1. MLAB 1227- Coagulation Keri Brophy-Martinez
Laboratory Testing in Coagulation

2. Lab Testing For Primary Hemostasis Disorders
Purpose
Evaluate platelet concentration and function
Tests
Bleeding time: discussed in lab
PFA-100
Platelet aggregometry

3. Lab Testing For Secondary Hemostasis Disorders
Purpose
Evaluates coagulation factors
Detects inhibitors
Screening Tests PT
APTT Discussed in lab
Fibrinogen
Thrombin Time

4. Thrombin Time Qualitative
Useful to detect parameters not detected by PT and aPTT; such as heparin, presence of FDPs, presence of dys/hypofibrinogenemia
Thrombin time is prolonged with these parameters
Measures conversion of fibrinogen to fibrin
Thrombin cleaves fibrinogen in undiluted plasma
Normal reference range seconds

5. Lab Testing For Secondary Hemostasis Disorders
If a screening test is prolonged, further testing must be performed to locate the specific cause of the abnormality
2 Possible Causes
Factor Deficiency
Circulating Inhibitors

7. Factor Deficiency Evaluation
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
Will determine whether a factor deficiency is present or a circulating inhibitor

8. 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
Clotting times tend to increase with time and incubation due to the loss of labile factors, so it is important to compare the results of the patient’s diluted sample with the normal pooled plasma

9. Mixing Study
If the procedure corrects the prolonged PT or APTT, 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

10. Mixing Study

11. 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 % 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

12. 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%, 10%, 20%, 30%, 40%, 50%, etc.
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.

13. 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.

14. Inhibitor Screens
When a PT or PTT is prolonged it must first be determined if the defect is due to a true factor deficiency or if it may be due to an abnormal circulating inhibitor (autoantibody to a factor). An inhibitor screen will rule out one or the other.

15. Inhibitor Screen Procedure
Based on the fact that if a specimen contains at least 50% of a normal level of factors, the PT or APTT will give a normal result.
Normal plasma (containing 100% of all factors and giving a normal APTT) is added in equal proportion to the unknown plasma (which has already given us an abnormal APTT result).
This 1:1 mixture we know contains at least 50% of all factors (because we added it) so we expect the APTT on this mixture to be normal.

16. Inhibitor Screen Procedure, cont’d.
If the APTT on the 1:1 mixture results in a “corrected” APTT (the patient sample was abnormal before but is normal now that we added normal plasma to it), then this indicates a factor deficiency was present in the original patient sample. The problem is not an autoantibody.
If the APTT on the 1:1 mixture does not correct the APTT, (the APTT is still abnormal even after normal plasma was added), then this indicates there is an autoantibody present. This antibody is not only binding the patient's factors, but the factors in the normal plasma that was added to the mixture as well.

17. Lupus Inhibitor Screen
Lupus inhibitor should be suspected in a patient with markedly prolonged PT and APTT, but no clinical symptoms or bleeding problems.
The 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. APTT results will then be normal

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

19. Anti Xa Assay Used to monitor LMWH Chromogenic assay Advantages
Less affected by biologic factors such as lupus anticoagulant, liver disease, or DIC
Less affected by pre-analytic factors, such as tube underfilling

20. Anti Xa Assay Procedure Excess FXa is added to patient PPP
Heparin/AT complexes in patient sample will bind FXa
Chromogenic substrate is added and any unbound FXa complexes the chromogen
Produces a yellow color, read spectrophotometrically
Results read off a standard curve

21. Factor XIII Necessary for the formation of a stable fibrin clot
Cross-linking by Factor XIII does not affect PT and APTT
F-XIII deficiency test indicated if screening tests are NORMAL
Patient exhibits delayed bleeding, poor wound healing, or bruising

22. F-XIII Test
Principle
Based on the observation that the fibrin clot has increased solubility because of the lack of cross-linking of the fibrin polymer in the absence of F-XIII
Patient PPP mixed with calcium chloride and allowed to clot for an hour at 37°C.
The clot is removed and placed in another tube containing urea
If the clot dissolves in less than 24 hours, there is less than 2% F-XIII activity

23. Lab Testing of the Fibrinolytic System
D-Dimer --Discussed in lab
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

24. Activated Clotting Time= ACT
Developed to monitor coagulation status and heparinization in immediate need situations.
Bedside test (POC)
The ACT uses tubes containing a negatively charged particulate activator of coagulation, such as kaolin.
When whole blood is drawn into the tube, the contact system is activated and clotting occurs.
This assay is particularly sensitive to heparin, but is affected by platelets, coagulation factors, and inhibitors.

25. References http://labmed.yale.edu/education/cme/casestu dies/6/7.aspx
McKenzie, Shirlyn B., and J. Lynne. Williams. "Chapter 40." Clinical Laboratory Hematology. 2nd ed. Boston: Pearson, Print.