Volume determination of human metaphase chromosomes by scanning force microscopy

The scanning force microscopy (SFM) yields the topography of the investigated surface. A procedure was developed which starts from this three-dimensional information to estimate the volume of a biological specimen. The volume of spread human metaphase chromosomes was determined in air and rehydrated in aqueous buffer. A difference of the determined volume of a air-dried metaphase chromosome set was found compared to values from electron microscopic investigations, and could be correlated with differences in the hydration state of the chromosomes. SFM-based relative volumes of air-dried chromosomes resembles literature data regarding volume range and distribution. Possible application of SFM-based relative volume measurements for chromosome classification purposes is discussed.     

Antibiotic susceptibility testing by flow cytometry

The first significant development in antibiotic susceptibility testing in recent years may be the flow cytofluorometric susceptibility test (FCST). The advanced analytical capability of the flow cytometer has provided the means to measure microbial diversity in culture. Membrane integrity and other indicators of microbial viability can be evaluated on a cell-by-cell basis. The FCST measures subtle dosage-response effects as well as the conventional minimum inhibitory and minimum bactericidal concentrations, simultaneously, in rapid tests which have the potential to supersede conventional techniques in terms of sensitivity and reproducibility.     

T-cell epitope mapping by flow cytometry

From March 1994 to September 1996, 39 patients underwent stenting of the unprotected left main coronary artery because of high surgical risk. Stenting appeared to improve clinical outcome, but there was a significant mortality rate at long-term follow-up.     

Detection of intracellular cytokines by flow cytometry

During the last years it has become increasingly clear that production of most cytokines is not confined to one cell type. Thus, a method to detect cytokines at the single cell level would be a helpful tool to study the contribution of different cells to cytokine production in heterogeneous cell populations. Recently, Sander et al. (1991) demonstrated that it is possible to detect intracellular cytokines by fixation with paraformaldehyde, permeabilization with saponin and subsequent indirect immunofluorescent staining using fluorescence microscopy. Here, we describe a modified method to increase the specific intracellular staining which enables us to detect IFN-gamma, IL-2 and IL-4 producing cells by single laser flow cytometry. The carboxylic ionophore monensin was used to interrupt intracellular transport processes leading to an accumulation of the cytokine in the Golgi complex. This resulting increase of the signal/noise ratio permitted us to detect weakly fluorescent cells such as IL-4 producing cells. While IL-4 was detected in approximately 1-3% of peripheral mononuclear cells from healthy donors, up to 30% of the cells produced IFN-gamma and nearly 50% IL-2 after phorbol ester and ionomycin stimulation. Microscopic and flow cytometric analysis showed a highly significant correlation. Using three-color flow cytometry it was possible to measure intracellular cytokines and cell surface markers simultaneously. Subpopulations of human T cells (e.g., CD4+ CD45R0-) producing a restricted cytokine pattern could be identified by cell surface staining and were characterized by their cytokine production. Consequently, there was no further need for cell sorting to determine cytokine producing subsets in heterogeneous cell populations. We have tested human T cell clones for intracellular cytokine production and found a high concordance to ELISA analysis of the supernatants. We conclude that detection of intracellular cytokines by flow cytometry is a rapid, easy and semiquantitative assay which may be used to study individual cells in heterogeneous populations as well as to screen homogeneous cells for their cytokine pattern. This method is particularly relevant in view of the accumulating evidence of the functional role that subsets of (T) cells may play in various diseases depending on the pattern of cytokines they produce.     

A comparison of cytogenetic studies and flow cytometry in breast carcinomas

The increased use of DNA-based typing techniques has improved the accuracy and reliability of HLA types assigned to patients requiring an allogeneic bone marrow transplant and their potential donors, facilitating better donor selection. The benefits of this technology, which will completely replace serologic typing within the next few years, have not yet been fully exploited since the typing information obtained in the form of DNA sequence polymorphisms is presently converted to an HLA allelic type for submission to a registry. Furthermore, within the registry, the allelic type may be further converted to a serologic antigen for use in the search and match process. Because the current nomenclature system makes selection of donors who are likely to provide optimal matches difficult, it is critical that DNA-based HLA typing data be recorded by registries as sequence polymorphisms tested as positive or negative rather than interpreted HLA types and that searching and matching utilize these polymorphisms. Until this transition is complete, we will not fully utilize this powerful HLA typing technology to best serve those patients seeking unrelated donors.     

Further studies on the immunopathology of atopic keratoconjunctivitis using flow cytometry

Atopic keratoconjunctivitis (AKC) is an ocular manifestation of systemic hypersensitivity. Although the pathogenesis of AKC is not fully understood, some previous data suggest that a decrease in numbers of suppressor T lymphocyte (Ts) and increase of Th, especially Th2 (the second subgroup of helper T lymphocyte), at the ocular surface may play an important role in the occurrence of the disease. In this study, the percentages of naive-Th (CD4/45RA+) and memory-Th (CD4/29+) cells, and the Th/Ts and memory-Th cells/naive-Th cells ratios were measured in the blood and tear samples of patients with AKC, atopic patients without ocular involvement and normal volunteers, using flow cytometry. Groups were compared using the Mann-Whitney U test. We found that patients with AKC had significantly higher memory-Th cell concentration, and Th/Ts and memory-Th cells/naive-Th cell ratios both in the tear and blood samples compared to normal subjects. While no significant difference existed between the tear samples of the atopic patients without ocular involvement and normal volunteers with respect to the above values, atopic patients had higher percentages of memory-Th cells and higher Th/Ts and memory-Th cells/naive-Th ratios in their blood than normal subjects. The percentages of memory-Th cells, and the Th/Ts and memory-Th cells/naive-Th cell ratios in the tear samples of AKC patients were also found to be higher than that of the atopic patients without ocular involvement, but no significant difference was present between the blood samples of these groups. The percentages of naive-Th cells did not show any significant difference between groups either in tear or blood samples. Since the mean memory-Th cells/naive-Th1 cells ratio in the tear samples of the patients with AKC was higher than in their blood samples, we propose that the localized accumulation of memory-Th2 cells, in addition to the increase of Th/Ts ratios in the external eye may cause AKC in atopic individuals.     

The study of apoptotic cells by flow cytometry

Programmed cell death (PCD) is of fundamental importance in the normal development of an animal. It also plays a key role in tumour and radiation biology. PCD produces a sequence of changes occurring in cells called apoptosis. The main elements of this apoptotic cascade are rapidly being elucidated. Flow cytometry has been used to follow many of these changes. It has also been used to quantify the number of apoptotic cells in a culture and, more recently, in clinical samples. In this review, the properties of apoptotic cells and the main features of the apoptotic cascade are described. How flow cytometry can be used to follow changes during the apoptotic cascade is then discussed.     

IgG anti-D MAbs in tests by flow cytometry

The faunistic results regarding intestinal parasitism by protozoa and helminths in 21 primate species (three Cebidae, thirteen Cercopithecidae, one Hylobatidae, one Lemuridae, three Pongidae) are reported. The primate species were housed in four separate galleries. Six faecal samples of each host species were subjected to coprological analysis. Fifteen parasite species were detected: 11 protozoa (Entamoeba coli, E. chattoni, E. hartmanni, Iodamoeba bütschlii, Endolimax nana, Giardia intestinalis, Chilomastix mesnilii, Enteromonas hominis, Trichomonas intestinalis, Balantidium coli, and Blastocystis hominis) and 4 helminths (Ancylostoma sp., Strongyloides fuelleborni, Strongyloides sp., and Trichuris trichiura). The results reveal certain parasitic similarities between host species housed in the same gallery; however, these primate species do not always carry identical parasite species.     

Phagosomal pH determination by dual fluorescence flow cytometry

This summary describes the psychiatric assessment of infants and toddlers (0-36 months) and supports the growth of infant and toddler psychiatry, a rapidly developing field. Infants and toddlers are brought to clinical attention because of concerns about emotional, behavioral, relational, or developmental difficulties. It is axiomatic that the infant or toddler must be understood evaluated and treated within the context of the family. A perspective that is developmental relational, and multidimensional and that borrows from the knowledge of multiple disciplines is essential. Collaborative efforts support the urgent need and incomparable opportunity to understand and to intervene early and preventively with young children and their families.     

Detection of myeloperoxidase by flow cytometry in acute leukemia

BACKGROUND: To evaluate long-term results of intraocular pressure after trabeculectomy for congenital glaucoma. METHODS: Data concerning 55 eyes (30 patients) who underwent trabeculectomy for congenital glaucoma were recorded. Mean age at diagnosis was 3.4 months (range: 2 days to 10 months). Mean follow up was 56.8 months. Associated anterior segment abnormalities, need for one or more new trabeculectomy procedures during follow up, and intraocular pressure at the last examination were noted. RESULTS: Of the 55 eyes, 48 met the success criteria (87.3%). A second and sometimes third or fourth trabeculectomy were necessary during follow up in 17 eyes (31%). Of the seven failures at final examination, six (85%) had been diagnosed and operated on before the age of 1 month, whereas 15 of the 48 eyes with good results (31.2%) were in this group (p < 0.02). Of the seven failures at final examination, six (85%) were operated on two to four times, whereas 10 of the 48 eyes with good results (20.1%) were in this group (p < 0.01). An associated anterior segment abnormality was present in 13 eyes (23%), and did not seem to influence the final outcome. CONCLUSION: Trabeculectomy is an effective procedure for long-term control of intraocular pressure in congenital glaucoma. The early diagnosis and surgical treatment correspond to a poor long-term prognosis, probably related to initially severe cases. In these cases, intraocular pressure is difficult to control despite repeated surgical procedures.     

Characterization of stromal progenitor cells enriched by flow cytometry

The progenitors for cells of bone, cartilage, fat, and muscle are thought to be derived from mesenchymal stem cells but despite extensive study of stromal cell differentiation, neither mesenchymal stem cells or the more committed, tissue-specific progenitors have been well-characterized. In this study we used flow cytometry to isolate from fetal rat periosteum a population of small, slowly cycling cells with low cytoplasmic granularity (S cells) that display stem cell characteristics. On plating, S cells exhibited a 90% higher labeling index with [3H]-thymidine compared to unsorted cells and when grown in culture generated cartilage, adipocyte, and smooth muscle phenotypes, in addition to bone. Only the S-cell population showed extensive self-renewal of cells with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphatase, or osteopontin. However, after attachment, S cells became immunopositive for collagens I, II, III, osteopontin, and also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. These studies show that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacity, and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric methods. These cells may be useful for regeneration of stromal tissues.     

Measurement of ceroid accumulation in macrophages by flow cytometry

The accumulation in macrophages of ceroid, an autofluorescent polymer composed of oxidised protein and lipid, can be monitored semiquantitatively by staining techniques. However, such methods are crude and give little information about the amount of ceroid within cells. Flow cytometry, however, can give a quantitative assessment of cellular ceroid accumulation in vitro. Recently, flow cytometry was explored as a method for measurement of the accumulation in macrophages of ceroid. The accumulation appeared to be diminished in the presence of the antioxidant, alpha-tocopherol. This is consistent with the role of lipoprotein oxidation in ceroid accumulation. Here the optimum wavelengths of emission and excitation, using both conventional fluorescence spectroscopy of cellular ceroid and flow cytometric measurements with a number of optical filters, are determined. The use of optimal wavelengths determined in these studies enhances overall sensitivity. The findings are discussed in the context of future investigation of cell-mediated lipid oxidation and its potential antagonists.     

双表型急性白血病流式细胞术免疫分型检测结果

目的 应用流式细胞术(FCM)检测双表型急性白血病(BAL)的免疫表型.方法 采用四色FCM检测23例BAL患者的免疫表型.结果 23例BAL患者中有10例(43.4%)表达cCD3,16例(69.6%)表达cCD79a,20例(87.0%)表达cMPO,14例(60.9%)表达TdT,19例(82.6%)表达CD34,20例(87.0%)表达CD117,同时表达髓系和B系抗原者13例(56.5%),均共同表达cCD79a和cMPO;同时表达髓系和T系抗原者7例(30.4%),均共同表达cCD3和cMPO;同时表达T系和B系抗原者3例(13.04%),均共同表达cCD3和cCD79a.结论 cCD3、cCD79a、cMPO为系列特异性抗原标志,对诊断及鉴别BAL具有重要意义,FCM是目前诊断BAL特异口靠的方法,在临床白血病治疗和预后方面有重要的指导意义.     

Automation of human sperm cell analysis by flow cytometry

Semen sample analysis is routinely performed by microscopical evaluation and manual techniques by laboratory operators; the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. Our aim was to automate sperm analysis with the use of flow cytometry for evaluation of cell counts and typing and with the use of a new membrane-permeant nucleic acid stain for evaluation of sperm viability. Statistical analysis of the comparison between manual and automated methods for sperm counts was performed by the Bland and Altman method; the mean difference was 0.243 x 10(6) sperms/ mL. The precision of the flow cytometric analysis was evaluated with whole sperm; the between-run CV was 7.5% and the within-run CV was 2.5%. Data observed suggest that flow cytometric sperm analysis, with high precision and accuracy and low costs, can be proposed for routine use in clinical laboratories.     

DNA analysis in hepatoblastoma by flow and image cytometry

BACKGROUND: In several types of tumors, including hepatocellular carcinoma, prognosis could be correlated with DNA ploidy. Few studies have been performed on hepatoblastoma with contradictory results. METHODS: Twenty-nine cases of nonpretreated hepatoblastoma were studied with flow cytometry and image cytometry for DNA index and proliferation index using paraffin-embedded tissue. RESULTS: Twenty-three (79.9%) tumors were diploid, and 6 (20.7%) were aneuploid (hyperdiploid). Patients with diploid tumors were younger than those with aneuploid tumors. With regard to stage, diploid tumors were almost equally distributed among stages (tumor, lymph node metastases, distant metastases), whereas aneuploid tumors tended to occur in higher stages (tumor, lymph node metastases, distant metastases). Diploid tumors had clearly a better prognosis than aneuploid tumors, although the difference was not statistically significant (flow cytometry, P = 0.06; image cytometry, P = 0.16). A more favorable prognosis was also noted for hepatoblastomas with low-proliferation index (< or = 7%), but the difference from tumors with high-proliferation index (> 7%) again was not statistically significant (P = 0.16). CONCLUSIONS: Although no statistically significant differences in prognosis between hepatoblastomas with diploid and aneuploid DNA content, respectively, were found, there is a clear tendency that diploid hepatoblastomas behave more favorably. The same is true for hepatoblastomas with low-proliferation index.     

Analyte and label binding assay read by flow cytometry

A new immunometric two-site sandwich assay is introduced, in which a label-scavenging binding partner is added to the sample in addition to the analyte-binding partner. The scavenger binding partner binds excess label antibody, giving a signal proportional to the amount of excess label antibody in the sample solution. A set of two calibration curves is obtained from the two binding partners simultaneously, and a combination of the two signals gives an unambiguous determination of the analyte concentration, even for high analyte concentrations where the hook effect may occur. Two-particle immunofluorometric assays developed for placental alkaline phosphatase and human chorionic gonadotropin on the basis of this principle and yielding signals measured by flow cytometry gave rapid results (2 h) and had working ranges in excess of 5 and 6 orders of magnitude for the respective analytes.