1. Iranian Blood Transfusion Organization Immunohematology Reference Laboratory ABO&Rh(D) Discrepancies Between Forward and Reverse Grouping Solving Blood Bank Puzzles Case Studies
Mostafa Moghaddam MS, MT ( ASCP ) BB Head of Immunohematology Reference Laboratory

2. Based on serologic specificity of the causative IgG antibody, HDFN is classified in to three categories:
Rh(D) hemolytic disease , due to anti-D alone or more rarely in combination with anti-C or anti-E . THIS IS THE MOST SEVERE TYPE and may result in fetal death.
“ Other HDFN” due to antibodies against other antigen in the Rh system, such as anti-c, anti-E, or anti-e , or the antigens in other blood group system, such as anti-K , anti-Fya and many others .Baby is usually mildly affected but sever HDFN has also been documented.
ABO HDFN ,due usually to anti-A,B in a group O woman , but rarely to anti-A or anti-B. THIS IS THE MOST COMMON FORM, BUT ALSO THE MILDEST.

3. Question: 1- Does an ABO discrepancy exist?
Situation : Unexpected discrepancy
A 27-year-old multiparous mom gave birth to a full- term girl
Baby's direct antiglobulin test was positive due to mom’s previous sensitization to anti-c
Blood bank further discovered an unexpected discrepancies in mom’s ABO blood grouping
ABO&Rh(D)
Interpretation
Auto Control Test
Antibody Screening Test
Rh(D)
Reverse Grouping
Forward Grouping
Patient
Case
DAT
Rh(D)Control
Anti-D
(IgM) B
Cell A1
Anti-
A,B
Anti-A A
O positive?!! 0
Positive 4+ 2+
Mom
O positive
Negative
Dad
A positive NT
Infant
NT: Not Tested
Question:
1- Does an ABO discrepancy exist?

4. Antibody Screening Test
Situation : A very rare ABO discrepancy
1- referred by S.F hospital
2- infant’s ABO blood group does not match with dad and mom’s ABO blood type
ABO&Rh(D)
Interpretation
Elution test
Auto Control Test
Antibody Screening Test
Rh(D)
Reverse Grouping
Forward Grouping
Patient
Case
DAT
Rh(D)Control
Anti-D
(IgM)
B cell
A1 Cell
Anti-
A,B B
Anti-A O+ NT 0
Negative 4+
Mom
AB=
Dad
AB+
A1 cells(AHG)2+
B cells(AHG)2+
Infant
Positive
NT: Not Tested
Question :
Is there an ABO discrepancy between Mom, Dad and Baby’s Blood Group

5. Antibody Screening Test
Situation : A very rare ABO discrepancy
ABO&Rh(D)
Interpretation
Elution test
Auto Control Test
Antibody Screening Test
Rh(D)
Reverse Grouping
Forward Grouping
Patient
Case
DAT
Rh(D)Control
Anti-D
(IgM)
B cell
A1 Cell
Anti-
A,B B
Anti-A O+ NT 0
Negative 4+
Mom
AB=
Dad
AB+
A1 cells(AHG)2+
B cells(AHG)2+
Infant
Positive
NT: Not Tested
Answer: AB infant was born to an O mother. The mother carries an O,O type and would have to pass an O to the infant, leaving the possibilities of O,O : A,O : or B,O type for the infant.
Probably due to :
1- blood specimen was switched and / or mislabeled
2- wrong infant was drawn
3 - infant was switched
4- clerical error of the mother’s blood type
5- procedural error made during the testing of the cord blood and mother’s specimen
6- surrogacy , IVF and other unusual circumstances were ruled out
7- the father’s blood specimen was confirmed as AB negative
8- Need further investigation since baby’s blood group discrepancy isn’t resolved

6. Situation : A very rare ABO discrepancy
Answer:
The infant was determined by the molecular testing ( PCR-RFLP testing and genomic DNA sequencing of the ABO transferase genes ) to be a unique cis- AB blood type , inherited from the father.

7. Situation : HDFN in an A2B Baby Case : C
A newborn baby appeared to be having complications after birth
Jaundiced skin
Low RBC count 1.66× 10 6 /µl
Low Hgb 7.4g/dl
High neonate bilirubin mg/dl
respiratory distress
Possible HDFN was suspected
Baby’s Jaundice was treated with UV lights
Mom was typed previously
Had an ABO discrepancy
Typed as a possible subgroup of A
Antibody screen test was negative

8. The Mother’s Testing Mother typing had an ABO discrepancy
Forward type looks like an A, reverse looks like an O
The A1 cells should be negative
Subgroup of A is investigated
A1 is the most common subtype of A by 80%
A2 is the second most common by 20%
A3, Ax, Am, and Ael are extremely weak and rare subgroups
ABO
Anti-A
Anti-B
Anti-D
A1 cells
B cells
Intr.
Expected 4+
Mom 1+ ?

9. Phenotype Occurrence Type Caucasians(%) Iranian A1 33 Total 32 ….? A210 B 9 24 O 44 36
A1B 3
Total 8….?
A2B 1

10. Mother is presumed to be an A2 type because she is
A1 (Pos contrl)
A2(Neg control)
Mother
Anti-A 4+
interpretation
A1+
A1=
Mother is presumed to be an A2 type because she is
negative with anti-A1

11. Compared to A1, A2 only has 1/5th the amount of antigen sites as A1.
Fewer antigen sites make it a weak A because there was
inefficient conversion of H antigen to A antigen.
Can test with anti H lectin to type A2.
A1 will not react with anti H while A2 will.
A2 individuals can develop an anti-A1 that is IgM in nature

12. Baby forward type, Ab screen tested with eluate , and DAT were performed from a heelstick
Only forward type is performed because baby has not
developed antibodies yet
ABO&Rh(D)
Anti-A
Anti-B
Intr.
Anti-D
Monoclonal control
DAT
Baby 3+ 4+ ?
Eluate test
SC I
SC II
SC III 0

13. Positive result is from the mother’s anti B that crossed the placenta
A and B cells are tested with the antibody screen to
determine if the mother’s ABO antibodies are crossing the placenta
A cells
B cells 3+
Positive result is from the mother’s anti B that
crossed the placenta
Babies do not develop ABO isoantibodies until 3 to 6 months, so if present, they are from the mom

14. Bilirubin Direct (0.0-0.3mg/dL)
RBC (at birth × /uL)
Day 1
01:15
05:17
17:30
Day 2
05:35
Day 3
05:00
1.66
4.58
3.75
3.60
3.57
Bilirubin Direct ( mg/dL)
Day 1
01:15
05:17
17:30
23:30
Day 2
05:35
Day 3
05:00
0.8
0.5
0.7
0.6
Bilirubin Total(0-1 day old mg/dL)
Day 1
01:15
05:17
17:30
23:30
Day 2
05:35
Day 3
05:00
18.6
9.8
12.7
10.5
9.5
6.1

15. Hemoglobin (14.5-22.5 g/dL) Platelets (150-400× 10 3 /uL) NRBC(0%)
Day 1
01:15
05:17
17:30
Day 2
05:35
Day 3
05:00
Day 4
04:00
7.4
14.1
11.6
11.2
10.8
12.7
Platelets ( × /uL)
Day 1
01:15
05:17
17:30
Day 2
05:35
Day 3
05:00
Day 4
04:00
214 57 66 69 77
106
NRBC(0%)
Day 1
01:15
05:17
17:30
Day 2
05:35
Day 3
05:00
202
128 49 22 4

16. A2B is a rare type; found in 1% of the population
A1 (Pos contrl)
A2(Neg control)
Baby
Anti-A1 4+
Interpretation
A1+
A1=
Baby typed as A2B
A2B is a rare type; found in 1% of the population
A2 is from the mother and B is from the father
Antigen typing was used again to determine the possible A2 type
25% of A2B individuals have an alloanti-A1 in their sera
Important to find compatible blood to prevent development of an
anti-A1

17. Anti-B in an A mother is typically IgM in nature
There have only been a few cases reported of IgG anti-B in A2 mothers causing HDFN in B type babies
IgG anti B can be naturally occurring, but is in small quantities
A higher titer IgG anti-B may be naturally occurring or develop after being sensitized to B antigens in previous pregnancies

18. Transfusion was performed to treat the anemia
Treatment
Transfusion was performed to treat the anemia
O negative type blood was used
Blood needs to be compatible with mother and baby since baby
has mom’s antibodies
AB Fresh Frozen Plasma
FFP needs to be compatible with baby and donor cells
Baby’s lab results improved to a better level after the
transfusion and use of phototherapy, and continued to reach normal levels as time progressed as the antibody decreased

19. A2 mother gives birth to an A2B baby who is experiencing HDFN
Summary
A2 mother gives birth to an A2B baby who is
experiencing HDFN
HDFN is determined to be caused by an IgG anti-B from the mother
Only IgG antibodies cross the placenta
Mother had a high titer of natural IgG anti-B 1024 possibly sensitized from a previous pregnancy
Baby was treated with phototherapy and a
transfusion.

20. Antibody Screening Test
Situation : Routine ABO subgroup
1- 28 years old female patient was referred by M.Y. hospital
2- blood bank was not able to determine patient ABO
ABO&Rh(D)
Interpretation
Auto Control Test
Antibody Screening Test
Rh(D)
Reverse Grouping
Forward Grouping
Incubation time
Case
DAT
Rh(D)Control
Anti-D
(IgM)
BCell
A2 Cell
A1 Cell
Anti-H
Anti-A,B
Anti-B
Anti-A1
Anti-A D
Unresolved positive
0 
Negative
Positive 4+ NT 3+ 1+ IS ?
C 15’°4
NT: Not Tested
IS: Immediate Spin
Your suggestions for ABO & Rh( D ) discrepancy?

21. Antibody Screening Test
Situation : Routine ABO subgroup
1- 28 years old female patient was referred by M.Y. hospital
2- blood bank was not able to determine patient ABO
ABO&Rh(D)
Interpretation
Auto Control Test
Antibody Screening Test
Rh(D)
Reverse Grouping
Forward Grouping
Incubation time
Case
DAT
Rh(D)Control
Anti-D
(IgM)
BCell
A2 Cell
A1 Cell
Anti-H
Anti-A,B
Anti-B
Anti-A1
Anti-A D
Unresolved positive
0 
Negative
Positive 4+ NT 3+ 1+ IS
A2B
C 15’°4
NT: Not Tested
IS: Immediate Spin
Answer:
Patient is a subgroup of A2B
Strong anti-A1 was observed in patient plasma
Transfusion recommendation?

22. Antibody Screening Test
Situation : DO I need RhIG or not !!?
referred by G hospital , 40years old mom Gravida : 3 para:3
Rh(D) mismatched with previous results
previously received RhIG
mom complains that she is Rh(D) negative and her husband is also Rh(D) negative !!!??
night shift reported mom’s Rh(D) as negative and her baby Rh(D) as positive but day shift reported mom as Rh(D)positive !!!
ABO&Rh(D)
Interpretation
Auto Control Test
Antibody Screening Test
Rh(D)
Reverse Grouping
Forward Grouping
Patient
Case
DAT
Rh(D)Control
Anti-D
(IgM/IgG) B
Cell
A2 Cell
A1 Cell
Anti-
A,B
Anti-A E
A negative
(night) 0
Negative 4+ NT
Mom
A positive (day) 3+
Dad
A positive
Infant
NT: Not Tested
Answer:
mom has a weak-Rh(D) reacting at AHG phase
Does she need to receive RhIG