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Bibliography

Bibliography

Clinical Research

Human Blood Stabilisation - Immunophenotyping

2011

KEY PAPER: CD4 Intragenic SNPs Associate With HIV-2 Plasma Viral Load and CD4 Count in a Community-Based Study From Guinea-Bissau, West Africa

Sample Type: Lymphocyte subset

Analysis Method: Flow Cytometry

Authors

Branwen Hennig , Digna Velez-Edwards, Maarten Schim van der Loeff, Cyrille Bisseye, Todd Edwards, Alessandra Tacconelli, Giuseppe Novelli, Peter Aaby, Steve Kaye, William Scott, Assan Jaye, Hilton Whittle, Scott Williams, Adrian Hill, Giorgio Sirugo

Publication

Journal of Acquired Immune Deficiency Syndromes (2011) 56, 1-8

A second, back-up method for lymphocyte subset measuring was also in place. For this method, 100 μl "TransFix" solution (Cytomark UK) 24 was added to 500 μl fresh blood and roller-mixed for 5 minutes, after which the sample was kept in a refrigerator at 4 degrees Celsius and transported to the MRC Laboratories in Fajara, The Gambia. When the routine method failed, the transfixed samples were stained with fluorochrome-conjugated monoclonal antibodies manually rather than by Q-Prep, and analyzed in the FACS calibur, using the MultiSet software (Becton Dickinson, Erembodegem, Belgium). Results were regarded as invalid if the CD3 % was < 45%, or if the sum of CD4 % and CD8 % was more than 10% different from the mean CD3 %. In order to investigate whether the results of the two methods (routine and back-up) were comparable, 13 samples were processed using both methods and results compared. The agreement was excellent (r2 = 0.89 for CD4 %). In addition, a Bland and Altman test 25 was performed to compare the two methods and indicated no difference between them (p = 0.3).

http://www.ncbi.nlm.nih.gov/pubmed/20924289

2016

KEY PAPER: First-in-Human Study in Healthy Subjects with FR104, a Pegylated Monoclonal Antibody Fragment Antagonist of CD28

Sample Type: Lymphocyte subsets

Analysis Method: Flow Cytometry

Authors

N. Poirier, G. Blancho, M. Hiance, C. Mary, T. Van Assche, J. Lempoels, S. Ramael, W. Wang, V. Thepenier, C. Braudeau, N. Salabert, R. Josien, I. Anderson, I. Gourley, J. Soulillou, D. Coquoz, B. Vanhove

Publication

J Immunol?DOI: https://doi.org/10.4049/jimmunol.1601538

This paper documents a phase I clinical trial to test FR104 (an antibody which inhibits CD28) in humans as a potential treatment of transplant rejection and autoimmune diseases. Blood samples were collected in TransFix/EDTA Vacuum Blood Collection Tubes (TVTs) to monitor lymphocyte subsets (CD45, CD3, CD28, CD45RO, CD4, CD25, CD127. CD69, CCR7) at time points ranging from day 0 to day 113, from a cohort of 64 healthy subjects.

Phlebotomy time points were initially very frequent post drug administration (0.5, 0.75, 1, 2, 4, 8 and 24 hours). TVTs gave the benefit of providing accurate snapshots of the immune profile of the subjects within this condensed testing period whilst giving the technician the flexibility to test the blood in a more leisurely timescale.

http://www.jimmunol.org/content/early/2016/11/12/jimmunol.1601538.abstract

2015

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

2008

Research needs and challenges in the development of HIV diagnostic and treatment monitoring tests for use in resource-limited settings

Sample Type: CD4+ cells

Analysis Method: Flow Cytometry

Authors

B. Cheng, A. Landay, V. Miller

Publication

Current Opinion in HIV & AIDS (2008) 3: 495-503

The aim of this article is to review research priorities for current and new technologies to diagnose HIV and to monitor treatment response, including technologies to enumerate CD4 cell counts in resource-limited settings.
The Authors note that blood preservatives may allow for the preservation of whole blood so that the sample can be shipped to a centralized laboratory with up to 15 days (TransFix; UK NEQAS, Shef?eld, South Yorkshire, UK) from collection to testing. Blood preservatives may play a very important role in improving access to CD4 cell count testing as they will allow the sample to be transported to a laboratory where the testing will be performed.

http://journals.lww.com/co-hivandaids/Fulltext/2008/07000/Research_needs_and_challenges_in_the_development.14.aspx

2002

Stabilised cellular immuno-fluorescence assay: CD45 expression as a calibration standard for human leukocytes

Sample Type: CD45+ cells

Analysis Method: Quantitative Indirect Immunofluorescence

Authors

A. Bikoue, G. Janossy, D. Barnett

Publication

Journal of Immunological Methods (2002) 266: 19-32

Quantitative immunofluorescence of CD45 expression is important in a wide variety of clinical studies however prior to this study, ranges of antibody binding capacity (ABC) in both fresh samples and stabilised control material had not been defined. As part of their work to establish the ABC range for stabilised control materials, the authors used TransFix to stabilise human whole blood for assessment. These investigations confirmed that TransFix stabilised human whole blood samples retain a CD45 ABC/lymphocyte level at a comparable value to fresh samples for 10-14 days when assessed using the quantitative indirect immunofluorescence (QIFI) test.

http://www.ncbi.nlm.nih.gov/pubmed/12133619

1999

KEY PAPER: TransFix: A clinical sample stabilising fluid for use in clinical haematology and immunology

Sample Type: Full haematological profile

Analysis Method: Flow Cytometry

Authors

D. Barnett, V.Granger, A.G. Pockley, J.M.saxton, I storie, L.Whitby & J.T.Reilly

Publication

Cytometry (1999) 38:88 (abstract)

Transportation of clinical samples between clinical sites raises concerns about sample integrity. TransFix was developed as a stabilising solution with minimal dilution effect and which retains sample integrity for up to 10 days. This facilitates the flow cytometer and haematological analysis on normal, Leukaemia and HIV patients. Phase 1 studies examined 40 patients specimens and found no loss of antigenicity over a 10 day period for the following antigens: CD2, CD3, CD4, CD5, CD7, CD10, CD11b, CD13, CD14, CD19, CD20, CD22, Cd23, CD33, Cd34, CD45, CD79b, HLA-Dr and surface bound immunoglobulin. A full haematological profile (including differential) was obtainable at day 7.

Human Blood Stabilisation - Other

2013

Quantification of circulating CD34+/KDR+/CD45 dim endothelial progenitor cells: analytical considerations

Sample Type: Circulating endothelial progenitor cells

Analysis Method: Flow Cytometry

Authors

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

Publication

International Journal of Cardiology, 167 (5): 1688-1695

A comprehensive review of characterisation of circulating endothelial progenitor cells with a particular focus on the considerations surrounding enumeration by flow cytometry and their impact on cardiovascular disease. The authors note TransFix can be used to prolong sample storage time for 7 days for this type of analysis.

http://www.sciencedirect.com/science/article/pii/S0167527312014222

2014

The role of tissue factor in activation coagulation system at pregnancy complications

Sample Type: Monocytes

Analysis Method: Flow Cytometry

Authors

M. Prochazka, L. Slavik L, J. Prochazkova, J. Ulehlova, M. Novák, P. Polak, R. Pilka

Publication

Thrombosis Research, 133: S109

The authors of this poster abstract propose a model to monitor activation of the coagulation system in preeclampsia and other pregnancy complications with Tissue Factor (TF) expression on monocytes by ?ow cytometry, and simultaneously ?xing the TF-induced thrombin generation in plasma. To determine expression of tissue factor (CD142) on monocytes, the authors used multicolour ?ow cytometry (CD45, CD14, CD16b, and CD142), and used TransFix to stabilise samples that could not be analysed within 2 hours.

https://www.mendeley.com/research/role-tissue-factor-activation-coagulation-system-pregnancy-complications/

CSF Stabilisation

Contribution of cerebrospinal fluid sCD19 levels to the detection of lymphoma and its impact on disease outcome

Analysis Method: Flow Cytometry

Authors

C. Muniz, L. Martin-Martin, A. Lopez

Publication

Blood, 123:1864-1869; doi: https://doi.org/10.1182/blood-2013-11-537993

The Authors treat CSF samples with TransFix for Flow Cytometry analysis within 24 hours. Samples are stained using the EuroFlow small sample tube (SST: CD20, CD45, CD8-surface membrane immunoglobulin (SmIg)lambda, CD56-SmIgkappa, CD4, CD19, SmCD3-CD14, CD38)

http://www.bloodjournal.org/content/123/12/1864.full

Differences in cerebrospinal fluid inflammatory cell reaction of patients with leptomeningeal involvement by Lymphoma and carcinoma

Sample Type: CSF (epithelial cell neoplasia and lymphomas)

Analysis Method: Flow Cytometry

Authors

Julia Illán, Marta Simo, Cristina Serrano, Susana Castañón, Raquel Gonzalo, María Martínez-García, Javier Pardo, Lidia Gómez, Miguel Navarro, Javier Pérez Altozano, Ruth Álvarez, Jordi Bruna, Dolores Subiráemail

Publication

Translational Research ,Volume 164, Issue 6, Pages 460–467

Dissemination of neoplastic cells into the cerebrospinal fluid (CSF) and leptomeninges is a devastating complication in patients with epithelial cell neoplasia and lymphomas. In this study, flow cytometry immunophenotyping was used to analyse CSF leukocyte populations in the CSF of patients diagnosed with leptomeningeal infiltration by solid tumors and lymphomas. CSF samples were collected in tubes containing ethylenediamine tetraacetic acid and an immunofixative reagent (TransFix) for safe transportation and were processed with a median of 3 days from extraction.

http://www.translationalres.com/article/S1931-5244(14)00122-4/pdf

2015

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

2014

KEY PAPER: Use of TransFix cerebrospinal fluid storage tubes prevents cellular loss and enhances flow cytometric detection of malignant hematological cells after 18 hours of storage

Sample Type: CSF (LHM)

Analysis Method: Flow Cytometry

Authors

A. H. de Jongste, J. Kraan, P. D. van den Broek, R. A. Brooimans, J. E. Bromberg, K. A. van Montfort, P. A. Sillevis Smitt, J. W. Gratama

Publication

Cytometry Part B: Clinical Cytometry Volume 86, Issue 4, pages 272–279, July 2014

In patients with suspected or proven leptomeningeal localised haematological malignancies (LHM) in CSF, rapid assessment is required as cell numbers decline quickly after lumbar puncture. The study aim was to examine leukocyte numbers and detection of LHM by flow cytometry in 99 CSF samples from patients with suspected or proven LHM after 30 minutes and 18 hours of storage in i) TransFix/EDTA CSF Sample Storage Tubes ii) serum-containing medium and iii) no stabilising agent (untreated). It was found that leukocyte numbers in TransFix stabilised CSF were higher than in the corresponding untreated samples at both time points. After 18 hours of storage at 4 degrees Celsius, the detection of LHM was enhanced in TransFix stabilised CSF. For all discordant paired observations, the level of suspicion of a patient having LHM was higher for the TransFix stabilised samples than for the untreated samples. The authors concluded that the use of TransFix/EDTA CSF Sample Storage Tubes prevents cellular loss and enhances flow cytometric detection of LHM after 18 hours of storage.

http://onlinelibrary.wiley.com/doi/10.1002/cyto.b.21097/full

Bone Marrow Stabilisation

2011

KEY PAPER: Immunophenotypic Characterization of Bone Marrow Mast Cells in Mastocytosis and Other Mast Cell Disorders

Sample Type: Bone marrow (mast cells)

Analysis Method: Flow Cytometry

Authors

L. Sánchez-Munoz, C, Teodósio, J.M. Morgado and L. Escribano

Publication

Recent Advances in Cytometry, Part B - Methods in Cell Biology (Book Series), Vol. 103. P333-359, Book published by Academic Press June 2011

Mastocytosis is a term used to designate a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow (BM), liver, spleen, and lymph nodes, among others. Recent advances in our understanding of mast cell biology and disease resulted in the identification of important differences in the expression of mast cell surface antigens between normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts lead to the inclusion of this immunophenotypic abnormality in the World Health Organization diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients lacking histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this chapter, we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, for their phenotypic characterization, and the criteria currently used for correct interpretation of the immunophenotypic results obtained. In cases in which it is not expected to perform sample processing within the first 24h following a bone marrow puncture, a stabilising solution such as TransFix should be used to avoid deterioration of cells.

http://www.ncbi.nlm.nih.gov/pubmed/21722810

2008

Standardization of Flow Cytometric Minimal Residual Disease Evaluation in Acute Lymphoblastic Leukemia: Multicentric Assessment Is Feasible

Sample Type: Bone Marrow (BCP- and T-ALLs)

Analysis Method: Flow Cytometry

Authors

Dworzak, M. N., Gaipa, G., Ratei, R., Veltroni, M., Schumich, A., Maglia, O., Karawajew, L., Benetello, A., Pötschger, U., Husak, Z., Gadner, H., Biondi, A., Ludwig, W.-D. and Basso, G.

Publication

Cytometry B Clin Cytom. 2008 Nov;74(6):331-40.

Flow cytometric (FCM) assessment of minimal residual disease (MRD) in acute lymphoblastic leukaemia (ALL) patients was assessed at 3 different sites in Italy and one in Germany to see whether standardization was possible.MRD-evaluation by FCM in ALL can be standardized for reliable multicentric assessment in large trials.In order to assess the inter-laboratory concordance, artificial samples were created using TransFix. TransFix was used to stabilise leukemic cells from nine patients (seven B cell ALL and two T cell ALL) and these cells were mixed into bone marrow preparations at different concentrations. These were then sent to the sites for FCM analysis. The concordance between all four centres was 98%. Therefore, TransFix was shown to be successful in stabilising leukemic cells.

http://www.ncbi.nlm.nih.gov/pubmed/18548617

2014

KEY PAPER: The impact of stem cells on electron fluxes, proton translation, and ATP synthesis in kidney mitochondria after ischemia/reperfusion

Sample Type: Bone marrow-derived stem cells

Analysis Method: Flow Cytometry

Authors

Beiral HJ1, Rodrigues-Ferreira C, Fernandes AM, Gonsalez SR, Mortari NC, Takiya CM, Sorenson MM, Figueiredo-Freitas C, Galina A, Vieyra A.

Publication

Cell Transplant. 2014 Feb;23(2):207-20

This study aims to show the administration of bone marrow-derived stem cells (BMSCs) can aid recovery of mitochondrial respiration following ischemia/reperfusion (I/R) injuries. The BMSCs were used immediately following isolation from the extracted bone marrow and some were retained for characterisation. 3x106 of the BMSCs were resuspended in PBS and stabilised using 1 part TransFix to 5 parts cell suspension for subsequent immunophenotyping, allowing the researchers to complete other time sensitive aspects of the study.

http://www.ncbi.nlm.nih.gov/pubmed/23211430


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