Tolerance induction between two different strains of parental mice prevents graft-versus-host disease in haploidentical hematopoietic stem cell transplantation to F1 mice.

Haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) has been employed worldwide in recent years and led to favorable outcome in a group of patients who do not have human leukocyte antigen (HLA)-matched donors. However, the high incidence of severe graft-versus-host disease (GVHD) is a major problem for Haplo-HSCT. In the current study, we performed a proof of concept mouse study to test whether induction of allogeneic tolerance between two different parental strains was able to attenuate GVHD in Haplo-HSCT to the F1 mice. We induced alloantigen tolerance in C3H mice (H-2k) using ultraviolet B (UVB) irradiated immature dendritic cells (iDCs) derived from the cultures of Balb/c bone marrow cells. Then, we performed Haplo-HSCT using tolerant C3H mice as donors to F1 mice (C3H×Balb/c). The results demonstrated that this approach markedly reduced GVHD-associated death and significantly prolonged the survival of recipient mice in contrast to the groups with donors (C3H mice) that received infusion of non-UVB-irradiated DCs. Further studies showed that there were enhanced Tregs in the tolerant mice and alloantigen-specific T cell response was skewed to more IL-10-producing T cells, suggesting that these regulatory T cells might have contributed to the attenuation of GVHD. This study suggests that it is a feasible approach to preventing GVHD in Haplo-HSCT in children by pre-induction of alloantigen tolerance between the two parents. This concept may also lead to more opportunities in cell-based immunotherapy for GVHD post Haplo-HSCT.

Anti-thymocyte globulin (ATG) differentially depletes naïve and memory T cells and permits memory-type regulatory T cells in nonobese diabetic mice.

BACKGROUND: ATG has been employed to deplete T cells in several immune-mediated conditions. However, whether ATG administration affects naïve and memory T cell differently is largely unknown. THE CONTEXT AND PURPOSE OF THE STUDY: In this study, we assessed how murine ATG therapy affected T cell subsets in NOD mice, based on their regulatory and naïve or memory phenotype, as well as its influence on antigen-specific immune responses. RESULTS: Peripheral blood CD4+ and CD8+ T cells post-ATG therapy declined to their lowest levels at day 3, while CD4+ T cells returned to normal levels more rapidly than CD8+ T cells. ATG therapy failed to eliminate antigen-primed T cells. CD4+ T cell responses post-ATG therapy skewed to T helper type 2 (Th2) and possibly IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) were less sensitive to ATG depletion and remained at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory T cell phenotype was significantly increased in ATG-treated animals. CONCLUSION: ATG therapy may modulate antigen-specific immune responses through inducing memory-like regulatory T cells as well as other protective T cells such as Th2 and IL-10-producing Tr1 cells.

Clinical outcome of autologous peripheral blood stem cell transplantation in opticospinal and conventional forms of secondary progressive multiple sclerosis in a Chinese population.

To evaluate clinical outcomes of autologous peripheral blood stem cell transplantation (APBCST) between opticospinal multiple sclerosis (OSMS) and conventional multiple sclerosis (CMS) during disease progressive stage in a Chinese population. Thirty-six secondary progressive MS patients, among whom 21 were with OSMS and 15 with CMS, underwent APBSCT and were followed up for an average of 48.92 months (range, 10-91 months). Peripheral blood stem cells were obtained by leukapheresis after mobilization with granulocyte colony-stimulating factor. Modified BEAM conditioning regimen (Tiniposide, melphalan, carmustin, and cytosine arabinoside) were administered. Outcomes were evaluated using the expanded disability status scale (EDSS). No maintenance treatment was administered if there was no disease progression. No treatment-related mortality occurred. Among the 36 patients, one OSMS patient dropped during the follow-up. Among the 22 relapse-free patients, 20 were with continuous neurological improvement without any relapse events, and two remained in neurologically stable states. Among the 13 relapse patients, seven had experienced of neurological relapse, but with no progression during the follow-up period; and six experienced neurological deterioration after transplantation and needed further immunosuppressant treatment. The confirmed relapse-free survival rate was 62.9% and progression-free survival rate was 83.3% after 91 months according to Kaplan and Meier survival curves. Eleven of the 20 OSMS patients (55%) and two of the 15 CMS patients (13.3%) stayed in disease active group (P = 0.014). For the 20 OSMS patients, the overall EDSS score decreased significantly after transplantation (P = 0.016), while visual functions had no significant improvement (P = 0.716). Progressive OSMS has a higher relapse rate than CMS following APBSCT.

Alterations of Signaling Pathways in Essential Thrombocythemia with Calreticulin Mutation.

PURPOSE: Though mutations of the calreticulin (CALR) gene have been identified in essential thrombocythemia patients, the detailed mechanisms for CALR mutations have not been completely clarified. Our study is aimed at characterizing alteration of protein expression in ET patients with mutated CALRdel52 and further recognizing possible involvement of signaling pathways associated with CALR mutations. PATIENTS AND METHODS: Protein pathway array was performed to analyze the expression levels of proteins involved in various signaling pathways in peripheral blood neutrophils from 18 ET patients with mutated CALRdel52 , 20 ET patients with JAK2V617F mutation and 20 controls. RESULTS: We found 20 proteins differentially expressed in ET patients with mutated CALRdel52 compared with healthy controls. These proteins were associated with molecular mechanisms of cancer in ingenuity pathways analysis (IPA) network. We identified top ten canonical pathways which including apoptotic pathways and cellular cytokine pathways might participate in pathogenesis of ET with mutated CALRdel52 . Additionally, there were 8 proteins found to be dysregulated differently between ET patients with mutated CALRdel52 and those with JAK2V617F mutation. These proteins might be related to the unique signaling pathways activated by CALRdel52 mutation which were different to JAK/STATs pathway by JAK2V617F mutation. CONCLUSION: Our study demonstrated that numerous alterations of signaling proteins and pathways in ET patients with mutated CALRdel52 . These findings could help to gain insights into the pathological mechanisms of ET.

Autologous peripheral blood stem cell transplantation for severe multiple sclerosis.

We describe the results of a clinical trial to evaluate the feasibility and toxicity of autologous hematopoietic stem cell transplantation (auto-HSCT) for patients with progressive multiple sclerosis (MS). Fifteen patients (all patients with secondary progressive MS) were enrolled. The median expanded disability status scale (EDSS) score at baseline was 6.0 (range, 4.5-7.5). Peripheral blood stem cells were obtained by leukapheresis after mobilization with granulocyte colony-stimulating factor. In 9 patients, CD34+ cell selection was performed with a CliniMACS cell selection system, and 6 patients accepted infusion of unmodified peripheral blood stem cells. The modified BEAM (carmustine, teniposide, cytarabine, and melphalan) was the sole conditioning regimen used. The adverse effects included infections, mucositis, transient hepatotoxicity, and diarrhea. Three patients had flares of neurologic deterioration during mobilization, 8 patients had the same manifestation during transplantation, and 2 patients had similar flares within 3 months of transplantation. Six patients experienced continuous neurologic improvement after transplantation, 5 patients experienced neurologic progression, and 4 patients had stabilization of their disease. The confirmed progression-free rate was 63.8% at 49 months. The results of lymphocyte purging were no better than for no purging. Auto-HSCT proved to be safe and beneficial for some MS patients. Further studies are needed to establish the merit of this procedure for MS patients.

Immunosuppressive CD11b+Ly6Chi monocytes in pristane-induced lupus mouse model.

Myeloid-derived suppressor cells with immunosuppressive functions have been described to be associated with one of the mechanisms by which malignant tumors escape immune surveillance. However, little is known about the role of myeloid-derived suppressor cells in autoimmunity. In the current study, when we attempted to characterize the peritoneal cells in pristane-induced lupus model, as reported previously, we observed that there were markedly increased CD11b(+)Ly6C(hi) monocytes. Surprisingly, this type of monocytes was almost phenotypically identical to the reported monocytic myeloid-derived suppressor cells. Further analysis on how these CD11b(+)Ly6C(hi) cells affected T cell response showed that they strongly suppressed T cell proliferation in vitro in a manner dependent on cell-cell contact, NO, and PGE2. In addition, we found that CD11b(+)Ly6C(hi) monocytes inhibited Th1 differentiation but enhanced development of forkhead box p3(+)CD4(+) regulatory T cells. Consistent with the in vitro experimental results, the in vivo adoptive cell transfer study showed that infusion of pristane-treated syngeneic CD11b(+)Ly6C(hi) monocytes significantly suppressed the production of anti-keyhole limpet hemocyanin antibodies induced by keyhole limpet hemocyanin immunization. In addition, we found that CD11b(+)Ly6C(hi) monocytes were also increased significantly in spleen and peripheral blood and showed immunosuppressive characteristics similar to their peritoneal counterparts. Our findings indicate that CD11b(+)Ly6C(hi) monocytes in a pristane-induced lupus mouse model are monocytic myeloid-derived suppressor cells instead of inflammatory monocytes, as demonstrated previously. To our knowledge, this is the first to describe myeloid-derived suppressor cells in a pristane-induced lupus mouse model, which may lead to a better understanding of the role of CD11b(+)Ly6C(hi) monocytes in this specific pristane-induced lupus model.

Potent antigen-specific immune response induced by infusion of spleen cells coupled with succinimidyl-4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SMCC) conjugated antigens.

In the present study, we report our recently developed new approach to inducing antigen-specific immune response. We use two nucleophilic substitution "click" chemistry processes to successfully couple protein antigens or peptides to mouse spleen cells or T cells by a heterobifunctional crosslinker, succinimidyl-4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SMCC) or sulfo-SMCC. SMCC and its water-soluble analog sulfo-SMCC contain N-hydroxysuccinimide (NHS) ester and maleimide groups, which allow stable covalent conjugation of amine- and sulfhydryl-containing molecules in trans. Protein coupling to cells relies on the free sulfhydryls (thiols) on cell surfaces and the free amines on protein antigens. Although the amount of protein coupled to cells is limited due to the limited number of cell surface thiols, the injection of spleen cells coupled with antigenic proteins, such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA), induces a potent antigen-specific immune response in vivo, which is even stronger than that induced by the injection of a large dose of protein plus adjuvants. In addition, short peptides coupled to purified splenic T cells also potently elicit peptide-specific T cell proliferation in vivo after injection. Further studies show that antigen-coupled spleen cell treatment leads to augmented IFN-γ-producing T cells. Our study provides a unique antigen delivery method that efficiently distributes antigen to the entire immune system, subsequently eliciting a potent antigen-specific immune response with enhanced IFN-γ production. The findings in the present study suggest that this antigen-cell coupling strategy could be employed in immunotherapy for cancers, infectious diseases as well as immune-mediated disorders.

[Rituximab combined with EPOCH regimen for treatment of diffuse large B cell lymphoma of the gastrointestinal tract: analysis of 4 cases].

We treated 4 with a diagnosis of diffuse large B cell lymphoma involving the gastrointestinal tract with rituximab combined with adjusted dose EPOCH (R-DA-EPOCH) scheme based on a comprehensive analysis of the onset process, clinical and pathological features, and prognosis of the patients, and evaluated their treatment response. Complete remission (CR) was achieved in 3 patients after the treatment and 1 patient with diabetes and hypertension died due to severe infection. R-DA-EPOCH regimen as the first-line treatment of gastrointestinal diffuse large B cell lymphoma has a good short-term efficacy, but its long-term efficacy awaits further evaluation in future studies with larger sample sizes.

New insights into the immunopathogenesis of systemic lupus erythematosus: the role of T follicular helper cells.

OBJECTIVE: To review the development of T follicular helper (TFH) cells and their role in systemic lupus erythematosus (SLE) pathogenesis, the effect of dendritic cells (DCs) on TFH cells in SLE, as well as the potential use of TFH cells as a new therapeutic target in clinical practice. DATA SOURCES: The data used in this review were retrieved mainly from the PubMed database (1989-2013). The terms used in the literature search were "T follicular helper cells," "systemic lupus erythematosus," and "dendritic cells." STUDY SELECTION: Relevant publications about the TFH cells development, the interaction between the TFH cells and the DCs, and the clinical applications of TFH cells were identified, retrieved, and reviewed. RESULTS: TFH cells, a novel distinct CD4+ T cell subset, are specialized in providing help to B cells in the formation of germinal centers (GCs) and long-term protective humoral immune responses. The development of TFH cells from naïve CD4+ T cell is a multistep process. As the pivot of immunoregulation, DCs are indispensable for TFH cells generation. In addition to receptor-ligand interactions between TFH cells and DCs, the cytokines secreted by DCs are also necessary for TFH cell generation. TFH cell dysregulation has been implicated in the development of SLE. More evidence from animal models of SLE and SLE patients suggests that TFH cells are necessary for pathogenic autoantibody production. Therefore, therapeutically targeting TFH cells can be a promising approach to treat antibody-mediated autoimmune diseases including SLE. CONCLUSION: TFH cells play a critical role in the pathogenesis of SLE, making them attractive therapeutic targets in clinical practice.

Anti-CD3 antibody treatment induces hypoglycemia and super tolerance to glucose challenge in mice through enhancing glucose consumption by activated lymphocytes.

Anti-CD3 antibody has been employed for various immune-mediated disorders. However, whether anti-CD3 administration leads to rapid metabolic alternation has not been well investigated. In the current study, we studied how anti-CD3 treatment affected blood glucose levels in mice. We found that anti-CD3 treatment induced immediate reduction of blood glucose after administration. Furthermore, a single dose of anti-CD3 treatment corrected hyperglycemia in all nonobese diabetic mice with recently diagnosed diabetes. This glucose-lowering effect was not attributable to major T cell produced cytokines. Of interest, when tested in a normal strain of mice (C57BL/6), the serum levels of C-peptide in anti-CD3 treated animals were significantly lower than control mice. Paradoxically, anti-CD3 treated animals were highly tolerant to exogenous glucose challenge. Additionally, we found that anti-CD3 treatment significantly induced activation of T and B cells in vitro and in vivo. Further studies demonstrated that anti-CD3 treatment lowered the glucose levels in T cell culture media and increased the intracellular transportation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2 deoxyglucose (2-NBDG) particularly in activated T and B cells. In addition, injection of anti-CD3 antibodies induced enhanced levels of Glut1 expression in spleen cells. This study suggests that anti-CD3 therapy-induced hypoglycemia likely results from increased glucose transportation and consumption by the activated lymphocytes.

[Genetic characteristics of human acute lymphoblastic leukemia cell line Molt-4].

This study was aimed to investigate the genetic characteristics of human acute lymphoblastic leukemia cell line Molt-4, and evaluate its application in measuring telomere length by Flow-FISH. Molt-4 cell line was cultured in suspension and subcultured regularly. Eight different passages of Molt-4 cells in exponential stage were selected.The growth curves were drawn by cell counting method, meanwhile calculating the population doubling times of cells,DNA ploidies were determined by flow cytometry,karyotypes were analyzed by G-banding and telomere lengths were measured by Southern blot. The results showed that the population doubling time of Molt-4 cell line was (1.315 ± 0.062) d, DNA ploidy index was (2.085 ± 0.0093) , and the telomere length was (32.05 ± 5.27) kb. There were no significant difference among different passages (P = 0.931,0.888 and 0.935 separately). The karyotypes showed that the chromosome numbers of Molt-4 cell line were from 91 to 99 in different metaphases, and the majority of them were hypertetraploid, and stable and recurrent structural abnormalities of chromosomes could be kept. It is concluded that the stable genetic characteristics and the longer telomere length of Molt-4 cell line makes it be a feasible control cells in measurement of telomere length by Flow-FISH.

[Evaluation of neutrophilic CD64 index as a diagnostic marker of bacterial infection in blood diseases].

This study was aimed to investigate the value of neutrophilic CD64 index (nCD64 index) as a diagnostic marker of bacterial infection in hematologic diseases. Experimental data of 232 patients with hematologic diseases were analyzed retrospectively. The nCD64 index was detected by flow cytometry and was compared with the levels of erythrocyte sedimentation rate (ESR), C reaction protein (CRP) and fibrinogen respectively. The results showed that the nCD64 index in clinical infection group were significantly higher than that in non-infection group and autoimmune disease group (P < 0.0001 respectively). The nCD64 index in blood culture positive group was also significantly higher than that in blood culture-negative group (P < 0.01). The result of ROC curve analysis showed that the optimal critical values of nCD64 index, ESR, CRP and Fib were 4.96, 21.5 mm/h, 8.56 mg/dl and 4.42 mg/dl, respectively. The sensitivity and specificity of nCD64 index were 0.928 and 0.933, while the sensitivities of ESR, CRP and Fib were 0.725, 0.754 and 0.594, and the specificities of CRP, ESR and Fib were 0.625,0.837 and 0.77, respectively. It is concluded that nCD64 index is possessed of much higher in sensitivity and specificity, compared with ESR, CRP and Fib in diagnosis of bacterial infection of hematologic diseases. nCD64 index can be used as an effective diagnostic marker for bacterial infection of hematologic diseases.

[Effect of granulocyte colony-stimulating factor on endothelial progenitor cell for coronary artery lesion in Kawasaki disease mice model].

OBJECTIVE: Number and function of endothelial progenitor cell (EPC) and coronary artery lesion in Kawasaki disease (KD) model were evaluated to investigate therapeutic efficacy of granulocyte colony-stimulating factor (G-CSF). METHOD: C57BL/6 mice were injected with L. casei cell wall extract (LCWE); 48 mice were divided into 3 groups randomly: KD model group; G-CSF treated model group and control group, 16 in each. G-CSF was subcutaneously injected from day 5 to day 9 after injection of LCWE. Coronary artery lesion, number of circulating EPC and the function of bone marrow EPC were evaluated. RESULT: In model group, inflammatory infiltration was found around coronary artery at 14 days. The number of circulating EPC was significantly decreased in model group (0.017% ± 0.008%) compared to control (0.028% ± 0.007%) (t = 2.037, P < 0.05). Disruption of elastin was consistently observed at 56 days. Stimulated by G-CSF, inflammatory infiltration was found around the coronary artery at day 14, while the number of circulating EPC (0.042% ± 0.015%) was increased significantly compared to models (t = 4.629, P < 0.05). At the day 56, the number of circulating EPC was decreased slightly (0.029% ± 0.012%), but still higher than the model group (t = 2.789, P < 0.05), and have no significant difference compared to controls (P > 0.05). Furthermore, there was no elastin disruption in the G-CSF group. In model group, bone marrow EPC's proliferation ability of absorbance (A value) was 0.38 ± 0.09 in thiazolyl blue assay, less than controls (0.61 ± 0.14, P < 0.01). Adhesion and migration function were down-regulated compared to controls [(3.1 ± 0.6) cells/HPF and (3.3 ± 0.6) cells/HPF vs. (6.4 ± 1.2) cells/HPF and (6.2 ± 0.5) cells/HPF, both P < 0.01]. In the G-CSF treated group, proliferation ability (A 0.58 ± 0.10), adhesion [(6.17 ± 1.13) cells/HPF], migration [(6.29 ± 0.42) cells/HPF] function were increased significantly compared to the model group (P < 0.01). CONCLUSION: G-CSF can up-regulate EPC number and function to prevent coronary artery lesion in mice model of KD.

Incipient Coombs' test negative autoimmune hemolytic anemia precedes non-Hodgkin's lymphoma.

The cases of lymphoma accompanied or preceded by Coombs' test positive autoimmune hemolytic anemia (AIHA) have been reported. However, Coombs' test negative AIHA prior to the diagnosis of lymphoma was rarely described. Herein, this article reports a case of non-Hodgkin's lymphoma (NHL) preceded about 1.5 years by Coombs test negative AIHA. A woman aged 69 was diagnosed with HA based on the history and laboratory tests. Further studies revealed that this patient was negative with Coombs' test for IgG, IgM, IgA and C3. After all possible causes of HA, especially malignancies were ruled out, the patient was diagnosed with Coombs' test negative AIHA and treated with prednisolone. The patient responded well initially to steroid treatment. Two recurrences of acute HA were presented at time of 10 months post steroid cessation, and immediately after an attempt to withdraw steroid, respectively, but the hemolysis was effectively controlled by reinstitution of prednisolone. At third recurrence, however, the patient was no longer responding to steroid, and was found with cervical lymphadenopathy. Coombs' test for IgG, IgM, IgA and C3 remained negative. B cell NHL was diagnosed by pathology. After receiving 6 cycles of CHOP chemotherapy, the patient was lymphoma free, but the hemolysis was not improved, however, which was effectively controlled by the following low dose-rituximab (RTX) therapy. The patient was still kept in a remission of lymphoma free of anemia. In conclusion, this report presented a very rare case of NHL with Coombs' test negative AIHA as initial major clinical manifestation.

Endothelial progenitor cell transplantation ameliorates elastin breakdown in a Kawasaki disease mouse model.

BACKGROUND: Coronary artery damage from Kawasaki disease (KD) is closely linked to the dysfunction of endothelial progenitor cells (EPCs). The aim of the present study was to evaluate the therapeutic effect of EPCs transplantation in KD model. METHODS: Lactobacillus casei cell wall extract (LCWE)-induced KD model in C57BL/6 mice was established. The model mice were injected intravenously with bone marrow-derived in vitro expanded EPCs. Histological evaluation, number of circulating EPCs and the function of bone marrow EPCs were examined at day 56. RESULTS: Inflammation was found around the coronary artery of the model mice after 14 days, Elastin breakdown was observed after 56 days. CM-Dil labeled EPCs incorporated into vessel repairing foci was found. At day 56, the number of peripheral EPCs in the KD model group was lower than in EPCs transplanted and control group. The functional index of bone marrow EPCs from the KD model group decreased in proliferation, adhesion and migration. Increased number of circulating EPCs and improved function were observed on the EPCs transplanted group compared with model group. CONCLUSION: Exogenously administered EPCs, which represent a novel strategy could prevent the dysfunction of EPCs, accelerate the repair of coronary artery endothelium lesion and decrease the occurrence of aneurysm.

Endothelial progenitor cell down-regulation in a mouse model of Kawasaki disease.

BACKGROUND: Cardiovascular complications of Kawasaki disease (KD) are a common cause of heart disease in pediatric populations. Previous studies have suggested a role for endothelial progenitor cells (EPCs) in coronary artery lesions associated with KD. However, long-term observations of EPCs during the natural progression of this disorder are lacking. Using an experimental model of KD, we aimed to determine whether the coronary artery lesions are associated with down-regulation of EPCs. METHODS: To induce KD, C57BL/6 mice were administered an intraperitoneal injection of Lactobacillus casei cell wall extract (LCWE; phosphate buffered saline used as control vehicle). Study groups included: group A (14 days following LCWE injection), group B (56 days following LCWE injection) and group C (controls). Numbers of circulating EPCs (positively staining for both CD34 and Flk-1 while staining negative for CD45) were evaluated using flow cytometry. Bone marrow mononuclear cells were cultured in vitro to expand EPCs for functional analysis. In vitro EPC proliferation, adhesion and migration were assessed. RESULTS: The model was shown to exhibit similar coronary artery lesions to KD patients with coronary aneurysms. Numbers of circulating EPCs decreased significantly in the KD models (groups A and B) compared to controls ((0.017 ± 0.008)% vs. (0.028 ± 0.007)%, P < 0.05 and (0.016 ± 0.007)% vs. (0.028 ± 0.007)%, P < 0.05). Proliferative, adhesive and migratory properties of EPCs were markedly impaired in groups A and B. CONCLUSION: Coronary artery lesions in KD occur as a consequence of impaired vascular injury repair, resulting from excess consumption of EPCs together with a functional impairment of bone marrow EPCs and their precursors.

[Multiparameter flow cytometric evaluation of bone marrow involvement in B cell non-Hodgkin's lymphoma].

The objective of study was to evaluate the clinical values of multiparameter flow cytometry (MPFC) and cytomorphology of bone marrow aspiration(BMA) in detecting bone marrow involvement in patients with B cell Non-Hodgkin's lymphoma (B-NHL). 96 bone marrow samples from the patients with B-NHL were measured by MPFC using CD45/SSC and CD20/SSC gating strategy combined with anti-kappa and anti-lamda monoclonal antibodies, and then compared with results acquired by cytomorphologic analysis of BMA. The results showed that the bone marrow involvement was confirmed by MPFC in 38 cases (39.6%), while it was detected by cytomorphologic analysis of BMA only in 12 cases (12.5%). There was a significant difference between the two methods (p<0.05). 12 positive cases detected by cytomorphologic analysis of BMA were also positive by MPFC. There was no difference of 3-year overall survival rate between negative and positive cases detected by MPFC, but their 4-year overall survival rate was 73.18+/-6.65% and 44.13%+/-19.55% respectively (p<0.05). It is concluded that the MPFC is a more sensitive method for detecting bone marrow involvement in patients with B-NHL than cytomorphologic analysis of BMA. The 4-year overall survival rate of the patients without bone marrow involvement was significant higher than those of patients with bone marrow involvement. Bone marrow involvement in B-NHL detected by MPFC can be useful for clinical evaluation and prognosis prediction.

[Diagnostic significance of immunophenotyping of bone marrow cells in myelodysplastic syndrome without an increase of marrow blasts].

This study was aimed to investigate the characteristics of immunophenotypes in the patients with myelodysplastic syndrome (MDS) without an increase of marrow blasts, and to confirm their diagnostic significance. Marrow cells from 222 patients with pancytopenia, dysplastic changes in one or more hematopoietic lineages and blast cells less than 5% were analyzed by multiparametric flow cytometry(FCM). The abnormal immunophenotypes were evaluated in asynchronous antigen expression (CD34 or CD117 in mature granulocytes or mature monocytes, HLA-DR in mature granulocytes), in cross-lineage antigen expression (CD7 or CD56 in granulocytes or monocytes), in aberrant light-scatter (CD45/SSC in mature granulocyte or monocyte) and in abnormal expression of differentiation antigen (CD13/CD16 pattern in granulocytes and HLA-DR under-expression in monocytes). The sensitivity and specificity of abnormal immunophenotypes were determined on diagnosis. Among 222 cases, 127 cases were diagnosed as MDS by traditional diagnostic method and 95 cases were non-MDS (drug-related neutropenia, autoimmune cytopenia and idiopathic thrombocytopenia). In mature granulocyte gate, the sensitivity of asynchronous, cross-lineage antigen expression, aberrant light-scatter of CD45/SSC and abnormal expression of differentiation antigen were 31.5%, 30.7%, 49.6% and 60.6% respectively, and the specificity were 100%, 100%, 88.4% and 52.6% respectively. In monocyte gate, the sensitivity of asynchronous, cross-lineage antigen expression, aberrant light-scatter of CD45/SSC and abnormal expression of differentiation antigen were 2.3%, 11%, 37% and 12.6% respectively. The specificity was 100% in all of them. Among 8 above mentioned items, sensitivity of more than 2 abnormalities was 77.9%, and specificity was 95.8%. The positive predictive value was 96.1%. It is concluded that the abnormal expression of asynchronous, cross-lineage antigen expression, aberrant light-scatter of CD45/SSC have a high specificity and a low sensitivity for diagnosis of MDS. The abnormal expressions of differentiation antigens have a high sensitivity and a low specificity; however, the detection of multiple expression abnormalities possesses the high sensitivity and specificity for diagnosis of MDS.

[Prognosticating relapse risk based on multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia].

The objective of this study was to investigate the prognosticating value of multiparameter flow cytometry in detection of minimal residual disease (MRD) and relapse risk of patients with acute myeloid leukemia (AML). Multiparameter flow cytometry (MPFC) analysis was used to detect the leukemia-associated aberrant immunophenotype (LAIP) of the pretreated patients with AML and to assess the levels of MRD after remission induction (Post-Ind MRD) and consolidation therapy (Post-Cons MRD). The results showed that the definite LAIP could be detected in 94.3% of the patients (115/122) with AML (except APL). Among 115 cases only one LAIP was identified in 15 cases (13.0%), but two or more LAIP were identified in other 100 cases (87.0%). The most frequent LAIP identified was cross-lineage antigen expression (40.9%). The percentages of asynchronous antigen expression, antigen over-expression and antigen lack expression were 20.9%, 27.0%and 34.8% respectively. MRD frequency was monitored in 41 AML patients with CR after remission induction chemotherapy and 2 or more cycles of consolidation chemotherapy. 24 patients were Post-Ind MRD(+) and 17 patients were Post-Ind MRD(-). The percentages of relapse in cases of Post-Ind MRD(+) and Post-Ind MRD(-) were 75.0% (18/24) and 29.4% (5/17) respectively after consolidation chemotherapy. The relapse free survival (RFS) times of the patients with Post-Ind MRD(+) and Post-Ind MRD(-) were 49.06 +/- 6.53 months and 11.92 +/- 1.64 months (p < 0.0001) respectively. 18 patients were Post-Cons MRD(+) and 23 patients were Post-Cons MRD(-). The percentages of relapse in cases of Post-Cons MRD(+) and Post-Cons MRD(-) patients were 100% (18/18) and 21.7% (5/23) respectively after consolidation chemotherapy. The RFS times of the patients with Post-Cons MRD(+) and Post-Cons MRD(-) were 41.74 +/- 5.52 months and 10.06 +/- 1.72 months (p < 0.0001) respectively. It is concluded that the levels of post-Ind MRD and post-Cons MRD identified in the patients with AML was highly associated with their RFS. The detection of MRD by MPFC provides prognostic information in AML patients.