1、Antibody Identification,Renee Wilkins, PhD, MLS(ASCP)cm CLS 325/435 School of Health Related Professions University of Mississippi Medical Center,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
2、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 patients serum If a person with an antibody is exposed to donor cells
3、 with the corresponding antigen, serious side effects can occur,Key Concepts,In blood banking, we test “knowns” with “unknowns”When detecting and/or identifying antibodies, we test patient serum (unknown) with reagent RBCs (known),Known: Unknown: Reagent RBCs + patient serum Reagent antisera + patie
4、nt RBCs,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 scree
5、n 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:
6、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,IS37AHG,Antibody ID Testing,A tube is labeled for each of t
7、he panel cells plus one tube for AC:,AC,1,2,3,4,5,6,7,8,9,10,11,1 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) 37C Phase,2 dr
8、ops of LISS are added, mixed and incubated for 10-15 minutes Centrifuge and check for agglutination Record results,(LISS) 37C Phase,2+,0,0,2+,0,0,2+,0,0,2+,0,0,0,0,IAT Phase (or AHG),Indirect Antiglobulin Test (IAT) were testing whether or not possible antibodies in patients serum will react with RB
9、Cs 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+,0,0,2+,0,0,2+,0,0,2+,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,
10、0,0,0,0,0,0,0,0,0,0,0,And dont forget.,.add “check” cells to any negative AHG !,All cells are negative at AHG, so add “Check” Cells,You have agglutinationnow what?,2+,0,0,2+,0,0,2+,0,0,2+,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,?,CC,Interpreting Antibody Panels,There are a few basic step
11、s to follow when interpreting panels “Ruling out” means crossing out antigens that did not react Circle the antigens that are not crossed out Consider antibodys usual reactivity Look for a matching pattern,An antibody will only react with cells that have the corresponding antigen; antibodies will no
12、t react with cells that do not have the antigen,Always remember:,Heres an example:,1. Ruling Out,2+,0,0,2+,0,0,2+,0,0,2+,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.,2. Circle antig
13、ens not crossed out,2+,0,0,2+,0,0,2+,0,0,2+,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,3. Consider antibodys usual reactivity,2+,0,0,2+,0,0,2+,0,0,2+,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,Lea is normally a Cold-Reacting antibody (IgM), so it makes sense that we see the reactio
14、n in the IS phase of testing; The E antigen will usually react at warmer temperatures,4. Look for a matching pattern,2+,0,0,2+,0,0,2+,0,0,2+,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,Yes, there is a matching pattern!,E doesnt match and its a warmer rx Ab,Interpretation,anti-Lea,Guidelines,
15、Again, its important to look at: Autocontrol Negative - alloantibody Positive autoantibody or DTR (i.e.,alloantibodies) Phases IS cold (IgM) 37 - cold (some have higher thermal range) or warm reacting AHG warm (IgG)significant! Reaction strength 1 consistent strength one antibody Different strengths
16、 multiple antibodies or dosage,About reaction strengths,Strength of reaction may be due to “dosage” If panel cells are homozygous, a strong reaction may be seen If panel cells are heterozygous, reaction may be weak or even non-reactive Panel cells that are heterozygous should not be crossed out beca
17、use antibody may be too weak to react (see first example),Guidelines (continued),Matching the pattern Single antibodies usually shows a pattern that matches one of the antigens (see previous panel example) Multiple antibodies are more difficult to match because they often show mixed reaction strengt
18、hs,Rule of three,The rule of three must be met to confirm the presence of the antibody A p-value 0.05 must be observed This gives a 95% confidence interval How is it demonstrated? Patient serum MUST be: Positive with 3 cells with the antigen Negative with 3 cells without the antigen,Our previous exa
19、mple fulfills the “rule of three”,2+,0,0,2+,0,0,2+,0,0,2+,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,3 Negative cells,3 Positive cells,Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin Panel Cells 8, 10, and 11 are negative for the antigen and did no
20、t give a reaction at immediate spin,What if the “rule of three” is not fulfilled?,If there are not enough cells in the panel to fulfill the rule, then additional cells from another panel could be used Most labs carry different lot numbers of panel cells,Phenotyping,In addition to the rule of three,
21、antigen typing the patient red cells can also confirm an antibody How is this done? Only perform this if the patient has NOT been recently transfused (donor cells could react) If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should resultWhy?,Remember
22、 Landsteiners Rule,Individuals DO NOT make allo-antibodies against antigens they have,Multiple antibodies,Multiple antibodies may be more of a challenge than a single antibody Why? Reaction strengths can vary Matching the pattern is difficult,So what is a tech to do?,Several procedures can be perfor
23、med to identify multiple antibodies Selected Cells Neutralization Chemical treatment Proteolytic enzymes Sulfhydryl reagents ZZAP,Selected Cells,Selected cells are chosen from other panel or screening cells to confirm or eliminate the antibody The cells are “selected” from other panels because of th
24、eir characteristics The number of selected cells needed depends on how may antibodies are identified,Selected Cells,Every cell should be positive for each of the antibodies and negative for the remaining antibodies For example: Lets say you ran a panel and identified 3 different antibodies: anti-S,
25、anti-Jka, and anti-P1 Selected cells could help,Selected Cells,These results show that instead of 3 antibodies, there are actually 2: anti-S and anti-Jka,Neutralization,Some antibodies may be neutralized as a way of confirmation Commercial “substances” bind to the antibodies in the patient serum, ca
26、using them to show no reaction when tested with the corresponding antigen (in panel),Neutralization,Manufacturers directions should be followed and a dilutional control should always be used The control contains saline and serum (no substance) and should remain positive A control shows that a loss o
27、f reactivity is due to the neutralization and not to the dilution of the antibody strength when the substance is added,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
28、 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,Enzymes (proteolyti
29、c),Can be used to enhance or destroy certain blood group antigens Several enzymes exist: Ficin (figs) Bromelin (pineapple) Papain (papaya) In addition, enzyme procedures may be One-step Two-step,Enzymes,Enzymes remove the sialic acid from the RBC membrane, thus “destroying” it and allowing other ant
30、igens 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 t
31、ested,Enzyme techniques,If there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen,Enzyme treament,Anti-K,Perfect match for anti-Fya,Duffy antigens destroyed Kell antigens not affected,Enzyme treatment,Sulfhydryl Reagents,Cleave the disulfide bonds of IgM mole
32、cules and help differentiate between IgM and IgG antibodies Good to use when you have both IgG and IgM antibodies (warm/cold) Dithiothreitol (DTT) is a thiol and will denature Kell antigens 2-mercaptoethanol (2-ME),ZZAP,A combination of proteolytic enzymes and DTT Denatures Kell, M, N, S, Duffy and
33、other less frequent blood group antigens Does not denature the Kx antigen Good for adsorption techniques “frees” autoantibody off patients cell, so that autoantibody can then be adsorbed onto another RBC,Autoantibodies.,Warm & Cold Reacting,Autoantibodies,Autoantibodies can be cold or warm reacting
34、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
35、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
36、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 significan
37、t allo-antibodies, so its important to differentiate between auto- and allo-antibodies,Cold autoantibodies,React at room temperature with most (if not all) of the panel cells and give a positive autocontrol The DAT is usually positive with anti-C3 AHG (detects complement) Could be due to Mycoplasma
38、pneumoniae, infectious mono, or cold agglutinin disease,Cold autoantibodies,Mini-cold panels can be used to help identify cold autoantibodies Since anti-I is a common autoantibody, cord blood cells (no I antigen) are usually included,Group O individual with cold autoanti-I,Group A individual with co
39、ld autoanti-IH,Anti-IH is reacting weakly with the cord cells (some H antigen present),Avoiding reactivity,Cold autoantibodies can be a nuisance at times. Here are a few ways to avoid a reaction: Use anti-IgG AHG instead of polyspecific. Most cold antibodies react with polyspecific AHG and anti-C AH
40、G because they fix complement Skipping the IS phase avoids the attachment of cold autoantibodies to the red cells Use 22% BSA instead of LISS,Other techniques,If the antibodies remain, then prewarmed techniques can be performed: Red cells, serum, and saline are incubated at 37 before being combined
41、Autoadsorption is another technique in which the autoantibody is removed from the patients serum using their own red cells The serum can be used to identify any underlying alloantibodies,Warm autoantibodies,More common that cold autoantibodies Positive DAT due to IgG antibodies coating the red cell
42、Again, the majority of panel or screening cells will be positive The Rh system (e antigen) seems to be the main target although others occur,Warm autoantibodies,Cause warm autoimmune hemolytic anemia (WAIHA)H&H How do you get a warm autoantibody? Idiopathic Known disorder (SLE, RA, leukemias, UC, pr
43、egnancy, infectious diseases, etc) Medications Several techniques are used when warm autoantibodies are suspected,Elution (whenever DAT is positive),Elution techniques “free” antibodies from the sensitized red cells so that the antibodies can be identified,Y,Y,Y,Y,Sensitized RBC,Positive DAT,Elution
44、,Y,Y,Y,Y,Y,Y,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 wit
45、h panel cells, the eluate usually remains reactive with all cells if a warm autoantibody is present,Elution Methods,Acid elutions (glycine acid) Most common Lowers pH, causing antibody to dissociate Organic solvents (ether, chloroform) Dissolve bilipid layer of RBC Heat (conformational change) Freez
46、e-Thaw (lyses cells),ABO antibodies,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 teste
47、d 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,Wash x3 after incubation,Centrifuge after incubating; and transfer serum to 2nd tube of treated cells; incubate and centrifuge again,
