TINOOD

• -Coagulation Instrumentation and Technology

  Advancements

  -Improved Accuracy and Precision

  -Random Access Testing

  -Improved Reagent Handling

  -Improved Specimen Management

  -Expanded Computer Capabilities

  -Quality Features of Automated Assay

  Performance

  -Specimen Quality Set Points
• Early Developments in Coagulation Testing (1822 - 1921)

  TILT TUBE

  "Koagulovis-kosimeter" marked the first clot detection instrument.

  ○ The apparatus measured blood viscosity change during clotting.

  ● Later developments led to the thromboelastograph (TEG) and sonar clot detection.
• Evolution of Plasma Coagulation Testing

  ● Plasma coagulation testing began in

  1920 with the addition of calcium

  chloride.

  ● Gram's method laid the foundation

  for PT and PTT assays.

  ● Initial testing involved manual

  methods like the tilt-tube technique.
• Advancements in Instrumentation (1950s - Present)

  ● The 1950s introduced the BBL Fibrometer, the first

  coagulometer.

  ● Subsequent developments included rolling steel ball and

  photo-optical measurements.

  ● Modern instrumentation integrates clot-based assays,

  chromogenic assays, and immunoassays for

  comprehensive testing.
• Observation-Based End-Point

  Detection

  ● Optical and Nephelometric

  Devices

  ● Utilizes "Observation" of Clot

  Formation

  ● Commonly Used in Traditional

  Coagulation Instruments
• Feel-Based End-Point

  Detection

  ● Mechanical and ViscosityBased Devices

  ● Detects Clot Formation by

  "Feel"

  ● Early Instruments Primarily

  Relied on Mechanical

  Principles
• Technological Advancements

  ● Transition to Photo-Optical

  Detection

  ● Fixed Wavelength between

  500 and 600 nm

  ● Integration of Viscoelastic

  End-Point Detection for

  Specialty Testing
• Modern Instrumentation

  ● Integration of Multiple EndPoint Mechanisms

  ● Incorporation of

  Chromogenic and

  Immunoassay-Based Testing

  ● Streamlining Laboratory

  Workflows for Routine and

  Specialized Testing Needs
• Photo-Optical Clot End-Point Detection

  ● Photo-optical (turbidometric) coagulometers detect changes in

  plasma optical density (OD) during clotting.

  ● Light of a specific wavelength passes through the sample,

  measuring light transmittance.

  ● Provides information on various aspects of the clotting

  process:

  ○ Time to clot

  ○ Kinetics of whole blood clot formation

  ○ Clot strength

  ○ Fibrinolytic activity
• Viscoelastic Instrumentation

  ● Viscoelastic instrumentation remains vital for

  assessing hemostasis.

  ● Detailed analysis of clotting dynamics and

  properties.

  ● Offers insights into coagulation disorders and

  thrombotic conditions.

  ● Instrumentation detailed in the Global Hemostasis

  Assessment section for comprehensive hemostasis

  evaluation.

  COLD BLOOD
• Chromogenic End-Point Detection

  ● Chromogenic methodology utilizes synthetic

  substrate conjugated to a chromophore, typically

  para-nitroaniline (pNA).

  ● Measures the activity of specific coagulation

  factors through enzymatic (protease) properties.

  ● Oligopeptide substrate cleaves the chromogenic

  substrate, releasing pNA, which is measured by a

  photodetector at 405 nm.
• Examples of Chromogenic Assays

  ● Direct Chromogenic Assay:

  ○ OD directly proportional to the activity of the analyte

  being measured.

  ○ Example: Protein C activity measured using a

  chromogenic substrate specific for protein C.

  ● Indirect Chromogenic Assay:

  ○ The protein being measured inhibits a target enzyme,

  which reacts with the chromogenic substrate.

  ○ Change in OD inversely proportional to the concentration

  or activity of the substance being measured.

  ○ Example: Heparin quantitation using the anti-factor Xa

  assay.
• Application and Benefits of Chromogenic Assay

  ● Application:

  ○ Measure specific coagulation factors' activity.

  ○ Assess inhibitors and quantitate substances like heparin.

  ● Benefits:

  ○ High specificity and sensitivity.

  ○ Quantitative assessment of coagulation factors.

  ○ Enables precise diagnosis and monitoring of coagulation

  disorders.
• Nephelometry Assay

  ● As fibrin polymers form, side scatter and forward-angle scatter increase.

  ● Timer stops when scatter reaches predetermined intensity, recording time to

  clot interval.

  ● Nephelometry adapted to measure dynamics or kinetics of clot formation.

  ● Continuous readings throughout clotting period produce clot curve or

  "signature."

  ● Originally applied to immunoassays, still used today to detect antigen-antibody

  complexes.

  ● Precipitation of immune complexes results in turbidity, scattering incident light
• Clot-Based Assays and Immunoassays on One

  Platform

  ● Integration of clot-based assays and immunoassays on a

  single platform enhances coagulation testing efficiency and

  versatility.

  ● Immunologic Light Absorbance End-Point Detection is a

  newer method for immunoassays in routine coagulation

  testing.

  ● Latex microparticles coated with antibodies react with

  antigens, forming agglutinates that absorb light,

  proportional to antigen levels.
• Improved Accuracy and Precision

  ● Visual methods necessitated duplicate testing to reduce

  the coefficient of variation, often exceeding 20%.

  ● Semiautomated instruments improved precision but still

  required duplicate testing due to manual pipetting.

  ● Fully automated instruments have significantly

  enhanced precision, eliminating the need for duplicate

  testing.

  ● Coefficients of variation of less than 5% are typically

  achieved, reducing material and reagent costs by half.
• Random Access Testing ● Automated coagulometers offer random access testing, allowing a variety of tests to be run in any order. ● Previous analyzers required batching, limiting the number of assays that could be run simultaneously. ● Current analyzers can run multiple tests limited only by reagent storage capacity and end-point detection methodologies. ● Benefits include improved turnaround times, reduced errors, and decreased labor costs.
• Improved Reagent Handling

  ○ Reduced reagent and specimen

  volumes

  ○ Automation enables tests with smaller

  volumes

  ○ Cost-effective, especially for pediatric

  patients
• Open Reagent Systems

  ○ Flexibility in selecting reagents

  ○ Compatibility with various manufacturers' reagents

  ○ Optimal performance without restricting choices
• Reagent Tracking

  ○ Record-keeping of lot numbers and

  expiration dates

  ○ On-board monitoring of reagent

  volumes

  ○ Reagent bar coding for efficient

  tracking
• Improved Specimen Management

  ○ Primary Tube Sampling W/o cover

  ■ Eliminates need for secondary tube

  separation

  ■ Accommodates multiple tube sizes

  ○ Closed-Tube Sampling

  ■ Enhanced safety and efficiency

  ■ Eliminates crossover between specimens

  ■ Reduces staff exposure to specimen

  hazards
• Enhanced Computer Capabilities in Coagulation

  Analyzers

  1. Data Storage and Retrieval

  ○ Hundreds of results stored and compiled

  ○ Multiple calibration curves accessible

  ○ Quality control files stored, eliminating manual

  logging

  2. Programming Flexibility

  ○ Preprogrammed test protocols

  ○ Easily changeable testing parameters

  ○ User-programmable additional tests for enhanced

  flexibility
• 3. Instrument Interfacing

  ○ Integration with laboratory information systems

  ○ Specimen bar coding for efficient tracking

  ○ Bidirectional interfaces for streamlined workflow

  4. Quality Features of Automated Assay Performance

  ○ Improved flagging capabilities for instrument performance and specimen quality

  ○ Reflex testing based on preset parameters or prior test results

  ○ Kinetics of clot formation graphing for enhanced analysis and troubleshootin
• :Point-of-care

  coagulation testing offers

  rapid and convenient testing

  options, but careful validation

  and understanding of

  differences between POC and

  central laboratory results are

  essential for accurate clinical

  decision-making
• ROTEM (Rotational Thromboelastometry) is a newer version of

  thromboelastography.

  ● Enhancements include:

  ○ Not sensitive to vibrations

  ○ Four test channels

  ○ Touch screen interface

  ○ Automated pipettor

  ● Similar to TEG, assesses clot formation and fibrinolysis using whole blood

  and various coagulation activators.

  ● ROTEM clot signature comparable to TEG.

  ● Reagents available for several coagulation assays.

  ● Results available within 10 to 20 minutes.

  ● Operator dependent and requires training and skill for accurate interpretation.
• Siemens PFA-100 Platelet Function Analyzer

  ○ Automated instrument for rapid results on

  platelet abnormalities.

  ○ Test cartridges stimulate platelet aggregation

  using collagen/epinephrine or collagen/ADP.

  ○ Measures the time for a platelet plug to occlude

  a microscopic aperture.

  ○ Successful in detecting von Willebrand disease

  and assessing aspirin therapy efficacy.
• Verify Now from Accriva Diagnostics

  ○ Optical detection system measuring platelet aggregation by

  microbead agglutination.

  ○ Uses disposable cartridges with fibrinogen-coated beads and

  platelet agonists.

  ○ Provides aspirin assay, glycoprotein IIb/IIIa inhibitor assay, and

  P2Y12 inhibitor assay.

  ○ Results available in about 10 minutes, suitable for near-patient

  testing
• Plateletworks Platelet Function Assay (Helena)

  ○ Can be run on any standard impedance cell counter.

  ○ Aggregation results based on platelet count before and after

  activation.

  ○ Blood collected via venipuncture directly into tubes provided

  in the kit.

  ○ Results obtainable in 2 minutes, ideal for presurgical

  screening and antiplatelet therapy monitoring
• Flow Cytometry for Platelet Function

  Analysis

  ● Flow cytometric assays offer advantages over conventional platelet

  aggregation studies.

  ● Whole blood testing provides a more physiologic evaluation.

  ● Requires minimal specimen volumes, suitable for pediatric and

  difficult-to-draw patients.

  ● Able to measure multiple cellular activation-dependent changes related

  to platelet function.

  ● Available for the diagnosis of heparin-induced thrombocytopenia (HIT),

  but currently laboratory developed tests (LDTs).
• Platelet Aggregometers

  • PAP-8E from BioData
  • Chrono-Log Whole Blood/Optical Lumi-Aggregation System
  • Multiplate Analyzer from Diapharma
  • AggRAM from Helena
• Multiplate Analyzer from Diapharma

  • Monitors platelet function by impedance, showing correlation with LTA in testing therapeutic efficacy
• PAP-8E from BioData

  • Eight-channel aggregometer with touch screen and low sample volume
• Classic platelet aggregometry developed by Born in 1962 measures light transmission induced by various agonists
• AggRAM from Helena

  • Modular system for platelet aggregation and ristocetin cofactor testing, using advanced optics for precision
• Chrono-Log Whole Blood/Optical Lumi-Aggregation System

  • Measures aggregation and platelet secretion
• New devices detect platelet aggregation

  • Based on whole blood impedance
  • Based on luminescence
  • Based on light scatter
• Non-PCR-based methods

  • Invader assay
• PCR-based assays

  Widely used for their accuracy in detecting point mutations and single-nucleotide polymorphisms
• PCR-based assays

  Polymerase chain reaction (PCR)
• Molecular testing in the coagulation laboratory has revolutionized the diagnosis of thrombophilia and related disorders