Clot-Based Procoagulant Screens

 Prothrombin time (PT): Used to monitor Coumadin/warfarin oral anticoagulant

therapy
Reagent: basic components of the prothrombin reagents =
thromboplastin/tissue thromboplastin prepared from suspended purified tissue factor
phospholipids mixed with a
buffered 0.025 M solution of calcium chloride

* = Principle: PPP mixed with PT reagents. This triggers fibrin polymerization by activating
plasma factor VII. Calcium and phospholipids (both required as cofactors) participate in the
formation of the tissue factor–factor VIIa complex, the factor VIIIa–factor IXa complex, and the
factor Va–factor Xa complex.

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant Screens
 Prothrombin time (PT)
Procedure:
2 parts tissue factor-phospholipid-calcium chloride reagent warmed at 37°C
Add 1 part PPP (also incubated at 37°C for at least 3 minutes)
Start timer.
Check for clot formation
Stop timer as soon as clot forms.
Can be done in duplicate.

Quality control
Commercial control or laboratory prepared controls used. Follow the same lab protocol as
for patient plasma.
Both normal control and prolonged control must be tested.
Must be done at the beginning of each shift depending on the laboratory protocol.

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant Screens
 Prothrombin time (PT)
Reporting results
• Seconds
• Duplicate values must be within 10% of their mean
• Control results:
 Normal control must be within the reference interval
 Prolonged controls must be within the therapeutic range for Coumadin
• Results reported to the nearest tenth of a second.
• International normalized ratio (INR): A calculated value. Reason is to
compensate for the inherent variations among thromboplastin reagents.
• Formula for INR: INR = (PTpatient/PTgeometric mean of normal)ISI

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant Screens
 Prothrombin time (PT)
Reporting results
• Formula for INR: INR = (PTpatient/PTgeometric mean of normal)ISI
• ISI: International sensitivity index
• Done by performing an orthogonal regression analysis comparing its PT
results on a set of plasmas, with the results obtained using the
international reference thromboplastin preparation

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant
Screens (Cont.)
PT
Reference range:
varies from site to site depending on patient population, type
of thromboplastins used, pH, type of instrument & purity of the
reagent
Determined by calculating the PT of healthy individuals
Established with each new lot of reagents, or at least once a
year
Typical range 12.6 to 14.6 seconds

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant
Screens (Cont.)
PT
Diagnostic value:
Prolonged in DIC, Liver disease, vitamin K deficiency (all
affect FVII activity)
Particularly sensitive to liver disease because FVII
Both FV & FVII reduced in Liver disease
Only FVII reduced in vitamin K deficiency.
Screening value
PT is not useful for establishing baseline values in Coumadin
therapy because the therapeutic target range for Coumadin
therapy is based on the INR
Chapter 41 Laboratory evaluation of Haemostasis
 Clot-Based Procoagulant
Screens (Cont.)
PT
Diagnostic value:
Limitations: See table 42-7

Chapter 41 Laboratory evaluation of Haemostasis

 PT

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant
Screens (Cont.)
APTT (aka Activated partial thromboplastin time)
Screening test for laboratory evaluation of inherited or

acquired procoagulant deficiencies of the intrinsic or common

pathway.
Used to monitor the effects of unfractionated heparin therapy

and to detect LA

Chapter 41 Laboratory evaluation of Haemostasis

Clot-Based Procoagulant Screens (Cont.)
 APTT
Principle:
Reagent contains phospholipid and a negatively charged activator.
Enzymatic action takes place on the phospholipid surfaces and the
activator provides the negatively charged surface for the activation of
factor XII.
FXII forms a complex with HMWK and pre-kallikrein (contact group)
After a specific incubation time of the PPP (control or patient plasma)
with the PTT reagent which allows activation of the contact factors,
calcium chloride is added and the time required for the fibrin clot to
form is recorded.
The contact activation factors initiate coagulation in vitro via the
intrinsic pathway but are not part of in vivo coagulation.
Chapter 41 Laboratory evaluation of Haemostasis
Clot-Based Procoagulant Screens (Cont.)
 APTT
 FVII & FXIII deficiencies do not affect PTT.

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant
Screens (Cont.)
APTT
Procedure:
Warm PTT reagent at 37°C
Add equal volume of warmed PPP
Incubate for 3 minutes
Add calcium chloride
Start timer
When clot forms, stop timer and record results in seconds
Manual method to be done in duplicate
Result must be within 10% of their mean

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant
Screens (Cont.)
APTT
Coagulation factor deficiencies detected:
FXI, FIX, FVIII, FX, FV, prothrombin & fibrinogen
Quality control: Similar to PTT but PTT reagent is used instead of PT
reagent and an incubation period is required.
Reference range: Normal control must be within the reference range
Labs often establish their own reference range for their patient
population
Typical range is 26-38 seconds
Prolonged PTT control must be within the therapeutic range for heparin
SEE TABLE 42-7 for specimen errors that may affect the test results

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant
Screens (Cont.)
APTT
Coagulation factor deficiencies detected:
Heparin therapy: Therapeutic range 60 to 100 seconds
Diagnostic value: PTT ordered when haemorrhagic disorder, recurrent
thrombosis or the presence of autoimmune disorder such as LA is
suspected
Prolonged PTT: Prothrombin, FV, FVIII, FIX, FX, FXI, FXII, OR
fibrinogen <100mg/dl ; presence of inhibitors eg FVIII or FIX inhibitor, LA,
interfering substance eg FDP or paraproteins in the case of myeloma.
Not sensitive to FVII deficiency

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant
Screens (Cont.)
 APTT mixing studies
 Lupus anticoagulants See chapter 39.
• Definition: Immunoglobulins against phospholipid-protein
complexes
• Nonspecific inhibitors because of their variety of target antigens
• Clinical features: venous thrombosis
• Method of detection: PTT mixing studies
 Specific inhibitors
• Definition: Ig directed against coagulation factors
• Examples: Anti-FVIII (most common) & anti-FIX
• Method of detection

Chapter 41 Laboratory evaluation of Haemostasis

 Method of detection
Very important to
know this

Mixing study employing a partial thromboplastin time (PTT) reagent with intermediate lupus anticoagulant sensitivity. Beginning at the top left,
when the PTT result exceeds the upper limit of the PTT reference interval, perform a thrombin clotting time (TT) to detect unfractionated heparin. If the TT
exceeds the TT reference interval, presume that heparin is present. Treat an aliquot of the specimen with Hepzyme and repeat the PTT. If the new PTT is normal,
assay the original sample for heparin. If the PTT remains prolonged, or if the TT was normal, proceed by mixing the patient plasma with control normal plasma
(NP) and perform a PTT on the mixture. If the PTT is still prolonged (uncorrected) in comparison to the NP, proceed to assay for the lupus anticoagulant. If the
PTT mixture corrects, prepare a new 1:1 mix and incubate at 37° C for 1 to 2 hours and repeat the PTT, comparing the result to incubated NP. If the incubated
PTT shows a correction, assay for a factor deficiency. If prolonged, assay for a lupus anticoagulant (Figs. 39-2 and 39-3) (a specific factor inhibitor such as
anti-factor VIII associated with bleeding will also be detected in this manner
Chapter 41 Laboratory evaluation of Haemostasis
 APTT

Chapter 41 Laboratory evaluation of Haemostasis

Clot-Based Procoagulant Screens (Cont.)

 Thrombin clotting time (TCT)

 Principle: Thrombin cleaves fibrinopeptides A & B
from fibrinogen to form fibrin
Procedure
 Incubate thrombin reagent at 37° for 3-10 minutes
 Warm PPP also at 37°C for 3-10 minutes
 Mix 2 part thrombin reagent + 1 part PPP. Start stop
watch and record time interval to clot formation.

Chapter 41 Laboratory evaluation of Haemostasis

 Clot-Based Procoagulant Screens (Cont.)

 Thrombin clotting time (TCT)

Quality control: Normal control results should fall within lab’s
reference interval
Abnormal control results should prolong to the range reached for
moderate hypofibrinogenaemia
Reporting results: 15-20 seconds
Interpretation of results:
insufficiency or ineffectiveness of fibrinogen
presence of antithrombotic substances

Chapter 41 Laboratory evaluation of Haemostasis

 TCT

Chapter 41 Laboratory evaluation of Haemostasis

 Coagulation Factor Assays

 Fibrinogen assay
 Principle: Clauss method; modification of the TCT
 PPP is diluted in Owren’s buffer (1:10 dilution). Reagent bovine thrombin
is added to the diluted PPP. The bovine thrombin reagent catalyzes the
conversion of fibrinogen to fibrin polymer. The fibrinogen concentration is
inversely proportional to clotting time.
 PPP is diluted to minimize the antithrombotic effects of heparin, FDP,
and paraprotein.

Chapter 41 Laboratory evaluation of Haemostasis

 Coagulation Factor Assays

 Fibrinogen assay
 Reference curve: Set up regularly depending on the lab protocol. Results
for controls and patient are determined from the reference curve
Test protocol:
 1:10 dilution of PPP in Owren’s buffer
 Incubate 200µl of diluted PPP at 37°C for 3 minutes.
 Add 100µl thrombin reagent, start timer and observe for clot formation
 If fibrinogen >480mg/dl make a 1:20 dilution of PPP and test. Multiply the
result by 2 to compensate for the dilution.

Chapter 41 Laboratory evaluation of Haemostasis

 Coagulation Factor Assays

 Fibrinogen assay
Quality control: Duplicate results must agree within a CV of <7%.
Normal and abnormal controls tested. Normal control must be within
reference range. Abnormal controls should be <100mg/dl.
Clinical utility: Hypofibrinogenaemia (fibrinogen < 220mg/dl) is associated with
DIC, and liver disease.
Congenital afibrinogenaemia gives prolonged clotting times
Dysfibrinogenaemia results same as hypofibrinogenaemia because
abnormal fibrinogen are hydrolyzed more slowly by thrombin than normal
fibrinogen
Limitations

Chapter 41 Laboratory evaluation of Haemostasis

 Coagulation Factor Assays

 Fibrinogen assay
Limitations: Heparin levels >0.6 units/ml, and FDP levels > 100µg/ml give
falsely lowered fibrinogen results.

Chapter 41 Laboratory evaluation of Haemostasis

Fibrinogen Assay

Chapter 41 Laboratory evaluation of Haemostasis

 Coagulation Factor Assays (Cont.)

 Single-factor assays based on APTT

Prolonged PTT commonly seen in single factor deficiencies of the
following: FVIII, FIX, FXI (Causes mild intermittent bleeding, aka
Rosenthal syndrome)
PPP that are FVIII depleted: All procoagulants have normal activity
except FVIII.
NP + PPP FVIII depleted plasma: If tested and result is normal,
implies FVIII deficiency.

Chapter 41 Laboratory evaluation of Haemostasis

 Coagulation Factor Assays (Cont.)

 Clinical utility: Reference interval is 50% to 186%

 ≤ 10% then symptoms for haemophilia is present
 Quality control: Normal control should be within reference range
 FVIII deficient control should be ≤ 10% FVIII activity
 Bethesda titer used to confirm the presence and quantify an anti–
factor VIII inhibitor.

Chapter 41 Laboratory evaluation of Haemostasis

 Test of Fibrinolysis

 Quantitative D-dimer assay

• Principle: Microlatex particles in buffered saline are coated
with monoclonal anti–D-dimer antibodies. When mixed with
patient plasma, these coated particles are agglutinated by
the patient’s D-dimer. Turbidity results which is then
measured using nephalometric or turbidometric technology
• Clinical application: To rule out various disease conditions eg
acute myocardial infarction, stroke etc.

Chapter 41 Laboratory evaluation of Haemostasis

 Test of Fibrinolysis (Cont.)

 Tissue plasminogen activator (TPA)

 Physiology of TPA
 Clinical significance of TPA
 Specimen collection
 Principle of assay

Chapter 41 Laboratory evaluation of Haemostasis

 Chapter Review

 What are some specimen collection

requirements for coagulation studies?
 What are some conditions that will give a
prolonged bleeding time?
 Discuss proper hemostasis specimen collection
 If the PT is normal and the APTT prolonged,
what factor deficiencies could be present?
 What are D-dimers, and what conditions will
cause increased levels?

Chapter 41 Laboratory evaluation of Haemostasis