Shawn T. Leonard MT(ASCP)Blood Centers of the Pacific

Antibody Exclusion“Crossing-Out”

Interpreting Panel Results

•Performing selected tests

•Determining the strength & pattern of agglutination

•Interpreting results

•Selecting suitable blood for the patient

Antibody Identification Antibody Identification Involves YOU as the

Expert!

•Define Antibody Exclusion

•List advantages of crossing out in Ab Identification

•Describe limitations of the cross-out process

•Identify helpful patient and reagent information

•Discuss AABB Standards on assigning Ab specificity

•Recognize homozygous vs. heterozygous expression

•Apply recommended rules for crossing out

Objectives

Systematic process by which panel results are interpreted to eliminate antibody specificities on the basis of non-reactivity of patient plasma with red cell samples that express the antigen.

-AABB Technical Manual 16th edition

““Antibody Exclusion” – Antibody Exclusion” – Immunohematology Immunohematology

definitiondefinition

Commonly known as…

““Antibody Exclusion” – Antibody Exclusion” – Immunohematology definitionImmunohematology definition

Crossing-OutCrossing-Out

1.1. Allows accurate ID of antibodies Allows accurate ID of antibodies 2.2. Eliminates additional antibody specificitiesEliminates additional antibody specificities3.3. Provides higher predictive valueProvides higher predictive value4.4. Supports initial hypothesis Supports initial hypothesis 5.5. Determines additional testingDetermines additional testing6.6. Decreases delay in patient careDecreases delay in patient care

Advantages of Crossing-Out

• A first approach

• May not lead directly to an answer

• A provisional step

• Converging evidence ???

Crossing Out

• Patient’s phenotype

• Ethnicity of the patient

• Ethnicity of panel cell donor

• Age of the panel cell

Helpful Information

Cross-Out protocol varies by institution

Recommended rules for Crossing-Out

To increase the probability of correct identification a p-value of <0.05 is observed

which gives a 95% confidence interval.

What about Ruling-In?

Standards for Immunohematology Reference Laboratories-6th Edition

“Assign specificity after demonstrating reactivity with at least two antigen-positive reagent red cell samples and non-reactivity with at least two antigen-negative reagent red cell samples.”

AABB IRL Standards

Recommended rules for Crossing-Out

It is best to rule out an antibody specificity on a cell with a double-dose expression

• Homozygous alleles - Jka/Jka (Double-Dose)

express Jk(a+b-)

• Heterozygous alleles - Jka/Jkb (Single-Dose)

express Jk(a+b+)

Rule #1

It is best to rule out an antibody specificity on a cell with a double-dose expression

Exceptions…

• K1 and P1

• C or E with Anti-D

Rule #1

It is better to rule out specificities with two unique cells rather than one

By chance, one may not react as well as expected due to:

•Variant antigen structure•Lack of antigen recognition by antibody•Weakened antigen expression•Antigen degradation•Tech error

Rule #2

“ Reverse of Rule-out ”- for the antibody that shows extreme variability or reacts in more than one phase of testing

•Anti-P1

•Anti-Lea

•Anti-Leb

Rule #3

Antibody Exclusion alone does not define which antibodies are or are not present

Additional Techniques :

• Selected Cells• Chemical Treatment

Proteolytic Enzymes - Papain, Ficin, Bromelin

Sulfhydryl Reagents - DTT & AET• NeutralizationP1 & Lewis

Rule #4

If antibodies to common blood group antigens cannot be ruled out, it may be necessary to consult a Blood Bank Reference Laboratory or provide antigen negative units.

When All Else FailsWhen All Else Fails

On the following example panels, try crossing out each blood group antigen with at least one homozygous representation.

Example PanelsExample Panels

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

GG

EE

LL

IATIAT

DD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

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

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

3 + 0 + + 0 0 + 0 + + 0 0 + 0 + + + + 3 00

4 + 0 0 + + 0 + 0 0 + 0 0 0 + 0 + 0 + 4 00

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

6 0 0 + + + 0 + + 0 0 + 0 + + + + + + 6 00

7 0 0 0 + + + + 0 + + + 0 + + + + + + 7 00

8 0 0 0 + + 0 + 0 + + + 0 + + + 0 + 0 8 00

9 0 0 0 + + 0 + + 0 + 0 + 0 + + + 0 + 9 00

10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 10 00

11 + + 0 0 + 0 + 0 + + 0 0 0 + + + + 0 11 4+4+

AC AC 00

(Panel A)Example #1

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

GG

EE

LL

IATIAT

DD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

1 + + 0 0 + 0 + + + + 0 0 + 0 + + 0 + 1 00

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

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

4 + 0 0 + + 0 + 0 0 + 0 0 0 + 0 + 0 + 4 00

5 0 + 0 + + 0 + 0 + + 0 + 0 +S 0 + 0 + 5 00

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

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

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

9 0 0 0 + + 0 + + 0 + 0 + 0 + + + 0 + 9 00

10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 10 00

11 + + 0 0 + 0 + 0 + + 0 0 0 + + + + 0 11 3+3+

AC AC 00

(Panel A)Example #2

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

GG

EE

LL

IATIAT

DD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

12 0 0 0 + + 0 + 0 + + 0 0 + + + + + 0 1 3+3+

13 0 0 0 + + + + + + 0 + 0 + +S + 0 0 + 2 00

14 0 0 0 + + 0 + + 0 0 + 0 + + + + 0 + 3 00

15 + 0 + + 0 0 + + 0 + + + 0 +S + + 0 + 4 2+2+

16 + 0 + + 0 0 + + + 0 + 0 + 0 + + + + 5 3+3+

17 + 0 + + 0 0 + 0 + + 0 + 0 + + 0 + + 6 3+3+

18 + + 0 0 + 0 + 0 + + 0 + 0 0 0 + 0 + 7 00

19 + + 0 0 + 0 + + 0 0 + 0 + + + 0 + 0 8 3+3+

20 + + + 0 + 0 + + 0 + + 0 + +S + + 0 + 9 2+2+

21 0 + 0 + + 0 + 0 + + + 0 + + + 0 0 + 10 00

22 + + 0 0 + + 0 0 + + 0 0 + + + + + + 11 3+3+

AC AC 00

(Panel B)Example #2

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

GG

EE

LL

IATIAT

DD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

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

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

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

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

5 0 + 0 + + 0 + 0 + + 0 + 0 +s 0 + 0 + 5 00

6 0 0 + + + 0 + + 0 0 + 0 + + + + + + 6 00

7 0 0 0 + + + + 0 + + + 0 + + + + + + 7 00

8 0 0 0 + + 0 + 0 + + + 0 + + + 0 + 0 8 00

9 0 0 0 + + 0 + + 0 + 0 + 0 + + + 0 + 9 00

10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 10 00

11 + + 0 0 + 0 + 0 + + 0 0 0 + + + + 0 11 4+4+

AC AC 00

(Panel A)Example #3

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

LISSLISS

3737oo

LISSLISS

IATIATDD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

1 + + + 0 + 0 + + 0 + 0 0 + + + + + 0 1 00 00

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

3 + 0 + + 0 0 + 0 + 0 + 0 + 0 0 + + 0 3 00 00

4 + 0 0 + + 0 + 0 0 + + 0 + + + + + 0 4 00 00

5 0 + 0 + + 0 + + 0 0 + 0 + + + 0 + 0 5 1+1+ 1+1+

6 0 0 + + + 0 + 0 + + + 0 + + + + 0 + 6 00 00

7 0 0 0 + + + + 0 + 0 + 0 + + + 0 + + 7 1+1+ 1+1+

8 0 0 0 + + 0 + + 0 + 0 + 0 0 0 + + + 8 00 00

9 0 0 0 + + 0 + + 0 + + + 0 0 + 0 0 + 9 1+1+ 1+1+

10 + + 0 0 + + + 0 + + 0 0 + + + + + 0 10 00 00

11 0 0 0 + + 0 + 0 + + 0 0 + 0 0 + + 0 11 00 00

AC AC 00 00

Example #4

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

LISSLISS

IATIATDD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

1 + + + 0 + 0 + + + + 0 + 0 + + + 0 + 1 w+w+

2 + + 0 0 + + + 0 + + + 0 0 0 0 + 0 + 2 00

3 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + + + 3 00

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

5 0 + 0 + + 0 + + + 0 + 0 + + + + 0 + 5 w+w+

6 0 0 + + + 0 + + 0 0 + 0 + + + + 0 + 6 00

7 0 0 0 + + + + 0 + 0 + + 0 0 + 0 + 0 7 00

8 0 0 0 + + 0 + + 0 0 + 0 0 + + 0 + 0 8 w+w+

9 0 0 0 + + 0 + 0 0 + + 0 + +S 0 + 0 + 9 2+2+

10 + + 0 0 + + 0 + + 0 + 0 + + + 0 + + 10 00

11 + + 0 0 + 0 + + 0 + + + 0 0 + + 0 + 11 00

AC AC 00

Example #5

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

RTRT

15’15’

PP11

Neut.Neut.

RTRT

15’15’DD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

1 + + + 0 + 0 + + + + 0 + 0 + + + 0 + 1 1+1+ 00

2 + + 0 0 + + + 0 + + + 0 0 0 0 + 0 + 2 00 00

3 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + + + 3 1+1+ 00

4 + 0 0 + + 0 + + 0 + + 0 + + 0 + 0 0 4 1+1+ 00

5 0 + 0 + + 0 + + + 0 + 0 + + + + 0 + 5 1+1+ 00

6 0 0 + + + 0 + + 0 0 + 0 + + + + 0 + 6 1+1+ 00

7 0 0 0 + + + + 0 + 0 + + 0 0 + 0 + 0 7 00 00

8 0 0 0 + + 0 + + 0 0 + 0 0 + + 0 + 0 8 1+1+ 00

9 0 0 0 + + 0 + 0 0 + + 0 + +S 0 + 0 + 9 2+2+ 00

10 + + 0 0 + + 0 + + 0 + 0 + + + 0 + + 10 1+1+ 00

11 + + 0 0 + 0 + + 0 + + + 0 0 + + 0 + 11 00 00

AC AC

(RT & P1 Neut.)Example #5

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

LISSLISS

3737oo

LISSLISS

IATIATDD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

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

2 + + 0 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 2 00 2+2+

3 + 0 + + 0 0 + 0 + 0 + 0 + + 0 + 0 + 3 1+1+ 2+2+

4 + 0 0 + + 0 + + 0 + + 0 + + + + 0 + 4 00 00

5 0 + 0 + + 0 + + + + 0 0 + + + 0 + + 5 00 2+2+

6 0 0 + + + 0 + + 0 0 + 0 + + + + 0 + 6 1+1+ 2+2+

7 0 0 0 + + + + 0 + + + + 0 + + + + + 7 00 00

8 0 0 0 + + 0 + + 0 + + + 0 0 0 + + + 8 00 00

9 0 0 0 + + 0 + + 0 + 0 + 0 + + + + + 9 00 2+2+

10 + + 0 0 + + + + + + 0 0 + + 0 + 0 + 10 00 2+2+

11 + + 0 0 + 0 + + 0 0 + 0 + 0 + 0 + 0 11 00 00

AC AC 00 00

Example #6

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSCC

EE

LL

LL

LISSLISS

IATIATDD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

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

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

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

4 + 0 0 + + 0 + 0 0 + 0 0 + + + + 0 0 4 00

5 0 + 0 + + + + + 0 + 0 0 + 0 0 + + 0 5 2+2+

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

7 0 0 0 + + + + + 0 + + + 0 + + + 0 + 7 2+2+

8 0 0 0 + + 0 + + 0 0 + 0 + + + 0 + + 8 00

9 0 0 0 + + 0 + 0 + + + 0 + + + 0 + 0 9 2+2+

10 + + + + 0 0 + 0 + + + 0 0 + 0 + 0 + 10 4+4+

11 + + 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 11 00

AC AC 00

Example #7

CC

EE

LL

LL

RHRH KellKell DuffyDuffy KiddKidd LewisLewis PP MNSMNSLL

II

SS

SS

IATIAT

EE

NN

ZZ

IATIAT

DD

TT

TTIATIAT

DD CC EE cc ee KK kk FyFyaa FyFybb JkJkaa JkJkbb LeLeaa LeLebb PP11 MM NN SS ss

1 + + 0 0 + + + + + + 0 + 0 + 0 + + 0 3+3+ 2+2+ 2+2+

2 + + 0 0 + 0 + 0 + 0 + 0 + + 0 + + + 2+2+ 00 2+2+

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

4 + 0 0 + + 0 + 0 0 + 0 0 + + + + 0 0 00 00 00

5 0 + 0 + + + + + 0 + 0 0 + 0 0 + + 0 2+2+ 2+2+ 00

6 0 0 + + + 0 + 0 + + + 0 + + 0 + + + 4+4+ 3+3+ 4+4+

7 0 0 0 + + + + + 0 + + + 0 + + + 0 + 2+2+ 2+2+ 00

8 0 0 0 + + 0 + + 0 0 + 0 + + + 0 + + 00 00 00

9 0 0 0 + + 0 + 0 + + + 0 + + + 0 + 0 2+2+ 00 2+2+

10 + + + + 0 0 + 0 + + + 0 0 + 0 + 0 + 4+4+ 3+3+ 4+4+

11 + + 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 00 00 00

AC 00

(Chemicals)Example #7