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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.


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


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


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


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)


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


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

18

Test selected RBC panelFocus on anti-D, -C, -E, -S, -Fya, -M ( -K and -Jkb already excluded)


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.


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.

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

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


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


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


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


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


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


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:


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


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


4+ 0 0 4+


3+ 3+ 0 3+ 0 3+ 3+

anti-Lua anti-Lub anti-Yta

0 3+ 3+

RBC phenotyping


4+ 0 0 4+


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


• 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


• BEFORE patient starts anti‐CD38:–Perform baseline type & screen–Baseline phenotype or genotype recommended

57


• 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

59

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

60

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

61

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


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



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



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


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’


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.)

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.

72

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


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+

80

Case #3 – initial panel

anti-IgG anti-C3 controlIS 4+ 0

RT 4+ 0

Chloroquine‐treated RBCs:anti-IgG

IS 0RT


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+

81

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

82

ZZAP denatures MNSs, Kk, Fy

adsorbed sera


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


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


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


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



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



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



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



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

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


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


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


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


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


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


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


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


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


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:


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?

[email protected]

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