Documente Academic
Documente Profesional
Documente Cultură
By Henok.B (MLT,HI)
January 2018
DDU
1
1. Understand Explain the basics of
immunohematology
2. Understand and explain ABO blood grouping
system
3. Explain the concepts regarding Cross-match
4. Perform and interpret laboratory tests for cross-
match
2
Immunohematology:
◦ is more commonly known as "blood banking“
3
◦ refers to immunologic reactions involving blood
components
4
The era of blood transfusion began when
William Harvey described the circulation of
blood in 1616.
5
In 1667, jean Bapiste Denys transfused,
6
Transfusions were prohibited from 1667 to
1818
◦ Due to the disastrous consequences resulting.
7
Karl Landsteiner
8
Two main postulates were drawn:
1.Each species of animal or human have certain
factors on the red cells that are unique to that
species, and
2.Each species have some common and some
uncommon factors to each other.
This landmark event initiated the era of
science based transfusion therapy and was
the foundation of immunohematology as a
science.
9
Concerned with the way in which the
different blood groups are inherited
10
Allomorphic genes (Alleles),and
Polymorphism
Each gene has its own locus, along the
length of the chromosome.
Certain inherited characteristic can be
represented by a group of genes, and the
locus can be occupied by only one of these
genes.
Such genes are called alleles or allomorphic
genes.
11
Mitosis: While body cells multiply they do so
by producing identical new cells with 46
chromosomes.
12
During fertilization when the egg and
sperm unite the fertilizer ovum receives 23
chromosomes from each sex cell.
13
Genotype versus phenotype
Phenotype
◦ Physical expression of inherited traits,
◦ Determined by reacting red cells with known
antisera
Genotype
◦ Actual genes inherited from each parent
◦ Can only be inferred from the phenotype .
◦ Family studies are required to determine the
actual genotype .
14
Phenotype Genotype
A AA, AO
B BB,BO
AB AB
O OO
15
Punnet square
Illustrates the probabilities of phenotypes
from known or inferred genotypes.
16
Table4.1.2. Punnet squares showing ABO inheritance
A O
A AA AO
O AO OO
17
In most cases blood group antigens are
inherited with co dominant expression.
18
Recessive or dominant inheritance patterns
◦ recessive
inheritance would require that the same alleles from
both parents be inherited to demonstrate the trait
◦ Dominant
expression would require only one form of the allele to
express the trait.
19
O gene is recessive, since it is expressed
only when both parents contribute the O
allele.
20
1.4.1 Red blood cell antigens
21
In biochemical terms these antigens may
take the form of:
◦ proteins,
◦ Glycoprotein,
◦ Glycolipids
22
Some of the red blood cell antigens are
more immunogenic than the others
Example
23
Is possessed by nucleated cells such as
leukocytes and tissues
24
The MHC system is important in the:
◦ graft rejection .
25
Platelet possesses inherited membrane
proteins that can also elicit an immune
response.
26
Antibodies to platelet antigens are the
major cause of :
◦ neonatal alloimmune thrombocytopenia,
27
Blood group antibodies are classified into:
◦ Natural and
◦ Immune antibodies.
28
Are RBC Abs in the serum of an individual
that are not provoked by previous RBC
sensitization.
29
Characteristics
They are mainly IgM type.
30
Do not react above the body temperature
31
Produced due to previous antigenic
stimulation either by transfusion or
pregnancy
Characteristics
Mainly IgG type
Do not exhibit visible agglutination in
saline, but in albumin medium .
◦ Incomplete antibodies.
32
Optimally react at 370C
◦ warm agglutinins.
33
The binding follow the law of mass action
and is a reversible process.
34
The amount of Ag - Ab complex formation
is determined by the association constant of
the reaction .
35
Properties that can influence the binding of Ag
and Ab
The goodness of fit (as a lock and key fit)
36
To determine a person’s blood type, some
sort of substance must be available to show
what antigens are present on the red cell.
37
Is highly purified solution of antibody.
38
The anti-sera used in Immuno hematology
are prepared in one of the two ways:
39
Anti-serum must:
◦ Be specific for the antigen to be detected
◦ Have sufficient titer to detect antigen
For Example
Anti-A should have a titer of at least
1/128 against A1 cells,
1/64 againstA2 cells, and
1/16 against AB cells
Anti-B should have a titer of at least 1/64
against B
cells
40
Be free from haemolysins, fat and rouleaux
41
Have a marked expiration date, and
42
The presence of Invitro antigen and antibody
interaction can be detected by:
◦ Hemolysis
◦ Precipitation
43
Agglutination:
◦ sensitization and
◦ lattice formation.
44
A-Sensitization-the first phase
45
1. The antigen - antibody ratio
46
B- Lattice formation – the second phase
47
Cross linking is influenced by Zeta potential
Zeta potential
48
Centrifugation
Treatment with proteolytic enzyme,
Others
◦ Low Ionic strength saline (LISS)
49
1. Define:
A. Antigen
B. Antibody
C. Immunogenicity
2. Identify some characteristics of the IgG subtypes
3. What are the characteristic differences between
Natural and Immune antibodies?
4. Which classes of antibodies predominate during
the primary immune response and secondary
immune response?
5. List the factors that affect antigen and antibody
interaction
6. List the methods that are routinely used in the
blood banking laboratory to enhance
agglutination reaction.
50
4.2 THE ABO BLOOD GROUP
SYSTEM
51
Karl Landsteiner in 1900
52
ABO antigens are widely distributed and
located
◦ on red blood cells
◦ lymphocytes
◦ platelets
◦ tissue cells
◦ bone marrow, and
◦ organs such as the kidneys.
53
Soluble forms of the antigens can be
54
ABO system Ags, which are intrinsic to the
RBC membrane
55
A% B% AB% O%
Asian 28 27 5 40
African 26 21 4 49
Nepalese 33 27 12 28
Caucasian 40 11 4 45
Ethiopian 31 23 6 40
56
The production of H antigen is genetically
controlled by the H gene, located on a
different chromosome from the ABO genetic
locus.
57
The Se gene genetically influences the
formation of ABO antigens in saliva,tears,
and other body fluids.
58
Ag building block for A, B, and H Ag is
oligosaccharide chain attached to either a
protein or lipid carrier molecule.
59
The H antigen is the only antigen in the H
blood group system .
The H-gene is assigned to H -locus on
chro.19
The H-locus has two significant alleles: H &
h.
◦ The H allele is a dominant allele,
frequency greater than 99.99%
60
Each gene (H, A and B genes) codes for the
production of a specific transferase
enzyme.
61
Note:
The formation of H antigen is critical to the
expression of A and B antigens
62
63
The gene for A and B Ags are located on
Chr.9.
64
65
The B-allele codes for D-galactosyl
transferase which transfers D-galactose
(Immunodominant sugar) to the H-antigen.
66
67
The O allele is considered non functional,
since the resulting gene product is an
enzymatically inactive protein.
68
Bombay phenotype (hh) individuals
69
◦ Can inherit the A and B genes, as they do not
make the H substance the A and B gene is not
expressed and so typed as group “O” unlike
group O individuals they lack H.
70
Do not have H antigen but possess trace
amounts of A or B antigen depending on the
ABO gene on chromosome 9.
◦ Small amount of H antigen is produced and almost
completely converted to A or B antigen if these
enzymes are present.
These individuals are termed Ah or
Bh respectively.
Cause -production of very weakly acting H
glycosyl transferase.
71
It is a temporary replacement of Blood
group A by group B due to infection with a
gram negative bacteria especially by
P.vulgaris
72
Present in individuals with no known
exposure to blood or blood products
73
Immunoglobulin class
74
Hemolytic properties and clinical significance
75
In vitro serologic reaction
directly agglutinate a suspension of RBC in
a physiologic saline
Optimally react in immediate spin phases at
room temperature (250C).
Agglutination reactions do not require an
incubation period
React without delay up on centrifugation.
76
Human anti- A, B from Group O individuals
possesses unique activities beyond mixtures
of anti- A and anti- B antibodies.
77
ABO phenotyping has two components:
1. Forward grouping
2. Reverse grouping
78
4.3.2. 1.Direct (cell grouping)
Testing of the red blood cells for the
presence of ABO Antigens (or forward
grouping).
79
Principle
When an antigen is mixed with its
corresponding antibody under the right
conditions it causes agglutination or
haemolysis of the red cells.
80
Clinical significance
81
Donor and recipient red blood cells must
be tested using anti- A and anti-B.
82
The ABO phenotype is determined when the
red blood cells are directly tested for the
presence or absence of either A or B
antigen.
83
ABO discrepancy
◦ occurs when ABO phenotyping of red blood cells
does not agree with expected serum testing
results for the particular ABO phenotype.
84
The methods for grouping
1. Tube method
2. Emergency tile or slide grouping
3. Tile or slide grouping of blood donors
85
1.Tube grouping method
Specimens
-Patient’s serum .
-Patient’s cells.
86
Tube grouping method….
87
Tube grouping method….
88
Procedure: (e.g. preparation of a 2% red blood cell
suspension of 10 ml volume)
89
Method
1. Take 4 tubes and label them 1-4.
2. Place each of the following in its
numbered tube.
Tube 1: 1 volume of anti- A sera.
1 volume of 3-5% suspension of
patient’s washed cells.
Tube 2: 1 volume of anti- B serum.
1 volume of 3-5% suspension of
patient’s
washed cells.
90
Tube 3: 1 volume of patient’s serum.
1 volume of 3-5% suspension of
washed
RBC (reagent RBC) containing
antigen A.
91
3.Mix the contents of each by gently taping
the tube with the finger and leave for five
minutes at room temp.
4. Centrifuge the tube for one minute at
1000 RPM for one minute.
5. Replace the tube in the rack in the same
position as before centrifugation, and read
the result by gently tapping each tube,
looking for agglutination or heamolysis.
92
6. Check the tube which shows no visible
agglutination by examining the contents
microscopically on a slide using a 10x
objective.
7. Decide what a blood group of a patient
is and record the result in the book
provided and one the patient’s form.
Controls
Anti- A and Anti- B should be controlled
using known A (preferably – A2) cells and B
cells.
93
Phenotype RBC reaction Serum or plasma reaction
A + O O +
B O + + O
AB + + O O
O O O + +
+= agglutination, O= no agglutination
94
4+ A single agglutinate with no free cell,
clear
supernatants
3+ Several large aggregates, some free
erythrocytes, clear supernatants.
2+ medium sized aggregates and some free
cells,
clear supernatant.
1+ A few small aggregates just visible
macroscopically, many free erythrocytes,
turbid
and reddish supernatant many.
95
+ Weak granularity in the cell suspension. A
few
macroscopic agglutinates but
numerous agglutinates microscopically.
Weak (+/_) Tiny aggregates that are barely
visible
macroscopically, many free erythrocytes,
turbid
and reddish supernatant
Mixed field Few isolated aggregates, mostly free
floating cells, supernatants appear red.
Negative No aggregates
96
2. Emergency tile or slide grouping
Emergency tile grouping is carried out using
a white glassed porcelain tile, Perspex tray
or slides.
97
Specimen
◦ Patient’s serum
◦ Patient’s cells
Reagents and equipments:
◦ Anti- A and anti-B serum
◦ Group O serum (anti-AB)
◦ Antigen A and antigen B (20% known RBC
suspension)
◦ Group AB serum( Control)
◦ Tile or slide
98
Method
1. Divide and mark a white tile as follows
a b c d e f
Anti- A Anti- B Anti- AB A - cell B- cell Control
99
Then add the following
a. 1 volume of anti- A serum
1 volume of 20% suspension of patient cell
b. 1 volume of anti- B serum
1 volume of 20% suspension of patient cell
C.1 volume of group O serum
1 volume of 20% suspension of patient cell
10
0
d. 1 volume of patient serum
1 volume of 20% suspension of A cell
e. 1 volume of patient serum
1 volume of 20% suspension of B cell
f. control
1 volume of group AB serum
1 volume of 20% suspension of patient washed
cells (there should not be agglutination here).
3. Mix the contents of each division, using a small
clean applicator stick for each.
4. After 2-3 minutes read the results and decide the
blood group.
10
1
10
2
3. Tile or slide grouping of blood donors
10
3
Specimen
◦ Whole blood
Reagents and equipments:
◦ Anti- A and anti-B serum
◦ Group O serum (anti-AB)
◦ Tile or slide
◦ Applicator stick
10
4
Method
1. Take two slides or a white tile and mark as
follows
Anti-AB
Anti-A Anti-B
10
5
2. Place in to each marked area (anti-A, anti-
B, anti-AB) one drop of donor capillary
blood.
3. In to the division marked
- Anti-A, place one drop of anti-A serum,
- Anti-B, place one drop of anti-B serum,
- Anti-AB, place one drop of anti-AB serum.
4. Mix the content of each division with small
clean applicator stick.
5. After two or three minute read the results
by naked eye and decide the blood group
the donor.
10
6
A. Gel Technology method
-It is a new and unique method
10
7
B. Micro plate testing methods
-A micro titer plate with 96 wells serves as
the substituted tube test to which the
principles of blood banking are applied.
10
8
May be indicated by the following
observations
10
9
Expected reactions in ABO red blood cell
testing and serum testing are missing
For example
◦ a group O is missing one or both reactions in
the serum testing with reagent A1 and B cells).
11
0
Can be technical or sample –related
problems.
2.8.1.Technical error
Identification or documentation error.
Reagent or equipment errors.
Standard operating procedure errors
11
1
ABO discrepancies that affect the ABO red
blood cell testing.
◦ Missing antigens
11
2
ABO discrepancies that affect the ABO
serum testing.
11
3
In 1940 Landsteiner & Wiener discovered a
human blood factor, which they called
Rhesus factor.
11
4
This discovery followed the detection of an
antibody occurred in the serum of a woman
who delivered a stillborn fetus by Levine &
Stetson in 1939.
11
5
They also postulated that the antibody had
arisen as the result of immunization of the
mother by a fetal antigen which had been
inherited from the father.
11
6
In 1940, Wiener & Peters showed that the
antibody (anti- Rh) could be found in the
serum of individuals who had transfusion
reactions following ABO group – compatible
transfusions.
11
7
In 1941, Levine & his co- workers showed
that not only could Rh negative mother
become immunized to an Rh positive fetus
in utero but also that the antibody could
then traverse the placenta and give rise to
erythroblastosis fetalis or Hemolytic Disease
of the New born (HDN).
11
8
Later work demonstrated that the animal or
rabbit anti-Rhesus and human anti-Rh are
not detecting the same antigen but the
system had already named the human
antibody anti-Rh.
11
9
The animal anti-rhesus was detecting
another antigen possessed by Rh positive &
Rh negative persons but in much greater
amount in Rh positives.
12
0
A theory originally postulated by Tippett, describes
two closely linked genes- RHD and RHCE- on
chromosome 1.
RHD determines the D Ag expression on the
surface of RBCs. D-negative individuals have no
genetic material at this site. An antithetical d allele
does not exist.
Adjacent to the RHD locus, the gene RHCE
determines the C,c,E and e antigens. The alleles at
this locus occupy the gene RHCE, RHCe, RHcE, and
RHce. These genes encode the red blood cell
Antigens CE,Ce, cE, and ce.
12
1
12
2
The assortment of other antigens in the Rh
system occurs as a result of variation
of these polypeptides embedded in the
cell membrane bilayer in unique
configurations.
12
3
Antigen Amino acid N0
C Serine 103
c Proline 103
E Proline 226
E Alanine 226
124
Phenotype- refers to the test results
obtained with specific antisera
12
5
D antigen
Is the most immunogenic antigen in the Rh
system.
12
6
General characteristics
12
7
Can cause a severe hemolytic transfusion
reaction in a recipient if transfused with
blood possessing the offending antigen .
12
8
Methods of Rh Grouping Technique
◦ Direct slide and
◦ Direct tube
12
9
Clinical significance
13
0
Principle
When an antigen is mixed with its
corresponding antibody under the right
conditions it causes agglutination or
haemolysis of the red cells.
13
1
Specimen
Washed RBC(40-50%)
Equipments and reagents
Anti- D serum
◦ Microscopic slide
◦ Microscope
◦ Applicator stick
◦ Albumin (Control)
13
2
1. Place a drop of anti-D on a labeled slide.
13
3
Interpretation of the test result
13
4
Specimen
Washed RBC(2-5%)
Equipments and reagents
Anti- D serum
◦ Test tube
◦ Centrifuge
◦ Microscope
◦ Albumin (Control)
13
5
1. Make a 2-5% red cell suspension.
13
6
5. Mix well and centrifuge at 2200-2800 rpm for 60
seconds.
13
7
It is a procedure performed before
transfusion to select donor’s blood that will
not cause any adverse reaction, (hemolysis
/agglutination)
13
8
Will not:
◦ prevent immunization of the patient
◦ guarantee normal survival of transfused
erythrocytes
◦ detect all unexpected antibodies in a patient’s
serum.
13
9
Two types
Major cross-match:
involves mixing recipient’s serum with the
donor’s red cells.
14
0
Minor cross match:
Involves mixing the donor’s serum with
patient’s red cells
14
1
Accurate Patient Identification
Proper sample collection and handling
14
2
In cases when the recipient possess a
clinically significant Antibody, donor units
must be:
◦ Cross- matched
14
3
Finally, during the actual transfusion :
14
4
The blood selected for cross-match should
be of the same ABO and Rh (D) group as
that of the recipient.
14
5
Whenever possible blood of the patients own
blood group should be given.
14
6
Group O patient.
Can only receive group O blood
Group AB patient.
Should receive from group AB, if not
possible can receive blood from group A,B,
and O.
14
7
When cross-matching is carried out, the
serum is tested against the cells.
14
8
When deciding on methods for cross-
matching, the following conditions are
required for Ag-Ab reactions.
14
9
The safe cross-matching of blood requires
that the donor’s cells be mixed with the
patient’s serum in three separate tubes,
using :
1. Saline
2. Albumin
3. Anti-human globulin reagents
15
0
The red cells from the donor are suspended
in saline and mixed with the patient’s
serum .
show the presence of any complete
antibodies
Agglutination in the saline tube is usually
caused by:
◦ anti-A or anti-B antibodies and
◦ Occasionally by Lewis, MNSs, Lutheran and kell
antibodies.
15
1
The red cells from the donor’s suspended in
saline, are mixed with the patient’s serum,
and albumin is added.
The tube is incubated at 370C
shows the presence of any incomplete
antibodies
the antibodies react in albumin or any other
protein medium
15
2
Agglutination in the albumin tube is often
caused by:
15
3
A more concentrated suspension of red
cells is mixed with the patient’s serum and
incubated at 370C and then AHG is added.
15
4
4.6. 5.1.Standard cross-match
Clinical significance
detects unexpected (irregular) antibodies in
the recipient/ donor serum
15
5
Principle
15
6
Type of specimen
15
7
Equipments and reagents
Test tubes
Centrifuge
Microscope
Microscopic slide
Normal saline
20% albumin
AHG (Coombs reagents)
15
8
1. Take 3 small tubes mark them 1,2 and 3, and add
to each the following
15
9
2. Incubate tube 1 and 2 for 30 minutes at
370C. Incubate tube 3 for 15 minutes at
370C.
16
1
Results
No hemolysis or agglutination is seen in
tube 1, 2 or 3
◦ the blood is compatible and can be issued with
the completed cross-match label.
If there is agglutination or hemolysis in any
of the tubes
◦ the blood is incompatible, and must not be
issued for the patient.
16
2
ABO incompatibility (anti A and Anti-B.)
Saline tube ………………….Shows strong
agglutination
Albumin tube ………………. Shows agglutination
Anti- globulin tube ………… show no agglutination
16
3
Performed when there is no enough time to
perform the standard cross match
16
4
Principle
Serum of the recipient / donor is tested
against the red cells of the donor/ recipient
in saline and albumin medium in order to
establish their compatibility
16
5
Type of specimen
16
6
Equipments and reagents
Test tubes
Centrifuge
Microscope
Microscopic slide
Normal saline
20% albumin
16
7
1. Take 2 small tubes, mark them 1 and 2
and add to each the following
16
8
2. Leave tube 1 at room temp for 15 minutes
incubate tube 2 for 15 minutes at 370C.
3. Centrifuge both tubes for one minute at
1000
rpm/min
4. Examine the tubes macroscopically for
hemolysis and microscopically for
agglutination.
16
9
Results
If no hemolysis or agglutination is seen in
either tube 1 or 2
◦ the blood is compatible and can be issued with
the emergency cross match.
17
0
Takes only 3 or 4 minutes
17
1
1. Take 2 volume of patient’s plasma on a
slide
17
2
Results
17
3
Sources of errors in cross-matching
Rouleaux
Auto agglutinins
Infected donor cells
Anti- A1
Over centrifugation
Dirty glass wares etc..
17
4
Only human blood and its components are
used for transfusion into humans
Transfusions are the introduction of either
◦ Whole blood
◦ blood components (RBCs, Plts, plasma or WBCs )
or
◦ blood derivatives (albumin, gamma globulin,
Factors VII,VIII,Von willebrand,or Immune
globulins and prothrombin) directly into the
blood stream.
17
5
Whole blood- complex tissue ,composed of
cells and plasma
17
6
The use of component therapy:
17
7
Contains all cellular elements and
coagulation factors
Freshly drawn w/b maintains all its
properties for a limited time.
Upon storage a number of changes occur,
Example:
◦ increase in O2 affinity and
◦ loss of viability of RBC.
17
8
To provide both O2 carrying capacity and
blood volume expansion
Example In the treatment of massive hemorrhage.
Used as a source material for blood
component preparation.
For exchange transfusion in new born.
◦ Whole blood less than 4-5 days old is often the
component of choice
17
9
Component make blood use more
economical by using one unit one can
treat:-
18
0
Is the product remaining after the removal
of most of the plasma from freshly drawn
whole blood by centrifugation.
18
1
The red cell prepared in this form contain
the same:
◦ red cell mass and
◦ oxygen carrying capacity as whole blood with
approximately one half of the volume.
18
2
Concentrated red cell (CRC) is used to treat
patients with symptomatic anemia
18
3
Plasma is a fluid portion of one unit of blood collected and
separated in a closed system and intended for intra venous
use.
18
4
Fresh Frozen plasma (FFP)
Is the plasma obtained from a unit of whole
blood after centrifugation
18
5
The indications for FFP are for:
18
6
FFP
Should be stored frozen at –300C or colder
for 12 months
◦ beyond this period the factors VII may have
decreased
◦ To maintain adequate levels of coagulation
factors
If not used in 12 months time may be re
designated and relabeled ”Plasma” and has
4 more years of shelf life at –180C or colder.
18
7
is deficient in labile coagulation factors.
18
8
OP is different from FFP in that it contains
high levels of K and ammonia since it is
prepared after long contact with red cells
18
9
Indications:
◦ In the treatment of stable coagulation factors
deficiencies
◦ For volume and protein replacement under special
circumstances
19
0
Cryo depleted plasma (CDP)
CDP is the supernatant plasma following
removal of cryo precipitate.
- albumin
◦ immunoglobulin and coagulation factors are the
same as that of FFP,
◦ fibrinogen concentration and levels of the labile
coagulation factors V and VIII are markedly
reduced.
19
1
CDP is indicated for patients requiring
volume expansion or protein replacement
when labile clotting factors are not required
19
2
Is conversion of whole blood into
concentrated red cells with in the first 6
hours of collection.
19
3
Each unit of PC
◦ contains 5.5x1010 platelet in 50-65ml of plasma
◦ represents 60-80% of the plate late present in a
unit of whole blood.
◦ increases the platelet count by approximately
5,000-10,000 / ml in an average adult.
19
4
Platelet should be stored at
◦ 1-60C with agitation for 72 hours if prepared in a
close system
◦ 20-240C for
19
5
Indications
19
6
PC of the same ABO group should be used
19
7
Is a cold- precipitated concentration of
factor VII, anti hemophilic factor (AHF),
obtained after the plasma has been thawed.
Contains:
◦ Factor VIII: C (pro coagulation factor)
◦ Factor VIII: VWF (Von-Willebrand’s factors)
◦ Fibrinogen factor XIII
◦ Fibronectin factor
19
8
Each unit has at least 80μl of factor VIII,
250 mg of fibrinogen in a volume of 15 to
20 ml.
Cryo:
19
9
Indications
In the management of classic hemophilia
(factor VIII deficiency)
In the management of factor XIII deficiency
In the management of von willebrand’s
disease and
source of fibrinogen for the treatment of
hypo fibrinogenemia.
20
0