February 2004 Bulletin

In-depth look at allograft safety

Overview of allograft soft tissue processing

By C. Thomas Vangsness Jr., MD

In 1999, 750,000 allografts were surgically implanted in the U.S. 1-4 Currently, musculoskeletal allografts provide the best solution to particular surgical procedures including revision surgery where an autograft is not available, or as a supplement when an ample supply of autograft tissue is not available.(5) Of the greatest concern to clinicians and patients is the potential for viral or bacterial disease transmission.

As the use of allograft tissue continues to increase, the safety of allografts will remain an issue of paramount importance. This article takes an in-depth look at the current problems with allografts and considerations for keeping them safe in the future.

Bacterial infection from musculoskeletal allograft tissue
Historically, the transmission of bacterial or viral infection from musculoskeletal tissue has been a rare event. Nevertheless, in March 2002, the Centers for Disease Control and Prevention (CDC) investigated 26 cases of allograft-associated infections, which are defined as any infection at the site of an allograft implantation occurring within 12 months of allograft implantation in an otherwise healthy patient with no known risk factors for surgical site infection, e.g. diabetes. (6)

The CDC investigations were precipitated by a 23-year-old Minnesota man who underwent reconstructive knee surgery in November 2001, and subsequently died three days after receiving a femoral condyle bone-cartilage allograft. The blood cultures obtained pre-mortem revealed Clostridium sordellii. Fresh femoral condyle and frozen meniscus grafts, both from the same allograft donor, were implanted into a second patient also undergoing reconstructive knee surgery. That patient developed septic arthritis and is currently recovering from the infection. Eight additional tissues from the same donor were implanted but no other infections were identified. This pattern of infection resulting from certain allograft tissues but not all originating from a single donor suggests that more effective tissue processing may reduce the risk of disease transmission after transplantation. (2,7)

Of the 26 cases of bacterial infections associated with musculoskeletal tissue allografts that were reported by the CDC, (13) patients were infected with Clostridium species (Table 1). A single tissue-processing facility processed 14 of the 26 grafts. While the Morbidity and Mortality Weekly Report (MMWR) referred to the tissue processor as Tissue Processor A, the New York Times attributed those cases to tissue processed by Cryolife, Inc. of Georgia.

The September 2003 case of aggressive invasive allograft infection, as reported in the December 5, 2003 MMWR, involved a healthy 17-year-old man who became infected with Streptococcus pyogenes after reconstructive knee surgery. (8) All the allograft implants responsible for these infections were processed aseptically and some even soaked in antimicrobial solutions; however, no other sterilization procedures, such as gamma irradiation, were used.

Although aseptic processing, in concept, minimizes contamination of tissue at the tissue bank, it does not eliminate contamination originating from the donor that might be inherent to the graft. (9) Therefore, it is essential for tissue processors to adhere to procedures that minimize donor infection, including the recovery of tissues from donors within safe periods of time and the application of tests to assess bioburden from the donor. Failure to adhere to established standards for tissue recovery time may result in the procurement of tissues from a contaminated donor, thereby increasing the risk for infection in tissue recipients.

Viral transmission from musculoskeletal allograft tissue
Historically, musculoskeletal tissue transplantation has resulted in several reported cases of viral infection, specifically human immunodeficiency virus (HIV),10-11 viral hepatitis, 13-14 and human T-lymphotropic virus (HTLV)15 (Table 2). It should be noted that these transmissions occurred before the implementation of guidelines for donor screening for viruses and bacteria, and before the availability of currently validated serological tests.

In April 2003, the CDC reported a case of Hepatitis C transmission from a patellar tendon allograft. Serological results from the initial donor screening revealed no detectable antibody to the Hepatitis C virus (HCV).16 During the ensuing investigation, a more sensitive HCV RNA assay was used on the donor’s serum, which confirmed that the donor was the probable source of the HCV infection. Further investigation determined that the donor tissue was the source of disease transmission in at least eight organ and tissue recipients from the 40 grafts that were transplanted from this donor.

It was noted that no cases of HCV transmission were reported in recipients of irradiated bone. The CDC stated that, although HCV transmission is rare, assessing the frequency and risk of transmission are important factors in determining whether additional prevention measures are warranted.

Aseptic processing techniques for allograft tissue
Aseptic processing is the most common method of allograft processing and refers to a methodology whereby a tissue bank restricts or minimizes contamination to the allograft tissue from the environment, processing personnel and equipment. As stated in the March 2002 issue of the CDC’s MMWR, “aseptically processed tissue should not be considered sterile, and health-care providers should be informed of the possible risk for bacterial infection.” (6) Organisms of high pathogenicity are generally endogenous, arising from the donor’s gastrointestinal tract or respiratory tract.(17) However, these clinically significant organisms are not reduced by simple antibiotic soaks.(18)

As a result, the tissue industry relies on donor screening, which includes serological testing for viruses, physical examination of the donor and medical history review, as well as bacterial and fungal cultures, to accept or reject allograft tissue for transplantation. Culturing is performed on allograft tissue to detect bacteria and fungi after aseptic tissue processing. However, studies have shown that the rates of sensitivity for these cultures are only 78 percent to 92 percent. Therefore, culturing alone is not a conclusive method for assuring allograft sterility. (19, 20) Furthermore, all tissue processors do not routinely culture for fungus growth.

Donor screening, although extremely effective in eliminating donors with active viral infections, has its limitations. As with all viral infections, there is a “window period” in which the donor does not have detectable antibodies to the virus, yet remains infectious. Using current screening and testing protocols, the risk of implanting tissue from an HIV-infected donor is less than 1:1,000,000.(3) However, the risk of implanting tissue from Hepatitis-B virus (HBV) or HCV-infected donors is significantly higher due to the greater prevalence of these viruses in the general population and the limitations of current testing methodologies. New DNA/RNA-based tests for HBV, HCV, and HIV drastically reduce the time frame to detectable contamination, but a window period does still exist. Human errors in screening and processing also can be factors in the reliability of the donor screening method. The blood supply is currently screened more rigorously using nucleic acid tests (NAT), while a licensed serology NAT test for tissues does not yet exist.

Due to the limitations of donor screening and microbial culturing, as well as the incidence of allograft-associated infections, the current aseptic processing practices can reduce but not eliminate transplantation of infectious tissue. A disinfection process that does not adversely affect the biological or biomechanical properties of allograft tissue would be the best way to reduce the risk of allograft-associated infection. (6)

Methods of allograft tissue disinfection
Sterilization has been defined as the process or act of inactivating or killing all forms of life, especially microorganisms. (21) Ideally, allograft tissue would be sterile, just like medical devices, and disease transmission would be improbable. Unfortunately, human cadaveric tissue is not as easily sterilized as metals or plastics. According to the Association for the Advancement of Medical Instrumentation, exposure to a validated sterilization process should achieve a sterility assurance level (SAL) of 10-6 for metallic and plastic devices.

However, for musculoskeletal allografts, the levels of bacteria, spores, fungi and viruses must all be eliminated to ensure sterility. Furthermore, blood is a significant pathogen reservoir and is reported to be the primary source for endogenous contamination of musculoskeletal tissue. (2, 22, 23)

Consequently, tissue processing is a critical component for ensuring allograft safety.(2) Practically, this has proven to be a difficult task given the complex structure of musculoskeletal tissue. A sterilization method must not affect biomechanical properties, must completely penetrate the tissue, and must not affect biocompatibility and incorporation characteristics.

A myriad of “sterilization” techniques for bacteria and spores have been attempted on allograft tissue. Historically, tissue banks use one of two methods to attempt disinfection; ethylene oxide gas or gamma irradiation, although a variety of other chemical antimicrobial solutions have been employed as well.(24) Some of these methods are more effective than others in eliminating microbiological contamination in and endogenous material such as blood and lipids, while not affecting material properties of allograft tissue (Table 3). Some approaches also have inherent risks, such as the residual toxic by-products of ethylene oxide if not adequately aerated. Further study is required to evaluate the effectiveness of these processing methods, as well as their effects on the biomechanical and biological properties of tissue.

Terminal processing techniques
There are several tissue-processing techniques used by various tissue banks that are currently available. To date, the safety and efficacy of these different processing techniques have not been scientifically established. There is no current single standard approach; however, all methods performed must be prepared, validated and in written protocol form to comply with regulations designed to prevent infectious disease transmission or cross-contamination during tissue processing.(25) This approach is under scientific review by the U.S. Food and Drug Administration (FDA).

The AllowashTM formula (Lifenet; Virginia Beach, Va.) utilizes ultrasonics, centrifugation, and negative pressure in combination with reagents such as biological detergents, alcohols and hydrogen peroxide. This combination aims to increase the solubilization and removal of components from processed allograft tissue that may act as reservoirs for potential bacterial, fungal and viral transmission, including bone marrow, blood elements, and lipids.

Cryolife, Inc. uses a slow freezing rate along with dimethyl sulfoxide (DMSO) or glycerol to remove water, a process known as cryopreservation. There is no secondary sterilization after this method; rather the tissue is incubated in a patented cocktail of bacterial antibiotics for 24 hours at 37 C before being frozen to –135 C.

The BioCleanseTM tissue sterilization process (Regeneration Technologies Incorporated; Alachua, Fla.) may be used in the final steps of processing and avoids the use of excessive heat, irradiation or ethylene oxide. This low-temperature chemical sterilization method claims to completely penetrate tissue and eliminate endogenous contamination from allografts.26-28 Tissue banks, such as Osteotech, Inc. of Eatontown, N.J., use low levels of gamma irradiation, usually in the ranges of 1.0 to 1.8 megarads. This technique can be performed before or after the normal aseptic processing. The Clearant Process® (Clearant, Inc; Los Angeles, Calif.) treats tissue with 50 kilograys of radiation, two to four times the dose recommended to avoid cell damage. The process claims to prevent radiation damage to cell proteins and devitalized tissue by freezing the sample, extracting the water, and adding stabilizers and antioxidants such as vitamins A and C.

Packaging techniques
Almost all of the major tissue banks follow their own unique processing methods with the common storage guidelines of the American Association of Tissue Banks (AATB) by deep freezing the tissue. These guidelines generally involve freezing the tissue at –80o C.(24) Some tissue banks may use freeze-drying (lyophilization) to allow storage at room temperature. The first process of lyophilization freezes the tissue, and then the water is reduced, first by sublimation (referred to as the primary drying process) and then by desorbtion (known as the secondary drying process) to values that will no longer support biological activity or chemical reactions.(29)

Many tissue banks label their packaging and allograft contents as sterile. This sterility labeling has not been confirmed or validated, and is currently under investigation by the Center for Biologics Evaluation and Research (CBER), the FDA branch that regulates biological products.

Safe allograft considerations for the future
With these current uncertainties, it is important that orthopaedic surgeons know the source of their allograft tissue. Allograft tissue processors are regulated and are required to follow mandatory federal and state regulations. The FDA and only two states, New York and Florida, require inspection and/or licensure of tissue-processing facilities. They also are currently the only agencies that can allow or prevent the distribution of allograft tissue. Yet 36 of the 154 tissue banks in the United States. have never been inspected, and only 50 of the remaining 118 have been inspected more than once.

This lack of FDA oversight is due to insufficient federal funds and resources needed to carry out the inspections.(30) Therefore, the FDA has proposed greater regulation and requirements for tissue banks. These proposed regulations, the Good Tissue Practices and Donor Suitability Determinations, should be enacted in 2004. A recent U.S. Senate hearing focused Congressional concern over the delayed finalization of the last two rules from the FDA. (25)

In the interim, the surgeon must consider several factors along with these FDA and state agencies’ regulations. All tissue banks should seek and receive voluntary accreditation, follow available peer-group and Federal standards, and perform their own extensive donor screening criteria. However, the Office of the Inspector General, Department of Health and Human Services, explains that many banks do not adhere to or seek accreditation because there has been no perceived benefit or incentive.(30)

Serological testing ideally should exceed the requirements of the FDA, AATB, or state agencies. Serological tests that should include NAT DNA/RNA-based testing used for living blood donors should be developed and licensed for cadaveric serum. Every surgeon should evaluate the specific terminal-processing method. This evaluation should be based on complete tissue penetration of the processing technique and any effects this method has on the biomechanical properties of allograft tissue. The final consideration involves the concept and labeling of sterility, which will be assessed by the FDA. If a graft is labeled sterile, it should have a statement of the Sterility Assurance Level by the tissue bank.

Safety is best assured by obtaining allografts from an accredited tissue bank or a processor that uses the most stringent standards. A current list of accredited tissue banks can be found at http://www.aatb.org.

C. Thomas Vangsness, Jr., MD, is professor of orthopaedic surgery and chief of sports medicine in the Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles.


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  9. CDC. Septic arthritis following anterior cruciate ligament reconstruction using tendon allografts – Florida and Louisiana, 2000. MMWR 2001; 50:1081-3.
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Tissue Banks Accredited by American Association of Tissue Banks *

Alabama Tissue Center, Inc.
Birmingham, Ala. 

Alamo Tissue Service, Ltd.
San Antonio, Texas 

Englewood, Colo.

American Red Cross National Tissue Services
Lorton, Va. 

American Red Cross Tissue Services North Central Area (Reg.2)
Eagan, Minn. 

American Red Cross Tissue Services Western Area
Costa Mesa, Calif.  

Andrology Laboratory & Sperm Bank
Cleveland, Ohio 

AppTec Laboratory Services
St. Paul, Minn.

Miami, Fla. 

BioGenetics Corp./The Sperm Bank of New York
Mountainside, N.J. 

Blood and Tissue Center of Central Texas
Austin, Texas 

Bone Bank Allografts
San Antonio, Texas 

California Cryobank, Inc. — Cambridge
Cambridge, Mass. 

California Cryobank, Inc. — Los Angeles
Los Angeles, Calif. 

California Cryobank, Inc. — Palo Alto
Palo Alto, Calif. 

California Transplant Services
Carlsbad, Calif. 

Central Florida Tissue Bank, Inc.
Orlando, Fla. 

Community Tissue Services (California)
Fresno, Calif. 

Community Tissue Services — (Dayton)
Dayton, Ohio 

Community Tissue Services — (Fort Worth)
Fort Worth, Texas 

Community Tissue Services — (Indiana)
Indianapolis, Ind. 

Community Tissue Services — (Northwest Ohio)
Toledo, Ohio 

Community Tissue Services — (Pennsylvania)
Philadelphia, Penn. 

Community Tissue Services — (Portland)
Portland, Ore. 

Comprehesive Tissue Centre University of Alberta Hospital
Edmonton, Alberta, Canada

Cryobiology, Inc.
Columbus, Ohio 

Cryogenic Laboratories, Inc
Roseville, Minn. 

DCI Donor Services, Tennessee
Nashville, Tenn. 

Doheny Eye and Tissue Transplant Bank
Los Angeles, Calif. 

Donor Alliance
Denver, Colo. 

Gift of Hope Organ and Tissue Donor Network
Elmhurst, Ill. 

The Hospital for Sick Children Tissue & Stem Cell Laboratory
Toronto, Ontario, Canada

Idant Laboratories
New York, N.Y. 

Indiana Organ Procurement Organization Eye and Tissue Bank
Indianapolis, Ind. 

Intermountain Tissue Center
Salt Lake City, Utah 

Interpore Cross International
Irvine, Calif. 

IsoTis OrthoBiologics
Irvine, Calif. 

Kentucky Organ Donor Affiliates
Louisville, Ky. 

Legacy of Life
San Antonio, Texas 

Cleveland, Ohio 

LifeCell Corporation
Branchburg, N.J. 

Lifeline of Ohio
Columbus, Ohio 

LifeLink Tissue Bank
Tampa, Fla. 

Virginia Beach, Va. 

Lifesharing Tissue Services
San Diego, Calif. 

Lost Mountain Tissue Bank
Kennesaw, Ga. 

Louisiana Organ
Procurement Agency
Metairie, La. 

Mercy Medical Center Surgical Bone Bank
Cedar Rapids, Iowa 

Mid-America Transplant Services
St. Louis, Mo. 

Mid-South Tissue Bank
Memphis, Tenn. 

Musculoskeletal Transplant Foundation
Edison, N.J. 

Nevada Donor Network, Inc.
Las Vegas, Nev. 

New England Organ Bank Tissue Banking Services
Newton, Mass. 
New York Cryo
Great Neck, N.Y. 

New York Tissue Services
Staten Island, N.Y. 

Northern California Transplant Bank
Oakland, Calif. 

Northwest Tissue Center
Seattle, Wash. 

Nu Med Technologies, Inc.
Tulsa, Okla. 

Ohio Valley Tissue and Skin Center
Cincinnati, Ohio 

Oklahoma Organ Sharing Network
Oklahoma City, Okla. 

Osteotech, Inc.
Eatontown, N.J. 

Regeneration Technologies, Inc.
Alachua, Fla. 

Regional Tissue Bank QEII Health Sciences
Halifax, Nova Scotia, Canada

ReproTech, Ltd.
Roseville, Minn. 

Rocky Mountain Tissue Bank
Aurora, Colo. 

RTI Donor Services
Allograft Resources Division
Madison, Wisc. 

RTI Donor Services Southeast Division
Marietta, Ga. 

RTI Donor Services — Southwest Division
Phoenix, Ariz.

Rubinoff Bone and Tissue Bank
Toronto, Ontario, Canada
ScienceCare Anatomical Body Donation Services (non-transplantable tissues)
Phoenix, Ariz. 

Shriners Hospital for Children Burns Institute Galveston
Galveston, Texas 

South Texas Blood and Tissue Center
San Antonio, Texas 

Southeast Tissue Alliance, Inc.
Gainesville, Fla. 

Sperm & Embryo Bank of New Jersey
Mountainside, N.J. 

SpinalGraft Technologies, LLC
Memphis, Tenn. 

Tissue Banks International National Processing Center
San Rafael, Calif. 

Tissue Bioengineering Laboratory — UC Davis Medical Center
Sacramento, Calif. 

Transplant Resource Center of Maryland
Baltimore, Md. 

Transplant Services Center — University of Texas SW Medical Center
Dallas, Texas 

Tutogen Medical, Inc.
Alachua, Fla. 

University of Miami — Tissue Bank Dept. of Ortho/Rehab
Miami, Fla. 

Wright Medical Technology, Inc.
Arlington, Tenn. 

* This information is provided for educational purposes and does not constitute an endorsement by AAOS. For more information about the AATB accreditation process, visit the organization's Web site at http://www.aatb.org.

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