Blood samples collected for analysis in a clinical laboratory in Hazebrouck (North) -
© Denis Charlet / AFP
Our partner The Conversation opens the doors to the molecular biology platform dedicated to the detection of the SARS-CoV-2 virus at the Rouen University Hospital Center.
It details the typical day of the lab, from the routine of sampling to analyzes, then to their interpretation and the reporting of results.
This report was conducted by Elodie Alessandri-Gradt, biologist, hospital practitioner in virology at Rouen University Hospital, Anaïs Soares, biologist, and Marie Gueudin, biologist (all three at the University of Rouen Normandie).
At the end of March 2020, as the first wave of the Covid-19 epidemic hit our country, the Ministry of Solidarity and Health officially established the establishment in several French cities of molecular biology platforms dedicated to detection of the SARS-CoV-2 virus by
polymerase chain reaction
(PCR), or
polymerase chain reaction
.
Élodie Alessandri Gradt, Anaïs Soares and Marie Gueudin open the doors to the one that was set up at the University Hospital of Rouen.
In one month, a laboratory was set up to take charge of thousands of PCR tests at the end of the first confinement.
Here's how a typical day unfolds.
6 a.m .: top start
The first team arrives, made up of six technicians and two administrative agents.
The samples to be analyzed are brought to the laboratory in an airtight packaging which allows secure transport, even in the event of an unexpected leak.
These are plastic tubes that contain a transport solution in which a swab is immersed.
This is used to collect, from the patient's nasopharynx, a sample of respiratory cells that may harbor the SARS-CoV-2 virus.
The genome of the latter being very fragile, the transport solution prevents degradation for several hours.
From there, the samples follow a precise circuit before the result is returned to the patient.
The initial step, called “pre-analytical”, consists in checking the conformity of the sample and making the result more reliable.
It is in particular imperative to ensure that the sample was collected in a container whose transport medium complies with the performance of the SARS-CoV-2 PCR, that the delivery times are acceptable to avoid too much degradation. important, or that the mandatory information on the sample and the patient's identity be completed.
Once validated, the sample is recorded in the laboratory's computer system to ensure its traceability.
7 am: start of analyzes
The sample goes into the first technical room.
Its packaging is opened under a microbiological safety station which ensures the protection of the handler.
The outer surface of the tube is then sprayed with decontaminating solution for five minutes, again to ensure the safety of the operator during the following steps.
Then the sample is heated at 56 ° C for 30 minutes to inactivate the virus it potentially contains.
When it leaves the room, it no longer presents any infectious risk for operators.
8 am: the robots come into action
The plates used for the PCR contain 96 wells © E. Alessandri-Gradt, A. Soares & M. Gueudin (via The Conversation)
Change of room.
The tubes are inserted in sets of 93 into a robotic pipette, which transfers the samples to a test plate pierced with 96 microwells.
The three empty wells receive control samples allowing the follow-up of the entire analysis process.
The plate is then placed in a second robot which separates the viral RNA from the other components of the sample.
Since SARS-CoV2 is an RNA virus, a step of converting RNA into DNA, known as reverse transcription (RT), is carried out by an enzyme upstream of the PCR.
We therefore speak more precisely of “RT-PCR” to designate the entire process.
(
N
b
: RNA - ribonucleic acid for - are molecules whose chemical structure is similar to that of DNA molecules - deoxyribonucleic acid consists of a single strand, the RNA molecule is more fragile than that of. DNA
)
A reaction mixture (mix) necessary for the polymerase chain reaction (PCR) is added.
PCR is the amplification of a target gene fragment using heat-sensitive enzymes.
Concretely, short sequences of nucleotides, the elementary “bricks” constituting the viral genome, are attached to the target gene by complementarity.
These “primers” are then completed by the addition (polymerization) of free nucleotides using an enzyme, Taq polymerase.
Schematic representation of the polymerase chain reaction (PCR) which amplifies the amount of genetic material present in a sample © Ygonaar / Wikimedia CC BY-SA
The extractor-distributor robot can handle two test plates at the same time, i.e. 192 independent reactions in 80 min.
At the height of activity, it operates without interruption.
10am: last analytical step
The plate is transferred to the last technical room, that of the thermal cyclers.
These devices alternate cycles of different temperatures allowing the enzymes to perform the PCR in 2 hours and 15 minutes and to detect a fluorescent signal in real time.
This is all the more intense as the viral RNA is amplified.
Three targets are specifically sought, two of which are in the viral genome.
The third is present in a reagent added to each sample.
Amplified even if the sample is negative, it is used to verify that the PCR worked correctly, to ensure that a negative result is not due to a problem during the reaction.
Amplification curve of a PCR reaction © E. Alessandri-Gradt, A. Soares & M. Gueudin (via The Conversation)
The appearance of fluorescence after a certain number of cycles, according to an exponential curve, indicates the positivity of the sample.
We speak of Ct of the sample (
cycle threshold
or cycle threshold): a sample very loaded with viral RNA will require few PCR cycles to detect a positive signal (it will have a low number of Ct).
Conversely, a sample with a low amount of viral RNA will induce a high Ct.
12h: interpretation and rendering of the result
This is the end of the analysis.
The technicians check that the technical validation criteria are met before depositing the results on the laboratory's computer server.
A biologist then intervenes to interpret all the results and validate the final interpretation, then the patient can consult his result on a secure server.
Either the two viral targets are amplified, in which case there is the presence of SARS-CoV-2 RNA, or only the non-viral target is amplified, and it can be concluded that there is no detection of viral RNA.
Our “Coronavirus” dossier
Certain results sometimes lead the biologist to carry out checks, the amplification of only one of the two viral targets, for example, the detection of a very weak signal or the failure of amplification of the non-viral target.
The sample concerned then undergoes a new cycle of analyzes.
Unfortunately, it is sometimes necessary to take a new sample to obtain a definitive result.
The biologist is also in charge of communicating with doctors and patients to obtain additional clinical information to better identify certain files and discuss the complexity of the results.
He is subject at all times to professional secrecy with regard to the results of the test.
12-2pm: it's the break!
But not for analysis.
A constant flow of samples should be maintained at the various technical stations while the staff take turns having lunch.
Everyone is responsible for ensuring strict compliance with hygiene instructions, the timing of the meal being a critical event in terms of the risk of contamination.
Many samples are waiting to be analyzed © Laurie Boquier / Rouen University Hospital (via The Conversation)
2 pm-3pm: rotation of technical teams
It's the end of the day for the first team, which passes the baton and transmits the important information of the day to the second team.
It is also the privileged moment to discuss with the whole team new instructions and any difficulties encountered.
4 p.m .: behind the scenes
The accreditation of the 24 laboratory technicians is provided by two engineers, during specialized training.
These engineers are also responsible for solving all the technical problems which do not fail to arise, such as the permanent adjustments of the robots, to be adjusted according to the shortages of consumables which oblige to adapt to the available references.
Finally, they ensure, with the help of a logistician, that the laboratory stocks are sufficient to avoid such a breakdown on a critical consumable.
A health manager ensures the management of human resources for non-medical personnel: hiring and development of the schedule in correspondence with the fluctuating activity of the laboratory, assessment of acquired skills, compliance with regulations (number of compulsory hours of rest, number of Sundays worked…).
Course of a day in a Covid laboratory © E. Alessandri-Gradt, A. Soares & M. Gueudin (via The Conversation)
Biologist and virologist, the manager of the platform ensures the link between the different laboratory staff, as well as with partner establishments.
He trains the team of biologists, takes decisions on possible critical problems and tries to anticipate the extremely variable flows of samples.
17-22h: continuous flow
At this time, many samples are flowing to the platform.
The team therefore remains fully mobilized to ensure their support as soon as possible.
For some, the remoteness of the sampling site (sometimes more than 100 kilometers) can impact the delay in rendering the result.
Analysis statistics of the Covid laboratory at Rouen University Hospital © E. Alessandri-Gradt, A. Soares & M. Gueudin (via The Conversation)
The series of PCR analyzes follow one another in continuous flow during the evening.
Some automated steps will even continue throughout the night.
The maximum analysis rate is 186 results every two hours, or up to 1,800 results per day, which implies optimal functioning at each stage of treatment.
More than 173,000 samples have thus been analyzed by the COVID platform of the Rouen University Hospital since May 2020.
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This analysis was written by Elodie Alessandri-Gradt, biologist, hospital practitioner in virology at Rouen University Hospital, Anaïs Soares, biologist, and Marie Gueudin, biologist (all three at the University of Rouen Normandy).
The original article was published on The Conversation website.
PCR remains the benchmark for COVID diagnosis.
The establishment and operation of a COVID laboratory on French territory represent an unprecedented challenge for all the teams concerned.
From taking the sample to rendering the result of the PCR analysis, the reliability of the diagnosis relies on the accuracy of the successive sample processing steps.
The logistics required for PCR analysis remain complex despite its automation.
However, despite the arrival of antigenic tests on the market which has enhanced the virus detection offer, this test remains to this day the reference diagnostic test for infection by SARS-CoV-2.
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