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34 Human Blood Stabilisation - Immunophenotyping articles
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TransFix: A clinical sample stabilising fluid for use in clinical haematology and immunology
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. -
TransFix for delayed flow cytometry of endothelial progenitor cells and angiogenic T cells
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. -
Stabilised cellular immuno-fluorescence assay: CD45 expression as a calibration standard for human leukocytes
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. -
Research needs and challenges in the development of HIV diagnostic and treatment monitoring tests for use in resource-limited settings
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, Shefield, 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. -
Performance of aged, TransFix treated blood in the Guava EasyCD4 and EasyCD8 Assays
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. -
No Differences in Levels of Circulating Progenitor Endothelial Cells or Circulating Endothelial Cells Among Patients Treated With Ticagrelor Compared With Clopidogrel During Non-ST-Segment-Elevation Myocardial Infarction
The aim of this study was to determine differences in the number of endothelial progenitor cells and/or circulating endothelial cells found in peripheral blood in patients treated with either ticagrelor or clopidogrel during non ST-segment elevation myocardial infarction In this multicentre, randomized study (NCT02244710), all samples were kept in TransFix to enable adequate shipping to a central laboratory for analysis within 5 days Markers included: CD34, CD133, CD45, CD146, CD31, KDR (CD309), 7AAD, and Syto-16. Generic 7AAD and Syto-16 markers were used to define cell viability and nuclear cells, respectively. -
New trends in affordable CD4+ T-cell enumeration by flow cytometry in HIV/AIDS
Inexpensive Antiretroviral Therapy (ART) might soon be available to treat human immunodeficiency Virus (HIV) infections in resource-restricted areas of the globe. The number of CD4+ T-cells in the blood is the single most important laboratory parameter to select patients for therapy at the right time and to monitor the effect of ART. Stabilised blood cell standards and blood stabilising fluid, referred to as TransFix have recently been introduced for Quality Assurance. As the fast changing scenery of Flow Cytometry diminished the influence of company-orientated and instrument-orientated Quality Assurance Schemes, more emphasis had to be paid to the use of stabilised standard biological samples in international Quality Assurance Schemes. Preservative fluids, such as TransFix have been introduced with two beneficial effects. Firstly, TransFix added to whole blood at 1:10 dilution stabilises whole blood to perform reliable lymphocyte subset analysis for as long at 10 days. The catchment areas for laboratories can therefore be extended to receive samples from further afield. Secondly. TransFix has an additional bonus for quality assurance and interlaboratory technical standardisation. Samples shipped and tested within the time limits of 10 days between continents can be fully utilised to check the performance of instruments, reagents, full technologies and other parameters. -
New concepts in affordable cd4+ t cell enumeration for resource-poor settings
Flow cytometers can provide accurate CD4+ T cell counts for monitoring HIV disease but remain too expensive for resource-poor settings. In this research they combined six modern concepts (i-vi) in flow cytometry (FCM) for more efficient and economical CD4 T cell enumeration. Volumetric flow cytometers were used with (ii) CD4, CD8 and CD45 generic monoclonal antibodies (iii) using the panleucogating protocol (iv) on TransFix stabilized blood samples. A simple protocol using generic MAb's, and with no hidden costs such as microbeads, allowed the accurate reporting of 16 parameters including leukocyte absolute counts and differentials, CD4 and CD8 absolute and percentage values, total CD4+CD8 T cell counts as well as CD4/CD8 ratios. Correlations for absolute CD4 counts and percentage values were R2>0.96, with virtually no bias. The correlation for CD8 counts was R2=0.997, with a bias of 5.2% in favour of the CD3+,CD8+ test but with no significant influence on CD4/CD8 ratios. One single assistant processed 400 samples per day, which extrapolates to a throughput of 100,000 CD4 tests per year. -
Monitoring of human immunodeficiency virus infection in resource-constrained countries
The reference standards used to monitor human immunodeficiency virus (HIV) infection are flow cytometric analysis of T lymphocyte subsets to provide the CD4+ T cell count and molecular assays to quantify plasma HIV load. Few laboratories in resource-constrained countries can afford to perform these tests. This review discusses the above assays and their role in addition to clinical monitoring in resource-constrained countries. It states that TransFix TM stabilises whole blood for CD4 cell analysis for 7 days at 37 degrees Celsius and <3 days at 42 degrees Celsius. This stabilisation allows the transport of samples from distant sites to laboratories. -
Method Validation and Study of Immunophenotype NK and T lymphocytes by flow cytometry
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. -
Method validation and immunophenotypical study of NK and T lymphocytes by flow cytometry
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. -
Longitudinal Follow Up of Immune Responses to SARS-CoV-2 in Health Care Workers in Sweden With Several Different Commercial IgG-Assays, Measurement of Neutralizing Antibodies and CD4+ T-Cell Responses
This study aimed to determine the previous infection of Health care workers in Sweden with Sars-Cov-19 and to compare this with the normal population. Different commercial IgG-assays were used along with measuring the neutralising antibodies and the CD4+ T-Cell responses. TransFix was used to fix the obtained samples which were stored until they underwent flow cytometry. Antibody panel: anti-CD3-V450 (#560365), anti-CD4-BV605 (#562658), anti-CD8-BV510 (#563919), anti-CD25-PE (#555432), anti-CD134-PECy7 (#563663), anti-CD69-FITC (#555530) and anti-CD137-APC (#550890) antibodies to the pre-fixed cells (all antibodies from BD Biosciences, Franklin Lake, NJ). -
Long-term effects of smallpox vaccination on expression of the HIV-1 co-receptor CCR5 in women
This study investigated whether smallpox vaccination is associated with a down-regulation of CCR5 on the surface of peripheral T-lymphocytes in healthy women in Guinea-Bissau. Cryopreservation of cells causes down-regulation of CCR5 on the cell surface. Antibody staining and analysis of surface CCR5 should therefore be performed on freshly isolated PBMCs. However, flow cytometry was not available in Guinea-Bissau during the study period. The Authors therefore validated the following method: Blood was sampled in special blood collection tubes containing TransFix (Data and description of TransFix validation is available from figshare https://doi.org/10.6084/m9.figshare.7139687.v1). -
Less expensive CD4+ T cell monitoring using panleucogating
Tests were carried out to compare the cost and accuracy of DP:lymphocyte gating using TriTEST with MultiSET software [LyG] and DP:panleucogating [PLG] and to evaluated TransFix to stabilize blood and extend the time between blood draw and testing.EDTA blood samples were analyzed within 24 hours by FACSCount, LyG and PLG. TransFix samples were evaluated after 3 and 7 days. Cells stabilized by TransFix, the correlation between results at day 3 and day 0 was excellent for PLG and FACSCount. Correlation between results at day 7 and day 0 were good only for PLG [R2=0.85]. In conclusion the PLG results correlated more closely than LyG with FACSCount results and TransFix allowed accurate testing for at least 3 days following blood draw. -
Interlaboratory variability of CD34+ stem cell enumeration. A pilot study to national external quality assessment within the Czech Republic
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. -
Interlaboratory Comparison of the TransFix®/EDTA Vacuum Blood Collection Tube with the 5 mL Cyto-Chex® BCT
An interlaboratory study was performed to compare the performance of the 3 mL TransFix®/EDTA Vacuum Blood Collection Tube (TVT), with the 5 mL Cyto-Chex® BCT tube (BCT). Both devices were tested for their intended use for collection and storage of whole blood specimens for immunophenotyping of leukocytes by flow cytometry for up to 14 days. Statistical concordance was demonstrated for both devices in relation to cell absolute count recovery. For cell marker signal, both devices exhibited a significant decrease in mean fluorescence intensity (MFI) for the detection of lymphocyte subsets and their target markers. There was a marked increase in autofluorescence observed for BCT stabilized lymphocytes whereas values for the TVTs were comparable to the control. There were eight instances of statistical equivalence between the level of antibody autofluorescence observed in the control tube and the TVT across both patient cohorts, versus two for the BCT. -
Implementation of highly sophisticated flow cytometry assays in multicenter clinical studies: considerations and guidance
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. -
Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes and red cells
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. -
Flow cytometric CD34+ stem cell enumeration: Lessons from nine years' external quality assessment within the Benelux countries
Assessment of interlab variability with regard to Flow Cytometric CD34+ Stem Cell enumeration. Authors discuss the use of TransFix in an EQA scheme to improve interlaboratory variability. -
First-in-Human Study in Healthy Subjects with FR104, a Pegylated Monoclonal Antibody Fragment Antagonist of CD28
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. -
Evaluation of TransFix, a commercial whole blood stabilizing reagent. This product reduces HIV replication
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
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 4 degrees Celsius, 22 degrees Celsius and 37 degrees Celsius. 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. -
Evaluation of leukocyte stabilisation in TransFix treated blood samples by flow cytometry and transmission electron microscopy.
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 -
Effects of benralizumab on airway eosinophils in asthmatic patients with sputum eosinophilia
The Authors evaluated the safety of benralizumab in adults with eosinophilic asthma and its effects on eosinophil counts in airway mucosal/submucosal biopsy specimens, sputum, bone marrow, and peripheral blood. One of the exploratory objectives in multicentre phase I study included evaluation of basophil in PB: Blood was collected in lithium-heparin and 0.4 mL of TransFix was added to stabilize and preserve cells for subsequent flow cytometric analysis. After shipping to a central testing facility (within 14 days) co-expression of IgE high-affinity receptor (FcεRI) and CCR3 with CD123 and chemoattractant receptor of type 2 helper T cells (CRTH2) were collectively consulted to identify the basophil population. -
Downregulation of the T-Cell Receptor by Human Immunodeficiency Virus Type 2 Nef Does Not Protect against Disease Progression
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). -
Double Blind, randomised, placebo-controlled intervention trial to evaluate the effects of Bifido longum CECT 7347 in children with newly diagnosed coeliac disease
The aim of this study was to conduct a 3-month double-blind, randomised, placebo-controlled intervention trial in children with newly diagnosed CD following a GFD in order to evaluate the effects of B. longum CECT 7347 administration on immune and anthropometric parameters, and on intestinal microbiota composition. For lymphocyte phenotyping, peripheral blood samples were collected in heparin tubes mixed with TransFix and sent immediately to IATA-CSIC at room temperature for cytometry analysis (CD45, CD3, HLA-DR,CD4, Foxp3, CD8) -
Direct Relationship between Virus Load and Systemic Immune Activation in HIV-2 Infection
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. -
Collection, Storage, and Preparation of Human Blood Cells
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. -
Clinical Samples: Selection, Shipping, and Processing
Flow Metric explores the various methods for selecting, shipping and processing samples. They mention various options for preservation-fixation solutions one of which being TransFix. -
CD45 assisted panleucogating for accurate, cost effective dual platform CD4+ T cell enumeration
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. -
CD4 Intragenic SNPs Associate With HIV-2 Plasma Viral Load and CD4 Count in a Community-Based Study From Guinea-Bissau, West Africa
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). -
Affordable CD4+ T cell counts by flow cytometry. II. The use of fixed whole blood in resource poor settings
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. -
Advances in CD4 cell enumeration in resource-poor countries
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. -
A proficiency testing program for flow cytometry: Istanbul experience
TransFix was used to prepare External Quality Assurance samples for CD4 Immune Monitoring