THROMBOSIS RESEARCH 66; 745-7551992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

MULTIMERIC ANALYSIS OF VON WILLEBRAND FACTOR BY VERTICAL SODI M DODECYL SULPHATE AGAROSE GEL ELECTROPHORESIS, VACUUM BLO NG TECHNOLOGY AND SENSlTlVE VISUALIZATION BY ALKALINE PHOSPHATA E ” ANTI-ALKALINE PHOSPHATASE COMPLEX

Peter Baillod, Beat Affolter, Gerhard H. Kurt, Rolf Pflugshaupt Diagnostics, Central Laboratory, Blood Transfusion Service, Swiss Red Cross, Wankdorfstrasse 10, CH-3000 Berne 22, SWITZERLAND

(Received 13.1 .1992; accepted in revised form 6.51992 by Editor M. Furlan) ABSTRACT To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.

INTRODUCTtON Von Willebrand factor (vWf) which is synthesized by endothelial cells and megakaryocytes exists in plasma as a series of multimers of which the size ranges from 450 to 10’000 kD (1). In primary haemostasis vWf plays an important role in platelet/subendothelium interaction. Furthermore, plasma vWf carries and stabilizes FVIII. In von Willebrand’s disease (#Id) the structural changes and the quantitative distribution of the multimers are important indicators for subtyping and therapy strategy (l-8). Commonly discontinuous SDS agarose gel electrophoresis is the preferred method to differentiate vWd-subtypes (9, 10) and accordingly, this method plays an important role in FVIII-concentrate quality control (11-16). The multimers are visualized by autoradiography (9) fs highly peroxidase mediated staining (17-19) or luminography (20, 21). Autoradiography sensitive but the hazards of gamma radiation are a disadvantage. Staining with peroxidase substrate is insensitive as compared to autoradiography. Luminescence assays show ‘an even higher sensitivity than autoradiography and the exposure time is much shorter (21). The transfer of the multimers to nitrocellulose membranes is often becoming a proiblem. In electroblotting the largest multimers are difficult to transfer to the membrane, particularly when Key words: von Willebrand factor multimers, von Willebrand’s disease, SDS-agarose electrophoresis, vacuum blotting, alkaline phosphatase anti-alkaline phosphatase complex, FVIII concentrate

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the transfer buffer contains methanol. If nylon membranes are used, methanol is not required, but due to high affinity of the membranes, background problems arise. The method described here consists of a vertical discontinuous SDS agarose system (low and high resolution) with an alkaline phosphatase anti-alkaline phosphatase staining system with even a higher sensitivity than luminography. The transfer was optimized by using a vacuum blotting system and high SDS concentration in the transfer buffer. This method is applied for vWf subtyping in clinical laboratories and for quality control of FVIII concentrates. It might also prove useful in research laboratories, whenever high sensitivity is required.

MATERIALSAND METHODS MATERIALS:

Reagents: HGT agarose for electrophoresis was purchased from FMC Corp., Rockland, ME. Rabbit anti-human vWf (cat. no.: A082), monoclonal mouse anti-rabbit (cat. no.: M737), rabbit anti-mouse (cat. no.: K670, 2”d bottle) and alkaline phosphatase anti-alkaline phosphatase complex (APAAP) (cat. no.: K670, 3rd bottle) were obtained from Dakopatts, Glostrup, Denmark. 5bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) were from Bio-Rad Labs, Richmond, CA. Bovine serum albumin (BSA), purity >96 % and leupeptin hemisulphate, purity >75 % were purchased from Fluka Corp.,Buchs, Switzerland. Aprotinin was from Bayer, Leverkusen, Germany. N-ethylmaleimide was obtained from ICN Biochemicals, Cleveland, USA. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), ethanolamine, glycine, methanol, magnesium chloride hexahydrate (MgCI?), sodium azide (NaN,), saccharose, hydrochloric acid, sodium dodecyl sulphate (SDS), Tnshydroxymethylaminomethane (Tris), polyoxyethylene sorbitan monolaurate (Tween 20) and bromophenol blue were all of analytical grade, the water was deionized and distilled. Consumables: nitrocellulose 0,2 urn transfer membrane was obtained from Schleicher & Schiill, Dassel, Germany. 3MM filter papers were purchased from Whatman, Maidstone, England.

EQUIPMENT:

Equipment for pouring the agarose gels: spacers 1.5 mm, 10 well combs, casting stand, clamp assemblies, inner glass plates 10 x 7.3 cm and outer glass plates 10 x 8.3 cm are available as an accessory of the mini Protean II electrophoresis system from Bio-Rad. Electrophoresis: Electrophoresis was performed with a power supply model 1000 / 500 and a mini-Protean II vertical electrophoresis cell from Bio-Rad. We supplied the electrophoresis cell with a PVC cooling pipe. The pipe was connected to a 2219 Multitemp II thermostatic circulator from Pharmacia LKB Biotech., Sweden.

Vacuum blotting unit: The blotting procedure was carried out with a vacuum blotter TransVac TE80 from Hoefer Scientific Instr., San Francisco, CA. The silicon rubber mask is available as an accessory of the blotting unit. Two rectangular holes 7.5 x 5.5 cm were cut into the mask. The blotting system was connected to a vacugene pump from Pharmacia. Incubation and washing: All washing and incubation steps were done on a horizontal platform shaker from Kijhner Corp., Basel, Switzerland. The shaker was equipped with a homemade thermoregulated metal block 20 x 25 cm. The metal block was connected to a commercially available thermostatic water circulator. Scanning:

All scans were performed on a TLC scanner II from Camag, Muttenz, Switzerland.

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SAMPLES:

Preparation of platelet poor plasma samples: In private practices blood was drawn into plastic tubes containing sodium citrate to achieve a final concentration of 0.011 M. The blood was sent to our laboratory by express mail. The plasma was prepared by centrifugation at 3000 g for 15 min at 22-25 OC. Plasma was separated from the sediment and centrifuged a second time for 10 min under the same conditions. The plasma was then divided into aliquots and stored at -70 OC until further use. For some experiments blood was drawn at our laboratory like above and immediately treated with protease inhibitors as published by Dent et al.(22). Commercial PVIII preparation: The studied FVIII concentrates were Haemate HS lot 663011 (500 IU) from Behring, Marburg, Germany and Faktor VIII-Konzentrat SRK, virusinaktiviert, lot 7.131.010.0 (500 IU) from Central Laboratory, Blood Transfusion Service, Swiss Red Cross, Berne, Switzerland. The factor VIII concentrates were reconstituted in 20 ml H,O dest. at 37 OC, divided into aliquots and stored at -70 OC until further use.

METHODS:

Preparation of agarose gels: The separating gels (low resolution 1.7 %, high resolution 3 %) 1.5 mm thick, were prepared by pouring melted HGT agarose in 0.375 M Tris buffer pH 8.8 containing 0.1 % SDS between the inner and outer glass plates fixed in the clamp assemblies. The casting stand was warmed up in an incubator to 80 OC prior to use. After pouring the separating gels, the casting stand was kept for 10 min at room temperature before coolinglit in the refrigerator for another 20 min. In the next step the gels were pushed up for about 1.3 ‘cm from the lower side with a spacer. The overlapping gel on the upper side was cut with a sharp knife along the inner glass plate. The clamp assemblies were put in vertical position to allow ithe gels to slip back to the lower side of the glass plates. After refixing the clamp assemblies in the casting stand the combs were placed between the glass plates 8 mm above the separating gels. Then the stacking gels (0.8 %) were prepared by pouring melted HGT agarose in 0.125 M Tris buffer pH 6.8 containing 0.1 % SDS into the remaining space. The casting stand has to be held slanted to avoid air bubbles. After the casting stand had been left for 115min at 4 OC the combs were removed by pushing the gels from the lower side and pulling the combs at the same time. If necessary the remaining stacking gel in the wells was sucked off by means of an injection needle connected to a vacuum pump. Then the gels were allowed to slip back to the lower side of the glass plates. In a final step the spacers were shifted about lmm towards the gels. Before running the electrophoresis the gels were kept at 4 OC inI a moist chamber, additionally wrapped in wet paper for at least 2 hours, but not more than 2 weeks. Specimen preparation: The vWf:Ag content of plasma samples and FVIII concentrates was quantified by rocket immunoelectrophoresis. Plasma samples were diluted from 1:4 to 1:3200 or to a specified vWf:Ag concentration with sample buffer containing 10 mM TrislHCI, 2 mM EDTA, pH 8.0, 3 % SDS, 10 % saccharose, 100 mg/l bromophenol blue. FVIII concentrates were diluted to 0.05 U/ml vWf:Ag with sample buffer. The samples were heated to 60 OC for 15 min before use. Sometimes the diluted and denatured samples were stored at -70 OC. Immediately prior to use they were incubated for 5 min at 37 OC.

Electrophoresis: Samples of 10 ul were applied to the inner 8 wells of the low ‘and high resolution gels by means of an Eppendorf pipette. The electrophoresis buffer contained 50 mM Tris, 2 mM EDTA, 384 mM glycine, 0.1 % SDS, pH 8.4 and was precooled to 12 OC. The electrophoretic separation was started at a constant voltage of 60 V until bromophenol blue could be observed as a distinct thin line, then it was continued at a constant voltage qf 13 V for about 16 hours until the dye front arrived 2 - 3 mm from the end of the gel. The buffer tank temperature was held at 14 OC.

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Vacuum blotting: One sheet of nitrocellulose membrane 0.2 urn and two 3MM filter papers Of the size 9 x 7 cm were wetted with transfer buffer containing 10 mM Tris/HCl, 150 mM NaCI, pH 7.4, 0.15 % SDS for at least 15 min. After placing first the filter papers, then the membranes and the silicon rubber mask on the metal screen of the vacuum blotting SyStern, the agarose gels were laid down on the top of the mask over the membrane avoiding air bubbles. The vacuum pump was started and when a vacuum of 60-70 mm H,O was reached, the buffer reservoir was filled with transfer buffer. The blotting was performed for 3-5 hours. Detection of the separated multimers: After blotting, the residual protein binding capacity was blocked by shaking the membranes in blocking buffer containing 10 mM Tris/HCI, 150 mM NaCI, pH 7.4, 2.5 % BSA, 0.05 % NaN, for 2 hours at 37 OC. After blocking, the membranes were incubated overnight in rabbit anti-human vWf diluted 1:lOOO in 25 ml blocking buffer at room temperature . The membranes were washed 3 times for 10 min in 10 mM Tris/HCI, 150 mM NaCI, pH 7.4, 0.05 % Tween 20, prewarmed to 37 OC. For further reactions the following antibodies were used: 1st mouse anti-rabbit (dil. 1:500), 2”d rabbit anti-mouse (dil. 1:50), 3ti APAAP (dil. 1:50), each diluted in blocking buffer. These antibody incubations were performed for 2 hours at 37 OC . Between the incubations the nitrocellulose sheets were washed 3 times as described before. During all blocking, incubation and washing steps, the membranes were gently shaken. Coloration was done by incubation of the membranes at room temperature in the substrate solution containing 45 ml 0.1 M ethanolamine pH 9.6, 5 ml 0.1 % NBT in 0.1 M ethanolamine pH 9.6, 750 ul 0.4 % BCIP in 66 % methanol 34 % acetone and 200 ul 1 M MgCI,. The reaction was stopped after sufficient coloration by rinsing the membranes in water. After staining, the dried membranes were scanned at 650 nm. For some experiments a direct staining procedure in the agarose gels was performed. After electrophoresis the gels were washed for 10 min in distilled water then pressed and dried. Compared to the procedure with the membranes the incubation and washing steps were prolonged to 6 hours and 30 min, respectively. Otherwise there were no changes.

RESULTS Figures 1 and 2 show the separation of patient plasmas in low and high resolution gels at agarose concentrations of 1.7 % and 3 % respectively. All samples were diluted 1:40 for the 1.7 % agarose gel. For the 3 % gel the vWf:Ag content was adjusted to 0.05 U/ml for samples 1, 2,3 and 5, sample 4 was diluted 1:4. Lane 1 shows normal multimeric pattern from a pool of 20 individuals with no evidence of vWd. Normally at least 18 to 20 bands are visible in the low resolution gels. Lane 2 is a sample from a patient with a type II vWd, which in the 1.7 % gel presents a marked reduction of high molecular weight multimers and an enhanced intensity of the two satellite bands. Also lane 3 shows a type II pattern. The leading band of the triplets has the same intensity as the central band and the high molecular weight multimers are decreased. Lane 4 is a patient with type I vWd (vWf:Ag < 0.1 U/ml). In comparison to the normal pattern it shows all bands markedly reduced. Lane 5 is a sample of a member of a family with a strong reduction in vWf:Ag and vWf:RiCo-activity (vWf:Ag 0.2 U/ml, vWf:RiCo 0.25 U/ml). This unclassified vWd type shows slight reduction of high molecular weight multimers and a diffuse triplet pattern. In the gel with 3 % agarose concentration the resolution is enhanced specially in the second multimer. In lanes 1, 2, 3 and 5 the multimers are resolved into 5 bands. The intensity of the different individual bands varies clearly from sample to sample. In type II of lane 2 the fastest and slowest satellite bands, in type II of lane 3 only the fastest one are markedly enhanced compared to normal plasma. Due to the very low vWf:Ag concentration, type I presents only the central and fastest moving band which shows an increased intensity compared to the fastest moving band of the higher multimers. In the unclassifed type the central band is between two slightly enhanced bands. The intensity of the slowest and fastest satellite band is decreased. In low and high resolution gels, the separation of the first multimer is not completed. In one experiment with a 3 % agarose gel the electrophoresis was stopped when the bromophenol blue

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front arrived about 1.2 cm before the end of the gel. In this case the first multimers were also separated into five bands (results not shown).

1

2

3

4

5

1

FIGURE 1

2

3

4

5

FIGURE 2

Low resolution vWf pattern

High resolution vWf pattern

(1.7 % separating

(3 % separating

gel)

1: normal plasma 2: type II 3: type II cathode: topof the gel /anode: bottom of the gel

Figure 3 presents the densitometric

4: type I [:

gel)

5: unclassified type 2”d multimer

scans of the same samples of the 1.7 % agarose gel. In

figure 4 only the second multimer of the 3 % gel was scanned to judge the structure of a quintuplet.

Because of absence of clear bands, the scan of type I is not shown.

Figure 5 shows a dilution series of a plasma pool with 0.8 U/ml vWf:Ag in a low resofution gel of 1.7 % agarose concentration. The sample dilution ranged from 1 :16 to 1:3200. In ~the 1:18 diluted sample 20 bands are visible. The 1 :1600 dilution still shows 16 multimers. To demonstrate the efficiency of the vacuum blot, two 1.7 % agarose gel electrophore$ses were done with normal plasma in a dilution series from 1 :16 to 1:512. One agarose gel was stained before and one after the transfer. The agarose gel which was stained before transfer, showed markedly enhanced background and less sensitivity compared to the detection on nitrocellulose membranes. The full pattern was visible up to a dilution of 1:256. The el which was stained after transfer showed only in the lowest dilution a trace of multimers in tRe region of 8000-l 0’000 kD. Figure 6 shows the scans of the first lane (1 :16) of the stained gels before and after the transfer. To study the risk of proteolytic degradation of vWf multimers, we compared the tability Of samples with and without addition of protease inhibitors. Figure 7 shows a plasma ith a norrecessed mal multimeric pattern in a 1:40 dilution. The samples in lane 1 and 2 were immediately after blood sampling, those in lane 3 and 4 after storing the whole bloot at room temperature for 72 hours. Before centrifugation and treatment with sample buffer, ‘the blood

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samples of lane 2 and 4 were treated with protease inhibitors and incubated for 30 min at room temperature. As figure 7 shows, there was no evidence of proteolytic degradation.

I\ .’it-l \_r,

.._.....1. ._...____.__._.--.____.___--.---...___. ‘\

l-

2

I

A w --___-_____----~____---.---~~~._~~~~~~.~_ _ P JIi

3

AAA 2 ....-...---.-..__.-~...____.--...________.-~ .._*.. ..I

.___.--__. ______-____ ____.___.__.._ ..__..___...____~s.

$

...L. .___ _._______ ______._ _________ ..___ ._....._L...... c&Kj&

_________._‘_‘___________~.._________> c&ode _------

______________-___

aode

FIGURE 3 Densitometric

FIGURE 4 scans of low resolution

Densitometric scans of high resolution vWf pattern of the second multimer (3 % separating gel)

vWf pattern (1.7 % separating gel) 1: normal plasma

---_ _____-----________ _____________________--__> a&

2: type II

3: type II

4: type I

5: unclassified type

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123456’ FIGURE 5

FIGURE 6

Dilution series of normal plasma separated in a low resolution gel

Scans of stained 1.7 % agarose gpls before and after vacuum transfer

1 :I :I 6, 2:1:80, 39 :400, 4:i :800, 5:l :1600,6:1:3200

1: vWf pattern of normal plasma stained in the agarose 2: remaining trace of vWf in the gel after vacuum transfer

cathode: top of the gel I anode: bottom of the gel

FIGURE 7

vWf pattern of normal plasma with and without addition of protease inhibitors 1: plasma immediatly processed after blood sampling (without addition of protease inhibitors) 2: plasma immediatly processed after blood sampling (with addition of protease inhibitors) 3: plasma prepared from whole blood which had been stored at room temperature for 72 hours before processing (without addition of protease inhibitors) 4: plasma prepared from whole blood which had been stored at room temperature for 72 hours before processing (with addition of protease inhibitors) cathode: top of the gel I anode: bottom of the gel

To see the differences in amount of high molecular weight multimers and strvcture of quintuplets, we compared two different FVIII concentrates with normal plasma. Figure 8 shows

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a low and high resolution gel with two different commercial factor VIII concentrates and a normal plasma from a person with no evidence of vWd. The concentrates were diluted to 0.05 U/ml vWf:Ag, the normal plasma to a concentration of 0.025 U/ml vWf:Ag. In the low resolution gel both concentrates have a slightly decreased intensity of the highest multimers and the fastest moving band of the triplets is increased. In the high resolution gel the multimers in both concentrates are resolved into 5 bands. In lane 1 the fastest and the slowest moving bands are enhanced. In the concentrate of lane 2 the intensity of the fastest and the second fastest band is increased. The fastest one shows even a higher intensity than the central band. These observations are confirmed in the densitometric scans in figure 9.

FIGURE 8

FIGURE 9

Low (left) and high (right) resolution vWf pattern of two factor VIII concentrates

Densitometric scans of high resolution vWf pattern of the second multimer of two factor VIII concentrates

cathode: [:

top of

the gel /anode:

bottom of the gel

2ndmultimer

1: F VIII concentrate, CL BTS SRC, (lot 7.131 .OlO.O)

2: F VIII concentrate, Behring HS, (lot 563011)

3: normal plasma

DISCUSSION The most frequently used electrophoresis technique to separate vWf multimers consists of a horizontal discontinuous SDS agarose electrophoresis (9, 10, 23). Compared to the horizontal method, the vertical system we used enabled better separation. Buffer contact with agarose is optimal, sample application is as easy as with polyacrylamide gel electrophoresis, the whole system can easily be cooled, the agarose cannot dry out. During electrophoresis the agarose shrinks a little, and direct buffer contact between anode and cathode might occur. This problem was solved by moving the spacers towards the agarose after the gel had solidified. Experiments with the more expensive, more purified and often used HGT(P) agarose from FMC Corp. resulted in no improvement of the resolution in our system. Repeated

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electrophoresis of the same plasma or F VIII concentrate sample showed always, in high as well as in low resolution separation, a reproducible pattern (results not shown). Electroblotting techniques are not well suited for vWf because electrophoretic mobility of vWf is low and mainly large multimers are difficult to transfer. The high electric current requires an efficient cooling system (24). The vacuum blotting system distinguishes itself by a high grade of efficiency and compared to capillary blotting (23) is more flexible and supervising is very easy. No damage is incurred to the agarose structure. High percent agarose may be blotted with higher vacuum. With our blotting technique 18-21 multimers from normal plasma were transferred from low resolution gel to nitrocellulose. If a direct staining method in theigel was used, a remaining trace peak in the region of the highest multimers was detected after transfer (figure 6). This transfer technique proved appropriate for agarose gels. By modifying the alkaline phosphatase anti-alkaline phosphatase (APAAP) staining; method originally developed for cell smears, cytocentrifuge preparations and cryostat sections 1(25,26), we achieved high sensitivity. In normal plasma with 0.8 U/ml vWf:Ag the multimers in a sample of 10 ul were still detectable up to a dilution of 1:1600 (figure 5). The system enables to enhance the sensitivity by loading up to 40 ul per sample. With chemiluminescence the detection limit is usually reached at a dilution of 1:500 (21). In our assays visualidation by streptavidin-biotin alkaline phosphatase did not result in a similarly high sensitivity, and background appeared increasingly. When we used a commercially available monoclonal antivWf antibody, instead of polyclonal anti-vWf, the detection of highly diluted vWf was slightly decreased. Comparisons between nylon and nitrocellulose membranes showed an increased background on the nylon membrane. With other blocking agents such as gelatin or non fat dry milk we also achieved satisfactory results. Compared to BSA, gelatin showed an augmented background, with non fat dry milk the sensitivity of multimer detection was diminished (results not shown). Direct detection of vWf multimers in agarose gel by immunoenzymatic staining method is also possible (19). Compared to staining on a nitrocellulose membrane with the APAAP staining technique, washing and incubation steps have to be considerably prolonged, background is increased whereas sensitivity is diminished. Detection limit in low resolution gel is reached at a dilution of 1:250. As our samples partially come from external medical practitioners, proteolytic degradation during transport may have occured. The same might also happen during denaturatibn in the sample buffer.Therefore we added a protease inhibitor mixture described by Dent et bl. (22) to freshly collected normal blood samples at different time intervals. In comparison to the untreated samples no difference could be observed (figure 7). The above described method has shown to be very useful for the analysis of vWf types (figures 1, 2, 3, 4) as well as for quality control of F VIII concentrates (figures 8, 9)., The high sensitivity of the staining and the efficiency of the transfer may easily be integrated into other already established systems.

ACKNOWLEGEMENTS The authors wish to thank Therese Hilfiker for translation and secretarial assistance. We also acknowledge Dr Hans Friedli and Dr Beat Perret for their critical review of the manuscript. We would also like to thank Prof lnge Scharrer, Klinikum der J.W. Goethe-Universitat, Zentrum der lnneren Medizin, Angiologie, Frankfurt, Germany, for generously supplying some of the plasma specimens.

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factor in factor VIII

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17 BUKH, A., INGERSLEV, J., STENBJERG, S., HUNDAHL MOELLER, N.P. The multimeric structure of plasma FVIII:RAg studied by electroelution and immunoperoxidase detection. Thromb Res 43,579-584,1984 18 LOMBARDI, R., GELFIC, C., RIGHETTI, P.G., LATTUADA, A., MANNUCCI, P.M. Electroblot and immunoperoxidase staining for rapid screening of the abnormalities of the multimeric structure of von Willebrand factor in von Willebrand’s disease. Thromb Haemostas X%246-249, 1986 19 AIHARA, M., SAWADA, Y., UENO, K., MORIMOTO, S., YOSHIDA, Y., DE SERRES, M., COOPER, H. A., WAGNER, R.H. Visualization of von Willebrand factor multimers by tmmunoenzymatic staining using avidin-biotin peroxidase complex. Thromb Haemostas 55, 863-267, 1986 20 SCHNEPPENHEIM, R., PLENDL, H., BUDDE, U. Luminography - an alternative assay for detection of von Willebrand factor multimers. Thromb Haemosfas SO, 133-l 36, 1988 21 BUDDE, U., SCHNEPPENHEIM, R., PLENDL, H., DENT, J., RUGGERI, Z. M., ZIMMERMAN, T.S. Luminographic detection of von Willebrand factor multimers in agarose gels and on nitrocellulose membranes. Thromb Haemosfas 63,312-315, 1990 22 DENT, J.A., GALBUSERA, M. RUGGERI, Z.M. Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit. J C/in Invest 88, 774-782, 1991 23 RAINES, G., AUMANN, H., SYKES, S., STREET, A. Multimeric analysis of von Willebrand factor by molecular sieving electrophoresis in sodium dodecyl sulphate agarose geli. Thromb Res 60,201-212,199O 24 YUKIHARU, T., HARRISON, J., ABILGAARD, C.F. Von Willebrand factor multimer analysis using a sensitive peroxidase staining method. Thromb Haemostas 62, 781-783, 1989 25 MASON, D.Y. lmmunocytochemical labeling of monoclonal antibodies munoalkaline phosphatase technique. Techniques in lmmunocytochemistry

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