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ZstatFlu™ "Technical" FAQ v1.8
Troubleshooting and Test Interpretation Issues
- How do I determine if my ZstatFlu™ test result is positive or negative?
- It's been 5 minutes and my ZstatFlu™ test still hasn't finished draining through the collection device! What's wrong?
- If the throat swab itself is visibly colored can I still perform the test?
- Some of the prefilter tubes in my specimen collection kit have liquid droplets in them. Is this normal?
- I just performed the positive control test, but did not get a positive, blue result. What should I do?
- My attempt to challenge the cut-off limits of the test yielded a negative result, even though the undiluted positive control test was positive. What does this mean?
- I've noticed that the test solution tends to foam as I squeeze the filtrate into the dry reagent tube. Is this a problem?
- After filtering the diluent and throat swab material into the dry reagent vial, I accidentally left the vial at room temperature for ten minutes before adding it to the heating block. Is the test invalid?
- I mistakenly added the red-capped Stop reagent to the throat swab specimen instead of the white-capped Diluent vial. Can I still salvage the test?
- I accidentally let the ZstatFlu™ test incubate in the heating block for 30 minutes, instead of 20 minutes. Are the test results valid?
- The heating block I received last year malfunctioned. Can I get a new one?
- I checked the temperature of my heating block with a calibrated thermometer, and the temperature is not 41 C. Should I still use it?
- Can I read the test results before the test solution has completely drained through the collection device?
- How often should I run the positive and negative controls?
- Do I have to perform the pH check?
- Does the sample need to incubate after adding the Stop reagent but before transferring the solution to the collection device?
- Do I have to transfer the solution with the transfer pipet provided, or can I simply pour the test solution directly into the collection device?
- How long should I wait after plugging in the heating block before I can begin incubating my test sample?
- When should the results of the ZstatFlu™ test be read?
- What's the difference between this year's "20-minute" ZstatFlu™ test and last year's "30-minute" test?
- What is the analytical sensitivity of the ZstatFlu™ test?
- What is the shelf life of the ZstatFlu™ test and components?
- I have some ZstatFlu™ kits left over from the 1998-99 flu season. Can I use them this year?
- What substances are known to interfere with the ZstatFlu™ test?
- Can I save part of the throat swab specimen for use in culture or for future screening?
- Can the throat swab specimens be frozen prior to testing?
- Can I use specimens other than throat swabs with the ZstatFlu™ test?
- One of my test kits is missing a component. How do I get a replacement?
Troubleshooting and Test Interpretation Issues
How do I determine if my ZstatFlu™ test result is positive or negative?
The criterion for a positive test result is "any shade of blue" appearing in the collection device. A positive test can be very lightly blue, or so dark that it appears nearly black. Most clinical positives are a light to medium shade of blue. A negative test is typically white or off-white in color. This does not mean, however, that a ZstatFlu™ negative is always white. The most common coloring observed in test negatives is white, off-white, beige, yellow-brown, olive, or any combination of these colors. Other "test-negative" colors are also possible. Since the patient specimen is drawn from the throat, the presence of mucus, blood, colored food or beverage, or medicinal substances in the mouth or throat can collect on the swab and transfer through the test process. If you have difficulty in determining whether your ZstatFlu™ test result is positive or negative, you can get help in several ways. First, we always encourage you to compare the patient test with corresponding positive and negative controls. If you are still unsure of the test outcome after this comparison, ZymeTx has established a visual reference database to assist the interpretation of ambiguous test results [Link]. We encourage you to visit this database and see for yourself what constitutes a positive and negative ZstatFlu™ test. Seeing is believing! Last, we are always willing to help directly, by email [Link], phone (1-888-817-1314), or fax (1-877-854-4187).
It's been 5 minutes and my ZstatFlu™ test still hasn't finished draining through the collection device! What's wrong?
Probably nothing. Although we introduced a filtration step prior to draining the test solution in the collection device to reduce the drain time, occasionally a sample will take more than a couple of minutes to drain. Results from testing at ZymeTx has revealed that roughly 90% of throat swabs drain within 5 minutes. However, it is important to remember that the throat swab is a biological sample that contains both viscous mucous and cellular debris and is highly variable in its composition from person-to-person. Swabs from select patients, especially those with heavy mucal secretions, can sometimes take several minutes to drain completely. If you find that your samples consistently take longer than 15 minutes to drain then you should report the problem to ZymeTx.
If the throat swab itself is visibly colored can I still perform the test?
Certain specimens can potentially alter the test interpretation (e.g., heavily colored medication, food and beverages, or blood). You should certainly consider this information in your interpretation of the results if a ZstatFlu™ test result obtained with a colored swab is any color other than white (for a test negative) or blue (for a test positive).
Some of the prefilter tubes in my specimen collection kit have liquid droplets in them. Is this normal?
Yes. The droplets contain specific chemicals necessary for proper test performance. Do not actively attempt to remove these droplets. You do not need to be concerned, however, if some of this liquid material clings to the tube cap and is disposed of with the cap. In some cases, you may observe that the tubes have a glassy, solid deposit - or you may not see anything at all. Either situation is not unusual, and poses no cause for concern.
I just performed the positive control test, but did not get a positive, blue result. What should I do?
First, re-check each step of the test procedure. Did you forget a step? Were any steps performed in the wrong order? Was the pH check successful? Re-familiarize yourself with each step, then repeat the positive control test. If you used the dry swab positive control and the test failed, you may need to treat the swab more vigorously. The dry swab must be thoroughly "massaged" in the diluent to ensure the release of the dried neuraminidase enzyme into solution. As with any throat swab, the positive control swab should be pinched in the prefilter tube and "wrung out" against the walls of the tube to extract as much liquid from the swab as possible. When using the red-capped liquid positive control, ZymeTx recommends that you add two (2) full drops to the diluent solution. If 2 drops do not produce a sufficiently dark positive test result, you can add additional drops of positive control as needed. If the test fails to show blue with any amount of positive control, do not use the reagents and contact ZymeTx. Patient results should only be reported when quality control testing produces the appropriate results. If you only have one set of controls and need to repeat the control test, contact ZymeTx for a replacement control kit.
My attempt to challenge the cut-off limits of the test yielded a negative result, even though the undiluted positive control test was positive. What does this mean?
The goal of the challenge assay is not to validate the test itself, but provide you with a visual reference for what a weak positive should look like. Since the response curve of the ZstatFlu™ test is steep near the limit of detection, the challenge assay may fail to present a visibly blue endpoint in some cases. This result does not mean that the test components are faulty. The more likely interpretation is that the limits for the challenge assay were a bit too ambitious. If you fail to observe any blue in the challenge assay in situations when the positive control is clearly blue, you may need to adjust the dilution factor slightly to achieve the appropriate weak positive result.
I've noticed that the test solution tends to foam as I squeeze the filtrate into the dry reagent tube. Is this a problem?
Not at all. Foaming is normal, the result of expelling liquid and air together with a chemical detergent through the filter tip.
After filtering the diluent and throat swab material into the dry reagent vial, I accidentally left the vial at room temperature for ten minutes before adding it to the heating block. Is the test invalid?
The neuraminidase assay is initiated once the filtrate is added to the substrate vial. This reaction is approximately three times slower at room temperature than at 41 C in the heating block. Leaving the filtrate at room temperature for a few minutes would not be expect to alter test performance. However, we strongly recommend that you add the reagent vial to the heating block as soon as possible after the filtration step and follow the instructions described in the package insert to ensure reliable results.
I mistakenly added the red-capped Stop reagent to the throat swab specimen instead of the white-capped Diluent vial. Can I still salvage the test?
No. The Stop reagent is designed to quickly and effectively terminate the test reaction by inactivating the neuraminidase enzyme. Once inactivated by the Stop solution the enzyme activity cannot be reliably recovered, thus invalidating the test.
I accidentally let the ZstatFlu™ test incubate in the heating block for 30 minutes, instead of 20 minutes. Are the test results valid?
The performance ZstatFlu™ test is only valid for a 20 minute incubation at 41 C. Testing at ZymeTx has indicated that an incubation extended to 25 minutes will not compromise the test performance. However, we cannot guarantee the test results after a 25 minute incubation. No ZstatFlu™ test should ever be incubated for less than 20 minutes or more than 25 minutes.
The heating block I received last year malfunctioned. Can I get a new one?
Absolutely. Contact ZymeTx at 1-888-817-1314.
I checked the temperature of my heating block with a calibrated thermometer, and the temperature is not 41 C. Should I still use it?
No! The ZstatFlu™ test is extremely sensitive to deviations from the current 40.5 ± 1.0 C specification. Temperatures below 40.5 C result in a loss of test sensitivity, whereas temperatures above 40.5 C increase the risk of false positives. If you find that your heating block consistently falls outside of the specification listed, then you should contact ZymeTx immediately and request a new heating block.
Test Procedural Issues
Can I read the test results before the test solution has completely drained through the collection device?
No. ZymeTx strongly recommends that you wait until all of the test solution has drained through the collection device before interpreting the results. A weakly positive test may appear negative until the last few drops of test solution have drained. Even if the test appears to be positive, it is always a good idea to carefully inspect the device to be certain of your interpretation. This level of inspection is not possible when liquid is still in the device.
How often should I run the positive and negative controls?
ZymeTx suggests that a positive and negative control be performed with each shipper (i.e. each shipment box), or whenever a new tester first begins to use the ZstatFlu™ product. Of course, controls should also be performed in accordance with your laboratory's standard quality control procedures or regulatory body guidelines.
Do I have to perform the pH check?
No. The pH check is an optional control step that confirms that the Stop reagent has been added to the test solution to stop the neuraminidase reaction. Similar to many other diagnostic tests, the pH check offers an "on-board control" that helps to ensure proper test execution.
Does the sample need to incubate for any specific length of time after adding the Stop reagent but before transferring the solution to the collection device?
No. Once the Stop reagent is added, the reaction vial should be inverted several times to ensure the contents are well mixed and gently "tapped" on a hard surface to bring down any liquid clinging to the walls of the vial. The liquid should then be transferred immediately to the collection device.
Do I have to transfer the solution with the transfer pipet provided, or can I simply pour the test solution directly into the collection device?
To ensure transfer of as much of the test solution as possible, ZymeTx recommends that you use the included transfer pipet. The opening of the dry reagent vial is not designed for efficient pouring. Test sensitivity will be affected if all of the solution is not transferred to the collection device.
How long should I wait after plugging in the heating block before I can begin incubating my test sample?
The ZstatFlu™ heating block has a "Z" indicator on the black temperature strip that changes color when the heating block reaches the proper operating temperature (41 C). Typically, 40 minutes is required for the heating block to reach this temperature once it is plugged in (longer times may be necessary to reach 41 C if the heating block has been stored at temperatures below room temperature). Note that the lightening bolt that illuminates within just a few minutes merely indicates that the unit is plugged in, and not that the proper temperature has been reached.
When should the results of the ZstatFlu™ test be read?
The results of the ZstatFlu™ test should be read immediately after the test solution has completely drained through the collection device. In some cases, the color of a flu-positive, blue endpoint can change (usually over a period of many hours to days) as the device filter dries. We have observed that some initially blue endpoints can develop an ambiguous greenish hue over 12-24 hours.
Specifications and Limitations
What's the difference between this year's "20-minute" ZstatFlu™ test and last year's "30-minute" test?
ZymeTx is pleased to introduce an improved 20-minute version of the ZstatFlu™ influenza diagnostic for the 1999-2000 flu season. This test contains an enhancer chemical that improves the intrinsic sensitivity of the diagnostic. This increase in test sensitivity means that samples incubated for 20 minutes are just as sensitive as samples incubated for 30 minutes with last year's test components! In addition, we have made significant advancements in the processing and manufacturing of the ZstatFlu™ substrate molecule. Taken together, these improvements translate to faster patient diagnosis, more clear-cut test positives and negatives, and even more reliable test results than before.
What is the analytical sensitivity of the ZstatFlu™ test?
The exact analytical sensitivity of the ZstatFlu™ test has not been determined for either the number of influenza virions (type A or B), or the multiplicity of infection (MOI). However, the visual threshold (limit of detection, or LOD) of the test is achieved with about 0.4 mU of neuraminidase enzyme activity. The LOD corresponds to less than 10 nmol of cleaved substrate.
What is the shelf life of the ZstatFlu™ test and components?
The shelf life for each ZstatFlu™ test kit can be determined from the expiration data given on the side of the box. For example, a date of 12/2000 indicates that the test components can be used until December 31, 2000.
I have some 30-minute ZstatFlu™ kits left over from the 1998-99 flu season. Can I use them this year?
ZymeTx has improved the 1998-1999 30-minute ZstatFlu™ test, resulting in the current 20-minute version. Additional quality measures have been taken to ensure reliable, consistent performance and extremely high test specificity. If you have any 30-minute ZstatFlu™ tests, ZymeTx will gladly exchange them for current 20-minute tests at no cost to you. Please contact our customer service representatives for more information (1-888-817-1314).
What substances are known to interfere with the ZstatFlu™ test?
The ZstatFlu™ test is a neuraminidase assay. As with any enzyme assay, harsh chemicals (acid, base, concentrated detergents) may impair enzyme activity and reduce test performance. We have also observed that high concentrations of proteins such as serum proteins can interfere with the test. The simplicity and portability of the ZstatFlu™ test was designed around a throat swab specimen that needs no additives beyond those included with the kit. Thus it is critical that no additional reagents or material, such as transport media, be added to the throat swab specimen prior to testing.
Can I save part of the throat swab specimen in for use in culture or for future screening?
No. ZymeTx makes no claims to test performance if the throat swab is stored in the prefilter tube beyond 24 hours. Within the first 24 hours of specimen collection, the specimen should not be immersed in any solution, except for the diluent once you are ready to actually perform the test. "Splitting" the sample with diluent will reduce in the sensitivity of the test and cannot be recommended.
Can the throat swab specimens be frozen prior to testing with ZstatFlu™?
No. However, throat swab specimens can be stored and transported to the laboratory for testing up to 24 hours after collection. Specimens should be handled within a temperature range of 0 C (32 F) to 40 C (104 F).
Can I use specimens other than throat swabs with the ZstatFlu™ test?
Although it may be possible to employ other specimen types at some future date, the currently marketed version of the ZstatFlu™ test is only approved for use with throat swabs.
One of my test kits is missing a component. How do I get a replacement?
Simply contact ZymeTx by email [Link], phone (1-888-817-1314), or fax (1-877-854-4187).
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ZymeTx Web Sites:
zymetx.com
flu101.com
fluwatch.com
©2003 - ZymeTx, Inc.
ZstatFlu®, ViraZyme®,
ViraSTAT®, and National Influenza Laboratory™ are trademarks of ZymeTx, Inc.