| ObjectiveAutoimmune Hemolytic Anemia(AIHA)/Evans syndrome can be divided into primary and secondary AIHA according to the etiology,40% of these occur secondary to Lymphoproliferative Disease(LPD).The emergence of Immunoglobulin(Ig)/T cell Receptor(TCR)gene clonal rearrangement suggests the monoclonal change of lymphocyte,which is an important diagnostic information for lymphoma.Ig/TCR gene clonal rearrangement can be disclosed on some patients with AIHA/Evans syndrome.Are there different clinical characteristics in this group of patients compared with patients without Ig/TCR gene clonal rearrangement?Bruton’s tyrosine kinase(BTK)is a downstream molecule of B cell Receptor(BCR)signal transduction,which’s important to the development of B cells.Does the expression of BTK in AIHA/Evans syndrome patients indicate clinical significance?This study compared the clinical features and effects between AIHA/Evans patients occur secondary to LPD and primary AIHA/Evans patients,and between AIHA/Evans syndrome patients with Ig/TCR gene clonal rearrangement and without.In addition,the study determined the expression level of BTK and Phosphorylated Bruton’s tyrosine kinase(p-BTK)in different B cell subgroup for AIHA/Evans patients.MethodsThe AIAH/Evans patients and normal controls in Tianjin Medical University General Hospital Department of Hematology from December 2012 to March 2018 were included in this study.Part Ⅰ Compare the clinical features and response between 17 cases of AIHA/Evans patients secondary to LPD and 62 cases of primary AIHA/ Evans patients.Summarize the clinical characteristics of AIHA/Evans patients secondary to LPD.Part Ⅱ The clonal rearrangement of Ig/TCR gene in 44 primary AIHA/Evans patients are determined by BIOMED-2 standard gene rearrangement polymerase chain reaction(PCR)amplification method.The differences of clinical characteristics and response between two groups are compared.Part Ⅲ Flow Cytometry(FCM)are used to detect the expression of BTK and p-BTK in peripheral blood CD5+CD19+、CD5-CD19+B cells in patients with primary AIHA/Evans syndrome,which are divided into hemolytic group(n = 16),remission group(n = 20)and control group(n = 15).ResultsPart Ⅰ:1.General features: Age of onset for patients with AIHA/Evans syndrome secondary to LPD(59.41±4.168)is older than primary group(47.18±2.261)(P=0.014).There exist no significant differences for gender distribution between secondary group(8 males,9 females r=0.89)and primary group(28 males,34 females r=0.82)(P=1.00)2.Hemolytic characteristics: The degree of hemolysis for the secondary group is less severe at the onset of the illness: Ret absolute value [(124.3±18.89)×109/L] is lower than primary group [(216.7±26.5)×109/L](P=0.048)and Hb(0.71±0.17)g/L is higher than primary group(0.32±0.04)g/L(P=0.035).3.Immunological characteristics: the ratio of CD3+CD4+/CD3+CD8+ for secondary group(1.81±0.41)is higher than primary group(1.05±0.12),(P=0.207),Ig G(881.36±101.50)mg/dl is lower than primary group(1137.68±70.49)mg/dl(P=0.048),Ig A(108.30±17.39)mg/dl is lower than primary group(179.1±16.9)mg/dl(P=0.013).4.Response: The time of treatment response for secondary group[(27.36±4.856)days] is longer than primary group [(14.26±1.134)days],(P=0.002).5.Prognosis: There is no difference for the relapse rate between the secondary group(33.3%)and primary group(25%)(P=0.685).The mortality in secondary group(23.53%)is higher than that in primary group(0%)(P=0.003).Part Ⅱ:1.Ig/TCR rearrangement positive patients accounted for 34.09%(15/44),Ig H rearrangement positive patients accounted for 53.33%(8/15),TCRγ rearrangement positive patients accounted for 71.43%(5/7),TCRβ rearrangement positive patients14.29%(1/7),both TCRβ and TCRγ rearrangement positive patients 14.29%(1/7).Ig/TCR rearrangement negative patients accounted for 65.91%(29/44).2.General character: Median age of patients with Ig/TCR rearrangement is60(16-81)years old(8 males,7 females r=1.14).Median age of patients without Ig/TCR rearrangement is 53(17-78)years old(10 males,19 females r=0.53).There is no significant difference about sex ratio between the two groups(P=0.378).In positive group,4 cases present with lymph node swelling,4 cases present with abnormal blood coagulation function and 5 cases present with splenomegaly which are 4cases,4cases and 5 cases in negative group respectively.3.Routine blood test: Hb of positive patients [(65.07±6.07)g/L] is lower than that of negative patients [(84.17±4.40)g/L],(P=0.015).RBC for positive patients[(1.82±0.23)×1012/L] is lower than negative group [(2.51±0.19)×1012/L],(P=0.032).4.Hemolytic characteristics: Ret% of positive patients [(16.45±3.54)%] is lower than negative group [(10.21±1.60)%],(P=0.013);there exist no differences for the level of FHb、Hp、LDH、TBIL、IBIL、TBIL between two groups.5.Immunological characteristics: The CRP of positive group and negative group is[((2.13±0.78),(1.03±0.32))mg/dl] respectively(P=0.025).The level of Ig G、Ig A、Ig E、CD5+CD19+/CD19+、CD3+CD4+/CD3+CD8+ do not differ significantly between two groups.6.The maintain of positive group [(18.87±2.28)days] is longer than negative group [(12.12±1.39)days](P=0.011).7.Prognosis: There exist no differences for relapse rate(33%,25%)and relapse time [(15.53±2.58)months],[(6.99±2.35)months] between two groups.Part Ⅲ:1.The level of CD19+CD5+BTK+/CD19+CD5+ B cell in hemolytic group[(44.56±7.79)%,P<0.0001] and remission group [(48.17±8.37)%,P<0.0001] are higher than that of control group [(2.98±0.53)%].The expression level of CD19+CD5+BTK+/CD19+ B cell in hemolytic group [(10.60±4.30)%,P<0.0001] and remission group[(14.63±5.43)%,P<0.0001] are higher than that of control group[(0.67±0.09)%].2.CD19+CD5+p-BTK+/CD19+CD5+ B cell in hemolytic group [(30.81± 7.16)%,P<0.0001] and remission group [(23.37±8.02)%,P<0.0001] are higher than that of control group [(1.71±0.58)%].CD19+CD5+p-BTK+ /CD19+B cell in hemolytic group[(3.33±0.94)%,P<0.0001] and remission group [(2.32±0.40)%,P<0.0001] are higher than that of control group [(0.34±0.06)%].3.CD19+CD5-BTK+/CD19+CD5-B cell in hemolytic group [(12.37± 5.81)%,P=0.003] and remission group [(21.75±7.98)%,P=0.002] are higher than that of control group [(0.37±0.13)%].CD19+CD5-BTK+ /CD19+B cell in hemolytic group[(5.26±2.43)%,P=0.003] and remission group [(15.98±6.26)%,P=0.001] are higher than that of control group [(0.26±0.09)%].4.The level of CD19+CD5-p-BTK+/CD19+CD5-B cell in hemolytic group[(1.22±0.52)%] and control group[(0.34±0.15)%] do not differ significantly(P=0.425);CD19+CD5-p-BTK+/CD19+CD5-B cell in remission group [(1.08±0.25)%] is higher than that of control group(P=0.017).CD19+CD5-p-BTK+/CD19+B cell in hemolytic group [(1.15±0.46)%] and control group [(0.26±0.12)%] do not differ significantly(P=0.152),CD19+CD5-p-BTK+/CD19+B cell in remission group [(0.89±0.20)%] is higher than that of control group(P=0.013).5.p-BTK/BTK of CD5+B cell in hemolytic group [(74.62±6.42)%] is higher than that of CD5-B cell [(29.63±10.19)%,P=0.001].p-BTK/BTK of CD5+B cell in remission group [(77.95±9.57)%] is higher than that of CD5-B cell [(26.29±6.86)%,P=0.006].However,there is no difference between CD5+ and CD5-B cell in control group [(54.89±9.56)%,(30.86±12.47)%,P=0.109].Conclusions1.Patients with AIHA/Evans syndrome secondary to LPD have delayed age of onset and less severe of hemolytic degree.The maintain of response is longer than that of primary patients,with poor prognosis,high mortality and high relapse rate.2.Anemia in Ig/TCR rearrangement positive AIHA/Evans syndrome patients is more severe at onset and duration of treatment is longer.However,hemolytic and immunological characteristics do not differ significantly between two groups.3.The expression level of both BTK and p-BTK in CD5+ and CD5-B cells for AIHA/Evans syndrome patients are higher than that in control group.The activation level of CD5+B cell is higher than CD5-B cells in peripheral blood. |
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