Hi! My facility performs antibody titers in Ortho pre-buffered anti-IgG cards and Ortho workstation using Universal Gel Protocol. I am not permitted to straight up share my procedure per facility rules, but I can copy+paste the language for the actual recipe. Note that we use double-dose antigen cells when possible, and ALWAYS maintain the dose of reagent cell throughout preg. I recommend purchasing Ortho's Panel B to always keep a double K. Even though Kell doesn't dose, until it does
Principle:
Antibody titration is a semi quantitative method of determining antibody concentration. Serial twofold dilutions of plasma are prepared and tested for antibody activity. The reciprocal of the highest dilution of serum or plasma that gives a 1+ reaction is referred to as the titer (i.e. 1 in 128 dilution; titer=128). In pregnancy, antibody titration is performed to identify women with significant levels of antibodies which may lead to HDFN, and, for low-titer antibodies, to establish a baseline for comparison with titers found later in pregnancy. The titer and the antibody specificity (in the absence of more invasive tests) guide the obstetrician’s decision to deliver the fetus to avoid fetal complications.
Process:
Prepare doubling dilution.
1. Prepare 11 tubes for the master dilution by labeling with the titer "2 4 8 16 32 64 128 256 512 1024 2048"
2. Add 250 uL saline to each tube using a calibrated pipette.
3. To master dilution tube #2, use a calibrated pipette to add 250 uL patient plasma.
4. Discard the pipette tip.
5. With a clean tip, return to master dilution tube #2 and use the pipette to mix contents. Depress and release the plunger in a slow and controlled manner (do not cause frothing) 8-10 times.
6. Express any residual fluid back into the tube and retrieve 250 uL from that tube to be placed in the next tube (tube #4).
7. After expressing the contents of the pipette into tube #4, discard the pipette tip. 8. Using a clean pipette tip, repeat the mixing step in tube #4, and transfer 250 uL of this mixture to tube #8. Discard the pipette tip. Get a clean pipette tip and continue this pattern until you have mixed the contents of tube 2048.
9. Visually inspect the volumes in the tubes. Tubes 2-1024 should have equal volumes (250 uL). Tube 2048 should have a double volume (500 uL). Perform the gel test
10.Label 2 gel cards with the patient’s identifying info.
11.Label 12 wells as follows "neat 2 4 8 16 32 64 128 256 512 1024 2048"
12.For each well, use a calibrated pipette to add 50µL 0.8% reagent red cell. See above for choosing red cell.
13.In the “neat” well, use a calibrated pipette to add 25µL plasma.
14.In the remaining wells, use a calibrated pipette to add 25µL of the master dilution corresponding to each well’s label.
15.Incubate at 37±2°C for the 15 minutes, but no longer than 40 minutes.
16.Centrifuge the gel cards at the preset conditions of 1032±10 RPMs for 10 minutes.
17.Read the front and the back of each microtube macroscopically and record reactions as described in the interpretation section of the corresponding MTS Gel Card package
Results: The titer is reported as the reciprocal of the highest dilution of serum at which 1+ agglutination is observed. A titer ≥64i (anti-D) is considered significant and may warrant monitoring for HDFN by cordocentesis, high-resolution ultra-sound, or examination of the amniotic fluid for bilirubin pigmentation.
Notes:
1. Titration studies should be performed upon initial detection of the antibody.
2. When the decision has been made to monitor the pregnancy by an invasive procedure such as amniocentesis, no further titrations are warranted.
3. For antibodies to low-incidence antigens, consider using paternal RBC’s having established that father carries the low-incidence antigen.
4. Failure to obtain the correct result may be cause by a. Incorrect technique, notably, failure to use separate pipette tips for each dilution. b. Failure to mix thawed frozen plasma.
5. The gel technique is more sensitive than earlier traditional tube methods and typically results 2 dilutions higher than the tube method. The historic literature describing the clinical importance of different titers tacitly assumes the tube method, so caution should be used when referencing texts that do not specify “by gel technique”.
Discarding your tips is a "best practice" from molecular assay pipetting techniques (my work history is in molec genetics before I went to BB). It may truly not make a difference on the macro scale for most blood banking, but we opted to retain the practice of fresh tips to avoid any potential carryover. My facility is a Safety Net hospital with a HUGE OB service line, compete with high-risk preg center, and they use rising titers as a benchmark to determine whether invasive procedures or invasive monitoring of the pregnancy is required. I'm also in the US where maternal mortality is very high, and most of my SOPs have extra protections for pregnant people or people who have the potential to become pregnant.