Titrations – Cold Autoantibodies
1.0Principle
Determination of the titre of an antibody in a patient's blood sample is performed by straightforward serial dilutions of the plasma. Cold reactive autoantibodies, if reactive at very high titres, may suggest a pathologic cold agglutinin disease.
2.0Scope and Related Policies
2.1All tests will be set up using warm separated (37°C) plasma.
2.2The antibody test – cold autoagglutinins procedure must be positive to proceed with this procedure.
3.0Specimen
EDTA anticoagulated red cells preferably less than 72 hours old.
4.0Material
Equipment:Serological centrifuge
Block for test tubes
Microscope
4°C refrigerator
Supplies:Test tubes – 10 x 75mm
Test tubes - 12 x 75 mm
Serological pipettes
Reagents:Normal saline
3% cell suspension of OI Cells (P1 neg screening cell)
5.0Quality Control – N/A
6.0Procedure
6.1 Check the suitability of the specimen to ensure that the specimen
label information matches the request form. See PA.002 - Determining Specimen Suitability.
6.2 Warm separate patient specimen by setting in 37°C
waterbath/heating block for a minimum of 15 minutes (with periodic mixing). Warm centrifuge cups in 37°C waterbath as well. Spin specimen in warmed cups approximately 2-5 minutes at 3000 rpm and place in 37°C waterbath immediately after spinning. With a pipette warmed to 37°C remove plasma into a clean, labeled tube avoiding any cells. Re-spin plasma sample to clear.
6.3 Prepare a master titre of the test plasma. Label 13 - 12 x 75mm
tubes numerically and with the first 3 letters of the patient's family name.
6.4Dispense 0.5mL of normal saline into each tube except # 1.
6.5Dispense 0.5mL of test plasma into tubes #'s 1 and 2. Mix contents of #2 ten times with pipette, discard pipette tip.
6.6 With a clean pipette tip transfer 0.5mL of contents in tube #2 to tube #3 and mix contents of tube #3 ten times.
6.7 With a clean pipette tip transfer 0.5mL from tube #3 to tube #4 and mix ten times.
6.8Repeat steps 6.5 - 6.6 until last tube is reached (#13), set this tube aside in case the endpoint of the titre continues past tube #12.
6.9Label one set of twelve 10 x 75mm tubes.
6.10Dispense 2 drops of each dilution (from master titre) to each appropriate tube.
6.11Dispense one vertical drop of OI P1 negative cells to each tube.
6.12Mix and incubate at 4°C for 1-2 hours. Centrifuge at 3400 rpm for 10-15 seconds.
6.13Examine each tube for agglutination macroscopically and record results.
7.0Reporting
7.1 Report the temperature and the reciprocal of the highest
dilution giving a grade 1 reaction.
e.g., Titre 16 OI cells at 4°C
8.0Procedural Notes
8.1It is critical that separate pipette tips are used when transferring the diluted plasma. This will minimize carryover of antibody.
8.2Do not use less than 0.5mL volumes for diluting, inaccuracies can occur with smaller volume.
9.0References
9.1Roback, JD. ed. AABB Technical Manual, 16th ed. Bethesda, MD: American Association of Blood Banks, 2008: pg 925-926.
/ Ontario Regional Blood Coordinating NetworkStandard Work Instruction Manual / SP.004
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