RESEARCH Open Access

Shipping blood to a central laboratory inmulticenter clinical trials: effect of ambienttemperature on specimen temperature, andeffects of temperature on mononuclear cell yield,viability and immunologic functionWalter C Olson1*, Mark E Smolkin2, Erin M Farris3, Robyn J Fink4, Andrea R Czarkowski5, Jonathan H Fink6,Kimberly A Chianese-Bullock1,7, Craig L Slingluff Jr1,7

Abstract

Background: Clinical trials of immunologic therapies provide opportunities to study the cellular and moleculareffects of those therapies and may permit identification of biomarkers of response. When the trials are performedat multiple centers, transport and storage of clinical specimens become important variables that may affectlymphocyte viability and function in blood and tissue specimens. The effect of temperature during storage andshipment of peripheral blood on subsequent processing, recovery, and function of lymphocytes is understudiedand represents the focus of this study.

Methods: Peripheral blood samples (n = 285) from patients enrolled in 2 clinical trials of a melanoma vaccinewere shipped from clinical centers 250 or 1100 miles to a central laboratory at the sponsoring institution. The yieldof peripheral blood mononuclear cells (PBMC) collected before and after cryostorage was correlated withtemperatures encountered during shipment. Also, to simulate shipping of whole blood, heparinized blood fromhealthy donors was collected and stored at 15C, 22C, 30C, or 40C, for varied intervals before isolation of PBMC.Specimen integrity was assessed by measures of yield, recovery, viability, and function of isolated lymphocytes.Several packaging systems were also evaluated during simulated shipping for the ability to maintain the internaltemperature in adverse temperatures over time.

Results: Blood specimen containers experienced temperatures during shipment ranging from -1 to 35C. Exposureto temperatures above room temperature (22C) resulted in greater yields of PBMC. Reduced cell recoveryfollowing cryo-preservation as well as decreased viability and immune function were observed in specimensexposed to 15C or 40C for greater than 8 hours when compared to storage at 22C. There was a trend towardimproved preservation of blood specimen integrity stored at 30C prior to processing for all time points tested.Internal temperatures of blood shipping containers were maintained longer in an acceptable range when warmpacks were included.

Conclusions: Blood packages shipped overnight by commercial carrier may encounter extreme seasonal temperatures.Therefore, considerations in the design of shipping containers should include protecting against extreme ambienttemperature deviations and maintaining specimen temperature above 22C or preferably near 30C.

* Correspondence: wco3j@virginia.edu1Human Immune Therapy Center, University of Virginia, Charlottesville, VA,USAFull list of author information is available at the end of the article

Olson et al. Journal of Translational Medicine 2011, 9:26http://www.translational-medicine.com/content/9/1/26

2011 Olson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

BackgroundCell-based immunological assays are integral to moni-toring the effects of immunotherapy clinical trials. Themain clinical specimen obtained for these assays iswhole blood collected in heparinized vacutainer tubesfrom which peripheral blood mononuclear cells (PBMC)are isolated. Assays of cellular immune responses toimmune therapy depend on functional and viablePBMC. It is critical that outside factors, other thanstudy parameters, do not introduce significant variabilityin the immune assays due to compromised PBMC integ-rity. Therefore, trials utilizing multiple clinical centerspresent challenges in how to best process and transportwhole blood and tissue samples.The need for specific guidelines for the shipment of

biological specimens is of great concern for the conductof multi-center clinical trails at the national and interna-tional level [1-3]. Both complex processing and delaybefore processing by individual laboratories increase thevariability in specimen performance [4]. In contrast,central laboratory processing lessens the variabilityintroduced by multiple processing protocols but is morecostly and may not be available for all investigators. Ittherefore becomes a critical issue in the design of multi-center clinical trials to determine whether biologicalspecimens should be processed immediately, the sameday, or after shipment to a central laboratory.Early studies have demonstrated how time and tem-

perature of storage affect lymphocyte viability and phe-notype when whole blood is stored overnight at 4C[5-7]. Storage at room temperature prior to processingalso affects viability and blastogenic responses [8] aswell as lymphocyte separation by Ficoll density centrifu-gation [9,10]. The importance of establishing standardshipping parameters has been stressed in the infectiousdisease setting, in which a profound impact of shippingwas noted on the lymphoproliferative responses tomicrobial antigens in both HIV-infected and healthydonors [11,12]. Single cell-based techniques such as ELI-spot assays [13-15], intracellular cytokine staining[16-19], and HLA-specific multimeric assays [20-22] arewidely used and depend on optimal conditions for speci-men handling in order to detect rare populations ofpeptide specific lymphocytes in response to immu-notherapy. Several studies have confirmed that cryopre-served PBMC can be used reliably in these assays[23-26]. Use of cryopreserved samples, however,depends on optimal sample handling before and aftercryopreservation. Some studies have defined optimaltime intervals between venipuncture and cryopreserva-tion [26-29] and optimal conditions for freezing [30].Also, handling and storage of cryopreserved PBMC havebeen evaluated, showing that fluctuations in sub-zerofreezing temperatures can alter the viability and function

of recovered lymphocytes; shipping conditions for frozensamples have also been addressed [31,32]. However, theeffect of ambient temperature changes during shippingor storage prior to cryopreservation has not beenaddressed.It has been suggested that an interval of whole blood

storage exceeding 8 hours (h) causes a significantdecrease in cellular immune function [27]. This findingprovides rationale for immediate isolation and cryopre-servation of PBMC at each participating clinical centerand indeed, optimization of cryopreservation media andof thawing practices has improved recovery of immuno-logical responses at the single cell level [25,30]. How-ever, processing of blood and cryopreservation of PBMCat off-site locations is expensive and requires oversightand quality control of the processing lab at each center.Thus, for many multicenter clinical trials of cancer vac-cines and other therapies, all off-site whole blood speci-mens are shipped to a central laboratory according to astandard operating protocol, and monitored strictly forquality control and quality assurance. Our concern thatshipping whole blood in different seasons, in various cli-mates, may impact PBMC viability and functionprompted this study. Specifically, we have addressed theeffect of shipping temperatures on cell viability, recoveryand function, and have modeled these in vitro whencontrolling for temperature.

MethodsBlood collection, processing and storagePatients blood specimens were derived from partici-pants enrolled in one of three studies. Participants wereenrolled in the clinical studies following informed con-sent, and with Institutional Review Board for HealthSciences Research approval (IRB-HSR# 10598, 10524,and11491) and review by the FDA (BB-IND# 9847 and12191). Patients blood specimens from 2 clinical trials(HSR# 1524(HSR# 10524 and 11491) were monitoredduring a 9 month period from late summer, throughfall, winter and early spring. Two hundred and eighty-five blood specimens collected at participating clinicaltrial centers in Houston, TX and Philadelphia PA, wereshipped to Charlottesville VA. Clinical laboratory ana-lyses, including complete blood counts (CBC) and differ-ential hematological counts, were performed at theindividual centers and the results incorporated into atrial database. An additional 60 ml of blood were col-lected in 10cc heparinized vacutainer tubes (BDBios-ciences, Franklin Lakes, NJ) and were shipped, ininsulated packaging, by overnight courier at ambienttemperature to the Biorepository and Tissue ResearchFacility (BTRF) at the University of Virginia (UVa) forprocessing and cryo-preservation, on the day theyarrived, for future immunological testing in cell-based

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assays. Shipments of patients blood specimens werecontinuously monitored using the TempCheck Sensor(Marathon Products, Inc., San Leandro, CA) to deter-mine the temperature range to which blood sampleswere exposed when shipped overnight by commercialcarrier, and to evaluate the effects of those temperatureson cell yield. Temperature gauges recorded the maxi-mum and minimum temperatures attained inside thepackages during shipment. Blood drawn at UVa wasprocessed either the same day or the following day,depending on when in the day it was drawn. Thevolume of blood collected, and the number of viablePBMC isolated were recorded by the BTRF. Thesevalues were used to determine the cell yield before cryo-preservation. In all cases, the PBMC fraction of wholeblood was collected from Leucosep (Greiner Bio-One,Monroe, NC) tubes following centrifugation for 10 min-utes at 1000 g.The expected cell yield for each sample was calculated

from the CBC and differential tests performed on wholeblood at the originating clinical laboratory. The absolutelymphocyte and absolute monocyte counts calculatedfrom the CBC and differential were combined and mul-tiplied by the volume of blood collected to represent theexpected total PBMC in the blood (expected cell yield).Additional File 1 provides a table of cell count datafrom each center. The table shows the calculated per-centage (mean, median, and quartiles) of lymphocytesand monocytes derived from differential and completecell counts. The number of PBMC isolated by Ficollseparation, divided by the expected cell yield providesthe ratio cell yield. Ratio cell yields of less than 1 areexpected due to losses in Ficoll separation. However,because the Ficoll separations were done by the samecentral laboratory and according to a consistent proto-col, differences in ratio cell yields in different subgroupsof specimens are primarily attributed to effects of ship-ping conditions.

Incubation conditions for whole bloodIn one set of experiments, approximately 7-8 ml wholeblood were collected into each of eleven heparinizedvacutainer tubes from six healthy donors according toIRB protocol 10598 and were labelled to define the tem-perature conditions to which they would be exposed.Each tube was incubated at various temperatures over a24 h period at conditions intended to model what mayhappen in overnight shipping conditions (Additional File2). After a 1-2 h equilibration period at room tempera-ture (RT, 22C), tubes from each sample were placed ineach of the 4 conditions: (a) temperature-controlledrefrigerated centrifuge set at 15C, (b) 22C as a controlcondition, (c) water bath set at 30C, or (d) water bathset at 40C. In addition, one tube was placed in a 50C

water bath for 2 h, but this condition invariably led tohemolysis and the samples were not evaluable. For eachtemperature condition (other than RT), one tubewas exposed to that low or high temperature for 2, 8 or12 h, and then each was returned to RT for the remain-ing 24 h study period. Thus, one tube served as anuntreated control and was at kept at RT for the whole24 h. After these incubations, PBMC were isolated fromeach blood sample by Ficoll density gradient asdescribed above. Viable cell numbers were determinedby trypan dye exclusion. PBMC were cryopreseved infreezing medium (90% FCS, 10% DMSO) overnight at-80C, then transferred to vapor phase liquid nitrogenfor 1-4 weeks before thawing for analysis.

ELIspot AssayCells producing IFNg after antigen specific and non-specific stimulation were enumerated by ELIspot assayas described previously [33,34]. In brief, PBMC werethawed in pre-warmed RPMI1640 (Invitrogen, CarlsbadCA) containing 10% human AB serum (HuAB; Gemini)and 100 Units/mL of DNase I (Worthington Biochem-ical Corp., Lakewood, NJ). Cells were centrifuged at350 g and adjusted to the desired cell density inRPMI 1640 supplemented with 10% HuAB serum andplated into PVDF-membrane plates coated with anti-interferon gamma antibody (Pierce-Endogen, ThermoScientific, Rockford IL). Phytohemagglutinin (PHA),phorbol myristate acetate (PMA and ionomycin wereobtained from Sigma-Aldrich (St. Louis, MO). A pool of35 MHC Class I restricted peptides consisting of pep-tides from cytomegalovirus, Epstein-Barr and influenzavirus proteins (CEF peptide pool; [35]; Anaspec, Fre-mont CA) or media alone were added in quadruplicateand cultures incubated overnight at 37C in a 5% CO2atmosphere. Spots were developed according to standardprotocol and enumerated on a BioReader 4000 (Bio-Sys,Karben, Germany) plate reader.

Flow cytometryCD3, CD4, CD8 and CD56 positive lymphocyte popula-tions were enumerated by flow cytometry using fluores-cent-labelled antibodies (BDBiosciences, San Diego, CA).Cells were washed, suspended in PBS (Invitrogen) con-taining 0.1% BSA (Sigma) and 0.1% sodium azide(Sigma). Titrated amounts of each reagent were added tocells, incubated, washed free of excess stain, and fixed inparaformaldehyde. To determine whether there was anincrease in apoptosis due to different storage conditions,thawed PBMC were incubated overnight at 37C in 5%CO2 in RPMI 1640 + 10% Human AB serum. The nextday, PBMC were surface stained with fluorescentlylabeled antibodies to CD3, CD4, and CD8, then stainedwith Annexin V according to manufacturers instructions

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(BDBioscience, San Diego, CA) and 7-AAD (EMD Che-micals, Inc., Gibbstown, NJ) to determine the level ofapoptosis [36-38]. Cells were acquired on a FACSCaliburflow cytometer maintained by the Flow Cytometry corefacility of the University of Virginia. Data were analyzedwith FlowJo software (Treestar, Ashland OR).

Testing of Blood Shipping PackagesThe standard shipping container used in our clinicaltrials was obtained from Safeguard Technologies Corp.(Conshohocken, PA). It consisted of a white corrugatedbox fitted with a hydrophilic foam-lined clear plasticsnap-lock case inserted into a plastic zip lock bag. Thiswas placed inside a cardboard shipping container linedwith 1 thick Styrofoam. An alternate packaging designwas provided by JVI (Charlottesville VA) and consistedof a 14 11 5 box of 200# corrugated cardboardinsulated with Control Temp Packaging foam of 1thickness. Inside was placed a 12 9 3 clamshelltype clear plastic box containing an 11 8.5 5/8foam vial holder.Each type of shipping container was tested for its abil-

ity to maintain temperature in cold ambient conditions(e.g.: during winter months). Forty heparinized vacutai-ners were filled with water and equilibrated to 37C.Ten vacutainers were placed inside each of 4 packages(2 of each type). Each package type received a gel packconditioned at either 37C or 22C which was thenplaced alongside the vacutainer holder. One probe of anindoor/outdoor thermometer (Taylor Precision Pro-ducts, Oak Brook IL) was placed inside the packagewhile another remained outside to monitor externalambient temperature. Packages were placed either in acold room at 4C for a minimum of 12 h or werehandled in a manner to model the experience of a pack-age being shipped via motor vehicle overnight in a non-heated compartment. Temperatures were recorded every15 minutes during the first hour, and 30-60 minutesthereafter.Additional testing of the JVI packaging material was

performed by R.N.C. Industries Inc. (Norcross GA30071) at high external package temperature. The clam-shell foam holder containing vials of liquid was placedinside the package. Two 12 oz Control Temp gel packsconditioned at 20C were placed in the clamshell ontowhich the foam vial holder (including the 1/4 foamabove and below) containing five 5/8 vials filled withwater conditioned at 20C was placed inside. The pack-age was closed, put at 45C and the internal packagetemperature was monitored for 48 hours using anOmega OMB-DAQ-55 USB data acquisition system,serial number #156772. T thermocouples were cali-brated 2 months earlier using a stirred water bathcalibration.

Statistical analysisThe MIXED procedure in SAS 9.1.3 (SAS Institute,Cary, NC) was used to analyze the effects of tempera-ture (3 levels) and duration (3 levels) on outcomesincluding ELIspot, phenotype, and viability. These effectswere modeled jointly (main effects plus interactions) foreach outcome measure and outcome measurementswere first normalized by division of the raw data by thedonor value at RT for 24 h. Since donors served asblocks and contributed an observation from each condi-tion (i.e. each combination of temperature and durationlevel), intra-donor correlation was modeled assuming acompound symmetry structure in the residual covar-iance matrix. Degrees of freedom were calculated usingthe Kenward-Roger method. To assess the effects of sto-rage under different temperature conditions on apopto-sis among CD4 and CD8 populations, a modelingscheme similar to the one above was performed usingcalculated logits as the outcome measure. This isdefined as the loge([pi/1-pi]/[pc/1-pc]) where p = theproportion of cells that are apoptotic or necrotic (asdefined by Annexin V and 7AAD staining); i = the sto-rage conditions of the whole blood specimen; and c =the storage condition of the control specimen at RT for24 hours. All tests were assessed at a = 0.05.

ResultsEffect of shipping temperatures and extreme changes intemperature on the cell yield for clinical trial specimensPackage temperatures were lowest in winter months andhighest in summer months, suggesting that the tempera-tures experienced during shipping varied by ambientseasonal temperatures (Figure 1A). The extreme tem-peratures ranged from about -1C to 35C with 91% fall-ing completely within the range of 4C and 32C.There was a trend to lower PBMC yields in colder

months from November through February (Figure 1B),although outliers were noted. Lower minimum temperaturewas associated with lower cell yield (p = 0.001, Figure 2A),whereas higher maximum temperature correlated withhigher cell yield (p = 0.04, Figure 2B). The range in shippingtemperatures during the winter was typically bounded by ahigh temperature of 22C, and during the warmer monthsby 22C as a low temperature. The maximum change(deviation) in temperature from 22C observed during ship-ment was determined using the high or low temperaturefurthest from 22C. This represents an estimate of thedegree of temperature fluctuation encountered during ship-ment and is plotted against the yield in Figure 2C, wherethere was a correlation with warmer temperatures (p