2 Antibody PresencePresence of an antibody may be indicated by the following serological tests:A discrepancy in the results of cell and serum ABH grouping.A positive test for unexpected antibodies.A positive direct Coomb’s test.An incompatible major cross match.
3 The Basics….. As we said in the previous lecture, Antibody Screens use 2 or 3 Screening Cells to “detect” if antibodies are present in the serumIf antibodies are detected, then they should be identified…
4 Why do we need to identify? Antibody identification is an important component of compatibility testingIt will identify any unexpected antibodies in the patient’s serumIf a person with an antibody is exposed to donor cells with the corresponding antigen, serious side effects can occur (i.e. transfusion reactions).
5 Key Concepts In blood banking, we test “knowns” with “unknowns”:- When detecting/screening and/or identifying antibodies, we test patient serum (unknown that contain blood bank antibodies) with reagent RBCs (known)Known: Unknown:Reagent RBCs + patient serumReagent antisera + patient RBCs
6 Reagent RBCsScreening Red Cells and Panel Red Cells are the same with minor differences:Screening red cellsAntibody detection/screeningSets of only 2 or 3 vialsPanel red cellsAntibody identificationAt least 10 vials/set
7 Antibody Panel vs. Screen An antibody panel is just an extended version of an antibody screenThe screen only uses 2-3 red cells:
8 Antibody PanelWhile antibody panel usually includes at least 10 panel cells: (8-16 group O RBCs)Group O red blood cells obtained from donors
9 Panel Red CellsEach 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
10 Panel Red CellsAn autocontrol (AC) should also be run with panels
11 Panel Red CellsThe same phases used in an antibody screen are used in a panel
12 Antibody ID TestingA tube is labeled for each of the panel cells plus one tube for AC
13 IS PhasePerform immediate spin (IS) and grade agglutination; inspect for hemolysisRecord the results in the appropriate space as shown.
14 (LISS) 37°C Phase2 drops of LISS are added, mixed and incubated for minutes.Centrifuge and check for agglutinationRecord results as previous but now fill the 37°C lane.Agglutination Viewer
17 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 vitroTo do this we use the Anti-Human Globulin reagent (AHG)Polyspecific AHGMonospecific Anti-IgGMonospecific Anti-Complement
18 IAT (AHG) PhaseWash red cells 3X with saline (manual or automated (cell washer))Add 2 drops of AHG and gently mixCentrifugeRead for agglutinationRecord reactions
22 Guidelines Again, it’s important to look at: Matching the pattern 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 dosageMatching the patternSingle antibodies usually shows a pattern that matches one of theMultiple antibodies are more difficult to match because they often show mixed reaction strengthsDTR – delayed transfusion reaction (donor cells are sensitized with recipient patient’s antibody)
24 Rule of threeThe rule of three must be met to confirm the presence of the antibodyHow is it demonstrated?Patient serum MUST be:Positive with 3 panel cells with the antigen, +ve reaction.Negative with 3 cells without the antigen and should not be reacting.
25 Our previous example fulfills the “rule of three”
26 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 usedBetter to carry different lot numbers of panel cells
27 Patient Antigen Typing (Phenotyping) In 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 (chimera)).If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…Why?
28 Remember Landsteiner’s Rule Individuals DO NOT make allo-antibodies against antigens they have
29 Multiple antibodiesMultiple antibodies may be more of a challenge than a single antibodyWhy?Reaction strengths can varyMatching the pattern is difficult
30 So what we have to do?Several procedures can be performed to identify multiple antibodiesSelected CellsNeutralizationChemical treatmentProteolytic enzymesSulfhydryl reagents
31 1- Selected Red CellsSelected 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.Every cell should be positive only for each of the antibodies and negative for the remaining suspicious antibodiesFor example:Let’s say you ran a panel and identified 3 different antibodies (you cannot rule out): anti-S, anti-Jka, and anti-P1Selected cells could help…
32 Selected Red Cells ….. Cont’d These are panel cells that are being used, I just didn’t include the rest of the antigram because it is not necessary.
33 2- Neutralization Common substances Some 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)Common substancesP1 substance (derived from hydatid cyst fluid)Lea and Leb substance (soluble antigen found in plasma and saliva)I substance can be found in breast milk**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
34 3- Again: Proteolytic Enzymes 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
35 EnzymesEnzymes remove the sialic acid from the RBC membrane, thus “destroying” it and allowing other antigens to be “enhanced”Antigens destroyed: M, N, S, s, DuffyAntigens enhanced: Rh, Kidd, Lewis, I, and POne-stageEnzyme is added directly to the serum/panel cell mixtureTwo-stagePanel cells are pre-treated with an enzyme, and washedPatient serum is added to treated panel cells and testedIf there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen
36 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)
38 Autoantibodies Autoantibodies can be cold or warm reacting A 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
39 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
40 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
41 Identifying autoantibodies Auto-antibodies can sometimes “mask” clinically significant allo-antibodies, so it’s important to differentiate between auto- and allo-antibodies
42 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
43 Avoiding reactivityCold Autoantibodies can be a trouble 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 AHG because they fix complementSkipping the IS phase avoids the attachment of cold autoantibodies to the red cellsUse 22% BSA instead of LISS
44 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
45 Warm autoantibodies More common that cold autoantibodies Positive DAT due to IgG antibodies coating the red cellAgain, the majority of panel or screening cells will be positive
46 Warm autoantibodies Cause warm autoimmune hemolytic anemia (WAIHA)… How do you get a warm autoantibody?IdiopathicKnown disorder (SLE, RA, leukemias, pregnancy, infectious diseases, etc)MedicationsSeveral techniques are used when warm autoantibodies are suspected…
47 Elution (whenever DAT is positive) Elution techniques “free” antibodies from the sensitized red cells so that the antibodies can be identified
48 Elution The eluate is a term used for the removed antibodies Testing the eluate is useful in investigations of positive DATsHDNTransfusion 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
49 Elution Methods Acid elutions (glycine acid) Most commonLowers pH, causing antibody to dissociateOrganic solvents (ether, chloroform)Dissolve bilipid layer of RBCHeat (conformational change)Freeze-Thaw (lyses cells)
50 AdsorptionAdsorption procedures can be used to investigate underlying alloantibodiesAfter the patient RBCs are incubated, the adsorbed serum is tested with panel cells to ID the alloantibody (if present)
51 Adsorption Two types: Autoadsorption No recent transfusionAutoantibodies are removed using patient RBCs, so alloantibodies can be identifiedAllogenic (Differential) adsorptionIf recently transfusedUses other cells with the patients serum
53 SummaryIf an unexpected antibody is detected in a patient’s serum or plasma it must be identified.Once identified the clinical significance must be determined.
54 SummaryIf the antibody is clinically significant antigen negative donors must be found and crossmatched for the patient, a Coomb’s crossmatch must be done.If the antibody is not clinically significant it is not necessary to provide antigen negative blood, but the donors must be compatible by the Coomb’s crossmatch.
55 Providing Compatible Donor Units Once an antibody has been identified, the next task is to provide appropriate units of RBCs for transfusion.When clinically insignificant antibodies are detected, use of crossmatch-compatible RBCs is appropriate.
56 Providing Compatible Donor Units No further testing is needed to confirm compatibility when the antibody is anti-M, anti-N, anti-Pi. Lea, or Leb.However, when a clinically significant antibody is identified, the blood must be cross-match compatible and confirmed as antigen-negative with reagent antisera.
57 ExampleKnowledge of the incidence of antigens is useful for determining how many units of blood to screen or crossmatch for patients with antibodies.If a patient with an anti-Jk(a-) needed 4 units of blood, how many units would need to be tested to find them?Jk (a+)= 0.77Jk (a-) = 0.234 units Jk(a-) blood needed = 17.4 units 0.23 incidence of Jk(a-)In this case, testing 17 or 18 random units should yield 4 Jk(a-) units.
58 Example 2The same calculations can be used when multiple antibodies are present if the antigen frequencies are first multiplied together.E.g. a patient with an anti-K and anti-Jka, 10 random units would need to be tested to find 2 that are compatible.Jk(a+) = K positive = 0.09Jk(a-) = K negative = 0.91Jk(a-) (0.23) X K negative (0.91) = 0.20 Jk(a-) and K negative2 units needed = 10 units 0.20 Jk(a-) and Kell negative