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Lab D > Lab 3 > Flashcards

Lab 3 Flashcards

1
Q

Definition of Haemostasis

A
  • Is the name of a group of processes initiated in the body in order to stop bleeding in case of tissue and/or blood vessel injuries.
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2
Q

Major group of hemostasis disorders:

A
  1. Vasculopathy 2. Thrombocytopathy 3. Coagulopathy
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3
Q

Definition of Thrombocytopathy

A

Decreased ability of platelets to aggregate and adhere to the site of injury, and formation of the primary thormbocyte -thrombus, the second step of haemostasis.

  • Thrombocytopenia: decreased amount of thrombocytes in the blood
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4
Q

Definition of Vasculopathy

A

decreased ability of vasoconstriction in case of blood vessel injury, the first step of the haemostasis process

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5
Q

Definition of Coagulopathy

A

Problems with the extrinsic-, intrinsic-, or common pathway of the coagultion cascade, which ends with the formation of a polymerised fibrin network, which keeps thrombocyte thrombi at the site of injury, the third and final step of haemostasis

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6
Q

Test performed by side of the animals:

A
  • Signs of increased bleeding tendency: on the skin and mucous membranes: anemia, petechia, ecchymosis, suffusion; in the thoracic cavity: haemothorax; in the abdominal cavity: haemoperotoneum; in the GI tract: haematemesis, melena.
  • Capillary resistance
  • Bleeding time (Buccal Mucosal Bleeding Time Test, BMBTT)
  • Appearance of the first fibrin strand (clotting time)
  • Appearance of the cot (clotting time on different surfaces)
  • Clot reaction time
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7
Q

How to test Capillary resistance:

A

By putting a ligature on the arm, above the elbow and checking the palmar side of the lower arm for petechie. After 3-5 min. of ligature 3 small petechie should appear normally.

  • If capillary function is not proper: more petechia appear.
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8
Q

When do you choose to test Bleeding time, Buccal mucosal bleeding time?

A

NOT for coagulopathies but for: Thrombocytopenias, thrombocythopathies and Vasopathies.

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9
Q

How to test BT, BMBT?

A

Make a 0.1-0.2 mm deep, 0.5cm long incision on the skin of the inner part of the external ear or the buccal mucosal surface. Vipe the blood drop flowing UNDER the wound with cotton or paper in 20-30 sec intervals. Dont touch the wound itself - you may remove all the small thrombocyte-thrombus and the bleeding time may be longer.

  • Measure the time from the appearance of the first drop of blood until the ceasing of bleeding.
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10
Q

Normal BMBT?

A

BT, BMBT is dependent on the thrombocytic function, the platelet cound and the capillary function. Normal: 3-5min

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11
Q

What is tested by Coagulationtime?

A

Coagulopathies!

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12
Q

Sampling method of the Coagulation time:

A

From fresh, native (without anticoagulants!) whole blood samples, immediately after taking them!

  • Samples should be taken with only one precise venipuncture (so we dont cause damage of the surrounding tissue
  • > this can cause increased tissue factor III release of the damaged cells, which leads to the initiation of the coagulation cascade during sampling!!).
  • Use the “two syringe method” (change syringe after the first drops, and use content of the second syringe.
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13
Q

Appearance of the first fibrin strand:

A

The first strand should appear within 1-2min.

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14
Q

Clotting time (CT) on watch glass:

A

Put the fresh blood sample on watch glass that was previously treated with paraffin or wax (otherwise scratches of the glass would activate the coagulation cascade).

  • check the complete coagulation time: normally 7-15min.
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15
Q

CT in plastic syringe:

A

Normal is 10-12 min

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16
Q

CT in glass tube:

A

Normal is 4-5min

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17
Q

CT in ACT(Activated clotting time) tube:

A

Fresh blood into a glass test tube that containes SiO2, put it in a thermostate (37*C) and check the time of the complete coagulation.

  • Coagulation should be checked by slowly moving the tube every 15-20 sec. Normal is 3 min.
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18
Q

What is the function of SiO2 in the ACT tube?

A

SiO2 activates Factor XII (Hagemann-factor, contact factor). Activated Factor XII activates Factor IX and Kallikreinogen, Kinigogen (fibronolytic pathway)

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19
Q

When is Platelet (thrombocytic) count used?

A

When the BT, BMBT is increased, or petechie are visible on the skin or mucouse membranes.

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20
Q

What sample should the Platelet (thrombocytic) count be measured from?

A

From anticoagulated blood. For this purpose Na2-, or K2 EDTA should be used.

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21
Q

Methods of Platelet (thrombocytic) count:

A

1: 0.1 ml EDTA into 0.9 physiological saline -> sedimentation for 2 hours.
2: Blood smear.
3: Automatic cell counters

22
Q

Major causes of Thrombocytopenia:

A

1: Decreased production of thormbocytes in the bone marrow
2: increased utilisation of thombocytes: DIC (disseminated intravascular coagulopathy)
3: increased destruction of thrombocytes: autoimmune thrombocytopenia (AITP)
4: increased sequestration of thrombocytes: in case of (chronic) splenomegaly
5: increased loss of thrombocytes: subacute/chronic bleeding

23
Q

What do you check with Clot retraction test?

A

Thrombocytc function. If the clot reaction is slower, or does not happen at all, we can suspect thrombocytopathy.

24
Q

Clot reaction test: what is the reason for clot reaction?

A

if you leave the blood in a tube for some hours, it will become smaller, and serum will appear around the clot. The reason fot this clot reaction is that platelets contain contractile protein called Thrombostenin.

25
Q

When do we perform Platelet aggregation test?

A

when we suspect thrombocytopathy, i.e von Willebrand disease. In this case we can use aggregometers to estimate the aggregating ability of platelets correctly.

26
Q

Preparation/method of the Platelet aggregation test:

A

Have to prepare a Citrated blood sample and the upper layer should be used for the analysis (this is the platelet rich plasma). Then the sample is placed into a cuvette and add drugs that causes exaggerated aggregation, such as ADP, epinephirne etc. These drugs will induce platelet aggregation which will cause the clearing up of the fluid in the cuvette. The speed and rate of clearing can be analysed by a spectrophotometer.

27
Q

Thrombocytic morphology:

A
  • 1-2 um diameter.
  • Their centre is the granulomer and the edge is the hyalomer part (zone).
  • Horse, sheep, cattles have the smallest platelets (3-5 fl) - dogs and swine have bigger: 7-8 fl - cats have biggest (10-15 fl)
28
Q

Major causes of thormbocytopathies:

A

1- improper development of platelets, fex beacuse of hereditary glucoprotein deficiencies.

2- von Willebrand´s disease.

3- Uraemia, liver failure, myelo-, and/or lymphoproliferative diseases, NSAID treatments etc.

29
Q

In case of thrombocytopenia, thrombocytopathies and vasopathies besides normal coagulation we cannot expect signs of severe bleeding disorder because?

A

these are prevented by the formation of polymerised fibrin strands (fibrin thrombus) at the end of the coagulation cascade.

30
Q

Coagulopathies can lead to severe bleedings because?

A

thrombocytic thrombi are not stable without a fibrin network, and in case of a big blood vessel injury, the power of blood flow can sweep away the thrombocyte-thrombus from the wound.

31
Q

What type of sample should be used in examination of Coagulopathies?

A

citrated blood (citrate prevents coagulation by binding calcium ions), which means 3,8% Na-citrate:blood = 1:9

32
Q

How to test Prothrombin time (PT):

A
  • must be performed within 1hr after sampling - Blood sample should be mixed with 3.8% sodium citrate in 9:1 dilution (4.5ml blood and 0.5ml Na-citrate).
  • then centrifugation in 3000 rmp for 10 min. and separate the decalcinated plasma from the sediment.
  • the plasma must be kept on 37*C
33
Q

The reagent (Simplastin) for Prothrombin time (PT) evaluation contains?

A

rat uterus tissue homogenate as tissue thromboplastin (Factor III) and CaCl2. The reagent must be kept on 37*C before usage.

34
Q

Evaluation of the Prothrombin Time (PT)?

A

can be performed by using coagulometer or in test-tube. 200ul reagent mixed with 100ul decalcinated (citrated) plasma and the time of coagulation should be noted. Normal PT: 10-15 sec.

35
Q

The Prothrombin Time (PT) gives information about?

A

the function of the extrinsic pathway, because the coagulation cascade is triggered by adding tissue factor (and calcium ion) to the decalcinated plasma sample. Factors involved in PT are: VII, X, V, II, I, XIII

36
Q

What type of sample should be used in examination of Activated partial thromboplastin time (APTT)?

A

decalcinated plasma (similar to PT).

37
Q

the reagent (silimat) in APTT contains?

A

rabbit brain homogenate as PF3 (platelet factor 3) and micronised silica as contact activator.

38
Q

How to test APTT?

A
  • 100ul decalcinated plasma should be mixed with 100ul reagent and the mixture should me kept on 37*C.
  • then 100ul of 0.025 mmol/l CaCl2 solution should be added and from this moment the time of coagulation should be noted.
  • Normal ATPP: 20-30 sec
39
Q

The Activated partial thromboplastin time (APTT) gives information about?

A

the function of the intrinsic pathway, because the cascade is triggered by providing surface activation (initiating the effect of free collagen, which appears on the inner surface of the vessels in case of blood vessel injury), and adding platelet factor 3 (PF3) and Ca2+ for the activation of factor X

40
Q

Factors involved in APTT?

A

XI, IX, VIII, X, V, II, I, XIII

41
Q

What type of sample should be used in examination of Thrombin time (TT) and what can be examined?

A
  • decalcinated plasma mixed with a reagent containing thrombin only.
  • in this case coagulation time depends on the concentration of fibrinogen and factor XIII in the plasma.
  • This test can also be used for estimation of fibrinogen concentration (suspecting a normal factor XIII level in the blood.
42
Q

Vitamin K is responsible for?

A

the gamma carboxylation of Proconvertin (Factor VII), Christmas (Factor IX), Stuart-Prower (Factor X) and the Prothormbin (Factor II), as they are Ca2+ dependent factors, so Vit K deficiency causes the inability of these factors to bind calcium.

43
Q

Fibrinolytic pathway is responsible for?

A
  • keeping the clot formation within normal limits. - Clot inhibitors (antithrombin III., alpha2-macroglobin, alpha2-antitripsin, heparin
  • the latter increases the binding of antithrombin III to thrombin) are able to bind to thrombin and neutralize it.
44
Q

Major alterations of the basic test in different haemostais disorders:

A
45
Q

Thrombin time:

  • intrinsic pathway problem
  • extrinsic pathway problem
  • common patway problem
A
46
Q

Diagnosis of von Willebrand disease -> table

A
47
Q

What happens after the complete coagulation?

A

The clot os not needed anymore, and should be broken down by fibrinolytic enzymes.

  • Free collagen fibers (at the site of vessel injury), kinogen and kallikrein activates factor XII (Hageman).
  • XIIa (activated form of factor XII) further activates the extrinsic pathway, and is also able to form kallikrein from prekallikrein.
  • kallikrein activates the kinogen system, also, forming bradikinin (activated form of kininogen), which is a mediator of pain.
  • Kallikrein is the most important activator of plasminogen.
  • Plasmin (activated form of plasminogen) is an endopeptidase, which can cleave fibrin strands into small pieces.
  • Before the total degradation of polymerised fibrin strands, increased level of fibrinolysis-products, fibrin dimers and monomers (called “fibrin degradation products or proteins, FDP) can be measured in the blood.
48
Q

Difference between detection of high FDP in the blood and D-dimer level in the blood.

A

FDP (Fibrin degradation products or proteins) is not a proper name for these proteins because some of them are the breakdown products of fibrinogen, also not just fibrin. Therefore high FDP in the blood is not necessarily the sign of increased fibrinolysis following increased clot formation, because these prodcuts can also originate from fibrinogen.

More accurate way to detected increased fibrinolysis is the examination of D-dimer level in the blood. This derivate is originated only from fibrin, and not fibrinogen.

49
Q

What are the FDP and D-dimer test based on?

A

on latex agglutination method, and fresh citrated (decalcinated) plasma samples should be used.

  • The reagent containes antibodies against FDP´s or D-dimers attached to latex particles.
  • the reagent is mixed with fresh citrated plasma sample and macroscopic agglutination can be seen as dark surface if enough FDP´s or D-dimers are present in the plasma.
50
Q

What can the FDP and D-dimer test help diagnose?

A

Disseminated Intravascular Coagulopathy (DIC)

  • DIC is a common acute disorder that require quick lab. diagnosis.
  • It is a secondary problem, caused by primary diseases
    eg: septicaemia, pancreatitis, widepsread burn injuries, or necrosis of big tumors, shock, polytraumatisation etc.
51
Q

What happens in case of DIC?

A
  • microthrombus formation and fibrinolysis are present at many different places in the body simultaneously, because of severe tissue damage or necrosis (initiation of the extrinsic pathway, by the release of tissue factor), and blood vessel injury (initiation of the intrinsic pathway), so coagulation factors and platetels are consumpted very quickly during this process.
  • This is why it is also called “consumptional coagulopathy”
  • DIC can be life threatening complicatig of the aforementioned diseases.
52
Q

Laboratory diagnosis of DIC:

A
  • Coagulation time: increased
  • Bleeding time: Increased
  • Platelet count: decreased
  • PT: increased
  • APTT: increased
  • TT: increased
  • FDP and D-dimer: increased

Appearance of schysocytes an/or burr-cells i blood smear.

Lab D (11 decks)

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