Lecture 14b: The Fibrinolytic System

THE FIBRINOLYTIC SYTEM

The blood also has a mechanism for the lysis of a clot. If thrombin is the active enzyme for fibrin formation, plasmin is the active enzyme for lysis or destruction of the fibrin (clot).

The fibrinolytic system has four components: plasminogen proactivator, plasminogen activators, plasminogen and plasmin. Plasminogen proactivator is a plasma protein with a molecular weight of approximately 95,000 daltons. The inert protein is a substrate for the proteolytic activity of XIIa. Thus, plasminogen proactivator is converted to a plasminogen activator by the action of XIIa.

Plasminogen proactivator is heterogeneous group of proteins which react with plasminogen to produce plasmin. Some of these activators are proteolytic enzymes found in lysozymes of most cells in the body.

Plasminogen is a single chain protein with a molecular weight of 85,000 daltons. Plasminogen is converted to plasmin by the action of plasminogen activators.

Plasmin is a potent proteolytic enzyme which hydrolyzes the argyl–lysyl bonds of fibrinogen and fibrin, resulting in the formation of fibrinogen / fibrin degradation products. In addition, plasmin also hydrolyzes Factors V and VII and other serum proteins.

Fibrinolysis results from the conversion of an inert plasma proenzyme (plasminogen) into a proteolytic enzyme (plasmin) whose main physiologic role presumably is the proteolytic dissolution of fibrin. Plasminogen and plasmin, together with activator and inhibitors of the process, comprise of fibrinolytic enzyme system. Fibrinolysis is usually considered to be the major physiologic means of disposing of fibrin after its hemostatic function has been fulfilled.

Plasminogen activators:

           

Fibrinokinases

        Cytokinases, like staphylokinase, streptokinase, urokinase

Other plasminogen activators present in other body fluids like milk, tears, saliva and semen

Inhibitors of fibrinolysis:

a. Antiplasmins – these are plasma proteins specifically neutralizing free plasmin, e.g. alpha–2–macroglobulin and alpha–1–globulin

b. Artificial inhibitors – EACA (epsilon–amino–caproic acid), tanexamic acid, trasylol

The proteolytic action of plasmin on fibrin of fibrinogen leads to the formation of a family of soluble protein fragments (degradation or split products) FDP or FSP.

FDP and complexes thereof profoundly impair the hemostatic process, and are a major cause of hemorrhage in intravascular, coagulation and fibrinogenolysis. Most FDP are inhibitors of coagulation. They are potent antithrombis and also from incoagulable or slowly coagulating complexes with fibrin monomer of fibrinogen. FDP are removed from the circulation by clearance mechanisms in the liver and reticuloendothelial system.

DISORDERS OF HEMOSTASIS

I.     DISORDERS CAUSED BY VASCULAR ABNORMALITIES

These disorders are caused by abnormality of the blood vessels or their supporting tissues.

A. Autoimmune Vascular Purpura

(1)   The Allergic Purpura

Syndrome characterized by a relatively distinctive purpuric eruption in association with various constitutional and localized symptoms. The disorder is the result of an autoimmune process.

(2)   Drug–induced vascular purpura

Purpura induced by iodides, quinine, procaine, penicillin, aspirin

(3)   Purpura fulminans

A unique disorder characterized by sudden onset fever, prostration, symmetric circumscribed ecchymoses and infarcts of the skin and frequently by gangrene of the extremeties. The major initiating factor appears to be diffuse vascular injury and intravascular coagulation. The term “purpura fulminans” applies to any severe purpura of rapid onset.

B. Purpura Associated with Infections

A wide variety of infections may produce purpura by means of vascular damage which results from direct endothelial injury by the infection agent, e.g. rickettsia, viruses, cocci.

C. Disorders caused by structural malformations of vessels and perivascular tissues

(1)   Hereditary hemorrhagic telangiectasia – vascular malformation involves vessels throughout the body which are dilated, tortuous and disorganized. Characterized by the presence of widespread telangiectasia lesion of the skin and mucous membrane

(2)   Hereditary disorders of connective tissue

Ehlers–Danlos syndrome and osteogenesis imperfecta – in these disorders, the abnormality is caused by qualitative and quantitative abnormalities of collagen and elastin.

(3)   Acquired disorders of connective tissue

(a)   Scurvy – associated with serious bleeding including persistent gingival bleeding and hemorrhage into the subcutaneous tissues and muscles. Petechiae often develop and most conspicuous around the hair follicles. Bleeding is attributed to a defect in the endothelial lining and perivascular tissues due to deficient synthesis of collagen and intercellular cement substance caused by deficiency in Vitamin C.

(b)   Senile purpura – chronic disorder of the elderly characterized by relatively distinctive red to purple ecchymotic spots on the forearm and back of hands and neck. The basic defect is degeneration and loss of collagen, elastin and fat.

D.    Miscellaneous vascular purpura

1. Autoerythrocyte sensitization and related disorders

2. Purpura in association with paraproteinemias

3. Purpura simplex – refers to mild purpuric manifestations in healthy persons. This is particularly common in women (devil’s pinches) during the menstrual period.

Laboratory findings in the disorders caused by vascular abnormalities

Capillary fragility test                                    –          usually positive

Bleeding time                                                 –          may be normal or prolonged

Platelet count                                                 –          usually normal

Other hemostasis and

blood coagulation tests         –          normal

Petechiae – round hemorrhagic discoloration in skin and mucous membrane which vary in size from 1 to 3 mm usually resulting from capillary bleeding and tend to occur especially in the lower extremities or other areas of high venous pressure.

Ecchymosis – refers to larger hemorrhagic discolorations which are red, purple, yellow, green or brown depending on the age of lesion.

Purpura – refers to hemorrhagic state characterized by skin and mucous membrane bleeding, i.e., petechiae and ecchymosis

Bleeding into deep tissue and joints is usually the result of defects in coagulation factors.

Petechiae and ecchymosis generally occur because of platelet disorders or vascular disorder.

II.    DISORDERS CAUSED BY PLATELET ABNORMALITIES

A. Quantitative Platelet Disorder

(1)   Thrombocytopenia – subnormal number of platelets in the circulating blood which is caused by:

(a)   Hypoproliferative thrombocytopenia

1.     Aplastic or hypoplastic marrow

2.     Infiltrative disease of marrow: carcinoma, leukemia, disseminated infection

3.     Specific megakaryocytic hypoplasia

(b)   Ineffective thrombopoeisis

1.     Folic acid deficiency

2.     Vitamin B₁₂ deficiency

(c)    Platelet sequestration

1.     Pooling of platelets in enlarged spleen

(d)   Increased platelet destruction

1.     Immune thrombocytopenia

2.     Disseminated intravascular coagulation

3.     Mechanical injury of platelets

(2)   Thrombocytosis – abnormally high number of platelets in the circulating blood. Thrombocytosis can be:

(a)   Reactive thrombocytosis – secondary to some other process

1. Infectious disorders

2. Malignancies

3. Iron deficiency anemia

4. Following surgical procedure

5. Inflammatory disorders (collagen vascular disease)

6. Following hemorrhage

(b)   Autonomous thrombocytosis – primary or idiopathic thrombocytopenia

1. Idiopathic thrombocythemia (primary thrombocytosis)

2. Myelofibrosis with myeloid metaplasia

3. Polycythemia vera

4. Chronic myelogenous leukemia

B.  Qualitative Platelet Disorder

(1)   Hereditary Qualitative Platelet Disorders

(a)   Thrombasthenia (Glanzmann’s disease)

Inherited disorder due to abnormality of surface membrane glycoprotein – it is characterized by epistaxis, menorrhagia, gingival bleeding and numerous ecchymosis.

Laboratory findings:

Platelet count and morphology – normal

Bleeding time – prolonged

Clot retraction – deficient

Platelet adhesion to glass beads – reduced

Platelet aggregation – decreased

Platelet factor 3 activity – abnormal

(b)   Thrombopathia

Hereditary disorder wherein the platelet aggregate normally but fail to release normal amounts of adenosine diphosphate (ADP)

Laboratory findings:

Platelet count – normal

Bleeding time – prolonged

Clot retraction – normal

Platelet adhesiveness test – reduced

Platelet aggregation – normal

Platelet factor 3 activity – abnormal

Prothrombin consumption test – abnormal

(c)    Thrombocytopathy

A hereditary disorder which refers to platelet dysfunction manifested by defective coagulant activity of the platelets that is deficient on PF₃ activity. It may be caused by deficient PF₃ content of the platelets or caused by unavailability or deficient release of PF₃, though the content is normal

Laboratory findings:

Platelet count – normal

Bleeding time – may be prolonged but often normal

Clot retraction – normal

Prothrombin consumption test – abnormal

Platelet Factor 3 activity – deficient or abnormal

(d)   Storage granule abnormality

(e)   Deficiencies of thromboxane generation

(f)    Defects in release reaction due to reduction in storage pool ADP or an inability to release ADP.

(g)   Platelet type von Willebrand’s disease

(2)   Acquired Qualitative Platelet Disorders

(a)   Patients with myeloproliferative disorders may present not only with abnormalities of platelet numbers but also with qualitative platelet abnormalities

(b)   Drug induced platelet dysfunction caused by anti–inflammatory agents, anti–depressant

(c)    Disorders associated with circulating FDP (fibrin degradation products)

(d)   Patients with uremia suffer impairment of primary hemostasis

(e)   Storage pool deficiency has been observed in patients with systemic lupus erythematosus (SLE)

III.  DISORDERS CAUSED BY ABNORMALITIES IN COAGULATION FACTORS

A. Hereditary Coagulation Disorders (Coagulopathies)

Hereditary disorders of coagulation usually are the result of a deficiency or abnormality of a single plasma protein

(1)   Fibrinogen disorder

(a)   Hereditary afibrinogenemia is the result of deficient biosynthesis of fibrinogen. The plasma fibrinogen concentration is usually less than 0.05 g/liter.

(b)   Hereditary hypofibrinogenemia – a mild bleeding syndrome

(c)    Congenital dysfibrinogenemia – results from qualitative abnormalities of fibrinogen molecule which involves the enzymatic polymerization and stabilization steps of thrombin–fibrinogen reactor.

(2)   Hereditary prothrombin deficiency

(a)   Constitutional dysprothrombinemia – caused by abnormal synthesis of prothrombin molecule

(b)   Hypoprothrombinemia – due to decreased synthesis of prothrombin

(3)   Factor V deficiency (parahemophilia) – also called Owren’s disease. This may be due to deficient biosynthesis of Factor V.

(4)   Hereditary Factor VII deficiency – due to abnormality in the synthesis of Factor VII

(5)   Hemophilia A or Factor VIII deficiency of Classical Hemophilia

This is inherited as a sex–linked recessive trait. The disorder is the result of the synthesis of a dysfunctional Factor VIII molecule. The defective gene is located in the X chromosome. In males, who lack a normal allele, the defect is manifested and who will not transmit disorder to his sons in the 2^nd generation but to his daughters who will be the carriers of the trait. In the succeeding generations the defect may be transmitted by the carrier mother to her sons and daughters.

(6)   Hemophilia B or Christmas disease or Factor IX deficiency

This is inherited as sex–linked recessive trait. Christmas disease is caused by any of several abnormal variants of the Factor IX molecule. The clinical manifestations of Christmas disease are identical to those of Hemophila

(7)   Hereditary Factor X deficiency or Stuart–Prower factor deficiency

This is due to molecular abnormality

(8)   Hereditary Factor XI deficiency or Hemophilia O or PTA deficiency

This is common among persons of Jewish parentage. The clinical manifestations are milder than hemophilia A or B

(9)   Hereditary Factor XII deficiency or Hageman trait

This is probably due to a deficient biosynthesis of Factor XII. With rare exceptions, Factor XII deficiency is unassociated with hemorrhagic symptoms even after trauma, surgery or childbirth

(10)   Hereditary Factor XIII deficiency

This disorder is an uncommon disorder wherein all the visual tests of coagulation give normal results and the disorder can be readily demonstrated by clot solubility tests, the normal clots being insoluble in 5 M urea or 1% monochloroacetic acid.

(11)    Fletcher factor deficiency

This is a rare disorder due to abnormality caused by deficiency in prekallikrein as in Hageman trait, none of the patient with Fletcher factor deficiency have excessive bleeding.

(12)   Deficiency in High Molecular Weight Kinninogen

This disorder is very rare and is caused by diminished HMWK

B. Acquired Disorder of Coagulation

These acquired coagulation disorders are more complex than the hereditary forms. In the acquired forms, deficiency of several factors usually is found and thrombocytopenia, abnormalities in platelet function, abnormal inhibitors of coagulation and vascular abnormality are commonly present.

(1)   Deficiency of the Vitamin K dependent factors namely: Factor II, VII, IX and X

Deficiencies of these four factors may develop in a variety of disorders in which there is a deficient intake or absorption of Vitamin K, as well as in disorders that impair the biosynthesis capacity of the liver.

Vitamin K deficient states that are associated with a variety of disorders:

(a)   Hemorrhagic Disease of the Newborn

(b)   Biliary obstruction (gallstones, stricturem fistula) either intrahepatic and extrahepatic

(c)    Malabsorption of Vitamin K, e.g. sprue, steatorrhea

(d)   Liver or hepatic disease

(e)   Nutritional deficiency

(f)    Action of drugs

(2)   Coagulation abnormalities associated with liver disease

Clotting abnormalities associate with hepatic dysfunction is markedly diverse because the liver plays a variety of roles in the coagulation system. The liver is the site of synthesis of most it not all coagulation factors.

(3)   Acquired von Willebrand disease

Deficiency in Factor VIII which is associated to tumor

(4)   Disorders associated with Diffuse Intravascular Coagulation

Diffuse Intravascular Coagulation (DIC) is a hemorrhagic syndrome which occurs following the uncontrolled activation of procoagulants and fibrinolytic enzymes in the microvasculature. In this disorder, fibrin is deposited in small vessels, causing tissue injury or necrosis. Disorders associated with DIC:

(a)   Infectious disease

(b)   Malignant disorders

(c)    Liver diseases

(d)   Obstetrical disorders

(e)   Neonatal disorders

(f)    Vascular disorders

(g)   Miscellaneous disorders, shock, burns and snake venom

Laboratory findings in Hereditary Coagulation Disorders

Disorder                                  PTT      PT        CT        PCT      Thrombin        TGT                 BT

                                                                                                Time

Hemophilia A                         A          N         A          A          N                     PD                    N

Hemophilia B                         A          N         A          A          N                     SD                    N

von Willebrands dse              vA        N         vA        vA        N                     vPD                  uA

Afibrinogenemia                    A          A          A          N         A                      N                     uA

Dysfibronogenemia                vA        A          uN       N         A                      N                     N

Hypoprothrombinemia         A          A          vA        –          N                     N                     N

Factor V deficiency                 A          A          vA        vA        N                     mild PD                       uN

Factor VII deficiency               N         A          N         N         N                     N                     N

Factor X deficiency                 A          A          A          A          N                     SD                    N

Factor XI deficiency                A          N         A          A          N                     mild PD           N

Factor XII deficiency               A          N         A          A          N                     mild PD           N

Factor XIII deficiency              N         N         N         N         N                     N                     N

Fletcher factor deficiency      A          N         A          –          N                     –                      –

HMWK                                    A          N         –          –          N                     –                      –

Platelet count and CRT are normal for all the above disorders.

A – abnormal; N – normal; u – usually; v – variable; PD – plasma deficient; SD – serum deficient

IV.   DISORDERS CAUSED BY ABNORMAL INHIBITORS OF COAGULATION        

Inhibitors of coagulation (Circulating anticoagulants)

The pathologic inhibitors of coagulation or circulating anticoagulants may be defined as abnormal endogenous components of blood that inhibit the coagulation of normal blood. They may act at virtually any stage in the process of coagulation. Some of these inhibitors are antibodies.

A. Specific Inhibitors

Antibodies to fibrinogen, Factor V, VIII, IX, X, XII and XIII

B. In Systemic Lupus Erythematosus, a unique type of coagulation inhibitor is commonly associated with it. These “lupus” anticoagulants are either of the IgG or IgM class

C. Miscellaneous inhibitor

Bleeding has been attributed to presence of antithrombins, FDP and abnormal proteins absorbed by fibrinogen or fibrin, acts as antithrombin and as inhibitor of fibrin polymerization and results in gelatinous, structurally abnormal clots.

D. Inhibitors associated with paraproteinemias

LABORATORY METHODS FOR THE STUDY OF HEMOSTASIS AND BLOOD COAGULATION

There is no single test which is suitable for the laboratory evaluation of the overall process of hemostasis and blood coagulation, but methods of varying complexity and utility are now available for assessing various components and functions individually.

Blood preparations often used for coagulation tests

1. Patient’s plasma

Mix 4.5 ml blood and 0.5 ml 0.1 M sodium oxalate

Centrifuge at 1,500 rpm for 5 minutes

A 1:10 dilution with saline solution is obtained by adding 0.1 ml plasma and 0.9 ml NSS

Store in refrigerator

2. Adsorbed normal plasma

Adsorption – process of removing prothrombin, Factor VII, IX and X from normal plasma by the use of certain insoluble salts of alkaline earth like aluminum hydroxide gel, barium sulfate and calcium phosphate. Factors XII, V and VIII are not adsorbed

Mix 2 ml plasma and 0.2 ml adsorbing agent

Centrifuge at 3,000 rpm for 5 minutes

Refrigerated plasma should be used within 2 hours

3. Aged normal plasma

Collect plasma in the usual manner

Incubate at 37^oC for 24 hours

Store in aliquots at 20^oC

4. Aged normal serum

Collect serum in the usual manner

Allow to stand at room temperature for 24 hours

Divide in aliquots and freeze

5. Platelet–rich plasma

Mix 9 parts of blood and 1 part of 3.8% sodium citrate

Centrifuge at 1,500 rpm for 5 minutes

6. Platelet–poor plasma

Mix blood as in number 5

Centrifuge at 3000 rpm for 30 minutes

CONSUMPTION, AGING AND ADSORPTION CHARACTERISTICS OF CLOTTING FACTORS

Factor              Present in Normal Plasma                 Present in Normal      Adsorbed

                        Fresh               Aged                                        Serum             BaSO₄ or Al(OH)₃

Factor VII        Yes                  Yes (3 days)                            Yes                              Yes

PTC                  Yes                  Yes                                          Yes                              Yes

AHF                 Yes                  No                                           No                               No

PTA                  Yes                  Yes                                          Yes                              No (partly)

Hageman        Yes                  Yes but adsorbed by glass     Yes                              No

Factor V          Yes                  No                                           No                               No

Stuart              Yes                  Yes                                          Yes                              No

Prothrombin   Yes                  Yes but gradually falls                       20% or less                 Yes

Fibrinogen      Yes                  Yes                                          No                               No

Test for vascular and platelet phases

A. Bleeding Time

Hemostasis in a small superficial wound, such as that produced by measuring bleeding time depends on the rate at which a stable platelet thrombus is formed and thus measures the efficiency of the vascular and platelet phase. The bleeding time leaves much to be desired in terms of reproducibility, since no two skin areas are exactly the same and it is impossible to produce a truly standard wound. Bleeding time is principally a measure of platelet plug formation. Factors which affect bleeding time include:

1. Elasticity of the out tissues

2. Ability of the blood vessels to constrict and retract

3. Mechanical and chemical action of platelets in the formation of hemostatic plug

Method of bleeding time determination

1.     Duke method

2.     Modified Ivy method

3.     Coply–Lalitch method            both methods involve the immersion of the wounded

4.     Adelson–Crosby method       finger in a sterile NSS warmed at 37^oC until bleeding

stops

Normal values:                       170 – 340 seconds

5. Macfarlane’s method – similar to Adelson–Crosby, only it uses the earlobe as the site of puncture

6. Aspirin Tolerance Test – this assesses the effect of a standard dose of aspirin on the Duke’s bleeding time

The bleeding time is prolonged in aspirin overdosage. Four aspirin tablets prolong the bleeding time of a normal individual and two tablets will lengthen it in a patient with von Willebrand’s disease

B.     Enumeration of platelets (platelet count)

C.     Test of specific platelet functions

1. Test for Adhesion of Platelets or Platelet Adhesiveness Test

a. In vivo method of Borchgrevink

It measures the adhesion of platelets to the wound surface. Platelets are enumerated in the capillary blood issuing from a bleeding time puncture and adherent platelets are expressed as a percentage of the venous platelet count. As estimated by this method, adhesion is influenced by all of the variables intrinsic to the bleeding time and by the hematocrit.

b. Salzmann’s method

Test for the retention of platelets within glass bead column (glass bead adhesion). In this method, venous blood is aspirated directly from the vein through a bead column and into a vacutainer. Results are expressed as the percentage of the venous platelet count retained.

c. Test for adhesion of platelets to collagen fibers

Platelet rich plasma containing EDTA is assessed for adhesion collagen in the absence of aggregation. Technique is based on enumeration of free and adherent platelets

2. Platelet Aggregation Test

Platelet aggregation is estimated qualitatively both by microscopic and macroscopic techniques. The most commonly used quantitative method employs the use of various aggregometers. These are instruments (photometers) modified so as to permit measurement of changes in optical density of a platelet suspension under condition of constant temperature and continuous agitation. Platelet aggregation usually is studied in suspension of citrated plasma (platelet rich plasma).

3. Platelet Factor 3 Availability Test

Platelet rich plasma is incubated with kaolin, causing the release of phospholipid (PF3) and activating the plasma contact factors. At appropriate intervals, samples of platelet–rich plasma are removed and added to a second tube containing kaolin – activated normal platelet poor plasma. A recalcification time is determined. Since the second tube will have a maximum contact activation of the plasma factors, the length of the recalcification time is inversely related to the amount of phospholipid released in the incubation mixture in the first tube. In normal individuals, there is a progressive shortening of the recalcification time.

D. Determination of clot retraction

It is essential in any hemorrhage study, to look at the clot, to observe its structure and to note its ability to retract, to squeeze out serum and to retain red cells. This simple and inexpensive test is most useful in diseases involving fibrinogen, platelets, and anticoagulants and in conditions characterized by abnormal proteins and increase in cells.

1. Qualitative estimation of clot retraction can be made by incubating a tube of clotted blood, in which retraction is normally apparent within two hours.

a. Hirschboeck or Castor oil method

b. Single tube method – blood in a test tube used for clotting time determination is saved and left at room temperature in order to note retraction, red cell fall – out and clot lysis. Note the shape and dry character of the clot, serum expressed and volume of red cell fall–out.

Defective clots

            Disease                                               Clot characteristics

Thrombocyte deficiencies

            Thrombocytopenia                            Clot non–retractile or retracts poorly

            Thrombasthenia                                Clot edematous and friable

Fibrinogen deficiencies

            Afibrinogenemia                                Blood does not clot

            Fibrinogenemia                                  Clot is small; increased red cell fall out

            Fibrinolysis                                         Clot is absent or moth–eaten and

                                                                        frayed; increased red cell fall out; serum

                                                                        will lyse Normal clots

Increased in blood constituents

            Thrombocythemia                             Defective retraction; clot flabby and

            Polycythemia                                      fragile; increase red cell fall–out

            Hyperproteinemia                             Rapid sedimentation of red cells; layered

                                                                        clot; Clot may not retract or may retract

                                                                        poorly

Delayed clotting

            Severe “hemophiloid” state              Slow clotting time with sedimentation of                                                                                 red cells; fibrin retractile clot

            Increase in anticoagulants                Increase in red cells and in fluid fall –

                                                                        out; clot may reform after initial partial

                                                                        clot removed

2. Quantitative methods for determining the extent of clot retraction

a. Stefanini method – similar with single tube method

b. Macfarlane method – in this method, blood is allowed to clot in a test tube containing a glass rod and retraction is observed after the incubation period. The clot attached to the rod is then removed and the serum extracted from the clot is measured. Retraction is expressed in terms of the volume of serum obtained from the % of original volume of blood. To calculate, use the following formula:

Volume of expressed serum        x          100      =          % expressed serum

Volume of whole blood

Normal values:     44 – 67%

E. Capillary fragility or capillary resistance test

Capillary fragility tests are tests of the ability of the small blood vessels to retain the red cell in their lumen under conditions of stress and trauma. Platelets and Vitamin C are important in the maintenance of normal capillary integrity resistance.

1. Tourniquet or Rumpel–Leede or Hess Test

Positive Pressure Technique

In the positive pressure test, a tourniquet or blood pressure cuff is placed above the elbow, retarding the venous return and creating condition of increased hydrostatic pressure and hypoxia within veins and capillaries. Abrupture of one or more blood vessels constitute a positive test.

Principle:

By partially obstructing the venous blood, the capillary pressure is increased, this will give rise to intravasation of blood which will be manifested in the form of small hemorrhages called petechiae.

Related methods:

a.     Quick method:      N.V.: 0 – 5 petechiae

b.     Gothin method:   N.V.: 0 – 8 petechiae

2. Suction cup or Petechiometer method

Negative Pressure Technique

In the negative pressure method trauma is created by means of suction cup applied to the skin

Principle

This method employs the use of a modified DaSilva Melle instrument. The cup is applied to the outer surface of the arm for a period of one minute and the resistance of the capillaries is expressed as the least negative pressure required to produce macroscopic petechiae

Related method:        Dalldorf method

N.V.:    less than 4 petechiae at 200 mmHg pressure

TEST OF THE COAGULATION PHASES (PHASES I AND II)

A. THE COAGULATION TIME

The coagulation time of whole blood is a procedure which tests the complete action of all plasma factors acting simultaneously. The time required for the blood to clot is a function of the combined factors favoring coagulation on one hand as opposed to the combined factors inhibiting coagulation on the other hand. Clotting time is a measure of the ability of the blood to clot and is not influenced by the platelet function other than PF – 3. Clotting measures only the time required for the formation of the first traces of thrombin suffice to produce a visible clot. This test is informative only if it is significantly prolonged.

1. Micromethods

a. Slide or drop method

b. Capillary or Dale and Laidlow method

N.V.:          2 – 4 minutes

2. Macromethods

These methods are superior for there is less contamination of the plasma with tissue fluids when blood is drawn from a vein.

a. Lee–White method or Whole blood clotting time

Principle:

The whole blood clotting time is the time required for freshly collected blood to form a firm clot in standardized glass tubes at 37^oC. Thus, the whole blood clotting time is a measure of the integrity of the intrinsic system

Normal values:     7 – 15 minutes

b. Howell’s method

Normal values:     10 – 30 minutes

c. Silicone tube method – Tocantin’s and Kazal’s method

This is the same as the whole blood clotting time with exception that the test is performed in silicone – coated tubes

Normal values:     20 – 40 minutes

3. Aggregated coagulation time of whole blood

Principle:

The activated coagulation time of whole blood is the time necessary for fresh blood to form a firm clot when incubated at 37^oC in the presence of surface contact activation. This assay like the whole blood clotting time, measures overall activity of the intrinsic clotting time.

Normal values:                       1 – 2 minutes

4. Plasma recalcification time

Recalcification of platelet poor plasma represents refinement of the glass tube whole blood coagulation time. It is more sensitive than the coagulation time of whole blood and may reveal abnormality which is not detectable by the determination of the clotting time of venous blood. If the coagulation time of recalcified plasma is significantly prolonged, one has a system which readily lends itself to substitution with various known substance. By mixing the plasma to be tested with plasma of patients with known deficiencies, the specificity of the deficient factors can be confirmed.

Normal values:           120 – 180 seconds

B. PARTIAL THROMBOPLASTIN TIME

This is a simple test of the intrinsic and common pathways of coagulation. When a mixture of plasma and a phospholipid platelet substitute is recalcified, fibrin forms at a normal rate only if the factors involved in the intrinsic pathway (Factors XII, XI, IX and VIII) and the common pathway (Factors X, V, II and I) are present in normal amounts. A short PTT may signify any of the various hypercoagulable states and high levels of any factor involved may mask deficiencies of others.

Normal values:     30 – 45 seconds

C. ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT)

The procedure in aPTT is similar to the original test (PTT) except that a standardized foreign surface is introduced to the plasma. Such foreign materials have to be inert and should not adsorb any coagulation factors from the plasma. Practically, the inorganic diatomaceous earths, kaolin and celite, are widely used. When incorporated into the reagent (cephalin) and added to the test plasma, they impart a standard, controlled foreign surface that activates Factor XII and XI in a precise way, thus speeding up the test formation of fibrin and allowing for a greater degree of reproducibility by eliminating the activation of glass contact. Another activator used is tannic acid derivative, e.g. ellagic acid.

D. DIFFERENTIAL TESTS OF ACTIVATED PARTIAL THROMBOPLASTIN TIME (DAPTT)

This is used to differentiate deficiency factor and disorder of circulating anticoagulants. This is done by mixing patient’s plasma and normal control plasma and running the activated partial thromboplastin time on the mixture. If the defect is corrected by addition of normal control plasma, the patient has factor deficiency; if not corrected, he has a defect due to circulating anticoagulants. To identify further the factor deficiency, mix the patient’s plasma with adsorbed plasma (with Factors V and VIII) or with serum (with Factors XI and XII), then rerun aPTT.

E. DIFFERENTIAL PARTIAL THROMBOPLASTIN TIME (DPTT)

This is the modification of aPTT which is done by mixing the patient’s plasma with commercially available correcting reagents, Factor VIII and IX reagents. If prolonged PTT is corrected with Factor VIII reagent – Hemophilia A; if corrected with Factor IX reagents – Hemophilia B, if partially corrected with either reagent – Hemophilia C.

F. PLASMA PROTHROMBIN TIME

The production of fibrin via the extrinsic and common pathways requires tissue thromboplastin and Factor VII, in addition to Factors X and V, prothrombin and fibrinogen. These pathways are measured by the plasma prothrombin time, in which plasma is recalcified in the presence of excess tissue thromboplastin. The prothrombin time usually will be prolonged if the plasma levels of any of the requisite factors are below 10% of normal.

Methods:

1. One–stage method of Quick, Stanley–Brown method

Tissue thromboplastin and calcium added to plasma causes formation of thrombin which reacts with fibrinogen to produce a clot. The thromboplastin added to the plasma takes the place of the tissue juice in formation of extrinsic thrombopastin. The protime therefore, is prolonged if there is deficiency of Factors V, VII or X or an especially severe deficiency of I or II.

2. Two–stage Prothrombin and Proconvertin Test (Owren and Aas)

It offers a combined estimation of the levels of prothrombin and proconvertin (Factor VII). Its advantages:

a. More sensitive than the one–step protime to minor deviations from normal.

b. Fresh specimens are not necessary, and the method can be used for mailed samples of blood.

c. Method is not affected by heparin in low concentrations

3. Owren’s Thrombotest Method

For the control of coumarin anticoagulant therapy, this is probably the most sensitive test

N.V.     The thrombotest clotting time of patients on adequate coumarin anticoagulant therapy

            Range from 50 to 100 seconds

4. Fibrometer method

Fibrometer (coagulation timer) is an electromechanical instrument that has been used extensively for the one–stage prothrombin time of Quick and for the Owren’s thrombotest. The instrument may also be used for determination of PTT and for PCT.

5. Micromethod (ProTime)

This is a microtechnique employed for children and the method uses micropipettes, the principle of the test is similar with the one–stage prothrombin time.

6. Related method – Stypven time (Russell’s Viper venom method)

The venom of the Russell’s viper contains a unique thromboplastic substance which initiates coagulation by the direct activation of Factor X and does not require Factor VII. The stypven time is used to distinguish deficiencies of Factor X and those of Factor VII.

Normal values:                       20 – 25 seconds

Prothrombin activity or index is reported in percentage, considering 100% as the maximum level:

ProTime (in seconds) of Control       x          100      =          % Prothrombin index or

ProTime (in seconds) of Patient                                                        activity

G. SERUM PROTHROMBIN TIME (PROTHROMBIN CONSUMPTION TEST)

Approximately 85 to 95% of the prothrombin in normal blood is utilized or consumed when blood clots, leaving relatively little residual prothrombin in the serum. The prothrombin that is not consumed and therefore remains in the serum is measured by supplying fibrinogen to the system and adding tissue thromboplastin and calcium. Normal serum which contains little residual prothrombin will clot relatively slowly, whereas serum that contains larger amounts of prothrombin will clot rapidly.

Principle:

During normal coagulation, thrombin production and prothrombin utilization continue after the blood or plasma has clotted. If the serum is tested 1 hour after coagulation, it will be found that all the prothrombin has been consumed. If there is a deficiency of any of the factors required for the coagulation of blood or plasma in glass, prothrombin will be incompletely consumed and more than normal will be present in the serum after 1 hour. The PCT is abnormal when any of the essential factors are below 2 or 3% of normal.

Normal value:       26 – 37 seconds

The prothrombin consumption test is best considered a test of platelet phospholipid activity. If the prothrombin time and the PTT are normal, a short PCT indicates a deficiency of platelet factor–3 from the thrombocytopenia or thrombopathia.

H. THROMBOPLASTIN GENERATION TIME (TGT)

The principle of this test lies on the knowledge that for normal thromboplastin activity to develop in blood, AHF, platelets, PTC, PTA, Factor V, the Stuart factor and ionized calcium are all necessary. Four reagents may be prepared that, separately contain some of the components and that, when added together, supply all the ingredients of this intrinsic thromboplastin – generating mechanism. The four reagents are:

1.     Adsorbed plasma

2.     Normal plasma

3.     Normal platelets

4.     Calcium

When these four reagents are mixed and incubated together, thromboplastin if formed, and the potency of this is then estimated by noting the time taken by small aliquots of the incubation mixture to induce clotting in a normal, platelet – free citrated plasma. The time taken for coagulation of the latter substrate plasma (normally up to 15 seconds) actually measures the rate of conversion of prothrombin to thrombin (by the thromboplastin generated in the original incubated mixture) and the conversion of fibrinogen to fibrin by the thrombin to produce.

By repeating the test, using platelets, adsorbed plasma and serum from the patient, the potency of the generated thromboplastin and the time required to develop maximal activity can be measured.

In order to detect which of thromboplastin ingredients is defective, the platelets, adsorbed plasma and serum of the patient and that of normal control are mixed in various combinations. Calcium is added and the rate and potency of the generated thromboplastin of each mixture is evaluated.

Assuming that other factors are normal, CA’s may be differentiated from other condition by substituting for adsorbed plasma in the TGT equal parts of the patient’s and normal adsorbed plasma. If the result of the test is then abnormal, it means that circulating anticoagulants are almost certainly present since all the defects would have been corrected by the inclusion of 50% volume of normal adsorbed plasma. CA’s are not adsorbed by barium sulfate or aluminum hydroxide.

Methods of TGT:

1.     Biggs and Macfarlane method

2.     Hick’s–Pitney kaolin modification of TGT

TESTS FOR STAGE III OF COAGULATION AND FOR FIBRINOLYSIS

1. Fibrinindex Test

When added to plasma containing fibrinogen, thrombin produces clotting. Thrombin is available commercially as Fibrindex

N.V.     Normal plasma begins to clot after 5 to 10 seconds. The firm clot is formed

            without serum after 30 to 60 seconds

2. Fi–test (Immunologic test)

This is a rapid slide test based on the agglutination of fibrinogen coated red blood cells by the latex anti–human fibrinogen reagent. Normally, presence of fibrinogen is indicated by agglutination

3. Fibrinogen Titer method

Serial dilutions of plasma are clotted with thrombin. The titer is the highest dilution in which a fibrin clot can be seen, and is related to the fibrinogen concentration and indirectly to the presence of circulating anticoagulants.

4. Assay of Plasma fibrinogen

Several accurate methods are now available for the quantitative assay of plasma fibrinogen; fibrinogen is usually converted into fibrin which is quantitated by gravimetric, nephelometric, chemical, immunologic and precipitation method

a.     Ellis and Stransky method

b.     Stinland method

c.      Turbidimetric method of Parfentjev, et.al.

d.     Ratnoff and Menzie method

e.     Fibrin clot method

5. Whole Blood’s Clot Lysis Time

Principle:

A clot dissolves as a result of plasmin activity. Normally, this does not occur in less than 72 hours because of the presence of plasma inhibitors which inactivate plasmin as it forms.

Normal value: Lysis of clot before 24 hours is abnormal

6. Euglobin Clot Lysis Time

Principle

Euglobin fraction of the plasma contains fibrinogen, plasminogen and all of plasminogen activators but only traces of antiplasmins. The lysis of a fibrin clot formed by the addition of thrombin is a measure of the fibrinolytic activity.

Normal value

Lysis in about 300 minutes or longer – normal

Lysis in 60 minutes or less – strong lysis

Lysis in 120 minutes – increase lytic activity

7. Diluted Blood Clot Lysis Time

Principle

Plasmin inhibitors lose activity on dilution. In this method, whole blood is diluted with a buffer solution and clotted by the addition of thrombin. Then the clot is observed for lysis.

Normal value: Blood should not lyse in less than 6 to 10 hours

8. Diluted Plasma Clot Lysis Time

Principle

In this method, serial dilutions of patient’s plasma and normal plasma are prepared. Thrombin is added to each test tube and is then observed later on for presence of clot, and eventually for lysis. Lysis within 12 hours means incomplete fibrinolytic activity.

9. Quantitative Assay of Fibrin – fibrinogen Degradation Products

The method for assay of these fragments is based on red cell hemagglutination inhibitors, staphylococcal agglutination and immunodiffusion.

10.  Plasma Protamine Paracoagulation Test

Principle

When a dilute solution of protamine sulfate is added to citrated plasma incubated at 37^oC, a precipitate forms in the presence of fibrin monomers or early fibrin degradation products.

Method:          Kidder’s method

TESTS FOR INHIBITORS OF COAGULATION (CIRCULATING ANTICOAGULANTS)

1. Plasma antithrombin test – this involves titration of plasma with decreasing amounts of thrombin to detect small amount of anticoagulants.

2. Plasma thrombin time – plasma is clotted by thrombin and the time take is dependent on the amount and quality of fibrinogen and inhibitors.

3. Assay for Lupus anticoagulants (Tissue inhibition test)

Method:          Schleider’s method

4. Assay for inhibitors of other factors

AUTOMATED METHODS FOR COAGULATION FACTORS

A variety of instruments have been developed which automatically detect the end point clotting time and are helpful in the performance of the one–stage prothrombin time and other screening tests:

Examples:       Thromboelastograph and Coagulogram