WORTHAM LABORATORIES, INC. DRAFT Thrombin Reagent

Transcription

1 WORTHAM LABORATORIES, INC. DRAFT Thrombin Reagent Intended Use Wortham Laboratories Thrombin Reagent is intended for thrombin to convert fibrinogen in the quantitative determination of fibrinogen in plasma samples. Summary and Principle The thrombin clotting time fibrinogen assay is based on the method described by Clauss. 1 In the presence of high concentrations of thrombin, the time required for clot formation in dilute plasma is inversely proportional to the fibrinogen concentrations. Reagents IVD For in vitro diagnostic use Composition: A frozen liquid preparation, of bovine thrombin containing 100 IU ml, 0.1% sodium azide, and stabilizers. Store unopened bottles at - 2 C. Thaw and mix well before using, and when stored at 2-4 C, the reagent is stable for 30 days. Stability studies have shown that Thrombin reagent can go through 30 repetitive freeze-thaw cycles before showing evidence of deterioration, and is stable for 1 year at - 2 C. Mix before each use. Provide some mechanism, such as a magnetic stirrer, to maintain adequate suspension during use. Warning: Thrombin Reagent contains sodium azide. Sodium azide under acidic conditions yields hydrozoic acid, and extremely toxic compound. Azide compounds should be flushed with large volumes of water. Those precautions are recommended to avoid deposits in metal pipes in which explosive conditions may develop. Specimen Collection One part 3.2% citrated solution and 9 parts whole blood is recommended for coagulation assays. Avoid hemolysis and contamination by tissue fluids. Samples that have less than 90% of the expected fill volume should be rejected. Centrifuge blood for 15 minutes at 2500xg. Test within 2 hours if samples are held at room temperature, C. 2 Procedure Materials Provided: Normal Fibrinogen Control, 1 x 5 ml bottle Thrombin Reagent, 1 x 5 ml bottle Fibrinogen Buffer, 4 x 100 ml bottle Note: Thrombin Reagent, Normal Fibrinogen Control, and Fibrinogen Buffer can also be purchased separately. Material Required But Not Provided: Fibrinogen Reference Plasma Precision pipettes: 0.1 ml and 0.2 ml Serological pipettes Test tubes Abnormal controls, such as Wortham Laboratories Low Fibrinogen Control Plasma

2 The Fibrinogen Assay Set and individual components are suitable for use with manual and mechanical methods of end-point clot detection. For manual and mechanical test methods: 1. Prepare a minimum of five (5) serial dilutions of the Fibrinogen Reference Plasma in Fibrinogen Buffer. Dilute plasma at least 1:3 to minimize interfering factors Dilute Normal fibrinogen quality control and patient samples 1:10 in Fibrinogen Buffer. 3. Prewarm 0.2 ml of each dilution to 37 C for 4 minutes. 4. Add 0.1 ml of Thrombin reagent to prewarmed dilution and time clot formation. Do not prewarm thrombin. 5. Establish a calibration curve from the clotting time of the serial dilutions of the Fibrinogen Reference Plasma. Determination of the fibrinogen result is made from reading the clotting time in seconds from the calculation curve. The frequency of curve preparation is partially determined by the method of clot detection used. Always prepare a new curve with each change in reagent lots, instrumentation, or when controls no longer fall within established ranges. 3 Results 1. Plasma diluted 1:10 represents 100% of the assayed value. The dilution factor indicates the relationship between the 1:10 dilution and other dilutions. For Example Only: Control = 286 mg/dl fibrinogen (Each laboratory must prepare curves with their reagents and instrumentation). Dilution Fibrinogen (mg/dl) Clotting Time log (mg/dl) log (sec) Mean (sec) 1:3 286 :3 = :5 286 :5 = : :10 = : :15 = : :30 =

3 Log Fibrinogen (mg/dl) 2. Calculate the mean of duplicate clotting time to the nearest 0.1 second. Use all five of the calibrator points to construct a log-log curve that test plots fibrinogen concentration vs clotting time. Draw a straight line of best fit. Constructing the curve with only the most linear points will produce the best recovery on control and patient samples. 3. Find the clotting time of quality control and patient samples on the curve and read the corresponding fibrinogen value. If clotting time for the 1:10 dilution falls outside the linear curve, prepare 1:5 or 1:20 dilutions as needed. If the sample is diluted 1:5, divide the result from the standard curve by 2; if the sample was diluted 1:20, multiply the curve result by 2 to get the final result. Limitations Interfering substances: EDTA and heparin are unsuitable anticoagulants. Oral contraceptives, estrogen, pregnancy, vitamin K, lipids, tissue extracts, and many classes of organic compounds. 6,7,8,9 Avoid patient samples that are hemolyzed and icteric, which may be inappropriate for each end point detection methods. 4 Acute inflammation reactions can elevate circulating fibrinogen. 4 High Fibrinogen Degradation Products (FDP) may prolong clotting times when the fibrinogen level is below 150 mg/dl. 4 Heparin does not interfere with therapeutic levels. However, very high heparin levels may cause low fibrinogen results. Batroxobin enzymes can be substituted for thrombin in this assay if heparin interference is suspected. 4 High paraprotein levels, thrombin antibodies, and drugs that activate the fibrinolytic system can interfere with fibrinogen assays. 4 Patients with qualitative fibrinogen abnormalities, the thrombin clotting time assay may indicate decreased fibrinogen. The quantitative results may be normal on these same samples if tested by 3,4 other methods.

4 Technique: The ratio of blood to sodium citrate anticoagulant Lipem K is 9:1. The sample should only contact nonwettable surfaces. The Fibrinogen Set and individual components are designed to work at 37 C. Ensure that all heating elements are functioning properly. Expected Values Laboratories should establish a normal control interval for fibrinogen measurement. Generally, the normal control is mg/dl. 5 Performance Characteristics Accuracy: Three lots of normal and low fibrinogen plasmas were tested in singlicate with Wortham Laboratories reagents on the BBL Fibrometer. A summary of the results follows: Control Dilution n Mean (sec) SD Normal 1: Low 1: Precision: Precision of the Normal and Low Fibrinogen Control result is dependent on many factors, such as the instrument, technique and the reagent used. A 1:6 and 1:8 dilution of 3 lots of the Normal and Low Fibrinogen Control respectively, were made and assayed in singlicate on the BBL Fibrometer. A summary of the results follow: Control n within-run n run-run Normal % CV % CV Low % CV % CV Reportable Range/Linearity: The Thrombin Reagent (100IU/ml) when diluted %, demonstrated linearity from 100 IU/ml to 10 IU/ml. Each laboratory should establish their own linearity using instrumentation, blood collection methods, and testing techniques used in the laboratory. References 1. Clauss, A. Acta Haemat; 17: , National Committee for Clinical Laboratory Standards. Collection, transport, and processing of blood specimens for coagulation testing and performing of coagulation assays. 2 nd edition. Approved quidelines. VCCLS Document H30-A, Villanova, PA, Musgrane, K.A.. Bicks, R.L., Quality Assurance in the Hemostasis Laboratory. In Bicks, R.L. et al editors; Hemotology: Clinical and Laboratory Practice; Vol 2, pp Mosly St. Louis, National Committee for Clinical Laboratory Standards; Procedure for determining fibrinogen in plasma. Approved guideline. NCCLS Document H30-A. Villanova, PA, Clinical Diagnosis and Management by Laboratory Methods; W.B. Saunders Company, 19 th edition; Scarabin, P., Alhenc-Gelss, M., etc; Arterio. Thromb, Vas, 1997; 17: Hemo Sense Technical Bulletin Barry, E.I., Triplett, D.A., Koepke, J., J Clin Path, 74:569, Wujastyb, J., Triplett, D.A.; Pathologist 37:398, 1983.

5 Ordering Information Cat No. Description Contents Thrombin reagent 100 IU/ml 10 ml Fibrinogen Control, Normal 10 ml Fibrinogen Buffer 100 ml WORTHAM LABORATORIES, INC. DOC: /07 CHATTANOOGA, TN USA Catalogue No