Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized clinical microbiology laboratories. Numerous studies have demonstrated the superiority of MALDI-TOF MS systems over conventional methods in identifying a wide range of microorganisms. Since a large number of clinical microbiology laboratories have adopted MALDI-TOFMS as a primary method, labs absolutely need to implement sufficient quality control (QC) practices to en-sure they report accurate identifications.

Until recently, few guidelines for using MALDI-TOF MS in clinical microbiology existed. However, in April 2017, the Clinical and Laboratory Standards Institute (CLSI) published a document, Methods for the Identification of Cultured Microorganisms Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (M58-Ed1), which lays out comprehensive recommendations for MALDI-TOF MS in clinical microbiology laboratories. This CLSI document, as well as recommendations from instrument manufacturers and regulatory agencies, all agree on the need for robust internal and external QC designed to account for the unique performance and limitations of MALDI-TOF MS.

Internal QC

Laboratories must perform internal QC before using MALDI-TOF MS to identify microorganisms. Internal QC consists of an automatic instrument calibration using a manufacturer-specified calibration standard. Depending on the system, calibrators include a manufactured extract of Escherichia coli (E. coli) or a specific E. coli calibration strain. Laboratories should ensure that they follow manufacturers’ specifications for preparing, using, and storing calibrators.

During calibration, the calibrator generates and automatically analyzes a mass spectrum to check the spectrum baseline and ensure the expected calibration peaks are present. Laboratories use these parameters to confirm their instrument settings are appropriate and their instruments will automatically adjust if necessary. The calibrator spectrum is also run against the reference database to ensure the correct identification is given with a level of confidence that meets the manufacturer’s specifications. Laboratories must perform calibration before every run.

In addition to ensuring a successful calibration, the College of American Pathologists (CAP) Microbiology Checklist requires that labs run a calibrator control each day of patient testing, when a new target is used, or more often if the manufacturer recommends it. The CAP checklist also re-quires that labs maintain a written procedure for operating and calibrating the instrument as well as all calibration records.

It is important for labs to not only document calibration results but also promptly investigate calibration failures. Spectral acquisition cannot occur until calibration is successful. Calibration failures of-ten result from user error, typically due to improper application of the calibrator. Labs can assess potential user error by reapplying and reanalyzing their calibrator. Calibration failure also occurs when the calibrator has been prepared improperly or when problems crop up with the matrix, reagents, tar-get, or instrument.

External QC

Laboratories should perform external QC using appropriate positive and negative controls. While most manufacturers do not, CAP requires that positive controls (either an appropriate control micro-organism or calibrator) be tested each day of patient testing.

For positive controls, labs should test well-characterized strains using the same methodology they use for patient isolates. For example, yeast typically require extraction prior to analysis, so labs should process yeast QC organisms using the same extraction methodology. Most laboratories should, at a minimum, test a bacterial QC organism on each day of testing. If laboratories are using MALDI-TOF MS to identify yeast, mycobacteria, Nocardia, or molds, appropriate QC organisms for each organism type should be run each day they test for these microorganisms. Labs must obtain correct, high-confidence identifications for all QC organisms. If a lab fails to identify a QC organism, it must investigate and suspend patient testing until the problem is resolved. If a Food and Drug Ad-ministration (FDA)-cleared platform is used, manufacturers may recommend specific American Type Culture Collection strains for use as positive controls.

CAP also requires that labs use manufacturer-recommended control microorganisms for FDA-approved platforms. While there are no specific QC organism recommendations for laboratories operating research-use-only platforms, laboratory directors should ensure appropriate control organ-isms are tested each day. Results of QC testing should be documented and periodically reviewed to assess not only instrument performance but also testing consistency among users.

Labs should also include a negative control with each run. Typically the negative control consists of reagents spotted directly on the target plate or slide. Matrix should be applied to a random blank spot on each target plate or slide to ensure there is no reagent contamination and, for systems that use a reusable target plate, to ensure that the target plate has been adequately cleaned between runs.

CAP requires labs that operate platforms with reusable targets test a blank negative control to en-sure adequate cleaning of the target. If an extraction is performed, the reagents used for the extraction can be spotted and overlaid with matrix to ensure no false-positive results are produced due to reagent contamination. Because of the implications of reporting organism identifications directly from blood cultures, labs should test lysis buffers and other reagents used for sample preparation to ensure they are free of contamination. Currently, there are no regulatory requirements for testing re-agents on a routine basis.

Ensuring Spectral Quality

Once controls are satisfactory, testing of patient samples can begin. To ensure they produce high-quality spectra, labs must follow recommendations for optimal culture conditions and sample preparation, as well as manufacturers’ recommendations for approved media types. If necessary, labs should validate additional media types. They should also use fresh isolates whenever possible. Spectral quality depends on placing an optimal quantity of microorganism on the target plate, and special spotting techniques and extractions might be necessary to identify certain microorganisms. Labs should consider analyzing all isolates in duplicate and have procedures in place to help re-solve discordant results between spots.

In addition, since MALDI-TOF MS cannot identify all organisms in polymicrobial cultures, labs should ensure cultures are pure. Ensuring purity is particularly important when microorganisms are identified directly from liquid cultures: Labs should report results as preliminary until purity can be confirmed. A robust training and competency assessment program is also essential to ensure testing staff are competent in performing identifications of commonly encountered microorganisms and in using and maintaining the instrument.

Reporting Identifications

Another important QC consideration involves interpreting and reporting of MALDI-TOF MS identifications. Spectral databases differ in composition depending on the manufacturer and whether they are FDA-cleared. Users also can develop custom databases. While manufacturers validate identifications from their FDA-cleared platforms and these identifications have been cleared for reporting, laboratories still must determine how to report them. Laboratories face choices such as reporting the identification to the genus, species, or complex level.

Reporting also may differ based on specimen source. For example, reporting species-level identifications for coagulase-negative staphylococci from certain sites may lead to clinicians attributing a higher degree of significance to a culture result.

A major challenge for laboratories operating research-use-only platforms is how to report unfamiliar or uncommon identifications. If these laboratories do not independently validate them, such identifications should always be confirmed with supplemental testing. However, validating targets for rarely encountered organisms is especially difficult due to problems obtaining an adequate number of isolates for validation studies.

In addition, labs should familiarize themselves with the identification limitations of their MALDI-TOF MS platform by reviewing technical bulletins, comments provided by the software, and from periodic review of the scientific literature. Unless they extensively validate lower confidence thresholds, labs should adhere to the manufacturer-recommended thresholds for genus and species-level identifications.

In light of the numerous considerations for interpreting and reporting of MALDI-TOF MS identifications, labs should develop reporting guidelines for their bench technologists to ensure accuracy and consistency in reporting. Labs also should participate in external proficiency testing programs to en-sure they are proficient in correctly generating and reporting identifications.

MALDI-TOF Limitations

While MALDI-TOF MS is a robust system for microorganism identification, it is not infallible. For ex-ample, MALDI-TOF MS cannot discriminate reliably between closely related microorganisms, and incorrect identifications can occur from user error (such as spotting the organism on the wrong spot on the target plate), analysis of mixed cultures, and other reasons.

Due to these limitations, labs should consider MALDI-TOF MS results as one component of the overall testing system for identifying microorganisms. Results should always be reviewed by a trained microbiologist and correlated with other characteristics, including growth requirements, colo-ny morphology, and Gram-stain. In addition, labs should ensure they maintain their instruments and update their database in keeping with manufacturers’ recommendations and retain all maintenance records in the laboratory.

As more laboratories abandon traditional methods in favor of MALDI-TOF MS, those that make the switch absolutely need to follow best practices for quality control by adhering to recommendations given by instrument manufacturers, regulatory agencies, and other guidelines.

Lori Bourassa, PhD, MPH, D(ABMM), is an assistant professor and assistant director of the clinical microbiology division at the University of Washington Medical Center in Seattle. +Email: [email protected]

CLN's Focus on Mass Spectrometry is supported by Waters Corporation.

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