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Antibody Identification. Mohammed Jaber. Antibody Presence . Presence 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.

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Antibody identification l.jpg

Antibody Identification

Mohammed Jaber


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Antibody Presence

Presence 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.


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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 serum

    • If antibodies are detected, then they should be identified…

present

Not present


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Why do we need to identify?

  • Antibody identification 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 (i.e. transfusion reactions).


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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 serum

Reagent antisera + patient RBCs


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Reagent RBCs

  • Screening Red Cells and Panel Red Cells are the same with minor differences:

    • Screening red cells

      • Antibody detection/screening

      • Sets of only 2 or 3 vials

    • Panel red cells

      • Antibody identification

      • At least 10 vials/set


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Antibody Panel vs. Screen

  • An antibody panel is just an extended version of an antibody screen

  • The screen only uses 2-3 red cells:


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Antibody Panel

  • While antibody panel usually includes at least 10 panel cells: (8-16 group O RBCs)


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Panel Red Cells

  • Group O red blood cells obtained from donors


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Panel Red Cells

  • 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


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Panel Red Cells

  • An autocontrol (AC) should also be run with ALL panels

Autocontrol

Patient RBCs + Patient serum


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Panel Red Cells

  • The same phases used in an antibody screen are used in a panel

  • IS

  • 37°C

  • AHG


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Antibody ID Testing

  • A tube is labeled for each of the panel cells plus one tube for AC:





AC

1 drop of each panel cell

+

2 drops of the patients serum

Do not forget to write patient name and ID, Lab No.

Patient cells



Patient serum


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IS Phase

  • Perform immediate spin (IS) and grade agglutination; inspect for hemolysis

  • Record the results in the appropriate space as shown:

2+

0

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Up to the Last tube


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(LISS) 37°C Phase

  • 2 drops of LISS are added, mixed and the mixture is incubated for 10-15 minutes

  • Centrifuge and check for agglutination

  • Record results as previous but now fill the 37°C lane.



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(LISS) 37°C Phase

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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 AHG

    • Monospecific Anti-IgG

    • Monospecific Anti-Complement


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IAT (AHG) Phase

  • Wash red cells 3X with saline (manual or automated (cell washer))

  • Add 2 drops of AHG and gently mix

    • Centrifuge

    • Read for agglutination

    • Record reactions


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IAT (AHG) Phase

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And don’t forget….

….add “check” cells to any negative AHG !


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All cells are negative at AHG, so add “Check” Cells


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You have agglutination…now what?

CC

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Interpreting Antibody Panels

  • There are a few basic steps to follow when interpreting panels

    • “Ruling out” means crossing out antigens that did not react

    • Circle the antigens that are not crossed out

    • Consider antibody’s (from the circled) usual reactivity

    • Look for a matching pattern


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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



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1. Ruling Out

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Cross out antigens that show NO REACTION in any phase; do NOTcross out heterozygous antigens that show dosage.


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2. Circle antigens not crossed out

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3. Consider antibody’s usual reactivity

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Lea 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


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4. Look for a matching pattern

E doesn’t match and it’s a warmer rxn Ab

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…Yes, there is a matching pattern!


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Interpretation:

anti-Lea


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Guidelines

  • Again, it’s 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 – multiple antibodies or dosage


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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 for an antigen should not be crossed out because antibody may be too weak to react (see previous example)


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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 strengths


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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 panel cells with the antigen, +ve reaction.

      • Negative with 3 cells without the antigen and should not be reacting.


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Our previous example fulfills the “rule of three”

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3 Positive cells

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3 Negative cells

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Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at IS

Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at IS


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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 good (I do not know if we are good or not!!) labs carry different lot numbers of panel cells


Patient antigen typing phenotyping l.jpg
Patient Antigen Typing (Phenotyping)

  • In addition to the rule of three, 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 (chimera)).

    • If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…Why?


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Remember Landsteiner’s Rule

Individuals DO NOT make allo-antibodies against antigens they have


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Multiple antibodies

  • Multiple antibodies may be more of a challenge than a single antibody

  • Why?

    • Reaction strengths can vary

    • Matching the pattern is difficult


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So what we have to do?

  • Several procedures can be performed to identify multiple antibodies

    • Selected Cells

    • Neutralization

    • Chemical treatment

      • Proteolytic enzymes

      • Sulfhydryl reagents

      • ZZAP


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1- Selected Red 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.


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Selected Red Cells ….. Cont’d

  • Every cell should be positive only for each of the antibodies and negative for the remaining suspicious antibodies

  • For example:

    • Let’s say you ran a panel and identified 3 different antibodies (you cannot rule out): anti-S, anti-Jka, and anti-P1

    • Selected cells could help…


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Selected Red Cells ….. Cont’d

Ag they have

Reaction pattern

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


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2- 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)


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Neutralization ….. Cont’d

  • Manufacturer’s directions should be followed and a positive dilutional control should always be used

    • The positive dilutional control contains saline and serum (no substance is added) and should remain positive with panel cells.

    • 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


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Neutralization ….. Cont’d

  • Common substances

    • P1 substance (derived from hydatid cyst fluid)

    • Lea and Lebsubstance (soluble antigen found in plasma and saliva)

    • Isubstance 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


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3- Again: Proteolytic Enzymes

  • 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


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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


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Enzyme techniques

  • One-stage

    • Enzyme is added directly to the serum/panel cell mixture

  • Two-stage

    • Panel cells are pre-treated with an enzyme, and washed

    • Patient serum is added to treated panel cells and tested


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Enzyme techniques

  • If there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen


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Enzyme treatment

Enzyme treatment

Anti-K

Perfect match for anti-Fya

  • Duffy antigens destroyed

  • Kell antigens not affected


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Summary

  • If an unexpected antibody is detected in a patient’s serum or plasma it must be identified.

  • Once identified the clinical significance must be determined.


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Summary

  • If 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 significantit is not necessary to provide antigen negative blood, but the donors must be compatible by the Coomb’s crossmatch.


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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.


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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.


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Example

  • Knowledge 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+)= 0.77

    Jk (a-) = 0.23

  • 4 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.


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Example 2

  • The 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+) = 0.77 K positive = 0.09

    Jk(a-) = 0.23 K negative = 0.91

  • Jk(a-) (0.23) X K negative (0.91) = 0.20 Jk(a-) and K negative

  • 2 units needed = 10 units 0.20 Jk(a-) and Kell negative



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