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PubMed1993

Prognostic significance of bromodeoxyuridine high labeled bladder cancer measured by flow cytometry: does flow cytometric determination predict the prognosis of patients with transitional cell carcinoma of the bladder.

We studied whether flow cytometric determination of proliferative cell activity estimated by the bromodeoxyuridine in vitro labeling technique provides significant prognosticators beyond the classical histological evaluation in the patient with bladder cancer. We evaluated 81 patients with transitional cell carcinoma of the bladder using fresh bladder specimens. Clinical followup of at least more than 24 months was requested, with the mean followup being 37.9 +/- 10.5 months. Tumor grade and stage were the histological prognostic parameters. Flow cytometric bivariate measurements of deoxyribonucleic acid (DNA) ploidy and bromodeoxyuridine labeled cell index were evaluated. Tumors with a bromodeoxyuridine labeled cell index of more than 10.7%, which was the value of the mean plus 2 times the standard deviation obtained in grade 1 bladder tumors, were designated as high bromodeoxyuridine labeled cell index tumors. A total of 51 patients with low bromodeoxyuridine labeled cell index tumors demonstrated a 98.0% 3-year actuarial survival rate estimated by the Kaplan-Meier method, compared to 42.9% for those with high index tumors. Multivariate survival analysis was performed with Cox's proportional regression model to study statistical individual prognostic values of histological and flow cytometric parameters. Histological tumor grade was the single most important prognostic factor (risk ratio 13.2, p < 0.05), with tumor stage being the second most important (risk ratio 9.2, p < 0.05), followed by bromodeoxyuridine labeled cell index status (risk ratio 6.9, p < 0.05). DNA ploidy status did not influence the clinical outcome. When grade 3 tumors were classified as low and/or high bromodeoxyuridine labeled cell index tumors, 3-year actuarial survival rates were 85.7% and 27.3%, respectively. These results indicate that DNA/bromodeoxyuridine bivariate analysis can be used as an effective adjunct to histological examination for prognostication and decision-making in treatment of bladder cancer patients.

Actuarial Analysis↗
PubMed2006

Comparison of immunophenotyping by slide-based cytometry and by flow cytometry.

BACKGROUND: Flow cytometry (FCM) is the gold standard for immunophenotyping of peripheral blood leukocytes (PBLs). Slide-based cytometry (SBC) systems, for example the laser scanning cytometer (LSC(R), CompuCyte), can give additional information (repeated staining and scanning, morphology). In order to adequately judge the clinical usefulness of LSC for immunophenotyping it is obligatory to compare it with FCM. AIM: The aim of this study was to systematically compare immunophenotyping by both FCM and LSC methods and to test the correlation of the results. METHODS: PBLs were stained with directly labeled monoclonal antibodies with the whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in parallel by a FACScan (BD-Biosciences) using standard protocols and by LSC with different triggers (forward scatter, CD45 FITC, or 7-AAD). For 7-AAD measurements by LSC, slides were additionally fixed with acetone before 7-AAD staining. RESULTS: Calculating the percentage distribution of PBLs obtained by LSC and by FCM showed very good correlation with regression coefficients close to 1.0 for the major populations and the lymphocyte sub-populations (neutrophils, monocytes, and lymphocytes; T-helper-, T-cytotoxic-, B-, NK-cells). The best trigger for LSC was 7-AAD. CONCLUSION: LSC can be recommended for immunophenotyping of PBLs especially in cases where only limited sample volumes are available or where additional analysis of the cells' morphology is important. The detection of rare leukocytes or weak antigens is limited; in these cases appropriate amplification steps for immunofluorescence should be engaged.

Dactinomycin↗
PubMed1989

Check samples for laboratory self-assessment in DNA flow cytometry. The National Cancer Institute's Flow Cytometry Network experience.

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.

Cell Count↗
PubMed1999

Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+ cells.

BACKGROUND: This study assesses the feasibility of a new volumetric cytometry system for the enumeration of CD34+ cells in apheresis components, peripheral blood, and cord blood samples in routine laboratory work. This system is compared with the following flow cytometry protocols: Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count fluorospheres. STUDY DESIGN AND METHODS: Correlation, linearity, and reproducibility studies were performed for the various methods. Clonogenic cultures were performed, as an external control, to assess the correlation between the number of CD34+ cells per microL and the number of colony-forming units per microL. RESULTS: The linear regression analysis demonstrated that the five methods were comparable (R2 ranged from 0.86 to 0.96 and slopes were close to 1). The CD34+ assay and the flow-count methods showed poor linearity for CD34+ cell counts below 10 cells per microL (R2 = 0.46 and 0.47). The reproducibility assay for a CD34+ count of 10 cells per microL showed a CV of 12 percent and 25 percent for the Milan and CD34+ assay methods, respectively. The mean CV among all five methods for the 46 evaluated samples was 20 percent. There was a strong correlation between the number of CD34+ cells per microL and colony-forming units per microL in cord blood and apheresis samples (r = 0.71-0.81). CONCLUSION: The CD34+ assay is useful in CD34 enumeration in cord blood, leukapheresis samples, and peripheral blood samples and provides comparable results to the Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count methods. Nevertheless, peripheral blood samples with low CD34 absolute counts (below 10 cells/microL) should be analyzed by alternative flow cytometry protocols. Even though the same operator performed the study in a single laboratory, the high inter-method CV suggests that differences in sample preparation and gating strategy are factors that increase variability. Protocols with fewer intermediate steps or fully automated protocols such as the CD34+ assay are expected to reduced intra- and inter-laboratory variability.

Antigens, CD34↗
PubMed2005

Survey for neoplasia in Macoma balthica from the Gulf of Gdansk by flow cytometry.

Using flow cytometry, 234 Macoma balthica were examined during a survey to determine frequency of neoplasia in the Gulf of Gdansk (Poland). Clams were collected in 4 locations and DNA content in gill tissue cells was determined by flow cytometry using propidium iodide staining. Cell permeabilization was induced by osmotic shock. Prevalence of neoplasia ranged from 9.6 to 26.7% depending on location. DNA content in aneuploid cells was higher than in normal dividing cells. The fluorescence value for aneuploid cells corresponded to tetraploid/pentaploid cells. Three stages of neoplasia were defined, based on the percentage of aneuploid cells determined by flow cytometry. Histopathological and cytogenetic analyses were also carried out on the same clams for comparative study. Proportions of normal and affected clams detected using flow cytometry were similar to those identified using both methods. In the present study, no clear relationship was demonstrated between prevalence of neoplasia and pollutant detection in the different sampling sites.

Aneuploidy↗
PubMed1990

Detection and quantitation of human immunodeficiency virus-infected peripheral blood mononuclear cells by flow cytometry.

A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells obtained from HIV-seropositive patients. Peripheral blood was obtained from patients attending an acquired immune deficiency syndrome clinic, and mononuclear cells were separated by centrifugation onto Ficoll-Hypaque. The cell layer at the interface was removed, washed in phosphate-buffered saline without Ca2+ and Mg2+, and fixed with 90% methanol, and intracellular HIV antigens were detected by indirect immunofluorescence with monoclonal antibodies to HIV antigens as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G F(ab')2 antibody as the secondary antibody. DNA content was determined by propidium diiodide staining after RNase treatment. These fluorochrome-treated cells were analyzed for two-color fluorescence by flow cytometry. The results showed that HIV-infected cells in peripheral blood that have been treated with monoclonal antibodies to the p24 or nef antigens of HIV can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells had a significant correlation (P = 0.0001) with the clinical status of the patient, i.e., those with a high percentage of p24 antigen-positive cells had a poorer prognosis than those with a lower percentage of p24 antigen-positive mononuclear cells. In addition, for those in Centers for Disease Control groups III and IV, there was an inverse correlation between the percentage of p24 antigen-positive mononuclear cells and the number of T4 cells. However, cell-associated antigen detection by flow cytometry did not correlate with detection of antigen in sera of HIV-seropositive patients by the standard antigen capture enzyme-linked immunosorbent assay. This lack of correlation was probably due to the presence of immune complexes in the sera of HIV-seropositive patients. These results suggest that flow cytometry can be used as a rapid, sensitive, and quantitative assay system for the determination of the antigen status of HIV-seropositive patients and that it may be more useful as an indicator of disease progression than the currently used antigen detection methods.

Antibodies, Monoclonal↗
PubMed1989

Expression of DNA polymerase alpha and Leu3a molecules in growing and saturated cultures of human leukemic cells: phenotype analysis of proliferative cells by flow cytometry.

A flow cytometric method to analyze phenotypes of proliferative cells was developed using human leukemic cell line MOLT 4. A nuclear protein, DNA polymerase alpha (pol alpha), was selected as a marker for proliferative cells, and Leu3a molecule as a cell-surface antigen phenotype marker of the cells. The procedure involved the simultaneous use of fluorescein-conjugated anti-pol alpha antibody, developed by us, and commercially available phycoerythrin-conjugated anti-Leu3a antibody. The optimal fixative for both proteins was phosphate-buffered 2% paraformaldehyde. The pol alpha-positive population in logarythmically growing MOLT 4 cells was estimated, by flow cytometry, to be ca. 95%. A sharp flow cytometry histogram with a strong pol alpha-linked fluorescence was observed. On the other hand, the pol alpha-positive population in the saturated culture was ca. 70%, with weaker pol alpha-linked fluorescence. Thus, the population of pol alpha-positive cells and the amount of pol alpha in cells was dependent on the cell density of the culture. In contrast, ca. 90% Leu3a-positive populations with similar flow cytometry histograms were seen in either growing or saturated states, suggesting that expression of Leu3a was independent of cell density. The flow cytometric method using fluorescein isothiocyanate-conjugated anti-pol alpha antibody is useful for detecting proliferative fractions of free tumor cells, such as leukemic cells. Furthermore, analysis of the phenotype of the proliferative or non-proliferative cells became easier by simultaneous labeling with antibodies against pol alpha and phenotype-specific proteins.

CD4 Antigens↗
PubMed1993

Assessment of fluorochromes for cellular structure and function studies by flow cytometry.

Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, such as fluorescence quenching and energy transfer, inherent to the staining, provide numerous applications in flow cytometry. The available fluorophores used in flow cytometry are classified according to their cellular incorporation and binding. Thus, probes are presented and discussed as follows: 1) dyes of cellular components (DNA, RNA, proteins, lipids); 2) probes of membrane potential; 3) fluorophores that are sensitive to their microenvironment (pH, calcium, etc); and 4) those used for measurement of enzymatic activities into cells.

Animals↗
PubMed2001

A dynamic inline sample thermoregulation unit for flow cytometry.

BACKGROUND: Flow cytometry is a valuable tool for the study of cellular and molecular interactions. We sought to develop instrumentation that would allow accurate and precise inline dynamic temperature control of flow cytometry samples in order to expand the analytical capabilities of flow cytometry. METHODS: Using a temperature controller, DC power supply, and a Peltier module, we designed a temperature control circuit that regulates the temperature of a heat transfer block. The heat transfer block surrounds the sample line to efficiently heat and cool the sample. We attached DNA oligomers to microspheres and observed the denaturation of fluorescently labeled complementary oligomers to verify that the sample was achieving the desired temperature. RESULTS: The inline thermoregulation unit can ramp between 30 and 95 degrees C (>2 degrees C per second) within 30 s repeatedly. The accuracy of the instrument was verified by comparing the observed melting temperatures of the oligomer sets to the predicted values. These values varied within 6% of the predicted and were reproducible over a variety of conditions. CONCLUSIONS: The apparatus we constructed accurately and dynamically controls the sample temperature of inline samples. Flow cytometers equipped with our inline thermoregulation unit will be able to conveniently use temperature as a robust experimental parameter.

Base Sequence↗
PubMed1995

Quantitation of cellular receptors by a new immunocytochemical flow cytometry technique.

Flow cytometry provides a rapid qualitative method for analyzing the binding of a fluorescent probe to a cell. To quantitate the binding of a probe using flow cytometry, one must be able to first calibrate the fluorescent signal with some known standard. We have compared a new one-step method with a previous two-step method for determining the number of binding sites (receptors) on the surface of cells using immunofluorescent staining of the cells and analysis by flow cytometry. Experimentally, recombinant chinese hamster ovary cells, expressing cell surface glycoprotein receptors, IIb/IIIa or Mac-1, were assayed using specific mouse monoclonal antibodies against these receptors. The two methods yielded comparable results and, depending on the cell type, detected anywhere from 100,000 to 300,000 antibody-binding sites per cell, respectively.

Animals↗
PubMed1989

Platelet crossmatching for kidney transplants by flow cytometry.

Although flow cytometry crossmatching is now performed by many kidney transplant centers, it yields a high false-positive rate, as evidenced in the present series by a 71% one-month success rate despite a positive crossmatch. In an attempt to reduce false-positive reactions while retaining the sensitivity of the flow cytometer, platelets were tested as targets. Whereas flow cytometry with T cells was not correlated with one-month failure rates, retrospective platelet crossmatching by flow cytometry showed that kidney failure within one month occurred in 53% of 19 patients with a positive platelet crossmatch compared with 15% of 48 patients with a negative platelet crossmatch. (P less than 0.002). Statistical significance in one-month outcome was also obtained when only first transplant patients were analyzed (P less than 0.04). The present results suggest that crossmatching with platelet targets may be a simpler way to avoid early primary nonfunction while minimizing false-positive reactions.

Blood Grouping and Crossmatching↗
PubMed2005

[The clinical significance of detecting minimal residual disease in acute childhood B-cell lymphoblastic leukemia with flow cytometry].

OBJECTIVE: Flow cytometry may be used to detect minimal residual disease (MRD) in acute lymphoblastic leukemia because leukemic cells often display aberrant phenotypes when compared to normal cells. The present study was designed to establish a flow cytometric method for detecting MRD in children with B-ALL and evaluate its clinical prognostic value. The investigators also aimed to study the value of the detection of MRD by flow cytometry in childhood B-ALL without effective antibody combinations. METHODS: Thirty-six cases of childhood B-ALL with effective antibody combinations were performed MRD analysis after induction therapy. The authors detected MRD in 6 cases without effective antibody combinations by the four-color antibody combinations consisting of CD(45)/CD(19)/CD(10)/CD(34) and CD(45)/CD(19)/CD(20)/CD(22) and detected the aberrance of the minor subsets of CD(19)(+) cells. RESULTS: (1) Forty-two cases of childhood B-ALL were screened for antibody combinations of interest and were identified in 86% (36/42) of the cases. The sensitivity of this method was 0.01%. (2) Patients with MRD levels > or = 0.01% at 9 and 12 months of therapy had significantly low disease-free survival compared with patients with MRD levels < 0.01%. (3) Six out of seven patients with recurrence in the BM had MRD levels > or = 0.1% prior to recurrence. Patients with MRD levels > or = 0.1% during chemotherapy had significantly low disease-free survival as compared with patients with MRD values < 0.1%. (4) Two out of seven patients with recurrence had positive results of the qualitative PCR prior to recurrence. (5) Five patients with recurrence had no shift of antigen expression at relapse except that a patient missed CD(13). (6) Detectable MRD was not found in six patients without effective antibody combinations. CONCLUSION: (1) Flow cytometry is a sensitive and specific method for detecting MRD of childhood ALL, and could predict the coming relapse. (2) Patients with MRD levels > 10(-3) had poor prognosis. (3) The levels of MRD at month 9 and 12 had prognostic value. (4) The value of antibody combinations consisting of CD(45)/CD(19)/CD(10)/CD(34) and CD(45)/CD(19)/CD(20)/CD(22) should be further investigated in patients without effective antibody combinations.

Adolescent↗
PubMed2000

Validation and quality control of immunophenotyping in clinical flow cytometry.

Clinical flow cytometry has evolved from two-parameter quantitative assessment of peripheral blood lymphocytes to six-parameter qualitative evaluation of bone marrow for hematopathology. Leukemia and lymphoma immunophenotyping represent an extremely important complement to morphology in the diagnosis and monitoring of hematopoietic malignancies. The complexity of five- and six-parameter analyses and the interpretation of the data rely on standardization and validation of the instrument, the reagents and the procedure. In addition, flow cytometry laboratories in the U.S. are required to document proficiency testing, sample preparation, method accuracy, specificity, sensitivity and precision. NCCLS and the U.S.-Canadian Consensus Conference have provided recommendations, but each laboratory is ultimately responsible for validating its own qualitative and quantitative procedures. This paper reviews procedures for validation and quality control of all aspects of the operation of a clinical flow cytometry service.

Cell Survival↗
PubMed1990

Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry.

A flow cytometry method was used to compare the amount of red cell (RBC)-bound IgG in 73 patients with and without immune hemolytic anemia (IHA). The positive results in 10 of the direct antiglobulin tests (DATs) were idiopathic, and those in 25 were due to methyldopa therapy; 38 of the 73 DAT-positive patients were babies born to women with IgG alloantibodies of potential clinical significance. Normal blood donors with (n = 30) and without (n = 121) positive DATs were also tested. RBCs that had been strongly sensitized (4+ indirect antiglobulin test) in vitro with different quantities of IgG anti-D, but that had similar antiglobulin test (AGT) titration scores, could easily be differentiated by flow cytometry. The mean percent fluorescence of RBCs, incubated with fluorescein-labeled anti-IgG, from neonatal patients with IHA was higher than that of RBCs from those without IHA, but there was no statistical difference in the other groups. There was considerable overlap in the respective ranges of percent fluorescence of RBCs from patients with and without IHA in all groups. It was not possible to define a clear quantitative threshold differentiating patients with IHA from those without. Although flow cytometry was more precise and reproducible than standard serology (e.g., AGT titration scores), correlations of the amount of RBC-bound IgG and in vivo hemolysis were similar.

Anemia, Hemolytic, Autoimmune↗
PubMed1991

The detection of platelet alloantibodies by flow cytometry.

A flow cytometric procedure was investigated for its ability to detect antibodies directed against blood group A, HLA, and PlA1 (HPA-1a) antigens. When type O sera were tested against platelets from blood group A donors, only 9 of 14 positive reactions were observed. Furthermore, the expression of blood group A varied more than 100-fold on platelets derived from individual donors. When anti-HLA-A2 and -B7 were evaluated, 11 of 11 individuals with HLA-A2 and -B7 antigens reacted. In contrast, when platelets from donors whose HLA antigens included HLA-B8 or -B12 were tested with anti-HLA-B8 or -HLA-B12, respectively, positive reactions were observed in only 3 of 7 instances, despite the fact that the lymphocytes reacted strongly. Platelets from 10 HLA-A2-positive donors, which had been stored for up to 20 months at -70 degrees C, were studied. In all cases, frozen-stored platelets reacted well with an anti-HLA-A2. Limited testing with an anti-PlA1 (anti-HPA-1a) showed equal reactivity with fresh and frozen platelets. Finally, the method was compared to a visual immunofluorescence assay using sera from patients who were refractory to platelet transfusions. The results agreed in 30 of 37 comparisons, and most discrepancies were resolved in favor of flow cytometry. It is concluded that flow cytometry is useful for detecting platelet alloantibodies and possibly for prospective platelet crossmatching, as HLA- and platelet-specific antibodies can be identified by using platelets stored frozen for several months.

ABO Blood-Group System↗