thermo scientific b·r·a·h·m·s ct-proavp lia for use in endocrinology february 2011

Thermo Scientific B·R·A·H·M·S CT-proAVP LIA for use in endocrinology

February 2011

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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?

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?

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What is Vasopressin and

where is it produced?

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Structure of Vasopressin

O

NH2

NH2-

O

NH2

-C

Arginine-Vasopressin (AVP) synonym: Vasopressin or antidiuretic hormone (ADH)

peptide hormone

9 amino acids

Disulfide bridge between two cysteine amino acids

C-terminal amidation

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Synthesis of Vasopressin

Figures adapted from: Golenhofen, Basislehrbuch Physiologie, Urban & Fischer; and Morgenthaler NG et al.: Clin Chem 2006Information: Russel IC and Glover PJ: Critical Care and Resuscitation 2002; Ranger GS: IJCP 2002; Oghlakian G and Klapholz M: Cardiology in Review 2009

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

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What is the physiological role of

Vasopressin?

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Vasopressin - physiological role

AVP:acts via V2-receptors in the kidney

-> water retention

Main role:

Regulation of water balance

Figure adapted from: Knoers NV N Engl J Med. 2005 May 5;352(18):1847-50

- Increased plasma osmolality - Decreased arterial circulating volume

AVP:Synthesis in the Hypothalamus

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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

Vasopressin (AVP) effects

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

http://upload.wikimedia.org/wikipedia/commons/a/a9/High_voltage_warning.svg

http://upload.wikimedia.org/wikipedia/commons/a/a9/High_voltage_warning.svg

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Vasopressin in stress situation

ACTH

AVP

STRESS

Cortisol

Myocardial infarction

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Why not simply measure Vasopressin?

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Quantification of Vasopressin is difficult

Vasopressin

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Vasopressin

Vasopressin

Receptor

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Vasopressin

Platelets

Vasopressin

Vasopressin

Receptor

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Vasopressin

Platelets

Vasopressin

Protease

Vasopressin

Vasopressin

Receptor

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Vasopressin

Platelets

Vasopressin

Protease

Vasopressin

Vasopressin

Receptor

Further problem: very unstable ex vivo (even frozen)

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Vasopressin

Platelets

Vasopressin

Protease

Vasopressin

Vasopressin

Receptor

Only specialized labs measure AVP (time to results several days)Not a single FDA approved AVP assay on the market

LIMITED CLINICAL USE

Further problem: very unstable ex vivo (even frozen)

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Morgenthaler NG et al., Clin Chem. 2006

Prohormone processing and assay

Signal Vasopressin Neurophysin II CT-proAVP

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Signal Peptidase

Vasopressin Neurophysin II CT-proAVP

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Signal Peptidase


Vasopressin

ProhormoneConvertase

CT-proAVP Neurophysin II

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Signal Peptidase


Vasopressin



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Signal Peptidase


Vasopressin



CT-proAVP very stable ex vivo

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Signal Peptidase


Vasopressin



CT-proAVP very stable ex vivo

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Is CT-proAVP produced together with Vasopressin?

Do both analytes show the same kinetics in vivo?

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r = 0.78LIA

Ass

ay

Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9.Jochberger S et al., Schock 2009 31: 132-138

Validation in: Jochberger S et al., Intensive Care Med 2009 35:489-497

Correlation of Vasopressin and CT-proAVP

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700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 18000123456789

10111213141516

Copeptin male 45 y, BMI 23

Copeptin female 23 y, BMI 19water

food

day time (hours)

Co

pep

tin

(p

mo

l/L)

t1/2: few minutes

CT-proAVP – like Vasopressin – is rapidly degraded in vivo

Morgenthaler et al. Clin Chem 2006

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700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 18000123456789

10111213141516



food

day time (hours)

Co

pep

tin

(p

mo

l/L)

t1/2: few minutes



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700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 18000123456789

10111213141516



food

day time (hours)

Co

pep

tin

(p

mo

l/L)

97.5 % percentile normals:

t1/2: few minutes



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Control Hypotonic salineinfusion

Hypertonic saline infusion / thirsting

n=8

CT-proAVP – Stimulation via osmoreceptors

CT-proAVP behaves like AVP

Szinnai et al. JCEM (2007)

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Balanescu S. et.al. JCEM 2011 in press

CT-proAVP correlates better with osmolality than Vasopressin

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0 1 2 3 4 5 6 7 8 9 10 11 12

0

100

200

300

400

500

reperfusionbleeding

baboon 157

baboon 118babbon 137baboon 150

MAP

0102030405060708090100110120130140

R21 R1R020 R4 R9

Time (hours)

CT

-pro

AV

P (

Co

pep

tin

) p

mo

l/Lm

m H

g

CT-proAVP- stimulation via baroreceptors/ hemorrhagic shock, model

CT-proAVP behaves like AVP

Morgenthaler et al. Shock 2007

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Which CT-proAVP levels should be expected in normals?

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Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9

Normal distribution

CT-proAVP is not age-related

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Bhandari SS et al, Clinical Science (2009) 116, 257–263

706 healthy volunteers

Significantly higher levels in males

CT-proAVP levels dependent on gender

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CT-proAVP: Influence of exercise

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CT-proAVP: Influence of exercise

97.5 % percentile normals:

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CT-proAVP LIA in the differential diagnosis of Diabetes insipidus

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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.

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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.

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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!

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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.orgAlso in French and Spanish language

http://www.diabetesinsipidus.org/

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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)

Differential Diagnosis of Diabetes insipidus

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CT-proAVP for Differential diagnosis of Diabetes insipidus

centralDI

primary Polidipsia

NephrogenicDI

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 urine- volume and – osmolalityability to respond to desmopressin intake

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Differential diagnosis of Diabetes insipidus

centralDI

primary Polidipsia

NephrogenicDI





low(~3 pmol/l)

high(>20 pmol/l)


no yes small


Diagnosis without water deprivation and

Desmopressin stimulation possible!

ability to concentrate urine during water deprivation , indirect measurement via urine- volume and - osmolality

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Differential diagnosis of Diabetes insipidus

centralDI

primary Polidipsia

NephrogenicDI





low(~3 pmol/l)

high(>20 pmol/l)


no yes small


Diagnosis without water deprivation possible!

Differential diagnosis of partial

DI possible

ability to concentrate urine during water deprivation , indirect measurement via urine- volume and - osmolality

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CT-proAVP course during water deprivation

0.00 4.00 8.00 12.00 16.00 20.000

1

2

3

4

5

6

7

8

time

CT

-pro

AV

P in

pm

ol/l

Mean value of CT-proAVP in primary polydipsia

Mean value of CT-proAVP in central DI

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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

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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 nephrogenic

insipidus totalis Diabetes insipidus

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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 DGEOctober 15, 2010 (Munich)

Paper accepted at JCEM Jan. 2011

Differential diagnosis of unclear cases after water deprivationer

um

-N

a+

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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 primary insipidus partialis polydipsia

2nd blood draw: Stimulated CT-proAVP differentiates safe between central DI partialis and Primary Polydipsia


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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

CT-proAVP>20 pmol/L

Central Diabetes insipidus totalis

Renal Diabetes insipidus

CT-proAVP>=2,6 - 20 pmol/L

CT-proAVP-Index<20

CT-proAVP-Index>=20

Central Diabetes insipidus partalis

Primary Polydipsia

Ratio of CT-proAVP-Delta (8 to16h) and Serum-Na+ (16h) = CT-proAVP-Index

CT-proAVP stimulated and Serum-Na+ (after 16 hours dehydration)

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Advantages for the diagnostic routine

• Significantly higher diagnostic accuracy for all variations of Diabetes insipidus and primary Polydipsia

• Considerably eased differential diagnosis of Polyuria-Polydipsia-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

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What about the performance of the LIA assay?

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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

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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

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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)

thermo scientific b·r·a·h·m·s ct-proavp lia for use in endocrinology february 2011

Documents

vasopressin ctproavp

ctproavp slide

difficult vasopressin

quantification of vasopressin

correlation of vasopressin

minutes ctproavp

ctproavp levels

hypothalamus slide