Evaluation of decontamination process of heart valveand artery tissues in European Homograft Bank (EHB):a retrospective study of 1,055 cases

Ye-Dong Fan • Beatrice Van Hoeck •

Vanda Holovska • Ramadan Jashari

Received: 28 January 2011 / Accepted: 4 April 2011 / Published online: 23 April 2011

� Springer Science+Business Media B.V. 2011

Abstract To evaluate the efficiency of decontami-

nation practice in European Homograft Bank (EHB),

the data of the cardiovascular tissues received during

recent 2 years were retrospectively analysed in this

study. After initial assessment, the tissues were incu-

bated in a 3-antibiotics’ cocktail at 4�C for 20–48 h.

The states of contamination were evaluated before and

after incubation with the focus on the differences in

donor type, tissue type, germ type and incubation time.

Amongst 1,055 eligible tissues, 77.2% were hearts and

22.8% were arteries. 82.2% of the tissues were

retrieved from the multi-organ donors (MOD), 15.4%

from the recipients of heart transplantation (RHT) and

2.4% from the non-heart beating donors (NHBD). The

initial contamination rate was 27.4% with a signifi-

cantly higher incidence in arteries. The RHT tissues

had the lowest contamination rate comparing to that of

MOD and NHBD. Staphylococcus species was the

major source of contamination. After antibiotic incu-

bation, 76.8% of the contaminated tissues were disin-

fected, which was significantly higher for the hearts

than the arteries. The RHT tissues had the highest

decontamination rate than that of MOD and NHBD

tissues. Propionibacterium acnes was detected in

48.1% of the remaining contaminated cases. The

average incubation time of the Propionibacterium-

positive tissues was significantly shorter than that of

decontaminated tissues. In conclusion, the current

decontamination protocol of EHB is sufficient for most

of the initially contaminated bacteria, whereas it is

inadequate for Propionibacterium acnes. This may be

related to the slow-growing nature of this bacterium

and thereby the relative shorter antibiotic incubation

time.

Keywords Heart valve and artery tissues �Decontamination � Propionibacterium acnes �Antibiotic incubation time

Introduction

Despite antiseptic practice during the process of donor

organ procurement and packaging, approximately 30%

of procured heart and arterial tissues are still contam-

inated with various microorganisms at the arrival of a

cardiovascular tissue bank. To prevent bacterial trans-

mission to allografts’ recipients, decontamination is

thus an important procedure commonly practiced in the

tissue banks around the world (van Kats et al. 2010;

Ireland and Spelman 2005; Gulbins et al. 2003; Dacey

2000). Although the setting of antibiotic treatment

does have a significant impact on decontamination

success, to date, no consensus exists on an optimal

formula. Several different decontamination protocols

Y.-D. Fan (&) � B. Van Hoeck � V. Holovska �R. Jashari

European Homograft Bank (EHB), International

Association, c/o Military Hospital Queen Astrid,

Brussels, Belgium

e-mail: yedong.fan@gmail.com

123

Cell Tissue Bank (2012) 13:297–304

DOI 10.1007/s10561-011-9255-3

are applied in the different tissue banks (Angell et al.

1976; Strickett et al. 1983; Kirklin et al. 1987; Barratt-

Boyes et al. 1987; Gall et al. 1995; Stolinski et al. 2006;

Jashari et al. 2007). The differences between these

protocols are mainly in the composition and concen-

tration of antibiotic cocktail, the temperature and the

duration of antibiotic incubation. In spite of these

variations, the reported decontamination rates from

different centres are comparably between 60 and 70%.

Approximately 30% of the potential allografts are

therefore lost purely due to decontamination failure. In

face to an increasing clinical demand for cardiovascu-

lar allografts, further efforts are needed to improve

decontamination efficiency. In addition, a bacterium

may become resistant to a certain type of antibiotic,

which makes a routine evaluation of decontamination

result an essential practice for a tissue bank.

Since 2005, our institution (the European Homo-

graft Bank, EHB) has used a modified decontamination

protocol with a 3-antibiotics’ cocktail of low concen-

tration including Lincomycin HCL, Polymixin B Sul-

phate and Vancomycin HCL. The proceeded heart

valve and artery tissues are incubated in the antibiotic

cocktail at the temperature of 4�C for between 20 and

48 h not exceeding 72 h. The preliminary results have

revealed that this 3-antibiotics’ cocktail achieved a

decontamination rate equivalent to that of the former

4-antibiotics’ cocktail (Jashari et al. 2007). However,

the study group of this previous report is rather small.

To evaluate the efficiency of the current decon-

tamination practice of EHB, in this study, the data of

all the heart and arterial segments received by EHB

during the period of 2006–2008 were retrospectively

reviewed. Following the inclusive criteria, the bacte-

riological statuses of all the qualified tissues were

compared before and after antibiotic incubation. The

initial contamination rate and decontamination rate

were analysed in relation to different tissue types,

donor types, germ types and incubation time.

Materials and methods

Tissue preparation

Procurement

According to the selection criteria (EU Directive 23,

2004; EATB standards, 2004), hearts and arteries

were procured from the networks of hospitals in

Belgium (7), France (3), Luxembourg (1), Germany

(2) and Switzerland (4) under strict aseptic condi-

tions. The tissues were retrieved from either the

multi-organ donors (MOD), or the recipients of heart

transplantation (RHT), or the non-heart-beating

donors (NHBD). The tissues of MOD and RHT were

procured under the standard sterile condition,

whereas the retrieval of NHBD tissues was often

performed in mortuaries under a clean condition. The

procured donor hearts or/and arteries were preserved

in a sterile triple bag with a cold sterile isotonic

solution and transported to EHB.

Tissue preparation

The tissue dissection was completed within 24 h after

retrieval from the MOD and RHT donors, and 24 h

after cardiac arrest of the NHBD donors. Tissue

preparation was carried out in a clean room of Class

A with vertical laminar flow (Basan b. v, Minervum

7020, 4817 ZL Breda, the Netherlands). The entire

process followed strictly the quality standards for

organs, tissues and cells required by the Council of

Europe, The Belgian Health Council, European

Association of Tissue Banks (EATB) and EHB.

After dissection, the tissues were macroscopically

examined for morphological abnormalities (including

congenital malformations, pathological conditions

and physical damage) and measured.

Decontamination

All the morphologically acceptable tissues were then

immersed in a solution of low-concentration antibi-

otic cocktail. Three different types of antibiotic were

added to 250 ml of Medium 199 with Hanks’ Salts, L-

Glutamine and 25 mM HEPES (Medium 199; Invit-

rogen, Belgium) to final concentration of Lincomycin

HCL 120 lg ml-1 (Lincocin 600 mg; Pfizer, Bel-

gium), Polymyxin B Sulphate 124 lg ml-1 (Poly-

myxini B Sulfas 5,000,000 IU; Fagron, France) and

Vancomycin HCL 50 lg ml-1 (Vancocin 500 mg;

GlaxoSmithKline, Belgium). The antibiotic incuba-

tion proceeded at the temperature of 4�C for mini-

mum 20 h. In case the tissue donor had been kept on

the respirator for longer than 24 h or the tissue was

procured under clean conditions, the antibiotic incu-

bation was prolonged up to 48 h.

298 Cell Tissue Bank (2012) 13:297–304

123

Cryopreservation

After decontamination, tissues were rinsed with

Medium 199, re-measured, transferred into a cryo-

protective solution (10% DMSO in Medium 199) and

sealed in a double pouch. The pouch was frozen in a

program-controlled-rate freezer (Planer Kryo 560-16,

Planer, Middlesex, UK) till -160�C and then stored

at the vapour phase of liquid nitrogen in a storage

tank (-150� to -186�C).

Microbiological control

Sampling

In addition to the other measures for quality control

such as serology and histology, specimens for bacte-

riological culture were routinely collected from all the

allografts at three different time points: (1) at the

beginning of tissue processing (A sample), 40 ml of

transport solution was collected directly from the bag

containing the human body material that was then

flushed through a 0.45 lm filter membrane. The filter

membrane was put into two jars with tissue medium;

(2) after antibiotic incubation (B sample), small

fragments of the incubated tissues were collected with

20 ml of the decontamination solution; (3) just before

final sealing, 10 ml of the cryo-protective solution was

collected from the inner pouch (C sample).

Testing

According to the EU regulation and Belgian legisla-

tion for cardiovascular allografts, the bacteriological

examinations were performed in two certified labo-

ratories (Central laboratory of the Military Hospital

Queen Astrid, Brussels; Laboratory for Control and

Analysis, Brussels).

All the samples were tested for the presence of

aerobic and anaerobic bacteria (Steritest; Millipore,

USA). The presence of fungi was also tested. The

tissues with positive microbiological results at B and/

or C sampling were rejected.

Data analysis

The data of all the hearts and arterial batches received

in EHB during the period of 2006–2008 were

retrospectively reviewed.

Exclusive criteria

1. All the tissues with morphological abnormalities;

2. All the tissues of serological positive for HIV1,

HIV2, HTLV, HBV, HCV, Syphilis and Q fever.

3. All the tissues of fungi positive.

For all the qualified cases, the bacteriological results

at both A and B samplings were analysed in relation

to the donor type, tissue type, germ type and

incubation time. The data of A sampling were

examined to evaluate the initial contamination states,

while it was further compared with that of B

sampling to evaluate the decontamination efficiency.

As it was extremely rare that a B-sampling-negative

tissue was tested positive at C sampling, the data of C

sampling was not further discussed.

Statistics

The data were expressed as mean ± SD. The Chi-

squared test was used to test the differences between

the different tissue groups. The Student’s t-test was

applied to compare the differences between the

different antibiotic incubation times. A P value of

\0.05 was regarded as statistically significant.

Results

Tissue and donor characteristics

A total of 1,055 heart and arterial tissues were eligible

for the study criteria. 77.2% of the tissues were from

the hearts (n = 814) and 22.8% were from the arteries

including arch of aorta, thoracic aorta, aortic bifurca-

tion, iliac artery and femoral artery (n = 241)

(Fig. 1a). 82.2% of the tissues were retrieved from

MOD (n = 868), 15.4% were from RHT (n = 162)

and only 2.4% were from NHBD (n = 25) (Fig. 1b).

Initial contamination

At bacteriological sampling A, a total 289 out of

1,055 tissues were positive and the initial contami-

nation rate was therefore 27.4%. The heart tissues had

an initial contamination rate of 25.3%, which was

significantly lower than 34.4% of the arterial tissues

(P = 0.005) (Fig. 2a).

Cell Tissue Bank (2012) 13:297–304 299

123

The initial contamination rate for the tissues from

RHT donors was 9.9%, which was significantly lower

than that of MOD tissues (30.8%; P \ 0.001) and

that of NHBD tissues (24%; P = 0.041) (Fig. 2b).

From the samples of the contaminated tissues, 36

different types of bacteria were identified (Table 1).

Staphylococcus species was the major source of

contamination and accounted for 61.6% of total

contamination (heart, 58.7%; artery, 68.7%). 9% of

the contaminated tissues were Propionibacterium

species positive (heart, 9.2%; artery, 8.4%) and

7.6% were Escherichia coli positive (heart, 7.8%;

artery, 7.2%) (Fig. 2c).

Decontamination

After antibiotic incubation, 222 out of 289 initially

contaminated tissues were bacteria free at sampling B

and the decontamination rate was therefore 76.8%.

The heart tissues had a decontamination rate of

80.1%, which was significantly higher than 68.7% of

the arterial tissues (P = 0.005) (Fig. 3a).

Fig. 1 Tissue and donor

characteristics. Amongst all

the eligible tissues

(n = 1,055), the majority

were hearts (a) and most of

them were procured from

the multi-organ donors

(MOD) (b). RHT the

recipients of heart

transplantation; NHBD the

non-heart beating donors

69.290.1

76

30.89.9

24

0%

20%

40%

60%

80%

100%

MOD RHT NHBD

PositiveNegative

72.6 74.765.6

27.4 25.334.4

0%

20%

40%

60%

80%

100%

Total Heart Artery

PositiveNegative

61.6%

9%

7.6%

21.8%

E. Coli

Propi.

Staph.

Others

(a)

(b)

(c)

Fig. 2 Initial

contamination. a Tissue

type. The heart tissues had a

significantly lower initial

contamination rate than that

of the arterial tissues

(P = 0.005). b Donor type.

The tissues from RHT

donors had the lowest initial

contamination rate than that

of MOD (P \ 0.001) and

NHBD (P = 0.041).

c Germ type. Amongst the

detected bacteria of 36

different types,

Staphylococcus species was

the major source followed

by Propionibacterium and

E. coli

300 Cell Tissue Bank (2012) 13:297–304

123

The initially contaminated RHT tissues were

100% decontaminated, which was significantly better

than that of MOD tissues (76.4%; P = 0.028). In

comparison, only 33.3% of the initially contaminated

NHBD tissues became bacterial free after antibiotic

incubation, which was significantly lower than that of

both RHT tissues (P \ 0.001) and MOD tissues

(P = 0.015) (Fig. 3b).

41 out of 1,055 tissues (3.9%) were negative at

sampling A, but tested positive after antibiotic

incubation (A - B ?). Amongst these 41 tissues,

28 were from the hearts and 13 were from the

arteries. Strikingly, 68.3% of these tissues (28/41)

were contaminated with Propionibacterium acnes

(Table 1).

Comparing to the 36 types of bacteria detected at

sampling A, 21 were completely eliminated after

antibiotic incubation (58%), while another 15

remained positive (42%). Peptostreptococcus and

Serratia plymuthica were the bacteria that were

newly identified at sampling B (Table 1). Amongst

the remaining 17 different types of bacteria, Propi-

onibacterium acnes was the most isolated germ type

with a positive rate of 48.1% (heart, 44.3%; artery,

55.3%). Staphylococcus species accounted for 19.1%

of total contamination (heart, 20%; artery, 18.4%).

Both Gram ? cocci and Streptococcus positive was

8.3%, while the former was more prominent for the

artery tissues (heart, 5.7%; artery, 13.2%) and the

latter for the heart tissues (heart, 11.4%; artery, 2.6%)

(Fig. 3c).

Antibiotic incubation time

Due to some technical reasons, 24 out of 1,055 tissues

(2.3%) had an antibiotic incubation time shorter than

the scheduled minimum 20 h, while other 61 tissues

(5.8%) were incubated longer than the scheduled

maximum 48 h. The average antibiotic incubation

time was 32.9 ± 11.5 (11–72) h for all the proceeded

tissues.

For the tissues that were free of bacteria after

decontamination process (n = 222), the average

incubation time was 33.3 ± 11.6 h, while it was

30.8 ± 11.0 h for the tissues that failed in decon-

tamination (n = 108; P = NS). For the Propioni-

bacterium species contaminated tissues, those

succeeded in decontamination (n = 16) had an

Table 1 The bacterial species detected before (Sample A) and

after (Sample B) antibiotic incubation

Bacteria identification Number of positive cases

Sample A Sample B

Acinetobacter haemolyticus 1 1

Bacillus gram negative 4

Bacillus gram positive 4 3

Bacillus subspecies 1

Brevundimonas vesicularis 1

Candida albicans 3 7

Citrobacter species 1

Corynebacterium subspecies 8 1

Enterobacter aerogenes 1

Enterobacter cloacae 1

Enterobacter faecalis 2 2

Escherichia coli 17

Flavobacterium subspecies 1

Gram 1 coccus 5 8

Hafnia alvei 1

Klebsiella oxytoca 2

Klebsiella pneumonia 2

Leclercia adecarboxylata 1

Micrococcus subspecies 8

Peptostreptococcus 2

Propionibacterium acnes 20 50

Propionibacterium avidum 1

Propionibacterium microaerophilum 2

Pseudomonas aeruginosa 4

Serratia plymuthica 1

Staphylococcus aureus 10 10

Staphylococcus capitis 17

Staphylococcus coagulase negative 22 2

Staphylococcus cohnii 5

Staphylococcus epidermidis 61 6

Staphylococcus hominis 18 1

Staphylococcus lugdunensis 2

Staphylococcus saprophyticus 2

Staphylococcus warneri 20 1

Streptococcus mitis 1 2

Streptococcus parasanguinis 3 1

Streptococcus pneumoniae 2

Streptococcus viridans group 7 3

Regular: The bacterial species that were eliminated after

decontamination (58%); Bold: the bacterial species that

remained positive after decontamination (42%); Bold Italic:

the bacterial species that were newly detected after

decontamination

Cell Tissue Bank (2012) 13:297–304 301

123

average incubation time of 38.6 ± 11.0 h, while it

was only 30.5 ± 11.1 h for the tissues that remained

positive after antibiotic treatment (n = 54;

P = 0.016).

Discussion

The data of the current study show that the initial

tissue contamination rate and the microbes’ spectrum

are comparable to that of the previous reports from

our and other cardiovascular tissue banks (Jashari

et al. 2007; van Kats 2010). Again the initial

contamination rate of the arterial tissues was signif-

icantly higher than that of the heart tissues. This may

attribute to the less sterile states of the abdominal

compartment or lower limbs where the arteries are

retrieved, which is especially relevant if a trauma is

involved. As the arteries are routinely removed after

the heart retrieve, a relatively longer exposure time

may be another factor for the higher contamination

rate. In order to avoid potential cross contamination,

it is therefore advisable to pack the arterial tissues

apart from the heart tissues at the procurement.

Likewise the tissues from the recipient of heart

transplantation (RHT) had the lowest initial

contamination rate in comparison to those from

either the multi organ donor (MOD) or the non-

heart-beating donor (NHBD). In different from our

previous report (Jashari et al. 2007), the NHBD

tissues of this study had an initial contamination rate

lower than that of the MOD tissues though not

significantly. The rather small group size of NHBD

tissues may be part of the reason for this discrepancy.

The spectrum of organisms varied with different

tissue types and as well donor types. The most

commonly isolated organism was Staphylococcus

species followed by Propionibacterium species and

Escherichia coli.

Although the decontamination rate of 76.8% under

the current protocol is similar to that of the other

tissue banks, 23.2% of the potential allografts were

lost purely due to the decontamination failure.

Strikingly, Propionibacterium acnes appeared to be

the bacterium most resistant to the present decon-

tamination procedure despite the fact that it is

generally sensitive to Lincomycin and Vancomy-

cin—two major components of our 3-antibiotics’

cocktail. It accounted for approximately half of the

decontamination failure. Propionibacterium acnes is

an aerotolerant anaerobic gram positive bacterium

that is often part of the normal flora of human skin.

76.8 80.168.7

23.2 19.931.3

0%

20%

40%

60%

80%

100%

Total Heart Artery

PositiveNegative

76.4

100

33.3

23.6

0

66.7

0%

20%

40%

60%

80%

100%

MOD RHT NHBD

PositiveNegative

48.1%

19.1%

8.3%

8.3%

16.2%

Propi. acnes

Staph.

Others

Gram+cocci

Strep.

(a)

(b)

(c)

Fig. 3 Decontamination

rate. a Tissue type. The

heart tissues had a

significantly higher

decontamination rate than

that of the arterial tissues

(P = 0.005). b Donor type.

The tissues from RHT

donors had the highest

decontamination rate than

that from MOD

(P = 0.028) and NHBD

(P \ 0.001). c Germ type.

Amongst the remaining

bacteria of 17 different

types, Propionibacteriumacnes was the most isolated

germ type followed by

Staphylococcus species,

Gram ? cocci and

streptococcus

302 Cell Tissue Bank (2012) 13:297–304

123

Although it has relatively low virulence, Propioni-

bacterium caused serious endocarditis and aortic root

abscess have been reported including infections of

native valves, prosthetic valves and annuloplasty

rings. Propionibacterium endocarditis tends to pursue

a very aggressive clinical course with extensive

valvular destruction, congestive heart failure, abscess

formation and systemic embolization (Kanjanauthai

and Kanluen 2008; Mohsen et al. 2001).

It is interesting to notice that, even under the same

regiment of antibiotic treatment, the decontamination

rate still varied between the different tissue types and

as well donor sources. This may indicate that

antiseptic effects of the antibiotic cocktail used in

EHB are yet potent enough to surpass the bioburden

of all the incoming bacteria.

Besides the composition or the concentration of

the antibiotic cocktail itself, the temperature during

the decontamination is another factor that may

influence the antibiotic’s efficiency. In a recent study,

Germain and co-investigators have demonstrated that

at the temperature of 4�C, most of the bacteria are

rather resistant to several commonly used antibiotics.

These bacteria including Propionibacterium would

completely be eliminated by the same antibiotic

treatment, if the temperature is raised up to 37�C

(Germain et al. 2010). Apparently, the major concern

in relation to conducting a routine antibiotic treat-

ment at high temperature is that the warm ischemia

damage may hamper the matrix quality of cardiovas-

cular tissues.

Relatively slow in growing is a notable biological

feature of Propionibacterium. Comparing to the

average 7 days for the organisms to grow, a pro-

longed aerobic and anaerobic culture for up to

3 weeks may be required to detect a slow growing

organism such as Propionibacterium (Mohsen et al.

2001). This may explain in part for the low success

rate in decontamination especially against this micro-

organism, as antibiotic is known to be effective only

at the time when bacteria present in the tissue are

actively replicating (Gall et al. 1995). This could also

be the reason why a certain number of tissues in this

study were originally bacteria free but became

detected positive after antibiotic incubation. Of most

important, the data of the current study did reveal that

the tissues that remained Propionibacterium acnes

positive had significantly shorter antibiotic incubation

time. As a certain elongation of antibiotic treatment

may avoid the potential tissue damage induced by

high temperature, this may be a better option to

increase decontamination efficiency. This observa-

tion is indeed worthy of further investigation.

In conclusion, through systematically reviewing

the microbiological data of 1,055 heart and artery

tissues processed in EHB, the initial contamination

rate was 27.4% with a significantly higher incidence

in the arteries than in the hearts. Under the current

decontamination protocol, the decontamination rate

was 76.8%. Propionibacterium acnes was the micro-

organism most resistant to the present antibiotic

treatment. The low decontamination rate of this

bacterium may be related to its slow-growing nature

and thereby the relative short antibiotic incubation

time. As elongating incubation time may be an

optimal approach to achieve higher decontamination

efficiency, we have recently modified our decontam-

ination process correspondingly and the preliminary

results are promising.

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