Monday, September 27, 2010 2:00 PM - American Society for ...

CD138, HLA-DR, IgM, and IgD. The contribution of Fc receptors was assessed by CD23, CD32, andCD16. Cellular activation was analyzed by CD69. HLA expression level was quantified by monoclonalantibodies to monomorphic epitopes on either class I or II (G46-2.6 or Tü39).Results: On CD3+ cell subsets CD4+, CD4+/CD25+, CD8+CD56-, CD8+CD56+ HLA class I expressionlevel correlated linearly to MCS in serum with class I DSA. In serum with class II DSA, CD19+ cellsshowed a high degree of correlation between HLA-DR expression and MCS. For CD3+ cell subsets, theCD69+ activated form of each subset had elevated HLA expression. While HLA class I and II expressionlevel varied on CD19+ cells, no one marker correlated with higher or lower HLA expression. However onCD19+ cells the level of Fc receptors (FcR) CD32 and CD23 correlated with bound IgG in sera withoutDSA. CD56+/CD16+ cells also showed a high degree of bound antibody in serum without DSA thatcorrelated with CD16 expression level.Conclusions: Different subsets of T and B cells have variable levels of HLA expression, which affect theMCS in and FCXM. This suggests that variation in relative proportions of these subsets could influenceFCXM results. Similarly, variation in FcR expression influences nonspecific antibody binding in a densitydependent manner.3-PPREDICTIVE VALUE OF USING LabScreen MIXED BEADS TO DETERMINE DILUTIONSNEEDED FOR QUANTITATIVE ANALYSIS ON LabScreen SINGLE ANTIGEN BEADS.P. Brailey, E. Portwood, A.L. Girnita. Transplantation Immunology Division, University of Cincinnati,Cincinnati, OH, USA.Aim: When analyzing solid organ transplant patients for HLA antibodies, the LabScreen Single AntigenBeads can be saturated with antibody. There are numerous consequences of bead saturation, with theinability to observe patient treatment response being one of the most significant. This inability requires theuse of dilution studies which are time and reagent intensive, and expensive. The aim of this study was todetermine if LabScreen Mixed Bead reagents could be used to determine the dilutions needed for SingleAntigen Bead analysis.Methods: LabScreen Mixed Beads (One Lambda, Inc.) were run against 4 renal patients at serial dilutionsfrom Neat to 1:1024 for both HLA Class I and Class II. The serum samples were first treated with DTT anddiluted using phosphate buffered saline (PBS). MFI values for either an immuno-dominant or donorspecific antibody were plotted on a graph. The graph was analyzed for the dilution values that were at thelower end of the linear portion of the graph. These values were used for dilutions of the LabScreen SingleAntigen Beads (One Lambda, Inc.) for that patient.Results: Mixed Bead dilution studies predicted that 3 of the 4 patients were diluted at 1:256 or 1:1024.LabScreen Single Antigen Bead studies at this dilution were successful for monitoring the patient’s therapyresponse. Mixed Bead dilution studies predicated for 1 patient successful dilution at 1:64, however thispatient actually required LabScreen Single Antigen Bead dilution at 1:2048.Conclusions: LabScreen Mixed Beads can be used to predict the dilutions needed for quantitative analysison LabScreen Single Antigen Beads for many patients. Care must be taken to observe that dilution studiesaccount for two linear curves on patients with extremely high levels of HLA antibodies.4-PUSE OF QUANTIPLEX BETWEEN INSTRUMENTS: WORTH THE BOTHER?Megan Brown, Anne Halpin, Luis Hidalgo, Patricia Campbell. Histocompatibility Laboratory, AlbertaHealth Services, Edmonton, AB, Canada.Aim: Many HLA laboratories use Luminex methodologies to identify HLA antibodies. QuantiplexBeads (One Lambda Inc) are reference beads for standardization of fluorescent signal. They are acquiredwith single antigen beads (LabScreen® products, One Lambda Inc) used to identify antibodies to HLAclass I (LSA1 beads). Quantiplex beads are intended to correct differences in instrument performance.Luminex output is measured as median fluorescence intensity (MFI); Quantiplex beads convert MFI tostandard fluorescence intensity (SFI) by creating a linear standard curve from Quantiplex bead values.We investigated whether Quantiplex beads provide standardization for the same samples run on twoLuminex instruments.