Principle: | Clinical Significance: | Specimen: | Materials: | Reagents: | Standardization: N.A. | Procedure: | Limitations: | Results Derivation: | Expected Result(s) | Quality Control: | References:
Titration is a semi-quantitative method of determining the antibody content (titer) of a given serum sample. Patient plasma/serum that is positive for an antibody(ies) is serially diluted and tested against red cells positive for the corresponding antigen. The titer is expressed as the reciprocal of the highest dilution that causes agglutination in the antiglobulin phase. In this test, the selected red blood cells, suspended in a hypotonic-buffered saline solution, are mixed with serially diluted patient plasma/serum, incubated, and centrifuged through a gel microtube containing anti-IgG. In the dilutions with measurable antibody, agglutinates of red cells will be trapped in the gel. In the dilutions with negligible antibody, the red cells will move freely through the gel, forming a button at the bottom of the microtube.
II. Clinical Significance:
Antibody titration is used to follow the antibody level in sensitized pregnant women as an indication for amniocentesis and possible interruption of the pregnancy. Testing paired titers collected 1 month apart is the recommended protocol. Each specimen must have its own antibody identification performed to confirm the antibody to be titered and to rule out newly formed antibodies.
Refer to the “Antibody Detection & Identification; MTS Gel Card” procedure.
Refer to the “Antibody Identification; MTS Gel Card” procedure.
Refer to the “Antibody Identification; MTS Gel Card” procedure.
VI. Standardization: N.A.
1. An Antibody Identification must be performed prior to all titers.
Note: If more than one antibody is identified, each clinically significant antibody must be titered individually. (If the patient has a record of having received RhIG (RhoGam), consult a Pathologist to determine the necessity of an Anti-D titer.)
2. If available, retrieve the previously drawn frozen sample to titer with the current sample.
Note: Paired titers (two separate collections) are preferred to document a change in titers. See the “Quality Control” section.
3. Select a screening cell or panel cell that is heterozygous (if possible) for the antigen that corresponds to the antibody to be tested. Record this cell in the comment field of the SafeTrace result entry screen (Panel A, cell#4, Lot#, Exp. Date).
Note: For Anti-D titers use an Rr cell (RH-hr column on the panel antigram).
4. Label six 12 x 75 mm test tubes to be used for serial dilutions as follows: 2, 4, 8, 16, 32, and 64. (The number represents the titer.)
Note: If a titer has been previously reported on this patient that is >64, label additional tubes to cover three tubes beyond the previous report.
5. Deliver 0.25 ml saline to each of the tubes with a graduated transfer pipette.
6. Add 0.25 ml patient's plasma/serum with a new graduated transfer pipette to tube 2.
7. Flick tube 2 to mix.
8. Aspirate 0.25 ml of the contents of tube 2 with a new graduated transfer pipette and deliver to tube 4.
9. Flick tube 4 to mix.
10. Continue this same technique through all six tubes using new graduated transfer pipettes for each new dilution. Save tube 64 in case further serial dilutions are necessary. (See note after VII 4.)
11. Label as many MTS Anti-IgG Gel Cards® as needed with a patient identifier (name, number, or initials) and the dilutions being tested. One set of wells is designated for the frozen prior specimen (refer to the Quality Control section) and one set is designated for the current specimen.
Note: If the titer is known to be high, choose the 6 dilutions that would show negative results in at least 3 gel microtubes (i.e.: previous titer=32, test tubes #8,16,32,64,128, 256).
12. Remove the foil seal from the entire card.
13. Set the DiaMed ID-Pipettor/ID TipMaster setting at 50 µl.
14. Mix the cell/diluent suspension again by slowly aspirating and dispensing the suspension with the Pipettor.
15. Aspirate enough of the well-mixed 0.8% screening cell/panel cell suspension with the DiaMed ID-Pipettor/ID TipMaster to discard a portion as a primer and still be able to deliver a 50 µl aliquot.
16. Deliver 50 µl of the 0.8% cell suspension to each microtube on the appropriately labeled Anti-IgG gel card(s).
17. Set the DiaMed ID-Pipettor/ID TipMaster to deliver 25 µl.
18. Aspirate enough of the patient plasma dilutions, with the DiaMed ID-Pipettor/ID TipMaster, to discard a portion as a primer and still be able to deliver a 25 µl aliquot.
19. Add 25 µl of the patient specimen dilution to the corresponding microtube(s).
20. Read and record the temperature of the gel card incubator.
21. Incubate the gel card at 37°C for 15 minutes. (Timing may be critical for duplication of previous test, don’t allow incubation time to exceed 15 min.)
22. Electronically retrieve the test. Refer to “Automatic Electronic Orders” or “Manual Patient Demographics & Orders: Retrieval”
23. Go to PW.
24. Click the Red Question Mark. Highlight the test.
25. Click RE. Verify the newly collected specimen.
26. At the end of the 15 minute incubation period, centrifuge the gel card for 10 minutes.
27. Read the front and the back of each microtube macroscopically against a white background and record the reactions on the Gel Card and in the fields in the computer. (Refer to the “Reading and Grading Reactions; Blood Bank” protocol.)
28. If doing a repeat test of a previous antibody titer, type in the specimen collection date of the frozen specimen in the comments section on the Repeat Previous Titer (RptPrvTitr) line.
29. If no previous specimen is available, enter NA in all test reaction fields for the RPTpvrTitr test.
30. If all tested dilutions give positive reactions, it will be necessary to test additional dilutions. These dilutions may be added by using Results (menu bar), Add Optional Subtests.
31. The Add Optional Subtest window opens. Choose the dilutions necessary to complete the testing.
32. The dilution options will appear in numerical order. Up to six additional dilutions will be available to choose.
33. Click OK and the new dilutions will appear on the result screen.
34. Some Titers are known to be higher than 64 and it is not necessary to test more than 6 dilutions. In these cases, it is possible to only test the higher dilutions. Example: Begin with 1:16 and test through 1:512. Add the additional subtests as described above and answer NT (not tested) to the first three dilution result boxes.
35. Record the Interpretation (may use “Find” – the binoculars icon).
Note: Use NA for the RPTpvrTitr test if no specimen is available. Save the results by clicking on the disk icon.
36. Exit by clicking on the small x in the upper right corner.
37. Locate any previous panel(s) completed on the patient to be tested, and include it with the current work-up and titer results for pathologist review.
38. Save the remaining undiluted plasma/serum for future titers.
A. Place the plasma/serum in a plastic tube labeled with the patient’s name and identifier(s), date, time and titer result.
B. Store at or below -18°C for up to twelve months.
IX. Results Derivation:
1. Report the titer result as the highest dilution showing agglutination.
EXAMPLE: A positive 1:16 dilution with a negative 1:32 dilution would be reported as "titer = 16". Report both the current sample titer and the saved sample titer, if a parallel could be done, noting date of draw for each.
2. Samples that no longer show reactivity are reported as <2.
X. Expected Result(s) and/or Critical Values: N.A.
XI. Quality Control:
1. Quality control is performed per protocol. Refer to the “Blood Bank Reagents; Quality Control” protocol. Instruments and equipment are tested as prescribed in the Maintenance and Function Verification procedures.
2. The initial titer of the test plasma/serum is only performed by itself on Doctor's orders. Paired titers (two separate draws) are preferred to document a change in titers. Subsequent draws of the same patient's plasma/serum are titered in conjunction with the plasma/serum most recently drawn, titered and frozen. This will help verify a true increase or decrease in titer. Different lot numbers of reagents and differing antigenic strengths of the selected cells contribute to the titer variations that may be seen. The titer of the stored plasma/serum must be within a range of +/- one dilution of the original titer of that sample. Notify the blood bank Supervisor or a Pathologist if the results of the stored sample do not fall within this range.
1. American Association of Blood Banks; AABB Technical Manual, Bethesda, MD; current edition.
2. Anti-Human Globulin Anti-IgG (Rabbit) MTS Anti-IgG Card package insert. Pompano Beach, FL: Micro Typing Systems, Inc.; current version.
3. MTS Diluent 2 Red Blood Cell Diluent package insert. Pompano Beach, FL; Micro Typing Systems, Inc.; current version.
4. SafeTrace Tx Patient Order Training Manual. 04/03/2001
i. August 1996 S. Rodriguez, N. Combs
ii. April 1997 M. Burger
iii. November 1999 M. Burger (Revised: III.,VII.6-22.,X.)
iv. June 2001 M. Burger (Revised: III., IV., XI.)
v. September 2003 M. Burger, N. Combs, S. Rodriguez
vi. November 2006 S. Rodriguez (Revised: II.;VII.1-4; IX.2; XI.; XII.)
vii. December 2006 S. Rodriguez (Revised: I.Note, II.5.,12.)
viii. June 2010 M. Burger, N Combs (Revised: combined two procedures)