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Bibliography by Year
2012
TransFix® for delayed flow cytometry of endothelial progenitor cells and angiogenic T cells
V. Y. Hoymans, A. H. Van Craenenbroeck, L.Bruyndonckx,
S. H. van Ierssel, C .J. Vrints, V. M. Conraads, E. M. Van Craenenbroeck
Microvascular Research, Pub. Online, http://dx.doi.org/10.1016/j.mvr.2012.08.007, 2012
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.
Method validation and immunophenotypical study of NK and T lymphocytes by flow cytometry
E. Konsta, Ε. Κώνστα
Pub. Online - http://phdtheses.ekt.gr/eadd/handle/10442/26992, 2012
Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials
A.A.P.Ng, BTK Lee, T.S.Y. Teo and M. Poidinger
Journal of Immunological Method, Pub. online http://dx.doi.org/10.1016/j.jim.2012.08.010, 2012
High dimensional flow cytometry is best served by centralized facilities. However, the difficulties around sample processing, storage and shipment make large scale international studies impractical. We therefore sought to identify optimized fixation procedures which fully leverage the analytical capability of high dimensional flow cytometry without the need for complex cell processing or a sustained cold chain. Whole blood staining procedure was employed to investigate the applicability of fixatives including Cyto-Chex® Blood Collection tube (Streck), Transfix® (Cytomark)
Current strategies in the diagnosis of diffuse large B-cell lymphoma of the central nervous system
Alexander Baraniskin, Martina Deckert, Gernot Schulte-Altedorneburg, Uwe Schlegel, Roland Schroers
British Journal of Haematology, Volume 156, Issue 4, pages 421–432, February 2012
Lymphomas can arise within the central nervous system (CNS) as primary CNS lymphoma (PCNSL) typically involving the brain and less often the leptomeninges, eyes, and spinal cord. In contrast to PCNSL, secondary CNS lymphoma (SCNSL) is considered to originate as quasi metastasis from systemic lymphoma spreading to the CNS. Both types of CNS lymphomas are predominantly tumours of the diffuse large B-cell type and represent aggressive diseases necessitating a rapid diagnosis. Following neuroimaging based on magnetic resonance imaging, stereotaxy and histopathological diagnosis of CNS lymphoma currently remain obligatory to plan treatment. However, progress in cytopathological, immunophenotypic, and molecular genetic analyses of the cerebrospinal fluid (CSF) has been achieved recently and potentially will facilitate lymphoma diagnosis in the future. This review describes the diagnostic procedures in patients with suspected CNS lymphomas, primarily PCNSL. In addition to a summary of the standard diagnostic work-up, an overview and discussion of current data on different techniques for evaluation of the CSF in CNS lymphoma are given.
R. Alvarez,
J. Dupuis,
1,
A. Plonquet,
C. Christov,
C. Copie-Bergman,
F. Hemery,
I. Gaillard,
T. El Gnaoui,
F. Kuhnowski,
M. Bedoui,
K. Belhadj,
P. Brugières and
C. Haioun
Annals of Oncology,
Volume 23, Issue 5 , pages 1274-1279, 2012
Central nervous system (CNS) relapse is an uncommon but dramatic complication of diffuse large B-cell lymphoma (DLBCL). Several studies have demonstrated the superiority of cerebrospinal fluid (CSF) flow cytometry (FCM), as compared with conventional cytology (CC), in detecting occult leptomeningeal disease. The clinical relevance of a positive FCM still has to be clarified. Makes reference to use of TransFix to stabilise and transport CSF samples.
Flow cytometry as a diagnostic tool in lymphomatous or leukemic meningitis
M.S. Ahluwalia, P.K. Wallace and D.M. Peereboom
Cancer, Volume 118, Issue 7, pages 1747 – 1753, 2012
In patients with neoplastic meningitis (NM), early diagnosis is highly desirable because the rapid institution of intrathecal therapy may mitigate the course of the disease. Cytology, long considered the "gold standard" for diagnosis, has low sensitivity because of both the paucity of cells in the cerebrospinal fluid (CSF) and morphological similarities between benign and malignant cells. A comprehensive review of the literature from 2005 through 2011 was performed that focused on diagnostic modalities for lymphomatous meningitis. Several studies demonstrated the sensitivity of flow cytometry to be several-fold higher than that of cytology for the detection of CSF leukemia/lymphoma. Makes reference to use of TransFix to stabilise and transport CSF samples.
Role of flow cytometry immunophenotyping in the diagnosis of leptomeningeal carcinomatosis
D. Subirá,
C. Serrano,
S. Castañón,
R. Gonzalo,
J. Illán,
J. Pardo,
M. Martínez-García,
E. Millastre,
F. Aparisi,
M. Navarro,
M. Dómine,
I. Gil-Bazo,
P. Pérez Segura,
M. Gil and
J. Bruna
Neuro-Oncology,
Volume 14, Issue 1, pages 43-52, 2012
Purpose: To explore the contribution of flow cytometry immunophenotyping (FCI) in detecting leptomeningeal disease in patients with solid tumors.
CSF samples obtained from a lumbar puncture (n = 77) and from an Ommaya reservoir (n = 1) were collected in EDTA tubes containing 0.2 mL of an immunofixative reagent (Transfix, Cytomark), necessary to guarantee safe transportation.
FCI seems to be a promising new tool for improving the diagnostic examination of patients with suspicion of LC. Detection of epithelial cells with a higher DNA content is highly specific of LC, but evaluation of the nonepithelial cell compartment of the CSF might also be useful for supporting this diagnosis.
C.Seliger, B. Schaerer, M. Kohn, H. Pendl, S. Weigend,
B.Kaspers, S. Härtle
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.
2011
EVALUATION OF FIXATIVES COMPATIBILITY WITH CD4 T CELL TECHNOLOGIES
T. Ding, P. Seely, C. Chabot, A. Martel, X. Yang, D. Bogdanovic, and M. Bergeron
International Clinical Cytometry Society October 14-18, 2011, Portland, Oregon, Page 50
CD4 T-cell enumeration remains the best surrogate marker for staging and monitoring the immune status of HIV patients under treatment in resource-limited countries. This preliminary study is evaluating the compatibility of commercial fixatives with various CD4 counting platforms. Compatibility was assessed on the capacity of each platform to measure CD4 in fixed blood; to generate an accurate measurement and; the degree of similarity between fixed and unfixed samples (cluster resolution). “Streck Cell Preservative” and “TransFix” fixatives solution were used as recommended. One-day-old fixed HIV-specimens were prepared in triplicate according to manufacturers' instruction and tested on their respective platforms: FACSCalibur, Epic-XL, FACSCount, Guava, CyFlow Counter, PointCare Now, and PIMA. The reference method was based on 4-color reagent using FACSCalibur with Flowcount beads.
All platforms tested could measure CD4 except PointCare. Accuracy (% difference) was <15% as compared to reference method. Fixed preparations had various degree of similarity. The results showed that fixatives are not fully compatible across the whole array of CD4 technologies and EQA provider must be aware of these limitations.
L. Escribano
Methods in Cell Biology (Book), 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.
Current strategies in the diagnosis of diffuse large B-cell lymphoma of the central nervous system.
Baraniskin, A., Deckert, M., Schulte-Altedorneburg, G., Schlegel, U. and Schroers, R.
British Journal of Haematology (2011). doi: 10.1111/j.1365-2141
Quote: Previous studies have shown that CSF samples often contain low cell numbers, which is explained by a rapid decay of viable cells in native CSF. With the availability of efficient stabilization methods, such as cellular fixation by Transfix, it is now possible to preserve CSF cells for up to 10 d (Canonico et al, 2004; Quijano et al,2009). Accordingly, it will be possible to analyse CSF samples from PCNSL patients in a central laboratory, preferably within a large multi-centre study.
Flow cytometry as a diagnostic tool in lymphomatous or leukemic meningitis
Manmeet S. Ahluwalia, Paul K. Wallace, David M. Peereboom
Cancer - Article first published online: 24 Oct 2011
Quote: Samples are typically transferred to the hospital without fixative, however the use of a fixative (TransFix/EDTA) may help prevent cellular degradation in CSF samples for hours or even days.
Clinical relevance of flow cytometric immunophenotyping of the cerebrospinal fluid in patients with diffuse large B-cell lymphoma
R. Alvarez, J. Dupuis,
A. Plonquet,
C. Christov, C. Copie-Bergman,
F. Hemery,
, Gaillard,
T. El Gnaoui,
F. Kuhnowski,
M. Bedoui,
K. Belhadj,
P. Brugières and
C. Haioun
First Ann Oncol (2011) doi: 10.1093 Published online: September 30, 2011
Central nervous system (CNS) relapse is an uncommon but dramatic complication of diffuse large B-cell lymphoma (DLBCL). Several studies have demonstrated the superiority of cerebrospinal fluid (CSF) flow cytometry (FCM), as compared with conventional cytology (CC), in detecting occult leptomeningeal disease. The clinical relevance of a positive FCM still has to be clarified.
Role of flow cytometry immunophenotyping in the diagnosis of leptomeningeal carcinomatosis
D. Subirá,
C. Serrano,
S. Castañón,
R. Gonzalo,
J. Illán,
J. Pardo,
M. Martínez-García,
E. Millastre,
F. Aparisi,
M. Navarro,
M. Dómine,
I. Gil-Bazo,
P. Pérez Segura,
M. Gil and
J. Bruna
Neuro Oncol (2011) doi: 10.1093 First published online: October 12, 2011
To explore the contribution of flow cytometry immunophenotyping (FCI) in detecting leptomeningeal disease in patients with solid tumors.
Cerebrospinal fluid (CSF) samples from 78 patients who received a diagnosis of epithelial-cell solid tumors and had clinical data suggestive of leptomeningeal carcinomatosis (LC) were studied. A novel FCI protocol was used to identify cells expressing the epithelial cell antigen EpCAM and their DNA content. Accompanying inflammatory cells were also described. FCI results (positive or negative for malignancy) were compared with those from CSF cytology and with the diagnosis established by the clinicians: patients with LC (n = 49), without LC (n = 26), and undetermined (n = 3). FCM samples were stabilised with TransFix.
CD4 Intragenic SNPs Associate With HIV-2 Plasma Viral Load and CD4 Count in a Community-Based Study From Guinea-Bissau, West Africa.
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
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 ºC 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).
Recent Advances in Cytometry, Part B: Advances in Applications (TransFix® stabilisation of bone marrow)
Author - Zbigniew Darzynkiewicz
Book published by Academic Press June 2011
2010
Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes and red cells.
Canonico B, Betti M, Luchetti F, Battistelli M, Falcieri E, Ferri P, Zamai L, Barnett D, Papa S.
Cytometry B, Clinical Cytometry. 2010 July: 78(4): 267-78
Quote: 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.
Quantification of circulating endothelial progenitor cells in human peripheral blood: Establishing a reliable flow cytometry protocol
Masouleh BM, Baraniskin A, Schmiegel W
Journal of Immunological Methods (2010) 357: 38-42
Clinical significance of occult cerebrospinal fluid involvement assessed by flow cytometry in non- Hodgkin's lymphoma patients at high risk of central nervous system.
Juan-Manuel Sancho, Alberto Orfao, Sandra Quijano, Olga García, Carlos Panizo, Elena Pérez-Ceballos, Guillermo Deben, Antonio Salar, Eva González-Barca, Natalia Alonso, Jose-Antonio García-Vela, Javier Capote, Francisco-Javier Peñalver, Mariano Provencio, Jesús Arias, Josefa Plaza, Dolores Caballero, Marta Morado, Evarist Feliu, Josep-Maria Ribera
European Journal of Haematology (2010) 85, Issue 4, 321-328
Quote: The specific techniques and procedures used for the analyses of CSF samples have been previously described in detail (21). Briefly, CSF samples were analysed in parallel using CC in the hospital of origin and centrally by FCM at the Cytometry Service of the University of Salamanca (Spain) using a standardised 11-parameter FCM immunophenotypic assay of CSF samples stabilised with 0.2 mL of Transfix added at the hospital of origin.
C.A .Renault, A. Traore, R.N. Machekan and
D. M. Israelski
Open AIDS Journal,
Volume 4: 171-175, 2010
Quote: Low-cost diagnostics are intrinsic to effective HIV/AIDS health service delivery. These results demonstrating the feasibility of microcapillary flow cytometry are promising, but novel technologies are urgently needed. Developments in low-cost diagnostic test methods for the monitoring of HIV-infected individuals are underway. Transfix is a blood stabilizing compound that permits accurate CD4+ T lymphocyte enumeration more than 48 hours after blood has been obtained, and it has been studied for use with the Guava system.
Interlaboratory variability of CD34+ stem cell enumeration. A pilot study to national external quality assessment within the Czech Republic.
D. LYSÁK1, T. KALINA, J. MARTÍNEK, Z. PIKALOVÁ, D. VOKURKOVÁ, M. JAREŠOVÁ, I. MARINOV, A. ONDREJKOVÁ, M. ŠPAČEK, O. STEHLÍKOVÁ
International Journal of Laboratory Hematology (2010) 32, e229–e236
Quote: The study took place between November 2007 and May 2009 and consisted of three surveys. Ten laboratories took part in the study, but only nine laboratories participated in the first survey. Each laboratory was assigned a unique number (ULN) to retain confidentiality. Two samples of mobilized peripheral blood were included in every survey. Samples were stabilized with TransFix (Cytomark Ltd, Buckingham, UK),divided in 1 ml aliquots and shipped overnight by courier to the participants. The blood was collected from patients or healthy donors who underwent stem cell mobilization after giving informed consent. All subjects were negative for infectious diseases (humanimmunodeficiency virus, hepatitis B and C, syphilis).The stability of the samples was assured for at least 3 days (CV up to 5% in the stability study).Each laboratory performed the CD34+ enumeration according to its local protocol and practice and reported the measured values (leukocyte count,CD34+ percentage and absolute count) and some methodological details. All data were analyzed in the co-ordinating laboratory, and the results sent to the participants. The absolute CD34+ count was assignedas an indicator of laboratory performance.
Effects of Cyclosporin A induced T-lymphocyte depletion on the course of avian Metapneumovirus (aMPV) infection in turkeys.
Dennis Rubbenstroth, Tina S. Dalgaard, Sonja Kothlow, Helle R. Juul-Madsen and Silke Rautenschlein
Developmental & Comparative Immunology (2010) 34, Issue 5, pages 518-529
2009
Evaluation of the blood stabilizers TransFix® and Cyto-Chex BCT for low-cost CD4 T-cell methodologies.
Plate MM, Louzao R, Steele PM, Greengrass V, Morris LM, Lewis J, Barnett D, Warrino D, Hearps AC, Denny T, Crowe SM.
Viral Immunology (2009) 22(5): 329-332
Quote: TransFix(TM) and Cyto-Chex((R)) BCT (blood collection tube) reagents have been shown to maintain whole blood integrity for delayed immunophenotyping by flow cytometry. We evaluated the ability of these blood-stabilizing reagents to preserve HIV-seropositive blood for delayed CD4(+) T-cell quantification utilizing the Dynal((R)) Biotech T4 Quant Kit. TransFix was added to EDTA-anticoagulated whole blood and tested at a 1:10 dilution over 7 d using the Dynal (n = 21) manual method. Compared to baseline analysis, a significant decrease in mean CD4(+) counts was observed over time. Cyto-Chex BCT-preserved samples (n = 20) were tested for CD4(+) counts by Dynal over 7 d, with storage at varying temperatures: room temperature (21 degrees C), 37 degrees C, and 37 degrees C with intermittent storage at 42 degrees C. A significant decline in mean CD4(+) counts was observed in samples at all temperatures compared to baseline (p < 0.05). Increases in temperature to and above 37 degrees C resulted in a greater decline in mean CD4(+) counts over time. Our findings indicated that neither TransFix or Cyto-Chex BCT was a suitable blood stabilizer when used for delayed CD4 (+) quantification with a low-cost manual CD4(+) bead-based method.
Identification of Leptomeningeal Disease in Aggressive B-Cell Non-Hodgkin's Lymphoma: Improved Sensitivity of Flow Cytometry
Quijano S., López A, Sancho JM, Panizo C, Debén G, Castilla C, García-Vela JA, Salar A, Alonso-Vence N, González-Barca J, Peñalver FJ, Plaza-Villa J,Morado M, José García-Marco, Jesús Arias, Javier Briones, Secundino Ferrer, Javier Capote, Concepción Nicolás, and Alberto Orfao
Journal of Clinical Oncology (2009) 27: 1462 - 1469.
Quote: We evaluate the sensitivity and specificity of a new 11-parameter flow cytometry (FCM) approach versus conventional cytology (CC) for detecting neoplastic cells in stabilized CSF samples from newly diagnosed aggressive B-cell non-Hodgkin’s lymphoma (B-NHL) at high risk of CNS relapse, using a prospective, multicentric study design.
For multi-parameter FCM analyses, CSF samples (median volume, 2.0mL; range, 0.5 to 4.0 mL) were directly collected into tubes containing EDTA and 0.2 mL of Transfix (Cytomark) and shipped overnight to the central FCM laboratory. Immediately on arrival at the central laboratory, the volume of the CSF sample was measured (after subtracting 0.2 mL corresponding to the Transfix solution) and recorded;
Downregulation of the T-Cell Receptor by Human Immunodeficiency Virus Type 2 Nef Does Not Protect against Disease Progression
Feldmann J, Leligdowicz A, Jaye A, Dong T
Journal of Virology (2009) 83(24): 12968-12972
Quote: 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).
W. A. Ramsdorf,
F. de S.F. Guimarães,
M. V. M. Ferraro,
J. Gabardo,
E. da Silva Trindade and
M. Margarete Cestari
Genetic Toxicology and Environmental Mutagenesism, Volume 673, Issue 1, Pages 78–81,2009
Establishment of experimental conditions for preserving samples of fish blood for analysis with both comet assay and flow cytometry
Ramsdorfa WA, Guimarãesb FSF, Ferraroa MVM, Gabardoa J, Trindadeb EDS, Cestaria MM
Mutation Research/Genetic Toxicology and Environmental Mutagenesis (2009) 673: 78-81
2008
R.A. Brooimans, C.S. Boyce and J. Popma
Cytometry Part A, Volume 73A, Issue 11, 992–1000, 2008
Quote: Major histocompatibility complex (MHC) multimers that identify antigen-specific T cells, coupled with flow cytometry, have made a major impact on immunological research. HLA Class I multimers detect T cells directed against viral, tumor, and transplantation antigens with exquisite sensitivity. This technique has become an important standard for the quantification of a T cell immune response. The utility of this method in multicenter studies, however, is dependent on reproducibility between laboratories. As part of a clinical study using a standardized two-tube three-color single-platform method, we monitored and characterized performance across multiple sites using tetramers against the T cell receptors (TCR) specific for MHC Class I.
Sites (labs) were qualified in Study 2 by training, proficiency testing, and a pre multicenter trial (all quality assessment studies) using stabilized samples. Common procedures were provided to each site. Pipetting precision and accuracy was determined. Three blood sample standards were stabilized in either TransFix (Cytomark Ltd, Buckingham, UK) or CytochexTM (Streck Laboratories, Omaha, NE) and tested within 7 days post stabilization. Three rounds of testing were completed for a total of nine samples. The study was performed in five separate laboratories, using seven different instruments and operators. After evaluation of this preliminary data, targets were established for intra-assay and inter-lab precision.
Flow Cytometric Immunophenotyping of Cerebrospinal Fluid
Kraan J, Gratama JW, Haioun C, Orfao A
Current Protocols Cytometry (2008) 45: 6.25.1-6.25.16
Quote: Leptomeningeal disease is an important adverse complication occurring in patients with B and T cell lymphomas and acute leukemias of lymphoid and myeloid origin. Recent reports suggest that multiparameter flow cytometry immunophenotypic assessment of spinal fluid samples could improve the efficiency of detection of CNS involvement, due to its high specificity and greater sensitivity. However, spinal fluid samples are frequently paucicellular with a rapidly decreasing cell viability. Staining of spinal fluid therefore requires dedicated sample storage/transport, staining, and preparation protocols. The Basic Protocol in this unit outlines a consensus multiparameter (3- to 8-color) flow cytometry immunophenotypic protocol for the evaluation of CNS involvement of cerebrospinal fluid (CSF) samples by neoplastic cells. A Support Protocol describing the simultaneous assessment of surface and cytoplasmic antigens is also provided. Finally, in the Alternate Protocol, we describe a method to calculate absolute numbers of both normal and pathological cell subpopulations by adding counting beads to the assay.
Research needs and challenges in the development of HIV diagnostic and treatment monitoring tests for use in resource-limited settings
Cheng B, Landay A, Miller V
Current Opinion in HIV & AIDS (2008) 3: 495-503
Long-term stabilized blood samples as controls for flow cytometric HLA-B27 screening: A feasibility study
Levering WHBM, Wind H, Granger V, Sintnicolaas K, Hooijkaas H, Reilly JT , Gratama JW, Barnett D
Cytometry Part B (2008) 74B: 169–181
Quote: Long-term stabilized blood samples are potentially useful as positive or negative procedure controls for flow cytometric HLA-B27 screening, and could serve as test samples in an external quality assessment (EQA) scheme. We evaluated long-term stabilized whole blood specimens as prepared for the UK NEQAS for Leucocyte Immunophenotyping EQA scheme (Sheffield, UK). METHODS: Peripheral blood samples were obtained from nine blood bank donors with known HLA-B typing. Short-term stabilization with TransFix was performed before shipment to Sheffield. Thereafter, long-term stabilization was performed
Flow cytometric determination of residual white blood cell levels in preserved samples from leukoreduced blood products
Palmer DS, Birch P, O'Toole J, Henderson D, Scalia V
Transfusion (2008) 48: 118–128
2007
CD44 deficiency is a consistent finding in childhood Burkitt's lymphoma and leukemia
Attarbaschi A, Mann G, Schumich A, König M, Pickl WF, Haas OA, Gadner H, Dworzak MN
Leukemia (2007) 21: 1110-1113
Quote: Although we found no impact of time delay between sampling and immunophenotyping on the expression levels of CD44, stabilization of the samples for transport (TransFix, Cytomark) will be very important to avoid loss of residual Burkitt's cells, which are especially prone to undergo spontaneous apoptosis.
Starting a proficienct A PROFICIENCY TESTING PROGRAM FOR FLOW CYTOMETRY: ISTANBUL EXPERIENCE
Demirel GY, Topbas F
Poster
Quote: TransFix was used to prepare External Quality Assurance samples for CD4 Immune Monitoring
Cell preparation (book)
DA McCarthy - Flow Cytometry
Cell Preparation, Flow Cytometry, 17-58, 2007
Advances in CD4 cell enumeration in resource-poor countries
Baum L, Crowe S. Landay AL
Current Opinion in HIV and AIDS (2007) 2, 157-245
Quote: The availability of antiretroviral medications increases the need for CD4 cell assays to monitor disease progression and the efficacy of therapy in resource-poor countries. Simplified flow cytometry is needed in centralized clinics, but there is a critical need for even less expensive easier methodology that can be used in smaller laboratories and in rural villages. TransFix can be used to stabilise samples for transportation to central laboratories.
Diagnostic accuracy and clinical utility of a simplified low cost method of counting CD4 cells with flow cytometry in Malawi: diagnostic accuracy study
MacLennan C, Liu MKP, White SA, van Oosterhout JJG, Simukonda F, Bwanali J, Moore MJ, Zijlstra EE, Drayson MT, Molyneux ME
British Medical Journal (2007) 335: 190
Quote: We have described an affordable accurate method of CD4 counting that has the potential to improve clinical decision making in the treatment of patients with HIV and service the whole of a country the size of Malawi using a limited number of instruments in regional centres. This arrangement could be facilitated by the use of blood stabilising agents such as Transfix, permitting delays in sending samples to regional centres. It remains to be seen whether such a service could be successfully implemented in such a resource poor setting.
2006
Identification of Neoplastic Infiltration of the Cerebrospinal Fluid (CSF) in Patients with Aggressive B-Cell Non-Hodgkin’s Lymphoma (B-NHL) without Clinical Evidence of Leptomeningeal Disease: A Comparative Analysis of the Utility of Flow Cytometry (FCM) Versus Conventional Cytology (CC).
Orfao A, Quijano S, Lopez A, Deben G, Salar A, Poderos C, Sancho JM, Vallejo C, Arias J, Garcia JA, Capote F, Morado M, Nicolas C, Briones J, Fernandez S,Carmona, L. Vazquez, and J.F. San Miguel
Blood (ASH Annual Meeting Abstracts) (2006) 108: 4605.
Quote: In all cases, the CSF samples were analysed simultaneously by CC at the institution of origin and FCM, centrally one institution. For the FCM analysis of the CSF, stabilised samples (Transfix, CYTOMARK) were systematically stained with the following combination of monoclonal antibodies: CD8-sIgl/CD56-sIgk/CD4-CD19/CD3/CD20/CD45 (FITC/PE/PERCPCY5.5/PECY7/APC/APCCY7). If the FCM test showed infiltration, an additional 6-color antibody panel was used for full phenotypic characterisation of the disease.
Evaluation of a low-cost method, the Guava EasyCD4 assay, to enumerate CD4-positive lymphocyte counts in HIV-infected patients in the United States and Uganda
Spacek LA, Shihab HM, Lutwama F, Summerton J, Mayanja H, Ronald A, Margolick J, Nilles TL, Quinn TC
Journal of Acquired Immune Deficiciency Syndrome (2006) 15;41(5): 607-10
2005
Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry
Pinto LA, MT Trivett, D Wallace
Cytometry Part B: Clinical Cytometry (2005) 63B, Issue 1: 47–55
Immunophenotyping of plasma cells in multiple myeloma
G.M. Manzanera, J.F.S.M. Izquierdo, A.O. de Matos
Methods in Molecular Medicine, Volume 113, 5-24, 2005
Performance of aged, TransFix® treated blood in the Guava EasyCD4 and EasyCD8 Assays
A Mergai et al
12th conference on retroviruses and opportunistic infection (2005) : Poster (www.millipore.com/publications)
Quote: Monitoring CD4+ and CD8+ T cell counts has become essential in monitoring healthy individuals and patients infected with HIV / AIDS. The study reported the compatibility of 15 day old TransFix® treated blood with the Guava EasyCD4 and EasyCD8 assays. The staining pattern for TransFixed® samples over 15 days was almost identical to the staining pattern at day 0. At 30 days the staining pattern had started to show signs of deterioration but accurate counts could still be obtained.
2004
Evaluation of leukocyte stabilisation in TransFix® treated blood samples by flow cytometry and transmission electron microscopy.
B. Canonico, L. Zamani, S.Burattini, V. Granger, F. Mannello, P.Gobbi, C. Felici, E. Falcieri, J.T.reilly, D.Barnett and S.Papa
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.
2003
Monitoring of human immunodeficiency virus infection in resource-constrained countries
S Crowe, S Turnbull and R Oelrichs
Clinical Infectious Diseases 37 (Supplement 1): S25-S35. 2003
New trends in affordable CD4+ T-cell enumeration by flow cytometry in HIV/AIDS
Janossy G, Jania IV, Brandob B
Clinical & Applied Immunology (2003) 4: 91-107
2002
New concepts in affordable cd4+ t cell enumeration for resource-poor settings.
Janossy G, Jani IV, NJ Bradley, Pitfield T
International Conference on AIDS (2002) 7-12; 14: abstract no. MoPeB3220.
Affordable CD4+-T-Cell Counting by Flow Cytometry: CD45 Gating for Volumetric Analysis
Janossy G., Jani IV, Bradley NJ, Bikoue A, Pitfield T, Glencross DK
Clinical Diagnostic Laboratory Immunology (2002) 9: 1085 - 1094.
Less expensive CD4+ T cell monitoring using panleucogating.
Wilja M, Janossy G, Glencross D, Barnett D
Poster: Barcelona Aids Meeting - July 2002
Evaluation of TransFix®, a commercial whole blood stabilizing reagent. This product reduces HIV replication.
Kim et al: Cytometry
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.
Evaluation of stabilised blood cell products as candidate preparations for quality assessment programs for CD4 T-cell counting.
M. Bergeron, A.Shafaie, T.Ding, S.Phaneuf, N.Soucy, F. Mandy, J.Bradley & J.Fahey
Cytometry (2002) 50, 86-91
Exceptionally robust cell preparations are needed for quality assessment programs. A suitable product must withstand the environmental stress related to transportation for a minimum of 6 day. Six preparations were evaluated by monitoring T-cell subset values for samples stored at 4ºC, 22ºC and 37ºC. Sample stability was tested daily up to day 7. Only TransFix® and ImmunoTrol were successful at stabilising blood samples across the full range of storage temperatures.
Stabilised cellular immuno-fluorescence assay: CD45 expression as a calibration standard for human leukocytes.
Bikoue et al
Journal of Immunological Methods (2002) 266: 19-32
2001
CD45 assisted panleucogating for accurate, cost effective dual platform CD4+ T cell enumeration.
Deborah Glencross, Lesley E. Scott, Ilesh V. Jani, David Barnett and George Janossy
Cytometry (2001) 50: 69-77
A higher accuracy and precision of CD4 counts was documented with Pan Leucogating compared with lymphocyte gating using stabilised blood controls provided by UK NEQAS. These results were verified with 183 fresh samples and 112 TransFixed samples. These observations showed that dual platform leucocyte counts should replace lymphocyte counts.
Affordable CD4+ T cell counts by flow cytometry. II. The use of fixed whole blood in resource poor settings.
I. Jani, G.Janossy, A.Iqbal, F. Mhalu, E.Lyamuya, G.Biberfield, D.Glencross, L.Scott, J.T.Reilly, V.Granger & D.Barnett
Journal of Immunological Methods (2001) 257: 145-154
The feasibility and precision of CD4 counting in resource poor areas was tested using TransFix®. It has been shown that immunological gating, including the primary identification of T, B and NK cells by virtue of their specific antibody binding capacity, is a robust method for absolute cell counting. With TransFixed samples primary CD4+ gating can be performed with great precision. There was also no difference in the precision of CD4+ counts performed on fresh cells in Tanzania and TransFixed cells in South Africa. TransFixed samples from smaller clinics can be batched over a period of one week for shipment to local or regional hospitals for analysis.
Low level leucocyte counting: a critical variable in the validation of leucodepleted blood transfusion components as highlighted by an external quality assurance study.
D. Barnett, K. Goodfellow, J. Ginnever, V. Granger, L. Whitby & J.T. Reilly
Clinical Laboratory Haematology (2001) 23: 43-51
Leucocyte counts of <5 x 106 per blood transfusion product are currently recommended in the UK in order to reduce transfusion related infections and febrile reactions. Routine leucocyte depletion requires the development of reliable internal and external quality assurance programmes. A stabilised low leucocyte blood control manufactured by UK NEQAS was used to determine inter and intra laboratory CV’s. The study highlighted the variability in low level leucocyte counting, especially within the critical range of 5-30 cells/μl. The results highlighted the need for a standardised protocol and nuclear staining reagent for the routine validation of leucocyte depleted blood products.
2000
Standardization of lymphocyte antibody binding capacity – a multi centre study.
D.Barnett, I.Storie, V.Granger, L.Whitbey, J.T.Reilly, S.Brough, S.Garner, J.Lawry, S.Richards, A.E.Bell and B.K.Shenton
Clinical Laboratory Haematology (2000) 22: 89-96
Quantitative flow cytometry is increasingly being used to characterise non-malignant and malignant disorders, inter and intra laboratory standardisation becomes an important issue. However the lack of standardisation methods and process controls with defined antibody binding values, limits direct comparison. The study showed that a standard protocol is required to achieve low CV’s. Also that stable whole blood can be used as a process control with defined antibody binding capacity values.
Reduction of intra and inter laboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training.
D.Barnett, V.Granger, J.Kraan, J.T.Reilly, S.Papa & J.W.Gratama
British Journal of Haematology (2000) 108: 784-792
The European Working Group on Clinical Cell Analysis has developed a single platform flow cytometric protocol for the enumeration of CD34+ stem cells. A standard protocol was used over 24 clinical sites together using a CD34+ stabilised whole blood control. The use of a common standardisation protocol and targeted training significantly reduced intra and inter laboratory CD34+ cell count variation.
1999
TransFix®: A clinical sample stabilising fluid for use in clinical haematology and immunology.
D. Barnett, V.Granger, A.G. Pockley, J.M.saxton, I storie, L.Whitby & J.T.Reilly
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.
Lymphocyte subset absolute counts in fixed blood samples.
Janossy et al
Afford CD4 (1999) TransFix® transfer experiments.
Absolute CD4+ T-lymphocyte and CD34+ stem cell counts by single platform flow cytometry: the way forward.
D. Barnett, V.Granger, L.Whitby, I.Storie and J.T.Reilly
British Journal of Haematology (1999) 106: 1059-1062
Stabilised blood controls for CD34 and CD4 were distributed to 280 laboratories worldwide, every 2 months for a year for absolute cell counts. Results were correlated by UK NEQAS. The study showed that the single-platform approach produced consistently lower inter laboratory CV’s for both CD4+ and CD34+ enumeration. Furthermore such technology is likely to prove the method of choice for the quality control of leucodepleted blood products.
1998
Quality assessment of CD34+ stem cell enumeration: experience of the United Kingdom National External Quality Assessment scheme (UK NEQAS) using a unique stable whole blood preparation.
D.Barnett, V.Granger, I.storie, J.Peel, R.Pollitt, T.Smart and J.T.Reilly
British Journal of Haematology (1998) 102: 553-565
CD34+ peripheral blood stem cell mobilisation and harvesting has replaced autologous bone marrow as a source of stem cells for transplantation. Timing and adequacy of harvests rely upon the accurate enumeration of circulating CD34+ cells. Previous EQA schemes reported inter-laboratory CV’s as high as 284%. A novel stabilised whole blood preparation was distributed to 91 laboratories in 21 countries and participants were required to determine the percentage and absolute values for CD34+ peripheral blood stem cells. Using this method inter-laboratory CV’s were reduced as low as 22% for both percentage and absolute counts.
1996
Evaluation of a novel stable whole blood quality control material for lymphocyte subset analysis: Results from the UK NEQAS immune monitoring scheme.
D. Barnett, V.Granger, P.Mayr, I.Storie, G.A.Wilson and J.T.Reilly Cytometry (Clinical Cytometry) (1996) 26: 216-222
The suitability of a novel stable whole blood preparation for use as both daily and longitudinal quality control was demonstrated through the UK NEQAS Immune Monitoring scheme. The stabilisation procedure ensures retention of leucocyte light scatter and immunological staining characteristics for up to 300 days. The preparation is also fully compatible with flow cytometer technology, incorporating either whole blood lysis or “no wash, no lyse” techniques. The ranges of inter-laboratory CV’s of the stabilised control were better than those previously obtained with fresh whole blood.
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