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