2.    Shapiro, H.M. (1995). Practical Flow Cytometry (3rd Edition) Wiley-Liss, New York, Chichester, Brisbane, Toronto, Singapore.  A brilliant book that remains practical. It has an added bonus with its engineering, aesthetic and intellectual attractions. See relevant pages 67-68,106-114, 134-139, 244-245, 274-282 and 360-363.

3.    Omega Optical Inc. (1998).  Fluorescence filter selection, 6th Edition.  Do not miss this exciting brochure if you wish to know the background.   Website:  www.omegafilters.com

4.    Haugland, R.P. (1999)  Handbook of Fluorescent Probes and Research Chemicals (7th Edition). Molecular Probes ISBN 0-9652240-4-X on CD-ROM.  Rich pallets for the various biological applications of fluorescence microscopy & cytometry. Website:  www.probes.com

5.    Roederer, M., De Rosa, S., Gerstein, R., Anderson, M., Bigos, M., Stovel, R., Nozaki, T., Parks, D. & Herzenberg, L. (1997). 8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity. Cytometry, 29, 328-39.  also  Lee, P.P., Yee,C., Savage, P.A., Fong, L., Brockstedt, D., Weber, J.S., Johnson, D., Swetter, S., Thompson, J., Greenberg, P.D., Roederer, M. & Davis, M.M. (1999) Characterization of circulating T cells specific for tumor associated antigens in melanoma patients. Nature Medicine, 5, 677-685.   If you want to see as many as 10 colours used for flow cytometry - or wish to know how the fluorochrome conjugations are done.  See Dr. Roederer's website:   www.drmr.com/abcon/

6.    Brando, B., Barnett, D., Janossy, G. Mandy, F., Autran, B., Rothe, G., Scarpati, B., D'Avanzo, G., D'Hautcourt, J-L., Lenkei, R., Schmitz, G., Kunkl, A., Chianese, R., Papa, S. & Gratama, J-W. for the European Working Party on Clinical Cell Analysis (EWGCCA)  (2000). Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. Cytometry (Communication in Clinical Cytometry) 42, 327-346.    This review is clinically and practically oriented (one can even learn how to pipette precisely - better now than never! - another simple and crucial issue) - while the recent volumes of the Journal of Immunological Methods (2000) 243, (1-2; editor H. Zola) are devoted to Flow Cytometry more from a scientific point of view.  pdf file

7.    Brando, B., Scarpati, B. & D'Avanzo, G. (2001). Development of simplified, inexpensive flow cytometry for CD4+ cell counting. This article is especially written for AffordCD4, 7, 1-4.   A testimony for the precision reached by clinical flow cytometry on the different machines such as FACSCalibur, Beckman Coulter XL, ORTHO Cytoron and DAKO Galaxy (as well as all the problems such as costs).  pdf file

8.    Sherman, G.G., Galpin, J.S., Patel, J.M., Mendelow, B.V. &  Glencross, D.K.  (1999). CD4+ T cell enumeration in HIV infection with limited resources. J Immunol Methods, 222, 209-17.   This was the first paper that revealed a genuine interest in cutting costs (from a laboratory with heavy routine service commitments on the African continent) whilst also offering sensible solutions (see also ref. 12).  pdf file 

9.    Loken, M.R., Brosnan, J.M., Bach, B.A. & Ault, K.A. (1990). Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry. Cytometry, 11, 453-9.   Leucocyte (CD45) reagents see all leucocytes  and in addition, discriminate between lymphocyts, monocytes and granulocytes by their differing expression of CD45 staining intensity (also see  ref. 12). 

10.   Mandy, F.F., Bergeron, M., Recktenwald, D. & Izaguirre, C.A. (1992). A simultaneous three-color T cell subsets analysis with single laser flow cytometers using T cell gating protocol. Comparison with conventional two-color immunophenotyping method. J Immunol Methods, 156, 151-62.    Routine analysis viewed from the angle of exquisiste immunological specificity (of CD3 reagent) - simple, logical and useful.

11.    Janossy, G., Jani, I.V. & Goehde, W. (2000) Affordable CD4+ T cell counts on 'single-platform' flow cytometers I. Primary CD4 gating. Brit.J.Haematol. 111, 1198-1208.   This paper sets out the concept of 'Primary CD4 gating' on volumetric flow cytometers.  pdf file

12.   Glencross, D., Scott L., W., Jani, I.V., Barnett, D. & Janossy, G. (2002). CD45 assisted PanLeucogating for Accurate, Cost Effective Dual Platform CD4+ T cell Enumeration.  In Press, Cytometry (CCC).  Use a dual platform system comprising any flow cytometer with any haematology  analyser (to generate a WBC count), and PanLeucogating performs brilliantly. Fact: dual-platform users are still firmly in the majority!. pdf file

13. Mercolino, T.J., Connelly, M.C., Meyer, E.J., Knight, M.D., Parker, J.W., Stelzer, G.T. & DeChirico, G. (1995). Immunologic differentiation of absolute lymphocyte count with an integrated flow cytometric system: a new concept for absolute T cell subset determinations. Cytometry, 22, 48-59.  All that the connoisseur  'public' ever wanted from automated absolute counting technology (including large auto-biosampler, volumetry, good clinical software with QC, etc.) - except that ORTHO has pulled the carpet (again, not the first time that this happened).

14. O'Gorman, M.R. & Nicholson, J.K. (2000). Adoption of single-platform technologies for enumeration of absolute T-lymphocyte subsets in peripheral blood. Clin Diagn Lab Immunol, 7, 333-5. 

See also :

• Schnizlein-Bick, C.T., Spritzler, J., Wilkening, C.L., Nicholson, J.K. & O'Gorman, M.R. (2000). Evaluation of TruCount absolute-count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus-positive adults. Clin Diagn Lab Immunol, 7, 336-343 and  

• Reimann K.A., O’Gorman M., Spritzler J., Wilkening C., Sabath D.E., Helm K., Campbell D.E., Nicholson J.K.A. and the NIAID DAIDS New Technologies Evaluation group (2000).   Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple Platform Methodologies: Evaluation of Beckman Coulter Flow-Count and the tetraONE System.  Clin Diagn Lab Immunol, 7: 344-351.   

Nice, but how affordable all this is for $1000-2400 per day?  Just watch how these costs mount up!  If support of that magnitude arrives at all, will the grant support from the West keep coming, once CD4 counts are "just a boring routine" - how it should be?

15. Jani, I., Janossy, G., Iqbal, A., Mhalu, F.S., Lyamuya, E.F., Biberfeld, G., Glencross D.K., Scott L.E., Reilly, J.J., Granger, V. & Barnett, D. (2001) Affordable CD4+ T cell counts on 'single-platform' flow cytometers II. The use of fixed whole blood in resource poor settings. Journal of Immunological methods 257: 145-154, 2001.   When blood stabilizers are used there is more time made available for the transported samples to reach their destination in a viable state - a crucial practical issue everywhere except in the richest countries.   pdf file

16. Barnett, D., Granger, V., Whitby, L., Storie, I. & Reilly, J.T.  Absolute CD4+ T-lymphocyte and CD34+ stem cell counts by single-platform flow cytometry: the way forward. Br J Haematol, 106, 1059-1062, 1999). .  A positive contribution from the UK NEQAS Quality Control centre - now of European dimensions. Such non-profit QC service is indispensible for training and for validating superb performance in laboratories of developing countries.

17. Mandy, F, Bradley, J. & Fahey, J.L. (2000) International program for Quality Assessment and Standardization for Immunity to HIV/AIDS. ISAC XX Intenational Congress, May 20-25, 2000.   Similarly important EQA and standardization issues related to HIV/ AIDS monitoring.

18. Janossy, G., Jani, I.V.,  Kahan, M., Barnett D., Mandy F. and Shapiro H.M. (2002). Precise CD4 T cell counting using Red Diode Laser Excitation: for Richer, for Poorer. Cytometry (CCC), in press, 2002  A new opening for flow cytometry with a laser that costs $200 or less.   pdf file

19. Shapiro, H.M. (2000). Cytometry in the Third World. 30th Oct.2000. Dr Shapiro's important announcement is reproduced here.   E-mail Dr. Shapiro: hms@shapirolab.com    pdf file

20. Janossy, G., Jani, I.V., Bradley N., Pitfield T and Glencross D.K. et al. (in press). Affordable CD4 T cell counts by Flow Cytometry III: CD45 gating for optimal Volumetric analysis. J. Imm. Methods. , in press (2002)  High volume output for CD4 reporting for minimal input and cost.   pdf file

21. Tibbe, AGJ., de Grooth BG,  Greve J, Liberti PA, Dolan GJ. and WMM Terstappenl. (2001). Optical tracking and detection of immunomagnetically selected and aligned cells. Nature Biotechnology, 17: 1210.  www.biotech.nature.com  A fresh approach to CD4 counting.   pdf file

22. Tibbe, AGJ., de Grooth BG,  Greve J, Liberti PA, Dolan GJ. and WMM Terstappenl. (2001). Cell analysis system based on immunomagnetic cell selection and alignment followed by Immunofluorescent analysis using compact disc technologies. Cytometry 43: 41-47.  www.biotech.nature.com  Once again this fresh approach using immunomagnetic cell selection is elegantly described.   pdf file

23. Jani, I., Janossy, G., Brown DWG and Mandy F.  Multiplexed Immunoassays by Flow Cytometry for Infectious Diseases in Resource Poor Settings (2002) . Lancet Infectious Diseases 2: 35-43.  Mulitiplex bead arrays that could really cut costs and time spent screening and diagnosing Infectious disease are summarised.   pdf file

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