Week 8 Flashcards
What are the three types of errors that can form in hemostasis - can cause either bleeding or clotting
Inability to clot- uncontrolled bleeding only when injured
tendency to clot or unregulated coagulation - lack of inhibitors only initiated by injury, in pts having thrombotic events
Inability to break down clot - issue with fibrinolytic system where the clotting stops but the clot stays and blocks blood flow
What is unregulated coag caused by
- lack of required inhibitors (increased tendency to clot)
leads to :
Thrombosis - that can cause
Stroke
Pulmonary embolism
Deep vein thrombosis
What does the inability to clot lead to and what is it caused by
-caused by of single or multiple coag factors or their inhibitors
Single factor - def in FVIII- patient cant form intrinsic tenase complex affecting thrombin production - leads to bleeding upon injury
Multiple factor def- due to liver disease which affects their concentration - lots of blood during injury
Inhibitors to factors- are the result of antibodies to factors or to phospholipids that neutralize the factor or complex - cannot participate in coagulation
Hemorrhage
Chronic inflammation
And can result in Transfusion dependence
How do we do lab testing for hemostasis
-assess in vitro coag by measuring the time it takes for a clot to form
-determines cause of excessive bleeding or clotting
Missing/deficient factors
Inhibitors of factors (and also those that break down the clot)
Abnormal Fibrinogen
Abnormal Thrombin
Fibrin Degradation Products
-is used to monitor anticoag therapy or the effectiveness of the therapy -common routine test
What is used for anticoag therapy
drugs like
Warfarin (Coumadin)
Unfractionated Heparin (UFH)
Low Molecular Weight Heparin (LMWH)
Direct-Acting Oral Anticoagulants (DOACs)
Direct Thrombin Inhibitor (DTI)
Aspirin (antiplatelet agent/drug)
how do anticoags work what will happen if too much or too little is given
Anticoagulants, work by suppressing coagulation and reduce thrombin formation.
With Coumadin or Heparin therapy (most common)-
If too much is given, person may bleed excessively
With too little, the person continues to clot
Therefore, the patient’s dosage or therapy needs to be well monitored
what are the comments associated with rejections
Tourniquet for more than a minute
sample storage at 1-6
sample storage at 25
Tourniquet for more than a minute - hemostatsis activates endothelial cells and increases vWf and Fib concentration which falsely decreases clot based results
sample storage at 1-6- fridge temps precipitate vWf factor multimers, activate FVII, PLTs, and destroy PLT integrity
sample storage at 25 -FV and FVIII deteriorate
how to collect a sample from vascular access devices
like IV, central line, dialysis catheters
-Line should be flushed with 5mL saline and the first 5mL of blood must be collected and discarded
-Frequently contaminated with heparin - used to keep the lines flowing or “open”
why is capillary testing not acceptable for coag testing
-Because it can be contaminated by connective tissue procoagulants (such as, tissue factor) or tissue fluid (relative to plasma or blood cells- no ‘milking’ of fingers)
POCT instruments exist for PT/INR using capillary samples (whole blood) - NEED FREE FLOWING PUNCTURE
what temp and how long is storage okay for
PT with no UFH
PTT with no UFH
PTT for monitoring UFH
PT when UFH is present
in household freezer
store for 6 months
PT with no UFH-15-25 but test in 24 hr upright and sealed
PTT with no UFH 15-25 but test in 4 hr upright and sealed
PTT for monitoring UFH- 15-25 separate plasma in an hour and test in 4
PT when UFH is present - 15-25 separate plasma in an hour and test in 4
in household freezer - -20 keep for 2 weeks
store for 6 months- -70 keep for 6 months to indefinite
what are the coag collection tubes
3.2% buffered Sodium Citrate
Sodium Citrate leaves all clotting factors available except Calcium
Citrate binds the calcium in the plasma and prevents coagulation, but not as strongly as EDTA
When coagulation tests are performed in the lab, calcium is added to allow clot formation. Citrate is used as an anticoagulant for clotting tests becauseit is has a low saturation level and its effects can be reversed by the calcium levels in the clotting reagent
what are the comments associated with rejections
short draw
sample clot
hemolysis
lipemia or icterus
short draw- Whole blood less than 90% of required. PT, PTT are prolonged
sample clot - sample inspected before centrifugation as clots affect hemostasis test result
hemolysis- pink or red plasma = in vitro activation of PLTs and coag. test interference. Hemolysis affects optical endpoint of coagulometer results
lipemia or icterus - optical intruments wont measure clots if sample is cloudy or colored. this type of sample will affect chromogenic substrate methods . Use electromechanical methods instead
how do you prepare the plasma you need to test
-plasma that has the coag factors is seperated from PLTs (phospholipid source) by centrifugation3000 x 15 at RT = PLATELET POOR PLASMA PPP <10 X 10^9
Plasma is then tested
-PT/INR and/or APTT ordered and tests completed
-To allow for clot formation, calcium and phospholipid are added back in for testing
-PT- rapid test
-APTT- longer test due to an incubation period
use a swing out bucket rotor so remixing is avoided
why isnt EDTA used if it also chelates calcium
Because FV and VIII are less stable in EDTA (more stable in sodium citrate).
EDTA will continue to chelate the calcium ions when added during coagulation testing.
sample collection types
Plasma
Anti-coagulated
All coagulation factors present
Serum
Clotted sample
Coagulation factors are used up during the clotting process:
Fibrinogen is predominantly consumed
Some other factors consumed - V, VIII and XIII
what are the in vitro coag assessment screening tests done in the lab
- invitro tests to measure the time it takes for a clot to form
Prothrombin Time (PT) &
Activated Partial Thromboplastin Time (APTT or PTT)
-these tests are starting points for excessive clotting or clotting disorders. Then you order the reflux tests to see what factors are decreased, deficient or absent.
what ration of blood to anticoag is needed for best results and when it affected
9:1 ratio affected when there is high HCT = High RBC = Less plasma which prolongs coag results
-if you have high HCT the citrate concentration is increased as it binds free ionized CA and when the clotting test reagents are added the excess citrate will bind to even more calcium which part of the reaction causing artificial increase in clotting time
When is PT/APTT ordered
- detection of mucocutaneous or anatomic bleed
-before surgery
-for routine monitoring of coag
PT/APTT may be ordered when a person who is not taking anticoagulant drugs has signs or symptoms of a bleeding disorder:
Nosebleeds (recurrent)
Bleeding gums
Bruising
Heavy menstrual period
Blood in the stool and/or urine
Arthritic-type symptoms (damage from bleeding into joints or other soft tissues)
When do we see instances of more RBC= LESS PLASMA = HIGH HCT in vivo
Cardiac patients have increased HCT
Decreased oxygen = increased RBC production to compensate
Neonates have high HCT and need corrected tubes (corrected for amount of blood and anticoagulant) to get proper results
Polycythemia Vera (rare)- marked increased RBC count (by virtue of this increase, there is less plasma and more RBCs - HCT will be increased as well)
When is PTT ordered
when is TT ordered
When is FG ordered
PTT ordered- prolonged clotting time low in all factors EXCEPT FVII and FXIII
TT ordered - when clotting prolonged by unfractionated heparin therapy, dysfibrinogenemia, and afibrinogenemia - qualitative
When is FG ordered - reduced in hypofibrinogenemia, and afibrinogenemia - QUANTITATIVE
When is HCT, HBA1C and RET ordered
when is PLT count ordered
When is PT ordered
HCT, HBA1C and RET ordered for chronic bleeding, hemolytic anemia, bone marrow response
PLT count ordered for thrombocytopenia
PT ordered when clotting time is prolonged, low in FII, FV, FVII, FX
What is PT- prothrombin time
what does it evaluate
what is the test sensitive to and insensitive to
-PT – clot-based routine coagulation test (time to clot is measured)
-Evaluates deficiencies or inhibitors of the factors in the extrinsic and common pathways:
Sensitive to
-FVII, FV, FX, FIB, PROTHOM deficiency
Insensitive to
FVIII, FIX, FXIII deficiency
-Results are used to monitor Warfarin/Coumadin therapy
What is PT prolonged with
-with (Prolonged PT/INR with normal APTT)
Congenital deficiency of FVII or congenital (or hereditary) single-factor deficiency of FX, V, prothrombin, or Fibrinogen
Disseminated intravascular coagulation (DIC)
**Liver disease (early)- FVII has shortest ½ life deceases quick because factors stay in des-gamma-carboxyl form
Vitamin K-deficiency (early)
-can be increased in presence of DTI or anti Xa
how do you distinguish between vitamin K deficiency and liver disease
-lab determines Fv and FVII, both are decreased in liver disease but only FVII is decreased in V K deficiency
how do you test for PT
PT reagent - source of Tissue Factor or Thromboplastin or Tissue Thromboplastin
ACL Elite PT reagent - RecombiPlasTin®
Recombinant Thromboplastin - commercially prepared
Requires reconstitution using diluent
Suspended in phospholipids
Mixed with buffered solution of calcium chloride (source of ionized Ca2+)
-PT reagent warmed to 37
-PPP warmed to 37 for 3-10 mins anything longer coag factors degrade, or are affected by evaporation or pH
-add reagent quickly
-clot forms, timer stops and time in secs noted
-clot detected visually or by electrical sensors
ranges are determined by the lab
What does the PT (thromboplastin) reagent do? IN VITRO testing
-Activates the extrinsic and common pathways
-Begins with FVII to Fibrin polymerization = Fibrin clot formation
-commercial reagent has Ca and phospolipids that also partake in the reaction to form
-TF:FVIIa complex (extrinsic tenase complex)
-Factor FXa:Va complex
-Thrombin activation, fibrin polymerization, and fibrin clot formation
What is Activated Partial Thromboplastin Time (APTT/PTT)
what does it assess and monitor
APTT – clot-based routine coagulation test
-used to assessfactors in the intrinsic and common pathway:
Factors XII, XI, IX, VIII, X, V, II (prothrombin), I (fibrinogen) – all factors except FVII
-monitor standard (unfractionated) Heparin anticoagulant therapy
what can interfere with Prothrombin Time & INR
-Alcohol (can slow dow breakdown of warfarin - causing a buildup in the body)
- antibiotics (increase PT/INR)
-Barbiturates, oral contraceptives, and hormone-replacement therapy (HRT) (decrease PT or prothrombotic)
-Foods that contain large amounts of vitamin K can interfere with Warfarin therapy
-pt history is important and a bleeding/thrombosis risk assessment is done by the doctor
how are PT results standardized if they change based on labs and thromboplastin variations
how is the calculation done?
-International Normalized Ratio (INR)
-allows for monitoring of Vitamin K antagonist therapy (such as, Coumadin/Warfarin) across different labs
-INR with PT result
ISI = International Sensitivity Index and PT in sec are used to calculate INR
-ISI shows how sensitive the reagent is to V K dependent factors it is compared to WHO ref standard
INR= pt PT/mean PT (from 20 normal plasma samples) to the power of ISI of the reagent used
What is APTT/PTT used for
-heparin therapy
-detect Specific anticoagulation factor antibodies (i.e., Lupus Anticoagulant and/or anti-factor VIII antibody) or non-specific inhibitors
- detect Coagulation factor deficiencies
-Determine cause of Unexplained bleeding or easy bruising
-Determine cause of recurrent miscarriages
-Determine if Disseminated Intravascular Coagulation (DIC) is present
-Determine reason behind recurrent thrombosis
-Prior to surgery when the surgery carries an increased risk of blood loss and/or when the person has a clinical history of bleeding
What is APTT prolonged with
-Congenital deficiency of FVIII, IX, or XI (usually a single factor deficiency)
-Def of FVIII & IX associated with bleeding
vWF disease has low FVIII and is associated with bleeding
-Def FXI variably associated with bleeding
-Contact factor def can elevate APTT but does not result in bleeding tendency
Acquired inhibitors, specific or non-specific
-Specific inhibitors directed against specific factors (commonly against FVIII)
-Non-specific inhibitors can be drugs (heparin, rivaroxaban, apixaban, or edoxaban) or antiphospholipid antibodies (lupus anticoagulants)
-Patients on Direct Thrombin Inhibitors
what reagents are part of the APTT lab testing
Reagent 1: Contact activator
Contact activator (negatively charged particulate activator, such as, silica, ellagic acid, or kaolin)
Phospholipids (PL) with NO TF or Ca2+
A ‘partial thromboplastin’ because it lacks Tissue Factor
ACL Elite: SynthASil®
Reagent 2: Calcium chloride
CaCl2 is added separately
ACL Elite: Calcium chloride
how is the 2 part lab test for APTT done
-PPP + R1 (contact activator + PL ) incubate for 3 min at37
Contact factor group activates FXI-FXIa which activates FIX-FIXa. Contact factor group= FXIIa + pre-K + HMWK. Intrinsic factor activation.
-R2 (CaCl2) added, mix at 37, start timing in sec
The Ca allows FIXa and FVIIIa to bind with phospholipid producing the Intrinsic tenase complex IXa:VIIIa + PL + Ca2+ that forms the Prothrombinase complex, Thrombin activation and clot formation.
-stop timing when you see fibrin strands
-stop fully when you or electrical sensors see a clot
each lab has their own reference values
-PT on heparin are 1.5-2.5X mean so multiply 1.5 for low end of mean and 2.5 for high end
-ranges must be re-established once a year
What interferes with APTT
Drugs: Antihistamines, ascorbic acid, chlorpromazine, heparin, and/or salicylates
Incorrect anticoagulant
Hematocrit that is markedly increased or decreased
Blood sample from heparin lock or heparinized catheter
how valid are the results if there is
UFH therapy
LUPUS Anticoag
Incorrect calibration, dilution of reagents
UFH therapy- use a reagent that is not sensitive to UFH one that has a UFH neutralizer like a polybrene
LUPUS Anticoag- PT and PTT results are invalid use a chromogenic assay instead
Incorrect calibration, dilution of reagents -correct the error and repeat
What is step 1 in investigating abnormal results
check sample
if coagulation test results appear short (or are more rapid) than RI’s, we do not investigate as they are not clinically significant.
-Was sample collected with correct anticoagulant & in a properly filled tube?
-ratio okay?
-Clotted / Hemolyzed / Lipemic / Icteric?
Clotted - means coagulation factors are all used up and we cannot test them
Hemolyzed/Lipemic/Icteric - may affect ability to measure clot on some analyzers and can falsely shorten or prolong testing
Check patient HCT ≥ 0.55 L/L?
-redraw and adjust anticoag volume used due to skew blood:plasma ratio = more RBC than plasma
What is step 2 in investigating abnormal results
Check Patient History
-compare with last results
-pf on Anticoag therapy? Need to reason for prolonged result if so release
-if not is there a diagnosis example liver disease affects PT then PT/APTT, V K PT/APTT affected
-if history or diagnosis correlates with result then release
What is step 3 in investigating abnormal results
Check Other causes
investigate if patient has:
Decreased fibrinogen levels
Specific single or multiple factor deficiency
Inhibitor or antibody to factors
Inhibitor (non-specific) or antibody interfering with test. because pt with SLE develop AB that affect APTT in vitro due to the incubation time (the time is enough for AB to neutralize PL in reagent)
-DO REFLEX AND ADDITIONAL TESTING
Thrombin Time
Fibrinogen
D-Dimer
Mixing Studies
Inhibitor testing
Factor Assays
What is Thrombin Time (TT)
what type of test is it
-assess deficiencies of fibrinogen or the presence of an inhibitor to thrombin (FIIa)
-referred to as Thrombin Clotting Time (TCT)
Recall, Thrombin polymerizes fibrinogen to form fibrin by cleaving fibrinopeptides A and B from plasma fibrinogen to form a detectable fibrin polymer
Qualitative test
This test bypasses all other factors and assesses the activity of fibrinogen
The less fibrinogen available the longer it takes to form a clot
What causes a prolonged TT
Congenital Fibrinogen deficiency
-Decreased fibrinogen level (quantitative - -hypofibrinogenemia or afibrinogenemia)
-Abnormal fibrinogen function (qualitative - dysfibrinogenemia)
Acquired by Heparin therapy, direct thrombin inhibitor therapy or contamination
Heparin contamination of a blood sample is suspected because samples are drawn through central line (heparin neutralization procedures are now used more commonly)
Acquired via Presence of an inhibitor of thrombin (FIIa) or fibrinogen
Acquired hypofibrinogenemia
Liver Disease
Massive hemorrhage
Abnormal fibrinolysis
increased products of clot breakdown like in DIC that affect fibrin polymerization
When is TT ordered
Bleeding or thrombotic episodes
Recurrent miscarriages
PT and APTT are unexpectedly both prolonged. Do TT to directly assess Fibrinogen function
how do you test for TT
PPP and reagent warmed at 37
-add reagent to PPP-Commercially prepared bovine Thrombin reagent . once mixed the thrombin will degrade
-start time with Thrombin addition and stop at clot
-test is Qualitative we only measure the time it took for the clot to form not how much fibrinogen is there or how much Fibrin is produced
-Thrombin time is most sensitive to fibrinogen and presence of inhibitors of thrombin (Factor IIa).
-Specimen errors that affect the PT and APTT also affect the TT.
What is the fibrinogen assay
Modification of the TT - estimates the concentration of functional fibrinogen
Measures the rate of conversion of fibrinogen to fibrin in a diluted sample with of excess thrombin
Clotting time correlates with levels of active fibrinogen that is present (inverse relationship)
When is a fibrin assay ordered
Acquired hypofibrinogenemia
-Bleeding episodes (consumption of fibrinogen)
-Consumption such as with traumatic injury or DIC (hyperfibrinolysis)
-Decrease production seen in severe Liver Disease
Assess inflammatory conditions
Fibrinogen is an acute phase reactant and can be non-specifically increased during acute or chron inflammation
Recurrent miscarriages
Suspected congenital fibrinogen deficiency or abnormality
Hypofibrinogenemia (rare congenital decreased FIB levels)
Dysfibrinogenemia (dysfunctional FIB)
PT and APTT test are both unexpectedly prolonged
when physician wants to evaluate pt risk of developing cardiovascular disease
how is the Fibriogen assay done
-ppp + bovine thrombin is prewarmed at 37
-PPP diluted with buffer like Owren buffer 1:10 (calibration curve determined with normal plasma)
-reagent added with concen of thrombin where thrombin cleaves fibrinogen to fibrin. So we are trying to see if there is any activity if Fibrinogen is at a low level
-measure time in seconds and compare with calibration curve. Time and Fib = inverse
When is a dilution needed in the fibrinogen assay
For low fibrinogen, a lower dilution may be needed
For high fibrinogen a higher dilution may be needed
In either case, result(s) are multiplied by the dilution factor to produce the correct result
curve is produced by the owren buffer dilutions
What is Ddimer
-specific product of digestion of cross-linked fibrin ONLY
-Marker of Thrombosis and Fibrinolysis
-breakdown products of fibrin clot digestion by Plasmin
-Plasmin cleaves fibrin, producing fibrin degradation products/fibrin fragments, like D-Dimer
-Ddimer has two D domain fragments from fibrin monomers, that are crosslinked by FXIIIa as clot forms
What can cause increased D dimer
-increased D Dimer indicated clot formation and breakdown
-not specific to thrombosis
Deep vein thrombosis (DVT)
Pulmonary embolism (PE)
Recent surgery or trauma
cancers
Acute or chronic infections or inflammatory diseases
DIC
Very heavy menstruation
Normal Pregnancy - levels rise in normal pregnancy however, very high levels are associated with complications
Healthy elderly patient
What can you tell between a negative and positive D dimer
-negative D-Dimer used to rule out the diagnosis of venousthromboembolism . So if there are no clot being formed or broken down then the pt does not have a thrombotic condition
- ## positive or elevated D-Dimer alone is not diagnostic of DIC, DVT or PE. They are non specific when elevated or positive
How do you do the testing for D Dimer
Quantitative Analysis- Turbidimetric Immunoassay:
D-Dimer Latex Reagent - polystyrene latex particles coated with a monoclonal antibody highly specific for the D-Dimer domain (epitope)
-ACL mixes control or pt plasma with Latex and Buffer = agglutination means Ddimers are present
Agglutination is measured by a decrease in transmitted light at 405nm
Degree of agglutination is directly proportional to concentration of D-Dimer in sample
how are D-Dimer Assay results reported
Positive (agglutination) or Negative(no agglutination) if the test is used for a qualitative assessment only
In quantitative or semi-quantitative measurements, D-Dimer reported in D-Dimer Units (DDU) or Fibrinogen Equivalent Units (FEU)
how do you do a qualitative analysis of D-Dimer
Slide Latex Immunoassay Procedure
-needs a pos and neg control with Reagent 3/4
-mix R1 with pt sample and rock the slide like Pathodx
-view agglutination
what are instrument methodologies used for high-volume coagulation and hemostasis testing
Endpoint detection - testing resulting in a finite endpoint and for routine coagulation testing this is clot formation
Mechanical
Photo-optical (turbidometric)
Nephelometric - our ACLs use this method
Chromogenic (amidolytic)
Immunologic
Viscoelastic
what is mechanical endpoint detection
-uses magnetic sensor that monitors the movement of a steel ball within the test plasma NOT AFFECTED by plasma
Two principles are used for the mechanical clot detection and what are the differences
Electromechanical I
-as fibrin clot forms , vicosity of test plasma and reagent increases slowing ball movement
-when the movement stops , timer stops = clotting time
Electromechanical II
-steel ball is put in inclined well and its position is detected by magnetic sensor
-the well rotates but the ball stays put
-when the fibron clot forms the ball is moved from position indicating clotting time
Photo-Optical Endpoint Detection
used by automated and semi auto coag instruments to detect clots
-Measures the change in plasma optical density (OD or light transmittance) during clotting (Turbidimetric analysis)
-Formation of fibrin strands causes the light to scatter = less arriving at the detector
-as clot get denser the more light is scattered OD increase
-A baseline OD is determined based on the color and clarity of the sample
-Baseline OD is subtracted from final OD – this minimizes the effects of icterus or lipemic samples
What is Nephelometric Endpoint Detection
-side scatter or forward-angle light scatter is measured
-A light-emitting diode produces incident light at a 600 nm
-A photodetector detects variations in light scatter at 90 degrees (side scatter) or at 180 degrees (forward-angle scatter)
-As fibrin polymers form, side scatter or forward-angle scatter rise
-timer stops when scatter reaches a predetermined density
-Continuous readings throughout and a clot curve is produced
What is chromogenic detection
-Measures specific coagulation factor activity, by exploiting Factors’ enzymatic (protease) properties
-colorless, synthetic substrate is attached to a chromophore like a protease structure
-Activated Factors cleaves the chromogenic substrate and OD of solution is measured at 405nm
-OD of solution proportional to protease activity
-chromophore is released and measured at a photodetector at 405nm - BILIRUN WILL AFFECT THE RANGE -THEY ARE BOTH YELLOW
-optical detection of the solution is proportional to protease activity - direct chromogenic assay
-Indirect is also used
What is Immunologic detection
-Based on Ab-Ag reaction with coagulation antigen
-latex coated AB against AG
-Monochromatic light of a greater wavelength passes through the suspension of latex micro-particles and absorbed light is measured
-Antigens - coagulation factors and proteins, such as D-Dimer
-after agglutination light is absorbed because the particles have wavelength larger than the light
-The absorbance is proportional to the agglutinate size which is proportional to the antigen level
What is Viscoelastic detection
Used for whole blood clotting - Thromboelastography
Provides information on:
Time to clot
Kinetics of whole blood clot formation
Clot strength
Fibrinolytic activity
What does the ACL Elite Clot Curve have
-as fibrinogen specimen converts to fibrin and a solid clot, the change in light transmission through the specimen is recorded.
baseline-recorded before clotting occurs
acceleration phase-reflects clotting and is normally steep because clotting is rapid.
deceleration phase-represents the decreasing rate of clot formation as all available fibrinogen converts to fibrin.
end-point-flat and stable, reflecting consumption of all fibrinogen.
-absorbance on the y axis gives the time interval of when the clot was detected
What are the sources of error for the ACL Elite Quality Control-
-preparation of lyophilized controls and reagents -make sure volumteric pipette is chip free
-no foam production
-dont use contaminated water
-calibrate analyzer
