Antibody_Identification_Case red cell.pdf

Antibody identification case study for red cell antibodies

1. The need is constant.
The gratification is instant.
Give blood.
TM
Antibody Identification
Case Studies
Karen Rodberg, MBA, MT (ASCP) SBB
Director, Immunohematology Laboratory
American Red Cross, Southern California Region

2. Commonly used problem-solving techniques:
–Proteolytic enzymes
–Reducing (thiol) reagents
–Titration and neutralization
–Adsorption
–Elution

3. 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

4. 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

5. 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

6. Other effects on RBCs
Effect on RBC antigens:
•Some antigens are denatured
•Some antigen-antibody
reactivity is enhanced
Therefore not used for routine
antibody detection, but very
useful in antibody identification
6

7. 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

8. Enzymes can denature or enhance:
Antigen denaturation(ficin/papain)
M, N, S, En
a
TS, En
a
FS,
Fy
a
, Fy
b
, Fy6, Ge2, Ge4, In
b
,
Ch, Rg, JMH, Pr, Xg
a
, s*, Yt
a
*
*variable
Antigen-antibody reactivity enhancement
P
1
, I, i, Lewis, Rh, Kidd, Colton,
Dombrock
8

9. Case Study #1 –case history
33 y.o. female obstetrical patient
Full term delivery
No prenatal care
4
th
pregnancy
9

10. Case Study #1
Rh type
Anti-D Cntl
00
Interpretation: Rh negative
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
I
++00++0+++0+0+0++0 1+ 3+
II
+0++00++0++++++0++ 0 3+
III
000++0+++0+00+++0+ 2+ 1+
A
BO group Anti-A Anti-B
A
1
RBCs
B
RBCs
0 0 4+ 4+
Interpretation: Group O
10

11. Case Study #1 –Initial Panel
Evidence of multiple alloantibodies – some reactivity at RT and
additional reactivity by indirect antiglobulin test.
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00+0+++0+00+0+0+ 2+ 3+
2++00+0++0+++0++00+ 0 3+
30+0++0++0+0+0+0++0 0 0
4+0++000+0+++0+0+0+ 0 3+
5+00+++0+++++++00+0 1+ 3+
6++0++0+0++0+0+0+0+ 1+ 3+
7+++++0+0++++0++00+ 1+ 3+
8++0++00+++0+++0+++ 1+ 3+
9000+++00++0+0+++0+ 1+ 0
10000++0+++0+0++0+0+ 2+ 1+
11+00++0++++0+0+0+0+ 1+ 3+
PT00
11

12. Begin exclusion with cell #3
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00+0+++0+00+0+0+ 2+ 3+
2++00+0++0+++0++00+ 0 3+
30+0++0++0+0+0+0++0 0 0
4+0++000+0+++0+0+0+ 0 3+
5+00+++0+++++++00+0 1+ 3+
6++0++0+0++0+0+0+0+ 1+ 3+
7+++++0+0++++0++00+ 1+ 3+
8++0++00+++0+++0+++ 1+ 3+
9000+++00++0+0+++0+ 1+ 0
10000++0+++0+0++0+0+ 2+ 1+
11+00++0++++0+0+0+0+ 1+ 3+
PT00
12

13. Examine RT reactivity
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00+0+++0+00+0+0+ 2+ 3+
2++00+0++0+++0++00+ 0 3+
30+0++0++0+0+0+0++0 0 0
4+0++000+0+++0+0+0+ 0 3+
5+00+++0+++++++00+0 1+ 3+
6++0++0+0++0+0+0+0+ 1+ 3+
7+++++0+0++++0++00+ 1+ 3+
8++0++00+++0+++0+++ 1+ 3+
9000+++00++0+0+++0+ 1+ 0
10000++0+++0+0++0+0+ 2+ 1+
11+00++0++++0+0+0+0+ 1+ 3+
PT00
Look first at Le
a
, Le
b
, P
1
, M, N – these antibodies most often react at RT
13

14. Examine RT reactivity
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00+0+++0+00+0+0+ 2+ 3+
2++00+0++0+++0++00+ 0 3+
30+0++0++0+0+0+0++0 0 0
4+0++000+0+++0+0+0+ 0 3+
5+00+++0+++++++00+0 1+ 3+
6++0++0+0++0+0+0+0+ 1+ 3+
7+++++0+0++++0++00+ 1+ 3+
8++0++00+++0+++0+++ 1+ 3+
9000+++00++0+0+++0+ 1+ 0
10000++0+++0+0++0+0+ 2+ 1+
11+00++0++++0+0+0+0+ 1+ 3+
PT00
Reactivity pattern matches anti-M, showing dosage,
but need non-reactive RBCs for exclusion.
14

15. Test same panel ficin‐treated
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
PEG
IAT
Ficin
IAT
1++00+0+++0+00+0+0+ 3+ 2+
2++00+0++0+++0++00+ 3+ 2+
30+0++0++0+0+0+0++0 00
4+0++000+0+++0+0+0+ 3+ 2+
5+00+++0+++++++00+0 3+ 2+
6++0++0+0++0+0+0+0+ 3+ 2+
7+++++0+0++++0++00+ 3+ 2+
8++0++00+++0+++0+++ 3+ 2+
9000+++00++0+0+++0+ 00
10000++0+++0+0++0+0+ 1+ 0
11+00++0++++0+0+0+0+ 3+ 2+
PT00
15

16. Test same panel ficin‐treated
(Some alloantibodies have already been excluded with cell #3)
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
PEG
IAT
Ficin
IAT
1++00+0+++0+00+0+0+ 3+ 2+
2++00+0++0+++0++00+ 3+ 2+
30+0++0++0+0+0+0++0 00
4+0++000+0+++0+0+0+ 3+ 2+
5+00+++0+++++++00+0 3+ 2+
6++0++0+0++0+0+0+0+ 3+ 2+
7+++++0+0++++0++00+ 3+ 2+
8++0++00+++0+++0+++ 3+ 2+
9000+++00++0+0+++0+ 00
10000++0+++0+0++0+0+ 1+ 0
11+00++0++++0+0+0+0+ 3+ 2+
PT00
16

17. Now exclude ficin‐resistant abys
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
PEG
IAT
Ficin
IAT
1++00+0+++0+00+0+0+ 3+ 2+
2++00+0++0+++0++00+ 3+ 2+
30+0++0++0+0+0+0++0 00
4+0++000+0+++0+0+0+ 3+ 2+
5+00+++0+++++++00+0 3+ 2+
6++0++0+0++0+0+0+0+ 3+ 2+
7+++++0+0++++0++00+ 3+ 2+
8++0++00+++0+++0+++ 3+ 2+
9000+++00++0+0+++0+ 00
10000++0+++0+0++0+0+ 1+ 0
11+00++0++++0+0+0+0+ 3+ 2+
PT00
17

18. Also helpful to phenotype patient
Anti-C Anti-E Anti-c Anti-e
0 0 4+ 4+
anti-S anti-s anti-K anti-Fy
a
anti-Fy
b
anti-Jk
a
anti-Jk
b
03+ 004+ 4+ 0
anti-M anti-N
04+
Patient can make alloanti-D, -C, -E, -S, -K, -Fy
a
, -Jk
b
, -M
18

19. Test selected RBC panel
Focus on anti-D, -C, -E, -S, -Fy
a
, -M ( -K and -Jk
b
already excluded)
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
PEG
IAT
Ficin
IAT
1+00++0++0+++0+0+++ 3+ 3+
D
20+00+0++0+++0+0+0+ 00
C
300+++0++0+0+0+0+++ 00
E
400+++00+0++00+0+0+ 00
S
5000+++0+0++++++0+0 00
Fya
600+++0+0++0+0+0+0+ 1+ 0
M
7+00++0+00+++0++00+ 3+ 3+
D
80+0++00+0+0+++0+++ 00
C
900++++000+0+0+++0+ 00
E
10000++0++0++0++0+0+ 00
S
11+00++0++0+0+0+0+0+ 00
Fya
12000++0+++0+0++0+0+ 2+ 0
M 19

20. Exclude and confirm antibody i.d.
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
PEG
IAT
Ficin
IAT
1+00++0++0+++0+0+++ 3+ 3+ D
20+00+0++0+++0+0+0+ 00C
300+++0++0+0+0+0+++ 00E
400+++00+0++00+0+0+ 00S
5000+++0+0++++++0+0 00Fya
600+++0+0++0+0+0+0+ 1+ 0M
7+00++0+00+++0++00+ 3+ 3+ D
80+0++00+0+0+++0+++ 00C
900++++000+0+0+++0+ 00E
10000++0++0++0++0+0+ 00S
11+00++0++0+0+0+0+0+ 00Fya
12000++0+++0+0++0+0+ 2+ 0M
Anti-D and anti-M confirmed
20

21. Case Study #1 conclusion
Anti-D and anti-M confirmed
Anti-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.

22. Commonly used problem-solving techniques:
–Proteolytic enzymes
–Reducing (thiol) reagents
–Titration and neutralization
–Adsorption
–Elution

23. 23Antigen Denaturation:
Effect of DTT (or AET) on RBCs Reduce disulfide bonds in structure of proteins
→ denaturation of antigens
Antigens destroyed:
Kell antigens
Knops
JMH, Yt
a
, Gy, Hy
Cromer (weakened)
Lutheran (weakened)
Vel (variable)
Reducing (thiol) reagents

24. Usefulness of Enzymes and DTT:
Ficin/ Possible Antibody
Papain DTT or Antibody in System
neg pos Fy
a
/Fy
b
; Ch/Rg; Ge2, Ge4
neg neg Indian; JMH
pos weak Cromer; Knops; Lutheran;
Dombrock; AnWj; MER2
variable neg Yt
a
pos neg Kell; LW
pos pos Rh; Jk3; Fy3; Diego; Colton;
Ge3; Ok
a
; I,i; P,LKE; At
a
; Cs
a
;
Er
a
; Jr
a
; Lan; Vel; Sd
a
,
Scianna

25. RBC antigens denatured by ZZAP
ZZAP is a combination of enzyme and DTT
(--frequently used for adsorptions)
Antigens denatured:
M, N, S, s*, Fy
a
, Fy
b
, Yt
a
*, Xg
a
,
JMH, Ch, Rg, En
a
TS, En
a
FS, Ge2, Ge4,
LW, Kell, Dombrock, Lutheran, and
Scianna system antigens
*variable

26. Commonly used problem-solving techniques:
–Proteolytic enzymes
–Reducing (thiol) reagents
–Titration and neutralization
–Adsorption
–Elution

27. Neutralization / inhibition
Antigens in soluble form can be used to
inhibit or neutralize reactivity to aid in
antibody identification
–A, B, H, Le
a
, Le
b
, P
1
(blood group substance)
–Ch, Rg (pooled normal plasma)
–Sd
a
(urine)

28. 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

29. Examples of titration / neutralization
dilution
1 2 4 8 16 32 64 128 256 512
plasma
1+1+1+1+±±±±±
0
albumin
1+1+1+1+±±±±±
0
plasma
0000000000
albumin
1+1+1+1+±±±±±
0
29

30. Neutralization with plasma
Neutralized anti-Ch, -Rg
Not neutralized anti-JMH, -Kn
a
,
-McC
a
, -Sl
a
,
-Yk
a
, -Cs
a
Possible other
alloantibodies
30

31. Case Study #2 –case history
59 y.o. woman with multiple myeloma,
transfused 3½ months earlier
No medication history given
Antibody i.d. requested, no blood
Patient discharged
Group O Rh Positive
Plasma:LISS-IgG = weak pos all RBCs
autocontrol = neg

32. Case Study #2
Anti-A Anti-B
A
RBCs
B
RBCs
IS
Anti-D
IS
Cntl
0 0 4+ 4+ 4+ 0
Anti-
IgG
Anti-
C3
10%
BSA
0

0

0
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
I++00++0+++0+0+0++0 0 1+
II+0++00++0++++++0++ 0 1+
III000++0+++0+00+++0+ 0 1+
ABO/Rh: DAT:
Antibody screen:

33. Initial Antibody Panel
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00++0+0+0+0+0++0 0 1+
2++00+000++++0++0++ 0 1+
3+0++00++0++++++0++ 0 1+
4000++00++0+00+000+ 0 1+
50+0++0+++++00++++0 0 1+
600+++0++0+++0+0+++ 0 1+
7++0+++0+0++00+00+0 0 0

8++++++00++0+0+++++ 0 1+
PT00

** Initial panel shows weak reactivity with 7 of 8 RBCs
Autocontrol negative, so we assume this is alloantibody

34. Antibody Panel also tested with 
ficin and DTT‐treated RBCs
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
Ficin
IAT
DTT
IAT
1++00++0+0+0+0+0++0 0 1+ 1+ 0

2++00+000++++0++0++ 0 1+ 1+ 0

3+0++00++0++++++0++ 0 1+ 1+ 0

4000++00++0+00+000+ 0 1+ 1+ 0

50+0++0+++++00++++0 0 1+ 1+ 0

600+++0++0+++0+0+++ 0 1+ 1+ 0

7++0+++0+0++00+00+0 0 0

0

0

8++++++00++0+0+++++ 0 1+ 1+ 0

PT00

**  IRL frequently tests ficin‐treated and DTT‐treated RBCs to characterize    
the antibody reactivity.  This antibody appears to be DTT‐sensitive.

35. Exclusion of antibodies
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
Ficin
IAT
DTT
IAT
1++00++0+0+0+0+0++0 0 1+ 1+ 0

2++00+000++++0++0++ 0 1+ 1+ 0

3+0++00++0++++++0++ 0 1+ 1+ 0

4000++00++0+00+000+ 0 1+ 1+ 0

50+0++0+++++00++++0 0 1+ 1+ 0

600+++0++0+++0+0+++ 0 1+ 1+ 0

7++0+++0+0++00+00+0 0 0

0

0

8++++++00++0+0+++++ 0 1+ 1+ 0

PT00

** 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.

36. Titration/Neutralization
tube #
123456789101112
Titer
Interp
dilution→
neat 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:10241:2048
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. 37Review: DTT-sensitive antigens associated
with antibodies with ‘HTLA’ characteristics 
Kell

Knops

LW

JMH

Indian

Dombrock

YT (variable)

Lutheran (variable)

Gerbich (variable)

Scianna (variable)

38. Selected Rare RBCs
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++++++0+0+0+0+++++ Yt(a-) 0 1+
2+0+++ 0++++0 +0++ JMH- 0 1+
3++00+0000+++0 +0++ Sc:-3 0 1+
4+00++00++0+00+000+Kp(b-) 0 1+
5++00+0+++++00 +++0 Ge:-3 0 1+
6000++0+++00+++0++0 Kn(a-) 0 1+
7++00+0+0++0+00++0+ K null 0 1+
8000++0+00+0+0++0+0LW(a-b-)0 1+
9+++++0+0++0+++0++0Lu(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+
12000++00+++++0++0++ Yk(a-) 0 1+ ** Selected cells focused on DTT‐sensitive antigens and antibodies that     
may have “HTLA” characteristics.

39. Selected Rare RBCs –exclusion:
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++++++0+0+0+0+++++ Yt(a-) 0 1+
2+0+++ 0++++0 +0++ JMH- 0 1+
3++00+0000+++0 +0++ Sc:-3 0 1+
4+00++00++0+00+000+Kp(b-) 0 1+
5++00+0+++++00 +++0 Ge:-3 0 1+
6000++0+++00+++0++0 Kn(a-) 0 1+
7++00+0+0++0+00++0+ K null 0 1+
8000++0+00+0+0++0+0LW(a-b-)0 1+
9+++++0+0++0+++0++0Lu(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+
12000++00+++++0++0++ Yk(a-) 0 1+ ** One example of Lu(a─b─) RBCs was non‐reactive 

40. Addi?onal rare Lu(a−b−) RBCs
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00++0+0+0+0+0++0Lu(a-b-)0 0

2 0 0 0 + + 0 0 + 0 + + Lu(a-b-) 0 0

3000++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

600+++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 commonalloantibodies could be excluded, 
including anti‐K.

41. RBC phenotyping
Anti-C Anti-E Anti-c Anti-e
4+ 0 0 4+
anti-S anti-s anti-K anti-Fy
a
anti-Fy
b
anti-Jk
a
anti-Jk
b
3+ 3+ 0

3+ 0

3+ 3+
anti-Lu
a
anti-Lu
b
anti-Yt
a
03+3+

42. RBC phenotyping
Anti-C Anti-E Anti-c Anti-e
4+ 0 0 4+
anti-S anti-s anti-K anti-Fy
a
anti-Fy
b
anti-Jk
a
anti-Jk
b
3+ 3+ 0

3+ 0

3+ 3+
anti-Lu
a
anti-Lu
b
anti-Yt
a
03+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. 43Summary –(note:  this was August 2014) •Plasma:
–DTT‐tt’d RBCs = neg
–Reactive RBCs: 
Yt(a−), JMH−, Kp(b−), K
0
, 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. 44Case #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 maybe 
related to medication

45. 452014 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. 46Hindsight is 20/20

47. 47Daratumumab •Daratumumab (DARA) is an IgG1κhuman 
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. 48Daratumumab •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. 49Daratumumab 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. 50DTT‐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, ‐Do
a
, ‐Do
b
)

51. 512015 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 Lu
b
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

52. Flow Cytometry
Untreated RBCs
% Pos = 62%
0.2M DTT‐tt’dRBCs
% Pos = 13%
RBC Background 
(Autofluorescence) 
RBCs + 
PE anti‐CD38

53. Flow CytometryResults ‐
CD38 Expression on Selected RBCs
0
10
20
30
40
50
60
70
80
90
100
Flow cytometry (% positive)
Fy(a+)
Fy(b+)
Fy(a‐b‐)     Lu(a‐b‐)       Cord      DTT‐tt'd DARA
Pts
#1
#2

54. 54AABB 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. 55AABB 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. 56AABB Association Bulletin #16‐02 •BEFORE patient starts anti‐CD38:
–Perform baseline type & screen
–Baseline phenotype or genotype 
recommended

57. 57AABB 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. 58Communication 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

59. 59Selecting 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

60. 60Panel of CD38-depressed RBCs 
Patients on DARA tend to have depression of
CD38 on their RBCs
1

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
1
Sullivan HC, et al. Transfusion 2016; 56 Suppl:25A
2
Velliquette RW, et al. Transfusion 2016; 56 Suppl:26A

61. 61Current 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

62. Example of current serology
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00++0+0+0+0+0++0 0 1+
2++00+000++++0++0++ 0 1+
3+0++00++0++++++0++ 0 1+
4000++00++0+00+000+ 0 1+
50+0++0+++++00++++0 0 1+
600+++0++0+++0+0+++ 0 1+
7+00+++0+0+++0+00+0 0 1+
8++++++00++0+0+++++ 0 1+
PT00


63. Example of current serology
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
Ficin
IAT
DTT
IAT
1++00++0+0+0+0+0++0 0 1+ 1+ 0

2++00+000++++0++0++ 0 1+ 1+ 0

3+0++00++0++++++0++ 0 1+ 1+ 0

4000++00++0+00+000+ 0 1+ 1+ 0

50+0++0+++++00++++0 0 1+ 1+ 0

600+++0++0+++0+0+++ 0 1+ 1+ 0

7+00+++0+0+++0+00+0 0 1+ 1+ 0

8++++++00++0+0+++++ 0 1+ 1+ 0

PT00


64. Example of current serology
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
Ficin
IAT
DTT
IAT
1++00++0+0+0+0+0++0 0 1+ 1+ 0

2++00+000++++0++0++ 0 1+ 1+ 0

3+0++00++0++++++0++ 0 1+ 1+ 0

4000++00++0+00+000+ 0 1+ 1+ 0

50+0++0+++++00++++0 0 1+ 1+ 0

600+++0++0+++0+0+++ 0 1+ 1+ 0

7+00+++0+0+++0+00+0 0 1+ 1+ 0

8++++++00++0+0+++++ 0 1+ 1+ 0

PT00

**  Using the DTT‐treated panel RBCs, all common alloscan be 
excluded, with the exception of anti‐K (and anti‐k).

65. Selected cell panel
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
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

4000++ +00+000+CD38−RBCs00

50+0++ 0+0++++0CD38−RBCs00

600+++ ++0+0+++CD38−RBCs00

7+00++ ++0+00+0CD38−RBCs00

8+++++ ++++0+++CD38−RBCs00


66. Selected cell panel –‘DARA RBCs’
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
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

4000++ +00+000+CD38−RBCs00

50+0++ 0+0++++0CD38−RBCs00

600+++ ++0+0+++CD38−RBCs00

7+00++ ++0+00+0CD38−RBCs00

8+++++ ++++0+++CD38−RBCs00

** Using the cord RBCs, KT’s RBCs, and CD38‐depressed RBCs we can 
also exclude anti‐K and anti‐k.

67. 67Transfusion 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.)

68. Commonly used problem-solving techniques:
–Proteolytic enzymes
–Reducing (thiol) reagents
–Titration and neutralization
–Adsorption
–Elution

69. Adsorption options
Remove auto-antibody to detect/rule out
alloantibodies
•Types
Autologous –only if pt not recently tx’d
Allogeneic -differential
•Methods
ZZAP
Enzyme
PEG
•Temperature (warm and/or cold)
69

70. Example of Adsorption
Add 1 vol of
adsorbing
RBCs
Empty
7ml tube
Add 1 vol of pt plasma
Mix
70

71. 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

72. ‘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.
72

73. Differential adsorption
Allogeneic adsorbing RBC selection
–Differential adsorption
▪Do not need to know pt RBC phenotype
▪RBCs from 3 donors whose RBC
phenotypes collectivelylack all common
clinically significant antigens
–D, C, E, c, e, S, s, K, Fy
a
, Fy
b
, Jk
a
, Jk
b

74. 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

75. 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 ++00+0+0+++0 + + 0 + +
#1 Ficin + + 0 0 + 0 0 0 0 + + 0 + 0 0 + +
#1 ZZAP++00+0000000 + 0 0 + +

76. 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 Jkb
A ++00+0+0+++0 + + 0 + +
B +0++0++++0+0+0+0+
C 000+++0+00+0 + + + + 0

77. 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 Jkb
A ++00+++++0+0 + 0 + + +
B +0++0++++0+0 + + + 0 +
C 000+++++00+0 + + + + 0
D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb
A ++00+00000+0 + 0 0 + +
B +0++000000+0 + 0 0 0 +
C 000++00000+0 + 0 0 + 0

78. 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 Jkb
A ++00+++++++0 + 0 + + +
B +0++0++++0+0 + + + 0 +
C 000+++++00+0 + + + + 0
D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb
A ++00+0000000 + 0 0 + +
B +0++00000000 + 0 0 0 +
C 000++0000000 + 0 0 + 0

79. Case study #3 –case history
60 year old Caucasian female
Dx: lymphoma
Multiple transfusions Jan –April 2010
History of Anti-K
Last transfusion 2 months ago, 2 units of K−
RBCs when only anti-K id’d
Hb 7.3 g/dl

80. Cell Typing: Reverse Typing:
Anti- A Anti- B anti-D control
A
1
B
004+0 4+4+
Case #3 –initial testing
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
I++00++0+++0+0+0++0 0 4+
II+0++00++0++++++0++ 0 4+
III000++0+++0+00+++0+ 0 4+
80

81. Case #3 –initial panel
anti-IgG anti-C3 control
IS 4+ 0

RT 4+ 0

Chloroquine‐treated RBCs:
anti-IgG
IS0

RT
DCEc eLe
a
Le
b
P
1
MNS sKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
Ficin
IAT
LISS
IAT
1++00++0+0+0+0+0++0 0 4+ 4+ 3+
2++00+000++++0++0++ 0 4+ 4+ 3+
3+0++00++0++++++0++ 0 4+ 4+ 3+
4000++0+0+0+00+000+ 0 4+ 4+ 3+
5000++0++++++0++++0 0 4+ 4+ 3+
6+00++0++0+++0+0+++ 0 4+ 4+ 3+
7++00++00+00++++00+ 0 4+ 4+ 3+
80+0+++00+++00+++++ 0 4+ 4+ 3+
9+0++000+0+0+0+0+0+ 0 4+ 4+ 3+
10000++0++0+0+0+0+++ 0 4+ 4+ 3+
11++0++0++++++0+0+++ 0 4+ 4+ 3+
PT
CDP-
ttd
04+
81

82. ZZAP‐treated RBCs for differential 
adsorptions
adsorbed sera
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00++00+0++0 R1 0
2+0++00+0++++0 R2 0
3000++++0++00+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6000+++++++00+ 3+ 3+ 3+
ZZAP adsorbed x2 double volume @ 37C for 30 minutes
82

83. ZZAP denatures MNSs, Kk, Fy
adsorbed sera
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6000+++++++00+ 3+ 3+ 3+
ZZAP treatment: combination of enzyme + DTT, so affects
antigens on adsorbing cells
83

84. R1 adsorbed serum
adsorbed sera
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6000+++++++00+ 3+ 3+ 3+
•R1 adsorbed serum: would contain anti –E, -c, -S, -s, -K, -k, -Fy
a
, -Fy
b
, Jk
b
84

85. R2 adsorbed serum
adsorbed sera
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6000+++++++00+ 3+ 3+ 3+
•R2 adsorbed serum: would contain –C, -e, -S, -s, -K, -k, -Fy
a
, -Fy
b
, -Jk
b
85

86. rr adsorbed serum
adsorbed sera
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6000+++++++00+ 3+ 3+ 3+
•rr adsorbed serum: would contain –D, -C, -E, -S, -s, -K, -k, -Fy
a
, -Fy
b
, -Jk
a
86

87. Additional selected RBCs
adsorbed sera Selected cell panel:
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6+0++00+++0++0 3+ 3+ 3+
7++00+0+0++0+0 0

00
8000++0++++++0 3+ 3+ 3+
9+00++0+0+0+0+ 0

00
10+0++00+0+0++0 2+ 02+
87

88. R1 column
adsorbed sera Selected cell panel:
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6+0++00+++0++0 3+ 3+ 3+
7++00+0+0++0+0 0

00
8000++0++++++0 3+ 3+ 3+
9+00++0+0+0+0+ 0

00
10+0++00+0+0++0 2+ 02+
88

89. R2 column
adsorbed sera Selected cell panel:
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6+0++00+++0++0 3+ 3+ 3+
7++00+0+0++0+0 0

00
8000++0++++++0 3+ 3+ 3+
9+00++0+0+0+0+ 0

00
10+0++00+0+0++0 2+ 02+
89

90. rr column
adsorbed sera Selected cell panel:
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0 2+ 02+
6+0++00+++0++0 3+ 3+ 3+
7++00+0+0++0+0 0

00
8000++0++++++0 3+ 3+ 3+
9+00++0+0+0+0+ 0

00
10+0++00+0+0++0 2+ 02+
90

91. Panel interpretation
adsorbed sera Selected cell panel:
DCEc eSsKkFy
a
Fy
b
Jk
a
Jk
b
R1 R2 rr
1++00+000000+0 R1 0
2+0++0000000+0 R2 0
3000++0000000+ rr 0
4++00++00+0+++ 000
5+0++00+0+0++0E+2+ 02+
6+0++00+++0++0E+ K+3+ 3+ 3+
7++00+0+0++0+0 0

00
8000++0++++++0K+3+ 3+ 3+
9+00++0+0+0+0+ 0

00
10+0++00+0+0++0E+2+ 02+
91

92. 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

93. Commonly used problem-solving techniques:
–Proteolytic enzymes
–Reducing (thiol) reagents
–Titration and neutralization
–Adsorption
–Elution

94. 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

95. Uses for elution
Investigation of + DAT
Autoimmune hemolytic anemia
Hemolytic transfusion reaction
Hemolytic disease of fetus/newborn
Drug-induced immune hemolytic
anemia
Antibody identification
Adsorption/elution, antibody separation
Preparation of antibody-free intact RBCs
(eg, for phenotyping, autoadsorption)
95

96. Elution methods to recover antibody
Heat (56C) ABO HDFN, IgM
agglutinating
antibodies
Easy, poor
recovery for
IgG
Luifreeze-
thaw
ABO HDFN onlyQuick, small
volRBCs
Acid Warm auto-&
alloantibodies
Easy, kits
available
Chemical/
organic
solvents
Warm auto-&
alloantibodies
Chemical
hazards
96

97. 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

98. ABO/Rh typing
DAT
Rh
phenotyping
Anti-A Anti-B
A

RBCs
B
RBCs
IS
Anti-D
IS
Cntl
0 0 4+ 4+ 4+ 0
Anti-
IgG
A
nti-
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

99. ABO/Rh typing
DAT
Rh
phenotyping
Anti-A Anti-B
A

RBCs
B
RBCs
IS
Anti-D
IS
Cntl
0 0 4+ 4+ 4+ 0
Anti-
IgG
A
nti-
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 R
o
r, but recently transfused,
so can’t say for sure. Mixed-field react ivity is evidence of two or more
RBC populations.
99

100. DCEc eLe
a
Le
b
P
1
MNSsKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
I++00++0+++0+0+0++0 0 2+
II+0++00++0++++++0++ 0 (+)
III000++0+++0+00+++0+ 00

0(+)
0(+)
Crossmatches:
Unit #1
Unit #2
Case Study #4 –antibody screen 
and crossmatches
(+)
microscopic positive
100

101. Case #4 –initial panel
DCEc eLe
a
Le
b
P
1
MNSsKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00++0+0+0+0+0++0 0 2+
2++00+000++++0++0++ 0 (+)
3+0++00++0++++++0++ 0 (+)
4+00++0+0+0+00+000+ 0 0

50+0++0++++++0++++0 0 2+
600+++0++0+++0+0+++ 0 (+)
7++00++00+00++++00+ 0 0

8000+++00+++00+++++ 0 (+)
9+0++000+s0+0+0+0+0+ 0 0

10000++0++0+0+0+0+++ 0 (+)
11+++++0++++++0+0+++ 0 (+)
PT00
101

102. Exclusion
DCEc eLe
a
Le
b
P
1
MNSsKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00++0+0+0+0+0++0 0 2+
2++00+000++++0++0++ 0 (+)
3+0++00++0++++++0++ 0 (+)
4+00++0+0+0+00+000+ 0 0

50+0++0++++++0++++0 0 2+
600+++0++0+++0+0+++ 0 (+)
7++00++00+00++++00+ 0 0

8000+++00+++00+++++ 0 (+)
9+0++000+s0+0+0+0+0+ 0 0

10000++0++0+0+0+0+++ 0 (+)
11+++++0++++++0+0+++ 0 (+)
PT00
102

103. DCEc eLe
a
Le
b
P
1
MNSsKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00++0+0+0+0+0++0 0 2+
2++00+000++++0++0++ 0 (+)
3+0++00++0++++++0++ 0 (+)
4+00++0+0+0+00+000+ 0 0

50+0++0++++++0++++0 0 2+
600+++0++0+++0+0+++ 0 (+)
7++00++00+00++++00+ 0 0

8000+++00+++00+++++ 0 (+)
9+0++000+s0+0+0+0+0+ 0 0

10000++0++0+0+0+0+++ 0 (+)
11+++++0++++++0+0+++ 0 (+)
PT00
103

104. DCEc eLe
a
Le
b
P
1
MNSsKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00++0+0+0+0+0++0 0 2+
2++00+000++++0++0++ 0 (+)
3+0++00++0++++++0++ 0 (+)
4+00++0+0+0+00+000+ 0 0

50+0++0++++++0++++0 0 2+
600+++0++0+++0+0+++ 0 (+)
7++00++00+00++++00+ 0 0

8000+++00+++00+++++ 0 (+)
9+0++000+s0+0+0+0+0+ 0 0

10000++0++0+0+0+0+++ 0 (+)
11+++++0++++++0+0+++ 0 (+)
PT00
104

105. DCEc eLe
a
Le
b
P
1
MNSsKkFy
a
Fy
b
Jk
a
Jk
b
RT
PEG
IAT
1++00++0+0+0+0+0++0 0 2+
2++00+000++++0++0++ 0 (+)
3+0++00++0++++++0++ 0 (+)
4+00++0+0+0+00+000+ 0 0

50+0++0++++++0++++0 0 2+
600+++0++0+++0+0+++ 0 (+)
7++00++00+00++++00+ 0 0

8000+++00+++00+++++ 0 (+)
9+0++000+s0+0+0+0+0+ 0 0

10000++0++0+0+0+0+++ 0 (+)
11+++++0++++++0+0+++ 0 (+)
PT00
Antibody identified: anti-Jk
a
showing dosage
105

106. Case #4 ‐eluate
DCEc eLe
a
Le
b
P
1
MNSsKkFy
a
Fy
b
Jk
a
Jk
b
Eluate
Last
Wash
1++00++0+0+0+0+0++0 3+0

2++00+000++++0++0++ 2+0

3+0++00++0++++++0++ 2+0

4+00++0+0+0+00+000+0

0

50+0++0++++++0++++0 3+0

600+++0++0+++0+0+++ 2+0

7++00++00+00++++00+0

0

8000+++00+++00+++++ 2+0

9+0++000+s0+0+0+0+0+0

0

10000++0++0+0+0+0+++ 2+0

11+++++0++++++0+0+++ 2+0

PT1+ mf0

Anti-Jk
a
also present in eluate –Why?
106

107. Patient’s RBC phenotype:
anti-S anti-santi-Kanti-Fy
a
anti-Fy
b
anti-Jk
a
anti-Jk
b
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

108. Case Study #4 -conclusions
The 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

109. Questions?
[email protected]
109