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HEMOSTASIS & COAGULATION INSTRUMENTATION

MLAB 1227 Coagulation

Keri Brophy-Martinez

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THE DISTANT PAST

Awareness of the concept of blood clotting in the 18^th century
From 1822- 1921 advances in testing included temperature control, detecting clots as resistance in blood, and using various sizes of glass tubes to observe clot formation
Early 1900s

Scientists monitor the length of time for whole blood to clot in glass tubes while being tilted
Eventually, platelet poor plasma becomes specimen of choice for the largest volume of coagulation testing
First clot detection instrument was developed- “Koaguloviskosimeter”

Measured change in blood viscosity

1920

Calcium chloride was added to citrated anticoagulated plasma at 37⁰C
Clot formation determined by visual inspection of the plasma as the tube was tilted- “tilt tube”
Nephelometers measure changes in light scatter over time to detect clot formation

1950s

BBL Fibrometer detects a plasma clot by electromechanical technology.

Lab assessment of blood clotting begin in the 18^th century with visual techniques, when a basin was used to collect the blood to observe how long it took to clot. More advances such as temperature control, detecting a clot as resistance in the blood, and the use of different sizes of glass tubes to visualize clot formation became the norm from 1822 to 1921. In the early 1900s, scientists monitored the length of time it took whole blood to clot in a glass tube while being tilted without the contents of the tube spilling out. This was coined the “tilt tube” method and was the basis for the Lee-White clotting time test- the first means to standardize assessment of the coagulation system in a written procedure. Plasma anticoagulated with sodium citrate came to replace whole blood in testing for the majority of coagulation tests. In 1910, the first instrument was developed to detect clots- the Koaguloviskosimeter. This instrument measured the change in the viscosity of blood as it clotted.

In 1920, Gram laid the groundwork for todays PT and PTT assays by adding calcium chloride to citrated anticoagulated plasma that was incubated at 37 degrees C. Plasma and reagents were added to a glass tube held in a water bath and clot formation determined by visual inspection of the plasma as the tube was tilted while looking for the white strands of fibrin monomer formation. A stopwatch was used to determine the time to clot. Also during this time period, nephelometers were used for testing. These devices measure 90degrees light dispersion of colloidal suspension, measuring changes in light scatter over time to detect clot formation.

In the 1950s, the BBL Fibrometer was developed that detected a plasma clot by electromechanical technology. This technology allowed labs to move away from manual to a semiautomated testing platforms.

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TODAYS INSTRUMENTATION

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LEVELS OF COAGULATION AUTOMATION

Level	Description	Examples
Manual	Reagents and specimens transferred manually to the instrument. Timer is started and stopped by the operator.	Tilt tube, Wire loop
Semiautomated	Reagents and specimens transferred manually to the instrument. Instrument initiates a timing device automatically once final reagent added. Instrument detect clots and stops time.	Fibrometer, Start 4, Cascade M-4, KC1
Automated	All reagents are automatically pipetted by the instrument. Timers are initiated and clot formation automatically detected.	ACL TOP, STA Compact, Sysmex CA series

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TO SEE OR TO FEEL…

Automation approaches to clot detection:

SEE

Optical/ Turbidometric
Nephelometric

FEEL

Mechanical
Viscosity based

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ENDPOINT DETECTION PRINCIPLES

Clot Based Assays	Chromogenic Assays	Immunoassays
Mechanical/ Physical	Spectrophotometric	Nephelometric
Photo-optical/Turbidometric		Spectrophotometric/ Light absorbance
Viscoelastic/ Physical

Coagulation instrumentation methodologies are classified into three groups based on the endpoint detection principle. The three groups are the clot based assays, the chromogenic assays and the immunoassays.

Each methodology has advantages and disadvantages. Clot-based assays are not highly specific, whereas chromogenic or immunological endpoint assays are single analyte specific as they do not rely on the coagulation cascade. Clot-based assays will show effects of substances that interfere in the coagulation cascade, whereas chromogenic assays allows for targeted evaluation of the analyte. Assays that use clot and chromogenic endpoints demonstrate the functional activity of the hemostatic protein, while immunoassays only quantitate the protein.

Mechanical clot-based methods, as opposed to photo-optical and chromogenic methods are not affected by icteric or lipemic samples.

In the next few slides we will examine these groups in more detail.

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CLOT BASED ENDPOINT DETECTION METHODS

Mechanical/Physical

Detect physical formation of fibrin strands
Examples:

BBL Fibromete-
Diagnostica Stago/AMAX/Tcoag
KC1 Delta- EVC

Uses a steel ball at the bottom of a cuvette that is held in place by a magnetic source.
While the cuvette continuously rotates, the technician adds the sample and reagents, which starts the timer. T
When true clot formation has occurred, the clot will incorporate the steel ball and pull it away from the magnetic source, stopping the timer.

Electromechanical clot detection systems measure a change in conductivity between two metal electrodes in plasma. Mechanical measurement methods detect the physical formation of fibrin strands. These fibrin strands attach to a moving mechanical device, which ultimately either completes or opens an electrical circuit. Any test that has fibrin formation as its endpoint can be determined with this method.

An example of an instrument using mechanical clot detection is the BBL Fibrometer. The probe of this instrument has one stationary and one moving electrode. During clotting, the moving electrode enters and leaves the plasma at regular intervals. When the clot forms, the fibrin strand conducts current between two electrodes, even when the moving electrode exits the solution. The current completes a circuit that stops the timer.

Another mechanical clot detection system uses a magnetic sensor that monitors the movement of a steel ball within the test plasma.

The KC1 Delta is a hemostasis instrument that detects clot formation using Trinity’s patented technique (The Ball Method), which is a descendent of the original Fibrometer. The Ball Method uses a steel ball at the bottom of a cuvette that is held in place by a magnetic source. While the cuvette continuously rotates, students add the sample and reagents, which starts the timer. The clot formation process begins and, because of the design of the analyzers, the students can actually view the clot formation taking place. When true clot formation has occurred, the clot will incorporate the steel ball and pull it away from the magnetic source, stopping the timer. This is one of the instruments used at the Eastview campus.

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CLOT BASED ENDPOINT DETECTION METHODS

Photo-Optical/ Turbidometric

Detects changes in light transmittance through plasma as the specimen clots, increasing turbidity.
Example- Cascade M-4-RRC

The basic premise of optical clot detection is that as the coagulation process occurs inside the cuvette, the optical opacity increases, thereby decreasing the amount of light transmitted through the plasma.

Formation of fibrin strands causes light to scatter, allowing less light to fall on the photodetector, generating a decrease in transmitted light. When the transmitted light decreases to a predetermined baseline, the timer stops, indicating a clot has been formed.

Because the change in transmittance (between the initial reading and the final reading) is used to calculate the results, the effect of adverse sample conditions (such as lipemic or icteric samples) can be minimized.

Most of the automated and semiautomated coagulation instruments in use since the 1970s use photo-optical clot detection.

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CLOT BASED ENDPOINT DETECTION METHODS

Viscoelastic/Physical “VET”

Whole blood based
Detects clot formation by increased blood viscosity created as the clot forms
Detects clot initiation through clot lysis
Measures the net effect of all hemostatic components interacting together during the clotting process
Demonstrates the hemostatic potential of a blood sample at a given point in time.
Assesses both bleeding and thrombosis risk

Examples

TEG- Thromboelastograph

ROTEM-Rotational thromboelastometry

TEG 5000

TEG 6s

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USES OF TEG^®/ROTEM

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 hypercoaguability
Differentiates surgical from pathological bleeding

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THROMBOELASTOGRAPHY (TEG^®)

Sample of whole blood is placed in a cup which has a pin carefully connected to a torsion wire.

As the cup rotates 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 lack of movement is reflected via either an optical or magnetic detector

A graphic presentation is produced

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TEG/ROTEM� TRACING

Parameter	TEG	ROTEM	What’s Happening	Who’s Involved
Clot initiation or clotting time	R	CT	Time of initial clot formation Initiation of thrombin generation	Thrombin
Clot kinetics	K	CFT	A measure of the speed to reach a specific level of clot strength	Plts, coag factors, thrombin & fibrinogen
Angle- clot kinetics & clot strength	α	α	Measures rate of clot formation, fibrin formation, cross-linking	Thrombin-activated plts, coag factors, fibrinogen, fibrin, FXIII
Clot strength	MA	MCF	Measures strength of clot	Plts, fibrinogen, fibrin, FXIII

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TYPICAL TEG GRAPH PATTERNS

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CHROMOGENIC ENDPOINT DETECTION METHODS

Chromogenic assays assess the FUNCTION of hemostatic proteins.

Uses a colorless substrate and a chromophore
Protease activity of the factor allows the substrate-chromophore complex to be cut
Color change results and the OD is measured at 405 nm.

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Chromogenic assays allow for specific coagulation factor activity or function to be measured.

Assays with a chromogenic endpoint apply basic enzymology. Enzymes are proteins that catalyze most of the chemical reactions in the body. The chemical compound on which the enzyme exerts its catalytic activity is called a substrate. Proteolytic enzymes act on their natural substrates by hydrolyzing a peptide bond to give subunits of the substrate. Chromogenic assays use a synthetic replica of the peptides of the natural substrate at the site of proteolytic hydrolysis or cleavage by the enzyme. The reaction mimics the specific enzyme-substrate reaction in the body.

Chromogenic assays are chemical reactions that have a color endpoint. This method uses a colorless substrate joined to a chromophore. Chromogenic methods allow specific coagulation factor activity to be measured by their proteolytic properties. The protease of the factor cleaves its corresponding substrate to release the attached chromophore, resulting in a color change of the solution. The OD of the solution is proportional to protease activity. This is measured by a photo detector at 405 nm.

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IMMUNOASSAY ENDPOINT DETECTION METHODS

Immunoassays indicate QUANTITY of the hemostatic parameter, NOT function of the parameter.

Nephelometric

Modification of photo-optical methods
Uses side & forward-angle light scatter
As fibrin polymers forms, side & forward light scatter, stopping the time when scatter reaches a predetermined intensity

Spectrophotometric/Light Absorbance/Immunologic

Based on antibody-antigen reactions
Increase in light absorbance is proportional to antigen level

Immunoassays indicate quantity and not function of the hemostatic parameter.

Nephelometry is a modification of photo-optical endpoint detection where the measurement of the amount of light scattered at a variety of angles after passing through a particulate solution. An instrument can utilize nephelometric clot detection by passing each optically clear cuvette through a fixed beam of light on a consistent revolution. For clot testing, a detector measures the light scatter from the fibrin strands as they form. The final clotting curve is based on consecutive readings per reaction, defining the entire clot curve to its completion.

With light absorbance, latex particles are coated with antibodies directed against select antigens. Monochromatic light passes through the suspension of latex particles. If the latex particles and antigen come in contact with each other, agglutination occurs, resulting in more light being absorbed. The increase in light absorbance is proportional to the size of the agglutinate, which is proportional to the antigen level.

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POINT OF CARE TESTING

Began in 1966 with the ACT to monitor heparin during surgery
Today PT/INR used to monitor warfarin therapy
POC Instruments

Handheld
Bedside, outpatient utility
CLIA waived
Use capillary whole blood

Examples

Abbott iSTAT
Roche CoaguCheck

iSTAT

CoaguCheck

POC or point of care coagulation testing has been in practice since 1966 when the ACT (activated clotting time) was used to monitor heparin during cardiac surgery. The fast turnaround time for the ACT was needed to adjust heparin to avoid bleeding and clotting during surgery. Today POC testing available for ACT, PT/INR, and PTT (although not widely accepted).

Point of care (POC) instruments are typically handheld devices that permit near patient testing during procedures, at the bedside, or for self testing for outpatients. The quick turnaround time for results, portability, and small specimen volume are advantages to using these types of instruments. Most POC instruments have CLIA waived status- meaning the performing of these tests can be delegated to anyone- not just limited to certified lab scientists.

POC analyzers use whole blood specimens from capillary punctures.

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PLATELET FUNCTION TESTING

Assess platelet function (qualitative defects)

Suspected when bleeding symptoms are present, but platelet count exceeds 50,000

Used to monitor antiplatelet therapy
Used to manage patients with a history of bleeding

Assessment of platelet function

Bleeding time
Platelet aggregation assays

Hemostasis labs not only provide coagulation factor testing, but test menus have expanded to include assays to evaluate platelet function. Qualitative or function defects are suspected when patients have bleeding symptoms, but the platelet count exceeds 50,000. The need to evaluate platelet function has increased over the years due to the increased need to monitor the efficacy of antiplatelet therapy such as aspirin, clopidodrel, and other ADP receptor antagonists used in patients with cardiac disorders. In addition, preop evaluation of platelet function is important in hemostatic management of patients that have a history of bleeding or are on antiplatelet medication.

Historically, platelet function has been assessed by the bleeding time test, platelet function analyzers, and platelet aggregation assays. Since we will be discussing the bleeding time in more detail in the lab, we will focus on platelet function analyzers and platelet aggregation assays in this unit.

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PLATELET FUNCTION ANALYZERS- PFA

Evaluates PRIMARY hemostasis

Uses stimulators of platelet adhesion and aggregation in an environment that stimulates an injured blood vessel wall.
More sensitive screening test than the bleeding time method
Offers increased sensitivity for platelet dysfunction and von Willebrand’s disease
PFA-100

Nonspecific test- not diagnostic for any single disorder

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PLATELET AGGREGOMETERS

Performed in specialized labs by experienced laboratory professionals
Assesses platelet adhesion, aggregation, secretion
Methods

Light transmittance utilizing photometry

Requires platelet RICH plasma

Impedance/ Light scatter

Requires whole blood

Impedance Method

Photometry Method

Platelet aggregometry is a challenging technique usually performed in specialized laboratories by experienced lab professionals. The testing is time consuming and requires attention to detail regarding sample collection, processing the samples and reagents, pH, time of storage, rate of stirring, and platelet count. Platelet adhesion, aggregation, and secretion are assessed using platelet aggregometry.

Platelet aggregometry can be performed by either photometry, using light transmittance or impedance/light scatter.

With photometry, this test requires platelet-rich plasma and measures the increase in light transmittance that occurs in direct proportion to platelet aggregation by various agonists, such as collagen, ADP, epinephrine, etc.

The impedance principle used whole blood. Upon activation, platelets become sticky and adhere to metal wire sensors(electrodes). The greater the platelet aggregate formation, the greater the impedance.

The picture in the top right corner is an example of a photometer using light transmittance. It is called the PAP-8E by Biodata.

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AGGREGATING REAGENTS�(AGONIST)

Thrombin
Collagen
ADP
Epinephrine
Ristocetin
Arachidonic Acid

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PLATELET AGGREGATION PATTERNS IN VARIOUS DISORDERS

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MOLECULAR GENETIC TESTING

Advantages

High sensitivity & specificity
Lack of interference from inhibitors

Current Platforms

Thrombophilia

Gene mutations of factor V
Prothrombin G20210A

Bleeding disorders

Von Willebrand disease

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REFERENCES

"Cascade M M4 Hemostasis Coagulation Analyzers Clotting Assays PTs APTTs Thrombins Fibrinogens Factor Assays - Discovery Diagnostics Canadian Distributor Helena Laboratories." Hematology Stainers Microbiology Stainers Cytocentrifuges Osmometers Sweat Collection Blood Temperature Indicators Fecal Occult Blood Platelet Aggregation. Discovery Diagnostics and JLS Web Designs, 8 Sept. 2008. Web. 14 Nov. 2010. <http://www.discovery-diagnostics.com/Cascade_M4.asp>.
"Fiche Produit - Stago." Homepage Stago Corporate - Stago. Web. 14 Nov. 2010. <http://www.stago.com/nc/products-services/catalogue/analyzers/fiche-produit/selection/type-analyzers/reference/58609/group/sta-compact/>.
"KC1 DELTA COAG ANALYZER 1/EA - Trinity Biotech # G05000." LabSource.com - Your Source for Science and Safety! 2009. Web. 14 Nov. 2010. <http://www.labsource.com/Catalog/Item.aspx?ItemID=1392043>.

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REFERENCES

Keohane, E. M., Butina, M., Mirza, K. M., & Walenga, J. M. ([Insert Year of Publication]). Rodak's Hematology (7th ed.). Elsevier Health Sciences (US).
McGlinchey, Kevin. "» More on Trinity’s KC1 and KC4 Educational Promotion." The Fritsma Factor: Your Interactive Hemostasis Resource. 5 Nov. 2009. Web. 14 Nov. 2010. <http://www.fritsmafactor.com/newfritsmafactor/?p=2044>.
McGlinchey, Kevin. "Coagulation Automation." Advance 19.6 (2010): 26-27. Print.
McKenzie, Shirlyn B. "Chapter 40." Clinical Laboratory Hematology. 2nd ed. Boston: Pearson, 2010. Web.
"PFA-100® System." Siemens Healthcare Worldwide. 2007. Web. 14 Nov. 2010. <http://www.medical.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1023065,1028378,1015818~a_langId~e_-111~a_productId~e_182047~a_storeId~e_10001.htm>.