BLOOD BANKING REAGENTS
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What is the first category of reagents?
RBCs with known antigens.
What is the second category of reagents?
Antisera with known antibodies.
What is the third category of reagents?
Antiglobulin reagents, which include anti-IgG (anti-immunoglobulin G) and/or complement.
What is the fourth category of reagents?
Potentiators to enhance antibodies.
What is Quality Control (QC)?
testing to determine the accuracy and precision of equipment, reagents, and procedures.
What are the components of a QC program?
A statement of the criteria for acceptable reagent performance.
Reagent product insert.
Documentation of reagent use.
Corrective actions for lack of performance.
What are polyclonal antibodies?
made from several different clones of B cells that secrete antibodies, recognizing multiple epitopes on the same antigen.
Can you provide an example of a polyclonal antibody?
antihuman globulin (AHG).
What are the advantages of polyclonal antibodies?
Inexpensive to produce.
Quick to produce (Purified antibody ready to use in under four months).
Easy to store.
Highly stable and tolerant of pH or buffer changes.
Higher overall antibody affinity against the antigen due to recognition of multiple epitopes.
Greater sensitivity for detecting proteins that are present in low quantities in a sample since multiple antibodies can bind to multiple epitopes.
What are the disadvantages of polyclonal antibodies?
Variability between different batches produced in different animals at different times.
Higher potential for cross-reactivity due to recognizing multiple epitopes.
What are monoclonal antibodies?
made from single clones of B cells that secrete antibodies with the same specificity and recognize a single epitope.
What technology is used to produce monoclonal antibodies?
hybridoma technology.
Can you provide examples of monoclonal antibodies?
Anti-A (murine mouse)
Anti-c
Anti-IgG, C3b, and AHG antibodies
What are the advantages of monoclonal antibodies?
Can produce large quantities of identical antibody.
Batch-to-batch homogeneity.
High specificity to a single epitope.
Reduced probability of cross-reactivity.
Provide better results in assays requiring quantification of protein levels.
What are the disadvantages of monoclonal antibodies?
Expensive to produce.
Requires significantly more time to develop the hybridized clone.
More demanding storage conditions for the clone.
Requires cell culture and purification capabilities.
More sensitive to pH and buffer conditions
What reagents are used to determine the ABO blood type?
Anti-A and anti-B reagents
What type of antibodies are used in ABO typing?
monoclonal, murine (mouse-derived) antibodies.
What is the purpose of anti-A and anti-B reagents in ABO typing?
They are used to detect weak reactions and determine if an individual possesses A and B antigens.
What medium are ABO typing reagents suspended in?
low-protein medium (6% albumin).
How do anti-A and anti-B reagents function in ABO typing?
Anti-A is directed toward the A antigen, and Anti-B is directed toward the B antigen on a patient’s red blood cells, determining their phenotype.
How are ABO typing reagents formulated for testing?
They are formulated to give a strong reaction (3+ to 4+).
At what phase is ABO testing performed?
It is performed in the immediate-spin (IS) phase at room temperature.
Why is confirmation testing necessary in ABO typing?
It ensures the presence of expected ABO antibodies in the patient’s plasma.
What antibodies does a patient have in relation to their ABO blood type?
A patient has antibodies to the ABO antigen(s) they lack (e.g., a type A person has anti-B).
Why is the D antigen considered the most important in the Rh blood group system?
Because of its increased immunogenicity, making it highly likely to stimulate an immune response.
What conditions can the D antigen be implicated in?
Hemolytic Disease of the Fetus and Newborn (HDFN) and hemolytic transfusion reactions.
What does the AABB’s Standards for Blood Banks and Transfusion Services require regarding D antigen typing?
It requires that all blood samples be typed for the D antigen along with ABO typing.
What are the characteristics of high-protein anti-D reagents?
They contain polyclonal antibodies and approximately 20% bovine albumin, which can promote false-positive agglutination.
What are the characteristics of low-protein anti-D reagents?
They contain monoclonal antibodies (IgM) or a monoclonal and polyclonal blend, with approximately 6% bovine albumin, reducing false positives.
What types of antibodies are used in low-protein anti-D reagents?
Monoclonal IgM or a mono-polyclonal blend (IgM and IgG).
Why have low-protein anti-D reagents replaced high-protein reagents?
They reduce false-positive agglutination and offer more accurate results.
What is the purpose of a low-protein reagent control?
It ensures that typing results are correct and confirms that any agglutination observed is due to specific antigen-antibody reactions.
What should a proper reagent control show?
It should show no agglutination, indicating a valid test result.
What can cause false-positive agglutination in D antigen typing?
Strong cold autoantibodies and protein abnormalities can lead to false-positive agglutination.
When is a separate reagent control required?
If RBCs agglutinate with all ABO antisera, ensuring that the reaction is valid.
When is a separate reagent control NOT required?
If any ABO low-protein reagent produces a negative result, a separate control is unnecessary.
What are commercial reagent RBCs used for?
They contain known antigens to confirm the presence of antibodies in patient serum or plasma.
How are reagent RBCs prepared?
They are selected from human donors and washed to remove antibodies to ensure accuracy in testing.
What procedures use commercial reagent RBCs?
ABO serum testing
Screening tests for unexpected antibodies
Antibody identification to determine specific antibodies in a patient's blood
What are A1 and B reagent cells used for?
They are used in ABO serum testing to detect the presence of anti-A and anti-B antibodies in a patient’s plasma.
Where do A1 and B cells come from?
They can be from a single donor or a pool of donors to ensure availability and consistency.
How are these reagent cells prepared?
They are washed and resuspended to a 2% to 5% concentration for accurate testing.
What is a key characteristic of A1 and B cells regarding Rh antigens?
They are negative for Rh antigens (D, C, and E) to avoid interference in testing.
When should A1 and B reagent cells not be used?
If the red cells darken,
agglutinate in the vial,
or show hemolysis, they should be discarded as they may give false results.
What are screening cells used for?
They are used in antibody screen (detection) tests to identify unexpected antibodies in a patient’s serum or plasma.
How are screening cells typically available?
They are available in sets of 2 or 3 vials, each containing either a single donor’s cells or two donors pooled together.
When are pooled screening cells used, and when are single-donor cells preferred?
Pooled cells can be used for donor testing
Single-donor cells are preferred for recipient testing because they tend to give stronger reactions.
What information comes with each lot of screening cells?
an antigram that provides the antigen profile of the cells.
What antigens must screening cells express?
antigens associated with the most clinically significant antibodies, such as D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, and Jkb.
What are panel cells used for?
Panel cells are used in antibody panels to identify antibodies in a patient’s serum or plasma.
How are panel cells typically available?
in vials containing 10 or more cells, as opposed to screening cells, which typically come in smaller sets.
What information comes with each lot of panel cells?
an antigram, which displays the antigenic profile of each vial of cells.
How do panel cells differ from screening cells?
While screening cells are used to detect unexpected antibodies, panel cells are more specifically used for identifying antibodies once an unexpected antibody has been detected.
What is the role of panel cells in antibody identification?
Panel cells help to identify specific antibodies by exposing the patient's serum to a known set of antigens, aiding in pinpointing the exact antibody present.
What is the principle of the antiglobulin test?
It detects antibody-coated RBCs by using commercial antibodies that specifically target human globulins. The reagents contain anti-IgG and/or anti-C3d, anti-C3b, leading to agglutination if antibodies or complement are present on the RBC surface.
What is the purpose of the Direct Antiglobulin Test (DAT)?
It helps diagnose conditions like
autoimmune hemolytic anemia (AIHA)
hemolytic disease of the newborn (HDFN),
drug-induced hemolysis
transfusion reactions.
How is the Direct Antiglobulin Test (DAT) performed?
AHG reagent is added after washing the RBCs. If agglutination occurs, it indicates that IgG or complement is bound to the RBCs, signaling RBC destruction and possibly anemia.
What is the purpose of the Indirect Antiglobulin Test (IAT)?
It is used to test for antibodies in serum that may react with RBC antigens.
How is the Indirect Antiglobulin Test (IAT) performed?
First, antibodies in the serum are incubated with RBCs at 37°C to allow binding. Then, the RBC suspension is washed and combined with AHG reagent to detect agglutination, which indicates the presence of sensitized RBCs.
What is Polyspecific AHG?
It contains both anti-IgG and anti-C3d antibodies. It can be derived from polyclonal or monoclonal sources. Agglutination with polyspecific AHG indicates that IgG or complement is coating the RBCs. If the result is positive, a differential DAT is performed to identify whether IgG or complement is responsible for sensitization.
What is the purpose of performing a differential DAT?
A differential DAT is performed to determine whether IgG or complement is responsible for coating the RBCs when the result is positive with polyspecific AHG.
What is Monospecific AHG?
It contains either anti-IgG or anti-C3b/C3d, but not both. It is used to differentiate between the presence of IgG and complement on the RBCs.
How does Anti-IgG in Monospecific AHG work?
Anti-IgG in monospecific AHG combines with human gamma chains on the RBCs to detect IgG antibodies that are bound to the RBCs.
What is the function of Anti-C3b/C3d in Monospecific AHG?
They specifically detect complement proteins on RBCs. This is important for detecting intravascular hemolysis caused by activation of the classical complement pathway.
What types of hemolysis can Anti-C3b/C3d in Monospecific AHG detect?
intravascular hemolysis and extravascular hemolysis that are a result of complement activation.
What are IgG-Sensitized Cells used for in the AHG test?
They are required by AABB as a control system when AHG results are negative. These cells, when added to a negative AHG test, should cause agglutination, helping confirm the test's accuracy.
What are IgG-Sensitized Cells made of
IgG-Sensitized Cells are typically type O RBCs prepared with IgG antibodies attached to the red blood cells.
What should happen when IgG-Sensitized Cells are added to a negative AHG test?
When IgG-Sensitized Cells are added to a negative AHG test, they should cause agglutination, demonstrating the AHG reagent's effectiveness.
What are the common causes of false-negative results in AHG testing?
Failure to add the AHG reagent.
Failure of the AHG reagent to react properly.
Failure to wash the RBCs adequately, which can lead to the removal of sensitizing antibodies or complement.
What is the zeta potential, and how does it influence agglutination?
It refers to the force of repulsion between red blood cells in a physiologic saline solution. This force influences the agglutination reaction by affecting how easily red cells can approach each other for antigen-antibody interactions.
How does IgM facilitate agglutination?
IgM antibodies facilitate agglutination between adjacent red cells because of their larger size, which allows them to span the distance between cells more easily. This makes IgM more effective in saline solutions, where the zeta potential might otherwise hinder agglutination.
Why is visible agglutination more difficult for IgG antibodies?
IgG antibodies are smaller than IgM antibodies and less able to span the distance between adjacent red cells created by the zeta potential. As a result, visible agglutination may not occur as readily when IgG antibodies are present.
What are antibody potentiators, and how do they help detect IgG antibodies?
They are commercially available reagents designed to enhance the reactivity of IgG antibodies. These reagents work by reducing the zeta potential of the red cell membrane, creating conditions that promote agglutination and increase antibody reactivity.
How do potentiators work in the agglutination process?
They enhance antibody uptake, which is the first stage of agglutination, and also promote direct agglutination, the second stage of the reaction. By improving both stages, potentiators help to detect the Ag-Ab complex more efficiently in routine testing.
What are potentiators in blood bank laboratories, and what is their function?
Reagents that enhance the detection of IgG antibodies by increasing their reactivity. They are used to improve antibody-antigen interactions during testing.
What are the major types of potentiators used in blood bank laboratories?
low-ionic-strength saline (LISS),
polyethylene glycol (PEG),
bovine serum albumin (BSA),
proteolytic enzymes (such as ficin and papain).
How does low-ionic-strength saline (LISS) function as a potentiator?
It increases the rate of antibody uptake by concentrating the antibody in the test environment, which enhances the reactivity of IgG antibodies.
How does polyethylene glycol (PEG) function as a potentiator?
It increases the rate of antibody uptake and concentrates the antibody in the test environment, which facilitates the agglutination process.
How do proteolytic enzymes like ficin and papain work as potentiators?
They (ficin and papain) remove negative charges from the red blood cell membrane, reducing the zeta potential and promoting better antigen-antibody interaction. These enzymes may also denature some red cell antigens, which can affect the reactivity of certain antibodies.
What is the role of bovine serum albumin (BSA) as a potentiator?
It reduces the repulsion between red cells but does not shorten the incubation time. It helps enhance the antibody reaction without affecting the timing of the test.
What is Bovine Serum Albumin (BSA), and in what concentrations is it available?
It is a reagent used in blood bank laboratories to enhance antibody reactions. It is available in 22% or 30% concentrations.
How does Bovine Serum Albumin affect antibody-sensitized cells?
It allows antibody-sensitized cells to come closer together than they would be able to in a saline solution, which favors agglutination.
How does Bovine Serum Albumin enhance the sensitivity of the Indirect Antiglobulin Test (IAT)?
By facilitating direct agglutination with Rh antibodies and improving overall antibody-antigen reactions, making it easier to detect small amounts of antibody.
What colors are associated with Murine Monoclonal Anti-A and Anti-B reagents?
Anti-A is blue, and Anti-B is yellow.
Why does a transfusion reaction cause a positive DAT?
A transfusion reaction occurs when donor red blood cells are coated with IgG, due to the recipient’s antibodies attacking the transfused cells.
How does hemolytic disease of the fetus and newborn (HDFN) result in a positive DAT?
Maternal IgG antibodies cross the placenta and coat the fetal red blood cells, leading to a positive DAT.
What is the cause of a positive DAT in autoimmune hemolytic anemia (AIHA)?
IgG or C3 binds to the patient’s red blood cells, due to the patient's own autoantibodies, leading to hemolysis.
How does a drug-related mechanism lead to a positive DAT?
It occurs when an IgG-drug complex attaches to red blood cells or when an immune complex forms with the drug, causing red cell destruction and a positive DAT.
What can cause red cells to be agglutinated before the washing step and the addition of antihuman globulin (AHG) reagent?
A potent cold-reactive antibody of patient origin can cause premature agglutination.
How can dirty glassware lead to a false-positive result?
Particles or contaminants in dirty glassware can interfere with testing and cause nonspecific agglutination.
How does improper centrifugation contribute to false-positive results?
Overcentrifugation can pack the red cell button too tightly, leading to nonspecific clumping that cannot be dispersed.
How can inadequate washing of red cells before adding AHG reagent cause false-negative results?
Unbound human serum globulins may neutralize the AHG reagent, preventing agglutination.
Why is it important to add AHG reagent immediately after washing?
If testing is interrupted or delayed, bound IgG or complement molecules may detach from coated red cells, leading to a false-negative reaction.
What can cause weak positive reactions to go undetected?
Technical errors in testing, such as improper observation or interpretation, may result in weak positive reactions being overlooked.
How can reagent issues contribute to false-negative results?
Loss of reagent activity due to improper storage, bacterial contamination, or contamination with human serum can prevent proper agglutination.
What happens if AHG reagent is not added during testing?
It can lead to a false-negative result.
How does improper centrifugation affect antiglobulin testing results?
Undercentrifugation may prevent optimal conditions for promoting agglutination, leading to false-negative results.
Why is red cell concentration important in antiglobulin testing?
If red cell suspensions fall outside the optimal 2%-5% concentration, agglutination reactions may not occur properly, affecting test results.