STANDARDISATION OF PROCALCITONIN...

STANDARDISATION OF PROCALCITONIN MEASUREMENT

Amandine BOEUF, PhD

BSAC Birmingham 21 March 2019

2 BSAC Birmingham 21 March 2019

“Novel materials and methods for the detection, traceable monitoring and evaluation of antimicrobial resistance”

AntiMicroResist EMPIR project

Coordinator : Jim Huggett (LGC)

Initiative for the standardisation

of Procalcitonin measurements

à Reduce unnecessary use of antibiotics

Procalcitonin (PCT) – Biomarker

• 116 amino acid polypeptide

• Precursor of calcitonin

• ä when bacterial inflammation

• Peak level achieved rapidly

• æ rapidly after end of injury

à Low PCT

concentration

(0.05 ng/mL)

à PCT

concentration may

rise to 100 ng/mL

à Specific biomarker for bacterial infection

from Lindsheid et al, Endocrinology, 2003;144(12):5578-5584

From https://www.biomerieux-diagnostics.com/vidasr-brahms-pct

Adapted from Meisner M. J Lab Med. 1999;23:263-272


Procalcitonin (PCT) – Antibiotic Stewardship

à Decision-making on antibiotic therapy initiation

PCT value < 0.01ng/mL

0.10 – 0.25 ng/mL

0.25 – 0.50 ng/mL

> 0.5 ng/mL

Bacterial infection ? Very unlikely unlikely likely Very likely

Antibiotic therapy ?

strongly discouraged discouraged encouraged strongly

encouraged

Clinical considerations

• Repeat PCT measurements every 1 – 2 days

• Continue antibiotic therapy regardless PCT level if patient is unstable, high risk or strong suspicion of pneumonia

• If PCT remains high, consider treatment failure

Patients with Lower Respiratory Tract Infection


Procalcitonin (PCT) – Antibiotic Stewardship

à Decision-making on antibiotic therapy reduction or discontinuation

PCT value< 0.25 ng/mL

Or PCT æ by 90%

0.25 – 0.50 ng/mLOr

PCT æ by ≥ 80%

0.50 – 1.00 ng/mL

> 1.00 ng/mL

Antibiotic therapy ? Discontinuation encouraged Discontinuation discouraged

Clinical considerations

• Repeat PCT measurements every 1 – 2 days

• Continue antibiotic therapy regardless PCT level if patient is unstable, high risk or strong suspicion of pneumonia

• If PCT remains high, consider treatment failure

Patients with confirmed Sepsis


Procalcitonin (PCT) – Antibiotic StewardshipPatients with Acute Respiratory Infection

ICUn = 598

EDn = 2,605

Primary caren = 1,008

All clinical settingsn = 4,221

- 65 % - 40 % - 23 %- 40 %

à Reduction of antibiotic exposure


Schuetz et al, Clin Infect Dis, 2012;55(5):651-662

Procalcitonin assays

LIA (Thermo Scientific/Brahms)KRYPTOR (Thermo Scientific/Brahms)

VIDAS (bioMérieux)ADVIA Centaur (Siemens)

ARCHITECT (Abbott)ELECSYS (Roche)LIAISON (DiaSorin)

Samsung IB (Samsung)Lumipulse G (Fujirebio)

AQT90 FLEX – PCT (Radiometer)Diazyme Procalcitonin (Diazyme)

Harmonization to KRYPTOR method

No data on calibration system

No higher order reference method and

material


à Need to standardise PCT

assays

Other methods

Brahms-like methods

SI-Traceable, commutable Reference Materials

Value Assignment with Reference method

Commutability assessment of frozen serum pools

Calibration of immunoassays

IDMS Reference Method for PCT quantification in serum

Production & purity assessment of primary calibrators (Peptides

and recombinant proteins)

ID-LC-MS/MS method

Protocol for sample preparation

Trueness assessment of immunoassays

Initiative for the standardisation of PCT measurements


MSPRM = Parallel Reaction MonitoringDigestion


OrbitrapFragmentation HCD Cell

Quadrupole mass filter

time

Proteins Peptides

LC

à Highly specific and sensitive quantification of a protein in biological matrix through quantification of selected peptides

LC-MS/MS method


Proteins Peptides

à2 tryptic peptides selected (quantification + confirmation)

Selection of peptides

• Proteotypic peptides : specific to the protein + good MS response• No PTMs• No missed cleavage• 5-20 amino acids

1) In silico digestion of PCT with different enzymes (trypsine, AspN, GluC,…)à Peptides of interest identified

2) Confirmation by LC-MS/MS analysis

- Precipitation- Detergent- SPE- Ultrafiltration- …

1% SDC 0.5% SDC

x 2


Sample preparation

Digestion+

Spike of labelled peptides

Proteins Peptides

Protein extractionMatrix simplification

LC-MS/MS

+


Sample preparationDigestion

+Spike of labelled

peptides

Proteins Peptides

Protein extractionMatrix simplification LC-MS/MS

+

Digestion conditions:- Enzyme- Time- Temperature- Denaturing agent- Reduction/alkylation- …

*

* Size marker

* *

Non optimised Optimised

Peptide 1

1) Synthetic peptides from Pepscan Presto

2) Intact mass measurement (LC-MS)

à Peptide/protein impurity profile


Primary calibrators

D:\Users\Data_201808\20180911_hhh_3032 09/13/18 04:32:13 blank

RT: 0.00 - 60.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative

Abundan

ce

5.665.81

5.885.636.076.17

45.226.34

6.77

6.94

7.41

7.597.69

7.87

8.07

8.14

8.318.378.428.518.71

8.90

9.279.34

9.67 46.08

9.8346.689.93

10.33 47.00

47.0810.73 44.78 48.2811.1711.60 48.53

48.6111.8748.7112.37

12.90 48.9249.7713.56

14.14 50.0251.54 52.2414.93 44.51

15.64 53.17 53.8716.75 44.0418.14 54.3241.7818.95 41.4620.48 21.83 23.28 25.01 54.8526.60 27.33 40.6928.86 31.95 32.772.98 4.32 57.1834.43 35.59 58.1839.532.780.57

NL:1.91E9TIC MS 20180911_hhh_3032


RT: 0.00 - 60.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative

Abundan

ce

45.22

46.11

46.68

46.78

46.91

47.07 48.35

48.5044.8248.59

48.76

48.99

49.6644.24 49.83

44.18 50.0250.3350.5344.053.00

50.8743.6242.49 51.7333.73 53.70 56.7641.49 57.0621.0010.26 17.96 23.8515.72 21.170.90 6.41 18.4414.11 28.003.43 5.26 24.022.35 26.40 29.5211.96 32.13 38.9334.087.61 9.95 37.75

NL:1.81E9TIC MS 20180911_hhh_2999

D:\Users\Data_201808\20180913_hhh_3054 09/13/18 21:00:43 FHT 10000ng/mL

RT: 0.00 - 60.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative A

bundance

4.834.79

4.93

5.00

5.15

5.31

5.45

45.01

5.72

5.77

47.835.88 45.93

47.9316.74

47.65 48.016.13

6.23

6.32 46.116.416.51

6.61

6.77

6.91

7.1548.31 49.43

7.51 49.687.68 49.787.80

49.927.978.46

50.2744.578.8050.489.34 44.179.78 50.76

10.56 51.2511.19 17.19 52.2741.63 43.9341.48 53.4613.462.89 17.3314.02 54.7918.14 59.0056.372.71 40.430.28 20.34 24.14 27.5522.68 26.30 33.9432.54 37.1529.22 35.7130.24

NL:1.98E9TIC MS 20180913_hhh_3054

Solvent sample

(DMSO 2%)

Blank sample

Peptide sample

Peptide 1

1) Synthetic peptides from Pepscan Presto

2) Intact mass measurement (LC-MS)

à Peptide/protein impurity profile


Primary calibratorsD:\Users\Data_201808\20180911_hhh_3022 09/12/18 18:14:42 blank

RT: 0.00 - 60.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

5.89 29.385.78 6.01

6.16 45.236.56

6.79

7.05

7.267.33

7.45

7.74

7.898.13

8.3746.138.56 46.82

8.74 46.97 48.479.099.39 48.59

44.829.87 48.75

10.41 48.8649.6811.24

12.10 50.2444.2513.46 51.78 52.5314.81 54.0516.35 43.7042.4641.2517.67 19.01 30.1020.76 25.2222.30 28.8525.57 55.0038.2631.032.99 33.61 55.8435.90 58.220.06 1.74 4.16

NL:1.87E9TIC MS 20180911_hhh_3022

20180911_hhh_3022 #12604 RT: 29.38 AV: 1 NL: 4.73E8T: FTMS + p ESI Full ms [120.0000-1500.0000]

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

543.2922z=3

814.4321z=2

544.6266z=3

652.1505z=5

567.3050z=3

522.2696z=2

825.4169z=2

1086.2373z=3

692.0694z=?

864.3997z=?

500.9376z=?

1017.2919z=?

786.4227z=?

1244.3768z=?

455.7036z=?

Spectra at 29.38 min

Peptide 1 sample

29.38

[M+2H]2+

[M+3H]3+

Peptide 1

1) Synthetic peptides

from Pepscan Presto

2) Intact mass

measurement (LC-MS)

à Peptide/protein

impurity profile

3) Amino Acid Analysis

à Quantification of

peptide


Primary calibrators


RT: 0.00 - 60.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

5.89 29.385.78 6.01

6.16 45.236.56

6.79

7.05

7.267.33

7.45

7.74

7.898.13

8.3746.138.56 46.82

8.74 46.97 48.479.099.39 48.59

44.829.87 48.75

10.41 48.8649.6811.24

12.10 50.2444.2513.46 51.78 52.5314.81 54.0516.35 43.7042.4641.2517.67 19.01 30.1020.76 25.2222.30 28.8525.57 55.0038.2631.032.99 33.61 55.8435.90 58.220.06 1.74 4.16

NL:1.87E9TIC MS 20180911_hhh_3022

20180911_hhh_3022 #12604 RT: 29.38 AV: 1 NL: 4.73E8T: FTMS + p ESI Full ms [120.0000-1500.0000]

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

543.2922z=3

814.4321z=2

544.6266z=3

652.1505z=5

567.3050z=3

522.2696z=2

825.4169z=2

1086.2373z=3

692.0694z=?

864.3997z=?

500.9376z=?

1017.2919z=?

786.4227z=?

1244.3768z=?

455.7036z=?

Spectra at 29.38 min

Peptide 1

sample

29.38

[M+2H]2+

[M+3H]3+

Standardisation of Procalcitonin assays (WG-PCT)


IFCC working group

1/ Develop and validate a reference measurement procedure for PCT absolute quantification by isotope dilution mass spectrometry (IDMS) in order to establish metrological traceability of results to the SI Units

2/ Document and understand the variability of results provided by the different commercially available PCT assays

3/ Evaluate the feasibility for standardization of PCT assays through common calibration with commutable calibrators value assigned with the IDMS reference measurement procedure

4/ If standardization of PCT assays appears desirable and feasible:

- Produce commutable calibrators value assigned with the IDMS reference method- Effectively recalibrate PCT assays;- Assess accuracy and comparability of PCT assays before and after recalibration- Evaluate the impact of assays recalibration and the need to revise clinical decision limits


IFCC working group on PCT standardisation

AcknowledgmentsLNE Biomedical and Organic Chemistry team, especially:

Huu Hien Huynh, Hélène Vaneeckhoutte, Chiara Giangrande, Vincent Delatour, Béatrice Lalere, Sophie Vaslin-Reimann

AntiMicroResist partners

IFCC WG-PCT

Joëlle Vinh


Thank you for your attention

STANDARDISATION OF PROCALCITONIN...

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