IGH for B-Cell Chronic Lymphocytic Leukemia (B-CLL), Somatic Hypermutation Analysis
Method Description Describes how the test is performed and provides a method-specific reference
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The method used is an unpublished Mayo method, but follows the guidelines put forth by the European Research Initiative on chronic lymphocytic leukemia (ERIC).(Unpublished Mayo method; ERIC recommendations on IGHV gene mutational status in chronic lymphocytic leukemia.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)
PDF Report Indicates whether the report includes an additional document with charts, images or other enriched information
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.