Somatic Hypermutation Analysis, IGH, Sequencing
Detection of the rate of mutation in the expressed IGH variable region of the B-cell tumor clone for use in prognostication between pre-germinal center and post-germinal center subsets of chronic lymphocytic leukemia.
RNA is extracted from white blood cells in bone marrow, and/or peripheral blood and reverse transcribed to cDNA. Multiplex PCR amplification of IGH transcripts is done using consensus VH primers. DNA sequencing of the PCR product is done and compared to germline IGH sequences in the EMBL database.
Requires that the B-cell clone comprise at least 10% of the leukocytes in the sample. Some samples with adequate clonal B-cells (approximately 10%) will be uninterpretable due to mutations in the primer binding sequences.
Five to 10 working days
- 10-30 ml peripheral blood in lavender top (EDTA) tube, sent on wet ice
- 2-5 ml of bone marrow aspirate in lavender top (EDTA) tube, sent on wet ice
- 15 µg of purified RNA, sent on dry ice
- 10 µg of cDNA, sent on dry ice
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