Lecture 8 - Lab. Evaluation of Haemostasis & Introduction to Bleeding Disorders

Question 1

What things must be done to a blood sample before we can test for blood coagulation?

Answer 1

We need to use citrated plasma, we obtain this by adding citrate to chelate the calcium. We only need to remove platelets to make the blood platelet poor

Question 2

Why do we use platelet poor blood to coagulation testing?

Answer 2

Plateltes will affect the test by producing extra phospolipid, and this will produce more variability in the test - therefore its better to use platelet poor blood to reduce variabiltiy

Question 3

Describe the APTT test

Answer 3

The activated partial thromboplastin time test is when we poor citrated, plasma poor blood into a test tube which has an activator, and we measure how long it takes for the blood to clot. The activator actives Xll to Xlla, and this activates Xl to Xla, then the reaction stops since Ca2+ is needed to go further. We leave the mixture in incubation for 5 minutes to let the Xa build up, and then we add calcium to produce the reaction, and it should take 22-34 secs to coagulate the blood.

When the fibrin begins to form, this is when the reaction has reached its end point.

Question 4

What are the two stages of the APTT test?

Answer 4

There is an acitvate and incubate stage, and then the second stage measures the functional activity of factors lX, Vlll, X, V, ll

Question 5

What is APTT useful for screening against?

Answer 5

Haemophilia, and liver disease where coagulation proteins may be decreased

Question 6

Which pathway does the Prothrombin time (PT) test measure?

Which pathway does the APTT measure?

Answer 6

PT - Extrinsic pathway

APTT - Intrinsic pathway

Question 7

Briefly describe the PT test

Answer 7

This test measures the extrinsic pathway. First we add tissue factor and calcium - this tests factor Vll

Question 8

How would low levels of Vll affect the PT and APTT?

Answer 8

If only Vll is decreased, PT will be prolonged and APTT will be normal

Question 9

If APTT and PT are prolonged, which factors must be deficient?

Answer 9

Either, or both, factors X and V will be deficient or prothrombin must be decreased

Question 10

If only APTT is prolonged, then which proteins may be deficient?

Answer 10

Xll, Xl, lX, Vlll

Question 11

What does a Fibrinogen and thrombin clotting time (TCT) test involve?

Answer 11

We add fibrinogen to plasma and it causes fibrinogen to clot quickly.

Fibrinogen assays/TCT’s are usful for screening against fibrinogen abnormalities, or for hepain (thrombin inhibitor)

Question 12

A deficiency in which factor is not clinically important?

Answer 12

Factor Xll

Factor Xll doesn’t cause a bleeding problem, its just important in inflammation, where it helps activates coagulation

Question 13

Describe how the activity of a specific factor is measured

Answer 13

Coagulation factor assays are usually performed as clotting bioassays

The acitivty of a factor is measured as a percentage of biological clotting activity (except fibrinogen)

The factor concentration in a ‘normal’ pool of plasma is 100%. 1% of any factor is equivalent to the activity present if normal plasma is diluted 1:100 in plasma that completely lacks that factor

The APTT and PT are prolonged if factor conc. is <40-50%

Question 14

How is fibrinogen measured?

Answer 14

Fibrinogen is assayed in mass concentration, and it has a reference range of 1.8-4.0 g/L

Question 15

At what factor concentration percentage do the APTT and PT start to become proloned?

Answer 15

if the factor conc. is <40-50%

Question 16

What are some causes of bleeding?

Answer 16

• Defective vessel wall (only a small number of conditions)
• Platelet disorders: Thrombocytopaenia (150-400e9/L) or there can be inherited conditions that cause defective platelet function, and some drugs affect platelet function
• Von Willebrand disease, where there are low levels or abnormal vWF molecule (and sometimes FVlll) - this causes reduced platelet aggregation and prolonged bleeding

- Defective coagulation, there are a large number of conditions, which can be either acquired or inherited

Question 17

What are the basic tests we can run for a patient with bleeding problems?

Answer 17

Can start with a blood screen - get a platelet count & look at morphology

And we can also run basic coagulation tests, such as APTT, PT, Fibrinogen, TCT

If appropriate, need to do specialist second stage tests

• vWF factor assays
• Platelet function studies
• Coagulation factor assays

Question 18

What is Disseminated Intravascular Coagulation?

Answer 18

DIC is the widespread activation of platelets and coagulation in blood

Its caused by either the release of procoagulant material into the circulation, or by widespraed focal or diffuse damage to endothelial cells

In mild DIC there are no adverse effects, but in moderate or severe DIC there is bleeding, and potentially thomboses and ischaemia

Question 19

What is the pathogenesis of Disseminated Intravascular Coagulation?

What procoagulant materials are released which causes DIC?

And what does this result in?

Answer 19

Tissue factor or other cell products are released inb blood vessels, and this can result from any cause of extensive damage (and some snake venom)

The release of these procoagulant causes platelet activation and aggregation, and the activation of coagulation, the formation of micro thrombi and secondary activation of fibrinolysis

Question 20

How are small fibrin deposits on blood vessels removed?

Answer 20

The endothelial cells secrete tPA to break down the fibrin

Question 21

During DIC, what is always activated to stop microthombi being formed?

Answer 21

Fibrinolysis, which clears fibrin form most small blood vessels, inadequate activation results in microvascular thomboses

Question 22

What laboratory findings may indicate DIC?

Answer 22

• Falling fibrinogen concentration - Prolonged TCT
• Falling platelet count
• High/rising level of fibrin degradation products (FDPs)

also may find:

• Reduced coagulation factors - prolonged APTT & PT - only in severe cases
• Protein C and Antithrombin may fall significantly
• RBC fragmentation seen in blood film (where RBC binds to fibrin then gets torn and continues to flow)

Question 23

How does Gram negative septicaemia cause disseminated intravascular coagulation?

Answer 23

The endotoxin producted activates monocytes (recognised by toll 4 receptors on monocyte surface) and this causes the monocyte to express tissue factor in the blood

This occurs in all cases of gram negative septicaemia - the most severe case is with meninococcal septicaemia as very high levels of endotoxin are released

Question 24

What are the control mechanisms against DIC?

Answer 24

Protein C, Antithrombin, Fibrinolysis

May be overcome by prothrombotic factors resulting in thromboses becoming clinically apparent

Question 25

How is acute DIC treated?

Answer 25

First we need to treat the root cause, and if we remove the cause then the DIC will stop e.g. infection, trauma/shock - we need to use antibiotics, and treat the shock Due to the depletion of platelets and coagulation factors during DIC, we may need to replace them to stop **secondary bleeding**. * For thombocytopenia & bleeding we give them platelet concentrate * For low fibrinogen and bleeding we give fibrinogen-rich blood component (Cryoprecipitate) * If a severe case with a prolonged APTT is not controlled by cryoprecepitate we give fresh frozen plasma

Question 26

Besides gram -ve septacaemia, what are some other causes of DIC?

Answer 26

Any severe or widespread tissue injury can cause DIC * Prolonged shock with secondary hypoxic tissue injury * Extensive tissue trauma - release of tissue products * Serve burns * Severe viral infections causing endothelial injury, eg dengue * or a traumatic brain injury where brain myelin (lipid) is released into blood

Question 27

Why is vitamin K needed?

Answer 27

To permit the addtion of carboxyl groups to factors ll, Vll, lX & X, and Proteins C & S

Question 28

What 3 groups of clinical conditions does vitamin K deficiency occur in?

Answer 28

Small bowel malabsorption disorders Liver disease (can't handle vitamin K very well, and the production of coagulation proteins is impaired) Neonate (newborn infant) & premature infant

Question 29

What are our sources of vitamin K?

Answer 29

Its a fat soluble vitamin which comes from vegetable sources and bacterial synthesis in the colon

Question 30

What are the causes of Vitamin K deficiency?

Answer 30

Low intake - uncommon Low - intake + antibiotics Malabsorption disorders (e.g. in a conditon with a decreased bile salt production, which is needed to emulsify fats, including fat soluble vitamin K, for absorption during small bowel digestion

Question 31

How long do our liver stores of Vitamin K last for?

Answer 31

Vit K stores usually last for 1-3+ weeks, and a deficiency can be detected by seeing a prolonged PT & APTT & a bleeding tendency NEED TO BE CAREFUL WITH ABDOMINAL SURGERY PATIENT WITH A WOUND INFECTION AND ANTIBIOTIC THERAPY WHO HAS NOT EATEN FOR 1-2 WEEKS

Question 32

In severe liver disease where there is reduced synthesis of coagulation factors, what can be given to increase vitamin K-dependant factors?

Answer 32

Injection of pharmacological doses of Vit K may improve coagulation status before liver biopsy or surgery

Question 33

Why are neonates Vitamin K deficient?

Answer 33

There is limited Vitamin K transported across the placenta, and usually for the first 24 hours there is no oral intake - we **must** given vitamin K at birth 0.5mg IM, or 3 oral doses over a time period

Question 34

Why do neonates need vitamin K at birth?

Answer 34

In premature infants there is an increased risk of intracerebral bleeding after birth due to the physical moulding of the head during the birth processes IM vitamin K must be given

Question 35

What is the mechanism of action for warfarin?

Answer 35

Inhibits epoxide reductase, preventing Vit K expoxide form returning to Vit K reduced form

Question 36

What are the benefits and potential problems with using warfarin?

Answer 36

Benefits * Useful for treating patients with a pathologically increased risk for clotting * Its taken orally, no injection is needed Potential problems * Risk of bleeding if excess anticoagulation * Risk of clotting if insufficent anticoagulation * *a narrow therapeutic window exists for warfarin - Careful clincal control need - with regular lab testing - use INR (standardised prothrombin time)*

Question 37

What is an INR?

Answer 37

International Normalised Ratio - The INR corrects for differences between test brands It is the ratio of the patients PT to a PT on normal plasma This ratio is then corrected using the international reference thromboplastin levels

Question 38

What are some common causes for warfarin treatment to go out control?

Answer 38

* Can occur when there is a changed Vit K intake in food - e.g. vomiting, binge eating, sudden changes in dietary patterns, eg when travelling * Decreased absroption of vitamin K - Can occur in gastroenteritis and other malabsorption conditions * Changes of medications - can cause increased drug clearance, e.g. can be displaced from albumin and cleared more quickly, or can be inducted be hepatic cytochrome So pretty much any cause for liver cell injury, including gastroenterits, heart failure, hepatitis, alcohol etc

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