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ABSTRACT
Introduction. Stored blood specimens collected in Ethylene Diamine Tetra Acetic Acid (EDTA) anti-coagulant tubes for CD4 T-Cell determinations are inevitable practices in Cameroon and many other countries worldwide. This study aimed to evaluate the reproducibility of results obtained from blood samples stored at room temperature (25° C) and 4° C within six days post collection. Methods. Whole blood samples collected in EDTA collection tubes were analyzed for CD4 absolute counts using the FACS Count Machine (Becton Dickinson, France). We Used the FACS Count reagent kit (CD4, CD8 and CD3 combine). Samples were strictly analyzed using the procedural manual of the kit .Freshly collected blood samples in day one served as control. Results. There was a significant correlation with results obtained at day 2 and 3 (47 and 72 hours).Day 4, 5, 6 (72, 96 and 120 hours) did not show any correlation with day one. Blood samples stored at -4°C degrees did not correlate with any other day. Conclusion. Reproducibility of CD4 cell count could only be evaluated up to 72 hours, whereas blood samples stored in the fridge at -4°C were not reproducible after day one. Our findings can serve as a guide to clinical laboratories and health care providers handling samples in rural and urban settings.
RÉSUMÉ
Introduction. La conservation des échantillons de sang prélevés dans les tubes contenant l’anticoagulant Ethylène Diamine-Tétra-Acétique (EDTA) pour la détermination du taux de lymphocytes T CD4 sont des pratiques courantes au Cameroun et dans de nombreux pays du monde .Cette étude visait à évaluer la reproductibilité des résultats obtenus à partir des échantillons de sang stockés à température ambiante (25°C) dans les six jours suivant la collecte. Méthodologie. Les échantillons de sang total collectés dans des tubes EDTA ont été analysés afin de déterminer le nombre absolu de CD4 en utilisant l’appareil FACS Count (Becton Dickinson, France).Nous avons utilisé le kit de réactifs FACS Count (CD4, CD8 et CD3 combinés). Les échantillons ont été analysés en respectant la procédure décrite par le fabriquant du kit. Les échantillons de sang fraîchement prélevés au jour 1 ont servi de témoins. Résultats. Nous avons trouvé une corrélation significative entre les résultats obtenus au jour 2 et 3 (47 et 72 heures). Il n’y avait pas de corrélation entre les jours 4, 5, 6 (72, 96 et 120 heures) et le jour un. Les résultats des échantillons de sang stockés à - 4°C n’ont été corrélés avec ceux d’aucun autre jour. Conclusion. La reproductibilité du nombre de cellules CD4 ne peut être évaluée que jusqu'à 72 heures, alors que les échantillons de sang stockés dans le réfrigérateur à - 4°C ne sont pas reproductibles après le premier jour. Nos résultats peuvent servir de guide aux laboratoires cliniques et aux prestataires de soins de santé manipulant des échantillons en milieu rural et urbain.
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References
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- Böhler T, Nolting J, Kamar N, Gurragchaa P, Reisener K, Glander P, et al. Validation of immunological biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs in humans . Drug Monit. 2007;29(1):77–86.
- World Health Organization (WHO). for enumerating CD4 T Lymphocytes in the context of HIV / AIDS Laboratory Guidelines for enumerating CD4 T Lymphocytes in the context of HIV / AIDS. 2007. 298-299 p.
- Westerman LE, Kohatsu L, Ortiz A, Mcclain B, Kaplan J, Spira T, et al. A Quality Management Systems Approach for CD4 Testing in Resource-Poor Settings. Am Soc Clin Pathol. 2010;134(10):556–67.
- INFO A. Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents. 2016.
- Baggaley R, Dalal S, Johnson C, Macdonald V, Mameletzis I, Rodolph M, et al. Beyond the 90-90-90 : refocusing HIV prevention as part of the global HIV response. J Int AIDS Soc. 2016;19(1):21348.
- Slingluff CL, Petroni GR, Chianese-Bullock KA, Smolkin ME, Hibbitts S, Murphy C, Johansen N, Grosh WW, Yamshchikov GV, Neese PY, Patterson JW, Fink R RP. Immunologic and clinical outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma in the adjuvant setting. Clin Cancer Res. 2007;13:6386–95.
- 2010 WHO. ANTIRETROVIRAL THERAPY FOR HIV INFECTION IN ADULTS AND ADOLESCENTS Recommendations for a public health approach 2010 revision. 2010.
- Sagnia B, Ngogang J, Iii FFA, Colizzi V, Montagnier L, Bélec L. Validation of a single-platform , volumetric , flow cytometry for CD4 T cell count monitoring in therapeutic mobile unit. J Transl Med. 2012;10(22):1–9.
- Olson WC, Smolkin ME, Farris EM, Fink RJ, Czarkowski AR, Fink JH, et al. Shipping blood to a central laboratory in multicenter clinical trials : effect of ambient temperature on specimen temperature , and effects of temperature on mononuclear cell yield , viability and immunologic function. J Transl Med. 2011;9(26):1–13.
- Olteanu H, Schur BC, Harrington AM, Kroft SH. Time and temperature stability of T-cell subsets evaluated by a dual-platform method. Am J Res. 2012;2(2):128–35.
- Jt T, Pk R, Da V, Seckinger D. The effects of anticoagulant and temperature on the measurements of helper and suppressor cells . Diagn Immunol. 1984;2(3):167–74.
- Ekong T, Am H, Gompels M, Brown A, Aj P. The effect of the temperature and duration of sample storage on the measurement of lymphocyte subpopulations from HIV-1-positive and control subjects . J Immunol Methods. 1992;151(6):217–25.
- Fajardo E, Metcalf C, Mbofana E, Vyve C Van, Munyaradzi D, Simons S, et al. Opportunities to improve storage and transportation of blood specimens for CD4 testing in a rural district in Zimbabwe using BD vacutainer CD4 stabilization tubes : a stability and diagnostic accuracy study. BMC Infect Dis. 2014;14(553):1471–2334.
- Ashmore LM, Shopp GM EB. Lymphocyte subset analysis by flow cytometry. Comparison of three different staining techniques and effects of blood storage. J Immunol Methods. 1989;118(89):209–15.
- Bourguignon P, Clément F, Renaud F, Le V, Koutsoukos M, Burny W, et al. Processing of blood samples in fl uences PBMC viability and outcome of cell-mediated immune responses in antiretroviral therapy-naïve HIV-1-infected patients. J Immunol Methods [Internet]. Elsevier B.V.; 2014;414:1–10. Available from: http://dx.doi.org/10.1016/j.jim.2014.09.001
- Seely P, Yang X, Diallo TO, Plews M. Compatibility of Stabilized Whole Blood Products with CD4 Technologies and Their Suitability for Quality Assessment Programs. PLoS One. 2014;9(8).
- Garraud O MT. Effect of blood storage on lymphocyte subpopulations. Journal of Immunological Methods. 1984; J Immunol Methods 1984; 1984;75(84):95–8.
- Weinberg A, Song LY, Wilkening CL, Fenton et al. O. Optimization of storage and shipment of cryopreserved peripheral blood mononuclear cells from HIV-infected and uninfected individuals for ELISPOT assays. J Immunol Methods. 2010;363(1016):42–50.
References
Jg S, Hr J, Green T, Ja F, Wooters M, Rm K, et al. Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clin Vaccine Immunol. 2007;14(10):2007.
Böhler T, Nolting J, Kamar N, Gurragchaa P, Reisener K, Glander P, et al. Validation of immunological biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs in humans . Drug Monit. 2007;29(1):77–86.
World Health Organization (WHO). for enumerating CD4 T Lymphocytes in the context of HIV / AIDS Laboratory Guidelines for enumerating CD4 T Lymphocytes in the context of HIV / AIDS. 2007. 298-299 p.
Westerman LE, Kohatsu L, Ortiz A, Mcclain B, Kaplan J, Spira T, et al. A Quality Management Systems Approach for CD4 Testing in Resource-Poor Settings. Am Soc Clin Pathol. 2010;134(10):556–67.
INFO A. Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents. 2016.
Baggaley R, Dalal S, Johnson C, Macdonald V, Mameletzis I, Rodolph M, et al. Beyond the 90-90-90 : refocusing HIV prevention as part of the global HIV response. J Int AIDS Soc. 2016;19(1):21348.
Slingluff CL, Petroni GR, Chianese-Bullock KA, Smolkin ME, Hibbitts S, Murphy C, Johansen N, Grosh WW, Yamshchikov GV, Neese PY, Patterson JW, Fink R RP. Immunologic and clinical outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma in the adjuvant setting. Clin Cancer Res. 2007;13:6386–95.
2010 WHO. ANTIRETROVIRAL THERAPY FOR HIV INFECTION IN ADULTS AND ADOLESCENTS Recommendations for a public health approach 2010 revision. 2010.
Sagnia B, Ngogang J, Iii FFA, Colizzi V, Montagnier L, Bélec L. Validation of a single-platform , volumetric , flow cytometry for CD4 T cell count monitoring in therapeutic mobile unit. J Transl Med. 2012;10(22):1–9.
Olson WC, Smolkin ME, Farris EM, Fink RJ, Czarkowski AR, Fink JH, et al. Shipping blood to a central laboratory in multicenter clinical trials : effect of ambient temperature on specimen temperature , and effects of temperature on mononuclear cell yield , viability and immunologic function. J Transl Med. 2011;9(26):1–13.
Olteanu H, Schur BC, Harrington AM, Kroft SH. Time and temperature stability of T-cell subsets evaluated by a dual-platform method. Am J Res. 2012;2(2):128–35.
Jt T, Pk R, Da V, Seckinger D. The effects of anticoagulant and temperature on the measurements of helper and suppressor cells . Diagn Immunol. 1984;2(3):167–74.
Ekong T, Am H, Gompels M, Brown A, Aj P. The effect of the temperature and duration of sample storage on the measurement of lymphocyte subpopulations from HIV-1-positive and control subjects . J Immunol Methods. 1992;151(6):217–25.
Fajardo E, Metcalf C, Mbofana E, Vyve C Van, Munyaradzi D, Simons S, et al. Opportunities to improve storage and transportation of blood specimens for CD4 testing in a rural district in Zimbabwe using BD vacutainer CD4 stabilization tubes : a stability and diagnostic accuracy study. BMC Infect Dis. 2014;14(553):1471–2334.
Ashmore LM, Shopp GM EB. Lymphocyte subset analysis by flow cytometry. Comparison of three different staining techniques and effects of blood storage. J Immunol Methods. 1989;118(89):209–15.
Bourguignon P, Clément F, Renaud F, Le V, Koutsoukos M, Burny W, et al. Processing of blood samples in fl uences PBMC viability and outcome of cell-mediated immune responses in antiretroviral therapy-naïve HIV-1-infected patients. J Immunol Methods [Internet]. Elsevier B.V.; 2014;414:1–10. Available from: http://dx.doi.org/10.1016/j.jim.2014.09.001
Seely P, Yang X, Diallo TO, Plews M. Compatibility of Stabilized Whole Blood Products with CD4 Technologies and Their Suitability for Quality Assessment Programs. PLoS One. 2014;9(8).
Garraud O MT. Effect of blood storage on lymphocyte subpopulations. Journal of Immunological Methods. 1984; J Immunol Methods 1984; 1984;75(84):95–8.
Weinberg A, Song LY, Wilkening CL, Fenton et al. O. Optimization of storage and shipment of cryopreserved peripheral blood mononuclear cells from HIV-infected and uninfected individuals for ELISPOT assays. J Immunol Methods. 2010;363(1016):42–50.