Objective To establish a novel method to detect autoantibodies against platelatespecific receptors by flow cytometric bead assay and study its clinical application. Methods The beads were coated with monoclonal antibodies SZ2, SZ22, SZ21 and 7E3 against platelet GP Ⅰ b, GP Ⅱ b, GP Ⅲa and GP Ⅱ b/Ⅲ a, respectively. Captured platelet glycoprotein and beads complex was detected by FITC labeled polyclonal goat antihuman immunoglobulin using flow cytometer. The platelet samples that reacted with antibodies (SZ2, SZ22, SZ21 and 7E3) negatively and positively were tested, respectively. Each sample was repeated 20 times to generate intra-day CV for the MFI and once a day for 8 days to generate inter-day CV values. The 85 ITP patients, 17 NITP patients and 50 controls from the First Affiliated Hospital of Soochow University during March 2006 to December 2008 were included in the studies. The sensitivity and specificity of these four platelet antibodies to diagnose ITP were analyzed using ROC curve. The results were compared with MAIPA. Results The CV of the intra-day-assay for samples negative to antibody SZ2, SZ22,SZ21 and 7E3 were 3.26%, 2. 86%, 1.65% and 4. 94%, respectively; While the CV of the intra-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 6. 16%, 4. 88%, 5.20% and 5. 85%,respectively. The CV of the inter-day-assay for samples negative to antibody SZ2, SZ22, SZ21 and 7E3 were 5. 86%, 4. 74%, 5.69% and 7.56%, respectively; While the CV of the inter-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 7.53%, 5.49%, 7.11% and 6.25%,respectively. The MFI for SZ2 in ITP group, NITP group and healthy control group were 1.49(0. 88-16. 24),1.12(1.00-1.33), 1.01 (0. 83-1.37), respectively, which showed significant differences (H = 36.89,P＜0.01). The MFI for SZ22 in the three groups were 1.55 (0.84-11.30), 1.13(1.03-1.29), 0.98(0. 85-1.24), respectively (H=28.41, P ＜0.01). The MFI of SZ21 were 1.50 (0.87-11.04), 1.13(0.97-1.32), 1.05 (0.85-1.48), respectively (H=54.42, P＜0. 01). The MFI for7E3 were 1.51(0. 84-9.81), 1.05(0.86-1.13), 1.03 (0.74-1.28), respectively (H =31.97, P ＜0.01). Based on ROC analysis, with cut-off values of 1.37, 1. 24, 1.48 and 1.28 for SZ2, SZ22, SZ21 and 7E3,respectively, the AUC were 0. 86, 0.90, 0. 87 and 0. 84, respectively. The sensitivities of the assays were 58. 82% (50/85), 52. 94% (45/85), 52.94% (45/85) and 51.76% (44/85), respectively. When all four antibodies were used, the sensitivity was increased to 74. 12% (63/85), which was higher than that of MAIPA [ 50. 59% (43/85) ,χ2 = 6. 78, P ＜ 0. 05) ]. Conclusion Flow cytometric bead assay can be used to detect four platelet-specific autoantibodies simultaneously, and may be a useful method to aid in the diagnosis of ITP.

Objective　To investigate the expression of LPS receptor-CD14(CD14) on the membrane of Kupffer′s cells (KCs) induced by Lipopolysaccharide (LPS )and its role in activation of KCs and production of cytokines. Methods　KCs were isolated by collagenase perfused Wistar rats and routinely cultured in 24-well dishes for 12 h. Cells were harvested and adjusted to a concentration of 1×106/ml/well and were devided into two groups. Group of LPS: KCs were induced with different concentration of LPS (0, 100 ng/ml, 1 μg/ml and 100 μg/ml). Group of PI-PLC: KCs were pre-incubated for 30 min with one unit of phophatidy linositol specific phospholipase C (PI-PLC) before different concentrations of LPS were added. KCs were cultured for 30 and 60 min respectively. Supernatants were then collected for measuring the level of TNFα and IL-6. Cells were stained by indirect immunofluorescent method ( rabbit anti-CD14 antibody and goat anti-rabbit IgG conjugated with FITC ) and analyzed with flow cytometer (FCM). The percentage and mean fluorescence intensity (FI) of CD14-positive cells were taken as the indexes. Results　In LPS group, after incubation of cells with increasing concentration of LPS, a significant increase in the percentage of CD14 positive KCs were found and the mean FI was stronger when compared with the control points or the group of PI-PLC. The levels of TNFα and IL-6 in supernatant also increased (P<0.01). In group of PI-PLC, decreased percentage of CD14 positive KCs and weakened mean FI were found when compared with group of LPS. The increasing production of TNFα and IL-6 slowed down in the group of PI-PLC. Conclusion　CD14 expression of KCs might be up-regulated by LPS with increase of some cytokines. The production of cytokines in KCs induced by LPS is partially inhibited by PI-PLC.

Objective To study the immunophenotypic feature of CD+2 adult B cell acute lymphoblastic leukemia (CD+2 B-ALL) and provide evidences for the diagnosis, therapy and prognosis. Methods The immunophenotypes of 18 cases of adult CD+2 B-ALL and 68 cases of adult CD-2 B cell acute lymphoblastic leukemia(CD-2 B-ALL) were assayed by a panel of monoclonal antibodies (McAbs) with FACS. Results The age of CD+2 B-ALL adults was younger compared to that of CD-2 B-ALL. The patients in the CD; group were similar to CD-2 group for the expression of most of the cell surface antigens assayed. However, it was notable that expression frequencies of CD10 [(73.78±26.67) %] in CD+2 B-ALL was higher than those in CD; B-ALL [(52.84±35.25) %] (t = 2.35, P<0.05) and CD33 [(15.46±27.41) %] were significantly lower than that of CD2 BALL [(31.15+27.72) %] (t = 2.16, P<0.05). Both groups showed high positive expression for CD34 72.2 % (13/ 18) and 80.9 % (55/68), respectively (χ2 = 0.64, P >0.05). The CD20 expression in CD+2 B-ALL was lower than that in CD-2 B-ALL significantly (χ2 = 11.38, P <0.05). The myeloid antigen (CD13 or CD33) expression in CD+2 B-ALL 44.4 % (8/18) was lower than that in CD-2 B-ALL 72.1 %(49/68) (χ2 = 4.86, P <0.05). Conclusion Patients in the CD+2 B-ALL were similar to CD-2 B-ALL for expression of most of the cell surface antigens assayed. CD+2 B-ALL showed low expression frequency of myeloid antigens (CD13, CD33) and CD20 antigens than CD-2 B-ALL. These results suggested that those with the CD+2 B-ALL immunophenotype generally presented with favorable prognostic features and usually could achieve good outcomes after treatment.

Objective To improve the recognition of aggressive natural killer-cell leukemia(ANKL)complicating muhiple organ failure(MOF).Methods A Fare case of ANKL was reported,and the related literatures were reviewed. Results One case of ANKL was diagnosed by bone marrow morphology and immunophenotypes of CD2,CD16,CD56,who developed multiple organ failure involving in liver,kidney,heart,lung,severe metabolic acidosis,tumour lysis syndrome and disseminated intravascular coagulation(DIC)in course of the disease and chemotherapy.VP regimen and symptomatic treatment were performed,and the disease could be stable shortly but died of the failure of lung and heart soon. Conclusion ANKL has a fulminant clinical course and diffuse infihration of tumor cells resulted in multiple organ failure with poor response to treatment and unfavorable prognosis.

AIM: To investigate the clinical significance in determination of the P- selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P- selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently - labeled SZ - 51 by direct FCM comparing with indirect FCM and enzyme- linked immunosorbent assay (ELISA). RESULTS: The level of P- selectin on platelet membrane is higher in DM (23.92 % + 15.83 % ), in hyperlipidemia ( 18.34 % + 9.46 % ) and in cerebral infarction ( 19.32 % + 10.38 % ) than normal subjects (3.38 % + 1.11% ) ( P < 0.01 ). In addition, similar results on P - selectin were obtained by indirect FCM and ELISA in patients with DM and cerebral infarction. CONCLUSION: FITC - labeled SZ - 51 - IgG can be used in FCM, and it would be a new and sensitive method in detecting platelet activation.

AIM: To investigate the clinical significance in determination of the P-selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P-selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently-labeled SZ-51 by direct FCM comparing with indirect FCM and enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of P-selectin on platelet membrane is higher in DM (23.92%?15.83%), in hyperlipidemia (18.34%?9.46%) and in cerebral infarction (19.32%?10.38%) than normal subjects (3.38%?1.11%) (P

AIM: To further investagate the mechanism of thrombus formation and develop a new remedy of anti-thrombus formation. METHODS: The amplified DNA fragment of vWF-A1 domain was inserted into expression vector with 6?his taq (pQE-31), the recombinant expression vect or was transformed into E coli (strain M15) and induced by IPTG. The recombinant fragment, comprising residues 449-728 of mature vWF subunit, designate rvWF-A1. It was purified by Ni-NTA agarose column and renatured by Tris buffer containin g GSH and GSSG. FACS and platelet aggregometer were employed to analyse the rvWF -A1 function of binding to platelet glycoprotein Ib and inhibiting ristocetin-in duced platelet aggregation. RESULTS: The rvWF-A1 was expressed successfully in E coli, comin g up to 30% of total bacterial protein. Its purify was over 95% through Ni-NTA a garose. It was identified to have ability to bind to GPIb, its biologic activity to inhibit ristocetin-induced platelet aggregation was observed, and the inhibi tive rate was 84 7%. CONCLUSION: The above results indicated that high-level expressi on of rvWF-A1 was successfully achieved in E coli and rvWF-A1 may be an effectiv e antithromotic agent in preventing thrombus formation.

Objective To investigate the clinical and laboratory features of acute promyelocytic leukemia (APL).Methotis 513 APL patients in the last two decades were retrospectively analyzed in this research.We investigated the clinical features including age,sex,abnormality of peripheral hemogram before treatment.therapeutic effect and follow-up and laboratory data such as morphology,immunology,cytogenetics and molecular biology(MICM).Results The median age of the APL patients was 33 years old and the ratio of male and female was 1.21:1.Before treatment,the median level of WBC was 4.3×109/L and the deteetion rate of abnormal promyelocyte on blood film was 85.8%;with immunophenotypie detection,the expression levels of CD117、CD34、HLA-DR、CD7、CD14 and CD19 in APL were found to be lower and the expression 1evels of CD2、CD33 and MPO higher than those in other subtypes of acute myelocytie leukemia(AML)(beth P<0.01).Specific abnormal chromosome t(15;17)was detected in 91.7%of the patients,of whom 75.9%had standard translocation of t(15;17),being the most common one and 15.8% of the patients had t(15;17)with additional abnormal chromosome.There was only 7.5%of the patients with nolnlal karyotype.However,the presence of both simple translocation and complex translocation was seldom seen.With molecular biological detection.PML/RARα fusion gene positive rate was 99.6%.In a relativelv long clinical follow-up,we found that the complete remission(CR)rate in APL patients was 84.7%.incidence of DIC was 13.4%and five-year survival rate was 30.7%.111e median count of WBC in CR group was lower than that non-remission group(P<0.01).There were no significant differences on expressions of CD34 and CD2 and changes of cytogenetics between the two groups(P>0.05).Conclusions Comprehensive evaluation of MICM could be of important significance in the diagnosis and prognosis iudgrnent for APL patients.The CR rate in these patients with high WBC eount was considerable low.

Objective Analysis of the expression of CD38,CD133 antigen and their clinical significance in myelodysplastic syndrome (MDS).Methods CD38 and CD133 antigen were analyzed by flow cytometry in 31 cases of MDS patients.Results CD38 was expressed in 18 cases (58.1% ),among them,12 cases were found to be myelodysplastic syndrome refractoryanermia ( MDS-RA ),accounting for 57.1%,6 cases were found to be MDS-RAEB,accounting for 66.7%.CD133 was expressed in 20 cases(64.5% ) ,among them,11 cases were found to be MDS-RA ( 52.4% ),1 case MDS-RAS,and 8 cases of MDS-RAEB,accounting for 88.9% .CD38 expressed significantly higher in MDS than anemia and relatively normal group ( P ＜ 0.05 ).CD133 expression in anemia groups was different from MDS-RA without statistical significance ( P ＞ 0.05 ),but was significantly different from relatively normal group (P ＜0.05).CD133 expression was significantly higher in these with MDS-RAEB than those in anemia and normal group ( P ＜ 0.05 ).Conclusions Combining with conventional antibodies,flow cytometry used in detection of CD38 ,CD133 ,could improve the diagnostic rate of MDS.

Objective To sudy the changes in mTOR signaling pathway in childhood aplastic anemia(AA) by detecting the expression levels of the molecules of mTOR signaling pathway in T cells,and to explore immunologoical pathogenesis of AA in children from T cell intracellular signal transduction pathway.Methods Peripheral blood samples were collected from 16 newly diagnosed severe AA(SAA) patients and 8 patiens treated with effective immunosuppressive therapy,and the findings were compared with those of 17 healthy children (normal controls) and CEM cells (positive controls).The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K in CD3 + T cells in peripheral blood were detected by flow cytometry(FCM).Results 1.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,pp70S6K of the newly diagnosed SAA group were higher than those of the normal control group (P ＜ 0.05),but were lower than the postive control group (CEM group) (P ＜ 0.05).The mean fluorescence intensity (MFI) of p-Akt of three groups was 8.04 ± 3.78,2.59 ± 1.01 and 20.23 ± 8.98 respectively ;p-TSC2 was 49.73 ± 19.49,16.10 ± 8.04 and 101.05 ± 29.78 respectively ; p-mTOR was 13.90 ± 9.32,2.92 ± 1.09 and 34.3 ± 19.03 ;p-4EBP1 was 142.69 ± 53.36,26.91 ± 13.70,256.01 ± 53.79 ; p-p70S6 K were 17.67 ± 10.48,3.69 ± 2.22,31.73 ± 12.85 respectively.2.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K of the effective treatment groups were lower than those of the newly diagnosed SAA group (P ＜ 0.05) ; the expressions of p-Akt,p-TSC2,p-mTORC1,p-p70S6K were similar to those of the normal control group(P ＞ 0.05),but the expressions of p-4EBP1 were higher(P ＜ 0.05).The MFI was followed by 3.28 ± 1.27,16.50 ± 10.91,3.54 ± 1.66,74.89 ± 49.69 and 4.21 ± 1.69.Conclusions 1.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K were increased in the newly diagnosed SAA patients,the mTOR signaling pathway was activated in SAA patients.2.The expressions of p-Akt,p-TSC2,p-mTORC1,p4EBP1,p-p70S6K were lower than those of the newly diagnosed SAA patients.The degree of activation of mTOR signaling pathway was associated with disease status.The signaling pathways may be involved in the T cells of AA of the immune abnormalities.