Coagulation Part 1: Hemostasis and Methods of Laboratory Evaluation

Question 1

describe the main steps of hemostasis (4)

Answer 1

1. vessel injury, followed by vasoconstriction that markedly reduces blood flow to the site of injury to prevent blood loss
2. primary hemostasis: platelets adhere to the exposed subendothelial collagen via vWF and spread to patch the defect, then become activated to release their granules and attract more platelets; the platelets adhere and aggregate to form the primary hemostatic plug (also via vWF)
3. secondary hemostasis: activation of the extrinsic and intrinsic pathways leading to the activation of the common pathway and production of thrombin; thrombin converts fibrinogen to fibrin that creates crosslinks within the primary hemostatic plug to form the stable secondary hemostatic plug
4. fibrinolysis: counterregulatory mechanisms are set into motion that limit further clotting at the site of injury, eventually leading to the breakdown of fibrin strands by plasmin and the resorption of the clot

Question 2

describe sample collection technique

Answer 2

serum versus plasma:
-serum is the liquid that remains after the clotting of blood, not used for coag testing bc lacks critical factors, consumed during clot formation
-plasma: liquid that remains when blood is prevented from clotting by anticoagulants; used for coag testing; citrate is the preferred anticoagulant or testing of secondary hemostasis

material:
-glass surfaces trigger coagulation cascade, plastic prevents

technique: fresh clean stick; trauma exposes blood to transcription factor, leading to platelet activation and clumping
-do NOT collect blood from a catheter (esp if heparanized)

Question 3

describe appropriate sample collection and handling procedures for laboratory tests evaluating hemostasis (tubes)

Answer 3

1. blue top tube: 3.2% sodium citrate:
   -coagulation testing (assessing secondary hemostasis);
   -usually centrifuged to obtain citrated plasma;
   -ratio of citrate to blood MUST be 1:9 (perfectly fill the tube); any deviation from this will give artifactual results (overcitrated prolongs coagulation, undercitrated shortens coag)
2. purple top tube: EDTA; CBCs and platelet counts (assessing primary hemostasis)
3. grey top tube: diatomaceous earth; activates collagen

Question 4

describe sample processing

Answer 4

1. anticoagulants like sodium citrate and EDTA inhibit coagulation by binding calcium
2. blood collected in sodium citrate (blue top) is usually centrifuged to obtain citrated plasma
   -does not contain cells or calcium
   -only contains coagulation factors and other unrelated blood chemical
   -during testing, we add calcium or other activators to the plasma to trigger coagulation
3. analysis should occur ASAP (within 30 min of plasma removal)
   -if not possible, freeze plasma

Question 5

describe the tests of primary hemostasis and what they evaluate (platelets)

Answer 5

1. quantitative: assess platelet number/mass

-blood smear estimates: prepare smear from EDTA anticoagulated whole blood, assess morphology, look for clumps at feathered edge (that may falsely decrease platelet count), perform platelet estimates in the monolayer; ALWAYS perform in a thrombocytopenic animal

-thrombogram: use EDTA anticoagulated whole blood samples; generated by automated hematology analyzer (impedence can be unreliable but flow cytometry based are fine); ALWAYS make a blood smear to verify automated platelet count

2. qualitative: assess platelet function

-buccal mucosal bleeding time: measures the time it takes to stop bleeding from a standardized cut in the mucosa (normal time is 3-4 min); causes of prolonged BMBT include thrombocytopenia (rule out first), platelet function defect, vasculitis, severe anemia
-only perform BMBT if platelet count in normal/increased to rule out thrombocytopenia!
-coagulation factors deficiency does NOT prolong BMBT unless a larger vessel is cut

-vWF antigen assay: quantifies the amount of vWF present

Question 6

describe the 4 aspects of a thrombogram

Answer 6

1. platelet count: may be falsely decreased due to platelet clumping, esp in cats
2. MPV: mean platelet volume; average size of platelets; increased MPV can indicate accelerated thromopoiesis (bone marrow is responding!)
3. PDW: platelet distribution width: similar to RDW on the erythrogram; shows size variability of platelets
4. PCT: plateletcrit; assessment of circulating platelet mass, calculated based on platelet number and MPV

Question 7

describe the tests of secondary hemostasis and what they evaluate (coagulation factors)

Answer 7

1. P(e)T: prothrombin time; tests extrinsic and common pathways; considered prolonged if >2sec;
   -prolonged due to inhibition or deficiency of factor 7 or a common pathway factor; a good screening test for vitamin K antagonism or deficiency (factors 2, 7, 9, 10 are vitamin K dependent but factor 7 has the shortest half life of those)
2. PTT/APTT: partial thromboplastin time; tests intrinsic and common pathways; also called activated PTT; prolonged if >2sec

both PT and PTT may be artifactually prolonged by dehydration or other causes of erythrocytosis or underfilling the citrate tube! both use citrated tube

3. ACT: activated clotting time; measures the time required for fibrin clot formation in whole blood;
   -prolonged could be due to inhibition or deficiency of any intrinsic or common pathway factors or marked thrombocytopenia
4. fibrinogen concentration: measured by heat precipitation; common in large animal CBCs; measured by immunoassays
5. individual factor analysis: measurement of specific factors to detect deficiencies, reported as % activity compared to a healthy control

Question 8

what is thrombopathia/thrombocytopathy?

Answer 8

abnormal platelet function; will present similar to severe thrombocytopenia but platelet count will be normal!

Question 9

what are products of fibrinolysis and how are they measured?

Answer 9

1. FDPs: fibrinogen and fibrin degradation products
   -formed by plasmin’s degradation of fibrinogen, fibrin monomers, cross-linked (stable) fibrin
   -measured by latex agglutination in dogs, cats, and horses
2. D-dimers: formed by plasmin degradation of cross-linked fibrin from the stable secondary hemostatic plug
   -detection of D-dimer epitope with monoclonal antibodies in dogs, cats, and horses

results:
1. increase FDPs only:
-rattlesnake envenomation
-excessive release of plasmin activator (t-PA); seen with hypotensive shock, heat stroke, surgical trauma

2. increased FDPs AND D-dimers:
   -disseminated intravascular coagulation
   -internal hemorrhage
   -thromboembolic disease