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Objective:To evaluate the performance of an artificial intelligent (AI)-based automated digital cell morphology analyzer (hereinafter referred as AI morphology analyzer) in detecting peripheral white blood cells (WBCs).Methods:A multi-center study. 1. A total of 3010 venous blood samples were collected from 11 tertiary hospitals nationwide, and 14 types of WBCs were analyzed with the AI morphology analyzers. The pre-classification results were compared with the post-classification results reviewed by senior morphological experts in evaluate the accuracy, sensitivity, specificity, and agreement of the AI morphology analyzers on the WBC pre-classification. 2. 400 blood samples (no less than 50% of the samples with abnormal WBCs after pre-classification and manual review) were selected from 3 010 samples, and the morphologists conducted manual microscopic examinations to differentiate different types of WBCs. The correlation between the post-classification and the manual microscopic examination results was analyzed. 3. Blood samples of patients diagnosed with lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, or myeloproliferative neoplasms were selected from the 3 010 blood samples. The performance of the AI morphology analyzers in these five hematological malignancies was evaluated by comparing the pre-classification and post-classification results. Cohen′s kappa test was used to analyze the consistency of WBC pre-classification and expert audit results, and Passing-Bablock regression analysis was used for comparison test, and accuracy, sensitivity, specificity, and agreement were calculated according to the formula.Results:1. AI morphology analyzers can pre-classify 14 types of WBCs and nucleated red blood cells. Compared with the post-classification results reviewed by senior morphological experts, the pre-classification accuracy of total WBCs reached 97.97%, of which the pre-classification accuracies of normal WBCs and abnormal WBCs were more than 96% and 87%, respectively. 2. The post-classification results reviewed by senior morphological experts correlated well with the manual differential results for all types of WBCs and nucleated red blood cells (neutrophils, lymphocytes, monocytes, eosinophils, basophils, immature granulocytes, blast cells, nucleated erythrocytes and malignant cells r>0.90 respectively, reactive lymphocytes r=0.85). With reference, the positive smear of abnormal cell types defined by The International Consensus Group for Hematology, the AI morphology analyzer has the similar screening ability for abnormal WBC samples as the manual microscopic examination. 3. For the blood samples with malignant hematologic diseases, the AI morphology analyzers showed accuracies higher than 84% on blast cells pre-classification, and the sensitivities were higher than 94%. In acute myeloid leukemia, the sensitivity of abnormal promyelocytes pre-classification exceeded 95%. Conclusion:The AI morphology analyzer showed high pre-classification accuracies and sensitivities on all types of leukocytes in peripheral blood when comparing with the post-classification results reviewed by experts. The post-classification results also showed a good correlation with the manual differential results. The AI morphology analyzer provides an efficient adjunctive white blood cell detection method for screening malignant hematological diseases.
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Objective:To investigate the role of methylation status of glutathione transferase mu5 (GSTM5) promoter region in the occurrence and development of myelodysplastic syndrome (MDS) and provide a new potential molecular marker for the early diagnosis of MDS.Method:Bone marrow blood samples were collected from 40 patients with initial diagnosis of MDS [5 cases of MDS with single dysplasia (MDS-SLD), 7 cases of MDS with multilineage dysplasia (MDS-MLD), 6 cases of MDS with ringed sideroblasts (MDS-RS), 13 patients with refractory with excess blast-1 (RAEB1), 9 patients with refractory with excess blast-2 (RAEB2)], 8 patients with AML secondary to MDS and 6 patients with non-malignant blood diseases(4 patients with iron deficiency anemia and 2 patients with nutritional megaloblastic anemia) in PLA General Hospital from October 2018 to June 2021. Methylation status of the promoter region of GSTM5 gene in three groups were detected by the Agena MassArray nucleic acid mass spectrometry. The Wilcoxon nonparametric test (non-normally distributed data, median (IQR)] was used to compare the methylation levels of GSTM5 gene in different groups. Receiver operating characteristic (ROC) curve was used to evaluate the specificity, sensitivity and accuracy of the test.Results:Cluster analysis showed that the methylation status of GSTM5 in MDS group was higher than that in control group [0.50 (0.27, 0.79) vs.0.29(0.10, 0.45), P<0.05]; The methylation status of GSTM5 in sAML group was significantly higher than that in MDS group [0.67 (0.36, 0.86) vs. 0.50 (0.27, 0.79), P<0.05].The differences in the methylation status of each CpG site were analyzed, and there were statistically significant differences between the MDS group and the control group at CpG_1, CpG_4, 5, CpG_6, 7, 8, CpG_11, CpG_13, 14, CpG_15, CpG_16, CpG_22 and CpG_24 sites ( P<0.05). The results of ROC curve analysis showed that the area under the CpG_6, 7, 8 site curves was the largest, with AUC=0.861(95% CI 0.717-1.000; P<0.05), and the sensitivity and specificity were 85% and 83%, respectively. By analyzing the relationship between GSTM5 methylation and MDS disease development, GSTM5 methylation levels were significantly increased in the higher bone marrow blast group and the high-risk subgroup (RAEB). Conclusion:Aberrant DNA promoter methylation of GSTM5 was a frequent event in MDS and may play an important role in the occurrence and development of MDS. It might be served as a promising biomarker in the diagnosis of MDS.
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Objective This study is aimed to investigate the prognostic significance of ring sideroblast ( RS) in MDS( Myelodysplastic Sydrome ) and evaluate the correlation of RS and other prognostic index.Methods A total of 198 patients with MDS between March 2009 and December 2015 in Chinese PLA′s Gerneral hospital were chosen for this study .Based on the ratio of RS in nucleated red blood cell , patients were first separated into myelodysplastic syndrome without ring sideroblast (MDS RS-) group, RS≥15%, and myelodysplastic syndrome with ring sideroblast ( MDS RS +) group, RS <15%. Then, according to the proportion of blasts in bone marrow nucleated cells above 5%or below, patients were further divided into myelodysplastic syndrome with low blasts without ring sideroblast ( MDS-LB RS-) group, myelodysplastic syndrome with low blasts and ring sideroblast ( MDS-LB RS+) group, refractory anemia with excess blast without ring sideroblast ( RAEB RS-) group and refractory anemia with excess blast and ring sideroblast ( RAEB RS+) groupe.All patients had completed the morphological , genetics , molecular biology examination at dignosis, and followed up by phone.The results of the overall survival (OS) analysis have been presented in a Kaplan-Meier curve and cox regression model .Last, according to the percentage of RS in nucleated red blood cell , patients were separated into RS <5%groupe, 5%-15%group, 15%-40%group, RS≥40%group, and analyse their survival prognosis by statistical methods .Results Comparing to MDS RS-group, the morbidity age, WBC and PLT count were significantly higher [61 ±1.91 vs 52 ±1.37, t=-3.555, P<0.01, 3.82(0.47-323)vs 2.6(0.6-59.7), z=-4.014, P<0.01;139.5(7-608) vs 60(3-724), z =-3.988, P<0.01], bone marrow eythroid hyperplasia and gigantocyte were more obvious in MDS RS+group[χ2 =11.032, P<0.01, χ2 =5.165, P<0.05]; the percentage of GATA1 gene and abnormal rate of poor prognosis gene ( MLL, NRAS, WT1 ) , either mutation or high gene expression , were higher in MDS-LB RS+group than that in MDS-LB RS-( P<0.05 ); Contrasting with RAEB RS-group, the karyotype is worse in RAEB RS +group[χ2 =4.966, P<0.05];Comparing to 15%-40%group, the OS were poorer in RS≥40%;MDS RS+patients were more prone to adverse prognosis than MDS RS-patients.Conclusion Compared to MDS RS-group, MDS RS +patients had worse prognosis;RS maybe correlate to morbidity age , eythroid dysplasia and gene abnormality in affecting the survival prognosis of MDS.
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Objective To study the changes of coagulation function and platelet-related parameters of patients with acute pancre-atitis(AP) and their clinical significance .Methods 36 patients with AP were served as the AP group ,38 healthy people ,the control group .Healthy people in the control group and patients in the AP group at the time of admission and remission were subjected to detection of serum amylase ,lipase ,leukocytes ,neutrophils ,lymphocytes ,platelets ,mean platelet volume(MPV) ,platelet distribution width(PDW),prothrombin time(PT),thrombin time(TT),activated partial thromboplastin time(APTT),prothrombin activity (PTA) ,international normalized ratio (INR) ,fibrinogen(FIB) and D-dimer(D-D) levels .Results Levels of serum amylase ,lipase , TT ,INR ,FIB ,and D-D of patients in AP group were significantly higher than those in the control group(P<0 .01) ,while their PT , PTA levels were significantly lower than those in the control group (P<0 .01) .Differences of leukocytes ,neutrophils ,lymphocytes and MPV of AP patients between at the time of admission and remission were statistically significant (P<0 .01) .Conclusion De-tections of coagulation function and platelet-related parameters changes of AP patients contribute to evaluation of the patients′con-dition and prognosis .
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Objective To determine the antigen-specific CTL in PBMC induced by a fusional family-gene vaccine of the immunoglobulin heavy chain variable gene framework region combined with the sequence of cytokine CM-CSF in vitro with MHC pentamer. Methods Peripheral blood samples were collected from two healthy donors and two patients. One was follicular lymphoma and another was hair cell leukemia. PBMC were isolated by density gradient centrifugalization with Ficoll and then subsequently differentiated into immature DCs (imDCs) induced by recombinant human GM-CSF and recombinant human IL-4. Gene gun was used to deliver the plasmids of the gene vaccine or the control plasmids into the imDCs. RT-PCR and ELISA assay were used to detect IgHVl-GM-CSF mRNA and GM-CSF in order to validate the transfection of the vaccine. After adding the cytokine cocktail, the imDCs became mature DCs. Then the mature DCs were co-cultured with lymphocytes from the blood samples for the induction of the antigen-specific CTL. The cultured cells were classified into vaccine group and control group and harvested at different time points of 0 d,7d, 17 d and 24 d after transfection. The subset of CD3+CD8+ T cells was analyzed by FCM assay. Finally, the CTL levels were detected with fluorescently labeled MHC pentamer antibody targeting vaccine epitopes. Results With the induction of cytokines, the imDCs with typical morphology were generated in PBMC. After delivering, the efficient expressions of the vaccine in the imDCs were determined by RT-PCR. And ELISA results also confirmed that GM-CSF was produced at a level of (28 ±6) ng/106 cells of the imDCs loaded with the vaccine, which was significantly different from that of control group (10 ± 3) ng/106 cells (t = 5. 191, P <0.01). FCM assay result showed that the CD3+ CD8+ T cells increased in a stepwise pattern during the culture. For control group, the levels at 0 d,7d, 17d and 24 d were ( 34. 24 ± 2. 72 )% , (46.06 ± 3.08)%, ( 65. 34 ± 4. 26 )% and (73.86 ±4.85 )% , respectively. For vaccine group, the results were (32. 28 ± 2. 08 ) % , (45. 32 ± 3. 81)% , ( 63. 37 ± 4. 21)% and (75. 01 ±3. 20)%. The differences between each time point had statistical significance (F = 176. 966 ,P <0.01) ,but there was no statistical differences between vaccine group and control group ( F = 0.657,P>0.05). The MHC pentamer analysis showed that the DCs loaded with IgHV1-GM-CSF fusional vaccine could efficiently induce the antigen-specific CTL response and the CTL levels increased gradually with the culture time, with the highest level of 4. 36% in the lymphoma blood and 3. 89% in the hair cell leukemia blood. Conclusions MHC pentamer assay could efficiently determine the antigen-specific CTLs response induced by the gene vaccine of family IgHV frame region in vitro. It could be a useful method for monitoring of anti-tumor cell immunity and evaluating of diagnosis and prognosis of the tumors in clinical application.
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Objective To explore the application of particle-gun technique in transfecting dendritic cells(DCs),and investigate the expression of DNA vaccine particle IgHV1-GM-CSF in DCs induced by particle-gun.Methods Monocytes were isolated from donor peripheral blood collected by Ficoll density gradient method and adherent culture,and then differentiated into immature DCs(imDCs) by rhGM-CSF and rhlL-4 induction.The plasmids for transfection were IgHV1-GM-CSF/pcDNA3.0 and pEGFP,which were prepared with endofree plasmid purification kit.The DCs were transfected with plasmid pEGFP by particle-gun at different transfection conditions,the green fluorescence positive cell and the total cell numbers were counted respectively under fluorescence microscope,and the transfection efficiency was calculated;the viable cell count was performed after trypan blue staining;the transfection efficiencies of particle-gun,liposome-mediated transfection and electroporation method were compared.The plasmids IgHV1-GM-CSF/pcDNA3.0 and pEGFP were cotransfected into imDCs by particle-gun under the optimum conditions,and then DNA was extracted,the expression of IgHV1-GM-CSF was determined by RT-PCR,and of GM-CSF by ELISA.Results The optimum conditions of particle-gun transfection in imDCs were: gas pressure at 120psi,PVP concentration in 0.02mg/ml,microcarrier loading in 0.5mg gold/shot,and DNA loading ratio in 1.5?g plasmid/mg gold.The transfection efficiencies of particle-gun and electroporation were 10.5%?2.4%(n=3) and 45.2%?5.6%(n=3) respectively,while that of liposome-mediated transfection was very low,and significant difference existed among the three methods(P
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Objective To approach the relationship between the membrane protein of platelet microparticles and myocardial infarction or hypertension. Methods The contents were examined of PAC-1~ + PMP and CD62P~+ PMP by activating platelet with 20?mol/L ADP in the blood collected from the patients with myocardial infarction (n=12) and hypertension (n=10). Results The contents of PAC-1~ + PMP and CD62P~+ PMP in myocardial infarction group (85.1%?3.7%, 85.9%?3.8%) were significantly higher than normal group (75.5%?4.4%, 76.3% 5.3%) (P
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Objective To observe the Ca 2+ concentration changes in the platelets activated by chemical agonist in different study group treated with aspirin.Methods We measured the Ca 2+concentration in the platelets activated by different concentration of ADP, thrombin, collagen, arachidonic acid and 5-HT with the flowcytometer in normal health (n=20).We measured the Ca 2+ concentration in the platelets activated by ADP, collagen, arachidonic acid and 5-HT in myocardial infarction (n=20) and hypertension (n=20).We measured the Ca 2+concentration in the platelets activated by arachidonic acid in myocardial infarction (n=20) treated with aspirin.Results As the concentrations of ADP, thrombin, collagen, arachidonic acid and 5-HT increased, the Ca 2+concentration in the platelets increased.There was the relation between agonist concentration and Ca 2+ concentration.Activated by the same agonist, the Ca 2+concentration in the platelets showed more increase in myocardial infarction and hypertension than in normal health.The Ca 2+ concentration show less increase in myocardial infarction treated with aspirin than in normal health.Conclusion The procedure which chemical agonist activating platelets is related to Ca 2+ concentration.The activity of Ca 2+ channels in platelets is high in myocardial infarction and hypertension.Examining the Ca 2+ concentration in platelets is helpful to supervise the aspirin therapy in myocardial infarction.
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Objective To identify and analyze the expression of microRNA miR-181a in HepG2.2.15 cell line transfected by full-length hepatitis B virus genome,and explore the potential role of miR-181a engaged in the development of HBV-related liver disease.Methods Based on the previous data from microRNA microarray,miR-181a specific probe was designed and synthesized.The expressive levels of miR-181a in HepG2.2.15 and its parent cell line HepG2 were analyzed using Northern blot analysis.Some putative targets of miR-181a were predicted by computational software RNAhybrid and confirmed by mRNA microarray.One of the targets was selected and flow cytometry analysis was used to further determine the difference of intracellular HLA-A2 level between the two cell lines.Results Northern blot analysis showed that the expressive level of miR-181a in HepG2.2.15 cell was significantly up-regulated compared with that in HepG2 cell.Some putative targets of miR-181a including C8A,IDH1 and HLA-A were predicted and miR-181a might down-regulate the expression of HLA-A gene via partial complement to the 3'-UTR of HLA-A gene.Consistency with the result of mRNA profile microarray,FCM analysis also showed a significantly lower expression of HLA-A2(43.9%)in HepG2.2.15 cell than that in HepG2 cell(96.6%).Conclusion The expressive level of miR-181a in HepG2.2.15 cell is significantly up-regulated and miR-181a might down-regulate its target gene HLA-A,which might be one of the molecular mechanisms that HBV may escape from the immune response and continue replication in hepatocytes.The knowledge is also helpful for understanding the mechanism of HBV-host microRNA interaction.
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Objective To morphologically evaluate the bone marrow (BM) smear specimens collected from the patients with chronic myelogenous leukaemia (CML) in accelerated phase and blastic phase receiving a short term Gleevec therapy, and to determine its clinical significance in evaluating the changes in disease condition to guide the treatment. Methods Sequential BM smear specimens of 16 Ph positive CML patients, including 9 in accelerated-phase and 7 in blastic-phase, were examined before and 3,6 and 9 weeks after Gleevec treatment (0.4/d or 0.6g/d, PO) with routine method. Periodic acid-Schiff reagent (PAS) staining was performed for a proper identification of abnormal erythroid precursor cells. Results The treatment rapidly caused following conspicuous BM changes in the process: both cellular proliferation and neutrophil granulopoiesis decreased significantly, while the accumulation of erythroid precursor cells increased obviously, and megakaryocytes decreased markedly at 3rd week. A few patients showed no such changes, but an accumulations of erythroid precursor cells, leading to misdiagnosis in some one third of the patients. In 21 percent of patients, in whom no erythroid cells were found, were classified into accelerated phase and blast phase based on both WHO classification system and FAB system. In fact the increase in red system was a bone marrow response to anemia caused by Gleevec therapy. If a condition of severe cellular proliferation of BM with a lower WBC and platelets appeared, most patients could not tolerate the therapy, then it should be ceased. Conclusion Initial treatment with Gleevec therapy exerts pronounced changes in BM morphology, which can be used as a simple and effective method to evaluate the outcome of the patients.
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Objective To study the rule of gene expression profiles of chronic myelogenous leukemia (CML), and to find new sensitive molecules which express specifically in peripheral blood mononuclear cells(PBMC) of CML patients, in order to further elucidate the potential molecular biological mechanisms leading to CML. Methods The expression of mRNA from PBMC of CML patients was compared with that of normal controls using a cDNA microarray. The bioinformatics analysis was used for every up- or down-regulated gene in CML. The new gene with unknown function and no homology to known genes in the database was confirmed, and electric polymerase chain reaction was conducted for the cloning of its full-length DNA in conjunction with Kozak rule and the exit of polyadenyl signal sequence. Sequence specific primers were designed to amplify the new gene from the mRNA of CML PBMC with the reverse transcription PCR (RT-PCR). Results Many genes commonly up- or down-regulated in CML cells were identified. Of these, we found a novel gene with unknown function with high expression in the patients cells. Its nucleotide sequence and corresponding protein-encoding amino acid had been determined, which contained 1 872nt and 624aa respectively. We named the new gene as human chronic myelogenous leukemia associated protein gene (CMLAP), and logged the sequence of the CMLAP gene into the GenBank with the accession number AY762229. Conclusion CMLAP gene expressed highly in CML PBMC was cloned and identified successfully by combining DNA chip technology and bioinformational technology. The gene was likely to play an important role in the disease and might serve as a molecular marker for CML. The findings will pave the way for the further study of the molecular mechanism of CML.
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Objective To establish a flow cytometric method for assay of platelet aggregation induced by shear stress(SIPA) and investigate its clinical value. Methods After the whole blood was subjected to a certain shear stress by a rotational viscometer, the platelets were activated and aggregated. Then the fluorescence antibody CD61 PerCP was added to stain the platelets. The free platelets and aggregates were counted respectively by flow cytometry, thereby the ratio of platelet aggregation was calculated. Results Shear stress-induced whole blood platelet aggregation varied with the increase of shear stress and time of stress. Only a small amount of platelet aggregation was detected at relatively low shear stress of 100S -1, and it was increased at 500S -1. However, a significant increase occurred with 2 000S -1. It approached to the maximum value (58.4%?5.3%) at 3 000S -1 with exposure of 3min, which was higher than the values at 100S -1(9.4%?1.3%)and 500S -1(26.4%?3.2%). At the same time, the aggregates increased in size and became more compact with increase in the shear stress and prolongation of time. High shear stress induced platelet aggregation(H-SIPA) was significantly enhanced in patients with AMI, cerebral infarction, TIA or diabetes, all of them were susceptible to coagulopathy, compared with normal control. Conclusion The results indicated that shear stress could induce platelet aggregation directly, and the degree of SIPA had positive correlation with the stress stength and exposure time.The measurement of H-SIPA was of some clinical value in the diagnosis of arterial thrombosis.