Where does your swab go? 

 


OUT ON PASS

Thaddeus C. Hinunangan, MD

Out on Pass is a borrowed term used in hospitals, where a patient is temporarily sent home for a respite, with promise to return for definitive treatment. Dr. Thaddeus C. Hinunangan is a physician by profession, and a writer by heart. His work was published in several anthologies and he also contributes to Philippine Daily Inquirer Opinion column.

For comments: thadhinunangan@gmail.com


Taking someone’s nasopharyngeal swab in a sealed, appropriate medium, properly labeled, packed in a ziplock, with outer labels and with the necessary documents is just step one.

At the receiving area, the outer containers are decontaminated, the documents double checked with outer labels. They are then grouped into batches and placed in a sealed, insulated container. If there are too many specimens, we place them in refrigerators until they can be processed.

From morning till afternoon to evening, specimens are processed by batch starting again with validation. The specimens are opened and brought out from their ziplocks in a Biosafety cabinet inside a negative pressure room. The medical technologist or pathology resident are in full gear Level 4 PPE. This is necessary to provide protection and contain infectious agents like viruses in the swabs. We make sure the names on the lists are exactly the ones on the specimen container label, we also make sure the samples have not been spilled or compromised- if so, the adulterated specimens are rejected. Validation can last up to an hour or more. Repeat runs are also included, and this takes time by searching the freezer for the specimens from previous runs. Depending on whether manual or automated nucleic acid extraction is used, the extraction can last anywhere between more than an hour (with automated extractor) to 4 hours (manual), again in full PPE in negative pressure. Imagine being in a sauna, wrapped in PVC or plastic. That’s how it feels. The Biosafety cabinets are usually very small workspaces with specific flow. You pipette amounts as small as 20 micro liters. If you make a mistake with your reagents, interchange samples, you can ruin a test or two, or worse, render an entire batch invalid. So for this 4 or 5 hours in the negative pressure room, with no bathroom breaks, with sweat dripping on your back, legs, face, you try to concentrate because nothing less than perfection is acceptable. The eluate (the final product in extraction) is transferred to labeled eppendorf tubes,and they are now taken to the amplification room in an insulated container. As for the remainder of the sample, they an aliqout is made to labeled cryotubes for storage in the freezer.

Another set of med techs work in the amplification room since we do not want any cross contamination. Those who do the extraction are not involved in the amplification. The PCR mix is usually prepared before the extraction is finished, and this will now be added with the eluate (with the extracted RNA) inside a biosafety cabinet in the amplification room. They are now ready to be placed inside the PCR machine, where the cyclical process of denaturation, annealing and elongation occurs. This takes roughly 2 hours.

Then pathology residents review the graphs to see whether the run was valid by checking negative and positive controls, NTC, then check each well for amplification of target genes. Each sample then is given an interpretation. The batch is now ready for signout with a Pathologist (a Molecular Pathologist or a Pathologist with special training for PCR). In simple terms, once we see those sigmoid curves in the graphs within the thresholds, it signifies there is amplification of the gene of interest, this means that the sample is positive.

The final step is updating the line lists, submitting the positive and negative line lists to DOH, backing up the PCR runs, and printing out the test results and releasing.

As you can see, many hours and effort is poured on each single test, from verification all the way to interpretation. Every step has been taken to make sure that the test is as accurate as it can be. That piece of paper represents the efforts of a multitude of laboratory and health professionals. For us pathology residents, we still do runs ourselves during Saturdays and Sundays when we are on Molecular path rotation, to make sure OB patients on labor, and surgical patients for OR the following week, ward patients and patients from other hospitals have their results available the soonest possible time.

Recently, I have seen a lot of posts on social media on people doing lateral flow assays (rapid antibody tests) on their own, which is a dangerous practice. I also received a lot of questions on how the tests are interpreted because it created confusion among them. Not only are people exposed to unnecessary risk when not done properly and correctly, the result interpretation can also cause anxiety especially when they are conflicting or doubtful. Decisions which affect lives of people are made based on these tests, which is why accuracy and precision are of utmost importance.

There is a lesson to be learned here. Leave the administration of these tests and interpretation to qualified health practitioners. If you do not have at least a good background in immunology, it would be easy to get confused about what positive IgG with a negative PCR means versus both positive IgM and positive PCR test, versus a negative IgM IgG and a positive PCR test. And again lateral flow assays are different from PCR, since they detect different things. PCR detects viral RNA, rapid test or lateral flow assays detect Immunoglobulins. Those results vary according to which phase of the illness the patient is in, depending on viral load, on whether the patient has mounted an immune response already. These are also correlated with clinical signs and symptoms to be interpreted meaningfully.

To add to the caveats, not all test kits are created equal. For example, not all brands of rapid tests are FDA approved. Moreover, some tests give only IgG, some IgM, some both. Ideally rapid tests have been validated by a laboratory (parallel testing is done with a machine-based, quantitative method like chemilluminescence or ELISA) because the sensitivity and specificity may not always be what they printed on the kit. In a good laboratory, the very first thing we do with a new assay is validate.

Don’t panic or despair, we have already been advised on what to do to minimize the spread of Covid. Not everyone needs to become a laboratory scientist or a doctor, but we can still do our part by observing physical distancing, wearing masks, appropriate quarantine and isolation when necessary. People’s behavior have a huge impact in this pandemic. PCR testing is very costly in terms of machine, reagents, labor, and expertise, and it serves mostly to guide treatment and make decisions on quarantine for people. At the end of the day it is our individual actions that make a difference on whether we are “flattening the curve” and beating Covid.

So before you go get tested, ask yourself if it is absolutely necessary, who will be performing the test, and what will be your course of action when you receive the result.

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