81219
CALR (Calreticulin) Gene Analysis, Common Variants in Exon 9
CPT 81219 involves the molecular pathology analysis of the calreticulin (CALR) gene, specifically targeting common variants located within exon 9. Calreticulin is a multifunctional protein primarily located in the lumen of the endoplasmic reticulum, where it serves as a calcium-binding chaperone. Somatic mutations in the CALR gene were identified as key drivers in the pathogenesis of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), particularly essential thrombocythemia (ET) and primary myelofibrosis (PMF). These mutations are almost exclusively localized to exon 9 and consist of various insertions or deletions (indels) that cause a +1 frameshift. This frameshift results in a mutant protein with a novel, positively charged C-terminus that lacks the endoplasmic reticulum retention signal (KDEL). The mutant CALR protein physically interacts with the thrombopoietin receptor (MPL), leading to its constitutive activation and subsequent stimulation of the JAK-STAT signaling pathway. This uncontrolled signaling drives megakaryocyte proliferation and excessive platelet production. The most common variants are Type 1 (a 52-bp deletion) and Type 2 (a 5-bp insertion). Testing for CALR mutations is a critical component of the diagnostic workup for patients suspected of having MPNs who lack the JAK2 V617F or MPL mutations. Diagnostic identification of these variants is integrated into the World Health Organization (WHO) classification criteria for myeloid malignancies. The procedure involves extracting genomic DNA from peripheral blood or bone marrow aspirates, followed by polymerase chain reaction (PCR) amplification of the exon 9 region and subsequent detection via fragment analysis or Sanger sequencing to characterize the specific insertion or deletion.
Clinical Indications
- Differential diagnosis of BCR-ABL1-negative myeloproliferative neoplasms (MPNs)
- Workup for essential thrombocythemia (ET) in patients negative for JAK2 V617F mutation
- Workup for primary myelofibrosis (PMF) in patients negative for JAK2 V617F mutation
- Evaluation of persistent, unexplained thrombocytosis (platelet count > 450,000/uL)
- Prognostic stratification of patients with myelofibrosis or essential thrombocythemia
- Distinguishing clonal MPNs from reactive or secondary thrombocytosis
Procedure Steps
- Collection of specimen: Obtaining 3-5 mL of peripheral blood in EDTA (purple top tube) or a bone marrow aspirate.
- DNA Extraction: Isolation of genomic DNA from the nucleated cells (leukocytes) of the specimen using standardized lysis and purification techniques.
- PCR Amplification: Utilizing specific primers to amplify the hotspot region of exon 9 within the CALR gene.
- Mutation Detection: Employment of fragment analysis (using capillary electrophoresis to detect size differences in DNA) or DNA sequencing (Sanger or NGS) to identify indels.
- Data Analysis: Comparison of the patient's DNA sequence/fragment size against reference wild-type CALR sequences to determine the presence of a +1 frameshift.
- Interpretation and Reporting: Classification of the mutation (e.g., Type 1, Type 2, or other) and correlation with clinical findings for a final diagnostic report.
Coding Guidelines
- Report CPT 81219 once per specimen, regardless of the number of specific variants within exon 9 being analyzed.
- This is a Tier 1 molecular pathology code; do not use the unlisted molecular pathology code (81479) if this specific code is applicable.
- Ensure the medical record supports the absence of other driver mutations (like JAK2) if following a sequential testing algorithm, although some labs perform these as panels.
- If the testing is performed as part of a larger next-generation sequencing (NGS) panel, refer to genomic sequence analysis codes (e.g., 81445 or 81450) instead of 81219.
- Genetic counseling is often recommended before and after this test, which should be billed using appropriate E/M or counseling codes.