Massachusetts On-Site Testing Program for Nursing Homes and Assisted Living

Assay overview

The CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in nasopharyngeal and oropharyngeal swabs collected from individuals who may have contracted the virus. Testing is limited to the Clinical Research Sequencing Platform at the Broad Institute which is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests.

The test was validated on 103 positive and 50 negative specimens representing nasopharyngeal (NP) and oropharyngeal (OP) swabs and found to have 100% accuracy with a lower limit of detection of 0.20 copies/µl.

Results are presumptive for the detection and identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in the nasopharynx and oropharynx during the acute phase of infection. Positive results are indicative of active infection with SARS-CoV-2 but do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. In addition, nucleic acid detection can persist following clearance of active viral replication. Laboratories within the United States and its territories are required to report all positive results to the appropriate public health authorities.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient treatment or other patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

Testing with the CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay is intended for use by trained and competency-certified clinical laboratory personnel, specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures. The CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay is only for use under the Food and Drug Administration’s Emergency Use Authorization.

Assay fact sheets

Collecting samples

Sample collection kit

Once the kit arrives at your institution, open the contents of the kit. Contents include:
  • Test requisition forms (1 per patient)
  • Nasopharyngeal swabs (1 per patient)
  • Tubes with media or saline (1 per patient)
  • Labels (2 per patient, one for the paper requisition form and one to place on a matching tube)
  • Biohazard bags to seal contents after you complete the test (1 per patient)
  • Ice packs for shipment back to CRSP - immediately freeze the ice packs for return shipping

Patient specimen collection

  1. Collect samples from one patient at a time.
  2. Specimens should be collected by trained personnel in accordance with manufacturer guidelines and in accordance with FDA approved collection devices (FAQs on Diagnostic Testing for SARS-CoV-2).
  3. Fill out the top of the Requisition Form by writing:
    • Nursing home facility name
    • Patient name (First and Last)
    • Date of birth
    • Specimen collection date
  4. Affix a barcode label on the requisition form in the space provided.
  5. Place the matching paired barcode label on tube containing media.
  6. Write the Patient Name or Date of Birth on the tube containing media (or use your own label).
  7. Collect swab sample from patient in accordance with established procedures.
  8. Break the swab tip into the tube and close tightly to prevent leaking.
  9. Place the tube in a sample bag and seal. (DO NOT place the requisition in with the tube in case of leakage).
  10. Collect all paper requisitions in a folder together and place in the box with the sealed bags.

Nonconformances

Samples may not be processed or significantly delayed if they arrive with any of the following nonconformances:

  • Non-scannable barcodes
  • Unlabeled tubes or tubes without 2 identifiers (barcode + patient name or DOB)
  • Tubes with insufficient media
  • Improperly capped tubes
  • Tubes with media that has congealed
  • Missing paper requisition form

Submitting samples to the Broad CRSP laboratory

  • After collecting samples from all patients, place all sealed bags in a box with ice packs and arrange courier pick up by calling 617-782-1130.
  • Provide the courier with information on where to pick up from (your facility) and let them know that they are delivering under Acct. #405 to the Broad Institute at the following address:

    CRSP Laboratory at the Broad Institute
    320 Charles Street
    Cambridge, MA 02141

Additional notes about sample collection and shipping

  • Specimens should be collected by trained personnel in accordance with manufacturer guidelines and in accordance with FDA approved collection devices (FAQs on Diagnostic Testing for SARS-CoV-2).
  • This test has currently been validated for use with NP and OP swabs.
    For a list of approved swabs, please visit FDA FAQ (Q: I am having trouble obtaining...).
  • VTM, UTM, M4, M5, M6, saline, and MTM are all acceptable transport media types.
  • Acceptable specimens should be transported in accordance with guidelines established by the Massachusetts state public health laboratory:
    • Refrigerate all samples at 2-8℃ prior to and during transport within 24 hrs.
    • If the specimen is to be submitted greater than 24 hrs post collection, freeze the specimens at -20℃ or below and then ship on dry ice.
    • Ship as a Category B (UN3373 - Biological Substances).
  • Specimen collection guidance is available here.

How you will receive results

Results are typically returned within 72 hours of receipt.

A physician's name was provided when the facility enrolled through the call center. All test result will be sent via secure email to that ordering physician.

The physician will receive two emails for each patient:
  1. One email that contains the password to access the results.
  2. A second email that has the results included as encrypted attachments in Excel spreadsheet format as well as in PDF format. Both files can be accessed using the password from the first email. Examples of what the attachments may look like are provided below.

Example Excel spreadsheet of results. Click here to download it.

Example PDF report of results.

Specimen rejection criteria

Regardless of option chosen above, samples may be rejected or significantly delayed if they arrive with any of the following nonconformances:

  • Non-scannable barcodes
  • Unlabeled, mislabeled or incomplete labeled specimen tubes
  • Tubes with insufficient media
  • Improperly capped tubes
  • Tubes with media that has congealed
  • Missing electronic manifest file
  • Incomplete electronic manifest file
  • Paper manifest not included in the box with samples
  • Specimens(s) deemed unacceptable due to the requirements of test; i.e. unvalidated specimen type, wrong swab type or placement in specimen tube
  • Suspected specimen contamination
  • Pre-analytic handling and transport requirements not met

Unsatisfactory/TNP (Test Not Performed) Result Code Information*

*updated July 28, 2020

Unsatisfactory reason codes that only apply to samples submitted in media are NOT italicized.

Possible Unsatisfactory/“Test Not Performed (TNP)” Reason Codes for samples submitted using dry, anterior nasal swab collection (aka NO media) OR in media are italicized.

TNP reasons found in CareEvolve are shown in bold/italics.

When the laboratory issues an unsatisfactory or TNP result in a report, the individual must be tested again and a NEW sample must be submitted to the laboratory in order to receive a confirmed test result.

Result Definition ("reason" or column J in the results file) Description
NEG 2019-novel Coronavirus (2019-nCoV) not detected by the qRT-PCR assay. Consider testing for other respiratory viruses or re-collecting for 2019-nCoV testing. Note: Optimum timing for peak viral levels during infections caused by 2019-nCoV have not been determined. Collection of multiple specimens from the same patient may be necessary to detect the virus. Applies to both assays with and without media.
INCONCLUSIVE Inconclusive for 2019-novel Coronavirus (2019-nCoV) by qRT-PCR with one of the two viral probes (either N1 or N2) being positive while the other is not detected after two attempts on this specimen. Consider re-collection of specimen. Note: Optimum timing for peak viral levels during infections caused by 2019-nCoV have not been determined. Collection of multiple specimens from the same patient may be necessary to detect the virus. Applies to samples submitted in media.
POS Positive for detection of 2019-novel Coronavirus (2019-nCoV) by qRT-PCR. Applies to both assays with and without media.
INVALID This specimen failed to produce a valid result after two attempts. An invalid result means no nucleic acids (viral or human) were detected by qRT-PCR. Consider re-collection of specimen. Applies to both assays with and without media.
CANCELED Testing canceled per submitter. Canceled by ordering provider.
UNSATISFACTORY_1 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: improper specimen transport medium. Applies to samples submitted in media. The media cannot be pipetted (too viscous).
UNSATISFACTORY_2 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: inappropriate timing of collection relative to specimen receipt. Specimens must be received within 72 hrs of collection unless frozen. Samples are received more than 72 hours after collection date
UNSATISFACTORY_3 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: sample tube unlabeled. Sample tube is unlabeled and cannot be matched to an order.
UNSATISFACTORY_4 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: insufficient volume. Applies to samples submitted in media. The tube is either empty or does not contain enough media volume to perform the test.
UNSATISFACTORY_5 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: tube sample data does not match the submission form. This is used when the order and tube label do not match.
UNSATISFACTORY_6 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: laboratory accident. This is used when a test can not be performed due to a laboratory error and the sample must be resubmitted.
UNSATISFACTORY_7 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: leaking/broken tube. This is used for broken or leaking tubes. Very similar to reason #10, but this is used for samples submitted in media.
UNSATISFACTORY_8 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: sample not received. This is used when we have an electronic or physical order in hand but do not receive a physical specimen.
UNSATISFACTORY_9 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: incomplete requisition. This is used when required elements of the requisition are missing and we are unable to obtain the information before the sample expires, or if there is not enough information to know who to reach out to for the missing information.
UNSATISFACTORY_10 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: Specimen tube received uncapped or broken. This is used in cases where the sample arrives in an uncapped or broken tube.
UNSATISFACTORY_11 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: Sample unsuitable for automated processing. The first step in processing is to add media to the swab. After adding media, some samples are too viscous to process. This most often happens when the patient does not blow their nose before collecting the sample, resulting in excessive mucus. This reason may also be used if we cannot read the barcode on the tube.
UNSATISFACTORY_12 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: Swab collection not to submission standards. This is used when the swab is upside down in the tube, there are multiple swabs in the tube, or when there is no swab in the tube.

Test method and limitations

Test method

This RT-PCR test, a high throughput version of the CDC 2019-nCoV Realtime RT-PCR test, was performed by the Clinical Research Sequencing Platform at the Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02141, CLIA #22D2055652. This test is not FDA-cleared but its performance characteristics were established by a CLIA-certified high-complexity laboratory in accordance with CLIA regulations. The test was validated on 103 positive and 50 negative specimens representing nasophryngeal (NP) and oropharyngeal (OP) swabs and found to have 100% accuracy with a lower limit of detection of 0.2 copies/µl. Subsequently, we validated oropharyngeal swabs (OP) which also showed 100% concordance with a second lab’s results. Further, we have validated the use of Saline as an alternate media (to VTM) for collection and transport of swabs.

Test limitations

Limitations include:

  • Negative results do not rule out SARS-CoV-2 infection, particularly in those who have been in contact with the virus. Follow-up testing with a molecular diagnostic should be considered to rule out infection in these individuals.
  • If the virus mutates in the RT-PCR target region, SARS-CoV-2 may not be detected or may be detected less predictably.
  • Inhibitors or other types of interference may produce a false negative result. An interference study evaluating the effect of common cold medications was not performed.
  • The test was validated for use with upper respiratory specimens obtained via nasopharyngeal or oropharyngeal swabs in VTM, UTM, M4, M5, M6, saline, and MTM media. The performance of this test has not been established for other specimens. Specimens collected using other FDA recommended Specimen Collection Materials listed in the FDA COVID-19 Diagnostic Technologies communication (March 26, 2020) are processed with the caveat that they were not all validated for use with this test and the result must be interpreted in this context. Furthermore, a false negative result may occur if a specimen is improperly collected, transported or handled.
  • False negative results may also occur if amplification inhibitors are present in the specimen or if inadequate numbers of organisms are present in the specimen. Optimum specimen types and timing for peak viral levels during infections caused by SARS-CoV-2 have not been fully determined. Collection of multiple specimens (types and time points) from the same patient may be necessary to detect the virus.
Quality control (to avoid and detect any contamination in the assay)
  1. Every qPCR run of 96 samples has NTC (no template control) in well H12.
  2. When preparing taqman mastermix, work space and all pipettes are DNA-zapped and UV treated (3 min).
  3. When preparing mastermix, empty PCR plates, empty qPCR plates, empty tubes, tips, and water for mastermix are UV-treated.
  4. Mastermix preparation occurs in a PCR-free clean room (no amplified PCR product in this room, no Viral RNA template in this room, and no positive control sample in this room.)
  5. Any opened positive control sample is prepared and physically stored in a separate room from where taqman mastermix plates are prepared.
  6. PPE (gowns, booties, gloves) is changed between the mastermix prep room and the room where positive control is added to the plate.
  7. No Template Controls (NTC) are prepared in a separate room from where the positive control is stored/prepared. NTC water and tubes are UV-treated before and after preparation.
  8. qPCR preparation steps are performed on an automated liquid handling platform. All automation for qPCR preparation is done using disposable, filtered tips.
  9. Before manually adding positive and negative controls to the RNA sample plates, bench space and pipettes are DNA-zapped.
  10. Plate-seals on extracted RNA sample plates are gently pierced with tips on the automated liquid handler when aspirating RNA and dispensing into the qPCR plates. This eliminates the possibility of spray between sample wells that can occur during manual removal of seals.
  11. qPCR occurs in a separate room from mastermix plate creation or RNA sample addition.
  12. Extraction and qPCR automation scripts are visually monitored to ensure no unexpected spraying or droplets.
  13. Each qPCR run is reviewed by a clinical supervisor before being reported out.

Contact information

For any questions you can contact us by email or phone.