Diskrepansi

Golongan darah
sistem ABO

Dr Samson Ehe Teron SpPK

Laboratorium Patologi Klinik
RSU Prof WZ Yohanes Kupang
Unit Transfusi Darah PMI NTT
 Diskrepansi
Suatu diskrepansi (konlik fakta) yang
muncul jika hasil uji sel tidak cocok
dengan hasil uji serum
Dengan kata lain uji tabung tidak cocok
dengan uji slide ( The Forward does NOT
match the reverse)
Hasil pemeriksaan golongn darah dapat
tidak sesuai dengan yang seharusnya
 Latar belakang
Penting mengenali ketidak cocokan hasil
pemeriksaan dan bagaimana memecahkan
masalahnya
Sistem ABO merupakan sistem golongan darah
yang paling penting dalam kaitan dengan
transfusi
Salah interpretasi diskrepansi dapat
mengakibatkan gangguan dan kerusakan organ
dan membutuhkan perawatan dan
penanganannya seumur hidup terhadap pasien
 ABO DISCREPANCIES

ABO discrepancies occur because of:

Intrinsic problems with the red cells or
the serum.
Test related problemsslide vs. tube
Technical errors.
 Kenapa ?
Kekuatan reaksi lebih lemah dari
yang diharapkan
Sebagian reaksi mungkin hilang saat
pemeriksaan reverse or forward
Mungkin ada reaksi Extra
ABO Antibodies

Generally IgM class antibodies

For Group A and Group B persons the predominant
antibody class is IgM
For Group O people the dominant antibody class is IgG
(with some IgM)
React best at room temperature (22-24oC) or below in
vitro.
Activates complement to completion at 37oC
Can cause acute Hemolytic Transfusion reactions
RBC Immune form: Predominantly IgG

Dr Samson
ABO Antibodies
Time of appearance:
Generally present within first 4-6
months of life
Reach adult level at 5-10 years of age
Level off through adult life
Begin to decrease in later years: >65
years of age
Dr Samson
A and B Subgroup
They both react strongly with reagent anti-A.
80% of group A individuals phenotype as A1
20% phenotype as A2

Reagent anti-A is a mixture of two Abs ;

anti-A which react with both A1 and A2 cells.

anti-A1 which reacts with A cells but not with
A2 cells in simple testing .

Dr Samson
A and B Subgroup
Qualitative difference due to ;

1-8 % of A2 and 22-35 % of A2B individuals

produce a readily identifiable anti-A1 in their
serum.

Quantitative difference
A2 cells carry 25 % as many A antigen sites
as do A1 cells
A1 individuals make A antigen from all type
II chains ( H1-4 ) .
A2 individuals produce A antigen only from
H1 and H2 precursors.

Dr Samson
A and B Subgroup
Differentiation between the A blood
subgroups
Reagent anti-A is a mixture of two Abs
The two Abs can be functionally separated by
adsorption with A2 cells.

Anti-A1-lectin: is another source of anti-A1.

lectins are seed extracts that agglutinate
human cells with some degree of specificity.
The seeds of the plant Dolichos biflorus
serve as the source of the anti-A1 lectin this
reagent agglutinate A1 or A1B cells but does
not agglutinate A2 or A2B cells.

Dr Samson
ABO Discrepancies
ABO discrepancies happen when
there is no match in results between
forward and reverse grouping.

ABO discrepancies are usually

technical in nature and can be
simply resolved by correctly
reporting the testing and carefully
checking reagents with meticulous
reading and recording of results.

Dr Samson
ABO Discrepancies
There are some ABO discrepancies
that can happen due to technical
errors and may lead to false positive
or false negative reactions.

False positive reactions are due to;

Un-calibrated centrifuges
Contaminated reagents
Dirty tubes or glassware

Dr Samson
ABO Discrepancies
False negative reactions can be due to
many causes

Failure to add serum or reagents

Use of incorrect reagents or samples
Cell suspension is too heavy or too
light
Inadequate identification of
samples or test tubes

Dr Samson
ABO Discrepancies

This type of discrepancy can be seen in

new born infants, elderly patients.

Patients with lymphoma.

Patients using immunosuppressive drugs.

Patients with immunodeficiency disease,

BM transplant.
Dr Samson
ABO Discrepancies

Resolving discrepancies

Eliminate all technical errors

Enhancing the reaction in
reverse grouping
Incubate the patients serum
with reagent cells at room temp.
for 15 mins.
Dr Samson
ABO Discrepancies
Some examples of discrepancies

Example 1
Forward grouping: anti-A =O, anti-B =O,
anti-AB= O
Reverse grouping: A1 cells= O, B cells =O
Blood group:
O
Possible discrepancy:
Missing Ab. Or group I discrepancy

Dr Samson
ABO Discrepancies
Group II discrepancies
These discrepancies are between forward
and reverse grouping due to weak reaction
or missing antigens.
This group is the least one. Can be caused by
some subgroups of A or subgroups of B or
both.

Also it can be present in patients with

leukaemia and hodgkins disease.

To resolve the problem wash the patients cells

with saline.

Dr Samson
ABO Discrepancies
Example 2

Forward grouping: anti-A = 4+,anti-B =O,

anti-AB =4+
Reverse grouping: A1 cells =1+, B cells =4+
Blood group:
A
Possible discrepancy:
Missing Ag. Or group II discrepancy

Dr Samson
ABO Discrepancies
Group III discrepancies

These discrepancies are between forward and

reverse grouping due to protein or plasma
abnormalities.

These can be caused by elevated levels of

globulin from certain diseases such as multiple
myloma, hodgekins lymphoma. Some are
caused by (Rouleaux formation).

Dr Samson
ABO Discrepancies
Rouleaux or red cells result from a stacking
of erythrocytes that adhere in a coin-link
fashion giving the appearance of
agglutination.
To resolve this kind of problem, washing the
patients red cells with saline or adding a
drop or two of saline to the tube in case of
rouleaux formation.
If the agglutination is true red cell clumping
will remain.
Cord blood must be washed 6-8 times in
forward grouping ONLY.

Dr Samson
ABO Discrepancies

Example 3

Forward grouping: anti-A 4+,anti-B 2+,anti-AB 4+

Reverse grouping: A1 cells 4+, B cells 4+
Blood group : A
Possible discrepancy

Rouleaux formation

Dr Samson
ABO Discrepancies

Group IV discrepancies
These kind of discrepancies are between forward and
reverse grouping due to miscellaneous problems.

Polyagglutination can occur due to exposure of hidden

erythrocyte Ag. (T antigen) in patients with bacterial or
viral infection.

Bacterial contamination in vitro or vivo produces an

enzyme that alters and exposes the hidden Ag. on red cell
leading to T activation.

Dr Samson
 Diskrepansi positif palsu
Adanya formasi rouleaux
Cold autoantibodi atau alloantibodi
Aktivasi sel T (Poliaglutinasi )
Aqquired B
Potentiator
Kontaminasi bakteri in vitro
 Diskrepansi negatif palsu
Lupa menambahkan reagensia
Reagen sudah kehilangan potensinya
Kegagalan mengenali hemolisis
Aglutinasi majemuk ( Mixed field
agglutination)
Fenotipe D varian
 Diskrepansi sejati
Adanya kelainan antigen dan atau antibodi
golongan darah
Kelainan eritrosit :
Atigen yang lemah atau tidak terdeteksi
Adanya antigen ekstra atau tambahan
Reaksi Mixed field
Kelainan serum akibat antibodi yang lemah atau
tidak terdeteksi dan adanya antibodi tambahan
 Kenapa ?
Kekuatan reaksi lebih lemah dari
yang diharapkan
Sebagian reaksi mungkin hilang saat
pemeriksaan reverse or forward
Mungkin ada reaksi Extra
 Intrinsic red cell problems
Weaker subgroups of A and B or AB such as A2,
Ax, A3, A2B and subgroups of B.

Variant A or B genes such as B(A). Must be

differentiated from a subgroup of AB.

A patient who has had a bone marrow transplant

or an A or B recipient recently transfused with
group O red cells.
 Intrinsic red cell problems

Acquired B. Usually associated with infections

from GI (gastro-intestinal) bacteria

Polyagglutinable state. The red cells are

unexpectedly agglutinated by human source
anti-A and Anti-B. Not seen with monoclonal
antisera.
Potent cold agglutinins
 Intrinsic problems in the serum
Anti- A or B is not usually detectable until 4-6 months of
age. Any reactivity is usually maternal IgG forms of anti-A
or B.

A2 or weaker subgroups of A or AB may produce anti-A1.

Anti-A1 will react with A1 cells but not A2 or O cells.

Allo-antibodies such as anti-M,-N,-P1, Lea or Leb

May react at IS (immidiate spin)or RT(room temperature)
and react with the A or B cells.
 Intrinsic problems with the serum
Abnormal concentrations of proteins or
infusion with high molecular weight plasma
expanders may show aggregation that is
difficult to distinguish from true agglutination.

Immunodeficient patient, due to disease or

therapy may have decreased levels of anti-A
or B.
 Intrinsic problems with the serum
Infusion of large volumes of compatible but not
type specific plasma.

Bone marrow transplants with compatible but

dissimilar ABO groups. Example: a group A
individual who receives group O marrow will
produce circulating O cells, but produce only anti-
B in the serum.

Potent cold aggutinins.

 ABO discrepancies
seen with test related problems

Abnormal proteins, infusion of

macromolecular solutions or improperly
collected cord blood (contaminated with
Whartons jelly) may cause false
agglutination of cells. This is especially
true when the cells are resuspended in
serum which is usual for slide typing.
 Problems with the test system

Serum may contain antibodies to the dyes used

to color anti-A and anti-B (USA) which may give
false positives with serum-suspended cells in the
slide test.
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
 Resolving AB0 discrepancies
Repeat the tests after washing patient cells
and the A and B cells several times.

Analyze the problem:

1. Unexptected + or unexpected results?

2. What is unusual or different?
3. Do you see spontaneous agglutination?
 Resolving ABO discrepancies
If the serum is unexpectedly positive, does the
agglutination look like rouleaux? Use saline-
replacement technique.

Should you try to prove anti-A1 in an A2

person. Type patient cells with anti-A1 lectin.

Test the serum with screening cells, and

another set of A1 and A2 cells.
 Resolving ABO dicrepancies
Is there strong hemolysis in reverse grouping
tubes? Add another drop of appropriate cell
and centrifuge again.

Is the serum typing unexpectedly negative?

Add several more drops of serum to all reverse
grouping tubes, incuding the 3 O screening
cells and an auto control. Incubate at RT or
colder.
 Resolving ABO discrepancies
If the discrepancy cannot be resolved , a
patient should be transfused with group O red
cells of the appropriate Rh type.

A donor units may not be labelled or released

for transfusion and should be discarded.
 Refer to examples of paper ABO discrepancies
And serological resolution
 Identifikasi Masalah

Kebanyakan masalah teknis

Salah label tabung
Lupa menambahkan reagensia
Salah satu usaha yang dilakukan :
ulangi tes dengan contoh darah yang
sama, Minta sampel baru, atau cuci
sel darah
Kadang kadang , ada diskrepansi
sejati berkaitan dengan masalah
pada sel darah merah atau serum
pasien
 Diskrepansi ?
Diskrepansi sejati , hasil harus
dicatat dan diberi label khusus
Interpretasi akan lambat sampai
diskrepansi dipecahkan
masalahnya
 Errors
atau
kesalahan yang terjadi
 Technical Errors
Kesalahan Klerikal
Salah label tabung
Salah identifikasi pasien
Dokumentasi interpretasi yang tidak akurat
Kesalahan pencatatan / menulis
Salah entri data
Masalah Reagensia dan peralatan
Menggunakan reagensia yang kedaluarsa
Alat Sentrifus yang tdak terkalibrasi
Reagen yang terkontaminasi dan hemolisis
Temperatur penyimpanan yang tidak sesuai
Kesalahan Prosedur
Reagen tidak ditambahkan
Petunjuk manufaktur tidak diikuti (GMP)
Konsentras Eritrosit tidak tepat
Endapan sel di Dasar tabung tidak di resuspensi sebelum ditetapkan gradasi
aglutinasi.
 Defisiensi faktor faktor yang terlibat
dalam sistem pembekuan
Serum tidak membeku :
Jumlah trombosit yang rendah
Terapi Anticoagulant (Heparin, Aspirin, dsb)
Defisiensi faktor pembekuan
Serum tidak jernih dan masih ada sisa fibrin sehingga
terlihat seperti ada agglutinasi
Thrombin dapat ditambahkan ke serum untuk
aktivasi pembentukan bekuan
Atau , menggunakan tabung EDTA
 Contaminated samples or reagents
Sample contamination
Microbial growth in tube
Reagent contamination
Bacterial growth causes cloudy or discolored
appearancedo not use if you see this!
Reagents contaminated with other reagents (dont
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

ABO discrepancies happen when there is no match in

results between forward and reverse grouping.

ABO discrepancies are usually technical in nature and

can be simply resolved by correctly reporting the testing
and carefully checking reagents with meticulous reading
and recording of results.

Dr Samson
ABO Discrepancies

There are some ABO discrepancies that can happen due

to technical errors and may lead to false positive or false
negative reactions.

False positive reactions are due to;

Un-calibrated centrifuges
Contaminated reagents
Dirty tubes or glassware

Dr Samson
 ABO Discrepancies
Problems with RBCs
Weak-reacting/Missing antigens
Extra antigens
Mixed field reactions
Problems with SERUM
Weak-reacting/Missing antibodies
Extra antibodies
 Grouping

Forward Reverse

Missing/Weak Extra Mixed Field Missing/Weak Extra

Young Cold
A/B Subgroup Acquired B O Transfusion Elderly
Immunocompromised Autoantibody

Disease Bone Marrow Cold

B(A) Phenotype
(cancer) Transplant Alloantibody

Rouleaux 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, Hodgkins disease)

Anti-A Anti-B A1 Cells B Cells

0 0 0 4+

Group O Group A

Since the forward and reverse dont 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

Anti-A Anti-B A1 B
Cells Cells
4+ 1+ 0 4+

EXAMPLE
 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)
 Acquired B
Bacteria (E. coli) have a deacetylating enzyme
that effects the A sugar.

Group A Acquired B
individual Phenotype

N-acetyl galactosamine Galactosamine now

resembles D-
galactose (found in
Bacterial enzyme removes Group B)
acetyl group
 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
Whartons 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 (sometimes B3)

Anti-A Anti-B A1 Cells B Cells

0 2+ mf 4+ 0
 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 the patients RBCs, but not with the transfused O cells.
~ (Antibody screen) Can also be seen post transfusion if a person makes an antibody to
antigen on donor cells; antibody agglutinates with donor cell, but not their on cells.
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
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 4C 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 with 2-
ME will also disperse cells
 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 37C and no
agglutination at AHG phase
Associated with:
Multiple meloma
Waldenstroms 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 (UMMC)

 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 AGGLUTINATION
Anti-A1 from
patient + A2 cells
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 A1 B
Cells Cells
4+ 0 2+ 4+
 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 (Bombays
dont 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 patients history:
age
Recent transplant
Recent transfusion
Patient medications
The list goes on.
Lets 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 6

Screening Autocontrol Conclusion

Cells (AC)
(I and II)
Patient Pos Neg Cold
Serum 1 alloantibody
Patient Pos Pos Cold
Serum 2 autoantibody
if alloantibody antibody ID techniques
if autoantibody special procedures (minicold panel, prewarming techniques); no
prior transfusions. If they have had a recent transfusion, then it could be an
alloantibody.
 Penyebab reAktivitas Eritrosit lemah atau hilang

Sub group ABO

Leukemia atau keganasan
Transfusi
Intra uterine fetal transfusion
Transplantasi
Excessive soluble blood group substance
 Penyebab mixedfield reaction
Recent transfusion
Transplantation
Fetomaternal bleeding
Twin or dispermic (Tetragametic) chimerism
 Penyebab extra antigen
Autoagglutinin / ecess protein coating red cell
Unwash red cells : plasma protein
Transplantasi
Aquired B antigen
B (A) phenomena
Out of group transfusion
 Penyebab weak/missing antibody
Age related ( (<4-6 months old , ederly)
ABO Subgroup
Hypogamammaglobulinemia
Transplantation
 Penyebab extra antibody
Cold autoantibody/Alloantibody
Serum antibody to reagent constituent
Excess serum protein
Trasfusion of plasma contituent
Transplantation
Inffusion of intravenous immune globuline
 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. (2009). Basic & Applied Concepts of
Immunohematology. St. Louis, MO: Mosby, Inc.
Rowley M, Cantwell, Milkins C. Laboratory Aspect of Blood Transfusion. In:
Bain BJ, Bates I, Laffan MA,Levis SM. Dacie and Levis Practical Hematology.
11 ed. China: Elsevier;2012
Davenport RD, Mintz PD . Transfusion Medicine . In McPherson RA, Pincus
MR. Henrys Clinical Diagnosis and Management by Laboratory
Methods.22nd ed . Philadelpia:2011.
Marcela Contreras, Geoff Daniels. Red Cell Immunohaematology . In
Hoffbrand Victor .A ea al . Post Graduate Haematology. 6th ed. Wiley
Blackwelll Oxford UK