Notifications
Clear all

COAGULATION SCREENING TESTS


ug
Posts: 33
 ug
Admin
Topic starter
(@mr-right)
Reporter
Joined: 4 months ago

 

  • Various tests are available for screening patients for disorders of haemostasis and basic tests which are important to the diagnosis of bleeding disorders include;
    • Bleeding time (BT)
    • Platelet count
    • Activated partial thromboplastin time (aPTT)
    • Prothrombin time (PT) and
    • Thrombin time (TT)
  • Other tests included
    • Mixing experiment
    • Factor essays

Basic Requirements for These Tests Include;

  • Patient plasma and control plasma
  • Water bath to keep temperature at 37°C
  • Refrigerators and freezers
  • Centrifuges
  • Reagents and Buffers
  • Plastic and glass tubes
  • Pipettes and pipette tips  Stopwatches and Stopclocks  Automatic Coagulometer, etc.

PRE-ANALYTICAL VARIABLES

  • Various stringent measures must be followed when carrying out tests for assessing haemostasis

Sample Collection

  • Sample collection must be done by a phlebotomist and sample taken in a well relaxed patient with minimal exercise and stress as this can affect factor VII, vWF and Fibrinolysis
  • Free flowing blood, if possible, avoid pressure cuff

Anticoagulants Used

  • Trisodium citrate as anticoagulant in a ratio of 1:9 or 1 in 10 (1 part of anticoagulant to 9 parts of blood) is recommended
  • Other anticoagulants like Oxalate, Heparin and EDTA are UNACCEPTABLE  Labile factors are unstable in Oxalate

 

Heparin and EDTA directly inhibit coagulation process and interfere with endpoint determination

  • APTT tests are more sensitive to the presence of Heparin

Time of Sample Collection

  • Time of sample collection is also crucial as fibrinolytic activity follow a circadian pattern

Preparation of PPP[1] Using Centrifugation Method

  • Usually centrifuge at 2000g for 15 min at 4°C

Storage of Samples

  • The sample must be kept frozen at -40°C or -80°C or in liquid Nitrogen -196°C
  • Gentle but thorough mixing of samples is essential after thawing[2] and before testing

THE FIRST LINE TESTS

  • Prothrombin time
  • Activated partial thromboplastin time
  • Thrombin time
  • Fibrinogen assay
  • Platelet count

PROTHROMBIN TIME

Principle

  • Prothrombin times is the time in seconds required for plasma to clot at 37°C when a tissue thromboplastin, an eternal coagulation factor and calcium are added
  • The test indicates the overall efficiency of the extrinsic clotting system and in addition, assess factors V, VII, X and Fibrinogen

Reagents

  • Patient and control plasma
  • Calcified tissue thromboplastin which must be reconstituted with de-ionized water

Method

  • The reconstituted thromboplastin will be warmed to 37°C in a water bath for 2 minutes
  • 1 ml of the test plasma will be dispensed into a glass tube placed in a rack in the water bath at 37°C also. The plasma will be warmed for 2 minutes

0.2ml of the warmed reconstituted calcified tissue thromboplastin will be added and the time taken for the clot to form recorded

  • The procedure will be carried out on the test and control in duplicate
  • PT will be expressed as the mean of the duplicate readings in seconds
  • Normal value – 11-16 sec (Rabbit thromboplastin) and 10-12 sec (recombinant human thromboplastin)

Causes of Prolonged PT

  • Administration of oral anticoagulant drugs
  • Liver disease
  • Vitamin K deficiency
  • DIC[3]
  • Factor II, V, VII, and X deficiency

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

Principle

  • The test measures the clotting time of plasma after the activation of contact factors, but without added tissue thromboplastin and it indicates the overall efficiency of the intrinsic pathway
  • To standardize the contact factors, the plasma is first pre-incubated with kaolin
  • And standardized phospholipids are provided to allow the clotting reaction to be performed on platelet poor plasma
  • The test depends on contact factors II, V, VIII, IX, X and fibrinogen
  • The test is sensitive to the presence of circulating inhibitors and heparin

Reagent

  • Test and control plasma
  • Standardized kaolin phospholipid reagent
  • Calcium chloride

Method

  • Calcium chloride should be pre-warmed for 3 minutes at 37°C in a water bath
  • 1ml of test plasma and 0.1ml of the kaolin phospholipid will be pipetted in a glass tube and incubated for 6 minutes
  • Then 0.1ml of the pre-warmed CaCl2 added and a stopwatch will be started simultaneously and the time taken for clot to form is recorded
  • Both test and the control samples must be done in duplicate and the result expressed as the mean of the paired clotting time

NR[4] – 30-40 seconds

Causes of Prolonged APTT

  • DIC
  • Liver dx
  • Massive transfusion
  • Administration of heparin or other anticoagulant
  • Deficiency of factor VIII

THROMBIN TIME

  • Thrombin is added to plasma and clotting time is measured
  • TT is affected by the concentration and reaction of fibrinogen and the presence of inhibitory substances, including FDPs[5]
  • N-R – 15-19 seconds

Causes of Prolonged Thrombin Time

  • Hypofibrinogenaemia
  • Dysfibrinogenaemia
  • Hypoalbuminaemia
  • Raised concentration of FDP as in DIC[6] or liver diseases
  • Presence of heparin which interferes with thrombin-fibrinogen reaction

FIBRINOGEN ASSAY

  • Various methods are available for fibrinogen assay
    • Clotting
    • Immunological
    • Physical and
    • Nephelometric techniques
  • Clauss technique is most commonly recommended

Dry Clot Technique of Ingram

  • Principle – fibrinogen in plasma is converted into fibrin by clotting with thrombin and calcium
  • The resulting clot is weighed
  • The quantity of clot formed is proportionate to the fibrinogen concentration
  • Result is expressed as g/l
  • N-R – 1.5-4g/l

 

INTERPRETATION OF RESULTS OF COAGULATION SCREENING TESTS

Condition                                         PT                 APTT          TT               FIB               PLT

Normal haemoglobin

N

N

N

N

N

Diseases of platelet function

 

 

 

 

 

Factor XIII

 

 

 

 

 

Diseases of vasculature

 

 

 

 

 

Mild vWD

 

 

 

 

 

Diseases of fibrinolysis

 

 

 

 

 

Factor VII deficiency

Prol[7]

N

N

N

N

Oral anticoagulant

 

 

 

 

 

Lupus anticoagulant

 

 

 

 

 

Mild II, V or X deficiency

 

 

 

 

 

Deficiency of VIII, IX, XI, XII,

HMWK, Pre-K

N

Prol

N

N

N

vWD

 

 

 

 

 

Lupus anticoagulant

 

 

 

 

 

Vitamin K deficiency

Prol

Prol

N

N

N

Oral anticoagulant

 

 

 

 

 

Deficiency of II, V or X

 

 

 

 

 

Liver diseases

Prol

Prol

Prol

N/Abn[8]

N

Heparin

 

 

 

 

 

Fibrinogen deficiency/disorder

 

 

 

 

 

Thrombocytopenia

N

N

N

N

L

Massive transfusion

P

P

N

N/Abn

L

DIC/Liver disease

P

P

P

L

L

 

CLOT SOLUBILITY TEST FOR FACTOR XIII

  • Fibrin clots formed in the presence of factor XIII and thrombin are stable as a result of cross-linking for at least 1hr in 5mol/l urea
  • Whereas clot formed in the absence of factor XIII dissolve rapidly

Euglobin Clot Lysis Time

 A test of Fibrinolysis

 

[1] PPP – Platelet Poor Plasma

[2] Melting/dissolving

[3] Disseminated Intravascular Coagulation

[4] NR – Normal Range

[5] FDP – Fibrin Degradation Protein

[6] DIC – Disseminated Intravascular Coagulation

[7] Prolonged

[8] Abn – abnormal