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Lecturer (IAEA expert mission)

Background Introduction

Current status of tissue banking

Major requirements are completely based on the safety of the grafts and associated standards including donor selection and process of processing activities. Currently major setbacks of graft healing identified. Radiation associated loss of biological, chemical and physical properties have to be fixed with the current knowledge of tissue banking.

Safety and sterility

When material exposed to radiation for sterilization, it automatically results stability reduction of the material. In Germany some studies shows that when bone allografts expose to radiation, 60% of the structural stability disappeared. Even ultra high molecular weight PE (UMWPE) loss its stability from 50%. Other than the radiation they have some other important factors affecting for the safety of biomechanical properties. Those are such as cooling rate, thawing rate and method of thawing. Type of cryoprecippitent and here mainly emphasis on the cell viability and safety ranged to 15-25% upto 90%.

Tissue Banking and tissue bank development

Tissue banking depend on the kind of co-operation which is available and development with the local hospital system or health care system. Better the cooperation and better the service rendered to the health care system and the hospital needs to create situations to become partners of health care system. The tissue banks listed as can cooperatively carry out the activities such as,

Daily Routine Activities:

Procurement, processing, sterilization, in house distribution, out door distribution. As a function for living materials, procurement can be either or both regional and national level activity of the tissue bank. It must be confidant to perform these activities comparatively to the health care system and the existing rules and regulations.

Musculoskeletel tissue banking:

This is the approach to the new chapter. Oscicles are the still fresh virgin to neuro-surgery needs. In case of duramater replace by pericardium. In Europe cattle get MCD related to the viral infection. Some of the leading chemical development of the brain cover or dura mater the reason.

Freeze drying;

This is the sublimation process to preserve structural, biological and other related properties of the graft. The recommended water content for tissue preservation was <5% dut to many reasons. Most appropriate reason is the dehydration process during the time of graft application. And also that reason studies reveals the stability breakdown also can be taken place due to over rehydration of organic and inorganic structure and associated properties of the tissue allograft.

Freeze drying technique:

Possibility to application of this technology for RBC freeze drying. After freeze drying these cells can be transplanted or transfuse.

Development activities mainly depend on following key events of the tissue bank.

Concentration on processing activities

Collection, retrieval activities and specialization of tissue banks and parallel other areas such as decentralization activities

Cooperation with other institutions in a possible levels including health care institutions

Other necessary remedial infrastructure activities

Laboratory facilities and standards of tissue and derived products and procedures

Standardization of procedures for cooperation with other institutions

Harmonization of tissue banks - this can be done as highest necessary quality and in a lowest possible level.

Safety level - is the inly guarantee of sterility of the tissue.

Goal - safe tissues means protectants against tissues and infectious diseases etc.

Tissues with required properties such as biological, biomechanical, physical and chemical properties (these properties are varies with the type of tissue.)

Regulations:

Standards are comparable to government standard level. This can be oftenly varies at that time we have to compromise the application level. Regulation of the government and recommendation may not be overlap and make all the applications confused. The reason is regulation comparable to law do not have any value if you are not able to perform any documentation by scientific documentation methods.

Validation studies:

Documented procedure for obtaining recording and interpreting the data required to show that a process will consistently comply with predetermined specifications. Therefore each and every single step has to be validated for accurate and precious application to the routine activities such as sterilization by gamma irradiation. Recommended range of dose for human tissues after concerning each possible negative barriers was 2.9 to 3.4 Mrad. The sterility of cortico-cancellous bone and soft tissue for transplantation is one of the requirement within the framework of all other quality assurance aspects of tissue banking. Therefore it is important for musculoskeletel tissue banks to conform to high standards of sterility and safety, in order to provide surgeons the best possible allograft. In Germany, DIZG uses ATCC of micro-organisms for their validation studies of sterility and safety. The objectives were

to provide the evidence that the production process will effectively inactivate or remove viruses which are either known to contaminate the starting materials, or which could conceivably do so.

to provide the indirect evidence that the production process might inactivate or remove novel or unpredictable virus contamination.

An important aspect here is the selection of viruses for the validation study. So these viruses has to be related to the possible contamination.

Certification:

This will certify that you are working according to the state-of-art standard. Main flexibility is the ability to cooperate and has to be done independent body.

Shipping:

The most important thing is that this has to be done by a qualified personals, working institute or a company. Safety of the product or tissue has to be guaranteed and able to provide necessary shipping storage temperature for the product. Future work is not only the routine production also the right identification of the need or demand also is considerable.

Methods of Cell and Tissue Preservation:

deep freezing by mechanical and electrical freezers

deep freezing by liquid Nitrogen

freeze drying

cryo preservation

other chemical and physical methods

Preservation of human cells and tissues by deep freezing

This depend on the following factors,

cooling rate and subsequent thawing rate

type of tissue

temperature

method of packaging

type of properties has to be preserved

Temperature preserve the cell or tissue by all the processes but when it goes to deeper without any protectants cell loss its viability due to structural damages. As ex: -800C particularly knee joint cells become non-viable. If the temperature enough to preserve the need range 400C - -800C, deep freezing is better. When the body cells lowering the temperature from 370C to 1960C with the cooling rate of one second per one degree of Celsius, that will take nearly 180 years to preserve. Deep freezing has a direct effect on healing and it does not act like a safe mediator during the process of healing.

Cryo preservation:

This method mainly apply for cells to keep viability.

Application temperature is less than 1800C.

Storage of cells or biological materials at vapor phase of liquid Nitrogen.

Possibility of cross contamination is more higher in liquid phase of liquid Nitrogen.

When heart valves are preserved by this method, it preserves only biomechanical properties.

Cryo selection termed selection of cells using their different septic temperatures. Septic temperatures are vary according to their sensibility. This method can be use to select different cell types such as WBC.

Especially this application is more widely useful for the patients like thalacemia and sickle cell anemia.

Other Methods:

Cartilage can be preserved nearly for two years using saline storage. Glycerol can be used to preserve skin for at least two years. Glycerol is not a sterilent, but it is a good preservative agent. HIV dies (85-98%) because of its fragile nature and weakness.

Quality Improvement application of new technology:

Improvements of available conventional methods.

Introduce bioengineered products - need to increase the physical properties Ex: shape improvements

These applications can be combine as a allo-composites.

Keratinocyte Cell Culture (KCC) and Melanocyte Cell Culture (MCC).

These applications protects/matching for the patients who have diabetes millitus and chronic ulcers as well. Growing melanocytes can also be used for "vititigo" disease. Mamo plasties of plastic surgery use these techniques and has a high "take" of KCC.

Introduce genetically engineered products such as hMBP and PDGFs.

Combinations:

There are tremendous outcome of these applications. Host bone and graft combine with chondrocytes and covered with the periosteum. Chondrocytes can be injected to the healing area. 3D chondrocyte culture, either artificial substitution or natural can be applied for the defected area. Application of osteoblasts is still under discussion. But usually surgeons take stem cells from the iliac crest to promote bone healing. Another application is the use of endothelial cells for the blood vessels and heart valves. Artificial or synthetic blood vessels can cover with these epithelial cells and help to prevent thrombosis.

Tissue bank can act as a source of following materials:

Cell line preparation and standardization of cell lines and primary cell lines. Establish primary cell culture collection.

Application of keratinocyte and stem cells in Ophthalmology.

Keratinocyte and fibroblasts based cosmetic industry applications.

Kidney cells related hepatocytes investigations.

Tissue bank as a good source of human materials, therefore can extract materials after clinically dissected.

Bovine and porcine based materials and industry. Ex: Bovine collagen, cell lines, calf periosteum and pig heart valves.

Ethical Aspects:

Donor sources cannot be commercialized but can perform services.

Controversies related to the removal and use of tissues and organs from executed persons.

Profit motivated trade: buying and selling of raw materials as well as processed allografts.

Use of fetal tissues

Artificial insemination and embryo transfer

DNA cloning

Laws/Guidelines/Regulations:

Which can influence for the activities of the tissue bank. European guidelines and standards has to be followed. European recommendations 1978 No R (94) 1 and R (79) 5 exchange II. In these guidelines, tissue banks/hospitals considered as a third party.

Non-profit making:

That activities related to banking of human tissues be deviled into separate functions such as organization, processing, preservation and storage, internal audit and distribution.

That these functions be carried out by non profit making institutions which are officially licensed by national health care administration and recognized by complement authorities.

 

Advance techniques in tissue banking

Responsibility of large bone segment production:

These responsibilities directly goes to the surgeon and he understands the graft as safe, standardized sterile product of the tissue bank.

Biomehanically stable:

Cannot be exposed to high dose of radiation. So we must compromise prior to and during sterilization. Testing has to be done as much as we can.

Sterilization:

Dose range 1.0 - 1.5 Mrad, but risk with virus infection. Need to preserve biomechanical stability and discuss with surgeon and discrepancy. Artificial materials are not bioactive at the healing process. Therefore it important to couple with growth factors and other possible advanced techniques with it.

Documentation:

Must be simple. It has to be clearly explained all steps.

Implementation of quality system:

Quality System, procedures and work instructions should be describe in clear and simple terms. This should also apply to process validation. The standards should be kept on the highest necessary level and lowest possible level.

Quality Assurance:

Activities to assure and verify confidence in the quality of the process used for the finished tissue and which document that the entire operation (ex: facilities, personnel, methods and research) is in conformity with the standards of tissue banking.

Quality Planing:

Activities that establish the objectives and requirements for quality and for the application of quality standards elements. ISO 8402, 1994; clause 3.3

Implementing a Quality System

Precondition: development of standards

Technical manual and quality manual

Introduce tissue bank quality system

Identification: specific to own demand

Production and planning:

product planing

managerial and operational planing

preparation of quality plan

provide provisions for quality improvements

Management system:

Methodology for tissue donation, procurement, tissue preparation, preservation, application and information feed back between supplier and surgeon.

Quality Management systems in tissue banks of IAEA

Accreditation and license:

Shipping such as WHO, ICAO, IATA

Temperature choices

Packaging and acceptable standards

Microbiological and toxicological attributes

Safety related factors

Tracking - each with identical implants or transplants and identical procedures, the very wide variety of host variables ensure that performance and acceptability will also variable.

Quality planing: - Follow ISO and European standards (CEN)

Total Sterility Assurance Level:

Total sterilization process depend on;

Prior to sterilization - raw materials, equipment, environment and personnel.

Sterilization process - control process of dose, choice of dose/facility

After sterilization - packaging integrity, release parameters

What is sterilization ?

The process where all types of microorganisms are either inactivated (unable to reproduce) or completely killed. The process renders the tissues sterile to achieve certain sterility assurance level (SAL) of 10-6 (final stage has to be "STERILE" - not semi or almost sterile conditions.)

Need for sterilization

Microbes are diverse group of life from, extremely small and nuisance. They are potentially to cause disease, ubiquitous in distribution, extremely small in size and invisible to naked eye. Hygienic practice in procurement, processing and packaging etc can only minimize types and number of microorganisms.

Processing validation and sterilization

All steps in the manufacture of the tissue implant/transplant that may affect its purity, potency, clinical efficacy or safety including but not limited to change original shape into desired shapes, sizes, removing extraneous and the clinical success "retarding tissue and chemical components, disinfecting, sterilizing, testing, labeling, packaging and storage.

Freeze dried tissue

Achieved by freezing tissue in such a way that mater is converted into ice in situ and subjected thereafter to a vacuum sufficient to facilitate sublimation of the ice thus formed. Residual water content less than 6% by volumetric analysis.

Glycerol (water phillic )/propylene glycol - can be used as a frozen protactants. Therefore preserve the natural condition including physical properties.

Frozen cell and tissues refrigeration:

Cooled to a solid state below 00C and stored below -180C. Cryopreserved cells and tissues are implies to cooled to a solid state with a cryoprotectant such as DMSO or Glycerol to prevent intreacellular ice formation and to maintain cell viability usually stored below -1500C in vapour phase.

Sterilization

Terminal sterilization - aseptic processing contamination control

The variables for controlling contamination during aseptic processing almost always exceed those of terminal sterilization, especially, when personnel are involved in the process of preparation, cleaning, cutting, filling or assembling the finish product and or package.

Sterilization of human tissues.

An effective method of sterilization of human tissues shall be able to eradicate all kinds of surface and interstitial microbes including bacteria, viruses and fungi. But it must not vitiate the essential biomechanical and biochemical nature of the tissue.

Method of Sterilization

Physical: Boiling, autoclaving, radiation

Chemical: Ethylene Oxide, gluteraldehyde, NaOCl

Combine: PAA with low pressure

Bioburden: Population of viable microorganisms on a product excluding viruses.

SAL: Probability of a viable microorganism being present on a product unit after sterilization.

Microbiological validation of radiation sterilization procedure

BS-EN-552 1994

ISO 11137

Requirements of ISO 11137

Describe the standards

Device and packaging material qualification

Dose setting method for radiation sterilization

Dosimeters, dosimetry and associated equipment

Bibliography

Quality System and Principles of GMP

Good Manufacturing Practices

This is a collective integrated set of principle helps tissue bankers as well, ensure there products are consistently design, manufactured, supplied and control to the quality standards required. This guideline meets the criteria of safety, efficacy and quality of the product or tissue allograft. In generally GMP guidelines common for all principles define by the GMP codes.

An integrated system of production and quality control.

QM with separate management responsibilities for production and QA.

Documentation for manufacture and QA

A recall procedure

A system of self audit and review of GMP.

Suitable premises and equipment

Appropriately qualified personnel

A policy on personnel hygiene, clothing and suitable sanitation program for premises and equipment.

Approved audited and formalized contract QC, manufacture and sterilization.

 

Definition of Quality:

Degree of excellence is the highest definition for the quality.

Quality Assurance(QA):

Positive statement intended to assure the confidence of the product, make certain and give a guarantee of the product.

Quality System (QS):

Clearly described set of policies. It has included actions of each step.

Quality Control(QC):

Operational activities or techniques to fulfill the requirements of product quality. This specifications testing procedures, sampling procedures and responsibility will broaden to prepare and maintain procedures and specs, inspections of products, raw materials, material purchasing, sample testing and follow up complaints.

Total Quality Management (TQM)

All procedures are well defined and documented. TQM provides assurance of all carried out according to required standards, customers satisfaction, fulfill legislative requirements etc.

Quality in tissue banking:

Traceability

Recipient needs

Donor characteristics

Design process as per clinical needs

Specification for each and every step

Minimum requirements needs to implement QS in tissue banking

Documentation

Manpower

Filing system

Traceabiliy

Process control

Validation of equipment, service, process, procedure, operator

Monitoring of rooms for confirmation of the system.

Maintenance of equipment and checking all of that has been validated.

Review of ISO 9000 and its implementation in the health care industry

These standards formulated by International Organization for Standards (ISO), Geneva, Switzerland. These standards harmonize local and national standards, to international standards which are adopted by all member countries. This series defines requirements for 20 system elements. Record what you do, check on the results, act on the difference, that is the "preventive".

Management Responsibility

Quality system

Contract review

Design control

Document and data control

Purchasing

Control of customer supplied products

Product identification and traceability

Process control

Inspection and testing

Control of inspection, measuring and test equipment

Inspection and test status.

Control of nonconforming products

Corrective and preventive action

Handling, storage, packaging, preservation and delivery

Control of quality records

Internal quality audits

Training

Servicing

Statistical technique

Four levels of ISO 9000 documentation

Quality Manual : Level I

Quality Procedures : Level II

Work Instructions/SOP's : Level III

Document records : Level IV

Quality Auditing

Quality Audit is a systematic and independent examination to determine whether quality activities and related results comply with planned arrangements and whether these arrangements are implemented effectively and are suitable to achieve objectives.

Ref:

ISO 10011 Guidelines for auditing QS

Part I: 1990 Auditing

Part II: 1991 Qualification criteria for QS auditors

Part III: 1991: Management of Audit program.

Quality audit - Objectives

To determine the conformity of the QS elements vs specified requirements

To determine the effectiveness of the quality system in meeting the quality objectives

To provide opportunity to improve the quality system.

Certification purpose

To meet regulatory requirements

Quality Audit - Advantages

To make us more confidant

Prepare checklists: premises, people, procedures, forms, plants, products etc.

Check the product thoroughly from starting material to final product.

Audit report actions

Reasons for Audit

To evaluate suppliers in view of a contractual relationship.

To verify that the organizations own quality system meets requirements and is been implemented

To evaluate an organization's own quality system against a standard (eg: ISO Guide 25/ISO 9001)

Types of Audit:

First party - an audit of the tissue bank/supplier's system that is perform by the suppliers management.

Second party - an audit of a vendor/subcontractor to verify that the suppliers quality system continues to meet specified contractual requirements and is being implemented.

Third party - an audit undertaken by independent certification/accreditation bodies.

Audit techniques

Personnel observation by auditors

Examination of documents

Communication with management, workers, contractors etc.

Test of system, procedures

Inspection of related lab, facility, instruments

Complaints from workers, customers

Documentation - write what you have to do

Implementation - do what you write,

Audit - ready for short and precious answer

Combine methods of sterilization - Per acetic acid

Instruments:

Tubes

Evacuation

Shaker

Intermittent vacuum reduction

Rinsing and pH control

Preservation (freeze drying, freezing, glycerol, )

Bacteria : 1 min - 0.005 - 0.05%

Viruses; 2 min - 0.05 - 0.1%

Fungi; 1 min 0.05% - 0.5%

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