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Clinical and Vaccine Immunology, August 2009, p. 1176-1186, Vol. 16, No. 8
1071-412X/09/$08.00+0     doi:10.1128/CVI.00342-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization{triangledown}

Adriana Weinberg,1* Lin-Ye Song,2 Cynthia Wilkening,2 Anne Sevin,2,¶ Bruce Blais,3 Raul Louzao,4 Dana Stein,4 Patricia Defechereux,5 Deborah Durand,6 Eric Riedel,7 Nancy Raftery,7 Renee Jesser,1 Betty Brown,8 M. Fran Keller,6 Ruth Dickover,9 Elizabeth McFarland,1 Terence Fenton,2 for the Pediatric ACTG Cryopreservation Working Group{dagger}

University of Colorado Denver School of Medicine, Denver, Colorado,1 Harvard School of Public Health Center for Biostatistics in AIDS Research, Boston, Massachusetts,2 University of Massachusetts Medical Center, Worcester, Massachusetts,3 New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey,4 University of California—San Francisco, San Francisco, California,5 University of California—San Diego, San Diego, California,6 The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania,7 Texas Children's Hospital, Houston, Texas,8 University of California—Los Angeles School of Medicine, Los Angeles, California9

Received 18 September 2008/ Returned for modification 24 November 2008/ Accepted 1 June 2009

The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4+ and CD8+ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at –70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at –70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

* Corresponding author. Mailing address: 4200 E. 9th Ave., Campus Box C227, Denver, CO 80262. Phone: (303) 315-4624. Fax: (303) 315-1787. E-mail: Adriana.Weinberg{at}uchsc.edu

{triangledown} Published ahead of print on 10 June 2009.

Deceased.

{dagger} Contributing members of the PACTG Cryopreservation Working Group are Bruce Blais, Robin Brody, Betty Brown, Patricia Defechereux, Ruth Dickover, Deborah Durand, Terence Fenton, Ambrosia Garcia-Louzao, Richard Hudson, Renee Jesser, M. Fran Keller, Linda Lambrecht, Erik Larson, Daniella Livnat, Raul Louzao, Elizabeth McFarland, Rebecca Peters, Tina Powell, Nancy Raftery, Eric Riedel, Howard Rosenblatt, Anne Sevin, Diane Smith, Lin-Ye Song, Dana Stein, Rodney Trout, Cynthia Wilkening, and Adriana Weinberg.

Clinical and Vaccine Immunology, August 2009, p. 1176-1186, Vol. 16, No. 8
1071-412X/09/$08.00+0     doi:10.1128/CVI.00342-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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