The Potential and Pitfalls of a Dye-Assisted Paper-Based Assay for Blood Grouping.
The most important role of pretransfusion testing is to ensure that a patient does not receive ABO-incompatible RBCs.  To avoid this situation, it is critically important to accurately determine a patient's blood group. Most healthcare facilities use redundant processes to ensure that the patient's blood group has been determined accurately.
Blood bank laboratories generally utilize 2 approaches to determine a patient's blood type. The "front type" detects the ABO antigens on erythrocytes, whereas the "back type" detects IgM antibodies to ABO antigens in the serum or plasma. Starting in the early 1900s, these tests have been performed by mixing erythrocytes and serum in tubes followed by visual assessment for the presence of agglutination. Although the tube methods are simple, many institutions have switched to alternative methods employing gel or solid-phase technology. These newer technologies can avoid the deficiencies inherent in tube methods such as variability in pipetting reactants and inconsistency in grading reaction strengths. The gel technology also uses lower sample volumes than tubes (2). Both approaches are appealing to blood banks because they have been largely automated.
All of the aforementioned approaches, i.e., tubes, gels, and solid phase, require multistep processes. First, the specimen is centrifuged to separate erythrocytes from plasma or serum, after which the types are performed. The front type is performed by detecting the binding of anti-ABO antibodies with RBCs from the specimen, whereas the back type is performed by detecting binding of anti-ABO antibodies in the patient's serum with RBCs of known ABO groups.
As a further safety measure, point-of-transfusion ABO tests have been suggested and are performed in France. Such tests are done at the bedside just before transfusion as an added safety measure. These methods detect binding of anti-ABO antibodies to patient erythrocytes and only take a few minutes because they do not require centrifugation (3). With these techniques, the antibody--erythrocyte interactions result in agglutination of the RBCs, which must be visually interpreted by the physician, nurse, or other healthcare provider. In one study, about 10%-14% of these results were misread by the transfusionists, although experienced professionals who regularly used these systems had error rates of 4%-9% (3). Given these high error rates, the benefits of such a system are limited, especially when compared with other modern safety systems.
The report by Zhang and colleagues describes an approach that was 99.9% accurate with 600 samples (1). Their system performs both front and back types on one test platform that uses several types of paper. Although the testing is conducted on one test platform, it employs two different approaches, one for the front type and another for the back type. The general approach of using the mobility of RBCs on paper to determine ABO groups has been previously described (4) and has been commercialized (http://www.hemokinesis.com/products/ bioactive-paper-tests/glif-abd).
The front type is performed by applying a specimen of whole blood and eluent to a cotton linter paper strip. The blood migrates by capillary action along the paper strip and encounters a portion of the strip at which anti-A or anti-B antibodies are bound to the paper. If the RBCs express the corresponding antigen, they become bound to that region and plasma alone continues to migrate along the strip to a detection zone. In contrast, if the RBCs do not express the ABO antigen recognized by the antibody, then both RBCs and plasma continue to migrate along the paper strip to the detection zone. In the detection zone, a yellow monoanionic dye, bromcresol green (BCG), is used to determine whether plasma alone or plasma with erythrocytes have migrated past the antibody-coated zone. BCG binds to human serum albumin to create a teal color if plasma alone is present, whereas whole blood produces a brown complex.
The principle underlying the method to determine the back type differs from that of the front type in that whole blood is separated into plasma and RBCs on a glass fiber II microglass fiber membrane (Jieyi) blood separation pad, which retains the RBCs but allows the plasma to pass through. The plasma then migrates along a cotton linter paper strip onto an area that contains group A or B RBCs that had previously been applied. If the plasma contains an antibody that binds to group A or B RBCs, then those RBCs migrate along the paper with the plasma to the detection zone. The RBCs do not migrate on the paper if the plasma lacks antibodies that bind to the RBCs. The presence of plasma alone or plasma with RBCs is then detected by detecting the color when BCG dye is used.
Zhang and colleagues produced a combination assay in which the front and back type strips were adjacent. The main advantage of the method is its speed; indeed, it produces a visual readout in 2 min. However, to get consistently reliable results, assay results from a spectrophotometer were interpreted with software trained using machine learning. It is unclear how much additional time was needed for the spectrophotometer to detect the color and the software to interpret the results.
This system would need refinement and advancement for general clinical utility. The requirement for a spectrophotometric readout precludes its use as a point-of-transfusion test performed at the patient's bedside. Additionally, although it could serve as a rapid ABO test performed in clinical laboratories, there are at least 3 barriers to widespread adoption in this setting.
The first barrier to blood bank adoption of this paper technology concerns the fact that almost all patient specimens are tested for the ABO type and an antibody screen. The antibody screen detects "unexpected" antibodies to minor non-ABO antigens. The most important antibodies detected in the antibody screen are IgG antibodies, which are smaller than the IgM antibodies detected in the back type. Enhancement techniques are normally required to detect relatively small IgG antibodies because they usually cannot agglutinate RBCs on their own.
RBC units cannot be cross-matched and released without the result of the antibody screen. The antibody screen takes longer than the ABO grouping and most automated platforms perform both the ABO group and the antibody screen simultaneously. The paper approach described by Zhang and colleagues does not perform an antibody screen, and it would need major modifications to perform the screen. Currently, the paper-based technique can detect anti-ABO antibodies, but those are normally high concentration, pentameric, high-avidity IgM antibodies that can cross-link RBCs. Although binding of these anti-ABO antibodies to RBCs facilitated migration of RBCs through paper substrates, IgG antibodies to other RBC antigens that are detected by antibody screens are often present in much lower concentrations and probably cannot promote migration of RBCs through paper.
The second barrier to blood bank adoption of this paper technology is related to the fact that hemolysis significantly impacts the assay. ABO types can be determined reliably on specimens that are no more than 1 week old, but as specimens age, free hemoglobin migrates in the plasma, causing interference. Indeed, highly consequential mis-typings occur once specimens are more than 12 days old. Blood banks sometimes need to determine ABO types on RBC units that have been stored for longer periods of time or on older patient specimens. The latter circumstance may result from pretransfusion specimens being collected up to several weeks before testing from outpatients who have not recently been pregnant or transfused.
The third barrier to blood bank adoption of this paper technology concerns the fact that there are minimal benefits from the speed of a rapid test. The current manual tube test to determine front and back types takes approximately 13 min, including centrifugation. Most blood bank specimens are provided hours or days before a transfusion and would not benefit from a more rapid test. Testing of emergency trauma patients would not usually benefit from the rapid test because emergency trauma patients are often provided RBC units even before the blood bank receives a specimen. Hence, only a small proportion of testing situations would benefit from the 2-min assay time offered by the paper-based test described by Zhang and colleagues.
The approach developed by Zhang and colleagues is unlikely to be adopted for clinical use currently. However, their innovative approach may have potential utility either as a rapid point-of-transfusion test or, with substantial improvements, as a laboratory test.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
(1.) Zhang H, Qiu X, Zou Y, Ye Y, Qi C, Zou L, et al. A dye-assisted paper-based point-of-care assay for fast and reliable blood grouping. Sci Transl Med. 2017;9(381)eaaf9209.
(2.) Langston MM, Procter JL, Cipolone KM, Stroncek DF. Evaluation of the gel system for ABO grouping and D typing. Transfusion 1999;39:300 -5.
(3.) Migeot V, Ingrand I, Salmi LR, Ingrand P. Reliability of bedside ABO testing before transfusion. Transfusion 2002;42:1348-55.
(4.) Khan MS, Thouas G, Shen W, Whyte G, Garnier G. Paper diagnostic for instantaneous blood typing. Anal Chem 2010;82:4158-64.
Steven R. Sloan  *
 Department of Laboratory Medicine, Boston Children's Hospital, Boston, MA.
* Address correspondence to the author at: Boston Children's Hospital, 300 Longwood Ave, Bader 406, Boston, MA 02115. Fax 617-730-0615; e-mail email@example.com.
Received May 11,2017; accepted July 12,2017.
Previously published online at DOI: 10.1373/clinchem.2017.274589
 Nonstandard abbreviations: RBC, red blood cell; BCG, bromcresol green.