Detection improvement of cytomegalovirus antigen in human peripheral blood using monoclonal antibodies and automated reading of cell preparations

Boland, Greet J.; Mesker, Wilma E.; Doorn, R. Van; Ploem-Zaaijer, J. J.; Tank, H. J.; Gast, Gijsbert C. de

doi:10.1111/j.1365-2362.1995.tb01979.x

Cited by 6 publications

(3 citation statements)

References 11 publications

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“…In an effort to automate the procedure, recent modifications of the antigenemia assay have included the use of flow cytometry (93) and image cytometry (28,95). With flow cytometry, the 1C3 MAb was superior to the C10-C11 pool; however, the overall sensitivity appeared to be somewhat lower than that of the cytospin method (93).…”

Section: Quantitative Viral Culturesmentioning

confidence: 99%

Quantitation of Cytomegalovirus: Methodologic Aspects and Clinical Applications

1998

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SUMMARY Cytomegalovirus (CMV) is an important pathogen in transplant recipients and human immunodeficiency virus (HIV)-infected individuals. Major progress has been made in developing quantitative detection methods for CMV in recent years. Due to their high sensitivity, these assays can detect CMV early, and quantitation may be useful in predicting the patient’s risk for disease and in monitoring the effect of antiviral therapy. This review discusses methodological aspects of currently used quantitative assays for CMV (i.e., viral culture techniques, antigen detection assays, DNA detection assays including PCR, branched-DNA assay, and the DNA hybrid capture assay) and addresses the correlation of systemic and site-specific CMV load and CMV disease in different populations of immunosuppressed patients as well as the response to antiviral treatment. To date, direct antigen detection and molecular techniques have largely replaced traditional culture-based techniques for CMV quantitation. In general, a high systemic CMV load is correlated with CMV disease. This correlation is strong in the HIV-infected population and in solid-organ transplant recipients but less clear in allogeneic marrow transplant recipients. Measuring the viral load at specific anatomic sites may be an alternative way to assess disease activity in situations where the systemic viral load correlates poorly with disease activity. A reduction of the systemic CMV load also correlates with a response to antiviral treatment, but more research is needed to evaluate the role of viral load as a surrogate marker for drug resistance. Due to the widespread use of quantitative CMV detection techniques to direct and monitor antiviral treatment, there is a great need for an assessment of the reproducibility of test results and better standardization of the assays.

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Section: Quantitative Viral Culturesmentioning

confidence: 99%

Quantitation of Cytomegalovirus: Methodologic Aspects and Clinical Applications

1998

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“…In radiation research, the rare number of mutant cells may be counted as a parameter related to the mutagenic effect in vivo (3,8,17). Finally, information on the number of virus-infected cells circulating in low frequencies in peripheral blood may provide clinical useful information such as the early detection of cytomegalovirus (CMV) reactivation in transplantation (4,14). In many of these applications, monoclonal antibodies directed to characteristic cellular constituents are used to stain the cells of interest, thereby increasing the sensitivity of detection compared to selection based on morphology only.…”

mentioning

confidence: 99%

“…Therefore, attempts have been made to automate the detection and quantification procedure (3,4,7,11,12,14). Basically, one distinguishes approaches based on flow cytometry (3,7,12) or on image cytometry (4,11,14,17). Despite a continuing improvement in speed, image-analysis systems are still slower than flow systems.…”

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confidence: 99%

Detection of immunocytochemically stained rare events using image analysis

et al. 1994

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The detection of rare-event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation.SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS-red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counterstaining based on ethyl green. Slides were automatically screened for cytokeratin-positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 x lo6 negative cells. A comparison between manual screening and automated screening for cytokeratin-positive cells showed a high level of correlation (0.9998).For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study.It is concluded that optimization of preparation and staining procedures for the detection of rare-event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events. 0 1994 Wiley-Liss, Inc.

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