Can a group 1 patient be included if already listed?

Yes, group 1 patients can already be on the waiting list.

Can a patient already on the waiting list who develops ACLF grade 2-3 be included in the study?

Yes. In this case, the date of ACLF 2-3 development will be considered the date of inclusion.

Can a group 2 patient be included if he/she is already listed?

Yes, group 2 patients can be in waiting list up to 3 months prior inclusion visit. The inclusion visit is mandatory

Which MELD score is considered to include a group 2 patient?

For Group 2, the MELD score should be higher than 20 at the time of listing and at inclusion visit.

Can a patient with a MELD > 20 at pre-screening but listed with a MELD score < 20 be included?

No, it is a screening failure.

What to do if a group 2 patient is not admitted to the hospital?

It can be assumed that the date of evaluation or inclusion is the hospital admission date.

How to register a temporary suspension from the waiting list?

There is no option to register it directly in the eCRF. Please register the visit for deslisting and continue with the visits. In these cases, the visit schedule will correspond to a Group3 patient.
Once you decide to include again the patient in waiting list you should complete the study termination form registering as a reason “Recruiting for second time after delisting”.

Can I enter into the study a patient who was temporarily suspended from the list?

Yes, as a new patient. The following variables should be Yes at Pre-screening form.

• Is the patient already listed or a candidate to enter in waiting list?
• Is the patient already included in the platform but delisted?

In toxic habits form, when is a patient considered former?

For alcohol, tobacco or recreational drugs consumption, a patient is considered 'former' if had quit the habit at least 6 months previous to inclusion.

Clinical events form at inclusion.

At inclusion, if a patient is admitted to the hospital because of a decompensation of cirrhosis, there should be some clinical event to be reported. For example, if a patient undergoes a paracentesis, ascites should be reported at the clinical event form.

Clinical events form between visits.

Decompensations between visits should be reported. No matter if the event finishes the day before the visit, it should be registered too.

When to collect the vital signs when there are several data collections from the same day?

They should be reported ideally at the time of sample collection. If not, always at the same time with a (+/-) 4 hours window.

Ammonium in the laboratory form: the only options and the beginning are arterial and venous, but no “not performed”.

At the end, the important thing is the value, so, please select whatever type of ammonium (or the common ammonium analyzed in your center) and enter the -99.

The laboratory report doesn’t provide the exact number of an analysis because it is out of range.

Please enter the most accurate data possible. For example, if the result is INR>8, please register 8.1 in order to not lose the data.

Which treatments should be reported?

All the treatments should be registered from 3 months prior to inclusion to 12 months after liver transplantation or at the end of the study. except for serums, K+, Ca++, Mg and P

I cannot find the treatment on the platform.

There are 2 options:

• You are not entering an active ingredient but a generic name.
• You have the autocomplete option activated in your browser.

I don’t know the start and end dates for a treatment.

It is mandatory to register the start and end dates for each treatment. If a specific registry is not possible, please enter at least an estimated date. If it’s not possible, just leave it blank.

I get an error in concomitant medications and procedures.

If all the procedures/treatments are closed at the visit, you will probably get an error for the platform. This message appears when procedures and treatments finish before the visit or occurred between two visits.

To avoid this error, please select YES, introduce the information, and if you get the error after select NO and then save the form.

I can’t find one specific translation of the Quality of Life scores.

Please contact the EF-Clif Data Management Center (chance_dm@efclif.com) and ask for the translation which is not present. We will ask the authors for the needed translations.

How can a CT-Scan be anonymized?

There are two different programmes to do it. Remember you cannot send any medical record or personal data.

• DICOM Anonymizer: https://sourceforge.net/projects/dicomanonymizer/ (it does not remove patient’s birthdate)
• FP IMAGE FOR WINDOWS: http://www.fpimage.com/ (use the option ‘Dicom Browser’).

Hospital stays form.

All hospital stays should be registered from 3 months prior to inclusion to 12 months after liver transplantation or end of the study. Each change of site (from ICU to regular ward for example) including discharge periods should be registered individually.

Is there a time gap to process the samples? What if the liver transplant surgery occurs at night or at weekends?

There is no established time gap. The best option is to process the samples immediately after collection but we know that this is really difficult in clinical practice.

That’s why in the eCRF there are variables asking about the time gaps between collection and processing and between processing and storage for every type of sample.

Is it mandatory to have a barcode scan to process the samples?

No, it is not necessary to use a barcode scan to process the samples. If the wilmut tubes are not moved from the initial position and not mixed within the plate or with other plates, the registry of the samples is really easy in the eCRF biobank form. Remember that each type of sample is stored in the same positions in the plates.

Which materials are not supplied?

The not supplied materials are: fetal calf serum (FCS), DMSO, DPBS without calcium and magnesium (DPBS-/-), isopropanol and the freezing container. All these reagents are used only in PBMCs isolation. Please purchase the materials and send the invoice back to the EF-Clif.

We are not familiar with the cell culture and the aseptic technique.

Here you can find some useful videos. Please contact us if you need more information.

https://www.youtube.com/watch?v=5Dqtp1B5j0g

https://www.youtube.com/watch?v=nr1tV_LuqJk

In the videos it is not said, but please close the reagents with parafilm (https://www.dilabsa.com/es/parafilm/) before taking them from the hood. Also remember to switch on the UV light 20 minutes before and after using the cabin.

How to aliquot Fetal Calf Serum?

It should be thawed slowly in the fridge. Please do aliquots (in the cabin using the aseptic technique and putting parafilm in the taps of the resulting aliquots) and store them again at -20ºC. In this way you will only have 1 aliquot at the same time in the fridge at 4ºC. In this way, if one aliquot becomes contaminated, the rest of them continue unaltered.

Which type of sample should be taken from the liver?

You need to take a needle biopsy.

How to process the liver biopsy?

A. About the frozen aliquot:

The handbook related to the reagent can be downloaded from:
https://www.qiagen.com/it/resources/resourcedetail?id=856b0ac5-eb56-4faa-915d-88221ee4c0bc&lang=en

Please take it into account when calculating the volume of reagent needed depending on the size of the biopsy.

About the processing, please incubate the tissue with the reagent overnight and afterwards remove it from the reagent and store at -80ºC.

5. Store the tissue submerged in Allprotect TIssue Reagent for up to 6 months at 2-8ºC, up to 7 days at 15-25ºC, or up to 1 day at 37ºC.
For archival storage at -20ºC, first incubate the tissue overnight in the reagent at 2-8ºC. Then transfer the tissue, in the reagent, to -20ºC for storage.
For archival storage at -80ºC, first incubate the tissue overnight in the reagent at 2-8ºC. Then remove the tissue from the reagent, and transfer it to -80ºC for storage.

Note: Lower temperatures are recommended for longer storage (e.g., 2-8ºC instead of room temperature or 37ºC; or -20ºC or -80ºC for longer storage). If analyzing proteins using downstream applications more sensitive than SDS-PAGE

B. About the paraffin block:

Please contact your histopathological department to process this aliquot after surgery. Remember you have to label this aliquot yourselves with only the patient code.

If it is put in formaldehyde before processing, follow the classical way of processing biopsies in your histopathological department. Classically, it’s 24 hours (it’s not strict) before putting this in the paraffin block. The variability among different centers is the reason why it is not described in the protocol.