Antibody IdentificationZEESHAN YOUSUFMEDICAL TECHNOLOGISTAGA KHAN UNIVERSITY HOSPITALKARACHI, PAKISTAN

The Basics..As you recall,Antibody Screens use 2 or 3 Screening Cells to detect if antibodies are present in the serumIf antibodies are detected, they must be identifiedpresentNot present

Why do we need to identify?Antibody identification is needed for transfusion purposes and is an important component of compatibility testingIt will identify any unexpected antibodies in the patients serumIf a person with an antibody is exposed to donor cells with the corresponding antigen, serious side effects can occur

Key ConceptsIn 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 serumReagent antisera+patient RBCs

Reagent RBCsScreening Cells and Panel Cells are the same with minor differences:Screening cellsAntibody detectionSets of 2 or 3 vialsPanel cellsAntibody identificationAt least 10 vials per set

Antibody Panel vs. ScreenAn antibody panel is just an extended version of an antibody screenThe screen only uses 2-3 cells:

Antibody PanelAn antibody panel usually includes at least 10 panel cells:

PanelGroup O red blood cells

PanelEach of the panel cells has been antigen typed (shown on antigram)+ refers to the presence of the antigen0 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

PanelAn autocontrol should also be run with ALL panels

AutocontrolPatient RBCs + Patient serum

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

Antibody ID TestingA tube is labeled for each of the panel cells plus one tube for AC:AC12345678910111 drop of each panel cell+2 drops of the patients serum

IS PhasePerform immediate spin (IS) and grade agglutination; inspect for hemolysisRecord the results in the appropriate space as shown:

2+00Last tube

(LISS) 37C Phase2 drops of LISS are added, mixed and incubated for 10-15 minutesCentrifuge and check for agglutinationRecord results

(LISS) 37C Phase2+002+002+002+0000

IAT Phase (or AHG)Indirect Antiglobulin Test (IAT) were testing whether or not possible antibodies in patients serum will react with RBCs in vitroTo do this we use the Anti-Human Globulin reagent (AHG)PolyspecificAnti-IgGAnti-complement

AHG PhaseWash cells 3 times with saline (manual or automated)Add 2 drops of AHG and gently mixCentrifugeReadRecord reactions

AHG Phase2+002+002+002+00000000000000000000000000

And dont forget..add check cells to any negative AHG !

All cells are negative at AHG, so add Check Cells

ISLISS 37AHGCC2+000000002+000000002+000002+00000000

You have agglutinationnow what?2+002+002+002+00000000000000000000000000??CC

Interpreting Antibody PanelsThere are a few basic steps to follow when interpreting panelsRuling out means crossing out antigens that did not reactCircle the antigens that are not crossed outConsider antibodys usual reactivityLook for a matching pattern

An antibody will only react with cells that have the corresponding antigen; antibodies will not react with cells that do not have the antigenAlways remember:

Heres an example:

1. Ruling Out2+002+002+002+00000000000000000000000000Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.

2. Circle antigens not crossed out2+002+002+002+00000000000000000000000000

3. Consider antibodys usual reactivity2+002+002+002+00000000000000000000000000Lea is normally a Cold-Reacting antibody (IgM), 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 pattern2+002+002+002+00000000000000000000000000Yes, there is a matching pattern!E doesnt match and its a warmer rx Ab

Interpretationanti-Lea

GuidelinesAgain, its important to look at:AutocontrolNegative - alloantibodyPositive autoantibody or DTR (i.e.,alloantibodies)PhasesIS cold (IgM)37 - cold (some have higher thermal range) or warm reactingAHG warm (IgG)significant!!Reaction strength1 consistent strength one antibodyDifferent strengths multiple antibodies or dosage

About reaction strengthsStrength of reaction may be due to dosageIf panel cells are homozygous, a strong reaction may be seenIf panel cells are heterozygous, reaction may be weak or even non-reactivePanel cells that are heterozygous should not be crossed out because antibody may be too weak to react (see first example)

Guidelines (continued)Matching the patternSingle 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 threeThe rule of three must be met to confirm the presence of the antibodyA p-value 0.05 must be observedThis gives a 95% confidence intervalHow is it demonstrated?Patient serum MUST be:Positive with 3 cells with the antigenNegative with 3 cells without the antigen

Our previous example fulfills the rule of three2+002+002+002+000000000000000000000000003 Negative cells3 Positive cellsPanel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spinPanel 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 usedMost labs carry different lot numbers of panel cells

PhenotypingIn addition to the rule of three, antigen typing the patient red cells can also confirm an antibodyHow 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 Landsteiners RuleIndividuals DO NOT make allo-antibodies against antigens they have

Multiple antibodiesMultiple antibodies may be more of a challenge than a single antibodyWhy?Reaction strengths can varyMatching the pattern is difficult

So what is a tech to do?Several procedures can be performed to identify multiple antibodiesSelected CellsNeutralizationChemical treatmentProteolytic enzymesSulfhydryl reagentsZZAP

Selected CellsSelected cells are chosen from other panel or screening cells to confirm or eliminate the antibodyThe cells are selected from other panels because of their characteristicsThe number of selected cells needed depends on how may antibodies are identified

Selected CellsEvery cell should be positive for each of the antibodies and negative for the remaining antibodiesFor example:Lets say you ran a panel and identified 3 different antibodies: anti-S, anti-Jka, and anti-P1Selected cells could help

Selected Cells These results show that instead of 3 antibodies, there are actually 2: anti-S and anti-Jka

Selected cellsSJkaP1ISLISS 37AHG#1+00002+#50+0003+#800+000

NeutralizationSome antibodies may be neutralized as a way of confirmationCommercial substances bind to the antibodies in the patient serum, causing them to show no reaction when tested with the corresponding antigen (in panel)

NeutralizationManufacturers directions should be followed and a dilutional control should always be usedThe control contains saline and serum (no substance) and should remain positiveA 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

NeutralizationCommon substancesP1 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 milkSda 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 (proteolytic)Can be used to enhance or destroy certain blood group antigensSeveral enzymes exist:Ficin (figs)Bromelin (pineapple)Papain (papaya)In addition, enzyme procedures may beOne-stepTwo-step

EnzymesEnzymes remove the sialic acid from the RBC membrane, thus destroying it and allowing other antigens to be enhancedAntigens destroyed: M, N, S, s, DuffyAntigens enhanced: Rh, Kidd, Lewis, I, and P

Enzyme techniquesOne-stageEnzyme is added directly to the serum/cell mixtureTwo-stagePanel cells are pre-treated with enzyme, incubated and washedPatient serum is added to panel cells and tested

Enzyme techniquesIf there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen

Enzyme treamentAnti-KPerfect match for anti-FyaDuffy antigens destroyedKell antigens not affectedEnzyme treatment

Sulfhydryl ReagentsCleave the disulfide bonds of IgM molecules and help differentiate between IgM and IgG antibodiesGood to use when you have both IgG and IgM antibodies (warm/cold)Dithiothreitol (DTT) is a thiol and will denature Kell antigens2-mercaptoethanol (2-ME)

ZZAPA combination of proteolytic enzymes and DTTDenatures Kell, M, N, S, Duffy and other less frequent blood group antigensDoes not denature the Kx antigenGood for adsorption techniquesfrees autoantibody off patients cell, so that autoantibody can then be adsorbed onto another RBC

Autoantibodies.Warm & Cold Reacting

AutoantibodiesAutoantibodies can be cold or warm reactingA positive autocontrol or DAT may indicate that an auto-antibody is presentSometimes the autocontrol may be positive, but the antibody screening may be negative, meaning something is coating the RBC

Getting a positive DATWe 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 informationIf the patient has been transfused, the patient may have an alloantibody coating the transfused cellsIf the patient has NOT been transfused, the patient may have an autoantibody coating their own cells

Identifying autoantibodiesAuto-antibodies can sometimes mask clinically significant allo-antibodies, so its important to differentiate between auto- and allo-antibodies

Cold autoantibodiesReact at room temperature with most (if not all) of the panel cells and give a positive autocontrolThe DAT is usually positive with anti-C3 AHG (detects complement)Could be due to Mycoplasma pneumoniae, infectious mono, or cold agglutinin disease

Cold autoantibodiesMini-cold panels can be used to help identify cold autoantibodiesSince anti-I is a common autoantibody, cord blood cells (no I antigen) are usually includedGroup O individual with cold autoanti-IGroup A individual with cold autoanti-IHAnti-IH is reacting weakly with the cord cells (some H antigen present)

Avoiding reactivityCold 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 AHG because they fix complementSkipping the IS phase avoids the attachment of cold autoantibodies to the red cellsUse 22% BSA instead of LISS

Other techniquesIf the antibodies remain, then prewarmed techniques can be performed:Red cells, serum, and saline are incubated at 37 before being combinedAutoadsorption is another technique in which the autoantibody is removed from the patients serum using their own red cellsThe serum can be used to identify any underlying alloantibodies

Warm autoantibodiesMore common that cold autoantibodiesPositive DAT due to IgG antibodies coating the red cellAgain, 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 autoantibodiesCause warm autoimmune hemolytic anemia (WAIHA)H&HHow do you get a warm autoantibody?IdiopathicKnown 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

YYYYSensitized RBCPositive DATElutionYYYYYYFrees antibodyAntibody ID

ElutionThe eluate is a term used for the removed antibodiesTesting the eluate is useful in investigations of positive DATsHDN Transfusion reactionsAutoimmune diseaseThe red cells can also be used after elution for RBC phenotyping if neededWhen tested with panel cells, the eluate usually remains reactive with all cells if a warm autoantibody is present

Elution MethodsAcid elutions (glycine acid)Most commonLowers pH, causing antibody to dissociateOrganic solvents (ether, chloroform)Dissolve bilipid layer of RBCHeat (conformational change)Freeze-Thaw (lyses cells)ABO antibodies

AdsorptionAdsorption procedures can be used to investigate underlying alloantibodiesZZAP 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)

AdsorptionTwo types:AutoadsorptionNo recent transfusionAutoantibodies are removed using patient RBCs, so alloantibodies can be identifiedAllogenic (Differential) adsorptionIf recently transfusedUses other cells with the patients serum

Wash x3 after incubationCentrifuge after incubating; and transfer serum to 2nd tube of treated cells; incubate and centrifuge again 2 tubesRemove serum and test for alloantibody

More reagents.Many of elution tests can damage the antigens on the RBCChoroquine diphosphate (CDP) and glycine acid EDTA reagents can dissociate IgG from the RBC without damaging the antigensVery useful if the RBC needs to be antigen typed

Chloroquine diphosphateQuinilone derivative often used as an antimalarialMay not remove autoantibody completely from DAT positive cellsPartial removal may be enough to antigen type the cells or to be used for autoadsorption of warm autoantibodies

THE END!!THANK YOU

*DTR delayed transfusion reaction (donor cells are sensitized with patients antibody)*These are panel cells that are being used, I just didnt include the rest of the antigram because it is not necessary.