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. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible.

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia.

BACKGROUND: Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare. OBJECTIVES: We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. METHODS: A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >10(4) colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1ß), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. RESULTS: Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1ß was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1ß and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). CONCLUSIONS: Low BALF IL-1ß in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship.

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. The sdAbs-QD conjugates achieved superior staining in a panel of lung cancer cell lines with differential HER2 expression. This shows their outstanding potential for the development of more sensitive assays for early detection of cancer biomarkers.

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. The simultaneous and automated detection of these biomarkers was performed using the BD Pathway 435 system. The ability of high-content imaging to detect dysplastic cervical cells was assessed using keratinocytes spiked with immunopositive SiHa cells and a high-grade squamous intraepithelial lesion (HSIL) LBC sample. The percentages of Ki-67- and ProExC-immunopositive objects correlated significantly with the percentages of spiked SiHa cells. The dysplastic cells of the HSIL sample could be detected using high-content cell analysis. In conclusion, high-content imaging allows the simultaneous and automated detection of Ki-67- and ProExC-immunopositive dysplastic cells in LBC specimens.

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. METHODS: We employ now lyse/no-wash procedure and bead-based determination of absolute cell counts. We use three-color antibody panels at appropriate compensation. Analysis of rare events and low antigen expression in the EPC experiment is strengthening by sequential gating with exclusion of dead cells, as well as by matching high-intensity fluorochromes to low-density markers and by implementing the fluorescence-minus-one control. RESULTS: Analysis of peripheral blood of ten healthy donors revealed median (IQR) value of 1.88 (1.35-2.85) viable CD45(dim)CD34)VEGFR2+ EPCs per microliter. Analysis of monocytes revealed 329.5 (264.5-374.8), 16.0 (8.0-22.2) and 26.5 (19.8-36.3) cells per microliter for classical CD14++(high))CD16⁻, intermediate CD14++CD16+(mid) and non-classical CD14+(low))CD16++ monocytes. CONCLUSION: Our current protocol provides quantitative information under a simple gating logic while using commonly accepted fluorochromes. This assay is therefore highly adapted for routine use.

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. Median VL decreased from 5.8 (4.6- 6.6) log10 copies/ml at baseline to 1.6 (1.6-2.3) log10 copies/ml after 12 months. Early CD4(+) T-cell recovery was accompanied by a reduction of the expression levels of CD95 and CD38 on T-cells. Out of 42 patients with complete virological follow-up under HAART, 19 (45%) achieved concordant good immunological (gain of ≥100 CD4(+) T-cells/µl above baseline) and virological (undetectable VL) responses after 12 months of treatment (intention-to-treat analysis). Neither a decreased expression of the T-cell activation markers CD38 and CD95, nor an increase in the percentage of naive T-cells reliably predicted good virological treatment responses in patients with good CD4(+) T-cell reconstitution. Repeated measurement of CD4(+) T-cell counts during HAART remains the most important parameter for immunologic monitoring. Substitution of repeated VL testing by determination of T-cell activation levels (e.g., CD38 expression on CD8(+) T-cells) should be applied with caution.

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. The percentages of naïve CD45RA(bright) CCR7(+) T cells were not significantly different between both study sites. Taking into consideration that relatively more HIV-1-infected patients in Nouna were in an advanced disease stage, no relevant differences were seen in T cell activation and maturation between patients at both study sites. As expected, the percentage of CD95(+) CD4(+) and CD38(+) CD8(+) T cells and the respective antigen density on these cells was significantly higher in patients than in controls in both settings. The percentage of naïve CD8(+) T cells was lower in HIV-1-infected subjects than in healthy controls irrespective of the study site, while a lower proportion of naïve CD4(+) T cells in patients compared with controls was seen only in Nouna. CONCLUSIONS: Environmentally triggered immune activation may contribute to the increased expression of the activation markers CD95 and CD38 on peripheral blood T cells from healthy adults living in rural versus urban settings in Burkina Faso. T cell activation is further increased in HIV-1-infected individuals due to T cell loss and high plasma viral load levels. The observed variations in T cell activation levels or the proportion of naïve T cells in our study patients, however, are not explained by differences in CD4(+) T cell counts or HIV-1 plasma viral load levels alone.

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. This observation is substantiated by numerous preclinical studies and retrospective studies done on patients with breast cancer. Against this background, the quantification of all HER receptor expressions at the same time would significantly extend the information content revealed by routine diagnosis of breast cancer tissues. Moreover, the knowledge of HER receptor coexpression profiles in primary tumor samples could provide the basis to design and develop highly specific antireceptor treatment strategies. Here, we report on a simultaneous flow cytometric detection of all four HER receptors on carcinoma cells isolated from primary breast cancer tissues and separated from nonepithelial cells by cytokeratin staining. Combined with DNA, i.e. ploidy quantification, the approach resulted in a six-parameter assay that could complement the diagnosis of a variety of diseases in which HER receptor expression has a pivotal impact on the degree of malignancy.

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. The number of Tie2(+)CD14(+)CD16(mid) angiogenic monocytes and CD34(+)VEGFR2(+)CD45(low/-) EPCs was assessed in the peripheral venous blood of patients with stable coronary artery disease by three-color flow cytometry using specific monoclonal antibodies conjugated to PerCP, PE, PE-Cy7, APC, and APC-Cy7. Scatter multigating with exclusion of dead cells was performed to dissect complex mononuclear cell populations. This analysis was further refined by matching bright fluorochromes (PE-Cy7, PE, APC) with dimly expressed markers (CD34, VEGFR2, Tie2), by automatic compensation for minimizing fluorescence spillover and by using fluorescence-minus-one (FMO) controls to determine positive/negative boundaries. Presuming a Gaussian distribution, we obtained average values (mean +/- SD) of 1.45 +/- 1.29% for Tie2(+)CD14(+)CD16(mid) monocytes (n = 11, range: 0.12-3.64%) and 0.019 +/- 0.013% for CD34(+)VEGFR2(+)CD45(low/-) EPCs (n = 17, range: 0.003-0.042%). The intra- and inter-assay variability was 1.6% and 4.5%, respectively. We have optimized a fast and sensitive assay for the flow cytometric quantification of circulating angiogenic monocytes and EPCs in cardiovascular medicine. This protocol may represent a basis for standardized analysis and monitoring of these cell subsets to define their normal range and prognostic/diagnostic value in clinical use.

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-Cy5.5 and anti-glycophorin A-fluorescein isothiocyanate label platelets (PLTs) and red blood cells (RBCs), respectively. No fixation, permeabilization, or washing steps were required. Validation was done according to guidelines of the International Conference on Harmonization and the National Committee for Clinical Laboratory Standards. Cell-free plasma was spiked with each cell type for accuracy, reproducibility, and linearity measurements. RESULTS: Results showed no carryover or drift under automated sample acquisition conditions. Nonspecific background was fewer than 0.3 cells per microL for residual WBCs (rWBCs), fewer than 2.7 cells per microL for rRBCs, and fewer than 85 cells per microL for rPLTs. Determinations of rWBC and rPLT counts were linear with a coefficient of variation of less than 12 percent for the imprecision. Owing to cross-linking of the anti-glycophorin A antibody, linearity and precision for rRBCs diverged up to 21 percent at a count of 6000 rRBCs per microL. In a 2-year period, five operators investigated 2666 quality control (QC) samples of fresh-frozen plasma on 108 working days. Maximum cell numbers found were 196 for rWBCs, 3960 for rRBCs, and 28,952 for rPLTs per microL. In 31 cases (1.2%) rWBCs were out of specification. No outlier was observed for rRBCs and rPLTs. Residual RBC cell numbers determined were always within the acceptable concentration range of the assay. CONCLUSION: These data demonstrate that the single-tube test is suitable for routine QC assessment of the cellular contaminants of therapeutic plasma according to the European recommendations.

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. This approach would overcome the sampling error observed in early sampling combined with culturing of bacteria and would, at least, prevent the transfusion of highly contaminated blood components leading to acute septic shock or even death of the patient. Flow cytometry has been demonstrated to be a rapid and feasible approach for detection of bacteria in platelet concentrates. The general aim of the current study was to develop protocols for the application of this technique under routine conditions. The effect of improved test reagents on practicability and sensitivity of the method is evaluated. Furthermore, the implementation of fluorescent absolute count beads as an internal standard is demonstrated. A simplified pre-incubation procedure has been undertaken to diminish the detection limit in a pragmatic manner. Additionally, the application of bacteria detection by flow cytometry as a culture method is shown, i.e., transfer of samples from platelet concentrates into a satellite bag, incubation of the latter at 37 degrees C, and measuring the contaminating bacteria in a flow cytometer.

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. Flow cytometric analysis was performed. A significant difference was observed in the mean+/-SD percentage of activated macrophages (CD14+/CD16+) between malignant (28.8%+/-13.0%) and benign conditions (17.3%+/-6.8%; P< .0001). A significant increase was detected in the percentage of PSA-containing macrophages in prostate cancer (17.7%+/-12.3%) vs benign disorders (3.6%+/-1.9%; P< .0001) and between localized (10.5%+/-3.4%) and metastasized prostate carcinoma (26.3%+/-14.3%; P= .0002). The new method for detecting circulating PSA-containing macrophages can be suitable for differentiating prostate cancer from benign conditions and, possibly, low-risk from more aggressive prostate cancer.

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. METHODS: Patients with Anisakis allergy (A.s.+, n = 37), patients reporting chronic urticaria or abdominal pain unrelated to fish ingestion (A.s.-, n = 51), and healthy controls (n = 12) were studied. Specific IgE to Anisakis simplex (A. simplex) was quantified with CAP-FEIA method, and basophil activation test was performed with three different concentrations of an Anisakis crude extract. Basophil gating was performed with CD123 and HLA-DR, and cellular activation was measured with CD63. RESULTS: A.s.+ patients showed significantly higher age and total IgE levels than did the A.s.- patients. Specific IgE to A. simplex correlated with the activated basophil percentages obtained with 15 microg/mL (r = 0.80; P < 0.001), 1.5 microg/mL (r = 0.84; P < 0.001), and 0.15 microg/mL (r = 0.82; P < 0.001) of A. simplex crude extract. Nine individuals (3 in the A.s.+ group and 6 in the A.s.- group) were nonresponders to basophil stimulation with anti-IgE. Five A.s.- patients showed positive IgE values to A. simplex while the basophil activation test was negative. According to the receiver operating characteristics curves performed between A.s.+ vs. A.s.- and A.s.+ vs. healthy controls, the cutoff for a positive basophil activation test was >or=21% (specificity = 96%, sensitivity = 100%), and 16% (sensitivity and specificity of 100%) respectively. When nonresponders were included in the A.s.+ vs. A.s.- analysis, sensitivity decreased to 95%. Multivariate logistic analysis showed that the specific basophil activation was a factor independently associated with clinical symptoms of A. simplex allergy. CONCLUSIONS: Detection of A. simplex-induced basophil activation by flow cytometry is a useful laboratory technique for the diagnosis of anisakidosis, supplementing specific IgE determinations.