PROOF OF CONCEPT
To evaluate various components of our prototype kit design, we performed a series of proof of concept tests with porcine red blood cells (pRBCs), which possess similar morphology and antigenic structures to human red blood cells. These tests include slide agglutination to test enzymatic cleavage, percent hemolysis tests to ensure cell health during conversion, as well as model tests to evaluate our proposed mechanisms that involve leukodepletion filters and nickel-sepharose beads.
Figure 1 - Prototype Experiments Overview
pRBCs share a number of similar characteristics with human RBCs (Table 1) (Smood et al., 2019), and possess antigens that closely mimic the A and B antigens of human RBCs (Fig 2)(Cooling, 2015). Because of these similarities, porcine blood is the most promising candidate for xeno transfusions--transfusions taking place across species (Smood et al.,2019), the applications of which we further explore in the Integrated Human Practices Page. The similar properties between pRBCs and human RBCs also make it an ideal substitute and model for human RBCs in our experiments, as human blood can contain blood borne viruses and other agents making it a health and safety risk when doing experiments with it.
Table 1 - Hematological similarities between pig and human blood. The RBCs have similar cell diameters and counts, while pig’s blood has a shorter lifespan. These properties make pRBCs an ideal substitute for human RBCs in our experiments. (Smood et al., 2019)
Figure 2 - Blood Group Antigens of a) pigs blood and b) human blood. Porcine RBCs have an A-O antigen system, meaning that they do not possess the B blood type antigen. Instead, porcine RBCs have a xenoantigen “α-gal,” that is present on all pRBCs, regardless of type. The structure of α-gal is nearly identical to the human B antigen, but lacks a fucose group branch. (Cooling, 2015)
Slide Agglutination Tests
The A-O antigen system of pRBCs means that the enzyme activity of NAGA can be evaluated through agglutination assays with pigs blood (Mujahid and Dickert, 2016). In theory, successful cleavage should eliminate agglutination when human anti-A serum is added.
We sourced porcine blood from a local farm in Hsinchu, a county in Taiwan, specializing in breeding pigs for scientific research purposes. We performed initial slide agglutination by adding human anti-A serum to blood type the pRBCs (Mujahid and Dickert, 2016). Previous studies have confirmed that porcine blood agglutinates well with human Anti-A, and thus should show visible clumps if the A antigen is present (Kresie, 2006). Our results demonstrated clear clumps in some samples, and no agglutination in others (Fig 3). We used this difference to blood type our RBCs either as A type or O type.
Figure 3 - Slide agglutination of porcine blood. Clear visible clumps were observed in the left well, indicating that the sample is blood type A. There are no clumps in the middle or right well, indicating that these samples are blood type O.
To test if our enzyme worked on pRBCs, we used slide agglutination to visualize our results (Fig 4). Following treatment with the NAGA enzyme, A-type pRBCs should cease to agglutinate with the addition of anti-A serum, assuming there is successful cleavage of the A antigen. Non-enzyme treated A-type pRBCs should continue to agglutinate as expected. Enzyme-treated A-type pRBCs, on the other hand, should not agglutinate and should resemble the results of O-type pRBCs, if we achieved successful blood type conversion.
Figure 4 - Enzyme activity and agglutination test theory. Following addition of NAGA enzyme to A-type pRBCs, cleavage of antigens and hence blood type conversion can be detected with the addition of Anti-A, which will cause agglutination if the A antigens are still present on the RBC surface. Successful blood type conversion should be reflected by the observation of visible differences in agglutination between enzyme-treated and PBS-treated pRBCs. The agglutination observed in enzyme-treated A-type pRBCs should be minimal, and correspond to the agglutination observed in O-type pRBCs, further suggesting successful blood type conversion.
Following enzyme treatment for 2 hours, we added human anti-A serum to the blood samples. We then visualized agglutination using 3 different qualitative methods (Fig 5). First, we spread the blood and antibody mixture on standard blood typing slides, to observe for macroscopic clumps. Our results show that enzyme-treated blood displayed less clumps compared to PBS-treated blood and results that resemble O-type pRBCs, indicating less agglutination and successful blood type conversion. We then observed the mixtures on a microscope slide with a glass coverslip. Once again, we saw more aggregation of RBCs in the PBS control treated samples than O-type pRBCs and enzyme-treated A-type pRBCs. Finally, we observed agglutination of the mixtures under a Nikon 20X magnification fluorescent microscope (Nikon H550S, Japan). We observed both more clumps and clumps larger in size in PBS-control treated samples (Fig 6). We observed very few clumps in enzyme-treated A-type pRBCs and O-type pRBCs, although O-type pRBCs were more concentrated. The corroborative results from these qualitative agglutination tests therefore provide convincing evidence of successful enzymatic cleavage by NAGA, demonstrating a proof of concept for our project.
Figure 5 - Post-enzyme treatment agglutination results visualized in blood typing wells, glass coverslips, and under light microscope at 20x. In all three methods of visualization, less agglutination was observed following enzyme treatment compared to treatment with PBS control. These results suggest successful A to O enzymatic conversion of pRBCs.
Figure 6 - RBCs clumps were observed at a much lower frequency when treated with enzymes, compared to samples treated with PBS control, suggesting successful enzymatic conversion.
Percent Hemolysis Tests
Monitoring cell hemolysis throughout the blood type conversion process is important to ensure that significant cell damage does not result from our methodology, and that our methodology is safe and viable. When we interviewed Dr. Hsiao, a hematologist, he emphasized that the life expectancy of our converted red blood cells should be sustainable for the patient, which guided us to design a Percent Hemolysis experiment to determine whether enzymatic conversion of RBCs increases cell lysis.
Luten et al observed an average RBC loss of 5-10% during a standard transfusion process. With increased storage time, this value can increase to 25%. We thus set our acceptable threshold for hemolysis at 10% (Bosman, 2013).
The cytoplasm of RBCs contains high amounts of hemoglobin. When RBCs rupture, hemoglobin is released into the plasma, giving the plasma its reddish tint. The degree of hemolysis can therefore be quantified by isolating the plasma of RBCs and detecting the amount of released hemoglobin using a spectrophotometer at 540 nm (Han, 2010).
We used xTractor Lysis Buffer (XTractorTM Buffer & XTractor Buffer Kit User Manual, n.d.) to lyse pRBCs to generate a fully lysed control sample. We used freshly washed pRBCs in PBS as an undamaged control sample. We derived a score for percent hemolysis by dividing the change in absorbance of the sample by the change in absorbance of a completely lysed cell, as represented by the equation below (Van Buren, 2020).
Figure 7 - Equation for calculating percent hemolysis
We monitored the percent hemolysis of pRBCs over time following the addition of NAGA enzyme or PBS medium (Fig 8). Our results show that the percent hemolysis remains within the acceptable threshold for at least 60 minutes of reaction time. Moreover, there is no major difference in hemolysis numbers of enzyme treated and PBS (medium) treated pRBCs, suggesting that the addition of the NAGA enzyme produces no additional hemolytic effects. Thus, these tests affirm the safety of our methodology.
Figure 8 - Percent hemolysis of a) enzyme-treated and b) PBS-treated pRBCs over time. In both treatments, the percent hemolysis remains within an acceptable threshold for at least 60 minutes, suggesting that our methodology is safe.
Model Hardware Tests
We wanted to ensure that RBCs are able to pass through our enzyme immobilized bead system and do not experience significant cell death during this process. Some studies have raised concerns that Nickel may negatively affect RBC function (De Luca, et al., 2007), (Tkeshelashvili, et al., 1989). As such, we tested the ability of pRBCs to flow through Nickel Sepharose beads. We found that with the application of air pressure, pRBCs were able to flow through the column at a constant rate (Fig 9). Furthermore, when we examined hemolysis after the passage of pRBCs through the beads, we found that it only increased by 5.7%, well within the percent hemolysis threshold defined by literature. This result suggests that nickel sepharose beads do not have significant adverse effects on RBC function.
Figure 9 - pRBCs were able to pass through a nickel sepharose bead column with application of pressure
In our prototype design, we also proposed using leukodepletion filters, with a 10–50 μm pore size, to separate agglutinated vs. non agglutinated RBCs, as a method to purify cleaved RBCs from uncleaved RBCs (Mizuno, 2013). To test our design, we used stainless steel mesh wire, with a pore size of 35 μm, which corresponds approximately to the pore size of leukodepletion filters. We attempted to pass non-agglutinated and agglutinated synthetic blood (Carolina, USA), which display similar agglutination mechanisms to real blood, through the mesh wire via pressurized suction (Fig 10). Our results show that the pore size of 35 μm was able to capture agglutinated RBCs, while letting non agglutinated RBCs pass through, confirming the viability of our prototype design.
Figure 10 - Non-agglutinated and agglutinated synthetic blood applied to stainless steel 400 mesh wire squares. Non agglutinated RBCs passed straight through the pores, while agglutinated RBCs were trapped. These results confirm that we are able to isolate non-agglutinated RBCs through pores of similar sizes, like those of leukodepletion filters.
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