Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.

Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs.

Highly sensitive single domain antibody-quantum dot conjugates for detection of HER2 biomarker in lung and breast cancer cells.

Despite the widespread availability of immunohistochemical and other methodologies for screening and early detection of lung and breast cancer biomarkers, diagnosis of the early stage of cancers can be difficult and prone to error. The identification and validation of early biomarkers specific to lung and breast cancers, which would permit the development of more sensitive methods for detection of early disease onset, is urgently needed. In this paper, ultra-small and bright nanoprobes based on quantum dots (QDs) conjugated to single domain anti-HER2 (human epidermal growth factor receptor 2) antibodies (sdAbs) were applied for immunolabeling of breast and lung cancer cell lines, and their performance was compared to that of anti-HER2 monoclonal antibodies conjugated to conventional organic dyes Alexa Fluor 488 and Alexa Fluor 568.

High-content imaging in cervical cancer screening.

A shift from conventional cytology to a molecular approach could improve cervical cancer screening. This proof-of-concept study aims to develop a high-content imaging platform for the simultaneous detection of multiple biomarkers for cervical disease. Liquid-based cytology (LBC) samples were used to optimize a dual ProExC/Ki-67 immunofluorescence staining protocol for SurePath-fixed cells.

A flow cytometric protocol for enumeration of endothelial progenitor cells and monocyte subsets in human blood.

Background: Accumulating evidence intensively advises circulating endothelial progenitor cells (EPCs) and monocyte subsets as surrogate cellular biomarkers in cardiovascular and cancer disease. However, a general standard on their quantification is still elusive, thus precluding a routine monitoring and comparative interpretation of clinical studies.

Objective: We intend to develop an advanced and express flow cytometric protocol for proper ex vivo quantification of monocyte subsets and EPCs in human blood.

Immune Reconstitution During the First Year of Antiretroviral Therapy of HIV-1-Infected Adults in Rural Burkina Faso.

There are no data on the outcome of highly active antiretroviral therapy (HAART) in HIV-infected adults in rural Burkina Faso. We therefore assessed CD4(+) T-cell counts and HIV-1 plasma viral load (VL), the proportion of naive T-cells (co-expressing CCR7 and CD45RA) and T-cell activation (expression of CD95 or CD38) in 61 previously untreated adult patients from Nouna, Burkina Faso, at baseline and 2 weeks, 1, 3, 6, 9 and 12 months after starting therapy. Median CD4(+) T-cell counts increased from 174 (10(th)-90(th) percentile: 33-314) cells/µl at baseline to 300 (114- 505) cells/µl after 3 months and 360 (169-562) cells/µl after 12 months of HAART.

Activation and maturation of peripheral blood T cells in HIV-1-infected and HIV-1-uninfected adults in Burkina Faso: a cross-sectional study.

Background: We wanted to explore to what extent environmental exposure to immune stimulants, which is expected to be more present in rural than in urban settings, influences T cell activation and maturation in healthy and in HIV-1-infected individuals in Burkina Faso in west Africa.

Methods: The proportion of circulating naïve T cells and the expression of the T cell activation markers, CD95 and CD38, were analyzed by immunophenotyping and three-colour flow cytometry in 63 healthy individuals and 137 treatment-naïve HIV-1-infected subjects from Ouagadougou (urban setting) and 26 healthy adults and 61 treatment-naïve patients from Nouna (rural).

Results: A slightly higher activation level of CD4(+) and CD8(+) peripheral blood T cells was seen in healthy adults living in Nouna than in those living in Ouagadougou.

Multichromatic phenotyping of HER receptor coexpression in breast tumor tissue samples using flow cytometry--possibilities and limitations.

The prognostic significance of HER2 expression in human breast carcinomas is beyond dispute nowadays. The HER family of receptor tyrosine kinases comprises four members (HER1/ErbB1/EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4) that act in concert via transactivation and consequently compose a functional signaling unit. Besides HER2 overexpression, coexpression of other HER receptors has substantial impact on course of disease and potential therapeutic benefit.

An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood.

Circulating adult CD34(+)VEGFR2(+) endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD14(+) monocytes coexpresses CD16 together with the angiopoietin receptor Tie2 and has been functionally implicated in tumor angiogenesis. However, clinically applicable protocols for flow cytometric quantification of EPCs and Tie2(+) monocytes in peripheral blood and a consensus on reference values remain elusive.

Flow cytometric assay for the simultaneous determination of residual white blood cells, red blood cells, and platelets in fresh-frozen plasma: validation and two years' experience.

Background: A fully automated single-tube assay with tubes (BD TruCOUNT, BD Biosciences) for absolute counting of residual cells in freshly prepared plasma by flow cytometry was developed (BD Plasma Count).

Study Design And Methods: The nucleic acid dye thiazole orange stains white blood cells (WBCs). The monoclonal antibodies anti-CD41a-peridinin chlorophyll protein-Cy5.

Bacteria detection by flow cytometry.

Since bacterial infection of the recipient has become the most frequent infection risk in transfusion medicine, suitable methods for bacteria detection in blood components are of great interest. Platelet concentrates are currently the focus of attention, as they are stored under temperature conditions, which enable the multiplication of most bacteria species contaminating blood donations. Rapid methods for bacteria detection allow testing immediately before transfusion in a bed-side like manner.

Circulating PSA-containing macrophages as a possible target for the detection of prostate cancer: a three-color/five-parameter flow cytometric study on peripheral blood samples.

A major problem of serum prostate-specific antigen (PSA) for predicting prostate cancer risk is diagnostic uncertainty. To detect circulating macrophages with phagocytized fragments (eg, PSA) of prostate tumor cells and determine if the number of circulating PSA-containing macrophages can help differentiate between benign and malignant prostate disease, we collected mononuclear cells from peripheral blood. After labeling the macrophages, phagocytized PSA was detected by incubating the cells with a phycoerythrin-conjugated PSA monoclonal antibody.

Detection of Anisakis simplex-induced basophil activation by flow cytometry.

Background: Laboratory diagnosis of anisakidosis is based on specific serum IgE detection. Recently, detection of allergen-induced basophil activation by flow cytometry has been proposed as a valuable tool for allergy diagnosis.

Objective: To evaluate if detection of Anisakis-induced basophil activation by flow cytometry is a useful tool in the diagnosis of Anisakis allergy.