Laboratory Investigation of Haemostatic Disorders Flashcards
Why does a patient present to a haemostasis clinic?
(4)
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
What are the two ways of preparing blood for analysis
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
Give three anti-coagulants used in blood
EDTA (pink or purple) -> for full blood count
Heparin (green) -> usually biochemistry
Other (serum samples, clot activator)
What is the basis of coagulation?
Coagulation requires the presence of Ca++
How does EDTA work?
Irreversible binding calcium
How does sodium citrate work
Binds the calcium but not as strongly as EDTA
How does acid-citrate dextrose (ACD) work?
(2)
A solution of citric acid, sodium citrate and dextrose in water
Used for tissue typing
How does heparin work?
Prevents the actions of thrombin (Factor II)
What are some pre-analytical variables for coagulation samples
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
What are the five main tests used to assess blood clotting function?
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
What is an INR
(5)
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
What is the function of using INR
(6)
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
What is prothrombin time (PT)
(6)
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
Why was INR put in place
(3)
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
What is considered a normal INR
1.1 or below
What is the INR effective therapeutic range for warfarin?
Between 2.0 and 3.0
What warfarin INR requires intervention?
(2)
INR greater than 10
Reduce warfarin and administer vitamin K
What six factors influence INR?
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
What is APTT
Activated Partial Thromboplastin Time
What is APTT?
(6)
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
How is the APTT activated
Caused by the addition of kaolin, phospholipid and CaCl2
What are the three steps to APRR?
Activation of coagulation with Silica
Incubation for 5 minutes at 37 degrees
Calcium is added and this triggers clot formation
What might cause a prolonged APTT and a normal PT
(5)
Deficiency of factor VIII, IX or XI
Inhibitor factor of VIII, IX or XI
Von Willebrand’s disease
Unfractionated heparin
Direct thrombin inhibitors
What would cause a normal APTT and prolonged PT?
(5)
Deficiency o f factor VII
Inhibitor of factor VII
Vitamin K deficiency
Liver disease
Warfarin
What would cause a prolonged APTT and prolonged PT
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
What would cause a prolonged APTT and prolonged PT
(5)
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
How can you overcome heparin contamination?
(3)
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
What is thrombin time?
(4)
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
What are four causes of abnormal Thrombin Time
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
What is reptilase time?
(6)
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
What would indicate dysfibrinogenaemia?
Abnormal TT
Abnormal RT
What would indicate elevated D dimers
Abnormal TT
Abnormal RT
What would indicate heparin in sample
Abnormal TT
Normal RT
What is fibrinogen?
(3)
Fibrinogen is the largest protein of the coagulation system
It is the substrate for the coagulation reaction
Normal range is 1.5-4g/L
How do we carry out a fibrinogen assay?
(5)
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
What are two congenital disorders of fibrinogen
Afibrinogenaemia
Dysfibrinogenaemia
What are four acquired disorders of fibrinogen
Quantitative or qualitative
DIC
Sever blood loss
Liver disease
When and why are correction/mixing studies/test carried out?
(6)
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
What does correction by mixing studies indicate
Factor deficiency
What does no correction by mixing studies indicate?
(2)
Lupus anticoagulant
Specific factor inhibitor
What are factor assays?
(5)
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
What are D-Dimer Test?
(3)
Fibrin split product
Circulating half life of 4-6 hours
Quantitative test have 80-85% sensitivity and 93-100% negative predictive value
What might cause a false positive D-Dimers?
(10)
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
What are D-Dimers?
(5)
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
When do we want to see high D-dimers?
After labour or surgery
We want to see clots being formed
What does elevated D-Dimers indicate?
(3)
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
When might D-Dimers be measured?
(3)
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
What might elevated D-dimer levels cause
(14)
DVT
PE
DIC
Old age Pregnancy
Pathological pregnancies
Coronary disease
Thrombolytic therapy
Cancer
Liver disease
Infection
Inflammation
haematoma
Peripheral arteriopathy
What can a D-Dimer test be used for
(5)
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
What is deep vein thrombosis
(6)
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
What is a pulmonary embolism
(9)
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
What is disseminated intravascular coagulation?
(8)
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)
What might cause DIC?
(3)
Labour
Malignancy
infection
What might cause acute DIC
Infection
Malignancy
Liver disease
Obstetrics
Trauma
Burns
Haemolytic reactions
Massive transfusion
Prosthetic devices
What might cause chronic DIC
(8)
Malignancy
Obstetrics
Myeloproliferative diseases
PNH
Vascular disease
Myocardial infarction
Inflammatory disease
How does DIC lead to end-organ damage?
(7)
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%
What are some laboratory features of DIC
PT APTT
Fibrinogen
D-Dimers
Thrombin Time
Platelet count
Blood film: Signs of haemolysis (schistocytes)
