Antibody Identification I - PDF Free Download

Antibody Identification I Amanda Smith, MLS(ASCP) CM Immunohematologist Laboratory of Immunohematology and Genomics April 18, 2017 New York Blood Center 1

Introduction Red cell alloantibodies other than naturally occurring anti-a or B are called unexpected red cell alloantibodies. Unexpected red cell alloantibodies can be found in 0.3% to 2.8% of the population, depending upon the group of patients or donors studied and the sensitivity of the antibody detection method used (albumin, LISS, PEG, IgG gel or Solid-Phase test). 2

Introduction (continued) Immunization to red cell antigens may result from pregnancy, transfusion, transplantation, or from injections with immunogenic material. Or, in some instances no specific immunizing event can be identified. 3

Goals of antibody identification investigations To identify the antibody or antibodies detected in serum or plasma antibody screening tests To determine the clinical importance of these antibodies To ensure availability of compatible units of RBCs for transfusion 4

Reagents for antibody identification Group O red cells for antibody screening of serum or plasma are commercially available and are offered as sets of either two or three vials of single-donor red cells. Reagent cells licensed by the FDA for this purpose must express the following antigens: D, C, E, c, e, M, N, S, s, P1, Le a, Le b, K, k, Fy a, Fy b, Jk a and Jk b. 5

Reagents for antibody identification (continued) The simplest serological method employs salinesuspended red cells that are mixed with the serum or plasma to be tested (1 drop of cells + 2 drops of serum/plasma). This test can be incubated at either room temperature (RT) or 37 C and can also include an antiglobulin phase (IAT). 6

Blood Bank Sample Patient sample received in hospital blood bank Serum/plasma antibody screen performed with two or three red cell panel kit Negative antibody screen Positive antibody screen No detectable irregular red cell antibodies Immediate spin (IS) crossmatch or electronic crossmatch Proceed to antibody identification Antibody/ies identified Full (IAT) antigen-negative (if required) crossmatch 7

Initial Antibody Screening Screen for Serum/plasma Antibody: Rh-hr Kell Kidd Duffy Lewis MNSs P LISS Cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37C AHG I + + 0 0 + + + + 0 0 + + 0 + 0 0 + + 0 0 II + 0 + + 0 0 + 0 + + 0 0 + 0 + + 0 0 2+ 2+ III 0 w 0 + + 0 + + 0 + + 0 + + + 0 + + 0 0 All screening cells non-reactive Non reactivity with any test cell indicates that serum/plasma antibody is not likely to be directed against an antigen present on that nonreactive test cell. One or more screening cells reactive Positive reactions can be indicated by hemolysis or agglutination 8

Initial Panel of RBCs Rh-hr Kell Kidd Duffy Lewis MNSs P LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37ºC AHG 1 + + 0 0 + 0 + + 0 0 + 0 + 0 + 0 + + 0 0 2 + 0 + + 0 + + 0 + + 0 0 + 0 + 0 + + 2+ 2+ 3 + 0 0 + + 0 + + + + + 0 + 0 + + + 0 0 0 4 0 0 0 + + 0 + 0 + + + 0 0 + 0 + 0 + 0 0 5 0 0 + + + + + + 0 0 + 0 + + 0 0 + 0 1+ 1+ 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0 7 0 0 0 + + + + + 0 + 0 0 + + + + 0 + 0 0 8 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 0 9 + 0 0 + + 0 + + + 0 + + 0 + 0 + + + 0 0 10 + 0 0 + + 0 + + 0 + + 0 0 + + 0 + 0 0 0 11 0 0 0 + + 0 + + + + + 0 + + 0 0 + + 0 0 AC/ Pheno 0 0 9

Initial panel of RBCs Rh-hr Kell Kidd Duffy Lewis MNSs P LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37ºC AHG Locate the line at the top of the antigram that defines each antigen in each blood group system. Move across the row of antigen headings and cross out any antigen present on a panel cell that fails to react with the serum being tested. 10

Initial Panel of RBCs Rh-hr Kell Kidd Duffy Lewis MNSs P LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37ºC AHG 1 + + 0 0 + 0 + + 0 0 + 0 + 0 + 0 + + 0 0 2 + 0 + + 0 0 + 0 + + 0 0 + 0 + 0 + + 2+ 2+ 3 + + 0 0 + 0 + + + + + 0 + 0 + + + 0 0 0 4 0 0 0 + + 0 + 0 + + + 0 0 + 0 + 0 + 0 0 5 0 0 + + + + + + 0 0 + 0 + + 0 0 + 0 1+ 1+ 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0 7 0 0 0 + + + + + 0 + 0 0 + + + + 0 + 0 0 8 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 0 9 + 0 0 + + + + + + 0 + + 0 + 0 + + + 0 0 10 + 0 0 + + 0 + + 0 + + 0 0 + + 0 + 0 0 0 11 0 0 0 + + 0 + + + + + 0 + + 0 0 + + 0 0 AC/ Pheno 0 0 11

Initial panel of RBCs (continued) Assess variability (or lack of variability) of reactions: Reactions at different phases of testing may indicate the presence of more than one antibody specificity, each with different reaction characteristics. Reactions that vary in strength may indicate that the antibody is reacting with more than one antigen present on the test cell. 13

Initial panel of RBCs (continued) Always include an autocontrol with the panel studies for comparison with reactions of test RBCs. Select the method that is most likely to produce detectable reactions: if reactions were observed at 37 C and in the IAT, continue by these techniques to identify antibody(ies). Consider the amount of serum or plasma available before beginning the investigation. 14

Two Directions Patient s RBCs Patient s Serum Combine Information 15

Patient s RBCs Phenotype the patient s RBCs for common antigens (Rh, Kidd, Duffy, Kell and Ss) to assist in ruling out antibody specificity But remember: Red cell phenotype results are unreliable when: Testing samples from a patient who has been recently transfused (transfused red cells may react with reagent antiserum causing incorrect interpretation of these tests). The patient received an allogeneic stem cell transplant The Direct Antiglobulin Test (DAT) is positive (all red cells positive in the DAT will be positive in the IAT). 16

Patient s RBCs Compare phenotype with results of initial panel Antigens that cannot be ruled out with negative panel reactions usually can be eliminated if the antibody maker s RBCs possess those antigens 17

Patient s RBCs (continued) Create and test selected cell panel to confirm antibody identity and to rule out all other commonly encountered antibody specificities At least 3 antigen positive RBC samples should react, and 3 antigen negative RBC samples should be nonreactive to establish acceptable probability of antibody specificity 18

Patient s Serum Note variation in reaction strength Compare variable reactivity to single and double dose of antigen expression Commonly encountered antibodies that demonstrate dosage: Anti-Jk a, -Jk b, -Fy a, -Fy b Example: Anti-Jk a reacts more strongly with Jk(a+b ) RBCs than with Jk(a+b+) RBCs Anti-S, -s, (-M, -N) Rh antibodies (but not anti-d) more often at 37 C than by IAT 19

Patient s Serum Use of several techniques optimizes chances of identification IAT/ 37 C for clinically significant antibodies (example: anti-d, -K, or E) RT (or lower): Anti-M, -N, -P1, -Le a and Le b have little clinical significance Enzyme treated panel cells Technique used to remove antigens from RBC membrane and can be useful if selected cells are not available or limited 20

Patient s Serum IAT using different potentiators (albumin, LISS, PEG, IgG gel or Solid-Phase test). Weakly reactive antibodies, or those showing dosage, are more easily detected using PEG or the IgG gel test HOWEVER PEG and the IgG gel test enhance some IgM antibodies reactive at low temperatures, e.g., anti-m, -Le a, -Le b, -P1 PEG and the IgG gel test also enhance autoantibodies 21

Patient s Serum: Use of Enzyme treated Panel Cells RBC treatment with ficin or papain Inactivates Fy a, Fy b ; S, s (variable), M, N; Xg a antigens (plus others of high incidence) Enhances reactivity of some antigens with corresponding antibodies, e.g., Rh; Jk a, Jk b ; Fy3; Le a, Le b ; P1; ABH; I; i and others Commercial ficin and papain treated panels available 22

Effect of enzyme on a RBC membrane protein NH 2 Antigen 1 The enzyme may remove some or all of the antigencarrying molecule Antigen 2 Outside RBC Lipid Layer COOH 23

Cases 1-3 24

Case 1: Patient Profile A 50 year-old female with lower GI bleeding was admitted. She received 2 units of RBCs 6 months ago during a total abdominal hysterectomy. No irregular blood group antibodies were detected at that time. Her current Hgb was 6.5 g/dl and there were orders to transfuse 3 units of RBCs. She typed group O D+ and the antibody screen was negative at 37 C and in the IAT. During the crossmatch procedure, the patient s serum reacted 1+ to 2+ at immediate spin with 3 of 3 ABO compatible RBC units. 25

Case 1: Screen for Serum Antibody Rh-hr Kell Kidd Duffy Lewis MNSs P LISS Cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37C AHG I + + 0 0 + + + + 0 0 + + 0 + 0 0 + + 0 0 II + 0 + + 0 0 + 0 + + 0 0 + 0 + + 0 0 0 0 III 0 w 0 + + 0 + + 0 + + 0 + + + 0 + + 0 0 26

Case 1: Initial Panel of RBCs Rh-hr Kell Kidd Duffy Lewis MNSs P SAL LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 IS 37ºC AHG 1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 0 0 2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 1+ 0 0 3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 0 0 0 4 + 0 + + 0 0 + 0 + + + 0 + + 0 + 0 + 2+ 0 0 5 0 0 + + + + + + 0 0 + 0 + 0 + 0 + + 0 0 0 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 2+ 0 0 7 0 0 0 + + + + + 0 + 0 0 + + + + 0 + 1+ 0 0 8 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 0 0 9 + 0 0 + + 0 + + + 0 + + 0 + 0 + + + 2+ 0 0 10 + 0 0 + + 0 + + 0 + + 0 0 + + 0 + 0 1+ 0 0 11 + + 0 0 + 0 + + + + + 0 + 0 + + + + 0 0 0 AC/ Pheno 0 0 0 + + 0 0 0 27

Case 1: Selected RBC Panel Rh-hr Kell Kidd Duffy Lewis MNSs P SAL LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 IS 37ºC AHG 1 + + 0 0 + 0 + + 0 0 + 0 + 0 + 0 + + 0 0 0 2 + 0 + + 0 + + 0 + + 0 0 + 0 + 0 + + 0 0 0 3 + 0 0 + + 0 + + + + + 0 + 0 + + + 0 0 0 0 4 0 0 0 + + 0 + 0 + + + 0 0 + 0 + 0 + 2+ 0 0 5 0 0 + + + + + + 0 0 + 0 0 + 0 0 + 0 2+ 0 0 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 2+ 0 0 29

Case # 1 Antibody Identified Anti-M identified in the serum 31

Case 2: Patient Profile A 60 year-old male with leukemia was admitted to the hem/onc floor with anemia. The Hgb level was 6.0 g/dl at admission. He was transfused 4 months ago with 2 units of RBCs. No irregular blood group antibodies were detected at that time. The attending physician ordered 3 units of RBCs to be transfused. The patient typed A D+ and the antibody screen was variably positive (1+ to 3+) in the IAT with 2 of 3 screening cells. The patient s RBCs were negative in the DAT. 32

Case 2: Screen for Serum Antibody Rh-hr Kell Kidd Duffy Lewis MNSs P LISS Cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37C AHG I + + 0 0 + + + + 0 0 + + 0 + 0 0 + + 0 0 II + 0 + + 0 0 + 0 + + 0 0 + 0 + + 0 0 0 3+ III 0 w 0 + + 0 + + 0 + + 0 + + + 0 + + 0 1+ 33

Case 2: Initial Panel of RBCs Rh-hr Kell Kidd Duffy Lewis MNSs P SAL LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 IS 37ºC AHG 1 + + 0 0 + 0 + + 0 0 + 0 + 0 + 0 + + 0 0 0 2 + 0 + + 0 + + 0 + + 0 0 + 0 + 0 + + 0 0 3+ 3 + 0 0 + + 0 + + + + + 0 + 0 + + + 0 0 0 1+ 4 0 0 0 + + 0 + 0 + + + 0 0 + 0 + 0 + 0 0 1+ 5 0 0 + + + + + + 0 0 + 0 + + 0 0 + 0 0 0 0 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0 0 7 0 0 0 + + + + + 0 + 0 0 + + + + 0 + 0 0 3+ 8 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 0 3+ 9 + 0 0 + + 0 + + + 0 + + 0 + 0 + + + 0 0 0 10 + 0 0 + + 0 + + 0 + + 0 0 + + 0 + 0 0 0 1+ 11 + + 0 0 + 0 + + + + + 0 + 0 + + + + 0 0 1+ AC/ Pheno 0 0 0 34

Case 2: Patient Phenotype Rh-hr Kell Kidd Duffy Lewis MNSs P D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 + + 0 + + 0 + + 0 0 + + 0 + + 0 + + 36

Case 2: Enzyme-treated RBC Panel Rh-hr Kell Kidd Duffy Lewis MNSs P Enzyme Treated Cells cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37 C AHG 1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 0 2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 0 0 3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 0 0 4 + 0 + + 0 0 + 0 + + + 0 + + 0 + 0 + 0 0 5 0 0 + + + 0 + + + 0 + 0 + 0 + 0 + 0 0 0 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0 7 0 0 0 + + + + + 0 + + 0 + + + 0 + + 0 0 8 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 0 9 + 0 0 + + 0 + + + 0 + + 0 + 0 + + + 0 0 10 + 0 0 + + 0 + + 0 + + 0 0 + + 0 + 0 0 0 11 + + 0 0 + 0 + + + + + 0 + 0 + + + + 0 0 37

Case 2: Selected RBC Panel Rh-hr Kell Kidd Duffy Lewis MNSs P LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37 C AHG 1 + + 0 0 + 0 + + 0 0 + + 0 0 + + 0 + 0 0 2 + 0 + + 0 + + 0 + 0 + 0 + + + 0 + + 0 0 3 0 0 0 + + 0 + + 0 0 + + 0 0 + + 0 0 0 0 4 + 0 0 + + 0 + + 0 0 0 0 + + 0 + 0 + 0 0 5 0 0 0 + + 0 + + 0 + 0 0 + 0 + 0 + 0 0 3+ 6 + 0 0 + + 0 + + 0 + 0 + 0 + 0 0 + 0 0 3+ 39

Case # 2 Antibody Identified Anti-Fy a 41

Case 3: Patient Profile A 47 year-old female with breast cancer submitted pre-operative blood samples prior to scheduled surgery. She was transfused 4 units RBCs 1 year ago due to bleeding during a total abdominal hysterectomy. At that time no unexpected blood group antibodies were detected. The patient typed B D+ and the patient s serum reacted weakly (1+) at 37 C with 1 of 3 screening cells and variably (2+ to 4+) in the IAT with 2 of 3 screening cells. The patient s RBCs were negative in the DAT 42

Case 3: Antibody Screen Rh-hr Kell Kidd Duffy Lewis MNSs P LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37ºC AHG I + + 0 0 + + + + 0 0 + + 0 + + 0 + + 1+ 4+ II + 0 + + 0 0 + 0 + + 0 0 + 0 + + 0 0 0 2+ III 0 0 0 + + 0 + + 0 + + 0 + + + 0 + + 0 0 43

Case 3: Initial RBC Panel Rh-hr Kell Kidd Duffy Lewis MNSs P SAL LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 IS 37ºC AHG 1 + + 0 0 + 0 + + 0 0 + 0 + 0 + 0 + + 0 0 0 2 + 0 + + 0 + + 0 + + 0 0 + 0 + 0 + + 0 1+ 4+ 3 + 0 0 + + 0 + + + + + 0 + 0 + + + 0 0 0 1+ 4 0 0 0 + + 0 + 0 + + + 0 0 + 0 + 0 + 0 0 2+ 5 0 0 + + + + + + 0 0 + 0 + + 0 0 + 0 0 1+ 4+ 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0 2+ 7 0 0 0 + + + + + 0 + 0 0 + + + + 0 + 0 1+ 4+ 8 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 0 0 9 + 0 0 + + 0 + + + 0 + + 0 + 0 + 0 + 0 0 2+ 10 + 0 0 + + 0 + + 0 + + 0 0 + + 0 + 0 0 0 0 11 + + 0 0 + 0 + + + + + 0 + 0 + + + + 0 0 1+ AC/ pheno 0 0 0 44

Case 3: Patient Phenotype Rh-hr Kell Kidd Duffy Lewis MNSs P D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 + 0 0 + + 0 + + 0 + + + 0 + + 0 + + 46

Case 3: Enzyme-treated RBC Panel Rh-hr Kell Kidd Duffy Lewis MNSs P Enzyme Treated Cells cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37 C AHG 1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 0 2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 2+ 4+ 3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 0 0 4 + 0 + + 0 0 + 0 + + + 0 + + 0 + 0 + 0 0 5 0 0 + + + 0 + + + 0 + 0 + 0 + 0 + 0 0 0 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0 7 0 0 0 + + + + + 0 + + 0 + + + 0 + + 2+ 4+ 8 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 0 9 + 0 0 + + 0 + + + 0 + + 0 + 0 + + + 0 0 10 + 0 0 + + 0 + + 0 + + 0 0 + + 0 + 0 0 0 11 + + 0 0 + 0 + + + + + 0 + 0 + + + + 0 0 47

Case 3: Enzyme-treated RBC Panel Rh-hr Kell Kidd Duffy Lewis MNSs P Enzyme Treated Cells cell D C E c e K k Jk a Jk b Fya Fy b Le a Le b M N S s P1 37 C AHG 1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 0 2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 2+ 4+ 3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 0 0 4 + 0 + + 0 0 + 0 + + + 0 + + 0 + 0 + 0 0 5 0 0 + + + 0 + + + 0 + 0 + 0 + 0 + 0 0 0 6 0 + 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0 7 0 0 0 + + + + + 0 + + 0 + + + 0 + + 2+ 4+ 8 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 0 9 + 0 0 + + 0 + + + 0 + + 0 + 0 + + + 0 0 10 + 0 0 + + 0 + + 0 + + 0 0 + + 0 + 0 0 0 11 + + 0 0 + 0 + + + + + 0 + 0 + + + + 0 0 48

Case 3: Selected RBC Panel Rh-hr Kell Kidd Duffy Lewis MNSs P LISS cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P1 37 C AHG 1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 0 2 + 0 + + 0 0 + + 0 0 + 0 + + + 0 + + 0 0 3 0 0 0 + + 0 + 0 + 0 + + 0 0 + 0 + 0 0 0 4 + 0 0 + + 0 + + 0 0 0 0 + + 0 + 0 + 0 2+ 5 0 0 0 + + 0 + + 0 + 0 0 + 0 + + 0 0 0 2+ 6 + 0 0 + + + + 0 + + 0 + 0 + 0 0 + 0 1+ 4+ 7 + 0 0 + + + + + 0 0 0 0 + + 0 0 + + 1+ 4+ 8 + 0 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 0 49

Case # 3 Antibody(ies) Identified Anti-K & anti-s 51

Antibody Identification: A Final Checklist Review each test phase individually Immediate spin, antiglobulin, enzyme Look for specific reaction pattern Antigen positive cells reactive; antigen negative cells nonreactive Be alert for variable strength reactions Multiple antibodies, dosage effect Rule out the presence of other antibody specificities known to react optimally in that test phase Based on antigens present (which should be present in double dose) on non-reactive cells 52

Additional Clues Some cells positive (same strength and phases), some cells negative: suspect single antibody All cells positive or some cells positive (different strengths and/or phases) some cells negative: suspect multiple antibodies Weak reactivity seen with some cells, all specificities eliminated: suspect weakly reactive antibody or antibody showing dosage 53

Additional Clues (continued) Only one cell positive on panel: suspect antibody to low incidence antigens. All cells positive (same strength and same phase): suspect antibody to high incidence antigen or possibility of multiple antibodies Review screening panel to make sure the answer fits with the initial result 54

Questions? 55