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Version: 12February2010
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Quality Monitoring of HIV-1 Infected and Uninfected Peripheral Blood
Mononuclear Cell Samples in a Resource Limited Setting
Robert E. Olemukan1, Leigh Anne Eller
1,2, Benson J. Ouma
1, Ben Etonu
1, Simon Erima
1,
Prossy Naluyima1, Denis Kyabaggu
1, Josephine H. Cox
3, Johan K. Sandberg
4, Fred
Wabwire-Mangen1, Nelson L. Michael
2, Merlin L. Robb
2, Mark S. de Souza
2,5, Michael A.
Eller1,2,4
Makerere University Walter Reed Project, Kampala, Uganda1, U.S. Military HIV
Research Program, Rockville, USA2, International AIDS Vaccine Initiative, New York,
USA3, Center for Infectious Medicine, Department of Medicine, Karolinska Institutet,
Karolinska University Hospital Huddinge, 14186, Stockholm, Sweden4, Armed Forces
Research Institute of Medical Sciences, Bangkok, Thailand5
Abstract word count: 224
Main text word count: 4,108
Display items: 4 figures, 2 tables
Running title: Quality monitoring of PBMC Processing in Uganda
Keywords: PBMC, HIV-1, Cryopreservation, Quality Assurance
Correspondence: Michael A. Eller, U.S. Military HIV Research Program, 13 Taft Court,
Suite 200, Rockville, MD 20850, USA. Tel: +1(301) 251-8304; Fax: +1(301) 762-4177;
Email: meller@hivresearch.org
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00492-09 CVI Accepts, published online ahead of print on 3 March 2010
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Abstract
Human immunodeficiency virus (HIV)-1 vaccine and natural history studies are critically
dependent on the ability to isolate, cryopreserve, and thaw PBMC samples with a high
level of quality and reproducibility. Here we characterize the yield, viability, phenotype
and function of PBMC from HIV-1 infected and uninfected Ugandans, and describe
measures to ascertain reproducibility and sample quality at the sites that perform
cryopreservation. We have developed a comprehensive internal quality control program
to monitor processing, including components of method validation. Quality indicators for
real time performance assessment included; time from venipuncture to cryopreservation,
time for PBMC processing, yield of PBMC from whole blood, and viability of the PBMC
before cryopreservation. Immune phenotype analysis indicated lowered B cell
frequencies following processing and cryopreservation in both HIV-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 and all specimens responded to SEB by interferon-gamma and
interleukin-2 production as measured by intracellular cytokine staining. Samples stored
for greater than three years did not decay with regard to yield or viability regardless of
HIV-1 infection status. These results demonstrate that it is possible to achieve the high
level of quality necessary for vaccine trials and natural history studies in a resource
limited setting, and provide strategies for laboratories to monitor PBMC processing
performance.
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INTRODUCTION
Cryopreserved peripheral blood mononuclear cells (PBMCs) are critical to studies
of HIV-1 infection and the development of an effective HIV vaccine. High quality
cryopreservation is particularly challenging and important in resource-limited settings
where natural history, therapeutic, and preventative protocols are being developed.
Cryopreservation and batch testing allow for simultaneous assessment of critical samples,
thereby reducing inter-assay variability. Since standard method validation procedures,
such as accuracy, precision, linearity, and reportable ranges, are not easily obtained for
PBMC processing, more studies are needed to understand the parameters influencing the
reproducibility of processing and quality of cryopreserved PBMC samples in resource
limited settings.
Separation of PBMCs should yield a pure population of mononuclear cells 95% ±
5% (18), with minimal contamination from red blood cells, granulocytes, and platelets.
Anticipated yields of mononuclear cells from normal donors are approximately 1-2 x 106
cells/ml of whole blood with a purity of 60-70% lymphocytes at >95% viability with
reduction of platelets to < 0.5% of original whole blood content (25). However, the data
on impact of cryopreservation on lymphocyte phenotypes is inconsistent (4, 31, 34, 39,
41). Additionally, there are differences in hematologic parameters and lymphocyte
subsets in African populations as compared to the western world (17, 26, 33, 44). Careful
examination of the impact of cryopreservation on yield and phenotype is therefore needed
for this region. Furthermore, studies indicate the ability to preserve function of
cryopreserved PBMC as compared to whole blood measured by proliferation assays (1,
12, 23, 46, 47), cytokine production (9, 28, 30, 32, 37), apoptosis (40), and HLA tetramer
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staining (2). This is particularly important for HIV-1 as many vaccine candidates are
designed to elicit T cell responses, and reliable PBMC based assays such as ELISpot and
multi-parameter flow cytometry are required to prioritize candidates for large scale
efficacy testing.
Some studies indicate that the functional integrity of cryopreserved PBMC
samples is maintained (22), while other reports suggest loss of function (38). Pre-
analytic variables, which have been closely examined, include time of blood draw to
initiation of processing, type of anti-coagulant, proper mixing of blood tubes, and
transport temperature (9, 13, 28, 45). Analytic variables that impact PBMC processing
include separation method, type of cryoprotectant medium, pre-chilling of the freeze
containers, cryopreservation parameters and temperature of thawing media (9, 15, 28, 30,
37). In addition to defining processing parameters, it is important to monitor quality
assurance for cryopreserved PBMC assays after cryopreservation (7). However, few
studies have addressed the importance of continuous monitoring of the processing and
cryopreservation of PBMC themselves, but focused on the procedures for thawing of
cells in labs that perform endpoint assays. We have developed a comprehensive internal
quality control program to monitor PBMC processing. This study describes this program
and the results achieved in a resource limited setting. Additionally, we describe
procedures to evaluate our laboratory methods for PBMC processing.
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METHODS
Study participants
All samples were obtained from participants enrolled in studies conducted in Kampala
(27), Kayunga (19), and Rakai (29) districts in Uganda as previously described. All
studies were reviewed and approved by the appropriate Institutional Review Boards in
the United States and Uganda with consent forms signed by all participants.
PBMC processing
Blood was collected into 8.5 ml acid citrose dextran (ACD) anti-coagulated tubes and
transported to the lab at room temperature. PBMCs were isolated from ACD whole blood
within 8 hours of collection by centrifugation at 800xg for 15 minutes through Ficoll-
hypaque PLUS (Pharmacia, Uppsala, Sweden) using Leucosep® tubes (Greiner Bio-One,
Frickenhausen, Germany). The PBMC layer was harvested and washed three times with
PBS by centrifugation at 250 times gravity for 10 minutes. Cell counts and viability were
determined by hemacytometer using trypan blue exclusion or by the Guava PCA machine
(Guava Technologies, Hayward, CA) using Guava ViaCount reagent. PBMC were
cryopreserved at a concentration of 107 cells/ml in freeze media (20% FBS, 10% DMSO,
1% Pen/Strep, 69% 1640-RPMI) using isopropyl "Mr. Frosty" freezing containers
(NALGENE® Labware, Thermo Fisher, Rochester, NY) to control the rate of freezing
overnight in a -80°C freezer, and 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 a 37°C water bath until a small ice crystal remained and washed with Complete
Media (CM): (RPMI 1640 supplemented with 10% FBS, 2% L-glutamine and 1%
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Penicillin/Streptomycin) added drop wise to avoid rapid osmotic change.
Lymphocyte immunophenotyping and hematology
Lymphocyte immunophenotyping was performed on EDTA anti-coagulated whole blood
or PBMC product post thaw using the FACS MultiSET™ System. Samples were run on a
dual laser flow cytometer using the single platform Multitest™ 4-color reagent in
combination with TruCount™ tubes and analyzed using MultiSET™ software (Becton
Dickinson, San Jose, CA). Absolute number and percentage of T (CD3+), helper T
(CD4+), cytotoxic T (CD8 +), B (CD19+) and NK (CD16+ or CD56+) lymphocytes were
determined. Complete blood count with 5-part differential was performed using the
Coulter AcT 5 diff (Beckman Coulter, Fullerton, CA) for whole blood and post
processing cell samples.
Intracellular cytokine staining
A standard intracellular cytokine staining assay (ICS) was performed on cryopreserved
peripheral blood mononuclear cells (PBMC), which had been thawed and rested
overnight. PBMCs (106) were incubated in 96-well round bottom plates with co-
stimulatory monoclonal antibodies anti-CD28 and anti-CD49d (Becton Dickinson, San
Jose, CA), a peptide pool from Cytomegalovirus, Epstein Barr virus and Influenza virus
(CEF) (14) and brefeldin A (Becton Dickinson, San Jose, CA). Samples had a negative
control of PBMCs stimulated without peptide and a positive control of Staphylococcal
enterotoxin B (SEB; Sigma-Aldrich, Munich, Germany). Cells were incubated for 6
hours at 37ºC in a 7% CO2 incubator and stored at 4ºC overnight. The next day, 20ul of
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20 mM EDTA was added to each well and incubated for 15 minutes. After centrifugation,
1x FACS lysing solution was added for 10 minutes at room temperature. Cells were then
washed once in wash buffer (0.5% BSA, 0.1% sodium azide in PBS), permeabilized in
200 µl 1x FACS Permeabilizing Solution for 10 minutes and washed three times. Cells
were incubated for 60 minutes with a 50 µl antibody cocktail containing anti-CD3-APC,
anti-CD4-FITC, anti-CD8-PerCpcy5.5, anti-interferon-gamma (IFN-γ)-PE and anti-
interleukin-2 (IL-2)-PE (Becton Dickinson, San Jose, CA). Cells were then washed three
times and fixed in 1% Paraformaldehyde. Flow cytometric analysis was performed using
a FACSCalibur flow cytometer where at least 20,000 CD3+/CD8+ and CD3+/CD4+
events each were acquired. Samples were analyzed using FlowJo Software version 8.5
(Tree Star, Ashland, OR). A positive ICS response was defined as at least 3-fold higher
than the mean unstimulated response and above 0.05.
HIV-1 Subtype Determination
All HIV-1 sero-positive participants’ plasma samples were tested for virus subtype using
previously described multi-region hybridization assay (MHA), version 2, for HIV-1
subtypes A, C, D, recombinants, and dual infections (3, 20, 21). HIV-1 subtype A, C or D
was assigned from five regions (gag, pol, vpu, env, and gp41) of the HIV-1 genome
based on reactivity of the probes, if at least 2 regions had probe reactivity. Samples with
probe reactivity of a single subtype in different regions were labeled as pure subtypes
Statistical analysis
All statistical analysis was performed using Graph Pad Prism Software version 5.0a for
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Mac OSX (GraphPad Software, La Jolla, CA). Direct comparison between two groups
was performed using the non-parametric Mann-Whitney U test for continuous data. For
paired observations the paired T test was used. P values of <0.05 were considered
significant. For descriptive statistics, mean with the standard deviation was presented
graphically while the median, mean and minimum to maximum range was reported
within the text. Reference range intervals were calculated as the range between the 2.5%
and 97.5% limit for the parameters tested. Thaw viability was reported as the percent of
viable cells at the time of thaw using the ViaCount assay and standard gating excluding
apoptotic cells. Overnight viability was reported as the percent of viable cells after
overnight incubation in CM at 37°C, 7%CO2, and 90% relative humidity using the
ViaCount assay and standard gating excluding apoptotic cells. Thaw and overnight yields
were cells divided by original vial cell content in similar conditions as for viability.
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RESULTS
PBMC Leucosep® processing and cryopreservation preserves lymphocytes. In order
to assess the technique of laboratory staff and the efficiency of the PBMC processing
procedure using Leucosep® tubes, 52 samples were assessed for whole blood and post-
processing concentrations of granulocytes, lymphocytes, monocytes, and platelets.
Basophils, eosinophils, and neutrophils were considered together as granulocytes. The
composition of white blood cells in whole blood was 50.3% (range: 28.4%-78.9%)
granulocytes, 40.4% lymphocytes (17.0%-58.7%), and 9.3% (2.0%-42.5%) monocytes
(Fig. 1A). After ficoll separation using Leucosep® tubes the mean percentages of these
cells were 8.8% (1.2%-45.8%) granulocytes, 69.9% (29.4%-92.6%) lymphocytes, and
21.4% (4.9%-56.3%) monocytes (Fig. 1B). Initial whole blood product (27 ml whole
blood) contained 103x106 (35-223x10
6) total platelets per ml, which decreased to 12x10
6
platelets/ml (0-100x106) following processing (Fig. 1C).
In order to assess the effect of processing and cryopreservation on PBMC
phenotype, we studied PBMCs from freshly drawn peripheral blood and after thaw of
samples stored in vapor phase of liquid nitrogen for approximately 1.5 years from 40
HIV-1 uninfected and 28 chronically HIV-1 infected donors. Whole blood immune cell
subsets median (range) in uninfected subjects were: 70% (47%-81%) CD3+, 22% (13%-
47%) CD8+, 42% (29%-57%) CD4+, 12% (5%-33%) CD16+ or CD56+, and 16% (9%-
26%) CD19+. Immediately following thawing and counting, PBMC immune subsets
were compared to those of whole blood and a statistically significant difference was
observed only for the percentage of B cells (P<0.001) (Fig. 1D). Whole blood immune
cell subsets median (range) in HIV-1 infected individuals were: 71% (47%-83%) CD3+,
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46% (27%-71%) CD8+, 20% (2%-43%) CD4+, 14% (4%-35%) CD16+ or CD56+, and
14% (6%-24%) CD19+. Comparing cell subsets between thawed PBMC samples and the
paired fresh PBMC samples, a statistically significant decrease was again observed only
for the B cell subset (P=0.007) (Fig. 1E).
Harvesting plasma before or after processing does not affect quality. In order to
assess the effect of harvesting plasma prior to and following ficoll separation, 10
uninfected donors specimens (each of 94 ml whole blood) were processed in parallel (47
ml each) to determine the final PBMC yield, viability, and purity. PBMC yield and
viability was not different between samples when plasma was collected before or after
ficoll separation, P = 0.506 and 0.085, respectively (data not shown). The median yield
(range) for plasma collection before and after ficoll separation was 54x106 PBMC (24–
67x106 PBMC) and 53x10
6 PBMC (27–71x10
6 PBMC), respectively. The median
viability (range) was 95% (90% – 100%) for plasma collection before ficoll and 94%
(91% - 98%) after ficoll. No difference was observed in platelet concentration between
harvesting plasma before or after ficoll separation; 67x106 (22–128x10
6)/ml and 69x10
6
(17–133x106)/ml respectively (P = 0.489). Similarly, contaminating granulocyte
concentration was 0.6x106 (0.4–2.6x10
6)/ml before, and 0.7x10
6 (0.5–1.1x10
6)/ml after
ficoll separation, with no statistical difference (P=0.706).
Establishing quality indicators for PBMC processing in Uganda. In order to monitor
the quality and performance of the processing laboratory in Uganda during the conduct of
a phase I/II HIV-1 vaccine trial, 4 processing quality indicators were acquired daily and
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collated monthly during the study: (1) total time from blood draw to cryopreservation, (2)
time for actual PBMC processing, (3) PBMC yield per ml of whole blood collected
before cryopreservation, and (4) PBMC viability before cryopreservation. 2310 samples
from both HIV-1 infected (n=115) and uninfected (n=2195) subjects were received in the
laboratory for PBMC processing. Samples were processed and cryopreserved from
March 2006 through August 2007. The median (range) total time from blood draw to
cryopreservation was 3.7 hours (3.0–7.2 hours), while the median (range) process time
from blood receipt in the lab to cryopreservation was 2.7 hours (2.2–6.0 hours). The
maximum number of samples processed in a month was 313. Cell yield per ml of whole
blood collected before cryopreservation was 1.3x106 (0.1–4.1x10
6) PBMC/ml whole
blood (Fig. 2A). Viability before cryopreservation was assessed and the overall median
(range) was 97% (75%–100%) (Fig. 2B).
Assessment of PBMC processing shows preservation of viability and function.
Assessment of PBMC processing, cryopreservation, and storage in liquid nitrogen was
made over the course of one year by assessing longevity and inter-vial precision in
selected samples. Samples from three HIV-1 uninfected individuals were assessed for
longevity following processing and cryopreservation, by thawing at month 1, 7, and 12.
Thawed samples were counted using the Guava ViaCount assay and cell concentration
and viability determined. Samples were rested overnight and then cell counts and
viability were repeated. Cells were assessed by ICS to measure the amount of IFN-γ and
IL-2 within the CD4 and CD8 T cell compartments in response to the superantigen SEB.
Mean viability after overnight rest was 93%, 92% and 98% at month 1, 7, and 12
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respectively, while mean recovery was 85%, 79%, and 80% respectively (Fig. 3A). CD4
T cells responses to SEB were 9.1%, 10.4%, and 10.0%, while CD8 T cells responses
were 10.7%, 9.6%, and 9.1% at months 1, 7, and 12 post cryopreservation, respectively
(Fig. 3A). Precision was assessed by examining PBMC from three individuals,
distributed into at least 3 vials each at 10x106 cells per aliquot. All samples showed
coefficients of variation less than 2% for thaw and overnight viability and recovery yield
(data not shown).
Monthly assessment of processing and cryopreservation was performed using 2-6
samples that were cryopreserved at the beginning of the month and thawed at the end of
the month. Cell counts and viabilities were determined both at the time of thaw and after
overnight rest. At the time of thaw the median (range) yield was 62% (32%-143%) and
viability was 93% (77%-98%), while after overnight rest the median (range) yield was
69% (26%-103%) and viability was 80% (65%-100%) (Fig. 3B). Stratifying the
responses by month showed fluctuations in mean recovery from the low of 49% in
September 2006 to the high of 126% in November 2006. The range of viability was from
the low of 85% in May and September 2006 to the high of 98% in November 2006 (Fig.
3C). Guava ViaCount was implemented in October 2006 in order to reduce the operator
variation from hemacytometer counting. No samples were processed for quality
monitoring during the months of February and March of 2007.
Monthly samples were also assessed for functional responses by conducting a 6-
hour stimulation, in the presence of the CEF peptide pool, SEB, or in the absence of
stimulation and ICS performed to assess IFN-γ and IL-2 in CD4 and CD8 T cells.
Cryopreserved specimens exhibited low background with no stimulation and retained
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functional responses to SEB. The median (range) combination IFN-γ/IL-2 response in the
absence of stimulation was 0.02% (0%-0.17%) in CD4 T cells and 0% (0%-0.13%) in
CD8 T cells. All 35 specimens (100%) had positive responses to SEB. The median
(range) IFN-γ/IL-2 response to SEB stimulation was 10.10% (2.15%-20.10%) in CD4 T
cells and 8.35% (1.03%-19.40%) in CD8 T cells (Fig.3D). Positive responses to the CEF
peptide pool were 6/35 (17%) and 24/35 (69%) of specimens in the CD4 T cell and CD8
T cell compartment, respectively. The median (range) of positive IFN-γ/IL-2 response to
CEF stimulation was 0.11% (0.06%-0.26%) in CD4 T cells and 0.36% (0.05%-7.17%) in
CD8 T cells.
PBMC maintain viability after long-term storage. The effect of long-term
cryopreservation (>3 years) was assessed in samples from HIV-1 uninfected (n=30) and
infected (n=29) individuals. Overall, the median (range) recovery was 88% (26%-173%)
and the viability was 94% (82%-98%) at time of thaw. After overnight rest the median
(range) percent recovery was 76% (22%-140%) and the viability was 95% (81%-98%)
(Fig. 4A). In the HIV-1 uninfected subjects after overnight rest the median (range)
percent recovery was 79% (22%-130%) and the viability was 96% (88%-98%) (Fig. 4B).
In 10 HIV-1 subtype A infected subjects the median (range) percent PBMC recovery was
68% (35%-87%) and the viability was 97% (92%-98%) after overnight rest (Fig. 4C). In
19 HIV-1 subtype D infected subjects, the median (range) percent recovery was 74%
(32%-140%) and the viability was 92% (81%-98%), after overnight rest (Fig. 4D). No
statistically significant difference was observed in the recovery and viability between the
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HIV-1 infected and uninfected individuals, or between the HIV-1 subtype A and subtype
D study participants.
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DISCUSSION
Many of the T-cell based assays used to assess potential HIV-1 candidate
vaccines have been standardized and evaluated to characterize vaccine immunogenicity
(7, 22, 43) but may not define correlates of protection (8, 35). T cells are linked to control
of HIV-1 infection and specific functional profiles are associated with slow HIV-1
disease progression in infected individuals (6). Common practice for many multi center
vaccine trials is to cryopreserve cells and ship to a central laboratory to reduce inter-
laboratory variation in assay performance. Additionally, cryopreserved cells allow for
carefully planned longitudinal analysis and case-control studies. Hematological
parameters have previously been shown to differ for populations in Uganda compared to
populations in industrialized countries (17, 26, 33, 44), and this could adversely affect
protocol endpoints designed to utilize white blood cells for immunologic phenotype or
functional analysis. Here we characterize the function, phenotype, viability, and yield, of
PBMC from HIV-1 infected and uninfected Ugandans and introduce a number of quality
measures that can be used to monitor the performance of collection, processing,
cryopreservation and storage of PBMC in real-time.
Studies from Uganda have used cryopreserved PBMC for cellular immunology
assessment; however minimal data has been presented on processing cell yields and post
thaw recoveries (5, 10, 11, 24). In a recent study conducted on PBMC from Tanzanian
subjects, yields of 1.1 X 106 cells were harvested per ml of whole blood with 96%
viability (37) compared to the 1.3 X 106 cells per ml of whole blood and 97% viability
we observed in Ugandan subjects using similar procedures and reagents. Our study
summarizes results from a much larger cohort of 2195 healthy individuals and proposes
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95% reference intervals (Table 1) for processing parameters in an effort to validate this
methodology in accordance with Clinical and Laboratory Standards Institute (CLSI)
guidelines, although for the granulocytes, monocytes, lymphocytes, and platelets the
recommended number of 120 samples was not achieved (36). Additionally, we observe
similar yields and viabilities in HIV-1 infected individuals, although some donors with
advanced disease can exhibit a high lymphopenia and poor separation from red blood
cells in the ficoll gradient resulting in extremely low yields and viabilities.
Separated PBMC should consist of a pure population of mononuclear cells 95%
(18), with yields of approximately 1-2 x 106 cells/ml of whole blood consisting of 60-
70% lymphocytes at >95% viability with reduction of platelets to < 0.5% of original
whole blood content (25). Performance characteristics using a porous high density
polyethylene barrier “frit” like a Leucosep® tube or equivalent Accuspin™ tube should
enable higher accuracy in harvesting mononuclear cells yielding a final product of 88%
lymphocytes, 9% monocytes, and 3% granulocytes at a viability of 98% (42). We do
observe slightly higher platelet contamination in the final product with a median
reduction to 1.16% of total original blood content but in general show that processing
with Leucosep® tubes gives results similar to manufacturer specifications allowing for
accurate preparation and planning for clinical studies requiring PBMC.
In order to assess the effect of processing and cryopreservation on immune
phenotype we compared T (CD4+ and CD8
+ T cells), B, and NK lymphocytes in fresh
whole blood or after ficoll processing, cryopreservation and thaw. Interestingly the
proportion of most subsets was preserved with the exception of B cells, consistent with a
previous multi-center study conducted in the US where after processing and
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cryopreservation, a reduction in the percentage of B cells was observed (39). Other
studies have shown increases or static levels of B cells (4, 31, 34, 41). Our data supports
the observation of reduced B cells after cryopreservation in both HIV-1 infected and
uninfected individuals. One potential explanation is that B cells may have different
density and could be centrifuged through the ficoll gradient and be included in the red
blood cell pellet. Another explanation could be that B cells are more susceptible to the
affects of cryopreservation and subsequently die. This loss is of great importance to B
cell immunologists who plan to study this subset from cryopreserved PBMC.
Processing PBMC is straightforward but many parameters need to be optimized in
order to maximize yield, recovery, viability and function. One important finding is that
contaminating granulocytes increase cell clumping upon thawing (16) and adversely
affect PBMC yield, function, and assay background responses (13). Another important
processing parameter is the time from blood draw to the time the PBMC are
cryopreserved. Several studies indicate that processing time of 24 hours or greater
negatively impacts the viability, recovery and function of PBMC and suggest that
processing and cryopreservation needs to occur within 8 hours (9, 13, 16, 28). We
confirm the feasibility of large scale processing in a resource limited setting with
transport to the laboratory from an off site clinic, with a time of blood draw to
cryopreservation of under 7.5 hours and a median time from receipt in the laboratory to
cryopreservation of 3.7 hours. Additional parameters that have minimal impact on PBMC
include shipping, centrifugal force for washing, concentration of cells in the vial,
anticoagulant used in collection of whole blood, using classical overlaying procedure, or
the size of the wash conical tube (9, 15, 37, 45) and here we show that harvesting the
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plasma before or after the ficoll gradient step also does not affect the overall quality of
PBMC, eliminating one centrifugation step, and reducing the processing time.
Other studies suggest that it is not the processing and cryopreservation procedures
that adversely affect PBMC, but how they are thawed and if rested overnight (13, 28, 30)
and suggest that viability needs to be greater than 70- 75% for consistent functional
assessment (15, 46, 47). In our study function is maintained in response to superantigen
and background responses remain low in unstimulated conditions. While unstimulated
and superantigen exposed PBMC were consistent, the antigen specific peptide set of CEF
was not consistent in generating positive responses. This may be in part due to
differences in the HLA alleles in African populations and the epitopes selected for the
CEF peptide pool. The lab has since transitioned to using a commercially available
CMVpp65 peptide set that generates a higher frequency of consistent responses in the
ICS assay making for a better positive control.
Preservation of PBMC for HIV vaccine trials are primarily evaluated by
centralized laboratories, which perform the immunogenicity assays. Some groups focus
on a central laboratory to serve as repository and evaluation unit (9, 28), while others
develop a quality assurance scheme that revolves around sample collection and
subsequent evaluation performed centrally (16). While these methods are a necessary
component to good PBMC practice (GPP), real time assessment of PBMC processing and
cryopreservation are needed. The network of labs in the US Military HIV Research
Program all adhere to centralized standard operating procedures for processing,
cryopreservation, and shipping of PBMC and participate in training across sites within
the network. Additionally, a new site goes through a validation and initialization period
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where a site must prove proficiency in processing and cryopreservation. Once qualified to
conduct PBMC processing a site is monitored both externally and internally. While our
PBMC quality management plan includes shipping to external sites for assessment
several times a year, here we outline several indicators that can be monitored by the
actual processing units including a plan to thaw PBMC and evaluate recoveries and
viabilities at regular intervals. It is critical that these procedures are done in such a
manner that does not impact the endpoint analysis for protocols and should be developed
early in study design to include blood for processing and cryopreservation assessment.
Table 2 summarizes our QC measures developed within the laboratory.
In summary, we show here that a standard procedure for processing of PBMC
using Leucosep® tubes in the resource limited setting of Uganda is practical and feasible
to conduct cellular immunology research studies. Through extant laboratory capacity, it is
possible to monitor parameters that may adversely affect PBMC functionality such as
granulocyte and platelet contamination. We also provide indicators to track performance
of technical staff and pre-analytical and analytical parameters including time from
venipuncture to cryopreservation, processing time, yield of PBMC /ml of whole blood
and viability. We also show comparable function, phenotype, viability, and yield, of
PBMC from HIV-1 infected and uninfected Ugandans that is preserved over time. Taken
together, this data suggests a prudent methodology to both validate and monitor the
method for processing and cryopreservation of PBMC.
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ACKNOWLEDGEMENTS
The authors would like to thank Kayunga, Rakai, and Kampala District research
participants for their valuable contribution and support during the conduct of these
studies. The Makerere University Walter Reed Project Staff for continued dedication
towards development of a safe and effective HIV-1 vaccine despite numerous hurdles.
Jeff Currier and lab at MHRP has made invaluable scientific, technical and logistical
support. Material has been reviewed by the Walter Reed Army Institute of Research.
There is no objection to its presentation and/or publication. This work was supported by a
cooperative agreement (W81XWH�07�2�0067) between the Henry M. Jackson
Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of
Defense (DOD). This research was funded, in part, by the U.S. National Institute of
Allergy and Infectious Diseases. The views expressed are those of the authors and should
not be construed to represent the positions of the U.S. Army or DoD.
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Figure Legends
FIGURE 1. PBMC were isolated from whole blood using Leucosep® tubes and the
quality of the procedure assessed. (A) Pie chart shows the mean proportion of
granulocytes 50.3% (28.4% - 78.9%), lymphocytes 40.4% (17.0% - 58.7%), and
monocytes 9.3% (2.0% - 42.5%) from 52 whole blood samples before processing (B) Pie
chart shows the mean proportion of granulocytes 8.8% (1.2% - 45.8%), lymphocytes
69.9% (29.4% - 92.6%), and monocytes 21.4% (4.9% - 56.3%) from 52 samples after
PBMC processing. (C) Bar chart showing the mean concentration of platelets (x 106) in
whole blood 1034 (35-223) or after processing 12 (0-100). The effect of processing and
cryopreservation on the immune phenotype was assessed and compared relative to
peripheral blood concentrations. (D) Box and whisker plots showing the median and 10-
90 percentiles of major immune cell subsets in 40 normal healthy individuals.
Statistically significant difference was observed for the percentage of B cells (P < 0.001).
(E) Box and whisker plots showing the median and 10-90 percentiles of major immune
cell subsets in 28 HIV-1 positive individuals. A statistically significant decrease was
observed for the percentage of B cells (P = 0.007).
FIGURE 2. Quality indicators were measured for PBMC processing conducted during a
phase I/II clinical trial from March 2006 through August 2007 (n=2310). PBMC were
received in the laboratory, processed using Leucosep® tubes and cryopreserved in LN
vapor. (A) Aligned dot plot showing the mean and standard deviation of PBMC yield
(cells x 106 per ml of whole blood processed) of samples processed each month over the
conduct of the clinical trial. The mean of all samples processed is solid line +/- 2 standard
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deviations above and below are shown with dotted lines. (B) Aligned dot plot showing
the mean and standard deviation of PBMC percent viability after processing of samples
each month over the conduct of the clinical trial. The mean of all samples processed is a
solid line +/- 2 standard deviations below are shown with a dotted line.
FIGURE 3. The MUWRP laboratory follows standard procedures for assessing quality
of PBMC collected and cryopreserved during protocol vaccine trials, including internal
assessment of longevity, precision, and functionality. (A) PBMC from 3 subjects were
assessed for the impact of cryopreservation on recovery, viability and functionality by
thawing the same samples at 1, 7, and 12 months post cryopreservation. Bar and line
chart showing the mean and standard deviation for overnight viability (red), overnight
recovery (blue), control positive response (IFN-γ/IL-2) to staphylococcal enterotoxin B
(SEB) for CD4 (white bars) and CD8 (black bars). (B) 35 samples were processed,
cryopreserved (approximately for 1 month), thawed, and assessed for recovery and
viability after thaw and overnight rest as part of a monthly internal quality assurance
procedure. Scatter plot showing the cumulative mean and standard deviation for all 35
samples. (C) Bar chart showing the mean and standard deviation thaw recovery and
viability for the 35 samples monitored by month (no samples processed February and
March 2007). (D) 35 samples were thawed, and assessed for function after thaw and
overnight rest in response to SEB or in the absence of stimulation. Scatter plot showing
the cumulative mean and standard deviation for all 35 samples for CD4 T cells (black)
and CD8 T cells (white).
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FIGURE 4. The effect of long-term cryopreservation (storage > 3 years) was assessed in
HIV infected and uninfected individuals. (A) Scatter plot showing the cumulative mean
and standard deviation for recovery and viability after thaw and overnight (O/N) rest on
30 HIV-1 uninfected samples. (B) Scatter plot showing the cumulative mean and
standard deviation for recovery and viability after thaw and overnight rest on 59 HIV-1
infected samples with HIV-1 subtype A (n = 10) in red and HIV-1 subtype D (n = 19) in
blue.
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FIGURE 1.
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FIGURE 2.
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FIGURE 3.
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FIGURE 4.
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Table 1. PBMC Processing Parameter 95% Reference Intervals
Test n Median Mean (StDev) Range
Lymphocyte (%) 52 71.7 69.9 (12.9) 32.9–90.1
Monocyte (%) 52 20.2 21.4 (9.7) 6.5–48.4
Granulocyte (%) 52 4.9 8.8 (8.3) 1.5–35.6
Platelet (106 cells) 52 10 12.12 (16.6) 0 – 88.2
Yield (cells/ml whole blood) 2195 1.3 1.3 (0.53) 0.4-2.6
Viability (%) 2195 97 96.7 (2.4) 91-100
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Table 2. Summary PBMC Quality Measures
Parameter Target Impact Additional
Reference(s)
Granulocyte 3.0 +/- 2.7% High 40
Lymphocyte 60-70% (using Leucosep®: 87.6 +/- 4.3%) High 24 (40)
Monocyte 9.1 +/- 3.8% Low 40
Platelets 0 – 88.2 / <0.5% of original content High 24
Viability > 95% Medium 24
Analytical
Yield 1-2 x 106 / ml whole blood Medium 24
ICS Various Targets including low background,
consistent strong positive controls, and
preserved antigen specific responses
High 1, 9, 28
Precision Code of variation < 5% Medium None
Long Term Storage in
liquid nitrogen
<10 years optimal, but indefinite storage has
been suggested
Low 12, 22
Transport Maintain cold chain High 13
Overnight viability >70-75% High 46
Post Analytical
Overnight recovery >50% High None
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