Lab 4 Haemostasis Flashcards
What is hemostasis?
The group of processes initiated in the body in order to stop bleeding in case of tissue/blood vessel injuries
What are the requirements for hemostasis tests?
They must be quick so they can be performed by the side of the animal, easy to perform and give good estimation
Describe the three phases of hemostasis
Phase 1: vessel reaction, platelet aggregation
Phase 2: thrombus formation
Phase 3: fibrinolysis
Give the three major groups of hemostasis disorders
Vasculopathy
Thrombocytopathy
Coagulopathy
What is vasculopathy
Decreased ability of vasoconstriction in case of blood vessel injury
What is thrombocytopathy
Decreased ability of platelets to aggregate and adhere to the site of injury, thrombocytopenia
What is coagulopathy
Problems with the extrinsic, intrinsic or common pathway for the coagulation cascade, which ends with the formation of a polymerized fibrin network
Give 5 hemostasis tests that can be performed by the side of the animal
- Capillary resistance (mainly humans)
- BMBTT
- Clotting time (first fibrin strand)
- Appearance of clot (different surfaces)
- Clot retraction time
Explain capillary resistance test
Ligature on arm, 3-5 mins: 3 petechie should appear
If more: fragile capillaries: vasculitis
Explain BMBTT
Buccal mucosal bleeding time
Make sharp incision in buccal mucosa/inside of ext ear
Wipe blood flowing under wound every 30 secs
Measure time from appearance of blood until bleeding ceases
Normal BT: 3 ish minutes
For vasculopathy and thrombocytopathies
Give the 5 CT tests
- Appearance of first fibrin strand
- CT on watch glass
- CT in plastic syringe
- CT in glass tube
- CT in ACT
- Appearance of first fibrin strand
Drops of blood on watch slide
Move needle back and forth through blood slowly
First fibrin strand: 1-2 mins
- CT on watch glass
Fresh blood sample on watch glass treated with paraffin/wax
Time for the blood to completely clot: 7-15 min
- CT in plastic syringe
Complete coagulation
10-12 min
- CT in glass tube
Glass test tube
Complete coagulation
4-5 min
- CT in ACT
Intrinsic pathway SiO2 (neg charged) Activates factor XII - contact factor XII activates IX and kallikreinogen, kininogen Normal: 3 mins
What 3 tests can be done to evaluate platelet count?
- Bürker chamber
- Blood smear in light microscope
- Automatic cell counter
What sample is used for platelet counts?
Na/K EDTA anticoagulated blood
Platelet count Bürker chamber
1:9 blood: saline solution
Count platelets in rectangles, and divide: average
Multiply by 10^9 = one litre of blood
Platelet count blood smear in light microscope
1000x magnification
one platelet in one view = 20x10^9/l
Platelet count automatic cell counter
5-30 fl size = platelets
ACC platelet errors giving false results
Young platelets (Cats, Spaniels): large: RBCs Small RBCs: can be mistaken for platelets
General platelet count
200-800 x10^9/l
5 major causes of thrombocytopenia
- Decreased production in BM: parvovirus, mycotoxins
- Increased utilization: DIC
- Increased destruction: autoimmune thrombocytopenia
- Increased sequestration: splenomegaly
- Increased loss: subacute bleeding
What tests are used to check thrombocytic function?
BMBT
Platelet count
Clot retraction test
Platelet aggregation test
Clot retraction test
Blood clot left in tube for hours: clot retracts, serum appears
Platelets contain a contractile protein thrombostenin
Within one hour: serum 25% of whole volume of initial clut
Platelet aggregation test
CITRATED blood sample, upper layer (platelet rich plasma)
Put sample in cuvette, add aggregating causing drugs (ADP, epinephrine)
Aggregated platelets: clear sample
Spectrophotometry
Thrombocytic morphology
1-2 um diameter, granulomer center and hyalomer edge
Cats have the biggest platelets
3 major causes of thrombocytopathy
- Improper development
- von Willebrand diesease
- Uraemia, liver failure, NSAIDS etc
If coagulation is normal, what can we expect in case of thrombocytopenia, thrombocytopathy and vasopathies?
No signs of severe bleeding disorder
Prevented by formation of fibrin thrombus
If platelet function and count is normal, and no vasopathies are present, what can we expect in case of coagulopathies?
Really severe bleeding disorders (suffusion, hematoma, hemothoax, hemoperitoneum
Thrombocytic thrombi are not stable without fibrin network
What test can be done to test the extrinsic+common coagulation cascade?
Prothrombin time
Explain PT, what sample is used
Na-Citrate 1:9
Decalcinated plasma + TF3 + CaCl2
Normal PT: 10-15 sec
Extrinsic coagulation cascade is triggered by tissue factor and Ca2+
What test can be done to test the intrinsic+common coagulation cascade?
Activated Partial Thromboplastin Time
Decalcinated plasma + PF3 + Micronised silica
Normal APTT: 20-30 sec
Intrinsic coagulation cascade is triggered by surface activation (which appears on inner surface of blood vessels), PF3 + Ca2+ activates factor X (common)
Explain thrombin time
Decalcinated plasma mixed with thrombin only
Coagulation time depends on conc of fibrinogen and factor XIII in plasma (fibrin stabilizing)
Intrinsic pathway problem
APTT, PT
What can cause it?
APTT increased
PT normal
Hemophilia A (Factor VIII deficiency) Hemophilia B (Factor IX deficiency) von Willebrand disease
Extrinsic pathway problem
APTT, PT
What can cause it?
APTT normal
PT increased
Factor VII deficiency
First stage of dicumarol/warfarin toxicosis
Common pathway problem
APTT, PT
What can cause it?
Both increased
Liver disease DIC Second stage of dicumarol toxicosis (vit K def) 1, 2, 5, 10, 13 factor deficiency Protein deficiency (cachexia)
Dicumarol toxicosis
Competitive vit K antagonist
Vit K: gamma carboxylation of factor 7, 9, 10, 2 (Ca2+ dependent factors)
Deficiency: factors cannot bind to Ca2+
Factor 7 has shortest half life: deficient first
= increased PT
then after a while = increased APTT too
FDP
D-dimer
Fibrin degradation products
Both from fibrin and fibrinogen
D-dimer: from fibrin only!
Clot inhibitors
Function
Antithrombin III
Alpha-2 macroglobulin
Alpha-1 antitrypsin
Heparin
Responsible for keeping the clot formation within normal limits
Bind to thrombin and neutralizes it
How are fibrinolytic enzymes activated?
Free collagen fibers, kallikrein and kininogen activates factor XII
Factor XII activates kallikrein which forms bradikinin (activated form of kininogen)
Bradikinin: potent mediator of pain
Kallikrein also activates plasminogen
Plasmin: endopeptidase: cleaves fibrin
What principles are FDP and D-dimer tests based on?
What sample?
Latex agglutination
Fresh citrated plasma
Contains antibodies against D-dimers/FDP = bound
Macroscopic agglutination can be seen if enough D-dimers/FDP are in the plasma
DIC
Disseminated intravascular coagulopathy
FDP/D-dimer test= early diagnosis
Usually secondary to diseases:
Septicaemia, pancreatitis, burn injuries, necrosis, shock
Microthrombus and fibrinolysis is present many places in the body simultaneously
Quick consumption of platelets and coag factors
Lab parameters of DIC
CT inc BT inc Platelet count dec PT, APTT, TT inc FDP/D-dimer inc Damaged RBCs
Diagnosis of von Willebrand
Deficiency of factor VIII
BT inc
Clot retraction dec
3 parts of factor VIII
von Willebrand: platelet adhesion and aggregation
VIIIc: antihemophilic factor
VIII related antigen: hapten bound to vW
