quality monitoring of hiv-1 infected and uninfected...

Version: 12February2010

of 34

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: [email protected]

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

on May 4, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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%


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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+,


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

HIV-1 infected and uninfected individuals, or between the HIV-1 subtype A and subtype

D study participants.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

REFERENCES

1. Allsopp, C. E., S. J. Nicholls, and J. Langhorne. 1998. A flow cytometric

method to assess antigen-specific proliferative responses of different

subpopulations of fresh and cryopreserved human peripheral blood mononuclear

cells. J Immunol Methods 214:175-86.

2. Appay, V., S. Reynard, V. Voelter, P. Romero, D. E. Speiser, and S. Leyvraz.

2006. Immuno-monitoring of CD8+ T cells in whole blood versus PBMC

samples. J Immunol Methods 309:192-9.

3. Arroyo, M. A., W. B. Sateren, D. Serwadda, R. H. Gray, M. J. Wawer, N. K.

Sewankambo, N. Kiwanuka, G. Kigozi, F. Wabwire-Mangen, M. Eller, L. A. Eller, D. L. Birx, M. L. Robb, and F. E. McCutchan. 2006. Higher HIV-1

Incidence and Genetic Complexity Along Main Roads in Rakai District, Uganda.

J Acquir Immune Defic Syndr 43:440-445.

4. Ashmore, L. M., G. M. Shopp, and B. S. Edwards. 1989. Lymphocyte subset

analysis by flow cytometry. Comparison of three different staining techniques and

effects of blood storage. J Immunol Methods 118:209-15.

5. Baker, C. A., K. McEvers, R. Byaruhanga, R. Mulindwa, D. Atwine, J.

Nantiba, N. G. Jones, I. Ssewanyana, and H. Cao. 2008. HIV subtypes induce

distinct profiles of HIV-specific CD8(+) T cell responses. AIDS Res Hum

Retroviruses 24:283-7.

6. Betts, M. R., M. C. Nason, S. M. West, S. C. De Rosa, S. A. Migueles, J.

Abraham, M. M. Lederman, J. M. Benito, P. A. Goepfert, M. Connors, M. Roederer, and R. A. Koup. 2006. HIV nonprogressors preferentially maintain

highly functional HIV-specific CD8+ T cells. Blood 107:4781-9.

7. Boaz, M. J., P. Hayes, T. Tarragona, L. Seamons, A. Cooper, J. Birungi, P.

Kitandwe, A. Semaganda, P. Kaleebu, G. Stevens, O. Anzala, B. Farah, S.

Ogola, J. Indangasi, P. Mhlanga, M. Van Eeden, M. Thakar, A. Pujari, S.

Mishra, N. Goonetilleke, S. Moore, A. Mahmoud, P. Sathyamoorthy, J.

Mahalingam, P. R. Narayanan, V. D. Ramanathan, J. H. Cox, L. Dally, D. K. Gill, and J. Gilmour. 2009. Concordant proficiency in measurement of T-cell

immunity in human immunodeficiency virus vaccine clinical trials by peripheral

blood mononuclear cell and enzyme-linked immunospot assays in laboratories

from three continents. Clin Vaccine Immunol 16:147-55.

8. Buchbinder, S. P., D. V. Mehrotra, A. Duerr, D. W. Fitzgerald, R. Mogg, D.

Li, P. B. Gilbert, J. R. Lama, M. Marmor, C. Del Rio, M. J. McElrath, D. R.

Casimiro, K. M. Gottesdiener, J. A. Chodakewitz, L. Corey, and M. N. Robertson. 2008. Efficacy assessment of a cell-mediated immunity HIV-1

vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-

concept trial. Lancet 372:1881-93.

9. Bull, M., D. Lee, J. Stucky, Y. L. Chiu, A. Rubin, H. Horton, and M. J.

McElrath. 2007. Defining blood processing parameters for optimal detection of

cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol

Methods 322:57-69.

10. Cao, H., P. Kaleebu, D. Hom, J. Flores, D. Agrawal, N. Jones, J. Serwanga,

M. Okello, C. Walker, H. Sheppard, R. El-Habib, M. Klein, E. Mbidde, P.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

Mugyenyi, B. Walker, J. Ellner, R. Mugerwa, and H. N. f. P. Trials. 2003.

Immunogenicity of a recombinant human immunodeficiency virus (HIV)-

canarypox vaccine in HIV-seronegative Ugandan volunteers: results of the HIV

Network for Prevention Trials 007 Vaccine Study. J Infect Dis 187:887-95.

11. Cao, H., I. Mani, R. Vincent, R. Mugerwa, P. Mugyenyi, P. Kanki, J. Ellner,

and B. D. Walker. 2000. Cellular immunity to human immunodeficiency virus

type 1 (HIV-1) clades: relevance to HIV-1 vaccine trials in Uganda. J Infect Dis

182:1350-6.

12. Costantini, A., S. Mancini, S. Giuliodoro, L. Butini, C. M. Regnery, G.

Silvestri, and M. Montroni. 2003. Effects of cryopreservation on lymphocyte

immunophenotype and function. J Immunol Methods 278:145-55.

13. Cox, J. H., G. Ferrari, R. T. Bailer, and R. A. Koup. 2004. Automating

procedures for processing, cryopreservation, storage, and manipulation of human

peripheral blood mononuclear cells. Journal of the Association for Laboratory

Automation 9:16-23.

14. Currier, J. R., E. G. Kuta, E. Turk, L. B. Earhart, L. Loomis-Price, S.

Janetzki, G. Ferrari, D. L. Birx, and J. H. Cox. 2002. A panel of MHC class I

restricted viral peptides for use as a quality control for vaccine trial ELISPOT

assays. J Immunol Methods 260:157-72.

15. Disis, M. L., C. dela Rosa, V. Goodell, L. Y. Kuan, J. C. Chang, K. Kuus-

Reichel, T. M. Clay, H. Kim Lyerly, S. Bhatia, S. A. Ghanekar, V. C. Maino, and H. T. Maecker. 2006. Maximizing the retention of antigen specific

lymphocyte function after cryopreservation. J Immunol Methods 308:13-8.

16. Dyer, W. B., S. L. Pett, J. S. Sullivan, S. Emery, D. A. Cooper, A. D. Kelleher,

A. Lloyd, and S. R. Lewin. 2007. Substantial improvements in performance

indicators achieved in a peripheral blood mononuclear cell cryopreservation

quality assurance program using single donor samples. Clin Vaccine Immunol

14:52-9.

17. Eller, L. A., M. A. Eller, B. Ouma, P. Kataaha, D. Kyabaggu, R. Tumusiime,

J. Wandege, R. Sanya, W. B. Sateren, F. Wabwire-Mangen, H. Kibuuka, M. L. Robb, N. L. Michael, and M. S. de Souza. 2008. Reference intervals in

healthy adult Ugandan blood donors and their impact on conducting international

vaccine trials. PLoS ONE 3:e3919.

18. GE_Healthcare. 2007. Ficoll-Paque Plus Intended Use for in vitro isolation of

lymphocytes. Uppsala, Sweden.

19. Guwatudde, D., F. Wabwire-Mangen, L. A. Eller, M. Eller, F. McCutchan,

H. Kibuuka, M. Millard, N. Sewankambo, D. Serwadda, N. Michael, and M. Robb. 2009. Relatively low HIV infection rates in rural Uganda, but with high

potential for a rise: a cohort study in Kayunga District, Uganda. PLoS ONE

4:e4145.

20. Harris, M., D. Serwada, N. K. Sewankambo, B. Kim, G. Kigozi, N.

Kiwanuka, J. B. Philips, F. Wabwire, M. Meehen, T. Lutalo, J. R. Lane, R.

Merling, R. H. Gray, M. J. Wawer, D. L. Birx, M. D. Robb, and F. E. McCutchan. 2002. Among 46 near full-length HIV Type 1 genome sequences

from Rakai District, Uganda, subtype D and AD recombinants predominate.

AIDS Res Hum Retroviruses 18:1281-1290.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

21. Hoelscher, M., W. E. Dowling, E. Sanders-Buell, J. K. Carr, M. E. Harris, A.

Thomschke, M. L. Robb, D. L. Birx, and F. E. McCutchan. 2002. Detection of

HIV-1 subtypes, recombinants, and dual infections in east Africa by a multi-

region hybridization assay. Aids 16:2055-64.

22. Horton, H., E. P. Thomas, J. A. Stucky, I. Frank, Z. Moodie, Y. Huang, Y. L.

Chiu, M. J. McElrath, and S. C. De Rosa. 2007. Optimization and validation of

an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific

T cells induced by vaccination. J Immunol Methods 323:39-54.

23. Jeurink, P. V., Y. M. Vissers, B. Rappard, and H. F. Savelkoul. 2008. T cell

responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics

of cell viability, cellular subsets, proliferation, and cytokine production.

Cryobiology 57:91-103.

24. Jones, N., M. Eggena, C. Baker, F. Nghania, D. Baliruno, P. Mugyenyi, F.

Ssali, B. Barugahare, and H. Cao. 2006. Presence of distinct subsets of cytolytic

CD8+ T cells in chronic HIV infection. AIDS Res Hum Retroviruses 22:1007-13.

25. Kanof, M. E., P. D. Smith, and H. Zola. 2003. Preparation of Human

Monoculear Cell Populations and Subpopulations. Current Protocols in

Immunology Supplement 19:Unit 7.1.

26. Karita, E., N. Ketter, M. A. Price, K. Kayitenkore, P. Kaleebu, A. Nanvubya,

O. Anzala, W. Jaoko, G. Mutua, E. Ruzagira, J. Mulenga, E. J. Sanders, M.

Mwangome, S. Allen, A. Bwanika, U. Bahemuka, K. Awuondo, G. Omosa, B.

Farah, P. Amornkul, J. Birungi, S. Yates, L. Stoll-Johnson, J. Gilmour, G.

Stevens, E. Shutes, O. Manigart, P. Hughes, L. Dally, J. Scott, W. Stevens, P. Fast, and A. Kamali. 2009. CLSI-derived hematology and biochemistry

reference intervals for healthy adults in eastern and southern Africa. PLoS ONE

4:e4401.

27. Kibuuka, H., D. Guwatudde, R. Kimutai, L. Maganga, L. Maboko, C.

Watyema, F. Sawe, D. Shaffer, D. Matsiko, M. Millard, N. Michael, F. Wabwire-Mangen, and M. Robb. 2009. Contraceptive use in women enrolled

into preventive HIV vaccine trials: experience from a phase I/II trial in East

Africa. PLoS ONE 4:e5164.

28. Kierstead, L. S., S. Dubey, B. Meyer, T. W. Tobery, R. Mogg, V. R.

Fernandez, R. Long, L. Guan, C. Gaunt, K. Collins, K. J. Sykes, D. V. Mehrotra, N. Chirmule, J. W. Shiver, and D. R. Casimiro. 2007. Enhanced

rates and magnitude of immune responses detected against an HIV vaccine: effect

of using an optimized process for isolating PBMC. AIDS Res Hum Retroviruses

23:86-92.

29. Kiwanuka, N., O. Laeyendecker, M. Robb, G. Kigozi, M. Arroyo, F.

McCutchan, L. A. Eller, M. Eller, F. Makumbi, D. Birx, F. Wabwire-

Mangen, D. Serwadda, N. K. Sewankambo, T. C. Quinn, M. Wawer, and R. Gray. 2008. Effect of Human Immunodeficiency Virus Type 1 (HIV-1) subtype

on disease progression in persons from Rakai, Uganda, with incident HIV-1

infection. J Infect Dis 197:707-13.

30. Kreher, C. R., M. T. Dittrich, R. Guerkov, B. O. Boehm, and M. Tary-

Lehmann. 2003. CD4+ and CD8+ cells in cryopreserved human PBMC maintain

full functionality in cytokine ELISPOT assays. J Immunol Methods 278:79-93.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

31. Kutvirt, S. G., S. L. Lewis, and T. L. Simon. 1993. Lymphocyte phenotypes in

infants are altered by separation of blood on density gradients. Br J Biomed Sci

50:321-8.

32. Kvarnström, M., M. C. Jenmalm, and C. Ekerfelt. 2004. Effect of

cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear

cells from patients with different cytokine profiles, analysed with three common

assays: an overall decrease of interleukin-4. Cryobiology 49:157-68.

33. Lugada, E. S., J. Mermin, F. Kaharuza, E. Ulvestad, W. Were, N. Langeland,

B. Asjo, S. Malamba, and R. Downing. 2004. Population-based hematologic

and immunologic reference values for a healthy Ugandan population. Clin Diagn

Lab Immunol 11:29-34.

34. Macey, M. G., C. J. Hyam, and A. C. Newland. 1988. Enumeration of

lymphocyte sub-populations: a comparative study of whole blood and gradient

centrifugation methods. Med Lab Sci 45:187-91.

35. McElrath, M. J., S. C. De Rosa, Z. Moodie, S. Dubey, L. Kierstead, H. Janes,

O. D. Defawe, D. K. Carter, J. Hural, R. Akondy, S. P. Buchbinder, M. N.

Robertson, D. V. Mehrotra, S. G. Self, L. Corey, J. W. Shiver, and D. R. Casimiro. 2008. HIV-1 vaccine-induced immunity in the test-of-concept Step

Study: a case-cohort analysis. Lancet 372:1894-905.

36. NCCLS. 2000. How to define and determine reference intervals in the clinical

laboratory; approved guideline-Second edition. National Committee for Clinical

Laboratory Standards, Wayne, PA C28-A2 Vol. 20 No. 13. 2ed.

37. Nilsson, C., S. Aboud, K. Karlén, B. Hejdeman, W. Urassa, and G. Biberfeld.

2008. Optimal blood mononuclear cell isolation procedures for gamma interferon

enzyme-linked immunospot testing of healthy Swedish and Tanzanian subjects.

Clin Vaccine Immunol 15:585-9.

38. Owen, R. E., E. Sinclair, B. Emu, J. W. Heitman, D. F. Hirschkorn, C. L.

Epling, Q. X. Tan, B. Custer, J. M. Harris, M. A. Jacobson, J. M. McCune, J. N. Martin, F. M. Hecht, S. G. Deeks, and P. J. Norris. 2007. Loss of T cell

responses following long-term cryopreservation. J Immunol Methods 326:93-115.

39. Reimann, K. A., M. Chernoff, C. L. Wilkening, C. E. Nickerson, and A. L.

Landay. 2000. Preservation of lymphocyte immunophenotype and proliferative

responses in cryopreserved peripheral blood mononuclear cells from human

immunodeficiency virus type 1-infected donors: implications for multicenter

clinical trials. The ACTG Immunology Advanced Technology Laboratories. Clin

Diagn Lab Immunol 7:352-9.

40. Riccio, E. K., I. Neves, D. M. Banic, S. Corte-Real, M. G. Alecrim, M.

Morgado, C. T. Daniel-Ribeiro, and M. d. e. F. Ferreira-da-Cruz. 2002.

Cryopreservation of peripheral blood mononuclear cells does not significantly

affect the levels of spontaneous apoptosis after 24-h culture. Cryobiology 45:127-

34.

41. Romeu, M. A., M. Mestre, L. Gonzalez, A. Valls, J. Verdaguer, M.

Corominas, J. Bas, E. Massip, and E. Buendia. 1992. Lymphocyte

immunophenotyping by flow cytometry in normal adults. Comparison of fresh

whole blood lysis technique, Ficoll-Paque separation and cryopreservation. J

Immunol Methods 154:7-10.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

42. SIGMA-ALDRICH. 2003. ACCUSPIN(TM) TUBES Procedure No. AST-1. St.

Louis, MO 63103 USA.

43. Tobery, T. W., S. A. Dubey, K. Anderson, D. C. Freed, K. S. Cox, J. Lin, M.

T. Prokop, K. J. Sykes, R. Mogg, D. V. Mehrotra, T. M. Fu, D. R. Casimiro, and J. W. Shiver. 2006. A comparison of standard immunogenicity assays for

monitoring HIV type 1 gag-specific T cell responses in Ad5 HIV Type 1 gag

vaccinated human subjects. AIDS Res Hum Retroviruses 22:1081-90.

44. Tugume, S. B., E. M. Piwowar, T. Lutalo, P. N. Mugyenyi, R. M. Grant, F.

W. Mangeni, K. Pattishall, and E. Katongole-Mbidde. 1995. Hematological

reference ranges among healthy Ugandans. Clin Diagn Lab Immunol 2:233-5.

45. Weinberg, A., R. A. Betensky, L. Zhang, and G. Ray. 1998. Effect of

shipment, storage, anticoagulant, and cell separation on lymphocyte proliferation

assays for human immunodeficiency virus-infected patients. Clin Diagn Lab

Immunol 5:804-7.

46. Weinberg, A., L. Y. Song, C. Wilkening, A. Sevin, B. Blais, R. Louzao, D.

Stein, P. Defechereux, D. Durand, E. Riedel, N. Raftery, R. Jesser, B. Brown, M. F. Keller, R. Dickover, E. McFarland, and T. Fenton. 2009. Optimization

and limitations of use of cryopreserved peripheral blood mononuclear cells for

functional and phenotypic T-cell characterization. Clin Vaccine Immunol

16:1176-86.

47. Weinberg, A., L. Zhang, D. Brown, A. Erice, B. Polsky, M. S. Hirsch, S.

Owens, and K. Lamb. 2000. Viability and functional activity of cryopreserved

mononuclear cells. Clin Diagn Lab Immunol 7:714-6.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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).


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

FIGURE 1.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

FIGURE 2.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

FIGURE 3.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

FIGURE 4.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


of 34

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


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

quality monitoring of hiv-1 infected and uninfected...

Documents