Report submitted by Wayne Chandler This report reviews examples of von Willebrand factor multimer analysis preformed by 7 different NASCOLA laboratories. We requested each performing site submit a scanned image of a typical gel from their laboratory. Scanned images and basic methodologic details were collected. Site identification was removed, and the resulting images and test details were combined into this document for review. von Willebrand factor antigen, factor VIII activity, ristocetin cofactor and collagen binding data were collected from the participating labs. Highlights: Most labs do not use stacking gels in front of separating gels 1% agarose is the most common separating gel used While most labs use unlabeled primary and labeled secondary antibodies, it is possible to use only a peroxidase labeled primary antibody, eliminating one step The most common visualization method is chemiluminescence Most labs run a normal plasma and in house type 2 vwd control Summary of data: 1. Electrophoresis: 7 different methods 2. Stacking gel % agarose: none (5 labs), 0.8% (2 labs) 3. Separating gel % agarose: 0.6%, 0.65%, 0.75%, 1.0% (3 labs), 1.3% 4. Primary antibody: polyclonal rabbit anti-human vwd (5 labs), peroxidase labeled antihuman vwd, monoclonal anti-human vwd 5. Secondary antibody: anti-rabbit-peroxidase (3 labs), anti-rabbit-alk phos, anti-mouseperoxidase, none (primary antibody labeled), Auro Probe anti-rabbit 1
6. Visualization method: chemiluminescence (5 labs), peroxidase stain, Auro Probe 7. Controls: normal plasma (all labs), in house type 2 vwd (6 labs), type 1 vwd, GK vwd Trait Severe 2
Multimer Example 1 1 2 3 4 5 6 7 8 Identification of lanes on multimer example 1 gel 1. Normal 2. vwd Type 2A 27% 29% <12% 3. Normal 4. Normal 5. Normal 6. Normal 7. Normal 8. vwd Type 2B 56% 90% 39% Electrophoresis system used Pharmacia Phast Stacking gel % agarose: NA Separating gel % agarose: 1.0% Primary (anti-vwf) antibody: Accurate Chemical rabbit-anti-human-vwf Secondary antibody: Sigma goat-anti-rabbit-alkaline phosphatase Visualization method: Alkaline phosphatase stain Controls: pooled normal plasma, known Type 2 3
Multimer Example 2 1 2 3 4 5 Identification of lanes on multimer example 2 gel 1. vwd Type 1, x2 <25% 2. vwd Type 1 <25% 3. vwd Type 2, x2 40% <25% 4. vwd Type 2 40% <25% 5. Normal Electrophoresis system used FischerBiotech Electrophoresis System (recirculating large horizontal system) Stacking gel % agarose: NA Separating gel % agarose: 0.6% Primary (anti-vwf) antibody: DakoCytomation polyclonal rabbit-anti-human vwf Secondary antibody: Amersham-ECL donkey anti-rabbit IgG Peroxidase Visualization method: Alpha Innotech ChemiGlow Chemiluminescence Substrate Alpha Innotech Fluorchem HD imaging system Controls: Precision Biologic Cryocheck normal reference plasma, in house Type 2 abnormal 4
Multimer Example 3 Id entification of lanes on multimer example 3 gel vwf Ag% FVIII% RCo% 1. Normal 115% 120% 100% 2. Normal 115% 120% 100% 3. Normal 115% 120% 100% 4. Normal 115% 120% 100% 5. Acquired vwd <6% 17% 14% 6. Normal 115% 120% 100% 7. GK vwd Trait Severe Coll Bind% E lectrophoresis system used Gibco-BRL Horizon 20-25, horizontal gel apparatus S tacking gel % agarose: NA Separating gel % agarose: 1% P rimary (anti-vwf) antibody: Dako rabbit-anti-human vwf S econdary antibody: Amersham anti-rabbit-peroxidase Visualization method: Controls: Kodak Image Station 2000R (luminescence) in house pooled normal plasma, George King vwd Trait Severe 5
Multimer Example 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Id entification of lanes on multimer example 4 gel 1. Normal 2. 37% 3. Type 1 6. 71% 58% 67% 9. 54% 49% 54% 77% 10. >300% 174% 141% 312% 11. 257% 172% 243% 324% 12. 122% 97% 79% 192% 13. >300% 103% 220% 234% 14. 120% 69% 36% 82% 15. Type 2 E lectrophoresis system used Home made S tacking gel % agarose: 0.8% Separating gel % agarose: 1% P rimary (anti-vwf) antibody: Dako rabbit-anti-human vwf S econdary antibody: Bio Rad goat-anti-rabbit-peroxidase Visualization method: Chemiluminescence Controls: known normal, Type 1, Type 2 6
Multimer Example 5 Identification of lanes on multimer example 5 gel 1. Normal Control 92% 2. ULM seen 104% 103% 152% 3. Normal 81% 109% 68% 4. Type 1 34% 47% 40% 5. Normal Multimer 53% 6. Type 1 or 2M 34% 44% 17% 7. Type 2 26% 27% 12% 8. Normal Control 92% 9. Normal 182% 117% 204% 10. Type 1 or 2M 22% 24% <10% 11. Prob Type 1 15% 20% 15% 12. Abn Control Type 2B 13. Type 2/Shifted 37% 51% 23% 14. Normal 93% 83% 81% 15. Normal Multimer 30% 16. Normal Multimer 17. Normal Control 92% 18. Normal Multimer 139% 19. Normal 63% 57% 55% 20. Normal 97% 98% 116% 21. Normal 66% 60% 61% 22. Normal 110% 86% 110% 23. Normal 97% 90% 84% 24. Normal Control 92% 7
Electrophoresis system used, multimer example 5 In house technique, horizontal gel in ISC-BioExpress Maxigel apparatus Stacking gel % agarose: NA Separating gel % agarose: 0.65% Primary (anti-vwf) antibody: Dako monoclonal anti-vwf Secondary antibody: Pierce, anti-mouse-peroxidase conjugate Visualization method: Blot, followed by chemiluminenscence Controls: Normal donor, genetic Type 2B 8
Multimer Example 6 Identification of lanes on multimer example 6 gel 1. Control lacking HWM multimers 2. Normal Multimer 44% 48% 42% 3. Acquired vwd 63% 57% 36% 4. Type 2 39% 48% 21% 5. Normal Multimer 6. Known severe vwd 3% 10% <10% 7. Control 50% normal plasma Electrophoresis system used Hoefer horizontal electrophoresis system Stacking gel % agarose: 0.8% Separating gel % agarose: 1.3% Primary (anti-vwf) antibody: Dako (primary antibody labeled) Secondary antibody: NA Visualization method: western blot with chemiluminescence detection Controls: 50% normal plasma, vwf concentrate lacking high molecular weight multimers 9
Multimer Example 7 Identifi cation of lan es on multimer exa mple 6 gel 1. Normal Control 2. Type 2B Control 320% 246% 258% 3. Normal 186% 233% 136% 4. Type 1 20% 48% 27% 5. Normal 207% 163% 133% 6. Normal Control 7. Normal 186% 233% 136% 8. Type 1 20% 48% 27% 9. Normal 207% 163% 133% 10. Normal Control E lectrophoresis system used Hoefer TE22 Mighty Small Stacking gel % agarose: NA Separating gel % agarose: 0.75% Primary (anti-vwf) antibody: rabbit anti-human vwf Secondary antibody: Auro Probe goat anti-rabbit Visualization method: Auro Probe with silver enhanced gold Controls: normal plasma, in house Type 2B 10