60 Antibody Titration

Principle

Antibody titration is a semi-quantitative method of determining antibody concentration. Serial, two fold dilutions of serum are prepared and tested for antibody activity.  The reciprocal of the highest dilution of plasma that gives a 1+ macroscopic reaction is referred to as the “titer” (ie 1 in 128 dilution; so the titer is 128). In pregnancy, antibody titration is performed to identify women with significant levels of antibodies that may lead to hemolytic disease of the fetus and newborn (HDFN) and, for low-titer antibodies, to establish a baseline for comparison with titers found later in pregnancy. The significance of titers has been sufficiently established only for anti-D (using a saline technique). Titration of non-Rh antibodies should be done only after a discussion about how the data will be used in the clinical management of the pregnancy.

 Specimen

1. Plasma (antibody) to be titrated.
2. As a quality control check, the previous sample on that patient is setup in parallel with the current sample.

Reagents 

1. 2-5% Group O reagent red blood cells. The selection of the most suitable phenotype of red cells to use is controversial. Some labs select double dose expression of the antigen corresponding to the red cell antibody and others select red cells with the phenotype that would be expected in the fetal circulation (ie red cells with a single dose of the antigen). Whichever cells are selected the laboratory should be consistent and use red cells of the same phenotype for future titrations to test the same patient’s serum.
2. Normal saline (dilutions may also be made with albumin if desired).

Procedure for Master Dilution

1. Label 10 test tubes according to the serum/plasma dilution (1 in 1-meaning one volume of plasma, undiluted; 1 in 2-meaning 1 volume of serum in a total volume of 2; etc.).
2. Deliver 0.5 ml. of normal saline to all test tubes except the first (undiluted).
3. Deliver 0.5 ml. of serum/plasma to each of the first two tubes (1 in 1 and 1 in 2).
4. Mix the 1 in 2 dilution.
5. Using a clean pipette, transfer 0.5 ml. of the 1 in 2 tube to the third tube (1 in 4).
6. Continue the same process, mixing and using a new pipette to transfer 0.5 ml. to the next tube. The final dilution will contain 1.0 ml. of diluted serum/plasma. Retain the full amount so the dilution can be continued, if necessary.

Procedure for Antibody Titration

1. Prepare Master Dilution tubes.
2. Label 10 more 10 X 75mm test tubes according to the serum/plasma dilutions, excluding the last tube dilution.
3. Using separate pipettes for each dilution, place 0.1ml (2 drops using BB transfer pipette) of each dilution in its’ respective tube.
4. Add 0.05 ml (1 drop) of 3-4% (commercial) appropriately selected reagent red cells to each tube.
5. Mix each tube gently and incubate at 37oC for 1 hour.
6. The prozone phenomenon may cause reactions to be weaker in the more concentrated serum preparations than in higher dilutions; to avoid misinterpretations of results, the tube containing the most dilute serum should be read first and proceed through the more concentrated samples down to the undiluted sample.
7. Examine the test results macroscopically, with the use of an optical aid. Grade and record results immediately while holding tube in hand.

Interpretation

1. The titer is reported as the reciprocal of the highest dilution that produces a 1+ macroscopic agglutination (reported as “32”, not “1 in 32”).
2. If there is agglutination in the tube containing the most dilute serum, the endpoint has not been reached, and additional dilutions should be prepared and tested.
3. If the undiluted tube gives a weak reaction, and all other dilutions are negative, the titer is reported as 1.
4. If, for example, the 1 in 2 dilution gives a 2+ reaction and the 1 in 4 dilution gives a weak reaction with all other reactions being negative, the titer should be reported as “2”.
5. In comparative studies, a significant difference in titer is two or more dilutions. Variations in technique can cause results that differ by one dilution in either direction. This is one reason the preceding patient sample is run in parallel to the current sample to allow for differing technologist interpretation.
6. A titer of ≥ 16 is considered significant and may warrant monitoring for HDFN (for the prenatal patient). It is recommended that repeat titrations be performed every 2 to 4 weeks.
7. Do not use enhancement techniques because falsely elevated titers may be obtained. LISS should not be used as a diluent in titration studies; nonspecific uptake of globulins may occur in serum-LISS dilutions. Gel testing is not recommended.

Notes

1. Since titration is a semi-quantitative technique, careful pipetting is essential.
2. The age, phenotype and concentration of the test cells will influence the results.
3. Completely reproducible results are virtually impossible to achieve. Comparisons are valid only when previous samples are tested along with the new sample. Frozen previous samples should be well mixed once thawed.