ABO DISCREPANCIES AND THEIR RESOLUTION

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ABO DISCREPANCIES AND THEIR RESOLUTION:

ABO DISCREPANCIES AND THEIR RESOLUTION Dr Satyam Arora

Discrepancies:

Discrepancies A discrepancy occurs when the red cell testing does NOT match the serum testing results. In other words, the forward does NOT match the reverse. Reaction strengths could be weaker than expected. Some reactions may be missing in the reverse or forward typing. Extra reactions may occur.

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There are mainly two causes of discrepancy Most of the time, the problem is technical errors Other times, there is a real discrepancy due to problems with the patient’s red cells or serum

Technical Errors:

Technical Errors Clerical errors Mislabeled vials Patient misidentification Errors due to inadequate details of previous transfusion on requision form Errors due to non-uniform identification number Reagent or equipment problems Using expired reagents Using an uncalibrated centrifuge Contaminated or hemolyzed reagents Incorrect storage temperatures Procedural errors Reagents not added Manufacturer’s directions not followed RBC suspensions incorrect concentration Cell buttons not resuspended before grading agglutination

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59.3% 15.4 % 7.3 % Sources of preventable errors related to transfusion Vox Sanguinis Volume 81 Issue 1 ,page 37-41,2001 R. R. Sharma , S. Kumar & S. K. Agnihotri Department of Transfusion Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India

ABO discrepancies:

ABO discrepancies

ABO Discrepancies:

ABO Discrepancies Group I discrepancies These discrepancies are between forward and reverse grouping due to weak reaction or missing antibodies . These kind of discrepancies are the most common .

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Group II discrepancies These discrepancies are between forward and reverse grouping due to weak reaction or missing antigens . This group is the least one . Can be caused by some subgroups of A or subgroups of B or both .

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Group III discrepancies These discrepancies are between forward and reverse grouping due to protein or plasma abnormalities. These can be caused by elevated levels of globulin from certain diseases such as multiple myeloma, Hodgkin’s lymphoma. Some are caused by Rouleaux formation

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Group IV discrepancies These kind of discrepancies are between forward and reverse groping due to miscellaneous problems. Cold reacting autoantibodies . Patients with RBCs of more than one ABO group due to RBC transfusion or marrow transplant. Unexpected non-ABO alloantibodies .

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May cause all + reactions

Reverse Grouping Problems (Group I discrepancies):

Reverse Grouping Problems ( Group I discrepancies)

Reverse Grouping:

Reverse Grouping Reverse grouping results affects by mainly 2 causes Missing or weak antibodies Extra antibodies

Reverse Grouping: Missing or Weak antibodies:

Reverse Grouping: Missing or Weak antibodies Newborns Do not form antibodies Elderly Weakened antibody activity Hypo- gammaglobulinemia Little or no antibody production (i.e. immuno -compromised) Patients with immunodeficiency disease, BM transplant and with lymphoma Patients on immunosuppressive drugs.

Resolving Weak or Missing antibodies:

Resolving Weak or Missing antibodies Determine patients age, diagnosis Incubate serum testing for 15 minutes (RT) to enhance antibody reactions If negative, place serum testing at 4°C for 5 minutes with autologous control ( Autocontrol , AC) ABO antibodies are enhanced on cold temperature

Reverse Grouping: Extra Antibodies:

Reverse Grouping: Extra Antibodies Cold antibodies ( allo - or auto-) Cold antibodies may include anti-I, H, M, N, P, Lewis Anti-A 1 in an A 2 or A 2 B individual

Cold antibodies:

Cold antibodies Sometimes a patient develops cold-reacting allo - or auto-antibodies that appear as “extra” antibodies on reverse typing Alloantibodies are made against foreign red cells Autoantibodies are made against ones own red cells. Cold reacting antibodies cause agglutination with red cells at room temperature and below. The autocontrol will be positive. Resolution: warming tube to 37° and washing red cells can disperse agglutination

Forward Grouping Problems (Group II discrepancies):

Forward Grouping Problems ( Group II discrepancies)

Red Cell Problems:

Red Cell Problems Affect the forward grouping results Missing or weak antigens Extra antigens Mixed field reactions

Forward Grouping: Missing or Weak antigens:

Forward Grouping: Missing or Weak antigens Anti-A Anti-B A1 Cells B Cells 0 0 0 4+ ABO Subgroups Disease (leukemia, Hodgkin’s disease) Least common type of discrepancy Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping) Group O Group A

Subgroups of A :

Subgroups of A Subgroups of A account for a small portion of the A population These subgroups have less antigen sites on the surface of the red blood cell As a result, they show weakened (or missing) reactions when tested with commercial antisera

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Testing Of Red Cells Naturally Occurring Antibody In Serum Substances in Saliva of secretors A transferase in Serum No. Of Antigen sites / Red cell × 10 3 Phenotype Anti-A Anti-B Anti-AB Anti-H Common Unexpected A 3 ++mf 0 ++mf +++ Anti-B Sometime Anti-A 1 A,H Weak Pos 30 A x wk/0 0 ++ ++++ Anti-B Always Anti-A 1 H Very Weak Pos 4 A end wk mf 0 wk mf ++++ Anti-B Sometime Anti-A 1 H Neg 3.5 A m 0 0 0 ++++ Anti-B No Anti-A 1 A,H Pos 0.2-1.9 A y 0 0 0 ++++ Anti-B No Anti-A 1 A,H Weak Pos _ A el 0 0 0 ++++ Anti-B Usually Anti-A 1 Sometime anti-A also H Neg 0.1 -1.4 Weak Subgroup of A

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4+ One large agglutinate with clear background 3+ Several large agglutinates with clear background 2+ Medium size agglutinates with clear background 1+ Small agglutinates with turbid background 0 No agglutination Mf Mixture of agglutinated & unagglutinated RBCs H Haemolysis Strength of Agglutination

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Strength of Agglutination 4+ 1+ 3+ 2+ 0

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Red cell Reactions With Anti-A and / Anti-AB Weakly agglutinated No agglutination (A 3 ,A x ,A end ) Adsorbes and elutes anti-A ( A m ,A y ,A el ) Mixed field ≤10% mf wk Rx n with Anti-AB A 3 A end Ax A sub.in saliva S mall amount H sub. A sub. in saliva in saliva A m A y A el

B Subgroup:

B Subgroup Subgroups of B are infrequent than the weaker subgroup of A, identified by anti-B and anti-A,B. Subgroups B3 , Bx , Bm and Bel .

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Testing Of Red Cells Naturally Occurring Antibody In Serum Substances in Saliva of secretors B transferase in Serum Phenotype Anti-A Anti-B Anti-AB Anti-H Common Unexpected B 0 ++++ ++++ ++ Anti-A None B,H Pos B 3 0 mf ++ ++mf +++ Anti-A None B,H Weak Pos B x 0 wk/0 + ++++ Anti-A Weak Anti-B H Neg B m 0 0 wk mf ++++ Anti-A None B,H Weak Pos B el 0 0 0 ++++ Anti-A Sometime a weak Anti-B H Neg Subgroups of B

Adsorption-Elution:

Adsorption-Elution Adsorption- In this an antibody with its antigen treated under optimal conditions so that the antibody will attach to the antigen, thereby removing the antibody from the serum Elution - In this cells that are coated with antibody are treated to disrupt the bonds between antibody and antigen, the free antibody is collected in an inert diluent such as saline or 6% albumin and tested with reagent cells for specificity.

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1 ml of antiserum and 0.5 ml of packed cells are added in a tube Similarly a set of control samples are also set up Resuspend the RBC and transfer tubes to 4 C for 2 hours Wash the RBCs six times with 4 C saline. Save the final wash supernatant for parallel testing with the eluate Add 1 ml of 6% BSA to each tube of packed RBCs and mix well Place the tubes at 56 C for 10 minutes and stir periodically Centrifuge the pack of cells and harvest the supernatant Test each eluate and final wash supernatant against the reagent RBCs (A, B and O cells)at RT and 4C Procedure (heat elution) Source – Judd’s methods in immunohematology 3 rd edition

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EXAMPLE - Adsorption- elution for weak A sub group Reagent used- ( 1) Donor packed cells ( 2) Polyclonal Anti-A sera ( sera from B group donor) ( 3) Bovine serum albumin 6% ( 4) 3 unpooled cells each of A,B,O group Results- With Final wash- A1 A2 A3 B1 B2 B3 O1 O2 O3 RT Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. 4* Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. 37* Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. A1 A2 A3 B1 B2 B3 O1 O2 O3 RT 4+ 3+ 3+ Neg. Neg. Neg. Neg. Neg. Neg. 4* 4+ 3+ 3+ Neg. Neg. Neg. Neg. Neg. Neg. 37* 4+ 3+ 3+ Neg. Neg. Neg. Neg. Neg. Neg. With Elute-

Forward Grouping: Extra Antigens:

Acquired B B(A) phenotype Rouleaux Polyagglutination Wharton’s Jelly Forward Grouping: Extra Antigens

Acquired B Phenotype:

Acquired B Phenotype Limited mainly to Group A 1 individuals with: Lower GI tract disease Cancer of colon/rectum Intestinal obstruction Gram negative septicemia (i.e. E. coli )

Acquired B:

Acquired B Bacteria ( E. coli ) have a de acetylating enzyme that effects the A sugar…. Group A individual N-acetyl galactosamine Acquired B Phenotype Bacterial enzyme removes acetyl group Galactosamine now resembles D-galactose (found in Group B)

Resolving Acquired B:

Resolving Acquired B Check patient diagnosis: Infection? Some manufacturers produce anti-B reagent that does not react with acquired B Test patients serum with their own RBCs The patients own anti-B will not react with the acquired B antigen on their red cell ( autologous testing) Treatment with acetic anhydrate (rehydrates the acquired B)

B(A) phenotype:

B(A) phenotype Similar to acquired B Patient is Group B with an apparent extra A antigen The RBC consists of high levels of galactosyltransferase which are agglutinated by selected potent monoclonal anti A reagents. Sometimes certain anti-A reagents will detect these trace amount of A antigen

Other reason for “extra” antigens:

Other reason for “ extra ” antigens Wharton’s Jelly – Gelatinous substance derived from connective tissue that is found in cord blood and may cause false agglutination Wash red cells 6 times or request new sample from heel, etc

Forward Grouping: Mixed Field Agglutination:

Forward Grouping: Mixed Field Agglutination Results from two different cell populations Agglutinates are seen with a background of unagglutinated cells All groups transfused with Group O cells Bone marrow/stem cell recipients A 3 phenotype

Rouleaux:

Rouleaux “stack of coins” appearance (microscopically) May falsely appear as agglutination due to the increase of serum proteins (globulins) Associated with: Multiple myeloma Waldenstrom’s macroglobulinemia Hydroxyethyl starch (HES), dextran

Resolving Rouleaux:

Resolving Rouleaux Remove proteins If the forward grouping is affected, wash cells to remove protein and repeat test If the reverse grouping is affected, perform saline replacement technique Reagent cells and patient serum centrifuged to allow antigen and antibody to react Serum is removed and replaced by an equal volume of saline (saline disperses cells) Tube is mixed, centrifuged, and reexamined for agglutination

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CASE 1 Pt.- J ,60 yr M, C.R. No.-653263 Diagnosis- Multiple Myeloma Forward Group on slide- AB+ *Forward Group on tube with washed cells- B+ Anti-A Anti-B Anti-D Anti-AB Neg. 4+ 4+ 4+ *Reverse Group on tube- React with all cells Ac Bc Oc Autocontrol 4+ 1+ 1+ 1+ *No reaction with screening cells

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RESOLUTION Reverse grouping with saline replacement tech. Ac Bc Oc Autocontrol 4+ Neg. Neg. Neg. Blood group is B Conclusion - Pt. serum having increase protein because of multiple myeloma causing rouleaux formation which cause group discrepancy which resolved by saline replacement tech.

CASE 2:

CASE 2 Anti-B Anti-A Anti-D Anti-AB 4+ 1+mf 4+ 4+ Forward Grouping- A wk B Rh (D) positive Reverse Grouping- Blood group AB A cell B cell O cell Neg. Neg. Neg. Conclusion - this is a case of AB+ blood group with weak A antigen which can be confirmed by saliva secretor status and transferase activity in serum. Donor unit No.-910429 Testing Of Red Cells Naturally Occurring Antibody In Serum Substances in Saliva of secretors A transferase in Serum No. Of Antigen sites / Red cell × 10 3 Phenotype Anti-A Anti-B Anti-AB Anti-H Common Unexpected A 3 ++mf 0 ++mf +++ Anti-B Sometime Anti-A 1 A,H Weak Pos 30 A x wk/0 0 ++ ++++ Anti-B Always Anti-A 1 H Very Weak Pos 4 A end wk mf 0 wk mf ++++ Anti-B Sometime Anti-A 1 H Neg 3.5 A m 0 0 0 ++++ Anti-B No Anti-A 1 A,H Pos 0.2-1.9 A y 0 0 0 ++++ Anti-B No Anti-A 1 A,H Weak Pos _ A el 0 0 0 ++++ Anti-B Usually Anti-A 1 Sometime anti-A also H Neg 0.1 -1.4 Possibility – weak sub group of A (type 2 discrepancy) {A 3 , A x or A end }

CASE 3:

CASE 3 Anti-B Anti-A Anti-D Anti-AB Anti –A1 Anti-H Neg. 1+mf 4+ 3+ Neg. 3+ Forward Grouping - Weak reaction with Anti-A Reverse Grouping- Blood group is A A cell B cell O cell Neg. 3+ Neg. Donor unit No.-907676 Testing Of Red Cells Naturally Occurring Antibody In Serum Substances in Saliva of secretors A transferase in Serum No. Of Antigen sites / Red cell × 10 3 Phenotype Anti-A Anti-B Anti-AB Anti-H Common Unexpected A 3 ++mf 0 ++mf +++ Anti-B Sometime Anti-A 1 A,H Weak Pos 30 A x wk/0 0 ++ ++++ Anti-B Always Anti-A 1 H Very Weak Pos 4 A end wk mf 0 wk mf ++++ Anti-B Sometime Anti-A 1 H Neg 3.5 A m 0 0 0 ++++ Anti-B No Anti-A 1 A,H Pos 0.2-1.9 A y 0 0 0 ++++ Anti-B No Anti-A 1 A,H Weak Pos _ A el 0 0 0 ++++ Anti-B Usually Anti-A 1 H Neg 0.1 -1.4

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Possibility – weak sub group of A (type 2 discrepancy) {A 3 , A x or A end } Conclusion - this is a case of A+ blood group with weak A antigen which can be confirmed by saliva secretor status and transferase activity in serum.

CASE 4:

CASE 4 Conclusion- This is a case of O+ blood group with weak Anti-A antibody which show reaction at cold Forward Grouping- Blood group is O+ Anti-B Anti-A Anti-D Anti-AB Neg. Neg. 4+ Neg. Reverse Grouping- weak reaction with A cell at 4*c Rx at Temp. A cell B cell O cell RT Neg. 4+ Neg. 4*c wk+ 4+ Neg. 37*c Neg. 4+ Neg. Donor unit No.-897654 Possibility – presence of weak Anti A ( type 1 discrepancy )

CASE 5 :

CASE 5 Donor no. 999372 Forward grouping – weak B antigen Rh D positive Anti-B Anti-A Anti-D Anti-AB MF Neg. 3+ Neg. Reverse grouping - B A cell B cell O cell 4+ Neg. Neg. Possibility – type 2 discrepancy (missing/ weak antigen)

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Adsorption elution revealed presence of weak B antigen On Saliva testing, the donor was found to be secretor for both B and H substance . Conclusion- Blood group is B 3 Rh D positive Testing Of Red Cells Naturally Occurring Antibody In Serum Substances in Saliva of secretors B transferase in Serum Phenotype Anti-A Anti-B Anti-AB Anti-H Common Unexpected B 0 ++++ ++++ ++ Anti-A None B,H Pos B 3 0 mf ++ ++mf +++ Anti-A None B,H Weak Pos B x 0 wk/0 + ++++ Anti-A Weak Anti-B H Neg B m 0 0 wk mf ++++ Anti-A None B,H Weak Pos B el 0 0 0 ++++ Anti-A Sometime a weak Anti-B H Neg

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Department of Transfusion Medicine PGIMER Chandigarh Registration No. 977603 Date : 15/12/2010 Sh. Sushil Kumar House No. 1484-A Sector 41-B Chandigarh Phone : 9872749303 Blood Group : A (weak Subgroup) Rh Positive Please Note : Weak A subgroup may be typed as O group For further clarification please contact at : 9914209487 RARE GROUP DONOR Rare donor card issued from our department

THANK YOU …..:

THANK YOU …..

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