Standard Operating Procedure

Subject APTT (Activated Partial Thromboplastin Time) – IL Elite Index Number Lab-8768 Section Laboratory Subsection Regional/Affiliate Category Departmental Contact Betty Tilleraas Last Revised 12/5/2019

References Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers for Medicare and Medicaid Services (CMS), and/or COLA.

Applicable To Employees of the Gundersen Health System clinical laboratories, Gundersen St. Joseph’s Health Services laboratories, Gundersen Boscobel Area Hospital laboratories and Gundersen Palmer Lutheran Hospitals and Clinics laboratories.

Detail INTENDED USE: The Activated Partial Thromboplastin Time (APTT) Assay is used as a screening test to evaluate the overall integrity of the intrinsic/common pathway. Results are reported in seconds. The APTT is used at Gundersen Health System to perform factor assays for the intrinsic/common pathway; namely Factors V, VIII, IX, XI, and XII. The APTT assay is not recommended for monitoring oral anticoagulant therapy nor is it sensitive to dysfunction.

PRINCIPLE: In the APTT test the incubation of plasma sample with an optimal quantity of phospholipid, a negatively charged contact activator and buffer initiates the activation of the intrinsic pathway. After incubation at 37oC for a specific period of time calcium is then added to trigger the coagulation process and the time required for clot formation is measured.

The principle of coagulometric (turbidimetric) clot detection is used in the system to measure and record the amount of time required for a plasma specimen to clot. This technique assesses coagulation endpoint by measuring change in optical density. Clot detection is based on the principle that light passing through a medium in which is converted to fibrin is absorbed by the fibrin strands. Light (671 nm) is transmitted through a sample onto a photodetector, which is positioned 180° incident to the source. Light absorption increases as fibrin clot formation progresses. Consequently, light transmittance through the sample continuously decreases and is measured by the photo detector. The corresponding electrical signal output from the photo detector changes according to the detected light. The signal output is processed via software through a series of algorithms to determine the clot point.

CLINICAL SIGNIFICANCE: The Activated Partial Thromboplastin Time (APTT) assay is a universally accepted screening procedure used to detect abnormalities in the intrinsic coagulation system. It can be used to detect deficiencies of Factors I, II, V, VIII, IX, X, XI, and XII, but is insensitive to platelet factor 3. In addition, it can be used to

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Standard Operating Procedure

detect lupus anticoagulants and is recommended when monitoring heparin therapy since it is sensitive to the presence of heparin.

SPECIMEN: One Blue tube containing 3.2% buffered sodium citrate. Stable 4 hours. Refer to Lab-6501 Coagulation Testing: Collection, Transport and Processing.

REAGENTS/MATERIALS: HemosIL SynthASil kit- APPT reagent: Buffered synthetic phospholipid reagent containing a colloidal silica activator, stabilizers and a preservative.

Calcium Chloride: Aqueous solution of calcium chloride (0.020 Mol/L) and a preservative.

Additional materials required IL Elite instrumentation – (Refer to instrument manual for operational instructions). Purified water, Type 1, Reagent grade deionized water, Buffered. HemosIL Normal & Abnormal controls – assayed. Micropipettes with disposable tips Factor Diluent Clean A Clean B Rinse Solution

Storage and Stability: On-board stability: 3 days. (15oC) Open and Stored at 2-8°C: 30 days

Store all HemosIL SynthASil kit components at 2- 8oC when not in use. Do not freeze. The expiration date printed on each container indicates the date beyond which the unopened product should not be used. Signs of deterioration are reflected by quality control results outside the established laboratory range. Refer to the package inserts located in the Coagulation Department procedure manual.

Preparation: Each vial of APTT reagent must be equilibrated at 15-25oC for 15 minutes and mixed thoroughly before used. The CaCl reagent is ready for use. SynthASil and CaCl should be treated as a set. If you add one new, the other should be added too.

Load APTT reagent into R2. Load CaCl reagent into R8.

Precautions and Warnings: Avoid contact with skin and eyes. Wear suitable protective clothing. Contains sodium azide. Use proper disposal procedures.

EQUIPMENT/INSTRUMENTATION: IL Elite

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Standard Operating Procedure

QUALITY CONTROL: Quality Control Levels: Normal control 1 Assayed and High Abnormal Control 3 Assayed Reconstitute with 1.0 ml deionized water. Set for 30 minutes. Mix. Load on analyzer.

Controls will be performed: 1. After start-up maintenance 2. Every 8 hours 3. After reagent bottle change 4. After major repairs 5. After new reagent lot calibration

Refer to Lab-0170 Quality Control.

Performance: If control values are out of range, do not report patient results. Determine which part of the instrumentation/reagent/control system is not functioning properly and correct it. After corrective measures are implemented and documented following good laboratory practice, retest the controls. If they are within range, patient samples can be tested and reported.

If it was determined that the instrument or reagent system failed, patient samples that were tested and reported in the interval between the time QC was last in range and when it was found to be out of range must be re-evaluated. Since coagulation samples are not stable more than 4 hours, repeat data and the scope of the problem will be reviewed by the laboratory management (technical leader, manager, or medical director). Any necessary corrective action or reports with notification of care providers will be determined by this team.

Implementation PROCEDURE: 1. Load bar-coded specimen in specimen rack with label facing out. 2. Specimens must be uncapped. 3. Click on Start/Resume button. Green Running man. 4. When testing is complete, results should cross interface. 5. Results with flags need to be assessed by the operator and manually validated. Clot curves will be evaluated.

PROCEDURE NOTES: Upper Limit: 250.0 seconds Lower Limit: 16.0 seconds Refer to Lab-0130 Critical Call Values

CALCULATIONS: The APTT screening assay is a clotting time endpoint result expressed in seconds.

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Standard Operating Procedure

INTERPRETATION: Normal Range APTT 26 – 36 seconds Critical Call: >130.0 seconds Therapeutic Range: 53.0 - 102.0 seconds

LIMITATIONS: The assay is sensitive to factors V, VIII, X, IX, XI, and XII at concentrations of 30-45% for each factor. Less than 1% of Factor VIII and IX is detectable.

Interfering substances: APTT results may be affected by many commonly administered drugs and further studies should be made to determine the source of unexpected abnormal results. No interference due to hemolysis up to 500 mg/dL. No interference due to bilirubin up to 26 mg/dL. No interference due to triglycerides up to 1000 mg/dL.

REVIEW AND CHANGES: This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as the maximum review date. Review will be done by the Supervisor, Manager, Technical Leader, Medical Director or designated person. Changes require retyping document or form and review by the medical director.

REFERENCES: 1. IL Elite operators manual. 2. HemosIL SynthASil package insert. 3. Laga, AC, Cheves, TA, Sweeney, JD. The Effect of Specimen Hemolysis on Coagulation Test Results. Am J Clin Pathol 2006; 126:748-755.

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