Improvement of Method for Detecting Peripheral Blood Neutrophil CD64 Index in Patients with Hematologic Malignancies / 中国实验血液学杂志

OBJECTIVE@#To improve the method for detecting the neutrophil CD64 (nCD64) index and to enhance the detection rate and accuracy of nCD64 index in patients with hematologic malignancies.@*METHODS@#The nCD64 index in peripheral blood of patients with hematologic malignancies combined with suspicious bacterial infection (255 cases-time) was detected by using array method. When the detection of nCD64 index in samples was interfered with abnormal cells in detection process of enrolled patients, the antibodies CD45, CD15 and CD10 were added into samples on the basis of routine detection by using the primary detection kit, in order to more accurately distinguish the neutrphils and obtain the nCD64 index. The nCD64 index as well as the efficiency of nCD64 index, PCT and CRP for diagnosis of sepsis before and after the improvement of deteation method were compared.@*RESULTS@#The samples of 60 cases were interfered with abnormal cells in detection process, out of which the samples of 18 cases failed in detection, but these samples of 18 cases all got the effective results of detection after the detection method was improved. The detection showed that the nCD64 index before and after the improvement as well as the PCT and CRP levels in sepsis group were higher than those in non-sepsis group(P＜0.05). After improvement of method, the efficiency of nCD64 index for diagnosis of sepsis was suprior to the PCT and CRP, the nCD64 index obtained after the improvement of method possessed the diagnosis efficiency same as the efficiency obtained before improvement of method, moreover the detection results were more reliable.@*CONCLUSION@#For the samples of patients with hematologic malignancies interfered with abnormal cells in the process of detecting the nCD64 index, the corresponding antibodies are added into detected samples according to the kinds of hemotologic diseases of patients, in order to more accurately gate the neutrophils in paripheral blood of patients, there by the detection rate and accuracy for detecting the nCD64 index are enhanced by accurately distinguishing the neutrophils.

Expression and clinical significance of PDCD5 in patients with acute myeloid leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to investigate the roles of PDCD5 (programmed cell death 5) in pathogenesis of acute myeloid leukemia (AML) and the relevance of PDCD5 with the clinical characteristics and prognosis of patients by testing the PDCD5 expression in adult AML patients.</p><p><b>METHODS</b>The mRNA and intracellular protein levels of PDCD5 from 36 newly diagnosed AML patients were analyzed by real-time fluorescence quantitative polymerase chain reaction (RQ-PCR) and flow cytometry (FCM), respectively. The correlation of mRNA levels and intracellular protein levels of PDCD5 with the clinical characteristics and survival time of patients were analyzed.</p><p><b>RESULTS</b>The intracellular protein expression levels of PDCD5 in AML patients were significantly higher than those in normal controls (P < 0.05). The PDCD5 mRNA levels were not significantly different between patients and controls (P > 0.05). The mRNA and protein levels of PDCD5 did not significantly correlate with sex, age, WBC count, FAB subtype, extramedullary infiltration, WT1 gene, NPM1 gene mutation and the patients response to induction therapy. The patients with positive FLT3/ITD mutation displayed higher protein levels of PDCD5 as compared with negative FLT3/ITD mutation patients (P < 0.05).</p><p><b>CONCLUSION</b>The intracellular protein of PDCD5 significantly increased in AML patients. However, the increased PDCD5 does not exert the pro-apoptotic effects on AML cells. The patients with positive FLT3/ITD mutation show higher protein levels of PDCD5.</p>

Expression and clinical significance of B-cell lymphoma 2 (BCL-2) in hyperleukocytic acute myeloid leukemia / 中国实验血液学杂志

This study was aimed to investigate the role of B-cell lymphoma 2 (BCL-2) in pathogenesis of hyperleukocytic acute myeloid leukemia (AML). The levels of intracellular BCL-2 in 48 AML patients were detected by flow cytometry (FCM). Serum levels of BCL-2 in 40 AML patients were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that the serum levels of BCL-2 in hyperleukocytic AML and non-hyperleukocytic AML patients were significantly higher than that in normal controls (P < 0.05), but intracellular BCL-2 levels were not significantly different, as compared with normal controls (P > 0.05). There were no difference of intracellular and serum BCL-2 levels between hyperleukocytic and non-hyperleukocytic AML patients (P > 0.05). The serum and intracellular levels of BCL-2 between hyperleukocytic AML, non-hyperleukocytic AML patients and normal controls were not statistically correlated. It is concluded that leukemic cells in AML patients produce and secrete too much BCL-2, which may be involved in the pathogenesis of leukemia disease. However, the anti-apoptosis effect of BCL-2 has no significant impact on the pathogenesis of hyperleukocytic AML.

Effect of heat stress on expression of gp96 in K562 cell line of the chronic myeloid leukemia and its significance / 中国实验血液学杂志

This study was purposed to investigate the effect of different heat stress conditions on expression level of heat shock protein gp96 in K562 cell line of chronic myeloid leukemia in order to provide experiment basis for seeking optimal heat stress condition increasing extraction amount of gp96 from K562 cells. The expression changes of gp96 in K562 cell line was detected by immunocytochemistry under 38, 40, 42, 44, 46 and 48 degrees C for 30 minutes in water, by flow cytometry under 40, 44, 48 and 52 degrees C for 30 minutes in water, by Western blot under 40, 44, 48 and 52 degrees C for 30 minutes in water. Immunocytochemistry assay showed that gp96 existed mainly in cytoplasm. The peak of gp96 expression was at 30 minutes after 48 degrees C in water. The result of flow cytometry was consistent to immunocytochemistry detection results under temperatures 40, 44, 48 and 52 degrees C (P < 0.01). Western blot showed that detection result was the same as the immunocytochemistry and flow cytometry detections. In conclusion, the expression of gp96 in K562 cell line reached peak at 30 minutes after 48 degrees C in water. This condition may be an effective preparative condition for extraction of gp96 from K562 cells.

Differentiation and function of human dendritic cells influenced by heat shock protein gp96 purified from K562 cells / 中国实验血液学杂志

This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influenced by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-sepharose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 from K562 cells lysate was isolated and purified. The identification of the purified protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from peripheral blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The results showed that 60-80 microg GP96 was purified successfully from 1 x 10(10) K562 cells. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to induce T cells proliferation. It is concluded that heat shock protein GP96 can be isolated and purified from K562 cells and could induce maturation of dendritic cell. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.

Dendritic cells cultured with human umbilical cord serum instead of fetal calf serum / 中国实验血液学杂志

To investigate whether the dendritic cells (DC) could grow up in cultural system with umbilical cord serum (UCS), the UCS was used in the culture instead of fetal calf serum. The phenotype of dendritic cells was detected by flow cytometry and the antigen presenting ability of DC in allo-MLR was measured by MTT assay. The results showed that DC grown in UCS (UCS-DC) had higher expression rate of CD86, CD83 and HLA-DR than that in grown in FCS (FCS-DC). (P < 0.05), and their expression of CD1a was lower than that of FCS-DC. The ability to induce T cell proliferation had no difference between UCS-DC and FCS-DC. It is suggested that dendritic cells with more mature phenotype had been produced in the medium containing UCS than those in the medium containing FCS, and UCS-DC possessed potent ability to stimulate proliferation of allogeneic T cells.