| Background: Rapid and accurate blood grouping plays a critical role in multiple scientific disciplines,particularly in the biological and medical sciences.Among the 35 officially recognized blood group systems,the ABO and Rhesus(Rh)systems receive the most attentions.As we know,the high mortality originating from mismatched ABO/Rh blood transfusion reactions has caused heavy social burden.Conventional methods for blood grouping can be categorized as serological phenotyping and genotyping.Despite the high accuracy,defects such as long turnaround times,labor-intensive operation,and technical training requirements to operate deprive their capacities of application in emergencies like car accidents,earthquakes,and war.To date,no reliable methodologies that have integrated forward and reverse(F&R)tests into one assay have been proposed,although there have been a few attempts in the past years.Here,an easy-to-interpret dye-assisted paper-based(DAP)assay for blood grouping is proposed,by integrating the F&R tests into a single Point-of-Care(POC)chip using a visual readout strategy.Object: A kind of rapid and accurate blood grouping assay was developed making use of paper-based diagnostic techniques,by which simultaneously forward and reverse blood grouping can be performed using only one just a drop of full blood.Meanwhile,simultaneously detection of various blood groups such as ABO/Rh is realized without centrifugation.At the same time,it provides an alternative strategy for blood groupingin shorter time,improving detection efficiency,by which time for rescue could be shortened and support for blood transfusion in an emergency could be gurantted.Additionally,the popularity of blood grouping in developing and resource scarcity areas could also be extended.Methods and Results:There were two parts in developing this rapid blood grouping technology.Part one: The establishment of dye-assisted paper-based(DAP)blood grouping technique.1.This research makes use of the mechanism that bromocresol green(BCG)dye immediately changes color in the presence of protein.Measuring the absorbance can prove the reliability of serum protein testing by BCG dye.When nonionic surfactant exists in acidic environment such as p H4.2,BCG dye can interact with plasma proteins and form blue-green complex with a characteristic absorption peak in 630 nm.Meanwhile,BCG dye can interact with whole bloodto generate brown complex with adistinctive absorption peak intensities in 530 nm,540 nm,and 570 nm.However,the reaction between BCG and blood alike materials caused no significant color change,so that the red color could be observed.2.In the later research,we had made strict selection of substrates and took the cotton linter paper and fiberglass paper as a substrate in the follow-up study.In principle,only serum can migrate along the porous chromatographic paper used in a paper-based assay after the RBCs are aggregated,whereas both serum and RBCs can migrate freely along the substrate if the RBCs remain non-aggregated.Based the above principle,an easy-to-interpret dye-assisted paper-based assay was designed,which read out the blood group in visual through color change.Further,optimal antigen and antibody concentrations were established to improve the analytical efficiency of the DAP assays.With a series of dilutions of seven antibodies(anti-A/B/D/C/c/E/e)introduced,a titer-dependent optical density profile was established.The results suggested that a 1:32 dilution was the optimal concentration for the anti-A,anti-B,and anti-E antibodies,a 1:64 dilution was optimal for the anti-D antibody,and a 1:16 dilution was sufficient for the other antibodies.The optimal red blood concentration for reverse blood grouping is 20%RBC.A Jieyi glass fiber II(GF2)micro-glass fiber was used as the plasma separation membrane in the reverse strip to separate plasma without centrifugation.At the same time,we characterized the results of the DAP blood grouping using fluorescein and spectrophotometry.3.Using the above BCG bio-sensing mechanism,we developed a series of blood-grouping formats that meet various clinical requirements: a fast ABO forward and ABD(ABO and Rh D)test,an ABO F&R format and an ABO/Rh format(A/B/D/C/c/E/e)for routine clinical tests,and a rare group format for specialized applications.Both ABO(A/B)and Rh(D/C/c/E/e)blood group antigens were differentiated within 30 s,whereas ABO F&R blood grouping required 2 min.By investigating on the reflectance spectra of DAP blood grouping,we found the reflectance curves of BCG-HSA complex was significantly different from that of BCG–whole blood complex.We detected the result of the series of blood-grouping formats through spectrophotometry,then reflectance spectra of different blood groups are obtained.Part two: Methodology evaluationand automatic identification of DAP technique.1.The effects of blood pH,ion intensity,hematocrit(HCT)value and human serum albumin(HSA)concentration on dye-assisted blood grouping assay were studied.It was found that the pH value,ion intensity and HSA concentration had no influence on color reaction.The change of HCT level affected the reflectance curve,which was inversely proportional to the reflectance curve,but the reflectance spectrum trend of different HCT level sample were identical.Furthermore,the conditions of test time,temperature,humidity,and light strengths were optimized to ensure the elimination of the interference of the detection environment.2.The data set of reflection spectra is analyzed by means of machine learning method.The classification algorithm was validated using 10-fold cross-validation tests.The proposed support vector machine(SVM)system demonstrated an accuracy of 99.9%,a sensitivity of 99.9%,and a specificity of 100% for the ABO F&R blood groups.The feasibility of the proposed DAP blood grouping was also validated by an area under the curve(AUC)of 0.999 and a Matthews correlation coefficient(MCC)of 0.999.3.Further,3550 clinical human blood samples were detected to validate the robustness of the proposed DAP blood-grouping platform.In contrast with the gel-card assay(combine with test tube method),an accuracy of 99.94% was obtained.Conclusions:The dye-assisted paper-based assay for rapid and automatic blood grouping holding the important clinical value and economic benefit in social development has been developed and validated clinically.This assay not only provides a new strategy for blood grouping but also can be used in time-and resource-limited situations,such as war zones,remote areas,and emergency circumstances.Characterized by an intensified and streamlined workflow capability,the proposed blood-grouping assay may be further developed into highly compact and fully automatic platforms that are highly efficient and economical,making large-scale manufacturing possible. |
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