Bibliography
Cellular Antigen Stabilising Reagent
TransFix® Bibliography
1. 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.guavatechnologies.com)
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.
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2. 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. Any days.
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3. 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.
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4. 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.
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5. 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
6. 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.
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7. 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.
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8. 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.
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9. 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.
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10. 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.
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11. 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.
12. Lymphocyte subset absolute counts in fixed blood samples.
Janossy et al
Afford CD4 (1999) TransFix® transfer experiments
13. 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.
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14. 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.
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15. 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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