MLAB 1227-CoagulationMLAB 1227-CoagulationKeri Brophy-MartinezKeri Brophy-Martinez

Preanalytical Issues in Coagulation Testing

ObjectivesObjectives1. 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.

Sample CollectionSample CollectionAnticoagulant 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

Sample CollectionSample CollectionSidenote: 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

Correction Formula: High Correction Formula: High HematocritsHematocrits

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

Site SelectionSite SelectionUntraumatic venipuncture is

required◦Traumatic venipunctures release

tissue factor and initiate coagulationFingersticks/Heelsticks are not

recommendedIndwelling IV line draws are

discouraged◦Contain heparin◦Falsely increased results

Order of DrawOrder of Draw

Evacuated tube system◦Blue top is 1st or 2nd tube. ◦ If 2nd tube drawn, 1st 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

Storage RequirementsStorage RequirementsProthrombin Time: PT

◦Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 18-24 oC 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 oC must be tested within 4 hours of collection

Storage RequirementsStorage RequirementsOther Assays

◦Fibrinogen, Thrombin Time, Factor Assays

◦Centrifuged with plasma remaining on top of cells in unopened tube kept at 18-24 oC must be tested within 4 hours of collection

Storage RequirementsStorage RequirementsOther 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 oC for two weeks -70 oC for six months

Transportation of Transportation of SpecimenSpecimenSend specimen on ice OR deliver

to lab ASAPSeparate cells from plasma

immediately via centrifugation

Error Consequence CommentShort draw<2.7 mL

PT/PTT falsely prolonged Anticoagulant to blood ratio exceeds 1:9

Failure to mix specimen after collection

PT/PTT falsely prolonged Blood clots form when anticoagulant & blood do not mix

Excess vigorous mixing PT/PTT falsely shortened Hemolysis and platelet activation cause start of cascade

Hemolysis PT/PTT falsely shortened Reject specimen

Improper storage: wrong temperature or held too long

PT/PTT falsely prolonged Must follow storage requirements

Chilling in refrigerator or placing on ice

PT falsely shortened Chilling to 4 oC activates factor VII.

Inadequate centrifugation

PTT loses sensitivity for lupus anticoagulants and heparin.Factor assays inaccurate

Desire platelet poor plasma

Prolonged tourniquet application

Falsely elevates vWF, factor VIII

Tourniquet causes venous stasis

Common Collection Common Collection Problems Problems

Common Collection Problems Common Collection Problems (con’t)(con’t)

Error Consequence Comment

Drawing coagulation tube PRIOR to other anticoagulant tubes

PT/PTT falsely affected Contamination

Probing the vein PT/PTT falsely shortened

Tissue thromboplastin is released activating coagulation

Heparin contamination from line draw

PTT falsely prolonged Heparin keeps the blood from clotting

Lipemia Test may not work Photo-optical methods affected

Icterus Test may not work Photo-optical methods affected

TermsTerms 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

TermsTermsPlatelet-Rich plasma(PRP)

◦Used in platelet function studies◦200-300 x 10 9 /L◦Specimen has been centrifuged for

30 minutes @ 50 xg

ReferencesReferencesCastellone, 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.