B L O O D C O M P O N E N T S

Evaluation of the automated collection and extended storage ofapheresis platelets in additive solution_3314 503..509

Lacey Johnson, Kelly M. Winter, Tanja Hartkopf-Theis, Samantha Reid, Matthew Kwok,

and Denese C. Marks

BACKGROUND: Collecting apheresis platelets (PLTs)into additive solution has many potential benefits. Thenew Trima software (Version 6.0, CaridianBCT) allowsautomated addition of PLT additive solution (PAS) aftercollection, compared to Trima Version 5.1, which onlycollects PLTs into plasma. The aim of this study was tocompare PLT quality during extended storage, aftercollection with the different Trima systems.STUDY DESIGN AND METHODS: Apheresis PLTswere collected using both Trima Accel apheresissystems. The test PLT units (n = 12) were collectedusing the new Trima Version 6.0 into PLT AS (PAS-IIIM), while the control units (n = 8) were collected intoautologous plasma using Trima Version 5.1. All unitswere stored for 9 days, and in vitro cell quality variableswere evaluated during this time.RESULTS: PLTs collected in PAS-IIIM maintained astable pH between 7.2 and 7.4, whereas plasma-storedapheresis units exhibited significantly increased acidityduring storage, due to lactate accumulation and bicar-bonate exhaustion. Plasma-stored PLTs also demon-strated a more rapid consumption of glucose. However,there was little difference in PLT activation or cytokinesecretion between PAS-IIIM and control PLTs.CONCLUSION: These data indicate that apheresis PLTconcentrates collected in PAS-IIIM, using Trima Version6.0 software, maintained acceptable PLT metabolic andcellular characteristics until Day 9 of storage.

In Australia, buffy coat–derived pooled platelets(PLTs) are currently prepared in approximately 70%additive solution (PAS-IIIM, SSP+), whereas apher-esis PLTs are collected and stored in 100% plasma.

Collection of apheresis PLTs into PLT additive solution(PAS) would allow standardization of PLT production inAustralia, as well as additional benefits. First, the use ofPAS allows more plasma to be recovered for fractionationand clinical use. Reducing the amount of plasma in a PLTcomponent also has the potential to reduce the numberof transfusion reactions, particularly allergic transfusionreactions and TRALI, where plasma proteins have beenimplicated.1,2 Further, PLTs in PAS are compatible withpathogen reduction technology (PRT).3

PAS-IIIM has been shown to be an effective substi-tute for plasma in both apheresis and buffy coat PLTconcentrates stored for up to 12 days.4-6 PAS-IIIM is athird-generation PAS containing acetate, a substratefor oxidative phosphorylation,7 phosphate to provideimproved buffering capacity as well as potassium, citrate,and magnesium.8 The composition of PAS-IIIM used inthis study is shown in Table 1. Storage of PLTs in this addi-tive has been shown to reduce the rate of glycolysis,leading to better retention of pH and hypotonic shockresponse (HSR) reactivity.9

ABBREVIATIONS: HSR = hypotonic shock response;

PAS(s) = platelet additive solution(s); PLTs = platelet;

PRT = pathogen reduction technology.

From Research and Development, Australian Red Cross Blood

Service, Sydney, Australia.

Address reprint requests to: Denese C. Marks, Applied and

Developmental Research, Research and Development, Austra-

lian Red Cross Blood Service, 17 O’Riordan Street, Alexandria,

NSW, 2015, Australia; e-mail: dmarks@redcrossblood.org.au.

The Australian governments fully fund the Australian Red

Cross Blood Service.

Received for publication November 16, 2010; revision

received July 4, 2011; and accepted July 6, 2011.

doi: 10.1111/j.1537-2995.2011.03314.x

TRANSFUSION 2012;52:503-509.

Volume 52, March 2012 TRANSFUSION 503

Currently, the Australian Red Cross Blood Service col-lects apheresis PLTs into autologous plasma, using theTrima Accel system with Version 5.1 software (Caridian-BCT, Lakewood, CO). The production of apheresis PLTs inadditive is possible by collection of hyperconcentratedPLT units with manual addition of PAS4,5,10 or the use ofalternate collection devices.11 The development of TrimaVersion 6.0 allows the collection of hyperconcentratedPLTs with automated addition of PAS at the end of collec-tion. Further, due to the addition of PAS, a larger volume ofplasma can be concurrently collected. The modificationsfrom Version 5.1 to 6.0 involve a software upgrade andalterations to the collection chamber (skimmer dam),which “scoops” up the PLTs and is thought to reducevariability in the collection process. In addition, TrimaVersion 6.0 also offers a new donor qualification algo-rithm, which may allow more donors to be eligible forplateletpheresis, particularly for a double collection,thereby improving productivity. How these changes to thecollection process affect PLT quality during storage areunknown.

The aim of this study was to assess and compare thein vitro quality of PLTs collected in PAS-IIIM, with TrimaVersion 6.0, to our standard apheresis PLTs currently col-lected in 100% plasma, for an extended storage period of9 days.

MATERIALS AND METHODS

PLT collection, storage, and samplingAll donors participating in this study met the AustralianRed Cross Blood Service “Guidelines for the Selection ofBlood Donors.” For collections using Version 6.0 software,donors were informed of the nature of the evaluation andgave written consent. This study was approved by the Aus-tralian Red Cross Blood Service Human Research EthicsCommittee before commencement.

Apheresis PLTs were collected into plasma and/or PAS(PAS-IIIM; SSP+; MacoPharma, Mouvaux, France) usingTrima Accel apheresis system (CaridianBCT) runningVersion 6.0 software (n = 12). A set volume of PAS-IIIM wasdelivered automatically at the end of the PLT donation.Control PLTs were collected into 100% plasma using aTrima Accel apheresis system running Version 5.1 software(n = 8), because this is the current process for apheresis

PLT collection at the blood service. The whole blood-to-citrate ratio was 10:1 for Trima 6.0 collections and 9:1for the control units. The target concentration was1600 ¥ 109/L PLTs for both collections. Contaminatingwhite blood cells were removed from all units by the leu-koreduction system of the cell separator. Plasma was col-lected concurrently during both collection types.

All PLTs were stored in bags integrated with the TrimaAccel collection kits (CaridianBCT) for 9 days at 20 to 24°Con a flat-bed shaker (Helmer, Inc., Noblesville, IN). Singleand double products were collected in both groups. Alldouble components were split into two therapeutic doses,and 1 unit was selected at random for sampling through-out the testing period.

Samples were taken from PLT components understerile conditions using a plasma transfer set (CharterMedical, Winston-Salem, NC) 2 hours after collection (Day0) and on Days 2, 5, 7, and 9 after collection. Approxi-mately 10 mL was removed on Day 0, and 5 mL on theremaining days of sampling.

Laboratory testingThroughout storage, the products were weighed and unitvolumes were calculated by dividing the unit weight by thespecific gravity of 1.025 for PLTs in plasma and 1.01 forPLTs in PAS-IIIM. To ensure sterility, a 5-mL sample, takenon Day 0, was inoculated into both aerobic (BPA) andanaerobic (BPN) bottles of a bacterial detection instru-ment (BacT/ALERT, bioMérieux, Durham, NC). Plasmacarryover was determined on Day 0, from the proteincontent of the supernatant, which was measured usingthe BCA protein assay kit according to the manufacturer’sprotocol (Pierce Biotechnology, Rockville, IL). Superna-tants from PLT concentrates were prepared by double cen-trifugation at 1600 ¥ g for 20 minutes and then 12,000 ¥ gfor 5 minutes at room temperature and frozen at -80°C.

The PLT concentration was determined using ahematology analyzer (CellDyn 3200, Abbott Diagnostics,Sydney, Australia). PLT swirl was determined to be presentor absent by visual inspection.12

The pO2, pCO2, pH, lactate, and glucose content ofPLT components were measured using a blood gas ana-lyzer, in conjunction with the CG4+ and glucose cartridges(iSTAT, Abbott Diagnostics, Sydney, Australia). The pH,pO2 and pCO2 were measured at 37°C, and bicarbonatewas automatically calculated by the iSTAT from the pHand pCO2 values. The pH was subsequently converted topH22°C using the following formula

pH 22 C 37 C37 C° − × ° − °[ ]( )0 015. .

Rates of glucose consumption and lactate generation werecalculated from iSTAT measurements and normalized to1012 PLTs per hour. Acetate concentration was measured

TABLE 1. Composition of PAS-IIIM (mmol/L)Na3-citrate 2H2O 12.77Na-acetate 3H2O 32.48NaH2PO4 2H2O 6.73Na2HPO4 21.49KCI 4.90MgCI2 6H2O 1.48NaCl 69.29pH 7.2

JOHNSON ET AL.

504 TRANSFUSION Volume 52, March 2012

using the acetic acid kit (acetate kinase format), accordingto the manufacturer’s instructions (Megazyme Interna-tional Ireland Ltd, Wicklow, Ireland). HSR was measuredusing an aggregometer (Helena Laboratories, Beaumont,TX). PLTs were diluted in ABO/RhD-matched fresh-frozenplasma, as described by VandenBroeke and colleagues.13

PLT viability was determined using two fluorescentdyes, calcein-AM and FM4-64, as previously described.14

The cells were analyzed by flow cytometry (FACSCanto II,Becton Dickinson, Franklin Lakes, NJ) within 1 hourof staining. Changes in PLT mitochondrial transmem-brane potential (Dy) were determined using the lipo-philic cationic fluorochrome 5,5″,6,6″-tetrachloro-1,1″,3,3″-tetraethylbenzimidazolylcarbocyanine iodide (JC-1;Biotium Inc., Hayward, CA).15

For measurement of phosphatidylserine exposure,PLTs (1 ¥ 106) were diluted in 100 mL of annexin V bindingbuffer and stained with 5 mL of annexin V-fluoresceinisothiocyanate (FITC; Biolegend, San Diego, CA). Thetubes were incubated for 15 minutes in the dark at roomtemperature. The cells were then diluted and analyzed byflow cytometry.

PLT glycoproteins were assessed by staining PLTs(3 ¥ 106) in Tyrode’s buffer for 20 minutes at room tem-perature with the following antibodies: anti-humanCD62P-phycoerythrin (PE; BD Biosciences, San Diego,CA), anti-human CD63-PE (Biolegend), anti-humanCD41a-FITC (BD Biosciences), anti-human CD61-FITC(Dako, Glostrup, Denmark), anti-human CD42b-PE (BDBiosciences), and anti-human CD47-PE (Biolegend). Anti-IgG1k-PE and anti-IgG1k-FITC were used as isotype con-trols (Biolegend). Samples were then washed, fixed, andanalyzed by flow cytometry.

Cytokines were measured from the supernatants ofPLT concentrates, prepared as described above. Commer-cially available enzyme-linked immunosorbent assay kitsfor CD40L, PLT factor 4 (PF4), PLT-derived growth factor(PDGF)-AB, CD62P (soluble P-selectin), RANTES, trans-forming growth factor (TGF)-b1, and epidermal growthfactor were used according to the manufacturer’s instruc-tions (R&D Systems Inc., Minneapolis, MN). All sampleswere tested in triplicate.

Statistical analysisData were analyzed using statistical software (SPSS,Version 17.0, SPSS, Inc., Chicago, IL). The mean values andstandard deviation (SD; n = 8 for plasma, n = 12 for PAS-IIIM) are presented. A mixed linear model was used todetermine whether there was a significant differencebetween PLTs stored in PAS-IIIM compared to plasma,across the entire storage period. The dependent variablewas the parameter being measured, and the fixed effectsof storage time and PLT storage medium (plasma or PAS-IIIM) were examined. Where a significant difference was

observed using this model (p < 0.05), further post hoccomparisons of PAS-IIIM versus plasma were performedat each sampling time, using an unpaired two-sided t testto determine where differences occurred during storage.

RESULTS

PLT componentsThe actual PLT product variables did not significantlydiffer between Trima Version 5.1 and Version 6.0 collec-tions. PLT volumes were 185.87 � 23.18 mL for plasmacontrols and 208.67 � 30.30 mL for Trima Version 6.0 aftercollection (p = 0.109). Plateletpheresis with Version 6.0allowed higher volumes of concurrent plasma to becollected (559.82 � 55.35 mL vs. 384.40 � 100.39 mL;p < 0.0001). The PLT concentration was similar betweencollection modalities and throughout storage (Table 2,p = 0.323). Importantly, all units were above the minimumrequirements of more than 200 ¥ 109/unit, set by theCouncil of Europe.16 The mean percentage of plasma car-ryover in the PLTs stored in PAS-IIIM and plasma was48.41 � 3.05% and 90.01 � 7.47%, respectively. Swirlingwas preserved during the storage period in all units, andall components tested negative for bacterial contamina-tion using the BacT/ALERT system.

PLT viabilityPLT viability remained high in both PLT groups through-out storage. There was no difference in the percentageof viable cells (calcein-AM positive) throughout storagewhen PLTs were stored in plasma or PAS-IIIM (Fig. 1,p = 0.659). Similarly, annexin V binding increased gradu-ally during storage of both PLT units, but was not signifi-cantly different between plasma or PAS-IIIM units (Fig. 1,p = 0.428).

PLT metabolismThe pH of PAS-IIIM units was maintained within anarrower range than the plasma-stored PLTs and alsoremained significantly higher from Day 5 of storage(Table 2, p < 0.0001). Further, the pH was maintainedwell above the Council of Europe recommendations(pH � 6.4) in all units.16

Glucose was present at the end of storage in bothcomponent types. Although the plasma-stored PLTs had asignificantly higher starting glucose concentration thanPLTs in PAS-IIIM (Table 2, p = 0.028), the rate of glucoseconsumption by PAS-IIIM PLTs was significantly lower.Consequently, total lactate production and the lactateproduction rate in control PLTs were significantly higherthan PAS-IIIM on Days 2, 5, and 7 (Table 2, p < 0.0001 andp = 0.006, respectively). The lactate concentration in some

EXTENDED STORAGE OF APHERESIS PLTs IN PAS-IIIM

Volume 52, March 2012 TRANSFUSION 505

of the control PLTs was above the upper limit(>20 mmol/L) of detection for the blood gas analyzer onDay 9, so could not be accurately measured. There wasalso a decline in bicarbonate concentration in controlPLTs from Day 2 (Table 2), and bicarbonate was undetect-able in 50% of the control units by Day 9. As expected, theacetate concentration was significantly higher in PAS-IIIMunits, compared to PLTs in plasma, which had a very lowacetate level. Further, in PAS-IIIM units, the acetateconcentration decreased at a constant rate throughoutstorage (Table 2).

All units exhibited a storage time–dependent increasein pO2 and decline in pCO2, although these changes wereless pronounced in the PAS-stored PLTs (Table 2). Thetransmembrane mitochondrial potential is essential formaintenance of oxidative phosphorylation,17 and therewas no difference in the percentage of PLTs with pola-rized mitochondria when stored in plasma or PAS-IIIM(Table 2, p = 0.633). Similarly, there was no significant dif-ference in the HSR of PAS-IIIM or plasma-stored PLTs(Table 2, p = 0.474).

PLT activationDuring storage, cell surface CD62P levels graduallyincreased in the plasma-stored PLTs, but were only signifi-cantly different on Day 9 (Fig. 2, p = 0.047). In contrast,CD62P expression in PLTs in the PAS-IIIM group washigher to begin with (p = 0.01), but did not increasethroughout storage (Fig. 2). The levels of CD42b (GPIba),CD41a (GPIIb), CD61, CD63, and CD47 on the PLT surface

TAB

LE

2.M

etab

olic

acti

vity

of

PLT

sco

llect

edin

PAS

-IIIM

or

pla

sma*

Sto

rage

time

and

trea

tmen

tP

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unt

(¥10

9 /L)

pH(2

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se(m

mol

/L)

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cose

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umpt

ion

Lact

ate

(mm

ol/L

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ctat

epr

oduc

tion

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arbo

nate

(mm

ol/L

)A

ceta

te(m

mol

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pO2

(mm

Hg)

pCO

2

(mm

Hg)

JC-1

pola

rized

(%)

HS

R(%

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sma

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Fig. 1. PLTs stored in PAS-IIIM ( ) have similar viability and

phosphatidylserine expression as plasma-stored PLTs (�).

PLTs were stained with calcein-AM and annexin V-FITC and

analyzed by flow cytometry. The data represent mean � SD

(error bars).

JOHNSON ET AL.

506 TRANSFUSION Volume 52, March 2012

were also examined and found to be comparable betweenthe control and PAS-IIIM units (data not shown).

The levels of PLT-derived cytokines were measured inthe PLT supernatants during storage (Table 3). There wasno significant difference in the level of any secreted cyto-kines measured, with the exception of RANTES, betweenplasma and PAS-stored PLTs. The concentration ofRANTES was initially higher in PAS-IIIM units, but equal-ized with plasma units by Day 2 of storage.

DISCUSSION

This is the first published study of the in vitro quality ofapheresis PLTs collected using the Trima Accel Version 6.0system. In this study, apheresis PLTs were collected andstored in PAS-IIIM, and the in vitro quality of PLT compo-nents was compared to standard apheresis PLT collectionsin plasma. The results demonstrate little difference in PLTviability and activation during storage in PAS-IIIM, whencompared to plasma. However, the metabolic variablesof pH, bicarbonate, acetate, glucose consumption, andlactate production were significantly different in PAS-stored PLTs during storage for 9 days.

There is a trend in Australia toward using single-donor apheresis PLTs rather than whole blood–derivedpooled PLTs, which carry the additional risks of exposingthe recipient to blood products from multiple donors aswell as increased risk of bacterial contamination.18 Theability to provide a standardized product with a reducedplasma content is therefore of importance. Furthermore,with the growing demand for plasma required for bothfractionation and clinical use, the ability to safely obtainmore plasma from each donation could be of great

Fig. 2. PLT CD62P expression during storage when stored in

PAS-IIIM ( ) or plasma (�). The cells were stained with the

CD62P-PE and analyzed by flow cytometry. The data represent

mean � SD (error bars). *p < 0.05.

TAB

LE

3.C

yto

kin

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nce

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atio

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PLT

con

cen

trat

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ng

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rag

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or

pla

sma*

Sto

rage

time

and

trea

tmen

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L(p

g/m

L)P

F4

(mg/

mL)

PD

GF

-AB

(ng/

mL)

Sol

uble

CD

62P

(ng/

mL)

RA

NT

ES

(ng/

mL)

TG

F-b

(ng/

mL)

EG

F(p

g/m

L)

Day

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a79

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1.56

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5.26

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9.23

4.97

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EXTENDED STORAGE OF APHERESIS PLTs IN PAS-IIIM

Volume 52, March 2012 TRANSFUSION 507

benefit. Collection of apheresis PLTs in PAS using TrimaVersion 6.0 could therefore have a great impact onmeeting these needs.

To collect a product with Version 6.0 that was equiva-lent in volume and final PLT concentration (1600 ¥ 1011/unit) to our current product, with the addition of PAS-IIIM, a minimum plasma carryover of 48% was necessary.Adjustments are being assessed to allow an increased ratioof PAS to plasma, as is typically seen with apheresis PLTstorage in PAS.11,19

PAS-stored PLTs showed improved maintenance ofpH, combined with a reduction in glucose consumptionand lactate production. This may be attributed to theacetate found in PAS-IIIM, which provides an alternatesource for energy production,20 as demonstrated by thedecrease in acetate concentration in PAS-IIIM PLTs duringstorage. Several studies have shown PAS-IIIM improves invitro PLT variables.9,10,21 In this study, glucose was con-sumed more rapidly in plasma-stored PLTs. Consequently,lactate was produced at a greater rate and bicarbonatewas exhausted, leading to the decrease in pH seen in theplasma-stored PLTs. The pH of PLTs in PAS-IIIM remainedvery stable throughout the entire storage period, due tothe improved buffering capacity of PAS-IIIM.7 Similar pH,lactate, and glucose results have recently been reported inPAS-IIIM–stored apheresis PLTs,22 although the PLT con-centrations reported in our study were higher than previ-ously published. Improvements to these variables may beof clinical importance as both pH and lactate productionin vitro have been correlated with better in vivo PLT recov-ery and survival using autologous PLT transfusions.23

The PLTs were assessed until Day 9 of storage,because previous data suggest that metabolic variablesare only adequately maintained in PAS-IIIM until thistime.6 Importantly, our data demonstrate alterations inmetabolic variables in plasma versus PAS-IIIM PLTs byDay 5 of storage, which is the current shelf life of PLTs inAustralia.

There was very little difference in PLT activationduring storage between the PLTs collected with eitherTrima system. However, the percentage of PLTs expressingCD62P was initially higher in the units collected into PAS-IIIM than plasma, which is similar to previous reports.4

There was also a higher concentration of RANTES presentin the supernatant of PAS-IIIM units on Day 0. Becauseboth CD62P and RANTES release are associated with PLTactivation,10,24 these results suggest that the new collectionprocess may result in slight PLT activation, which thennormalizes by Day 2. Further to this, there was no signifi-cant difference in the expression of other markers ofactivation throughout storage, including CD62P, CD63,phosphatidylserine, and cytokine release, between con-trols and PAS-IIIM PLT units. These results are in contrastto other reports, which suggested higher activation occursin PLTs stored in PAS.11,24 However, it is well established

that different apheresis systems result in variations in PLTquality.25,26 In addition, the minimal PLT activation seen inthis study may be due to the higher plasma carryover,compared to other studies.11,24

In summary, the data presented here indicate thatapheresis PLT concentrates collected and stored in PAS-IIIM, using Trima Version 6.0 software, have similar invitro activation characteristics as PLTs collected andstored in 100% plasma on Day 5 of storage. However, aph-eresis PLTs collected and stored in PAS-IIIM showed lessdeterioration in metabolic variables when stored for 9days. These results suggest that extension of the storagetime may be attainable, if problems with bacterial con-tamination were appropriately addressed, such as withPRT. However, in vivo studies are still required to deter-mine whether apheresis PLTs stored in PAS-IIIM improvetransfusion efficacy.

ACKNOWLEDGMENTS

We acknowledge Di Duggen and Alice North, apheresis nurses at

Elizabeth Street Donor Centre, for organizing and booking suit-

able platelet donors and for their care in making sure the platelets

were collected and packed in time for our assays. We also thank

Ron Halse, Donor Centre Manager at Elizabeth Street, and Rod

Jones, Donor Services Manager, for accommodating this research

study. We acknowledge the Australian governments that fully

fund the Australian Red Cross Blood Service for the provision of

blood products and services to the Australian community.

CONFLICT OF INTEREST

The authors have no conflict of interest to disclose.

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