VET 131

Hemostasis


Required reading
: 

 Hendrix, “Laboratory Procedures for Veterinary Technicians”, pages. 27-73

Voigt. “Hematology Techniques and Concepts for Veterinary Technicians”, Chapter 12, pages 103 - 110


Objectives

After completing this unit, you should be able to:

1.                   Define hemostasis
2.                   Describe the vascular response to an injury
3.                   Describe the platelet response to an injury
4.                   Describe the mechanisms involved in blood coagulation
5.                   Identify three major imbalances affecting hemostatis
6.                   Describe the production and morphology of platelets
7.                   Describe procedures involved in doing a platelet estimate and platelet count
8.                   Describe five tests used to screen for coagulation defects


Sites on the Web

 http://labtestsonline.org/images/coag_cascade.pdf

Identifies all of the steps in blood coagulation (coagulation cascade) - American Association for Clinical Chemistry

  

Introduction

When blood vessels are damaged a number of processes occur that are designed to stop the loss of blood.  The process of stopping blood flow is called hemostasis and involves a number of mechanisms.  These mechanisms include a vascular response, a platelet response and blood coagulation.

 

Vascular response 

When a small artery or arteriole is damaged, the smooth muscle cells in the wall of the vessel contract.  The contraction decreases the diameter of the blood vessel and limits the flow of blood.  The contraction can last for several minutes to several hours.   

Capillaries and most veins lack smooth muscle and, therefore, do not contract when damaged. When these vessels are injured, blood loss is controlled by contraction of surrounding tissues.

 

Platelet response
(platelet plug formation)
 

Blood platelets, also called thromobocytes, are small irregular cytoplasmic fragments.   They are produced in the bone marrow by the fragmentation of large multinucleated cells called megakaryocytes.   Once released into circulation, platelets stay in the vessels and live for 5 to 10 days.

When a blood vessel is damaged, platelets become “sticky” and adhere to the vessel wall and to each other.  The “sticky” platelets release the following chemicals:
    1.      ADP - causes more platelets to clump together forming a platelet plug 
    2.      Serotonin and histamine - maintains contraction of the blood vessels
    3.      PF3 (platelet factor 3 ) – a phospholipid which is important in starting blood coagulation

 The platelet plug may be enough to stop the loss of blood in small blood vessels. 

 

Blood coagulation

 There are many steps involved in blood coagulation.  The coagulation mechanism is a series of reactions which ultimately results in fibrinogen (a blood protein) being converted to fibrin (an insoluble fiber.)  Twelve blood clotting factors have been identified (eleven blood proteins and one element, calcium.)  The steps in the “Coagulation Cascade” are illustrated on page 106 of Voigt and at http://labtestsonline.org/images/coag_cascade.pdf

 

Summary of Coagulation Cascade

 cascade.JPG (16917 bytes)

 There are three pathways involved in the coagulation cascade

1.       Extrinsic pathway contains a few steps and occurs rapidly.  It is activated by injured tissue which releases a factor that activates tissue thromboplastin which initiates formation of prothrombinase.

2.      
Intrinsic pathway contains many steps and takes several minutes.   It is activated by a phospholipid (PF3) released by “sticky” platelets when a blood vessel is injured.  The pathway eventually ends up with an activation of thromboplastin which initiates formation of prothrombinase. 

3.      
Common pathway contains the final steps ending with a blood clot.  Prothrombin and fibrinogen are blood proteins.  Prothombinase converts prothrombin to thrombin and thrombin converts fibrinogen to fibrin.  The fibrin forms a network that traps cells forming a blood clot.  The clot functions as a barrier to further blood loss and stays in place until the injury has healed.

Anticoagulants are chemicals that prevent blood from clotting.  Heparin is an anticoagulant that inhibits the formation of thrombin. All other anticoagulants, for example EDTA, work by binding calcium ions.  Calcium is required in many of the steps in the “coagulation cascade.”  Refer to Table 4.2 (page18) in Voigt for a listing of anticoagulants and their modes of action.

A blood clot that forms in a blood vessel is called a thrombus.  If it detaches and circulates in the blood stream it is called an embolus.  A scab is a contracted and dried up blood clot that forms on the skin.

  

Clot retraction, repair and fibrinolysis

Once a blood clot is formed it is stabilized and made tighter.  Blood platelets release a factor (F13) which stabilizes the fibrin and a chemical (thrombosthenin) which is responsible for shrinking the blood clot (retraction).   If the clot region becomes damaged, tissue fibroblasts and endothelial cells repair the damage

The retraction of a blood clot can be seen by looking at a red tube of blood (lacks anticoagulants).  Within 2 to 5 minutes of blood collection, the blood will coagulate and a clot will form.  After a period of time the clot will shrink to half of its original size.  As the clot contracts, serum is squeezed out.

Fibrinolysis is the breakdown of the fibrin in a blood clot.  When an injured vessel is being repaired, there is a delicate balance between fibrin deposition and lysis.  Blood flow through the vessel needs to be maintained while repair is taking place.  Once the vessel is repaired, the clot can be totally dissolved.   Fibrinolysis is catalyzed by plasmin, a protein digesting enzyme whose activation is controlled by tissue cells and endothelium.

  

Hemostatic Defects

Defects in the hemostatic mechanisms result in problems with blood clotting and subsequent blood loss.  Van Willebrand’s Disease is a hereditary disease where there is the lack of the protein necessary for adherence of platelets to damaged cells.  This slows down the clotting process.  Van Willebrand’s disease is most common canine hereditary bleeding disorder.

A decrease in the number of circulating platelets (thrombocytes) is called thrombocytopenia.  Thrombocytopenia can be caused by abnormal platelet production, accelerated platelet removal, infectious agents (e.g. FIV in cats) or toxicity to drugs (e.g. antibiotics, chemotherapeutic agents.)  

An increase in the number of circulating platelets is called thrombocytosis.   Although thrombocytosis rarely occurs because of an over proliferation of platelets, it may be found as a response to blood loss, drug therapy, neoplasia or GI disorders.   Exercise or epinephrine injection can cause a transient increase in the number of circulating platelets.

Vitamin K is necessary for the formation of several clotting factors.  Vitamin K deficiency or antagonism causes hemostatic defects.  Coumarin, also called warfarin, is a Vitamin K antagonist which is a component of some rat poisons.   The effect of ingestion of these poisons by pets is occasionally seen in veterinary clinics.

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Platelet/Coagulation Tests

A platelet estimate should be routinely made on all blood film examinations.  Five to ten platelets per oil immersion field is considered adequate.  If the number of platelets appears to be low, a more detailed examination of the blood smear is recommended.   Look for clumped platelets at the feathered edge of the slide. 

platelets.JPG (65597 bytes)       Platelets

A closer estimate of platelet count can be made by counting the number of platelets in 10 – 20 oil immersion fields.  If the average of these field counts is between 5 and 25, the number of platelets is recorded as “adequate.”  Platelet counts in normal animals range from 200,000 to 500,000/cu mm.  A Unopette system is available for the dilution and counting of platelets.

Most veterinary clinics send out their samples for coagulation tests.  Blood collected for these tests should be collected carefully, placed in an ice bath and immediately sent out to a local commercial lab for testing.  If plasma is to be mailed to a commercial lab, it should be rapidly frozen in dry ice.   Some of the following tests can be performed in a veterinary hospital.

An examination of the Clot Retraction in a red tube of blood can be a screening test for platelets and blood clotting factors.  Within two to four hours after collection, a blood clot should be tight with a quantity of serum expressed.  If the clot is not tight, further testing should be considered

A Bleeding Time Test is a simple test to determine how long it takes for bleeding to cease.  A puncture wound is made on a clean site (e.g. mucous membrane of upper lip) and the time recorded when blood first appears.  The blood is carefully blotted every 30 seconds, with care begin taken that the wound site is not touched.   The time is recorded when bleeding stops.   Bleeding time for most animals is 3 minutes (3 – 5 minute normal range.)   Animals with von Villebrand’s disease can have a bleeding time of over 10 minutes.

The Capillary Tube Method involves collecting blood in a capillary tube that does not contain anticoagulant.  The timer is started when the blood first enters the tube.  The outside of the tube is carefully wiped and every 30 seconds a piece of the tube is broken.   The time is recorded when a strand of fibrin appears between the two pieces of capillary tube.  Clotting time vary with species with the dog having a time between 2 – 4 minutes, the cat between 2 – 5 minutes and the horse 3 – 15 minutes.    

The Lee-White Method is a test for the coagulation factors and involves measuring whole blood clotting times.  The technician collects 3 ml of blood in a plastic syringe and carefully places 1 ml in each in three glass or silicone tubes (blood clots faster in glass surfaces).  The tubes are placed in a 37° water bath.  The first tube is tilted every 30 seconds until coagulation occurs, then the second tube is tilted every 30 seconds until the blood coagulates.  The third tube is then tilted every 10 – 20 seconds until it coagulates.  The stopwatch is started when blood first enters the syringe and is stopped when the third tube coagulates.   Normal coagulation times are quite variable (3 – 30 minutes.)  Each laboratory must establish reference values for normal animals.

The Activated Clotting Time (ACT) is the ideal screening test.  Diatomaceous earth initiates clotting in this test.  Commercially available ACT tubes are prewarmed in a 37° water bath.   Two ml of blood is carefully drawn with a plastic syringe and injected into the ACT tube.  The tube is inverted 5 times to mix it and returned to the water bath.  After one minute the tube is removed and tilted every 5 seconds.   For this test the stopwatch is started when the blood is injected into the ACT tube and stopped when there is first evidence of clotting.  Normal values are between 60 and 90 seconds.

 

 Assignment

Perform a UA and CBC on two patients and report your findings on Lab Report 10. 

Use one of your slides and perform a platelet count.

Answer the questions on Unit 9 - Hemostasis

Email your UA's, CBC's and answers to Dr. Durham