LAB DIAGNOSIS OF HEMOSTASIS 1 & 2

Lab diagnosis of hemostasis 1 & 2 Handling of specimen for hemostasis evaluation

Hemostasis dependent upon : specimen storage
: vessel wall integrity - whole blood specimen: kept at room temperature
: adequate numbers of platelets (18-24°C)
: proper functioning platelets - avoid storage at: 1-6°C- activation of F7, platelets,
: adequate levels of clotting factors cryoprecipitation of VWF
: proper function of fibrinolytic pathway : more than 24°C- deterioration of
F8
Primary : injury-> vessel wall + platelet-> formation of
platelet plug Specimen storage temperature & time
↓
Secondary: activation of plasma coagulation factors->
formation of stable fibrin clot
↓
Dissolution of fibrin clot by fibrinolysis
- tests for platelets: platelet count
: bleeding time
- tests for coagulation factors: prothrombin time
: activated partial
thromboplastin time : specimen thawing- specimen (platelet poor plasma)
: thrombin time must be thawed rapidly at 37°C
: fibrinogen assay - test is performed within 1 hr after
removed from freezer
Requirement of specimen collection for - specimen may not be frozen &
hemostasis evaluation thawed more than once
Pre-analytical variables
Patient management: not need to fasting Pre-analytic errors
: patient on medication : problems with blue-top tube
: may hv to stop for a period of - partial fill tubes
time as instructed by doctor - vacuum leak acid citrate evaporation
Specimen collection tubes: 3.2% buffered sodium : problem with phlebotomy
citrate tube - heparin contamination
- wrong label
Selection of needles - slow fill
: adult with good veins, specimen not more than 25ml - underfill
- 20/21G, thin walled, 1/1.25 inches long - vigorous shaking
: adult with good veins, specimen not less than 25ml : biological effects
- 19G,1/1.25 inches long - hematocrit not less than 55/not more than 15
: child/adult with small, friable/hardened veins - lipemia, hyperbilirubinemia, hemolysis
- 23G, winged-needle set : lab errors- delay in testing
- prolonged incubation at 37°C
Rules of collection - freeze/thaw deterioration
: specimen for hemostasis est must be collected
first/immediately after non-additive tube Approach of primary hemostasis test
: ratio of blood to anticoagulant must be 9 parts blood Screening test of platelet function
to 1 part anticoagulant : tests for screening of platelet function are usually
: specimen must be gently inverted at least 5 times performed at same time as global assays of
few seconds after collection coagulation pathway function
: tourniquet must be removed within 1 min after : bleeding time (BT)
application to avoid stasis : platelet function analyser (PFA-100) closure time
: allow blood to flow gently down the tube
Bleeding time (BT)
: first described by Duke (1910), modification made by
Ivy in 1941
: used to detect significant detect in platelet function
: assesses the integrity of primary hemostasis at small
vessel- interaction between platelets & vascular wall
- subsequent formation of platelet plug
 LAB DIAGNOSIS OF HEMOSTASIS 1 & 2
Principles
: incision is made on volar surface of forearm & the
time the incision bleeds is measured
: cessation of bleeding indicates formation of platelet
plugs that depends on
- sufficient platelet number
- platelets adherence to sub-endothelium
- platelet aggregation
BT- Ivy’s method
: place sphygmomanometer cuff around the upper arm
above the elbow
: inflate to 40mmHg & keep this pressure throughout
the test
: clean the underside of forearm with 70% alcohol &
allow to air dry
: ensure the chosen area is devoid of visible superficial
veins/scar with about 3 fingers distance from the
elbow
: puncture the skin using sterile lancet at 2 different
spots with 1 cm distance
: start stopwatch once first puncture is done
: blot off blood from each wound using edge of filter
paper at 30 secs interval without direct contact with
wound
: stop timing when all wounds hv stop bleeding
: record the time taken as BT
* reference interval: 2-7 mins
* interpretation
: causes of prolonged BT- thrombocytopenia
- von willebrand disease
- platelet function disorder
- disorders of blood vessels
BT- Surgicutt
: modification of Ivy’s method
: it is sterile, standardized, easy to use, disposable
device
: makes a uniform, surgical incision
(5mm long x 1mm deep)
PFA-100 closure time (CT)
: measures platelets function under flow conditions
that
create high shear similar to flow through blood vessel
: whole blood pass through aperture cut into
membrane coated with collagen & either epinephrine
(CEPI)/ADP(CADP)
: system measures the ability of platelets to occlude
the aperture & reports the closure time
: PFA test result is dependent on platelet function,
plasma von willebrand factor level, platelet number
* referene interval: 78-199 secs for CEPI cartridge
: 55-137 secs for CADP cartridge
* prolonged CT interpretation
: thrombocytopenia
: von willebrand disease
: platelet function abnormality
Approach of secondary hemostasis test
Coagulation screening test
: coagulation screening tests are performed when
coagulopathy is suspected hereditary/acquired
: tests are designed to isolate specific compartment of
coagulation cascade- to gives clues o where
abnormality exists
: prothrombin time (PT)
: activated partial thromboplastin time (APTT)
: thrombin time (TT)
: fibrinogen assay
Prothrombin time (PT)
: background/purpose
- PT measures clotting time of plasma in presence of
optimal concentration of tissue extract
- it indicates overall efficiency of extrinsic pathway &
common pathway of coagulation cascade
- it measures prothrombin & other factors like
fibrinogen, FV (factor 5), FVII (factor 7), FX (factor
10)
- used to monitor patients on warfarin, liver disease &
vit K deficiency
- most sensitive to factor 7 deficiencies, moderately
sensitive to factor 5 & factor 10 deficiencies
: principle
- thromboplastin activates extrinsic coagulation
system in presence of phospholipid & calcium ion
- when phospholipid-calcium-thromboplastin reagent
is added to PPP, the clotting mechanism is initiated
by activating factor 7 & subsequent reaction
resulting in formation of solid gel clot within specific
period of time in secs
- clotting time is dependent on concentration of
prothrombin, fibrinogen, factor 5,7,& 10
: procedure
- prepare platelet-poor-plasma, centrifuge wholeblood
at 100g for 15 min
- warm both PPP & calcium-thromboplastin reagent
to
37°C in water bath for 3 mins
- pipette 100μL of PPP into test tube
- transfer 200μL of calcium-thromboplastin reagent
into test tube containing PPP
- start stopwatch immediately after adding reagent
into test tube
- mix gently while holding test tube with lower end of
tube submerged in water bath
- tilt & shake test tube continuously & observe for clot
formation
 LAB DIAGNOSIS OF HEMOSTASIS 1 & 2
: referene interval
- male: 10.23-16.33 secs
- female: 10.72-16.71 secs
: common cause of prolonged of PT
- administration o oral anticoagulant drugs (vit K
antagonist)
- deficiency of factor 5, 7, 10, prothrombin, fibrinogen
- vit K deficiency
- liver disease
- disseminated intravascular coagulation (DIC)
Activated partial thromboplastin time (APTT)
: background/purpose
- measures clotting time of plasma after activation of
contact factors bu without added tissue
thromboplastin & indicates overall efficiency of
intrinsic &common pathway
- is performed to to monitor patients on heparin
therapy & to detect lupus anticoagulant (LA),
anticoagulation factor Ab & factor deficiencies
: principle
- when kaolin-phospholipid reagent (contact activator)
is added to PPP, the clotting mechanism is initiated
by activating factor 12 to factor 12a & factor 11 is
cleaved to factor 11a
- addition of calcium continues the coagulation
reaction, results in solid gel clot formation within
specific period time in secs
: procedure
- prepare PPP
- warm PPP, kaolin-phospholipid & calcium chloride
reagent to 37°C in water bath
- pipette 100μL of PPP into test tube
- transfer 100μL of kaolin-phospholipid reagent into
test tube containing PPP
- mix gently & incubate at 37°C water bath for 3 mins
- add 100μL of calcium chloride into test tube & mix
content while holding the test tube with lower end of
tube submerged in water bath
- start stopwatch immediately after adding calcium
chloride into test tube
- tilt & shake test tube continuously & observe for clot
formation
- stop timing when fibrin is formed & record time
: reference interval
- male: 26.51-37.11 secs
- female: 25.8-39.54 secs
: common causes of prolonged APTT
- stop timing when fibrin clot is formed & record time
Thrombin time (TT)
: background/purpose
- TT test measures time taken for fibrinogen to
convert into fibrin after addition of thrombin
- reagent: thrombin solution
: principle
- when thrombin reagent is added to PPP, fibrinogen
is converted to fibrin forming solid gel clot within
specific period of time in secs
: procedure
- prepare PPP
- warm PPP & thrombin reagent to 37°C in water bath
- pipette 200μL of PPP into test tube
- transfer 200μL of thrombin reagent into test tube
containing PPP
- start stopwatch immediately after adding reagent
into test tube
- mix gently while holding test tube with lower end of
tube submerged in water bath
- tilt & shake test tube continuously & observe for clot
formation
- stop timing when fibrin clot is formed & record time
: reference interval
- 15-20 secs
: common causes of prolonged TT
- hypofibrinogenemia as found in DIC
- ↑FDP as seen in liver disease
- heparin therapy
- dysfibrinogenemia
- hypoalbuminemia
Fibrinogen assay (Clauss technique)
: fibrinogen (Fib) is a blood coagulating protein with its
highest content in plasma, is one of the acute phase
proteins, synthesized by liver cells & megakarocytes
: fibrinogen (Fib) is closely associated with
consumption coagulopathy, liver disease,
nephropathy syndrome, CVS disease, diabetes,
malignant tumors
: high-level of Fib is one of the dangerous factors
contributing to coronary heart disease
: principle
- Fib assays are quantitative techniques to measure
the amount of functional Fib present in plasma
- the assay is based on Clauss assay that is the
reference method
- procedure is determination based on fibrinogen
activity but results are converted to concentration by
comparison with control plasma results
- in Fib procedure, thrombin is added to various
dilutions of known concentrations of Fib to produce
thrombin-clotting time in secs
- clotting times are then plotted on graph, with known
concentration (x-axis) vs clotting time (y-axis)
 LAB DIAGNOSIS OF HEMOSTASIS 1 & 2

- DIC
- liver disease
- heparin administration/contamination
- deficiency of factors other than factor 7

: procedure
- standard curve is constructed using reference
plasma by preparing series of dilutions in buffer to
give a range of Fib concentrations
- clotting time of each of these dilution is established
in duplicates, add thrombin
- place clotting time against Fib concentration on
graph
- 1:10 concentration is considered to be 100%
(normal)
- test sample is diluted 1:10 dilution
- perform the TT test & determine clotting time
- Fib level is determined from standard curve

: preparation of calibration curve

- the calibration curve is prepared from reference
standard
- make dilutions of Fib standard with Imidazole buffer
as follows

: reference interval
- 1.8-3.6g/L

: interpretation
- low level: inherited dysfibrinogenemia
: DIC
: severe liver disease
- elevated level: moderately severe liver disease
: pregnancy
: chronic inflammation