Detecting Antibodies The Antibody Screen CLS 422 Clinical Immunohematology I

Objectives Explain the purpose of performing an antibody screen. Discuss the antigen characteristics important in the composition of screening cells. Describe the phases of antibody detection. Describe the types of antibodies that can be encountered in each of the phases of antibody detection.

Objectives State the difference between an alloantibody and an autoantibody. List factors affecting the antigen-antibody reaction in the indirect antiglobulin test. Discuss the action of potentiators. Compare and contrast methods for performing an antibody screen. Assess sources of error affecting the indirect antiglobulin test. Interpret the results of an antibody screen.

The Antibody Screen Used to detect “irregular” antibodies. Maximize detection of clinically significant antibodies Minimize detection of insignificant antibodies Patient’s serum or plasma is tested against reagent red blood cells (RBCs) with known antigens (screen cells). Irregular or unexpected antibodies are antibodies other than the expected antibodies of the ABO system. Only 0.3 – 2% of the population will have an irregular antibody. This is an example of an indirect antiglobulin test. It allows detection of “incomplete” antibodies.

Allo vs. Auto Alloantibody – antibody directed against antigens that the individual does not possess Immune Naturally-occurring Autoantibody – antibody directed against one’s own antigens Auto control – patient’s RBCs tested against patient’s plasma Immune antibodies are formed in direct response to a foreign antigen following exposure (transfusion, transplantation, pregnancy) Naturally-occurring antibodies are formed following exposure to something in the environment that is similar in structure to the target RBC antigen. Autoantibodies typically react with all RBCs tested in addition to the patient’s own RBCs (positive autocontrol)

What makes an antibody clinically significant? The ability to cause decreased RBC survival. If the antibody activates complement, there may be intravascular RBC lysis. There may be extravascular destruction of antibody-coated RBCs by the macrophages of the RES. Hemolytic Transfusion Reaction (HTR) Hemolytic Disease of the Fetus & Newborn (HDFN) HTR- antigen positive RBCs are transfused to a recipient with an antibody directed against that antigen. HDFN- antigen positive fetal cells are coated with maternal antibody, and cleared by the fetal RES.

Who needs to be tested? OB patients – looking for antibodies that may cause HDFN in the fetus. Patients who need an RBC transfusion – looking for antibodies in the recipient that could destroy the donor RBCs (HTR). Blood, organ and tissue donors – avoid passive antibody transfer; evaluate as source of anti-serum and rare RBCs. Antibody screen is a better test for antibody detection (and ultimately patient/donor compatibility) than the crossmatch alone because screen cells: Express most clinically significant antigens (donor RBCs may not) Have homozygous antigen expression to assist in detecting antibodies showing dosage (donor RBCs may not) Are in a preservative to protect antigen integrity (donor RBC antigen expression will weaken as cells age) Crossmatch = patient serum/plasma vs. donor RBC cell suspension.

An Application of the Indirect Antiglobulin Test Patient’s plasma (unknown antibody) is tested against reagent RBCs (known antigen). Y Y Y Incubated at 37oC to allow antibody to sensitize RBCs. Antiglobulin phase to allow sensitized RBCs to agglutinate. AABB Standards require that antibody detection tests include a 37C incubation phase, and an antiglobulin (AHG) phase. The 37C incubation allows for antibodies to sensitize the antigen positive cells. The AHG phase allows the cross-linking of antigen-antibody complexes, resulting in visualization of the reaction. Standards also requires that the AHG reagent contain anti-IgG. (It is acceptable to use monospecific anti-IgG, or polyspecific AHG that has both anti-IgG and C3.) May add an enhancement reagent before incubation. If the test is negative at AHG (no antibodies sensitizing the RBC), Coombs Control Cells must be added to validate the test results. Optional - May perform an immediate spin phase before incubation to detect cold IgM antibodies. Since IgM antibodies are usually insignificant, this step is frequently omitted.

Screen Cell Composition Sets of 2 to 4 different cells Group O Rh Positive and Rh Negative cells Other major blood group antigens are represented Each vial of RBCs is from a different donor and possess different antigens from the other cells in the set. When performing the antibody screen by tube, cells are suspended in a diluent/preservative (2-5% suspension). It is preferable to have antigens from the major blood groups expressed homozygously on at least 1 screen cell in the set. When performing compatibility testing, each screen cell is tested individually. When screening donor blood, cells may be pooled into 1 vial.

Antigen Profile (Antigram) D C c E e Cw K k Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xg I + II III If the patient’s serum/plasma contains an antibody directed against one of the antigens on the screen cell, the RBCs will agglutinate (or hemolyze)= positive reaction. We can not be certain which antigen is the target of the antibody. Each screen cell set comes with an antigram that lists the antigens present on the RBCs in that vial. A + indicates the antigen is present. A 0 indicates the antigen is absent. Ideally, there will be cells of the Rh, Kell, Duffy, Kidd, and MNSs systems that have homozygous antigen expression. The antigram is lot specific. In the Immucor three cell screen set, cell I is always R1R1, cell II is R2R2, and cell III is rr.

Phases of Testing Immediate spin (I.S.) (optional phase) 37oC AHG Lewis, M, N, P1, cold autoantibodies (I, H, IH) May see D, E, K, strong cold antibodies Rh, Kell, Kidd, Duffy, Ss Immediate spin (or room temperature) phase is not required. Many institutions do not include this phase in order to avoid detecting the clinically insignificant cold antibodies. An enhancement reagent may be added before incubation at 37C to promote sensitization and agglutination. Antibodies that activate complement may show hemolysis at 37C. While incubation is required, some methods do not allow for evaluation of results at this phase. May see cold antibodies at AHG phase, if they activated complement, and if you are using an AHG reagent containing anti-complement.

Specimen Plasma Serum EDTA is the specimen of choice for most institutions. Many automated instruments require an anticoagulated specimen (especially if performing the ABO/Rh along with the screen, i.e. Type and Screen)

Antibody Screen Tube Method ID I 37C inc ID II ID III √ √ √ One drop of a 2 -5% suspension of reagent red blood cells (screen cells) is added to two drops of patient’s serum or plasma. There may be centrifugation and reading of results at this phase, but this is not required. The tube is incubated at 37C (an enhancement medium may be added immediately before incubation). The length of incubation is dependant on the medium. Following incubation, there may be centrifugation and reading of results. This is not required. The cells are washed with saline a minimum of 3 times, to remove any unbound antibody. Following the final wash, two drops of AHG reagent (must contain anti-IgG) are added to the dry cell button. The tube is centrifuged and results are read. The tube may be read microscopically, depending on the test medium. Coombs Control Cells are added to each negative test. The tubes are centrifuged and results read.

Antibody Screen Gel Method 1. Patient’s plasma is added to the reaction chamber along with reagent RBCs. The screen cells are Group O and have various antigens present, as described for the tube method. The reagent cell concentration is 0.8%, and the cells are suspended in LISS. 2. Card is incubated at 37C for 15 to 60 minutes. No immediate spin phase. Card is centrifuged for 10 minutes. Red blood cells are forced through the anti-IgG layer and into the gel. No wash step necessary following incubation as unbound antibodies are too light to be forced into the gel, and will remain in the reaction chamber. If an antigen/antibody reaction has taken place, the sensitized cells will be “bridged” by the anti-IgG as the cells pass through the AHG reagent layer. The agglutinates formed will be too large to pass through the pores between gel particles, and will be trapped within the gel. If no antigen/antibody reactions occurred, the individual cells will pass through the pores in the gel, and will pellet out at the bottom of the microtubule.

Antibody Screen Gel Method Reactions are graded. Remain stable. Lends itself well to automation.

Ortho Provue Courtesy Ortho-Clinical Diagnostics Raritan, NJ The Ortho Provue uses gel methodology to perform ABO/Rh, antibody detection and identification, crossmatching, and some phenotyping. Courtesy Ortho-Clinical Diagnostics Raritan, NJ

Antibody Screen Solid Phase Adherence Method RBC antigens are bound to the sides of a microtiter well. Different wells possess different antigens, and thus are different “screen cells”. Antigens are not on intact red blood cells.

Antibody Screen Solid Phase Adherence Method The patient’s serum is added to each well along with a color enhanced LISS. The microtiter plate, containing multiple wells, is incubated at 37C. Following incubation, the plate is washed with saline. Indicator cells, which are RBCs coated with anti-IgG, are added to each well, and the plate is centrifuged. If antigen/antibody reactions have occurred, the anti-IgG from the indicator cells will crosslink, and there will be a diffuse pattern of red cells in the microtiter well. If there was no reaction, the indicator cells will pellet to the bottom of the well during centrifugation.

Immucor Automation ABO and Rh are performed using direct agglutination in a microtiter well. Screens, panels and DATs are performed using solid phase adherence. Among the tests that can be performed by the Galileo are: ABO Rh Antibody screen Antibody panel DAT Newest model is the Echo…a table top version with capabilities similar to the Galileo.

Reporting Results IS 37 AHG CC I 3+ II III 3+ II III Reporting is “all or nothing”, rather than reporting the results of each cell at each phase. If all cells are nonreactive at all phases, and the Coombs Control Cells are positive, the screen is reported as negative. Negative screen - The most common clinically significant antibodies have been eliminated. Your workup is complete!  If the patient requires RBC units, an immediate spin or computer crossmatch may be used, providing there is no previous history of antibodies.

Reporting Results IS 37 AHG CC I 2+ II III 2+ II III If one or more cells react at any phase of testing, the screen is reported as positive. An antibody panel should be performed to determine the specificity of the antibody present. Get patient history of transfusion, pregnancy, medications, diagnosis. It is possible that more than one antibody is in the specimen!

Are We Done Now? IS 37 AHG CC I 2+ II III 1+ III 1+ NO – all cells should be tested through all phases, even if a positive reaction has already been observed. The persistence and strength of the reaction through various phases can assist in identifying the antibody present.

How Would you Report This Screen? 37 AHG CC I 1+ 3+ II 2+ III Positive

How Would you Report This Screen? 37 AHG CC I 2+ II III Negative Remember that the IS phase is not mandatory.

How Would you Report This Screen? 37 AHG CC I 2+ II 1+ III Positive This is a cold antibody.

How Would you Report This Screen? 37 AHG CC I II 2+ III Inconclusive – the Coombs Control Cells for Screen Cell I did not react, which invalidates the test with that cell. Testing on screen cell I must be repeated from the beginning.

How Would you Report This Screen? (Performed Using Gel) 37 AHG CC I II III Negative Remember that not every method is read at all 3 phases. Also, only the tube method requires the use of Coombs Control Cells.

How Would you Report This Screen How Would you Report This Screen? (Performed Using Solid Phase Adherence) IS 37 AHG CC I II 2+ III Positive

Factors Affecting the Antibody Screen These factors will affect ANY application of the indirect antiglobulin test!

Antigen/Antibody Ratio Postzone Prozone Equivalence Ratio is usually 2 drops of serum or plasma to 1 drop of RBC suspension.

Reaction Temperature IgG antibodies have a very narrow temperature range - 37oC + 1oC. IS phase = room temperature 20-25oC; will detect IgM antibodies

Reaction Time IgG antibodies are incomplete Require incubation at 37oC to react Length of incubation dependant on test medium IgG requires incubation at 37oC. Length of incubation can be anywhere from 10 to 15 minutes in a test environment using LISS or PEG enhancement to 1 hour in a saline test environment. If the incubation time is cut short, there will not be enough time to allow sensitization and/or lattice formation. If incubation is prolonged, the agglutinates may revert to the free antigen / free antibody state.

Test Medium May add potentiators to overcome the effects of shielding and the zeta potential Albumin LISS PEG Enzymes Albumin lowers the zeta potential, which allows RBCs to approach each other more closely, which in turn allows for more cross-linking of RBC antigens. Decreases incubation time needed for ag/ab reactions to occur (15-30 minutes) LISS lowers zeta potential and removes shielding, which promotes sensitization as well as agglutination. Incubation time 10-15 minutes. PEG does everything LISS does, plus it removes water from the test system, which concentrates the antibodies, making it more likely that antigen and antibody will encounter each other. Incubation time ≈ 15 minutes. Enzymes alter the structure of the RBC membrane by cleaving sialic acid residues. Expression of some antigens is enhanced, while other antigens are destroyed by this method.

Quantity and location of Antigen Dosage – When an antibody reacts with greater strength to a cell that has homozygous antigen expression than it does to a cell that has heterozygous antigen expression. Remember that cells with homozygous antigen expression have twice as much antigen available to participate in the reaction than do cells with heterozygous antigen expression. Antigens that extend from the surface of the RBC membrane are more readily available to react than those that pass in and out of the membrane.

pH Optimal 6.5 to 7.5

Sources of Error in the Antibody Screen False Negative Results These errors may be found in other applications of the indirect antiglobulin test.

Y Improper Wash Y Y Y Y Y Y Y Y Y Y Y Affects tube and solid phase tests- If unbound antibodies are not removed, they will react with the anti-IgG in the AHG reagent. There will not be enough anti-IgG to cross-link the sensitized RBCs. Y Y

Failure to… Add plasma (the antibody source) Add reagents Make it a habit to add plasma to tube before adding RBCs. Add reagents Follow manufacturer’s directions Recognize hemolysis as a positive reaction Forgetting to add enhancement, and then “under” incubating. Failure to add AHG…may purchase reagent with green dye added to make it more obvious when it has been added. Follow directions regarding cell suspension concentration, amount of reagent to use and incubation time, etc. Hemolysis – red supernatant with no cell button (or reduced in size)

Work Quickly Y Y Y Y Y Y Y If the addition of AHG reagent following the wash is interrupted, the IgG bound to the RBCs may dissociate. There will either not be enough IgG on the cells to allow for cross-linking, or the free IgG will neutralize the anti-IgG in the AHG reagent.

Other causes of false negative results AHG reagent neutralized Using expired reagents Under-centrifugation Complement-dependant antibody/plasma specimen Under-centrifugation does not allow RBCs to come close enough together to cross-link. Complement will not be activated when using an anticoagulated specimen… the calcium required for activation is chelated by the anticoagulant.

Sources of Error in the Antibody Screen False Positive Results

False Positives Over-centrifugation Contaminated reagents Debris Rouleaux Over-centrifugation packs the RBCs into a tight button that may be difficult to dislodge; may break off in “chunks” of aggregated cells…not agglutinated. Saline especially prone to bacterial contamination. Reading results microscopically may lead one to mistake debris or rouleaux for agglutination. Rouleaux will not bee seen at AHG because the excess protein will be removed during the wash step.

Limitations of the Antibody Screen Will not detect ABO incompatibility Will not detect antibodies to antigens that are not present on the screen cells May not detect antibodies exhibiting dosage May not detect antibodies that are low in titer

Primary vs. Secondary Humoral Response IgG IgM IgM Delayed Hemolytic Transfusion Reactions happen because the antibody titer was below the detection threshold of the antibody screen method used. Studies have shown that 42% of antibodies decrease in titer to a point that the antibody is no longer detectable 5 years later (unless the patient is re-stimulated). IgG First exposure Second exposure

Today’s Lab… The Type & Screen (ABO, Rh, and Antibody Screen)