Human Retrovirus Protocols pp 183-189
Detection of HTLV-1 Gene on Cytologic Smear Slides
In this chapter we describe a method for the detection of human T-cell leukemia virus type 1 (HTLV-1) genes in cytologic smears by polymerase chain reaction (PCR). First, already-stained and covered slides should be immersed in xylene for removal of cover slips. After passage through a descending ethanol series, slides are ready for DNA extraction. If the neoplastic cells on slides are mixed with nonneoplastic lymphocytes, cells of interest are isolated by microdissection. Two easy methods to dissect the samples using hydrophobic and hydrophilic mounting media are detailed. Second, microdissected cells are collected in microtubes and digested with proteinase K. The cells that did not undergo the microdissection are digested and dissolve in the proteinase K solution on the slides. Last, the template DNA is extracted from the solution and provided to PCR. We use two sets of primers for detection of HTLV-1 genes, and the products of amplification by PCR that correspond to the pX and tax regions are expected to be 127 and 159 base pairs long, respectively. Although this method does not provide proof of the monoclonal integration of HTLV-1 genes, it can be applied when adult T-cell leukemia/lymphoma is suspected cytologically but fresh samples for Southern blotting are unavailable.
Key WordsCytologic samples HTLV-1 gene pX region tax region polymerase chain reaction microdissection