Antibody Detection & Identification Review

CLS 423Clinical Immunohematology

Advanced Antibody Identification Module

Objectives

After viewing this presentation, the student will be able to:

• Apply appropriate exclusion/inclusion techniques to antibody identification

• Select cells to be tested in order to complete an antibody identification

• Prove within 95% confidence that the antibody identification is correct

• Select appropriate positive and negative controls for antigen typing

Vial D C c E e Cw K k Kpa

Jsa Fya

Fyb

Jka Jkb

Lea

Leb

P1 M N S S Lua

Xga

Pt

AHG

I + + 0 0 + 0 + + 0 0 + 0 + 0 0 + + + + + 0 0 0 3+

II + 0 + + 0 0 0 + 0 0 + + 0 + + 0 + + 0 0 + 0 + 0

III 0 0 + 0 + 0 0 + 0 0 0 + 0 + 0 + + 0 + 0 + 0 + 0

• Assume that testing was performed using the tube technique with LISS enhancement.– Immediate spin (IS) and 37oC phases showed no reactivity with all 3

screen cells.– Coombs Control Cells (CC) added to tubes II and III after the AHG

phase were 2+ positive.

Antibody Screen Results

How would you interpret the results of the antibody screen?

• Positive

• Negative

• Unable to interpret

(Click on one of the buttons to register your choice)

How would you interpret the results of the antibody screen?

• Positive

• Negative

• Unable to interpret

CORRECT!Click here to continue

How would you interpret the results of the antibody screen?

• Positive

• Negative

• Unable to interpret

Incorrect.

The antibody screen in this case is positive.

(3+ reaction with screen cell I)

What is the next appropriate step?

• Antigen type patient’s red blood cells• Perform a panel using patient’s plasma

or serum• Repeat the screen using prewarmed

patient’s serum and screen cells• No additional testing is necessary

(Click on one of the buttons above to register your choice)

No additional testing is necessary

Sorry…there is more work to be done to resolve this case.

Click here to review the other options

Antigen Type Patient’s RBCs

• This step is premature.• The antibodies should be identified first.• Which action would you like to take?

– Perform a panel– Repeat the screen using prewarmed

technique– No additional testing is necessary

(Click on one of the buttons above to register your choice)

Prewarm Technique

• This action is not appropriate in this case.

• There is no evidence of interference from a cold antibody.

• Which action would you like to take?– Antigen type patient’s red blood cells– Perform a panel– No additional testing is necessary

Perform a Panel

• This would be the most useful step to take next in resolving this case.

Initial Panel

Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 + 0 0 + + + 0 + 0 0 + + 3+

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

3 + 0 + + 0 0 0 + 0 0 0 + + + 0 + + 0 + 0 + 0 + + 2+

4 + 0 + 0 + 0 0 + 0 0 0 0 0 + 0 + 0 + + 0 0 0 + + 0 3+

5 0 + + 0 + 0 0 + 0 0 + + 0 + 0 + + + + + + 0 + 0 0 3+

6 0 0 + + + 0 0 + 0 0 0 + + + 0 + + + + 0 + 0 + + 2+

7 0 0 + 0 + 0 + + 0 0 + + + + + 0 0 + 0 + + 0 + + 3+

8 0 0 + 0 + 0 0 + 0 0 0 + + 0 0 + + 0 + + + 0 + + 3+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0 3+

10 0 0 + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + + 0 3+

11 + + 0 0 + 0 0 + 0 0 + 0 + + 0 + 0 0 + + + 0 + + 2+

PtCells

0 3+

“THE RULES”

• Exclude using only cells that failed to react with the serum (negative)

• Exclude an antibody when the cell has homozygous antigen expression

• Exceptions:– K, Kpa, Jsa, and Lua can be ruled out when they are

present on a cell with heterozygous or homozygous expression

– If anti-D is in the plasma, then C and E can be excluded using a cell with heterozygous expression

• If anti-D is not in the plasma, C and E must be ruled out using a cell with homozygous antigen expression

Find a cell with … Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 + 0 0 + + + 0 + 0 0 + + 3+

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

3 + 0 + + 0 0 0 + 0 0 0 + + + 0 + + 0 + 0 + 0 + + 2+

4 + 0 + 0 + 0 0 + 0 0 0 0 0 + 0 + 0 + + 0 0 0 + + 0 3+

5 0 + + 0 + 0 0 + 0 0 + + 0 + 0 + + + + + + 0 + 0 0 3+

6 0 0 + + + 0 0 + 0 0 0 + + + 0 + + + + 0 + 0 + + 2+

7 0 0 + 0 + 0 + + 0 0 + + + + + 0 0 + 0 + + 0 + + 3+

8 0 0 + 0 + 0 0 + 0 0 0 + + 0 0 + + 0 + + + 0 + + 3+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0 3+

10 0 0 + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + + 0 3+

11 + + 0 0 + 0 0 + 0 0 + 0 + + 0 + 0 0 + + + 0 + + 2+

PtCells

0 3+

•Homozygous expression of E

3

Find a cell with … Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 + 0 0 + + + 0 + 0 0 + + 3+

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

3 + 0 + + 0 0 0 + 0 0 0 + + + 0 + + 0 + 0 + 0 + + 2+

4 + 0 + 0 + 0 0 + 0 0 0 0 0 + 0 + 0 + + 0 0 0 + + 0 3+

5 0 + + 0 + 0 0 + 0 0 + + 0 + 0 + + + + + + 0 + 0 0 3+

6 0 0 + + + 0 0 + 0 0 0 + + + 0 + + + + 0 + 0 + + 2+

7 0 0 + 0 + 0 + + 0 0 + + + + + 0 0 + 0 + + 0 + + 3+

8 0 0 + 0 + 0 0 + 0 0 0 + + 0 0 + + 0 + + + 0 + + 3+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0 3+

10 0 0 + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + + 0 3+

11 + + 0 0 + 0 0 + 0 0 + 0 + + 0 + 0 0 + + + 0 + + 2+

PtCells

0 3+

• Heterozygous expression of M

2, 4, 5, 6, 9

Homozygous vs. Heterozygous

• Homozygous – only one allele of the pair is present on the cell C + c -

• Heterozygous – both alleles of the pair are present on the cell C+ c+

Initial Panel Results

Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 + 0 0 + + + 0 + 0 0 + + 3+

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

3 + 0 + + 0 0 0 + 0 0 0 + + + 0 + + 0 + 0 + 0 + + 2+

4 + 0 + 0 + 0 0 + 0 0 0 0 0 + 0 + 0 + + 0 0 0 + + 0 3+

5 0 + + 0 + 0 0 + 0 0 + + 0 + 0 + + + + + + 0 + 0 0 3+

6 0 0 + + + 0 0 + 0 0 0 + + + 0 + + + + 0 + 0 + + 2+

7 0 0 + 0 + 0 + + 0 0 + + + + + 0 0 + 0 + + 0 + + 3+

8 0 0 + 0 + 0 0 + 0 0 0 + + 0 0 + + 0 + + + 0 + + 3+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0 3+

10 0 0 + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + + 0 3+

11 + + 0 0 + 0 0 + 0 0 + 0 + + 0 + 0 0 + + + 0 + + 2+

PtCells

0 3+

Take a moment to determine what antibodies can be excluded based on the results of the initial panel.

Initial Panel Results

Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 + 0 0 + + + 0 + 0 0 + + 3+

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

3 + 0 + + 0 0 0 + 0 0 0 + + + 0 + + 0 + 0 + 0 + + 2+

4 + 0 + 0 + 0 0 + 0 0 0 0 0 + 0 + 0 + + 0 0 0 + + 0 3+

5 0 + + 0 + 0 0 + 0 0 + + 0 + 0 + + + + + + 0 + 0 0 3+

6 0 0 + + + 0 0 + 0 0 0 + + + 0 + + + + 0 + 0 + + 2+

7 0 0 + 0 + 0 + + 0 0 + + + + + 0 0 + 0 + + 0 + + 3+

8 0 0 + 0 + 0 0 + 0 0 0 + + 0 0 + + 0 + + + 0 + + 3+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0 3+

10 0 0 + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + + 0 3+

11 + + 0 0 + 0 0 + 0 0 + 0 + + 0 + 0 0 + + + 0 + + 2+

PtCells

0 3+

Initial Panel Results

• What antibodies are you unable to exclude?– C, E, Cw, K, Kpa, Jsa, Fyb, Jka, N, S, & Lua

• Does any single antibody explain all of the positive results?– NO

• What is the significance of the auto control result?– No autoantibodies are present

Can any additional antibodies be excluded based on the antibody screen results?

Vial D C c E e Cw K k Kpa

Jsa Fya

Fyb

Jka Jkb

Lea

Leb

P1 M N S S Lua

Xga

Pt

AHG

I + + 0 0 + 0 + + 0 0 + 0 + 0 0 + + + + + 0 0 0 3+

II + 0 + + 0 0 0 + 0 0 + + 0 + + 0 + + 0 0 + 0 + 0

III 0 0 + 0 + 0 0 + 0 0 0 + 0 + 0 + + 0 + 0 + 0 + 0

•If so, what are they?

E, Fyb, & N

C, Cw, K, Kpa, Jsa, Jka, S, & Lua

have not been excluded

• What is the next appropriate step to take in order to complete the antibody identification? – Antigen type the patient’s red blood cells– Fill the rule of 3 & 3 to provide 95% confidence in

the antibody identification– Test selected cells to eliminate or confirm the

presence of the antibodies that were not excluded from the initial panel

– Treat panel cells with enzymes and repeat testing

Antigen type the patient’s RBCs

• Antigen typing for this many antigens will be more time consuming than other steps that could be taken.

• Antigen typing for this many antigens is generally not cost effective.

• Anti-sera for typing some of these antigens (Cw, Kpa, Jsa, & Lua) may not be readily available

• What step would you like to take instead? – Fill the rule of 3 & 3 to provide 95% confidence in the

antibody identification– Test selected cells to eliminate or confirm the presence of

the antibodies that were not excluded from the initial panel– Treat panel cells with enzymes and repeat testing

Fill the rule of 3 & 3

• There are too many antibodies that have not been excluded to begin examining the probability that these reactions are caused by antibodies and not just by random chance.

• What step would you like to take instead?– Antigen type the patient’s red blood cells– Test selected cells to eliminate or confirm the

presence of the antibodies that were not excluded from the initial panel

– Treat panel cells with enzymes and repeat testing

Enzyme Panel

• C, Cw, K, Kpa, Jsa, Jka, S, & Lua have not been excluded– C, Cw, and Jka show stronger antigen expression when cells are

treated with enzymes– S is destroyed by enzymes– K, Kpa, Jsa, and Lua are unaffected by routine enzymes

• May not provide adequate separation of antibodies in this case

• There is a better choice for this case• What step would you like to take instead?

– Antigen type the patient’s red blood cells– Fill the rule of 3 & 3 to provide 95% confidence in the antibody

identification– Test selected cells to eliminate or confirm the presence of the

antibodies that were not excluded from the initial panel

Selected Cells

• This would be a useful step to take at this point

• C, Cw, K, Kpa, Jsa, Jka, S, & Lua have not been excluded

• All but Lua are clinically significant • Cw, Kpa, Jsa & Lua are low prevalence

antigens. It may be difficult to find cells that are antigen positive to use for exclusion

Selected Cells Cell #

D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga

AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 0 + 0 + + + 0 + 0 0 + +

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

3 + 0 + + 0 0 + + 0 0+

+ + 0 0 + + 0 + 0 + 0 + +

4 + 0 + 0 + 0 0 + 0 0 0 0 + 0 0 + 0 + + 0 0 0 + +

5 0 + + 0 + 0 0 + 0 0 0 + 0 + 0 + + + + + + 0 + 0

6 0 0 + + + 0 0 + + 0 0 + 0 + 0 + + 0 + + 0 0 + +

7 0 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 0 + + 0 + +

8 0 0 + 0 + 0 + + 0 0 0 + 0 + 0 + + + + 0 + 0 + +

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0

10 + + + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + +

11 + + 0 0 + 0 0 + 0 0 + + + + 0 + 0 0 + + + 0 + +

PtCells

These cells are available for use in a selected cell panel.Take a moment to decide which cells you would like to test.

The yellow highlighted cells would make a good selected cell panel for this case

Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 0 + 0 + + + 0 + 0 0 + +

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

3 + 0 + + 0 0 + + 0 0+

+ + 0 0 + + 0 + 0 + 0 + +

4 + 0 + 0 + 0 0 + 0 0 0 0 + 0 0 + 0 + + 0 0 0 + + 3+

5 0 + + 0 + 0 0 + 0 0 0 + 0 + 0 + + + + + + 0 + 0

6 0 0 + + + 0 0 + + 0 0 + 0 + 0 + + 0 + + 0 0 + + 0 2+

7 0 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 0 + + 0 + +

8 0 0 + 0 + 0 + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + 2+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0

10 + + + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + +

11 + + 0 0 + 0 0 + 0 0 + + + + 0 + 0 0 + + + 0 + +

PtCells

Other cells on this panel could be run, but would have more overlap in antigens. Additional cells may need to be tested as a result.

Selected Cell Panel

Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 0 + 0 + + + 0 + 0 0 + +

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

3 + 0 + + 0 0 + + 0 0+

+ + 0 0 + + 0 + 0 + 0 + +

4 + 0 + 0 + 0 0 + 0 0 0 0 + 0 0 + 0 + + 0 0 0 + + 3+

5 0 + + 0 + 0 0 + 0 0 0 + 0 + 0 + + + + + + 0 + 0

6 0 0 + + + 0 0 + + 0 0 + 0 + 0 + + 0 + + 0 0 + + 0 2+

7 0 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 0 + + 0 + +

8 0 0 + 0 + 0 + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + 2+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0

10 + + + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + +

11 + + 0 0 + 0 0 + 0 0 + + + + 0 + 0 0 + + + 0 + +

PtCells

Selected Cell Results

• What antibodies are present in this specimen?– Anti-K and anti- Jka

• Are there clinically significant antibodies that still cannot be excluded?– Anti- Jsa

– Anti-Lua is not excluded, but is generally considered insignificant

What to do when an antibody cannot be excluded?

• Type the patient for the antigen.– If the patient is antigen positive, he should not be able to make

the antibody.• If the antibody is to a low prevalence antigen (like the

anti-Jsa in this case), it may not be possible to exclude it with the available reagents.– In most cases, a compatible antiglobulin crossmatch is

considered “proof” that a unit of red blood cells would be safe to transfuse the patient (“crossmatch compatible” blood vs. antigen negative blood)

• If the unit was positive for the low prevalence antigen, and the patient had the antibody, the crossmatch should be incompatible.

– It may be necessary to send the specimen to a reference laboratory to prove or exclude an antibody to a low prevalence antigen.

List the phenotypes of cells that need to be tested in order to prove this ID

with 95% confidence?

• 3 cells that are K+ Jka – that react +• 3 cells that are K- Jka + that react +• 3 cells that are K- Jka – that react –• Has this been filled with the cells that have

already been tested?– NO

• What cells still need to be tested?– Need one more cell that is K+ Jka – that reacts +

What additional step should be taken to confirm this ID?

– Absorb out the anti-Jka using a Jka + K- cell to “unmask” the anti-K

– Antigen type the patient’s red blood cells– Fill the rule of 3 & 3 – Perform a DAT using the patient’s red blood

cells

(Click on one of the buttons above to register your choice)

Absorption

• Not necessary at this point, since the selected cell panel has already demonstrated these 2 specificities

• What other action would you take?– Antigen type the patient’s red blood cells– Fill the rule of 3 & 3 – Perform a DAT using the patient’s red blood

cells

(Click on one of the buttons above to register your choice)

Rule of 3 & 3

• This is the 95% confidence rule.• If there is a cell available, one more cell

that is K+ Jka – should be tested to fill this rule. We would expect this cell to react +.

• What other step should be performed?– Absorb out the anti-Jka using a Jka + K- cell to

“unmask” the anti-K– Antigen type the patient’s red blood cells– Perform a DAT using the patient’s red blood

cells

Perform DAT

• Not necessary in this case.

• Auto control would have been positive if the patient had a positive DAT.

• What other action would you take?– Absorb out the anti-Jka using a Jka + K- cell to

“unmask” the anti-K– Antigen type the patient’s red blood cells– Fill the rule of 3 & 3

Antigen Typing

• What would be the expected results of antigen typing?

• K…– Neg

• Jka …– Neg

Using this panel, select positive and negative controls for K antigen typing

Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 + 0 0 + + + 0 + 0 0 + + 3+

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

3 + 0 + + 0 0 0 + 0 0 0 + + + 0 + + 0 + 0 + 0 + + 2+

4 + 0 + 0 + 0 0 + 0 0 0 0 0 + 0 + 0 + + 0 0 0 + + 0 3+

5 0 + + 0 + 0 0 + 0 0 + + 0 + 0 + + + + + + 0 + 0 0 3+

6 0 0 + + + 0 0 + 0 0 0 + + + 0 + + + + 0 + 0 + + 2+

7 0 0 + 0 + 0 + + 0 0 + + + + + 0 0 + 0 + + 0 + + 3+

8 0 0 + 0 + 0 0 + 0 0 0 + + 0 0 + + 0 + + + 0 + + 3+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0 3+

10 0 0 + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + + 0 3+

11 + + 0 0 + 0 0 + 0 0 + 0 + + 0 + 0 0 + + + 0 + + 2+

PtCells

0 3+

K Controls

• Positive Control– Cell 2 or 7

• Negative Control– Any of the remaining cells

Using this panel, select positive and negative controls for Jka antigen typing

Cell # D C c E e Cw K k Kpa Jsa Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga AHG CC

1 + + 0 0 + 0 0 + 0 0 + 0 + 0 0 + + + 0 + 0 0 + + 3+

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

3 + 0 + + 0 0 0 + 0 0 0 + + + 0 + + 0 + 0 + 0 + + 2+

4 + 0 + 0 + 0 0 + 0 0 0 0 0 + 0 + 0 + + 0 0 0 + + 0 3+

5 0 + + 0 + 0 0 + 0 0 + + 0 + 0 + + + + + + 0 + 0 0 3+

6 0 0 + + + 0 0 + 0 0 0 + + + 0 + + + + 0 + 0 + + 2+

7 0 0 + 0 + 0 + + 0 0 + + + + + 0 0 + 0 + + 0 + + 3+

8 0 0 + 0 + 0 0 + 0 0 0 + + 0 0 + + 0 + + + 0 + + 3+

9 0 0 + + + 0 0 + 0 0 + + + 0 0 + + + + + + + + 0 3+

10 0 0 + 0 + 0 0 + 0 0 + 0 0 + + 0 + + 0 0 + 0 + + 0 3+

11 + + 0 0 + 0 0 + 0 0 + 0 + + 0 + 0 0 + + + 0 + + 2+

PtCells

0 3+

Jka Controls

• Positive– Cell 3, 6, 7, or 11– Must be heterozygous in order to detect the

weakest amount of antigen present

• Negative– Cell 2, 4, 5, or 10

How would you prepare packed RBC units for this patient?

• Antigen type units for K & Jka

• Perform an antiglobulin crossmatch

If the physician ordered 6 units of packed RBCs for this patient, how many units would need to be antigen typed in order to find compatible units?

6/(.33 x .91) = 19.98

# units ordered /(freq. Jka - x freq. K-)

Type 20 units to find 6 antigen negative units

The EndPlease complete the 8 Antibody

Identification – Advanced Methods case studies and the assigned TracQ

cases.