pt, ptt, fib, inr by acl 7000 - welcome to project...

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Author: Penny Stevens Document Number: Equ21-01Effective (or Post) Date: 13-Jan-09

Review History Date of last review: 27 July 2010Reviewed by: Heidi Hanes

SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering their use in other applications. If you have any questions contact SMILE..

Copy _____ of ___ Effective Date:

HEMATOLOGY SECTION

G. COAGULATION

G.1. ACL 7000 ANALYZER - PROTHROMBIN TIME (PT), ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) AND INR TESTING

G.1.1. PRINCIPLE:

1. The ACL 7000 analyzer is a fully automatic microcomputer-controlled, microcentrifugal instrument capable of performing the following tests:

1.1. PT-FIB (Prothrombin Time and Fibrinogen Level).

1.2. APTT (Activated Partial Thromboplastin Time).

1.3. PT-FIB/APTT (all three tests run simultaneously).

1.4. D-dimer.

1.5. Thrombin Time

1.6. Single Factors (II, V, VII, VIII, IX, X, XI, XII).

2. The ACL 7000 methodology utilizes spectrophotometry, which is based on the change of light scatter associated with the formation of a fibrin clot. The ACL method is not influenced by inhibitors and will reflect the true fibrinogen concentration

3. The following tests are performed at this hospital: PT (Prothrombin Time) - INR and APTT (Activated Partial Thromboplastin Time).

4. The Prothrombin Time (PT) test evaluates the extrinsic and common pathway of clotting which involve factors II, V, VII, and X.

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CoagulationG.1. ACL 7000 Analyzer - . Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), and INR Testing

4.1. The PT is performed by adding a source of tissue extract (typically from brain or lung) to the control or patient plasma, then adding an excess of calcium and measuring the time for clot detection optically.

4.2. The PT test is commonly used for the monitoring of coumadin drug therapy.4.3. Common causes of a prolonged PT test are vitamin K deficiency, liver disease,

specific coagulation deficiencies, Disseminated Intravascular Coagulation (DIC), circulating anticoagulants, presence of fibrin (or fibrinogen) split products, some dysproteinemias, and oral anticoagulant therapy.

5. The activated Partial Thromboplastin Time (aPTT or PTT) evaluates the activity of the coagulation factors involved in the intrinsic coagulation system which involves factors VIII, IX, XI and XII.

5.1. The PTT test is performed by adding a standardized phospholipid preparation as a platelet substitute (an activator) to the control or patient plasma, then adding an excess of calcium and optically measuring the time for clot detection.

5.2. The PTT test is commonly used for monitoring heparin drug therapy.

5.3. The PTT will be prolonged by intrinsic pathway factor deficiencies or by inhibitors, such as lupus anticoagulants, directed against the involved factors or complexes.

5.4. The PTT is also used to monitor low molecular weight heparin using Factor Xa. This test is sent to Bioscentia.

6. International Normalized Ratio (INR):

6.1. The ratio of the patient PT time to that of the geometric mean PT of the normal reference range is useful in monitoring patients receiving oral anticoagulant therapy. However, different thromboplastins exhibit different sensitivities. Therefore, the PT ratio is affected by the reagent used and the method of clot detection. As a result, the INR was developed to improve the consistency of oral anticoagulant therapy. An international reference thromboplastin reagent has been developed for use in standardizing the PT ratio. Manufacturers of thromboplastins calibrate each lot number of this reagent against the standard WHO (World Health Organization) thromboplastin reagent. The results of this calibration are used to develop the International Sensitivity Index (ISI) for each lot of reagent. This number allows for calculation of the INR, which is calculated as follows:

6.1.1. INR EQUATION:

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ISI Patient PT expressed in seconds INR = Geo. Mean PT of Normal Reference Range Expressed in seconds

6.1.2. “Patient PT expressed in seconds” is the PT as resulted by the ACL.

6.1.3 Geo. Mean PT of the Normal Patient Reference Range expressed in seconds is determined by our normal reference interval. This number is a constant that changes only if a new reference interval is established.

6.1.4 The ISI is provided by the thromboplastin reagent manufacturer. This value can be found on the package insert for the reagent and is lot specific. The ISI will change with each lot of reagent.

6.2. ACL 7000 Ratio Factor:

6.2.1 The ACL 7000 analyzer automatically calculates the INR value. However, in order for the calculation to be accurate, an INR ratio factor must be calculated and entered into the ACL. If the ratio is not calculated and input into the ACL, the geometric mean from the 100% point of the PT calibration curve will be used as the denominator in the INR calculation. This will result in a denominator change with every PT reagent calibration.

6.2.2. ACL INR ratio factor calculation:

(PT Calibration Geo. Mean)/ (Geo. Mean of Ref. Range PT) = ACL INR Ratio

6.2.2.1. The PT Calibration Geo. Mean is the geometric mean of the 100% point of the PT calibration curve.

6.2.2.2. The Geo. Mean of the normal patient reference range PT is currently 11.7. This value will change only if a new normal reference interval is established.

6.2.3 ACL INR ratio factor entry: Once the ratio has been calculated, it must be entered into the ACL through the following menus:

6.2.3.1. Press the PROG key and press the “” or “” keys to select SET UP and press ENTER.

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6.2.3.2. Select RATIO ADJUSTMENT and press ENTER. Press “” to continue.

6.2.3.3. Enter the ratio as calculated above (7.2.2) for PT and press ENTER. Do not change the values entered for APTT or TT. Both values must remain at 1.0. Press “<=” to return to the previous screen and save the ratio data that was entered.

6.3. ACL INR verification: After the ratio change, the ACL INR calculation must be verified by manual calculation. Verification is also required when the PT reagent is recalibrated, with the implementation of a new PT reagent lot, if a new PT reference range is established, if any factor in the calculation changes or, at a minimum, annually. All INR verifications and all PT reagent calibrations must be performed using the worksheet included in Appendix 3.

G.1.2. SPECIMENS:

1. Specimen Type:

1.1. The anticoagulant of choice for coagulation studies is 3.2% Sodium Citrate (Blue Top Tube).

1.2. Citrated plasma preserves the labile clotting factors V and VIII and is the most satisfactory for platelet aggregation studies.

1.3. Citrated plasma samples are also more sensitive to the effects of heparin and therefore are preferred for monitoring heparin therapy.

2. Specimen Collection:

2.1. Identify the patient. Collect supplies used for appropriate venipuncture technique.

2.2. Draw a clearing tube or a serum type tube prior to drawing the sodium citrate (blue top tube).

2.3. The ratio of blood to anticoagulant is critical for valid coagulation test results. The standard ratio for sodium citrate specimens is nine (9) parts of blood to one (1) part of anticoagulant, (9:1 ratio).

2.4. The order of collection is to draw the EDTA and heparin containing tubes AFTER the citrate coagulation specimen.

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2.5. Flush indwelling catheter lines with 5 mL of saline or 6-times the line volume (dead space volume of the catheter). Draw and discard the first 5 mL’s of blood prior to filling the coagulation tube. Do not collect coagulation tubes from IV lines that have been flushed with heparin. Heparin contamination will result in specimen dilution and erroneously elevated patient results.

3. Specimen Handling, Storage and Transportation:

3.1. Platelet Poor Plasma Check:

As soon as possible, centrifuge the sample for the appropriate time and speed as determined from a platelet poor plasma check.

3.1.1. Platelet poor plasma checks will be performed semi -annually to ensure that the centrifuge time and speed are sufficient to remove all of the platelets from the sample. It is performed as follows:

3.1.2 Set the centrifugation speed to 3,000 rpm and centrifuge a sample for 10 minutes.

3.1.3. Set the Cell-Dyn analyzer to background mode and perform a standard background check to ensure the reading for platelets is 0.00 K/uL.

3.1.4. Aspirate the plasma from the sodium citrate sample in the background mode on the Cell-Dyn instrument. The value must be less than <10.0 K/uL for 3 consecutive backgrounds. If it is not, increase the speed to 3,500 rpm and repeat the above procedures. If the platelet count is still too high, the centrifuge time can be increased as needed. The sufficient time and speed may vary based upon the centrifuge.

3.1.5. Document the centrifuge time and speed on the centrifuge maintenance log and on the centrifuge. Use the RPM and time determined from the platelet poor plasma check as the centrifuge minimum standard for all coagulation specimens. This standard applies only to the centrifuge upon which the platelet poor plasma check was performed.

3.2. Separate the plasma from the cells within 30 minutes after centrifugation of the sample and place the plasma sample in a plastic capped tube using a plastic transfer pipette.

3.3. If the specimen will be tested within two to four hours after collection, it may be kept at room temperature.

3.4. Freeze the specimen if it cannot be processed within four (4) hours after collection.

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3.4.1. Plasma can be frozen at -20ºC or lower for at 7 days or at -80ºC for six (6) months without loss of most factors.

3.4.2. Thaw rapidly in a 37ºC water bath. Remove the plasma sample as soon as it is thawed and perform the test immediately. Samples are viable for a maximum of two (2) hours at room temperature after they have been thawed.

3.4.3 Plasma samples cannot be re-frozen.3.5. Keep plasma samples capped at all times except when testing. This prevents the

loss of Carbon Dioxide (CO2) and a resultant pH change of the plasma. Changes in the pH of the samples can affect results by causing prolongation of clotting times.

3.6. Outlying Clinics will freeze specimens completely prior to shipping (with a transmittal list) unless a PT test ONLY is ordered. See note below.

NOTE: Specimens for PT testing from outlying clinics can be sent on transmittal list in an unopened sodium citrate tube and shipped at room temperature. The cap must be secured with parafilm and the sample must be annotated as “uncentrifuged & unopened” and include the collection time. PT’s are stable for 24 hours in an unopened sodium citrate tube. Samples requested for APTT are only viable for 4 hours. Centrifuge, separate, and freeze all APTT’s thoroughly prior to shipment.

4. The following specimens are not suitable for testing:

4.1. Clotted specimens

4.2. Lipemic, icteric or hemolyzed plasma samples

4.3. Under- or over-filled samples. Use the fill guide in appendix 5 to determine acceptability.

NOTE: Reject and request another specimen. Document all action taken in CHCS.

G.1.3. EQUIPMENT & REAGENTS

1. Equipment:

1.1. ACL 7000 Analyzer

1.2. Centrifuge

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1.3. Reagents reservoirs

1.4. Sample Tray

1.5. Sample cups (0.5mL or 2mL capacity)

1.6. Rotors

1.7. Pipettes (1mL and 10mL capacity)

1.8. Wooden applicators sticks

1.9. Plastic test tubes with caps

1.10. Plastic transfer pipettes

1.11. CHCS labels

2. Reagents:

2.1. Reference Solution

2.2. IL PT-FIB HS reagent

2.3. IL APTT-SP reagent

2.4. IL Calcium Chloride reagent

2.5. IL Normal and Abnormal Low controls

2.6. IL Calibration plasma

2.7. IL Sample Diluent

G.1.4. CALIBRATION:

1. The PT-FIB Calibration is required in order to perform patient testing. Do not perform patient testing without a valid PT-Fibrinogen calibration curve. The calibration curve is determined using the target fibrinogen value from the calibration plasma. The 100% point of the PT calibration curve is used to calculate the INR.

2. Calibration is necessary for every new reagent lot (thromboplastin), new lot of reference emulsion, new lot of rotors, when indicated by QC information, after major maintenance or service, when recommended by manufacturer or every six months.

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3. Equipment & Reagents:

3.1. IL Sample Diluent

3.2. 2 mLs of IL Calibration Plasma

3.3. Thromboplastin Reagent

3.4. 2 large 2 mL sample cups

3.5. Pipette (1 mL capacity)

4. Calibration Procedure:

4.1. Reconstitute 2 vials of lyophilized Calibration Plasma with one (1) mL of Reagent Grade Water each.

4.2. Invert gently to mix. Do not shake.

4.3. Maintain the calibration plasma at room temperature for 30 minutes before use. Calibration plasma is stable for two hours from reconstitution.

4.4. Review Calibration Data:

4.4.1. Access the CAL DATA PROGRAM: This program is used to verify current calibration values and cannot be edited.

4.4.1.1 Press the PROG key and using the “” or “” keys select CALIBRATION DATA and press ENTER.

4.4.1.2. Select PT-FIB using the “” or “” keys and press ENTER. This screen indicates the last saved calibration.

4.4.1.3. The screen “PT-FIB analytical calibration conditions” is displayed. Press “” key to see CAL DATA and GRAPHS.

4.4.1.4. Press PRT to print the calibration data for PT and FIB. Select “” key to continue and PRT to print the PT calibration graph. Select “” key again and press PRT to print the FIB calibration graph. Press “<=” 2-times to return to the CALIBRATION DATA menu. Keep the

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print out of the previous calibration for the Hematology Supervisor.

4.4.2. If “PT-FIB not calibrated” frame appears, the instrument is not calibrated. Proceed as follows with calibration:

4.5 PT-FIB Calibration:

4.5.1. Press PROG to return to the READY menu.

4.5.2. Select PT-FIB using the “” or “” and press ENTER.

4.5.3. The "Check" frame is displayed. Press “” to start the calibration cycle.

4.5.4. Enter the following data:

4.5.4.1. Enter the N.P. LOT Number. This is the lot number of the calibration plasma and is thromboplastin reagent specific.

4.5.4.2. Enter the Normal Plasma Fibrinogen value in mg/dL. This value can be obtained from the calibration plasma package insert.

4.5.4.3. Enter the reference emulsion lot number.

4.5.4.4. Enter the thromboplastin lot number. This is the lot number of the PT-Fibrinogen reagent.

4.5.4.5. Enter the ISI value. This value can be obtained from the PT-Fibrinogen package insert.

4.6. After all the reference values have been entered, press ENTER.

4.7. Empty the Thromboplastin reagent into reservoir number 1 (PT-FIB) of the instrument.

4.8. Empty the cephalin reagent into reservoir 2 (APTT-SP), and the calcium chloride reagent into reservoir number 3 (CaCl2 ) on the instrument.

4.9. Make sure that the IL Reference Solution level is sufficient to complete the calibration procedure (1.5cm of solution as a minimum).

4.10. Load a new rotor into the rotor holder.

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4.11. Fill one 2 mL sample cup with IL Sample Diluent and place it in the "DILUENT" position in the sample tray.

4.12. Fill another 2 mL sample cup with both vials of reconstituted IL Calibration Plasma and place it in the “POOL” position on the sample tray.

4.13. Press “” key to start the Calibration.

4.14. At the end of the analysis, the calibration data, to include results and graphics, will be printed and stored in the memory of the instrument.

4.15. The acceptable limits for the Calibration are as follows:

PT FIBRINOGEN

100% Standard CV <1.5% Normal Plasma Neat CV <8.0% 50% Standard CV <2.0% Normal Plasma 1 in 2 CV <12.0% 25% Standard CV <2.0% Normal Plasma 1 in 4 CV <12.0% R2 >0.980 R2 >0.980

4.16. The ACL 7000 Analyzer highlights any parameter that falls outside the specified limits. If this occurs, the CALIBRATION CAN NOT BE ACCEPTED, and the procedure must be performed again. Repeat the procedure as outlined above.

4.17. If the calibration is within acceptable limits, press “<=” to save and ENTER to confirm.

4.18. Keep the calibration data printout as part of the quality control and include it the monthly quality control packet.

G.1.5. QUALITY CONTROL:

1. The normal control and the abnormal control will be run on each shift if PT or PTT testing is performed.

2. Normal and abnormal controls are stable for 24 hours after reconstitution. Always ensure that the posted lot numbers and expiration dates match the vials of controls in use.

3. Properly label vials with the reconstitution date, expiration date and initials of tech.

4. Preparation of the controls:

4.1 Reconstitute the lyophilized controls with one (1) mL of Reagent Grade Water.

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4.2. Invert gently to mix. Do not shake.

4.3. Maintain controls at room temperature for 30 minutes before using.

4.4. Label each vial of control with the time, expiration date, and tech initials.

4.5. Lyophilized Normal and Abnormal controls remain stable until the indicated expiration date if kept at 2 to 8ºC. Do not use controls after the indicated expiration date.

5. Run the controls on the ACL 7000 in the same manner as patient samples.

6. Verify that the controls are within range by documenting and initialing the ACL 7000 control log and certify the results in CHCS.

7. The control results must be within the specified (SD) limits listed below.

8. Procedure for out of range control:

8.1. If one level of control is outside + 2SD but within + 3SD, and the other control or controls is or are within + 2SD patient results can be reported. This can only be allowed once every 20 days or runs. This is the 12S rule.

8.2. If one level of control is outside + 3SD, do not report patient results. Perform troubleshooting action: check reagent levels and expiration dates. Repeat the control again. If it is still outside + 3SD, reconstitute new controls, check instrument maintenance/cleaning, and repeat. Refer to ACL 7000 Preventive Maintenance SOP. This is the 13S rule.

8.3. If both levels of control are outside + 2SD, do not report patient results. Perform troubleshooting action: check reagent levels and expiration dates. .Repeat both controls. If they are still outside + 2SD, reconstitute new controls, check instrument maintenance/cleaning and repeat. Stop, reject run, and take corrective action. This is the 22S rule.

8.4. If results are still outside limits, notify the Hematology Supervisor immediately. Corrective action must taken before reporting patient results

8.5. Out of range controls will be recorded on the appropriate (normal/abnormal control) ACL-7000 Service Troubleshooting Log along with the corrective action taken.

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8.6. Each time that the control lot number changes, each control level will be run 20 times in order to establish laboratory ranges.

9. REAGENTS PREPARATION:

9.1. IL PT-FIB HS reagent:

9.1.1. Reconstitute each vial of IL RecombiPlastin with 8 mL’s of reagent grade water. Pipette exactly 8 mL’s.

9.1.2. Immediately after reconstitution, mix the contents by gentle inversion to ensure complete resuspension (DO NOT SHAKE)

9.1.3. Label the reconstituted reagent vial with the date and initials of tech placing reagent in use. Maintain the reagent at room temperature for 30 minutes before using. Mix well again before use (DO NOT SHAKE). A teflon coated magnetic stir bar must be inserted into the reagent reservoir for continuous mixing action.

9.1.4. Stability after reconstitution:

9.1.4.1. 8 Hours at 15ºC (on the ACL 7000 with continuous stirring)

9.1.4.2. 3 days at 2 to 8ºC (in the original bottle).

9.1.4.3. DO NOT FREEZE.

9.1.4.4. Lyophilized IL PT-Fib HS reagent remains stable until the indicated expiration dates if kept at the indicated temperatures. Do not use reagents after the expiration date.

9.2. IL APTT-SP Reagent:

9.2.1. The IL APTT-SP reagent comes ready for use. No further preparation is necessary. Shake the APTT-SP regent vigorously for 15 seconds before use. The APTT-SP and CaCL2 reagents are provided as a set and must be replaced as a set. Do not replace the APTT-SP reagent without also replacing the CaCL2 reagent. A Teflon coated magnetic stir bar must be inserted into the APTT-SP reagent vial only for continuous mixing action. Do not place a stir bar into the CaCL2

reagent.

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9.2.2. When a new bottle of reagent is opened, label it with the open date, expiration date and initials of tech placing reagent in use.

9.2.3. Opened vials are stable for 30 days if stored at 2 to 8ºC and for 5 days if kept on the analyzer. DO NOT FREEZE.

9.2.4. Unopened vials are stable until the indicated expiration date if kept at 2 to 8ºC. Do not use reagents after the expiration date.

9.3. IL Calcium Chloride:

9.3.1. The IL Calcium Chloride comes ready for use. No further preparation is necessary. Mix well by inversion before use.

9.3.2. When a new bottle of reagent is opened, label it with the opened date, expiration date and initials of tech placing reagent in use. The APTT-SP and CaCL2 reagents are provided as a set and must be replaced as a set

9.3.3. DO NOT FREEZE.

9.3.4. Reagent remains stable until the indicated expiration date if kept at 2 to 8ºC and for 5 days if kept on the analyzer. Do not use reagents after the expiration date.

G.1.6. PROCEDURE:

1. PT testing will be batched. Testing will be performed at the beginning of each shift or as necessary for STAT and ASAP requests.

2. Prior to performing coagulation testing on patient samples, verify the patient identification. Any discrepancy must be investigated before processing the specimen.

3. Check the specimen for clots, visually or using two wooden applicator sticks.

4. Ensure the specimen is labeled and label a sample cup, if one is to be used.

5. From the READY status of the instrument, select the desired test (i.e., PT-FIB, PT-FIB/APTT or APTT) by using “” or “” keys and press ENTER.

6. The "Check" frame is displayed.

7. Empty the PT-FIB HS (thromboplastin) bottle content into reservoir number 1 (PT-FIB) on the instrument.

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8. Empty the APTT-SP (cephalin) bottle content into reservoir number 2 (APTT-SP) on the instrument.

9. Empty the Calcium Chloride bottle content into reservoir number 3 (CaCl2) on the instrument.

10. Ensure that the Reference Solution volume is sufficient to perform testing (1.5-cm of solution as a minimum).

11. Press “” to continue.

12. Load the sample tray with sodium citrate tubes or sample cups. For PT-FIB/APTT testing, only nine (9) samples can be programmed to run at one time. For PT-FIB testing, 18 samples can be programmed and loaded on the sample tray.

13. Select a load list, enter the number and press enter.

14. Program the load list according to the load positions in the sample tray. Use the HCX number for patient samples.

15. Load a fresh rotor on the instrument.

16. Press the commands key to start analysis.

17. At the end of analysis the "Results" frame is displayed and results will be printed out for patient samples. Control results will not print on the laser printer.

18. TAR the patient and control results into CHCS. Review the results that are to be filed and certified.

19. Clean the instrument surfaces with 10% bleach. Return the reagents to their original vials and store at 2-8 °C.

G.1.7. INTERPRETATIONS & REPORTING RESULTS:

1. The PT and PTT tests are reported in seconds along with the reference ranges.

2. The ACL 7000 Analyzer will automatically calculate the INR value. This value will appear beside the PT result.

3. Enter patient results in CHCS through “Transfer Auto Instrument Results” (TAR) or “Enter Results by Accession Number (ERA)”.

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4. Expected Results:

4.1. The normal PT range for non-medicated patients at this hospital with the PT-FIB HS reagent is 10.0 – 13.0 seconds and the geometric mean is 11.7 seconds. For non-medicated patients, PT results greater (>) than 17 seconds are critical. Call the provider, on-call provider or the ward/clinic RN to report all critical results, unless the patient is a known coumadin clinic patient. Refer to the Critical Value SOP and document the action taken in the LIS.

4.2. The normal PTT range for non-medicated patients at this hospital is 21.0 to 35.0 seconds. PTT results greater (>) than 40 seconds are critical results. Call the provider, on-call provider or the ward/clinic RN to report all critical results. Refer to the Critical Value SOP and document the action taken in CHCS.

4.2.1. Heparin therapy is monitored using the APTT test. We recommend a goal APTT of 1.5 to 2.5 times the patient’s baseline level for a reference interval. Low molecular weight heparin is monitored using Factor Xa and is sent to [your reference lab]. Reference ranges vary based on dosage: Therapeutic dosage (1x Daily) = 0.80 – 1.20 U/mL and (2x Daily) = 0.40 – 0.80 U/mL; Prophylactic dosage (1x Daily) = 0.15 – 0.40 U/mL.

4.2.2. Heparin Curve information: The heparin curve was performed using Catalog No. P5771 (25,000 Units per 500mL = 50 Units per 1 mL. Issued from HDB Inpatient Pharmacy. Therapeutic Range: 0.2 – 0.4 u/mL.

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Heparin u/mL APTT sec

0.0 27.50.1 30.60.2 36.10.3 45.10.4 64.20.5 81.90.6 96.80.7 105.00.8 125.00.9 158.01.0 177.0


Heparin Curve Data

0.020.040.060.080.0

100.0120.0140.0160.0180.0200.0

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

APTTsec

4.3. The INR for a normal patient is 0.9 to 1.1. For non-medicated patients, INR values greater (>) than 1.5 are critical.

4.4. For standard oral anticoagulant therapy, the recommended therapeutic range for the INR is 2.0 to 3.0.

4.5. For high-dose oral anticoagulant therapy, the recommended therapeutic range the INR is 2.5 to 3.5. For documented coumadin patients, INR values less than (<) 1.7 and greater (>) than 4.0 are critical. Call the provider, on-call provider or the ward/clinic RN to report all critical results. Refer to the Critical Value SOP and the critical procedure on page 16 of this SOP. Document all action taken in CHCS.

4.6. The highest reportable value for an INR is 6.0. Do not report any INR values greater than 6.0. All values determined to be greater, must be reported as TNP with the following comment: INR > 6.0.

4.7. Critical Coagulation Notification Procedures:

4.7.1. Verify the patient’s anticoagulant status. This can be performed using the following methods:

4.7.1.1. Review the Coumadin Clinic Patient lists located in the coagulation book.

4.7.1.2. Search the LIS prescription menu for active Warfarin orders.

4.7.1.3. Obtain verbal verification from the provider.

4.7.2. If the critical result is due to incorrect order entry, i.e. the provider ordered a normal plasma sample type instead of anticoagulant plasma sample or vice versa, proceed as follows:

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4.7.2.1. Reorder the test and indicate the correct sample type.

4.7.2.2. Cancel the order using the ^CAO option in the LIS.

4.7.2.3. Enter a cancellation comment, reference the reorder, and the reason for reorder.

4.7.2.4. Enter a comment in the new order indicating that test was reordered for the cancelled accession, that the patient is or is not on coumadin, how verification was established, and that the original order was entered incorrectly in LIS.

4.7.2.3. Example: “Reordered for HCX999. Patient is a documented coumadin patient per the Lab coumadin list. Incorrect sample type indicated during original order entry.”

4.7.3. If the order was entered correctly and the value is critical, contact and call the critical value as follows:

Order Location Time of Day Contact Phone Number

Main Lab

Monday – Friday 0830 - 1700Coumadin Clinic

371-2674Saturday – Sunday 0800 - 1300Federal Holidays 0800 - 1300Training Holidays 0800 - 1600Outside Normal Duty Hours Internal Med on call HCP Number provided by SDO

Outlying Clinics Duty Hours Ordering Provider Provider or Clinic NumberOutside Normal Duty Hours Clinic Commander Listed on Call Roster

Cardiology Monday – Friday 0800 - 1700 Cardiology Staff 486-5115 or 7927Outside Normal Duty Hours LAB Staff 486-7114 or 7499

All Other Locations Duty Hours Ordering Provider Provider or Clinic NumberOutside Normal Duty Hours LAB Staff 486-7114 or 7499

G.1.8. PROCEDURAL NOTES:

1. PROG-ACQUISITION TIME Procedure (Extended Mode):

1.1. This procedure must be followed for those specimens with a result of “No Coag” or “Coag Error” after appropriate trouble shooting has been attempted and documented.

1.2. From PROG Menu, select SET-UP, and ACQUISITION TIME by using the “” or “” keys.

1.3. Select the test that needs to be performed on the extended mode. Only one test can be performed at a time.

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1.4. Press the “” or “” keys to change from standard to extended mode, press ENTER to confirm, and “” key to continue.

1.5. Press “PROG” to return to the Ready menu, select the test that needs to be performed on the extended mode, and press ENTER. It is important that the appropriate test panel is selected because regardless of the acquisition time selected, the STANDARD mode will be used if the PT-FIB/APTT panel is chosen. In order for the machine to operate in the extended mode, you must select either PT-FIB or APTT, accordingly. Select the appropriate test and press ENTER. At the top left side of the screen, the selected test will be displayed followed by an (E) indicating extended mode.

1.6. Continue with step 11 to 18 from the Procedure Section of this SOP to complete patient testing. After test completion, change the acquisition time back to ‘standard’ using the procedure identified above.

1.7. Review the results print out for information displayed to the right of the results and below the statement “No Cal Verification”. If the extended mode was used “Extend acq. time” will be displayed.

1.8. If the sample does not coagulate within the extended mode acquisition time, resulting in a “No Coag” or “Coag Error” result, you must perform patient testing with NORMAL CONTROL in the SAME RUN to ensure adequate needle (probe) function.

2. PARTICULAR INDICATIONS:

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TEST INDICATION PROCEDURE

PT “No Coag” or “Coag Error” Coag Errors may sometimes be followed by a number. Use the same procedure for all “coag errors” and “no coag” results.

The sample does not coagulate within the standard acquisition time:

1. Verify that there is sufficient patient sample, reagent and reference emulsion.

2. Visually inspect the rotor cuvette to ensure that the specimen did not clot prior to the instrument reading.

3. Repeat the sample and normal control (in the same run) in EXTENDED mode. (See para. 1, page 17)

4. If the result is still “No Coag or Coag Error” and the control is within range, result the patient values as >169. If the control does not coagulate, verify probe function and perform probe cleaning as identified in the maintenance SOP, H.5

If the fibrinogen result is <60 mg/dL, the sample may already be coagulated. Check the sample for clots using two wooden sticks and check the rotor cuvette visually. If the sample is clotted, reject it and request another. Document action taken in CHCS.

APTT “No Coag” or“Coag Error”

Coag Errors may sometimes be followed by a number. Use the same procedure for all “coag errors” and “no coag” results

The sample does not coagulate within the programmed acquisition time:

1. Verify that there is sufficient patient sample, reagent and reference emulsion

2. Visually inspect the rotor cuvette to ensure that the specimen did not clot prior to the instrument reading.

3. Repeat the sample and normal control (in the same run) in EXTENDED mode. (See para. 1, page 17.) If the result is still “No Coag or Coag Error” and the control is in range, report the APTT as >249 seconds. If the control does not coagulate, verify probe function and perform probe cleaning as identified in the maintenance SOP, H.5.

If the fibrinogen result is <60 mg/dL, the sample may already be coagulated. Check the sample for clots using two wooden sticks and check the rotor cuvette visually. If the sample is clotted, reject it and request another. Document action taken in CHCS.

3. ACL 7000 LINEARITY LIMITS:

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TEST MEASURED VALUES

PT 62 seconds on standard modePT 169 seconds on extended modePTT 119 seconds on standard modePTT 249 seconds on extended modeFIBRINOGEN 40 to 800 mg/dL

4. SOURCES OF ERROR:

4.1. A non-traumatic venipuncture is absolutely necessary. Any contamination with tissue thromboplastin leads to falsely decreased results. Tissue thromboplastin is a potent clot-activating substance that activates the extrinsic pathway of clotting causing erroneous results.

4.2. The ratio of blood to anticoagulant is critical for valid coagulation test results. If there is incomplete filling of the sample tube, the amount of unbound citrate in the citrate-plasma mixture causes a false prolongation of clotting times, particularly in PT and PTT tests. Likewise, if the tube is overfilled, the result may be falsely shortened.

4.3. Do not use samples that have visible hemolysis. Hemolysis can cause clotting factor activation and falsely decrease end point measurements.

4.4. Do not use lipemic or icteric plasma samples. Lipemia and icterus interfere with the spectrophotometric measurements resulting in falsely decreased end point determinations.

4.5. Contaminated or expired reagents will give inaccurate results. If this it is suspected, replace the reagents.

4.6. Inadequate preparation of the controls or reagents will cause inaccurate results.

4.7. Materials used for collecting, transporting, and storing blood samples for coagulation studies should have a “noncontact” surface. Therefore, the inside surface of the tubes should not be of material (such as glass or soda lime) that will activate the coagulation factors, causing a shortening of both the intrinsic and extrinsic pathways. The recommended materials for coagulation studies are plastic, polystyrene, or silicone-coated tubes.

4.8. Samples with a fibrinogen levels <25 mg/dl should be suspected of being a serum sample and rejected.

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4.9. Physically examine all coagulation specimens after centrifugation and visually estimate the hematocrit using a sodium citrate hematocrit guide. Additionally, annotate CBC specimens with hematocrits (greater than) >55% on the “Elevated Hematocrit Specimen Log”. Redraw the specimen, if an elevated hematocrit is observed using a reduced volume of sodium citrate, adjusted for the elevated hematocrit. Refer to SOP, G.4. ADJUSTING SODIUM CITRATE CONCENTRATIONS FOR PATIENTS WITH HEMATOCRITS >55%.

G.1.9. APPENDICES:

1. SOP Validation Form and SOP Change Control2. SOP Approval3. PT Calibration and INR Verification Worksheet4. ACL 7000 Troubleshooting logs – Normal and Abnormal Controls5. Intrinsic, extrinsic, and common pathways in the coagulation cascade.6. Beckton Dickenson Vacutainer Fill Guide

G.1.10. REFERENCES:

1. ACL 7000 Operator's Manual, Instrumentation Laboratory Inc., Rev. 4, January 2001.

2. NCCLS Document H21-A4, Volume 11 No.35, Collection, Transport, and Processing of blood for Coagulation Testing, Fourth Edition, 2003.

3. Ratnoss, Oscar D., Disorders of Hemostasis, Third Edition, W.B. Saunders Book Publisher, 1996, Pages 59 to 65.

4. Beutler, Ernest, Hematology, Fifth Edition, McGraw-Hill Book Publisher, 1995, Pages L82 to L88.

5. Lacy CF, Armstrong LL, et. al., Drug Information Handbook, 2003, 11th edition, Heidelberg pharmacy.

6. Yuan, Shan, et. al., The physiology of hemostasis, common bleeding disorders and their laboratory evaluation, Coagulation Training, www.medtraining.org

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http://www.medtraining.org/


SOP VALIDATION

SOP NAME: G.1. Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and INR by ACL 7000

Clear and specific title and principle: yes / noComments:

All necessary supplies, equipment, and materials are listed: yes / noComments:

SOP is sufficiently detailed to be understood but not overly complex: yes / noComments:

SOP text adequately describes process/procedure: yes / noComments:

SOP accomplishes purpose: yes / noComments:

Reviewed by: (Name & Title)

Signature: __________________ Date: __________________

SOP CHANGE CONTROL

Date Change QA OIC Med. Dir.

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SOP APPROVAL

SIGNATURE DATEPREPARER

QA COORDINATOR

LABORATORY OIC

MEDICAL DIRECTOR

ANNUAL REVIEW

REVIEWER SIGNATURE DATE REVIEWER SIGNATURE DATE

DOCUMENT COPY CONTROL DATE: _______________# COPIES __________LOCATIONS

1 10

2 11

3

4

5

6

7

8

9

SUPERSEDES: ____________

DATE SOP RETIRED: __________

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