Quantitation of baculovirus particles by flow cytometry

A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures. The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye. Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities. In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions. FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml. Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures. The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA). Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA). Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers. It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.     

Quantitation of fetal-maternal hemorrhage by flow cytometry. A simple and accurate method

A simple and objective assay was developed for the detection and quantitation of fetal-maternal hemorrhage with the use of flow cytometry. In vitro prepared control mixtures of 10%, 2%, 1%, 0.5%, 0.25%, 0.125%, and 0.06% D+ RBCs in D- RBCs were tested (8-11) different times by flow cytometry and gave mean % D+ results of 11.10%, 1.90%, 0.92%, 0.45%, 0.24%, 0.11%, and 0.05%. The coefficient of variation of preparing and testing these mixtures ranged from 11.0 to 15.9% for the 10-0.125% mixtures. Thus, flow cytometry was accurate, reproducible, and sensitive. Flow cytometry was compared with Du tests, rosette tests, and acid elution. The Du test was highly variable because it was not sensitive enough to detect a significant bleed (approximately 0.6%) in some cases and too sensitive (necessitating quantitation of an insignificant bleed) in others. The rosette test was too sensitive. Acid elution and flow cytometry results did not always agree; acid elution results were approximately twice as high as flow cytometry. The authors believe flow cytometric detection of D+ red blood cells to be more accurate than the detection of fetal hemoglobin by acid elution techniques, which is known to have poor reproducibility. Postpartum samples from 56 D- women who delivered D+ babies were tested. Fifty-two had fetal bleeds less than 0.3% by acid elution and flow cytometry; all had negative Du test results, but there were two false positive results with the use of the rosette technique. Four had significant bleeds (greater than or equal to 0.6%); in all four cases the flow cytometry results were lower than the acid elution results. The authors were able to quantitate a bleed of fetal RBCs, which were D+ only by the Du test, in a D- mother with the use of flow cytometry, and D+ RBCs in a mother whose RBCs were of the rare DVI mosaic phenotype. This would not have been possible with the use of the standard Du or rosette techniques.     

Detection of minimal residual T cell acute lymphoblastic leukemia by flow cytometry.

We have developed a flow cytometric assay for the determination of cellular expression of terminal deoxynucleotidyl transferase (TdT) and applied this to the detection of minimal residual T cell acute lymphoblastic leukemia (T-ALL). The flow cytometric assay for TdT demonstrated requisite specificity: TdT was localized to the nucleus, and was detected in MOLT3 T lymphoblasts, clinical T-ALL samples, and normal bone marrow B lymphoid precursors, but in neither the KG1a myeloid leukemia cell line nor normal myeloid cells. Co-expression of TdT and the pan T cell marker CD5 was used to quantify T lymphoblasts. 0.25 +/- 0.13% of normal adult bone marrow CD5+ cells were TdT+; these may represent early T lymphoid precursors. When admixed with normal bone marrow, CD5+TdT+ leukemic cells could be detected above background levels at an added concentration of 0.035% (95% confidence interval 0.028-0.43%). Long term follow-up of a large number of patients will be required to determine the clinical significance of a minimal burden of leukemic cells.     

T cell receptor usage by HLA-DQw8-specific T cell clones.

To investigate whether T cells recognizing the same HLA molecule may demonstrate homology in parts of their TCR, five different HLA-DQw8-specific T lymphocyte clones (TLC) were studied. The TCR alpha and -beta genes of four alloreactive, HLA-DQw8-specific and one antigen-specific TLC were sequenced. All TLC used different V alpha and V beta genes. However, four of the TLC shared a certain CDR1 beta motif and all five used either J beta 2.3 or -2.5. In addition, two used the same J alpha. The results indicate a possible preferential usage of certain TCR structures by T cells specific for DQw8.     

Sendai virus-specific T cell clones II. Induction of interferon production by Sendai virus-specific T helper cell clones

Interferon (IFN) was detected upon co-culture of cloned Sendai virus (SV)-specific T lymphocyte with SV-presenting syngeneic stimulator cells. The antiviral activity was defined as IFN-alpha, beta. The T cell clones, upon contact with antigen-presenting stimulator cells, stimulated adherent cells present in the stimulator cell population to secrete IFN. Induction of IFN production was independent from interleukin 2 production by the T cell clones.     

Quantitation of the host cell infiltration kinetics of the nonimmunogenic colon 26 tumor by multiparameter flow cytometry

In this study, we examined the kinetics of host cell infiltration into the nonimmunogenic Colon 26 tumor. We found that 1 x 10(4) cells were required to produce tumors in 100% of mice. The vivo doubling time was 42.5 h, and a barely palpable tumor contained 8 x 10(6) cells. No evidence of concomitant immunity was found. The number of host cells infiltrating the in vivo tumors increased at the same rate as the number of tumor cells, but averaged only 22% of total cells. Cycling T lymphocytes were present in the host cell infiltrate of this tumor. In addition, approximately 50% of in vivo Colon 26 cells were Thy-1.2 positive. The observed characteristic of low immunogenicity makes it a useful murine model for studying human malignant tumors.     

Red cell age by flow cytometry

A method is presented for the use of red cell markers to assess the age of red cells in clinical samples. The reticulocyte count and its variants are already in clinical use to measure the number of young circulating red cells, but tools have not been put into place for studying the overall distribution of red cell age. These data could be of significant value, not merely for hematologic investigations, but as a part of infectious disease, renal, and toxicologic studies.     

Quantitation of spermatogenesis by DNA flow cytometry from fine-needle aspiration cytology material

DNA flow cytometry (FCM) was performed from fine-needle aspiration cytology (FNAC) of testis in 15 cases of male infertility to quantitate spermatogenesis. The results were correlated with FNAC findings. DNA FCM showed a ploidy relationship of haploid (1N) > diploid (2N) > tetraploid (4N) in cases of normal spermatogenesis. A ploidy relation of 2N > 1N > 4N was observed in cases of hypospermatogenesis or maturation arrest. In Sertoli cell-only cases, there were only 2N populations of cells. With the help of DNA FCM, a rapid and objective assessment of spermatogenesis is possible from FNAC of the testis.     

Determination of natural killer cell function by flow cytometry.

Natural killer cells (NK cells) are a subset of peripheral blood lymphocytes that mediate non-major histocompatibility complex-restricted cytotoxicity of foreign target cells. The "gold standard" assay for NK cell activity has been the chromium release assay. This method is not easily performed in the clinical laboratory because of difficulties with disposal of radioactive and hazardous materials, short reagent half-lives, expense, and difficulties with assay standardization. We describe a flow cytometric assay for the clinical measurement of NK cell activity. This study compared the chromium release assay and the flow cytometric assay by using clinically relevant specimens. There were no significant differences between the two assays in the measurement of lytic activity for 17 peripheral blood specimens or in reproducibility in repeated samplings of healthy individuals. We also established a normal range of values for NK activity in healthy adults and identified a small cluster of individuals who have exceptionally high or low levels of NK activity. The flow cytometric assay was validated by testing specimens from subjects expected to have abnormally low levels of NK activity (pregnant women) and specimens from healthy individuals in whom the activity of NK cells was enhanced by exposure to interleukin-2 or alpha interferon. Treatment with these agents was associated with a significant increase in NK activity. These results confirm and extend those of others, showing that the flow cytometric assay is a viable alternative to the chromium release assay for measuring NK cell activity.     

Quantitation and estimation of lymphocyte subsets in tissue sections. Comparison with flow cytometry

A quantitation method for lymphocyte subsets in immunoperoxidase-stained frozen tissue sections was compared with flow cytometry in 23 cases of non-Hodgkin's lymphoma. Close correlations were obtained, demonstrating the accuracy of the technic. Weak intensity of fluorescence and fragility of the tumor cells during the fluorescence-activated cell sorter (FACS) analyses were the most likely explanations for a number of the discrepancies observed. The tissue quantitation method was precise, particularly at low values, where it was better than the FACS. A simpler and faster estimation method employing categories within 10 percentage units was also tested in this study; this method correlated as well with the FACS as the quantitation method and gave the best interobserver correlations.     

A comparison of TRECs and flow cytometry for naive T cell quantification

Assessment of thymic output by measurement of naive T cells is carried out routinely in clinical diagnostic laboratories, predominantly using flow cytometry with a suitable panel of antibodies. Naive T cell measurements can also be made using molecular analyses to quantify T cell receptor excision circle (TRECs) levels in sorted cells from peripheral blood. In this study we have compared TRECs levels retrospectively with CD45RA⁺CD27⁺ T cells and also with CD45RA⁺CD31⁺ T cells in 134 patient samples at diagnosis or during follow‐up. Both panels provide naive T cell measurements that have a strongly positive correlation with TRECs numbers and are suitable for use with enumerating naive T cell levels in a clinical laboratory.     

Quantitative flow cytometry for the analysis of T cell receptor Vbeta chain expression.

Detailed characterisation of the T cell receptor (TCR) repertoire expressed by peripheral blood lymphocytes has been used to study specific T cell responses in disease conditions. The methods have mostly involved molecular biology analysis of transcribed gene products isolated from T cell subsets or individual clones. Extensive characterisation of the TCR Vbeta chain repertoire by flow cytometry is now possible due to the recently increased availability of specific monoclonal antibodies. However, there are major logistical problems inherent in this analysis relating to the number of cells required to obtain accurate results and the vast amounts of data generated. To reduce these factors to a practical level, we have performed a detailed study to define the limits of precision of cell subset analysis by flow cytometry. Maximal achievable precision was obtained by analysing 10(4) lymphocytes; no significant improvement was obtained by analysing greater numbers of cells up to 10(5) cells, even for cell subsets present at frequencies as low as 0.5%. Careful application of these precision profiles will also permit more effective use of clinical research samples for flow cytometry when the availability of cells is limited.     

Immunoglobulin-specific T-B cell interaction. III. B cell activation by immunoglobulin-recognizing T cell clones

Immunoglobulin (Ig)-specific T-B cell interactions were studied in the model of T cell recognition of Ig kappa chain Ig kappa-1b allotype in Ig kappa-1-congenic rats. Using Ig kappa-1b-recognizing major histocompatibility complex (MHC)-restricted T helper clones from August rats we have shown that Ig kappa-1b+ B cells from congenic August.1b rats presented Ig kappa-1b epitope of the processed self-synthesized Ig to T clones. This interaction was found to be a bidirectional regulatory event inducing specific MHC-dependent proliferation of both interacting T cell and B cell as well as Ig(Ig kappa-1b) synthesis. Small Ig kappa-1b+ B cells were capable of inducing T clone proliferation and becoming activated in response to the same T clone. Limiting dilution analysis suggested that every tenth cell in Ig kappa-1b+ B cell population is involved in this interaction. The bystander activation of Ig kappa-1a+ B cells by T clones in the presence of irradiated Ig kappa-1b+ spleen cells, if observed, was less than the level of specific Ig kappa-1b+ B cell proliferation. In contrast to a 20-fold increase of Ig(Ig kappa-1b) levels upon stimulation of Ig kappa-1b+/1a+ B cell population from heterozygous (August x August.1b)F1 rats by T clones, a "nonspecific" increase of Ig(Ig kappa-1a) was not observed. This result demonstrates the requirement for direct T-B contact for B cell activation to occur. The data suggest a great functional potency of T-B interactions mediated by T cell recognition of Ig-derived peptide/MHC class II complexes on the B cell surface. The implication of the data for idiotypic regulation enables us to propose the existence of putative idiopeptidic network T-B cell interactions.     

Antigen-specific directional target cell lysis by perforin-negative T lymphocyte clones.

Despite a number of reports indicating that perforin, a pore-forming protein, is the primary effector molecule mediating specific target cell lysis by cytotoxic T lymphocytes (CTL), several lines of evidence suggest the existence of perforin-independent mechanisms. We established class II-restricted, soluble protein-specific CD4+ T cell clones with killing function which do not express a detectable amount of perforin and perforin mRNA. Nevertheless, these clones induced cytolysis and DNA fragmentation of target cells in a specific and highly directional manner which was not inhibitable by antibody against TNF/lymphotoxin. These data not only indicate the existence of cytotoxic T cell subsets which do not utilize perforin, but also suggest that perforin is not mandatory for specific target lysis by T cells.     

New technologies in cell analysis by flow cytometry

Automated flow cytometry provides an efficient, sensitive, objective, and quantitative means to analyze cells and their components in suspension. Two major diagnostic applications are the identification of cells in the blood by monoclonal antibodies to surface molecules and the characterization of tumor cell ploidy by nuclear DNA analysis. New technologies permit analysis of small quantities of cells (as in fine-needle aspirates), multiple simultaneous markers, oncogene products, and paraffin-embedded tissue. The technique has become the standard for cells normally in suspension (blood, other fluids). For cells readily obtained in suspension (fine-needle aspirates, nuclei), flow cytometry offers a practical alternative to computer-assisted microscopy.     

单链抗体经流式细胞仪检测细胞表面受体

用流式细胞仪检测两个自然杀伤细胞抑制受体的单链抗体（ＮＫＡＴ２－ｓｃＦｖ；ＮＫＡＴ４－ｓｃＦｖ），识别细胞表面受体分子的能力。单链抗体是吸附固相的抗原筛选一噬菌体抗体库而获得。结果显示，含ＮＫＡＴ２－ｓｃＦｖ的细菌上清及胞质提取物均可识别到表达在细胞表面的ＮＫＡＴ２受体分子，但含ＮＫＡＴ４－ｓｃＦｖ的细菌上清及胞质提取物均未成功     

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.