Solid-Phase Red Cell Adherence Assay For Red Cell Antigen And Antibody Detection - Term Paper Example

First Name Surname Instructor Course Date Introduction Detection and identification of the antibodies are purposefully done as a compatibility test during blood transfusions to avoid health complications that arise if an individual with antibodies is exposed to donor cells with corresponding antigens (5). The basis of the red cell antigen and antibody detection is that antibodies only react with the cells that contain the corresponding antigens (15). The blood group system was identified due to the visible agglutination of erythrocytes with the anti sera (6). Other blood group antibodies like those that are against the rhesus antigens belong to the class of IgG, and they usually do not lead to the direct clumping of the erythrocytes (1). The most implemented methods in this assay include the gel technique that involves the use of microtubules filled with buffer, gel, and reagent. The test is usually reproducible and has a high sensitivity. Coomb’s test is employed in the recognition of the incomplete antibodies (3). The direct antiglobulin test is usually employed in identification of the within the circulating red blood cells for instance, in the cases of autoimmune hemolytic anemia and a mismatched blood transfusion (16). The indirect antiglobulin test is however used in the detection of the blood group antibodies that are present in the plasma. The Indirect antiglobulin test has been employed in the detection of almost all the clinically essential red cell antibodies (4). Erythrocytes Assay Process Figure 1. (22) Solid Phase Red Cell Adherence Assay Method (SPRCA) The recent development in the pre-transfusion compatibility tests led to the adoption of the SPRCA This serological method is more efficient and cost effective as compared to the previous ones. The tubeless method also possesses a great sensitivity and specificity in the blood serology. The method is easily adapted to the automated platforms within the blood serology that begun in the 1960s. Automation in performing serologic tests was adapted to minimize the errors in sample identification, transcription errors, and to improve the objectivity and reproducibility of the immunohematology results. The SPRCA assay technique was developed based on the research study that was conducted in the 1980s for detection and identification of the unpredicted antibodies. The technique has been employed in antigen phenotyping, antibody screening, cross matching, and identification(14). The solid phase testing system uses microtiter plates that have wells coated with the stroma from the reagents of red blood cells of known phenotypes. The standard reagent red blood cell coatings are made either in-house or by the manufacturer (23). Most people have preferred the use of SPRCA technique since its result interpretation is less subjective; it requires small sample sizes and reagent volumes, and due to its stable results that can allow even a review by another person at a later time. SPRCA process involves the immobilization of one of the components of the antigen-antibody reactions into a solid medium to react with the unbound antigen or antibodies. The indicator cells at the end point are the red blood cells that are either added or are components of the antigen-antibody reaction (24). The red blood cells are usually treated with bromelin before they are added to the wells covered with anti A, B, and D sera (23). The technique works based on the principle that the red cell membranes that bind to the surface of its polystyrene micro-titration strip wells can trap the Immunoglobulin G (IgG) antibodies within the patient’s sera (26). During centrifugation, the erythrocytes with the antigen spread out while the negative ones adhere to the bottom of the plates forming a smaller cell button. The formation of the anti-IgG-IgG complexes when the patient’s IgG antibodies are attached to the cell membranes is indicative of a positive test. When SPRCA has used in the antibody cross-matching tests, the patient’s erythrocytes bind to the capture-R select test walls and are incubated (21). If positive, the recipient releases antibodies to the donor’s red cells antigens while a negative result has no detectable immunoglobulin G or antibody to the donor antigen (9). In phenotyping tests, the donor red cells get attached to the capture-r select test wells. The red blood cells that are bound are then incubated with the selected antiserum, and a parallel autologous control is also run. The presence of a corresponding antigen with the negative autolgous control is indicative of a positive result. The negative result in both the antiserum and the autolgous control points out the lack of a corresponding antigen on the donor’s erythrocytes. SPRCA has been associated with some challenges when being used. Among the limitations, that the assay method possesses when used in cross-matching of the immunoglobulin G includes the capability of the capture-R select to identify only the incompatibilities due to the IgG antibodies. The red blood cell cross-match is not meant to detect the incompatibilities resulting from IgM antibodies like the ABO incompatibilities (21). In most scenarios where the identification of the incompatibilities due to the IgM antibodies are crucial then the cross-match immediate spin is applied. Another possible limitation that has been reported with the assay method is the errors that result from the bacterial and chemical contamination of the materials used during the testing process. Mistakes have also been reported to arise from the improper centrifugation and timing of the incubation duration. Centrifugation affects the assaying process as it may result into a false positive or negative result due to the failure in the semi-automated tests or over-centrifugation manuals (7). The deceleration process during centrifugation also interferes with the results of the assays. Long deceleration times have been associated with the false negative reactions while short deceleration periods contribute to false results, especially if the centrifuge braking is not properly observed. It is, therefore, proper that the optimum spin speeds and the spin times are set, and a constant performance evaluation of the SPRCA centrifuge reviewed and maintained (20). Advances in the SPRCA SPRCA assay was discovered in the 1950s when the hemagglutination based assays showed different challenges. Among the challenges that the hemagglutination assays had includes the rouleaux formation that interfered with the assaying process (19). The technique also required special potentiators and a special method for the detection of the antibodies. Hemolysis, challenges in the automation of the hemagglutination test processes and lack of an objective endpoint were also among the problems noted by the hemagglutination assays. The first adopted assaying alternative in 1955 was the mixed agglutination technique by Coombs and Bedford. The assay method was meant to identify blood groups on the platelets. The assay method worked based on matching of antibodies to a similar antigens. Later on, the assaying method was implemented in the detection of the blood group antigens present in the HeLa cells, leucocytes, and the human epidermal cells (18). A report by Vogel and Shelokov in 1957 on the hemadsorption that applied the principles of hemagglutinating of the surface adsorbed viruses was then released. In the test, the two scientists found out that the erythrocytes could clump the culture cells containing influenza virus. The first SPRCA assay was conducted in 1959 by a scientist known as Hongman. The principle of the direct antiglobulin test, however, was first demonstrated in 1908 (2) . The test indicated antibodies in the living organisms and the complement coatings of the erythrocytes. In the test, the erythrocytes from the rabbit was sensitized using goat anti-rabbit serum (17). A strong agglutination was observed from this reaction with the addition of the rabbit anti-goat serum. The test applied the ideologies of the mixed agglutination and hemadsorption. The test that aimed at identifying the blood group antigens on the tissue culture grown cells involved sensitization of the kidney cells with the anti A, D, and B specific antibodies. The process was then followed by the addition of the saline suspension of erythrocytes onto the sensitized monolayers and incubation (13). Hogman then displaced the unbound red blood cells by rocking the cell monolayer. A microscopic examination of the monolayer was meant to reveal the adsorbed red blood cells (25). The result was able to also detect the A and B antigens while it was incapable of indicating the Rh D antigen when viewed microscopically. The Hogman’s assaying technique was later used in the examination of the existence of the antigens in the fetal lung, heart, kidney, skin, and liver after culturing them. Numerous limitations were, however, reported regarding the Hogman's assaying method. Among the problems identified includes the sharing of antigens between the cells that are studied and the indicator erythrocytes. Coomb then developed the mixed antiglobulin test as a way of solving the problem with the Hogman’s technique in 1945. In the mixed antiglobulin method, both the cell types shared the adsorbed antibody. Coomb tried to demonstrate that the antibodies for the blood groups are present in the serum or on the prepared erythrocytes. The Coomb's mixed antiglobulin test was largely employed in the identification of platelet-specific antibodies (10). Later in 1961, the hemadsorption and mixed antiglobulin techniques were modified to give a technique that was termed as mixed-haemadsorption. In this technique, the antibodies that are specific to a particular virus adhered on the single layered viral antigens . These were identified using the antiglobulin-coated sheep erythrocytes (11). The mixed-haemadsorption method was later applied in many tests including in the study of the species-specific autoantibodies and alloantibodies. In 1972, it was implemented in the identification of the platelet-specific antibodies and the detection of antigens on the platelets. Graphical representation of the SPRCA Results Figure 2 (18) The SPRCA methods were meant to demonstrate the adherence of the antibodies onto the immobilized single layered cells. Later in 1977, Catt and Tregear were able to identify that the antibodies can also adsorb onto the polystyrene surfaces, and they were capable of separating the B lymphocytes from the heterogeneous spleen cell population using the immobilized anti-immunoglobulin G (IgG) process (4). Many advances have since taken place, especially in the immobilization and dying of the single layers red blood cells that are utilized in the SPRCA assays. The technological changes have resulted in the designing of the reagent erythrocyte for the antibody screening and identification that are not highly affected when in the room temperatures. The tests that were conducted using SPRCA assays appeared to be more sensitive and faster as compared to the conventional hemagglutination tests (22). Another advantage of this advancement is on the red cell antigens, which were equally found to be more stable and reactive when stored for a long period in the room temperature (12). The red cell antigens were found to be capable of staying for up to 120 days before getting unreactive. Studies Comparing SPRCA with Other Red Cell Antigen and Antibody Detection Techniques Many scientists have been interested in determining the most appropriate assay technique that may be implemented in the detection of the red cell antigen and antibody. The gel-based systems have gained popularity in the laboratory sector over the classic tube tests since their introduction in the late 1980s (3). Most scientists prefer the gel based micro-column tests since they are easier to use and are equally more accurate as compared to the tube tests. Little amounts of the red blood cell samples and serum are also utilized in the process. Most researchers have also confirmed that the gel tests are more sensitive in detecting the patient’s erythrocytes alloantibodies within the serum that can be undiagnosed making it the most appropriate test for the determination of the hemolytic transfusion reactions. A comparative study on the accuracy of the SPRCA found tha it is capable of indicating the presence of human leucocyte antigen (HLA) antibodies in the patient’s sera. There were, however, some false negative results that were indicated by the SPRCA. The SPRCA sensitivity was rated at 84.21 percent while its specificity was at 95.83 percent, making the flow cytometry technique the most sensitive antibody screening method. Another study comparing the precise nature of gel microcolumn assay with the automated SPRCA system for the detection of antibodies showed that the concordance instances with the two techniques were 88.2 percent (15). The study concluded that the performance of SPRCA technique in identifying antibodies and antigens eluted from the erythrocytes is comparable to the gel micro-column technique used in the identification of both peripheral blood and the cord erythrocytes antibodies. Conclusion Identification of the erythrocytes antigen and antibodies is one of an essential compatibility tests done in the clinical setup. The antibodies to the antigens ABO occurs naturally in the human beings and can be identified after a person’s third month of life using different techniques (8). Though techniques like direct antiglobulin tests and the gel micro-column tests can be utilized in identifying these blood components, the red cell adherence assay method is one of the most implemented techniques in the clinical laboratories since it is cheaper and more efficient. The principle that the SPRCA technique utilizes has been modified since the 1950s, when the hemagglutination assays became unreliable and automated platforms in the blood bank serology were considered as the best alternative.Various research studies and improvements have since been made to the original technique that was only based on matching of antigens and antibodies. Despite improvements in SPRCA technique, some studies still discredit its specificity and sensitivity based on some tests, and therefore more improvements should still be made to enhance its specificity and sensitivity in the erythrocyte assays. References 1. Casina TS. In search of the Holy Grail: comparison of antibody screening methods. 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