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The page below is a sample from the LabCE course Overview Of Major Antigens of the Rh Blood Group System. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Reagents For Use In Rh Typing

Given the need to provide Rh compatible blood for transfusion expediently, the goal in testing is to select reagents that will allow for D typing red cell samples as fast and accurately as typing for ABO group. These reagent antisera may come from a variety of sources including saline reactive, high or low-protein based anti-D, chemically-modified antisera, and more recently, those prepared using monoclonal methods. Each variety has distinct advantages and disadvantages as it relates to detection of the D antigen.
The first typing reagents available for D typing were saline reactive antisera containing IgM immunoglobulins. This type of preparation has the advantage of being low-protein, allowing for its use with red cells already coated with antibody, as in patients with warm autoantibodies coating their red cells. Use of a high-protein reagent would result in a false-positive reaction owing to the tendency for the cells to agglutinate on their own without addition of anti-D. The main disadvantages of saline-based reagents include short supply of the resource, the high cost of production, and the requirement for longer incubation time. In addition, these IgM immunoglobulins are not suitable for weak-D typing.
High-protein reagents were developed in the 1940s and contained IgG anti-D. Potentiators and macromolecules (bovine albumin, dextran) were used as additives to optimize reactivity and allow for direct agglutination in slide and tube tests for the D antigen. These reagents provide for a shorter incubation time, the ability to perform weak D and slide testing using the same reagent, and have polyspecificity to recognize multiple epitopes of the RhD protein. Unfortunately, the potentiators can cause false-positive reactions during testing, therefore a control reagent was required to be carried out in parallel with each Rh test. If the control was positive, the Rh test was considered invalid.
Chemical modification of the IgG anti-D molecule by breaking the disulfide bonds was developed in the 1970s. This allows the antibody to link to the antigen sites on the red cells in a low-protein environment. The reagents can be used for slide or tube testing and do not require a manufactured Rh control for samples that test A, B, or O. If a given sample appears to be AB Rh-positive or when Rh testing is performed without ABO testing, a separate saline control or 6-8% albumin control must be used. A negative reaction in the control test ensures that reactions observed with the anti-Rh reagent is not the result of spontaneous agglutination. Using these types of reagents, fewer false positive reactions are obtained.
With growing production of monoclonal antibodies, Rh monoclonal reagents have become more widely available. These reagents are made from single clones of myeloma-hybridized cells that maximize the production of antibodies. Due to the complexity of the Rh protein the monoclonal anti-D reagents are usually a combination of monoclonal anti-D reagents from several different clones. This "cocktail" ensures reactivity with a wide spectrum of Rh-positive red cells. Several manufacturers also include blends of IgG and IgM anti-D in order to enhance the ability to visualize reactions at immediate spin and allow for indirect antiglobulin testing for weak D.