Antibody Identification Part 1

Part 1: The Basics

• Prerequisites
A Blood Bank Guy Video Podcast • Geography of a panel
• Antibody ID method
• Case examples
D. Joe Chaffin, MD
March 2012

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Tuesday, March 6, 12 1 Tuesday, March 6, 12 2

Prerequisites When Do We I.D.?

• Blood Group Overview • Following a positive antibody screen
• General facts • Patients, prenatals, donors
• Podcast from December 2011 • When testing suggests a new antibody
• Pretransfusion Testing • To confirm a previously identified
• Testing methodologies antibody (per facility SOP)
• Antibody screen
• Podcast from February 2012
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 Definitions What’s a Panel?
• Alloantibody • Just an expanded antibody screen
• Antibody against RBC antigens • Uses group O reagent RBCs
not present on patient’s own RBCs • RBCs from 8-20 donors
• Autoantibody • Patient serum or plasma
• Antibody against RBC antigens • IS / 37 C / AHG if tubes
present on patient’s own RBCs • AHG only if gel or solid phase
• Reactions documented on a sheet
that outlines every RBC’s phenotype

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Geography
• Let’s take a close look at a panel
• Important to know your way around
• There are variations, but this is a
general guide

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Tuesday, March 6, 12 7 Tuesday, March 6, 12 8
 A series of 8-20 group O A series of 8-20 group O
donor reagent red cells, donor reagent red cells,
each tested for all main each tested for all main
antigens (“phenotyped”) antigens (“phenotyped”)

Rh-hr (or “Hr”) = Rh-hr (or “Hr”) =

Modified Wiener Rh Modified Wiener Rh
genotype for donor genotype for donor

R1: DCe r’: dCE R1: DCe r’: dCE

R2: DcE r”: dcE R2: DcE r”: dcE
R0: Dce r: dce R0: Dce r: dce
RZ: DCE ry: dCE RZ: DCE ry: dCE

Other stuff: Other stuff:

-Donor ID -Donor ID
-Special Antigen Type -Special Antigen Type

Co(b+) Pt. phenotype results

+ 0 +
Tuesday, March 6, 12 9 Tuesday, March 6, 12 10

D, C, c, E, e, Fya, Fyb, Jka, Jkb, Jk(a+b–): Assume “double dose” Jka

K, k, Lea, Leb, M, N, P1, S, s
Jk(a+b+): “Single dose” Jka and Jkb
Full donor phenotype Jk(a–b+): Assume “double dose” Jkb
D phenotype for each donor,
C phenotype for each donor,etc...

*
Tuesday, March 6, 12 11 Tuesday, March 6, 12 12
 Jka Jka Jkb
Jka Jkb Jkb IS 37 AHG IS = Immediate spin
37 = 37 C Incubation
0 1+ 3+ AHG = Anti-human globulin
IAT = Indirect antiglobulin test
0 1+ 3+ (=AHG)

0 1+ 3+
0 1+ 3+ This is tube testing!
0 0 0 LISS
Cell 1 FyaCell 2 FyCell
a
5 Albumin
0 0 0
Fya Fy Saline
0 0 0
0 0 0 PEG
0 0 0
0 0 0
0 1+ 3+

Tuesday, March 6, 12 13 Tuesday, March 6, 12 14

IS AHG IS = Immediate spin AHG IS = Immediate spin

37 = 37 C Incubation 37 = 37 C Incubation
0 3+ AHG = Anti-human globulin 3+ AHG = Anti-human globulin
IAT = Indirect antiglobulin test IAT = Indirect antiglobulin test
0 3+ (=AHG) 3+ (=AHG)

0 3+ This is also tube testing! 3+

0 3+ 3+
This could be:
(often with PEG)
0 0 0
-Tube testing
0 0 0
OR
-Gel testing
0 0 0
OR
0 0 0
-Solid-phase testing
0 0 0
0 0 0
0 3+ 3+

Tuesday, March 6, 12 15 Tuesday, March 6, 12 16

 3+
0
IS = Immediate spin IS = Immediate spin
0 37 = 37 C Incubation 37 = 37 C Incubation
0 AHG = Anti-human globulin 3+ AHG = Anti-human globulin
IAT = Indirect antiglobulin test IAT = Indirect antiglobulin test
0 (=AHG) 3+ (=AHG)

0 3+
This could be: 3+ This is almost always:
0
-Tube testing 0 -Gel testing
3+
OR 0 OR
Pos Control 4+
-Gel testing -Solid-phase
Neg Control 0 0
OR
0
-Solid-phase testing
0
0
3+

Tuesday, March 6, 12 17 Tuesday, March 6, 12 18

3+
3+
0
0
0
All other results
are suspect Ya Gotta Have a Plan...
0
0 • Consistent approach minimizes error
0 • Most are simple, but cutting corners
3+ If the AC is positive: increases risk for dumb mistakes
0 • Use this or your own system, but use
the same approach every time!

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Tuesday, March 6, 12 19 Tuesday, March 6, 12 20
 General Process History
• Check history • Both in real life and on exams
• Check autocontrol • History can give you a clue and keep
• Look at general pattern you from doing something dumb
• Look at what’s NOT there (cross-outs)
• Up to 70% of cases impacted by
• Look at what IS there history
• Use special techniques as necessary
• Ensure statistical significance

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Tuesday, March 6, 12 21 Tuesday, March 6, 12 22

History History
• Clinical history examples: • Clinical history examples:
• Anti-D in pregnant pt; consider RhIG • Consider racial profiling (in a good
• Recent bacterial infection; consider way, of course!)
antibiotic induced warm autoab • African-Americans: Lack of Duffy
• Recent viral illness; consider auto- antigens
anti-I or -i (consider age) • Asians: Almost all D+
• Recent transfusion; consider newly • Whites: May lack high freq antigens
developing antibody
• ITP; consider IV RhIG in D+ patient
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Tuesday, March 6, 12 23 Tuesday, March 6, 12 24
 History General Process
• Consider serologic history, if known • Check history
• Previous phenotyping will help, but • Check autocontrol
be careful! • Look at general pattern
• Transplants, transfusions, errors • Look at what’s NOT there (cross-outs)

• In real world, can use this info for • Look at what IS there
targeting the panel and cells chosen • Use special techniques as necessary
• Ensure statistical significance

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Tuesday, March 6, 12 25 Tuesday, March 6, 12 26

Positive Autocontrol
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+
3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
• More details next time...
• Question 1: Is DAT positive?
4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0 • Question 2: What’s the patient history?
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
• Possibilities include:
• Autoantibodies (warm and cold)
8 rr

9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
• Recent transfusion/DHTR
3+
• Drug-induced issues
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11
AC AC 0

• Passively acquired antibodies

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Tuesday, March 6, 12 27 Tuesday, March 6, 12 28
 General Process Pattern
• Check history • Are reactions:
• Check autocontrol • Uniform or variable?
• Look at general pattern • Against all, most, or rare cells?
• Look at what’s NOT there (cross-outs)
• Present in what phases?
• Look at what IS there
• Use special techniques as necessary
• Ensure statistical significance

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Tuesday, March 6, 12 29 Tuesday, March 6, 12 30

Variability Cells Reactive

• Uniform reactions suggest a single • With negative autocontrol...
antibody • A mixture of reactive and
• Variable reactions suggest either: nonreactive cells suggests:
• Multiple antibodies OR • A single alloantibody OR
• Single antibody with dosage • Multiple alloantibodies
• A single reactive cell suggests:
• A single alloantibody against a
low-prevalence antigen

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Tuesday, March 6, 12 31 Tuesday, March 6, 12 32
 37/AHG: Rh

Cells Reactive AHG: K, Fy, Jk, S/s

Variable: Lu, Xg
IS (RT): Le, MN, P

• With negative autocontrol... 37 C and AHG V AHG Only RT Var. RT AHG RT

• All or virtually all cells reactive

suggests:
• Multiple alloantibodies
• A single alloantibody against a
high-prevalence antigen

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Tuesday, March 6, 12 33 Tuesday, March 6, 12 34

AC neg AC neg
1. Allo vs high prev

Uniform Variable All + antigen (IgG or IgM)

Warm Cold Warm Warm/Cold Cold

All + +&– All + +&– Many/most + Many/most + Many/most +

1. Multiple IgG allos 1. IgG/IgM allos (multiple) 1. Multiple IgM allos

1. IgG allo vs high prev 1. Single IgG allo 1. IgM allo vs high prev 1. Single IgM allo
2. Single IgG allo (dosage) 2. Strong IgM binding 2. Single IgM allo (dosage)
2. Multiple IgG allos 2. Multiple IgG allos 2. Multiple IgM allos
3. HTLA-like

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Tuesday, March 6, 12 35 Tuesday, March 6, 12 36
 General Process
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+
3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
+ 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
• Check history
•
4 R0r

5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0
Check autocontrol
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0 • Look at general pattern
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
• Look at what’s NOT there (cross-outs)
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
• Look at what IS there
9 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
•
rr

10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
Use special techniques as necessary
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+ • Ensure statistical significance
AC AC 0
1. Gel or solid-phase most likely (probably gel)
2. No autoantibody obvious
3. Uniform pattern; single antibody most likely www.bbguy.org
Tuesday, March 6, 12 37 Tuesday, March 6, 12 38

“Cross-outs”
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+
3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+
AC AC 0
1. Gel or solid-phase most likely (probably gel)
2. No autoantibody obvious
3. Uniform pattern; single antibody most likely www.bbguy.org
Tuesday, March 6, 12 39 Tuesday, March 6, 12 40
 “Cross-outs”
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+
3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+
AC AC 0

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Tuesday, March 6, 12 41 Tuesday, March 6, 12 42

Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+ 1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+ 2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+
3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+ 3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+ 4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0 5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0 6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0 7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0 8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0 9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0 10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+ 11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+
AC AC 0 AC AC 0

Tuesday, March 6, 12 43 Tuesday, March 6, 12 44

 Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+ 1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+ 2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+
3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+ 3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+ 4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0 5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0 6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0 7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0 8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0 9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0 10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+ 11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+
AC AC 0 AC AC 0
1. Gel or solid-phase most likely (probably gel)
2. No autoantibody obvious
3. Uniform pattern; single antibody most likely
Tuesday, March 6, 12 45 Tuesday, March 6, 12 46

General Process General Process

• Check history • Try first for a single antibody to
• Check autocontrol explain all reactions
• Look at general pattern
• Look at what’s NOT there (cross-outs)
• Look at what IS there
• Use special techniques as necessary
• Ensure statistical significance

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Tuesday, March 6, 12 47 Tuesday, March 6, 12 48
 DC E Cw V Kpa Jsa Lua

General Process
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+
3

4
R2R2

R0r
+ 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
+ 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
• Try first for a single antibody to
5 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0
explain all reactions
• Failing that...
r’r

6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
• Depending on pattern, hypothesize:
• Two antibodies in same phase
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+
AC AC 0
1. Gel or solid-phase most likely (probably gel)
2. No autoantibody obvious
3. Uniform pattern; single antibody most likely www.bbguy.org
Tuesday, March 6, 12 49 Tuesday, March 6, 12 50

DC E Cw V Kpa Jsa Lua

General Process
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 4+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 4+
3

4
R2R2

R0r
+ 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
+ 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
• Try first for a single antibody to
5 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 2+
explain all reactions
• Failing that...
r’r

6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
• Depending on pattern, hypothesize:
• Two antibodies in same phase
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 4+
• One warm and one cold antibody
AC AC 0

1. Gel or solid-phase most likely (probably gel)

2. No autoantibody obvious
3. Variable pattern; multiple antibodies most likely www.bbguy.org
Tuesday, March 6, 12 51 Tuesday, March 6, 12 52
 Fyb
“Colds”: Lea, Leb, M, N, P
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell IS 37 IAT Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell IS 37 IAT
1 R1R1 + + 0 0 + 0 + 0 +
0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 0 0 0 1 R1R1 + + 0 0 + 0 + 0 +
0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 0 0 0
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0+ +0 0 + 0 + 2 0 0 0 2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0+ +0 0 + 0 + 2 0 0 0
3 R2R2 0 0 0 3 0
+ 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 R2R2 0 0 0 3 0
+ 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0
4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 0 0 0 4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 0 0 0
5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 1+ 1+ 3+ 5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 1+ 1+ 3+
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 1+ 1+ 1+ 6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 1+ 1+ 1+
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0 0 3+ 7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0 0 3+
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 +
0 +
0 + 0 + + + + 0 + 0 8 1+ 1+ 0 8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + 0 + 0 + + + + 0 + 0 8 1+ 1+ 0
0 +
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0 0 0 9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0 0 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 1+ 1+ 1+ 10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 1+ 1+ 1+
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 0 0 3+ 11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0
+ + 0 0 + + 0 0 + 0 + 11 0 0 3+
AC AC 0 0 0 AC AC 0 0 0

1. Tube testing for sure 1. Tube testing for sure

2. No autoantibody obvious 2. No autoantibody obvious
3. Highly variable pattern, both warm and cold 3. Highly variable pattern, both warm and cold
Tuesday, March 6, 12 53 Tuesday, March 6, 12 54

Fyb , Lea
“Colds”: Lea, Leb, M, N, P

General Process
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell IS 37 IAT
1 R1R1 + + 0 0 + 0 + 0 +
0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 0 0 0
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0+ +0 0 + 0 + 2 0 0 0
3

4
R2R2

R0r
0 0 0 3 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 4 0
0 0
0 0
• Try first for a single antibody to
5 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 1+ 1+ 3+
explain all reactions
• Failing that...
r’r

6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 1+ 1+ 1+
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0 0 3+
• Depending on pattern, hypothesize:
• Two antibodies in same phase
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + 0 + 0 + + + + 0 + 0 8 1+ 1+ 0
0 +
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0 0 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 1+ 1+ 1+
• One warm and one cold antibody
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0
AC
+ + 0 0 + + 0 0 + 0 + 11
AC
0 0 3+
0 0 0 • Consider multiple warms and colds
1. Tube testing for sure • Next podcast!!
2. No autoantibody obvious
3. Highly variable pattern, both warm and cold www.bbguy.org
Tuesday, March 6, 12 55 Tuesday, March 6, 12 56
 General Process Phenotyping
• Check history • Helps confirm identification of
• Check autocontrol alloantibody by demonstrating lack
• Look at general pattern of antigen
• Look at what’s NOT there (cross-outs) • Is a TOOL in confirmation, not sole
• Look at what IS there measure of confirmation
• Use special techniques as necessary
• Ensure statistical significance

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Tuesday, March 6, 12 57 Tuesday, March 6, 12 58

DC E Cw V Kpa Jsa Lua

Adsorption
Rh-hr Kell Duffy Kidd P Lutheran MNSs Lewis Results
Cell Rh-hr D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S s Lea Leb Cell

1 R1R1 + + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 4+
2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 4+
3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
+ 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
• Removing antibodies from sample by
4 R0r
incubation with antigen-positive RBCs
5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 2+
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0 “Autoadsorption”
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0 AutoAb
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
Patient Patient
K– Patient K–
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 4+
K–
Autoantibody AutoAb
Patient
+
AC 0 0 AC 0 Anti-K K– Anti-K
1. Gel or solid-phase most likely (probably gel)
2. No autoantibody obvious
3. Variable pattern; multiple antibodies most likely p. 7 www.bbguy.org
Tuesday, March 6, 12 59 Tuesday, March 6, 12 60
 Adsorption Elution
• Removing antibodies from sample by • Removal of RBC-bound antibodies
incubation with antigen-positive RBCs • Heat, cold, chemical (glycine)
“Alloadsorption” “Elution”
A
K Anti -K nti-
ti- -C nti C K+C+S–
Anti-KK+C+S– An K+C+S– A K+C+S–
Anti-K
Anti-CK+C+S–
nti
-K
An
ti- + ti-
K Anti Treat Anti-C
+ or...
A K+C+S– C An K+C+S– C
- K+C+S–
Anti-SK+C+S– Anti-S
“Adsorbed Serum”

p. 7 www.bbguy.org p. 7 www.bbguy.org
Tuesday, March 6, 12 61 Tuesday, March 6, 12 62

Proteolytic Enzymes General Process

• Enzymes such as ficin and papain may • Check history
change Ag expression/Ab binding • Check autocontrol
• Look at general pattern
• Look at what’s NOT there (cross-outs)

M M M M M • Look at what IS there

M M • Use special techniques as necessary
• Ensure statistical significance

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Tuesday, March 6, 12 63 Tuesday, March 6, 12 64
 Probability Probability
• Basic idea: Ensure what you are • For practical purposes, preliminary
seeing is not pure chance ID only requires ONE double-dose
• Traditional interpretation: rule out to establish lack of identity
• Ag present: 3 positive reactions • This does NOT mean your work is
• Ag absent: 3 negative reactions done, however
• AABB Standards for IRLs, 7th ed: • Still need to use selected cells to
rule in/out specific Abs (per OP)
• 5.3.3 requires only 2 of each
reaction to assign specificity

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Tuesday, March 6, 12 65 Tuesday, March 6, 12 66

DC E Cw V Kpa Jsa Lua

Cell Rh-hr D C E
Rh-hr
c e f Cw V K
Kell Duffy Kidd P Lutheran MNSs
k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb P1 Lua Lub M N S
Lewis
s Lea Leb Cell
Results
Let’s do some
+ + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + 1 3+

together!
1 R1R1

2 R1R1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 + 2 3+
3 R2R2 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + + + 0 0 3 3+
4 R0r + 0 0 + + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 0 4 3+
5 r’r 0 + 0 + + + 0 0 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + 0 5 0
6 r”r 0 0 + + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 6 0
7 rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 7 0
8 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + + + + 0 + 0 8 0
9 rr 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + 9 0
10 rr 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + + 0 0 + + 0 10 0
11 R1R1 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + 0 + 11 3+
AC AC 0
1. Gel or solid-phase most likely (probably gel)
2. No autoantibody obvious
3. Uniform pattern; single antibody most likely www.bbguy.org
Tuesday, March 6, 12 67 Tuesday, March 6, 12 68
 Thanks!
This podcast is not a medical consultation. Please
• Monica LaSarre (Bonfils) consult your provider before making any medical
• Tuan Le (Bonfils) decisions, and do not use the information presented
here for anything other than educational purposes.
• Colleen Chiappa (Bonfils)
• Kevin Elman (N. Co. Med Center) Please do not reproduce or rebroadcast
• Cami Melland (Bonfils) this podcast without my permission!
• The immortal Connie Howard
(Walter Reed) Seriously! You’re getting it for free, right?

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Tuesday, March 6, 12 69 Tuesday, March 6, 12 70