2 Antibody DetectionThe test used to detect antibodies is called an antibody screenAntibody screens are used for:Patients needing a transfusionPregnant womenCases of transfusion reactionsBlood and plasma donorsUses patients plasma/serum against reagent red cells to detect unexpected antibodiesUnexpected antibodies are found in addition to the expected anti-A and/or anti-B
3 Unexpected antibodies may be: Unexpected antibodies are a result of red cell stimulation (transfusion, HDN)Unexpected antibodies may be:Clinically significant (IgG)Not clinically significant (IgM)Antibody Screen
4 Clinically significant antibodies Usually IgGReact best at 37° and AHG phase (IAT)Clinically significant antibodies are associated with hemolytic transfusion reactions (HTR) and hemolytic disease of the newborn (HDN)
5 Performing an antibody screen Patients plasma/serum is incubated with screening cellsAfter incubation, an IAT is performed (indirect antiglobulin test) using AHG reagentThis will detect any IgG antibodies.
6 Screening CellsScreening cells are single or pooled donor group O cells, however, single-donor vials offer increased sensitivityWhy group O? so anti-A and anti-B won’t reactScreening cells come in sets of 2 or 3 vials eachEach vial (donor) has been phenotyped for each antigen18 antigens are required on at least one of the vials: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb
7 Screening cellsScreening cells come with a sheet of paper called an antigramScreening cells are an already prepared 2-5% RBC suspensionAn antigram (2 or 3 cells) will list the antigens present in each vialA reaction to one or more cells indicates the presence of an unexpected antibody
9 The technologist should be aware that some antigens demonstrate dosage An attempt should be made to used screening cells that are homozygous for the clinically significant antigens (Rh, Duffy, Kidd). Just be aware that different strengths can occurHomozygous antigens will react strongerHeterozygous antigens will react weaker
10 Examples Fya Fyb SCI + 4+ SCII Fya Fyb SCI + 2+ SCII 4+ If patient’s serum contains anti-Fya, there will be a stronger reaction because SCI is homozygous for the Duffy antigenFyaFybSCI+4+SCIIFyaFybSCI+2+SCII4+In this case, the person has anti-Fyb. The antibody reacts weaker with SCI (heterozygous) and stronger with SCII (homozygous)
11 Screening CellsScreening cells may also contain low incidence antigens like V, Cw, and KpaThe presence of these antigens is not required for screening cells
12 Pretransfusion Screening Screening for antibodies is normally performed prior to blood transfusion to detect antibodies that react at body temperature (37°)Colder reacting antibodies (RT and below) are therefore considered insignificant and just cause interference when performing lab testingThe only important thing to remember concerning cold antibodies is that they may bind complement if a persons body temperature becomes lowOpen-heart surgeryHypothermia
13 Autocontrol Tests patient serum with their own red cells Some labs may or may not perform an autocontrol (AC) with the screen…depends on the hospitalHowever, the AC should be run with the antibody panel…we’ll discuss this laterAC is incubated with the antibody screen (or antibody panel)If a lab uses an AC with the screen and it is positive, they may run a DAT (patient cells + AHG) to detect in vivo coating
14 AutocontrolThe AC and DAT can help in determining whether the antibodies are directed against the patient’s cells or transfused cells (allo or autoantibody).ScreenIf PositiveAntibodyPanel (w/AC)If PositiveDAT
15 PotentiatorsUsed in antibody detection and identification to enhance antigen-antibody reactionSaline (may only enhance if incubated long time)Low-ionic strength solution (LISS)…commonBovine serum albumin (BSA)Polyethylene glycol (PEG)Proteolytic enzymes (can destroy some antigens)
16 Potentiators Albumin LISS PEG Serum/cell mixture should incubate at least minutes;doesn’t enhance warm autoantibodiesLISSIncubation time of 10 minutes;lowers ionic strength allowing better reaction; sensitive and quick!PEGPolyethylene glycolEnhances warm autoantibodies;does not react well with insignificant antibodies (IgM)
17 Testing Techniques – Saline Tube Simplest to perform.Mix serum or plasma with saline suspended RBCs, centrifuge and read, incubate at RT or 37C.Used in crossmatching to detect ABO incompatibility.In antibody tests used to detect IgM antibodies which react preferentially at RT: anti-M, -N, -P1, -Le and –I
18 Testing Techniques – Bovine Albumin Tube Utilized to enhance agglutination of IgG antibodies since 1945.Decreases amount of time required for incubation.Controversy: Decrease zeta potential (affects second stage of agglutination) or due to function of ionic strength of albumin diluents does it increase uptake of antibody onto cells?Many antibodies have enhanced reactivity when albumin is added to test system.
19 Testing Techniques – LISS Tube Low Ionic Strength Saline shortens incubation time.Increases antibody uptake onto cell, enhancing agglutination.Several important factors to consider:Incubation time and sensitivity subsequent to AHG depends upon desired ionic conditions.Adding additional serum will increase ionic strength, must not be done.MUST adhere to manufacturer’s instructions.
20 Testing Techniques – PEG Tube Polyethylene Glycol (PEG) is a water soluble, neutral polymer which is an effective potentiator of antigen-antibody reactions.Advantages over albumin include:Increases rate of detection of clinically significant antibodies.Decreases detection of clinically insignificant antibodies.May decrease need for other enhancement techniques.ProcedureSerum or plasma added to RBCs, perform IS.Add PEG and incubate at 37C – IS NOT READ AFTER 37CWash and add AHG.
21 Testing Techniques – Enzymes Tube More appropriate for antibody ID than routine testing.GREATLY enhance reactivity of Rh antibodies.CANNOT be only method used as M, N, S, Fy and other antigens are destroyed, those antibody specificities would not be detected.Enzymes used includePapainBromelainTrypsinFicin – MOST POPULAR
22 ProcedureAntibody screening tests using a test tube method are performed in a variety of ways. American Association of Blood Banks Standards requires that these tests detect clinically significant antibodies and that they include a 37°C incubation and an AHG test. Generally, testing includes the following steps:Appropriately label each tube.Add 2 drops of patient serum to each tube.Add 1 drop of appropriate screening cells to each tube.Centrifuge, then gently resuspend the cell button and read for agglutination or hemolysis. Record results. It should be noted that this step is optional because most significant antibodies are IgG and do not cause agglutination of saline-suspended RBCs.
23 Add 2 drops of enhancement reagent to each tube (may vary with enhancement reagent used). Incubate at 37°C for 15 to 30 minutes, according to the manufacturer's recommendation for the enhancement reagent being used. During the incubation, antibody in the patient serum will bind to antigens on the reagent RBC. This is called the sensitization phase.Centrifuge, then gently resuspend the cell button and read for agglutination or hemolysis. Record results.Fill all tubes with saline, centrifuge, and discard supernatant. This is called washing, and it removes unbound IgG that neutralizes the AHG reagent.Repeat step 8 two or three times to remove unbound antibody completely.Add 2 drops of AHG to each tube (polyspecific or anti-IgG).
24 Centrifuge, then gently resuspend the cell button and read for agglutination or hemolysis. Tests that are macroscopically negative are usually checked for microscopic agglutination. Record results.Add 1 drop of Coombs control cells (or "check cells") to all negative tests.Centrifuge and read for agglutination. Repeat test if agglutination is not observed.
26 InterpretationAgglutination or hemolysis at any stage of testing is a positive test result, indicating the need for antibody identification studies. However, evaluation of the antibody screen and autologous control results can provide clues and give direction for the identification and resolution of the antibody or antibodies.The investigator should consider the following questions:In what phase(s) did the reaction(s) occur?Is the autologous control negative or positive?Did more than one screening cell sample react, and, if so, did they react at the same strength and phase?Is hemolysis or mixed-field agglutination present?Are the cells truly agglutinated, or is rouleaux present?
27 In what phase(s) did the reaction(s) occur? Antibodies of the IgM class react best at low temperatures and are capable of causing agglutination of saline-suspended RBCs (immediate spin reading). Antibodies of the IgG class react best at the AHG phase. Of the commonly encountered antibodies,anti-N. anti-I, and anti-PI are frequently IgM,whereas those directed against Rh. Kell. Kidd, and Duffy antigens are usually IgG.Lewis and M antibodies may be IgG, IgM, or a mixture of both.Interpretation
28 Is the autologous control negative or positive? A positive antibody screen and a negative autologous control indicate that an alloantibody has been detected.A positive autologous control may indicate the presence of autoantibodies or antibodies to medications.If the patient has been recently transfused, the positive autologous control may be caused by alloantibody coating circulating donor RBCs.Evaluation of samples with positive autologous control or DAT results is often complex and may require a lot of time and experience on the part of the investigator.
29 Did more than one screening cell sample react, and, if so, did they react at the same strength and phase?More than one screening cell sample is positivewhen the patient has multiple antibodies,when the antibodies' corresponding antigen is found on more than one screening cellwhen the patient's serum contains an autoantibody.A single antibody specificity should be suspected when all cells react at the same phase and strength.Multiple antibodies are most likely when cells react at different phases and strengthsAnd autoantibodies are suspected when the autologous control is positive. .
30 Is hemolysis or mixed-field agglutination present? Certain antibodies-such as anti-Lea, anti- Leb, anti P+P1+Pk, and anti-Vel-are known to cause in vitro hemolysis.Mixed-field agglutination is associated with anti-Sda and Lutheran antibodies.
31 Are the cells truly agglutinated, or is rouleaux present? Serum from patients with altered albumin-to-globulin ratios (e.g., patients with multiple myeloma) or who have received high-molecular-weight plasma expanders (e.g.. dextran) may cause nonspecific aggregation of RBCs, known as rouleaux.Rouleaux is not a significant finding in antibody screening tests, but it is easily confused with antibody-mediated agglutination.Knowledge of the following characteristics of rouleaux helps in differentiation between rouleaux and agglutination:Cells have a "stacked coin" appearance when viewed microscopically.Rouleaux is observed in all tests containing the patient's serum, including the autologous control and the reverse ABO typing.
32 Rouleaux does not interfere with the AHG phase of testing because the patient's serum is washed away prior to the addition of the AHG reagent.Unlike agglutination, rouleaux is dispersed by the addition of 1 to 3 drops of saline to the test tube.
33 Limitations Very effective in detecting antibodies If negative, then the crossmatch should be compatibleAntibody screening tests are designed to detect significant RBC antibodies, but they cannot detect all such antibodies. Antigens with frequencies of less than 10 percent (e.g., Cw. Lu-, Kpa) are not usually represented on screening cells, and, as a result, their corresponding antibodies are not detected in routine screening tests.Antibody screening tests may also yield negative results when the titer or concentration of antibody drops below detectable limits.
34 Limitationsantibody levels decrease over time when the individual is no longer exposed to the corresponding antigen. If the level of an RBC antibody drops too low, results of antibody screening tests and crossmatches will appear negative and may lead to transfusion of donor units that carry the corresponding antigen.Re exposure to the RBC antigen will elicit a secondary immune response, resulting in a dramatic increase in the antibody titer and possible immunologic destruction of the transfused RBCs. this is called a delayed hemolytic transfusion reaction (DHTR) because it occurs days or weeks after the transfusion.The student should keep in mind that proper performance and interpretation of antibody detection tests minimize the risk of DHTRs.
35 Patient History GET THE HISTORY!! Mixed red cell populations from a previous transfusion can remain for up to 3 monthsPatient may have come from another hospitalSome diseases are associated with antibodiesSome antibodies occur at a higher frequency in some racesGet diagnosis, age, race, etc…Lupus or carcinoma associated with warm autoantibody; cold autoantibody - pneumonia
36 Example 1 Screening Cell IS 37°C AHG CC* I II 2+ ND IgG antibody II2+NDIgG antibodySingle specificityNote: This positive reaction is mixed-field in nature because half of the RBCs in the mixture lack IgG on their surface (screening cells) and are free cells, whereas half of the RBCs have IgG (Coombs control cells) and are agglutinated."CC: Coombs Control Red Blood CellsND: Not Done
37 Example 2 Screening Cell IS 37°C AHG CC I 3+ II 2+ IgG antibody 3+II2+IgG antibodyMultiple specificities
38 Example 3 Screening Cell IS 37°C AHG CC I 1+ II 3+ IgM antibody II3+Neg AHG, add CCIgM antibodySingle specificity showing dosage
39 Example 4 Screening Cell IS 37°C AHG CC I 2+ II IgG antibody 2+IIIgG antibodyAllo or autoantibody?(don’t know without further testing)