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Special Specimen Collection and Handling

Infectious Disease

The proper collection of a specimen for culture is the most important step in the recovery of pathogenic organisms responsible for infectious disease. A poorly collected specimen may lead to failure in isolating the causative organism(s) and/or result in the recovery of contaminating organisms.

• Infectious Diseases Collection and Transport Media
• Specimen Types for Molecular Infectious Disease Tests

Basic Concepts for Specimen Collection

• Collect the specimen from the actual site of infection, avoiding contamination from adjacent tissues or secretions.
• Collect the specimen at optimal times (e.g., early morning sputum for AFB culture).
• Collect a sufficient quantity of material. Use appropriate collection devices (i.e., sterile, leak-proof specimen containers) and transport media (i.e., anaerobe transport vials, Amies or Stuart’s, for bacterial culture; Cary–Blair for stool culture; M4 for viral, chlamydia, and ureaplasma cultures).
• Whenever possible, collect specimens prior to administration of antimicrobials.
• Properly label the specimen and complete the test request form. The source of specimen is required.
• Minimize transport time. Maintain an appropriate environment between collection of specimens and delivery to the laboratory.
• If appropriate, decontaminate the skin surface. Use 70–95 percent alcohol (ALC) and 1–2 percent tincture of iodine (TOI) to prepare the site. Allow a contact time of two minutes to maximize the antiseptic effect.
• Package each specimen separately in its own sealable transport bag.
• Isolates submitted on agar plates or as mixed cultures are unacceptable.
• Slides must be labeled individually on the side the sample is inoculated.

Abscess

• Decontaminate the surface with 70–95 percent ALC and 1–2 percent TOI.
• Collect purulent material aseptically from an undrained abscess using a sterile needle and syringe. Open miliary abscesses with a sterile scalpel and collect the expressed material with a sterile needle and syringe.
• Expel air from the syringe, remove the needle, and cap the syringe. Alternatively, transfer 5–10 mL of theaspirated material to an anaerobic transport vial. Transport immediately.
• Swabs are a poor choice because they dry easily and there is limited amount of material obtained. Swabs are not acceptable for mycobacterial or fungal cultures.

Blood (bacteria only)

• Gather the collection blood bottles or tubes needed.
• Swab the tops of each blood culture bottle and/or the stopper of an Isolator^® or SPS tube with alcohol. Do not allow alcohol to pool, as it could enter the system and kill organisms. Allow to dry while preparing the patient.
• Cleanse the skin with 70–95 percent ALC.
• Cleanse the skin with 1–2 percent TOI. Move in an ever-increasing circular pattern, starting at the point of projected needle insertion.
• Apply a tourniquet proximal to the point of venous entry. The venipuncture site should not be palpated following disinfection unless sterile gloves are worn.
• Use a sterile needle and syringe or closed system blood-collection tubing. For fastidious microorganisms, use the Vacutainer^® system for Isolator tubes.
• Collect blood. The volume of blood collected is critical. Inoculate the bottles or tubes without changing needles.
• For adult bacterial culture, inoculate aerobic bottle with 10 mL of blood or the anaerobic bottle with 7 mL of blood. If less than 17 mL is collected for two bottles, inoculate the aerobic bottle with 10 mL and inoculate the anaerobic bottle with the remainder.
• For pediatric specimens, inoculate 1–3 mL blood into a pediatric bottle.
• For fungal and AFB cultures, inoculate 5 mL blood into a Bactec^™ Myco/F Lytic bottle.
• The adult Isolator tubes will accommodate 9.5–10 mL blood. Allow the vacuum to draw in the proper amount of blood. Do not force the blood into the tube.
• The SPS vacutainers hold 8 mL or 3 mL.
• Invert tubes several times after specimen collection.
• Remove the iodine from the skin after collection of the specimen.
• Label and transport specimens immediately. Do not refrigerate. Hold at room temperature or at 35°C. Note: Isolator tubes should be used only for isolation of histoplasma and unusual fastidious or intracellular bacteria such as Brucella sp., Francisella sp., Bartonella sp., etc.

Body Fluids, Sterile (except urine and CSF )

• Prepare the skin as for blood cultures.
• Collect the fluid using a sterile needle and syringe.
• Submit 10 mL of the specimen for analysis. Transport the specimen in a capped syringe, or
• For aerobic and anaerobic organisms, use an anaerobic transport vial to ensure the survival of anaerobic organisms.
• 10 mL of peritoneal fluid may be added to a blood culture bottle. Peritoneal fluid is the only body fluid that may be cultured in blood culture media.
• For viral isolation, send 3 mL or less fluid in viral transport medium or a sterile vial.
• If tuberculosis or fungal infections are suspected, larger volumes are required. Collect in sterile container.
• Immediately transport fungal specimens at 2–8°C, viral specimens at 2–8°C, and all other specimens at ambient temperature.

Bone Marrow

• Physicians should wear gowns, masks, and gloves during specimen collection.
• Prepare skin as for blood cultures.
• Drape the surrounding skin with sterile linen.
• Aspirate the marrow percutaneously using a sterile needle and syringe.
• Transfer 3–5 mL to a sterile tube containing SPS for bacterial AFB and fungal cultures. EDTA is required for viral cultures and molecular tests.
• Transport specimens immediately at ambient temperature.

Bordetella pertussis Culture, DFA , and PCR

Culture

• Allow the transport medium (Regan–Lowe) to equilibrate to ambient temperature.
• Use two swabs on a flexible wire handle to collect the specimen. One swab is used to inoculate the transport medium. The other swab is used to prepare the smears for the DFA test. (Calcium alginate swabs cannot be used for PCR testing.)
• Seat the patient comfortably. Tilt the head back.
• If available, insert a nasal speculum. Press the swab through the nares until resistance is met due to contact with the nasopharynx.
• Rotate the swab gently and allow the swab to maintain contact with the nasopharynx for 20–30 seconds or until coughing is induced.
• Place the swab into the transport medium. Label the tube with the patient’s name and identification number. Leave the swab embedded in the tube during transport. Before transport, incubate the tube 24–48 hours at 35°C in humidifier. Transport the specimens at ambient temperature.

DFA Stain

• Repeat the procedure with the second swab. Gently roll the swab onto two glass slides. Label both slides with the patient’s name, identification number, and the date on the side the specimen is inoculated.
• Transport the slides (placed in a microscope slide mailer) at ambient temperature.
• If slides are not available, swab may be transported in bacterial transport media.

PCR

• Preferred specimen is nasal wash or dry swab. Do not use calcium alginate.
• Transport frozen.

Bronchial Brush/Washing/Lavage

• This technique should be performed by an experienced individual. Descriptions of the methodology are readily available in literature.
• Transport in a sterile container at 2–8°C for cultures or frozen for molecular tests.

Buffy Coat (virus and molecular tests)

• Cleanse the skin as for blood cultures.
• Collect blood in a 5 mL EDTA (lavender–top) tube. Use the pediatric size (approximately 3 mL) only when absolutely necessary. Do not use pediatric tubes to collect from adults or children over 2 years of age.
• Invert tubes several times after specimen collection.
• Remove the iodine from the skin after collection of the specimen.
• Document an order that buffy coat preparation is requested.
• Refrigerate immediately. Transport whole blood (EDTA) specimens. Do not attempt to separate the buffy coat from the whole blood specimen.

Bullae, Cellulitis, Vesicles

Bullae, Vesicles

• Cleanse the skin as for blood cultures.
• Aspirate the fluid/purulent material using a sterile needle and syringe.
• If an aspirate is obtained, place in appropriate viral or bacterial transport.
• If no material is obtained, unroof vesicle or bullous lesion and use a swab to collect cells from the base of the lesion. Place in appropriate viral or bacterial transport media.

Cellulitis

Swabs and leading-edge aspirates with or without injection of saline fail to yield etiologic agents in the majority of cases. If an unusual organism is suspected, a leading-edge (advancing margin) punch biopsy is the recommended specimen of choice.

Cerebrospinal Fluid

• Physicians should wear gowns, masks, and gloves to collect the specimen. Because an open tube is held to collect the fluid, other personnel should stand away or wear masks in order to avoid respiratory contamination.
• Decontaminate the skin with 1–2 percent TOI, followed by 70–90 percent ALC using an increasingly outward circular movement.
• Drape sterile linen over the skin surrounding the puncture site.
• Insert the needle. Collect the fluid into three sterile leak-proof tubes. Collect an adequate volume of fluid as recommended below.
  • Bacterial culture: > 1 mL
  • Fungal culture: 8–10 mL<
  • Molecular: > 1 mL
  • Mycobacterial culture: 8–10 mL
  • Viral culture: > 2 mL
• Cap the tubes tightly. Submit the third tube for culture to reduce the possibility of contamination due to skin flora. Transport immediately.
  • Transport other bacterial cultures at ambient temperature. If a delay in transport occurs, incubate at 37°C or leave the fluid at ambient temperature for transport.
  • Freeze specimens for molecular (PCR) analysis.
  • For viral culture, if volume is greater than 3 mL, refrigerate and transport at once. If volume is less than 3 mL, add fluid to M4 viral transport media and transport at 2–8ºC.

Cervix (Endocervix) for HPV Testing

• Use a Digene^® Cervical Sampler, available from ARUP. This kit includes a cervical brush and a tube of transport medium for HPV testing.
• Do not use cervical brush with pregnant women.
• Collect Pap smear specimen before obtaining specimen for DNA testing. Collect DNA specimen prior to application of acetic acid or iodine if a colposcopy will be performed.
• Remove excess mucus from the cervical os and surrounding ectocervix using a cotton or Dacron swab. Discard the swab.
• Insert the brush 1–1.5 cm into the os of the cervix until the largest outer bristles of the brush touch the ectocervix. Rotate it three full turns in a counter–clockwise direction. DO NOT INSERT BRUSH COMPLETELY INTO THE CERVICAL CANAL.
• Remove the brush from the canal. Avoid touching the bristles to the outside of the tube or to any other object.
• Insert brush to bottom of the transport tube. Snap off shaft at score line and cap tube securely.
• Transport the tube at 2–8°C.
• Cervical biopsy: Fresh cervical biopsies up to 5 mm in cross-section must be placed immediately into the DigeneSpecimen Transport Medium and frozen.
• Alternatively, liquid–based specimens may be collected and transported (SurePath^™, ThinPrep^® Pap Test^™, or AutoCyte^®). Place each individual specimen in a sealed bag.

Chlamydia/Gonorrhea

• Chlamydia and gonorrhea testing is available by several methods. A DNA-amplification method that detects Chlamydia trachomatis or Neisseria gonorrhoeae nucleic acid in urogenital specimens is the preferred diagnostic method. The nonamplified direct DNA probe is also available, but, in general, it is less sensitive than an amplified test. Culture for Chlamydia trachomatis or Neisseria gonorrhoeae is the method of choice in cases of treatment failure and sexual abuse and for nongenital sources. Test orders will be changed to match test-specific transport media submitted.
• Specimens for all of the above can be collected following the procedures below. Test-specific collection and transport kits are required for DNA tests and are available from ARUP.
  • Females (endocervical:)
    • Place patient in the lithotomy position.
    • Insert speculum and visualize the cervical os.
    • Remove excess mucus from cervical os and surrounding mucosa using the large swab provided in the kit. Discard this swab.
    • Insert second swab from kit, 1 to 1.5 cm into endocervical canal.
    • Rotate swab for 30 seconds in endocervical canal to ensure adequate sampling.
    • Withdraw swab carefully, avoiding any contact with vaginal mucosa.
  • Males (urethral):
    • Do not allow patient to urinate for at least one hour prior to collection.
    • If purulent discharge is present, collect discharge directly on swab.
    • If no discharge is present, insert smaller swab 2–4 cm into urethra. Rotate gently to ensure contact with all urethral surfaces. Leave inserted for two to three seconds. Rotate gently while withdrawing swab.
  • Place swab into the test–specific transport tube.
    • Break swab shaft to fit tube, if required.
    • Cap tube tightly.
    • Transport at 2–8°C.
    • For culture, inoculate specimen as specified below.
  • For culture of N. gonorrhoeae, use calcium alginate or Dacron swabs for specimen collection. Cotton fibers contain fatty acids that are inhibitory to the gonococcus. Avoid swabs with wood sticks.
    • Inoculate culture directly onto a Modified Thayer- Martin agar plate. Direct plating is necessary because the gonococcus is extremely susceptible to drying. Use one plate for each culture obtained. Roll the swab in a Z pattern across the agar surface.
    • Label the plate with patient identification and specimen site.
    • Place the plate and a CO2 generating tablet in a plastic bag. Do not place the tablet on the plate itself. Seal the bag.
    • Transport immediately at ambient temperature. If there is any delay in transport, incubate at 37°C.
    • Cervical culture: Refer to Cervix (Endocervix) for Culture.
    • For male patients, also submit a slide of urethral material for Gram stain. Gram stains of specimens from the endocervix are not as reliable.
  • Rectal culture:
    • Moisten a swab with sterile water and insert the swab into the anal canal just beyond the anal sphincter.
    • Allow 10–30 seconds for absorption of the organisms onto the swab.
    • Withdraw swab gently and inoculate plate as described above.
    • Stool is not an acceptable specimen for gonorrheal culture.
  • If disseminated gonococcal infection is suspected, culture blood and suspicious sites such as petechiae or joint fluid.

Cutaneous (fungus only)

• Hair
  • Scrape the scalp with a blunt scalpel.
  • Place specimen in a dry sterile container.
  • Transport at ambient temperature.
  • The following specimens are also acceptable:
    • Hair stubs
    • Contents of plugged follicles
    • Skin scales
    • Hair plucked from the scalp with forceps

  Note: Cut hair is NOT an acceptable specimen.

• Nails
  • Cleanse the nail with 70–95 percent ALC.
  • Remove the outermost layer by scraping with a scalpel.
  • Place specimen in a dry, sterile container.
  • Transport at ambient temperature.
  • The following specimens are also acceptable:
    • Clippings from any discolored or brittle parts of nail
    • Deeper scrapings and debris under the edges of the nail
• Skin
  • Cleanse the skin with 70–95 percent ALC.
  • Collect epidermal scales with a scalpel, at the active border of the lesion.
  • Place specimen in a dry, sterile container.
  • Transport at ambient temperature.

Ear

• External ear cultures are processed as superficial wounds.
• Middle ear fluid will be processed as a sterile body fluid. If the diagnosis is otitis media, the specimen of choice is middle ear fluid collected by tympanocentesis.

Eye

• Cleanse the skin around the eye with a mild antiseptic.
• Purulent conjunctivitis:
  • Collect purulent material with a regular cotton swab.
  • Place the swab into transport media and transport at ambient temperature or 2–8°C for viral cultures.
• Corneal infections:
  • Swab the conjunctiva as described above.
  • Collect multiple corneal scrapings and inoculate directly onto bacterial agar media (chocolate agar, potato dextrose agar, and sheep blood agar) or viral transport media.
  • Transport at ambient temperature or 2–8°C for viral cultures.
• Intraocular fluid:
  • Collect fluid by surgical needle aspiration.
  • Transport bacterial cultures at ambient temperature, viral cultures at 2–8°C, or frozen for molecular tests.

Nasopharyngeal Aspirates/Washings (virus only)

• For aspirate, attach mucus trap to suction pump and catheter, leaving wrapper on suction catheter. Turn on suction and adjust to suggested pressure.
• Without applying suction, insert catheter into the nose, directed posteriorly and toward the opening of the external ear. Note: depth of insertion necessary to reach posterior pharynx is equivalent to distance between anterior nares and external opening of the ear.
• Apply suction. Using a rotating movement, slowly withdraw the catheter.
• Transport at 2–8°C for viral cultures or frozen for molecular tests.
• For washings, suction 3–5 mL of sterile saline into a new sterile bulb.
• Insert bulb into one nostril until nostril is occluded.
• Instill saline into one nostril with one squeeze of the bulb and immediately release bulb to collect recoverable nasal specimen.
• Empty bulb into suitable dry, sterile specimen container or add 3 mL or less to viral transport media (M4).
• Transport at 2–8°C.

Nasopharyngeal Swab

• Seat the patient comfortably and tilt the head back.
• Insert a nasal speculum.
• Insert a nasopharyngeal swab (on a malleable wire) through the speculum into the nasopharyngeal area.
• Rotate the swab gently and allow to remain for 20–30 seconds.
• Remove the swab and place in a non-growth-promoting transport medium (such as the culturette container from which the original swab has been removed). Place swab in M4 media for viral cultures.
• Transport at ambient temperature or 2–8°C for viral cultures.

Notes:

• Transport media must be used because the swab tip is small and vulnerable to drying. The organisms likely to be present are fastidious.
• For infants, special bulb suction procedures are available.
• If unusual organisms such as Bordetella pertussis are suspected, special culture media is necessary for collection and transport. (Refer to Bordetella pertussis Culture and DFA .)

Nose

Note: This is an inappropriate specimen for anything other than assessment of staphylococcal colonization.

• Collect anterior nares culture with a regular cotton swab. In small children, use a nasopharyngeal swab to facilitate collection.
• Transport at ambient temperature.

Prostate

• Cleanse the glans with soap and water.
• Obtain prostate fluid by digital massage through the rectum.
• Collect fluid using a sterile swab.
• Transport at room temperature.
• Alternatively, a urine specimen obtained immediately before and after massage may be submitted for culture.

Skin

Refer to Abscess, Bullae, Cellulitis, Vesicles, and Wounds.

Sputum

• Assure patient cooperation to get an adequate specimen. ARUP will determine the number of squamous epithelial cells present for specimen adequacy.
• Instruct the patient as follows:
  • Rinse mouth with tap water to remove food particles and debris.
  • Have patient breathe deeply and cough several times to receive deep specimen.
  • Patient should expectorate into dry, sterile container.
• If patient is unable to produce sputum, induce using saline nebulization. Consult respiratory therapy for assistance.
• Transport immediately at ambient temperature. Refrigerate if a delay of more than one hour is anticipated; freeze for molecular tests.

Stool, Feces

• Collect specimen in a clean bed pan or use plastic wrap placed between the toilet seat and the bowl. Do not submit feces contaminated with urine or toilet water.
• Immediately transfer specimen into a clean, dry container or the appropriate preservative.*
• Transport unpreserved stool refrigerated or frozen.

Notes:

• Only loose or diarrheal stools are recommended for routine bacterial and C. difficile cultures.
• Place the specimen in an appropriate stool preservativeor transport media immediately after collection. For ova and parasite, use 10 percent formalin and modified PVA; for routine stool culture, use Cary-Blair transport media.
• If a stool specimen is not available, the following are suitable alternatives for culture:
  • A swab of rectal mucusr
  • A rectal swab inserted one inch into the anal canal (not acceptable for Rotavirus/ Adenovirus EIA)

Throat

• Use a cotton or Dacron swab.
• Use a tongue blade and an adequate light source to ensure proper visualization.
• Reach behind the uvula and swab:
  • Both tonsillar fauces
  • The posterior pharynx
  • Any ulceration, exudate, lesion, or area of inflammation
• Place the swab into the transport media and transport at ambient temperature or 2–8°C for viral cultures.

Tissues

• Tissue collection is an invasive procedure and requires surgery by a trained physician.
• Collect tissue aseptically. Include material from both the center and the edge of the lesion. Submit actual tissue specimen, not swab of tissue surface.
• Place the specimen in a sterile container on sterile gauze moistened with sterile nonbacteriostatic saline.
• Transport immediately at ambient temperature, in a manner to ensure recovery of anaerobic organisms. For virology cultures, do not allow the tissue to dry. Transport tissue suspended in viral transport media (M4) at 2–8°C, or frozen for molecular tests.
• Do not submit tissue in formalin.

Urethra

Refer to Chlamydia/Gonorrhea.

Urine

• Instructions for female patients to collect midstream urine for bacterial culture:
  • Remove undergarments.
  • Wash hands thoroughly with soap and water, rinse them, and dry them on a disposable paper towel or shake off excess water.
  • Spread labia with one hand and keep itcontinuously apart.
  • Take the open sterile cup in the other hand without touching the rim or inner surface of the cup or lid.
  • Void 20 to 25 mL into the toilet and catch a portion of the rest of the urine in the container without stopping the stream. Do not touch the legs, vulva, or clothing with the cup.
  • Place the lid on the cup.
• Instructions for male patients to collect midstream urine for bacterial culture:
  • Wash hands.
  • Retract the foreskin completely.
  • Void 20 to 25 mL into the toilet and catch a portion of the remaining urine in the cup without stopping the stream. Do not touch the cup with the penis.
  • Place the lid on the cup.
• First-void urine for nucleic acid amplification tests:
  • Patient must not have urinated during the previous two hours.
  • Collect the first 10–50 mL of the urine stream in a clean, empty plastic cup.
  • Place the lid on the cup.
  • Transport urine refrigerated in test–specific transport media.
• Suprapubic aspiration:
  • This is not a routine technique and is best performed by an experienced individual. Descriptions of the method are readily available in literature.
  • These specimens are acceptable for anaerobic culture, and should be submitted in an anaerobic environment if an anaerobic culture is requested.
• Indwelling catheter urine:
  • Do not collect urine from the drainage bag because growth of bacteria outside the catheter may have occurred at this site.
  • Clean the catheter with an alcohol pad.
  • Use a sterile needle and syringe to puncture the tubing. Aspirate the urine directly from the tubing.
  • Transfer the urine to a sterile specimen container.
  • Urine catheter tip cultures are not acceptable.
• Specimen handling:
  • Label the container immediately and refrigerate at 2–8°C within 10 minutes of collection or transfer > 2 mL urine into a boric acid transport tube.

Wounds

• For closed wounds, refer to Abscess and Bullae, Cellulitis, Vesicles.
• For open wounds:
  • Clean the sinus tract opening of the wound surface mechanically, without using a germicidal agent, to remove as much of the superficial flora as possible.
  • Attempt to culture the base or edges of the wound to avoid collecting normal flora organisms.
  • The following are preferred specimens for sinus tracts:
    • Aspiration material obtained by needle or catheterization.
    • Curettings from the lining of the sinus tract.
  • Specimen swabbings of sinus tracts are acceptable only if the above cannot be obtained. Swabs of sinus tracts may not accurately reflect underlying disease process.
  • Do not submit cultures of superficial lesions for anaerobic culture. Biopsy of advancing margin of wound is the preferred specimen for anaerobes, mycobacteria, and fungi.

Viral Transport Media (M4)

Some specimens can be submitted without utilizing a transport media with a reasonable expectation of virus viability. Specimens in this category include sterile fluids such as cerebrospinal fluid, pleural fluid, blood or bone marrow submitted in EDTA, urine, as well as some nonsterile specimens such as nasopharyngeal washings, sputum, bronchoalveolar lavage, and feces. Whenever there is a question of stability, the specimen should be placed in a suitable virus transport media such as Microtest M4. Refer to specific test in the Laboratory Test Directory for more information.

• Tissue and biopsy material can be placed directly into the viral transport media. Each specimen need not be more than 1–2 cm in diameter.
• Abscess material, bullae, pustules, vesicles, lesions, and skin scrapings can be collected on the swab and placed directly into viral transport media. If the material has been aspirated, place no more than 3 mL (equal to the amount of transport media) in the vial of M4.
• CSF should be submitted in a sterile container.
• Urine should be submitted in a sterile container.
• Bronchoalveolar washings, nasopharyngeal washings, sputums, and other sterile body fluids can be submitted in sterile containers or no more than 3 mL placed in the M4 tube.
• Stool should be submitted in a sterile container, or a small aliquot the size of a walnut can be placed in the M4 tube.
• Blood and bone marrow should be submitted in an EDTA tube. Do not extract the buffy coat.

Viral transport media (M4) criteria is the same for other liquid viral transport media such as those available from Bartels, Syva, etc. labeled for viral/chlamydia transport. Swabs that are made of calcium alginate and wood are known to interfere with the recovery of some viruses. These can also act as PCR inhibitors and are not appropriate for this type of testing.

Not all types of viral transport media have been validated for all testing; some may require a disclaimer, dependent on the assay.