Absolute CD4 T-Cell Counting in Resource-Poor Settings: Direct Volumetric Measurements Versus Bead-Based Clinical Flow Cytometry Instruments

Flow cytometry is an accurate but expensive method to determine absolute CD4 cell counts. We compared different methods to measure absolute CD4 counts in blood samples from HIV-infected and uninfected subjects using a research/clinical flow cytometer (FACScan); a dedicated clinical instrument (FACSCount); and a volumetric, mobile, open-system flow cytometer equipped with 3 fluorescence and 2 light scatter detectors (Cyflow SL blue). The FACScan and Cyflow were used as single-platform instruments, but they differ in running cost, which is a central factor for resource-poor settings. Direct volumetric and bead-based CD4 measurements on the Cyflow were compared with 2 bead-based single-platform CD4 measurements on the FACSCount and on FACScan (TruCount) in “Le Dantec” Hospital, Dakar, Senegal, using whole blood samples from 102 HIV+ and 28 HIV− subjects. The agreement between the various measurement methods was evaluated by Bland-Altman analysis. Volumetric CD4 measurements on the Cyflow using a no-lyse-no-wash (NLNW) procedure and a lyse-no-wash (LNW) procedure correlated well with each other (R2 = 0.98) and with CD4 measurements on the FACSCount (R2 = 0.97) and FACScan (R2 = 0.97), respectively. Red blood cell lysis had no negative effect on the accuracy of absolute CD4 counting on the Cyflow. An excellent correlation was observed between bead-based CD4 measurements on the Cyflow and CD4 measurements on the FACSCount (R2 = 0.99) and FACScan (R2 = 0.99). Rigid internal and external quality control monitoring and adequate training of technicians were considered essential to generate accurate volumetric CD4 measurements on the Cyflow.

[1]  A. Beckett,et al.  AKUFO AND IBARAPA. , 1965, Lancet.

[2]  W. Bablok,et al.  A New Biometrical Procedure for Testing the Equality of Measurements from Two Different Analytical Methods. Application of linear regression procedures for method comparison studies in Clinical Chemistry, Part I , 1983, Journal of clinical chemistry and clinical biochemistry. Zeitschrift fur klinische Chemie und klinische Biochemie.

[3]  W Bablok,et al.  Comparison of Several Regression Procedures for Method Comparison Studies and Determination of Sample Sizes Application of linear regression procedures for method comparison studies in Clinical Chemistry, Part II , 1984, Journal of clinical chemistry and clinical biochemistry. Zeitschrift fur klinische Chemie und klinische Biochemie.

[4]  D. Altman,et al.  STATISTICAL METHODS FOR ASSESSING AGREEMENT BETWEEN TWO METHODS OF CLINICAL MEASUREMENT , 1986, The Lancet.

[5]  A. Landay,et al.  Precision and accuracy of absolute lymphocyte counts. , 1989, Clinical immunology and immunopathology.

[6]  A Orfao,et al.  Performance evaluation of the FACSCount System: a dedicated system for clinical cellular analysis. , 1996, Cytometry.

[7]  J K Nicholson,et al.  Use of CD45 fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometry. , 1996, Cytometry.

[8]  D Barnett,et al.  Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis. , 2000, Cytometry.

[9]  George Janossy,et al.  Affordable CD4(+) T-cell counts on 'single-platform' flow cytometers I. Primary CD4 gating. , 2000 .

[10]  W. Göhde,et al.  Affordable CD4+ T‐cell counts on ‘single‐platform’ flow cytometers I. Primary CD4 gating , 2000, British journal of haematology.

[11]  M D Kazatchkine,et al.  Comparative assessment of five alternative methods for CD4+ T-lymphocyte enumeration for implementation in developing countries. , 2001, Journal of acquired immune deficiency syndromes.

[12]  Lesley E Scott,et al.  CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration. , 2002, Cytometry.

[13]  Jonathan AC Sterne,et al.  Prognosis of HIV-1-infected patients starting highly active antiretroviral therapy: a collaborative analysis of prospective studies , 2002, The Lancet.

[14]  George Janossy,et al.  Precise CD4 T-cell counting using red diode laser excitation: for richer, for poorer. , 2002, Cytometry.

[15]  Robert Oelrichs,et al.  Monitoring of human immunodeficiency virus infection in resource-constrained countries. , 2003, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[16]  W. Göhde,et al.  Individual Patient-Dependent Influence of Erythrocyte Lysing Procedures on Flow-Cytometric Analysis of Leukocyte Subpopulations , 2003, Transfusion Medicine and Hemotherapy.

[17]  Yves Traoré,et al.  Simplified Volumetric Flow Cytometry Allows Feasible and Accurate Determination of Cd4 T Lymphocytes in Immunodeficient Patients Worldwide , 2003, Antiviral therapy.

[18]  S. Kapiga,et al.  Comparison of a New, Affordable Flow Cytometric Method and the Manual Magnetic Bead Technique for CD4 T-Lymphocyte Counting in a Northern Nigerian Setting , 2005, Clinical Diagnostic Laboratory Immunology.