When to draw blood samples? 

• Donor’s suitability (anamnesis, questionnaire)
• Drawing blood samples at the same time as a donation or within and not further than the following 7 days
• Avoiding drawing blood after or during hemodilution as it could cause false negative tests

How to draw blood samples?

• Draw two test tubes of whole blood (vacutainer purple top), add EDTA anticoagulant and swirl gently. These samples are going to be sent to the Musculoskeletal Tissue Bank BTM IOR
• DO NOT use heparin as an anticoagulant because it can inhibit PCR test results
• Send the blood samples promptly to the laboratory taking care of fractioning and storage of serums
• You can carry out first-level screening on the samples following your iter, however, it MUST be carried out at the same time as the donation or within and not further than the following 7 days. If for any reason, you are unable to respect the abovementioned procedure, our personnel can carry out the screening on your behalf once the plasma and the frozen femoral heads will be delivered to our laboratory

Microbiology Screening

In the case of a positive test result for any of the following, the subject will not be suitable for donation, and the tissue will not be utilised for transplantation purposes:

• Anti-Hepatitis B Surface Antigen (HBsAg)
• Anti-Hepatitis B core antibodies (HBcAb)
• Anti-Hepatitis C Virus antibodies (anti-HCV)
• Anti-HIV 1 & 2 antibodies (anti-HIV 1 and 2)
• TPHA or other tests resulting positive for anti-treponema antibodies
• PCR test resulting positive for SARS CoV-2
• Any other additional testing issued by the National Transplant Centre

Please note: A positive test for HBcAb is not an exclusion factor. A negative HBV DNA will be needed to certify the suitability for donation and it can be verified by the Musculoskeletal Tissue Bank BTM IOR.

Stability of the samples

The stability of samples is influenced by high temperatures. Furthermore, plasma samples are generally subjected to molecular biology methods that are easily a source of contamination. Here are the guidelines to avoid contamination:

• Separate the plasma within 6 hours from the drawing of blood samples. It is however possible to store the whole blood samples for no further than 6 hours between + 2° and + 25° C and for no further than 72 hours between 0° and +4° C
• Whole blood samples must not be frozen
• Once the plasma has been separated it can be stored for over 72 hours between + 2° and + 8° C (both before freezing or after thawing)
• For longer periods it must be kept at -70° or -80° C in sterile test tubes with a polypropylene top.
• Plasma must not undergo more than 2 freeze-and-thaw cycles

Blood fractionation

• Spin at 3000rpm for 20 minutes
• Mandatory use of sterile gloves to avoid contamination which could compromise NAT assays 
• Mandatory use of sterile tools (cryovials test tubes and tips)
• Once opened, the tools must be kept clean; keep the cryovial and tip holder bags closed; pipettes must be handled only with gloves and must be kept clean.
• Perform the fractionation in a dedicated area. Surfaces must be kept clean and protected by tissue paper
• Prepare 4 cryovial test tubes: two 5ml tubes and two 3ml tubes, keep them closed and apply the label with the donor’s unique reference number.
• Open only the test tubes of the samples you want to fractionate
• Open only the test tubes at the moment of fractionation
• By using the designated filtered pipette tips, fractionate the plasma in the 4 cryovial test tubes as follows: aliquot at least 2ml of plasma in the two 5ml cryovial test tubes and put the leftover plasma in the other two 3ml cryovial test tubes. Please note that each fractionated plasma sample should come from both the primary test tubes
• In the case in which you are working with samples from different donors, it is preferable to open only the test tubes of the sample you are fractionating to avoid mix-ups. You can do so by checking the unique reference numbers and details of the donors on the samples and the primary test tubes
• Do not draw the particulate matter as it will contaminate the plasma
• Signal the presence of hemolysis or lipemic samples
• Utilise only cryovial test tubes with a screw-in external top. The test tubes with a screw-in internal top can cause serum overflow at the moment of thawing. We recommend a cryovial test tube with a screw-in external polypropylene top, sizes 5ml and 3ml. Please note that the Musculoskeletal Tissue Bank BTM IOR is available to supply the test tubes upon request

Labelling 

• Do not use labels that tend to come off during thawing, nor labels too thick as they might increase the dimension of the test tube, which will not fit in the designated cryopreservation trays. This would force personnel to attempt the removal of the label and therefore risking damaging it and making it possibly difficult to identify the sample
• Do not use labels that are too large as the sight of the plasma inside the test tube must be visible at all times
• Write on the labels only with water-resistant pens
• Give standard operative instructions with an accurate personal data check to avoid pre-analytical errors

Sending Us the Samples

• You must make sure to maintain the cold chain during transportation
• Transportation for long distances, especially in the hottest times of the year, requires the use of dry ice
• You must guarantee the delivery of the sample to us WITHIN A MONTH from the date of the drawing, so as not to compromise the NAT assays