CLINICAL AND VACCINE IMMUNOLOGY, June 2010, p. 910–918 Vol. 17, No. 61556-6811/10/$12.00 doi:10.1128/CVI.00492-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Quality Monitoring of HIV-1-Infected and Uninfected PeripheralBlood Mononuclear Cell Samples in a Resource-Limited Setting�

Robert E. Olemukan,1 Leigh Anne Eller,1,2 Benson J. Ouma,1 Ben Etonu,1 Simon Erima,1Prossy Naluyima,1 Denis Kyabaggu,1 Josephine H. Cox,3 Johan K. Sandberg,4

Fred Wabwire-Mangen,1 Nelson L. Michael,2 Merlin L. Robb,2Mark S. de Souza,2,5 and Michael A. Eller1,2,4*

Makerere University Walter Reed Project, Kampala, Uganda1; U.S. Military HIV Research Program, Rockville, Maryland2;International AIDS Vaccine Initiative, New York, New York3; Center for Infectious Medicine, Department of Medicine,

Karolinska Institutet, Karolinska University Hospital Huddinge, 14186 Stockholm, Sweden4; andArmed Forces Research Institute of Medical Sciences, Bangkok, Thailand5

Received 3 December 2009/Returned for modification 11 January 2010/Accepted 24 February 2010

Human immunodeficiency virus type 1 (HIV-1) vaccine and natural history studies are critically dependenton the ability to isolate, cryopreserve, and thaw peripheral blood mononuclear cell (PBMC) samples with ahigh level of quality and reproducibility. Here we characterize the yield, viability, phenotype, and function ofPBMC from HIV-1-infected and uninfected Ugandans and describe measures to ascertain reproducibility andsample quality at the sites that perform cryopreservation. We have developed a comprehensive internal qualitycontrol program to monitor processing, including components of method validation. Quality indicators forreal-time performance assessment included the time from venipuncture to cryopreservation, time for PBMCprocessing, yield of PBMC from whole blood, and viability of the PBMC before cryopreservation. Immunephenotype analysis indicated lowered B-cell frequencies following processing and cryopreservation for bothHIV-1-infected and uninfected subjects (P < 0.007), but all other major lymphocyte subsets were unchanged.Long-term cryopreservation did not impact function, as unstimulated specimens exhibited low background andall specimens responded to staphylococcal enterotoxin B (SEB) by gamma interferon and interleukin-2production, as measured by intracellular cytokine staining. Samples stored for more than 3 years did not decaywith regard to yield or viability, regardless of HIV-1 infection status. These results demonstrate that it ispossible to achieve the high level of quality necessary for vaccine trials and natural history studies in aresource-limited setting and provide strategies for laboratories to monitor PBMC processing performance.

Cryopreserved peripheral blood mononuclear cells (PBMC)are critical to studies of HIV-1 infection and for the devel-opment of an effective HIV vaccine. High-quality cryo-preservation is particularly challenging and important inresource-limited settings, where natural history, therapeu-tic, and preventative protocols are being developed. Cryo-preservation and batch testing allow for simultaneous as-sessment of critical samples, thereby reducing interassayvariability. Since standard method validation measures, suchas accuracy, precision, linearity, and reportable ranges, arenot easily obtained for PBMC processing, more studies areneeded to understand the parameters influencing the repro-ducibility of processing and quality of cryopreserved PBMCsamples in resource-limited settings.

Separation of PBMC should yield a pure population ofmononuclear cells (95% � 5%) (18), with minimal contami-nation from red blood cells, granulocytes, and platelets. An-ticipated yields of mononuclear cells from healthy donors areapproximately 1 � 106 to 2 � 106 cells/ml of whole blood, witha purity of 60 to 70% lymphocytes at �95% viability, with areduction of platelets to �0.5% of the original whole-bloodcontent (25). However, the data on the impact of cryopreser-

vation on lymphocyte phenotypes are inconsistent (4, 31, 34,39, 41). Additionally, there are differences in hematologic pa-rameters and lymphocyte subsets in African populations com-pared to populations in the industrialized world (17, 26, 33,44). Careful examination of the impact of cryopreservation onyield and phenotype is therefore needed for this region. Fur-thermore, studies indicate an increased ability to preserve thefunction of cryopreserved PBMC compared to cells in wholeblood, as measured by proliferation assays (1, 12, 23, 46, 47),cytokine production (9, 28, 30, 32, 37), apoptosis (40), andHLA tetramer staining (2). This is particularly important forHIV-1, as many vaccine candidates are designed to elicit T-cellresponses, and reliable PBMC-based assays such as enzyme-linked immunospot (ELISPOT) assay and multiparameterflow cytometry are required to prioritize candidates for large-scale efficacy testing.

Some studies indicate that the functional integrity of cryo-preserved PBMC samples is maintained (22), while other re-ports suggest a loss of function (38). Preanalytic variableswhich have been examined closely include time from blooddraw to initiation of processing, type of anticoagulant, propermixing of blood tubes, and transport temperature (9, 13, 28,45). Analytic variables that impact PBMC processing includeseparation method, type of cryoprotectant medium, prechillingof the freeze containers, cryopreservation parameters, andtemperature of the thawing medium (9, 15, 28, 30, 37). In

* Corresponding author. Mailing address: U.S. Military HIV ResearchProgram, 13 Taft Court, Suite 200, Rockville, MD 20850. Phone: (301)251-8304. Fax: (301) 762-4177. E-mail: meller@hivresearch.org.

� Published ahead of print on 3 March 2010.

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addition to defining processing parameters, it is important tomonitor quality assurance for cryopreserved PBMC assays af-ter cryopreservation (7). However, few studies have addressedthe importance of continuous monitoring of the processing andcryopreservation of PBMC themselves but have focused in-stead on the procedures for thawing of cells in labs that per-form end-point assays. We have developed a comprehensiveinternal quality control program to monitor PBMC processing.This study describes this program and the results achieved in aresource-limited setting. Additionally, we describe proceduresto evaluate our laboratory methods for PBMC processing.

MATERIALS AND METHODS

Study participants. All samples were obtained from participants enrolled instudies conducted in the Kampala (27), Kayunga (19), and Rakai (29) districts inUganda as previously described. All studies were reviewed and approved by theappropriate institutional review boards in the United States and Uganda, withconsent forms signed by all participants.

PBMC processing. Blood was collected into 8.5-ml acid citrose dextran (ACD)-anticoagulated tubes and transported to the lab at room temperature. PBMC wereisolated from ACD-whole blood within 8 h of collection by centrifugation at 800 �g for 15 min through Ficoll-Hypaque Plus (Pharmacia, Uppsala, Sweden), usingLeucosep tubes (Greiner Bio-One, Frickenhausen, Germany). The PBMC layer washarvested and washed three times with phosphate-buffered saline (PBS) by centrif-ugation at 250 � g for 10 min. Cell counts and viability were determined with ahemacytometer, using trypan blue exclusion, or by a Guava PCA machine (GuavaTechnologies, Hayward, CA), using Guava ViaCount reagent. PBMC were cryopre-served at a concentration of 107 cells/ml in freeze medium (20% fetal bovine serum[FBS], 10% dimethyl sulfoxide [DMSO], 1% penicillin-streptomycin, 69% RPMI1640), using isopropyl “Mr. Frosty” freezing containers (Nalgene Labware, ThermoFisher, Rochester, NY) to control the rate of freezing overnight in a �80°C freezer,and were transferred the next day for long-term storage in liquid nitrogen vapor at�130°C. In order to assess cryopreserved specimens, cells were rapidly thawed in a37°C water bath until a small ice crystal remained and were washed with completemedium (CM) (RPMI 1640 supplemented with 10% FBS, 2% L-glutamine, and 1%penicillin-streptomycin) added dropwise to avoid a rapid osmotic change.

Lymphocyte immunophenotyping and hematology. Lymphocyte immunophe-notyping was performed on EDTA-anticoagulated whole blood or PBMC prod-uct postthawing. Samples were run on a dual-laser flow cytometer using thesingle-platform Multitest four-color reagent in combination with TruCount tubesand were analyzed using MultiSET software (Becton Dickinson, San Jose, CA).Absolute numbers and percentages of T (CD3�), helper T (CD4�), cytotoxic T(CD8�), B (CD19�), and NK (CD16� or CD56�) lymphocytes were deter-mined. Complete blood counts with a five-part differential were performed usinga Coulter AcT 5 differential instrument (Beckman Coulter, Fullerton, CA) forwhole blood and after processing of cell samples.

ICS assay. A standard intracellular cytokine staining (ICS) assay was per-formed on cryopreserved PBMC which had been thawed and rested overnight.PBMC (106) were incubated in 96-well round-bottom plates with costimulatoryanti-CD28 and anti-CD49d monoclonal antibodies (Becton Dickinson, San Jose,CA), a peptide pool from cytomegalovirus (CMV), Epstein-Barr virus, andinfluenza virus (CEF pool) (14), and brefeldin A (Becton Dickinson, San Jose,CA). Samples included a negative control of PBMC stimulated without peptideand a positive control of staphylococcal enterotoxin B (SEB; Sigma-Aldrich,Munich, Germany). Cells were incubated for 6 h at 37°C in a 7% CO2 incubatorand were stored at 4°C overnight. The next day, 20 �l of 20 mM EDTA wasadded to each well and incubated for 15 min. After centrifugation, 1� fluores-cence-activated cell sorter (FACS) lysing solution was added for 10 min at roomtemperature. Cells were then washed once in wash buffer (0.5% bovine serumalbumin [BSA], 0.1% sodium azide in PBS), permeabilized in 200 �l 1� FACSpermeabilizing solution for 10 min, and washed three times. Cells were incubatedfor 60 min with a 50-�l antibody cocktail containing anti-CD3–allophycocyanin(anti-CD3–APC), anti-CD4–fluorescein isothiocyanate (anti-CD4–FITC), anti-CD8–PerCpcy5.5, anti-gamma interferon (IFN-�)–phycoerythrin (PE), and anti-interleukin-2 (IL-2)–PE (Becton Dickinson, San Jose, CA). Cells were thenwashed three times and fixed in 1% paraformaldehyde. Flow cytometric analysiswas performed using a FACSCalibur flow cytometer, with at least 20,000 CD3�

CD8� and CD3� CD4� events (each) being acquired. Samples were analyzedusing FlowJo software, version 8.5 (Tree Star, Ashland, OR). A positive ICS

response was defined as a response at least 3-fold higher than the mean unstimu-lated response and above 0.05.

HIV-1 subtype determination. All HIV-1-seropositive participants’ plasmasamples were tested for virus subtype by the previously described multiregionhybridization assay (MHA), version 2, for HIV-1 subtypes A, C, and D, recom-binants, and dual infections (3, 20, 21). HIV-1 subtype A, C, or D was assignedbased on five regions (gag, pol, vpu, env, and gp41) of the HIV-1 genome, basedon reactivities of the probes if at least 2 regions had probe reactivity. Sampleswith probe reactivity for a single subtype in different regions were consideredpure subtypes.

Statistical analysis. All statistical analyses were performed using Graph PadPrism software, version 5.0a, for Mac OS X (GraphPad Software, La Jolla, CA).Direct comparison between two groups was performed using the nonparametricMann-Whitney U test for continuous data. For paired observations, the paired ttest was used. P values of �0.05 were considered significant. For descriptivestatistics, the mean with standard deviation is presented graphically, while themedian, mean, and minimum-to-maximum range are reported within the text.Reference range intervals were calculated as the range between the 2.5% and97.5% limits for the parameters tested. Thaw viability was reported as thepercentage of viable cells at the time of thaw, using the ViaCount assay andstandard gating excluding apoptotic cells. Overnight viability was reported as thepercentage of viable cells after overnight incubation in CM at 37°C, 7% CO2, and90% relative humidity, using the ViaCount assay and standard gating excludingapoptotic cells. Thaw and overnight yields were calculated as numbers of cellsdivided by the original vial cell content under similar conditions to those forviability.

RESULTS

Leucosep processing and cryopreservation of PBMC pre-serve lymphocytes. In order to assess the technique of labora-tory staff and the efficiency of the PBMC processing procedureusing Leucosep tubes, 52 samples were assessed for whole-blood and postprocessing concentrations of granulocytes, lym-phocytes, monocytes, and platelets. Basophils, eosinophils, andneutrophils were considered together as granulocytes. Thecomposition of white blood cells in whole blood was 50.3%(range, 28.4% to 78.9%) granulocytes, 40.4% (17.0% to58.7%) lymphocytes, and 9.3% (2.0% to 42.5%) monocytes(Fig. 1A). After Ficoll separation using Leucosep tubes, themean percentages were 8.8% (1.2% to 45.8%) granulocytes,69.9% (29.4% to 92.6%) lymphocytes, and 21.4% (4.9% to56.3%) monocytes (Fig. 1B). Initial whole-blood product (27ml whole blood) contained 103 � 106 (35 � 106 to 223 � 106)total platelets per ml, which decreased to 12 � 106 platelets/ml(0 to 100 � 106) following processing (Fig. 1C).

In order to assess the effect of processing and cryopreserva-tion on PBMC phenotype, we studied PBMC from freshlydrawn peripheral blood and from thawed samples that hadbeen stored in the vapor phase of liquid nitrogen for approx-imately 1.5 years from 40 uninfected and 28 chronically HIV-1-infected donors. Whole-blood immune cell subsets in unin-fected subjects were 70% (47% to 81%) (median [range])CD3�, 22% (13% to 47%) CD8�, 42% (29% to 57%) CD4�,12% (5% to 33%) CD16� or CD56�, and 16% (9% to 26%)CD19�. Immediately following thawing and counting, PBMCimmune subsets were compared to those of whole blood, anda statistically significant difference was observed only for thepercentage of B cells (P � 0.001) (Fig. 1D). Whole-bloodimmune cell subsets in HIV-1-infected individuals were 71%(47% to 83%) (median [range]) CD3�, 46% (27% to 71%)CD8�, 20% (2% to 43%) CD4�, 14% (4% to 35%) CD16� orCD56�, and 14% (6% to 24%) CD19�. Comparing cell sub-sets between thawed PBMC samples and the paired fresh

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PBMC samples, a statistically significant decrease was againobserved only for the B-cell subset (P 0.007) (Fig. 1E).

Harvesting plasma before or after processing does not affectquality. In order to assess the effects of harvesting plasma priorto and following Ficoll separation, 10 uninfected donor spec-imens (94 ml whole blood [each]) were processed in parallel(47 ml for each sample) to determine the final PBMC yield,viability, and purity. PBMC yield and viability were not differ-ent between samples when plasma was collected before orafter Ficoll separation (P 0.506 and 0.085, respectively [datanot shown]). The median yields (ranges) for plasma collectionbefore and after Ficoll separation were 54 � 106 PBMC (24 �106 to 67 � 106 PBMC) and 53 � 106 PBMC (27 � 106 to 71 �106 PBMC), respectively. The median viability (range) was95% (90% to 100%) for plasma collection before Ficoll sepa-ration and 94% (91% to 98%) for plasma collection after Ficollseparation. No difference was observed in platelet concentra-tion for harvest of plasma before and after Ficoll separation(67 � 106 [22 � 106 to 128 � 106]/ml and 69 � 106 [17 � 106

to 133 � 106]/ml, respectively [P 0.489]). Similarly, thecontaminating granulocyte concentration was 0.6 � 106 (0.4 �106 to 2.6 � 106)/ml before Ficoll separation and 0.7 � 106

(0.5 � 106 to 1.1 � 106)/ml after Ficoll separation, with nostatistical difference (P 0.706).

Establishing quality indicators for PBMC processing inUganda. In order to monitor the quality and performance ofthe processing laboratory in Uganda during the conduct of aphase I/II HIV-1 vaccine trial, the following four processingquality indicators were acquired daily and collated monthlyduring the study: (i) total time from blood draw to cryopreser-vation, (ii) time for actual PBMC processing, (iii) PBMC yieldper ml of whole blood collected before cryopreservation, and(iv) PBMC viability before cryopreservation. A total of 2,310samples from both HIV-1-infected (n 115) and uninfected(n 2,195) subjects were received in the laboratory for PBMCprocessing. Samples were processed and cryopreserved fromMarch 2006 through August 2007. The median (range) totaltime from blood draw to cryopreservation was 3.7 h (3.0 to7.2 h), while the median (range) process time from bloodreceipt in the lab to cryopreservation was 2.7 h (2.2 to 6.0 h).The maximum number of samples processed in a month was313. The cell yield per ml of whole blood collected beforecryopreservation was 1.3 � 106 (0.1 � 106 to 4.1 � 106)PBMC/ml whole blood (Fig. 2A). Viability before cryopreser-

FIG. 1. PBMC were isolated from whole blood by use of Leucosep tubes, and the quality of the procedure was assessed. (A) Pie chart showingmean (range) proportions of granulocytes (50.3% [28.4% to 78.9%]), lymphocytes (40.4% [17.0% to 58.7%]), and monocytes (9.3% [2.0% to42.5%]) from 52 whole-blood samples before processing. (B) Pie chart showing mean (range) proportions of granulocytes (8.8% [1.2% to 45.8%]),lymphocytes (69.9% [29.4% to 92.6%]), and monocytes (21.4% [4.9% to 56.3%]) from 52 samples after PBMC processing. (C) Bar chart showingthe mean concentration of platelets (106) in whole blood (1,034 [35 to 223]) or after processing (12 [0 to 100]). The effect of processing andcryopreservation on the immune phenotype was assessed and compared to peripheral blood concentrations. (D) Box and whisker plots showingthe median and 10th and 90th percentiles for major immune cell subsets in 40 normal healthy individuals. A statistically significant difference wasobserved for the percentage of B cells (P � 0.001). (E) Box and whisker plots showing the median and 10th and 90th percentiles for major immunecell subsets in 28 HIV-1-positive individuals. A statistically significant decrease was observed for the percentage of B cells (P 0.007).

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vation was assessed, and the overall median (range) was 97%(75% to 100%) (Fig. 2B).

Assessment of PBMC processing shows preservation of vi-ability and function. Assessments of PBMC processing, cryo-preservation, and storage in liquid nitrogen were made overthe course of 1 year by assessing longevity and interval preci-sion in selected samples. Samples from three uninfected indi-viduals were assessed for longevity following processing andcryopreservation by thawing of samples at months 1, 7, and 12.Thawed samples were counted using the Guava ViaCount as-say, and cell concentration and viability were determined. Sam-ples were rested overnight, and then cell counts and viabilitydeterminations were repeated. Cells were assessed by ICS tomeasure the amounts of IFN-� and IL-2 within the CD4 and

CD8 T-cell compartments in response to the superantigenSEB. Mean viability after the overnight rest was 93%, 92%,and 98% at months 1, 7, and 12, respectively, while meanrecovery was 85%, 79%, and 80%, respectively (Fig. 3A). CD4T-cell responses to SEB were 9.1%, 10.4%, and 10.0%, whileCD8 T-cell responses were 10.7%, 9.6%, and 9.1% at months1, 7, and 12 postcryopreservation, respectively (Fig. 3A). Pre-cision was assessed by examining PBMC from three individu-als, distributed into at least 3 vials each, at 10 � 106 cells peraliquot. All samples showed coefficients of variation of �2%for thaw and overnight viabilities and recovery yields (data notshown).

Monthly assessments of processing and cryopreservationwere performed using 2 to 6 samples that were cryopreservedat the beginning of the month and thawed at the end of themonth. Cell counts and viabilities were determined both at thetime of thaw and after an overnight rest. At the time of thaw,the median (range) yield was 62% (32% to 143%) and viabilitywas 93% (77% to 98%), while after overnight rest, the median(range) yield was 69% (26% to 103%) and viability was 80%(65% to 100%) (Fig. 3B). Stratifying the responses by monthshowed fluctuations in mean recovery, from the low of 49% inSeptember 2006 to the high of 126% in November 2006. Therange of viability was from the low of 85% in May and Sep-tember 2006 to the high of 98% in November 2006 (Fig. 3C).The Guava ViaCount assay was implemented in October 2006in order to reduce the operator variation from hemacytometercounting. No samples were processed for quality monitoringduring the months of February and March 2007.

Monthly samples were also assessed for functional responsesby conducting a 6-hour stimulation in the presence of the CEFpeptide pool or SEB or in the absence of stimulation, and ICSwas performed to assess IFN-� and IL-2 levels in CD4 andCD8 T cells. Cryopreserved specimens exhibited low back-ground with no stimulation and retained functional responsesto SEB. The median (range) combination IFN-�/IL-2 responsein the absence of stimulation was 0.02% (0% to 0.17%) in CD4T cells and 0% (0% to 0.13%) in CD8 T cells. All 35 specimens(100%) had positive responses to SEB. The median (range)IFN-�/IL-2 response to SEB stimulation was 10.10% (2.15% to20.10%) in CD4 T cells and 8.35% (1.03% to 19.40%) in CD8T cells (Fig. 3D). Positive responses to the CEF peptide poolwere seen in 6/35 (17%) and 24/35 (69%) specimens from theCD4 T-cell and CD8 T-cell compartments, respectively. Themedian (range) positive IFN-�/IL-2 response to CEF stimula-tion was 0.11% (0.06% to 0.26%) in CD4 T cells and 0.36%(0.05% to 7.17%) in CD8 T cells.

PBMC maintain viability after long-term storage. The effectof long-term cryopreservation (�3 years) was assessed in sam-ples from uninfected (n 30) and HIV-1-infected (n 29)individuals. Overall, the median (range) percent recovery was88% (26% to 173%) and the viability was 94% (82% to 98%)at the time of thaw. After overnight rest, the median (range)percent recovery was 76% (22% to 140%) and the viability was95% (81% to 98%) (Fig. 4A). For the uninfected subjects afterovernight rest of samples, the median (range) percent recoverywas 79% (22% to 130%) and the viability was 96% (88% to98%) (Fig. 4A). For 10 HIV-1 subtype A-infected subjects, themedian (range) percent PBMC recovery was 68% (35% to87%) and the viability was 97% (92% to 98%) after overnight

FIG. 2. Quality indicators were measured for PBMC processingconducted during a phase I/II clinical trial from March 2006 throughAugust 2007 (n 2,310). PBMC were received in the laboratory,processed using Leucosep tubes, and cryopreserved in liquid nitrogenvapor. (A) Aligned dot plot showing the mean and standard deviationfor PBMC yield (cells [106] per ml of whole blood processed) forsamples processed each month over the conduct of the clinical trial.The mean for all samples processed is shown with a solid line, and thelevel of 2 standard deviations is shown with dotted lines. (B) Aligneddot plot showing the mean and standard deviation for PBMC percentviability after processing of samples each month over the conduct ofthe clinical trial. The mean for all samples processed is shown with asolid line, and the level of 2 standard deviations is shown with a dottedline.

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rest (Fig. 4B). For 19 HIV-1 subtype D-infected subjects, themedian (range) percent recovery was 74% (32% to 140%) andthe viability was 92% (81% to 98%) after overnight rest (Fig.4D). No statistically significant difference in recovery and via-bility was observed between the HIV-1-infected and unin-fected individuals or between the HIV-1 subtype A- and sub-type D-infected study participants.

DISCUSSION

Many of the T-cell-based assays used to assess potentialHIV-1 candidate vaccines have been standardized and evalu-ated to characterize vaccine immunogenicity (7, 22, 43) butmay not define correlates of protection (8, 35). T cells arelinked to control of HIV-1 infection, and specific functionalprofiles are associated with slow HIV-1 disease progression ininfected individuals (6). Common practice for many multi-center vaccine trials is to cryopreserve cells and ship them to a

FIG. 4. The effect of long-term cryopreservation (storage for �3years) was assessed for HIV-infected and uninfected individuals.(A) Scatter plot showing the cumulative mean and standard deviationfor recovery and viability after thaw and overnight (O/N) rest for 30uninfected samples. (B) Scatter plot showing the cumulative mean andstandard deviation for recovery and viability after thaw and overnightrest for 59 HIV-1-infected samples with HIV-1 subtype A (n 10)(red) and HIV-1 subtype D (n 19) (blue).

FIG. 3. The Makerere University Walter Reed Project laboratory follows standard procedures for assessing quality of PBMC collected andcryopreserved during protocol vaccine trials, including internal assessment of longevity, precision, and functionality. (A) PBMC from 3 subjectswere assessed for the impact of cryopreservation on recovery, viability, and functionality by thawing the same samples at 1, 7, and 12 monthspostcryopreservation. The bar and line charts show the means and standard deviations for overnight viability (red), overnight recovery (blue), andcontrol positive responses (IFN-�/IL-2) to SEB for CD4 (white bars) and CD8 (black bars) cells. (B) Thirty-five samples were processed,cryopreserved (for approximately 1 month), thawed, and assessed for recovery and viability after thawing and overnight rest as part of a monthlyinternal quality assurance procedure. The scatter plot shows the cumulative mean and standard deviation for all 35 samples. (C) Bar chart showingthe mean and standard deviation for thaw recovery and viability for the 35 samples monitored, by month (no samples were processed in Februaryand March 2007). (D) Thirty-five samples were thawed and assessed for function after thaw and overnight rest in response to SEB or in the absenceof stimulation. The scatter plot shows the cumulative mean and standard deviation for all 35 samples for CD4 T cells and CD8 T cells.

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central laboratory to reduce interlaboratory variation in assayperformance. Additionally, cryopreserved cells allow for care-fully planned longitudinal analysis and case-control studies.Hematological parameters have previously been shown to dif-fer for populations in Uganda compared to populations inindustrialized countries (17, 26, 33, 44), and this could ad-versely affect protocol end points designed to utilize whiteblood cells for immunologic phenotype or functional analysis.Here we characterized the function, phenotype, viability, andyield of PBMC from HIV-1-infected and uninfected Ugandansand introduced a number of quality measures that can be usedto monitor the performance of collection, processing, cryo-preservation, and storage of PBMC in real time.

Studies from Uganda have used cryopreserved PBMC forcellular immunology assessment; however, minimal data havebeen presented on processing cell yields and postthaw recov-eries (5, 10, 11, 24). In a recent study conducted on PBMCfrom Tanzanian subjects, a yield of 1.1 � 106 cells was har-vested per ml of whole blood, with 96% viability (37), com-pared to the 1.3 � 106 cells per ml of whole blood and 97%viability we observed in Ugandan subjects, using similar pro-cedures and reagents. Our study summarizes results for a muchlarger cohort of 2,195 healthy individuals and proposes 95%reference intervals (Table 1) for processing parameters in aneffort to validate this methodology in accordance with Clinicaland Laboratory Standards Institute (CLSI) guidelines, al-though for granulocytes, monocytes, lymphocytes, and plate-lets, the recommended number of 120 samples was notachieved (36). Additionally, we observed similar yields andviabilities for HIV-1-infected individuals, although some do-nors with advanced disease exhibited a high level of lymphope-nia and poor separation from red blood cells in the Ficollgradient, resulting in extremely low yields and viabilities.

Separated PBMC should consist of a pure population ofmononuclear cells (95%) (18) with yields of approximately 1 �106 to 2 � 106 cells/ml of whole blood, consisting of 60 to 70%lymphocytes at �95% viability, with a reduction of platelets to�0.5% of the original whole-blood content (25). Performancecharacteristics obtained using a porous high-density polyethyl-ene barrier “frit” like a Leucosep tube or equivalent Accuspintube should enable higher accuracy in harvesting mononuclearcells, yielding a final product of 88% lymphocytes, 9% mono-cytes, and 3% granulocytes at a viability of 98% (42). We didobserve slightly higher platelet contamination in the final prod-uct, with a median reduction to 1.16% of the total originalblood content, but in general we showed that processing withLeucosep tubes gives results similar to manufacturer specifi-cations, allowing for accurate preparation and planning forclinical studies requiring PBMC.

In order to assess the effects of processing and cryopreser-vation on immune phenotype, we compared T (CD4� andCD8� T cells), B, and NK lymphocytes in fresh whole blood orafter Ficoll processing, cryopreservation, and thawing. Inter-estingly, the proportions of most subsets were preserved, withthe exception of B cells, consistent with a previous multicenterstudy conducted in the United States in which a reduction inthe percentage of B cells was observed after processing andcryopreservation (39). Other studies have shown increases orstatic levels of B cells (4, 31, 34, 41). Our data support theobservation of reduced B-cell numbers after cryopreservationfor both HIV-1-infected and uninfected individuals. One po-tential explanation is that B cells may have a different densityand could be centrifuged through the Ficoll gradient and beincluded in the red blood cell pellet. Another explanationcould be that B cells are more susceptible to the effects ofcryopreservation and subsequently die. This loss is of greatimportance to B-cell immunologists who plan to study thissubset from cryopreserved PBMC.

Processing of PBMC is straightforward, but many param-eters need to be optimized in order to maximize yield, recov-ery, viability, and function. One important finding is that con-taminating granulocytes increase cell clumping upon thawing(16) and adversely affect PBMC yield, function, and assaybackground responses (13). Another important processing pa-rameter is the time from blood draw to the time the PBMC arecryopreserved. Several studies indicate that a processing timeof 24 h or greater negatively impacts the viability, recovery, andfunction of PBMC and suggest that processing and cryopreser-vation need to occur within 8 h of blood draw (9, 13, 16, 28).We confirmed the feasibility of large-scale processing in aresource-limited setting with transport to the laboratory froman off-site clinic, with a time of blood draw to cryopreservationof under 7.5 h and a median time from receipt in the laboratoryto cryopreservation of 3.7 h. Additional parameters that haveminimal impact on PBMC include shipping, centrifugal forcefor washing, concentration of cells in the vial, anticoagulantused in collection of whole blood, use of a classical overlayprocedure, and the size of the conical wash tube (9, 15, 37, 45),and here we showed that harvesting the plasma before or afterthe Ficoll gradient step also did not affect the overall quality ofPBMC, eliminating one centrifugation step and reducing theprocessing time.

Other studies suggest that it is not the processing and cryo-preservation procedures that adversely affect PBMC, but howthey are thawed and if they are rested overnight (13, 28, 30),and that viability needs to be �70 to 75% for consistent func-tional assessment (15, 46, 47). In our study, function was main-tained in response to superantigen, and background responsesremained low under unstimulated conditions. While unstimu-lated and superantigen-exposed PBMC were consistent infunction, the antigen-specific CEF peptide set was not consis-tent in generating positive responses. This may be due in partto differences in the HLA alleles in African populations andthe epitopes selected for the CEF peptide pool. The lab hassince transitioned to using a commercially available CMV pp65peptide set that generates a higher frequency of consistentresponses in the ICS assay, making for a better positive control.

Preservation of PBMC for HIV vaccine trials is evaluatedprimarily by centralized laboratories, which perform immuno-

TABLE 1. PBMC processing parameter 95% reference intervals

Parameter (n) Median Mean (SD) Range

Lymphocytes (%) (52) 71.7 69.9 (12.9) 32.9–90.1Monocytes (%) (52) 20.2 21.4 (9.7) 6.5–48.4Granulocytes (%) (52) 4.9 8.8 (8.3) 1.5–35.6Platelets (106 cells) (52) 10.0 12.1 (16.6) 0.0–88.2Yield (cells/ml whole blood) (2,195) 1.3 1.3 (0.5) 0.4–2.6Viability (%) (2,195) 97.0 96.7 (2.4) 91.0–100

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genicity assays. Some groups focus on a central laboratory toserve as a repository and evaluation unit (9, 28), while othersdevelop a quality assurance scheme that revolves around sam-ple collection and subsequent evaluation performed centrally(16). While these methods are a necessary component to goodPBMC practice (GPP), real-time assessments of PBMC pro-cessing and cryopreservation are needed. The network of labsin the U.S. Military HIV Research Program all adhere tocentralized standard operating procedures for processing,cryopreservation, and shipping of PBMC and participate intraining across sites within the network. Additionally, a newsite goes through a validation and initialization period, duringwhich a site must prove proficiency in processing and cryo-preservation. Once qualified to conduct PBMC processing, asite is monitored both externally and internally. While ourPBMC quality management plan includes shipping to externalsites for assessment several times a year, here we outline sev-eral indicators that can be monitored by the actual processingunits, including a plan to thaw PBMC and evaluate recoveriesand viabilities at regular intervals. It is critical that these pro-cedures be done in such a manner that they do not impact theend-point analysis for protocols, and they should be developedearly in study design to include blood for processing and cryo-preservation assessment. Table 2 summarizes the quality con-trol measures developed within our laboratory.

In summary, we show here that a standard procedure forprocessing of PBMC by use of Leucosep tubes in the resource-limited setting of Uganda is practical and feasible for conductof cellular immunology research studies. Through extant lab-oratory capacity, it is possible to monitor parameters that mayadversely affect PBMC functionality, such as granulocyte andplatelet contamination. We also provide indicators to track theperformance of technical staff and preanalytical and analyticalparameters, including time from venipuncture to cryopreser-vation, processing time, yield of PBMC/ml of whole blood, andviability. We also show comparable functions, phenotypes, vi-abilities, and yields of PBMC from HIV-1-infected and unin-fected Ugandans with preservation over time. Taken together,these data suggest a prudent methodology to both validate andmonitor the method for processing and cryopreservation ofPBMC.

ACKNOWLEDGMENTS

We thank Kayunga, Rakai, and Kampala District research partici-pants for their valuable contributions and support during the conductof these studies. We thank the Makerere University Walter ReedProject staff for continued dedication toward development of a safeand effective HIV-1 vaccine, despite numerous hurdles. Jeff Currierand the lab at MHRP have given invaluable scientific, technical, andlogistical support.

Material has been reviewed by the Walter Reed Army Institute ofResearch. There is no objection to its presentation and/or publication.The views expressed here are those of the authors and should not beconstrued to represent the positions of the U.S. Army or DOD.

This work was supported by a cooperative agreement (W81XWH-07-2-0067) between the Henry M. Jackson Foundation for the Ad-vancement of Military Medicine, Inc., and the U.S. Department ofDefense (DOD). This research was funded in part by the U.S. NationalInstitute of Allergy and Infectious Diseases.

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TABLE 2. PBMC quality measures

Parameter Target Impact Reference(s)

Analytical parametersGranulocytes (%) 3.0 � 2.7 High 40Lymphocytes (%) 60–70 (with Leucosep tubes, 87.6 � 4.3) High 24, 40Monocytes (%) 9.1 � 3.8 Low 40Platelets (%) 0–88.2 (�0.5% of original content) High 24Viability (%) �95 Medium 24Yield (cells/ml whole blood) 1 � 106–2 � 106 Medium 24

Postanalytical parametersICS Various targets, including low background, consistently strong

positive controls, and preserved antigen-specific responsesHigh 1, 9, 28

Precision Code of variation of �5% MediumLong-term storage in liquid nitrogen (yr) �10 is optimal, but indefinite storage has been suggested Low 12, 22Transport Maintain cold chain High 13Overnight viability (%) �70–75 High 46Overnight recovery (%) �50 High

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