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Flow cytometry: role in monitoring transitional cell carcinoma of bladder.

The ability to detect and monitor the course of transitional cell carcinoma (TCC) of the bladder using DNA histograms obtained from flow cytometry was studied. Voided urine and barbotage specimens were collected from patients with active TCC or a past history of TCC. These specimens were submitted to routine cytologic and flow cytometric analyses. Samples were considered to be positive if they met one or more of three criteria: if they had aneuploid or tetraploid peaks, if the DNA index of the major G1 peak was shifted more than 10 per cent from that of diploid cells, or if 15 per cent or more of the cells fell to the right of the major diploid G1 cell population thereby constituting a significant hyperdiploid cell population. Using these methods for patients with active disease, the detection rate was 91 per cent. In patients with a past history of TCC, positive histograms preceded the appearance of visible tumor in one third of the cases. Flow cytometry proved to be an excellent way of following patients with a past history of TCC or of screening patients suspected of having active disease. Following this protocol, few biologically active tumors go undetected. However, in 112 patients without a history of bladder cancer, the false positive or suspicious rate was 38 per cent. Before flow cytometry can be recommended as a widespread screening method for patients thought to be at risk of TCC of the bladder developing, this suspicious group will have to be eliminated.

Carcinoma, Transitional Cell↗

Glucose uptake, perfusion, and cell proliferation in head and neck tumors: relation of positron emission tomography to flow cytometry.

The uptake of 18F-Deoxyglucose (FDG) was studied in vivo in relation to the proliferation rate of human head and neck tumors. Forty-two patients with histologically proven squamous-cell carcinoma of the head and neck and four patients with metastases of head and neck tumors were examined with PET and FDG prior to surgery. In 35 of these patients, a flow cytometric analysis of the DNA content and the proliferation rate was done using one-dimensional flow cytometry rate was done using one-dimensional flow cytometry (DAPI staining). In 17 cases, perfusion studies with 15O-labeled water were performed. Twenty-seven specimens were evaluable by flow cytometry. The analysis of the distribution of the FDG uptake revealed two groups, showing a high and a lower uptake pattern. In both groups the FDG uptake and the proliferation rate were correlated with an r-value of 0.64 and 0.8 respectively. However, the slope of the regression function was flat. No correlation was found between the perfusion and the proliferation rate. It is suggested that these differences in uptake in histologically identical tumor populations may correspond to differences at the molecular level, e.g., differences in the amount of the glucose carrier, perhaps caused by oncogenic transformation.

Carcinoma, Squamous Cell↗

The sensitivity of flow cytometry compared with conventional cytology in the detection of superficial bladder carcinoma.

Three groups of patients with superficial bladder carcinoma were studied: 110 patients had carcinoma in situ (TIS), 54 had noninvasive papillary transitional cell carcinoma (TA), and 64 had transitional cell carcinoma infiltrating the lamina propria (T1). One to six conventional cytologic examinations per patient (mean 2.8) were obtained during the 24 hour period before biopsy. For all tumor categories combined, the sensitivity was 49%, 54%, 62%, and 66% for one, two, three, and four cytologic examinations per patient, respectively. Cytologic sensitivity varied according to tumor category; it was greater in the TIS and T1 categories than in the TA category. To compare with the cytologic results, a subset of 103 patients had bladder irrigation performed immediately before the biopsy which was subsequently analyzed by flow cytometry. The sensitivity of a single flow cytometric examination for all tumor categories combined was 78%. As with the cytologic examinations, the sensitivity of flow cytometry varied with tumor category; it was greater in TIS and T1 categories than in the TA category. The sensitivity of one flow cytometric examination was superior (binomial test, P less than or equal to 0.05) to that of one or two cytologic examinations for TA tumors and to that of one, two, or three cytologic examinations for TIS, T1, and all tumor categories combined. Only three of 103 patients had their cancers detected by cytologic examination and not by flow cytometry.

Carcinoma in Situ↗

Computational analysis of flow-cytometry antigen expression profiles in childhood acute lymphoblastic leukemia: an MLL/AF4 identification.

Precursor B-acute lymphoblastic leukemia (pB-ALL) is a heterogeneous disease and multiparameter flow cytometry, molecular genetics, and cytogenetic studies have all contributed to classification of subgroups with prognostic significance. Recently, gene expression microarray technology has been used to investigate lymphoblastic leukemias, demonstrating that known and novel pB-ALL subclasses can be separated on the basis of gene expression profiles. The strength of microarray technique lays in part in the multivariate nature of the expression data. We propose a parallel multiparametric approach based on immunophenotypic flow-cytometry expression data for the analysis of leukemia patients. Specifically, we tested the potential of this approach on a data set of 145 samples of pediatric pB-ALL that included 46 samples positive for mixed lineage leukemia (MLL) translocations (MLL+) and 99 control pB-ALLs, negative for this translocation (MLL-). The expression levels of 16 marker proteins have been monitored by four-color flow cytometry using a standardized diagnostic panel of antibodies. The protein expression database has been then analyzed using those univariate and multivariate computational techniques normally applied to mine and model large microarray data sets. Marker protein expression profiling not only allowed separating pB-ALL cases with an MLL rearrangement from other ALLs, but also demonstrates that MLL+ leukemias constitute a heterogeneous group in which MLL/AF4 leukemias represent a homogenous subclass described by a specific expression fingerprint.

Adolescent↗

Feasibility of an easily applicable method of ZAP-70 measurement in chronic lymphocytic leukemia in the routine flow cytometry setting: A methodological approach.

Zeta-associated protein 70 (ZAP-70), determined by flow cytometry, has been advocated a surrogate marker of immunoglobulin (Ig)V(H) unmutated status in B chronic lymphocytic leukemia (CLL). The aim of the current study was to test the applicability of an easy flow cytometry protocol for ZAP-70 measurement in CLL samples. Samples from 61 CLL patients and 44 normal subjects were analyzed using a commercial ZAP-70 monoclonal antibody (1E7.2 clone) conjugated with phycoerythrin (PE) and Alexa 488 fluorochromes. Modifications of the published methods led to the structure of a simplified in-house method of ZAP-70 measurement. A three-color approach was used with CD19, CD3 gating comparing with the isotype control provided by the same manufacturer. The cutoff levels for ZAP-70 positivity were defined from a receiver operator characteristic curve in relation to the IgV(H) mutational status and from the ln normalized mean value +2 SD of normal controls. Using the 20% cutoff value for ZAP-70 positivity in CLL patients defined by the literature, ZAP-PE had a sensitivity of 55% and a specificity of 98% in predicting the IgV(H) mutational status, whereas the corresponding values for ZAP-Alexa were 55% and 84%, respectively. Using the 7% cutoff value for CD38 positivity, the sensitivity was 55%, whereas the specificity was 76%. ZAP-70-positive patients showed a shorter time to disease progression in comparison with ZAP-70-negative patients (p < 0.001). In conclusion, the 100% specific prediction of mutational status is accompanied by reduced sensitivity, thus limiting ZAP-70's applicability either as a single marker or combined with CD38 for the assessment of the mutational status of CLL.

ADP-ribosyl Cyclase 1↗

Flow cytometry in the diagnosis of lymphomas.

Because of recent advances in instrumentation, immunological reagents, and methods for measuring a variety of cell constituents and functions, there has been increasing interest in clinical applications of flow cytometry. Several commercially available flow cytometers and a growing list of monoclonal antibodies make it feasible for clinical laboratories to apply this technology for phenotyping leukocytes in leukemias, lymphomas, immunological disorders, transplantation, and infectious diseases. The ability to measure cell constituents such as DNA, RNA, proteins, enzymes, and hormone receptors has added to the list of applications. Use in conjunction with simultaneous phenotyping with monoclonal antibodies makes such measurements even more valuable. Phenotyping specimens from a large number of patients with lymphomas and leukemias has demonstrated the value of flow cytometry in diagnosis and patient management.

AIDS-Related Complex↗

[The value of multiparameter flow cytometry in diagnosis of lymphocytic leukemia and bone marrow involvement of non-Hodgkin's lymphoma].

BACKGROUND & OBJECTIVE: Lymphocytic leukemia and bone marrow involvement of non-Hodgkin's lymphoma (NHL) can be diagnosed by bone marrow morphology or lymph node biopsy combined with bone marrow examination. The data of original and differentiation status of tumor can be analyzed by immunophenotype of bone marrow. These are necessary for diagnosis and treatment of lymphocytic malignancy. This study was designed to investigate the value of multiparameter flow cytometry in diagnosis of leukemia and bone marrow involvement of NHL. METHODS: The samples from 11 cases of untreated leukemia bone marrow and 41 cases of untreated NHL with bone marrow involvement and 2 cases of bone marrow whose biopsy could not be obtained due to huge mass in mediastinum and abdomen were detected by multiparameter flow cytometry using antibodies of T, B, Myeloid cell series. Three-color staining was done by CD45 combined with two cell series or special phase antibodies. Using CD45/SSC set gate to identify blast cells from mature cells. The samples were analyzed using five parameters [forward scatter (FSC), side scatter(SSC), McAb1-FITC, McAb2-PE, and CD45-cychrome]. RESULTS: Immunophenotype and diagnosis of 11 cases of leukemia were further confirmed by flow cytometry (FCM). Of 41 cases of NHL with bone marrow involvement, 33 cases (80.5%) lymph nodes immunophenotype by pathology diagnosis were consistent with bone marrow immunophenotype by FCM, 8 cases (19.5%) were inconsistent, but right diagnosis were made by combining with clinical presentation, pathology, bone marrow morphology and FCM. Another 2 cases with huge mediastinal mass and abdominal mass were diagnosed as T-NHL and B-NHL by bone marrow morphology and FCM without lymph node biopsy. CONCLUSION: Multiparameter flow cytometry of bone marrow can further ascertain the diagnosis of leukemia and NHL with bone marrow involvement. It also gives us data of cells lineage and differentiation status for leukemia and NHL. It is helpful for diagnosis, differential diagnosis, and treatment.

Adolescent↗

An automated method to prepare cell suspensions from human biopsy samples for immunophenotyping by flow cytometry.

A comparison was made between a manual and automated method for preparing single cell suspensions from various types of human biopsy samples. The automated method uses the shearing action of fluid movement generated by a reciprocating paddle system. When compared on 11 samples, the automated instrument always isolated cells in less time, usually requiring only 15-30 seconds in contrast to 10-15 minutes for the manual method. The time comparisons involved "set up" time of the required minor equipment as well as the time to make a complete single cell suspension from the portion of the biopsy sample sent to the Flow Cytometry Lab so extra cells could be used for other purposes and cryogenically stored for future reference. Cells isolated by the automated method from various B-lymphocytic and T-lymphocytic malignancies were still viable and could be successfully immunophenotyped by flow cytometry. The immunophenotyping results were compared for both cell isolation methods on seven of these samples, and the results were comparable. The automated technique has now been used to satisfactorily immunophenotype more than 50 biopsy samples. The automated method should represent a significant aid for clinical flow cytometry laboratories performing immunophenotyping tests by efficiently preparing single cell suspensions in a few seconds instead of minutes. Furthermore, the automated method uses a closed sterile bag system that helps minimize the exposure of personnel to potential infectious material present in biopsy samples and prevents external contamination of cell suspensions. The automated technique proven successful for immunophenotyping may also be helpful for related procedures involving hematopoietic malignancies such as DNA content analysis and cell functional assays by flow cytometry, as well as other assays such as tissue typing, gene probe, and in vitro chemosensitivity assays.

Antigens, CD↗

Plasmodium species: flow cytometry and microfluorometry assessments of DNA content and synthesis.

Fluorescence intensities were established by flow cytometry of different erythrocytic stages of Plasmodium berghei after staining of their DNA with Hoechst-33258 or Hoechst-33342. Parasites were obtained from highly synchronized infections or in vitro cultures. Most fluorescence measurements were performed using a low cost, clinical flow cytometer, equipped with a mercury arc lamp. Cells infected with P. berghei could be readily distinguished from uninfected cells on the basis of Hoechst-DNA fluorescence and single, double, and triple ring infected cells were separated clearly. The relative fluorescence intensities of different developmental stages (merozoites, ringforms, trophozoites, schizonts, and gametocytes) corresponded closely to the relative DNA contents of these stages as measured by microfluorometry. Flow cytometry appeared to be a sensitive and rapid method to measure DNA synthesis during asexual development; a C50 value of 5 microM of aphidicolin, a specific inhibitor of DNA synthesis, was established. Vital staining of parasites in culture was possible with both Hoechst dyes. After removal of Hoechst-33258, normal in vitro development of the stained parasites was observed. After Hoechst staining, the haploid ringforms of P. vivax showed slightly less fluorescence (15%) than ringforms of P. berghei and P. falciparum. No differences in fluorescence intensity were observed, however, by direct microfluorometry after Feulgen-pararosaniline staining, indicating that all three species have the same DNA content.

Base Composition↗

Pleomorphic xanthoastrocytoma: DNA flow cytometry and outcome analysis of 12 patients.

BACKGROUND: Pleomorphic xanthoastrocytoma (PXA) is an astrocytic tumor occurring primarily in childhood and adolescence with some malignant histologic features but a relatively slow clinical course. However, some tumors progress more rapidly and can undergo malignant degeneration. The authors attempted to determine whether various histologic features or tumor cell proliferative indices might help identify lesions at risk for early progression and distinguish PXAs from malignant gliomas. METHODS: In a retrospective study of 12 patients with PXA, the tumor's histologic features and DNA flow cytometric parameters were compared with their clinical course. DNA flow cytometry values for the S- and G2-phase of the PXAs also were compared with control group samples of malignant and low grade astrocytomas. RESULTS: Of the 12 tumors at initial diagnosis, 5 were considered typical PXAs whereas 7 had some atypical features (4 with paucity of reticulin fibers, 1 with focal necrosis, and 2 with both atypical reticulin and focal necrosis). During the follow-up period (range, 3.75-11 years; mean, 6.8 years), 2 patients had recurrences; 1 atypical reticulin PXA progressed to glioblastoma after 6.5 years and the 1 tumor with focal necrosis recurred at 6 months and again at 2 years with typical histologic features. DNA flow cytometry parameters of the typical PXA group were similar to values for malignant astrocytoma and significantly higher than values for control specimens of low grade astrocytomas. There were no distinctive DNA flow cytometric features that could distinguish this last tumor from others with a more benign clinical course. CONCLUSIONS: Measurements of the S-phase and G2-phase obtained from DNA flow cytometry and atypical histologic features cannot reliably identify PXA patients at risk for early progression and overall are significantly higher than values obtained for low grade gliomas. Therefore, frequent (i.e., two to three times per year) postoperative clinical and radiologic examinations are necessary to judge the appropriateness of adjuvant therapy in patients with PXA. The paradox of slow growth but DNA flow cytometry consistent with aggressive malignant lesions may represent a cell-cycle arrest mechanism in these lesions that could be verified in subsequent studies.

Adolescent↗

Studies of chromaffin granule functioning by flow cytometry: transport of fluorescent epsilon-ATP and granular size increase induced by ATP.

Flow cytometry techniques, usually employed to characterize cellular populations, are reported here to be a valuable tool to approach the study of subcellular organelle functioning. Chromaffin granules rendered fluorescent by using an antibody against their membrane protein, synaptophysin, are detectable by flow cytometry. Moreover, these storage granules are able to transport the fluorescent ATP analogue, epsilon-ATP (1,N6-ethenoadenosine 5'-triphosphate), and the resulting granular fluorescence increase can also be followed by this technique. The saturation studies show a non-hyperbolic kinetic behaviour, with a two step curve. The K0.5 values were 0.26 and 2.5 mM and Hill numbers 1 and 6 respectively. In addition, an unexpected granular size increase, which was dependent on the epsilon-ATP concentration, occurred together with the fluorescence increase. Other nucleotide triphosphate substrates of V-ATPase, such as ATP or GTP, but not the non-hydrolyzable analogue ATP gamma S (adenosine 5'-O-(3-thiotriphosphate), mimic this effect, which exhibited sigmoidal saturation curves with K0.5 values of 1.8 and 3.1 mM for ATP and epsilon-ATP respectively. The V-ATPase inhibitors, suramin, EGTA or EDTA significantly reduced the granular size increase in the presence of ATP. Extragranular addition of noradrenaline has no effect by itself on the granular size, but significantly reduced the granular size increase induced by ATP. This effect was reversed by the amine transport inhibitor reserpine. The granular size increase induced by ATP was more effective in the presence of Cl- than Br- or I-. Moreover, no increase occurred in the presence of F- or acetate. The Cl- channel blockers were poorly effective, and only 2-(phenylamino)-benzoic acid (DPC) exhibited an effect on the ATP-induced granular size increase.

Adrenal Glands↗

Benzoxazinone derivatives: new fluorescent probes for two-color flow cytometry analysis using one excitation wavelength.

A new class of fluorescent dye which upon excitation at 488 nm turns red is shown to be probe-suitable for using in flow cytometry alone or in conjunction with fluorescein derivatives. 7-dimethylamino 3-(p-formylstyryl) 1,4 benzoxazin 2-one is suitable for rendering microorganisms, such as Plasmodium merozoites and cells detectable by flow cytometry, allowing a dual fluorescence analysis when the cells are labelled with suitable fluoresceinylated ligands such as fluorescein labeled neoglycoproteins or antibodies. The synthesis of the new benzoxazinone derivatives is described: p-[beta-(7-dimethylamino 1,4 benzoxazin 2-one 3-yl)-vinyl]-phenylpropenoic acid can be easily activated as a hydroxysuccinimide derivative and linked to amino groups of polypeptides. Hydrophilic polypeptides such as poly-L-lysine or glycosylated polymers combined with this new fluorescent dye are shown to be helpful in analyzing cell surface receptors, in dual fluorescence flow cytometry analysis, using a single excitation wavelength and two sets of compounds labeled with the new benzoxazinone derivative and with fluorescein isothiocyanate, respectively. The new benzoxazinone derivative has a high molar absorbance, a good quantum yield fluorescence when it is bound to hydrophilic polypeptides and its fluorescence intensity is not dependent on pH in the physiological pH range.

Animals↗

Monitoring responses by use of five-color flow cytometry in subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine.

OBJECTIVE: To monitor by use of 5-color flow cytometry the antigen-specific responses of subsets of peripheral T cells in cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine and to compare results with those for 2 established cell-mediated immunity assays. ANIMALS: 45 female Holstein cattle with negative results for MAP in skin tests conducted at time of inoculation with MAP. PROCEDURES: Cattle were allocated to 4 groups. Cattle of group 1 (n = 12) were 0 to 3 months old and inoculated with a killed MAP vaccine. The 10 cattle of group 2 were the same age as those in group 1 but were not inoculated with MAP vaccine. The 11 cattle of group 3 were 9 to 12 months old and inoculated with killed MAP vaccine. The 12 cattle of group 4 were the same age as those in group 3 but were not inoculated with MAP vaccine. RESULTS: Flow cytometry identified T-cell subsets that responded specifically to the recall antigen. Results of assays for CD25 expression and wholeblood interferon-gamma had the strongest correlation with results for skin tests as well as results with each other. Intracellular expression of interferon-gamma was not correlated as well with results for the other tests. CONCLUSIONS AND CLINICAL RELEVANCE: Flow cytometry can be useful for characterizing the immune response after administration of MAP vaccine and should be evaluated with regard to its sensitivity and specificity when used in detecting cattle naturally infected with MAP.

Animals↗

Duchenne muscular dystrophy due to familial Xp21 deletion detectable by DNA analysis and flow cytometry.

We report two male cousins with Duchenne muscular dystrophy (DMD) in whom cytogenetic studies have shown a small interstitial deletion at Xp21. The lesion is readily detectable in patients and carriers by flow cytometry which indicates that approximately 6000 kb of DNA are deleted in each case. The DNA markers OTC, C7, and B24 are present in the deleted X chromosome but 87-8, 87-1, and 754 are absent. Despite apparently identical deletions one affected boy has profound mental handicap while the other is only mildly retarded. The results confirm the assignment of familial DMD to Xp21 and illustrate the value of flow cytometry in improving the precision of chromosome analysis. We have also undertaken flow cytometry in a cell line from a previously reported DMD patient with a de novo Xp21 deletion who had, in addition, chronic granulomatous disease, retinitis pigmentosa, and the McLeod syndrome. The results indicate that the amount of DNA deleted from the X is similar in both families despite the striking differences in phenotype.

Child↗

Fixation and long-term storage of human lymphocytes for surface marker analysis by flow cytometry.

A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4 degrees C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analyzing lymphocytes that express T-cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.

Antibodies, Monoclonal↗

Monitoring caspase activity in living cells using fluorescent proteins and flow cytometry.

A molecular probe was developed to monitor caspase activity in living cells by flow cytometry. It consists of CFP and YFP with a peptide linker containing two caspase-cleavage sites (LEVD). Its expression resulted in intense fluorescence resonance energy transfer (FRET), whereas cleavage of this linker by caspases eliminated FRET because of physical separation of the CFP and YFP moieties. Using flow cytometry, cells expressing this probe exhibited two patterns, strong FRET and diminished or absent FRET. The appearance of diminished FRET was inhibited by a pan-caspase inhibitor z-VAD or D->A mutations in the LEVD sequence and was markedly increased by apoptosis-inducing agents, etoposide and camptothecin, or overexpression of a caspase 8-red fluorescent protein fusion protein. Importantly, this probe's ability to monitor caspase activity was comparable with results obtained with fluorogenic substrates or fluorochrome-labeled inhibitors of caspases. Specific caspase inhibitors indicated the probe was highly sensitive to cleavage by caspase 6 and 8, less sensitive to caspase 4, and resistant to other caspases. Activation of caspase 8 by Fas engagement markedly increased the probe's cleavage, whereas treatment of caspase 8-deficient cells with anti-Fas did not increase cleavage. However, staurosporine induced cleavage of the probe in caspase 8-deficient cells by a mechanism that was inhibited by overexpression of bcl-x. Taken together, the data indicate that this caspase-sensitive probe can be used to monitor the basal and apoptosis-related activities of caspases, including an initiator caspase, caspase 8, and effector caspases, such as caspase 6.

Apoptosis↗

Application of flow cytometry in transplantation medicine.

Immunological rejection remains a major barrier to successful organ transplantation. Consequently, immunosuppressive intervention to prevent or control the rejection process renders the transplant recipient susceptible to infectious diseases. Flow cytometry has become a useful tool for monitoring immunological responses in transplant recipients. There are three areas of clinical transplantation immunology that may benefit from this technology. First, characterizing and classifying alloreactive antibodies by flow cytometry identifies high-risk donor and recipient combinations with greater precision. Second, the ability to detect subtle changes in the cellular components of the immune system cytometrically may facilitate the differential diagnosis of rejection, infection, and iatrogenic toxicity. Finally, the ease with which flow cytometry determines the adequacy or inadequacy of immunosuppressive therapy through T cell receptor analyses serves to maximize the beneficial effects of engraftment.

Flow Cytometry↗

Significance of flow cytometry in the diagnosis and treatment of bladder tumors.

The results of application of flow cytometry (FCM) in analysis of 139 bladder irrigations of DNA contents in tumor cells from 52 cases of bladder tumors are reported. A comparison was made between this assay and cytological examinations as well as pathological grading. The results suggested that the accuracy of cytological analysis was apparently lower than the cell DNA measurement by flow cytometry, the higher the tumor pathological grading and the deeper the infiltration, the higher the heteroploid DNA contents in the tumor cells. Treatment of high heteroploid bladder tumor required radical cystectomy while the efficiency of simple electrosection or partial cystectomy appeared inferior. When FCM and the bladder tumor pathological grading had a high degree of correlation, especially in the high grading tumor, recurrence was high even when chemical treatment with bladder instillation of BCG was adopted. However, FCM cannot substitute for cystoscopy.

Adult↗