Avoiding room temperature storage and delayed cryopreservation provide better postthaw potency in...

T R A N S P L A N T A T I O N A N D C E L L U L A R E N G I N E E R I N G

Avoiding room temperature storage and delayedcryopreservation provide better postthaw potency in

hematopoietic progenitor cell grafts

Laura Jayne Fry, Sergio Querol Giner, Susana G. Gomez, Melanie Green, Sally Anderson,

Jackie Horder, Stephanie McArdle, Robert Rees, and Jose Alejandro Madrigal

BACKGROUND: Conditions for maintaining hematopoi-etic progenitor cells (HPCs) before cryopreservationremain controversial. An understanding of the impact oftime and temperature during nonfrozen storage cancontribute to the maintenance of the quality of products,improving transplantation outcomes. The objective ofthis study was to determine the influence on cellpotency of thawed products from three sources ofHPCs after prolonged storage at different temperaturesbefore cryopreservation.STUDY DESIGN AND METHODS: Viable cell countsby flow cytometry and colony-forming unit (CFU) recov-eries were assessed on cord blood (CB), mobilizedperipheral blood stem cell (PBSC), and bone marrow(BM) samples over 72 hours using two different storageconditions, refrigerated (4-8°C) or room temperature(19-22°C). To determine the effects of delayed freezingon progenitor recoveries, paired samples were evalu-ated before and after cryopreservation.RESULTS: All samples maintained at refrigerated tem-peratures resulted in higher recoveries than those atroom temperature in all variables assessed. Specifically,when assessing for CFU yields after thawing, theimpact of time on BM resulted in a significant loss assoon as 24 hours (n = 10, 36.4 � 28.0%, p = 0.003).This decrease was also observed for PBSCs and CBbut at 48 hours of fresh storage (PBSCs n = 11,32.7 � 26.2%, p = 0.006; CB n = 10, 39.6 � 26.4%,p = 0.001).CONCLUSION: Our data suggest that HPC productsare better maintained at refrigerated temperaturesbefore cryopreservation. Delaying cryopreservationshould be minimized to avoid significant losses in cellpotency.

Hematopoietic progenitor cell (HPC) trans-plants are routinely used to restore thehematopoietic system after high-dose che-motherapy.1,2 HPCs can be obtained from

three different sources: bone marrow (BM), mobilizedperipheral blood stem cells (PBSCs), and umbilical cordblood (CB). HPC grafts can be infused after a short storageperiod after collection, although more frequently they arecryopreserved, such as in autologous transplantation andsome cases of some allogeneic procedures particularlywhen CB samples are used.

A delay between harvest of the cells and their cryo-preservation is becoming usual as processing laboratoriescan be some distance from the collection centers. Jansenand colleagues3 reported that the median transport timefor HPC products collected in Europe to the UnitedStates is 37 hours (range, 19-50 hr). Worryingly it has alsobeen reported that PBSC and BM products with anincreased transport time can lead to slower platelet (PLT)

ABBREVIATIONS: 4°C = refrigerated temperature (4-8°C);

20°C = room temperature (19-22°C); 7AAD = 7-

aminoactinomycin D; BM = bone marrow; CB = cord blood;

PBSC(s) = mobilized peripheral blood stem cell(s); t = 0 = Time

Point 0.

From the Anthony Nolan Cell Therapy Centre and the John van

Geest Cancer Research Centre, Nottingham Trent University,

Nottingham, UK; Banc de Sang i Teixits, Barcelona, Spain; City

Hospital, Nottingham, UK; University College Hospital, London,

UK; and the Anthony Nolan Research Institute, Royal Free

Hospital, London, UK.

Address reprint requests to: Laura Jayne Fry, Anthony Nolan

Cell Therapy Centre, Nottingham Trent University, Clifton

Campus, Clifton Lane, Nottingham NG11 8NS, UK; e-mail:

[email protected].

Received for publication May 31, 2012; revision received

September 27, 2012, and accepted October 4, 2012.

doi: 10.1111/trf.12006

TRANSFUSION 2013;53:1834-1842.

1834 TRANSFUSION Volume 53, August 2013

engraftment,4,5 suggesting a potential impact of the con-servation conditions on the transplantation outcomes.Optimal fresh storage conditions have not as yet beenestablished and the current literature remains inconclu-sive; according to current literature CB, PBSCs, and BM allhave optimal storage temperatures of either refrigeratedtemperature (4-8°C [4°C]) or 25°C depending on whichgroup conducted the study and over what time period.6-27

Quality variables used when assessing the potencyof these stem cell products usually consist of identify-ing the numbers of viable cells assessed using7-aminoactinomycin D (7AAD) and numbers of colony-forming units (CFUs). In our study we focus on CFUs afterthaw as a main graft potency surrogate because this hasbeen found to be the best current predictive marker ofengraftment with a minimum of 12.0 ¥ 104 CFU-GM/kgrequired for rapid neutrophil and PLT engraftment.28-30 Nostudies have evaluated all three sources of HPCs in thesame laboratory with the same quality variables. This isbecoming particularly important when more centralizedfacilities for cell processing are being developed.

This study investigates the effect of temperature onthe fresh storage of CB, BM, and PBSCs, to determine themaximum time these cells can be stored before losingpotency and to determine the effects that different storageconditions have on the ability to freeze and thaw the cells.

MATERIALS AND METHODS

Sample collectionThis study was approved by the Trent Ethics Committeeand authorized by the Nottingham University HospitalR&D office. After informed consent of the donor wasobtained, umbilical CB was collected by trained staff atKing’s College Hospital, London into CB collection bagscontaining 25 mL CPD (Grifols, Barcelona, Spain). Elevenmobilized peripheral blood samples (nine autologous andtwo allogeneic) were collected from consented patients atCity Hospital, Nottingham. A 1-mL volume of blood fromeach donor was diluted to 10 mL with autologous plasmadue to ethical reasons. Ten undiluted BM samples werecollected for this study (one autologous and nine alloge-neic), four from City Hospital, Nottingham and six fromUniversity College Hospital, London; the anticoagulantused was heparin. After collection immediate transportwas arranged using a cooled environment (4-15°C). Allsamples were received by the Anthony Nolan Cell TherapyCentre within 4 hours of collection.

Fresh storage, cryopreservation, andthawing protocolSamples were split into seven 1-mL aliquots, one of whichwas assessed immediately upon arrival as the base line orTime Point 0 (t = 0) and then immediately cryopreserved.

The remaining six aliquots were split and stored with threeat room temperature (19-22°C [20°C]) and three at 4°C. Atset time points (24, 48, and 72 hr from t = 0), an aliquotwas taken from each storage condition and assessed, andthe remaining sample was cryopreserved.

A cryopreservation medium was added to the cellsresulting in a final concentration of 10% DMSO and 1%dextran (Origen Biomedical, Helsingborg, Sweden). Thecells were frozen overnight in a freezing container(Nalgene Mr. Frosty, Thermo Fisher Scientific, Leicester-shire, UK) in a -80°C freezer before being transferred tothe vapor phase of liquid nitrogen tanks and stored for aminimum of 24 hours.

The aliquots were thawed rapidly in a water bath at37°C and an equal volume of thaw medium (7.5%dextran 40, 5% AB serum [Lonza, Basel, Switzerland],5 mmol/L MgCl2 [BDH, VWR International Ltd, Lutter-worth, UK], 0.63% sodium citrate [BDH], 1000 IU/mLDNase-1 [Calbiochem, Merck Chemicals Ltd, Nottingham,UK]) was added dropwise followed by culture medium(RPMI, 2.5% AB serum, 500 mmol/L l-glutamine) toachieve a final dilution of 1:5. A schematic representationof the experimental protocol can be found in Fig. 1.

Cell enumeration and viability assaysCD45+ and CD34+ cell enumeration was assessed basedon the International Society of Hematotherapy and GraftEngineering (ISHAGE) guidelines.31 A total of 0.6 ¥ 106

cells were stained with anti-CD45-fluorescein isothiocy-anate and CD34-phycoerythrin–conjugated antibodies(both BD Biosciences, Oxford, UK) and treated with FACSlysing solution (BD Pharm Lyse, BD Biosciences) for 15minutes in the dark. Viability was assessed using 7AADand count beads (Perfect, Cytognos, Salamanca, Spain)for enumeration; 2000 bead events were analyzed foreach sample. Flow cytometry was performed on a flowcytometer (either BD FACSCalibur or FACSCanto, both BD

Collection of 10-mL sample

(CB, BM, or PBSCs)

Split sample into 7 × 1-mL

aliquots

1-mL aliquot

Baseline analysis (t = 0)

and cryopreservation with

10% DMSO 1% dextran

3 x 1-mL aliquots

Fresh storage at

refrigerated temperature

(4-8°C ) (4°C)

3 x 1-mL aliquots

Fresh storage at room

temperature

(19-22°C ) (20°C)

Fresh analysis of aliquots

and cryopreservation at

set time points (24, 48,

and 72 hours after t = 0)

Thawing of aliquots

and postthaw analysis

Fig. 1. Schematic representation of the experimental protocol.

OPTIMAL PROGENITOR CELL STORAGE BEFORE CRYOPRESERVATION

Volume 53, August 2013 TRANSFUSION 1835

Biosciences) and analyzed using the accompanying soft-ware (CellQuest Pro, BD Biosciences). The gating strategyfor viable cell enumeration is shown in Fig. 2.

Hematopoietic progenitor functional assayCFU assay was performed using methylcellulose-basedmedium (Methocult GF H84434, Stem Cell Technologies,Vancouver, British Columbia, Canada). After flow cyto-metry analysis, a total of 450 CD34+ cells were seeded into3 mL of Methocult and the sample volume was made up to300 mL with Iscove’s modified Dulbecco’s medium (StemCell Technologies). After thorough mixing, 1.1 mL wasplated in duplicate. After 14 days’ incubation at 37°Cin humidified air and 5% CO2, CFU colonies were scoredby a single operator by examination under an invertedmicroscope.

Statistical analysisResults are expressed as mean � standard deviation (SD)and ranges. Graphs represent the mean of the normalizeddata � standard error. The data points were normalizedagainst the baseline (t = 0) for each sample due to largevariations in cell counts between samples, these valuestherefore represent actual cell yield compared to baselineat each specific testing point. Comparisons of cell countsat different time points and temperatures were performedusing the t test and p values of less than 0.05 were consid-ered to be significant.

RESULTS

Ten CB, 11 PBSC, and 10 BM samples were used in thisstudy. The mean viable cell counts assessed by flow cytom-etry and CFUs after collection (t = 0) are shown in Table 1,and the mean viable cell counts after cryopreservation at

Fig. 2. Gating strategy used to identify viable CD45+, CD34+, granulocytes, and MNCs.

FRY ET AL.


t = 0 are shown in Table 2. At the time of collection thecell concentration varied between samples and was12.93 ¥ 1012 � 7.02 ¥ 1012/L for CB, 20.88 ¥ 1012 �

6.25 ¥ 1012/L for PBSCs, and 29.70 ¥ 1012 � 8.86 ¥ 1012/Lfor BM. The viability of the samples, assessed using7AAD, gave viabilities of 95.67 � 8.48%, 96.74 � 3.78%,and 94.63 � 3.07% for CB, PBSC, and BM samples,respectively.

The effect of storage temperature and time onCD45+7AAD– cellsTo elucidate the optimum storage condition for the threesources of HPCs, numbers of viable CD45+ cells assessedby 7AAD were monitored and the effects of the two tem-peratures compared. The results are shown in Fig. 3.

In CB the CD45+7AAD– cell count shows a significantdifference between the two storage temperatures after72 hours with recoveries of 86.78 � 11.64% at 20°C and94.14 � 9.57% at 4°C (p = 0.0004). This significant declineat 72 hours was also observed in viable granulocytes(p = 0.02) and mononuclear cells (MNCs; p = 0.0008)identified by flow cytometry through CD45 expressionand side scatter (data not shown). This gradual decreasewas not reflected in the postthaw samples without anydifferences seen over the time period.

The mean PBSC CD45+7AAD– cell count also showeda difference between the two storage temperatures after

72 hours (p = 0.03). Postthaw cellsstored at 20°C for 24 hours before cryo-preservation had a higher CD45+7AAD–recovery (p = 0.009); however, after 48hours, cells stored at 4°C had the higherrecovery over 20°C and became signifi-cantly greater after 72 hours (p = 0.02).

Although no significant differencewas identified in BM CD45+7AAD– cells,a trend toward higher recoveries afterstorage at 4°C was observed in freshsamples, with a recovery of 77.55 �

15.62% at 20°C and 97.24 � 40.65% at4°C (p = 0.24).

All three sample types maintainedat 4°C in fresh storage and before cryo-preservation resulted in higher CD45+7AAD– recoveries after thaw. We contin-ued by assessing the effects of pro-longed storage time at 4°C.

CB CD45+7AAD– cell counts show agradual decrease in the samples over the72-hour period but was not significant(p = 0.08 for fresh and p = 0.58 forthawed). However, the granulocytepopulation declined by a significantamount after 72 hours (p = 0.02).

Mean PBSC CD45+7AAD– cell count decreased bya significant amount after 48 hours of 4°C storage(89.75 � 13.32% recovery, p = 0.03). This loss wasobserved earlier in thawed samples resulting in asignificant decrease after 24 hours with a recovery of91.39 � 10.53% (p = 0.04). This decrease was observed inthe granulocyte cell population (p = 0.01).

BM CD45+7AAD– recovery resulted in no significantdifference after 72 hours of fresh storage. However, whenassessed after thawing a significant loss in CD45+7AAD–cells was detected after 72 hours with a recovery of68.66 � 26.85% (p = 0.005). This decrease was alsoobserved in the MNC population (p = 0.02).

The effect of storage temperature and time onCD34+7AAD– cellsTemperature assessment of CD34+7AAD–, in Fig. 4A,showed a trend toward higher recoveries in fresh CBsamples stored at 4°C. However, a significant differencewas observed in the thawed samples after 48 hours ofstorage before cryopreservation (74.95 � 24.89% recoveryafter 20°C storage vs. 90.14 � 21.34% after 4°C storage,p = 0.046).

An increased loss of CD34+7AAD– cells was observedin PBSC samples after 72 hours in cells stored at 20°C over4°C both in fresh and in thawed samples (p = 0.01 for freshsamples and p = 0.005 after thaw).

TABLE 1. Mean viable cell and CFU count in samples assessed atfresh t = 0

HPC source

Cell count (¥1012/L)

CD45+7AAD– CD34+7AAD– CFUs

CBMean � SD 12.21 � 6.58 74.99 � 115.50 14.16 � 10.23Range 5.83-25.85 11.03-392.30 4.58-41.33

PBSCsMean � SD 21.10 � 6.64 234.46 � 325.55 41.35 � 43.93Range 9.48-32.60 75.71-1205.60 7.50-168.00

BMMean � SD 27.97 � 17.26 484.29 � 453.89 104.91 � 102.53Range 8.48-66.50 93.87-1427.20 16.50-297.75

TABLE 2. Mean viable cell and CFU count in samples assessed aftercryopreservation at t = 0

HPC source

Cell count (¥1012/L)

CD45+7AAD– CD34+7AAD– CFUs

CBMean � SD 10.55 � 6.96 64.95 � 119.58 7.98 � 8.16Range 3.38-23.63 8.01-401.76 1.56-30.00

PBSCsMean � SD 18.62 � 5.46 208.84 � 278.36 15.92 � 9.93Range 6.99-28.32 56.96-1026.19 6.25-33.75

BMMean � SD 12.70 � 10.77 252.98 � 258.00 83.18 � 98.89Range 2.42-41.43 41.99-900.00 2.50-273.75



No significant difference was observed between BMsamples stored at 20 or 4°C throughout the 72 hours;however, those at 4°C had higher mean recoveries.

When assessing the effects of time on the HPCsources at 4°C, CB showed a decrease in the freshCD34+7AAD– cell counts in the first 24 hours after collec-tion (recovery of 81.82 � 19.24%, p = 0.03). A significantreduction was also observed after 72 hours in freshsamples as well as in the thawed samples (86.27 � 16.39%,p = 0.03, for fresh samples; 86.16 � 18.29%, p = 0.04, forthawed samples).

A significant reduction in PBSC CD34+7AAD– cellswas observed after 48 hours in both fresh and thawedsamples (90.03 � 14.84%, fresh recovery, p = 0.05;86.80 � 14.13%, thaw recovery, p = 0.005). This significantdifference continued to samples stored for 72 hoursbefore cryopreservation (p = 0.002).

In BM a significant loss was observed after 48 hours offresh storage at 4°C (p = 0.03) and later in the thawedsamples after 72 hours (p = 0.003).

The effect of storage temperature and time onCFU assessmentThe main objective of our study was to determine howthese variables affect graft potency through CFU assess-ment. Comparing the two storage temperatures on theCFU recovery of fresh HPC products shows a trend towardhigher recoveries when stored at 4°C (data shown inFig. 5). However, the difference was shown to be signifi-cant in the postthaw samples. In CB a significant differ-ence was observed after 72 hours (recovery of38.50 � 31.65% and 54.12 � 34.22% for 20 and 4°C,respectively, p = 0.02), 48 hours for PBSC (45.56 � 20.23%

Fig. 3. Mean CD45+7AAD– recovery in HPC products during prolonged fresh storage at different temperatures and the effects of

prolonged storage before cryopreservation in (A) fresh CB cells, (B) postthaw CB cells, (C) fresh PBSCs, (D) postthaw PBSCs,

(E) fresh BM cells, and (F) postthaw BM cells. Error bars correspond to the SEM. *p < 0.05 between storage temperatures.

†p < 0.05 in cells stored at refrigerated temperatures compared to t = 0. (- · -) 20°C; (—) 4°C.

FRY ET AL.


for 20°C vs. 67.34 � 26.19% for 4°C, p = 0.01), and 24 hoursfor BM samples (40.51 � 11.52% for 20°C vs. 63.60 �

28.03% for 4°C, p = 0.02), suggesting that 4°C is theoptimum temperature to maintain all three HPC samplesafter collection.

For samples stored at 4°C, time had a continuousinfluence on the potency of the HPCs both in fresh and inpostthaw data. A significant reduction in CFUs from thoseat t = 0 in CB samples stored at 4°C was observed after 48hours in both fresh (61.82 � 24.36% recovery, p = 0.0008)and thawed samples (60.40 � 26.42% recovery, p = 0.001).

Although a decline was observed, no significant lossof CFU was observed in fresh PBSC samples stored at 4°C.However, cryopreservation after 48 hours of fresh storageresulted in a thaw CFU recovery of 67.34 � 26.19% com-pared to those frozen at t = 0 (p = 0.006).

BM samples resulted in a significant reduction inCFUs after 48 hours of 4°C storage (67.25 � 31.25%

recovery compared to fresh t = 0, p = 0.009), whereas a sig-nificant reduction was observed after 24 hours in the post-thaw samples (63.60 � 28.03% recovery compared to thawt = 0, p = 0.003).

Table 3 summarizes the main results regardingbanking showing the CFU percentage losses after delayingcryopreservation.

DISCUSSION

This study has identified the effects of time and differentstorage temperatures before cryopreservation on threeHPC sources. Higher recoveries were obtained, in all celltypes assessed, for samples maintained at refrigeratedtemperatures before cryopreservation. The differencebetween the two storage temperatures was significant inCD45+ cells after 72 hours for PBSC samples; in CD34+cells after 48 hours for CB and 72 hours for PBSCs; and in

Fig. 4. Mean CD34+7AAD– recovery in HPC products during prolonged fresh storage at different temperatures and the effects of

prolonged storage before cryopreservation in (A) fresh CB cells, (B) postthaw CB cells, (C) fresh PBSCs, (D) postthaw PBSCs,

(E) fresh BM cells, and (F) postthaw BM cells. Error bars correspond to the SEM. *p < 0.05 between storage temperatures.

†p < 0.05 in cells stored at refrigerated temperatures compared to t = 0. (- · -) 20°C; (—) 4°C.



CFUs after 24 hours for BM, 48 hours for PBSCs, and 72hours for CB.

The assessment of time showed continuous reduc-tions in the numbers of viable cells and CFUs before andafter thaw; however, there were slight differences betweenthe sources of HPCs. CB postthaw viable CD45+ cell recov-eries showed a gradual but not significant decline over the

72 hours; however, PBSC and BM samples gave a signifi-cant difference compared to samples frozen at t = 0 afterdelays of 24 hours (p = 0.04) and 72 hours (p = 0.005),respectively.

The effect of an increased delay before cryopreserva-tion on viable CD34+ cell recovery in PBSC samplesresulted in a significant difference after 48 hours(p = 0.005); a significant loss occurred later in CB and BMsamples at 72 hours (p = 0.04 and p = 0.003, respectively).

CFU recovery declined significantly after a 24-hourdelay before cryopreservation in BM samples and aftera 48-hour delay in CB (p = 0.001) and PBSC samples(p = 0.003). However, although losses in CFUs were notsignificant in CB and PBSC samples until 48 hours, 19.59%of CFUs were lost in CB and 21.88% were lost in PBSCsamples after a delay of 24 hours.

CB units are routinely frozen and banked beforetransplantation; these data suggest that units should be

Fig. 5. Mean CFU recovery in HPC products during prolonged fresh storage at different temperatures and the effects of prolonged

storage before cryopreservation in (A) fresh CB cells, (B) postthaw CB cells, (C) fresh PBSCs, (D) postthaw PBSCs, (E) fresh BM cells,

and (F) postthaw BM cells. Error bars correspond to the SEM. *p < 0.05 between storage temperatures. †p < 0.05 in cells stored at

refrigerated temperatures compared to t = 0. (- · -) 20°C; (—) 4°C.

TABLE 3. Percentage CFU loss after prolongedstorage at 4°C before cryopreservation

HPC source

Storage time (hr)

24 48 72

CB 19.59 � 30.24 39.62 � 26.42 45.88 � 34.22p value 0.071 0.001 0.002

PBSCs 21.88 � 40.15 32.66 � 26.19 39.46 � 17.86p value 0.141 0.006 0.0002

BM 36.40 � 28.03 52.21 � 11.68 75.74 � 8.73p value 0.003 9.1 ¥ 10–7 5.1 ¥ 10–9

FRY ET AL.


maintained at refrigerated temperatures and cryopre-served as soon after collection as possible (preferablywithin the first 24 hr but no later than 48 hr) to retain thehighest possible potency of the cells. This range is insupport of the current recommendations of NetCord-FACT standards32 and in agreement with other studies.6-12

The PBSC results confirm earlier reports that the viabilityof the cells within samples are maintained best whenstored at refrigerated temperatures.18-21 The fresh BMresults are consistent with those obtained by othergroups.24-27 However, upon thawing, BM samples resultedin low viable cell and CFU recoveries, suggesting that it isbetter to maintain BM samples in a fresh state for up to 72hours than to cryopreserve.

In this study we used 1-mL aliquots in cryovials tocompare different conditions on the same sample.However, in this experimental set-up, the cryopreserva-tion protocol is not consistent with that used in clinicalprocessing, in which larger volumes are cryopreservedwith the use of controlled rate freezers. Although compar-ing these two methods of cryopreservation in CB samplesresulted in 7.27% fewer CFUs recovered when freezingsmall volumes, we still consider the pattern of recoveriesto be representative.

In conclusion, this study highlights the importance ofminimizing the delay between collection of a sample andcryopreservation or infusion into a patient. Thawed cellproducts resulted in significantly decreased CFU recover-ies when cryopreservation had been delayed for 24 hoursin BM samples and 48 hours for both CB and PBSCsamples. Because the CFU dose given to a patient is thebest current predictive marker of engraftment, this studyshows that delays in cryopreservation can increase risk foran unsuccessful transplant.

ACKNOWLEDGMENTS

The authors thank Terie Duffy and all the midwives at Kingscord,

King’s College Hospital, London, for collecting CB samples. We

also acknowledge Linda Barber in the Department of Haemato-

logical Medicine at King’s College Hospital, London, for use of

laboratory equipment.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest relevant

to the manuscript submission to TRANSFUSION.

REFERENCES

1. Appelbaum FR, Sullivan KM, Buckner CD. Treatment of

malignant lymphoma in 100 patients with chemotherapy,

total body irradiation, and marrow transplant. J Clin Oncol

1987;5:1340-7.

2. Eaves CJ. Peripheral blood stem cells reach new heights.

Blood 1959;82:1957-9.

3. Jansen J, Nolan PL, Reeves MI, Morgan JA, Akard LP,

Thompson JM, Dugan MJ, Hanks SG. Transportation of

peripheral blood progenitor cell products: effect of

ambient temperature. Cytotherapy 2010;12:919-23.

4. Jansen J, Hanks SG, Akard LP, Morgan JA, Nolan PL, Dugan

MJ, Reeves MI, Thompson JM. Slow platelet recovery after

peripheral blood progenitor cell transplantation from unre-

lated donors. Bone Marrow Transplant 2009;43:499-505.

5. Lazarus HM, Kan F, Tarima S, Champin RE, Confer DL,

Frey N, Gee AP, Wagner JE, Horowitz MM, Eapen M. Rapid

transport and infusion of hematopoietic cells is associated

with improved outcome after myeloablative therapy and

unrelated donor transplant. Biol Blood Marrow Transplant

2009;15:589-96.

6. Tron de Bouchony E, Pelletier D, Alcalay D, Bruneau J,

Predeau M, Brizard A, Magnin M. Hematopoietic progeni-

tor content of fetal cord blood collected using citrate-

phosphate-dextrose: influence of holding temperature and

delays. J Hematother 1993;2:271-3.

7. Shlebak AA, Marley SB, Roberts IA, Davidson RJ, Goldman

JM, Gordon MY. Optimal timing for processing and cryo-

preservation of umbilical cord. Bone Marrow Transplant

1999;23:131-6.

8. Rogers I, Sutherland A, Holt D, Macpate F, Lains A, Hollow-

ell S, Cruickshank B, Casper RF. Human UC-blood

banking: impact of blood volume, cell separation and cryo-

preservation on leukocyte and CD34+ cell recovery. Cyto-

therapy 2001;3:269-76.

9. Hubel A, Carlquist D, Clay M, McCullough J. Short-term

liquid storage of umbilical cord blood. Transfusion 2003;43:

626-32.

10. Hubel A, Carlquist D, Clay M, McCullough J. Cryopreserva-

tion of cord blood after liquid storage. Cytotherapy 2003;5:

370-6.

11. Hubel A, Carlquist D, Clay M, McCullough J. Liquid

storage, shipment, and cryopreservation of cord blood.

Transfusion 2004;44:518-25.

12. Tsagias N, Kouzi-Koliakos K, Karagiannis DH, Alamdari V,

Koliakos G. Time and temperature before processing influ-

ence the recovery of umbilical cord blood hematopoietic

progenitors. Transfusion 2007;47:1550-2.

13. Rubinstein P, Rosenfield RE, Adamson JW, May WS. Stored

placental blood for unrelated bone marrow reconstitution.

Blood 1993;81:1679-90.

14. Campos L, Roubi N, Guyotat D. Definition of optimal con-

ditions for collection and cryopreservation of umbilical

cord hematopoietic cells. Cryobiology 1995;32:511-5.

15. Kurtz J, Shalini S, Greco N, Moroff G. Assessment of cord

blood hematopoietic cell parameters before and after cryo-

preservation. Transfusion 2007;47:1578-86.

16. Broxmeyer HE, Gluckman E, Auerbach A, Douglas GW,

Friedman H, Cooper S, Hangoc G, Kurtzberg J, Bard J,

Boyse EA. Human umbilical cord blood: a clinically useful

source of transplantable hematopoietic stem/progenitor

cells. Int J Cell Cloning 1990;8:76-91.



17. Broxmeyer HE, Kurtzberg J, Gluckman E, Auerbach AD,

Douglas G, Cooper S, Falkenburg JH, Bard J, Boyse EA.

Umbilical cord blood hematopoietic stem and repopulat-

ing cells in human clinical transplantation. Blood Cells

1991;17:313-29.

18. Moroff G, Seetharaman S, Kurtz JW, Greco NJ, Mullen MD,

Lane TA, Law P. Retention of cellular properties of PBPCs

following liquid storage and cryopreservation. Transfusion

2004;44:245-2.

19. Sugrue MW, Hutcheson CE, Fisk DD, Roberts CG, Mageed

A, Wingard JR, Moreb JS. The effect of overnight storage of

leukapheresis stem cell products on cell viability, recovery,

and cost. J Hematother 1998;7:431-6.

20. Jestice HK, Scott MA, Ager S, Tolliday BH, Marcus RE.

Liquid storage of peripheral blood progenitor cells for

transplantation. Bone Marrow Transplant 1994;14:991-4.

21. Ruiz-Arguelles GJ, Ruiz-Arguelles A, Perez-Romano B,

Marin-Lopez A, Larregina-Diez A, Apreza-Molina MG.

Filgrastim-mobilized peripheral-blood stem cells can be

stored at 4 degrees and used in autografts to rescue high-

dose chemotherapy. Am J Hematol 1995;48:100-3.

22. Petzer AL, Gunsilius E, Zech N, Clausen J, Hoflehner E,

Nussbaumer W, Gastl G. Evaluation of optimal survival of

primitive progenitor cells (LTC-IC) from PBPC apheresis

products after overnight storage. Bone Marrow Transplant

2000;25:197-200.

23. Beaujean F, Pico J, Norol F, Divine M, Le Forestier C,

Duedari N. Characteristics of peripheral blood progenitor

cells frozen after 24 hours of liquid storage. J Hematother

1996;5:681-6.

24. Lasky LC, McCullough J, Zanjani ED. Liquid storage of

unseparated human bone marrow evaluation of hemato-

poietic progenitors by clonal assay. Transfusion 1986;26:

331-4.

25. Mangalik A, Robinson WA, Drebing C, Hartmann D, Joshi

JH. Liquid storage of bone marrow. Exp Hematol 1979;7:

76-94.

26. Kohsaki M, Yanes B, Ungerleider JS, Murphy MJ Jr. Non-

frozen preservation of committed hematopoietic stem cells

from normal human bone marrow. Stem Cells 1981;1:111-

23.

27. Antonenas V, Garvin F, Webb M, Sartor M, Bradstock KF,

Gottlieb D. Fresh PBSC harvests, but not BM, show tem-

perature related loss of CD34 viability during storage and

transport. Cytotherapy 2006;8:158-65.

28. Haas R, Witt B, Mohle R, Goldschmidt H, Hohaus S, Freu-

hauf S, Wannenmacher M, Hunstein W. Sustained long-

term hematopoiesis after myeloablative therapy with

peripheral blood progenitor cell support. Blood 1995;85:

3754-61.

29. Rowley SD, Zuehlsdorf M, Braine HG, Colvin OM, Davis J,

Jones RJ, Saral R, Sensenbrenner LL, Yeager A, Santos

GW. CFU-GM content of bone marrow graft correlates

with time to hematologic reconstitution following

autologous bone marrow transplantation with

4-hydroperoxycyclophosphamide-purged bone marrow.

Blood 1987;70:271-5.

30. Yoo KH, Lee SH, Kim HJ, Sung KW, Jung HL, Cho EJ, Park

HK, Kim HA, Koo HH. The impact of post-thaw colony-

forming units-granulocyte/macrophage on engraftment

following unrelated cord blood transplantation in pediatric

recipients. Bone Marrow Transplant 2007;39:515-21.

31. Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Suther-

land RS. Single platform flow cytometric absolute CD34+cell counts based on the ISHAGE guidelines. Cytometry

1998;34:61-70.

32. International NetCord Foundation and Foundation for the

Accreditation of Cellular Therapy. NetCord-FACT interna-

tional standards for cord blood collection, banking, and

release for administration. 4th ed. 2010. [cited 2012 Nov

16]. Available from: URL: www.factwebsite.org.

FRY ET AL.


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