DHA Standard Operating Procedures Template

Standard Operating Procedures Title: Antibody Titration

Ownership: Effective Date: 04/10/2014 Code:
Pathology & Genetics Department. Revision Due Date:04/10/2017 DHA/PGD/COM/TS/016
Revision No: 03 Type:
First Edition Date: 04/10/2008 Technical

Applies to:
Rashid Hospital Dubai Hospital Latifa Hospital Hatta Hospital PHC
1.0Purpose:
To describes the method of measuring the relative amount antibody of an identified antibody.

2.0 Principle:
Titration is a semi quantitative method used to determine the concentration or the relative amount of antibody in
a serum/plasma sample. Serial two fold dilution of serum/plasma is prepared and tested for a specific antibody.
The reciprocal of the highest dilution of the tested sample that gives 1+ reaction is referred to as the titer (1 in
128 dilution; titer = 128).
It is a measure of the strength/concentration of an antibody. It can provide information to compare the strength of
antigen expression on different cell samples. Antibody titers are useful in prenatal evaluation of hemolytic
disease of the fetus and newborn (HDFN). It is used to estimate antibody activity in alloimmunized pregnant
women to determine whether and when to perform amniocentesis or other complex invasive investigation of fetal
condition.

3.0 Special Safety Precautions:

3.1 Refer to Lab Safety Manual & apply the universal safety precautions- as indicated.
3.2 All reagents should be treated as potentially infectious.
3.3 Wear protective lab gown & gloves throughout the procedure.
3.4 Special protective measures, conditions for clinical waste disposal & disinfection should be followed in
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accordance to Lab Infection Control Guidelines.

4.0 Specimen:
Aseptically drawn serum or plasma sample separated as much as possible from the red cells. If not tested
immediately, samples can be stored between 2-8ºC for 2 days, or frozen at -30ºC for 1 year. Blood specimens
exhibiting gross haemolysis or contamination should not be used. Clotted sample must be cleared by
centrifugation at 1500g for 10 minutes or by acceptable alternative g force and time. Test the current sample in
parallel with the most recent previously tested sample from the current pregnancy.

5.0 Reagents /Supplies:

Tube method
5.1 Polyspecific AHG (IgG+C3d).
5.2 Commercial antibody screening red cells that expresses the antigen(s) corresponding to the antibody
specificity.
5.3 Coombs Control (IgG-coated RBC).
5.4 Isotonic Saline Solution (0.9% NaCl).
5.5 10 or 12 X 75 mm test tubes.
5.6 Test tube rack.
Card Method
a. LISS//Coombs Card (Anti-IgG+C3d) - stored between 18-25 ºC.
b. Isotonic saline solution (0.9% Nacl)
c. 0.8% Red cell suspension that express the antigen corresponding to the antibody specificity.
d. ID- Working Table
e. Pipette & Disposable Tips
f. Tubes for suspension

6.0 Equipment:

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Tube Method
6.1 Automatic adjustable pipettes.
6.2 Calibrated Centrifuge.
6.3 Water bath, at 37 ± 2 °C.
Gel Card Method
a. ID-Centrifuge
b. ID-Incubator 37°C

7.0 Quality Control:

a. Confirm the validity of all negative Coombs results with IgG-coated cells.
b. Reagents must be stored between 2-8ºC, inspected & tested each day of use with controls.
c. It's advisable to minimize the reagent time outside refrigerator between uses.
d. Don't use reagents beyond expiry dates & don't use any damaged or leaking reagents.
e. Standardization of equipments used is a must.
f. Follow the reagent manufacturer's QC instructions.
g. Test the preceding sample in parallel with the most recent sample.
h. Prepare the dilution using a separate pipette for each tube.

8.0 Procedure:
Tube Method.
1 Check name & ID number of the sample to be tested.
2 Allow the reagents to reach room temperature (18 -25 ºC).
3 Select commercially prepared screening red cells, which should express the antigen
corresponding to the antibody specificity present in the sample.
4 For titration assisting in early detection of Hemolytic Disease of the fetus and Newborn,
choose cells with a double-dose expression of antigen to which the serum contains
antibody (use R2R2 cells when titrating anti-D).
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5 When tittering serum/plasma with multiple antibodies, choose cells that are positive for
only one antigen for each titer at a time (e.g. K+E- and K-E+ cells for the corresponding
antibodies).
6 Prepare master dilution. Label ten test tubes of 10 x 75 mm according to the serum/plasma
dilution (1, 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024). The 1:1 dilution means one
volume of serum/plasma undiluted, and the 1:2 dilution means one volume of
serum/plasma in a final volume of two.
7 Using a 0.5 mL pipette, place 0.5 mL saline into the bottom of all tubes except the first
one.
8 Using a 0.5 mL pipette, deliver 0.5 mL serum/plasma into tubes 1 and 2.
9 Using a clean 0.5 mL pipette, mix the contents of tube 2 several times. Transfer 0.5 mL of
mixture to the next tube (1:4).
10 Continue the same process for all the subsequent serial dilutions, using a clean pipette to
mix and transfer each dilution (serial dilution).
11 Remove one volume of diluted serum/plasma from the final tube (1:512) and save it for
use if further dilutions are required.
12 Label another ten tubes as in step 8.6. Place 0.1 mL (100ul) of each master dilution into
appropriately labeled test tubes.
13 Add 0.1 mL of commercially prepared 3-5% red cell suspension to each dilution.
14 Mix well, and test by the serologic technique appropriate to the identified antibody as
follows:
8.13.1) Saline Titer:

8.13.1.1 Follow the previous steps (1 to 14).

8.13.1.2 Gently agitate contents of each tube; incubate at room temperature for 1 hour.
8.13.1.3 Centrifuge at speed designated for saline phase.
8.13.1.4 Starting with the last tube (1024) gently dislodge cell button and observe for

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agglutination or hemolysis – as in Attachment 3: Grading Tube Agglutination
Reaction.
8.13.1.5 Grade and record results. Report the highest dilution that gives 1+ macroscopic
agglutination.

8.13.2) Coombs (AHG)Titer:

8.13.2.1 Follow the previous steps (1 to 12).
8.13.2.2 Gently mix and incubate at 37°C for 1 hour. Do not add any enhancement media. For HDFN
purposes, do not use enhancement techniques (albumin, PEG, LISS or enzyme treated red cells
because falsely elevated titers may be obtained.
8.13.2.3 Wash the red cells four times with saline; completely decant the final wash supernatant and add two
drops antihuman globulin.
8.13.2.4 Centrifuge at speed and time designated for Antiglobulin phase.
8.13.2.5 Observe the highest dilution that produces w+ macroscopic agglutination. Refer to as in
Attachment 3: Grading Tube Agglutination Reaction.
8.13.2.6 Grade and record results in Transfusion Serviced daily registers, as appropriate and Sunquest
(laboratory system).
8.13.2.7 Add IgG-coated cells to all negative reactions.

8.13.3) Gel Card Method

8.13.3.1 Follow the previous step of 8.1 to 8.11
8.13.3.2 Label Coombs anti-IgG card according to the serial dilution.
8.13.3.3 Remove the aluminum foil from as many microtubes as required by holding the ID-Card in the
upright position.
8.13.3.4 Pipette 50 uL of the red cell suspension to all the microtubes.
8.13.3.5 Add 25 uL of each master dilution to corresponding microtubes.
8.13.3.6 Incubate the ID-Card for 15 minutes at 37oC in the ID-Incubator

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8.13.3.7 Centrifuge the ID-Card for 10 minutes in the ID-Centrifuge
8.13.3.8 Observe the highest dilution that shows 1+ reaction, this will be the endpoint.
8.13.3.9 Read and record the results at Transfusion Services daily registers and Sunquest (laboratory system)

09. Calculation: NA

10.0 Expected values:

Tube # 1 2 3 4 5 6 7 8 9 10 11
Serum Dilution 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024
Titer 1 2 4 8 16 32 64 128 256 512 1024
NOTE: Titer is the reciprocal of the highest serum/plasma dilution that causes macroscopically apparent
agglutination, i.e. 1+ reaction.

11.0 Interpreting/ Reporting Results:

11.1. Read & Grade the agglutination reaction -as in Attachment 3: Grading Tube Agglutination Reaction.
The titer is the reciprocal of the highest serum/plasma dilution that causes macroscopically apparent
agglutination, i.e. 1+ reaction.
11.2. If there is agglutination in the last diluted tube, the titer endpoint has not been reached and additional
dilutions should be prepared and tested.
11.3. In comparative studies, a significant difference is three or more dilutions, since variation in technique
and inherent variability can cause duplicate tests to give results that differ by one dilution in either
direction. E.g. titer of 32 may -on replicate tests- show endpoint of 64 or 16.
11.4. Titer values alone can be misleading without evaluating the strength of agglutination.
11.5. Antibodies with high-titer, low-avidity characteristics generally have a titer greater than 64 with most
tubes show consistently weak reactivity.
11.6. Enter the antibody titer result in blood bank records and computer.

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Example for Interpreting Result of Antibody Titer :

Tube # 1 2 3 4 5 6 7 8 9 10

Serum/plasma Dilution 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 Titer

Agglutination Strength 4+ 3+ 3+ 3+ 2+ 2+ 2+ 1+ ± 0 128

12.0 Procedure Notes:

12.1. Grade the strength of agglutination reaction- as described in Attachment 3: Grading Tube
Agglutination Reaction.
12.2. Use a separate pipette for each tube when preparing dilutions. Failure to do so will result in
falsely high titers due to carry-over.
12.3. Uniformity of cell suspensions is very important to ensure comparability of results.
12.4. Passively acquired anti-D (RhIG) rarely achieves a titer above 4.
12.5. The critical titer for anti-D is usually 32 (may be 16).
12.6. Critical titer for antibodies other than D has not been well defined, although a critical titer of 32 is
often used.
12.7. The critical titer for anti-K may be lower than anti-D, typically a value of 8.
12.8. Antibodies are unstable in the diluted stage. Perform test immediately after dilution of
serum/plasma.
13.0 Method limitations:
13.1 Only qualified personnel should perform the procedure.
13.2 Technical variables greatly affect the results and care should be taken to achieve the most uniform
possible practices.
13.3 Careful pipetting is essential. Use pipettes with disposable tips that can be changed after each

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dilution.
13.4 Optimal time and incubation temperature and time and centrifugation force must be used
consistently.
13.5 Completely reproducible results are virtually impossible to achieve.
13.6 Comparisons are valid only when specimens are tested concurrently.
13.7 Measurements are more accurate with large volumes than small volumes.
13.8 Prozone phenomenon may cause reactions to be weaker in the more concentrated serum/plasma
preparations, than in higher dilutions. To avoid misinterpretation of results it may be preferable to
examine first the tube containing the most dilute serum and proceed through more concentrated
tubes, to the undiluted tube (from 1:1024 to 1:1).
13.9 It is imperative to work with clean supplies and non-contaminated reagents- bacterial or other
contamination.
13.10 Storage conditions & expiry date must be observed.
13.11 Certain drugs are known to cause positive reaction in anti-human globulin procedures.
13.12 Some pathological conditions are also reported as causing positive reaction in anti-human globulin
procedures
13.13 Bacterial or other contamination of materials used can cause false positive or false negative results
13.14 Fibrin residues in the red cell suspension may trap non-agglutinated cells presenting a fine pink line
on top of the gel while most the cells are on the bottom of the microtube after centrifugation
13.15 Too heavy or too weak red cell suspensions can cause aberrant results.

14.0 Tools/Attachments Form:

14.1 Attachment 1: Grading Tube Agglutination Reactions.

15.0 References:
15.1 Manufacturer’s instructions leaflet.
15.2 AABB Technical Manual, 16th Edition, 2008.

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16. Revision History

New issue Part revision Complete revision

Date Status Change Reference Section

04/10/2008 Deleted New
Modified
Added
14/09/2010 Deleted 3.2: added.
Modified 4.0: 5 ml removed.
Added 8.13.1.4, 8.13.2.5, 8.13.3.6, 11.1, 12.1: AABB Technical

Reference attachment added. Manual,2008

14.1: Attachment added.

15.2: Reference edition changed.
20/09/1012 Deleted Hatta hospital is included
Modified AWH changed to Latifa
Added 2.0 phrase relative amount added, sentences 4
and 6 added.
4. aseptically and separated from the red cell as
much as possible added, sentences 4-5 added AABB Technical Manual

5.0 tube method added 16th edition

6.0 tube method added Manufacturer’s

8.0 serial dilution of 0.1ml change to 0.5ml Instructions leaflet

8.6 master dilution added

8.12 added
8.13.2.2 sentences 2 added
8.13.2.3 Wash the red cells four times with

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saline; completely decant the final wash
supernatant added
8.13.3 gel card method added
12.9 deleted
13.11 – 13.5 added
04/10/2014 Deleted 2. Principle edited
Modified 4. last sentence added
Added 7.7 & 7.8 added
8.3 rephrased AABB Technical
8.5 edited Manual 16th edition
8.6 1024 added Manufacturer’s
8.12 100 ul added Instructions leaflet
New template used
Search words: Standard Operating Procedures

17. Search words: Standard Operating Procedures.

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Attachment 1
GRADING TUBE AGGLUTINATION REACTIONS

 The purpose of grading reactions is to allow comparison of reaction strengths.

 This is beneficial in detecting multiple antibody specificities or antibodies exhibiting
dosage.
 Grading agglutination reactions gives an indication of the relative amount of antigen
or antibody present.
 All tubes tests should be graded.
 The technique used in the resuspension of the cells will affect the grading of the
reaction.

Interpretation of Agglutination Reactions

 4+ = one solid agglutinates.

 3+ = several large agglutinates.
 2+ = medium-size agglutinates; clear background.
 1+ = small agglutinates; cloudy background.
 1+W = very small agglutinates, cloudy background.
 ± or w+ = Barely visible agglutination; cloudy background.
 0 = negative, no agglutination.
 MF = mixed field. Mixture of agglutinated & unagglutinated red.
 H = Complete hemolysis (a positive reaction).
 PH = Partial hemolysis, some red cells remain.

Refer to the below illustrations for comparison.

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References:

 AABB Technical Manual, 16th Edition, 2008.

http://faculty.matcmadison.edu/mljensen/BloodBank/lectures/Basic_Laboratory_techniques&Re
agents.htm

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