Monitoring bacterial contamination in equine platelet concentrates ...

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EQUINE VETERINARY JOURNAL Equine vet. J. (2010) 42 (1) 63-67 doi: 10.2746/042516409X455221

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Monitoring bacterial contamination in equine platelet concentrates obtained by the tube method in a clean laboratory environment under three different technical conditions M. E. ÁLVAREZ, C. E. GIRALDO and J. U. CARMONA* Grupo de Investigación Terapia Regenerativa, Departamento de Salud Animal, Universidad de Caldas, Calle 65 No 26-10, Manizales, Caldas, Colombia. Keywords: horse; autologous platelet concentrates; bacteriological quality control; regenerative therapy

Summary

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Reasons for performing study: There is a growing interest in the use of autologous platelet concentrates (PCs) as treatment for chronic musculoskeletal diseases in horses. However, there is no information on the risk of bacterial contamination during their preparation. Objectives: To: 1) assess the risk of bacterial contamination in equine PCs obtained by the tube method under 3 technical conditions: a) in a laminar flow cabinet, in a clean laboratory environment both with (b) and without (c) Bunsen burner; 2) identify the critical points of the process of PCs preparation with risk of bacterial contamination; and 3) identify the potential bacterial contaminants in the process and their antibiotic susceptibility. Methods: Bacteriological samples were taken from: the skin (shaved or unshaved) of the venipuncture site in 15 horses, both before and after being disinfected; hands and throat of the operator; caps of the tubes where the blood was processed; environment where the equine blood samples were collected; laboratory environment; laminar flow cabinet; bacteriological stove; and PCs obtained under 3 technical conditions. Results: Bacteria were isolated from nonaseptically prepared equine skin, hands and throat of the operator, and the place where the blood samples were taken. Bacteria were not isolated from tube caps, laboratory environment, laminar flow cabinet or PCs. The isolated bacteria were normal biota from equine skin, human skin and throat, and environmental contaminants. Of the isolated bacteria, 23% were resistant to penicillin, 19% to ampicillin, 2.12% to ceftiofur, 3.2% to sulphamethoxazole/trimethoprim and 1.1% to enrofloxacin. Resistance to amikacin and gentamicin was not seen. Conclusions and potential relevance: Uncontaminated PCs can be obtained by the tube method in a clean laboratory environment without the need for either a laminar flow cabinet or a Bunsen burner. It is mandatory to perform the procedure following strict aseptic technique.

Introduction There is growing interest in the use of both autologous cell and growth factor-oriented therapies as treatment for chronic musculoskeletal diseases in horses (Sutter 2007). Osteoarthritis, superficial digital flexor tendon tendinopathy and suspensory ligament desmopathy have been treated with autologous platelet concentrates (PCs) (Carmona et al. 2007; Argüelles et al. 2008; Waselau et al. 2008), bone marrow aspirates (Herthel 2001) and mesenchymal stromal cells (Smith et al. 2003). However, despite the spread of the use of these regenerative therapies (especially PCs) and the basic molecular knowledge that supports their clinical utilisation (Smith et al. 2006; Schnabel et al. 2007), there is no information on the risk of bacterial contamination during their preparation for injection into synovial sheaths (Argüelles et al. 2008) or joints (Carmona et al. 2007). These structures are very susceptible to bacterial contamination and infection could result in severe consequences. Protocols for obtaining and clinically applying cell or growth factor-based therapies emphasise the need to prepare these ‘biodrugs’ under sterile conditions in a laminar flow cabinet (Carmona et al. 2007; Argüelles et al. 2008). This situation restricts the preparation and therapeutic use of PCs and cell-based therapies to specialised institutions only and, on the other hand, limits their use under field conditions. The aims of this study were to: 1) evaluate the risk of bacterial contamination of equine PCs obtained by the tube method (Argüelles et al. 2006) and prepared under 3 different technical laboratory conditions, using: a) a laminar flow cabinet, b) a clean laboratory environment using a Bunsen burner and c) a clean laboratory environment without Bunsen burner; 2) identify the critical points for bacterial contamination during equine PCs preparation; and 3) identify the possible contaminant bacteria during the preparation process and determine their antimicrobial susceptibility. The hypothesis tested was that PCs processed by tube method in clean environment is contamination free of bacteria.

*Author to whom correspondence should be addressed. [Paper received for publication 18.02.09; Accepted 07.04.09]

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Materials and methods The study was approved by the Ethical Committee of the Universidad de Caldas. Horses Fifteen clinically normal mature Argentinean Creole horses were used in the study: 13 geldings, 1 stallion and 1 mare, age 6–22 years (mean 14 years). All animals were stabled at a riding school and were fed and managed under the same conditions. Study design The critical points associated with high risk for bacterial contamination during the preparation of equine PCs were identified as follows: 1) technical procedure: a) disinfection of venipuncture site (with or without previous shaving), b) blood collection, c) blood manipulation and packaging in tubes without addition of plasma of previous centrifugation, and d) procurement of the PC (final therapeutic product); 2) operator: a) hands and b) throat; 3) horse: flora from venipuncture site (skin); 4) environmental conditions related to PC preparation: a) environmental contaminants (bacteria) during blood collection and b) preparation of PCs under each of the 3 technical conditions studied. Serial sampling (with swabs) was carried out for bacteriological screening from the skin of each horse (shaved or unshaved), before and after its disinfection. The disinfection protocol included a scrub with chlorhexidine digluconate-based (0.5% w/v) liquid soap for 8 min, followed by an alternated cleaning with 10 sterile gauze impregnated with chlorhexidine digluconate solution 0.5% w/v, and 10 sterile gauze impregnated with ethyl alcohol 70%. A final cleaning using 2 sterile saline solution impregnated gauze was performed. Previously, smears from operator’s hands and throat were taken for bacterial culture. The same operator always performed all the procedures related to preparation of PCs. The operator used sterile gloves, surgical mask and hat during the venipuncture and a sterile surgical gown during the preparation of PCs. Smears for bacteriological culture from cap tubes used for both blood collection (citrate tubes) and packaging of plasma from the first centrifugation (tubes without additive) were performed. A microbiological analysis was conducted in the environment where the horses’ blood samples were obtained, in the environment of the 3 technical conditions evaluated and in the microbiological stove where all bacteriological cultures were performed. Finally, a microbiological study of PCs obtained under each of the 3 technical conditions evaluated was done. All bacteriological cultures were performed in triplicate. Collection and processing of operator samples Smears from the posterior pharynx were taken with sterile cotton swabs. The samples were sown immediately on 5% sheep blood agar plates and incubated at 37°C in an atmosphere of 5% CO2 during 24 h. Negative cultures were re-incubated for 48 h. This procedure was performed to determine if the operator was a carrier of Streptococcus pyogenes. Smears from the operator’s hands were taken with sterile cotton swabs previously damped in a 2 ml screw cap tube containing a sterile diluent of 0.1% peptonated water. Cotton swabs were rotated on the surface of the palms, between © 2009 EVJ Ltd

Monitoring bacterial contamination in equine platelet concentrates

fingers and nails of the operator and then washed in the diluent. The excess of liquid was drained on the tube’s wall. Samples were homogenised with a Vortex mixer and aliquots of 0.1 ml sown on 5% sheep blood agar plates and MacConkey’s medium. Plates were incubated at 37°C in an atmosphere of 5% CO2 during 24 h. Negative cultures were re-incubated for an additional 48 h. These samples were taken to detect if the skin of the operator’s hands carried Staphylococcus aureus, E. coli or other faecal coliforms. All blood agar colonies that presented a typical b-haemolysis and growth of Staphylococcus aureus were counted one day after incubation. Plates containing 20–200 isolated colonies were chosen for the microbiological analysis. In addition, lactose fermentation reactions in MacConkey agar characteristic of faecal coliforms were considered in the study. The identification of colonies was performed using commercial systems API Staph1 and Crystal Enteric/Non-fermenter ID kit2. Collection and processing of bacterial samples from horses’ skin Bacteriological samples were collected in the same fashion as the samples from the operator’s skin. The venipuncture sites (shaved or unshaved) were sampled with sterile cotton swabs before and after skin disinfection. Samples were processed 1 h after their collection. Aliquots of 0.1 ml from samples were sown on thioglycollate broth, chocolate agar and MacConkey agar. The number of colonyforming units/ml (CFUs/ml) was determined by seeding 3 zigzag lines in 5% sheep blood agar plates using decreasing dilutions (0.25, 0.1 and 0.05 ml) of supernatant of homogenised samples. CFUs were counted manually after 24 h of aerobic incubation at 37°C. The highest growth recorded for any sample was limited to 10000 CFUs/ml by the highest dilution (Zubrod et al. 2004). The preliminary identification of positive cultures was performed by microscopic examination of the colonies using Gram staining. Final identification was performed using commercial systems (Crystal Enteric/Non-fermenter ID Kit, API 20 Strep1, API Staph and API Coryne1). Collection and processing of bacteriological samples from the caps of the tubes both with and without anticoagulant These samples were collected, processed and evaluated in the same fashion as the operator’s bacteriological sampling process. Samples were also sown on thioglycollate broth, chocolate agar and MacConkey agar. Blood collection and preparation of the platelet concentrates One side of the neck was selected randomly for shaving and blood collection from the jugular vein. The site for venipuncture was disinfected following the aseptic protocol described previously. Blood was drawn from each horse and deposited in 36 tubes with sodium citrate 3.2% (4.5 ml blood/tube). Eighteen tubes were collected from each jugular vein and 6 citrate tubes used for each experiment. The order of preparation of PCs for each of the 3 technical conditions evaluated was chosen randomly. Whole blood was collected via a 21 gauge butterfly catheter and placed in citrate tubes. After centrifugation at 120 g for 5 min, the first supernatant plasma fraction (50%), adjacent to the buffy coat, was removed under aseptic conditions in either a class II laminar flow cabinet or in clean laboratory environment with or without Bunsen burner. This fraction was then centrifuged at 240 g for 5 min and 25% of

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the first fraction collected (as previously described by Argüelles et al. 2006). This fraction was drawn into sterile syringes and considered as a PC. PCs were prepared in the same laboratory (temperature 22°C, 70% relative humidity, airstream free and disinfected with standard procedures), according to the criteria established by Ritter et al. (2003).

penicillin, ampicillin, trimethoprim/sulphamethoxazole, enrofloxacin, ceftiofur, amikacin and gentamicin were evaluated. Statistical analysis

Platelet concentrates obtained were processed 1 h after their preparation. After homogenisation, the PCs were seeded, using decreasing dilutions of 0.25, 0.1 and 0.05 ml, on thioglycollate broth, chocolate agar and MacConkey agar. Furthermore, PCs were seeded on a 5% sheep blood agar plate and incubated at room temperature. The number of CFUs/ml was determined by seeding 3 zigzag lines in 5% sheep blood agar plates using decreasing dilutions described previously of supernatant homogenised samples. CFUs were counted manually after 24 h of aerobic incubation at 37°C. Negative cultures were re-incubated for additional 48 h. The highest growth recorded for any sample was limited to 10,000 CFUs/ml by the highest dilution (Zubrod et al. 2004).

Data were analysed with a commercial statistical software (SPSS 15.0)3 using nonparametric paired comparison (Wilcoxon signedrank) test to determine any statistical differences between the number of CFUs/ml isolated from the nonaseptically prepared equine skin (either shaved or unshaved), and the operator’s hands. As 60% CFUs/ml of the intact nonaseptically prepared equine skin presented counts over 10,000 CFUs/ml (right censored data), this number of colonies was considered the highest detection value in such cases. A Chi-squared test was performed to determine whether there were differences related to the distribution of bacteria between nonaseptically prepared equine skin (either shaved or unshaved) or between right and left hands of the operator. Statistical analysis was performed only for the number of CFUs/ml of these variables, because bacterial growth was not present under the other critical conditions evaluated. A P value ⱕ0.05 was accepted as a statistically significant difference for all the tests. Data are presented as median (range).

Collection and processing of environmental bacterial samples

Results

Environmental samples were collected by removing the lid of a new 100 mm diameter Petri dish with 5% sheep blood agar. Agar was exposed to the environment (place of blood collection, laboratory environment, microbiological incubator and laminar flow cabinet) for 10 min. The agar plates were placed approximately 1 m away from the horse or the operator in the laboratory (as appropriate) and incubated for 24 h in an aerobic atmosphere at 37°C. Negative cultures were re-incubated for additional 48 h. The identification of positive cultures was conducted by using commercial kits previously described.

A total of 4290 bacteriological cultures were performed. Bacterial growth was observed only in the nonaseptically prepared equine skin (either shaved or unshaved), hands and throat of the operator, and the environment where equine blood samples were obtained. No bacteria were detected on disinfected skin of the venipuncture site (shaved or unshaved), tube caps, clean laboratory environment, microbiological stove, laminar flow cabinet or in PCs. Bacteria species isolated from the horses’ skin, hands and throat of the operator, and the environment of the place of blood collection, as well as their antimicrobial susceptibility, are presented in Tables 1 and 2. Bacteria isolated from the skin (shaved or unshaved) before disinfection were predominantly normal biota (Staphylococcus spp., coagulase-negative Streptococcus spp., Corynebacterium spp., Moraxella spp. and Enterobacteria). The number of CFUs/ml found in the intact (nonshaved) skin of the horses without disinfection (10,000; range 1300–10,000 CFUs/ml) was

Bacteriological processing of the platelet concentrates

Antibiograms A test for antimicrobial susceptibility by the semi-quantitative Kirby-Bauer method (Bauer et al. 1966) was conducted in bacteria identified previously. The antimicrobial susceptibilities of the bacteria to antibiotics commonly used in equine practice, such as

TABLE 1: Bacteria isolated from horses, operator and environment in the study Horse

Genus and species Staphylococcus aureus Staphylococcus epidermidis Streptococcus bovis Streptococcus viridans Escherichia coli Enterobacter aerogenes Enterobacter sakazakii Enterococcus faecalis Corynebacterium spp. Acinetobacter baumanii Moraxella spp. Citrobacter freundii Bacillus subtilis

Operator

Skin without shaving and without disinfection

Shaved skin without disinfection

X X X X X X X

X X

X X X X

X X

Throat

Environment

Hands

Place where blood samples were collected

X X X

X X X X X X

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Monitoring bacterial contamination in equine platelet concentrates

significantly greater (Pⱕ0.001) than the number of colonies found on the nonaseptically prepared shaved skin (1900; range: 500–9600 CFUs/ml). However, the distribution of bacteria isolated was similar to the nonaseptically prepared equine skin, either shaved or unshaved. There was no statistically significant difference for CFUs/ml between the left hand (185, range: 20–200 CFUs/ml) and the right hand (50, range: 20–200 CFUs/ml) from the operator. Bacteria isolated from the operator’s hands and throat were normal biota (Table 1). Bacteria commonly found on nonaseptically prepared equine skin and operator’s hands were Staphylococcus aureus and Staphylococcus epidermidis. Streptococcus viridans was present in nonaseptically prepared equine skin (shaved or unshaved) and operator’s throat. Of the 13 bacterial species isolated, 23% were resistant to penicillin, 19% to ampicillin, 2.12% to ceftiofur, 3.2% to sulphamethoxazole/trimethoprim and, 1.1% to enrofloxacin. Resistance to amikacin and gentamicin was not seen (Table 2). An isolated strain of Staphylococcus epidermidis from the operator’s hands presented resistance to penicillin, ampicillin, sulphamethoxazole/trimethoprim and enrofloxacin, unlike the same bacterial species isolated from the skin of the horses, which were susceptible to all antibiotics tested. Bacillus subtilis was the only environmental pollutant isolated at the place where equine blood samples were collected (20, range: 30–200 CFUs/plate). Discussion The results of this study showed that potential sources of bacterial contamination for equine PCs obtained by the tube method (Argüelles et al. 2006) and processed under 3 different techniques, were indigenous biota of the skin of the horses, the biota of the throat and hands of the operator and environmental contaminants where equine blood samples were collected. Although bacteria were not found in equine PCs tested, it is possible that the most critical point for bacterial contamination is the aseptic preparation of the equine skin of the venipuncture site. This situation has been described for bacteriological quality control of human platelet concentrates (Lee et al. 2002; te Boekhorst et al. 2005; Walther-Wenke et al. 2006). It was not possible to find any publication related to bacteriological quality control of PCs used for regenerative therapy

procedures (at least not for haematological clinical usage) in either man or animals; nor was information found in relation to bacteriological quality control for bone marrow aspirates or mononuclear cells from equine fat used for clinical purposes. This issue is very important in relation with the control of quality of human platelet gels used for therapeutic purposes in some countries e.g. Italy (Borzini et al. 2006). On the other hand, some researchers recommend the use of prophylactic antibiotics after injecting expanded mesenchymal cells from equine bone marrow aspirates (Smith et al. 2003; Guest et al. 2008). Other researchers do not recommend using prophylactic antibiotics when autologous equine PCs are clinically used (Carmona et al. 2007; Argüelles et al. 2008), although this issue is of great controversy in both human and veterinary medicine. Bielecki et al. (2007) demonstrated that human platelet gel has in vitro antibacterial effect against Staphylococcus aureus and Escherichia coli, but not against Klebsiella pneumoniae, Enterococcus faecalis or Pseudomonas aeruginosa; moreover, the latter pathogen seems to grow when platelet gel is added to the culture media (Bielecki et al. 2007). An in vitro study on susceptibility of various pathogenic bacteria to equine autologous PCs should be performed to expand this kind of knowledge. Bacteria isolated from the skin of the horses in this study coincided with the normal biota and some environmental bacteria described previously by Nagase et al. (2002) and Zubrod et al. (2004). Bacteria isolated from the hands and throat of the operator were similar to those in previous reports (Pittet et al. 1999; Nagase et al. 2002; Hull and Chow 2007). Environmental bacteria isolated in the present study were similar to those reported by Zubrod et al. (2004) (Table 1). An important finding was the isolation, from the hands of the operator, of strains of Staphylococcus epidermidis that showed resistance to antibiotics commonly used in equine practice (Table 2). The use of sterile implements and proper aseptic technique (Pittet et al. 1999) was helpful in preventing the bacterial contamination of the equine PCs during its preparation. Proper hand disinfection and the use of sterile gloves (Pittet et al. 1999) are strongly recommended for preventing bacterial contamination of PCs when they are used therapeutically in the horse, especially intra-articularly (Carmona et al. 2007) or for intrasynovial purposes (Argüelles et al. 2008). It is important to bear in mind that

TABLE 2: Total isolates and number of bacterial strains that presented antimicrobial resistance in the study Antimicrobial resistance Genus and species Staphylococcus aureus Staphylococcus epidermidis1 Streptococcus bovi Streptococcus viridans Escherichia coli Enterobacter aerogenes Enterobacter sakazakii Enterococcus faecalis Corynebacterium spp. Acinetobacter baumanii Moraxella spp. Citrobacter freundii Bacillus subtilis Total strains

Penicillin 0/24 2/52 0/2 0/14 14/14 2/2 12/12 4/4 0/22 2/2 0/16 8/8 0/26

(0%) (3.8%) (0%) (0%) (100%) (100%) (100%) (100%) (0%) (100%) (0%) (100%) (0%)

44/188 (23%)

Ampicillin 0/24 2/52 0/2 0/14 10/14 2/2 8/12 4/4 0/22 2/2 0/16 8/8 0/26

(0%) (3.8%) (0%) (0%) (72%) (100%) (66%) (100%) (0%) (100%) (0%) (100%) (0%)

36/188 (19%)

Ceftiofur 0/24 0/52 0/2 0/14 0/14 0/2 0/12 2/4 0/22 2/2 0/16 0/8 0/26

(0%) (0%) (0%) (0%) (0%) (0%) (0%) (50%) (0%) (100%) (0%) (0%) (0%)

4/188 (2.1%)

SXT 0/24 2/52 0/2 0/14 2/14 0/2 0/12 0/4 0/22 2/2 0/16 0/8 0/26

(0%) (3.8%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (100%) (0%) (0%) (0%)

6/188 (3.2%)

Enrofloxacin 0/24 2/52 0/2 0/14 0/14 0/2 0/12 0/4 0/22 0/2 0/16 0/8 0/26

(0%) (3.8%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%)

2/188 (1.1%)

Amikacin 0/24 0/52 0/2 0/14 0/14 0/2 0/12 0/4 0/22 0/2 0/16 0/8 0/26

(0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%)

0/188 (0%)

SXT: Sulphamethoxazole/trimethoprim. 1Microbial drug resistance found on operator’s hands for penicillin, ampicillin, SXT and enrofloxacin.

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Gentamicin 0/24 0/52 0/2 0/14 0/14 0/2 0/12 0/4 0/22 0/2 0/16 0/8 0/26

(0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%) (0%)

0/188 (0%)

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colony counts as low as 33 CFUs/ml of Staphylococcus aureus can produce infectious arthritis in horses (Gustafson et al. 1989). An insignificant bacterial contamination in a PC could trigger serious consequences in the synovial tissue treated, especially when contaminants are bacteria resistant to conventional antibiotics, such as some of the bacteria observed in this study. It was observed that if the horse’s skin is properly disinfected, its shaving is not a critical factor associated with the contamination of the blood used for the PC preparation. These results are comparable to the findings of a study by Hague et al. (1997), who showed that the shaving of equine skin is not a critical point associated with risk of infection for arthrocentesis sites when they are aseptically prepared (Hague et al. 1997). The disinfection protocol used for the skin of the horses in this study is often used by equine practitioners (Freeman 2006), although other disinfection protocols including povidone-iodine, or iodine tincture in combination with alcohol, could also be used (Hague et al. 1997; Zubrod et al. 2004; Freeman 2006) for preparing equine PCs. Finally, the hypothesis of the study was validated, since it is not necessary to use a laminar flow cabinet or a Bunsen burner to prepare sterile PCs if the environment of the laboratory (or room) remains clean, free of dust and airstream. Equine clinicians can use the tube method for preparing therapeutic PCs with minimal technical requirements in field conditions. Acknowledgements The authors thank Dr Manuel Navarro-Gonzalez at the Australian National University and, Dr Piero Borzini at the Blood Transfusion Centre, Department of Haematology and Transfusion Medicine, Ospedale Santi Antonio e Biagio, Alessandria, Italy for helpful comments on the manuscript. They also thank Catalina López, MVZ for her technical assistance and to the Escuela de Carabineros ‘Alejandro Gutiérrez’ of the National Police of Colombia. This research was funded by the Vicerrectoría de Investigaciones y Postgrados of the Universidad de Caldas, Columbia. Manufacturers’ addresses 1

bioMérieux, Marcy l’Etoile, France. Becton Dickinson Microbiology Systems, Cockeysville, Maryland, USA. SPSS, Chicago, Illinois, USA.

2 3

References Argüelles, D., Carmona, J.U., Pastor, J., Iborra, A., Viñals, L., Martínez, P., Bach, E. and Prades, M. (2006) Evaluation of single and double centrifugation tube method for concentrating equine platelets. Res. vet. Sci. 81, 237-245. Argüelles, D., Carmona, J.U., Climent, F., Muñoz, E. and Prades, M. (2008) Autologous platelet concentrates as a treatment for musculoskeletal lesions in five horses. Vet. Rec. 162, 208-211. Bauer, A.W., Kirby, W.M., Sherris, J.C. and Turch, M. (1966) Antibiotic susceptibility testing by a standardized single disc method. Am. J. clin. Pathol. 45, 493-496.

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Carmona, J.U., Argüelles, D., Climent, F. and Prades, M. (2007) Autologous platelet concentrates as a treatment of horses with osteoarthritis: a preliminary pilot clinical study. J. equine vet. Sci. 27, 167-170. Freeman, D.E. (2006) Sterilization and antiseptics. In: Equine Surgery, 3rd edn., Eds: J.A. Auer and J.A. Stick, Saunders Elsevier, St Louis. pp 112-123. Guest, D.J., Smith, M.R. and Allen, W.R. (2008) Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study. Equine vet. J. 40, 178-181. Gustafson, S.B., Mcilwraith, C.W. and Jones, R.L. (1989) Comparison of the effect of polysulfated glycosaminoglycan, corticosteroids, and sodium hyaluronate in the potentiation of a subinfective dose of Staphylococcus aureus in the midcarpal joint of horses. Am. J. vet. Res. 50, 2014-2017. Hague, B.A., Honnas, C.M., Simpson, R.B. and Peloso, J.G. (1997) Evaluation of skin bacterial flora before and after aseptic preparation of clipped and nonclipped arthrocentesis sites in horses. Vet. Surg. 26, 121-125. Herthel, D.J. (2001) Enhanced suspensory ligament healing in 100 horses by stem cells and other bone marrow components. Proc. Am. Ass. equine Practnrs. 47, 319-321. Hull, M.W. and Chow, A.W. (2007) Indigenous microflora and innate immunity of the head and neck. Infect. Dis. Clin. North Am. 21, 265-282. Lee, C.K., Ho, P.L., Chan, N.K., Mak, A., Hong, J. and Lin, C.K. (2002) Impact of donor arm skin disinfection on the bacterial contamination rate of platelet concentrates. Vox Sang. 83, 204-208. Nagase, N., Sasaki, A., Yamashita, K., Shimizu, A., Wakita, Y., Kitai, S. and Kawano, J. (2002) Isolation and species distribution of staphylococci from animal and human skin. J. vet. med. Sci. 64, 245-250. Pittet, D., Dharan, S., Touveneau, S., Sauvan, V. and Perneger, T.V. (1999) Bacterial contamination of the hands of hospital staff during routine patient care. Arch. Int. Med. 159, 821-826. Ritter, M., Schwedler, J., Beyer, J., Movassaghi, K., Mutters, R., Neubauer, A. and Schwella, N. (2003) Bacterial contamination of ex vivo processed PBPC products under clean room conditions. Transfusion 43, 1587-1595. Schnabel, L.V., Mohammed, H.O., Millar, B.J., Mcdermott, W.G., Jacobson, M.S., Santangelo, K.S. and Fortier, L.A. (2007) Platelet rich plasma (PRP) enhances anabolic gene expression patterns in flexor digitorum superficialis tendons. J. orthop. Res. 25, 230-240. Smith, J.J., Ross, M.W. and Smith, R.K. (2006) Anabolic effects of acellular bone marrow, platelet rich plasma, and serum on equine suspensory ligament fibroblasts in vitro. Vet. comp. Orthop. Traumatol. 19, 43-47. Smith, R.K., Korda, M., Blunn, G.W. and Goodship, A.E. (2003) Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment. Equine vet. J. 35, 99-102. Sutter, W.W. (2007) Autologous cell-based therapy for tendon and ligament injuries. Clin. Tech. Equine Pract. 6, 198-208. Te Boekhorst, P.A., Beckers, E.A., Vos, M.C., Vermeij, H. and van Rhenen, D.J. (2005) Clinical significance of bacteriologic screening in platelet concentrates. Transfusion 45, 514-519. Walther-Wenke, G., Doerner, R., Montag, T., Greiss, O., Hornei, B., Knels, R., Strobel, J., Volkers P. and Däubener, W. (2006) Bacterial contamination of platelet concentrates prepared by different methods: results of standardized sterility testing in Germany. Vox Sang. 90, 177-182. Waselau, M., Sutter, W.W., Genovese, R.L. and Bertone, A.L. (2008) Intralesional injection of platelet-rich plasma followed by controlled exercise for treatment of midbody suspensory ligament desmitis in Standardbred racehorses. J. Am. vet. med. Ass. 232, 1515-1520. Zubrod, C.J., Farnsworth, K.D. and Oaks, J.L. (2004). Evaluation of arthrocentesis site bacterial flora before and after 4 methods of preparation in horses with and without evidence of skin contamination. Vet. Surg. 33, 525-530.

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Author contributions All authors contributed to the initiation, conception, planning, execution and writing for this study. The microbiology was by M.E.A., and statistics by C.E.G. and J.U.C.

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