SEQUENCE BASED TYPING OF BLOOD SPOTS ON FILTER PAPER AFTER EXTENDED STORAGE.
DA Fici, S Mahan, J Williamson, R Houranieh, S. Mitchell, ZL Awdeh, The Center for Blood Research and CBR Laboratories, Boston, MA 02115. 

---

The significant increase in accuracy that can be achieved with the modern molecular methods to define the products of the HLA genes is the major contributing factor for the remarkable and rapid transition to this type of methodology. A significant advantage intrinsic to all molecular methods has been the level of efficiency possible due to the limited restriction of sample requirements. The cost of sample collection, storage and transportation has dramatically decreased. We tested whole blood samples collected in ACD or EDTA that were spotted onto filter paper cards "untreated" and treated with FTA. Long term storage effects on this type of matrix were evaluated by testing samples that were stored for thirty months (provided by FITZCO, Inc.). Sample dots were prepared and used as the template for PCR amplification. The amplicons were then directly sequenced for HLA-Class I or HLA-Class II polymorphisms using the Visible Genetics Open Gene System. Sequence specific oligonucleotide probe hybridization patterns confirmed the sequence obtained. Genomic DNA samples showed complete sequence homology when tested in parrallel. The samples stored on cards not treated with FTA were less robust in the PCR, but generated sufficient product to sequence. This simple method for sample collection and storage at room temperature does maintain accuracy and improve efficiency.