10.07.

17

AACC 2017 – San Diego, CA – July 31, 2017

Clinical Endocrine Assays: What Endocrinologists Will Ask You

Insulin-like Growth Factor I (IGF-I)

Assays: Issues, interpretation and
improvements

Martin Bidlingmaier, MD
Endocrine Laboratory
Medizinische Klinik und Poliklinik IV
Ludwig Maximilians University
Munich - Germany

Bidlingmaier - IGF-I assays

Speaker Financial Disclosure information

• Grant/Research Support: IDS, Roche, Diasorin, Novartis, Pfizer,

OPKO, Chiasma, Genexine, Antisense, ONO, Ionis

• Salary/Consultant Fees: Roche, Genescience, Genexine

• Board/Committee/Advisory Board Membership: Sandoz,

OPKO, Chiasma, Versartis

• Stocks/Bonds: None

• Honorarium/Expenses: IDS, Diasorin, Roche, Siemens, Pfizer,

Novartis, IPSEN, Sandoz

• Intellectual Property/Royalty Income: CMZ

2 July 31, 2017

Bidlingmaier - IGF-I assays

Outline
Clinical use of IGF assays

Common problems for clinicians

Analytical issues with IGF assays

• Calibration and interference
• Variability between methods
• Long term stability
Interpretation issues with IGF assays
• Biological confounders
• Reference intervals

3 July 31, 2017

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Clinical use of IGF assays

4 July 31, 2017

Bidlingmaier - IGF-I assays

Biochemical diagnosis of growth hormone

(GH) related disorders

simple in theory…

GH GH resistance GH excess
deficiency (receptor defect) (acromegaly)

hGH ê é éé

IGF-I ê êê éé

5 July 31, 2017

Bidlingmaier - IGF-I assays

IGF-I in response to rhGH treatment in healthy

young adults
GH IGF-I

6 July 31, 2017 Keller et  al.  EJE  (2007)  156.  647-­653

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Bidlingmaier - IGF-I assays

IGF-I in response to rhGH treatment in healthy

young adults
IGF-I

• increase after ≈6 hours

• inter-individual variability

• females require higher

doses than males to
achieve same delta IGF-I

7 July 31, 2017 Keller et  al.  EJE  (2007)  156.  647-­653

Bidlingmaier - IGF-I assays

IGF-I in response to surgical treatment of

acromegaly

• on average,
IGF-I declines
within one
week post
surgery
• inter-
individual
variability

Feelders  RA  et  al.

JCEM  90:  6480–
6489,  2005)

8 July 31, 2017

Bidlingmaier - IGF-I assays

Intra-individual fluctuations in IGF-I following

surgical treatment of acromegaly

• OGTT nadir
predictive in
week 1
• fluctuations in
IGF-I post
surgery
• time-point of
stabilization
variable – up
to 12 weeks

Feelders  RA  et  al.

JCEM  90:  6480–
9 July 31, 2017
6489,  2005)

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Common problems for clinicians

10 July 31, 2017

Bidlingmaier - IGF-I assays

Correlation between peak GH and IGF-I in GHD

Hartman ML et al., JCE&M 2002

correlation exists but

is very modest!

Juul A et al., JCE&M 1997

11 July 31, 2017

Bidlingmaier - IGF-I assays

Overlapping IGF-I between healthy and GHD

Juul A et al., JCE&M 1997 Biller BMK et al., JCE&M 2002

12 July 31, 2017

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Bidlingmaier - IGF-I assays

Biochemical assessment of GH excess:

Conflicting results from GH and IGF-I

35% GH/IGF-I discordant

24% high IGF-I/normal GH

11% high GH/normal IGF-I

Biology or analytical issues?

13 July 31, 2017

Alexopoulou et al. JCEM 2008

Bidlingmaier - IGF-I assays

Biochemical assessment of GH excess:

Conflicting results from GH and IGF-I

Elevated  IGF  with  “normal”  GH  frequently  reported

14 July 31, 2017

Schilbach et al., Pituitary 2017

Bidlingmaier - IGF-I assays

Biochemical assessment of GH excess:

Conflicting results from OGTT and IGF-I

Separate  disease  entity?

Change  in  assays?
Change  in  patients  characteristics?

15 July 31, 2017 Butz et al., Pituitary 2016

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Bidlingmaier - IGF-I assays

GH assay results in clinical routine:

Lack of comparability
Same sample sent to 36 labs. The labs used 14 different assays
4.0
3.5
3.0
GH (mU/L)

2.5
2.0
1.5
The choice of the assay/lab makes
1.0
0.5
the diagnosis?!
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Method
Pokrajac A et al. Clin Endocrinol (Oxf) 2007;67:65–70
16 July 31, 2017

Analytical issues
with IGF assays

17 July 31, 2017

Bidlingmaier - IGF-I assays

IGF-I results using different assays:

German EQAS 3/2013
2  samples  analyzed by  all  laboratories  (n>150)
(each  dot  represents  the  2  results  from  1  lab)

800  ng/mL

200  ng/mL

18July 31, 2017

www.rfb.bio

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Bidlingmaier - IGF-I assays

IGF-I results using different systems:

German EQAS 3/2013
2  samples  analyzed  by  all  laboratories
(each  dot  represents  the  2  results  from  1  lab)

IDS iSYS Siemens Diasorin

n=37
Immulite Liaison
n=31 n=66

19 July 31, 2017

Bidlingmaier - IGF-I assays

IGF-I results using different systems:

German EQAS 3/2013
2  samples  analyzed  by  all  laboratories
(each  dot  represents  the  2  results  from  1  lab)

IDS iSYS Siemens Diasorin

n=37
Immulite Liaison
n=31 n=66

new calibrator 02/274 old calibrator 87/518 new calibrator 02/274

20 July 31, 2017

Bidlingmaier - IGF-I assays

‘Longitudinal’ problems with an IGF-I assay

21 July 31, 2017

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Bidlingmaier - IGF-I assays

‘Longitudinal’ problems with an IGF-I assay

22 July 31, 2017

Bidlingmaier - IGF-I assays

‘Longitudinal’ problems with an IGF-I assay

Mayo Clinic University of Virginia

20072008 2009 2010 2011 2012 2008 200 2010 2011 2012
9
40 30

30
Percentage (%)

Percentage (%)

20

20

10
10 Above the reference
intervalthe reference interval
Below

0 0
1 10 20 30 1 5 10 15
IGF-1 reagent lot number IGF-1 reagent lot number

Significant increase in the percentage of results above

normal range

23 July 31, 2017

Algeciras-Schimnich et al. Clin Chem 2013

Bidlingmaier - IGF-I assays

‘Longitudinal’ problems with an IGF-I assay

‘Traditional’  tests  for  acceptability  of  new  reagent  lots  did  not  
detect  the  problem!

Standard  acceptance test All  patients  mean/median

2007 2008 2009 2010 2011 2012 2007 2008 2009 2010 2011 2012
1.2 180

*
Lot-­to-­lot  comparison  slope

160 * *
1.1 * *
*
IGF-­1  (ng/mL)

*
140 *
1.0 *
120

0.9
100

0.8 80
1 10 20 30 1 10 20 30
IGF-­1  reagent  lot  number IGF-­1  reagent  lot  number

24 July 31, 2017

Algeciras-Schimnich et al. Clin Chem 2013

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Bidlingmaier - IGF-I assays

Mass spectrometry assays for IGF-I

25 July 31, 2017

Kam et al., eJIFCC 2016

Bidlingmaier - IGF-I assays

Mass spectrometry assays for IGF-I

Imprecision  and  linear  range  of  LC-­MS  based  IGF-­I  assays

26 July 31, 2017

Kam et al., eJIFCC 2016

Bidlingmaier - IGF-I assays

Mass spectrometry assays for IGF-I

Interlaboratory agreement  of  IGF-­I  measured  by  mass  spec

27 July 31, 2017 Cox et al., Clin Chem 2014

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Bidlingmaier - IGF-I assays

IGF-I control pools over a 29 months period

Pooled patients sera, Munich Endocrine Laboratory
(IDS iSYS IGF-I assay, October 2014 – March 2017)

800
700
CV 6.5%
600
500
400
300
CV 4.3%
200
100
CV 5.0%
0
10/8/2014 1/16/2015 4/26/2015 8/4/2015 11/12/2015 2/20/2016 5/30/2016 9/7/2016 12/16/2016 3/26/2017

28 July 31, 2017

Interpretation issues
with IGF assays

29 July 31, 2017

Biological confounders

30 July 31, 2017

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Bidlingmaier - IGF-I assays

IGF and age

the older the

GHD patient,
the more likely
IGF-I is normal!

Aimaretti G et al., Clinical Endocrinology (2003) 59, 56–61

31 July 31, 2017

Bidlingmaier - IGF-I assays

GH/IGF-I and fasting (36 hours)

GH increases but IGF-I tends to (moderately) decrease

32 July 31, 2017

Maccarion et al., Int J Obesity 2001

Bidlingmaier - IGF-I assays

IGF-I and BMI

32  women:  anorexia nervosa (n=11),  normal-­weight (n=11),  obese (n=10)

Pooled hourly overnight serum samples

GH

IGF-I

33 July 31, 2017

Brick  DJ  et  al.  Eur J  Endocrinol.  2010  Aug;;163(2):185-­91

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Bidlingmaier - IGF-I assays

IGF-I and BMI: Obesity

normal weight
obese

34 July 31, 2017 Galli G  et  al.

Obes Surg.  2012  Aug;;22(8):1276-­80

Bidlingmaier - IGF-I assays increase  after  weight  loss  induced  by  

IGF-I and BMI: Obesity laparoscopic  adjustable  gastric  banding

Δ normal weight
Ÿ obese

note: variable
response between
individuals – not all
increase!

35 July 31, 2017 Galli G  et  al.

Obes Surg.  2012  Aug;;22(8):1276-­80

Bidlingmaier - IGF-I assays

Beyond biology: Impact of IGF-I assay method

on observed correlation to BMI
§same set of samples/same cohort (population based, n=4000)
§IGF-I measured by 2 assays
§significant correlation in both assays for males
§signficant correlation for females only for IDS iSYS, not for Nichols!
IDS iSYS Nichols IDS iSYS Nichols

36 July 31, 2017 Müller  C.,  Wallaschofski H.  et  al,  JCEM,  2014,  99(8):2804–2812

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Bidlingmaier - IGF-I assays

Impact of gender?

Females (n=8353) Males (n=6697)

37 31 May 2012

Centiles:
peak IGF-I LMS method
during puberty higher
and 6 months earlier in girls
37 July 31, 2017 Bidlingmaier et al. JCEM 2014

Bidlingmaier - IGF-I assays

Impact of gender?

Females (n=8353) Males (n=6697)

38 31 May 2012

at old ageCentiles: LMS method

(>60) upper limit of
normal higher in males
38 July 31, 2017 Bidlingmaier et al. JCEM 2014

Bidlingmaier - IGF-I assays

Oral vs. transdermal estrogens: Effect on IGF-I

in response to endogenous and exogenous GH

postmenopausal
women
IGF decreased
with oral,
unchanged with
transdermal E2

Weissberger AJ  et  al.  JCEM.  1991;;72(2):374-­81

GHD females
Higher increase in
IGF-I on
trandermal E2

39 July 31, 2017

Wolthers,  T.,  et  al.  AJP  Endo  Metab 281,  E1191–E1196.

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Bidlingmaier - IGF-I assays

Renal failure

§ Total IGF-I unchanged

§ Free IGF-I (and IGF-I bioactivity)
markedly reduced
§ IGFBP-2 and -3 markedly elevated

40 July 31, 2017

Frystyk,  J.,  et  al.,  Kidney  Int,  1999.  56(6):  p.  2076-­84.

Bidlingmaier - IGF-I assays

Abnormal GH secretion in patients with Type 1

diabetes mellitus
§ GH secretion normal/enhanced in
insulin-treated diabetes
Ø Random GH can be
inappropriately high
§ Insulin deficiency induces GH
resistance
Ø IGF-I can be inappropriately
low in subjects with diabetes
(most studies in T1D)

Frara  S.  Et  al  Trends  in  Endocrinology  

&  Metabolism  2016
41 July 31, 2017
Kamenicky et  al  Endocrine  Rev  2014

Bidlingmaier - IGF-I assays

Beyond biology: IGF-I assay problems in

patients with diabetes

different  IGF-­I  assays  

can  nicely  correlate  in  
healthy  individuals…..

….but  can  give  very  

different  results  in  
patients  with  diabetes!

Chestnut  RE,  Quarmby V

Journal  of  Immunological  
42 July 31, 2017
Methods  259  2002.  11–24

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Reference intervals

43 July 31, 2017

Bidlingmaier - IGF-I assays

Reference intervals need to be method specific

700 samples from healthy adults

Assay A Assay B Assay C Assay D

44 July 31, 2017

Ranke et al., Clin Chem Lab Med 2003

Bidlingmaier - IGF-I assays

Do laboratories appreciate the importance of

reference intervals for IGF-I?
same  sample  -­ 23  laboratories  -­ 6  different  assays

14  different  reference  intervals…..

45 July 31, 2017

Pokrajac et al. Clinical Endocrinology 2007

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Bidlingmaier - IGF-I assays

Published reference data for IGF-I – different in cohort

size, different in statistics (examples)
participants
study (male/female) statistical method
log transformation, multiple linear regression
Löfqvist et al. 2001 468 (468, 0) (best linear model: age, gender and puberty)
Brabant et al. 2003 3961 (1469, 2492) polynomial modelling
Ranke et al. 2003 427 (0, 427) log transformation, linear regression
Elmlinger et al. 2004 1584 (881, 703) local regression analysis
Ivan et al. 2005 88(?, ?) not detailed
square root transformation, least linear
Bereket et al. 2006 807 (807, 0) regression
Massart 2007 693 (?, ?) not detailed
Kong et al. 2007 1692 (1692, 0) LMS
Aimaretti et al. 2008 547 (0, 547) grouped linear regression
Friedrich et al. 2008 2499 (0, 2499) linear and quantiles regression
log transformation, means for different age-
Granada et al. 2008 405 (0, 405) groups
Brugts et al. 2008 426 (0, 426) linear regression (with bioactivity)

46 July 31, 2017

Bidlingmaier et al. JCEM 2014

Bidlingmaier - IGF-I assays

Published reference data for IGF-I – different in cohort

size, different in statistics (examples)
participants
study (male/female) statistical method
log transformation, multiple linear regression
Löfqvist et al. 2001 468 (468, 0) (best linear model: age, gender and puberty)
Brabant et al. 2003 3961 (1469, 2492) polynomial modelling
Ranke et al. 2003 427 (0, 427) log transformation, linear regression
Elmlinger et al. 2004 1584 (881, 703) local regression analysis
Ivan et al. 2005 88(?, ?) not detailed
square root transformation, least linear
Bereket et al. 2006 807 (807, 0) regression
Massart 2007 693 (?, ?) not detailed
Kong et al. 2007 1692 (1692, 0) LMS
Aimaretti et al. 2008 547 (0, 547) grouped linear regression
Friedrich et al. 2008 2499 (0, 2499) linear and quantiles regression
log transformation, means for different age-
Granada et al. 2008 405 (0, 405) groups
Brugts et al. 2008 426 (0, 426) linear regression (with bioactivity)

47 July 31, 2017

Bidlingmaier et al. JCEM 2014

Bidlingmaier - IGF-I assays

How to calculate reference intervals?

(n=579) (n=540)

??
??

Alberti et al. Clin Chem 2011)

?? ??

48 July 31, 2017

Brabant et al. Horm Res 2003

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Bidlingmaier - IGF-I assays

IGF-I reference intervals for the IDS iSYS IGF-I

(international multicenter study)

Females (n=8353) Males (n=6697)

Centiles:
49July 31, 2017 LMS method Bidlingmaier et al. JCEM 2014

Bidlingmaier - IGF-I assays

IGF-I reference intervals: No statistical model

perfectly fits all age groups!

250

200 p<0.0001
IGF-I (ng/ml)

150

100

50

0
cord blood 12 months
(n=146) (n=324)

50 July 31, 2017

Bidlingmaier et al. JCEM 2014

Bidlingmaier - IGF-I assays

Reference intervals for IGF-I: Same cohort,

same mathematics, different assays
§ Cross-sectional multicenter cohort study (VARIETE)
§ IGF-I measured by 6 immunoassays in the same 911 healthy
adults
§ Age 18–90 years; BMI 19 and 28 kg/m2
§ Exclusion if „medical conditions or medications … might affect
IGF-I serum levels“
§ Construction of reference intervals using LMS method
§ Assessment of assay concordance (Bland-Altman plots) for both
IGF-1 raw data and standard deviation scores (SDS)

51 July 31, 2017

Chanson et al. JCEM. 2016

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Bidlingmaier - IGF-I assays

Reference intervals for IGF-I: Same cohort,

same mathematics, different assays

52 July 31, 2017

Chanson et al. JCEM. 2016

Bidlingmaier - IGF-I assays

Reference intervals for IGF-I: Same cohort,

same mathematics, different assays

53 July 31, 2017

Chanson et al. JCEM. 2016

Bidlingmaier - IGF-I assays

Reference intervals for IGF-I: Same cohort,

same mathematics, different assays

IGF-I ng/mL

scatter >100%
scatter <30%
bias with concentration
no trend

IGF-I SDS

SDS do not automatically agree much

better than absolute concentrations!
54 July 31, 2017
Chanson  P et  al.  J Clin Endocrinol Metab.  2016  Sep;;101(9):3450-­8

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Bidlingmaier - IGF-I assays

Summary:

• Calibration, removal of IGFBP3 interference, long term

stability of assay performance and quality of reference
intervals are crucial for IGF assay quality
• Both, immunoassays and MS assays must be (and can be)
designed to fulfil the quality criteria outlined in the
consensus statement

• Keep in mind potential technical issues, but also biological

confounders for appropriate interpretation
• Size and selection of the reference population as well as
statistical model influence suitability of “reference ranges”

55 July 31, 2017

Thank you!

Martin Bidlingmaier MD
Endocrine Laboratory
Medizinische Klinik und Poliklinik IV
Klinikum der Universität München

email: martin.bidlingmaier@med.uni-muenchen.de
phone: +49 89 440052277

56 July 31, 2017

57 July 31, 2017

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USC
UNIVERSITY
AACC Symposium
Clinical Endocrine Assays:
OF SOUTHERN
CALIFORNIA
What Endocrinologists Will Ask You
7/31/17

Thyroglobulin (Tg) and Tg Autoantibodies -

used as Tumor Markers for
Differentiated Thyroid Cancer (DTC)
Carole Spencer MT, Ph.D, FACB
Professor of Medicine
Department of Medicine
University of Southern California Spencer
email: cspencer@usc.edu USC 2017

DISCLOSURE

Nothing to disclose

Spencer
USC 2017

Guidelines mandate TgAb be measured with every Tg test:

because TgAb status can change (TgAb- to TgAb+, or vice versa)

}
Thyroglobulin (Tg) measurement
measured
as a panel
Thyroglobulin antibodies (TgAb)

75% 25%

The absence of TgAb When present, the trend in

authenticates that the Tg TgAb concentrations can
measurement is free from serve as a surrogate
TgAb interference tumor-marker
Spencer
2015 ATA Guidelines. Haugen et al Thyroid 26, 1-133, 2016 USC 2017

20
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Tg and TgAb Measurements - DTC Tumor Markers

Technical Challenges that Impact Clinical Utility:

1 There are 3 different classes of Tg method:

• Immunometric assay (IMA)
• Radioimmunoassay (RIA)
• Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)

Significance

These different classes of method have intrinsically

different Sensitivities, Specificities and Propensity for
Interference by Heterophile Abs and TgAb. Spencer
USC 2017

Spencer
Tg Methodologies USC 2017

Immunometric Assay
(IMA)
(1990 – present) 2,4

Only valid in absence

of TgAb (75% patients) 1

Radioimmunoassay
(RIA) 2,4
(1973 - present)

Used for TgAb+ sera

Liquid Chromatog.
Tandem Mass Spec.
LC-MS/MS • extensive specimen prep.
(2009 - present) 3,4,5
Poor clin. sensitivity: >40% • trypsin digestion may fail
Used for TgAb+ sera TgAb+ with Dz. have UD Tg-MS to yield target peptide(s)
1 Baloch 2 from polymorphic tumor Tgs
Thyroid 2003;13:1-126; Spencer JCEM 90:5566, 2005
3 Hoofnagle JCEM 98:1343, 2013 4 Spencer JCEM 99:4589, 2014 5Netzel JCEM 100:E1074 ,2015

Tg and TgAb Measurements - DTC Tumor Markers

Technical Challenges that Impact Clinical Utility:

2 The new more sensitive (Second-Generation) IMA methods

(2G-Tg-IMA) can reliably measure the subnormal Tg levels

typically seen after Tx. (serum Tg the 0.1-1.0 ng/mL range).

Significance

The use of a 2G-Tg-IMA method obviates the need

for rhTSH-stimulated Tg testing. Spencer

USC 2017

21
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More Sensitive (2nd Generation) 2G-Tg-IMA Methods

can Monitor Subnormal Serum Tg Concentrations
50
40
mean intact
euthyroid reference range
10 ~ 13.0 (12-15 g)
thyroid gland
serum
Tg 2
ng/mL
1 1
1st generation functional sensitivity RIAs / IMAs / LC-MS/MS
0.5
subnormal (Tx.) range

0.1 0.1
2nd generation functional sensitivity 2G-Tg-IMA
0.05

Spencer
Spencer et al. Curr Opin Endo Diab Obes 5:394, 2014 USC 2017

2G-Tg IMA has Become the Standard of Care (all major US labs):

Obviates the Need for Recombinant Human TSH (rhTSH) Stimulation

Basal-Tg/rhTSH-Tg correlation 1.0

(n = 1029)
10,000 0.9
rhTSH-Tg = 17.6*basal Tg -12.9 * AUC = 0.95
r = 0.72, p < 0.0001 0.8

1,000
0.7 Basal Tg < 0.15 ng/mL
Sensitivity

0.6

0.5 NPV = 98.6%

100
rhTSH-Tg 0.4
PPV = 47.8%
ng/mL
0.3
10
0.2
rhTSH cut-off
2 0.1
1
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
functional 1 - Specificity
0.1
sensitivity
Spencer Nat Clin Pract Endocr Metab 4:223, 2008
Grebe Expert Rev Endocrinol Metab 4:25, 2009
Spencer Thyroid 20:587, 2010
Malandrino et al JCEM 96:1703, 2011
0. 1 10 100 Chindris JCEM 97:2714, 2012 Spencer
1 basal Tg ng/mL Trimboli Horm Metab Res 45:664, 2013 USC 2017

Interpretation of Tg & TgAb Measurements

Key Issues:

3 Current Guidelines* recommend monitoring

the serum 2G-Tg-IMA trend as a prognostic indicator
(measured during L-T4 Rx. with TSH at target).
.
Significance
The serum 2G-Tg-IMA trend is a better clinical indicator

than using fixed Tg cut-offs (i.e. Tg “detectability”

or serum Tg less than 1 ng/mL).

Spencer
USC 2017
* Haugen et al Thyroid 26; 1-133, 2016

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Prognostic Value of Monitoring the Serum 2G-Tg-IMA Trend

Low-Risk Disease-Free PTC patients (no RAI Rx.)

(constant L-T4 dose to maintain TSH < 0.5 mIU/L)
1000 1000

100 100

10 10
Serum Serum
Tg 2GIMA Tg 2GIMA
ng/mL ng/mL
1 expect Tg < 0.5 ng/mL (non-elevated TSH) 1
0.5 0.5

0.1 0.1
functional functional
sensitivity sensitivity
below assay
functional sensitivity
0.01 0.01
-3 0 3 6 9 1 3 years 5 7 9 11 13
months
Angell and LoPresti Thyroid 24:1127,2014 Spencer
thyroidectomy Spencer et al Curr Opin Endoc Diab 21:394, 2014 USC 2017

2G-Tg-IMA Sensitivity is Needed for Reliable Detection of DTC Recurrences

1993
RAI
TSH < 0.1 mIU/L

100

PET +

10
surgery
serum MRI +
Tg 2GIMA surgery
ng/mL
UZT-
1

2G-
TgIMA
4-year Detection
doubling time Range

0.1

FS = 0.05
0 5 10 15 20 25
years after Tx.
FNA PTC+ Tx. 3cm PTC with 4/8 +LNs
Spencer
USC 2017

Serum Tg Doubling-Time (Tg-DT on L-T4 Rx.) is a Useful Prognostic Indicator

Tg-DT = < 1 yr Tg-DT = 1-3 yrs

1000 100
Thyroglobulin (ng/mL)
Thyroglobulin (ng/mL)

100
100

10
80
10

60
Survival (%)

1 1
0 2 4 6 8 0 2 4 6 8

Times (years) Times (years)

40
Tg-DT = ≥ 3 yrs Tg-DT = -21.6 yrs
100 100
Thyroglobulin (ng/mL)
Thyroglobulin (ng/mL)

Tg-DT <1 year 20

Tg-DT 1-3 years
Tg-DT ≥ 3 years
10 10
Tg-DT no rising trend 0

2 4 6 8 10 12 14
Time (years)
1
0 2 4 6 8
1
0 2 4 6
08
Times (years) Times (years) Miyauchi Thyroid 21:707, 2011 Spencer
Wong Ann Surg Oncol 19:3479, 2012 USC 2017

23
 10.07.17

Tg and TgAb Measurements - DTC Tumor Markers

Technical Challenges that Impact Clinical Utility:

4 It is critical to monitor the serum Tg trend

using the same manufacturers method.

Significance

Different Tg methods can report up to a 2-fold

difference in the Tg value reported for the same serum.

Spencer
USC 2017

Tg in Sera, Especially Tg Secreted by Tumors, is Heterogeneous – the cause of

2-Fold Differences in Serum Tg Measured by Different Methods!
1299
1075

1,000
A
901
765
799 777
672 732
500 614 579

40
method
mean serum Tg 29.2
30 26.2
ng/mL(± 2sd)
B
20 17.5 16.5 16.4

C 13.3 10.
4
10

D 8.1 4.4
2.9
UK NEQAS QC program
(51 Labs. using 10 methods) 1 1.9

Spencer
Spencer and Fatemi COED 21:394,2014 USC 2017

In the Absence of TgAb

Constant (non-elevated) No
TSH maintained Thyroid Injury

Trend in Trendin
Basal Tgbasal
(SameTgMethod!)

Changes in Efficiency of
Tumor Mass that individual’s
tumor to secrete Tg

Baudin et al JCEM 88;1107, 2003

Tuttle et al EMCNA 37:419, 2008 Spencer
Giovanella et al Clin Endocrinol 68:659, 2008 USC 2017

24
 10.07.17

Tg and TgAb Measurements - DTC Tumor Markers

Technical Challenges that Impact Clinical Utility:

5 Immunometric Assay (IMA) is the methodology

most prone to interferences from Heterophile Abs and

Tg autoantibodies (TgAb).

Significance

Most Labs restrict Tg-IMA testing to TgAb-negative sera,

and use Tg-RIA or Tg-LC-MS/MS for TgAb-positive sera.

Spencer
USC 2017

Only the Physician (and not the Lab!) can Suspect an Interference
TgAb, HAb/HAMA and high dose dietary Biotin can interfere with Tg (and TgAb)

• Test Result is clinically unexpected

• Inconsistent with other clinical correlates

• Inconsistent with other biochemical tests

• Odd results in more than one method

• Significant or unexplained change from a previous test

• Patients with autoimmune or chronic diseases are more at risk

• Recent immunization, blood transfusion or monoclonal Ab therapy

• Veterinarians and those who come into contact with animals

Ismail Clin Med 7:357, 2007
Tate Clin Biochem Rev 25:105, 2004 Spencer
Sturgeon Ann Clin Biochem 48:218, 2011 USC 2017

HAb/HAMA interference characterized by a blunted/absent

rhTSH-Tg response (< 1.5 fold response)
10
5/1034 (0.5% Tg tests were HAMA+)

Tg
ng/mL

0.1

0.01
rhTSH basal blocker blocker Tg RIA
functional Tg Tg X1 X2 Spencer
sensitivity Spencer et al Thyroid 20:587, 2010 USC 2017

25
 10.07.17

Graves’ Hyperthyroid Patients with TgAb have Paradoxically low/UD Tg

Graves‘ Hyperthyroidism

1,000

100

Serum Tg
ng/mL

10
TgAb-negative
TgAb-positive

Spencer
Marriotti JCEM 80:468, 1995
IMA # 1 2 USC 2017

Many Labs reflex Tg testing to different methodologies

based on the TgAb status of the specimen (+ or neg)
Maximize clinical sensitivity Minimize TgAb Interferences

reflex
Tg testing
?
Highest NO TgAb YES Resistant
Functional present in to
Sensitivity 75% serum 25% Interferences

2GTg-IMA

Spencer
USC 2017

Tg and TgAb Measurements - DTC Tumor Markers

Technical Challenges that Impact Clinical Utility:

6 Tg measured by any methodology can be unreliable

when TgAb is present!

Significance
In the presence of TgAb, the absolute Tg concentration

can be unreliable (by any method) whereas a rising

trend in Tg-RIA or Tg-IMA suggests disease progression.

Spencer
* Haugen et al Thyroid 26; 1-133, 2016 USC 2017

26
 10.07.17

Tg-RIA resistance to TgAb interference is seen in patients with Dz. whose TgAb status (+/-) changes.
10,000 1,000
Patient A TgAb

1,000 100

Tg-RIA

100 10
Tg-IMA serum Tg
TgAb ng/mL
Negative TgAb
kIU/L
10 1

0.5 FS Tg-RIA (USC)

1 0.1 FS Tg-IMA
(Beckman)
Kronus TgAb FS 0.
4
0.
1 2 4 6 8 10 12
Tx. RAI Sx. Sx. lung mets. yrs after Tx. deceased

100
Patient B 100
Tg-IMA

TgAb
10 10
serum Tg
Tg-RIA ng/mL

TgAb 1 1
Tg-RIA kIU/L
0.5 FS Tg-RIA (USC)
Kronus TgAb FS 0.
4 Tg-IMA
Tg-IMA
0.1 0.1 FS Tg-IMA
Negative TgAb
(Beckman)
RAI Sx. Sx. lung mets. deceased
Spencer , CA
Thyroid Manager 3 4 5
Spencer
1 yrs after Tx.
2017 Tx. 2 USC 2017

Current Controversial Question:

Do the new Tg LC-MS/MS tests
overcome the problem of TgAb
interference that causes falsely low
or undetectable Tg-IMA values
that can mask disease?
Spencer
USC 2017

>
33/47 (70%) TgAb+ Patients with Structural Disease (11% with Distant Mets)
had Undetectable Tg using Mass Spectrometry (Tg-LC-MS/MS)

10

Serum Tg
μg/L

(n=35) (n=32) (n=25) (n=1)

0.1

below assay (n=28)

functional sensitivity

0.01
Tg2GIMA Tg-LC-MS/MS Methods
Tg-RIA
5 site collaborative study ARUP MAYO QUEST Spencer
Spencer et al ITC 2015 USC 2017

27
 10.07.17

Clinical Sensitivity of Serum Tg Testing in TgAb+ Patients with Structural Dz.

100 100
Same detection limit (0.5 ng/mL) used for all methods
92 88
8 8
% with0 0
UD Tg • The higher the TgAb concentration the77more76likely that Tg-LC-MS/MS
will be undetectable in TgAb+ patients with disease.
6 6
05 58 58
• Tg-LC-MS/MS less clinically sensitive than 2GTg-IMA (offers no advantage). 0
56 5
0 0
4 44 44 Azmat et al Thyroid 27:74, 4
0 2017 0

2 23 2
0 4 0
2
0 0
Mayo Quest ARUP
Tg Method 2GTg- IMA (Beckman) USC Tg-RIA
LC-MS/MS Methods
median TgAb
Spencer et al JCEM 99:4589, 2014 (n=52) 23% UD Tg-LC-MS/MSMS 185
Netzel et al JCEM 100: E1074, 2015 (n=57) 44% UD Tg-LC-MS/MSMS 288 kIU/L
Spencer et al ITC 2015 (n=41) >75% UD Tg-LC-MS/MSMS 829 Spencer
Azmat et al Thyroid 27:74, 2017 (n=16) 44% UD Tg-LC-MS/MSMS not avail. USC 2017

Tg-TgAb Complexes may have a Faster Metabolic Clearance than Free Tg

TgAb is Absent TgAb is Present

Thyroid Thyroid
Tissue Tissue

Tg Tg + TgAb epitope spec.?

TgAb
affinity?
capacity?
Tg
Tg TgAb

hepatic
ASGPR receptor?

t1/2 ~ 3 days t1/2 ~ 3 days ? increased clearance

of Tg complexed with TgAb
Spencer
Weigle et al J Immunol 98:1105, 1967 USC 2017

Tg and TgAb Measurements - DTC Tumor Markers

Technical Challenges that Impact Clinical Utility:

7 The TgAb trend (measured by same method) can

reflect the status of disease.

Significance

Current Guidelines* suggest that the TgAb trend

is a more reliable (surrogate) tumor-marker

than serum Tg measurement.

Spencer
USC 2017
* Haugen et al Thyroid 26; 1-133, 2016

28
 10.07.17

For Disease-Free Patients, the Higher the Initial TgAb, the Longer it takes for TgAb to become Undetectable
Disease-Free PTC Patients Spencer and Fatemi COEDO 21:394,2014
1000
(a) TgAb concentrations (b) % change from initial (0-3mos) TgAb 150

100 neg.UTZ

neg.UTZ 100
Trend in % change from
serum TgAb 10 initial TgAb
(kIU/L) neg.UTZ

50
1 neg.UTZ
FS = 0.4
10

Tx. 2 4 yrs. post Tx.8 10 Tx. 2 4 yrs. post Tx.8 10

PTC Patients with Active Disease
1000
(c) TgAb concentrations (d) % change from initial (0-3mos) TgAb 150

100 lung mets.

LN +
LN +
100
Trend in % change from
LN +
serum TgAb 10 LN + initial TgAb
(kIU/L)
50
1 LN +

FS = 0.4
10

Tx. 2 4 8 10 Tx. 2 4 8 10
Spencer
disease detected yrs. post Tx. yrs. post Tx. USC 2017

TgAb Levels Respond to Tg Antigen Sensed by the Immune System

1,00
0 radioiodine lymph node recurrence lymph node recurrence
Rx. diagnosed by FNA diagnosed by FNA biopsy
biopsy
TgAb concentration kIU/L

10 lymph node surgery lymph node surgery

0

1
0

0.4
below assay detection limit

1 2 4 5 6 1 2 3 4
pre- Tx. years
months
op

Chung et al. Clin Endocrinol 57:215, 2002; Kim et al. JCEM 93:4683, 2008
Spencer JCEM 96:3615, 2011; Verburg Thyroid 23, 1211, 2013 Spencer
Spencer Curr Opin Endocrinol Diabetes Obes 21:394, 2014 Haugen Thyroid 26:1, 2016 USC 2017

TgAb Methods Differ in Sensitivity and Specificity Spencer

USC 2017
1. Different methods report different numeric values (can differ 100-fold!)
2. MCO cutoffs for “positive TgAb” set too high (for AITD not TgAb interference of Tg)
3. Optimal cutoff for TgAb “positivity” is the assay’s functional sensitivity not the MCO
4. Method insensitivity: Sera TgAb+ by one method may be “negative” by another.

144 DTC sera (evidence of TgAb IMA interference with Tg2GIMA)

TgAb
kIU/L
1,000

MCO
Manufacturer Cut-Offs
100
(MCO) are set to detect
MCO
thyroid autoimmunity Functional Sensitivity Functional Sensitivity
& are too high to detect
TgAb interference 10
with Tg measurement MCO

1 MCO Functional Sensitivity

Functional Sensitivity

0.1
Assay Kronus/RSR Roche Beckman Siemens
Reference
Spencer et al JCEM 96:1283, 2011

29
 10.07.17

Optimal DTC Monitoring Depends on the Patient’s TgAb Status

Patients WITHOUT TgAb detected:

Monitor the basal Tg TREND (TSH < 1.0 mIU/L) using a 2GTg-IMA
method (FS ≤ 0.10 μg/L).

Patients WITH TgAb detected:

1° monitor the serum TgAb TREND as a surrogate tumor-marker

2° monitor the Tg TREND using Tg-RIA or ? Tg-LC-MS/MS

(studies are needed to investigate why Tg -LC-MS/MS methods
fail to detect Tg in > 40% of patients with structural disease).
Spencer
* Haugen et al Thyroid 26; 1-133, 2016 USC 2017

Spencer
Tg and TgAb Measurements - DTC Tumor Markers USC 2017
Technical Challenges that Impact Clinical Utility:
There are three different classes of Tg method: IMA, RIA, LC-MS/MS.
1
Methods differ in sensitivity, specificity and propensity for interferences
2G-Tg-IMAmethods reliably measure subnormal Tg levels (0.1-1.0 ng/mL).
2
The use of a 2G-Tg-IMA method obviates the need for rhTSH stimulation

ATA GL recommend monitoring Tg trend (constant L-T4 dose/TSH at

3
target)
Serum 2G-Tg-IMA trend = better clinical indicator than Tg “detectability”
4 It is critical to monitor the Tg trend using the same manufacturers method.
Different Tg methods can report up to a 2x differences in a serum Tg value
IMA methodology – is the most prone to TgAb interferences
5
Labs restrict IMA to TgAb(-) sera, using Tg-RIA or Tg-LC-MS/MS for TgAb+

When TgAb is present, absolute Tg levels can be unreliable (by any

6
method).
A rising trend in serum Tg (RIA or IMA) suggests disease progression.
7 The TgAb trend (measured by same method) reflects disease status.
The TgAb trend is a more reliable “surrogate” tumor marker than serum
Tg.

More information on is
available in two webinars
available on USC laboratory
website:

www.thyroidlab.com/updates

30
 10.07.17

Cortisol  in  Hair

Dr.  Stan  Van  Uum

Professor,  Western  University
Division  of  Endocrinology  and  M etabolism
Division  of  Clinical  Pharmacology July  2017

Outline
• Overview  measuring  cortisol  in  hair
• Cushing  syndrome
– Cortisol  in  saliva
– Cortisol  in  hair
• Adrenal  insufficiency
– Cortisol  in  saliva
– Cortisol  in  hair
• Areas  for  further  research
• Conclusion

31
 10.07.17

How  to  measure  Stress?

Acute  stress Chronic  stress

• Questionnaires • Questionnaires
• Biological  parameters: • Biological  parameters??
– Blood  pressure
– Heart  rate
– Sweating
– Cortisol
– …

Cortisol:  Circadian  Rhythm

How  to  assess  integrated  cortisol  production  over  longer  time??

32
 10.07.17

Effect  Relocation  Stress  on  Cortisol  

in  Rhesus  Macaques

Study  in  20  male  rhesus  monkeys

Measure  cortisol  concentrations  in  hair

Compared  with  cortisol  in  saliva

Determine  effect  of  relocation =  MAJOR  STRESS

Davenport    et  al.

Gen  Comparat Endocrinol
2006,  147:  255-­‐261

Effect  Relocation  Stress  on  Cortisol  

in  Rhesus  Macaques

Baseline After 1 year Linear

move after move trend P
value
Cortisol saliva 0.12±0.01 0.33±0.05 0.01
(µg/dL)
Cortisol hair 81.7±7.5 129.6±15.5 75.8±8.7 <0.01
(pg/mg)
Davenport    et  al.
Results  are  presented  as  mean+SEM Gen  Comparat Endocrinol
2006,  147:  255-­‐261

Questions  Regarding  Measurement  of  

Cortisol  in  Human  Hair  
• What  is  appropriate  methodology?
• Does  is  reflect  systemic  cortisol,  incl.changes?
• Is  hair  cortisol  stable  along  the  hair  shaft?
• Is  hair  cortisol  stable  over  time?
• How  is  cortisol  incorporated  in  hair?
• Association  with  health/disease?

33
 10.07.17

Methodology

Measuring  Cortisol  in  hair

• Mostly  done  using  saliva  based  cortisol  assay

• Extraction  overnight  – 16  hours  optimal
• Discussion  about  need  of  fine  mincing  of  hair
• More  recently  exploration  of  LC-­‐MS-­‐MS

34
 10.07.17

Hair  cortisol  measurement

• Variation  coefficient  6%
• Same  samples  repeat  measurements  (6  
weeks)    11%  variation
• Most  

Effect  of  
Requirements  for   Retrospective   Dynamic  
Sampling   circadian  
Sample samples  storage  and   cortisol  level   Collecting  Method Response  
invasiveness cortisol  
transportation measurement Measurable
fluctuation

Serum Immediately   Highly  invasive Not  possible Venipuncture Affected Possible

measured  or  
freezing

Saliva Immediately   May  cause   Not  possible Spitting,  Use  of   Affected Possible
measured  or   discomfort saliva  collecting  
freezing tubes.

Urine Immediately   Could  be  difficult   Not  possible 24  hrs  urine   Not  affected Not  Possible
measured  or   in  certain  patient   collection
freezing populations

Hair Room  temperature/   Painless,  Not   Possible Small  samples   Not  affected Not  Possible
long  shelf-­‐life invasive from  the  posterior  
vertex  of  the  head

Cushing’s  
Syndrome

Effect  of  too  

much  cortisol  

35
 10.07.17

Example  15y  ♀ Cushing’s

syndrome  (over  2  years)
Weight  gain
Diabetes
Hypertension
Changed  
appearance

Results  – Cushing’s  Syndrome

Hair:
Sensitivity:  86%
Specificity:  98%

Manenschijn,  van  Rossum,  et  al  2012

Sensitivity  
Sensitivity:
and  Specificity
86%

Specificity: Specificity:
98% 98%                        93%                          93%

36
 10.07.17

Hair  can  look  into  the  past,  depending  on  

how  long  the  hair  is

• University  Student  
• 1  year  into  a  Masters
• Why  the  recent  increase?

Hair  cortisol  along  hair  shaft:

Thomson    et  al.

Exp Clin Endocrinol Diabetes
2010,  118  (2):133-­‐8

Hair  cortisol  in  cyclic  Cushing’s  Disease

Cyclic  Cushing’s  Syndrome

Cyclic  Cushing’s  Syndrome

Manenschijn,  van  Rossum,  et  al  2012

37
 10.07.17

Hair  cortisol  in  cyclic  Cushing’s  Disease

Cyclic  Cushing’s  Syndrome

Manenschijn  et  al,  JCEM  Oct  2012

Results

Cushing’s  Disease

Manenschijn,  van  Rossum,  et  al  2012

Cyclical  CS  Patient

300 75.9   75.9  ng/g  Cut-­‐off  for  CS
Cutoff • 86%  Sensitivity
250
Cortisol  Conventration  (ng/g)

31.1   • 98%  Specificity

Manenschijn  et  al.  
200 Cutoff J  Clin  Endocrinol  Metab  (2012)  
Patient doi:  10.1210/jc.2012-­‐1852  

150
31.1  ng/g  Cut-­‐off  for  CS
100 • 93%  Sensitivity
• 90%  Specificity
50 Wester  et  al.    
Eur J  Endocrinol (2017)  
doi:  10.1530/EJE-­‐16-­‐0873
0

Months  (back  in  time)  /  Hair  Segment  (cm  from  

scalp)

38
 10.07.17

Cyclical  CS  Patient

700
600
500
HCC  (ng/g)

400
300
200
100
0

Hair  Segment  (cm)

Fig  1.    Proposed  mechanisms  for  incorporation  of  

cortisol  into  hair  via  blood  (A),  sebum  (B),  and  sweat  (C)  

7cm  distal  to  scalp

Direction  of  hair   6cm  distal  to  scalp

growth
5cm  distal  to  scalp
medulla
cortex
cuticle 4cm  distal  to  scalp
Universally  
agreed  to  be  
3cm  distal  to  scalp reliable  up  to  at  
least  5cm  from  
2cm  distal  to  scalp 3  months’   scalp
exposure
scalp
1cm  distal  to  scalp 1  month’s  
exposure

3-­‐5mm  beneath  scalp;

lag  time  of  1-­‐2  weeks
C B
A Russell  et  al.
=  cortisol Psychoneuroendocrinol.
2011,;37:  589-­‐601

Cortisol  incorporation  in  hair:  

considerations:
1)  Local  cortisol  production:  levels  come  down  
quickly – suggesting  acute  response.  Does  not  
explain  sustained elevation  in  groups  with  
systemic  hypercortisolism
2)  other  corticosteroids,  e.g.  testosterone,  
dexamethasone  etc.  are  present  in  hair,  there  is  
no  sign  of  them  being  produced  locally.  
Biologically  one  would  expect  similar  molecules  to  
behave  similarly

39
 10.07.17

Cortisol  in  Human  Hair  &  Chronic  Stress  

• Chronic  pain  is  a  source  of  major  stress
• We  recruited  patients  form  a  pain  clinic  
with  severe  chronic  pain  necessitating  
chronic  opioid  use
• Control  group  of  healthy  controls
• Measurement  of  hair  cortisol  and  
subjective  stress  (Perceived  Stress  Scale)

Van  Uum    et  al.

Stress    2008  (11)  483-­‐8

Hair  Cortisol  &  Chronic  Pain  

Van  Uum    et  al.

Stress    2008  (11)  483-­‐8

40
 10.07.17

Long-­term  cortisol  levels  in  shift  workers  and  day  workers  in  total  and  divided  into  age  
groups,  based  on  the  median  age  of  shift  workers.  *,  P  <  0.001  (unadjusted).  

Manenschijn L  et  al.  JCEM  2011;;96:E1862-­E1865

©2011  by  Endocrine  Society

Too  Little  Cortisol  is  a  Problem…

Abdominal pain
Diarrhoea
Vomiting Addison’s
Weight Loss Disease
Muscle Weakness
Low Blood Pressure
Dehydration
Death

Too Little Cortisol

is a Problem…

Addison’s Disease

Abdominal  pain
Diarrhoea
Vomiting
Weight  Loss
Muscle  Weakness
Low  Blood  Pressure
Dehydration
Death

41
 10.07.17

Diagnosis  – Other  Diagnostic  Tests

-­‐ Our  patient  gave  a  hair  sample  of  ~36  cm,  providing  historical  data  for  3  years.
-­‐ The  graph  suggests  decline  in  cortisol  production  3  years  before  her  ICU  admission,  and  matches  
symptomology  gathered  on  history.  

Results
Addison’s  Disease

Manenschijn,  van  Rossum,  et  al  2012

Hair  analysis  Along  Hair  shaft:

Patient  on  High  Dose  Hydrocortisone

150  mg/day

Effect  tapering  of  

hydrocortisone  
(cortisol),  
90  mg/day
measured  in  one  
hair  sample

Thomson    et  al.

Exp Clin Endocrinol Diabetes
2010,  118  (2):133-­‐8

42
 10.07.17

Study  Package  – study  on  monitoring  

hydrocortisone  treatment
• Letter  of  information &  consent  form
• Personal  information  form
• Perceived  stress  scale  (PSS)  questionnaire
• Hair  sampling  instructions  (brochure)
• Hair  collection  form
• Pre-­‐stamped  envelope

39  patients  from  
Canada

78  patients  from  the  USA

43
 10.07.17

Hair  cortisol  in  Adrenal  Insufficiency:

Hair  cortisol  and  hydrocortisone  dose

Men Women

Hair  cortisol  and  the  risk  for  acute  

myocardial  infarction  in  adult  men

Pereg  et  al.

Stress  2011:14(1):73-­‐81

44
 10.07.17

Hair  cortisol  and  the  risk  for  

acute  myocardial  infarction  in  
adult  men

Pereg  et  al.

Stress  2011:14(1):73-­‐81

Pereg  et  al.

Stress  2011:14(1):73-­‐81

Is  Cortisol  in  Hair  Stable  over  Time?

Anthropology…..

Webb    et  al.

J  Archeol Science    
37  (2010)  807-­‐812

Is  Cortisol  in  Hair  Stable??

Anthropology

Webb    et  al.

J  Archeol Science    
37  (2010)  807-­‐812

45
 10.07.17

Hair  Cortisol  Analysis:

Advantages  &  Disadvantages
• Non-­‐invasive  sample  collection • Approximate,  long-­‐term  
measurements,  not  exact
• Increased  detection  window  (months  
à ?years!) • Potential  contamination
• Historical  information  (past  exposure)
• Variability
• In  case  of  debatable  results,  one  can   • Hair  growth  rate
obtain  another  hair  sample,  while  it  is   • Colour,  thickness,  length
impossible  to  obtain  another   • Sample  collection
urine/blood  sample  from  the  same  
time  period
• Able  to  be  sent  through  the  mail,  easy  
storage  and  temperature  requirements

Current  view  on  hair  cortisol:

1. Reflects  chronic  systemic  cortisol  exposure

2. Stable  along  > 6  cm  hair  shaft
3. Changes  along  with  changes  in  systemic  
cortisol/stress
4. Not  reflecting  subjective  stress
5. May  convey  risk  for  health  problems
6. Normal  range  still  to  be  determined
7. Best  method  still  to  be  determined
8. Exact  mechanism  of  incorporation  still  not  clear

Acknowledgement  

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 10.07.17

Acknowledgements
Graduate  Students: Collaborators
•Sanjog Kalra •Lisa-­‐Ann  Fraser
•Brittany  Sauve •Terri  Paul
•Steven  Thomson •Pat  M orley  Forster
•Rachel  Gow •Emily  Webb
•Evan  Russell •Chris  W hite
•Michael  Gref •Andrew  Nelson
•Baset Elzagallaai
•Theodore  Friedman
Co-­‐PIs
•Gideon  Koren •David  Pereg
•Michael  Rieder
•M.  Kramer
•All  patients  and  volunteers
•All  patients  and  volunteeers
•Liesbeth van  Rossum

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