Clinical Study on Dynamic Detection of Minimal Residual Disease in Acute Myeloid Leukemia by Multiparameter Flow Cytometry / 中国实验血液学杂志

OBJECTIVE@#To investigate the prognostic significance of dynamic detection of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) by 8-color flow cytometry.@*METHODS@#MRD of 282 AML patients who achieved remission after initial therapy was detected by 8-color flow cytometry. MRD threshold for predicting recurrence was determined by receiver operating characteristic (ROC) curve, and time from MRD-positive to clinical recurrence was analyzed. The differences in overall survival (OS) time and relapse-free survival (RFS) time of patients with different MRD-changes were compared, and the related factors of recurrence in patients with MRD-negative were analyzed by univariate and logistic regression analysis.@*RESULTS@#ROC curve determined that the MFC-MRD threshold for predicting the recurrence of AML was 0.105%, and the recurrence rate of MRD-positive patients was significantly higher than that of MRD-negative patients [52.45% (75/143 cases) vs 35.97% (50/139 cases), P=0.005]. The patients in MRD persistent positive group and negative to positive group recurred earlier than those in positive to negative group and negative-positive fluctuation group (P<0.005). Survival analysis showed that OS and RFS time of patients with MRD persistent positive were significantly shorter than those of patients with MRD persistent negative, positive to negative, and negative-positive fluctuation (P<0.005). There was no significant difference in OS and RFS between MRD negative to positive group and MRD persistent positive group (P>0.005), either between MRD persistent negative group and MRD positive to negative group (P>0.005). Among 139 MRD-negative patients, 50 recurred. Univariate and logistic regression analysis showed that the risk of recurrence increased with the increase of white blood cells level (95%CI: 1.000-1.013, P=0.045). The risk of recurrence in patients without hematopoietic stem cell transplantation (HSCT) was 9.694 times higher than that in patients who received HSCT (95%CI: 1.720-54.651, P=0.010), and in the high-risk group was 5.848 times higher than that in the low-risk group (95%CI: 1.418-24.121, P=0.015).@*CONCLUSION@#The prognosis of AML patients with different MRD changes is significantly different. No matter MRD-positive or MRD-negative at the initial remission, dynamic detection of MRD after treatment is more helpful to accurately guide treatment.

Killing Effect of A CD7 Chimeric Antigen Receptor-Modified NK-92MI Cell Line on CD7-Positive Hematological Malignant Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the killing effect of NK-92MI cells modified by chimeric antigen receptor (CD7-CAR) and specifically targeting CD7 to CD7 hematological malignant cells.@*METHODS@#Three types of hematological malignant tumor cells, including 5 cases of CD7 acute T-lymphoblastic leukemia (T-ALL), 10 cases of acute myeloid leukemia (AML) and 6 cases of T-cell lymphoma were collected, centrifuged, cultured and used to detect the expression levels of tumor cell surface targets; 7-AAD, CD56-APC, CD3-FITC, IgG Fc-PE flow cytometry were used to detected the transfection efficiency of NK-92MI and CD7-CAR-NK-92MI cells, killing efficiencies of CD7-CAR-NK-92MI cells to CD7 hematological tumor cells in vitro were determined by flow cytometry using PE Annexin V Apoptosis Detection Kit. Secretion differences of NK-92MI and CD7-CAR-NK-92MI cytokines interleukin (IL)-2, interferon (IFN)-γ, and granzyme B detection were estimated by using CBA kit.@*RESULTS@#The killing efficiencies of CD7-CAR-modified NK-92MI cells to CD7 T-ALL, AML, T-cell lymphoma tumor cells were significantly higher than those of NK-92MI cells without genetical modification. The difference showed statistically significant (P＜0.05). The level of IFN-γ and granzyme B were significantly increased among cytokines secreted by CD7-CAR-modified NK-92MI cells as compared with those of NK-92MI cells without genetical modification (P＜0.05) .@*CONCLUSION@#CD7-CAR-modified NK-92MI cells have significantly improved killing efficiency against CD7 T-ALL, AML and T lymphoma cells, and shows specific targeting effects, which provides a clinical basis for the treatment of CD7 hematological malignancies.

Research on the C-terminal domain of ADAMTS13 regulates its cleaving activity / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the influence of C-terminal domain of ADAMTS13 on its cleaving activity.</p><p><b>METHODS</b>The full-length wild-type (WT) and C-terminal domain truncated type (TT, TSP8 + CUB domains were deleted) of human ADAMTS13 recombinant protein were transfected into and permanent expressed on Hela cells. Western blot and R-CBA were used to directly detect the activities of the two recombinant proteins under the static and stressed condition respectively. ELISA was used to compare the binding abilities of the two proteins by coating with vWF.</p><p><b>RESULTS</b>The recombinant proteins were identified by Western blot with anti-his-tag or anti-ADAMTS13 antibodies. With pretreatment of 1.5 M urea, the enzyme activity of TT was significantly higher than that of WT, and so did in binding ability with vWF While, only WT could cleave vWF under high stress.</p><p><b>CONCLUSION</b>The distal carboxyl-terminal TSP8 together with CUB domains of ADAMTS13 may affect the enzyme activity by regulating the binding of ADAMTS13 to vWF in different conditions, and they are very important for the enzyme activity under high stress force condition.</p>

Evaluation and clinical application of a new method for detecting ADAMTS13 activity / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, so not easily adapted to routine laboratories. ADAMTS13 cleaves von Willebrand factor (VWF) within the domain A2, located between domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structures of VWF before and after the cleavage. Using this hypothesis we try to establish a new and simple method to determine ADAMTS13 activity.</p><p><b>METHODS</b>First, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies, SZ-129 and SZ-125, which specifically recognize the VWF A1 and A3 domains by using a two-site sandwich ELISA. Compared with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibody in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated.</p><p><b>RESULTS</b>Plasma ADAMTS13 activities in normal people and TTP, acute myocardial infarction (AMI), and idiopathic thrombocytopenic purpura (ITP) patients determined by the new assay were (89.75 +/- 7.93)%, (17.63 +/- 18.71)%, (68.55 +/- 18.08)%, (85.83 +/- 9.84)%, respectively. Results were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays. The new assay can easily discriminate a TTP plasma sample from a non-TTP plasma sample (P < 0.01), and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activity of ADAMTS13 autoantibody ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient.</p><p><b>CONCLUSION</b>This new and simple assay for ADAMTS13 activity could be used routinely in the clinic to determine the activity of ADAMTS13.</p>