Solid phase red cell adherence assay for red cell antigen and antibody detection Essay Example
- Category:Formal science & Physical science
- Document type:Assignment
In this section, the review of the existing literature of solid phase red cell adherence assay (SPRCA) antigens of red cell and detecting antibodies. Since SPRCA is a method used in antibody detection, other methods will also be explored for the sake of grasping the concept of detecting antibodies. Other methods that will be discussed in this section are CAT and TUBE, as well as how they relate to SPRCA. We will look the advantages and the shortcomings of each of the methods closely.
Solid Phase Red cell Adherence Assay Development
The technique of immunologic agglutination was realized by Stillmark in 1888 when he saw that the Ricinus communis seed extracts agglutinated erythrocytes . Agglutination of bacteria by serum was observed by Durham . Later in the early 20th century, it was pragmatic that all antibodies could be used in erythrocytes agglutination. Landsteiner could discover the ABO blood grouping then . For over many years past, agglutination has remained the widest method of blood grouping for detecting how the antibodies interact with the antigens of red cells. Currently, blood centers and hospitals frequently use either automated or manual agglutination techniques to perform Rh grouping and undertake ABO, to identify antibodies, screen antibodies, and cross matching.
TUBE methods of standard agglutination are still in use in most medical laboratories and blood centers. Despite its advantages of maintaining simplicity, versatility, and reliability, this approach has significant drawbacks that call for its disapproval . Some of the limitations of this method are that identifying the sample, dispensing of reagent and the samples, and the evaluation of results are all done manually. Additionally, every test performed for serum needs different reagent additives and conditions of reaction to enable agglutination.
Other fields of research laboratory medicine have gone over most limitations of using manual techniques for testing manually by introducing automated systems. For instance, continuous flow auto analyzer introduced in the clinical medicine has modernized the field. Modern instruments such as the auto grouper 16C, auto analyzer, the Groupamatic GM360and the mini Groupamatic from Hoffman-LaRoche Company have effectively mechanized the antibody detection, and grouping [5-8]. Nonetheless, they have proved not to be effective in antibody detection. These methods are costly and complicated to use. Therefore, they are uncommon for use in large blood centers and medical laboratories.
It is because of this drawback that other methods of agglutinations had to be introduced. Wegmann and Smithies in 1966 explained the first agglutination tests carried by ABO testing and Rh grouping . They used V-bottom micro-plates. This system of agglutination gave a notable rise in sensitivity and a significant reduction in the amount of the antiserum required as well as the cells compared to the tests carried through tubes. Crawford also explained a system of micro-plating that is useful for blood grouping and detecting antibodies . His methods, though used U-bottom wells previously treated with plasma and albumin. The use of ficin and centrifugation significantly reduced the incubation times. The improved saving on time, resources and reagents were achieved without neglecting sensitivity. The method of antibody detection and Rh grouping were also described by Parkers and his associates  using flexible polyvinyl plates with V-bottom bores. The conclusion they drew was that microplate system of antibody cluster was more sensitive, save in time and cheaper than the tube systems of agglutination. Between U-bottom and the V-bottom, the U-bottom is more preferred for it requires less agitation in resuspending the cells. Many blood centers and hospitals are presently carrying improvements on these microplate systems .
Following the acceptance received for the manual microplate antibody detection systems, there were attempts to mechanize them. For instance, in 1982, Nieri and Innocenti used microplate spectrophotometer designed to a computer that is personal to determine the tests for agglutination . Later, it was showed by Bowley and his associates  how a standard spectrophotometer connected to a particular machine could sense the results from agglutination and transmit them to the individual by showing the blood group. The tilting forward of the spectrophotometer was done to make sure that the covered cells could move away from the visible way. The time taken for this exercise was approximately one hour. The automated results related to the perception readings in almost 99% of the donor samples summing up to 20,000. The remaining percentage of rejection was because the antibodies of reagent A and B red cells reacted irregularly. These findings were strengthened by Severns and others , who explained almost similar undertaking but with relatively less impressive outcomes. These half automated microplate systems of agglutination stood for meaningful progress towards the blood bank serology automation. Their primary advantage was their ability to remove the errors of manual transcription, and at the same time saving on time with considerably uncomplicated and cheaper equipment.
Although these advancements were welcomed, there were still limitations with agglutination automation. Issues related to non-conforming antibodies, rouleaux, and additives which are exogenous were not completely resolved. Serverns  also noted that there was a problem with the pigmented sera. However, the main limitation was there was never an objective endpoint. Densitometers were unable to differentiate between adverse results from the weak reactions of agglutination. To get around this issue, a large number of cell controls in each microplate had to be performed to get a wider range of values of positive and negative absorbance. Additionally, another disadvantage was that these methods could successfully automate the processes of ABO and Rh grouping.
Adoption of SPRCA
Regarding the above limitations, there was a need for new approaches that would be cheaper automation of all serologic tests that were carried out in blood centers and laboratories in the hospitals. Earlier, in trying to make microplate testing automatic, agglutination as a measure of red cell and antibody reaction was neglected and adopt an automated, solid-phase technology that could be applied in almost all immunology research field. Solid Phase Red Cell Adherence Assay is a tubeless method that had been designed to improve specificity and sensitivity in serology of grouping the blood . Solid phase technology has been accepted due owing to the extensive use of platforms that are fully automated. SPRCA use red cells as indicators, and they are a replacement for routine tests done by hemagglutination.
In SPRCA tests, one of the reactants, the antibody or the antigen is made immobile on a solid phase before the trial begins. During the experiment, excess reactants combine with the immobilized element and therefore be made part of the solid phase. The support period is not participants in the trial, but they are set to restrict the reactants from extending certain boundaries. Making the reactants immobile is a way of making sure that the bound part is separable from those which are free .
Rosenfield and associates  showed that solid phase technology could stand for the routine blood grouping test processes by hemagglutination. The study showed that in a low ionic setting, specific antibodies rapidly tied to monolayers of red blood cells that had been irrevocably combined to a matrix. To assess the quantity of the bound antibody, the immobilized red blood cells which were coated with antibodies were first lysed with water without impurities. Anti-IgG and a pure suspension of Red blood cells which were sensitized by IgG were added. The monolayer was allowed to combine with the stimulated cells through the passages created by the molecules of Anti-IgG. The absence or presence of antibody was indirectly determined by taking the quantity of hemoglobin released via the lysis of red blood cells that are tied residually and bound in IgG.
Figure 1: Snapshot of Immobilized Components and indicator particles.
Studies from the literature have shown that solid phase microplate assays using intact red blood cells as signs can efficaciously substitute the tests for hemagglutination . SPRCA can also be employed in for routine RH and ABO tests and detecting antibodies. For the case of ABO and Rh testing, solid phase technology is likely to identify the weak parts of antigens in the A, B or D classes. Whereas, by hemagglutination processes detect these antigens by support procedures such as incubation, which is extended, including phases of antiglobulin, elution, or adsorption, the solid phase assays directly recognize the weakened antigens. Discrepancies in the grouping are removed by use of solid phase methods. It is also time-saving which is in the mode of hemagglutination while detecting the D antigens.
Again, solid phase processes are helpful in detecting medically vital antibodies to antigens of Red cells. Studies carried out showed that only a small amount of significant antibodies did not react with red cells that had antigens . In some instances, the desired results were attained when the experiments were carried out repeatedly as shown in the figure below
Figure 2: comparison of systems.
In some cases, the desired reactions were produced when the incubation time was increased to fifteen minutes.
The reality that the solid phase assay does not detect the IgM antibodies is an added advantage of the system. The IgM antibodies are often encountered in the transfusions of the blood. Their identification is time-consuming which could be used to detect and identify the specificities. Other nuisance antibodies such as anti-Kn are not detected by the systems, but they react in both the tube and solid phase systems . If patient antibodies of IgG have attached to the red cell membranes, the indicator coated with the IgG form complexes of IgG-anti-IgG. Consequently, the cells that work as indicators tend to the wells as the second layer which is immobilized and make a conflux that is dispersed, that consists a test which is positive. However, where the tests are negative, the indicator cells rest at the bottom of the well, making a minute, different cell button.
Previous studies of the SPRCA tests are bound to its effectiveness in cross-matching the platelets. However, its recent research shows that solid phase tests are also helpful if PAIgG detection in immune thrombocytopenia . When the method is evaluated by easy it is to use, its efficiency and cost friendly, and the results compared to PSIF (Platelet Suspension Immunofluorescence), the results showed that SPRCA had an absolute sensitivity of 65% while PSIF had 53%. The sensitivity level of the solid phase method is better as compared to other methods in use.
Table I: Performance Characteristics of the SPRCA Compared to the PSIF
|Ease of performance||Moderate|
Additionally, there are improvements through increased patient texting through SPRCA. Therefore the sensitivity level is expected to rise . The direct method is made possible for implementation on routine basis because the platelets of the patients for the SPRCA tests can be got from EDTA blood, which is always in platelet count orders and CBC. Also, for batch testing, PRP can be frozen.
Both the ability the PRP and EDTA collection give benefits in testing directly with the solid phase method over other methods. In other methods, it is a requirement that PRP is harvested from the collected blood in ACD, which must be used within a day . This practice prevents batching which is a standard feature in solid phase technology. The direct test is more sensitive than the long test, and this is what SPRCA offers and rarely performed in other methods of antibody detection.
Concerning comfortable performance and interpretation, the studies have exposed that solid phase methods are outstanding. TUBE and CAT process requires experience in using them, especially in reading and interpretation of the results. Understanding for the solid phase systems is apparent and straightforward, and the SPRCA kit avails reasonable tool control to ensure the validity of the results. With increased objectivity in the reading of the results and increased control on the quality, the SPRCA tests are more reproducible and reliable than other tests .
The cost of reagents for SPRCA kit are relatively higher than for the tube method, but this cost is covered by the fact that labor cost is reduced significantly. SPRCA performance is exemplary, with it requiring less than an hour, compared to more than three hours for other methods, like SPRCA. Again, no special equipment should be used with solid methods, but PSIF method requires a microscope .
Because of the advantages outlined above and the previous studies that show the benefits of SPRCA, it is recommendable to use solid phase technologies for laboratory medicine. For its reliability, cost effectiveness, sensitivity, the blood centers and medical labs that need an easy to interpret, easy to read test to perform antibody detection, and IgG associated with the platelet should adopt this method. The related organization should, however, consider the cost of the SPRCA kits which is relatively higher; for this, it is recommended they do a cost analysis to check the viability of adopting the modern technology of tubeless testing.
In brief, this section has covered the development of the antibody and antigen development technologies from their inception in 1888. We have briefly checked the disadvantages of TUBE technologies and the rationale for adopting the Solid Phase Red Cell Assay techniques in antibody detection in the red cell antigen. We have seen that the SPRCA techniques are cost-efficient, objective in their reading, the stability of results in profound and that it only requires a small quantity of samples and reagents for the experimentation.
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