Table 1 Technical considerations for determination of the IGHV somatic hypermutation status of clonotypic IGHV-IGHD-IGHJ gene rearrangements in CLL
From: Immunoglobulin gene sequence analysis in chronic lymphocytic leukemia: updated ERIC recommendations
Item | Recommendations | Remarks |
|---|---|---|
Material | ||
Anticoagulants | EDTA (or CPT) | |
Cells/tissue | Blood, bone marrow, tissue biopsy | Purification of B cells usually not necessary unless low fraction of leukemic cells |
Nucleic acid | gDNA or cDNA | cDNA useful when mutations within the IGHJ gene impair amplification |
Production of template for sequencing | ||
Primers | 5′: leader | VH FR1, VH FR2 and VH FR3 primers are not acceptable |
3′: IGHJ or IGHC | IGHC primers (on cDNA) useful when mutations within IGHJ gene impair amplification | |
Amplification | Multiplex PCR | individual PCR reactions (for each 5′ primer) may be useful when more than one rearrangement found |
Detection of IGH rearrangement | GeneScan or PAGE electrophoresis | Agarose gel electrophoresis strongly discouraged (lack of resolution) |
Cloning | Not necessary | Except in rare circumstances (more than one rearrangement not isolated by simplex PCR) |
Sequencing | ||
Methodology | Direct, both strands | Both strands mandatory for high-quality sequence |
Sequence alignment | IMGT/V-QUEST (www.imgt.org) | Adjustable parameters: (1) search for insertions/deletions; (2) number of accepted D genes |
IGHV identity (%) | Automatic or adjusted | Adjusted: use option ‘search for insertions/deletions’ when low % identity |
Stereotypic subset identification | ARResT/AssignSubsets (bat.infspire.org/arrest/ericll.org/pages/services/tool) | Applicable for the current 19 major BcR stereotyped subsets in CLLa |
