Overview: Overview: Antigen-antibody reactions are commonly used in immunological assays. In a traditional agglutination assay, the end product is a visible antibody-antigen complex that settles as visible particulate matter in the solution. In other cases, the reaction is invisible and wants the use of a specific enzyme or isotopic probes that can amplify the signal created by the antigen-antibody reaction, so that they can be detected with the naked eye or an ELISA/RIA reader1. Hemagglutination is a visible antigen -antibody assay in which soluble antibodies react with antigen on the surface of (red blood cell) RBC to form a visible agglutination (clumping together of RBCs). The antibody is referred to as agglutinin and the antibody on the RBC surface is referred to as agglutinogen. The ABO blood group antigens are examples of RBC agglutinogen1. The ABO blood grouping is an example of an agglutination reaction that was conventionally performed using a test tube or a slide 1.
Agglutination reaction is quite rapid and provides an immediate result. However, this simple reaction is influenced by a number of exogenous factors like pressure, temperature, agitation, pH, type of antibody, concentration of antigen and antibody 2. For this reason, hemagglutinin reaction is done with a serially diluted antibody solution, so as to determine the titer that gives rise to the agglutination reaction. The antigen -antibody complex bond, is highly reversible 3. The equilibrium of the antigen-antibody complex is more unstable in liquid and is relatively stronger in solid phase assays. Thus, the solid phase red cell assay has replaced traditional hemagglutinin assay in blood banks and other application 3. Solid-phase immunoassays are amenable to automation and the antigen-antibody reaction can be quantified used spectrophotometer 3. For these reasons, solid phase assay is much suited than test tube based assay in labs that have a high turnover of samples 3. This literature review examines a vast body of scientific evidence to synthesize knowledge on the advances in SRPCA (Solid phase red cell adherence assay for red cell antigen) and the challenges for future.
Review of literature
Hemolytic transfusion reaction and pathobiology
Hemolytic transfusion reaction (HTR) following transfusion of blood or blood products, occurs from presence of antigen or antibodies that are incompatible with the recipient 4. This reaction accounts for 20-60% of the death occurring from transfusion of blood and blood products 4. Errors that happen during blood collection, labelling, storing, testing procedures, account for most cases of hemolytic transfusion reaction. Both intravascular and extravascular hemolysis can occur in HTR 4.
The antigen-antibody complex formed by incompatible blood transfusion, activates Hagman factor (factor 12) 4. Hagman factor activates the kinin system forming active bradykinin. Bradykinin causes vasodilation and increases capillary permeability 4. Vasodilation leads to lowering of blood pressure. The antigen -antibody complex also activates the complement pathway 4. Activated complements not only mediates hemolysis, but also causes the release of histamine and serotonin from the mast cell. These biomolecules contribute to bronchospasm symptom. The hemoglobin released from the lysed RBCs, are initially bound to albumin 4. However, following saturation of albumin, free haptoglobin circulates in the blood and is excreted by the kidney. The free haptoglobin causes damage to the kidney 4. The systemic hypotension and vascular constriction, decreases blood flow to the kidney, aggravating the damage done to the kidney 4. The antigen-antibody complex is found deposited in kidney glomeruli. The chemokines released during the immune reaction cause fever, child, gastric disturbances, nausea, diarrhea, restlessness and headache. Disseminated intravascular hemolysis, often accompanies hemolytic transfusion reaction. The products of hemolysis activate the intrinsic pathway of coagulation.
Based on the rate at which hemolysis occurs, transfusion reaction is classified as acute and delayed 5. In acute reaction there is immediate destruction of donor red blood cells. It often occurs from improper typing, errors in cross checking the blood sample, and other clerical mistakes 5. Acute reaction can be either febrile or nonfebrile. Febrile acute reaction occurs from pyrogenic molecules released from the white blood cell of the recipient and the donor 5. Bacterial sepsis can also result in acute febrile reaction. In delayed hemolytic transfusion reaction, the hemolysis occurs after 3-10 days following blood transfusion 5. Delayed response is caused by amnestic or primary antibodies that respond specifically to unique antigens on RBCs. It is common in patients who receive multiple transfusion 5. Certain antibodies that developed in the recipient from previous transfusion, can react with antigens in subsequent transfusion. Rh incompatibility is a classic example of delayed hemolytic transfusion reaction 5. The antigens that elicit delayed antibody response are often weakly immunogenic and are difficult to detect in routine blood typing test 5. All most all cases of hemolytic blood transfusion are preventable. While most hemolytic transfusion reaction occurs from major ABO antibody related incompatibilities, a minor but significant amount of transfusion reaction occur from incompatibility related to other red cell antigens 5. Errors that occurs from misidentification of antibodies are also responsible for transfusion reaction.
Antibodies associated with acute and delayed hemolytic blood transfusion
Acute hemolytic transfusion reaction can occur from in A, B and O antibody incompatibility 6. Anti-Kpa alloantigen that goes undetected also gives rise to acute extravascular HTR 6. Anti-A response is seen in patients with type B blood group and Anti-B response is seen in patient with type A blood group 6. Both Anti -A and Anti-B response is seen in patient with type O blood group 6. RhD antibody incompatibility is often a cause for delayed type HTR. There are 40 different types of RhD antigens detected in humans 6. While majority in the population are positive for RhD antigen, a few lack RhD antigen and thus produce Anti-RhD response when transfused with RhD positive red blood cell. ABO and RhD antigens are red cell specific antigens. Anti-RhD develops in an individual when exposed for the first time to a new RhD antigen, and the amnestic antibodies from the first transfusion can cause delayed type hemolytic reaction in subsequent transfusions 6. IgG and IgA class of immunoglobulin are the major contributors of HTR 6.
Techniques for testing of clinically significant antibodies in the blood
Compatibility testing is done to ensure that that there is no HTR following the blood transfusion. The common compatibility tests include ABO group/RhD type testing, antibody screening and cross matching 8. Over the years, there has been improvement in techniques used for blood typing, screening of antibodies, and for compatibility testing. These technologies not only improved the accuracy of the assay outcome, but also enabled automatization and reduced the reagent quantity 8. Indirect anti-globulin test (IAT) is routinely used to test for antibodies in the recipient sera that could be incompatible with the antigens of the donor RBC 8. This technique is useful in testing for any unexpected allo or autoantibody in the recipient sera that could cross react with the donor RBC. IAT can be used to detect antibodies in amniotic fluid and in other tissue fluids 9. It can be used for detecting antigen in the RBC using a known antibody in the sera, or the vice versa. The sensitivity of this assay is affected by factors like serum: cell ratio, incubation temperature, incubation time, buffer composition, washing step and centrifugation speed 9. Standardization and technology enhancement has enabled better control over these factors and helped in reducing error that may occur from manual operation. IAT is not sensitive enough to detect low titer of antibody 9. Further, in traditional method, the patient serum is tested against two or more separate sample cells obtained from the recipient, so as to obtain reliant test results 1. New column based IAT techniques with improved buffer compositions are designed to improve the test results and avoid excessive false positive reactions that are common with traditional IAT 1.
Antibody screening is done to identify non ABO red cell antibodies that can have a negative effect on transfused red blood cells 9. There has been improvement in antibody screening modalities like: gel based and solid based technology that have improve sensitivity. The main goal of these improvements are to maximize the detection of clinically relevant antibodies and decrease testing time1. IAT is an antibody screening technique that was traditionally used, but it has low sensitivity in detecting antibodies with very low titer 10. Use of low strength ionic buffers as a suspending medium for RBCs have helped to maximize antibody detection 11, when compared to the medium that contains albumin 12. It was also found effective in reducing the time of incubation. The latest modification to IAT buffer is the use of low ionic salt buffers for suspending the RBCs 11. Use of precision samplers for dispensing sample and other buffers in the reaction, has not only improved precision, but also improved standardization and reproducibility1.
SRPCA is yet another technology advancement made to antibody screening assays, and it employs immobilized monolayer RBCs that are stable at room temperature 13. This technology enables fast turnover for routine antibody screening in blood banks. The test panel has different RBC antigens immobilized to the individual wells of the microplate. The solid phase assay, enables simultaneous evaluation of more than 100s of sample in a single microtiter plate. The solid phase red cell assay panel, has not only improved the sensitivity, but also reduced the time taken to do hemagglutination test like IAT 13. The dried red cell antigens are more stable on the solid phase of the ELISA plate, when compared to IAT test that employs red cell suspended in liquid. Rosentifeld of Paris, identified the potential of solid phase assay for serological applications as early as 1978 14. Later in 1989, Immucor, U.S.A, developed the first solid phase assays that employed red blood cells 14. Following this period, a number of improvements were made and today solid phase assays have become popular in serological testing due to their specific, sensitive and effective nature. Further solid phase assay is easily amenable to automation.
Many blood banks and hospitals use SRPCA for screening donors blood for antibodies. The positive results are further confirmed using polyethylene glycol based tube technique. Polyethylene glycol based assays were found to reduce the incidence of false negative results that common in SRPCA 15. As blood samples are valuable, losing samples to false positive assays could be very costly. As PEG assays lack specificity, when compared to SRPCA, they are not suitable to screen the thousands of samples that arrive for screening in a blood bank. Instead, the few positive reactions that show a low titer reaction are further confirmed with PEG based tube assays 15.
An ideal antibody screening test must not only be sensitive in identifying maximum number of antibodies, but also be cost effective. SRPCA is used in initial screening of donor’s plasma. Though solid phase assays are amenable to automation, automated assays were found to have less sensitivity, when compared to manual performed solid phase assays. PEG based assay was intermediate between manual and automated SRPCA 16. Polyethylene glycol helps to detect weak RBC antigen antibody reaction. 20 % of PEG (M.Wt: 4000) was found optimal for detecting weak reactive antibodies 16. Though PEG improves sensitivity, it reduces specificity 17. Thus Solid phase assay that employs low ionic salt solution based reagents are used for routine application. PEG based IAT can detect clinically superior antibodies that goes undetected with a conventional low ionic buffer based SRPCA 17.
Cid et al., compared the agglutination gel micro-column developed by three different manufacturers: DG-Gel, Dia Med-ID and Ortho BioVue. DG-gel column and Ortho BioVue have nearly 100% sensitivity and specificity in detecting the antibody reactivity to RBC antigen 18. This was an ideal feature expected from such column 18. The control group in this study included 3024 samples with unknown antibodies and 100 samples with antibodies of known specificity18. The same column in the hands of another researcher produced slightly reduced sensitivity. Bio-Vue and Dia Med was tested on 3000 patient samples 19. The rate of detecting clinically relevant antibodies was 91.6% by Dia Med-ID and 92.2% by BioVue 19. Though these columns are much efficient than conventional micro Colum, a sample margin of error can lead to loss of value blood sample, which is already scares.
Advantage and disadvantages of different RBC antigen assays
In a traditional test tube based red cell agglutination assay, the RBCs of the donor are mixed with the plasma of the recipient, along with the enhancement solution. The mixture is incubated so as to allow antibody and complement RBC antigen binding. The RBC bound to the antibody are precipitated following the centrifugation step. The agglutination and hemolysis of the reaction mixture indicates positive reaction. In negative test, there is no agglutination. Tube based assays were later replaced by gel based assay. Gel column made of dextra acrylamide are used to for the purpose. The gel column was impregnated with antiglobulin that traps RBCs bound to antibody. The mixture of patient serum and RBC reagent in enhancement buffer was placed on top of the column. The gel based technique, avoided centrifugation based separation of the RBC bound antibody, as in test tube based assays. The negative RBCs escape through the bottom of the gel, while the RBC that are bond to the patient’s antibodies in the sera are retained Gel based assays enable grading of the agglutination reaction on an arbitrary visual scale. It required lesser reagent, and the bias associated with subjective reading of agglutination reaction was overcome with gel based assays. 1
Solid phase red cell adherence assay uses microtiter plates coated with known RBC antigens. The microtiter plates with coated antigen is usually supplied by the manufacturer. It is also possible to coat the pate in-house. In the lab, the patient plasma is mixed with low ionic suspension solution and then is added to the well coated with RBC antigen 13. Once the antibody in the plasma binds to the antigen coated to the palate, the unbound antibodies are removed by washing. The presence of bound antibody is detected by a second antibody that is bound to a probe. Solid phase assays have better sensitivity and can be easily automated 13. The results are easy to interpret and can be quantified by spectrometer. Solid phase assays require less time and lesser reagents20. Solid phase assay can be done in a fully automated platform, without human intervention. With automation, it is easy to manage the quality of testing 20. SRPCA automation, reduces the incidence of human error and transcription errors20. The current challenge in automation is the high error rates that makes it necessary to employ more than one test to arrive at a conclusive decision 20.
Conclusion: In spite of the advancement in antibody screening technologies, there is no single technique that can detect all of the incompatible antibodies in the donor’s sera. Thus, a series of test are required to ensure that the recipient receives a compatible blood transfusion. Though SRPCA based assay helped improve the quality of antibody screening by improving sensitivity of the test, by enabling automation and reducing testing time, the high incidence of error (false negative and false positive) in the procedure is still a matter of concern. It not only causes loss of valuable blood sample; it also gives rise to incompatibility issues in blood transfusion.
Therefore, the aim of the study is to identify factors that reduce the sensitivity of automated SPSS in routine antibody screening of patient sample.
The hypothesis of the study will be: Proper instrument selection, and improvement in testing competencies can help to reduce error in automated solid phase red cell assay. Through proper planning and consideration of key variables, it is possible to reduce error in automated SRPCA.
Figure 1: The Solid phase red cell adherence assay for red cell antigen is similar to a protein microarray in which the red cell antigens are bound to the microtiter wells. The density of the antigen in each well are the same. The intensity of the signal generated at the end of the assay will depend on the titer of antibody in the patient serum. Samples that have low titer of antibodies are further confirmed using PEG based solid phase assay.
Picture taken from www.bioscience.org
References
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