Week 8-Haemostasis and Coagulation Tests

Haemostasis

• Process of stopping loss of blood from blood vessels

• – Include Coagulation (clot formation) and Fibrinolysis (dissolution)

  • – An interrelated and interdependent system

Hemostasis involve four major phases

– 1. Vasoconstriction / vascular spasms

– 2. Platelet activation / plug formation

– 3. Coagulation / clotting

– 4. Fibrinolysis

Vasoconstriction / vascular spasms→First phase of hemostatsis

• blood vessels narrow

• Broken blood vessels attract platelets (collagen exposure)

• Anchored platelets (adhesion) release serotonin

  • causes blood vessel muscles to spasm (contract)

• Spasms narrow the blood vessel, decreasing blood loss/flow

Platelet Activation→Second phase of hemostatsis

• undergo Adhesion + Aggregation

• Initiated by damaged or irregular blood vessels

• Platelets adhere to damaged blood vessel due to collagen (exposed when vessel breaks, platelts have special binding site)

  • – platelet adhesion

  • cause platelet to activate, and turn to its dendritic form

• Anchored platelets release chemicals to attract more platelets

  • – platelet aggregation

  • in dendrite form release chemicals to attract other platelets

    • like Adenosine Triphosphate (ADP)

      • inducing aggregation to the site

    • Thromboxane

      • cause platelets to interlink and activate exponentially

    • induces activation of thrombin

      • stimulate platelet activation further (positive feedback loop)

      • and induce fibrin to stabilize clots

• Aggregate and stick to each other to form a plug

• Stop bleeding in a small wound

Coagulation / Clotting→third phase of hemostatsis

• Intrinsic & Extrinsic pathways merging into a Common pathway

Factors in the Intrinsic pathway: (what is used in a PTT)

Involves factors

• XII, (7)

• XI, (6)

• IX, (4)

• VIII, (8)

• [X, V, II, and I] (10. 5, 2, 1); (the common pathway)

• Each activated factor activates the next in the series

• Initiated by contact with surface of blood vessel lining

• actor XII is activated; each activated factor activates the next in the series

  • XII→(need fletcher) XI→(need Ca++ to activate) IX→ (need Ca++ and PF3 to activate) VIII

    • PF3: platelet factor

PTT (partial prothrombin time)

• Used to detect congenital and acquired deficiencies of the above factors and to monitor heparin therapy

  • factors XII, XI, IX, VIII, [X, V, II, and I]

• Each activated factor activates the next in the series

• FOR TESTING OF THE INTRINSIC PATHWAY

Extrinsic pathway INVOLVES what factors

• Factors

  • VII,

  • [X, V, II, I]

• Thromboplastin (factor III) is released from tissue and damaged blood vessels, and activates the next factor (factor VII)

  • factor III+Ca++→ activated VII and activating extrinsic pathway

PT (prothrombin time)

• Used to monitor Warfarin/Coumadin therapy

• Thromboplastin (factor III) is released from tissue and damaged blood vessels, and activates the next factor

• Ca++ is needed to activate factor VII

Factors in the common pathway

Factors X, V, II, and I

• • Activated factors VII (by the Extrinsic) or VIII (by Intrinsic)

  • Activate the next factors in the series

• Factor 10 activate thrombin, allowing further activation of platelets, and fibrin

• Is done to perform the following process (ACTIVATES THROMBIN

  • Fibrinogen (I) {with Thrombin)→ Fibrin (monomer) {with calcium)→ Fibrin (polymer) {with factor III) → Stable fibrin clot (secondary hemostatsis)

Overview of how Instrinsic and Extrinsic Pathway lead to the common pathway

• Factor VIII (intrinsic) and/or Factor VII (extrinsic) activate factor X

• X→V→II (prothrombin)→ Thrombin → XIII

• Thrombin can activate Fibrinogen (I)→Fibrin (monomer) → to form a stable fibrin clot

  • (factor XIII cross links the fibrin fibres)

  • XIII can direcrly bind to fibrin and form the stable clot

Fibrinolysis

• Breakdown of fibrin after tissue is repaired

• Initiated by the conversion of plasminogen to plasmin

  • based on negative feedback loop

• Blood clots activate Tissue plasminogen activator (TPA),

  • this increases Plaminogens conversion into Plasmin

  • PLASMIN Dissolves blood clot

• This more blood clot means increase activation of plasmin

Problems in Haemostasis

• Insufficient or delayed clotting

• Intravascular clotting:

Thrombus and/or thrombosis

(clot on vessel wall)

embolus

Clot can break off which travels through the blood

lot can lodge in the brain (stroke), heart (cardiac infarction), lung (pulmonary embolism), calves (deep vein thrombosis or phlebitis)

defect of X chromosome (Inherited Disorders Bleeding disorders)

• Haemophilia A

  • factor VIII deficiency

• Haemophilia B

  • factor IX deficiency (Christmas disease)

Chromosome 4 defect

Haemophilia C

• factor XI deficiency

Von Wildebrand’s disease {Inherited Disorders: (Bleeding disorders)}

• Platelet do not aggregate and do not activate factor VIII

Thrombocytopenia {Inherited Disorders: (Bleeding disorders)}

Low platelet count – can also cause a bleeding disorder

Vitamin K deficiency (Acquired Blood Disorders)

Factors ll, Vll, lX and X are vitamin K dependent

lead to clotting issues

Disseminated Intravascular Coagulation (DIC) (Acquired Blood Disorders)

most often due to infection

clots shut down organs; result in death

Heparin; Coumadin (Warfarin) (Acquired Blood Disorders)

Affect patient ability to clot, could bleed to death

Aspirin (Acquired Blood Disorders)

has anti-platelet properties

Liver Disease(Acquired Blood Disorders)

Most coagulation factors are synthesized in the liver

e.x cirrosis

Coagulation Tests

• Bleeding Time

• Prothrombin Time – PT

• (Activated) Partial Thromboplastin Time – APTT or PTT

Bleeding Time

• Screening test for platelet function, capillary integrity and clotting factors

• Method

  • • Standard puncture made,

    • and blood blotted by filter paper to prevent external clotting

  • Platelet count of < 50×10⁹ - may prolong the bleeding time

• old test not readily used

(Miekle Modified) Ivy Method – Bleeding Time Test

• Blood pressure cuff on the patient’s forearm inflated to 40 mm Hg

• Standard puncture on the forearm by a template device (2 incisions 5 mm wide and 1 mm deep)

• Template device (Simplate or Surgicut) retractable blade

• Start a stop-watch when the puncture is made

• Record the time at which the bleeding stops

Normal Range for bleeding time

• Normal Range 1 - 9 minutes

Critical Value for bleeding time

• Critical Value > 12 min

Quality Control for Bleeding time

• Do not touch the incision when blotting blood droplets (will remove clots forming)

• Record if the patient used aspirin in past 2 weeks (as it affects platelets functionality)

• Proper PPE

• Follow Standard Precautions

Prothrombin Time test – PT

• Used to monitor oral anticoagulant therapy (Coumadin)

• Specimen: citrated plasma (light blue top tube)

• Centrifuge whole blood for 15 min at 3000 rpm (need platelet poor plasma)

• Separate plasma and refrigerate; freeze plasma if not tested within 4 hours

• Note: Some factors deteriorate rapidly – Factors VIII, VII, X, V

• Test Extrinsic pathway III → VII & common pathway → X→ V→ II→ I (→ ) these steps require Ca++)

• PT increased by deficiency of factors VII, X, V, II (prothrombin) and I (fibrinogen)

• PT increased in liver disease

• Factors ll, Vll, lX and X are vitamin K dependent

what factors deterioate rapidly even if tube is clsoed

Factors VIII, VII, X, V

PT – Test procedures

• Reagent – thromboplastin (tissue factor III; of the extrinsic pathway) and Ca++

• Reagent and specimen are incubated for 5 minutes at 37ºC

• Incubate no longer than 10 mins

– Factor VIII deteriorates, Reagent evaporates (and make sure to keep covered)

• Reagent is added to plasma and time to form a clot is measured

• Perform in duplicate: PT result must be within 10%

Factors that increase PT (clinical reasons)

• deficiency of factors VII, X, V, II (prothrombin) and I (fibrinogen)

• PT increased in liver disease

• Vitamin K deficency

  • factors ll, Vll, lX and X are vitamin K dependent

Manual PT: Tilt Tube

• Mix plasma and reagent and tilt tubes

• Examine visually for clot to form (based on the viscosity of the plasma mixed with reagent)

Automated PT

• Instrument monitors tube for change in optical density or electrical resistance and plasma viscosity

– End point fibrin clot

is affected by

• colored plasmas

• hemolyzed plasma

• lipemic plasma

• billirubin

• turbing reagents

SEMI –AUTOMATED PT (Stago)

• Viscosity based detection of clotting

• using amplitude of oscillating steel ball in specialized cuvette

• insensitive to and unaffected

  • colored plasmas

  • hemolyzed plasma

  • lipemic plasma

  • billirubin

  • turbing reagents

• The increase viscosity is measured through the motion of a stainless steel ball

• Constant pendulum swings of the ball are created by an electromagnetic field that is applied alternately on opposite sides of the cuvette by two independent driving coils

• As soon as the plasma starts to clot (as of coagulation process being initiated by addition of the clot starting reagent),

  • the viscosity of the plasma starts to increase, and this change in plasma viscosity affects ball movement, slowing it down

• •End product – solid clot

Result reporting of PT

• PT, in sec, is converted to an International Normalized Ratio (INR)

INR (International Normalized Ratio)

• is a way of standardizing the results of prothrombin time tests, regardless of the testing method

• Helps the doctor to understand results in the same way even when they come from different labs and different test methods

• treatment with blood-thinning medicine (anticoagulant therapy) will be standardized

• In some labs, only the INR is reported and the PT (Sec) is not reported

Calculations for INR/ PT ratio

PT _{patient sec} / PT _{mean normal cont. sec})^ISI

ISI is specific to thromboplastin used

Normal Range of PT/INR

• PT 10 - 14 sec

• INR 0.8 - 1

Therapeutic Range of PT/INR (if comadin or heparin given)

• PT 16 - 18 sec

• INR 2 - 3

Critical Range of PT/INR

• PT > 25 sec

• INR > 4

abnormal prothrombin time is often caused by:

• liver disease or by treatment with blood thinners

• Some Drugs that can affect the PT result:

  • Aspirin, (decrease; double check)

  • Vit. K supplement (increase)

  • antibiotics

  • and the birth control pill

(Activated) Partial Thromboplastin Time, APTT or PTT

• Used to monitor intravenous anticoagulant therapy (Heparin)

• Specimen same as PT (3.2% Na Citrate - plasma)

• If frozen (-200C or -700C), plasma should be thawed rapidly at 37⁰C, then gently mixed and tested immediately

• Mixing is critical before testing in order to resuspend proteins that may have been precipitated by freezing

• Most useful routine screening tests for the intrinsic pathway and common pathways

• Intrinsic pathway XII→XI→IX→VIII

• Common pathway →X→V→II→I

– (→these steps require Ca++)

• Used to monitor intravenous anticoagulant therapy (Heparin)

Testing for APTT or PTT

Two reagents

• Cephaloplasmin (phospholipid) with contact activator

• CaCl2

• Both reagents and specimen must be equilibrated for 5 minutes at 37ºC

• Cephaloplasmin (phospholipid) is added to the specimen and factor XII is activated after 3 minutes

– CaCl2 is added to complete the clotting cascade

• done in dublicate

Normal Range PTT

25 - 35 sec

Therapeutic Range PTT

.5 to 2x normal control

Critical Value PTT

> 60 - 100 sec

Quality Control for PTT

Normal and Abnormal Control plasma must be run every 8 hours

Manual Duplicate results:

<40 sec: must be within ±2 seconds or repeat

>40 sec: must be within ±3 seconds or repeat

Source of Errors Associated with specimen

– Order of draw

– Inappropriate ratio of anticoagulant to blood

– Clotted, hemolyzed or lipemic samples

– <10,000 platelets x109/L (false high)

– Delay in testing or processing

– Inappropriate storage

Source of Errors Associated with storage

Incorrect preparation of reagents

– Failure to properly store reagents (or not warm them up)

– Use of reagents beyond reconstituted stability time or expiration date

– Contaminated reagents

Source of Errors Associated with procedure

– Incorrect temperature

– Incorrect incubation times (↑ incubation time = ↓PTT due to contact activation and > 5min heating will result in loss of heat-labile factor V)

– Incorrect volumes of sample, reagents or both

Automated Coagulation Instrument Maintenance

• Practice proper PPE

• Always wear non-latex examination gloves when performing

maintenance work or inspection

• Each instrument is different. Follow the specified manufactures tools and parts

Maintenance Schedule-of autmoated PT

Daily

• • Probe cleaning reagent is cycled through the instrument –system maintenance menu

• • Check consumables (cuvettes, etc.)

Weekly

• • Clean sample area, reagent wells and light path

Monthly

• Clean and replace filters, change waste containers

Instrument Calibration

• Six months or when QC is off (as needed)

Reagent Change

• As needed Controls

• Make fresh daily

Point of Care Coagulating Tests

• defined as medical diagnostic testing performed outside the clinical laboratory in close proximity to where the patient is receiving care

• typically performed by non-laboratory personnel and the results are used for clinical decision making

• POCT devices are often ‘hand held’ or may be small portable analyzers

• generally more expensive than in lab testing but is appropriate and cost effective in some clinical settings

  • Testing is performed near the patient and informs immediate decisions for clinical management of the patient

  • FAST TAT

  • Laboratory ensure program quality

Example POCT tests

 blood glucose

 urine dipsticks

 blood gases

 chemistry

 hematology

 coagulation

 cardiac markers

 pregnancy tests

Quality Assurance for Point of Care Test Operators

• Training for all POCT operators who perform Point-of-Care Testing (POCT)

• To meet accreditation standards POCT operators must be knowledgeable about the types of Point-of-Care Testing (POCT) they are responsible for

• POCT operators are accountable to complete training

• Training can be In house or online

• POCT operators have documented evidence of training

• POCT operators are qualified to perform their responsibilities and perform tests according to the manufacturer’s instructions to obtain accurate and reliable results.

• A Policies & Procedures Manual is available to POCT operators

• Assess POCT operator competency as per SOP and accreditation

Criteria for Result Reporting (general)

• Run QC for accuracy and precision

• Test results are reported only when all performance specifications for a test are within QC acceptable limits

• Remedial actions are taken and documented when required

• When instrument problems are detected, all patient results obtained since the last acceptable quality control run are evaluated to determine if they have been adversely affected

• Reported result include pertinent information required for interpretation

• Patient specimen is analyzed only after meter function checks are OK

• Calibrate the system for each new box of test strips

Example of a POCT coagulatin test→CoaguChek XS System

• PT/INR in one minute

• Finger-prick or venous sample (minimum 8 ul)

• Test strip has reagent and code

• A blood drop is applied within 15 sec of collection

• The meter starts the test and the blood mixe with the ingredients on the test strip

• When the meter determines that the blood has clotted, it stops the measurement and calculate the result

• Results are stored in the meter

• Battery operated

Safety Precaution

• All samples must be regarded as potentially infectious.

• POCT operators must follow PPE

Maintenance

Daily

• • Check expiry date of strips

  • Place the meter on a flat surface, free of vibrations

  • Ensure that internal QC results are acceptable during testing.

  • Follow the policies and procedures for collecting and handling

  samples, testing patient samples, and reporting test results.

  • Report or log patient tests result

  • Clean and disinfect POC system

Monthly

• • Review QA Checklist. Include all the actions associated with the erroneous result

Every 3- 6 Months

• • Perform an external evaluation of accuracy, such as split sample testing or have two operators test the same patient and compare results

As Needed:

• Train new POCT operators

QUALITY ASSURANCE (ASSESSMENT)

• QA involves the entire testing process including safety, personnel, specimen collection and handling, performing the test, recording results and finally reporting patient test results to providers

• Develop a corrective action plan and implement the corrections

• The QA process involves investigation, identification and resolution of any problems with subsequent development of policies that will prevent recurrence

• These new policies will be periodically reviewed to ensure the actions taken corrected the initial problem over time

Pre-analytic Errors

• Patient ID

• Sample collection

• Test Ordering

• Incomplete Requisition

• Leaving the test strip exposed to light for extended time

• Selecting the correct site

• Specimen accessing