Current Treatment Patterns of Aplastic Anemia in China: A Prospective Cohort Registry Study.

Aplastic anemia (AA) is a hematologic disease characterized by pancytopenia and hypocellular bone marrow, potentially leading to chronic anemia, hemorrhage, and infection. The China Aplastic Anemia Committee and British Committee for Standards in Haematology guidelines recommend hematopoietic stem-cell transplantation (HSCT) or immunosuppressive therapy (IST) comprising antithymocyte globulin (ATG) with cyclosporine (CsA) as initial treatment for AA patients. With limited epidemiological data on the clinical management of AA in Asia, a prospective cohort registry study involving 22 AA treatment centers in China was conducted to describe the disease characteristics of newly diagnosed AA patients and investigate real-world treatment patterns and patient outcomes. Of 340 AA patients, 72.9, 12.6, and 3.5% were receiving IST, traditional Chinese medicine, and HSCT, respectively, at baseline; only 22.2% of IST-treated patients received guideline-recommended ATG with CsA initially. Almost all patients received supportive care (95.6%) as blood transfusion (97.8%), antibiotics (63.7%), and/or hematopoietic growth factors (58.2%). Overall, 64.8% achieved a partial or complete response, and 0.9% experienced relapse. No new safety concerns were identified; serious adverse events were largely unrelated to the treatment regimen. These results demonstrate the need to identify and minimize treatment barriers to standardize and align AA management in China with treatment guideline recommendations and further improve patient outcomes.

Human erythrocyte lifespan measured by Levitt's CO breath test with newly developed automatic instrument.

Existing standard techniques for erythrocyte (RBC) lifespan measurement, such as quantitation of labeling with isotopes or biotin, are cumbersome and time-consuming. Given that endogenous CO originates mainly from degraded RBCs, a team lead by Levitt developed a CO breath test to enable more efficient RBC lifespan estimation. The purpose of this study was to evaluate the reliability of Levitt's CO breath test method with our newly developed automatic instrument. RBC lifespan measurements conducted by Levitt's CO breath test method were conducted in 109 healthy subjects and 91 patients with chronic hemolytic anemia. In healthy subjects, the RBC lifespan was 126 ± 26 days, similar to values obtained with classical standard labeling methods. RBC lifespan did not differ significantly between males and females or between juveniles and adults, and did not correlate with age. To our knowledge, this datum represents an RBC lifespan average for the largest sample to date. In subjects with hemolytic anemia, RBC lifespan was 29 ± 14 days, which is significantly shorter than that of the healthy subjects (p = 0.001). Using 75 days as a cut-off, diagnostic accuracy for hemolytic anemia in the present study sample was 100%. In conclusion, the present results indicate that Levitt's CO breath test is an ideal method for human RBC lifespan measurement, and the newly developed automatic instrument is reliable and convenient for clinical practice.

[Effects of hermap gene on p-STAT5 kinases in signal transduction pathway during erythroid differentiation].

OBJECTIVE: To study the effects of hermap gene on kinases in erythroid signal transduction pathway and investigate the mechanism of hermap on erythroid differentiation. METHODS: The K562 cells expressing hermap and hermap-siRNA respectively were established for up- and down-regulating the expression of hermap gene. These K562 cells were then induced by Ara-C to erythroid differentiation and analyzed at 0, 24, 48, 72 and 96 h, respectively, for cell morphology and biphenylamine staining positive cells, determination of CD235a, CD36, kinases p-STAT5, p-Akt, p-MAPK and p-c-JUN by FCM; and quantification of hermap gene and γ (Aγ,Gγ) globin gene by FQ-PCR. RESULTS: With up-regulating hermap gene and inducing by Ara-C, K562 cells were changing to low ratio of nucleus to cytoplasm, cytoplasm colour from basophilic to pinkish or amethyst tinge, increase of number of biphenylamine positive cells and expression of CD235a, CD36, γ (Aγ,Gγ) globin gene, hermap gene and p-STAT5 from 0 to 96 h. At 0, 24, 48, 72 and 96 h of culture, the positive rates of p-STAT5 cells were detected of 0.46%, 4.54%, 20.01%, 23.65% and 33.08%, respectively. This results demonstrated that there was a positive correlation between expression of p-STAT5 and hermap gene expression (P < 0.05). CONCLUSION: hermap gene can stimulate erythroid differentiation of Ara-C induced K562 cells mainly through JAK/STAT5 signal transduction pathway.

[Analysis of TCR gene rearrangement for identification of T cell leukemia clone by using fine-tiling aCGH].

AIM: To establish a new method which analyzes T cell receptor (TCR) gene rearrangement for identification of T cells acute lymphoblastic leukemia (T-ALL) clone, it will provide the basis for the study of T-ALL including the chromosome translocation involving TCR loci. METHODS: Total DNA was isolated from peripheral blood mononuclear cells (PBMC) of one case with T-ALL. Using the fine-tiling array comparative genomic hybridization (fine-tiling aCGH) to analyze the genomic DNA differences of the case and control group, we could find the breakpoints and their position in the chromosomes. According to the preliminary results, we could design the specific primers for the positions of the breakpoints relative to sequence. Furthermore, the ligation-mediated PCR (LM-PCR) and sequence analysis were used to identify the TCR gene rearrangement. And TCR gene expression was detected by RT-PCR. RESULTS: The fine-tiling aCGH results of the T-ALL showed that the TCRα/δ locus of chromosome 14 appeared four breakpoints, corresponding to TCR Vδ1, Vδ2, Jδ1 and Jδ2. By LM-PCR, sequencing and sequence analysis, TCR gene of the case of T-ALL was involved in Vδ1Dδ2Dδ3Jδ1, Vδ2Dδ3Jδ2 rearrangement. RT-PCR results also confirmed the expression of these TCR gene rearrangements. CONCLUSION: The results demonstrated that fine-tiling aCGH and LM-PCR techniques could be used to identify the TCR gene rearrangement as one of the best perfect methods. And it was also a way to find some fusion genes involving in TCR gene.

[Establishment of the cell line K562 with stable expression of hermap and hermap-siRNA].

In order to establish K562 line with stable expressions of hermap and hermap-siRNA, amplified hermap and hermap-siRNA were cloned into pEGFP-c1 and pRNAT to acquire hermap-pEGFP-c1 and hermap-siRNA-pRNAT, respectively. These two plasmids were electrotransferred into K562 cells, then were followed by culturing with G418. The result showed that the transfer rate of hermap-pEGFP-c1-K562 and hermap-siRNA-pRNAT-K562 plasmids were 10.0% and 9.3%, respectively. After selective culture by G418, these two cell lines were still able to express GFP. It is concluded that the eukaryotic expression plasmids containing hermap and hermap-siRNA have been successfully constructed, and the cell lines of hermap-K562 and hermap-siRNA-K562 are established, definitely contributing to further functional investigation on HERMAP and its interaction with other proteins.

[Effects of human ERMAP-siRNA on erythroid differentiation of K562 cells induced by Ara-C].

In order to investigate the potential role of human ERMAP gene in erythropoiesis, the ERMAP-dsDNA was designed, ERMAP-shRNA expressing plasmids was constructed, and ERMAP-shRNA/K562 cell was established. Cell morphology, biphenylamine staining, expression of cell surface antigens as well as quantitative level of human ERMAP gene were observed during K562 cells differentiating toward erythroid lineage induced by Ara-C. The results showed that at 72 hours after Ara-C treatment, ERMAP-shRNA/K562 cell size became large with increasing cytoplasm content. The percentage of biphenylamine positive cells increased from 1.17% to 2.04% (p < 0.05), but still lower than that in group K562 + Ara-C. The percentage of CD36(-)/CD235a(+) increased from 8.83% to 11.28%, CD36(+)/CD235a(+) increased from 1.23% to 2.64%, and CD36(+)/CD235a(-) increased from 0.59% to 1.47% respectively, which were all lower than that in group K562 + Ara-C at either time point. At the same time, the level of ERMAP expression increased slowly from 2.52 x 10(-3) to 4.53 x 10(-3), which was also significantly lower than that of group K562 + Ara-C. It is concluded that the ERMAP-shRNA inhibits the Ara-C-induced erythroid differentiation of K562 cells, which further suggests that there is relationship between hERMAP and erythroid differentiation and development.

[Study on the ecological association between natural radioactivity and childhood leukemia in Guangdong province].

OBJECTIVE: This study was undertaken to evaluate the ecological association between terrestrial natural radionuclide, indoor radon concentration, natural radioactivity and leukemia incidence among children under 18 years of age. METHODS: Data were gathered from the disease surveillance program and literature reading while software SPSS 13.0 was used to calculate the Spearman's correlation. RESULTS: The incidence rates of childhood (0-18 year) leukemia showed significant differences in different places with the highest as 3.13/10(5) in Jiangmen area and the lowest as 0.42/10(5) in Maoming area. The incidence in Jiangmen was 7.45 times higher than that in Maoming. There was a rank correlation between the incidence of childhood leukemia and the mean concentrations of natural radio-nuclides in soil (226Ra and 232Th), with a positive correlation observed for overall leukemia (r(s) = 0.70, P = 0.011; r(s) = 0.66, P= 0.02 for 226 Ra and 232Th respectively) and acute lymphoblastic leukemia (ALL) (r(s) = 0.66, P = 0.019; r(s) = 0.64, P = 0.025 for 226 Ra and 232Th respectively). Associations between the incidence of childhood leukemia and the indoor gamma radiation dose rate, the total annual average effective dose equivalent from natural background radiationwere also analyzed (both r(s) = 0.59, P = 0.042). CONCLUSION: The natural radioactivity was likely to be a causative factor for childhood leukemia in Guangdong.

[Expression of human ermap gene in umbilical cord blood mononuclear cells during differentiation and development towards erythroid lineage].

The aim of study was to explore the potential of human erythroid membrane associated protein (ERMAP) gene in erythroid cell differentiation and development, mononuclear cells (MNCs) were isolated from umbilical cord blood and induced to erythroid cell differentiation by SCF, IL-3 and EPO. The cell morphology was observed by using optical microscopy, the positive rate of cells was counted by biphenylamine staining and the ratios of CD36+/CD235a-, CD36+/CD235a+, CD36-/CD235a+ cells were detected by flow cytometry, the change of human ermap gene expression level was analyzed by using fluorescent quantitative PCR (FQ-PCR). The results showed that the ermap gene expression level increased while MNCs were induced to erythroid lineage after treatment with SCF, IL-3 and EPO. It is concluded that the human ermap gene plays an important role in differentiation and development of erythroid cells.

[Detection of minimal residual disease in children leukemia patients by using PCR - review].

MRD detection in children leukemia has a potential importance to predict clinical outcome and to modify treatment protocols of the diseases. Although some patients with leukemia have achieved complete remission according to the clinical and morphological criteria, there are still very low numbers of malignant cells that can not be discriminated by morphology and remained in bone marrow, which is called minimal residual disease (MRD) and is the main reason leading to relapse. MRD detection has an important significance for designing treatment protocols. Several methods of MRD detection have been developed. These include conventional cytogenetics, fluorescence in situ hybridization (FISH), flow-cytometric immunophenotyping (FCM), Southern blot and polymerase chain reaction (PCR) techniques, etc. Each of these techniques has its advantages and disadvantages, so not all of them are suitable for clinical MRD detection because of several inherent disadvantages, such as limited sensitivity, time-consuming, high cost, or requiring high-quality DNA or RNA. For example, the sensitivities of conventional cytogenetics, FISH, FCM and Southern blot approaches for MRD monitoring are 10(-1) - 10(-2), 10(-2), 10(-3) - 10(-4) and 10(-1), respectively. Relatively, PCR can reach a good sensitivity of 10(-4) - 10(-6), and show more advantages, such as fast, specific, simple and low-cost, as well as minimal amounts of DNA or RNA for detection, etc., so PCR has its specific features for MRD detection. In this review, the progress on the detection technique for screening leukemia specific marker by muitiplex PCR and FQ-PCR in recent years are summarized.

[Expression of human ERMAP gene in fetal tissues].

Human ERMAP (hERMAP) is a novel gene coding for erythroid membrane-associated protein, which may play a role in erythropoiesis. To explore the role of hERMAP in fetal hematopoiesis, 29 fetuses of inevitable abortion with the fetal ages of 9 - 36 weeks were collected, and the total RNAs were isolated from the liver, spleen, kidney, heart, lung, thymus, brain, bone marrow and skeletal muscle, the RNAs from which at 25(+5) week fetal age were used to detect hERMAP expressions in different fetal tissues with Northern blot, and the hERMAP in various fetal tissues were quantified by FQ-PCR. As a result, Northern blot showed that hERMAP was expressed in liver and bone marrow at 25(+5) fetal age, but not in the other organ tissues. FQ-PCR results indicated that the hERMAP had been still expressed in 9 - 36th week fetal liver, increased starting from 12th-week, reaching a peak between 18 - 20th week and declining slowly starting from 21st-week. In the fetal bone marrow, expression of the hERMAP began from 15th week, reached a highest level between 27 - 32nd week and fell rapidly from 33rd week, the expression level of which was lower than that in liver. The low level expression of this gene was observed in some specimens of kidney, heart, lung, thymus, brain and skeletal muscle. It is concluded that the expression of hERMAP in fetal liver approximately consistent with haematopoiesis of fetal liver, and the expression of this gene in bone marrow aged 15 - 32nd fetal weeks coincides with haematopoiesis of fetal bone marrow. It suggests that the function of the hERMAP is possibly related with the migration of erythroid cells to liver and bone marrow during the fetal development process.

[Detection of mdr1 gene by real-time fluorescence quantitative polymerase chain reaction using Taq Man-MGB probe].

OBJECTIVE: Primer Express 2.0 software was used to design the primers and the MGB probe. With the plasmid pHaMDR1/A containing mdr1 cDNA as the template, we established a real-time fluorescent quantitative polymerase chain reaction system, which, at the template concentration of 3.061 x 10(3) to 3.061 x 10(9) cps/ml, had a correlation coefficient of 0.988243 between template concentration and threshold cycle value. This PCR method allows sensitive, specific and quantitative detection of human mdr1 gene.

[Preliminary study on the safety and pharmacodynamic action of low dose L-asparaginase].

OBJECTIVE: To investigate the safety and therapeutic effect of low dose (1000 U/m(2)) L-asparaginase (L-Asp) in the treatment of children with acute lymphoblastic leukemia (ALL). METHODS: Six patients were treated with low dose L-Asp after previously suffered severe side effects from standard dose L-Asp (5000 - 10,000 U/m(2)). Twenty-eight blood samples were obtained randomly from 5 of them. Plasma asparagine concentration was detected by reverse phase-high performance liquid chromatography (RP-HPLC). RESULTS: All the patients treated with low dose L-Asp showed no any toxic symptoms. The plasma asparagine levels in the patients were all above 5 micromol/L except case 4 (4.91 micromol/L) before receiving L-Asp, and were all decreased below 0.5 micromol/L five days after receiving low dose L-Asp, except case 3 (3.70 micromol/L), the results being like that of receiving standard dose L-Asp. CONCLUSION: Low dose L-Asp has definite efficacy for childhood ALL, while avoids serious side effects from standard dose L-Asp.

[RNA isolation from human embryonic tissues].

To investigate the method of RNA isolation from human embryonic tissues and the factors influencing the quality of RNA, the RNA from human embryonic tissues obtained with drug-induced labor or non-drug induced labor were isolated by using grind with liquid nitrogen or homogenizer without liquid nitrogen. The results showed that the positive rates of RNA integrity in grind with liquid nitrogen group and in homogenizer without liquid nitrogen group were 68.42% and 29.79% respectively, and there was significant difference between these two groups; however, there was no statistic difference in positive rate of RNA integrity, OD(260)/OD(280) ratio and beta-actin gene expression level between the drug-induced labor group and non-drug induced labor group. It is concluded that pulverize of tissue in liquid nitrogen remains the integrity of RNA isolated and may be applied for RNA isolation from human embryonic tissues. The quality of RNA is not affected by different methods of induction of maternal labor.

[Quantification of human ermap by using real-time FQ-PCR].

To develop a real-time FQ-PCR method for quantifying human ermap, a set of primers and a fluorescent probe were designed by primer express 2.0. pBluescriptSK(+) plasmid contained ermap cDNA was transcribed to generate calibration standards for quantification. A real time FQ-PCR method was established. The results showed that when the concentrations of DNA to be amplified were ranged from 1.725 x 10(7) to 1.725 x 10(10) cps/ml, there was a good correlation between template concentration and cycle threshold, and the correlation coefficient reached to -0.999376. In conclusion, real time FQ-PCR which is specific, sensitive and accurate can be used to further research on human ermap.

[Study on the expression of human ERMAP gene in erythropoietic and macrophage differentiation of K562 cells].

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.

[Expression of human ERMAP gene in different cell lines].

To investigate the pattern of human ERMAP gene expression in different cell lines, 15 cell lines derived from hematopoietic tumor, somatic tumor and normal tissue were chosen and were cultured; cells were harvested after culture for 12, 24, 36, 48, 60 and 72 hours; the expression of the human ERMAP was detected by using fluorescent quantitative PCR. The results showed that human ERMAP gene expression was positive in K562 cell line at interval of 12 and 24 hours, and the expression levels were (5.092 +/- 2.331) x 10(6) cps/microl RNA, (5.328 +/- 3.916) x 10(6) cps/microl RNA, respectively; ERMAP gene expression was also positive in ECV304 cell line at interval of 24 hours, and the expression level was (0.84 +/- 0.12) x 10(6) cps/microl RNA; and its expression was negative in other 13 cell lines. It is concluded that human ERMAP gene expression in ECV304 cell line was found first, and its expression in K562 cell line was further confirmed.

[Research on the pharmacokinetics and pharmacodynamics of L-asparaginase during its treatment of childhood acute lymphoblastic leukemia].

OBJECTIVE: To investigate the changes in the activity of Escherichia coli asparaginase (L-asp) and the concentration of asparagines (ASN) in the plasma of the acute lymphoblastic leukemia (ALL) children receiving L-asp containing chemotherapeutic protocol to explore more reasonable usage of L-asp in the treatment of childhood ALL. METHODS: L-asp containing hemotherapy regimen of VDLP was used, in which L-asp (10,000 U/m(2)) was administered intravenously every other day for 10 doses in 15 children with ALL. A total of 340 peripheral blood samples were collected at scheduled time points during the therapy and plasma L-asp activity (by spectrophotometric assay) and asparagines concentration (by RP-HPLC) were measured. RESULTS: During the administration of L-asp, the plasma L-asp activity was increasing gradually peaked after eight doses and then decreased gradually, while the plasma concentration of asparagines maintained in complete or nearly complete depletion status. After the therapy courses finished, a plasma L-asp activity above 100 U/L with asparagines almost complete depletion status was lasting for about seven days. CONCLUSION: The current L-asp containing chemotherapeutic protocols in which L-asp was administered in a dose of 10 000/m(2) intravenously every other day, are efficient enough for the depletion of plasma ASN.