The BQC19’s primary mission is to give the scientific community access to biological samples, and clinical and experimental data, from individuals having developed COVID-19 and control subjects.
The BQC19 has established laboratory procedures for the collection of biological samples; these have been adhered to at each of its participating institutions. You will find the procedures set out in detail here.
The minimum required in blood sampling is one ACD (acid citrate dextrose) tube of plasma and one PAXgene© tube. In certain cases, only one plasma ACD tube could be collected (limited set).
An optimal collection of samples includes:
One PAXgene© tube;
Three ACD tubes with samples;
One red top tube or SST tube (Serum Separation Tubes).
The tubes must be kept at room temperature prior to processing. The ideal timeframe for processing is less than six hours between the drawing of blood and the processing of the sample. A timeframe of less than 12 hours is acceptable for most analyses. If the time elapsed is more than 12 hours, a significant number of analyses will become more difficult to execute or less reliable. The processing time is noted on worksheets and also noted in the database that compiles the inventories.
Biological samples collected
The BQC19 can provide several types of biological samples from blood collected:
RNA extract from PAXgene© tube;
DNA extract from whole blood on ACD;
Serum, from red top tubes or SST tubes;
Plasma on ACD;
Peripheral Blood Mononuclear Cells (PBMCs) from plasma on ACD;
Other biological samples, in a very limited and occasional manner (EDTA plasma or heparin plasma) drawn from remainders of clinical samples;
Pediatric samples: The pediatric Management Framework allows for the collection of other, more specific, types of samples in very limited quantities.
Biological samples are distributed according to the proportions established in the Management Framework relative to provincial collection and local collection by participating institutions.
The BQC19 requires the transmission of results of analyses carried out using these samples, so as to be able to add them to its collection of data, and subsequently disseminate them to the scientific community.
Whole blood is collected in PAXgene© tubes; 2.5 ml of whole blood is collected in an environment that is conducive to the preservation of RNA. Other than for storage purposes, there is no handling of the tubes.
They are kept for 24 h at room temperature, then transferred for 24 h to a -20 ºC environment prior to being kept at -80 ºC for long-term storage until extraction.
RNA is extracted from PAXgene© tubes in accordance with QIAGEN procedures. The volume of eluate collected is 80 µl with an average concentration of 115.7 ng/µl.
The BQC19 is performing transcriptomic analyses, and these will be available via access to the BQC19’s data. The data will be enhanced over time with the addition of new results.
RNA extracts will be made available to the scientific community. Access to these extracts shall be requested through a sample access request. Samples will be provided as a total quantity of RNA in ng according to the needs of the researcher and the quantities available.
The collection of blood drawn for the purposes of DNA extraction is performed using ACD collection tubes. Collection for DNA analyses is only carried out once during the study, that is, when the participant is recruited.
The whole blood in all the tubes is transferred into one tube so that it may be combined. Two 0.5 ml samples are collected in cryopreservation tubes for DNA extraction. These two aliquots are then transferred for storage at -80 ºC. The remainder of the whole blood is used for plasma collection.
The samples are then conveyed to the McGill Genome Centre for DNA extraction.
The extraction is carried out using 0.4 ml of whole blood. It is performed using a Perkin Elmer Chemagic method with a CMG-1091 kit. The final elution volume is 200 µl.
The available quantities of DNA have an average concentration of de 43.51 ng / µl, with an average quantity of 8.198 ng.
The BQC19 is carrying out genomic analyses. The results will be available though access to data, and will be enhanced with the addition of supplementary genomic data.
DNA extracts will also be made available to the scientific community. Access to the extracts shall be obtained through a request for access to samples. Samples will be provided as a total quantity of DNA in ng according to the needs of the researcher and the quantities available.
Plasma is collected with ACD tubes. This method is favoured as it produces a greater yield of PBMCs. The BQC19 also collects occasional plasma specimens on heparin and on EDTA. Quantities of these are limited. They come from remaining clinical specimens that the BQC has been able to bank.
All plasma is handled according to the same protocol. The tube of whole blood is centrifuged at 850 g for 10 minutes at room temperature, with no braking. The plasma is then aspirated, avoiding the aspiration of the interface of the white cells between the plasma and the packed red blood cells.
The plasma, in aliquots of 0.25 et 0.5 ml, is then transferred into cryopreservation tubes of 1.5 to 2 ml. If the total volume collected is significant, 1 ml aliquots can be constituted. The samples are then transferred to a -80 ºC environment for long-term storage.
The number of aliquots and the volume will vary and will depend on the clinical situation of those participants who have developed a severe form of COVID-19. The total volume will range from 4.5 ml to 15 ml. The number of aliquots collected during follow-ups is less variable and their total volume is greater.
The BQC19 is carrying out proteomic, metabolomic and immuno-serological analyses as well as basic laboratory analyses; the results will be made available through access to data. These results will be enhanced from time to time as new analytical results are added.
The plasmas will be placed at the disposal of the scientific community. Access to the samples shall be obtained through a sample access request. Plasma will be provided in aliquots of 0.25 ml, 0.5 ml or 1 ml according to the needs of the researcher and the quantities available.
Peripheral Blood Mononuclear Cells (PBMCs)
PBMCs are collected following the centrifugation carried out for plasma collection. Two methods can be used, the Ficoll-Hypaque method or the alternative SEPMATE method. You will find a detailed explanation of the handling required for PBMC collection here.
Once the PBMCs have been isolated, they are frozen in an FBS 20% DMSO solution in 1 to 2 ml cryopreservation NALGENE tubes. The final concentration is adjusted to 10 million cells per ml. The tubes are transferred to a -80 ºC environment in a "Mr. Frosty" freezing container for 24 h, then transferred to liquid nitrogen tanks for long-term storage.
The possibilities for collection of PBMCs vary. The median number of aliquots is two, with one set aside for provincial collection efforts.
The distribution of the total quantity of PBMCs (in millions of cells) at the BQC19 is as follows (including local collection):
Access to the samples shall be obtained through a sample access request. PMBCs will be provided in aliquots of approximately 10 million cells per ml, according to the needs of the researcher and the quantities available.
The BQC19 has no serum samples for severely ill participants from the first wave of the COVID-19 pandemic. Serum can be collected with SST tubes (containing a clot activator and a gel separator) or with red top tubes. The volume of the collection tubes ranges from 4 to 10 ml, depending on the type of tube used.
The same protocol is used for the handling of all serum tubes. The tube of whole blood is centrifuged at 2000 g for 10 minutes at room temperature with braking.
The serum is then transferred in aliquots of 0.25 and 0.5 ml into cryopreservation tubes of 1.5 to 2 ml. If the total volume collected is significant, 1 ml aliquots can be constituted. The samples are then transferred to a -80 ºC environment for long-term storage.
The number of aliquots and the volume will vary; they will depend on the clinical situation of participants who have developed severe illness. The number of aliquots collected during follow-ups is less variable and their total volume is greater.
Access to the samples shall be obtained through a sample access request. Serum will be provided in aliquots of 0.25 ml, 0.5 ml or 1 ml, according to the needs of the researcher and the quantities available.