Antibody Identification Renee Wilkins Ph D MLS ASCP cm CLS

Antibody Identification Renee Wilkins, Ph. D, MLS(ASCP)cm CLS 325/435 School of Health Related Professions University of Mississippi Medical Center

The Basics…. . ¡ As you recall, l l 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: Reagent RBCs + Reagent antisera + ¡ Unknown: patient serum 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: l Screening cells Antibody detection ¡ Sets of 2 or 3 vials ¡ l 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) l l + 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 ¡ Autocontrol Patient RBCs + Patient serum An autocontrol should also be run with ALL panels

Panel ¡ The same phases used in an antibody screen are used in a panel • IS • 37° • AHG

Antibody ID Testing ¡ 1 2 A tube is labeled for each of the panel cells plus one tube for AC: 3 4 5 6 7 8 1 drop of each panel cell + 2 drops of the patients serum 9 10 11 AC

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 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) ¡ l l l Polyspecific Anti-Ig. G Anti-complement

AHG Phase Wash cells 3 times with saline (manual or automated) ¡ Add 2 drops of AHG and gently mix ¡ l l l Centrifuge Read Record reactions

AHG Phase 2+ 0 0 0 0 0 0 0 0

And don’t forget…. …. add “check” cells to any negative AHG !

IS 2+ 0 0 LISS AHG 37° 0 0 0 0 0 0 CC All cells are negative at AHG, so add “Check” Cells

You have agglutination…now what? CC 2+ 0 0 0 ? ? 0 0 0 0 0 0

Interpreting Antibody Panels ¡ There a few basic steps to follow when interpreting panels 1. 2. 3. 4. “Ruling out” means crossing out antigens that did not react Circle the antigens that are not crossed out Consider antibody’s usual reactivity 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

Here’s an example:

1. Ruling Out 2+ 0 0 0 0 0 Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage. 0 0 0

2. Circle antigens not crossed out 2+ 0 0 0 0 0 0 0 0

3. Consider antibody’s usual reactivity 2+ 0 0 0 0 0 0 0 0 Lea is normally a Cold-Reacting antibody (Ig. M), so it makes sense that we see the reaction in the IS phase of testing; The E antigen will usually react at warmer temperatures

4. Look for a matching pattern E doesn’t match and it’s a warmer rx Ab 2+ 0 0 0 …Yes, there is a matching pattern! 0 0 0 0 0 0

Interpretation anti. Lea

Guidelines ¡ Again, it’s important to look at: l l l Autocontrol ¡ Negative - alloantibody ¡ Positive – autoantibody or DTR (i. e. , alloantibodies) Phases ¡ IS – cold (Ig. M) ¡ 37° - cold (some have higher thermal range) or warm reacting ¡ AHG – warm (Ig. G)…significant!! Reaction strength ¡ 1 consistent strength – one antibody ¡ Different strengths – multiple antibodies or dosage

About reaction strengths…… ¡ Strength of reaction may be due to “dosage” l l ¡ If panel cells are homozygous, a strong reaction may be seen If panel cells are heterozygous, reaction may be weak or even nonreactive Panel cells that are heterozygous should not be crossed out because antibody may be too weak to react (see first example)

Guidelines (continued) ¡ Matching the pattern l l 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 strengths

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? ¡ l Patient serum MUST be: Positive with 3 cells with the antigen ¡ Negative with 3 cells without the antigen ¡

Our previous example fulfills the “rule of three” 3 Positive cells 3 Negative cells 2+ 0 0 0 0 0 0 0 0 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 not 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, antigen typing the patient red cells can also confirm an antibody ¡ How is this done? ¡ l l 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 result…Why?

Remember Landsteiner’s Rule Individuals DO NOT make allo-antibodies against antigens they have

Multiple antibodies may be more of a challenge than a single antibody ¡ Why? ¡ l l Reaction strengths can vary Matching the pattern is difficult

So what is a tech to do? ¡ Several procedures can be performed to identify multiple antibodies l l l 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 their 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: ¡ l l Let’s say you ran a panel and identified 3 different antibodies: anti-S, anti-Jka, and anti-P 1 Selected cells could help…

Selected Cells Selected cells S Jka P 1 IS LISS AHG 37° #1 + 0 0 2+ #5 0 + 0 0 0 3+ #8 0 0 + 0 0 0 These results show that instead of 3 antibodies, there 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, causing them to show no reaction when tested with the corresponding antigen (in panel) ¡

Neutralization ¡ Manufacturer’s directions should be followed and a dilutional control should always be used l l The control contains saline and serum (no substance) and should remain positive A control shows that a loss of reactivity is due to the neutralization and not to the dilution of the antibody strength when the substance is added

Neutralization ¡ Common substances l l P 1 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 (Ig. G), an important reason to use neutralization techniques

Enzymes (proteolytic) Can be used to enhance or destroy certain blood group antigens ¡ Several enzymes exist: ¡ l l l ¡ Ficin (figs) Bromelin (pineapple) Papain (papaya) In addition, enzyme procedures may be l l One-step Two-step

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 l ¡ Enzyme is added directly to the serum/cell mixture Two-stage l l Panel cells are pre-treated with enzyme, incubated and washed Patient serum is added to panel cells and tested

Enzyme techniques ¡ If there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen

Enzyme treatment Enzyme treament Anti-K Perfect match for anti-Fy a • Duffy antigens destroyed • Kell antigens not affected

Sulfhydryl Reagents Cleave the disulfide bonds of Ig. M molecules and help differentiate between Ig. M and Ig. G antibodies ¡ Good to use when you have both Ig. G and Ig. M antibodies (warm/cold) ¡ l l 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 other less frequent blood group antigens ¡ Does not denature the Kx antigen ¡ Good for adsorption techniques ¡ l “frees” autoantibody off patient’s cell, so that autoantibody can then be adsorbed onto another RBC

Autoantibodies…. Warm & Cold Reacting

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 l l 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 alloantibodies, so it’s 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 -C 3 AHG (detects complement) ¡ Could be due to Mycoplasma 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 cold 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 a few ways to avoid a reaction: l l l Use anti-Ig. G AHG instead of polyspecific. Most cold antibodies react with polyspecific AHG and anti-C AHG 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: l ¡ Red cells, serum, and saline are incubated at 37° before being combined Autoadsorption is another technique in which the autoantibody is removed from the patients serum using their own red cells l The serum can be used to identify any underlying alloantibodies

Warm autoantibodies More common that cold autoantibodies ¡ Positive DAT due to Ig. G antibodies coating the red cell ¡ 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? ¡ l l l ¡ Idiopathic Known disorder (SLE, RA, leukemias, UC, pregnancy, 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 Positive DAT Y Y YY Y Y Sensitized RBC Elution 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 l l l ¡ ¡ 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

Elution Methods ¡ Acid elutions (glycine acid) l l ¡ Organic solvents (ether, chloroform) l ABO antibodies Most common Lowers p. H, causing antibody to dissociate Dissolve bilipid layer of RBC Heat (conformational change) ¡ Freeze-Thaw (lyses cells) ¡

Adsorption procedures can be used to investigate underlying alloantibodies ¡ ZZAP or chloroquine diphosphate can be used to dissociate Ig. G 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: l Autoadsorption No recent transfusion ¡ Autoantibodies are removed using patient RBCs, so alloantibodies can be identified ¡ l Allogenic (Differential) adsorption If recently transfused ¡ Uses other cells with the patients serum ¡

Remove serum and test for alloantibody 2 tubes Wash x 3 after incubation Centrifuge after incubating; and transfer serum to 2 nd tube of treated cells; incubate and centrifuge again

More reagents…. Many of elution tests can damage the antigens on the RBC ¡ Choroquine diphosphate (CDP) and glycine acid EDTA reagents can dissociate Ig. G from the RBC without damaging the antigens ¡ l Very useful if the RBC needs to be antigen typed

Chloroquine diphosphate Quinilone derivative often used as an antimalarial ¡ May not remove autoantibody completely from DAT positive cells ¡ Partial removal may be enough to antigen type the cells or to be used for autoadsorption of warm autoantibodies ¡

THE END!!