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QQuality AssessmentEnzyme Analysis for Lysosomal Storage Diseases
ERNDIM / EUGT meeting 5-6 October 2006, Prague
Results of 1st “large scale” pilot
LaboratoriesRotterdam
Hamburg/HeidelbergDr, Zoltan Lukacs/Dr. Friederike Bürger
Quality AssessmentEnzyme Analysis for Lysosomal Storage Diseases
• Inter-laboratory variation─ Many participants: > 30
• Intra-laboratory variation─ Analyse two pairs of identical control samples (blind)
• Proficiency testing of enzyme deficiencies─ Which samples are best?
- Blood samples: most widely used, but impractible- Fibroblasts: good but laborious- EBV lymphoblasts: easy to obtain, not widely used
QA-pilot for LSD’sthe aims
QA-pilot for LSD’sthe set up
• Samples, without clinical information─ Leukocytes─ EBV lymphoblasts─ Fibroblasts
• Enzymes: easy ones─ 4MU-substrates─ Simple colorimetric assays
• Shipping: economic─ Send at room temp., postal service
- Lyophilised enzymes, stable at room temp. for 5 days- Fibroblasts
• Data entry through existing ERNDIM programmes─ “Metabolite presentation” (www.erndimqa.nl Lysosomal Enzymes)
QA-pilot for LSD’sthe participants
• Questionnaire to members (85 from 21 countries)─ 40 labs want to join a QA-pilot─ 65% want to include DBS in future QA-schemes
• Samples sent, data returned─ 40 labs received samples─ 36 labs entered data on www.erndimqa.nl
QA-pilot for LSD’sthe samples
• 10 samples─ 4 leukocytes (two duplicate samples)─ 4 EBV lymphoblasts─ 2 fibroblasts
• 10 easy enzymes─ specific enzyme activity (e.g.. nmol/h/mg)─ normalise to % of mean control
• 2 separate experiments─ test reproducibility
QA-pilot for LSD’sControl fibroblasts, specific enzyme activity
Deficient?
Range of 18 labs: 4 – 460 nmol/h/mg
QA-pilot for LSD’sControl fibroblasts, specific enzyme activity
Deficient?
Range of 18 labs: 40 – 127 % of Mean
QA-pilot for LSD’sControl fibroblasts, % of participants mean control
How do these values relate to the control range of the participant?
QA-pilot for LSD’sGaucher fibroblasts, specific enzyme activity
SD scores (nmol/h/mg) not useful for enzyme deficiencies
QA-pilot for LSD’sGaucher fibroblasts, specific enzyme activity
QA-pilot for LSD’sGaucher fibroblasts, % of participants mean control
>3SD: 4 or 400 % of Mean? deficient or normal?
Range of 18 labs: 0.1 – 11 % of Mean
QA-pilot for LSD’sGaucher fibroblasts, % of participants mean control
>3SD: 4 or 400 % of Mean? deficient or normal?
SD scores (% mean control) with cut-offnot useful for enzyme deficiencies
beta-Hexosaminidase A
0
500
1000
1500
2000
0 1 2 3 40
20
40
60
80
100
120
140
160
180
200
beta-Hexosaminidase (A+B)
0
2000
4000
6000
8000
0 1 2 3 40
50
100
150
200
250
300
350
400
beta-Galactosidase
0
50
100
150
200
250
300
0 1 2 3 40
20
40
60
80
100
120
140
160
nmol
/ h
/ mg
β-Hexosaminidase A+B
% o
f mea
n co
ntro
l
β-Hexosaminidase A β-Galactosidase
Control leukocytes Specific enzyme activity (left) versus % of mean control (right)
beta-Hexosaminidase A
0
500
1000
1500
2000
0 1 2 3 40
20
40
60
80
100
120
140
160
180
200
beta-Hexosaminidase (A+B)
0
2000
4000
6000
8000
0 1 2 3 40
50
100
150
200
250
300
350
400
beta-Galactosidase
0
50
100
150
200
250
300
0 1 2 3 40
20
40
60
80
100
120
140
160
nmol
/ h
/ mg
β-Hexosaminidase A+B
% o
f mea
n co
ntro
l
β-Hexosaminidase A β-Galactosidase
Control leukocytes Specific enzyme activity (left) versus % of mean control (right)
ASA 0 degrees
0
100
200
300
400
500
0 1 2 3 40
25
50
75
100
125
150
175
ASA 37
0
50
100
150
200
250
300
350
0 1 2 3 40
20
40
60
80
100
120
140
160
% o
f mea
n co
ntro
l
Arylsulfatase A (0°) Arylsulfatase A (37°)
nmol
/ h
/ mg
% o
f mea
n co
ntro
l
nmol
/ 17
h /
mg
Control leukocytes Specific enzyme activity (left) versus % of mean control (right)
ASA 0 degrees
0
100
200
300
400
500
0 1 2 3 40
25
50
75
100
125
150
175
ASA 37
0
50
100
150
200
250
300
350
0 1 2 3 40
20
40
60
80
100
120
140
160
% o
f mea
n co
ntro
l
Arylsulfatase A (0°) Arylsulfatase A (37°)
nmol
/ h
/ mg
% o
f mea
n co
ntro
l
nmol
/ 17
h /
mg
}right-data is missing
Control leukocytes Specific enzyme activity (left) versus % of mean control (right)
BHEX-A EBV
0
500
1000
1500
2000
2500
0 1 2 3 40
50
100
150
200
250
BHEX-T EBV
0
1000
2000
3000
4000
5000
6000
0 1 2 3 40
20
40
60
80
100
120
140
160
180
200
BGAL EBV
0
50
100
150
200
250
0 1 2 3 40
20
40
60
80
100
120
nmol
/ h
/ mg
β-Hexosaminidase A+B
% o
f mea
n co
ntro
l
β-Hexosaminidase A β-Galactosidase
Control EBV lymphoblasts Specific enzyme activity (left) versus % of mean control (right)
ASA 37 EBV
0
20
40
60
80
100
120
0 1 2 3 40
20
40
60
80
100
120
140
160
% o
f mea
n co
ntro
l
ASA 0 EBV
0
50
100
150
200
250
300
350
0 1 2 3 40
50
100
150
200
250
% o
f mea
n co
ntro
l
nmol
/ 17
h / m
g
Arylsulfatase A (0°) Arylsulfatase A (37°)
nmol
/ h
/ mg
Control EBV lymphoblasts Specific enzyme activity (left) versus % of mean control (right)
EBV lymphoblasts are suitable for
QA for enzyme analysis
Stability of 20 lysosomal enzymes in lyophilised leukocyte pellets (n=4)
0
25
50
75
100
10, 1 or 3 weeks at room temperature
enzy
me
activ
ity re
mai
ning
(%)
Sph
ingo
mye
linas
e
a-M
anno
sida
se
Asp
.glu
cosa
min
idas
e
a-Id
uron
idas
e
b-G
luco
sida
se
Glc
NAc
-6S
sul
fata
se
Aryl
sulfa
tase
B
a-G
alac
tosi
dase
a-N
Ac-
gala
ctos
amin
idas
e
a-F
ucos
idas
e
b-G
alac
tosi
dase
Gal
acto
cere
bros
idas
e
a-G
luco
sida
se
N-a
cety
ltran
sfer
ase
Ary
lsul
fata
se A
b-H
exos
amin
idas
e T
b-H
exos
amin
idas
e A
b-M
anno
sida
se
b-G
lucu
roni
dase
a-G
luco
sida
se
Reproducibility of enzyme analysisin duplicate leukocyte samples
Blind duplicate samples were analysed in 2 experiments- 10 different enzymes / sample- 2 couples of identical samples- 20 matched pairs of enzyme activities / laboratory
difference between 2 activitiessum of 2 activities Variation =
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Reproducibility, duplicate leukocyte samples%
dev
iatio
n fo
rm m
ean
activ
ity
Laboratory number
no d
ata
no d
ata
no d
ata
no d
ata
no d
ata
no d
ata
no d
ata
no d
ata
90% [10,190]
40% [60,140]
20% [80,120]
10% [90,110]
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Reproducibility, duplicate leukocyte samples%
dev
iatio
n fo
rm m
ean
activ
ity
Laboratory number
no d
ata
no d
ata
no d
ata
no d
ata
no d
ata
no d
ata
no d
ata
no d
ata
< 10%
< 20%
< 40%
> 40%
no data 8 labs
12 labs
9 labs
3 labs
4 labs
Urgently needed:
QA for enzyme analysisof LSD’s
4 patient samples were included
• 3 EBV lymphoblasts- Tay-Sachs (β-hexosaminidase A)- Sandhoff (β-hexosaminidase A+B)- GM1-gangliosidosis (β-galactosidase)
• 1 fibroblast cell line- Gaucher type II (β-glucosidase)
Proficiency testing Lysosomal Storage Diseases
SandhofEBV7
0
5
10
15
20
25
0.00 1.00 2.00
GM1EBV5
0
5
10
15
20
25
0.00 1.00 2.00
% o
f mea
n Q
A-p
ilot c
ontr
ols
β-Hexosaminidase A+B β-Galactosidase Tay Sachs
EBV6
0
5
10
15
20
25
0.00 1.00 2.00
β-Hexosaminidase A264%
112%
EBV lymphoblasts form patients% of QA-pilot controls
SandhofEBV7
0
5
10
15
20
25
0.00 1.00 2.00
GM1EBV5
0
5
10
15
20
25
0.00 1.00 2.00
% o
f mea
n Q
A-p
ilot c
ontr
ols
β-Hexosaminidase A+B β-Galactosidase Tay Sachs
EBV6
0
5
10
15
20
25
0.00 1.00 2.00
β-Hexosaminidase A264%
112%
EBV lymphoblasts form patients% of QA-pilot controls
< 13%< 12%
0
2
4
6
8
0 1 2 3 40
2
4
6
8
10
12
% o
f mea
n co
ntro
l
Normal β-glucosidase from QA-pilot:4, 19, 113 – 460 nmol/h/mg (n=18)
Fibroblasts from Gaucher II patient
nmol
/ h
/ mg
< 11%
QA for LSD’sWhat needs to be done
• Report results to participants
• Website adjustment for enzyme analysis─ show all data, without cut-off (e.g. > 3SD)─ show data on reproducibility of duplicate samples─ show track records of all laboratories
• More information from participants─ which enzyme assay? [substrate, pH]─ which homogenisation procedure? [sonication, freeze-thaw]─ enzyme assay in: [full-homogenate, supernatant]
• Increase compliance of participants─ 10% of participants did not return any data─ 21 - 41% of participants did not return all data
QA for LSD’sPlans for 2007 / 2008 and after
• Towards a new ERNDIM activity: QA-for LSD ─ Start regular ERNDIM programme in 2009─ Prolong the pilot phase with 2 years (2007 and 2008).
- How to obtain permanent support for making the samples? - Make samples for coming QA-pilots on larger scale
Leukocytes, EBV lymphoblasts, fibroblasts, DBS
- Determine costs for participants for of regular ERNDIM QA-programmeno cost for participants in the pilot phase (estimated participants: 50)
- Start with DBS enzyme analysis, collect blood from patients - Assist to improve poor performance.
• QA pilots for 2007 / 2008Genzyme will financially support the coming 2 years.
─ February 2007- small pilot, leukocytes, EBV lymphoblasts (fibroblasts)
─ February 2008 :- big pilot, leukocytes, EBV lymphoblasts (fibroblasts)
one shipment, many samples, 4 independent experiment
Enduring QA-Programme forEnzyme Analysis for Lysosomal Storage Diseases