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PubMed1996

Cell cycle analysis of normal, atrophic, and hyperplastic breast epithelium using two-color multiparametric flow cytometry.

We performed two-color flow cytometric synthesis phase fraction (SPF) determinations on cytokeratin-labeled benign epithelial populations from 142 breast specimens (41 mastectomy, 70 diagnostic biopsy, 31 reduction mammoplasty). There was wide variability of SPF, ranging from 0.1 to 3.5%, with a frequency distribution skewed to higher values (mean 0.75%, median 0.5%). The mean SPE for women less than 29 years was 0.91%, vs. 0.89% for 30-42 years, 0.66% for 43-49 years, and 0.56% for > or = 50 years (P = 0.05). Histologically atrophic tissue samples exhibited a mean SPF approximately half that of morphologically normal tissue from premenopausal age women (0.79% vs. 0.36%, P = 0.02). Tissues showing histologically proliferative fibrocystic features had a greater mean SPF than non-proliferative fibrocystic tissues (0.59% vs. 0.92%); however, due to the wide spread of values within each of these categories, this difference was not statistically significant and neither group was significantly different from 'normal' tissue samples. Patients with histologically normal breast tissue, though, were significantly younger (mean = 34.6 years) than those with fibrocystic changes (non-proliferative mean = 53.4 years vs. proliferative mean = 42.8 years, P = 0.005). Synchronous right- and left-sided specimens obtained from reduction mammoplasty demonstrated significantly correlated SPF determinations (R = 0.77). We conclude that selective analysis of epithelial populations using two-color flow cytometry provides cell cycle information in benign breast tissue which is analogous to that obtained by labor-intensive nucleotide labeling studies. This study also confirms the biologic variability and age-dependence of breast epithelial proliferation. Finally, the data imply that derangements of cell proliferation in fibrocystic conditions are heterogeneous, complex and incompletely correlated with histologic parameters such as hyperplasia.

Adult↗

PubMed1988

The flow cytometry of melanocytic skin lesions.

DNA flow cytometry was performed on formalin fixed, paraffin embedded melanocytic naevi. DNA aneuploidy was detected in all three types of naevus but was significantly more frequent in those naevi accepted as precursors of malignancy: that is, dysplastic and congenital pigmented hairy naevi. It may be that the presence of DNA aneuploidy has prognostic significance in these naevi. Technical problems were encountered in the analysis of data from melanocytic lesions so that caution is recommended in interpretation of studies using formalin fixed tissue.

Aneuploidy↗

PubMed1999

Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.

Bacteria↗

PubMed1986

Flow cytometry: methodology and applications in pathology.

Flow cytometry offers pathologists a powerful new method which can be applied especially for diagnosis, prediction of prognosis and measurement of response to therapy of tumours. It also has a wide variety of other applications in research and its use for investigation of non-neoplastic disease has only just begun. The object of this brief review is to stimulate interest in this technique and to outline some of the most interesting applications that may be of use to pathologists in the near future.

Cell Cycle↗

PubMed1988

Detection of platelet-associated immunoglobulin in immune thrombocytopenia by flow cytometry.

The development of a flow-cytometry (FCM) technique for the direct detection of antiplatelet globulins bound to the surface of platelets (platelet-associated immunoglobulins, or PAIg) is described and utilized to evaluate patients with idiopathic thrombocytopenic purpura (ITP). Elevated levels of platelet-bound globulin were detected by FCM in the ITP patients studied. When plasma was analyzed by FCM for the presence of antiplatelet antibodies, only one patient demonstrated elevated levels. The use of FCM in the direct analysis of PAIg may result in earlier diagnosis and better management of immune thrombocytopenias.

Blood Platelets↗

PubMed1985

DNA flow cytometry in human testicular cancer.

DNA flow cytometry revealed aneuploid tumour stemlines in 19 of 20 primary testicular cancers without significant difference of the ploidy values between seminomas and non-seminomas. In 7 of 8 analyzable histograms the S-phase activity was 22-51%. A metastatic mature teratoma had 6% cells in S-phase. These results support the clinical observation that testicular cancer is usually a rapidly growing human tumour. The high percentage of aneuploidy in testicular cancer may be of clinical value in the diagnosis of this malignancy.

DNA, Neoplasm↗

PubMed1983

[Intraclonal and interclonal differences of ploidy in transplantable rat rhabdomyosarcoma RA-2 detected using DNA flow cytometry].

DNA contents were determined using flow cytometry in cells of 71 clones of the rat transplantable rhabdomyosarcoma passed through more than 70 cycles of selection for increased malignancy. 58 clones (82%) consisted of cells with diploid DNA content, whereas 13 clones (18%) displayed not only diploid, but also near tetraploid cell subpopulations, the latter covering from 20 to 60% of the total number of cells measured. The data obtained show the existence of intra- and interclonal karyotypic heterogeneity in populations of tumor clonogenic cells.

Animals↗

PubMed2000

Sequential use of paraformaldehyde and methanol as optimal conditions for the direct quantification of ZEBRA and rta antigens by flow cytometry.

A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixation-permeabilization procedures were compared. The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies, were found to be the optimal conditions in these cell lines. Our procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.

Animals↗

PubMed1993

A stochastic model for cell debris in flow cytometry.

An important problem in analyzing flow cytometry DNA measurements, especially for tumor samples, is the presence of background noise, mainly resulting from cellular debris. Several models have been proposed for subtracting this contribution from DNA content histograms. In the present paper we propose a model for background debris distribution based on a specific mechanism for the DNA fragmentation process of the cell nucleus. In particular, we suggest that the fragmentation is more likely to occur in the sites which are closer to the end points of the DNA chain. This accounts for the observed higher frequency of small-sized fragments in experimental DNA distribution. The proposed model depends on two parameters describing the intensity and the shape of the background. It has been successfully validated against four series of experimental data of DNA distribution with increasing level of background.

Cell Fractionation↗

PubMed2000

The role of automated urine particle flow cytometry in clinical practice.

Urine particle flow cytometers (UFC) have improved count precision and accuracy compared to visual microscopy and offer significant labor saving. The absence of an internationally recognized reference measurement procedure, however, is a serious drawback to their validation. Chamber counting by phase contrast microscopy of supravitally-stained uncentrifuged urine is considered the best candidate for reference. The UF-100 (Sysmex Corporation, Japan) identifies RBC, WBC, squamous epithelial cells, transitional epithelial and renal tubular cells (SRC), bacteria, hyaline and inclusional casts, yeast-like cells, crystals and spermatozoa, using argon laser flow cytometry. Evaluations have established acceptable linearity over useful working ranges, with an imprecision that is consistently and significantly less than microscopy, and with negligible carry-over. Comparisons of UFC with chamber counts, quantitative urine microscopy, sediment counts, test strips, bacterial culture and urine density are reviewed. Clinical studies include diagnosis and monitoring of urinary tract infection; localization of the sites of hematuria; and diagnosis, monitoring and exclusion of renal disease. The most popular approach is to combine test strips with UFC for primary screening either always by both methods or by using test strips for analytes unrelated to particles analyzed by UFC. Expert systems now exist combining both test modalities based on user definable decision rules. The implementation of such a strategy significantly reduces microscopy review and saves time and expense without diminishing clinical utility.

Bacteriuria↗

PubMed1998

Development of a rapid and sensitive quantitative assay for rotavirus based on flow cytometry.

A very sensitive and accurate flow cytometry (FC) based method have developed to quantitate rotavirus infection in MA104 cells. Confluent cell monolayers were infected with serial dilutions of rotavirus SA11. After infection, the cells were recovered with the aid of trypsin and then reacted with monoclonal antibody M60 (specific for the rotavirus outer capsid protein, VP7), followed by a second antibody (anti-mouse IgG-FITC). A FACScan FC was used to estimate the number of infected cells, as well as the level of infection. Viral infection was optimised by varying the concentration of trypsin used in the maintenance medium. The FC method enables many cells to be screened quickly for infectivity, and can detect low levels of virus. This method can be adapted to monitor the presence of other viruses in clinical and environmental samples without the need for prolonged periods of adaptation to growth in tissue culture.

Animals↗

PubMed2004

Use of an automated hematology analyzer and flow cytometry to assess bone marrow cellularity and differential cell count.

The automated analysis of bone marrow aspirates was performed to estimate the bone marrow cellularity and differential cell count. Total nucleated cell count (TNC) was measured using an automated hematology analyzer. TNC of the bone marrow correlated well with the bone marrow cellularity estimated by microscopic examination (r = 0.590, p <0.001). The bone marrow cellularity was readily confirmed as <30%, from 30 to 70%, or >70% according to TNC. Differential count of bone marrow cells was done with the combination of CD45 monoclonal antibody and propidium iodide using flow cytometry. Excellent correlations were obtained for the population distributions of normoblasts, eosinophils, lymphocytes, and blasts between the results of flow cytometry and manual differential counts. The percentage of mature granulocytes in flow cytometry showed good correlation with the manual percentage of metamyelocytes + neutrophils. The percentage of immature granulocytes by flow cytometry showed good correlation with the manual percentage of blasts + promyelocytes + myelocytes. These results demonstrate that analysis of bone marrow aspirates using an automated hematology analyzer is valuable in the determination of bone marrow cellularity. Moreover, as flow cytometry provides objective findings for percentages of major cell populations, it can serve as a method to automate bone marrow differentials.

Antigens, CD↗

PubMed2001

Value of flow cytometry in the evaluation of head and neck fine-needle lymphoid aspirates: a 3-year retrospective review of a community-based practice.

OBJECTIVE: To study the clinical efficacy of flow cytometry in patients suspected of having a lymphoma with head and neck pathology. STUDY DESIGN: Retrospective review of 49 patients with lymphoid aspirates having concurrent flow cytometry studies. SETTING: Private practice office-based study. CONCLUSION: Fine-needle aspiration with concurrent flow cytometry is a useful, convenient adjunct to the diagnosis of head and neck non-Hodgkins lymphomas. SIGNIFICANCE: Flow cytometry is a significant improvement over fine-needle aspiration derived cytologic diagnosis alone. Tissue open biopsy may be avoided altogether in some patients with non-Hodgkins lymphoma.

Adolescent↗

PubMed2006

9-color and 10-color flow cytometry in the clinical laboratory.

CONTEXT: The development of commercial flow cytometers capable of detecting more than 10 simultaneous fluorescent signals presents opportunities for improved diagnosis and monitoring of patients with leukemia and lymphoma. OBJECTIVE: To describe instrument and reagent characteristics necessary for successful 9-color and 10-color flow cytometry in a clinical setting. DESIGN: Systematic review of issues related to instrument settings, reagent performance, and general principles of panel construction. RESULTS: Nine-color and 10-color flow cytometry offers the possibility for increased accuracy in population identification, the ability to obtain detailed information from paucicellular specimens, improved laboratory efficiency, and the means to consistently detect abnormal populations at low levels. Careful attention to details of instrument and reagent performance allows for the development of panels suitable for screening of samples for leukemia and lymphoma in a clinical setting. CONCLUSIONS: The characteristics of this technique are particularly well suited to the analysis of leukemia and lymphoma and have the potential to revolutionize and standardize this type of analysis in the clinical laboratory.

Cell Separation↗

PubMed1990

Measurement of alloantibody by flow cytometry.

A new method based on flow cytometry has been developed to determine IgG alloantibodies in the serum of immunized rodents. The method utilizes target lymphoid cells, diluted serum and labeled anti-mouse or anti-rat IgG antibodies. In serum from highly immunized animals alloantibodies could be demonstrated up to a dilution of (3 x 10(4))-1 which makes the test approximately 300 times more sensitive than a simultaneously performed complement-dependent cytotoxicity assay (CDCA). Moreover a linear relationship between the amount of alloantibody and the log value of the mean fluorescence of the target cells was found. This linearity permits the comparison of alloantibody production between individual samples by comparing the mean fluorescence values. The reproducibility of the assay was excellent since the coefficients of variation for all dilutions were less than 15%. By using two-color fluorescence the method can discriminate alloantibodies directed against class I and class II MHC antigens. Finally, in addition to its high sensitivity and good reproducibility, the method was found to be at least twice as fast as CDCA.

Animals↗

PubMed1994

Ploidy pattern and cell cycle in breast cancer as detected by image analysis and flow cytometry.

Both image analysis (IA) and flow cytometry (FCM) may be applied to detect ploidy pattern and cell cycle fractions. However, they have different performance characteristics and may yield different results. The two approaches are applied in this study to 66 breast cancers: IA on imprints and FCM on fresh tissue. The percent coefficient of variation (CV) ranged from 2.0 to 7.0 (mean 5.5; SD 1.1) in IA and from 2.0 to 7.0 (mean 4.4; SD 1.1) in FCM. The values were well correlated. With regard to ploidy pattern, the agreement between the two methods was 92.4%; disagreements were due to four cases being aneuploid by IA but not detected by FCM and one case being aneuploid by IA but tetraploid by FCM. This suggests that IA is capable of detecting aneuploidy with more sensitivity than FCM. In diploid cases, the percent values of cells in G0/G1, S-phase (SPF), and G2M phase were concordant and well correlated. In aneuploid cases, IA was more sensitive than FCM in detecting aneuploid fraction as well as G2M phase, whereas FCM was more sensitive than IA in detecting SPF. A good correlation was found between the DNA indexes (DIs) obtained with the two methods.

Aneuploidy↗

PubMed2001

Application of CD45/SSC gating multiparameter flow cytometry in the classification of acute leukemia--an analysis of 139 cases.

In order to study the significance of flow cytometry immunophenotyping in the diagnosis of acute leukemia, CD45/SSC gating multiparameter flow cytometry (FCM) was utilized to analyze the immunophenotypes of 139 cases of acute leukemia. 139 cases of acute leukemia were enrolled in our hospital from April 1998 to April 2000. Morphological analysis and FCM immunophenotypic tests were conducted on all cases. Our results showed that CD45/SSC gating multiparameter flow cytometry immunophenotyping could reflect the origin of leukemic cells specifically. It is one of the important methods for the diagnosis of ALL, AML, and HAL. CD45/SSC gating multiparameter FCM analysis is a good technique for immunophenotyping. FCM immunophenotypic analysis can help improve the diagnosis and classification of acute leukemia, and extend the use of FCM in clinical practice.

Adolescent↗

PubMed1999

DNA quantitation of distal bile duct carcinoma measured by image and flow cytometry.

OBJECTIVE: To evaluate discrepancies between flow cytometry (FCM) and image cytometry (ICM), ploidy incidence and relation between DNA ploidies and survival in distal bile duct carcinomas (DBDCs). STUDY DESIGN: Forty-four archival tumor samples from patients with DBDC who underwent subtotal pancreatoduodenectomy from 1985 to 1996 were examined for DNA ploidy using FCM and ICM. RESULTS: Overall, 59% (26/44) of the tumors were aneuploid by at least one of the two techniques. We detected more cases of aneuploidy with ICM than FCM in formalin-fixed, paraffin-embedded DBDCs, 62% (21/34) versus 33% (13/40), respectively. When results could be compared, moderate strength of agreement (kappa = .45) was demonstrated. No correlation was found between DNA ploidy by FCM, ICM or combined FCM-ICM and survival time (P = .80, P = .35, and P = .54, respectively). CONCLUSION: Approximately 59% of DNA histograms contained aneuploid cell populations. Although ICM, as compared to FCM, is more sensitive in assessing the ploidy status of DBDC, both methods were complementary. Most discrepancies between FCM and ICM were due to the dilution of aneuploid populations by non-neoplastic diploid cells. DNA ploidy assessment in DBDC did not offer the possibility of improving the ability to predict survival.

Adult↗