Ultrastructure and Raman Spectral Characteristics of Two Kinds of Acute Myeloid Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the Raman spectral characteristics of leukemia cells from 4 patients with acute promyelocytic leukemia (M) and 3 patients with acute monoblastic leukemia (M), establish a novel Raman label-free method to distinguish 2 kinds of acute myeloid leukemia cells so as to provide basis for clinical research.</p><p><b>METHODS</b>Leukemia cells were collected from bone marrow of above-mentioned patients. Raman spectra were acquired by Horiba Xplora Raman spectrometer and Raman spectra of 30-50 cells from each patient were recorded. The diagnostic model was established according to principle component analysis (PCA), discriminant function analysis (DFA) and cluster analysis, and the spectra of leukemia cells from 7 patients were analyzed and classified. Characteristics of Raman spectra were analyzed combining with ultrastructure of leukemia cells.</p><p><b>RESULTS</b>There were significant differences between Raman spectra of 2 kinds of leukemia cells. Compared with acute monoblastic leukemia cells, the spectra of acute promyelocytic leukemia cells showed stronger peaks in 622, 643, 757, 852, 1003, 1033, 1117, 1157, 1173, 1208, 1340, 1551, 1581 cm. The diagnostic models established by PCA-DFA and cluster analysis could successfully classify these Raman spectra of different samples with a high accuracy of 100% (233/233). The model was evaluated by "Leave-one-out" cross-validation and reached a high accuracy of 97% (226/233).</p><p><b>CONCLUSION</b>The level of macromolecules of Mcells is higher than that of M. The diagnostic models established by PCA-DFA can classify these Raman spectra of different cells with a high accuracy. Raman spectra shows consistent result with ultrastructure by TEM.</p>

The effect of transmission electron microscopy on diagnosis of acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze coincidence rate of acute myeloid leukemia (AML) sub-typing between transmission electron microscopy (TEM) and clinical discharge diagnosis.</p><p><b>METHODS</b>Reviewing sub-typing results of TEM, light microscopy, flow cytometric analyzing, molecular biological detection and karyotype in 793 AML cases, comparing their coincidence rates with discharge diagnosis to reveal advantages of AML sub-typing by TEM.</p><p><b>RESULTS</b>General coincidence rates of TEM, light microscopy, flow cytometric analyzing, molecular biological detection and karyotype on AML sub-typing were 63%, 59%, 52%, 47%, 26% and 23% respectively, and clinical coincidence rates of TEM on M1, M2a, M4 and M5, M6, M7, t (8; 21) and t (15; 17) were 39%, 34%, 17%, 74%, 50%, 73%, 87% and 89% respectively.</p><p><b>CONCLUSION</b>TEM has a higher coincidence rate in general AML sub-typing, especially strong screenings on t (15; 17), t (8; 21), M7, M5 and M6, but lower coincidence rates on M1, M2a and M4 sub-typing than other methods.</p>

Increasing sensitivity of leukemia cells to imatinib by inhibiting NHE1 and p38MAPK signaling pathway / 中国实验血液学杂志

This study was aimed to investigate whether the inhibition of NHE1 activity and intracellular acidification can reverse resistance of leukemia cells to the imatinib and to explore downstream signal molecule networks of BCR/ABL in the cells of chronic myelocytic leukemia (CML) patients. The mRNA and protein expression of P-glycoprotein (Pgp) and the drug accumulation were assayed after acidifying the primary leukemia cells of patients or K562/DOX and K562/G01 cells. The effects of intracellular acidification of primary leukemia cells on the phosphorylation level changes of ERK1/2 and p38 MAPK were analyzed by Western blot. The results showed that the intracellular concentration of drugs in the advanced patients increased and the sensitivity of K562/DOX and K562/G01 cells to imatinib was enhanced after intracellular acidification or treatment with NHE1 inhibitor cariporide. With downregulation of intracellular pH, the phosphorylation of p38 MAPK decreased in advanced patients and the phosphorylation of ERK1/2 increased within 3 min and then decreased after 30 min. SB203580, the specific inhibitor of p38 MAPK, displayed a synergistic effect with the inhibitor of NHE1 to downregulate the mRNA and protein expression of Pgp. It is concluded that the inhibiton of NHE1 can significantly decrease the protein expression of Pgp in K562/DOX and K562/G01 cells, increase the accumulation of Rhodamine123 and doxorubicin in the cells of advanced patients and enhance the sensitivity of cells to imatinib in which the p38 MAPK signal transduction pathways involves.

A mouse model of myelofibrosis induced by high dose of recombinant human erythropoietin / 中国实验血液学杂志

Tn order to set up a mouse model of myelofibrosis (MF) induced with high dose recombinant human erythropoietin (rhEPO). 60 mice were collected and divided into EPO and control groups, the former was injected with rhEPO and the latter with normal saline intraperitoneally. 5 mice from each group were executed on day 6, 30, 60, 90, 120 and 150 respectively. Their WBC count, Hb level, MCV, RDW and platelet amount were measured by automatic blood cell analyzer; CD34(+) cell ratio in bone marrow were analyzed by flow cytometry; liver and spleen coefficients were measured; pathological changes of liver, spleen, femur were observed by HE staining and reticular fibers staining; cortex thickness, femoral canal diameter and lumbar spine density were determined by computerized tomography (CT). The results indicated that as compared with normal control group in EPO induced group, WBC count was increased slightly in whole period, but without statistic significance (p > 0.05), Hb level and RDW increased at day 6 and 30 significantly (p < 0.05), MCV increased at day 6 significantly (p < 0.05), but platelet amount decreased significantly at all time points (p < 0.05). Most mice in EPO-induced group had hepatomegalia and their liver and spleen coefficient increased significantly at day 60 (p < 0.05), while most mice had splenomegaly and its coefficient was increased significantly at all time-points (p < 0.05). CD43(+) cell ratio of EPO group increased significantly in whole period (p < 0.05). CT scanning displayed femoral cortical thickening, medulla canal narrowing and lumbar spine density increasing at day 150, meanwhile, HE staining and reticular fiber staining showed the fatty degeneration or vacuolization in liver, splenomegaly with megakaryocytic proliferation, femur bone marrow fibrosis and osteosclerosis. It is concluded that the mouse induced by high dose of rhEPO displays the myelofibrosis associated with splenic extramedullary hemopoiesis, and this study is useful to establish a practical MF model, and to explore its pathological mechanism.

Characteristics and clinical significance of CD73 expression in subtypes of leukemia / 中国实验血液学杂志

The study was purposed to investigate the expression of CD73 on bone marrow nucleated cells (BMMNC) in various leukemia subtypes and its relationship with cell differentiation of leukemia. Immunocytochemistry staining and Wright-Giemsa staining of BMMNC from 75 cases of leukemia, 11 cases of myelodysplastic syndrome (MDS), 13 cases of non-leukemic patients and 9 healthy adults were performed, and the CD73(+) ratio in BMMNC and its relationship with differentiation of leukemia cells were analyzed. The results showed that the ratios of CD73(+) in BMMNC of com-B ALL, pre-B ALL and PLL were significantly higher than those in B-CLL (p < 0.05). CD73(+) ratios in AML subtypes of M(1), M(2a), t (8; 21), t (15; 17), M(4) and M(5) were markedly higher than those in MDS respectively, but in M(6) and MDS were lower and had no statistical difference between them. CD73(+) ratios in T-ALL, B-CLL, M(6), MDS, non-leukemia patients and healthy adults were close to each other and all of them were lower than those in B-ALL and other AML subtypes. It is concluded that the expression of CD73 is associated with leukemia subtype, differentiation and development. The higher differentiation of leukemia cells, the lower of CD73 expression in myeloid and B lymphoid leukemia, but T-ALL does not meet this pattern.

Correlation between ultrastructural abnormality of bone marrow cells and anemia and neutropenia in myelodysplastic syndrome / 中国实验血液学杂志

The aim of study was to investigate the relationship of anemia and neutropenia with ultrastructural abnormalities of erythroblasts and young neutrophils in bone marrow of patients with myelodysplastic syndrome (MDS). Anemia parameters and peripheral neutrophil amount of 74 patients with MDS were measured by automatic hemocyte analyzer. According to Hb value and neutropenia degree, MDS patients were divided into 4 groups: normal, mild, middle and severe anemia or neutropenia. The morbid rate and apoptosis rate of erythroblasts and young neutrophils in bone marrow were measured by transmission electron microscopy (TEM). The results indicated that 68 out of 74 patients were consistent with anemia diagnostic criteria, and 51 out of 68 patients were with neutrocytopenia. TEM showed different abnormal features of erythroblasts and young neutrophils in all patients. The morbid rates of erythroblasts in normal, mild, middle and severe anemia groups were 37 ± 14.7%, 24 ± 9%, 32 ± 16% and 34 ± 21% respectively, while apoptotic rates of erythroblasts in normal, mild, middle and severe anemia groups were 2.25 ± 1.03%, 4.43 ± 2.60%, 8.78 ± 4.04% and 11.67 ± 4.57% respectively. The morbid rate and apoptotic rate of erythroblasts were correlated negatively with Hb and HCT value (p < 0.05). The apoptotic rates of bone marrow young neutrophils in 4 groups with different degree of neutropenia were 6.00 ± 2.67%, 9.50 ± 4.42%, 13.00 ± 3.54% and 17.00 ± 2.39%, which correlated negatively with peripheral neutrophil quantity (p < 0.01). Morbid rates of neutrophils in normal, mild, middle and severe anemia groups were 12.25 ± 16.31%, 13.5 ± 10.01%, 23 ± 8.59% and 51.67 ± 19.67% respectively, which positively correlated with its apoptotic rates (p < 0.01). It is concluded that anemia and neutropenia in patient with MDS are correlated with apoptosis and morbid rate of erythroblasts and young neutrophils in bone marrow, which may result in ineffective hematopoiesis.

Comparison of effect between homologous recombinant gene knockout and siRNA gene silence in cell lines / 中国实验血液学杂志

The objective of this study was to compare the effects between knocking-out Sam68 gene by homologous recombination method and silencing the gene by siRNA silencing technique in DT40 cell line. Gene targeting technique was used to isolate Sam68 gene-deleted chicken DT40 cells. Meanwhile, Sam68 gene silencing cells was obtained by using stable expression of siRNA plasmid pSilencer-Sam68. Then, the function of these two cell lines were analyzed by comparing with wild-type DT40 cell line. The results showed that the growth retardation in Sam68 gene knocked-out cell line was observed due to elongation of the G2/M phase, but which could not be found in Sam68 gene silencing cell line. It is concluded that in accordance with study of protein function in living cells, use of gene knockout technique for cell line can provide the experimental results more real than those resulting from gene silence technique.

Effect of intracellular acidification on P-glycoprotein in drug-resistant K562/A02 cells / 中国实验血液学杂志

The aim of this study was to investigate the effect of intracellular acidification on the P-gp in K562/A02 cells. Confocal laser microscope was used to determine the intracellular acidification. MTT assay was used to detect the cytotoxicity of intracellular acidification on K562 and K562/A02 cells. Flow cytometry was applied to measure the influence of intracellular acidification on the activity of P-gp. The P-gp expression at protein and mRNA levels were determined by Western blot and real-time RT-PCR respectively. The results indicated that intracellular acidification had no obvious cytotoxicity on K562 and K562/A02 cells. The function of P-gp in K562/A02 cells weakened along with decrease of intracellular acidification, the intracellular acidification significantly increased the accumulation of Rhodamine 123 (Rh 123) and suppressed the efflux of Rh 123 mediated by P-gp. The intracellular acidification also inhibited the expression of P-gp in K562/A02 cells at protein and mRNA levels which showed intracellular acidification with time-dependence. It is concluded that the intracellular acidification can inhibit the expression and function of P-gp in K562/A02 cells.

Clinical and hematological features of congenital dyserythropoietic anemia type I / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the clinical and laboratory features of patients with congenital dyserythropoietic anemia type I (CDA-I), and improve the clinical diagnostic accuracy.</p><p><b>METHODS</b>The clinical and hematological features of 5 patients diagnosed as CDA-I in our hospital between July 2002 and July 2007 were analyzed retrospectively, and the related literatures was reviewed.</p><p><b>RESULTS</b>Five CDA-I patients, 1 male and 4 females, all had a long history of varied degree of chronic anemia. One patient had congenital malformations, 3 jaundice and 4 hepatosplenomegaly. Bone marrow specimens invariably showed hypercellularity due to erythroid hyperplasia with megaloblastic changes, irregularly shaped nuclear, and chromatin bridges in 0.2% to 0.6% of all erythroblasts. All the 5 patients' bone marrow erythroblasts showed spongy heterochromatin appearances (swiss-cheese) with electron microscopy examination. There was no morphologic abnormality in the granulocytes and megakaryocytes. Serum ferritin levels were increased in 3/4 patients. One patient had been misdiagnosed as hereditary spherocytosis and performed splenectomy in the local hospital with no improvement in Hb level.</p><p><b>CONCLUSIONS</b>CDA-I is a rare congenital anemia characterized by ineffective erythropoiesis, jaundice, hepatosplenomegaly and iron overload, and may be misdiagnosed. Keeping these manifestations in mind should avoid misdiagnosis.</p>

Expression of a tumor related gene chp2 in leukemia cells / 中国实验血液学杂志

The purpose of this study was to investigate the expression feature of a human tumor related gene chp2 in leukemia primary cells and leukemia cell lines, real-time quantitative PCR (RQ-PCR) was performed to detect the expression level of chp2 gene in peripheral blood mononuclear cells from 10 healthy individuals (as control) and 24 cases of leukemia, and in 4 kinds of leukemia cell lines. The results showed that the detection rate of chp2 gene in 10 normal controls was 80%, positive expression was (0.744 +/- 0.682) x 10(5) cps/microl. The expression levels of chp2 mRNA leukemia primary cells and leukemia cell lines were significantly higher than that in the normal control (p < 0.05). The expression levels of chp2 mRNA were higher in AML cells (7 cases), CML cells (6 cases), ALL cells (7 cases) and CLL cells (4 cases), and their expression levels were (11.637 +/- 5.588), (6.122 +/- 3.785), (4.262 +/- 2.561) and (3.434 +/- 1.974) x 10(5) cps/microl respectively. Gene chp2 positively expressed in four kinds of leukemia cell lines, and the expression levels in K562 cells, Jurkat cells, HL-60 cells and M07e cells were (5.243 +/- 1.852), (4.463 +/- 1.621), (4.137 +/- 1.837) and (2.578 +/- 1.137) x 10(6) cps/microl respectively. The expression level in leukemia cell lines was higher than that in primary cells. It is concluded that the human tumor related gene chp2 expression in leukemia primary cells and leukemia cell lines significantly increase, which may play an important role in growth process of leukemia cells.

Ultrastructural analysis of 5' nucleotides distribution in acute myeloid leukemia subtypes / 中国实验血液学杂志

5' nucleotides (5'NT), a purine degradative enzyme, is capable of hydrolyzing nucleotide and acting as a phosphotransferase simultaneously. It has critical role in maintaining nucleotide metabolism balance. The present study was aimed to investigate the expression of 5'NT in bone marrow granulocytes (BMGs) from patients with acute myeloid leukemia (AML) and healthy donors comparatively. The BMGs were isolated from bone marrow of 33 patients with AML and 6 healthy donors by using lymphocyte isolating solution. The reactivity of 5'NT was detected by electron microscope and cytochemistry of cytidine monophosphate (CMP). The positive BMG ratio and their index were calculated on the base of ultrastructural observation semiquantitatively. The results indicated that electron microscopy revealed plasma membrane reacting pattern of CMP. Most BMGs from normal donors were CMP negative or exhibited lower active degree. All cases of M(0), M(1), M(2) and t (8; 21) showed high positive percentages and high indexes of BMGs, but no statistic differences between them. APL of t (15; 17) shared lower percentages and indexes than other subtypes. There was no significant difference between APL and normal donors statistically. In conclusions, the results suggested the expression of 5'NT may be associated with BMG differentiation in AML, and APL of t (15; 17) may be a highly differentiated leukemia subtype.

Ultrastructural characteristics of megakaryocytes in 11 patients with acute megakaryocytic leukemia / 中国实验血液学杂志

The purpose of study was to investigate the ultrastructural features of leukemic megakarocyte (LMK) in patients with acute megakaryocytic leukemia (M(7)). Analyzing the ultrastructure characteristics of LMK and positive ratio of platelet peroxides (PPO) in 11 patients with M(7) were analyzed on basis of transmission electron microscopic observation retrospectively. The results showed that the diameter of LMK in 7 out of 11 cases was less than 20 microm, in 2 cases of them, the LMK diameter was from 10 to 15 microm and their PPO positive ratio was more than 50%, most LMK displayed regular shape, less protrusions, irregular nucleus, high nuclear/cytoplasm ratio, tiny granules, undeveloped demarcation membrane system (DMS) and irregular tubules in cytoplasm; in 5 out of those 7 cases the diameter of LMK was about 20 microm, PPO positive cell count was from 8% to 22%, most showing round or horseshoe nuclei, more or less heterochromatin, no DMS and granules were found in LMK in 3 cases and 2 cases occasionally. In other 5 out of 11 cases, the diameter of LMK was from 20 to 40 microm and PPO positive ratio was from 16% to 80%, in which smaller LMKs were similar to those in former cases in shape, and the larger LMK had irregular protrusions, varied nuclear/cytoplasm ratio, more heterochromatin, prominent nucleolus, some of them contained developed DMS, tubules and alpha-granules. It is concluded that most patients with M(7) are predominant of LMK in stage-I and minority contained LMK in II or III stage simultaneously. The differentiation degrees of LMK are different in individual and various cases.

Ultrastructural characteristics of congenital dyserythropoietic anemia-type I / 中国实验血液学杂志

The study was aimed to investigate the ultranstructural feature and diagnostic criteria of congenital dyserythropoietic anemia-type I (CDA-type I). Nucleated red cells in bone marrow from two patients with CDA-type I were analyzed by transmission electron microscopy (TEM). The results indicated that the erythropoietic/granulopoietic ratio was markedly increased with megaloblastic morphology in all stage of erythrocyte. Most proerythroblast showed of irregular nuclei, while the Swiss-cheese-appearance of the heterochromatin was usually found in basophilic and polychromatic erythroblast. About half of orthochromatic erythroblast illustrated karyolysis and karyorrhexis. Some orthochromatic erythroblast exhibited karyolysis and plasmolysis simultaneously. The inter-nuclear chromatin bridge between separated erythroblasts was seldom found by TEM. The nuclear membrane and rough endoplasmic reticulum were destructed at all stage of erythrocytes in different degree. In conclusion, the megaloblastic erythrosis was the main characteristic of CDA-type I, and then nuclear membrane disruption in polychromatic erythroblast and karyolysis or karyorrhexis in orthochromatic erythroblast. The universal breakdown of cytoplasm membranous system was fundamental pathogenesis of CDA-type I.

Ultrastructural characteristics of malignant T cell in T cell lymphoma / 中国实验血液学杂志

In order to investigate the ultrastructural features of malignant T cell (MTC) in bona marrow aspirate (BMA) from patients with T Cell Lymphoma, the antigen expression of MTC was analyzed by flow cytometry, and the ultrastructural features of MTC in BMA from 13 T-cell lymphoma patients with bone marrow involvement (BMI) were observed by transmission electron microscopy. The results indicated that the sizes of MTC were uneven in every patient and their diameter were between 12 and 28 microm, in 6 out of 13 cases sizes of MTC were slightly uneven but in 7/13 cases sizes of MTC were significantly uneven. The heterochromatin of MTC was less than that of normal T cell and nucleolus diameter was from 2 to 8 microm in all cases. The nuclear contour of MTC was strikingly irregular in 10 out of 13 cases. The MTC had plenty of cytoplasm in 8 out of 13 cases and displayed many microvilli or processes on MTC surface in 7 out of 13 cases, while MTC in 6 out of 13 cases contained more Golgi's apparatuses, secretary vacuoles, dense granules and intermediate filaments. In 8 out of 13 cases mitochondria apparently swelled. It is concluded that the size of MTC increase unevenly in all patients. MTC nuclear contour in most cases is irregular by folding, indenting, and twisting, which often correlated with arising of paranuclear intermediate filaments. Processes and microvilli on surface and Golgi's apparatus, secretary vesicles, dense granules as well as intermediate filament in cytoplasm of MTC develop synchronously, meanwhile, mitochondria of MTC strikingly swell in most cases.