Mixed Lymphocyte Culture Assay

Mixed lymphocyte culture (MLC) is a test method for determining the degree of compatibility between a receptor and a donor's major histocompatibility antigen (HLA antigen), and is commonly used for tissue matching prior to organ transplantation. Mixed lymphocyte culture is a two-way mixed culture of donor and recipient lymphocytes, or inactivation of donor lymphocytes to a mixed culture, and the extent of cellular response is negatively correlated with the degree of donor-recipient compatibility. Basic Information Specialist classification: growth and development check classification: immunological examination Applicable gender: whether men and women apply fasting: not fasting Tips: People with high rejection should not do this check. Normal value The morphological counting method conversion rate is <10%. The isotope counting method compares cpm to obtain P>0.05. Clinical significance Abnormal results: In kidney transplantation, the conversion rate of living donors should be <10%; the conversion rate of cadaveric donors should be >10%. Low-reaction matching can only be used in the same batch of experiments. A lymphocyte conversion rate of 10% is compatible between tissues of different bodies. The lymphocyte mixed culture test is used to detect the immune status of the patient. When the number of T lymphocytes is reduced, the conversion rate is also lowered. It can be used as a matching method for organ and bone marrow transplantation to select a suitable donor. If the conversion rate is high, it is used for the difference between the recipient and the recipient. The conversion rate is low and the success rate after transplantation is high. Need to detect people: People with impaired immunity or missing. Precautions At the time of examination: This test can be used as a method for screening donors for organ transplantation. Those with low conversion rate have high success rate and those with high conversion rate have low success rate. Inspection process 1. Preparation of donor lymphocytes: Fresh peripheral venous blood was added to a centrifuge tube containing a lymphocyte separation solution with a specific gravity of 1.077 in a ratio of 1:1, and gently applied to the liquid surface. Centrifuge at 2000 r/min for 20 min to absorb the intermediate halo lymphocyte layer. Wash twice. Trypan blue staining, counting plate count. Adjust the cell density to 2×109/L, divide into 3 tubes, centrifuge separately, and leave a precipitate. Add 250 μL of mitomycin to each tube (final concentration 50 mg/L) And 1.75 mL of physiological saline, water bath at 37 ° C for 40 min, and washed twice with physiological saline. Adjust the cell density to 1 × 10 10 /L with RPMI1640 solution containing 100 mL / L fetal bovine serum. Add the antibody to block the antigen. Mark as D. 2. Preparation of receptor lymphocytes: Fresh peripheral venous blood was added to a centrifuge tube containing a lymphocyte separation solution with a specific gravity of 1.077 in a ratio of 1:1, and gently applied to the liquid surface. Centrifuge at 2000 r/min for 20 min to absorb the intermediate halo lymphocyte layer. Wash twice. Adjust the cell density to 1×1010/L with RPMI1640 medium containing 120 mL/L fetal bovine serum. 3. Mixed lymphocyte culture: The prepared R and D were added to the 96-well plate as follows, and cultured in a 50 mL/L CO2 incubator for 24 h at 37 ° C. Group A: R50 μL + D 50 μL; Group B: R50 μL + D50 μL + IL 2 purification and neutralization of mAb 15 μL ; Group C: R50μL+IL?2 purified and neutralized mAb 15μL; Group D: R50μL. Each group had 3 duplicate wells, and the blank control group was 100μL of RPMI1640 medium. Not suitable for the crowd Not suitable for people: people with high rejection. Adverse reactions and risks There are no related complications and hazards.