-D- phenotype screening by polybrene method / 中国输血杂志

【Objective】 To develop a novel screening reagent for -D- phenotype preliminary screening based on the difference in RhD antigen expression level of -D- phenotype and normal RhD phenotype. 【Methods】 RhD antigen expression of -D-phenotype and Rh D-- gene carrier were detected by flow cytometry. By adjusting the concentration of polybrene in the screening system, the red blood cells with high RhD antigen expression level agglutinated, and the preliminary screening of the -D-phenotype and its gene carriers was realized. 【Results】 According to the quantitative results of immunofluorescence intensity (MFI) analysis by flow cytometry, the expression level of RhD antigen in -D- phenotype cells (284 360±16 698, n=3) was about 3 times normal RhD positive cells (98 642±35 908, n=9)(P<0.01), while RhD antigen expression level of RhD-- gene carrier (181 109±39 455, n=4) was about 2 times normal RhD positive cells(P<0.01). RhD antigen expression (144 538±227 445, n=7) of the positive cells screened by 15 μL 3% fresh red blood cell suspension and screening system 35 μL (1 μL IgG anti-D, 29 μL polybrene polybrene, and 5 μL low ionic strength solution) was about 1.5 times normal RhD positive cells. 【Conclusion】 The polybrene preliminary screening system, which can be used for high-throughput screening of -D- phenotype, is a reliable technical method for frequency study of this phenotype.

Endothelial Progenitor Cells' Classification and Application in Neurological Diseases

The therapeutic effects of endothelial progenitor cells (EPCs) on ischemic stroke have been extensively studied in recent years. However, the differences in early EPCs and endothelial outgrowth cells (EOCs) are still unclear. Clarifications of their respective properties and specific functioning characteristics contribute to better applications of EPCs in ischemic diseases. In this review, we discuss cellular origin, isolation, culture, surface markers of early EPCs and EOCs and relevant applications in neurological diseases. We conclude that EOCs possess all haracteristics of true endothelial progenitors and have potent advantages in EPC-based therapies for ischemic diseases. A number of preclinical and clinical applications of EPCs in neurological diseases are under study. More studies are needed to determine the specific characteristics of EPCs and the relevant mechanisms of EPCs for neurological diseases.

Flow cytometric analysis of Mixed phenotype acute leukemia: Experience from a tertiary oncology center.

Introduction: Mixed phenotype acute leukemia (MPAL) is a rare subset of acute leukemia where the blasts exhibit lineage specifi c antigens of more than one lineage. Flow cytometric immunophenotyping is essential for the diagnosis of MPAL and the accurate diagnosis highly depends on the panel of markers used. The precise incidence of MPAL is uncertain as various institutions use different combinations of antibodies to assign the blasts to a particular lineage. Aim: The aim was to study the immunoprofi le of acute leukemia including aberrant antigen expressions and to study the incidence, clinical features, laboratory fi ndings, and immunophenotype of MPAL in our institution. Materials and Methods: All cases of acute leukemias in which fl ow cytometric analysis during 1-year period from July 2012 to July 2013 were included in this study. Results: During the study period, fl ow cytometric analysis of 506 cases was performed. B lymphoblastic leukemia was the most common subtype of acute leukemia. CD13 was the most common aberrant antigen expression in acute lymphoblastic leukemia and CD7 was the most common aberrant antigen expression in acute myeloid leukemia. A diagnosis of MPAL was made in 15 cases, which accounted for 2.96% of all leukemias. 9 cases were diagnosed as T/myeloid, 5 cases as B/myeloid and 1 case as B/T. Conclusion: Mixed phenotype acute leukemia is a rare subset of acute leukemia. Flow cytometry is critical in establishing a diagnosis of MPAL. The panel of antibodies used is important in the identifi cation of the “mixed” phenotype. Cytoplasmic markers (cytoplasmic MPO, cytoplasmic 79a, cytoplasmic 22 and cytoplasmic CD3) should be included in the primary flow cytometric panel.

Minimal Residual Disease Detection in Acute Leukemia Patients by Flow Cytometric Assay of Cross-lineage Antigen Expression / 대한진단검사의학회지

BACKGROUND: It has been demonstrated that flow cytometric detection of minimal residual disease (MRD) has a prognostic significance in the treatment of patients with acute leukemia. We investigated the significance of flow cytometric MRD detection for the first time in Korea. METHODS: We analyzed the results of MRD detection in morphologically complete remission bone marrow aspirates from 89 patients with newly-diagnosed or relapsed acute leukemia, in which leukemic cells had cross-lineage antigen expression. Patients were grouped based on MRD frequencies: > or =1.0%, high MRD; <1.0%, low MRD. RESULTS: Forty-seven ALL patients consisted of 10 with high and 37 with low MRD levels. Patients with high MRD levels showed a tendency of more frequent relapse than those with low MRD levels (40.0% and 13.5%, respectively) (P=0.08). High MRD group showed a tendency of short relapse-free survival (RFS) and overall survival (OS), although the differences were not statistically significant. Forty-two AML patients consisted of 16 with high and 26 with low MRD levels. There were no correlations between the MRD levels and relapse rate, RFS or OS. AML patients with high MRD levels showed significantly higher rate of unfavorable cytogenetic risk categories and lower rate of favorable risk categories (P=0.03). CONCLUSIONS: MRD detection by flow cytometric assay of cross-lineage antigen expression would be useful in predicting treatment outcome in patients with ALL rather than AML. We expect that the establishment of the standardization of methods, time to test or antibody combination would be achieved through further trials in this country.

Construction of The Antigen Expression Vector Used for Library-library Screening / 生物化学与生物物理进展

Selectively infective phage (SIP) technology was developed for screening interacting protein-protein pairs. The in vivo SIP strategy would in principle be suitable for??library-library??selections, and the co-packaged polyphage may be a suitable approach. In order to construct the antigen expression vector which can be co-packaged into polyphage with phage displaying vectors, plasmid TG10 was chosen as the basic vector which is compatible with antibody display vector. The interval sequence of phage genome was amplified with PCR and cloned into TG10 to provide the packaging signal. It was named pTMI and it can be packaged into phage particles in 1011 level. The N1N2 region of gene ￠? was amplified and cloned into pTMI under the control of lac promoter to give pTMIN. Promoter trc was synthesized and replaced the lac promoter to give pTTMIN which permits the fusion expression of antigen with N1N2. To test its ability for fusion expression, gene code for ten-peptide of c-myc was synthesized and inserted into pTTMIN downstream to N1N2. After induction expression, the results of ELISA and SDS-PAGE showed that it has been expressed successfully. When pTTMIN was transfected into cell carrying antibody display vector p3MHHB3, it was copackaged into phage particles in 0.3% to 55% after rescuing with helper phage VCSM13.From the results it can concluded that the antigen expression vector was constructed successfully and it can be used for library-library screening in theory.

Unusual Antigen Expression of Acute Leukemia / 대한혈액학회지

BACKGROUND: The recent advances in flow cytometric technology and the development of monoclonal antibodies have led to the important insights into the cell lineage and maturation stage of leukemia. The increased use of immunophenotyping in acute leukemia revealed the unusual anigen expression and biclonal or biphenotypic acute mixed lineage leukemia (AMLL). However, the data on their frequency and prognostic significance are still conflicting. METHODS: The immunophenotyping of leukemic cells (HLA-DR, CD10, CD19, CD20, CD22, CD3, CD5, CD7, CD13, CD33, CD61, TdT, cytoplasmic Ig, surface Ig) was performed by flow cytometry in 115 cases of acute leukemia between January 1994 and August 1996. Double-color immunofluorescent staining was performed in the cases expressing unusual antigens. RESULTS: 51 cases (44.3%) of 115 acute leukemias showed unusual antigens expression. These included 27 cases (38.6%) of 70 AML, 13 cases (43.3%) of 30 B-lineage ALL, 4 cases (50%) of 8 T-LL and 7 AMLL cases (6.1%) of 115 acute leukemias. CD7 (28.6%) and CD19 (11.4%) are expressed in AML, and CD13 (36.7%) and CD33 (26.7%) are expressed in ALL. Among 7 cases of AMLL, we could obtain the clinical data of 5 cases. The 4 cases of 5 AMLL failed to respond to induction chemotherapy or died before or during induction chemotherapy, and only one case showed partial remission. CONCLUSION: The unusual antigen expressions of acute leukemic cells are frequently observed, and the identification of relatively rare AMLL is very important, because AMLL showed poor response to the chemotherapy.

Inhibition of Anti-EB Virus Liquor on EB virus antigen expression and its cytotoxicity / 中草药

Object To study the inhibitory effect of Anti-EB Virus Liquor (AEVL) on EB (Epstein-Barr) virus antigen expression and its cytotoxicity. Methods The effect of AEVL on Raji cell early antigen (EA) expression and B 95-8 cell virus capsid antigen (VCA) expression was assayed by indirect fluorescent technique; the cytotoxicity on nasopharyngeal carcinoma CNE 2 cells was determined by MTT method. Results At the non-toxic concentration, AEVL had markedly inhibitory effect on Raji cell EB-virus, IC 50 was 0.667 mg/mL and showed powerfully inhibitory effect on B 95-8 cell EB-virus VCA expression, IC 50 was 0.89 mg/mL; and it had strongly inhibitory effect on B 95-8 cell EB virus VCA expression stimulated by sodium n-butyric acid, IC 50 was 1.4 mg/mL. The IC 50 of AEVL on human nasopharyngeal carcinoma CNE 2 cell was 7.57 mg/mL. Conclusion AEVL could inhibit EB virus antigen expression and have cytotoxicity on nasopharyngel carcinoma cells at high concentration.

Acute leukemias with unusual immunophenotypes

Over a two-year period, immunophenotypic patterns of 266 acute leukemia cases were analyzed using a panel of tests including TdT, SmIg and 9 surface antigens by the immunofluorescence stains for the assessment of the incidence and grade of phenotypic ambiguity (lineage infidelity) and the possible clinical significance of unusual immunophenotypes. Immunophenotypes were classified into four groups according to the degree of ectopic antigen expression. We classified as Group A (91.7%, 244 of 266 cases) those expressing conventional pattern without ectopic antigen. Group B (3.0%, 8 of 266 cases) was defined to have at least two lineage specific markers and single ectopic antigen. Such a "low grade deviation" did not prevent a definite immunodiagnosis. Group C (4.2%, 11 of 266 cases) revealed a promiscuous coexpression of markers related to different lineages, including two cases (0.8%, 2 cases) of biphenotypic leukemia. Group D (1.1%, 3 cases) included unclassifiable immunophenotypes with no antigen or HLA-DR only expression. Both patients with biphenotypic leukemia and one patient with unclassifiable immunophenotypes failed to respond to induction chemotherapy, suggesting a poor prognosis in these patients. The incidence of acute myelogenous leukemia (AML) cases with one or more ectopic surface antigens was 10 (8.1%) of the 124 AML cases. Ectopic antigen expression was seen in 5 (4%) of the 125 B-lineage acute lymphoblastic leukemia (ALL) cases and 3 (25%) of the 12 T-ALL cases. It is concluded that nearly 95% of cases of acute leukemia cases can be diagnosed accurately with immunophenotyping alone including patients with a mild degree of deviation from expected antigenic patterns.(ABSTRACT TRUNCATED AT 250 WORDS)