IJZRO>fBI?SISRESEARCH Printed in Great
BRIEF
Vol.
Efritain
12,
pp.
677-680,
Pergamon
C 0 81 M UN
I CA
T IO
Press,
1978
Lrd.
S
A SIMPLIFIED ASSAY FOR VOX WILLEBRAXD FACTOR
Georges E. Rivard and Madeleine B. Daviault Centre de Recherche Phdiatrique de 1'HGpital Sainte-Justine Montrdal, Canada (Received
3.10.1977; in Accepted by Editor
revFsed form 17.1.1978. K.M. Brinkhous)
IWTRODUCTION Since the demonstration in 1971 by Howard and Firkin (1) that ristocetin can induce platelet aggregation in normal, hemophilic, and afibrinogenemic platelet-rich plasma (PRP) but not in PRP of some patients with von Willebrand's disease, our understanding of Factor VIII and of the different clinical conditions in which it is absent or abnormal has increased significantly. Weiss et al. (2) developed a quantitative assay for the plasma factor necessary for ristocetin-induced platelet aggregation. That assay has been used extensively, with minor modifications, by virtually every investigator in this field. In 1975, Allain et al. (3) and Brinkhous et al. (4) described a macroscopic aggregation test using paraformaldehyde-fixed platelets. This test is as sensitive as Weiss' test and has the advantage of being performed on fixed platelets which can be stored for over one month at 4OC: unfortunately, the macroscopic aggregation test has an end point which is difficult to read because the size of the platelet aggregates varies as the concentration of the measured factor changes. We report herein a modification of the macroscopic aggregation test which makes it easier to read and renders it suitable for utilization in laboratories with minimal experience in coagulation methodology. Reisner et al. (5) have presented preliminary report using a somewhat analogous procedure.
MATERIAL AND METHODS Blood specimens were obtained with plastic material and were anticoagulated by mixing 9 ml of blood with 1 ml of balanced citrate (0.04M citric acid and 0.06M sodium citrate). Factor VIII activity (VIII:C) was measured by a one-stage activated partial thromboplastin time assay(6). Factor VIII related antigen (VIII:RAg) was measured by immunoelectrophoresis (7) using a commercially available antibody (Hoechst Behring Diagnostics, Batch 5701B). Weiss' method(2) was used as a reference assay for von Willebrand factor. We initially used blood obtained from fasting donors and processed as described in the original method(3). We rapidly realized that short-dated 677
platelet-rich plasmas (PR?) from blood bank gave as good results as fresh platelets. All data presented in this work were obtained with such dated platelets. plasma prepared by the Canadian Qed Cross from Platelet-rich blood anticoagulated with C?D (63ml of citrate-phosphate-dextrose and 45Oml of blood) and kept at 220C with constant agitation for more than 72 hours but less than 5 days was used. The PRP were centrifuged at 3000 g for 20 minutes at 22OC. The platelet button was washed by resuspending 3 times in titrated saline (0.15M NaCl, 0.006M sodium citrate, pH 7.1) and centrifuging at 135Og for 20 minutes. After the final centrifugation the supernatant was removed ant the platelet button was resuspended in paraformaldehyde (9 parts of 4 percent paraformaldehyde in O.135M monosodium phosphate, one part of ACD, pH 6.9). The platelets were fixed for 48 hours at 4oC. Paraformaldehyde was then removed by centrifuging at 1350 g for 20 minutes. Platelets were resuspended three times in phosphate buffer 0.15M pH 7.3 and centrifuged at 1350 g for 20 minutes. After the third wash , platelets were resuspended in the same phosphate buffer at a concentration of 1.0 x 10e6 platelets per cubic millimeter; sodium azide was added at a final concentration of .0002 percent. The platelet suspension was distributed in small aliquots and stored at 4oC in capped plastic tubes for up to six weeks. Using a wax marking pencil, circles were made in the center of clean new glass slides. With a short Pasteur pipet (14.5 cm), 2 drops of the platelet suspension, one drop of the specimen to be tested appropriately diluted in phosphate buffer, and one drop of ristocetin ( Abbott, North Chicago, Ill.) were put in the center of the circle and mixed with a wood stick. The platelet aggregation of all the dilutions of a single plasma was observed simultaneously at room temperature under constant agitation and illumination with a Rh Typing Box (Clay-Adams, NY A2220). Multiples of l/5 dilution of normal plasma were done in phosphate buffer (0.15M pH7.3). A nepative control was done by replacing the dilution of plasma by the phosphate buffer. The last dilution still giving a visible aggregate determined the sensibility of a given set of reagents. If, for example, that last dilution was l/.80, the assay was said to be sensitive to 1.25% of VIII:WF (loo/SO ~1.25). An unknown plasma tested in those conditions and giving a greatest positive dilution at l/5 was said to contain 6.25% of VIII:WF (1.25 x 5 = 6.25). One unit of von Willebrand factor was defined as the amount of the factor present in 1 ml of a pool of plasmas from at least 10 normal male donors. The results were expressed as percent of the normal pool. Statistical analysis of the results was done by one was analysis of variance(8).
RESULTS
Different concentrations of ristocetin as well as of platelets were used to determine the optimal conditions for producing a sensitive reaction which would be easy to read; final concentrations of 4 x lo5 platelets/mm3 and ristocetin 1.5mg/ml gave the best results. As the concentration of von Willebrand factor decreases, the time required to produce macroscopically visible aggregates increases; after 3 minutes at room temperature, a reaction mixture negative for aggregates will remain negative. Dosage of von Willebrand factor on forty normal volunteers gave a mean of 92.1% (S.D. i 32). Table 1 gives results of dosage of von Willebrand factor on plasma from patients with hemophilia and with von Villebrand's disease of different clinical severity. Different dilutions of stored plasma from 13 controls and 6 patients with von Willebrand's disease were assayed blindly on three different days by herein described method and once by the technique of Weiss(2). There was no statistically significant difference between the mean results of those four determinations (F test observed = 0.06, F test expected = 4.20, with an a of 0.01).
.ASS.?IY
FOR
von
W’ILLEBKQZD
F.;CT:>R
TABLE 1 Hemophilia vs von Willebrand Hemophilia
S.F.
M.K. P.C. A.D. N.L.
VIII:C (X)
WIII:RAg (X)
VIII:'WF (X)
1.5 3.0 1.5 2.5 0.9
174 140 128 133 112
100 75 125 75 150
4.4 6.0 5.0 47.0 18.0 25.0 34.0
BRIEF
Vol.
Efritain
12,
pp.
677-680,
Pergamon
C 0 81 M UN
I CA
T IO
Press,
1978
Lrd.
S
A SIMPLIFIED ASSAY FOR VOX WILLEBRAXD FACTOR
Georges E. Rivard and Madeleine B. Daviault Centre de Recherche Phdiatrique de 1'HGpital Sainte-Justine Montrdal, Canada (Received
3.10.1977; in Accepted by Editor
revFsed form 17.1.1978. K.M. Brinkhous)
IWTRODUCTION Since the demonstration in 1971 by Howard and Firkin (1) that ristocetin can induce platelet aggregation in normal, hemophilic, and afibrinogenemic platelet-rich plasma (PRP) but not in PRP of some patients with von Willebrand's disease, our understanding of Factor VIII and of the different clinical conditions in which it is absent or abnormal has increased significantly. Weiss et al. (2) developed a quantitative assay for the plasma factor necessary for ristocetin-induced platelet aggregation. That assay has been used extensively, with minor modifications, by virtually every investigator in this field. In 1975, Allain et al. (3) and Brinkhous et al. (4) described a macroscopic aggregation test using paraformaldehyde-fixed platelets. This test is as sensitive as Weiss' test and has the advantage of being performed on fixed platelets which can be stored for over one month at 4OC: unfortunately, the macroscopic aggregation test has an end point which is difficult to read because the size of the platelet aggregates varies as the concentration of the measured factor changes. We report herein a modification of the macroscopic aggregation test which makes it easier to read and renders it suitable for utilization in laboratories with minimal experience in coagulation methodology. Reisner et al. (5) have presented preliminary report using a somewhat analogous procedure.
MATERIAL AND METHODS Blood specimens were obtained with plastic material and were anticoagulated by mixing 9 ml of blood with 1 ml of balanced citrate (0.04M citric acid and 0.06M sodium citrate). Factor VIII activity (VIII:C) was measured by a one-stage activated partial thromboplastin time assay(6). Factor VIII related antigen (VIII:RAg) was measured by immunoelectrophoresis (7) using a commercially available antibody (Hoechst Behring Diagnostics, Batch 5701B). Weiss' method(2) was used as a reference assay for von Willebrand factor. We initially used blood obtained from fasting donors and processed as described in the original method(3). We rapidly realized that short-dated 677
platelet-rich plasmas (PR?) from blood bank gave as good results as fresh platelets. All data presented in this work were obtained with such dated platelets. plasma prepared by the Canadian Qed Cross from Platelet-rich blood anticoagulated with C?D (63ml of citrate-phosphate-dextrose and 45Oml of blood) and kept at 220C with constant agitation for more than 72 hours but less than 5 days was used. The PRP were centrifuged at 3000 g for 20 minutes at 22OC. The platelet button was washed by resuspending 3 times in titrated saline (0.15M NaCl, 0.006M sodium citrate, pH 7.1) and centrifuging at 135Og for 20 minutes. After the final centrifugation the supernatant was removed ant the platelet button was resuspended in paraformaldehyde (9 parts of 4 percent paraformaldehyde in O.135M monosodium phosphate, one part of ACD, pH 6.9). The platelets were fixed for 48 hours at 4oC. Paraformaldehyde was then removed by centrifuging at 1350 g for 20 minutes. Platelets were resuspended three times in phosphate buffer 0.15M pH 7.3 and centrifuged at 1350 g for 20 minutes. After the third wash , platelets were resuspended in the same phosphate buffer at a concentration of 1.0 x 10e6 platelets per cubic millimeter; sodium azide was added at a final concentration of .0002 percent. The platelet suspension was distributed in small aliquots and stored at 4oC in capped plastic tubes for up to six weeks. Using a wax marking pencil, circles were made in the center of clean new glass slides. With a short Pasteur pipet (14.5 cm), 2 drops of the platelet suspension, one drop of the specimen to be tested appropriately diluted in phosphate buffer, and one drop of ristocetin ( Abbott, North Chicago, Ill.) were put in the center of the circle and mixed with a wood stick. The platelet aggregation of all the dilutions of a single plasma was observed simultaneously at room temperature under constant agitation and illumination with a Rh Typing Box (Clay-Adams, NY A2220). Multiples of l/5 dilution of normal plasma were done in phosphate buffer (0.15M pH7.3). A nepative control was done by replacing the dilution of plasma by the phosphate buffer. The last dilution still giving a visible aggregate determined the sensibility of a given set of reagents. If, for example, that last dilution was l/.80, the assay was said to be sensitive to 1.25% of VIII:WF (loo/SO ~1.25). An unknown plasma tested in those conditions and giving a greatest positive dilution at l/5 was said to contain 6.25% of VIII:WF (1.25 x 5 = 6.25). One unit of von Willebrand factor was defined as the amount of the factor present in 1 ml of a pool of plasmas from at least 10 normal male donors. The results were expressed as percent of the normal pool. Statistical analysis of the results was done by one was analysis of variance(8).
RESULTS
Different concentrations of ristocetin as well as of platelets were used to determine the optimal conditions for producing a sensitive reaction which would be easy to read; final concentrations of 4 x lo5 platelets/mm3 and ristocetin 1.5mg/ml gave the best results. As the concentration of von Willebrand factor decreases, the time required to produce macroscopically visible aggregates increases; after 3 minutes at room temperature, a reaction mixture negative for aggregates will remain negative. Dosage of von Willebrand factor on forty normal volunteers gave a mean of 92.1% (S.D. i 32). Table 1 gives results of dosage of von Willebrand factor on plasma from patients with hemophilia and with von Villebrand's disease of different clinical severity. Different dilutions of stored plasma from 13 controls and 6 patients with von Willebrand's disease were assayed blindly on three different days by herein described method and once by the technique of Weiss(2). There was no statistically significant difference between the mean results of those four determinations (F test observed = 0.06, F test expected = 4.20, with an a of 0.01).
.ASS.?IY
FOR
von
W’ILLEBKQZD
F.;CT:>R
TABLE 1 Hemophilia vs von Willebrand Hemophilia
S.F.
M.K. P.C. A.D. N.L.
VIII:C (X)
WIII:RAg (X)
VIII:'WF (X)
1.5 3.0 1.5 2.5 0.9
174 140 128 133 112
100 75 125 75 150
4.4 6.0 5.0 47.0 18.0 25.0 34.0
