Finding the Phenotype of a

Recently Transfused Warm

Autoantibody Patient

Kathy Evans MS, MT(ASCP)BB

Blood Bank Master Tech at UKHC Good Samaritan Hospital

August 18, 2017

gkev@uky.edu

Background

• Indiana Blood Center IRL, Indianapolis, IN

• UK HealthCare Good Samaritan Hospital, Lexington, KY

Objectives

• Identify the key differences between a typical patient and

a patient that presents with a warm autoantibody in the

Blood Bank.

• Describe the additional testing required to complete a

warm autoantibody.

• Discuss the practical use for molecular testing for recently

transfused patients with a warm autoantibody.

Single Alloantibody

BLOOD TYPE

Anti-A Anti-B Anti-A,B Anti-D Ctrl A1 C B C Interp

4+ 0 4+ 4+ 0 0 4+ A positive

IAT SCREEN

Cell Antigens Gel

D C E c e K k Fya Fyb Jka Jkb M N S s

I + + 0 0 + 0 + + + 0 + + + 0 + 0

II + 0 + + 0 0 + + 0 + 0 0 + 0 + 3+

III 0 0 0 + + + + 0 + + + + 0 + 0 0

INTERPRETATION: IAT Positive

Case Patient: Atypical, Andy

IAT PANEL

Cell Antigens Testing

D C E c e K k Fya Fyb Jka Jkb M N S s Gel

1 + + 0 0 + 0 + 0 + + 0 + + 0 + 0

2 + + 0 0 + 0 + + + + 0 0 + 0 + 0

3 + 0 + + 0 0 + + + + + + 0 0 + 4+

4 + 0 0 + + 0 + 0 0 + 0 + + 0 + 0

5 0 + 0 + + 0 + 0 + + 0 + + 0 + 0

6 0 0 + + + 0 + 0 0 + 0 + + 0 + 3+

7 0 0 0 + + + + 0 + 0 + + 0 + + 0

8 0 0 0 + + 0 + + 0 + + + + 0 + 0

9 0 0 0 + + 0 + + 0 + + + + 0 + 0

10 0 0 0 + + 0 + + 0 0 + 0 + + 0 0

AC 0

INTERPRETATION: Anti-E

Additional testing

• Panel with Autocontrol

• Direct Antiglobulin Test

• Elution

• IgG removal

• Cell separation*

• Extended red cell phenotype

• Auto-adsorption or Allo-adsorption*

• Auto-checks

Looks like a Warm

Autoantibody

Case Patient: Transfused, Teddy

BLOOD TYPE

Anti-A Anti-B Anti-A,B Anti-D Ctrl A1 C B C Interp

4+mf 0 4+mf 4+ 0 0 4+ A positive

IAT SCREEN

Cell Antigens Gel

D C E c e K k Fya Fyb Jka Jkb M N S s

I + + 0 0 + 0 + + + 0 + + + 0 + 4+

II + 0 + + 0 0 + + 0 + 0 0 + 0 + 4+

III 0 0 0 + + + + 0 + + + + 0 + 0 4+

INTERPRETATION: IAT Positive

DAT SCREEN

Poly IgG IgG IgG Ctrl C3 C3

ctrl

Interp

3+mf 3+mf 0 0 0 positive

Rh Phenotype

C E c e Ctrl

2+mf 1+mf 4+ 4+ 0

Looks like a Warm

Autoantibody

Case Patient: Transfused, Teddy

IAT PANEL

Cell Antigens Testing

D C E c e K k Fya Fyb Jka Jkb M N S s Gel PEG LISS

1 + + 0 0 + 0 + 0 + + 0 + + 0 + 4+ 4+ 3+

2 + + 0 0 + 0 + + + + 0 0 + 0 + 4+ 4+ 3+

3 + 0 + + 0 0 + + + + + + 0 0 + 4+ 4+ 3+

4 + 0 0 + + 0 + 0 0 + 0 + + 0 + 4+ 4+ 3+

5 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 + + 4+ 4+ 3+

8 0 0 0 + + 0 + + 0 + + + + 0 + 4+ 4+ 3+

9 0 0 0 + + 0 + + 0 + + + + 0 + 4+ 4+ 3+

10 0 0 0 + + 0 + + 0 0 + 0 + + 0 4+ 4+ 3+

AC 4+ 4+ 3+

Elution

Patient’s Red Cells Eluate

IgG IgG

IgG

IgG IgG

Case Patient: Transfused, Teddy

Acid wash

ELUTION

Cell Antigens Testing

D C E c e K k Fya Fyb Jka Jkb M N S s ELU LW

SC I + + 0 0 + 0 + + + 0 + 0 + + 0 4+ 0

SC2 + 0 + + 0 + + 0 + + 0 0 + 0 + 4+ 0

SC3 0 0 0 + + 0 + + 0 + + + 0 0 + 4+ 0

IgG Coated Red Cells

• When your red cells are coated with IgG

• EGA (EDTA glycine acid)

• CDP (Choloroquin diphosphate)

Y Ls

IgG

IgG

Ls Ls

Ls

Ls Ls

Patient coated red cells Patient treated red cells IgG removed

Recently transfused

• Why is “recently transfused” important to know?

• Cell Separation Techniques:

Retic harvest

Hypotonic cell wash

Impact of Cell Separation

on Phenotype testing:

6mm

Rh Phenotype

C E c e Ctrl

2+mf 1+mf 4+ 4+ 0

Rh Phenotype

C E c e Ctrl

0 0 4+ 4+ 0

Extended phenotype testing

Case Patient: Transfused, Teddy

IgG REMOVAL

Patient 0

Control Donor 0

BLOOD TYPE

Anti-A Anti-B Anti-A,B Anti-D Ctrl A1 C B C Interp

4+ 0 4+ 4+ 0 0 4+ A positive

DAT SCREEN

Poly IgG IgG IgG Ctrl C3 C3

ctrl

Interp

3+ 3+ 0 0 0 positive

Rh Phenotype

C E c e Ctrl

0 0 4+ 4+ 0

Extended Phenotype

K k Fya Fyb Jka Jkb S s M N Lea Leb

0 NA 0 0 0 3+ 0 4+ NA NA NA NA

Auto-Adsorption

• ZZAP treated patient cells

• Patient’s plasma

• Depending on strength of reactions, could be up to 4 serial

adsorptions (45 minutes each)

Patient

Patient

A B

C

Bound and unbound IgG

Alloantibody if present

Allo-Adsorption

• ZZAP treated known cells (R1R1, R2R2, rr)

• Patient’s plasma

• Depending on strength of reactions, could be up to 4 serial

adsorptions each (45 minutes each)

Patient

rr

R2R2

R1R1

A C B

Auto-IgG and Allo-IgG

Transfused WAA Allo-adsorption

ALLO-ADSORPTION PANEL (x4)

Cell Antigens Testing

D C E c e K k Fya Fyb Jka Jkb M N S s R1

LISS

R2

LISS

rr

LISS

1 + + 0 0 + 0 + 0 + + 0 + + 0 + 0 0 0

2 + + 0 0 + 0 + + + + 0 0 + 0 + 0 0 0

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

4 + 0 0 + + 0 + 0 0 + 0 + + 0 + 0 0 0

5 0 + 0 + + 0 + 0 + + 0 + + 0 + 0 0 0

6 0 0 + + + 0 + 0 0 + 0 + + 0 + 0 0 0

7 0 0 0 + + + + 0 + 0 + + 0 + + 3+ 3+ 3+

8 0 0 0 + + 0 + + 0 + + + + 0 + 0 0 0

9 0 0 0 + + 0 + + 0 + + + + 0 + 0 0 0

10 0 0 0 + + 0 + + 0 0 + 0 + + 0 3+ 3+ 3+

PT* 0 0 0

INTERPRETATION: Warm Autoantibody

Case Patient: Transfused, Teddy

Auto-Checks

Case Patient: Transfused, Teddy

IgG IgG

Treated cells

AUTO CHECKS (after cell separation)

DAT IgG PEG-AHG LISS-AHG Gel Eluate

0 3+ 3+ 4+ 3+

Transfused WAA Common

Issues

• Quantity

Not a Sufficient Quantity of patient red

cells

• Quality

-Ability to provide red cell separation

based on when the last transfusion

occurred

-The patient is not producing retics

-IgG coating red cells too heavy to be

removed

Advantages to Molecular

• Can provide extended antigen typing otherwise difficult to conclude with serological testing

• Not effected by recent transfusions

• Future alloantibodies generally limited to absent antigens

• Provide information about “self-antibodies”

• Serologic typing discrepancy or ambiguity

Patient Population

• Anemias (sickle cell, thalassemia), autoantibodies, anti-

CD38, complex multiple unidentified or high frequency

antigen alloantibodies, antibodies with no or little typing

sera available such as Do, Js, Kp, V

Conclusions:

• The key differences between a typical patient and a patient that presents with a warm autoantibody in the Blood Bank are; panreactivity seen in the antibody screen and panel, a positive autocontrol, and a positive DAT.

• Additional testing required to complete a warm autoantibody include; direct antiglobulin testing, elution, IgG removal, extended phenyotyping, cell separation, and adsorptions.

• Antigen typing by Molecular method on recently transfused patients with a warm autoantibody can provide conclusive extended antigen typing dependent of transfusion status and bound IgG on the patient’s red cells. This will aid in future transfusions for these patients.

References

• Technical Manual AABB Bethesda Maryland

• The Blood Group Antigen Facts Book Marion E Reid &

Christine Lomas-Francis

• Modern Blood Banking and Transfusion Practices Denise

M Harmening

• Beadchip Molecular Immunohematology JoAnne M

Moulds