Andy Durham BSc.BVSc.CertEP.DEIM.DipECEIM.

MRCVS

8"

""cor.sol"g/dL!

7"

PPID

6"
5"
4"
3"

!!!!normal!response!
19h!:!<!1g/dL!
!!!!!!!!!!!!!!<!27!nmol/L!

normal

2"

27"nmol/L,"1"g/dL"

1"
0"
0"

4"

8"

12"

.me"hours"
40 g/kg DEX

16"

20"

PPID (n)

Normal (n)

Cut-off

Sensitivity

Specificity

Accuracy

42

18

27 nmol/L

100%

100%

100%

17

25

27 nmol/L

65%

76%

71%

Frank et al 2006

27 nmol/L

66%

100%

89%

Beech et al 2007

Sensitivity = % PPID cases that test positive

Specificity = % normal horses that test negative
Accuracy = % correct test results

Dybdal et al 1994

1. Collect)into)EDTA)tube)
2. Chill)sample)within)3)hours)of)collec<on))
3. Centrifuge)as)soon)as)possible)
4. Keep)chilled)during)shipping)to)laboratory)
5. Observe)seasonal)reference)intervals!

200
150
100
50

plasma ACTH pg/mL

250

PPID (n) Normal (n) Cut-off

Sensitivity Specificity Accuracy Ref

Van der Kolk et al 1995

24

55 pg/mL

100%

100%

100%

10

35 pg/mL

67%

100%

88%

Beech et al 2007

25

23

35 pg/mL

71%

96%

83%

Beech et al 2011

84%

97%

90%

Sensitivity = % PPID cases that test positive

Specificity = % normal horses that test negative
Accuracy = % correct test results

PPID!is!not!an!all<or<nothing!disease!

Histopathologic!grading!

Grade

Description

Interpretation

normal

normal

mild hypertrophy/hyperplasia

normal age effects

marked hypertrophy/hyperplasia

mild PPID

+microadenoma

moderate PPID

+macroadenoma

marked PPID

Miller!et)al)2008!

120!horses,!mean!age!21!y!

48!normal,!72!PPID!

Grade

Clinical signs present

Interpretation

normal

normal age effects

mild PPID

moderate PPID

marked PPID

Dianne!McFarlane,!Oklahoma,!ACVIM!forum!2012!

120!horses,!mean!age!21!y!

48!normal,!72!PPID!

Grade

Clinical signs present

Interpretation

normal

39

normal age effects

mild PPID

moderate PPID

marked PPID

Dianne!McFarlane,!Oklahoma,!ACVIM!forum!2012!

120!horses,!mean!age!21!y!

48!normal,!72!PPID!

Overall!15/72!(21%)!PPID!cases!had!suggesSve!clinical!signs!

Grade

Clinical signs present

Interpretation

normal

39

normal age effects

(2/34) 6%

34

mild PPID

(3/27) 11%

27

moderate PPID

(10/11) 91%

11

marked PPID

Dianne!McFarlane,!Oklahoma,!ACVIM!forum!2012!

ACTH!cuto!58"pg/mL!(=!29!pg/mL!on!Liphook!analyser!)!

CorSsol!cuto!27"nmol/L"

Grade ACTH

pg/mL

Post-dex Cortisol

nmol/L

Interpretation

normal

normal age effects

mild PPID

moderate PPID

marked PPID

Dianne!McFarlane,!Oklahoma,!ACVIM!forum!2012!

ACTH!cuto!58"pg/mL!(=!29!pg/mL!on!Liphook!analyser!)!

CorSsol!cuto!27"nmol/L"

Grade ACTH

pg/mL

Post-dex Cortisol

nmol/L

Interpretation

52

normal

36

normal age effects

mild PPID

moderate PPID

marked PPID

Dianne!McFarlane,!Oklahoma,!ACVIM!forum!2012!

ACTH!cuto!58"pg/mL!(=!29!pg/mL!on!Liphook!analyser!)!

CorSsol!cuto!27"nmol/L"

all!grade!5!PPID!cases!tested!posiSve;!grades!3,!4!oYen!negaSve!

Grade ACTH

pg/mL

Post-dex Cortisol

nmol/L

Interpretation

52

normal

36

normal age effects

41

18

mild PPID

50

12

moderate PPID

268

76

marked PPID

Dianne!McFarlane,!Oklahoma,!ACVIM!forum!2012!

 We"are"probably"missing"early"PPID"cases"

Grade

Description

Interpretation

normal

normal

mild hypertrophy/hyperplasia

normal age effects

marked hypertrophy/hyperplasia

mild PPID

+microadenoma

moderate PPID

+macroadenoma

marked PPID

1750
1500

mean normal

ACTH pg/mL

1250
1000
750
500
250
0

1 mg TRH iv

10

20
30
40
Time post-TRH (mins)

50

60

Data!from:!!McFarlane!et!al!2005,!!
!Beech!et!al!2007,2011!

1750

1250

mean normal

ACTH pg/mL

1500

mean PPID

1000
750
500
100!pg/mL!

250
0

1 mg TRH iv

10

35!pg/mL!

20
30
40
Time post-TRH (mins)

50

60

Data!from:!!McFarlane!et!al!2005,!!
!Beech!et!al!2007,2011!

PPID (n)

Normal
(n)

Time
post-TRH

Cut-off

10

10 mins

100 pg/mL

100%

10

30 mins

35 pg/mL

25

23

30 mins

35 pg/mL

Accuracy

Ref

100%

100%

Beech et al
2007

100%

89%

94%

Beech et al
2007

95%

91%

94%

Beech et al
2011

Sensitivity Specificity

Sensitivity = % PPID cases that test positive

Specificity = % normal horses that test negative
Accuracy = % correct test results

52!TRH!sSmulaSon!tests!(0,!10!mins)!

15th!March!2012!<!30th!July!2012!

1140 pg/mL

ACTH (post-TRH) (pg/mL)

700
600
500
400
300
200
100
0

20

40

60

80

100

120

140

160

Basal ACTH (pre-TRH) (pg/mL)

180

200

220

52!TRH!sSmulaSon!tests!(0,!10!mins)!

15th!March!2012!<!30th!July!2012!

1140 pg/mL

ACTH (post-TRH) (pg/mL)

700
600
500
400
300
200
100
0

20

40

60

80

100

120

140

160

Basal ACTH (pre-TRH) (pg/mL)

180

200

220

Dose!of!pergolide?!
!
!
!
!
!
!

Orth!et)al)1982 !
Peters!1995
!!
Watson!et)al)1998
Donaldson!et)al)2002!
Sgorbini!et)al)2004
Schoc!et)al)2001 !

!!10.0! !
!!0.85! !
!1.8<2.8
!1.7<5.5!
!6.0<8.0!
!!!!2.0! !

!g/kg!sid!!
!g/kg!bid!
!g/kg!sid!!
!g/kg!sid!!
!g/kg!sid!!!
!g/kg!sid!!

Improvement!in!test!results!aYer!pergolide?!
! !
!
!
!
!ODST!
!ACTH!
!
!
!
!
!
!

Peters!1995!!
!
Watson!et)al)1998 !
Donaldson!et)al)2002!
Perkins!et)al)2002! !
Sgorbini!et)al)2004!
Schoc!et)al)2001 !

!!!!7/9!
!!!!6/6!
!
!
!
!
!
!
!!!!7/20!

!!14/20!
!!!!3/5!
!!!!2/2!

Submissions!to!Liphook!laboratory!2010<2011!

ACTH!measured!before!and!aYer!pergolide!

Good"response"
return"to"reference"range"(29"or"47"pg/mL)"

Par.al"response"
<50%"baseline"but">RR""

Poor"response"
>50%"baseline"

 ACTH decreased in 91.8% cases

Wilcoxon P<0.0001

36%

648 cases
Median interval

46 days

Median ACTH pre tx

113 pg/mL

Median ACTH post tx

41 pg/mL

Mean % reduction

-62%

Good = reference range

Partial = <50% baseline
Poor
= >50% baseline

4-12w
25%

38%

37%

36%

Good = reference range

Partial = <50% baseline
Poor
= >50% baseline

> 6m
4-12w
25%

31%
42%

37%

27%
38%
4-12 weeks

> 6 months

Mean ACTH post-tx

45 pg/mL

63 pg/mL

ACTH % reduction

-62%

-33%

75% of cases have an initial substantial

response to pergolide (good or partial)
after 6 months this falls to <60%
disease progression?
tachyphylaxis?
selection bias (poorer responders more likely to be retested?)

retest every 3-6 months?

 To!determine!the!most!appropriate!Sme!to!retest!basal!plasma!
ACTH!concentraSon!following!pergolide!therapy!
If!too!early!!waste!of!Sme!and!money!
If!too!late!!risks!further!laminiSs!acacks!if!PPID!is!not!controlled!

 39!horses!and!ponies!!
samples!collected!12!March!<!9!May!2011!
basal!plasma!ACTH!>!50!pg/mL!(reference)interval)<)29)pg/mL))
treated!with!pergolide!!
monitoring!blood!samples!collected!as)frequently)as)possible!
followed!up!for!up!to!3!months!

Deni.on"of"responses:"(ACTH)reference)interval)<)29)pg/mL)"
Good!response:

!plasma!ACTH!<35!pg/mL!

ParSal!response:

!plasma!ACTH!>35!pg/mL,!and!

!<50%!iniSal!plasma!ACTH!

!
Poor!response:!
!

!plasma!ACTH!>35!pg/mL,!and!
!>50%!iniSal!plasma!ACTH!

.me"

Cases"responding"
average!

range)

1 week

63%

46-78%

2 weeks

80%

73-88%

4 weeks

91%

85-97%

the!majority!of!cases!that!respond!to!pergolide!will!do!so!
within!the!rst!week!of!treatment!
only!1/33!cases!(3%)!was!known!to!have!denitely!not!
responded!by!4!weeks!
recommend!that!ACTH!is!rechecked!4!weeks!aYer!starSng!
pergolide!treatment!and!dosage!adjusted!if!response!is!
disappoinSng!

 23!PPID!cases!!
o
o
o
o

started!pergolide!Nov!08!!Jan!09!
iniSal!dose!2!g/kg!
3!month!check!<!dose!increased!to!4!g/kg!if!no!response!
6!month!check!

8!cases!were!non<responders!at!3!and!6!months!but!carried!on!
treatment!at!4!g/kg!
o 2!years!later!<!5!had!responded!
o 3!years!later!<!6!had!responded!
Hal!Schoc,!Michigan,!ACVIM!forum!2012!

Diagnosis!
ACTH!generally!preferred!to!ODST!for!rouSne!diagnosis!
we!are!almost!certainly!missing!early!cases!
TRH!sSmulaSon!test!(of!ACTH)!!
o might!detect!cases!earlier?!
o indicated!with!borderline!results!(20<40!pg/mL)?!
o we!dont!yet!know!seasonal!eects!on!TRH!test?!

addiSonal!PPID!tests!need!to!be!explored!

Monitoring!
recheck!monthly!and!adjust!dosage!as!required!
maximum!dose!=!10!g/kg!(or!budget)!
non<responders!at!maximum!dose:!
o carry!on!with!aordable!dosage!as!might!eventually!respond!
o try!adding!cyproheptadine!0.25!mg/kg!q!12hrs!

when!stable!and!controlled,!try!to!decrease!dose!and!
retest?!

How!to!be!a!becer!PPID!detecSve!
Dr Catherine McGowan

QuesSons!
Which!diagnosSc!test!for!rst!choice?!
Is!<MSH!becer!than!ACTH?!
Eect!of!season?!
How!prevalent!is!PPID?!
What!are!the!rst!&!most!common!signs?!
Do!I!test!early!or!wait!for!laminiSs?!!

IntroducSon!
Diagnosis!of!PPID!can!be!achieved!by!dynamic!or!
basal!tests!
Basal!tests!increasingly!popular!
Simplicity!and!availability!to!eld!pracSSoners!
Good!sensiSvity!and!specicity?!
Small!selected!groups!not!well!evaluated!

Seasonal!variaSons!have!caused!concern!
Recently!seasonally!adjusted!reference!ranges!
reported!(Copas!and!Durham!2011)!

Clinical!gold!standard!for!this!study!
Owner<reported!hirsuSsm!+!3!or!more!signs!
HirsuSsm!=!owner!detected!delayed!shedding!
and/or!failure!to!shed!and/or!a!long!haircoat!

Clinical!gold!standard!for!this!study!
+3!veterinary!clinical!examinaSon!signs!and/or!
owner!reported!historical!signs!
owner!reported!Cushings!
muscle!wasSng!(pot!belly)!
laminiSs!or!hoof!changes!
bulging!supraorbital!fat!
depression/lethargy!

Aims!
to!evaluate!basal!plasma!<MSH!and!ACTH!for!
the!diagnosis!of!PPID!in!a!populaSon!of!horses!
aged!!15!years!SE!QLD,!Australia!including:!
the!correlaSon!between!<MSH!and!ACTH!
the!relaSonship!of!<MSH!and!ACTH!to!clinical!
signs!of!PPID!
Development!of!seasonally!adjusted!cut<o!values!
!

Methods:!!
Study!1!populaSon!
Owner"based"survey"
ParScipants!recruited!from!South!East!QLD!
Via!mail!outs!in!2003!and!2005!

Target!group!was!the!EFA!(now!EA)!
denable!owner!numbers!
best!case!scenario!

Completed!(useable)!quesSonnaires!from!536!
owners!for!974!horses!!15!years!of!age!

Methods:!!
Study!2!populaSon!
Veterinarian!clinical!examinaSon!and!
diagnosSc!tesSng!
sub<sample!of!347!horses!from!survey!
populaSon!was!selected!
restricted!to!EFA!members!who!reside!in!
dened!geographical!regions!of!QLD!

Materials!and!Methods!
Endocrinology!
plasma!ACTH!(Immulite!1000)!
plasma!<MSH!(RIA,!Euro!diagnositca)!

Materials!and!Methods!
Youden!index!values!used!to!obtain!seasonally!
adjusted!cut<o!values!for!<MSH!and!ACTH!

Percent

Adjusted!to!maximise!both!sensiSvity!and!
specicity!based!on!esSmated!populaSon!
History of hirsutism and 3 or more signs
prevalence!(13%)!
100
90
80
70
60
50
40
30
20
10
0

82.5
80.0
Sensitivity (%)
Specificity (%)

3.3911
2

Log n ACTH
Season excluding Autumn

Results!
Veterinary!clinical!examinaSon!data!for!340!horses!
325!horses!with!complete!dataset!including!ACTH!!
Thoroughbred
6%

Pony

4% 2%

7%

34%

Quarterhorse
Arabian

12%

Warmblood
17%

18%

Australian>stock
horse
Crossbred
Standardbred

Age!&!breed!distribuSon!
29
Age in years

27
25
23
21
19
17
15

4
2

Pearsons r = 0.79
P<0.001
No differences between horses and ponies

Loge a-MSH

Results!

4
Loge ACTH

4
3

Pearsons r = 0.69
P<0.001

Loge ACTH

Results!

10

History of Hirsutism and Total number of Signs

15

Results!
Normal!

7!

Loge"UMSH"

6!

HirsuSsm!and!3!or!more!
signs!

5!
4!
3!
2!
1!
0!
Spring!

Summer!

Autumn!

Season"

Winter!

Error!bars!represent!!
95%!Condence!!
Intervals!(CI)!
!

Autumn!=!March,!April,!May!
Each!dierent!from!the!other!months!p!<!0.05!!

Results!
Normal!

7!
6!

HirsuSsm!and!3!or!
more!signs!

Loge"ACTH"

5!
4!
3!
2!
1!
0!
Spring!

Summer! Autumn!

Season"

Winter!

Error!bars!represent!!
95%!Condence!!
Intervals!(CI)!
!

Autumn!=!March,!April,!May!
Each!dierent!from!the!other!months!p!<!0.05!!

Plasma!<MSH!cut!o!values!
Variable

alpha MSH

alpha MSH

Season

Spring, Summer, Winter Autumn

Sample size
Positive group
Negative group :
Area under the ROC curve
(95% CI)
Significance level P (Area
=0.5)
Criterion (cut-off) at max
Youden Index
Sensitivity (95% CI)

249
22
227
0.79
(0.74- 0.84)
<0.0001

69
5
64
0.98
(0.92 - 1.00)
<0.0001

52.0 pmol/L

165.4 pmol/L

59.09 (36.4 - 79.3)

100 (47.8 - 100.0)

Specificity (95% CI)

92.95 (88.8 - 95.9)

96.87 (89.2 - 99.6)

Plasma!ACTH!cut!o!values!
Variable

ACTH

ACTH

Season

Spring, Summer, Winter Autumn

Sample size
Positive group
Negative group :
Area under the ROC curve
(95% CI)
Significance level P (Area =0.5)
Criterion (cut-off) at max
Youden Index
Sensitivity (95% CI)

237
20
217
0.85
(0.80 - 0.89)
<0.0001
29.7 pg/ml

67
4
63
0.98
(0.91 - 1.00)
<0.0001
>77.4 pg/ml

80 (56.3 - 94.3)

100 (39.8 - 100.0)

Specificity (95% CI)

82.49 (76.8 - 87.3)

95.24 (86.7 - 99.0)

Discussion!
Good!correlaSon!between!<MSH!and!ACTH!
!

Increased!values!with!increased!numbers!of!
signs!supports!that!PPID!is!a!progressive!
syndrome!associated!with!aging!

Discussion!and!Conclusions!
Seasonal!eect!despite!laStude!(<27.3<24.5)!

Peak!in!Autumn!only!not!late!summer!as!for!greater!
laStudes!

Non!Autumn!cut<o!value!for!ACTH!idenScal!to!
previous!research!(Copas!and!Durham!2011)!
But!higher!Autumn!cut!o!

For!both!tests!sensiSvity!greatly!increased!in!the!
Autumn!(100%)!
Natural!sSmulaSon!test?!

Discussion!and!Conclusions!
<MSH!had!lower!sensiSvity!for!diagnosis!of!
PPID!(non<Autumn)!

59%!versus!80%!ACTH!
also!slightly!lower!negaSve!predicSve!value!versus!
ACTH!
Although!posiSve!predicSve!value!higher!than!
ACTH!(becer!specicity)!
<MSH!also!more!pronounced!seasonal!eect!

Conclusions!
<MSH!and!ACTH!are!highly!correlated!with!each!
other!and!clinical!and!historical!indicators!of!PPID!
ACTH!has!becer!sensiSvity!and!negaSve!
predicSve!values!
More!likely!to!detect!disease!and!negaSves!are!truly!
negaSve!

Autumn!season!appropriate!for!region!with!
appropriately!adjusted!references!ranges!may!
represent!a!natural!sSmulaSon!test!

QuesSons!
Which!diagnosSc!test!for!rst!choice?!
Is!<MSH!becer!than!ACTH?!
Eect!of!season?!
How!prevalent!is!PPID?!
What!are!the!rst!signs?!
Do!I!test!early!or!wait!for!laminiSs?!!

Results:!Study!1!!
Owner<reported!signs!!
Prevalence!of!hirsuSsm!16.7%!(163/974)!
Long!hair!coat/delayed!shedding/failure!to!shed!

Prevalence!of!laminiSs!4.8%!(47/974)!
Only!1.6%!(16/974)!owners!reported!Cushings!
syndrome!(or!synonym)!as!a!known!condiSon!
in!their!horse!

Results:!Study!2!
Prevalence!of!owner<reported!hirsuSsm!
14.2%!(48/340)!
Prevalence!of!owner!reported!laminiSs!6.2%!
(21/340)!
Similar!to!study!1!

Prevalence!
Prevalence!of!PPID!from!seasonally!adjusted!
ACTH!values!21.2%"(69/325)!
95%!CI!(16.9<26.1)!

Elevated!<MSH!(seasonally!adjusted)!11.7%!
(38/325)!
Elevated!either!22.2%!(72/325)!

Signalment!
Univariable!analysis:!
Only!age!(OR!1.2;!95%CI!1.13<1.28)!
Not!gender,!breed!or!horse!versus!pony!
Owner<reported!hirsuSsm!more!likely!in!ponies!
(McGowan!et!al.!2010)!

in!a!mulSvariable!model!of!age,!breed!and!sex!with!every!
clinical!sign!
hirsuSsm!more!obvious!for!ponies?!

Historical!signs!
Univariable!analysis:!
History!of!hirsuSsm!
!OR!10.2!

History!of!!laminiSs!
OR!4.7!

Clinical!signs!
Univariable!analysis:!
HirsuSsm!OR!6.2!
Bulging!supraorbital!fat!OR!3.0!
Wasted!topline!OR!2.4!
Pot!belly!OR!2.3!
LaminiSc!rings!OR!4.0!

Results:!Study!2!
Univariable!analysis:!haematology!and!
biochemistry!
No!dierences!detected!

!!
PCV!

P P I D!
Non<PPID!
Median! IQ!range!
median! IQ!range!
Ref""range!
0.38!
0.34!<!0.42!
0.39! 0.36!<!0.43! 0.35!<!0.43!

Fibrinogen!

3.0!

3.0!!4.0!

3.0!

2.0!!4.0!

2.0!<!4.0!

GGT!

14!

12!<!19!

14!

11!<!16!

7!<!41!

T o t a l!
Protein!

71!

67!<!75!

69!

66!<!72!

57!<!79!

Clinical!syndrome:!Blood!tests!
PPID Positive

10

Percent

20

30

PPID Negative

.2

.3

.4

.5

.6

.2

.3

PCV
Percent
normal packedcellvolume
Graphs by Seasonally Adjusted ACTH

.4

.5

.6

Clinical!syndrome:!Blood!tests!
PPID Positive

10
5
0

Percent

15

20

PPID Negative

10

15

White Cell Count

Percent
normal whitecellcount
Graphs by Seasonally Adjusted ACTH

10

15

Clinical!syndrome:!Blood!tests!
PPID Positive

10
5
0

Percent

15

20

PPID Negative

10

15

Neutrophils
Percent
normal neutrophils
Graphs by Seasonally Adjusted ACTH

10

15

Clinical!syndrome:!Blood!tests!
PPID Positive

10
5

Consistent with decreased lymphocyte pool

0

Percent

15

20

PPID Negative

Lymphocytes
Percent
normal lymphocytes
Graphs by Seasonally Adjusted ACTH

Clinical!syndrome:!Blood!tests!
PPID Positive

20
10
0

Percent

30

40

PPID Negative

Fibrinogen
Percent
normal fibrinogen
Graphs by Seasonally Adjusted ACTH

Clinical!syndrome:!Blood!tests!
PPID Positive

20
10
0

Percent

30

40

PPID Negative

20

40

60

GGT
Percent
normal ggt
Graphs by Seasonally Adjusted ACTH

20

40

60

PPID!versus!Aged!Controls!
Basal!hyperinsulinaemia!(32%)!
!20!mIU/L!

LaminiSs!occurred!at!a!low!frequency!(6%)!
Although!10%!had!divergent!rings!
Horses!with!PPID!more!likely!to!have!suered!
from!laminiSs!and!hyperinsulinaemia!
But!hyperinsulinaemia!was!equally!associated!
with!laminiSs!in!non<PPID!horses!(66%;!OR!5.5)!

Results:!Study!2!
MulSvariable!analysis:!only!2!factors!
ROC!curve!0.80!

Owner!reported!history!of!hirsuSsm!OR!7.80!
(3.67!<!16.57)!
Age!OR!1.18!(1.11!<!1.25)!!
risk!of!developing!PPID!increasing!by!18%!per!year!
of!life!over!15!years!

Discussion!
Prevalence!is!high!20%!
Corresponds!with!level!of!hirsuSsm!study!
group!1!(17%)!

Also!the!degree!of!hirsuSsm!detected!in!the!UK!
22%!(Ireland!et!al!2011)!

OwnerMreported)signs!of!hirsuSsm!(delayed!
shedding!and/or!long!hair!coat!and/or!failure!
to!shed)!are!the!best!indicator!for!PPID!

Discussion!and!Conclusions!
No!evidence!of!inammatory!or!stress!leucogram!
for!PPID!horses!
RelaSve!lymphopaenia!in!aged!horses!may!be!
associated!with!immunosenescence!
But!not!signicantly!associated!with!age!

!
Could!horses!with!lymphopaenia!represent!a!
subgroup!of!horses!that!age!less!well?!

Discussion!and!Conclusions!
Aged!horses!with!clinical!or!historical!signs!consistent!
with!PPID!should!be!tested!and!appropriate!treatment!
insStuted!
RouSne!blood!evaluaSon!of!limited!use!to!support!
diagnosis!of!PPID!
Abnormal!ndings!in!either!aged!horses!or!those!with!PPID!
should!be!invesSgated!as!concurrent!disease!