1. MLAB 1227-Coagulation Keri Brophy-Martinez Preanalytical Issues in Coagulation Testing

2. Objectives 1. Describe special precautions regarding specimen collection and processing for coagulation testing and determine the appropriateness of specimens. 2. State the type of anticoagulant tube used for coagulation testing. 3. Discuss the function of the sodium citrate anticoagulant in coagulation testing. 4. Recognize the appropriate order of draw in terms of coagulation testing. 5. State and apply the formula used to correct for high hematocrits. 6. Cite storage requirements for the prothrombin and activated prothrombin time assays. 7. Define platelet poor plasma. 8. Define platelet rich plasma. 9. State the purpose of the blood to anticoagulant ratio in the sodium citrate tube.

3. Sample Collection Anticoagulant of choice ◦ 3.8% or 3.2% Sodium Citrate  3.2 % Preferred as the standard measure due to stability and closeness to the plasma osmolality ◦ Anticoagulant/blood ratio is critical (1:9)  Exact amount of blood must be drawn. No short draws are acceptable, this will falsely increase results due to presence of too much anticoagulant  CLSI guideline is 90 % of calibrated volume ◦ Purpose of the anticoagulant is to bind or chelate calcium to prevent clotting of specimen

4. Sample Collection Sidenote: Samples with High hematocrits ◦ CLSI recommends adjusting anticoagulant ratio for patients with hematocrits exceeding 55% ◦ High hematocrits may cause falsely prolonged test results due to an over- anticoagulated sample

5. Correction Formula: High Hematocrits C= (1.85 x 10 -3 ) (100-H)V Where: C= volume of sodium citrate V=volume of whole blood drawn H= patient’s hematocrit Example: Patient has a hematocrit of 60%, and blood is to be drawn into a 2.7 mL blue top-tube. Patients Hct= 60% V= 2.7 mL C= (1.85 x 10 -3 )(100-60) * 2.7mL = 0.2 mL

6. Site Selection Untraumatic venipuncture is required ◦ Traumatic venipunctures release tissue factor and initiate coagulation Fingersticks/Heelsticks are not recommended Indwelling IV line draws are discouraged ◦ Contain heparin ◦ Falsely increased results

7. Order of Draw Evacuated tube system ◦ Blue top is 1 st or 2 nd tube. ◦ If 2 nd tube drawn, 1 st top must be anticoagulant free (i.e. red top) Butterfly system ◦ Discard tube must be utilized  Why?  To avoid clotting  To rid specimen of air in the tubing which can cause an inaccurate blood-anticoagulant ratio

8. Storage Requirements Prothrombin Time: PT ◦ Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 18-24 o C must be tested within 24 hours of collection Activated Partial Thrombin Time: APTT ◦ Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at18-24 o C must be tested within 4 hours of collection

9. Storage Requirements Other Assays ◦ Fibrinogen, Thrombin Time, Factor Assays ◦ Centrifuged with plasma remaining on top of cells in unopened tube kept at 18-24 o C must be tested within 4 hours of collection

10. Storage Requirements Other general notes ◦ Perform coagulation tests ASAP  Specimen may deteriorate rapidly (especially factors V and VIII) ◦ If the testing is not completed within specified times, plasma should be removed from the cells and placed in a frost free freezer  - 20 o C for two weeks  -70 o C for six months

11. Transportation of Specimen Send specimen on ice OR deliver to lab ASAP Separate cells from plasma immediately via centrifugation

12. ErrorConsequenceComment Short draw <2.7 mL PT/PTT falsely prolongedAnticoagulant to blood ratio exceeds 1:9 Failure to mix specimen after collection PT/PTT falsely prolongedBlood clots form when anticoagulant & blood do not mix Excess vigorous mixingPT/PTT falsely shortenedHemolysis and platelet activation cause start of cascade HemolysisPT/PTT falsely shortenedReject specimen Improper storage: wrong temperature or held too long PT/PTT falsely prolongedMust follow storage requirements Chilling in refrigerator or placing on ice PT falsely shortenedChilling to 4 o C activates factor VII. Inadequate centrifugationPTT loses sensitivity for lupus anticoagulants and heparin. Factor assays inaccurate Desire platelet poor plasma Prolonged tourniquet application Falsely elevates vWF, factor VIIITourniquet causes venous stasis Common Collection Problems

13. Common Collection Problems (con’t) ErrorConsequenceComment Drawing coagulation tube PRIOR to other anticoagulant tubes PT/PTT falsely affectedContamination Probing the veinPT/PTT falsely shortenedTissue thromboplastin is released activating coagulation Heparin contamination from line draw PTT falsely prolongedHeparin keeps the blood from clotting LipemiaTest may not workPhoto-optical methods affected IcterusTest may not workPhoto-optical methods affected

14. Terms Platelet –Poor plasma (PPP) ◦ <10 x 10 9 /L ◦ Specimen has been centrifuged for 15 minutes @ 1500 xg ◦ The reasons for plasma to be PP are:  Contains platelet factor 4  heparin neutralizer- results in falsely decreased results  Contains phospholipid  affects lupus anticoagulant and factor assay testing  Results in a false normal result  Contains factor 8 and von Willebrand antigen  Results in falsely decreased results

15. Terms Platelet-Rich plasma(PRP) ◦ Used in platelet function studies ◦ 200-300 x 10 9 /L ◦ Specimen has been centrifuged for 30 minutes @ 50 xg

16. References Castellone, D. (n.d.). Coagulation Dos and Dont's. Advance for Administrators of Clinical Laboratories, 13(1), 1-2. McKenzie, Shirlyn B., and J. Lynne. Williams. "Chapter 40." Clinical Laboratory Hematology. Boston: Pearson, 2010. Print.