1. Laboratory Testing in Coagulation Coagulation Keri Brophy-Martinez

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

3. PFA-100: Platelet Function Assay In-vitro system that stimulates the in- vivo function of platelets in primary hemostasis: adhesion, activation and aggregation Aids in detection of platelet dysfunction Whole blood is passed through an aperture in a membrane coated with agonists Closure of aperture with platelet plug results in “closure time” Replacing the BT in many labs due to variability

4. Platelet Aggregation Studies Foundation of platelet function testing Disadvantages: Time consuming Requires attention to detail Tests the platelets ability to respond and aggregate In the presence of in-vitro platelet aggregating agents, normal platelets will aggregate producing characteristic patterns of aggregation. (Refer to Coag Automation ppt. from lab) Agonists include epinephrine, collagen, ADP, ristocetin, and arachidonic acid

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

6. Qualitative Useful to detect parameters not detected by PT and aPTT; such as heparin, presence of FDPs, presence of dys/hypofibrinogenemia Measures conversion of fibrinogen to fibrin Thrombin cleaves fibrinogen in undiluted plasma Normal reference range 10-16 seconds

7. 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

8. Factor Deficiency Evaluation First Considerations PT and/or APTT must be prolonged Patient history and symptoms must be considered Reflex Testing (follow-up tests) Mixing Study Will determine whether a factor deficiency is present or a circulating inhibitor

9. 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

10. 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

11. Mixing Study

12. 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

13. Factor Assay Procedure How is it done? 1. 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. 2. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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

19. Factor Deficiency vs. Circulating Inhibitor Deficiency or Inhibitor Immediate PT or APTT after Mixing Study Mixing study following 2 hour incubation Factor deficiencyCorrection Lupus-like anticoagulantNo correction F-VIII inhibitorCorrection No correction

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

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. Thrombelastography= TEG Global Testing: analyzes the entire hemostasis process Non-invasive instrument designed to analyze a whole blood sample to assess a patient’s clinical hemostatic condition Primarily used during surgical procedures Basic Principle Monitors hemostasis in its entirety Clot initiation through clot lysis Net effect of all hemostatic components interacting together Activated blood maximizes thrombin generation and platelet activation in vitro Demonstrates hemostatic potential of a blood sample at a given point

26. TEG Normal TEG

27. TEG Advantages Differentiates surgical from pathological bleeding Reduces the use of unnecessary blood products

28. Historical Tests In other words, might see on Registry **Student will not be tested on this material for MLAB 1227 1.Clot Retraction Test Normal blood clots 30-60 minutes after collection pg 897 2.Russel’s Viper Venom Test Stypven time LA or aPL test Reference value: <1:2 ratio of patient serum to normal control pg 906

29. References http://labmed.yale.edu/education/cme/casestu dies/6/7.aspx 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, 2010. Print.