CLS 3311 Advanced Clinical Immunohematology

1
CLS 3311Advanced Clinical Immunohematology

• ABO Discrepancys

2
ABO Discrepancy

• 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)
• Strength of reaction
• Weak or missing
• Additional reactions
• Abnormal reactions

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.
• HINT
• 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?

4
Patient A Additional reaction with anti-B and
patients cells. Patient B Weak reaction with
patients serum and A1-cells. Patient C
Additional reaction with patients serum and
A1-cells. Patient D Missing reactions with
patients serum A1-cells
5
Problems with The Forward Grouping

• Chimera Two cell populations
• Mixed Field reactions observed
• Causes Recent transfusion (O cells to an A
  patient), Bone marrow transplant
• Testing using Serum or plasma suspended Patient
  RBCs
• Can cause non specific aggregation of rbcs
• Increased serum proteins Multiple Myeloma
  patient
• Contamination in cord blood sample Whartons
  jelly
• Infusion of macromolecular solutions Dextran,
  etc.

6
Problems with The Forward Grouping

• Potent cold reacting autoagglutinins
• Antibody coats patient RBC and agglutinate
  spontaneously in the diluent
• Weakened Antigen Expression
• Subgroups of A or B
• Some leukemias, Hodgkin's disease
• Excess soluble A and B blood group substances
• Carcinoma of the stomach and/or carcinoma of the
  pancreas

7
Problems with The Forward Grouping

• Extra ABO antigens

• Acquired B Antigen
• Microbial deacetylating enzymes such as E. coli
  cleave off the N-Acetyl of the Group A
  N-acetyl-D-galactosamine immunodominant sugar.
  The remaining D-galactosamine becomes similar
  enough to the Group B D-galactose immunodominant
  sugar that it DOES react with reagent anti-B.
• Secondary to bowel obstruction or carcinoma of
  the bowel

8
Problems with The Forward Grouping Extra ABO
antigens

• Polyagglutinable state
• Exposure of crypt or buried antigens (T, Tk,
  etc.) by inheritance or bacterial enzymes RBCs
  agglutinate with most sera.
• Exposure of T, Tn and Tk (etc.) antigens.
  Antibodies to these antigens are present in
  virtually all human antisera. If using human
  source anti-A and anti-B these cells will
  agglutinate.

9
Problems with The Reverse Grouping

• Plasma or incompletely clotted serum
• Non specific red cell aggregation

• Still has fibrinogen and can cause fibrin
  formation (rbcs can be caught up and appears as
  clumping)
• Increased serum proteins (Multiple Myeloma,
  etc.), infusion of high molecular weight plasma
  expanders

10
Problems with The Reverse Grouping

• Unexpected antibodies to reagent RBCs
• Antibodies to constituents of the reagent RBCs
  diluent

• Cold reacting antibodies such as anti-M, -N or -I
• Antibody reacts with diluent and non-specifically
  involves red blood cells in reaction causing
  clumping.

11
Problems with The Reverse Grouping Unexpectedly
Weakened Antibodies

• Immunodeficient due to therapy or disease
• Immunosuppressive drugs
• Certain leukemias (CLL) or lymphomas (malignant
  lymphomas) have hypogammaglobulinemia
• Age related
• Very young lt6 months of age
• Very old gt65 years of age
• Dilutional Effect
• Plasma Exchange, Transfusion, etc. dilutes out
  patient antibodies

12
Popular LAB CAUSES Of ABO Discrepancies

• Poorly labeled specimen OR test tubes
• Patient RBC suspension too heavy or light
• Wrong specimen put in Patients labeled test
  tubes
• Oh? Is hemolysis really a Pos. Rxn?
• Wrong results recorded on Pt. Form
• Didnt follow manufacturers instructions
• Poor centrifugation over or under!

13
Popular LAB CAUSES Of ABO Discrepancies

• Didnt add
• Patient Serum
• Reagents
• Correct Reagent
• Reaction Reading
• Shaking tubes while looking elsewhere
• Shaking tubes too hard
• Shaking tubes too gently or not completely re-
  suspending cell button

14
ABO Discrepancy

• When an ABO Discrepancy is encountered
• Results must be recorded, but interpretation of
  the ABO group must be delayed until the
  discrepancy is resolvedby you!
• Begin follow up by getting an accurate patient
  history age, medications, diagnosis, etc.
• Repeat testing to rule out tech errors such as
  mislabeling, adding reagents, wrong patient
  sample, etc.

15
Resolving ABO Discrepancies

• Repeat testing on the same sample
• Repeat testing using saline suspended and/or
  washed patient red blood cells. Saline
  Replacement.

• From the beginning re-label tubes, re-drop
  patient and reagent drops, etc.
• 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.

16
Resolving ABO Discrepancies

• Weak or missing reactions?
• Mislabeled or contaminated specimen

• Incubate test system at room temperature for
  15-30 minutes! Get patient history.
• Redraw Patient!!
• ALL of the above any labeling error may account
  for the problem and needs to be redrawn.
• Drawn above an IV?

17
Resolving ABO Discrepancies

• Call the floor!!!

• Get patient history.
• Recent transplant two cell populations
• Recent transfusion two cell populations and/or
  dilutional effect
• Patient medication
• etc., etc., etc.

18
Resolving ABO Discrepancies

• Test patient cells with anti-A1 (Dolichos
  biflorus), anti-A,B or anti-H (Ulex europaeus)
• Test patient serum with A1 or A2 cells

• For suspected subgroups of A
• Ditto!

19
Resolving ABO Discrepancies

• Review Antibody Screening tests
• Allo antibody or cold reactive allo or auto Ab
• Incubate tests and controls for 10-30 minutes
  room temperature

• Can react with reagent A1 and B cells
• Should strengthen weakened ABO antibody
  reactivity! WHY?

20
Problem Reverse grouping - weakened patient
antibody
Causes Age related (gt65, infant),
immunosuppressed or immunocompromised,
Resolution Incubate Room Temperature 15-30
minutes and respin. Check Patient history.
21
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.)
22
Problem Weak forward anti-A and 1 reaction with
A1 Cells.
Causes 1)Subgroup of A A2 with anti-A1. 2)
Unexpected cold reacting antibody to antigen on
reagent A1 cells.
Resolution1) Test patient cells with anti-A1
lectin and with patient serum test A2 cells 2)
Antibody screen should demonstrate unexpected
cold reacting antibody.
23
ABO Discrepancy Cases

• The following slides are patient reactions
  including the ABO forward and reverse grouping
  using monoclonal reagent anti-A and anti-B and
  reagent red blood cells. The Indirect
  Antiglobulin Test (IAT) is included.
• You need to email me your results PRIOR to
  posting them on the discussion board. All
  Students must post their cases on the same day to
  insure integrity of work. I will email the
  deadline to each student. Following student
  posting I will post my results. We can then
  discuss any questions you may have.

24
Case 1
MF stands for mixed field reaction.
25
Case 1 Write up.

• List the abnormal results.
• How does the Forward grouping correlate with the
  IAT?
• What is the suspected ABO Group of the patient?
  Support you answer.
• What action would you take to resolve this case?

26
Case 2
27
Case 2 Write up.

• List the abnormal results.
• How does the Forward grouping correlate with the
  IAT?
• What is the suspected ABO Group of the patient?
  Support you answer.
• What action would you take to resolve this case?

28
Case 3
29
Case 3 Write Up

• List the abnormal results.
• How does the Forward grouping correlate with the
  IAT?
• What is the suspected ABO Group of the patient?
  Support you answer.
• What action would you take to resolve this case?
  Explain your choice of tests.

30
Case 4
31
Case 4 Write up.

• List the abnormal results.
• How does the Forward grouping correlate with the
  IAT?
• What is the suspected ABO Group of the patient?
  Support you answer.
• What action would you take to resolve this case?

32
Case 5
33
Case 5 Write up.

• List the abnormal results.
• How does the Forward grouping correlate with the
  IAT?
• What is the suspected ABO Group of the patient?
  Support you answer.
• What action would you take to resolve this case?