Introduction: Flow cytometric cross match is performed to identify antibodies against foreign HLA (Human Leucocyte Antigen) on exposure. This method is nearly 10 to 100 times more sensitive than the traditional CDC-XM (Complement Dependant Cytotoxicity Cross Match), which is considered to be the gold standard and is highly specific.
Donor directed HLA antibodies in the recipient sera is a risk factor for allograft rejection or loss. The titre of these antibodies is proportional to the risk of graft outcome.
Principle: In FCXM, the donor mononuclear cells are incubated with the patient’s serum. This is followed by the addition of fluorescent labelled anti-human immunoglobulin reagent, which are polyclonal antibodies specific to Fc portion of IgG. Antibodies are also added to identify B cells and T cells.
Method: A three colour, single lase (488 nm) flow cytometer is sufficient to perform the flow cytometric cross match. At our centre we use BD FACSCalibur flow cytometer having a green laser and 3 fluorescent channels. The anti-IgG antibodies are tagged with FITC, while CD3 is tagged to PerCP and CD22 to PE. The negative serum helps to identify the cut-off to measure the median channel shift of the patient’s serum. The positive control is used to check for the validity of the test. The mononuclear cells are separated by density gradient method, using LymphoPrep.
We have performed over 200 cases of flow cytometric cross match (FCXM) in the last 2 years, and have seen a spectrum of cases showing positivity for B cells and/ or T cells. We would like to share our experience of these cases with the fellow medical scientists at AIMS NSM.