Detection of intracytoplasmic antigens by flow cytometry

A new technique is described for the detection of intracellular antigens by immunofluorescence and flow cytometry. The technique utilizes lysolecithin (lysophosphatidylcholine), a naturally occuring phospholipid, to permeabilize cell membranes and allow antibodies to reach intracellular antigens. The technique is rapid and sensitive, and retains sufficient integrity of the cells being treated to enable differentiation of cell types on the basis of light scatter (e.g., lymphocytes from monocytes). Permeabilization of cells following lysolecithin was assessed using standard techniques including trypan blue exclusion, propidium iodide staining, and hydrolysis of fluorescein diacetate. Lysolecithin treatment was accompanied by only minimal increases in non-specific background fluorescence, and no increase in autofluorescence. Our studies have demonstrated that lysolecithin treatment of human mononuclear cell populations permits flow cytometric analysis of cytoskeletal structures, including intermediate filaments, as well as cytoplasmic immunoglobulin. Studies currently in progress in our laboratory have demonstrated broad intracellular reactivity with some monoclonal antibodies identifying leukocyte differentiation and tumor-associated antigens. These findings contrast with the more restricted expression of these antigens on the cell surface and demonstrate not only the value of the lysolecithin technique but also the importance of the study of intracellular antigens in our overall understanding of the specificity, distribution, synthesis, and function of cellular antigens.     

Leukocyte detection in human semen using flow cytometry

This study set out to establish a new method, using flow cytometry, to evaluate leukocytes in semen. Ejaculates of 59 males, asymptomatic for genitourinary infections, were examined. Routine semen analyses were carried out as well as peroxidase and polymorphonuclear granulocyte-elastase detection. Leukocytes were detected combining flow cytometry and monoclonal antibodies (anti-CD45, anti-CD53). This technique reliably assessed the total number of leukocytes and differentiated subpopulations even at low concentrations. The peroxidase test and elastase determination showed good specificity, but only moderate sensitivity versus flow cytometry combined with monoclonal antibodies. No significant association was observed between semen parameters and leukocytospermia whether evaluated by conventional methods or flow cytometry except for a moderate correlation between spermatozoa and CD53-positive cell concentrations. A first comparison of data from patients grouped on the basis of leukocytospermia (>10(6) white blood cells, WBC/ml) or non-leukocytospermia revealed no significant differences in semen parameters; lowering the threshold value for leukocytospermia to 2x10(5) WBC/ml, sperm concentration was reduced in the group with a low number of WBC identified by monoclonal antibodies. Flow cytometry using monoclonal antibodies was seen to be a simple, reproducible method that enables leukocytes in semen to be accurately detected and to identify WBC subpopulations without preliminary purification procedures.     

Binding of human C-reactive protein to monocytes: analysis by flow cytometry.

An opsonic role has been proposed as a major function of C-reactive protein (CRP) in humans. In support of this hypothesis, recent radiolabelled ligand binding studies have provided evidence for the presence of specific receptors for soluble human CRP on human phagocytic cells, including neutrophils and monocytes. In order to confirm specific binding of CRP to monocytes and to quantify the percentage of such cells capable of expressing binding sites, we employed a sensitive biotin-avidin fluorescence assay to study the CRP-monocyte interaction. It was observed that 67% of monocytes bound biotinylated CRP in a dose-dependent manner, that the binding was calcium dependent, and that it could be inhibited by 60% in the presence of a greater than 20-fold excess of competing native CRP. In other experiments, neither IgG nor heat-aggregated IgG inhibited the binding of CRP to monocytes; and no significant binding to lymphocyte population could be detected. These studies confirm the ability of human CRP to bind to a majority of human monocytes in a calcium-dependent and specific manner, and provide further support for a biologically important interaction of this acute-phase protein with phagocytic cells.     

DNA flow cytometry of histological material from human gastric cancer

DNA flow cytometry was performed on fixed embedded histological material from 56 radical gastrectomies for human gastric cancer. The ploidy and proportion of cells in the DNA synthetic phase of the cell cycle (S phase fraction) were estimated and the results correlated with histological features. DNA aneuploidy was encountered in 73 per cent of cases. Aneuploid and diploid tumours both had significantly higher fractions of cells in S phase than normal mucosa. S phase fractions for aneuploid tumours were higher than diploid tumours. There was a trend for tumours exhibiting an infiltrating mode of growth and poor tubule formation to have a diploid DNA content and low S phase fraction. No difference was observed between the results obtained from tumours with or without lymph node metastases. Early uniform fixation greatly improved the quality of the flow cytometry results measurable by a reduction in the mean coefficient of variation.     

Detection of Mycobacterium-specific interferon-gamma-producing human T lymphocytes by flow cytometry

Flow cytometry has proven to be a useful tool for the investigation of cytokine synthesis by selected cell subpopulations. While most reports have used mitogen stimulation or long-term cultures with antigen, we describe here a novel method to allow the detection of rare mycobacterial antigen-specific cytokine synthesizing cells within one day. The most important feature of this method is the use of an FITC-conjugated isotype-matched control antibody to identify and exclude cells which fluoresce non-specifically. With this technique, we demonstrate interferon-gamma (IFN-gamma) staining in 785 cells per 1 x 10(5) T cells counted, in mycobacterial antigen-stimulated peripheral blood mononuclear cells from a BCG-vaccinated subject. In comparison, only 14 IFN-gamma-staining T cells were seen in the cultures not stimulated by mycobacterial antigen. Less than 10 cells per 1 x 10(5) T cells are stained by an irrelevant control antibody. Specific responses are detectable after 12 h of in vitro culture, and peak at 24 h. In volunteer health care workers, IFN-gamma staining correlated with IFN-gamma production using a published ELISPOT assay (r=0.927). IFN-gamma staining was also higher in PBMC from mantoux skin test-positive volunteers, compared to cells from skin test-negative subjects (p=0.0045). Flow cytometry following short-term culture can thus be used for enumeration of antigen-specific IFN-gamma synthesizing cells.     

用流式细胞术检测胃癌p53蛋白的表达

目的：检测并比较胃癌及癌前病变p53蛋白表达水平,探讨p53蛋白在胃癌发病中的意义。方法;采用间接免疫荧光标记,流式细胞术分析。以DNA指数、增殖指数、荧光指数为分析指标。结果：胃癌、不典型增生及肠上皮化生的荧光指数（FI）值与正常胃粘膜相比差异均有显著性（P＜0．05）,前三者与浅表性胃炎相比差异也都有显著性（P＜0．05）。不同病变间的比较,可见胃癌FI高于不典型增生及肠上皮化生（P＜0．05）。不典型增生p53蛋白阳性率为27％,胃癌为68％,在不典型增生及胃癌病例中,其异倍体的FI值、增殖指数（PI）值和p53蛋白阳性率与二倍体者相比差异都有显著性（P＜0．05）。结论：胃癌组织p53蛋白的表达水平高于癌前病变及正常胃粘膜组织,随病变向恶性转化,p53蛋白、PI及异倍体率均增高。检测p53蛋白表达水平对胃癌的诊断具有一定意义。     

DNA flow cytometry of thymomas

Forty-seven thymomas have been examined by DNA flow cytometry and results correlated with histology, stage, associated clinical features and survival. Twenty-five cases were DNA diploid, 14 were aneuploid and no results could be obtained for eight cases. The presence of aneuploidy correlated with more advanced (stage II and III) disease and the presence of myasthenia gravis and was more frequent in epithelial predominant thymomas. Tumour recurrence was more frequent in DNA aneuploid tumours, stage II/III disease and epithelial predominant neoplasms. Multifactorial analysis showed that DNA aneuploidy was predictive of tumour recurrence independent of the effects of stage and histology.     

Fluorescence polarization assay by flow cytometry

Fluorescence polarization measurement on cell suspensions provides a highly sensitive means for detecting subtle changes in the cells, such as occur early after lymphocyte activation or on malignant transformation. We review here the principles of fluorescence polarization, its measurement by a commercially available flow cytometer and application of such assays especially in cellular immunology.     

Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4: detection by flow cytometry

Background IgE-stimulated cultured basophils from atopic subjects are capable of secreting interleukin-4 (IL-4). We describe a flow-cytometric technique which identified intracellular IL-4 in unstimulated basophils unseparated from peripheral blood mononuclear cells (PBMC) in both atopic (AT) and nonatopic (NC) volunteers.
Methods Freshly isolated PBMC were fixed in 4% paraformaldehyde (PFA). Surface staining with 22E7, a noncompetitive anti-Fc-a antibody, allowed identification of basophils. Permeabilization by 0.1 % saponin allowed staining of intracellular cytokines with specific monoclonal antibodies (mAbs). Two series of experiments utilizing different protocols and anticytokine mAbs were performed. The first protocol required a two-stage fluorochrome staining technique. The availability of fluorochrome-conjugated mAbs allowed a simpler, one-stage labelling procedure for the second protocol.
Results With the first protocol, IL-4 (but not IFN-y), immunoreactivity was detectable in a majority (median 77%) of peripheral blood basophils from both AT and NC subjects (N = 8). Basophil IL-4 immunoreactivity was again evident in experiment 2 but did not differ significantly between AT and NC subjects - either evaluated as percentage of IL-4+ basophils (AT median=66%, NC median=38.4%, P=0.41) or lL-4-specific mean fluorescence (AT median=0.85, NC median=0.3, P=0.07).
Conclusions This simple technique allowed the study of intracellular cytokine expression in unstimulated blood basophils. It demonstrated constitutive basophil expression of lL-4 (but not IFN-y) in all subjects, with no significant increases in atopies.     

DNA and chromatin structure: Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry

Flow cytometry analysis was used for the acurate and objectiveevaluation of sperm chromatin condensation and chromatin stabilityof sperm nuclei. It was also possible to determine the influenceof incubation on sperm chromatin. Different types of spermatozoawere studied: unprocessed spermatozoa at 1 and 45 min afterejaculation, after swim-up (migrated), spermatozoa incubatedfor 6 h in non-capacitating conditions (aged), or in B2 medium(capacitated) or B2 medium followed 1 h later with A23187 (reacted).All types of spermatozoa were analysed before and after treatmentwith various decondensation agents: sodium dodecyl sulphate(SDS), SDS plus EDTA and SDS plus disulphide-reducing agent[dithiotreitol (DTT)). Sperm nuclei were enzymatically isolatedand stained with propidium iodide. Three flow cytometric parameterswere then measured: forward light scatter (cellular size), sidelight scatter (cellular complexity) and fluorescence (uptakeof propidium iodide). Fluorescence was the most suitable parameterto study the degree of condensation and resistance to decondensationof DNA in the spermatozoa. Unprocessed spermatozoa 1 min afterejaculation underwent decondensation by all assessed treatments(anionic detergent, chelating or disulphidereducing agents).Unprocessed spermatozoa 45 min after ejaculation and migratedspermatozoa did not undergo decondensation with SDS treatment,but decondensation occurred after treatment with SDS + EDTAor SDS + DTT. Spermatozoa incubated for 6 h under both non-capacitating(aged spermatozoa) and capacitating conditions (capacitatedspermatozoa) and reacted spermatozoa were decondensed only aftertreatment with SDS + DTT. In conclusion, the post-ejaculationand incubation time have to be taken into account when clinicalinterpretation of the effect of different treatments on spermchromatin condensation is made.     

Seminal haploid cell detection by flow cytometry in non-obstructive azoospermia: a good predictive parameter for testicular sperm extraction

BACKGROUND: Testicular sperm extraction (TESE) associated with ICSI gives patients suffering from non-obstructive azoospermia (NOA) the possibility of becoming a father. The success rate of TESE based on sperm recovery is approximately 50%, and the commonly used non-invasive parameters are not predictive enough. Only the invasive testis biopsy has a good prognostic value. The aim of this study was to evaluate the prognostic value of the detection of seminal haploid cells by flow cytometry (FCM) in order to avoid unnecessary testicular biopsy. METHODS: For 37 NOA patients undergoing testicular biopsy, we measured testis size, serum FSH and inhibin B levels and carried out seminal cytology, seminal FCM analysis and histological examination. RESULTS: Sperm were found in 18 biopsies. These results were correlated with cytology, FCM analysis and the histological examination. FCM was more sensitive than cytology (100 versus 59%) but less specific (67 versus 83.5%) whereas the histological observation of complete spermatogenesis appeared to be less sensitive (50%) but more specific (100%). CONCLUSION: Detection of seminal haploid cells by FCM appears to be an interesting non-invasive technique which can predict TESE results and improve the management of NOA patients.     

IgG anti-D MAbs in tests by flow cytometry

Seventy-two IgG anti-D Mabs were studied by indirect immunoflorescence and flow cytometry (FCM) with R₁r, rr and various D variant phenotype red blood cells (RBCs) (D^IIIb, D^VI, D^VII, Rh33 and uncategorized variant ISBT number 48). The results were compared with those obtained by indirect antiglobulin test (IAT).     

Antisperm antibodies detection by flow cytometry is affected by aggregation of antigen-antibody complexes on the surface of spermatozoa

Flow cytometry (FCM) analysis of live antibody-coated spermatozoa subjected to immunofluorescence staining (FCM test) is considered an objective method for the quantitative detection of antisperm antibodies (ASA). But the cross-linking of cell surface antigen (Ag) with bivalent antibodies and/or antigen-antibody (Ag-Ab) complexes with second antibodies may induce the reorganization of surface components (patching and capping) and result in their shedding from the sperm surface. The present study estimates the relationship between aggregation of Ag-Ab complexes on the sperm surface and the results of indirect FCM test. Swim-up spermatozoa of normozoospermic men were incubated with ASA-positive sera from infertile patients and with second antibodies fluorescein isothiocyanate (FITC)-labelled goat anti-human IgG polyclonal antiserum under different conditions and then analysed by FCM and fluorescence microscopy. It was shown that low temperature, cytochalasin B, excess or lack of the primary and/or secondary antibodies and sperm fixation by paraformaldehyde may inhibit aggregation and shedding of Ag-Ab complexes and dramatically increase ASA quantity determined on the sperm surface. However, inhibition of aggregation on the live sperm surface was observed only in a minority of ASA-positive samples and was poorly reproducible using semen of different donors. A high probability of Ag-Ab complex shedding from the sperm surface during experimental manipulation limits the use of indirect FCM test for quantitative ASA determination.     

Immunophenotyping of cytologic specimens by flow cytometry

Immunophenotyping by flow cytometry is well established as an ancillary technique in the diagnosis of hematopoietic neoplasms. However, flow cytometry is rarely performed on cytologic specimens because most cytologists are more comfortable with direct microscopy and believe that there is inadequate cellularity for analysis. Paradoxically, cytologic material is usually cell suspensions making it ideal for flow cytometry. In order to evaluate the usefulness of immunophenotyping cytologic specimens by flow cytometry, we retrospectively reviewed all cytologic specimens submitted to our flow cytometry unit from 1988 to 1991. Thirty-one cerebrospinal fluid specimens were analyzed. There were inadequate cells for analysis in 15 cases. Five showed a monoclonal proliferation; 11 were nondiagnostic. A range (r) of one to six cell surface markers were performed. Thirty-two body cavity fluids were analyzed: 7 peritoneal, 19 pleural, 2 pericardial, and 4 bronchoalveolar lavage. There were cells to analyze in all cases. Seven had a monoclonal proliferation; 25 were nondiagnostic (r = 4-21 markers performed). One hundred eighteen fine needle aspirates (FNA) were reviewed; 58 FNA were radiologically guided, 60 were superficial lesions. There were inadequate cells for analysis in two cases. Sixty-one demonstrated a monoclonal proliferation; 55 were nondiagnostic (r = 1-22 markers performed). We conclude that immunophenotyping by flow cytometry is of limited value for cerebrospinal fluid analysis and that knowledge of previous immunophenotyping studies is essential for correct analysis; analysis of body cavity fluids is easily performed but less often demonstrates a monoclonal proliferation. Immunophenotyping by flow cytometry is a valuable adjunctive technique for FNA and yields adequate cells for analysis.     

Detection of antisperm antibodies by immobilization and cytotoxicity tests using flow cytometry

All-Union Research Institute for Maternal and Child Health Care, Ministry of Health of Russia. Institute of Human Morphology, Russian Academy of Medical Sciences, Moscow. (Presented by Academician of the Russian Academy of Medical Sciences A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 10, pp. 398–399, October, 1992.     

Application of flow cytometry for biomarker-based cervical cancer cells detection

The Pap test used for cervical cancer screening is subjective, labor-intensive, and has relatively low sensitivity and specificity for the detection of underlying clinically significant lesions. The objective of this study is to develop a biomarker/flow cytometry-based approach for cervical cancer screening. Immunofluorescence technology to quantify cervical cell expression of two biomarkers p16(INK4A) and Mcm5 was developed and evaluated by both microscopy and flow cytometry. The capability of using biomarker/flow cytometry approach to detect rare-event dysplastic cells in a large background of benign epithelial and inflammatory cells was evaluated. The results indicate that flow cytometry could detect 0.01% dysplastic cells in a background of normal cervical epithelial cells with the combination of the two biomarkers. Thirty-two clinical specimens were used to test the biomarker/flow cytometry-based approach and the results were compared with the liquid-based cervical cytology. The experiment yielded 100% sensitivity and 93% specificity with reference to the liquid-based cervical cytology. This study indicates the promise of using multi-color fluorescence flow cytometry for biomarker-based cervical cancer screening. This molecular-based, potentially high-throughput and automated method is expected to provide an alternative/auxiliary means of cervical cancer screening.     

Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry

We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser₆₅ to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.     

Andrology: Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry

Inasmuch as caput epididymal and even testicular spermatozoaare now being used to generate pregnancies by direct injectioninto the oocyte, differences in the chromatin of spermatozoafrom proximal and distal locations in the epidldymis were studied.Acridine Orange staining was used to investigate chromatin structurein human spermatozoa which had left the testis and were undergoingmaturation in the epididymis. Measurement of green and red fluorescenceIntensities of human spermatozoa by flow cytometry demonstrateda decrease in binding of Acridine Orange to DNA as the spermatozoatraversed the epididymis. Using spermatozoa from the cauda epididymisas the standard, the percentages of spermatozoa from the efferentduct, proximal corpus epididymis, midcorpus epididymis, distalcorpus epididymis, proximal cauda epidldymis and distal caudaepididymis that had matured with regard to chromatin condensationwere 28 ± 5, 39 ± 3, 49 ± 5, 64 ±5, 69 ± 6 and 74 ± 4% respectively. It may beconcluded that eggs fertilized by ejaculated spermatozoa receivea more highly condensed form of chromatin than that receivedby eggs Inseminated with proximal epididymal or testicular spermatozoa.