Blood Phenotyping Assay On A Magnetic Bead Labeled Array Or In A Fluorescence-encoded Microsphere Labeled Suspension Array

Background and ObjectivesBlood grouping make it possible to transfuse safely.With medical development,it has been affirmed that more and more blood group antigens from different blood group systems are closely associated with safe blood transfusion.More blood group antigens should be identified rapidly to improve personalized health care services.However,conventional methods to phenotype the blood groups were single-targeted and time-consuming,which poses an obstacle to determine large-scale blood group antigens.In this study,two alternative methods combining conventional microarrays with new markers of magnetic beads or fluorescence-encoded microspheres were developed,which aims at phenotyping the blood groups rapidly and multiply.MethodsIn the assay on the magnetic bead labeled array,Polymer Slide used as the substrate was modified with 3D aldehyde polymeric layer to provide abundant of activated covalent sites for binding antibodies.The washed RBCs were lysed with Tris-HCl(0.01 M,p H=7.3~7.5)and 0.2 ?l of the lysed RBCs was loaded onto the antibody-coupled sites.Subsequently,0.2 ?l C1q-coupled beads were also loaded onto the sites to recognize the antibody-antigen complex and the unbonded beads were separated under magnetic force.In quantitative analysis,each site was imaged by CCD microscopy and signal intensity was quantified by photoshop.By introducing the fluorescence-encoded microspheres to mark the antibodies and the magnetic beads to mark the hemagglutinin,the performance of the method was improved,which was called fluorescence-encoded microspheres labeled suspension array.In the assay,1 ?l whole blood sample(106 RBC),5 ?l fluorescence-encoded microsphere labeled antibodies,and 5 ?l magnetic bead labeled hemagglutinin were add into 100 ?l normal saline.One minute later,the suspended microspheres in the media were classified and counted by Luminex 100 after separation under magnetic force.ResultsIn the assay on the magnetic bead labeled array,it was found that PBS-Glycerinum(40% glycerinum in the PBS)was efficient to avoid the coffee-ring formation and the magnetic beads labeled C1 q was efficient to recognize the antibody-antigen complex.In addition,the liquid or semi-liquid environment was necessary in the assay.The C1 q can couple to magnetic beads efficiently with the coupling reagent of NHS and EDAC.The antibodies used in the assay should be at 100 ?g/ml in a volume of 0.2 ?l and 105 RBCs should be enough for a single detection point.The separation with magnetic pin should be performed at a length of 4~6 cm and the assay should be accomplished in 10~20 min.By analyzing the 108 cases,it was demonstrated that the sensitivity to group A,B,D,M,N antigens were 93.18%,83.72%,92.39%,93.24% and 90.36% respectively and all of the specificities were 100.00%.In the assay in the fluorescence-encoded microspheres labeled suspension array,it was found that the antibodies in the 100 ?l serum decreased 0.17,0.61,0.40,0.31 and 0.94?g respectively after coupled with 106 microspheres,which means 100,380,1600,1400,3770 thousand respective antibody molecules coupled on single microsphere.For a stable result,2000~10000×6 microspheres should be added into media and 106 RBCs were enough in the assay(microspheres vs RBCs =1︰100~20).The assay should be accomplished in 2 min.By analyzing the 276 cases,it was demonstrated that all of the sensitivities and specificities to group A,B,D,M,N antigens were 100.00%.In addition,the weak aggregation RBCs(1+,grouped with microcolumn gel test)had similar test performance with the strong aggregation(1+ vs 4+,P=0.889)and the stored RBCs(stored at 4 ℃ for 6 months)had similar test performance with the fresh(stored vs fresh,P=0.804).ConclusionTwo methods for multiply blood grouping were preliminarily established.The assay on the magnetic beads labeled microarray was multiple-targeted flexible and cost-saving,as well as be fit to lysed or free blood group materials determination.After introducing the fluorescence-encoded microspheres to mark the antibodies,the sensitivity and specificities has a significant improvement,and batch test can be implemented in a faster speed.