IGH Somatic Hypermutation Analysis, B-Cell Chronic Lymphocytic Leukemia (B-CLL)
Method Description Describes how the test is performed and provides a method-specific reference
RNA is extracted from B-cell chronic lymphocytic leukemia specimens and converted to cDNA using reverse transcription. PCR is then used to amplify the IGH gene rearrangements with indexed multiplex 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. A check gel is performed to make sure that there is amplified product. If no amplified product is visible a second PCR is performed with a different multiplex primer set targeting the framework 1 (FR1) regions of the V genes. FR1 PCR rearrangements are shorter in length than leader primer products and result in slightly truncated 5' V-region sequence coverage. The amplified product is then purified and the DNA concentration measured. Pooled patient samples (identifiable by the index bar codes) are subjected to sequencing on the Illumina-MiSeq platform. FASTQC sequence data is subsequently analyzed using proprietary software to identify the IGHV rearrangement and the unique sequence. Results are compared to a germline IGVH sequence database by the software to calculate the percent identify of the tumor IGHV rearrangement to the closest germline sequence. 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.(Warren EH, Matsen IV FA, Chou J: High-throughput sequencing of B- and T-lymphocyte antigen receptors in hematology. Blood 2013 Jul 4;122(1):19-22; Schumacher JA, Duncavage EJ, Mosbruger TL, et al: A Comparison of Deep Sequencing of TCRG Rearrangements vs Traditional Capillary Electrophoresis for Assessment of Clonality in T-Cell Lymphoproliferative Disorders. Am J Clin Pathol 2014 Mar;141(3):348-359; Blachly JS, Ruppert AS, Zhao W: Immunoglobulin transcript sequence and somatic Hypermutation computation from unselected RNA-seq reads in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2015;112(14):4322-4327)
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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.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Up to 2 weeks
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test