Patent Application
20080076114
The linear increase in the reference curve shows: the behavior of the agglutination reaction of the platelet fragments of the invention is proportional to the VWF activity in bovine plasma.
Correlation of the assay results (VWF activity) were determined with a conventional platelet assay and with the novel platelet fragment assay for the same bovine plasma samples. The correlation coefficient of R2=0.9879 confirms the linear relation between the assay results in the two assays.
Native platelets were obtained by centrifuging a platelet apheresis concentrate from human whole blood in several sample vessels in a precooled centrifuge at 1500 g with brake off at 4° C. for 10 minutes. The supernatants were removed and the sediments were taken up in 30 ml portions of cooled citrate buffer (9.07 g/l disodium hydrogen phosphate, 1.65 g/l potassium dihydrogen phosphate, 0.38% sodium citrate, 1.2 g/l NaCl). The resuspension and homogenization took place with the aid of an Ultra-Turrax disperser (Janke und Kunkel GmbH, Staufen, Germany) (10 sec at 13 500 rpm). The platelet count was then adjusted to 1 000 000 platelets/μl by dilution with citrate buffer.
The platelet suspension was incubated in an ice bath for 30 minutes. Then 20 ml aliquots were treated with a Branson Sonifier 250 (Branson, Danbury, USA) ultrasonic probe with continuous cooling on ice for 15 seconds in the following way: duty cycle: 100%, output control: 7. Immediately thereafter, formaldehyde was added so that the final formaldehyde concentration was 1%. Fixation at 2-8° C. overnight (for about 14 hours) was followed by dialysis with buffer (9.07 g/l disodium hydrogen phosphate, 1.65 g/l potassium dihydrogen phosphate, 1.2 g/l NaCl) until the formaldehyde content had fallen to a concentration of less than 0.001%.
To enrich the particularly advantageous fraction, first the fragment suspension was homogenized by using the Ultra-Turrax disperser, and then 20 ml aliquots were centrifuged at 1500 g at 20° C. for 10 minutes. The supernatants were removed and centrifuged at 4000 g at 20° C. for 40 minutes. The sediments were taken up in 1 ml portions of phosphate buffer (0.907 g/l disodium hydrogen phosphate, 0.165 g/l potassium dihydrogen phosphate) and treated with a shaker until the sediment was completely detached from the base. Several sediments resuspended in this way were combined in a 14 ml plastic tube and homogenized using an Ultra-Turrax disperser at 13 500 rounds/minute for 5 to 10 seconds.
Immediately before use of the fragment suspension for determining the VWF activity, NaCl was added so that the NaCl concentration in the suspension was 2 g/l.
The following steps were carried automatically in a BCT® (Dade Behring Marburg GmbH, Marburg, Germany) coagulation analyzer:
50 μl of platelet-poor bovine citrated plasma were mixed with 100 μl of a platelet fragment suspension prepared as in example 1 to give a reaction mixture, and incubated at 37° C. for 15 seconds.
The extinction of the reaction mixture at 620 nm was measured over a period of 90 seconds without mixing the reaction mixture during the measurement. The kinetics found in this way for the aggregation reaction were used to determine the change in extinction (mE/minute), and the maximum change in extinction (Vmax of the increase in extinction) was found.
A calibration plot was constructed by diluting normal bovine plasma (VWF activity=100%) with VWF-deficient plasma in a dilution series. The undiluted normal plasma, and the various dilutions having different VWF activities, were analyzed by the method described.
In a comparative experiment, the same bovine plasma dilutions were assayed in a conventional agglutination assay in which fixed whole platelets were mixed with the sample. As is evident from
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2006 045 550.9 | Sep 2006 | DE | national |
