Blood Specimens: Coagulation

Sample Collection for Coagulation Testing

1. Collection Tube. Blood should be collected in a blue-top tube containing 3.2% buffered sodium citrate.

Note: Please examine specimen collection and transportation supplies to be sure they do not include expired containers.

2. High Hematocrit Samples. Patients with elevated hematocrits have a relatively low amount of plasma for a given whole blood (collection) volume. This tends to increase the plasma citrate concentration effectively. If the patient has a known hematocrit >55%, the amount of citrate in the collection tube must be decreased according to the formula below.

The equation in CLSI H21 is as follows:

C = (1.85 x 10⁻³) (100−Hct) (V_blood)

Where:

C is the volume of citrate remaining in the tube.

Hct is the patient's hematocrit.

V is the volume of blood added to the evacuated tube.

Example:

Patient hematocrit = 60%

Total volume = 5 mL (standard citrated plasma collection tube volume)

C = (0.00185) x (100-60) x 4.5

C = (0.00185) x 180 (or 40 x 4.5)

C = 0.333

3. Order of Draw. A discard tube is not required prior to collection of coagulation samples, except when using a safety winged blood collection device (ie, "butterfly"), in which case a discard tube should be used. When noncitrate tubes are collected for other tests, collect sterile and nonadditive (red-top) tubes prior to citrate (blue-top) tubes. Any tube containing an alternate anticoagulant should be collected after the blue-top tube. Gel-barrier tubes and serum tubes with clot initiators should also be collected after the citrate tubes.

4. Venipuncture Technique. To avoid activation of the sample, the venipuncture should be clean, with minimal trauma. The tourniquet should be in place only as long as is needed to identify a vein and should never be in place for longer than one minute. Severely hemolyzed samples are not acceptable.

5. Safety winged blood collection kits (butterfly) must use a discard lead tube prior to collecting specimen tube to submit for testing. Failure to use a discard tube may lead to underfilling of the evacuated tube.

6. Fill Volume. Evacuated collection tubes must be filled to completion to ensure that a 9:1 blood-to-anticoagulant ratio is achieved. Under-filling of citrate collection tubes results in an increased anticoagulant-to-blood ratio and can extend clot-based coagulation assays. Note: Never combine two underfilled tubes together.

Please print and use the Volume Guide for Coagulation Testing to ensure proper draw volume.

7. Mixing. The sample should be mixed immediately by three to six complete gentle end-over-end inversions to ensure adequate mixing of the anticoagulant with the blood.

8. Plasma Processing. Process the sample as soon as possible (preferably within 30 minutes of collection). Centrifuge at an adequate speed and duration to achieve platelet-poor plasma (<10,000/μL). After centrifuging the sample, transfer plasma, being careful not to approach the buffy coat, using a plastic pipette into a LabCorp PP transpak frozen purple tube with screw cap (LabCorp N° 49482). Note that glass should not be used because glass can activate the clotting cascade. Label each tube “plasma, citrate.” The specimen should be frozen immediately and maintained frozen until tested. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested.

Platelet-poor Plasma (PPP) Collection for Lupus Anticoagulant Testing

Lupus anticoagulants (LA) are are nonspecific antibodies that extend clot-based coagulation assays as the result of their interaction with phospholipid in the reaction mixture. Platelets in plasma samples can act as a source of phospholipid and mask the effects of LA. For this reason, it is important to prepare platelet-poor plasma (PPP) for LA testing. PPP should have a platelet count <10,000/μL. PPP samples should be collected by double centrifugation.

1. Centrifuge for 10 minutes, and carefully remove two-thirds of the plasma using a plastic transfer pipette, being careful not to disturb the cells.

2. Deliver plasma to a plastic transport tube, cap, and recentrifuge for 10 minutes.

3. Use a second plastic pipette to remove the plasma, staying clear of the platelets at the bottom of the tube.

4. Transfer plasma using a plastic pipette into a LabCorp PP transpak frozen purple tube with screw cap (LabCorp N° 49482). Label each tube "plasma, citrate."

The specimen should be frozen immediately and maintained frozen until tested. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested.

Clearing (Flushing) the Volume of Intravenous Lines Before Drawing Samples for Hemostasis Testing

Collection of blood for coagulation testing through intravenous lines that have been previously flushed with heparin should be avoided, if possible. If the blood must be drawn through an indwelling catheter, possible heparin contamination and specimen dilution should be considered.

When obtaining specimens from indwelling lines that may contain heparin, the line should be flushed with 5 mL of saline, and the first 5 mL of blood or six times the line volume (dead space volume of the catheter) should be drawn off and discarded before the coagulation tube is filled. For those samples collected from a normal saline lock (capped off venous port), twice the dead space volume of the catheter and extension set should be discarded.

References

Adcock DM, Kressin DC, Marlar RA. Are discard tubes necessary in coagulation studies? Lab Med.1997; 28:530-533.

Brigden ML, Graydon C, McLeod B, Lesperance M. Prothrombin time determination. The lack of need for a discard tube and 24-hour stability. Am J Clin Pathol. 1997 Oct; 108(4):422-426.

Clinical and Laboratory Standards Institute. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays; Approved Guideline—Fifth Edition. Wayne, Pa: CLSI; 2008.

Konopad E, Grace M, Johnston R, Noseworthy T, Shustack A. Comparison of PT and aPTT values drawn by venipuncture and arterial line using three discard volumes. Am J Crit Care. 1992 Nov; 1(3):94-101.

Laxson CJ, Titler MG. Drawing coagulation studies from arterial lines; an integrative literature review. Am J Critical Care. 1994 Jan; 3(1):16-24.

Lew JK, Hutchinson R, Lin ES. Intra-arterial blood sampling for clotting studies. Effects of heparin contamination. Anaesthesia. 1991 Sep; 46(9):719-721.