UNIT VIII: LABORATORY EVALUATION – a.

Micro Methods
SECONDARY HEMOSTASIS ACTIVATION OF 1. Slide or Drop Method
COMMON PATHWAY AND CONVERSION OF 2. Capillary or Dale and Laidlaw’s Method
PROTHROMBIN TO THROMBIN b. Macro Methods
1. Lee-White Method/ Whole Blood Clotting
Outline Time
c. Activated Coagulation Time of Whole Blood
I. TEST OF PHASE II OF COAGULATION
d. Plasma Recalcification Time
a. The Coagulation Test
b. Partial Thromboplastin Time e. Activated Recalcification Time
c. Activated Partial Thromboplastic Time (APTT)
d. Differentiated Tests of Activated Partial SLIDE METHOD
Thromboplastic Time (DAPTT)
e. Differential Partial Thromboplastin Time (DPTT) • Perform capillary puncture
f. Prothrombin Time (PROTIME) • Start timing
g. Serum Prothrombin Time/ Prothrombin Consumption
Test (PCT)
• Wait for 30 second
h. Thromboplastin Generation Test • Check for the fibrin strand
II. TEST OF PHASE III OF COAGULATION • Interval: 30 seconds
a. Thrombin Time
b. Fibrindex Test
c. Fit-test (Immunological Test)
d. Fibrinogen Titer Method
e. Assay of Plasma Fibrinogen

TEST OF PHASE II OF COAGULATION

A. The Coagulation Test

B. Partial Thromboplastin Time
C. Activated Partial Thromboplastic Time (APTT)
D. Differentiated Tests of Activated Partial
Thromboplastic Time (DAPTT)
E. Differential Partial Thromboplastin Time CAPILLARY TUBE METHOD
(DPTT)
• Uses capillary tube with no anticoagulant (blue
F. Prothrombin Time (PROTIME)
band)
G. Serum Prothrombin Time/ Prothrombin
• Stand for 2 minutes then break every 30 seconds
Consumption Test (PCT)
• Check for fibrin strand
H. Thromboplastin Generation Test

THE COAGULATION TEST Macro Methods – superior for there is less contamination
of the plasma with tissue fluids when blood is drawn from
• Tests the composite action of all plasma factors a vein.
acting simultaneously.
LEE-WHITE METHOD OR WHOLE BLOOD CLOTTING
• Secondary hemostasis – involves clotting factors
TIME
• Clotting time is a measure of the ability of the
blood to clot and is not influenced by the platelet • First in-vitro procedure that employed the
functions other than PF3. It also measures only principle that the time interval from the initiation of
the time required for the formation of the traces of clotting to visible clot formation reflects the
thrombin sufficient to produce a visible clot. condition of the coagulation mechanism
• Prolonged clotting time indicates a coagulopathy • Principle: The whole blood clotting time is the
(coagulation deficiency) time required for freshly collected blood to form a
o PF3 – phospholipid needed in the intrinsic firm clot in standardized glass tubes at 37̊C. Thus,
coagulation pathway; needed in the the whole blood clotting time is a measure of the
activation of the coagulation integrity of the intrinsic system.
o PF3 is released when platelet is activated • Involves 3 tubes that are agitated; result to be
that will then participate in thrombin formation reported is from tube #3 because of less agitation
• Normal values: 5-10 minutes
• Advantages ACTIVATED RECALCIFICATION TIME
o More accurate and standard method
o Test can be run with control • Modification of PRT with two activators
• Disadvantages • Employs the use of 0.1 ml of platelet-rich plasma
o It is also a rough method • 0.1 ml of 0.025 M calcium chloride – activator
o There can be contamination of syringe or • 0.1 ml of 1% celite – activator
tube or with tissue fluid • Normal value: less than 50 seconds
o Vigorous agitation of the tubes should be
PARTIAL THROMBOPLASTIN TIME
avoided as it shortens the clotting time
• Simple test for the INTRINSIC and COMMON
ACTIVATED COAGULATION TIME OF WHOLE
pathways of coagulation.
BLOOD (ACT)
• Checks for deficiencies in intrinsic/ common
• Principle: The activated coagulation time of coagulation pathway
whole blood is the time necessary for fresh blood • PTT is also called activated partial thromboplastin
to form a clot when incubated at 37̊C in the time (APTT)
presence of “surface contact” activation. This
ACTIVATED PARTIAL THROMBOPLASTIN TIME
assay, like to whole blood clotting, measures
(APTT)
overall activity of the intrinsic clotting system.
• Utilizes a particulate clot activator in the test tube • A test for the deficiencies of factors in the
which speeds the clotting process INTRINSIC system (Factor VIII, IX, XI, XII)
• Uses activator diatomite (12 mg) – algae – o Deficiency in prothrombin, factor V, VIII, IX,
stimulates the clotting factor X, XI, XII, fibrinogen (≤100 mg/dL)
• Used to check patients undergoing high-dose • Cannot measure Factor VII and XIII
heparin • Performed to monitor the effects of unfractionated
• Reference value of those having heparin therapy heparin therapy and to detect Lupus
is 140-185 seconds anticoagulants (LA) and specific anticoagulation
• Normal values: 1-2 minutes factor antibodies such as anti-FVII antibody
• PTT has been the standard method for
FACTORS THAT MAY AFFECT THE RESULTS
monitoring unfractionated heparin therapy (used
• The effects of surgery in treating venous thrombosis, pulmonary
• Body temperature embolism, MI, etc.)
• Other medicines you are taking (Coumadin, • Prolonged in congenital and acquired
Warfarin) procoagulant deficiencies except FVII and FXIII
• Getting IV (intravenous) fluids, which can dilute deficiencies
your blood • Factors whose deficiencies are associated with
• Platelet counts and platelet function hemorrhage and are reflected in prolonged PTT
• Coagulation factor deficiencies results, taken int the order of reaction, are XI, IX,
VIII, X, and V; prothrombin; fibrinogen
PLASMA RECALCIFICATION TIME • Factor VII and XIII deficiencies have no effect on
PTT; PTT is not as sensitive to Vit K def. or
• Uses 0.025M calcium chloride Coumadin therapy as PT
• Can either use citrated PPP or PRP, but not • Deficiencies of FXII, PK, HMWK prolong the PTT,
whole blood but do not cause bleeding
• Is a measure of intrinsic coagulation mechanism • DIC prolongs PTT, but is not definitive for DIC
• Screens platelet function • Vit K def. → diminished factors II, VII, IX, X →
• More sensitive method than the coagulation time prolonged PTT
of whole blood. • Normal values: 25-35 seconds
• May reveal abnormality which is not detectable by • If PT is prolonged (RV: 12.6-14.6 secs), there are
the determination of the clotting time of venous defects in extrinsic/common pathway – Factor VII
blood.
• If PTT is prolonged with prolonged PT – defects
• As platelet number increases, the reading in common pathway
shortens
• Procedure:
• Normal PRT: 90-250 seconds o 50 or 100 uL of warmed reagent (37°C) is
• Ref. value PPP: 130-240 seconds mixed with an equal volume of warmed PPP
• Ref. value PRP: 100-150 seconds o Incubated, usually 3 minutes

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 o 50 or 100 uL of warmed 0.025M CaCl2 is • Specimen of choice is citrated PPP
forcibly added to the mixture and timer is • Phospholipid is extracted from rabbit’s brain; now
started is synthetically produced
o If PTT is performed manually, test is done in • Activators provide a surface that mediates a
duplicate and the results must match within conformational change in plasma FXII that results
10% in its activation. Activators used in the PTT
reagent that will enhance the coagulation process
FACTORS THAT INTERFERE WITH THE VALIDITY
include:
OF CLOT-BASED RESULTS
1. Kaolin
Clot-based tests include PT, APTT, TT, ACT 2. Celite
3. Silica
1. Hemolysis – releases ADP resulting to platelet 4. Ellagic acid
activation
2. Increased HCT – recollect sample DIFFERENTIAL TESTS OF ACTIVATED PARTIAL
3. Lipemic – interferes with the reading of the THROMBOPLASTIN TIME (DAPTT)
machine
• Used to differentiate factor deficiency (intrinsic,
extrinsic, common) and disorder of circulating
anticoagulants (autoantibodies against FV and
FVIII

DIFFERENTIAL PARTIAL THROMBOPLASTIN TIME

(DPTT)

• Another modification of APTT which is done by

mixing the patient’s plasma with commercially
available correcting reagents, Factor VIII and IX
reagents
• If prolonged:
o PTT is corrected with Factor VIII (hemophilia
A)
o PTT is corrected with Factor IX (hemophilia
B)

PROTHROMBIN TIME (PROTIME)

• Measures the EXTRINSIC and COMMON

pathway of coagulation. It is used to monitor oral
anticoagulant therapy. This can detect
• Anticoagulant volume must be adjusted when prothrombin, fibrinogen, Factors V, VII and X
HCT is >55% to avoid false prolonged results deficiencies.
• Invert 5 times after collection, but must be gentle • Used to detect defects in vitamin K-dependent
• Reject clotted and visibly hemolyzed samples factors
• Polybrene are incorporated to thromboplastin • Specimen of choice is citrated PPP
reagents to neutralize heparin • Reagent, thromboplastin or tissue
thromboplastin, is prepared from recombinant or
Contact Factors affinity-purified tissue factor suspended in
phospholipids mixed with a buffered 0.025M
calcium chloride
o When mixed with citrated PPP, it triggers
fibrin polymerization by activating FVII
Ca2+, PL
• Prolonged in deficiencies of fibrinogen,
Ca2+, PL prothrombin, and factors V, VII, VIII, X
• Most sensitive to FVII deficiencies; moderately
severe to FV and FX; sensitive to severe
fibrinogen and prothrombin deficiencies;
insensitive to FVIII and FIX deficiencies
• Performed when any coagulopathy is suspected
• Reference value: 12.6-14.6 seconds

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 • PT results are reported to the nearest tenth of a • If patient is in Coumadin therapy, it will suppress
second; if assay is duplicated, results are the production of FVII
averaged and is reported • Heparin therapy → prolonged PT
• PT is prolonged in congenital single-factor • Increase in either PT or PTT – mixing studies can
deficiencies of FV, FVII, FX; profound be done
prothrombin deficiency; fibrinogen deficiency – • PT is prolonged in DIC, liver diseases (decreased
≤100 mg/dL FVII and FV), vitamin K deficiency
• If PT is prolonged, but PTT and thrombin clotting o All affect FVII activity → prolonged PT
time (TCT) are normal, FVII activity may be o Vit K def. in severe malnutrition, use of broad-
deficient spectrum antibiotics, parenteral nutrition,
• PT is not affected by FVIII and FIX deficiency malabsorption syndromes
because the concentration of tissue factor in the o Vit K levels are low in newborns
reagent is high, and those factors are bypassed o To distinguish between Vit K def. and liver
in thrombin generation disease, FV and FVII levels are determined
• No data support PT as a screening test for ▪ FV and FVII reduced in liver disease
individuals at low risk of bleeding ▪ FVII is only reduced in Vit K def.
• 50 or 100 uL aliquot of test sample
METHODS:
• At least 3-10 minutes only of incubation time
1. One Stage Method of Quick (Stanley Brown since coagulation factors start to deteriorate
Method) beyond this time and the pH changes
2. Two-stage Prothrombin and Proconvertin • 100 or 200 uL reagent is directly and quickly
Test (Owren and Aas) added to the PPP aliquot and a timer is started;
3. Owren’s Thrombotest Method timer is stopped once clot forms
4. Fibrometer Method
TWO-STAGE PROTHROMBIN AND PROCONVERTIN
5. Micromethod (Protime)
TEST (OWREN AND AAS)
6. Related Method-Stypven Time (Rusell’s
Viper Venom Time) • It offers a combined estimation of the levels of
7. Prothrombin Activity or Index prothrombin and proconvertin.
8. International Normalized Ratio • Advantages:
o It is more sensitive with Stanley Brown
ONE STAGE METHOD OF QUICK (STANLEY BROWN
Method.
METHOD)
o Fresh specimens are not necessary, and the
• Principle: Tissue thromboplastin and calcium method can be used for mailed samples of
added to plasma react to fibrinogen to form a clot. blood.
The thromboplastin added to the plasma takes o The method is not affected by heparin.
the place of the tissue juice formation of extrinsic
OWREN’S THROMBOTEST METHOD
thromboplastin. The prothrombin time is therefore
prolonged if there is a deficiency of Factors V, VII • For the control of Coumarin anticoagulant
or X or a very severe deficiency of Factor I and II. therapy, this is considered as the most sensitive
test.
• Coumarin suppresses cyclooxygenase

FIBROMETER METHOD

• It is an electromechanical semi-automated
instrument that has been used extensively in one-
stage prothrombin method of Quick.

MICROMETHOD (PRO TIME)

• This is microtechnique employed for children and

the method uses micropipettes; the principle of
the test is similar with one-stage prothrombin time
or Stypven Time.
• If prolonged – defects in Factor VII
• If PT and PTT are both prolonged – defects in RELATED METHOD – STYPVEN TIME (RUSELL’S
common pathway VIPER VENOM TIME)

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 • It is used to distinguish deficiencies of Factor X THROMBOPLASTIN GENERATION TEST
and those of Factor VII. It is also used to detect
1. Bigg’s and Macfarlane Method
deficiencies in prothrombin, fibrinogen and
2. Hick’s-Pitney Kaolin Modification Method
factors V and X. It differs from prothrombin time
in that deficiencies in factor VII are not detected. TEST OF PHASE III OF COAGULATION
• Not used in the present
• Venom is from Vipera russelli (Daboia russelli 1. Thrombin Time
viper) – bypasses the action of FVII that directly 2. Fibrindex Test
activates FX 3. Fit-test (Immunological Test)
• Prolonged PT and ST – defects in FX 4. Fibrinogen Titer Method
• Prolonged PT and normal ST – defects in FVII 5. Assay of Plasma Fibrinogen

PROTHROMBIN ACTIVITY OR INDEX THROMBIN TIME / THROMBIN CLOTTING TIME (TCT)

• Manual PT measures only PT of the patient; • Measures the availability of functional fibrinogen
automated machine can measure PT activity, INR • Prolonged if fibrinogen is decreased (NV is 200-
• Reported in percentage, with 100% as the 400 mg/dL), impaired fibrinogen function, and in
maximum level. afibrinogenemia
• Sensitive test in detecting heparin inhibition
𝑃𝑟𝑜 𝑡𝑖𝑚𝑒 (𝑖𝑛 𝑠𝑒𝑐𝑠 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) • Affected by circulation anticoagulants
𝑃𝑇 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = × 100
𝑃𝑟𝑜 𝑡𝑖𝑚𝑒 (𝑖𝑛 𝑠𝑒𝑐𝑠 𝑜𝑓 𝑝𝑎𝑡𝑖𝑒𝑛𝑡) • Test for the deficiency or inhibition of fibrinogen
• Principle: Commercially prepared thrombin
INTERNATIONAL NORMALIZED RATIO
reagent is added to citrated plasma, and the time
• This method of reporting has been proposed to required for clot formation is measured.
monitor patients on oral anticoagulant therapy • Normal values: 10-20 seconds
• Used in Coumadin monitoring, to compensate for • Procedure:
the inherent variations among thromboplastin o Thrombin reagent is warmed to 37°C for 3-10
reagents minutes (thrombin deteriorates during
• It is defined as the prothrombin time ratio had the incubation and must be used within 10 mins)
test been performed using international standard o 100 uL of PPP is incubated at 37°C for 3-10
thromboplastin reagent. minutes
o 200 uL of thrombin is pipetted into PPP, start
𝐼𝑆𝐼
𝑃𝑇𝑝𝑎𝑡𝑖𝑒𝑛𝑡 a timer, and record the interval to clot
𝐼𝑁𝑅 = ( ) formation
𝑃𝑇𝑔𝑒𝑜𝑚𝑒𝑡𝑟𝑖𝑐 𝑚𝑒𝑎𝑛 𝑜𝑓 𝑛𝑜𝑟𝑚𝑎𝑙
• Prolonged when fibrinogen level is <100 mg/dL
PTpatient = PT of the px in seconds (hypofibrinogenemia); presence of antithrombotic
PTgeometric mean of normal = PT of the geometric mean of the materials such as FDP’s, paraproteins, or
reference interval heparin; afibrinogenemia, dysfibrinogenemia
• TCT is part of the PTT mixing study protocol and
ISI = international sensitivity index
is used to determine whether heparin is present
• International sensitivity index whenever the PTT is prolonged
o Standard for PT reagent
o >1.0 lower sensitivity
o <1.0 high sensitivity
o Most responsive thromboplastin reagents
have ISIs near 1 according to WHO

SERUM PROTHROMBIN TIME/ PROTHROMBIN

CONSUMPTION TEST (PCT)

• Best considered as a test of platelet phospholipid

activity. If the prothrombin time and the PTT are
normal, a short PCT indicated a deficiency of PF3
due to thrombocytopenia (low platelet) or
thrombopathia (type of platelet coagulopathy;
bleeding or coagulation).

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 FIBRINDEX TEST

• Commercially available test wherein upon

addition of plasma containing fibrinogen,
thrombin produces clotting.
• Normal value:
o Plasma at 5-10 seconds
o Firm clot without serum at 30-60 seconds

FIT-TEST (IMMUNOLOGICALTEST)

• 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.

FIBRINOGEN TITER METHOD

• Serial dilutions of plasma are diluted 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.

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
quantified by gravimetric, nephelometric,
chemical, immunologic and precipitation
methods.

METHODS:

a. Ellis and Stransky Method

b. Stirland’s Method
c. Turbidimetric Method of Partfantjev et.al.
d. Ratnoff and Menzie Method
e. Fibrin Clot Method
f. Duckert’s Test/ 5M urea solubility test – tests for
FXIII

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