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Use of hyaluronidase as an adjunct to local anaesthetic eye blocks

  1. Heinrich Rüschen1,*,
  2. Lee Adams1,
  3. Catey Bunce2

Editorial Group: Cochrane Anaesthesia Group

Published Online: 28 FEB 2013

Assessed as up-to-date: 13 JAN 2013

DOI: 10.1002/14651858.CD010368

How to Cite

Rüschen H, Adams L, Bunce C. Use of hyaluronidase as an adjunct to local anaesthetic eye blocks (Protocol). Cochrane Database of Systematic Reviews 2013, Issue 2. Art. No.: CD010368. DOI: 10.1002/14651858.CD010368.

Author Information

  1. 1

    Moorfields Eye Hospital NHS Foundation Trust, Department of Anaesthesia, London, UK

  2. 2

    Moorfields Eye Hospital NHS Foundation Trust, Research and Development Department, London, UK

*Heinrich Rüschen, Department of Anaesthesia, Moorfields Eye Hospital NHS Foundation Trust, 162 City Road, London, EC1V 2PD, UK.

Publication History

  1. Publication Status: New
  2. Published Online: 28 FEB 2013




  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
  9. Sources of support

Description of the condition

Local anaesthesia for ophthalmic surgery can be provided by regional injection block or topical anaesthesia alone. The most frequently used anaesthetic injections are retrobulbar, peribulbar and sub-Tenon's block.

During any local anaesthetic injection for ophthalmic surgery, the objective is to deliver local anaesthetic fluid to the sensory and motor nerve fibres in the orbit. There is a large variation in the techniques and instruments in use to achieve this. Some of these techniques have been compared in systematic reviews, for example peribulbar versus retrobulbar block (Alhassan 2011) and topical anaesthesia alone versus sub-Tenon's block (Davison 2007).

Unfortunately there is always a proportion of blocks that fail to provide adequate analgesia or akinesia. In an attempt to improve the quality of the anaesthetic block, various adjuncts to the local anaesthetic fluid have been introduced.


Description of the intervention

The addition of hyaluronidase to local anaesthetic fluid with the intention of improving the speed of onset of analgesia and akinesia was first described by Atkinson 1949. Subsequently hyaluronidase has commonly been added to local anaesthetic fluid, but there has never been a systematic analysis of the use of hyaluronidase for this purpose.


How the intervention might work

For any local anaesthetic block to work, the local anaesthetic fluid needs to spread through the orbital cavity to reach the relevant motor and sensor fibres. A complex system of connective tissue membranes divides the orbital space, thereby impeding the spread of local anaesthetic fluid (Koornneef 1988). Hyaluronidase (hyaluronoglucosaminidase) is an enzyme that facilitates the spread of local anaesthetic fluid through these membranes by temporarily depolymerising hyaluronic acid and chondroitin sulphate, two components of the connective tissue matrix (Girish 2007; Vickers 1984). It hydrolyses hyaluronic acid by splitting the glucosamine bond between C1 of the glucosamine moiety and C4 of glucuronic acid. This temporarily decreases the viscosity of the cellular cement and promotes diffusion of injected fluids (Vitrase® Prescribing Information 2010).

Hyaluronidase was first described by Duke-Elder 1929 and first extracted by Meyer 1934. Preparations contain purified ovine testicular hyaluronidase as a dehydrated sterilized solid for reconstitution before use. Brand names of animal-derived hyaluronidase include Hydase™, Vitrase®, Amphadase®, Wydase® and Hyalase®. Apart from a preparation of ovine testicular hyaluronidase, a recombinant human hyaluronidase is also available, as Hylenex®. It is produced by genetically engineered Chinese hamster ovary cells containing a DNA plasmid encoding for a soluble fragment of human hyaluronidase (Hylenex® Prescribing Information 2008). The exact chemical structure of this enzyme is unknown. The approximate molecular weight is 61,000 Daltons (Borders 1968).

Hyaluronidase also alters the pH of a local anaesthetic due to the presence of phosphate buffers within the preparation. The pH of plain bupivacaine solution is changed from 5.3 to 6.3 following the addition of hyaluronidase and it may maintain local anaesthetic solubility during the process of alkalinization as described by Roberts 1993. This alkalinization may also explain any improved anaesthesia and akinesia. 

A reduction in time to onset of surgical anaesthesia is considered desirable in order to facilitate patient throughput. The action of hyaluronidase may promote rapid onset of anaesthesia and akinesia.The minimum and maximum effective doses of hyaluronidase are not known. The doses used range from 0.75 IU/ml to 300 IU/ml (Dempsey 1997).


Why it is important to do this review

The use of hyaluronidase increases the cost of the anaesthetic and has been associated with adverse allergic reactions in a number of reported cases. For example, Kempeneers 1992 described five patients who developed an orbital pseudotumour as a complication of retrobulbar anaesthesia. Allergic reactions can range from local reactions to anaphylactic shock. Generally, when hyaluronidase is added to local anaesthetic agent, the wider spread of the local anaesthetic solution increases its absorption. This shortens the duration of action of the local anaesthetic and tends to increase the incidence of systemic reactions (Hylenex® Prescribing Information 2008). Some hyaluronidase products contain bovine ingredients and, due to the at least theoretical concerns about transmissible spongiform encephalopathies, the World Health Organization (WHO) has issued guidelines regarding the use of bovine materials in the manufacture of biological and pharmaceutical products (WHO Guidelines on Tissue Infectivity 2006).

The absence of hyaluronidase in ophthalmic regional blockade has also been associated with adverse events. An interruption in hyaluronidase supply was associated with a cluster of postoperative diplopia described by Brown 1999. It was postulated that the absence of hyaluronidase caused the local anaesthetic to loculate in close proximity to the extraocular muscles and cause clinically significant myotoxicity.

The use of hyaluronidase must therefore be justified and data must be available for clinicians to make an informed decision regarding the efficacy of hyaluronidase addition. The outcome measures of this meta-analysis should allow justification (or not) for use of adjuvant hyaluronidase to improve the quality of anaesthesia and analgesia.



  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
  9. Sources of support

To ascertain if adding hyaluronidase to local anaesthetic solutions for use in ophthalmic anaesthesia results in a reduction of perceived pain during the operation.



  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
  9. Sources of support

Criteria for considering studies for this review


Types of studies

We will include all randomized and quasi-randomized controlled clinical trials (RCTs), either published or unpublished.

We will include the studies if they compare equal volumes and concentrations of local anaesthetic with and without adjuvant hyaluronidase administered with the injection.

We will include studies when other adjuvants such as adrenaline are used only if the adjuvant is present in both the control and hyaluronidase intervention.


Types of participants

We will include adult patients (18 years and older) presenting for ophthalmic surgery under ophthalmic anaesthetic block.

We will include patients receiving sub-Tenon's, peribulbar, retrobulbar or other types of injection of local anaesthesia.

We will include patients receiving sedation, but this fact will be clearly documented.

We will exclude patients who receive general anaesthesia.

We will include 'first eye' operations only.


Types of interventions

Ophthalmic local anaesthetic blocks comparing the use of adjuvant hyaluronidase to an otherwise equal anaesthetic and surgery without hyaluronidase.

We will consider any dose of hyaluronidase in the intervention group and any dose or type of local anaesthetic agent.

We will include studies that use any number of injections to anaesthetise the eye, as long as the number of injections is equal in the treatment and control groups.


Types of outcome measures


Primary outcomes

1. Intraoperative pain, as measured by analogue rating scales


Secondary outcomes

2. Incidence of harm

3. Patient and surgical satisfaction, as documented by scoring systems

4. Economic outcomes or cost calculations if undertaken by any studies


Search methods for identification of studies


Electronic searches

We will search the most recent issue of the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library); MEDLINE (1992 to date); EMBASE (1992 to date); CINAHL (1992 to date) and Web of Science (1992 to date).

Please see Appendix 1 for our MEDLINE search terms.

We will combine the sensitive strategies described in Section 6.4 of the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011).

We will incorporate any new terms that are identified into the search strategy. We will report the modified search strategy in full in the final review.

We will assess retrieved studies for any free text terms or MeSH terms for adjuvant hyaluronidase compared to controls.

We will not apply any language restrictions.


Searching other resources

We will search conference proceedings.

We will screen the reference lists of all eligible trials and reviews and use any trials that are found to fit the inclusion criteria.

We will search the registers at and for relevant trials.

We will contact specialists in the field, authors of the included trials and pharmaceutical manufacturers for any unpublished data.


Data collection and analysis

We (LA and HR) will independently review the trials identified from the search strategy. We (LA and HR) will document the reason for each trial being excluded. We will list the included trials in a table 'Characteristics of included studies'. We will also include a table 'Characteristics of excluded studies' for the possibly relevant studies found not to fulfil the inclusion criteria.

We will resolve any disagreements between LA and HR by input from CB.


Selection of studies

Using the results of the above searches, we will screen all titles and abstracts for eligibility. Two authors (LA and HR) will independently perform this screening. Each of these authors will document the reason for each trial exclusion.

We will resolve any disagreements between LA and HR by input from CB.


Data extraction and management

Two authors (LA and HR) will independently extract and collect data on a paper form. A copy of this form is in Appendix 2. We (LA and HR) will resolve any discrepancies in the data extracted by discussion, with CB as final arbiter. In the case of additional information being required, HR will contact the first author of the relevant trial.


Assessment of risk of bias in included studies

In order to assess the risk of bias we will independently assess the studies included in the review according to the criteria described by Higgins 2011. We will assess the following aspects as being at either 'low risk', 'high risk' or 'unclear risk' of bias. We will assess risk of bias for the following components of each trial.

1. Random sequence generation.

2. Allocation concealment.

3. Blinding of participants and personnel.

4. Blinding of outcome assessment.

5. Incomplete outcome data.

6. Selective reporting.

7. Other bias.

We will include a risk of bias table as part of the characteristics of included studies based upon the Cochrane Collaboration tool for assessing risk of bias, from Chapter 8 of the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011). See Appendix 2 (Data collection form) and Appendix 4 (Risk of bias table).


Measures of treatment effect

1. Intraoperative pain will be noted as visual analogue scores (VAS pain scores) when available and interpreted as continuous data. We will use the greatest intraoperative score reported. For these results, we will use the standardized mean difference (SMD) as effect measure and 95% confidence interval (CI). If authors have documented non-normality of their data we will extract medians and interquartile ranges and collate this information.

In the case of absolute numbers of patients experiencing pain where no rating scale is used (dichotomous data as pain or no pain) we will use the risk ratio (RR) as a measure of effect.

To avoid multiplicity issues, we do not plan to meta-analyse other outcomes, but we believe it of value to place collated information on these outcomes in the public domain for others to use in their own reviews.

2. Adverse events (allergic reactions, injury to globe or extraocular muscle, adverse changes in intraocular pressure, complications or cancellation of surgery and conversion to general anaesthesia) will be reported as a narrative. We do not expect a significant number of adverse events due to their rarity in the ophthalmic surgery population.

3. Patient and surgical satisfaction scores, if present, will be noted as a narrative. We will note as narrative if validated tools were used.

4. We will collate any economic information provided by the included studies.


Unit of analysis issues

We anticipate that most trials will involve one eye per patient, and even if both eyes are included our outcomes are primarily measured at the patient rather than eye level. It is very unlikely that both eyes will be operated on simultaneously with different anaesthetic procedures. Since prior surgery influences how a patient reacts, we wish to include first eye only studies.


Dealing with missing data

We will attempt to contact authors for them to provide missing data. We will impose a time limit of two months and follow up on one occasion. Irrespective of the type of data, we will report dropout rates in the 'Characteristics of included studies' section and make note of whether or not authors have compared characteristics of patients who have complete data sets against those that did not. We intend to meta-analyse results which have been analysed by adhering to the intention-to-treat principles, however we acknowledge that authors may present available case data only. Should this be the case, we will meta-analyse available case analyses but comment on this and examine, using sensitivity analyses, whether or not imputing for missing cases impacts greatly on the interpretation of findings.


Assessment of heterogeneity

We will assess all studies for clinical and methodological heterogeneity. We will examine the I2 statistic and its confidence interval as a means to assess inconsistency between studies, as advised by the Cochrane Handbook for Systematic Reviews of Interventions. We will use the thresholds advised by Higgins 2011 for the interpretation of I2. If we find substantial inconsistency (for example I2 > 50%), we will attempt to investigate causes for this by exploring factors such as the type and duration of surgery and anaesthetic intervention (hyaluronidase dosage, type and volume of anaesthetic fluid). If there is substantial heterogeneity we will not meta-analyse our data but will instead present a narrative summary.


Assessment of reporting biases

We will assess publication bias and small study effects in a qualitative manner using a funnel plot. We will test for funnel plot asymmetry if there are greater than 10 studies included in the meta-analysis.


Data synthesis

We will perform the analysis using Review Manager software (RevMan 5.1).

Providing we do not detect heterogeneity, we will meta-analyse our primary outcome only. We intend to use a random-effects model for our meta-analysis although, if there are very few studies (three or less), we may use a fixed-effect model simply because there will be insufficient data to adequately estimate random effects. If heterogeneity is detected, we will not conduct a meta-analysis but will present a narrative summary of the findings.


Subgroup analysis and investigation of heterogeneity

We are not planning any subgroup analysis.


Sensitivity analysis

If there are an adequate number of studies, we will carry out sensitivity analyses to explore the robustness of the results to key methodological decisions we have made in our review. We will examine whether or not excluding studies at risk of bias impacts on our findings and, if we have missing data, we will impute and examine whether or not analysing intention-to-treat data differs considerably from the available case meta-analysis.



  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
  9. Sources of support

We thank Jane Cracknell, Managing Editor of the Cochrane Anaesthesia Review Group (CARG), and Karen Hovhannisyan (CARG Trials Search Coordinator). We would also like to thank Andrew Smith (content editor); Marialena Trivella (statistical editor); Mahmoud B Alhassan, Richard Wormald, Jacques Ripart (peer reviewers) for their help and editorial advice during the preparation of this protocol for the systematic review.



  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
  9. Sources of support

Appendix 1. MEDLINE search terms

1     exp Ophthalmologic Surgical Procedures/ 

2     ((Ophthalm* or eye*) adj5 (operat* or surg*)).mp. 

3     exp Anesthetics, Local/ or exp Anesthesia, Local/

4     exp Cataract Extraction/ 

5     (SubTenon or Sub-Tenon).af. 

6     ((retrobulbar or lid) adj3 block*).mp.

7     1 or 2 or 3 or 4 or 5 or 6

8     (Hyalase or Vitrase or Hydase or Vitrase or Amphadase or Wydase or Hylenex Hyaluronidase).af. 

9     exp Hyaluronoglucosaminidase/

10     8 or 9

11     7 and 10 

12     ((randomised controlled trial or controlled clinical trial).pt. or randomized.ab. or placebo.ab. or drug therapy.fs. or randomly.ab. or trial.ab. or groups.ab.) not (animals not (humans and animals)).sh.

13     11 and 12 

NOTE MEDLINE search terms will be adapted to search other databases (EMBASE, CENTRAL, CINHAL, Web of Sceience)


Appendix 2. Data collection form

Cochrane Anaesthesia Review Group

Study Selection, Quality Assessment & Data Extraction Form

First authorJournal/Conference Proceedings etcYear


Study eligibility

RCT/Quasi/CCT  (delete as appropriate)Relevant participantsRelevant interventionsRelevant outcomes


Yes / No / Unclear

Yes / No / Unclear

Yes / No / Unclear

Yes / No* / Unclear


* Issue relates to selective reporting when authors may have taken measurements for particular outcomes, but not reported these within the paper(s). Reviewers should contact trialists for information on possible non-reported outcomes & reasons for exclusion from publication. Study should be listed in ‘Studies awaiting assessment’ until clarified. If no clarification is received after three attempts, study should then be excluded.


Do not proceed if any of the above answers are ‘No’. If study to be included in ‘Excluded studies’ section of the review, record below the information to be inserted into ‘Table of excluded studies’.





Freehand space for comments on study design and treatment:

References to trial

Check other references identified in searches. If there are further references to this trial link the papers now & list below. All references to a trial should be linked under one Study ID in RevMan.

Code each paperAuthor(s)Journal/Conference Proceedings etcYear

AThe paper listed above  

BFurther papers  


Participant characteristics

 Further details

Age (mean, median, range, etc) 

Sex of participants (numbers / %, etc) 

Dose of hyaluronidase 


Trial characteristics

Methodological quality

Allocation of intervention

State here method used to generate allocation and reasons for gradingGrade (circle)


Adequate (Random)

Inadequate (e.g. alternate)


Concealment of allocation

Process used to prevent foreknowledge of group assignment in a RCT, which should be seen as distinct from blinding

State here method used to conceal allocation and reasons for gradingGrade (circle)





Person responsible for participants careYes / No

ParticipantYes / No

Outcome assessorYes / No

Other (please specify)Yes / No


An intention-to-treat analysis is one in which all the participants in a trial are analysed according to the intervention to which they were allocated, whether they received it or not.

All participants entering trial 

15% or fewer excluded 

More than 15% excluded 

Not analysed as ‘intention-to-treat’ 





Were withdrawals described?    Yes  ?              No ?        not clear  ?  


Is attrition reported? Yes ? no ?

Total number randomized

Number in hyaluronidase group

Number in control group


Discuss if appropriate and note reasons for attrition


Is there a possibility of selective outcome reporting Yes? No?

Discuss if appropriate

Data extraction

Outcomes relevant to your review

 Reported in paper (circle)

Intraoperative painYes / No

Adverse EventsYes / No

Satisfaction scoringYes / No

For Continuous data


Code of paper




Unit of measurement
Intervention groupControl groupDetails if outcome only described in text

nMean (SD)nMean (SD) 

Intraoperative VAS pain score

(include range)

For Dichotomous data

Code of paperOutcomesIntervention group (n)

n = number of participants, not number of events
Control group (n)

n = number of  participants, not number of events

 Intraoperative Pain (where not VAS)  



Other information which you feel is relevant to the results

Indicate if: any data were obtained from the primary author; if results were estimated from graphs etc; or calculated by you using a formula (this should be stated and the formula given). In general if results not reported in paper(s) are obtained this should be made clear here to be cited in review.

 Adverse events recorded here





Freehand space for writing actions such as contact with study authors and changes


 Surgical specialty: (CIRCLE)

1. Anterior segment.

2. Glaucoma.

3. Vitreoretinal surgery.


References to other trials

Did this report include any references to published reports of potentially eligible trials not already identified for this review?

First authorJournal / ConferenceYear of publication


Did this report include any references to unpublished data from potentially eligible trials not already identified for this review? If yes, give list contact name and details





Trial characteristics Summary

 Further details

Single centre / Multicentre 

Country / Countries 

How was participant eligibility defined?


How many people were randomized? 

Number of participants in each intervention group 

Number of participants who received intended treatment 

Number of participants who were analysed 

Local anaesthetic Drug dose (s) used 

Dose of hyaluronidase 

Median (range) length of follow-up reported in this paper (state weeks, months or years or if not stated) 

Time-points when measurements were taken during the study 

Time-points reported in the study 

Time-points you are using in RevMan 

Trial design 



Appendix 3. Characteristics of excluded studies

Reason for exclusion noted as narrative only:


Appendix 4. Risk of bias table


Low risk/high risk/unclear risk

1. Random sequence generation

2. Allocation concealment

3. Blinding of participants and personnel

4. Blinding of outcome assessment

5. Incomplete outcome data

6. Selective reporting

7. Other bias


Contributions of authors

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
  9. Sources of support

Conceiving the review: Lee Adams (LA)

Co-ordinating the review: HR

Undertaking manual searches: Heinrich Rüschen (HR)

Screening search results: LA and HR

Organizing retrieval of papers: HR

Screening retrieved papers against inclusion criteria: LA and HR

Appraising quality of papers: LA and HR

Abstracting data from papers: LA and HR

Writing to authors of papers for additional information: HR

Providing additional data about papers: HR

Obtaining and screening data on unpublished studies: HR

Data management for the review: HR

Entering data into Review Manager (RevMan 5.1): HR

RevMan statistical data: HR and Catey Bunce (CB)

Other statistical analysis not using RevMan: CB

Double entry of data: LA and HR

Interpretation of data: LA and HR

Statistical inferences: CB

Writing the review: HR

Securing funding for the review: LA HR and CB

Performing previous work that was the foundation of the present study: LA

Guarantor for the review (one author): HR

Person responsible for reading and checking review before submission: HR


Declarations of interest

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
  9. Sources of support

All authors: none known.


Sources of support

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
  9. Sources of support

Internal sources

  • Moorfields Eye Hospital, UK.
    Salary and facilities


External sources

  • No sources of support supplied


Additional references

  1. Top of page
  2. Abstract
  3. Background
  4. Objectives
  5. Methods
  6. Acknowledgements
  7. Appendices
  8. Contributions of authors
  9. Declarations of interest
  10. Sources of support
  11. Additional references
Alhassan 2011
Atkinson 1949
  • Atkinson WS. Use of hyaluronidase with local anaesthesia in ophthalmology; preliminary report. Archives of Opthalmology 1949;42:628-33. [MEDLINE: 15393388]
Borders 1968
Brown 1999
Davison 2007
Dempsey 1997
  • Dempsey GA, Barrett PJ, Kirby IJ. Hyaluronidase and peribulbar block. British Journal of Anaesthesia 1997;78:671-4. [MEDLINE: 9215017]
Duke-Elder 1929
  • Duke-Elder WS. The nature of the vitreous body. British Journal of Ophthalmology, Monograph Supplement 4 1929;LVIII:155.
Girish 2007
Higgins 2011
  • Higgins JPT, Green S (editors). Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [updated March 2011]. The Cochrane Collaboration, 2011. Available from
Hylenex® Prescribing Information 2008
  • Halozyme Therapeutics, Inc. Hylenex® Recombinant prescribing information. Drug Label - archived 10/2008:1.
Kempeneers 1992
  • Kempeneers A, Dralands L, Ceuppens J. Hyaluronidase induced orbital pseudotumour as complication of retrobulbar anesthesia. Bulletin de la Société Belge d'Ophtalmologie 1992;243:159-66. [MEDLINE: 1302146]
Koornneef 1988
  • Koornneef L. Eyelid and orbital fascial attachments and their clinical significance. Eye 1988;2:130-4. [MEDLINE: 3197870]
Meyer 1934
RevMan 5.1
  • The Nordic Cochrane Centre, The Cochrane Collaboration. Review Manager (RevMan). 5.1. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2011.
Roberts 1993
  • Roberts J, MacLeod B, Hollands RH. Improved peribulbar anaesthesia with alkalinization and hyaluronidase. Canadian Journal of Anaesthesia 1993;40(9):835-8. [MEDLINE: 8403178]
Vickers 1984
  • Vickers MD, Schneiden H, Wood-Smith FG. Drugs in Anaesthetic Practice. 6th Edition. London: Butterworths, 1984.
Vitrase® Prescribing Information 2010
  • ISTA Pharmaceuticals. Vitrase® - hyaluronidase, ovine injection, solution prescribing information. Drug Label - archived 01/2010;1:1.
WHO Guidelines on Tissue Infectivity 2006
  • World Health Organization. WHO Guidelines on Tissue Infectivity Distribution in Transmissible Spongiform Encephalopathies. Vol. 1, Geneva: WHO Press, 2006.