目的 探讨脑脊液流式细胞术(CSF-FCM)在诊断非霍奇金淋巴瘤(NHL)脑脊膜转移(LM)中的价值.方法 回顾性分析3例影像学检查及脑脊液(CSF)病理检查均不典型的继发中枢神经系统淋巴瘤(SCNSL)的临床资料并复习相关文献.结果 病例1诊断为睾丸弥漫大B细胞淋巴瘤,查体无神经系统症状,CSF细胞学检查阴性,但CSF-FCM检到少量单克隆B淋巴细胞(CD19+B细胞10个,表达kappa,不表达lambda),予R-CHOP-HD-MTX化疗及腰穿鞘内注射治疗,患者停化疗7月复发,脑组织活检证实中枢复发.病例2诊断为小B细胞淋巴瘤(脾边缘区淋巴瘤可能性大),查体有神经系统症状,CSF细胞学检查阴性,CSF-FCM检测到单克隆B细胞占CSF有核细胞65.8％,表达CD19 kappa,不表达lambda,确诊NHL脑脊膜累及.病例3确诊小B细胞淋巴瘤(淋巴浆细胞淋巴瘤可能),停化疗8月后出现神经系统症状,骨髓检查﹑CSF细胞学检查及脑活检均未明确诊断,CSF-FCM提示脑脊液单克隆B细胞增多,表达CD45dim+CD19+CD20dim+CD5-CD10-CD22dim+CD2部分+FMC7部分+KAPPA-LAMBDA+CD200+CD79b+CD25+CD103-CD11C-CD38部分+,确诊NHL的脑脊膜受累.结论 所有存在CNS受累风险的淋巴瘤患者均应接受腰椎穿刺(LP)检查,CSF的流式细胞术分析有助于隐匿性脑脊膜受累的诊断.CSF样本的全面评估还包括二代测序基因检测、免疫球蛋白重链(IgH)或T细胞受体基因重排检测及细胞因子检测.

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.

目的 分析2例早期前T淋巴母细胞白血病/淋巴瘤(early T-cell precursor acute lymphoblastic leukemia/lymphoma,ETP-ALL/LBL)患者的临床及实验室资料,结合文献复习,提高对该病的认识.方法 回顾性分析我院诊治的2例ETP-ALL/LBL患者病例资料,并进行文献检索分析.结果 2例患者均为中年,女性,以淋巴结肿大为主要表现起病.淋巴结活检病理诊断为T-淋巴母细胞淋巴瘤(T-LBL),流式细胞术免疫分型符合ETP-ALL/LBL(病例1为胸水标本,病例2为淋巴结标本).病例1先后给予VDCLP方案及Hyper-CVAD方案化疗,病例2给予BFM-90方案化疗,2例患者化疗后均无效,因疾病进展死亡.结论 ETP-ALL/LBL具有独特的免疫表型特点,病理诊断时注意加做早期标记和髓系抗体;ETP-ALL/LBL常规化疗效果较差,复发率高,预后差,强化疗及早期行造血干细胞移植可以改善预后.

随着多发性骨髓治疗方法快速发展,对于该病的诊断、微小残留病灶监测及预后的判断有了越来越多的方式及方法,多参数流式细胞术免疫表型分析,通过细胞免疫表型的异质性及胞内轻链的克隆性,能够准确、客观的鉴别出良性及恶性浆细胞比例以及抗原表达水平,不仅为多发性骨髓瘤患者的诊断提供依据,同时能够追踪患者的微小残留病灶,这对预测多发性骨髓瘤患者的预后密切相关,全文将对多参数流式细胞术免疫表型分析在多发性骨髓瘤诊断、微小残留病灶监测及预后方面进行综述.

2016年更新版《WHO造血和淋巴组织肿瘤分类》中[1],将伴有骨髓原始红细胞≥30％,原始髓系＜20％,既往有放化疗史,定义为治疗相关伴红系明显增生的急性髓系白血病(therapy-related acute myeloid leukemia,T-AML)的范畴.在急性红白血病(acute erythroleukemia,AEL)分类中,相比于2008年的标准[2],仅保留了纯红细胞白血病(pure erythroid leukemia,PEL),诊断标准为骨髓红系＞80％,原始红细胞≥30％,骨髓或外周血非红原始细胞＜20％、无放化疗史、无WHO重现性细胞遗传学异常.但如何诊断骨髓有核红＞80％,原始红细胞≥30％,既往伴有化疗史的患者,在更新分类中并未阐明.本文报道1例骨髓有核红＞80％,原始红细胞≥30％,既往伴有化疗史的患者.分析患者临床、实验室特征及治疗过程,文献复习T-AML与PEL特征及诊断.

Background: Somatic mutations in WT1 and TET2 were separately perceived as contributors to hematopoietic disorders and thought to have a mutually exclusive effect in acute myeloid leukemia (AML). Here, we report a case in which WT1 and TET2 mutations co-existed at different stages in an AML patient with t(9;11)(p21.3;q23.3), and without abnormal WT1 expression, which did not synchronize with the patient's tumor state. Hence, the origins and prognostic value of these two mutations were investigated. Methods: Bone marrow (BM) and buccal mucosal cells were obtained from a 27-year-old male AML patient, and next-generation sequencing (NGS), targeting multiple genes, was performed after DNA extraction. Single nucleotide variant (SNV) and insertion-deletion (Indel) associated with AML were identified in these two samples by using SAM tools. Then, peripheral blood or buccal mucosal cells were obtained from the patient’s relatives, and NGS targeting multiple genes was applied. Besides, the relationship between overall survival (OS) and alteration of these two genes in AML patients was analyzed by using cBioPortal and UALCAN databases, respectively. Results: SNV of WT1 (NM_024426:exon7:c.1109G>C；p.Arg370Pro) and TET2 (NM_001127208:exon11:c.5530G>A；p.Asp1844Asn) were present in the BM of the patient, and these two mutations were also observed in his buccal mucosal cells, which suggested that they were germline mutations. Variation analysis of samples from other relatives indicated that WT1 (NM_024426:exon7:c.1109G>C;p.Arg370Pro) and TET2 (NM_001127208:exon11:c.5530G>A;p.Asp1844Asn) were also present in his father and mother, respectively. The patient’s brother also carried TET2 (NM_001127208:exon11:c.5530G>A；p.Asp1844Asn) mutations. Bioinformatic analysis suggested that mutations in the WT1 and TET2 genes are both associated with a poor prognosis of AML, and abnormal TET2 expression is associated with a poorer prognosis than abnormal WT1 expression. Conclusion: It is firstly report that WT1 p.Arg370Pro and TET2 p.Asp1844Asn mutations co-existed in an AML patient with t(9;11)(p21.3;q23.3) and no WT1 abnormal expression, which highlight the importance of the identification and prognostic evaluation of germline mutations in clinical practice for AML.

Erythroid proliferations in myeloid neoplasms (MN﹣EP) is a group of heterogeneous diseases characterized by nucleated red blood cell proliferation and ineffective hematopoiesis in the bone marrow. Initial French﹣American﹣British (FAB) classification was used to diagnose and classify MN﹣EP according to morphological criteria. However, with the development of flow cytometry, cytogenetics and molecular biology techniques, FAB classification is obviously insufficient, which can not meet the clinical needs. Therefore, the World Health Organization (WHO) classification has introduced more accurate diagnostic classification standards and revised them several times. This paper reviews the research progress of MN﹣EP diagnostic classification.

Objective To investigate the usage of T cell receptor β chain variable region(TCRVβ) in peripheral blood of NMO and NMOSD patients.Method Hospitalized patients of Xuanwu Hospital, which were divided into 3 groups. Two experimental groups: NMO group of 7 patients, NMOSD group of 23 patients, and the control group: 30 healthy volunteers. To took 2mL EDTA anticoagulated peripheral blood from subjects, then to analysis the usage of TCRVβ by flow cytometry. We got the statistic of the qualitative and quantitative expression of the total 24 subfamilies of TCRVβ.Results The expansion of the TCRVβ in the disease groups were significantly higher than the control group(P<0.05), there were no significant different between disease groups(P>0.05). Compared to the control group, CD4+TCRVβ5.1 and CD8+TCRVβ13.6 were significantly expanded in NMO group, CD4+TCRVβ2 and CD8+TCRVβ2 were significantly expanded in NMOSD group, CD8+TCRVβ11 were significantly reduced in NMO group; CD4+TCRVβ11 and CD8+TCRVβ11 were significantly reduced in NMOSD group. The CD8+TCRVβ13.6 in NMO group were significantly expanded than NMOSD group, CD8+TCRVβ9, 17 in NMO group were significantly reduced than NMOSD group. Receiver operating characteristic(ROC) curve analysis indicated that CD8+TCRVβ11 has some diagnostic value for NMO and NMOSD, CD4+TCRVβ2 and CD8+TCRVβ11 have some diagnostic value for NMOSD. Conclusion The early diagnosis of NMO and NMOSD can be partly solved by detecting some TCRVβ fragments.

Objective To investigate the diagnostic value of T cell receptor β chain variable region (TCRVβ) in peripheral blood of clinically isolated syndrome (CIS ) , relapsing-remitting multiple sclerosis (RRMS ) and secondary-progressive multiple sclerosis (SPMS ) patients. Methods Hospitalized patients of Xuanwu Hospital form 2012.08.20 to 2014.03.20 ,who met the inclusion and exclusion criteria ,were divided into 4 groups. Three experimental groups :CIS group of 30 patients , RRMS group of 40 patients , SPMS patients of 10 cases , and the control group:30 healthy volunteers. Sex and age were well matched. We took 2 mL EDTA anticoagulated peripheral blood from the subjects , then analyzed the polymorphism of TCRVβby flow cytometry. We got the statistic of qualitative and quantitative expression of TCRVβ (total of 24 subfamilies) . The usage of TCRVβcan be descripted by the number of the clonal expanded TCRVβfragments (expression ≥10％ of all TCRVβ) and expansion fraction (the accumulation of expansion degree ) . Results TCRVβ was significantly higher in the disease groups than the control group (P<0.01) , there were no significant difference between disease groups (P> 0.05) . Quantitative and qualitative analysis of TCRVβ:CD4+ TCRVβ 2 was significantly expanded in diseases groups ;CD8+ TCRVβ11 was significantly reduced in disease groups , CD4+TCRVβ21.3 of the CIS group was significantly decreased than the SPMS group. Receiver operating characteristic(ROC) curve analysis indicated that CD8+ TCRVβ11 fragments had some diagnostic value for MS , and CD4+TCRVβ21.3 fragments had some diagnostic value for SPMS. Conclusions The early diagnosis of MS and SPMS can be partly facilitated by detecting some TCRV β fragments.

目的 探讨毛细胞白血病（HCL）的临床特征及诊疗方法。 方法 回顾性分析3例HCL患者的临床资料并复习相关文献。 结果 3例HCL患者中，2例以淋巴细胞增多起病，病情进展缓慢，1例以发热症状起病，病情凶险，短期内死亡。2例为典型HCL表型，免疫表型为CD19+、CD20+、CD11c+、CD103+、CD25+；1例为变异型HCL，免疫表型为CD19+、CD20+、CD103+、CD11c+、CD25-。2例典型表型HCL患者中，1例应用克拉曲滨治疗未缓解后死亡，1例先后予脾脏切除及干扰素治疗。1例变异型HCL患者予克拉曲滨治疗未缓解，换用利妥昔单抗治疗后缓解。 结论 HCL是一类不常见的慢性B淋巴细胞增殖性肿瘤，流式细胞术免疫表型分析有利于明确诊断及分型。克拉曲滨治疗可以使大多数HCL达到持久完全缓解，但是变异型HCL对克拉曲滨反应差，需积极二线药物如利妥昔单抗治疗。

Objective To induce alloantigen immune tolerance between C3H mice and Balb/c mice by infusing the ultraviolet B(UVB)radiation immature dendritic cells (imDC) and and study the immunological mechanisms of this process.Methods (①) The authors induced alloantigen tolerance in C3H mice (H-2k) by intravenous injecting ultraviolet B (UVB) irradiated Balb/c immature dendritic cells (UVB-Balb/c imDC) derived from the cultures of Balb/c bone marrow cells.(②)Detection of immune tolerance induced regulatory T cells,interleukin-10 (IL-10) and interferon-γ(IFN-γ) in mice in order to study the tolerance mechanism.Results (①)C3H mice immunized with UVB-Balb/c imDC can produce immune tolerance to Balb/c antigens and can not produce anti-Balb/c antibody.(②)C3H mice immunized with UVB-Balb/c imDC can not clear the Balb/c spleen cells in vivo.(③)The T cells of C3H mice immunized with UVB-Balb/c imDC can secrete more IL-10 and produce more FOX-P3 + regulatory T cells than the control group in vitro.Conclusion (①)Immunization of C3H mice with UVB-Balb/c imDC allows C3H mice to fully tolerate Balb/c antigen.(②)The possible cause of immune tolerance to Balb/c antigens in C3H mice is due to the increased secretion of IL-10 from T cells and the production of more FOX-P3 + regulatory T cells.

Objective To investigate the clinical value of low-dose decitabine (DAC) in elderly patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) patients with intermediate-or high-risk. Methods Low-dose DAC (10 mg/d, 7 days) combined with CAG regimen were given to 19 elderly patients with AML and intermediate- or high-risk MDS patients. The efficacy and adverse reactions were evaluated after a course of treatment, and the patients were followed up for survival. Results After a course of treatment, 8 patients achieved complete remission (CR), 7 patients achieved partial remission (PR). After 4 courses of treatment, 68.4 % (13/19) of patients achieved CR, the overall response rate reached 78.9% (15/19). Fewer side effects were seen associated with chemotherapy. After 42 months of follow-up, there were 12 survival cases, the median survival time was 13.5 months (3-42 months). Conclusion Low-dose DAC combined with CAG regimen have a better efficacy, higher safety, and lower economic burden for elderly AML patients and intermediate- or high-risk MDS patients, which is beneficial to greatly improve patients' compliance.

Objective To evaluate the effectiveness and safety of subcutaneous injection of bortezomib in patients with polyneuropathy,organomegaly,endocrinopathy,monoclonal gammopathy and skin changes(POEMS)syndrome. Methods Since January 2013,patients who were newly diagnosed with POEMS syndrome in hematology department of Xuanwu Hospital of Capital Medical University were given treatments with subcutaneous injection of bortezomib 1. 3 mg/( m2 · d)on day 1,4,8,11 and dexamethasone 20 mg/ d on day 1,2,4,5,8,9,11,12 for a period of 28 days. The treatments included no more than 4 cycles. The effects and adverse reactions/ events of the treatments were observed. Results Until April 2015,6 patients with POEMS syndrome were admitted. These patients aged from 37 to 66 years included 1 male,5 female. All patients first presented with polyneuropathy,accompanied by monoclonal gammopathy,organomegaly,edema,endocrinopathy and pigment in skins. Optic disc edema was found in 4 patients and 3 patients had bone lesions. After 2 to 4 cycles of treatments,peripheral neuropathy was improved in 5 patients. Monoclonal gammopathy disappeared in 5 patients. Organomegaly, edema, endocrinopathy and skin changes recovered to different degree in all 6 patients. Recovery of optic disc edema was found in all 4 patients. Adverse reactions/ events happened in 6 patients during the therapy. Peripheral neuropathy of 1 and 2 grade were reported in 3 patients. Three patients developed 1 and 2 grade of gastrointestinal reactions during treatments. Three patients developed respiratory tract infection. One patient developed mild anemia. One patient developed abnormal liver function. No patient discontinued therapy because of adverse events. Conclusion Subcutaneous injection of bortezomib may be an effective and safe treatment for POEMS syndrome.

目的 探讨免疫性血小板减少症(immune thrombocytopenia,ITP)患者外周血B细胞、成熟B细胞、滤泡辅助性T细胞及树突状细胞亚群的变化,及其相互关系及Tfh细胞在ITP发病中的作用.方法 采用流式细胞术检测35例初治ITP患者及30例正常人外周血B细胞及成熟B细胞比例、Tfh细胞数量及树突细胞亚群.结果 ITP患者外周血CD19+B细胞及成熟B细胞比例分别为(13.49±9.05)％和(81.96±11.99)％,均高于对照组[(9.12±4.60)％和(75.42±12.86)％,P＜0.05].ITP患者Tfh细胞(CD4+CXCR5+ICOS+PD-1+)比例为(4.27±3.88)％,明显高于对照组(1.02±0.54)％,差异有统计学意义(P＜0.01).ITP患者浆细胞树突细胞比例为(2.06±1.65)％,明显低于对照组(5.07±3.48)％,差异有统计学意义(P＜0.01).结论 ITP患者存在Tfh细胞数量异常,可能导致B细胞比例升高,在ITP的发病机制中起重要作用.

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.

目的 研究自身免疫性溶血性贫血(autoimmune hemolytic anemia,AIHA)患者外周血滤泡辅助性T细胞(T follicular helper cells,Tfh细胞)数量的变化,探讨Tfh细胞在AIHA发病机制中的作用.方法 采用流式细胞术的方法检测分析26例AIHA患者及30例正常人(对照组)外周血B细胞、成熟B细胞及CD4+T细胞比例,Tfh细胞数量.结果 AIHA患者外周血CD19+B细胞百分比为(17.46±16.65)％,高于对照组(9.12±4.60)％(P=0.011);成熟B细胞百分比为(87.33±7.80)％,明显高于对照组(76.52±13.90)％ (P =0.001).AIHA患者CD4+T细胞为(25.47±11.75)％,较正常对照组(34.36 ±7.21)％明显减低(P =0.001).AIHA患者Tfh细胞比例为(6.72±5.38)％,明显高于对照组(1.52±1.05)％(P=0.000).结论 Tfh细胞数量的扩增参与了AIHA病理过程,并在其发病过程中起重要作用.

Somatic mutation of calreticulin (CALR) has been found in about 25% patients with essential thrombocythemia (ET), where is the second most common mutated gene after JAK2V617F.Recent data have shown that thrombopoietin receptor MPL function is essential in mutant CALR-mediated cellular transformation. However, the mechanisms for CALR mutant-driven MPN have not been fully elucidated.

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.

Objective To explore the meaning of midkine (MK) levels in serum in different development stage of acute leukemia,and to explore the relationship between MK and WT1.Methods The levels of the MK in serum of 86 cases of acute leukemia and 30 cases of normal people were detected by ELISA.Real-time quantitative PCR (RQ-PCR) method was used to determine the expression of WT1 at mRNA level in 15 AML patients.Results The MK level in serum in the new diagnosed group was higher than that in the complete remission group and the normal control group [7.52 (5.44,10.55) ng/ml vs 3.52 (1.56,5.20) ng/ml vs 2.44 (1.89,3.12) ng/ml].There' s no statistical difference between midkine level in new diagnosed acute B cell leukemia (B-ALL) group and acute myeloid leukemia (AML) group [7.88 (5.78,15.78) ng/ml vs 6.25 (4.59,16.33) ng/ml].The clear correlation was found between the level of serum MK and quantities of marrow WT1 gene (r =0.529,P =0.043).Conclusions The level of MK in serum of acute leukemia patients is increased at the time of new diagnosis and decreased at complete remission.ELISA may be a way to measure the status of AL.The location of MK gene is adjacent to WT1 gene and MK' s clinical significance is similar to WT1' s,furthermore,there is a clear correlation between MK in serum and WT1 of marrow in quantities.

Objective To investigate the clinical characteristics and methods of diagnosis and treatment of granular lymphocytic leukemia (LGLL).Methods Clinical data of 3 patients with LGLL were retrospectively analyzed and relevant literature was reviewed.Results 3 patients were all onset with lymphocytosis,whose conditions progressed slowly.The diagnosis of 2 patients was T-LGLL with immunological characteristics of CD3+ CD4 CD8+ CD56-CD57+.The other patient' s diagnosis was NK-LGLL,whose immunological characteristic was CD3-CD4-CD8-CD56+ CD57-.Two of them didn' t need any treatment.One of them was treated with cyclosporine because of agranulocytosis and recurrent infection.Conclusions LGLL is a group of heterogeneous diseases,which clinical characteristic and prognosis are different.Flow cytometric immunopheotype,TCR Vβ analysis and TCR gene rearrangement are helpful to diagnosis.