Coagulation studies

Question 1

Steps in investigations of abnormal coagulation profile

Answer 1

• Rule out pre analytical, analytical and post analytical issues
• If only PT/INR and APTT are prolonged check for warfarin therapy.
• Fibrinogen and D-dimer levels can exclude DIC.
• TCT will exclude heparin, dabigatran or paraprotein.
• An anti-Xa assay is useful to exclude LMWH, UFH, rivaroxaban or apixaban.
• Mixing test may or may not be useful here as some inhibitors a time dependant.
• If all these are normal, running specific clotting factor assay at a wide range of dilutions are useful.

Question 2

Bethesda assay principle

Answer 2

• Inhibitors are antibodies that form against specific clotting factors (e.g. f8, f9)
• They may be time-dependent (e.g. f8) or immediate acting (e.g. f9)
• They can develop in patients with haemophilia (allogenic), or without a bleeding disorder (autoantibodies)
• Assay to quantify amount of a factor VIII inhibitor present.
• Serial dilutions of the patient sample are incubated for 2hrs at 37ºC with a known amount of factor VIII.
• Amount of FVIII in the incubation mixture is corrected for the expected deterioration of FVIII after 2hrs by comparing:
  o FVIII in the normal plasma + patient plasma mixture
  o to that of a mixture of normal plasma and diluent (referred to as the control tube).
• 1 Bethesda Unit corresponds to 50% residual Factor VIII after 2hrs incubation at 37°C
  o Aka the amount of inhibitor required to inactivate half of the Factor VIII present in the patient plasma/NPP test mixture.
• Results are expressed as Bethesda units/mL (normal range 0 – 0.5 BU/mL)

Question 3

Sample requirements for bethesda Assay

Answer 3

• PPP
• Patient plasma heated to 58°C for 90mins then centrifuged to remove precipitated proteins.
  o In patients receiving treatment with a factor VIII concentrate, the measurement of inhibitors can be problematic as residual factor VIII may be present
  o Heat treatment/inactivated PPP  to a FVIII:C and  FVIII:Ag of <0.10 IU/ml.
  o Igs are heat resistant so the inhibitor level in the plasma sample is unaffected.
• This resulting supernatant is free of all clotting factors, but still contains the IgG inhibitor in an active form.

Question 4

Bethesda Assay Method

Answer 4

• Control Tube 1: Equal parts of commercial FVIII deficient plasma is mixed NPP (buffered to pH7.4) .
• Patient Tube 2: Equal parts of heat inactivated PPP is mixed with NPP
• Serial dilutions are made of the heat inactivated PPP with the commercial FVIII deficient plasma to titrate the antibody.
• All tubes are covered and incubated at 37 degrees for 2 hrs. Time to allow the inhibitor to fully express itself.
  The use of the control corrects for the deterioration in f8 and f5 during the incubation period.)
• A factor 8 assay is now performed on all tubes using a standard one stage factor VIII assay
• The percent residual FVIII per patient tube is calculated using the following equation:
• “Residual factor activity is defined as the relative percentage factor activity of the test mixture compared to the control mixture”
• Only residual factor VIII% between 25% and 70% should be taken from the graph.
• A log-linear plot reflecting the correlation between residual activity FVIII% and inhibitor level is used to determine the patient inhibitor level
• The dilution factor for each tube is applied to the graph reading to obtain BU/ml which is then multiplied by the dilution factor
• residual FVIII levels between 25% and 70% should derive comparable inhibitor levels from the graph once corrected for dilution.
• Where increasing dilutions do not give acceptable duplicates the antibody is displaying non linearity. In this instance the dilution closest to residual factor VIII of 50% has been found to be the most clinically useful.

(The incubation step is not required for a f9 inhibitor, since these are immediate acting.)

Question 5

Factor VIII inhibitor detection by ELISA 

Answer 5

• Recombinant f8 is immobilized on a microtitre plate, and diluted patient plasma is added to wells and incubated
• If an anti-f8 antibody is present, it will bind to the immobilized f8
• The plate is washed, and any unbound material is removed
• Labelled anti-IgG is added to the wells and incubated
• The unbound anti-IgG is removed by washing, and substrate (which reacts with the label of the IgG) is added that can be measured at an optical density at 405 nm, from which the presence of a f8 antibody can be established
• This method picks up both neutralizing and non-neutralizing antibodies, unlike the Bethesda, which only picks up neutralizing antibodies

Question 6

APTT (Activated Partial Thromboplastin) summary

Answer 6

• Clot-based test that evaluates the overall efficiency of the classical intrinsic pathway – APTT reagent = Actin FS (purified
  soy phosphatides)
• Sample: platelet-poor plasma (PPP) – citrate tube
• The APTT measures the clotting time of plasma after the addition of a contact activator (reagent used is Actin FS), phospholipid and CaCl2 – but without tissue thromboplastin
• It depends on the contact factors within the classical intrinsic pathway (8,9,10, 12), as well as X, V, prothrombin and fibrinogen
• It is also sensitive to the presence of circulating anticoagulants (inhibitors) and heparin

Question 7

APTT/PT/Fibrinogen and TCT QC

Answer 7

Internal QC:
- commercial normal and abnormal controls, tested with every new vial, at 7am (startup) and then 8 hourly

All other tests must have controls run with each batch, change of reagent vials or at least every 24 hours. EG: DDimer, Lupus, APCR, VWF

External QC:
- RCPA QAP (2 samples, 4/year)

Question 8

Warfarin MOA

Answer 8

Warfarin competitively inhibits the vitamin K epoxide reductase complex subunit 1 (VKORC1).

–> blocks the gamma-carboxylation of glutamic acid residues of the Vitamin K−dependent coagulation factors II, VII, IX and X.

Question 9

Heparin MOA

Answer 9

UFH is a polysaccharide with MW of 3-30 kDA.
Works indirectly by binding to AT –> causing a conformational change –> converts AT from a slow to a rapid inactivator of coagulation factors.

LMWH is purified from UFH has a more uniform size between 3.5 – 5 kDA  

UFH is able to accelerate (up to 2000 fold) the inhibitory effect of antithrombin,

Heparin AT complex inactivate factors IIa (thrombin), Xa, IX, XI, XII and plasmin.

UFH, LMW hepаrin, and fondaparinux all inactivate factor Xa, but unfractionated heраrin also inhibits thrombin (due to the long heparin chain)
Fondaparinux appears to have nearly pure anti-factor Xa activity.

Question 10

Heparin Resistance

Answer 10

Requirement of unusually large doses of heparin in order to achieve APTT in therapeutic range.
- eg > 35 000 units of heparin in 24 hours, excluding initial bolus and infusion rate of >400 units/hour in CAGS patients

Some predictors of heparin resistance:
- baseline AT activity level <60%,
- platelet count >300,
- age >;65

Question 11

Causes of Heparin resistance

Answer 11

• Antithrombin deficiency
  > sepsis, liver disease, ECMO, bypass, congenital
• Heparin-binding plasma proteins ( factor 8, vWF, acute phase reactants)
• Increased heparin clearance

Question 12

Laboratory investigation of heparin resistance

Answer 12

Ensure correct collection of sample. Not from heparin line/arm

Recommendation is Anti-Xa as excludes interaction from upstream effects

APTT
–> PPP, Actin FS (contact factor activator), calcium and phospholipid.
–> measured by either optical of mechanical endpoints
–> usually first test to identify heparin resistance

Anti-Xa
–> Citrated patient plasma is added to an excess of factor Xa;
heparin and AT in the patient plasma will inhibits factor Xa;
residual Xa activity is then measured via cleavage of a chromogenic substrate and is inversely proportional to the anticoagulant present

UFH target is 0.3-0.7
LMWH target is 0.5-1.0

Advantages over APTT include being insensitive to low factor levels (e.g. in DIC), high factor 8 levels (e.g. in critical illness), or antiphospholipid antibodies

Chromogenic assays are affected by haemolysis, bilirubin and lipids

ATIII levels
–> Plasma is incubated with heparin and excess prothrombin (2a)
–>ATIII in the patient plasma complexes with the heparin —> inhibiting factor 2a

Residual factor 2a cleaves a chromogenic substrate, which is measured optically at 405nm, and is inversely proportional to the ATIII concentration in the sample

Alternative reagents include bovine factor 2a; or bovine factor 10a

Will overestimate ATIII levels in the presence of an anti-Xa DOAC if the 10a method is used (and in the presence of dabigatran if the 2a method is used)

Question 13

Management of heparin resistance

Answer 13

little evidence for antithrombin replacement in acquired antithrombin deficiency  

Recommendation to continue uptitrating heparin to therapeutic antiXa levels (0.3 - 0.7)  

Question 14

HITS

Answer 14

Type 1
Non-immune mediated response to heparin therapy
Much more frequent that type 2
Occurs in 10-30% of patients receiving heparin
Typically presents with mild thrombocytopenia within the first 2 days of treatment
Self limited, direct effect of heparin and normalisation of platelet count occurs spontaneously without discontinuation of heparin therapy

Type 2
Immune mediated
Highly prothrombotic state resulting from pathogenic antibodies to platelet factor 4/heparin (PF4/H) complexes
Commonly develops 5-10 days after exposure to heparin

Question 15

Clinical suspicion of HITs

Answer 15

4T score used to evaluate the pre test probability of HITs and in correlation with the screening test
(3 or lower - NPV of 0.998)

Score based on degree of thrombocytopenia and nadir
Timing of onset after exposure to heparin therapy
Presence of thrombosis
Other causes of thrombocytopenia likely/unlikely

Question 16

Management of HITs

Answer 16

Discontinue heparin-containing agent 

Administer parenteral alternative to heparin

Warfarin may be commenced only once platelet count >150 or stable baseline –> due to risk of limb gangrene/skin necrosis secondary to profound protein C depletion in setting of anti-PF4/heparin Abs 

Warfarin reversal recommended if patient already warfarinised 

AC continued for minimum of 4 weeks (if no thrombosis), 3 months with thrombosis 

Question 17

Laboratory investigations in HIT

Answer 17

FBE + film
Coagulations
> thrombocytopenia and DIC and consideration of other differentials

Lateral flow immunoassay

HITs Acustar (reference lab)

Serotonin release assay (interstate/NSW)

Question 18

HIT - Lateral flow immunoassay
Method

Answer 18

Sampled - Citrate collection
Serum sample

STic EXPERT HIT test strip is a lateral flow immunoassay for the detection of IgG Ab to
PF4/polyanion-complexes in plasma or serum.

Rapid nano-particle based lateral flow immunoassay.

IF there are IgG Abs present they bind to ligand labelled PF4/polyanion complex and migrate through the pad.
Gold nanoparticles which carry an anti-ligand Ab bind to the ligand labelled Pf4/polyanion strip complex during migration and then immobilized on test strip –> colored line appears.

Control line at end of strip captures the excess god particles.

Sensitivity/ Specificity:
100% Sensitivity
93% Specificity

Question 19

Acustar HIT

Answer 19

Send to reference lab

Batch run
Sensitive but not specific
This is a chemiluminiscent assay

Detection of anti-PF4 antibodies 
Magnetic particles are coated with PF4/polyvinyl sulfonate and capture PF4/heparin antibodies .
Magnetically seperate and wash.
An anti-human IgG which is labelled to isoluminal binds the PF4/heparin antibodies and a luminescent reaction is initiated by addition of a trigger reagent .

RLUs are directly proportional to the PF4-heparin IgG concentration in the sample.

The light emission is measured optically 

Comes with two commercial controls (one high, one low), and two commercial calibrators 

Pitfalls:
May not detect all anti-PF4/heparin antibodies
Cannot differentiate between pathogenic antibodies and clinically insignificant Abs

Question 20

Serotonin release assay

Answer 20

Confirmatory testing

Functional assay that measures release of serotonin from platelets in response to heparin DEPENDENT antibodies.

Washed donor platelets are incubated with radioactive (14C) serotonin + patient serum added + heparin –> measure serotonin release

IF HIT antibodies are present –> Therapeutic (low dose) heparin will lead to serotonin release
followed by a LACK of serotonin release with supratherapeutic (high dose)

This is because a true HIT antibody causes serotonin release from plts at low dose and at much higher doses the heparin should prevent true HIT antibodies from complexing on the platelet surface, thus no serotonin release.

This improves both the sensitivity and specificity of the test.

Results can be negative, indeterminate, low positive, positive

95% specific and sensitivie

Question 21

Activated Protein C Resistance

Answer 21

APCR ~90% of cases are due to the Factor V Leiden [FVL] mutation
a point missense mutation within the F5 gene resulting in the replacement of the Arginine 506 residue by a Glutamine [R506Q] 🡪 abolition of an APC inactivation cleavage site in Factor Va. Hence resistance to APC

This amino acid substitution slows the rate of FVa inactivation by approximately 10-fold –> in increased thrombin generation and a hypercoagulable state.

Factor V Leiden = most common cause of inherited thrombophilia
~20% to 50% of cases

Heterozygous = 5-10 fold increased risk of thrombosis.
Homozygous = 50-100 fold.

Question 22

APCR Pefakit - method
Sysmex CS5100/CN6000

Functional clotting assay using PPP

Answer 22

Differs from other function APC resistance test by acting specifically on prothrombinase complex level (Factor Xa + Factor Va).
> reduces upstream factors and independent from calcium

Utilizes two different snake venom proteases which do not require calcium:
- RVV-Va ( venom of Daboia russeli) activates FV to FVa, the version that is readily inactivated by APC of the reagent.
- Noscarin (venom of Notechis scutatus) activates FX to FXa but only in the presence of FVa from the sample.

The presence of an APC resistant version of FV in the sample leads to shorter clotting times while normal FV provides longer clotting times.

Test is insensitive to the presence of the Direct Xa Inhibitors because the snake venoms bypass Factor Xa.  Also independent of phospholipid and therefore, the test is unaffected by the presence of a Lupus Anticoaglant
Interference from UFH, LMWH and pentasaccharide in the blood sample is precluded by a heparin inhibitor (presence of polybrene).

Affected by DABIGATRAN

Question 23

APCR Pefakit method

Answer 23

Sample plasma is prediluted with dilution plasma (Factor V depleted) and incubated at 37°C + RVV-Va (produce FVa) 🡪 addition of Noscarin (FX to FXa only in presence of FVa) 🡪 clotting times are recorded and the ratios:
(clotting time in the presence of APC / clotting time in the absence of APC) are calculated.

if there is APC resistance than the clotting time in presence of APC would be same/similar to that in the absence of APC

if <2.6 equivocal requiring haematologist input

<2.0 abnormality suspected;
Heterozygous 1.5-1.8
Homozygous < 1.5

Any patients where we cannot achieve a reasonable APCR Ratio – should be referred for
a FVL

Internal QC - normal and positive control daily

Question 24

Causes of acquired APC-R include:

Answer 24

Pregnancy
In association with oral contraceptive use
The presence of a Lupus Anticoagulant
Elevated Factor VIII levels
Elevated Factor II [Prothrombin] level
Reduced Protein S levels
Anti-Protein C antibodies
Some malignancies
- Increasing age

Question 25

Factor V Leiden mutation > light mix FVL

Answer 25

Allows the detection of the Factor V G1691A single nucleiotide polymorphism. PCR used a 110bp fragment of Factor V gene amplied with specific primer PCR fragment analysed using internally labelled simple probe oligomer binding to the region spanning the mutation site. Melt curve analysis- temperature slowly increase Probe leaves at a specific temperature causing fluorescence Any mismatch covered by the probe destabilizes the hybrid at a lower temperature.

Question 26

Sample requirements for FVL - light mix

Answer 26

EDTA/citrate All specimens for FVL at MPS is first testing for APCR in haem. If specimen fails to demonstrate activated protein C resistance--> not tested for FVL Each run have 3 standards and No template Control (NTC): Homozygous Wild type Heterozygous

Question 27

Interpretation of FVL melt curves

Answer 27

Question 28

Melt curves - principle

Answer 28

DNA of interest is amplified by standard PCR The analysis begins by melting apart the DNA strands of the final DNA product at 95° C and then allowing them to re-anneal as they cool. When a mixture of wild-type and mutant DNA is present in the sample, heteroduplexes are formed, and these can be detected by slowly ramping the temperature back up to 95° C in the presence of a fluorescent dye that binds preferentially to double-stranded DNA. As the temperature increases, the heteroduplexes unravel at a slightly lower temperature than the homoduplexes of wild-type/wild-type or mutant/mutant DNA. Thus, the presence of a mutation shifts the melting point of the overall population, and this shift is readily detected by a decrease in fluorescence as the DNA strands separate.

Question 29

Prothrombin Gene mutation testing

Answer 29

Almost exactly the same test as Factor V gene mutation Allows for detection of prothrombin gene mutation G20210A from genomic human DNA extracted from peripheral blood Using PCR methodology, a 106bp fragment of the factor 2 gene is amplified with specific primers The PCR fragment is analysed using an internally labelled probe oligomer binding to the region spanning the mutation site. Controls of WT, HO and HT

Question 30

Prothrombin Gene mutation

Answer 30

2nd most common inherited thrombophilia  1-2% of Europeans cauasians   Cause: single nucleotide substitution (G20210A) in promoter region of gene for F2 (prothrombin) --> increased prothrombin  Prothrombin increased by 30% in heterozygotes and 70% in homozygotes   2-4x increased risk of VTE through life  Slightly less than FVL  Present in ~5% of 1st unprovoked VTE  

Question 31

Protein C Deficiency 

Answer 31

Protein C is a vitamin K-dependent anticoagulant protein synthesized in the liver  Protein C circulates as a zymogen and exerts its anticoagulant function after activation to activated protein C (aPC)   via factor 2a (thrombin)  > rate of activation of protein C to APC is increased 20,000 fold by the proteins thrombomodulin and endothelial protein C receptor Primary role of aPC is to inactivate coagulation factors Va and VIIIa  Most patients with inherited protein C deficiency are heterozygous.  Types of Protein C Deficiency  : Type I (reduced protein C Ag and activity levels)  >Most commonly cause by a missense or nonsense mutation   > Plasma protein C level is approx 50% of normal  Type II (reduced protein C function with normal Ag level)   > Qualitative defect  > Due to alternated AA sequence from various point mutations 

Question 32

Factors influencing Protein C

Answer 32

Reduced protein C : - DIC  - Liver disease  - Infection   - Uraemia  - Cancer/chemotherapy (i.e. asparaginase)   - Warfarin  - Vit K deficiency  - AutoAbs   Increased protein C  - Nephrotic syndrome  - Hyperlipiaemia  - Normal aging 

Question 33

Protein S

Answer 33

Cofactor for activated protein C, which:  > Inactivates procoagulant factors Va and VIIIa, reducing thrombin generation  > Enhances fibrinolysis   Can directly inhibit prothrombin activation via interactions with other coagulation factor  Protein S deficiency impairs normal control mechanism, increasing the risk of thrombosis  Circulates in two states:  > Free (has cofactor activity)  40% > Bound to complement component C4b binding protein  (no fucntion) (60%)

Question 34

Protein S deficiency

Answer 34

Protein S deficiency is an autosomal dominant condition due to mutations in the PROS1 gene, a large gene on Chr 3.   Majority are heterozygotes  Rare homozygotes  or compound heterozyotes have more severe phenotype   Types of Protein S Deficiency : Type I Deficiency (reduced total protein S, free protein S and protein S function)  > Classic type   > Typically, total protein S is ~50% of normal, free protein S as low as 15% of normal   > Most mutations are missense or nonsense mutations  Type II Deficiency (normal total and free protein S; reduced protein S function)  > Rare, case reports only   Type III (selectively reduced free protein S and protein S function; normal total protein S)   > Quantitative defect   Considerations in Paediatrics : > Severe thrombotic complications, including neonatal purpura fulminans, have been reported in newborns with very low protein S levels; this is rare and typically due to homozygous deficiency 

Question 35

Causes for a reduced Protein S

Answer 35

Genetic  Pregnancy, OCP  DIC   Acute thrombosis   HIV  Nephrotic syndrome   Liver disease  L-asparaginase chemotherapy 

Question 36

Protein C assay

Answer 36

Berichrom - CHROMOGENIC assay Platelet poor plasma - citrate collection Protein C is the patient sample is activated using a specific snake venom activator (protac). A chromogenic substrate is added and cleaved by Protein C, inducing colour change which is quantified by measuring the increase in absorbance at 405nm. NOTE: this detects the active portion of APC as well as the non-carboxylate molecules synthesized in vitamin K deficiency - therefore, this chromogenic result will be higher than if a coagulometric methos is used **to obtain a complete picture of the cause of a protein C deficiency it is therefore advisable to also use the coagulometric method and the antigenic determination technique. Commercial normal and abnormal controls performed with each run. Calibration with new lot or if controls are out of range, Limitations:

Question 37

Protein S assay

Answer 37

Innovance - IMMUNOTURBIDIMETRIC Platelet poor plasma - citrate collection A suspension of latex microparticles covalently bound to monoclonal antibodies specific for free protein S is mixed with the test plasma. An antigen-antibody reaction results in microparticle agglutination, which induces an increase in turbidity of the reaction medium The change in turbidity is detected by photometry, which is proportional to the concentration of free protein S. Commercial normal and abnormal controls performed with each run. Calibration with new lot or if controls are out of range,

Question 38

Test methods available for Protein C

Answer 38

ELISA – measures levels but no functional defects   Chromogenic (what MPS use) – sensitive for low levels and MOST functional defects (but not if there is a defect in phospholipid binding)   Clot based (APTT based) assay – based can lead to misleadingly low protein C levels in the presence of a factor V leiden mutation and other causes of protein C resistance   No single test is sensitive for ALL abnormalities  

Question 39

Preanalytical issues in Protein C assay

Answer 39

Sample Collection: WBIT Correct phlebotomy technique Correct sample volume Correct sample collection tube Sample Handling Storage (i.e. warmed sample, cold sample) Mixing and centrifugation (hard spin, soft spin, no spin) Transport conditions and delays in transport - protein C has a very short half life so plasma must be frozen immediately after centrifuging (stable for 6 months at -20 degrees) Half life of 6 hours - this is also why warfarin has a procoagulant effect when first initiated as it has a shorter half life than other VitK dependent plasma proteins Activated protein C only has a half life of 15 minutes Patient Factors: Age (babies have low levels) Acute clot lowers levels and may have a rebound increase after this Liver disease lowers levels Haemolysis Turbidity Lipemia Paraproteinemia Uremia Icterus Cross-reactivity

Question 40

Causes for reduced protein S

Answer 40

The total and free protein S levels are lower in women than in men. During pregnancy there is a decrease in free protein S levels. Protein S level is decreased in the following: - congenital deficiencies - inflammatory syndromes - hepatic disorders (hepatitis, cirrhosis) - oral anticoagulant therapy - oral contraceptive use - L-asparaginase treatment

Question 41

Antithrombin

Answer 41

Major physiological inhibitor of thrombin and factor Xa primarily, but also of IXa and XIa. Antithrombin is encoded by the gene SERPINC1, is synthesised in liver cells and has a half-life of 2-3 days. Heterozygous antithrombin deficiency is found in approximately 2% of cases of thrombosis. Acquired causes of low antithrombin are much more common than hereditary antithrombin deficiency. In the presence of heparin, antithrombin exerts a powerful and immediate inhibitory effect on thrombin of approximately 1000-fold.

Question 42

Antithrombin III

Answer 42

Innovance ATIII assay - CHROMOGENIC > quantifies functionally active ATIII platelet poor plasma (PPP) - citrate tube PPP is incubated with a known excess of factor Xa, in the presence of heparin. Excess uninhibited factor Xa then cleaves a specific chromogenic substrate --> release of dye. This is measured via absorbance and release of dye measured is INVERSLY proportional to the ATII level in the sample ATIII deficiency may be inherited

Question 43

Fibrinolytic pathway

Answer 43

PAI-1 and PAI-2 INHIBIT fibrinolysis - they inhibit conversion of plasminogen to plasmin

Question 44

Euglobulin Clot Lysis Time (ECLT) - not routinely done

Answer 44

Principle > historical marker of fibrinolysis. > most reflective of activity of PAI-1 Venous blood is collected into chilled tubes with trisodium citrate and place on ice Sample centrifuged at 4 degrees Plasma collected and diluted with acetic acid and incubated on ice for 15 minutes Precipitate forms (the euglobulin fraction of plasma) which contains plasminogen, plasminogen activators (primarily t-PA) and fibrinogen The supernatant is collected and centrifuged in a refrigerated centrifuge at 4 degrees The precipitate is dissolved in buffer with calcium chloride added at 37 degrees This is then clotted with thrombin and the time to clot lysis is determined by inspection every 15 minutes A control sample is collected at the same time as the patient sample and must be run in parallel

Question 45

Causes of prolonged CLT

Answer 45

Causes of prolonged CLT >Diabetes - high BSLs glycosylated lysine (present on plasminogen), and hence leads to reduced clot degradation. Causes of shortened CLT > Blood vessel injury or surgery >Prostate cancer >Liver cirrhosis >Fibrinogen deficiency >ITP

Question 46

TXA MOA

Answer 46

inhibiting the breakdown of blood clots (fibrinolysis) by blocking the lysine binding sites on plasminogen, thus preventing plasminogen from converting to plasmin and stabilizing the fibrin clot.

Question 47

Factor 13

Answer 47

Factor XIII (FXIII), also known as fibrin-stabilizing factor --> crucial role in the coagulation cascade by stabilizing fibrin clots through covalent cross-linking, making them resistant to fibrinolysis and strengthening the clot.

Question 48

Antithrombin level variation

Answer 48

Antithrombin levels can be decreased by oral contraceptives and pregnancy. > liver disease > recent/active thrombosis > DIC > nephrotic syndrome Antithrombin levels are lower in premenopausal women but higher in postmenopausal women, compared with men. A marked diminution of this level is observed after the thirteenth week of pregnancy and in the postpartum period. In men the Antithrombin level decreases with age. In children the Antithrombin level is normally low until the age of 6 months, after which it is the same as adults.

Question 49

D-Dimer Assay - INNOVANCE immunoturbidimetric assay

Answer 49

Polystyrene particles covalently coated with a monoclonal antibody (8D3)14 are aggregated when mixed with samples containing D-dimer. The D-dimer cross-linkage region has a stereosymmetrical structure, i.e. the epitope for the monoclonal antibody occurs twice. Consequently, one antibody suffices in order to trigger an aggregation reaction, which is then detected turbidimetrically via the increase in turbidity Limitations - haemolysis - hyperbilirubinaemia - lipemia- heterophilic antibdoies

Question 50

High D Dimer approach

Answer 50

For all high results: that have the 1 /10 dilution result differing from the second diluted result (either higher or lower), by a factor of 2 i.e.: > 100%, results MUST be referred to a Haematologist urgently for reporting. Send to STAGO for mechanical method and not subject to htereophile Abs. High DD results of >1.0 are to be phoned to the referring Doctor.

Question 51

Causes for increased D Dimer

Answer 51

Elevated D-dimer levels are observed in all diseases and conditions with increased coagulation activation, such as: - thromboembolic disease - disseminated intravascular coagulopathy (DIC) - acute aortic dissection - myocardial infarction - malignant diseases - obstetric complications - third trimester of pregnancy - surgery or polytrauma

Question 52

Fibrinogen

Answer 52

3 pairs of polypeptide chains >Two Aa, TwoBb and Two y Activation of fibrinogen by THROMBIN cleaves FpA and FpB which exposes 'knobs' in the E domain that can spontaneously interact/link with D-domains to form fibrin polymers. With Factor 13 - CROSS LINKING of these fibrin polymers then occurs to strength the clot

Question 53

Clauss Assay for Fibrinogen

Answer 53

Siemens CS5100 Venous blood collected into 3.2% sodium citrate to make a dilution of 1:9 Transport to laboratory unspun and at room temp Centrifuged 3000RPM for 10 minutes Dilute plasma mixed with a high concentration of thrombin. Diluted to reduced the effect of ‘inhibitor substances’ eg heparin or elevated levels of FDPs. High concentration of thrombin (human derived) , to ensure clotting times are independent of thrombin concentrations over a wide range of fibrinogen levels. Clotting time is plotted on calibration curve --> ,is inversely proportional to the fibrinogen concentration of the plasma. Clotting time obtained in this manner is compared with that of a standardized fibrinogen preparation. Detects both hypofibrinogenemia and dysfibrinogenemia. Low fibrinogens < 2.0g/L will automatically repeat Results beyond the limits of the calibration curver (belwo or above) will automatically repeat with following dilution: <0.8g/L - 4/1 dilution >4.5g/L - 1/2 dilution Phone notification if <1.0g/L LOQ = <0.3g/L

Question 54

Limitations in Clauss Assay

Answer 54

Clauss method for measuring Fibrinogen → Cross-linking of Fibrin by Factor XIIIa is not required for the Clauss Fibrinogen assay and so the Fibrinogen levels will be normal in severe Factor XIII deficiency. High concentrations of UFH or dabigatran Increase in plasma turbidity (BIL) A normal APTT/PT does not necessarily mean that the fibrinogen is normal Criteria for rejection: -Grossly hemolyzed -Clotted -> 24 hours old -Not stored correctly -Under filled or overfilled tubes

Question 55

Andexnet Alfa

Answer 55

Recombinant modified factor Xa protein Acts as a deocy and sequesters rivaroxaban or apixaban inhibiting them from binding to natural factor Xa. It has been modified so that it cannot assemble into proteombinase complex and removing any potential anti-coagulant effects. It also binds tissue factor inhibitor pathway (TPFI), a peptide that inhibits factor Xa,

Question 56

Emicizumab

Answer 56

Emicizumab is a humanised bispecific monoclonal antibody that bridges Factor 9a and Factor 10 to restore the missing function of factor 8. It interferes with APTT and APTT based assays: - APTT: reduced - APTT-based single factor assays: increased -Chromogenic FVIII assays (human FIXa and FX): increased -APTT-based Bethesda assays: false negative -Also affects APC resistance and APTT-based protein S assays Unaffected by emicizumab: - extremely small effect on INR; no effect on PT-based single factor assays - TCT and Clauss fibrinogen - Chromogenic assays for antithrombin, protein C, and anti-Xa based chromogenic assays

Question 57

Interpretation of TEG tracing

Answer 57

Question 58

TEG 6 principle

Answer 58

Principle: -TEG6 is a cartridge based system that mixes patient whole blood with various reagents to help guide blood management -Our TEG6 process is outlined below --Collect patient whole blood in a citrate tube --Utilises a number of measurements to help guide blood management 1. R time -> time for initiation of coagulation to occur 2. alpha angle -> rate at which a clot is formed 3. Maximum amplitude -> the strength of clot that is formed 4. Fibrinolysis time -> tapering of the graph at the end of coagulation -Blood is added to cartridge that has 4 different reagents: -- vibrations pulsed through the blood can measure its density and fluidity and assess it's tensile strength (i.e. whether a clot has formed, and how strong that clot is) - modern analyser no longer use a ball in cup method -The 4 channels **1. CFF** -Channel best used to asses **fibrinogen** -Maximum amplitude should be assessed, and if it is low, fibrinogen should be replaced. **2. CRT** -Channel best used to assess platelet function and fibrinogen -If the MA in the CFF channel is normal, and the MA in the CRT channel is low, then the patient needs a **platelet transfusion** **3. CK** -Channel best used to assess for heparin activity in comparison to the CKH graph -if prolonged R time compared to CKH, indicates heparin activity. Advised of **protamine** administration **4. CKH** -Has a heparinase in it that neutralises heparin activity -If R time prolonged in CKH channel, indicates a **factor deficiency and FFP **should be transfused 5. If there is evidence of increased fibrinolysis (i.e. tapering of the graph) then this can indicate the need for tranexamic acid

Question 59

Viscoelastic testing values

Answer 59

R time (time taken for clot formation) --> if prolonged --> due to factor deficiency/heparin on board (may suggest to give more FFP/factor replacement) --> if shortened --> hypercoaguable state Alpha angle (rate of clot formation) --> reflection of fibrinogen level or function (may suggest to give more cryo) Maximum amplitude of pin oscillitation - reflection of clot strength (comprised of both platelets and fibrinogen function/presence) --> low MA = thrombocytopenia or plt dysfunction OR hypo/dysfibrinogenaemia (may suggest to give more plts) EPL/LY30 (lysis time) -> reflection of there is excess fibrinolysis --> hence role for TXA

Question 60

What is Viscoelastic testing?

Answer 60

Point of care test that provides global information on the dynamics of clot development, stabilization and dissolution that reflect in vivo haemostasis. Main utility: minimize hyperfibrinolysis assoc with trauma and surgery. Minimize subsequent transfusion Citrated whole blood is added to a heated cuvette at 37C and recalcified with CaCl2. Within the cup, a pin is suspended connected to a detector system   - TEG: cup oscillates in a limited arc; as clot forms, rotation of cup is transmitted to pin   - ROTEM: pin oscillates in a limited arc; as clot forms impedes rotation of the pin   As blood clots, fibrin-platelet strands form between the cup and the pin that impedes rotation  With the onset of fibrinolysis, the clot dissolves and movement of the pin is affected, reflected in the trace   Cand add different activating agents to measure specific variables: > Heparinase to inhibit heparin in sample > tissue factor - activate extrinisic pathway, more info re PT > phospholipid - activate intrinsic pathwya, similar info re APTT >addition of plt inhibitor - blocking plt contribution to provide more info re functional fibrinogen component

Question 61

ROTEM/TEG interpretation flow chart

Answer 61

Question 62

Limitations of viscoelastic testing

Answer 62

Impaired detection to primary haemostasis: no detection of aspirin/clopidogrel, insensitive to presence of vWD   Poor sensitivity to LMWH or oral anticoagulants   Thrombocytosis: a platelet count >1000 may lead to incomplete platelet inhibition on FibTem test and can be misinterpreted as a falsely high fibrinogen   Overall poorly standardized  

Question 63

QC of ROTEM/TEG

Answer 63

Internal QC: - System involves continuous self-monitoring that monitors performance of axis movement, sensors and temperatures with pre-specified target ranges  - All users must run daily internal QC, which is reviwed, signed and filed   External QC  : - Level 1 (normal control) and Level 2 (abnormal control) controls are provided as pre-made cartridges by the company   Recommendations for running external controls:   - Minimum once a week  - After preventive maintenance is performed   - Whenever a new reagent is being used   - Whenever a value from service menu is out of range 

Question 64

ADAMTS 13 Acustar

Answer 64

AcuStar ADAMTS13 activity assay is a two-step immunoassay to quantify ADAMTS-13 activity in plasma using magnetic particles as the solid phase and chemiluminescence in the detection system Samples are mixed with assay buffer and magnetic particles coated with a recombinant VWF fragment ADAMTS13 in the sample cleaves the substrate bound to the particles proportionally to its activity After magnetic separation and a wash step, a labelled mAb directed against the cleaved peptide is added and incubated. A resulting chemiluminescent reaction is measured as relative light units by the AcuStars optical system The RLUs are directly proportional to the ADAMTS13 activity in the sample

Question 65

ADAMTS 13 ELISA

Answer 65

ELISA is a enzyme linked immunoasborant assay, Sample is added to a plate which is coated with vWF.    Plasma is added and ADAMTS13 in the pts sample cleaves the vWF substrate.   A horseradish peroxidase (HRP) conjugated antibody directed against cleavage site is added and incubated before the subsequent addition of a HRP substrate which generates colour at 450nm proportion to the amount of Ab binding.