PARATHYROID HORMONE –

JUST WHEN YOU THOUGHT YOU

UNDERSTOOD IT............?

Alison Almond, Chris Isles, Ken

Donaldson, Sue Robertson, Simon
Walker, Andy Ellis, Ewan Bell, Scottish
Biochemist Clinical and Research
Network
 A COMPARISON OF PTH ASSAYS
• Background to the study
• ‘The Troll’s Guide’ to PTH Biochemistry
• History of PTH assays
• Comparison of PTH assays
• Clinical implications
• The way forward…..other than to the bar!
 METABOLISM OF PTH
PTH
NH2 COOH

1 84

ACTIVE END INACTIVE END

LIVER

NH2 COOH
1 34 35 84
ELIMINATED IN LIVER RE-ENTERS CIRCULATION.
EXCRETED BY KIDNEYS
 HISTORY OF PTH ASSAYS

NH2
1
PTH
Y 84
COOH

• 1960’S- Berson described first RIA using single

radiolabelled antibody directed at the C-terminal of
PTH.
 HISTORY OF PTH ASSAYS

NH2
1
Y 84
COOH

• 1960’S- Berson described first RIA using single

radiolabelled antibody directed at the C-terminal of
PTH.
• Problems-picked up all C-terminal fragments so PTH
measurements correlated poorly with bone histology
(Andress DL et al.Journal of Clinical Endocrinology
and Metabolism 1986;63:1163-1169)
 HISTORY OF PTH ASSAYS(2)
SOLID PHASE
I125 ie.plastic bead

NH2
1
84
15
Y
PTH
34
Y COOH

• 1980’s-2-site IRMA. C-terminal antibody + solid

phase,attached to radio-labelled antibody to N-
terminal (epitopes 15-34).2nd generation or so called
‘intact PTH’.Proto-type-Nichols ‘Allegro’.
 HISTORY OF PTH ASSAYS(2)
SOLID PHASE
I125 ie.plastic bead

NH2
1
84
7 15
Y
PTH
34
Y COOH

• 1980’s-2-site IRMA. C-terminal antibody + solid

phase,attached to radio-labelled antibody to N-
terminal (epitopes 15-34).2nd generation or so called
‘intact PTH’.Proto-type-Nichols ‘Allegro’.
 “..once GFR<30 ml/min/1.73msq reached PTH had
decreasedfrom 79% to 68%, and non-(1-84)PTH had
increased from 21% to 32%.....in haemodialysed patients
...non-(1-84)PTH accounted for 50% of the iPTH.”

Brossard et al.Clinical Chemistry2000; 46:697-703

 HISTORY OF PTH ASSAYS(3)
SOLID PHASE
I125 ie.plastic bead

NH2 Y 1
84
6
PTH
15 34
Y COOH

• 1992-2-site IRMA. C-terminal antibody + solid

phase,attached to radio-labelled antibody to N-
terminal , this time to epitopes 1-6 .3rd generation, so
called ‘Bio-intact PTH’ or ‘whole PTH’.
SINGLE PATIENT STUDY -
METHOD
 SINGLE PATIENT STUDY -
METHOD
• 21 haemodialysis patients from DGRI
consented to participate.
 SINGLE PATIENT STUDY -
METHOD
• 21 haemodialysis patients from DGRI
consented to participate.
• 5mls of EDTA plasma was taken from each
patient before dialysis
 SINGLE PATIENT STUDY -
METHOD
• 21 haemodialysis patients from DGRI
consented to participate.
• 5mls of EDTA plasma was taken from each
patient before dialysis
• Specimens were centrifuged, separated and
divided into 5 aliquots of 1ml and frozen at
DGRI lab.where one sample from each patient
was retained.
 SINGLE PATIENT STUDY -
METHOD
• 21 haemodialysis patients from DGRI consented
to participate.
• 5mls of EDTA plasma was taken from each
patient before dialysis
• Specimens were centrifuged, separated and
divided into 5 alliquots of 1ml and frozen at DGRI
lab.where one sample from each patient was
retained.
• 4 remaining sets were sent to UK NEQAS , RIE
who retained one and distributed other 3 on dry
ice to GRI,Borders and Kirkcaldy.
 SINGLE PATIENT STUDY -
METHOD
• 21 haemodialysis patients from DGRI consented to
participate.
• 5mls of EDTA plasma was taken from each patient
before dialysis
• Specimens were centrifuged, separated and divided
into 5 alliquots of 1ml and frozen at DGRI lab.where
one sample from each patient was retained.
• 4 remaining sets were sent to UK NEQAS , RIE who
retained one and distributed other 3 on dry ice to
GRI,Borders and Kirkcaldy.
• All specimens were analysed on the same day and
results returned to UK NEQAS for analysis.
 ONE PATIENT – 5 LABORATORIES
DiaSorin Liaison (GRI)
53.8

Beckman Access (BORDERS)

126.4

Roche Elecsys (DGRI)

139.9

Siemens Centaur (KIRKCALDY)

163.57

Siemens Immulite (RIE)

183.47

0 20 40 60 80 100 120 140 160 180 200

PARATHYROID HORMONE LEVEL (pmol/L

 ONE PATIENT – 5 LABORATORIES
DiaSorin Liaison (GRI) 3.4 FOLD DIFFERENCE
53.8

Beckman Access (BORDERS)

126.4

Roche Elecsys (DGRI)

139.9

Siemens Centaur (KIRKCALDY)

163.57

Siemens Immulite (RIE)

183.47

0 20 40 60 80 100 120 140 160 180 200

PARATHYROID HORMONE LEVEL (pmol/L

IN EACH OF THE 17 OTHER PATIENTS.....

1.4 -4.2 FOLD

DIFFERENCE
HIGHEST AND LOWEST PTH FOR EACH PATIENT
200

175 COMPARISON OF
HIGHEST vs LOWEST
150 PTH FOR EACH
PATIENT (n=18)
PTH 125
(pm/L)
100

75

50

25

0
LOWEST HIGHEST
HIGHEST AND LOWEST PTH FOR EACH PATIENT
200

175 COMPARISON OF
HIGHEST vs LOWEST
150 PTH FOR EACH
PATIENT (n=18)
PTH 125
(pm/L)
100
80 75
8/18 CROSSED
THRESHOLD FOR
50
SURGERY/MIMPARA
25

0
LOWEST HIGHEST
 HIGHEST AND LOWEST PTH FOR EACH PATIENT
200

175 COMPARISON OF
HIGHEST vs LOWEST
150 PTH FOR EACH
PATIENT (n=18)
PTH 125
(pm/L)
100
80 75
8/18 CROSSED
THRESHOLD FOR
50
SURGERY/MIMPARA
30
RA 25
target 14
0
LOWEST HIGHEST
 Assay variations meant,in total,15 out of 18
patients (83%)would have required changes to
their treatment....
 Assay variations meant,in total,15 out of 18
patients (83%)would have required changes to
their treatment....
*2 at risk of ABD by lowest reading would have
had treatment increased by highest reading.
 Assay variations meant,in total,15 out of 18
patients (83%)would have required changes to
their treatment....
*2 at risk of ABD by lowest reading would have
had treatment increased by highest reading.

*5,on target by lowest reading would have had

treatment intensified by highest reading
 Assay variations meant,in total,15 out of 18
patients (83%)would have required changes to
their treatment....
*2 at risk of ABD by lowest reading would have
had treatment increased by highest reading.

*5,on target by lowest reading would have had

treatment intensified by highest reading

*8 would have had medical therapy by lowest

reading and surgery by highest.
“....up to 11 of 34 patients were classified in different
(K/DOQI) categories with some assays....PTH variability
caused by the nature of the assay...is large enough to
potentially influence clinical decision making.”
JOLY ET AL. Am J Kidney Disease Apr 2008
 PTH ASSAYS USED IN SCOTLAND
ASSAY

SIEMENS IMMULITE (RIE)

SIEMENS CENTAUR (KIRKCALDY)
ROCHE ELECSYS (DGRI)
DIASORIN LIAISON (GRI)
BECKMANS ACCESS (BORDERS)
 PTH ASSAYS USED IN SCOTLAND
ASSAY NORMAL RANGE

SIEMENS IMMULITE (RIE) 1.2 -7.6 pmol/L

SIEMENS CENTAUR (KIRKCALDY) 1.2 - 7.6 pmol/L
ROCHE ELECSYS (DGRI) 1.6 - 6.9 pmol/L
DIASORIN LIAISON (GRI) 1.0 – 6.5 pmol/L
BECKMANS ACCESS (BORDERS) 1.2 – 7.6 pmol/L
On average the Diasorin Liason assay
gave readings 2.38 times lower than the
Siemens Immulite assay ,yet clinicians
using the 2 different methods are aiming
for almost exactly the same target range
(13-26 pmol/L vs 15-30 pmol/L) .
 RENAL ASSOCIATION –PTH
“The target range should increase from the normal range
with CKD stages 1-3,to between the top of the normal
range and twice the normal range for stage 4 CKD, and
to between 2 and 4 times the upper limit of normal in
CKD stage 5,not on dialysis. These targets should apply
to transplant patients also.”
(Good Practice)

“In dialysis patients the target range for PTH should be

between 2 and 4 times the upper limit of normal for the
PTH assay used.”
(Good Practice)
 WHERE ARE WE NOW?
“…measuring PTH and ‘an unknown amount of
other things which have an undetermined
effect’,…..using machines that measure
different amounts of the different things,in a
way that we can’t quantify,yet use the same
normal range…..aiming to achieve a target
based on work done with an old assay that is
no longer used!”
SO,WHERE FROM HERE?
 SO,WHERE FROM HERE?
• Confirm study findings with a larger
population.
 SO,WHERE FROM HERE?
• Confirm study findings with a larger
population.
• Calibrate machines with plasma from CRF
patients
 SO,WHERE FROM HERE?
• Confirm study findings with a larger
population.
• Calibrate machines with plasma from CRF
patients
• Introduce correction factors for each assay.
 SO,WHERE FROM HERE?
• Confirm study findings with a larger
population.
• Calibrate machines with plasma from CRF
patients
• Introduce correction factors for each assay.
• Await automated 3rd generation PTH assay.
‘ So the answer
isn’t 42 then?....’
“.......and DON’T even get me STARTED on
Calcium!!!!!!”
IN SUMMARY -

 DIASORIN LIAISON CONSISTANTLY GAVE THE LOWEST PTH

MEASUREMENT.
 METABOLISM OF PTH
PTH
NH2 COOH

1 84

ACTIVE END INACTIVE END

 DIASORIN LIAISON CONSISTANTLY GAVE THE LOWEST PTH
MEASUREMENT.

HIGHEST READING WAS CONSISTANTLY FROM ONE OF THE

SIEMENS ANALYSERS.
HIGHEST AND LOWEST PTH FOR EACH PATIENT
200

175 COMPARISON OF
HIGHEST vs LOWEST
150 PTH FOR EACH
PATIENT (n=18)
PTH 125
(pm/L)
100
80 75
8/18 CROSSED
THRESHOLD FOR
50
SURGERY/MIMPARA
25
15 2/18 CROSSED
0 LOWER
LOWEST HIGHEST TREATMENT
THRESHOLD
 DIASORIN LIAISON CONSISTANTLY GAVE THE LOWEST PTH
MEASUREMENT.

HIGHEST READING WAS CONSISTANTLY FROM ONE OF THE

SIEMENS ANALYSERS.

RANGE OF ASSAY VARIATION WAS FROM 1.4 FOLD TO 4.2

FOLD.
 METABOLISM OF PTH
PTH
NH2 COOH

1 84

ACTIVE END INACTIVE END

NH2 COOH
1 34 35 84
 DIASORIN LIAISON CONSISTANTLY GAVE THE LOWEST PTH
MEASUREMENT.

HIGHEST READING WAS CONSISTANTLY FROM ONE OF THE

SIEMENS ANALYSERS.

RANGE OF ASSAY VARIATION WAS FROM 1.4 FOLD TO 4.2

FOLD.

HIGHER PTH LEVELS WERE NOT ASSOCIATED WITH THE

LARGER VARIATIONS.
ALL 17 OTHER PATIENTS SHOWED

1.4 -4.2 FOLD DIFFERENCE

BETWEEN THE LOWEST PTH

MEASUREMENT AND THE HIGHEST PTH
MEASUREMENT
 METABOLISM OF PTH
PTH

NH2

1 28 34
BIOLOGICALLY ACTIVE
 METABOLISM OF PTH
PTH
NH2 COOH

1 84

ACTIVE END INACTIVE END

NH2 COOH
1 34 35 84

NH2 COOH
1 6 7 15 34
ACTIONS OF PARATHYROID HORMONE

.....IN HEALTH .......IN CHRONIC RENAL FAILURE

• Increase calcium
reabsorption from gut
• Increase tubular
reabsorption from kidney
• Increase 1-OH of inactive
D3
• Calcium resorption from
bones
• Enhance PO4 excretion
ACTIONS OF PARATHYROID HORMONE

.....IN HEALTH .......IN CHRONIC RENAL FAILURE

• Increase calcium
reabsorption from gut
• Increase tubular • X
reabsorption from kidney
• Increase 1-OH of inactive • X
D3
• Calcium resorption from
bones • X
• Enhance PO4 excretion
WHAT IS PTH ?
 SYNTHESIS OF PTH

Pre-pro-PTH
N-terminal C-terminal

31 aa
Pro-PTH
N-terminal C-terminal

6 aa

C-terminal
PTH N-terminal
1 7 84
Biologically active end
HIGHEST AND LOWEST PTH FOR EACH PATIENT
200

175
8/18 ie 44%
150 crossed
threshold for
PTH (pm/ 125 surgery
L)
100
80
75

50

25

LOWEST HIGHEST
 METABOLISM OF PTH
PTH
NH2 COOH

1 84

ACTIVE END INACTIVE END

LIVER

COOH
35 84
RE-ENTERS CIRCULATION.
EXCRETED BY KIDNEYS
 METABOLISM OF PTH
PTH
NH2 COOH

1 84

ACTIVE END INACTIVE END

LIVER

NH2 COOH
1 34 35 84
ELIMINATED IN LIVER RE-ENTERS CIRCULATION.
EXCRETED BY KIDNEYS