Vasculitis Sample and Data Collection and Management Protocol V7

Rare Kidney Disease Registry & BioResource: Data and Sample Collection Manual 2014

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Rare Kidney Disease Registry & Bioresource Protocol

Data and Sample Collection Manual Version 7

Supported by:

http://www.sfi.ie/index/

http://twitter.com/tcddublin/status/263987809457676288/photo/1


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Table of Contents

I. Part A. Study Introduction ......................................................................................... 4

A.1 Abbreviations................................................................................................................ 4

A.2 Investigator Signature Page ........................................................................................... 5

A.3 Site Responsibilities ............................................................................................................ 6

A.4 Rare Kidney Disease & Vasculitis Group Statement ........................................................ 6

A.5 Study Contact Details .................................................................................................... 7

Overall study Principal Investigator: ............................................................................................... 7

Renal Inflammation Group Senior Scientist, Scientific Advisor and Biobank Supervisor: .............. 7

Biobank Coordinator: ...................................................................................................................... 7

Overall study Lead Research Nurse: ............................................................................................... 7

Local study contacts Tallaght Hospital: ........................................................................................... 7

Local study contacts St Vincent’s University Hospital: ................................................................... 8

Local study contacts St. James’s Hospital: ...................................................................................... 8

Local study contacts Beaumont Hospital: ....................................................................................... 8

Local study contacts Galway University Hospital: .......................................................................... 8

Local study contacts Cork University Hospital: ............................................................................... 8

A.6 Study Overview ............................................................................................................. 9

Investigation of rare diseases ......................................................................................................... 9

Systemic Vasculitis: networked investigation of a “Common-Rare” disease ................................. 9

Novel genetic kidney diseases: centralised study of “Rare-Rare” kidney disease ........................ 10

Study Design.................................................................................................................................. 11

Study Methodology. ...................................................................................................................... 11

A.7 Patient Eligibility ......................................................................................................... 12

Inclusion Criteria: .......................................................................................................................... 12

Exclusion Criteria:.......................................................................................................................... 13

A.8 Participant Recruitment .............................................................................................. 13

Patient group: ............................................................................................................................... 13

Control group: ............................................................................................................................... 14

A.9 Informed Consent ....................................................................................................... 15

Informed consent procedure: Patients ......................................................................................... 15

Informed consent procedure: Controls ........................................................................................ 16

A.10 Sample Collection Summary ..................................................................................... 16

A.11 Ethical Considerations ............................................................................................. 16

Confidentiality ............................................................................................................................... 17

A.12 Data Collection & Management ............................................................................... 17

A.13 Patient Information Log - Completion Guidelines ......................................................... 18

A.14 CRF Completion Guidelines .......................................................................................... 18

A.15 Data Entry into Patient’s Medical Records .................................................................... 19

A.16 Study Monitoring/Quality Review................................................................................ 19

Governance ................................................................................................................................... 19

Rare Kidney Disease and Vasculitis Registry Steering Group Members: ...................................... 20


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II. Part B: Sample and data management ..................................................................... 21

B.1. Sample & Data Management-Part 1, Collection: ............................................................... 21

Patient Registration pack: ............................................................................................................. 21

Healthy control Registration pack: ............................................................................................... 21

Disease control Registration pack: ................................................................................................ 22

Visit pack: ...................................................................................................................................... 22

Control Recruitment Envelope...................................................................................................... 22

Sample Collection Components-All Packs will contain the following: .......................................... 23

PaxGene Tubes .............................................................................................................................. 23

Data Collection .............................................................................................................................. 24

Sample Collection ......................................................................................................................... 25

Post-sample collection .................................................................................................................. 26

B.2. Sample & Data Management-Part 2, Processing: .............................................................. 27

B.3. Sample & Data Management-Part 3: Sample transfer ....................................................... 28

Transfer of Samples from Local Sites to Trinity Biobank at IMM and enrolment into the Central

Rare Kidney Disease Bio Resource. ............................................................................................... 28

B.4. Sample & Data Management-Part 4: Barcoding System. ................................................... 28

Notes on the Barcode Labels ........................................................................................................ 31

III. Part C. Safety Guidelines ...................................................................................... 34

C.1 Safety Guidelines for Blood Collection, including specific guidelines for use of Paxgene

RNA tubes .............................................................................................................................. 34

C.2 Safety Guidelines for Urine Collection .......................................................................... 36

C.3 Safety Guidelines for Handling of Biological Materials .................................................. 36

C.4 Safety Guidelines for Handling of High Risk Samples .................................................... 37

C.5 Safety Guidelines for Disposal of Sharps ...................................................................... 38

C.6 Safety Guidelines for Handling Chemical Hazards ......................................................... 38

C.7 Safety Guidelines for Handling Dry Ice ......................................................................... 39

C.8 Safety Guidelines for Handling Liquid Nitrogen ............................................................ 39

IV. D: Standard Operating Procedures: ...................................................................... 41

D1. Standard Operating Procedure for elective recruitment and clinical data entry .................. 41

D.2: Standard Operating Procedure: Recruitment at a peripheral site without research nurse or

CRF facilities ........................................................................................................................... 47

D2.1: Recruitment at peripheral sites outside Dublin .................................................................. 48

D2.2: Recruitment of acute untreated patients in Beaumont Hospital:....................................... 48

D.3: Standard Operating Procedure: Sample Collection ........................................................... 49

Priority Patients ............................................................................................................................ 49

D.3.1 Urine Sample Collection ...................................................................................................... 53

D.3.2 Blood Sample Collection ...................................................................................................... 53

D.3.3 Plasma Exchange Sample Collection on acute patients at Tallaght Hospital. ..................... 56

D.4: Standard Operating Procedure: Sample Processing .......................................................... 56

D.4.1: Receipt of the samples at laboratory: ................................................................................ 56


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D4.2: Notes on Centrifugation and the importance of setting the centrifuge to G or RCF and not

RPM ............................................................................................................................................... 57

D4.3: Processing of Urine for Proteomics ..................................................................................... 57

D4.4: Processing of urine for metabolomics ................................................................................. 58

D4.5 Processing of Urine for Exosome Analysis ........................................................................ 59

D.4.6 Processing of EDTA Plasma Sample for Proteomics and DNA Extraction. .......................... 61

D.4.7: Processing of Serum for Metabolomics ............................................................................. 63

D.4.8: Processing of PAXgene tube for RNA analysis. ................................................................... 64

D.4.9: Processing of 2 x EDTA tubes for PBMC preparation. ........................................................ 65

D.4.10 Sample Processing BDP100 Plasma ................................................................................... 66

D.4.11 Sample Processing Summary ............................................................................................. 67

D.5: Standard Operating Procedure: Sample Transportation after processing at local sites ....... 68

D.6: Standard Operating Procedure: Sample Storage at the RKD Biobank ................................ 68

D.7: S.O.P. Emergency Response in case of main Biobank Freezer malfunction......................... 68

E. References ......................................................................................................................... 70

Appendix I: Paxgene tube ....................................................................................................... 71

Appendix II: BD P100 tube ...................................................................................................... 71

Appendix III: Paper CRF .......................................................................................................... 71

Appendix IV: Tallaght specific information .............................................................................. 71

Appendix VI: SOP: Distiller to Freezerworks Data Export ......................................................... 71

I. Part A. Study Introduction

A.1 Abbreviations

ANCA Anti neutrophil cytoplasm antibodies

BBV Blood-borne viruses such as HIV, Hep B, Hep C

CKD Chronic Kidney Disease

CRF Case Report Form

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid (DNA)

ECRF Electronic Case Report Form

ESKD End Stage Kidney Disease

FCS Fetal Calf Serum

HEPB/C Hepatitis B or C


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HIV Human Immune Deficiency virus

ICF Informed Consent Form

PBMC Peripheral Blood Mononuclear cells

PI Principal Investigator

PIL Patient Information Leaflet

SOP Standard Operating Procedure

A.2 Investigator Signature Page

I agree to conduct the study according to this protocol (subject to any amendments), and with all

locally applicable regulatory requirements.

I agree to conduct the study in person or to supervise the study.

I agree to ensure that all who assist me in the conduct of the study have access to the study protocol

plus any amendments and are aware of their obligations

Name of Chief Investigator: Professor Mark Little

Title: Consultant Nephrologist

Signature of Chief Investigator: Date:

___________________________ _____________


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A.3 Site Responsibilities

Each individual site is responsible for study implementation and associated patient and

study site management. These responsibilities include:

Patient protection: ensuring the dignity, rights and safety of the patient are upheld

and maintained.

Accurate patient identification.

Achieving target recruitment.

Timely and accurate data collection and entry.

Ensuring that all study protocols are adhered to.

Sample collection, transportation, processing and storage at local sites.

A.4 Rare Kidney Disease & Vasculitis Group Statement

All research conducted by the Vasculitis Study Group will adhere to the ethical and scientific

quality standards of Good Clinical Practice. This study manual has been prepared to provide

reference and guidance to support the collection of bio-specimens from patients for

research. Please refer to these instructions to guide you through the specimen and data

collection procedures.

The following are core guidance principals:

• The safety, rights and wellbeing of all study participants are of primary importance.

• The right to privacy and confidentiality for all study participants will be adhered to in

accordance with national data protection guidelines.

• Studies will be conducted in compliance with the study protocol, which will have

been authorised by an appropriate ethics committee.

• No clinical study will commence without prior approval from the local research

ethics committee.

• The approved study protocol will be adhered to at all times. In the event of a study

amendment, it will not be implemented without prior approval from the local

research ethics committee.

• All personnel conducting the study will have had an appropriate level of education,

training and experience necessary for study procedures.

• All data will be recorded, handled and stored for study purposes in accordance to

the principals of Good Clinical Practice and study protocol requirements to ensure

accurate reporting, interpretation and verification of data.


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A.5 Study Contact Details

Overall study Principal Investigator:

Prof Mark Little (Nephrology lead)

Trinity Health Kidney Centre, Tallaght Hospital, Tallaght, Dublin 24

Email: [email protected]

Renal Inflammation Group Senior Scientist, Scientific Advisor and Biobank Supervisor:

Dr Fionnuala Hickey

Room 2.13 The Institute for Molecular Medicine, St. James Hospital Dublin 8.


Biobank Coordinator:

Ms. Valerie Logan

Room 1.35, The Institute for Molecular Medicine, St. James Hospital Dublin 8.


Overall study Lead Research Nurse:

For any data collection or procedural queries:

Clinical Research Nurse, Ms Emily Naylor,

St James’s Hospital/TCD


Alternatively contact Deputy Lead Research Nurse:

Clinical Research Nurse, Ms. Ann Marie O’ Sullivan

St James’s Hospital/TCD


Local study contacts Tallaght Hospital:

Dr Peter Lavin

Consultant Nephrologist, Tallaght Hospital, Dublin 24


Local Research Nurse:

mailto:[email protected]






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Ms Emily Naylor, [email protected]

Local study contacts St Vincent’s University Hospital:

Dr Eamonn Molloy (Rheumatology Lead)

Dept of Rheumatology, St Vincent’s University Hospital, Dublin 4


Local Research Nurse: Ms. Mairead Murray


Local study contacts St. James’s Hospital:

Prof Con Feighery

Consultant Immunologist, St James’ Hospital, Dublin 8


Local Research Nurse:

Ms Emily Naylor, [email protected]

Local study contacts Beaumont Hospital:

Dr Mary Keogan

Consultant Immunologist, Beaumont Hospital, Dublin 9


Local Research nurse:

Ms Claire Foley, [email protected]

Local study contacts Galway University Hospital:

Prof Matt Griffin

Professor of Transplant Immunology, Galway University Hospital


Local Research Nurse: To be confirmed

Local study contacts Cork University Hospital:

Dr Mike Clarkson

Consultant Nephrologist, Cork University Hospital








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Local Research Nurse: To be confirmed.

A.6 Study Overview

Investigation of rare diseases

People with rare diseases (those with a prevalence <5 in 10,000) are served poorly by health

providers. As most doctors see few cases of a given rare disease in their entire career,

significant delays in diagnosis are usual, with patients receiving treatment for other

conditions before a unifying diagnosis is made. Even then, care is fragmented and poorly

coordinated: 80% of rare diseases are genetic in origin affecting multiple organ systems,

requiring input from multiple specialties on multiple hospital sites, which often lack the

specific expertise to deal with these unusual conditions.

Furthermore, research into rare diseases is extremely challenging. Even large centres will

see very few patients with a given rare disease, so it is virtually impossible to develop a

sufficiently large cohort for meaningful study. Individually, rare diseases do not have a large

impact nationally, so they are ignored by funding agencies, which favour supporting heart

disease and cancer research. However, 1 in 17 people will be affected by a rare disease at

some point in their life, equating to 250,000 people in Ireland. Therefore, collectively they

represent a very significant burden to health services.

To share expertise and resources, networked collaboration between units is essential. For a

country the size of Ireland, a national network would be of the right size and would allow

study of epidemiology across the whole country. Such a network would revolve around a

robust patient registry, with capture of detailed longitudinal clinical data across multiple

units and linked collection of biological samples to facilitate in depth study of a large cohort

from all parts of Ireland.

Systemic Vasculitis: networked investigation of a “Common-Rare” disease

We intend implementing and developing the UKIVAS vasculitis patient registry and bio-

resource in Ireland. Prof Little is co-founder and chair of this Kidney Research UK funded

multi-centre UK initiative, which seeks to recruit and investigate patients with systemic

vasculitis. This affects about 50 per million population per year, as opposed to affecting a

handful of people in the country, so is considered a “common-rare disease”. It causes severe

multi-organ dysfunction, including irreversible kidney failure, lung haemorrhage, stroke and

sino-nasal destruction. The prevalence is 300/million and hence can only be studied by a

coordinated international network of centres.



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The primary aims of this initiative are to address fundamental questions about vasculitis

epidemiology, facilitate conduct of phase II/III interventional studies by allowing easy

identification of a suitable cohort, monitor use of novel biologic agents, compile sufficiently

large cohorts to study immunogenetics, rapidly assess the clinical utility of new biomarkers

for development in clinical trials as surrogate end-points, and to characterise ‘difficult to

define’ disease subgroups including ANCA negative vasculitis and polyarteritis overlap

syndromes.

UKIVAS is on target to enrol 2000 prevalent patients with systemic vasculitis across the UK,

allowing us to address questions not previously possible. We envisage that Ireland will link

with the UKIVAS initiative. The first step in this is to implement and test sample and data

acquisition across the Dublin centres with an interest in vasculitis. If 50% of prevalent

patients are recruited, this would add about 600 cases to UKIVAS, with an additional 100

incident cases per year.

This registry would allow:

access to a sufficient number of patients to permit testing of new therapies

(subject to ethical approval) which would not otherwise be possible,

a means of monitoring the most cost effective implementation of expensive

new biologic agents

compilation of enough cases to facilitate study of genetic factors

contributing to disease

rapid assessment of novel disease biomarkers

performance of health services research informing development of a model

of care that improves on the current fragmented approach.

Novel genetic kidney diseases: centralised study of “Rare-Rare” kidney disease

Kidney failure is a major focus of the Irish healthcare system, with over 4000 people either

with a kidney transplant or receiving dialysis in 2011. In 2011, 20% of hospital discharges

had a diagnosis of dialysis or end stage kidney disease (ESKD); approximately one third of

these people with kidney failure suffer from a “rare kidney disease”. In those with “rare

kidney disease”, some may have several affected first degree family members but the

diagnosis remains unknown or the condition undescribed. Indeed, such families may be the

only cases in the country. This type of “rare-rare” kidney disease can only be studied

through an infrastructure dedicated to genetic investigation of sporadic cases through a

centralised clinic, which is set up to network with other centres around the world, thereby

developing sufficiently large kindred sets to permit identification of the causative genetic

mutation(s). This service will be coordinated through the Rare Kidney Disease Clinic at

Tallaght hospital under the guidance of Dr Peter Lavin.


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Study Design

The primary objective of this study is to understand more about rare kidney diseases by

forming a national network of interested units.

The aims of the study are to:

1. Establish a registry for “Rare Kidney Diseases”.

2. Use the registry to study outcomes for systemic vasculitis and other rare kidney

diseases.

3. Collect and store biological samples (serum, urine, leucocytes and DNA samples) and

use these in concert with the registry data to identify susceptibility genes and

proteins for the disease.

4. Establish a control group through collection of samples from unrelated healthy

controls, unaffected family members, or from the spouse/partner of patient.

Clinical data entry will be via a Dublin Centre for Clinical Research developed online

database. The endpoints in this study will include patient survival and renal survival.

Secondary endpoints will include time to End Stage Kidney Disease and relapse following

treatment.

The registry will be linked to the UKIVAS registry via an online portal. Longitudinal clinical

data are recorded using a specifically designed application. The linked biological samples will

make it possible to identify susceptibility genes and proteins leading to these diseases.

Study Methodology.

Prevalent patients will be identified from existing databases in various centres around the

country and from known cohorts of potential genetic renal disease. The goal is to

amalgamate these in an ethically robust fashion. As the study progresses, recruitment will

also begin at the various Clinical Research Centres (CRCs) in Dublin (Immunology and

Rheumatology clinics in St James’ Hospital, St Vincent’s Hospital, Beaumont Hospital), Cork

and Galway subject to local ethics approval.

Biological samples will be processed using a standard operating procedure (SOP) at each site

and ultimately stored centrally at the TCD biobank. Rare Kidney Disease patients will be

identified by cross reference of national immunology records (or ANCA status) and renal

histopathology records (to identify characteristic renal histology). Appropriate inpatients in

participating hospitals will also be identified.

Patients will also be asked to invite unaffected related and married-in (spousal) controls to

participate. These control patients will be screened for disease and consented in the normal


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fashion. Their participation is vital to the success of identifying disease causing genes as

examining their DNA can help differentiate disease causing gene changes from harmless

familial changes.

A.7 Patient Eligibility

Inclusion Criteria:

Patient Group

1. Diagnosis of one of the following forms of systemic vasculitis:

(i) Small vessel vasculitis (ANCA associated):

a.Microscopic polyangiitis (including renal limited vasculitis)

b.Granulomatosis with polyangiitis (Wegener)

c.Eosinophilic granulomatosis with polyangiitis (Churg Strauss)

d.ANCA vasculitis unclassified

(ii) Small vessel vasculitis (Immune complex)

a.anti-GBM disease

b.Cryoglobulinemic vasculitis

c.IgA vasculitis (Henoch-Schonlein)

(iii) Medium vessel vasculitis:

a.Classical PAN

b.Kawasaki disease

(iv) Large vessel vasculitis:

a.Giant cell arteritis

b.Takayasu’s arteritis

(v) Variable vessel vasculitis:

a.Behcet’s disease

b.Cogan’s syndrome

(vi) Single organ vasculitis:

a.Isolated aortitis

b.Primary cerebral angiitis

OR

2. Diagnosis with Rare Kidney Disease (those with a prevalence <5 in 10,000 or

restricted to certain known families).

AND


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3. Capable of understanding and complying with the requirements of the protocol,

including ability to attend study clinic

4. Capable of understanding and willing to provide signed and dated written, voluntary

informed consent before any protocol specific procedures are performed.

Control Group

1. Unaffected family members, ideally both parents (to enable formation of “trios” for

genetic studies).

2. Spouse/partner living in same environment.

3. Unrelated healthy control - an individual who does not have the disorder or disease

being studied and does not share the same environment. (These unrelated healthy

control Individuals will not be recruited at the Beaumont Hospital site). Please note

the importance of aiming to recruit age-matched healthy controls. Ideally, to match

the Vasculitis Patient Cohort, the Healthy controls should be above 50 years old.

4. Disease Control Individual- diagnosis of a renal disease other than Vasculitis and in

most cases also on immunosuppressive medication. (Disease control recruitment will

be restricted to the RKD clinics at which the named Principal Investigators and study

leads are present)

5. Capable of understanding and complying with the requirements of the protocol,

including ability to attend study clinic.

6. Capable of understanding and willing to provide signed and dated written, voluntary

informed consent before any protocol specific procedures are performed.

Exclusion Criteria:

Pregnant Women

A.8 Participant Recruitment

Patient group:

Patients with rare kidney disease and vasculitis are eligible to participate as above. With

respect to vasculitis, the aim is to invite >90% of incident patients in Dublin to participate

(about 20 incident patients per year). Recruitment of patients outside Dublin will be more

challenging and will rely upon the efforts of local champions. We hope to recruit a further

30 incident vasculitis patients per year from outside Dublin. Over 5 years, this will give 250

incident patients, which will be combined with a further 250 prevalent patients.

It is possible to derive important data from the patients in remission but that it is the acute,

untreated patient from whom the very high quality data are derived. Patients in remission

can be “corralled” into specific clinics and can therefore be recruited and sampled


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efficiently. Acute untreated patients with vasculitis arrive unannounced as an inpatient at

any time. There is usually a clinical imperative to initiate treatment with high dose steroids,

which makes assays on samples derived from that patient difficult to interpret. Therefore, a

high level of urgency is attached to these potential recruits and it would be very unlikely

for a research nurse to be available to lead the recruitment and sampling. It is for this

reason that most existing sample collections around the world are virtually deficient in this

patient group.

Recruitment of acute untreated patients will require a trade-off between simplicity and a

robust processing protocol. We aim to recruit the majority of such patients by:

1. Making the process of recruitment as simple as possible to allow clinicians with only

a peripheral interest in the endeavour to contribute. This will entail removal of the

need for local processing and clinical data capture. All that will be required is consent

of the patient and obtaining a blood and urine sample.

2. Management of the ethics approval in given centres by the lead research nurse,

removing the need for the local clinicians to become involved in this.

3. A robust cold courier mechanism initiated and coordinated by the lead DCCR

research nurse to bring collected samples to a central processing site in the TCD

biobank.

4. A visit by the research nurse to the unit in question in the weeks following

recruitment to ensure complete and accurate clinical data capture.

Control group:

We aim in addition to recruit 300 controls (non-affected family members or spouse /partner

of patient, healthy controls and diseased controls). As mentioned above, unrelated healthy

control individuals will not be recruited at the Beaumont Hospital site. Disease control

recruitment will be restricted to the RKD clinics, at which the named Principal Investigators

and study leads are present. Patients will be invited to circulate a Participant Information

Leaflet and a “Consent to be Contacted” letter to unaffected related and married-in

(spousal) controls who may wish to participate. Unaffected relatives would be screened for

the disease as it is possible that they may have subclinical manifestation of the disease, or

they could theoretically develop the disease at some stage in the future. Screening will

involve urinalysis: a negative result for blood, nitrite and protein will allow for recruitment

onto the study when combined with self-reported absence of known history of kidney

disease.

Informed consent guidance and regulation ensure that the study participants are given

sufficient information about the study and procedures and are allowed to choose freely

whether or not to participate.


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A.9 Informed Consent

In line with the International Conference on Harmonisation of technical requirements for

registration of pharmaceuticals for human use and the publication of the Guideline for Good

Clinical Practice:

The rights, safety and wellbeing of the subjects are the most important consideration

and should prevail over interests of science and society.

Freely given informed consent should be obtained from every subject prior to

participation.

Note: This study is NOT a clinical trial

Informed consent will be obtained from all patients. Consent will encompass the nature of

the study, consent to review and collate the information from medical records, phlebotomy,

urine sample, renal biopsy (if appropriate). Patients will be informed of the risks associated

with phlebotomy. Additionally, they will be informed that DNA and proteins will be

extracted from blood, plasma and urine samples and that a small portion of the biopsy

sample will be used for experimental studies; this will not affect their diagnosis or

treatment. They will not be required to have a biopsy for the purpose of the study. They will

also be informed that they will not have access to the results of our analysis of their

individual specimens and will be reassured regarding confidentiality

Informed consent procedure: Patients

• Inpatients will be given Patient Information Leaflets and Consent forms along with

an explanation of the study. They will be given a period of 24 hours to reflect before

being asked to sign the consent forms.

• In the outpatient clinic setting, information sheets will be included in the

appointment letter. Copies will also be available at the relevant clinic. If patients

need more time than allowed in the initial consultation to decide whether to

participate, it can be deferred until a subsequent visit.

• Patients identified as meeting the study criteria will be approached in a friendly

manner by the research nurse whilst in the waiting area. The research nurse will

identify themselves and explain about the research study and escort the patient to

an interview room.

• Informed consent will be obtained by one of the lead investigators or a fully qualified

member of the research team such as a research nurse or research registrar.

• The nature and objectives of the study will be explained to the patient. Risks and

benefits of participation will be discussed and patients will be made aware that

participation or otherwise will not alter their healthcare in any way.

• The patient will decide for themselves whether they wish to participate in the study

or not. Their decision making process will be free from coercion or undue pressure


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and the patient will be aware that they are free to withdraw from the research

process at any time. Once satisfied that the patient fully understands the nature and

objectives of the study as outlined in the PIL/ICF and is capable of giving informed

consent, the patient can be consented and enrolled in the study.

• A site code and unique study number will be issued and written on the consent form.

• The consenting patient, research nurse and/or doctor will sign the ICF.

• The patient should date their own ICF.

• The Research Nurse will photocopy the form twice, maintaining one copy in the CRF,

one copy in the patient’s medical chart, and giving one copy to the patient. At some

sites a carbon copy book may be used to produce 3 copies of the signed consent

forms if a photocopying machine is unavailable.

• If the patient decides to withdraw from the study at any stage, the research

nurse/research team member will document this decision clearly in the patient’s

medical notes and CRF and ECRF detailing the reason if known.

• If during the study, it is discovered that the patient was enrolled but did not meet

the inclusion criteria, this will be documented clearly in the CRF and ECRF and the

principal investigator will be informed.

Informed consent procedure: Controls

Participant Information Sheet will be provided to patient for unaffected relatives or

spouses, inviting them to make contact with research team for donation of clinical

samples. Alternatively, unrelated healthy controls will be given the PIL to peruse at

their leisure.

If interested in participating, these controls will be invited to attend the research

centre for collection of demographic and basic clinical data, and donation of clinical

samples.

Informed consent will be obtained as above before any study-related procedure is

carried out.

A.10 Sample Collection Summary

Patients in the disease group may have repeat blood and urine samples taken during routine

clinic visits on up to four occasions in five years. If a patient is identified as experiencing a

relapse of disease it may warrant repeating the process of taking blood and urine samples.

Patients will be informed of this prior to signing the consent form.

A.11 Ethical Considerations

It is possible that a genetic predisposition to develop disease will be discovered during the

course of this research. In patients who already have presented with the disease and are

engaging in clinical care there is little ethical issue.


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However, it may be possible that a family member who was previously considered

unaffected caries a disease gene. Unaffected individuals will be informed of this risk on an

information sheet and during the consent process. This raises a number of ethical issues:

It is possible to carry a disease phenotype and not manifest the disease. This

depends on the penetrance of the phenotype.

It is also possible that there may be no immediate therapy available for any disease

gene identified.

In these situations, it is imperative to balance the benefit of telling the individual their

genotype with the potential anxiety caused and whether they wish to know and the

potential for intervention.

In such a case, a generic letter would be written to all of the family members with

information specific to the gene identified in non-medical language. All individuals would

be invited to make an appointment to discuss their results. Individuals who would wish

to discuss their results would have genetic counselling prior to this and they would then

have the genetic tests confirmed in a clinical genetics setting. They would then be

discussed at an appropriate level of detail by the PIs and an appropriate medical

screening plan along with any appropriate investigation or care plan would be made at

that stage.

Confidentiality

A unique patient identifier (study ID) should be recorded on the patient’s consent form, CRF

and any study related documents. This will consist of a unique study specific number, which

will be pre-generated and will serve as the unique identifier for both patient samples and

clinical data held outside the study site.

Identifiable patient information will not leave the study sites.

The data and samples collected will be coded with this unique identifier and marked with a

barcode. All data collected will be stored in a local recruitment log located in a password

secured folder, or folder with access restricted to principal investigator and research nurse,

on an encrypted server behind a hospital or university firewall. The key that allows re-

identification of coded data will be held securely by the site principal investigator.

A.12 Data Collection & Management

A study site file will be prepared containing all critical documents pertinent to the study,

including:

Protocol

Ethics approval letters


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Patient Logs

• PIL/IC documents

• CRF template

• QOL Questionnaires

• Specimen logs

• GP Letter template

Patient Case Report File will contain:

Completed CRFs

Completed Quality of life forms

GP Letter

A.13 Patient Information Log - Completion Guidelines

This document contains patient identifiable information and is a link between an individual

and their unique anonymised study number. The log should be held securely on the host

site. These data are essential so that the patient can be followed-up and longitudinal clinical

information linked to subsequent samples.

The following information will be collected:

• Full name (First name, surname)

• Date of birth

• Hospital MRN

• Contact phone number

• Email address

• Site code

• Study specific number

• Full name of GP

• Address of GP

• Phone number of GP

A.14 CRF Completion Guidelines

All information should be recorded, handled and stored in a way that allows its accurate

reporting, interpretation and verification. The confidentiality of records that could identify

subjects should be protected, respecting the privacy and confidentiality. Data recorded in

the CRF, which are derived from source documents, should be consistent with the source

documents. Hard copies of all CRF forms should be stored securely at each of the study sites

by the Research Nurse until batch transfer to a secure locked storage unit at the Central


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Registry site at Trinity Biobank. Anonymised clinical registry data will be recorded

electronically in the eCRF (https://www.studies.dccr.ie/distiller22/login.php).

A.15 Data Entry into Patient’s Medical Records

When a patient is approached to participate in this study and gives their consent,

information pertaining to the study enrolment should be documented in their medical

notes.

• Clearly state the name of the study “Rare Kidney Disease Registry and Bio-resource”.

• Clearly state that the patient gave informed consent and include a copy of the ICF.

• Clearly state the date the consent was given.

• Clearly indicate what samples were collected. If there were any issues with samples

being collected, please detail these.

A.16 Study Monitoring/Quality Review

Study files (CRF and eCRF) will be reviewed periodically by the PI. All data points should be

completed. The lead Research Nurse will act as a liaison person between the various

hospital sites and the PI to ensure that:

• Study is being conducted according to protocol and any applicable amendments, if

they arise

• Data are collected and recorded accurately.

• Informed consent is obtained before any study related procedures are carried out.

• Study patients meet with the eligibility criteria.

• Study site file is up to date

Periodically, as well as at the end of the study, the PI will review the eCRF, and will assess

the accuracy and completeness of the data collected. Further follow-up may be necessary at

this point.

Governance

The registry and bio-resource will be managed by a steering group comprising a

representative from those units that contribute >10% of cases plus a lay member and will be

chaired by a respected independent clinician with experience of this work. This group will

work to maintain and build this resource for the public good in accordance with its purpose.

They will retain full control of all access to and uses of this resource. Any application for

access will be reviewed to ensure that it is consistent with the participants’ consent, that

they have the relevant ethics approval and that there is a sound scientific basis.

https://www.studies.dccr.ie/distiller22/login.php


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Rare Kidney Disease and Vasculitis Registry Steering Group Members:

Prof. Eoin Gaffney, Consultant Histopathologist (retired from active clinical service)

Prof. Mark Little, Consultant Nephrologist, Study Chief Investigator

Dr. Fionnuala Hickey, Research Fellow, Trinity Health Kidney Centre Senior Scientist, Biobank

Scientific Advisor and Supervisor.

Dr. Peter Lavin, Consultant Nephrologist, Rare Kidney Disease lead

Dr. Eamonn Molloy, Consultant Rheumatologist, Rheumatology lead

Prof. Matt Griffin, Professor of Transplant Immunology, Galway University Hospital

Dr. Mary Keogan, Consultant Immunologist, Beaumont Hospital

Prof. Conleth Feighery, Consultant Immunologist, St. James Hospital

Prof. Peter Conlon, Consultant Nephrologist, Beaumont Hospital

Dr. Antoinette Perry, Senior Research Fellow, Clinical Medicine- Prostate Cancer Group T.C.D.

Ms Julie Power- Patient Representative from Vasculitis Ireland Awareness Group.

Mr Anthony White – Patient representative


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II. Part B: Sample and data management

B.1. Sample & Data Management-Part 1, Collection:

1. Research Nurse along with Lead Clinician identifies suitable patients to recruit. For acute,

untreated patients, the potential recruit will be identified by the local clinician and the lead

clinician and research nurse will be informed by text, phone call, whatsapp or email.

2. For electively recruited patients, each will be assigned a registration sample collection

pack sequentially as they arrive at clinic. There are 4 types of Data and Sample Collection

Packs depending on whether the individual is a patient, a healthy control or a disease

control, and whether the visit is a registration visit or a subsequent visit. The primary goal

following recruitment of a patient with vasculitis is to obtain one full sample set at the

time of acute disease, and a further full set at the time of remission. Provision has been

made to obtain up to 4 sequential samples over 5 years.

Note that there are no packs for acute, untreated recruits in peripheral hospitals. These

employ locally supplied clinical blood tubes and the samples are married to Biobank ID

labels during processing at the central biobank site.

The types of packs are:

Patient Registration Packs

Healthy Control Registration Packs

Disease Control Registration Packs

Subsequent Visit Packs

Patient Registration pack:

Pre-generated numbered labels for forms and sample tubes for Patient Registration

Patient Information Leaflet

Consent Form

EQ5D Quality of life questionnaire

Registry and Bio Resource Data Collection Case Report Form which will include

sample details section.

Vasculitis Ireland Awareness Contact details cards are in all registration packs for

newly diagnosed patients and their families.

Healthy control Registration pack:

Pre-generated numbered labels for forms and sample tubes for Healthy Control

Registration


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Participant Information Sheet

Additional Information sheet for individuals participating in the Vasculitis registry for

Healthy control purposes


Consent Form

Additional Consent Form for control individuals (attached to the main consent form)

Disease control Registration pack:

Pre-generated numbered labels for forms and sample tubes for Disease Control

Registration





Additional Information sheet for individuals participating in the Vasculitis registry for

Disease control purposes

Consent Form

Additional Consent Form for control individuals (attached to the main consent form)

Visit pack:


Registry and Bio Resource Data Collection visit Case Report Form, which will include

sample details section. This form is to be used for Patients /Disease control

individuals/Healthy Control Individuals registered onto the study previously who are

having a follow up visit.

Control Recruitment Envelopes are also available to the Research Nurse to be given to

Patients at the discretion of the Research Nurse to be passed on to a relative for control

recruitment containing:

Additional Information sheet for individuals participating in the Vasculitis

registry for control purposes


Consent Form


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Additional Consent Form for control individuals (attached to the main

consent form)


Stamped addressed envelope addressed with Research Nurse’s address.

Sample Collection Components-All Packs will contain the following:

Blood Collection system- BD Vacutainer Safety-Lok Blood Collection Set with pre

attached holder. Catalogue number: 368654

9ml EDTA Tube for DNA and Plasma (For certain patients specified by lead

clinician a BD P100 tube for plasma for Proteomic studies will also be collected)

9ml Plain Tube for Serum

2 x 9ml EDTA tube for PBMCs

2.5ml Paxgene tube for RNA (note: this will not be included in every pack but will

be available at each site for certain cases).

250 ml Yellow capped Urine Tube

2x 50ml tubes for collection of Urine for Metabolomics and Proteomics.

In Registration Packs: Barcoded labels with a unique barcode specific for each

patient assigned with the Main Study ID number assigned to the patient.

It may not be feasible to obtain all samples on all recruited individuals. In particular, the

paxgene and BD100 tubes are expensive, and the PBMC sample requires substantial

processing. These samples will be included at the discretion of the local PI. When the

opportunity to recruit an untreated patient with acute vasculitis arises, all efforts should be

made to obtain all possible samples as these patients have the greatest potential for

scientific investigation.

PaxGene Tubes

Paxgene tubes were initially collected at all sites from all individuals but from 1st September

2014 this will be restricted to:

All new acutes

Age-matched healthy controls- specifically all healthy controls over 50 years of age.

Return visits of patients for whom we already have a Paxgene tube from an ‘Active’ encounter

A field stating whether or not revisit Paxgene tubes are to be collected has been added to

Distiller. The research nurse should check this for each patient for any return visits to

determine if a Paxgene tube is to be taken.


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Please note: EDTA for Plasma and DNA should be collected at every encounter. Previously,

we only collected samples for DNA at the first visit but it has been decided to collect for

DNA extraction from each encounter so that investigation into epigenetic mechanisms of

disease will be possible.

3. The Research Nurse will recruit the patient and bring them to a separate room for sample

and data collection. For acute untreated recruits in peripheral sites, the local clinician will

obtain consent and samples.

Oragene Saliva kits for DNA Samples When a patient or control who consents to take part in the

study cannot attend a research clinic for sample collection they may be sent an Oragene Saliva Kit

for donation of saliva for DNA extraction along with a stamped addressed envelope for return of the

sample to the biobank.. Patients will be sent a cover letter along with the kit and will be called in

advance to let them know that a kit is in the post to them to ensure they are completely happy

about taking part in this way.

Data Collection

Registration:

At Registration, the Research Nurse will obtain informed consent as described above, fill in

the consent forms and ensure they are signed and dated by both parties. They will ensure

the quality of life form is filled in and fill in the case report form. They will attach one of the

unique identifier barcoded labels with the Main study ID number assigned to it onto the

case report form in the space allocated for it, as detailed below.

[Note if the patient is a control subject this will be recorded on the case report form and if

they are a relative/linked these data will be recorded and described in the pedigree

assignment space on the form]


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Fig. 1: Case Report Form, indicating where to attach Main Study ID Barcode Label.

Sample Collection

The blood and urine samples will be taken (as per the collection protocol described below)

and a barcode label with the unique identifier barcoded labels with the Main Study ID

number assigned will be attached to all samples. Management of the clinical sample

collection will vary from site to site, depending on local practices. For acute untreated

recruits in peripheral sites the samples will be collected by the team caring for the patient

using locally sourced blood tubes. The over-riding principal is that, wherever possible, one

should avoid having to perform venepuncture more than once. If necessary arrangements

should be made with the local clinical phlebotomy service for the research nurse to obtain

the clinical samples at the time of research sampling (with handover of the clinical samples),

or for the phlebotomist to obtain the research samples at the time of clinical phlebotomy

(with rapid handover of research samples to the research nurse).

The research nurse will then detach the sample processing details (last) page of the case

report form and place it along with the remaining barcoded labels into a new plastic pocket.

This will be transported along with the samples to the lab to the bio-bank technician or

Research Nurse to be filled in as processing takes place.

All the remaining forms are returned to the original plastic pocket with the CRF assigned to

the patient. The research nurse is responsible for storing this file and entering all data

required onto the UKIVAS site (for vasculitis recruits) and the patient recruitment log. After

initial sample processing and storage, consent forms and eCRFs are stored at the local site,

with a view to intermittent batch transferring both samples and documentation to the

central study site at the TCD biobank.


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Post-sample collection

At the registration visit, at the discretion of the research nurse, the patient will be handed

the envelope included in the pack to be passed on to a relative for recruitment of the

control patients.

The research nurse will also maintain a record of patients enrolled and some patient basic

details on a secure recruitment log spreadsheet to include: Main Study I.D., UKIVAS I.D, Date

recruited/Visit Date, Name, DOB, Address, GP details, samples collected, whether or not it is

an acute Vasculitis Patient, study site and any Controls recruited for backup/re-identifier

purposes.

The research nurse will enter some of the details from this form onto the UKIVAS Registry

which will then generate a UKIVAS ID Number. This UKIVAS ID Number will then be written

on the case report form. If IT systems onsite permit, it may be possible to enter these data

on site at the clinic. Otherwise, they can be entered at a more convenient time.

The research nurse or lead clinician will then fill in the distiller entry.

Subsequent Visit Encounters:

All of the above sample and data collection procedure is repeated, the only difference being

that consent and information leaflets are not required at subsequent encounters and that

the barcode labels are not included in sample collection packs so the paperwork and

samples must be handwritten. The main study ID and encounter date form the identifiers

for the encounter paperwork and samples for the system, so it is imperative that the date of

visit is correct. Handwrite the main study ID and date of visit clearly on the paperwork and

all samples and aliquots.

Healthy Control Registration Encounters:

All Healthy controls should have a urine sample tested, ideally with the following dipsticks:

Siemens Multistix 10 SG REF 2300 (0353597).


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It is important that the dipstick used is capable of recording nitrite, blood and protein to

ensure that there is no urinary tract infection.

B.2. Sample & Data Management-Part 2, Processing:

Samples will be processed at each local site if facilities are available. For acute, untreated

recruits at peripheral sites, samples will be collected by courier and brought to the central

TCD lab for processing. Whether or not the biobank technician or the research nurse carries

out the processing at each site will depend on the availability and expertise of staff at the

particular site. This decision will be made on a site by site basis with the staff involved and

the PI at the site initiation visit. If a research nurse is aided by a technician to do the

processing they should aim to recruit or sample 4-6 patients per clinic when they are fully

trained in. If there is no technician available, the research nurse should aim to collect one to

two samples early on in the clinic and proceed directly to the lab for processing.

If a biobank technician is on site, and if local clinic timings allow, samples may be collected

and transported to the lab on a batch basis (2 patients’ samples per batch); this will be

discussed and organised between the research nurse and biobank technician on a site by

site basis.

If a biobank technician is on site to carry out the processing the research nurse will call to

inform them that samples are ready for processing. If not, the research nurse will proceed

directly to the lab. Processing will be carried out as described in the Standard Operating

Procedures in section D.

The samples will be frozen at -80 (or initially -20 for Paxgene tube & BDP100) at the local

site and entered into a simple inventory system by the biobank technician/research nurse

who processed the samples. This will usually involve placing all the samples from one

patient encounter to be stored at -80 together in a purposely supplied Ziploc Bag, writing

the main study ID and the visit date on the front of the bag and placing it in a box marked

RKD samples in the -80 Freezer. The BDP100 plasma and Pax gene tubes will be stored in

labelled boxes in the -20 Freezer. This is temporary storage until routine batch transfer of

samples to Trinity Biobank at the IMM, St. James Hospital, takes place. This system allows

quicker placement on the inventory system at the Biobank.

The files will be retained securely by the research nurse/biobank technician, transported,

and filed at the Trinity Biobank.

(Note that if the samples have been collected at St. James Hospital this will permit for them

to be entered directly into the biobank software by the Biobank Technician at this stage).


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All files including consent forms will be stored in a lockable secure filling cabinet until study

ends.

B.3. Sample & Data Management-Part 3: Sample transfer

Transfer of Samples from Local Sites to Trinity Biobank at IMM and enrolment into the

Central Rare Kidney Disease Bio Resource.

Once enough samples to warrant a collection visit are accumulated at a local site, transfer to

Trinity Biobank while maintaining correct transport and storage temperatures will be

arranged (using dry ice). The sample files and coded CRFs should accompany the samples.

For acute untreated recruits at peripheral sites, samples will be collected by a courier within

12 hours of sampling and transferred at 4oC to central TCD biobank site for processing.

Immediately upon arrival onsite, the biobank technician will unpack the samples ensure all

have barcoded labels, and input samples onto the central biobank inventory system. For

samples from peripheral sites a Main study ID will be assigned.

Once per month a data import of the basic fields from distiller database will be exported to

automatically populate the central registry inventory system. Periodically, the research

nurse will also cross-reference distiller data with UKIVAS data to identify inconsistencies.

If required, the biobank technician will arrange/carry out PBMC separation and DNA

extraction, and will barcode and store all generated aliquots appropriately on the inventory

system, in the central freezer space/liquid nitrogen tank at the IMM.

B.4. Sample & Data Management-Part 4: Barcoding System.

When the patient is first registered on the study, they are assigned their Main study ID and

barcode label. This is their ‘Main study ID’ code for the study. This ‘Main study ID’ code and

barcode label will be recorded on the patients file by the research nurse on the green sticker

(SOP D1), and also recorded in the Research Nurse’s enrolment log.

This number will also be written by the research nurse on all case report forms for each

encounter for that patient/control individual as indicated on the case report form below in

Figure 2A. It should be written on all of the pages of the case report form on the upper left

hand side, and on the indicated field on the first page. The date of visit should also be

clearly written on the first page of the case report form and also on the sample details page

as illustrated below in Figure 2 B. The type of recruit and the Encounter number should also

be recorded as indicated below.


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A:

B:

Figure 2. Case Report Form, A: First Page, B: Sample Details page, indicating where to

write Main Study I.D., Date of Visit, Type of Recruit and Encounter Number.


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The research nurse will be assigned the data collection packs for the registration visits with

pre-assigned bar-coded labels displaying the main study ID. These packs will be taken from

the storage box in sequential order by the research nurse. This Main Study ID is recorded in

the patient’s medical file and also on the recruitment log. If the individual returns to the

clinic and has a repeat visit for the purpose of the study, the research nurse will look up this

Main Study ID on the patient’s medical file or the recruitment log, and record the Main

Study ID number on the visit set of samples and paperwork. At the visit they will handwrite

clearly the Main Study ID and the Visit Date on the paperwork, and aliquots must be

clearly labelled with Main Study ID, Date of Visit and Sample type with lab marker to link

these follow up samples to a given registration visit.

The Main study and Visit Date form the Identifiers for the Visit Encounter Paperwork and

samples for our system so it is imperative that the date of Visit is correct.

The pre-assigned Main Study ID’s are summarised in table B.1.

Site Recruit type Site

Code

Code

Tallaght Hospital Vasculitis TA 000001->000500

Tallaght Hospital Healthy Control TA 002001->002500

Tallaght Hospital Disease Control TA 002501->003000

St. James Hospital Vasculitis SJ 004001->004500

St. James Hospital Healthy Control SJ 006001->006500

St. James Hospital Disease Control SJ 006501->007000

Beaumont Hospital Vasculitis BH 008001->008500

Beaumont Hospital Healthy Control BH 010001->010500

Beaumont Hospital Disease Control BH 010501->011000

St. Vincent’s Hospital Vasculitis SVH 012001->012500

St. Vincent’s Hospital Healthy Control SVH 014001->014500

St. Vincent’s Hospital Disease Control SVH 014501->015000

Galway University Hospital Vasculitis UHG 016001->016500

Galway University Hospital Healthy Control UHG 018001->018500

Galway University Hospital Disease Control UHG 018501->019000

Cork University Hospital Vasculitis CUH 020001->020500


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Table B.1: Patients Barcoding and Labelling

Notes on the Barcode Labels

Please note that there are two main types of labels in the packs:

Paperwork labels that only have the Main Study ID and a barcoded form of the study

ID- These should be used for paperwork and any sample that will be processed

further and discarded before storage.

Sample and aliquot labels that have Main Study ID, aliquot ID, study code

aliquot/sample type and aliquot number in barcode form- these should be used only

for tubes/aliquots that are to be stored in the Biobank, they should not be placed on

tubes that will be discarded during processing.

These are illustrated in figure 3 below. Please ensure that you label the samples and aliquots

with the correct labels according to the sample/aliquot type as described in the sample

collection and processing SOP’s. The small circular labels are for the top of the aliquot

cryovials.

Cork University Hospital Healthy Control CUH 022001->022500

Cork University Hospital Disease Control CUH 022501->023000

Limerick University Hospital Vasculitis LUH 028200->028500

Limerick University Hospital Healthy Control LUH 030001->030500

Limerick University Hospital Disease Control LUH 030501->031000

Waterford Regional Hospital Vasculitis WRH 032001->032500

Waterford Regional Hospital Healthy Control WRH 034001->034500

Waterford Regional Hospital Disease Control WRH 034501->035000

Mater Hospital Vasculitis MH 036001->036500

Mater Hospital Healthy Control MH 038001->038500

Mater Hospital Disease Control MH 038501->039000

Our Lady of Lourdes Hospital Drogheda Vasculitis DH 040001->040500

Our Lady of Lourdes Hospital Drogheda Healthy Control DH 042001->042500

Our Lady of Lourdes Hospital Drogheda Disease Control DH 042501->043000

Letterkenny General Hospital Vasculitis LGH 044001->044500

Letterkenny General Hospital Healthy Control LGH 046001->046500

Letterkenny General Hospital Disease Control LGH 046501->047000


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B

Figure 3: A. Different Label types, B. Layout of Sample Aliquot labels.


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Figure 5. Summary Schema of

Sample and Data Management


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III. Part C. Safety Guidelines

Please Note: There are also more detailed and specific safety statements and guidelines

available at each of the sites Clinical Research Centres that should be adhered to - these

include specific safety instructions on the use of the biohazard safety hoods, centrifuges

and biohazard spill kits, which vary from site to site depending on the model. Safety

Training Records and Registration with the TCD Health and Safety Department should be

completed for all staff processing at Trinity College sites.

C.1 Safety Guidelines for Blood Collection, including specific guidelines for use

of Paxgene RNA tubes

Responsibility

It is the responsibility of the research personnel carrying out this procedure to ensure that

all steps are completed both competently and safely.

Procedure

1. Research personnel will have completed the appropriate training and be deemed

competent in the procedure of venepuncture prior to any blood collection in accordance

with local policy and procedures.

2. Research personnel will have been deemed competent to respond in the event of

fainting or any other adverse event during or after the blood collection procedure.

3. Research personnel will greet the research participant, identify themselves, and then

explain the blood collection procedure to the research participant.

4. The research participant will be approached in a friendly calm manner and their

cooperation will be gained prior to blood collection.

5. The research participant will be correctly identified prior to blood collection by

asking them to give their name and date of birth.

6. The research participant will be positioned safely and comfortably in the chair/couch

provided for venepuncture, ensuring that the protective arm is in the correct position to

support the research participant in the event of fainting or any other adverse event.

7. The research participant’s mouth will be free from food or gum prior to

venepuncture.

8. All sample containers and equipment needed to competently and efficiently carry

out the venepuncture will be assembled prior to the procedure.

9. Research personnel will wear gloves at all times during venepuncture.


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10. Research personnel will use appropriate barrier protection, such as gloves, gowns,

masks, and protective eyewear to prevent exposure to skin and mucus membranes when

working with known infectious research participants.

11. Research personnel will ensure that needles should never be broken, bent or

recapped.

12. Research personnel will take care to prevent needle stick injuries when using and

disposing of needles. Local hospital site policy and procedures should be followed in the

event of a needle stick injury and research personnel will familiarise themselves with these.

13. Blood collection tubes will not be labelled in advance of venepuncture.

14. A unique study identification number and/or bar-coded label will be applied to all

blood samples immediately after venepuncture. The time of collection will also be recorded

in the study specific documentation.

15. A puncture-resistant incineration container will be placed as close to the use-area as

practical.

16. Using gloves, blood spillages will be covered with Milton or a suitable

decontamination agent such as ‘Virkon’’, ‘Bio cleanse’’, or ‘‘Presept’’ granules, mopped up

with paper towels and discarded into puncture-resistant incineration containers.

17. Transfer of specimens collected using a syringe and needle to a blood collection tube

is not recommended as this additional manipulation of sharps such as hollow bore needles

increases the potential for needle-stick injury.

18. Transfer of specimens from a syringe to an evacuated tube using a non-sharps device

should be performed with caution for the reasons outlined below:

Depressing the syringe plunger during transfer can create a positive pressure,

forcefully displacing the stopper and sample, causing splatter and potential

blood exposure.

Using a syringe for blood transfer may also cause overfilling, or under filling

of tubes, resulting in an incorrect blood-to additive ratio and potentially

incorrect analytical results.

Evacuated tubes are designed to draw the volume indicated. Filling is

complete when vacuum no longer continues to draw, though some tubes

may partially fill due to plunger resistance when filled from a syringe. The

laboratory should be consulted regarding the use of these samples.

19. If blood is collected through an intravenous (I.V.) line, ensure that the line has been

cleared of I.V. solution before beginning to fill the blood collection tubes. This is critical to

avoid erroneous laboratory/analytical results from I.V. fluid contamination.

20. Overfilling or under filling of tubes will result in an incorrect blood-to-additive ratio

and may lead to incorrect laboratory/analytical results or poor product performance.


36 | P a g e

21. During collection it is important to avoid possible backflow from blood collection

tubes that contain chemical additives which may result in the possibility of an

adverse patient reaction.

22. The Pax Gene RNA blood collection tube has specific collection guidelines which are

outlined clearly in the SOP D.1.2 for blood collection. These guidelines should be

adhered to and an instructional video of the safety precautions can be viewed at:

http://www.preanalytix.com/videos/rna-tube-collection-video/

23. If the BD P100 tube is used there are also specific guidelines for blood collection using this

tube (appendix II).

C.2 Safety Guidelines for Urine Collection

1. Research personnel will have been deemed competent in the procedure of urine

collection.

2. Research personnel will wear gloves at all times during specimen collection.


masks, and protective eyewear to prevent exposure to skin and mucus membranes

when working with known infectious research participants.

4. Specimen containers will not be labelled in advance of urine collection.

5. A unique study identification number and/or bar-coded label will be applied to all

specimens immediately after collection.

6. The time of collection will also be recorded in the study specific documentation or

data management system.

7. Research personnel will ensure that the lids of the specimen containers are securely

replaced so that leakage does not occur during transport.

8. Using gloves, all spillages will be covered with Milton or a suitable decontamination

agent such as ‘Virkon’’, ‘Bio cleanse’’, or ‘‘Presept’’ granules, cleaned up with paper

towels and discarded into puncture-resistant incineration containers.

C.3 Safety Guidelines for Handling of Biological Materials

1. Research personnel will be aware of the correct procedures for the handling of

biological materials.

2. All research personnel will have received the appropriate immunizations as per local

policy and procedures prior to working with potentially infectious materials. Regular

controls of immunization status will be performed as per local policy and

procedures.



37 | P a g e

3. All biological materials collected will be treated as being potentially infectious for

blood-borne diseases regardless of their known infectious status. All samples

received should be assumed to be potentially hazardous and thus should be

processed in a class 2 biosafety cabinets. Samples from patients known to be

infected with BBV’s should be processed, on their own, and all surfaces wiped down

with 4% Virkon. The cabinets should be wiped down cabinets with 1% Virkon before

and after use, discarding waste in clinical waste bags or bins as appropriate and

avoiding the use of sharps.

4. Universal precautions will be applied to all blood, body fluids, and tissue specimen

collections, regardless of their infectious status even when they do not contain

visible blood.


masks, and protective eyewear to prevent exposure to skin and mucus membranes

when working with biological materials.

6. Gloves will be changed after the handling of each biological material or when

contaminated and will be disposed of correctly in the appropriate waste disposal

bins provided.

7. Hands will be washed immediately after removing gloves, using a hand-washing

technique defined by local standardized procedures.

8. Specimens of biological materials (e.g., blood) will be placed in a secure secondary

container to prevent breakage and leakage during transport and transported in

accordance with the relevant regulations.

C.4 Safety Guidelines for Handling of High Risk Samples

This describes the procedure for safe handling of HIGH RISK blood samples that are received with information indicating that they are or may be High Risk Blood Samples. These samples may be highlighted as being high risk by:

Presence of a red spot sticker on the referral form and/or blood tube

Patient is/suspected to be an IV drug user (IVDA – intravenous drug abuse)

Patient is/suspected to be HepC/HepB/HIV/

Sample is/suspected to be a high risk sample All blood samples received by the laboratory are considered to be potentially high risk

samples. Safety policies and procedures in place for the handling of all blood samples, as

detailed in this safety guidelines section, have been developed with the aim of protecting all

lab workers against infection from Hep C, Hep B and HIV. All new staff/students shall receive

an overview of blood sample handling safety from an experienced member of staff during

their induction and all new staff/students are required to familiarise themselves with the


38 | P a g e

safety procedures detailed in the safety guidelines before they embark on any work that

involves the handling of blood samples. Safe working practices include the use of the correct

PPE, handling and processing all samples as if they were potentially infectious in the class 2

biological safety cabinets and completing the Hep B course of vaccination.

All samples are processed in the same way, regardless as to whether or

not they are flagged as being high risk samples

C.5 Safety Guidelines for Disposal of Sharps

1. Research personnel will be responsible for the safety and disposal of used sharps.

2. Sharps will be placed in the sharps container as soon as possible after use and will not be

left lying around.

3. Sharps containers will be placed as near as possible to the site of use.

4. Sharps containers will not be overfilled and will be securely closed. Used and sealed

sharps containers will be stored in a location which will prevent risk of injury to staff,

research participants and other personnel, while awaiting collection by appropriate

personnel. Research personnel will never discard needles or other sharps, including plastic

pipette tips into polythene bags.

C.6 Safety Guidelines for Handling Chemical Hazards

1. All research laboratory personnel will receive the appropriate training and education to

develop and implement work practices to minimize personal and co-worker exposure to the

chemicals in the laboratory. Based on the realization that all chemicals inherently present

hazards in certain conditions, exposure to all chemicals shall be minimized.

2. Research laboratory personnel will be required to read and understand the Material

Safety Data Sheet (MSDS) and SOP for the chemical and/or process that they are working

with. This will allow research laboratory personnel to be familiar with the symptoms of

exposure for the chemicals with which they work and the precautions necessary to prevent

exposure.

3. General precautions to be followed for the handling and use of all chemicals are as

follows:

Skin contact with all chemicals shall be avoided.

Research personnel will wash all areas of exposed skin prior to leaving the

laboratory.

Mouth suction for pipetting or starting a siphon is prohibited.


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Eating, drinking, smoking, gum chewing or application of cosmetics in areas where

laboratory chemicals are present shall not be permitted. Hands shall be thoroughly

washed prior to performing these activities.

Storage, handling and consumption of food or beverages shall not occur in chemical

storage areas, laboratories or refrigerators, nor shall any glassware or utensils used

for laboratory operations be used in the handling of food or beverages.

Any chemical mixture shall be assumed to be as toxic as its most toxic component.

Substances of unknown toxicity shall be assumed to be toxic.

C.7 Safety Guidelines for Handling Dry Ice

1. Avoid contact with skin and eyes when handling dry ice as it can cause severe burning and

frostbite within seconds. NEVER handle dry ice with bare hands. Dry ice should not be put in

the mouth or ingested.

2. When handling dry ice wear insulated gloves, safety glasses or goggles, long sleeves, long

pants and shoes. Tongs may be used to handle large blocks of dry ice as required.

3. Never store dry ice in glass or other sealed/air-tight containers as it would be liable to

cause an explosion. Do not store dry ice in a confined space, only store in a well-ventilated

area. Do not store dry ice in a freezer/fridge.

4. Do not use dry ice in a confined area. Do not place on a tilted surface or on laminated

counter tops as it may destroy bonding agents. Only work with dry ice on a solid wooden

board.

5. Do not dump dry ice; allow it to sublime in a well-ventilated area where there is no

opportunity for the gas to build up. Do not dispose of dry ice in sewers, sinks, or toilets—the

extreme cold may damage pipes.

6. If a dry ice spillage occurs isolate the area. Put on personal protective equipment, i.e.

gloves, safety goggles and long sleeves. Using a dust pan and brush; carefully collect the spilt

dry ice and dispose of in the usual disposal area.

7. Carbon dioxide monitors that alarm when levels are too high should be used in areas

where dry ice will be stored or used.

C.8 Safety Guidelines for Handling Liquid Nitrogen

1. Liquid nitrogen is very cold (-168oC) and can quickly freeze the skin. Only persons trained

in the safe handling should be allowed use liquid nitrogen.

2. Users must first read the relevant Material Safety Data Sheet for liquid nitrogen.

3. Personal protective equipment must be worn, including protective gloves specifically

designed for cryogenic handling, a closed lab coat, a face shield, and shoes when working

with liquid nitrogen.


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4. Pour liquid nitrogen slowly and carefully to minimize splashing and rapid cooling of the

receiving container. Never overfill containers.

5. Use tongs when placing in or removing items from liquid nitrogen.

6. Use dip sticks to check liquid depth in Dewar’s. Do not use fingers.

7. The area for use should be well ventilated.

8. Oxygen sensors and alarms should be put in place to detect a drop in ambient oxygen if a

spill occurs.


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IV. D: Standard Operating Procedures:

D1. Standard Operating Procedure for elective recruitment and clinical data entry

Purpose

This SOP describes the procedure for recruitment and clinical Data Entry

Responsibility

It is the responsibility of the research nurse and local lead investigator to ensure that all

steps are completed competently and safely.

Procedure

1. Participants can be recruited as Patients, Healthy Controls or Disease Controls.

2. Prior to the collection of clinical data or any study samples being obtained informed

consent is obtained (see A.9).

3. A pack containing a set of labels with a unique identifier is sequentially assigned to the

participant. Each unique identifier refers to a site code and is specific to the recruiting

centre. The pack used will depend on whether recruitment is for one of the following :

a) Patient Registration Visit

b) Healthy Control

c) Disease Control Registration Visit,

d) Return Visit

NB: Return Visit packs do not contain labels. The patients Unique ID allocated at the

registration visit remains the only ID throughout the study. At return visits the

unique ID and date of visit is applied to all written documentation and labelling of

samples. Sample type should also be noted on samples. The Research Nurse will

have access to this by reference to the recruitment log.

4. The method in which the research nurse interacts with the participant will vary at each

site, depending on how the clinic operates locally. A suggested method is for the

research nurse to approach the patient as they arrive at clinic. The patient should have

received a patient information leaflet in the post prior to this. The research nurse will

ask the patient if they have had time to consider participating. If they are willing to

provide informed consent the research nurse will escort the patient to a private area to

explain the study in more detail and provide an opportunity for any questions to be

asked.


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5. Following obtaining informed consent the research nurse will collect the demographic

data for the CRF. They will explain to the patient that a portion of the urine sample

they have provided for clinic will be used for the study.

6. The patient will then be escorted back to the waiting area for their appointment. The

Research Nurse will explain that after seeing the Doctor they will come back to the

Research Nurse for blood draw to include clinical and research bloods. This is to

prevent the patient being exposed to multiple blood draws.

7. SOP D.2 Blood Sample Collection will be adhered to for the process of obtaining the

research samples. Any clinical bloods requested by the medical team will be collected

simultaneously. The research nurse will arrange locally with the lab the process of

ensuring the delivery of clinical samples to the lab.

8. If necessary in order to obtain the clinical samples the Research Nurse will liaise with

phlebotomy staff to obtain the correct blood bottles and the local ‘Laboratory

Medicine User Manual’ (if available locally). The User Manual will ensure that clinical

samples are taken in accordance with local hospital laboratory guidelines.

9. The research nurse will collect the urine sample as per S.O.P:D2.1.

10. The research nurse completes the following: affixes unique study number sticker to all

pages of CRF, consent form and EQ5D. Documents date of visit.

11. Note the EQ5D must also have the Main Study ID and Date of Visit Recorded on each

page.

12. Persons completing the form initial all pages.

13. Fills in basic identifiable demographics on the front page of the CRF: name, address,

MRN, D.O.B., contact number and email.

14. Indicates type of participant, linked to registered participant, GP details, Encounter

number and samples collected on CRF.

15. Completes Registration Fields on distiller

Baseline Characteristics- Common

Baseline Characteristics-Vasculitis

Pre-existing co-morbidity

Diagnosis - Vasculitis

Treatment: Continuing Medications

Treatment-Intermittent Pulse administration

Treatment Plasma Exchange

Biopsy

Complications

16. Complete Encounter data fields on Distiller )

Disease Assessment Common


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Disease Assessment Vasculitis

BVAS (specific training on this can be provided by Prof Little or Dr Clarkson)

Investigations: Common

Investigations: Vasculitis

Note the CRF section 2 contains space for dating when each section of distiller is completed

as the information becomes available from lab reports etc. –this is to help the research

nurse keep track when completing distiller and ensure all data fields are filled in.

17. Note: The following information can be retrieved from the Medical Notes,

Correspondence letters and/or consultation with patient’s medical team at the clinic.

Document any previously recorded ANCA IF Result

Document any previously recorded ANCA ELISA specificity

Document Systems involved at any point

Document induction treatment received

Document any previously received maintenance treatment.

Document whether a biopsy has been performed and whether there was

evidence of vasculitis.

Document whether patient has end stage renal disease.

Document whether patient is a control recruit.

Document whether the control is a relative of a patient.

Document the date of the formal diagnosis.

Document clinical diagnosis.

18. Notes for Encounter Data Fields on Distiller

Document all clinical samples obtained. Specify on Part C - page 4 of the form if

there are any issues with sample collection.

Document disease activity since last return. Consult with patient’s medical team

to clarify this. Note that, for patients with vasculitis, if disease activity is not

“remission”, there should be at least one active BVAS item recorded, and if

disease activity is “remission” then BVAS will be zero.

Complete renal assessment section.

Ensure weight is recorded at visit.

The “disease assessment” and “current immunosuppression” fields within the

encounter section are absolutely critical.

Diagnosis Confidence


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Establish Diagnosis Confidence (Table 1). The Diagnosis Confidence table is

accessible on both Distiller and UKIVAS, and can therefore be entered

retrospectively. e.g. A patient who has only one organ involved i.e pulmonary

haemorrhage, a CT scan which is inconclusive, and a negative ELISA would have a

‘possible diagnosis’

No. Of Organs Histology ANCA Diagnosis

with Clinical Features or Radiology ELISA Confidence

Diagnostic Positive Definite

Negative Definite

One Equivocal Positive Probable

Negative Possible

None Positive Probable

Negative Possible

Table 1: Diagnosis Confidence Table

Investigations

Blood results are specific to the Research Visit +/- 7 days. If clinical bloods have

been taken as part of the routine clinical care these should be followed up and

documented as part of the clinical data for the research visit.

Complete ANCA result specific to visit +/- 14 days.

Identify whether results are normal, pending, abnormal or have not been

performed.

Please note that recording of lab values greatly enhances the research potential

of the linked samples, so every effort should be made to complete them, even if

they were taken on a different day.

Treatment

Document whether patient is on Oral or Pulsed IV Steroids (Very important!)

Document Immunosuppressive medication. (Very important!)

Document whether patient is under the influence of Rituximab. If the patient has

received Rituximab in the past 9 months, or if CD19 positive B cell count is <0.005

cells/uL, they are considered to be under the influence of the drug.

Document if the patient is receiving any prophylaxis treatment.

Current Complications


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A complication is defined as an event occurring as an unintended consequence of therapy.

Complications are captured under 3 elements: “Leukopenia”, “Infection” and “Other

Complications”.

Complications of Therapy

Document if the patient has experienced any complications related to the therapy they have

received. The threshold for inclusion is the requirement for the intervention of a medical

professional (ie: a self-limiting coryzal illness would not be included).

Infection

If the patient has experienced an infection document the type of infection and grade it

using the Infection Severity Score 1-5 using table 3 or the CTCAE table (viewable on the

online clinical data entry system).

Leukopenia

If the patient has had an episode of leukopenia (total white cell count obtained from full

blood count) document this and grade the event from 1-4 as per table 2. Note that an

episode of leukopenia is scored once, even if several white cell count estimations are

performed. The nadir white cell count is considered for scoring purposes.

Table 2 Leukopenia Score.

Other Complications

Document other complications that have occurred. Identify the type of event and grade

it as per Table 3. Examples include fragility fracture, cataracts, new onset diabetes

mellitus and allergic reaction to a medication.

Total white cell count (WCC) Grade

WCC 3.1-4x109/L 1

WCC 2.1-3x109/L 2

WCC 1.1-2x109/L 3

WCC <1.1x109/L 4


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Adverse Event Reporting Guidelines (Grade 1-5 refers to the severity of the AE. Descriptions of severity for each AE are based on the following as a general guideline)

Grade 1 Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not indicated.

Grade 2 Moderate; minimal, local or non-invasive intervention indicated; limiting age-appropriate instrumental ADL*.

Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization indicated; disabling; limiting self care ADL**.

Grade 4 Life-threatening consequences; urgent intervention indicated.

Grade 5 Death related to AE.

Table 3 Adverse Event Grading

19. It may be necessary to consult with the patient’s medical doctor in order to accurately

complete the CRF. The Research Nurse may also refer to the pocket guide for Research

Nurses on the Rare Kidney Disease Registry and Bio-Resource. If in doubt, don’t enter

the data, check with the doctor.

20. Section 3 of CRF: Sample Collection. The research nurse documents the time samples

were taken on the Sample Data Collection Form. In the case of issues with sample

collection, e.g if a smaller gauge needle was used for blood draw, this must be

documented. Also, note if the sample was haemolysed, if there was a delay with

putting the sample on ice or if the addition of protease inhibitors was delayed.

21. On completion of recruitment and sample collection the following documentation is

required:

Completed Green Sticker - placed on Front of medical notes with date of

recruitment and unique study identifier.


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Completed White Sticker - applied to medical notes

22. Samples are transported to the lab for sample processing (see D.2 SOP: Sample

Processing).

23. Following clinic the research nurse sends a letter to the participants GP if they have

agreed to this in the consent form.

24. The participant is inputted onto the local recruitment log. All sites will use a generic

electronic recruitment log and ensure access to this either via PC/Laptop. This version

can be obtained from the lead research nurse.

25. The sample processing sheet and EQ5D will be batched to the central biobank with the

samples at a later date.

Registration on UKIVAS

26. UKIVAS ID is allocated when inputted into UKIVAS Database. This may be done at clinic

if access is available or retrospectively. This registers the patient on the UKIVAS

database which covers the UK and Ireland.

27. The UKIVAS registry is accessed via a specifically developed application. This should be

located on a shared folder accessible to any team member with responsibility for data

entry. Login IDs are allocated by the registry admin centre in Oxford:

https://research.ndorms.ox.ac.uk/public/ukvas/contact-us/.

28. Data can be amended after entry. The patient identifiable details are held within the

application at the study site. The central server in Oxford does not see any identifiable

data. It is therefore very important to ensure that this application is not accidentally

deleted, as this will render the patients unidentifiable locally.

D.2: Standard Operating Procedure: Recruitment at a peripheral site without

research nurse or CRF facilities

In order to obtain samples from patients presenting acutely before receiving

immunosuppression, it is necessary to engage the peripheral sites where these patients may

present. The procedure is simpler, does not use a specially prepared recruitment pack and

compromises on patient to freezer processing time.


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D2.1: Recruitment at peripheral sites outside Dublin

1. Upon making a clinical diagnosis of acute vasculitis, obtain consent from the patient

for enrolment in the registry and biobank. Consent and Quality of life documents are

available to download at: www.medicine.tcd.ie/thkc/research/thkc-ethics-docs.php

2. Inform RKD study team of the potential recruit by phoning, texting or emailing Prof.

Little, ([email protected] or tel: 086-6096068) or by placing a message on the “Acute

Vasculitis” WhatsApp group.

3. Obtain blood samples: 3 x 9mL EDTA / full blood count tubes and 1 x 9mL serum

tube, using locally available blood tubes

4. Obtain urine sample: as large a volume as possible in one or more 30mL sterilins or

any urine container. (Aim to collect as much urine as possible)

5. Record the time of sampling in the patient’s notes and write the time of voiding on

the urine containers and the time of collection on the blood sample. If the urine is

from a catheter bag please note this on the side of the container also.

6. If available, place samples in a fridge; alternatively, store samples along with consent

form at the nurse’s station until collection. Clearly write on the side of the blood

tubes and the urine container whether the samples were stored at room

temperature or were stored at in the fridge before pick up by the courier.

7. Sample collection by the contracted courier company will be arranged by lead PI or

research nurse. This will ideally be within 4 hours of sampling, but may occur the

following morning if the patient was recruited during the night.

8. The samples will be brought to the central TCD lab for processing (SOP D4)

9. 7-10 days later, the lead research nurse will visit the site to obtain a clinical dataset.

This should be collated after reference to the medical notes and discussion with the

medical team caring for the patient.

10. A local recruitment log with identifiable patient data should be maintained at each

peripheral site.

D2.2: Recruitment of acute untreated patients in Beaumont Hospital:

1. Where possible during office hours, follow generic recruitment protocol (SOP D1),

with slight modifications outlined below. If this is not possible, follow SOP D2.1.

2. Sample collection packs are available in the Drs. Office on St. Peters Ward, on the

shelves above the computer desk.

3. If there is no one from the biobank available to go to Beaumont, transport the

samples to St. James Hospital using an alternative transport link system from

Beaumont Pathology labs to the Central Pathology Labs at St. James Hospital as

follows:

4. Samples for shipment should be brought to Beaumont pathology lab before midday.

http://www.medicine.tcd.ie/thkc/research/thkc-ethics-docs.php



49 | P a g e

5. Samples should be transported at room temperature in appropriate transport

containers (if not at hand please ask staff at Beaumont Pathology Labs for assistance

in packaging appropriately in sample transport containers -tubes can lie on their

sides) and should not be refrigerated. Time to processing is important so they should

be included in the earliest available transport.

6. Upon arrival in the central pathology laboratory (CPL) in St. James’, the sample

should be left at room temperature (tubes can lay on their sides) and a member of

the renal inflammation group (RIG) should be contacted.

7. A nominated member of the RIG team will come to the CPL and collect the samples.

8. Local queries: Peter O’Leary ([email protected])

9. Contacts on arrival in CPL:

a. Fionnuala Hickey – 087 920 6721

b. Valerie Logan - 0876560962

10. Samples taken after 12 noon will generally have to be transported on the following

day and should be stored in a fridge overnight. This should be documented on the

Sample Details sheet.

D.3: Standard Operating Procedure: Sample Collection

The research nurse will have completed the appropriate training and be deemed competent

in the procedure of venepuncture prior to any blood collection in accordance with local

policy and procedures and will also be competent to respond in the event of fainting or any

other adverse event during or after the blood collection procedure. Sample collection packs

will be prepared in advance of the clinic (Items are shown in fig.1 below).

Priority Patients

At Certain times Priority Cases will be identified by the Lead Clinician.

These will usually be untreated acute patients that will be of particular value to the

biobank. In these circumstances a 8.5ml BD P100 proteomics tube may be used i along

with the EDTA for plasma. It is important for the research nurse to be aware that they

should make a particular effort try to collect all samples and capture all data on these

patients because of this value to the registry. This includes collecting urine for exosomes

if possible.

All necessary sample collection components are provided to the Research Nurse in Sample

Collection Packs the components of which are illustrated in figure 6 below.


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Summary of order of sample collection


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Sample Collection Pack Components.

A: Blood Collection:


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B: Urine Collection:

Blood tube descriptions

EDTA Plasma purple top tube. (ethylene diamine tetra acetic acid).

A colourless crystalline acid that acts as a strong chelating agent, forms a sodium salt used

to keep blood samples from clotting before tests are run: an anticoagulant.

Can be used to collect blood for DNA extraction, PBMC preparation and Plasma.

Serum Collection Tube Red top: contains clotting activators to encourage the clotting

process to enable collection of serum 30 minutes after collection.

BD™ P100

BD™ P100 tubes contain proprietary stabilizers that immediately solubilize during blood

collection, enhancing recovery and preservation of plasma analytes such as proteins and

polypeptides.

PAXgene Blood RNA (clear top with rubber/brown insert)

The PAXgene RNA tube will be used for blood collection, RNA stabilization, specimen

transport and storage. It is prefilled with an RNA stabilization reagent to provide immediate

RNA stabilization. The blood cell lysis in the tube simplifies subsequent RNA purification. It

also allows for consistent blood draw volume and blood-to-additive ratio.


53 | P a g e

D.3.1 Urine Sample Collection

1. As part of normal clinical care the patient will be asked to provide a urine sample.

They will be asked to provide an additional 250ml mid-stream urine for study

purposes (The research nurse should offer a glass of water at the patient’s first

arrival to the clinic to improve the ability of the patient to provide this volume of

urine). The urine sample should be placed on ice.

2. Label the sides of the urine tubes with the unique Main study ID barcoded labels

provided in the assigned collection pack for that patient.

3. Add 10 ml of the urine to the red capped 50ml tube for Proteomics. Label this tube

with the barcoded paperwork label with Main Study ID and write ‘Urine-P’ on the lid

and sides of the tube.

4. Add a protease inhibitor tablet (Roche Complete Ultra Mini Protease inhibitor tablet

Catalogue Number 05892791001). Invert the tube. Place this tube immediately on

ice.

5. Add 10 ml of the urine into the second red capped 50ml tube. Label this tube with

the barcoded paperwork label with Main Study ID. Write ‘Urine-M’ on the sides and

the lid of the tube. Place this tube immediately on ice.

6. Store the remaining urine in the yellow capped urine collection container, at room

temperature labelled with the barcoded paperwork label with Main study ID and

write Urine-E on the side and lid.

7. Record the time of collection in the designated sample details section of the study

Case Report Form as indicated in figure 5.

D.3.2 Blood Sample Collection

1. Position the patient safely and comfortably in a chair/couch, ensuring that the arm is

in the correct position to draw blood.

2. Ensure that the patient’s mouth is free from any food or gum.

3. All sample containers and equipment will be assembled prior to the procedure.

4. Research nurse will wear gloves and appropriate barrier protection at all times

during the venepuncture and will follow the standard local safety guidelines for

blood collection as well as the special safety guidelines for collection of Pax gene

tubes as described below and in Appendix i.

5. If the P100 tube for proteomics is to be used the Research Nurse should familiarise

with the specific instructions for this tube in Appendix ii.

A video instruction of the safety precautions should also be viewed at:


6. Order of Draw:



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a. Collect 9ml of blood directly into a vacutainer blood collection tube

containing serum clot activator (red top),

b. a second 9ml of blood into an EDTA tube (purple top) for PBMC preparation,

c. a third 9ml of blood into an EDTA tube (purple top) for PBMC preparation

and

d. a fourth 9ml of blood into a vacutainer blood container containing EDTA

(purple top) for plasma and DNA, (also BD P100 proteomics tube, green top,

if indicated).

e. Finally 2.5ml should be collected into the PAX gene RNA tube (if required).

7. If the PAX gene Blood RNA Tube is the only tube to be drawn, a small amount of

blood should be drawn into a “Discard Tube” prior to drawing blood into the

PAXgene Blood RNA Tube. Otherwise, the PAXgene Blood RNA Tube should be the

last tube drawn in the phlebotomy procedure.

8. Hold the PAXgene Blood RNA Tube vertically, below the blood donor’s arm, during

blood collection and make sure tube additives do not touch stopper or end of the

needle during venepuncture.

9. Allow at least 10 seconds for a complete blood draw to take place. Ensure that the

blood has stopped flowing into the tube before removing the tube from the holder.

10. Filling the blood collection tube to the black mark on the tube label indicates that

the correct amount of blood has been drawn. Under-filling or over-filling of the tube

may affect laboratory results due to the incorrect blood/additive ratio.

11. Immediately after blood collection, gently invert all of the blood collection tubes 10

times to mix the additives with the blood and to prevent formation of fibrin which

may affect subsequent analysis.

12. Write the time of collection on the serum tube and the 2x EDTA for PBMCs.

13. Label the sides of the tubes with the unique Main Study ID barcoded paperwork

labels provided in the assigned collection pack for that patient and affix the

corresponding barcoded paperwork label in the appropriate box on the Case Report

form.

14. DO NOT PLACE THE SERUM (red top), PAXgene or EDTA tube for PBMC preparation

ON ICE. Place the Serum (red top) tube at room temperature for 30 minutes to clot.

Store the PAXgene® Blood RNA Tube upright at room temperature while

transporting to the lab. Store the 2x EDTA tube for PBMC preparation at room

temperature until transfer to a scientist trained in PBMC preparation unless

otherwise discussed and arranged with the scientist.

15. Place the remaining EDTA tube (for plasma and DNA) immediately into a

polystyrene/’cooler’ box filled with ice/ice packs to maintain it at 4°C until processing

and storage at -80°C.


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16. Record the time of collection in the sample details section of the study Case Report

Form as indicated in figure 7 below.

17. Once the urine and blood samples are collected, the samples are transported to the

lab for processing. In some cases it may be decided to batch collect one or two

patient’s samples and pass them over for processing together, however this will

depend on local factors and how the clinic is run and will be discussed with research

nurses on a site by site basis.

Note: Venous blood samples may be obtained via direct venepuncture or via other

available venous access (e.g., an existing peripheral intravenous line or hep-lock) – as

long as the hospital staff follows their protocol for first withdrawing blood to flush

the line.

Figure 7: Sample Details Section of the Data Collection Case Report Form.


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D.3.3 Plasma Exchange Sample Collection on acute patients at Tallaght Hospital.

When a suitable acute patient is identified on the ward please aim to collect the first bag of

plasma exchange fluid.

It is hoped that in addition to the plasma exchange sample on acute patients a complete set

of samples would also be collected within 1-2 days of the plasma exchange collection and a

patient CRF would be filled in on distiller, in addition to a plasma exchange data form.

If however the plasma exchange collection does not coincide with collection of other

samples please ensure that a CRF form with data collection relating to the time of plasma

exchange collection is filled in on Distiller.

1. Take a study plasma exchange form from sample pack store, or printed from shared

Tallaght folder. A clear plastic pocket with the plasma exchange forms will also be

posted on the fridge in the dialysis unit so that the forms are easily available.

2. Record time and date of collection.

3. Recruit the patient to the study and assign a main study ID (if not already recruited).

4. Record if the patient was given medication before the collection of PEX and if so

record what medication was given.

5. Write main study ID on the plasma exchange form.

6. Write in permanent marker the main study ID, date and whether or not this is the

first plasma exchange on the bag of plasma exchange in permanent marker.

7. Place bag and form in the fridge in the dialysis unit.

8. Email [email protected] to advise that there is a plasma exchange sample to be

collected. Please include patient initials, DOB and assigned main study ID in the email

as well as any info on whether it is the first plasma exchange bag and whether or not

the patient had received any treatment. Plasma exchange fluid will be aliquoted into

8 x 50 ml tubes and 8-10x 15ml tubes, labelled with Main Study ID, date of

collection, and date of freezing. Plasma exchange samples are stored long term at -

20 degrees.

D.4: Standard Operating Procedure: Sample Processing

D.4.1: Receipt of the samples at laboratory:

It is the responsibility of the biobank technician or research nurse carrying out this

procedure to ensure that all steps are completed competently and safely.

1. Research laboratory personnel/research nurses will be trained in the receipt and

inspection of samples on arrival at the laboratory. All specimens will be inspected

upon arrival, verifying that the correct specimen was received and that the

information on the sample form, specimen collection container and sample


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identification, match each other. During this process of verification, appropriate

handling temperature will be maintained.

2. If the integrity of the sample container is compromised, the proper amount of

sample is not present, or the sample containers are not adequate this will be

documented in the appropriate section, in the sample data section of the Case

Report Form.

3. When the sample arrives in the biobank it must be registered and recorded in

accordance with local laboratory procedure. The date and time that the specimen is

received will be recorded in the Case Report Form.

D4.2: Notes on Centrifugation and the importance of setting the centrifuge to G or RCF

and not RPM

It is important that care is taken to ensure that you are spinning all samples at G or RCF

centrifuge setting and not RPM- The biobank technician will show all processors how to

do this during their training but if there are any doubts please ask!

D4.3: Processing of Urine for Proteomics

1. 10 ml of urine is received on ice in a red capped 50ml tube labelled urine with ‘P’

clearly written on the lid. Note: It should also be recorded on the CRF whether or not

protease inhibitor was added.

2. Invert a few times to ensure the protease inhibitor tablet is dissolved and mixed. It

may take a few minutes for the tablet to dissolve so let the tube sit on ice and gently

invert occasionally, until tablet is dissolved.

3. Spin for 10 minutes at 2000G at 4̊oC. This can be spun at the same time as the urine

for metabolomics (D3.4 below).

4. While taking care not to disturb the pellet, transfer the supernatant to a 5ml white

tube with green insert and 4 x 1ml green vials with green inserts. Discard the pellet

and the 50 ml tube (see Figure 8).

5. The supernatant will be aliquoted into 4x 1ml cryovials with green caps and 1x5ml

cryovial with white cap and green insert.

6. The cryovials will all be labelled with the barcoded labels marked ‘urine-P’.


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7. Place aliquots at -80 (see notes below on voiding to freezing time*).

Figure 8: Processing and Aliquoting Urine for Proteomics

D4.4: Processing of urine for metabolomics

1. A red capped 50ml tube containing 10ml of urine will be received on ice.

2. Spin the tube for 10 minutes at 2000G at 4̊ degrees.

3. While taking care not to disturb the pellet, transfer the supernatant into 4x1ml

cryovials with blue caps and 1x5ml cryovial with white cap and white insert.

4. The cryovials will all be labelled with the barcoded labels marked ‘urine-M’.

5. If more than 10ml of Urine was collected for metabolomics the additional urine

should be aliquoted into 5ml cryovials with white caps and inserts. Handwrite the

Main study ID and Visit date on these samples as labels will not be available for

these additional tubes.

6. Place the aliquots at -80 as soon as possible (see notes below on voiding to freezing

time*).


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Figure 9: Processing and Aliquoting of Urine for Metabolomics

D4.5 Processing of Urine for Exosome Analysis

1. The sample is received at room temperature and should be maintained at room

temperature throughout processing apart from the centrifugation step.

2. Invert the urine collection cup a few times to gently re-suspend the cells.

3. Carefully pour the urine into two (or more if required) 50ml tubes. Write the Main

Study ID and Urine-E on the tubes side and lid.

4. Centrifuge the tubes for 10 minutes at 2000G at 4̊ degrees to pellet any cells.

Immediately after spin is finished remove the tubes from the cold centrifuge and

place back at room temperature.

5. With a 6 ml pipette, or by decanting remove the supernatant and aliquot it into fresh

red capped 50 ml tubes.

6. Discard the tube and the pellet.

7. Label the new tubes containing supernatant with the barcoded urine-E labels. For

subsequent visits write the Main study ID, Date of Visit, and Urine-E on the tubes.

8. Store at -80 within 6 hours of collection time.

9. Record the time of freezing and total volume collected in the sample details section

in the study Case Report Form.


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10. Ensure that you tick that the sample was centrifuged and the pellet discarded in the

box indicated on the Sample Data Sheet as displayed below:

Figure 10: Processing and Aliquoting of Urine for Exosome Analysis

8. * The voiding to freezing time for all samples should ideally be a maximum of 2-3

hours with the exception of the Urine for Exosome analysis which can be stored at

room temperature for up to 6 hours. All sample collection and freezing times are

recorded. If, on occasion, the voiding to freezing time extends over 3 hours, the

samples are still valuable for many types of analysis; this timing may not be crucial


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for some purposes however it is important to let the Biobank Technician know if this

time is consistently extending to over 4 hours and never within 3 hours. This would

indicate that there may be a problem with local clinic timings or processing that

would have to be solved. The biobank technician will be monitoring the arm to

freezing times at each site to ensure that there is no problem and assessing if

changes need to be made in this regard.

D.4.6 Processing of EDTA Plasma Sample for Proteomics and DNA Extraction.

1. The EDTA tube for plasma and DNA (or BD P100 tube if indicated) is received on ice

and should remain on ice/at 4̊C throughout processing.

2. The arm to freezing time should ideally be a maximum of 2 hours. All sample

collection times, and freezing times are recorded and this enables the researchers to

request only samples with an arm to freezing time of 2hours if this is what is

necessary for their analysis. This proteomics sample is one of the most prone to

degradation and so it is imperative that the sample is processed and frozen if

possible within 2 hours.

3. Centrifuge the plasma tube at 2000G for 10 minutes at 4̊oC. Record the time of

spinning in the Sample Data Section of the Case Report Form. This spin can be done

at the same time as the serum spin, D3.7 below.

4. The Blood will separate into 3 layers: a clear, upper plasma layer; a lower red blood

cell layer; and a thin interface containing the white blood cells (buffy coat) as

indicated in Figure 13 below.

5. Using a plastic transfer pipette, collect the plasma (clear liquid) from the EDTA tube

for plasma, being very careful not to disturb the white blood cell layer and transfer it

into a 15ml red capped tube.

6. Label this tube with Main study ID

7. Centrifuge the 15ml red-capped tube containing the plasma again at 2000g for 10

minutes to remove all platelets.

8. Remove the 15ml tube from the centrifuge after the 2nd spin is complete and using a

P1000 pipette, -distribute the plasma among 3 cryovials: one with purple lid and

purple insert (will have protease inhibitor added) and two with white lids and purple

inserts (no protease inhibitor added)

9. Write “NO PI” clearly on the sides of the tubes to which protease inhibitor has not

been added.

10. Add 10µl of Protease Inhibitor solution to the vial with the purple lid and purple

insert and mix by inversion at least 8 times. Write “PI” on the side of this aliquot

tube.

11. Label each cryovial with the barcoded label marked ‘Plasma’ or in the case of visit

samples label with Main study ID, Plasma and Date of Visit.


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12. Transfer the tubes to a -80oC Freezer for storage. Record the time of freezing in the

sample details section of the Case Report Form, and record how many vials had

protease inhibitor added. Also Record that 2nd spin has been done as illustrated

below:

13. When the plasma has been aliquoted do not discard the EDTA tube. Label it with the

barcoded label marked “EDTA DNA”, or in the case of Visit Samples write the Main

Study ID, Date of Visit and EDTA DNA on the tube and store at -80 for DNA

extraction. The biobank technician will arrange for DNA extraction to be carried out

at a later date and aliquots of normalised DNA will be barcoded and labelled and

stored at -20̊C.


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Figure 11: Processing and Aliquoting of Plasma Sample and storage of EDTA DNA

sample for DNA Extraction.

D.4.7: Processing of Serum for Metabolomics

1. The red capped serum tube for metabolomics is received at room temperature and

should remain at room temperature throughout processing apart from the 10 minute

spin at 4 degrees..

2. Centrifuge the serum tube at 2000G for 10 minutes at 4̊oC. Remove from Centrifuge

once spin is over and place back at room temperature.

3. Record the time of spinning in the Sample Data Section of the Case Report Form.

4. The Blood will separate into 2 layers: a clear, upper serum layer; a lower red blood cell

layer.

5. Using a plastic transfer pipette, collect the serum (clear liquid) from the serum tube,

being careful not to disturb the red cell clot.

6. Distribute the serum among 3 x 1ml cryovials and cap with yellow lids.

7. Label each cryovial with the barcoded label marked ‘Serum’

8. Transfer the tubes immediately to a -80̊C Freezer for storage.

9. Record the time of freezing in the Sample details section of the Case Report Form.


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Figure 12: Processing and Aliquoting of serum sample

D.4.8: Processing of PAXgene tube for RNA analysis.

1. The Paxgene sample is received at room temperature and should be kept upright at

all times.

2. Ensure tube is labelled with the appropriate barcoded label. Hold specimen at room

temperature for a minimum of 2 hours before storage.

(If it is not suitable due to time constraints to maintain the Paxgene tube at room

temperature for two hours it may be stored at room temperature for up to 72hours

before placing at -20. However the Paxgene tube must be stored at room

temperature for a minimum of 2 hours before freezing at -20.)

The Paxgene tube should remain at -20 at the local site until subsequent transfer to

the central biobank for long-term storage at -80.

3. The time and date of freezing at -20 is recorded on the sample details section of the

Case Report form.


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D.4.9: Processing of 2 x EDTA tubes for PBMC preparation.

Please Note: All labels for the PBMC aliquots are pre-printed and included in the registration

packs. These must be transported to St. James along with the 2 x EDTA tubes to the

processing scientist.

1. Adjust temperature of “Mr Frosty” to 4°C.

2. Transfer the blood sample (~9ml) from each EDTA tube into a 50ml tube. Wash the residual

blood from each EDTA tube with 9ml of PBS (GIBCO Invitrogen catalogue number: 14190-

094) and add to the new tube to ensure maximum sample collection and 1:1 dilution of

blood with PBS.

3. In two additional tubes pipette 9ml of lymphoprep (Axis Shield catalogue: 1114545).

4. Slowly pipette the 18mls of diluted blood sample into each tube containing lymphoprep (2:1

ratio of diluted blood to lymphoprep). The blood should sit as a layer above the

lymphoprep.

5. Centrifuge the sample at 400g for 25 minutes at room temperature with 0 Acceleration and

0 Brake.

6. Remove sample from centrifuge and discard top layer which contains plasma, taking care

not to disrupt the buffy coat layer directly beneath the plasma.

7. Hold a Pasteur pipette above the buffy coat layer and gently pipette out the cells into an

additional tube. Circle the tube with the pipette to remove all cells.

8. Add PBS to fill the tube containing the cells. Centrifuge at 800g for 5 minutes.

9. Remove the supernatant and resuspend cell pellet in 50ml PBS.

10. Centrifuge at 400g for 10 minutes.

11. Remove supernatant and resuspend cell pellet in media (Complete RPMI – see recipe

below).

12. Count the cells using a haemocytometer according to the following formula:NO. OF CELLS IN

ONE LARGE SQUARE X DILUTION FACTOR X 10,000 = Number of cells/ml

13. Place cells on ice or in fridge for a few minutes to cool.

14. Dilute cells with media to give a concentration of ~10x106 cells/ml

15. Label required number of cryovials (will depend on number of cells obtained) with labels

provided with blood samples or manually label to include patient ID, date, No. of cells per

vial and processor initials

16. Add 500μl of cold cells (~5x106 cells) to cryovials on ice. Quickly add 500μl of cold freezing

mix (see recipe below)

17. Place in “Mr Frosty” and store in a -80˚C freezer. NOTE: Freezing media contains DMSO

which is toxic to cells at ambient temperature, so once added to cells tubes should be

transferred to -80C freezer with minimum delay.

18. The time and date of freezing is recorded on the sample details section of the Case Report

form.


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19. Next day, transfer cells from “Mr Frosty” to normal freezer box and eventually to liquid

nitrogen for long term storage.

A. Complete RPMI media recipe:

· 500ml RPMI media (GIBCO Invitrogen catalogue number: 61870)

· 5ml Pen/Strep (GIBCO Invitrogen catalogue number: 15140-026)

· 5ml Fungizone (GIBCO Invitrogen catalogue number: 15290-122)

· 50ml FCS (Biosera-from Labtech catalogue number: FB1001/500)

B. Cold freezing mix recipe:

80%FCS FCS (Biosera-from Labtech catalogue number: FB1001/500)

20%DMSO Dimethyl sulfoxide (Sigma Aldrich Catalogue number: D8418)

D.4.10 Sample Processing BDP100 Plasma

A box of BD P100 Proteomics tubes will be available at designated storage areas at each site.

These are extremely expensive and are only to be used for acute patients with the

permission of Prof. Mark Little (via email, text message or telephone).

1. Please ensure you have read the safety guidelines for blood collection that are

specified in the protocol appendix IIa.

2. Take the blood sample as per instructions and record collection time on the sample

details page.

3. When the sample is collected invert it 8-10 times to mix. Transport to the lab at

room temperature and spin/centrifuge it a.s.a.p. and within 2-4 hours for 20minutes

at 2500G at room temperature.

4. After centrifugation aliquot into 1ml volume aliquots in the red capped vials

enclosed in the box.

5. Label sample with the Main Study ID, encounter date, and sample type specifically

BD P100 plasma.

6. Freeze the plasma aliquots at -20 degrees in the in the box labelled BD P100

PLASMA ACUTE PATIENTS.

7. Record the freezing time in the sample details page.

8. The sample will be transferred to -80 once received at the central biobank.


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D.4.11 Sample Processing Summary

Serum

Transport at room temp. Centrifuge at 4◦C at 2000G for 10 minutes

Aliquot ~1mL plasma into each of 3 yellow capped cryovials.

Label with appropriate barcoded label. (Serum)

Store in Patient Encounter bag in -80◦C Freezer

EDTA: Plasma & DNA

Transport on ice. Centrifuge at 4◦C at 2000G for 10 minutes. Remove plasma taking care not to disturb buffy coat to a new 15ml tube. Respin at 2000G for 10 minutes at 4 degrees. Remove plasma with P1000 pipette.

Aliquot final ( spun twice) ~1mL plasma into 1 violet capped cryovials with purple insert, and 2x white capped cryovials with purple inserts. Add (1X) 10µl of Sigma Protease Inhibitor to 1x purple capped tubes with purple insert, no Pi to be added to 2x white capped tubes with purple inserts.

Label with appropriate barcoded label. (Plasma) Write PI on the purple capped tube with PI added and No PI on the white capped tubes with no PI added. Ensure remaining tube with packed cells including intact buffy coat is labelled with the appropriate barcoded label. (EDTA -DNA).

Store all in Patient Encounter bag in -80◦C Freezer

Paxgene tube for RNA

No Initial Processing. Transport at room temp.

Ensure tube is labelled with the appropriate barcoded label. (Pax-RNA)

N.B. Hold specimen at room temperature (18◦C-25◦C) for a minimum of two hours before storage.

Store in open plastic rack in -20◦C Freezer for 24hours and then transfer to -80◦C. NB: DO NOT STORE IN POLYSTYRENE RACK

2x EDTA tube for PBMC prep.

No Initial Processing. Transport at room temp. Record time of collection on tubes.

Ensure tubes are labelled with the appropriate barcoded label and time of collection (EDTA for PBMC)

N.B. Hold specimen at room temperature until transfer to a scientist trained in PBMC preparation.

Urine for proteomics

Transport on Ice. Protease Inhibitor tablet is added at clinic. Centrifuge tube at 4◦C at 2000G for 10 minutes

Aliquot ~1mL urine supernatant into each of 4 green capped cryovials and Aliquot ~5mL urine into 1 white capped cryovials with green insert.

Label with appropriate barcoded label. (Urine-P)


Urine for metabolomics

Transport on ice. Centrifuge tube at 4◦C at 2000G for 10 minutes

Aliquot ~1mL urine into each of 4 blue capped cryovials with white inserts and Aliquot ~5mL urine into 1 white capped cryovials with blue insert.

Label with appropriate barcoded label. (Urine-M)


Urine for Exosome Analysis

Transport at room temp. Pour urine contents left in Urine Collection Container into a 50ml tube. Centrifuge at 2000G for 10 minutes at 4 degrees. Remove supernatant to a fresh 50ml tube and store. Discard Pellet.

Write Exosome on lid of tube.

Label with appropriate barcoded label. (Urine-E)



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BDP100 Transport at room temp. Centrifuge at room temperature at 2500G for 20 minutes

Aliquot into 1ml volume aliquots in the red capped vials

Handwrite label with Main Study Id, Visit Date and sample type: P100PLASMA

Store in RKD labelled box at -20 and arrange with Biobank technician transfer to -80.

Table B.1: Sample Processing Summary Table-Quick Reference Guide

D.5: Standard Operating Procedure: Sample Transportation after processing at

local sites

Samples will be stored at their local hospital sites until intermittent transport to the TCD

biobank.

• Samples will be transported on dry ice to ensure against freeze/thaw to maintain the

integrity of the samples.

• Samples will be transported in bulk.

The biobank technician will liaise with the local Research Nurse at the relevant sites

to arrange sample pick-up.

No samples are to be removed from the -80C freezer until the time of pick-up.

A log of samples being transported should be provided with the specimens at the

time of collection.

D.6: Standard Operating Procedure: Sample Storage at the RKD Biobank

The specimens should reach suitable storage the -80C̊/-20̊C freezer as soon as

possible.

The visit samples are barcoded at the Biobank.

All samples stored will be checked in using the central biobank inventory system.

All sample data are entered onto the inventory database.

If there are any issues with the samples or data the Biobank technician will feed this

back to the research nurse to gain clarity.

If any aliquots are removed from the freezer, they must be checked out in the data

management system.

Under normal circumstances, biobank staff only are permitted to access to the

Registry samples.

D.7: S.O.P. Emergency Response in case of main Biobank Freezer malfunction.

The Biobank Freezers are monitored the XILTRIX monitoring system. This system sends a

text message and an email to Mark Little, Fionnuala Hickey and Valerie Logan in the event of

a power loss or if the temperature rises to above -60 degrees.


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If the temperature rises above -50 degrees a back-up CO2 system will also kick in.

Please note-there is a door switch on the freezer which prevents the CO2 from firing if the

door is open. You need to be careful if you are working on the -80 freezer for extended

periods that that nothing is left on this switch thus the freezer thinking the door is closed

may fire CO2 out at you which could cause injuries.

Also, If you come across the freezer and it is above -50 and the C02 had been activated,

before gaining access to the samples you must first close the C02 cylinder valve at the CO2

bottle-the turn direction will be marked on the valve.

For safety you should not open the door for 1 hour after doing this to allow all CO2 to

dissipate.

Please ensure all ventilation is functioning in any room that contains CO2.

Decant -80 Freezers are situated in the IMM Room 0.38 in the Basement. There are also

some decant -30 Freezers in Room 0.44 in the basement.

At times further decant freezers may be temporarily situated on the corridor of the first

floor by Freezer room 2.17 and there are at times a-80 Freezer empty or some space

available in the Biobank Freezer room 1.35 Ground Floor.

If you come across a freezer malfunctioning at any of the sites please inform a member of

the biobank team and take a record of the highest temperature that the freezer reaches

while containing the samples. Please ensure you are aware of the location of any decant

freezers at the site you work at by communicating with your lab manager.


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E. References

Safety, Health and Welfare at work Act (Biological Agents) 1994.

WHO Guidance on regulations for the transport of Infectious Substances.

Molecular Medicine Ireland Guidelines for Standardised Biobanking. First Edition 2010.

F. Sample Collection and Processing Consumables

Item Description Catalogue Number Supplier

9ml EDTA tubes (pack of 100) 455/045 Cruinn Diagnostics

9ml serum tubes 455092 Cruinn Diagnostics

2.5ml Pax gene RNA tubes 762165 BD

BD Vacutainer Safety Lok blood collection set 21g Needle 7" tubing, Pre Attached Holder (Pack of 25) 368654 BD

2ml cryopure tube external thread yellow ( 1 box of 500 vials) 72.379.004 Sarstedt Ltd.

2ml cryopure tube external thread violet ( 1 box of 500 vials) 72.379.007 Sarstedt Ltd.

Violet colour coded inserts- cyrotubes 65.386.007 Sarstedt Ltd.

2ml cryopure tube external thread Red (1 box of 500 vials) 72.379.002 Sarstedt Ltd.

Red colour coded inserts- cryotubes 65.386.002 Sarstedt Ltd.

2ml cryopure tube external thread green (1 box of 500 vials) 72.379.005 Sarstedt Ltd.

Green colour coded inserts- cryotubes 65.386.005 Sarstedt Ltd.

Yellow colour coded inserts- cryotubes 65.386.004 Sarstedt Ltd.

White inserts 65.386 Sarstedt Ltd.

Blue 1.8ml crypure tubes ( pack of 500 vials) 72.379.006 Sarstedt Ltd.

Transfer pipettes 86.1171.001 Sarstedt Ltd.

50 ml PP Skirted Sterile Base tube with assembled screwcap 62.559.001 Sarstedt Ltd.

15ml PP Conical sterile Base tube with assembled screwcap 62.554.002 Sarstedt Ltd.

100ml urine collection tube 75.562.105 Sarstedt Ltd.

Protease Inhibitor tablets (30 in each kit)

Roche Diagnostics Ireland Ltd

Protease Inhibitor (liquid 1ml) P8340 Sigma

Table F.1 Sample collection and processing consumables


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Appendix I: Paxgene tube

Appendix II: BD P100 tube

Appendix III: Paper CRF

Appendix IV: Tallaght specific information

Appendix VI: SOP: Distiller to Freezerworks Data Export

Vasculitis Sample and Data Collection and Management Protocol V7

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