4.1
#109
COST-EFFECTIVE HLA AMBIGUITY RESOLUTION USING PEL-FREEZ® AMBISOLV™ PRIMER MIXES.
C. Carozza BS , G. T. Hill, MS , L. Mead BS , L. Gallagher BS , L. Scott BSMT, CHS and S. Hsu Ph.D. . Philadelphia PA, American Red Cross, Penn-Jersey Region, 19123, Histocompatibility/Molecular Genetics Department .

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One major obstacle facing laboratories that perform intermediate-level HLA-DNA typing by SSOP is the resolution of generic ambiguities. Each week, our laboratory must resolve Class I ambiguities for 5% - 15% of our total sample volume, which substantially increases the amount of time, labor, and money required to complete typing. Conventionally, generic ambiguities are resolved using group specific amplification, in-house designed SSP primers, or commercially-available SSP trays. In the past, we have used all three strategies, but since 1999 we have primarily utilized commercial SSP trays. While an effective method, only a few primer sets included on the tray are necessary to resolve the SSOP ambiguities. Pel-Freez® Ambisolv™ Primer Mixes allow laboratories to customize cost-effective trays for specific ambiguity resolution. In addition to decreasing overall reagent costs, thermocycler space is conserved, gel electrophoresis is minimized, and analysis time is reduced. Previously, the resolution of eight HLA-B*51 vs. B*52 ambiguities would have required four 96-well thermocyclers, and electrophoresis of eight gels. With Ambisolv™ Primer Mixes, only thirty-two wells of a 96-well thermocycler are needed, and one gel is analyzed. Additionally, the required DNA volume is reduced by approximately 90%; this is critical for typing of blood-spotted filter paper samples, which yield limited DNA. The currently available Ambisolv™ Primer Mixes allow efficient resolution of our ten most common Class I ambiguities. The flexibility afforded by these mixes will allow laboratories to meet the increasing demand of ambiguity resolution due to new typing methods and continued allelic expansion.