# Why are COVID nucleic acid tests sometimes reported with non-integer CT values?

I saw a positive COVID test, and the CT value was not reported as an integer value. Instead, it was reported with two decimal point precision. I expected the CT value to be the number of PCR cycles.

I searched many combinations of "PCR", "CT", "integer" and "decimals" and I found many resources that explain the basics of PCR and how the integer CT is derived. However, I am still unclear on the decimal precision.

How the decimals are obtained, possibly some normalization?

• Welcome to Medical Sciences! Please take the tour and read the help center. For reasons mentioned in this post and in How to Ask, we require questions to demonstrate some degree of prior research. See this list of helpful resources. Please help us to help you and edit your question to provide more information on what you have read on this subject, what made you ask this question, and any problems you are having understanding your research. If you found nothing, what did you Google? Mar 19, 2022 at 22:50
• I googled many combinations of PCR, CT, integer and decimals in three languages and I found many ressources that explain the basics of PCR and how the integer CT is derived. My assumption that there is some normalization is based on the indication of the report that the CT value is calculated as a quotient of two unspecified quantities. Mar 20, 2022 at 9:45
• @user11235 I would also expect the Ct value to be an integer. Maybe they did a fit of the RT-PCR signal and estimated where it would intersect the threshold? This way, you could get a non-integer value. However, I'm afraid the best chance is to probably call the test provider if you have contact details and ask them about it. Could be average of multiple tests (but this is not done standardly), could be the mathematically predicted intersection I mentioned (also not done standardly). Mar 20, 2022 at 12:18

## 1 Answer

Viral nucleic acid amplification tests use real time PCR to detect viral genomic material. If the genetic material is present, a reaction will occur that generates fluorescence. If sample produces a reaction that reaches a specified fluorescence "threshold" by a predetermined cycle, it will be deemed positive.

The cycle at which a reaction reaches the threshold, is the cycle threshold (CT) value.

If we plot the fluorescent signal generated during the reaction against the number of cycles, it generally follows a sigmoid curve.

Consider this hypothetical plot: Source: Ian Campbell

As we can see, the black sample fluorescence line crosses the orange threshold line during the 20th cycle. It doesn't happen at the exact beginning of the cycle, but about two thirds of the way through it.

In this case the CT value (dashed blue line) is 20.63.

The reality is that the precision of this value is meaningless, as variability in sample preparation and stochasticity lead to much more difference than a 100th of a cycle.

For more precision, dilution experiments can be performed, but these aren't really necessary / feasible in a clinical testing scenario. See this guide from a real-time PCR thermocycler vendor for more.