Vox Sanguinis (2002) 82, 3238

ORIGINAL PAPER
Blackwell Science Ltd

2002 Blackwell Science

Microbial contamination of cord blood stem cells

Sanquin Blood Supply Foundation, Blood Bank Leiden-Haaglanden, Leiden, the Netherlands Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands

. Honohan1, H. Olthuis1, A. T. Bernards2, J. M. van Beckhoven1 & A. Brand1

1 2

Background and Objectives After storage, low levels of contaminating bacteria in standard blood components can reach bacteraemic levels, causing severe transfusionassociated sepsis. For cord blood (CB), the signicance of low levels of contaminating bacteria and the optimal detection method is unknown and not supported by available guidelines. Materials and Methods Spiking experiments and testing of various subfractions of CB units were used to determine the behaviour of bacteria during centrifugation, freezing and 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 concentration of bacteria in any particular fraction and supports the possibility of culture of waste fractions. Dimethylsulphoxide (DMSO) and freezing does not affect the viability of bacteria 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. Eighty ve 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 colonyforming unit (CFU)/ml. Such levels of bacteria of low pathogenicity are expected to be of clinical importance only when CB is expanded in vitro for a prolonged period of 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 components may be contaminated with low levels of bacteria (< 10 colonyforming units [CFU]/ml) [13]. The storage temperature and shelf-life of platelet concentrates (22 C for 5 days) and red cell concentrates (46 C for up to 42 days) allow the number of certain bacteria to reach levels of > 105106 CFU/ml, which several reports suggest to be of clinical signicance [47]. Despite a strict disinfection protocol, cord blood (CB) units carry a greater risk of being contaminated with low levels of bacteria. In contrast to standard blood components, the

Correspondence: ine Honohan, Blood Bank Leiden-Haaglanden, Postbus 2184, 2301 CD Leiden, the Netherlands E-mail: aine.honohan@sanquinbblh.nl

storage temperature of CB (150 C) does not allow the growth of bacteria. Although a relatively small number of CB transplantations (1500) has been performed, there have been no reports of immediate or delayed adverse reactions associated with contaminating bacteria [8]. For CB bank purposes several different methods are employed to reduce the volume to as low as 20 ml prior to cryopreservation. Netcord Fahct Standards request a microbial culture to be performed on a sample of the CB unit after processing [9]. With respect to product quality it is questionable if compliance with these standards is acceptable; the culture volume is strongly reduced compared to standard culture methods, resulting in reduced sensitivity of the test. Furthermore, sampling a relatively large volume of the nal product further reduces the limited number of stem cells available. If a future perspective is to include ex vivo expansion and genetic manipulation of CB stem cells, the detection

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of low numbers of micro-organisms may be important as these techniques create conditions in which low numbers of contaminating bacteria may reach levels of clinical signicance. The aim of this study was to dene a suitable sample and an adequate sample volume for the detection of low numbers of contaminating bacteria in CB. We investigated the inuence of volume reduction by hydroxyethyl starch (HES) sedimentation on the distribution of contaminating bacteria in CB units and the effect of dimethylsulphoxide (DMSO) and cryopreservation on the viability of bacteria after thawing.

Spiking experiments of CB units collected for experimental purposes

The inuence of the addition of HES, and the effect of centrifugation, on the distribution of bacteria in deliberately spiked CB units were investigated in order to establish a suitable sample and an adequate sample volume for detection of low numbers of bacteria. The effect of 10% DMSO and cryopreservation on the viability of the bacteria was also investigated. Nine fresh CB units were inoculated within 24 h of collection with one of three reference strains (coagulase-negative Staphylococcus ATCC 12228; Bacillus cereus ATCC 14579; or Streptococcus agalactiae ATCC 13813) (American Type Culture Collection [ATCC], Mijdrecht, the Netherlands). Before spiking, 5-ml samples were removed from each unit and cultured to ensure baseline sterility. Bacterial suspensions were prepared in phosphate-buffered saline (PBS) and serially diluted 10-fold. The CB units were inoculated in the range of 2426 CFU/ml (median 40 CFU/ml), as established by count plates prior to volume reduction. From each of the spiked units, 02-, 10-, 25- and 50-ml sample volumes of the SCC after addition of DMSO, and 10-, 25- and 50-ml samples of the EF and PF, were cultured. The PF and SCC were cultured post-thaw from four of the nine units spiked with low numbers of bacteria (in the range of 240 CFU/ml), including all three reference strains. The PF and SCC were cultured immediately after thawing; DMSO was not removed from the SCC. From the SCC, 02- and 1-ml samples were cultured post-thaw; from the PF 1-ml samples were cultured post-thaw.

Materials and methods

Collection and processing of CB
CB was collected for banking or for experimental purposes from consenting donors before delivery of the placenta by trained midwives. The umbilical vein was cleaned with 70% alcohol and subsequently disinfected with 100 mg/ml povidoneiodine solution, after which the CB was collected into a bag containing 25 ml of citratephosphatedextroseadenine (CPDA) anticoagulant solution (Baxter Fenwal, Utrecht, the Netherlands Pl146-CPDA-1-35 ml, modied to 25 ml). CB units were stored at 46 C on arrival at the blood bank and cryopreserved within 24 h of collection. The CB units were volume reduced in a closed system, as described by Rubinstein et al. [10]. In brief, HES was added to the anticoagulated CB at a volume ratio of 1 : 5. As a result of centrifuging (50 g) the CB/HES mixture in the original collection bag, a stem cell-rich supernatant was separated from the residual erythrocyte fraction (EF). The stem cell-rich supernatant was then expressed from the bag to a plasma transfer bag and centrifuged to sediment the cells. Surplus supernatant plasma fraction (PF) was transferred into a waste bag. Finally, the sedimented stem cells were resuspended in supernatant plasma to a volume of 20 ml and designated as stem cell concentrate (SCC). The SCC was cryopreserved in 5 ml of 50% DMSO in 5% (wt/vol) Dextran-40 (Mr 40 000 Da) to give a nal concentration of 10% DMSO. CB collections with a volume of > 120 ml (including anticoagulant) were processed into two SCCs in separate bags. SCCs collected for banking were frozen by controlled rate freezing and stored at 150 C. SCCs from spiked units were frozen and stored at 86 C. The EF and PF were sampled for microbial culture. The residual PF was frozen and stored at 86 C; the remaining EF was used for human leucocyte antigen (HLA) typing.

Bacterial contamination of EF and PF from units collected for banking

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

Contamination level of PF and SCC post-thaw

From routine screening of CB units, 94 of 740 units were culture positive prior to cryopreservation. The PFs from 56 of the 94 positive units were available for testing post-thaw. Immediately after thawing, 20 ml of the PF was cultured in standard aerobic and anaerobic culture bottles (Organon Teknika, Turnhout, Belgium). To establish the number of viable bacteria, an additional 5 ml was plated onto four agar plates. The SCC (25 ml/including 5 ml of DMSO medium),

Thawing of PF and SCC

The PF was thawed rapidly in a 37 C water bath and cultured immediately. The SCC was removed from storage at 86 C or 150 C and allowed to stand at room temperature for 15 min before being rapidly thawed in a 37 C water bath. The SCC was cultured either before or after removal of DMSO.
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34 . Honohan et al.

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 Precryopreservation CB units (n) 9 EF 27/27 positive; median: 40 CFU/ml (range: 2426 CFU/ml) N/A PF 27/27 positive; median: 40 CFU/ml (range: 2 426 CFU/ml) 8/8 positive; median: 16 CFU/ml (range: 2 40 CFU/ml) SCC 36/36 positive; median: 40 CFU/ml (range: 2 426 CFU/ml) 24/25b positive; median: 16 CFU/ml (range: 2 40 CFU/ml)

Post-thaw

4a

CB, cord blood; CFU, colony-forming units; EF, erythrocyte fraction; N/A, not available for culture; PF, plasma fraction; SCC, stem cell concentrate. a Four of nine units were spiked at lower levels: 2 40 CFU/ml. b 0ne 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.

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 a laminar ow cabinet. Standard BacT/Alert culture bottles (Organan Teknika) were lled to the recommended volume (10 ml), except where stated otherwise. The culture bottles were incubated at 35 C and monitored for up to 14 days using the BacT/Alert continuous monitoring blood culture system (Organon Teknika). All culture-positive bottles were subcultured and isolates were identied by conventional microbiological procedures. Establishment of the number of viable bacteria post-thaw was performed on the preferred agar (sheep blood or chocolate) for the micro-organism isolated prior to cryopreservation. All agar plates were incubated at 35 C for 6 days and colony counts were performed on two occasions. All isolates were frozen in glycerol and stored at 70 C.

precryopreservation, were positive. One post-thaw sample of 02 ml from a unit spiked with < 3 CFU/ml remained culture negative (Table 1). The three strains used in this study were viable post-thaw after cryopreservation in 10% DMSO and storage at 86 C.

CB units for banking Contamination rate of EF and PF

Between March 1998 and December 2000, the EF and PF from 740 CB units (collected from 16 participating maternity centres) were cultured. A unit was dened as positive after conrmation of at least one positive culture bottle. The EF/ PF samples from 94 of the 740 units (127%) were contaminated prior to cryopreservation. Culturing a volume of 20 ml gave a signicantly higher number of positive results than culturing a 10-ml volume (P = 0024). There was no signicant difference in the number of positive results between 20- and 40-ml culture volumes (Table 2). The culture samples from the 94 contaminated units comprised 54 pooled EF/PF fractions, 20 unpooled EF and PF fractions, and 20 CB units of > 120 ml yielding two EF/PF pairs. Only one bottle was positive from 28 of the 54 pooled fractions. Only one fraction was positive from 14 of the 20 unpooled fractions; in eight cases 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 microbes isolated from EF/PF are shown in Table 3. Fourteen units yielded isolates we categorized as being potential pathogens, and 80 units yielded isolates we categorized as being of low pathogenicity.

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

Results
Spiking experiments
Table 1 shows the precryopreservation and post-thaw microbial culture results of CB units spiked with three reference strains. All cultures from all fractions cultured in volumes ranging from 02 to 5 ml for SCC and 105 ml for PF and EF

Post-thaw contamination level of the PF and SCC

The PFs from 56 of the 94 positive CB units were cultured post-thaw. Twenty-three PFs were culture negative, indicative
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Table 2 Culture results of 10, 20 and 40 ml samples of equal volumes of the erythrocyte fraction (EF) and plasma fraction (PF) of volumereduced cord blood units collected for banking

Period of testing March 1998March 1999 April 1999December 2000 April 1999December 2000b Total
a

Sample volume (ml) 10 20 40

No. cultured 216 445 79 740

No. positive 18 65 11 94

Percentage positive 83 146a 139 127

95% CI 4971317 1127 1862 6942490

Signicantly higher than with the 10-ml volume; P = 0024 (odds ratio 188; 95% condence interval [CI] 109326). b See the Materials and methods.

Table 3 Micro-organisms isolated from 94 cord blood units collected for banking Potential pathogensa (no. of contaminated units) Escherichia coli (1) Enterococcus faecalis (1) Streptococcus agalactiae (5) Bacteroides spp. (2) Candida albicans (1) Haemophilus inuenzae (1) Strictly anaerobic Gramnegative rods (3) Micro-organisms of low pathogenicityb Coagulase-negative Staphylococcus Diphtheroid rods Propionibacterium spp. Streptococcus viridans Lactobacillus Non-haemolytic Streptococcus Bidobacterium spp. 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

the corresponding prefreeze EF/PF and PF post-thaw. The microbial load of the SCC (standard volume 25 ml) ranged from 1 to 204 CFU/25 ml (median: 13 CFU/25 ml). More than one micro-organism was isolated from four SCC (UD2126, 1570, 1403 and 2837). The microbial load of the SCC corresponded well with the PF post-thaw (148 CFU/5 ml; median: 2 CFU/5 ml). Identication of the micro-organisms revealed at 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 discrepancy was observed between the EF/PF, the PF post-thaw and the SCC in all eight units. S. agalactiae, coagulase-negative staphylococcus (CNS) and diphtheroid rods remained viable after cryopreservation in 10% DMSO and storage at 150 C. Neither Haemophilus inuenzae (UD2566) nor non-haemolytic streptococcus (UD1403, UD2126) were recovered post-thaw from the SCC or PF.

n = 14. Micro-organisms of low pathogenicity were also isolated from a number of units. b n = 80. Multiple micro-organisms of low pathogenicity were isolated from a number of units.

Discussion
CB units spiked before processing showed no preferential localization of bacteria after centrifugation and thereby established the EF and PF (after volume reduction by HES sedimentation) as suitable samples for estimating the bacterial contamination rate of the SCC (Table 1). A sample volume of 1 ml of EF or PF was suitable for detecting a contamination level of 2 CFU/ml. Culturing the EF and PF avoids the unnecessary loss of the already limited number of stem cells. We observed a contamination rate of 13% from the routine culture of CB units for banking. A number of CB banks are now reporting a reduction in the bacterial contamination rate to < 1% [1113] and < 5% [14], after extensive training in collection procedures and the introduction of stringent disinfection protocols. However, as the results of our study support those of a previous study [15], which dened the volume of the sample cultured as being the single most important factor governing the sensitivity of the blood culture, these contamination rates must be viewed in the light of the volume cultured. The London, Dsseldorf and Galician CB Banks

of a contamination level of < 1 CFU/20 ml. Micro-organisms were isolated from the 33 remaining PF. Twenty-two of the 33 PF showed a low contamination level of 1 CFU/1020 ml and 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 same micro-organism(s) was identied from the EF/PF and PF post-thaw; in four of the 28 cases an additional microorganism was isolated from the PF post-thaw and in eight of the 28 cases at least one of the organisms isolated from the EF/PF was not found in the PF post-thaw. A completely different micro-organism was isolated from the prefreeze EF/PF and PF post-thaw in ve of 33 cases. The SCC was cultured post-thaw from eight CB units, selected because of a relatively higher contamination level, as determined from culturing the PF post-thaw (148 CFU/5 ml). Table 4 shows the culture results of eight SCC post-thaw and
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36 . Honohan et al.

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 Contamination level (CFU/25 ml) 204 170 PF post-thaw Contamination level (CFU/5 ml) 12 36

UD no. 1625

Isolate a. S. agalactiae b. S. agalactiae

Isolate S. agalactiae S. agalactiae

Isolate S. agalactiae S. agalactiae E. coli CNS Diphtheroid rods CNS CNS Diphtheroid rods CNS Diphtheroid rods

1639

a. CNS b. CNS

CNS CNS CNS Diphtheroid rods CNS Diphtheroid rods

9 12 16

5 1 1

2126

a. CNS Diphtheroid rods Non-haemolytic strep. b. CNS Diphtheroid rods Non-haemolytic strep. CNS a. negative

21

2491 1570

Micrococcus S. agalactiae Diphtheroid rod Micrococcus Non-haemolytic strep. S. agalactiae Micrococcus Diphtheroid rods Diphtheroid rods CNS S. agalactiae

1 6

CNS NT

48 NT

b. S. agalactiae

NT

NT

2566 1403

H. inuenzae Diphtheroid rods Non-haemolytic strep. S. agalactiae CNS

2 19

Diphtheroid rods Diphtheroid rods

1 1

2837

13

S. agalactiae

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 identication number. E. coli, Escherichia coli; H. inuenzae, Haemophilus inuenzae; strep., streptococcus; S. agalactiae, Streptococcus agalactiae.

culture 1, 2 and 15 ml, respectively [12 14]. The volume cultured by the Milano CB Bank is not specied [11,16], which makes it difcult to place any value on the reported contamination rate. In contrast to other CB banks, we observed an increase in the contamination rate from 87 to 14% by increasing the culture volume from 10 to 20 ml (Table 2). Culturing 20 ml of the EF/PF gave a signicantly higher percentage of positives than culturing 10 ml (P = 0024). There was no signicant difference in the contamination rate after culturing 20 ml or 40 ml, which suggests that a sample volume of 20 ml is sufcient to detect the majority of contaminants in CB. The culture results from pooled and unpooled EF and PF from 94 contaminated units indicated a low microbial load. The PF was evaluated post-thaw from 56 of the 94 contaminated units; in 45 cases the microbial load was 1 CFU/20 ml.

From eight of 11 units with a relatively higher contamination level (148 CFU/5 ml of plasma post-thaw), the SCC showed a microbial load of 2204 CFU/25 ml (median: 13 CFU/25 ml; Table 4), verifying that CB units were contaminated with very low numbers of bacteria. Results from EF and PF cultured separately 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 (ve of eight), was indicative of true contamination (Table 4). The few discrepancies observed after identication of the species may have been the result of several causes. These include: exogenous contamination of the culture bottle (most probable for UD2194 post-thaw plasma); a lower number of contaminating micro-organisms than the detection level of
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the sample cultured; or non-survival of the strain following cryopreservation in 10% DMSO. This study shows that a signicant number of micro-organisms remain viable, even when the initial contamination level is low. It is probable that discrepancies in culture results can occur at the low contamination levels observed in this study. Post-thaw culture results from spiking experiments show that 10% DMSO and cryopreservation at 86 C had no measurable effect on the viability of the three reference strains used in this study. Post-thaw culture results from CB units collected for banking showed that S. agalactiae, CNS and diphtheroid rods survive in 10% DMSO and cryopreservation at 150 C. Escherichia coli, Enterococcus faecalis and Candida albicans were isolated from the EF/PF from three different CB units but not from the corresponding PF post-thaw. H. inuenzae was isolated from the EF/PF from a fourth unit, but not from the corresponding PF post-thaw or from SCC. Given the spiking results, these bacteria were probably present at a number below the detection level of the sample volume cultured. These four micro-organisms are potential pathogens, and the presence of even low numbers of potentially pathogenic micro-organisms is a criterion for discarding units from our bank. A recent report of septic shock caused by Bacillus cereus after unrelated bone marrow (BM) transplantation highlights the implications of pathogenic bacteria in stem cell therapy [17]. The potential risks of not detecting pathogens in culture where the sample volumes are low should be reviewed. Of the 94 contaminated units, 85% were contaminated with micro-organisms of low pathogenicity (Table 2). Information available from BM and peripheral blood progenitor cells (PBPC) data indicate that no recognized clinical sequelae results from the infusion of bacteria of low pathogenicity in neutropenic and immunosuppressed recipients [1821]. A number of CB banks have recently adopted a policy to release units for banking that are contaminated with bacteria of low pathogenicity [11,12]. The recently published Netcord-Fahct Standards [9] accept this policy. However, as these standards do not specify the culture method and/or the minimum test volume for demonstrating or excluding bacterial contamination, the suitability of such recommendations is doubtful. We suggest that these standards should be extended to include a maximum acceptable contamination level and to exclude the release of units contaminated with potential pathogens. At our centre this would mean discarding 18% of units as a result of contamination with potential pathogens. Our CB bank has adopted the policy of releasing units contaminated with microorganisms of low pathogenicity with a report of the contamination level. An antibiotic sensitivity test can be performed on the frozen isolate(s) upon selection for transplantation. In conclusion, CB is contaminated with bacteria at such a low level that the volume cultured (up to 20 ml) is directly related to the contamination rate. These low numbers of
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contaminants can be detected without the loss of stem cells by culturing the EF/PF after volume reduction by HES sedimentation. In 85% of the contaminated units, micro-organisms of low pathogenicity were not identied as a reason to prohibit the release of such CB for transplantation. However, the trend towards ex vivo expansion and genetic manipulation of CB stem cells will challenge the contamination rate of CB banks employing microbial culture procedures with a low sensitivity. Further investigations are needed to evaluate whether these in vitro techniques create conditions in which low levels of contaminating bacteria may reach levels of clinical signicance, and to evaluate the role of addition of antibiotics, as often used in in vitro culture [22].

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