In the Microbiology lab I teach, blood typing is one of the labs that my students enjoyed doing as it directly applies to their own life. Our blood consists of red blood cells (RBCs), white blood cells, platelets and serum (the liquid, non-cellular portion). On the surface of the RBCs, there are specific proteins attached, sort of like ID cards. These are called antigens. For example, if your blood group is B+ve, then you have the ‘B’ antigen and ‘D’ antigen on your RBCs (See Fig 1). You might have also heard that you can only accept blood from another person who has the same blood type as you. Why? That is because in the serum or non-cell part of your blood there are other proteins called as antibodies whose job is to detect any antigens/ID cards that are foreign and destroy them. It is therefore crucial to be able to quickly test if blood types are compatible between a donor and a recipient, even if they share the same blood type to avoid any unnecessary complications resulting from destruction of RBCs. In the lab class, my students mixed synthetic blood samples (which would contain the antigens) and various serum antibodies and observed for a reaction which can be visualized as small clumps or in scientific terms called as agglutination. Sometimes the agglutination was not easily visible leading to some confusion about the blood typing. However, in a large hospital/diagnostic center setting, a reasonably quick, effective and reliable method is of utmost importance as there can be no room for confusion and, mistakes would be costly. Such methods exist already but a newly published paper in Science Translational Medicine shows the discovery of a new assay that allows one to find out their blood type within 2 mins by observing a color change on a piece of paper.

blood_types

Traditionally, to be able to test for the presence of antigens on the RBCs and the antibodies in the serum, the blood is first spun at a high speed to separate the blood components. The USP of this new paper-based method is that there is no prior blood preparation needed. The method takes advantage of the presence of a protein called human serum albumin (HSA) in the serum of the blood and its ability to react with a dye called bromocrecol green (BCG). The HSA-BCG dye complex produces a teal color. Whole blood which contains both RBCs and HSA in the serum reacts with the dye to give a brown color. Both reactions absorb light at specific wavelengths and produce distinct spectra which the authors find especially useful to automate this process and ensure accuracy by removing the need for manual color distinction. Let us visualize this using our previous example of B blood type which will have B antigen on the RBCs and antibodies ready to destroy the A antigen in the serum (Fig. 1). As the blood moves along the paper, imagine its constituents undergoing reactions progressively. First, the B antigens on the RBCs would react with the immobilized anti-B antibody on the paper. Now the blood is free of RBCs and the serum contains antibodies against the A antigen and HSA. Next, the antibodies against A would react with the A type red cells confirming that it is indeed B blood group. Finally, devoid of any RBCs and antibodies, the serum now only contains HSA which reacts with the BCG dye resulting in a teal color. If the sample was not the B blood type, then HSA and BCG dye would react to form a brown color and none of the other reactions would occur. The whole process takes about 2 minutes and just a few microliters of blood.  Using this Dye Assisted Paper-based method (DAP), the authors tested more than 3500 blood samples and achieved 99% accuracy in blood typing and the method showed a similar accuracy when performed side by side with the commonly used gel card method of blood typing.

Since this is a new paper-based method, the authors conduct many quality control tests to show that their method is robust and reproducible. For instance, they test a bunch of different papers and membranes for suitability to effectively separate the RBCs and serum. They also test various dilutions of RBCs (antigens) and antibodies to determine the ideal ratio needed to produce reproducible results and the stability of the immobilized antibodies on the paper (6months). They examine if longer interactions between antigens and antibodies affect color change (maximum intensity was seen at 10mins), investigate how long after blood collection the test result is reliably accurate (7 days), if the pH of the blood influences the reaction (no) and whether various temperature, light and humidity conditions during experiment affect the results (no). Although they suggest ways to overcome challenges of testing blood with varying number of RBCs (think anemic conditions or newborns who typically have higher number of RBCs), they acknowledge their inability to predict if the DAP method would be affected by samples from people with any changes in their serum proteins due to disease or medications. They also realize that their method needs to be made better at detecting some of the rare RBC antigens that are weakly expressed.

The DAP assay appears to be a better, easier and quicker mouse trap for blood typing and will probably replace some of the current blood typing methods in the near future.

 

References

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

Image courtesy – inline image, gel card method of blood typing – cover image