Assessment of a five-color flow cytometric assay for verifying automated white blood cell differentials / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>White blood cell (WBC) counts and differentials performed using an automated cell counter typically require manual microscopic review. However, this last step is time consuming and requires experienced personnel. We evaluated the clinical efficiency of using flow cytometry (FCM) employing a six-antibody/five-color reagent for verifying automated WBC differentials.</p><p><b>METHODS</b>A total of 56 apparently healthy samples were assessed using a five-color flow cytometer to verify the normal reference ranges of WBC differentials. WBC differentials of 622 samples were also determined using both a cell counter and FCM. These results were then confirmed using manual microscopic methods.</p><p><b>RESULTS</b>The probabilities for all of the parameters of WBC differentials exceeded the corresponding normal reference ranges by no more than 7.5%. The resulting WBC differentials were well correlated between FCM and the cell counter (r > 0.88, P < 0.001), except in the case of basophils. Neutrophils, lymphocytes, and eosinophils were well correlated between FCM and standard microscopic cytology assessment (r > 0.80, P < 0.001). The sensitivities of FCM for identification of immature granulocytes and blast cells (72.03% and 22.22%, respectively) were higher than those of the cell counter method (44.92% and 11.11%, respectively). The specificities of FCM were all above 85%, substantially better than those of the cell counter method.</p><p><b>CONCLUSION</b>These five-color FCM assays could be applied to accurately verify abnormal results of automated assessment of WBC differentials.</p>

Characteristics and monitoring of minimal residual disease in patients with T acute lymphoid leukemia by flow cytometry with two 4-color combinations of fluorescent antibodies / 中国实验血液学杂志

The purpose of this study was to establish a method for the monitoring of minimal residual disease (MRD) in bone marrow samples of the children with T acute lymphoid leukemia (T-ALL), and to evaluate its value in clinical application. The immuno-phenotype of the leukemic cells were detected by flow cytometry with two sets of 4-color combinations of antibodies against TdT/CD5/cCD3/HLA-DR(+)CD19(+)CD33 and CD34/CD5/cCD3/HLA-DR(+)CD19(+)CD33 in 32 cases of de novo T-ALL and were compared with the results in 10 normal controls. The antibody combination in regions of the two-parameter plots where the leukemic cells appeared were different from the normal cells was screened as the effective combination which was used to monitor MRD in the bone marrows of the T-ALL children after the inductive treatment. The results indicated that the respective effective frequencies of antibodies against TdT/CD5/cCD3/HLA-DR(+)CD19(+)CD33 and CD34/CD5/cCD3/HLA-DR(+)CD19(+)CD33 were 90.6% and 62.5%. 32 cases of childhood T-ALL were successively screened for antibodies combinations of interest and were identified in 100% (32/32) of these cases. After inductive treatment, the positive rate in 129 times of MRD monitoring was 19.4% (25/129) by flow cytometry and 5.43% (7/129) by FAB morphology. It is concluded that monitoring MRD in patients with T-ALL by flow cytometry with two 4 color combinations of fluorescent antibodies is an quick and effective method. The sensitivity of this method is high and it may be of important significance for the treatment and prognostic evaluation in childhood T-ALL.

Effect of pax5 gene blocked by RNAi on biological characteristics of B cell malignant lymphoma / 中国实验血液学杂志

This study was aimed to investigate the biological characteristics of B hematological tumor cells such as proliferation, immunological phenotype and apoptosis by silencing pax5. The specific pax5 small hairpin RNA (shRNA) was synthesized by in vitro transcription. For evaluating the inhibition efficiency, the expression change at mRNA and protein levels were assessed by real-time RT-PCR and Western blot respectively. To detect the biological characteristics of pax5-silenced hematological tumor cells, the immunological phenotype, apoptosis and cell proliferation were measured by using real-time RT-PCR, MTT assay and flow cytometry respectively. The results showed that two shRNA were synthesized, both of which were effective to block pax5 expression. After being blocked by RNAi the immunological phenotype of pax5-silenced lymphoma cells was changed, the expressions of CD19 mRNA and protein were reduced, but the expression of IgM was not changed. As compared with control group, the effect on proliferation and apoptosis of lymphoma cells not could be detected after pax5 silencing. It is concluded that the pax5 plays important role in late differentiation of B cells, and may participate in signal transduction of lymphoma cells, but the effect on proliferation and apoptosis of lymphoma cells were not detected after RNAi, which need to be elucidated further.

Expression of retinoblastoma protein in child acute leukemia cells and its clinical significance / 中国实验血液学杂志

The research was aimed to detect the expression levels of retinoblastoma protein (pRb) in child acute leukemia cells, and to explore its possible association with leukemia cells cycle, the risk of disease, minimal residual disease (MRD) monitoring and prognosis of B-ALL. Flow cytometry (FCM) was used to detect the expression of pRb in 89 cases of acute leukemia (including 25 AML, 10 T-ALL and 54 B-ALL) and bone marrows from 7 normal children (control group). Meanwhile the cell cycle in some cases was analyzed. The results showed that (1) the FCM could accurately detect the expression of pRb in acute leukemia cells; (2) the high level of pRb expression was frequent in all types of child acute leukemias. In the same case, the expression of pRb was significantly increased in leukemia cells when compared with non-leukemia cells. And no detectable pRb protein was found in partial cases of acute leukemia; (3) there was a close relation between expression of pRb and the cell cycle of leukemia cells, the number of G(1) phase cells in pRb positive case of B-ALL was more than that in pRb negative case (92% vs 77%); (4) in B-ALL, the level of pRb expression in MRD positive group was significantly lower than that in MRD negative group (P < 0.05), but pRb expression was stable in non-leukemia cells during therapy; (5) pRb expression was related to the early response to therapy in B-ALL, the expression of pRb was significantly increased in sensitive group when compared with insensitive group (P < 0.05). It is concluded that high level or absence of pRb expression can be found in child acute leukemia cells. The expression of pRb is positively related to cell cycle of leukemia cells, MRD monitoring and the early response to therapy. In short, the detection of pRb expression level can guide the therapy and the evaluation of prognosis in B-ALL.

Expression of the transcription factor PAX5 in childhood acute leukemic cells / 中国实验血液学杂志

To investigate transcription factor PAX5 expression characteristics in childhood acute leukemic cells, expression levels of PAX5 and CD19 mRNA in 6 hematological tumor cell lines and bone marrow cells of 6 normal children, 58 de novo patients and 4 relapse acute leukemic children, including 39 cases of B-ALL, 10 cases of T-ALL and 13 cases of AML, were detected by a real-time RT-PCR. The results showed that PAX5 and CD19 mRNA expression levels were 2.35% and 2.52% in Namalwa (B-cell lines) respectively, but almost not detectable in other T- and myeloid cell lines. Among clinical samples, expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML (P = 0.029 and P = 0.013 respectively). PAX5 expression was significantly lower in T-ALL and AML than that in normal controls. The difference of PAX5 mRNA expression levels between T-ALL and AML was not significant. Individual difference of PAX5 mRNA expression levels in children with B-ALL was great. Moreover, PAX5 mRNA expressions in de novo and relapse patients with B-ALL were significantly higher than those in remission (P = 0.011 and P = 0.006 respectively). As binding sites for B-cell specific activator protein have been identified in the promoter regions of CD19, the study found that in B-ALL, there was clear correlation between the expression levels of PAX5 and CD19, which was also studied by real-time RT-PCR. It is concluded that PAX5 transcripts are readily detectable and quantifiable in clinical materials with B-ALL by real-time RT-PCR. The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.

Mechanism of resistance of Pseudomonas aeruginosa to carbapenems / 上海第二医科大学学报

Objective To investigate the mechanism of resistance of clinical isolates of Pseudomonas aeruginosa to carbapenems.Methods Fifty-eight strains of imipenem-and meropenem-resistant Pseudomonas aeruginosa were isolated.Modified K-B technique was adopted in the susceptibility test by adding ?-lactamases inhibitors and efflux pumps inhibitors to M-H agar plates.The refined three-dimensional extract test was used to detect ?-lactamases.The genes of metallo-enzyme IMP,VIM,as well as outer membrane porin D2(OprD2) were analyzed with polymerase chain reaction.Results Among the 58 strains of carbapenems-resistant Pseudomonas aeruginosa,53 produced over-expressed active efflux and continuously produced large amont of AmpC enzyme,15 of which were accompanied by the loss of OprD2,and 1 of which were accompanied by extended spectrum ?-lactamases(ESBLs).Among the 5 strains which neither produced over-expressed active efflux nor ?-lactamases,only 1 was found with OprD2 gene deletion.Metallo-enzyme was not detected in any of the 58 strains.Conclusion The mechanism of resistance of Pseudomonas aeruginosa to carbapenems was mainly the production of the over-expressed active efflux combined with the continuous production of large amount of AmpC enzyme.Sixteen of the strains were accompanied by the loss of OprD2 gene.

?-lactamase production in multidrug-resistant Pseudomonas aeruginosa / 上海第二医科大学学报

Objective To study the production of ?-lactamase in multidrug-resistant Pseudomonas aeruginosa and to guide the proper use of antibiotics in clinical practice. Methods The modified three-dimensional extract test was employed to detect ?-lactamase in 30 multidrug-resistant Pseudomonas aeruginosa strains screened from antimicrobial susceptibility test in our hospital,and isoelectric focusing electrophoresis was performed on the enzyme-producing strains. Results No metalloenzyme was detected in all the 30 strains.Twenty-six strains produced ?-lactamase,among which 25 continuously yielded large amount of AmpC enzyme and the other one both AmpC enzyme and extended-spectrum ?-lactamases(ESBLs).86.7% of multidrug-resistant Pseudomonas aeruginosa produced enzyme. ConclusionThe majority of the multidrug-resistant Pseudomonas aeruginosa in our hospital yielded large amount of AmpC enzyme in a continuous way.The modified three-dimensional extract test can eliminate some interference such as the decrease of outer membrane permeability and overexpression of efflux pump,facilitating the effective and accurate detection of ESBLs in Pseudomonas aeruginosa.

Changes of plasma biomolecules in patients with acute cerebral infarction before and after treatment / 上海第二医科大学学报

Objective To study the changes of plasma biomolecules in patients with acute cerebral infarction(ACI) and the potential influences from lumbrukinase and aspirin(ASA). Methods Seventy patients with ACI were randomly divided into three groups according to the ways of administration: lumbrukinase group(n=26),ASA group(n=24)and lumbrukinase + ASA group(n=20).Plasma levels of P-selectin,D-dimer(D-D),tissue plasminogen activator(t-PA),plasminogen activator inhibitor-1(PAI-1),plasmin-antiplasmin complex(PAP),thrombin-antithrombin complex(TAT),homocysteine(Hcy) and thrombin-activatable fibrinolysis inhibitor(TAFI) were measured in these patients with ACI and normal individuals(n=20) by ELISA method.Changes of these parameters were detected before and after the treatment,and the diagnostic efficiencies for ACI were compared by receiver characteristic curve(ROC).Results Before the treatment,all the parameters in patients with ACI were significantly higher than those in the healthy controls(P0.05).However,there were significant changes of PAP in the lumbrukinase group(P

Investigation of the Correlation between Deep Vein Thrombosis and Clinical Laboratory Tests in the Regulation of Hemostasis / 上海第二医科大学学报

Objective To investigate the correlation between deep vein thrombosis(DVT) and clinical laboratory tests in the regulation of hemostasis. Methods Endothelin-1 (ET-1), thrombomodulin (TM), P-selectin, prothrombin fragment _ 1+2 (F_ 1+2 ), thrombin-antithrombin complex (TAT), thrombus precursor protein (TpP) and D-Dimer (D-D) were measured in DVT patients (n=72) and normal individuals (n=20) with ELISA method. The sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and accuracy were evaluated for each of the items, and the area of the underlying receiver operating characteristic curve (AUC ROC ) was used to appraise diagnostic efficiency. Results Except ET-1, there were significant differences between the DVT group and normal control group in the other items. The AUC ROC of P-selectin, F_ 1+2 , TpP and D-D was bigger than that of ET-1, TM and TAT (P0.05). The TpP had the highest sensitivity, NPV and accuracy, and the D-D had the highest specificity and PPV. Conclusion The increase of D-D and TpP could reflect the occurrence of continuous coagulation and fibrolysis respectively. Thus the combination of measuring TpP and D-D could provide better laboratory evidence for the diagnosis of patients with DVT.

Prognostic significance of lymphocyte function associated anti-gen-3 (CD58) in childhood B cell-acute lymphocytic leukemia / 中国实验血液学杂志

This study was aimed to investigate the value of CD58 in evaluation of early therapeutic effect on childhood B-ALL. The expression features of CD58 in 135 cases of childhood B-ALL were analyzed by four-color flow cytometry; MRD detection protocol for B-ALL using CD58/CD10/CD34/CD19 combination was established; the correlation between the expression features of CD58 and MRD detection was analyzed for the early therapeutic response in childhood B-ALL. The results showed that the mean value of CD58 MFI in 135 cases of B-ALL was 113.08 +/- 63.33, which was significantly higher than that in 15 cases of normal bone marrow controls (14.68 +/- 5.26, P < 0.01). In addition, CD58 was over expressed in 51.9% (70/135) of B-ALL patients, indicating that CD58 could be an effective marker in MRD detection. The CD58/CD10/CD34/CD19 was the second most effective combination next to TdT/CD10/CD34/CD19 in B-ALL MRD detection with flow cytometry. Meanwhile, the positive rate of MRD detection by flow cytometry was significantly lower in CD58 over expression group (P < 0.05). It is concluded that CD58 may be used as an indicator for detection of MRD in B-ALL patients, which would enrich the combination of MRD detection. The CD58 over expression may be considered as a marker of a favorable prognosis in childhood B-ALL.

Characterization of CD10 expression and its significance in minimal residual disease detection in childhood B-acute lymphoblastic leukemia / 中国实验血液学杂志

To observe the expressions of CD10 in childhood B-acute lymphoblastic leukemia (B-ALL) and to define the role of CD10 in minimal residual disease (MRD) detection. 58 cases of childhood B-ALL were studied in this program. Four-color flow cytometry was used to analyze the characteristics of B-ALL phenotypes. The four-color fluorochrome labeled antibody combinations of CD10 with other markers were used to detect MRD. The results showed that CD10 overexpression (CD10(bright)) was detected in 65.5% (38/58) of B-ALL patients and a strong correlation between CD10(bright) and CD34 expression was also observed, i.e. CD10(bright) expression most frequently happened in B-ALL with high percentage of CD34 positive cells. In detection of MRD, CD10(bright), combined with other markers, could effectively distinguish normal cells with leukemic cells, even if there was no any other marker that can be used. It is concluded that CD10(bright) expression correlates with high expression of CD34 in B-ALL, it is a good marker for MRD detection. The combination of CD10 and other markers can be applied in B-ALL MRD detection with flow cytometry.

Detection of minimal residual disease in childhood B-ALL by flow cytometry / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a flow cytometric method for detecting minimal residual disease (MRD) in children with B-ALL and evaluate its clinical application.</p><p><b>METHODS</b>Fifty-eight childhood B-ALL cases entered this study and 30 MRD analyses were performed after remission induction therapy. Four-color fluorochrome labeled monoclonal antibodies were used to analyze the cell immunophenotypes. Cells from normal bone marrow were used as controls. The leukemic cell populations located in flow cytometry dot plots different from those of normal were considered to be the markers of interest in the first step screening, and then used to monitor MRD step after therapy.</p><p><b>RESULTS</b>Fifty-eight cases of childhood B-ALL were screened for antibodies combinations of interest and were identified in 89.7% (52/58) of these cases. The four-color antibody combinations consisted of CD(10)/CD(34)/CD(19) plus another effective marker such as CD(38), CD(65), CD(66c), CD(21). The sensitivity of this method was 0.01%, much higher than microscopic inspection. In 8 cases whose bone marrow microscopically showed no residual leukemic cells, the percentage of leukemic cells were identified with this method of 0.028%, 1.430%, 3.050%, 0.015%, 5.660%, 2.700%, 0.027%, and 0.069%, respectively.</p><p><b>CONCLUSION</b>The application of flow cytometry in MRD monitoring can significantly improve the detection sensitivity in childhood B-ALL, thus facilitate the further treatment decision and follow-up.</p>

Evaluation and cell warning information for clinical application of five-part differential hematology analyzer / 中华检验医学杂志

Objective To investigate the technical charactenstics ot SYSMEX XS1000i 5-part differential automated hematological analyzer and its clinical applications.Methods 209 samples were analyzed with the analyzer of XS1000i and compared to the results from Beckman-Coulter LH750 analyzer.The main parameters from XS1000i,such as precision within-run,day to day precision and carryover contamination rates etc,were recorded respectively in 509 samples to compare the difference between the instrumental and optical examination.Results All variation coefficients of precision from XSI000i were within the manufacturer.The carryover contamination rates of WBC,RBC,Hb,HCT and PLT were 0.19%,0.93%,0,-0.88%,-0.76%,respectively.The parameters of XS1000i were correlated with the results of LH750 except basophil granulocyte.The correlation coefficients of WBC,RBC, HGB and PLT were 0.994 5,0.996 8,0.997 0 and 0.974 6 ,respectively.The sensitivity of warning flags in immature leukocytes was 100% and the specificity was 69.7%,the sensitivity of warning flags in atypical lymphocyte was 100% and the specificity was 66.7%.Especially in 3 leukocytopenia that was induced by chemotherapy in patients with leukemia who had only few immature cells existed in the peripheral blood,the parameters of XS1000i were positive,and correlated with the results of detection of minimal residual disease with flow cytometry.Conclusions The warning system of XSIO00i provides more valuable information for manual microscopic examination.If it is combined with flow cytometry,the advantages in detection of residual leukemic cell will be fully displayed.