In order to meet the needs of the flow cytometry for the simultaneous analysis of multiple fluorescence wavelengths and small volume, the design method of flow cytometry spectrum analysis system is presented by analyzing the characteristics of Dyson structure. And according to the method, a flow cytometry spectrum analysis system is disigned with Dyson type.The system's spectral range is 400 nm to 800 nm, the defocused spot size is less than the pixel size 24μ mm, the ransfer function value is above 0.8 at the Nyquist cut-off frequency 21 lp/mm,the spectral resolution is less than 3 nm, and the overall size is 83.54 mm×85.60 mm.The system has good optical performance and small volume, which meets the needs of the flow cytometry fluorescence spectral analysis. The outstanding innovation of this system is the application of Dyson light splitting structure and EMCCD detector which is high speed and high sensitivity.
Equipment Design ; Flow Cytometry ; instrumentation

OBJECTIVETo prepare stable chicken red blood cells for the calibration of flow cytometry.

METHODSThe traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2+-free HBSS treatment and low-speed centrifugation. The effect of fluorescence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed cells.

RESULTSChicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [(98.5∓3.5)% vs (93.5∓2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [(6.0∓0.3)% to 6.2∓0.4% vs (8.6∓0.5)% to (13.1∓1.4)%, P<0.01].

CONCLUSIONThe chicken red blood cells isolated using the modified method can be applicable for calibration of flow cytometry.

Animals ; Calibration ; Chickens ; Erythrocytes ; cytology ; Flow Cytometry ; instrumentation ; methods

Flow Cytometry ; instrumentation ; Indicators and Reagents ; Investigational New Drug Application

BACKGROUND: The usefulness of the CytoDiff flow cytometric system (Beckman Coulter, USA) has been studied in various conditions, but its performance including rapidity in detecting and counting blasts, the most significant abnormal cells in the peripheral blood, has not been well evaluated. The objective of this study was to evaluate the performance of the CytoDiff differential counting method in challenging samples with blasts. METHODS: In total, 815 blood samples were analyzed. Samples flagged as "blasts" or "variant lymphocytes" and showing <10% blasts by manual counts were included. In total, 322 samples showed blasts on manual counts, ranging from 0.5% to 99%. The CytoDiff method was performed by flow cytometry (FC500; Beckman Coulter, USA) with a pre-mixed CytoDiff reagent and analyzing software (CytoDiff CXP 2.0; Beckman Coulter). RESULTS: The average time required to analyze 20 samples was approximately 60 min for manual counts, and the hands-on time for the CytoDiff method was 15 min. The correlation between the CytoDiff and manual counts was good (r>0.8) for neutrophils and lymphocytes but poor (r<0.8) for other cells. When the cutoff value of the CytoDiff blast count was set at 1%, the sensitivity was 94.4% (95% CI; 91.2-96.6) and specificity was 91.9% (95% CI; 89.0-94.1). The positive predictive value was 88.4% (95% CI; 84.4-91.5) (304/344 cases) and negative predictive value was 96.2% (95% CI; 93.9-97.7) (453/471 cases). The CytoDiff blast counts correlated well to the manual counts (r=0.9223). CONCLUSIONS: The CytoDiff method is a specific, sensitive, and rapid method for counting blasts. A cutoff value of 1% of at least 1 type of blast is recommended for positive CytoDiff blast counts.
Adult ; Female ; Flow Cytometry/*instrumentation ; Humans ; Leukocyte Count ; Leukocytes/*cytology ; Lymphocytes/cytology ; Male ; Neutrophils/cytology

This study was purposed to investigate the efficacy of different whole flow lysis reagents for lysis of red blood cells in flow cytometric analysis. The expression of immunocytes was detected by flow cytometry after lysis of red blood cells using commercial reagents (Optilyse C, FACS Lysing Solution) and self-made red blood cell lysis reagents (RBC Lysis Buffer), the detection results were analyzed comparatively. The results showed that there was no significant difference in the percentage of CD3e(+), CD3e(+)CD4(+), CD3e(+)CD8a(+), CD3e(-)CD19(+), CD3e(-)NK1.1(+) and Gr-1(+) cells between 3 different lysis reagent groups. However OptiLyse C solution was suitable to Gr-1(+) cell detection, but did not suit to Foxp3(+) Treg detection. The self-made RBC Lysis Buffer and FACS Lysing Solution were suited to Foxp3(+) Treg detection. It is concluded that the use of self-made RBC Lysis Buffer for flow cytometry can get the lysis efficiency of commercially available lysis solutions when samples are prepared in accordance with standardized procedure. The self-made RBC Lysis Buffer not only can satisfy experimental requirements, but also can reduce the experimental costs.
Animals ; Erythrocyte Count ; Erythrocytes ; immunology ; metabolism ; Flow Cytometry ; instrumentation ; methods ; Immune System ; immunology ; Indicators and Reagents ; analysis ; Mice ; Mice, Inbred C57BL

OBJECTIVEThis study is aimed at evaluating the utility of the portable CD4 analyzers (PIMA).

METHODSThe paired finger prick blood (25 µl) and 5 ml venous blood samples were collected from 196 HIV infected patients, who came to Yunnan CDC voluntary counseling and testing (VCT) clinic for CD4 test services, from May to August, 2012. The absolute CD4 cell counts were measured by PIMA (using venous and finger-prick blood) and by Calibur (using venous blood) as the reference. The PIMA and Calibur CD4 results were compared using the Wilcoxon matched-pairs test, and the Spearman's rank correlation coefficients were estimated. The Bland-Altman plots were used to assess the consistency of the two methods.

RESULTSThe median absolute CD4 counts of 196 venous blood samples obtained by PIMA and by Calibur were 268 (range:169-403) cells/µl and 302 (range:181-474) cells/µl respectively, which showed significant difference (Z = -7.31, P < 0.01). The median absolute CD4 counts measured by PIMA and by Calibur (using 188 finger-prick and venous blood samples respectively) were 271 (range: 165-450) cells/µl and 304 (range:188-476) cells/µl, which also showed significant difference (Z = -7.60, P < 0.01). The CD4 counts obtained by PIMA CD4 analyzer (using venous and finger-prick blood) showed strong positive correlation with the CD4 counts obtained by the reference method (using venous blood), and the r values were 0.94 and 0.92 respectively (P < 0.01) . The mean biases (limit of agreement) were -38.7 (-210.9-133.5)cells/µl and -45.4 (-221.8-131.0) cells/µl, respectively.Using 350 CD4 counts as the threshold for ART treatment initiation, the sensitivity and specificity of PIMA were 99.1% and 79.3% for venous blood samples, and 97.2%and 78.5% for finger-prick blood samples, respectively.

CONCLUSIONThe CD4 counts obtained by PIMA are lower than that obtained by Calibur, while the sensitivity is high.

Adolescent ; Adult ; Aged ; CD4 Lymphocyte Count ; instrumentation ; methods ; Child ; Female ; Flow Cytometry ; instrumentation ; methods ; HIV Infections ; blood ; Humans ; Male ; Middle Aged ; Sensitivity and Specificity ; Young Adult

No abstract available.
Adult ; Aged ; Aged, 80 and over ; Blood Chemical Analysis/instrumentation ; Blood Platelets/*cytology/physiology ; Female ; Flow Cytometry/*instrumentation/standards ; Humans ; Male ; Middle Aged ; Platelet Count/*instrumentation/standards ; Reference Values ; Republic of Korea ; Young Adult

No abstract available.
Antigens, CD34/*metabolism ; Automation ; Blood Cells/*cytology/metabolism ; Cell Count/*instrumentation/standards ; Flow Cytometry/standards ; Hematopoietic Stem Cells/*cytology/metabolism ; Humans

The study was to investigate the hemorheologyic changes of group A blood recipient transfused with large amounts of group O whole blood, by erythrocyte count, sympexis index, erythrocyte deformation index, erythrocyte rigid index and whole blood reduced viscosity, 60 ml of group A whole blood were added with 9, 12, 15 and 18 ml of group O whole blood (which corresponds to the 4 000 ml whole blood added with 600, 800, 1 000 and 1 200 ml whole blood). The mixed blood was incubated at 37 degrees C with mixing at 80 times per minute. Samples were taken from the mixed blood at 30 minutes, 2, 4, 8, 12 and 24 hours after culture, and the hemorheology of the mixed whole blood was determined by FASCO-series type 3020B automatic rheograph apparatus. The results showed that there were no differences of erythrocyte count, sympexis index, erythrocyte deformation index, erythrocyte rigid index and whole blood reduced viscosity among all different kinds of mixed whole blood, and there was no difference of sympexis index at different times, but there were obvious differences of erythrocyte count, erythrocyte deformation index, erythrocyte rigid index and whole blood reduced viscosity. It is concluded that blood transfusion of 1,200 ml group O whole blood to a recipient with 50 kg of body weight but with different blood type in emergent situation may exert no harm to the erythrocytes of recipient in a short term.
ABO Blood-Group System ; Blood Group Incompatibility ; blood ; prevention & control ; Blood Transfusion ; Erythrocyte Count ; Erythrocytes ; cytology ; Flow Cytometry ; instrumentation ; methods ; Hemodynamics ; Hemorheology ; Humans

In order to find a method suitable for purifying large amount of CD34(+) cells, from 5 cases who accepted autologous peripheral blood stem cell transplantation, CD34(+) cells were collected and enriched by using Isolex 300i (Nexell). Phenotypes were detected by flow cytometry and the biological viability were assayed by the colony-forming experiments and cell expansion experiment in vitro. The results showed that the number of mononuclear cells first collected was about (3.5 - 6.0) x 10(10) and (0.55 - 1.2)% of cells were CD34 positive. The number of positive production was about (2.0 - 3.0) x 10(8); the CD34(+) cells purity was (75 - 85)% and the yield was (40 - 65)%. The CD34(+) cells of positive production could expand up to 2 - 3 times when cultured with SCF + IL3 + FL + TPO + EPO in vitro. The results of colony-forming experiments demonstrated that the CD34(+) cells collected has enough colony-forming ability. All results showed the enriched CD34(+) cells with biological viability. In conclusion, the CD34(+) immunomagnetic isolation apparatus Isolex300i is suitable to clinical application for a large amount of CD34(+) cell enrichment.
Antigens, CD34 ; immunology ; Colony-Forming Units Assay ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Immunomagnetic Separation ; instrumentation ; methods ; standards ; Neoplasms ; blood