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VDSP Commutability Study Results: Total 25(OH)D nmol/L
Commutability of SRM 972a, and CAP ABVD Survey and DEQAS Test Materials in the Selected Measurement Systems
November 14, 2013
Karen W. Phinney, Ph.D., Joyce Merkel, M.S., R.D.,
Christopher T. Sempos, Ph.D., And the VDSP Members
Structural Forms for 25(OH)D
Immunoassays utilize
antibody against
similar regions of the
two analytes
25-hydroxyvitamin D2 25-hydroxyvitamin D3
The two primary metabolites of interest differ only in the side chains
Molecular Formula: C28H44O2 C27H44O2
Molecular Weight: 412.65 g/mole 400.63 g/mole
Total 25-Hydroxyvitamin D or 25(OH)D
• Total 25(OH)D is defined as
Total 25(OH)D = 25(OH)D2 + 25(OH)D3*
• Units: ng/mL or nmol/L where:
ng/ml * 2.5 ≈ nmol/L
* Assumes that Vitamin D2 and D3 are of equal biological value.
Vitamin D Status Measurement
NB: Total 25(OH)D does not include epimer concentrations!
Protocol Overview: # 1
• Same analysis protocol as for VDSP interlaboratory comparison study (this study is an add on to it)
• 50 single donor patient samples, blinded
• 1 NIST SRM (4 levels), 5 CAP ABVD and 8 DEQAS test materials, non-blinded
• All samples run in duplicate on 3 different days
• NIST and Ghent reference measurement procedures (RMP) used to determine “Target Values” for patient samples and test materials.
Protocol Overview: # 2
• DEQAS, CAP and NIST test materials were analyzed together with patient samples to minimize effects that can confound commutability assessment such as lot-to-lot variability, variability in specimen preparation etc.
• Due to sample volume limitations samples sent to selected laboratories (3 MS labs, 17 IA labs).
Approach to Assessing Commutability
Calculate mean from 6
replicates for each
patient sample
Plot patient sample means (Y)
vs.
mean target values (X)
Calculate and plot OLS
regression line, &
95% prediction interval
Calculate mean from
6 replicates for each
test material
Plot test material
means over target
values in same graph
Test materials with values inside the 95% prediction interval
are considered commutable
Results for test materials,
i.e. SRM 972a (Levels 1-
4), & DEQAS & CAP
CTM, are shown
separately vs. Ghent and
NIST RMP Target Values
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Samples
DEQAS SAMPLES
VDSP Commutability Study: L03_D001
Approach to Assessing Commutability
Calculate mean from 6
replicates for each
patient sample
Plot patient sample means (Y)
vs.
mean target values (X)
Calculate and plot OLS
regression line, &
95% prediction interval
Calculate mean from
6 replicates for each
test material
Plot test material
means over target
values in same graph
Test materials with values inside the 95% prediction interval
are considered commutable
Results for test materials,
i.e. SRM 972a (Levels 1-
4), & DEQAS & CAP
CTM, are shown
separately vs. Ghent and
NIST RMP Target Values
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Samples
DEQAS SAMPLES
VDSP Commutability Study: L03_D001
VDSP Commutability Studies
• Reference Material and EQA/PT Materials Tested:
• NIST 972a – (4)
• CAP – (5)
• DEQAS - (8)
• Participating Labs:
• 20 Total: 4 LC-MS/MS and 16 Immunoassay
• 2 dropout
• 5 – Open reporting
• 4 – Anonymous reporting
• 2 – Results not to be used
• 7 - TBA
Suggested Assay Performance
Limits Based on Biological Variation*
*Stöckl D et al. Clin Chim Acta 2009;408:8-13
Measurements CV (%) Bias (%)
Reference Labs ≤ 5% ≤ 1.7%
“Routine” Labs ≤ 10% ≤ 5%
VDSP Commutability Studies
Lab # Mean CV%
Lab # Mean CV%
1 7.5 12 14.8
2 6.2* 13 3.3
9 15.0 16 3.9
10 4.3 17 4.3
11 10.3
*Baseline study results. No replicates measured in commutability study.
Organization of Slides
• Results are given below in turn for 9 Laboratories (1, 2, 9-13, 16, 17) as follows:
• Plots of Lab Mean (Y) vs. Ghent or NIST (X) final assigned target values first for SRM 972A superimposed
• Repeat sequence for CAP ABVD Survey and then for DEQAS test materials
SRM972a
Levels 1-4
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 1: SRM 972a Levels 1-4
VDSP Commutability Study: Immunoassay
n 50
R2 0.92
Term Coefficient
Intercept -4.64 Slope 1.15
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 2: SRM 972a Levels 1-4
VDSP Commutability Study: Immunoassay
n 50 R2 0.88
Term Coefficient
Intercept -1.60
Slope 1.06
Lab 2 Outlier vs. Ghent Target Values [nmol/L]
Lab 2 Ghent Target Values
Sample DT DT D3 3-Epimer D2
SRM 972a Level 3 65 85 51 3 34
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 9: SRM 972a Levels 1-4
VDSP Commutability Study: Immunoassay
n 50 R2 0.80
Term Coefficient
Intercept -17.70
Slope 1.35
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 10: SRM 972a Levels 1-4
VDSP Commutability Study: LC-MS/MS
n 50 R2 0.99
Term Coefficient Intercept -2.14
Slope 1.10
Lab 10 Outlier vs. Ghent Target Values [nmol/L]
Lab 10 Ghent Target Values
Sample DT DT D3 3-Epimer D2
SRM 972a Level 4 149 77 76 75 < LOQ
DEQAS 5 113 52 51 63 < LOQ
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 11: SRM 972a Levels 1-4
VDSP Commutability Study: Immunoassay
n 50 R2 0.83
Term Coefficient
Intercept -10.92
Slope 1.02
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 12: SRM 972a Levels 1-4
VDSP Commutability Study: Immunoassay
n 50
R2 0.88
Term Coefficient
Intercept -11.64 Slope 0.96
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 13: SRM 972a Levels 1-4
VDSP Commutability Study: LC-MS/MS
n 50 R2 0.998
Term Coefficient
Intercept 0.08
Slope 1.03
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 16: SRM 972a Levels 1-4
VDSP Commutability Study: LC-MS/MS
n 50
R2 0.998
Term Coefficient
Intercept -0.08 Slope 1.01
Lab 16 Outlier vs. Ghent Target Values [nmol/L]
Lab 16 Ghent Target Values
Sample DT DT D3 3-Epimer D2
SRM 972a Level 4 70 77 76 75 < LOQ
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
SRM 972a Sample
Lab 17: SRM 972a Levels 1-4
VDSP Commutability Study: Immunoassay
n 49 R2 0.85
Term Coefficient
Intercept -13.01
Slope 1.44
CAP ABVD
5 Samples
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 1: CAP ABVD Survey Samples
VDSP Commutability Study: Immunoassay
n 50 R2 0.93
Term Coefficient
Intercept -4.39
Slope 1.12
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 2: CAP ABVD Survey Samples
VDSP Commutability Study: Immunoassay
n 50
R2 0.88
Term Coefficient
Intercept -1.74 Slope 1.08
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 9: CAP ABVD Survey Samples
VDSP Commutability Study: Immunoassay
n 50
R2 0.80
Term Coefficient
Intercept -17.23 Slope 1.32
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 10: CAP ABVD Survey Samples
VDSP Commutability Study: LC-MS/MS
n 50
R2 0.99
Term Coefficient
Intercept -2.55
Slope 1.12
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 11: CAP ABVD Survey Samples
VDSP Commutability Study: Immunoassay
n 50 R2 0.84
Term Coefficient
Intercept -10.93
Slope 1.01
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 12: CAP ABVD Survey Samples
VDSP Commutability Study: Immunoassay
n 50 R2 0.87
Term Coefficient
Intercept -11.75
Slope 0.98
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 13: CAP ABVD Survey Samples
VDSP Commutability Study: LC-MS/MS
n 50
R2 0.998
Term Coefficient Intercept 0.42
Slope 1.01
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 16: CAP ABVD Survey Samples
VDSP Commutability Study: LC-MS/MS
n 50 R2 0.998
Term Coefficient
Intercept -0.41
Slope 1.03
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
CAP Sample
Lab 17: CAP ABVD Survey Samples
VDSP Commutability Study: Immunoassay
n 49 R2 0.86
Term Coefficient
Intercept -12.97
Slope 1.42
DEQAS
8 Samples
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 1: DEQAS Survey Samples
VDSP Commutability Study: Immunoassay
n 50
R2 0.92
Term Coefficient
Intercept -4.64 Slope 1.15
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 2: DEQAS Survey Samples
VDSP Commutability Study: Immunoassay
n 50 R2 0.88
Term Coefficient
Intercept -1.60
Slope 1.06
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 9: DEQAS Survey Samples
VDSP Commutability Study: Immunoassay
n 50 R2 0.80
Term Coefficient
Intercept -17.70
Slope 1.35
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 10: DEQAS Survey Samples
VDSP Commutability Study: LC-MS/MS
n 50 R2 0.99
Term Coefficient Intercept -2.14
Slope 1.10
Lab 10 Outlier vs. Ghent Target Values [nmol/L]
Lab 10 Ghent Target Values
Sample DT DT D3 3-Epimer D2
SRM 972a Level 4 149 77 76 75 < LOQ
DEQAS 5 113 52 51 63 < LOQ
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 11: DEQAS Survey Samples
VDSP Commutability Study: Immunoassay
n 50 R2 0.83
Term Coefficient
Intercept -10.92
Slope 1.02
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 12: DEQAS Survey Samples
VDSP Commutability Study: Immunoassay
n 50
R2 0.88
Term Coefficient
Intercept -11.64 Slope 0.96
10
40
70
100
130
160
190
Test
Lab M
ean T
ota
l 25(O
H)D
(nm
ol/L)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 13: DEQAS Survey Samples
VDSP Commutability Study: LC-MS/MS
n 50 R2 0.998
Term Coefficient
Intercept 0.08
Slope 1.03
10
40
70
100
130
160
190
Te
st
La
b M
ea
n T
ota
l 2
5(O
H)D
(n
mo
l/L
)
10 40 70 100 130 160 190
Ghent Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 16: DEQAS Survey Samples
VDSP Commutability Study: LC-MS/MS
n 50
R2 0.998
Term Coefficient
Intercept -0.08 Slope 1.01
10
40
70
100
130
160
190
Test
Lab M
ean T
ota
l 25(O
H)D
(nm
ol/L)
10 40 70 100 130 160 190
NIST Mean Target Total 25(OH)D (nmol/L)
95% PI
Fitted Line
Observed
DEQAS Sample
Lab 17: DEQAS Survey Samples
VDSP Commutability Study: Immunoassay
n 49 R2 0.85
Term Coefficient
Intercept -13.01
Slope 1.44
VDSP Contributors • ODS: Overall Coordination
• Paul Coates • Joseph Betz • Claudia Faigen • Joyce Merkel • Mary Frances Picciano* • Christopher Sempos** • Anne Thurn • Elizabeth Yetley
• International Organizations: • AACC - Gary Myers • IFCC - Ian Young • CAP
• Andrew Hoofnagle • Nataliya Polyakov • Sharon Burr
• DEQAS • Graham Carter • Julia Jones • Jayne Shannon
Reference Laboratories • USA – NIST
• Stephen Wise • Lane Sander • Karen Phinney • Susan Tai • Johanna Camara • Mary Bedner • Katrice Lippa • Carolyn Burdette
• Belgium: Ghent University • Linda Thienpont • Linde De Grande • Katleen Van Uytfanghe
• CDC Standardization-Certification Program • Hubert Vesper • Yasamin Rahmani
* Deceased
** VDSP Coordinator
VDSP Contributors: Surveys • Australia
• Juanita Pettit • Andy Liu • Grahame Caldwell
• Canada • Steven Brooks • Kurtis Sarafin • Evan Green
• Germany • Michael Thamm • Thea Riedel • Christa Scheidt-Nave • Gert Mensink
• Ireland • Kevin Cashman • Mairead Kiely • Albert Flynn • Michael Kinsella
• Mexico • Ricardo Martín Robledo Pérez • Simón Barquera • Mario Efraín Flores Aldana • Salvador Villalpando
• South Korea • Kyungwon Oh • Chae Lim Jung
• United Kingdom • Alison Tedstone • Ann Prentice • Lorna Cox • Gail Goldberg • Kate Guberg
• USA – CDC • Rosemary Schleicher • David Lacher • Christine Pfeiffer
VDSP Contributors: Scientific Consultants
• Australian National University • Robyn Lucas
• Cornel University • Patsy Brannon
• Food Standards Australia New Zealand • Janis Baines
• Loyola University Chicago • Ramón Durazo • Holly Kramer
• Neil Greenberg Consulting Services, LLC • Neil Greenberg
• Queen’s University, Canada • Glenville Jones
• Stanford University • Lu Tian
• University of Minnesota • John Eckfeldt
• University of Otago, NZ • Murray Skeaff
• University of Washington • Andrew Hoofnagle
• University of Wisconsin-Madison • Hector DeLuca • Neil Binkley • Gary Lensmeyer
• Virginia Commonwealth University • Greg Miller • Lorin Bachmann
Thank you!
Questions -
Discussion
