clinical test 2 final draft

Coagulation

The formation of a clot

Platelets

• aka thrombocytes

• Small, anucleate, discoid-shaped. Light blue-staining cells that may have multiple, fine, pink-to-purple granules in the cytoplasm. They play a crucial role in blood clotting and wound healing.

1/3 of the total blood platelets are located where

in the spleen

2/3 of the total blood platelets are where

circulating in the bloodstream

The production of platelets is called

thrombopoiesis

Produced in the bone marrow by

Megakaryocytes.

Thrombopoietin

stimuli for megakaryocyte to differentiate into thrombocyte precursor

platelet parent cell

is the megakaryocyte, which undergoes fragmentation to produce platelets.

Megakaryocyte

• A large bone marrow cell responsible for the production of platelets through a process of fragmentation.

• Has multiple copies of DNA in the nucleus

What breaks off into small chunks while still in bone marrow and is sent into circulation as platelets

is the megakaryocyte, which fragments to produce platelets.

Some platelets are stored in BLANK for release as needed

the spleen.

What type of cell is this?

Megakaryocytes

Thrombocytes are commonly referred to as

platelets

Thrombocytes are not complete cell; they lack a BLANK, but are frequently listed as one of the cell types in peripheral blood

nucleus

Most thrombocytes are BLANK than RBCs

smaller

PLTS have numerous, small, purple/pink BLANK scattered throughout the cytoplasm

granules

Occasionally, BLANK platelets are seen in blood smears

Megakaryocytes

How many Thrombocytes are seen per high-power field

7-10

How are actual counts for thrombocytes performed

by using a hemocytometer or automated analyzers that evaluate blood samples.

Thrombocytopenia can be caused by

Trauma, Hemorrhage, rodenticide poisoning, immune-mediated, medications, neoplasia, and Infectious causes like Babesia, Ehrlichia, rickettsia, Toxoplasma, FELV, FIV

When should you be concerned about thrombocytopenia

• Normal for dogs and cats should be > 200,000/microliter

• when platelets numbers <100,000/microliter is a red flag and may indicate serious underlying conditions such as severe disease, bone marrow disorders, or significant blood loss.

• < 20,000-50,000/microliter patient is at risk for spontaneous bleeding and requires immediate veterinary attention.

Thrombocytosis can have other causes like

Physiologic causes like stress, exercise, and pathologic causes like neoplasia, splenectomy, trauma, etc.

Hemostasis

The ability of the body’s systems to maintain the integrity of the blood and blood vessels by preventing excessive bleeding or clotting involves a complex interplay of vascular, platelet, and coagulation factors.

Primary hemostasis is

The formation of a platelet plug

Mechanical hemostats of platelets is

aggregation and adhesion

Secondary hemostasis is

the cascade of coagulation factors that leads to the stabilization of the platelet plug by forming fibrin strands.

chemical hemostasis

involves the biochemical processes that regulate blood coagulation and the formation of a stable clot.

Tertiary hemostasis is

clot degradation or clot breakdown

Hemostasis involves

• A number of complex pathways that are interrelated

• platelets

• coagulation factors

The mechanical phase of hemostasis

• The mechanical phase is initiated when the blood vessel is ruptured or torn (collagen is exposed)

  • The exposed subendothelium ( collagen) of the vessel is charged

• Platelets are attracted to this charged surface, which undergoes morphologic and physiologic changes. This charge causes platelets to adhere to each other and the endothelium to form a temporary plug, leading to the initiation of the coagulation cascade.

• This plug formation requires the von Willebrand factor, which will stabilize the plug

• The adhesion and aggregation of the platelets trigger the chemical phase

The steps of the mechanical stage of hemostasis

1. A blood vessel is ruptured or torn

2. The exposed vessel subendothelium is a charged surface composed of collagen fibers, and platelets are attracted to it.

3. Platelets congregate at the site of injury

4. Platelets undergo morphologic and physiologic changes, causing the platelets to adhere to each other as well as the blood vessel endothelium. This forms an initial platelet plug.

5. Platelets release the initiating factor for the chemical phase of homeostasis

The chemical phase ( secondary phase)

• The coagulation cascade involves several factors

  • a cascade of reactions occurs to activate clotting factors

• Clotting factors are mostly plasma proteins synthesized by the liver ( vitamin K is produced by the liver)

  • Vitamin K is needed to synthesize factors II, VII, IX, X

• Intrinsic and extrinsic pathways will result in a fibrin mesh or clot

The final phase of Hemostasis

the degradation of the fibrin clot ( fibrinolysis), which restores normal blood flow and tissue integrity.

What factors does vitamin K synthesize?

Factors II, VII, IX, and X

Plug formation

• A damaged blood vessel initiates the primary process of clot formation

• The platelets adhere to the damaged vessels and to each other

• Platelets change shape, and this allows platelets to intertwine with each other to create a platelet plug.

• The intertwining of platelets helps stop bleeding and causes the release of hormones to help attract more platelets and cause vasoconstriction of the blood vessel

steps for the formation of a clot

1. Platelets

2. The von Willebrand factor

3. clotting factors

4. Fibrinogen

What is the end point of the secondary hemostasis?

The formation of a stable fibrin clot.

What are the defects in a coagulation factor characterized by

• A large hemorrhage

• With a large hemorrhage, the platelet plug can form but is not stabilized with fibrin

The clot breakdown tertiary hemostasis

• The process by which unwanted clots are removed is fibrinolysis, which involves the degradation of fibrin by plasmin.

• Once the hemorrhage has been controlled and vascular repair has begun, the clot degrades

• Fibrin is broken down by fibrin degradation products ( FDPS)

• Antithrombin and trans plasminogen activator ( a drug)

What is Antithrombin?

• Role: It is a natural protein made by the liver that acts as an anticoagulant (blood thinner).

• Function: It turns off specific proteins that cause blood to clot, mainly thrombin and factor xa

what is Tissue Plasminogen Activator (tPA)?

• Role: It is an enzyme involved in fibrinolysis, which is the body's process of dissolving unwanted blood clots.

• Function: It changes an inactive protein called plasminogen into plasmin. Plasmin then chews up the mesh (fibrin) that holds a blood clot together

• a drug

The ability for blood to clot depends on what?

• Vascular integrity

• Adequate numbers and normally functioning platelets

• Coagulation factors

• liver issues

  • Any alteration in these parameters can result in a bleeding disorder

What happens if there is an interruption with the hemostatic process?

• It can lead to excessive bleeding or thrombosis, as the balance between clot formation and breakdown is disrupted. This disruption can result in conditions such as hemophilia or thrombophilia.

Coagulopathy

Any disorder of blood coagulation

Coagulation testing

• Blood samples should be collected carefully, with minimal tissue damage and minimal venous stasis

• Samples should never be collected through indwelling catheters

What is the blood tube preferred for coagulation testing

• Sodium citrate

• Works by chelating calcium ions to prevent clotting ( same as EDTA)

What should you do if a clotting disorder is suspected for a platelet count?

A manual count should be performed because the automated reader will misread clumped platelets

Hemostatic Defects

• Bleeding disorders may be caused by congenital or acquired defects in coagulation proteins, platelets, or the vasculature.

• Most bleeding disorders found in veterinary species are secondary to another disease process

What are signs of hemostatic deficiencies in coagulation proteins

• They usually involve delayed deep tissue hemorrhage and hematoma formation

  • A general oozing of blood that does not stop

• Some signs of this can be petechiae and ecchymotic hemorrhage

what is this?

• Petechia

• A sign that there is a problem with primary hemostasis

what is this?

• Ecchymotic hemorrhage

• A sign that there is a problem with primary hemostasis

What are these pictures a sign of?

Hemostatic disorders

what does the majority of congenital coagulation factor disorders involve

They involve a deficiency or abnormality of a single factor

When are the signs of a congenital coagulopathy noticed?

will be noticed before 6 months of age

von Willebrand factor

is a blood glycoprotein involved in hemostasis, crucial for platelet adhesion and aggregation. A deficiency or dysfunction can lead to von Willebrand disease, a common bleeding disorder.

Von willebrand’s Disease

• The most common inherited disorder of hemostasis caused by a deficiency or dysfunction of von Willebrand factor, leading to bleeding symptoms.

• When an animal has a decreased amount or lack of this factor, a bleeding disorder will result

What breeds are affected by Von willebrand’s Disease

• Doberman, GSD, and labs

• There are 54 breeds affected in total in the U.S.

What will you see with Von Willebrand’s disease

• You will see prolonged bleeding after procedures, gingival bleeding, epistaxis, and hematuria.

• They will have a normal number of platelets, but reduced platelet function. (The platelets do not stick together)

• Other signs may include easy bruising, excessive bleeding during heat cycles, and spontaneous bleeding in severe cases.

What is the test used for Von Willebrand’s disease?

The test that is used is the BMBT ( buccal mucosal bleed time)

Is there a cure for Von Willebrand’s Disease

• There is no cure

• It’s best to avoid bleeding events

Should dogs with Von Williebrand’s disease be breed?

No, affected dogs should not breed as it may pass the condition to offspring, increasing the risk of bleeding disorders.

Predisposed breeds should be tested before breeding to ensure offspring will be clear.

What can be done for patients who have Von Willebrand’s disease before surgery/

They can be given blood products before the surgery to help them manage their clotting deficiencies and minimize bleeding risks.

What is an alternative test for the BMBT test

• toenail bleeding time

• The toenail is clipped just past the quick causing bleeding

• Keeping the animal undisturbed, monitor for bleeding to cease

• The normal time should be < 5 minutes

How is the BMBT test performed

• The BMBT test is performed under sedation. An incision is made in the mucosal gums using a standardized lancet

• The patient is kept in lateral recumbency, and a stopwatch and non-blotting paper are used

• The stopwatch is stopped when the patient stops bleeding.

  • This is considered to be the clotting time

secondary coagulation disorders

• are a result of decreased production and consumption, and increased destruction of platelets

• This can be viral, bacterial, parasitic, ingestion of certain medications, or toxic substances

Thrombocytopenia

• refers to a decreased number of platelets and is the most common coagulation disorder seen in small animal veterinary practices

• The causes are often unknown

• Infections with certain bacterial, viral, and parasitic agents can result in thrombocytopenia.

What are the other causes thrombocytopenia?

It can also occur from a bone marrow depression, which reduces the production of platelets due to autoimmune disease that increases the rate of platelet destruction

What happens when a condition affects liver function?

It can lead to coagulopathy due to impaired synthesis of clotting factors and thrombocytopenia.

Immune mediated thrombocytopenia

• The immune system is destroying circulating platelets

• It is life-threatening

• Causes bleeding that leads to anemia

  • Signs: petechiae, ecchymosis, epistaxis, melena, hematuria, dyspnea

    • platelet count is usually less than 10,0000-50,000 platelets/microliter

  • The treatment: corticosteroids, immunosuppressants

• Relapses are common.

Thrombocytosis

• An increased number of platelets

• either due to increased production in the bone marrow or decreased removal in the spleen

Thrombocytosis can be caused by what?

• stress leading to the release of epinephrine

• disease in the bone marrow

• signs of the disease in the body that cause an increase in demand

• following surgery

• acute/chronic blood loss

• infections

Hemophillia

• a clotting factor deficiency ( can be caused by different factors that are deficient)

• Hemophilia A ( factor VIII deficient)

• Hemophilia B ( factor IX)

• Bleeding times are prolonged- in pups, prolonged bleeding after tooth eruption

• Cannot be cured

• Plasma transfusion used during bleeding episodes

• These animals cannot be bred

What is hemophilia A associated with?

• It is associated with a deficiency of clotting factor VIII, which leads to prolonged bleeding times. This condition primarily affects males and can be inherited in an X-linked recessive pattern.

What is hemophilia B associated with?

• It is associated with a deficiency of clotting factor IX, resulting in similar symptoms as hemophilia A, including prolonged bleeding times. This condition also primarily affects males and is inherited in an X-linked recessive pattern.

Feline aortic thromboembolism or feline saddle thrombus

• A feline-only condition

• Too much hemostasis; anticoagulants missing

• Thromboembolic lodging where the aorta branches at the pelvis into the R?L iliac arteries

• Obstructs blood flow to the rear legs

• Causes tissue damage

• most occur due to underlying heart disease

• usually both rear legs are hard, cold, paralyzed, painful, and have cold paw pads

• the cats will be vocalizing, panting, tachypneic, and have no pulse in the rear legs

• Temperature can be an indicator of prognosis: 98.9

what is the treatment for Feline aortic thromboembolism

blood thinners, control the heart disease, and surgery

Rodenticide ingestion

• Warfarin, Pindone, and/or brodifacoum are types of rodenticides

• These will inhibit the function of vitamin K.

• Vitamin K is required for synthesis and activation of some coagulation factors.

  • This creates a deficiency in several necessary components of the coagulation cascade.

What is the most common type of toxicity in dogs and cats?

Rodenticide toxicity

What are the clinical signs of rodenticide toxicity?

They will appear 2-5 days after ingestion, hematomas, petechial, ecchymosis, hemorrhage, anemia, hematuria, and hemoptysis

What is the treatment for rodenticide toxicity?

emesis if recent ingestion ( within 1-2 hours), charcoal, blood transfusion, and vitamin K

What helps with the absorption of Vitamin K?

• Is the presence of dietary fat. It enhances the bioavailability of vitamin K.

• Feed the patient a fatty meal when giving vitamin K

Disseminated Intravascular Coagulation ( DIC Death is coming)

• An uncontrolled coagulation and fibrinolysis ( clot breakdown)

• A hypercoagulable state at first

• Massive consumption of clotting factors and platelets; inability to clot

• Always due to secondary causes

• Needs multiple clotting tests to diagnose

• Has a very grave prognosis; very few survive.

What can cause Disseminated Intravascular Coagulation?

electric shock, heat stroke, sepsis, burns, neoplasia, pancreatitis, and sometimes rodenticide in advanced stages

Things to keep in mind for collecting samples for coagulation testing

• careful technique ( clean sticks) to minimize tissue damage

• Patient excitement can affect results

• With stress, it can cause splenic contraction to falsely elevate platelets

• Must be collected in proper order if multiple samples are needed

• Fill the coagulation tube first and fill it with the proper ratio

What must be run along with the coagulation test?

A CBC must be run since thrombocytopenia’s can affect the coagulation testing results

What should be done if the coagulation test cannot be performed right away

The test should be run as soon as possible. If it can’t be done within 2 hours of collection, the plasma needs to be separated and frozen in plastic tubes or shipped on dry ice

The BMBT test

• Patients may tolerate this test without sedation; however, we can’t have the animal pawing at the lancet site, while others will need to be sedated

• The upper lip is tied back, then a spring-loaded lancet (1 mm incision) is used on the buccal mucosa at the level of the premolars

• Blot the incision to catch the drips until a clot forms (1- 5 minutes) with standard blotting/filter paper

• Do not TOUCH the incision made with the blotting paper; it is supposed to be used to wick the blood away

• The time bleeding stops is the endpoint

• This test is performed on patients who have normal platelet counts

What are the supplies needed for a BMBT

Stop watch, gauze ( to tie lip back), special spring loaded lancet ( has a measurement on it), filter paper used to blot the blood.

Where must the Von Willebrand factor tests be sent

must be sent out to the reference laboratory

The Von Willebrand antigen Factor

• Performed at Cornell University Laboratory

• This test is often based on the results of the von Willebrand factor antigen assay

• The test measures the amount of concentration of vWF in a blood sample

• Each dog’s results are expressed as a percentage vWF:Ag compared to a 100% standard

  • Dogs having a low plasma percentage vWF:Ag ( below 50%) are at risk for transmitting or expressing the vWD trait

• The most severely affected dogs have a marked reduction in plasma vWF:Ag with values of less than 15%

What tube is used for the Von Willebrand Antigen Factor

• The sodium citrate tube

• The plasma is spun down and submitted

Activated Clotting time

• is a test that can be performed in the vet clinic

• The blood sample is added to a special ACT tube containing a substance ( diatomaceous earth and kaolin) that activates the coagulation factors

  • The tubes need to be prewarmed by placing them in a water bath at 37 degrees and then checked at 60 seconds

• Both the intrinsic and common pathways of the clotting cascade can be tested this way

• A severe decrease in these clotting factors or a severe decrease in platelet numbers will lead to an increase in the length of time it takes the blood to clot

• The ACT is not very sensitive to a milder decrease in clotting factors

Not used very often anymore

The Activated clotting time Evalutes what

• Elevates the secondary hemostasis ( all factors except factor VII)

• The test can be affected significantly by platelet numbers (<10,000)

• A CBC should be performed before this test

How to perform the ACT test

• The blood sample is placed in the tube and is immediately put in the water bath

• A normal dog should form a clot in 60-90 seconds

• Not used too much anymore due to sensitivity

What do the tubes used to perform ACT contain

contains diatomaceous earth, can be gray or white

Whole blood clotting time

• This test is less accurate than activated clotting time

• 3cc of whole blood are collected in a plastic syringe

• 1 mL is placed into 3 tubes that have been rinsed with saline and placed in a water bath at 37 degrees

• The tubes are checked by tilting at 30-second intervals

• time that the clot forms in the third tube is the crude clotting time ( Usually 2-10 minutes for dogs)

PT and PTT coagulation tests

• Performed on automated analyzers

• Most samples are sent out to reference labs or an in-house lab in large hospitals

• PT stands for Prothrombin Time

• aPTT stands for activated partial thromboplastin time

Prothrombin time ( PT)

• Usually performed by an automated analyzer

• Has the shortest half-life, so abnormalities will be seen first in PT

• Evaluates the clotting factors for the extrinsic and common pathways

• Reagent added to blood sample in the blue-top tube that causes recalcification

• Normal time is 7-10 seconds for a clot to occur

• See prolongation of PT in severe liver disease, DIC, rat poison toxicity, or clotting factor deficiency

• Elevations 24 hours with rat poison

What is the Prothrombin time (PT) testing for

evaluates the clotting factors for the extrinsic and common pathways

what is the normal value for Prothrombin time

• Canine is 6.4-7.4 seconds

• Feline is 7-11.5 seconds