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Blood Grouping Techeniques
Blood Grouping Techeniques
An error in ABO or Rh grouping of a patient or a crossmatch incompatibility, can have the most serious
effects on the patient and may even be fatal. It is therefore essential to perform all the technical
procedures very carefully and precisely.
In addition, it is very important to use recommended controls with each serological technique used.
Reagents can deteriorate due to improper storage or contamination and such errors can only be detected
by correct application of controls for that reagent.
The various basic serology techniques used in a blood transfusion laboratory are
ABO grouping
Rh grouping
Antiglobulin testing
Antibody detection and identification
Pretransfusion testing
Specialized serology procedures
o Antibody titration
o Saliva testing for A,B and H substance
o Preparation and use of lectins
o Preparation and use of enzymes
o Preparation and use of LISS
o Investigation of autoimmune haemolytic anaemia
ABO Grouping
Aim of this subsection is to make the medical officers understanding the principle of ABO grouping so that
they can perform accurate basic blood grouping test.
Principle
The principle of ABO grouping is based on a specific agglutination reaction between antigens on red cells
and IgM antibodies in the tyuping serum. Cell grouping is determined by testing an individual’s red cells
with known blood grouping reagents and the corresponding serum grouping with known A cells, B cells
and 0 cells.
1. ABO grouping tests should be done at room temperature (2Oo24oC) or lower; testing in hot
environment weakens the raction.
2. Routine ABO grouping must include both cell and serum testing as each serves as a check on the
other.
3. Antisera used in the ABO grouping must be used as per the manufacturer’s instructions.
4. Adequate controls should be put with each batch of tests for quality control of the reagents,
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alternately once a day the reagents must be checked with appropriate cells. Before use all the reagents
must be checked grossly to rule out any turbidity or contamination.
5. Tubes, slides, tiles, microplates or gel cards should be labelled properly. One should not rely on the
colour of the dye to identify the antiserum.
7. Serum should be added before adding cells and each tube should be examined after serum has been
added to ensure that none has been missed.
9. Concave mirror (agglutination viewer) or microscope may be used to examine reactions that appear
negative by the naked eye.
It is essential to use reliable grouping reagents to obtain correct results. Commercially prepared
polyclonal (human source) and monoclonal (tissue culture) antisera are available. Monoclonal antisera
give more specific reactions and are very sensitive for weaker reactions.
Adequate quality control must be done before purchasing commercial antisera. Each batch of antisera
must be checked against A1,A2, (if available, otherwise A group) B and 0 cells before use and should be
put to routine use onlyw hen unequivocal results are obtained.
The potency of any antisera deteriorates rapidly if kept for too long at ambinet temperature, groupign
sera should therefore, be kept at 4°C or as directed by the manufacturer when not in use. Frozen antisera
must be completely thawed before use and no refreezing should be done.
1. Properly labelled samples of clotted blood collected in screwcapped plain test-tube are most suitable
for ABO grouping. Ths amples should be stored at 4°C and preferrably be tested within 48 hours.
3. The blood sample may be centrifuged at 1000-3000 rpm for 3 minutes for adequate serum separation.
4. Separated serum should be placed in a prelabelled tube for serum grouping and other serological tests.
5. Cells from the test sample should be washed in 0.9% nromal saline and a 2-5% cell suspension should
be prepared.
Preparation of reagent red cells in addition to patients’ and donors’ red cells is required daily while
performing the serum grouping and cell grouping.
When performing an ABO serum grouping, it is important to ensure that the cells prepared are fresh and
well-selected
‘A’ cells
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Pool known fresh A group cells from at least 3 donors (to include Al subtype). Wash three times with
normal (0.9%) saline, make a 2-5% suspension in saline for use.
‘B’ cells
Pook known fresh B group cells from 1-2 donors. Wash three times with normal saline, make a 2-5%
suspension in saline for use.
‘0’ cells
Pool known fresh C group cells from 1-2 donors. Wash three times with saline and make a 2% suspension
in saline for use.
Cells are washed in isotonic saline to remove all traces of plasma or serum. They must then be accurately
diluted to give a standard suspension in saline by either using a graduated tube (Wintrobe’s tube) or by
counting drops to make a 2-5% cell suspension.
When prepared, the cells should be checked against anti-A and anti-B. The cells should be kept at 4°C in
the refrigerator when not in use.
If low-ionic strength saline solution (LISS) is being used in the laboratory as an enhancing medium, then
the last wash should be in LISS and the cells are thereafter suspended in LISS to make a 2-3% cell
suspension.
The test cells also should be washed at least once with saline and suspended to make a 2-5% suspension
in saline. When dealing with large number of samples, adequate precautions must be taken to avoid
mislabelling of tubes and the cells must be dispensed in already labelled tube for washing.
Positive Negative
Anti-A A2 (A2B) B
Anti-A Al A2
Anti-B B(Al,B) A2
Anti-AB A2B(A2) 0
Al A2 B AB 0
Anti-A + + - + -
Anti-Al + - - + -
Anti-B - - + + -
Anti-AB + + + + -
Newer techniques
This technique may be used for emergency ABO grouping tests or for preliminary grouping particularly in
an outdoor camp, however it should always be supplemented with a cell and serum grouping using any
one of the other above mentioned techniques.
Slide or tile testing is not recommended for routine use because it is not reliable for
Disadvantages
Procedure
1. Place 1 drop of anti-A and 1 drop of anti-B reagent separately on a labelled slide or tile.
2. Add 1 drop of 20% test red cell suspension to each drop of the typing antiserum (the suspension may
be prepared by adding 20 parts of red cells to 80 part of normal saline).
3. Mix the cells and reagent using a clean stick. Spread each mixture evenly on the slide over an area of
10-15 mm diameter.
4. Tilt the slide and leave the test for 2 minutes at room temperature (22°-24°C). Then rock again and
look for agglutination.
Tube Testing
Test tubes either of glass or plastic may be used, of lOx75mm size. The tube technique is more sensitive
than slide technique for ABO grouping.
- It allows for fairly long incubation without drying up of the tubes’ contents.
- Centrigugation involved enhances the reaction allowing weaker antigens and antibodies to be detected.
- Simplicity of reading and grading of results.
- Clean and more hygienic.
- Requires smaller volume of reagents.
Procedure
Saline agglutination test for cell and serum grouping by tube method
Reagents required
2. Set up 6 tubes correctly labelled with donor/patient no. Anti A, Anti-B, Anti-AB, Ac, Bc and Cc.
4. Add I drop of anti-A in tube labelled A, anti-B in tube labelled B, and anti-AB in tube labelled AB.
5. Add 1 drop of 2-5% test cell suspension in the three tubes A,B and AB.
6. Add 2 drops each of the test serum in tubes labellaed Ac,Bc and Cc.
7. Add I drop each of reagent A cells in labelled tube Ac, B cells in labelled tube Bc and 0 cells in tube
labelled Oc.
8. Mix all the 6 tubes and centrifuge at 1000 rpm for 1 minute.
9. Resuspend cell button by gently shaking the tubes and read against well-lit background.
To record the difference in the strength of reaction, it is necessary to have a system of grading or scoring
the reactions, as depicted in figure.
Grades Description
4+ 1 big clump
3+ 2 or 3 clumps
2+ many small clumps with clear supernatent
1+ many small clumps with turbid supernatent
w granular suspension
Zero or - smooth suspension
partial or complete haemolysis
H
(positive reaction)
Interpretation
Precautions
2. While reading the result, shake very gently to dislbdge the button.
Microplate Technique
Microwell plate consists of a small tray with 96 small wells each of which can hold about 200-300u1 of
reagent. Microplate technology is gaining widespread popularity due to increasing workload in blood
transfusion laboratories and recent availability of packaged automated system.
a. U-type well
b. V-type well
c. Flat-bottom
The U-type well is generally used in red cell serological work as it is easier to read the results in U-
bottom plates.
Flat bottom plates are useful in ELISA technology but are unsuitable for liquid-phase blood group
serology.
U-well are preferred to V-well plates for resuspension technique because of the case of suspension prior
to reading and their superior optical quality when using automated plate reader.
- Small volumes and low concentration of sera and red cells are used, making it cost-effective.
- Easy handling of a microplate, which can replace 96 test tubes.
- Batching of samples can be achieved with considerable economy in space and time.
- If larger laboratories acquire microplate hardware items e.g. reagent dispenser, sample handler and cell
washer it may further reduce the operation time.
- Large batches of plates can be predispensed with antisera and reagent red cells before testing.
- The technique of microplate grouping may be automated by on-line data capture in larger laboratories,
which may help in
---- reduction in reading and transcription errors
---- saving in staff time
---- use of bar codes for samples and microplate identification
---- integration into a comprehensive computer system for storage of data.
Microplate
-Pipettes
-Manual / automated single or multi-channel dispenser
-Variable volume dispenser (20 - 50 ul)
-Centrifuges
-Bench-top centrifuge with head for carrying microplates
-Microplate shaker
-Two/four place shakers
-Reading aid
-Automated plate readers
-Automated plate reader based on ELISA reading system
Column technique
Recently interest has been generated in use of column technology for serological testing. The aims of this
technology is to standardize red cell agglutination reactions and trap the agglutinates to permit simple
and reliable reading.
The column consists of special microtubes containing a sephadex gel matrix. Red cells and serum or red
blood cells alone are dispensed into the microtubes, incubated if necessary and then centrifuged under
strictly controlled parameters.
The gel within the microtubes acts as a sieve, unagglutinated cells form a button at the bottom of the
microtube and agglutinated red blood cells are trapped in the gel.
These depend on the immobilization of one of the reactant so that during testing the immobilized
component captures additional reactant from the liquid phase and binds them to solid phase. These
techniques have been successfully developed for a range of serological testing including red cell
grouping, antiglobulin testing and antibody detection.
- Wash cord red cells 3-4 times with saline to minimize errors due to Wharton’s Jelly.
- Reactions in cell grouping may be weak as ABO antigens are not fully developed at birth.
- Corresponding ABO antibodies are usually absent, therefore, only cell grouping is recommended till 6
months of age.
Dolichus biflorus lectin reacts specifically with Al antigen and causes agglutination. It is stored at 4-8°C
and may be frozen at -20°C for prolonged storage.
The group A and AR blood groups can be subtyped into Al, A2 and A2B and A2B. Approximately 8% of A2
individuals and 30% A2 B individuals develop naturally occurring anti-Al antibodies. Therefore, it may be
necessary to determine the subgroups of A for both, donors and the patients. If a patient with anti-Al
requires blood transfusion, it will be necessary to select blood from A2 donors.
Procedure
Interpretation
Controls
Perform the test using known Al cells and A2 cells instead of patients’ cell. These will act as positive and
negative controls.
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Errors during ABO blood grouping usually present as discrepancies in the cell and serum grouping. The
important factors leading to such problems could be due to
Reagents
Blood sample
The sample should be as fresh as possible. Venous blood may be drawn in a clean container and stored at
4°C (if not tested within 12 hours).
Use serum (instead of plasma) for reverse grouping as plasma may lead to non-detection of weak or
complement binding antibodies.
All reagents which need to be restandardized for microplate use should be tested for potency and
specificity. High concentration of proteins or polymers such as ficoll, polyvinyl pyrrolidine (PVP) and
dextran cause red cells to stick to surface of the well.
Antisera for use in microplate technique should be diluted to take care of the dye and to decrease the
effect of additives. Colour of the antisera should be light or colourless otherwise it will interfere with the
results on plate reader.
In routine laboratory, ABO and Rh (D) grouping is usually performed in parallel. ABO monoclonal and
Anti-D IgM reagents will give excellent results when diluted in phosphate buffer saline containing 1- 3%
bovine serum albumin.
Polyclonal anti-D sera available for sLide and rapid tube test are usually unsuitable for microplate use.
Usually a dilution of 1:20 of ABO antisera and 1:10 of anti-D antisera give good results, however
individual laboratories should standardize the technique depending on the antisera in use.
The cells can be suspended in phosphate buffer saline (PBS), low ionic strength saline solution (LISS) or
isotonic saline. A 3% suspension of red cells may be used.
Enzymes techniques are commonly used in mocroplate blood grouping as the forces involved in
resuspending negative reactions in the resuspension technique may lead to fragmentation of weak
agglutinates. One-stage (bromelain or papain-cystein) enzyme method is likely to give better results for
the weaker phenotypes.
Procedure
o Add I drop of 2-3% cell suspension of known A cell, B cells and 0 cells to the well of row
F-i, G0i and li-i through 12.
1 2 3 4 5 6 7 8 9 10 11 12
Anti-A A
Anti-B B
Anti-AB C
Anti-D1 D
Anti-D2 E
A Cells F
B Cells G
O Cells H
II.
o Starting with test samples 1, dispense 1 drop of serum to well F,G and H vertically i.e. row
I
o Transfer 1-2 drop of red blood cells to saline tube labelled-I and prepare 2-3% washed
suspension of test cells.
o Repeat with all test and control samples till-12.
III.
o Dispense 1 drop of 2-3% cells from test sample 1 to well A,B,C,D, and E inrowl vertically
i.e. row 1.
o Repeat with all test samples and control samples till 12.
o Gently tap the microplate and incubate at room temperature (22-24°C) for 1 hour.
If the microplate centrifuge is available, spin after 15 minute incubation at bog for 40 seconds.
IV.
o Resuspend the red cells using a microplate shaker. Overshaking will reduce the strength of
agglutination and this can be avoided by switching off the microplate shaker as soon as a
known negative reaction is fully suspended and positive reactions dislodged.
o Record results
o Results are preferrably read by two technician independently, one reads while the other
records results in the register and then they reverse roles.
o Typical reaction pattern may be interpreted visually or using an automated or semi-
automated reader.
Interpretation
A positive reaction will show the button of cells having a strongly crenated edge, while a negative result
will show a compact button with smooth edge that streams on tilting or gets suspended on shaking.
A gentle shake of the plate will often assist in clarifying doubtful results.
If a microplate centrifuge and shaker is not available, after the one hour incubation, tilt the microplate at
an angle of 600 and leave for 5 minutes to allow negative reactions to stream. (same can be done in V-
bottom plates also).
Predispensed plates
A multichannel pipette or automated microplate dispenser may be used, which can save considerable
time. Predispensed plates should be kept covered at 4°C until required. In order to avoid evaporation of
reagents, predispensed microplates should not be stored for more than 24 hours.
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Rh Grouping
Rh grouping in routine use for donors and patients involves testing for Rh (D) antigen only, however tests
for other important Rh antigens e.g. C,c,E and e may be done for Rh genotyping.
The method of Rh grouping mainly depends on the type of reagents available, for which the
manufacturers’ instructions have to be strictly followed.
Potentiating or enhancing substances such as albumin, enzymes and AHG reagent are used to bring about
agglutination with human IgG anti-D.
Anti-D serum (IgG) for saline or rapid tube test (high protein medium) This contains
macromolecular additives and give reliable results.
Anti-D for saline tube test 2 types
o Anti-D IgM
II Monoclonal antibodies
These antibodies are highly specific, react equally well at 20°C as well as 37°C and are reliable for slide
and rapid test tube technique.
IgM anti-D monoclonal reagent cannot be used for Du testing by indirect antiglobulin test (IAT) while IgM
+ IgG monoclonat reagent and blend of IgM monolconal and IgG polyclonal can be used for Du testing.
It is preferrable to use potent anti-D reagents from two different batches of different firms following the
manufacturers’ recommended technique (anti-Di, anti-D2)
Known 0 Rh (D) positive and 0 Rh (D) negative cells may be used as controls with monoclonal anti-D
reagent.
Alternatively, AB serum or diluent control provided with the anti-D reagent or 22% bovine serum albumin
may be used as negative control with the test cells
Rh (D) Grouping
In most of the blood transfusion laboratories, Rh (D) grouping is performed along with the ABO grouping
and same techniques as used for ABO grouping may also be employed for Rh typing ie.e
1. Slide technique
2. Tube technique - Tube sedimentation’
(saline, albumin, - Tube spin
enzyme and Al-IG test)
3. Microplate technique
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Slide Technique
This technique may be used in emergency Rh (D) typing if a centrifuge is not available. The slide test is
not recommended for routine test as it may not pick up weak reactions, thus giving negative results.
Tube Technique
Procedure
2. Place 1 drop of anti-D (Dl) in cleaned tube labelled Dl and place 1 drop .of anti-D (D2) from a different
manufacturer in a clean tube labelled D2.
5. Mix well and centrifuge at 1000 rpm for 1 minute (in case of using IgG anti-D, incubate at 37°C for
10mm. and centrifuge (spin tube method) or incubate at 37°C for 60 minutes (sedimentation method).
6. Resuspend the cell button and look for agglutination. All negative results must be confirmed under
microscope.
Interpreation
Positive test : Agglutination in anti-D (both tubes) and smooth suspension in control tube.
Negative test : Smooth suspension in all the tubes (test and control)
In such case, the test should be repeated using saline IgM anti-D.
For all microscopically negative reactions in donor grouping, Du testing should be performed, hereas
some workers suggest that if the two anti D reagents used are potent and specific, it is not necessary to
perform Du testing.
Principle
Albumin increases the dielectric constant of the medium and thus reduces the zeta potential. Due to this
effect, the electrical repulsion between the red blood cells is less and the cells agglutinate. Mostly 22%
bovine albumin is used, as higher concentrations can cause rouleaux formation.
Albumin addition technique Procedure 1. Add 1 drop of anti-D to a labelled tube. 2. Add 1 drop of 2-5%
test red cell suspension. 3 Add 1 drop of 22% bovine albumin. 4. Mix and incubate at 37°C for 15-20
minutes. 5. Centrifuge at 1000rpm for 1 minute. 6. Gently resuspend the cell button and observe for
agglutination. 7. Confirm all negative reactions under microscope. Albumin layering technique
Procedure
1. Place 1 drop of anti-D in a labelled tube.
4. Allow 1 drop of 22% albumin to run down the inside wall of the tube. Albumin will form a layer on top
of the red cells. Do not mix.
6. Examine for agglutination after gentle shaking and confirm all negative results under microscope.
Proteolytic enzymes such as papain, trypsin, bromelain and ficin digest the cell membrane partially and
expose Rh antigens to react with IgG antibodies. When the membrane is partially removed, it brings
about a loss of negative electric charge on the red cell which is responsible for keeping the cells a set
distance apart, hence small IgG molecules are able to span the gap between cells and bring about
agglutination. (for further details see Subsection 3.6 : Specialized serological techniques.)
Procedure
2. Add 1 drop of 2-5% saline suspension of test red cell (put appropriate controls).
5. Agglutination of test cells indicates positivity while no agglutination shows a negative test (Read
macroscopically only).
Procedure
2. To .1 drop of washed packed test cells, add 2 drop of diluted papain solution.
4. Wash the red cells 3-4 times with normal saline and prepare a 2-5% cell suspension.
8. Agglutination of cells indicates a positive reaction and no agglutination indicates a negative reaction
(Ready only macroscopically)
d. Indirect antiglobulin test (Du Testing)
If using IgG anti-D in routine saline agglutination Rh (D) grouping proceed from step 6 in all negative
reactions otherwise perform the following procedure :
3. Add 2-5% washed test red cell suspension to both the tubes.
6. Gently suspend the cell button and look for agglutination, if positive test, no need to proceed as the
sample is Rh (D) positive.
7. If negative, wash the cells 3-4 times with saline and decant the last washing.
8. Add 1-2 drop of anti-human globulin reagent (AI-IG-Coombs’ reagent). Mix gently and centrifuge at
1000 rpm for 1 minute.
9. Resuspended the cell button gently, examine for agglutination and record the results.
10. All negative reactions should be confirmed by adding known IgG sensitized cells, re-centrifuge and re-
examine for agglutination. The presence of agglutination confirms the test result. (For details, refer to
Subsection 3.3 Antiglobulin test)
Interpretation
Agglutination in test sample and negative reaction in control sample shows a positive test and the sample
is labelled Rh (d) positive.
In haemolytic disease of the newborn, the baby’s red cells may be coated with immunoglobulin and a
saline reactive Rh antiserum is usually necessary for testing.
When the cells are heavily coated with antibody, no free antigenic sites remain for reaction, resulting in a
negative test. This is suspected when the infant’s cells show a positive direct antiglobulin test (DAT) and
a negative test with anti-D reagent.
In such instances it is recommended that the antibody should be eluted by gentle elution (heating at 45°C
for 30 minutes) to expose the antigenic sites before testing.
Inaccurate or incorrect results in Rh grouping may occur due to technical errors. These can result from
defects in equipment, reagents, specimen and techniques or through wrong interpretation.
Resolving Rh Problems
3. Check patient rçcords for history, diagnosis, pregnancy, medication and previous transfusion.
6. Perform alternative procedures such as washing of red cells with warm saline, enzyme treatment of red
cells, absorption / elution studies.
The purpose of pretransfusion testing is to minimize the risk of blood transfusion to a patient by selecting
blood and blood components that will have acceptable survival when transfused and will not cause
destruction of the recipients red cells.
The term compatibility testing or pretransfusion testing refers to a set of procedures required before
blood is issued as being compatible.
1. Identification of recipient
2. Labelling of tubes before puffing in sample Name, Age, Date of Birth, l.D.No. Ward/Bed No. and Date
3. Nature of specimen
* Clotted blood sample - serum sample
* EDTA blood sample - for DAT
* Sample should not be haemolysed
* Sample should be less than 3 days old
4. Test on Neonate
- Request Form
Transfusion request form must contain sufficient information for definite identification of the recipient. It
should include:
- Name
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- Age/Date of birth
- Sex
- Registration number (1.D.No.)
- Ward/Bed No.
- Address
For proper selection of blood and blood components; and for record maintenance, the request form
should also provide information on:
6. Storage of sample
Perform ABO and Rh(D) grouping using a recommended technique. Any discrepancy must be resolved
before blood is issued. In emergency situations, if time does not permit detailed testing, issue C group
red blood cells.
Selection of Blood
ABO & Rh (D) grouping and screening for transfusion transmitted diseases must be done on all donor
blood or blood component before the compatibility test is performed.
1. Whenever possible select ABO group specific blood or blood component for the recipient.
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2. When group specific bipod is not available, use alternate ABO compatible blood or blood component.
3. Select blood of same Rh (D) type as that of recipient particularly in women of child bearing age.
4. If Rh(D) negative blood is not available, give Rh (D) positive blood followed by Rh (D)
immunoglobulins.
5. Change over to group specific blood after administering group nonspecific blood.
6. If recipient’s serum contains unexpected antibodies, identify the antibody if possible before selecting
the appropriate blood.
When this is not possible, perform compatibility testing by indirect antiglobulin technique with multiple
(>10) ABO and Rh group specific donor units to find compatible blood.
2. Massive transfusion (transfusion of blood equal to or more than the recipients blood volume within a
period of 24 hours) select 10-14 days old blood.
3. Compromised heart and brain circulation select <10-14 days old blood.
The function of a crossmatch is to detect in vitro incompatibilities between the donor’s cells and the
patient’s serum.
If antibody screening of recipient’s serum is negative, an ABO immediate spin crossmatch (ISXM) is
sufficient to check for errors that may have occured in ABO grouping of the recipient and the donor.
If antibody screening has not been done, perform crossmatch using antiglobulin technique.
Reagents required
Procedure
3. Add 1 drop of 2-5% saline suspension of donor red cells from each donor unit to appropriate tube.
6. No agglutination should occur if the donor’s and recipient’s blood are compatible.
Do not proceed further if agglutination occurs since it indicates ABO mismatch. Regroup the recipient and
the donor.
If no agglutination occurs in the immediate spin crossmatch and no antibody screening has been
performed, proceed to the antiglobulin crossmatch.
Enzyme technique
Reagents required
1. 1% papain-cystein solution
2. Recipient’s serum.
Procedure
1. Mix 2 drops of patient’s serum with 1 drop of papain-cystein enzyme and 1 drop of 2-5% washed saline
suspension of donor’s red cells.
Interpretation
Agglutination indicates that the unit is incompatible and absence of agglutination indicates a compatible
unit. Antiglobulin technique
Reagents required
2. Recipient’s serum.
Procedure
1. Mix and incubate the tubes from step 6 of immediate spin test for 45 minutes at 3 7°C.
Page 18 of 17
3. Proceed to IAT.
Enhancing techniques e.g. albumin, enzyme and LISS can be used with AI-IG test to increase the
sensitivity.
It is recommended to use two enhancing techniques for increasing the sensitivity of compatibility test.
Interpretation
Use of LISS allows reduction in incubation time with relatively little loss of sensitivity. Instead of
suspending cells in saline, make to the correct strength suspension using LISS. The serum to cell ratio
must be kept to unity. Increasing the serum to cell ratio reduces the sensitivity of LISS tests.
LISS may be used with Albumin, Enzyme or IAT. When incubation time is reduced, the second stage of
haemagglutination must be assisted by gentle centrifugation. Great care must be exercised in reading the
tests to distinguish between true agglutination and aggregation.
Precautions
Use the attached tubing with the blood bag for obtaining donor red cells in major crossmatch.
Always place the sample from the segment of tubing from the blood bag into a prelabelled test tube and
match the donor unit no on the bag and the test tubes.
1. Emergency transfusion
After the blood has been issued, full routine procedure should be adopted.
Minimum testing
2. Massive transfusion
a. When time does not allow for compatibility testing, issue C Rh (D)-negative blood.
b. Do ABO & Rh (D) grouping of the patient and change over to the ABO and Rh (D) group-specific blood
as soon as possible. Issue the blood after doing an immediate spin crossmatch.
c. After the emergency has been dealt with, antibody screening on pretransfusion samples may be
performed. If negative, additional units can be issued after immediate spin crossmatch.
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d. If antibody screening detects presence of antibody, additional compatibility testing may be done using
AHG technique.
3. Infants
a. Determine ABO and Rh (D) group of the infant. Only cell grouping is necessary in infants less than 6
months.
b. Perform a direct antiglobulin test (DAT) on baby’s cells.
c. Screen maternal serum for expected antibodies.
d. If baby’s DAT and maternal antibody screening are negative:
* For neonates, select group ‘C’ Rh type specific blood using mother’s (if ABO compatible) or baby’s
serum for compatibility testing. Transfusing group ‘C’ blood will also ensure compatible blood in ABO-HDN
without any additional tests.
* For infants, select ABO and Rh group specific blood for compatibility test using infant’s serum.
* If baby’s DAT and maternal antibody screening are positive.
* Identify the antibody in the maternal serum.
* Alternatively perform compatibility testing with multiple ABO and Rh specific donor units by the AI-IG
technique against maternal and infant’s serum.