Blood Group Antibody Identification

Jim Perkins, M.D.NorthShore University Health System

Blood Banks

© 2006, James T. Perkins, all rights reserved

• Results which prompt ABID

• Initial identification tests

• Analyzing a panel (forming a hypothesis)

• Confirmatory tests:– “Rule out” cells

– “Rule in” cells

– Antigen typing

– Elution/adsorption

– Neutralization tests© ‘06, James T. Perkins

Standard test results:Normal Person

• Forward and Reverse ABO types match

• Antibody screen negative

• Crossmatch is negative with ABO

compatible RBCs

• (Direct Antiglobulin Test negative)© ‘06, James T. Perkins

Identify cause of abnormality when:

• Antibody screen is positive

• Forward and reverse ABO typing don’t match

• Crossmatch positive; negative antibody screen

• (Positive DAT)

© ‘06, James T. Perkins

Typical problem

<A <B A cells B cells >D Interpretation

0 0 + + + O Pos

IS 37o, 30’ AHG CC Interpretation

OI 0 0 0 2+Positive

OII 0 0 3+ NT© ‘06, James T. Perkins

Initial ABID Tests

• ABID panel– Same IAT technique as antibody screen

• DAT

• More specimen• Patient history

– What is the urgency of transfusion?– Has the patient been pregnant or transfused?– Is there an autoimmune disease?

© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

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

Antibody Identification panel

© ‘06, James T. Perkins

Analyzing a panel

“Crossout” all antigens NON-REACTIVE in “double dose”

Corresponding ANTIBODIES are “ruled out”

© ‘06, James T. Perkins

C+c+ (C/c)

Single dose C

C+c- (C/C)

Double dose C© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

Hypothesis

“The positive antibody screen was due to anti-E”

Anti-E explains all of the reactions.

(Note “dosage effect”)© ‘06, James T. Perkins

When is the hypothesis regarding the antibody specificity“Proved?”

1. 3 antigen positive cells are reactive

2. 3 antigen negative cells are non-reactive

3. Clinically significant and common antibodies are “ruled out”Rh (anti-D, -C, -c, -E, -e)Rh (anti-D, -C, -c, -E, -e) Kell (anti-K, -k)Kell (anti-K, -k)

Duffy (anti-FyDuffy (anti-Fyaa, -Fy, -Fybb)) Kidd (anti-JkKidd (anti-Jkaa, -Jk, -Jkbb))

MNSs (anti-M, -N, -S, -s)MNSs (anti-M, -N, -S, -s) Lewis (anti-LeLewis (anti-Leaa, -Le, -Leb)b)

P system (anti-P1)P system (anti-P1)

4. The patient LACKS the corresponding antigen© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

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

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

+ + 0 0 + + + + 0 0

Need to rule out anti-S

“Rule-out cells” are NON-reactive cells

© ‘06, James T. Perkins

Antigen TypingHypothesis: ALLO-anti-E

Patient CCPos

controlNeg

controlCC

Anti-E 0 2+ 4+ 0 2+

© ‘06, James T. Perkins

Immunohematologic diagnosis: alloanti-E

1. 3 E positive cells are reactive

2. 3 E negative cells are non-reactive

3. The following antibodies are “ruled out”Rh (anti-D, -C, -c, -e)Rh (anti-D, -C, -c, -e) Kell (anti-K, -k)Kell (anti-K, -k)Duffy (anti-FyDuffy (anti-Fyaa, -Fy, -Fybb)) Kidd (anti-JkKidd (anti-Jkaa, -Jk, -Jkbb))MNSs (anti-M, -N, -S, -s)MNSs (anti-M, -N, -S, -s) Lewis (anti-LeLewis (anti-Leaa, -Le, -Leb)b)

P system (anti-P1)P system (anti-P1)

4. The patient LACKS the E antigen

© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 4+

2 + + 0 0 + 0 + 0 + 4+

3 + 0 + + 0 0 + + 0 0

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 3+

7 0 0 + 0 + 0 + + + 0

8 0 0 + + + 0 + 0 + 0© ‘06, James T. Perkins

Hypothesis: anti-C plus anti-K

Need to “RULE IN” both antibodies

“Rule-in cells” are REACTIVE cells

D C c E e K k S s AHG

+ + 0 0 + 0 + 0 + 4+

+ + 0 0 + 0 + 0 + 4+

0 0 + 0 + + + 0 + 3+

© ‘06, James T. Perkins

Antigen TypingHypothesis: ALLO-anti-C and -anti-K

Patient CCPos

controlNeg

controlCC

Anti-C 0 2+ 4+ 0 2+

Anti-K 0 2+ 4+ 0 2+

© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 4+

2 + + 0 0 + 0 + 0 + 4+

3 + 0 + + 0 + + + 0 0

4 + 0 + + 0 0 + 0 + 0

5 0 0 + 0 + 0 + 0 + 4+

6 0 0 + 0 + + + 0 + 4+

7 0 0 + 0 + 0 + + + 4+

8 0 0 + + + 0 + 0 + 4+© ‘06, James T. Perkins

Antigen TypingHypothesis: ALLO-anti-e

Can’t rule out: anti-C

Patient CCPos

controlNeg

controlCC

Anti-C 3+ NT 4+ 0 2+

Anti-e 0 2+ 4+ 0 2+

Conclusion: anti-C ruled out© ‘06, James T. Perkins

What if the DAT was positive?

Detects: In vivo sensitization by antibody or complement

Application: Autoimmune Hemolytic Anemia

Hemolytic Transfusion Reaction

Hemolytic Disease of Newborn

Drug/RBC Antibodies

Passive Immunization (esp. plts.)

© ‘06, James T. Perkins

Consider the following test results:

• Group O, Rh positive

• Antibody screen positive

• Antibody identification; serum anti-e

• Direct antiglobulin test positive

• The patient HAS the e antigen (not

transfused)

© ‘06, James T. Perkins

Eluate

Poly Anti-IgG Anti-C3

+ + O

DAT

e+

e-

e-

e+

e+

Conclusion: Auto-anti-e © ‘06, James T. Perkins

Autoanti-e in serum and eluate.

D C c E e K k S s Serum Eluate

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

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

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

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

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

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

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

0 0 + + + 0 + 0 + 2+ 3+© ‘06, James T. Perkins

D C c E e K k S s Serum Eluate

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

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

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

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

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

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

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

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

Most Autoantibodies are “Pan-agglutinins”

© ‘06, James T. Perkins

D C c E e K k S sAuto-

abAnti-

K

+ + 0 0 + + + 0 + 4+ 1+

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

+ 0 + + 0 + + + 0 4+ 1+

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

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

0 0 + 0 + + + 0 + 4+ 1+

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

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

Auto-antibody can “mask” allo-antibody

© ‘06, James T. Perkins

Auto-antibody + Allo-antibody

© ‘06, James T. Perkins

Separate serum and RBCs

© ‘06, James T. Perkins

Remove auto-antibody from the RBCs

ZZAP: DTT

Papain

PBS

Chloroquine© ‘06, James T. Perkins

Return serum to the RBCs

© ‘06, James T. Perkins

Incubate

© ‘06, James T. Perkins

Separate again

Adsorbed serum

© ‘06, James T. Perkins

D C c E e K k S sRaw

Serum

Ads

serum

+ + 0 0 + + + 0 + 2+ 1+

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

+ 0 + + 0 + + + 0 2+ 1+

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

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

0 0 + 0 + + + 0 + 2+ 1+

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

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

Autoadsorbed serum may reveal an “underlying” ab

© ‘06, James T. Perkins

+

Neutralizable Antibody

© ‘06, James T. Perkins

+

incubate

+

Neutralization Test

© ‘06, James T. Perkins

D C c E e K k Lea Leb Raw serum

HPC

neut.

+ + 0 0 + + + 0 + 0 0

+ + 0 0 + 0 + + 0 0 0

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

0 0 + + + 0 + 0 + 0 0

Neutralization may demonstrate allo-ab specificity

© ‘06, James T. Perkins

D C c E e K k Lea Leb Raw serum

Neut. serum

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

+ + 0 0 + 0 + + 0 0 0

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

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

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

0 0 + 0 + + + 0 0 0 0

0 0 + 0 + 0 + + 0 0 0

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

Neutralized serum may reveal an “underlying” ab

© ‘06, James T. Perkins

ABO discrepancy

<A <B A cells B cells >D Interpretation

4+ 0 1+ 4+ 3+ ?

IS 37o, 30’ AHG CC Interpretation

OI 1+ W+ 0 2+ Positive

(? ab)OI 0 0 0 2+© ‘06, James T. Perkins

D C c E e K k S s AHG

1 + + 0 0 + + + 0 + 0

2 + + 0 0 + 0 + 0 + 0

3 + 0 + + 0 + + + 0 0

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

5 0 0 + 0 + 0 + 0 + 0

6 0 0 + 0 + + + 0 + 0

7 0 0 + 0 + 0 + + 0 0

8 0 0 + + + 0 + 0 + 0

Antibody against a low-frequency antigen

© ‘06, James T. Perkins

D C c E e K k S s AHG

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

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

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

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

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

6 0 0 + 0 + + + 0 + 3+

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

AC (Patient cells + patient serum) 0

Antibody against a high-frequency antigen

© ‘06, James T. Perkins

Key question:

“Does my answer explain all of the reactions?”

© ‘06, James T. Perkins