SECONDARY HEMOSTASIS
- Formation of irreversible clot (Fibrin)
- Coagulable cascade: Coagulation Pathway and
Coagulation Factors
- Coagulation pathways and Activators:
a. Extrinsic: Activated by tissue thromboplastin
b. Intrinsic: Activated by Collagen
c. Common: both intrinsic and extrinsic by
formation of prothrombinase complex
Coagulation pathway
Coagulation factors:
- Synthesize in Liver except III and IV
- Circulating on its inactive/ zymogen form except III
and IV
- All coagulation factor including PK and HMWK, active
forms are in Serine Proteases except:
a. Factor 1 (Fibrinogen): main substrate
b. Factor V and VIII: as cofactors
c. Factor XIII (Transglutaminase/Transaminase): for
fibrin plug stabilization
 COAGULATION FACTORS

NUMERAL PREFERRED NAME SYNONYMS GENETIC DISORDERS AND OTHER

INFORMATION

I Fibrinogen - Fibrinogen - Congenital Afibrinogenemia/

Hypofibrinogenemia/
Dysfribrinogenemia
- Familial renal Amyloidosis
- NV: 200-400 mg/dL
- Inc PT & APTT: <80 mg/dL

II Prothrombin - Prothrombin - Prothrombin deficiency

- Thrombophilia

III Tissue factor - Tse Thromboplastin - Present in tissues and are insoluble
IV Calcium
V Proaccelerin - Labile Factor - Owrensen’s disease
- Accelerator globulin (aCg) - Leiden dse
- Parahemophilia
- Activated protein C Resistant

VII Proconvertin - Stable factor - Congenital Factor VII deficiency

- SPCA (Serum Prothrombin - Hemopilia
Conversion accelerator) - 1st to be affected by Warfarin
- Autoprothrombin I

VIII Antihemophilic Factor - Antihemophilic globulin - Hemophilia A

- Antihemophilic Factor A - Classic Hemophilia
- Platelet Cofactor 1 - Royals Disease
- VWF: largest and heaviest mol in
plasma

IX Plasma Thromboplastin - Christmas factor - Hemophilia B

Component - Antihemophilic Factor B - Christmas Factor Dse
- Platelet Cofactor 2

X Stuart-Prower Factor - Stuart Factor - Congenital Factor X

- Prower Factor - Stuart Power disease
- Autoprothrombin III

XI Plasma Thromboplastin - Anti-hemophilic Factor C - Hemophilia C

Antecedent - Rosenthal syndrome
- 50% hemophilia seen in Askenazi
Jews
XII Hageman Factor - Glass factor - Hereditary angioedema type II
- Contact Factor - NO BLEEDING TENDANCY

XIII Fibrin-Stabilizing Factor - Laki-Lorand Factor - Congenital Factor XIII deficiency

- Fibrinase - Associated with POOR HEALING
- Plasma Transglutase/
Tranglutaminase
- Fibrinoligase

Prekalikrein (PK) - Fletcher factor - Kininogen deficiency

High Molecular Weight - Fitzgerald Factor - Kininogen deficiency
Kininogen - Contact Activation
cofactor
- William’s factor
- Flaujeac factor
 FACTOR VIII SUBTYPES
- Circulates in blood bound to Vwf

1. Factor VIII/Factor VIIIC/ Factor VIII:C 4. Factor VIII: vWF

- Refers to procoagulant portion - termed as Von Willebrand Factor
- Measured by standard factor VIII assays and APTT required for normal platelet adhesion
- Markedly decreased in hemophilia A
5. Factor VIII: Ag 5. vWF: Ag (Previously: Factor VIIIR: Ag)
- Refers to antigenic properties - antigenic portion of the Von Willebrand Factor
- Measured by immunoassays measured by immunoassays
3. Factor VIIIR:RCo (Restocetin Cofactor)
- refers to portion responsible for platelet aggregation
in the presence of restocetin

GROUP OF CLOTTING FACTORS

A. ACCORDING TO THE COAGULATION PATHWAY

Intrinsic Coagulation Pathway Extrinsic coagulation Pathway Common Coagulation pathway
8 9 11 12 37 2 5 10

B. ACCORDING TO PROPERTIES
Fibrinogen Group Prothrombin Group Contact group
1 5 8 13 2 7 9 10 11 12 PK HMWK
 Vitamin K independent  Vitamin K dependent  Vitamin K independent
 Calcium Dependent  Calcium Dependent  Calcium Dependent
- Absent in serum (consumed - Present in serum except factor 2 - Present in serum
during coagulation) - Most stable factors - Moderately stable
- Most labile group and largest - Gamma Carboxylation of - Activated by Negatively
group because of VIII Glutamic Acid Charged Surface
- Activated by Thrombin (Thrombin - Adsorbed From plasma using the  In vivo: Collagen and
Sensitive group) following: Endothelium
- Increase in Pregnancy,  Barium Sulfate  In vitro: Kaolin Glass tube
Inflammation, Stress,  Aluminum Hydroxide
Contraceptive - Inhibited By Oral Contraceptive
(Warfarin and Coumadin)

OTHERS
Extrinsic Tenase Tissue factor, Factor VII, Platelet Phospholipids, Calcium
Intrinsic Tenase Xa, VIIIa, PL, Ca
Prothrombinae group Xa, Va, PL, Ca
Labile Factor 5, 8
Cold temperate Factors 7, 11

REES ECKER COUNTING

Improved Neubauer Counting Chamber

- Has two ID sides and both sies are counted - Manual WBC count: 4 corners (Large squares: 1mm 2)-subdivided
- Chamber is 3x3 mm (divided into 9 square into 16 squares (0.0025mm 2)
mm) - Manual RBC count: Middle central square-subdivided into 25
- Depth: 0.1mm squares (0.04mm2)
- Depth factor: 1/10
Pipets
RBC THOMA PIPET WBC THOMA PIPET

Color of beads 0.5,1,101 0.5, 1, 11

Markings RED WHITE
Volume (bulb) 100 10
DF: total volume/sample volume
Alternative: Plain microhematocrit tube (Blue)= 20:10
 Cells counted Diluting fluid
WBC - 1% ammonium oxalate Dilution objective Area counted
- 3% acetic acid (weak acid) 1:20 LPO 10x 4mm2 (4 large squares)
- 1% Hydrochloric acid 1:100 LPO 10x 9mm2 (9 large squares)
- Turks sol (3% Gentian Violet)/ Note:
Methylene blue - When WBC count <30/uL use Plt dilution factor
1:100 counted at the center large squares in 40x
HPO
- NRBC in sample are not lysed by WBC DF and ay be
counted as WBCs and caused false Inc count and
Must be corrected

Platelet 1% ammonium oxalate Dillution Objective Area counted

1:100 HPO 40x 1mm2 1 large squares)

RBCs (rarely performed) - Hayems solution Dillution Objective Area counted

- Gowers 1:100 HPO 40x 0.2mm (0.04mm2)
- Teigison’s fluid (5 small squares a the central
- Bethel’s large squares)
- Dacies (Formol Citrate)
- 0.85% NaCl
- 3.8% Sodium Citrate

REES ECKER DILUTING FLUID COMPONENTS

- BRILLIANT CRESYL BLUE
- SODIUM CITRATE
- DISTILLED WATER
- 1% AMMONIUM OXALATE

Methods
1. By automated hematology analyzers.
2. Direct smear also gives information about platelets’ size, shape, and clumping.
3. Direct count from the peripheral blood smears.
- Count platelets on oil objective in 10 fields and multiply by 2000, giving a rough idea about the count.
 Platelets in 10 field X 2000 = Total platelets.

The manual method of platelets count:

1. Take 20 µL of blood.
2. Add 1.8 mL of 1% ammonium oxalate.
 Ammonium oxalate will lyse the RBCs and WBCs while Platelets will remain intact.
3. Leave for 15 minutes for complete lysis of RBCs.
4. Mount the Neubauer chamber.
5. Leave the chamber for 15 minutes in the high humidity.
6. Count the large central square labeled as P.

FORMULA
- Original:

No cells/uL= cells counted x Dilution Factor

Total area (mm2) x depth (0.1)

- Semi shortcut:

RBC = Cell counted x 10x DF x No of square counted

WBC = cell counted x 10x DF
No of square counted

PLT (x 10^9/L) = average number of plts x 100 x 1 mm2/0.1 mm x 10^6

Ex. When a 1:20 dilution is used the four large squares on side yields count 21, 34, 10, 28, 29. On the other side yielded
11, 23,24,29, 30. What is the leucocyte count?
 EVALUATION OF SECONDARY HEMOSTASIS

Error in both pathway Error in Extrinsic Pathway Error in Intrinsic pathway Specimen consideration and
processing
Problem in Problem in Problem in - Sample for Coagulation: PPP
Common pathway Intrinsic Pathway Extrinsic pathway (Platelet Poor Plasma)
 10-15x10^9/L
 2000-2500rpm for 15 mins
- Coagulation sample must be
processed w/in 1 hr at RT 18-
24hrs
 PT: 24 hrs
 APTT: 4 hrs

COMMON TESTS FOR EVALUATION OF SECONDARY HEMOSTASIS

1. APTT (Activated Partial Thromboplastin Time) 2. PT (Prothrombin time)

- to monitor INTRINSIC and COMMON pathway - To monitor EXTRINSIC AND COMMON pathway
- to monitor WARFARIN (COUMADIN) therapy - To monitor HEPARIN therapy
- affected by circulating Anticoagulant
NV: 35-45 seconds NV:10-14 seconds
Reagents: Reagents:
- Platelet substitute (Phospholipids): to substitute for PF3 - Thromboplastin-0.025M CaCl2 or SIMPLASTIN
- Negative Charge Activator (To activate Contact Materials:
pathway): - Controls
 Kaolin, Ellargic acid, Celites, Silica - Test tubes (12x75 mm)
- 0.025M CaCl2 - Pipets
Materials: - 27C water bath
- Controls
- Test tubes (12x75 mm)
- Pipets
- 27C water bath
Procedure: Procedure:
- 0.1 mL of PPP is added to 0.1 mL of APTT reagent - Aliquot of plasma and control are warmed
- Mixture is incubated at 37C according to the method being used
- After incubation 0.1 mL of warmed CaCl2 is added - Warm PT reagent by incubating at 37C for 3-5 mins
- Start the time after addition of CaCl2 - 0.2 mL of PT reagent is added to 0.1 mL of plasma (px
- Time for clotting to occur is then recorded or control)
- Start time after addition of simplastin
- Time for clotting to occur is then recorded.
Reporting: APTT is reported in seconds to the nearest tenth Reporting: can be in several ways
along with the reference value 1. Px time w/ w/ the control time ( in seconds)
2. Px time (in secs) w/ the reference Range
3. Prothrombin ratio: Px PT time x100
Mean of the reference Range
4. INR (International Normalized Ratio)

INR (INTERNATIONAL NORMALIZED RATIO)

- standardized way of reporting PT ratio of Px under Oral ISI (International Sensitivity Index)
PT Anticoagulants - perform to determine the
- introduced to minimize the - NV: 1 sensitivity of reagent
differences in PT results due to - 1-2.5: under warfarin  <1: more sensitive
different reagent-instrument  >1: less sensitive
combinations
- formula: (Px PT time/ Mean of
RV)ISI
- ISI (International Sensitivity
Index)
- INR of 2-3 must be achieved

THERAPEUTIC ANTICOAGULANTS
HEPARIN WARFARIN
- Most commonly used Intravenous anticoagulant - Most commonly used Oral anticoagulant
- Inhibits thrombin - Brandname: coumadin
- Incase of Overdose Administer: Protamine sulfate - Inhibits: 2,7,9,10 synthesis
- Incase of Overdose administer:
• Vit K supplement • Fresh Frozen Plasma
• Coagulation Factor Concentrates
 ELECTRICAL IMPEDENCE OPTICAL
- Data Clot 2 - Coag-A-Mate X2 and Coag-A-Mate-2001
- Fibrinometer - Electra 700 and 750
- Koagulab 40-A

COAGULATION INSTRUMENTATION AND PRINCIPLE UTILIZED

3. STYPVEN TIME (RUSSEL’S VIPER VENOM TIME) 4. DILUTE RUSSEL’S VIPER VENOM TIME (dRVVT)
- Detection of Common Pathway deficiencies - for detection of LUPUS ANTICOAGULANT
(1,2,5,10)
- For differentiation of Factor X and VII def NV: 30-35secs
- Uses coagulant properties of Russel’s viper venom
from snake Vipera Russel
NV: 20-25 secs
5. THROMBIN TIME (affected w/ heparin circulating 6. REPTILASE TIME (unaffected with heparin, circulating
anticoagulants and Antithrombins) anticoagulants and Antithrombins)
- Inc TT= - Uses Reptilase enzyme (from snake Bothrops atrox)
 Dec Fibrinogen (I) and Impaired Fibrinogen capable of converting Fibrinogen to Fibrin.
function
 Heparin inhibition/Presence (sensitive) NV: 10-15 secs
 Fibrin(ogen) Degradation Product (FDP) TT Reptilase time
 Streptokinase Hypofibrinogenemia Inc Inc
Immunologic Inc Normal
NV: 15-18 secs antithrombins
Heparin therapy Inc Normal

1. CLOTTING TIME
A. Slide/Drop Method
- Perform skin Puncture and wipe first drop of blood
- Start timer as soon as the second drop of blood
appears
- Transfer 2nd drop of blood at the center of slide
- Pass the tip of needle thru the drop of blood every
30 secs and not for the formation of fibrin strands
- Stop time as soon as fibrin strands are seen
B. Lee and White/ Whole Blood Clotting Method
- Place 3 test tube in 37C water bath
- Place 1mL blood into each 3 tubes
- Start time as soon as blood enters tube 1
- Gently tilt tube 3 every 30 seconds and observe for
clotting
- After clotting is observe in tube 3, observe tube 2
then tube 1
- when no flowing of blood is observed upon lifting
tube 1 stop timer
- clotting time is the time It takes for blood to
coagulate in tube 1
- NV: 7-15 mins

2. DUCKER’S TEST/ 3 M UREA TEST/ 1% MONOCHLOROACETIC ACID

- for detection of Factor XIII deficiency
- Fibrin stabilized by XIII are not soluble in 5M urea
- Normal: if clot (sufficient factor XIII)
- Abnormal: if No Clot (Factor XIII deficiency)