**12. Laboratory coagulation tests**

#### *Platelet count*

The normal range is 150-400 x109 / L. Thrombocytopenia can occur as a result of decreased bone marrow production or excessive destruction. Typically, traumatic bleeding, purpura and easy bruising occur at platelet counts less than 50 x109/L; spontaneous bleeding may occur at platelet counts below 20x109/L. There is not a strong evidence base for a transfusion trigger in thrombocytopaenia (30), however the consensus is that a platelet count of >50 x109 should be achieved prior to invasive procedures (31) with a higher target of >100 x109 prior to neurosurgery.

#### *Platelet function*

Platelet function can be impaired by low pH, hypothermia, myeloproliferative disease, uraemia, non-steroidal anti-inflammatory drugs and antiplatelet drugs. As well as having a sufficient number of platelets, adequate platelet function is also necessary for haemostasis. Platelet function is not measured in standard laboratory tests, but it is possible to measure platelet activity using methods such as aggregometry. Platelet function can be inferred from


Fig. 6. Antiplatelet Agents, their mechanisms of action and implications for Cardiac Surgery

**Antiplatelet Drug Name Mechanism of Action Implications for Cardiac** 

Prevents Thromboxane A2 production (by irreversibly acetylating cyclooxygenase-1) therefore reducing platelet aggregation

Their metabolites covalently bind to the P2Y12 receptor, the main platelet receptor responsible for ADPinduced platelet aggregation.

These agents change P2Y12 receptor conformation, causing a reversible, concentration-dependent receptor inhibition.

messengers. Also stimulates prostacyclin release and inhibits thromboxane 2A

Fig. 6. Antiplatelet Agents, their mechanisms of action and implications for Cardiac Surgery

therefore increasing intraplatelet concentrations of cyclic AMP which reduces the activation of cytoplasmic second

Compete with ligand binding of fibrinogen to glycoprotein IIb/IIIa receptor, therefore block the final common pathway for platelet aggregation. Given intravenously as loading bolus followed by infusion.

production

Dipyridamole Inhibits phosphodiesterase,

Aspirin = Acetylsalicylic

Clopidogrel, Ticlopidine,

Direct-acting P2Y12

Cangrelor, Ticagrelor,

Glycoprotein IIb/IIIa receptor antagonists: Abciximab, Tirofiban,

Eptifibatide

Thienopyridine derivatives:

Prasugrel

inhibitors:

Elinogrel

acid

**Surgery**

Antithrombotic effect starts within 30min of oral loading dose and lasts for lifespan of the platelet (8–10 days). Is often continued perioperatively with some

degree of platelet dysfunction expected.

Active within 2hrs of oral loading dose. Lower risk of GI-bleeding than aspirin. Expect platelet dysfunction if not discontinued at least 7 days pre-operatively (23).

These are in phase 3 of development(24)

Short half-life so usually given as a slow-release

Platelet function can take 2 days to recover following discontinuation of Abciximab infusion. Strategies to reduce blood

2) Platelet transfusion (less effective if given when free drug still in circulation) 3) Fibrinogen or

antifibrinolytics may be of

loss may include: 1) Delaying surgery for 12hrs post-abciximab, and 2hrs post-tirofiban or

eptifibatide.

benefit

preparation.

thromboelastography (see later section). Bearing in mind the lack of routine platelet function measurement prior to cardiac surgery, it is important to identify clinical clues to impaired platelet activity. A patient's medication history should be noted. Some patients are advised to stop antiplatelet agents in advance of elective surgery. In those taking clopidogrel up to the day of surgery, platelet dysfunction should be anticipated as should the need for platelet transfusion.

### *Prothrombin time (PT) and international normalized ratio (INR)*

Blood is sampled into a tube containing EDTA or citrate, both of which chelate calcium and prevent the blood clotting en route to the laboratory. The sample is centrifuged in the lab to yield platelet-poor plasma. The test starts when calcium and thromboplastin (consisting of tissue factor and phospholipid) are added to the plasma, and is complete when fibrin strands are formed. The time taken is the PT. The INR puts the measured PT in context by comparing it with the PT of a standardised plasma sample. An INR of 1.0 is normal; higher INRs represent coagulation that is impaired secondary to pathology or drugs. PT and INR are considered to be tests of the extrinsic pathway, with FVII deficiency having the greatest impact on raising the INR/PT. Other causes of abnormal PT and INR are shown in the table. Of note, the large amount of TF used to trigger coagulation in this test negates the effect of TFPI and renders the test independent of FVIII, FIX and FXI, meaning patients with haemophilia can have normal PT and INR results.

#### *Activated partial thromboplastin time (APTT) and aptt ratio (APTTR)*

The blood sample for this test is prepared in the same way as that for the PT. The resultant platelet-poor plasma is triggered to coagulate by mixing it with the APTT reagent and replacing the chelated calcium. The APTT reagent consists of phospholipid (as a substitute for the platelet membrane) and negatively-charged particles that cause contact activation. The time taken for fibrin strands to form is the APTT. The APTT ratio is calculated in a similar manner to the INR, with normal APTTR being 1.0 and higher values representing coagulopathy. APTT is prolonged by deficiencies of the Intrinsic or Common pathways, meaning any procoagulant factor deficiency apart from FVII and FXIII will prolong the


Fig. 7. Causes of Prolonged PT and INR

APTT. APTT is used to titrate unfractionated heparin anticoagulation. It is also a screening tool for Haemophilia A and B. Prolonged APTT does not necessarily predict clinical bleeding, for example FXII deficiency greatly prolongs APTT but is not associated with bleeding tendency.


*Thrombin clotting time (TT or TCT) and hemoclot®* 

TT measures the time taken for fibrin to form when thrombin is added to the plasma sample. Prolongation of TT suggests low fibrinogen levels, dysfibrinogenaemia or inhibition of thrombin. Thrombin can be inhibited by unfractionated heparin or direct thrombin inhibitors. Fibrin degradation products inhibit fibrin cross-linking; at high concentrations these can also prolong the TT.

Adding Protamine sulphate to the sample will negate the effect of heparin and FDPs. Adding Toluidine Blue will negate the effect of heparin but not FDPs. Another technique for samples containing heparin is to perform a "Reptilase Test", where a snake venon product 'Reptilase' is used to activate fibrinogen. Unlike thrombin, reptilase is not inhibited by heparin.

Hemoclot® is a sensitive, diluted TT assay.

These tests are used to assess the effectiveness of fibrinolytic therapy and may be useful in quantifying the effects of direct thrombin inhibitor drugs.

### *Ecarin clotting time (ECT)*

This is a specific assay of thrombin generation. Ecarin comes from snake venom, and specifically activates prothrombin (FII). ECT can be used to monitor anticoagulation during Cardiopulmonary Bypass but is not widely utilised outside a research setting (13)

#### *Fibrinogen*

Fibrinogen (FI), the precursor of fibrin, is synthesised by hepatocytes and normal plasma concentration is 1.5-4.0g/L. Levels vary seasonally within an individual, and causes of abnormal fibrinogen level or function are shown in the tables below. Fibrinogen can be depleted by haemorrhage and consumed by coagulation during or after cardiac surgery, and is routinely quantified by the Clauss method in this setting. The PT-derived fibrinogen level (PT-Fg) is more convenient but less reliable. When investigating congenital fibrinogen defects, more complex and time-consuming tests are employed.

There is evidence that the low preoperative fibrinogen concentrations correlate with higher peri-operative blood loss in coronary artery bypass surgery (32, 33). Pre-operative prophylactic fibrinogen infusion may be beneficial, although larger trials are required to determine safety and efficacy of this practice (34). Pre-operative hyperfibrinogenaemia is associated with higher all-cause mortality (35), possibly because raised fibrinogen levels are a marker of inflammation, reflecting the presence of a systemic disorder (eg sepsis, acute coronary syndrome).

In major blood loss treated with packed red blood cells and crystalloid/colloid intravascular volume resuscitation, fibrinogen is the first coagulation factor to become depleted (36). It is therefore a useful marker for severity of haemorrhage (37).

Fibrinogen quality is assessed when thromboelastometry is performed in the presence of a platelet inhibitor, a test available commercially as "FIBTEM" (see ROTEM section).

A prolonged Thrombin Time will also detect low fibrinogen levels.

Options for replacing fibrinogen are fibrinogen concentrate reconstituted to 20g/L, Fresh Frozen Plasma containing 1.6-2g/L fibrinogen, or cryoprecipitate containing 17g/L. The traditional target of replacement of fibrinogen aimed for a level of 1g/L, but higher targets of 1.5-2g/L may be more appropriate in patients undergoing cardiac surgery (38).

It remains to be established whether it is better to administer fibrinogen concentrate prophylactically, or to reserve treatment only for patients who are bleeding. There is also debate regarding the most suitable goal for fibrinogen replacement, which may be fibrinogen quantity (eg Clauss assay) or fibrin quality (eg. FIBTEM).

#### *The Clauss assay*

192 Perioperative Considerations in Cardiac Surgery

APTT. APTT is used to titrate unfractionated heparin anticoagulation. It is also a screening tool for Haemophilia A and B. Prolonged APTT does not necessarily predict clinical bleeding, for example FXII deficiency greatly prolongs APTT but is not associated with

Specific Clotting Factor Inhibitors eg. Factor VIII inhibitors develop in up to

Lupus Anticoagulant This is a non-specific inhibitor of the intrinsic

Dilution artefact Underfilling of the sample tube, or raised

TT measures the time taken for fibrin to form when thrombin is added to the plasma sample. Prolongation of TT suggests low fibrinogen levels, dysfibrinogenaemia or inhibition of thrombin. Thrombin can be inhibited by unfractionated heparin or direct thrombin inhibitors. Fibrin degradation products inhibit fibrin cross-linking; at high concentrations

Adding Protamine sulphate to the sample will negate the effect of heparin and FDPs. Adding Toluidine Blue will negate the effect of heparin but not FDPs. Another technique for samples containing heparin is to perform a "Reptilase Test", where a snake venon product 'Reptilase' is used to activate fibrinogen. Unlike thrombin, reptilase is not inhibited by

These tests are used to assess the effectiveness of fibrinolytic therapy and may be useful in

This is a specific assay of thrombin generation. Ecarin comes from snake venom, and specifically activates prothrombin (FII). ECT can be used to monitor anticoagulation during

Fibrinogen (FI), the precursor of fibrin, is synthesised by hepatocytes and normal plasma concentration is 1.5-4.0g/L. Levels vary seasonally within an individual, and causes of abnormal fibrinogen level or function are shown in the tables below. Fibrinogen can be depleted by haemorrhage and consumed by coagulation during or after cardiac surgery,

Cardiopulmonary Bypass but is not widely utilised outside a research setting (13)

Contamination of sample with heparin Sample drawn from vein upstream of

FVII is not involved in the intrinsic pathway. Completion of the test does not require FXIII-dependent cross-linking of fibrin.

heparin in the pressure-bag of an arterial line

pathway so prolongs APTT. Paradoxically, patients with lupus anticoagulant actually

haematocrit will increase the proportion of

10% of patients with haemophilia A

heparin infusion, or contaminated by

have a prothrombotic tendency.

citrate (or EDTA) to plasma

bleeding tendency.

FVII or FXIII)

**Causes of Prolonged APTT and APTTR Notes** 

*Thrombin clotting time (TT or TCT) and hemoclot®* 

Hemoclot® is a sensitive, diluted TT assay.

quantifying the effects of direct thrombin inhibitor drugs.

these can also prolong the TT.

*Ecarin clotting time (ECT)* 

heparin.

*Fibrinogen* 

Procoagulant factor deficiency (except

A high concentration of thrombin is added to diluted plasma and the clotting time is measured. A reference curve has been plotted using plasma of known fibrinogen concentrations. The fibrinogen concentration of the test sample is determined by comparing the clotting time with the reference curve. The clotting time can be determined automatically using systems that detect changes in light scattering, or light absorption as fibrin is formed. Artefacts can occur when turbid or lipaemic plasma causes abnormal light scattering, and when free haemoglobin or bile cause abnormal light absorption. Heparin will affect clot formation, making the Clauss assay inaccurate, so every effort should be made to obtain a heparin-free sample (avoid sampling within 4 hours of low-molecular weight heparin administration, caution when sampling from arterial lines flushed with heparin). If a heparin-free sample cannot be obtained (eg during Cardiopulmonary Bypass) then ionexchange resins or heparinase can be used to negate the heparin effect. Fibrin Degradation Products (FDPs) affect fibrinogen activity in vivo, and also affect the time taken to complete the Clauss assay, so this assay naturally reflects the any potential impact of FDPs.

#### *PT-derived fibrinogen assays (PT-Fg)*

This method indirectly measures fibrinogen by analysing the change in light scattering or optical density as fibrin is formed in the sample during the PT test. The advantage of this method is that it can give a measurement of fibrinogen any time the PT is tested without additional expense. The test may be inaccurate in samples with high fibrinogen and, of particular relevance to Cardiac Surgery, the test may be less reliable than Clauss in the investigation of bleeding diathesis. FDPs have less effect on the PT-Fg than on the Clauss



assay; an adequate fibrinogen measurement using PT-Fg may not reflect adequate fibrinogen activity if there is a significant quantity of FDPs in circulation.

#### *Clottable protein*

194 Perioperative Considerations in Cardiac Surgery

Seasonal change Higher fibrinogen levels in winter

Congenital Afibrinogenaemia Grossly decreased fibrinogen synthesis, with low or

Congenital Hypofibrinogenaemia Mild-to-moderate reduction in circulating fibrinogen

Congenital Dysfibrinogenaemia Immunoassays may detect fibrinogen, but tests that

asymptomatic

Decompensated liver disease Hepatocellular failure leads to decreased fibrinogen

function.

administration Systemic Thrombolytic Therapy Usually leads to gross reduction in fibrinogen levels

and high risk of haemorrhage

Haemodilution From massive blood transfusion or fluid

**Notes** 

Occurs in 20% of population, leads to 7-10% higher fibrinogen levels than GG genotype (39)

**Notes** 

undetectable plasma fibrinogen. Patients have haemorrhagic diathesis, prolonged clotting times

levels. Potential for haemorrhagic problems, but

depend on production of fibrin strands will give lower fibrinogen results. Patients may have haemorrhagic tendency, thrombophilia or be

synthesis. Excessive glycosylation of fibrinogen leads to an acquired dysfibrinogenaemia. Excess Fibrin Degradation Products (FDPs) also disrupt fibrinogen

microcirculation leads to consumption and depletion of fibrinogen. FDP concentration is also raised.

Generalised fibrin formation throughout the

and abnormal platelet function

patient may be asymptomatic.

**Causes of Elevated Plasma Fibrinogen Concentration** 

Pregnancy and Oral Contraception

Genetic Polymorphism of the beta fibrinogen gene promoter (G-455A)

**Causes of Decreased Plasma Fibrinogen Concentration** 

**Inherited Fibrinogen Defects (rare):** 

**Acquired hypofibrinogenaemia** 

Viral Hepatitis

Coagulation (DIC)

Disseminated Intravascular

Increased age Female Sex

Smoking Acute exercise

Acute phase reaction

Disseminated malignancy

This test is too time-consuming and labour-intensive to be used routinely, but does give very accurate results and can be used for reference assays.

### *Testing for specific coagulation factor abnormalities*

Clinical or laboratory evidence of abnormal coagulation that is not explained by haemorrhage, known pathology, or iatrogenic causes may require investigation of specific factor abnormalities. Uncommonly, individuals may have a factor deficiency or a factor inhibitor (the most common example is lupus anticoagulant). Consultation with a haematologist followed by specialised laboratory tests may be indicated.

#### *Other measurements in coagulopathy*

For effective coagulation, supportive management to maintain homeostasis is required. Temperature <34°C, pH <7.1, ionised Calcium <0.9mmol/L and Haematocrit <30% have all been shown to adversely affect haemostasis (40).

In coagulopathy, hypocalcaemia should be corrected because of calcium's key role in the activation of FX, FII, FXIII and Fibrinogen. Both haemodilution and chelation of serum calcium by citrate in units of packed red blood cells can contribute to hypocalcaemia in massive transfusion. A value for ionised Calcium can be rapidly obtained from many blood gas analysis machines, and the reference range is 1.0 – 1.3 mmol/L (no correction calculation required). Total Calcium ranges from 2.1 – 2.7mmol/L (corrected for hypoalbuminaemia may be necessary).

Prevention of heat loss and active warming may be required to raise core temperature, acidotic patients may benefit from buffering, and haematocrit should be maintained with transfusion to optimise coagulation.

When transfusing Packed Red Cells it is preferable to use blood that has been stored for the minimum possible duration, ideally less than two weeks. Transfusion of older blood has been associated with greater incidence of complications and higher mortality (41).
