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Bibliography

Bibliography

Basic Research

Human Blood Stabilisation - Immunophenotyping

2010

Direct Relationship between Virus Load and Systemic Immune Activation in HIV-2 Infection

Sample Type: T cell activation markers

Analysis Method: Flow Cytometry

Authors

A. Leligdowicz, J. Feldmann, A. Jaye, M. Cotton, T. Dong, A. McMichael, H. Whittle, S. Rowland-Jones

Publication

J Infect Dis (2010) 201 (1): 114-122

This paper reports a study into the relationship between virus load and systemic immune activation in HIV 2 infection. TransFix was used to stabilise whole blood for 2-14 days and used for determination of T cell surface activation marker expression with anti-HLA-DR, CD38, CD4, and CD8.

https://academic.oup.com/jid/article/201/1/114/870467/Direct-Relationship-between-Virus-Load-and

2009

Downregulation of the T-Cell Receptor by Human Immunodeficiency Virus Type 2 Nef Does Not Protect against Disease Progression

Sample Type: T-cells

Analysis Method: Flow Cytometry

Authors

Jérôme Feldmann, Aleksandra Leligdowicz, Assan Jaye, Tao Dong, Hilton Whittle, and Sarah L. Rowland-Jones

Publication

Journal of Virology (2009) 83(24): 12968-12972

T-cell activation marker expression. Fresh whole blood was stabilized in a 5:1 ratio with TransFix (Cytomark) for 2 to 14 days and used for determination of T-cell surface activation marker expression using anti-HLA-DR-fluorescein isothiocyanate-, CD38-phycoerythrin (PE)-, CD4-peridinin chlorophyll protein-, and CD8-allophycocyanin-titrated monoclonal antibodies (BD Pharmingen).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786859/

2004

KEY PAPER: Evaluation of leukocyte stabilisation in TransFix treated blood samples by flow cytometry and transmission electron microscopy.

Sample Type: Lymphocytes

Analysis Method: TEM, Flow Cytometry

Authors

B. Canonico, L. Zamani, S.Burattini, V. Granger, F. Mannello, P.Gobbi, C. Felici, E. Falcieri, J.T.reilly, D.Barnett and S.Papa

Publication

Journal of Immunological Methods (2004) 295: 67-78

The study evaluated the effects of TransFix on leucocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology. The results showed that this short term stabilisation method is particularly suitable for the analysis of human lymphocytes. Good results were obtained with respect to antigen definition and absolute cell counting procedures

http://www.ncbi.nlm.nih.gov/pubmed/15627612

2002

KEY PAPER: Evaluation of TransFix, a commercial whole blood stabilizing reagent. This product reduces HIV replication

Sample Type: CEM 13D cells

Analysis Method: P24 AG assay

Authors

Kim et al

Publication

Clinical Cytometry (2002) 50: 281

Samples treated with TransFix retain morphology and cell surface expression over time. Shipping and handling of HIV+ samples involved inherent elevated costs and a risk of exposure to the HIV virus. The study showed that HIV+ blood samples showed a 1-log reduction in HIV replication as measured by HIV p24 antigen production. The results indicate that TransFix has the ability to cause significant reduction in HIV replication.

2010

KEY PAPER: Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes and red cells

Sample Type: Lymphomonocytic cells (blood)

Analysis Method: SEM, TEM, Western blotting, and ?ow cytometry (biomolecular and morphological aspects), FC, Tunel, and electrophoresis (DNA behavior)

Authors

Canonico B, Betti M, Luchetti F, Battistelli M, Falcieri E, Ferri P, Zamai L, Barnett D, Papa S.

Publication

Cytometry B, Clinical Cytometry. 2010 July: 78(4): 267-78

Cytometric performance is suboptimal in aged unfixed specimens because of apoptosis that affects light scatter properties. Our findings highlight that lymphomonocytic cells are well stabilized even at suboptimal temperature and cell density. TransFix is able to abolish any apoptotic features and acts as an optimal blood preservative for appropriate preanalytical stabilization.

http://onlinelibrary.wiley.com/doi/10.1002/cyto.b.20510/full

2009

KEY PAPER: Method validation and immunophenotypical study of NK and T lymphocytes by flow cytometry

Sample Type: NK and NKT cells

Analysis Method: Flow Cytometry

Authors

E. Konsta, E. Kwvota

Publication

National and Kapodistrian University of Athens

The purpose of this study was the clarification of the importance of the phenotypic markers oh human NK and NKT cells definition in subjects with a significant increase of these cells percentage. Specimens with malignant populations (B-Chronic Lymphocytic Leukaemia, Acute Myeloblastic Leukaemia and Myelodysplastic Syndrome) were studied in order to evaluate the suitability of a blood stabilising reagent (TransFix) in routine laboratory practise. The study indicates that TransFix can be used reliably in flow cytometric analysis – light scatter characteristics and immunophenotype are well preserved in TransFix-treated specimens stored at 4-10 degrees Celsius, particularly concerning the blasts and lymphocyte populations for 15 days.

http://phdtheses.ekt.gr/eadd/handle/10442/26992

Method Validation and Study of Immunophenotype NK and T lymphocytes by flow cytometry

Sample Type: NK and NKT malignant populations (B-Chronic Lymphocytic Leukaemia, Acute Myeloblastic Leukaemia and Myelodysplastic Syndrome)

Analysis Method: Flow Cytometry

Authors

E. P. Konstas

Publication

PhD Thesis (2009) National and Kapodistrian University of Athens (NCAA)

The purpose of this study was to clarify the importance of the phenotypic markers of human NK and NKT cells in subjects with a significant increase of these cells. One of the aims was to study subjects with malignant populations (B-Chronic Lymphocytic Leukaemia, Acute Myeloblastic Leukaemia and Myelodysplastic Syndrome) in order to evaluate the suitability of a blood stabilizing reagent (TransFix®) in routine laboratory practice. The Author found that TransFix® stabilising reagent can be used reliably in flow cytometric analysis; light scatter characteristics and immunophenotype are well preserved in TransFix®-treated specimens stored at 4-10?C particularly concerning the blasts and lymphocytes populations for 15 days.

https://phdtheses.ekt.gr/eadd/handle/10442/26992

2012

TransFix for delayed flow cytometry of endothelial progenitor cells and angiogenic T cells

Sample Type: Endothelial progenitor cells (EPC) and angiogenic T cells

Analysis Method: Flow Cytometry

Authors

V. Y. Hoymans, A. H. Van Craenenbroeck, L.Bruyndonckx, S. H. van Ierssel, C .J. Vrints, V. M. Conraads, E. M. Van Craenenbroeck

Publication

Microvascular Research, Volume 84, Issue 3, November 2012, Pages 384–386

Endothelial progenitor cells (EPC) and angiogenic T cells have not been validated for use in studies that involve delayed sample processing and analysis. Here, we report our results for the flow cytometric enumeration of circulating EPC and angiogenic T cells using TransFix-treated whole blood obtained from adult patients with cardiovascular disease and healthy volunteers. Both cell types promote neovascularization and vascular homeostasis. As such they have been put forward as novel diagnostic markers for endothelial dysfunction and may add prognostic information in patients with cardiovascular disease. Our findings indicate that by the addition of TransFix cellular antigen stabilizing reagent to whole blood, analyses can be postponed up to 7 days after blood collection. Therefore, this procedure may facilitate laboratory workflow, as well as the organization of multicenter studies, which requires analyses to be conducted in a central core laboratory.

http://www.sciencedirect.com/science/article/pii/S0026286212001562

Human Blood Stabilisation - Circulating Tumour Cells

2013

4EVER: Assessment of circulating tumor cells with a novel, filtration-based method, in a phase IIIb multicenter study for postmenopausal, HER2-negative, estrogen receptor-positive, advanced breast cancer patients

Sample Type: Circulating tumour cells

Analysis Method: Fluorescence scanning microscopy

Authors

P. A. Fasching et al

Publication

J Clin Oncol 31, 2013 (suppl; abstract 591)

The presence of circulating tumor cells (CTC’s) has been shown to be of diagnostic relevance for patients with early and advanced breast cancer (BC) The usefulness of CTC assessments depends upon accurate cell counts and the corresponding analysis of molecular targets. Patient blood samples were preserved using TransFix vacuum blood collection tubes and CTC’s were detected by image analytics after fluorescence scanning microscopy. The clinical trial is ongoing.

http://meetinglibrary.asco.org/content/117629-132

2016

KEY PAPER: Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer

Sample Type: CTCs, circulating tumour cell clusters (CTC clusters), CTCs of epithelial-mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs).

Analysis Method: Immunofluorescence staining, Cell sorting, Sanger sequencing

Authors

Z. Mu, N. Benali-Furet, G. Uzan, A. Znaty, Z. Ye, C. Paolillo, C. Wang, L. Austin, G. Rossi, P. Fortina, H. Yang, M. Cristofanilli

Publication

Int. J. Mol. Sci (2016) 17(10): 1665

This paper describes the use of the Screencell method of size-exclusion isolation of CTCs, circulating tumour cell clusters (CTC clusters), CTCs of epithelial-mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs). Blood samples collected in TransFix tubes and analysed within 72 hours. Cytological Analysis was performed by immuno?uorescence staining of cytokeratins (CK-8, 18 and 19), DAPI, CD45 for CTCs, and EMT-CTCs were stained for Vimentin and N-Cadherin. Cell sorting was performed by the DEPArray™ system. TP53 and ESR1 Mutations in CTCs were analysed by Sanger sequencing.

http://www.mdpi.com/1422-0067/17/10/1665/html

2015

Development of a method to measure acetylated histone H4 in the nuclei of circulating myeloid cells as a surrogate tissue for the pharmacodynamics of HDAC inhibitors in the treatment of solid tumors

Sample Type: Circulating myeloid cells

Analysis Method:  imaging flow cytometer

Authors

J. David, W. Wong, G. Veal

Publication

AACR Cancer Res (2015) 75(15 Suppl):Abstract nr 5374

This conference abstract details the development and validation of an assay to measure the proportion of myeloid cells in peripheral blood with nuclei localised acetylated Histone H4 detectable by immunofluorescence and imaging flow cytometry. Blood samples treated with the potential anti­cancer agent sodium valproate were then stabilised in TransFix, stained by anti­acH4 and DAPI then analsysed by an Imagestream MkII imaging flow cytometer.

http://cancerres.aacrjournals.org/content/75/15_Supplement/5374.short

2016

High-resolution imaging for the detection and characterisation of circulating tumour cells from patients with oesophageal, hepatocellular, thyroid and ovarian cancers

Sample Type: Circulating tumour cells

Analysis Method:  imaging flow cytometer

Authors

B. M. Dent, L. F. Ogle, R. L. O'Donnell, N. Hayes, U. Malik, N. J. Curtin, A. V. Boddy, E. R. Plummer, R. J. Edmondson, H. L. Reeves, F. E. B. May, D. Jamieson

Publication

International Journal of Cancer (2016) 138(1): 206-216

The purpose of this study was to develop and validate a novel, widely applicable method for detection and characterisation of circulating tumour cells from 4 tumour types. Blood samples were collected in TransFix collection tubes and stained for immunofluorescence by the following membrane antibodies and nuclear stains: EpCAM, cytokeratins 4, 5, 6, 8, 10, 13 and 18, survivin and CD45, and DAPI or DRAQ5. Circulating tumour cells were enriched using an EasySep human CD45 depletion kit, and were analysed using an ImageStream X Flow cytometer.

http://onlinelibrary.wiley.com/doi/10.1002/ijc.29680/full

Human Blood Stabilisation - Other

2015

CD26 Expression on T Helper Populations and sCD26 Serum Levels in Patients with Rheumatoid Arthritis

Sample Type: T helper cells

Analysis Method: Flow Cytometry

Authors

O. J. Cordero, R. Varela-Calviño, T. López-González, C. Calviño-Sampedro, J. E. Viñuela, C. Mouriño, I. Hernández-Rodríguez, M. Rodríguez-López, B. Aspe de la Iglesia, J. M. Pego

Publication

PloS One 10(7):e0131992

The Authors studied dipeptidyl peptidase IV (DPP-IV, CD26) expression in different T helper cells and serum soluble DPP-IV/sCD26 levels in rheumatoid arthritis (RA) patients, correlated these with disease activity score (DAS), and examined how they were affected by different therapies. The authors concluded that, according to their CD26 expression, different cell subsets could serve to monitor RA course, and an uncharacterized T helper CD26- subset, not targeted by therapies, should be monitored for early diagnosis. Blood cells were collected using TransFix Vacuum Blood Collection Tubes and stored at 4°C until use.

http://www.ncbi.nlm.nih.gov/pubmed/26177310

2014

Enumeration of circulating fibrocytes for clinical use is asthma by oprimized single-platform flow cytometry assay

Sample Type: Circulating fibrocytes

Analysis Method: Flow Cytometry

Authors

L Bianchetti, M Isgro, MA Marini, M Schmidt

Publication

BBA Clinical, Volume 1, June 2014, Pages 52–58

Elevated numbers of circulating fibrocytes is emerging as an easily accessible biomarker for asthma. In this report, blood samples were taken from 44 patients with asthma of various severity and 14 age-matched healthy individuals. Fibrocytes were quantified using flow cytometry. Blood samples were either processed within four hours or mixed with the cellular surface antigen-stabilizing agent TransFix and stored at 4 degrees Celsius for up to 96 hours before processing. Robust intra-assay variability demonstrated a good agreement between the fibrocyte counts measured in fresh whole blood and the fibrocyte counts obtained in stored whole blood at 48 and 96h.

http://www.sciencedirect.com/science/article/pii/S2214647414000099

Evaluation of TransFix® mediated stabilisation of adipose-derived stromal vascular fraction for delayed flow cytometry analysis

Sample Type: Adipose-derived stromal vascular fraction

Analysis Method: Flow Cytometry

Authors

Karsten,E., Sung,J., Morgan,C., Herbert,B., Vesey,G.

Publication

Open Journal of Regenerative Medicine (2014), 3, 54-63

In multi-centre studies where stromal vascular fraction (SVF) is used for immediate treatment, post-hoc assessment at a central or core facility of cell numbers, cell viability and immunophenotype may be necessary. Additional time taken to ship and store these samples can lead to time-dependant loss of cell markers and a reduction in cell viability. This paper provides an assessment of the suitability of TransFix for the purpose of SVF stabilisation across 77 consenting patients. The data shows that following treatment with TransFix the samples remain stable for up to seven days providing accurate total nucleated cell counts, stable populations of CD90, CD31 and CD45 markers and the potential to back calculate cell viability in the original sample. The authors also report an apparent increase in cell numbers post-stabilisation that is hypothesised to be due to a reduction in cell clumping as a result of treatment with TransFix.

http://www.scirp.org/journal/PaperInformation.aspx?paperID=49498#.VLPTzCusWTI

2015

Platelet leukocyte aggregates and markers of platelet aggregation, immune activation and disease progression in HIV infected treatment naive asymptomatic individuals

Sample Type: Platelet monocyte and platelet neutrophil aggregates

Analysis Method: Flow Cytometry

Authors

B. B. Nkambule, G. Davison

Publication

Journal of Thrombosis and Thrombolysis, 40 (4): 458-467

The primary aim of this study was to determine the levels of platelet monocyte and platelet neutrophil interactions in HIV treatment naive individuals. This was achieved by measuring the levels of platelet monocyte and platelet neutrophil aggregates and assessing the association between platelet aggregates; markers of immune activation and disease progression. Measurement of Platelet CD62P (platelet activation marker), CD36 (platelet aggregation marker), PMAs (platelet monocyte aggregates, CD14+CD42b+), and PNAs (platelet neutrophil aggregates, CD16+CD42b+) was performed by flow cytometry. TransFix was added to whole blood samples at the time of red blood cell lysis to keep the consequential platelet activation to a minimum, limiting artefactual platelet activation induced by ADP which is released upon the lysis of red blood cells.

https://link.springer.com/article/10.1007/s11239-015-1212-8

2013

Relationships between emerging cardiovascular risk factors, z-BMI, waist circumference and body adiposity index (BAI) on adolescents

Sample Type: Circulating endothelial cells

Analysis Method: Flow Cytometry

Authors

I. B. F. Dias, D. G. Panazzolo,M. F. Marques, B. D. Paredes, M. G. C. Souza, D. P. Manhanini,V. Morandi, P. T. V. Farinatti, E. Bouskela, L. G. Kraemer-Aguiar

Publication

Clin Endocrinol (2013) 79(5): 667-674

The body adiposity index (BAI) has been recently proposed as an alternative index to body mass index (BMI) and waist circumference (WC) to evaluate adiposity in adults. This study aimed to determine the correlation between BAI and emerging cardiovascular risk markers including circulating endothelial cells (CECs) in normal weight and obese adolescents. Percentage of CECs in peripheral blood were assessed by fow cytometric analysis: 400 uL cellular stabilization solution (TransFix) was added to a 4 ml of peripheral blood to preserve cell surface antigens.

http://onlinelibrary.wiley.com/doi/10.1111/cen.12195/full

2016

The Association of High Prevalence of Trophozoites in Peripheral Blood with Lower Antibody Response to P. falciparum Infected Erythrocytes among Asymptomatic Children in Sudan

Sample Type: Malaria infected erythrocytes

Analysis Method: Flow Cytometry

Authors

S. N. Mohamed, D. A. Hassan, A. M. El Hussein, I. M. Osman, M. E. Ibrahim, H. S. Mohamed, B. Y. Nour, N. H. Abdulhadi

Publication

Mediators of Inflammation Article ID 7987686

The Authors of this paper examined the antibody reactivity of autologous plasma from symptomatic and asymptomatic malaria infected children against the infected erythrocytes' surface antigens using flow cytometry. The Authors use TransFix to stabilise variant surface antigens (VSA) for flow cytometric analysis.

https://www.hindawi.com/journals/mi/2016/7987686/abs/

Animal Blood Stabilisation

2012

KEY PAPER: A rapid high-precision flow cytometry based technique for total white blood cell counting in chickens

Sample Type: Chicken White Blood Cell

Analysis Method: Flow Cytometry

Authors

C.Seliger, B. Schaerer, M. Kohn, H. Pendl, S. Weigend, B.Kaspers, S. Härtle

Publication

Veterinary Immunology and Immunopathology, Volume 145, pages 86–99, 2012

TransFix (Cytomark) was used to stabilise samples to demonstrate that samples can be shipped and stored prior to analysis. TransFix was used to stabilise and transport the avian blood samples. The automated analysis of total white blood cell count and white blood cell differentials is routine in research and clinical diagnosis in mammalian species. In contrast, in avian haematology these parameters are still estimated by conventional microscopic procedures due to technical difficulties associated with the morphological peculiarities of avian erythrocytes and thrombocytes. Both cell types are nucleated and fairly resistant to cell lysis, a prerequisite for automated leukocyte quantification and differentiation by commercial instruments. By using an anti-CD45 monoclonal antibody in combination with selected subset specific markers we have established a simple (no-lyse no-wash single-step one-tube) flow cytometry based technique for high precision chicken blood cell quantification.

http://www.sciencedirect.com/science/article/pii/S016524271100417X

2010

KEY PAPER: Effects of Cyclosporin A induced T-lymphocyte depletion on the course of avian Metapneumovirus (aMPV) infection in turkeys

Sample Type: Turkey T-lymphocytes

Analysis Method: Flow Cytometry

Authors

Dennis Rubbenstroth, Tina S. Dalgaard, Sonja Kothlow, Helle R. Juul-Madsen and Silke Rautenschlein

Publication

Developmental & Comparative Immunology (2010) 34, Issue 5, pages 518-529

The avian Metapneumovirus (Ampv) causes an economically important acute respiratoroy disease in turkeys. While antibodies were shown to be insufficient for protection against aMPV-infection, the role of T-lymphocytes in the control aMPV-infection is not clear. In this study, the role of T-lymphocytes in aMPV-pathogenesis in a T-Cell suppression model in turkeys was investigated. Blood samples were collected with a syringe and were immediately transferred to S-Monovette EDTA-tubes. A total volume of 400ul EDTA-treated blood was mixed with 80ul fixation reagent TransFix, resulting in a 1.2-FOLD dilution of the sample. Samples were then stored for up to 1 day until further analysis.

2017

Vaccination Against Histomonosis Limits Pronounced Changes of B-cells and T-cell Subsets in Turkeys and Chickens

Sample Type: Chicken and Turkey T-cell and B-cell subsets

Analysis Method: Flow Cytometry

Authors

T. Mitraa, W. Gernerb, F. A. Kidanea, P. Wernsdorfa, M. Hessa, A. Saalmüllerb, D. Liebharta

Publication

Vaccine (2017) 35(33): 4184-4196

The protozoan parasite histomonas is causative agent of histomonosis in Turkeys and chickens and causes a high mortality rate in Turkeys. Experimental vaccination has shown to protect these species. TransFix was used to stabilise 0.75ml and 1ml whole blood samples in order to analyse monocytes/macrophages and heterophils via Flow cytometry.

http://www.sciencedirect.com/science/article/pii/S0264410X17308228

Other Sample Types

2012

Effects of nano-TiO 2 aerosols showing two distinct agglomeration states on rat lungs

Sample Type: Bronchoalveolar lavage fluid

Analysis Method: Haemocytometer, cytospin, Bio-Plex Luminex assay

Authors

A Noel, K Maghni, C Dion, KJ Wilkinson, S. Hallé, R. Tardif, G. Truchon

Publication

Toxicology Letters, Volume 214, Issue 2, 17 October 2012, Pages 109–119

The study examined inflammation resulting from the inhalation of nono-Ti02 aerosols by rats. After exposure to inhalation the rats were anaesthetized with isoflurane and sacrificed by exsanguination. Fluids from the BAL (BALF) were collected with 0.9% saline. The lungs were washed with a total of 21ml of 0.9 % PBS in 5 steps. These samples were centrifuged to isolate the cells and the cells from the five washes combined. The cell pellet was re-suspended in 1ml of 0.9% PBS containing 1% BSA and 200ul of TransFix. TransFix stabilized cell fractions were examined using a range of techniques to determine the degree of inflammation resulting from inhalation of nano-Ti2 aerosols. The techniques used to examine the TransFix stabilized leucocytes included; total cell counts using a haemocytometer, differential cell counts for lymphocytes using the cytospin cell staining method and inflammation markers were quantified (IL-1 , IL-6, TNF-, MIP-1 and MCP-1) using the Bio-Plex Luminex assay.

http://www.sciencedirect.com/science/article/pii/S0378427412012647


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