A standardised protocol for blood and cerebrospinal fluid collection and processing for biomarker research in ataxia

Abstract The European Spinocerebellar Ataxia Type 3/Machado‐Joseph Disease Initiative (ESMI) is a consortium established with the ambition to set up the largest European longitudinal trial‐ready cohort of Spinocerebellar Ataxia Type 3/Machado‐Joseph Disease (SCA3/MJD), the most common autosomal dominantly inherited ataxia worldwide. A major focus of ESMI has been the identification of SCA3/MJD biomarkers to enable future interventional studies. As biosample collection and processing variables significantly impact the outcomes of biomarkers studies, biosampling procedures standardisation was done previously to study visit initiation. Here, we describe the ESMI consensus biosampling protocol, developed within the scope of ESMI, that ultimately might be translated to other neurodegenerative disorders, particularly ataxias, being the first step to protocol harmonisation in the field.

This polyQ expansion confers a toxic gain-of-function to mutant ataxin-3 that leads to the formation of neuronal intranuclear inclusions, neuronal dysfunction and degeneration of specific brain regions, particularly the spinal cord, the brainstem and the cerebellum [6][7][8].
SCA3/MJD is a progressive disease that leads to severe disability and premature death, and there is an unmet need for a therapy that can stop or delay disease progression.
In this context, in 2016, the European Spinocerebellar Ataxia As biosample collection and processing variables have been shown to significantly impact the outcomes of biomarkers studies [20][21][22], the standardisation of sample biobanking and processing, especially in the context of multicentric studies, is crucial to ensure that biosample analysis is not compromised by preanalytical factors [23][24][25]. Still, previous multicentric studies in the ataxia field did not have a common biosampling protocol [26][27][28]. To obtain high-quality biological samples and reduce sampling variability between centres, a biosample collection and processing manual was developed and implemented at all ESMI sites.
Here, we describe the ESMI consensus biosampling protocol established with the aim of guiding clinical research personnel on standardised collection, processing and biobanking of biological samples with application on biomarkers clinical research. Although this protocol was developed within the scope of ESMI, its applicability might be translated to other ataxias being the first step to protocol harmonisation in the ataxia field. This protocol could also be envisioned as a biosampling manual for other neurodegenerative disorders.

Ethical considerations
The biosampling procedures described here require the informed consent of participants prior to biosample collection. Ethical committees must previously approve the study protocol that is carried out in accordance with local and international regulations.

Study visits
In the ESMI protocol, annual visits are scheduled and biosamples are collected at each visit. Biosampling is performed at the time of clinical evaluation, which also follows a standardised assessment.
If biosamples cannot be collected at the same time as clinical evaluation, they should be collected within 1 month of the clinical visit.

Biosamples
Blood and CSF are collected as described in Table 1 • A major focus of ESMI has also been the identification of SCA3/MJD biomarkers for future interventional studies.
• To obtain high-quality biological samples and reduce sampling variability between centres, a biosample collection and processing manual was developed and implemented at all ESMI sites.
• The ESMI biosample collection and processing manual has been used on an international scale and might be translated to other neurodegenerative disorders.

Collection tubes
Collection tubes were carefully selected according to the type of biosamples being collected. Tubes should not be supplemented or replaced by tubes from other suppliers unless approval to do so is granted by the Biosample Coordination Site. A description of the different types of collection tubes and appropriate storage conditions, before sample collection, is presented in Table 2 (more detailed information about each tube can be obtained from the supplier).

Equipment and materials
In order to collect and process samples consistently across all centres and to ensure the highest sample quality, sites must have access to the following equipment: • Swing-out rotor-type centrifuge at room temperature; • À20 C freezer; • À80 C freezer.
Each centre is responsible to ensure that collection blood kits are compatible with the tubes described in Table 2. A list of the material necessary for blood collection and processing can be found in the supporting information Annex I.

Collection tubes labelling
Each collection tube should be unequivocally labelled prior to blood collection. Each centre should use its local labelling system and codes, but efforts must be made to evade handwriting in labelling to avoid T A B L E 1 Biosampling description. Type of biosample and required collection tubes and blood volumes for each purpose. any source of misunderstanding. Labels should be printed and pasted on the tubes. The following recommendations should be followed when labelling the tubes: • Confirm patient identity before collecting the blood; • Place the label vertically on the tube; • Avoid misaligned labelling; • Ensure the label has completely adhered to the tube.

Aliquot labelling
After blood sample processing, cryovials will be used to store PBMCs and aliquots of plasma/serum/whole blood. Each cryovial should be unequivocally labelled before blood processing. Each centre will use their local labelling system, but efforts should be done to avoid handwriting in the labelling to avoid misspelling words.

Records
To have a written record of each biosample collection procedure, a biosample identification form (supporting information Annexes IV and V) must be filled out every time biosampling is collected.
The biosample identification form contains the following information: • Research centre; • Visit number; • Date of the collection; • Hour of the collection; • Identification number of the sample/patient; • Gender and age of the patient; • Number of CAG repeats; • Disease onset; • SARA score; • Time of the last meal; • Number of tubes collected; • Any occurrence out of ordinary during collection and processing.
Additionally, the back of the sample identification form might be used to add any relevant information/observations related to sample collection and processing.

Participants preparation
Efforts must be made to ensure the procedure is as easy and painless as possible. Technicians must remain calm and project an attitude of competence and confidence even when faced with the most nervous or inquiring participants. Approximately 67 mL of blood is collected from each participant. The technician should inform any participant who is concerned about the volume of blood collected in this protocol that the total amount drawn is almost seven times less than the volume usually collected in a blood donation.
Note: this biosampling protocol was developed for adult participants, as SCA3 is an adult-onset disease.

Time for blood collection
Blood samples should be collected during the morning between 8:00 and 12:00, preferably fasting. The time of blood collection and the last meal must be recorded in the biosample identification form.

Order of tube collection
Technicians should be previously familiarised with the different tubes.
The biosample collection protocol, and a blood collection tray with the tubes in the correct order, should be prepared in advance.
To guarantee the collection of all the different biosample types, tubes should be filled with the recommended volumes of blood in the following order: The number of collected tubes must be registered in the blood biosample identification form (fill out the checklist with X for each tube collected).

Venipuncture and blood collection
Blood collection should be performed according to recommended guidelines for standard venipuncture. General recommendations should be followed, such as the following: • Assess participant disposition.
• Place the donor's arm in a downward position.
• Hold collection tubes in a vertical position, below the donor's arm during blood collection.
[CRITICAL STEP]: Make sure tube additives do not touch the stopper or end of the needle during venipuncture.
• Release the tourniquet as soon as blood starts to flow into the tube.
• Make sure the tubes are totally filled.
• Immediately invert the tube, in accordance with the number of inversions recommended for each tube type (see Table 3).
[CRITICAL STEP]: Tubes must be immediately mixed by complete inversion according to Table 3.
• Tubes should then be stored upright, at room temperature, until processing.
A representative picture of a workflow of the venipuncture procedure can be seen in the supporting information Annex VII.
Processing of blood biosamples [CRITICAL STEP]: Blood processing must start as soon as possible and within a timeframe of 2 h after blood collection to ensure high-quality samples. If sample processing starts after a period of 2 h of collection, it must be reported in the sample identification form.
Specific instructions for blood processing for each blood collection tube are described below.
PAXgene Tube (#1 and #2) • Incubate tubes upright, at room temperature, for at least 2 h and no more than 6 h.
• Freeze tubes at À20 C for 24 to 72 h.
• Store tubes at À80 C.
A representative workflow of PAXgene blood processing is shown in Figure 1A.
CPT (#3 and #4) • Centrifuge tubes at room temperature in a balanced, swing-out rotor-type, for 30 min at 1700 RCF, with the brake off.
• After centrifugation, plasma will become evident at the top layer, while PBMCs and platelets will appear as a whitish ring just under the plasma layer.
A. For plasma collection • Discard the supernatant; • Resuspend the PBMCs pellet in 1 mL of PBS; • Transfer to a cryovial and centrifuge at 300 RCF, at room temperature, with the brake on; • Discard the supernatant removing all the PBS from the cell pellet; • Freeze the cryovials with the cell pellet at À80 C.
Option 2: • Discard the supernatant removing all the PBS from the cell pellet; • Freeze the 15 mL polypropylene tube with the cell pellet at À80 C.
A representative workflow of CPT blood processing is shown in Figure 1B.
SST (#5, #6 and #7) • Allow blood to clot in a vertical position for a minimum of 30 min and no more than 2 h.
• Centrifuge tubes in a balanced, swing-out rotor-type centrifuge, at room temperature, at 1100 RCF for 10 min, with the brake off.
• Remove the BD Hemogard™ Closure. Aspirate the serum layer with a 10 mL syringe, using a 16G needle. Alternatively, a sterile Pasteur pipette can be used to transfer serum into the 10 mL syringe.
• Replace the needle with a 0.8 μm filter and push the serum through the filter drop by drop into a 15 mL polypropylene tube.
Note: When aspirating into the syringe/Pasteur pipette, be sure not to disturb the red cell layer/buffy coat with the tip of the needle.
• Divide serum into 500 μL cryovial aliquots and freeze immediately at À80 C.
Note: Less than 500 μL aliquots should be saved to the extent that local conditions allow.
A representative workflow of SST blood processing is shown in Figure 2A.

PPT (#8 and #9)
• Centrifuge tube in a balanced, swing-out rotor-type centrifuge at room temperature at 1100 RCF for 10 min, with the brake off.
• Aspirate the plasma layer into a 10 mL syringe using a 16G needle.
Alternatively, a sterile Pasteur pipette can be used to transfer plasma into the 10 mL syringe.
• Replace the needle with a 0.8 μm filter and push the plasma through the filter drop by drop into a 15 mL polypropylene tube.
Note: When aspirating into the syringe/Pasteur pipette, be sure NOT to disturb the red cell layer/buffy coat with the tip of the needle.
• Divide plasma into 500 μL cryovial aliquots and freeze immediately at À80 C.
Note: Less than 500 μL aliquots should be saved to the extent that local conditions allow.
A representative workflow of PPT blood processing is presented in Figure 2B.
Note: Less than 500 μL aliquots should be saved to the extent that local conditions allow.
• Freeze directly at À80 C.
A representative workflow of EDTA blood processing is shown in Figure 2C. An overview table for blood collection and processing can be found in the supporting information Annex VIII.

Lumbar puncture and CSF collection
The lumbar puncture should be performed according to each centre's recommended standard operating procedure, by trained personnel.
The following recommendations should be followed: • Assess participant disposition; • Confirm participant identity; • Ensure antiseptic cleansing and anaesthesia; • In case of bleeding at the puncture site, discard the first 1 mL of CSF and record the event on the CSF biosample identification form; • Collect between 12 and 15 mL of CSF in a polypropylene tube; • Mix the CSF gently by turning the tube upside down three to four times (cap on).
F I G U R E 2 Protocol for serum separator tube, plasma preparation tube and EDTA tube processing. Workflow summarising the protocol for serum separator tube (A), plasma preparation tube (B) and EDTA tube (C) processing to obtain serum, plasma and whole blood for biomarker studies, respectively. This picture was drawn specifically for this publication and is not included in European Spinocerebellar Ataxia Type 3/Machado-Joseph Disease Initiative (ESMI) biosampling manual. Created with BioRender.com (agreement number: DF250CCVQ0).
Processing of CSF biosamples [CRITICAL STEP]: The CSF processing must start as soon as possible and within a timeframe of 2 h after CSF collection to ensure the high quality of samples. If sample processing starts after a period of 2 h, it must be reported in the biosample identification form.
Specific instructions for CSF processing are the following: • Centrifuge the tube in a balanced, swing-out rotor-type centrifuge, at room temperature, for 10 min at 1100 RCF.
• Aspirate the CSF and divide the fluid into 500 μL cryovial aliquots.
Note: Less than 500 μL aliquots should be saved to the extent that local conditions allow.
• Freeze immediately at À80 C.

Reporting
The biosample identification form (supporting information Annexes IV and V) should be filled immediately after blood/CSF collection and processing. Any deviation from this protocol or any occurrence during collection and processing (e.g. haemolysis during collection, timeframe for blood processing not respected and sample contamination) must be reported in the biosample identification form.

Storage of biosamples
Storage of biomaterials should be done locally in appropriate cooling devices at À80 C, after appropriate processing.

FINAL REMARKS
The collection of biological samples is an integral part of clinical research studies. Biological samples are, however, subject to different collecting, processing and storage conditions that can significantly alter their molecular composition, affecting experimental results and the ability to reproduce accurate scientific data [29][30][31].
Therefore, the development of guidelines or standardised protocols for biosample collection and processing is extremely important to ensure that variation in measurement values reflects true biological differences between samples rather than differences in biosampling procedures.
Taking this into consideration, we present here the standardised protocol of the ESMI consortium for the collection, processing and storage of blood and CSF samples from SCA3/MJD patients and control subjects.
The ESMI project was successfully kicked off with a meeting in Bonn (Germany) in May 2016. The first version of this manual was written in June 2016 and implemented over a trial period of 6 months in 6 research centres. It is important to highlight that the type and number of collected tubes, and processing procedures, were defined in accordance with the project aims. These included (1) [32,33], CPT tubes for PBMCs isolation [34,35] and SST and PPT tubes for serum and plasma isolation, respectively, for protein analysis and for EVs isolation [36]. A K2EDTA tube was also included to collect whole blood for genotyping, including the number of CAG repeats in the ATXN3 gene. A filtration step was included in plasma and serum processing as required for EV isolation [36].
In December 2016, a second version of the protocol was written, implemented and followed in a total of 11 research centres (six con-  After signing a material transfer agreement (MTA), samples were transferred between sites for biomarker studies approved by the ESMI Executive Board. The standardisation of these procedures has been extremely valuable and already allowed us to develop an assay for ataxin-3 quantification [33] and to establish neurofilaments as biomarkers for MJD/SCA3 [37][38][39]. Biosamples have since been used in many other studies that are still ongoing and profit from the adherence to this protocol.
In 2020, a third version of the ESMI manual (the one presented in this manuscript) was implemented to maintain the collection and processing of biological samples during clinical visits, with fewer resources.
In this last version of the manual, the quantity and type of tubes used were revised. The number of PAXgene tubes was decreased from four to two, and the number of serum and plasma tubes was increased from two to three and one to two, respectively. Despite their extensive use in transcriptomic biomarker studies [40][41][42], PAXgene tubes are expensive, which restricts their use. Conversely, PPT and SST tubes are more cost-affordable and allow serum and plasma collection, two of the most clinically relevant and used biofluids. In addition, the aliquot volume of biosamples was also decreased from 1000 to 500 μL. Nowadays, analytical technologies are more sensitive, requiring lower volumes of input material. The storage of small volumes is thus preferred to avoid repeated freeze/thaw cycles of larger aliquots [29]. Aliquots of smaller volumes also increase biomaterials availability for a larger number of studies. As lack of storage space was one of the major hurdles reported by research centres, the presented version also included the optional suggestion to discard low-volume aliquots of samples. In this last version, aligned with what was referred to above, partners were requested to store all collected materials (even small volumes), to the extent that local conditions allow.
Biomarker research is a continuous process that begins with the discovery phase followed by a validation process, ultimately leading to implementation in a clinical setting [43]. This is an extensive and

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analysed in this study.

ETHICS STATEMENT
No ethical approval is needed.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/nan.12892.

SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.