Download - Immunohematology Case Studies Program Handouts/BB Case Stu… · Immunohematology Case Studies
Immunohematology
Case Studies
All Content © 2015 Immucor, Inc.
September 29 RhD Molecular Testing
October 31 HLA: Transfusion and
Transplant
December 6 Running a Remote
Transfusion Service
Future Webinars
Link to register:
https://immucor.webinato.com/register
All Content © 2015 Immucor, Inc. All Content © 2015 Immucor, Inc. All Content © 2015 Immucor, Inc.
Continuing Education
• PACE, Florida and California DHS
• 1.0 Contact Hours
• Each attendee must register to receive CE at: https://www.surveymonkey.com/r/BBcasestudies
• Registration deadline is September 15, 2017
• Certificates will be sent via email only to those
who have registered by September 29, 2017
All Content © 2015 Immucor, Inc. All Content © 2015 Immucor, Inc. All Content © 2015 Immucor, Inc.
Presentation Recording
• Session will be recorded and posted.
– Access information will be sent to each
registrant when the recording becomes
available
• CE credits will be issued to anyone who
listens to the recording within one year of
the original presentation date (today).
All Content © 2015 Immucor, Inc. All Content © 2015 Immucor, Inc. All Content © 2015 Immucor, Inc.
• Course content is for information and illustration
purposes only. Immucor makes no
representation or warranties about the accuracy
or reliability of the information presented, and
this information is not to be used for clinical or
maintenance evaluations.
• The opinions contained in this presentation are
those of the presenter and do not necessarily
reflect those of Immucor.
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!
All Content © 2015 Immucor, Inc.
We like you!
Like us on social media!
Thierry PEYRARD
PharmD, PhD, European Specialist in Clinical Chemistry and Laboratory
Medicine
Director, National Immunohematology Reference Laboratory
National Institute of Blood Transfusion – Paris – France
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
All Content © 2015 Immucor, Inc.
September 29 RhD Molecular Testing
October 31 HLA: Transfusion and
Transplant
December 6 Running a Remote
Transfusion Service
Future Webinars
Link to register:
https://immucor.webinato.com/register
All Content © 2015 Immucor, Inc.
Continuing Education
• PACE, Florida and California DHS
• 1.0 Contact Hours
• Each attendee must register to receive CE at:
https://www.surveymonkey.com/r/BBcasestudies
• Registration deadline is September 15, 2017
• Certificates will be sent via email only to those
who have registered by September 29, 2017
All Content © 2015 Immucor, Inc. All Content © 2015 Immucor, Inc. All Content © 2015 Immucor, Inc.
Thank you!