Subject(s)
Leukemia, Myeloid/genetics , Mutation , Myeloproliferative Disorders/genetics , Tumor Suppressor Protein p53/genetics , Acute Disease , Adult , Aged , Aged, 80 and over , Female , Humans , Immunohistochemistry/methods , Leukemia, Myeloid/diagnosis , Leukemia, Myeloid/metabolism , Male , Middle Aged , Myeloproliferative Disorders/diagnosis , Myeloproliferative Disorders/metabolism , Staining and Labeling/methods , Tumor Suppressor Protein p53/biosynthesis , Young AdultABSTRACT
OBJECTIVES: Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods. METHODS: Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF. RESULTS: Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased. CONCLUSIONS: Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.
Subject(s)
Blast Crisis/genetics , Leukemia, Myeloid, Acute/genetics , Mutation , Tandem Repeat Sequences , fms-Like Tyrosine Kinase 3/genetics , Alleles , Electrophoresis, Capillary , High-Throughput Nucleotide Sequencing , Humans , Polymerase Chain ReactionSubject(s)
Bone Marrow/pathology , Diagnosis, Differential , Multiple Myeloma/diagnosis , Multiple Myeloma/pathology , Anemia/etiology , Back Pain/etiology , Biomarkers/metabolism , Biopsy , Bone Marrow/metabolism , Female , Flow Cytometry , Hodgkin Disease/pathology , Humans , Immunoglobulin kappa-Chains/metabolism , Immunohistochemistry , Middle Aged , Multiple Myeloma/complications , Multiple Myeloma/diagnostic imaging , Reed-Sternberg Cells/pathology , Syndecan-1/metabolism , Thrombocytopenia/etiologyABSTRACT
OBJECTIVES: Evaluation of the peripheral blood smear (PBS) is an essential diagnostic test in current medical practice. We aimed to evaluate the use of digital microscopy for the examination of PBS as an option to provide expert interpretation to remote sites and in "on-call" situations. METHODS: We collected 100 Wright-Giemsa-stained PBS slides representing normal and abnormal findings seen at a community-based hospital. Four hematopathologists independently evaluated the cases using conventional light and digital microscopy. RESULTS: When comparing digital vs light microscopy, most of the cellular features evaluated showed at least a moderate degree of agreement in at least three of the reviewers. Discrepancies in final diagnosis were identified in a minority of the cases, most of which were attributed to the poorer resolution of digital microscopy at high magnification (×400). CONCLUSIONS: These results support the limited use of digital microscopy for evaluation and triage of peripheral blood smears as a practical option to obtain expert opinion in locations where experienced staff is not available on site. Our results indicate that while digital microscopy is well suited for basic triage of these blood smears, limitations in quality of imaging at higher magnification as well as large file size may limit its utility in certain settings and situations.
