During testing, laboratories are increasingly aware that proper collection, preservation, and transport of samples is an important factor in the success of test results. During the testing process, there may be questions about blood samples: whole blood, serum, and plasma, which sample is more suitable for pathogen nucleic acid detection
First, let's look at how serum, plasma, and whole blood are prepared.
Whole Blood:All blood treated with anticoagulation is whole blood, i.e. it includes all components of the blood.
Plasma:Anticoagulants are added to the blood, and the yellowish supernatant obtained after centrifugation to remove blood cells is plasma.
Serum:The blood is not treated with anticoagulation, allowing it to clot on its own, and after a period of time to sit or centrifuge with a centrifuge, the coagulated part of the blood separates from some clear, yellowish liquid called serum.
The main difference between serum and plasma is that plasma contains fibrin and clotting factors, while serum does not.
Trivia:The use of plasma samples for PCR testing requires the use of EDTA anticoagulant tubes, and the use of heparin anticoagulant tubes is strictly prohibited. Because heparin is a strong inhibitor of Taq enzyme and it is difficult to remove it all during nucleic acid extraction, heparin anticoagulants can severely inhibit PCR amplification and may lead to detection failure.
Selection of blood samples
Normally the genomic DNA of pigs is present in white blood cells, with a portion of free DNA in non-cellular components.
The pathogen that causes the disease of pigs can be detected in blood samples when viremia or bacteremia appears, in this case, the pathogenic pathogen is present in a large number of non-cellular components in the blood, and the pathogen can be detected by testing blood samples.
Some viruses can replicate in white blood cells, while others are captured by immune cells that swim in the bloodstream. Theoretically, the leukocyte + plasma test can achieve higher sensitivity. However, the sensitivity of whole blood samples is not very good.
Whole blood vs plasma
Serum and plasma are cleaner than whole blood, which can improve the purity and yield of nucleic acid samples. In addition, there are hemoglobin and other interferences that seriously affect the PCR reaction in whole blood, which affects PCR amplification and leads to a decrease in detection efficiency.
In order to verify the above hypothesis, 6 whole blood samples were taken, some of which were used as whole blood samples, and the other part was centrifuged and the supernatant was taken as plasma samples, and nucleic acid extraction was carried out at the same time, and PCR detection was performed simultaneously, and the test results were as follows:
According to the experimental results, for the same sample, the plasma sample was 298-4.52 ct values,Indicates that plasma is a more appropriate sample type than whole blood, but which one is more appropriate for plasma or serum samples?
Serum vs plasma
In the process of serum separation and precipitation, a large number of viruses will be adsorbed by fibrin and coagulation factors, and precipitated during centrifugation, losing a part of the virus. Plasma samples reduce the loss of this part and are more suitable for the detection of pathogenic nucleic acids in blood samples by fluorescence quantitative PCR.
Ahmed Elnagar, a researcher at the Friedrich-Loeffler Institute in Germany, compared the real-time fluorescence PCR detection of EDTA blood, serum, oral swabs, spleen tissue homogenates, and bite ropes collected at different time points after non-plague infection, and analyzed the best sample materials for the accurate diagnosis of ASFV by real-time fluorescence PCR method. As can be seen from the results, EDTA anticoagulant blood has a lower CT value.
Considering the sensitivity of detection,EDTA anticoagulant (plasma) is the best sample type for non-plague.
For details, see EDTA anticoagulant blood is the best sample material for real-time fluorescence PCR detection of African swine fever virus (ASFV) (click on the blue text to view the original article).
Conclusion
Therefore, for pathogen detection, in order to improve the sensitivity of detection, when testing blood samples, using plasma samples as the samples to be tested can significantly improve the detection sensitivity of pathogens and reduce the probability of missed detection.