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Antibody Identification
Case Studies
Karen Rodberg, MBA, MT (ASCP) SBBDirector, Immunohematology LaboratoryAmerican Red Cross, Southern California Region
Commonly used problem-solving techniques:
–Proteolytic enzymes–Reducing (thiol) reagents–Titration and neutralization–Adsorption–Elution
Enzyme use for antibody i.d.
• Antibody identification tool- Weak or equivocal reactions- Suspected multiple antibodies- Characterize unknown specificity
• Adsorption studies- Remove or separate antibodies
3
Proteolytic Enzymes
Proteases: cleave bonds in appropriate amino acid chains of membrane bound proteins or glycoproteins
Ficin (figs)Papain (papayas)Bromelin (pineapples)Trypsin (bovine/porcine pancreas)-Chymotrypsin (pancreas)Pronase (Streptomyces griseus)
4
Effects of enzyme-treatment of RBCs
• Proteases remove sialic acid-bearing glycoproteins → reduction in:
- Cell surface negative charge - Steric hindrance- Membrane-bound water
• Results in:RBCs closer together so IgG can
span distance → agglutinate
5
Other effects on RBCs
Effect on RBC antigens:• Some antigens are denatured • Some antigen-antibody
reactivity is enhancedTherefore not used for routine antibody detection, but very useful in antibody identification
6
Example – MNS System
- Cleave [ ] large portions of glycoproteins (e.g., GPA, GPB) from RBC- Site of action is enzyme-specific- Carbohydrates attached to the portion of protein affected will also be removed
7
Enzymes can denature or enhance:
Antigen denaturation (ficin/papain)M, N, S, EnaTS, EnaFS, Fya, Fyb, Fy6, Ge2, Ge4, Inb, Ch, Rg, JMH, Pr, Xga, s*, Yta**variable
Antigen-antibody reactivity enhancementP1, I, i, Lewis, Rh, Kidd, Colton, Dombrock
8
Case Study #1 – case history
33 y.o. female obstetrical patientFull term deliveryNo prenatal care4th pregnancy
9
Case Study #1
Rh type
Anti-D Cntl
0 0
Interpretation: Rh negative
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
I + + 0 0 + + 0 + + + 0 + 0 + 0 + + 0 1+ 3+II + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 3+
III 0 0 0 + + 0 + + + 0 + 0 0 + + + 0 + 2+ 1+
ABO group
Anti-A Anti-BA1
RBCsB
RBCs
0 0 4+ 4+
Interpretation: Group O
10
Case Study #1 – Initial Panel
Evidence of multiple alloantibodies – some reactivity at RT and additional reactivity by indirect antiglobulin test.
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + 0 + + + 0 + 0 0 + 0 + 0 + 2+ 3+2 + + 0 0 + 0 + + 0 + + + 0 + + 0 0 + 0 3+3 0 + 0 + + 0 + + 0 + 0 + 0 + 0 + + 0 0 04 + 0 + + 0 0 0 + 0 + + + 0 + 0 + 0 + 0 3+5 + 0 0 + + + 0 + + + + + + + 0 0 + 0 1+ 3+6 + + 0 + + 0 + 0 + + 0 + 0 + 0 + 0 + 1+ 3+7 + + + + + 0 + 0 + + + + 0 + + 0 0 + 1+ 3+8 + + 0 + + 0 0 + + + 0 + + + 0 + + + 1+ 3+9 0 0 0 + + + 0 0 + + 0 + 0 + + + 0 + 1+ 0
10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 2+ 1+11 + 0 0 + + 0 + + + + 0 + 0 + 0 + 0 + 1+ 3+PT 0 0
11
Begin exclusion with cell #3
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + 0 + + + 0 + 0 0 + 0 + 0 + 2+ 3+2 + + 0 0 + 0 + + 0 + + + 0 + + 0 0 + 0 3+3 0 + 0 + + 0 + + 0 + 0 + 0 + 0 + + 0 0 04 + 0 + + 0 0 0 + 0 + + + 0 + 0 + 0 + 0 3+5 + 0 0 + + + 0 + + + + + + + 0 0 + 0 1+ 3+6 + + 0 + + 0 + 0 + + 0 + 0 + 0 + 0 + 1+ 3+7 + + + + + 0 + 0 + + + + 0 + + 0 0 + 1+ 3+8 + + 0 + + 0 0 + + + 0 + + + 0 + + + 1+ 3+9 0 0 0 + + + 0 0 + + 0 + 0 + + + 0 + 1+ 0
10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 2+ 1+11 + 0 0 + + 0 + + + + 0 + 0 + 0 + 0 + 1+ 3+
PT 0 0
12
Examine RT reactivity
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + 0 + + + 0 + 0 0 + 0 + 0 + 2+ 3+
2 + + 0 0 + 0 + + 0 + + + 0 + + 0 0 + 0 3+
3 0 + 0 + + 0 + + 0 + 0 + 0 + 0 + + 0 0 0
4 + 0 + + 0 0 0 + 0 + + + 0 + 0 + 0 + 0 3+
5 + 0 0 + + + 0 + + + + + + + 0 0 + 0 1+ 3+
6 + + 0 + + 0 + 0 + + 0 + 0 + 0 + 0 + 1+ 3+
7 + + + + + 0 + 0 + + + + 0 + + 0 0 + 1+ 3+
8 + + 0 + + 0 0 + + + 0 + + + 0 + + + 1+ 3+
9 0 0 0 + + + 0 0 + + 0 + 0 + + + 0 + 1+ 0
10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 2+ 1+
11 + 0 0 + + 0 + + + + 0 + 0 + 0 + 0 + 1+ 3+
PT 0 0
Look first at Lea, Leb, P1, M, N – these antibodies most often react at RT
13
Examine RT reactivity
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + 0 + + + 0 + 0 0 + 0 + 0 + 2+ 3+
2 + + 0 0 + 0 + + 0 + + + 0 + + 0 0 + 0 3+
3 0 + 0 + + 0 + + 0 + 0 + 0 + 0 + + 0 0 0
4 + 0 + + 0 0 0 + 0 + + + 0 + 0 + 0 + 0 3+
5 + 0 0 + + + 0 + + + + + + + 0 0 + 0 1+ 3+
6 + + 0 + + 0 + 0 + + 0 + 0 + 0 + 0 + 1+ 3+
7 + + + + + 0 + 0 + + + + 0 + + 0 0 + 1+ 3+
8 + + 0 + + 0 0 + + + 0 + + + 0 + + + 1+ 3+
9 0 0 0 + + + 0 0 + + 0 + 0 + + + 0 + 1+ 0
10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 2+ 1+
11 + 0 0 + + 0 + + + + 0 + 0 + 0 + 0 + 1+ 3+
PT 0 0
Reactivity pattern matches anti-M, showing dosage, but need non-reactive RBCs for exclusion.
14
Test same panel ficin‐treated
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka JkbPEGIAT
FicinIAT
1 + + 0 0 + 0 + + + 0 + 0 0 + 0 + 0 + 3+ 2+2 + + 0 0 + 0 + + 0 + + + 0 + + 0 0 + 3+ 2+3 0 + 0 + + 0 + + 0 + 0 + 0 + 0 + + 0 0 04 + 0 + + 0 0 0 + 0 + + + 0 + 0 + 0 + 3+ 2+5 + 0 0 + + + 0 + + + + + + + 0 0 + 0 3+ 2+6 + + 0 + + 0 + 0 + + 0 + 0 + 0 + 0 + 3+ 2+7 + + + + + 0 + 0 + + + + 0 + + 0 0 + 3+ 2+8 + + 0 + + 0 0 + + + 0 + + + 0 + + + 3+ 2+9 0 0 0 + + + 0 0 + + 0 + 0 + + + 0 + 0 0
10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 1+ 011 + 0 0 + + 0 + + + + 0 + 0 + 0 + 0 + 3+ 2+
PT 0 0
15
Test same panel ficin‐treated
(Some alloantibodies have already been excluded with cell #3)
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka JkbPEGIAT
FicinIAT
1 + + 0 0 + 0 + + + 0 + 0 0 + 0 + 0 + 3+ 2+
2 + + 0 0 + 0 + + 0 + + + 0 + + 0 0 + 3+ 2+
3 0 + 0 + + 0 + + 0 + 0 + 0 + 0 + + 0 0 04 + 0 + + 0 0 0 + 0 + + + 0 + 0 + 0 + 3+ 2+5 + 0 0 + + + 0 + + + + + + + 0 0 + 0 3+ 2+6 + + 0 + + 0 + 0 + + 0 + 0 + 0 + 0 + 3+ 2+7 + + + + + 0 + 0 + + + + 0 + + 0 0 + 3+ 2+8 + + 0 + + 0 0 + + + 0 + + + 0 + + + 3+ 2+9 0 0 0 + + + 0 0 + + 0 + 0 + + + 0 + 0 0
10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 1+ 011 + 0 0 + + 0 + + + + 0 + 0 + 0 + 0 + 3+ 2+
PT 0 0
16
Now exclude ficin‐resistant abys
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka JkbPEGIAT
FicinIAT
1 + + 0 0 + 0 + + + 0 + 0 0 + 0 + 0 + 3+ 2+2 + + 0 0 + 0 + + 0 + + + 0 + + 0 0 + 3+ 2+3 0 + 0 + + 0 + + 0 + 0 + 0 + 0 + + 0 0 04 + 0 + + 0 0 0 + 0 + + + 0 + 0 + 0 + 3+ 2+5 + 0 0 + + + 0 + + + + + + + 0 0 + 0 3+ 2+6 + + 0 + + 0 + 0 + + 0 + 0 + 0 + 0 + 3+ 2+7 + + + + + 0 + 0 + + + + 0 + + 0 0 + 3+ 2+8 + + 0 + + 0 0 + + + 0 + + + 0 + + + 3+ 2+9 0 0 0 + + + 0 0 + + 0 + 0 + + + 0 + 0 0
10 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 1+ 0
11 + 0 0 + + 0 + + + + 0 + 0 + 0 + 0 + 3+ 2+
PT 0 0
17
Also helpful to phenotype patient
Anti-C Anti-E Anti-c Anti-e
0 0 4+ 4+
anti-S anti-s anti-K anti-Fya anti-Fyb anti-Jka anti-Jkb
0 3+ 0 0 4+ 4+ 0
anti-M anti-N
0 4+
Patient can make alloanti-D, -C, -E, -S, -K, -Fya, -Jkb, -M
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Test selected RBC panelFocus on anti-D, -C, -E, -S, -Fya, -M ( -K and -Jkb already excluded)
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka JkbPEGIAT
FicinIAT
1 + 0 0 + + 0 + + 0 + + + 0 + 0 + + + 3+ 3+ D2 0 + 0 0 + 0 + + 0 + + + 0 + 0 + 0 + 0 0 C3 0 0 + + + 0 + + 0 + 0 + 0 + 0 + + + 0 0 E4 0 0 + + + 0 0 + 0 + + 0 0 + 0 + 0 + 0 0 S5 0 0 0 + + + 0 + 0 + + + + + + 0 + 0 0 0 Fya6 0 0 + + + 0 + 0 + + 0 + 0 + 0 + 0 + 1+ 0 M7 + 0 0 + + 0 + 0 0 + + + 0 + + 0 0 + 3+ 3+ D8 0 + 0 + + 0 0 + 0 + 0 + + + 0 + + + 0 0 C9 0 0 + + + + 0 0 0 + 0 + 0 + + + 0 + 0 0 E
10 0 0 0 + + 0 + + 0 + + 0 + + 0 + 0 + 0 0 S11 + 0 0 + + 0 + + 0 + 0 + 0 + 0 + 0 + 0 0 Fya12 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 2+ 0 M
19
Exclude and confirm antibody i.d.
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka JkbPEGIAT
FicinIAT
1 + 0 0 + + 0 + + 0 + + + 0 + 0 + + + 3+ 3+ D
2 0 + 0 0 + 0 + + 0 + + + 0 + 0 + 0 + 0 0 C
3 0 0 + + + 0 + + 0 + 0 + 0 + 0 + + + 0 0 E
4 0 0 + + + 0 0 + 0 + + 0 0 + 0 + 0 + 0 0 S
5 0 0 0 + + + 0 + 0 + + + + + + 0 + 0 0 0 Fya
6 0 0 + + + 0 + 0 + + 0 + 0 + 0 + 0 + 1+ 0 M
7 + 0 0 + + 0 + 0 0 + + + 0 + + 0 0 + 3+ 3+ D
8 0 + 0 + + 0 0 + 0 + 0 + + + 0 + + + 0 0 C
9 0 0 + + + + 0 0 0 + 0 + 0 + + + 0 + 0 0 E
10 0 0 0 + + 0 + + 0 + + 0 + + 0 + 0 + 0 0 S
11 + 0 0 + + 0 + + 0 + 0 + 0 + 0 + 0 + 0 0 Fya
12 0 0 0 + + 0 + + + 0 + 0 + + 0 + 0 + 2+ 0 M
Anti-D and anti-M confirmed20
Case Study #1 conclusion
Anti-D and anti-M confirmedAnti-D is likely an alloantibody, but cannot be distinguished serologically from passive anti-D (antenatal RhIg), although this patient had no prenatal care.Anti-M is likely naturally occurring and not clinically significant.
* Transfusion recommendation: Group O Rh negative RBCs* Do cord blood studies on neonate (ABO/Rh, DAT) and watch baby’s bilirubin, etc.
Commonly used problem-solving techniques:
–Proteolytic enzymes–Reducing (thiol) reagents–Titration and neutralization–Adsorption–Elution
23
Antigen Denaturation:Effect of DTT (or AET) on RBCs
Reduce disulfide bonds in structure of proteins → denaturation of antigens
Antigens destroyed:Kell antigensKnopsJMH, Yta, Gy, HyCromer (weakened)Lutheran (weakened)Vel (variable)
Reducing (thiol) reagents
Usefulness of Enzymes and DTT:
Ficin/ Possible AntibodyPapain DTT or Antibody in Systemneg pos Fya/Fyb; Ch/Rg; Ge2, Ge4 neg neg Indian; JMHpos weak Cromer; Knops; Lutheran;
Dombrock; AnWj; MER2variable neg Yta
pos neg Kell; LW pos pos Rh; Jk3; Fy3; Diego; Colton;
Ge3; Oka; I,i; P,LKE; Ata; Csa; Era; Jra; Lan; Vel; Sda,Scianna
RBC antigens denatured by ZZAP
ZZAP is a combination of enzyme and DTT(--frequently used for adsorptions)
Antigens denatured:M, N, S, s*, Fya, Fyb, Yta*, Xga, JMH, Ch, Rg, EnaTS, EnaFS, Ge2, Ge4, LW, Kell, Dombrock, Lutheran, and Scianna system antigens
*variable
Commonly used problem-solving techniques:
–Proteolytic enzymes–Reducing (thiol) reagents–Titration and neutralization–Adsorption–Elution
Neutralization / inhibition
Antigens in soluble form can be used to inhibit or neutralize reactivity to aid in antibody identification–A, B, H, Lea, Leb, P1
(blood group substance)–Ch, Rg (pooled normal plasma)–Sda (urine)
Titration / neutralization
• Titrate to help classify HTLA-type reactivity ("high-titer, low avidity")
‘HTLA’ ≠ clinically insignificant
• Neutralization or inhibition with plasma or other blood group substance
Examples of titration / neutralizationdilution 1 2 4 8 16 32 64 128 256 512
plasma 1+ 1+ 1+ 1+ ± ± ± ± ± 0
albumin 1+ 1+ 1+ 1+ ± ± ± ± ± 0
plasma 0 0 0 0 0 0 0 0 0 0
albumin 1+ 1+ 1+ 1+ ± ± ± ± ± 0
29
Neutralization with plasma
Neutralized anti-Ch, -Rg
Not neutralized anti-JMH, -Kna, -McCa, -Sla, -Yka, -Csa
Possible other alloantibodies
30
Case Study #2 – case history
59 y.o. woman with multiple myeloma, transfused 3½ months earlierNo medication history givenAntibody i.d. requested, no bloodPatient dischargedGroup O Rh PositivePlasma: LISS-IgG = weak pos all RBCs
autocontrol = neg
Case Study #2
Anti-A Anti-BA
RBCsB
RBCsIS
Anti-DIS
Cntl0 0 4+ 4+ 4+ 0
Anti-IgG
Anti-C3
10% BSA
0 0 0
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
I + + 0 0 + + 0 + + + 0 + 0 + 0 + + 0 0 1+II + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 1+
III 0 0 0 + + 0 + + + 0 + 0 0 + + + 0 + 0 1+
ABO/Rh: DAT:
Antibody screen:
Initial Antibody Panel
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 1+
2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 1+
3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 1+
4 0 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + 0 1+
5 0 + 0 + + 0 + + + + + 0 0 + + + + 0 0 1+
6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 1+
7 + + 0 + + + 0 + 0 + + 0 0 + 0 0 + 0 0 0
8 + + + + + + 0 0 + + 0 + 0 + + + + + 0 1+
PT 0 0
** Initial panel shows weak reactivity with 7 of 8 RBCsAutocontrol negative, so we assume this is alloantibody
Antibody Panel also tested with ficin and DTT‐treated RBCs
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
Ficin IAT
DTT IAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 1+ 1+ 0
2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 1+ 1+ 0
3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 1+ 1+ 0
4 0 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + 0 1+ 1+ 0
5 0 + 0 + + 0 + + + + + 0 0 + + + + 0 0 1+ 1+ 0
6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 1+ 1+ 0
7 + + 0 + + + 0 + 0 + + 0 0 + 0 0 + 0 0 0 0 0
8 + + + + + + 0 0 + + 0 + 0 + + + + + 0 1+ 1+ 0
PT 0 0
** IRL frequently tests ficin‐treated and DTT‐treated RBCs to characterize the antibody reactivity. This antibody appears to be DTT‐sensitive.
Exclusion of antibodies
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
Ficin IAT
DTT IAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 1+ 1+ 0
2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 1+ 1+ 0
3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 1+ 1+ 0
4 0 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + 0 1+ 1+ 0
5 0 + 0 + + 0 + + + + + 0 0 + + + + 0 0 1+ 1+ 0
6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 1+ 1+ 0
7 + + 0 + + + 0 + 0 + + 0 0 + 0 0 + 0 0 0 0 0
8 + + + + + + 0 0 + + 0 + 0 + + + + + 0 1+ 1+ 0
PT 0 0
** Using the DTT‐treated RBC panel, all common allos can be excluded, except anti‐K.Anti‐k can be excluded using cell #7, but is not a “common” alloantibody.
Titration/Neutralization
tube # 1 2 3 4 5 6 7 8 9 10 11 12
Titer Interp
dilution→
neat 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048
Case # / Antibody ↓
diluent ↓
2014-2413
AB plasma 1+ 1+ 1+ 1+ 1+ w+ w+ w+ 0 0 0 0 128
2014-2413
6% albumin 1+ 1+ 1+ 1+ 1+ w+ w+ w+ 0 0 0 0 128
RBC + AB pool control 0
** Antibody appears to have “HTLA” characteristics, and is not neutralized.
37
Review: DTT-sensitive antigens associated with antibodies with ‘HTLA’ characteristics
Kell Knops LW JMH Indian Dombrock YT (variable) Lutheran (variable) Gerbich (variable) Scianna (variable)
Selected Rare RBCs
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + + + + + 0 + 0 + 0 + 0 + + + + + Yt(a-) 0 1+2 + 0 + + + 0 + + + + 0 + 0 + + JMH- 0 1+3 + + 0 0 + 0 0 0 0 + + + 0 + 0 + + Sc:-3 0 1+4 + 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + Kp(b-) 0 1+5 + + 0 0 + 0 + + + + + 0 0 + + + 0 Ge:-3 0 1+6 0 0 0 + + 0 + + + 0 0 + + + 0 + + 0 Kn(a-) 0 1+7 + + 0 0 + 0 + 0 + + 0 + 0 0 + + 0 + K null 0 1+8 0 0 0 + + 0 + 0 0 + 0 + 0 + + 0 + 0 LW(a-b-) 0 1+9 + + + + + 0 + 0 + + 0 + + + 0 + + 0 Lu(a+b-) 0 1+
10 0 0 0 + + + + 0 + + 0 + 0 + + 0 Lu(a-b-) 0 0
11 + + + + + 0 + + + + + + + 0 + 0 + + Do(b-) 0 1+12 0 0 0 + + 0 0 + + + + + 0 + + 0 + + Yk(a-) 0 1+
** Selected cells focused on DTT‐sensitive antigens and antibodies that may have “HTLA” characteristics.
Selected Rare RBCs – exclusion:
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + + + + + 0 + 0 + 0 + 0 + + + + + Yt(a-) 0 1+2 + 0 + + + 0 + + + + 0 + 0 + + JMH- 0 1+3 + + 0 0 + 0 0 0 0 + + + 0 + 0 + + Sc:-3 0 1+4 + 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + Kp(b-) 0 1+5 + + 0 0 + 0 + + + + + 0 0 + + + 0 Ge:-3 0 1+6 0 0 0 + + 0 + + + 0 0 + + + 0 + + 0 Kn(a-) 0 1+7 + + 0 0 + 0 + 0 + + 0 + 0 0 + + 0 + K null 0 1+8 0 0 0 + + 0 + 0 0 + 0 + 0 + + 0 + 0 LW(a-b-) 0 1+9 + + + + + 0 + 0 + + 0 + + + 0 + + 0 Lu(a+b-) 0 1+
10 0 0 0 + + + + 0 + + 0 + 0 + + 0 Lu(a-b-) 0 0
11 + + + + + 0 + + + + + + + 0 + 0 + + Do(b-) 0 1+12 0 0 0 + + 0 0 + + + + + 0 + + 0 + + Yk(a-) 0 1+
** One example of Lu(a─b─) RBCs was non‐reactive
Addi onal rare Lu(a−b−) RBCs
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 Lu(a-b-) 0 0
2 0 0 0 + + 0 0 + 0 + + Lu(a-b-) 0 03 0 0 0 + + 0 + + 0 + + + 0 + 0 + + Lu(a-b-) 0 1+4 + + 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + Lu(a-b-) 0 0
5 + 0 + + 0 0 + + + + + 0 + + + + + 0 Lu(a-b-) 0 0
6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + Lu(a-b-) 0 0
** Antibody specificity appears to be anti‐Lu3 …but one example of Lu(a─b─) RBCs reacted weakly…
Additional common alloantibodies could be excluded, including anti‐K.
RBC phenotyping
Anti-C Anti-E Anti-c Anti-e
4+ 0 0 4+
anti-S anti-s anti-K anti-Fya anti-Fyb anti-Jka anti-Jkb
3+ 3+ 0 3+ 0 3+ 3+
anti-Lua anti-Lub anti-Yta
0 3+ 3+
RBC phenotyping
Anti-C Anti-E Anti-c Anti-e
4+ 0 0 4+
anti-S anti-s anti-K anti-Fya anti-Fyb anti-Jka anti-Jkb
3+ 3+ 0 3+ 0 3+ 3+
anti-Lua anti-Lub anti-Yta
0 3+ 3+
** Individuals who make anti‐Lu3 would be expected to be Lu(a−b−)This pa ent is Lu(a−b+) so should not be able to make an ‐Lu3.
43
Summary – (note: this was August 2014)
• Plasma:–DTT‐tt’d RBCs = neg–Reactive RBCs: Yt(a−), JMH−, Kp(b−), K0, LW(a−b−), Kn(a−), McC−, Yk(a−), Do(b−), Lu(a+b−)–6/7 Lu(a−b−) = neg• Anti‐Lu3 specificity?• No, pa ent’s RBCs type Lu(a−b+)
44
Case #2 ‐ Preliminary conclusions
• Medications not listed on request form; called hospital: patient on daratumumab
• IRL Report:–Antibody to Lutheran‐related high incidence antigen; unable to further identify the specificity–All common alloantibodies excluded–Reactivity in patient’s plasma appearing to have Lutheran‐related specificity may be related to medication
45
2014 AABB Meeting (October)
• Hannon JL, et al. Transfusion 2014;54Suppl: 162A (abstr) [Transfusion 2015;55:2770]– 3/6 myeloma patients with positive IATs after DARA (PEG 1+, solid phase 1‐4+)
• Chapuy CI, et al. Transfusion 2014;54Suppl: 157A (abstr) [Transfusion 2015;55:1545‐54]– 5/5 DARA‐treated myeloma patients with positive IATs (weak‐1+, tube & solid‐phase)
– DTT pretreatment of reagent RBCs a “robust method” to negate DARA interference
46
Hindsight is 20/20
47
Daratumumab
• Daratumumab (DARA) is an IgG1κ human monoclonal antibody to CD38
• CD38 ‐ type II transmembrane glycoprotein– Expressed on immune cells, e.g., T lymphocytes; also widely distributed on non‐immune cells, e.g., RBCs, platelets, neurons…..
– Functions include: • Receptor that mediates adhesion & signaling • Ectoenzyme that contributes to intracellular calcium mobilization
48
Daratumumab
• FDA approved Nov. 16, 2015 (Darzalex™, Janssen)– “indicated for the treatment of patients with multiple myeloma who have received at least three prior lines of therapy including a proteasome inhibitor (PI) and an immunomodulatory agent or who are double‐refractory to a PI and an immunomodulatory agent.”
– accelerated approval based on response rate– 16 mg/kg; weekly (weeks 1‐8), every 2 weeks (weeks 9‐24), every 4 weeks (week 25 on)
49
Daratumumab Product Insert
• Interference with Serological Testing:– DARA binds to CD38 on RBCs, resulting in positive indirect antiglobulin tests (IATs), i.e., antibody screens & crossmatches
– DARA‐mediated positive IATs may persist for up to 6 months after the last DARA infusion*
– DARA bound to RBCs masks detection of antibodies to minor antigens
– ABO and Rh blood type determinations are not impacted
50
DTT‐treated reagent RBCs
• Chapuy CI, et al. Transfusion 2015;55:1545‐54• Chapuy CI, et al. Blood 2015;126:3567 (abstr)– DTT more efficient than trypsin– Advantage: DTT is inexpensive & already used by blood banks
– Disadvantage: some antigens are disrupted by DTT treatment (CROM, DO, IN, JMH, KEL, KN, LW, LU, RAPH, YT)• Provide K− blood to DARA pa ents• Rarely a potentially clinically significant antibody could be missed (e.g., anti‐k, ‐Doa, ‐Dob)
51
2015 AABB Meeting – DARA can be Mistaken for Lutheran or Knops Antibody• Aye T, et al. Transfusion 2015;55 Suppl:28A– 5/6 pts nonreac ve with most Lu(a−b−) RBCs–Using flow cytometry, showed nonreactive Lu(a−b−) RBCs had low levels of CD38– RBCs from one in‐house donor, with weak expression of Lub and very low levels of CD38, were nonreactive with all 6 patients’ plasma
• Velliquette RW, et al. Transfusion 2015; 55 Suppl:26A– DARA can also be mistaken for anti‐Kn
Flow CytometryUntreated RBCs% Pos = 62%
0.2M DTT‐tt’d RBCs% Pos = 13%
RBC Background (Autofluorescence)
RBCs + PE anti‐CD38
Flow Cytometry Results ‐CD38 Expression on Selected RBCs
0
10
20
30
40
50
60
70
80
90
100
Flow
cytom
etry
(% positive)
Fy(a+)Fy(b+)
Fy(a‐b‐) Lu(a‐b‐) Cord DTT‐tt'd DARAPts
#1
#2
54
AABB Association Bulletin #16‐02
• Jan. 15, 2016• Positive IATs may occur in all media & by all methods (gel, tube, solid phase); usually weak (1+) but stronger in solid phase (up to 4+)
• Adsorptions with untreated or ZZAP‐treated RBCs don’t eliminate interference
• Anti‐CD38 doesn’t interfere with IS crossmatch; variably interferes with DATs & autocontrols
• Anti‐CD38 may cause small Hb decrease in vivo (1 g/dL) but severe hemolysis not observed
55
AABB Association Bulletin #16‐02
• If patient’s history of anti‐CD38 unknown:– ABO/RhD typing = no issues– Antibody detection (screen) test = all cells pos– Antibody identification panel = all cells pos, autocontrol may be neg
– DAT = pos or neg– AHG crossmatches = all units pos– Post adsorptions = all cells still pos
• Thus, 1) delays in issuing blood, & 2) clinically significant alloantibodies could be masked
56
AABB Association Bulletin #16‐02
• BEFORE patient starts anti‐CD38:–Perform baseline type & screen–Baseline phenotype or genotype recommended
57
AABB Association Bulletin #16‐02
• AFTER patient starts anti‐CD38:– DTT‐treated RBCs can be used for Ab screen/ID• Provide K− units, unless pa ent known to be K+• Abs to other DTT‐sensitive agns can be missed, but are infrequent• If DTT‐treated Ab screen neg, may use electronic or IS crossmatch (ABO/D compat, K‐matched)
– For patients with known alloabs, phenotypically or genotypically matched units may be provided; AHG xmatches will still be incompatible; some clinically sig abs may be missed, but infrequently
– AHG crossmatch with DTT‐tt’d donor cells may be performed
58
Communication is Critical
• Patients should be advised to inform healthcare providers that they are taking anti‐CD38 prior to receiving blood transfusions
• Hospital Transfusion Services & Immunohematology Reference Labs need to be informed that patients have received anti‐CD38
• Patients should have type and screen performed prior to starting anti‐CD38
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Selecting Blood for Transfusion
• Anti‐CD38 not removed by adsorptions• Proposed solutions:
1. Treat reagent RBCs with DTT or trypsin to denature/remove cell surface CD38
2. Use results of phenotyping & genotyping to select antigen‐matched units
3. Inhibit anti‐CD38 using anti‐idiotype or soluble CD38
4. Test a panel of antigen‐typed group O cord RBCs
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Panel of CD38-depressed RBCs
Patients on DARA tend to have depression of CD38 on their RBCs1
NYBC published abstract in 2016 suggesting the use of a panel of “DARA RBCs”2
Phenotype DARA RBCs if DAT− and constuct a selected cell panel
1Sullivan HC, et al. Transfusion 2016; 56 Suppl:25A2Velliquette RW, et al. Transfusion 2016; 56 Suppl:26A
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Current SoCal IRL Approach
• Hope for a good medication history or accurate diagnosis on request form
• Review hospital’s panel if submitted– If hospital tests by solid phase or gel, reactions will probably be stronger than by tube
• If the serology is suggestive of DARA (i.e. weak to moderate reactivity by PEG and DTT‐sensitive) then test cord RBCs and CD38 depressed RBCs (e.g., from DARA patients)
• Exclude or identify alloantibodies
Example of current serology
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 1+
2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 1+
3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 1+
4 0 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + 0 1+
5 0 + 0 + + 0 + + + + + 0 0 + + + + 0 0 1+
6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 1+
7 + 0 0 + + + 0 + 0 + + + 0 + 0 0 + 0 0 1+
8 + + + + + + 0 0 + + 0 + 0 + + + + + 0 1+
PT 0 0
Example of current serology
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
Ficin IAT
DTT IAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 1+ 1+ 0
2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 1+ 1+ 0
3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 1+ 1+ 0
4 0 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + 0 1+ 1+ 0
5 0 + 0 + + 0 + + + + + 0 0 + + + + 0 0 1+ 1+ 0
6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 1+ 1+ 0
7 + 0 0 + + + 0 + 0 + + + 0 + 0 0 + 0 0 1+ 1+ 0
8 + + + + + + 0 0 + + 0 + 0 + + + + + 0 1+ 1+ 0
PT 0 0
Example of current serology
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
Ficin IAT
DTT IAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 1+ 1+ 0
2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 1+ 1+ 0
3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 1+ 1+ 0
4 0 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 + 0 1+ 1+ 0
5 0 + 0 + + 0 + + + + + 0 0 + + + + 0 0 1+ 1+ 0
6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 1+ 1+ 0
7 + 0 0 + + + 0 + 0 + + + 0 + 0 0 + 0 0 1+ 1+ 0
8 + + + + + + 0 0 + + 0 + 0 + + + + + 0 1+ 1+ 0
PT 0 0
** Using the DTT‐treated panel RBCs, all common allos can be excluded, with the exception of anti‐K (and anti‐k).
Selected cell panel
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 + + + 0 + 0 + + 0 0 + 0 0 + 0 KT RBCs 0 0
2 + + 0 0 + + + 0 + + 0 + + cord RBCs 0 0
3 + 0 + + 0 + + 0 + + + + + cord RBCs 0 0
4 0 0 0 + + + 0 0 + 0 0 0 + CD38− RBCs 0 0
5 0 + 0 + + 0 + 0 + + + + 0 CD38− RBCs 0 0
6 0 0 + + + + + 0 + 0 + + + CD38− RBCs 0 0
7 + 0 0 + + + + 0 + 0 0 + 0 CD38− RBCs 0 0
8 + + + + + + + + + 0 + + + CD38− RBCs 0 0
Selected cell panel – ‘DARA RBCs’
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 + + + 0 + 0 + + 0 0 + 0 0 + 0 KT RBCs 0 0
2 + + 0 0 + + + 0 + + 0 + + cord RBCs 0 0
3 + 0 + + 0 + + 0 + + + + + cord RBCs 0 0
4 0 0 0 + + + 0 0 + 0 0 0 + CD38− RBCs 0 0
5 0 + 0 + + 0 + 0 + + + + 0 CD38− RBCs 0 0
6 0 0 + + + + + 0 + 0 + + + CD38− RBCs 0 0
7 + 0 0 + + + + 0 + 0 0 + 0 CD38− RBCs 0 0
8 + + + + + + + + + 0 + + + CD38− RBCs 0 0
** Using the cord RBCs, KT’s RBCs, and CD38‐depressed RBCs we can also exclude anti‐K and anti‐k.
67
Transfusion recommendations
• If no alloantibody, random ABO/Rh compatible units may be transfused; select least‐reactive (agn‐neg, if appropriate).
• Recommend K− if an ‐K cannot be excluded• If hospital has a phenotype/genotype, antigen‐negative units may be transfused without repeated serological investigations. (Disadvantage: may cost more than the workup, depending on phenotype.)
Commonly used problem-solving techniques:
–Proteolytic enzymes–Reducing (thiol) reagents–Titration and neutralization–Adsorption–Elution
Adsorption options
Remove auto-antibody to detect/rule out alloantibodies• Types
Autologous – only if pt not recently tx’dAllogeneic - differential
• MethodsZZAPEnzymePEG
• Temperature (warm and/or cold)
69
Example of Adsorption
Add 1 vol of adsorbing RBCs
Empty 7ml tube
Add 1 vol of pt plasma
Mix
70
Example of Adsorption Procedure, continued
Incubate at 37C for 30 min
37C
Centrifuge Harvest ads plasma to fresh tube & discard ads RBCs
Test ads plasma
71
‘Pre’‐prepared ZZAP‐treated RBCs• Our laboratory does so many adsorptions on behalf
of our local hospitals that it is more efficient for us to prepare these adsorbing cells ahead of need, and have them available for use.
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Differential adsorption
Allogeneic adsorbing RBC selection– Differential adsorption
▪ Do not need to know pt RBC phenotype▪ RBCs from 3 donors whose RBC
phenotypes collectively lack all common clinically significant antigens–D, C, E, c, e, S, s, K, Fya, Fyb, Jka, Jkb
Example of adsorbing cells
Allogeneic adsorbing RBC selection– Differential
▪ Example:Donor #1: E─c─S─K─Fy(a─)Donor #2: C─e─s─K─Jk(b─)Donor #3: D─ C─E─Fy(b─)Jk(a─)
▪ Note: treatment of the adsorbing RBCs with enzymes or ZZAP destroys certain antigens which changes the adsorbing RBC phenotype making selection easier
Untreated vs treated RBCs
Adsorption treatment comparison
D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb#1 UT + + 0 0 + 0 + 0 + + + 0 + + 0 + +
#1 Ficin + + 0 0 + 0 0 0 0 + + 0 + 0 0 + +#1 ZZAP + + 0 0 + 0 0 0 0 0 0 0 + 0 0 + +
Selection of adsorbing RBCs for PEG
Untreated RBCs for PEG Adsorption
D C E c e M N S s K k Lea Leb Fya Fyb Jka JkbA + + 0 0 + 0 + 0 + + + 0 + + 0 + +B + 0 + + 0 + + + + 0 + 0 + 0 + 0 +C 0 0 0 + + + 0 + 0 0 + 0 + + + + 0
Ficin-treated adsorbing RBCs
Ficin RBCs for Enzyme Adsorption
Before treatment
After treatment
D C E c e M N S s K k Lea Leb Fya Fyb Jka JkbA + + 0 0 + + + + + 0 + 0 + 0 + + +B + 0 + + 0 + + + + 0 + 0 + + + 0 +C 0 0 0 + + + + + 0 0 + 0 + + + + 0
D C E c e M N S s K k Lea Leb Fya Fyb Jka JkbA + + 0 0 + 0 0 0 0 0 + 0 + 0 0 + +B + 0 + + 0 0 0 0 0 0 + 0 + 0 0 0 +C 0 0 0 + + 0 0 0 0 0 + 0 + 0 0 + 0
ZZAP-treated adsorbing RBCs
RBCs for ZZAP Adsorption
Before treatment
After treatment
D C E c e M N S s K k Lea Leb Fya Fyb Jka JkbA + + 0 0 + + + + + + + 0 + 0 + + +B + 0 + + 0 + + + + 0 + 0 + + + 0 +C 0 0 0 + + + + + 0 0 + 0 + + + + 0
D C E c e M N S s K k Lea Leb Fya Fyb Jka JkbA + + 0 0 + 0 0 0 0 0 0 0 + 0 0 + +B + 0 + + 0 0 0 0 0 0 0 0 + 0 0 0 +C 0 0 0 + + 0 0 0 0 0 0 0 + 0 0 + 0
Case study #3 – case history
60 year old Caucasian femaleDx: lymphomaMultiple transfusions Jan – April 2010 History of Anti-KLast transfusion 2 months ago, 2 units of K− RBCs when only anti-K id’dHb 7.3 g/dl
Cell Typing: Reverse Typing:
Anti- A Anti- B anti-D control A1 B
0 0 4+ 0 4+ 4+
Case #3 – initial testing
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
I + + 0 0 + + 0 + + + 0 + 0 + 0 + + 0 0 4+
II + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 4+
III 0 0 0 + + 0 + + + 0 + 0 0 + + + 0 + 0 4+
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Case #3 – initial panel
anti-IgG anti-C3 controlIS 4+ 0
RT 4+ 0
Chloroquine‐treated RBCs:anti-IgG
IS 0RT
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
FicinIAT
LISSIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 4+ 4+ 3+2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 4+ 4+ 3+3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 4+ 4+ 3+4 0 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 0 + 0 4+ 4+ 3+5 0 0 0 + + 0 + + + + + + 0 + + + + 0 0 4+ 4+ 3+6 + 0 0 + + 0 + + 0 + + + 0 + 0 + + + 0 4+ 4+ 3+7 + + 0 0 + + 0 0 + 0 0 + + + + 0 0 + 0 4+ 4+ 3+8 0 + 0 + + + 0 0 + + + 0 0 + + + + + 0 4+ 4+ 3+9 + 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + 0 4+ 4+ 3+
10 0 0 0 + + 0 + + 0 + 0 + 0 + 0 + + + 0 4+ 4+ 3+11 + + 0 + + 0 + + + + + + 0 + 0 + + + 0 4+ 4+ 3+
PTCDP-
ttd 0 4+
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ZZAP‐treated RBCs for differential adsorptions
adsorbed sera
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr
1 + + 0 0 + + 0 0 + 0 + + 0 R1 0
2 + 0 + + 0 0 + 0 + + + + 0 R2 0
3 0 0 0 + + + + 0 + + 0 0 + rr 0
4 + + 0 0 + + 0 0 + 0 + + + 0 0 0
5 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
6 0 0 0 + + + + + + + 0 0 + 3+ 3+ 3+
ZZAP adsorbed x2 double volume @ 37C for 30 minutes
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ZZAP denatures MNSs, Kk, Fy
adsorbed sera
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr
1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 0
2 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 0
4 + + 0 0 + + 0 0 + 0 + + + 0 0 0
5 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
6 0 0 0 + + + + + + + 0 0 + 3+ 3+ 3+
ZZAP treatment: combination of enzyme + DTT, so affects antigens on adsorbing cells
83
R1 adsorbed serum
adsorbed sera
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr
1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 0
2 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 0
4 + + 0 0 + + 0 0 + 0 + + + 0 0 0
5 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
6 0 0 0 + + + + + + + 0 0 + 3+ 3+ 3+
• R1 adsorbed serum: would contain anti –E, -c, -S, -s, -K, -k, -Fya, -Fyb, Jkb
84
R2 adsorbed serum
adsorbed sera
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr
1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 0
2 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 0
4 + + 0 0 + + 0 0 + 0 + + + 0 0 0
5 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
6 0 0 0 + + + + + + + 0 0 + 3+ 3+ 3+
• R2 adsorbed serum: would contain –C, -e, -S, -s, -K, -k, -Fya, -Fyb, -Jkb
85
rr adsorbed serum
adsorbed sera
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr
1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 0
2 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 0
4 + + 0 0 + + 0 0 + 0 + + + 0 0 0
5 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
6 0 0 0 + + + + + + + 0 0 + 3+ 3+ 3+
• rr adsorbed serum: would contain –D, -C, -E, -S, -s, -K, -k, -Fya, -Fyb, -Jka
86
Additional selected RBCs
adsorbed seraSelected cell panel:
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 02 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 04 + + 0 0 + + 0 0 + 0 + + + 0 0 05 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+6 + 0 + + 0 0 + + + 0 + + 0 3+ 3+ 3+7 + + 0 0 + 0 + 0 + + 0 + 0 0 0 08 0 0 0 + + 0 + + + + + + 0 3+ 3+ 3+9 + 0 0 + + 0 + 0 + 0 + 0 + 0 0 0
10 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
87
R1 column
adsorbed seraSelected cell panel:
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 02 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 04 + + 0 0 + + 0 0 + 0 + + + 0 0 05 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+6 + 0 + + 0 0 + + + 0 + + 0 3+ 3+ 3+7 + + 0 0 + 0 + 0 + + 0 + 0 0 0 08 0 0 0 + + 0 + + + + + + 0 3+ 3+ 3+9 + 0 0 + + 0 + 0 + 0 + 0 + 0 0 0
10 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
88
R2 column
adsorbed seraSelected cell panel:
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 02 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 04 + + 0 0 + + 0 0 + 0 + + + 0 0 05 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+6 + 0 + + 0 0 + + + 0 + + 0 3+ 3+ 3+7 + + 0 0 + 0 + 0 + + 0 + 0 0 0 08 0 0 0 + + 0 + + + + + + 0 3+ 3+ 3+9 + 0 0 + + 0 + 0 + 0 + 0 + 0 0 0
10 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
89
rr column
adsorbed seraSelected cell panel:
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 02 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 04 + + 0 0 + + 0 0 + 0 + + + 0 0 05 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+6 + 0 + + 0 0 + + + 0 + + 0 3+ 3+ 3+7 + + 0 0 + 0 + 0 + + 0 + 0 0 0 08 0 0 0 + + 0 + + + + + + 0 3+ 3+ 3+9 + 0 0 + + 0 + 0 + 0 + 0 + 0 0 0
10 + 0 + + 0 0 + 0 + 0 + + 0 2+ 0 2+
90
Panel interpretation
adsorbed seraSelected cell panel:
D C E c e S s K k Fya Fyb Jka Jkb R1 R2 rr1 + + 0 0 + 0 0 0 0 0 0 + 0 R1 02 + 0 + + 0 0 0 0 0 0 0 + 0 R2 0
3 0 0 0 + + 0 0 0 0 0 0 0 + rr 04 + + 0 0 + + 0 0 + 0 + + + 0 0 05 + 0 + + 0 0 + 0 + 0 + + 0 E+ 2+ 0 2+6 + 0 + + 0 0 + + + 0 + + 0 E+ K+ 3+ 3+ 3+7 + + 0 0 + 0 + 0 + + 0 + 0 0 0 08 0 0 0 + + 0 + + + + + + 0 K+ 3+ 3+ 3+9 + 0 0 + + 0 + 0 + 0 + 0 + 0 0 0
10 + 0 + + 0 0 + 0 + 0 + + 0 E+ 2+ 0 2+
91
Case #3 conclusions
• The patient has anti‐E in addition to anti‐K, plus a warm autoantibody
• Transfusion recommendations – give E─ K─ units compatible with adsorbed sera, or least incompatible with unadsorbed serum
92
Commonly used problem-solving techniques:
–Proteolytic enzymes–Reducing (thiol) reagents–Titration and neutralization–Adsorption–Elution
Purpose of elution
Cause dissociation of antigen and antibody from antigen-antibody complexes. The objective is to:
Recover antibody in a usable form or
Recover intact RBCs free of antibody (Ig removal)
94
Uses for elution
Investigation of + DATAutoimmune hemolytic anemiaHemolytic transfusion reactionHemolytic disease of fetus/newborn Drug-induced immune hemolytic anemia
Antibody identificationAdsorption/elution, antibody separationPreparation of antibody-free intact RBCs (eg, for phenotyping, autoadsorption)
95
Elution methods to recover antibody
Heat (56C) ABO HDFN, IgM agglutinating antibodies
Easy, poor recovery for IgG
Lui freeze-thaw
ABO HDFN only Quick, small vol RBCs
Acid Warm auto- & alloantibodies
Easy, kits available
Chemical/ organic solvents
Warm auto- & alloantibodies
Chemical hazards
96
Case Study #4 – case history
Pt is a 65 year old male who had cardiac bypass surgery about 2 ½ weeks ago. During surgery he was transfused 2 units of RBCs and has received 1 unit per week since then. His hemoglobin and hematocrit are still gradually dropping, so 2 more units of RBCs are ordered for transfusion today. His antibody screen was previously negative, but now it is weakly positive with 2 of the 3 screening cells. Both of the units being crossmatched are weakly incompatible. Should you just crossmatch a couple more units (the floor keeps bugging you), or first identify the antibody?
97
ABO/Rh typing
DAT
Rh phenotyping
Anti-A Anti-BA
RBCsB
RBCsIS
Anti-DIS
Cntl
0 0 4+ 4+ 4+ 0
Anti-IgG
Anti-C3
10% BSA
0 1+ 0
Anti-C Anti-E Anti-c Anti-e
1+ mf 1+ mf 4+ 4+
• What is ABO/Rh?
• What is DAT?
• What is Rh probable genotype?
•What does the mixed-field (mf) reactivity indicate?
Case Study #4 – initial testing
98
ABO/Rh typing
DAT
Rh phenotyping
Anti-A Anti-BA
RBCsB
RBCsIS
Anti-DIS
Cntl
0 0 4+ 4+ 4+ 0
Anti-IgG
Anti-C3
10% BSA
0 1+ 0
Anti-C Anti-E Anti-c Anti-e
1+ mf 1+ mf 4+ 4+
• What is ABO/Rh? O Positive
• What is DAT? DAT + with complement only
• What is Rh probable genotype? Might be Ror, but recently transfused, so can’t say for sure. Mixed-field reactivity is evidence of two or more RBC populations.
99
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
I + + 0 0 + + 0 + + + 0 + 0 + 0 + + 0 0 2+II + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 (+)III 0 0 0 + + 0 + + + 0 + 0 0 + + + 0 + 0 0
0 (+)0 (+)
Crossmatches:Unit #1Unit #2
Case Study #4 – antibody screen and crossmatches
(+) microscopic positive
100
Case #4 – initial panel
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 2+2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 (+)3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 (+)4 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 0 + 0 0
5 0 + 0 + + 0 + + + + + + 0 + + + + 0 0 2+6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 (+)7 + + 0 0 + + 0 0 + 0 0 + + + + 0 0 + 0 0
8 0 0 0 + + + 0 0 + + + 0 0 + + + + + 0 (+)9 + 0 + + 0 0 0 +s 0 + 0 + 0 + 0 + 0 + 0 0
10 0 0 0 + + 0 + + 0 + 0 + 0 + 0 + + + 0 (+)11 + + + + + 0 + + + + + + 0 + 0 + + + 0 (+)PT 0 0
101
Exclusion
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 2+2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 (+)3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 (+)4 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 0 + 0 0
5 0 + 0 + + 0 + + + + + + 0 + + + + 0 0 2+6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 (+)7 + + 0 0 + + 0 0 + 0 0 + + + + 0 0 + 0 0
8 0 0 0 + + + 0 0 + + + 0 0 + + + + + 0 (+)9 + 0 + + 0 0 0 +s 0 + 0 + 0 + 0 + 0 + 0 0
10 0 0 0 + + 0 + + 0 + 0 + 0 + 0 + + + 0 (+)11 + + + + + 0 + + + + + + 0 + 0 + + + 0 (+)PT 0 0
102
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 2+2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 (+)3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 (+)4 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 0 + 0 0
5 0 + 0 + + 0 + + + + + + 0 + + + + 0 0 2+6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 (+)7 + + 0 0 + + 0 0 + 0 0 + + + + 0 0 + 0 0
8 0 0 0 + + + 0 0 + + + 0 0 + + + + + 0 (+)9 + 0 + + 0 0 0 +s 0 + 0 + 0 + 0 + 0 + 0 0
10 0 0 0 + + 0 + + 0 + 0 + 0 + 0 + + + 0 (+)11 + + + + + 0 + + + + + + 0 + 0 + + + 0 (+)PT 0 0
103
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 2+2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 (+)3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 (+)4 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 0 + 0 0
5 0 + 0 + + 0 + + + + + + 0 + + + + 0 0 2+6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 (+)7 + + 0 0 + + 0 0 + 0 0 + + + + 0 0 + 0 0
8 0 0 0 + + + 0 0 + + + 0 0 + + + + + 0 (+)9 + 0 + + 0 0 0 +s 0 + 0 + 0 + 0 + 0 + 0 0
10 0 0 0 + + 0 + + 0 + 0 + 0 + 0 + + + 0 (+)11 + + + + + 0 + + + + + + 0 + 0 + + + 0 (+)PT 0 0
104
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb RTPEGIAT
1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 0 2+2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 0 (+)3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 0 (+)4 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 0 + 0 0
5 0 + 0 + + 0 + + + + + + 0 + + + + 0 0 2+6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 0 (+)7 + + 0 0 + + 0 0 + 0 0 + + + + 0 0 + 0 0
8 0 0 0 + + + 0 0 + + + 0 0 + + + + + 0 (+)9 + 0 + + 0 0 0 +s 0 + 0 + 0 + 0 + 0 + 0 0
10 0 0 0 + + 0 + + 0 + 0 + 0 + 0 + + + 0 (+)11 + + + + + 0 + + + + + + 0 + 0 + + + 0 (+)PT 0 0
Antibody identified: anti-Jka showing dosage
105
Case #4 ‐ eluate
D C E c e Lea Leb P1 M N S s K k Fya Fyb Jka Jkb EluateLast
Wash1 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + + 0 3+ 0
2 + + 0 0 + 0 0 0 + + + + 0 + + 0 + + 2+ 0
3 + 0 + + 0 0 + + 0 + + + + + + 0 + + 2+ 0
4 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 0 + 0 0
5 0 + 0 + + 0 + + + + + + 0 + + + + 0 3+ 0
6 0 0 + + + 0 + + 0 + + + 0 + 0 + + + 2+ 0
7 + + 0 0 + + 0 0 + 0 0 + + + + 0 0 + 0 0
8 0 0 0 + + + 0 0 + + + 0 0 + + + + + 2+ 0
9 + 0 + + 0 0 0 +s 0 + 0 + 0 + 0 + 0 + 0 0
10 0 0 0 + + 0 + + 0 + 0 + 0 + 0 + + + 2+ 0
11 + + + + + 0 + + + + + + 0 + 0 + + + 2+ 0
PT 1+ mf 0
Anti-Jka also present in eluate – Why?
106
Patient’s RBC phenotype:
anti-S anti-s anti-K anti-Fya anti-Fyb anti-Jka anti-Jkb
1+ mf 4+ 0 1+ mf 3+ mf 1+ mf 3+ mf
4+ 4+
4+ 4+
Unit # 1
Unit # 2
Next steps
Both units are Jk(a+b+)
Patient needs Jk(a−) RBCs
107
Case Study #4 - conclusionsThe patient has made alloanti-Jka which is present in both serum and eluate.
Transfusion recommendations: Provide Jk(a−) units
Alert patient’s physician that his hemoglobin may continue to drop slowly as Jk(a+) RBCs are cleared from circulation. It may not be evidence that the patient is bleeding.
108
Questions?