UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

HEMOSTASIS AND THROMBOSIS

OVERVIEW

 Circulatory hemostasis (arresting of bleeding) is achieved through the process of balancing bleeding (hemorrhage)
and clotting (thrombosis)
 Major components
o Vascular System
o Thrombocytes
o Coagulation Factors
o Fibrinolysis and Tissue Repair
 Minor Components
o Complement System
o Kinin System
o Serine Protease inhibitors
 Process
o Blood Vessel Spasm
o Platelet Plug Formation
o Contact among damaged BV, blood platelet, and coagulation proteins
o Formation of blod clot on site of injury
o Fibrinolysis

BLOOD VASCULATURE

 Vessel Wall is divided into three coats or tunics

o Tunica intima
 Forms the glistening surface of endothelium that lines the lumen of blood and lymphatic vessels
and the heart
 Single layer of endothelial cells (simple squamous epithelium) thickened by subendothelial
connective tissue layer containing elastic fibers
o Tunica media
 Thickest
 Composed of smooth muscle and elastic fibers
o Tunica adventitia
 Fibrous connective tissue that contains autonomic nerve endings and the vasa vasorum (small
networks of blood vessels that supply nutrients to tissues of the wall
 Arteries and Veins
o Arteries
 Have thickest wall
o Veins
 Irregular lumen
 Larger
 Thin walled with weaker middle coat
 Fewer nerves
 Arterioles and venules
o Arterioles – microscopic continuations of arteries that give off branches called metartioles which in turn join
the capillaries
o Venules – microscopically sized veins that connect the capillaries to veins
 Capillaries
o Constitute the major vessels of microcirculation along with arterioles and venules
 Physiology
o Vasoconstriction
 Short-lived reflex reaction of the smooth muscle in the vessel produced by sympathetic branches of
the ANS
 Narrowing/stenosis of blood vessel to decrease flow of blood in injured smaller vessels and
surrounding vascular bed
 May be sufficient to close severed capillaries
o Endothelium
 Contains collagen and elastin which regulates
permeability of inner vessel wall and provides
principal stimulus for thrombosis after an injury
 Collagen initiates contact activation of
factor XII
 Rich with plasminogen activator
 Plasminogen – ensures rapid lysis of
fibrin clots
 Prostacyclin – inhibits platelet aggregation and
adhesion
 Weibel-Palade body – organelle that stores vWF

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

THROMBOPOIESIS

Megakaryoblast

Promegakaryocyte

Megakaryocyte

Thrombocyte

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

PRIMARY HEMOSTASIS : PLATELET FUNCTION

 Passive Surveillance
o Monitor vessel lining for small holes or gaps, platelets plug holes without activation of coagulation system
 Formation of primary hemostatic plug
 Provides phospholipid surface for secondary hemostasis
 Promotion of healing by stimulation of smooth muscle cells and fibroblasts
 PLATELET ADHESION
o Major interaction is the binding of platelet receptor glycoprotein Ib (GPIb)/IX to vWF, which binds to
collagen
o vWF: Stored in a-granules in platelets and Weibel-Palade bodies in endothelial cells
o Important step that triggers several events leading to platelet activation
 PLATELET ACTIVATION
o Results to shape change, alteration of orientation of phospholipids, expression of new receptors and
changes in biochemistry
 appearance of pseudopods
 will convert to original shape if stimulus is not sufficient
 Receptor expression:GPIb/IXon surface, increase in number of GPIIb/IIIa receptors on surface
 Microtubules, microfilaments, and intermediate filaments reorganize so that organelles are centrally
located in the activated platelet
o May also result from exposure to agonists
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Collagen and Thrombin – strong agonists

 ADP and Epinephrine – weak agonists
 Requires presence of TXA2 and platelet aggregation
 Thromboxane 2
 Synthesized from arachidonic acid by cyclooxygenase and thromboxane synthase
 stimulates platelet granule secretion, enhances vasoconstriction;
 if blocked, secretion is impaired; aspirin blocks cyclooxygenase
 Arachidonic acid
 PLATELET SECRETION
o Requires adenosine triphosphate (ATP); open canalicular system fuses with granular membrane, and
contents of a-granules and dense bodies are released to the outside of the platelet
 PLATELET AGGREGATION
o vWF binding to GPIb/IX activates an intracellular signaling pathway that results in the activation of
GPIIb/IIIa, which then binds to fibrinogen
o Fibrinogen forms bridges to other GPIIb/IIIa receptors on other activated platelets, resulting in platelet
aggregates; Ca2+ is needed for aggregation
o Fibrinogen and Ca2+ are delivered locally from granules and dense tubular system
o Primary: Loose aggregation, reversible if stimulus is not sufficient
o Secondary: Irreversible provided sufficient stimulus; occurs after internal ADP release, TXA2 synthesis and
release, further stimulation then occurs

SECONDARY HEMOSTASIS : COAGULATION FACTORS

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

COAGULATION CASCADE

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

HEMOSTASIS LABORATORY ASSESSMENT

COAGULATION PROTOCOLS AND PROCEDURES

 Replication Analysis is frequently performed but it does not enhance accuracy nor precision.
 Use of Sodium Citrate anticoagulant
o 3.2% Sodium Citrate – choice
 Closer osmolality to plasma
o 9:1 ratio of sample to anticoagulant
o Excess citrate binds the calcium subsequently added in the testing procedure. If the citrate is not adjusted
for blood collection, the sample will produce falsely prolonged clottingtimes because of inadequate
recalcification
 If single sample is to be withdrawn using evacuated tube, small amount of blood be allowed to enter plastic needle
holder before collecting blood to ensure enough blood volume (vaccum from tube might be consumed if air enters)
 Nonwettable plastic evacuated tubes should be used to prevent activation of Factors XII and VII by glass
 If using syringe in withdrawing blood, two barrels must be used.
o Acquire 1 mL on first, then remove barrel and connect 2nd barrel
 Once a sample is in vitro
o Factors V and VIII deteriorate quickly
o PTT must be done within 24 hrs
o APTT must be completed within 4 hrs
o Hold sample only for a maximum of 2 hrs at 4 degrees Celsius
o If testing can not be conducted asap
 Seprate plasma from cells
 Centrifuge 20 mins at 2500 rpm
 Store plasma at -20 degrees celsius for 2months or at -70 degrees celsius for 6 months
 Samples in coagulation studies
o Platelet Poor Plasma
 Citrated whole blood is centrifuged for 15 mins at 2500 g
 < 10 x 109/L Platelets
 Essential for technical reasons
 platelets contain platelet factor 4 (PF4), which neutralizes heparin (thus affecting sample’s
testing for the presence of heparin)
 platelets contain phospholipids, which affect lupus anticoagulant testing and factor assay
testing (especially if the sample is frozen and thawed).
 platelets contain proteases, which, when released during the thawing of a frozen sample,
alter results for von Willebrand factor testing
 can be stored at 18–24°C or 2–8°C for up to 4 hours before testing.
 If the testing cannot be completed within the 4 hours, the PPP should be stored at -20degC for up
to 1 week or -70degCfor up to 6 months.
 Frozen samples must be thawed rapidly at 37°C because excessive heating ( > 5 minutes) can
result in the loss of factor V and F-VIII.
o Platelet-Rich Plasma
 Obtained to perform platelet function studies
 Citrated blood on polystrene tube centrifuged for 10 minutes at 200 g at room temperature
 Contains 200-300 x 109 / L platelet
 can be stored at room temperature, and testing should be completed within 3 hours.
 Plasma Adsorption
o Reagent used in classical mixing studies (along with aged serum and Factor VIII/IX defiecient plasma)
o Mixing Studies are done to identify possibility of factor deficiency in samples with prolonged PT/aPTT
 Correction of PT/aPTT strongly suggests a deficiency of a coagulation factor
 Nowadays, mixing studies only use plasma to determine whether or not factor defieciency exists
o Uses Barium Sulfate or Aluminum Hydroxide
o Adsorbed plasma contains (1, 5, 8, 11, 12)
 Factors I, V, VIII, XI, and XII
 Aged Serum
o Also used in claassical mixing studies
o Clotted blood mixed with sodium citrate
o Before use dilute with 0.85 NSS (1 part serum to 4 parts NSS)
o Aged serum contains (7, 9, 10, 11, 12)
 Factors VII, IX, X, XI, and XII

ASSESSMENT OF PRIMARY HEMOSTASIS

1. Bleeding Time
 Measures ability of small blood vessels to control free flow of blood after injury
 Measures the function of platelets and integrity of blood vessel walls
 In vivo measurement of platelet aggregation and adhesion on locally injured vascular endothelium
 Prolonged in THROMBOCYTOPENIA, von Willebrand disease, and aspirin ingestion
 General Reference Range is 1-9 minutes
 Methods
o Duke’s Method
 Pricking of earlobe/3rd/4th Finger
 Blot every 30 seconds
 Reference Range : 2 to 4 minutes

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

o Ivy’s Method
 Application of sphygmomanometer cuff inflated at 40 mmHg on upper arm
 Prick volar surface of forearm on three sites adjacent to each other (2-3 mm depth)
 Blot every 30 seconds
 Reference range : 1-7 minutes
o Copley-Lalitch Method
 Prick fingertips 6 mm depth
 Immerse pricked finger on NSS warmed at 37 degrees celsius until bleeding stops
 Normal Value : < 3 minutes
2. Platelet Aggregation Studies
 Measures ability of platelets to aggregate in the presence of agonists
 Measurement uses photooptical instrument (turbidimeteric aggregometer)
 Aggregation is measured by a decrease in optical density after addition of an agonist is added to a platelet
rich plasma
o Monophasic curve – ADP (used as doubled dose) Ristocetin, Arachidonic acid
o Biphasic Curve – Epinephrine (double dose)
 Patients must refrain from aspirin containing products for 7-10 days before testing
 Specimen must be tested within 4 hours
 Lumiaggregation – extension of aggregation
o Measures ATP from dense granules in PRP/WB via luminiscence

3. Clot Retraction Time

 Directly proportional to platelet count and quality
 Inversely proportional to hematocrit and fibrinogen levels
 Abnormal if platelet count is <100 000/mm3
 Decreased on Anemia and Hypofibrinogenemia
 Rapid dissolution of clot is seen on cases of DIC and Increased Fibrinolysis
 No Clot Retraction on Glanzmann Thrombasthemia
 Methods
o Hirshboeck Method / Castor Oil method
 Endpoint : Extrusion
 Range : 15-45 minutes
o Stefanini / Test Tube Method
 3-5 mL of venous blood
 Wasserman tube with cotton plug
 Water bath 37 deg C
 Observe 1 hr-2hrs-16hrs-18hts-24hts
 Expected Result
 Starts in 1 hr and ends within 18-24 hrs
o MacFarlane Method
 Uses calibrated centrifuge tubes and 5 mL venous blood
 Place a glass rod into tube then Water bath 37 deg C
 Observe 5-10 mins interval
 Expected Result
 Clot will stick to the glass rod
 Read Volume of serum using the graduations
 CRT % = vol serum / vol blood used x 100

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Normal : 44% to 67%

ASSESSMENT OF SECONDARY HEMOSTASIS

SCREENING TESTS

detect a factor deficiency only when the factor decreases to a level of 25–40% of the normal

1. Prothrombin Time (PT)

 Evaluates coagulation factors of the extrinsic pathway and the common pathway
 Thromboplastin is added to citrated PPP or control PPP
o Commercially prepared (usual source is rabbit brain)
o Stable for 7 days (2-8 deg C) or 24 hrs at RT or 37 deg C
 General Reference Interval : 10 to 13 seconds
 Use for monitorng oral anticoagulant therapy
 Prolonged in
o Deficiency of Factors I, II, V, VII, X
o Presence of inhibitor
 Internationalized Normalized Ratio (INR)
o INR = (PT RESULT / MEAN OF REF RANGE)ISI
o Therapeutic Goal : 2.0 – 3.5
2. Activated Partial Thromboplastin Time (aPTT)
 Evaluates coagulation factors of the instrinsic pathway and the common pathway
 can be used as a screening test for the detection of circulating inhibitors of blood coagulation (lupus
anticoagulants)
 most commercial APTT reagents have good sensitivity for F-VIII deficiency,
 most common procedure used to monitor the effectiveness of standard (unfractionated) heparin therapy.
 partial thromboplastin reagent simulates activated platelet surfaces by providing phospholipid surfaces on
which enzymatic reactions in the coagulation cascade can occur, plus an activator (kaolin, celite, micronized
celite, or ellagic acid) that provides the negatively charged surface for the activation of F-XII.
 after a specific incubation time of citrated plasma (control or patient) with the APTT reagent which allows
optimum activation of the contact factors, calcium chloride is added, and the time required for a fibrin clot
to form is recorded
 General Reference Interval : 28 – 35 seconds
 Deficiency of F-XII, PK, or HK can result in a markedly prolonged APTT in the absence of clinically significant
bleeding
3. Thrombin Time
 Measures the conversion of fibrinogen to fibrin by adding excess thrombin to undiluted plasma
 interference with the conversion of fibrinogen to fibrin has three major reasons:
o the presence of hypofibrinogenemia or dysfibrinogenemia,
o the presence of heparin, and
o the presence of fibrin degradation products (FDP).
o In rare cases, autoantibodies against thrombin (e.g., induced by topical thrombin application or the
use of fibrin sealants) and myeloma proteins can also interfere with fibrin formation and result in
an abnormal TT
 General reference interval : 10 – 16 seconds

TESTS TO IDENTIFY SPECIFIC FACTORE DEFICIENCIES

1. MIXING STUDIES
 Screening test for circulating inhibitor
 Px ppp mixed with normal pooled plasma
o Historically, it uses 3 reagents (aged serum, adsorbed plasma, and factor vii/ix deficit plasma)
 Correction of prolonged PT/aPTT indicates factor deficiency
o Otherwise, a circulating inhibitor is present

 Classical
o Deficiencies corrected by Aged Serum only
 Factor VII / IX/ X
o Deficiencies Corrected by Adsorbed Plasma only
 Factors I / V / VIII
o Deficiencies Corrected bt Aged Serum and Adsorbed Plasma
 Factors XI / XII
o All deficiencies are corrected by Factor VIII/IX defiecient plasma except for
 Factors VIII / IX deficiencies
o All deficiencies are corrected by Aged Plasma except
 Factors V and VIII – the (labile factors)
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

2. Reptilase Time
 Not affected by heparin
 Reptilase is a serine protease found in the venom of the Bothrops atrox snake. This thrombin-like enzyme
cleaves fibrinopeptide A from fibrinogen, whereas thrombin cleaves both fibrinopeptide A and B.
 General reference interval : 18 – 22 sec
 An increase to 25 sec or longer is considered significant
o indicates dysfibrinogenemia, hypofibrinogenemia, or afibrinogenemia.

3. Specific Factor Assay

 Patient plasma and factor-deficient plasma are mixed and measured using PT or PTT (depending on factor
suspected).
 Results measured against factor-specific standard curve to determine percentage of factor present.
 Normal Range : 5% to 150%
4. Prekalikrein Screening Test
 Correction of the prolonged APTT of the patient’s citrated plasma to normal, or near the upper limit of the
normal APTT reference range, after a 10-minute incubation period (before adding calcium chloride)
suggests PK deficiency.
 The longer incubation period increases contact activation of F-XII in the absence of PK. The normal control
plasma APTT should remain within or near its control range following the extended incubation period.
 Kaolin, celite, or silica is the activator of choice for the APTT reagent. Prolonged incubation with ellagic acid
might not correct the APTT in PK deficiency.
 PK deficiency can be confirmed by performing a specific factor assay using PK-deficient substrate.
 As with the F-VIII assay, a quantitative chromogenic substrate assay can quantitate the level of PK in
plasma samples.
5. Factor XIII Urea Solubility
 Clot solubility is measured in the presence of urea or monocholoroacetic acid in 24 hr.
 Clot dissolution corresponds to factor XIII level of 1%-2%.

TESTS for vWF

1. vWF Functional Test

 In the presence of ristocetin, VWF induces platelet agglutination, which a platelet aggregometer can
measure.
 Decreased RIPA in patient PRP occurs in patients with VWD caused by VWF deficiency or BSS (VWF
receptor/GPIb/IX deficiency.
 Platelet aggregation test assessing ability of patient’s platelets to aggregate in the presence of ristocetin;
slope of aggregation compared to slopes of standard curve dilutions; also called ristocetin cofactor assay
(RCoF).
 The quantitative test for VWF activity uses ristocetin to induce VWF to bind to the glycoprotein Ib/IX
receptor (VWF receptor) on formalin-fixed platelets rather than the patient’s own platelets. This test system
adds ristocetin to formalin-fixed platelets suspended in patient plasma (the source of VWF) and uses a
platelet aggregometer to measure the resultant platelet agglutination.
o The slope of the agglutination is plotted versus the percent activity of a reference plasma.
o Other modifications of the assay use the time elapsed expressed in millimeters on the graph
instead of slope of agglutination.
2. vWF Antigen Test
 Levels measured using antibody against vWF
 methodologies include ELISA, EIA, immunoturbidometry

TESTS for Circulating Inhibitor

1. Lupus-like Anticoagulant Assay

 immunoglobulins, usually of the IgG class, that are directed against the protein component of protein-
phospholipid complexes
 Two separate screening tests
o Dilute Russel Viper Venom Time (dRVVT) / Stypven time
 Venom activates F-X
 In the presence of lupus anticoagulant, the DRVVT result will be prolonged because of
phospholipids in the reagent.
o LA-sensitive aPTT
 Criteria for LAs:
o 1. Prolongation of phospholipid-dependent coagulation reaction

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

o 2. Demonstration that it is an inhibitor and not a factor deficiency

o 3. Demonstration of inhibitor against phospholipid inhibitor (DRVVT)
2. Bethseda Titer
 Patient plasma and normal pooled plasma are mixed (several different dilutions) and incubated at
37
C for 2 hr, and factor level is measured.
 Presence of inhibitor will inactivate patient factor, resulting in decreased activity.
 Used to assess inhibitor levels for patients receiving factor concentrates
 Most often for patients with severe hemophilia (factors VIII or IX).
 Inhibitor suspected when patient fails to respond to treatment

ASSESSMENT OF FIBRINOLYTIC SYSTEM

1. D-Dimer Test
 specific marker (fragment) resulting from plasmin degradation (lysis) of the fibrin clot, which has been
cross-linked by F-XIIIa.
 Excellent marker of DIC
 also elevated in pulmonary embolism, deep vein thrombosis, arterial thromboembolism, recent trauma or
surgery, cirrhotic liver disease, and renal failure.
 should not be used in patients who are taking anticoagulant therapy
 The assay can be performed on plasma (citrated, ethylenediaminetetraacetic acid [EDTA], or heparinized)
and serum.
 fibrinogen and fibrin do not cross-react with the D-dimer monoclonal antibodies used in these tests.
 utilizes monoclonal antibodies against the D-dimer fragment, which can be used in three separate
methodologies
o Semiquantitative
 macroscopic latex agglutination
 Most procedures test both undiluted plasma and a plasma sample, diluted 1:2 in buffer
which allows a semiquantitation of results.
 Lack of macroscopic agglutination in either sample corresponds to a D-dimer level of <0.5
ug/mL
 Macroscopic agglutination in the undiluted sample but not in the 1:2 dilution corresponds
to a D-dimer of 0.5-1.0 ug/mL
 If both samples show agglutination, the result is > 1.0 ug/mL
 Normal individuals have <0.5 ug/mL
o ELISA
 More sensitive and specific for D-Dimer
o Turbidimetric Automated Microscopic Latex Agglutination
 Antibody-coated latex particles aggregate in the presence of D-dimer, resulting in
increased turbidity of the mixture.
 The increase in scattered light is proportional to the amount of D-dimer in the sample
 This assay is extremely sensitive and has an increased detection range.
 a negative test can be used to rule out deep vein thrombosis (DVT), venous
thromboembolism (VTE), and pulmonary embolism (PE).
2. Fibrin Degradation Products
 Blood sample on special collection tube containing thrombin to clot sample and ensure all fibrinogen are
removed and a fibrinolytic inhibtor to prevent in vitro fibrinogenolysis
 patient’s serum is mixed with latex particles coated with specific antibodies that recognize FDP on a glass
slide for a specific amount of time (e.g., 2 minutes).
 reaction mixture is observed macroscopically for the presence of agglutination.
 Semiquantitative
o Most procedures require the preparation of two dilutions for each sample (i.e., 1:2 and 1:8), which
allows a semiquantitative determination of FDP.
o Macroscopic agglutination in both dilutions corresponds to an FDP level of more than 20 ug/mL
o No macroscopic agglutination in either dilution indicates an FDP level less than 5 ug/mL
o agglutination observed in the 1:2 dilution but not in the 1:8 dilution corresponds to an FDP level
between 5ug/mL and 20 ug/mL
o Normal : < 5 ug/mL
 The test is nonspecific because it also is abnormal in the following conditions associated with increased
FDPs: liver disease, alcoholic cirrhosis, kidney disease, cardiac disease, postsurgical complications,
carcinoma, myocardial infarction, pulmonary embolism, DVT, eclampsia, and DIC
3. Euglobin Clot Lysis
 euglobulin protein fraction is precipitated from plasma in an acid solution (1% acetic acid).
 The precipitate contains fibrinogen, plasminogen, and plasminogen activators but no fibrinolytic inhibitors
(which remain in the supernant).
 With the removal of the fibrinolytic inhibitors from the test system, the fibrinolytic system’s action is more
easily demonstrated.
 The precipitate is redissolved, clotted with thrombin, and observed for clot lysis at 37°C.
 The fibrin clot generated by thrombin serves as the substrate for plasmin generated from plasminogen by
plasminogen activators (also activated by thrombin).
 The end point is the time in minutes required for complete degradation of the clot into small fibrin strands
or particles
 Normal : longer than 90 mins
 Shortened lysis times indicate increased fibrinolytic activity, liver disease

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 If ifibrinogen is < 100 mg/dL : false positive

ASSESSMENT OF HYERCOAGULABLE STATES

1. Antithrombin (AT) Assay

 powerful and immediate antiprotease action in the presence of heparin
 two-part Chromogenic Assay
o the first part, plasma is incubated with a known excess of thrombin in the presence of heparin
(heparinized buffer). AT neutralizes a proportional amount of the thrombin in the presence of
heparin.
o The second part of the assay determines the residual thrombin activity by its enzymatic activity on
an appropriate substrate (fibrinogen tagged with a chromophore, such as p-Nitroanilin [pNA],
which when released produces a yellow color that is measured at 405 nm).
o The amount of thrombin neutralized in the first reaction step is proportional to the amount of AT
present in the test sample; thus, the residual amount of thrombin (as measured by the pNA
release) is inversely proportional to the test sample’s AT level
o reference (standard) curve is prepared by plotting the AT activity (%) for each reference plasma
dilution prepared against its corresponding absorbance.
o Normal : 80% to 120%
 Immunologic methods
o EIA, ELISA, RID, LIA
2. Protein C
 Functional Assay
o Patient plasma mixed with protein C–deficient plasma, aPTT reagent (with an activator) and calcium
chloride are added, time to clot is measured
o If functional, APC will inactivate factors Va and VIIIa causing prolongation of aPTT result; time
compared to standard curve to determine percent function
o A reference (standard) curve is prepared by plotting the PC activity for each reference plasma dilution
assayed in conjunction with the patient samples against its clotting time.
o Normal : 60% to 150%
o Chromogenic method available
 PC is incubated with a specific activator and the amount of APC measured is based on its
enzymatic activity on a chromogenic substrate.
 The enzymatic activity releases pNA from the chromogenic substrate, and pNA is measured
spectrophotometrically at 405 nm.
 The absorbance of pNA is directly proportional to the amount of APC.
 A reference (standard) curve is prepared by plotting the protein C activity (%) for each
reference plasma dilution against
its corresponding absorbance.
 The results for patients and
controls are obtained from this
curve using the respective
readings.
 Antigenic Assay
o Immunologic assays

3. Protein S
 Circulating protein S (PS) exists in two forms: free (40%) and bound to C4b binding protein (60%).
 Only the free protein S serves as a cofactor for APC, enhancing its anticoagulant activity.
 The functional free form is what clot-based assays measure.
 Laboratory evaluation of PS also can include assays of total PS antigen (ELISA) and free PS (immunoassays
using monoclonal antibody specific for the free form).
 Functional Assay
o based on the ability of PS to serve as a cofactor for the anticoagulant effect of APC.
o A typical clot-based procedure for measuring the cofactor activity of PS requires four reagents
 PS-deficient plasma
 Purified activated PC
 Purified F-Va
 Substrate of aPC
 CaCl2
o Patient PPP is mixed with PS-deficient plasma
o APC and activated F-Va reagents are added to this mixture, which is incubated at 37°C.
o Following incubation, calcium chloride is added to initiate clot formation. The clotting time is
proportional to the PS activity in the sample (i.e., the higher the level of PS, the longer is the clotting
time).

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Like PC clot-based assays, the presence of factor V Leiden, APC resistance, elevated F-VIII levels, and a lupus
anticoagulant can cause false positive PS test results if clotting (functional) assays are used to diagnose PS
deficiency. Inherited deficiencies (autosomal dominant disorder) can involve a decrease in the protein or a
dysfunctional protein.

 The clot-based procedure for PS detects both quantitative and qualitative deficiencies of PS, but immunologic
methods do not detect qualitative deficiencies.
4. Activated protein C resistance (APCR)
 Screening for factor V leiden mutation
 Patient tested in the presence and absence of APC
 Normal
o Cleaving and inactivation of Va and VIIIa
 Affected Px
o Shortened Clotting time
5. Plaminogen
 measured by using a chromogenic assay based on the conversion of plasminogen to plasmin by an excess of
streptokinase (SK), which acts as an activator.
 The first step in this assay involves incubating patient plasma with a known excess of SK, which forms a
complex (plasminogen-SK) that causes plasmin-like activity.
 The second step determines the amount of the complex by its enzymatic activity on a chromogenic substrate.
The enzymatic activity results in the release from the chromogenic substrate of pNA, which is measured
spectrophotometrically at 405 nm
 The pNA absorbance is directly proportional to the plasminogen quantity.
 A reference (standard) curve is prepared by plotting the plasminogen activity (%) for each reference plasma
dilution against its corresponding absorbance. The results for patients and controls are obtained from this
curve using the respective absorbance readings.
 deficiencies characterized by decreased antigenic and functional levels as well as qualitative deficiencies
characterized by dysfunctional protein.
 Acquired deficiencies are associated with DIC, liver disease, and leukemia.
 Measuring circulating plasminogen levels is useful in monitoring hepatic regeneration of plasminogen after
discontinuation of treatment with SK and in controling and adjusting the rate of infusion of FFP being given
to the patient.
 The general reference interval for plasminogen levels is 74–124%.

DISORDERS OF HEMOSTASIS AND THROMBOSIS

DISORDERS OF PRIMARY HEMOSTASIS

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FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

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FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

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FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

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FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

DISORDERS OF SECONDARY HEMOSTASIS

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FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

References

Graeter, L. J., In Hertenstein, E. G., In Accurso, C. E., & In Labiner, G. H. (2015). Elsevier's medical laboratory
science examination review.

McKenzie, S. B., Williams, J. L., & Landis-Piwowar, K. (2015). Clinical laboratory hematology.

McPherson, R. A., Pincus, M. R., & Henry, J. B. (2017). Henry's Clinical diagnosis and management by laboratory
methods. St. Louis: Elsevier.

Turgeon, M. L. (2017). Clinical Hematology: Theory & Procedures.

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