Lecture 18 Coagulation Instrumentation

Question 1

What are the various end-point detection principles used to measure coaguation?

Answer 1

1. Mechanical Clot End-Point Detection
2. Photo-Optical (turbidometric)
3. Nephelometry
4. Chromogenic
5. Immunologic

See slide 19 for advantages and disadvantages of each.

Question 2

Describe a mechanical method of end-point collection?

Answer 2

1. Uses a magnetic sensor that monitors the movement of a steel ball within a reagent/patient plasma sample
2. In this method the steel ball will oscillate (rock) back and forth during testing
3. When the clot forms oscillation decreases to predefined rate and the timer stops indicating clotting time

Question 3

What is a variation on the mechanical steel ball for end point method from the oscillating steel ball?

Answer 3

Place the steel ball on a stationary incline within the well. When ball is swept away by fibrin there is a break in the circuit and timer stops

Question 4

Describe the photo-optical (turbidometric) method of end-point detection?

Answer 4

1. Photo-Optical measurement detects a change in Optical density (OD) during clotting
2. As the clot forms decreased transmittance (less light) will fall onto the sensor
3. A baseline optical density is taken at the start of the test to take into account individual sample variations and is subtracted from the final clotted OD. This also helps to minimize the interfering effects of icteric and lipemic samples

Question 5

How do manufacturers using the photo-optical method try to filter out the effects of lipemia and icteric samples?

Answer 5

Many manufacturers use multiple wavelengths which additionally discriminate and filter out the effects of lipemia and icteric samples

Question 6

Describe the nephelometric end-point detection method. What is the advantage?

Answer 6

1. Forward and side-scattered light 90-degree are measured during and at the end of clot formation
2. Scattered light will increase with polymer and clot formation and timer will stop when predetermined intensity is reached
3. Because measurements are taken during clotting a signature clot curve can be obtained which can be helpful in certain situations

Question 7

What is the acceleration area and deceleration area on the nephelometric plot curve to measure clotting?

Answer 7

Acceleration area - readings taken while clot is forming
Deceleration area - time when the clot formation begins to slow down

Question 8

What is the baseline and end-point on the nephelometric plot curve to measure clotting?

Answer 8

Baseline - Readings take after the reagent and sample are mixed but before the clot starts forming.
Endpoint - Readings taken after the clot is complete.

Question 9

What kind of instrument malfunction flags is available on automatic coagulometers?

Answer 9

Examples are:
- Temp error
- Photo-optics error
- Mechanical movement error
- Probe not aspirating

Question 10

What kind of sample quality flags is available on automatic coagulometers?

Answer 10

Examples are:
- Lipemia
- Hemolysis
- Icterus
- Abnormal clot formation
- No end-point detected.

Question 11

What is PT Derived Fibrinogen?

Answer 11

1. The height of the light scatter or optical density from the baseline is proportional to the fibrinogen concentration.
2. Modern photo-optical coagulometers can compare the response of a test plasma with that of a standard (of known fibrinogen concentration) and extrapolating the fibrinogen level.

Question 12

What is the PT derived fibrinogen values used for?

Answer 12

PT Derived fibrinogens are not usually reported, rather low values are reflexed to perform the more accurate Clauss Fibrinogen assay

Question 13

What are two tests that determine deficiencies in fibrinogen?

Answer 13

Two popular tests for fibrinogen assay are the:
1. Thrombin based Modified Thrombin Time also known as the Claus Fibrinogen Assay
2. Immunologic method.

Question 14

What is the advantage of the modified thrombin time over the immunologic method for measuring fibrinogen?

Answer 14

1. Modified Thrombin Time: measured both quantitative and qualitative defects.
2. Immunologic Method: measures fibrinogen molecule. Not sensitive to qualitative defect such as dysfibrinogenemia.

Question 15

How is the Clauss Fibrinogen Modified Thrombin Time performed?

Answer 15

Requires a curve be established by making 1:5, 1:15 and 1:40 dilutions and further modified by using a high concentration of thrombin

● Dilutions of patient’s and control plasma are made and read against graph.

Question 16

What makes the Clauss Fibrinogen Modified Thrombin Time less sensitive to Fibrinogen Degradation Products and heparin?

Answer 16

Two modifications (dilutions and ↑thrombin concentration) makes this test less sensitive to Fibrinogen Degradation Products (FDP’s) and heparin.

Question 17

Describe the chromogenic method of measuring end-point?

Answer 17

Chromogenic methodology uses a substrate conjugated to a chromophore (color producing substance) which will react specifically with the target analyte, in the case presented below Protein C

Question 18

What is the difference between direct chromogenic assay and a indirect chromogenic assay?

Answer 18

Direct measures the activity of the analyte in proportion to increasing color.

Indirect measures the activity of the analyte inversely proportional to the amount of colour produced.

Question 19

What is the immunologic end-point method based on?

Answer 19

1. Are based on antigen-antibody reactions
2. Latex microparticles are coated with antibodies directed against a specific antigen
3. In the presence of the target antigen agglutinates will form causing absorbance of light
4. The increase in measured light absorbance is proportional to the size of the agglutinates which is proportional in turn to the antigen level

Question 20

What is an example of the immunologic end-point testing being used?

Answer 20

Example of this technology is the D-Dimer test which used to be manual can now be quickly and efficiently performed on a automated analyzer.

Question 21

What are advantages of automated coagulation testing?

Answer 21

1. Improved Accuracy and Precision
2. Random Access testing
3. Improved reagent handling
   - ~50% of volume compared to manual testing
4. Open Regent Systems and Reagent Tracking - Can use reagents from various vendors, keeps track of status
5. Primary Tube Sampling and Closed-Tube Sampling - use original Na Citrate tube
6. Automatic dilutions
7. Computer capabilities
8. Flagging
9. Reflex Testing

Review slide 22 on special features on current coagulameters.

Question 22

What is an advantage of being able to use the original Na Citrate tube on an automated coag analzyer?

Answer 22

Primary Tube Sampling can place the original Na Citrate tube on analyzer saving valuable time and reducing labelling errors. Cap piercing closed-tube sampling saves time, preserves pH and reduces dangerous aerosols and spillage

Question 23

What kind of computer capabilities are offered on coagulation automated machines?

Answer 23

Offer the ability to store results in lab and validate them electronically to hospital wards and Doctor offices. Controls can be recorded and monitored with electronic Levy-Jennings charts and files. Calibration records can be stored and easily accessed

Question 24

How can automated coagulation machines help with reflex testing

Answer 24

Coagulation instruments can be automatically programmed to run additional testing if initial results exceed linearity or follow-up tests need to performed. The operator does not need to initiate the follow-up action

Question 25

How does Point of Care Coagulation Testing iStat work?

Answer 25

1. Whole blood is added to a reagent strip containing Thromboplastin
2. In the iStat PT/INR the Thrombin generated converts a substrate Phenylalanine - Pipecolic acid - Arginine – NH - C6H4 - NH - C6H4 - OCH3 to electroactive compound NH3+ - C6H4 - NH - C6H4 - OCH3.
3. The formation of the electroactive compound is detected by an electrochemical sensor and the time of detection is measured. This is regarded as an Amperometric method.

Question 26

What can the PFA-100 analyzer do?

Answer 26

The PFA-100 is an example of platelet function analyzer capable of detecting von Willebrand disease and abnormal platelet aggregation

Question 27

How does the PFA-100 work to measure vWF and platelet aggregation?

Answer 27

Whole citrated blood is aspirated through a small aperature by high sheer stress through a cartridge containing Collagen/Epinephrine or Collagen/ADP

The Collagen portion of the cartridge will adhere the platelets and the ADP or Epinephrin which are aggregating agents will cause platelet aggregation

The time to block the small aperature is measured and is referred to as the closure time

See figure on slide 24.

Question 28

What are reasons to use (indications) the PFA-100 for Platelet Testing in case of bleeding?

Answer 28

BLEEDING:
1. Screening for VWD and platelet dysfunction
2. Transfusion medicine: donor screening, transfusion efficacy
3. Menorrhagia: screening for platelet defect/VWD
4. Surgery: patients with high risk for platelet dysfunction or vWD

Question 29

What are reasons to use (indications) the PFA-100 for Platelet Testing in case of thrombosis?

Answer 29

THROMBOSIS:
1. Detection of aspirin non-responsiveness and platelet hyperactivity

Question 30

How can molecular techniques help detect coagulation disorders? Which conditions is it most helpful in?

Answer 30

Thrombotic Conditions can be detected using a variety of molecular techniques such as Polymerase Chain Reaction (PCR)
Factor V Leiden which is a genetic mutation resistant to Protein C regulation causing thrombosis due to Factor V hyperactivity
Prothrombin Mutation is genetic defect that leads to increased amounts of Prothrombin

Question 31

Which conditions is molecular techniques so far have not been as helpful in detecting?

Answer 31

Molecular techniques for Hemophilia and von Willebrand disease are currently available but limited. Further research and development may see more common use of molecular techniques for these and other conditions