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January 18, 2017

Tissue Banks & Cleanrooms: The Ultimate Price of Contamination Issues

Cooler for human organFrom the earliest times in human civilization, healers and physicians have dreamed of curing the sick and repairing the injured by transplanting organs or bones. The most common procedure, allogenic skin grafting – the process of transplanting skin from a donor to a patient – is now widely used to resurface burns, help close large open wounds, treat bed sores or ulcers, and prevent skin infections. But another grafting technique using bone was first described in medical literature as far back as 1668 when Job van Meeneren – a surgeon in Amsterdam – successfully restored a cranial defect by grafting bone from a deceased canine donor.(1) From the successes of George Pollack and J.H. Girdner in the late 1800s and the first use of refrigeration in tissue preservation in 1903, the stage was set for a new type of surgery – and all without the benefit of modern cleanroom facilities.

But the technology necessary for wide scale tissue transplants developed when clinicians realized that the same processes used for skin grafts could be used to treat bone defects or for tumor removal. The main obstacle to success, however, was in sourcing – and storing – harvested living tissue for later use. In the early part of the twentieth century, the problem seemed intractable but the 1930 establishment of the first blood bank brought the possibility of tissue banks one step closer. By1949, the first American tissue bank was founded in Maryland by George Hyatt. Three years later, Rudolph Klen opened Hardec Kralove, a bank based in Czechoslovakia, with the United Kingdom following suit in 1955 in Leeds.(2) By the early 1980s, tissue banks had sprung up in far-flung regions such as Myanmar (formerly Burma) and Thailand, and by 1986, more than three hundred nonprofit bone banks were in operation worldwide.(3)

Although banks initially operated on the basis of altruism with donations being sensitively solicited from the recently bereaved, the need for tissue and organ donations always outstripped the supply. In an attempt to encourage organ and tissue donation, the Uniform Anatomical Act was drafted by the National Conference of Commissioners on Uniform State Laws in 1968, establishing a donor card scheme that would, in effect, allow the bearer to legally donate their own organs after death and remove the burden of a decision from grieving next-of-kin. This nascent donor card scheme later combined with the Required Request legislation of 1986 that mandated hospitals create policies to request organ donation from the relatives of viable donors. Now, according to OrganDonor.gov, an initiative of the U.S. Department of Health and Human Services, 130 million citizens over the age of 18 are registered as donors – that’s a full 51% of the potential donor base in the U.S.(4)

So what exactly are tissue banks and what do they collect?

Harvesting cells with a cell scraperTissue banks are usually either housed at educational facilities or are for-profit entities. Universities with on-site cleanrooms may house small tissue collections for use in surgical training programs, but the larger banks tend to be commercial enterprises that collect a variety of tissues for third-party clients. Collecting not only whole body donations – frequently through ‘Willed Body Donation’ programs – commercial tissue banks also typically harvest skin, bones and organs, from a broad variety of anatomical and physiological systems – lymphatic, digestive, respiratory, integumentary, reproductive, and cardiovascular systems, for instance.(5) In addition, amnion – a substance collected from the placenta following delivery – is also harvested for use as a temporary cover for large burns or chronic wounds, and long bones are frequently recovered from the limbs of amputees. Human allografts (grafts from donor to recipient of the same species) are used to treat bone defects or to aid in joint reconstruction, and living tissue samples enable the creation of artificial tissues within an organic matrix for use in drug studies, to research the normal/abnormal biochemistry of cells, or for the creation of proteins from cell cultures.

According to the National Center for Biotechnology Information (NCBI), the overt sale of organs or tissues is illegal, but that has done nothing to slow the growth of tissue banks for one very simple reason.(6) Tissue banks charge a fee not for the tissues themselves, but for their services in the areas of public education, harvesting, processing, storing, preparation, and transportation. And when we look a little closer at both the technology and the resources involved, it is easy to see why these fees are applied. Take, for instance, the processes involved in tissue collection…

To ensure the optimal viability of the materials, samples for skin grafts are harvested as soon after death as possible. This process involves multiple layers of tissue bank expertise – from outreach to removal to processing and storage. For instance, on the death of a potential donor, the more than 100 tissue banks accredited by the American Association of Tissue Banks (AATB) will work with National Funeral Directors Association (NFDA) to ensure that best practices are followed in maintaining cadavers prior to tissue donation. These include both medical guidelines – such as the need to ligate major arteries and ensure prosthetic replacement for bones harvested – and support in understanding social/cultural guidelines and religious or ethnic considerations.(7) Once the donation passes from the funeral director, viable samples will be harvested. These might include skin, organs, eyes, femoral head removal, and other bone samples, but let’s look at the process for just two of these – skin and bone.

With all tissue processing, aseptic and sterile conditions must be maintained, and a class 10,000 cleanroom with laminar air flow is ideal.

In the case of skin removal, the targeted area is first prepared using povidone iodine and isopropopyl alcohol, with sterile sheets draping the surrounding area. Split-level thickness skin samples – those involving the removal of both the epidermis and the dermis for use in treating large areas – are removed by electric dermatome before being washed in saline and transferred to sterile, sealed containers. In order to avoid any possibility of cross-contamination, it is critical to store skin samples harvested from different body areas – the leg, the arm, or the back, for example – in different and easily identifiable containers. For each section of skin harvested an additional small segment is collected separately to be tested for viability. All containers are labeled with the time and date of collection, site of harvest, size of sample, and a unique ID.

Containers with tissue samples intended for allogenic grafting are usually stored using refrigeration, since alternate method of tissue preservation such as irradiation, freeze drying, or immersion in glycerol render the material non-viable for transplant. Bone samples are pressure washed using ethanol, acetone, or hydrogen peroxide to eliminate marrow and cellular debris before being shaped into blocks or stored as granules or powder. After preparation, they are stored in liquid nitrogen at temperatures of zero Celsius (32 degrees Fahrenheit) or are freeze dried.

And the importance of following correct preparation and storage procedures cannot be overstated in respect of future transplant success. Contrary to the popular Hollywood portrayal of cryogenic or suspended animation procedures, deep freeze systems are likely to simply fail, exposing the subject to a swift and unexpected thawing. Commercial cryo facilities are fitted with multiply-redundant alarm systems and fail-over temperature backups to guard against power disruptions, and the temperature of each inner chamber is recorded separately, with 24/7 monitoring and surveillance in place. Why different chambers? Tissue samples have different ‘life expectancies’ depending on their future usage. For those designated for use within two to three weeks of harvesting, a relatively balmy 4 degrees Celsius (39 degrees Fahrenheit) is an ideal storage temperature. But if tissues are to remain viable for longer, much cooler temperatures must be used. For instance, in order to store samples for up to six months, the freezing chamber must reach -70 to -80 degrees Celsius (-94 to -112 degrees Fahrenheit), and the exceedingly chilly environment of -180 to -196 degrees Celsius (-292 to -321 degrees Fahrenheit) will maintain viability for up to ten years.

And all of this requires state-of-the-art facilities. In an article submitted to the Indian Journal of Plastic Surgery, R.P. Narayan describes the ideal tissue bank as being a dedicated class 10,000 cleanroom that is able to segregate clean, sterile, and non-sterile  areas even to the point of them having separate access points. Movement into and within the sterile areas should always be unidirectional, with the flow moving from the employee scrub and change area to the surgical theater for tissue retrieval operations or via a corridor to a sterile tissue processing room. Quarantine and general tissue storage areas should be situated within the sterile confines, with no access either from the cadaver receiving areas or from the general access areas such as the administrative offices. For all three zones, separate air conditioning should prevent air mixing, and HEPA filtration should be combined with positive air pressure ventilation in sterile and aseptic rooms. Additionally, facilities should offer in-house laboratories to perform serology, tissue typing, and cell culture testing in order to minimize tissue travel outside of cleanroom facilities.

This sounds like an ideal scenario and one that would minimize the potential for contamination. But are these facilities infallible, and what happens if this ideal is not maintained? In the case of Brian Lykins, a college student from St Cloud, MN, the results were tragic…

the clostridiumIn 2001, Brian Lykins underwent surgical treatment to repair the cartilege in his knee. A not uncommon procedure, this transplant, however, would ultimately lead to a fatal infection and complete cariovascular collapse of the otherwise fit and healthy 23-year old. Given the extreme outcome of the procedure, the Centers for Disease Control (CDC) were notified and an inquiry got underway. The transplant tissues used in the surgery were provided by CryoLife, a tissue bank based in Kennesaw, GA, that also specializes in cardiac allografts, pediatric heart valves, and BioGlue® – a surgical adhesive.(8) But along with the transplant tissue, Lykins also received a catastrophic dose of Clostridium sordellii, a bacterium normally found only in soil or on farmed animals but now coating what surgeons had assumed to be sterile tissues. Massive infection and death followed within three days of surgery.

And tragically this was not the only case. As the CDC inquiry continued, investigators came across no fewer than 41 incidents of contaminated tissue sourced from CryoLife, none of which had previously been reported because tissue banks were not required to divulge such ‘adverse reaction.’ And perhaps the most damning fact is that when several other cases of contaminated tissues were investigated it was found that they had come from the same donor cadaver.(9)

So the problem was two-fold: firstly, current Good Management Practices (cGMP) had not been followed in screening the donor for potential infections; and secondly, procedures for harvesting and storing the tissues had failed catastrophically. And they would continue failing. Lykins death occurred in 2001 and the similar cases leading up to it occurred in the late 1990s. But in spite of regulatory intervention the company continued to eshew cGMPs, culminating in the 2013 receipt of an FDA warning letter in which investigators set out a litany of infractions that should already have been corrected. For instance, CryoLife’s policy of discarding solely contaminated tissues but retaining the rest of the cadaver seemed to be an obvious breach of protocol as indeed the serious failings in terms of aseptic processing SOPs. According to the later report, ‘poor asecptic techniques were observed of Microbiology Technician performing sterility testing’ and also that an ‘[e]mployee’s head covering was not attired during the filling process of the BioGlue Adhesive while working in a fume hood’, and equally that ‘[e]mployees were not trained on the validation requirements prior to protocol executive […] Training is not documented as required in the protocol.’(10)

And this dire warning letter was received a full 12 years after the death of Brian Lykins, a tragedy that ultimately sparked a recall of all of CryoLife’s human allograft tissues (with the exception of the neonate and pediatric heart values) and resulted in the loss of two-thirds of its market value between May and July 2002.(11) In addition, it came more than a decade after CryoLife, then one of the largest life-science companies in the U.S., was found to have also allowed heart valves contaminated with Candida Tropicalis, Candida Albicans, and Staphylococcus Epidermidis into the medical supply chain.(12) So despite knowing that there were problems as far back as the late 1990s, the company was still receiving critical FDA and CDC attention in 2013.

So what does that mean for patients and those of us involved in contamination control? As the 2011 paper ‘Cleanrooms and tissue banking how happy I could be with either GMP or GTP?’ by J. Klykens of the Cell and Tissue Banks, University Hospital Leuven (Belgium) et al concludes

“A controlled environment is mandatory for tissue and cell processing. A monitoring program based on at rest measurements is feasible for all cell and tissue banks and it is a strong element in the control of the environment in which safe allografts have to be processed.” (13)

In other words, contamination control and active monitoring of the ‘three Ps’ – processes, procedures, and personnel – is absolutely critical to the safety of tissues for transplant. As we’ve indicated in previous articles, knowledge of cGMPs combined with the creation of strict SOPs is the only way to minimize the potential for contamination and to prevent a large-scale danger to human health. And also, of course, ensuring that everyone within your company – from the most junior technician to the most senior executive – actually adheres to these living documents is also critical because, as was demonstrated in the case of Brian Lykins – a student with so much still ahead of him, someone’s life ultimately depends upon it.

Do you have thoughts on the safety and future of tissue banks? Have you ever received a donation of human tissue from a deceased donor? We would love to hear your opinions and experiences – just submit them in the comments below!

References:

  1. See ‘Repairing holes in the head: a history of cranioplasty.’ Sanan A, Haines, SJ, Neurosurgery. 1997 Mar; 40(3):588-603
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3495391/
  3. https://www.mtf.org/news_history_of_transplantation.html
  4. https://www.organdonor.gov/statistics-stories/statistics.html
  5. http://tissue4research.com/index.php/tissue-types/list-of-tissues-and-organs-available
  6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1071143/
  7. http://www.aatb.org/sites/default/files/NFDA-AATB%20Best%20Practices%20for%20Cooperation%20-%202013.pdf
  8. http://www.cbsnews.com/news/what-killed-brian-lykins/
  9. ibid
  10. http://www.fda.gov/ICECI/EnforcementActions/WarningLetters/2013/ucm341423.htm
  11. http://www.bizjournals.com/atlanta/stories/2002/07/08/daily54.html
  12. http://wiki.legalexaminer.com/topic/cryolife-recall-overview.aspx
  13. http://orbi.ulg.ac.be/bitstream/2268/140648/1/Klijkens-Cleanrooms%20and%20tissue%20banking%20how%20happy%20I%20could%20be%20with%20either%20GMP%20or%20GTP.pdf
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