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PubMed2003

Serodiagnosis of human plague by a combination of immunomagnetic separation and flow cytometry.

BACKGROUND: Plague is a severe, highly communicable bacterial disease caused by Yersinia pestis. It is still endemic in more than 20 countries worldwide. Although known as a devastating disease for centuries, laboratory confirmation of clinical suspected cases is still problematic. No standardized and internationally approved test system is commercially available. The aim of this study was the introduction and evaluation of a combination of immunomagnetic separation and flow cytometry for the serodiagnosis of human plague. METHODS: Paramagnetic polystyrene beads were coated with purified F1 capsular antigen (F1 CA) and reacted with sera from plague patients, from 26 laboratory personnel vaccinated against plague and from 102 healthy blood donors (HBD). After incubation with fluorescein isothiocyanate-conjugated anti-human rabbit IgG, particle-associated fluorescence was detected by flow cytometry. RESULTS: Anti-F1 CA antibodies could be demonstrated in all patients with bacteriologically confirmed plague and in 22 sera (84.6%) from vaccinees. Only one serum in the HBD group showed a weakly positive reaction. The total assay time was less than 2 h. CONCLUSIONS: Compared with a recently published combination of an anti-F1 CA enzyme-linked immunosorbent assay (ELISA) and immunoblot, the new assay showed the same sensitivity as the ELISA and almost the same specificity (99.0 versus 100%) as the immunoblot. Allowing a rapid, reliable, and quantitative analysis, immunomagnetic separation combined with flow cytometry might replace both conventional immunoassays.

Antibodies↗

PubMed1993

Detection of two early gene products of Epstein-Barr virus by fluorescence flow cytometry.

The intracellular expression of two early gene products of Epstein-Barr virus (EBV) was evaluated by fluorescence flow cytometry. Two Burkitt's lymphoma cell lines were superinfected by EBV from the P3HR-1 cell line. Twelve to 96 hr after superinfection the cells were fixed with paraformaldehyde and made permeable by saponin treatment. Monoclonal antibodies to the diffuse (D) and restricted (R) components of the early antigen (EA) complex were applied to the cells followed by fluorochrome-conjugated goat antibodies specific for the heavy chain isotypes of the monoclonal antibodies. Fluorescence flow cytometry revealed a clear separation in fluorescence intensity between cells containing EA-R or EA-D and negative cells. A major proportion of EA-positive cells displayed both antigens. In addition, a significant fraction expressed either EA-R or EA-D but not both. Expression of EA occurred more rapidly and peaked earlier in Daudi cells than in Raji cells. This apparent difference in EA expression between the two cell lines, however, was much less pronounced when one considered the proportion of cells expressing EA-R only, EA-D only, or both EA-R and EA-D, as a percentage of the total EA expression. Parallel fluorescence microscopy experiments revealed that the variation of the ratio of EA-R to EA-D expression with time correlated well between the two methods.

Antibodies, Monoclonal↗

PubMed1990

Single-cell entrapment and microcolony development within uniform microspheres amenable to flow cytometry.

A method is presented for encapsulating single microbial cells in small spheres suitable for analysis and sorting by flow cytometry. The entrapped cells are able to multiply and form colonies contained within their respective microspheres. The system is based on ejecting the cells suspended in a gellable liquid through an orifice vibrating at ultrasonic frequencies, thus shearing the cell-containing jet into uniform droplets. When low-melting-temperature agarose was used, the droplets could be gelled into solid spheres during flight by appropriately directed colling air streams. This gelling was accompanied by significant dehydration, resulting in a twofold decrease in bead diameter and a corresponding increase in agarose concentration. Nevertheless, the microbeads obtained were highly uniform and had diameters which could be precisely controlled in the range of 10 to 40 microns. A variety of bacterial and yeast species were entrapped in agarose beads by using this system. In all cases the cells were able to develop into microcolonies containing as many as several hundred cells. This system enables one to apply the powerful method of flow cytometry to the analysis and sorting of whole microbial colonies. Potential applications of this technology in various areas of microbiology are considered.

Bacteria↗

PubMed2006

Detecting fetomaternal hemorrhage by flow cytometry.

PURPOSE OF REVIEW: The aim of this review is to summarize the most recent developments in the area of detection of fetomaternal hemorrhage by flow cytometry. RECENT FINDINGS: Maternal red blood cell chimerism is readily detectable by flow cytometry. Fetal and maternal red blood cells differ in their content of fetal hemoglobin (alpha2gamma2). Fetal red blood cells contain fetal hemoglobin, and normal maternal red blood cells contain some percentage of fetal hemoglobin in a background of normal adult hemoglobin. All blood group systems with allelic differences between mother and fetus are readily applicable for detection of fetomaternal hemorrhage by fetal hemoglobin. SUMMARY: Fetal hemoglobin for detection of fetomaternal hemorrhage is an accurate clinical diagnostic procedure for investigation of anemia in fetus and newborn.

Chimera↗

PubMed1985

The use of monoclonal antibodies and flow cytometry to detect peripheral blood and bone marrow involvement of a diffuse, poorly differentiated lymphoma.

Using monoclonal antibodies and flow cytometry, we were able to characterize the phenotype of a diffuse, poorly differentiated lymphoma and to isolate subpopulations of cells from the blood and bone marrow that expressed the malignant phenotype even though the patient exhibited no absolute lymphocytosis. Because the circulating clone reacted with the anti-T cell monoclonal antibody T101, we initiated serotherapy with T101 as part of a phase I study. A 10 mg infusion of T101 resulted in the rapid clearance of normal T cells from circulation, but the clone showed evidence of modulation and was not cleared. Twenty-four hours following infusion, all cell populations had returned to pre-treatment levels. Our study suggests that, by using monoclonal antibodies and flow cytometry, blood and bone marrow involvement of a lymphoma can be demonstrated in patients without absolute lymphocytosis, a finding which may influence the staging and treatment of the disease.

Antibodies, Monoclonal↗

PubMed1984

Estimation of the frequency of malformed sperm by slit scan flow cytometry.

We have investigated the utility of Slit Scan Flow Cytometry (SSFCM) for measuring the frequencies of malformed sperm heads in control and mutagen treated B6C3F1/CRL mice. In SSFCM, fluorescence profiles of sperm heads stained with the DNA-specific fluorescent dye acriflavine were recorded for sperm flowing lengthwise through a 2.5-microns-thick laser beam. Malformed sperm were detected as having fluorescence profiles that differed substantially from an average fluorescence profile for sperm from untreated mice. Specifically, a sum of squared difference (SSD) value was calculated for the fluorescence profile of each sperm according to the equation (Formula: see text) where c(i) and t(i) are the ith values for the fluorescence profiles from control and test sperm, respectively. Profiles whose SSD exceeded a threshold value of 20 were considered to be from malformed sperm. We measured fluorescence profiles for 500 sperm per mouse from five control mice, five mice injected intraperitoneally daily for 5 days with a total of 375 mg/kg of body weight methyl methane sulfonate (MMS), and for 30 mice injected intraperitoneally daily for 5 days with total doses of procarbazine ranging from 125 mg/kg to 1,250 mg/kg. Sperm were collected from the caudae epididymides 35 days after the last injection. Frequencies of malformed sperm in these samples were also estimated by visual analysis. All samples were analyzed in double blind fashion. The visual and SSFCM malformed sperm frequencies for the samples from control, MMS-treated, and procarbazine-treated mice were correlated (r = 0.83). A dose effect was seen with both the visual and SSFCM estimates for the sperm from the procarbazine-treated mice.

Animals↗

PubMed1986

Flow cytometry of uveal melanomas.

Tumor cell kinetic parameters were determined for 36 uveal melanomas retained in fixed tissue sections using flow cytometric techniques and computerized morphometry. By flow cytometry the majority of cells comprising the 36 tumors were in the G0/G1 phase (55.7%). The DNA index was 1.40 +/- 0.57 units. Using Spearman's rank order correlation test, the correlation between DNA index and the inverse standard deviation of nucleolar area was found to be statistically significant. The potential usefulness of flow cytometry in predicting tumors of high malignant potential is discussed.

Cell Cycle↗

PubMed1999

An augmentation of Fas (CD95/APO-1) antigen induced by radiation: flow cytometry analysis of lymphoma and leukemia cell lines.

An augmentation of Fas antigen induced by radiation was examined using flow cytometry. Six cell lines established from lymphomas or leukemias (HD-70, FLAM-76, CML-C-1, CML-C-2, DL-40 and DL-95) were used in this study. Each cell line was distributed to two dishes. The cells in one dish were irradiated at 10 Gy with cobalt-60 gamma rays. The control cells were not irradiated. At 6 h, 24 h, and 48 h after treatment, irradiated and non-irradiated cells of each cell line were stained with fluorescein isocyanate (FITC)-conjugated anti-human Fas antibody, and analyzed with flow cytometry. Mean fluorescence intensity (MFI) values of irradiated or non-irradiated cells were examined. MFI rates (MFI value of irradiated cells/MFI value of non-irradiated cells) of each cell line were calculated at each investigated time point, and an augmentation of the Fas antigen expression with radiation was evaluated. FLAM-76 did not express Fas antigen at any time. An augmented expression of Fas antigen due to irradiation was observed in the other five cell lines. These findings strongly suggest that radiation can augment Fas antigen expression in certain tumor cells. Further studies using Fas ligand or specific anti-Fas antibody are needed.

Antibodies↗

PubMed1986

[Study of the DNA content of 72 cases of bladder tumors using flow cytometry. Relation with anatomoclinical data].

The DNA content of 72 bladder tumors and 20 healthy bladders was studied with flow cytometry. Reproducibility of DNA measurement was systematically verified with the use of 3 controls: calf thymocytes, mouse hepatocytes and a mixture of human lymphocytes with trout red blood cells. For each tumor or normal bladder, a first sample containing urothelial cells only and a second sample with the addition of trout red blood cells were systematically examined. In the first group of patients with healthy bladders the percentage of urothelial cells with a DNA content greater than G0 G1 (2N) was on average 7.9% +/- 2.8%. Among the second group of 72 tumors, 2 DNA profiles were remarkable: in a first group (39/72; 55%) DNA profile was unimodal (single diploid peak); in a second group (33/72; 45%) DNA profile was bimodal (association of a diploid peak with an aneuploid one). Comparison of flow data with pathology data (WHO classification) clearly demonstrated that the presence of an aneuploid peak, characterizes grade elevation and infiltration. Hence, 1/16 (6%) of grade I; 7/75 (28%) of grade II; 25/31 (81%) of grade III tumors show an aneuploid peak, as well as 11/44 (25%) of Ta; 9/14 (64%) of T1; 5/6 (83%) of T2, and 8/8 (100%) of T3 tumors. Demonstration of an aneuploid peak with flow cytometry particularly in the group of patients having non infiltrating Ta tumors (11/44 in our series) could further either lead to a closer follow-up of this high risk group of patients or to the early recommendation of a non conservative treatment.

DNA, Neoplasm↗

PubMed2000

Quantitative flow cytometry for the differential diagnosis of leukemic B-cell chronic lymphoproliferative disorders.

We have investigated whether the quantitative flow cytometry is an useful tool to better characterize B-cell chronic lymphoproliferative disorders (CLDs). Peripheral blood samples from 104 patients with leukemic B-cell disorders and 20 healthy donors were analyzed. Directly phycoerythrin-conjugated CD19, CD20, CD22, CD23, CD79b, and CD5 monoclonal antibodies (MoAbs) and QuantiBRITE pre-calibrated beads were used to calculate the number of antigen molecules per cell, expressed as antibody binding capacity (ABC). As compared to normal controls, in chronic lymphocytic leukemias (CLL) all MoAbs tested, with the exception of CD23 and CD5, showed lower ABC levels. In prolymphocytic leukemias (PL), CD5 and CD23 antigens were constantly absent while lower CD19 and CD22 ABC levels were observed. Hairy cell leukemias (HCL) displayed very high levels of CD20 and CD22. Of interest, splenic lymphomas with villous lymphocytes (SLVL) could be discriminated from HCL for higher CD79b and lower CD19 ABC values. Finally, higher CD20 levels were detected in follicular lymphomas (FL), whereas higher CD79b and CD5 levels characterized mantle cell lymphomas (MCL). Seven out of 61 CLL cases were defined as morphologically atypical. When compared with typical forms, lower levels of CD19 and CD23 and higher CD20 and CD22 ABC values were detected. However, we failed to demonstrate quantitative differences between atypical CLL and MCL. Our results suggest that quantitative flow cytometry may be a useful additional tool to better identify some types of B-cell CLDs.

Adolescent↗

PubMedDate not supplied

A new sensitive and specific combination of CD81/CD56/CD45 monoclonal antibodies for detecting circulating neuroblastoma cells in peripheral blood using flow cytometry.

PURPOSE: Intensive chemoradiotherapy followed by peripheral blood stem cell transplantation has been introduced to treat children with advanced neuroblastoma (NBL). Detection of NBL cells in peripheral blood (PB) is important to prevent reinfusion of NBL cells. Several immunologic methods have been proposed for detecting NBL cells in hematologic samples. The development of a sensitive and specific combination of monoclonal antibodies (MoAbs) for detecting small numbers of NBL cells in PB using flow cytometry remains an important challenge. METHODS: Twenty-one clinical samples from NBL tissues or smears containing NBL cells were examined for reactivity against CD81, CD56, and CD9 using an immunocytochemical technique. The expressions of CD81, CD56, CD9, and antihuman disialoganglioside GD2 MoAb (GD2) in five NBL cell lines were assayed by flow cytometry. For the evaluation of sensitivity, five NBL cell lines were added to normal PB and the detection level of the combination of CD81/CD56/CD45 MoAbs was compared with that of CD9/CD56/CD45 MoAbs (reported previously). One hundred thirty-three normal PB samples were examined to determine the sensitivity and specificity of this method. RESULTS: All NBL cell lines showed strong positivity with CD81 and CD56 MoAb. However, CD9 MoAb was weakly positive against the five NBL cell lines. GD2 MoAb reacted strongly with four NBL cell lines, although almost the entire cell population of the SK-N-SH NBL line failed to bind the GD2 MoAb. In vitro experiments using NBL cell lines demonstrated that tumor cells added to normal PB cells could be detected by flow cytometry using CD81/CD56/CD45 MoAbs even at a concentration of 0.005%. Through comparative studies, the combination of CD81/CD56/CD45 MoAbs was found to be more sensitive and specific than that of CD9/CD56/CD45 MoAbs for detecting small numbers of NBL cells using the above cell lines. CONCLUSIONS: Triple-color flow cytometric analysis using CD81/CD56/CD45 MoAbs is useful for detecting NBL cells in PB. Further studies testing this approach using samples of PB with NBL contamination are needed to test this approach in patients.

Adolescent↗

PubMed1995

Flow cytometry in node-positive breast cancer: cancer and leukemia group B protocol 8869.

This report describes a companion flow cytometry study (Cancer and Leukemia Group B (CALGB)--8869) using tumors derived from patients enrolled in a large randomized clinical trial (CALGB-8541) performed on 1,572 patients with early stage, node-positive breast cancer. The CALGB initiated an adjuvant breast cancer trial in 1985 to determine if dose intensification (dose of drug per unit time) of chemotherapy was related to relapse-free and overall survival. Patients were randomized by pretreatment clinical variables to one of three different dosage regimens of chemotherapy. Using a tumor enrichment procedure, 442 paraffin-embedded blocks were analyzed by flow cytometry, and S-phase fraction (SPF) was analyzed by three different methods. Ploidy analysis was performed using standard procedures. Tissue from 90% of the patients was suitable for ploidy analysis, whereas only 68% could be assessed for SPF. With a median follow-up time of 80 months, our results show that ploidy status had no clinical utility, whereas high SPF predicted poorer overall survival. The rectangular fit model for SPF was more predictive of outcome than both the area fit model and a computer fit model (modfit) for SPF. In univariate analysis, patients with a low SPF (< 10%) had a better prognosis than those patients with a high SPF (> 10%), but they responded equally well to the different treatment regimens. Patients with high SPF (> 10%) had longer relapse-free and overall survival to high dose chemotherapy compared to low or standard dose chemotherapy. Multivariate analysis indicated that treatment intensity as well as the number of positive nodes, tumor size, steroid receptor status, and c-erb B-2 expression were significant in predicting overall and disease-free survival. The multivariate analysis, however, revealed that SPF was significant in predicting overall but not disease-free survival, but there was no longer any relationship among SPF, dose intensity, and outcome.

Breast Neoplasms↗

PubMed1993

The significance of a positive flow cytometry crossmatch test in primary kidney transplantation.

This study was conducted to determine the efficacy of the T cell flow cytometry crossmatch (T-FCXM) test in 841 first cadaver donor transplants. Results showed one-year graft survival rates were 82% for T-FCXM-negative patients, compared with 75% for T-FCXM-positive patients (P = 0.01). Early one-month graft failure was 13 percentage points higher in those with a positive T-FCXM than those with a negative T-FCXM. The positive crossmatch patients also had more frequent immunological failures. A positive T-FCXM was found in 39% of the sensitized patients (PRA > 10%) and 8% of those who had not been sensitized. Patients with a positive T-FCXM in either category had a 74% graft survival rate. Thus, most of the T-FCXM-positive results occurred in patients with complement-fixing antibodies. It is suggested that flow cytometry crossmatching (FCXM) be used prospectively, despite the fact that many patients with a positive crossmatch did have successful transplants (TXs). In the current climate of a cadaver kidney scarcity and large recipient waiting pools, utilization of kidneys for patients with the highest probability of success seems a most prudent policy.

Antibodies↗

PubMed2007

Assessing immunophenotyping performance: proficiency-validation for adopting improved flow cytometry methods.

BACKGROUND: The continuous improvement and evolution of immune cell phenotyping requires periodic upgrading of laboratory methods and technology. Flow cytometry laboratories that are participating in research protocols sponsored by the NIAID are required to perform "switch" studies to validate performance before methods for T-cell subset analysis can be changed. METHODS: Switch studies were conducted among the four flow cytometry laboratories of the Multicenter AIDS Cohort Study (MACS), comparing a 2-color, lyse-wash method and a newer, 3-color, lyse no-wash method. Two of the laboratories twice failed to satisfy the criteria for acceptable differences from the previous method. Rather than repeating more switch studies, these laboratories were allowed to adopt the 3-color, lyse no-wash method. To evaluate the impact of the switch to the new method at these two sites, their results with the new method were evaluated within the context of all laboratories participating in the NIH-NIAID-Division of AIDS Immunology Quality Assurance (IQA) proficiency-testing program. RESULTS: Laboratory performance at these two sites substantially improved relative to the IQA standard test results. Variation across the four MACS sites and across replicate samples was also reduced. CONCLUSIONS: Although switch studies are the conventional method for assessing comparability of laboratory methods, two alternatives to the requirement of repeating failed switch studies should be considered: (1) test the new method and assess performance on the proficiency testing reference panel, and (2) prior to adoption of the new methods, use both the old and the new method on the reference panel samples and demonstrate that performance with the new method is better according to standard statistical procedures. These alternatives may help some laboratories' transition to a new and superior methodology more quickly than if they are required to attempt multiple, serial switch studies.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Assessment of interlaboratory variability in analytical cytology. Results of the College of American Pathologists Flow Cytometry Study.

In 1986, the College of American Pathologists introduced an interlaboratory proficiency testing program for clinical laboratories that perform flow cytometry. Specimens consisting of fresh human lymphocytes isolated from blood or tissue or neoplastic cells grown in culture were mailed to participants by overnight mail. Results of analyses for cell surface antigens on lymphocytes and for cellular DNA content were reported to the College of American Pathologists Computer Center and the summary data were mailed to participants. The results obtained to date indicate no reagent- or instrument-related biases for measurements of cell surface antigens or for cellular DNA content. Interlaboratory variability in enumerating lymphocytes of a given subset varies inversely with the percentage of such cells in each specimen. In our experience, it is feasible and practical to conduct an interlaboratory proficiency survey in clinical flow cytometry.

Antigens, Surface↗

PubMed2003

Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry.

Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to gfp was constructed, resulting in a luxR-PluxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.

4-Butyrolactone↗

PubMed2003

The use of flow cytometry to detect expression of subunits encoded by 11 Salmonella enterica serotype Typhimurium fimbrial operons.

The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome contains 13 putative fimbrial operons termed agf (csg), fim, pef, lpf, bcf, saf, stb, stc, std, stf, sth, sti and stj. Evidence for in vitro expression of fimbrial proteins encoded by these operons is currently only available for agf, fim and pef. We raised antisera against putative major fimbrial subunits of S. Typhimurium, including AgfA, FimA, PefA, LpfA, BcfA, StbA, StcA, StdA, StfA, SthA and StiA. Elaboration of StcA on the bacterial surface could be detected by flow cytometry and immunoelectron microscopy after expression of the cloned stcABCD operon from a heterologous T7 promoter in Escherichia coli. To study the expression of fimbrial antigens in S. Typhimurium by flow cytometry, we constructed strains carrying deletions of agfAB, pefBACDI, lpfABCDE, bcfABCDEFG, stbABCD, stcABC, stdAB, stfACDEFG, sthABCDE or stiABCDE. Using these deletion mutants for gating, expression of fimbrial antigens was measured by flow cytometry in cultures grown in vitro or in samples recovered 8 h after infection of bovine ligated ileal loops with S. Typhimurium. FimA was the only fimbrial antigen expressed by S. Typhimurium after static growth in Luria-Bertani (LB) broth. Injection of static LB broth cultures of S. Typhimurium into bovine ligated ileal loops resulted in the expression of BcfA, FimA, LpfA, PefA, StbA, StcA, StdA, StfA and StiA. These data show that in vivo growth conditions drastically alter the repertoire of fimbrial antigens expressed in S. Typhimurium.

Animals↗

PubMed2005

Quantification of CD59- mutants in human-hamster hybrid (AL) cells by flow cytometry.

Mutation assay is an important approach in evaluating the genotoxic risk of potentially harmful environmental chemicals. The human-hamster hybrid (A(L)) cell mutagenesis system, based on the complement/antibody-mediated cytotoxicity principle, has been used successfully to evaluate the mutagenic potential of a variety of environmental toxicants. The A(L) cells contain a standard set of CHO chromosomes and a single human chromosome 11, which expresses several cell surface proteins including CD59 encoded by the CD59 gene at 11p13.5. A modified mutation assay by flow cytometry was developed to determine the yield of CD59- mutants after either radiation or chemical treatment. After incubation with phycoerythrin-conjugated mouse monoclonal anti-CD59 antibody, the CD59- mutant yields were determined by quantifying the fluorescence of the cells using flow cytometry. This method is faster and eliminates the commonly encountered toxicity problems of the complements with the traditional complement/antibody assay. By comparing the mutant fractions of radiation or chemically treated A(L) cultures using the two methods, we show here that the flow cytometry assay is an excellent substitute in providing an efficient and highly sensitive method in mutant detection for the traditional complement/antibody assay.

Alkylating Agents↗