Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials

Abstract

Interferon-gamma (IFN-gamma) ELISpot and intracellular cytokine staining (ICS) assays are routinely employed in clinical HIV vaccine trials to identify antigen-specific T cells in cryopreserved peripheral blood mononuclear cells (PBMC). Several parameters involved in blood collection, processing and shipping may influence immunological function of the resulting cells, including anticoagulant type, time from venipuncture to PBMC isolation/cryopreservation, method of PBMC isolation and procedure for sample shipping. We examined these parameters in single and multiple site studies, and found the length of time from venipuncture to cryopreservation is the most important parameter affecting performance of T cells in immunological assays. Comparing blood processed at 24 h after venipuncture with that processed within 8 h, we observed on average a modest reduction in PBMC viability ( approximately 8% decrease), a greater loss in cell recovery ( approximately 32%), and between 36-56% loss in IFN-gamma T cell frequencies by ELISpot assay. We also describe three cold shipping methods that maintain immunological function in appropriately cryopreserved PBMC. These data indicate that cryopreservation of PBMC should occur within 8 h of venipuncture for optimal performance. This narrow window for specimen processing has important implications in selecting and monitoring clinical sites with laboratory capacity to perform these procedures in future clinical trials.

Figure 1

Overview of the four study designs examining the effect of different anticoagulants, PBMC processing methods, processing times and shipping methods on cell recovery, viability, and function in immunological assays.

Figure 2

Comparison of different cryopreservation media on percent viability of PBMC. Blood was anticoagulated with either ACD, EDTA, or Heparin. PBMC were isolated within 8 or 24 hours using Ficoll density centrifugation, and then cryopreserved in either 10% DMSO/FBS (left panel) or 10%DMSO/12.5% HSA in RPMI (right panel). Percent viability of PBMC in different cryopreservation media was calculated using the Guava counter immediately after fresh isolation, after thawing cryopreserved PBMC, and after overnight incubation at 37°C of thawed PBMC. Filled shapes represent PBMC isolated within 8 hours, open shapes indicate PBMC isolated within 24 hours. Symbols denote type of blood anticoagulant: ● ACD; ▲ EDTA; ■Heparin. Error bars represent the standard error of the mean for the percent viability.

Figure 3

CMV-specific T cell responses detected by IFN-γ ELISpot in 11 representative donors, stratified by high (A) and low to intermediate (B) IFN-γ SFC frequencies. Blood was taken with one of three anticoagulants (ACD, EDTA, Heparin), and PBMC were isolated by Ficoll centrifugation or with Accuspin tubes. Cryopreservation of PBMC occurred within 8 or 24 hours of venipuncture, as indicated. Data shown are background subtracted. *Sample not available.

Figure 3

CMV-specific T cell responses detected by IFN-γ ELISpot in 11 representative donors, stratified by high (A) and low to intermediate (B) IFN-γ SFC frequencies. Blood was taken with one of three anticoagulants (ACD, EDTA, Heparin), and PBMC were isolated by Ficoll centrifugation or with Accuspin tubes. Cryopreservation of PBMC occurred within 8 or 24 hours of venipuncture, as indicated. Data shown are background subtracted. *Sample not available.

Figure 4

Functional activity of PBMC isolated at different time points, in different anticoagulants and by different isolation methods as measured by intracellular cytokine staining (ICS). A) Responses from individuals with strong, and B) weak and intermediate responses to CMV peptide are graphed according to isolation method. Data are gated on CD3+ CD8+ lymphocytes.

Figure 4

Functional activity of PBMC isolated at different time points, in different anticoagulants and by different isolation methods as measured by intracellular cytokine staining (ICS). A) Responses from individuals with strong, and B) weak and intermediate responses to CMV peptide are graphed according to isolation method. Data are gated on CD3+ CD8+ lymphocytes.

Figure 5

Comparison of % PBMC viability from donors at four sites processed either with Accuspin tubes (left panel) or Ficoll centrifugation (right panel). Box plots represent quartiles, dark bar represent the median, and whiskers represent 95% and 5% values for the group. Responses are shown with use of different anticoagulants (EDTA or heparin) and within 8 and 24 hours of cryopreservation.

Figure 6

Representative dot plots of ICS data from cryopreserved PBMC shipped using 3 different methods: shipped on liquid nitrogen after 24 hr at −70°C (LN), shipped on dry ice after 24 hr at −70°C (DI) or shipped on dry ice after 3 weeks at −70°C (−70 3 WK). Top row shows data from un-stimulated cultures. Bottom row shows data from PBMC stimulated with CEF peptide for 8 hr. Data are gated on CD3+ CD8+ lymphocytes. Examination of CD3+CD8- population as a surrogate for CD4+ cells demonstrated no difference in IFN-γ production between the three freezing methods (data not shown).