Detection of intracytoplasmic cytokines by flow cytometry

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.     

Characterization of hematologic malignancies by flow cytometry

The quantitative assessment of cellular DNA and RNA content by flow cytometry to provide useful information for both diagnosis and prognosis of patients with hematologic malignancies is reviewed. While the characterization of cell surface antigens seems to be more germane to questions of the normal cell counterpart (stage) of malignant transformation and the biology of regulation of proliferation and differentiation by cell-cell contact and humoral factors, DNA-derived and RNA-derived parameters were surprisingly sensitive in the distinction of major morphologic groups, drug sensitivity and long-term prognosis. Our findings to date in the study of leukemias, lymphomas and myelomas are summarized.     

A kinetic study of membrane immunoglobulin capping by flow cytometry

A flow cytometric procedure has been developed for performing kinetic studies on the capping of membrane immunoglobulin (mIg) on B lymphocytes. Mouse B cells were stained with fluorescein-conjugated antimouse-Ig antisera and subjected to pulse-shape (width, peak, and area) analyses prior to, during, and after ligand-induced redistribution of mIg. It was found that ring-stained, patched, and capped cells could be discriminated based on the width of the electronic signal curve generated as the cells passed through the laser beam. Additionally, endocytosis and or shedding of the cap could be correlated with a change in the area under the curve. Using these two parameters (width and area), the effects of temperature, cross-linking, and several pharmacological agents on the capping process were examined. Through the use of flow cytometry, the inhibitory effects of various perturbants could be localized to discrete stages of the capping process.     

Detection of hemoglobin variants in erythrocytes by flow cytometry

BACKGROUND: With the emergence of fetal hemoglobin (Hb F)stimulating agents as potential treatments for sickle-cell disease and thalassemias, procedures to monitor the effect of these agents on Hb F levels in individuals will be needed. We developed a rapid procedure that detects fetal hemoglobin in erythrocytes (F cells) using a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against Hb F. METHODS: Ten microliters of washed blood was fixed in formaldehyde and glutaraldehyde, then permeabilized in a Triton X-100/PBS solution containing a FITC-labeled monoclonal antibody to Hb F. The blood was analyzed by flow cytometry to determine the percentage of F cells. RESULTS: Nearly 200 Hb F-containing samples were analyzed by this protocol and demonstrated good correlation to percent Hb F results determined by high pressure liquid chromatography (HPLC). In addition, a number of samples were fixed and permeabilized using this method as well as a previously-described method that uses dimethyl 3,3'dithiobispropionimadate (DTBP) as a fixative as well as a different anti-Hb F monoclonal. Good correlation (r = 0.96, r2 = 0.93, P<0.001) was observed between the two protocols. CONCLUSIONS: This procedure is easy, reproducible, and gives accurate F cell results. It can be used to measure a wide range of F cell percentages and may also be used to dual-stain Hb F along with other hemoglobin variants and erythrocyte surface antigens.     

Detection of bladder carcinoma in females by flow cytometry and cytology

The sensitivity of bladder wash flow cytometry (BWFCM), voided urinary cytology (VUC), and cytology of catheterized urine obtained at the time of cystoscopy (CUC) were reviewed on all women evaluated for bladder cancer at Memorial Sloan-Kettering Cancer Center between June 1985 and December 1986. This comprised sixty-four episodes of pathologically proven bladder cancer in 48 women. Considering positive and suspicious results jointly the sensitivities of BWFCM, CUC and 3 VUC were 75%, 64% and 56%, respectively. If only positive results were considered (i.e., suspicious results considered as negative), the sensitivities of BWFCM, CUC and 3 VUC were 64%, 31% and 32%, respectively. The sensitivities of these tests are less than for a predominantly male population, presumably related to the presence of squamous epithelium and greater frequency of pyuria. However, bladder wash flow cytometry and conventional cytology are still a very valuable addition to cystoscopic examination, and the combination of BWFCM with conventional cytology is more sensitive than either procedure alone.     

Detection of acid-beta-galactosidase activity in viable human fibroblasts by flow cytometry

The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was used to detect acid beta-galactosidase in intact cultured human fibroblasts. The accumulation of intracellular fluorescein, as measured by flow cytophotometry was linear with the incubation time in three control strains. The two fibroblast strains from patients with acid beta-galactosidase deficiency did not show an accumulation of intracellular fluorescence. Within one control cell population there was a positive correlation between the amount of accumulated intracellular fluorescein fluorescence and the specific acid beta-galactosidase activity as measured biochemically on sorted cells from different zones of the fluorescence distribution. No correlation was found between the specific acid beta-galactosidase activity and the fluorescein fluorescence of three different control cell strains.     

Detection and discrimination of individual viruses by flow cytometry.

A new flow cytometer with a very small observation volume has been developed to detect individual viruses with good resolution, and has been used to discriminate between two types of viral particles based on differences in their light scattering. Measurements of light scattering and fluorescence made with such an instrument can provide a basis for quantitative analysis and sorting of viruses and other particles in the micron and submicron size range.     

Comparative analysis of surface membrane immunoglobulin determination by flow cytometry and fluorescence microscopy

The analysis of membrane surface immunoglobulin (SmIg) on B lymphocytes was carried out in 59 normal individuals and nine patients with B-cell non-Hodgkin's lymphomas by conventional immunofluorescence microscopy and flow cytometry. Five channel settings of a cytofluorograph were evaluated (100, 150, 200, 250, 300) and the mean and standard deviation of the percent positive cells were calculated and compared to the mean and standard deviation of the microscope reading. On the basis of the relative fluorescence reactivity, we were able to determine a fluorescence intensity at which the results of flow cytometry and fluorescence microscopy were comparable. In normal individuals, for cells expressing surface Ia, the channel giving similar results to that of fluorescence microscopy was 150; for kappa and lambda chains, channel 200; for Fab'PV, channel 200; and for IgM, channel 250. In patients with B-cell non-Hodgkin's lymphomas, for cells expressing surface Ia the channel giving similar results to that of fluorescence microscopy was 100; for kappa, channel 100; for lambda, channel 200; for Fab'FV, channel 150; and for IgM, channel 150. Flow cytometric analysis of SmIg appears to be superior to fluorescence microscopy in efficiency, and has the added advantages of being a rapid, sensitive, and objectively quantitative methodology.     

Detection of minimal residual disease in acute leukemia by flow cytometry.

Patients with acute leukemia in clinical remission may still have up to 10(10) residual malignant cells (the upper limit of detection by standard morphologic techniques). Sensitive techniques to detect minimal residual disease (MRD) may allow better estimates of the leukemia burden and help the selection of appropriate therapeutic strategies. Flow cytometry and polymerase chain reaction have emerged as the most promising methods for detecting submicrospopic levels of leukemia. Flowcytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. It affords the detection of one leukemic cell among 10,000 normal bone marrow cells, and can be currently applied to at least two thirds of all patients with acute leukemia. Prospective studies in large series of patients have demonstrated a strong correlation between MRD levels during clinical remission and treatment outcome. Therefore, MRD assays can be reliably used to assess early response to treatment and predict relapse. In this review, we discuss methodologic aspects and clinical results of flowcytometric detection of MRD in patients with acute leukemia.     

Detection of JNK and p38 activation by flow cytometry analysis

JNK and p38 protein kinases are involved in the signal transduction of apoptotic stimulus. JNK and p38 are activated by dual phosphorylation on threonine and tyrosine residues. Different techniques such as Western blotting (WB) and confocal microscopy analysis have been developed to detect the activation by using antibodies that recognize the phosphorylated forms of both enzymes. However, these techniques are time consuming, not quantitative, and dependent on subjective interpretation. Herein, we describe a flow cytometry-based analysis to detect JNK and p38 activation. Using human primary lymphocytes and Jurkat CD4(+) T cells stimulated with PMA/ionomycin, we demonstrate activation (phosphorylation) of JNK and p38, which is further confirmed by two additional established techniques (WB and confocal microscopy). Flow cytometry analysis is shown to be more sensitive than WB to detect JNK and p38 activation, which can be quantitated and enables us to study their activation within cell populations.     

Normospermic versus teratospermic domestic cat sperm chromatin integrity evaluated by flow cytometry and intracytoplasmic sperm injection

Teratospermia (>60% of morphologically abnormal spermatozoa) is well documented in felids. Even morphologically normal spermatozoa from teratospermic ejaculates have reduced ability to undergo tyrosine phosphorylation, acrosome react, and bind and penetrate oocytes compared with normospermic (<40% abnormal spermatozoa) counterparts. However, it is unknown whether fertilization deficiencies originate at a nuclear level. This study examined whether fertilization failure also was attributable to abnormal sperm chromatin, using the sperm chromatin structure assay (SCSA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Aliquots of unprocessed and swim-up-processed (to isolate morphologically normal spermatozoa) spermatozoa from teratospermic and normospermic domestic cats were analyzed by the flow cytometric SCSA. Swim-up-processed sperm were incubated with in vivo-matured oocytes or used for ICSI. Teratospermic ejaculates expressed more (P < 0.05) chromatin heterogeneity (abnormal chromatin structure) than their normospermic counterparts, both in unprocessed and swim-up-processed samples. Fertilization success in vitro was higher (P < 0.05) from normo- compared with teratospermic inseminates. Similar (P > 0.05) proportions of oocytes fertilized after ICSI using spermatozoa from normo- and teratospermic cats. Results reveal that teratospermia in the cat is expressed at the nuclear level as increased sperm chromatin heterogeneity, but ICSI showed that this does not apparently affect fertilization rates if the zona pellucida and oolemma can be bypassed.     

Detection of anti-phosphatidylethanolamine antibodies using flow cytometry.

BACKGROUND: The finding that lupus anticoagulant (LA) is significantly associated with anti-phosphatidylethanolamine (PE) activity has led to great interest in its relation to the clinical features of the antiphospholipid syndrome (APS). Considerable variability has, however, been reported in the prevalence of anti-PE antibodies in APS patients using enzyme-linked immunosorbent assay (ELISA) methodology. The lack of standardization and differences in technique may in part explain these discrepancies. PE binds variably to different types of microtiter wells, reflected in the consequent detection, or lack of detection, of anti-PE antibodies. This study describes the use of flow cytometry as an alternative method for the detection of anti-PE antibodies. METHODS: Six LA-positive plasma samples were used in this original study. Polystyrene beads were coated with PE overnight. These were subsequently incubated with patient plasma. Both IgG and IgM binding were detected by flow cytometry using a cocktail of fluorescently labelled anti-human Ig isotypes. RESULTS: When these results were compared with those from ELISA, flow cytometric analysis provided an apparent enhanced detection of anti-PE antibodies. It was found that 6/6 were IgM anti-PE positive by flow cytometry, whereas 5/6 were IgM by ELISA; 2/6 negative for anti-cardiolipin antibodies by ELISA were positive by flow cytometry; and 2/6 positive for antiphosphatidylcholine antibodies in cytometry were negative by ELISA. CONCLUSIONS: With appropriate quantification, this method may be more sensitive than ELISA in detecting anti-PE antibodies in plasma samples of patients with APS.     

Detection of distinct subpopulations of Langerhans cells by flow cytometry and sorting

Flow cytometry was found to be a very appropriate tool for the study of Langerhans cells (LC), which represent a minor cell population (2-3%) of human epidermis, and allowed us to obtain new phenotypic, functional, and cell cycle data on these rare cells. The phenotypic analysis of cell surface antigens demonstrates the existence of two subpopulations of LC: the former is HLA-DR+ and OKT 6+ (about 90% of total HLA-DR+ cells) and the latter is HLA-DR+ and OKT 6- (about 10% of total HLA-DR+ cells). These subpopulations of LC are both able to stimulate the proliferation of peripheral blood lymphocytes (PBL) in the presence of keratinocytes i.e., in mixed skin lymphocyte reaction (MSLR). Analysis of the cell cycle could be performed on OKT 6+ LC. Results show that they can be found in the various phases of the cell cycle, suggesting that the large majority of Langerhans cells are able to proliferate in situ in normal human epidermis.     

Detection of endometrial cancer by flow cytometry using two monoclonal antibodies.

BACKGROUND: To develop a supplementary diagnostic method for endometrial cancer by measuring the reactivity of various endometrial lesions with two monoclonal antibodies. METHODS: We investigated the reactivity of various endometrial lesions with two monoclonal antibodies (MSN-1 and MSN-3) by flow cytometry (one-color and two-color methods). RESULTS: The two-color method appeared to be suitable for use in place of simultaneous performance of the one-color methods with MSN-1 and MSN-3. The positivity rate for normal endometrium was 16.0% with the two-color method, which was lower than the rate of 30.0% obtained with concomitant used of the one-color methods. The positivity rate for endometrial cancer was high, 84.0%, with the two-color method. The positivity rate was 85.7% for well-differentiated endometrial cancer, 71.4% for moderately differentiated cancer, and 100.0% for poorly differentiated cancer; thus, the rate was high irrespective of the cellular differentiation. CONCLUSIONS: The two-color method is more useful than the one-color method as a supplementary diagnostic procedure for endometrial cancer.     

Detection of a Plasmodium vivax erythrocyte binding protein by flow cytometry.

BACKGROUND: The malaria parasite Plasmodium vivax preferentially invades reticulocytes. It is therefore relevant for vaccine development purposes to identify and characterize P. vivax proteins that bind specifically to the surface of reticulocytes. We have developed a two-color flow cytometric erythrocyte binding assay (F-EBA) that has several advantages over traditional erythrocyte binding assays (T-EBAs) used in malaria research. We demonstrate the use of F-EBA using the P. vivax Duffy binding protein region II (PvDBP-RII) recombinant protein as a model. This protein binds to all erythrocytes that express the Duffy receptor (Fy) and discriminates binding between normocytes and reticulocytes. METHODS: F-EBAs were performed by incubating freshly isolated Aotus nancymai, Macaca mulatta, Saimiri boliviensis, and human erythrocytes with PvDBP-RII, a fluorescent anti-His tag detection antibody, and thiazole orange before flow cytometric analysis. T-EBAs employing immunoblot detection with an anti-His antibody were performed concomitantly. RESULTS: PvDBP-RII bound to A. nancymai, M. mulatta, and human Fy+ erythrocytes, but not human Fy- erythrocytes, by F-EBAs and T-EBAs. However, F-EBAs exhibited higher sensitivity and better concordance between experiments compared with T-EBAs. CONCLUSIONS: F-EBA is a rapid, simple, and reliable method for quantifying the ability of malaria proteins to bind to the surface of erythrocytes. F-EBA can discriminate binding between erythrocyte subpopulations without enrichment protocols and may be more reliable and sensitive than T-EBAs in identifying novel erythrocyte binding proteins.     

Detection of terminal deoxynucleotidyl transferase (TdT) by flow cytometry in leukemic disorders

We applied a new technique to the detection of intracellular TdT in 26 leukemic patients, including 16 non-T acute lymphoblastic leukemia (ALL), four T-ALL, one T-lymphoblastic lymphoma in leukemia phase, one undifferentiated leukemia, one de novo lymphoblastic phase of chronic myeloid leukemia, and three acute monocytic leukemias (AMOL). Mononuclear cell suspensions were incubated in saponin to permeabilize the cell membrane. The cells were then stained by indirect immunofluorescence (IF) using anti-human TdT monoclonal antibodies and were analyzed by flow cytometry (FCM). The TdT results were compared with those obtained by biochemical TdT assay (26 cases), immunoperoxidase determination (PAP) (12 cases), and fluorescence microscopy (seven cases). The results obtained by PAP and fluorescence microscopy were 100% concordant with those obtained by FCM and biochemical assay. TdT determination by FCM allows the analysis of large numbers of cells in a fast, objective, and reliable manner, as compared with biochemical assay, PAP, and fluorescence microscopy determinations.     

DNA analysis by flow cytometry

Accurate quantification of DNA from cells of several species is possible with flow cytometry. When one species is used as a reference, cytometric readings from two or more different species can be compared to obtain relative percent DNA or DNA indices. Differences in DNA from the male and female of the same species also can be measured. The method allows rapid screening of chromosomal abnormalities among large clinical populations, and evaluation of errors of sex determination such as XY sex reversal.     

DC-based immunotherapy of B-cell malignancies

B-cell malignancies are a group of diseases for which vaccination protocols have been thoroughly studied over the last few years. All different vaccination protocols share the goal of inducing or augmenting tumor-specific immune responses in the tumor-bearing host, in order to potentially achieve therapeutic benefit in these otherwise ultimately fatal diseases. Attention has been drawn to the use of DC-based immunotherapy protocols relying on the unique properties of these powerful APCs. This review focuses on DC-based immunotherapy experience gained so far in B-cell malignancies, and discusses published and on-going clinical trials in follicular NHL and multiple myeloma, and preclinical results in CLL and Waldenstr?m's macroglobulinemia. This will form the basis for a discussion of perspectives of DC vaccination in this group of human malignancies.     

Detection of toxic phytoplankton species by immunochemical particle analysis based on flow cytometry

Particulate suspended matter in oceanic, coastal, and estuarine regions can be specifically marked immunochemically with a fluorescent probe using antisera recognizing antigens present on their surface. Of the particulate matter, phytoplankton is a major component. Toxic species that may form harmful blooms can be a direct threat to aquaculturing tourism, sea-life and man. In order to detect such species in natural fixed phytoplankton populations, immunochemical tagging has been combined with flow cytometric evaluation Microalgal cells can be labeled with a fluorescent probe (fluorescein isothiocyanate, FITC, is recommended). Labeled cells are counted using a flow cytometer. This method has proved to be applicable in a monitoring programme in the North Sea.