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Haematology and Transfusion Science > HS3 - Haemostasis 3 > Flashcards

HS3 - Haemostasis 3 Flashcards

0
Q

What is the anticoagulant EDTA used for, and how does it exert its effect?

A
  • Cell counts

- powerful Ca++ chelator

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1
Q

What is the anticoagulant citrate used for, and how does it exert its effect?

A
  • Clotting and platelet studies

- Ca++ chelator

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2
Q

What is the anticoagulant heparin used for, and how does it exert its effect?

A

Thrombin inactivator (may activate platelets)

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3
Q

How does the anticoagulant hirudin exert its effect?

A

Thrombin inactivator (expensive)

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4
Q

How does the anticoagulant PPACK exert its effect?

A

Thrombin inactivator

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5
Q

In what ratio is citrate added to blood?

A

1 volume of citrate is added to 9 volumes of blood

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6
Q

How is plasma prepared for coagulation tests?

A
  • blood is citrated
  • centrifuged at 200g for 10 minutes for platelet-rich plasma
  • centrifuged at 2000g for 10 minutes for platelet-poor plasma
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7
Q

How is the recalcification time test performed?

A
  • Ca++ is added to citrated PPP
  • hook used to pull up fibrin strands
  • time taken for fibrin strands to form is measured (130-240 s)
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8
Q

What are the inadequacies of the recalcification time test?

A
  • Insensitive to all but gross deficiencies

- affected by platelets, degree of contact with glass

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9
Q

How is the Prothrombin time (Quick test) performed?

A
  • PT reagent (Ca++, and thromboplastin) added to citrated PPP
  • Hook used to catch fibrin strands
  • Time to coagulation measured (10-15s)
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10
Q

What factors does the Prothrombin time measure?

A

Factors I, II, V, VII, and X

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11
Q

What is the Prothrombin time usually used to monitor?

A

Oral anticoagulants, eg, warfarin.

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12
Q

What is the results of the Prothrombin test usually reported as?

A

International normalised ratio (INR)

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13
Q

How is the Activated partial thromboplastin time (APTT) test carried out?

A
  • aPTT reagent (Ca++, kaolin, phospholipid) is added to PPP
  • Hook is used to catch fibrin strands
  • Time to fibrin formation is measured (20-45 s)
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14
Q

What does PT reagent contain?

A
  • Ca++

- Thromboplastin

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15
Q

What does aPTT reagent contain?

A
  • Ca++
  • Kaolin
  • Phospholipid
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16
Q

How do you perform a mixed test for a specific coagulation facto eg to test for FX deficiency?

A
  • patient plasma is mixed with an equal volume of FX deficient plasma
  • If patient FX deficient, clotting time is long as no FX in mix
  • If patient FV deficient, clotting time is normal (both FX and FV in mix)
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17
Q

How is the D dimer assay performed?

A
  • Latex beads coated with mAb against D-dimer reacts with D-dimers in blood sample causing an agglutination reaction.
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18
Q

Name 8 platelet function tests.

A
  • Count.
  • Aggregation.
  • Ristocetin-induced agglutination.
  • Adhesion tests.
  • Platelet function analyser (PFA-100)
  • Thromboelastograph
  • template bleeding time.
  • Flow cytometry
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19
Q

What is the name of the counting chamber used for performing a manual count of platelets?

A

Neubauer

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20
Q

Automatic counting of platelets is carried out in which machine?

A

Coulter automated cell counter

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21
Q

How does a Coulter counter work?

A
  • Cancer aspirates ethics volume.

- Blip recorded as each cell passes through aperture.

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22
Q

Aggregometry is used to measure platelet aggregation. How is this carried out?

A
  • ADP added to platelet rich plasma.
  • Platelets aggregate so tube becomes clear.
  • Light transmission detected by photodiode, Plotted against time.
  • Can measure maximum amount of aggregation, speed of aggregation, etc.
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23
Q

What is primary aggregation?

A

1st wave of aggregation triggered by weak stimulus

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24
Q

What is secondary aggregation, and what is it dependent upon?

A

sustained aggregation dependent upon positive feedback provided by platelet release reaction (ADP, 5-HT), and production of thromboxane A2.

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25
Q

Name 4 commonly used aggregating agents used in diagnosis.

A
  • Collagen
  • ADP
  • Adrenaline
  • Ristocetin
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26
Q

ristocetin is used in aggregometry tests, what is it and how does it work?

A
  • Antibiotic

- triggers binding of VWF to GPIb/IX/V in absence of shear forces

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27
Q

What positive feedback mechanisms make platelets sustain aggregation ?

A

Platelet release reaction, release of

  • ADP
  • 5HT
  • TxA2
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28
Q

Under low shear conditions, what receptor do platelets primarily bind to?

A
  • Collagen
29
Q

Under conditions of high shear platelets bind primarily to which receptor ?

A
  • VWF ( Von Willebrand Factor)
30
Q

What is the shear rate ?

A

The shear rate is the rate of change of velocity with each incremental change in the radial distance

31
Q

What is the formula for the shear rate ?

A

Shear rate = dv/dr

32
Q

In what vessels is the shear rate low?

A
  • veins

- wide vessels

33
Q

In what vessels is the shear rate high?

A
  • arteries

- Narrow vessels

34
Q

What happens to VWF molecules under high shear conditions?

A
  • becomes unravelled, more binding sites exposed.
35
Q

What effect does Ristocetin have on VWF ?

A

Unravels it even in the absence of shear forces and causes agglutination.

36
Q

What the test of ADP + collagen added to PRP tell you?

A
  • That aggregation is working.

- measures platelets binding together via fibrinogen

37
Q

What does the Ristocetin test tell you?

A

If platelets are able to bind to VWF

38
Q

What type of reaction is caused by the Ristocetin test and why?

A

Agglutination, because it works on dead platelets.

39
Q

There are two laboratory tests for agglutination which utilise the old antibiotic Ristocetin, name these two tests.

A
  • Ristocetin Induced Platelet Agglutination (RIPA)

- Ristocetin cofactor activity (RICOF)

40
Q

What does the ristocetin induced platelet agglutination (RIPA) test measure?

A

The degree of platelet agglutination when

ristocetin is added to patient PRP

41
Q

What is the ristocetin cofactor activity (RICOF) test measure?

A

The degree of platelet agglutination when
ristocetin is added to washed normal
platelets resuspended in patient plasma

42
Q

What does the ristocetin induced platelet agglutination (RIPA) test tell us?

A

Either platelets are not binding VWF all there is no VWF present, but the test will not identify which

43
Q

What is the ristocetin cofactor activity (RIOCOF) test tell us?

A

Platelets used normal and so the must be something wrong with the plasma such as Von Willebrand disease (no VWF).

44
Q

What does the Glass bead retention time test detect?

A

Platelets have an adhesion defect

45
Q

How is the Glass bead retention time test carried out?

A
  • Blood/PRP passed through column packed with glass beads
  • platelets stick to the beads
  • platelets collected at the end of the column if a large amount then adhesion defect
46
Q

What does the Glass bead retention time test depend on in order to work?

A

Functioning VWF and GPIb/IX/V complex

47
Q

What does the parallel plate flow chamber measure?

A

Adhesion

48
Q

How is adhesion measured using a parallel plate flow chamber?

A
  • Tube with collagen adsorbed on one of the sides
  • blood/PRP passed through Tube
  • fluorescent labelling of the platelets
  • look under fluorescent microscope and see how many platelets have stopped to the collagen
49
Q

What is a cone and plate viscometer used for?

A

Looking at what happens to platelets under high shear and low shear conditions (not used today)

50
Q

How is a test with the code and plate viscometer carried out?

A

Cone shape is plunged into a beaker of PRP and rotated at high speeds

51
Q

When using the cone and plate viscometer where does the area of high shear occur?

A

Near the narrow gap

52
Q

When using the cone and plate the spirometer where does the area of low shear occur?

A

At the sides of the vessel

53
Q

What is platelet function analyser designed to measure?

A
  • Aggregation

- Adhesion

54
Q

The cartridge used in the platelet function analyser has a collagen coated membrane which is also impregnated with what?

A

ADP or adrenaline

55
Q

How is platelet function analyser test carried out?

A
  • Anticoagulated blood sucked through cartridge
  • platelets activated on contact with collagen on membrane
  • platelets aggregate and block the aperture
  • resistance plotted against time
57
Q

What conditions is the platelet function analyser test sensitive to?

A
  • Low platelet counts
  • Platelet defects
  • Adhesion problems (VWD)
58
Q

How is a thromboelastograph test carried out?

A
  • Cylinder with a stylus attached is inserted into a tube of blood
  • Tube rotated
  • As blood clots cylinder and stylus moves and marks paper
59
Q

As a Thromboelastograph is carried out what happens to the movement of the of the stylus on the paper as the blood clots?

A

Movement gets bigger as clot forms and then gets slower again (traces an hour-glass shape)

60
Q

How is a template bleeding time carried out?

A
  • Small incision made in the skin of the patient’s forearm
  • Cut dealt with filter paper every 15 seconds
  • Time to cessation of bleeding recorded
61
Q

How long does it normally take for cessation of bleeding during a template bleeding time test?

A

2 - 9 minutes

62
Q

The template bleeding time is sensitive to what problems?

A

Defects in platelet function

63
Q

What substance can moderately lengthen the bleeding time in a template bleeding time test?

A

Aspirin

64
Q

What disorders are the template bleeding time test not sensitive to?

A

Clotting disorders

65
Q

How is Flow Cytometry performed?

A
  • Suspension of single cells aspirated in single file through a laser beam
  • detected by forward scatter detector, right angle scatter detector and fluorescent detector
66
Q

What does the forward scatter detector measure in flow cytometry?

A

Cell size

67
Q

What does the right angle scatter detector measure in flow cytometry?

A

Cell granularity

68
Q

How can the number of platelets in the sample be measured using flow cytometry?

A
  • monoclonal antibodies against CD41 labelelled with FITC Fluorochrome
  • labelled antibody mixed with sample
  • Antibody binds to platelets
  • Antibodies glow bright green when viewed through a flow cytometer
69
Q

How is Glannsmanns Thrombasthaenia diagnosed using flow cytometery?

A
  • !st Perform test with irrelevent antibody (wont bind, low signal)
  • Mix Normal blood with labbeled anti-body strong green signal
  • Mix patient sample no GPIIb on surface so weak green signal
70
Q

What receptor do patients with Glansmanns Thrombasthaenia lack?

A

Fibrinogen

Haematology and Transfusion Science (10 decks)

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