Transcript A 1 and B cells

ABO grouping is required for all of the following
individuals:
 Blood Donors-since it can be life threatening to
give the wrong ABO group to the patient.
 Transfusion recipients-since we need to know the
donor blood is ABO compatible.
 Transplant Candidates and Donors-ABO antigens
are found in other tissues as well. Therefore the
transplant candidates and donors must be
compatible.
Newborns (sometimes) If the baby is
demonstrating symptoms of Hemolytic Disease of
the Newborn, the ABO group needs to be
determined along with Rh and others.
 Paternity testing Since the inheritance of the ABO
Blood Group System is very specific, this serves as
one of the first methods to determine the
likelihood that the accused father is the father or
not.
 Prenatal Patients-To determine whether the
mothers may have babies who are suffering from
ABO-HDN. It is also beneficial to know the ABO
group should she start hemorrhaging.


Patient Identification
› The patient MUST be positively identified and
›
›
›
›
preferably banded. Some institutions use specific
Blood Bank arm bands.
Ask patient to state his/her name.
Responsible party should identify patient if he/she
cannot.
Verify information by comparing to ID band.
Resolve any differences before proceeding with
the blood draw.
 Labeling
of Sample
› The information on sample MUST match information on ID
band, which would also needs to be consistent with the
order.
› Information on samples MUST include the following:
 Name (last, first, middle initial) and no nicknames.
 Unique identification number such as medical record
number or possibly social security number.
 Date and time sample drawn along with the signature or
unique identifier of phlebotomist (on sample or on orders)
 Gender and birth date desirable but not mandatory . The
date of birth provides another unique identifier along with
the medical record number and full name of the patient.
 Mislabeled
Samples
› Do NOT accept any sample not properly labeled.
› The following are what would warrant an improperly
labeled specimen:
 Missing information
 Incorrect information
 Information on sample not matching information on
orders
› Improperly labeled samples must be discarded if the
problem cannot be resolved.
› In the case of an emergency blood draw on a patient
who is unidentified at that time, the blood specimen
must also discarded when both name and medical
record number have changed

Technical guidelines
› ABO grouping tests should be done at room temperature
›
›
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›
(2Oo24oC) or lower; testing in hot environment weakens the
reaction.
Routine ABO grouping must include both cell and serum
testing as each serves as a check on the other.
Antisera used in the ABO grouping must be used as per the
manufacturer’s instructions.
Adequate controls should be put with each batch of tests for
quality control of the reagents, alternately once a day the
reagents must be checked with appropriate cells.
Before use all the reagents must be checked grossly to rule out
any turbidity or contamination.
› Tubes, slides, microplates or gel cards should be
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labeled properly.
One should not rely on the colour of the dye to
identify the antiserum.
The glassware used should be dry and clean.
Serum should be added before adding cells and
each tube should be examined after serum has been
added to ensure that none has been missed.
Results should be recorded immediately after
observation.
Concave mirror (agglutination viewer) or microscope
may be used to examine reactions that appear
negative by the naked eye.
Blood sample for ABO grouping

›
Properly labelled samples of clotted blood collected
in screwcapped plain test-tube are most suitable for
ABO grouping. Ths amples should be stored at 4°C
and preferrably be tested within 48 hours.
›
Haemolysed samples are not suitable for testing.
›
The blood sample may be centrifuged at 1000-3000
rpm for 3 minutes for adequate serum separation.
›
Cells from the test sample should be washed in 0.9%
nromal saline and a 2-5% cell suspension should be
prepared.

Most samples for blood banking are drawn into a
red top tube - serum is preferred.

No clot activation tube should be used since the
patient's red cells may also need to used and no
other chemicals should be present

Antigens on cells are stable longer (months) in a clot
tube.

For antibody detection, plasma has the
theoretical disadvantage, compared with serum,
of:
› Containing anticoagulants that inhibit complement
activation and therefore possibly interfere with the
detection of some complement-activating antibodies.
› False positive may form in case of plasma due to the
formation of small fibrin clots.
A few tests require an EDTA sample if complement
is not to be activated.
 Serum must be tested while fresh to ensure good
complement activity.

 The
techniques used in Blood Bank
involves
› Specific antigens carried on red
blood cells
› Specific antibodies for certain red cell
antigens.




In almost all blood bank techniques we have red cells
with antigens present.
These red cells may either reagent red cells with
known antigens, patient red cells, or donor red cells.
The reagent red cells are commercially prepared and
have all the red cell antigens identified.
When we use red cells where the antigens have
already been determined, we can identify the
possible antibodies present.
 The
reagent cells used for blood banking
include the following:
› A1 and B cells for confirmation of the ABO type in all
patients and donors other than newborn babies
› Antibody identification cell panel are O cells with the
specific antigens known. Usually there are between 8
and 12 different cells in a cell panel. The pattern of
positive and negative reactions help identify the
antibody.

It is essential to use reliable grouping reagents to obtain correct
results.

Commercially prepared polyclonal (human source) and
monoclonal (tissue culture) antisera are available

Adequate quality control must be done before purchasing
commercial antisera. Each batch of antisera must be checked
against A1,A2, (if available, otherwise A group) B and 0 cells
before use.

The potency of any antisera deteriorates rapidly if kept for too
long at ambient temperature, grouping sera should therefore, be
kept at 4°C or as directed by the manufacturer when not in use.

Frozen antisera must be completely thawed before use and no
refreezing should be done.

This technique may be used for emergency ABO grouping tests or
for preliminary grouping particularly in an outdoor camp, however
it should always be supplemented with a cell and serum grouping
using any one of the other above mentioned techniques.

Slide or tile testing is not recommended for routine use because it is
not reliable for
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›

weakly reactive antigens on cells
serum grouping with low titre anti-A or anti-B
Disadvantages
›
Less sensitive than the tube test
›
Drying up of the reaction mixture can cause aggregation of
cells, giving false positive results.
›
Weaker reactions are difficult to interpret.
Test tubes either of glass or plastic may be used, of
lOx75mm size. The tube technique is more sensitive than
slide technique for ABO grouping.
Advantages of tube testing
› It allows for fairly long incubation without drying up of the
tubes’ contents.
› It allows to distinguish hemolysis.
› Centrigugation involved enhances the reaction allowing
weaker antigens and antibodies to be detected.
› Simplicity of reading and grading of results.
› Clean and more hygienic.
› Requires smaller volume of reagents.
Serum grouping
Result
Cell grouping
Oc
Bc
Ac
Anti-AB
Anti-B
Anti-A
A
-
+
-
+
-
+
B
-
-
+
+
+
-
O
-
+
+
-
-
-
AB
-
-
-
+
+
+
Oh or
any
other
irregular
antibody
+
+
+
-
-
-
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

Patient
A
B
C
D
Anti-A
4+
0
4+
0
Anti-B
1+
4+
4+
3+
A1 Cells
0
1+
1+
0
B Cells
4+
0
0
0
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

Most of the time, the problem is technical
› 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
 If a real discrepancy is encountered, the
results must be recorded
 However, the interpretation is delayed until
the discrepancy is RESOLVED

It is important 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

 Clerical
errors
› Mislabeled tubes
› Patient misidentification
› Inaccurate interpretations recorded
› Transcription error
› Computer entry error
› Mix up in samples
1. Contaminated sample or reagent
 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
 Incorrect storage temperatures
2. hemolyzed reagents
3. Using expired reagent
4. Using an uncalibrated centrifuge
› 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
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Reagents not added
Manufacturer’s directions not followed
RBC suspensions incorrect concentration
Cell buttons not resuspended before grading
agglutination
Warming during centrifugation
Rough dislodgment of the button while
reading the result
Inaccurate speed and time of centrifuge
Too many cells in your cell suspension can lead
to decreased or negative reactions since there
are too many cells for the number of antibodies
present in the reagents.
Failure to detect weak results can occur if you
are not watching the reactions while you are
shaking them out or if you shake too hard.
 Failure to detect hemolysis can be a definite
problem. Remember a positive reaction can
be hemolysis as well as agglutination since the
antigen-antibody reaction can bind
complement. When complement is bound it
can lead to hemolysis that is also an indication
of a positive reaction.
 Dirty glassware can cause the cells to artificially
clump.


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

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

Problems with RBCs
Weak-reacting/Missing antigens
Extra antigens
Mixed field reactions

Problems with SERUM
Weak-reacting/Missing antibodies
Extra antibodies

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.
A.
B.
C.
D.
Cell Typing – Additional Reactions
Cell Typing – Missing Reactions
Serum Typing- Additional Reactions
Serum Typing- Missing Reactions
Grouping
Forward
Missing/Weak
Extra
Reverse
Mixed Field
Missing/Weak
A/B Subgroup
Acquired B
O Transfusion
Disease
(cancer)
B(A) Phenotype
Bone Marrow
Transplant
Extra
Young
Elderly
Immunocompromised
Rouleaux
Cold
Autoantibody
Cold
Alloantibody
Rouleaux
May cause all + reactions
Anti-A1
ANTI-A
4+

ANTI-B
4+
A CELL
0
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.
ANTI-A
4+
•
•
•
•
ANTI-B
2+
A CELL
0
B CELL
4+
Microbial deacetylating enzymes such as E. coli cleave
off the N-Acetyl of the Group A1 N-acetylgalactosamine
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
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)
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)
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

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.

Wharton’s jelly
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

Sensitized RBCs
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

Albumin
Decrease zeta potential
Cause autoagglutination
ANTI-A
ANTI-B
A CELL
B CELL
0
0
0
4+
Weak variants of both A & B
Carry poorly expressed Ags
May not produce expected reactions with antiA & anti-B
 They are categorized according to the strength
and pattern of reaction with anti-A, anti-H &
anti-A,B
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

ANTI-A
ANTI-B
A CELL
B CELL
0
0
0
4+
• Weak Ags may be found on RBCs of some
people with diseases (Leukemia)
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
O
O
 Chimera: Two
cell populations
› Natural Chimera:
A
A
O
O A
 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)

Chimera will produce Mixed Field Agglutination

Agglutinates are seen with a background
of unagglutinated cells
 Mixed
Field Agglutination is also seen in
A3 phenotype (sometimes B3)
Anti-A
Anti-B
0
2+ mf
A1 Cells B Cells
4+
0
 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
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
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ANTI-A
ANTI-B
A CELL
B CELL
4+
0
2+
4+
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
Reverse Grouping
Extra 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 with 2-ME will also disperse cells
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)
 Associated with:

› Multiple meloma
› Macroglobulinemia
› Hydroxyethyl starch (HES), dextran, etc
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)
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

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+
Infants develop ABO Abs by 3-6 months of
age
 Serum typing before this time:

› Weak reaction
› Negative reaction
Have low levels of immunoglobulins
 Anti-A & anti-B may not be detected

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
 Deterioration
of Ags on A or B cells used
for serum typing
› Weak
› Negative reaction
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


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

Get the patient’s history
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age
Recent transplant
Recent transfusion
Patient medications
Anti-A
Anti-B
A1 Cells
B Cells
3+
0
0
1+
Problem: Reverse grouping weakened patient antibody
Causes: Age related or weakened immune system
Resolution: Incubate at Room Temperature 15-30
minutes and respin. Check patient history
Anti-A
Anti-B
A1 Cells
B Cells
3+
1+
0
4+
Problem: 1+ reaction with anti-B. Appears to have additional
antigens
Causes: Acquired B antigen
Resolution: Patient history – bowel obstruction, carcinoma of
colon/rectum(E. coli)
Anti-A
Anti-B
A1 Cells
B Cells
2+
0+
1+
4+
Problem: Weak forward with 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
Resolution:1) test patient cells with anti-A1 lectin and with patient serum test with A2 cells
2) an unexpected cold antibody would be detected in the antibody screen
Anti-A
Anti-B
A1 Cells
B Cells
0
0
0
3+
Problem: missing antigen in forward grouping. Patient appears as group A in
reverse grouping
Causes: A subgroup
Resolution: extend incubation time because this may enhance the reaction.
Test with a polyclonal or monoclonal blend of anti-A,B (may contain subgroup
antigens)…..
Anti-A
Anti-B
A1 Cells
B Cells
0
2+mf
3+
0
Problem: strength of anti-B is weaker than expected; reverse indicates a
group B individual
Causes: Group B individual transfused with group O cells
Resolution: recent transfusion? Bone marrow/stem cell transplant? Find
what ABO type the patient was prior to transfusion
Anti-A
Anti-B
A1 Cells
B Cells
4+
4+
0
1+
Problem:: Forward shows AB individual, Reverse shows weaker “extra” reaction with
B cells (looks like a group A)
Causes: Possible cold allo- or autoantibody (patient may have an antibody to another
blood group system; A1 and B cells may have the antigens to these antibodies) (allo:
P, M, N, Lewis) (auto: I or IH)
Resolution: screen for antibodies using Screening Cells and an autocontrol (we’ll talk
later about Ab screens
Anti-A
Anti-B
A1 Cells
B Cells
0
0
0
0
Problem:Reverse grouping, missing patient antibody (probably
group O with no antibodies)
Causes:Age related or weakened immune system
Resolution:Incubate at Room Temperature 15-30 minutes and
respin. Check patient history
Anti-A
Anti-B
A1 Cells
B Cells
4+
2+
4+
4+
Blood group : A
Possible discrepancy: Rouleaux formation
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Example 8


Anti-A
Anti-B
A1 Cells
B Cells
4+
0
1+
4+
Blood group: A
Possible discrepancy: Missing Ag.
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