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2015

Human Blood Stabilisation - Immunophenotyping

Collection, Storage, and Preparation of Human Blood Cells

Sample Type: Monocytes, T lymphocytes, B lymphocytes, neutrophils, and platelets

Analysis Method: Flow Cytometry

Authors

P. K. Dagur, J. P. McCoy

Publication

Current Protocols in Cytometry 73 (5): 5.1-5.1.16

Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non-flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils, and platelets, prior to flow cytometric analysis. In some instances, such as shipping specimens from a distant site, it may be necessary to store blood for brief periods of time prior to staining. Several commercially available reagents, such as TransFix can be used to treat blood for storage. As an example of using these, a protocol for using Transfix is provided in this publication.

http://onlinelibrary.wiley.com/doi/10.1002/0471142956.cy0501s73/abstract

Implementation of highly sophisticated flow cytometry assays in multicenter clinical studies: considerations and guidance

Analysis Method: Flow Cytometry

Authors

U. Sommer, J. Morales, A. Groenewegen, A. Muller

Publication

Bioanalysis, 7(10): 1299-1311

A discussion of the current challenges and solutions surrounding the design of multicentre studies involving flow cytometry analysis of biomarkers. Using TransFix/EDTA Vacuum Blood Collection tubes for sample collection is highlighted as a convenient approach for immunophenotyping assays.

https://www.bioanalysis-zone.com/wp-content/uploads/2015/11/Bioanalysis-Review-Paper-PPD-Novartis-2015.pdf

Human Blood Stabilisation - Circulating Tumour Cells

A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay

Sample Type: Circulating rare cells (CTCs, CMCs, CECs, CSCs)

Analysis Method: Automated microfluidic filtration and multiplex immunoassay

Authors

M. J. M. Magbanua, M. Pugia, J. S. Lee, M. Jabon, V. Wang, M. Gubens, K. Marfurt, J. Pence, H. Sidhu, A. Uzgiris, H. S. Rugo, J. W. Park

Publication

PLoS ONE 10(10): e0141166

In this study, the performance of a novel approach for detection and enumeration of multiple rare cell populations was evaluated in the blood of metastatic breast and lung cancer patients using an automated microfluidic filtration and multiplex immunoassay strategy. Different circulating rare cell populations were detected and enumerated, including circulating tumour cells (CTCs), circulating mesenchymal cells (CMCs), circulating endothelial cells (CECs), and putative circulating stem cells (CSCs). Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.
The highly controlled filtration process and the multi-step staining parameters were optimised to minimise the detection of false positives in healthy donor blood. Use of TransFix®, along with controlled shipping and storage conditions contributed to the high rate of reportable results (98%).
Blood was collected into tubes containing K3EDTA and 0.45mL Transfix® in customised TransFix/EDTA Vacuum Blood Collection Tubes (TVT-09-50-45).

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141166

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

Human Blood Stabilisation - Other

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

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

Quantification of Cells with Specific Phenotypes I: Determination of CD4+ Cell Count Per Microliter in Reconstituted Lyophilised Human PBMC Prelabeled with Anti-CD4 FITC Antibody

Sample Type: Human peripheral blood mononuclear cells

Analysis Method: Flow Cytometry

Authors

Stebbings R, Wang L, Sutherland J, Kammel M, Gaigalas AK, John M, Roemer B, Kuhne M, Schneider RJ, Braun M, Engel A, Dikshit DK, Abbasi F, Marti GE, Sassi MP, Revel L, Kim SK, Baradez MO, Lekishvili T, Marshall D, Whitby L, Jing W, Ost V, Vonsky M, Neukammer J.

Publication

Cytometry Part A (2015): 87A: 244-253

A surface-labelled lyophilised lymphocyte (Sll) preparation has been developed using human peripheral blood mononuclear cells prelabelled with a fluorescin isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ counting, including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. The sLL reference material was prepared from a pool of three buffy coats from normal human donors, and the PBMCs were separated and labelled with anti-CD4 FITC monoclonal antibody, washed and resuspended in a fixative solution comprised of 10% TransFix.

http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22614/abstract

CSF Stabilisation

Detection of central nervous system involvement in childhood acute lymphoblastic leukemia by cytomorphology and flow cytometry of the cerebrospinal fluid

Sample Type: CSF (lymphoblastic leukaemia)

Analysis Method: Flow Cytometry

Authors

S. Ranta, F. Nilsson, A. Harila-Saari, L. Saft

Publication

Pediatric Blood & Cancer (2015) 62(6): 951

The authors retrospectively compared flow cytometric immunophenotyping (CFI) of CSF with cytomorphology (CM) at diagnosis or relapse of childhood acute lymphoblastic leukaemia (ALL). They concluded that FCI of CSF increased the detection rate of CNS involvement of ALL approximately two times compared to cytomorphology, and that patients with low level CNS involvement may bene?t from additional intensi?ed systemic or CNS-directed therapy. CSF specimens were processed immediately on receipt in the laboratory or ?xed in Trans?x and processed the next morning. The authors note that one of the main problems in analysing CSF is the rapid cell death of leukocytes after sampling. Therefore, the FCI and CM results also always re?ect the time from sampling until the start of analysis. Routine use of stabilization media or ?xative immediately after sampling could further facilitate detection of malignant cells.

http://onlinelibrary.wiley.com/doi/10.1002/pbc.25363/full

Diagnostic and prognostic significance of flow cytometry immunophenotyping in patients with leptomeningeal carcinomatosis

Analysis Method: Flow Cytometry

Authors

D. Subira, M. Simo, J. Illian, C Serrano, S. Castanon, R. Gonzalo, J. J. Granizo, M Martinez-Garcia, M. Navarro, J. Pardo and J. Bruna

Publication

Clinical & Experimental Metastasis (2015) 32: 383-391

A multi-center study with eight patient recruitment centers throughout Spain that used TransFix CSF tubes to stabilise CSF samples from 166 patients. The study examined the sensitivity and specificity of flow cytometry (FC) diagnosis for leptomeningeal carcinomatosis. Compared with Cytology, FC showed greater sensitivity and negative predictive value, but lower specificity and positive predictive value. The multivariate analysis revealed that the percentage of CSF EpCAM positive cells predicted an increased risk of death.

http://www.ncbi.nlm.nih.gov/pubmed/25795393

Infiltration of CNS by acute leukaemia: Analysis of fresh and TransFix stabilised CSF

Sample Type: CSF (acute leukaemia blasts)

Analysis Method: Flow Cytometry

Authors

U. Johansson, M. Crawford, M. Hughes, K. Day, D. Harrison, T. Almond

A research study to determine whether acute leukaemia blasts present in CSF can be successfully stabilised by TransFix has concluded that Transfix preserved light scatter and key antigen expression patterns allowing analysis of diagnostic and follow up CSF specimens for patients with CNS infiltration. Full evaluation of the antibody panels to be used on transfixed samples is also required since some antigens are expressed dimly.

https://www.cytomark.co.uk/downloads/Infiltration_of_CNS_by_acute_leukemia_ESSCA_Oct_2015.pdf

Recommendations for quality assurance in multiparametric flow cytometry: first consensus of the Brazilian Group of Flow Cytometry (GBCFLUX)

Analysis Method: Flow Cytometry

Authors

R. P. Correia, A. C. A. Bortolucci, A. C. W. Lopes, A. F. Sandes, A. Paula de Azambuja, M. A. Viana, M. M. Sales, M. Yamamoto, N. S. Bacal

Publication

Jornal Brasileiro de Patologia e Medicina Laboratorial 51(6): 389-396

The Brazilian Group of Flow Cytometry have the objective of contributing to technical and scientific advances in Brazilian clinical and research laboratories. In this publication, they present consensus recommendations to ensure the process quality, technical standardization, and reproducibility of results for all FC working groups in Brazil. For quality control of the pre-analytical phase, they recommend to collect CSF directly into TransFix, which can then be stored at 2-8°C for 48-72 hours.

https://dx.doi.org/10.5935/1676-2444.20150061


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