Quality Assessment Enzyme Analysis for Lysosomal ... - ERNDIM

Quality Assessment
Enzyme Analysis for Lysosomal Storage Diseases
ERNDIM / EUGT meeting 5-6 October 2006, Prague
Results of 1 st “large scale” pilot

Quality Assessment
Enzyme Analysis for Lysosomal Storage Diseases
Laboratories
Rotterdam
Hamburg/Heidelberg
Dr, Zoltan Lukacs/Dr. Friederike Bürger

• Inter-laboratory variation
─ Many participants: > 30
QA-pilot for LSD’s
the aims
• 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’s
the 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’s
the 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’s
the 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’s
Control fibroblasts, specific enzyme activity
Deficient?

QA-pilot for LSD’s
Control fibroblasts, specific enzyme activity
Deficient?
Range of 18 labs: 4 – 460 nmol/h/mg

QA-pilot for LSD’s
Control fibroblasts, % of participants mean control
Range of 18 labs: 40 – 127 % of Mean

QA-pilot for LSD’s
Gaucher fibroblasts, specific enzyme activity
How do these values relate to the control range
of the participant?

QA-pilot for LSD’s
Gaucher fibroblasts, specific enzyme activity
SD scores (nmol/h/mg) not useful for enzyme deficiencies

QA-pilot for LSD’s
Gaucher fibroblasts, % of participants mean control
>3SD: 4 or 400 % of Mean? deficient or normal?

QA-pilot for LSD’s
Gaucher fibroblasts, % of participants mean control
>3SD: 4 or 400 % of Mean? deficient or normal?
Range of 18 labs: 0.1 – 11 % of Mean
SD scores (% mean control) with cut-off
not useful for enzyme deficiencies

nmol / h / mg
2000
1500
1000
500
0
Control leukocytes
Specific enzyme activity (left) versus % of mean control (right)
beta-Hexosaminidase A
0 1 2 3 4
200
180
160
140
120
100
80
60
40
20
0
8000
6000
4000
2000
0
beta-Hexosaminidase (A+B)
0 1 2 3 4
400
350
300
250
200
150
100
50
0
300
250
200
150
100
50
0
beta-Galactosidase
β-Hexosaminidase A β-Hexosaminidase A+B β-Galactosidase
0 1 2 3 4
160
140
120
100
80
60
40
20
0
% of mean control

nmol / h / mg
2000
1500
1000
500
0
Control leukocytes
Specific enzyme activity (left) versus % of mean control (right)
beta-Hexosaminidase A
0 1 2 3 4
200
180
160
140
120
100
80
60
40
20
0
8000
6000
4000
2000
0
beta-Hexosaminidase (A+B)
0 1 2 3 4
400
350
300
250
200
150
100
50
0
300
250
200
150
100
50
0
beta-Galactosidase
β-Hexosaminidase A β-Hexosaminidase A+B β-Galactosidase
0 1 2 3 4
160
140
120
100
80
60
40
20
0
% of mean control

nmol / 17 h / mg
500
400
300
200
100
0
Control leukocytes
Specific enzyme activity (left) versus % of mean control (right)
ASA 0 degrees
0 1 2 3 4
175
150
125
100
75
50
25
0
350
300
250
200
150
100
50
0
ASA 37
Arylsulfatase A (0°) Arylsulfatase A (37°)
% of mean control
nmol / h / mg
0 1 2 3 4
160
140
120
100
80
60
40
20
0
% of mean control

nmol / 17 h / mg
500
400
300
200
100
0
Control leukocytes
Specific enzyme activity (left) versus % of mean control (right)
ASA 0 degrees
0 1 2 3 4
175
150
125
100
75
50
25
0
350
300
250
200
150
100
50
0
ASA 37
Arylsulfatase A (0°) Arylsulfatase A (37°)
% of mean control
nmol / h / mg
}right-data is missing
0 1 2 3 4
160
140
120
100
80
60
40
20
0
% of mean control

nmol / h / mg
2500
2000
1500
1000
500
0
Control EBV lymphoblasts
Specific enzyme activity (left) versus % of mean control (right)
BHEX-A EBV
0 1 2 3 4
250
200
150
100
50
0
6000
5000
4000
3000
2000
1000
0
BHEX-T EBV
0 1 2 3 4
200
180
160
140
120
100
80
60
40
20
0
250
200
150
100
50
0
BGAL EBV
β-Hexosaminidase A β-Hexosaminidase A+B
β-Galactosidase
0 1 2 3 4
120
100
80
60
40
20
0
% of mean control

nmol / 17 h / mg
350
300
250
200
150
100
50
0
Control EBV lymphoblasts
Specific enzyme activity (left) versus % of mean control (right)
ASA 0 EBV
120
100
80
60
40
20
0
ASA 37 EBV
Arylsulfatase A (0°) Arylsulfatase A (37°)
0 1 2 3 4
250
200
150
100
50
0
% of mean control
nmol / h / mg
0 1 2 3 4
160
140
120
100
80
60
40
20
0
% of mean control

EBV lymphoblasts are suitable for
QA for enzyme analysis

enzyme activity remaining (%)
100
75
50
25
0
Asp.g lucosaminidase
Stability of 20 lysosomal enzymes in
lyophilised leukocyte pellets (n=4)
a-Iduronidase
b-Glucosidase
GlcNAc-6S sulfatase
Arylsulfatase B
a-Galactosidase
a-NAc-g alactosaminidase
a-Fucosidase
Sp hing omyelinase
b-Galactosidase
0, 1 or 3 weeks at room temperature
1
Galactocerebrosidase
a-Glucosidase
N -acetyltransferase
Arylsu lfa ta se A
b-Hexosaminidase T
b-Hexosaminidase A
b -Mannosidase
a-Mannosidase
b-Glucuronidase

Reproducibility of enzyme analysis
in 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
Variation =
difference between 2 activities
sum of 2 activities

% deviation form mean activity
100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
Reproducibility, duplicate leukocyte samples
4
90% [10,190]
40% [60,140]
20% [80,120]
10% [90,110]
no data
5
6
7
no data
8
9
no data
10
11
12
13
14
15
16
17
18
19
20
21
Laboratory number
no data
22
23
no data
24
25
26
27
28
29
30
31
no data
no data
32
33
34
35
no data
36
37

% deviation form mean activity
100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
Reproducibility, duplicate leukocyte samples
4
no data
5
6
no data 8 labs
7
> 40%
< 40%
< 20%
< 10%
no data
8
9
no data
10
12 labs
9 labs
3 labs
4 labs
11
12
13
14
15
16
17
18
19
20
21
Laboratory number
no data
22
23
no data
24
25
26
27
28
29
30
31
no data
no data
32
33
34
35
no data
36
37

Urgently needed:
QA for enzyme analysis
of LSD’s

Proficiency testing
Lysosomal Storage Diseases
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)

% of mean QA-pilot controls
25
20
15
10
5
0
EBV lymphoblasts form patients
β-Hexosaminidase A
264%
Tay Sachs
EBV6
112%
0.00 1.00 2.00
% of QA-pilot controls
25
20
15
10
5
0
Sandhof
EBV7
0.00 1.00 2.00
25
20
15
10
5
0
GM1
EBV5
β-Hexosaminidase A+B β-Galactosidase
0.00 1.00 2.00

% of mean QA-pilot controls
25
20
15
10
5
0
EBV lymphoblasts form patients
β-Hexosaminidase A
264%
Tay Sachs
EBV6
112%
0.00 1.00 2.00
% of QA-pilot controls
25
20
15
10
5
0
Sandhof
EBV7
0.00 1.00 2.00
25
20
15
10
5
0
GM1
EBV5
β-Hexosaminidase A+B β-Galactosidase
< 13%
< 12%
0.00 1.00 2.00

Fibroblasts from Gaucher II patient
Normal β-glucosidase from QA-pilot:
4, 19, 113 – 460 nmol/h/mg (n=18)
nmol / h / mg
8
6
4
2
0
< 11%
0 1 2 3 4
12
10
8
6
4
2
0
% of mean control

• Report results to participants
QA for LSD’s
What needs to be done
• 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’s
Plans 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-programme
no 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 / 2008
Genzyme 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 for
Enzyme Analysis for Lysosomal Storage Diseases