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Results for “Flow Cytometry”

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PubMed1996

Genotypic analysis of primary head and neck squamous carcinoma by combined fluorescence in situ hybridization and DNA flow cytometry.

Concurrent DNA flow cytometric (FCM) and fluorescence in situ hybridization (FISH) analyses were prospectively performed on 24 primary untreated head and neck squamous carcinomas for characterization of the genotypic and phenotypic DNA aberrations of these neoplasms. Eleven tumors (42.0%) manifested DNA diploidy (DI = 1.00) and 15 (58.0%) had DNA aneuploidy (DI < or > 1.00) by FCM. Fluorescence in situ hybridization results showed aneusomy in the majority of DNA diploid and in all DNA aneuploid tumors. The extent of the abnormalities for individual chromosomes and the number of involved chromosomes in a given DNA diploid or aneuploid tumor were significantly different. Overall, a statistical correlation between the FCM DNA index (DI) and the magnitude of the chromosomal aberration by FISH was found. Our results also show a significant association between the DI and histologic differentiation and stage of disease in these neoplasms. In conclusion, (1) chromosomal aneusomy characterizes most DNA diploid (DI = 1.00) and all DNA aneuploid (DI < or > 1.00) head and neck squamous carcinomas; (2) polysomy is the most prevalent finding; (3) loss of the Y chromosome in tumors from male patients is a consistent feature; (4) the FCM DI reflects net chromosomal gains or losses in these neoplasms; and (5) DNA aneuploidy is associated with tumor aggressiveness.

Adult↗

PubMed1991

An improved method for the detection of soluble interleukin 2 receptors in liver transplant recipients by flow cytometry.

A new flow cytometric method (FCM) for detection of soluble interleukin 2 receptor (sIL-2R) in serum was established. Using this method, results from one to forty samples can be obtained in less than 2 hr as compared with 5 hr in an ELISA. Comparing over 300 sera samples tested by both methods, we found the FCM to be as specific and sensitive as the ELISA, but even more reproducible. Regression analysis of ELISAt on FCMt showed there is a strong relationship between the two assays and they mimic each other over time. We studied the daily serum levels of sIL-2R by FCM in 18 liver allograft recipients: 7 were in the stable group, 6 in the rejection group, and 5 had both rejection and infection. Low or decreasing sIL-2R levels correlated with absence of rejection. In patients with rejection episodes, a typical elevation of sIL-2R was followed by a rapid decrease of sIL-2R after successful response to rejection therapy. Patients with multiple complications displayed elevated sIL-2R levels. In conclusion, because of its rapid turnover time and accuracy, the FCM has the potential to determine the appropriate timing of post-transplant liver biopsies.

Antibodies, Monoclonal↗

PubMed1994

Prognostic significance of flow cytometry deoxyribonucleic acid analysis of human astrocytomas.

Flow cytometry was used to determine the deoxyribonucleic acid ploidy and proliferative activity of 230 astrocytomas. The relationships among survival, ploidy, proliferation, histological features, and clinical variables were analyzed. Multivariate analysis confirmed the independent prognostic significance of the S-phase fraction (P < 0.01), ploidy (P = 0.04), age at diagnosis (P < 0.001), extent of surgery (P < 0.01), and sex (P = 0.03). Three groups with significantly different survival were defined based on S-phase fraction ranges of < 3%, 3%-5.9%, and > or = 6%. The strong correlation between the S-phase fraction and survival confirmed the importance of quantitative proliferation assays in predicting tumor behavior and demonstrated that specific reference ranges can be defined for clinical application. The weaker association between ploidy and survival leaves the usefulness of the determination of ploidy with flow cytometry in doubt.

Adolescent↗

PubMed2000

[Possibilities and uses of flow cytometry in the framework of clinical diagnostics in dogs and cats].

Flow cytometry has become a routine method in human medicine in the last years. Important applications in clinical diagnostics are measurement of DNA-content, cell cycle analysis, immunophenotyping of lymphocytes and cell function measurements. Flow cytometry is at present rarely applied in veterinary medicine. This article reviews basic principles of flow cytometry and demonstrates possibilities of flow cytometric analysis in oncology and diagnostics of anaemia and immunodeficiency in dogs and cats.

Animals↗

PubMed1992

Evaluation of in vivo breast fine needle aspirates by flow cytometry: an efficacy study.

BACKGROUND: Malignancy of the breast is frequently diagnosed through fine needle aspiration. In the hands of a skilled aspirator and cytopathologist, this can be a highly accurate procedure. PURPOSE: This study was undertaken to evaluate whether sufficient residual cells in the bore of the needle could be harvested and analyzed efficiently by flow cytometry analysis. The goal was then to determine the value of routine flow cytometry as an adjunctive technology in the interpretation of breast fine needle aspirations. METHODS: Cells were rinsed from the needles of 83 consecutive diagnostic fine needle aspirates after preliminary inspection had confirmed adequate material was obtained for cytopathology. Cells were washed, and nuclei prepared by detergent treatment. After ribonuclease treatment, DNA was stained with the fluorescent marker propidium iodide. DNA content per cell was determined by flow cytometry by measurement of right-angle fluorescence. RESULTS: Less than 4% of the samples were rejected for inadequate cell numbers. Flow cytometry criteria for evidence of malignancy included the presence of a DNA aneuploid population or an elevated rate of proliferation (13% or higher) of a diploid population. Accuracy of flow cytometry was based on cytopathologic interpretation in all cases except two which were based on results of excisional biopsy. The sensitivity of the flow cytometry analysis was 76%; the specificity was 100%, with results from flow cytometry pivotal in the correct diagnoses for two patients whose cytopathologic results were equivocal. Analysis of histograms indicated acceptable coefficients of variation for all populations. Gating analysis indicated the suitability of the material for this type of study, with an average of 85% of the events selected, or "gated in." Low recoveries were associated with the presence of necrotic debris in the sample. CONCLUSION: Flow cytometry can be a valuable adjunctive technology, capable of providing the cytopathologist with additional information regarding the character of cells analyzed.

Biopsy, Needle↗

PubMed1998

Image cytometry of breast carcinomas that are DNA diploid by flow cytometry: time to revise the concept of DNA diploidy?

OBJECTIVE: To investigate whether breast carcinomas found to be DNA diploid by flow cytometry (FCM) are still diploid if reassessed by image cytometry (ICM). STUDY DESIGN: In a series of 286 breast cancers analyzed by FCM there were 100 (35%) cancers that were classified as DNA diploid. Fourteen of the 100 diploid cases were selected for further analysis with ICM because the patient had died of breast cancer within 11-84 months after the diagnosis (a group with unfavorable outcomes), and 19 cases were selected at random from the cases who had no recurrence of cancer during follow-up of six or more years (a favorable group). RESULTS: Eleven (33%) of the 33 cases turned out to be DNA nondiploid, with a DNA index > or = 1.2 when analyzed by ICM. Nine of the 11 DNA aneuploid samples by ICM were found among the 14 patients with unfavorable prognoses and only 2 among the 19 patients with favorable outcomes (P = .002). The five-year survival rate of the women with DNA diploid cancer by both methods was 86%, whereas that of patients with DNA aneuploid cancer by ICM was 36% (P = .002). CONCLUSION: The results show that some breast carcinomas classified as DNA diploid based on FCM are not DNA diploid by ICM and that such carcinomas are associated with poorer outcomes than the ones that are DNA diploid also by ICM. The prognostic significance of DNA ploidy in breast cancer may need to be reexamined in studies where both FCM and ICM are used.

Breast Neoplasms↗

PubMed1992

Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems.

Flow cytometry was used to count bacterial cells from diverse origins: one strain of E. coli, one sample of lake water, and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results of this study showed that flow cytometry was a reliable technique for counting a mixture of bacteria in samples from aquatic ecosystems.

Escherichia coli↗

PubMed1995

Flow cytometry related to hematopoietic malignancy.

Flow cytometry is used in the workup of hematological malignancies to establish the immunophenotype of a given malignancy and/or to determine DNA ploidy and S-phase fractions. Flow cytometric data is used as an adjunct to routine morphology and can have diagnostic, confirmatory and prognostic relevance. Samples that can be analyzed include blood, bone marrow, lymph nodes, fine needle aspirates and effusions. DNA analysis can be performed not only on fresh and frozen tissue but also on archival material.

DNA, Neoplasm↗

PubMed1996

Flow cytometry analysis of pituitary adenomas.

UNLABELLED: Using flow cytometry, DNA content and index, and/or proliferative capacity (measuring proliferating cell nuclear antigen PCNA) in operated pituitary tumors, control pituitaries obtained at necropsy, and experimental pituitary hyperplasia induced in rats were analyzed. Simultaneous measurement of cell ploidy and proliferation differentiated normal pituitary (diploid DNA index and negative PCNA) from pituitary hyperplasia (diploid DNA index with intensely positive PCNA, between 30 and 72% of cells). In the tumors 83% (19/ 23) were positive for PCNA (between 3 and 84%) and 73% (17/23) aneuploid; only 1 tumor was diploid and negative for PCNA. CONCLUSIONS: Differentiation between normal and abnormal (neoplastic or hyperplastic) pituitary is possible by flow cytometry, but in the adenomas no correlation with postoperative clinical outcome was observed.

Adenoma↗

PubMed2001

Cytochemical staining and flow cytometry methods applied to the diagnosis of acute leukemia in the pediatric population: an assessment of relative usefulness.

BACKGROUND: Cytochemical staining has been used in the diagnosis of acute leukemia for more than 20 years. The general availability of flow cytometers and an extensive panel of antibody reagents useful for characterizing blood cell lineage question the usefulness of continuing routine use of the cytochemical staining for the diagnosis of acute leukemia. PATIENTS AND METHODS: Test results were evaluated in 122 (n = 122; 112 with acute lymphocytic leukemia and 10 with acute myeloid leukemia) patients selected from among 320 patients with acute leukemia at Texas Children's Hospital in 1997 and 1998. Results were selected for review if the clinical encounter represented the initial diagnostic work-up and if data were available from cytochemical staining and flow cytometry studies. RESULTS: Cell lineage classification derived from flow cytometry and cytochemical stains were in agreement in all cases. Definitive diagnoses were feasible using flow cytometry results alone in 120 of 122 patients (98.4%) as compared with only 99 of 122 patients (81.2%) when only cytochemical staining results were considered. In two patients with inconclusive flow cytometry results, cytochemical staining alone provided information sufficient for diagnosis. CONCLUSIONS: Results from this study indicate that with few exceptions, flow cytometry studies alone provide sufficient information for diagnosis and management of acute leukemia in children. Nevertheless, cytochemical staining should be available for those cases in which flow cytometry results fail to allow a definitive diagnosis. A modified testing protocol is recommended.

Acute Disease↗

PubMed1999

A limited antibody panel can distinguish B-precursor acute lymphoblastic leukemia from normal B precursors with four color flow cytometry: implications for residual disease detection.

Multiparameter flow cytometry may be used to detect minimal residual disease in acute leukemia because leukemic cells often display aberrant phenotypes when compared to normal cells. One limitation of this approach in B-precursor ALL is that leukemic phenotypes are often qualitatively similar to normal marrow B progenitors, though it has long been recognized that the latter show a predictable pattern of antigen expression with differentiation. In this study we used four-color flow cytometry to define precisely the patterns of normal antigen expression on a series of normal bone marrows using two different four-color combinations of antibodies: CD19-APC/CD45-perCP/CD20-PE/CD10-FITC; and CD19-APC/CD45-perCP/CD9-PE/CD34-FITC. A series of dual parameter displays were created in which normal B precursors occupied predictable regions. We then tested these antibody combinations on a series of 82 cases of B-precursor ALL and found that in 76/82 cases (93%) the first combination demonstrated an abnormal population on at least one of the dual parameter displays, and that 72/77 cases tested (94%) showed an abnormality with the second combination. When taken together, 81/82 cases (99%) showed an abnormality. When purified blasts were serially diluted into normal marrows we found a sensitivity of detection of 1 cell in 10(4) normal marrow cells provided sufficient CD19+ cells were acquired to visualize the abnormal population as a discrete cluster. Because the pattern of antigen expression in normals is very reproducible, it is possible to create a fixed set of geometrical regions to define the normal; this makes analysis of an unknown sample very straightforward. We conclude that our approach could be employed as a simple method for the detection of minimal residual disease in B-precursor ALL, and unlike many other methods should prove applicable to virtually all cases of this malignancy.

Antibodies, Monoclonal↗

PubMed1994

The value of quantitative DNA flow cytometry of testicular fine-needle aspirates in assessment of spermatogenesis: a study of 137 previously maldescended human testes.

In order to assess the suitability of DNA flow cytometry of fine-needle aspirates for quantifying spermatogenesis, the results from DNA flow cytometry were compared to histological evaluation of testicular biopsies taken concomitantly from 171 previously maldescended testes. In 137 of 171 cases, sufficient material for flow cytometric as well as histological evaluation was obtained. Histological analysis of surgical biopsy specimens revealed spermatogenesis including the spermatid stage in 117 of the 137 gonads. In six of the 117 gonads no haploid cells were found using flow cytometry. On the other hand, surgical biopsies failed to reveal spermatogenesis in five cases in which the corresponding aspirates contained haploid cells. Both methods therefore seem equally sensitive in detection of spermatogenesis. Other types of histological patterns also corresponded to distinct DNA histograms. Thus, in 11 of 12 cases with Sertoli-cell-only pattern in all tubules, at least 95% of the cells had a diploid DNA content. Furthermore, predominance of tubules with maturation arrest at the primary spermatocyte level corresponded to an increased proportion of tetraploid cells. When compared to surgical biopsy, DNA flow cytometry of testicular fine-needle aspirates is a more objective, easy and rapid method, which is more convenient for the patient. This study has indicated that DNA flow cytometry is a suitable method of quantitative assessment of spermatogenesis. One of the first target groups might be men with azoospermia. In such men, DNA flow cytometric analysis of fine-needle aspirates and surgical biopsy are apparently of equal sensitivity in detecting gonads with spermatogenesis. We conclude that DNA flow cytometry may become an alternative method for the quantification of spermatogenesis.

Adult↗

PubMed1999

The use of flow cytometry in the diagnosis and monitoring of malignant hematological disorders.

Flow cytometry is a modality with ever increasing application in modern hematological practice. This is due to the rapidity of obtaining results, ease of use and increasing power to detect abnormal populations of cells. The major uses of flow cytometry in malignant hematology are in the diagnosis, classification and monitoring of diseases such as leukemia, lymphoma and myeloma. The technique is now used also to detect disease-specific populations of cells in paroxysmal nocturnal hemoglobinuria. This review describes the use of flow cytometry in many disease states.

Cell Separation↗

PubMed2004

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Adult↗

PubMed1994

Prognostic significance of flow cytometry deoxyribonucleic acid analysis of human oligodendrogliomas.

Flow cytometry was used to determine the deoxyribonucleic acid ploidy and proliferative activity of 60 oligodendrogliomas and oligoastrocytomas. The relationships among survival, ploidy, proliferation, histological features, and clinical variables were analyzed. Survival was strongly associated with the S-phase fraction (P < 0.001). Three groups with significantly different survival rates were defined, based on S-phase fraction ranges of < 3%, 3 to 5.9%, and > 6%. Significant associations between survival and age at diagnosis (P < 0.001), tumor grade (P < 0.001), and extent of surgery (P < 0.01) were found also. The poor correlation between mitotic figures and survival in oligodendrogliomas that has been reported previously emphasizes the need for an accurate method to measure proliferative activity. Our study demonstrated the usefulness of the flow cytometry-determined S-phase fraction in this regard and demonstrated that specific reference ranges could be defined for clinical application. In contrast, the determination of ploidy by flow cytometry was not useful in the evaluation of oligodendrogliomas.

Adolescent↗

PubMed2004

Immunophenotypic analysis of hematogones (B-lymphocyte precursors) and neoplastic lymphoblasts by 4-color flow cytometry.

Hematogones are identified by 4-color flow cytometry in most bone marrow specimens. They are more commonly found and are generally present in higher numbers in children. There is a general decline in hematogones with increasing age but a broad range exists at all ages and marrow from some adults contains relatively high numbers. They are often increased (> 5%) in regenerating marrow and in some clinical conditions, particularly various types of cytopenias and neoplastic diseases. Hematogones may morphologically resemble the neoplastic lymphoblasts of precursor B ALL and their immunophenotype also has features in common with neoplastic lymphoblasts. Distinguishing hematogones from neoplastic lymphoblasts may be problematic in post-chemotherapy and post-bone marrow transplant regenerating marrow. With 4-color flow cytometry using optimal antibody combinations the distinction can nearly always be made. Hematogone populations always exhibit a continuous and complete maturation spectrum of antigen expression typical of the normal evolution of B-lineage precursors; they lack aberrant or asynchronous antigen expression. The neoplastic lymphoblasts in precursor B ALL deviate from the normal B-lineage maturation spectrum and exhibit maturation arrest and over-, under-, and asynchronous expression of antigens observed on normal B-cell precursors and they often aberrantly express myeloid-associated antigens.

Adolescent↗

PubMed2005

Performance analysis of a dual-buffer architecture for digital flow cytometry.

BACKGROUND: Most current commercial flow cytometers employ analog circuitry to provide feature values describing the pulse waveforms produced from suspended cells and particles. This restricts the type of features that can be extracted (typically pulse height, width, and integral) and consequently places a limit on classification performance. In previous work, we described a first-generation digital data acquisition and processing system that was used to demonstrate the classification advantages provided by the extraction of additional waveform features. An improved version of the system is discussed in this paper, focusing on dual-buffering to ensure increased pulse capture. A mathematical model of the system is also presented for performance analysis. METHODS: The second-generation system incorporates fast digitization of analog pulse waveforms, instantaneous pulse detection hardware, and a novel dual-buffering scheme. A mathematical model of the system was developed to theoretically compute the capture-rate performance. RESULTS: The capture rate of the system was theoretically analyzed and empirically measured. Under typical conditions, a capture rate of 8,000 pulses/s was experimentally achieved. CONCLUSIONS: Based on these results, the dual-buffer architecture shows great potential for use in flow cytometry.

Algorithms↗

PubMed2003

Performance of plate-based cytokine flow cytometry with automated data analysis.

BACKGROUND: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates. RESULTS: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis. CONCLUSION: When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses.

Algorithms↗