Thermo Scientific
B·R·A·H·M·S CT-proAVP LIA
for use in endocrinology
February 2011
Vasopressin & CT-proAVP - FAQs

What is Vasopressin (CT-proAVP) and where is it produced?

What is the physiological role of Vasopressin?

Why not simply measure Vasopressin?

Is CT-proAVP produced together with Vasopressin?
Do both analytes show the same kinetics?
2

Which CT-proAVP levels should be expected in normals?

Thermo Scientific B·R·A·H·M·S CT-proAVP LIA in the Differential
Diagnosis of Diabetes insipidus

What about the performance of the
Thermo Scientific B·R·A·H·M·S CT-proAVP LIA Assay?
What is Vasopressin
and
where is it produced?
3
Structure of Vasopressin
O
O
NH2
-C
NH2-
Arginine-Vasopressin (AVP)
 synonym: Vasopressin or antidiuretic hormone (ADH)
 peptide hormone
 9 amino acids
 Disulfide bridge between two cysteine amino acids
 C-terminal amidation
4
NH2
Synthesis of Vasopressin
 Synthesis as a precursor hormone
(pre-pro-vasopressin) in the hypothalamus
 Cleavage and transport in granules
down the axons
 Storage in granules in the posterior pituitary
 Release into nearby capillaries upon
appropriate stimulation
Figures adapted from: Golenhofen, Basislehrbuch Physiologie, Urban & Fischer; and Morgenthaler NG et al.: Clin Chem 2006
Information: Russel IC and Glover PJ: Critical Care and Resuscitation 2002; Ranger GS: IJCP 2002; Oghlakian G and Klapholz M: Cardiology in Review 2009
5
What is the physiological
role of Vasopressin?
6
Vasopressin - physiological role
Main role:
Regulation of water balance
- Increased plasma osmolality
- Decreased arterial circulating volume
AVP:
Synthesis in the
Hypothalamus
AVP:
acts via V2-receptors in
the kidney
-> water retention
Figure adapted from: Knoers NV N Engl J Med. 2005 May 5;352(18):1847-50
7
Vasopressin (AVP) effects
receptor location
effect
V2
kidney
water retention
V1a
vascular smooth
muscle cells
strong vasoconstriction
V1b
endocrine cells
(e.g. pituitary)
regulation of ACTH
secretion during stress
Effects of AVP dependent on concentration :
 maximal antidiuretic effect: below 15 pg/ml
 vasoconstrictor effect at higher concentrations
 very little effect on blood pressure at physiological levels!
Singh Ranger G, Int J Clin Pract 2002; 56(10):777-782
8
Vasopressin in stress situation
Myocardial infarction
STRESS
AVP
ACTH
Cortisol
9
Why not simply measure
Vasopressin?
10
Quantification of Vasopressin is difficult
Vasopressin
11
Quantification of Vasopressin is difficult
Vasopressin
Receptor
Vasopressin
12
Quantification of Vasopressin is difficult
Vasopressin
Receptor
Vasopressin
Vasopressin
Platelets
13
Quantification of Vasopressin is difficult
Protease
Vasopressin
Vasopressin
Receptor
Vasopressin
Vasopressin
Platelets
14
Quantification of Vasopressin is difficult
Protease
Vasopressin
Vasopressin
Receptor
Vasopressin
Vasopressin
Platelets
Further problem: very unstable ex vivo (even frozen)
15
Quantification of Vasopressin is difficult
Protease
Vasopressin
Vasopressin
Receptor
Vasopressin
Vasopressin
Platelets
Further problem: very unstable ex vivo (even frozen)
16
Only specialized labs measure AVP (time to results several days)
Not a single FDA approved AVP assay on the market
LIMITED CLINICAL USE
Prohormone processing and assay
Signal
Vasopressin
Neurophysin II
CT-proAVP
Morgenthaler NG et al., Clin Chem. 2006
17
Prohormone processing and assay
Signal
Vasopressin
Neurophysin II
CT-proAVP
Neurophysin II
CT-proAVP
Signal Peptidase
Vasopressin
Morgenthaler NG et al., Clin Chem. 2006
18
Prohormone processing and assay
Signal
Vasopressin
Neurophysin II
CT-proAVP
Neurophysin II
CT-proAVP
Signal Peptidase
Vasopressin
Prohormone
Convertase
Vasopressin
Neurophysin II
CT-proAVP
Morgenthaler NG et al., Clin Chem. 2006
19
Prohormone processing and assay
Signal
Vasopressin
Neurophysin II
CT-proAVP
Neurophysin II
CT-proAVP
Signal Peptidase
Vasopressin
Prohormone
Convertase
Vasopressin
Neurophysin II
CT-proAVP
Morgenthaler NG et al., Clin Chem. 2006
20
Prohormone processing and assay
Signal
Vasopressin
Neurophysin II
CT-proAVP
Neurophysin II
CT-proAVP
Signal Peptidase
Vasopressin
Prohormone
Convertase
Vasopressin
Neurophysin II
CT-proAVP
CT-proAVP very
stable ex vivo
Morgenthaler NG et al., Clin Chem. 2006
21
Prohormone processing and assay
Signal
Vasopressin
Neurophysin II
CT-proAVP
Neurophysin II
CT-proAVP
Signal Peptidase
Vasopressin
Prohormone
Convertase
Vasopressin
Neurophysin II
CT-proAVP
CT-proAVP very
stable ex vivo
Morgenthaler NG et al., Clin Chem. 2006
22
Is CT-proAVP produced together
with Vasopressin?
Do both analytes show the same
kinetics in vivo?
23
LIA Assay
Correlation of Vasopressin and CT-proAVP
Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9.
24
r = 0.78
Jochberger S et al., Schock 2009 31: 132-138
Validation in: Jochberger S et al., Intensive Care Med 2009 35:489-497
Copeptin (pmol/L)
CT-proAVP – like Vasopressin – is rapidly degraded in vivo
Copeptin male 45 y, BMI 23
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
700
water
food
800
900
t1/2: few minutes
25
Copeptin female 23 y, BMI 19
1000 1100 1200 1300 1400 1500 1600 1700 1800
day time (hours)
Morgenthaler et al. Clin Chem 2006
Copeptin (pmol/L)
CT-proAVP – like Vasopressin – is rapidly degraded in vivo
Copeptin male 45 y, BMI 23
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
700
water
food
800
900
t1/2: few minutes
26
Copeptin female 23 y, BMI 19
1000 1100 1200 1300 1400 1500 1600 1700 1800
day time (hours)
Morgenthaler et al. Clin Chem 2006
CT-proAVP – like Vasopressin – is rapidly degraded in vivo
Copeptin (pmol/L)
97.5 % percentile normals:
Copeptin male 45 y, BMI 23
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
700
water
food
800
900
t1/2: few minutes
27
Copeptin female 23 y, BMI 19
1000 1100 1200 1300 1400 1500 1600 1700 1800
day time (hours)
Morgenthaler et al. Clin Chem 2006
CT-proAVP – Stimulation via osmoreceptors
CT-proAVP behaves like AVP
Control
n=8
28
Hypotonic saline
infusion
Hypertonic saline
infusion / thirsting
Szinnai et al. JCEM (2007)
CT-proAVP correlates better with osmolality than Vasopressin
Balanescu S. et.al. JCEM 2011 in press
29
CT-proAVP- stimulation via baroreceptors/ hemorrhagic shock, model
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
500
400
baboon 118
babbon 137
baboon 150
baboon 157
MAP
300
200
100
0
0
1
2
0
1
2
bleeding
3
4
5
6
7
8
Time (hours)
R0 R1 R2
R4
9
mm Hg
CT-proAVP (Copeptin) pmol/L
CT-proAVP behaves like AVP
10 11 12
R9
reperfusion
Morgenthaler et al. Shock 2007
30
Which CT-proAVP levels should
be expected in normals?
31
CT-proAVP is not age-related
Normal distribution
Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9
32
CT-proAVP levels dependent on gender
Significantly higher levels in males
706 healthy volunteers
Bhandari SS et al, Clinical Science (2009) 116, 257–263
33
CT-proAVP: Influence of exercise
Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9
34
CT-proAVP: Influence of exercise
97.5 % percentile normals:
Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9
35
CT-proAVP LIA in the differential
diagnosis of Diabetes insipidus
36
What is Diabetes insipidus ?
• Diabetes Insipidus (DI) is a disorder in which there is
an abnormal increase in urine output, fluid intake and
often thirst (polyuria-polydipsia-syndrome).
• Urine output is increased because it is not
concentrated normally -> the urine is not yellow but
pale, colorless or watery.
• Diabetes Insipidus is divided into three types, each of
which has a different cause and must be treated
differently.
37
Types of Diabetes insipidus
• Central Diabetes Insipidus (also known as neurogenic
DI): The most common type of DI is caused by a lack of
vasopressin.
Treatment: various drugs including a modified
vasopressin known as desmopressin or DDAVP
• Nephrogenic Diabetes insipidus (also known as renal
DI): is caused by an inability of the kidneys to respond to
the "antidiuretic effect" of normal amounts of vasopressin.
Treatment: It cannot be treated with DDAVP and,
depending on the cause, may or may not be curable
by eliminating the offending drug or disease.
38
Types of Diabetes insipidus
• Central Diabetes Insipidus (also known as neurogenic
DI): The most common type of DI is caused by a lack of
vasopressin.
Treatment: various drugs including a modified
vasopressin known as desmopressin or DDAVP
• Nephrogenic Diabetes insipidus (also known as renal
DI): is caused by an inability of the kidneys to respond to
the "antidiuretic effect" of normal amounts of vasopressin.
Treatment: It cannot be treated with DDAVP and,
depending on the cause, may or may not be curable
by eliminating the offending drug or disease.
Diagnostic Challenge:
All types of Diabetes insipidus also as partial forms existing!
39
Types of Diabetes insipidus (II)
• primary polydipsia : occurs when vasopressin is
suppressed by excessive intake of fluids.
• most common type of polyuria-polydipsia-syndrome
• most often caused by an abnormality in the part of the
brain that regulates thirst or by psychogenic illnesses
(psychogenic polydipsia)
• difficult to differentiate from central DI because it mimics
DI.
Interested in more?: http://www.diabetesinsipidus.org
Also in French and Spanish language
40
Differential Diagnosis of Diabetes insipidus
41

Clinical Challenges: Differential diagnosis of patients with
polyuria-polydipsia syndrome

State-of-the art diagnosis:
1. Stimulation of AVP release via a Water deprivation test
2. Indirect measurement of AVP release by monitoring of urine
osmolality and - volume during water deprivation
(ability to concentrate urine).
3. Additional Desmopressin administration to differentiate
nephrogenic DI from central DI.

Direct AVP measurement becomes not the diagnostic
reference standard because of its methological limitations
(instability of analyte and uncomfortable assay handling)
CT-proAVP for Differential diagnosis of Diabetes insipidus
42
central
DI
primary
Polidipsia
Nephrogenic
DI
Urine Volume/ fluid
intake
Excessive
Excessive
Excessive
Urine- Osmolality
low
low
low
CT-proAVP basal
low (< 2.6
pmol/l)
low
(~3 pmol/l)
high
(>20 pmol/l)
CT-proAVP increase
after thirsting
no
yes
small
State-of-the-art
diagnosis
ability to concentrate urine during water
deprivation , indirect measurement via urinevolume and – osmolality
ability to respond to desmopressin intake
Differential diagnosis of Diabetes insipidus
central
DI
Urine Volume/ fluid
intake
Urine- Osmolality
43
primary
Polidipsia
Nephrogenic
DI
Excessive
Excessive
Excessive
Diagnosis without water
deprivation and Desmopressin
stimulation
low
low possible!
low
CT-proAVP basal
low (< 2.6
pmol/l)
low
(~3 pmol/l)
high
(>20 pmol/l)
CT-proAVP increase
after thirsting
no
yes
small
State-of-the-art
diagnosis
ability to concentrate urine during water
deprivation , indirect measurement via urinevolume and - osmolality
Differential diagnosis of Diabetes insipidus
central
DI
Urine Volume/ fluid
intake
Nephrogenic
DI
Excessive
Excessive
Diagnosis without water
deprivation possible!
low
low
Excessive
CT-proAVP basal
low (< 2.6
pmol/l)
high
(>20 pmol/l)
CT-proAVP increase
after thirsting
Differential yes
no
diagnosis of partial
DI possible
State-of-the-art
diagnosis
ability to concentrate urine during water
deprivation , indirect measurement via urinevolume and - osmolality
Urine- Osmolality
44
primary
Polidipsia
low
(~3 pmol/l)
low
small
CT-proAVP course during water deprivation
8
7
CT-proAVP in pmol/l
6
5
4
Mean value of CT-proAVP
in primary polydipsia
3
Mean value of CT-proAVP
in central DI
2
1
0
0.00
4.00
8.00
12.00
time
45
16.00
20.00
Superiority of CT-proAVP in diagnosing Diabetes insipidus
Conclusion:
Current state-of -the
art - method WDT
gives no reliable
results in the
differential diagnosis
of polyuria-polydipsia
syndrome!
CT-proAVP is
superior to the
current method of
choice and revives
the concept of the
direct test in the
polyuria- polydipsia
syndrome.
Fenske W. et.al. Copeptin in the differential diagnosis of the polyuria- polydipsia syndrome
– revisiting the direct and indirect water deprivation tests. JCEM accepted January 2011
46
CT-proAVP: Diagnosis of central DI totalis and nephrogenic DI
in the 1st blood draw
basal CT-proAVP [pmol/l] (fasting, in the morning after 8h
dehydration)
< 2.6
>20
Sensitivity (%)
95
100
Specificity (%)
100
100
Central Diabetes
insipidus totalis
nephrogenic
Diabetes insipidus
Fenske W. et.al. Copeptin in the differential diagnosis of the polyuria- polydipsia syndrome
– revisiting the direct and indirect water deprivation tests. JCEM accepted January 2011
47
erum -Na+
Differential diagnosis of unclear cases after water deprivation
Best separation of
primary polydipsia and
partial central DI
(in contrast to current
methods including AVP
measurements)
specificity 100%
sensitivity 86%
Poster:
Fenske W: 14th Annual meeting of the
neuroendocrinology section of the DGE
October 15, 2010 (Munich)
Paper accepted at JCEM Jan. 2011
48
2nd blood draw: Stimulated CT-proAVP differentiates safe
between central DI partialis and Primary Polydipsia
Index
Δ CT-proAVP [8h-16h] x 1000 [pmol/L/mmol/L]
S-Na+ [16h]
<20
>20
Sensitivity (%)
100
86
Specificity (%)
86
100
central Diabetes
insipidus partialis
primary
polydipsia
Fenske W. et.al. Copeptin in the differential diagnosis of the polyuria- polydipsia syndrome
– revisiting the direct and indirect water deprivation tests. JCEM accepted January 2011
49
Reliable differential diagnosis of DI with the help of CT-proAVP
Suspicion of Diabetes insipidus
with Polyurie-Polydipsie-Syndrome
CT-proAVP basal
(in the morning, fasting, after 8h dehydration)
CT-proAVP
<2,6 pmol/L
Central Diabetes
insipidus totalis
CT-proAVP
>=2,6 - 20 pmol/L
CT-proAVP
>20 pmol/L
CT-proAVP stimulated and Serum-Na+
(after 16 hours dehydration)
Ratio of CT-proAVP-Delta (8 to16h)
and Serum-Na+ (16h) = CT-proAVP-Index
50
CT-proAVP-Index
<20
CT-proAVP-Index
>=20
Central Diabetes
insipidus partalis
Primary
Polydipsia
Renal Diabetes
insipidus
Advantages for the diagnostic routine
• Significantly higher diagnostic accuracy for all variations of
Diabetes insipidus and primary Polydipsia
• Considerably eased differential diagnosis of PolyuriaPolydipsia-Syndrome
• Reduced physical and psychical exposure of the patient due
to simplified WDT and redundancy of desmopressin stimulation
• Support of safe therapeutic decisions with highly sensitive
measurement values
• Overall cost reduction due to reduced complexity, less lab
consulting and no prescription of desmopressin
51
What about the performance of
the LIA assay?
52
Reminder: Why not measure AVP directly?
• AVP is very unstable in plasma even at -20 °C storage (sample
transport frozen or blood collection directly in the lab)
• AVP is largely attached to platelets
• AVP assays performed with the required accuracy are available
in only a few selected laboratories (non of them FDA cleared)
• Sample extraction necessary
• Time to result up to 72 hours
• Sample volume 1-4 ml plasma
• No reliable clinical results
53
Advantages Thermo Scientific B·R·A·H·M·S CT-proAVP LIA
• sample volume only 50µl
• for plasma and serum
• one-step procedure (time to result 3 hours)
• stable analyte (at room temperature)
• highest sensitivity
• sandwich-immunoassay
• clinical superiority shown
54
CT-proAVP LIA assay parameters
Sample type
serum, plasma
Volume
50 µl
Incubation time
2 hours
Stability at RT
minimum 8 hours
Stability at 2-8°C
14 days
Freezing and thawing
No influence up to 3 cycles
Analytical assay sensitivity
< 0,4 pmol/L
FAS (20% CV)
< 1 pmol/L
Direct measuring range
0,4 - 1250 pmol/L
Data taken from IFU (instructions for use)
55