Transcript ABO Discrepancies & other problems

ABO Discrepancies &
other problems
Prepared By
Ahmad Shihada Silmi Msc, FIBMS
Medical Technology Dept
Islamic University of Gaza
Importance
 It
is important for students to recognize
discrepant results and how to (basically)
resolve them
 Remember, the ABO system is the most
important blood group system in relation to
transfusions
 Misinterpreting ABO discrepancies could
be life threatening to patients
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
Why?
 Reaction
strengths could be weaker than
expected
 Some reactions may be missing in the
reverse or forward typing
 Extra reactions may occur
Patient
Anti-A
Anti-B A1 Cells B Cells
1
4+
1+
0
4+
2
0
4+
1+
0
3
4+
4+
1+
0
4
0
3+
0
0
What do you do?
 Identify
the problem
 Most of the time, the problem is technical



Mislabeled tube
Failure to add reagent
Either repeat test on same sample, request a
new sample, or wash cells
 Other
times, there is a real discrepancy
due to problems with the patient’s red cells
or serum
Discrepancy ?
 If
a real discrepancy is encountered, the
results must be recorded
 However,
the interpretation is delayed until
the discrepancy is RESOLVED
Errors
Technical Errors

Clerical errors






Reagent or equipment problems





Mislabeled tubes
Patient misidentification
Inaccurate interpretations recorded
Transcription error
Computer entry error
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
Clotting deficiencies

Serum that does not clot may be due to:




Low platelet counts
Anticoagulant therapy (Heparin, Aspirin, etc)
Factor deficiencies
Serum that does not clot completely before
testing is prone to developing fibrin clots that
may mimic agglutination
 Thrombin can be added to serum to activate
clot formation
 OR, tubes containing EDTA can be used
Contaminated samples or reagents
 Sample

contamination
Microbial growth in tube
 Reagent



contamination
Bacterial growth causes cloudy or discolored
appearance…do not use if you see this!
Reagents contaminated with other reagents
(don’t touch side of tube when dispensing)
Saline should be changed regularly
Equipment problems
 Routine
maintenance should be performed
on a regular basis (daily, weekly, etc)
 Keep instruments like centrifuges,
thermometers, and timers calibrated

Uncalibrated serofuges can cause false results
Hemolysis
 Detected
in serum after centrifugation (red)
 Important if not documented
 Can result from:

Complement binding
• Anti-A, anti-B, anti-H, and anti-Lea

Bacterial contamination
Red
supernatant
ABO discrepancies
ABO Discrepancies
 Problems



Weak-reacting/Missing antigens
Extra antigens
Mixed field reactions
 Problems


with RBCs
with serum
Weak-reacting/Missing antibodies
Extra antibodies
Grouping
Forward
Missing/Weak
A/B Subgroup
Reverse
Extra
Mixed Field
Acquired B
Missing/Weak
O Transfusion
Extra
Young
Elderly
Immunocompromised
Disease
(cancer)
B(A) Phenotype
Bone Marrow
Transplant
Rouleaux
Cold
Autoantibody
Cold
Alloantibody
Rouleaux
May cause all + reactions
Anti-A1
Forward Grouping
Problems
Red Cell Problems
 Affect



the forward grouping results
Missing or weak antigens
Extra antigens
Mixed field reactions
Forward Grouping:
Missing or Weak antigens
 ABO
Subgroups
 Disease (leukemia, Hodgkin’s disease)
Anti-A
Anti-B
A1 Cells
B Cells
0
0
0
4+
Group O
Group A
• Since the forward and reverse don’t match, there must be a
discrepancy (in this case, a missing antigen in the forward grouping)
Subgroups of A (or B)
 Subgroups
of A account for a small portion
of the A population (B subgroups rarer)
 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
 Resolution: test with Anti-A1, Anti-H, and
anti-A,B for A subgroups
Forward Grouping:
Extra Antigens





Acquired B
B(A) phenotype
Rouleaux
Polyagglutination
Wharton’s Jelly
Anti-A Anti-B
4+
1+
A1
Cells
0
EXAMPLE
B
Cells
4+
Acquired B Phenomenon

Cause: there are two causes of acquired B phenomenon:
In vivo, patients with bacterial infections and often cancer of the
colon or rectum may develop
a false B-like antigen.
The mechanism: The bacterial produce a deacetylase (enzyme)
which chemically alters the terminal sugar of A antigens (Nacetyl-D-galactosamine) into D-galactosamine.

Because the terminal sugar of the B antigen is galactose, anti-B
antisera will cross react with the B-like D-galactosamine
antigen. Because of this, in vivo, only group A people can
develop an acquired B-like antigen. The condition is transient
and disappears when the infection is cured.
Acquired B
 Bacteria
(E. coli) have a deacetylating
enzyme that effects the A sugar….
Group A
individual
N-acetyl galactosamine
Bacterial enzyme
removes acetyl group
Acquired
B
Phenotype
Galactosamine
now resembles
D-galactose (found
in Group B)
Another mechanism

In vitro, blood specimens can get an acquired B-like
antigen if they are bacterially contaminated. This is
because the membranes of some bacteria (e.g., E.
coli and P. vulgaris ) have determinants which are
chemically similar to the B antigen.

In this case, anti-B antisera is actually reacting with
the bacterial antigens which have attached to the red
cells.

In vitro, both group O and group A cells can acquire
the B-like antigen. Note: most examples of acquired
B phenomenon detected in the blood bank happen in
vivo to group A people only.
ES4 Anti-B antisera

The use of monoclonal ABO typing antisera
(specifically an anti-B clone designated "ES4")
initially caused an increase in acquired B
phenomenon.
because

The ES4 monoclonals can detect even a small
number of galactosamine molecules on red cells.
However, the reactions are particularly sensitive to
pH and can be reduced (not eliminated totally) if the
pH is lowered, something that the manufacturers
have done.
Typical reaction pattern
 The
reactions with anti-B are weaker than
expected (e.g., 1+ or 2+). The patient's
autocontrol is negative even though anti-B
is present (patient is group A).

The patient's own anti-B will not recognize
and agglutinate the B-like antigen, but
everyone else's anti-B (including the typing
sera) will.
Resolution of acquired B

Check the past records in case the patient is a known group
A.
 Check the diagnosis for bacterial infection (with or without
Cancer of the colon or rectum).
 Test the red cells with anti-B reagent acidified to pH 6.0
 If using human polyclonal reagents, redo the ABO group
using monoclonal anti-A and anti-B typing sera, which may
resolve the problem.
 If using monoclonal reagents, redo the ABO group using
human polyclonal anti-A and anti-B typing sera.
 Do autologous control it should give negative result.
 Try secretor status studies (usually not necessary). If the
patient is group A and a secretor, he will secrete A and H
antigens only.
Implication in blood transfusion

Group A people (especially children with a small
blood volume) who have acquired B
phenomenon should receive group A washed
red cells (or group O washed red cells).

The red cells should be washed to remove all
traces of donor anti-B which can react with the
patient's B-like antigens.
Acquired B Phenotype

Limited mainly to Group
A1 individuals with:




Lower GI tract disease
Cancer of colon/rectum
Intestinal obstruction
Gram negative septicemia
(i.e. E. coli)
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)
B(A) phenotype
 Similar
to acquired B
 Patient is Group B with an apparent extra
A antigen
 The B gene transfers small amounts of the
A sugar to the H antigen
 Sometimes certain anti-A reagents will
detect these trace amount of A antigen
 Resolution: test with another anti-A
reagent from another manufacturer
Other reasons for “extra” antigens

Polyagglutination – agglutination of RBCs with
human antisera no matter what blood type


Due to bacterial infections
Expression of hidden T antigens react with antisera
Rouleaux – extra serum proteins
 Wharton’s Jelly – gelatinous substance derived
from connective tissue that is found in cord blood
and may cause false agglutination (Remember:
only forward typing is performed on cord blood)


Wash red cells or request new sample from heel, etc
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
A3 phenotype
Anti-A
Anti-B
0
2+
A1 Cells B Cells
4+
0
Mixed Field Agglutination
Reverse Grouping
Problems
Reverse Grouping
 Affect


the reverse grouping results
Missing or weak antibodies
Extra antibodies
Reverse Grouping:
Missing or Weak antibodies
 Newborns

Do not form antibodies until later
 Elderly

Weakened antibody activity
 Hypogammaglobulinemia

Little or no antibody production (i.e.
immunocompromised)
 Often
shows NO agglutination on reverse
groupings
Missing or Weak antibodies
Example
Anti-A Anti-B A1 cells B cells
Tentative group
#1
+4
-
-
-
A
#2
#3
-
+4
-
-
-
B or AB
O
#1: Patient is a newborn: Anti-A and anti-B are not present at birth and
develop about 3-6 months of age. (Usually the reverse group is not done
when grouping newborns.)
#2: Patient is very elderly: Anti-A and anti-B levels decrease in old age
because levels of immunoglobulins decrease. Because the levels may only be
decreased and not totally missing, further investigation can be done. (Note: It
would be unusual for an elderly person to totally lack ABO antibodies in the
absence of an immune disorder.)
#3: Patient has a- or hypogammaglobulinemia: Anti-A and anti-B will be
weak or missing in patients with a gammaglobulinemia or
hypogammaglobulinemia.
Resolution

Check the age of the patient
 Repeat the ABO group at 4°C [anti-A and anti-B
react best at 4°C].
 QC required: because all persons have a
harmless auto-anti-I reactive at 4°C, include an
autocontrol. (Auto-anti-I may agglutinate the A1
cells, the B cells, and the patient's own cells at
4°C.)
 Check the diagnosis.
 If undiagnosed, have gammaglobulin levels
tested
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 (a.k.a.
Autocontrol, AC)
 This is called a “mini-cold” panel and
should enhance the reactivity of the
antibodies
Reverse Grouping:
Extra Antibodies
 Cold

antibodies (allo- or auto-)
Cold antibodies may include anti-I, H, M, N, P,
Lewis
 Rouleaux
 Anti-A1
in an A2 or A2B individual
Cold antibodies

Sometimes a patient will develop 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; breaking the IgM
bonds.
Rouleaux





Can cause both extra antigens and extra
antibodies
“stack of coins” appearance
May falsely appear as agglutination due to the
increase of serum proteins (globulins)
Stronger at IS and weak reaction at 37°C and no
agglutination at AHG phase
Associated with:



Multiple meloma
Waldenstrom’s macroglobulinemia (WM)
Hydroxyethyl starch (HES), dextran, etc
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 (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)
*some procedures suggest only 2 drops of saline.
Extra Antibodies
Example Anti-A Anti-B A1 cells B cells Tentative group
#1
+4
-
+1
+4
A
#2
+4
+4
+2
-
AB
#3
-
+4
+4
+1
B
Anti-A1 in A2 or A2B people: examples #1 and #2 illustrate the presence
of anti-A1. The autocontrol (not shown) would be negative.
Irregular IgM Alloantibodies: All three examples could represent the
presence of irregular IgM alloantibodies such as anti-M, -N, -Lea, -Leb, or
-P1. The A1 cells (or B cells) may be agglutinating because they are
positive for the corresponding antigen. The autocontrol (not shown)
would be negative.
Example Anti-A Anti-B
A1 cells B cells
Tentative group
#1
+4
-
+1
+4
A
#2
+4
+4
+2
-
AB
#3
-
+4
+4
+1
B
Rouleaux: providing both cells in the reverse grouping show agglutination
(examples #1 and #3), the discrepancy could be due to Rouleaux. The
autocontrol (not shown) would be positive.
Causes: Rouleaux is a type of false agglutination caused by an increase in
serum globulins. This can occur in diseases such as multiple myeloma or
macroglobulinemia or can be caused by infusion of macromolecular
substances such as dextran or polyvinyl pyrollidone (PVP), which are used
as blood volume expanders.
Autoanti-I: Many people have a harmless autoanti-I that is IgM and reacts
best at 4°C. The harmless autoanti-I of most people will not react above 10°15°C, but some people have an autoanti-I that can react at RT and cause
unexpected agglutination in both cells of the reverse serum group (examples
#1 and #3).
Anti-A1
 Sometimes A2
(or A2B) individuals will
develop an anti-A1 antibody
 A2 (or A2B) individuals have less antigen
sites than A1 individuals
 The antibody is a naturally occurring IgM
 Reacts with A1 Cells, but not A2 Cells
+ A1 cells
Anti-A1 from
patient
+ A2 cells
AGGLUTINATION
NO AGGLUTINATION
Resolving anti-A1 discrepancy
2



steps:
Typing patient RBCs with Anti-A1 lectin
Repeat reverse grouping with A2 Cells instead
of A1 Cells
Both results should yield NO agglutination
Anti-A Anti-B
4+
0
A1
Cells
2+
B
Cells
4+
Resolution of discrepancies caused
by anti-A1

First step:
We must determine if the person is group A1 or group A2.
(If group A1, the discrepancy with the A1 cells is NOT due
to anti-A1).
To do this, we antigen type the person's red cells with the
anti-A1 lectin which is Dolichos biflorus . If the red cells
agglutinate, the person is group A1. If the red cells do not
agglutinate, the person is not group A1, and probably is
group A2 assuming the red cells reacted strongly (3+ or
4+ with anti-A).
Resolution of discrepancies caused by
anti-A1

Second step:
If the person appears to be group A2, we must
prove that the extra antibody is anti-A1, and not
some other IgM irregular antibody.
To do this we test the person's serum against a panel
of 3 A1 cells and 3 A2 cells. If anti-A1 is present,
only the A1 cells should agglutinate.
Resolution of discrepancies caused by
anti-A1

NOTE:
3 A1 and 3 A2 cells are required in order to ensure that
the antibody reacting is anti-A1 and not some other
antibody.
With 3 cells of each group we can achieve a statistical
probability of 95% that the right antibody has been
identified.
For example, if only one A1 cell and one A2 cell were
tested, by chance, another antibody like anti-M or antiP1 could react with the A1 cells (if they were M+ or P1+),
but not the A2 cells (if they were M- or P1-).
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 (Bombay’s don’t have
H and will not react)
Finding the problem…





Forward type tests for the
antigen (red cell)
Reverse type tests for the
antibody (serum)
Identify what the patient
types as in both Forward
& Reverse Groupings
Is there a weaker than
usual reaction?
Is it a missing, weak, or
extra reaction??
Resolving ABO Discrepancies
 Get





the patient’s history:
age
Recent transplant
Recent transfusion
Patient medications
The list goes on….
Let’s practice !
Example 1
Anti-A
Anti-B
A1 Cells
B Cells
3+
0
0
1+
Problem:
Causes:
Resolution:
Example 2
Anti-A
Anti-B
A1 Cells
B Cells
3+
1+
0
4+
Problem:
Causes:
Resolution:
Example 3
Anti-A
Anti-B
A1 Cells
B Cells
2+
0+
1+
4+
Problem:
Causes:
Resolution:
Example 4
Anti-A
Anti-B
A1 Cells
B Cells
0
0
0
3+
Problem:
Causes:
Resolution:
Example 4
Anti-A,B
Patient RBC
1+
• Probably a subgroup of A (Ax)
• if the result was negative (0), adsorption or elution
studies with anti-A could be performed (these will
help determine what A antigens)
Example 5
Anti-A
Anti-B
A1 Cells
B Cells
0
2+mf
3+
0
Problem:
Causes:
Resolution:
Example 6
Anti-A
Anti-B
A1 Cells
B Cells
4+
4+
0
1+
Problem:
Causes:
Resolution:
Example 7
Anti-A
Anti-B
A1 Cells
B Cells
0
0
0
0
Problem:
Causes:
Resolution:
Example 8
Screening
Cells
(I and II)
Patient
Pos
Serum 1
Patient
Pos
Serum 2
Autocontrol
(AC)
Conclusion
Neg
Cold
alloantibody
Cold
autoantibody
Pos
• if alloantibody – antibody ID techniques
• if autoantibody – special procedures (minicold panel, prewarming techniques
References


Rudmann, S. V. (2005). Textbook of Blood Banking and Transfusion
Medicine (2nd Ed.). Philadelphia, PA: Elsevier Saunders.
Blaney, K. D. and Howard, P. R. (2000). Basic & Applied Concepts
of Immunohematology. St. Louis, MO: Mosby, Inc.