HEMOCLOT THROMBIN INHIBITORS # CK002K Clotting assay for the quantitative measurement of hirudin and other direct thrombin inhibitors in plasma
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1 HEMOCLOT THROMBIN INHIBITORS # CK002K Clotting assay for the quantitative measurement of hirudin and other direct thrombin inhibitors in plasma For in vitro use only For research use only Last revision : 08/06/ Intended use The HEMOCLOT THROMBIN INHIBITORS kit is proposed for the quantitative measurement of hirudin and other direct thrombin inhibitors in plasma, with a clotting method based on the inhibition of a constant and defined concentration of thrombin. 2. Assay principle For measuring hirudin, or any other direct thrombin inhibitor, in plasma, first, the diluted tested plasma is mixed with a normal pooled human plasma. Clotting is then initiated by adding a constant amount of highly purified human thrombin, in the α form. The clotting time measured is directly related to the concentration of hirudin in tested plasma. 3. Assay sample Plasma prepared form citrated anticoagulated blood, or any biological sample where hirudin (or any other direct thrombin inhibitor) activity must be measured. The assay can also be performed on EDTA anticoagulated plasma. 4. Reagents Each kit contains: R1: 3 vials of 1 ml of normal pooled citrated plasma, lyophilised (Reagent 1). R2: 3 vials of 1 ml of highly purified human calcium thrombin (in the α form), stabilised with additives, and lyophilised (Reagent 2). Nota : Use only reagents from a same kit lot. Do not mix reagents from kits with different lots. 5. Reagents and material required, but not supplied Pipettes with dispensing volumes of 20 µl, 50 µl and 100 µl. Pipette with a variable dispensing volume from 50 µl to 1,000 µl. Semi-automatic or automatic coagulation instrument, or fibrometer of electromagnetic water bath. Distilled water 9 g/l NaCl physiological saline solution. Normal plasma (or normal pooled plasma) or calibration kits for hirudin testing (#SC020K and SC020L). 6. Reagent preparation and stability In their original package, and before any use, when stored at 2-8 C, the reagents are stable until the expiration date printed on the kit. Preparation: R1: Normal pooled plasma: Restore the normal pooled plasma vial with 1 ml distilled water; let for 15 min. at room temperature; mix gently until complete dissolution of the content. This plasma is stable for: 24 hours at room temperature (18-25 C). 48 hours at 2-8 C. 2 months, frozen in its original vial or in a plastic tube, at 20 C or below (before use, thaw in a water bath at 37 C for at least 15 min.). Nota: Plasmas used for the pool preparation were tested with registered methods and found negative for HIV antibodies, HBs Ag and HVC antibodies. However, no assay may warrant the total absence of infectious agents. Any product of human origin must then be handled with all the required cautions, as being potentially infectious. R2: Human calcium Thrombin: Restore the vial with 1 ml distilled water and let for 15 min. at room temperature; mix gently until complete dissolution of the content. The restored human calcium thrombin is stable for at least: 24 hours at room temperature (18-25 C). 48 hours at 2-8 C. 2 months frozen in its original vial or in a plastic tube at 20 C or below (before use thaw in a water bath at 37 C for at least 15 min.). Nota: Thrombin is prepared by activation of purified prothrombin extracted from human plasma. Plasmas used for prothrombin preparation were tested with registered methods and found negative for HIV antibodies, HBs Ag and HVC antibodies. However, no assay may warrant the total absence of infectious agents. Any product of human origin must then be handled with all the required cautions, as being potentially infectious. 7. Sample collection and preparation Blood (9 vol.) must be collected on 0.109M trisodium citrate anticoagulant (1 vol.); plasma supernatant is decanted following a 20 min. centrifugation at 2,500 g; citrated plasma must be tested within 8 hours when stored at room temperature (18-25 C), or can be used within 24 hours if kept at 2-8 C, or it can be frozen at 20 C or below for up to 6 months. Just before use, the plasma must be thawed for 15 min. in a water bath at 37 C. Thawed plasma must be used within 4 hours, at room temperature (18-25 C). 8. Tested plasma Tested plasma must be used diluted 1:8 (i.e. 100 µl of plasma and 700 µl of diluent) or 1:20 (i.e. 100 µl of plasma and 1900 µl of diluent) in 9 g/l physiological saline solution, or in Owren Koller buffer, according to the assay variant used: - 1:8 for the low concentration range - 1:20 for the high concentration range D /CK/002K
2 9. Protocol According to the expected hirudin concentration in tested plasma, the low range or the high range protocol must be used. Nota: Calibration of the assay can be performed with normal plasma supplemented with hirudin or with the Plasma Hirudin Standard Low (#SC020K) and Plasma Hirudin Standard High (SC020L) kits (low or high range). When other direct thrombin inhibitors are used, a calibration curve can be prepared by spiking the inhibitor in normal plasma. Alternatively, inhibition can be expressed as hirudin equivalent. 9.1 Low hirudin concentration range: This protocol is proposed for testing hirudin in citrated plasma, and is used for hirudin concentrations of about 1 µg/ml. It must be adapted to the thrombin inhibitor used. Calibration curve: The plasma Hirudin Standard kit, low range (SC020K), can be used for preparing the calibration curve. If a homemade calibration is used, prepare 1 ml of normal citrated plasma (or a normal plasma pool) containing 2 µg/ml of hirudin, using preferably the hirudin used for patient's treatment. Using this preparation, or with the calibration kit, the calibration curve is obtained as follows: Hirudin µg/ml Normal plasma with 2 µg/ml hirudin (µl) Normal plasma (µl) g/l physiological saline solution (µl) The calibration plasmas, already diluted 1:8, are obtained. With the Plasma Hirudin Standard Low kit (#SC020K), the same calibration range can be prepared using the lyophilised plasmas at 0 and 2 µg/ml hirudin concentration. Tested plasma: Tested plasma must be diluted 1:8 (i.e. 100 µl of plasma and 700 µl of diluent) in Owen Koller type buffer or in a physiological saline solution (9 g/l sodium chloride). Assay: Preincubate thrombin at 37 C. In a test tube, or in a cuvette, introduce: 100 µl of normal pooled plasma (Reagent 1) 50 µl of calibration solution or of tested plasma diluted 1:8. Incubate for 1 Min. at 37 C, then introduce: 100 µl of thrombin (Reagent 2), preincubated at 37 C, starting the stop watch. Record the clotting time High hirudin concentration range: This protocol is used for hirudin concentrations in plasma of about 4 µg/ml. Calibration curve: The plasma Hirudin Standard kit, high range (SC020L), can be used for preparing the calibration curve. If a homemade calibration is used, prepare 1 ml of normal citrated plasma (or a normal plasma pool) containing 5 µg/ml of hirudin, using preferably the hirudin used for patient's treatment. Using this preparation or the calibration kit, the calibration curve is obtained as follows: Hirudin µg/ml Normal plasma with 5 µg/ml hirudin (µl) Normal plasma (µl) g/l physiological saline solution (µl) The calibration curve, already diluted 1:20, is obtained. With the Plasma Hirudin Standard high kit (#SC020L), the same calibration range can be prepared using the lyophilised plasmas at 0 and 5 µg/ml hirudin concentration. Tested plasma: Tested plasma must be diluted 1:20 (i.e. 100 µl of plasma and 1900 µl of diluent) in Owen Koller type buffer or in a physiological saline solution (9 g/l sodium chloride). Assay: Preincubate thrombin at 37 C. In a test tube, or in a cuvette, introduce: 100 µl of normal pooled plasma (R 1) 50 µl of calibration solution or of tested plasma diluted 1:20. Incubate for 1 Min. at 37 C, then introduce: 100 µl of thrombin (R 2), preincubated at 37 C, starting the stop watch. Record the clotting time. 10. Expression of results On a linear graph paper, plot on abscissae the hirudin (or other direct thrombin inhibitor) concentrations and on ordinates the corresponding clotting times. On the calibration curve obtained, interpolate directly the corresponding hirudin concentration for the tested plasma. 11. Interferences and characteristics: The HEMOCLOT THROMBIN INHIBITORS reagents do not contain heparin inhibitors. Presence of heparin or of other anti-thrombin substances, different from the one to be tested, may interfere in the assay and prolong the clotting time. Therefore, any anti-thrombin activity present in the tested plasma is not masked and this allows avoiding any underestimation of an existing hypocoagulability, as the result from the presence of an anti-thrombin substance. 12. Complementary Information : The assay is optimised for hirudin concentration, expressed in µg/ml. The specific activity for hirudin drugs can vary from product to product or from lot to lot (from < 10,000 ATU*/mg to > 15,000 ATU*/mg). The curves are constructed respectively to the hirudin concentration. If a calibration by hirudin activity, expressed in ATU*/mL, is needed, or when a different thrombin inhibitor is used, the user must take into account the specific anti-thrombin activity of the preparation used. *ATU: Anti-Thrombin Unit D /CK/002K
3 a link between research and specialized applications for the biomedical diagnosis un lien entre recherche et applications spécialisées pour le diagnostic biomédical ZAC Neuville Université 155 Rue d Eragny Neuville sur Oise (France) Tel: Fax: Web site: MATERIAL SAFETY DATA SHEET 1. IDENTIFICATION: Name: HEMOCLOT THROMBIN INHIBITORS Product number: CK002K Kit composition: - 3 vials of normal human pooled citrated plasma, lyophilised (powder) - 3 vials of human thrombin, lyophilized (powder) Manufacturer: HYPHEN BioMed ZAC Neuville Université 155 rue d Eragny Neuville sur Oise (France) Tel: Fax: APPLICATION/INTENDED USED: This kit should be used only by suitable trained personnel, wearing the appropriate laboratory protective clothing, particularly eye and skin protection. It must be used according strictly to the instructions of package insert, and for the indicated purpose. The kit should be stored within the manufacturer's box at the specified temperature (2-8 C) and handled as per pack insert instructions. The information provided in this material safety data sheet is believed to be correct and does not purport to be all inclusive and shall be used only as guide. HYPHEN BioMed and its appointed agents/distributors or OEM contractors shall not be held liable for any damage resulting from or from contact with the products included in the kit. 3. COMPOSITION/INFORMATION ON INGREDIENTS: Normal human citrated pooled plasma. Freeze dried powder contains human plasma (about 80 mg desiccated), hepes (<10mg), glycine (<25mg), ciprofloxacine (<0.001%). Page 1 / 4 Creation: Feb 07
4 Human thrombin: Contains human thrombin (<1 µg), Glycine (<20mg), Bovine Serum Albumin (<15mg), calcium chloride (<2mg), sodium chloride (<10mg), hepes (<10mg), calcium gluconate (25mg), PEG6000 (<12 mg) and ciprofloxacine (<0.001%). 4. TOXICITY HAZARDS: Reagents Chemical compounds CAS N % or weight Classification LD50 (oral) Human Plasma Human plasma Glycine Ciprofloxacine > 75 % < 25% < % S:22-24/25 Thrombin Thrombin Glycine BSA Calcium Chloride Sodium Chloride Calcium gluconate PEG6000 Ciprofloxacine 5. HEALTH HAZARDS DATA: < 0.001% < 20% < 15 % < 2.5% < 15 % < 25 % < 15 % < 0.001% S:22-24/25 R R S22-24/25 R36/ LD50(rats) 42.2 mg/kg LD50(rats) 3.75 g/kg LD50 (rats) >50g/Kg All the above listed chemicals or biologicals may be harmful by inhalation, ingestion, or skin adsorption. Nasal irritation, eye reddening, and allergic reactions may result from overexposure. First aid: - If swallowed, wash out mouth with water provided person is conscious. Medical advice is necessary. - In case of contact with eyes, flush with copious amounts of water, for at least 15 minutes. Assure adequate flushing by separating the eyelids with fingers. - In case of inhalation, remove victim to fresh air, and seek medical advice. - In any case of overexposure, call a physician. Viral Safety: - Thrombin is extracted from human plasma, and human plasma have been tested for HIV, HBs:Ag and HVC, with registered methods. However, all human sourced material should be treated as potentially hazardous and the appropriate handling and disposal procedures must be adhered to. - BSA was prepared from bovine plasma; which was tested for the absence of infectious agents, and collected from animals free from BSE. However, no test may totally exclude the absence of infectious agents. As any product of bovine origin, this reagent must be used with all the cautions required for handling a material potentially infectious Page 2 / 4 Creation: Feb 07
5 6. FIRE AND EXPLOSION HAZARD DATA: Flammability: Restored solutions are aqueous and non-flammable. Lyophilised vial is non flammable, only carton boxes, dry chemical, interiors inserts are flammable. Extinguishing Media: Carbon dioxide, dry chemical powder or appropriate foam. Special fire fighting procedures: Wear self-contained breathing apparatus and protective clothing to prevent contact with skin. 7. REACTIVITY DATA: Stability: Stable Hazardous combustion or decomposition products: Carbon monoxide, carbon dioxide, nitrogen oxide. Hazardous polymerisation: Does not occur. 8. SPILL, LEAK AND DISPOSAL PROCEDURES: Steps to be taken if material is released or spilled: - Sweep up, place in a bag and hold for waste material. Avoid raising dust. Ventilate area and wash spill site after material pickup is complete. - Waste disposal method: Comply with all federal, state and local environmental regulations on waste handling and disposal. 9. ECOLOGY INFORMATION: Do not empty plasma or thrombin into waters or drains. Comply with state and local environmental regulations. Usually wasted biological material is stocked in hermetic specific containers for incineration by specialized companies. 10. PRECAUTIONS TO BE TAKEN IN HANDLING AND STORAGE: Must be only used by suitable trained and informed personnel. Wear chemical resistant gloves, chemical safety goggles and protective laboratory clothing. 11. TOXICOLOGICAL INFORMATION: This reagent is intended for in vitro research use only, by experienced and suitably trained personnel. There is no special risk when used in these conditions. Products may be toxic following skin or eye contact, inhalation or ingestion. For toxicity of components, refer to chapters 4 and 5. Page 3 / 4 Creation: Feb 07
6 12. WASTE DISPOSAL CONSIDERATIONS: Any waste product or reagent must be discarded according to local considerations. Do not reuse vials or containers. Biohazard risk is mentioned on the insert. 13. PHYSICAL AND CHEMICAL PROPERTIES: Lyophilized powder. It does not present any specific physical or chemical reactivity (stable compound). 14. TRANSPORT AND STORAGE INFORMATION: This reagent must be shipped adequately packaged and protected from any break during transportation. It can be shipped at ambient temperature for a short period, not exceeding 7 days. It must be stored in a cold room at 2-8 C upon receipt. No special regulation for transporting this product. General rules for in vitro research should apply. Local, State and Federal regulations for this kind of product must be respected. The reagent must be stored in an appropriate refrigerated area, specifically dedicated for in vitro research kits. All the storage constraints are indicated on the labels and on the insert. 15. QUALITY MAGEMENT INFORMATION: This reagent is designed, manufactured, controlled and followed according to the quality management system (based on ISO 9001:2000 and ISO 13485:2003) developed by HYPHEN BioMed. 16. OTHER INFORMATION: For in vitro research use only. Disclaimer: The information reported on this MSDS is believed to be accurate and represents the best information available to us. However, we make no warranty of merchantability or any other warranty, expressed or implied, with respect to such information, and we assure no liability resulting from its use. Users should make their own investigations to determine the suitability of the information for their particular purposes. Page 4 / 4 Creation: Feb 07
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