Diagnostic Application of Flow Cytometry in Phosphotyrosine Level Detection and Disease Status Evaluation in Patients with Bcr-abl Positive Leukemia.
Authors: Sun Xue-mei , Chen Jun-hao , Chen Lei-lei , Xu Jing-yan , Yang Yong-gong , Xie Wei 
Published: October 2019

Abstract
Currently, karyotype analysis for chromosome translocation of 9 and 22 and FISH or gene amplification-based technique for bcr-abl fusion gene have been the major strategies for the diagnosis and disease status evaluation of bcr-abl positive leukemias. However, the method for aberranttyrosine kinase level, which is the reflection of cellular function, is limited to the detection of the level of phosphorylated tyrosine by Western blot. This method is technically laborious, costly and sometimes leads to feigned results. We thus tried to set up a flow cytometry based technique to evaluate its possible utilization in the diagnosis and the evaluation of the disease status in bcr-abl positive leukemias. Mononuclear cells from heparinized bone marrow of patients were collected. After treatment with hemolysin, 106 cells were fixated, permeabilized and then incubated with fluorescein isothiocyanate (FITC) conjugated anti-phosphotyrosine monoclonal antibody (PY20). Fluorescence conjugated microspheres were put in the system as a calibration factor in order to provide a quantitive value for the sake of comparison with different detections. The geomean value of fluorescent microsphere was adjusted to 20 in each case and the ratio of the geomean value of each detected sample to 20 was read as relative value of phosphotyrosine level. This relative value was used in statistical analysis. The human leukemia cell line Mo7e and its derivation Mo7e P210 expressing human bcr-abl fusion gene were employed as negative and positive samples to optimize the experimental conditions. Phosphotyrosine protein in fresh bone marrow cells from patients with chronic myelogenous leukemia (CML) in different phases, bcr-abl positive acute lymphoblastic leukemia (ALL) and normal controls were evaluated by semi-quantitative analysis. A total of 32 CML, including 22 in chronic phase(CP), 3 in accelerated phase(AP) and 7 in blast crisis(BC) were enrolled in this study. Four patients with bcr-abl positive ALL were also included. It was found that there was a significantly different geomean value between Mo7e and Mo7e P210. The relative values of phosphotyrosine levels were different between bcr-abl positive samples and normal controls. The value was 756±144, 865±96, 847±105 and 819±225 with CML patients in CP,AP,BC groups and ALL group respectively, while it was only 18±7 in control group. Statistical analysis showed a significant difference among these five groups. The comparison between each two groups showed a significant difference between each bcr-abl positive group and control group (P< 0.01). There were also significant differences between the AP/CML, BC/CML or ALL group and CP/CML group(P< 0.01). We observed a trend that the value of a given patient escalated with disease progression. In a patient who was on imatinib treatment for one month, there existed two peaks of cells with different fluorescent intensity, each representing a bcr-abl positive or negative cell group, coincident with the FISH result of 158 bcr-abl positive cells in 1000 detected cells. We concluded that flow cytometry analysis of the phosphotyrosine level could be used as an adjuvant technique for the diagnosis and differential diagnosis of bcr-abl positive leukemias. It may also play an important role in the evaluation of disease status or in the judgment of therapeutic efficacy during patient follow-up.