Abstracts 151

C-7.5 #289

A NEW ASSAY SYSTEM FOR THE ESTIMATION OF RESIDUAL T CELL SUBSETS IN T-CELL DEPLETED BONE MARROW Y Kawanishi, N Flomenberg, CA Keever. Bone Marrow Transplantation Program, Medical College of Wisconsin, Milwaukee, WI.

Nearly all methods for T cell depletion (TCD) of allogeneic donor marrow reduce the incidence and severity of acute graft-versus-host Disease (aGvHD). For some forms of TCD, the advantage of less aGvHD is offset by an increased incidence of graft rejection and leukemia relapse. The degree of TCD as well as the specific subsets of T cells which are removed may be important variables in the overall outcome of the transplant. Most assays used to monitor TCD do not provide information regarding the T cell subsets which remain. For this purpose we have developed an assay system which can discriminate between cells which are: TCRctl3 +, TCRv~ +, CD4 + or CD8 + directly in the wells of a limiting dilution culture plate. A series of 7 dilution's selected to contain 50-1 untreated marrow cells and 1000-10 TCD cells per 20/al are prepared. The dilution's are performed using RPMI medium with 10% human serum, 5/ag/ml PHA, 20 U/ml rlL-2 and 2.5 x 105/ml irradiated feeder PBMC. Each dilution is added to the inner 40 wells of two 72 well Terasaki plates. The plates are incubated at 37°C for 11-14 days in a well humidified atmosphere of 5% CO2 and are scored by microscopic examination for total colony growth. After scoring 5/A of mAb precoated Dynabeads at 0.3 mg/ml are added to the growing wells. The plates are incubated with rotation, at 4°C for 30 min, allowed to settle for 10 min, and are then fixed to a template of samarium cobalt magnets before washing away nonadherent cells with PBS. Wells in which 10 or more cells have bound 2 or more beads are scored as positive. We have used this assay to assess the predictive value of total marrow cells, LDA clonable cells, TCRctI~ + T cells, or TCR,t6 + T cells/kg body weight for aGvHD in patients receiving marrow treated with complement and T loB9, a mAb which has selective specificity for TCRctl 3+ cells. Preliminary results show a dose response correlation of aGvHD only with TCRc~I 3+ cells. This assay system should premit the use of both quantitative and qualitative aspects of TCD marrow in the assessment of clinical outcome.

C-7.5 #290

AUTOMATED HLA-B27 TESTING USING THE FACSPREP/FACSCAN SYSTEM.

W M Reynolds, P R Evans, A C Lane, W M Howell, Wessex

Histocompatibility Group, Southampton University Hospitals NHS Trust, Southampton, UK.

Using the FACSPrep automatic sample preparation system we have developed a test for B27 typing using whole blood which is

both rapid, reproducible and permits large numbers of samples to be screened simultaneously. We have used both an indirect

monoclonal antibody staining technique (Ind. MoAb) and direct

staining (Dir.MoAb). Results were assessed using median channel shift (CS) from the negative control. In 248 cases, usin~

Ind.MoAb staining with HLA-B27, the B7 samples were found to

have a significantly lower CS value than the B27 samples. In

60% of these cases there was a clear distinction between B27 and B7. All B27 and B7 negative samples had a CS of 0 (p <0.001). To further evaluate the status of the remaining 40% 126 samples were dual stained with CD3 and HLA-B27 by Dir.Mo~b staining. From these Dir°MoAb stained samples we can show that all B27 and B7 negative samples have a low CS value (15 maximum), B7 samples have an intermediate CS value (20-55 approx.) and B27

samples have the highest CS values (70 upwards). This approach eliminates, as B27 negatives, 63.5% of samples tested, i.e.

those with a CS of <15. Confirmation of B27 status in the remainder was obtained using PCR-SSOP.