Antibody Case StudiesAntibody Case StudiesTh G d th B d d th U lThe Good, the Bad and the UglyKaren Hodgins MLT ARTGHEST SymposiumGHEST SymposiumSeptember 29, 2012

Case 1Case 1

ED from Saint John New BrunswickJ 77 yr old female with unstable angina Urgent cardiac surgery required Urgent cardiac surgery required A Rh Positive

P i A i E d i Previous Anti-E and anti-c R1R1, Kk, Jk(a+b+), Fy(a+b+), M-, N+, S- All panel cells reactive

Initial Panel (Initial Panel (ImmucorImmucor 10)10)Initial Panel (Initial Panel (ImmucorImmucor 10)10)

D C c E e K k Fya Fyb Jka Jkb Lea

Leb M N S s G

el icin

y y J J a b G F

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

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

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

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

0 + + 0 + 0 + 0 + + + 0 + 0 + 0 + 2 30 0 0 0 0 0 0 3

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

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

0 0 + 0 + 0 + + 0 + 0 0 + + 0 + + 2 30 0 + 0 + 0 + + 0 + 0 0 + + 0 + + 2 3

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

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

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

Enzyme treated cellsEnzyme treated cellsEnzyme treated cellsEnzyme treated cells Enzyme cleaves glycophorins A and and B which

carry most of the negatively charged sialic acidcarry most of the negatively charged sialic acid◦ Decreased zeta potential◦ Cells can move closer together◦ Removes some antigens completely and exposes

other Ficin – figsg Papain – Papayas Bromelin – Pineapples

P S i l Pronase – Streptomyces griseus culture supernatant.

Case 1Case 1Case 1Case 1

DAT negativeg Destroyed by ficin Antigens destroyed by ficin Antigens destroyed by ficin◦ M,N,S◦ Fy◦ Fy◦ JMH◦ Ch◦ Ch◦ Rg

Extra Cell PanelExtra Cell PanelExtra Cell PanelExtra Cell Panel

D C c E e K k Lea Leb M N S s Ch Rg Gel

Ch/Rg Inhibition

t nt 0 0

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

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

test

con

1/10

1/10

2 2 0 1

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

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

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

2 2 0 0

2 2 0 1

2 2 0 1

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

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

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

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

0 0 + 0 + 0 + 0 + + + 0 0

ChidoChido/Rodgers Inhibition/Rodgers InhibitionChidoChido/Rodgers Inhibition/Rodgers Inhibition Inhibition/neutralization technique

U d f b d d d Used to confirm antibody identity and to rule out other allo antibodies◦ Source: Plasma (pool of 4 patients same blood Source: Plasma (pool of 4 patients same blood

group)◦ Soluble antigen present in plasma will bind with

antibody thus inhibiting antibody from attaching antibody thus inhibiting antibody from attaching to red cells◦ 6% albumin control must show positive results

for test to be valid (1:2 dilution)for test to be valid (1:2 dilution)◦ High titre anti-Rg may need additional dilutions to

allow inhibition to work.

ChidoChido/Rodgers/RodgersChidoChido/Rodgers/Rodgers Chido/Rodgers not true blood group

tiantigens Located on the C4d of complement which

are adsorbed on to the red cells from the are adsorbed on to the red cells from the plasma

Rg antigen occurs in >98% population Absent or weak on cord RBCs Anti-Rg usually IgG and reacts best using IAT

and does not bind complementand does not bind complement Does not cause HDN or Transfusion

ReactionThe Blood Group Antigen Facts Book 2nd Edition: Marion E. Reid and Christine Lomas-Francis

Case 1 ConclusionCase 1 ConclusionCase 1 ConclusionCase 1 Conclusion

Anti-Rgg Anti-E Anti-c Anti-c Patient phenotype:

R◦ Rg-

Case 2Case 2Case 2Case 2

DM from Windsor 80 yr old female pre-op Last transfused 6 months ago Last transfused 6 months ago ◦ Antibody screen negative at that time

O Positive O Positive

Initial Panel (Initial Panel (ImmucorImmucor 10)10)Initial Panel (Initial Panel (ImmucorImmucor 10)10)

D C c E e K k Fya Fyb Jka Jkb Lea

Leb M N S s G

el icin

y y J J a b G F

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

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

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

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

0 + + 0 + 0 + + + + 0 0 + + 0 0 + 3 40 0 0 0 0 0 0 3

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

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

0 0 + 0 + 0 + + 0 0 + 0 + + 0 + + 3 40 0 + 0 + 0 + + 0 0 + 0 + + 0 + + 3 4

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

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

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

Case 2Case 2Case 2Case 2 DAT positive Due to IgG Eluate non-specific R2R2, Jk(a-b+), Fy(a+b+), S-s+, K-◦ Cells EGA treated to test for Fy and s antigen

? Warm auto antibody, anti-e + ? Allo absorption using R2R2 S-, K-, Jk(a-) cell WARM used to pre-treat cells before

adsorption

Case 2Case 2Case 2Case 2

D C c E e K k Fya Fyb Jka Jkb Lea

Leb M N S s A

lloy y J J a b A

+ + 0 + + 0 + + 0 + + 0 + + + 0 +

+ + 0 0 + 0 + + + + 0 0 + 0 + 0 +

+ 0 + + 0 0 + + + 0 + + 0 + + 0 + 0

+ 0 + 0 + 0 + + 0 + + 0 + + 0 + 0

0 + + 0 + 0 + + + + 0 0 + + 0 0 +0 0 0 0 0 0 0

0 0 + + + + + 0 + 0 + 0 + 0 + 0 +

0 0 + 0 + + + 0 + 0 + 0 + 0 + 0 +

0 0 + 0 + 0 + + 0 0 + 0 + + 0 + +0 0 + 0 + 0 + + 0 0 + 0 + + 0 + +

0 0 + 0 + 0 + 0 0 + + 0 + + + 0 0

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

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

Case 2Case 2Case 2Case 2

Allo absorbed plasma indicates presence p pof other antibody(ies)

Anti-e “interfering” few e negative cells to t e te e g ew e egat ve ce s to perform exclusions

Remove anti-e: Absorb onto rr K- S- Remove anti-e: Absorb onto rr, K-, S-, Jk(a-)◦ After 9 absorptions R1R1 K- S- Jk(a-) non-◦ After 9 absorptions R1R1 K-, S-, Jk(a-) non-

reactive

Case 2Case 2Case 2Case 2

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

llo abs

D C c E e K k Fy Fy Jk Jk a b M N S s A e a

0 0 + 0 + 0 + 0 + 0 + 0 + + 0 + 0 0

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

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

+ 0 + + 0 0 + + + 0 + + 0 + + + + 0

+ 0 + + 0 0 + + 0 0 + 0 + 0 + + + 0+ 0 + + 0 0 + + 0 0 + 0 + 0 + + + 0

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

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

+ + 0 0 + 0 + + 0 0 + 0 + + 0 0 + 0

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

0 0 + + + 0 + + + + 0 0 + + + 0 + 20 0 0 0 0 0 2

+ + + + + + + 0 + 0 + + 0 + 0 0 + 2 1

Case 2 ConclusionCase 2 ConclusionCase 2 ConclusionCase 2 Conclusion

Conclusion◦ Warm Autoantibody◦ Anti-e◦ Anti-Jka

◦ Anti-K

Case 3Case 3Case 3Case 3

TL from Moncton NB 56 yr old male pre-op O Negative O Negative All Panel cells reacting

Case 3Case 3Case 3Case 3

D C c E e K k Fya Fyb Jka Jkb Lea

Leb M N S s G

el icin

y y J J a b G F

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

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

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

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

0 + + 0 + 0 + + 0 + + 0 + + 0 + 0 2 20 0 0 0 0 0 0

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

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

0 0 + 0 + 0 + + 0 + 0 0 + 0 + + + 2 20 0 + 0 + 0 + + 0 + 0 0 + 0 + + + 2 2

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

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

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

Case 3Case 3Case 3Case 3 DAT Negative Phenotype: rr, Fy(a-b+), Jk(a+b+), Ms, kk Decrease using ficin (variable results)g ( ) ?? Ch/Rg, ◦ Inhibition: antibody not inhibited.y

Other high incidence antigens tested◦ Kn(a-), Yt(a-), Hy-, Lu(a-b-), Cs(a-), Sc: 1,2( ) ( ) y ( ) ( )◦ All still reactive

Case 3Case 3Case 3Case 3

DTT treated cells: no changeg a-chymotrypsin treated cells: no change ZZAP treated cells: non-reactive ZZAP treated cells: non-reactive Ficin treated cells: non-reactive (variable)

could there be multiple antibodies?could there be multiple antibodies? Where do we go from here…….

DithiothreitolDithiothreitolDithiothreitolDithiothreitol Reducing agent Cleves disulphide bonds of IgM antibodies,

0.01MA / b d ◦ Antigen/antibody reaction cannot occur

Cleves disulphide bonds within the structure of an antigen 0 2Mstructure of an antigen, 0.2M◦ Kell antigens very susceptible to DTT◦ Also Knops, Dombrock, Lutheran, Cartwright, Also Knops, Dombrock, Lutheran, Cartwright,

Lwa, and JMH◦ Helpful to categorize high incidence antibodies

αα--ChymotypsinChymotypsin treated cellstreated cellsαα ChymotypsinChymotypsin treated cellstreated cells

Enzyme cleves proteins at specific amino y p pacids (carboxyl terminus of leucine, phenylalanine to name a fewp y

Effects glycophorin B, Duffy glycophorinplus other glycophoirnsp g y p

Cromer antigen (Cra)- sensitive to this enzyme but not to Papain/ficinenzyme, but not to Papain/ficin.

In combination with DTT can narrow down the search for antigen specificitydown the search for antigen specificity

Case 3Case 3Case 3Case 3 Absorption/elution technique◦ Used unit that matched patient’s phenotype.◦ Ficin treated unit and tested against plasma Results: non-reactive

◦ Absorbed ficin treated unit with patient’s plasma f 30 i t t 37Cfor 30 minutes at 37C DAT after 30 minutes: +/-

◦ Tested absorbed plasma and eluate made from Tested absorbed plasma and eluate made from absorbed cells against high incidence cells

Case 3Case 3Case 3Case 3

No antibody eluteda y Absorbed plasma

one negative cell??Abs

plas

ma

Elu

ate

Pt.

Pla

sma

Did this work?? Set up plasma against

P

Sc: 1,2 2 0

Ge: -2-3-4 0 0 0 p p gnegative cell: non-reactive!!!

Hy- Wk 0

At(a-) 2 0

Cs(a-) 1 0

More Ge negative cells tested

( )

Co(a-) 2 0

ConclusionConclusionConclusionConclusion

a

Appears to anti-Ge

Pt

plas

ma

Ge 0

pp These cells also ruled

out all other Ge- 0

Ge-1,-2,-3 0

Ge-1,-2,-3 0

common clinically significant antibodies

Ge-1,-2,-3 0

Ge-2,-3,-4 0 Cells frozen for

future testing

GerbichGerbich Blood Group SystemBlood Group SystemGerbichGerbich Blood Group SystemBlood Group System

Most Populations

Melanesians

Ge:2,3,4 (Ge+) > 99.9% 50-90%

Ge-negative

Ge:-2,3,4 (Yus type) Rare Not FoundGe: 2,3,4 (Yus type) Rare Not Found

Ge:-2,-3,4(Gerbich type) Rare 10-50%

Ge:-2,-3,-4 (Leach type) (Null) Rare Not Found

The Blood Group Antigen Facts Book 2nd Edition: Marion E. Reid and Christine Lomas-Francis

GerbichGerbich Blood Group Blood Group SystemSystemGerbichGerbich Blood Group Blood Group SystemSystem

The Blood Group Antigen Facts Book 2nd Edition: Marion E. R id d Ch i ti L F iReid and Christine Lomas-Francis

Case 4Case 4Case 4Case 4

RS chemo patientp Metastatic colon cancer involving lung,

liver and boneve a bo e A Positive Last Transfusion February 2012 Last Transfusion February 2012 1 unit requested March 2012 Antibody screen negative

Case 4Case 4Case 4Case 4

Electronic Pre Post

crossmatch Transfusion started

Temperature 36.7 37.1

BP 135/72 189/95

Pulse 97 140

@1230 completed @1350

SpO2 99% Unable to obtain

Reaction noted @ 1400 • Chills/rigors

• DyspneaDyspnea• Tickle in throat

Case 4Case 4Case 4Case 4

Post transfusion◦ A Positive◦ Ab Screen Negative

Cel

l

elua

te

Pre

–tx

’d

Post

tx’d

Unit 4 3 3◦ DAT positive due to

IgGEl t ti

Wr(a+) 4 3

Wr(a+) 4 3

W ( +) 4 3◦ Eluate non-reactive◦ Transfused Unit gel

crossmatch

Wr(a+) 4 3

incompatible◦ Antibody to low

i id ti incidence antigen

ConclusionConclusionConclusionConclusion

Acute Hemolytic Transfusion Reaction ydue to anti-Wra

◦ AHTR causes ABO incompatibility Other blood group incompatibility Rare cases of high titre antibody group O platelets

transfused to a non-O recipient

ConclusionsConclusionsConclusionsConclusions◦ RBC alloantibodies Patient immunized from previous exposures Low levels of allo-ab in patient Error in screening Limitations in screening cells Uncrossed blood Uncrossed blood

◦ Outcome Less than 10% are fatal Less than 10% are fatal Over 50% result in no morbidity

AHTR◦ Accelerated destruction of rbc in a transfused patient ◦ Accelerated destruction of rbc in a transfused patient

occurring during or within 24 hrs after a blood transfusion◦ Characterized by sudden onset of fever, chills, facial

flushing, chest pain or low back pain, hypotension and flushing, chest pain or low back pain, hypotension and dyspnea

◦ May result in severe complications ie renal failure and DIC◦ Leading cause of death associated with transfusionea g cause o eat assoc ate w t t a s us o◦ Estimated to occur in ~ 1 in 12,000 to 1 in 33,000 red cell

units transfused◦ Fatal AHTR occurs in 1 in 600,000 to 1 in 800,000 units , ,

transfused

The Wright Blood GroupThe Wright Blood GroupThe Wright Blood GroupThe Wright Blood Group Identified in 1953 as cause of HDN Diego blood group system Antigens◦ Wra occurs in <0.01% of population◦ Wrb occurs in 100% of populationA i W a Anti- Wra

◦ Resistant to ficin/papain◦ Can be IgM or IgG◦ Can be IgM or IgG◦ React at RT or IAT◦ Does not bind complementDoes not bind complement

The Wright Blood GroupThe Wright Blood GroupThe Wright Blood GroupThe Wright Blood Group

Anti- Wra

◦ transfusion reactions: none to severe/immediate or delayed/hemolytic◦ HDN: mild to severe◦ Can be naturally occurring◦ Found in 1% to 2% of blood donors◦ Common specificity in patients with AIHAp y p

The Blood Group Antigen Facts Book 2nd Edition: Marion E. Reid and Christine Lomas-p gFrancis

Questions?Questions?Questions?Questions?

ReferencesReferencesReferencesReferences

Judd’s Methods in Immunohematology, 3rdJ gyEdition, 2008

Applied Blood Group Serology, Peter D. pp e oo G oup Se o ogy, ete . Issitt, David J Anstee, Fourth Edition, Apr 1998

The Blood Group Antigen Facts Book, Marion E Reid Christine Lomas-Francis Marion E Reid, Christine Lomas Francis, 2nd edition, 2007