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Results for “Flow Cytometry”

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PubMed2001

Outcome of kidney transplants in patients known to be flow cytometry crossmatch positive.

BACKGROUND: The clinical significance of the flow cytometry crossmatch has been addressed in several retrospective studies, but the results have been controversial. There are no prospective studies in which patients known to be antibody positive underwent transplantation. METHODS: The flow cytometry crossmatch was performed prospectively in 1130 renal transplant recipients. A decision to perform transplantation was based on whether the positive results were on T or B cells, in the current or peak specimen, and taking into account the presence or absence of other immunological risk factors. One hundred antibody-positive patients received a transplant. Graft survival and rejection episodes were analyzed in this group and compared with 100 crossmatch-negative patients matched for age, sex, race, and time of transplantation. RESULTS: The incidence of rejection at 1 month was higher in antibody-positive patients (26%) than in antibody-negative patients (12%, P<0.01). Early rejection seemed to be more frequent in antibody-positive patients regardless of whether the antibodies were current or historic, or against T or B cells. There were more steroid-resistant rejections in antibody-positive than in antibody-negative patients. However, biopsy specimens showed that vascular lesions that can be associated with humoral rejection were not more frequent in the antibody-positive patients than in the controls. There were no differences in graft survival between the two groups. CONCLUSIONS: Low-level preformed alloantibodies detected by flow cytometry represent a risk of rejection even for patients purposely selected for having no additional immunological risk factors. The risk seems to be due to donor-specific memory rather than to a direct effect of the antibodies. The results indicate that flow cytometry provides useful information to assess donor-recipient compatibility.

B-Lymphocytes↗

PubMed2002

Rapid detection of aneuploidy in Musa using flow cytometry.

We report a procedure for the rapid and convenient detection of aneuploidy in triploid Musa using DNA flow cytometry. From a population of plants derived from gamma-irradiated shoot tips, plants were selected based on aberrant morphology and their chromosome numbers were counted. Aneuploids plants with chromosome numbers 2n=31 or 32 were found as well as the expected triploid plants (2n=3x=33). At the same time, the nuclear DNA content of all plants was measured using flow cytometry. The flow cytometric assay involved the use of nuclei isolated from chicken red blood cells (CRBC), which served as an internal reference standard. The relative DNA content of individual plants was expressed as a ratio of DNA content of CRBC and Musa (DNA index). In order to estimate the chromosome number using flow cytometry, the relative DNA content of plants with unknown ploidy was expressed as a percentage of the DNA content of triploid plants. The classification based on flow cytometry fully agreed with the results obtained by chromosome counting. The results indicated that flow cytometry is a convenient and rapid method for the detection of aneuploidy in Musa.

Aneuploidy↗

PubMed2000

Flow-cytometry-enhanced fine-needle aspiration biopsy of the spleen.

OBJECTIVE: Flow cytometry is proving useful in the evaluation of lymphoproliferative disorders. In a case series, the authors investigated the safety of cross-sectional fine-needle aspiration biopsy of the spleen under sonographic guidance, and the usefulness of flow cytometry in analysis of biopsy samples. METHODS: Five patients underwent fine-needle biopsy with freehand sonographic guidance. Samples were analyzed on a flow cytometer. RESULTS: Through cytologic examination enhanced by flow cytometry, 2 cases of lymphoma, 1 case of metastatic transitional cell carcinoma, and 1 case of focal splenic hemangioma were diagnosed. Normal lymphocytes were demonstrated in 1 case, in which long-term follow-up of splenomegaly showed that this was related to cirrhosis and portal hypertension in a patient with a history of treated non-Hodgkin's lymphoma. CONCLUSION: Flow-cytometry-enhanced fine-needle aspiration of the spleen is a safe and useful tool for the interventional radiologist. In our institution, it evolved as the result of effective teamwork between diagnostic radiologists and pathologists. Flow cytometry promises to be increasingly useful in the diagnosis and management of lymphoproliferative diseases.

Aged↗

PubMed2000

Leukocyte detection in human semen using flow cytometry.

This study set out to establish a new method, using flow cytometry, to evaluate leukocytes in semen. Ejaculates of 59 males, asymptomatic for genitourinary infections, were examined. Routine semen analyses were carried out as well as peroxidase and polymorphonuclear granulocyte-elastase detection. Leukocytes were detected combining flow cytometry and monoclonal antibodies (anti-CD45, anti-CD53). This technique reliably assessed the total number of leukocytes and differentiated subpopulations even at low concentrations. The peroxidase test and elastase determination showed good specificity, but only moderate sensitivity versus flow cytometry combined with monoclonal antibodies. No significant association was observed between semen parameters and leukocytospermia whether evaluated by conventional methods or flow cytometry except for a moderate correlation between spermatozoa and CD53-positive cell concentrations. A first comparison of data from patients grouped on the basis of leukocytospermia (>10(6) white blood cells, WBC/ml) or non-leukocytospermia revealed no significant differences in semen parameters; lowering the threshold value for leukocytospermia to 2x10(5) WBC/ml, sperm concentration was reduced in the group with a low number of WBC identified by monoclonal antibodies. Flow cytometry using monoclonal antibodies was seen to be a simple, reproducible method that enables leukocytes in semen to be accurately detected and to identify WBC subpopulations without preliminary purification procedures.

Adult↗

PubMed2003

The potential role of flow cytometry in the diagnosis of small cell carcinoma.

CONTEXT: Virtually no information exists in the medical literature on the immunophenotyping of small cell carcinoma by flow cytometry. CD56, or neural cell adhesion molecule, is widely expressed by small cell carcinoma and easily measured by flow cytometry. OBJECTIVE: To determine the potential usefulness of flow cytometry in the diagnosis of small cell carcinoma. DESIGN AND SETTING: Retrospective data and archival material on 27 patients were obtained from community hospitals. Specimens (needle aspirations and tissue biopsies) from all patients demonstrated cytomorphologic and flow cytometric features consistent with small cell carcinoma. All measurements were performed at a large reference laboratory. Routine 3- and 4-color flow cytometry using a lymphoma antibody panel, including anti-CD56, was performed. Anti-cytokeratin antibody was also used in the last 12 cases. Immunohistochemical staining with a panel of conventional markers for neuroendocrine neoplasms was performed on available tissue for purposes of confirmation of small cell carcinoma. PATIENTS: Twenty-seven patients whose tissue specimens showed a clearly defined population of CD45-CD56+ cells by flow cytometry and cytomorphologic features consistent with small cell carcinoma. INTERVENTIONS: Needle aspiration (n = 3) and tissue biopsy (n = 24) from a variety of sites. RESULTS: CD56 positivity by flow cytometry was 100 to 1000 times that of the matched isotype control in 25 cases and 10 to 100 times that of the control in 2 cases. Cytokeratin positivity by flow cytometry was found in 12 of 12 cases. Immunohistochemical staining showed positivity for at least 1 cytokeratin and 1 or more neuroendocrine markers in 26 of 27 cases and confirmed the diagnosis of small cell carcinoma. CONCLUSIONS: Routine flow cytometry can identify a neuroendocrine phenotype that shows a strong correlation with confirmatory immunohistochemical markers in cases exhibiting cytomorphologic features of small cell carcinoma. Flow cytometry appears to complement and may possibly be a satisfactory alternative to immunohistochemical staining when small cell carcinoma is suspected.

Biomarkers, Tumor↗

PubMed2003

Fine-needle aspiration biopsy and flow cytometry immunophenotyping of lymphoid and myeloproliferative disorders of the spleen.

BACKGROUND: Flow cytometry (FC) is a useful adjunct to fine-needle aspiration biopsy (FNAB) in the evaluation of lymphoproliferative disorders. The application of FC to FNAB of the spleen (sFNAB) is reported. METHODS: Flow cytometry was performed on 18 sFNAB collected over 3 years. The series comprised 10 cases of non- Hodgkin lymphomas (NHL), 2 cases insufficient for diagnosis, 2 cases of reactive hyperplasia (RH), and 4 cases of myeloid metaplasia (MM). FNAB was performed under ultrasound guidance using a 22-gauge needle. One or two passes were sufficient to prepare a conventional smear that was immediately evaluated to select the cases studied and to prepare a cell suspension for FC. The following fluoresceinated antibodies were used: CD3, CD19/kappa/lambda, FMC7/CD23/CD19, Bcl-2, and CD13/HLA-DR. In six cases, cytospins were also prepared for immunocytochemistry and were tested for CD20 (L26), CD45Ro, and kappa and lambda light chain expression. RESULTS: Flow cytometry contributed to the diagnosis of all cases of NHL by assessing light chain restriction. The specific subtype was also diagnosed by CD19/CD5 and CD 19/CD10 coexpression in two cases. Flow cytometry quantified the percentage of myeloid cells in MM cases and contributed to the cytologic diagnosis showing a polyclonal light chain expression in RH cases. Immunocytochemistry was effective and concordant in four cases. Patients tolerated the sFNAB well and no complications were reported. Cytologic and FC diagnoses were confirmed by follow-up and by histologic evaluation in cases in which splenectomy was performed for therapeutic purposes. CONCLUSION: Flow cytometry applied to sFNAB corroborates the cytologic diagnosis in lymphoid and myeloproliferative disorders of the spleen and allows therapeutic decisions avoiding splenectomy.

Adult↗

PubMed1997

Detection of antisperm antibodies in seminal plasma by flow cytometry: comparison with the indirect immunobead binding test.

OBJECTIVE: To compare flow cytometry with the established indirect immunobead binding test (IBT) for the detection of antisperm antibodies in seminal plasma. DESIGN: A prospective, comparative study. SETTING: University-based andrology unit. PATIENT(S): One hundred and fifty-eight men with suspected male factor subfertility. INTERVENTION(S): Seminal plasma samples were incubated with antisperm antibody-negative donor sperm. Surface-bound antibody was detected with fluorescence-labeled antihuman antibody in the flow cytometry assay or with immunobead-labeled antihuman antibody in the IBT. MAIN OUTCOME MEASURE(S): The percentage of sperm that tested positive for surface-bound antibody was determined in the two assays. Seminal plasma was antisperm antibody-positive when > or = 20% of the sperm were antibody-bound, and clinically significant levels were present when > or = 50% of the sperm were antibody-bound. RESULT(S): Of 71 samples that were negative by the IMT, 66 (93%) also were negative by flow cytometry. Of 63 samples that had > or = 50% immunobead binding, 55 had equivalent results by flow cytometry. Overall statistical analysis showed a good correlation between the two assays. CONCLUSION(S): There is a good correlation between the indirect IBT and indirect flow cytometry for the detection of antisperm antibodies in seminal plasma.

Autoantibodies↗

PubMed2001

Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture.

Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods.

Animals↗

PubMed1990

Flow cytometry: illuminating microbiology.

By means of flow cytometry, a technique whereby a hydrodynamically directed stream of cells is passed through a focus of exciting light, one can measure cell size and the macromolecular content of individual bacteria. The sensitivity and versatility of the flow cytometer make it a powerful tool in studies of the bacterial cell cycle, in identifying and characterizing bacterial infections, and in selecting bacteria of desired qualities. We review some of these applications of flow cytometry and conclude that this method holds great promise in many other areas of microbiology.

Bacteria↗

PubMed2001

Flow cytometry: an 'old' tool for novel applications in medical genetics.

Flow cytometry was originally established as an automated method for measuring optical or fluorescence characteristics of cells or particles in suspension. In the meantime, flow cytometers have become user-friendlier, less expensive instruments with an increasing importance in clinical diagnostics. Besides the classical fields of application, such as immunophenotyping blood cells or analyzing the cell cycle status by measuring the DNA content, novel flow cytometric methods have been developed to identify and to quantify disease-related gene sequences. Here we give an overview of current and future applications, including the detection of viral sequences via microsphere-based PCR assays and the analysis of single nucleotide polymorphisms, reflecting individual phenotypic traits. Furthermore, flow cytometry allows the quantification of gene expression changes as well as the isolation of differentially expressed gene sequences. Flow cytometry is also convenient for multiplex analyses, e.g. when hybridizing DNA samples to a mixture of various microsphere populations each coated with different DNA probes. Last but not least, the use of magnetic beads in combination with flow cytometers coupled with automated devices enables molecular diagnostics on a large scale. Overall, this review demonstrates flow cytometry as a rapid, sensitive, and reproducible tool applicable to a wide range of medical genetic approaches.

DNA↗

PubMed2001

Using light scatter signal to estimate bacterial biovolume by flow cytometry.

BACKGROUND: In the past decade, flow cytometry has become a useful and precise alternative to microscopic bacterial cell counts in aquatic samples. However, little evidence of its usefulness for the evaluation of bacterial biovolumes has emerged in from the literature. METHODS: The light scattering and cell volume of starved bacterial strains and natural bacterial communities from the Black Sea were measured by flow cytometry and epifluorescence microscopy, respectively, in order to establish a relationship between light scattering and cell volume. RESULTS: With the arc-lamp flow cytometer, forward angle light scatter (FALS) was related to cell size in both the starved strains and natural communities, although regression parameters differed. We tested the predictive capacity of the FALS verous cell size relationship in a bacterial community from the North Sea. That analysis showed that a reliable bacterial biovolume prediction of a natural bacterial community can be obtained from FALS using a model generated from natural bacterial community data. CONCLUSIONS: Bacterial biovolume is likely to be related to FALS measurements. It is possible to establish a generally applicable model derived from natural bacterial assemblages for flow cytometric estimation of bacterial biovolumes by light scatter.

Aeromonas hydrophila↗

PubMed2006

In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow.

Flow cytometry is a well-established, powerful technique for studying cells in artificial flow in vitro. This review covers a new potential application of this technique for studying normal and abnormal cells in their native condition in blood or lymph flow in vivo. Specifically, the capabilities of the label-free photothermal (PT) technique for detecting and imaging cells in the microvessel network of rat mesentery are analyzed from the point of view of overcoming the problems of flow cytometry in vivo. These problems include, among others, the influences of light scattering and absorption in vessel walls and surrounding tissues, instability of cell velocity, and cells numbers and positions in a vessel's cross-section. The potential applications of this new approach in cell biochemistry and medicine are discussed, including molecular imaging; studying the metabolism and pathogenesis of many diseases at a cellular level; and monitoring and quantifying metastatic and apoptotic cells, and/or their responses to therapeutic interventions (e.g., drug or radiation), in natural biological environments.

Animals↗

PubMed1991

Dynamics of human neutrophil aggregation evaluated by flow cytometry.

In order to directly monitor neutrophil aggregation, we have developed a simple particle counting technique using flow cytometry. Flow times were used to determine aggregation from changes in the total number of particles per unit volume (%PA(T)), while fluorescent signals emitted from glutaraldehyde-fixed neutrophils were used to measure changes in the total number of singlet neutrophils (%PA(S)). Flow cytometrically-determined %PA values were found to be virtually identical to values determined from microscopy. We show that aggregation parameters can be evaluated and compared simply from measures of changes in total particle count without a need for distinguishing singlets from multiplets. A number of aggregation parameters are introduced and related to the initial cell concentration (N(o)) and stir speed (shear). Aggregation of neutrophils, following stimulation with 0.5 microM FMLP (N-formyl-methionyl-leucyl-phenylalanine), showed a latent time (tl) of 4 +/- 1.5 sec independent of N(o) or stir speed; had a forward rate of aggregation (vf) which was proportional to the initial cell concentration and the stir speed; plateaued for greater than or equal to 60 s; and showed partial to complete reversal by 7.5 min. Above a critical stir speed, the extent and duration of aggregation varied inversely with the stir speed. The stir and N(o) dependence of the aggregation parameters studied suggest the existence of subpopulations of neutrophils with distinct efficiencies of aggregation.

Blood Cell Count↗

PubMedDate not supplied

Cytochemical assessments of the nuclear DNA distribution pattern by means of image and flow cytometry in thyroid neoplasms and in non-neoplastic thyroid lesions. A comparative methodological study.

The two most prevalent techniques for cytochemical DNA assessment of the nuclear DNA distribution pattern in neoplastic cells are image cytometry (ICM) and flow cytometry (FCM). The aim of the present study was to compare the results of nuclear DNA assessments, obtained by means of these two methods, in fresh surgical biopsy specimens from the thyroid gland, both in neoplasms and in nonneoplastic lesions. Material for DNA analysis was taken preoperatively by fine needle aspiration (FNA) biopsy from 13 papillary thyroid carcinomas and analyzed by the two methods. Surgical specimens were taken from 48 papillary thyroid carcinomas (one of which showed low differentiated papillary and anaplastic giant cell formations at autopsy), 2 follicular carcinomas, 66 follicular adenomas, 7 medullary carcinomas as well as from the nodules of 17 non-toxic colloid goitres and 19 specimens from the diffusely hyperplastic thyroid parenchyma in patients with hyperthyroidism. For the ICM assessments, FNA biopsies or imprints were made from the macroscopically identified fresh thyroid biopsy specimens; for the FCM assessments FNA specimens from the same region were used. In 155 out of the 159 specimens the results obtained by means of the two methods were the same. The DNA distribution pattern in 106 of the neoplasms and in all the 36 non-neoplastic lesions were of the "euploid" type (i.e. "diploid" or diploid/tetraploid"), whereas that of 17 neoplasms were of the "aneuploid" type. Fifteen of the histopathologically benign follicular adenomas showed a cytochemical DNA distribution pattern that by means of FCM was of the "aneuploid" type.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1996

DNA ploidy analysis by laser scanning cytometry (LSC) in colorectal cancers and comparison with flow cytometry.

We evaluated laser scanning cytometry (LSC) by comparing nuclear DNA ploidy determined by LSC and by flow cytometry (FCM) in 77 samples of human colorectal cancer from 48 patients. Both methods revealed an aneuploid peak in 30 (62.5%) of the cases, although two samples that were aneuploid by LSC were diploid by FCM and two others were diploid by LSC and aneuploid by FCM. The concordance rate for nuclear DNA ploidy was 91.7% in the 48 patients and 87.0% for the 77 samples. The DNA index was also highly correlated between two methods (r2 = 0.97, P < 0.001). We concluded that LSC provides DNA histograms equivalent to FCM for surgical specimens and has potential clinical application in pathology.

Aneuploidy↗

PubMed1991

Applications of flow cytometry to artificial insemination: a review.

Flow cytometry is a technique in which sub-populations of cells can be analysed and separated according to the staining pattern seen with various fluorescent markers. This review describes some of the ways in which flow cytometry can be applied to the investigation of sperm populations, either as a means of quality control of semen or to examine the characteristics of different sub-populations of sperm within an ejaculate. These methods can replace or augment existing subjective assessments of semen characteristics. Using this technique it is possible to produce aliquots of sexed sperm for insemination or for in vitro fertilisation. An objective assessment can be made of the effects of environmental stress on male physiology by monitoring changes in semen quality.

Animals↗

PubMedDate not supplied

[Flow cytometry, basic principles and applications--with special reference to pediatrics].

Flow cytometry in clinical applications and research is the result of knowledge and techniques concerning cyto and fluorochemistry, laser technology, monoclonal antibodies and computer processing. Flow cytometry can assess multiple physical and biological cell properties, in a relatively short period of time. It is specially important, that specimens can concern different types of material: fresh solid tissue, embedded in paraffin, blood, bone marrow, body fluids. Under optimal conditions (standarization of method and quality control) precise and objective analysis of a large number of cells is provided. An experienced team and cooperation with clinicians and pathologists are indispensable for correct clinical interpretation of flow cytometry data. The aim of this study mainly concerns: general principles of flow cytometry operation, clinical applications--especially in paediatrics. It is also an encouragement to use this valuable and modern instrument in clinical and research work.

Adolescent↗

PubMed1989

Quantitation of fetal-maternal hemorrhage by flow cytometry. A simple and accurate method.

A simple and objective assay was developed for the detection and quantitation of fetal-maternal hemorrhage with the use of flow cytometry. In vitro prepared control mixtures of 10%, 2%, 1%, 0.5%, 0.25%, 0.125%, and 0.06% D+ RBCs in D- RBCs were tested (8-11) different times by flow cytometry and gave mean % D+ results of 11.10%, 1.90%, 0.92%, 0.45%, 0.24%, 0.11%, and 0.05%. The coefficient of variation of preparing and testing these mixtures ranged from 11.0 to 15.9% for the 10-0.125% mixtures. Thus, flow cytometry was accurate, reproducible, and sensitive. Flow cytometry was compared with Du tests, rosette tests, and acid elution. The Du test was highly variable because it was not sensitive enough to detect a significant bleed (approximately 0.6%) in some cases and too sensitive (necessitating quantitation of an insignificant bleed) in others. The rosette test was too sensitive. Acid elution and flow cytometry results did not always agree; acid elution results were approximately twice as high as flow cytometry. The authors believe flow cytometric detection of D+ red blood cells to be more accurate than the detection of fetal hemoglobin by acid elution techniques, which is known to have poor reproducibility. Postpartum samples from 56 D- women who delivered D+ babies were tested. Fifty-two had fetal bleeds less than 0.3% by acid elution and flow cytometry; all had negative Du test results, but there were two false positive results with the use of the rosette technique. Four had significant bleeds (greater than or equal to 0.6%); in all four cases the flow cytometry results were lower than the acid elution results. The authors were able to quantitate a bleed of fetal RBCs, which were D+ only by the Du test, in a D- mother with the use of flow cytometry, and D+ RBCs in a mother whose RBCs were of the rare DVI mosaic phenotype. This would not have been possible with the use of the standard Du or rosette techniques.

Erythrocyte Count↗