standardisation of procalcitonin...
TRANSCRIPT
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
3 BSAC Birmingham 21 March 2019
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
4 BSAC Birmingham 21 March 2019
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
5 BSAC Birmingham 21 March 2019
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
6 BSAC Birmingham 21 March 2019
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
7 BSAC Birmingham 21 March 2019
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
8 BSAC Birmingham 21 March 2019
MSPRM = Parallel Reaction MonitoringDigestion
9 BSAC Birmingham 21 March 2019
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
10 BSAC Birmingham 21 March 2019
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
11 BSAC Birmingham 21 March 2019
Sample preparation
Digestion+
Spike of labelled peptides
Proteins Peptides
Protein extractionMatrix simplification
LC-MS/MS
+
12 BSAC Birmingham 21 March 2019
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
13 BSAC Birmingham 21 March 2019
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
D:\Users\Data_201808\20180911_hhh_2999 09/11/18 23:42:49 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
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
14 BSAC Birmingham 21 March 2019
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
15 BSAC Birmingham 21 March 2019
Primary calibrators
D:\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+
Standardisation of Procalcitonin assays (WG-PCT)
16 BSAC Birmingham 21 March 2019
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
17 BSAC Birmingham 21 March 2019
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
18 BSAC Birmingham 21 March 2019
Thank you for your attention