Elevation of viral load by PCR and use of plasma preparation tubes for quantification of human immunodeficiency virus type 1

It has become very common to quantify human immunodeficiency virus type-1 and PCR Tubes are also extensively used to regularly monitor the general response to antiretroviral drugs. 

Extremely sensitive viral loads are also used in the HIV-1 and RNA present in peripheral areas of the human blood. 

What is the role of frozen plasma in quantification?

Besides the essential quantification role played by strip PCR tubes, studies have also reported the essential role of PPTs or Vacutainer plasma preparation tubes. At the time PPT was being used by the scientists working at the laboratory, an undetectable viral load was discovered. It was also when alongside the thin-wall PCR  tubes, plasma preparation tubes were stored at around 70 degree Celsius. 

Despite the dropping number of undetectable viral loads and thus the analysis, the results depicted by control analysis were fortunately satisfactory.  With everything given,  scientists diverted their focus on investigating the analytical variables which could explain the descent of HIV-1 and a load of undetectable viral particles.

Here, the viral load testing was put parallel to each other by putting them in secured specimens. This simultaneously processed and then stored the data in primary PPTs or plasma in situ. Furthermore, the data was stored in PPT sample aliquots which are later transferred into the secondary standard of transport tube before freezing.

Where and how plasma samples were tested?

The plasma samples taken for the test were subjected to experimentation. For doing so, plasma samples were taken from the patients infected with HIV type-1. The samples were taken from the patients admitted to La Paz University Hospital, Madrid Spain.

For paired plasma sampling, scientists compared the viral load obtained for as many as 51 patients.

The 51 PPT sample aliquots were placed inside the secondary tubes before they were put away for freezing. 5 millilitres of blood from every patient is collected in PPTs by a standard venipuncture. After this is done, the samples are maintained at normal room temperature until it is centrifuged at around 1100×g for at least 10 minutes of a couple of hours of collection.

This entire process followed by 1.5 mL of plasma aliquotation in the secondary tube.  These plasma samples are then stored at around -70 degree Celsius until everything starts thawing at room temperature. For a more sensitive assay, plasma is subjected to intense centrifugation for one complete hour. As a result, the sediment of the virus is produced and the supernatant plasma is eliminated or discarded. The viral pellet is then lysed, precipitated, extracted and then redissolved. 

More about the plasma extraction

During the above-mentioned experiment, the scientists divided the patients into three major subgroups. It was primarily done based on viral load results. 

The net HIV type-1 viral load was in the ratio of 3.1 log copies per millimetre. A difference as significant as this was of great importance in terms of statistics.

The total percentage of undetectable viral load in aliquoted plasma was thought to be around 21.6%.

What does the comparison of HIV type 1 reveal?

There were a total of 11 patients with HIV type-1 load. That was when the PPTs were being aliquoted before freezing. 

However, when the scientists started to use PPTs for every patient, amazingly it was nobody among patients had shown any undetectable viral load. 

Interestingly, after the scientists froze the samples, the viral load was shown to reach a much higher rate. Thus a huge proportion of some primary PPTS was compared to the corresponding PPT aliquots. 

This whole experimental and comparison exercise confirmed that by using the primary PPTs to transfer the frozen plasma samples, the HIV viral load increases to around 50 copies.

The influence of this high variability at such a low end of the assay is extremely imperative because it helps to interpret the transition or changes in the viral loads under the clinical practice. 

Success in these treatment cases is predominantly based on the viral load response. This response further dictates the decisions about therapy. 

The recent studies by scientists have depicted a high increase of HIV and RNA samples and it is compiled in EDTA tubes as compared to standard PPT tubes.

Bibliography

1. García Bujalance, Guevara, L. d., J. González-García, J.R. Arribas, F. Zamora, & Department of Microbiology and Parasitology, La Paz University Hospital, Paseo de la Castellana 261, 28046 Madrid, Spain b Department of Internal Medicine, La Paz University Hospital, Paseo de la Castellana 261, 28046 Madrid, Spain. (2007, Feburary 7). Elevation of viral load by PCR and use of plasma preparation tubes for quantification of human immunodeficiency virus type 1. Science Direct. 

https://sci-hub.do/https://pubmed.ncbi.nlm.nih.gov/17363096/