Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Nathan A Ledeboer...

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)


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”)



• 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



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


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)


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


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


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

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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

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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

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


< 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.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%


< 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.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

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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

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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

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Questions

?

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