Wednesday, June 28, 2017
Project co-funded by the European Commission in the framework of the 2nd Health Programme
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B.3.1. ACTIVITIES

B.3.1.1. Reception

See generic requirements in section A.5.1.5.

B.3.1.2. Access to the processing facilities

See generic requirements in section A.5.1.2.

B.3.1.3. Processing

B.3.1.3.1.General

See generic requirements in section A.5.1.3

B.3.1.3.2. Processing methods

Proof is required that the validated procedures are performed by the tissue bank staff in a uniform manner in accordance with the approved standard operating procedures (e.g. standardization of corneal endothelial cell counting among the processing staff is required.)

B.3.1.3.2.1. Processing of tissues and cells

See generic requirements in section A.5.

B.3.1.3.2.1.1. Cornea processing

The preparation of the cornea should be performed with either excision of the corneoscleral button from enucleated whole eyes in vitro or excision of the corneoscleral button from the donor eyes in situ.  Preparation of cornea for lamellar keratoplasty (anterior or posterior) may be performed using manual or automated methods.  Lasers may be used to prepare corneal tissue in which unique tissue architecture is required.  This can include shaping for penetrating, anterior or posterior keratoplasty.

Inspection of the endothelium is mandatory at the end of the storage period and a cell loss during storage must be taken into account, except for tissue designated for emergency or anterior lamellar grafting.

B.3.1.3.2.1.2. Sclera processing

Sclera should be prepared using aseptic techniques after removal of the corneoscleral button from the bulbus.  The remaining contents (vitreous, lens, iris, choridal and retinal tissue) and adnexa (remnants of muscles, conjunctiva) should be removed.  If requested the tissue should be cut into pieces.

B.3.1.3.2.2. Decontamination of tissues and cells

For decontamination of ocular tissue, antibiotics may be used in the preservation medium.

When whole eye is delivered to the eye bank, the globe should be rinsed in a disinfectant solution (e.g. povidone-iodine) and thoroughly rinsed with a balanced electrolyte solution prior to further processing for cornea and sclera preparations.

For scleral tissue, decontamination in a gentamicin bath for 20 minutes before storage in glycerin, or a quarantine period in ethanol 70% for 14 days before renewal of the ethanol 70% is recommended.

B.3.1.4. Quality control

The quality control tests on corneal grafts should consider at least the following minimum quality criteria:

a)     Endothelial characteristics (cell density and morphology);

b)    Morphology and integrity of the cornea layers;

c)     Free visual area and diameter of corneal button;

d)    No evidence of microbiological growth or malignant cells.

See generic requirements in section A.5.1.4.

B.3.1.4.1. Microbiological control
B.3.1.4.1.1. General Principle

See generic requirements in section A.5.1.4.1

Additionally, antimicrobial effects of antibiotics in the culture medium / preservation solution should be taken into account while choosing and validating the microbiological test method.

B.3.1.4.1.2. Methods

B.3.1.4.1.2.1. Microbiological controls for organ cultured corneas

During preservation in organ culture at least one sterility test of the culture medium surrounding the donor cornea is to be performed.  For this purpose, a sensitive microbiological method to detect bacteria and fungi is to be applied.  The test should be performed several days into the storage period, and / or at a point near distribution of the cornea e.g. at the time of terminal evaluation and transfer of the cornea into transport medium.  The medium sample should not be collected for microbiological testing any earlier than on the third day of preservation, due to the known fact that donor corneas are not sterile and a significant growth of microorganisms should be awaited.  Any positive microbiological culture should have the organism identified to at least the genus level.

The culture medium is to be regularly inspected for cloudiness, which may indicate contamination.  If visible cloudiness or decolouration of the culture medium should occur, suitable microbiological testing should be initiated.

All used media should be stored at +31°C for four (4) weeks after transplantation of the donor cornea and inspected regularly for cloudiness.  In the case of suspected contamination, suitable microbiological testing should be initiated immediately.  Alternatively, the remaining culture medium may be tested for bacterial and fungal contamination readily after the tissue has been removed from the container.  Further, the transplanting organization should be informed immediately to allow a short-term control examination and treatment of the recipient, if necessary.  Only donor corneas without indication of contamination of the culture medium showing at least one normal finding of a sensitive microbiological test may be released for application in humans.

Instructions for surgeons to perform a microbiological test of either the donor tissue remaining after corneal trephination (i.e. the ‘donor rim’) and / or of the storage medium in which the cornea is received, is also recommended.  If either of these tests are performed, the eye bank may request the results to be reported to them by the surgeon and be retained as part of the records for that tissue.  Eye banks should request positive results in cases of post-operative infection that are, in the opinion of the surgeon, likely to be attributable to the donor tissue to be reported to the Eye Bank as an adverse reaction.

B.3.3.4.1.2.2. Microbiological controls for corneas in hypothermic storage

In hypothermic storage (short-time culture), a microbiological diagnosis of the culture medium is not feasible for several reasons and therefore does not have to be made.  However, for such donor corneas, documented recommendation from the cornea bank to the transplanting organization is required to apply a sensitive microbiological method for detection of bacteria and fungi in the culture medium and in the remaining tissue at the time of transplantation.

B.3.1.4.1.2.3. Microbiological controls for sclera

A sensitive microbiological method to detect bacteria and fungi is to be applied for scleral tissue before storage.

B.3.1.4.2.Other controls
B.3.1.4.2.1.Other controls for cornea

The cornea should be examined both macroscopically as well as microscopically.  To be able to select the tissue for the specific surgical use for which it is intended, it is necessary to check the condition of the epithelium (intended use: penetrating keratoplasty / full-thickness graft, superficial or deep anterior lamellar graft, limbal graft), the corneal stroma (intended use: penetrating keratoplasty / full-thickness graft, superficial or deep anterior lamellar graft) whose transparency is crucial; and the endothelium, which is essential for maintaining corneal transparency (intended use: penetrating keratoplasty / full-thickness graft, posterior lamellar graft).

B.3.1.4.2.1.1.Macroscopic evaluation of corneal quality

Corneal evaluation should begin with a gross optically unaided examination in situ where the cornea is to be inspected for transparency, epithelial defects, foreign objects, contamination and scleral colour e.g. jaundice and corneal pathology.  This examination may be aided by use of a penlight or portable slit-lamp.  Careful in situ examination is especially important  for corneoscleral rim excisions in situ since this is the only opportunity (other than a good medical history) to determine if there are any congenital abnormalities of the anterior chamber, iris or lens or if there has been previous anterior segment surgery such as cataract surgery and lens implantation.

 

 

B.3.1.4.2.1.2.Slit lamp evaluation of corneal quality

In the process of donor cornea preservation, a slit lamp examination to exclude any visible epithelial, stromal and endothelial pathological changes (scars, oedema, significant arcus, striae, epithelial defects, endothelial guttae or disease, polymegethism, pleomorphism, infiltrates or foreign bodies) is highly recommended, but not mandatory.  The slit-lamp enables a more accurate observation of the cornea, revealing earlier stages of pathology that are visible grossly.  For slit lamp examination, the eye or cornea should be allowed to reach room temperature.  This makes the endothelium easier to visualize and more normal in appearance.

With slit lamp, the cornea and limbal area should be inspected for features which may preclude use of tissue e.g. signs of corneal pathology or post mortem artefacts.  The epithelium should be inspected for integrity and overall condition specifically abrasions, defects and foreign bodies.  The stroma should be examined for overall clarity, amount of oedema and stromal folding.

B.3.1.4.2.1.3.Microscopic evaluation of corneal quality

The condition of the corneal endothelium is crucial to evaluating the suitability of corneal tissue for penetrating keratoplasty, as it is primarily the layer responsible for the maintenance of corneal hydration and transparency.  During preservation, a microscopic examination (e.g. specular microscopy, phase contrast microscopy, transmitted light microscopy) of the endothelial cell layer is to be performed at least once.  n the case of organ-cultivated dehydrated donor corneas this should be done shortly before the planned transplantation.

Endothelial cell examination and evaluation is not required for those corneas intended to be used only for anterior lamellar procedures.

Evaluation should include an assessment of endothelial cell morphology (pleomorphism, polymegathism) and determination of endothelial cell density.  In this examinationn the total endothelial area is to be analysed according to a validated and regularly checked method, either counting directly with the help of a graticule or afterwards on a photograph or with a calibrated software programme.  Cell counting should be done at different areas, centrally and paracentrally up to 5-6 mm from the centre.

For ease of cell counting with light microscopy, the endothelial cell borders are to be made visible by induction of swelling of the intercellular space with a hypotonic solution.  The induction of the swelling and the swelling pattern is dependent on type of medium and time of preservation.  The use of a vital stain (e.g. trypan blue) may help to recognize dead or necrotic cells and denuded Descemet’s membrane.

B.3.1.4.2.1.4.Pachymetric evaluation of corneal thickness

Eye Banks may choose to perform pachymetry and / or endothelial evaluation after lamellar or laser assisted preparation of tissue.

B.3.1.4.2.2. Evaluation of scleral quality

There are no absolute criteria for evaluation of scleral quality; however, scleral shells should be visually examined for gross defects before storage and distribution.

B.3.1.5. Packaging and labelling

See generic requirements in section A.5.1.5.

B.3.1.6. Storage/ preservation

B.3.1.6.1. General

See generic requirements in section A.6

Additionally, it must be recognized that while maximum storage times are recommended for the various methods of tissue preservation, in most cases it is preferable to transplant tissues before these maximum times are approached in order to optimize surgical outcome.  Availability of tissue, clinical requirements and surgical need may determine the storage time for each individual tissue.

B.3.1.6.2. Methods of preservation and storage

Preservation/storage methods for recovered donor ocular tissue are presented in chapter B.2.3.6.

B.3.1.6.2.1. Preservation and storage of corneal tissue

For the preservation and storage of donor corneas, eye banks should adhere to the subsequent minimal storage conditions necessary to maintain the required biological properties of the donor cornea.  For each kind of storage condition the maximum duration is to be specified. Among other features, when selecting the time interval, the potential deterioration of the required cell and tissue characteristics needs to be considered.

 

 

 

B.3.1.6.2.1.1. Organ culture of cornea

Organ cultured corneas are to be stored in a closed system at temperature between +30 and 38°C at normal room air; or in an open system (with gassing) at a temperature between +30 and 38°C at normal room air with 5% +/- 1% CO2 addition.

Prior to transplantation, the cornea is to be transferred into a de-swelling / transport medium in order to thin it.  In de-swelling / transport medium (containing an agent of osmolarity,(e.g. dextran) the cornea is to be stored in a closed system at temperature between +15 and 40°C at normal room air; or in de-swelling / transport medium (containing an agent of osmolarity, (e.g. dextran) in an open system (with gassing) at a temperature between +15 and 38°C at normal room air with 5% +/- 1% CO2 addition.

Principally, especially during transport, storage temperatures of less than 1°C and more than 40°C must be strictly avoided, as they may affect the corneal tissue and consequently jeopardize the safety for the recipient.

The maximum recommended storage time for corneas in organ culture medium is thirty five (35) days including the storage in de-swelling / transport medium, with the latter not exceeding a period of six (6) days.  For emergency transplants, a deviation from these storage periods is possible, with the periods to be reviewed by the person responsible on a case-by-case basis.

B.3.1.6.2.1.2. Hypothermic storage of cornea

Corneas for hypothermic storage / short-time culture should be stored in a closed system at normal room air at a temperature between + 1 and +10°C.  The maximum period for hypothermic storage is fourteen (14) days.

B.3.1.6.2.1.3. Corneal cryopreservation

The maximum recommended storage time by corneal cryopreservation is two years.  However, this may be extended upon the approval of the Medical Director and agreement with the transplanting surgeon.

B.3.1.6.2.2. Preservation and storage of scleral tissue

Sclera is used surgically as a structural tissue where viability is not important.  Several methods of preserving and storing sclera, including the use of ethyl alcohol (70% or greater), sterile glycerin, or formalin in room temperature; Optisol-GS or saline with antibiotics in +4°C; and cryopreservation as freeze dried or frozen, are acceptable.  Eye banks should preserve scleral tissue using aseptic techniques, using one of these methods.  A preservation and expiry date for scleral tissue should be specified.

B.3.1.6.3. Storage area

See generic requirements in section A.6

B.3.1.7. Documentation and release

B.3.1.7.1. General

See generic requirements in section A.5.1.6.1.

B.3.1.7.2. Release

Before tissues are released for transplantation, all pertinent records concerning donor screening, testing and quality control should be reviewed and found to be complete and accurate.  Final release for transplantation is to be performed and signed by the person responsible.  The documentation for the release of donor tissue is to demonstrate that all pertinent specifications were met, especially that all effective notification forms, pertinent medical records, processing records and test results were inspected.

B.3.1.7.2.1. Release of cornea

The eye bank should establish and document their criteria for release of tissue for transplantation, which should include (but is not limited to), the following:

a)     Full donor screening for contraindications with normal result;

b)    Negative serological or molecular biological diagnosis of the donor;

c)     Acceptable microbiological test result of the culture medium (except for hypothermic storage);

d)    No relevant, biomicroscopically detectable pathological changes (relevant changes are: stromal cloudiness located centrally and thus being optically relevant, stromal thinning located in the transplant area, unless proven to be of traumatic genesis, stromal changes of infectious genesis, Descemet´s membrane detachment);

e)     Sufficient quantity, vitality and morphology of endothelial cells (deviations from the normal range are: large, central multicellular necroses and cell count of less than 2000-2200 endothelial cells per mm², distinct polymegatism, distinct pleomorphism, signs of significant cell loss during organ culture, distinct granulation / vacuolization, guttae in the endothelial cell layer or cornea guttata);

For all corneal transplantations (elective and emergency penetrating keratoplasty, elective posterior lamellar keratoplasty, elective and emergency stroma patch, tectonic keratoplasty) only a donor cornea with a central endothelial cell count of at least 2000 endothelial cells per mm2 may be released.

Donor corneas with an endothelial cell count of less than 2000 endothelial cells per mm2 in the final examination may be used for all corneal transplantations except for elective penetrating keratoplasty, elective posterior lamellar keratoplasty and elective penetrating limbal keratoplasty.  These restricted applications in recipients are only permissible upon consent of the person applying the corneal transplant.  Containers containing such corneas are to be provided with a label with an appropriate text (e.g. ‘for emergency PKP’, ‘for anterior lamellar keratoplasty only’).

Until requirements a) – e) have been met, the donor cornea should be under quarantine.  In this respect, the quarantine period for organ cultivated donor corneas should not be shorter than ten (10) days, since the valid result of a sensitive microbiological test method is not to be expected any earlier.  Deviations from the quarantine period are possible as an exception, under the precondition that all above requirements are met, if the corneal transplant is needed for a case of urgent medical emergency.

B.3.1.7.2.2. Release of sclera

The Eye Bank should establish and document their criteria for release of sclera for transplantation, which should include (but is not limited to), the following:

a)     Full donor screening for contraindications with normal result;

b)    Negative serological or molecular biological diagnosis of the donor;

c)     Acceptable microbiological test result of the tissue prior to storage;

d)    Sufficient scleral quality.

B.3.1.7.3. Processing file contents

See generic requirements in section A.5.1.6.3.

B.3.1.7.4. Availability for inspection

See generic requirements in section A.5.1.6.4.

B.3.1.7.5. Traceability

See generic requirements in section A.5.1.6.5.

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