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

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.

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?

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

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

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.

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- 18.6 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

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+ ?

Phenotype Occurrence Type Caucasians(%) Iranian A1 33 Total 32 ….? A2 10 B 9 24 O 44 36 A1B 3 Total 8….? A2B 1

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

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

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

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

Bilirubin Direct (0.0-0.3mg/dL) RBC (at birth 3.9-0.65× 10 3 /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 (0.0-0.3mg/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 1.4-8.7 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

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 (150-400× 10 3 /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

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

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

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

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.

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?

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?

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