Laboratory Investigation of Haemostatic Disorders

Question 1

Why does a patient present to a haemostasis clinic?
(4)

Answer 1

Patient presents with personal history of bleeding of bleeding or thrombosis

Family history of bleeding or thrombosis

Unexpected results in coagulation screen

Prior to surgery

Question 2

What are the two ways of preparing blood for analysis

Answer 2

Whole blood from finger-pricks, for immediate, on site-analysis -> used in coagulation clinics or home tests

Blood samples are collected in laboratory test tubes (vacutainers) for remote analysis -> for laboratory testing, usually stored in an anti-coagulant

Question 3

Give three anti-coagulants used in blood

Answer 3

EDTA (pink or purple) -> for full blood count

Heparin (green) -> usually biochemistry

Other (serum samples, clot activator)

Question 4

What is the basis of coagulation?

Answer 4

Coagulation requires the presence of Ca++

Question 5

How does EDTA work?

Answer 5

Irreversible binding calcium

Question 6

How does sodium citrate work

Answer 6

Binds the calcium but not as strongly as EDTA

Question 7

How does acid-citrate dextrose (ACD) work?
(2)

Answer 7

A solution of citric acid, sodium citrate and dextrose in water

Used for tissue typing

Question 8

How does heparin work?

Answer 8

Prevents the actions of thrombin (Factor II)

Question 9

What are some pre-analytical variables for coagulation samples

Answer 9

Underfilling/overfilling of sample container, HCT concentration

Delay in sample analysis (< 4 hours)

Collection of blood through a line contaminated with Heparin, recognition of the interference of drugs on haemostasis

Clean venepuncture, needle gauge from 22-19

Patients should be relaxed and in a warm atmosphere

Venous blood, minimum stasis, no venous occlusion

Tri-sodium citrate, 105mmol or 109mmol, Ratio 1:9

Question 10

What are the five main tests used to assess blood clotting function?

Answer 10

Platelet count (done on EDTA FBC sample)

Prothrombin Time (PT), INR: Assess function of the Extrinsic Pathway (Tissue Factor Pathway)

Activated Partial Thromboplastin Time (APTT) to assess function of the intrinsic pathway

Thrombin time

Fibrinogen

Question 11

What is an INR
(5)

Answer 11

International Normalised Ratio

Used for people on warfarin

Narrow therapeutic window which needs to be monitored

Uses the PT of patient/normal patient -> to the power of the ISI value

ISI -> international sensitivity index

Question 12

What is the function of using INR
(6)

Answer 12

Takes into account that each lab might use different PT reagents

Only used to monitor warfarin

Makes sure your not under or over dossing

Needs to be between 2 and 3

If INR is a little high might be advised to skip a dose

If INR very high might be administered vitamin K

Question 13

What is prothrombin time (PT)
(6)

Answer 13

Sensitive to factor II, V, VII, X and fibrinogen (I) (extrinsic system(

Thromboplastin is added to the patients plasma, along with calcium chloride which activates factor VII and the extrinsic pathway continues

The time it takes for the clot to form is recorded

The test is done in a glass test tube at 37 degrees

Normal range of 10-14 seconds, all laboratories must determine their own normal range

This test is used to monitor warfarin therapy

A prolonged PT may indicate a disorder of clotting processes

Question 14

Why was INR put in place
(3)

Answer 14

PT result on a normal individual will vary according to the type of analytical system employed

Due to variations between different batches of tissue factor used in the reagent to perform the test

Each manufacturer assigns an ISI value for any TF they manufacture, this indicates how their batch of TF compares to an international reference tissue factor

Question 15

What is considered a normal INR

Answer 15

1.1 or below

Question 16

What is the INR effective therapeutic range for warfarin?

Answer 16

Between 2.0 and 3.0

Question 17

What warfarin INR requires intervention?
(2)

Answer 17

INR greater than 10
Reduce warfarin and administer vitamin K

Question 18

What six factors influence INR?

Answer 18

Drugs
Illness especially liver disease
Nutritional intake e.g. cabbage, spinach are rich in vitamin K and therefore can affect the INR
Smoking, alcohol consumption
Physical and mental stress
Climatic variations during travel

Question 19

What is APTT

Answer 19

Activated Partial Thromboplastin Time

Question 20

What is APTT?
(6)

Answer 20

The test measures the clotting time of plasma after the activation of the contact factors (Prekalikren, high molecular weight kininogen, XI and XII)

The APTT is termed ‘partial’ due to the absence of tissue factor from the reaction mixture

It measures the factors XII, XI, X, IX, VIII, V, II and I (intrinsic pathway)

Activation is caused by the addition of kaolin, phospholipid and CaCl2

The reference range normally reported is between 21-35 seconds and depends on the reagents used, each lab establishes their own range

APTT testing is used to monitor heparin therapy

Question 21

How is the APTT activated

Answer 21

Caused by the addition of kaolin, phospholipid and CaCl2

Question 22

What are the three steps to APRR?

Answer 22

Activation of coagulation with Silica
Incubation for 5 minutes at 37 degrees
Calcium is added and this triggers clot formation

Question 23

What might cause a prolonged APTT and a normal PT
(5)

Answer 23

Deficiency of factor VIII, IX or XI

Inhibitor factor of VIII, IX or XI

Von Willebrand’s disease

Unfractionated heparin

Direct thrombin inhibitors

Question 24

What would cause a normal APTT and prolonged PT?
(5)

Answer 24

Deficiency o f factor VII
Inhibitor of factor VII
Vitamin K deficiency
Liver disease
Warfarin

Question 25

What would cause a prolonged APTT and prolonged PT

Answer 25

Deficiency of prothrombin, fibrinogen, factor V, or factor X Inhibitor of prothrombin, fibrinogen, factor V or factor X Supratherapeutic doses of heparin or warfarin Liver disease Disseminated intravascular coagulation Argatroban

Question 26

What would cause a prolonged APTT and prolonged PT (5)

Answer 26

Deficiency of prothrombin, fibrinogen, factor V, or factor X Inhibitor of prothrombin, fibrinogen, factor V or factor X Supratherapeutic doses of heparin or warfarin Liver disease Disseminated intravascular coagulation Argatroban

Question 27

How can you overcome heparin contamination? (3)

Answer 27

Use reptilase time test to confirm this is heparin coagulation prolonging the APTT The reptilase isn't sensitive to heparin This is purified from snake venom

Question 28

What is thrombin time? (4)

Answer 28

Can sometimes be part of a coagulation screen, depending on the laboratory Thrombin is added to plasma and fibrinogen is converted to fibrin Time taken for clot formation is measured Normal Range is 15-23 seconds

Question 29

What are four causes of abnormal Thrombin Time

Answer 29

Dysfibrinogenaemia (abnormal form of fibrinogen) -> Congenital, liver disease or neonate Hypofibrinogenaemia (decreased fibrinogen) -> DIC or congenital deficiency Increased levels of Fibrin Degradation Products -> DIC/liver disease Heparin contamination -> exclude with reptilase time

Question 30

What is reptilase time? (6)

Answer 30

If there is elevated TT, elevated APTT and normal PT Need to confirm if prolonged results are due to heparin contamination through RT RT is similar to TT except Bothrops atrox, a thrombin-like enzyme purified from snake venom is used Heparin is an anticoagulant which works by increasing the power of anti-thrombin Reptilase is unaffected by anti-thrombin (heparin) so the RT will be normal A normal RT in conjunction with a prolonged TT is diagnostic of the presence of Heparin in a sample

Question 31

What would indicate dysfibrinogenaemia?

Answer 31

Abnormal TT Abnormal RT

Question 32

What would indicate elevated D dimers

Answer 32

Abnormal TT Abnormal RT

Question 33

What would indicate heparin in sample

Answer 33

Abnormal TT Normal RT

Question 34

What is fibrinogen? (3)

Answer 34

Fibrinogen is the largest protein of the coagulation system It is the substrate for the coagulation reaction Normal range is 1.5-4g/L

Question 35

How do we carry out a fibrinogen assay? (5)

Answer 35

Plasma is diluted in Owrens buffer Thrombin is added to diluted plasma Fibrinogen is converted to fibrin The fibrin undergoes polymerisation to form a fibrin mesh Activated factor XIII stabilises this mesh to form a visible clot

Question 36

What are two congenital disorders of fibrinogen

Answer 36

Afibrinogenaemia Dysfibrinogenaemia

Question 37

What are four acquired disorders of fibrinogen

Answer 37

Quantitative or qualitative DIC Sever blood loss Liver disease

Question 38

When and why are correction/mixing studies/test carried out? (6)

Answer 38

May be performed when a prolonged PT or the APTT is found In order to narrow down the cause, control normal plasma is mixed with the patient's plasma and the test is repeated If a correction is made, then the control normal plasma which contains all the coagulation factors which was added to the patient's plasma has corrected the deficiency in the patient Addition of control normal plasma to patient plasma Look for correction Test at T0 and T60 after incubation at 37 degrees Celsius

Question 39

What does correction by mixing studies indicate

Answer 39

Factor deficiency

Question 40

What does no correction by mixing studies indicate? (2)

Answer 40

Lupus anticoagulant Specific factor inhibitor

Question 41

What are factor assays? (5)

Answer 41

If a factor deficiency is suspected this is confirmed by carrying out a factor assay for that particular factor An abnormal level of any coagulation factor is verified by repeat assay Patient is tested on a number of occasions Three abnormal factor levels: patient registered as deficient Siblings and family are then tested

Question 42

What are D-Dimer Test? (3)

Answer 42

Fibrin split product Circulating half life of 4-6 hours Quantitative test have 80-85% sensitivity and 93-100% negative predictive value

Question 43

What might cause a false positive D-Dimers? (10)

Answer 43

Pregnant patients Malignancy Advanced age > 80 years Haemorrhage Hepatic impairment Less than 1 week post partum Surgery within 1 week Sepsis CVA Collagen vascular diseases

Question 44

What are D-Dimers? (5)

Answer 44

Specific degradation product of cross-linked fibrin Released when cross-linked fibrin is degraded by plasmin Indirect measurement of thombin generation and subsequent clot formation The only marker of thrombotic disorders that indicate the presence of stabilised fibrin Marker of activation of coagulation and fibrinolysis

Question 45

When do we want to see high D-dimers?

Answer 45

After labour or surgery We want to see clots being formed

Question 46

What does elevated D-Dimers indicate? (3)

Answer 46

Indicates the occurrence of recent thrombotic event It doesn't differentiate between appropriate thrombosis (wound healing) or inappropriate thrombosis (pathological thrombi) A normal D-Dimer Concentration excludes thrombo-embolic events such as DVT and PE with a very high probability

Question 47

When might D-Dimers be measured? (3)

Answer 47

Patient with suspected venous thromboembolism Doctor determines clinical probability according to clinical decision rule Determined as high clinical probability D-Dimer measured Imaging examination to confirm of refute diagnosis

Question 48

What might elevated D-dimer levels cause (14)

Answer 48

DVT PE DIC Old age Pregnancy Pathological pregnancies Coronary disease Thrombolytic therapy Cancer Liver disease Infection Inflammation haematoma Peripheral arteriopathy

Question 49

What can a D-Dimer test be used for (5)

Answer 49

Rules out the presence of a thrombus Rules out deep vein thrombosis Rules out pulmonary embolism Used to determine if further testing is necessary to help diagnose diseases and conditions that cause hypercoagulability A D-dimer level may be used to help diagnose disseminated intravascular coagulation (DIC) and to monitor the effectiveness of DIC treatment

Question 50

What is deep vein thrombosis (6)

Answer 50

Clot in the lower limbs Pains in deep veins at the back of the calf Oedema of the limbs Pulmonary embolism Platelets and fibrin Jelly like mass of fibrin and red cells that may detach and form an embolism

Question 51

What is a pulmonary embolism (9)

Answer 51

Clot that gets stuck in the pulmonary circulation Most occur as a result of a DVT Clot travels via leg veins to lung Chest pain Breathlessness Cold clammy skin Tachycardia Hypotension A large embolus can be immediately fatal

Question 52

What is disseminated intravascular coagulation? (8)

Answer 52

Excessive and widespread activation of coagulation Consumption of coagulation factors and inhibitors Activation of the fibrinolytic system and an increase in fibrin degradation products Systemic generation of thrombin and plasma activity Formation of fibrin-platelet thrombus leading to microvascular ischaemia Plasmin generation leads to the breakdown of the fibrin clot and the cleavage of fibrinogen Loss of regulatory mechanisms Defibrination and haemorrhagic diathesis (rarely associated with thrombosis)

Question 53

What might cause DIC? (3)

Answer 53

Labour Malignancy infection

Question 54

What might cause acute DIC

Answer 54

Infection Malignancy Liver disease Obstetrics Trauma Burns Haemolytic reactions Massive transfusion Prosthetic devices

Question 55

What might cause chronic DIC (8)

Answer 55

Malignancy Obstetrics Myeloproliferative diseases PNH Vascular disease Myocardial infarction Inflammatory disease

Question 56

How does DIC lead to end-organ damage? (7)

Answer 56

Impaired blood flow caused by microvascular thrombosis Ischemia reperfusion injury Systemic inflammatory response syndrome Multiple organ dysfunction syndrome Kidneys-renal damage seen in 25% of DIC cases in one series Liver - hepatic dysfunction in 19% Lungs - respiratory dysfunction in 16%

Question 57

What are some laboratory features of DIC

Answer 57

PT APTT Fibrinogen D-Dimers Thrombin Time Platelet count Blood film: Signs of haemolysis (schistocytes)