Research ArticleDiagnostics

A dye-assisted paper-based point-of-care assay for fast and reliable blood grouping

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Science Translational Medicine  15 Mar 2017:
Vol. 9, Issue 381, eaaf9209
DOI: 10.1126/scitranslmed.aaf9209
  • Fig. 1. Mechanism of the dye-based visual readout for fast blood grouping.

    (A) Chemical structures of BCG (C21H14Br4O5) and HSA and the principle of the BCG-HSA reaction. (B) Color changes observed when BCG reacted with plasma (teal), whole blood (brown), PBS (no color change), and red ink (dark red). (C) Relative absorbance values for BCG (black line), PBS-plasma (red dashed line), PBS–whole blood (gray line), the BCG-plasma complex (red line), and the BCG–whole blood complex (blue line).

  • Fig. 2. Schematic of the fast blood-grouping devices.

    Designs, testing procedures, and results of our (A) ABO forward strip, (B) F&R assay, and (C) ABO and Rh group multiplex antigen assays. I and II represent the forward blood-grouping observation windows; III and IV represent the reverse blood-grouping observation windows. The observation zone is located between the two dotted lines as shown in the platforms of the ABO and Rh group assays (C). A strip without antibody (BCG only) was used for quality control (QC).

  • Fig. 3. Characterization of the results of the DAP blood grouping using fluorescein and spectrophotometry.

    Confocal images of (A) agglutinated RBCs, (B) nonagglutinated RBCs, (C) nonagglutinated RBC-HSA, and (D) HSA within the paper. RBCs, green; HSA, red; cotton linter paper, blue. (E) Divergent waveforms of the reflectance curves for BCG–red ink, BCG-plasma, BCG–whole blood, BCG-PBS, plasma, and whole blood. Circle regions denote the shared characteristic peaks at 410 and 630 nm for both BCG-plasma and BCG–whole blood samples. Arrows are the newly appeared peaks for BCG–whole blood mix when compared with BCG-plasma mix.

  • Fig. 4. Evaluating the feasibility of detecting stored samples (type A).

    (A) Relative reflectance curves of blood samples stored for 0 to 20 days before testing using the F&R assay. I and II represent the observation windows for the forward blood grouping; III and IV represent the observation windows for the reverse blood grouping. (B) FHB concentration in samples stored for 0 to 20 days before testing. (C) Relative reflectance of the detection zones (I, II, III, and IV) at 410 nm for samples stored for 0 to 20 days before testing.

  • Fig. 5. Results of the visual readout for fast blood grouping.

    Images of (A) forward strip and (B) F&R assays showing ABO grouping for A, B, AB, and O as indicated by the presence of teal color in the observation zone (red dashed box). Images of multiplexed assays testing (C) ABO/Rh and (D) rare blood systems. The sample in (C) is ABO (A)/Rh (ccDee); the samples in (D) show Kell (Kk+), P (P2), Kidd (Jka+Jkb+), MNS (MNss), Lewis (Lea−Leb+), and Duffy (Fya+Fyb−).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/381/eaaf9209/DC1

    Fig. S1. Schematics of the DAP assays.

    Fig. S2. Improved performance of the DAP assay through optimized paper structure design and antibody immobilization.

    Fig. S3. Optimized concentrations of reagents, RBCs, and antibodies.

    Fig. S4. Stability of the immobilized antibodies and the optimal reaction time of the DAP assay.

    Fig. S5. Stability of the BCG-plasma reaction according to pH and ionic strength.

    Fig. S6. Results of samples with different HCT and HSA levels using the F&R assay.

    Fig. S7. Influence of different blood storage times on the final results.

    Fig. S8. Stability of the BCG-plasma reaction according to environmental factors.

    Fig. S9. Relative reflectances of invalid samples.

    Fig. S10. The receiver operating characteristic (ROC) curves and AUC of the samples.

    Fig. S11. Images of the visual readout and reflectance spectra using the ABO forward and ABD assays.

    Fig. S12. Images of the visual readout and reflectance spectra using the ABO F&R assay.

    Fig. S13. Images of the visual readout and reflectance spectra using the ABO/Rh assay.

    Fig. S14. Image of the visual readout and reflectance spectrum of a rare blood groups assay.

    Fig. S15. The reproducibility of the DAP F&R assay.

    Fig. S16. A brief instruction book for using the ABO forward strip.

    Fig. S17. Confocal microscopic images of the FITC-RBCs prepared by using either the modified staining reagents or the traditional DMSO.

    Table S1. Parameters of four types of plasma separation membranes.

    Table S2. Monoclonal antibody details.

    Table S3. Ionic strength change from blood samples of uremic patients.

    Table S4. Accuracy of the ABO forward assay (n = 800) and ABD assay (n = 50).

    Table S5. Accuracy of the ABO F&R assay.

    Table S6. Accuracy of the ABO/Rh assay (n = 1200).

    Table S7. Accuracy of other rare blood group assay (n = 100).

    Table S8. Definitions of TP, FP, TN, and FN used in the machine-learning method.

    Table S9. Individual level data for N < 20.

    Movie S1. The testing procedure of fast ABO forward assay.

    Movie S2. The testing procedure of ABO and Rh group multiplex antigen assay.

  • Supplementary Material for:

    A dye-assisted paper-based point-of-care assay for fast and reliable blood grouping

    Hong Zhang, Xiaopei Qiu, Yurui Zou, Yanyao Ye, Chao Qi, Lingyun Zou, Xiang Yang, Ke Yang, Yuanfeng Zhu, Yongjun Yang, Yang Zhou, Yang Luo*

    *Corresponding author. Email: luoyang{at}tmmu.edu.cn

    Published 15 March 2017, Sci. Transl. Med. 9, eaaf9209 (2017)
    DOI: 10.1126/scitranslmed.aaf9209

    This PDF file includes:

    • Fig. S1. Schematics of the DAP assays.
    • Fig. S2. Improved performance of the DAP assay through optimized paper structure design and antibody immobilization.
    • Fig. S3. Optimized concentrations of reagents, RBCs, and antibodies.
    • Fig. S4. Stability of the immobilized antibodies and the optimal reaction time of the DAP assay.
    • Fig. S5. Stability of the BCG-plasma reaction according to pH and ionic strength.
    • Fig. S6. Results of samples with different HCT and HSA levels using the F&R assay.
    • Fig. S7. Influence of different blood storage times on the final results.
    • Fig. S8. Stability of the BCG-plasma reaction according to environmental factors.
    • Fig. S9. Relative reflectances of invalid samples.
    • Fig. S10. The receiver operating characteristic (ROC) curves and AUC of the samples.
    • Fig. S11. Images of the visual readout and reflectance spectra using the ABO forward and ABD assays.
    • Fig. S12. Images of the visual readout and reflectance spectra using the ABO F&R assay.
    • Fig. S13. Images of the visual readout and reflectance spectra using the ABO/Rh assay.
    • Fig. S14. Image of the visual readout and reflectance spectrum of a rare blood groups assay.
    • Fig. S15. The reproducibility of the DAP F&R assay.
    • Fig. S16. A brief instruction book for using the ABO forward strip.
    • Fig. S17. Confocal microscopic images of the FITC-RBCs prepared by using either the modified staining reagents or the traditional DMSO.
    • Table S1. Parameters of four types of plasma separation membranes.
    • Table S2. Monoclonal antibody details.
    • Table S3. Ionic strength change from blood samples of uremic patients.
    • Table S4. Accuracy of the ABO forward assay (n = 800) and ABD assay (n = 50).
    • Table S5. Accuracy of the ABO F&R assay.
    • Table S6. Accuracy of the ABO/Rh assay (n = 1200).
    • Table S7. Accuracy of other rare blood group assay (n = 100).
    • Table S8. Definitions of TP, FP, TN, and FN used in the machine-learning method.
    • Legend for table S9
    • Legends for movies S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S9 (Microsoft Excel format). Individual level data for N < 20.
    • Movie S1 (.mp4 format). The testing procedure of fast ABO forward assay.
    • Movie S2 (.mp4 format). The testing procedure of ABO and Rh group multiplex antigen assay.

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