Transcript ABO Discrepancies: Donor Testing Laboratory Results

Donor ABO Blood Grouping
Discrepancies
Heather Hume
Greg Denomme
CBS HO Serology Laboratory
Feb 20, 2009
1
GAL
GAL
GLNAC
GAL
GAL
GLNAC
GL
GAL
GALNAC
GL
GAL
Group ‘O’ Antigen
GLNAC
GAL
Group ‘B’ Antigen
GL
Group ‘A’ Antigen
3
Common A, B, and O Alleles: exons 6 - 7
261
526
703 796/803
1060
C
A1
A2
Exon 6 Exon 7
B1
G
O1
4
Basic ABO Grouping
Forward grouping
Reverse grouping
ABO group
Anti-A
Anti-B
A cells
B cells
neg
neg
pos
pos
O
pos
neg
neg
pos
A
neg
pos
pos
neg
B
pos
pos
neg
neg
AB
5
ABO Blood Grouping Discrepancies
• Missing reactivity?
– apparent absent antigen
– apparent absent antibody
• Additional reactivity?
– apparent extra antigen
– apparent extra antibody
6
ABO Blood Grouping Discrepancies
Most Common Causes
• Patients
– decreased antibody levels
• physiologic - newborns, elderly
• immunodeficiency - congenital or acquired
– auto- or alloantibodies
– recent transfusion
– HSCT
• Donors
– unusual blood group - genetic polymorphisms
7
Donor Testing Laboratory Results
Case #1
PK7200 Data Log Results
Donor #2
ABO
Rh
-B
-A
-A,B
D1
D2
Control
A1
B
???
Pos
-
?
+
+
+
-
-
+
Prediluted Olympus reagents used
DTL Manual Bench Results
Method
I.S.
-B
-A
-A,B
A1
A2
B
O
-
-
2+
-
-
3+
-
-
1+
2+
-
-
3+
-
Donor #2
RT 30’
Immucor Gamma-clone reagents used
8
National Reference Laboratory
Investigation
Red Cell Testing Results
Donor #2
-A Ortho
Bioclone
-B Ortho
Bioclone
-A,B Ortho
Bioclone
-D Immucor
DAT
Polyspecific
-A1 Dolichos
Bifloris
-H Ulex
Eropeus
NA=not applicable
I.S.
RT 60’
4oC 60’
-
1+
2+
-
NA
NA
-
1+
2+
4+
NA
NA
-
NA
NA
-
NA
NA
3+
NA
NA
9
National Reference Laboratory
Investigation
Plasma Testing Results
Donor#2
I.S.
RT 60’
4oC 60’
Group O
-
-
1+
Group O
-
-
1+
Group A1
-
-
1+
Group A1
-
-
1+
Group A1
-
-
1+
Group A2
-
-
1+
Group A2
-
-
1+
Group A2
-
-
1+
Auto
-
-
1+
10
Comparability Study
Donor RBCs
Method
-A Ortho
Bioclone
-A DBL
Novoclone
-A Immucor
Gamma-clone
-A Biotest
Seraclone
-AB Ortho
Bioclone
-AB DBL
Novoclone
-AB Immucor
Gamma-clone
-AB Biotest
Seraclone
-AB CBS
Human
w=weak
Ax RBCs
Ael RBCs
(Neg control)
A3 RBCs
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
1w
1+
2+
1w
1+
2+
2+
2+
3+
-
-
-
-
1w
2+
-
w
2+
1+
2+
3+
-
-
-
-
1+
2+
-
w
2+
1+
2+
3+
-
-
-
1w
1+
2+
1w
1+
2+
2+
2+
3+
-
-
-
1w
1+
2+
1w
1+
2+
2+
2+
3+
-
-
-
-
1+
1+
-
1+
1+
1+
2+
2+
-
-
-
2+
3+
3+
2+
3+
3+
3+
3+
4+
-
-
-
1w
1+
2+
1w
1w
2+
2+
2+
3+
-
-
-
1+
2+
2+
1+
2+
2+
2+
3+
3+
-
-
-
11
Conclusion Case #1
• Anti-A1 lectin neg and anti-H lectin pos
• Reverse grouping as a Group A individual
• Autoantibody present at 4oC 60’ incubation
• Comparability Study helped determine a possible Ax
subgroup
• All results confirms subgroup of A
12
Ax : 646A, 681A [novel changes]
261
526
646 681 703 796/803
A1
Ax
B1
G
O1
13
Donor Testing Laboratory Results
Case #2
PK7200 Data Log Results
Donor #5
ABO
Rh
-B
-A
-A,B
D1
D2
Control
A1
B
???
Pos
-
-
-
+
+
-
-
+
Prediluted Olympus reagents used
DTL Manual Bench Results
Method
I.S.
-B
-A
-A,B
A1
A2
B
O
-
-
-
-
-
2+
-
-
-
-
2+
-
3+
-
Donor #5
RT 30’
Immucor Gamma-clone reagents used
14
National Reference Laboratory Investigation
Red Cell Testing Results
Donor #5
-A Ortho
Bioclone
-A CBS
Human
-B Ortho
Bioclone
-A,B Ortho
Bioclone
-A,B CBS
Human
-D Immucor
DAT
Polyspecific
-A1 Dolichos
Bifloris
-H Ulex
Eropeus
I.S.
RT 60’
4oC 60’
-
-
-
-
-
-
-
NA
NA
-
-
-
-
-
-
4+
NA
NA
-
NA
NA
-
NA
NA
4+
NA
NA
NA=not applicable
15
National Reference Laboratory Investigation
Plasma Testing Results
Donor #5
I.S.
RT 60’
4oC 60’
Group O
-
-
NA=not applicable
Group O
-
-
-
Group O
-
-
-
Group A1
-
1+
2+
Group A1
-
1+
3+
Group A1
-
1+
3+
Group A2
-
-
2+
Group A2
-
-
2+
Group A2
-
-
1+
Group B
3+
NA
NA
Auto
-
-
-
16
Comparability Study
Donor RBCs
Method
-A Ortho
Bioclone
-A Immucor
Gamma-clone
-A Biotest
Seraclone
-A CBS
Human
-AB Ortho
Bioclone
-AB DBL
Novoclone
-AB Immucor
Gamma-clone
-AB CBS
Human
w=weak
MF=mixed field
Ax RBCs
O Cells
(Neg control)
Ael RBCs
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
-
-
-
1w
1+
2+
-
-
-
-
-
-
-
-
-
w
w
1MF
-
-
-
-
-
-
-
-
-
1w
1w
1w
-
-
-
-
-
-
-
-
-
-
-
w
-
-
-
-
-
-
-
-
-
2+
2+
2+
-
-
-
-
-
-
-
-
-
1+
1MF
1MF
-
-
-
-
-
-
-
-
-
1+
3+
4+
-
-
-
-
-
-
-
-
-
w
2+
2+
-
-
-
-
-
-
17
National Reference Laboratory
Investigation
Results of Eluate Testing
Donor
I.S.
37oC 60’
IAT
Last Wash
Group O
-
-
-
-
Group O
-
-
-
-
Group O
-
-
-
-
Group A1
-
1+
1+
-
Group A1
-
1+
1+
-
Group A1
-
1+
1+
-
Performed fixation/elution with Donor RBCs and Human anti-A
18
Conclusion Case #2
• Anti-A1 lectin neg and anti-H lectin pos
• No reactivity with Anti-A and Anti-A,B reagents
• No reactivity with A2 RBCs in the reverse grouping
• Anti-A present in the plasma at 4oC
• Comparability Study showed no A antigen prompting
further investigation of Ael subgroup
• Anti-A was fixated and a weak anti-A was eluted from
donor RBCs
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Ael : 467T[novel], 646A[Ax], 681A[Ax}
261 467 526
646 681 703 796/803
A1
Ael
B1
G
O1
20
Donor Testing Laboratory
Results Case #3
PK7200 Data Log Results
Donor #3
ABO
Rh
-B
-A
-A,B
D1
D2
Control
A1
B
???
POS
-
-
+
+
+
-
-
+
Prediluted Olympus reagents used
DTL Manual Bench Results
Method
-B
-A
-A,B
A1
A2
B
O
I.S.
-
-
MF
3+
3+
4+
2+
RT 30’
-
-
MF
3+
3+
4+
4+
Donor #3
Immucor Gamma-clone reagents used
MF=mixed field
21
National Reference Laboratory Investigation
Red Cell Testing Results
Donor #3
-A Ortho
Bioclone
-A CBS
Human
-B Ortho
Bioclone
-A,B Ortho
Bioclone
-A,B CBS
Human
-D Immucor
DAT
Polyspecific
-A1 Dolichos
Bifloris
-H Ulex
Eropeus
I.S.
RT 60’
4oC 60’
-
-
w
-
-
-
-
NA
NA
-
-
w
-
1+
2+
4+
NA
NA
-
NA
NA
-
NA
NA
-
NA
NA
w=weak
NA=not applicable
22
National Reference Laboratory Investigation
Plasma Testing Results 1
Donor #3
I.S.
RT 60’
4oC 60’
Group O
1w
1+
2+
Group O
1w
1+
2+
Group O
1w
1+
2+
Group A1
1w
1+
2+
Group A1
1w
1+
4+
Group A1
-
1+
2+
Group A2
1w
1+
2+
Group A2
-
1+
3+
Group A2
-
1+
3+
O Cord I-
-
-
1+
Auto
-
-
-
w=weak
23
National Reference Laboratory
Investigation
Plasma Testing Results 2
Donor #3
I.S.
Saline 37oC
IAT
I.S.
RT 60’
4oC 60’
Group O
1w
-
w
NA
NA
NA
Group O
1w
-
1+
NA
NA
NA
Group O
1w
-
1+
NA
NA
NA
Group Oh
-
-
-
-
-
w
Group Oh
-
-
-
-
-
-
Group Ah
-
-
-
-
-
-
Group A1
-
-
w
NA
NA
NA
Group B
4+
NA
NA
NA
NA
NA
Auto
-
-
-
-
-
-
w=weak
NA=not applicable
24
National Reference Laboratory
Investigation
Plasma Testing Results 3
Donor #3
I.S.
Saline 37oC
IAT
PEG IAT
Group O
NA
NA
NA
w
Group O
NA
NA
NA
1+
Group A1
NA
NA
NA
1+
Group A1
NA
NA
NA
w
Group Oh
-
-
-
-
Group O (I-)
-
1+
2+
vw
Group A1(I-)
-
vw
1+
NT
Group Ax
NA
NA
NA
w
Auto
-
-
-
-
w=weak
NA=not applicable
v w=very weak
NT=not tested
25
Comparability Study
Donor RBCs
Method
-A Ortho
Bioclone
-A DBL
Novoclone
-A Immucor
Gamma-clone
-A Biotest
Seraclone
-A CBS
Human
-AB Ortho
Bioclone
-AB DBL
Novoclone
-AB Immucor
Gamma-clone
-AB Biotest
Seraclone
-AB CBS
Human
w=weak
MF=mixed field
Ax RBCs
O Cells
(Neg control)
A3 RBCs
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
I.S
RT 60’
4oC
60’
-
-
w
1w
2+
2+
3+
3+MF
3+MF
-
-
-
-
-
w
-
w
1+
1+
2+MF
3+MF
-
-
-
-
-
-
w
w
1+
1+
2+MF
3+MF
-
-
-
-
-
-
w
w
w
3+MF
3+MF
3+MF
-
-
-
-
-
-
-
-
-
-
-
w
-
-
-
-
-
w
1w
2+
2+
3+
3+
3+MF
-
-
-
-
-
1w
1+
1+
2+MF
3+
3+MF
3+MF
-
-
-
1+
3+
2+
3+
4+
4+
3+
4+MF
4+MF
-
-
-
-
-
-
w
w
1+
2+
2+MF
2+MF
-
-
-
-
1+
2+
1+
2+
3+
2+
3+MF
3+MF
-
-
-
26
Flow Cytometry Results
Testing perfomed by Jill R. Storry, Ph.D., FIBMS of Blodcentralen Skane in the UK
Flow cytometric analysis of the donor RBCs, shown in red, with anti-H (left) and anti-A (right)
27
Conclusion Case #3
• Donor reverses as a Group A individual as per the PK7200 results
and MF reactions in the forward suggest Subgroup of A (slide 11)
• DTL results also MF reaction in the forward grouping and reverse
grouping reaction with Group O cells suggests interfering antibody
present in the plasma possible cold agglutinin (slide 11)
• Cold antibody present in plasma
• Presence of anti-H and excludes anti-I and other antibodies to the
major blood group antigens
• The comparability study concludes that this donor has some A
antigen expressed.
• Flow Cytometry results confirm that donor is Ah with weak A antigen
present on the RBCs
28
ABO(H) Antigens
• Type 2 precursor chain:
– fucosyltransferase 1 (FUT1) H substance
• Type 1 precursor chain:
– fucosyltransferase 2 (FUT2) H substance
H Substance:
– N-acetylgalactosaminyltransferase: A
– galactosyltransferase: B
• Bombay phenotype
– FUT1 deletion, FUT2 deletion (se/se)
• Para-Bombay phenotype
– FUT1 deletion, Secretor
29
Donor Testing Laboratory Results Case #4
Unit returned to CBS - Labelled B pos, tested AB pos at hospital
PK7200 Data Log Results
Donor #4
ABO
Rh
-B
-A
-A,B
D1
D2
Control
A1
B
B
POS
+
-
+
+
+
-
+
-
Prediluted Olympus reagents used
DTL Manual Bench Results
Method
-B
-A
-A,B
A1
A2
B
O
I.S.
4+
-
4+
4+
3+
-
-
RT 30’
4+
MF
4+
4+
3+
-
-
Donor #4
Immucor Gamma-clone reagents used
MF=mixed field
30
National Reference Laboratory Investigation
Red Cell Testing Results
Donor #4
-A Ortho
Bioclone
-A DBL
Novoclone
-A Gamma
Gammaclone
-A Biotest
Seraclone
-A CBS
Human
-AB Ortho
Bioclone
-D Immucor
DAT
Polyspecific
-A1 Dolichos
Bifloris
-H Ulex
Eropeus
I.S.
RT 60’
4oC 60’
-
vw
w
-
-
vw
-
-
w
-
-
vw
-
-
vw
4+
NA
NA
4+
NA
NA
w
NA
NA
-
NA
NA
-
NA
NA
v w=very weak
w=weak
NA=not applicable
31
National Reference Laboratory
Investigation
Plasma Testing Results
Donor #4
I.S.
RT 60’
4oC 60’
Group O
-
-
-
Group O
-
-
1+
Group O
-
-
1+
Group A1
4+
4+
4+
Group A1
4+
4+
4+
Group A1
4+
4+
4+
Group A2
4+
4+
4+
Group A2
4+
4+
4+
Group A2
4+
4+
4+
Group B
-
-
1+
Auto
-
-
2+
32
Conclusion Case #4
• Numerous donations and historically a Group B+
individual
• Forwards and reverses as a Group B individual on this
donation
• Mixed field reactions seen with anti-A reagents
• 1/100 Group B individuals reacts with a blend of 2
monoclonal anti-A, one clone is used for its rapid
reactions with A1 and A2 RBCs and the second clone for
its ability to react strongly with Ax RBCs
• B(A) activity
33
B(A) activity
• The wild-type B glycoslytransferase transfers
very little A sugar onto H substance
– not detectable using antibody probes!
• Some B glycosyltransferases transfer more A
sugar onto H substance.
– B cells phenotype as B with some moAbs and
weakA,B with other moAbs
– The B glycosyltransferase was found to contain an
703G (235Gly), normally associated with the A1
– represented by the acronym BABB
• Can make anti-A
34
B(A) : 703G[A1]
261
526
703 796/803
A1
B1
B(A)
G
O1
35
Donor Testing Laboratory Results
Case #5
PK7200 Data Log Results
Donor #2
ABO
Rh
-B
-A
-A,B
D1
D2
Control
A1
B
???
Pos
-
-
-
+
+
-
?
+
Prediluted Olympus reagents used
DTL Manual Bench Results
Method
I.S.
-B
-A
-A,B
A1
A2
B
O
-
-
-
w
-
4+
-
NA
-
-
1+
w
NA
-
Donor #2
RT 30’
Immucor Gamma-clone reagents used
36
O2 : 526G[B], 802A[novel]
261
526
802
703 796/803
A1
B1
G
O1
O2
The (nondeletional) O2 allele produces an
enzygme with weak glycosyltransferase A activity
37
Donor Testing Laboratory Results
Case #6
PK7200 Data Log Results
Donor #2
ABO
Rh
-B
-A
-A,B
D1
D2
Control
A1
B
???
Pos
-
+
+
+
+
-
-
-
Prediluted Olympus reagents used
DTL Manual Bench Results
Method
I.S.
-B
-A
-A,B
A1
A2
B
O
-
4+
4+
-
-
-
-
-
NA
NA
-
-
-
-
Donor #2
RT 30’
Immucor Gamma-clone reagents used
38
Sudbury case (Bel ?): B + 641G[novel]
261
526
641
703 796/803
A1
B
Bel
G
O1
39
References
• Westhoff CM, Reid ME. ABO and related antigens and
antibodies. In Blood Banking and Transfusion Medicine
Basic Principles and Practice 2nd ed. Hillyer CD et al
eds. Churchill Livingstone, Philadelphia, PA. 2007: 6979.
• ABO, Lewis and P groups and Ii antigens. In Mollison's
Blood Transfusion in Clinical Medicine 11th ed. Klein HG,
Anstee DJ eds. Blackwell Publishing Inc, Malden, MA.
2005:114-162.
• Investigating ABO typing problems. In Judd's Methods in
Immunohematology 3rd ed. Judd WJ, Johnson SJ,
Storry JR eds. AABB Press, Bethesda, MA. 2008: 515558
40