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Laboratory Testing in Coagulation

MLAB 1227- Coagulation

�Keri Brophy-Martinez

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Platelet Function Tests

Purpose

Evaluate platelet function (qualitative)
Evaluate primary hemostasis

Tests

Bleeding time test
Platelet Function Assays
Platelet aggregometry
Von Willebrand Factor Activity Assays
Heparin- Induced Thrombocytopenia Assays

Laboratory testing to evaluate primary hemostasis includes tests for platelet function. In testing for platelet function, the technician must be aware that a wide variety of drugs can affect platelet function for the entire life span of the platelet which is 7-10 days. The most common primary hemostasis tests are the Bleeding Time which will be discussed in lab, platelet function assays, platelet aggregometry, Von Willebrand factor activity assays, and heparin- induced thrombocytopenia assays.

Platelet function assays and platelet aggregometry will be discussed in more detail in the next unit. Von Willebrand factor activity assays and heparin-induced thrombocytopenia assays are typically considered specialized testing in hemostasis and will not be elaborated on in this unit. Please refer to your textbook for further information on these assays.

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Clot-Based Screening Tests

Purpose

Evaluates coagulation factors- secondary hemostasis
Detects inhibitors

Screening Tests

PT
APTT
Fibrinogen
Thrombin Time

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Prolonged Clotting Times…�what does that mean?

First Considerations

PT and/or APTT must be prolonged
Patient history and symptoms must be considered

Bleeding tendency important to note

Reflex Testing (follow-up tests)

Mixing Study

Determines whether a factor deficiency is present or a circulating inhibitor

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Mixing Study

Also referred to as a circulating inhibitor screen
Principle

Patient plasma is diluted with normal pooled plasma to demonstrate factor levels
The normal plasma provides the missing factor in the patient plasma
50% activity is generally ample to produce a normal PT or APTT

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Inhibitor or Factor Deficiency?

Prolonged aPTT or PT

As an example:

The patient aPTT is 67.2 seconds, with a reference range for normal as 25.5-35.5 seconds.

The aPTT of the normal plasma is 29.0 seconds. The technologist performs a 1:1 mixing study and the aPTT result is 55.0 seconds.

Because the aPTT result did not correct, the most likely cause is an inhibitor.

A second illustration:

The Protime of our patient is 17.5 seconds, with a reference range for normal as 11.5- 13.2 seconds. The Protime of the normal plasma is 12.2 seconds. The technologist performs a 1:1 mixing study and the Protime result is 11.3 seconds. Because the Protime “corrected” back within normal range, the initial patient protime is most likely caused by a factor deficiency.

If the procedure corrects the prolonged PT or APTT within 10% of the reference interval, the defect is in the procoagulant family. Mixing study corrects=Factor deficiency

If the procedure does not correct the PT or APTT, the defect is due to an inhibitor Mixing study does NOT correct=Inhibitor

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Inhibitor Screens

Mixing study did NOT correct
Root cause= INHIBITOR
Presence of an IgG autoantibody

Binds patient’s factors

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Lupus Anticoagulant

Lupus inhibitor should be suspected in a patient with markedly prolonged PTT, but no clinical symptoms or bleeding problems.
Abnormal antibody reacts mildly in vivo with thrombotic events, but reacts stronger in vitro by binding to the phospholipids in the reagents used for coag testing. Commercially prepared reagents are available that do not contain phospholipids and should be used to perform the APTT on these patients.

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Factor Assays

Principle

Ability of the patient’s plasma to correct a prolonged PT or APTT of a known factor deficient plasma
Normal activity range is 50-150% or 50% factor activity

Determines type of factor deficiency and activity
Targets either

PT: Factors VII, X,V, III and II
APTT: Factors XII, XI, IX and VIII

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Factor Assay Procedure

How is it done?

Commercially prepared Factor deficient plasmas are used that contain 100% of all factors except the one in question. A series of these plasmas is usually used which contain various levels of the factor. For example 0%, 20%, 40%, 80%
As a control to compare results to, normal plasma (containing 100% of all factors) is added to the commercially prepared factor deficient plasma in the same way.

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Factor Assay Procedure, cont’d.

3. The patient mixture results and the normal control mixture results are then compared to quantitate the factor level in the patient plasma.

4. A factor assay curve is the basis for plotting patient clotting times at various dilutions.

5. Results of the patient are expressed as a percent of the normal plasma. A patient with a normal factor level should be 50%-150% of the normal control plasma.

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Factor Deficiency vs. Circulating Inhibitor

Deficiency or Inhibitor	Immediate PT or APTT after Mixing Study	Mixing study following 2 hour incubation
Factor deficiency	Correction	Correction
Lupus-like anticoagulant	No correction	No correction
F-VIII inhibitor	Correction	No correction

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Factor XIII

Necessary for the formation of a stable fibrin clot
PT and APTT testing is NORMAL
F-XIII deficiency indicated if screening tests are NORMAL
Patient exhibits delayed bleeding, poor wound healing, or bruising

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Fibrinolytic System Assays

Detects active fibrinolysis
Assays

D-Dimer
FDP

Detects fibrin/fibrinogen degradation products
Requires a special collection tube that contains thrombin and a fibrinolytic inhibitor
Patient serum is mixed with latex particles that detect FDPs and slide is observed for agglutination

Plasminogen
Tissue Plasminogen Activator
Plasminogen Activator Inhibitor-1

FDP assays, including D-dimer are used to detect active fibrinolysis, which indirectly implies thrombosis. Normally, FDPs circulate in concentrations of less than 2 ng/mL. Fibrinolysis generates FDPs and D-dimers in concentrations greater than 200 ng/mL.

D-dimer is a specific marker or fragment resulting from plasmin degradation of the factor XIII stabilized fibrin clot. The D-Dimer test is performed on most coagulation instruments and will be discussed further in the lab. The D-dimer is more common than the FDP test because this test requires an agglutination kit and a special collection tube that contains thrombin to clot the sample and ensure that ALL fibrinogen has been removed and a fibrinolytic inhibitor to prevent IN VITRO fibrinolysis and fibrinogenolysis. Patient serum is mixed with latex particles coated with specific antibodies that recognize FDP on a glass slide for a specific amount of time. The reaction mixture is observed macroscopically for the presence of agglutination.

Plasminogen, tissue plasminogen activator, and plasminogen activator inhibitor-1 are also assays that can detect fibrinolysis. These assays will not be further discussed in this unit as the D-dimer is more commonly ordered for fibrinolytic assessment.

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References

http://labmed.yale.edu/education/cme/casestudies/6/7.aspx
Keohane, E. M., Butina, M., Mirza, K. M., & Walenga, J. M. ([Insert Year of Publication]). Rodak's Hematology (7th ed.). Elsevier Health Sciences (US).
McKenzie, Shirlyn B., and J. Lynne. Williams. "Chapter 40." Clinical Laboratory Hematology. 2nd ed. Boston: Pearson, 2010. Print.