ABID-antibody identification PDF

ADVANCED ANTIBODY IDENTIFICATION: CASE STUDIES Fardus Aljunibi  REVIEW passement id copy (1) - Mentimeter THE BASICS….. ▪ As you recall, ▪ Antibody Screens use 2 or 3 Screening Cells to “detect” if antibodies are present in the serum ▪ If antibodies are detected,...

ADVANCED ANTIBODY IDENTIFICATION: CASE STUDIES Fardus Aljunibi  REVIEW passement id copy (1) - Mentimeter THE BASICS….. ▪ As you recall, ▪ Antibody Screens use 2 or 3 Screening Cells to “detect” if antibodies are present in the serum ▪ If antibodies are detected, they must be identified… present Not present WHY DO WE NEED TO IDENTIFY? ▪ Antibody identification is needed for transfusion purposes and is an important component of compatibility testing ▪ It will identify any unexpected antibodies in the patient’s serum ▪ If a person with an antibody is exposed to donor cells with the corresponding antigen, serious side effects can occur KEY CONCEPTS ▪ In blood banking, we test “knowns” with “unknowns” Known: Unknown: Reagent RBCs + patient serum Reagent antisera + patient RBCs ▪ When detecting and/or identifying antibodies, we test patient serum (unknown) with reagent RBCs (known) REAGENT RBCS ▪ Screening Cells and Panel Cells are the same with minor differences: ▪ Screening cells ▪ Antibody detection ▪ Sets of 2 or 3 vials ▪ Panel cells ▪ Antibody identification ▪ At least 10 vials per set ANTIBODY PANEL VS. SCREEN ▪ An antibody panel is just an extended version of an antibody screen ▪ The screen only uses 2-3 cells: ANTIBODY PANEL ▪ An antibody panel usually includes at least 10 panel cells: PANEL ▪ Group O red blood cells  PANEL ▪ Each of the panel cells has been antigen typed (shown on antigram) ▪ + refers to the presence of the antigen ▪ 0 refers to the absence of the antigen Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka  PANEL ▪ An autocontrol should also be run with ALL panels Autocontrol Patient RBCs + Patient serum PANEL ▪ The same phases used in an antibody screen are used in a panel IS 37° AHG ANTIBODY ID TESTING ▪ A tube is labeled for each of the panel cells plus one tube for AC: 1 2 3 4 5 6 7 8 9 10 11 AC 1 drop of each panel cell + 💧 2 drops of the patients serum IS PHASE ▪ Perform immediate spin (IS) and grade agglutination; inspect for hemolysis ▪ Record the results in the appropriate space as shown: 2+ 0 0 Last tube (LISS) 37°C PHASE ▪ 2 drops of LISS are added, mixed and incubated for 10-15 minutes ▪ Centrifuge and check for agglutination ▪ Record results (LISS) 37°C PHASE 2+ 0 0 0 0 0 2+ 0 0 2+ 0 2+ 0 0 IAT PHASE (OR AHG) ▪ Indirect Antiglobulin Test (IAT) – we’re testing whether or not possible antibodies in patient’s serum will react with RBCs in vitro ▪ To do this we use the Anti-Human Globulin reagent (AHG) ▪ Polyspecific ▪ Anti-IgG ▪ Anti-complement AHG PHASE ▪ Wash cells 3 times with saline (manual or automated) ▪ Add 2 drops of AHG and gently mix ▪ Centrifuge ▪ Read ▪ Record reactions AHG PHASE 2+ 0 0 0 0 0 0 0 0 2+ 0 0 0 0 0 0 0 0 2+ 0 0 0 0 0 2+ 0 0 0 0 0 0 0 0 0 0 0 AND DON’T FORGET…. ….add “check” cells to any negative AHG ! IS LISS AHG CC 37° 2+ 0 0 ✓ All cells are negative at 0 0 0 ✓ AHG, so 0 0 0 ✓ add 2+ 0 0 ✓ “Check” 0 0 0 ✓ Cells 0 0 0 ✓ 2+ 0 0 ✓ 0 0 0 ✓ 2+ 0 0 ✓ 0 0 0 ✓ 0 0 0 ✓ YOU HAVE AGGLUTINATION…NOW WHAT? CC 2+ 0 0 ✓ 0 0 0 ✓ 0 0 0 ✓ 2+ 0 0 ✓ 0 0 0 ✓ 0 0 0 ✓ 2+ 0 0 ✓ 0 0 0 ✓ 2+ 0 0 ✓ 0 0 0 ✓ 0 0 0 ✓ 0 0 0 ✓ ?? INTERPRETING ANTIBODY PANELS ▪ There are a few basic steps to follow when interpreting panels 1. “Ruling out” means crossing out antigens that did not react 2. Circle the antigens that are not crossed out 3. Consider antibody’s usual reactivity 4. Look for a matching pattern ALWAYS REMEMBER: An antibody will only react with cells that have the corresponding antigen; antibodies will not react with cells that do not have the antigen  PRINCIPLES AND PROCEDURES OF THE TESTS “Why are there so many different procedures for ABID?” – The protocols that laboratories choose will affect what they detect. – Protocols should be tailored to the experience of the staff and the general patient population encountered. – Media needs to be taken into account Gel Solid Phase Tube – Saline – Albumin – LISS – PeG – Enzymes  CLINICAL SIGNIFICANC E AND PREVALENCE An antibody is considered significant if it has been associated with – HDFN – HTR – Notable decreased survival of RBCs – The degree of clinical significance varies among antibodies with the same specificity Most commonly identified alloantibodies: – Anti-D – Anti-E – Anti-K  ANTIBODY REACTIVITY IN VARIOUS MEDIA Albumin LISS PeG Gel Solid Phase May enhance Some examples Newly forming Increased Increased Rh and anti-P1 of anti-K do not IgM antibody detection of detection of antibodies react well in may not react antibodies that antibodies that during the 37ºC LISS are not are not spin phase Some examples clinically clinically of anti-Jka not significant significant detected Can enhance Can enhance Can enhance clinically benign clinically benign clinically benign autoantibodies autoantibodies autoantibodies Sources: Sally Rudmann, Ed. Serologic Problem-Solving: A Systematic Approach for Improved Practice. 2005. AABB Press. John D. Roback, Ed. AABB Technical Manual, 17th Edition. Denise Harmening, Ed. Modern Blood Banking and Transfusion Practices, 6th Edition.  PRINCIPLES AND PROCEDURES OF THE TESTS Microscopic evaluation of macroscopically negative test tube reactions? Use of the autocontrol in antibody screening and panels? Two- or three- cell screens? Screen and panel—methods the same? Variations in ruling out – Homozygous for C, c, E, e, Duffy, Kidd, MNSs How many strikes? – Heterozygous ok? How many strikes? Variations in ruling in – 2/2 rule? 3/3 rule? Etc.  PRINCIPLES AND PROCEDURES OF THE TESTS Gather relevant patient information Observe and evaluate results – Phase of reactivity: immediate spin, 37C incubation, AHG – Incompatible crossmatches? Strength – Hemolysis? Pattern – Most or all cells positive, autocontrol negative – 1 or 2 cells positive, autocontrol negative – Panreactivity – Variability—an antibody showing dosage effect, multiple antibodies, or antigen showing variable expression from one panel cell to another – Weak, variable reactivity Physical Appearance  KNOWLEDGE OF ANTIBODY SPECIFICITIES Anti-D, -E, and -K antibodies most common in U.S. Anti-C, -c, -e, -Jka, -Jkb –Fya, -S, -s sometimes seen Anti-Fya and anti-Fyb rarely exist as single alloantibodies Temperature (IgM = cold reactive, usually not clinically significant) ”Lemon Pie is best served cold” Lewis M, N, P1  KNOWLEDGE OF ANTIBODY SPECIFICITIES Usually clinically significant: – ABO, Rh, Kidd, Duffy, S, s, U, P Rarely (if ever) cause clinically obvious symptoms: – Bg (HLA), Ch/Rg (C4), Leb, JMH, Xga Sometimes: – Cartwright (Yt), Lutheran (Lu), Gerbich (Ge), Dombrock (Do), M,N, Lea, Vel, LW, Ii, H, Ata, Inb, Mia, Csa  RULING OUT A tool in the process, not infallible It is always preferable to rule out an antibody specificity on a homozygous cell It is better to rule out specificities with two unique cells rather than one. There is no reason to routinely rule out antibodies to low-incidence antigens. – Screening cells may not detect these – Patients rarely form these antibodies – Transfusion probability – Ethnicity/Geography matters: Dia 10% in Asians, 36% South American Indian Even if a specificity is ruled out by the laboratory’s SOP, it does not mean the antibody is not present  Ruling In p value is a calculation of the number of antigen-positive cells that react and the number of antigen-negative cells that do not react.1,3 AABB’s IRL Standards require two antigen-positive cells that are reactive and two antigen-negative cells that are nonreactive Donor Cell Patient Reaction K+ + K+ + K- 0 K- 0  Ruling In with Multiples Multiple specificities must be ruled in independently of each other: Donor Cell Patient Reaction E+K- E+K- + E-K+ + + E-K+ + E-K- 0 E-K- 0  STEPS Go to the first panel cell with a negative reaction, “rule out” or exclude the specificities of antibodies directed against antigens present on the cell. – (Rule out when the antigen is positive and the patient did not react) – Some antibodies demonstrate dosage.  DOSAG E Some antibodies may react so weakly with antigens with heterozygous expression, they might not be detected. For antibodies in the following blood groups, it may be prudent to rule out with panel cells that have a homozygous expression of antigen: – Rhesus (C, c, E, e) – Kidd – Duffy – MNSs  DOSA GE Mother Father  DOSA Mother GE Father  DOSA GE Anti-Jka may not react with a heterozygous “single dose” cell It may only react with a cell that has “double the dose” of Jka antigens  EXAMPLES Rh System C, c E, e Duffy System Fya, Fyb Kidd System Jka, Jkb MNSs System M, N S, s READY TO GO?  STEP 1. GATHER ALL RELEVANT DATA 37-year-old male patient, he is A+ Received 4 units of pRBCs during previous hospital admission under a trauma name, 3 months ago Scheduled for surgery tomorrow. what test should be run in this case to determine the most compatible unit for him?  CASE Rh-Hr 1 Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 8 + + + + + 0 + + + + + 0 + + 0 + AC  CASE Rh-Hr 1 Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  CASE Rh-Hr 1 Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  CASE Rh-Hr 1 Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  CASE Rh-Hr 1 Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  CASE Rh-Hr 1 Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  CASE Rh-Hr 1 Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  CASE Rh-Hr 1 Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  THE NEXT STEP What alloantibody or alloantibodies have not been ruled out? Anti-E Anti-Fya Which of the following is or are most likely? Look closely at the pattern of reactivity.  ANTIBODY IDENTIFICATION PANEL Rh-Hr Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  ANTIBODY IDENTIFICATION PANEL Rh-Hr Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  CONFIRMATION STEPS Anti-E is the most likely antibody reacting However, we still have not ruled out anti-Fya The patient could have anti-Fya underlying the reactions of anti-E We need to select another cell that is E antigen negative, and Fy(a+b-) HOMOZYGOUS for Duffy A  SELECTED CELL FROM A DIFFERENT PANEL Rh-Hr Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 3 + 0 + 0 + + + + 0 + 0 + 0 + + + 4 + + + + + 0 + 0 0 + + 0 + 0 + + 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 6 0 0 + + + 0 + 0 0 + 0 0 + 0 0 + 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 8 0 + + 0 + 0 + + + + + 0 + + 0 + AC  SELECTED CELL FROM A DIFFERENT PANEL Rh-Hr Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 3 + 0 + 0 + + + + 0 + 0 + 0 + + + 4 + + + + + 0 + 0 0 + + 0 + 0 + + 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 6 0 0 + + + 0 + 0 0 + 0 0 + 0 0 + 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 8 0 + + 0 + 0 + + + + + 0 + + 0 + AC  SELECTED CELL FROM A DIFFERENT PANEL Rh-Hr Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 3 + 0 + 0 + + + + 0 + 0 + 0 + + + 0 0 ✓ 4 + + + + + 0 + 0 0 + + 0 + 0 + + 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 6 0 0 + + + 0 + 0 0 + 0 0 + 0 0 + 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 8 0 + + 0 + 0 + + + + + 0 + + 0 + AC  SELECTED CELL FROM A DIFFERENT PANEL Rh-Hr Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 3 + 0 + 0 + + + + 0 + 0 + 0 + + + 0 0 ✓ 4 + + + + + 0 + 0 0 + + 0 + 0 + + 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 6 0 0 + + + 0 + 0 0 + 0 0 + 0 0 + 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 8 0 + + 0 + 0 + + + + + 0 + + 0 + AC  RULE OF 3 Criteria: – At least 3 panel cells with E antigen reacted (positive result) with patient’s sample – At least 3 panel cells lacking E antigen did not react (negative result) with the patient’s sample Does our example fulfill these criteria?  ANTIBODY IDENTIFICATION PANEL Rh-Hr Kell Duffy Kidd P MNSs Results AH D C c E e K k Fya Fyb Jka Jkb P1 M N S s 37 G CC 1 + 0 + + + 0 + 0 0 + + + + + 0 + 0 2+ 2 + 0 + 0 + 0 + 0 0 + 0 + + 0 0 + 0 0 ✓ 3 + 0 + + 0 0 + 0 0 + 0 + 0 + + + 0 2+ 4 0 + 0 + + 0 + 0 0 + + 0 + 0 + + 0 0 ✓ 5 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 2+ 6 0 0 + 0 + 0 + 0 0 + 0 0 + 0 0 + 0 0 ✓ 7 0 0 + 0 + + + 0 + 0 + 0 0 + + 0 0 0 ✓ 8 + + + + + 0 + + + + + 0 + + 0 + 0 2+ AC 0 0 ✓  RULE OF 3 At least 3 true positives and 3 true negatives: Following this rule gives us a P value of 0.05 95% chance that the antibody we have identified is correct.  RULE OF 2 Clinical utility of P value in ABID1,3 At least 2 true positives and 2 true negatives: – AABB IRL Standards – Confirmation that the antibody(ies) identified are present – All other clinically significant alloantibodies are ruled out  BEFORE REACHING A FINAL CONCLUSION Is the final answer a “unicorn?” Are there extra reactions not explained by the final answer? Is the result consistent with the available data? Have all of the alternatives not included in the final result been ruled out? Has enough evidence been collected to establish a high degree of confidence?  RESU LT Anti-E identified. All other clinically significant alloantibodies have been ruled out. Donor units lacking E antigen should appear crossmatch compatible through the indirect antiglobulin test (IAT). SO WHAT A TECH CAN DO? ▪ Several procedures can be performed to identify multiple antibodies ▪ Selected Cells ▪ Neutralization ▪ Adsorption ▪ Elution ▪ Chemical treatment ▪ Proteolytic enzymes ▪ Sulfhydryl reagents ▪ ZZAP ADSORPTION ▪ Adsorption procedures can be used to investigate underlying alloantibodies ▪ ZZAP or chloroquine diphosphate can be used to dissociate IgG antibodies from the RBC (may take several repeats) ▪ After the patient RBCs are incubated, the adsorbed serum is tested with panel cells to ID the alloantibody (if present) ADSORPTION ▪ Two types: ▪ Autoadsorption ▪ No recent transfusion ▪ Autoantibodies are removed using patient RBCs, so alloantibodies can be identified ▪ Allogenic (Differential) adsorption ▪ If recently transfused ▪ Uses other cells with the patients serum ELUTION (WHENEVER DAT IS POSITIVE) Elution techniques “free” antibodies from the sensitized red cells so that the antibodies can be identified Y Elution Y Sensitized Y Y Y RBC YY Y Y Y Positive DAT Frees antibody Antibody ID ELUTION ▪ The eluate is a term used for the removed antibodies ▪ Testing the eluate is useful in investigations of positive DATs ▪ HDN ▪ Transfusion reactions ▪ Autoimmune disease ▪ The red cells can also be used after elution for RBC phenotyping if needed ▪ When tested with panel cells, the eluate usually remains reactive with all cells if a warm autoantibody is present ENZYMES ▪ Enzymes remove the sialic acid from the RBC membrane, thus “destroying” it and allowing other antigens to be “enhanced” ▪ Antigens destroyed: M, N, S, s, Duffy ▪ Antigens enhanced: Rh, Kidd, Lewis, I, and P ENZYME TECHNIQUES ▪ One-stage ▪ Enzyme is added directly to the serum/cell mixture ▪ Two-stage ▪ Panel cells are pre-treated with enzyme, incubated and washed ▪ Patient serum is added to panel cells and tested NEUTRALIZATION ▪ Some antibodies may be neutralized as a way of confirmation ▪ Commercial “substances” bind to the antibodies in the patient serum, causing them to show no reaction when tested with the corresponding antigen (in panel) NEUTRALIZATION ▪ Common substances ▪ P1 substance (sometimes derived from hydatid cyst fluid) ▪ Lea and Leb substance (soluble antigen found in plasma and saliva) ▪ I substance can be found in breast milk ▪ Sda substance derived from human or guinea pig urine **you should be aware that many of these substances neutralize COLD antibodies; Cold antibodies can sometimes mask more clinically significant antibodies (IgG), an important reason to use neutralization techniques AUTOANTIBODIES ▪ Autoantibodies can be cold or warm reacting ▪ A positive autocontrol or DAT may indicate that an auto-antibody is present ▪ Sometimes the autocontrol may be positive, but the antibody screening may be negative, meaning something is coating the RBC GETTING A POSITIVE DAT ▪ We have focused a lot on the IAT used in antibody screening and ID, but what about the DAT? ▪ The direct antiglobulin test (DAT) tests for the in vivo coating of RBCs with antibody (in the body) ▪ AHG is added to washed patient red cells to determine this WHAT CAN THE DAT TELL US? ▪ Although not always performed in routine pretransfusion testing, a positive DAT can offer valuable information ▪ If the patient has been transfused, the patient may have an alloantibody coating the transfused cells ▪ If the patient has NOT been transfused, the patient may have an autoantibody coating their own cells IDENTIFYING AUTOANTIBODIES ▪ Auto-antibodies can sometimes “mask” clinically significant allo-antibodies, so it’s important to differentiate between auto- and allo-antibodies ACTIVITY https://www.mentimeter.com/app/presentation/n/al9 ekp7hna2edafg19p7pyf6jgkzmud6/edit  CASE 4 28-year-old male with sickle cell anemia. First visit to your hospital system—no record. Has received many transfusions throughout his lifetime, including about 3 weeks ago. Admitted due to sickle cell crisis triggered by the high altitude. Solid phase screen was positive—reflexed to antibody ID in tube using LISS. CASE 4 CASE 4 CASE 4 SELECTED CELLS CASE 4 SELECTED CELLS  Case 4 Review of the workup ✔ Is the final answer a “unicorn?” ✔ Are there extra reactions not explained by the final answer? ✔ Is the result consistent with the available data? ✔ Have all of the alternatives not included in the final result been ruled out? ✔ Has enough evidence been collected to establish a high degree of confidence? Thank you! Questions?