Syazana Akmal Sharifudin

Scientific Officer
Immunohematology Division
National Blood Centre

Kursus Immunohematologi
19-20 Ogos 2015
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 Importance of Blood Group System
 Routine ABO Blood Grouping Testing
 Resolving Discrepancies in ABO Blood Grouping
 Standard Operating Procedure for ABO
Discrepancies in Blood Banks
 Selection of blood groups for component and
exchange transfusion in neonates

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.

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ISBT System Simbol Epitope/ Carrier Chromosome
001 ABO ABO Carbohydrate 9

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Percentage of Distribution (app)

5%

15%

20% 60%

Group O Group A
Group B Group AB

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 The differences in human blood are due to the
presence or absence of certain protein molecules
called antigens and antibodies

 The antigen are located on the surface of RBC and

the antibodies are in blood plasma. Individuals have
different types and combinations of these
molecules

 The blood group you belong to depends on what you

have inherited from your parents.

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 RBC

Glucose

Galactose
Precursor
Substance
(stays the N-acetylglucosamine
same)
Galactose

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 RBC

Glucose

Galactose
H antigen
N-acetylglucosamine

Galactose

Fucose
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 RBC

Glucose

Galactose

A antigen N-acetylglucosamine

Galactose

N-acetylgalactosamine
Fucose
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 RBC

Glucose

Galactose

B antigen N-acetylglucosamine

Galactose

Galactose
Fucose
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o Highly expressed on surface of a variety of
human cells and tissues, including:
- Epithelium, sensory, neurons, platelet, vascular
endothelium, kidney, heart, bowel pancreas and
lung.
o Also found on all body secretion such as
saliva, tears, milk, urine, bile, amniotic fluids &
pathologic fluids.
o Although ABH antigen have been detectable as
early as 5-6 weeks gestation, weaker expression
is expected in newborns (25-50%).

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 Naturally occurring, directed against the
missing ABO antigen
 Generally IgM class antibodies – cold reacting
antibodies that bind complement
 Time of appearance:
- Generally present within first 4-6 months of
life
- Reach adult level at 5-10 years of age
- ABO Titer in healthy adults varies from 4-
2048
- Begin to decrease in later years: >65 years of
age
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*1mL of RC lyse per minute
 ABO Hemolytic disease in newborn
 Causes:“O” mother & “A” or “B” baby
 Group O individual has anti-A, anti-B &
anti-AB
 RBC Immune form: Predominantly IgG
 Knowing the IgG anti-AB, anti-A & anti-
B allow the prediction or diagnosis of
HDN
 Positive DAT result may indicate HDN-
Heat elution (ABO)/Acid elution
(alloantibodies)
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 Clinically, ABO is the most important of the
red cell antigen system identified to date
 Determination of ABO compatibility
between donor and recipient is the basis of
all the pre-transfusion testing.
 A person must receive ABO matched blood
because ABO incompatibilities are the
major cause of fatal transfusion reactions

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 To catch ABO incompatibilities, grouping is
done in two steps:
 Forward grouping

 Reverse grouping -Used as

confirmatory for the forward method.
*Each grouping serves as a check on the
other

 Reverse is not required in 2 circumstances:

- For confirmation testing of
labeled, previously typed donor red cells
- In Infants younger than 4 months of age

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 TUBE METHOD GEL TESTING MICROTITER
Agglutination is Unagglutinated PLATE (AUTO)
visually red cells pass Passive
detected by the through a matrix Agglutination
adhesion of red but agglutinated where
cells are retained agglutination is
postcentrifuge visualized by
pellet spread patterns

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Reagent Quality Control
1. A statement of criteria for acceptable reagent
performance
2. Document of reagent use
3. Appropriate corrective action

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Name Type Physical Type of Titre Avidity Intensity
Appearance Red Cell
Anti-A Monoclonal Clear, no A ≥1:256 3-4 sec 4+
turbidity, A2 ≥1:128 5-6 sec 3+
precipitate,
particles by A2B ≥1: 64 5-6 sec 3+
visual B -- -- Neg
inspection & O -- -- Neg
Blue colour
liquid

The AABB requires the institutional of QC program

to ensure that all reagent function as expected. It
is generally performed on each reagent at least
once each day of use
 Washing cells to be tested removes serum or
plasma which may contain
 proteins that interfere with
testing, causing non-specific agglutination
or rouleaux formation.
 Washing also removes fibrinogen, which
may cause small clots.
 Preparation of a 3-5% cell suspension
provides cells in an optimum concentration
to detect weak antibodies.
 QC must be done once each day of use
(3+, 4+)

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• The ratio of serum to cells markedly affects
the sensitivity of agglutination tests.

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 Sufficient (1ml, 3ml or 10ml)
 Correct Tube (Anticoagulant-EDTA/ Plain
Tube w/o gel)
 Non-hemolysed – may cause technologist
to miss hemolysis as a positive endpoint in
testing
 Specimen not drawn from IV site – dilute
antibodies/may cause rouleaux
 Specimen, form & tube tallied
 Current results agree with historical
Non-hemolysed & hemolysed
sample
results
 Previous history of
transfusion/pregnancies
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 Put 2 drops of antisera anti-A, anti-B, anti-AB and
anti-D in each labeled tube

Put one drop of patient’s RC supension (3% – 5%) in

each tube.

2:1

Tubes labeled with respective antisera & patient ID no.

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Put 2 drops of patient’s plasma/ serum into labelled tube.

Put one drop of ABO cell into the labelled tube correctly.

2:1
Tubes labeled with respective known cell & patient ID no.
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 Mix the tubes well for a few sec
(leave for RT@3-5min) then
centrifuge at 3500rpm for 15 sec.

 Gently,tilt & roll the tubes and

observe one by one.

 Examine the tube and look for

agglutination appearance (button
face up).

 Grade the agglutination and record

the result immediately.

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 Partial or Complete
hemolysed is a positive
reaction
Mixed Field Agglutination (Post Transfusion)
•ABO Testing
Can be seen in A, B and AB individuals who have received O units.
The antisera reacts with patient RBC but not transfused O cell
•Antibody Screening
Can also be seen post transfusion if a person makes an antibody to
antigen on donor cells; antibody agglutinates on donor cell, but
not their own cell.

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 Forward Grouping Reverse Grouping Interpretation

Anti-A Anti-B Anti-AB A-cell B-cell O-cell ABO result

4+ 0 4+ 0 3+ 0 A

0 4+ 4+ 3+ 0 0 B

4+ 4+ 4+ 0 0 0 AB

0 0 0 3+ 3+ 0 O

0 0 0 3+ 3+ 3+ ? Bombay
Type of ABO Gelcards

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CAT vs tube method

 Standardization
 Stability
 No wash steps
 Consistent &
reproducible result
 Less sample volume

Cost
Time
Specific incubator &
centrifuge

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 Biorad (Diamed) Grifols

Patient Red cell suspension Red cell suspension (5% v/v) in

(5% v/v) in ID-Diluent 1 Grifols Diluent
(modified bromelin).
FORWARD

Add 10µl or 12.5µl of the red Add 10µl of the red cell into
cell into A/B/D/ctrl mirotube A/B/D/ctrl mirotube

Pippete 50µl ID-DiaCell A1, 50µl Pippete 50µl Serigrup Diana A1,
ID-DiaCell B 50µl Serigrup Diana B
REVERSE

Add 25µl serum/plasma into A1 Add 25µl serum/plasma into A1

/ B cell microtube / B cell microtube

*Phos. Buffer Saline pH 7.5 ± 0.2 can be use as an alternative solution.

Not suitable of Rh Phenotyping
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Figure 2: Correct procedure when
pipetting red cell & plasma into gelcard

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 Drying, discoloration, bubbles, crystals, other artifacts,
opened or damaged seals may indicate product
alteration
 Diamed: ID Incubator 15min, ID Centrifuge 10min
 Grifols: DG THERM incubator 15min, DG SPIN
centrifuge 9 min

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 If the control microtube is positive the results cannot be use!!
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 Microplate techniques can be used to test for
antigens on red cells and for antibodies in serum.

 A microplate can be considered as a matrix of 96

“short” test tubes; the principles that apply to
hemagglutination in tube tests also apply to tests in
microplate.
 Add reagent and patient sample( red cells/ serum)
 Agitate & Incubation RT 10min,
 Centrifugation 900rpm 10min
 Red cell resuspension,
 Reading of results
 Interpretation of results

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Olympus PK7300 – Donor Blood Grouping Confirmation
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 Correlate ABO testing with the expected
result and define various ways to resolve an
ABO discrepancies
 Identify various clerical and technical errors
that can affect ABO Interpretation
 Discuss the effect of disease on the
expression of ABH antigen.
 Forward Grouping Reverse Grouping

Anti-A Anti-B group A1-Cells B-Cells Group

0 0 O? 0 + A?

0 0 O? + 0 B?

+ + AB? 0 + A?

+ + AB? + + O?

Forward does not match the reverse

ABO Discrepancies MUST be Resolved
Patients : Resolve before any blood component transfused
Donors: Resolve before any blood is labeled with a blood type.
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 Reject Is the sample labeled appropriately?
sample, recollect No
Yes

Consider Sources of technical error

Yes
Proceed to pre- Is discrepancy resolved?
transfusion testing
No

Proceed to serologic discrepancy resolution

Obtain or review patient history

What is discrepant about this testing?

Weak or Missing antigen Inform

Weak or missing antibody supervisor/
Unexpected antigen reaction TMS/MO
Unexpected antibody reaction
False positive False negative
• Failure to add serum
Others
• Un-calibrated
centrifuges or reagents • Clerical Errors
• Contaminated • Use of incorrect • A mixed up in
reagents reagents or samples samples
• Dirty tubes or • Cell suspension is too • Missed observation
glassware heavy or too light of hemolysis
• Fibrin clots • Inadequate • Failure to follow
identification of manufacturer’s
samples or test tubes
instruction

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Initial steps to resolve discrepancy include:
 Clerical check of specimen and paperwork
 Determine patient’s age
 Diagnosis
 History of transfusion, transplants or
pregnancies
 Current medications

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Weak/ Missing Red Extra Red Cell Mixed field red cell
Cell reactivity Activity activity

Autoagglutinins/ Fetomaternal
ABO subgroup
excess protein hemorrhage

Excessive soluble
Acquire B Transplanted
blood group
substance
antigen bone marrow

Transplantation Transplantation
Exchange
transfusion

Transfusion / Unwashed Recent

IUT red cells transfusion

Twin or
Leukemia/ B(A)
dispermic
Malignancy phenomenon
chimerism

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 Weak/Missing Extra serum
serum reactivity activity

Age related (<4-6

Cold autoantibody
months, elderly)

ABO Subgroup Cold Alloantibody

Hypogamma
Excess serum protein
globulinemia

Transfusion of
Transplantation
plasma component

Infusion of
intravenous IG

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Transfusion delayed until
discrepancy resolved

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 Forward Grouping Reverse Grouping
Anti-A Anti-B Anti-A,B A1 Cells B Cells
0 3+ 4+ 0 0

Group I discrepancies
 These discrepancies are between forward and
reverse grouping due to weak reaction or missing
antibodies.

 These kind of discrepancies are the most common.

 The reason for the missing antibody or weak

reaction is that the patient has depressed antibody
production or cannot produce the ABO antibodies.

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 Some of the common populations with
discrepancies in this group:
a) Newborns and Elderly patients
b) Patient with leukemias (CLL)
Hypogamma-
c) Patients with lymphomas globulinemia
d) Patient using immunosuppressive drugs
e) Patient with Bone Marrow transplant
f) Patients with congenital
agammaglobunolinemia
g) Patient with immunodeficiency disease

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 Eliminate all technical errors
 Enhance the reverse group reaction by
incubating serum with A and B cells at RT for 15
to 30 min
 If no reaction, incubate serum-cell mixture at
40C for 15 to 30 min
 An auto-control and an O cell control must
always tested concurrently – cold agglutinins
 Rare Case: Chimerism-presence of 2 cell
populations in a single individual. Occurs in twins
or dispermy

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 Forward Grouping Reverse Grouping
Anti-A Anti-B Anti- A1 B Cells O cell Auto
A,B Cells C
Patient Z 0 3+ 4+ 0 0 0 0
30’ RT 0 3+ 4+ 0 0 0 0
30’ 4C 0 3+ 4+ 1+ 0 0 0
 Forward Grouping Reverse Grouping
Anti-A Anti-B Anti-A,B A1 Cells B Cells
0 1+ 1+ 4+ 0

Group II discrepancies
These discrepancies are between forward and
reverse grouping due to weak reaction or missing
antigens.
 This group is the least frequently encountered.

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 Some of the common populations with discrepancies
in this group:
a) Subgroups of A and/or subgroups of B
b) Leukemia
c) Hodgkin’s disease
d) Stomach or Pancreas CA – Excess amounts of Blood
group-specific soluble substances present in plasma
e) “Acquired B” Phenomenon –limited to group A1
individual with : Lower GI tract disease, Intestinal
Obstruction or malignancy of stomach &
Intestine, Gram negative septicemia (E. coli)
f) Antibodies to low incidence antigens in reagent A or
anti-B

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 Forward Grouping Reverse Grouping
Anti-A Anti-B Anti-A,B A1 Cells B Cells
4+ 2+ 4+ 0 4+
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 Incubate test mixture at RT for 30 minutes, if
negative result, reduce temp to 40C
 Washing patient’s cell with saline - remove
excess amount of BGSS that neutralize reagent.

Forward Grouping Reverse Grouping

Anti-A Anti-B Anti-A,B A1 Cells B Cells
Patient A 0 1+ 1+ 4+ 0
-Washing 0 3+ 4+ 4+ 0

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 Acquired anti-B antigen not agglutinate with
Anti-B at pH > 8.5 or pH <6.0 (using acidified
human anti-B/ autologous RBC / modified BS-1
lectin)

Forward Grouping Reverse Grouping

Anti-A Anti-B Anti-A,B A1 Cells B Cells
Patient B 4+ 2+ 4+ 0 4+
-Acidified 4+ 0 4+ 0 4+
anti-B
*
 Rare Case- additional antibody in the reagent
antisera present on the corresponding low
incidence antigen present in patient RC
–use different lot no. reagent
 Others:
-Patient have received HPC transplant: Document
findings in record
-Seek medical direction in determining type of
blood product to transfuse
 Forward Grouping Reverse Grouping
Anti-A Anti-B Anti-A,B A1 Cells B Cells
4+ 2+ 4+ 2+ 4+

Group III discrepancies

 These discrepancies are between forward and reverse
grouping due to protein or plasma abnormalities, or
pseudoagglutination, attributal to:
a) Elevated levels of globulin from certain diseases such
as multiple myloma, Waldenström’s
macroglobulinemia, other plasma cell dyscrasias, and
certain moderately advance cases of Hodgekin’s
lymphomas.

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b) Elevated levels of fibrinogen
c) Plasma expanders such as dextran and polyvinyl
pyrrolidone
c)Wharton’s jelly – gelatinous substance derive
from connective tissue that is found from cord
blood
 Some are caused by Rouleaux formation-
Rouleaux or red cells result from a stacking of
erythrocytes that adhere in a coin-link fashion
giving the appearance of agglutination.

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 Washing the patient’s red cells with saline or
saline replacement technique in case of rouleaux
formation (multiple myeloma, reverse albumin-
to-globulin ratio or given plasma expanders) &
repeat the test.

 If the agglutination is true red cell clumping will

remain.

 Cord blood must be washed 6-8 times & perform

forward grouping only or request new sample
from heel etc.
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Group IV discrepancies
 These kind of discrepancies are between forward
and reverse groping due to miscellaneous
problems and have the following causes:
A) Cold Reactive antibodies (allo and auto)
B) Warm autoantibodies
C) Unexpected alloantibodies
D) Polyagglutination – agglutination RBC with
human antisera due to bacterial infection or
expression of T antigens with antisera

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 Forward Grouping Reverse Grouping
Anti-A Anti-B Anti-A,B A1 Cells B Cells O cells AC
4+ 4+ 4+ 2+ 2+ 2+ 2+

 Patients probable group : AB

 Caused By potent cold autoantibodies
 Yield Positive DCT or ICT
 Incubate patient’s red cell at 370C , washed with 370C
saline 3x and re-typed
 Further Ix: treat RBC with DTT to disperse IgM
agglutination
 A and B cell warm at 370C, mixed with serum and read
at 370C
 If necessary, convert to AHG phase
- (recommend: IgG reagent)
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 Forward Grouping Reverse Grouping
Anti-A Anti-B Anti-A,B A1 Cells B Cells
1+ 1+ 1+ 4+ 4+

 Patient’s probable group : O

 Caused By Warm Autoantibodies / Alpha
methyldopa / Transfusion Reaction Yielding
Antibody Coated Red Cells or Positive DCT
 Gentle heat elution at 450C may remove
sufficient antibody that the RBC can reliably
typed with anti-A and anti-B

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 Forward Grouping Reverse Grouping
Anti-A Anti-B Anti-A,B A1 Cells B Cells
4+ 4+ 4+ 1+ 1+

 Patients probable group : AB

 Reverse grouping cells posses other antigens in
addition to A and B, and it is possible that other
unexpected antibodies present in patient’s
serum (e.g anti-M).
 Panel should be performed with patient serum.
 A1 and B cell negative for the corresponding
antigen can be use for the reverse grouping
 Repeat at 37ºC

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 Occasionally weak subgroups of A may
present practical problems.
 If Ax donor is mistyped as group O, this is
potentially dangerous
 The group O patient posses anti-A,B which
agglutinates and lyses Ax red cells.
 Occasional problems also arise when A2 or
A2B individuals demonstrate Anti-A1 in their
serum.

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 Degree of red cell agglutination by anti-A and
anti-A1
 Degree of red cell agglutination by human
and some monoclonal antisera
 Degree of H antigen (anti-H lectin and Ulex
europaeus) expression
 Presence or absence of anti-A1
 Presence of A and H in saliva
 Further Investigation:
 Adsorption and elution studies
 Family (pedigree) studies
Red Cell Red Cell Reactions with Antisera Serum reactions with reagent red
Phenoty or Lectins cells
ping Anti-A Anti-B Anti- Anti-H A1 cell B cell O cell Saliva
A,B (Secret
or)
A1 4+ 0 4+ 0 0 4+ 0 A&H
A2 4+ 0 4+ 2+ 0/2+ 4+ 0 A&H
A3 2+ mf 0 2+ mf 3+ 0/2+ 4+ 0 A&H
Ax 0/± 0 1-2+ 4+ 0/2+ 4+ 0 H

B 0 4+ 4+ 0 4+ 0 0 B&H
B3 0 1+ mf 2+ mf 4+ 4+ 0 0 B&H
Bx 0 0/± 0/2+ 4+ 4+ 0 0 H
B(A) ±/2+ 4+ 4+ 0 4+ 0 0 B&H
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 Present as an alloantibody in serum of 1%-8% A2
individuals and 22% to 35% of A2B individual
 Group O serum contains a mixture of Anti-A and
Anti-A1
 Anti-A1 is considered clinically significant if
reactive at 37ºC.
 To resolve an ABO discrepancy caused by anti-
A1, red cells should be tested with Dolichos
biflorus lectin, which agglutinates A1 but not A2
 Group A2 patients with an anti-A1 that is reactive
at 37ºC should be transfused with group O or A2
red cells only.
 Group A2B : O,A2, A2B or B red cells

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 Classical Bombay (Oh) Para-Bombay Phenotype

Homozygous for nonfunctional H Homozygous for a nonfunctional H

gene (hh) and secretor (sese) genes gene (hh), but inherited one
functional secretor gene (Se)

Oh red cells type as H negative with Lack serologically detectable H

anti-H lectin, Ulex europaeus and antigen but carry small amount of A
monoclonal Anti-H and/or B antigen. Designated “Ah”,
“Bh” and ABh”.

Posses natural isohemagglutinins to The sera contain anti-H, anti-HI or

A, B, and H. Reactive with all both
screening & panel cell.
Serum not reactive with Oh red Can occur in group O individual.
cells from other individuals
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 Red Cell Red Cell Reactions with Serum reactions with
Phenotyping Antisera or Lectins reagent red cells

Anti-A Anti-B Anti- Anti-H A1 B cell O cell Saliva

A,B cell (Secret
or)
Classical 0 0 0 0 4+ 4+ 4+ -
Bombay
Para-Bombay A ± 0 ± 0 1+ 4+ 4+ A&H

Para-Bombay B 0 ± ± 0 4+ 1+ 4+ B&H

*Literature on Para-bombay often have different description from one individual to another.
Further readings:
Reid ME, Lomas-Francis C: The Blood Group Antigen Factbook, 2nd ed. London, Academic Press,
Elsevier, 2004
Spitalnik PF, Spitalnik SL: Human carbohydrate blood group systems, 3rd ed. Philadelphia,
Lippincott Williams and Wilkins, 2002
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 Autoanti-H and Autoanti-HI
- May Cause ABO discrepancies
- Clinically in-significant. Can occur in Group A1, A1B
- Transfuse group specific or group O RBC should have a
normal in-vivo survival

 Alloanti-H (Bombay & Para Bombay)

- Highly Clinically significant
- Predominantly of IgM isotype and exhibit a broad
thermal range (4-37 ºC) with all red cells except
Oh
- Capable of activating complement and causing
red cell hemolysis
 Must transfused with (Oh) RBC

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76
77
 Must
Report !

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 Paed Pack group O Paed FFP group AB

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 Blood Group of Mother
O A B AB
O O O O O

Blood A EO A EO A
Group
Neonate B EO EO B B
AB EO A B AB

Fresh Irradiated Whole Blood (filtered if available)

**Emergency O (EO) blood is group O RhD positive whole blood with low
titers of Anti-A and Anti-B, negative for hemolysin test

Safe O is group O RhD positive packed cells – Emergency Cases

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not ET
ABO blood group of ABO group of plasma ABO group of
patients to be issued in order platelet to be
of preference issued

Unknown Issue AB if urgent Issue O if urgent

O O, AB, A, B O

A A, AB A

B B, AB B

AB AB (A or B if AB A or B
unobtainable)

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 AABB Technical Manual , 18th Edition
 Modern Blood Banking & Transfusion Practice, 4th Edition
 Textbook of Blood Banking and Transfusion Medicine, 2nd Edition
 CLS 422 Clinical Immunohematology I
 American Red Cross: ABO discrepancies
 Transfusion Practice Guidelines for Clinical and Laboratory
Personnel, National Blood Center (Un-publish, 2014)
 SOP Transfusion Medicine Unit 2015
 SOP Red Cell Serology Unit 2015

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 All Immunohematology Staff and
Organizing Committee of
Immunohematology Course/Workshop 2015

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