Immunohematology

Case Studies

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Immunohematology Case Studies

Rebecca Coward, MT(ASCP)SBBCM

WakeMed Health and Hospitals

Case 1

Patent DC: 26 year old Latin American female

G5P2 at 37w4d

Presents with contractions

Forward Reverse

Anti-

A

Anti

-B

Anti-

D1

Anti-

D2

Mono

Ctrl

A1 B

Patient

DC 4+ 0 0 0 0 1+ 4+

Echo

R800 D C E c e M N S s P1 K k Fya Fyb Jka Jkb IAT

R1R1 + + 0 0 + 0 + 0 + + + + 0 + + 0 3+

R2R2 + 0 + + 0 0 + + + 0 + + + + 0 + 4+

rr 0 0 0 + + + 0 + 0 + 0 + + 0 + 0 0

POS

Ctrl 4+

Rh MNS LU P Lewis Kell Duffy Kidd SP

Lot

DN096 D C E c e V M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IAT

1 r’r 0 + 0 + + 0 + 0 + + 0 + + 0 + 0 + 0 + 0 + 0

2 r’’r 0 0 + + + 0 + 0 + 0 0 + + 0 + 0 + + + 0 + 0

3 r’r 0 + 0 + + 0 + + 0 + 0 + + 0 + 0 + + + + + 0

4 r’’r 0 0 + + W 0 0 + 0 + 0 + + 0 + 0 + 0 + 0 + 0 5 rr 0 0 0 + + 0 + + + + 0 + + + 0 + + + + + 0 0 6 rr 0 0 0 + + 0 0 + 0 + + + + 0 + 0 + + 0 + 0 0 7 rr 0 0 0 + + 0 + 0 0 + 0 + + + 0 0 + 0 + + + 0 8 rr 0 0 0 + + 0 0 + + + 0 + + 0 0 0 + + 0 0 + 0 9 rr 0 0 0 + + 0 + + + + 0 + + 0 + + + 0 + 0 + 0 10 rr 0 0 0 + + 0 + 0 + + 0 + + 0 + 0 + + 0 + 0 0 11 rr 0 0 0 + + 0 0 + 0 + 0 + 0 + 0 0 + 0 + + + 0 12 rr 0 0 0 + + 0 0 + + + 0 + + + 0 0 + + 0 0 + 0 13 rr 0 0 0 + + 0 + + + + 0 + 0 0 + 0 + 0 + 0 + 0 14 R2R2 + 0 + + 0 0 + 0 + 0 0 + 0 0 + 0 + + + + + 3+

Pos Ctrl / / / / / / / / /

/ / / / / / / / / / / / 4+

Neg Ctrl / / / / / / / / /

/ / / / / / / / / / / / 0

Capture-R Extend II

DAT- Echo

DAT= 0

Ctrl= 3+

ABO Discrepancy Workup

Repeat ABO/Rh in tube

Forward Reverse

Anti-A Anti-B Anti-D A1 B

Patient

DC 4+ 0 0 1+ 4+

Anti-A1 workup

A1 cells A2 cells O cells

IS Lot IS Lot IS Lot

Cell #1 1+ 111108 0 112795 0 42994-SC1

Cell #2 0 111104 0 112793 0 42994-SC2

Cell #3 1+ 111099 0 112741 0 42994-SC3

Anti-A1 lectin Lot

IS

Patient DC cells 4+ 980012

Positive Control 4+ 111108

Negative Control 0 112795

Pre-warm

*Use pre-warm methods with caution!

Note: no rouleaux noted with microscopic review

A1 B Auto

Prewarm

30’ 37°C

W+ 4+ 0

42994 D C E c e M N S s P1 K k Fya Fyb Jka Jkb IS 15’

RT

15’

4C

R1R1 + + 0 0 + + + + + + 0 + 0 + 0 + 0 0 2+

R2R2 + 0 + + 0 0 + 0 + + + + + + + 0 0 1+ 3+

rr 0 0 0 + + + 0 + 0 0 0 + + 0 + 0 0 0 0

Auto

Ctrl 0 0 0

Cold Screen

Rh MNS LU P Lewis Kell Duffy Kidd

D C E c e V M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IS 15’ RT

15’ 4C

1 R1R1 + + 0 0 + 0 + 0 + + 0 + + + 0 0 + + 0 0 + 0 0 W+

2 R1R1 + + + 0 + 0 0 + 0 + 0 + + 0 0 + + + + + 0 0 1+ 2+

3 R1R1 + + 0 0 + 0 + 0 + 0 0 + + 0 + 0 + + + + 0 0 W+ 2+

4 R1R1 + + + 0 + 0 + + 0 + 0 + 0 0 + 0 + + 0 + + 0 0 0 5 r’r 0 + 0 + + 0 0 + + + 0 + 0 0 + 0 + 0 + 0 0 0 0 0 6 R1R1 + + 0 0 + 0 + + + + + + 0 + 0 + + + + + 0 0 0 0 7 r’’r 0 0 0 + + 0 + 0 + + 0 + 0 + 0 0 + 0 + + 0 0 0 0

Cold Screen

P1 • P1PK blood group system

• Expression of antigen is variable

– Shows dosage based on zygosity

– Expression weakens with in-vitro storage

– Expressed more weakly on cord cells (than adult RBCs)

• P1 expression is weakened/inhibited In(Lu) phenotype

Caucasians Blacks Cambodian/

Vietnamese

P1 79% 94% 20%

Anti-P1

• IgM (IgG is rare)

• Detected at RT or lower

• May be neutralized

– Hydatid cyst fluid

– Pigeon egg white

– Echinococcus cyst fluid

• Naturally occurring- found in many P2 donors

• Not considered clinically significant (no HTR/HDFN)

Case 1 Summary

• Anti-D due to RhIg administration

• Anti-P1 causing ABO discrepancy

• No phenotyping or titer performed

Case 2

Patient RP: 45 year old male; race unknown

Trauma 1; head on motor vehicle collision

Trauma 1 cooler issued:

– 4 O pos uncrossmatched RBCs

– 2 A plasma

Forward Reverse

Anti-A Anti-B Anti-D1 Anti-D2

Mono Ctrl

A1 B

Patient

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

ECHO

R866 D C E c e M N S s P1 K k Fya Fyb Jka Jkb IAT

R1R1 + + 0 0 + 0 + 0 + + 0 + + 0 + 0 1+

R2R2 + 0 + + 0 + 0 + + + 0 + + + 0 + 0

rr 0 0 0 + + + + + 0 + + + 0 + 0 + 0

POS

Ctrl 4+

Rh MNS LU P Lewis Kell Duffy Kidd SP

Lot

ID339 D C E c e V M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IAT

1 RzR1 + + + 0 + 0 + + 0 + 0 + + 0 + 0 + + 0 + + 1+

2 R1wR1 + + 0 0 + 0 + 0 + 0 0 + + 0 + 0 + + 0 + + 0

3 R2R2 + 0 + + 0 0 + + 0 + + + + 0 + 0 + + 0 + 0 1+

4 Ror + 0 0 + + 0 0 + 0 + + + + 0 + 0 + + 0 + + 0 5 r’r 0 + 0 + + 0 0 + 0 + 0 + + 0 + 0 + + + + + 2+ 6 r’’r 0 0 + + + 0 + + 0 + 0 + + 0 + + + 0 + + + 1+ 7 rr 0 0 0 + + 0 0 + 0 + 0 + + + 0 0 + 0 + 0 + 0 8 rr 0 0 0 + + 0 0 + 0 + 0 + 0 + 0 + 0 0 + 0 + 0 9 rr 0 0 0 + + 0 + + 0 + 0 + + 0 0 0 + + 0 0 + 0 10 rr 0 0 0 + + 0 + 0 0 + 0 + + + 0 0 + 0 0 + + 1+ 11 rr 0 0 0 + + 0 + 0 + + 0 + + + 0 0 + 0 + + 0 0 12 rr 0 0 0 + + 0 + + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 13 rr 0 0 0 + + 0 + 0 + + 0 + + 0 + 0 + + 0 + 0 1+

14 R1R1 + + 0 0 0 0 + 0 0 0 0 + + + 0 0 + + + + + 2+

Pos Ctrl / / / / / / / / / / / / / / / / / / / / / 4+

Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 0

Capture-R Ready-ID

Antigen Typing

• Transfusion history is unknown

Anti-Jka

Patient RP cells 0

Positive Control 3+

Negative Control 0

ECHO

R866 D C E c e M N S s P1 K k Fya Fyb Jka Jkb IAT

R1R1 + + 0 0 + 0 + 0 + + 0 + + 0 + 0 1+

R2R2 + 0 + + 0 + 0 + + + 0 + + + 0 + 0

rr 0 0 0 + + + + + 0 + + + 0 + 0 + 0

POS

Ctrl 4+

Antibody ID Review

Rh MNS LU P Lewis Kell Duffy Kidd SP

Lot

ID339 D C E c e V M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IAT

1 RzR1 + + + 0 + 0 + + 0 + 0 + + 0 + 0 + + 0 + + 1+

2 R1wR1 + + 0 0 + 0 + 0 + 0 0 + + 0 + 0 + + 0 + + 0

3 R2R2 + 0 + + 0 0 + + 0 + + + + 0 + 0 + + 0 + 0 1+

4 Ror + 0 0 + + 0 0 + 0 + + + + 0 + 0 + + 0 + + 0 5 r’r 0 + 0 + + 0 0 + 0 + 0 + + 0 + 0 + + + + + 2+ 6 r’’r 0 0 + + + 0 + + 0 + 0 + + 0 + + + 0 + + + 1+ 7 rr 0 0 0 + + 0 0 + 0 + 0 + + + 0 0 + 0 + 0 + 0 8 rr 0 0 0 + + 0 0 + 0 + 0 + 0 + 0 + 0 0 + 0 + 0 9 rr 0 0 0 + + 0 + + 0 + 0 + + 0 0 0 + + 0 0 + 0 10 rr 0 0 0 + + 0 + 0 0 + 0 + + + 0 0 + 0 0 + + 1+ 11 rr 0 0 0 + + 0 + 0 + + 0 + + + 0 0 + 0 + + 0 0 12 rr 0 0 0 + + 0 + + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 13 rr 0 0 0 + + 0 + 0 + + 0 + + 0 + 0 + + 0 + 0 1+

14 R1R1 + + 0 0 0 0 + 0 0 0 0 + + + 0 0 + + + + + 2+

Pos Ctrl / / / / / / / / / / / / / / / / / / / / / 4+

Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 0

Antibody ID Review

????

PEG Screen

PEG tube

22122 D C E c e M N S s P1 K k Fya Fyb Jka Jkb IS IAT CC

R1R1 + + 0 0 + 0 + 0 + + 0 + + 0 + 0 1+ 0 3+

R2R2 + 0 + + 0 + 0 + + + 0 + + + 0 + 1+ 0 3+

rr 0 0 0 + + + + + 0 + + + 0 + 0 + 1+ 0 3+

AC 1+ 0 3+

Cold Screen

Tube

D C E c e M N S s P1 K k Fya Fyb Jka Jkb IS 15’

RT

15’

4C

A1 0 1+ 3+

A2 0 1+ 3+

SC1 + + 0 0 + 0 + 0 + + 0 + + 0 + 0 1+ 2+ 3+

SC2 + 0 + + 0 + 0 + + + 0 + + + 0 + 1+ 2+ 3+

SC3 0 0 0 + + + + + 0 + + + 0 + 0 + 1+ 2+ 3+

AC 1+ 2+ 3+

LISS Screen

LISS tube

22122 D C E c e M N S s P1 K k Fya Fyb Jka Jkb IS 37C IAT CC

R1R1 + + 0 0 + 0 + 0 + + 0 + + 0 + 0 1+ 0 0 3+

R2R2 + 0 + + 0 + 0 + + + 0 + + + 0 + 1+ 0 0 3+

rr 0 0 0 + + + + + 0 + + + 0 + 0 + 1+ 0 0 3+

AC 1+ 0 0 3+

Case 2 Summary

• Solid phase platform may have increased sensitivity to Kidd antibodies

– Clinical significance of “solid-phase” only antibodies has been debated

– Kay, et al. Anti-Jka that are detected by solid-phase red blood cell adherence but missed by gel testing can cause hemolytic transfusion reactions. Transfusion 2016;56:2973-2979.

Case 2 Summary

• Capture-R assays are designed primarily for the detection of IgG antibodies

• Cold antibodies detected by Capture-R

– May have an IgG component

– Indicator cells may carry antigen toward which the antibody is directed

– Antibody may link the Indicator cells to the immobilized RBC layer by binding to antigens on both

Case 2 Summary

• Impact of ABID on transfusion recommendation and future transfusions

– Screening for antigen negative blood

– Extended crossmatches

Case 3

Patient AS: 18 year old African American female

Past Medical History: sickle cell anemia (SSA) SS–type

Presents with pain

Previous antibody ID: anti-S, warm autoantibody. Patient receives C-, E-, K-, S- RBCs per hospital protocol for SSA

Forward Reverse

Anti-A Anti-B Anti-D1 Anti-D2

Mono Ctrl

A1 B

Patient AS 0 0 4+ 4+ 0 4+ 3+

ECHO

R780 D C E c e M N S s P1 K k Fya Fyb Jka Jkb IAT

R1R1 + + 0 0 + + + 0 + 0 0 + + 0 0 + 4+

R2R2 + 0 + + 0 0 + 0 + + 0 + + + + + 4+

rr 0 0 0 + + + 0 + 0 + + + 0 + + 0 4+

POS

Ctrl 4+

Rh MNS LU P Lewis Kell Duffy Kidd SP

Lot

ID310 D C E c e V M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IAT

1 RzR1 + + + 0 + 0 0 + + + 0 + + 0 + 0 + 0 + 0 + 4+

2 R1wR1 + + 0 0 + 0 + + 0 + 0 + + 0 + 0 + 0 + + + 4+

3 R2R2 + 0 + + 0 0 + + 0 + 0 + + 0 0 0 + 0 + 0 + 4+

4 Ror VS+ + 0 0 + + + 0 + + 0 0 + + 0 + 0 + 0 0 + 0 0 5 r’r 0 + 0 + + 0 + + + + 0 + 0 0 + 0 + 0 + 0 + 4+ 6 r’’r 0 0 + + + 0 + 0 + 0 0 + + 0 + + + 0 + + + 4+ 7 rr 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 0 4+ 8 rr 0 0 0 + + 0 + + 0 + 0 + + 0 + + + 0 + + + 4+ 9 rr 0 0 0 + + 0 + 0 0 + 0 + 0 0 + 0 + + 0 + + 4+ 10 rr 0 0 0 + + 0 + + + + 0 + + + 0 0 + + 0 0 + 4+ 11 rr 0 0 0 + + 0 + 0 + 0 0 + + 0 + + + + + + 0 4+ 12 rr 0 0 0 + + 0 0 + 0 + + + 0 + 0 0 + 0 + + + 4+ 13 rr

Di(a+) 0 0 0 + + 0 + 0 + + 0 + + 0 + 0 + 0 0 + 0 3+

14 R1R2 + + + + + 0 + + + + 0 + + + 0 0 + 0 + + + 4+

Pos Ctrl / / / / / / / / / / / / / / / / / / / / / 4+

Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 0

Capture-R Ready-ID

DAT - Tube

IgG = 0

CC = 3+

PEG tube Selected cell panel

PEG tube

D C E c e M N S s P1 K k Fya Fyb Jka Jkb IS IAT CC

R1R1 + + 0 0 + + + 0 + 0 + + + + 0 + 0 2+ NT

R1R1 + + 0 0 + 0 + 0 + + 0 + + 0 + 0 0 3+ NT

R2R2 + 0 + + 0 + + 0 + + 0 + + 0 + 0 0 2+ NT

r’r 0 0 0 + + + + 0 + + + + 0 + + + 0 2+ NT

rr 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 1+ NT

AC 0 0 2+

Rh MNS LU P Lewis Kell Duffy Kidd SP

Lot

ID310 D C E c e V M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IAT

1 RzR1 + + + 0 + 0 0 + + + 0 + + 0 + 0 + 0 + 0 + 4+

2 R1wR1 + + 0 0 + 0 + + 0 + 0 + + 0 + 0 + 0 + + + 4+

3 R2R2 + 0 + + 0 0 + + 0 + 0 + + 0 0 0 + 0 + 0 + 4+

4 Ror VS+ + 0 0 + + + 0 + + 0 0 + + 0 + 0 + 0 0 + 0 0 5 r’r 0 + 0 + + 0 + + + + 0 + 0 0 + 0 + 0 + 0 + 4+ 6 r’’r 0 0 + + + 0 + 0 + 0 0 + + 0 + + + 0 + + + 4+ 7 rr 0 0 0 + + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 0 4+ 8 rr 0 0 0 + + 0 + + 0 + 0 + + 0 + + + 0 + + + 4+ 9 rr 0 0 0 + + 0 + 0 0 + 0 + 0 0 + 0 + + 0 + + 4+ 10 rr 0 0 0 + + 0 + + + + 0 + + + 0 0 + + 0 0 + 4+ 11 rr 0 0 0 + + 0 + 0 + 0 0 + + 0 + + + + + + 0 4+ 12 rr 0 0 0 + + 0 0 + 0 + + + 0 + 0 0 + 0 + + + 4+ 13 rr

Di(a+) 0 0 0 + + 0 + 0 + + 0 + + 0 + 0 + 0 0 + 0 3+

14 R1R2 + + + + + 0 + + + + 0 + + + 0 0 + 0 + + + 4+

Pos Ctrl / / / / / / / / / / / / / / / / / / / / / 4+

Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 0

Capture-R Ready-ID

OSH Information

• TAS 2 weeks prior- negative with S negative selected cell panel

• Received 1 unit XM compatible RBCs

• Phenotype shared:

Group O; D+C-E-c+e+; K-, Fy(a-b-), Jk(a+b+), M+N+S-s+; P1+

• WARM identified 2007

• Anti-S identified 2012

Peg tube Selected cell panel

PEG tube

D C E c e M N S s P1 K k Fya Fyb Jka Jkb IS IAT CC

rr VS+

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

Ror U-

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

R1R1 + + 0 0 + + 0 0 + + 0 + 0 0 + 0 0 0 2+

R2R2 + 0 + + 0 0 + 0 + + 0 + 0 0 + 0 0 0 2+

Reference Lab Results

• Anti-Fy3 confirmed in current sample

• Anti-Sla is ruled out

• Recommendation: transfuse pheno-similar cells

– Negative for C, E, K, Fya, Fyb, S

• Transfusion Service where patient receives routine care was notified of ABID

• Clinicians at WM were made aware of ABID and potential delays in procuring blood for future transfusions

HEA BeadChip Analysis

Fy3

• Absent from Fy(a-b-) RBCs

• Resistant to enzymes

– anti-Fya and anti-Fyb sensitive to papain/ficin

• Expressed on cord cells and expression increases after birth (HDFN- mild/rare)

Fy3 Occurrence

Caucasians 100%

Blacks 32%

Asians 99.9%

Anti-Fy3

• IgG

• Clinically significant: HTR (mild/moderate-delayed); HDFN (mild-rare)

• Formation of anti-Fy3 is usually preceded by anti-Fya

• Anti-Fy3 produced by blacks does not react (or weakly reacts) with cord cells

Anti-Fy3

Anti-Fy3 vs anti-Sla

• Sla frequency is 50-60% in Blacks

• Sla is expressed weakly on cord cells

• Most Fy(a-b-) cells are also likely Sl(a-)

• Sla is weakened/sensitive to papain/ficin

Anti-Fy3 vs anti-Fy5

• Anti-Fy3 agglutinates Rhnull RBCs

• Anti-Fy5 does not agglutinate Rhnull RBCs

Case 3 Summary

• Even with previous antibody ID with panagglutinin, the possibility of a new antibody to HFA should always be considered

• There is power in the pheno-matched reagent cell

• What to do with “extra” reactivity in SPRCA?

– Miller NM, et al. Patient factors associated with unidentified reactivity in solid-phase and polyethylene glycol antibody detection methods. Transfusion 2017;57:1288-1293.

– 19% of patients studied with UID; had autoantibody or alloantibody identified on a subsequent screen or panel

– Patient AS developed warm autoantibody on subsequent sample

Case 4

Patient DG: 19 year old African American female

No pertinent past medical history

Presents to the ED for generalized weakness, decreased appetite. At-home pregnancy test positive

TAS Echo

Forward Reverse

Anti

-A

Anti-

B

Anti-

D1

Anti

-D2

Mono

Ctrl

A1 B

Patient

DG 0 4+ 0 0 0 1+ 0

Echo

D C E c e M N S s P1 K k Fya Fyb Jka Jkb IAT

R1R1 + + 0 0 + 0 + 0 + + + + 0 + + 0 0

R2R2 + 0 + + 0 0 + + + 0 + + + + 0 + 0

rr 0 0 0 + + + 0 + 0 + 0 + + 0 + 0 0

POS

Ctrl 4+

ABO Reconfirm- Tube

Forward

Anti-A Anti-B Anti-D

Patient DG 0 4+ 2+

Rh discrepancy workup

Weak D- tube

Anti-D CC

Patient DG 3+ NT

Control 0 3+

DAT Echo

DG 0

Other troubleshooting

• Instrument

• Tech

• Reagent

• Call Immucor!

Immucor Investigation

• Testing by Echo

– ECHO Confirm Assay

– ECHO Weak D Assay

– ECHO DAT

• Testing by tube

– D hemagglutination assay

– Weak D

• Immucor DX Reference Laboratory

– RHD and RHCE BeadChip Analysis

ECHO Testing

• Testing by Echo

– ECHO Confirm Assay- Group B, D-negative

– ECHO Weak D Assay- 4+

– ECHO DAT- negative

Tube Testing

• Testing by tube

– D hemagglutination assay

– Weak D

Anti-D1 Anti-D2 Monoclonal

Control

IS 0 0 0

Weak D 1+ 1+ 0

RHD BeadChip assay

• Weak D type 4.0 or 4.3 (hemizygous or homozygous) “partial D”

• Anti-D has been observed (allo/auto?)

• Consider patient D negative for the purposes of transfusion and/or RhIg administration

• As a donor, consider patient D positive

• Further classification could be accomplished by sequencing

RHCE BeadChip assay

• Predicted phenotype: C- c+ E- e+

• Alleles detected: ce(48C)/ce(48C, 733G)

ce(48C) - encodes a “normal” e that may react weakly with some monoclonals

ce(48C, 733G) - encodes partial c, partial e, V+, VS+, hrB-

Case 4 Summary

• Weak reactivity detected by tube (manual) tests may not always be reproducible using automated hemagglutination assays

• D typing discrepancies often point to altered D expression

• Variant RHD alleles are often inherited with variant RHCE alleles

• Workup and management of patients with Rh variants is not standardized

References

• The Blood Group Antigen FactsBook. 3rd ed. Reid, Lomas-Francis, Olsson

• Human Blood Groups. 3rd ed. Daniels

• Technical Manual. 18th ed. Fung, et al.

• Rhesus Base http://www.rhesusbase.info/ last accessed 8/21/17

Thank you!

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Thierry PEYRARD

PharmD, PhD, European Specialist in Clinical Chemistry and Laboratory

Medicine

Director, National Immunohematology Reference Laboratory

National Institute of Blood Transfusion – Paris – France

tpeyrard@ints.fr

Immunohematology Case Studies

Presented as a Webinar for the Immucor Global User-Group

August 30th, 2017

Short presentation of the French National Immunohematology Reference Laboratory

Case studies

Discussion

PRESENTATION OUTLINE

CNRGS: THE FRENCH NATIONAL IMMUNOHEMATOLOGY

REFERENCE LABORATORY (IRL)

• Centre National de Référence pour les Groupes Sanguins

• Department of the French National Institute of Blood Transfusion (INTS)

• Only official national IRL in France

• Staff: 40 people

INTS headquarter

CNRGS

MAJOR MISSIONS

• Complex case solving: referrals from continental France, French overseas territories and foreign countries (serology, molecular testing)

• National Rare Blood Program

• Manufacturing of the National Reference Identification Panel of Reagent-RBCs

• Contribution to the control of performance of immunohematology reagents according to the European "CE-marking" scheme

• Scientific research

• Continuing education and university teaching

CASE STUDIES

CASE STUDY 1

• 37 year old pregnant patient, 30th week of gestation

• RBC antibody screen positive => Antibody identification

RH KEL FY JK LE MNS P LU DO YT CO XG

IAT IAT-

papain 1 2 3 4 5 8 1 2 3 4 1 2 1 2 1 2 1 2 3 4 1 1 2 19 1 2 1 2 1 2 1

1 0 + 0 0 + 0 0 + 0 + + + + + 0 0 + 0 + 0 + 0 + + + + 0 + 0 + ++++ -

2 0 0 + + + 0 0 + 0 + + + + 0 0 + 0 + 0 + + 0 + + + + 0 + 0 + - -

3 0 0 0 + + 0 + + 0 + + 0 0 + 0 + + 0 + + + + + 0 + + + + + 0 + ++++ -

4 0 0 0 + + 0 0 + 0 + 0 + + + + 0 + + + + + + + + + 0 + 0 + 0 + ++ -

5 0 0 0 + + 0 0 + 0 + 0 + + + 0 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + ++++ -

6 0 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + 0 + 0 + + + + + 0 + 0 + ++ -

7 + 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 + 0 + 0 + - -

8 + + 0 0 + + 0 + 0 + + 0 0 + 0 + + + + 0 + + + 0 + + 0 + 0 + ++ -

9 + + 0 0 + 0 + + 0 + + + + 0 0 + 0 + + + 0 0 + 0 + + 0 + 0 0 - -

10 + + 0 0 + 0 0 + 0 + + 0 + + 0 + 0 + 0 + + 0 + + + 0 + 0 + - -

11 + 0 0 + + 0 0 + 0 + + + + 0 + 0 + 0 + 0 0 0 + 0 + + 0 + 0 + ++++ -

12 + + + 0 + 0 0 + 0 + + + + 0 0 + + + 0 + + 0 + + + 0 + 0 + 0 + ++ -

13 + + 0 0 + 0 0 + + + + + + 0 0 + + + 0 + 0 0 + 0 + + 0 + 0 0 ++ -

14 + + 0 0 + 0 + + 0 + + + + + 0 + + 0 + 0 + 0 + 0 0 + + 0 + 0 0 ++++ -

15 0 0 0 + + 0 0 + 0 + + 0 + 0 0 + 0 + 0 + + 0 + + + + 0 + 0 0 - -

Autocontrols - -

• 37 year old pregnant patient, 30th week of gestation

• RBC antibody screen positive => Antibody identification

RH KEL FY JK LE MNS P LU DO YT CO XG

IAT IAT-

papain 1 2 3 4 5 8 1 2 3 4 1 2 1 2 1 2 1 2 3 4 1 1 2 19 1 2 1 2 1 2 1

1 0 + 0 0 + 0 0 + 0 + + + + + 0 0 + 0 + 0 + 0 + + + + 0 + 0 + ++++ -

2 0 0 + + + 0 0 + 0 + + + + 0 0 + 0 + 0 + + 0 + + + + 0 + 0 + - -

3 0 0 0 + + 0 + + 0 + + 0 0 + 0 + + 0 + + + + + 0 + + + + + 0 + ++++ -

4 0 0 0 + + 0 0 + 0 + 0 + + + + 0 + + + + + + + + + 0 + 0 + 0 + ++ -

5 0 0 0 + + 0 0 + 0 + 0 + + + 0 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + ++++ -

6 0 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + 0 + 0 + + + + + 0 + 0 + ++ -

7 + 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 + 0 + 0 + - -

8 + + 0 0 + + 0 + 0 + + 0 0 + 0 + + + + 0 + + + 0 + + 0 + 0 + ++ -

9 + + 0 0 + 0 + + 0 + + + + 0 0 + 0 + + + 0 0 + 0 + + 0 + 0 0 - -

10 + + 0 0 + 0 0 + 0 + + 0 + + 0 + 0 + 0 + + 0 + + + 0 + 0 + - -

11 + 0 0 + + 0 0 + 0 + + + + 0 + 0 + 0 + 0 0 0 + 0 + + 0 + 0 + ++++ -

12 + + + 0 + 0 0 + 0 + + + + 0 0 + + + 0 + + 0 + + + 0 + 0 + 0 + ++ -

13 + + 0 0 + 0 0 + + + + + + 0 0 + + + 0 + 0 0 + 0 + + 0 + 0 0 ++ -

14 + + 0 0 + 0 + + 0 + + + + + 0 + + 0 + 0 + 0 + 0 0 + + 0 + 0 0 ++++ -

15 0 0 0 + + 0 0 + 0 + + 0 + 0 0 + 0 + 0 + + 0 + + + + 0 + 0 0 - -

Autocontrols - -

• Anti-M, with dosage effect

• Negative autocontrols => probable allo-anti-M but M/N typing required to conclude

• MNS phenotype M-N+ => alloantibody

RH KEL FY JK LE MNS P LU DO YT CO XG

IAT IAT-

papain 1 2 3 4 5 8 1 2 3 4 1 2 1 2 1 2 1 2 3 4 1 1 2 19 1 2 1 2 1 2 1

1 0 + 0 0 + 0 0 + 0 + + + + + 0 0 + 0 + 0 + 0 + + + + 0 + 0 + ++++ -

2 0 0 + + + 0 0 + 0 + + + + 0 0 + 0 + 0 + + 0 + + + + 0 + 0 + - -

3 0 0 0 + + 0 + + 0 + + 0 0 + 0 + + 0 + + + + + 0 + + + + + 0 + ++++ -

4 0 0 0 + + 0 0 + 0 + 0 + + + + 0 + + + + + + + + + 0 + 0 + 0 + ++ -

5 0 0 0 + + 0 0 + 0 + 0 + + + 0 0 + 0 + 0 + 0 + + 0 + + 0 + 0 + ++++ -

6 0 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + 0 + 0 + + + + + 0 + 0 + ++ -

7 + 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 + 0 + 0 + - -

8 + + 0 0 + + 0 + 0 + + 0 0 + 0 + + + + 0 + + + 0 + + 0 + 0 + ++ -

9 + + 0 0 + 0 + + 0 + + + + 0 0 + 0 + + + 0 0 + 0 + + 0 + 0 0 - -

10 + + 0 0 + 0 0 + 0 + + 0 + + 0 + 0 + 0 + + 0 + + + 0 + 0 + - -

11 + 0 0 + + 0 0 + 0 + + + + 0 + 0 + 0 + 0 0 0 + 0 + + 0 + 0 + ++++ -

12 + + + 0 + 0 0 + 0 + + + + 0 0 + + + 0 + + 0 + + + 0 + 0 + 0 + ++ -

13 + + 0 0 + 0 0 + + + + + + 0 0 + + + 0 + 0 0 + 0 + + 0 + 0 0 ++ -

14 + + 0 0 + 0 + + 0 + + + + + 0 + + 0 + 0 + 0 + 0 0 + + 0 + 0 0 ++++ -

15 0 0 0 + + 0 0 + 0 + + 0 + 0 0 + 0 + 0 + + 0 + + + + 0 + 0 0 - -

Autocontrols - -

• New blood sample investigated a few weeks later in a second laboratory: anti-M confirmed

• But the phenotype this time was found to be M+N+!

• Strong reaction for M (4+), equivalent to the M+N- control RBCs

=> Blood samples referred to our reference laboratory

• M-N+ type found in our laboratory, with two different sources of reagents

• Control with the anti-M of laboratory #2 who found a 4+ reactivity => confirmation of the M+ type!

Patient M+ or M- ? Auto- or allo-anti-M ?

Typical issue of cross-reactivity interference. Some reagents may more or less react with another antigen than the test antigen.

Some widely used anti-M clones (e.g. 2514E6 and M-11H2) strongly crossreact with the low-prevalence He antigen (Henshaw, MNS6)

7-10% of people of African descent are He+ => not "extremely rare" in some countries!

• The anti-M clones that crossreact with He are, however, considered the most peformant anti-M

• Of note, anti-M clone BS57 does not crossreact with He

• Some anti-M clones (E3, E6, 425/2B) also crossreact with the low-prevalence Mg antigen (MNS11) => less problematic because this antigen is very rare in all populations (except in Switzerland but prevalence still <1%)

CONCLUSION

• Beware of possible crossreactions with some monoclonal antibodies, responsible for false positive results

• Always carefully read the package insert of the manufacturer and limitations of the reagent

• May explain discrepancies between phenotype and genotype

• May also explain apparent parentage exclusion (mother and father previously typed as M-N+ with a non-involved reagent, and child typed as M+N+ with a crossreacting reagent with He)

CASE STUDY 2

• 25 year old female sickle cell patient

• No history of recent transfusion

• Group O, D+C-E-c+e+, K-

• Positive antibody screen and

identification

++

++ ++

++++

++++

++

++ ++

++

IAT Papain

Autocontrols

• Pattern of reactivity consistent with

an anti-N (dosage effect)

• Negative autocontrols

alloanti-N? Exceptional antibody!

Is this really an alloanti-N?

• N is papain and trypsin sensitive…

++

++ ++

+++

+++

++

++ ++

++

IAT Pap

Autocontrols

Tryp

++

++

++

++ ++

++

++++

++++ ++++ ++++ ++++ ++++ ++++

++++

++++

Strongly positive reactions on trypsin-treated RBCs!

Extended phenotype of the patient

Fy(a-b-), Jk(a+b-), M+N-S-s-

Rare S-s- phenotype (1-2% in Africans, up to

35% in Equatorial Africa)

50% are U- (no glycophorin B)

50% are U+var (altered glycophorin B,

weak/partial U)

Possible anti-U alloimmunization,

either in U- or U+var

GPA

M/N

GPB

S/s/U

Trypsin

sensitive

Trypsin

resistant

GPA 106

molecules/cel

l

GPB 2.105

molecules/c

ell

Example of

a M+N+S+s+

individual

M

N

S

s

GPA(N)

GPB

Same 26 amino acids than those

which define N on GPA(N) => ‘N’

or N-like

N is a high-prevalence antigen!

N

N

GPA

GPB

Why is it

possible

to type for

N on

GPA?

Same 26 amino acids than those

which define N on GPA(N) => ‘N’

or N-like

N is a high-prevalence antigen!

Number of GPA

molecules >> GPB

=> anti-N diluted

enough to react

only with N on GPA

GPA

GPB

Number of GPA

molecules >> GPB

=> anti-N diluted

enough to react

only with N on GPA

But N typing

quite often shows

weak or unclear

reactions…

Possible detection of ‘N’ on GPB if anti-N too concentrated or

Number of GPB molecules above the average, e.g. in S+s-

(express 50% more GPB than S-s+) => highest risk in M+N-S+s-

(6% Caucasians, 2% Africans)

GPA

No GPB

=> No ‘N’

=>

"True" N-

Patient

M+N-S-s-

U-

M

M

May form an antibody to a high-prevalence antigen named

anti-’N’ or anti-N-like (anti-MNS30), that ressembles anti-N

when starting to develop (first reacts with N on GPA because

GPA expression >> GPB)

=> rare blood N-S-s-U- required !

GPA

Altered GPB

=> No ‘N’ in

>90% of

cases =>

"True" N-

Patient

M+N-S-s-U+var

M

M

Rare blood N-S-s-U+var or N-S-s-U- required!

GPB

Presence of the He (MNS6) antigen =>

abolishes ‘N’ expression

GPA

Altered GPB

=> No ‘N’ in

>90% of

cases =>

"True" N-

Patient

M+N-S-s-U+var

M

M

Rare blood N-S-s-U+var or N-S-s-U- required!

GPB (one

dose) Presence of the He (MNS6) antigen =>

abolishes ‘N’ expression

CONCLUSION

• Alloanti-N is exceptional in

Caucasians as they are all virtually ‘N’

positive (N on GPB) => most anti-N

are autoantibodies directed to

glycophorin A/B and correspond in N-

patients (28%) to the so-called

« mimicking alloantibodies »

• In addition to the M/N type, the

discovery of an anti-N must

systematically lead to a Ss typing,

especially in Africans

• Beware of the presence of any anti-N

in a N- patient of African descent

- If S-s- => rare MNS:-30 type!

- If not S-s- (S+s- for example), can

also be a rare MNS:-30 type because

some rare African GPB variants may

also loose ‘N’!

=> A MNS genotype must be

performed in any case of anti-N

discovered in an African patient,

whatever the Ss phenotype

CONCLUSION

CASE STUDY 3

IAT IAT-

Papai

n +

++

++

++

++

+

+

++

+

+++

+++

+ +++

+ +++

+ +++

+ +++

+++

+++

+

+++

-

-

-

-

-

-

-

-

-

- -

-

Pregnant woman, group O, D-C-E-c+e+, K-. 4th pregnancy. 26th week of

gestation

Negative autocontrols

What do you think?

IAT IAT-

Papai

n +

++

++

++

++

+

+

++

+

+++

+++

+ +++

+ +++

+ +++

+ +++

+++

+++

+

+++

-

-

-

-

-

-

-

-

-

- -

-

Pregnant woman, group O, D-C-E-c+e+, K-. 4th pregnancy. 26th week of

gestation

Anti-D + Anti-C pattern of reactivity

• Anti-D immunoprophylaxis. Batches

of anti-D often contain a small

amount of anti-C => But anti-C too

reactive here

• After investigation, no anti-D

immunoprophylaxis performed here:

consistent with a real anti-D+C

alloimmunization => logically no need

to inject anti-D between 28th-32nd

week of gestation because anti-D

alloimmunization considered being

present

HYPOTHESES

• But may also be an anti-G!

• G is a common epitope between D and

C antigens => anti-G ressembles anti-

D+C!

• Several possible configurations Anti-G

Anti-C+G

Anti-D+G

Anti-C+D+G

• Essential in pregnancy to know if

alloanti-D present or not. If not =>

anti-D injection!

HYPOTHESES

• Adsorption of plasma on a D-C+E-

c+e+ (r’r) RBC

• Only anti-C and anti-G will adsorb

onto r’r RBCs and anti-D, if present,

will remain free in the adsorbate =>

antibody identification in the

adsorbate

• If no anti-D found in the r’r adsorbate,

anti-D immunoprohylaxis is required!

HOW TO RULE OUT THE

PRESENCE OF ANTI-D

• Any anti-D+C pattern of reactivity in a

pregnant woman must lead to the

systematic search for the presence of

a real alloanti-D (risk of the D

reactivity being osberved due to anti-

G!)

• If not considered, no anti-D

immunoprophylaxis will be carried

out, whereas the patient is not yet

alloimmunized to the D antigen… =>

may seriously affect obstetrical future

CONCLUSION

THANK YOU FOR YOUR ATTENTION

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