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THE PRIMER :: MOLECULAR DIAGNOSTICS


has just gone up by a factor of two, say from 200 U/ml to 400 U/ml. Usually with a qualita- tive IC, there will be some accepted rules or guidelines as


to what sort of a shift in IC CT (CP


) is allowed before all analyte


results are called into question or discarded as not reportable. For the mathematically inclined, it’s interesting to con- sider how a drop in per-cycle PCR efficiency relates to loss of overall sensitivity. Recall that an ideal PCR is a doubling (x2) per cycle, the theoretical yield of PCR product for N thermo- cycles is 2^N. Inhibition acts to reduce 2 to a smaller num- ber, say 1.8. That’s only a 10 percent loss in efficiency, but the exponential nature of the process amplifies that error. Using some hard numbers, say 35 cycles, a perfect PCR would yield 2.44e10 products, while the 10 percent inhibited assay only yields 1.27e9 products—or about a 19 fold loss. If this were our above example with an ideal LOD of 200 U/ml, it’s now 3843 U/ml.


Figure 1. Theoretical PCR yield vs per-cycle efficiency Legend. Theoretical amplicon yields at 35 cyles vs per-cycle PCR efficiency, at 35 cycles.


In either context, the bottom line is that there’s potential for there to be some degradation of assay per- formance before it’s going to fully supress internal control signals, and this is a window of opportunity for false negative results.


Reaction volumes—size matters There was a promise above that we’d return to the issue of sample size. In this context we mean “amount of template/extract volume put in the reaction,” as opposed to the size of the original specimen. I was recently approached by the sales representa- tive for an unnamed company, who proceeded to wax lyrical about their new PCR platform with 1.2 µl reac-µl reac-


reac-


tion volumes; not nearly as tiny as digital PCR range, but certainly smaller than reactions volumes on most common platforms. If we take that to be around 25 µl—make it 24 µl for simplicity—it’s 20 times smaller.


Of course, there are some real advantages to this. Per reaction reagents cost is lowered, and ther- mal mass is smaller, meaning shorter dwell times needed in thermo- cycling, and concurrent faster whole reaction times. However, all other


things being equal—including ratio of template to PCR reagents per reaction—then using this platform one can only put 1/20 as much extract in the reaction as you could in a 24 µl reaction and simplistically, we’ve just raised our LOD by 20x as compared to running the larger vol- ume reaction. In the early days of real-time PCR, there were even such things as 100 µl reactions which would allow for sampling ~80x more input material per reaction. This particular line of logic is mostly pertinent to the LOD value observed in the assay validation, and where the balance between reagent cost savings and required LOD lies. For what are expected to be high copy number analytes, small reaction volumes can be great—but apply- ing them if low range sensitivity is required may be counterproductive. Like most fields, different tools are better suited for different jobs.


Sequence variation Finally, what about target genetic variability? MDx assay developers go to great lengths to try to find ideal, well conserved/consensus primer binding sites. These help ensure that biological pressure on the target analyte/organism limits variation off from these sequences but it’s not an absolute guarantee. Especially if the analyte is an emerging organism, one for which there are necessarily limited example sequences to base primer design on, it’s within the


realm of possibility that you could encounter an organism with critical sequence variations under primer and/or probe binding sites. Depend- ing on where and what these nucleo- tide changes are, they may cause very significant losses in assay sensitivity and lead to false negative results.


Absence of proof is not proof of absence—traditional aphorism Fortunately, the single most likely reason for a well designed, well vali- dated, and properly run MDx assay to return a negative result is that the sample doesn’t contain the analyte in question. All of the above however should serve to remind the end user of any assay that while rare, false negatives can and do occur. In the MDx context we considered there are a number of factors which can contribute to this. All of this goes to underline the importance of inter- preting lab results in real context and being ready to question them if they really don’t seem to fit. In such cases retesting or use of an alternate secondary assay may be wise.


John Brunstein, PhD, serves as an Editorial Advisory Board member for MLO. John is also President and CEO for British Columbia-based PathoID, Inc., which provides consulting for development and validation of molecular assays.


MLO-ONLINE.COM MARCH 2019 39


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