LESSON 6: Fibrinogen, Fibrinolysis and D-Dimer Test
OUTLINE INTRODUCTION
I Overview of the Stages of Secondary Hemostasis
II Introduction
A. Activation of Plasminogen to Plasmin
B. Role of Plasminogen and Plasmin in Normal and
Abnormal Fibrinolysis
C. Fibrinogen Degradation by Plasmin
D. Naturally occurring Coagulation and Fibrinolytic
Inhibitors
E. Pathologic Fibrinolysis
F. Laboratory Evaluation of Fibrinolysis
i Whole Blood Clot Lysis Time
ii Euglobulin Lysis Time
iii Protamine Sulfate Test
iv Ethanol Gelatin Test
v Latex D-Dimer Assay
OVERVIEW OF THE STAGES OF SECONDARY
HEMOSTASIS
STAGE 1: GENERATION OF THROMBOPLASTIN
• Three Pathways:
a. Intrinsic Pathway (Factors XII, XI, IX and VIII)
o All these factors are present in plasma
o Factor XII, in vivo – contact to collagen
o Factor XII, in vitro – contact to glass
b. Extrinsic Pathway (Factors III and VII)
o All these factors are absent in plasma
o What is the factor involved in the Extrinsic
Pathway?
o Factor VII only – because Factor III is
only a Tissue Factor that helps
activate Factor VII
c. Common Pathway (Factors X and V)
STAGE 2: CONVERSION OF PROTHROMBIN TO THROMBIN
• Factor II to Factor IIa or Thrombin
STAGE 3: CONVERSION OF FIBRINOGEN TO FIBRIN CLOT
• Factor I to Fibrin clot
• Fragments X and Y referred to as early degradation
Fibrinolysis is the defense against occlusion of blood products.
• Fragments D and E are late degradation products.
vessels, but it is also important that bleeding does not recur
because of premature lysing of the clot. • Fragment X is the first and largest formed.
Diagram: Degradation of cross-linked fibrin by plasmin and
I. ACTIVATION OF PLASMINOGEN TO PLASMIN Degradation of fibrinogen and non-crosslinked fibrin by plasmin (on
the last page)
• Fibrinolysis is dependent on the enzyme PLASMIN
which can digest or destroy fibrinogen, fibrin, and
IV. NATURALLY OCCURRING COAGULATION AND
factors V and VIII.
• A zymogen known as PLASMINOGEN, which normally
FIRBRINOLYTIC INHIBITORS
is present in plasma, is converted to plasmin by the • The counterforce of the naturally occurring
coagulation and fibrinolytic inhibitors are necessary
action of specific enzymes called PLASMINOGEN
ACTIVATORS. It is stored and transported in to achieve a balance between activated clotting
eosinophils. Increased concentrations are found in factors and fibrinolytic enzymes.
association with inflammation.
Diagram: Activation of the Fibrinolytic System (on the last page) A. NATURALLY OCCURRING COAGULATION
INHIBITORS
II. ROLE OF PLASMINOGEN AND PLASMIN IN 1. Antithrombin III: synthesized in the liver
NORMAL AND ABNORMAL FIBRINOLYSIS 2. Alpha 2 Macroglobulin: inhibits both coagulation and
• Under normal circumstances, plasminogen is part of fibrinolysis; inhibits kinin system
clot because of tendency of fibrin to absorb 3. Alpha 1 antitrypsin: potent inhibitor of factor Xia.
plasminogen from the plasma. 4. C1 Inactivator
• When plasminogen activators perform, plasmin is 5. Protein C and S: these are Vitamin K dependent
formed within the clot, gradually dissolves the clot
while leaving time for tissue repair. B. NATURALLY OCCURRING FIBRINOLYTIC
• When pathologic coagulation processes involved, INHIBITORS
excessive free plasmin is released to the plasma. In 1. Alpha 2 antiplasmin: major inhibitor
this situation, antiplasmin is depleted, and plasmin
2. Alpha 2 macroglobulin
begins to destroy components like fibrinogen, factor 3. Thrombospondin
V and VIII and other factors.
4. Plasminogen Activator inhibitor 1 and 2
III. FIBRIN(OGEN) DEGRADATION BY PLASMIN
V. PATHOLOGIC FIBRINOLYSIS
• After degradation of fibrinogen or fibrin by plasmin,
1. Primary Fibrinolysis
fragments produced called FIBRIN(OGEN)
• Excessive amounts of plasminogen activators
DEGRADATION PRODUCTS (FDP) or FIBRIN(OGEN)
from damaged cells/malignant cells
SPLIT PRODUCTS (FSP).
• Converts plasminogen to plasmin in the
• These degradation products are removed by the
absence of fibrin formation
reticuloendothelial system and other organs.
• No formation of fibrin polymer, D-Dimer, and
fibrin monomer.
DIVINA GRACIA A. MARGES | BSMT 3-3 1
 LESSON 6: Fibrinogen, Fibrinolysis and D-Dimer Test

2. Secondary Fibrinolysis 2. EUGLOBULIN LYSIS TIME NOTE!

• DIC: Uncontrolled, inappropriate formation of • Screening procedure for the measurement of • Manual Detection of Fibrin Clot formation = Tilt
fibrin within the blood vessels fibrinolytic activity. tube or Wire loop
• Formation of fibrin polymer, D-Dimer, and fibrin • Euglobulin are proteins that precipitate when plasma • Visible Detection of Fibrin Clot formation = Tilt
monomer is diluted with water and acidified. tube
✓ Most specific test for DIC: formation of D-Dimer. • Include plasminogen, plasmin, fibrinogen, and • Lipemic sample must be rejected for the
✓ The initial laboratory profile in DIC includes a platelet plasminogen activators. Laboratory Evaluation of Fibrinolysis
count, blood film examination, PT, PTT, D-dimer, and
• Normally, clot lysis does not occur in less than 1 hr. • Use electromechanical instrument for lipemic
fibrinogen assay. samples.
• Clot lysis in less than 1 hr can be a sign of abnormal
✓ Two types of Disseminated Intravascular Coagulation:
fibrinolytic activity.
• Acute DIC / Uncompensated DIC – all
laboratory tests are ABNORMAL REFERENCES
3. PROTAMINE SULFATE TEST
o Prolonged PT, APTT, TT Notes from the discussion of Mr. Raffy C. Lacorte, RMT
o Decreased Fibrinogen (<100 mg/dL) • Detects the presence of fibrin monomer in plasma
Cavite State University powerpoint presentation: Lesson 6
o (+) D-Dimer Test • Formation of fibrin strands or gel-like clots
– Fibrinogen, Fibrinolysis and D-Dimer Test
• Chronic DIC / Compensated DIC – all laboratory (Paracoagulation).
Clinical Hematology: Principles, Procedures, Correlations E.
tests are NORMAL, except D-Dimer Test • Normally, there should be no fibrin monomers
Anne Stiene-Martin, Cheryl A. Lotspeich-
o (+) D-Dimer Test present in plasma.
Steininger,John A. Koepke
NOTE! • Test specific for DIC or secondary fibrinolysis
Rodak&#39;s Hematology: Clinical Principles and
formation of D-Dimer – most specific test for DIC Applications 6th Edition by Elaine Keohane;
4. ETHANOL GELATIN TEST Catherine N. Otto; Jeanine Walenga Hematology: Principles
Table No. 1 Anticipated Results of Disseminated • Detects fibrin monomers in the plasma and Procedures by Barbara A. Brown
Intravascular Coagulation (DIC) Primary Laboratory Profile • Screening procedure to be utilized as an aid in the
TEST REFERENCE VALUE IN DIC diagnosis of DIC
INTERVAL • Procedure:
Platelet count 150,000- <150,000/uL o Obtain plasma. Add NaOH and ethanol.
450,000/uL o Detects: precipitates or gel-like clots
Prothrombin 11-14 secs >14 secs
Time 5. LATEX D-DIMER ASSAY
Partial 25-35 secs >35 secs
• Presence of cross-linked D-Dimer indicates stable
Thromboplastin
fibrin clot has been lysed and will be found in
Time
pulmonary embolism, deep vein thrombosis, DIC with
D-dimer 0-240 >240 ng/mL, often
secondary fibrinolysis and sickle cell disease.
ng/mL 10,000 to 20,000
• D-dimer test is positive in DIC as soon as 4 hours after
ng/mL
onset. Fibrinogen levels may decrease in 4 to 24 hrs.;
Fibrinogen 220-498 <220 mg/dL, often
platelets decrease up to 48 hrs. after onset.
mg/dL higher, because
fibrinogen is an acute
Table No. 2 Differentiation of Tests
phase reactant
TEST PRIMARY SECONDARY
FIBRINOLYSIS FIBRINOLYSIS
VI. LABORATORY EVALUATION OF FIBRINOLYSIS WBCLT <48 hours <48 hours
1. WHOLE BLOOD CLOT LYSIS TIME (WBCLT) ELT <1 hour <1 hour
• Test for increased fibrinolysis PST NEGATIVE POSITIVE
• Clot should remain intact for approximately 48 hrs at EGT NEGATIVE POSITIVE
37oC LDA NEGATIVE POSITIVE
• Clot lysis prior to 48 hrs is indicative of excessive
systemic fibrinolysis.

DIVINA GRACIA A. MARGES | BSMT 3-3 2

 LESSON 6: Fibrinogen, Fibrinolysis and D-Dimer Test

Exogenous Plasminogen Activators (Therapeutic Activators)

• Treatment of thrombosis (ex. Streptokinase, Urokinase, Tissue like PA)

DIVINA GRACIA A. MARGES | BSMT 3-3 3

 LESSON 6: Fibrinogen, Fibrinolysis and D-Dimer Test

The one being measured in D-Dimer

DIVINA GRACIA A. MARGES | BSMT 3-3 4

 Supplementary Notes: Direct and Indirect Platelet Count, and Platelet Disorders

OUTLINE PLATELET COUNT INDIRECT METHODS • Count the platelets in the 4 large corner squares (as in
I Platelet Count DAMESHEK WBC count)
A. Platelet Count Indirect Methods
i Dameshek • Prick the puncture site. Gently, place a drop of diluting
ii Fonio’s fluid over the puncture wound and gently press the GUY AND LEAKE’S
iii Olef’s finger so that a small amount of blood wells up into • Diluting fluid is composed of: crystal violet, sodium
B. Platelet Count Direct Methods (Light Microscopy) the drop of diluting fluid. The proportion of blood to citrate, distilled water formalin (40%)
i Rees and Ecker’s the diluting fluid should be 1:5. • Suck diluting fluid to mark 1 of RBC pipet
ii Guy and Leake’s
• Transfer the mixture into a slide and cover it with a • Suck blood to .5 mark and followed by diluting fluid
C. Platelet Count Direct Methods (Phase Microscopy)
i Brecker-Cronkite coverslip again up to 101 mark
ii Unopette • Allow the platelets for 15-45mins. • Shake the pipet for 3-5mins., charge and then let
iii Tocantin’s • Count the RBCs and platelets under OIO until 250 stand for at least 3mins.
iv Nygard’s RBCs have been counted • Count platelets in the intermediate squares in the
v Walker and Sweeney’s central large square for RBC count
vi Van Allen’s
FONIO’S
II Platelet Count Estimation
III Disorders of the Primary Hemostasis • Place a large drop of diluting fluid on the finger to be PLATELET COUNT DIRECT METHODS ((PHASE
A. Vascular Disorders pricked then puncture. Then press the blood from the MICROSCOPY)
IV Platelet Disorders puncture site. BRECKER-CRONKITE
A. Thrombocytopenia •
B. Thrombocytosis
Mix one part blood and 5 parts magnesium sulfate • Diluting fluid used: 1% ammonium oxalate
• Make a smear using the mixture • Procedure: Same with Rees and Ecker except that
C. Defect in Platelet Adhesion
D. Defect in Platelet Secretion • Allow the smear to dry, then apply staining fluid. platelets are counted with the use of phase contrast
E. Defect in Platelet Aggregation • Examine under OIO and count the RBCs and platelet microscope
until 1000 RBCs are counted
PLATELET COUNT UNOPETTE
• Reasons why platelets are hard to count: OLEF’S
o Platelets adhere to foreign surfaces (like skin • Best indirect method TOCANTIN’S
and dried walls of pipets)
o Platelets easily disintegrate PLATELET COUNT DIRECT METHODS (LIGHT NYGARD’S
o They are hard to differentiate from debris MICROSCOPY)
o Platelets are unevenly distributed in the blood REES AND ECKER’S
because they tend to clump WALKER AND SWEENEY’S
• Diluting fluid is composed of: Briliant cresyl blue,
• Significant bleeding usually does not occur until the
sodium citrate, distilled water
platelet count is less than 60 VAN ALLEN’S
• Rinse RBC pipet first with rees and ecker’s fluid by
• Indirect methods: platelets are counted in relation to • Reported as %
sucking in and out of the pipet
1000 RBCs in the blood smear (not reliable)
• Suck blood to .5 mark of RBC pipet
• Direct Methods: whole blood is diluted with platelet PLATELET COUNT ESTIMATION
• Suck diluting fluid to 101 mark
diluting fluid in an RBC pipet and counted in a
• Shake the pipet for 3-5mins • To determine approximate number if platelets per
hemacytometer
• Discard first few drops then charge the counting field, examine the thin area of the slide using OIO
chambers and let it stand for 3 mins.

DIVINA GRACIA A. MARGES | BSMT 3-3 1

 Supplementary Notes: Direct and Indirect Platelet Count, and Platelet Disorders

• A normal blood smear should demonstrate • Characterized by long THROMBOCYTOPENIA

approximately 8 to 20 platelets per field extremities and
• Estimates may be reported using the following table: arachnodactyly (spider GROUPS ASSOCIATED CONDITIONS
fingers) Decreased • Fanconi Syndrome
Table No. 1 Senile purpura • Disease of old people platelet • TAR syndrome
ESTIMATED PLATELET REPORTED AS: • Bruised area in the forearms of production • Viral Infections (dengue)
COUNT the elderly • Leukemias
0 - 49,000 /uL Marked decrease • patients due to the • May–Hegglin anomaly
50,000 - 99,000 /uL Moderate decrease degeneration of collagen • Megaloblastic anemia
100,000 - 149,000/ uL Slight decrease Henoch – • Allergic purpura • WAS (Wiskott–Aldrich)
150,000 – 199,000 /uL Low normal Schonlein • Non thrombocytopenic syndrome
200,00 – 400,000 /uL Normal Purpura purpura Decreased • Idiopathic thrombocytopenic
401,000 – 599,000 /uL Slight increase • Due to immunologic damage platelet survival purpura
600,000 – 800,000 / uL Moderate increase to the endothelial cells • Thnrombotic
Above 800,000 /uL Marked increase • Characterized by: GI bleeding thrombocytopenic purpura
and joint swelling • Hemolytic Uremic Syndrome
DISORDERS OF THE PRIMARY HEMOSTASIS • Most commonly seen in • DIC
children Increased platelet • Gaucher disease
GENERAL LABORATORY RESULT Purpura • Results from streptococcal sequestration • Myelofibrosis
Normal results • Platelet count Fulminans infection Dilutional • Multiple transfusions
• Platelet function test • Characterized by: hemorrhagic thrombocytopenia
• Coagulation test manifestation
Abnormal result • Bleeding time Waterhouse – • Characterized by: severe THROMBOCYTOSIS
• Rumple – Leede’s test Friedrichsen capillary damage following
Syndrome meningococcal specimen GROUPS ASSOCIATED CONDITIONS
VASCULAR DISORDERS Kasabach – • Congenital hemagioma Reactive thrombocytosis • Recovery from
Meritt • Characterized by tumors *Moderate increase in splenectomy
Syndrome composed of blood vessels
DISORDER DESCRIPTION platelet counts • Acute blood loss
that swell and bleed at the •
Hereditary • Osler – Weber Rendu Disease Major surgery
surface
Hemorrhagic • Inherited as an autosomal Autonomous • Essential
Telanagectasia dominant trait
Scurvy • Ascorbic acid deficiency thrombocytosis thrombocytemia
• Most common inherited • Characterized by defective *Markedly increase • Polycythemia vera
synthesis of collagen and
vascular bleeding disorder • Chronic
hyaluronic acid
• Characterized by: chronic myelogenous
dilataion of the walls of leukemia
capillaries (skin and mucous PLATELET DISORDERS • Myeloid metaplasia
membrane) • Quantitative Platelet Disorders Increased platelet • Gaucher disease
• o Thrombocytopenia – decreased count
Ehlers – Danlos Cutis – hyperelastica sequestration • Myelofibrosis
Syndrome • Inherited as autosomal o Thrombocytosis – Increased count
dominant trait • Qualitative Platelet Disorders
DEFECT IN PLATELET ADHESION
• Characterized by hyper o Defect in platelet adhesion
extensible joints and o Defect in platelet secretion
DISORDER DESCRIPTION
hypersplenic skin o Defect in Platelet aggregation
Bernard Soulier Syndrome • Deficiency in GpIb
Marfan’s • Inherited as autosomal Von Willebrand disease • Deficiency in vWF
Syndrome dominant trait

DIVINA GRACIA A. MARGES | BSMT 3-3 2

 Supplementary Notes: Direct and Indirect Platelet Count, and Platelet Disorders

DEFECT IN PLATELET SECRETION

DISORDER DESCRIPTION
Gray Platelet • Absence in alpha granules
syndrome contents
• Platelets are light gray and large
Storage pool • Decrease dense granules contents
disorder • May be considered as an isolated
problem or in association to:
o Hermansku-pudlack –
characterized by albinism
o Chediak–Higashi –
characterized by albinism
o WAS – characterized by
severe eczema and small
platelets

DEFECT IN PLATELET AGGREGATION

DISORDER DESCRIPTION
Glanzmann’s • Characterized by prolonged
thrombosthemia bleeding time
• Abnormal clot retraction
Aspirin Intake -

REFERENCES
Cavite State University powerpoint presentation:
Supplementary Notes – Direct and Indirect Platelet
Count, and Platelet Disorders

DIVINA GRACIA A. MARGES | BSMT 3-3 3