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Unit 12 > Haemostasis and coagulation practical > Flashcards

Haemostasis and coagulation practical Flashcards

1
Q

What is meant by primary and secondary haemostasis?

A

Primary - interaction between blood vessels, platelets and vWF, leads to initial platelet plug to act as a barrier to blood loss.

Secondary - coagulation biochemistry generates firbin strand which strengthen the clot.

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2
Q

What word can be used to describe endothelial cells that have been activated and promote blood clotting?

A

Prothrombotic

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3
Q

What is the main function of platelets during haemostasis?

A

Interact with vWF to form the initial barrier to blood loss
Allow platelet-platelet interaction to propagate the thrombus
Provide a negatively charged lipid surface to support key reaction of coagulation biochemistry.
Promote vasoconstriction
Promote vessel repair
Maintain the molecular integrity of endothelial cell junctions.

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4
Q

What is the normal sample prothrombin time?

A

10-15 seconds

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5
Q

What are some of the indication of an elevated prothrombin time?

A

Indicates a deficienct in the extrinsic or common pathway.
May be due to a vitamin K deficient, liver disease, drug side effects etc.

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6
Q

What is an INR ratio?

A

International Normalised Ratio
A standardised form of the PR.
Compares the patients PR to a normal PT from the same lab, gives results as a ratio.
1 = the same as normal
Elevated = longer time for blood to clot in patient
Different labs may use different reagenets to measure PT, reagent effectiveness varies between batches therefore a single PT value can be highly inaccurate.

INR = PT patient/ PT normal.

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7
Q

How is INR used to monitor patients on anticoagulants?

A

Mainly used for patients on warfarin as this has a low therapeutic index meaning it is easy to overdoes and risk excessive bleeding
INR values can vary but a value of 2.5 (+_0.5) is normally a good range for patients on anti-coagulatns.

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8
Q

What are the three main classifications of anti-thrombotic drugs?

A

1) Vitamin K-dependent anticoagulants (warfarin)
2) Heparin and heparin-like compounds - indirectly inhibit thrombin and Fxa (require injection or infusion)
3) Direct acting oral anticoagulants (NOAC or DOAC) directly inhibit coagulation factors usually thrombin or FXa.

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9
Q

What is thromboplastin and modified thromboplastin?

A

Terms originating from labarotiry practise rather than physiology
Thromboplastin - mixture of TF and phospholipids (normally prepared from brain or placenta)
Partial thromboplastin - phospholipids, this meant coagulation was only activated when comes in contact with glass surface or other contact activators such as kaolin.

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10
Q

What is the difference in what the results of an activated partial thromboplastin time (aPTT) and a Prothrombin time (PT)indicate?

A

PT - extrinsic pathway and common pathway
aPTT - intrinsic pathway and common pathway.

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11
Q

What is the aPTT in a normal sample?

A

25-35 seconds

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12
Q

What may be some of the causes behind a prolonged aPTT?

A

Coagulation factor deficienties
Inhibitors of the coagulation pathway
Normally prolonged by heparin-like anticoagulants and direct thrombin inhibitors.
Antiphospholipid syndrome

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13
Q

How do we monitor patients being treated with unfractionated heparin?
Why?

A

aPTT
Expect value to be 1.5-2.5 times there normal.
UFH - limited bioavailability and high variable anticoagulant response, meaning higher risk of haemorrhagic side effects,

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14
Q

Are patients on low-molecule weight heparin monitored? Why/why not?

A

Do not need monitoring
has a specific molecular weight distribution that determines anticoagulant activity and duration of action
Predictable dose response and fewer non-haemorrhagic side effects therefore do not need as much monitoring.
Mainly only activates AT-3, has lower affinity for other coagulation enzymes.

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15
Q

Draw a table to show what coagulation factors aPTT and PT look for?

A
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16
Q

Describe how to complete a prothrombin time experiment?

A

PT reagent already contains CaCl2 - incubate at 37 degrees in the water bath
Pipette 0.1ml (normal plasma) in 5ml conical tube and incubate at 37 degrees for 2 minutes
Pipette 0.2ml PT reagent into plasma tube and start timer
Mix tube and leave in water bath for 7-8 seconds.
Remove from water bath, wipe exterior tilt back and forth gently until a visible clot is formed (mixture gelatinize and turn cloudy)
Stop timer immediately when clot starts and record the time.
Repeat steps until 3 norm, and 3 patient samples
Calculate a mean

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17
Q

Describe the experiment to calculate an activated Partial Thromboplastin Time?

A

aPTT reagent and CaCl2 seperate tubes should be incubated at 37 degrees
Pipette 0.1 ml (normal plasma) into a 5ml conical tube, incubate for 5 minutes at 37 degrees
Add 0.1ml of CaCl2 to the tube containing plasma/apTT reagent and simultaneously start the stopwatch
Gently swirl the tube in the water bath for 20 seconds.
Remove from water bath, wipe exterior and tilt back and forth gentil until a visible clot is formed
Stop timer
Record and repeat for 3 norm and 3 patient samples - calculate a mean and compare.

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18
Q

What are the consequences of a patient with a reduced Packed Cell Road/ hemetocrit?

A

Reduced RBC count
Reduced blood oxygen carrying capacity
Increased CO and HR to compromise
Viscosity of blood will also be reduced.
More likely to have turbulent blood flow

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19
Q

What are the different layers in blood?
What proportion of blood do they make up?

A

Plasma - 54
Buffy coat - less than 1%
Erthrocytes - 45% males, 42% in females
Is separated into these three layers from top to bottom by centrifuging

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20
Q

What is the plasma components of blood?

A

The cell free component
is 90% water, 8% plasma proteins mostly made in the liver and 2% dissolved solutes (nutrients, gases, hormones, waste and ions)
Made from proteins, molecules and ions
Pale yellow due to bilrubin

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21
Q

What makes up the buffy coat layer of blood?

A

Leukocytes and thrombocytes
Note there are more platelets but each platelet has a smaller volume than a WBC, in total they are equal in volume.

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22
Q

What other terms can be used for the erythrocyte components of blood?

A

The packed cell volume
The haematocrit.

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23
Q

What are the normal blood count ranges in a healthy adult?

A
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24
Q

What is the role of albumin?

A

Exerts osmotic pressure to maintain water balance

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25
Q

What is the role of alpha and beta globulins?

A

Transport proteins that bind to lipids, ions and fat-soluble vitamins

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26
Q

What is meant by gamm globulins?

A

Antibodies released by plasma cells during an immune response

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27
Q

What are some examples of clotting proteins?

A

Fibrinogen and prothrombin

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28
Q

Describe the structure of platelets

A

Anucleate cells
Smallest cell in the body (1/1o of rbcs)
Bi-convex shaped
Contains granules and functional mitochondria
Lifespan in approximately 7-10 days

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29
Q

What is the role of platelets during primary haemostasis?

A

Accumulate at site on injury activated by vWF binding to glycorptein VI on the surface. Platelets also bind directly to subendothelial collagen via another glycoprotein on their surface which activates them
Conformational platelet change causes platelets to project pseudopodia across tissue increasing surface area to interact with each other and cover wound
Bind to each other by a process called aggregation, aided by fibrinogen having a bi-functional receptor for platelets GPIIb/IIIa on surface (up regulated when activated)
Activated platelets release chemical mediators. ADP (from dense granules) and Thromboxane A2 to activate more platelets and encourage to aggregating to growing plug.

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30
Q

What is the role of platelets during secondary haemostasis?

A

Flip phosphatidylserine from inner to outer plasma membrane this has procoagulant activity
Acts as a scaffold for Ca2+ and coagulation factors to accumulate, this supports the coagulation cascade, promoting fibrin formation and secondary haemostasis.

31
Q

How are platelets produced?

A

Multipotent Haemopoietic stem cell develops into a common myeloid progenitor cells
Megakaryoblast are produced
Thrombopoietin acts as a hormone to regulate the production of platelets.
Stimulates megakaryoblast to become magakaryocyte through repeated mitosis but NOT cytokinesis
The plasma membrane invades the cytoplasm to divide in into compartments.
The cell ruptures and the compartments released are platelets.
The plasma membrane reforms around to seal these fragments.

32
Q

What is meant by formed elements in blood?

A

Cells, cell fragments or cell remenants
In the blood there are three formed elements - rbcs, wbcs and platelets

33
Q

What is another name for a platelet?

A

Thrombocyte

34
Q

What section of the vessel lumen are platelets normally found in?

A

Closer to the vessel wall - due to their smaller mass are pushed outwards
So they are immediately available if the vessel wall is injured

35
Q

What alternative names are used for tissue factor?

A

Thromboplastin
FIIIa

36
Q

What terms can be used to indicate low and high platelet count?

A

Thrombocytopenia - low platelet count
Thrombocytothaemia - high platelet count

37
Q

What is the difference between bleeding time and coagulation time?

A

Bleeding time is measured in via and tends to be depdents on the formation of a primary haemostatic pulg
Coagulation time is measured in vitro, tends to measure a certain part of the coagulation cascade, timer is stopped with a fibrin fibre starts to form.

38
Q

How long do you need to wait between giving blood?

A

12 weeks for males
16 weeks for females
To ensure haemoglobin levels remain within the normal range, are depleted when blood is taken, must increase against as rbcs reproliferate and make up the population again.

39
Q

What is the difference between haemostasis and thrombosis?

A

Haemostasis tends to be physiological
Thrombosis tends to be pathological
Both are the formation of clots (platelet adhesion, aggregation and coagulation).

40
Q

What are the key differences between arterial and venous thrombosis?

A

Arterial thrombosis - platelet rich hence termed white thrombi, mainly found in cerebral and coronary arteries

Venous thrombosis - fibrin rich and trap large number of rcs hence termed (red thrombi), FOrm mainly in deep veins and tend to become disloged and cause pulmonary embolism.

41
Q

What is a bleeding diathesis?

A

A tendency to bleed excessively
For example inherited von Willebrand Disease and Haemophilia A.

42
Q

How can anemia cause a bleeding risk?

A

Reduced rbc count
Without large erthrocytes in the centre of the vessel, platelets are not pushed to the outside edges of the vessel, so are not as incontact with the vessel wall so respond to endothelial injury less efficiently.

43
Q

What is the purpose of vascular spasm?

A

Reduces the speed and volume of blood flow.

44
Q

What platelet released factors are vasoconstrictors?

A

Seratonin and thromboxana A2.

45
Q

What is the importance of the platelet granules in haemostasis?

A

Dense granules - contain ADP (aggregation and activation by binding to purinoceptors - inhibited by drugs clopidogrel and ticagrelor)
Alpha granules - vWF, fibrinogen, factor V, GPIIb/IIIa.
De Novo syntheses - release arachidonic acid from phospholipid membranes, converted by COX and thromboxane synthetase to TxA2 which promotes platelet activation and vasoconstriction.

46
Q

What makes up the prothrombinase complx?

A

The factors that convert prthrombin to thrombin
Includes FXa and Va,

47
Q

How does fibrin limit the continued growth of a clot?

A

Fibrin binds thrombin
Prevents thrombin from escaping an stimulating earlier septs in the coagulation cascade
Unbound thrombin is also inactivated in the plasma by antithrombin 3 and protein C

48
Q

Draw a diagram to represent the coagulation cascade.

A
49
Q

Why is it physiologically beneficial to have so many steps in the coagulation pathway?

A

More steps provides more control over a pathway, particularly when there is positive feedback, this can prevent the pathway being triggered in uninjured areas.

Also coagulation factors are enzymes so can active many substrate factors, this results in rapid growth in the number of activated coagulation factors, this enables a strong coagulation cascade response and rapid thrombus formation.

50
Q

What treatment is given to patients with haemophilia who have a bleed?

A

Transfusion of fresh plasma
Injections of the synthesesied missing coagulation factor VIII
This is expensive and only provides relief for several days
If often very inconvenient for the patient.

51
Q

What factors can convert plasminogen to plasmin?

A

Tissue plasminogen activate (tPA)
Urokinase
Activated factor XII and thrombin

52
Q

How does atherosclerosis increase the risk of a thrombus formation?

A

Causes turbulent blood flow
Results in endothelial damage
Increased risk of thrombus formation

53
Q

How does dehydration increase the risk of thrombus formation?

A

Blood is more viscous
Slower blood flow
Increases likelhood of platelet adhesion to the endothelial walls.

54
Q

What compounds are used in vitro to prevent blood clotting during blood collection?

A

Citrate and EDTA

55
Q

What does aspirin act as an anti-coagulant?

A

Class: NSAID
Pharmacology: irreverisble inhibits COX-1 and COX-2 enzymes so blocks the production of prostaglandins particularly thromboxane A2 in platelts
This decreases platelet aggregation decreasing the risk of thrombus formation
**

56
Q

How do citrates and EDTA prevent blood coagulation?

A

Binds Ca2+

57
Q

What anti-coagulant has a rapid affect interfereing with blood clotting with administered intravenously?

A

Heparin

58
Q

Why is it important for people who take warfarin to monitor their VitK intake?

A

Warfarin inhibits Vkreductase enzyme, decreases the amount of active vitamin K (reduced form) in the body
Their are many VitK dependent enzymes, not just those involved in the clotting cascade, for example
Ensuring the person has a high dietary VitK ensures that these enzymes are still functional.
But must be careful not to take too much Vit K that the warfarin is not as effective.

59
Q

How do we prevent spontaneous coagulation of the blood?

A

By endogenous anti-coagulants
These are mainly produced by undamaged endothelial cells
Although antithrombin 3 on of the most important is produced in the liver.

60
Q

How do endothelial cells prevent spontaneous coagulation and how do they cause fibrinolysis?

A

Inhibit platelet aggregation - producing PGI2 and NO, and form physical smooth barrier from underlying collagen
Inhibit coagulation by producing - thrombomodulin, heparin sulphate (membrane bound) and TFPI (membrane bound and released into serum).
Promote fibrinolysis by synthesising tissue plasminogen activator (tPA)

61
Q

How can thrombin prevent coagulation?

A

Binds to protease-activated receptor on endothelium causes production of heparin sulphate NO and PGI2.

62
Q

How does thrombomodulin prevent thrombus formation?

A

Expressed on the surface of endothelial cells - binds to and inactivates thrombin hence preventing inappropriate fibrin formation and platelet aggregation
Bound thrombin can then activate Protein C
Protein C (and co-factor Protein S) then inhibit FVa and FVIIIa found earlier in the intrinsic and common pathway of the coagulation cascade.

63
Q

What function in haemostasis does antithrombin inhibit?

A

Thrombin
Inactivates factor IIa, Xa, XIa, XIIa
This inhibits the intrinsc pathway and coagulation.

64
Q

What factors inhibit the first step of haemostasis, platelet plug formation?

A

Smooth endothelial surfaces
Heparan sulfate
PGI2 (also inhibits the release of platelet granules)

65
Q

What experiment could be done to measure bleeding time?

A

Patient anterior forearm
Inflate BP cuff to 40mmHg to maintain pressure
Alcohol wipe arm and allow to air dry
Using a sterile lancet puncture the arm around 2 inches below the cuff, start timer
Allow to bleed for 30 seconds
Blot blood with filter paper
Repeat until stops bleeding
Record time
Do not touch the arm with the filter paper.

66
Q

What procedure can we use to test bleeding time?

A

Glass slide
Lancet to finger prick patient
Blood on slide
Start timer.
Leave 30 seconds
Use toothpick to spread out drop and pick upwards after placing in blood
Look for thin fibrin strings forming
Repeat every 5 seconds until observed
Record time

67
Q

Why do we often test bleeding time and clotting time?

A

Bleeding time - in vivo function - primary platelet plug
Coagulatin time - in vitro - reliant on fibrin - secondary haemostasis
Tests different aspects of a thrombus formation.

68
Q

How do you set up a hemocytometer?

A

Complete finger prick, wipe off first drop, use the larger second drop, fill the rbc pipette up to 0.5 mark with blood
Fill pipette to 101 mark with hayems solution
Holding horizontally, twist the pippetee to mix and dilute the blood sample for 2 minutes
Set up the hemocytometer with a cover slip, fill the stage with blood using a hanging drop between the cover slips and counting chamber using the pipette, will fill the stage by capillary action.

69
Q

How do we count cells on a hemocytometer?

A

Use the middle small sqaure
Specifically the top left, top right, bottom left, bottom right and central smaller square
Count all the platelets/rbcs starting and working across the smallest squares within these are rows for guidance
Count cells that touch the middle line in the top left border
Do not count cells that touch the middle line in the bottom or right border.

70
Q

How to calculate the concentration of cells from in a hemocytometer?

A

Each small square has a volume of 0.1mm3 or 0.1ul
As counter 5 out of 25 squares multiply cell count by 5
Then multiply by 200 (dilutation factors from rbcs to haymens)
Then multiply be 10 to get the number of cells in 1ul

71
Q

What are some common causes of thrombocytothaemia?

A

Infection/inflammatory condition
Anemia
Cancer
Contraceptive pill
Absence of a spleen
Recovery from intense exercise or alcohol consumption.

72
Q

What can cause a platelet count to be below the reference range?

A

Thrombocytopenia
Bone marrow disroders
Cerain drugs - aspirin and ibuprofen
Autoimmune disroders
Cirrhosis
Cancer
Von Willebrand disease
Long term bleeding problems such as ulcers.

73
Q

What conditions in characterised by an elevated aPTT and a FVIII deficiency?

A

Haemophilia.

Unit 12 (23 decks)

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