CD33SNP Genotyping by Real Time PCR
A real-time polymerase chain reaction (RT-PCR) assay of the CD33 rs12459419 (dbSNP) polymorphism, leveraging TaqMan chemistry to differentiate between C and T alleles. The CC genotype appears to have a substantial response to Gemtuzumab Ozogamicin (GO), whereas, CT or TT genotypes are associated with a suboptimal response to GO. The c.41C>T (p.Ala14Val) variant alters the exonic splicing enhancer (ESE) binding site for SRSF2 which has been associated to skipping of exon2, resulting in the shorter CD33 isoform (D2-CD33), which lacks the IgV domain. Detection of CD33 isoforms without exon2-encoding IgV domain is clinically important because this domain contains the immune-dominant epitope (hP67.6) which is used for diagnostic immunophenotyping, and the target for the antibody that is conjugated to calicheamicin in GO and other CD33 targeted therapies.
DNA extracted from white blood cells in bone marrow, peripheral blood, or other sources (e.g., saliva, buccal cells, FFPE biopsy) are analyzed through RT-PCR reactions using allele-specific TaqMan probes, with genotypes called with TaqMan® Genotyper software V1.4.0 (ThermoFisher Scientific). Assay Performance Characteristics:
- Method: Qualitative.
- Analytical Sensitivity: 100%.
- Limit of Detection (LOD): 0.5 ng.
- Reference: Cell lines and previously analyzed patient samples with known genotypes. This test is designed to determine the genotype of the CD33 rs12459419 polymorphism as CC, CT or TT.
The assay is validated for use in pre-transplant setting only. Genotyping of host DNA in the presence of multiple genomes (transfusion, transplant) is not supported and may likely be non-informative. Correlation with any history of granulocyte transfusion and stem cell transplant is recommended. The assay is performed on a sample that may be involved by acute myeloid leukemia and a loss/gain of one or both CD33 alleles on chromosome 19 (19q13.41) in leukemic cells may result in an altered or inconclusive genotype. Genotyping using an alternative sample without copy number changes involving CD33 may be required for such cases. Correlation with cytogenetics findings for chromosomal alterations is recommended.
5 to 7 working days
- 30 ml peripheral blood in EDTA (purple/pink top) tube, refrigerated
- 2-5 ml of bone marrow aspirate in EDTA (purple/pink top) tube, refrigerated
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