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2002

Human Blood Stabilisation - Immunophenotyping

KEY PAPER: Evaluation of stabilised blood cell products as candidate preparations for quality assessment programs for CD4 T-cell counting

Sample Type: T-cell subsets

Analysis Method: Flow Cytometry

Authors

M. Bergeron, A.Shafaie, T.Ding, S.Phaneuf, N.Soucy, F. Mandy, J.Bradley & J.Fahey

Publication

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 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.

http://www.ncbi.nlm.nih.gov/pubmed/12116350

KEY PAPER: Evaluation of TransFix, a commercial whole blood stabilizing reagent. This product reduces HIV replication

Sample Type: CEM 13D cells

Analysis Method: P24 AG assay

Authors

Kim et al

Publication

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.

Less expensive CD4+ T cell monitoring using panleucogating

Sample Type: CD4+ cells

Analysis Method: Flow Cytometry

Authors

Wilja M, Janossy G, Glencross D, Barnett D

Publication

Poster: Barcelona Aids Meeting - July 2002

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.

http://www.iasociety.org/Abstracts/A5137.aspx

New concepts in affordable cd4+ t cell enumeration for resource-poor settings

Sample Type: CD4, CD8 and CD45+ cells

Analysis Method: Flow Cytometry

Authors

Janossy G, Jani IV, NJ Bradley, Pitfield T

Publication

International Conference on AIDS (2002) 7-12; 14: abstract no. MoPeB3220

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.

http://www.iasociety.org/Abstracts/A2320.aspx

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


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