Effects of Leukapheresis on Hemostatic Function in Patients with Hyperleukocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To analyze and compare the effects of leukapheresis on hemostatic function in patients with hyperleukocytic leukemia.@*METHODS@#A total of 139 patients with AML, ALL and CML who underwent leukapheresis from June 2009 to February 2020 and did coagulation test before and after operation were included in this study. The clearance efficiency of each group and the difference among three groups were evaluated, as well as hemostatic function including platelet counts, coagulation indicators, CDSS score and incidence of adverse events. The difference of hemostatic function caused by leukapheresis in different leukemia patients were compared.@*RESULTS@#After leukapheresis, the WBC counts were decreased significantly in the three groups of patients (P<0.001), and the clearance efficiency was highest in ALL patients. However, the platelet counts also were decreased significantly (AML:P＜0.001, ALL: P＜0.001, CML: P＜0.01) in the three groups of patients, particularly for acute leukemia patients with a positive correlation with WBC clearance efficiency(r=0.284). After leukapheresis, fibrinogen decreased, PT and APTT prolonged. For acute leukemia patients, higher CDSS score was related to an elevated incidence of bleeding events (P<0.05).@*CONCLUSION@#Leukapheresis is an effective method to decrease the leukemic burden, but it is necessary to monitor the impact on hemostatic function. It is recommended to assess the CDSS socre for acute leukemia patients, in order to identify the predictive value for bleedings.

Diagnostic Value of G and GM Tests in Acute Leukemia Patients with Invasive Fungal Disease / 中国实验血液学杂志

OBJECTIVE@#To analyze the diagnostic value of (1, 3) -β-D-glucan and galactomannan (GM) tests in the patients with acute leukemia complicated by invasive fungal disease, and explore the application of serological detection (G/GM) and lung CT for early diagnosis of invasive fungal disease (IFD).@*METHODS@#A total of 493 patients with acute leukemia complicated by high risk invasive fungal infection, also receival G and GM tests, in Department of hematology of our hospital from June 2016 to December 2016 were selected and were divided into IFD-confirmed group (62 cases) including confirmed and clinical diagnesed IFD, and IFD-unconfirmed group (431 cases) including suspected IFD and non-IFD according to the diagnostic criteria of IFD. The results of G and GM tests in patients of 2 groups were analyzed, then the diagnostic efficacy of G and GM done and combination evaluated. In addition, 26 patients whose lung CT negative at hospitalization, moreover, presentation of changes in lung by CT during hospitalization and serological G and GM test positive were selected, and the difference of time between serological that postive and presentation of changes in lung by CT were compared for the estimation of early diagnotic value.@*RESULTS@#The positive rate of (1, 3) -β-D-glucan in IFD-confirmed group and IFD-unconfirmed group was 56.5% and 10.4%, respectively. Meanwhile, that of galactomannan test was 41.9% and 9.0%, respectively. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of (1, 3) -β-D-glucan was 56%, 90%, 44% and 92%, and that of galactomannan was 42%、91%、40% and 93%, respectively. Moreover, the combination of (1, 3) -β-D-glucan and galactomannan could raise the sensitivity to 69% and specificity to 98%. The positive results of serological detection (G/GM) come earlier about five days than CT changes.@*CONCLUSION@#Both (1, 3) -β-D-glucan and galactomannan test have high sensitivity and specificity, and the combination of them can improve the diagnostic efficacy, and make the clinical antifungal therapy more precisely. In the early clinical diagnosis of IFD, the positive results of serological detection coming earlier than lung CT.

Clinical study of unrelated umbilical cord blood transplantation for 25 patients with hematological diseases / 中国实验血液学杂志

This study was purposed to investigate the engraftment, graft-versus-host disease (GVHD), transplantation related mortality (TRM), relapse and survival in hematologic patients received unrelated umbilical cord blood transplantation (UCBT). A total of 25 patients with hematological disease underwent UCBT, including 8 pediatric and 17 young adult patients. Among them 3 cases received single unit of UCBT and 22 cases received double units of UCBT. For donor/recipients human leukocyte antigen (HLA) matching: HLA 6/6 loci matched in 9 cases, HLA 4-5/6 loci matched in 16 cases. There were 19 patients with hematologic malignancies, including 3 cases in the period of disease progression and 6 cases of non-hematologic malignancies. Conditioning regimens were TBI/Cy ± Flu ± ATG or BuCy ± Flu ± ATG for 21 patients and Cy+Flu+ATG for 4 patients. For prophylaxis of acute graft-versus-host disease (aGVHD) the regimen of cyclosporine (CsA) as dominant drug was used. The results showed that among 16 patients (80.0%) achieved engraftment, 20 patients survived for more than 42 d after transplantation. The cumulative neutrophil recovery rate on day 42 after transplant was 64.0%, with a median time of 17.0 d;the cumulative platelet recovery rate on day 100 after transplant was 60.0 %, with a median time of 35.0 d. The cumulative rate of grade II-IV and III-IV aGVHD after transplantation 100 d was 44.0% and 30.7%, respectively. Until the end of the follow-up, the cumulative rate of TRM was 54.3%. For all the patients, overall survival rate was 42.7%. Out of 17 evaluable patients with hematologic malignancies 7 cases (41.2%) survived to date, and only 1 case relapsed, so event-free survival rate was 35.3%. Out of 5 evaluable patients with non-hematologic malignancies, 4 patients survived and 2 patients were in stable engraftment state, 2 cases with autologous hematopoietic recovery. Among 3 cases of hematologic malignancies at advanced stage, only 1 case survived to date. It is concluded that HLA-4-6/6 loci matched UCBT is an effective option to treat hematological diseases. Double cord blood transplantation (dUCBT) can overcome the disadvantage of insufficient cells of single cord blood UCBT to treat overweight children and adult.

Quantitative monitoring of multi-donor chimerism after multi-donor allogeneic hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was aimed to establish a model for detecting the donor chimerism rate following the multi-donor hematopoietic stem cell transplantations, and simplify its calculation method. Patients with hematologic disease receiving allogeneic hematopoietic stem cell transplantation including single-donor and multi-donor were selected in this study and the donor cell chimerism rates were detected, using STR-PCR combined with capillary electrophoresis. The results indicated that the peaks of the sister alleles coming from the same individual were confirmed to have the approximate areas and can be replaced each other in the situation of mixed chimerism. In the calculation model, the value between reference chimerism and approximate chimerism have no significant difference using the hypothetical peak areas, and the result was confirmed to be accepted basing on typical measurement error between sister alleles (5% - 20%). It is concluded that the areas of share peaks can be replaced by non-share peaks and this conclusion can be used to calculate the double-donor CHM (DD-CHM)(%). Compared to the D alleles, R alleles show more strategic importance because it can lead to more accurate result and allowed simplifying the arithmetic calculations for DD-CHM(%).

Three times spontaneous remission of severe aplastic anemia following granulocyte transfusion from related donors: a case report and literature review / 中国医学科学杂志(英文版)

Aplastic anemia (AA) is a bone marrow failure disease caused by abnormal activation of T lymphocytes, resulting in the apoptosis of hematopoietic cells and bone marrow failure. Currently, hematopoietic stem cell transplantation (HSCT), immunosuppressive - therapy (IST), and supportive care (e.g. transfusion adjuvant therapy, hematopoietic growth factors, and prevention of infection) are the main treatments of AA. Granulocyte transfusion has recently been accepted as an useful adjuvant therapy of HSCT and intensive IST. This article reported a severe AA patient who failed to respond to IST, but achieved spontaneous remission three times after granulocyte transfusions from related donors. Such cases have rarely been reported. Existence of human leukocyte antigen (HLA) cross between the patient and his relatives may influence the T cell-mediated immunity, which might explain this patient's recovery.

Establishment of murine allogeneic umbilical cord blood cell transplantation model and study on the mechanism of keratinocyte growth factor enhancing immune reconstitution after transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the impact and mechanism of keratinocyte growth factor (KGF) on immune reconstitution post murine allogeneic umbilical cord blood transplantation (UCBT).</p><p><b>METHODS</b>Perpheral blood (PB) from 19.5-day embryos post-conception (E 19.5 d) mice was used as umbilical cord blood (UCB) graft. Thirty-two BALB/c mice were randomly assigned to 4 groups with 8 mice each in the first cohort UCBT. Mice were infused with PBS (control group) or 1 x 10(6) (group 1A), 2 x 10(6) (group 1B), 3 x 10(6) UCB mononuclear cells (MNCs) (group 1C), respectively. Twenty-four BALB/c mice were randomly assigned to 3 groups with 8 mice each in the second cohort UCBT. Mice were injected with 1 x 10(6) (group 2A), 2 x 10(6) (group 1B) or 3 x 10(6) (UCB) MNCs (group 2C). All mice received platelet transfusion on +8d. Sixteen BALB/c mice were randomly assigned to 2 groups with 8 mice each in the third cohort UCBT. Mice were injected s.c. with KGF (group 3) or PBS (control group) before TBI. All mice were injected with 2 x 10(6) UCB MNCs and were supported with platelet transfusion on +8 d. The survival time, splenic lymphoid cell subsets, sjTREC assay were observed after UCBT.</p><p><b>RESULTS</b>The 100-day survival of mice were 2, 3 and 3 in group 1A, 1B, 1C and 7, 8, 8 in group 2A, 2B, 2C, respectively. The splenic T, NKT, NK and B cell counts on +35 d were (9.57 +/- 0.74) x 10(6), (0.64 +/- 0.06) x 10(6), (1.43 +/- 0.10) x 10(6) and (19.13 +/- 1.50) x 10(6) in control group, respectively; while were (13.47 +/- 0.74) x 10(6), (0.89 +/- 0.03) x 10(6), (1.79 +/- 0.04) x 10(6) and (20.50 +/- 0.91) x 10(6) in group 3, respectively, being significantly higher than in control group. The sjTREC level was 182.2 +/- 10.7copies per 10(5) cells in control group; while was 224.2 +/- 9.6 copied per 10(5) cells in group 3, being significantly higher than in control group (P = 0.019).</p><p><b>CONCLUSIONS</b>Peripheral blood from E19.5d is rich in hematopoietic stem cells. A murine allogeneic UCBT model with platelet support on +8 d is established. KGF treatment can enhance thymic output and improve T cell immune reconstitution after UCBT.</p>

Preliminary study on 5-azacytidine anti-myeloma activity in vitro / 中国实验血液学杂志

This study was aimed to investigate the effect of 5-azacytidine (5-AZA) on XAF1 expression in myeloma cells and efficacy of 5-AZA treatment for myeloma in vitro. XAF1 expression was analyzed by semi-quantitative PCR. Methylation-specific PCR (MSP) was used to detect the methylation status of XAF1 promoter CpG islands. RPMI 8226 and XG-7 cells were treated with 0-5 micromol/L of 5-AZA. Expression of XAF1 mRNA variants was confirmed by gel electrophoresis. The results indicated that the untreated RPMI 8226 cell expressed XAF1 mRNA transcript 1 and transcript 2, untreated XG-7 cells did not express XAF1 mRNA. Hypermethylation of XAF1 promoter CpG islands could be detected in both cell lines. Both cell lines expressed full-length XAF1 transcript after being treated with 2.5 micromol/L of 5-AZA for 72 hours. 5-AZA treatment led XAF1 promoter CpG island to hypomethylation in both cell lines. 5-AZA exerted anti-myeloma activity in a time- and concentration-dependent manner. The IC(50) value of XG-7 cells treated with 5-AZA for 48 hours was 2.6 micromol/L. 1.0, 2.0, 2.5 and 5.0 micromol/L of 5-AZA treatment for 48 hours induced (34.3 +/- 8.0)%, (54.8 +/- 3.1)%, (64.1 +/- 3.4)%, (81.0 +/- 4.1)% apoptosis in XG-7 cell line respectively. The combination of 1.0 - 4.0 micromol/L of 5-AZA with 1.0 - 4.0 micromol/L of arsenic trioxide (ATO) exhibited synergistic toxicity in myeloma cells with all CI values less than 1.0. It is concluded that lack of XAF1 expression and abnormal expression of XAF1 in myeloma cell lines are associated with the hypermethylation of XAF1 gene promoter CpG island. 5-AZA treatment can induce the expression of XAF1 mRNA and protein in myeloma. 5-AZA exerts anti-myeloma activity via apoptosis at clinically achievable concentrations. The findings suggested that 5-AZA and ATO may be an effective combination in the therapy of patients with multiple myeloma.

Clinical study of allogeneic hematopoietic stem cell transplantation for relapsed/refractory acute lymphocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the efficacy and toxicity of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for relapsed/refractory acute lymphocytic leukemia (ALL).</p><p><b>METHODS</b>Forty-seven patients with relapsed/refractory ALL received allo-HSCT, which containing 19/47 from HLA-identical sibling donors (sib-HSCT), 18/47 from HLA-identical unrelated donors (URD-HSCT) and 10/47 from haplo-identical donors (Hi-HSCT). Conditioning regimens included "TBI plus Cyclophosphamide (Cy) (42/ 47)" or "busulfan (Bu) plus Cy (5/47)". Cyclosporine (CsA) combined with a short-course Methotrexate (MTX) were used for graft versus host disease (GVHD) prophylaxis. In addition, patients receiving URD-HSCT or Hi-HSCT were given mycophenolate mofetil (MMF) and anti-thymocyte immunoglobulin (ATG). Patients with molecular or cytogenetic relapse tendency on minimal residual disease (MRD) monitoring received donor lymphocyte infusion (DLI).</p><p><b>RESULTS</b>All patients tolerated the therapy well except for mucositis. Renal dysfunction occurred in 2 patients on CsA therapy. Epilepsy occurred in 1 patient, fatal infectious complications in 9 (including 3 interstitial pneumonia), grade III-IV acute GVHD (aGVHD) in 7, chronic GVHD (cGVHD) in 22 and hemorrhagic cystitis (HC) in 4 patients. Thirteen patients relapsed after transplantation. The median time of hematopoietic reconstitution was + 17 ds. Nineteen patients received DLI, and 6 of them had no disease progression. With a median follow-up duration of 43 (10-77) months, the estimated 5-year overall survival (OS) and disease free survival (DFS) rates were 49.65% and 46.55%, respectively.</p><p><b>CONCLUSION</b>Allo-HSCT is an effective therapy for relapsed/refractory ALL. Relapse after transplantation, fatal infection, and severe acute GVHD are the main causes for failure. DLI might decrease the relapse rate after transplantation.</p>

Rosiglitazone and all-trans retinoic acid inhibit human myeloma cell proliferation via apoptosis signaling pathway modulation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of artificial ligand of peroxisome proliferators activated receptors (PPARs), rosiglitazone (RGZ) and all trans-retinoic acid (ATRA) on human myeloma cell line growth in vitro and in vivo.</p><p><b>METHODS</b>U266 and RPMI 8226 cells were treated with different concentration of RGZ in the presence or absence of ATRA and the results were studied by 3H-TdR thymidine incorporation (for cells proliferation), Annexin V-PI staining and caspase-3 activity assay (for cells apoptosis), RT-PCR (for FLIP, XIAP and survivin mRNA expression), and tumor formation test in BALB/c nude mice.</p><p><b>RESULTS</b>Exposure to RGZ induced proliferation inhibition in a dose-dependent manner in both U266 (r = 0.991, P < 0.01) and RPMI 8226 cells (r = 0.961, P < 0.01). A combination of RGZ with ATRA could enhance the inhibition effect (P < 0.001 in U266, P < 0.01 in RPMI8226). 10 micromol/L of RGZ induced apoptosis of (9.8 +/- 1.7)% in U266 cells and (10.7 +/- 3.3)% in RPMI8226 cells, in a time and dose dependent manner and combined with ATRA intensified the apoptosis induction effects (P < 0.01 in both cell lines). The FLIP, XIAP and survivin mRNAs were expressed in both cell lines and their levels decreased significantly after cultured with RGZ. The addition of RGZ + ATRA in the culture further decreased the levels. Caspase-3 activity increased substantially with the increase of RGZ concentration and the addition of RGZ + ATRA in the culture medium showed similar synergism effect on caspase-3 activation (P < 0.01). The xenograft of U266 cells in BALB/c nude mice were inhibited by RGZ and so did more by the combination of RGZ and ATRA (P < 0.01).</p><p><b>CONCLUSION</b>The down-regulation of FLIP, XIAP and Survivin induced by RGZ can activate caspase-3, whereby induced apoptosis and proliferation inhibition in myeloma cells. ATRA can enhance these effects of RGZ.</p>

Differentiating effect of PPARgamma ligand rosiglitazone and all trans-retinoic acid on myeloma cells and its possible mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of PPARgamma ligand (rosiglitazone, RGZ) as well as combined with all trans-retinoic acid (ATRA) on human myeloma cells and try to explore the possible mechanism.</p><p><b>METHODS</b>Human myeloma cell lines U266 and RPMI-8226 cells were treated with RGZ in the presence or absence of ATRA. Cell proliferation was evaluated by [(3)H] thymidine incorporation, cell cycle distribution and CD49e expression were analyzed by flow cytometry, morphology changes were evaluated by Wright-Giemsa staining, and p27(Kip1) and p21(Waf1) expression was detected by Western blotting.</p><p><b>RESULTS</b>The exposure to RGZ induced proliferation inhibition in both cell lines in a dose-dependent manner. After cultured with 5 micromol/L RGZ, the proportion of U266 and RPMI-8226 cells in phase G(0)/G(1) was (45.2 +/- 6.7)% and (40.3 +/- 7.3)%, respectively (P < 0.05). The proportion of the cells in phase G(2)/M and S was (52.2 +/- 7.4)% and (57.4 +/- 9.5)%, respectively (P < 0.05). These changes were more evident when the RGZ concentration was increased to 10 micromol/L. A combination of RGZ with ATRA enhanced the growth inhibition and cell cycle arrest effects of RGZ. The RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression. The expression of p27(Kip1) and p21(Waf1) in myeloma cells was up-regulated by RGZ and this change was more apparent when RGZ was used in combination with ATRA.</p><p><b>CONCLUSION</b>RGZ can induce cell cycle arrest and cell differentiation in myeloma cells which maybe caused by up-regulation of p27(Kip1) and p21(Waf1) expression. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs may show a synergistic effect on myeloma cells.</p>

Relationship between CMV reactivation and KIR haplotype/HLA-Cw genotype in patients after unrelated-donor hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the relationship between CMV reactivation and KIR haplotype or HLA-Cw genotype in patients after unrelated-donor hematopoietic stem cell transplantation (HSCT).</p><p><b>METHODS</b>From January 2003 to December 2008 the HLA-Cw/KIR genotype of 48 patient-donor pairs were determined by polymerase chain reaction with sequence specific primers (PCR-SSP) and sequence specific nucleotide (PCR-SSOP). Posttransplant CMV reactivation was performed by immune histochemically assay.</p><p><b>RESULTS</b>Of 48 patients, 15 were transplanted from unrelated donors with an antigen mismatch for HLA Cw and 33 patient-donor pairs were matched for HLA-Cw. The CMV reaction rate was 66.7% for HLA-Cw mismatch group and 48.5% for HLA-Cw match group (chi(2) = 1.39, P = 0.2375). Thirty-seven donor-patients pairs belonged to group C1 and 11 to group C2, and CMV reaction rate was 64.9% in group C1 and 18.2% in group C2 (chi(2) = 18.13, P < 0.0001). Twenty-six patients received graft from KIR haplotype A (group A donor) and 22 from KIR haplotype B donors (group B donor) and CMV reaction rate was 57.7% in group A donor and 50.0% in group B donor (chi(2) = 0.28, P = 0.5941). The number of donor activating KIRs (aKIRs) was less than that of recipient aKIRs in 34 patient-donor pairs in which the CMV reaction rate was 70.6%, and the number of donor aKIRs was more than that of recipient aKIRs in 14 patient-donor pairs in which the CMV reactivation was 14.3%. There was a significan difference between the two group (chi(2) = 12.44, P = 0.0004).</p><p><b>CONCLUSION</b>KIR and HLA-Cw genotypes influence the rate of CMV reactivation following non-T cell deleted unrelated donor hematopoietic cell transplantation.</p>

Experimental study on IL-2- and IL-15 application in allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the impact of IL-2- and IL-15-activated donor natural killer (NK) cell infusion on graft-versus-host-disease (GVHD) and graft-versus-leukemia (GVL) effect post allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>The C57BL/6 mice splenic NK cells were selected by microbeads, and then expanded in the media containing IL-2 and IL-15. The killing activity of NK cells was detected. In the leukemia mouse model, recipients (BALB/c) were intravenously inoculated with EL9611 leukemia cells 8 days before transplantation. Lethally irradiated BALB/c recipient mice were transplanted with 5 x 10(6) bone marrow cells (BMCs), or 5 x 10(6) BMCs plus 1 x 10(7) splenocytes with or without 1 x 10(7) activated NK cells. Additionally, NK cell infusion group mice were intraperitoneally injected with a mixture of IL-2 and IL-15 post transplant. Survival time, GVHD occurrence, lineage chimerism, TRBV spectra-typing were observed post transplant.</p><p><b>RESULTS</b>The purity of isolated splenic NK cells was 95.7% - 97.1%. The killing activity of NK cells after activation was increased by 3 times. GVHD did not occurred in allogeneic BMCs infusion group, whereas did from 1 week after transplant in allogeneic BMCs + splenocytes infusion group. The severity of GVHD in total body irradiation (TBI) experimental group was significantly lower than in splenocytes infusion group (P < 0.05). The survival time was 9.5 - 14.0 d in TBI alone conditioning group. In leukemia mouse model, 100 day survival rate was 10% the rest of them were died of leukemia while in experimental group, the more than 100 days survival rate was 80% (P < 0.01). PB NK cells at 2 week post-transplant were 4.8% in experimental group and 2.8% in control group. NK cells recovery in experimental group was earlier than that in control group (P < 0.05). TRBV reconstitution was faster in experimental group than in control group, moreover, the number of TRBV family expression was more in experimental group than in control group which mainly expressed monoclone or oligo-clone.</p><p><b>CONCLUSIONS</b>Donor alloreactive NK cells can be efficiently expanded and activated with IL-2 and IL-15. Donor activated NK cell infusion and IL-2, IL-15 treatment can promote immune reconstitution, mitigate GVHD and reduce leukemia relapse.</p>