lab pro - coagulation and PLTs

hemostasis

ability of the bodys systems to maintain the integrity of blood and blood vessels

• endothelial cells, platelets, coagulation factors, complex pathways

endothelial cells

• line all blood vessels

• cells retract when injured; expose proteins from tissues that interact with platelets to promote adhesion

• release molecules that promote activation of clotting factors

• healthy tissues have anticoagulant activity (thrombi)

platelets

• essential for hemostasis, form a primary platelet plug (intial clot)

• injured endothelium promote adhesion, aggregation, swelling, and secretion by PLT’s

• activated PLTs promote activation of other platelets

how many platelets need to be present for an animal to bleed

<40,000 /uL

thrombocytopenia

most common defect in primary hemostasis

what venipuncture tool can affect platelet counts

too large a needle gauge

what happens in the mechanical phase (primary phase)

• initiated when. blood vessel is injured

• exposed endothelium is charged and secretes proteins to promote PLT adhesion

• PLTs attracted to charged surface undergo morphologic and physiologic changes

• activated PLTs secrete factors to begin clotting cascade

• required the von Willebrand factor → stabilizes the plug

• adhesion and aggregation of PLTs trigger the chemical phase

what are coagulation factors

they are part of the chemical phase, and stabilize the platelet plug by formation of fibrin, which begins the coagulation cascade

what do deficiencies in clotting factors lead to

the primary clot degrades and bleeding resumes

purpose of the coagulation cascade

stopd bleeding in more severe hemorrhage

extrinsic pathway

• uses tissue clotting factors

• create fibrin clot outside blood vessel

intrinsic pathway

• uses plasma clotting factors

• react to form fibrin clot inside the vessel

chemical phase (secondary phase)

includes fibrinogen, thrombin, and prothrombin in the formation of fibrin

fibrinogen

protein precursor to fibrin that circulates in the plasma

thrombin

major enzyme involved in formation of fibrin

• converts fibrinogen to fibrin

• promotes PLT aggregation

• activates several clotting factors

• inhibits breakdown of fibrin 

prothrombin

innactive form of thrombin that circulates in the blood

fibrinolysis

process of breaking down a clot

• occurs simultaneously with coagulation; keeps clot at site of injury

• once vessel is repaired, removes the clot and promotes blood flow through healed vessel

rare diseases (sepsis) can cause increased fibrinolysis

which pathway requires more factors before the formation of fibrin

intrinsic pathway

how can excited patients affect sample collection

• more tissue damage

• increase PLT count and activation

• increased levels of VWF and factors 1,5, and 8

how to collect a sample for a coagulation test

• have to be done with one poke to reduce trauma to vessels

• never use an indwelling catheter, may have fibrinogen, fibrin and PLTs around catheter

• Vacutainers or monovette’s are preferred

why are Vacutainers or monovette’s preferred collection method

have the proper ratio of anticoagulent:blood → 1 part citrate : 9 parts blood

how to transfer a sample

• label and transfer ASAP

• hold at room temperature, tightly capped, kept upright, avoid vibrational trauma

if unable to test within 2 hours,

• must be centrifuged for 15 min, 2500 rpm

• separate plasma, freeze plasma or ship on dry ice

what do coagulation tests detect

• specific phases of the pathways

• can use fresh, or citrate anticoagulated blood

what is the goal of the mechanical phase

to reduce size of the hole, and ceate turbulence of blood flow

Buccal Mucosal Bleeding time (BMBT)

• assess PRIMARY hemostasis

  • von Willebrands disease

  • thrombocytopenia

• normal limits = 1-5 min of bleeding time

• patient not required to be sedated

von Willebrand factor

• assesses PRIMARY hemostasis

• vWF required for PLT adhesion

• deficiency means PLT will not adhere to the subendothelium

• performed if PLT function defects are evident

activated clotting time (ACT)

• assesses SECODNARY/INTRINSIC hemostasis

• can evaluate every clinically significant clotting factor except Factor 7

dogs = 71-102 seconds

cats = 70-120 seconds

manual method for ACT

• pre-warm tube to 37 degrees celcius (stimulates body temp)

• collect 2mL via venipuncture (use gray top tube)

• timed from collection to presence of clot 

  • observed at 60 sec, then every 5 sec until clot formed

• normal = 60-90 seconds

which are the most common tests done in clinic

the aPTT and PT

when do aPTT and PT coagulaton tests become prolonged

when 75% of factors are missing

• if patient has lost 75% of liver function, it decreases clotting factor production

where are clotting factors produced

the liver

activated partial thromboplastin time (aPTT)

• evaluates INTRINSIC and common clotting factors

• uses the CoagDx analyzer

• collection in vacutainer is critial

• requires excellent, atraumatic venipuncture

• perform test within 4 hours

• prolonged time can indicate factor 8 or 9 deficiency

Dogs: 71-102 seconds

Cats: 70-120 seconds

Prothrombin time (PT)

• evaluates EXTRINSIC and common clotting factors

• uses the CoagDx analyzer

• collection in vacutainer is critial

• requires excellent, atraumatic venipuncture

• perform test within 4 hours

• prolonged time can indicate Factor 7 deficiency

Dog: 12-17 seconds

Cats: 15-23 seconds

what does a prolonged time in both the aPTT and PT suggest

liver disease, DIC, vitamin K antagonism (rat poisoning), inherited coagualation diseases

disseminated intravascular coagulation (DIC)

results in procoagulation (PLT gone wild)

• thrombic and fibrinolytic phases

• begins with uncontrolled clotting, ends with uncontrolled bleeding

• is always secondary to underlying disorder

what happens in the thrombic phase of DIC

• cause systemic activation of PLTs and coagulation casacade throughout capillaries

• causes microthrombi → very small blood clots

• consumption of PLTs and clotting factors

what is microthrombi

 very small blood clots form in capillaries, arterioles, venules

• results in multiple organ failure, prevents blood flow to all organs

• schistocytes seen on blood film and thrombocytopenia (< PLT)

what happens in the fibrinolytic phase of DIC

• fibrin degradation (suppress thrombin)

• PLT and clotting factors become depleted → uncontrolled bleeding begins

PIVKA

Proteins Induced by Vitamin K Absence

• vitamin K required to activate coagulation factors 2,7,9,10

• more sensitive than prothrombin

• results may be prolonged within 6 hours of ingestion of rodenticides

what are platelets

small cytoplasmic fragments shed from megakaryocytes in bone marrow

thrombocytopenia

decreased platelets

thrombocytosis

increased platelets

how can platelet counts be inaccurate

can be inaccurate because of clumping and overlaps

• done manually by observing morphologic changes on blood smear

  • aggregation

  • giant PLTs

• reticulated PLT = newly released PLT with high levels of RNA

platelet estimates procedure

indirect measurements of PLT numbers using blood smears

• evaluated in the monolayer

  1. start on 40x to observe clumps

  2. change to 100x (oil immersion)

  3. count PLTs seen in 10 FOV

  4. multiply the avg. in 10 fields by 15,000 to get plt/uL

• each PLT seen = 15000 plt

• normal = 10-15 plt per view

reticulocyte count procedure

• mix EDTA blood with equal amount of drops of NMB stain in small tube

• let mixture stand for 15 min

• prepare 2 blood films

• count 500 RBC on each slide (1000 total)

  • note number of reticulocytes

• calculate % of reticulocytes

corrected WBC count

done if any nucleated RBCs are present in a whole sample, will not be lysed

• if 5 or more nRBC are seen per 100 WBC differential, total WBC count must be corrected to account for nRBC