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Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass
Spectrometry
Nathan A LedeboerAssistant Professor of Pathology
Medical College of Wisconsin andMedical Director, Microbiology and Molecular Pathology
Dynacare Laboratories and Froedtert HospitalMilwaukee, WI
1
Outline
• Overview of Technology• Are MALDI-TOF instruments all they described to
be?• How can I justify the cost?• Applications in Development• What is the Impact for Patient Care• The Future of Mass Spectrometry• Conclusions• Questions
2
Disclosures• Dr. Ledeboer will discuss products that are not
FDA cleared• Financial Disclosures
– Consultant:• Nanosphere, Inc• ThermoFisher Scientific, Inc• LabCorp• Cepheid (Scientific Advisory Board)• iCubate
– Honoraria• Bruker Daltonics• Meridian
3
OVERVIEW OF TECHNOLOGY
4
Bruker Biotyper vs Vitek MS
5
Direct Smear Method:• Touch colony with transfer device, such as toothpick• Transfer a small amount onto spot• Let air dry• Cover with 1 µL of MALDI matrix, let air dry• Analyze
Step 1: Target Preparation, continued
Research use only – not for use in diagnostic procedures
dH2O
Ethanol-Formic Acid Extraction (if required – low score value after direct smear)
Research use only – not for use in diagnostic procedures
Inactivation of pathogens
Ethanol Ethanol Formic acid,acetonitrile
Analyze supernatant
10 min
Target plate
Analyte (organism)
1 µL Matrix
Matrix Assisted Laser Desorption/Ionization
Matrix: HCCA (a-Cyano-4-hydroxycinnamic acid)Solvent: Acetonitrile, TFA (trifluoroacetic acid)
• Lyses cell walls and extract protein• Separates protein molecules (proteins are “sticky”)
Research use only – not for use in diagnostic procedures
Matrix Assisted Laser Desorption/Ionization
• Laser light pulses
• Matrix molecules readily absorb laser light (photon energy), creating an excited energy state
• The matrix is acidic, and donates positive charge to the analytes
Research use only – not for use in diagnostic procedures
Matrix Assisted Laser Desorption/Ionization
Matrix
• Localized heating causes micro-explosion of material
• Collisions with neutral sample facilitate charge transfer to/from excited matrix molecules
• Ions “desorb” from the target surface
Research use only – not for use in diagnostic procedures
Drift region
Detector
TOF – Time of Flight
m/z
Intensity
• Following acceleration, the charged ions are allowed to drift through a free field toward the detector
• The speed of travel (time of flight) is proportional to the ion’s mass (smaller ions reach the detector first)
Research use only – not for use in diagnostic procedures
MALDI: Results output
Raw profile spectrum Refined profile spectrum
12
Results are analyzed by a computer, cleaned-up and the spectrum is searched against a database with known spectra.
2.0-3.00 Secure genus and species identification
1.7-1.99 Probable genus identification
0.0- 1.69 Unreliable identification
MALDI identification result
ARE MALDI-TOF INSTRUMENTS ALL THEY ARE DESCRIBED TO BE?
15
MALDI publications:
More than 200 peer reviewed publications, mainly in high-ranking microbiology journals as of September 2012
Topics include:
- Particular groups of bacteria - (e.g. anaerobes, Listeria, Neisseria, Yeast...)
- Routine application - Rare and difficult to analyse microorganisms (e.g. Prothotheca)- Highly pathogenic bacteria (e.g. Francisella, Brucella)- Blood culture direct analysis - Urine direct analysis- Sub-typing- Resistance or Virulence Factors - Mycobacteria- Filamentous Fungi
17Benagli C et al. PLoS One. 6(1).
In a study by Benagli et al., the authors compared performance of MALDI-TOF to biochemical ID and resolved discrepancies with sequencing. The results follow.
MALDI ID discrepant sequenced*
Bacteroides fragilis 179 5 2Bacteroides thetaiotaomicron 43 5 4Bacteroides ovatus 15 6 1Bacteroides vulgatus 20 1 1Bacteroides uniformis 5 3 2Bacteroides eggerthii 1 0Bacteroides nordii 4 3 1Bacteroides salyersiae 1 0Bacteroides massiliensis 2 0------------------------------------------------------------------------------------------------------
270 23 11 *only IDs with log(score)<2.5
277 Clinical Bacteroides Isolates from a European study: 270 isolates (97,5%) identified with significant score, 7 isolates not in Reference Library (e.g. Bacteriodes distasonis)
16S rDNA Sequenceing Confirmed 10 of 11 Discrepant MALDI Results, 1 Case Only “Bacteroides spec.“
Nagy et al., Clin Microbiol Infect 2009; 15: 796–802
Clinical Application - Bacteroides species
Marklein et al., JCM, Vol. 47 2009
Clinical Application: Yeast
No incorrect Yeast Identification by the Respective Molecular Fingerprint
Bruker Biotyper vs Vitek MS
20
21
System and application (no. of isolates tested)
Saramis Biotyper
% Correctly identified P value % Correctly identified P value
Biotyper
Routine (986)a 83.8 vs 92.7 <0.001 NAd NA
86.9 vs 95.5 <0.001 NA NA
12.8 vs 3.2 <0.001 NA NA
0.3 vs 1.2 <0.01 NA NA
Vitek MS
Routine (986)a 83.8 vs 93.2 <0.001 92.7 vs 93.2 0.608
86.9 vs 93.6 <0.001 95.5 vs 93.6 <0.05
12.8 vs 5.8 <0.001 3.2 vs 5.8 <0.01
0.3 vs 0.4 1 1.2 vs 0.4 <0.05
Martiny, et al, JCM, 2012, 50
Bruker Biotyper vs Vitek MS
22
Property Microflex LT Vitek MS RUO Vitek MS IVD RemarksUser friendliness Ready-to use Matrix solution No Yes Yes
Facility of preparing smear Very easy Easy Easy For Vitek-MS systems, matrix solution must be deposed each two spots Disposable targets Yes Yes Yes
Reusable targets Yes No No Software Easy to use Not easy to use Very easy to useTime for 96 identifications
Time to prepare work list (min) <5 5–10 NDa
Time to load target and make vacuum 2 5
Time for analysis (min) 40 55
Time for 16 identifications (min) ND ND 15 No ID before success of QC at end of run (each 16 IDs)
Quality IVD Yes No Yes
RUO Yes Yes No Need for validation before clinical reporting
Quality management Easy Easy Very easyCostc
Device + NAb ++ Reactants +++ NA + Based on catalog prices Maintenance ++ NA +++Implementation Noise Silent Noisy Noisy Size Smaller Bulkier BulkierConnectivity Via LIS NA Via MylaCapacity 1 × 96 4 × 48 4 × 48
Martiny, et al, JCM, 2012, 50
23
Time/ test
(hour)FTE
Cost/test*Supply
Cost/testTotal Cost
Rapid Biochemicals 0.10 $4.14 $0.29 $4.43
Automated Biochemicals 0.14 $5.79 $9.59 $15.38
Long Biochemicals 0.33 $13.65 $5.32 $18.97
Sequencing 0.73 $30.19 $20.02 $50.21
Mass Spectrometry 0.05 $2.07 $0.24 $2.31
*FTE cost/hour $41.35
Slide courtesy of Robin Patel, MD
24
Cost Savings Comparison
Instrument Included
* Assumes a 3 year depreciation of instrument and an instrument cost of $200,000
Tests per Year 5000 10000 20000 30000 40000 50000Instrument
Add*$13.33 $6.66 $3.33 $2.22 $1.66 $1.33
MALDI-TOF ID Consumables and Labor
$2.31 $2.31 $2.31 $2.31 $2.31 $2.31
Biochemical ID Consumables and Labor
$15.38 $15.38 $15.38 $15.38 $15.38 $15.38
Difference per test
-$0.26 $6.41 $9.74 $10.85 $11.41 $11.74
Cost Savings per year -$1,300.00 $64,100.00 $296,337.55 $448,005.18 $471,128.03 $484,753.99
Return on Investment (in years) 6.06 3.12 0.67 0.45 0.42 0.41
No Instrument
Tests per Year 5000 10000 20000 30000 40000 50000
MALDI-TOF ID Consumables and Labor
$2.31 $2.31 $2.31 $2.31 $2.31 $2.31
Biochemical ID Consumables and Labor
$15.38 $15.38 $15.38 $15.38 $15.38 $15.38
Difference per test
$13.07 $13.07 $13.07 $13.07 $13.07 $13.07
Cost Savings per year $65,350.00 $130,700.00 $397,652.14 $539,670.76 $539,670.76 $539,670.76
Return on Investment (in years) 3.06 1.53 0.50 0.37 0.37 0.37
Cost-effectivenesss of switching to MALDI-TOF MS for routine bacterial identification
Galliot O, Blondiaux N, Lorez C, Wallet F, Lemaitre N, Herwegh S and Courcol R
September 2009 Switched from conventional
biochemicals (Vitek 2 and API) to MALDI-TOF MS (Bruker)
Cost analysis performed
October 2008-September
2009
October 2009-September
2010
Isolates Tested 33,320 38,624
Biochemical Costs
$193,754 $5,374
MALDI-TOF - $15,836
TOTAL $193,754 $21, 210
Avg Cost/ID $5.81 $.54
JCM epub ahead of print
Annual Savings = $177, 090 “allowed decrease of 89.3% of the cost of bacterial identification in the first year.”In addition:
Decreased waste from 1,424kg to 44kg Decreased subculture media of $1,102
Decreased sequencing cost of $1,650
COMING APPLICATIONS
26
MALDI– blood culture direct analysis
27
MALDI – blood culture direct analysis
< 1.63,9%11,7%9,1%11,7%< 1.7
1.6 - 1.810,4%5,2%27,3%20,8%1.7 - 2.0
> 1.885,7%83,1%63,6%67,5%> 2.0
4000300040003000
< 1.63979< 1.7
1.6 - 1.88421161.7 - 2.0
> 1.866644952> 2.0
4000300040003000
NEW - ThresholdsORIGINAL - Thresholds
77 samples
< 1.65,4%7,5%11,8%11,8%< 1.7
1.6 - 1.88,6%9,7%15,1%26,9%1.7 - 2.0
> 1.886,0%82,8%73,1%61,3%> 2.0
4000300040003000
< 1.6571111< 1.7
1.6 - 1.88914251.7 - 2.0
> 1.880776857> 2.0
4000300040003000
NEW - ThresholdsORIGINAL - Thresholds
93 samples
Detailed results and effect of adopted processing
No false positive result in the “yellow“ and “green“ log(score) range3000/4000 –detection boundary increased for protein to reduce background
Proof Point: Blood Culture Innovative Extraction
99% of tested strains were correctly identified directly from Blood Culture
2 ml
Positive culture
~60 min.
Sample Preparation
Water75% EtOH Water
Water50ul
95°C30 min
100%EtOH
50ul ACN + silica
beads, vortex 1 min.
FA50ul
2 ml
Positive culture
~60 min.
Sample Preparation
Water75% EtOH Water
Water50ul
95°C30 min
100%EtOH
50ul ACN + silica
beads, vortex 1 min.
FA50ul
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 50
2
4
6
8
10
12
14
16
18
15 Min
30 Min
Day
s to
pos
itivi
ty
M. che/abs gr M. fortuitum gr MAI MTB
Heat Kill Test
Following heat inactivation step, TREK Myco bottles were inoculated and held 6 weeks.
2 ml
Positive culture
~30 min.
Sample Preparation
Water75% EtOH Water
Water50ul
95°C30 min
100%EtOH
50ul ACN + silica
beads, vortex 1 min.
FA50ul
AFB15-A 0:A9 MS, BaselineSubtracted, Smoothed
0
2000
4000
6000
8000
Inte
ns.
[a.u
.]
Mycobacterium sp AFB015 MCW 0:H8 MS, BaselineSubtracted, Smoothed
0
1000
2000
3000
4000
5000
Inte
ns.
[a.u
.]
5400 5600 5800 6000 6200 6400 6600 6800m/z
No Bead-beat step
Bead-beat step
Effect of Bead-Beat on MALDI Spectrum
Mycobacteria v1.0 Database
HPLC Sequencing ID #Middlebrook 7H10 Trek Myco
< 1.7 1.7 – 2.0 > 2.0 < 1.7 1.7 – 2.0 > 2.0
MAI (14) M. avium 14 0 4 10 0 4(1) 10
M. intracellularae
MTBC (6) M. tuberculosis* 6 0 3 3 0 0 6
M. chelonae/ abcessus
(9)
M. abscessus 5 0 1 4 0 1 4
M. chelonae 3 0 1 2 0 1 2
M. immunogenum 1 0 1 0 0 0 1
M. fortuitum (16)
M. fortuitum 13 1 9 3 0 4 9
M. peregrinum 2 1 1 0 1 0 1
M. porcinum 1 1 0 0 1 0 0
M. peregrinum M. conceptionense
M. xenopi (6) M. xenopi 6 1 2 3 2 2 2
M. szulgai (1) M. szulgai 1 0 1 0 1 0 0
HPLC Sequencing ID #Middlebrook 7H10 Trek Myco
< 1.7 1.7 – 2.0 > 2.0 < 1.7 1.7 – 2.0 > 2.0
M. gordonae M. gordonae 3 0 0 3 0 0 3
M. mucogenicum
(14)
M. mucogenicum 1 0 1 0 0 0 1
M. phocaicum 2 0 2 0 0 0 2
M. llatzerense 5 0 3 2 0 1 4
M. peregrinum 1 0 0 1 0 0 1
M.muco/phocaicum 5 2 2 1 0 1 4
M. phocaicum
M. malmoense M. malmoense 1 0 0 1 0 0 1
M. phlei (1) M. phlei 1 1 0 0 1 0 0
M. smegmatis M. porcinum 1 0 0 1 0 0 1
Total 72 7 (9.7%)
31 (43.1%)
34 (47.2%)
6(8.3%)
14 (19.4%)
52 (72.2%)
Agreement (species) 80.0% 96.6% 100% 83.3% 92.9% 100%
HPLC Sequencing ID #Middlebrook 7H10 Trek Myco
< 1.7 1.7 – 2.0 > 2.0 < 1.7 1.7 – 2.0 > 2.0
M. gordonae M. gordonae 3 0 0 3 0 0 3
M. mucogenicum
(14)
M. mucogenicum 1 0 1 0 0 0 1
M. phocaicum 2 0 2 0 0 0 2
M. llatzerense 5 0 3 2 0 1 4
M. peregrinum 1 0 0 1 0 0 1
M.muco/phocaicum 5 2 2 1 0 1 4
M. phocaicum
M. malmoense M. malmoense 1 0 0 1 0 0 1
M. phlei (1) M. phlei 1 1 0 0 1 0 0
M. smegmatis M. porcinum 1 0 0 1 0 0 1
Total 72 7 (9.7%)
31 (43.1%)
34 (47.2%)
6(8.3%)
14 (19.4%)
52 (72.2%)
Agreement (species) 85.7% 96.4% 100% 83.3% 92.3% 100%
Middlebrook 7H10
• 90.3% (65/72) with acceptable ID score (>1.7) 98.3% (59/60) correct species ID
Trek Myco
• 91.7% (66/72) with acceptable ID score (>1.7)
98.4% (60/61) correct species ID
Data Summary
Time:
Clinical Impact
t = 0
t = 3-5 days t = 1 day t = 1-2 days
t = 1 hr
ID Filamentous Fungi - workflow
1. Direct Transfer of “Front Mycelium“ (1 min)
if successful: ID is FINISHED
e.g. A.niger
2. Ethanol Extraction of “Front Mycelium“ (10 min)
if successful: ID is FINISHED
3. Broth Cultivation (approx. 1 additional day) & extraction ID is possible for agar adhering filamentous fungi ID is possible for slow or fast sporulating fungi ID is possible for every kind of filamentous
fungi6585,2
1
6744,0
5
6016,9
3
8049,5
5
4536,9
6
9074,5
2
0
1000
2000
3000
4000
Inte
ns.
[a.u
.]
4000 5000 6000 7000 8000 9000 10000 11000 12000m/z
ALL matches against the SAME Filamentous Fungi DB
IMPACT ON PATIENT CARE
42
Work flow and delay for matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry identification of bacteria in this study.
Seng P et al. Clin Infect Dis. 2009;49:543-551 © 2009 by the Infectious Diseases Society of America
44Tan KE, et al, JCM, In Press – Kindly provided by K. Carroll, MD
45
Organism-group n
Mean # of days isolate
identified earlier
Proportion identified earlier by MALDI-protocol,
by number of days of workup
<0da 0db 1d 2d 3d 4d 5d 6d >6d
(days) (%) (%) (%) (%) (%) (%) (%) (%) (%)
S. aureus 109 1.35 1.8 66.1 28.4 2.8 0.9
Other Staphc 26 1.19 7.7 65.4 26.9
BHSd 72 0.60 1.4 38.9 58.3 1.4
VGSe 7 0.57 42.9 57.1
S. anginosus 17 1.12 41.2 29.4 5.9 23.5
S. pneumoniae 6 0.33 66.7 33.3
Other GPCf 6 3.33 33.3 16.7 16.7 16.7 16.7
Enterococcus sp. 78 1.64 1.3 51.3 34.6 9.0 2.6 1.3
Enterobacteriaceae 284 1.34 2.8 69.4 23.2 2.1 1.1 1.1 0.4
P. aeruginosa 77 1.82 41.6 49.4 2.6 1.3 2.6 2.6
Other NF GNBg 39 2.59 2.6 30.8 35.9 2.6 15.4 5.1 5.1 2.6
Haemophilus sp. 10 1.40 80.0 20.0
Other GNCBh 7 0.14 85.7 14.3
Corynebacterium sp. 9 1.67 22.2 33.3 22.2 22.2
Other GPRi 8 4.13 12.5 37.5 12.5 37.5
Anaerobic GNj 26 2.54 3.8 65.4 7.7 19.2 3.8
Anaerobic GPk 14 2.64 21.4 14.3 28.6 7.1 7.1 14.3 7.1
C. albicans 52 0.04 3.8 92.3 1.9 1.9
Other Candida sp. 56 1.93 8.9 67.9 7.1 3.6 3.6 3.6 5.4
Other yeasts 8 3.75 25.0 37.5 12.5 12.5 12.5
All organisms 911 1.45 0.4 13.5 52.7 23.6 3.7 2.7 1.1 1.1 1.1
Tan KE
, et al, JCM
, In Press – K
indly provided by K. C
arroll, MD
Patient Impact
• Retrospective chart review of patients with positive blood cultures obtained during validation of MALDI-TOF Biotyper and Sepsityper Kit
• Time to ID (MALDI-TOF v traditional culture methods) and start/stop times of antibiotics reviewed
• Theoretical reduction in empiric antibiotic duration and cost difference (AWP) calculated
46Paul J., et al. IDSA. 2011
Time to Antimicrobial De-Escalation
47
Traditional ID MALDI-TOF Direct ID
Difference (in hours) P
Emperic Antibiotic
Duration ( IQR, hrs)
66.6 (44.4-87.5)
15.5(1.4-21.3)
51.6(41.8-71.6) <0.01
Antibiotic Cost (USD) $245.24 $88.48 $156.76 <0.01
Paul J., et al. IDSA. 2011
THE FUTURE OF MASS SPECTROMETRY
48
Electrospray Mass Spectrometry
50
Characteristic
MALDI-TOF LC-ESI-QqQ-MS
Ionization Soft ionization with matrix Soft ionization with solvents and electronebulization
Fragmentation No (intact molecules) Yes
Sample Solid form (or liquid allowed to dry on target) Liquid form (downstream of a liquid chromatography step)
Molecules Mainly proteins, large glycopeptides, oligonucleotides, carbohydrates Different molecules, especially peptides
Turnaround time 20-30 s per sample at laser frequency of 50 Hz to generate a spectrum
Minutes or hours depending on liquid chromatography adsorption/elution times
Minutes from sample preparation to result
Throughput Disposable target with multiple spots (48 to 96) No batch mode at the moment due to LC step
Reagent Chemical matrix Chemical reagents for chromatographic separation and elution
External calibrant Internal calibrantMaterial Disposable target Chromatographic column and precolumn
Vials for LC injectionQuantification Not well suited Fully adapted
Specificity Depending on MS specificity and proteins tested Usually higher than MALDI when selected reaction monitoring (MS2) is used
Sensitivity Bacterial ID: 105 CFU using fingerprint approach To be exploredUrine sample after purification without culture: 105 CFU/ml
Integration into microbiology lab workflow Yes (IVD-compliant systems) No (research applications)
Today's clinical microbiology applications
Microbiology: identification of bacteria, yeast, and molds None
Quantitative assays for small molecules, such as vitamin D (outside microbiology field)
Van Belkum, et al, JCM, 2012, 50
Microcantilevers
Conclusions• Data demonstrates excellent performance of
MALDI-TOF MS for identification of bacteria and yeast from plates and from positive blood cultures.
• High Capital Cost can be overcome by consumable savings
• Use of technology can result in a significant reduction in laboratory turnaround and significant antimicrobial cost savings
• MALDI-TOF and current technologies represent the beginning of protein revolution
52
Questions
?