【作者】 杨瑾；

【导师】 邢莉民；

【作者基本信息】 天津医科大学 ， 内科学 血液病， 2022， 硕士

【摘要】 自身免疫性溶血性贫血(Autoimmune Hemolytic Anemia,AIHA)是由于患者体内生成大量抗红细胞自身抗体,从而引发红细胞破坏过多、骨髓代偿不足,最终导致贫血;免疫性血小板减少症(Immune thrombocytopenia,ITP)由于机体免疫功能异常,产生自身抗体或细胞因子,破坏巨核细胞或导致巨核细胞产板不良;埃文斯综合征(Evans Syndrome)指AIHA和ITP先后发病或同时发病,B细胞不仅数量异常,其功能也存在异常,B细胞在AIHA和Evans综合征发病过程中发挥重要作用。我们前期研究发现B细胞,尤其是CD5~+B细胞与自身免疫性溶血性贫血/埃文斯综合征有关,CD5~+B细胞可以通过天然抗体的自身反应性、细胞因子的产生和增强的抗原提呈能力来诱发疾病。我们证明在自身免疫性溶血性贫血/埃文斯综合征患者体内CD5~+B细胞数量增多,且数量增多与患者疾病的严重程度呈正相关、与临床治疗效果呈负相关。B细胞成熟抗原(B-cell maturation antigen,BCMA)主要表达在人体B细胞发育阶段的晚期阶段和浆细胞表面,是一种促进B细胞成熟的标志性蛋白,BCMA与肿瘤坏死因子受体相关因子(TNFR-associated factor,TRAF)相互作用并激活TRAF依赖的NF-κB、JNK和p38 MAPK通路,促进并维持B细胞和浆细胞的活性和功能。跨膜激活剂及钙调亲环素配体相互作用分子(transmembrane activator and calcium modulator and cyclophilin ligand interactor,TACI)主要特征是表达在B细胞表面,尤其是活化B细胞表面,除了可以促进T细胞非依赖性免疫反应,还可以抑制B细胞的活化和扩增,同时促进浆细胞的存活。B细胞激活因子受体(B-cell activation factor receptor,BAFF-R)主要存在于初始和记忆B细胞表面,对未成熟B细胞的存活和成熟至关重要。BCMA、TACI和BAFF-R都作为增殖诱导配体(a proliferation-inducing ligand,APRIL)及B细胞激活因子(B-cell activation factor,BAFF)的受体参与调节B细胞的存活、增殖和活化,BAFF-APRIL系统在部分恶性肿瘤、自身免疫性疾病和炎症性疾病中存在异质性表达,如BCMA在多发性骨髓瘤(Multiple Myeloma,MM)及系统性红斑狼疮(Systemic Lupus Erythematosus,SLE)患者体内升高,而ITP患者体内BCMA的表达水平降低,并且BCMA在不同的自身免疫性疾病中表达水平也不同。自身免疫性溶血性贫血/埃文斯综合征患者外周血B细胞BCMA的水平、BCMA在B细胞尤其是CD5~+B细胞表面的表达水平、血清中游离BCMA(sBCMA)、游离TACI(sTACI)及游离BAFF-R(sBAFFR)的浓度尚未见相关报道,本论文拟对AIHA/Evans患者B细胞表面及胞内BCMA水平进行检测,并检测血清中sBCMA、sTACI和sBAFFR的浓度,了解BCMA在自身免疫性溶血性贫血/埃文斯综合征患者中的水平及其与疾病的相关性。本论文分为三部分。第一部分自身免疫性溶血性贫血/埃文斯综合征患者外周血B细胞BCMA mRNA水平及临床意义目的:了解自身免疫性溶血性贫血/埃文斯综合征患者外周血B细胞BCMA mRNA水平及临床意义。方法:共检测19例自身免疫性溶血性贫血/埃文斯综合征患者,溶血发作组11例,中位年龄46(18-65)岁,男女比3:8;溶血缓解组共8例,中位年龄50(22-73)岁,男女比3:5。健康志愿者8名,中位年龄26(24-27)岁,男女比1:1。首先使用磁珠分选法提取出外周血CD19~+B细胞,流式细胞仪检测分选细胞纯度,采用RT-PCR检测各组分选出的CD19~+B细胞细胞中BCMA mRNA水平,实验结果使用2-ΔΔCt法进行相对定量分析。结果:1、自身免疫性溶血性贫血/埃文斯综合征患者外周血CD19~+B细胞中BCMA mRNA水平(0.188±0.297)显著低于健康对照者(1.055±0.335,P<0.0001)。自身免疫性溶血性贫血/埃文斯综合征溶血发作组患者外周血CD19~+B细胞内BCMA mRNA的相对表达量为(0.076±0.052),溶血缓解组为(0.342±0.421),健康对照组为(1.055±0.335)。其中,溶血发作组BCMA mRNA水平低于健康对照组(P=0.0002),溶血缓解组BCMA mRNA水平低于健康对照组(P=0.0072),溶血发作组和缓解组之间无统计学差异。2、自身免疫性溶血性贫血/埃文斯综合征患者外周血B细胞中BCMA mRNA水平与RBC、Hb、Ret%、Ret#、LDH、TBIL、DBIL、IBIL、IgG、IgM、IgE、IgA、C3、C4、CRP、FHb及Hp等均无明显的相关性(P均大于0.05)。3、自身免疫性溶血性贫血/埃文斯综合征患者外周血CD19~+B细胞中BCMA mRNA水平与接受临床治疗3周后Ret%(R=-0.72,P=0.024)、Ret#(R=-0.79,P=0.009)和IBIL(R=-0.82,P=0.003)呈负相关性。结论:自身免疫性溶血性贫血/埃文斯综合征患者外周血B细胞中BCMA mRNA水平远低于健康对照组,与患者的溶血指标、免疫指标没有相关性,与短期疗效呈负相关。第二部分自身免疫性溶血性贫血/埃文斯综合征患者外周血B细胞表面BCMA的表达水平及临床意义目的:了解自身免疫性溶血性贫血/埃文斯综合征患者外周血B细胞表面BCMA的表达水平及临床意义。方法:采用流式细胞术(FCM)检测各分组B细胞表面BCMA的表达。共检测27例自身免疫性溶血性贫血/埃文斯综合征患者,溶血发作组共17例,中位年龄54(33-77)岁,男女比8:9。溶血缓解组10例,中位年龄42(18-63)岁,男女比3:7。健康志愿者12名,中位年龄29(24-45)岁,男女比1:2。结果:1、自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组CD19~+BCMA~+/CD19~+B细胞比值[(7.62±6.31)%]低于缓解组[(14.52±3.26)%,P=0.01]及健康对照组[(16.30±6.54)%,P<0.001]。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+BCMA~+/CD19~+B细胞比值与TBIL(R=-0.76,P<0.0001)、DBIL(R=-0.57,P=0.0032)、IBIL(R=-0.77,P<0.0001)呈负相关。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+BCMA~+/CD19~+B细胞比值与接受临床治疗3周后TBIL(R=-0.78,P=0.007)、DBIL(R=-0.67,P=0.050)呈负相关性。2、自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组CD19~+CD5~+BCMA~+/CD19~+CD5~+B细胞比值[(10.90±6.85)%]低于缓解组[(22.14±6.06)%,P=0.01]及健康对照组[(21.07±8.57)%,P=0.02]。自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组CD19~+CD5~+BCMA~+/CD19~+B细胞比值[(3.91±2.94)%]明显低于缓解组[(4.90±2.22)%,P<0.01]及健康对照组[(8.19±4.33)%,P<0.05]。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+CD5~+BCMA~+/CD19~+CD5~+B细胞比值与总胆红素(TBIL,R=-0.49,P=0.012)、间接胆红素(IBIL,R=-0.58,P=0.002)呈负相关。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+CD5~+BCMA~+/CD19~+B细胞比值与TBIL(R=-0.39,P=0.05)、IBIL(R=-0.50,P=0.009)呈负相关。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+CD5~+BCMA~+/CD19~+CD5~+B细胞比值与接受临床治疗3周后RBC(R=0.61,P=0.019)和Hb(R=0.70,P=0.005)呈正相关性。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+CD5~+BCMA~+/CD19~+B细胞比值与接受临床治疗3周后RBC(R=0.63,P=0.015)和Hb(R=0.69,P=0.007)呈正相关性,与Ret%(R=-0.81,P=0.022)及Ret#(R=-0.76,P=0.037)呈负相关性。3、自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组CD19~+CD5-BCMA~+/CD19~+CD5-B细胞比值[(6.23±6.71)%]显著低于缓解组[(14.36±4.24)%,P=0.003]及健康对照组[(14.47±5.28)%,P<0.01]。自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组CD19~+CD5-BCMA~+/CD19~+B细胞比值[(7.27±4.58)%]低于健康对照组[(11.23±4.52)%,P<0.05]。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+CD5-BCMA~+/CD19~+CD5-B细胞比值与Hb(R=0.41,P=0.04)呈正相关性,而与TBIL(R=-0.81,P<0.0001)、DBIL(R=-0.63,P=0.0008)、IBIL(R=-0.79,P<0.0001)呈负相关。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+CD5-BCMA~+/CD19~+B细胞比值与IgM(R=0.52,P=0.03)呈正相关性,与TBIL(R=-0.39,P=0.05)呈负相关。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+CD5-BCMA~+/CD19~+CD5-B细胞比值与接受临床治疗3周后RBC(R=0.74,P=0.002)和Hb(R=0.60,P=0.021)呈正相关性。自身免疫性溶血性贫血/埃文斯综合征患者CD19~+CD5-BCMA~+/CD19~+B细胞比值与接受临床治疗3周后TBIL(R=-0.79,P=0.009)及IBIL(R=-0.70,P=0.043)呈负相关性。4、自身免疫性溶血性贫血/埃文斯综合征患者溶血缓解组CD19~+CD5~+BCMA~+/CD19~+CD5~+B细胞比值[(22.14±6.06)%]高于CD19~+CD5-BCMA~+/CD19~+CD5-B细胞比值[(14.36±4.24)%,P=0.004]。健康对照者CD19~+CD5~+BCMA~+/CD19~+CD5~+B细胞比值[(21.07±8.57)%]高于CD19~+CD5-BCMA~+/CD19~+CD5-B细胞比值[(14.47±5.28)%,P=0.03]。自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组CD19~+CD5~+BCMA~+/CD19~+B细胞比值[(3.91±2.94)%]低于CD19~+CD5-BCMA~+/CD19~+B细胞比值[(7.27±4.58)%,P=0.02]。自身免疫性溶血性贫血/埃文斯综合征患者溶血缓解组CD19~+CD5~+BCMA~+/CD19~+B细胞比值[(4.90±2.22)%]低于CD19~+CD5-BCMA~+/CD19~+B细胞比值[(10.31±3.06)%,P<0.001]。5、自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组CD19~+CD5~+BCMA~+/CD19~+B细胞比值[(3.91±2.94)%]低于CD19~+CD5-BCMA~+/CD19~+B细胞比值[(7.27±4.58)%,P=0.02]。自身免疫性溶血性贫血/埃文斯综合征患者溶血缓解组CD19~+CD5~+BCMA~+/CD19~+B细胞比值[(4.90±2.22)%]低于CD19~+CD5-BCMA~+/CD19~+B细胞比值[(10.31±3.06)%,P<0.001]。健康对照者CD19~+CD5~+BCMA~+/CD19~+B细胞比值[(8.19±4.33)%]与CD19~+CD5-BCMA~+/CD19~+B细胞比值[(11.23±4.52)%,P=0.11]无统计学差异。结论:自身免疫性溶血性贫血/埃文斯综合征溶血发作组患者B细胞表面BCMA显著降低,CD5~+B细胞BCMA低于CD5-B细胞水平,BCMA水平与短期疗效呈负相关。第三部分自身免疫性溶血性贫血/埃文斯综合征患者血清中sBCMA、sTACI和sBAFFR的浓度及临床意义目的:了解自身免疫性溶血性贫血/埃文斯综合征患者血清sBCMA、sTACI和sBAFFR的浓度及临床意义。方法:采用酶联免疫吸附实验(ELISA)检测各组血清中sBCMA的浓度。检测标本来自28例自身免疫性溶血性贫血/埃文斯综合征患者,溶血发作组共14例,中位年龄55(16-77)岁,男女比3:4。溶血缓解组14例,中位年龄52(28-69)岁,男女比1:2。健康志愿者10名,中位年龄29(24-51)岁,男女比2:3。结果:1、自身免疫性溶血性贫血/埃文斯综合征患者血清中sBCMA浓度[(5.11±2.18)]显著低于正常对照组[(9.94±4.40),P=0.003]。自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组血清中sBCMA浓度[(5.12±1.95)]低于正常对照组[(9.94±4.40),P=0.001],溶血缓解组血清中sBCMA浓度[(5.10±2.46)]也低于正常对照组[(9.94±4.40),P=0.001]。自身免疫性溶血性贫血/埃文斯综合征患者外周血血清中sBCMA浓度与C3(R=-0.49,P=0.022)、CRP(R=-0.55,P=0.008)呈负相关,与接受临床治疗3周后及3月后临床指标无明显相关性。2、自身免疫性溶血性贫血/埃文斯综合征患者血清中sTACI浓度[(1.06±0.93)]显著低于正常对照组[(3.77±4.25),P=0.002]。自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组血清中sTACI浓度[(1.03±1.09)]低于正常对照组[(3.77±4.25),P=0.008],溶血缓解组血清中sBCMA浓度[(1.12±0.39)]也低于正常对照组[(3.77±4.25),P=0.04]。AIHA/Evans综合征患者外周血上清液中sTACI浓度与Hp(R=0.81,P=0.011)呈正相关性,与Ret%(R=-0.40,P=0.040)和IgM(R=-0.42,P=0.039)呈负相关性,与接受临床治疗3周后及3月后临床指标无明显相关性。3、自身免疫性溶血性贫血/埃文斯综合征患者血清中sBAFFR浓度[(3.07±2.91)]高于正常对照组[(2.13±1.63)],但差距不存在统计学意义。自身免疫性溶血性贫血/埃文斯综合征患者溶血发作组患者血清中sBAFFR浓度[(3.75±3.34)]高于正常对照组[(2.13±1.63)],溶血缓解组血清中sBCMA浓度[(1.72±0.77)]略低于正常对照组[(2.13±1.63)],溶血发作组患者血清中sBAFFR浓度[(3.75±3.34)]高于溶血缓解组[(1.72±0.77)],但均无统计学差异。结论:自身免疫性溶血性贫血/埃文斯综合征患者血清中sBCMA的浓度显著减低,与免疫反应有一定相关性。血清sTACI的浓度减低,与溶血程度负相关。血清sBAFFR的浓度与健康对照无差别。更多还原

【Abstract】 Autoimmune hemolytic anemia（AIHA）is a kind of anemia caused by excessive destruction of red blood cells and insufficient compensation of bone marrow caused by the production of red blood cell autoantibodies.Immune thrombocytopenia（ITP）produces autoantibodies or cytokines due to abnormal immune function,which destroys megakaryocytes or leads to megakaryocytic dysplasia.Evans syndrome refers to the onset of AIHA and ITP successively or at the same time.B cells not only have abnormal number,but also have abnormal function.B cells have an important effect on the pathogenesis of AIHA and Evans syndrome.Our previous studies found that B cells,especially CD5⁺B cells,are associated with autoimmune hemolytic anemia/Evans syndrome.CD5⁺B cells can induce disease through the autoreactivity of natural antibodies,the production of cytokines and enhanced antigen presentation^[1].We proved that the number of CD5⁺B cells increased in patients with autoimmune hemolytic anemia/Evans syndrome,and the increase was positively correlated with the severity of the disease and negatively correlated with the effect of clinical treatment^[2].BCMA is mainly expressed in the late stage of human B cell development and on the surface of plasma cells.it is a marker protein that promotesB cell maturation^[3].BCMA interacts with TRAF and activatesTRAF-dependent NF-κB,JNK and p38 MAPK pathways to promote and maintain the activity and function of B cells and plasma cells^[4,5].TACI are mainly expressed on the surface of B cells,especially activated B cells,which can not only promote T cell independent immune response,but also inhibit the activation and expansion of B cells and promote the survival of plasma cells^[6].Both BCMA and TACI are involved in the regulation of B cell survival,proliferation and activation as receptors of APRIL and BAFF.BAFF-APRIL system is heterogeneously expressed in some malignant tumors,autoimmune diseases and inflammatory diseases,such as increased BCMA in patients with multiple myeloma and systemic lupus erythematosus.However,the expression level of BCMA in ITP patients decreased,and the expression level of BCMA was different in different autoimmune diseases^[4,9].The level of BCMA in B cells of patients with autoimmune hemolytic anemia/Evans syndrome,the expression level of BCMA on B cells,especially CD5⁺B cells,and the concentration of sBCMA、sTACI and free sBAFFR in serum have not been reported.In this study,to understand the level of BCMA in patients with autoimmune hemolytic anemia/Evans syndrome and its correlation with the disease,we detect BCMA levels on the surface and intracellular of B cells in AIHA/Evans patients,and the concentrations of sBCMA,sTACI and sBAFFR in serum.This thesis is divided into three parts.Part 1 BCMA mRNA level and clinical significance of peripheral blood B cells in patients with autoimmune hemolytic anemia/Evans syndromeObjective:To investigate the level and clinical significance of peripheral blood B cell BCMA mRNA in patients with AIHA/Evans syndrome.Methods:A total of 19 patients with AIHA/Evans syndrome and 11 patients with hemolysis attack were detected,median age 46（18-65）years old,the ratio of male to female was 3:8.There were 8 cases in hemolysis remission group,the median age was 50（22-73）years old,3 males and 5 females.There were 8 healthy volunteers with a median age of 26（24-27）years old,including 4 males and 4 females.Firstly,magnetic beads sorting method was used to extract CD19⁺B cells from peripheral blood,and flow cytometry was used to detect the purity of the selected cells.Finally,RT-PCR was used to detect the level of BCMA mRNA in CD19⁺B cells selected by each group.The experimental results were analyzed by 2^-ΔΔCtmethod.Results:1.The level of BCMA mRNA in CD19⁺B cells of patients with AIHA/Evans syndrome（0.188±0.297）was significantly lower than that in normal controls（1.055±0.335,P<0.0001）.The relative expression of BCMA mRNA in CD19⁺B cells of patients with hemolysis attack of AIHA/Evans syndrome was（0.076±0.052）,that of hemolysis remission group was（0.342±0.421）,and that of healthy control group was（1.055±0.335）.Among them,the level of BCMA mRNA in hemolysis attack group was lower than that in normal control group（P=0.0002）,and the level of BCMA mRNA in hemolysis remission group was lower than that in healthy control group（P=0.0072）.There was no obvious difference between hemolysis attack group and remission group.2.There was no significant correlation between BCMA mRNA level and RBC,Hb,Ret%,Ret#,LDH,TBIL,DBIL,IBIL,IgG,IgM,IgE,IgA,C3,C4,CRP,FHb and Hp in patients with AIHA/Evans syndrome.3.The level of BCMA mRNA in CD19⁺B cells of patients with AIHA/Evans syndrome was negatively correlated with Ret%（R=-0.72,P=0.024）,Ret#（R=-0.79,P=0.009）and IBIL（R=-0.82,P=0.003）after 3 weeks of clinical treatment.Conclusion:The level of BCMA mRNA in peripheral blood B cells of patients with AIHA/Evans syndrome was much lower than that of healthy controls,which was not correlated with hemolysis index and immune index,but negatively correlated with short-term curative effect.Part 2 Expression level and clinical significance of BCMA on peripheral blood B cells in patients with autoimmune hemolytic anemia/Evans syndromeObjective:To investigate the expression level and clinical significance of BCMA on peripheral blood B cells in patients with AIHA/Evans syndrome.Methods:Flow cytometry（FCM）was used to detect the expression of BCMA on B cells in each group.A total of 27 patients with AIHA/Evans syndrome and 17patients with hemolysis attack were examined,the median age was 54（33-77）years old,8 males and 9 females,the ratio of male to female was 8:9.There were 10 cases in hemolysis remission group,the median age was 42（18-63）years old,3 males and7 females,the ratio of male to female was 3:7.There were 12 healthy volunteers,with a median age of 29（24-45）years old,4 males and 8 females,with a male-to-female ratio of 1:2.Results:1.The percentage of CD19⁺BCMA/CD19⁺B cells in hemolytic attack group[（7.62±6.31）%]was lower than that in remission group[（14.52±3.26）%]and healthy control group[（16.30±6.54）%,P<0.001].The proportion of CD19⁺BCMA/CD19⁺B cells in patients with AIHA/Evans syndrome was negatively correlated with TBIL（R=-0.76,P<0.0001）,DBIL（R=-0.57,P=0.0032）and IBIL（R=-0.77,P<0.0001）.The CD19⁺BCMA⁺/CD19⁺B cell ratio in patients with AIHA/Evans syndrome was negatively correlated with TBIL（R=-0.78,P=0.007）and DBIL（R=-0.67,P=0.050）after 3 weeks of clinical treatment.2.The percentage of CD19⁺CD5⁺BCMA/CD19⁺CD5⁺B cells in hemolytic attack group of AIHA/Evans syndrome[（10.90±6.85）%]was lower than that in remission group[（22.14±6.06）%,P=0.01]and healthy control group[（21.07±8.57）%,P=0.02].The percentage of CD19⁺CD5⁺BCMA/CD19⁺B cells in hemolytic attack group of AIHA/Evans syndrome[（3.91±2.94）%]was significantly lower than that in remission group[（4.90±2.22）%,P<0.01]and healthy control group[（8.19±4.33）%,P<0.05].The proportion of CD19⁺CD5⁺BCMA/CD19⁺CD5⁺B cells in patients with AIHA/Evans syndrome was negatively correlated with TBIL（R=-0.49,P=0.012）and IBIL（R=-0.58,P=0.002）.The proportion of CD19⁺CD5⁺BCMA/CD19⁺B cells in patients with AIHA/Evans syndrome was negatively correlated with TBIL（R=-0.39,P=0.05）and IBIL（R=-0.50,P=0.009）.The CD19⁺CD5⁺BCMA⁺/CD19⁺CD5⁺B cell ratio in patients with AIHA/Evans syndrome was positively correlated with RBC（R=0.61,P=0.019）and Hb（R=0.70,P=0.005）after 3 weeks of clinical treatment.The ratio of CD19⁺CD5⁺BCMA⁺/CD19⁺B cells in patients with AIHA/Evans syndrome was positively correlated with RBC（R=0.63,P=0.015）and Hb（R=0.69,P=0.007）after 3 weeks of clinical treatment,and negatively correlated with Ret%（R=-0.81,P=0.022）and Ret#（R=-0.76,P=0.037）.3.The percentage of CD19⁺CD5^-BCMA/CD19⁺CD5^-B cells in hemolytic attack group of AIHA/Evans syndrome[（6.23±6.71）%]was significantly lower than that in remission group[（14.36±4.24）%,P=0.003]and healthy control group[（14.47±5.28）%,P<0.01].The proportion of CD19⁺CD5^-BCMA/CD19⁺B cells in the hemolytic attack group of AIHA/Evans syndrome[（7.27±4.58）%]was lower than that of the healthy control group[（11.23±4.52）%,P<0.05].The proportion of CD19⁺CD5^-BCMA/CD19⁺CD5^-B cells in patients with AIHA/Evans syndrome was positively correlated with Hb（R=0.41,P=0.04）,but negatively correlated with TBIL（R=-0.81,P<0.0001）,DBIL（R=-0.63,P=0.0008）and IBIL（R=-0.79,P<0.0001）.The proportion of CD19⁺CD5^-BCMA/CD19⁺B cells in patients with AIHA/Evans syndrome was positively correlated with IgM（R=0.52,P=0.03）and negatively correlated with TBIL（R⁼-0.39,P=0.05）.The CD19⁺CD5^-BCMA⁺/CD19⁺CD5^-B cell ratio in patients with AIHA/Evans syndrome was positively correlated with RBC（R=0.74,P=0.002）and Hb（R=0.60,P=0.021）after 3 weeks of clinical treatment.The CD19⁺CD5^-BCMA⁺/CD19⁺B cell ratio in patients with AIHA/Evans syndrome was negatively correlated with TBIL（R=-0.79,P=0.009）and IBIL（R=-0.70,P=0.043）after 3 weeks of clinical treatment.4.The percentage of CD19⁺CD5⁺BCMA/CD19⁺CD5⁺B cells in hemolytic remission group of patients with AIHA/Evans syndrome was higher than that of CD19⁺CD5^-BCMA/CD19⁺CD5^-B cells[（22.14±6.06）%vs（14.36±4.24）%,P=0.004].The proportion of CD19⁺CD5⁺BCMA/CD19⁺CD5⁺B cells in healthy controls[（21.07±8.57）%]was higher than that of CD19⁺CD5^-BCMA/CD19⁺CD5^-B cells[（14.47±5.28）%,P=0.03].The proportion of CD19⁺CD5⁺BCMA/CD19⁺B cells in hemolytic attack group[（3.91±2.94）%]of patients with AIHA/Evans syndrome was lower than that of CD19⁺CD5^-BCMA/CD19⁺B cells[（7.27±4.58）%,P=0.02].The percentage of CD19⁺CD5⁺BCMA/CD19⁺B cells in hemolytic remission group[（4.90±2.22）%]of patients with AIHA/Evans syndrome was lower than that of CD19⁺CD5^-BCMA/CD19⁺B cells[（10.31±3.06）%,P<0.001].5.The ratio of CD19⁺CD5⁺BCMA⁺/CD19⁺B cells in patients with AIHA/Evans syndrome[（3.91±2.94）%]was lower than that of CD19⁺CD5^-BCMA⁺/CD19⁺B cells[（7.27±4.58）%,P=0.02].The ratio of CD19⁺CD5⁺BCMA⁺/CD19⁺B cells in hemolytic remission group[（4.90±2.22）%]of patients with AIHA/Evans syndrome was lower than that of CD19⁺CD5^-BCMA⁺/CD19⁺B cells[（10.31±3.06）%,P<0.001].There was no significant difference between CD19⁺CD5⁺BCMA⁺/CD19⁺B cell ratio[（8.19±4.33）%]and CD19⁺CD5^-BCMA⁺/CD19⁺B cell ratio[（11.23±4.52）%,P=0.11]in healthy controls.Conclusion:In AIHA/Evans syndrome hemolytic attack group,B cell surface BCMA decreased significantly,CD5⁺B cell BCMA level was lower than CD5^-B cell level,and BCMA level was negatively correlated with short-term efficacy.Part 3 The concentration and clinical significance of serum sBCMA、sTACI and sBAFFR in patients with autoimmune hemolytic anemia/Evans syndromeObjective:To understand the concentration and clinical significance of serum sBCMA、sTACI and sBAFFR in patients with AIHA/Evans syndrome.Methods:The concentration of sBCMA、sTACI and sBAFFR in serum of each group was detected by ELISA.The samples were collected from 28 patients with AIHA/Evans syndrome and 14 patients with hemolysis attack,the ratio of male to female was 3:4.There were 14 cases in hemolysis remission group,the median age was 52（28-69）years old,4 males and 8 females,the ratio of male to female was 1:2.There were 10 healthy volunteers,the median age was 29（24-51）years old,4 males and 6females,the ratio of male to female was 2:3.Results:1、The serum sBCMA concentration in patients with AIHA/Evans syndrome[（5.11±2.18）]was significantly lower than that in normal controls[（9.94±4.40）,P=0.003].The serum sBCMA concentration in the patients with AIHA/Evans syndrome[（5.12±1.95）]was lower than that in the normal control group[（9.94±4.40）,P=0.001],and the serum sBCMA concentration in the hemolytic remission group[（5.10±2.46）]was also lower than that in the normal control group[（9.94±4.40）,P=0.001].The concentration of sBCMA in the supernatant of peripheral blood of patients with AIHA/Evans syndrome was negatively correlated with C3（R=-0.49,P=0.022）and CRP（R=-0.55,P=0.008）,but not with clinical indexes after 3weeks and 3 months.2、The serum sTACI concentration in patients with AIHA/Evans syndrome[（1.06±0.93）]was significantly lower than that in normal controls[（3.77±4.25）,P=0.002].The concentration of sTACI in serum of patients with AIHA/Evans syndrome in hemolytic attack group[（1.03±1.09）]was lower than that in normal control group[（3.77±4.25）,P=0.008].The serum sBCMA concentration in the hemolytic remission group[（1.12±0.39）]was also lower than that in the normal control group[（3.77±4.25）,P=0.04].The concentration of sTACI in the supernatant of peripheral blood of patients with AIHA/Evans syndrome was positively correlated with Hp（R=0.81,P=0.011）,and negatively correlated with Ret%（R=-0.40,P=0.040）and IgM（R=-0.42,P=0.039）.There was no significant correlation with clinical indexes 3 weeks and 3 months after clinical treatment.3、The serum sBAFFR concentration in patients with AIHA/Evans syndrome[（3.07±2.91）]was higher than that in the normal control group[（2.13±1.63）],but the difference was not statistically significant.The serum sBAFFR concentration of patients with AIHA/Evans syndrome in hemolytic attack group[（3.75±3.34）]was higher than that in normal control group[（2.13±1.63）].The serum sBCMA concentration in the hemolysis remission group[（1.72±0.77）]was slightly lower than that in the normal control group[（2.13±1.63）],and the serum sBAFFR concentration in the hemolysis attack group[（3.75±3.34）]was higher than that in the hemolysis remission group[（1.72±0.77）],but there was no statistical difference.Conclusion:The serum concentration of sBCMA in patients with AIHA/Evans syndrome was significantly reduced,which was related to immune response.The decrease of serum sTACI was negatively correlated with the degree of hemolysis.The concentration of serum sBAFFR was not different from that of healthy controls.更多还原