The Science, Technology, and Trends of Coagulation Analyzers

The science behind the human body’s immune response after a blood vessel is damaged is very complex and highly coordinated. After the blood vessels are damaged, the bleeding must stop and then the blood must clot. While the body’s immune response works to stem the bleeding, multiple factors and mechanisms jump into play. In the clinical world, the study of clotting factors, bleeding or excessive bleeding, and the monitoring of such effects is an enormous field. Not only are there diseases that cause reduced clot formation, but prevention of blood coagulation—the exact opposite—is required postsurgery for wounds to heal.

Coagulation monitoring, especially perioperative, is important to diagnose potential causes of hemorrhage, to guide hemostatic therapies, and to predict the risk of bleeding during consecutive surgical procedures.

Initiation of Hemostasis

Hemostasis—in which blood changes from a fluid to a solid state—is a complex mechanism, In general, intact blood vessels contain endothelial cells that prevent clot formation via tissue plasminogen factor, inactivating thrombin, and ADP. However, when blood vessels are damaged, the body establishes a few basic mechanisms in order to stop bleeding or hemostasis:

• First, in the short-lived stage of primary hemostasis, there is a reflex reaction that promotes constriction of the vessels, reducing blood loss.
• Then, the exposed collagen from the damaged site accelerates platelet adhesion to the site. This enables the release of cytoplasmic granules that contain vasoconstrictors, such as serotonin, ADP, and Thromboxane A2.
• ADP enables more platelet aggregation to the damage site. Thromboxane A2 promotes aggregation, further degranulation, and, ultimately, vasoconstriction. Platelet aggregation occurs less than half a minute postinjury.
• This allows for the formation of a platelet plug, which ultimately allows for hemostasis to enable coagulation. Secondary hemostasis occurs after that and is responsible for stabilizing the soft clotting and maintaining vasoconstriction.

Formation of a blood clot:
The damaged tissues release multiple coagulation factors (produced in the liver). This takes place in a cascade mechanism:

• Factor III, which activates Factor VII in the presence of Ca(II).
• Factor XII from active platelets activate Factor XI. These steps initiate the extrinsic and intrinsic mechanisms of clot formation.
• Eventually, Factor X is activated via cascade reactions promoted by Factor VII and Factor XI.
• Active Factor X then activates prothrombin activator, in the presence of Factor III, Factor V, Ca (II), and platelet thromboplastic factor PF3.
• This is followed by the formation of thrombin from prothrombin via prothrombin activator. Thrombin then converts fibrinogen to fibrin, which initially forms a loose mesh, but in the presence of Factor XIII, forms further covalent crosslinks, allowing the fibers to aggregate densely, trapping platelets and RBCs, to form a blood clot.

Technologies for Coagulation Analysis

In the recent past, the numbers of ordered coagulation screening tests have increased tremendously. This, in turn, has improved the efficiency of automated coagulation testing, enabling the generation of high-throughput, accurate, and precise analyzers with minimal human error in measurement.

Initial coagulation analyzers were operated mechanically, using a hook to detect a clot in the cuvette. This has now been replaced by simultaneous detection of clotting factors via clotting, colorimetric, and immuno principles.

Current technologies use automated platelet function analyzers, flow cytometers, PCR, and microarrays. Combinations of all these technologies eliminate preanalytical and postanalytical handling, yielding accuracy and increased productivity.

A quick and standard test is the PT/INR test. A prothrombin time (PT) test (traditional) shows activation via mechanical or optic means when the sample is mixed with thromboplastin. Activated thrombin converts fibrinogen to fibrin, and clots (extensive and local) are detected. The test results are reported as an International Normalized Ratio (INR) in seconds. The INR is then recommended during oral anticoagulant therapy monitoring. INR is determined as the ratio of patient prothrombin time (s) ISI, to mean normal prothrombin time (s).

While PT/INR tests are routine, the value of these tests have been questioned in the acute perioperative setting since there is a delay of nearly an hour from sampling to results. Tests are determined in plasma rather than in whole blood, while no information is available on platelet function. Additionally, assays are run at 37°C rather than at patient body temperature.

Coagulation Analysis Products

The point-of-care products from Abbott Diagnostics, Abbott Park, Ill, include the i-STAT PT/INR in vitro diagnostic test for quantitative monitoring of oral anticoagulant therapy monitoring. While limited in its capability to evaluate individual factor deficiencies, it works well to identify the coagulation end point when used in conjunction with the i-STAT Portable Clinical Analyzer and i-STAT 1 Analyzer. The cartridge is designed to accept sample volumes between 20 and 45 µL.

Since 1994, the POC for PT/INR systems from Roche Diagnostics, Indianapolis, have enabled health care professionals to allow closer patient monitoring and care management, especially in the case of Warfarin (Coumadin®) therapy. According to Courtney Sweeney of Roche Diagnostics, the CoaguChek® XS Plus System uses exclusive smart technology to help ensure accurate PT/INR results, and offers new QC and data-management capabilities for health care professionals who manage anticoagulation patients at the point of care. The system has data management streamlined to store 1,000 patient and 500 optional QC test results. Sample volumes of about 8 µL are small, easy to use, and convenient.

Other products related to coagulation analysis focus primarily on hemostasis and give not only PT/INR data but also information on the coagulation factors. Beckman Coulter, Brea, Calif, in partnership with Instrumentation Laboratory (IL), Lexington, Mass, recently released its latest analyzer in hemostasis testing, ACL TOP 500 CTS (closed tube sampling) for hemostasis, to be used in mid- to high-volume laboratories with more than 800 samples tested per run and 240 test results per hour. Its prothrombin test is delivered in about 3 minutes, while other clotting, immunoturbimetric, and chromogenic tests can also be performed in this analyzer.

In addition, Beckman Coulter has improved the HemosIL D-Dimer HS test to be run on the ACL TOP analyzers to exclude venous thromboembolism (VTE) in outpatients, eliminating or reducing interference from rheumatoid factor, bilirubin, hemoglobin, and lipemia. Results from the combination of the hemostasis analyzer and the D-dimer test has enabled Beckman Coulter analyzers to yield results in about 10 minutes.

Another player in the hemostasis field is Siemens Healthcare Diagnostics, Deerfield, Ill. Working with its partner, Sysmex Corp, Mundelein, Ill, it has developed a new generation of hemostasis analyzers, the Sysmex® CS-2000i System, which is designed to maximize efficiency and reduce cost and time while focusing on preanalytical sample integrity.

Siemens is also developing new reagents and analyzers, according to Jackie Hauser, the company’s senior marketing manager, US hemostasis. It released the Siemens Innovance® D-Dimer assay in 2009, available on BCS®/BCS® XP, Sysmex® CA-560, CA-1500, and CA-7000 automated coagulation systems.

According to Hauser, “This assay allows labs of all sizes the opportunity to efficiently perform D-Dimer testing in their coagulation department.” The Innovance D-Dimer test assay has received clearance for diagnostic exclusion of pulmonary embolism. In addition, the Innovance Antithrombin assay was recently released, adding to Siemens’ portfolio of thrombosis risk assays.

Diagnostica Stago (US), Parsippany, NJ, which calls itself “the coagulation company,” has a plethora of coagulation analyzers that can be used for small-, mid-, and large-volume sample runs. Using what is termed as the “Diagnostica Viscosity Detection System,” the analyzers reduce interference from lipemia, icterus, or hemolysis, increasing accuracy and precision. When viscosity changes due to clot formation are used as a method of detection, the natural thickening/clotting of a reaction is monitored in real time as change takes place. The final result, then, is an accurate depiction of the patient sample’s clotting ability.

According to Larry Wright, the company’s US systems product manager, these products make the company stand apart from others. The STA-R Evolution is a large-volume analyzer with lab automation; STA Compact® is a medium-volume benchtop analyzer; STA Compact CT is used for clotting assays only; Start® 4 is a semiautomated version for low-volume runs; while the STA Satellite® is a fully automated benchtop version capable of clotting, chromogenic, and immunologic assays. The reagents used are standardized across lines, and accessory software is consistent as well, providing automatic validation for patient results.

While coagulation analysis and monitoring tests, and POC analyzers, bring results quickly and reliably to the clinician for health care monitoring, several concerns have been raised regarding the sample processing time and blood collection site—even how patient age and gender may alter the test result. Therefore, in order to interpret results accurately during a run, a standardized test with controls needs to be run on the same assay and analyzer.

The need for such products has given companies such as Bio-Rad Laboratories Inc, Hercules, Calif, an opportunity to provide coagulation controls like the Lyphochek© Coagulation Control. This control is intended for use as a QC plasma control to monitor citrated coagulation systems. The control can be used in any lab providing coagulation analysis and is flexible enough for use in low-, medium-, or high-volume sample analyzers.

In addition, this coagulation control can be used in a variety of instruments and assays, including Diagnostica Stago STA Neoplastine CI + / Roche STA Neoplastin Plus, IL HemosIL PT-Fibrinogen, IL HemosIL PT-Fibrinogen HS Plus, IL HemosIL PT-Fibrinogen Recombinant, IL HemosIL RecombiPlas Tin, IL HemosIL RecombiPlas Tin 2G, Siemens Dade Innovin, Siemens Dade Thromboplastin C Plus, Siemens Thromborel S, Trinity Biotech TriniCLOT PT Excel, and Trinity Biotech TriniCLOT PT Excel S. Compared to other coagulation controls, which have an 8-hour shelf life, Bio-Rad’s Lyphocheck Coagulation Control has a 2-day shelf life.

Direction of Coagulation Products

While most coagulation analyzers are heading toward accuracy, ease of use, and timely results, focus has shifted to better bioinformatics and integrated software across multiple platforms and analyzers. “Informatics is an area of growing interest for clinical laboratories,” Hauser states. “Our customers are looking for IT solutions that will help them leverage data management, improve process flow in their lab, and monitor instrument events—for example, error flags and service events.” The idea behind their products is to yield enhanced diagnostic value for clinicians.

Hauser also notes that clinical labs look for a full continuum of hemostasis instrumentation to meet small-, medium-, and large-volume lab needs. Therefore, it is essential within Integrated Delivery Network systems to provide product fits across a broad range of services.

Bioinformatics also plays an important role in Diagnostica Stago’s (US) new product portfolio. Wright notes that the newly released STA Coag Connect, its new software accessory, is a perfect example of innovation in enabling the consolidation, enhancement, and standardization of patient sample results and QC.

“Since Diagnostica Stago (US) analyzers use the same high-quality reagents, it allows for standardization throughout hospitals, saving time and reducing inventory management cost,” he adds. This, in combination with the software, standardizes results and allows for quality control across runs, samples, and analyzers.

Conclusions

Coagulation analyzers have come a long way from measuring the optical density of a clot in a cuvette. Now, not only are there multiple factor analyses across runs that are standardized, but improved and ever-precise software allows for hospital runs to be standardized, easy to use, and quality controlled. Monitoring of perioperative patients using coagulation analyzers is now significantly faster, easier, and more accurate.

The direction that this field is now moving is toward integrated analyzing software, high robustness of devices, a wide variety of ranges in these analyzers, and increased precision of results with reduced analysis time.

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Madhushree Ghosh, PhD, is a San Diego-based science and health writer.