Laboratory Evaluation of Coagulation and Fibrinolysis Flashcards
Things to consider:
- Tissue thromboplastin contamination
- Inappropriate container
- Improper temperature: labile factors
- Hemolysis
Inappropriate container
a) Glass surface
b) Polystyrene tubes/ glass tube with silicone- preferred
- preferred
Polystyrene tubes/ glass tube with silicone
@room temp: _______
V & VIII
@ref temp: _________
VII & XI
PT plasma capped stability for 24 hours @: _____
RT (18-24oC)
APTT samples stable for 4 hours @: ___________
RT (18-24oC)
Factor assays stable for 4 hours @: ___________
RT (18-24oC)
Hemolysis
-prolonged tourniquet application
-moisture/contamination
-inappropriate needle bore
-Frothing of sample
-improper transfer from barrel to tube
-excessive mixing mixing
▪ Adult (≤25 mL):
G20 -21; 1 - 1.25 in.
▪ Adult (≥25 mL):
G19; 1 or 1.25 in.
▪ Child or adult with small, friable, or hardened veins:
G23
▪ Syringe w/ winged-needle set:
G20, 21, or 23 (mostly)
: used in blood donations
▪ G16-18
Type of Syringe:
Plastic/Silicone coated- single draw
2-way needle and holder-multiple draw
Winged-needle sets
Anticoagulants:
A) Sodium oxalate
B) Trisodium citrate
4) Heparin
3) EDTA
: Interfere with Spectrophotometric reading (OD)
A) Sodium oxalate
: -Preserves FV and FVIII
B) Trisodium citrate
- Ratio of 9:1 (blood: anticoagulant)
B) Trisodium citrate
B) Trisodium citrate Prolonged
- Incomplete filling/high hematocrit
B) Trisodium citrate
- Two concentrations:
3.2% and 3.8%
-acts with antithrombin III to inhibit all stages of coagulation
4) Heparin:
-used for plt retention test
4) Heparin:
-unsuitable for coagulation
3) EDTA
-inhibits thrombin-fibrinogen reaction
3) EDTA
-Factor V is unstable in its presence
3) EDTA
Evacuated tube arrangement: 2-way needle collection
a. Red stopper
b. Blue stopper (3.2%)
c. Lavander stopper
d. plastic syringe (for additional tests)
1) Effects of pH: _______
PROLONG THE CT
2) Temperature: (as mentioned during the first slide)
PPP preparation: __________ for _________
2000 x g for 10 mins
Storage: 4 deg within 2 hours/ rapid freezing @:_______________
-20oC
PRP preparation: __________ for________@___
60-100 x g for 10 mins @ RT
Principle: when venous blood is put into a glass tube, it will form a solid clot.
Lee and White Method
-the time response is a measure of overall intrinsic and common pathway
Lee and White Method
-Insensitive to factor deficiencies.
Lee and White Method
Lee and White Method NV:
7-15 mins
Principle: Presence of an activator (DIATOME) and by keeping the blood constant at 37 deg celsius, the test is more reliable and rapid.
Activated Clotting Time
Activated Clotting Time NV:
75-120 sec.
Principle: Time required for blood to clot after the addition of calcium
Plasma Recalcification Time (PRT)
-Modified Lee and White
Plasma Recalcification Time (PRT)
Plasma Recalcification Time (PRT) NV:
PRP: 100-150 sec
PPP: 130-240 sec
Routine screening of coagulation disorders in intrinsic system and common pathway
Activated partial thromboplastic time (APTT/PTT)
- Detects the presence of circulating anti-coagulant
Activated partial thromboplastic time (APTT/PTT)
- Monitors heparin therapy
Activated partial thromboplastic time (APTT/PTT)
Principle: Measures all coagulation factors needed for the generation of Intrinsic prothrombinase except for Calcium and PPL
Activated partial thromboplastic time (APTT/PTT)
When Calcium is added with incomplete thromboplastin (plt substitute- CEPHALIN), intrinsic prothrombinase is generated.
Activated partial thromboplastic time (APTT/PTT)
Activated partial thromboplastic time (APTT/PTT) Specimen:
Citrated PPP
Activated partial thromboplastic time (APTT/PTT) Reagent:
0.025M CaCL2
Phospholipid (plt sub)
Activated partial thromboplastic time (APTT/PTT) Activators:
kaolin, celite or ellagic acid
Activated partial thromboplastic time (APTT/PTT) NV:
25-35 seconds/ 22-34 seconds
Prolonged PTT:
- Deficiency in intrinsic/common pathway factors
- Presence of inhibitor
- <60 mg/dL fibrinogen
- Increased FSP
-screening for the extrinsic and common pathway coagulation.
PROTHROMBIN TIME (Quick’s Test)
-monitors oral anticoagulant with COUMADIN
PROTHROMBIN TIME (Quick’s Test)
PROTHROMBIN TIME (Quick’s Test)
Principle: When tissue extract of (?) is added to (?), along with (?), it reacts with (?) to activate (?).
thromboplastin (complete)
PPP
Calcium
FVII
FX
PROTHROMBIN TIME (Quick’s Test) Sample:
Citrated PPP
PROTHROMBIN TIME (Quick’s Test) Reagents:
Thromboplastin 0.025M CaCL2
INR=
International Normalized Ratio
-standardized way of reporting PT in monitoring anticoagulant therapy
International Normalized Ratio
-calculated as a ratio of the px PT to a control PT (standardized for the potency of the thromboplastin rgt)
International Normalized Ratio
ISI=
International Sensitivity Index (calibration index)
Reference Values for INR
2.0-3.0
2.5-3.5
2.0-3.0
▪ in prevention and treatment of viscous thrombosis
▪ treatment of pulmonary embolism
▪ prevention of stroke in myocardial infarction
▪ peripheral arterial disease
▪ prevention of systemic embolism in atrial fibrillation
▪ cardia valve replacement (tissue valves)
2.5-3.5
▪ in prevention of recurrent MI
▪ reduction of mortality in MI
▪ mechanical prosthetic heart valve (high risk)
PROTHROMBIN TIME (Quick’s Test)
NV:
10-12 seconds
PROTHROMBIN TIME (Quick’s Test) Prolonged in:
-extrinsic and common pathway factor deficiencies
- Fibrinogen of <100 mg/dL
- Dysfibrinogenemia
- Vit K deficiency and certain liver disease
- Increased FDP’s
- False prolongation: heparin contamination
PROTHROMBIN TIME (Quick’s Test) Shortened in:
-Traumatic venipuncture
is a thromboplastin-like substance
Venom “Vipera ruselli”
STYPVEN/RUSELL’s VIPER VENOM
Principle: Addition of the (?) bypasses the activation of(?) and directly activates (?).
venom
VII
FX
STYPVEN/RUSELL’s VIPER VENOM NV:
6-10 seconds
What is the function of Dilute Russel Viper Venome time (dRVVT)?
Detects the presence of lupus anticoagulant
INDIRECT TESTS
Otherwise known as “Fibrinogen Deficiency Test”
THROMBIN TIME
THROMBIN TIME
Principle: Addition of thrombin bypasses all coagulation pathways except:
polymerization of Fibrinogen
.-measures the availability of functional fibrinogen
THROMBIN TIME
-measures the conversion of fibrinogen to fibrin
THROMBIN TIME
THROMBIN TIME NV:
17-25 seconds
Prolonged TT:
Fibrinogen level: 75-100 mg/dL
Impairment of fibrinogen
Presence of Heparin and FDP
Normally prolonged in newborn and multiple myeloma
REPTILASE TIME NV:
18-20 seconds
REPTILASE TIME Prolonged in:
Fibrinogen deficiency
Presence of FSP
Heparin Therapy; NF1 Prolonged
THROMBIN TIME
Heparin Therapy; NF1 Normal
REPTILASE TIME
FSP: NF1 Prolonged
THROMBIN TIME
REPTILASE TIME
Hypofibrinogenemia Greatly prolonged
THROMBIN TIME
Hypofibrinogenemia Prolonged
REPTILASE TIME
Dysfibrinogenemia Prolonged
THROMBIN TIME
Dysfibrinogenemia Greatly prolonged
REPTILASE TIME
Test for FXIII deficiency
DUKERT TEST (5M Urea Solubility Test)
DUKERT TEST (5M Urea Solubility Test)
Principle: clot formed in (?) is insoluble in (?) during a (?).
normal plasma
5M urea
24 hr incubation
Indicator: If Factor XIII is deficient, the clot is dissolved in less than 24 hours by urea
DUKERT TEST (5M Urea Solubility Test)
Clot + 5M Urea
NORMAL
FXIII Deficiency
Excessive fibrinolysis
Clot +NaCl
NORMAL
FXIII Deficiency
Excessive fibrinolysis
-screening procedure for the assessment of fibrinolysis
EUGLOBULIN TEST
-Euglobulin fraction:
Plasminogen, activators of plasminogen, fibrinogen
-EUGLOBULIN TEST
Principle: Plasma euglobulins are precipitated with (?). Precipitates are redissolved and clotted with (?). The clot is incubated and the time for complete lysis @ (?) is measured.
1 %Hac
thrombin
37 deg celcius
EUGLOBULIN TEST NV:
2-4 hours
: abnormal fibrinolytic activity
<2 hours
EUGLOBULIN TEST Increase fibrinolytic activity is seen on:
-circulatory collapse
-adrenalin injection
-sudden death
-pulmonary surgery
-pyrogen reaction
-obstetric complications
: Rapid clot breakdown
Primary
-increase in the circulating tPA binding to fibrin
Primary
-Excess tPA
Primary
-Excess tPA 2:
decreased hepatic clearance
decreased fibrinolytic inhibitors
Secondary: Secondary to
systemic hypercoagulability
-systemic or microvascular
Secondary
-not localized
Secondary
-commonly assoc with DIC
Secondary
-detects fibrin monomers
PROTAMINE SULFATE TEST and ETHANOL GELATION TEST
-distinguishes primary vs secondary fibrinolysis
PROTAMINE SULFATE TEST and ETHANOL GELATION TEST
-screening procedure in diagnosing DIC
PROTAMINE SULFATE TEST and ETHANOL GELATION TEST
PROTAMINE SULFATE TEST and ETHANOL GELATION TEST
Principle: (?) displaces the (?) from (?) and spontaneously form (?) (paracoagulation)
Protamine sulfate/ 50% ethanol
secondary FDPs
fibrin monomer
gel-like clots
PROTAMINE SULFATE TEST and ETHANOL GELATION TEST Normal:
no gel formation
arises from the degradation of cross linked fibrin
D-d
-measures fibrinolysis but not fibrinogenolysis
D-Dimer Test
-uses latex bead with monoclonal ab
D-Dimer Test
-NV: <200 ng/mL
D-Dimer Test
D-Dimer Test Increased in:
DIC
Thrombosis
Phlebitis
Test for FSP
THROMBO-WELCOTEST
-THROMBO-WELCOTEST
Whole blood is added to (?’ to ensure complete clotting and (?) after incubation, the px serum is diluted and mixed with (?) that have been coated with (?).
thrombin
soya bean enzyme inhibitor
latex particles
anti-fibrin split products
caused by traumatic phlebotomy
TISSUE THROMBOPLASTIN CONTAMINATION
: contains tissue factor (TF) released from injured cells that causes premature activation of extrinsic pathway (starts coagulation prior to testing)
tissue thromboplastin
e.g., release of [?] during probing, through and through puncture to the vein
TF
: probing is detrimental to the results
sodium citrate in coagulation tests (PT, APTT)
: probing can be considered
EDTA
TISSUE THROMBOPLASTIN CONTAMINATION
effect:
falsely shortened test result
Effect: use of plastic centrifuge tubes
alsely shortened coagulation time
room V and VIII easily deteriorates
Room Temperature (18-24 C)
stable for 24 hrs:
• PT plasma (remained capped until use)
stable for 4 hrs:
• APTT samples
• factor assay samples
VII and XI deteriorates
Ref/Cold Temperature
prevention: by pulling the plunger in sync with the speed of blood flow to the tube
frothing of sample
improper transfer from barrel to tube (frothing on top layer of blood)
• remedy: [?] (remove cap and let the blood run to the walls of tube)
recap
• transfer of blood from syringe to the rubber ([?]): only done in microbiology (bacteria is still present in hemolyzed samples)
SPS in yellow tube or goldtop
blue top:
EDTA:
4-5x
8x
Short draw (< 2.7 mL)
PT/PTT falsely prolonged
Failure to mix specimen after collection
PT/PTT falsely prolonged
Excess vigorous mixing
PT/PTT falsely shortened
Hemolysis
PT/PTT falsely shortened
Improper storage: wrong temperature or held too long
PT/PTT falsely prolonged
Chilling in refrigerator or placing on ice
PT/PTT falsely shortened
Inadequate centrifugation
PTT loses sensitivity for lupus anticoagulants and heparin.
Prolonged tourniquet application
Falsely elevates vWF, factor VIII
Drawing coagulation tube after to other anticoagulant tubes
PT/PTT falsely affected
Probing the vein
PT/PTT falsely shortened
Heparin contamination from line draw
PT/PTT falsely prolonged
Lipemia
Test may not work
Winged-needle sets needle gauge:
G23
for difficult or fragile veins; commonly used on babies, young children, and the elderly to draw blood or to administer medication using an IV
Winged-needle sets
▪ disadvantage: insoluble complex or precipitate of calcium oxalate interfere with spectrophotometric reading (optical density) incorporated in the automations of hematology coagulation machines
SODIUM OXALATE
combine with calcium in the blood to form insoluble complex or precipitate of calcium oxalate which causes depletion of calcium necessary for coagulation of blood
SODIUM OXALATE
such as in the use of PPP that is often recalcified (addition of calcium)
SODIUM OXALATE
less commonly used in hematology
SODIUM OXALATE
self-filling tube (due to vacuum)
TRISODIUM CITRATE
: presence of excess unbound citrate (prolongs the test)
incomplete filling
: may cause underfilling
high hematocrit (viscous blood)
(blood: anticoagulant)
9:1 ratio
coagulation reagents are sensitive to
citrate
concentrations:
- 3.2% (light blue)
- 3.8% (black)
3.2% (light blue)
- formulation:
0.105 M (glass) or 0.109 M (plastic)
- preferred for coagulation tests (PT and APTT)
3.2% (light blue)
3.8% (black) formulation:
0.129 M
overanticoagulation with high hematocrit blood samples (extra unbound citrate)
3.8% (black)
not used on coagulation studies (thrombin is inhibited)
HEPARIN
action: chelation of calcium
EDTA
FV is unstable in its presence (may cause deterioration)
EDTA
: plain discard tube to contain initial blood volume that may be contaminated with tissue thromboplastin)
a. Red stopper
: all other following tubes affect coagulation tests
b. Blue stopper (3.2%)
: chelates calcium thus must follow after tubes for coagualation tests (citrates)
c. Lavender stopper
: for additional tests (emergency purposes)
d. Plastic Syringe
any changes in the pH can
prolong clotting/coagulation time
uncapping and capping of the tube mediates the loss of carbon dioxide making the sample suffer from
alkalosis
rough test for intrinsic and common pathway (not reliable)
LEE AND WHITE METHOD
addition of an activator (diatome) in the blood must cause enhanced clotting time
ACTIVATED CLOTTING TIME
with the same value in Lee and White method (7-15 mins) indicates problem in coagulation
ACTIVATED CLOTTING TIME
normal PPP and PRP contains all necessary components to generate fibrin clot except
calcium
: x calcium; ✓ PPL
- PRP
: x calcium; x PPL
- PPP
PLASMA RECALCIFICATION TIME
recalcification (?) must coagulate and form fibrin clot
addition of calcium to PRP and PPP
one of the most important tests
ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT/PTT)
to determine/differentiate whether the patient has factor deficiency or just under heparin therapy
APTT
- used as a treatment for clotting to ensure normal blood flow
heparin therapy
- inhibits thrombin by acting on antithrombin-III
heparin therapy
targets of heparin:
- FII - FX
may be caused by heparin therapy and not by factor deficiency
• prolonged APTT/PT
• fraction of plasma which contains TF and PPL
thromboplastin
thromboplastin
• two types:
complete: provides both TF and PPL
incomplete: only provides PPL
: provides both TF and PPL
complete thromboplastin
: only provides PPL
incomplete thromboplastin
• in APTT: incomplete thromboplastin is used thus called
partial thromboplastin time
refrigerated thus, shift it in a body temperature
APTT
reagent does not need incubation, just invert it to maintain the suspension and prevent concentration of reagent at the bottom.
APTT
APTT Deficiency in intrinsic/common pathway factors:
XI, IX, VIII, V, X, II, I
circulating inhibitor: autoantibodies such as lupus anticoagulant
APTT
: a powerful inhibitor of thrombin in APTT
E dimer/Fragment E
: inhibit thrombin (no cleaving of fibrinogen into fibrin) in APTT
presence of FSP
use of PRP: presence of numerous platelets will try to react potentially thus falsely prolonging the test
APTT
TEST FOR EXTRINSIC AND COMMON PATHWAY
involves factors:
except
VII, TF, X, V, II, I
FXIII
: vitamin K antagonists
coumadin and warfarin
coumadin and warfarin consequently affects Vitamin K-dependent factors:
FII, FVII, FIX, FX (prothrombin group)
DO NOT INCUBATE SAMPLES AT 37 C FOR LONGER THAN 5 MINUTES TO AVOID THE LOSS OF FV and FVIII.
Tilt Tube Technique
Expected PT (Manual): presence of coagulum in 12-15 seconds (if not within the range, defective reagent)
Tilt Tube Technique
Note: A control plasma should be run together with the test to validate the reagents used
Tilt Tube Technique
the closer to 1, the more sensitive the reagent
International Sensitivity Index (ISI)
optimal ISI:
1.3-1.5
the PT reagent is calibrated against WHO thromboplastin reagent
International Sensitivity Index (ISI)
: first to disappear (very labile in vivo) with intake of oral anticoagulant and during liver disease
Factor VII
• affects both PT (falsely prolonged) and APTT (more prominent; prolonged) in PT
heparin contamination
Shortened due to traumatic venipuncture (probing): contamination of tissue factor
PT
assay similar to the prothrombin time
STYPVEN TIME/RUSSELL’S VIPER VENOM TEST
“Vipera ruselli/ Daboia ruselli” with a thromboplastin-like action (has [?])
TF and FVIII
contains a potent activator of FX which in the presence of PPL, prothrombin, and calcium ions cleaves fibrinogen to fibrin
“Vipera ruselli/ Daboia ruselli”
triggers coagulation at the level of factor X
STYPVEN TIME/RUSSELL’S VIPER VENOM TEST
: prolonged Russell viper venom clotting time (> 10 secs)
FX deficiency
- no clotting occurs even with the addition of venom (containing TF and FVII) to PPP
FX deficiency
- prolonged PT and PTT
FX deficiency
: does not prolong (normal) the Russell viper venom clotting time and results to formation of clot
FVII deficiency
• inhibits most coagulation factors
lupus anticoagulant
lupus anticoagulant
• usually targets
FV, FVIII and FX
• its presence causes massive bleeding
lupus anticoagulant
• involved in DIC
lupus anticoagulant
addition of thrombin does not require the activation of other coagulation factors in the cascade to cleave fibrinogen
THOMBIN TIME
THOMBIN TIME
sample:
reagents:
sample: citrated PPP
reagents: standardized thrombin solution and calcium
estimates fibrinogen concentration (thrombin time is proportional to the rate of fibrinogen polymerization
THOMBIN TIME
also used to monitor heparin therapy (more accurate than APTT)
THOMBIN TIME
sensitive to presence of increased levels of FSP
THOMBIN TIME
: considers heparin, intrinsic and common pathway factors
- APTT
: determine thrombin concentration and heparin
- thrombin time
- lacks protein fibrinogen or liver only starts to produce it
newborns
- has abnormal proteins (paraproteins) that impairs fibrinogen
multiple myeloma
used to confirm presence of fibrinogen deficiency in patients undergoing heparin therapy as it only affects thrombin ( [?]is a thrombin-like substance only thus not affected)
reptilase
absence of fibrin polymerization with the addition of reptilase indicates
fibrinogen deficiency
confirm presence of FSP through test for fibrinolysis
REPTILASE TIME
FXIII stabilizes fibrin clot so plasmin cannot dissolve it easily
DUKERT TEST (5 M UREA SOLUBILITY TEST) — TEST FOR FACTOR XIII
DUKERT TEST (5 M UREA SOLUBILITY TEST) — TEST FOR FACTOR XIII
: same mechanism with plasmin
urea
no dissolution of clot with NaCl salts
DUKERT TEST (5 M UREA SOLUBILITY TEST) — TEST FOR FACTOR XIII
high concentrations of salt could precipitate protein
DUKERT TEST (5 M UREA SOLUBILITY TEST) — TEST FOR FACTOR XIII
denatured proteins (thru heat or high CHON concentration) will initiate structure modification to become more stable (not easily destroyed)
DUKERT TEST (5 M UREA SOLUBILITY TEST) — TEST FOR FACTOR XIII
salts modified the protein structure (especially fibrinogen which has highest concentration) and makes clot solid as it is precipitated already (insoluble clot)
DUKERT TEST (5 M UREA SOLUBILITY TEST) — TEST FOR FACTOR XIII
plasmin keeps on working in fibrinogen to produce FDP or fibrin degradation products
PRIMARY FIBRINOLYSIS
not cleared by macrophages thus continued activation of plasminogen (increases plasmin)
PRIMARY FIBRINOLYSIS
plasmin simultaneously acts with clotting (normal)
SECONDARY FIBRINOLYSIS
coagulation in the brain but fibrinolysis is activated on other body parts (not useful)
SECONDARY FIBRINOLYSIS
keeps on activating plasmin but not used as coagulation occurred in other area
SECONDARY FIBRINOLYSIS
D-dimer is seen: indicates in vivo fibrinolysis
SECONDARY FIBRINOLYSIS
obstetric complications (pregnancy)
SECONDARY FIBRINOLYSIS
increased plasminogen or plasmin
Abnormal fibrinolytic activity: (<2 hours)
opposite to coagulation tests
Abnormal fibrinolytic activity: (<2 hours)
abnormal results cause prolong coagulation; abnormal test when lysis of fibrin is shortened
EUGLOBULIN TEST
displaces the secondary FDPs from fibrin monomer and spontaneously form gel-like clots (paracoagulation)
Protamine sulfate / 50% ethanol
when there is paracoagulation, this means that the d-dimer is still crosslinks with other fibrin monomers
PROTAMINE SULFATE TEST AND ETHANOL GELATION TEST
PROTAMINE SULFATE TEST AND ETHANOL GELATION TEST
gel-like clots:
presence of FDPs
confirmation after performing reptilase time
PROTAMINE SULFATE TEST AND ETHANOL GELATION TEST
specific marker for fibrin formation
D-DIMER TEST
the detection of D dimer is not good
D-DIMER TEST
• plasmin should stop at FDPs and be removed from the circulation
D-DIMER TEST
• it is not safe that clots are dissolved into FDP due to anticoagulant properties
D-DIMER TEST
presence of D dimer:
absence of D dimer:
flocculation
no flocculation
D-DIMER TEST
normal values:
<200 ng/mL
anti-FPS will be added to the blood
THROMBO-WELLCOTEST
• presence of FPS:
• absence of FPS:
THROMBO-WELLCOTEST
flocculation (small aggregates)
no flocculation
confirmatory test after protamine sulfate test wherein there is abnormal result and when the D-dimer test is negative
THROMBO-WELLCOTEST
tests for abnormal fibrinolytic activity
EUGLOBULIN TEST
determines what causes abnormal fibrinolytic activity or distinguishes primary vs secondary fibrinolysis
PROTAMINE SULFATE TEST AND ETHANOL GELATION TEST
PROTAMINE SULFATE TEST AND ETHANOL GELATION TEST
No gel formation Primary fibrinolysis (?)
Gel formation (positive) Secondary fibrinolysis (?)
excess tPA; administer inhibitors needed
presence of FDPs
to confirm if D-dimer is the cause of the abnormality (if negative)
D-DIMER TEST
to confirm other FSP products (X, Y) which may cause the abnormality
THROMBO-WELLCOTEST
(determine specific factor deficiency)
• factor assay samples
its presence is specific of fibrin formation
D-d
