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 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 enzyme activity remaining (%) 100 25 50 75 0 Asp.g lucosaminidase Stability of20lysosomalenzymesin a-Iduronidase lyophilised leukocyte pellets(n=4) b-Glucosidase GlcNAc-6S sulfatase Arylsulfatase B 0, 1 or 3 weeks at room temperature 3weeks 1 or room at 0, a-Galactosidase a-NAc-g alactosaminidase a-Fucosidase Sp hing omyelinase 1 b-Galactosidase Galactocerebrosidase a-Glucosidase a-Glucosidase N -acetyltransferase Arylsulfatase 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 difference between 2 activities Variation = sum of 2 activities % deviation form mean activity 100 10 20 30 40 50 60 70 80 90 0 0 1 2 3 Reproducibility, duplicateleukocytesamples 4 10% [90,110] 20% [80,120] 40% [60,140] 90% [10,190] 5 no data 6 7 8 no data 9 10 no data 11 12 Laboratory number 13 14 15 16 17 18 19 20 21 22 no data 23 24 no data 25 26 27 28 29 30 31 32 no data 33 no data 34 35 36 no data 37 % deviation form mean activity 100 10 20 30 40 50 60 70 80 90 0 0 1 2 3 Reproducibility, duplicateleukocytesamples 4 5 no data 6 no data 8labs 7 < 10% < 20% < 40% > 40% 8 no data 9 10 12 labs no data 3 labs 4 labs 11 9 labs 12 Laboratory number 13 14 15 16 17 18 19 20 21 22 no data 23 24 no data 25 26 27 28 29 30 31 32 no data 33 no data 34 35 36 no data 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) EBV lymphoblasts form patients % of QA-pilot controls β-Hexosaminidase A Tay 264%Sachs Sandhof GM1 EBV6 β-HexosaminidaseEBV7 A+B β-GalactosidaseEBV5 25 112% 25 25 20 20 20 15 15 15 10 10 10 % of mean QA-pilot controls 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 264%Sachs Sandhof GM1 EBV6 β-HexosaminidaseEBV7 A+B β-GalactosidaseEBV5 25 112% 25 25 20 20 20 15 15 < 13% 15 < 12% 10 10 10 % of mean QA-pilot controls 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% 10 6 8 Normal β-glucosidase from QA-pilot: 4, 19, 113 – 460 nmol/h/mg (n=18) 4 6 nmol / h / mg 4 % of mean control 2 2 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.