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COAMATIC® Protein C - 82 2098 63
ENGLISH - Insert revision 12/2002
Intended use
Specimen collection
This kit is for the quantitative determination of Protein C activity in human citrated plasma.
Nine parts of freshly drawn venous blood are collected into one part trisodium citrate.
Plot the absorbance (A) for the standard samples against their protein C activity on Background and summary
Centrifugation: 2000 x g for 10-20 minutes at 20-25°C.
linear graph paper. Plot A on the Y axis and % protein C on the X axis. Connect the Refer to NCCLS document H21-A2 for further instructions on specimen collection, standard points with the best fitting straight line.
Protein C is a vitamin K dependent plasma protein which plays an important role in the anticoagulant regulatory mechanisms. It circulates as a zymogen and is converted to an Samples are evaluated based on this standard curve. An example of a typical standard Quality control
active serine protease, activated Protein C (APC), by the action of thrombin in presence curve (microplate method) is shown below.
of thrombomodulin. APC regulates the coagulation system by proteolytic cleavage and Normal and abnormal controls are recommended for a complete quality control inactivation of activated factors V and VIII. Hereditary and/or acquired Protein C program . Each laboratory should establish its own mean and standard deviation and deficiency has been shown to be a risk factor for development of venous thrombosis.
should establish a quality control program to monitor laboratory testing. Controls should Measurement principle
be analyzed at least every 8 hour shift in accordance with good laboratory practice.
Refer in Westgard et al for identification and resolution for out of control situations .
Protein C in plasma is activated by a specific enzyme from Southern Copperhead Snake venom. The amount of activated protein C is determined by the rate of hydrolysis of thechromogenic substrate S-2366. The pNA release measured at 405 nm is proportional to Protein C results are reported in activity (%).
the Protein C level in the range from 0-120% of normal plasma.
Expected values
The Protein C levels measured in 95 healthy individuals, 53 males and 42 females, Protein C → APC A 405-490 nm
aged between 19 and 64 were in the range 70 -149%. Average 103%, SD 17%.
All Performance Characteristics included in this package insert are referred to 1. S-2366 6 mg
Cobas Mira . Detailed instrument settings including instructions for Protein C %
Lyophilized chromogenic substrate pyroGlu-Pro-Arg-pNA·HCl.
preparation of the reagents for a variety of automated instruments are 2. Protein C activator 1.2 U
Performance Characteristics
Lyophilized venom enzyme from Southern Copperhead Snake (Agkistrodon Calibration
Contortrix Contortrix), with bovine serum albumin (stabilizer) and Ciprofloxacin Limitations/interfering factors
A standard curve is obtained by analyzing different dilutions of calibrated normal plasma Addition of anti-human Protein C antibodies to normal plasma inhibits >98% of the in water to obtain levels 0, 25, 50, 75 and 100% activity.
amidolytic activity. Heparin levels ≤ 3 IU/mL do not interfere in the assay. Aprotinin Avoid contact with skin and eyes (S24/25).
Microplate method
inhibits activated Protein C. A low Protein C activity in aprotinin treated patients is thus Dilution of samples and controls
Wear suitable protective clothing (S36).
Sample blank activities should be determined and subtracted in plasmas from patients on Streptokinase therapy as well as in plasmas where contact factor activation is This product is for in vitro diagnostic use.
suspected, such as in plasmas from DIC patients or from individuals on oral Reagent preparation
contraceptives where cold activation may occur. The sample blank activity is determined For microplate and test tube techniques reconstitute each reagent with 7.2 mL of water by substituting the Protein C activator with the same volume of sterile water.
(see REAGENTS 3.) Replace the stopper and swirl gently. Make sure of the complete Microplate Application
reconstitution of the product. Keep reagent at 15-25°C for 10-30 min and invert before Add diluted samples/controls/standards to wells The repeatability of the method has been determined on Cobas Mira.
NOTE: Other reagent reconstitution volumes may apply for automated methods.
Reagents are not interchangeable between lots.
Reagents required but not provided
3. Deionized water, filtered through 0.22 µm or NCCLS type II water .
4. Human normal plasma with a Protein C activity of 100%.
n=number of replicates per series, N=number of series Different standard dilutions are obtained by diluting normal plasma with water (seeREAGENTS 3). A lyophilized preparation of human normal plasma, calibrated Read the absorbance at 405 nm. Dual wavelength mode reading (405 and 490 nm) is These results have been obtained at Chromogenix laboratories and should be against International standard for Protein C is recommended.
preferable to compensate for differences between microplate wells.
considered as examples only. Other laboratories are requested to establish their own 5. Acetic acid 20% or citric acid 2%.
Refer to LIMITATIONS/INTERFERING FACTORS for information regarding sample Materials required but not provided:
— Spectrophotometer 405 nm (405 nm and 490 nm for microplate procedure) Test tube method
The assays show a strong correlation with Coatest Protein C: % PC (microplate) = 8.2 + 0.99x (Coatest) % PC (Cobas Mira) = -3.7 + 0.98x (Coatest) *NOTE: Do not use microplates intended for coating! Recommended measuring range
Storage conditions and stability
Refer to LIMITATIONS/INTERFERING FACTORS for information regarding sample The relationship between released pNA, measured as absorbance at 405 nm, and the The sealed reagents are stable at 2-8°C until the expiry date printed on the label.
level of Protein C is linear in the 0-120% range of normal plasma.
Detection limit
Stability after reconstitution: 3 months at 2-8°C in the original vial.
The assays allow detection of at least 5% Protein C.
Stability after reconstitution: 3 months at 2-8°C in the original vial.
WARNING: Do not use reagents beyond the expiry date printed on the package label.
Discard if the substrate solution appears yellow.
Cobas Mira ∆Abs per 1% Protein C activity: 0.0013 ∆Abs
Microplate method: 2 x 144 Test tube method: 2 x 36
Instrumentation Laboratory Company - Lexington, MA 02421-3125 (USA)Instrumentation Laboratory SpA - V.le Monza 338 - 20128 Milano (Italy) Bibliography / Literatur / Bibliografía / Bibliographie / Bibliografia /Bibliografia / Litteratur / Litteraturförteckning /
1. STOCKNER K et al. Protein C activators in snake venoms. Behring Inst Mitt 79, 37- 2. EXNER T and VAASJOKI R. Characterisation and some properties of the Protein C activator from Agkistrodon Contortrix venom. Thromb Haemost 59, 40-44 (1988).
3. PABINGER I. Clinical relevance of Protein C. Blut 53, 63-75 (1986).
4. VINAZZER H and PANGRAZ U. Protein C: Comparison of different assays in normal and abnormal plasma samples. Thromb Res 46, 1-8 (1987).
5. WENDEL H P et al. Aprotinin in therapeutic doses inhibits chromogenic peptide substrate assays for Protein C. Thromb Res 74, 543-548 (1994).
6. National Committee for Clinical Laboratory Standards. Specifications for reagent water used in the clinical laboratory, NCCLS Approved Standard: ASC-3.
7. National Committee for Clinical Laboratory Standards.
Collection, transport and processing of blood specimens for coagulation testing andperformance of coagulation assays, NCCLS Document H21-A2; vol. 11 No. 23.
8. ZUCKER S, CATHEY M H, WEST B. Preparation of Quality control specimens for coagulation. Am J Clin Pathol 53, 924-927 (1970).
9. WESTGARD J O, BARRY P L. Cost-effective quality control: Managing the quality and productivity of analytical process. AACC press (1988).
Symbols used / Verwendete Symbole / Símbolos utilizados / Symboles utilisés / Simboli impiegati / Símbolos utilizados / Anvendte symboler / Använda Symboler /
Instrumentation Laboratory Company - Lexington, MA 02421-3125 (USA)Instrumentation Laboratory SpA - V.le Monza 338 - 20128 Milano (Italy)


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