A somatic 52-bp deletion in the CALR gene is detected in buccal epithelial cells. (A) Table summarizing the detection of CALR mutation by NGS and Sanger sequencing in various tissues. (B-C) NGS of diagnostic BM (B) and buccal cells (C) visualized through Integrated Genome Viewer. Sequence reads are aligned to the CALR locus. Gray bars represent read alignments. White gaps in alignment represent areas of deletion. (D) Cytospin preparation of buccal cells stained with Diff-Quick (Romanowski) stain. (E-G) Paraffin-embedded buccal cell block stained with hematoxylin and eosin (E), cytokeratin AE1/AE3 (F), and CD45 (G). Original magnification ×40 in panels D-G. (H) The CALR locus was amplified by polymerase chain reaction in PB, buccal mucosa, BM, CD3-selected PB lymphocytes, CD15-selected PB myeloid cells, and hair follicles from the patient and in a normal tissue control obtained from an unrelated donor. The amplified product was separated by electrophoresis on an agarose gel and stained with ethidium bromide. The wild-type and 52-bp–deleted CALR bands are indicated. (I) Sanger sequencing traces of the wild-type (upper panel) and mutant (lower panel) buccal sample, mapped to the reference CALR sequence. Bu, buccal mucosa; H, hair follicles; Ly, CD3-selected PB lymphocytes; My, CD15-selected PB myeloid cells; NC, normal tissue control.