring sTfR in DBS. When used in combination with the MN 7-plex, all seven biomarkers can be simultaneously measured using eluates from the PCDs.

Fig 1. Sample panels and experiment design.

Fig 2. Schematic illustrating the plasma collection disc in operation.

The schematic illustrates a single PCD. Photos on the left show the PCD with the the filter disc placed on a manifold for multiple samples and the addition of 35 μL of whole blood to each disc (top), a PDC detail view prior to whole blood being added (middle), and the discs after blood has been absorbed and finally the plasma collection disc exposed after the whole blood treated filter has been removed (bottom). The disc is removed with sterile forceps and is ready for use or can be stored until required.

Fig 3

A. Lin’s concordance correlation coefficient (CCC) plots comparing the analyte measurements in paired wet plasma (x-axes) and DBS samples (y-axes). AGP, α-1-acid glycoprotein; CRP, C-reactive protein; FER, ferritin; HRP2, histidine rich protein 2; RBP4, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin; DBS, dried blood spot; rho_c, CCC. B, Lin’s CCC plots comparing the analyte measurements in paired wet plasma (x-axes) and the adjusted PCD (y-axes). AGP, α-1-acid glycoprotein; CRP, C-reactive protein; FER, ferritin; HRP2, histidine rich protein 2; RBP4, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin; DBS, dried blood spot; rho_c, CCC.

Fig 4

A. Bland Altman plots of the difference in results derived from wet plasma versus DBS types measured using the 7-plex assay. Average of the wet plasma and dry sample type (x-axes) are plotted against the difference between measurements from the 7-Plex assay for paired wet plasma and dry sample types (y-axes). B. Bland Altman plots of the difference in results derived from wet plasma versus the adjusted PCD. For both Figures, the horizontal lines indicate line of perfect agreement (green), mean (purple), and ± 2standard deviations of the difference (red). AGP, α-1-acid glycoprotein; CRP, C-reactive protein; FER, ferritin; HRP2, histidine rich protein 2; RBP4, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin; DBS, dried blood spot.