Automation in Hematology Part (3)
Automation in Coagulation
Screening test:
PT, APTT, Fibrinogen, and D- dimer.
Tests which look for abnormalities in coagulation & offers direction for selecting the specific test and help in the diagnosis and treatment
Specific test:
Platelet Aggregation Studies, Specific coagulation factor assay, Antithrombin, Protein C activity, etc.,
Manual Methods
- Reagents and samples are added manually.
- The temperature is maintained by a water bath.
- Values are measured manually by using a stopwatch.
- Leading to human error and is difficult to do when there is a large workload.
Semi-Automated Analyzer
- Has a mechanism to automatically initiate timing device upon addition of the final reagent and internal mechanism for detecting Clot formation.
-Both reagents and samples are added manually by the operator.
- A semi-automated analyzer may or may not internally maintain the temperature.
Automated analyzer
- Human errors are eliminated(sample identification, the addition of sample and reagents, and endpoint detection)
- Maintains and check the temperature.
- Perform auto dilutions mainly for the single factor assays.
- Storage of large no. of patient data and control results.
- Generates flagging for sample abnormality and instrumental malfunctioning.
- Batch processing of multiple samples for a single test or many tests on a single sample can be performed.
Clotting assay
Functional assay
- In this sample and reagent are mixed to form a clot.
- The endpoint of the reaction i.e., the clot is detected by
▪ Mechanical
▪ Optical detection
▪ Electrochemical
Mechanical method
Magnetic steel ball method.
- The sample is injected into a cuvette that has a small steel ball inside.
- The cuvette continuously moves when the reagent and the sample are added.
- The fibrin strands start to form and attach to the moving ball.
- There is a gap in contact with the magnetic sensors when the steel ball becomes incorporated into a fibrin clot as the cuvette.
- Clotting time is recorded.
Optical detection method
• As the plasma sample clots, it becomes more optically dense and the amount of light falling on a photosensitive detector decrease.
• The drop or change in light is determined as the endpoint = turbidimetric method.
Nephelometric method
- The nephelometer utilizes a light-emitting diode at a high wavelength (>600 nm) to find variations in light scatter as fibrin clot is formed.
- When the light rays encounter insoluble complexes such as fibrin strands, they are scattered at 900 angles.
Chromogenic Method
• Chromogenic or amidolytic methodology is formulated on the use of a specific color-producing substance = chromophore.
• Normally used chromophore is para-nitroaniline (405nm)
Immunologic Method
• Immunologic assays are based on Ag-Ab reactions.
• Latex Microparticles are coated with a specific Ab directed against Ag.
• A beam of monochromatic light is then passed through the suspension of micro latex particles.
• Genesis of agglutination leads to an increase in turbidity of the test solution and an increase in Absorbance which, in turn, is proportional to the antigen level present in the sample, which is taken from a standard curve.
Electrochemical
• The INRatio one-use test strip has a sample well where blood is added, three channels through which the blood sample flows to reach the testing areas.
• Reagents start the coagulation process.
• The device noted a change in electrical resistance when blood clots.
Platelet Function Analyzers
- Uses stimulators of platelet adhesion and aggregation in an environment that stimulates an injured blood vessel wall.
- Better screening test than the bleeding time method.
- Not definite test- not diagnostic for any single disorder.
- The instrument adds citrated blood to a reservoir with collagen/epinephrine on a bioactive membrane.
- A pressure sensor determines the formation of a platelet plug on the membrane.
- The time it takes to close the aperture in the membrane with the platelet plug is recorded.
- The result is a function of platelet count, platelet activity, VWF activity.
Platelet Aggregometry
• Platelet aggregometry involves a series of tests carried out on whole blood or platelet-rich plasma, using several agonists (platelet activators).
• The agonist has put into the suspension and a dynamic measure of platelet clumping is recorded.
• Simultaneously to platelet aggregation, a luminometry test can be performed. In that case, ATP liberate is assayed using a luminescent marker.
Thrombelastography (TEG)
•Sample of citrated whole blood is placed in a cup that has a pin carefully connected to a torsion wire.
•As the cup spin in a back and forth movement, the aggregates formed within the cup cause the wire to become more rigidly placed and reflects the strength of the aggregates formed within the cup.
•The movement or no movement is reflected via either an optical or magnetic detector
•A graphic presentation is produced.
TEG Graphic Result
R=Time of latency from the start of the test to initial fibrin formation
K=Time taken to reach a certain level of clot strength Alpha angle = quantify the speed at which fibrin build up and cross-linking takes
MA = maximum amplitude place
Uses of TEG
- Illustrates function and dysfunction in the Hemostatic system.
- Allows physicians to give appropriate amounts of FFP, Cryo, and platelets to control hemorrhage.
- Reduces unnecessary use of blood products.
- Allows effective management of hypercoagulability.
- Differentiates surgical from pathological bleeding.
Automation in ESR
Fully automated analyzer
- No reagents required – no waste.
- straight use of primary EDTA tubes – no contact with blood.
- Therefore, the EDTA tube can be used unchanged for further analyses after erythrocyte sedimentation.
- Compatible with all the commonly used EDTA tubes in the market.
- Photometric infrared (950nm) reading prevents interference produced by lipids or bilirubin in the sample.
High-Performance Liquid Chromatography (HPLC)
• HPLC is a chromatographic technique that can differentiate a mixture of compounds.
• Type of liquid chromatography where the sample is pushed through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer.
Principle:
Depends on the interchange of charged groups on the ion exchange material with charged groups on the Hb molecule.
Cation Exchange Cartridge
Carboxyl groups attached to a resin base
Whole blood + hemolysate
Positively charged hemoglobin fragments in the hemolysate link to the carboxyl groups at varying strengths.
Starting Gradient:
Low Ionic Strength Buffer
- The gradient begins with a low % of Buffer.
- At this gradient, hemoglobin particles with an ionic strength less than the buffer gradient, such as HbF, are replaced from the cartridge and pass into the detector.
Ending Gradient:
High Ionic Strength Buffer
- As the percentage of the Ionic Strength of Buffer increases, the more hemoglobin fragments will be displaced
- Once the gradient is 100% all remaining hemoglobin fragments, including any variant hemoglobins such as S, D, and C, will be removed.
• Separated components pass through dual-wavelength detectors.
• Absorbance is measured at 415 nm and the data is displayed as a chromatogram.
• Finally it is converted into peaks as per the retention time
• Separated Hb’s with % is displayed.
Retention time: Time in minutes from the sample injection to the max point of elution peak of Hb.
 Automation in Coagulation Screening test: PT, APTT, Fibrinogen, and D- dimer. Tests which look for …){kind=link}
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