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In 1900 karl landstainer discovered the blood groups ABO and classifieds human blood into A,B and O
Groups. A fourth blood Group AB was discovered by landstainer’s associates. Von Decastello and sturli
in 1902.
The four groups are determined by the presence or absence of blood group antigens
(agglutinations) on the red cells and accordingly an individual’s group A,B,AB, or O (O is donates the
absence of A or B antigens). In addition, it has been shown that corresponding to antigens A and B, these
are naturally occurring antibodies anti-A and B (agglutinations) in the plasma/serum of individually
whose red cells lack the corresponding antigen.
Group ‘A’ individually have anti-B, Group-B individually anti-A, Group ‘O’ Individually have
both anti-A and anti-B and Group AB individually have no agglutination in the plasma/Serum.
In 1991 von lungern Hirszfeid showed that Group ‘A’ could be divided into two principal sub Groups
A1 and A2 on the basis of this the ABO system is classified into six main Groups A1,A2,B A1,B1, A2 B
and O.
ABH ANTIGENS:
A, B and H antigens are present not only on the red cells but are also widely distributed through out the
body tissues except in the central nervous system.
A,B and H antigen city is determined by specific sugars linked to the terminal portion of
oligosaccharides (short chain sugars) these are present on glycoproteins or glycolipids.
In the red cell membrane both glycolipids and glycoprotein’s with ABH activity are present. In
the plasma only glycolipids in soluble form are found. Cell membranes of endothelial and epithelial
cells have both glycolipids and glycoproteins.
Secretor Status
A, B and H Substances are also found in the Secretions of 80 % of the population the ability to
secrete B and H substances is determined by the presence of the secretor gene(se) in either the
homozygous se se or heterozygous se se state, which is inherited independently of the ABO and Hh
genes. Normally all secretors secrete H, in addition A and / or B sub stances.
Showing Sector Status
SUB GROUPS OF A and AB: A and AB have divided into Subgroup A1, A2, A, B and A2 B depending
up on the reaction with the extract of a lactin Dolichos biflorus seeds or human Anti-A1 serum. Anti-A
sera very seldom differentiate between A1 and A2. both human anti-A1 and lactin anti-A1 agglutinate
A1 and A1B cells but not A2 and A2 B cells. 20% of persons with A antigens in the A or AB group are
A2 (or) A2 B.
It is not necessary to classify group A patients or donors as A1 or A2 except when the individual serum
contains anti-A1, anti-A1, occurs in the serum of 1-8% of A2 group person and 22-35% of A2 B groups
person. Anti-A1 courses discrepancies between ABO cells and serum test and may also causes
crossmatch incompatibilities but is considered clinically significant if it reacts at 37°C.
Weak Subgroup of A:
Sub group weaker than A2 occurs in frequently they are characterized by the declining number of A
antigen sites or red cells and reciprocal increased in H reactivity. Weaker variants of A are mainly A3,
Ag, Am and A. Intermediate.
Classification of weak A subgroups based on.
1. Degree of agglutination by anti-A, Anti-A1 and Anti-AB
2. Degree of H reactivity on the red cells.
3. Presence or absence of anti-A1 in the serum.
4. Presence of A and H substance in saline of secretors.
A B AB H A1 A1 A2 B O
A1 +4 - +4 +,- +4 - - +4 - A&H
A2 +4 - +4 +2 - - +4 - A&H
A1 and 2 +4 - +4 +3 +2 - - +4 - A&H
A3 + - +2mg +3 - - +4 - A&H
2mf
Ag -/w - +1/+2 +4 - +2 -/w +4 - H
Am -/w - -/w +4 - - - +4 - A&H
B - +4 +4 - - +4 +4 - - B&H
B2 - -/w -/+ +4 - +4 +4 - - H
Bm - - -/w +4 - +4 +4 - - B&H
Anti-A
The antibody anti-A is found in group B and group O individuals and reacts well with A1 and A2 cells
but not as well with weaker subgroups of A.
Anti-B
The antibody anti-B is found in group A and O individuals, and reacts almost with all B group cells but
less effectively with weaker variants of B group.
Anti-AB
An antibody anti-AB is found in group O individuals. Serum from O group individuals is particularly
useful in detecting some weak A and B antigens.
Anti-A1
This is found in 1-8% of A2 and 22 to 35% of A2 B individuals. It is usually active at room temperature
or below and is rarely clinically significant, except when it reacts at 37°C.
Anti-H
Anti-H very rarely occurs as cold reactive agglutinin in individuals with very low levels of H antigens
on their cells and have little clinical significance. However, anti-H found in Bombay blood group (Oh)
is an alloantibody and is clinically significant. It occurs as a heamolysis and agglutinates cells at 37
A B O Grouping:
PRACTICAL ASPECTS OF ABO GROUPING
(1) Routine ABO grouping must include both cells and serum testing as each test serves as check on
the other.
(2) ABO grouping tests should be done at room temperature or lower; testing at 37°c weakens
reaction.
(3) Anti-sera used in the ABO grouping must be used as per the instruction of manufacturer.
(4) Controls should always be run with respect to ABO grouping. Most laboratories have quality
control of antisera once a day in order to eliminate the need to run individual controls every time
the reagents are used.
(5) Tubes, slides or micro plates should be labeled properly. One should not rely on the colored dyes
to identify the regent antiserum.
(6) Serum should always be added before adding cells and examine each tube after serum has been
added to ensure that none has been missed.
(7) Tubes or slides should be clean and dry.
(8) Optical aid should be used to examine reactions that appear negative by the naked eye.
(9) Results should be recorded immediately after observation.
Techniques
Blood Sample
1. Clearly labeled samples of clotted blood in sterile plain tubes are best for ABO and Rh tests. The
sample can be stored at 4°Cand should be tested within 48 hours. No sign of heamolysis should
be there.
2. If serum not completely separated or become clear, centrifuge the blood sample at 1000-3000
rpm for 3 min.
3. One to two milliliters of serum is pipetted into a pre-labeled tube (identification double checked)
for serum grouping and other tests.
4. Take about 1 ml of cells into pre-labelled tube (identification double checked) and add 0.9% of
normal saline and mix. Wash the cells thrice with normal saline and make 2-5% cell suspension
in normal saline.
ABO typing should be performed using the manufacturer’s directions. There are three basic techniques:
1. Slide test
2. Tube sedimentation
3. Spin-tube.
Slide testing
It is used for emergency purpose ABO grouping for preliminary test. Slide test is not recommended as a
routine test because it is not reliable for weakly reactive antigens on cells and for serum typing tests
with low titre anti-A and anti-B. This is so because the slide test is less sensitive than the tube test. The
other disadvantage is that drying of the reaction mixture can cause aggregation of cells that may be
misinterpreted as agglutination.
Method
1. The slide test may be performed on any non-porous clean surface, but it is usual to use glass or
ceramic opaque tiles or microscopic slides.
2. Put one drop of anti-A serum and one drop of anti-B serum separately on the labeled plate or
slide.
3. Add drop of about 20% red cell suspension to each drop of typing serum.
4. Mix the cells and reagent using a clean stick. Spread each mixture evenly on the slide over an
area of 15-mm-diameter.
5. Rock-rotate the slide or plate and leave the test for 2min at room temperature (20°-24°C). Then
rock again and look for agglutination.
6. Record the results.
Tube testing
The tube technique is recommended because it is easy to perform and advantageous because the
centrifugation involved enhances the reaction, allowing weaker antigens/antibodies to be detected and
because the contents can be protected from drying and smaller amounts of reagents are required. Ideally
cell typing and serum typing should be performed separately by different workers who check each
other’s results by a ‘call-back procedure’. Tests in which discrepancies are identified, must be repeated.
However, in smaller blood banks one trained worker is likely to perform all tests and discrepancies
normally do not occur.
Cell grouping
Method
1. Prepare an appropriate 2-5% cell suspension in normal saline .It is advisable to wash the cells 2-
3times in saline ,especially in case of cord blood or specimens in poor conditions
2. Set up three rows of clean test tubes and label them. Add two volumes (drops) of anti-A in the
tube labeled A two volumes (drops) of anti-B in tube labeled B and two volumes (drops) of anti-
AB in tube labeled AB.
3. Add one volume (drop) of test cell suspension in each tube.
4. Mix the contents of each tube by gentle shaking and leave at room temperature (20°-24°C) for
30-60 min. in urgent cases, centrifuge the tubes at 1000 rpm for one minute after 5-10 min
incubation at R.T. (spin tube method).
5. Observe the supernatant fluid for the presence of heamolysis against a well-lighted background.
6. Gently disperse the cell button and check for agglutination against a well-lighted background.
7. Where no agglutination is seen macroscopically examine the contents under microscope.
8. Record the results immediately. Table 4.5
Serum grouping
Use a similar tube technique to test patients’/donors’ serum against saline suspensions of pooled 2-3
samples of group A cells, B cells and O cells.
Method
1. Label three tubes A cell, B cell and O cell.
2. Place two volumes (drops) of the test serum in each tube.
3. Add one volume (drop) of A cells to tube labeled A, one volume (drop) of B cells to tube labeled
B and one volume (drop) of O cells to tube labeled O.
4. Mix the contents of each tube by gentle shaking and leave at R.T. for 30-60 min. in urgent cases,
centrifuge after 5-10 min incubation at R.T. (spin tube method).
5. Observe the supernatant fluid for the presence of heamolysis against well lighted background.
6. Gently disperse cell button and see for agglutination.
7. All negative results must be examined under microscope.
8. Record the results immediately. (Table 4.5)
Reaction of test red cells with Reaction of serum with pooled cells Interpretation
(blood group of test cells)
Anti-A Anti-B Anti-AB A B O
+ + + - - - AB
+ - + - +/H - A
- + + +/H - - B
- - - +/H +/H - O
- - - +/H +/H + Oh
Controls
It is generally unnecessary to run controls each time ABO grouping is performed; however, reagent anti-
sera and cells used should be controlled on a daily basis as a part of routine quality control programme.
There are several sub groups of A, as already described but the main division is into A1, A2, A1 B and
A2 B. Eighty per cent of group A and AB blood will be agglutinated by anti-A1 serum and are classified
as A1 and A1B respectively. Those negative with anti-A1 are classified as A2, A2 B.
Anti-A1 reagents can be obtained from three main sources:
1. Human anti-A1 (prepared by absorption of group B anti-A serum with A2 cells).
2. The lactin (plant agglutinin) Dolichos biflorus which is specific for A1 antigen.
3. Anti-A1 found as a naturally occurring antibody in the serum of A2 and A2 B individuals.
Method
1. Place 1 volume each of Anti-A1 reagent into three clean test tubes.
2. To the first add 1 volume of 2% saline suspension of the donor’s / patient’s red cells.
To the second tube add 1 volume of known A cells. To the third tube, add 1 volume of known A2
cells (A1 and A2 cells as positive and negative controls should always be included).
3. Mix the contents of each tube by gentle shaking and leave at room temperature for 30-60 min. In
case of emergency centrifuge the tube after 5-10 min. at 1000 rpm for 1 min.]
Read results and record.
Reaction of red cells with anti - sera Reaction of serum with cells interpretation
Anti-A Anti-B Anti-AB Anti-A1 A B O
+4 - +4 +4 - +/H - A1
+4 - +4 - - or +/H* +/H - A2*
+4 +4 +4 +4 - - - A1 B
+4 +4 +4 - - or +/H* -/H - A2B*
Reading
Gently shake the tube and examine. Those showing agglutination are labeled as A1 and A1B and those
not showing agglutination as A2 and A2B.
STEP-I
1. Label a row of test tubes, according to the serum dilution, usually 1:1 through 1:152.
2. Deliver 0.1 ml or 1 volume of saline into all tubes except the first tube.
3. Add 0.1 ml or 1 volume of serum to tubes 1 and 2 (dilution 1:1 and 1:2).
4. Mix the contents of tube 2 with a clean pipette and then transfer 0.1 ml or 1 volume from the
mixture to tube 3 (1:4 dilution).
5. Continue the same technique, through all dilutions and remove 0.1 ml or 1 volume from the
dilution tube with dilution of 1:152 and discard or save for further dilution if required.
DILUTION METHOD
STEP-II
6. Add 0.1 ml or 1 volume of 2-5% saline suspension of appropriate red cells to each tube.
7. Incubate in the appropriate manner according to the antibody being tested. In case of anti-A and
anti-B, incubate at room temperature for 60 min.
8. Gently dislodge the red cells and observe macroscopically for agglutination. The agglutination
titre is recorded as the reciprocal of the highest serum dilution in which there is (W)
agglutination. Thus a serum which gives weak (W) agglutination at 1:256 is said to have a titre
of 256. in describing the titre of the serum, it is usual to ignore the diluting effect of the cells
suspension.
AVIDITY
Speed and strength of agglutination is termed as avidity. The test is done by mixing one drop of anti-
serum with one drop of 10% cell suspension on a slide or title and rocking gently at room temperature
(RT). The time for a clearly visible reaction (+1) and then for strong (+4) reaction to occur is recorded
with the help of a stopwatch.
These antibodies are highly specific and react equally well at 20°C as well as at 37°C and are reliable
for slide for rapid test tube technique.
The same method has been used to produce anti-A, anti-B, anti-AB, etc.
Rh Grouping
Procedure
Rh (D) typing should be performed according to the instructions given by the manufacturers of the
anti-sera or as described below, There are three methods:
1. Slide test
2. Tube sedimentation
3. Spin tube
The blood sample should be collected with or without anticoagulant in a sterile tube and stored at 4°C. it
should be tested within 48 hours. No sign of heamolysis should be there.
Slide Testing
This may be used for emergency Rh (D) typing if centrifuge is not available. The slide test is not
recommended for routine tests because is it not reliable especially for weakly reactive cells and it also
has the disadvantage that driving of the reaction mixture can cause aggregation of the cells that may be
misinterpreted as agglutination.
Method
1. Place one drop of reagent anti-Rh (D) on a labeled slide.
2. Place one drop of control medium or 22% albumin on another labeled slide.
3. Put one drop of 40-50% red cells suspended in plasma or serum on both the slides.
4. Mix the cell suspension and reagent, using a clean stick for each slide and spread the mixture
evenly on the slide over an area of 15 mm diameter.
5. Place both slides on a view box surface (lighted), tilt gently and continuously for two minutes.
Observe for agglutination.
Interpretation
A positive test has agglutination with anti-Rh (D) in the ‘test’ and smooth suspension of the cells in the
control. A negative test has a smooth suspension of cells in both the ‘test’ and control. If there is
agglutination in the control, the test results must be considered invalid and the test with saline-reacting
anti-D must be performed.
Tube testing
Method
1. Place 1 drop of anti-Rh (D) serum in a tube labeled ‘test’.
2. Place 1 drop of control diluting reagent or 22% albumin in a tube labeled control.
3. Add 1 drop of 2-5% cell suspension in plasma or serum in each tube.
4. Mix well and keep at 37°C for one hour (sedimentation method).
5. Gently re-suspend the cell button and observe for agglutination. All negative results must be
confirmed under microscope.
Interpretation
A) The interpreted result may be false positive in the following causes:
i) The anti-Rh (D) used may contain antibodies of other specificities in addition to anti-Rh (D).
Some workers routinely perform the test in duplicate using antisera from two different
sources.
ii) Immunological coating of the patient’s cells of factors in patient’s serum causing cellular
aggregation can cause false agglutination in the control tube containing immunologic inert
reagent. Serum factors can be washed by washing the cells in normal saline. If after washing
the agglutination persists in control test, then it is most likely the cells are coated with
immunoglobulin. In such cases the cells are tested with saline reactive antiserum.
iii) Antisera and regents may be contaminated with bacteria, foreign substance or another
antiserum.
iv) Poly-agglutinable red cells may cause false agglutination with any reagent containing human
serum because the antibodies anti-T and anti-Tn which agglutinate these surface altered cells
are present in most adult human sera.
B) The interpreted results may be false negative in the following cases:
Saline agglutinating anti-Rh sera should be used when the control of high protein anti-D gives a positive
test that persists even when washed red cells are used. Cells giving a positive direct antiglobulin test can
usually be tested with saline-reacting anti-D serum. These sera are not suitable for the slide test. They
are also not suitable for D* test as IgM antibody generally reacts poorly in the indirect antiglobulin test.
Method
1. Place one drop of saline reactive anti-D in a properly labeled ‘test’ tube.
2. Add one drop 2-5% cell suspension of well-washed red cells with normal saline.
3. Mix gently and incubate at 37°C usually for 15-30 min.
4. Centrifuge usually 1000 rpm for 1 min.
5. Gently re-suspend the cell button and observe for agglutination. Negative result must be
confirmed under microscope.
Note: A saline suspension of known Rh (D) positive (O R1 R2) and Rh (D) negative (Orr) cells should
be run parallel with the test as controls. Ensure that the concentration of cells in the controls is
comparable to that of the test cells. Observe the controls before reading the test.
Interpretation
Agglutination in the test indicates that the red cells are Rh (D) positive. The result is valid only if the
results of controls are satisfactory.
These reagents work equally well at 20°-37°C and are reliable for emergency Rh (D) typing by slide or
immediate spin tube technique besides the routine sedimentation tube technique.
Methods
Methods for Rh (D) typing with monoclonal regents are the same as described for anti-Rh (D) sera for
slide or rapid tube test, or saline-reacting anti-D tube method.
Controls
Testing for D
Cells of low-grade D possess the D antigen but expressed so weakly that they are not directly
agglutinated by most anti-D sera D can be detected by antiglobulin test. (IAT)
All anti-D sera, especially anti-D sera for saline tube test and IgM monoclonal sera are not suitable for
testing as IgM react poorly in the IAT. The manufacturer’s package insert will tell if the reagent can be
used for D testing. Polyclonal anti-D (human IgG anti-D); IgM and IgG anti-D monoclonal reagent, and
IgM anti-D (human IgG anti-D) can be used to detect D by IAT.
Method
1. Take one drop of anti-Rh (D) serum in a clean labeled ‘test’ tube.
2. Take one drop of appropriate control reagent in labeled tube.
3. Add 2-5% of cell suspension to be tested to both the tubes.
4. Mix and incubate both the tubes at 37°C for 15-30 min.
5. Centrifuge at 1000 rpm for 1 min.
6. Gently re suspend the cell button and examine for agglutination. If there is strong
agglutination of cells in ‘test’ tube, then sample is Rh (D) positive and there is no need to
proceed with antiglobulin phase of test.
7. If no agglutination or doubtful reaction observed, wash the cells 3-4 times with saline can
decant the last washing.
8. Add 1-2 drops of anti-globulin reagents (Coombs serum). Mix gently and centrifuge at
1000 rpm for 1 min.
9. Resuspend the cell button gently and examine for agglutination and record the test result.
10. If the test is negative, the reaction may be confirmed by adding known IgG sensitized
cells, re centrifuge and re-examine for agglutination. The presence of agglutination
confirms the test result.
Interpretation
Agglutination in the ‘test’ tube and none in ‘negative control’ tube constitutes a positive test result and
the blood is accordingly labeled D”. if the negative control test is positive no valid interpretation of D
test is made.
A best general control for the DAT / DCT and IAT / ICT is the addition of IgG sensitized OR h (D)
Positive cells to any AHG test that are non-reactive. A positive result indicates the following.
1. The AHG reagents has remained active and not been neutralized.
2. The red cells have been adequately washed.
3. The final volume of saline did not exclusively dilute the AHG serum.
4. AHG reagent was added to the test tubes.
Preparation of O Rh (D) Positive Sensitized cells
Procedure
1. Select a dilution of anti-Rh (D) sera that costs the O Rh (D) Positive cells at 37°C in vitro but
does not agglutinate them one has to determine by experience to what extent anti-D serum
should be diluted to give sensitized cells (No agglutination).
2. Add 5% washed cell suspension of O Rh (D) positive cells equal to the volume of diluted anti-D
serum.
3. Mix, incubate at 37°C for 30 minutes.
4. Look for the agglutination (if there is agglutination the procedure is repeated by taking more
diluted anti-D serum).
5. If there is no agglutination wash the cells three times with a large volume of saline. Decant the
supernatant saline completely every time. Make 5% suspension of sensitized cells in saline.
6. Add 1 drop AHG serum to 1 drop of the 5% washed sensitized cells.
7. Mix, spin immediately at 100 rpm for 1 min.
8. Cells should show +2 agglutination (if there is no agglutination the whole procedure in repeated
by taking less diluted anti-D serum).
Procedure
1. Place one drop of 2-5% cell suspension to be tested in a clean labeled tube.
Note: The clotted sample should be as fresh as possible (not more than 24 hold) other wise the
sample may be taken in EDTA to prevent the uptake of complement in vitro).
2. Wash the red cells 3-4 times in a large volume of saline care should be taken for adequate
removal of the supernatant after each wash. Completely decant the final supernatant wash.
3. Add 1-2 drop of poly specific AHG serum immediately.
4. Mix centrifuge at 1000 rpm for 1 min.
5. Gently shake the tube to dislodge the cell button and examine for agglutination using an optical
aid and record the result.
6. Leave an apparently non-reactive test tube at room temperature for 5 min. Centrifuge and read
again. Though this step is optional all manufacturers recommend it when maximum sensitivity
for complement or IgG is desired. The additional step should be never be substituted for the
immediate reading because reaction due to IgG coating may become weaker after incubation.
7. Add one drop of IgG coated red cells to any test that is non-reactive mix, centrifuge at 1000 rpm
for 1 min. look for agglutination if a negative result (no age) is obtained the test result is invalid
and whole test should be repeated.
INTERPRETATION
DAT is positive when agglutination is observed either after immediate spin or after spin following RT
incubation.
DAT is negative when no agglutination is seen at either phase provided IgG coated red cells are added in
step 7 of test.
A negative DAT does not necessarily mean absence of coating globulin poly specific reagents detect
approximately 500 molecules of IgG per red cell but auto-immune haemolytic anaemia has been
reported with an IgA coating below this level.
Procedure
1. Place two or four drops of the test serum in a tube (sample should be fresh for detecting
complement-binding antibodies otherwise fresh AB serum should be added to it).
2. Add one drop of 4-5% suspension of washed cells (i.e. donors / patients red cells or screening
red cells etc)
3. Mix and incubate at 37°C for 30-60 min.
4. Centrifuge at 1000 rpm for 1 min
5. Examine for haemolysis or agglutination using an optical aid; record results. Agglutination at
this stage indicates the presence of saline reacting antibodies.
6. If no agglutination is seen wash three or four times in large amount of saline Decant supernatant
in each wash as completely as possible.
7. Add 1 – 2 drops of AHG serum.
8. Centrifuge at 1000 rpm for 1 min.
9. Gently shake the tube to dislodge the bottom and examine for agglutination using an optical aid.
10. Leave an apparently non-reactive test at RT for 5 min centrifuge and read again as in DAT.
11. Add 1 drop of IgG coated red cells to any test that is non-reactive. Mix, centrifuge at 1000 rpm
for one minute. Look for agglutination if a negative result (no agglutination) is obtained the test
result is in valid and the whole test should be repeated.
Interpretation
IAT is positive when agglutination is observed either after immediate spin or after spin following RT
incubation.
IAT is negative who no agglutination is seen at either phase, provided IgG coated cells are added in step
2 of IAT test.
Temperature
Most IgG antibody and red cell reaction occurs optimally at 37°C. incubation at lower temperature
decreases the rate of association between antigen and antibody, while incubation at higher temperature
may damage red cells or antibody molecules
Incubation time
For saline or albumin techniques 30 min incubation at 37°C is adequate to detect most of the clinically
significant coating antibodies. For some weak reactive in LISS medium incubation time is 10-15 min.
Suspending Medium
The sensitivity of IAT can be increased with the addition of 22% BOVINE ALBUMIN or ENZYME or
LISS.
Procedure
ENZYME-IAT
Enzymes after the configuration of erythrocyte surface and increase the mobility of antigen and
clustering of the antigenic site, which increase the chance of effective antigen antibody collisions.
Besides they reduce the negative charge on the red cell surface. For details see chapter on screening and
identification for antibodies.
Procedure
Mostly one stage method with papain cystein is conducted for determining antibodies by enzyme-IAT.
1. Add one drop of papin cystein solution in step 2 of saline-IAT
2. Incubation at 37°C for 30 min
3. Proceed further as step 4 in saline-IAT
Note: For the preparation of papain-cystein solution sec chapter on special methods.
Methods
Stage-I
Serum 1 – 2 drops
Cell suspension 2 – 4 % 1 drop
Bovine albumin 22% 2 drops
Mix and incubation 37°C 15-20 minutes and centrifuge at 1000 rpm for 1-2 min.
Gently re suspends the cell button and observes agglutination. Control all negative result under m/s
Stage-II
Serum 2 – 3 drops
2 – 4 cell suspension 1 drop
Mix at 37°C incubate for 30 min
Centrifuge at 1000 rpm at 1 – 2 min
Without disturbing allow two drops of 22% albumin bovine inside of the test tube.
Incubate at 37°C for 10 – 20 min.
Gently re suspend the cell button and observe agglutination / haemolysis.
UNEXPECTED ANTIBODIES
These antibodies are screened and detected in pre-transfusion testing of patient’s blood and during
antenatal care in mother’s blood.
If the unexpected antibodies are detected during screening test or compability tests the technologist has
two choices;
1. To identify the antibody (antibodies) and select blood that lacks the corresponding antigens (s)
for cross-matching.
2. To perform cross-matching using several other units an attempt to find units that fail to react
with the recipient’s serum. These units are regarded as compatible and issued for transfusion.
3. These second course of action is much less desirable than the first and should be taken in
emergency only.
Screening of Antibodies
The candidates of antibody screening are
i) Patients requiring a transfusion.
ii) Donors blood
iii) Antenatal patients
Donors Blood
Every donor’s blood should be screened for antibodies. The presence of antibodies in donor’s blood may
cause a mild transfusion reaction of decrease of patient red cells.
Antenatal patients
All antenatal patients should be screened for a antibodies besides anti Rh (D), other antibodies also
which could be problem for both the mother and the child.
Early detection of an antibody will give time to the pediatrician and the blood bank to prepare for
intrauterine or exchange transfusion in the baby or for an emergency transfusion to the mother.
All the screening cells are group ‘O’ it should be remembered that ABO in compabiilty will not be
detected unless a cross-match is also carried out. Thus cross matching and antibody screening both are
every necessary.
To maintain the optimum strength of the antigens the two cells OR1 R1 and OR2 R2 should be used
separately (and, of course, in conjunction with each other) and not pooled. This will increase the chance
of detecting the weak antibodies.
Cell panels are commercially available in developed countries; how ever they can also be prepared by
the individual laboratory institutions that prefer to prepare their own panel, should determine the full
phenotype of individuals on staff and bleed them regularly, or freeze large donations from these
individuals in glycerol. For freezing or red cells in glycerol see chapter on storage and preservation of
blood.
1. Saline test
1. Set up 2 tubes of (size 10x75 mm)
2. Add 2 drops of patients serum to each tube
3. Add 1 drop of screening cells to each appropriate tube
4. Mix and incubate at RT (20-25°C) for 1 hour.
5. Shake the tubes gently and read microscopically negative readings should be checked
microscopically.
6. Result should be scored and recorded.
7. Any haemolysis must be noted as this indicates and positive result.
2. Enzyme test
Procedure
1. Set up 2 tubes.
2. Place 2 drops of patient’s serum in each tube.
3. Add one drop of papin cystein to each tube.
4. Add one drop of screening cells to each tube.
5. Mix well and incubate at 37°C for 1 hour.
6. Shake the tubes gently and read microscopically negative readings should be scored and
recorded.
7. Result should be scored and recorded.
Note: Any haemolysis must be noted as this indicates agglutinations for preparation of papain cystein
solution, see chapter on special methods.
Procedure
1. Setup 2 tubes
2. Place 2-4 drops of patient’s serum in each tube.
3. Add 1 drop of screening cells to each tube.
4. Mix and incubate at 37°C for 30-60 min.
5. Examine for agglutination and for haemolysis and record the result.
6. If there is no agglutination or haemolysis. Wash the contents of both tubes 3 – 4 times in large
volume of saline Decand each wash the completely as possible.
7. Add 1 – 2 drops of antiglobulin serum to each tube.
8. Mix well and centrifuge at 1000 rpm for 1 min.
9. Gently shake the tube to dislodge the cell button and examine for agglutination / haemolysis
macroscopically negative readings should be checked microscopically.
10. Add 1 drop of IgG anti-D coated cells in non-reactive tests. Mix and centrifuge at 1000 rpm for 1
min and examine for agglutination. This acts as control for AGT. If a negative result no
agglutination) is obtained the result is in valied and whole test should be repeated.
ANTIBODY IDENTIFICATION
Before beginning the antibody (ies) identification, it is use full to review the following.
1. Medical history-diagnosis.
2. History of transfusion or pregnancy.
3. Drug therapy (including Rh immunoglobulin)
4. Result of previous testing.
1. Saline test
Purpose: The saline test at RT is used to identify IgM agglutinations, namely anti-M, anti-N, anti-Lea,
anti Leb, anti-I, anti-H, anti-P, etc.
Procedure:
1. Setup the appropriate number of (10x75) test tube for panel cell, auto control, two cord cell)
2. Place 2 drops of patient’s serum in each tube.
3. Add 1 drop of Panel cell, patient’s cell suspension for auto control, two group O cord cell
suspension [one Rh (D) + one Rh (D) - ] to each appropriate tubes.
4. Mix and incubate at RT (20-25°) for 1 hour
5. Shake the tube gently and read macroscopically negative results should be checked
microscopically.
6. Result should be a cored and recorded.
7. Any haemolysis must be noticed as this indicates agglutinations many lewis antibodies produce
haemolysis in saline when fresh serum is used.
Note: The above procedure can be performed at a lower temperature (4°C) which tends to enhance the
reaction of many cold antibodies.
It auto agglutination is due to cold agglutinings, reaction will commonly be negative at 37°C. if the
serum fails to react against the cord cells. There may be anti-I.
ENZYME TEST
Purpose: Enzyme tests are useful for the identification of warm reacting (IgG or complement binding)
allo-and auto-antibodies. This technique serves to enhance the reaction of Rah Lewis and kidd
antibodies. Enzymes may weaken or inactivate certain antigens like M.N.S. Fya and Fyb.
Method
1. Set up appropriate number of 10x75 mm tubes.
2. Place 2 drops of patient’s serum in each tube.
3. Add 1 drop of papain cystein solution to each tube.
4. Add 1 drop of panel cell, patient’s cell suspension for the auto control and 2 group ‘O’ cord cell
suspension (ORh (D) + ORh (D)-) to each appropriate tubes.
5. Mix well and incubate at 37°C for one hour.
6. Shake the tube gently and read macroscopically. Negative reading should be checked
microscopically.
7. Result should be scored and recorded.
Procedure
1. Set up appropriate number of tubes. (for pond cells, auto control, 2 group ‘O’ cord cells)
2. Place 2 – 4 drops of patient’s serum in each tube.
3. Add one drop of panel cell, patient’s washed cell suspension for the auto-control and 2 group O
cord washed cell suspension {O Rh (D) +, O Rh (D) -} to each appropriate tubes.
4. Mix and incubate at 37°C for 30-60 min.
5. Examine for agglutination and haemolysis record the result.
6. If there is no agglutination or haemolysis, wash the contents of each tube 3 to 4 times in large
volume of normal saline; decant wash as completely as possible.
7. Add 1 – 2 drops of antiglobulin serum to each tube.
8. Mix well.
9. Centrifuge at 1000 rpm for 1 min.
10. Gently shake the tube to discord the cell button and examine for agglutination and / or
haemolysis macroscopically negative reading should be checked microscopically.
11. Add 1 drop of IgG anti-D coated cells in non-reactive tests: Mix and centrifuge at 1000 rpm for
1 min and examine for agglutination. This acts as control for AGT. If negative result (no
agglutination) is obtained the result is invalid and whole tests should be repeated.
Interpretation of result
To define the antibody (ies) that has been found. The result of the three techniques must be looked at
individually and then as whole. By what techniques do reactions occur? For identification cross out all
the antigenic determinates occurring on the panel cells that did not react with test serum beginning with
the first cell and proceeding to the end of the panel see examples 9.1 and 9.2
Interpretation
Agglutination or haemolysis indicates a positive result (incompatible)
Note: Immediate spin test is acceptable but the incubation improves sensitivity of the test if the recipient
has weakly reactive anti-A or anti-B or if the donor’s red cells have weak expressions of an antigen eg
A2 B red cells.
Method
1. Put 2 drops of patient’s serum in a labeled tube
2. Add 1 drop of 2-4% saline suspended red cells of donor.
3. Incubate for 30 – 60 min at 37°C.
4. Centrifuge at 1000 rpm for 1 min. check for haemolysis/agglutination.
5. Wash the cells three times with normal saline.
6. Perform AHG test.
7. Add IgG coated red cells to negative AHG test.
8. Centrifuge and check for agglutination-if there is no agglutination tests in valid.
For details see chapter on AHG test.
ENZYME TECHNIQUE
For method see chapter special methods
ALBUMIN TECHNIQUE
For method see chapter on special method.
Issue of blood
1. Cross match result should be sent along with the blood to be issued. The cross match report form
must include.
Note: If blood issued before compability test are completed label should clearly state uncross matched
blood.
Immunological effects
2. Technical Errors
i) Error in blood grouping and cross-matching.
ii) Incompatibility not detected in cross-matching due to improper method.
iii) Weak antibodies not detected by routine tests.
iv) Destruction of recipient red cells. By the donor antibodies.
It occurs when donor blood has antibodies against antigen (S) in recipient.
It is not serious as donor antibodies are diluted. It occurs in the transfusion of group ‘O’ blood to group
A1 group B (or) group AB recipients. This usually results from the indiscriminate use of group ‘O’
blood which has to been screened to ascertain the anti-A and anti-B titre and haemolysis.
1. SHOCK PHASE
Fever, chill
Byring sensation at the site of transfusion
Pain in chest, lumbar region of back
Haemorrhage
Shock
3. ANURIC PHASE
Renal failure
Oliguria
Anuria
Uraemia
4. RECOVERY PHASE
Patient phases large amount of urine. In the beginning urine is glomerular filtrate with a low
concentration of urea. After some days the tubular functions return and diuresis may have effect on
blood urea and creatinine. There may be excessive loss of potassium.
In case of suspected transfusion reaction-the transfusional must take the following actions
1. Stop transfusion and inform the patient’s physicians.
2. Keep the intravenous line open with infusion of normal saline or other suitable
intravenous infusion.
3. Recheck all labels, forms and identity of the patient to determine if the patient received
the correct blood or component.
4. Send the fresh blood sample of the patient, care fully drawn to avoid mechanical
haemolysis, to the having remaining blood or component, the administrative set attached
and all related labels and form.
5. Preserve the urine passed after transfusion to check the presence of haemoglobin
produced by the lysis of red cells.
Laboratory investigations
1. Check identity of the patient donor blood and all relative and all relevant papers to ensure that
there was no clerical error.
2. Compare the patients are and transfusion specimen for the colour of serum of plasma.
i) Pink or red discolouration in post-transfusion sample indicates the presence of free
haemoglobin due to the destruction of red cells.
ii) Yellow or brown discolouration in samples drawn 4-10 hours after the transfusion
indicates increased bilirubin.
3. Repeat ABO Rh (D) testing in the patients pre-and post transfusion blood sample, blood from the
bag or from a segment of the table still attached to the unit, to check any error in ABO and Rh
(D).
4. Direct antiglobulin test
i) If direct AGT is negative the red cells are not coated with IgG antibody there is no in
compability.
ii) If antibody coated donor-in compatible cells are not immediately destroyed the direct
AGT on the post reaction sample will be positive.
iii) If the patients blood sample is drawn after several hours of the suspected reaction the
antibody coated donor-in compatible cells are destroyed. The direct AGT will be
negative.
iv) In non-immunologic reactions the direct AGT will be negative.
DEFINITION
• Mole, gram-molecular weight: weight in grams of a substance is equal to the molecular weight
of a substance.
• Molar solution: a one molar (IM) solution contains one mole of solute in a titre of solution. The
solvent is generally distilled water unless other wise indicated.
• Normal solution: a one normal (IM) solution contains one gram-equivalent weight of solute in a
liter of solution.
• Gram equivalent weight: weight in gram of substance which will produce or react with / more of
hydrogen ion.
• Percentage solution: the percentage of a solution gives the weight or volume of solute in 100
units of total solution.
Normal Solution
1N NaoH
Molecular weight of NaoH = 23+1+16 + 40 g
1N NaoH requires 1x40 = 40 g of solution (NaoH)
Made up to 1000 ml of D/W. one mole of NaoH dissociates with one mole H; so grame equivalent
weight and gram molecular weight are same.
Percent Solution
0.9% of Nacl required 0.9 gm solute made up to 100 ml of D/W.
Normal Saline
Saline for use in blood group serology should have Nacl 9 gms/L D/W.
Nacl-9 gms
D/W mark to 1 liter
pH 5.5 – 8.0
Prepare working buffer solutions of the desired pH by mixing appropriate volumes of the two solutions.
A few examples are.
pH Solution-A Solution-B
7.0 32 ml 68 ml
7.2 24 ml 76 ml
7.4 18 ml 82 ml
7.6 13 ml 87 ml
7.7 9.5 ml 90.5 ml
Blood Sample
1. Fresh blood or free flowing capillary blood added to any solid anticoagulant (1 mg EDTA
1 ml) can be used. Measurement can be carried on blood which has been stored at 4°C.
2. Fresh capillary blood can also be used if added immediately to reagent solution.
Reagent (Diluent)
Modified Drabkin’s reagent
Potassium ferricyanide – 200 mg
Potassium cyanide – 50 mg
Potassium dihydrogen phosphate – 140 mg
Nomidet P40 (shell chemical co) 1 ml
D/W – 1 liter
Other non ionic detergents which can be used in place of nomidet include sterox SE (Harleco) 0.5 ml
Triton X-100 (Rohm and Haas) 1ml and saponic 218 (alcoac Inc)
1 ml pH should be 7.0 – 7.4.
The reagent should be clear and pale yellow in colour when measured as blank in a photometric
calorimeter at a wave length of 540 nm, the absorbance must read zero.
Method
1. Switch on the photoelectric calorimeter and wait for 15-20 min to warm up before use.
2. Add 20 µl of blood to 5 ml of diluent stopper the tube containing the solution and invert
it several times. Allow to stand at RT for 5-10 min to ensure the completion of the
reaction. This solution of HICN is ready to be compared with standard.
3. Select filter of wavelength 540 nm.
4. Set the colorimeter at zero against blank
5. Measure the absorbance value of the test solution prepared as in step 2.
Interpretation
1. Record the absorbance value directly in the calorimeter calibrated for direct reading of Hb% (or
mg/dl)
2. If the calorimeter is not meant for taking direct reading of haemoglobin g/dl record the
absorbance reading and haemoglobin can be calculated from the following formula.
g/dl of Hb = OD of the test / OD of the std x conc. of std g/dl
Precautions
1. Blood should not be clotted.
2. The reagent should be discarded, if it becomes turbid.
3. The mixture of blood and reagent should be clear turbidity is due to contamination and give false
result.
4. Pipette should be accurate to take 20 µl of blood.
5. Standard solution should be discarded at the end of day on which ampoule in opened.
Methods
Methods
The 2 stage technique is used for detection and the one stage technique issued for detection and
identification of IgG antibodies and for cross matching also the two stage techniques is more sensitive
than the one stage technique. In one stage technique protealytic effect of enzymes inhibited by the serum
the method in which one volume of serum is placed in a test tube one volume of enzyme solution (low)
is layered carefully on top of the serum mixing between enzymes and serum. The tube one incubated for
1 hour and the cells then examined for agglutination.
b) If desired this suspension can be centrifuge dafter storage for 24 hours at 4°C with occasional
agglutination. The clear supernatant is slightly less active than the original suspension.
c) For use 100 ml of the stock solution is added 109 volumes of M/15 phosphate buffer, pH 7.3
prepared by adding 3 volumes of M/15 Na2 HPO4 (9.46 g/l) to 1 volume of M/15 – M – KH2
PO4 (9.07 g/l)
ii) Preparation of working solution (pH 6.2) at the time of preparing papain cystein reagent.
Na2HPO4 KH2PO4
1 Volume 4 Volumes for pH 6.2
4 volumes 1 volume for pH 7.4
0.4 volume 9.6 volumes for pH 5.4
Methods
1. Take 1 volume serum in a test tube
2. Add 1 volume of papain cystein reagent.
3. Add 1 volume of 2% cell suspension
4. Incubate at 37°C for 1 hour
5. See for agglutination (read microscopically)
Thus the rate and degree of antibody uptake is increased 2 to 4 folds in comparison with normal saline.
However all antibodies are not equally response to LISS solution (anti-A and anti-B usually remain un
effected.
1. 18 g of glycine is dissolve in about 500 ml of distilled water (sodium glycinate is not available
commercially).
2. The pH is adjusted to 6.7 by dropwise addition of 1 N NaoH.
3. 20 ml of phosphate buffer (0.15) pH 6.7 as added to the glycine solution.
4. 1.79 g of Nacl dissolve in 100 ml of distilled water is added to the solution.
5. The solution is made up to 1 liter with distilled water, mix thoroughly
6. Adjust pH to 6.7 with 1N NaoH
7. Dispense into 100 ml amounts.
It is sterilized by seitz filtration or autoclaving for storage at 4°C stored at 20°C to avoid pacterial
growth. Alternatively a low conc. of sodium azide-0.1 g/l may be added.
Quality Control
A) Non-Serological
1. pH should be within the range 6.65-6.85
2. conductivity should be 3.6-3.7 mmol/cm at 23°C
3. Osmolarity 270-285 mmol.
Serological
A weak IgG anti-D (0.25 iu/ml) should give a +/++ reaction with R1 red cells by the routine LISS-ANG
test. This examination should be carried out in paralled test using the previous batch of LISS.
Method (LISS-IAT)
1. Wash the red cells twice in normal saline
2. Wash there cells one in LISS
3. Make 2-3% cell suspension LISS.
4. Take equal volume of serum and LISS suspended cell in a tube.
5. Incubate at 37°C for 15 min in routine and 5 min in emergency.
6. Centrifuge examines the supernatant for haemolysis resuspend the cells and observe for
agglutination. Grade and record results.
7. Wash the cells 3 times in large amount of normal saline decant supernatant the each wash as
completely as possible.
8. Add 1-2 drops of AHG serum
9. Centrifuge at 1000 rpm for 1 min (500 g for 1.5s)
10. Gently shake the tube to dislodge the solution N and examine for agglutination using optical aid
and record the result.
11. Leave atapparently non-reactive test at room temp for 5 min centrifuge and read again as in
DAT.
12. Add 1 drop of IgG coated red cells to any test that is non-reactive mix centrifuge at 1000 rpm for
1 min. look for agglutination if a negative result (no agglutination) is obtained the test result is
invalid and few hole test should be repeated.
Interpretation
It is same as in DAT or IAT
Haemolysis or agglutination is a positive result.
Precautions
1. For routine work LISS should be used at ambient temp as could LISS straight from 4°C storage
increases un wanted cold antibody reaction.
2. Red cell should be washed 2 in normal saline to free there from serum before adding LISS to
them traces of residual serum will results in non-specific uptake of auto logous serum
complement
3. Equal volume of serum and LISS suspend 2-3% cell should be used.
4. The to be should be shake gently to see agglutination
5. Bottle of LISS in used should be discarded after 48 hours.
Uses of LISS
1. Screening identification and quantitation of antibody.
2. It is useful in emergency cross-matching due to sort incubation time.
3. useful in electine surgery patient serum is kept after doing blood group
4. Antibody screen and cross matching is done on request at the time of operation.
Application
To remove an unwanted antibody from serum for the purpose of antibody identification or to prepare
reagent antisera
Method
1. Wash a large volume of red cells to be used in the absorption, 3 times in normal saline these cells
must possess the antigen corresponding to the antibody that is to be absorbed. And shall lack the
antigen corresponding to another antibody if any that is not to be absorbed shall lack the antigen
corresponding to another antibody if any that is not to be absorbed i.e remain in the serum.
2. After the final wash centrifuge the cell so that they are tightly pached and removed as much of
saline as possible.
3. Divided the cells into two aliquots.
4. Add serum equal to the volume to packed cells in one aliquot.
5. Incubate at the optional temp at which antibody to be absorbed will react to 30 – 60 mm mixing
occasionally.
6. Centrifuge and recover the serum.
7. Test the serum to see that absorption is complete. If not repeat the procedure using another
aliquot of packed red cells always test to ensure that the antibody that is to remain in the serum is
sufficiently reach before continuing with repeat absorption.
1. Wash 1 ml of cells to be tested at least 3 times with saline Discard the supernatant after last
wash.
2. Add 1 ml of anti-A1 to red cells is weak variant of a is suspected or 1 ml of anti-B if weak
variant of B is suspected.
3. Mix the cells with antisera and incubate at room temperature for one hour.
4. Centrifuge the mixture and discard the supernatant antisera.
5. Wash the remaining red cells for a minimum of 5 times with a large volume of salin (10 ml or
more) save the supernatant of the fifth was to test for free antibody.
6. Add an equal volume of saline to the washed and packed cells and mix
7. Elute the absorbed antibody by placing the tube at 56°C water bath for ten min and mix the red
cell saline mixture at least one during this period.
8. Centrifuge and remove the cherry colored elute and discard the cells.
Testing of ELUTE
1. If anti-A was used, test the elute against three different samples A1 cells and 3 group O cells at
room temp at 37°C and with antiglobulin serum.
2. If anti-B was used test the elute against 3 sample of B cells and 3 group fo O cells at room temp
at 37°C and with antiglobulins serum.
3. Test the fifth saline was in the same manner to show that washing has removed all antibody not
bound to the cells.
Interpretation
If the elute agglutinates or reacts with antiglobulin testing with specific A or B cells and does not react
with O cells tested have active A or B antigen or their surface capable of binding with specific antibody.
If the elute also reacts with O cells it indicates non-specific reactivity in elute and the results are not
valid.
If the fifth saline wash material is reactive with A and B cells the results of the test made on elute are not
valid because it indicates that active antibody was present in the medium un attached to the cells being
tested.
Haemolysin test
The haemolysin test detects incomplete IgG, ABO antibodies by their ability to haemolyse appropriate
red cells in the presence of complement, it is not describe. To include substantial amount of such
antibodies in grouping sera because they can complete with the complete (IgM) antibodies and may
inhibit agglutination (prozon effect)
Haemolysin test issued to determine the ability of anti-A and anti-B jin group O subjects to cause the
haemoysin of cells and therefore to determine their safety when group O is transfused out cross
matching or when group is give to an individual of an other blood group.
Method
1. Place 2 drop of fresh serum not more than 24 hold in each of he tubes labeled A and B. if seru is
un avoid by more than 24 hour old add an equal volume of fresh. Human AB serum free from
lysine as a source of complement which is important to reaction.
2. Add 2 drops of fresh 2-5% saline suspension of washed A cells to tube A and 2 drops of B cells
suspension to tube B.
3. Mix gently and incubate of 37°C for 2 hours
4. Centrifuge and examine the supernatant against a well lighted white back ground to detect
haemolysis.
5. Score the degree of lysis according to the intensity of the supernant colour (pink ro red) and cell
button size washed packed red cells.
Note: use of weaker cell suspension or large amount of serum will increase incidence of haemolytic
activity.
Alternative Method
1 volume of 50% suspension of washed A1 or B cells in suspended in 9 volumes of fresh test anti-A or
anti-AB sera the mixture is incubated at 37°C for 2 hours then centrifuge and the supernatant is
inspected for haemolysis.