Quality Assessment Enzyme Analysis for Lysosomal Storage Diseases ERNDIM / EUGT Meeting 5-6 October 2006, Prague

QQuality Assessment Enzyme Analysis for Lysosomal Storage Diseases ERNDIM / EUGT meeting 5-6 October 2006, Prague

Results of 1st “large scale” pilot Quality Assessment Enzyme Analysis for Lysosomal Storage Diseases

Laboratories Rotterdam

Hamburg/Heidelberg Dr, Zoltan Lukacs/Dr. Friederike Bürger QA-pilot for LSD’s the aims

• 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’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 Æ 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

Å Deficient? QA-pilot for LSD’s Control fibroblasts, specific enzyme activity

Range of 18 labs: 4 – 460 nmol/h/mg

Å Deficient? 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? Æ deficient or normal? QA-pilot for LSD’s Gaucher fibroblasts, % of participants mean control

>3SD: 4 or 400 % of Mean? Æ deficient or normal?

Range of 18 labs: 0.1 – 11 % of Mean

SD scores (% mean control) with cut-off not useful for enzyme deficiencies Control leukocytes Specific enzyme activity (left) versus % of mean control (right)

beta-Hexosaminidase A beta-Hexosaminidase (A+B) beta-Galactosidase

β-Hexosaminidase A β-Hexosaminidase A+B β-Galactosidase 2000 200 8000 400 300 160

180 350 140 250 160 1500 6000 300 120 140 200 250 100 120

1000 100 4000 200 150 80

80 150 60 nmol / h / mg 100 60

500 2000 100 40 % of mean control 40 50 50 20 20

0 0 0 0 0 0 01234 01234 01234 Control leukocytes Specific enzyme activity (left) versus % of mean control (right)

beta-Hexosaminidase A beta-Hexosaminidase (A+B) beta-Galactosidase

β-Hexosaminidase A β-Hexosaminidase A+B β-Galactosidase 2000 200 8000 400 300 160

180 350 140 250 160 1500 6000 300 120 140 200 250 100 120

1000 100 4000 200 150 80

80 150 60 nmol / h / mg 100 60

500 2000 100 40 % of mean control 40 50 50 20 20

0 0 0 0 0 0 01234 01234 01234 Control leukocytes Specific enzyme activity (left) versus % of mean control (right)

ASA 0 degrees ASA 37

Arylsulfatase A (0°) Arylsulfatase A (37°) 500 175 350 160

140 150 300 400 120 125 250

100 300 100 200 h / mg

17 80

75 150 200

nmol / h / mg 60 nmol / % of mean control of mean % 50 control of mean % 100 40 100 25 50 20

0 0 0 0 01234 01234 Control leukocytes Specific enzyme activity (left) versus % of mean control (right)

ASA 0 degrees ASA 37

Arylsulfatase A (0°) Arylsulfatase A (37°) 500 175 350 160

140 150 300 400 120 125 250

100 300 100 200 h / mg

17 80

75 150 200

nmol / h / mg 60 nmol / % of mean control of mean % 50 control of mean % 100 40 100 25 50 20

right-data is missing 0 0 0 } 0 01234 01234 Control EBV lymphoblasts Specific enzyme activity (left) versus % of mean control (right)

BHEX-A EBV BHEX-T EBV BGAL EBV

β-Hexosaminidase A β-Hexosaminidase A+B β-Galactosidase 2500 250 6000 200 250 120

180

5000 100 2000 200 160 200

140 4000 80

1500 150 120 150

3000 100 60

1000 100 80 100 nmol / h / mg 2000 40 60 % of mean control of mean % 500 50 40 50 1000 20

0 0 0 0 0 0 01234 01234 01234 Control EBV lymphoblasts Specific enzyme activity (left) versus % of mean control (right)

ASA 0 EBV ASA 37 EBV

Arylsulfatase A (0°) Arylsulfatase A (37°) 350 250 120 160

140 300 100 200 120 250 80 100 150 200 h / mg 60 80 17 150 100 60 nmol / h / mg nmol / 40 % of mean control of mean %

100 control of mean % 40 50 20 50 20

0 0 0 0 01234 01234 EBV lymphoblasts are suitable for QA for enzyme analysis Stability of 20 lysosomal enzymes in lyophilised leukocyte pellets (n=4)

100

50 elinase y alactosaminidase om g g lucosaminidase g

25 hin p a-NAc- S a-Glucosidase Arylsulfatase B a-Galactosidase a-Fucosidase b-Galactosidase GlcNAc-6S sulfatase Galactocerebrosidase a-Glucosidase b-Glucosidase Asp. a-Iduronidase lsulfatase A acetyltransferase y enzyme activity remaining (%) Mannosidase - - N b b-Hexosaminidase A b-Hexosaminidase 0 Ar b-Hexosaminidase T 1 a-Mannosidase b-Glucuronidase 0, 1 or 3 weeks at room temperature 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

difference between 2 activities Variation = sum of 2 activities 100 Reproducibility, duplicate leukocyte samples

90 90% [10,190]

40% [60,140] 80

20% [80,120] 70 10% [90,110] 60

20 % deviation form mean activity

10 no data no data no data no data no data no data no data 0 no data 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 Laboratory number 100 Reproducibility, duplicate leukocyte samples

90 no data 8 labs > 40% 12 labs 80 < 40% 9 labs 70 < 20% 3 labs < 10% 4 labs 60

20 % deviation form mean activity

10 no data no data no data no data no data no data no data 0 no data 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 Laboratory number 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) EBV lymphoblasts form patients % of QA-pilot controls β-Hexosaminidase A Tay Sachs Sandhof GM1 264% EBV7 EBV6 25β-Hexosaminidase A+B 25 β-GalactosidaseEBV5 25 112%

20 20 20

15 15 15

10 10 10

% of mean QA-pilot controls % of mean QA-pilot 5 5 5

0 0 0 0.00 1.00 2.00 0.00 1.00 2.00 0.00 1.00 2.00 EBV lymphoblasts form patients % of QA-pilot controls β-Hexosaminidase A Tay Sachs Sandhof GM1 264% EBV7 EBV6 25β-Hexosaminidase A+B 25 β-GalactosidaseEBV5 25 112%

20 20 20

15 15 15 < 13% < 12%

10 10 10

% of mean QA-pilot controls % of mean QA-pilot 5 5 5

0 0 0 0.00 1.00 2.00 0.00 1.00 2.00 0.00 1.00 2.00 Fibroblasts from Gaucher II patient

8 12 < 11%

Normal β-glucosidase from QA-pilot: 4, 19, 113 – 460 nmol/h/mg (n=18) 4 6 nmol / h / mg 4 % of mean control

0 0 01234 QA for LSD’s What 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’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