Performance evaluation of a novel reticulocyte identification method that uses metachromatic nucleic acid staining based on a crossover analysis of emission DNA/RNA light (RNP Determination™) in hematology analyzer Celltac G.

INTRODUCTION: Assessing the percentage of reticulocytes (%Retic) is useful for diagnosing and treating blood diseases that present with anaemia. The Celltac G+™ hematology analyzer (HA) uses a novel reticulocyte identification method that involves metachromatic nucleic acid staining with acridine orange and crossover analysis of emission light of DNA/RNA (determination of red cells, nucleic acid-containing cells, and platelets, RNP Determination™). The red and green fluorescence generated by stained single-stranded RNA and double-stranded DNA express immaturity and morphological abnormality of erythrocytes by detecting erythrocyte RNA and DNA content. METHODS: The basic performance of the test automated analyzer (TAA) Celltac G+ was evaluated and compared with the flow cytometry reference method and the comparative automated analyzer (CAA) XN-1000/2000™. In addition, its precision, limit of quantity (LoQ), linearity, analytical measurement interval (AMI), accuracy, and comparability and the effects of interfering substances were evaluated. RESULTS: Evaluation of %Retic by the TAA demonstrated good precision and linearity. The AMI was confirmed from 0.02 to 8.23, and the LoQ in %Retic as the coefficient of variation within an 11% limit (SD, within a 0.01 limit) was 0.14. TAA correlated well with the reference method and routine HA (CAA). Some deviations were found between TAA and CAA in DNA measurements of erythrocytes from abnormal samples. CONCLUSION: Celltac G+ uses a novel measurement principle and can assess erythrocyte immaturity independent of DNA contents. It represents a new HA that provides novel, useful information on immaturity and morphological abnormality of erythrocytes.

Detection of residual disease in chronic myeloid leukemia utilizing genomic next generation sequencing reveals persistence of differentiated Ph+ B cells but not bone marrow stem/progenitors.

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring BCR-ABL1 fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were BCR-ABL1-negative. Moreover, in some patients BCR-ABL1 was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.