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The Truth (and definition) of Pooling Samples

There are many labs across the country that will pool samples, or offer to pool samples together in order to decrease the cost of testing. This is popular with PCR testing and on-site test mechanisms. Seems like a no brainer, right? Save money while finding PI’s. I’m in!

Not so fast, though. While pooling samples may save you money up front, it can greatly decrease accuracy of the test. In this article, I will dive into what pooling is exactly and how it effects your test.

Before we get started, I want to point out the differences between PCR and on-site tests (ACE tests). PCR is a very sensitive test that replicates strands of DNA. It is an extremely accurate test if used correctly, but costs anywhere from 3 – 10x as much as ACE. On the other hand, ACE tests capture the virus that is already present within a sample. ACE tests are also extremely accurate if ran correctly but more cost effective. We only use ACE in the CST lab.

Pooling is where you use one single test for multiple samples at one time. Instead of using 1 mL of solution per animal, you use 0.125 mL’s of 8 animals to equal the same amount of solution required to run the test. Here is a picture to help visualize.

As you can see, Sample #1 is positive for BVD-PI. Now, look at the amount of virus particles (black dots). When you test as a single sample, you have more than an adequate amount of BVD virus particles to ensure the test comes out correctly but when you pool samples the number of virus particles is greatly decreased. There have been two different studies that have shown dilution of a sample can cause missed PI animals, one by Dr. Julia Ridpath and one by Dr. Susan Cleveland.

Dr. Kraus with Spur Ridge Vet Hospital conducted a study where he sent identical BVD-PI samples to two different labs to be tested. One lab used pooled PCR technology, the other used ACE technology and ran single samples. The labs did not know which samples were positive, if any. What Dr. Kraus found was the lab that pooled PCR missed 67% of the samples that the individually tested ACE found positive (8 out of 12 positives).

Central States Testing used to pool samples until 2006 when we noticed some pools came out negative but when you break them down and run them as single tests you found a PI. We noticed this occurred more often in pens where there was a current or recent sickness. This leads us to believe there is something besides straight dilution of a sample causing false negative outcomes. There could be some type of interference between certain negative animals and positive animals. From this moment on, we were dedicated to keep our tests as economically priced as possible, so producers would be less inclined to pool samples together.

Today, there is an option of testing by yourself, on site with quick results. This gives producers a great advantage as they are able to identify and remove PI’s very quickly. One downside is the test is expensive. So, to combat the price of the test, producers started pooling samples. We decided to run an internal study evaluating the effects of pooling with this particular test. Keep 2 things in mind: 1) This will happen to any ACE test, including ours. 2) We wanted to show producers how pooling just 2 animals can give you a false negative test (a positive animal that comes out negative).

We chose particular samples for this study, some negatives that we suspected had inhibiting abilities for positive samples. Keep in mind, there is a greater chance for animals with inhibiting abilities in pens where there is a current or recent sickness.

For this study we used 6 sets of tests, one pooled 1:1 (one positive, one negative) the other ran the positive sample as a single test. The test missed 4 out of 6 positives when pooled 1:1.

The national average prevalence rate we see is around 0.4%, or 4 PI’s per 1,000 head for 400-600lb cattle. Sometimes when I tell people this, they will smile and say “we must buy good cattle because we only find 1 – 2 PI’s per thousand.” I then ask how they test and every single one has told me that they pool samples.

Here is a chart based on some data we have accrued over the years. This chart shows the estimated percent of false negative results (how many PI’s weren’t found) based off pool size and inhibitor probability. Inhibitor probability is based off of the current and recent herd health status (sickness), cattle source and other factors. If you have a sick pen of co-mingled cattle, your inhibitor probability sky-rockets. If you pool 4 samples together with a 2-4% inhibitor probability rate, you could miss 7% to 15% of your PI’s.

Hopefully this article will help you see that pooling samples together may not be cost effective, as the PI’s you WILL miss will still be in your general cattle population.

P.S. stay tuned as we have some exciting research coming out in the future that dives deep into the effects of pooling.

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