Group I Discrepancies These are associated with unexpected reactions in the reverse grouping due to weakly reacting or

missing antibodies. Discrepancies in this group includes

I. Infants less than 4-6 month of age2,3: Anti-A and anti-B agglutinins (IgM) produced by the infant can
first be demonstrated at 36 months1, 4. Anti-A and anti-B if present in cord sera are usually IgG and are
of maternal origin. Reverse ABO grouping in infants is not warranted before 6 months of age2.
II. Elderly patients : Earlier studies showed a progressive decrease in anti-A and -B agglutinin titers
with age, with low levels (titer 4 or less) being common in subjects aged 80 years or more . However in a
study by Maur et al8 decline in titres was not observed.
5, 6

5, 7

III. Severe hypogammaglobulinemia: Anti A and Anti B are often present in very low concentration in
patients with inherited immunodeficiency and in rare X-linked Wiskott- Aldrich syndrome . Anti-A and antiB may also be present in very low concentration in patient immunosuppressed by therapy or disease and
in patients undergoing intensive plasma exchange.
9

IV. ABO incompatible HPC transplantation: ABO incompatible HPC transplantation with induction of
tolerance e.g. group A patient receiving group O bone marrow will have circulating group O red cell but
will only produce Anti B antibody.
V. In chimerism: That is, a person with dual population of cells from more than one zygote. Presence of
two populations of red cells of different ABO group may lead to absence of antibodies . Twin chimerism
occurs when hematopoietic stem cells migrate between vascular bridges which allow mixing of blood
between two fetuses. chimeric twins have immune tolerance; they do not make against A ro B antigens
that are absent from their own red cells but present on cells of engrafted twins.
1

VI. Pediatric patients receiving long term parentral and entral nutrition which is sterile and free of
bacteria . It is believed that the immunizing source for such naturally occurring antibodies is gut and
environmental bacteria which have been shown to possess ABO like structures on their
lipopolysaccharide coats .
10

Resolution of group I discrepancies

a. It can be resolved by enhancing weak or missing reaction by incubating the patients serum with
reagent A1 and B cells at room temperature for 15-30 minutes
b. If there is still no reaction after centrifugation, serum cell mixture is incubated at 4 0 C for 15-30 minutes
Note: an autocontrol and O cell control must always be tested concurrently to detect reactivity of other
commonly occurring cold agglutinins e.g. anti I
Group II discrepancies
These are associated with unexpected reactions in forward grouping due to weakly reacting or missing
antigen. Discrepancies in this group includes
I. Subgroups of A or B : subgroups of A antigen ( Ax, Am, Ay, A ) which are not agglutinated or weakly
agglutinated by most anti A. subgroups of B antigen (B , B , B ) ) which are not agglutinated or weakly
agglutinated by most anti B. Both can present as group II discrepancies.
11

el

el

II. Leukaemia may yield weakened A or B antigen : In acute leukaemia, the A antigen may be
weakened . Sometimes the blood appears to contain a mixture of group A and group O cells or of A1
and weak A . In other cases the red cells react weakly with anti-A, even behaving like A or A . In a
patient with erythroleukaemia, of group B, 60% of the cells were not agglutinated by anti-B and appeared
to be group O, but were really very weak B, when separated from the normal B cells they would absorb
anti-B .
12

13

14, 15

14

15
m

12

III. Acquired B , B(A) and A(B) phenotypes: Acquired B results from the action of bacterial
deacetylase, which converts N-acetylgalactosamine to ?-galactosamine, which is very similar to
galactose, the chief determinant of B. The second type of acquired B that may be called the passenger
antigen type is caused by adsorption of B-like bacterial products on to O or A cells but occurs only in
vitro .
16,

17

18

IV. Out of group transfusion or ABO mismatched hematopoietic progenitor stem cell
transplantation: ABOcompatible but not identical transfusion of red cell (e.g. group O red cells
transfused to group A or B person) results in artificially induced chimerism. ABO mismatched
hematopoietic stem cell transplant (e.g. group O person transplanted with group A or B marrow, group A
or B person transplanted with group O marrow).
V. Neutralization of anti A and anti B typing reagent by high concentration of A or B soluble
substances in serum with serum or plasma suspended red cell
VI. Chimerism in fraternal twins, mosaicism arising from dispermy: Tetra-gametic or dispermic
chimeras present with chimerism in all tissues and are more frequently identified because of infertility and
rarely because of mixed populations of red cells
Resolution of group II discrepancies
a. Weaker reactions with antisera can be resolved by enhancing reaction of antigen with respective
antisera by incubating test mixture at room temperature for 15-30 minutes
b. Sub groups causing group discrepancies can be resolved by adsorption elution studies
c. Acquired B phenomenon can be resolved by lowering P of monoclonal antisera. Anti B in the serum
of acquired B person does not agglutinate autologous red cells (autocontrol negative). Secretor status of
person can resolve acquired B, saliva of acquired B person contains A substance not B substance. Serum
of acquired B person contains A substance.
H

d. High concentration of A or B substance causing group discrepancies can be resolved by saline

washing of red cells
Group III discrepancies
These are associated with protein or plasma abnormalities, rouleaux formation and pseudoagglutination.
Discrepancies in this group includes
I. Elevated level of globulin from e.g. multiple myeloma, waldenstorm macroglobulinemia, Hodgkin
lymphoma.
II. Elevated level of fibrinogen.
III. Small fibrin clot in plasma or incompletely clotted serum can be mistaken for red cell agglutinates of
reverse grouping. Principal: patients sample with abnormal concentration of serum proteins, altered

serum protein ratio, or high molecular weight volume expanders can aggregate reagent red cells and can
mimic agglutination. Rouleaux are red cell aggregates that adhere along their flat surfaces, giving a
stacked coin appearance microscopically. Rouleaux will disperse when suspended in saline. True
agglutination is stable in the presence of saline.
Group IV discrepancies
These discrepancies are because of miscellaneous problems. These can be due toI. Recent transfusion of out of group plasma containing component.
II. Cold alloantibodies (e.g. anti M) or autoantibodies (e.g. anti I), P dependent autoantibodies, a reagent
dependent antibody (e.g. EDTA, paraben) leading to unexpected positive eaction.
H

III. Recent infusion of IvIg which can contain ABO isoagglutinins.

IV. Mix field agglutination with circulating red cell of more than one ABO type.
V. Polyagglutination (e.g. T activation) resulting from inherited or acquired abnormalities of red cell
membrane with exposure of auto cryptantigen . The T determinant is normally covered
by N acetylneuraminic acid and can therefore be described as a cryptantigen. The antigen can be
exposed by the action of bacterial or viral neuraminidases Anti-T and anti-Tn present in the serum of all
subjects except infants, are presumably formed as a reaction to T and Tn present in many Gramnegative
bacteria and vaccines . Very many organisms, including pneumococci, streptococci,
staphylococci,clostridia, E. coli, Vibrio cholerae and influenza viruses are capable of producing this
effect in vitro. T activation may occur in vivo. Usually, this polyagglutinability occurs as a transient
phenomenon, disappearing within a few weeks or months of the time when it is first observed In the past,
T activation was almost always detected by finding discrepancies between the results of testing red cells
and sera in the course of ABO grouping. Nowadays, monoclonal anti-A and -B are widely used and so T
activation seldom causes trouble in blood grouping.
20

20

Resolution of group IV discrepancies

a. Cold autoantibodies causing false positive reaction in forward grouping can be eliminated by washing
of red cell with warm saline. If warm saline fails to resolve, removal of autoantibody with acid glycine
EDTA or chloroquine can be used
b. Autoagglutination causing false positive reaction in reverse grouping can be resolve by incubating at
37 c for 30-60 minutes. It can also be resolved by incubating red cells in presence of either dithiothreitol
or 2-mercaptoethanol.
c. Unexpected alloantibodies in the patients serum other than ABO isoagglutinins causing group
discrepancy is resolved by as soon as antibody is identified (e.g. anti M), reverse grouping should be
repeated by A1 and B cell that are negative for that antigen.
d. Unexpected ABO isoagglutinins (e.g. anti A1 in A2 or A2B) producing group discrepancies can be
resolved by repeating reverse grouping using at least 3 A1, A2, B, O cell along with autocontrol. Patients
red cell can be typed with anti A1 lectin from dolichos biflorus to determine subgroups of A antigen.
e. A reagent dependent antibody (anti acriflavine antibody against acriflavi used in Anti B) causing group
discrepancy should be resolved by washing persons red cell with normal saline for at least 3 times.

CLS 422 Clinical Immunohematology I Student Lab Page 1 of

9
ABO Discrepancies - Handout
ABO DISCREPANCIES
I. Steps for Resolution of ABO Discrepancies
- ABO interpretation must be delayed until the discrepancy is resolved.
- In a crisis situation Group O, Rh compatible packed red blood cells (RBCs) may be
issued after a physician signs a release form.
A. Perform clerical check of specimen and paperwork. -85% of fatal transfusion
reactions are caused by clerical errors!
1. Specimen label to test request
2. Current results with historical type
3. Clerical errors may be caused by:
a. Inadequate identification of patient and/or specimen at the time of collection
b. A mix-up of samples in the laboratory
c. Mislabeled tubes/ gel card
d. Recording/ transcription errors
e. Wrong patient record
B. Check patients age, diagnosis, transfusion/transplant and pregnancy history, and
medications.
C. Repeat all tests; wash RBC suspension if not previously washed (may require
multiple
washes, or warm saline wash).
D. Technical errors:
1. Failure to add reagents
a. Reagents have color added to aid in visualization
b. Add plasma or anti-serum to tube first, and then add cells, to aid in

visualization CLS 422 Clinical Immunohematology I Student Lab Page 2 of

9
ABO Discrepancies - Handout
2. Failure to follow manufacturers instructions
a. Check daily reagent QC to insure proper reactivity
b. Confirm that reagents are not expired
3. Contaminated reagents
a. If contaminated with bacteria, may get false positives
b. If reagent has been neutralized, may get false negatives
c. Human source reagents may be contaminated with an antibody to another RBC
antigen (usually a low frequency antigen), giving false positive results try a
different lot of antiserum, or a monoclonal reagent
4. Uncalibrated centrifuge
a. Too fast or too long a time= false positive
b. Too slow or too short a time = false negative
5. Incorrect temperature
a. Too warm = false negative
b. Too cold = false positive (interference from cold auto antibodies)
6. Inappropriate concentration of cell suspension
a. Too weak = false negative (prozone)
b. Too heavy = false negative (postzone)
7. Delay in reading reactions
a. Weak reactions dissipate
b. Interference from cold agglutinins
8. Missed observation of hemolysis; missed observation of weak or mixed-field
reactions CLS 422 Clinical Immunohematology I Student Lab Page 3 of
9
ABO Discrepancies - Handout
9. Fibrin/ debris interpreted as agglutination
E. Perform additional testing to resolve discrepancy.

F. Collect a new specimen when specimen appears to be :

1. From the wrong patient
2. Diluted/ contaminated with IV fluid
3. From a traumatic draw
II. Red Blood Cell Problems: Forward Grouping
A. Unexpected weak or negative reactions least common form of discrepancy
1. Weakened expression due to age:
a. Newborns - Antigens not fully developed at birth
b. Elderly - Antigen strength may diminish
c. Red blood cells lose antigen sites as they age
2. Weakened expression due to disease:
a. Leukemia
b. Hodgkins lymphoma
c. Cancer of stomach, pancreas, ovarian cyst which may produce excess blood
group
substance in sufficient concentration to neutralize typing sera
3. Rare subgroups of A or B
a. Give weaker than normal reactions with conventional (human source) antisera,
stronger with monoclonal antisera
b. A3 mixed field agglutination with conventional anti-A and anti-A,B
c. Ax reacts with anti-A,B but not conventional anti-A. May react with monoclonal
anti-A reagents CLS 422 Clinical Immunohematology I Student Lab Page 4 of
9
ABO Discrepancies - Handout
4. Resolution:
a. Check age and diagnosis of patient
b. Incubate at RT for 15-30 min
c. Incubate 4o
C for 15-30 min, including an autocontrol
d. Switch to a different reagent conventional vs. monoclonal, monoclonal that uses

a different clone
e. Treat patients RBCs with enzymes to enhance antigen expression.
f. Read microscopically
g. For weak subgroups of A, testing may include:
1) Confirmation that a subgroup of A is present by testing RBCs with anti-A1
lectin
2) Using anti-A,B in testing is more sensitive to A antigen than human source
anti-A in some instances
3) Secretor studies
4) Adsorption/elution studies
B. Unexpected Positive Reactions
1. Rouleaux
a. Characteristic stack of coins appearance to red blood cells resembling
agglutination
b. Caused by elevated globulin levels in serum
c. Common in multiple myeloma or when patient has received plasma expanders
d. Resolved by washing RBCs well
2. Whartons jelly in cord bloods Gelatinous material that can trap red blood cells,
appearing like agglutination. Wash cells a minimum of 2 times before testing. CLS
422 Clinical Immunohematology I Student Lab Page 5 of
9
ABO Discrepancies - Handout
3. Red blood cells coated with cold autoantibodies
a. Wash cells with warm saline
b. Use prewarm technique antibodies elute from cells at 37o
C
1) Add 1 drop of anti-A or 1 drop of anti-B to appropriately labeled tube(s).
2) Add 3 4 drops of patients RBC suspension a third tube.
3) Place all of the tubes in a 37o
C heat block and incubate for 3-5 minutes.

4) Following incubation, add one drop of heated cells to the heated anti-sera.
5) Immediately centrifuge, read and record results.
c. Treat cells with chloroquine or EGA to remove autoantibody
4. Polyagglutinable red blood cells
a. Patients cells react with all antisera, including control (may be 6% albumin or
Rh-hr control).
b. Types of polyagglutination
1) T activation (T is for Thomsen)
a) Alteration of RBC membrane by bacterial or viral enzymes that act to
expose hidden T antigen
b) Exposed T antigen reacts with anti-T present in all normal human serum,
including conventional anti-A and anti-B typing sera (not seen as
frequently when using monoclonal reagents)
2) Tn activation
a) Abnormality of red cell membrane- not bacterial
b) Permanent
3) Other types of polyagglutination
a) Tk, Th, and VA activation - bacterial
b) Cad and NOR inherited
c. Resolution:
1) Switch to monoclonal reagents
2) Polybrene will not agglutinate T activated cells
3) Secretor studies
4) Adsorption/elution studies
5) Identified by testing with lectins
5. Acquired B phenomenon; B-like antigen CLS 422 Clinical Immunohematology I
Student Lab Page 6 of
9
ABO Discrepancies - Handout
a. Group A1 RBCs acquire B-like antigens in vivo due to bacterial enzymes,

deacetylases, which change acetyl-galactosamine to galactosamine

b. Patients group A cells absorb the B-like bacterial polysaccharide, which reacts
with anti-B (may show mixed field agglutination)
c. Usually seen in:
1) Intestinal obstruction
2) Carcinoma of colon or rectum
3) Lower GI tract disorders
4) Septicemia
5) Wound infections
d. Organisms responsible:
1) E coli
2) Clostridium
3) Proteus vulgaris
e. Resolution:
1) Testing with monoclonal anti-B reagent (Some will not react with acquired B
antigens check manufacturers directions)
2) Test an autocontrol Patients own anti-B will not react with acquired Blike
antigens
3) Acidified anti-B reagent, pH 6.0, will not react with acquired B-like
antigens
4) Secretor studies - only A substance present in secretions
5) Treat cells with acetic anhydride to reacetylate them, restoring A antigen
6. Chimerism: dual population of cells, mixed-field agglutination seen
a. In fraternal twins, two cell populations exist through life - expected reverse group
antibodies do not develop. Caused by vascular anastomosis or two sperm
fertilizing the same egg.
b. Artificial Chimerism caused by:
1) Transfusion of non- ABO specific blood. For example, intrauterine
transfusion, or transfusion of O Rh Negative units in trauma situations
2) Hematopoietic Progenitor Cell (HPC) transplant, where the donors

ABO is different from the recipients

3) Fetal-maternal bleed CLS 422 Clinical Immunohematology I Student Lab Page 7
of
9
ABO Discrepancies - Handout
7. Other problems:
a. Patient antibody to acriflavine (yellow dye) in anti-B reagent or caprylate
(preservative) - use reagents that do not contain these
b. Membrane modification due to drugs - check history on patient
III. Serum Problems - Reverse Grouping
A. Weak or missing antibodies most common form of discrepancy
1. Decreased antibody titer seen in:
a. Newborns (Antibodies usually detected at 4-6 months of age)
b. Elderly
c. Leukemia & lymphomas
d. Immunosuppressive drugs
e. Congenital agammaglobulinemia & immunodeficiencies
f. HPC transplant from a donor with an ABO group different from the
recipients
g. Plasma exchange
h. Dilution due to IV fluid
i. Chimerism - two cell populations in a single individual
1) Chimeric twins
2) Transfusion with non-ABO specific red cells
3) Fetomaternal bleeding
2. Resolution
a. Check patients age and diagnosis
b. Incubate patients plasma and reagent RBCs 15 - 30 minutes at room
temperature
c. Incubate at 40
C for 15 - 30 minutes. Must include an autocontrol; may include

screen cells to rule out reactions due to cold agglutinins CLS 422 Clinical
Immunohematology I Student Lab Page 8 of
9
ABO Discrepancies - Handout
d. Increase plasma from 2 drops to 4, 5 or 10 drops vs. 1 drop of RBC suspension
e. Treat reagent A and B cells with enzymes to enhance antigen expression
f. Read microscopically
B. Unexpected positive reactions:
1. Rouleaux Resolve using saline replacement technique
a. Spin tube containing patient plasma and reagent red blood cells a second time
b. Carefully remove from centrifuge; remove plasma using a pipette.
c. Add 2 drops of saline, using a pipette
d. Read for agglutination
1) True agglutination will persist
2) Rouleaux will be dispersed
2. Cold autoantibodies
a. Specificities:
1) Anti-I most common - reacts with all adult cells; does not react with cord
blood cells
2) Anti-H in A1 or A1B patient
b. Resolution:
1) Prewarm technique (2 tubes with patients plasma, 1 tube with 2 -3 drops A1
cells, 1 tube with 2-3 drops B cells, warm to 37o
C and proceed with testing as
described previously)
2) Cold autoabsorption
a) Incubate an equal volume of washed patient RBCs and patient plasma
together at 4o
C for up to one hour.
i. Every 15 minutes during incubation, mix tube and check for

agglutination. CLS 422 Clinical Immunohematology I Student Lab Page 9 of

9
ABO Discrepancies - Handout
ii. If agglutination is present, centrifuge tube, harvest plasma, and
incubate on a new set of patient cells.
b) After one hour of incubation (with no observable agglutination),
centrifuge tube and harvest plasma.
c) Test an autocontrol to ensure that all the autoantibody has been removed
from the plasma.
d) Test adsorbed plasma using usual method.
3. Cold alloantibodies, such as anti-M or anti- P1
a. Reverse grouping cells possess other antigens in addition to A and B, therefore
other unexpected antibodies in patients plasma may react with them.
b. Perform antibody identification.
c. Repeat reverse grouping using RBCs that are negative for corresponding antigen.
4. Anti-A1
a. Found in some A2 and A2B people, and in almost all other subgroups of A
b. Confirm patient is A1 antigen negative by testing patients RBCs with anti-A1
lectin
c. Test A2 reverse grouping RBCs against the patients plasma to get a valid reverse

grouping (also test screen cells in order to exclude an alloantibody)