|Values are valid only on day of printing.|
mRNA is extracted from specimens containing B-cell chronic lymphocytic leukemia and converted to cDNA using reverse transcription. PCR is then used to amplify the IGH gene rearrangements with primers designed to include a portion of the leader segment, all of the variable (V) and diversity (D) segments, and a portion of the joining (J) segment. The predominant amplified fragment is sequenced and evaluated for its ability to produce a functional protein. If functional, the most similar germ line IGH V sequence is selected using the IMGT (ImMunoGeneTics) database and homology is evaluated. Mutations in the entire V segment of the patient sequence are identified and a mutation percentage is calculated. Rearrangements containing a mutation frequency of 2% or greater are interpreted as mutated. Rearrangements containing a mutation frequency less than 2% are interpreted as unmutated.
In some cases, a clonal IGH rearrangement cannot be detected using PCR primers to the leader sequence and primers to the FR1 region of the V segment are substituted. This results in an incomplete V segment sequence for mutation analysis, which is adequate for most cases, but is inadequate in cases with a borderline mutation frequency. These borderline cases will be identified in the report with a comment.
The method used is an unpublished Mayo method, but follows the guidelines put forth by the European Research Initiative on chronic lymphocytic leukemia (ERIC).(Unpublished Mayo method; ERIC recommendations on IGHV gene mutational status in chronic lymphocytic leukemia. Leukemia 2007;21:1-3; Fais F, Ghiotto F, Hashimoto S, et al: Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest 1998 October 15;102(8):1515-1525; Giudicelli V, Chaume D, Lefranc MP: IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucl Acids Res 2005 January 1;33[Database issue]:D256-D291)