Related Experiment Video
Updated: Dec 20, 2025
Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis
Published on: August 26, 2020
Mutant specific anti calreticulin antibody (CAL2) immunohistochemistry as a screening test for calreticulin (CALR)
Khaliqur Rahman1, Dinesh Chandra1, Manish Kumar Singh1
1Department of Hematology, SGPGI, Lucknow, Uttar Pradesh, India.
Background:
About 50 different CALR frameshift mutations have been identified in BCR-ABL1 negative MPN, all leading to the development of common new protein C terminus. Antibody targeting this terminal epitope can be useful to identify this driver mutation using immunohistochemistry.
Materials And Methods:
CALR mutation analysis was carried out in 51 JAK2V617F negative cases, PMF (n = 22) and ET (n = 29). PCR followed by fragment analysis was performed for molecular detection of CALR mutation. Bone marrow biopsy specimens of corresponding patients were subjected to IHC using mutation specific antibody CAL2. Staining pattern and intensity were observed. Staining of <2% of background nonmegakaryocytic (non- MK) cells were regarded as Pattern A, while staining of more than 2% of background nonmegakaryocytic (non-MK) was regarded as pattern B.
Results:
CALR mutation was noted in 40.9% (9/22) and 41.4% (12/29) of JAK2V617F negative PMF and ET, respectively. All CALR mutated cases, irrespective of the mutation type, showed a positive IHC staining in the megakaryocytes with moderate to bright intensity. All CALR wild-type cases were negative on IHC. (Concordance rate- 100%). Pattern A was noted in 40% cases, while pattern B was noted in 60% cases. Pattern A staining had significantly higher chances of having type 1 mutation as compared to pattern B. In contrast, pattern B had a nonsignificant trend toward higher bone marrow cellularity and marrow fibrosis.
Conclusion:
CAL2 IHC detects all types of CALR mutation. This can act as a sensitive, specific, rapid, and cost-effective screening test for CALR mutation analysis.
Insights
Calreticulin (CALR) mutations are key drivers in myeloproliferative neoplasms. Immunohistochemistry using the CAL2 antibody offers a sensitive, specific, and rapid method for detecting these CALR mutations.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- Calreticulin (CALR) frameshift mutations are identified in BCR-ABL1 negative myeloproliferative neoplasms (MPN).
- These mutations result in a common neo-epitope at the C-terminus of the protein.
- Antibody-based detection of this neo-epitope can identify CALR driver mutations via immunohistochemistry (IHC).
Purpose of the Study:
- To evaluate the utility of mutation-specific antibody CAL2 for immunohistochemical detection of CALR mutations.
- To assess the correlation between CALR mutation status and IHC staining patterns in JAK2V617F negative primary myelofibrosis (PMF) and essential thrombocythemia (ET).
Main Methods:
- CALR mutation analysis was performed on 51 JAK2V617F negative PMF (n=22) and ET (n=29) cases using PCR and fragment analysis.
- Bone marrow biopsy specimens were subjected to IHC using the CAL2 antibody.
- Staining patterns (Pattern A: <2% non-megakaryocytic cells; Pattern B: >2% non-megakaryocytic cells) and intensity were evaluated.
Main Results:
- CALR mutations were detected in 40.9% of PMF and 41.4% of ET cases.
- All CALR-mutated cases showed positive IHC staining in megakaryocytes, while wild-type cases were negative (100% concordance).
- Pattern A staining correlated with CALR type 1 mutations, while Pattern B showed a trend towards higher cellularity and fibrosis.
Conclusions:
- The CAL2 antibody-based IHC test accurately detects all types of CALR mutations.
- This IHC method serves as a sensitive, specific, rapid, and cost-effective screening tool for CALR mutation analysis in MPN.
More Related Videos
05:45Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5
Published on: April 26, 2019
10:21Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells
Published on: January 5, 2018