Practical Blood Bank Lab 2 ABO Discrepancies Slide 2 ABO
Discrepancy When the results of the forward grouping (patient
cells) is not matching the results of the reverse grouping (patient
serum) or abnormal reactivity is present (i.e. Mixed Field) then we
called this ABO discrepancy. The Discrepancy will be noticed by:
Strength of reaction Weak or missing. Additional reactions Abnormal
reactions Slide 3 HINT ABO forward and reverse reactions are
typically very strong: 3+ to 4+. Weaker reactions should
immediately send up red flags indicating that something is wrong.
Since production of ABO antigens is genetically controlled they are
less vulnerable to problems than does the production of ABO
antibodies. Therefore we see more problems in which grouping:
Forward or Reverse? Slide 4 PatientAnti-AAnti-BA 1 -CellsB-Cells
A4+1+04+ B0 1+0 C4+ 1+0 D03+00 Patient A: Additional reaction with
anti-B and patients cells. Patient B: Weak reaction with patients
serum and A 1 -cells. Patient C: Additional reaction with patients
serum and A 1 -cells. Patient D: Missing reactions with patients
serum A 1 -cells Slide 5 Forward Grouping Problems Slide 6 Missing
or Weak antigens Since the forward and reverse dont match, there
must be a discrepancy (in this case, a missing antigen in the
forward grouping) Subgroups of A and B. Solution : test with Anti-A
1, Anti-H, and anti-A,B for A subgroups Slide 7 Extra Antigens
Anti-AAnti-BA1 Cells B Cells 4+1+04+ Acquired B B(A) phenotype
Rouleaux Polyagglutination Whartons Jelly EXAMPLE Slide 8
Solutions: 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) B(A) phenotype Test with another anti-A
reagent from another manufacturer Polyagglutination, Rouleaux,
Whartons Jelly Wash red cells or request new sample from heel, etc
Slide 9 Can be seen in A, B and AB individuals who have received O
units. Can also be seen post transfusion if a person makes an
antibody to antigen on donor cells. Mixed Field agglutination Mixed
Field Agglutination (Post transfusion) Slide 10 Reverse Grouping
Problems Slide 11 Unexpectedly Weakened Antibodies Immunodeficient
due to therapy or disease Immunosuppressive drugs Certain leukemias
(CLL) or lymphomas (malignant lymphomas) have hypogammaglobulinemia
(Little or no antibody production) Age related Very young: 65 years
of age (Weakened Abs Activity) Dilutional Effect Plasma Exchange,
Transfusion, etc. dilutes out patient antibodies Slide 12 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 4C for 5 minutes with
autologous control (a.k.a. Autocontrol, AC) This is called a
mini-cold panel and should enhance the reactivity of the antibodies
Slide 13 Extra Antibodies Cold antibodies (allo- or auto-) Cold
antibodies may include anti-I, H, M, N, P, Lewis The autocontrol
will be positive. Resolution: warming tube to 37 and washing red
cells can disperse agglutination; breaking the IgM bonds with 2-ME
will also disperse cells Rouleaux Stronger at IS and weak reaction
at 37 C and no agglutination at AHG phase Solutions Solutions
Anti-A 1 in an A 2 or A 2 B individual Slide 14 Resolving Rouleaux
If the forward grouping is affected, wash cells to remove protein
and repeat test If the reverse grouping is affected, perform saline
replacement technique (more common) Cells (reagent) and serum
(patient) centrifuged to allow antigen and antibody to react (if
present) Serum is removed and replaced by an equal volume of saline
(saline disperses cells)* Tube is mixed, centrifuged, and
reexamined for agglutination (macro and micro) Slide 15 Anti-A 1
Sometimes A 2 (or A 2 B) individuals will develop an anti-A 1
antibody A 2 (or A 2 B) individuals have less antigen sites than A
1 individuals The antibody is a naturally occurring IgM Reacts with
A 1 Cells, but not A 2 Cells Slide 16 Resolving anti-A 1
discrepancy Anti-AAnti-BA1 Cells B Cells 4+02+4+ 2 steps: Typing
patient RBCs with Anti-A 1 lectin Repeat reverse grouping with A 2
Cells instead of A 1 Cells Both results should yield NO
agglutination Slide 17 Others The Bombay phenotype (extremely RARE)
results when hh is inherited These individuals do not have any
antigens and naturally produce, anti-A, anti-B, anti-A,B, and anti-
H Basically, NO forward reaction and POSITIVE reverse Resolution:
test with anti-H lectin (Bombays dont have H and will not react)
Slide 18 Popular LAB CAUSES Of ABO Discrepancies 1. Poorly labeled
specimen OR test tubes 2. Patient RBC suspension too heavy or light
3. Wrong specimen put in Patients labeled test tubes 4. Oh? Is
hemolysis really a Pos. Rxn? 5. Wrong results recorded on Pt. Form
6. Didnt follow manufacturers instructions 7. Poor centrifugation:
over or under! Slide 19 Popular LAB CAUSES Of ABO Discrepancies
Didnt add: 1. Patient Serum 2. Reagents 3. Correct Reagent Reaction
Reading: 1. Shaking tubes while looking elsewhere 2. Shaking tubes
too hard 3. Shaking tubes too gently or not completely re-
suspending cell button Slide 20 ABO Discrepancy When an ABO
Discrepancy is encountered: 1. Results must be recorded, but
interpretation of the ABO group must be delayed until the
discrepancy is resolvedby you! 2. Begin follow up by getting an
accurate patient history age, medications, diagnosis, etc. 3.
Repeat testing to rule out tech errors such as mislabeling, adding
reagents, wrong patient sample, etc. Slide 21 Resolving ABO
Discrepancies 1. Repeat testing on the same sample 2. Repeat
testing using saline suspended and/or washed patient red blood
cells. Saline Replacement. 1. From the beginning: re-label tubes,
re-drop patient and reagent drops, etc. 1. Many labs make the
patients red blood cell suspension with the patients serum/plasma.
If the patient has increased plasma proteins it can cause
non-specific red cell aggregation. Slide 22 3. Weak or missing
reactions? 4. Mislabeled or contaminated specimen: 3. Incubate test
system at room temperature for 15-30 minutes! Get patient history.
4. Redraw Patient!! Redraw Patient a) ALL of the above: any
labeling error may account for the problem and needs to be redrawn.
b) Drawn above an IV? Slide 23 Call the floor, Contact the patient
!!! 1. Get patient history. a) Recent transplant: two cell
populations b) Recent transfusion: two cell populations and/or
dilutional effect c) Patient medication d) etc., etc., etc. Slide
24 5. Test patient cells with anti-A 1 (Dolichos biflorus),
anti-A,B or anti-H (Ulex europaeus) 6. Test patient serum with A 1
or A 2 cells 5. For suspected subgroups of A 6. Ditto! Slide 25 7.
Review Antibody Screening tests 1. Allo antibody or cold reactive
allo or auto Ab 8. Incubate tests and controls for 10-30 minutes
room temperature 7. Can react with reagent A 1 and B cells 8.
Should strengthen weakened ABO antibody reactivity! WHY? Slide 26
Anti-AAnti-BA 1 -CellsB-Cells 3+001+ Resolution: Incubate Room
Temperature 15- 30 minutes and respin. Check Patient history.
Problem: Reverse grouping - weakened patient antibody Causes: Age
related (>65, infant), immunosuppressed or immunocompromised,
Slide 27 Anti-AAnti-BA 1 -CellsB-Cells 3+1+04+ Problem: 1+ Reaction
with Anti-B. Appears to have additional antigens. Causes: Acquired
B antigen. Resolution: Patient history bowel obstruction, carcinoma
of the bowel. (E. coli deacetylation of the Group A antigen.) Slide
28 Anti-AAnti-BA 1 -CellsB-Cells 2+01+4+ Problem: Weak forward
anti-A and 1+ reaction with A 1 Cells. Causes: 1.Subgroup of A A 2
with anti-A 1. 2.Unexpected cold reacting antibody to antigen on
reagent A 1 cells. Resolution: 1.Test patient cells with anti-A 1
lectin and with patient serum test A 2 cells 2.Antibody screen
should demonstrate unexpected cold reacting antibody. Slide 29 Lets
practice ! Slide 30 EXAMPLES of ABO Discrepancies and Possible
Resolution Forward:Reverse: Screening Anti-AAnti-BA 1 CellsB
CellsCellsAutocontrol:Possible CausesPossible Resolutions 1 000000
Group O newborn; elderly patient; low immunoglobulin levels
Incubate tests at 4C, check age of patient 2 4+ 2+ Rouleaux; cold
autoantibody Wash RBCs and repeat testing; test for cold antibodies
3 4+01+4+00 Probable A 2 subgroup with anti-A 1 Test with anti-A 1
and anti- H lectins and A 2 cells 4 3+4+1+000 Probable A 2 B
subgroup with anti-A 1 Test with anti-A 1 and anti- H lectins and A
2 cells 5 004+ 0 Probable O h (Bombay)Test with anti-H lectin; may
sent to reference lab for confirmation 6 4+2+04+00 Probable
acquired B phenotype Investigate patient history; test with anti-B
lectin if available 7 4+ 2+0 0 Probable alloantibodyPerform
antibody identification (antibody panel) 8 04+ 1+ Probable group B
with cold autoantibody Test for cold antibodies and identify if
appropriate Adapted from Table 3-11: Flynn, J. C. (1998).
Essentials of Immunohematology. Philadelphia: W.B. Saunders
Company. Slide 31 Example 1 Anti-AAnti-BA1 CellsB Cells 3+001+
Problem: Causes: Resolution: Slide 32 Example 2 Anti-AAnti-BA1
CellsB Cells 3+1+04+ Problem: Causes: Resolution: Slide 33 Example
3 Anti-AAnti-BA1 CellsB Cells 2+0+1+4+ Problem: Causes: Resolution:
Slide 34 Example 4 Anti-AAnti-BA1 CellsB Cells 0003+ Problem:
Causes: Resolution: Slide 35 Example 4 Anti-A,B Patient RBC1+
Problem: Causes: Resolution: Slide 36 Example 5 Anti-AAnti-BA1
CellsB Cells 02+ mf3+0 Problem: Causes: Resolution: Slide 37
Example 6 Anti-AAnti-BA1 CellsB Cells 4+ 01+ Problem: Causes:
Resolution: Slide 38 Example 7 Anti-AAnti-BA1 CellsB Cells 0000
Problem: Causes: Resolution:
