Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 44. Hemostasis and coagulation instrumentation

Debra A. Hoppensteadt, JoAnn Molnar*

OUTLINE

Historical Perspective

Assay End-Point Detection Principles

Mechanical End-Point Detection

Photo-Optical End-Point Detection

Nephelometric End-Point Detection

Chromogenic End-Point Detection

Immunologic Light Absorbance End-Point Detection

Advances in Coagulation Technology

Improved Accuracy and Precision

Random Access Testing

Improved Reagent Handling

Improved Specimen Management

Expanded Computer Capabilities

Other Automated Features

Specimen Quality Set Points

Instrument Malfunction Flags

Advantages and Disadvantages of Detection Methods

Point-of-Care Testing

Whole-Blood Clotting Assays

Platelet Function Testing

Platelet Aggregometers

Platelet Function Analyzers

Molecular Coagulation Testing

Selection of Coagulation Instrumentation

Currently Available Instruments

Objectives

After completion of this chapter, the reader will be able to:

1. Describe testing methodologies previously considered as specialized that are now routinely available on coagulation analyzers.

2. Identify testing applications for various coagulation analyzers.

3. Explain the methods of clot detection used by each type of coagulation analyzer presented.

4. List common instrument flags that alert operators to specimen and instrument problems.

5. Describe the advantages and disadvantages of each method of clot detection.

6. Distinguish the characteristics of manual, semiautomated, and automated coagulation analyzers.

7. Identify key performance characteristics that should be evaluated when selecting the most appropriate coagulation analyzer for an individual laboratory setting.

8. Explain the purpose of incorporating platelet function testing analyzers into the routine coagulation laboratory.

9. Identify the role of platelet aggregation in the coagulation laboratory.

10. Describe the methods available for molecular testing in the clinical lab and the analytes that can be measured using these techniques.

11. Develop a model plan of action for objective evaluation of coagulation analyzers for purchase.

12. Explain the main purpose of point-of-care coagulation testing.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 35-year-old white man was admitted to the hospital with abdominal pain and tenderness, malaise, and a low-grade fever. A tentative diagnosis of cholecystitis was made, with possible surgical intervention considered. Pertinent medical history included tonsillectomy at age 6 and appendectomy at age 18 with no abnormal bleeding symptoms noted. The patient reported that he was taking no medications at this time. In anticipation of surgery, routine coagulation studies were ordered. When the specimen arrived in the laboratory, it was centrifuged, and it was noted that the plasma had a whitish, milky appearance. The specimen was processed by an automated analyzer using photo-optical end-point detection methodology, and the following results were obtained:

Test

Results

Reference Interval

Flags

Prothrombin time

16.7 sec

10.9–13.0 sec

Lipemia

Partial thromboplastin time

> 150 sec

30.6–35.0 sec

Lipemia

Fibrinogen

245 mg/dL

190–410 mg/dL

Lipemia

Because the laboratory’s policy is to retest after all abnormal coagulation results, the prothrombin time and partial thromboplastin time assays were repeated, and similar values were obtained.

1. Should the operator report the test results as shown?

2. What action should the operator take to address the lipemia flagging of this specimen?

3. Would you expect this patient to be at risk for bleeding based on these test results?

T he coagulation laboratory is an ever-changing environment populated by automated analyzers that offer advances in both volume and variety of tests.1 Hardware and software innovations provide for random access testing with multitest profiles. In the past, the routine coagulation test menu consisted of prothrombin time (PT) with the international normalized ratio (INR), partial thromboplastin time (PTT; also referred to as the activated partial thromboplastin time [APTT]), fibrinogen, thrombin time, and D-dimer assays. More specialized testing was performed in tertiary care institutions or reference laboratories employing medical laboratory scientists with specialized training. With the introduction of new instrumentation and test methodologies, coagulation testing capabilities have expanded significantly, so many formerly “specialized” tests can be performed easily by general medical laboratory staff. New instrumentation has made coagulation testing more standardized, consistent, and cost effective. Automation has not advanced, however, to the point of making coagulation testing foolproof or an exact science. Operators must develop expertise in correlating critical test results with the patient’s diagnosis and when monitoring antithrombotic therapy. Good method validation of procedures, cognitive ability, and theoretical understanding of the hemostatic mechanisms are still required to ensure the accuracy and validity of test results so that the physician can make an informed decision about patient care.

Historical perspective

Visual clot-based testing began in the eighteenth century. The first observation of blood clotting was from blood taken from the vein of a dog that was completely “jellied” in about 7 minutes. In 1780, Hewson measured that human blood clotted in 7 minutes using a basin to collect the blood. With the discovery of the microscope, scientists were able to observe visible clot formation and turbidity.

Many advances took place from 1822 to 1921. These included temperature control during clot formation, passing objects such as a fine needle through the blood to detect resistance, and using different sizes and shapes of glass tubes to view clot formation. In the early 1900s, researchers monitored the length of time it took whole blood to clot in a glass tube while it was being tilted, a precursor to the Lee-White clotting time (1913). These early clotting time tests depended on observing the clot directly (visually) or microscopically.

In 1910, the first clot detection instrument, the “Koaguloviskosimeter,” was developed by Kottman. This apparatus measured the change in viscosity of blood as it clotted. This process generated a voltage change that was recorded by a direct readout system. Voltage changes were plotted against time to measure clot formation.

Except for point-of-care testing and whole-blood platelet aggregometry, citrated plasma (usually platelet-poor plasma, plasma with a platelet count of less than 10,000/μL) has now replaced whole blood in coagulation instruments. However, the principle of interval to clot formation lives on.12

Plasma coagulation testing began in 1920 when Gram added calcium chloride to anticoagulated plasma at 37° C. He measured the increasing viscosity of the blood during fibrin monomer polymerization, a principle used today in thromboelastography (TEG) and sonar clot detection, laying the groundwork for the PT and PTT (Chapter 42).3

In these early days and for many years hence, coagulation testing was typically performed by adding plasma and reagents to a glass tube held in a 37° C water bath. Clot formation was determined by visual inspection of the plasma as the tube was tilted, and a stopwatch was used to determine the time to clot formation. This is referred to as the “tilt-tube technique.”

Nephelometers, developed in 1920, were the first instruments for coagulation testing. These devices measure 90-degree light dispersion of a colloidal suspension. As plasma clots, a change in light scatter can be measured over time—a principle still in use today. Subsequent twentieth-century developments in clot detectors include manual loops, an electromechanical end-point using a movable electrode (BBL Fibrometer) or a rolling steel ball (Diagnostica Stago ST-4), and photo-optical clot detection (Coag-A-Mate, originally manufactured by General Diagnostics).

The 1950s saw the development of the BBL Fibrometer, an instrument that can still be found in coagulation laboratories, although it is no longer being manufactured. This instrument employed an electromechanical clot detection methodology that allowed laboratories to transition from the manual tilt tube or wire loop method to a more accurate semiautomated testing process.

Current coagulation instruments apply many of the clot detection principles of these early analytical systems. They either “observe” the clot formation (optical, nephelometric devices) or they detect the clot by “feel” (mechanical, viscosity-based devices). Although the detection principles remain the same, current instrumentation has been enhanced to eliminate variations in pipetting and end-point detection. They also allow multiple analyses to be performed simultaneously on a single specimen.4

The introduction of new coagulation methodologies as will be described below has further improved testing capabilities in the coagulation laboratory. Refinement of these methodologies has allowed the use of synthetic substrates and measurements of single proenzymes, enzymes, and monoclonal antibodies, which increases the ability to recognize the causes of disorders of hemostasis and thrombosis.5

Assay end-point detection principles

The available coagulometers are automated or semiautomated. Semiautomated coagulometers require the operator to deliver test plasma and reagents manually to the reaction cuvette and limit testing to one or two specimens at a time. These are relatively inexpensive instruments, but their use requires considerable operator expertise.

Fully automated analyzers provide pipetting systems that automatically transfer reagents and test plasma to reaction vessels and measure the end-point without operator intervention (). Multiple specimens can be tested simultaneously. Table 44-1 Automated coagulometers are expensive, and laboratory staff require specialized training to operate and maintain them. Regardless of technology, all semiautomated and automated analyzers offer better coagulation testing accuracy and precision than the manual methods.

TABLE 44-1

Levels of Coagulation Automation

Level

Description

Examples

Manual

All reagents and specimens are transferred manually by the operator. Temperature is maintained by a water bath or heat block; external measurement by operator may be required. End-point is determined visually by the operator. Timer is initiated and stopped by the operator.

Tilt tube 

Wire loop

Semiautomated

All reagents and specimens are transferred manually by the operator. Instrument usually contains a device for maintaining constant 37° C temperature. Analyzer may internally monitor temperature. Instrument has mechanism to initiate timing device automatically on addition of final reagent and mechanism for detecting clot formation and stopping the timer.

Fibrometer 

STart 4 

Cascade M and M-4 

BFT-II 

KC1 and KC4

Automated

All reagents are automatically pipetted by the instrument. Specimens may or may not be automatically pipetted. Analyzer contains monitoring devices and internal mechanism to maintain and monitor constant 37° C temperature throughout testing sequence. Timers are initiated and clot formation is detected automatically.

ACL TOP 

STA-R Evolution 

STA Compact and Compact CT 

Sysmex CA-530, CA-560, CA-620, CA-660, CA-1500, CA-7000 

BCS XP 

CoaLAB

Instrument methodologies used for coagulation testing are classified into five groups based on the end-point detection principle:

1. Mechanical

2. Photo-optical (turbidometric)

3. Nephelometric

4. Chromogenic (amidolytic)

5. Immunologic

Historically, clot detecting instruments were limited to a single type of end-point detection system such as the mechanical or photo-optical detection. Photo-optical detection in instruments that read at a fixed wavelength between 500 nm and 600 nm has become the most commonly used system in today’s clinical instruments. With the advancements in coagulation testing, a second type of instrument was designed to read at 405 nm to perform the chromogenic (colorimetric) assays. With advancements in technology, changes made it possible to automate advanced procedures. However, laboratories were required to purchase multiple analyzers if they wanted to offer the wider range of clot-based and chromogenic testing methods. Since 1990, instrument manufacturers have successfully incorporated multiple detection methods into single analyzers, which allows a laboratory to purchase and train on only one instrument while still providing specialized testing capabilities.4 Immunologics have recently been incorporated into coagulation laboratories for specific analyte measurements.

Mechanical end-point detection

Electromechanical clot detection systems measure a change in conductivity between two metal electrodes in plasma. The BBL Fibrometer was the first semiautomated instrument to be used routinely in the coagulation laboratory. The probe of this instrument has one stationary and one moving electrode. During clotting, the moving electrode enters and leaves the plasma at regular intervals. The current between the electrodes is broken as the moving electrode leaves the plasma. When a clot forms, the fibrin strand conducts current between the electrodes even when the moving electrode exits the solution. The current completes a circuit and stops the timer.6

Another mechanical clot detection method employs a magnetic sensor that monitors the movement of a steel ball within the test plasma. Two principles are used for the mechanical clot detection in the routinely used coagulation instruments. In one system, an electromagnetic field detects the oscillation of a steel ball within the plasma-reagent solution.3 As fibrin strands form, the viscosity starts to increase, slowing the movement (Figure 44-1). When the oscillation decreases to a predefined rate, the timer stops, indicating the clotting time of the plasma. This methodology is found on all Diagnostica Stago analyzers.

Image 

FIGURE 44-1 Viscosimetric (electromechanical) clot detection in a Diagnostica Stago analyzer. A steel ball oscillates in an arc from one side of the cuvette to the other. Movement is monitored continuously within a magnetic field. As the sample clots, viscosity rises and movement of the steel ball is impeded. Variation in amplitude stops the timer, and the interval is the clotting time.

In the second system, a steel ball is positioned in an inclined well. The position of the ball is detected by a magnetic sensor. As the well rotates, the ball remains positioned on the incline. When fibrin forms, the ball is swept out of position. As it moves away from the sensor, there is a break in the circuit, which stops the timer. This technology can be found on AMAX and Destiny instruments distributed by Tcoag US, a division of Diagnostica Stago, as well as on the original Hemochron ACT instruments.

Mechanical methods are not affected by icteric or lipemic plasma. Mechanical methods also provide a sensitive end-point able to detect weak clots such as those formed in plasmas with low fibrinogen or a factor XIII deficiency where clots are not stabilized.

Photo-optical end-point detection

Photo-optical (turbidometric) coagulometers detect a change in plasma optical density (OD, light transmittance) during clotting. Light of a specified wavelength passes through plasma, and its intensity (OD) is recorded by a photodetector. The OD depends on the color and clarity of the sample and is established as the baseline. Formation of fibrin strands causes light to scatter, allowing less light to fall on the photodetector, thus generating an increase in OD. When the OD rises to a predetermined variance from baseline, the timer stops indicating clot formation. Because the baseline OD is subtracted from the final OD, effects of lipemia and icterus are minimized. Many optical systems employ multiple wavelengths that discriminate and filter out the effects of icterus and lipemia. Most of the automated and semiautomated coagulation instruments developed since 1970 use photo-optical clot detection (Figure 44-2).

Image 

FIGURE 44-2 Photo-optical (turbidometric) clot detection. Polychromatic light is focused by a collimator and filtered to transmit a selected wavelength. Monochromatic light is transmitted by fiber optics and focused on the reaction cuvette. As fibrin forms, opacity increases and the intensity of light reaching the sensor decreases. Collim. , Collimator.

Nephelometric end-point detection

Nephelometry is a modification of photo-optical end-point detection in which 90-degree or forward-angle light scatter, rather than OD, is measured. A light-emitting diode produces incident light at approximately 600 nm, and a photodetector detects variations in light scatter at 90 degrees (side scatter) and 180 degrees (forward-angle scatter). As fibrin polymers form, side scatter and forward-angle scatter rise ().Figure 44-34,78 The timer stops when scatter reaches a predetermined intensity, and the interval is recorded.

Image 

FIGURE 44-3 Nephelometric clot detection. A, Light from below passes through the sample in a cuvette to the detector above. B, As fibrin polymerizes, light is deflected and is detected at an angle from the original path.

Nephelometry can be adapted to dynamic clot measurement. Continuous readings are taken throughout clotting, measuring the entire clotting sequence to completion and producing a clot curve or “signature.”

Nephelometry was first applied to immunoassays. As antigen-antibody complexes (immune complexes) precipitate, the resulting turbidity scatters incident light.9 In reactions in which the immune complexes are known to be too small for detection, the antibodies are first attached to microlatex particles. In coagulation, coagulation factor immunoassays employ specific factor antibodies bound to latex particles. Nephelometry provides a quantitative, but not functional, assay of coagulation factors. Nephelometry is often employed in complex automated coagulometers because it allows for both clot-based assays and immunoassays. Nephelometry-style analyzers can be used to produce high-volume multiple-assay coagulation profiles.

Chromogenic end-point detection

Chromogenic (synthetic substrate, amidolytic) methodology employs a synthetic oligopeptide substrate conjugated to a chromophore, usually para-nitroaniline (pNA) (Chapter 42). Chromogenic analysis is a means for measuring specific coagulation factor activity because it exploits the factor’s enzymatic (protease) properties. The oligopeptide is a series of amino acids whose sequence matches the natural substrate of the protease being measured. Protease cleaves the chromogenic substrate at the site binding the oligopeptide to the pNA, freeing the pNA. Free pNA is yellow; the OD of the solution is proportional to protease activity and is measured by a photodetector at 405 nm (Figure 44-4).

Image 

FIGURE 44-4 Method used in the DiaPharma chromogenic factor X assay as an example of a direct chromogenic assay. The method involves two stages. In stage 1, the activator Russell viper venom (RVV) activates factor X (FX) in the presence of calcium to factor Xa (FXa). In stage 2, the generated FXa hydrolyzes the chromogenic substrate S-2765, liberating the chromophore group para-nitroaniline (pNA). Free pNA is yellow in color. The color is then read photometrically at 405 nm. The amount of FXa generated, and thus the intensity of the color, is proportional to the FX activity in the sample over the assay range.

Assays based on a chromogenic end-point (rather than a clot-based assay) are useful to evaluate specimens from patients who have circulating inhibitors or who are on anticoagulant treatment because the inhibitors do not interfere in the chromogenic assay. For clot-based assays, the entire coagulation cascade is part of the test system. For chromogenic assays, the test is isolated to the specific chemical reaction in question.

The activity of coagulation factors and many other enzymes are measured by the chromogenic method directly or indirectly:

• Direct chromogenic measurement: OD is proportional to the activity of the substance being measured—for instance, protein C activity measured by a synthetic chromogenic substrate specific for protein C.

• Indirect chromogenic measurement: The protein or analyte being measured inhibits a target enzyme. It is the target enzyme that has activity directed toward the synthetic chromogenic substrate. The change in OD is inversely proportional to the concentration or activity of the substance being measured—for example, heparin in the anti-factor Xa assay.

Immunologic light absorbance end-point detection

Immunologic assays are the newest assays available in coagulation laboratories and are based on antigen-antibody reactions similar to those used in nephelometry as described previously. Latex microparticles are coated with antibodies directed against the selected analyte (antigen). Monochromatic light passes through the suspension of latex microparticles. When the wavelength is greater than the diameter of the particles, only a small amount of light is absorbed.14 When the coated latex microparticles come into contact with their antigen, however, the antigen attaches to the antibody and forms “bridges,” which causes the particles to agglutinate. As the diameter of the agglutinates increases relative to the wavelength of the monochromatic light beam, light is absorbed. The increase in light absorbance is proportional to the size of the agglutinates, which in turn is proportional to the antigen level.

Immunoassay technology became available on coagulometers in the 1990s and is used to measure a growing number of coagulation factors and proteins, such as D-dimer. These assays, which used to take hours or days to perform using traditional antigen-antibody detection methodologies such as enzyme-linked immunosorbent assay or electrophoresis, now can be done in minutes on an automated analyzer.

Advances in coagulation technology

Significant advances have been made in the capability and flexibility of coagulation instrumentation. Instruments previously required manual pipetting, recording, and calculating the results, which necessitated significant operator expertise, intervention, and time. Current technology allows a “walkaway” environment in which, after specimens and reagents are loaded and the testing sequence is initiated, the operator can move on to perform other tasks.

Clot detection methods have remained consistent, but with the advent of chromogenic- and immunologic-based assays other instrumentation needed to be incorporated into the coagulation laboratory. Multiple methodologies became incorporated into single analyzers to expand their test menu options. From instruments that performed only clot-based assays, clinical laboratory instruments were developed that could perform both clot-based and chromogenic-based assays on one platform.12The next step was the development of a single instrument that could perform clotting and chromogenic and immunologic assays. Additional advances have included improved specimen and reagent storage and processing, increased throughput, and enhanced data management and result traceability.

Improved accuracy and precision

In the days of visual methods, coagulation assays were performed in duplicate to reduce the coefficient of variation, which generally exceeded 20%. Semiautomated instruments have improved upon precision, but the requirement for manual pipetting of plasma and reagents continues to necessitate duplicate testing. With the advent of fully automated instruments, precision has improved to the extent that single testing can be performed with confidence, halving material and reagent costs. Coefficients of variation of less than 5%, and even less than 1% for some tests, have been achieved. Initial accuracy and precision are established by in-lab method validation for all instrument and reagent combinations.

Random access testing

Automated coagulometers now provide random access testing. Through simple programming, a variety of tests can be run in any order on single or multiple specimens within a testing sequence. Previous automated analyzers were capable of running only one or two assays at a time, so batching was necessary. The disadvantage was that specimens with multiple orders had to be handled multiple times. For current automated analyzers, the ability to run multiple tests is limited only by the number of reagents that can be stored in the analyzer and the instrument’s ability to interweave tests requiring different end-point detection methodologies simultaneously, such as clot-based, chromogenic, and immunologic methods. Random access promotes profiling.

Improved reagent handling

Reduced reagent and specimen volumes. 

Automated and semiautomated coagulometers now have the capability to perform tests on smaller sample volumes. Traditionally, PT assays required 0.1 mL of patient plasma and 0.2 mL of thromboplastin/calcium chloride reagent. PTT was measured using 0.1 mL of plasma, 0.1 mL of activated partial thromboplastin, and 0.1 mL of calcium chloride. Current analyzers can perform the same tests using one half or even one quarter the traditional volumes of reagents and patient specimens. This promotes the use of smaller specimen volumes, especially from pediatric patients or those from whom specimens are difficult to draw, and further reduces reagent costs.

Open reagent systems

A variety of reagents from numerous distributors are available for coagulation testing, and laboratory directors want the flexibility of selecting the reagents that best suit their needs without being restricted in their choices by the analyzers being used. Recognizing that the ability to select reagents independently of the test system is a high priority, instrument manufacturers have responded by developing systems that provide optimal performance with alternative manufacturer’s reagents, provided that the reagents are compatible with the instrument’s methodology.

Reagent tracking

Many automated instruments keep records of reagent lot numbers and expiration dates, which makes it easier for the laboratory to maintain reagent integrity and comply with regulatory requirements. Additional features often include on-board monitoring of reagent volumes with flagging systems to alert the operator when an insufficient volume of reagent is present in relation to the number of specimens programmed to be run. Reagent bar coding supports record keeping because it tracks reagent properties and enables the operator to load coagulometers without stopping specimen analyses.

Improved specimen management

Primary tube sampling

Many coagulometers encourage the operator to place the primary collection tube on the instrument after centrifugation, which eliminates the need to separate the plasma into a secondary tube. In addition, instruments often accommodate multiple tube sizes. Significant time savings occur as a result of elimination of the extra specimen preparation step, and errors resulting from mislabeling of the aliquot tube are reduced.

Closed-tube sampling

Closed-tube sampling has improved the safety and efficiency of coagulation testing. After centrifugation, the tube is placed on the analyzer without removing the blue stopper. The cap is pierced by a needle that aspirates plasma without disturbing the red blood cell layer. Not only does closed-tube sampling save staff time, it also reduces the risk of specimen exposure through aerosols or spillage. Closure also promotes plasma pH stabilization. When closed-tube sampling is used, specimens are visually checked for clots after centrifugation by looking for the presence of fibrin strands. For example, if the assay result is a short clotting time (or the corresponding coagulation tracing available on some instruments) is abnormal then the sample will be rimmed with wooden sticks to determine if a clot is present.

Flagging for specimen interferences

Some analyzers monitor the quality of the test specimen for interfering substances or unusual testing characteristics, such as hemolysis, lipemia, bilirubinemia (icterus), abnormal clotting patterns, or results that fall outside the linear range of the reference curve (values above the top point or below the bottom point of the calibration line). Flags warn the operator of potential errors so that problems can be resolved in a timely manner (see later).

Automatic dilutions

Many instruments perform multiple dilutions on patient specimens, calibrators, or controls, eliminating the need for the operator to perform this task manually and reducing the potential for dilution errors. These conditions can be automatically programmed into the individual test setups on the analyzers being used.

Expanded computer capabilities

The computer circuitry of analyzers now incorporates internal data storage and retrieval systems. Hundreds of results can be stored, retrieved, and compiled into cumulative reports. Multiple calibration curves can be stored and accessed. Quality control files can be stored, which eliminates the time-consuming task of manually logging and graphing quality control values. Westgard rules can be applied, and failures are automatically flagged. Some analyzers feature automatic repeat testing when failures occur on the initial run. The quality control files can be reviewed or printed on a regular basis to meet regulatory requirements.

The programming flexibility of modern analyzers has enhanced the laboratory’s opportunities to provide expanded test menus. Most advanced analyzers are preprogrammed with several routine test protocols ready for use. Specimen and reagent volumes, incubation times, and other testing parameters do not need to be predetermined by the operator but can be changed easily when necessary. Additional tests can be programmed into the analyzer by the user whenever needed, which allows for enhanced flexibility of the analyzer and reduces the need for laboratories to have multiple instruments.

Instrument interfacing to laboratory information systems and specimen bar coding capabilities have become a priority as facilities of all sizes endeavor to reduce dependence on manual record keeping. Bidirectional interfaces improve efficiency through the ability of the instrument to send specimen bar code information to the laboratory information systems and receive a response listing the tests that have been ordered. This eliminates the need for the operator to program each specimen and test.

Other automated features

A few additional features offered by current coagulation analyzers should be mentioned:

Improved flagging capabilities alert the operator when preset criteria have been exceeded (Box 44-1). Flags may indicate instrument malfunction such as cuvette jams, low reagent volume, and temperature errors, or a problem with the results such as values that exceed critical limits, inability to detect an accurate end-point, or values outside of the linear range.

BOX 44-1

Warning Flags Available on Coagulometers

Instrument malfunction flags

Temperature error

Photo-optics error

Mechanical movement error

Probe not aspirating

Sample quality flags

Lipemia

Hemolysis

Icterus

Abnormal clot formation

No end-point detected

Reflex testing is the automatic ordering of tests based on preset parameters or the results of prior tests. Instruments may make additional dilutions if the initial result is outside of the linearity limits, or supplementary tests can be run automatically if clinically indicated by the initial test result. The first result does not need to wait for review by the operator before follow-up action is taken.

Graphing of clot formation is provided on analyzers such as the ACL TOP (Instrumentation Laboratory). The graph is generated by an algorithm, a formula used to convert raw optical measurements into a clotting time. Besides determining the clotting time, it also smooths the raw data into a visible curve and uses the curve to check for clot integrity. Multiple checks are performed to ensure an accurate and reproducible result. Should the data not meet all of the acceptable criteria, an error flag is generated. The clot curve is examined to troubleshoot potential technical aberrations. The clot formation graph may also be used as a “signature” that correlates with the disease state. Figure 44-5 shows an example of a typical clot curve.

Image 

FIGURE 44-5 Clot signature produced by the ACL TOP analyzer (Instrumentation Laboratory). The clot curve consists of baseline, acceleration phase, deceleration phase, and end-point. The baseline is recorded before clotting occurs. Acceleration reflects clotting and is normally steep because clotting is rapid. The deceleration phase represents the decreasing rate of clot formation as all available fibrinogen converts to fibrin. The end-point is flat and stable, reflecting consumption of all fibrinogen. A key component in evaluating the clot curve is the y-axis, absorbance, which provides the clot interval when it reaches a defined minimum. Absorbance adjusts to compensate for baseline fibrinogen and interferences such as lipemia or icterus.Source: (Image courtesy of Beckman-Coulter, Brea, CA.)

Specimen quality set points

Specimen quality flags, such as the following, can be included on some coagulation instrumentation:

• Clotted: will cause falsely shortened clotting times because of premature activation of coagulation factors and platelets that generate FVIIa and thrombin.

• Lipemia: may cause falsely prolonged clotting times on OD instruments because of interference with light transmittance.

• Hemolysis: may cause falsely shortened clotting times because of premature activation of coagulation factors and platelets that generate FVIIa and thrombin.18

• Icterus (bilirubinemia): indicates liver dysfunction that may lead to prolonged clotting times because of inadequate clotting factor production; also may interfere with OD instruments.

• Abnormal clot formation: may lead to falsely elevated clotting times because of instrument inability to detect an end-point.

• No end-point detected: indicates that the instrument was unable to detect clot formation; the specimen may need to be tested using an alternate methodology.

Instrument malfunction flags

Instrument malfunction flags, such as the following, can be included on some instrumentation:

• Temperature error

• Photo-optics error

• Mechanical movement error

• Probe not aspirating

• No end-point detected

• Specimen track error

• Cuvette jams

Advantages and disadvantages of detection methods

Photo-optical end-point detection may be confounded by icterus or lipemia, which erroneously prolongs the clotting time because the change in OD is masked by the color or turbidity of the specimen. Some coagulation instruments may be unable to employ synthetic reagents because they are more translucent than reagents used to optimize the end-point detectors.19

All coagulation tests that use clot-based end-points need special considerations when interpreting results. There are many preanalytical variables that affect coagulation function, and the integrity of the entire coagulation cascade is relevant to the final test result. Thus these assays are not highly specific. This would not be a function of chromogenic or immunologic end-point assays that do not rely on the cascade but rather are single analyte specific. Table 44-2 summarizes the advantages and disadvantages of each detection methodology.

TABLE 44-2

Advantages and Disadvantages of Detection Systems

End-Point Detection Method

Advantages

Disadvantages

Mechanical

No interference from specimen lipemia or bilirubinemia (icterus) 

Ability to use specimen and reagent volumes as small as 25 μL in some instruments 

Able to detect weak clots

Reliance on the integrity of the entire coagulation cascade 

Inability to observe graph of clot formation

Photo-optical

Good precision 

Increased test menu flexibility and specimen quality information when multiple wavelengths are used 

Ability to observe graph of clot formation with some instrumentation

Interference from lipemia, hemolysis, bilirubinemia, and increased plasma proteins; this issue has been addressed by some manufacturers with readings from multiple wavelengths 

May not detect short clotting times owing to long lag phase 

May not detect small friable clots that are translucent

Chromogenic

Ability to measure proteins that do not clot 

More specific than clot-based assays 

Expanded menu options to replace clottable assays affected by preanalytical variables such as heparin, thrombin inhibitors (e.g., argatroban, dabigatran) or FXa inhibitors (e.g., rivaroxaban) 

Most automated systems now have cost-effective chromogenic capabilities

Limited by wavelength capabilities of some instruments 

May need large test volume to be cost effective

Immunologic

Ability to automate tests previously available only with manual, time-consuming methods, such as enzyme-linked immunosorbent assays 

Expanded test menu capabilities

Limited number of automated tests available 

Higher cost of instruments and reagents 

May need to have additional instruments available to run routine tests in laboratories without automated coagulation analyzers that have random access capability

Nephelometric

Ability to measure antigen-antibody reactions for proteins present in small concentrations

Limited number of tests available 

Higher cost of reagents 

Need for special staff training

Point-of-care testing

Point-of-care coagulation testing has been used since 1966 in the setting of cardiac surgery using the whole-blood activated clotting time (ACT) for heparin monitoring in the operating room (Chapter 43). Today point-of-care testing has expanded beyond the ACT. Most point-of-care instruments are handheld and permit near-patient testing, bedside testing, self-testing, and testing of infants. The instantaneous turnaround time of results, small sample volume, and portability of the instruments are conveniences appreciated by physicians and patients.

Anticoagulation clinics can be high-volume users of point-of-care testing. Patients on oral anticoagulant therapy with vitamin K antagonists need to be monitored monthly (Chapter 43). Anticoagulation clinics provide this service in the outpatient setting using the prothrombin time/international normalized ratio (PT/INR) test on a point-of-care instrument. Quick test resulting allows for dose adjustments at the same clinic visit, eliminates patient waiting time, and allows time for patient education.

Other point-of-care coagulation tests include the PTT and thrombin clotting time with other assays such as fibrinogen likely available in the future.

summarizes the variety of FDA-cleared point-of-care instruments and assays available.Table 44-320 Various end-point detection techniques are employed. Each instrument uses individual patented technology. The newer versions of these point-of-care instruments include touch screen, wireless transmission of results in real-time, and micro-blood volumes.

TABLE 44-3

A Comparison of Point-of-Care Instruments for Coagulation Testing

Manufacturer/Instrument

Year

Testing

Specimen

Clot Detection

Includes QC

Abbott/iSTAT

2000

PT/INR, PTT, ACT

Whole Blood

Electrogenic

Yes

Alere/INRatio/INRatio2

2003/2008

PT/INR

Fingerstick

Electrochemical, impedance

Yes

Helena/Cascade POC

2000

PT/INR, PTT, ACT, low molecular weight heparin

Whole Blood/Fingerstick

Photo-mechanical

Yes

Helena/Actalyke Mini II

2004

ACT

Whole Blood

2-point electromechanical

Yes

Helena/Actalyke XL

2002

ACT

Whole Blood

2-point electromechanical

Yes

Instrumentation Lab/Gem PCL Plus

2003

PT/INR, PTT, ACT

Whole Blood/Fingerstick

Mechanical end-point monitored optically

Yes

ITC/ProTime Micro Coagulation System

1995/2003/2006

PT/INR

Fingerstick

Mechanical clot

Yes

ITC/Hemochron Signature Elite

2005

PT/INR, PTT, ACT

Whole Blood/Fingerstick

Mechanical clot

Yes

ITC/Hemochron Signature +

2002

PT/INR, PTT, ACT

Whole Blood/Fingerstick

Mechanical clot

Yes

ITC/Hemochron Response

2000

PT/INR, PTT, ACT, TT

Whole Blood/Fingerstick

Mechanical clot

Yes

Medtronics/HMS Plus

1999

ACT, protamine titration

Whole Blood

Mechanical clot

Yes

Medtronics/ACT Plus

2003

ACT

Whole Blood

Mechanical clot

Yes

Roche/CoaguCheck XS PT Test System

2007

PT/INR

Whole Blood/Fingerstick

Amperometric

No

Roche/CoaguCheck XS Plus PT Test System

2007

PT/INR

Whole Blood/Fingerstick

Amperometric

Yes

Roche/CoaguCheck XS Pro PT Test System

2010

PT/INR

Whole Blood/Fingerstick

Amperometric

Yes

CoaguSense PT/INR Monitoring System

2010

PT/INR

Fingerstick

Mechanical

Yes

Point-of-care coagulation analyzers employ capillary (fingerstick) or anticoagulated whole blood (venipuncture). Typically a 10- to 50-μL sample is transferred to a test cartridge and the cartridge is inserted into the test module. Other instruments require nonanticoagulated whole blood, so higher volumes of blood are needed.

Before point-of-care instruments are placed in service, laboratory scientists validate the units against a reference method in a central lab using the plasma-based assays (Chapter 5). Because point-of-care assays and the plasma-based central laboratory assays can show a weak correlation, care must be taken to understand the differences between point-of-care and central lab results and to ensure that clinical decisions are consistent. In the case of the anticoagulation clinic, an INR that exceeds 4.0 or any unexpected INR change is confirmed with a venipuncture blood specimen tested by the plasma-based assay in the central lab.

Whole-blood clotting assays

The TEG Thromboelastograph Hemostasis Analyzer System (Haemoscope, Niles, IL, a division of Haemonetics) is an operator-dependent system that provides global hemostasis assessment. The TEG assesses both bleeding and thrombosis risk in patients. TEG analysis is useful in patients with hepatic disease, having liver transplant surgery, undergoing cardiac surgery, obstetrics patients, and trauma patients. Computerization of TEG facilitates its usefulness in intraoperative hemostasis management, where it helps to predict the need for and to monitor clotting factor administration, platelet transfusion, fibrinolytic therapy, and antiplatelet therapy with medications such as aspirin or clopidogrel.

Citrated whole blood is pipetted into a cylindrical cup that oscillates by 4.75°. A stationary pin with a diameter 1 mm smaller than the cup’s is suspended by a torsion wire in the cup. Kaolin (or another activator) is added to trigger clotting. As the blood clots, fibrin links the pin to the cup, and viscoelasticity changes are transmitted to the pin. The resulting pin torque generates an electrical signal from the torsion wire that is plotted as a function of time to produce a TEG tracing (). The tracing is analyzed to determine the speed, strength, and stability of clot formation and the downstream effect of fibrinolysis. Viscoelasticity depends on procoagulant activity, cellular components (red blood cells, white blood cells, and platelets), and fibrinolysis and the interactions between these components. The trace furnishes real-time information about the evolving clot from platelet activation to initial fibrin formation, fibrin cross-linkage, and fibrinolysis.Figure 44-621 This is the only available whole blood assay that provides a comprehensive, global evaluation of functional hemostasis.

Image 

FIGURE 44-6  Thromboelastograph (TEG) tracing from the TEG 5000 Hemostasis Analyzer System. The TEG® analyzer produces results that document the interaction of platelets with proteins (enzymes, inhibitors, cofactors) of the coagulation pathway and the fibrinolytic system as a whole blood clot forms and eventually breaks down. The R value reflects the initial generation of thrombin and fibrin formation. The K value and angle reflect the rate of initial clot formation mediated by thrombin-activated platelets, fibrin generation, fibrin polymerization, and the developing strength and stabilization of the clot due to platelet function, fibrinogen level, and FXIII. The MA value is an indicator of fibrin-platelet interaction, and overall clot firmness and stability. LY is a kinetic measure of time to and extent of clot lysis. Source: (This image is used with permission of the Haemoscope Corporation, a division of Haemonetics, Niles, IL.)

A newer version is the ROTEM (Tem Innovations GmbH, Munich, Germany). The enhancement of the ROTEM is that it is not sensitive to vibrations. In the ROTEM, a whole-blood sample is placed in a cuvette, and a suspended pin is immersed into the blood. One of various activators is added to trigger clotting. The pin rotates (the cup is stationary), and upon clot formation, the increased tension from fibrin binding the cup to the pin is detected by sensors. ROTEM parameters are the same as those described above for TEG.22 ROTEM reagents are available to assess factor and fibrinogen deficiency, platelet function, fibrinolysis, and anticoagulant influence on hemostasis.

Platelet function testing

The demand for rapid, cost-effective methods for the evaluation of platelet function has increased due to the need to monitor the efficacy of antiplatelet therapy such as aspirin, clopidogrel, and glycoprotein IIb/IIIa inhibitors used in cardiovascular patients. In addition, preoperative evaluation of platelet function is important in hemostatic management, particularly if the patient has a history of bleeding or if the patient is on antiplatelet medication.23 Platelet function testing has been a challenge for the clinical laboratory because of the lack of reliable, accurate, and easy-to-perform methods. In addition, specimen procurement for platelet function testing plays an important role in the reliability and accuracy of the test. Platelet function historically has been assessed by the bleeding time test and platelet aggregation assays (Chapter 42). The bleeding time is technically demanding and is highly dependent on the technician performing the test. In addition, it fails to correlate with intraoperative bleeding risk and thus has been discontinued in most institutions. New platelet aggregometers and several new devices are making it easier to assess platelet function.

Platelet aggregometers

Classical platelet aggregometry using the light transmission principle was developed in the 1960s by Born. This test system measures the increase in light transmission that occurs in direct proportion to platelet aggregation (Figures 42-3 and 42-4) induced by various agonists (e.g., collagen, adenosine diphosphate [ADP], epinephrine) that stimulate different platelet receptors. The test sample is patient platelet-rich plasma (PRP) produced by differential centrifugation of a whole-blood specimen to isolate platelets in plasma with platelet-poor plasma (PPP) as a control. Since its inception, platelet aggregation has been the primary assay to determine alterations in platelet function.

Several newer devices to detect platelet aggregation based on whole-blood impedance, luminescence, and light scatter have since been developed. compares the newest FDA-cleared platelet aggregometers. Table 44-4

TABLE 44-4

A Comparison of Platelet Aggregometers

Parameter

Bio/Data Corp.

ChronoLog Corp.

Helena Laboratories

Instrument name (first year sold)

Platelet Aggregation Profiler, Model PAP-8E (2005)

Whole Blood–Optical Lumi–Aggregation System, Model 700-2/700-4 (2006)

AggRAM (2005)

Operational type

Batch, random access

Batch, random access

Batch, random access

Reagent type

Open reagent system, assay kits, reagents, controls, diluents, buffers

Open reagent system, assay kits, reference plasma, controls

Open reagent system

Operates on whole blood or spun plasma

Spun plasma, optional centrifugation with PDQ to obtain PRP in 3 minutes

Whole blood, spun plasma

Spun plasma

Plasma volume/test

225 μL

225 μL

225 μL

Model type

Benchtop

Benchtop

Benchtop

Number of channels

8

2–4

4–8

Time required for maintenance by lab staff

Weekly:15 minutes; monthly: 30 minutes

30 minutes when optical calibration required

Daily: 15 minutes; weekly: 15 minutes; monthly: 1 hour

The Multiplate Analyzer, also called the Whole-Blood Multiple Electrode Platelet Aggregometer (MEA; Dynabyte, Munich, Germany, distributed by DiaPharma Group, West Chester, OH), monitors platelet function by impedance.28 The multiplate has 5 channels with duplicate electrodes per channel. Small whole blood sample volumes are required (300 μL). The multiplate analysis is based on the principle that upon activation, platelets become sticky and adhere to metal sensor wires (electrodes) (Figure 42-5). The change in electrical resistance between the electrodes is detected and recorded. Three parameters are calculated from each sample: aggregation, area under the curve, and velocity.28 Tests for the therapeutic efficacy of aspirin, clopidogrel, and glycoprotein IIb/IIIa antagonists using arachidonic acid, ADP, and TRAP, respectively, are pending FDA approval. Other applications for platelet function testing are available on this device.

The AggRAM (Helena Laboratories Corporation, Beaumont, TX) is a modular system for platelet aggregation and ristocetin cofactor testing that has advanced laser optics utilizing a laser diode measuring at a wavelength of 650 nm to enhance precision of the measured aggregation tracing.29 The AggRAM has four channels capable of micro-volume testing, customized result reporting, and internal quality control programs.

The PAP-8E from BioData Corporation (Horsham, PA) is an eight-channel platelet aggregometer with a touch screen and on-screen procedure templates (Figure 44-7). It has a programmable pipette and an optional bar code scanner. The PAP-8E utilizes light transmission aggregometry and requires a low sample volume (225 μL). BioData also has a Platelet Function Centrifuge that has been validated for platelet function testing (Figure 44-8).30 PRP is prepared in 30 seconds, and PPP can be prepared in 120 seconds. Platelet-free plasma (PFP) is prepared in 180 seconds.

Image 

FIGURE 44-7 PAP-8E from BioData Corporation. An eight-channel aggregometer with a touch screen and a programmable pipet. This instrument requires a sample volume of 225 μL per test and allows for the analysis of routine platelet aggregation testing, measurement of ristocetin cofactor activity, the monitoring of patients with platelet function abnormalities, and the management of antiplatelet therapy. Source: (Photo courtesy of BioData Corp, Horsham, PA.)

Image 

FIGURE 44-8 The PDQ platelet function centrifuge from BioData Corporation is an optional unit to the PAP-8E. The centrifuge is validated for platelet function testing. The PDQ provides platelet-rich plasma, platelet-poor plasma and platelet-free plasma within 5 minutes. Source: (Photo courtesy of BioData Corp, Horsham, PA.)

Chrono-Log Corporation (Havertown, PA) has a Whole Blood–Optical Lumi–Aggregation System. The Model 700 aggregometer provides platelet aggregation in whole blood or PRP, while simultaneously measuring secretion (Figure 42-6). This aggregometer uses electrical impedance in whole blood and optical density for measuring luminescence. It can be configured as either a two- or four-channel aggregometer.31 Either disposable or reusable electrodes can be used for impedance measurements. In addition, Chrono-Log has several other optical and impedance-based instruments. The advantage of whole-blood aggregometry is that it is a more physiological test due to the ability to measure platelet aggregation in the presence of erythrocytes and leukocytes.

Platelet function analyzers

The PFA-100 Platelet Function Analyzer (Siemens, Deerfield, IL) is a rapid, automated instrument that is sensitive to quantitative and qualitative platelet abnormalities. Test cartridges contain membranes coated with collagen/epinephrine or collagen/ADP to stimulate platelet aggregation. Whole blood is aspirated under controlled flow conditions through a microscopic aperture in the membrane. The time required for a platelet plug to occlude the aperture is an indication of platelet function.3233 The PFA-100 system is successful at detecting von Willebrand disease and the efficacy of aspirin therapy.3334

The Accumetrics VerifyNow System (San Diego, CA) is an optical detection system that measures platelet-induced aggregation by microbead agglutination. The system employs a disposable cartridge that contains lyophilized fibrinogen-coated beads and a platelet agonist specific for the test. Whole blood is dispensed from the blood collection tube into the assay device, with no blood handling required by the operator. The instrument provides an aspirin assay using arachidonic acid as the test reagent, a glycoprotein IIb/IIIa inhibitor (abciximab, tirofiban, eptifibatide) assay using thrombin receptor activation peptide (TRAP) as the test reagent, and a P2Y12 inhibitor (clopidogrel, prasugrel, ticagrelor) assay using ADP as the test reagent.

The Plateletworks platelet function assay is available from Helena Laboratories (Beaumont, TX). This assay kit can be run on any standard impedance cell counter found in the hematology laboratory. Aggregation results are based on a platelet count before (high count) and after (lower count) platelet activation using one of the agonist-filled tubes provided in the kit. Testing requires 1 mL of whole blood for baseline count and 1 mL for each additional agonist-containing reagent tube. Results can be obtained in 2 minutes. The Plateletworks platelet function kit can be used for presurgical screening and to monitor antiplatelet therapy.39

Molecular coagulation testing

Molecular testing in the coagulation laboratory is available for patients with thrombophilia (Chapter 39). Molecular testing has become readily available for gene mutations of factor V (FV Leiden) and prothrombin (prothrombin G20210A). Testing for methylene-tetrahydrofolate reductase (MTHFR) is also commonly performed for patients with thrombophilia. However, the clinical utility of MTHFR testing is not clear, and the American College of Medical Genetics and Genomics (ACMG) has recommended that MTHFR testing should not be routinely performed for the workup of patients with thrombophilia.40

There are several methods available for the clinical laboratory. Cost and labor usually are considered when evaluating which test system to use in the clinical laboratory. The most common methods used are polymerase chain reaction (PCR)-based assays. PCR is accurate for the detection of both point mutations and single-nucleotide polymorphisms. FV Leiden and prothrombin G20210A, and the MTHFR mutations have been shown to be detectable using this method (). A common method to analyze PCR products is restriction fragment-length polymorphism (RFLP) analysis. However, RFLP analysis is not a high-throughput method and is not suitable for high volume laboratories. Other methods that are PCR-based and some non-PCR-based methods, which no longer require restriction digestion, are also used.Table 44-5A popular non-PCR based method is the Invader assay which uses allele-specific hybridization in a high-throughput format.

TABLE 44-5

Molecular Techniques for the Evaluation of Hypercoagulable States

Assay

Accuracy

Throughput

Current Clinical Applications

PCR/RFLP

Good

Limited

Factor V Leiden, prothrombin G20210A, MTHFR

PCR/ARMS

Excellent

Intermediate

Factor V Leiden, prothrombin G20210A, MTHFR

Light cycler

Excellent

Intermediate

Factor V Leiden, prothrombin G20210A, MTHFR

Array technology

Excellent

Very high

Factor V Leiden, prothrombin G20210A, MTHFR

Invader assays

Excellent

Limited

Factor V Leiden, prothrombin G20210A, MTHFR

Ligand-based technologies

Excellent

Very high

Factor V Leiden, prothrombin G20210A, MTHFR

MTHFR, Methylene-tetrahydrofolate reductase.

In order to obtain rapid and reliable results, sequence-specific primers, allele-specific oligonucleotides, hybridization, rapid-cycle PCR using LightCycler instrumentation, and nanochips have become available for molecular testing. The major advantages of molecular testing are the increase in sensitivity and specificity and lack of interference by anticoagulants or inhibitors.4142

Molecular diagnostics in hemophilia and von Willebrand disease are currently limited; however, this is an area that is under development. There is a strong potential for utilization of these assays for the diagnosis and classification of the subgroups of von Willebrand disease. Molecular diagnostics also may have a role in the diagnosis of hemophilia and the mutations involved in both factor VIII and factor IX genes.42 Testing for these disorders will become more accessible with the rapid decrease in cost of sequencing and availability of high-throughput next generation sequencing technologies.

The role of molecular diagnostics in thrombophilia workups will continue to grow due to the identification of new genetic mutations and polymorphisms in coagulation disorders. The challenge is for the laboratory to determine which tests to offer and their relevance to patient care.

Selection of coagulation instrumentation

In today’s laboratory, more than ever before, cost effectiveness, testing capabilities, and standardization are top priorities. As an increasing number of tests become available, laboratories must determine what tests to incorporate to provide guidance to physicians in diagnosis and treatment. Identification of testing needs based on patient population should be the first step in the process. The decisions regarding which tests are the most appropriate for the clinical situations encountered by each laboratory should be made in conjunction with the medical staff. When that input has been obtained, the laboratory can determine the availability and cost of instruments that would meet those requirements.

An instrument should be matched to the anticipated workload. It may not be necessary to purchase a highly sophisticated analyzer capable of performing a large menu of tests if the setting is a small hospital laboratory ordering very few of the more “esoteric” test options available on the instrument under consideration. A batch analyzer with high throughput may be more appropriate for this situation. The option to send out esoteric tests and/or low-volume tests to a reference lab is always available.

Instrument selection criteria may include, but are not limited to, the following:

• Instrument cost

• Consumables cost

• Service response time

• Reliability and downtime

• Maintenance requirements and time

• Operator ease of use

• Breadth of testing menu

• Ability to add new testing protocols

• Reagent lot to lot variation

• Throughput for high-volume testing

• Laboratory information systems (LIS) interface capabilities

• Footprint (the space the instrument occupies; benchtop or floor model)

• Special requirements (water, power, waste drain)

• Flexibility in using other manufacturers’ reagents

• Availability of a training program and continued training support

When the choices have been narrowed based on the most desirable criteria, consideration should be given to additional features. Because no instrument has all the desired features, prioritizing helps the laboratory focus on the capabilities that would be the most advantageous for them. summarizes several of these specialized features. Box 44-2

BOX 44-2

Specialized Coagulometer Features

Random access: A variety of tests can be performed on a single specimen or multiple specimens in any order as determined by the operator.

Primary tube sampling: Plasma is directly aspirated from an open or capped centrifuged primary collection tube on the analyzer.

Cap piercing: Analyzer aspirates plasma from the closed centrifuged primary collection tube.

Bar coding: Reagents and specimens are identified with a bar code; eliminates manual information entry.

Bidirectional laboratory information system (LIS) interface: Analyzer queries the host computer (LIS) to determine which tests have been ordered. Results are returned to the LIS after verification.

Specimen and instrument flagging: Automated alerts indicate problems with specimen integrity or instrument malfunction.

Liquid level sensing: Operator is alerted when there is inadequate specimen or reagent volume. An alert is also given when the instrument fails to aspirate the required sample volume. Volume is verified each time a specimen or reagent is aspirated.

On-board quality control: Instrument stores and organizes quality control data; may include application of Westgard rules for flagging out-of-range results; instrument may transmit quality control data to the LIS.

Stat capabilities: Operator can interrupt a testing sequence to place a stat specimen next in line for testing.

On-board refrigeration of specimens and reagents: Refrigeration maintains the integrity of specimens and reagents throughout testing and allows reagents to be kept in the analyzer for extended periods, which reduces setup time for less frequently performed tests.

On-board specimen storage capacity: Indicates the number of specimens that can be loaded at a time.

Reflex testing: Instrument can be programmed to perform repeat or additional testing under operator-defined circumstances.

Patient data storage: Test results can be stored for future retrieval; clot formation graphs may be included.

Throughput: Number of tests that can be processed within a specified interval, usually the number of tests per hour; depends on test mix and methodologies.

Total testing (dwell) time: Length of time from specimen placement in the analyzer until testing is completed; depends on the type and complexity of the procedure.

Graph of clot formation: Operator can visualize how the clot is formed over time.

Currently available instruments

A variety of coagulometers address the increasing demand for test volume, random access testing, and test variety. All analyzers perform routine testing quickly and efficiently. The challenge lies in determining which instruments should be considered for a particular laboratory setting and in developing an organized approach for their evaluation. lists several of the coagulation analyzers currently available, the type of end-point detection offered, and selected specialized features highlighted by the manufacturers in their product information.Table 44-64445

TABLE 44-6

Comparison of the Available Coagulation Analyzers

Instrument Name/ Manufacturer

Sample Handling System

FDA-Cleared Clot-Based Tests

FDA-Cleared Chromogenic Tests

FDA-Cleared Immunologic Tests

Methodologies Supported

Number of Different Assays Onboard Simultaneously

Standard Specimen Volume PT/PTT

Detection of Hemolysis/Turbidity

Onboard Patient Dilutions

American 

Labor 

Lab A.C.M.

Cuvette, semi automated

PT, PTT, fibrinogen, any clot-based assay

None

None

Clot detection, optical

2

Manual pipetting

No/no

No

Diagnostica Stago 

STA Satellite

Carousel—primary tube

PT, PTT, fibrinogen

Heparin (UFH, LMWH), AT

D-dimer

Clot detection, mechanical; chromogenic; immunologic

80

5 μL

No/no (mechanical method)

Yes

Diagnostica Stago 

STA-R Evolution

Rack with continuous access

PT, PTT, TT, fibrinogen, reptilase, factors, protein C, protein S, lupus anticoagulant, DRVVT screen and confirm

Heparin (UFH, LMWH), protein C, AT, plasminogen, antiplasmin

D-dimer, VWF, total and free protein S, AT antigen

Clot detection, mechanical; chromogenic; immunologic

200

5 μL

No/no (mechanical method)

Yes

Diagnostica Stago 

STA Compact, CT

Continuous specimen access—primary tube

PT, PTT, TT, fibrinogen, reptilase, factors, protein C, protein S, lupus anticoagulant, DRVVT

None

None

Clot detection, mechanical

80

5 μL

No/no (mechanical method)

Yes

Diagnostica Stago 

Start4 Hemostasis

Manual

PT, PTT, TT, fibrinogen, reptilase, factors, protein C, protein S, lupus anticoagulant

None

None

Clot detection

1

25 μL

No/no (mechanical method)

No

Diagnostica Stago 

STA Compact

Continuous specimen access—primary tube

PT, PTT, TT, fibrinogen, reptilase, factors, protein C, protein S, lupus anticoagulant, DRVVT screen and confirm

Heparin (UFH and LMWH), protein C, AT, plasminogen, antiplasmin

D-dimer, VWF, total and free protein S, AT antigen

Clot detection; chromogenic; immunologic

80

50 μL/5 μL

No/no (mechanical method)

Yes

Diagnostica Stago 

STA Compact Plus

Continuous specimen access—primary tube

PT, PTT, TT, fibrinogen, reptilase, factors, protein C, protein S, lupus anticoagulant, DRVVT screen and confirm

Heparin (UFH and LMWH), protein C, AT, plasminogen, antiplasmin

D-dimer, VWF, total and free protein S, AT antigen

Clot detection, mechanical; chromogenic; immunologic

80

50 μL/5 μL

No/no (mechanical method)

Yes

Diagnostica Stago 

Destiny Plus

Continuous rack loading

Open system: all clottable assays; PT, PTT, fibrinogen, TT, factors, venom time, protein C, protein S, aPCR, lupus screen and confirm

Open system: all chromogenic assays (protein C, AT FIIa and FXa based, heparin anti-FXa, plasminogen

Open system: all latex immunoassays (D-dimer)

Clot detection, mechanical and optical (turbidometric); chromogenic; immunologic

10

25 μL/10 μL

Not necessary

Yes

Diagnostica Stago 

Destiny Max

Continuous rack loading

Open system: all clottable assays; PT, PTT, fibrinogen, TT, factors, venom time, protein C, protein S, aPCR, lupus screen and confirm

Open system: all chromogenic assays (protein C, AT FIIa and FXa based, heparin anti-FXa, plasminogen)

Open system: all latex immunoassays (D-dimer)

Clot detection, mechanical and optical; chromogenic; immunologic

Unlimited

25 μL/10 μL

Not necessary

Yes

Helena Laboratories 

Cascade M-4

Manual

PT, PTT, fibrinogen, TT, factors II, V, VII to XII

None

None

Clot detection, optical, turbidometric

4

100 μL

No/no

No

Helena Laboratories 

Cascade M

Manual

PT, PTT, fibrinogen, TT, factors II, V, VII to XII

None

None

Clot detection, optical, turbidometric

1

100 μL

No/no

No

Instrumentation Laboratory 

ACL 300

Continuous rack loading

PT, PTT, fibrinogen, TT, factors, FVIII (with VWF)

 

D-dimer HS

 

500

 

No/no

Yes

Instrumentation Laboratory 

ACL 500

Continuous rack loading

PT, PTT, fibrinogen, TT, factors, lupus (SCT and DRVVT), protein C, protein S, aPCR-V, FVIII (with VWF)

Heparin anti-FXa, protein C, AT, plasminogen, antiplasmin

D-dimer, D-dimer HS, VWF (activity and antigen), free protein S, FXIII antigen, homocysteine

Clot detection, LED optical; chromogenic; immunologic (turbidometric)

500

PT and PTT: 50 μL; FVIII: 25 μL

No/no

Yes

Instrumentation Laboratory 

ACL 700

Continuous rack loading

PT, PTT, fibrinogen, TT, factors, lupus (SCT and DRVVT), aPCR-V, protein C, protein S, FVIII (with VWF)

Heparin anti-FXa, protein C, AT, plasminogen, antiplasmin

D-dimer, D-dimer HS, VWF (activity and antigen), free protein S, FXIII antigen, homocysteine

Clot detection, LED optical; chromogenic; immunologic

500

PT and PTT: 50 μL; FVIII: 25 μL

No/no

Yes

Instrumentation Laboratory 

ACL Elite Series

Tray-primary tubes

PT, PTT, fibrinogen, TT, factors, protein C, protein S, lupus (SCT and DRVVT), aPCR-V

Heparin anti-FXa, protein C, AT, plasminogen, antiplasmin, FVIII

D-dimer, VWF (activity and antigen), free protein S, FXIII antigen, homocysteine

Clot detection, LED optical (nephelometric); chromogenic; immunologic

22

PT and PTT: 60 μL; FVIII: 18 μL

No/no

Yes

Siemens 

BFT II

Manual

PT, PTT, fibrinogen

None

None

Turbodensitometric

1

50 μL

No/no

No

Siemens 

CA-1500

10-Tube position sample rack × 5

PT, PTT, fibrinogen, TT, reptilase time, factors, DRVVT screen and confirm, FV Leiden, protein C clot, protein S activity

Innovance AT, Berichrom AT, plasminogen, FVIII chromogenic, antiplasmin, protein C chromogenic, heparin

Innovance D-dimer

Clot detection, optical, turbidometric; chromogenic; immunologic

15

50 μL/5 μL

No/yes

Yes

Siemens 

CA-7000

Rack

PT, PTT, fibrinogen, TT, reptilase time, factors, DRVVT screen and confirm, FV Leiden, protein C clot, protein S activity

Innovance AT, Berichrom AT, plasminogen, FVIII chromogenic, antiplasmin, protein C chromogenic, heparin

Innovance D-dimer

Clot detection, optical, turbidometric; chromogenic; immunologic

20

50 μL/5 μL

No/yes

Yes

Siemens 

BCS XP

10-Tube position sample rack

PT, PTT, fibrinogen, TT, reptilase time, factors, DRVVT screen and confirm, FV Leiden, protein C clot, protein S activity

Innovance AT, Berichrom AT, plasminogen, FVIII chromogenic, antiplasmin, protein C chromogenic, heparin

Innovance D-dimer

Clot detection, optical (xenon flasher lamp); chromogenic; immunologic

> 100

50 μL/20 μL min. 100 μL (includes dead volume)/50 μL

Yes/no

Yes

Siemens 

CA-600

10-Tube position sample rack

PT, PTT, fibrinogen, TT, reptilase time, protein C clot, factor assays

Innovance AT, Berichrom AT, protein C chromogenic, heparin

Innovance D-dimer

Clot detection, optical; turbidometric; chromogenic; immunologic

5

50 μL/5 μL

No/yes

Yes

LABiTech GmbH 

CoaData 2004

Semiautomated manual pipette-auto start

PT, PTT

None

None

Clot detection, optical; turbodensitometric

1

50 μL

No/no

No

aPCR, Activated protein C resistance; AT, antithrombin; DRVVT, dilute Russell viper venom test; F, factor; LMWH; low molecular weight heparin; HS, high sensitivity; PT, prothrombin time; PTT, partial thromboplastin time; SCT, silica clotting test; TT, thrombin clotting time; UFH,unfractionated heparin; VWF, von Willebrand factor.

Summary

• Advanced technology used in semiautomated and automated analyzers has greatly improved coagulation testing accuracy and precision.

• End-point detection methodologies employed by modern coagulation analyzers include mechanical, photo-optical, nephelometric, chromogenic, and immunologic methods.

• Advances in end-point detection methodologies have greatly expanded the testing capabilities available in the routine coagulation laboratory.

• Markedly improved instrument precision and reduced reagent volume requirements have led to substantial cost savings in coagulation testing.

• Instrument manufacturers have incorporated many features that have enhanced efficiency, safety, and diagnostic capabilities in hemostasis testing.

• Coagulation analyzer flagging alert functions warn the operator when sample or instrument conditions exist that might lead to invalid test results so that appropriate actions can be taken to ensure test accuracy.

• Each method of end-point detection has advantages and disadvantages that must be recognized and understood to ensure the validity of test results.

• Several methods to evaluate platelet function are available for both general platelet function testing and antiplatelet drug monitoring.

• The role of molecular diagnostics will continue to grow to identify new mutations and polymorphisms associated with bleeding and clotting disorders.

• Coagulation testing has been incorporated into the arena of point-of-care testing primarily to enhance the patient’s and physician’s ability to monitor oral anticoagulant therapy.

• A systematic approach to the evaluation and selection of a new coagulation analyzer should be developed and followed to determine the best instrument for a specific laboratory setting.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. The photo-optical method of end-point detection can be described as:

a. Measurement of a color-producing chromophore at a wavelength of 405 nm

b. Measurement of the change in OD of a test solution as a result of fibrin formation

c. Application of an electromagnetic field to the test cuvette to detect the decreased motion of an iron ball within the cuvette

d. Measurement of the turbidity of a test solution resulting from the formation of antigen-antibody complexes using latex particles

2. Modern coagulation analyzers have greatly enhanced the ability to perform coagulation testing as a result of which of the following?

a. Maintenance of a level of accuracy and precision similar to that of manual methods

b. Increase in reagent volume capabilities to improve sensitivity

c. Automatic adjustment of results for interfering substances

d. Improved flagging capabilities to identify problems in sample quality or instrument function

3. Which of the following is considered to be an advantage of the mechanical end-point detection methodology?

a. It is not affected by lipemia in the test sample

b. It has the ability to provide a graph of clot formation

c. It can incorporate multiple wavelengths into a single testing sequence

d. It can measure proteins that do not have fibrin formation as the end-point

4. Which of the following methods use the principle of changes in light scatter or transmission to detect the end-point of the reaction?

a. Immunologic, mechanical, photo-optical

b. Photo-optical, nephelometric, mechanical

c. Photo-optical, nephelometric, immunologic

d. Chromogenic, immunologic, mechanical

5. Which of the following is a feature of semiautomated coagulation testing analyzers?

a. The temperature is maintained externally by a heat block or water bath

b. Reagents and samples usually are added manually by the operator

c. Timers are automatically started as soon as the analyzer adds reagents to the test cuvette

d. The end-point must be detected by the operator

6. When a sample has been flagged as being icteric by an automated coagulation analyzer, which method would be most susceptible to erroneous results because of the interfering substance?

a. Mechanical clot detection

b. Immunologic antigen-antibody reaction detection

c. Photo-optical clot detection

d. Chromogenic end-point detection

7. Platelet function testing has been incorporated into the routine coagulation laboratory in recent years as a result of:

a. Increased use of drugs that stimulate platelet production in patients receiving chemotherapy

b. The convenience of being able to do the testing on the same instrument that performs the coagulation testing

c. Increased therapeutic use of aspirin in the treatment of heart disease

d. Increased outpatient/outreach testing that prevents the laboratory from having access to patients to do bleeding time tests

8. All of the following are performance characteristics to consider in the selection of a coagulation analyzer except:

a. Location of the manufacturer’s home office

b. Instrument footprint

c. Ease of use for the operator

d. Variety of tests the instrument can perform

9. The PFA-100 measures platelet function by:

a. Detecting the change in blood flow pressure along a small tube when a clot impairs blood flow

b. Detecting the aggregation of latex beads coated with platelet activators

c. Graphing the transmittance of light through platelet-rich plasma over time after addition of platelet activators

d. Detecting the time it takes for a clot to form as blood flows through a small aperture in a tube coated with platelet activators

10. Point-of-care coagulation testing is used mainly:

a. To monitor patients receiving oral anticoagulant therapy

b. To monitor patients taking platelet inhibitors such as aspirin

c. To provide a baseline for all subsequent patient test result comparisons when the patient starts any kind of anticoagulant therapy

d. To monitor obstetric patients at risk of fetal loss

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*The authors acknowledge the substantial contributions of David L. McGlasson, MS, MLS(ASCP), to Chapter 47 of the fourth edition of this textbook, many of which continue in this edition.