Coagulation Instrumentation and Technology

Hemostasis and Coagulation Instrumentation

Overview

  • The clinical coagulation laboratory is continuously evolving with advancements in automated analyzers.
  • Routine coagulation tests include Prothrombin Time (PT) with International Normalized Ratio (INR), Partial Thromboplastin Time (PTT), fibrinogen, and thrombin time assays.
  • Specialized testing, once confined to tertiary care or reference labs, is now more accessible due to new instrumentation and methodologies.
  • New instrumentation has standardized coagulation testing, making it more consistent and cost-effective.
  • While automation has improved coagulation testing, it isn't foolproof; expertise in correlating test results with patient diagnosis and antithrombotic therapy monitoring remains crucial.
  • Good method validation, cognitive ability, and a theoretical understanding of hemostatic mechanisms are vital for accurate test results and informed clinical decisions.

Historical Perspective of Blood Clot Assessment

  • Visual clot-based testing began in the 18th century.
  • In 1780, Hewson measured human blood clotting time as 7 minutes using a basin to collect blood.
  • The discovery of the microscope allowed scientists to observe visible clot formation and turbidity.
  • From 1822 to 1921, advances included temperature control during clot formation, resistance detection via a fine needle, and the use of glass tubes to view clot formation.
  • In the early 1900s, researchers monitored the time it took whole blood to clot in a tilted glass tube, a precursor to the Lee-White clotting time (1913).
  • These early tests depended on direct visual or microscopic observation of the clot.
  • In 1910, Kottman developed the “Koaguloviskosimeter,” the first clot detection instrument, measuring the change in the viscosity of blood as it clotted.

Assay End-Point Detection Principles

  • Instrument methodologies for high-volume coagulation and hemostasis testing are classified into six groups based on the end-point detection principle:
    • Mechanical
    • Photo-optical (turbidometric)
    • Nephelometric
    • Chromogenic (amidolytic)
    • Immunologic
    • Viscoelastic
  • Coagulation instruments either "observe" clot formation (optical and nephelometric devices) or detect the clot by "feel" (mechanical and viscosity-based devices).
  • Original instruments typically had a single end-point detection system based on the mechanical principle.
  • Photo-optical detection, reading at a fixed wavelength between 500 and 600 nm, is the most common end-point in clinical coagulation instruments today.

Mechanical Clot 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 used routinely in coagulation labs.
  • During clotting, the moving electrode enters and leaves the plasma at regular intervals, breaking the current between the electrodes.
  • When a clot forms, the fibrin strand conducts current between the electrodes even when the moving electrode exits the solution, completing a circuit that stops the timer.
  • Another method uses a magnetic sensor that monitors the movement of a steel ball within the test plasma.

Photo-Optical Clot End-Point Detection

  • Photo-optical (turbidometric) coagulometers detect a change in plasma optical density (OD; light transmittance) during the clotting process.
  • Light of a specified wavelength passes through clots, a change in light scatter can be measured over time, a principle still in use today.
  • The 1950s witnessed the dawn of the modern era of instrumentation in coagulation testing with the development of the first coagulometer, the BBL Fibrometer.
  • This instrument used a movable electrode that detected a plasma clot via electromechanical methodology.
  • This advancement allowed laboratories to transition from the manual tilt tube or the manual wire loop method to a more accurate semiautomated testing process.

Viscoelastic Clot Detection

  • The viscoelastic technique used to detect clot formation in the past is still used today in particular instruments for whole blood clotting.
  • These assays are not high volume, but their design provides much information about the entire blood clotting process.
  • Depending on the instrument and assay, information can be obtained on the time to clot, kinetics of whole blood clot formation, clot strength, and fibrinolytic activity.

Chromogenic End-Point Detection

  • Chromogenic (synthetic substrate, amidolytic) methodology uses a synthetic small peptide substrate conjugated to a chromophore, usually para-nitroaniline (pNA).
  • Chromogenic analysis is used to measure the activity of a specific coagulation factor because it exploits the factor’s enzymatic (protease) properties.
  • The oligopeptide substrate is a series of usually three amino acids whose sequence matches the natural substrate of the factor being measured.
  • The hemostatic factor cleaves the chromogenic substrate at the site binding the oligopeptide to the pNA, freeing the pNA.
  • Free pNA is yellow (bound pNA is clear).
  • The OD of the solution is proportional to protease activity and is measured by a photodetector at 405 nm.
  • Simple spectrophotometers can be used for end-point detection.

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.
  • The timer stops when scatter reaches a predetermined intensity, and the time to clot interval is recorded.
  • Nephelometry can be adapted to measure the dynamics or kinetics of clot formation.

Immunologic Light Absorbance End-Point Detection

  • Immunologic assays are the newest assays available for routine coagulation testing.
  • These assays are based on antigen-antibody reactions.
  • In slight contrast to the nephelometry principle described previously, which uses a light scatter end-point, another common means to detect an end-point in an immunoassay is to use light absorbance.
  • 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.
  • When the coated latex microparticles come into contact with their antigen, however, the antigen attaches to the antibody and “bridges” are formed, which causes the particles to agglutinate.

Advantages and Disadvantages of Detection Methods

Mechanical
  • Advantages:
    • No interference from specimen lipemia or bilirubinemia (icterus).
    • Ability to use specimen and reagent volumes as small as 25 L in some instruments.
    • Able to detect weak clots.
  • Disadvantages:
    • Reliance on the integrity of the entire coagulation cascade.
    • Inability to observe graph of clot formation.
Photo-Optical
  • Advantages:
    • 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.
  • Disadvantages:
    • Interference from lipemia, hemolysis, bilirubinemia, and increased plasma proteins; this issue has been addressed by some manufacturers with readings from multiple wavelengths.
Chromogenic
  • Advantages:
    • Ability to measure proteins that do not clot.
    • More specific than clot-based assays.
    • Most automated systems now have cost-effective chromogenic capabilities.
  • Disadvantages:
    • Limited by wavelength capabilities of some instruments.
    • May need large test volume to be cost effective.
Immunologic
  • Advantages:
    • Ability to automate tests previously available only with manual, time-consuming methods, such as enzyme-linked immunosorbent assays.
    • Expanded test menu capabilities.
  • Disadvantages:
    • 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
  • Advantages:
    • Ability to measure antigen-antibody reactions for proteins present in small concentrations.
  • Disadvantages:
    • Limited number of tests available.
    • Higher cost of reagents

Coagulation Instrumentation and Technology Advancements

  • Coagulometers are manual, semiautomated, or automated.
  • Manual and 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.
  • Manual result recording and calculation of the results are also done.

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 improved upon precision, but the requirement for manual pipetting of plasma and reagents continued to necessitate duplicate testing.
  • With the advent of fully automated instruments, precision improved to the extent that duplicate testing is no longer necessary, halving material and reagent costs.

Random Access Testing

  • Automated coagulometers 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.

Improved Reagent Handling

Reduced Reagent and Specimen Volumes
  • Automated and semiautomated coagulometers now have the capability to perform tests on smaller specimen volumes.
Open Reagent Systems
  • Laboratory directors want the flexibility of selecting reagents that best suit their needs and prefer not to be restricted in their choices by the analyzer being used.

Improved Specimen Management

Primary Tube Sampling
  • The design of many coagulometers encourages the operator to place the primary specimen collection tube on the instrument after centrifugation, which eliminates the need to separate the plasma into a secondary tube.
Closed-Tube Sampling
  • Closed-tube sampling of specimens has improved the safety and efficiency of coagulation testing.
  • After centrifugation, the operator places the primary blood collection tube on the analyzer without removing the blue stopper.
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.

Expanded Computer Capabilities

  • The computer circuitry of coagulation 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.

Quality Features of Automated Assay Performance

Flagging
  • Improved flagging capabilities alert the operator when preset criteria have been exceeded for instrument performance and specimen quality.
Reflex Testing
  • Reflex testing is the automatic ordering of tests based on preset parameters or the results of prior tests.

Selection of Coagulation Instrumentation

  • In today’s clinical 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.

Point-of-Care Coagulation Testing

  • Point-of-care (POC) instruments are handheld devices that permit near-patient testing during clinical procedures or surgeries, bedside testing for hospitalized patients, and self-testing for outpatients.
  • In addition, the small specimen volume is an advantage for testing in infants.
  • The instantaneous turnaround time of results, portability of the devices, and small specimen volume are conveniences appreciated by both physicians and patients.

Global Hemostasis Assessment

  • Thromboelastography was developed in 1948, and to this day follows the same general format.
  • This technique uses the viscoelastic property of blood clotting described previously.
  • The assay provides information on the entire kinetic process of whole blood clot formation.

Platelet Function Testing

  • In addition to coagulation factor testing, clinical hemostasis laboratories have expanded their test menu to include assays that evaluate platelet function.
  • 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

Molecular Coagulation Testing

  • Molecular testing in the coagulation laboratory is available for patients with thrombophilia.

Future Technologies for the Coagulation Laboratory

  • With advances in technology, novel techniques that can potentially be used for coagulation testing are being developed.
  • Lateral flow assays are a paper-based platform for the detection and quantitation of analytes in a complex mixture using antibody detection.
  • This technology provides rapid results at a low cost.