TITLE: DToL Tissue and Blood Sampling Standard Operating Procedure: Chordata: Vertebrata (general)
AUTHORS: Inez Januszczak, Nancy Holroyd

[DESCRIPTION]
This Standard Operating Procedure (SOP) contains guidance on how to sample tissue and blood from Vertebrata specimens submitted to the Darwin Tree of Life (DToL) project. 

Please note this SOP covers sampling tissue and blood either during an appropriate veterinary procedure or post-mortem. It is applicable if the submitter/centre in question has opportunities to sample tissue from animals that are being either being euthanized for clinical purposes, undergoing veterinary procedures. or eligible biobanked carcasses (storage dependent).

This SOP does not cover procedures regarding bio banked, pre - dissected tissue or blood. 

This SOP is covers animals in general - please refer to the DToL Chordata: Vertebrata: Aves and DToL Chordata: Vertebrata: Fish tissue sampling SOPs for more a more detailed breakdown for those groups.

This is version one of this SOP, and may be subject to change in the future. 

[GUIDELINES]
Tissue/blood sampling post mortem 

If the centre in question has opportunities to sample tissue from animals that are being either being euthanized for clinical purposes, undergoing veterinary procedures, or eligible biobanked carcasses (storage dependent), such samples would be very useful for the Darwin Tree of Life (DToL) project. 

Regulatory compliance considerations 

Animal Welfare - the samples must only come from animals undergoing veterinary care, euthanized for health reasons or biobanked carcasses that are eligible for dissection/ tissue preparation. 

Permissions may be requested depending on the species in question. 

 

Logistical Considerations 

A -80 freezer is required for tissue sampling (suggested brand Fryka B35-85 under bench, ETA 2-3 weeks 80kg. 58x76x54cm). Preserving the tissue directly on dry ice is also an option.

Prior to submission
 
1. It is important to check whether DToL would require the sample. 
 
The submitter/centre in question would be shared a list, which states the species required. As this is opportunistic sampling, the list should be regularly checked to see what species are needed.  

 

2. Sampling

Aside from the specimen itself, labelled cryotubes that are able to withstand -80 degrees cold storage will be required (for the tissue) as well as ziplock/ plastic bags to store the tubes and carcasses. 

Each specimen requires two identical ID numbers; one will be used to label the ziplock bag of tubes containing the samples, the other to label the remaining carcass (if applicable).  

The Natural History Museum (NHM) is able to provide unique ID numbers, otherwise any matching ID will be appropriate, preferably a series of numbers. 

The NHM is able to provide cryotubes with unique IDs if this aids the archiving. 

 

If the specimen (in any of the listed scenarios) is suitable: 
 
a. Take a photo of the specimen – a dorsal and ventral photograph, with the specimen ID clearly visible in the frame of the photo. This photo will act as a photographic voucher if it is not possible to retain to the rest of the carcass post dissection.  Label the photograph with the given Specimen ID. 

b. Dissect out multiple (ideally minimum of 3, up to 8) samples per tissue below, maximum 0.5g per tube (around pea-sized) as is practical from the size and condition of specimen: 
 
Muscle 
Heart 
Liver 
Brain 
Inject out blood IF POSSIBLE  

Please note listed tissues are ranked in order of priority. 

 

c. Put tissue immediately into a clearly labelled tube (with a unique tube ID) and place immediately into the -80 freezer/dry ice.  Place all tubes into a ziplock bag  - keeping tissues separate can also help with the labeling. Ensure each ziplock bag of samples has its own specimen ID label. 

d. Put the rest of the carcass into a ziplock bag, double bag the carcass, then label with the second identical specimen ID.  

e. Add the appropriate data to an Excel spreadsheet, clearly linking the specimen ID, tube label and tissue type. A draft spreadsheet is attached to this protocol.

f. Ensure the tissues are stored at -80 degrees.

g. If it is possible to store the rest of the carcass in minimum –20 conditions, then it should be stored. The NHM is interested in obtaining the rest of the carcass as a specimen voucher, regardless of its condition after postmortem. 

h. Email the DToL NHM sampling coordinator regarding the samples obtained.  
 
3. Submission 
 
Once the submitter/center has collected at least 5 sets of samples from 5 specimens, and thus potentially 5 carcasses, they should contact NHM and Sanger DToL Sampling team who will arrange for their collection.  

 

When submitting/ considering a submission, please contact: 
 
Inez Januszczak, DToL NHM Sampling Coordinator: Inez.januszczak@nhm.ac.uk 
The DToL NHM Sampling team: darwintreeoflife@nhm.ac.uk 
Molly Carter, DToL Sanger/Tree of Life Coordinator: mc39@sanger.ac.uk 
 


[STEPS]