120 Abstracts

C-7.1 #227

THE USE OF THE DENSITY GRADIENT SOLUTION REVIVA-A-CELL TO REDUCE FALSE REACTIONS IN THE MICROLYMPHOCYTOTOXICITY ASSAY. J Kadushin, M Pickarski, D Lindemuth, W Hillman and P McGrath, Gen Trak Inc., Plymouth Meeting, PA.

A new density gradient media (DGM) product has been used to significantly reduce contamination of lymphocyte preparations (used for HLA typing and other procedures) by non-viable cells and platelets. The DGM is a proprietary composition of tissue culture media and a silica particle suspension that form a solution capable of separating cell populations according toslight differences of relative specific gravity. This media is successfully used for removing dead T and B lymphocytes, monocytes, and other non-viable cell populations from thawed or degraded cell preparations. Platelets and cellular debris are also removed by the same procedure, which is easy to use and can be performed in less than 15 minutes. In one study, 19 of 20 frozen lymphocyte preparations significantly increased viability after a single treatment with DGM. The average pre-treatment viability of the 20 samples was 83.9%, with a range of 21 to 96%. After treatment, average viability improved to 94.6%, with a range of 81 to 99%. This treatment results in the decrease of false positive reactions caused by contaminating non-viable cells, and in fewer false negative reactions due to competitive antibody binding by contaminating platelets and cellular debris.

C-7.1 #228

COMPARISON OF SEROLOGIC DR TYPING TECHNIQUES (IMMUNOMAGNETIC BEADS VERSUS NYLON WOOL) WITH RESTRICTION FRAGMENT LENGTH , POLYMORPHISM. CM Kroning, PJ Santrach, SR DeGoey, SB Moore. Mayo Clinic, Rochester, MN.

Immunomagnetic cell isolation (beads) has been replacing nylon wool (NW) for serologic DR typing. We examined the accuracy of these two methods in comparison to typing by restriction fragment length polymorphism (RFLP).

For 1255 patients, typing was performed using B cells isolated by a NW method followed by standard NIH mierolymphocytotoxieity (MCT). For 475 patients, MCT with two color fluorescence was done using B cells separated with beads. All 1730 patients had RFLP typing performed on TaqI digested DNA hybridized with DR beta, DQ beta, and DQ alpha eDNA probes. DR antigen assignment by RFLP was compared to that by either serologic method for each patient.

Concordance with RFLP

Discordance with RFLP

Serology Indeterminant

RFLP Indeterminant

Serology with Beads Serology with NW No. (%) No. (%)

418 (88.0%) 902 (71.9%)

47 (9.9%) 220 (17.5%)

10 (2.1%) 124 (9.9%)

0 9 (0.7%) X 2 = 54.46, p<0.001

Significant differences between the two serologic methods were seen in these patient subgroups: bone marrow and kidney transplant recipients. No significant differences were noted in other solid organ recipients or any donor group.

Serologic DR typing with immunomagnetie cell isolation is more accurate and more likely to be successful than typing with nylon wool-separated B cells.