| Objective: Aplastic anemia (AA) and myelodysplastic Syndrome (MDS) are common hematologic diseases.In AA peripheral blood pancytopenia was caused by bone marrow failure and reduction of hematopoietic tissue. In MDS the ineffective hematopoiesis and dysplasia was caused by the abnormal hematopoietic stem cell clone. MDS and AA are two totally different disease, the pathogenesis of the two is not very clear at present, treatment options and prognosis are quite different, but both are mainly manifested in the clinical ,such as hemophthisis,heamorrhage and infection caused by pancytopenia. It is difficult to diagnosis atypical cases because there is no specific marker for diagnosis . Diagnosis of MDS, especially for those whose proportion of primitive cells is not obvious increased and who have no clonal chromosomal abnormalities is essentially exclusive diagnosis. About 10% of MDS patients with treatment may reduce the performance of bone marrow hyperplasia, about 1 / 4 of patients with no obvious ill-blood, and only 40% ~ 70% of the existence of cytogenetic abnormalities [1].AA can be expressed as bone marrow hyperplasia active ,also appears dyshaematopoiesis ,even appears cytogenetic abnormalities. Therefore, it is difficult to identification hypoplastic MDS and AA.Recent studies had found that the expression frequeney of human Leukocyte antigen DR15(HLA-DR15)was higher inAA,MDS than normal.There was a significant correlation between HLA-DR15 positive and the response to IST。HLA-DR15 positive patients show higher efficieney to IST and more dependence to cyclosporineA(CsA). In addition, most scholars believe that AA is the major link in the incidence of primary / secondary hematopoietic stem / progenitor cells abnormal in quality or quantity. The disease of MDS exist heterogeneity, the nature of his illness is a hematopoietic stem / progenitor cells in malignant clones and generation. CD34+ is the characteristic markers of hematopoietic stem / progenitor cell .Study the characteristics of CD34+ with MDS and AA have a certain significance in diagnosis and differential diagnosis. In recent years, the role of CD4+ CD25+Treg in regulating immunosuppression is importance as a hot research that CD4+CD25+ T cells may be involved in the regulation of autoimmune diseases, play a certain role in the pathogenesis of AA and MDS. Some people found that in some hematological malignancies, such as leukemia, MDS patients, the leval of fetal hemoglobin in erythroblasts increased, but the expression is not increased in the AA, may be this have some significance to identify AA and MDS.This study was testing the expression of HLA-DRB1*1501 in AA, MDS patients and compared with normal control group in order to understand the characteristics of HLA-DRB1*1501 in AA and MDS.IST efficacy of HLA-DRB1*1501 Positive and negative groups were observed Preliminarily to AA and MDS.At the same time , The level of CD34+,CD4+CD25+Treg cell and HbF were measured by Flow cytometry(FCM) ,to know the action of these makers in immunopathogenesis. Immunohistochemistry was used to detect the expression of Fetal hemoglobin .We studied the expression of these makers in patients whose post-treatment in 6 months for parallel the date.Through joint detection of multiple targets, to guide clinical identification and diagnosis purposes.Methods: The study included 14 cases of alastic anemia(AA),20 cases of myelodysplastic Syndrome (MDS) and 30 cases normal control(NC).There were 14 cases of alastic anemia(8males and 6 females,aging form 14to 66with a midian age of 26),including chronic aplastic anemia(CAA)9cases and 5cases of acute aplastic anemia (SAA). There were 20 cases of myelodysplastic syndrome (9males and 11 females,aging form 27 to 75 with a midian age of 58), including refractory anemia(RA) 8cases,refractory anemia with excess blasts (RAEB)12cases.Take 30 nomal cases as controls. (15males and 15 females,aging form 20 to 75 with a midian age of 55.HLA-DR15 was detected by the technique of polymerase chain reaction-sequence specific primers (PCR-SSP) in peripheral blood of all groups.The level of CD34+,CD4+CD25+Treg cell and HbF were measured by the method of Flow cytometry(FCM) . Immunohistochemistry was used to detect the expression of Fetal hemoglobin . We studied the expression of these makers in 14 cases of AA patients whose post-treatment in 6 months .Finally, collect the clinical date to parallel.Results: 1 The expression rates of HLA-DRB1*1501 in all groups1.1 The expression rates of HLA-DRB1*1501 in AA patients was 64.28%, The expression rates of HLA-DRB1*1501 in normal control group was 13.33%. The expression rates of HLA-DRB1*1501 in AA patients was significant higher than that of normal controls (P<0.01), RR value was11.7,RR value>1。1.2 The expression rates of HLA-DRB1*1501 in MDS patients was 45.0%.It was significant higher than that of normal controls (P<0.01), RR value was 5.318,RR value>1。The expression rates of HLA-DRB1*1501 in AA patients was significant higher than that in MDS patients,statistically the difference is significant(P<0.01).1.3 The efficiency of IST to14 cases of AA patienis was 42.86%(6/14),in which HLA - DRB1*1501 positive group was 66.67% (6/9)and negative group was 0 , the difference between the two groups has statistically significant(P<0.05).2 The level of CD34+were measured in all groups2.1 The level of the ratio of CD34+ positive cells in bone marrow mononuclear cells in patients with AA (0.224±0.106) was significantly lower than that of normal controls (1.183±0.468) , MDS-RA(0.753±0.295)and MDS-RAEB(3.760±1.228),the difference was statistically significant. (P<0.05).2.2 The level of the ratio of CD34+ positive cells in bone marrow mononuclear cells in patients with RA(0.753±0.295)was significantly lower than that of MDS-RAEB(3.760±1.228)(P<0.01), The level of the ratio of CD34 positive cells in bone marrow mononuclear cells in patients with MDS-RAEB(3.760±1.228)was significantly higher than that of RA(0.753±0.295),normal controls(1.183±0.468) (P<0.01).2.3 After 6 months treatment ,the date of the ratio of CD34+ positive cells in bone marrow mononuclear cells in patients with AA (0.701±0.286)were significantly higher than before treatment(0.224±0.106).3 The level of CD4+CD25+Treg were measured in all groups3.1 The level of CD4+CD25+Treg in patients with AA(6.00±1.47) was significantly lower than that of normal controls(8.52±1.50)and MDS -RAEB(10.19±1.54).But compared with MDS-RA(6.45±1.26),the two groups has no significance.(p>0.05).3.2 The expression levels of CD4+CD25+Treg were increase gradually in RA,RAEB groups;patients with MDS-RA(6.45±1.26) group had obviously lower than those in the MDS-RAEB (10.19±1.54)groups and normal controls(8.52±1.50)(P<0.01);patients with RAEB had significantly higher CD4+CD25+Treg than the RA,RCMD groups and normal controls(P<0.01).3.3 After treatment of CsA for 6 months, the level of CD4+CD25+Treg in patients with AA(7.12±2.78) were significantly higher than before treatment.4 The expression of Fetal hemoglobin4.1 The expression of Fetal hemoglobin with FCMThe level of Fetal hemoglobin in patients with AA(54.993±8.775)was significantly higher than that of normal controls(48.190±7.978), the difference between the two groups was statistically significant. (P<0.05), The level of Fetal hemoglobin in patients with MDS (62.140±10.409)was significantly higher than that of normal controls(48.190±7.978), the difference between the two groups was statistically significant. (P<0.05). The level of Fetal hemoglobin in patients with MDS (62.140±10.409)was significantly higher than that in AA(54.993±8.775). The difference between the two groups was statistically significant. (P<0.05).4.2 The expression of Fetal hemoglobin with ImmunohistochemistryHbF-positive erythroblasts were observed in 11 out of 20 MDS patients (RA 6/8 ,RAEB 5/12). Two out of 11 AA patients were found to be HbF-positive . 5 14 cases of AA,the expression leval of CD34+ is low . HbF-positive erythroblasts were only 2 cases.8 cases of MDS-RA,both CD34+ and HbF positive were 4cases.There were only 1 cases that the two indicators are both negtive. There were only 1 cases that CD34+ positive and 2 case HbF- positive. 12 cases of MDS-RAEB,both CD34+ and HbF positive were 4 cases.There were only 2 cases that the two indicators are both negtive. There were 5 cases that CD34+ positive and 1 case HbF- positive.Conclusions: 1 The expression rates of HLA-DRB1*1501 in AA and MDS was significantly higher than that of normal controls, statistically the difference is significant.In AA and MDS group ,the higher RR value show that there was a positive association between the diseases and HLA- DRB1*1501.Since the higher RR value show the stronger association.The strongest association between the diseases and the expression of HLA-DRB1*1501 existed in AA patients. HLA-DRB1*1501 is susceptibility gene in AA and MDS.So the HLA-DRB1*1501 gene could be used to identify AA and MDS,evaluating clinical prognosis.2 The level of the ratio of CD34+ positive cells in bone marrow mononuclear cells in patients with AA was significantly lower than that of normal controls (P<0.01), the difference between the two groups has statistically significant . It may indicate that the etiological factors of aplastic anemia have a relationship with bone marrow hematopoietic stem cell / progenitor cell defects. The level of the ratio of CD34+positive cells in bone marrow mononuclear cells in patients with MDS-RA has no significant difference with normal controls.The level of the ratio of CD34+positive cells in bone marrow mononuclear cells in patients with MDS-RAEB was significantly higher than normal controls, suggesting that the abnormal clone of MDS-RAEB occurred in hematopoietic stem / progenitor cells.The difference between MDS-RA and MDS-RAEB was also statistically significant, given the CD34+ have a relationship with progress and prognosis of disease .3 The level of CD4+CD25+Treg in patients with AA was significantly lower than that of normal controls,suggesting that AA patients had significantly abnormal immune regulation and may be have a close relationship with the occurrence and develop of AA; The leval of CD4+ CD25+ Treg in patients of MDS would increase with the increase of prognosis risk, suggesting that the immune abnormalities in MDS is a contributing factor of disease development. But the immune states of MDS exist heterogeneity ,to exact the role of immunifaction in MDS remains to be further studied.4 Using immunohistochemistry and flow cytometry methods have proved that the level of fetal hemoglobin in patients with MDS was significantly higher than AA patients, suggesting that the fetal hemoglobin has changed in pathogenesis of hematonosis.This can be used as a new indicators to identify AA and MDS.5 Joint CD34+ and HbF two indicators to identify MDS-RA and AA, diagnosis has improved significantly.6 Follow-up 14 cases of AA,made them to take medicine of CsA immunosuppressive therapy. Then measure the indicators of post-treatment and discover that the efficitive power of HLA-DRB1*1501-positive group was higher than the negative group, statistically significant difference between the two groups, suggesting that HLA-DR15 may be effective treatment of CsA predicted gene.Otherwise, after take medicine of CsA immunosuppressive therapy,the expression leval of CD34+and CD4+CD25+Treg was significantly higher than before treatment,suggesting that those indicators have some action in identifing AA and MDS.
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