**4.2 Processing and cryopreservation of musculoskeletal tissues**

At the end of the uptake, the tissues are sent to BTME chilled in coolers with temperature monitoring throughout the period of transportation. The processing step is preceded by a

Cryopreservation – A Viable Alternative in

environment (cleaning maintenance).

Preparation for Use of Allografts in Knee Ligament Reconstruction 97

of the nursing staff that performs the procedure, but also other professionals who access the

Fig. 4. Processing team in activity in the controlled área (ISO 5: Class 100).

The BTME conducts various types of processing of these tissues with the purpose to use in orthopedic and dental surgeries, each of which requires a specific plan. For the processing of fresh frozen tissues it is performed what we call mechanical processing, ie removal of

planning of activities needed for its implementation, such as provision of materials and instruments, convocation of the processing team, definition of preparation and dimensioning according to the need for service (queue) requests from orthopedic and dental surgeons. This step is performed in the operating room properly rated (class 100 or ISO 5) equipped with integrated laminar flow (Figure 3). The room also has an antechamber and pass-through and all environments have strict control of air particles and positive pressure for quality assurance of tissues processed there.

Fig. 3. Tissue cryopreservation área. Ultrafreezer with a temperature of -80ºC.

In addition, specific attire is required of the professional team that should only use nonwoven clothes to avoid dispersion of particles that emit the cotton clothes (Figure 4). Not only the non-woven attire is required but the team's behavior should be differentiated. Thus, sudden movements, use of cosmetics and hair exposure should be avoided during the permanence in this room. Ensuring an appropriate approach is not only a result of training

planning of activities needed for its implementation, such as provision of materials and instruments, convocation of the processing team, definition of preparation and dimensioning according to the need for service (queue) requests from orthopedic and dental surgeons. This step is performed in the operating room properly rated (class 100 or ISO 5) equipped with integrated laminar flow (Figure 3). The room also has an antechamber and pass-through and all environments have strict control of air particles and positive pressure

Fig. 3. Tissue cryopreservation área. Ultrafreezer with a temperature of -80ºC.

In addition, specific attire is required of the professional team that should only use nonwoven clothes to avoid dispersion of particles that emit the cotton clothes (Figure 4). Not only the non-woven attire is required but the team's behavior should be differentiated. Thus, sudden movements, use of cosmetics and hair exposure should be avoided during the permanence in this room. Ensuring an appropriate approach is not only a result of training

for quality assurance of tissues processed there.

of the nursing staff that performs the procedure, but also other professionals who access the environment (cleaning maintenance).

Fig. 4. Processing team in activity in the controlled área (ISO 5: Class 100).

The BTME conducts various types of processing of these tissues with the purpose to use in orthopedic and dental surgeries, each of which requires a specific plan. For the processing of fresh frozen tissues it is performed what we call mechanical processing, ie removal of

Cryopreservation – A Viable Alternative in

tissue and two years for soft tissues and tendons.

Fig. 6. and 7. Patellar tendon allograft transplant.

**4.4 Quality control and distribution** 

procedure (Figures 6 and 7).

actions.

**4.3 Tissue cryopreservation** 

(Amatuzzi et al., 2000).

Preparation for Use of Allografts in Knee Ligament Reconstruction 99

In the room of cryopreservation tissues are stored according to their status in the process. Thus, there is a space for tissues in analysis or in quarantine (where they remain for about 60 days until the results of all examinations) and those already released for use. Both rooms are equipped with ultrafreezers with temperatures ranging from 85 to 110 degrees below zero

The room is also equipped with air-conditioning system, own power generator and the unfreezing protection of carbon dioxide (CO2 Backup), and a rigorous system of monitoring the temperature, with printed record of temperature for 24 hours and alarm system via satellite, which guarantees the right temperature and early detection of complications.

Depending on the outcome of the analysis, the tissues are transferred to the room of material released for use. The maximum period of cryopreservation is five years to bone

By the time of transplantation, all tissues processed are subjected to rigorous quality assurance criteria. It requires the evaluation of all data pertinent to the donor, test results, maintenance and control of equipment, materials and instruments used in all phases of each

All processes are computerized through the System Manager of the Tissue Bank, a program designed to record all the steps which allows the rescue and traceability of each graft processed and delivered. Through a coding is possible to identify the donor, lot, expiration,

Given the need of retrieval of information, as the evidence of an adverse effect, you can quickly and safely obtain the information and implementation of corrective and preventive

For a lot of graft in analysis to be released for use nurses must analyze the results of all tests performed: NAT serology or PCR for HIV, HBC and HCV, General Culture, Anaerobic Culture and Culture of Fungi, pathology reports and radiographic findings. These reports of

and status of the tissue examination (under review, released, deleted and used).

adventitial tissue such as blood, periosteum, subcutaneous tissue, muscle, fascia and fibrotic tissue. Then, these tissues are immersed in emulsifying solutions based on hydrogen peroxide and alcohol under ultrasonic agitation (Figure 5).

Fig. 5. Tissue's chemical processing. Ultrasonic clining with emulsifying solution.

Then, a sampling of these resultant solutions, of bone marrow of long bones and fragments of each tissue processing are subjected to microbiological examination (General Knowledge, and Culture of Anaerobic Fungi). Furthermore, it is also obtained samples to histopathological analysis.

Finally starts the procedure of packaging of all the processed grafts which are measured (length, height, weight, volume, perimeter) and kept in sterile triple wrappers, vacuum sealed and properly identified as tissue in analysis. The label contains information from the donor, examination, lot number, item, expiration date, type of conservation and barcode.

Once all the tissues are identified, they are x-rayed at the very BTME and referred to cryopreservation.

The bones can also be processed in its lyophilized form, where all water is removed with the tissue still frozen. The process involves placing the tissue in a lyophilizer chamber where ice crystals sublimate by the action of the high pressure, not passing through the liquid phase and thus maintaining the viability of bone matrix. The result is a dry tissue, conservable at room temperature that must receive final sterilization by irradiation.

At the end of the processing it is performed the documentation of the procedure in the Processing and archived in the donor's chart. The stock of tissues can be kept either frozen or dried, if necessary, according to the same standards used by the Global Association of Tissue Banks . Other forms of processing have been investigated in order to reduce costs related to storage and maintenance. The glicerolization of bone tissue is presented as a processing methodology capable of maintaining the viability of the matrix and prevent bacterial growth, and allows storage at room temperature (Giovani et al., 2006).

#### **4.3 Tissue cryopreservation**

98 Current Frontiers in Cryopreservation

adventitial tissue such as blood, periosteum, subcutaneous tissue, muscle, fascia and fibrotic tissue. Then, these tissues are immersed in emulsifying solutions based on hydrogen

Fig. 5. Tissue's chemical processing. Ultrasonic clining with emulsifying solution.

Then, a sampling of these resultant solutions, of bone marrow of long bones and fragments of each tissue processing are subjected to microbiological examination (General Knowledge, and Culture of Anaerobic Fungi). Furthermore, it is also obtained samples to

Finally starts the procedure of packaging of all the processed grafts which are measured (length, height, weight, volume, perimeter) and kept in sterile triple wrappers, vacuum sealed and properly identified as tissue in analysis. The label contains information from the donor, examination, lot number, item, expiration date, type of conservation and barcode.

Once all the tissues are identified, they are x-rayed at the very BTME and referred to

The bones can also be processed in its lyophilized form, where all water is removed with the tissue still frozen. The process involves placing the tissue in a lyophilizer chamber where ice crystals sublimate by the action of the high pressure, not passing through the liquid phase and thus maintaining the viability of bone matrix. The result is a dry tissue, conservable at

At the end of the processing it is performed the documentation of the procedure in the Processing and archived in the donor's chart. The stock of tissues can be kept either frozen or dried, if necessary, according to the same standards used by the Global Association of Tissue Banks . Other forms of processing have been investigated in order to reduce costs related to storage and maintenance. The glicerolization of bone tissue is presented as a processing methodology capable of maintaining the viability of the matrix and prevent

room temperature that must receive final sterilization by irradiation.

bacterial growth, and allows storage at room temperature (Giovani et al., 2006).

peroxide and alcohol under ultrasonic agitation (Figure 5).

histopathological analysis.

cryopreservation.

In the room of cryopreservation tissues are stored according to their status in the process. Thus, there is a space for tissues in analysis or in quarantine (where they remain for about 60 days until the results of all examinations) and those already released for use. Both rooms are equipped with ultrafreezers with temperatures ranging from 85 to 110 degrees below zero (Amatuzzi et al., 2000).

The room is also equipped with air-conditioning system, own power generator and the unfreezing protection of carbon dioxide (CO2 Backup), and a rigorous system of monitoring the temperature, with printed record of temperature for 24 hours and alarm system via satellite, which guarantees the right temperature and early detection of complications.

Depending on the outcome of the analysis, the tissues are transferred to the room of material released for use. The maximum period of cryopreservation is five years to bone tissue and two years for soft tissues and tendons.

#### **4.4 Quality control and distribution**

By the time of transplantation, all tissues processed are subjected to rigorous quality assurance criteria. It requires the evaluation of all data pertinent to the donor, test results, maintenance and control of equipment, materials and instruments used in all phases of each procedure (Figures 6 and 7).

Fig. 6. and 7. Patellar tendon allograft transplant.

All processes are computerized through the System Manager of the Tissue Bank, a program designed to record all the steps which allows the rescue and traceability of each graft processed and delivered. Through a coding is possible to identify the donor, lot, expiration, and status of the tissue examination (under review, released, deleted and used).

Given the need of retrieval of information, as the evidence of an adverse effect, you can quickly and safely obtain the information and implementation of corrective and preventive actions.

For a lot of graft in analysis to be released for use nurses must analyze the results of all tests performed: NAT serology or PCR for HIV, HBC and HCV, General Culture, Anaerobic Culture and Culture of Fungi, pathology reports and radiographic findings. These reports of

Cryopreservation – A Viable Alternative in

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Camanho, GL; Croci, AT; Santos, L; Helito, CP. (2009) Use of Allograft in Ligamentar Reconstruction of Knee. *Acta Ortop Bras,* 17(5), 265-8 ISSN 2176-7521. Fideler, BM; Vangsness, CT Jr; Lu, B; Orlando, C; Moore T. (1995) Gamma irradiation: effects

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examinations are assessed and ultimately released by the Technical Director of the Tissue Bank.

Besides examinations, evaluation of the printed record of temperature during the storage period is considered. The temperature oscillations are quickly detected and reported to the team members from the BTME even remotely by cell phones. In addition, audible alarms at strategic points in the hospital and the presence of CO2 backups, ensure system reliability.

After the release of each lot, the nurses carry a detailed examination of integrity of each tissue during the replacement of tissues in analysis labels to released labels and posterior transfer of the sector. The logistics of storage of tissues in ultrafreezers considers the type of tissue and speed up the search.

We emphasize that for a rigorous quality control all steps of each procedure are carried out through check-lists with double checking and approval.All relevant data of the donor or lot records are filed in a single file and stored at the BTME for a minimum of 25 years.

A serum bank with plasma samples of donors are offered by BTME if necessary examination of counterproof.
