Flow Cytometer Performance Characterization, Standardization and Calibration against CD4 on T Lymphocytes Enables Quantification of Biomarker Expressions for Immunological Applications


There is an urgent need for developing a procedure for biomarker standardization and relative quantificationin clinical laboratories. Measuring the expression levels of cell antigens is critical for the diagnosis of many diseases, e.g. leukemia, lymphoma and immunodeficiency diseases. One of the most significant challenges in flow cytometry is obtaining inter-laboratory and intra-laboratory consistent and reproducible results across multiple cytometer platforms and locations longitudinally over time. To obtain measurement consistency, the target flow cytometer voltages should be optimized to segregate the negative population from the electronic noise, and to keep the brightest positive population within the dynamic range of each detector. Then target values should be determined and transferred to selected cytometers. In this study, we optimized a procedure for instrument standardization across three different flow cytometer platforms from the same vendor and in two different locations. The biomarker quantification was implemented on standardized instruments using CD4 expression on T lymphocytes with a known amount of antibody bound per cell as a quantification standard. Our results on blood cell subset typing and CD19 quantification demonstrated that consistent and reliable results could be accomplished between instruments using the developed procedure. Quantitating the expression levels of certain cell biomarkers relative to a known reference marker before, during, and after therapy would provide important information for monitoring antibody-based therapy and could be potentially used to adjust dosing. Presently, we are implementing this protocol to quantify critical disease biomarkers, and making necessary modifications to the procedure to include instruments from different instrument manufacturers.

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Degheidy, H. , Bauer, S. , Marti, G. and Wang, L. (2014) Flow Cytometer Performance Characterization, Standardization and Calibration against CD4 on T Lymphocytes Enables Quantification of Biomarker Expressions for Immunological Applications. Journal of Biomedical Science and Engineering, 7, 756-768. doi: 10.4236/jbise.2014.79074.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Van de Loosdrecht, A.A., Alhan, C., Bene, M.C., Della Porta, M.G., Drager, A.M., Feuillard, J., et al. (2009) Standardization of Flow Cytometry in Myelodysplastic Syndromes: Report from the First European Leukemia Net Working Conference on Flow Cytometry Inmyelodysplastic Syndromes. Haematologica, 94, 1124-1134.
[2] Bjorklund, E., Matinlauri, I., Tierens, A., Axelsson, S., Forestier, E., Jacobsson, S. et al. (2009) Quality Control of Flow Cytometry Data Analysis for Evaluation of Minimal Residual Disease in Bone Marrow from Acute Leukemia Patients during Treatment. Journal of Pediatric Hematology/Oncology, 31, 406-415.
[3] Dworzak, M.N., Gaipa, G., Ratei, R., Veltroni, M., Schumich, A., Maglia, O., et al. (2008) Standardization of Flow Cytometric Minimal Residual Disease Evaluation in Acute Lymphoblastic Leukemia: Multicentric Assessment Is Feasible. Cytometry Part B: Clinical Cytometry, 74, 331-340.
[4] Irving, J., Jesson, J., Virgo, P., Case, M., Minto, L., Eyre, L., et al. (2009) Establishment and Validation of a Standard Protocol for the Detection of Minimal Residual Disease in B Lineage Childhood Acute Lymphoblastic Leukemia by Flow Cytometry in a Multicenter Setting. Haematologica, 94, 870-874.
[5] Letestu, R., Rawstron, A., Ghia, P., Villamor, N., Leuven, N.B., Boettcher, S., Buhl, A.M., Duerig, J., Ibbotson, R., Kroeber, A., Langerak, A., Le Garff-Tavernier, M. and Mockridge, I. (2006) Evaluation of ZAP-70 Expression by Flow Cytometry in Chronic Lymphocytic Leukemia: AMulticentric International Harmonization Process. Cytometry Part B (Clinical Cytometry), 70B, 309-314.
[6] Kraan, J., Gratama, J.W., Keeney, M., D’Hautcourt, J.L. (2003) Setting up and Calibration of a Flow Cytometer for Multicolor Immunophenotyping. Journal of Biological Regulators Homeostatic Agents, 17, 223-233.
[7] Shankey, T.V., Forman, M., Scibelli, P., Cobb, J., Smith, C.M., Mills, R., et al. (2006) An Optimized Whole Blood Method for Flow Cytometric Measurement of ZAP-70 Protein Expression in Chronic Lymphocytic Leukemia. Cytometry Part B: Clinical Cytometry, 70, 259-269.
[8] Kalina, T., Flores-Montero, J., van der Velden, V.H.J., Martin-Ayuso, M., Bottcher, S., Ritgen, M., Almeida, J., Lhermitte, L., Asnafi, V., Mendonca, A., de Tute, R., Cullen, M., Sedek, L., Vidriales, M.B., Perez, J.J., teMarvelde, J.G., Mejstrikova, E., Hrusak, O., Szczepanski, T., van Dongen, J.J.M. and Orfao, A. (2012) On Behalf of the EuroFlow Consortium. EuroFlow Standardization of Flow Cytometer Instrument Settings and Immunophenotyping Protocols. Leukemia, 26, 1986-2010.
[9] Hultin, L.E., Matud, J.L. and Giorgi, J.V. (1998) Quantitation of CD38 Activation Antigen Expression on CD8+ T cells in HIV-1 Infection Using CD4 Expression on CD4+ T Lymphocytes as a Biological Calibrator. Cytometry, 33, 123-132.
[10] Wang, L., Gaigalas, A.K., Marti, G.E., Abbasi, F. and Hoffman, R.A. (2008) Toward Quantitative Fluorescence Measurements with Multicolor Flow Cytometry. Cytometry Part A, 73A, 279-288.
[11] Wang, L., Gaigalas, A.K. and Yan, M. (2011) Quantitative Fluorescence Measurements with Multicolor Flow Cytometry. Flow Cytometry Protocols, 3rd Edition, Humana Press/Springer, New York, 53-65.
[12] Poncelet, P., Poinas, G., Corbeau, P., Devaux, C., Tubiana, N., Muloko, N., Tamalet, C., Chermann, J.C., Kourilsky, F., and Sampol, J. (1991) Surface CD4 Density Remains Constant on Lymphocytes of HIV-Infected Patients in the Progression of Disease. Research in Immunology, 142, 291-298.
[13] Davis, K.A., Abrams, B., Iyer, S.B., Hoffman, R.A. and Bishop, J.E. (1998) Determination of CD4 Antigen Density on Cells: Role of Antibody Valency, Avidity, Clones, and Conjugation. Cytometry, 33, 197-205.
[14] Wang L., Abbasi F., Ornatsky O., Cole K.D., Misakian M., Gaigalas A.K., He, H.-J., Marti, G.E., Tanner, S. and Stebbings, R. (2012) Human CD4+ Lymphocytes for Antigen Quantification: Characterization Using Conventional Flow Cytometry and Mass Cytometry. Cytometry Part A, 81A, 567-575.
[15] Technical Bulletin of BD Biosciences: BD_FACSDiva_Stndrd_App_Setup_TechBulletin.pdf
[16] Wang, L., Abbasi, F., Gaigalas, A.K., Vogt, R.F. and Marti, G.E. (2006) Comparison of Fluorescein and Phycoerythrin Conjugates for Quantifying CD20 Expression on Normal and Leukemic B-Cells. Cytometry Part B: Clinical Cytometry, 70B, 410-415.

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