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Expires: January 15, 2025
PresenterRobin Patel, M.D., is the Division Chair of Clinical Microbiology in the Department of Laboratory Medicine and Pathology in Rochester, Minnesota. She holds the academic rank of Professor of Medicine and MMicrobiology.
Contact us: mcleducation@mayo.edu.
Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic. In this month’s "Hot Topic," Dr. Robin Patel, discusses how matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF) works for bacterial identification, including the strengths and limitations of this technology, and Mayo Clinic’s experience with the technology in the clinical laboratory. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patient’s health care.
Thank you for the introduction Dr. Binnicker.
Dr. Patel reports grants from CD Diagnostics, BioFire, Curetis, Merck, Contrafect, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, EnBiotix, Contrafect and The Medicines Company. Dr. Patel is or has been a consultant to Curetis, Specific Technologies, Selux Dx, GenMark Diagnostics, PathoQuest, Heraeus Medical, and Qvella; monies are paid to Mayo Clinic. In addition, Dr. Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued. Dr. Patel receives travel reimbursement from ASM and IDSA and an editor’s stipend from ASM and IDSA, and honoraria from the NBME, Up-to-Date and the Infectious Diseases Board Review Course.
The objectives of this presentation are: 1. to explain how matrix-assisted laser desorption ionization time of flight (also referred to as MALDI-TOF) mass spectrometry works as a clinical microbiology laboratory application, 2. to explain the role of MALDI-TOF mass spectrometry in clinical microbiology, and 3. to highlight our experience with MALDI mass spectrometry.
Mass spectrometry measures particles based on their mass-to-charge ratio. To do this, a sample (in the described method, the whole organism, either a bacterium or a fungus) is exposed to an ion source, and its particles (in the described methods, proteins) are ionized, separated based on their mass-to-charge ratio, detected, and then the generated mass spectrum (in the described method) is compared against a library of mass spectra.
Mass spectrometry requires an ion source, mass analyzer, and detector. There are multiple possible ion sources. In the remote past, ionization required molecules in the gas phase, limiting analysis to volatile compounds or those that could be rendered volatile. Large nonvolatile polar molecules, such as proteins, were not easily analyzed and, therefore, mass spectrometry was not used for protein analysis. With the arrival in the late 1980s of matrix-assisted laser desorption ionization, mass spectrometry based on microbial proteomics has become possible. MALDI is a soft ionization technique allowing molecules to remain relatively intact during ionization. Large proteins can be measured as little protein fragmentation occurs. Following ionization, the ions are separated, enabling measurement of mass. Using the approach covered in this presentation, ions are separated by time of flight in a flight tube.
I will first go over how MALDI-TOF mass spectrometry is practically done in the clinical microbiology laboratory, starting from a colony. Commercial systems for clinical microbiology laboratories are available from bioMérieux, Inc. and Bruker Daltonics, Inc. The example shows the latter because it is the system with which I have had the most experience.
A colony may be picked directly from a plate and smeared onto a target plate. Then, 1 to 2 µL of a “matrix” consisting of, for example, alpha-cyano-4-hydroxycinnamic acid dissolved in acetonitrile and trifluoroacetic acid, is added and dried on the plate.
Some organisms require preparatory extraction to generate spectra of sufficient quality to enable microbial identification. For example, the isolate of interest may be placed into a microcentrifuge tube with 70% ethanol, mixed, and centrifuged, with the supernatant decanted, the pellet dried, 70% formic acid and acetonitrile added, and the mixture vortexed and centrifuged again. The supernatant can then be deposited onto a target plate, dried, and overlain with matrix.
Alternatively, on-plate formic acid treatment can be performed; this is also referred to as “on-plate extraction” or “extended direct transfer.” Using this approach, whole cells from colonies are moved to the target plate and then exposed to a formic acid solution, either by adding the formic acid solution prior to colony transfer or by overlaying the transferred colony with formic acid solution. This is then dried, overlain with matrix, and the process continued.
The target plate is placed into the plate chamber of the mass spectrometer, the plate chamber is closed, and analysis is performed. Target plates have multiple spots, so multiple isolates can be prepared and analyzed together, at about 2 to 3 minutes per sample.
Let us look at the details. The sample is mixed with the matrix and co-crystalized onto the target plate (the “matrix-assisted” component of matrix-assisted laser desorption ionization). The matrix “buffers” the sample, preventing its decomposition and enabling transformation of laser light into heat.
A laser is applied (the “laser” component of matrix-assisted laser desorption ionization).
The matrix absorbs energy from the laser, releasing it into the sample as heat. This causes the sample to desorb and form singly charged ions (the “desorption ionization” component of matrix-assisted laser desorption ionization).
Next, the mass of the ions is analyzed. This is accomplished using a flight tube, the lighter ions traveling faster and, therefore, being detected earlier than the heavier ions. In the described method, particles are typically singly charged. The net result is generation of a mass spectrum in which the mass-to-charge is plotted against signal intensity. Only highly abundant proteins that are of low mass and ionize readily are detected. These are typically ribosomal proteins, although the specific nature of the analyzed proteins is not part of the analysis.
The mass profile is used as a fingerprint or mass spectrum (as shown on this slide) to compare with those of well-characterized organisms in a database. The spectrum typically includes genus- and species-specific peaks so that with a comprehensive library of spectra, the genus and often the species of the organism is determined using bioinformatics.
Matrix-assisted laser desorption ionization time of flight mass spectrometry has changed our workflow, which historically involved rapid biochemicals, an automated phenotypic identification system, long-tubed biochemicals, and 16S ribosomal RNA gene sequencing . . .
. . . to a matrix-assisted laser desorption ionization time of flight mass spectrometry-based approach, which typically abrogates the need for many other identification tools traditionally used.
The next few slides show examples of the types of organisms that can be identified with MALDI-TOF mass spectrometry based on the current FDA-approved/cleared systems.
There are many aerobic Gram-positive bacteria that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
There are multiple Enterobacteriaceae that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems. Note that the Escherichia coli and Shigella species cannot be differentiated.
There are also many Gram-negative bacilli that are not Enterobacteriaceae that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
There are several fastidious Gram-negative bacteria that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
There are multiple anaerobes that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
There are numerous mycobacteria and Nocardia species that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
Additionally, there are various yeasts that are FDA-approved/cleared on the Vitek MS and MALDI Biotyper CA systems.
And finally, there are filamentous fungi that are FDA-approved/cleared on the Vitek MS system.
Bruker also has a research use only (RUO) database, as well as other RUO databases, including a filamentous fungi library, a mycobacteria library, and a “security relevant” database. bioMérieux likewise has an RUO database called VITEK MS RUO. In addition, users can construct their own databases, as we have done at Mayo Clinic.
Matrix-assisted laser desorption ionization mass spectrometry has a number of strengths. It is automated, green, it doesn’t require specific expertise in mass spectrometry, and it has a rapid turnaround time and high throughput capability. It only requires a single colony and is associated with a low exposure risk due to sample inactivation. Although not covered in today’s presentation, this approach is cost-effective and has demonstrated high inter-laboratory reproducibility. It has broad applicability (covering all types of bacteria, including anaerobes as well as fungi). Finally, the system is open and adaptable by the user.
There are limitations to matrix-assisted laser desorption ionization mass spectrometry. No susceptibility information is provided, and the technology is not generally useful for direct testing of clinical specimens. Some organisms require repeat analyses and additional processing. The acceptable score or percentage cutoffs for identification of genera and species, which were not covered in this presentation, vary between studies. Some closely related organisms are not differentiated. Comparison of data from the two companies’ instruments is not feasible. Laboratories acquiring the needed equipment will suffer financial loss on existing equipment. And finally, instrument downtime can be problematic if institutions only have single systems.
