A novel method based on nonparametric regression with a Gaussian kernel algorithm identifies the critical components in CHO media and feed optimization.

As the composition of animal cell culture medium becomes more complex, the identification of key variables is important for simplifying and guiding the subsequent medium optimization. However, the traditional experimental design methods are impractical and limited in their ability to explore such large feature spaces. Therefore, in this work, we developed a NRGK (nonparametric regression with Gaussian kernel) method, which aimed to identify the critical components that affect product titres during the development of cell culture media. With this nonparametric model, we successfully identified the important components that were neglected by the conventional PLS (partial least squares regression) method. The superiority of the NRGK method was further verified by ANOVA (analysis of variance). Additionally, it was proven that the selection accuracy was increased with the NRGK method because of its ability to model both the nonlinear and linear relationships between the medium components and titres. The application of this NRGK method provides new perspectives for the more precise identification of the critical components that further enable the optimization of media in a shorter timeframe.

Enhancing Immune Response and Heterosubtypic Protection Ability of Inactivated H7N9 Vaccine by Using STING Agonist as a Mucosal Adjuvant.

Influenza vaccines for H7N9 subtype have shown low immunogenicity in human clinical trials. Using novel adjuvants might represent the optimal available option in vaccine development. In this study, we demonstrated that the using of the STING agonist cGAMP as a mucosal adjuvant is effective in enhancing humoral, cellular and mucosal immune responses of whole virus, inactivated H7N9 vaccine in mice. A single dose of immunization was able to completely protect mice against a high lethal doses of homologous virus challenge with an significant dose-sparing effect. We also found that intranasal co-administration of H7N9 vaccine with cGAMP could provide effective cross protection against H1N1, H3N2, and H9N2 influenza virus. Furthermore, cGAMP induced significantly higher nucleoprotein specific CD4+ and CD8+ T cells responses in immunized mice, as well as upregulated the IFN-γ and Granzyme B expression in the lung tissue of mice in the early stages post a heterosubtypic virus challenge. These results indicated that STING agonist cGAMP was expected to be an effective mucosal immune adjuvant for pre-pandemic vaccines such as H7N9 vaccines, and the cGAMP combined nasal inactivated influenza vaccine will also be a promising strategy for development of broad-spectrum influenza vaccines.

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines.

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 µL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 µL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.

Overcoming nutrient limitations for cell-based production of influenza vaccine.

Metabolic analysis for medium optimization represents a very useful strategy in the process development of production of vaccines in cells. During influenza vaccine production, viruses hijack host cells and take advantage of host's metabolism. As a consequence, the nutritional demand of host cells should undergo a profound change, and usually more nutrients such as glucose and amino acids should be consumed. As such, the maintaining media used in virus production processes often cannot provide sufficient nutrients, and novel methods are urged to be established to address this severe issue of nutritional limitation. A detailed study on impacts of influenza virus on cell death and metabolism, with a profound analysis of nutritional requirements during virus production process, followed by a rational medium optimization is expected to be the most straightfoward and effective strategy. This would ensure a balanced and adequate nutritional supply, which should minimize cell death and improve both cell-specific virus yield and total influenza virus production. Such a metabolic analysis-based medium optimization would lay a solid foundation for the development of cell culture technology in influenza vaccine production.

Rational design of medium supplementation strategy for improved influenza viruses production based on analyzing nutritional requirements of MDCK Cells.

Influenza vaccine production using cell culture technology has become popular nowadays. However, to meet the ever increasing demand of influenza vaccine, it is prerequisite to improve the yield of influenza virus in cells. To achieve this, in the present study, the nutritional requirements of MDCK cells in the virus production process were analyzed and a nutrient-feeding strategy was developed accordingly. Based on the consumption rates and corresponding concentration optimization, glucose and fast metabolized amino acids were supplemented into the maintaining medium at the time of infection. Compared with the non-supplemented culture, the average cell specific death rate during 0-48 h post-infection was 0.013 h(-1), which was 40.91% lower in the nutrient-supplemented culture. Total virus titer, HA antigen protein concentration and cell-specific virus yield were (1.88±0.23)×10(3) HA units/50µL, 11.70±0.22 µg/mL and (10.06±1.16)×10(3) virions/cell, respectively, which were 84.04±22.50%, 31.46±2.87% and 86.64±25.81% higher than those in the control, respectively. These data showed that the appropriate supplementation of nutrients during virus production process could reduce cell death, and improve cell-specific virus yield and total influenza virus output. This study laid foundation for the development of cell culture technology for influenza vaccine production.

ERIC-PCR genotyping of Pseudomonas aeruginosa isolates from haemorrhagic pneumonia cases in mink.

BACKGROUND: Pseudomonas aeruginosa is a significant pathogen of mink and the cause of haemorrhagic pneumonia, an acute fatal disease in farmed mink. RESULTS: Among 90 P. aeruginosa isolates from haemorrhagic pneumonia in mink from 16 farms in Shandong province, China, 43 genotypes were identified by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), with a diversity index of 0.96. The most prevalent ERIC-PCR types were type 18, found in 16 isolates, and type 39, found in 15 isolates. Four serotypes were detected, with serotype G (55.6 per cent) being the most frequent. CONCLUSIONS: These results showed that there was a high degree of clonal diversity among mink P. aeruginosa clinical isolates in this study.

[Acute myeloid leukemia with t(11;12)(p15;q13) translocation: two cases report and literature review].

OBJECTIVE: To investigate the clinical and laboratory features of acute myeloid leukemia (AML) with t(11;12)(p15;q13) translocation. METHODS: Two cases of AML with t(11;12)(p15;q13) translocation were reported and the related literatures were reviewed. RESULTS: The diagnosis of AML-M3 was supported by morphological, cytochemical staining and electron microscope tests. A rare t(11;12)(p15;q13) translocation, but not classical t(15;17)(q22;q12) translocation and PML- RARα fusion gene, was detected in both cases. Both of the patients were refractory to differentiation induction therapy such as retinoic acid and arsenic trioxide. CONCLUSION: AML is a group of heterogeneous disease derived from hematopoietic stem cell. Cytogenetic characteristic is important for diagnosis, prognosis stratification and therapy selection. Because of the heterogeneity of clinical and molecular features, it is unsuitable to classify AML with t(11;12)(p15;q13) as AML with recurrent cytogenetic aberration. This group of disease may benefit from allogeneic hematopoietic stem cell transplantation.

An investigation into the relationship between metabolic responses and energy regulation in antibody-producing cell.

Energy-efficient metabolic responses were often noted in high-productive cultures. To better understand these metabolic responses, an investigation into the relationship between metabolic responses and energy regulation was conducted via a comparative analysis among cultures with different energy source supplies. Both glycolysis and glutaminolysis were studied through the kinetic analyses of major extracellular metabolites concerning the fast and slow cell growth stages, respectively, as well as the time-course profiles of intracellular metabolites. In three cultures showing distinct antibody productivities, the amino acid metabolism and energy state were further examined. Both the transition of lactate from production to consumption and steady intracellular pools of pyruvate and lactate were observed to be correlated with efficient energy regulation. In addition, an efficient utilization of amino acids as the replenishment for the TCA cycle was also found in the cultures with upregulated energy metabolism. It was further revealed that the inefficient energy regulation would cause low cell productivity based on the comparative analysis of cell growth and productivity in cultures having distinct energy regulation.

[Study on the role of fluorescence in situ hybridization in cytogenetic evaluation of myelodysplastic syndrome].

OBJECTIVE: To exploit the role of bone marrow (BM) and peripheral blood (PB) fluorescence in situ hybridization (FISH) in cytogenetic evaluation of myelodysplastic syndrome (MDS). METHODS: The metaphase cytogenetics and BM interphase FISH were prospectively compared in 112 cases of de novo MDS. At the same time, comparison of BM and PB FISH was conducted in 56 cases. RESULTS: The differences between metaphase cytogenetics and BM FISH were observed in 22 (54%) of 41 cases with clonal karyotypic abnormalities, most of differences were caused by the limitation of FISH probe panel which could not target all of the regions with aberrations. Only 6 (27%) of 22 differences were involved in our probe regions, the FISH results did not change their cytogenetic risk categories. BM FISH testing was abnormal in 15 (21%) of 71 cases with normal karyotypes, FISH testing was abnormal in 14/51 (27%) and 1/20 (5%) cases with fewer than 20 normal metaphases or more than 20 normal metaphases. Comparison of FISH results of PB and BM samples showed abnormal PB FISH results in 21 (72%) of 29 cases with abnormal BM FISH results, and in 1 (4%) of 27 cases with normal BM FISH results. CONCLUSION: BM FISH should be used to MDS cases with fewer than 20 normal metaphases. Although PB FISH testing is limited by a relatively high false negative rate, it is a reasonable choice to cases with failure of BM aspiration.

[Clinical and laboratory investigation of patients with hematologic malignancies harboring der(1;7)(q10;p10)].

OBJECTIVE: To investigate the clinical and laboratory characteristics of patients with various hematological malignancies harboring der(1;7)(q10;p10). METHODS: Bone marrow samples were collected and undergone short-time unstimulated culture and R-banding, and karyotyped by conventional cytogenetic assay (CCA). Megalokaryocytes were detected by streptavidin-AKP (SAP). Retrospective analyses including the clinical and laboratory data were performed. RESULTS: Nineteen of the 21 patients were male. Most of the patients are of older age. Thirteen cases (61.9%) were der(1;7)(q10;p10) without additional aberrations, 8(38.1%) patients had additional aberrations. Sixteen out of the 18 cases (88.9%) who underwent SAP analysis had diminutive megalokaryocyte, and lymphoid megalokaryocyte was found in 10 cases (55.6%). The der(1;7) patients manifested poor response to treatment. CONCLUSION: The der(1;7) patients demonstrated distinct male predominance, older age at diagnosis, and some clinically distinctive features. These patients showed poor prognosis. The cytogenetic abnormality, i.e., der(1;7)(q10;p10), can be used as a prognostic indicator.

[Analysis of effectiveness and prognostic factors for (m)HAD regimen as induction therapy in acute monocytic leukaemia].

OBJECTIVE: To analyze the treatment outcome and impact of cytogenetic abnormalities on the response and survival of acute monocytic leukaemia (AMOL) patients received (m)HAD regimen as induction chemotherapy. METHODS: Seventy-nine AMOL patients were treated with (m)HAD regimen as induction therapy (HHT 2 mg/m(2), d 1-7; Ara-C 100 mg/m(2), d 1-7 and increasing to 1.5 g×m(-2)×(12 h)(-1), d 5-7 in some patients; DNR 40 mg/m(2), d 1-3). The treatment outcome and prognostic factors were analyzed. RESULTS: (1) The complete remission (CR) rate was 79.7% (63/79), partial remission (PR) rate was 6.3% (5/79), overall rate was 86.0%. (2) The chromosome karyotypes were analyzed in 75 patients, of whom 43 with normal karyotypes (NCR) and 30 abnormal karyotypes (ACR). For the cytogenetic prognostic groups, 49 patients were intermediate, 18 poor and 6 unknown. The CR, 1-year and 3-year overal survival (OS) rates in NCR group were significantly higher than those in ACR group (P < 0.05); but there was no significantly statistical difference in disease free survival (DFS) between the two groups (P > 0.05). The CR, 1-year OS, 3-year OS and 1-year DFS and 3-year DFS rates in intermediate prognostic group were significantly higher than those in poor prognostic group (85.7% vs 61.1%, 75.9% vs 51.3%, 65.4% vs 25.6%, 82.2% vs 66.7%, and 77.9% vs 26.7%, respectively) (P < 0.05). (3) Chromosome karyotype and the number of consolidation therapy courses had more important influence on survival in COX analysis. CONCLUSION: (m)HAD regimen as induction chemotherapy for AMOL patients achieves a high CR rate. It has an important influence on survival for the patients to received adequate consolidation therapy. The frequency of cytogenetic abnormalities in AMOL is similar to that in other AMLs. The prognosis of AMOL patients with chromosome karyotype in intermediate prognostic group is significantly better than that in poor prognostic group.

A training set selection strategy for a universal near-infrared quantitative model.

The purpose of this article is to propose an empirical solution to the problem of how many clusters of complex samples should be selected to construct the training set for a universal near infrared quantitative model based on the Naes method. The sample spectra were hierarchically classified into clusters by Ward's algorithm and Euclidean distance. If the sample spectra were classified into two clusters, the 1/50 of the largest Heterogeneity value in the cluster with larger variation was set as the threshold to determine the total number of clusters. One sample was then randomly selected from each cluster to construct the training set, and the number of samples in training set equaled the number of clusters. In this study, 98 batches of rifampicin capsules with API contents ranging from 50.1% to 99.4% were studied with this strategy. The root mean square errors of cross validation and prediction were 2.54% and 2.31% for the model for rifampicin capsules, respectively. Then, we evaluated this model in terms of outlier diagnostics, accuracy, precision, and robustness. We also used the strategy of training set sample selection to revalidate the models for cefradine capsules, roxithromycin tablets, and erythromycin ethylsuccinate tablets, and the results were satisfactory. In conclusion, all results showed that this training set sample selection strategy assisted in the quick and accurate construction of quantitative models using near-infrared spectroscopy.

[The significances of 13q14 deletion for development and prognosis of multiple myeloma].

OBJECTIVE: To determine the incidence and clinical significance of chromosome 13q14 deletion in multiple myeloma (MM). METHODS: Bone marrow samples were collected from 132 newly diagnosed MM patients referred to our hospital. Interphase fluorescence in situ hybridization (i-FISH) combined with magnetic activated cell sorting (MACS) were performed on chromosome 13q14 (RB-1). RESULTS: (1) i-FISH was used to investigate CD138-enriched bone marrow MM cells and revealed a 13q14 deletion rate of 51.5% (68/132), while conventional cytogenetic (CC) analysis revealed 13q deletions/monosomy 13 (Δ13) only of 5.0%(6/120). (2) Univariate analysis showed that 13q14 deletion rate by i-FISH > 25%, bone marrow plasma cells > 50%, ISS stage and ß(2)-MG ≥ 5.5 mg/L were associated with shorter overall survival (OS). Multivariate analysis revealed that 13q14 deletion rate by i-FISH > 25% was an independent unfavorable factor (P = 0.042). (3) Patients treated with bortezomib had a much better response than those treated with traditional chemotherapy (P = 0.001). There was no significant difference in OS between patients received bortezomib with and without 13q14 deletion (P > 0.05), indicating that bortezomib could reverse the poor prognosis of 13q14 deletion. CONCLUSION: (1) i-FISH followed CD138 cell sorting appears to be a highly sensitive method for detecting 13q14 deletion. (2) 13q14 deletion rate by i-FISH > 25% is an independent unfavorable factor. (3) Bortezomib could reverse the poor prognosis of 13q14 deletion.

[Influence of immunomagnetic sorting on detecting genetic aberration of multiple myeloma cells by interphase fluorescence in situ hybridization].

This study was to aimed investigate the influence of immunomagnetic sorting on detecting the genetic aberrations of multiple myeloma (MM) by interphase fluorescence in situ hybridization (FISH) and to explore the detection method suitable to use in our country. The genetic aberrations of immunomagnetically sorted and unsorted bone marrow cells from the same MM patients were detected by interphase FISH and the detectable rate of genetic aberration was compared. The types of probes included 13 q14 (RB-1) and 14q32 (IGH). The 42 and 22 sorted and unsorted marrow samples from MM patients were detected by using 13q14 probe and 14q32 probes respectively, the results indicated that the 13q14 deletion was found in 9 of 42 (21.4%) unsorted marrow samples and in 25 of 42 (56.8%) CD138(+)-sorted marrow samples. The 13q32 rearrangement was found in 7 of 22 (31.8%) unsorted marrow samples and in 14 of 22(63.6%) CD138(+)-sorted marrow samples. Both of the difference was statistically significant (p = 0.001 and p = 0.035 respectively). Percentages of cytogenetic alterations detected in unsorted bone marrow cells correlated positively with percentage of plasma cells tested by bone marrow smears or flow cytometry. When percentage of plasma cells tested by bone marrow smears exceed 50%, or by flow cytometry exceed 10%, there was no difference between 2 methods. It is concluded that immunomagnetic sorting of CD138(+) cells increases the probability of detection of the 13q14 deletion and 14q32 rearrangement in bone marrow samples. The low detectable rate of genetic aberration in unsorted bone marrow cells is associated to the low percentage of plasma cells in bone marrow samples, higher percentage of plasma cells can partly overcome the shortage of unsorted detection method. When percentage of plasma cells tested by bone marrow smears exceed 50%, or by flow cytometry exceed 10%, there was no difference between 2 methods.

[Clinical and cytogenetic features and their influencing factors of core binding factor acute myeloid leukemia].

OBJECTIVE: To discuss the clinical and cytogenetic features of core binding factor (CBF) acute myeloid leukemia (AML) patients and the main factors that influence the prognosis. METHOD: Totally 130 CBF AML patients were followed up and their clinical features, immunophenotype, chromosome karyotype, treatment regimen, overall survival (OS), and relapse-free survival (RFS) were analyzed. RESULTS: The overall complete remission (CR) rate was 96.1%, among which the CR rate after the first treatment course was 77.2%. The overall median OS was 51.64 (0.26-132.5) months, while the median RFS did not reach 1.18-96.62 months. The 3-year OS was 50% and the 5-year OS was 41%; the 3-year RFS was 59% and the 5-year RFS was 54%. Patients who were over 45 years and those with chromosome karyotype of 9q- tended to have poorer prognosis. During the consolidating chemotherapy, patients who had received two or more courses of intermediate-dose Ara-C therapy had better prognosis and longer survival. AML patients with inv (16) /t (16; 16) had a significantly higher OS than those with t (8; 21) (P = 0.046), while the RFS showed an opposite finding (P = 0.038). CONCLUSIONS: Age, chromosomal karyotype, and consolidating chemotherapy are the main factors that influence the survival and prognosis of CBF AML patients. Two or more courses of intermediate-dose Ara-C during consolidating chemotherapy can obviously prolong the OS and RFS of CBF AML patients. AML patients with a chromosomal karyotype of inv (16) /t (16; 16) have longer OS and better prognosis than those with t (8; 21).

[Clinical and laboratory studies of 11 acute myeloid leukemia patients with t(7;11) (p15;p15) translocation].

OBJECTIVE: To investigate clinical and laboratory characteristics of acute myeloid leukemia (AML) patients with t(7;11)(p15;p15). METHODS: Eleven patients with t(7;11)(p15;p15) were retrospectively reviewed involved in cell morphology, immunophenotype, cytogenetics as well as clinical features and prognosis. RESULTS: Eight patients out of the eleven were female, six patients were AML-M2a, two M4, two M5, and one M6. All the 11 cases expressed CD33, 10 expressed CD117 and CD13, HLA-DR and CD34 was expressed in 7 and 6 patients, respectively. Karyotypes of all the patients were t(7;11) (p115;p15), additional trisomy 8 were found in only one patient. FLT3-ITD was positive in one of nine patients who were analysed for FLT3-ITD and FLT3-TKD. Two patients were alive, and one lost to followed up, while the rest of eight were dead. CONCLUSION: The t(7;11) (p15;p15) abnormalities is one of rare chromosomal translocation in patients with AML. AML patients with t(7;11) (p15;p15) have clinical features of anemia, thrombocytopenia, higher white blood cell, and poor prognosis.

[Study on prognostic significances of different cytogenetic risk categories in patients with primary myelodysplastic syndromes].

OBJECTIVE: To analyze significances of different cytogenetic categories for prognostic stratification in patients with primary myelodysplastic syndromes (MDS). METHODS: Chromosomal abnormalities of 532 primary MDS patients were categorized according to cytogenetic categories of International Prognostic Scoring System (IPSS), Revised IPSS (IPSS-R), and German-Austrian (G-A). Prognostic impacts of different cytogenetic categories and frequent isolated anomalies were investigated. RESULTS: Of 532 patients, 346(65%) patients had clonal cytogenetic abnormalities, including 200(38%) patients had 1 abnormality, 61(11%) patients had 2 abnormalities, and 85(16%) patients had complex abnormalities. Trisomy 8 was the most frequent karyotype abnormality, occurring in 31% of the patients with clonal cytogenetic abnormalities, other frequent anomalies were -7/del(7q)(13%), del(20q)(12%), del(5q)(9%), -18(5%), -21(5%), i(17q)(5%), -Y(4%), -17(4%), +21(4%), -13/del(13q)(4%), and -22(4%). The proportion of poor karyotypes of IPSS was higher in RAEBI and RAEBII among the World Health Organization classifications than in subgroups with less than 5% blasts. The follow-up data were available for 310 patients with a median follow-up duration of 14.5 months. Median survival was 59 months for patients with normal karyotypes and 26 months for those with abnormal karyotypes. According to IPSS cytogenetic categories, the median survivals of good-risk subgroup, intermediate-risk subgroup and poor-risk subgroup were 59, 43 and 12 months, respectively (P < 0.01). For IPSS-R cytogenetic groups, the median survivals of good-risk subgroup, intermediate-risk(int-risk) subgroup, poor-risk and very poor-risk subgroup were 59, 36, 15, and 10 months, respectively (P < 0.01). According to G-A classification, the median survivals of good-risk subgroup, int-1-risk subgroup, int-2-risk subgroup and poor-risk subgroup were 59, 44, 15, and 11 months, respectively (P < 0.01). In frequent isolated karyotypic abnormalities, +8 had a median survival of 44 months, i(17q) had a median survival of 12 months, and -7/del(7q) had a median survival of 14 months. CONCLUSION: In comparison with IPSS and G-A categories, IPSS-R cytogenetic categories are more sophisticated, and can stratify prognosis effectively, but prognostic significances of some karyotypes in IPSS-R still need to be confirmed.

Detailed understanding of enhanced specific productivity in Chinese hamster ovary cells at low culture temperature.

The specific productivity of tumor necrosis factor receptor-immunoglobulin G1 Fc fusion (TNFR-Fc) (q(TNFR-Fc)) in Chinese hamster ovary (CHO) cells at 30°C was approximately 5-fold higher than that at 37°C. To investigate reasons for increased q(TNFR-Fc) at low culture temperature, TNFR-Fc mRNA levels were determined by real-time PCR. It was found that like q(TNFR-Fc), the relative TNFR-Fc mRNA level was increased by lowering culture temperature, and more importantly, the kinetics of the increase in TNFR-Fc mRNA levels were in accordance with the changes in q(TNFR-Fc). The results demonstrated that the increased transcriptional level of TNFR-Fc was responsible for the increased q(TNFR-Fc) at low culture temperature. Enhanced levels of mRNA could derive from increased gene copy number, improved mRNA stability, or enhanced transcriptional rate. There was not a big change of gene copy number by lowering culture temperature. The transcriptional rate of TNFR-Fc was slightly decreased at 30°C, compared to 37°C. However, mRNA stability of TNFR-Fc was significantly improved by lowering culture temperature. The half-life of TNFR-Fc mRNA was 5.55 h at 30°C, whereas that was 3.69h at 37°C. Taken together, the reasons for the increased q(TNFR-Fc) in CHO cells at low culture temperature were mainly the enhanced TNFR-Fc mRNA levels, which resulted from the improved mRNA stability, rather than the changes in the gene copy number or the transcriptional rate.

[Probe of classification of adult acute lymphoblastic leukemia].

OBJECTIVE: To investigate the biologic features of adult acute lymphoblastic leukemia (ALL), and reclassified our ALL patients according to the 2008 WHO classification. METHODS: Immunophenotype and cytogenetic/molecular genetic results were obtained by flow cytometry, R-banding and RT-PCR, respectively. RESULTS: (1) A total of 412 newly diagnosed and previously untreated adult ALL patients, were 239 males and 173 females. Among 410 patients with available immunophenotypic results, 357 were B-ALL and 53 T-ALL. Myeloid antigen (MyAg) was higher expression in B-ALL than in T-ALL, and was correlated with the expression of CD34. (2) 93 Ph + ALL patients, mainly CD10 ALL, was associated with high WBC count and MyAg and CD34 expression. MLL rearrangement was found in 12 cases, mainly pro-B ALL. (3) 299 cases could be analysed, according to the 2008 WHO classification of ALL, including 126 B-ALL with recurrent genetic abnormalities, and 120 B-ALL not otherwise specified. Among the 126 B-ALL with recurrent genetic abnormalities, 92 were Ph + ALL, 10 MLL + ALL, 11 hyperdiploid, 9 hypodiploid, 3 E2A-PBX +, and 1 TEL-AML1 +. Patients with Ph +, MLL +, hypodiploid or E2A-PBX + were associated with older age, higher WBC count, higher HGB, higher peripheral blasts and higher LDH level as compared with other patients. CONCLUSION: Combination of immunophenotype and cytogenetic-molecular profiles can provide a further detailed classification of B-ALL.

[Identification of complex chromosomal aberrations in acute leukemia by using conventional cytogenetics combined with multiplex fluorescence in situ hybridization].

OBJECTIVE: To explore the value of multiplex fluorescence in situ hybridization (M-FISH) technique in the detection of the complex chromosomal aberrations (CCAs) and marker chromosomes in acute leukemia (AL). METHODS: M-FISH was performed in 11 AL patients with R-banding CCAs or marker chromosomes to define the unrecognized chromosomal aberrations and the constitution of marker chromosomes, and to identify small and cryptic translocations. RESULTS: In the 11 AL cases studied, 27 numerical and 41 structural chromosomal abnormalities were detected by conventional cytogenetics (CC), among which 3 chromosomal gains and 9 chromosomal losses as well as 12 structural abnormalities were confirmed by M-FISH, and another 15 chromosomal losses were revised by M-FISH as derivative chromosomes. M-FISH detected 3 additional chromosomal gains that were undetected by CC. The other 29 structural abnormalities including 17 marker chromosomes were characterized by M-FISH. A total of 33 structural abnormalities were detected by M-FISH, in which 6 were unreported before, i.e. t(5q-;16)(? q14;q24), der(9)(Y::9::Y::9), der(7) (7::8::9), ins(20;21), der(11) (11::21::20) and der(3)t(3p-;13)(3p-;q21), most of which resulted from unbalanced translocations. Almost all chromosomes were involved in CCAs, the more common ones were chromosome 17, 5, 7, 15, 11 in AML and 8, 9, 14, 22 in ALL. CONCLUSION: Combining M-FISH with CC can raise resolution of the latter, which justifies its clinical application for the detection of CCAs and marker chromosomes.