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Red Cell Serology Introduction

Red cell serological reactions form the basis of laboratory testing in a blood transfusion service.
Antigen-antibody reactions in blood group serology are usually detected by

• Haemagglutination
• Complement fixation
• Neutralization
• Absorption and Elution
• Precipitation

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.

ABO Grouping Techniques

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.

Practical Aspects of ABO Grouping

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.

6. The glassware used should be dry and clean.

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.

8. Results should be recorded immediately after observation.

9. Concave mirror (agglutination viewer) or microscope may be used to examine reactions that
appear negative by the naked eye.

ABO grouping sera

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.

Blood sample for ABO grouping

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.

2. Haemolysed samples are not suitable for testing.

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 Red Cells for ABO Testing

Preparation of reagent red cells in addition to patients’ and donors’ red cells is required daily
while performing the serum grouping and cell grouping.

Reagent red cells

When performing an ABO serum grouping, it is important to ensure that the cells prepared are
fresh and well-selected
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‘A’ cells

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.

Patient and donor cells

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.

Control cells for ABO grouping

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 + + + + -

Blood grouping - Methods & Procedure

Three manual methods can be used when performing blood grouping:
- Glass slide or white porcelain tile
- Glass test tube
- Microwell plate or microplate

Newer techniques

- Column technique (sephadex gel)

- Solid phase tests

Slide or Tile Testing

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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

- weakly reactive antigens on cells

- serum grouping with low titre anti-A or anti-B

Disadvantages

- Less sensitive than the tube test

- Drying up of the reaction mixture can cause aggregation of cells, giving false positive results.
- Weaker reactions are difficult to interpret.

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.

5. Record the results.

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.

Advantages of tube testing

- 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

1. Cell grouping / forward grouping

2. Serum grouping / reverse grouping

Reagents required

1. Known antisera (anti-A, anti-B, anti-AB), monoclonal or polyclonal.

2. Red cell suspension - reagent cells (Ac, Bc and Cc); test red cells (patients or donor)

Cell and serum grouping

1. Spin test sample to separate serum.

2. Set up 6 tubes correctly labelled with donor/patient no. Anti A, Anti-B, Anti-AB, Ac, Bc and Cc.

3. Prepare once washed 2-5% suspension of the test cells.

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.

10. Record results according to grades of agglutination.

Defining the Stregth of Reaction (Grading of Agglutination)

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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

Cell grouping Serum grouping

Anti-A Anti-B Anti-AB Ac Bc Oc Result
+ - + - + - A
- + + + - - B
- - - + + - O
+ + + - - - AB
Oh or any other irregular
- - - + + +
antibody

Precautions

1. Always use throughly cleaned test tubes for the test.

2. While reading the result, shake very gently to dislbdge the button.

3. Use correct speed and time centrifugation to avoid erroneous results.

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.

Three types of microplates are available

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.

Advantages of Microplate ABO grouping

- 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.

Equipment required for microplate grouping

Microplate

- Untreated rigid polystyrene microplates

-Pipettes
-Manual / automated single or multi-channel dispenser
-Variable volume dispenser (20 - 50 ul)
-Centrifuges
-Bench-top centrifuge with head for carrying microplates
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-Microplate shaker
-Two/four place shakers
-Reading aid
-Automated plate readers
-Automated plate reader based on ELISA reading system

Blood Grouping - Newer Techniques

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.

The Sephadex gel may be

• Neutral
• Specific reagent
o AHG
o Anti-A
o Anti-B
o Anti-D
o Anti-Kell, etc.

Image : Reading and grading of haemagglutination in gel system

Advantages of gel system

- Superior in sensitivity to conventional tube test without loss of specificity.

- Easy to use.
- Requires small amount of red cells and serum for testing (1O-40m1), making it ideal for
neonatal and paediatric use.
- Reactions are easily visible and can be graded
- Reactions using gel techniques are stable for at least 48 hours and the gel cards can be
photocopied providing a permanent record for future reference.

Sold phase tests

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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.

Grouping of Cord or Newborn Infant Blood

- 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.

Testing for Al and A2 Subgroups

Anti-Al reagent is used to differentiate Al and A2 subgroups.

Sources of anti-Al reagent

• Lectin - Dolichus biflorus (most commonly used)

• Naturally occurring anti-Al in A2 and A2B individuals
• Human anti-Al(by adsorption of group B anti-A serum by A2 cells)

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

1. Place 1 drop of anti-Al reagent into a clean, dry test tube.

2. Add 1 drop of 2-4% saline suspension of patient’s cells.

3. Mix and leave at RT for 30-60 minutes.

4. Gently agitate and examine for agglutination.

Interpretation

Reactions showing agglutination indicate Al blood group.

Controls

Perform the test using known Al cells and A2 cells instead of patients’ cell. These will act as
positive and negative controls.

Errors Encountered in ABO Grouping

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

• factors related to red cells

• factor related to serum
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• factors related to technical procedure

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.

Blood grouping antisera

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.

Reagent and test red cells

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

Prepare a worksheet for proper layout of samples.

• I.
o Arrange test samples in serial number i.e 1-10
o Separate cells and serum of the test samples and arrange them as shown below
o Using a fine tipped Pasture pipette, dispense 1 drop each of anti-A, anti-B, anti-AB,
anti-Di and anti-D2 (anti-D from two different firms or batches) into each well of
row A,B,C,D,E, respectively.
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.

Micropiate A4BO & Rh (D) grouping

1 2 3 4 5 6 7 8 9 10 11 12
Anti-A A
Anti-B B
Anti-AB C
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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.

Rh Grouping

Practical Aspects of 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
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manufacturers’ instructions have to be strictly followed.

Reagents for Rh (D) Grouping

I Polyclonal human anti-D serum (IgG)

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

Anti-D IgG - Chemically modified

II Monoclonal antibodies

• IgM anti-D monoclonal reagent

• IgM and IgG anti-D monoclonal reagent
• Blend of IgM monoclonal + IgG polyclonal reagent

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)

III Controls for Rh (D) grouping

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

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
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a. Saline Agglutination test for Rh (13) Typing

Procedure

1. Prepare 2-5% washed red cell suspensionof test sample.

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.

3. Place 1 drop of 22% bovine albumin/control reagent in another tube labelled C.

4. Add 1 drop of 2-5% test cell suspension to each tube.

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)

Test is considerable invalid if

- both test and control tubes show a positive reaction.

- conflicting results in tubes Dl and D2.

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.

b. Albumin technique for Rh (D) typing

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 can be used in

= Albumin addition technique additive to serum & cell mixture.

- Albumin layering technique layered on cell button

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
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1. Place 1 drop of anti-D in a labelled tube.

2. Add 1 drop of 2-5% test red cell saline suspension.

3. Incubate at 37°C for 45-60 minutes.

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.

5. Incubate further at 37°C for 15-20 minutes.

6. Examine for agglutination after gentle shaking and confirm all negative results under
microscope.

c. Enzyme agglutination technique for Rh (D) typing

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.)

One stage enzyme technique (papain-cystein)

Procedure

1. To 1 drop of anti-D (IgG), add 1 drop 1% papain-cystein enzyme.

2. Add 1 drop of 2-5% saline suspension of test red cell (put appropriate controls).

3. Mix and incubate at 37°C for 45 minutes.

4. Centrifuge at 1000 rpm for 1 minute and record the results.

5. Agglutination of test cells indicates positivity while no agglutination shows a negative test
(Read macroscopically only).

Two-stage enzyme technique (Pre-treatment of test red cells with exzyme)

Procedure

1. Prepare a 1% papain solution and dilute it to 1:3 using normal saline.

2. To .1 drop of washed packed test cells, add 2 drop of diluted papain solution.

3. Incubate at 37°C for 15 minutes.

4. Wash the red cells 3-4 times with normal saline and prepare a 2-5% cell suspension.

5. To 1 drop of anti-D (IgG), add I drop of enzyme-treated red cell suspension.

6. Mix & incubate at 37°C for 45 minutes.

7. Centrifuge at 1000 rpm for 1 minute and record the result.

8. Agglutination of cells indicates a positive reaction and no agglutination indicates a negative

reaction (Ready only macroscopically)
d. Indirect antiglobulin test (Du Testing)
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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 :

1. Take 1 drop of anti-D (IgG) in a cleaned labelled test tube (T).

2. Take 1 drop of appropriate diluent control in another tube (C).

3. Add 2-5% washed test red cell suspension to both the tubes.

4. Mix and incubate at 37°C for 45-60 minutes.

5. Centrifuge at 1000 rpm for 1 minute.

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.

Microplate technique for Rh (D) grouping

As described in ABO grouping

Rh(D) Grouping In Haemolytlc Disease of the Newborn

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.

Erroneous Results in Rh Grouping

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

1. Perform clerical checks for validity of labels and requisition forms.

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2. Obtain a fresh blood sample of patient.

3. Check patient rçcords for history, diagnosis, pregnancy, medication and previous transfusion.

4. Check equipment and reagents for proper quality control.

5. Check the antisera and controls.

6. Perform alternative procedures such as washing of red cells with warm saline, enzyme
treatment of red cells, absorption / elution studies.

7. Carry out family studies.

Pretransfusion Testing (Compatibility Testing)

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.

Collection, Identification and Storage of Patient Sample

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

* Baby’s blood specimen for ABO & Rh(D) grouping

* Mother’s serum for crossmatching

5. Identification of blood sample in the blood transfusion laboratory.

- Request Form

Transfusion request form must contain sufficient information for definite identification of the
recipient. It should include:

- Name
- 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:
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• Type of requirement (Routine/Emergency/Group & Screen)
• Clinical diagnosis
• Obstetric history
• Indication for transfusion
• Number of units required
• Which component required
• Date and time when required
• History of previous transfusion and transfusion reaction
• Previous tests and unexpected antibody
• The blood sample alone with the request form should be signed by a doctor or a
phelebotomist collecting the sample.
• Laboratory personnel should check the information on the request form with the sample
and record the time of receiving the request form.

6. Storage of sample

- For 7 days at 2-6°C in sealed tubes

7. Checking previous transfusion records

• ABO & Rh(D) group

• presence of unexpected antibodies
• any problem in compatibility testing
• previous transaction
• any transfusion reaction

ABO and Rh (D) Grouping of Recipient

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.

If problem with Rh (D) typing, give Rh (D) negative blood.

Screening and identification of Unexpected Antibodies

* Unpooled C group cells should be used.

* C cells with known antigen profile preferred.
* Antibody identification if antibody screen positive.
* Selection of blood lacking corresponding antigen for compatibility testing.

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.

2. When group specific bipod is not available, use alternate ABO compatible blood or blood
component.

Component ABO group of recipient Alternate compatible gorup

1. Whole blood O,A,B,AB None (only ABO group specific blood)
2. Red cell concentrate O None use only 0 group
A O
B O
AB A,B or O
3. Plasma O A,B, or AB
A AB
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B AB
AB Note use only AB
4. Platelet concentrate All ABO group Compatible with red cells are preferred

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.

Special Transfusion Requirement

1. Exchange transfusion in the newborn select <5 days old 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.

Compatibility Testing (Major Crossmatch)

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.

Routine procedure for compatibility testing

• ABO and Rh (D) group

• Antibody screen
• Crossmatch
o Saline RT 37”C
o Albumin or
o Enzyme
o Antiglobulin test (using Broad Spectrum AHG)

Techniques of compatibility test

Immediate spin technique (Saline technique)

Reagents required

1. 2-5% suspension of donor’s red cells.

2. Recipient’s serum.
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Procedure

1. Label 1 tube for each donor sample to be crossmatched.

2. Add 2 drops of recipient’s serum to each tube.

3. Add 1 drop of 2-5% saline suspension of donor red cells from each donor unit to appropriate
tube.

4. Spin tubes at 1000 rpm for 1 minute.

5. Look for agglutination.

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.

2. Incubate at 37°C for 45 minutes.

3. Read results after centrifugation at 1000 rpm for 1 minute.

Interpretation
Agglutination indicates that the unit is incompatible and absence of agglutination indicates a
compatible unit. Antiglobulin technique

Reagents required

1. Anti-human globulin reagent (Al-IC)

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.

2. Spin and look for agglutination. Record the results.

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
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No agglutination should be seen at any stage in compatibility testing. Blood is compatible if no

agglutination occurs.

Use of low ionic strength saline (LISS) solution

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.

Minor Cross match

Minor cross match using patient’s red cells and donor’s serum is not essential, if every donor
serum is screened for irregular antibodies at least with pooled 0 cells by saline and enzyme
techniques.

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.

Pretransfusion Testing in Special Situations

1. Emergency transfusion

During an emergency it is best to do as much as the time allows.

After the blood has been issued, full routine procedure should be adopted.
Minimum testing

. Rapid ABO grouping of patient and donor unit

. Immediate spin crossmatch (ISXM) technique

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.

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.
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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.