Supplementary Materials 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@tmmu.edu.cn

Published 15 March 2017, Sci. Transl. Med. 9, eaaf9209 (2017)

DOI: 10.1126/scitranslmed.aaf9209

The 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).

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

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

Other Supplementary Material for this manuscript includes the following:

(available at

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

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.

Fig. S1. Schematics of the DAP assays. The structures of the ABO forward strip (A) and ABD format

(B). Ain red color (anti-A coatedstrip), B in red color (anti-B coatedstrip), D in red color (anti-D coated

strip), QC in red color (Quality Control, without antibody). (C) The structures of theABO F&R format.

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

antibody immobilization. (A) Migration rates of blood (20 µl) on the NC membrane, filter paper,

fiberglass paper and cotton linter paper in 15 sec, 30 sec and 120 sec. (B) Agglutinated blood fixation

on filter paper, fiberglass paper and cotton linter paper. Blood from type A donors spotted on Anti-A

antibodies adsorbed to filter paper, fiberglass paper and cotton linter paper and chromatographically

eluted with 0.9% NaCl buffer for 30 sec. (C) Confocal images of fiberglass paper and cotton linter

paper structure captured using a 20x lens.Blue color lines denote the autofluorescence of these paper

fibers. Scale bar is 20 μm. (D) Validation of the antibody immobilization. Ten microliter of type A

(or type B) blood was loaded on anti-A (or anti-B) coated strip and the RBC agglutination was

observed after the strips was eluted by PBS buffer. Dashed box indicates the antibody coated zone. The

differernt plasma separation pattern represents differenct plasma generating capability of plasma

separation membranes for reverse typing (red indicates the blood). (E) Schematics illustrating plasma

separation direction according to membrane structure. GR and PES membranes employ downward

infiltration; GF2 and MF1 membranes employ parallel infiltration. A same-sized cotton linter pad was

placed beneath the separation pad for absorption of plasma and then attached to the test paper on the

right owing to the vertical separation of plasma using GR/PES. In contrast, for GF2 and MF1, the test

paper was directly attached to the separation pad on the right without the additional cotton linter pad.

(F) The plasma generation of 200 µL whole blood using four types of plasma separation membranes

(GR, PES, GF2, and MF1) at 15 sec, 30 sec, 120 sec.The observed plasma wick distance is indicated

bythe blue dashed box.

Fig. S3. Optimized concentrations of reagents, RBCs, and antibodies. (A) Sensitivity tests of

theABO reverse typingby serially dilutingreagent red blood cell (5%, 10%, 20%, 40%, 80%)carrying

known antigens (A1 cells, B cells). GF2 plasma separation membrane was used to filter the plasma of

type A sample in the middle. (B) Antibody concentration optimization for anti-A, anti-B, anti-C, anti-c,

anti-D, anti-E, anti-e.Color intensity of thenonagglutinated reagent red cell (concentrated from

thecommercialreagent to a hematocrit level of 45%) on paper (blue box zone) was recorded. The color

intensity was obtained by subtracting the intensities of the red (R)/green (G)/blue (B) color channels

using ImageJ 1.44p software (color intensity=0.229R+0.587G+0.114B). Data are means ± SD (n = 8

per fold).

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

(A) Agglutination was observed after loading typeA, typeB blood sample onanti-A coated strips stored

for 10-180days. Black dashed boxesdenote the antibody zones. (B) Images of the the ABO format

assay forreaction times of 30-600 sec. (C) Colorintensities ofthe detection zones in B after different

observing time.Data are means ± SD (n =3).

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

BCG-plasma reaction color intensity according to sample pH.No significant difference in color

intensity has been observed between samples with different pH conditions(p=0.78, one-way ANOVA,

n=5).(B) BCG-plasma reaction color absorbance spectra according to sample ionic strengths. Samples

with different ionic strengthes and normal samples demonstrated no statistically significant difference

in absorbance (A630) (* P=0.84versus normal samples). Data are means ± SD (n = 10).

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

of reflectanceof indipendent samples with (A) normal HCT level, (B) low HCT level, (C) high HCT

level, and (D) the measured reflectivities of testing area at 410 nm. No significant differences of

relative reflectivity (zones I, III, and IV)were observed between samples with different HCT values

(P=0.79 for zone I, P=0.66 for zone III, and P=0.96 for zone IV, one-way ANOVA). A difference of

relative reflectivity (zone II) was observed between samples with low and high HCT values (P=0.006,

one-way ANOVA with LSD test), while no significant differences were observed between low and

normalHCT values (P=0.11, one-way ANOVA with LSD test) and between high and normal HCT

values (P=0.15,one-way ANOVA with LSD test). The spectrum of reflectance with (E) normal HSA

level, (F) low HSA level, (G) high HSA level, and (H) the measured reflectivities of the testing area at

410 nm.No significant difference of relative reflectivity (zone I, II, III, and IV) was observed between

samples with low and normal HSA value (P=0.79 for zone I, P=0.94 for zone II, P=0.89 for zone III,

and P=0.85 for zone IV, one-way ANOVA). Data are means ± SD (n = 5).

Fig. S7. Influence of different blood storage times on the final results. (A) The migration distance

of blood in antibody-coated cotton linter strip after storing the wholeblood for 4-16 days (4℃, use

EDTA asanticoagulant). (B) The migration distance of blood in a ABO F&R strip after different

storage time (4 days, 8 days,12 days,and 16 days).

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

the BCG-plasma reaction in different ambient (A) temperature, (B) humidity, and (C) light strength.

Fig. S9. Relative reflectances of invalid samples. These invalid samples include (A) BCG-red ink, (B)

BCG-PBS, and (C) BCG only on the cotton linter paper strip. n=5

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

feasibility of the proposed DAP blood grouping was validated by an area under the curve (AUC) of

overall samples (AUC=0.999) from the outward box. Pink line represents the ROC curve of the

overeall samples. The insertedthree boxes represent the ROCs of each group: upper left for teal

(AUC=0.999, blue), upper right for brown (AUC=1.000, green), lower left for invalid samples

(AUC=1.000, red).

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

assays. (A) Images of type A, type B, type AB, and type O blood samples using the ABO forward

assay. Red dashed boxes (I and II) represent the observing windows and s in black represents the

sample loading zone. (B) The corresponding reflectance spectrum of type A, type B, type AB, and type

O blood samples using the ABO forward assay (data from the red observing windows). (C) Images and

(D) the corresponding reflectance spectrum of a type B and RhD sample in aABD format assays.

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

Visual result and (B) the measured reflectance spectrum of type A, type B, type AB, and type O

bloodsamples.

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

readoutof a type A and Rh (ccDee) blood sample in the ABO/Rh format assay. Balck box indicates the

detection zone in each strip. (B) The corresponding reflectance spectrum (detection zone in panel A)

for a type A and Rh (ccDee) blood sample in the ABO/Rh format assay. (C) Visual results oftwoweak

D samples on the paper stripusing the indirect Coombs' test. Sample 1 and sample 2 are representatives

of two different blood samples. Columns A, B, and D in the gel-card (left) represent corresponding

detection columns for type A, type B, and Rh (D). Columns IgG, IgG+ IgM are applied for indirect

Coombs' test (middle). A similar control experiment was conducted in the DAP strip, anti-human

antibodies (IgG+IgM) have been immobilized on the strip in advance (right) and the elution was

applied after different reaction time (5 min vs. 3 min). Longer reaction time (5 min) induces shorter

wicking distance in both samples.

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

(A).The observation zones arethe strips between the two dashed black circles. (B) The corresponding

reflectance spectrumofrare blood groups assay at the observing zones.

Fig. S15. The reproducibility of the DAP F&R assay. Relative reflectance of a sample (type B) in

three duplicates within one day and assayed consecutively for an additional 8 days. The colorful solid

lines represent the results of three duplicates at four observing windows (I, II, III, and IV).

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. (A) Modified staining reagents, (B) traditional DMSO.

FITC-RBCs shown in green. Black square frame denotes the amplified image of the red frame. Red

arrows indicate the red blood cells susceptable to deformation and fragmentation when prepared using

traditonal DMSO.

Supplementary Tables

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

Membrane

Vivid Cobetter JY biotech Whatman

（GR） （PES HD 1.8B） （GF2） （MF1）

Material

asymmetric

polysulfone

polyether sulfone fiberglass fiberglass

Thickness(μm) 330 +/- 20 350 850 367

Plasma generation ≥ 80% ≥70% ≥ 75% ≥73%

Hemolysis levels no hemolysis no hemolysis no hemolysis no hemolysis

Plasma separation time (s/cm2) ≤ 2 minutes ≤ 2 minutes ≤ 2 minutes ≤ 2 minutes

Blood volume recommendations (μL/cm2) 40-50 50-60 90-100 60-70

The detailed parameters refer to manufacturer's instructions.

Table S2. Monoclonal antibody details.

Blood

group

Antibody Clones

optimal titer

(Diluted folds)

Type

ABO

A BIRMA-1 625(1/32) IgM

B LB-2 625(1/32) IgM

Rh

C P3X25513G8 1250(1/16) IgM

c H48 1250(1/16) IgM

D LDM1, ESD1M 312.5(1/64) IgM/IgM

E DEM1 625(1/32) IgM

e P3GD512, MS-63 1250 (1/16) IgM

Kell

K MS56 1250 (1/16) IgM

k Lk1 1250 (1/16) IgM

P P1 650 625 (1/32) IgM

Kidd

Jka P3HT7 1250 (1/16) IgM

Jkb P3 143 1250 (1/16) IgM

MNS

M 2514E6 1250 (1/16) IgM

N 1422C7 1250 (1/16) IgM

S P3S13JS123 1250 (1/16) IgM

s P3YAN3 1250 (1/16) IgM

Lewis

Lea 13643B9 1250 (1/16) IgM

Leb GX336 625 (1/32) IgM

Duffy

Fya DG-FYA-02 1250(1/16) IgM

Fyb SpA264LBg1 1250 (1/16) IgM

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

Reference ranges are as follows: K (3.5~5.5 mmol/L); Na (135~150 mmol/L); Cl (96~111 mmol/L); Ca (2.2~2.65

mmol/L); Pi (0.81~1.55 mmol/L); Mg (0.6~1.1mmol/L); **Higher than the reference value; *** lower than the

reference value.

Sample K Na Cl Ca Pi Mg

1 4.53 131.60*** 99.80 2.31 1.37 1.20**

2 4.68 141.98 95.10*** 2.67** 2.55** 1.27**

3 5.94** 152.13** 94.50*** 2.17*** 2.33** 1.17

4 6.79** 148.32 99.30 1.92*** 2.54** 1.71**

5 6.38** 145.10 105.20 2.57 2.95** 1.11**

6 6.05** 133.70*** 107.10 2.42 1.05 1.05

7 4.25 132.80*** 95.00*** 3.34 2.37** 1.19**

8 6.73** 130.14*** 104.90 2.31 1.89** 1.07

9 5.90** 134.60*** 99.70 2.69** 2.31** 2.64**

10 4.10 133.50*** 95.00*** 3.38** 2.01** 1.08

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

Blood group (ABO)

Gel card assay Forward assay

Accuracy* (%)

Anti-A Anti-B Anti-A Anti-B

A (n=256) 256 0 256 0 100%

B (n=196) 0 196 0 196 100%

AB (n=66) 66 66 66 66 100%

O (n=282) 0 0 0 0 100%

Total (n=800) 322 262 322 262 100%

Blood group Gel card assay ABD Accuracy (%)

ABO

A 15 15 100.00%

B 16 16 100.00%

AB 5 5 100.00%

O 14 14 100.00%

Rh D 50 50 100.00%

*𝑎𝑐𝑐𝑢𝑟𝑎𝑐𝑦 =𝑇𝑃+𝑇𝑁

𝑇𝑃+𝑇𝑁+𝐹𝑃+𝐹𝑁

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

Blood group

(ABO)

Gel card assay F&R assay

Accuracy (%)

Anti-A Anti-B Antigen-A Antigen-B Anti-A Anti-B Antigen-A Antigen-B

A (n=425) 425 0 0 425 424 0 0 425 99.76%

B (n=357) 0 357 357 0 0 357 357 0 100%

AB (n=160) 160 160 0 0 160 160 0 0 100%

O (n=458) 0 0 458 458 0 0 458 458 100%

Total (n=1400) 585 517 815 883 584 517 815 883 99.93%

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

Blood group Gel card assay ABO/Rh

Accuracy

(%)

ABO

A 390 389 99.92%

B 288 288 100.00%

AB 102 102 100.00%

O 420 421 99.76%

Rh

C 1041 1041 100.00%

c 589 589 100.00%

D 1196 1196 100.00%

E 486 486 100.00%

e 1098 1098 100.00%

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

Blood group Gel card assay

Rare blood

groups assay

Accuracy (%)

Kell

K+ 0 0 100%

k+ 100 100 100%

P P1+ 0 0 100%

Kidd

Jka+ 66 66 100%

Jkb+ 87 87 100%

MNS

M+ 90 90 100%

N+ 79 79 100%

S+ 1 1 100%

s+ 100 100 100%

Lewis

Lea+ 9 9 100%

Leb+ 76 76 100%

Duffy

Fya+ 85 85 100%

Fyb+ 52 52 100%

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

Gel card assay Dye-based assay

TP RBC agglutinated RBC agglutinated, the detection zone turned teal

FP RBC non-agglutinated RBC agglutinated, the detection zone turned teal

TN RBC non-agglutinated RBC non-agglutinated, the detection zone turned brown

FN RBC agglutinated RBC non-agglutinated, the detection zone turned brown

TP = true positives, FP = false positives, TN = true negatives, and FN = false negatives .

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.