ABO & Rh Discrepancies

Definition When the results of the forward grouping (patient cells) do not correspond to the results of the reverse grouping (patient serum) or abnormal reactivity is present (i.e. Mixed Field) i.e. the forward does NOT match the reverse

Patient Anti-A Anti-B A-Cells B-Cells A 4+ 1+ B C D 3+ B C D 3+ Patient A: Additional reaction with anti-B and patients cells. Patient B: Weak reaction with patients serum and A-cells. Patient C: Additional reaction with patients serum and A-cells. Patient D: Missing reactions with patients serum A-cells

ABO Discrepancies must be resolved In RECIPIENTS the discrepancies must be resolved before any blood component is transfused. If not resolved before blood is needed, transfuse Group O (O NEGATIVE if there is a discrepancy in the Rh type also). In DONORS the discrepancies must be resolved before any blood is labeled with a blood type.

Categories of ABO Discrepancies Cell Typing – Additional Reactions Cell Typing – Missing Reactions Serum Typing- Additional Reactions Serum Typing- Missing Reactions

A- Cell Typing: Additional Reactions

1- Polyagglutinable Red Cells ANTI-A ANTI-B A CELL B CELL 4+ The removal of red cell N-Acetyl neuraminic acid by bacterial enzymes expose the T-Ag on the cell membrane. Antibodies to T-antigens are naturally present in most human sera. This Ab can also be found as a contaminant in some ABO typing reagents. This cause unexpected agglutination of T Ags on red cells.

2- Acquired Antigens ANTI-A ANTI-B A CELL B CELL 4+ 2+ Microbial deacetylating enzymes such as E. coli cleave off the N-Acetyl of the Group A N-acetyl-galactosamine The remaining galactosamine becomes similar to the Group B galactose Anti-B react with this B-like Ag causing agglutination A-like Ag can also be acquired

N-acetyl galactosamine Acquired B Phenotype Group A individual N-acetyl galactosamine Acquired B Phenotype Bacterial enzyme removes acetyl group Galactosamine now resembles D-galactose (found in Group B)

3- Non-specific Agglutination Wharton’s Jelly – gelatinous substance derived from connective tissue that is found in cord blood and may cause false agglutination Wharton’s Jelly Coats newborn cord cells and the child's type may appear AB.  We do not do a reverse on newborn blood since they have not made any anti-A or anti-B yet. mucopolysaccharides (hyaluronic acid and chondroitin sulfate). It also contains some fibroblasts and macrophages

If the baby types as an AB  recheck by washing cells several times and re-testing since you need to make sure you have removed the Wharton's Jelly and the baby is truly an AB.  Better ALWAYS wash cord blood at least 4 to 5 before determining the type of the baby, or request new sample from heel

4- Sensitized Red Blood Cells Albumin in the ABO typing reagent can reduce the zeta potential Effectively decreases the distance between red cells. If the red cells are coated with antibody, false positive agglutination can occur

B- Cell Typing Missing Reactions

1- Subgroups Weak variants of both A & B Carry poorly expressed Ags ANTI-A ANTI-B A CELL B CELL 4+ Weak variants of both A & B Carry poorly expressed Ags May not produce expected reactions with anti-A & anti-B They are categorized according to the strength and pattern of reaction with anti-A, anti-H & anti-A,B

2- Altered Antigen Expression ANTI-A ANTI-B A CELL B CELL 4+ Weak Ags may be found on RBCs of some people with diseases (Leukemia)

3- Chimera Chimera: Two cell populations Natural Chimera: In utero exchange of erythropoetic tissue between non-identical twins Strength of reaction with ABO typing depends on the percentage of A or B cells in blood Temporary Chimera: following blood transfusion of ABO compatible, but not identical blood ( A received O cells)

4- Excessive amounts of group specific substances Patients with certain types of cancer Large amounts of soluble A or B Ags Inhibit anti-A or anti-B typing reagent Can be resolved by washing RBCs prior to ABO typing

C- Serum Typing Additional Reactions

1- Alloantibodies Abs other than anti-A or anti-B Can agglutinate A or B cells if express specific Ag Abs commonly cause this discrepancy anti-M, -N, -S, -Lea, Leb, -P1, A1 Can be identified by testing serum with a panel of O cells that have been phenotyped for these Ags

2- Autoantibodies IgM autoantibodies can cause false-positive results in cell & serum grouping Problem can be solved by washing cells with warm saline prior to testing In serum typing, autoabsorption can be performed Or serum typing at 37oC

3- Rouleaux formation ANTI-A ANTI-B A CELL B CELL 4+ 2+ Rouleaux may also give false positive cell typing if strong enough Looks like agglutination macroscopically This phenomenon is due to alteration in serum protein concentration caused by: elevated levels of gammaglobulins elevated levels of fibrinogen Also seen with plasma expanders (dextran) Cell grouping- can be avoided by washing RBCs Serum grouping- addition of saline

D- Serum Typing Missing Reactions

1- Newborns Infants develop ABO Abs by 3-6 months of age Serum typing before this time: Weak reaction Negative reaction

2- Patients with Hypogamma-globulinemia Have low levels of immunoglobulins Anti-A & anti-B may not be detected

3- Chimera Twins that have chimeric blood group can lack A & B Abs Chimera with 98% O cells & 2% B cells Group as O Serum contain only anti-A

4- Reagent Problems Deterioration of Ags on A or B cells used for serum typing Weak Negative reaction

Rh Discrepancies Rh –ve persons mistyped, & transfused with Rh +ve blood have 70% chance of becoming immunized False +ve reactions can be identified by testing an Rh control with the patient’s red cells each time an Rh typing is performed The test is not valid if the control causes agglutination Rh Control: The Rh control is an autocontrol that reveals whether or not the patient's red cells are agglutinating in the absence of the D antigen, i.e., are clumping whether they are D-positive or D-negative. The control consists of testing the patient's cells with an Rh control medium (supplied by the manufacturer) that contains everything that is present in the anti-D typing sera except the anti-D.

Causes of false positive reactions Positive direct antiglobulin test Polagglutinable red cells Cold agglutinins or Rouleaux formation

1- Positive direct antiglobulin test The presence of Ab on patient’s red cells can cause a false +ve reaction with slide and tube anti-D High protein medium reduces zeta potential allowing red cells to move closer The cell bound Ab can cross link cells and cause agglutination

2- Polagglutinable red cells Rh –ve red cells that are polyagglutinable due to T or Tn activation Agglutination occurs if anti-T or anti-Tn present in the anti-D reagent Most anti-D reagents do not contain these antibodies

3- Cold agglutinins or Rouleaux formation Rh typing is performed using serum suspended red cells If individual’s serum contains cold agglutinin or abnormal protein, false positive reactions can occur