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1

Haemostasis:

process of stopping loss of blood from blood vessels

2

Steps in Haemostasis:

1. vasoconstriction: blood vessels narrow
2. platelet activation: adhesion + aggregation (platelet plug formation)
• Initiated by damaged or irregular blood vessels
• Adhesion- Platelets adhere to damaged blood vessel
• Aggregation- stick to each other to form a plug
3. clotting cascade: intrinsic + extrinsic pathways (blood clotting)
4. Fibrinolysis breakdown of fibrin initiated by the conversion of plasminogen to plasmin - LOOK AT LECTURE

3

APTT PATHWAY…………Intrinsic

• each activated factor activates the next in the series)
Initiated by contact with surface of blood vessel lining factor XII (12) is activated followed by (XI) 11 then (IX)9 using Ca then (VIII)8 using Ca and PF3 (platelet factor 3)
Contains factor XII, XI, IX, VIlI
Calcium is also needed for the activation

4

PT PATHWAY……Extrinsic

Thromboplastin (factor III) (comes from the skin) is released from damaged blood vessels and activates the next factor
Contains tissue factor and factor VII (7) using calcium

5

Common pathway

Check the lecture
Intrinsic and Extrinsic activates factor X (in common pathway)
Factors circulate in inactive forms until they are activated.

6

Stable Fibrin Clot

Fibrinogen (I) Fibrin (monomer) Fibrin (polymer) Stable fibrin clot

hemophiliacs cant clot

7

Problems in haemostatis:

insufficient or delayed clotting
intravascular clotting

8

intravascular clotting

thrombus (clot on vessel wall) can break off and become an embolus which travels through the blood
clot can lodge in the brain (stroke), heart (cardiac infarction), lung (pulmonary embolism), calves (deep vein thrombosis or phlebitis - when people are in a plane sitting)

9

Disorders of Haemostasis
Inherited Disorders:

Common Haemophilia A- factor VIII and VII:C deficiency – x chromosome defect (mostly men have disease and women are mostly carriers)
Haemophilia B- factor IX deficiency (Christmas disease) – x chromosome defect
Haemophilia C- factor XI deficiency chromosome 4 defect
Von Wildebrand’s disease- defect of platelets, platelet do not aggregate and do not activate factor VIII (proconvertin) – epistaxis (nose bleed symptoms) autosomal inherited by both men and women

10

Acquired Disorders

• Vitamin K deficiency (clotting factors II- Prothrombin , VII-Proconvertin , IX- Christmas factor , X- Stuart Power Factor)
• Disseminated Intravascular Coagulation (DIC):most often due to infection extra bleeding because of injury- thrombosis or bleeding
• Heparin – inhibits thrombin activation and factor X-
• Coumadin (warfarin) (anticoagulant – thrombosis) – acts to decrease Vit K dependent factors to allow for clotting
• Aspirin – prevents platelets from making PF3

11

Coagulation Tests
Bleeding Time:

Test analyzes how quickly small blood vessels in your skin close up and stop bleeding.
Test evaluates 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- may prolong the bleeding time

12

Miekle Modified Ivy Method –Bleeding Time Test (Done by technologists)

• blood pressure cuff on patient forearm inflated to 40 mm Hg
• standard puncture on forearm by template device (2 incisions 5mm wide and 1 mm deep)
• Template device (Simplate or Surgicut) retractable blade
• Start stop watch when puncture made
• Record time at which bleeding stops

NR 1-9 minutes
Critical Value > 12 min

13

Bleeding Time Test QC

• Do not touch incision when blotting blood droplets
• Record if patient has used aspirin in past 2 weeks (platelet survival is 10 -14 days)
• Proper PPE
• Follow Standard Precautions

14

Prothrombin Time Test- PT

• Used to monitor oral anticoagulant therapy (Coumadin) - common blood thinner - inactivates factor 7 so patient does not clot easy- too much bleed out not enough too many clots
• Specimen citrated plasma (blue top tube)
• Need Platelet Poor Plasma--Centrifuge 15 min at 3000 rpm, separate plasma and refrigerate; freeze plasma if not tested within 4 hours
• Reagent ( thromboplastin)= (tissue factor III-phospholipid) and CaCl++
• PPP plasma and thromboplastin are incubated for 5 minutes at 37ºC

Incubate no longer than 10mins-why?
• Factor VIII deteriorates
• Reagent evapourates
• Reagent is added to plasma and time to form a clot is measured
• Perform in duplicate: PT result must be within 10%
Tests extrinsic pathway III -> (needs Ca) VII & common pathway-> X ->(needs Ca) V-> II-> I
PT increased by deficiency of factors VII, X, V, II (prothrombin) and I (fibrinogen)
PT increased in liver disease

15

PT- Manual method: Tilt tube

• mix plasma and reagent
• tilt tubes
• examine visually for clot to form

16


Automated method

• The instrument monitors tube for change in optical density or electrical resistance
• End point fibrin clot

17

Principle of clot detection: SEMI –AUTOMATED

• The increase of 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.
• However 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.

18

Prothrombin Time Test

Results: 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.
• This helps the doctor to understand results in the same way even when they come from different labs and different test methods.
• Using the INR system, treatment with blood-thinning medicine (anticoagulant therapy) will be standardized.
• In some labs, only the INR is reported and the PT is not reported
• ISI, International Sensitivity Index: specific to thromboplastin used (usually 1.0)

19

Ranges for PT and INR

Normal Range PT = 10-14 sec
INR = 0.8-1
Therapeutic Range PT = 16-18 sec
INR = 2-3
Critical Value PT > 25 sec
INR> 4

An abnormal prothrombin time is often caused by liver disease or by treatment with blood thinners.
Some Drugs that can affect the PT result: Aspirin, Vit K supplement, antibiotics and the birth control pill

20

(Activated) Partial Thromboplastin Time, APTT or PTT

• Specimen same as PT (3.2% Na citrate)
Two reagents
(1) cephaloplasmin (phospholipid) with contact activator and (2) CaCl2

Both reagents and specimen are equilibrated for 5 minutes at 37ºC
Cephaloplasmin (phospholipid) is added to specimen and factor XII is activated after 3 minutes
CaCl2 is added to complete the clotting cascade

PTT is the most useful routine screening tests for the intrinsic pathway and common pathways
Intrinsic XII->XI->(require Ca++)IX->(require Ca++)VIII and common->X-> (require Ca++)V->II->I
Used to monitor intravenous anticoagulant therapy (Heparin)

21

QC (Activated) Partial Thromboplastin Time, APTT or PTT

Manual method
Duplicate results:
<40 sec: must be within ±2 seconds or repeat
>40 sec: must be within ±3 seconds or repeat

22

Sources of Error/Troubleshooting:(Activated) Partial Thromboplastin Time, APTT or PTT

1. Associated with specimen

a. Order of draw
b. Inappropriate ratio of anticoagulant to blood
c. Clotted, hemolyzed or lipemic samples
d. <10,000 platelets x10^/L
e. Delay in testing or processing
f. Inappropriate storage

23

Sources of Error/Troubleshooting:(Activated) Partial Thromboplastin Time, APTT or PTT

2. Associated with storage

a. Incorrect preparation of reagents
b. Failure to properly store reagents
c. Use of reagents beyond reconstituted stability time or expiration date
d. Contaminated reagents

24

Sources of Error/Troubleshooting:(Activated) Partial Thromboplastin Time, APTT or PTT

Associated with procedure

a. Incorrect temperature
b. Incorrect incubation times (↑ incubation time=↓PTT due to contact activation and > 5min heating will result in loss of heat-labile factor V)
c. Incorrect volumes of sample, reagents or both

Quality Control: Normal and Abnormal Control plasma must be run every 8 hours

25

PTT normal range

Normal Range PTT = 25-35 sec
Therapeutic Range PTT = 1.5 to 2 x normal control
Critical Value PTT > 60 -100 sec

26

Interpretation of PT and PTT Results
PT result - prolonged
PTT normal

Liver disease, decreased vitamin K, decreased or defective factor VII

27

Interpretation of PT and PTT Results
PT result - normal
PTT - prolonged

Decreased or defective factor VIII, IX, or XI, von Willebrand disease, or lupus anticoagulant present

28

Interpretation of PT and PTT Results
PT result - prolonged
PTT - prolonged

Decreased or defective factor I, II, V or X, severe liver disease, disseminated intravascular coagulation (DIC)

29

Interpretation of PT and PTT Results
PT result - normal
PTT - normal

May indicate normal haemostasis; however PT and PTT can be normal in conditions such as mild deficiencies in other factors and mild form of von Willebrand disease. Further testing may be required to diagnose these conditions.

30

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