Objective: To analyze the laboratory characteristics in patients with acute megakaryoblastic leukemia(AMKL).Methods: The immunophenotypes of leukemia cells in 28 patients with AMKL were analyzed by means of 4 tubes of 8 color panel. Meanwhile, bone marrow morphology,cell chemistry,chromosome karyotype and gene were examined.Results: Among 28 AMKL patients, the highest positive rates were macrokaryocyte-associated antibodies: CD41a, CD61, CD42b, CD36. The positive rates were 81.48%, 92.86%, 72.00% and 70.83%, respectively. Among them, 53.57% of the patients expressed CD41a, CD61 and CD42b, and 82.14% of the patients expressed at least two kinds of antibodies. The positive expression rates of CD117, CD34, CD38 and HLA-DR were 64.29%, 42.86%, 64.29% and 46.15% respectively, which were lower in AMKL patients than in non-APL patients (P< 0.01). There was no significant difference between the positive expression rates of CD13 and CD33 in AMKL and non-APL AML patients. CD15, CD64, CD14, CD300e and MPO, cCD79a and cCD3 were all negative. Compared with non-Down syndrome-related AMKL (non-DS-AMKL), the expression of CD7 and CD11b was higher in Down syndrome-related AMKL (DS-AMKL) (P < 0.05). Among AMKL patients, 17 (65.4%) had complex chromosome karyotypes and 5 had + 21 chromosome abnormalities; only 5 had normal karyotypes. Twenty-five leukemia patients were screened for fusion genes. WT1 gene expression increased in 24 patients (96%) and 12 patients (70.58%) with EVI1 gene expression increased (53.93 < 37.98%). Four patients were positive for fusion genes (2 MLL-AF9, 1 TLS-ERG and 1 P210 BCL/ABL).Conclusion: 82.14% of AMKL patients express at least two megakaryocyte-related markers. The expression of myeloid progenitor cell markers is relatively low, most of which are complex chromosomal karyotype abnormalities. The abnormal expression rates of WT1 and EVI1 are higher.
Objective: To investigate the characteristics and prognostic significance of Ki-67 antigen expression at diagnosis in adult t(8;21) acute myeloid leukemia (AML).Methods: Bone marrow samples of 57 adult t(8;21) AML patients were collected at diagnosis from July 2012 to February 2019 in our hospital, their frequencies of Ki-67 in CD34 + cells were detected by flow cytometry method, and the relationships between Ki-67 and the biological characteristics, therapeutic effect and relapse were analyzed. Results: Of all patients tested, the median percentage of CD34 +Ki-67 + cells were 30.5% (range: 10.0%~65.8%); A receiver operating characteristic (ROC) curve was used to identify the optimal cutoff levels of CD34 +Ki-67 + cells, higher frequencies of CD34 +Ki-67 + cells were significantly related to higher rate of c-KIT mutation and lower WT1 transcript level at diagnosis (P=0.001; P=0.042). Of all patients followed up, higher frequency of CD34 +Ki-67 + cells was significantly related to a higher 1-year cumulative incidence relapse (CIR) rate (P=0.035). In addition, both lower WT1 transcript level at diagnosis and higher level of minimal residual diseases (MRD, < 3-log reduction of the RUNX1-RUNX1T1 transcript level after the second consolidation therapy) were significantly related to a higher 1-year CIR rate (P<0.000 1; P=0.041), and patients with c-KIT mutation and white blood cell count>10×10 9/L at diagnosis tended to have higher 1-year CIR rates, respectively (P=0.091; P=0.054). Patients simultaneously with high frequency of CD34 +Ki-67 + cells and high MRD levels have a significantly higher 1-year CIR rate than others (P<0.000 1). Conclusion: In adult patients with t(8;21) AML, high frequency of CD34 +Ki-67 + cells at diagnosis may be a poor prognostic factor, combination of MRD levels and frequency of CD34 +Ki-67 + cells at diagnosis may better predict relapse than MRD alone.
Objective: To explore the expression of CD11c antigen in patients with chronic lymphocytic leukemia(CLL),and its clinical diagnostic value,as well as the correlation of CD11c with genetic abnormalities.Methods: 200 cases of CLL,49 cases of MCL were detected for CD11c expression rate and mean fluorescence intensity(MFI)by Multi parameter flow cytometry (FCM).And the relationship between CD11c expression and prognostic markers (ZAP-70 and CD38) expression in 200 CLL patients were compared.At the same time,the fluorescence in situ hybridization(FISH) was used to detecte P53 deletion, 13q14 deletion, ATM deletion, 6q23 deletion,+12 and IGH rearrangement.Then the genetic abnormalities were compared between CD11c - and CD11c + CLL patients. Results: The CD11c positive rate in CLL patients was 49.5%(99/200),MFI median 2.06(1.00 to7.34).While the CD11c positive rate in MCL patients was 6.12%(3/49).MFI median 2.00(1.97 to 2.54).The positive rate of CD11c expression were significantly higher in CLL patients than in MCL patients(x 2=30.62,P<0.05).The positive rate of ZAP-70 and CD38 were significantly higher in CD11c +CLL patients than CD11c -CLL patients(x 2=15.472,P<0.05;x 2=11.556,P<0.05). The results of P53 deletion, 13q14 deletion,ATM deletion,6q23 deletion,+12 and IGH rearrangement were all not significantly different between CD11c - and CD11c + CLL patients. Conclusion: CD11c has significant value for assistant diagnosis of CLL,especially for the differential diagnosis between CLL and MCL.
Objective: To assess if therapeutic drug monitoring (TDM)of imatinib used for chronic myelogenous leukemia (CML) patients led to improve clinical outcome and reduce side effects compared to empiric adjustments.Methods: Patients followed between 2013 to 2018 at CML Center of Xiangya Hospital are performed. Primary outcomes are composite remission at 3, 6, 12 and 18 months in those with empiric adjustments versus TDM-guided optimization.Results: There are 51 patients who are included in the study. Among them, 35 are in the experimental group and 16 are in the control group. The results show that when taking imatinib 400mg/d, the serum level is 568.00~3 989.66ng/ml, the mean (standard deviation): 1 716.46ng/ml (788.96); when taking imatinib 300mg/d, serum level is 720.89~1 497.11ng/ml, mean (standard deviation): 971.67ng/ml (204.02). The serum level at the time of achieving a stable molecular response is higher than that the time when the stable molecular response is not achieved. There are significant differences in adverse reaction ratings between the two groups. The incidence of adverse reactions of grade III and above in the experimental group is significantly smaller than that of the control group.Conclusions: There is a large individual difference in the steady serum level of imatinib, and this individual difference is related to efficacy and adverse reactions. TDM can reduce the adverse effects while ensuring efficacy in the treatment of CML patients. The results still require further validation of large sample clinical trials. The reasons for individual differences in imatinib drug metabolism require further investigation of large sample pharmacogenetics studies.
Objective: To explore the importance and clinical indications of high-sensitivity assay detecting PNH clones by flow cytometry and the incidence and clinical significance of PNH clones in patients with hematopenia.Methods: FLAER combined with CD24 or CD14 was used to detect the percentage of PNH clones in granulocytes and monocytes, and CD59 was used to detect the percentage of PNH clones in erythrocytes of 20 healthy volunteers and 1 095 patients with hematopenia. Meanwhile, CD59 negative percentage in granulocytes and monocytes of 31 patients was detected by traditional CD59 method.Results: According to background of volunteers and numbers of cells, limited of detection (LOD) of granulocytes, monocytes and erythrocytes were 0.04%, 0.10% and 0.05%, respectively. The median percentages of FLAER -CD24 - granulocytes, FLAER -CD14 - monocytes and CD59 - erythrocytes were 0.02%, 0.02% and 0.03% in 827 patients, separately. The proportions of PNH clones in granulocytes were above LOD in 318 patients. Among of 180 patients with PNH clones below 1.00%, the consistent rates of granulocytes with monocytes and erythrocytes were from 43% to 45%. When the percentages of PNH clones in granulocytes were more than 1.00%, the consistency rates of granulocytes with monocytes and erythrocytes were around 90.00%. Comparing the percentage of CD59 negative granulocytes and monocytes with that of FLAER and anchor protein double negative, the former was significantly lower than the latter (P< 0.000 1 and P=0.000 9). The percentages of FLAER single negative granulocytes were higher than that of FLAER and anchor protein double negative cells in 26.85% patients. The similar results occured in monocytes of 24.54% patients. The patients with PNH clones below 0.10% were affected more easily. PNH clones were 90.30% (44.49%~99.05%) in 36 patients with PNH and 1.30% (0.10%~96.07%) in 50 patients with MDS or AA, separately (P<0.000 1). When PNH clone was 41.81%, the sensitivity and specificity of PNH diagnosis were 100.0% and 96.0%, respectively. Conclusion: In this study, the LOD of granulocyte and erythrocyte was 0.04% and 0.05% respectively. When the proportion of PNH clones in granulocytes is more than 1.00%, the positive results of monocytes and erythocytes are more consistent. PNH disease is more likely when the proportion of PNH clones is higher than 41.81%.
Objective: To establish normal reference range of peripheral blood lymphocyte subsets in healthy adults in Shanxi Province and provide theoretical basis for studying immunity status and assessing immune function in cancer patients.Methods: Selected 1 238 healthy adults in Shanxi Province and used six-color monoclonal antibody to detect and analysis absolute and percentage counts of peripheral blood lymphocyte subsets.Results: The expression of peripheral blood lymphocytes levels was determined, and it was found that CD3 +T、CD4 +T、CD8 +T、NK、CD19 cell percentage and absolute counts and CD4/CD8 ratio dissimilarity with a P value <0.05 was considered statistically significant ;and there was no statistical significance in CD8 + T cell percentage counts、CD4 +T cell and CD19 cell absolute counts between different genders,CD3 +T cell、CD8 +T cell、NK cell percentage and absolute counts、CD4 +T cell、CD19 cell percentage counts dissimilarity with a P value <0.05 was considered statistically significant. Conclusion: The refe -rence range of peripheral blood lymphocyte subsets in healthy adults in Shanxi Province was preliminarily established ,which provided reference for the evaluation of immunity function, tumor immunotherapy and diagnosis.
Objective: To investigate the effect of peripheral blood lymphocyte subsets and clinical characteristics on the prognosis of hematologic malignancies.Methods: The peripheral blood lymphocyte subsets of 64 patients with newly diagnosed hematologic malignancy were detected by flow cytometry. Diseases included Acute myeloid leukemia (AML)、Acute lymphoblastic leukemia (ALL)、Hodgkin’s lymphoma (HL)、non-Hodgkin’s lymphoma as bone marrow infiltration (NHL). We analyzed the difference of peripheral blood lymphocyte subsets between 30 normal patients and the experimental group. And We analyzed the relationship between the changes of peripheral blood lymphocyte subsets and prognosis in 21 patients with acute leukemia who underwent continuous dynamic monitoring in 64 patients with malignant hematological diseases.Results: No significant difference was shown in lymphocyte subsets among different age groups of adult patients with malignant hematopathy. The percentage of CD3 +CD8 + T lymphocytes and T-reg cells in patients with malignant hematopathy increased, while the percentages of CD16 +/CD56 +NK cells, and CD4 +/CD8 + ratio were all decreased. The number of CD3 +T lymphocyte, CD3 +CD4 + lymphocyte, CD3 +CD8 + lymphocyte, CD19 + lymphocytes, CD16 +/CD56 + lymphocyte and CD4 +/CD8 + ratio were all decreased. There were some differences in peripheral blood lymphocyte subsets between acute leukemia and malignant lymphoma patients and control group. The proportion of T-reg cells in non-remission group of acute leukemia was significantly higher than that in remission group of first course of treatment of acute leukemia and control group. The proportion of T-reg cells in relapsed acute leukemia group was remarkably higher than that in sustained remission group and control group. In 21 patients with acute leukemia, we found that T-reg cells in remission group gradually decreased during chemotherapy and gradually approached the control group during the third to sixth courses of treatment. T-reg cells in non-remission group increased gradually or continued to exceed 10% in the course of chemotherapy, which was significantly higher than remission group. Furthermore, T-reg cells shown a trend of first subtraction and then addition during the chemotherapy in recurrence patients. Conclusion: The immune function of patients with malignant hematological diseases is significantly lower than that of healthy people, and accompanied by immune dysfunction. In addition, different types of diseases and different disease states brought about different degrees of immune disorders.the proportion of T-reg cells can be used to predict the curative effect and recurrence in patients with hematologic malignancies, for providing guidance basis for clinical treatment and medication intensity.
Objective: To analyze the clinical significance of CD177 + neutrophils in ulcerative colitis (UC) by comparing the expression differences of CD177 + neutrophils in peripheral blood of UC patients with normal controls. Methods: Peripheral blood of 30 UC patients and 20 normal controls were collected, and the expression of neutrophils CD177 + was detected by flow cytometry, and compare the expression difference of CD177 in the two groups. Results: The expression of CD177 + neutrophils in peripheral blood of UC patients was significantly higher than the normal control group (P<0.01), the expression of CD177 + neutrophils in peripheral blood of UC patients was significantly higher than that of the mild UC patients (P<0.05), and the count of CD177 + neutrophils in peripheral blood of UC patients was correlated with Mayo score (r=0.384,P=0.036). Conclusion: The expression of CD177 + neutrophils in UC patients was significantly increased and closely related to the activity of the disease, which may reflect the clinical activity of UC.
Objective: To explore the effects of TNF-α on apoptosis and autophagy of human placenta fetal derived mesenchymal stem cells (hfPMSCs) and the regulation of autophagy on apoptosis.Methods: The expression of CD73, CD90, CD105, CD14, CD34 and CD45 was identified by flow cytometry analysis for fPMSCs cultured in serum-free medium. hfPMSCs were treated with TNF-α at a final concentration of 20 g /L for 24h. Annexin V/PI double staining assay detected cells apoptosis; Each group of total protein was extracted, the expression of autophagy marker protein LC3 Ⅰ/Ⅱ was tested by Western blot; mRFP-GFP-LC3 adenovirus was used to infect fPMSCs, the formation of the puncta light was observed by confocal fluorescence microscopy; hfPMSCs were infected with Atg5 interfering lentivirus (si-Atg5) and negative control lentivirus (si-NC), and Annexin V/PI double staining was used to detect cells apoptosis.Results: The cultured cells possessed typical MSCs morphology and belong to CD73 +CD90 +CD105 +/ CD14 -CD34 -CD45 - cells. Annexin V/PI staining results showed that after TNF-αtreatment for 24 h, the apoptosis number and rate of hfPMSCs were higher than those in the control group (P<0.05). Compared to untreated group, TNF-α increased the deposition number of LC3 Ⅱ (P<0.05),and induced more aggregation of puncta light in cytoplasm.Compared: with the control group, TNF-αinduced apoptosis of hfPMSCs was significantly elevated in autopahgy inhibition group (P<0.05). Conclusion: TNF-αinduced autophagy can inhibit the apoptosis of hfPMSCs, play an important protective function.
Flow cytometry, which measures cells,particles or aggregates in flow, is able to provide a large number of single-cell measurements with statistical significance within a short time. After more than 70 years of development, it has become an indispensable technology in many fields including biology, medicine and environmental surveillance. This technology was put forward in the 1940s and formed in the 1970s. In the following 4 decades, the detection performance, multi-parameter measurement ability and sorting ability have been significantly improved. Especially its application fields have expanded rapidly from bacteria measurement, environmental microorganism detection, conventional cell function detection to the diagnosis and monitoring of many clinical diseases, and up to the cutting-edge fields of cancer immune mechanism research, immunotherapy and biopharmaceuticals discovery. At present, a series of new technologies and new types of corresponding flow cytometers have been developed. Although their working principles are different more or less from the traditional method, the concept of obtaining large-scale single cell multi-parameter measurements has never changed. Currently the field of flow cytometry is on the edge of a major development and revolution.
Cytomorphological test is a traditional experimental diagnostic technique for hematological diseases, which is convenient, fast and practical. It is the most intuitive basis for pathological diagnosis, while the current optical microscopy technology can not fully meet the needs of accurate diagnosis of hematological tumors. How to develop, research and improve the related technologies and maximize the advantages of morphology is a problem worthy of our discussion and research. The application of cytomorphological technology in early diagnosis, curative effect, prognosis evaluation and disease recurrence of Hematological neoplasms is systematically reviewed. It is believed that Cytomorphological technology will bring new opportunities for the diagnosis and treatment of hematological system tumors.
Mixed-phenotype acute leukemia (MPAL) is a rare type of acute leukemia, in which the blasts express lineage specific antigens of more than 1 lineage. The incidence of MPAL comprises 2% to 5% of all cases of acute leukemia. Clonal chromosomal abnormalities can be detected in about 59% to 91% of MPAL patients, such as t(9;22)(q34;q11)/BCR-ABL1 and t(v;11q23)/KMT2A-rearrangment, which play a prominent role in the diagnosis and prognosis of these disorders. More recently, molecular approaches have been useful in further characterizing this group of diseases, such as whole genome sequencing, whole exome sequencing and next generation sequencing. ZNF384 fusions are common in B/My MPAL and WT1 mutations are common in T/My MPAL, which provide potential biological insights and may have clinical implications for this disease. This review aims to provide a brief overview of the recent advances in MPAL genetics.
Precision treatment is the present therapeutic strategy to leukemia, and accurate diagnosis and prognostic stratification is the basis. At present, leukemia typing is based on the combination of morphology, immunotyping, cytogenetics and molecular biology. In the past decades, due to the identification of a lot of new molecular abnormalities, molecular detection is becoming more and more important in the diagnosis and treatment of leukemia. The basic techniques for molecular detection are PCR and sequencing. The common leukemia related molecular markers include fusion gene, gene mutation and gene overexpression, and they have shown important clinical significances in the diagnosis, the selection of target therapy, the monitoring of measurable residual disease (MRD) and prognosis.
Currently, cell therapy of Chimeric antigen receptors (CAR) has been widely used in the treatment of leukemia and lymphoma.CD19 and CD22 targeting CAR-T have shown significant efficacy in the treatment of recurrent and refractory acute B-lymphoblastic leukemia (RR-B-ALL) and other hematologic diseases. However, the progress is slow in the treatment of T-lineage tumors. This review introduces the current domestic and international use of CAR cell technology and CRISPR/Cas9 gene coding technology to design T-ALL CAR cell and the preliminary exploration of CAR cell immunotherapy in the treatment of T-lineage acute lymphoblastic leukaemia.
