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PubMed2001

Cyclin D1 by flow cytometry as a useful tool in the diagnosis of B-cell malignancies.

The translocation (11;14)(q13;q32) and its molecular counterpart the BCL-1 rearrangement are features observed in mantle cell lymphoma (MCL) and less commonly in other B-cell disorders. This rearrangement leads to cyclin D1 overexpression, which may be the main pathogenic event in these tumours and is therefore recognised as a diagnostic marker. We developed a flow cytometry method to detect cyclin D1 overexpression using the monoclonal antibody (MoAb) 5D4, and characterised its frequency in 93 B-cell malignancies. The competitive reverse transcriptase polymerase chain reaction (RT-PCR) for cyclin D1, D2 and D3 was then performed on 40 of these cases to assess the validity of the flow cytometry method. Fluorescence in situ hybridisation (FISH) to detect t(11;14)(q13;q32) was carried out on 31 cases and results were compared with cyclin D1 expression by flow cytometry. Twenty five cases showed cyclin D1 expression using 5D4, including MCL (12/13, 92%), chronic lymphocytic leukaemia (CLL) (4/30), B-prolymphocytic leukaemia (B-PLL) (1/4), splenic lymphoma with villous lymphocytes (SLVL) (4/13), hairy cell leukaemia (HCL) (1/7) and other B-non Hodgkins Lymphoma (B-NHL) (3/15). There was a good correlation between flow cytometry results and RT-PCR in 36/40 cases (90%), and with FISH for t(11;14) in 25/31 cases (80%). We concluded that the detection of cyclin D1 expression by flow cytometry in cell suspensions could be applied routinely to the study of B-lymphoproliferative disorders and may be of value for their diagnosis and management.

Biomarkers, Tumor↗

PubMed1988

Use of allophycocyanin allows quantitative description by flow cytometry of alveolar macrophage surface antigens present in low numbers of cells.

Autofluorescence has limited quantitative description by flow cytometry of certain alveolar macrophage surface antigens. Autofluorescence is most significant when macrophages are excited by blue light and emission is monitored in the green-yellow light range. Because of recent advances in flow cytometry and in the development of fluorescent labeling compounds, such as allophycocyanin, autofluorescence can be minimized by the use of red excitation and emission wavelengths. We have developed a three-step labeling technique that discretely identifies the minor subpopulation of normal rat alveolar macrophages that express Class II major histocompatibility (Ia) antigens. Using this technique, we observed that alveolar macrophage Ia antigen expression increases at least fourfold after intravenous administration of bacillus Calmette-Guerin to previously primed rats. Additionally, we observed that in vitro treatment of alveolar macrophages by exposure to gamma interferon results in an increase in Ia antigen expression. This new method is significant because flow cytometry is a powerful tool with which to analyze quickly, accurately, and reproducibly alveolar macrophage surface antigens.

Animals↗

PubMed1999

Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry.

BACKGROUND: The primary source of interference in immunofluorescence measurements by flow cytometry is background autofluorescence. METHODS: Using human lung fibroblasts (HLFs) as an autofluorescent cell model, unfixed HLFs and HLFs fixed in methanol, ethanol, formaldehyde, paraformaldehyde and glutaraldehyde were analyzed by phase-sensitive flow cytometry to compare their fluorescence intensity and lifetime histograms. Based on these results, a surface antigen on HLFs was labeled with a fluorescein isothiocyanate (FITC) conjugated antibody and fixed in glutaraldehyde, and the cells were analyzed by conventional and phase-resolved methods. RESULTS: The lifetimes of unfixed and ethanol-, methanol-, paraformaldehyde- and formaldehyde-fixed HLFs were in the 1.7-1.9 nanosecond (ns) range, with coefficients of variation 25-35%. Since the autofluorescence lifetime histograms of unfixed and fixed HLFs partially overlapped the 3.5 ns lifetime histogram of FITC-labeled microspheres, which were used to approximate FITC-antibody labeling of HLFs, the ability to resolve FITC-labeled probe, based on differences in the FITC and autofluorescence lifetimes, was severely limited. When HLFs labeled with an FITC-antibody cell-surface marker were fixed in glutaraldehyde (autofluorescence lifetime 0.9-1.4 ns, coefficient of variation approximately 11%) and analyzed by phase-resolved methods, the results showed that FITC-antibody labeling could be readily resolved from background autofluorescence. CONCLUSIONS: Phase-sensitive detection improves the immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells. Cytometry 37: 275-283, 1999. Published 1999 Wiley-Liss, Inc.

Antigens, Surface↗

PubMed1988

Diagnostic value of DNA flow cytometry combined with fine needle aspiration biopsy in lymphomas.

The nuclear DNA content of cells from 45 malignant lymphomas and from 60 benign lymph nodes obtained by fine needle aspiration was analysed to investigate the diagnostic value of DNA flow cytometry combined with routine diagnostic cytology in lymphomas. DNA aneuploidy was found in 43 per cent of lymphomas of high grade malignancy (NCI Working Formulation) but only rarely in lymphomas of intermediate- or low-grade malignancy or in Hodgkin's disease, and never in benign lymph nodes. The median percentage of proliferative cells (S + G2/M) was 22.6 per cent in diploid high-grade lymphomas, 15.3 per cent in intermediate-, and 8.1 per cent in low-grade lymphomas, as compared with 4.9 per cent in benign lymph nodes (P less than 0.0001). If the presence of DNA aneuploidy or more than 12 per cent of proliferative cells is used as a criterion for malignancy, the diagnostic accuracy of DNA flow cytometry in detecting lymphoma is 81 per cent. DNA flow cytometry suggested correct diagnosis in 10 of the 19 false positive, false negative, or indeterminate cytological findings encountered during the study. It is concluded that DNA flow cytometry combined with fine needle aspiration biopsy has diagnostic value in lymphomas, but false negative results are common especially in low-grade lymphomas; the method should therefore be used in conjunction with light microscopy.

Adolescent↗

PubMed1999

Automated reticulocyte counting and immature reticulocyte fraction measurement. Comparison of ABX PENTRA 120 Retic, Sysmex R-2000, flow cytometry, and manual counts.

We evaluated reticulocyte counting and measurement of immature reticulocyte fraction (IRF) with the ABX PENTRA 120 Retic blood analyzer on 300 blood samples. Reticulocyte counts were compared with those obtained by visual counting of 2,000 RBCs, by the TOA (Kobe, Japan) Sysmex R-2000 and a flow cytometry method. The parameters analyzed were the percentages of reticulocytes on all analyzers and the IRF with different modalities. The Retic Count kit (Becton Dickinson, San Jose, CA) was used with the Coulter (Hialech, FL) XL, and a mean channel of fluorescence (MCF) was calculated to fit the reticulocyte maturation. Reticulocyte counting with the ABX (Montpellier, France) PENTRA 120 Retic showed excellent precision and linearity with no significant carryover. Reticulocyte counts were stable after blood storage for 72 hours at 4 degrees C but not at room temperature (RT). IRF parameters values were stable for only 8 hours at 4 degrees C and 6 hours at RT. Comparisons of the methods showed good intraclass correlation (RI) for reticulocyte percentages between ABX PENTRA 120 Retic and Sysmex R-2000, ABX PENTRA 120 Retic and flow cytometry, Sysmex R-2000 and flow cytometry, and ABX PENTRA 120 Retic and manual counting. IRF values were correlated between fluorescence rates and RNA content, but in each case, low RI values were found, showing that Sysmex and ABX IRF values were not concordant. We obtained a significant correlation between mean fluorescence index and the MCF measured by flow cytometry, but the 2 methods were not concordant using the RI. The ABX PENTRA 120 Retic is a good instrument for analyzing reticulocyte count and percentage and allows a good analysis of IRF with several modalities.

Acute Disease↗

PubMedDate not supplied

Past and present concepts in flow cytometry: a European perspective.

The development of flow cytometric instrumentation, methods and research concepts in Europe has been a continuous driving force for the general scientific advancement in this area over the years. This review addresses early European concepts of continuing interest with regard to instrumentation, data analysis, clinical and eperimental DNA analysis, cell function and microbiology at their worldwide first appearence while flow cytometric immunology and immunophenotyping will be covered separately. Flow cytometry represents an efficient approach to the enormous complexity of molecular cell architecture and cell function by the analysis of apparent molecular cell phenotypes in heterogeneous cell samples. The present merger of flow and image cytometry into the method independent cytomics discipline increases the potential of cell analysis very significantly. It opens the way for predictive medicine as well as for predictive cytopathology and predictive cytology in everyday clinical and medical practice. Current progress is driven by joint advances in molecular fluorescence technologies and instrument development. This complements the analysis of genome sequence information in an efficient way.

Apoptosis↗

PubMed1998

Characteristics and dynamics of bacterial populations during postantibiotic effect determined by flow cytometry.

Changes in bacterial ultrastructure after antibiotic exposure and during the postantibiotic effect (PAE) have been demonstrated by electron microscopy (EM). However, EM is qualitative and subject to individual interpretation. In contrast, flow cytometry gives qualitative and quantitative information. The sizes and nucleic acid contents of Escherichia coli and Pseudomonas aeruginosa were studied during antimicrobial exposure as well as during the PAE period by staining the organisms with propidium iodide and analyzing them with flow cytometry and fluorescence microscopy. The effects of ampicillin, ceftriaxone, ciprofloxacin, gentamicin, and rifampin were studied for E. coli, whereas for P. aeruginosa imipenem and ciprofloxacin were investigated. After exposure of E. coli to ampicillin, ceftriaxone, and ciprofloxacin, filamentous organisms were observed by fluorescence microscopy. These changes in morphology were reflected by increased forward light scatter (FSC) and nucleic acid content as measured by flow cytometry. For the beta-lactams the extent of filamentation increased in a dose-dependent manner after drug removal, resulting in formation of distinct subpopulations of bacteria. These changes peaked at 20 to 35 min, and bacteria returned to normal after 90 min after drug removal. In contrast, the subpopulations induced by ciprofloxacin did not return to normal until > 180 min after the end of the classically defined PAE. Rifampin resulted in formation of small organisms with low FSC, whereas no distinctive characteristics were noted after gentamicin exposure. For P. aeruginosa an identifiable subpopulation of large globoid cells and increased nucleic acid content was detected after exposure to imipenem. These changes persisted past the PAE, as defined by viability counting. Swollen organisms with increased FSC were detected after ciprofloxacin exposure, even persisting during bacterial growth. In summary, for beta-lactam antibiotics and ciprofloxacin, the PAE is characterized by dynamic formation of enlarged cell populations of increased nucleic acid content, whereas rifampin induces a decrease in size and nucleic acid content in the organisms. Flow cytometry is an ideal method for future studies of bacterial phenotypic characteristics during the PAE.

Anti-Bacterial Agents↗

PubMed2000

A three-colour flow cytometry technique for measuring trophoblast intracellular antigens: the relative expression of TAP1 in human cytotrophoblast and decidual cells.

Flow cytometry is conventionally used to measure cell-surface antigen expression. However, many antigens are found within the cytoplasm, and it is necessary to fix and permeabilize cells to enable antibodies to gain access to them. In this study we have established the conditions for studying intracellular antigens in human trophoblast cells by flow cytometry using an antibody to TAP1 (a key molecule in the process of Class I MHC assembly). We have previously shown by immunocytochemistry that TAP1 expression is apparently greater on Class 1 positive extravillous cytotrophoblast than on any other fetal or maternal tissue. However, as immunohistochemistry is not quantitative we have used three-colour flow cytometry to measure the expression of TAP1 in different trophoblast populations. Villous and extravillous cytotrophoblast were identified in first trimester and term placental and decidual digests on the basis of their expression of cytokeratin and Class I MHC antigens. The level of expression of TAP1 for each population was investigated using a commercial kit that determines the number of antibody-binding sites per cell. TAP expression was found to be three- to fivefold higher in extravillous cytotrophoblast, confirming our previous findings. The techniques developed here are directly applicable to the measurement of other intracellular molecules in trophoblast, in particular cytokines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Analysis of DNA content in multidrug-resistant cells by image and flow cytometry.

Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells.

Animals↗

PubMed2006

Detection of malignant haematopoietic cells in the cerebrospinal fluid by conventional cytology and flow cytometry.

Morphological evaluation of cerebrospinal fluid (CSF) samples by light microscopy is the method of choice for the detection of central nervous system involvement in patients with haematological malignancies. In this retrospective study, we assessed the value of three-colour immunophenotyping by flow cytometry in addition to conventional cytology in 28 patients with and 17 patients without pre-existent haematological malignancy in whom CSF analysis was performed because of neurological abnormalities (n = 37) or as part of routine staging procedures (n = 8). Four samples could not be analysed by flow cytometry because of insufficient cell numbers. CSF involvement was detected in a total of 18 patients, in 12 by both cytology and flow cytometry, and in three each by either cytology or flow cytometry alone. Discordant results were particularly frequent in patients with low CSF cell numbers and B cell neoplasms. When combined with conventional cytology, flow cytometric analysis improves the detection rate of CSF involvement in patients with haematological malignancies. It is particularly useful in B cell neoplasms with low CSF cell numbers.

Flow Cytometry↗

PubMed1989

Offspring from inseminations with mammalian sperm stained with Hoechst 33342, either with or without flow cytometry.

The technique of vital staining with a fluorochrome and subsequent flow cytometry has been employed to investigate the DNA content of living sperm. There have been several reports of chromosomal damage caused by staining cells with a bis-benzimidazole dye. Hoechst 33342, either with or without subsequent flow cytometry. Therefore the use of this technique might be expected to affect adversely the fertilising capacity of inseminated sperm, or to result in the production of congenital deformities in the young. Insemination experiments to date have resulted in the production of more than 400 offspring in four species, including 5 successive generations of rabbits, where stained or sorted sperm were used. All progeny were observed to be normal by anatomical criteria. There was no evidence to suggest that sperm treated in this manner were unable to fertilise oocytes in vivo or that embryonic development was affected.

Animals↗

PubMed1991

Use of flow cytometry for determination of differential leukocyte counts in bovine blood.

A flow cytometric method was developed to perform differential leukocyte counts on bovine blood. Blood specimens from 50 healthy Holstein cows were analyzed by use of a flow cytometer. The method entailed diluting blood with phosphate-buffered, hypotonic saline solution containing acridine orange, and performing a step-wise, 3-parameter analysis on the bases of cell size, cellular granularity, and granulocyte fluorescence. Initially, proportions of monocytes, granulocytes, and lymphocytes were determined by creating appropriate windows on dot plots of cell size (determined by forward light scatter) vs cellular granularity (determined by the logarithm of side light scatter). Eosinophils were resolved by analysis of granulocytes as dot plots of logarithms of green vs red fluorescence ascribed to acridine orange. Proportions of eosinophils and neutrophils were computed from data so generated. Microclumps of platelets spuriously affected counts of some granulocytes, particularly eosinophils. Differential leukocyte counts determined by flow cytometry generally compared favorably with those obtained by use of the conventional microscopic method, using Wright-stained blood films. Mean neutrophil and eosinophil counts determined by the 2 methods did not differ significantly, but lymphocyte counts determined by flow cytometry were significantly higher than those determined by microscopy (P less than 0.01). Correlation coefficients for counts of neutrophils, eosinophils, and lymphocytes determined by the 2 methods ranged from 0.519 to 0.833. Correlation between monocyte counts was low (r = 0.147), although mean monocyte counts determined by the 2 methods did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

Flow cytometry immunophenotyping and polymerase chain reaction-site-specific primers genotyping for HPA-1 alloantigens in an Italian blood donor population.

BACKGROUND AND OBJECTIVES: There is increasing interest in the development of rapid and reliable techniques for human platelet alloantigen (HPA) typing. This study investigates the reliability of flow cytometry for large-scale immunophenotyping of platelet alloantigens. MATERIALS AND METHODS: We used flow cytometry and polymerase chain reaction-site-specific primer (PCR-SSP) for the characterization of the human platelet antigen 1 (HPA-1) mosaic in blood donors. RESULTS: By using specific alloantisera and immunofluorescence labelling 9 (2.6%) out of 351 samples were HPA-1a-negative. To confirm this antigenic phenotype, all of the latter samples were submitted to PCR-SSP analysis, showing an HPA1-b/b genomic pattern. In HPA-1a-positive donors, flow cytometry was unable to distinguish HPA-1a/b heterozygous from HPA-1a/a homozygous subjects who were clearly identified by genotyping. CONCLUSIONS: Flow cytometry is a valuable tool for large-scale screening to identify HPA-1a-negative persons, whereas genotyping is the assay of choice for zygosity testing, antenatal diagnosis, and for thrombocytopenic alloimmunized patients.

Adult↗

PubMed1997

Quality assurance of stem cell enumeration by flow cytometry. Canadian QASE Study Group.

The enumeration of CD34+ cells by flow cytometry is commonly employed to assess progenitor/stem cell numbers in peripheral blood, cord blood, and apheresis products used for peripheral blood stem cell transplantation (PBSCT). Until recently, the enumeration of CD34+ cells has evolved in the absence of any procedural guidelines or quality assurance programs. We established a voluntary quality assurance for stem cell enumeration (QASE) program for Canadian laboratories involved in PBSCT. The goals of this study were 1) to survey current flow cytometric procedures for CD34 enumeration; 2) to evaluate inter-institutional reproducibility of currently employed assays; and 3) to evaluate the impact of adoption of a common method on inter-laboratory variability. Fourteen to nineteen laboratories participated in this program by enumerating CD34+ cells in shared cord blood samples. The survey showed a wide variation in flow cytometric methodology which in part contributed to the wide range in results obtained between different centres. Major differences in methodology included the number of events counted, use of isotype control and gating strategy. Participation in this program and adoption of a common methodology increased level of agreement for any given result although this trend did not reach statistical significance (P = 0.13). The results of this survey emphasize the need for a quality assurance program for stem cell enumeration by flow cytometry.

Antigens, CD34↗

PubMed1992

Flow cytometry of oesophageal mucosal biopsies; epidermal growth factor receptor, and CD15.

Flow cytometry may be used to examine the properties of single or isolated cells. We have shown that it is possible to disaggregate and label oesophageal epithelial cells for two surface markers, CD15 and epidermal growth factor receptor. We have previously demonstrated these markers in oesophageal squamous cells using immunoperoxidase techniques. These labelled disaggregated cells could then be measured by flow cytometry.

Adult↗

PubMed2005

Quality control in neutrophil granulocyte (PMN) concentrates by flow cytometry.

BACKGROUND: In peripheral blood, chemotaxis, phagocytosis, and oxidative burst of polymorphonuclear cells (PMNs) can be assessed by flow cytometry, whereas function tests, i.e., quality control in PMN concentrates designed for neutropenia therapy, are lacking. METHODS: PMN concentrates (n=6) harvested from healthy donors who had been premedicated with granulocyte colony-stimulating factor (G-CSF) and dexamethasone were stored undiluted (control, C; n=6) and diluted 1:4 (D; n=6) with autologous plasma for 72 h. Commercial flow cytometry function tests were performed to quantify changes in chemotaxis, phagocytosis, and oxidative burst of PMNs over time. RESULTS: Median levels of phagocytosis and oxidative burst levelled at 86% (82-94) and 98% (83-100) in C on the day of apheresis, respectively, but deteriorated to 15% (0-24) and 0% within 72 h; in D these parameters remained close to 90%. Median levels of chemotaxis were comparable in C (69%, 65-74) and D (74%, 70-84) at baseline. No migration was detected in C after 72 h; however, D retained approximately 63% (13-76) migration capacity. CONCLUSION: Quality control in PMN concentrates is practical using flow cytometry and commercial test kits. While phagocytosis and oxidative burst may be maintained for 72 h in vitro, chemotaxis of apheresed PMNs is already reduced on the day of apheresis.

Chemotaxis, Leukocyte↗

PubMed1993

Purification of human muscle satellite cells by flow cytometry.

To purify satellite cells directly from human muscle biopsies, we have developed a method based on size separation of dissociated cells by flow cytometry. Immediately after tryptic dissociation of human muscle biopsies and elimination of erythrocytes, microscopic observation and flow cytometry analysis of cell suspensions revealed two populations of cells differing in size and nucleocytoplasmic ratio. Clonal cultures of these two cell types with a manual procedure demonstrated that only the small cells were myogenic satellite cells. Flow cytometry-sorting and analysis of the small cell population showed that (1) all sorted cells contained desmin immediately after dissociation and plating; (2) more than 98% of the cells expressed the 5.1.H11 epitope after 2 weeks of proliferation in culture; and (3) 90% of the sorted cells were able to form myotubes when cultivated at low density or in clonal cultures. Thus, human muscle satellite cells can be directly purified from human muscle samples using flow cytometry.

Adolescent↗

PubMed2007

Monitoring survival and function of transfused platelets in Bernard-Soulier syndrome by flow cytometry and a cone and plate(let) analyzer (Impact-R).

BACKGROUND: Bernard-Soulier syndrome (BSS) patients may repeatedly require transfusion of platelets (PLTs). The hemostatic competence of transfused PLTs requires monitoring. STUDY DESIGN AND METHODS: Flow cytometry and a cone and plate(let) analyzer (Impact-R, DiaMed) were used to monitor survival and function of transfused PLTs in a 7-year-old girl with BSS undergoing surgery. Flow cytometry was applied to differentiate autologous PLTs from transfused PLTs by staining for CD42b. The Impact, which measures PLT adhesion and aggregation in response to high shear stress, was used to evaluate PLT function. RESULTS: Transfused PLTs were detectable by flow cytometry for 1 week after transfusion. While the patient's PLTs did not respond to high shear stress before transfusion, a normal response was documented by the Impact on the day after transfusion and 1 week thereafter. CONCLUSION: Transfused PLTs were detectable by flow cytometry, and their functional activity was demonstrated by the Impact.

Bernard-Soulier Syndrome↗