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Chapter 27 : Cryopreservation of Peripheral Blood Mononuclear Cells

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Cryopreservation of Peripheral Blood Mononuclear Cells, Page 1 of 2

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Abstract:

The use of cryopreserved peripheral blood mononuclear cells (PBMC) for the studies of immune reconstitution in human immunodeficiency virus (HIV)-infected patients permits the selection of samples from well-characterized study subjects. The goal of the cryopreservation is to freeze and thaw the PBMC without compromising cell viability. Both glycerol and dimethyl sulfoxide (DMSO) can provide this function, but only DMSO has been extensively studied for cryopreservation of PBMC. Other critical steps of the cryopreservation procedure include inhibition of cell metabolism while in the presence of DMSO, rate-controlled freezing to avoid cell dehydration, rapid thawing, and resuspension in serum-containing medium, which protects the cells against osmotic trauma. Lymphoproliferative responses measure mainly CD4-dependent immune responses. Severely impaired CD4 responses confer broad immunodeficiency. Natural killer (NK) cell activity, in contrast to cytotoxic T lymphocytes (CTL), is more liable to freezing and thawing. Enumeration of PBMC subsets by flow cytometry provides important information on the immune capacity of the host. As such, flow cytometry-based immune phenotyping is a standard procedure in the evaluation of immunodeficiency disorders. The distribution of major surface markers, such as CD3, CD4, CD8, CD19, and CD14, is essentially unchanged by cryopreservation. Definition of the T-cell receptor repertoire has become increasingly prevalent in studies of the maturation and senescence of the immune system. The two techniques seem to yield similar results when performed with fresh and cryopreserved PBMC.

Citation: Weinberg A. 2006. Cryopreservation of Peripheral Blood Mononuclear Cells, p 241-244. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch27

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Figures

Image of FIGURE 1
FIGURE 1

CMV-specific responder cell frequency (RCF) in cryopreserved versus fresh PBMC from HIV-infected patients (gray triangles) and uninfected controls (solid squares). The diagonal represents the slope of equivalence between fresh- and frozen-cell assays.

Citation: Weinberg A. 2006. Cryopreservation of Peripheral Blood Mononuclear Cells, p 241-244. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch27
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Image of FIGURE 2
FIGURE 2

Effect of viability on cryopreserved PBMC LPA results. Data were derived from samples from HIV-infected patients. The column on the left shows that when all samples were analyzed, the LPA results significantly increased with greater viability. A breakpoint in the distribution pattern can be observed at a viability of 70%. The column on the right shows that when only samples with a viability of ≥70% were analyzed, the LPA results were independent of the PBMC viability. SI, stimulation index.

Citation: Weinberg A. 2006. Cryopreservation of Peripheral Blood Mononuclear Cells, p 241-244. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch27
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Image of FIGURE 3
FIGURE 3

CMV-specific IFN-γ in cryopreserved versus fresh PBMC from HIV-infected patients (gray triangles) and uninfected controls (solid squares). The diagonal represents the slope of equivalence between the fresh- and frozen-cell assays.

Citation: Weinberg A. 2006. Cryopreservation of Peripheral Blood Mononuclear Cells, p 241-244. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch27
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Image of FIGURE 4
FIGURE 4

Effect of cryopreservation on flow cytometric immune phenotyping. Data were derived from HIV-infected subjects. Cell membrane markers CD95 (A) and CD62L (B) were used. The diagonal represents the slope of equivalence between the fresh- and frozen-cell assays, which give identical results.

Citation: Weinberg A. 2006. Cryopreservation of Peripheral Blood Mononuclear Cells, p 241-244. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch27
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Download as Powerpoint

References

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1. Autran, B.,, G. Carcelain,, T. S. Li,, C. Blanc,, D. Mathez,, R. Tubiana,, C. Katlama,, P. Debre, and , J. Leibowitch. 1997. Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science 277:112116.
2. Hviid, L.,, G. Albeck,, B. Hansen,, T. G. Theander, and , A. Talbot. 1993. A new portable device for automatic controlled-gradient cryopreservation of blood mononuclear cells. J. Immunol. Methods 157:135142.
3. Kleeberger, C. A.,, R. H. Lyles,, J. B. Margolick,, C. R. Rinaldo,, J. P. Phair, and , J. V. Giorgi. 1999. Viability and recovery of peripheral blood mononuclear cells cryopreserved for up to 12 years in multicenter study. Clin. Diagn. Lab. Immunol. 6:1419.
4. Reimann, K. A.,, M. Chernoff,, C. Wilkening,, C. Nickerson,, A. Landay, and the ACTG Immunology Advanced Technology Laboratories. 2000. Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from HIV-1-infected donors: implications for multicenter clinical trials. Clin. Diagn. Lab. Immunol. 7:352359.
5. Sleasman, J. W.,, B. H. Leon,, L. F. Aleixo,, M. Rojas, and , M. M. Goodenow. 1997. Immunomagnetic selection of purified monocyte and lymphocyte populations from peripheral blood mononuclear cells following cryopreservation. Clin. Diagn. Lab. Immunol. 4:653658.
6. Ventakaraman, M. 1992. Cryopreservation-induced enhancement of interleukin-2 production in human peripheral blood mononuclear cells. Cryobiology 29:165174.
7. Weinberg, A.,, L. Zhang,, D. Brown,, A. Erice,, B. Polsky,, M. S. Hirsch,, S. Owens, and K. Lamb for ACTG Team 360. 2000. Viability and functional activity of cryopreserved mononuclear cells. Clin. Diagn. Lab. Immunol. 7:714716.
8. Weinberg, A.,, R. Betensky,, L. Zhang, and , G. Ray. 1998. Effect of shipment, storage, anticoagulant and cell separation on lymphoproliferative responses in HIV-infected patients. Clin. Diagn. Lab. Immunol. 5:804807.

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