ObjectiveTo investigate the current situation of immunophenotyping of acute leukemia by flow cytometry in China (except Taiwan, Hongkong and Macau), and to provide theoretical evidence for the formulation of guidelines of immunophenotyping of leukemia by flow cytometry.MethodsThe Chinese Hospital Knowledge Database(CHKD) was used to retrieve the1994-2009 published papers on immunophenotyping of acute leukemia by flow cytometry.All of 380 retrieved papers were analyzed by the GraphPad Prism 4. ResultsThe number of published papers in China increased yearly and changed gradually from large cities to small and medium-sized cities in local distribution. The most often reported area was Beijing, followed by Jiangsu and Guangdong. In specimen processing, the ratios of using mononuclear cell labling method and whole blood labling-lysing-washing method were 34.2 ％ (113/330) and 65.8 ％ (217/330) respectively. The ratios of using single color, two color, three color and four color labling method were 15.4 ％ (53/344), 13.7 ％ (47/344), 54.1％ (186/344) and 16.3 ％ (56/344), respectively. The number of fluorescent antibodies was used from less than 10 to more than 20.Most of them were between 11 to 16,which was 48.6 ％ (167/344) of total number of published papers. In data acquisition and analysis, the ratios of using FSC/SSC gating and CD45/SSC gating were 34.2 ％ (113/330) and 65.8 ％ (217/330), respectively. There were some differences in the positive criteria of lymphatic and myeloid and cytoplasm antigens.The main positive criteria of lymphatic, myeloid antigen was ≥20 ％, which was 64.8 ％ (223/344) and 95.4 ％ (328/344) . The main positive criteria of cytoplasm antigen was ≥ 10 ％, which accounted for 61.2 ％ (156/255). Conclusion A common view has been reached in specimen processing,gating method,data acquisition and analysis in immunophenotyping of acute leukemia by flow cytometry. But there were still some difference in the number of fluorescent antibodies, panels and evaluation of positive criteria of antigens.

To explore the necessity of teaching of elective course:Flow Cytometry and its application to clinical medicine in medical students,sixty nine medical students including sevenyear program undergraduate,master and Ph.D candidates were surveyed with a community questionnaire. More than 95% students thought it was very necessary to set up the course because they knew little about the course and it would be very helpful in their future clinical and research work.

Objective To evaluate the utility of flow cytometry (FCM) in diagnosis and subclassification of non-Hodgkin lymphoma (NHL). Methods The samples of lymph nodes biopsy from 59 cases clinically suspected of NHL were detected by flow cytometry; and clonal lymphocytes and their immunophenotypes were identified analyzed. The concordance between the results of flow cytometry and histopathology was analyzed. Results Among the 59 cases, flow cytometry was able to identify aberrant clonal lymphocytes in 24 of 28 NHL cases identified by histopathology, the neoplastic lymphocytes ranged from 4.28 % to 89.10 %; 23 cases were diagnosed as B-NHL and 1 case was diagnosed as T-NHL. Compared with histopathology, the accuracy of FCM was 85.71% in diagnosis of NHL. The specificity and sensitivity of FCM was 100 % and 92% in diagnosis of B-NHL. The accuracy of flow cytometry immunophenotyping in classification of 24 cases of NHL was consistent with that of histopathology. Conclusion Flow cytometry could be an ancillary technique in diagnosis of NHL by identifying aberrant clonal lymphocytes, and enable identification of B-NHL subtype.

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 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.

Objective To investigate the relationship between JAK2 V617F mutation and vascular embolism diseases,in order to provide important basis for clinical diagnosis and treatment and prevention of embolism.Methods Patients who were hemoglobin ＞ 160 g/L,platelets ＞ 300×109/L treated in department of neurology,heart and vascular surgery in Xuanwu Hospital of Capital Medical University were collected.Vessel embolism and JAK2 V617F mutation situation and correlation were retrospectively analyzed.Results Among the total 56 cases,JAK2 V617F gene mutation positive rate was 37.50 ％ (21/56),the incidence of embolism was 40.07 ％ (23/56),there was correlation between JAK2 V617F mutation and embolism (P =0.014).Conclusion JAK2 V617F mutation is helpful to early diagnosis and treatment of myeloproliferative neoplasm,reduce thrombosis complication,improve the quality of life.

OBJECTIVESTo evaluate the specificity of three-color flow cytometry in childhood acute lymphoblastic leukemia (ALL) immunophenotyping.

METHODSImmunophenotyping was performed by three-color flow cytometry analysis using CD(45)/SSC gating.

RESULTSThe percentage of blasts was correlated better with leukemic cell count compared with that of FSC/SSC, and the false positive results were low. Among eighty six cases of ALL, 95.3% was B-ALL, in which common-ALL and Pro-B-ALL were 76.8% and 6.1%, respectively, and 2.3% was T-ALL. CD(34)(+) and myeloid-associated antigen expression were observed in 57.0% and 34.9% of the cases, respectively, among which Pro-B-ALL was the commonest. CD(33) was more commonly expressed than CD(13) in Pro-B-ALL cases, but no difference in the expression between these two antigens in other subtypes.

CONCLUSIONGating of CD(45)/SSC eliminated effection of normal cells to blasts in bone marrow, with which the immunophenotyping results were more reliable.

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.