Lecture Topics:

INTRODUCTION TO IONIZING RADIATION

Nature of ionizing radiation and history.

Natural radiation discovered by Becquerel in February 1896, Marie Curie discovered radium in 1903 and X-ray by Roentgen 1895. Unstable molecules become stable after disintegration the energy rays recognized as radiation. This can be either natural or artificial. Electromagnetic and High-energy particles are the examples for that.

Radiation Units and Gamma rays:

Gray (J/kg), Kilo Gray and rads are commonly used for radiation measurements. 100 rads equivalent to one gray and absorb dose rate is indication of absorbed dose per unit time.

Properties of Gamma rays:

Co-60: half life 5.37 years, energy 1.17 MeV and 1.33

Caesium-137: half life 30 years and energy 0.66 MeV.

Source strength is measured by curies. One curie indicates 3.7 X 10¹⁰ disintegration per second. After bombarden on Co-59, gamma rays will produce. Cs made up from nuclear waste and half life is longer. Due to those reasons Cs are much more economical than Co-60. But Cs has a contamination potential due to its nature of the source is pencils like.

With Aqueous system (tissues >80%)

Observed reactions are mainly due to the interaction of ionizing radiation with aqueous system. This is applicable for irradiation of tissues having >80% water. Main effects are taking place due to ionization (electron aq), recombination (H₂O*) and decomposition (H*, OH*). Further free radicals (H*, OH*) lead some adverse effects in the aqueous system. Finally effect on the tissue or tissue product.

Organic Molecules:

Radiation effects will lead chemical changes of the product. Also has a killing effect on microorganisms.

Radiolysis of water:

When glycerol is added for the preservation process, it react with free radicals and protect the tissue. The amount of radiolytic products formed due to existing energy of radiation is influenced by many factors can be determined. Thermal G value means value of free radicals. Anna Closka a Polish Scientist measured free radicals by using ESR technique. Heat also can produce free radicals. Therefore measurement of temperature during sterilization is highly important.

GOOD RADIATION PRACTICE

Requirements of Good radiation practice:

This is important for GMP followed by international standards. Therefore standard operating procedures and validated methods are highly applicable.

Irradiator commissioning:

Magnitude of distribution and reproducibility of absorb dose according to the product density is result safe application. Therefore calibration or dosimetry, dose mapping, calculation of DUR and current activity are important with radiation survey auditing.

Dosimeters:

For gamma radiation, Fricky dosimeters can be used to calibrate other dosimeters. For the commissioning of Gamma and electron beam, Cerric/Cerrous and Cellulose triacetate can be used respectively. International Dose Assurance Service (IDAS) helps for intercomparison by exchanging reference dosimeters.

Primary (reference) dosimeter eg. Fricke (ferous sulfate solution for gamma) or graphite calorimeter for electron beam) must be used to compare the response of the routine dosimeter in the production environment.

Routine dosemeter eg. Ce/ce (ceric-cerous sulfate solution for gamma) or CTA (cellulose triacetate for electron beam) is calibrated for use in commissioning of irradiator and routine.

Traceable to international comparison eg IDAS.

	Placement of dosimeters throughout a representative product load during validation.
	Identify positions for placing routine dosimeters eg maximum and minimum dose.
	Identify Dose Uniformity Ration (DUR) = maximum dose/ minimum dose

Take the measurement of chamber and or product container. Place the dosimeters and sketch the lay out. Irradiate for pre-calculated/determined time of exposure or doses. Read out the dosimeters and take absorbed values. Mark (mapping) it on the sketch/select minimum and maximum doses. Adoption of corrected practices. Ex: Dose Uniformity Ration.

Mainly product and product packaging material concern. Most ideal packaging material is polyethylene. Physic-chemical and biocompatibility tests will help for evaluation. During irradiation, changes may lead product contamination as well as direct toxic effects to the product.

For the validation, pattern of work or art of work has to be established. Length of irradiation, product density, loading interval, arrangement of product within the container, placement of dosimeters, adding GO NO GO indicators and documentation has to be added to the evaluation. For the process control, documents must to be clearly indicated the procedure of receiving, storage, returning products. Color change indicators and irradiation batch numbers, records of absorbed dose measurements and certification of minimum dose delivery.

Lecture Topic: EFFECT OF RADIATION ON MICROORAGNIMS

Five major groups

	Bacteria Neither plants nor animals, Free-living or parasitic, Source : Soil, water, organic materials, human, animal, plant
	Viruses Subcellular particles, Obligate intracellular parasites, Source: Human, animal, plant or bacterial hosts.
	Fungi (moulds and yeasts) Some resemblance to plants, but not photosynthetic, Free-living or parasitic Source: Decaying organic matter, soil, fruit juice, plant, animal, human
	Protozoa Animals, Free-living or parasitic, Source: Soil, water
	Algae Green plants, Free-living, Source: Sea, fresh water, soil

In the field of radiation sterilization, it is important to measure the level of microorganisms in a given product or package. Radiation can damage them by direct or indirect methods.

interaction between the ionizing radiation and critical biological molecules, results in excitation, lesion and scission of DNA polymeric structure.

DN - 2 polynucleotide chains (double helix)

base sequence

hydrogen bonds

- Breaks in sugar phosphate back bone (single or double strand break)

- Base damage (change / loss)

due to chemical changes and depends on water content. Drying or freezing of the tissue

can reduce or remove the mechanism.

Radiolysis of water/Free radical formation

H₂O--------> H^• + OH^• + e-_aq + H₂ + H₂O₂ +H₂⁺0

Radicals react with organic molecules: amino acids, proteins, mitochondrials, transport components, DNA bases, DNA chains, lipids

Other molecules: oxygen, water, medium, chemicals.

Indirectly radiolysis of amino acids, protein, mitochondria, transport componenets, DNA bases, DNA chains, lipids may lead formation of free radicals and radiolysis of water and reaction of free radicals with these components lead indirect dominant damage. In this case predominant damage can be repaired.

Effect of Radiation will depend on following factors:

	Types and species, cell cycle
	Sensitive, Moderate resistant, Resistant, Highly resistant
	Virus 100 kGy vs Man 5 kGy
	Bacterial spores more resistant > vegetative
	Non dividing > dividing/ growing
	Efficient repair mechanisms.
	Moisture or water content
	More water, more radiolytic products, more damage
	Interrelated with oxygen presence, more peroxy species,
	Keep low water content, 4 to 7 % or < 10 %

Increase by 10 %, reduce D₁₀ by 50%,

Synergistic effect of combined treatment heat + radiation In frozen more resistant due to immobilised radicals

3 to 5 x sensitive than anoxia condition. React with e-_aq and H• , produce perhydroxyl,

superoxide radicals react with organic radicals, produce peroxy species.

Protective effect

Dried serum, broth, grease films, sucrose, other complex substrates.

Protective action: glycerol, thiourea, sulphoxide, cysteine

- free radicals scavenger, deplete oxygen, block radiolysis,

Sensitizer: iodoacetic acid, potassium iodide - with radiolyic product to increase strand breaks.

No differences between gamma sources to give significant differences in bacterial inactivation. Differences between gamma and electron at very high dose rate, oxygen depletion, more resistant

Commercial plant : no significant effect on microbes.

Mainly three types of curves explain the nature of microbes when they expose to radiation. Mainly there survival and the given dose have a significant relationship. For more sensitive microbes, exponential curve is linear. Certain repair at the low dose (predominant damage) - shouldered (at low dose) curve. Mixed populations, flawed experiments have a resistant tail - Concave Curve (resistant tail).

The dose requirements for the reduction of 90% given population of microbes. From the response or inactivation curve, D10 value can be determined from linear gradient or by using following Stumbo equation (Stumbo, 1950).

D₁₀ = D/logN₀ - logN_n

D = Radiation dose

N0 = Initial number of microorganisms

Nn = Population count after irradiation dose

Log N₀ - logN_n = No of log cycles at the dose respose curve

In the case of human tissues, safety and efficacy directly related with the SAL and shelf life and keeping quality also goes with the final flora and water content.

Methods of application of Microbiological tests that design to ensure the process meets certain defined microbiological quality standards. This concept we are using as a quality control concept. Those concepts are;

Bioburden estimation (total number of viable microorganisms on a package or a product prior to radiation sterilization. Isolation and identification are also important consideration here)

Sterility tests: Old method to Microbiological techniques. But it is important to measure sterility each and every batch after sterilization. Mostly going with other sterilization techniques. Performed to determine any viable microorganism (for release product after sterilisation other than radiation). An expected probability of a single viable microorganism being present on individual product (for verification of sterilisation dose under ISO 11137) .

Most active stage (mitosis at cell division) of microorganisms are more susceptible to radiation. Previous research shows following findings related to the microorganisms and ionizing radiation.

"Generally, the lethal effects of ionozing radiation on living cells is attributed primarily to the energy deposition in the critical components such as DNA which carries genetic information of the cell, and perhaps the cell membrane to which DNA and other vital cellular components are attached (Gina, 1968; Coggle, 1973; Gauhan and Goudie, 1974)"

This killing effect of radiation may be due to direct and indirect effect. The later is more prominent and very much influenced by environmental factors including Oxygen, water, medium, temperature and chemicals present during irradiation(Tallentire 1973; Gardner and Pell, 1986; Whitby, 1993).

Lecture Topic: EFFECT OF RADIATION ON TISSUE ALLOGRAFTS

These effects have direct relationship with radiation condition (state) and method of processing. Decrease of immunogenicity, increase of resorpbility comes under desirable effects and reduces of biomechanical properties; decrease of osteoinductive capacity can classify under undesirable effects.

Fibril structure changes take place > 100 kGy. In tendons, reduce tensile strength by 1/3 at 180 kGy. Collagen derived membranes increases its solubility by 50% at 35kGy and amino acids (content) in collagen will not change at 50-1000 kGy.

Bone has a dramatic structural and chemical changes due to irradiation dose and the state of irradiation. Therefore following properties have found changing due to that irradiation process;

Structure

Deep frozen state 1- 50 kGy nonstructural effects. But freeze dried state leading to micro cracks.

Elasticity

Deep frozen state at 25 kGy no significant changes of elasticity. But freeze dried state leading to decrease the elasticity.

Compressive failure stress;

No effect at 10, 31, 50 kGy. Rehydration improves to normal. Remodelling has an effect when radiation dose 10, 15 kGy before process. No effect on osteoconduction. Irradiated bone matrix with gelatin - 25 kGy destroy osteoinduction but not significant change in induction mechanism. At 50 kGy destroy osteoinduction.

Demineralized irradiated at 25 kGy reduce the induction capacity and 40 -50 kGy enhanced bone induction appeared. Irradiated frozen at 35 - 50 kGy, new bone formation occur. But freeze dried irradiated bones resorption 5 weeks after implant and no osteoinduction. Radiation <30 kGy reduce immunogenicity and possible BMP. Role of BMP is not clear.

Radiation effect on heart valves:

Irradiated at 29, 32 kGy frozen state effect biomechanical effect of aortic wall. Irradiated at 25, 33 kGy freeze dried state, reduce tensile strength, crack formation and become brittle due to damage in gel structure.

Radiation effect on skin and amnion.

Irradiated freeze dried reduce tensile strength by 25% (25kGy). 33 kGy increases solubility (will resorp) by 5-15%. 25 kGy decreased permeability by 50%.

For amnions; 17 kGy and freeze dreid result lower tensile strength than irradiated air dry. Irradiated air dried state no changes in tensile strength and elongation after 20 months storage at room temperature.

Radiation effect on fascia lata

25 kGy decrease permeability and longer time to reach steady state. Take time to stabilize when rehydrate.

Radiation effects on tendons

25 kGy no changes in histological pattern. Comparably fresh at 6 months after implantation. 20/40 kGy no effect on biomechanical properties even six months after surgery.

HIV does not transmit from freeze dried tissues with and without radiation (Life Net, 1985)

Recipients HIV Positive

Fresh Organs                04                  04

Fresh Cornea                02                  00

Frozen Bone                  04                04

Freeze dried bone         47                00

Soft tissues and irradiated tissues: Contradictory results on inactivation dose (D10 value) wide range 0.25 - 25 kGy to 56 kGy.

Research data shows:

Pasteurization at 56⁰C for 30 minutes to inactivate HIV (Spire et al 1985).

Pasteurization inactivates all microbes (Yusof et al, 1994).

Proper screening (medical reports, detection methods) and inactivation process can eliminate HIV in tissues.

Radiation Sterilization Dose:

Radiation sterilization of tissue allograft is not a substitute for proper screening, handling or processing but it can provide an additional safety measure against infections (Czitrom, 1992). Therefore sterilization dose can be calculated by using Stumbo equation. First we have to calculate D10 value for each and every microorganisms. With the known D10, sterilization dose can be easily calculated with pre calculated or assayed bioburden estimation. Therefore gamma radiation is still an effective method to sterilize human tissues. In contrasting <25 Kgy has no damaging effect on tissue and its constituents. Better choose processing method wisely, depend on the functional roll of the tissue. Combination of radiation and freeze dry is still the best procedure (weight bearing tissues are better to replace by deep frozen tissues).

Gamma irradiation upto 30 kGy: No effect on biomechanical properties of freeze dried amnions. But freeze drying reduces to some extents.

Polyethylene and Aluminium laminated foil: No effect.

Significant reduction by 50% in tensile strength and elongation, of irradiated (15, 30 kGy) freeze dried amnions after 12 months storage at room temperature.

to identify effect of processing, radiation and storage on biomechanical and physical properties

to validate shelf life of air dried amnion

Effects of Radiation (17kGy) & Storage (RT) on Air Dried Amnions

Air dried is comparable to freeze dried amnions, better in tensile strength after storage.

Air Dried amnions for routine procesing.

2 years shelf life given is acceptable, no effect on biomechanical properties.

Biomechanical characteristic has no clinical significance.

No change in appearance and packaging after 5 years.

Air dried amnion can be stored up to 5 years at RT, Sterility is guaranteed as long as packaging is maintained

Lecture Topic: SELECTION OF STERILIZATION DOSE

Sterilization process has to be balanced process of biologically and physico-chemically. Final result will be effective to kill microorganisms and tolerable/minimal destructive effects respectively.

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

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" - no semi or almost sterile conditions).

Microbes are diverse group of life from, extremely small and nuisance.

	Potentially cause diseases
	Ubiquitous distribution
	Extremely small size, invisible to naked eye.

Hygienic practice in procurement, processing, packaging etc can only minimize types and number of microorganisms.

To achieve high sterility assuarance level of low bioburden tissue product will helpful for safe clinical application. And using irradiation, this can be easily achieved.

Following criteria will helpful for the dose determination.

Can reduce by reducing bioburden.

Determination of radiation dose with biological indicators and radio sensitivity.

Good Manufacturing Practices (GMP).

Type of microorganisms.

Type of packaging materials.

1940’s - Experimental irradiation of sutures, B.pumilus spores, at 25 kGy 100% kiiling

1950’s - UK quatitative approach. At 25 kGy D₁₀ = 2 kGy, inactivation factor of 10^x

1960’s - Denmark & Scandinivian countries

<50 viable mo       - 35 kGy Inflexible for

50 - 500                  - 45 kGy polymeric

500 - 1000               - 50 kGy materials

1980’s - GMP, reduce bioburden to reduce dose

1990’s - Dose decided by manufacturers: quality (bioburden, type of microbes), SAL

Dose Setting:

Based on:

Initial number of microorganisms on product

Mixed population with D₁₀ of probability model

Verification dose experiment at SAL 10^-2

....is TOTAL NUMBER or COUNT of viable microorganisms (Virus, Bacteria, Yeast, Mould) on a packaged product prior to sterilisation process

COUNT = Bioburden

ISO 11137 :

Based on

Natural Bioburden resistance to determine SAL dose

Sterility Test after a series of incremental doses

D₁₀ Value is the dose required to reduce a given population of microorganism to 10% of initial number (=90% killing) or by one log cycle

D₁₀ = D / [log N_o - log N]

or

D = [log N_o - log N] x D₁₀

where

D Radiation Dose (kGy)

N_o Initial number (Bioburden, cfu/item)

N Number of microorganisms after dose D

...is expected probability of surviving microorganisms on individual product after exposure to valid sterilisation process

For tissue grafts, SAL is

10^-6 for product come into contact with open tissue

N or count after dose D

Describe the standard.

Device and packaging materials qualification

Dose setting methods for radiation sterilization.

Dosimeters, dosimetry and associated equipment

STERILISATION is a process for which efficacy cannot be verified by retrospective inspection and testing of the product.

To achieve:

exposure to validated accurately controlled sterilisation process, microbiological status of raw materials and components, microbiological barrier properties of the packaging, control of the environment (manufacture, assembly, packaging and storage).

SAL is a function of the end use of the product specifications.

Expressed as 10^-n , 2 levels:

10^-3 for products which do not contact with compromised tissues (swabs, serum tubes, bioassay dishes, centrifuge tubes, surgical drapes).

10^-6 for products which are in contact with compromised tissues / used to invade natural body barriers (blood lancets, medical implants, surgical gloves, syringe).

Most authorities require SAL 10^-6 regardless of end use….

3 steps:

The knowledge of the number and resistance to radiation of the natural microbial population on/in the product.

Then used for the determination of the sterilisation dose. The selected dose is capable of achieving the preselected SAL.

2 approaches:

(i) Bioburden information - ISO11137 Method 1

(ii) Information obtained by incremental dosing -

ISO11137 Method 2.

Substantiate the dose - ISO(TR) 13409:1996(E)

“Sterilization of Health Care Products -

Radiation Sterilization - Substantiation of 25 kGy as a sterilization dose for small or infrequent production batches”

Access to Microbiology Laboratory with QS/ certified to Guide 25.

Testing be performed in accordance with

ISO11737-1 Microbiological Methods Part 1 Estimation

ISO11737-2 Microbiological Methods Part 2 Sterility performed in the validation (draft / AAMI 1991).

Access to Radiation Facility capable of delivering accurate and precise doses ranging from 1 kGy.

This is a collective integrated set of principles help tissue bankers as well, ensure there products are consistently design, manufactured, supplied and control to the quality standards required. This guideline meets the criteria's of safety, efficacy and quality of the product or tissue allograft. In generally GMP guide lines common for all healthcare products both sterile and unsterile only effect the quality. There are 10 major principles defined 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 and trained personnel.

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

Documented manufacturing procedures with attention to manufacturing requirements and controls.

Approved, audited and formalized contact QC, manufacture and sterilization.

Originally ISO 11137 or other standards or GMP are not designed for Human Tissues. But these grafts also can consider under medical products category other than very few differences. Therefore we use these guidelines for human tissues until the developments of separate standards for human tissues.

Degree of excellence is the highest definition for the quality.

Positive statement intended to assure the confidence of the product, make certain and give a guarantee of the product. (this is an integration of GMP and QC).

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

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 purchased, sample testing and follow up complaints.

All procedures are well defined and documented. Organizational structure, responsibilities, procedures and processes, resources to implement and traceability and sustainability. Therefore TQM provides assurance of all carried out according to required standards, customer's satisfaction, fulfill legislative requirements etc. Finally all everything provide objective evidence to prove that those have been implemented and seek by audit.

Quality in tissue banking - need to establish for following purposes:

Traceability

Recipient needs

Donor Characteristics

Design process as per clinical need

Write specifications (how you do) for each and every step.

Validation of equipment, service, process, procedure, operator, change in any of these, monitoring of rooms for confirmation of the system. Maintenance of equipment and checking all of that has been validated. As a part of the irradiation process control, it is necessary to establish the dose distribution in each irradiation facility and for each type of product. A dose mapping exercise which characterizes the distribution of dose throughout a product must be carried out in which the actual product or simulated material of homogeneous density is extremely mapped by placing dosimeters at various positions within the product and throughout the container (IAEA, 1990).

isos (a greek word) means “equal”

formulated by International Organisation for Standard (ISO), Geneva, Switzerland

series on quality system

Objective:

to harmonise local and national standards to international standards which are adopted by all member countries

Defines requirements for 20 system elements

1. Management responsibility

2. Quality system

3. Contract review

4. Design control

5. Document and data control

6. Purchasing

7. Control of customer-supplied products

8. Product identification & traceability

9. Process control

                    10. Inspection and testing

11. Control of inspection, measuring and test equipment

12. Inspection and test status

13. Control of nonconforming product

14. Corrective and Preventative action

15. Handling, storage, packaging, preservation and delivery

16. Control of quality records

17. Internal quality audits

18. Training

19. Servicing

                    20. Statistical techniques

Quality audit is a systematic and independent examinations 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.

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 an opportunity to improve the quality system (QS).

To meet regulatory requirements.

Certification purpose.

To make us more confident.

Prepare checklists: premises, people, procedure, forms, plants, products.

Implement/auditing: check the product thoroughly from starting material to final product.

Audit report and actions.

To evaluate suppliers in view of a contractual relationship.

To verify that the organisation’s own quality system meets requirements and is being implemented.

To verify that the suppliers quality system continues to meet specified contractual requirements and is being implemented.

To evaluate an organization’s own qualtiy system against a standard (e.g. ISO Guide 25 / ISO 9001).

FIRST PARTY An audit of the tissue bank/supplier’s system that is performed by the supplier’s management (own personnel)

SECOND PARTY An audit of the vendor /subcontractors 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

PERSONAL OBSERVATION by auditors

EXAMINATION of the documents

COMMUNICATION with management, workers, contractors etc

TEST of systems, procedures

INSPECTION of related lab, facility, instruments

COMPLAINTS from workers, customers

DOCUMENTATION

Write what you have to do

IMPLEMENTATION

Do what you write

Record & Compile what you do

AUDIT

Ready for short and precise answer

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