Procedure:

Hemochron®Response System

Clinical and Laboratory Standards Institute (CLSI) Formatted Procedure

Approved by ______Date ______

Created by ______Date ______

Reviewed by ______Date ______

Reviewed by ______Date ______

Reviewed by ______Date ______

Reviewed by ______Date ______

Contact Person ______

Contact Number(s) ______

PURPOSE

The Hemochron®Response Whole Blood Coagulation System is a dual-well microprocessor-controlled coagulation testing instrument with an integral test type barcode reader, RS232 communication interfacecapability, and a printer. The system runs coagulation tests such as: Activated Clotting Time (ACT), ActivatedPartial Thromboplastin Time (APTT), Prothrombin Time (PT) and other specialty tests that are specific to the RxDx® to include: Heparin Response Time (HRT), Protamine Response Time(PRT), Protamine Dose Assay - O (PDAO), Thrombin Time (TT), Heparin Neutralized Thrombin Time (HNTT)

The HemochronRxDx®Analysis Module is a supplementary module for the HemochronResponse Whole Blood Coagulation System that provides automated calculations for use during cardiopulmonarybypass surgery and cardiac catheterization procedures. These calculations are used to determine the doses of heparin to be administered before and during theseprocedures, the dose of protamine needed to reverse the effects of heparin after the procedure iscompleted, and the patient's clotting time and residual heparin level after protamine has beenadministered.

PRINCIPLE

The Hemochron clot detection module contains two test wells into which disposable unitized coagulation test tubes can be inserted. The test tubes containreagents for a particular test and a magnet. Immediately after the sample is added to the test tube,the “START” button is pressed, the test tube is agitated, and the test tube is placed into the test well by theoperator. Once in the test well, the tube containing the sample is automatically rotated at a controlled speed and incubated at 37 °C ±1.0 °C.

When a fibrin clot begins to form, it causes the magnet in the test tube to be displaced. Two magnetic detectors located in the test well continuously monitor the precise magnet position. When a specificdisplacement of the magnet occurs, the elapsed time between the beginning of the test and the clotendpoint is displayed as the coagulation time (in seconds).

The instrument also emits an audible beepwhen clot formation occurs, indicating the end of the test.The coagulation time is displayed on the LCD screen.

The Analytic Measurement Range (AMR): 22-1500 seconds

Activated Clotting Time (ACT)

The ACT is performed by adding a clotting activator such as Celite®, silica, kaolin, or glass particles to a blood sample and then measuring the length of time required for clot formation. The particular clotting activatorthat is used influences the time required for clot formation. Celite (diatomaceous earth) is the standard ACTreagent used for high level heparin monitoring because of its excellent activating properties.

However,serine protease inhibitors such as aprotinin that may be administered to certain patients to decrease postoperative bleeding can prolong the Celite-activated ACT. When aprotinin is on-board, a kaolin-activatedACT tube should be used.

Activated Partial Thromboplastin Time (APTT)

The OneStepAPTT test measures the intrinsic coagulation pathway and involves all coagulation factors except Factors VII and III (tissue factor). The APTT test improves the earlier Partial Thromboplastin Test (PTT) test through use of a contact activatingsubstance (kaolin) which initiates activation of Factor XII and acts as a Platelet Factor 3 substitute to provide for lowlevel heparin monitoring.

Prothrombin Time (PT)

The PT test measures the extrinsic coagulation pathway and is sensitive to coagulation Factors VII, X, V, II, and fibrinogen. PT results may be abnormal in patients with liver disease or Vitamin K deficiency, and thetest is widely used to monitor warfarin anticoagulant therapy.

RxDx Analysis Mode

Prior to administration of heparin, an Activated Clotting Time (ACT) and Heparin Response Time (HRT) test are run on the HemochronResponse Whole Blood Coagulation System. These tests measure the patient'sbaseline clotting time and determine how the patient will respond to heparin. The patient blood volume caneither be directly entered (if available) or calculated by the system based upon the patient's height, weight, and gender.

1. The initial heparin dose (referred to as the bolus heparin dose) necessary to reach target time is then calculated by thesystem. During this process, the case is in the heparin mode.

2. Once the patient has received heparin, additional ACT or High Dose Thrombin Time (HiTT) tests are run tomeasure the coagulation status, determine the efficacy of heparin, and calculate additional heparin dosesthat may be needed to maintain the target clotting time in the patient. The case is now in the additionalheparin mode.

3. After the cardiac surgery or cardiac angioplasty procedure is completed, the effects of administered heparincan be reversed by administration of protamine.

Like heparin, protamine must be administered in thecorrect dose to prevent dangerous bleeding or thrombosis. For this reason, a Protamine Response Time(PRT) test is performed, and the results of this test and the most recent ACT test are used to determine thedose of protamine that is required. The case is now in the protamine mode.

Once the protamine has been administered, the patient's clotting time and residual heparin level aremeasured, using an ACT and a Protamine Dose Assay - O (PDAO) test to determine the post-protamineclotting time and residual heparin level in the patient's blood. HeparinNeutralized Thrombin Time (HNTT) tests can also be run after protamine administration to identify thepresence of heparin rebound or abnormal fibrinogen function.

Since Celite®(diatomaceous earth) is sensitive to the presence of serine protease inhibitors such asaprotinin, it is recommended that kaolin test tubes are used instead of Celite test tubes when aprotinin

High Dose Thrombin Time (HiTT)

The Hemochron®HiTT is a quantitative, one-stage high dose thrombin time test for the purpose of monitoring high levels of heparin anticoagulation. The HiTT employs a specific concentration of thrombin so that high heparinlevels may be monitored. The HiTT can be used in conjunction with, or as an alternative to, the Activated Clotting Time (ACT) test to monitor heparinization during cardiopulmonary bypass surgery. The test may be used in the presence ofantifibrinolytic drugs, such as aprotinin (Trasylol®), which appear to preserve platelet function and reduce post-operative bleeding.

Protamine Response Test(PRT)

The Hemochron® PRT is designed for in vitro diagnostic protamine dose Response evaluation. The Hemochron PRT200 and the PRT400tubes are recommended for use in invasive cardiology proceduresrequiring protamine for neutralization of heparin anticoagulation with the PRT 400 used for extremely high heparin concentrations. The kaolin- activatedPRT assays may also be used for patientsreceiving aprotinin. ThePRT200 and PRT400 should be used with established Hemochron proceduralguidelines with HemochronResponse. The dose of protamine sulfate required to neutralize heparin aftercardiopulmonary bypass surgery is commonly based on a ratio of protamineto the amount of heparin infused during the procedure, or on protaminetitration.

Protamine Dose Assay-Orange(PDAO)

The Hemochron®PDAO provides a convenient method for protamine titration using the principles of in vitro dose-responseheparin neutralization and verification of heparin neutralization.Titration analysis performed using PDAOquantifies the milligrams of protamine per milliliter of patient blood necessary to neutralize the patient’s residual heparin load and return the Activated Clotting Time (ACT) to the pre-heparinized baseline value. The PDAO test is available with either Celite®(diatomaceous earth) or kaolin activators. The Celite-activatedPDAO test is not intended for use with the protease inhibitor aprotinin (Trasylol®, Bayer Corporation), which maybe administered to reduce post-operative bleeding, especially during cardiopulmonary bypass surgery, and canprolong the Celite-activated PDAO. Kaolin is unaffected by moderate doses of aprotinin. The kaolin-activated PDAOcan be used as an alternative for protamine dose response evaluation in patients receiving aprotinin.

The postsurgicalpatient should be monitored with the ACT and PDAO to determine the need to infuse additional protamineto compensate for latent heparin effect, which may possibly result in heparin rebound.

Thrombin Time (TT)

The Hemochron®TT is designed for use as a screening assay to evaluate the level of circulating fibrinogen and detection of heparin. The thrombin time is commonly employed to detect suspected heparin anticoagulation and to quantify circulating fibrinogen. The measurement of fibrinogen is useful in the diagnosis of consumptive coagulopathy or intravascular coagulation and the monitoring of thrombolytic therapy.

Heparin Neutralized Thrombin Time (HNTT)

The Hemochron®HNTT is designed to be solely indicative of circulating fibrinogen and is not affected by the presence of heparin. In addition to sensitivity to fibrinogen level, the thrombin time is sensitive to the presence of heparin. Heparin prolongs the thrombin time through neutralization of the thrombin in the test tube. The results of a coagulation assay, such as the thrombin time, performed on a blood specimen in which heparin has been neutralized, reflect the hemostatic condition of the patient. Such an assay may be referred to as the heparin-neutralized thrombin time (HNTT).

SAFETY

Universal precautions should be observed through all phases of the testing procedure. Refer to the current versions of CLSI Document M29– Protection of Laboratory Workers from Occupationally Acquired Infections and CLSI Document GP17 - Clinical Laboratory Safety for Information on Laboratory Safety and Special Requirements.

SPECIMEN

A. Patient Preparation

There is no patient preparation needed prior to testing.

B. Sample Collection

The ACT test is performed using fresh whole blood collected with a syringe from the patient. Refer to the most recent version of CLSI Document H21 - Collection, Transport, and Preparation of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline for the collection of specimens for coagulation testing.

When sampling through indwelling blood lines, flush access port thoroughly following institutional procedures.

1.Identify patient as directed by policy

2.Collect a blood sample from the patient by venipuncture or an indwelling catheter lineto allow for 2cc volume required for ACT testing.

3.If using a syringe with needle:

a.Use a 23 or larger gauge needle

b.Safely remove needle and discard in a sharps container.

c.Optional: attach a transfer needle no less than 23 gauge to the syringe.

d.After dispensing the sample, discard syringe and transfer needle in labeled sharps container.

C. Sample Storage

Specimens must be processed immediately. There is no storage prior to testing.

D.Unacceptable Sample Types

Samples with any of the following characteristics should be discarded immediately, and a fresh whole bloodsample must be collected prior to performing any test on theHemochron instrument.

  • If a non-citrated test, citrated tube used for collection
  • Sample collected into a pre-heparinized syringe
  • Sample contamination with tissue thromboplastin
  • Sample contamination with indwelling intravenous (I.V.) solutions
  • Sample contamination with alcohol cleansing solution
  • Samples with visible clotting or debris accumulation

EQUIPMENT/ MATERIALS/ REAGENTS

A.Equipment

1.Hemochron®ResponseInstrument

2.AC/DC Power Module ITC Part Number HR1283

B.Materials

  1. Fresh whole blood sample
  1. Biohazard disposal receptacle
  1. Sharps disposal receptacle

C.Reagents

ACT Celite

FTCA–(Part Number FTCA510: black top tube)

  • Flip-top non-evacuated glass test tube contains diatomaceous earthas an activator.

ACT Kaolin

FTK-ACT–(Part Number FTK-ACT: gold top tube)

  • Flip-top non-evacuated glass test tube contains kaolin as the activator

ACT for Low Levels of Heparin

P214–(Part Number P214: top tube; white top tube)

  • Flip-top plastic test tube with glass beads as the activator

APTT

OneStepAPTT forWhole Blood

APTT – (Part Number A103: navy blue top tube)

  • Each tube contains a lyophilized preparation of colloidal kaolin, a platelet factor 3 substitute, stabilizers and buffers
  • Thimerosal (0.02%) is added as a preservative.

CAUTION: Reagents containingthimerosal should be discarded in accordance with your institutions policy concerning disposal of medical waste.

PT

PT for Fresh Whole Blood

PT – (Part Number A201: dark purple top tube)

  • Each tube containsa lyophilized preparation of acetone-dried rabbit brain thromboplastin, stabilizers, and buffers.
  • Sodium azide (0.05%) added as a preservative

PT for Citrated Whole Blood

Cit PT – (Part Number A202: light purple top tube)

  • Each tube contains alyophilized preparation of acetone-dried rabbit brain, calcium salt, stabilizers and buffers.
  • Sodium azide (0.05%) added as a preservative

CAUTION: Reagents containing sodium azide should be discarded in accordance with your institutions policy concerning disposal of medical waste.

D.Reagents – RxDx System

PRT 200 (ITC Part Number R-PRT 200: peach top tube)

  • Each tube contains protamine sulfate(0.04 mg), diatomaceous earth, stabilizers and buffers.

PRT 200K (ITC Part Number R-PRT 200K: peach top tube with Kaolin label)

  • Each PRT200K tube contains protamine sulfate(0.04 mg), kaolin, stabilizers and buffers.

PRT 400 (ITC Part Number R-PRT 400: red top tube)

  • Each PRT400 tube contains protamine sulfate(0.08 mg), diatomaceous earth, stabilizers and buffers.

PRT 400K (ITC Part Number R-PRT400K: red top tube with Kaolin label)

  • Each PRT400K tube (red top) contains protamine sulfate(0.08 mg), kaolin, stabilizers and buffers.

HiTT– (ITC Part Number A501:turquoise top tube)

  • Each tube contains a lyophilized preparation of human thrombin, snake venom, protamine sulfate, calcium salts,stabilizers and buffers.
  • Thimerosal (0.02%) is added as a preservative.

CAUTION: Reagents containingthimerosal should be discarded in accordance with your institutions policy concerning disposal of medical waste.

HNTT–(ITC Part Number A401 – brown top tube)

  • Each tube contains lyophilized human thrombin, calcium salts, protamine sulfate, stabilizers and buffers.

TT – (ITC Part Number A301 – rose top tube)

  • Each tube contains a lyophilized preparation of human thrombin, calcium salts, stabilizers and buffers.

HRT- Kaolin– (ITC Part Number R-480 PK: light green top tube with Kaolin label)

  • Each R-HRT480PK tube contains the equivalent of 6 USP unitsof porcine mucosal heparin (3 units/ml in blood), kaolin, stabilizers and buffers.

HRT – Celite – (ITC Part Number R-HRT480P: light green top tube)

  • Each R-HRT480P tube contains the equivalent of 6 USP units ofporcine mucosal heparin (3 units/ml in blood), diatomaceousearth, stabilizers and buffers.

PDAO– (ITC Part Number PDAO: orange top tube)

  • Each PDAO tube contains protamine sulfate (0.01mg), diatomaceous earth, stabilizers and buffers.

PDAOK – (ITC Part Number PDAOK: orange top tube with Kaolin label)

  • Each PDAOK tube contains protamine sulfate (0.01mg), kaolin, stabilizers and buffers.

D.Reagent Storage

Test tubes of all types are stable when stored at room temperature (15-30°C or 59-86ºF) and used prior to expiration date on carton and/or case.

QUALITY CONTROL

A.Self-Check

The Responseinstrument performs a “self-check” every time it is activated and a test isperformed. When a test is initiated by pressing START, system checks are automatically performed and include:

  • Verification of adequate battery power to complete a 1500 second test.
  • Verification that a tube has been inserted and that the test well is functioning properly. If anyrotation or temperature parameters are not appropriate, the test is terminated and an errormessage is displayed.
  • For barcoded tubes, the test type and expiration data are read. The test type will be displayed onthe screen. If the expiration date has passed, the test is aborted and an error message displayed. Once a barcode is read, the user cannot change the test type.
  • Verification that the test well is warmed to 37± 1.0 ºC. If this temperature is not achieved or isexceeded, an appropriate error message will be displayed and testing is prohibited.
  • Verification that the internal timers function correctly for each test. If the system timer andassay timer disagree at the end of a test, a real-time clock error message is displayed and thetest result is not reported.

B.Electronic Quality Control(EQC)

The Electronic System Verification (ESV) should be performed once every 8 hours while the instrument is in operation. Both wells are to be tested if both are in use.

1.A prompt is displayed if entry of an OID or PIN is required. Enter your OID or PIN and press “YES”.

2. A prompt is displayed to enter the serial number of the ESV tube. The serial number of the lastESV used is displayed.

NOTE: If needed, enter the ESV serial number (up to 9 characters) located on the back of the ESV tube. Press “BACKSPACE”to undo an entry for retyping.

3.Insert the multilevel ESV tube intotest well of the instrument.

4.Simultaneously press both the “START” button on the Response and the button that corresponds to the level of testing to be performed (100, 300, or 500 seconds).

5.The green detector light on the instrument and the red lighton the ESV tube will illuminate indicating start of the test.

6.When the verification tube reaches its pre-programmed time, it will electronically

simulate a test endpoint as indicated by a“beep” tone and display the test result.

7.Record the time displayed according to institutional policy.

8.Repeat steps 2-5 with the next test level of the ESV tube.

9.Results are acceptable if within 10 seconds of the selected times.

10.The ESV may be manually tagged if the barcode label is not read.

NOTE:Contact ITC if the results are not within range.

Call toll-free in the U.S. (800) 631.5945.

If calling from outside the U.S 001.732.548.5700.

Contact ITC by e-mail:

C.Optional Temperature Verification (TVT)

1.Select the well to be tested.

2.In the Menu program, first page select “#2- QC Selects“

3.Press “#3 – Temp Check” and insert the Temperature Probe.

4.A prompt is displayed if entry of an OID or PIN is required. Enter your OID or PIN and press “YES”.

5.When the instrument “Beeps”, record the temperature according to policy.

6.The expected range is 37 °C ±1.0 °C

NOTE:Contact ITC Technical Support if the temperature is not within specifications:

Call toll-free in the U.S. (800) 631.5945.