False-Positive Investigation Toolkit: Pre-Analytic, Analytic, and Post Analytic Prevention Practices

What to know

False-positive Mycobacterium tuberculosis complex (MTBC) results can be caused by pre-analytic, analytic (laboratory practices), and post-analytic errors. Information from each of these three areas will be described for readers to refer to and utilize during potential false-positive investigations.

Introduction

In any mycobacteriology laboratory, false-positive results can occur from the inadvertent transfer of bacilli from one specimen or culture to another specimen or culture through cross-contamination, mislabeling errors, specimen mix-ups, or the use of nonsterile reagents.

Pre-Analytic Prevention Practices

Specimens submitted to a laboratory should be properly collected, labeled, stored, and transported. Each of these processes can affect specimen quality, accuracy of test results, and can result in false-positive results. It may be helpful for laboratories to create specimen collection and submission guidelines for submitters that includes information regarding acceptable specimen types, minimum volumes, collection techniques, labeling, storage, transport, and laboratory rejection criteria1.

It is important to properly collect adequate specimens of acceptable quality for laboratory testing. Contamination can be introduced at the time of collection. For optimal sputum specimens, patients should be instructed on proper specimen collection techniques to ensure a quality specimen is collected.

The material should be mucoid and not saliva or spit. Specimens should not be overtly bloody. The minimum volume of sputum collected should be 3mL2. The specimen should be collected completely inside a sterile container and material should not be on the outside of the container. For guidance on other specimen types, please refer to Table 3 in CLSI M48, 2nd Edition2.

Each specimen container, labeled prior to time of collection, should have the correct patient name and a unique identifying number (two patient identifiers) clearly written or printed on the specimen container. It is best practice for the submitter to match the patient specimen to the specimen test order requisition form. Patient name and identifiers on the collection container should match what is written or printed on the test order requisition form.

Specimen containers should be properly closed using the container threads and sealed using laboratory film or another suitable material before transport or shipping. Packaging and shipping for diagnostic mycobacteriology specimens should be in accordance with Department of Transportation (DOT) and the International Air Transport Association (IATA) packaging requirements for UN 3373 Biological Substance, Category B.

Proper storage and prompt transport of diagnostic specimens (e.g., sputum, other specimen types) from the collection site or submitter to the testing laboratory is extremely important for quality test results and efficient result reporting. Collection sites should refrigerate diagnostic specimens that cannot be transported immediately in order to reduce overgrowth of contaminating organisms. Specimens should be delivered to the laboratory as soon as possible (within 24 hours of collection being optimal) and specimens should not be batched for shipment if at all possible3. Specimens collected at the end of the day or on a Friday, when transport may not be available, should be stored in the site's refrigerator and then transported with a cold pack and delivered to the laboratory the following morning or after the weekend4.

Laboratory Prevention Practices - Analytic

Laboratories that perform mycobacteriology testing should ensure that appropriate practices and procedures are in place to avoid cross-contamination. Best practices are those methods, techniques, or processes that have been generally accepted through experience and research to be reliable and lead to an accurate result. These processes may reduce the likelihood of false-positive test results, or could simplify investigating, identifying, or resolving potential false-positive situations.

When processing specimens from patients that are documented as known acid-fast bacilli (AFB) positive, there is an increased opportunity for the carryover of organisms from positive to negative specimens. Production of aerosolized particles during processing of smear-positive specimens, use of MTBC as a positive control, or contamination of reagents are major causes of cross-contamination5. The use of best practices should reduce the opportunity for transfer from positive to negative specimens. Laboratory policies and procedures should be designed to minimize the risk of potential cross-contamination:

  • Use of separate biological safety cabinets (BSC) if possible: When feasible, do not use the same BSC for processing initial diagnostic specimens and known positive patient specimens or isolates to prevent cross-contamination56. If only one BSC is available, ensure that diagnostic specimens are not handled at the same time as culture growth and that the BSC is decontaminated between processing batches.
  • Use individual aliquots of reagents for each specimen being processed: Never use a common flask or beaker to dispense reagents78910. Use individual or daily aliquots of processing reagents and buffers and discard any leftover reagents not used that day4.
  • Avoid processing large batches of specimens: Processing large numbers of specimens at one time increases the odds that aerosolization from a positive specimen with MTBC will contaminate adjacent specimens111. Process specimens in a batch size small enough to maintain the timing of the digestion- decontamination step. Additionally, only process the number of specimens than can be placed in a centrifuge at one time.
  • Process known AFB smear-positive clinical specimens either after or separately from other specimens: AFB smear-positive specimens are more likely than smear-negative specimens to be a source of cross-contamination during batch processing 12. Separately processing specimens from patients previously known to be smear- or culture-positive can significantly reduce the rate of false-positive results in a laboratory13. If processing separate batches is not feasible, the laboratory should consider placing these specimens at the end of the processing batch.
  • A negative control should be included with each batch of cultures that are inoculated to monitor for contamination4 : The negative control may be 5 to 10 mL of sterile water, broth, or buffer placed at the end of the processing batch and should be processed in the same manner as patient specimens. A note of caution: a negative control can be negative yet cross-contamination within a batch may have still occurred. The negative control would likely pick up a large contamination issue but not necessarily an isolated issue.
  • Do not process patient specimens in the same batch as laboratory proficiency testing (PT) samples or quality control (QC) strains: Batch processing highly smear- and culture-positive strains used for proficiency surveys with clinical specimens can result in cross-contamination.12 Process PT and QC strains in separate batches and preferably not in the same BSC used for patient specimens.
  • Use caution when using positive controls: M. tuberculosis is a hardy organism that can survive in the laboratory and contaminate patient clinical specimens. Cross-contamination of patient specimens in a laboratory that used the avirulent control strain H37Ra in the same BSC on a different day than specimen processing has been documented.1415 Laboratories should be sure to follow decontamination practices after use of positive controls.
  • Handle specimen containers properly during processing:
    • Leave a space between tubes in the rack.
    • Keep specimen tubes tightly closed and clean the outside of each tube before vortexing or shaking.
    • When adding reagents, open only one tube at a time.
    • Pour reagents slowly down the side of the inside of the tube to avoid splashing and creating aerosols.
    • Never touch the lip of the tube with the reagent ontainer.
    • After mixing or vortexing, wait five minutes before opening tubes to allow aerosols to settle.
    • Open tubes gently to avoid aerosol generation.
    • Use a discard container that is splash proof when pouring off reagents.
  • BSCs must be used properly16 :
    • Do not impede or disturb the airflow during specimen processing.
    • Place materials as far back inside the BSC as is comfortable, without blocking the rear grill.
    • Place aerosol-generating equipment (e.g., vortex mixers, mini-centrifuges) towards the rear of the cabinet.
    • Place discard pans to one side of the cabinet interior.
    • Use absorbent pads soaked in disinfectant on the work surface to minimize splatter and aerosol generation in case of spillage.
    • Decontaminate work surfaces before and after using the BSC.

A patient sputum specimen with a positive AFB smear result with a subsequent culture that is negative for growth with no clinical evidence of pulmonary TB or the patient is known to be on therapy should be considered suspicious for a potential false-positive smear result.17 Some patients can shed dead AFB long after cultures are negative.18 AFB microscopy is not specific and will detect nontuberculous mycobacteria (NTM) and acid-fast environmental contaminants. It is essential not to introduce environmental contaminants when preparing reagents and during wash steps of the procedure. Patient AFB smears should only be examined and reported when control slides are acceptable.

To avoid false-positive AFB smear readings:19

  • Use new, clean, greaseless, and unscratched slides.
  • Match patient identifier on the slide with clinical specimens.
  • Label slide with material that stays permanently affixed during the staining procedure (e.g., graphite or diamond tip pencil).
  • Use a negative control slide to monitor environmental contaminants during the staining process.
  • Use a positive quality control slide to ensure that staining occurs as expected.
  • Avoid using bulk containers of reagents and carboys of water.
  • Use filtered, distilled, or deionized water.
  • Ensure slides do not touch on the staining rack. Do not use staining jars.
  • Filter stains if precipitates are present.
  • Wipe oil from lens after each AFB-positive smear is read if an oil immersion lens is used.
  • Ensure that microscopists are competent before reading and reporting AFB smear results.

For molecular testing that uses targeted amplification, it is important to prevent contamination of patient specimens with amplified nucleic acid (DNA) from other patient specimens20. Assays that do not use targeted amplification still have the potential for cross-contamination between patient specimens. Laboratorians can reduce the risk of molecular contamination through:

Use of separate workspaces for molecular testing allowing for separation of the different testing procedures and minimizing the risk of backflow contamination of amplicons.

Ideally, these workspaces would be separate rooms, but at the very least should be defined areas. The distinct molecular areas should be: the "reagent preparation room/area," the "sample preparation room/area," and the "amplification detection room/area.20"

  • The "reagent preparation room/area" and "sample preparation room/area" should both be considered clean areas.
  • The "reagent preparation room/area" should be reserved for only reagent preparation and the room must not house any DNA, RNA, or patient specimens.
  • The "sample preparation room/area" is where specimen preparation, nucleic acid extraction, and reaction set-up occur. This room should be under negative pressure to prevent the spread of aerosolized specimens, controls, or calibrators.
  • The two pre-amplification rooms/areas ("reagent preparation room/area" and "sample preparation room/area") should be cleaned before and after use with a 10-20% bleach solution followed by ethanol or clean distilled water or as recommended by equipment manufacturers for specific equipment or work spaces1621.
  • The "amplification detection room/area" is the post-amplification room where nucleic acid amplification and analysis occur. This room is considered "dirty" due to the manipulation of amplicons and should also be under negative pressure to minimize the risk of contamination.
  • Routine laboratory cleaning of all rooms can reduce the risk of contamination from patient specimen DNA/RNA, controls, or amplicons.

One of the most important practices to reduce contamination during molecular testing is to implement unidirectional workflow.

This practice ensures that work moves from clean (pre-amplification) to dirty (post- amplification) areas and decreases the potential for carryover or backflow contamination of amplicons.

Laboratorians should limit their movements to unidirectional workflow: moving through the clean rooms ("reagent preparation room/area" and "sample preparation room/ area") to the dirty room ("amplification detection room/area") without going directly back to a previous room. Movement backwards between the rooms could result in introduction of DNA/ RNA or amplicons into a clean room, thus causing contamination and potentially false-positive results.

It is best practice to have dedicated equipment and personal protective equipment (PPE) for molecular testing within each of the defined rooms listed above21.

Dedicated equipment and PPE will reduce contamination from exposure to patient specimens or amplicons. Specifically, pipettes, racks, ice buckets, and cooling blocks should be dedicated to specific tasks within each room and should use filtered tips to prevent aerosolization and contamination of pipettor barrels. PPE should be disposable when possible and gloves should be changed frequently. Many laboratories utilize a color-coding system to ensure that equipment and PPE are maintained in the appropriate rooms.

Additionally, laboratorians should utilize best practices to prevent aerosolization and to minimize cross-contamination. If patient specimens, quality controls, or calibrators from the "sample preparation room/ area" are introduced to the "reagent preparation room/area" or if amplicons from the "amplification detection room/area" are introduced to either clean room, testing results can be compromised leading to contamination and questionable results. Thus, it is important to monitor clean rooms for potential contamination through the use of wipe tests (performed at least monthly and when troubleshooting) collected from various laboratory surfaces in the clean rooms.

If a polymerase chain reaction (PCR) target is found, the area is considered contaminated and must be decontaminated immediately. An investigation must be performed to try to determine the source or practice that led to the contamination in order to prevent it from recurring in the future.

Laboratorians should use quality control measures to ensure testing runs are as expected. Specifically, no-template controls (also known as blank controls) are necessary.

No-template controls should consist of water, buffer, or specimen transport medium that is assayed with the testing reagents, but without patient specimens or nucleic acid. These negative controls are used to detect contamination of reagents or background signal. Controls ensure that the assay performed as expected and that patient results are accurate, reliable, and reportable.

Post-Analytic Prevention Practices

The accurate verification and reporting of laboratory results are essential. Post-analytic practices include all steps taken to verify and review results, to communicate results, and to provide easily interpretable results to submitting laboratories or healthcare providers. Laboratorians can reduce common post-analytic errors by:

  • Reviewing test results to ensure the correct result was reported for the correct patient.
  • Ensuring that data entry or transcription errors did not occur when results were entered into the laboratory information management system (LIMS).
  • Verifying that the correct report was sent to the submitting laboratory and healthcare provider.
  • Monitoring periodically whether reports of laboratory results and associated reporting language were received and interpreted correctly by the submitting laboratory or healthcare provider. This in turn also allows for laboratory review of report design, format and ease of use.
  • Reviewing quality control results.
  • Ensuring quality control procedures were followed (e.g., secondary review).
  • Cross-referencing genotypes for samples in question to any known positives processed the same day or to the H37Rv/Ra control strain.
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Content Source:
Division of Tuberculosis Elimination ; National Center for HIV, Viral Hepatitis, STD, and Tuberculosis Prevention; Centers for Disease Control and Prevention
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