The effect of thalassemia on erythrocyte reference intervals in a representative Han Chinese adult population.

BACKGROUND: The data from apparently healthy individuals with thalassemia has been demonstrated to have an effect on the reference intervals for the erythrocyte indices in areas with a high incidence of thalassemia. METHODS: Six clinical centers screened apparently healthy individuals using a questionnaire and a physical examination. Then, the qualified reference individuals were selected by hematological indices and a genotypic thalassemia diagnosis. Statistical comparisons were conducted for the erythrocyte reference intervals in the Chinese population with and without thalassemia. The constituent ratios and the mean (SD) of erythrocyte indices according to the thalassemia genotype were calculated. The relationship between the MCV values and the thalassemia genotype was also estimated. RESULTS: 4,636 reference individuals were included using hematological indices and genotypic thalassemia screening. The results of the erythrocyte reference intervals for individuals in Guangzhou with thalassemia demonstrated that the RBC, MCV, and MCH values significantly differed by gender compared with other regions (p < 0.01). In contrast, for individuals without thalassemia, the results tended to be similar and clinically acceptable. In addition, the results of the erythrocyte indices revealed significant differences among α-thalassemia patients, ß-thalassemia patients, and the control group. CONCLUSIONS: Apparently healthy individuals with thalassemia in the high prevalence zone of thalassemia could not be excluded by the questionnaire, physical examination or laboratory indices (Fe < 6 µmol/L, Hb < 90 g/L). The screening of genotypic thalassemia based on the MCV or MCH values to exclude unqualified individuals is the most effective way to obtain accurate and reliable reference intervals for the erythrocyte indices.

[Establishment of hematological malignancy model in ex vivo cell culture].

The aim of this study was to develop an ex vivo cell culture system for establishing the hematological malignancy model. Mouse bone marrow cells were transfected with GFP-expressed retroviral vectors encoding various leukemia/lymphoma-associated fusion proteins (TEL-PDGFR, Rabaptin5-PDGFR, p210BCR-ABL, AML1-ETO, NPM-ALK). After transfection, the cells were cultured in IMDM containing 10% FCS without growth factors, or with one of the following growth factor combinations: (1) murine c-kit ligand (KL) plus human flt3 ligand (FL); (2) IL-3, thrombopoietin, G-CSF, and hyper-IL-6 (3/T/G/H6); (3) KL/FL plus 3/T/G/H6. The flow cytometry was used to detect the ability of combinations of growth factors to complement the oncogene fusion protein to support self-renewal of the transfected cells. The results showed that the transfected cells could be amplified sustainably in the logarithmic growth way. The indicated combination of c-Kit ligand (KL) with flt-3 ligand (FL) supported the self-renewal of the marrow cells transfected with vectors encoding TEL-PDGFR, Rabaptin5-PDGFR, AML1-ETO and NPM-ALK, in addition to KL/FL, the self-renewal of p210 BCR-ABL transfected-marrow cells also required IL-3. The morphology of cells emerged from culture can be the predictor of the corresponding oncogene-associated malignancy. It is concluded that this study establishes a culture system ex vivo which provides a generalized method for studying hematological malignancies, and may facilitate the screening for therapeutic agents.

[Long-term expansion of T cell progenitors by JAK3 gene transduced primitive hematopoietic cells in vitro].

OBJECTIVE: To explore whether activation of the JAK3-signaling pathway can stimulate long-term expansion of the earliest T cell progenitors from transduced primitive hematopoietic cells and evaluate their potential ability of committed differentiation. METHODS: A retrovirus vector (RV) containing JAK3 gene, two binding sites for chemical inducers of dimerization (AP20187), and green fluorescence protein (GFP), MGI-F(2)JAK3, was constructed. The RV vector MGI-F(2)JAK3 was then transduced into murine bone marrow hematopoietic cells. The transduced murine bone hematopoietic marrow cells were divided equally into four groups blank control group (No drug group), AP20187 group (added with AP20187 only), SCF group (added with stem cell factor only), and AP20187 + SCF group. Cytometry was used to detect the GFP marker and observe the survival of cells. The murine bone hematopoietic marrow cells expanded for 50 days were divided into 2 groups: one group was washed to remove the cytokines to observe their survival, and AP20187 + SCF was added into the culture fluid of other group. Cytometry and CELLQuest v3.1 software analysis were used to analyze the phenotype of the cells of AP20187 + SCF after 50 days' expansion. The C-kit(hi) CD44(+)CD25(-)TN cells after 50 days' expansion were further cultured under the condition of SCF + IL7 + IL3 for 5 days. The differentiation rate of Thy1.2 positive cells was observed by cytometry. Five Ly5.2 mice underwent radiation of the dose of 600 cGy, and then injected with the expanded cells into the thymus. Three weeks later the mice were killed and their thymus glands were taken out to prepare suspension of single cells to undergo cytometry to observe the proportions of GFP positive CD3 and CD4 mature T lymphocytes. RESULTS: One week later the cells of the No Drug group all died, the cells of the AP20187 group and SCF group died 2 - 4 weeks later, however, the cells of the AP20187 + SCF continued to grow and expanded up to 10(12)-fold after 50 days' culture. The cells of the AP20187 + SCF group with the cytokines washed died 2 more weeks later, and those with the cytokines washed and added with AP20187 + SCF continued to grow. The phenotype of the expanded proportion was identified as the earliest T cell progenitors expressing C-kit(hi) CD44(+) CD25(-)TN (triple negative). These progenitors could differentiate into Thy1.2 + T cells in the presence of SCF + IL-7 + IL-3 culture condition. 32% - 96% of the mice thymus cell were GFP positive, 5% +/- 0.8% of the thymus cells were GFP + CD(3) double positive, and 11.0% +/- 2.1% of the T lymphocytes were GFP + CD4(+) double positive. CONCLUSION: AP20187 combined with SCF mediating the activation of JAK3 signaling can dramatically expand the earliest T cell progenitors subpopulation, and acquires the capacity to induce the differentiation into T mature cells. This system may help understand the T cell biology and provide a fundamental basement for gene therapy to immunodeficiency disease in the future.