FREQUENTLY ASKED QUESTIONS FOR DIAGNOSTICS
GENERAL
- How do you choose the preferred sample types. Can I submit a sample that is not on the list?
- There are multiple sample types per pathogen/panel listed on your submission forms. Is it necessary to send all of the sample types?
- What do you mean by “pool” samples?
- What is the specificity/sensitivity/efficiency of your assays?
- Why is serum not an acceptable sample type?
- How long can I hold a sample before shipping?
- Do you have an IDEXX account?
- How do I ship a sample to you?
- Is it worthwhile to send a sample collected after the animal has started treatment?
- What is your turn-around time?
- Why is whole blood not recommended for the testing of Borrelia burgdorferi (Lyme Disease)?
QUALITY CONTROL (QC)
- What does it mean when a sample fails QC?
- What are the possible reasons for failure to pass QC?
- What techniques do you use to ensure sample quality?
- What procedures are in place to ensure the accuracy and quality of your laboratory’s work/tests?
BILLING
- What types of payment are acceptable?
- What is a FEIN# and why do you require it for billing?
- I submitted a sample a couple weeks ago and still have not received a bill. How long does it typically take to receive an invoice?
LARGE ANIMAL
- Do you test for the neurotropic strain of Equine Herpesvirus 1 (EHV-1)?
- Why do you recommend submitting dual samples (whole blood and nasal swab) for EHV-1 testing?
- Do you test for EPM (Equine Protozoal Myeloencephalitis)?
- What is the most accurate method for diagnosis of Streptococcus equi (S.equi)? How do I interpret the bacterial load?
SMALL ANIMAL
- Do you test for Feline Immunodeficiency Virus (FIV)?
- Why isn’t Mycoplasma felis offered as part of the feline respiratory panel?
- Does the Chlamydia felis/psittaci assay detect the isolate that infects guinea pigs/hamsters?
ANSWERS
GENERAL
How do you choose the preferred sample types. Can I submit a sample that is not on the list?
The preferred sample types are those suggested by our veterinarians to be the most likely to host the specific pathogen of interest. You may submit other sample types that are not on the list. It is important to note, however, that the likelihood of detecting the pathogen in other sample types may not be as great. If you do decide to use a different sample type, it is a good idea to call the lab before collecting the specimen to make sure we have a means of processing the sample.
There are multiple sample types per pathogen/panel listed on your submission forms. Is it necessary to send all of the sample types?
No, it is not necessary to send each listed sample type. It is up to you to send the sample type that you think is most suitable for diagnosis based on your patient’s clinical signs/symptoms. If you do send multiple types, please indicate on the form if you would like them pooled or tested separately (see question 3). If you wish to test them individually we will charge you per sample type as outlined below.
What do you mean by “pool” samples?
We pool multiple samples by combining them and testing the combined mixture for the specific pathogen(s) of interest. This is a great option if you are not sure where the pathogen might be present as you can combine as many sample types as you like. It is important to note, however, that because we pool all of the samples, should the PCR yield a positive result, we can not trace back and determine which sample type hosted the pathogen. Also, pooling samples may diminish pathogen detection. For billing, if you pool samples you will only be charged for one test, instead of one test per sample type. The example below outlines these two options; pooling multiple sample types, or testing each sample type individually.
| Option A: Pool samples | |||
| Patient Name: | Sample type(s): | Test requested: | Price: |
| “Harley the Horse” | POOLED whole blood, nasal swab | Equine Influenza A | $57.00 |
|
Total: |
$57.00 | |
| Option B: Samples tested individually | |||
| Patient Name: | Sample type(s): | Test requested: | Price: |
| “Harley the Horse” | whole blood | Equine Influenza A | $57.00 |
| “Harley the Horse” | nasal swab | Equine Influenza A | $28.50* |
Total: |
$85.50 | ||
*half price for the same test on a different sample type for the same patient. |
What is the specificity/sensitivity/efficiency of your assays?
Each assay is 99-100% specific to the available sequence information, sensitive enough to detect as few as five copies of the gene, and 95-100% efficient. Our primer/probe sequences are checked annually to make sure we use the most up to date sequence information.
Why is serum not an acceptable sample type?
Serum separates the blood cells from the plasma. To ensure that our tests yield accurate results, the DNA contained within the blood cells must be tested, and is why we only accept whole blood EDTA (purple topped tube).
How long can I hold a sample before shipping?
We recommend freezing the sample if you need to hold it over the weekend or for longer periods of time (up to a week). If freezing is not an option, refrigeration can keep a sample fresh for up to five days.
While IDEXX delivers to our facility every morning, we do not have an IDEXX account number. We recommend calling the IDEXX office in West Sacramento if you have questions in regards to shipping.
How do I ship a sample to you?
Complete shipping instructions can be found in our sample submission packet (available through FAX or on our website. All samples need to be shipped on ice in a Styrofoam container via overnight delivery. This preserves the integrity of the DNA/RNA during outside temperature fluctuations. If possible, please put the sample submission form in a separate plastic bag to protect it from condensation from the ice pack or leaky samples. We receive deliveries from FedEx, IDEXX, DHL, UPS, and USPS. We DO NOT recommend submitting samples through USPS. Many times the packages are retained at the main post office on central campus for several days before being distributed. All tubes should be wrapped in something padded (i.e. bubble wrap, vet wrap, paper) to prevent tubes from breaking during shipping.
Is it worthwhile to send a sample collected after the animal has started treatment?
It is up to the discretion of the veterinarian. While antibiotics or other various treatments will not inhibit the PCR process, there is a chance that treatment will diminish the presence of the pathogen(s) of interest beyond detection. If possible, a pre-treated sample that has been kept cold (either in the freezer or refrigerator) would be preferable.
What is your turn-around time?
In most cases, if we receive the sample by 11am, we are able to FAX results by 5pm the same day. If there is a large sample load that day, results will be faxed the next morning. There are a few exceptions, however. If your sample does not pass quality control (see below) then extra time is needed for retesting. Also, paraffin samples take 24-48 hours due to intricate processing techniques. Please keep in mind any time zone differences as well. If you are in dire need of results and will be out of the office, we will be more than happy to call you if you let us know ahead of time.
Why is whole blood not recommended for the testing of Borrelia burgdorferi (Lyme Disease)?
Whole blood is not a preferable sample type for PCR-based detection for the presence of Borrelia burgdorferi DNA because spirochetes may be present for a limited time in blood, so the negative PCR result does not rule-out Lyme disease. Tissues most likely to harbor B. burgdorferi are lymph nodes in the region of the tick bite, muscle, fascia, and synovial (joint) membrane. Less frequently, the spirochetes end up in meninges.
QUALITY CONTROL (QC)
What does it mean when a sample fails QC?
The DNA, cDNA of every diagnostic sample is run with a standard housekeeping gene to ensure successful nucleic acid extraction. If the housekeeping gene is negative, or very weak (Cycle Threshold value greater than 30), we re-extract the nucleic acids from the back up sample, and run it through PCR a second time. If the housekeeping gene for the backup sample is also negative or weak, we consider the sample to “fail quality control”; that is, there are not enough cells in the sample to accurately test for the presence of a pathogen. You have the option of submitting another sample if the package is shipped correctly. Recently, our facility has introduced a new methodology to reduce these instances (see question 3 below).
What are the possible reasons for failure to pass QC?
Samples fail quality control for a variety of reasons, most of which depend on the sample type, or how the sample is handled. The longer a sample sits without being processed, the more likely the DNA/RNA will degrade. The sample should be shipped on an ice pack to preserve the integrity of the nucleic acids.
What techniques do you use to ensure sample quality?
Samples with typically low cell concentrations (mainly urine, CSF, and joint fluid) can fail quality control with our standard housekeeping gene. In these cases, if the sample fails quality control the first time, we spike the sample before the DNA/RNA extraction with an unrelated gene from a potato. After extraction, we test for the presence of this gene. The PCR should yield a positive result if the DNA/RNA extraction was successful and guarantees we would detect a pathogen if it is present.
What procedures are in place to ensure the accuracy and quality of your laboratory’s work/tests?
Below is a condensed version of our quality assurance procedures. These control measures include, but are not limited to the following:
- All researchers are extensively trained in the areas of laboratory safety, sample preparation, reagent preparation, and TaqMan® data analysis. Training records are held onsite.
- The TaqMan® Core Facility has physically separate rooms for sample receiving/preparation, assay preparation, amplification, and analysis. Separation of workflow greatly decreases the risk of cross contamination between samples and reagents.
- All samples are tested with a standard housekeeping gene. This ensures proper nucleic acid extraction and absence of inhibitors.
- Positive and negative controls are run for the diagnostic assays daily. This guarantees the assays are made correctly and no contaminants are present.
- Various areas within the lab are swabbed on a biweekly basis and tested for any pathogens that have tested positive within the previous two week interval. This ensures work areas are free from contamination.
*This is a small portion of the Quality Assurance program in place at the TaqMan® Core Facility. A complete guide can be found on our website.
BILLING/PAYMENT
What types of payment are acceptable?
We do not accept cash or credit card payments. Personal or business checks are okay if made out to “The Regents of UC Davis”. If sending a check with your sample, please call prior to submission to find out the exact charge.
What is a FEIN# and why do you require it for billing?
A Federal Tax Identification Number (FEIN# or FTID#) is a nine digit number; two digits followed by a dash and seven more digits (ex: 12-3456789). Every facility is required to have one by law as it is used to identify your company by the Internal Revenue Service (IRS). We require a Federal Tax Identification Number (FEIN#) for all first time clients in order to set up an account in our University accounting database. You may either call us with the FEIN# or fill in the field marked “Your Federal Tax ID (FTID) #” on the sample submission form. Please keep in mind that FEIN#’s are public domain, and should not be regarded as the equivalent to your company’s social security number. Once an account is created for your company, an invoice will be mailed with the amount due and the address of the Cashier’s Office where the check should be sent.
I submitted a sample a couple weeks ago and still have not received a bill. How long does it typically take to receive an invoice?
Because the University has so many checks and balances in regards to billing, receiving an invoice can take up to three weeks. If you need to know the exact cost of our services so you can bill your client, please feel free to call ahead of time and ask for a quote.
LARGE ANIMAL
Do you test for the neurotropic strain of Equine Herpesvirus 1 (EHV-1)?
Yes, we do run a test that will differentiate between the neurotropic and non-neurotropic strains of EHV-1. All samples submitted for EHV-1 testing will be run with these two assays. Should you receive a positive EHV-1 result, the report will also include the strain type (neurotropic/non-neurotropic) and a viral load concentration. Guidelines for interpreting the viral load (see next question) will be included at the bottom of the report. It is highly recommended to call the lab and get the contact information for our equine specialist who can further explain the results.
Why do you recommend submitting dual samples (whole blood and nasal swab) for EHV-1 testing?
The disease stage can be determined using the individual results for each sample type. At different times of the infection the nasal swab and the whole blood can be positive or negative. The following is what we include in the report if the nasal swab and/or the whole blood samples are positive:
PCR results for EHV-1 are expressed qualitatively (positive or negative) and quantitatively in the case of a positive result. The viral load of a sample is calculated by the absolute number of EHV-1 (glycoprotein B gene) per million cells (either blood cells or nasopharyngeal cells). We are in the process of validating thresholds in order to better understand the viral kinetics of diseased and subclinical horses. Diseased horses (neurological or febrile) commonly have high (greater than 1x10^4) viral loads in blood and nasal secretions. Subclinical horses commonly have low (less than 1x10^3) to moderate (1x10^3 – 1x10^4) viral loads only in nasal secretions. The absolute number should be used judiciously and allow comparisons between samples taken at different time points. Viral load ranges are only suggestions and are not currently well-defined.
Do you test for EPM (Equine Protozoal Myeloencephalitis)?
We do have assays designed to detect the causing agents of EPM, Sarcocystis neurona and Neospora hughesi. It is important to note, however, that these pathogens are rarely found in the blood or cerebral spinal fluid of an infected horse. Therefore, PCR testing of neurologic horses using either blood or CSF is not recommended, as this will generate false negative results. Concrete diagnosis of EPM relies on the detection of antibodies against the two Apicomplexan parasites in blood and/or CSF using the quantitative serological IFAT. The Immunology Lab at the Veterinary Medical Teaching Hospital at UC Davis offers such antibody testing (530-752-7373).
What is the most accurate method for diagnosis of Streptococcus equi (S.equi)? How do I interpret the bacterial load?
You have one of two options for diagnosing S.equi:
- Submit a nasal swab or guttural pouch wash for PCR testing. Retesting at a later time to determine carrier state is recommended.
- Perform a single culture.
We include a calculated bacterial load with the results if the horse is positive for S.equi. The bacterial load represents the number of S.equi cells per million nasopharyngeal cells. Even though we do not have guidelines established for interpretation, the bacterial load is meant to give you an idea of how strongly present the bacteria is. We recommend testing a positive S.equi patient at a later date to see if the bacteria has decreased or increased over time/treatment.
SMALL ANIMAL
Do you test for Feline Immunodeficiency Virus (FIV)?
As you may or may not be aware of, the TaqMan Core Facility previously tested for FIV free of charge. While we could not charge for the test, we still ran it because we firmly believed it was an important diagnostic tool for differentiating between active infection and exposure/vaccine.
With that said, thanks to the legalities of patenting we are no longer allowed to offer a PCR test for FIV. At this time, we are aware of only one facility in the country that does offer this test: Auburn University, Alabama. Because we have not used their facility, nor have we confirmed the accuracy of their tests, we cannot endorse them in any way. We do recommend calling their facility and inquiring about their test: http://www.vetmed.auburn.edu/index.pl/mdcontact
Why isn’t Mycoplasma felis offered as part of the feline respiratory panel?
Instances of false positives are extremely high (~50%) in clinical cases when veterinarians submit corneal or nasal swabs for M.felis. To prevent misdiagnosis, and therefore unnecessary treatment, only paraffin or fresh tissue should be submitted.
Does the Chlamydia felis/psittaci assay detect the isolate that infects guinea pigs/hamsters?
Yes, the assay will pick up the caviae strain of Chlamydia that commonly infects guinea pigs/hamsters.