Antibody Exclusion
“Crossing-Out”
Interpreting Panel Results
Shawn T. Leonard MT(ASCP)
Blood Centers of the Pacific
Antibody Identification
Involves YOU as the
Expert!
• Performing selected tests
• Determining the strength & pattern of agglutination
• Interpreting results
• Selecting suitable blood for the patient
Objectives
• 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
“Antibody Exclusion” –
Immunohematology
definition
Systematic process by which panel
results are interpreted to eliminate
antibody specificities on the basis of nonreactivity of patient plasma with red cell
samples that express the antigen.
-AABB Technical Manual 16th edition
“Antibody Exclusion” –
Immunohematology definition
Commonly known as…
Crossing-Out
Advantages of Crossing-Out
1. Allows accurate ID of antibodies
2. Eliminates additional antibody specificities
3. Provides higher predictive value
4. Supports initial hypothesis
5. Determines additional testing
6. Decreases delay in patient care
Crossing Out
•
•
•
•
A first approach
May not lead directly to an answer
A provisional step
Converging evidence ???
Helpful Information
•
•
•
•
Patient’s phenotype
Ethnicity of the patient
Ethnicity of panel cell donor
Age of the panel cell
Recommended rules for
Crossing-Out
Cross-Out protocol varies by institution
What about Ruling-In?
To increase the probability of correct
identification a p-value of <0.05 is observed
which gives a 95% confidence interval.
AABB IRL Standards
“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.”
Standards for Immunohematology Reference Laboratories-6th Edition
Recommended rules
for Crossing-Out
Rule #1
It is best to rule out an antibody specificity on
a cell with a double-dose expression
• Homozygous alleles
-
Jka/Jka
(Double-Dose)
-
Jka/Jkb
(Single-Dose)
express Jk(a+b-)
• Heterozygous alleles
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 #2
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 #3
“ 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 #4
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
• Neutralization
P1 & Lewis
When All Else Fails
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.
Example Panels
On the following example panels, try
crossing out each blood group antigen with at
least one homozygous representation.
(Panel A)
Example #1
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E
L
L
G
E
L
IAT
1 + + 0
0
+
0
+
+
+
+
0
0
+
0 +
+
0
+
1
4+
2 + + 0
0
+
+
+
0
+
0
+
+
0
+ +
+
+
+
2
4+
3 + 0 +
+
0
0
+
0
+
+
0
0
+
0 +
+
+
+
3
0
4 + 0
0
+
+
0
+
0
0
+
0
0
0
+ 0
+
0
+
4
0
5 0 + 0
+
+
0
+
0
+
+
0
+
0 +s 0
+
0
+
5
4+
6 0 0 +
+
+
0
+
+
0
0
+
0
+
+ +
+
+
+
6
0
7 0 0
0
+
+
+
+
0
+
+
+
0
+
+ +
+
+
+
7
0
8 0 0
0
+
+
0
+
0
+
+
+
0
+
+ +
0
+
0
8
0
9 0 0
0
+
+
0
+
+
0
+
0
+
0
+ +
+
0
+
9
0
10 0 0
0
+
+
0
+
+
+
0
+
0
+
+ 0
+
0
+ 10
0
11 + + 0
0
+
0
+
0
+
+
0
0
0
+ +
+
+
0 11
4+
AC
AC
0
Example #2
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
(Panel A)
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E
L
L
G
E
L
IAT
1 + + 0
0
+
0
+
+
+
+
0
0
+
0 +
+
0
+
1
0
2 + + 0
0
+
+
+
0
+
0
+
+
0
+ +
+
+
+
2
3+
3 + 0 +
+
0
0
+
0
+
+
0
0
+
0 +
+
+
+
3
3+
4 + 0
0
+
+
0
+
0
0
+
0
0
0
+ 0
+
0
+
4
0
5 0 + 0
+
+
0
+
0
+
+
0
+
0 +S 0
+
0
+
5
0
6 0 0 +
+
+
0
+
+
0
0
+
0
+
+ +
+
+
+
6
3+
7 0 0
0
+
+
+
+
0
+
+
+
0
+
+ +
+
+
+
7
3+
8 0 0
0
+
+
0
+
0
+
+
+
0
+
+ +
0
+
0
8
3+
9 0 0
0
+
+
0
+
+
0
+
0
+
0
+ +
+
0
+
9
0
10 0 0
0
+
+
0
+
+
+
0
+
0
+
+ 0
+
0
+ 10
0
11 + + 0
0
+
0
+
0
+
+
0
0
0
+ +
+
+
0 11
3+
AC
AC
0
(Panel B)
Example #2
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E
L
L
G
E
L
IAT
12 0 0
0
+
+
0
+
0
+
+
0
0
+
+ +
+
+
0
1
3+
13 0 0
0
+
+
+
+
+
+
0
+
0
+ +S +
0
0
+
2
0
14 0 0
0
+
+
0
+
+
0
0
+
0
+
+ +
+
0
+
3
0
15 + 0 +
+
0
0
+
+
0
+
+
+
0 +S +
+
0
+
4
2+
16 + 0 +
+
0
0
+
+
+
0
+
0
+
0 +
+
+
+
5
3+
17 + 0 +
+
0
0
+
0
+
+
0
+
0
+ +
0
+
+
6
3+
18 + + 0
0
+
0
+
0
+
+
0
+
0
0 0
+
0
+
7
0
19 + + 0
0
+
0
+
+
0
0
+
0
+
+ +
0
+
0
8
3+
20 + + +
0
+
0
+
+
0
+
+
0
+ +S +
+
0
+
9
2+
21 0 + 0
+
+
0
+
0
+
+
+
0
+
+ +
0
0
+ 10
0
22 + + 0
0
+
+
0
0
+
+
0
0
+
+ +
+
+
+ 11
3+
AC
AC
0
(Panel A)
Example #3
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E
L
L
G
E
L
IAT
1 + + 0
0
+
0
+
+
+
+
0
0
+
0 +
+
0
+
1
4+
2 + + 0
0
+
+
+
0
+
0
+
+
0
+ +
+
+
+
2
4+
3 + 0 +
+
0
0
+
0
+
+
0
0
+
0 +
+
+
+
3
4+
4 + 0
0
+
+
0
+
0
0
+
0
0
0
+ 0
+
0
+
4
4+
5 0 + 0
+
+
0
+
0
+
+
0
+
0 +s 0
+
0
+
5
0
6 0 0 +
+
+
0
+
+
0
0
+
0
+
+ +
+
+
+
6
0
7 0 0
0
+
+
+
+
0
+
+
+
0
+
+ +
+
+
+
7
0
8 0 0
0
+
+
0
+
0
+
+
+
0
+
+ +
0
+
0
8
0
9 0 0
0
+
+
0
+
+
0
+
0
+
0
+ +
+
0
+
9
0
10 0 0
0
+
+
0
+
+
+
0
+
0
+
+ 0
+
0
+ 10
0
11 + + 0
0
+
0
+
0
+
+
0
0
0
+ +
+
+
0 11
4+
AC
AC
0
Example #4
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E LISS LISS
L 37o IAT
L
1 + + +
0
+
0
+
+
0
+
0
0
+
+ +
+
+
0
1
0
0
2 + + 0
0
+
0
+
0
+
0
+
0
+
+ +
0
0
+
2
1+
1+
3 + 0 +
+
0
0
+
0
+
0
+
0
+
0 0
+
+
0
3
0
0
4 + 0
0
+
+
0
+
0
0
+
+
0
+
+ +
+
+
0
4
0
0
5 0 + 0
+
+
0
+
+
0
0
+
0
+
+ +
0
+
0
5
1+
1+
6 0 0 +
+
+
0
+
0
+
+
+
0
+
+ +
+
0
+
6
0
0
7 0 0
0
+
+
+
+
0
+
0
+
0
+
+ +
0
+
+
7
1+
1+
8 0 0
0
+
+
0
+
+
0
+
0
+
0
0 0
+
+
+
8
0
0
9 0 0
0
+
+
0
+
+
0
+
+
+
0
0 +
0
0
+
9
1+
1+
10 + + 0
0
+
+
+
0
+
+
0
0
+
+ +
+
+
0 10
0
0
11 0 0
+
+
0
+
0
+
+
0
0
+
0 0
+
+
0 11
0
0
AC
0
0
AC
0
Example #5
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E
L
L
LISS
IAT
1 + + +
0
+
0
+
+
+
+
0
+
0
+ +
+
0
+
1
w+
2 + + 0
0
+
+
+
0
+
+
+
0
0
0 0
+
0
+
2
0
3 + 0 +
+
0
0
+
0
+
+
0
0
0
+ 0
+
+
+
3
0
4 + 0
0
+
+
0
+
+
0
+
+
0
+
+ 0
+
0
0
4
1+
5 0 + 0
+
+
0
+
+
+
0
+
0
+
+ +
+
0
+
5
w+
6 0 0 +
+
+
0
+
+
0
0
+
0
+
+ +
+
0
+
6
0
7 0 0
0
+
+
+
+
0
+
0
+
+
0
0 +
0
+
0
7
0
8 0 0
0
+
+
0
+
+
0
0
+
0
0
+ +
0
+
0
8
w+
9 0 0
0
+
+
0
+
0
0
+
+
0
+ +S 0
+
0
+
9
2+
10 + + 0
0
+
+
0
+
+
0
+
0
+
+ +
0
+
+ 10
0
11 + + 0
0
+
0
+
+
0
+
+
+
0
0 +
+
0
+ 11
0
AC
0
AC
Example #5
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
(RT & P1 Neut.)
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E
L
L
P1
RT Neut.
15’ RT
15’
1 + + +
0
+
0
+
+
+
+
0
+
0
+ +
+
0
+
1
1+
0
2 + + 0
0
+
+
+
0
+
+
+
0
0
0 0
+
0
+
2
0
0
3 + 0 +
+
0
0
+
0
+
+
0
0
0
+ 0
+
+
+
3
1+
0
4 + 0
0
+
+
0
+
+
0
+
+
0
+
+ 0
+
0
0
4
1+
0
5 0 + 0
+
+
0
+
+
+
0
+
0
+
+ +
+
0
+
5
1+
0
6 0 0 +
+
+
0
+
+
0
0
+
0
+
+ +
+
0
+
6
1+
0
7 0 0
0
+
+
+
+
0
+
0
+
+
0
0 +
0
+
0
7
0
0
8 0 0
0
+
+
0
+
+
0
0
+
0
0
+ +
0
+
0
8
1+
0
9 0 0
0
+
+
0
+
0
0
+
+
0
+ +S 0
+
0
+
9
2+
0
10 + + 0
0
+
+
0
+
+
0
+
0
+
+ +
0
+
+ 10 1+
0
11 + + 0
0
+
0
+
+
0
+
+
+
0
0 +
+
0
+ 11
0
AC
AC
0
Example #6
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E LISS LISS
L 37o IAT
L
1 + + +
0
+
0
+
0
+
0
+
0
+
+ +
0
+
+
1
1+
2+
2 + + 0
0
+
0
+
0
+
+
0
0
+
0 0
+
0
+
2
0
2+
3 + 0 +
+
0
0
+
0
+
0
+
0
+
+ 0
+
0
+
3
1+
2+
4 + 0
0
+
+
0
+
+
0
+
+
0
+
+ +
+
0
+
4
0
0
5 0 + 0
+
+
0
+
+
+
+
0
0
+
+ +
0
+
+
5
0
2+
6 0 0 +
+
+
0
+
+
0
0
+
0
+
+ +
+
0
+
6
1+
2+
7 0 0
0
+
+
+
+
0
+
+
+
+
0
+ +
+
+
+
7
0
0
8 0 0
0
+
+
0
+
+
0
+
+
+
0
0 0
+
+
+
8
0
0
9 0 0
0
+
+
0
+
+
0
+
0
+
0
+ +
+
+
+
9
0
2+
10 + + 0
0
+
+
+
+
+
+
0
0
+
+ 0
+
0
+ 10
0
2+
11 + + 0
0
+
0
+
+
0
0
+
0
+
0 +
0
+
0 11
0
0
AC
0
0
AC
Example #7
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
C
E
L
L
LISS
IAT
1 + + 0
0
+
+
+
+
+
+
0
+
0
+ 0
+
+
0
1
3+
2 + + 0
0
+
0
+
0
+
0
+
0
+
+ 0
+
+
+
2
2+
3 + 0 +
+
0
0
+
0
0
0
+
0
+
0 +
0
+
+
3
2+
4 + 0
0
+
+
0
+
0
0
+
0
0
+
+ +
+
0
0
4
0
5 0 + 0
+
+
+
+
+
0
+
0
0
+
0 0
+
+
0
5
2+
6 0 0 +
+
+
0
+
0
+
+
+
0
+
+ 0
+
+
+
6
4+
7 0 0
0
+
+
+
+
+
0
+
+
+
0
+ +
+
0
+
7
2+
8 0 0
0
+
+
0
+
+
0
0
+
0
+
+ +
0
+
+
8
0
9 0 0
0
+
+
0
+
0
+
+
+
0
+
+ +
0
+
0
9
2+
10 + + +
+
0
0
+
0
+
+
+
0
0
+ 0
+
0
+ 10
4+
11 + + 0
0
+
0
+
0
0
+
0
+
0
+ 0
+
0
+ 11
0
AC
0
AC
Example #7
C
RH
E
L
D C E c
L
Kell
Duffy
Kidd
Lewis P
(Chemicals)
MNS
e K k Fya Fyb Jka Jkb Lea Leb P1 M N S
s
L
I
S
S
IAT
IAT IAT
E
N
Z
D
T
T
1 + + 0
0
+
+
+
+
+
+
0
+
0
+ 0
+
+
0
3+
2+ 2+
2 + + 0
0
+
0
+
0
+
0
+
0
+
+ 0
+
+
+
2+
0
3 + 0 +
+
0
0
+
0
0
0
+
0
+
0 +
0
+
+
2+
3+ 2+
4 + 0
0
+
+
0
+
0
0
+
0
0
+
+ +
+
0
0
0
0
0
5 0 + 0
+
+
+
+
+
0
+
0
0
+
0 0
+
+
0
2+
2+
0
6 0 0 +
+
+
0
+
0
+
+
+
0
+
+ 0
+
+
+
4+
3+ 4+
7 0 0
0
+
+
+
+
+
0
+
+
+
0
+ +
+
0
+
2+
2+
0
8 0 0
0
+
+
0
+
+
0
0
+
0
+
+ +
0
+
+
0
0
0
9 0 0
0
+
+
0
+
0
+
+
+
0
+
+ +
0
+
0
2+
0
2+
10 + + +
+
0
0
+
0
+
+
+
0
0
+ 0
+
0
+
4+
3+ 4+
11 + + 0
0
+
0
+
0
0
+
0
+
0
+ 0
+
0
+
0
0
AC
0
2+
0