Antibody Identification

ZEESHAN YOUSUF MEDICAL TECHNOLOGIST AGA KHAN UNIVERSITY HOSPITAL KARACHI, PAKISTAN

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 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 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:

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 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)

+ 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

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

IS

37
AHG

Antibody ID Testing

A tube is labeled for each of the panel cells plus one tube for AC:
3 4 5 6 7 8 9 10 11

1 drop of each panel cell

+

AC

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 drops of LISS are added, mixed and incubated for 10-15 minutes Centrifuge and check for agglutination Record results

(LISS) 37C Phase

2+ 0 0 0 0 0 2+ 0 0 2+ 0 2+ 0 0

IAT Phase (or AHG)

Indirect Antiglobulin Test (IAT) were testing whether or not possible antibodies in patients serum will react with RBCs 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 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 0

And dont forget.

.add check cells to any negative AHG !

IS 2+ 0 0 2+ 0 0 2+ 0 2+ 0 0

LISS AHG 37 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

CC
All cells are negative at AHG, so add Check Cells

You have agglutinationnow what?

CC

2+ 0 0 2+ 0 0 2+ 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 0

??

Interpreting Antibody Panels

There are 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 antibodys 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

Heres an example:

1. Ruling Out
2+ 0 0 2+ 0 0 2+ 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 0

Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.

2. Circle antigens not crossed out

2+ 0 0 2+ 0 0 2+ 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 0

3. Consider antibodys usual reactivity

2+ 0 0 2+ 0 0 2+ 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 0

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

4. Look for a matching pattern

E doesnt match and its a warmer rx Ab

2+ 0 0 2+ 0 0 2+ 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 0

Yes, there is a matching pattern!

Interpretation
antiLea

Guidelines

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 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 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

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?

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

2+ 0 0 2+ 0 0 2+ 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 0

3 Positive cells

3 Negative 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 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?

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 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 performed 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 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:

Lets say you ran a panel and identified 3 different antibodies: anti-S, anti-Jka, and anti-P1 Selected cells could help

Selected Cells
Selected cells S Jka P1 IS LISS AHG 37

#1
#5

+
0

0
+

0
0

0
0

0
0

2+
3+

#8

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, causing 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 of 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 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 (proteolytic)

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 antigens 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 Panel cells are pre-treated with enzyme, incubated and washed Patient serum is added to panel cells and tested

Two-stage

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-Fya

Duffy antigens destroyed Kell antigens not affected

Sulfhydryl Reagents

Cleave the disulfide bonds of IgM molecules 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 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 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

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 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 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 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 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

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 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, 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

Sensitized RBC

Y
Positive DAT

Frees antibody

Y
Antibody ID

Y
Elution

Elution

The eluate is a term used for the removed antibodies Testing the eluate is useful in investigations of positive DATs

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

HDN Transfusion reactions Autoimmune disease

Elution Methods

Acid elutions (glycine acid)

Most common Lowers pH, causing antibody to dissociate

Dissolve bilipid layer of RBC

Organic solvents (ether, chloroform)

ABO antibodies

Heat (conformational change) Freeze-Thaw (lyses cells)

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 tested 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

2 tubes
Wash x3 after incubation Centrifuge after incubating; and transfer serum to 2nd tube of treated cells; incubate and centrifuge again

Remove serum and test for alloantibody

More reagents.

Many of elution tests can damage the antigens on the RBC Choroquine diphosphate (CDP) and glycine acid EDTA reagents can dissociate IgG from the RBC without damaging the antigens

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!!
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