The Prothrombin Time UMC Clinical Laboratory Sciences Hematology CLS 322

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The Prothrombin Time UMC Clinical Laboratory Sciences Hematology CLS 322 Powered By Docstoc
					Hematology CLS 322 Student Laboratory Prothrombin Time (PT)
Principle The PT is the time needed for plasma to clot after adding calcium and tissue factor. The complex formed between plasma Factor VII and tissue factor, in the presence of calcium, directly activates Factor X. This assay measures the coagulation factors of the extrinsic pathway with the exception of Factor XIII. Measures: Factors III, VII, X, V, II, I Specimen Collection & Preparation Blood should be anticoagulated with sodium citrate in the ratio of one part anticoagulant (3.8% Na Citrate) to nine parts freshly collected blood. Centrifuge the blood as soon as possible after collection for a minimum of 10 minutes at 1000 rcf. Remove plasma to a plastic tube and keep refrigerated until ready to test. Patient plasma should be tested within two hours of collection and neither patient nor control plasma should stand at 37C for more than five minutes. Reagents Thromboplastin-C Distilled Water Equipment and Supplies Fibrometer Fibrometer Heating Block Pipet Pipet Pipet Pipet 20 ml 0.1 ml 0.2 ml 1 ml volumetric

KimWipes Reaction Cups Stopwatch

PT Procedure 1. Reconstitute Thromboplastin-C reagent according to label on vial. 2. 3. 4. 5. 6. Reconstitute Controls (Levels I-III) with 1 ml of distilled water. Label one small test tube "Thromboplastin-C". Pre-incubate 2 ml of Thromboplastin-C for 5 minutes in the fibrometer heating block. Place 4 reaction cups in the fibrometer heating block. Fill one reaction cup with distilled water and place in a holding well adjacent to the fibrometer probe. Pipet 0.1 ml of patient plasma into the first reaction cup. Begin stopwatch as plasma is being pipetted into the reaction cup. At 30 second intervals pipet 0.1 ml of patient plasma in each of the remaining reaction cups. Shortly prior to 1 minute time elapsed, place first reaction cup into the reaction well. Aspirate 0.2ml Thromboplastin-C into the pipet. Switch button on pipet to "ON" and dispense 0.2 ml of Thromboplastin-C into the reaction well cup. [Or leave "OFF" and depress fibrometer timing bar as you dispense the thromboplastin.] Lift probe when timer stops. Clean probe electrodes with distilled water and KimWipes. Wipe straight down. Record results from digital readout. Depress fibrometer reset button. At 30 second intervals dispense 0.2 ml of Thromboplastin-C into each of the remaining reaction cups after having placed them in the reaction well. [Turn pipet switch "OFF" when aspirating and back "ON" to dispense.] Repeat Steps 10-15 for each of the remaining reaction cups. Note: All prothrombin time determinations must be performed in duplicate and the results averaged.

7.

8. 9.

10.

11. 12. 13. 14. 15.

16.

Matching Criteria for the PT 1. Precision: (reproducibility between duplicate samples) within 1 second. 2. Accuracy: (closeness to UMC Laboratory results) within 2 seconds. Reference Range 10-14 seconds depending upon anticoagulant and thromboplastin reagent and sensing. Interpretation The Prothrombin Time is prolonged in deficiencies of fibrinogen, Factor V, Factor VII or Factor X. The test is often used to monitor coumadin therapy.

Activated Partial Thromboplastin Time (APTT)
Principle The APTT is the time needed for plasma to form a fibrin clot following the addition of calcium and a phospholipid reagent. A contact activating reagent is included in the phospholipid reagent. The assay measures all of the coagulation factors except Factor VII and Factor XIII. Measures: Factors XII, XI, IX, VIII, X, V, II, I Reagents Actin Calcium Chloride Distilled Water Equipment and Supplies Fibrometer Fibrometer Heating Block Pipet (0.1 ml) Pipet (1.0 ml) volumetric KimWipes Reaction Cups Stopwatch Procedure 1. 2. Reconstitute Controls (Levels I - III) with 1 ml of distilled water. Label one small test tube "CaCl". Place it in the fibrometer heating block. Pre-incubate 1-2 ml of CaCl2 for at least 5 minutes in the block. Fill a reaction cup with distilled water and place in a holding well adjacent to the fibrometer probe. Place 4 reactions cups in the fibrometer heating block. Add 0.1 ml of Actin to each of the reaction cups. Pipet 0.1 ml of patient plasma into the first reaction cup. Begin stopwatch as plasma is being pipetted into the reaction cup. At 1 minute intervals pipet 0.1 ml of plasma in each of the remaining reaction cups. Shortly prior to 3 minutes time elapsed place first reaction cup in reaction well. Aspirate 0.1ml of CaCl2 into the pipet.

3. 4. 5. 6.

7. 8.

(APTT Procedure, continued) 9. Switch button on pipet to "ON" and dispense 0.1 ml of CaCl2 into the reaction well cup. [Or leave "OFF" and depress fibrometer timing bar as you dispense the CaCl2.] Lift probe when timer stops. Clean probe electrodes with distilled water and KimWipes. Record results from digital readout. Depress reset button. At 1 minute intervals (1.5 min for abnormals) dispense 0.1 ml of CaCl2 into each of the remaining reaction cups after having placed them in the reaction well. Repeat steps 9-14 for each of the remaining reaction cups. Note: All activated partial thromboplastin determinations must be performed in duplicate and the results averaged.

10. 11. 12. 13. 14.

15.

Matching Criteria 1. Precision: (reproducibility between duplicate samples) within 2 seconds for 20-30 second range within 3 seconds for 40-50 second range within 4 seconds for 60 and above second range 2. Accuracy: (closeness to UMC Laboratory results) same as Precision criteria.

Reference Range - less than 40 seconds

Interpretation Prolonged values can occur with any factor deficiency with the exception of Factor VII and Factor XIII. The APTT is often used for the monitoring of heparin therapy.
PT&PTT.DOC Wednesday, November 04, 2009


				
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