What Lies Beneath?A case study of an auto antibody and underlying HTLA

Kelli McGrathRed Cell Reference Lab QLD

Clinical presentation 65 year old woman presents at hospital for bowel resection

Referred to ARCBS due to a “strong auto antibody.”

Hospital reports patient’s DAT is 4+ with IgG

No previous history with ARCBS

Red Cell Reference Results Initial Screen

Red Cell Reference Results

Patient phenotype:

O R2R2, K-, Fy(a+b+), Jk(a+b-), MNSs.

DAT: IgG =10 C3d =0

Red Cell Reference Results

Results of the initial screen indicate adsorption is needed.

3 cell differential allo-adsorption

3 x 30min at 37oC using enzyme treated cells with PEG added

Rhesus MNSs Le Lu Kell Fy Jk SpecialRh D C E c e Cw M N S s P1 a b a b K k Kpa a b a b Type

R1wR1 + + - - + + + + - + - - + - + - + - - + - + Co(a+b-)

R2R2 + - + + - - + - - + +++ - + - + - + - - + + + Co(a+b-)

rr - - - + + - + - + + +++ - - - + + + - + - + -

Red Cell Reference Results

Red Cell Reference Results

Red Cell Reference Results So Far

Auto antibody + ? Second antibody that is destroyed in Enzyme – left behind Possibly HTLA Ch/Rg Knops JMH Cost

Need to exclude clinically significant antibodies

Plasma adsorbed with:

Yka-Kna-McCa-

Kanno - JMH- Csa- Yta- Cha-

*R1R1 cells

3 - 5 - 8 -

*rr cells 5 5 5 8 5 -

Adsorbed plasma tested against HTLA negative cells

C4 coated cells

Application: Anti-Ch/Rg antibodies are directed against the C4d fragment of the fourth component of complement. These antibodies will be enhanced when incubated with erythrocytes that have been coated in vitro with excess C4.

Required materials FNHS 10% sucrose Red cells to be coated

Washed packed cells are incubated with 10% sucrose solution and FNHS @37oC for 30mins, washed 3 times and resuspended in PBS.

Red cell Reference Lab results

Adsorbed with:

C4 coated cells

Uncoated cells

R1R1 10 3rr 10 5

C4 coated cellsAdsorbed plasma + donor plasma

-

What do we know?

Sample contains: Auto-antibody Anti-Ch or anti-Rg

Last step? Inhibition studies to distinguish between anti-Ch and anti-Rg

Rg(-) plasma Ch(-) plasma

Chido Rogers

Rg(-) plasma Ch(-) plasma

Chido

Anti-Chido

Rogers

+ patient plasma + patient plasma

Rg(-) plasma Ch(-) plasma

Chido Rogers

C4 Coated

Anti-Chido

C4 Coated

Inhibition

Titre PBS Control Pt + Ch(-) plasma Pt + Rg(-) plasmaNeat + + 02 + + 04 0 0 08 0 0 016 0 0 032 0 0 064 0 0 0128 0 0 0

Contains Rg Ag Contains Ch Ag

Patient outcome

Report : Auto-AntibodyAnti-Ch antibody

Recommend Transfusion with O R2R2 K- Jkb- units

Thank you

Acknowledgements: Australian governments fund the Australian Red Cross Blood Service for

the provision of blood, blood products and services to the Australian community

Red Cell Reference Lab, Brisbane