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Vox Sanguinis(2002) 82, 3238

ORIGINAL PAPER 2002 Blackwell Science

BlackwellScienceLtd

Microbial contamination of cord blood stem cells. Honohan1, H. Olthuis1, A. T. Bernards2, J. M. van Beckhoven1& A. Brand1

1Sanquin Blood Supply Foundation, Blood Bank Leiden-Haaglanden, Leiden, the Netherlands

2Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands

Background and Objectives After storage, low levels of contaminating bacteria instandard blood components can reach bacteraemic levels, causing severe transfusion-associated sepsis. For cord blood (CB), the significance of low levels of contaminatingbacteria and the optimal detection method is unknown and not supported by availableguidelines.

Materials and Methods Spiking experiments and testing of various subfractions of CBunits were used to determine the behaviour of bacteria during centrifugation, freezingand thawing. For routine testing of CB, different volumes were compared for the

detection of potential pathogens and micro-organisms of low pathogenicity.Results Centrifugation, as applied to CB fractionation, does not show concentrationof bacteria in any particular fraction and supports the possibility of culture of wastefractions. Dimethylsulphoxide (DMSO) and freezing does not affect the viability ofbacteria under the conditions used in this study. Owing to the low contamination level,a large sample volume of 20 ml was more sensitive than a 10-ml sample volume. Eightyfive per cent of the isolated strains can be considered to be of low pathogenicity.

Conclusion When an optimal waste fraction sample volume of 20 ml was cultured,the contamination rate of CB was found to be 13%, with low levels of < 1 colony-forming unit (CFU)/ml. Such levels of bacteria of low pathogenicity are expected tobe of clinical importance only when CB is expanded in vitrofor a prolonged periodof time.

Key words: cord blood, low microbial load, low-pathogens, microbial contamination.

Received: 5 June 2001,

accepted 14 July 2001

Introduction

It has been well established that standard blood componentsmay be contaminated with low levels of bacteria (< 10 colony-forming units [CFU]/ml) [13]. The storage temperature andshelf-life of platelet concentrates (22 C for 5 days) and redcell concentrates (46 C for up to 42 days) allow the numberof certain bacteria to reach levels of > 105106CFU/ml, which

several reports suggest to be of clinical significance [47].Despite a strict disinfection protocol, cord blood (CB) unitscarry a greater risk of being contaminated with low levels ofbacteria. In contrast to standard blood components, the

storage temperature of CB (150 C) does not allow thegrowth of bacteria. Although a relatively small number of CBtransplantations (1500) has been performed, there have beenno reports of immediate or delayed adverse reactions associ-ated with contaminating bacteria [8].

For CB bank purposes several different methods areemployed to reduce the volume to as low as 20 ml priorto cryopreservation. Netcord Fahct Standards request a

microbial culture to be performed on a sample of the CB unitafter processing [9]. With respect to product quality it isquestionable if compliance with these standards is acceptable;the culture volume is strongly reduced compared to standardculture methods, resulting in reduced sensitivity of the test.Furthermore, sampling a relatively large volume of the finalproduct further reduces the limited number of stem cellsavailable. If a future perspective is to include ex vivoexpan-sion and genetic manipulation of CB stem cells, the detection

Correspondence: ine Honohan, Blood Bank Leiden-Haaglanden, Postbus2184, 2301 CD Leiden, the NetherlandsE-mail: [email protected]

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2002 Blackwell Science Ltd. Vox Sanguinis(2002) 82, 3238

Microbial contamination of CB stem cells 33

of low numbers of micro-organisms may be important as thesetechniques create conditions in which low numbers of con-taminating bacteria may reach levels of clinical significance.

The aim of this study was to define a suitable sample andan adequate sample volume for the detection of low numbersof contaminating bacteria in CB. We investigated the influ-ence of volume reduction by hydroxyethyl starch (HES)

sedimentation on the distribution of contaminating bacteriain CB units and the effect of dimethylsulphoxide (DMSO) andcryopreservation on the viability of bacteria after thawing.

Materials and methods

Collection and processing of CB

CB was collected for banking or for experimental purposesfrom consenting donors before delivery of the placenta bytrained midwives. The umbilical vein was cleaned with 70%alcohol and subsequently disinfected with 100 mg/ml

povidoneiodine solution, after which the CB was collectedinto a bag containing 25 ml of citratephosphatedextrose-adenine (CPDA) anticoagulant solution (Baxter Fenwal, Utrecht,the Netherlands Pl146-CPDA-1-35 ml, modified to 25 ml). CBunits were stored at 46 C on arrival at the blood bank andcryopreserved within 24 h of collection. The CB units were volumereduced in a closed system, as described by Rubinsteinet al. [10].In brief, HES was added to the anticoagulated CB at a volumeratio of 1 : 5. As a result of centrifuging (50g) the CB/HES mixturein the original collection bag, a stem cell-rich supernatant wasseparated from the residual erythrocyte fraction (EF). The stemcell-rich supernatant was then expressed from the bag to aplasma transfer bag and centrifuged to sediment the cells.Surplus supernatant plasma fraction (PF) was transferred intoa waste bag. Finally, the sedimented stem cells were resuspendedin supernatant plasma to a volume of 20 ml and designatedas stem cell concentrate (SCC). The SCC was cryopreserved in5 ml of 50% DMSO in 5% (wt/vol) Dextran-40 (Mr40 000 Da)to give a final concentration of 10% DMSO. CB collectionswith a volume of > 120 ml (including anticoagulant) wereprocessed into two SCCs in separate bags. SCCs collected forbanking were frozen by controlled rate freezing and storedat 150 C. SCCs from spiked units were frozen and stored at86 C. The EF and PF were sampled for microbial culture.The residual PF was frozen and stored at 86 C; the remain-

ing EF was used for human leucocyte antigen (HLA) typing.

Thawing of PF and SCC

The PF was thawed rapidly in a 37 C water bath and culturedimmediately. The SCC was removed from storage at 86 Cor 150 C and allowed to stand at room temperature for15 min before being rapidly thawed in a 37 C water bath.The SCC was cultured either before or after removal of DMSO.

Spiking experiments of CB units collected for

experimental purposes

The influence of the addition of HES, and the effect of cen-trifugation, on the distribution of bacteria in deliberatelyspiked CB units were investigated in order to establish asuitable sample and an adequate sample volume for detection

of low numbers of bacteria. The effect of 10% DMSO andcryopreservation on the viability of the bacteria was alsoinvestigated.

Nine fresh CB units were inoculated within 24 h of collec-tion with one of three reference strains (coagulase-negativeStaphylococcusATCC 12228; Bacillus cereusATCC 14579;or Streptococcus agalactiaeATCC 13813) (American TypeCulture Collection [ATCC], Mijdrecht, the Netherlands).Before spiking, 5-ml samples were removed from each unitand cultured to ensure baseline sterility. Bacterial suspen-sions were prepared in phosphate-buffered saline (PBS) andserially diluted 10-fold. The CB units were inoculated in the

range of 2426 CFU/ml (median 40 CFU/ml), as establishedby count plates prior to volume reduction. From each of thespiked units, 02-, 10-, 25- and 50-ml sample volumes ofthe SCC after addition of DMSO, and 10-, 25- and 50-mlsamples of the EF and PF, were cultured. The PF and SCC werecultured post-thaw from four of the nine units spiked withlow numbers of bacteria (in the range of 240 CFU/ml),including all three reference strains. The PF and SCC werecultured immediately after thawing; DMSO was not removedfrom the SCC. From the SCC, 02- and 1-ml samples werecultured post-thaw; from the PF 1-ml samples were culturedpost-thaw.

Bacterial contamination of EF and PF from units

collected for banking

CB units were routinely screened for the presence of aerobicand anaerobic microbes by culturing the EF and PF sepa-rately or pooled. Initially, a total sample volume of 10 ml (EF5 ml/PF 5 ml) was cultured and 20 ml for units (> 120 ml)processed to two SCC. Subsequently the volumes wereincreased to 20 and 40 ml, respectively. In all cases, equalvolumes of the EF and PF were cultured.

Contamination level of PF and SCC post-thawFrom routine screening of CB units, 94 of 740 units were cul-ture positive prior to cryopreservation. The PFs from 56 ofthe 94 positive units were available for testing post-thaw.Immediately after thawing, 20 ml of the PF was cultured instandard aerobic and anaerobic culture bottles (OrganonTeknika, Turnhout, Belgium). To establish the number ofviable bacteria, an additional 5 ml was plated onto four agarplates. The SCC (25 ml/including 5 ml of DMSO medium),

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from eight of 11 CB units with a relatively high contamination

level (post-thaw contamination level of 148 CFU/5 ml PF),was cultured on a series of agar plates.

General microbiology procedures

All sampling was carried out under aseptic conditions in alaminar flow cabinet. Standard BacT/Alert culture bottles(Organan Teknika) were filled to the recommended volume(10 ml), except where stated otherwise. The culture bottleswere incubated at 35 C and monitored for up to 14 daysusing the BacT/Alert continuous monitoring blood culturesystem (Organon Teknika). All culture-positive bottles were

subcultured and isolates were identified by conventionalmicrobiological procedures. Establishment of the number ofviable bacteria post-thaw was performed on the preferredagar (sheep blood or chocolate) for the micro-organismisolated prior to cryopreservation. All agar plates were incu-bated at 35 C for 6 days and colony counts were performedon two occasions.

All isolates were frozen in glycerol and stored at 70 C.

Statistical analysis

Statistical analysis was carried out using the two-tailedFishers exact test.

Results

Spiking experiments

Table 1 shows the precryopreservation and post-thaw micro-bial culture results of CB units spiked with three referencestrains. All cultures from all fractions cultured in volumesranging from 02 to 5 ml for SCC and 105 ml for PF and EF

precryopreservation, were positive. One post-thaw sample of

02 ml from a unit spiked with < 3 CFU/ml remained culturenegative (Table 1). The three strains used in this study wereviable post-thaw after cryopreservation in 10% DMSO andstorage at 86 C.

CB units for banking

Contamination rate of EF and PFBetween March 1998 and December 2000, the EF and PFfrom 740 CB units (collected from 16 participating maternitycentres) were cultured. A unit was defined as positive afterconfirmation of at least one positive culture bottle. The EF/PF samples from 94 of the 740 units (127%) were contamin-ated prior to cryopreservation. Culturing a volume of 20 mlgave a significantly higher number of positive results thanculturing a 10-ml volume (P = 0024). There was no signific-ant difference in the number of positive results between20- and 40-ml culture volumes (Table 2). The culture samplesfrom the 94 contaminated units comprised 54 pooled EF/PFfractions, 20 unpooled EF and PF fractions, and 20 CB unitsof > 120 ml yielding two EF/PF pairs. Only one bottle waspositive from 28 of the 54 pooled fractions. Only one fractionwas positive from 14 of the 20 unpooled fractions; in eightcases only the EF was positive and in six cases only the PF.Only one of the pooled EF/PF pairs was positive from 12 of

the 20 CB units with a volume of > 120 ml. The microbesisolated from EF/PF are shown in Table 3. Fourteen unitsyielded isolates we categorized as being potential pathogens,and 80 units yielded isolates we categorized as being of lowpathogenicity.

Post-thaw contamination level of the PF and SCCThe PFs from 56 of the 94 positive CB units were culturedpost-thaw. Twenty-three PFs were culture negative, indicative

Table 1 Precryopreservation and post-thaw bacteriological culture results of nine cord blood (CB) units spiked with one of three reference strains

CB fractions

Time of culture CB units (n) EF PF SCC

Precryopreservation 9 27/27 positive; 27/27 positive; 36/36 positive;

median: 40 CFU/ml median: 40 CFU/ml median: 40 CFU/ml

(range: 2426 CFU/ml) (range: 2426 CFU/ml) (range: 2 426 CFU/ml)

Post-thaw 4a N/A 8/8 positive; 24/25bpositive;

median: 16 CFU/ml median: 16 CFU/ml

(range: 2 40 CFU/ml) (range: 2 40 CFU/ml)

CB, cord blood; CFU, colony-forming units; EF, erythrocyte fraction; N/A, not available for culture; PF, plasma fraction; SCC, stem cell concentrate.aFour of nine units were spiked at lower levels: 240 CFU/ml.b0ne 02-ml SCC sample from a unit contaminated with < 3 CFU/ml was negative.

Sample sizes were as follows. EF: 10, 25 and 50 ml precryopreservation. PF: 10, 25 and 50 ml precryopreservation; 10 ml post-thaw. SCC: 02, 10, 25 and

50 ml precryopreservation; 02 and 10 ml post-thaw.

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of a contamination level of < 1 CFU/20 ml. Micro-organismswere isolated from the 33 remaining PF. Twenty-two of the33 PF showed a low contamination level of 1 CFU/1020 mland 11 of the 33 PF a contamination level of 148 CFU/5 ml(median: 2 CFU/5 ml). In 28 of the 33 positive PF, the samemicro-organism(s) was identified from the EF/PF and PFpost-thaw; in four of the 28 cases an additional micro-

organism was isolated from the PF post-thaw and in eight ofthe 28 cases at least one of the organisms isolated from theEF/PF was not found in the PF post-thaw. A completelydifferent micro-organism was isolated from the prefreezeEF/PF and PF post-thaw in five of 33 cases.

The SCC was cultured post-thaw from eight CB units,selected because of a relatively higher contamination level, asdetermined from culturing the PF post-thaw (148 CFU/5 ml).Table 4 shows the culture results of eight SCC post-thaw and

the corresponding prefreeze EF/PF and PF post-thaw. Themicrobial load of the SCC (standard volume 25 ml) rangedfrom 1 to 204 CFU/25 ml (median: 13 CFU/25 ml). More thanone micro-organism was isolated from four SCC (UD2126,1570, 1403 and 2837). The microbial load of the SCC corre-sponded well with the PF post-thaw (148 CFU/5 ml; median:

2 CFU/5 ml). Identification of the micro-organisms revealedat least one micro-organism of the same species in the SCC,PF post-thaw and EF/PF from six CB units (UD1625, 1639,2126, 1570, 1403 and 2837). At least one additional discrep-ancy was observed between the EF/PF, the PF post-thaw andthe SCC in all eight units. S. agalactiae, coagulase-negativestaphylococcus(CNS) and diphtheroid rods remained viableafter cryopreservation in 10% DMSO and storage at 150 C.NeitherHaemophilusinfluenzae(UD2566) nor non-haemolyticstreptococcus(UD1403, UD2126) were recovered post-thawfrom the SCC or PF.

DiscussionCB units spiked before processing showed no preferentiallocalization of bacteria after centrifugation and therebyestablished the EF and PF (after volume reduction by HESsedimentation) as suitable samples for estimating the bac-terial contamination rate of the SCC (Table 1). A samplevolume of 1 ml of EF or PF was suitable for detecting acontamination level of 2 CFU/ml. Culturing the EF and PFavoids the unnecessary loss of the already limited number ofstem cells.

We observed a contamination rate of 13% from the routineculture of CB units for banking. A number of CB banks are

now reporting a reduction in the bacterial contamination rateto < 1% [1113] and < 5% [14], after extensive training incollection procedures and the introduction of stringent dis-infection protocols. However, as the results of our study sup-port those of a previous study [15], which defined the volumeof the sample cultured as being the single most importantfactor governing the sensitivity of the blood culture, thesecontamination rates must be viewed in the light of the volumecultured. The London, Dsseldorf and Galician CB Banks

Period of testing

Sample

volume (ml)

No.

cultured

No.

positive

Percentage

positive 95% CI

March 1998March 1999 10 216 18 83 4971317

April 1999December 2000 20 445 65 146a 1127 1862

April 1999December 2000b 40 79 11 139 6942490

Total 740 94 127

aSignificantly higher than with the 10-ml volume; P = 0024 (odds ratio 188; 95% confidence interval

[CI] 109326).bSee the Materials and methods.

Table 2 Culture results of 10, 20 and 40 mlsamples of equal volumes of the erythrocyte

fraction (EF) and plasma fraction (PF) of volume-

reduced cord blood units collected for banking

Table 3 Micro-organisms isolated from 94 cord blood units collected forbanking

Potential pathogensa(no. of contaminated units)

Micro-organisms oflow pathogenicityb

Escherichia coli(1) Coagulase-negative Staphylococcus

Enterococcus faecalis(1) Diphtheroid rods

Streptococcus agalactiae(5) Propionibacteriumspp.

Bacteroidesspp. (2) Streptococcus viridans

Candida albicans(1) Lactobacillus

Haemophilus influenzae (1) Non-haemolytic Streptococcus

Strictly anaerobic Gram-

negative rods (3)

Bifidobacteriumspp.

Anaerobic Gram-positive rods

Fine anaerobic Gram-positive rods

Microaerophilic Gram-positive rods

Gram-negative rods (non-fermenters)

Gram-positive coccus

Anaerobic Gram-positive cocci

a

n= 14. Micro-organisms of low pathogenicity were also isolated from anumber of units.bn= 80. Multiple micro-organisms of low pathogenicity were isolated from

a number of units.

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culture 1, 2 and 15 ml, respectively [1214]. The volume culturedby the Milano CB Bank is not specified [11,16], which makesit difficult to place any value on the reported contaminationrate. In contrast to other CB banks, we observed an increasein the contamination rate from 87 to 14% by increasing theculture volume from 10 to 20 ml (Table 2). Culturing 20 ml

of the EF/PF gave a significantly higher percentage of positivesthan culturing 10 ml (P = 0024). There was no significantdifference in the contamination rate after culturing 20 mlor 40 ml, which suggests that a sample volume of 20 ml issufficient to detect the majority of contaminants in CB.

The culture results from pooled and unpooled EF and PFfrom 94 contaminated units indicated a low microbial load.The PF was evaluated post-thaw from 56 of the 94 contaminatedunits; in 45 cases the microbial load was 1 CFU/20 ml.

From eight of 11 units with a relatively higher contaminationlevel (148 CFU/5 ml of plasma post-thaw), the SCC showeda microbial load of 2204 CFU/25 ml (median: 13 CFU/25 ml;Table 4), verifying that CB units were contaminated with verylow numbers of bacteria. Results from EF and PF culturedseparately showed a similar suitability of both fractions for

culture.The consistency by which the same bacteria were isolated

from the EF/PF, post-thaw PF (28 of 33) and SCC (five ofeight), was indicative of true contamination (Table 4). Thefew discrepancies observed after identification of the speciesmay have been the result of several causes. These include:exogenous contamination of the culture bottle (mostprobable for UD2194 post-thaw plasma); a lower number ofcontaminating micro-organisms than the detection level of

Table 4 Culture results of eight cord blood (CB) units contaminated with > 1 colony-forming units (CFU)/ml: comparison of prefreeze erythrocyte fraction/plasma fraction (EF/PF) and post-thaw stem cell concentrate (SCC) and PF

EF/PF prefreeze SCC post-thaw PF post-thaw

UD no. Isolate IsolateContaminationlevel (CFU/25 ml) Isolate

Contaminationlevel (CFU/5 ml)

1625 a. S. agalactiae S. agalactiae 204 S. agalactiae 12

b. S. agalactiae S. agalactiae 170 S. agalactiae 36

E. coli

1639 a. CNS CNS 9 CNS 5

Diphtheroid rods

b. CNS CNS 12 CNS 1

2126 a. CNS CNS 16 CNS 1

Diphtheroid rods Diphtheroid rods Diphtheroid rods

Non-haemolytic strep.

b. CNS CNS 21 CNS 5

Diphtheroid rods Diphtheroid rods Diphtheroid rods


2491 CNS Micrococcus 1 CNS 48

1570 a. negative S. agalactiae 6 NT NT

Diphtheroid rod

Micrococcus


b. S. agalactiae S. agalactiae 4 NT NT

Micrococcus

2566 H. influenzae Diphtheroid rods 2 Diphtheroid rods 1

1403 Diphtheroid rods Diphtheroid rods 19 Diphtheroid rods 1

Non-haemolytic strep. CNS

2837 S. agalactiae S. agalactiae 13 S. agalactiae 2

CNS

a. and b., fractions from CB units of > 120 ml in volume; CNS, coagulase-negative staphylococcus; EF/PF: erythrocyte fraction/plasma fraction (prefreeze);

NT, not tested; PF, plasma fraction (post-thaw); SCC, stem cell concentrate (standard volume 25 ml); UD, unique identification number.

E. coli, Escherichia coli; H. influenzae, Haemophilus influenzae; strep., streptococcus; S. agalactiae, Streptococcus agalactiae.

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the sample cultured; or non-survival of the strain followingcryopreservation in 10% DMSO. This study shows that a sig-nificant number of micro-organisms remain viable, evenwhen the initial contamination level is low. It is probable thatdiscrepancies in culture results can occur at the low contam-ination levels observed in this study.

Post-thaw culture results from spiking experiments show

that 10% DMSO and cryopreservation at 86 C had nomeasurable effect on the viability of the three referencestrains used in this study. Post-thaw culture results from CBunits collected for banking showed that S. agalactiae, CNSand diphtheroid rods survive in 10% DMSO and cryopreser-vation at 150 C. Escherichia coli, Enterococcus faecalisand Candida albicanswere isolated from the EF/PF fromthree different CB units but not from the corresponding PFpost-thaw. H. influenzaewas isolated from the EF/PF from afourth unit, but not from the corresponding PF post-thaw orfrom SCC. Given the spiking results, these bacteria wereprobably present at a number below the detection level of the

sample volume cultured. These four micro-organisms arepotential pathogens, and the presence of even low numbersof potentially pathogenic micro-organisms is a criterion fordiscarding units from our bank. A recent report of septicshock caused by Bacillus cereusafter unrelated bone marrow(BM) transplantation highlights the implications of patho-genic bacteria in stem cell therapy [17]. The potential risks ofnot detecting pathogens in culture where the sample volumesare low should be reviewed.

Of the 94 contaminated units, 85% were contaminatedwith micro-organisms of low pathogenicity (Table 2). Infor-mation available from BM and peripheral blood progenitorcells (PBPC) data indicate that no recognized clinical sequelaeresults from the infusion of bacteria of low pathogenicityin neutropenic and immunosuppressed recipients [1821]. Anumber of CB banks have recently adopted a policy to releaseunits for banking that are contaminated with bacteria of lowpathogenicity [11,12]. The recently published Netcord-FahctStandards [9] accept this policy. However, as these standardsdo not specify the culture method and/or the minimum testvolume for demonstrating or excluding bacterial contamina-tion, the suitability of such recommendations is doubtful. Wesuggest that these standards should be extended to include amaximum acceptable contamination level and to exclude therelease of units contaminated with potential pathogens. At our

centre this would mean discarding 18% of units as a resultof contamination with potential pathogens. Our CB bank hasadopted the policy of releasing units contaminated with micro-organisms of low pathogenicity with a report of the contam-ination level. An antibiotic sensitivity test can be performedon the frozen isolate(s) upon selection for transplantation.

In conclusion, CB is contaminated with bacteria at such alow level that the volume cultured (up to 20 ml) is directlyrelated to the contamination rate. These low numbers of

contaminants can be detected without the loss of stem cells byculturing the EF/PF after volume reduction by HES sedimenta-tion. In 85% of the contaminated units, micro-organisms oflow pathogenicity were not identified as a reason to prohibitthe release of such CB for transplantation. However, the trendtowards ex vivoexpansion and genetic manipulation of CBstem cells will challenge the contamination rate of CB banks

employing microbial culture procedures with a low sensitivity.Further investigations are needed to evaluate whether thesein vitrotechniques create conditions in which low levels ofcontaminating bacteria may reach levels of clinical signifi-cance, and to evaluate the role of addition of antibiotics, asoften used in in vitroculture [22].

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