Coagulation

1. 3.2% citrate plasma, shipped frozen, is the only acceptable sample type.
   All other anticoagulants (heparin, EDTA, oxalate) are NOT acceptable.
2. Proper blood to anticoagulant ratio is required

• VACUTAINER® tubes must be filled to completion to ensure the proper 9:1 blood to anticoagulant ratio is achieved.
• Routine collection requires 4.5-mL blood added to 0.5-mL sodium citrate. For patients with normal hematocrits, no adjustment is necessary. If the patient has a known hematocrit above 55%, adjust the amount of anticoagulant in the collection tube before drawing the blood according to the CLSI (formerly NCCLS) guidelines below:

3. To avoid contaminating the sample with tissue thromboplastin or heparin, follow the guidelines below. These substances may alter results.

• The venipuncture must be clean, with no trauma.
• Hemolyzed samples are not acceptable.
• The first 5 mL of blood drawn from a patient should not be used for coagulation testing.
• If drawn through an indwelling catheter, flush with 5 mL of saline and discard the first 5 mL of blood collected before collecting the specimen for coagulation testing. Blood should not be collected from heparinized lines.

4. Mix the sample gently by inverting the tube gently at least but not more than four times immediately after filling.

5. Process the sample as soon as possible (within 60 minutes). Do not store whole blood samples refrigerated prior to processing. Spin down the specimen at a speed and time required to produce platelet poor plasma (<5,000 - 10,000/uL). This can be accomplished by centrifuging at 1500 x g for 15 minutes.

6. Preparing samples for transport:

• Transfer plasma into a plastic tube using a plastic Pasteur pipette. Do not use glass tubes or glass Pasteur pipettes as glass can activate the clotting cascade.
• Label each tube “plasma.” Submit a plasma aliquot for each and every coagulation assay requested (one tube for each test). If possible, submit one additional plasma aliquot for repeat and/or test additions.
• If you are requesting other tests that require serum, please label these tubes as “serum”.

7. Ship samples for testing on dry ice. Samples must remain frozen in transit.

8. We highly recommend quick-freezing the sample to keep coagulation factors intact. This can be achieved by one of the following methods:

• Freeze with liquid nitrogen.
• Freeze in a mixture of dry ice and methanol.
• Freeze in a -70°C freezer.

An important step in the diagnosis of the lupus anticoagulant (LA) is appropriate specimen collection and processing. The more platelet free the sample, the greater the sensitivity of most test systems to the presence of LA. It is imperative that the laboratory take extra precautions in preparing platelet poor plasma (PPP).

Ideally, PPP should have a platelet count of less than 10,000 /uL. Although the sample collection process described above should yield PPP, the following double-spin technique can also be used:

• Spin down specimen at 1500g for 15 minutes.
• Transfer the plasma to a plastic tube with a plastic Pasteur pipette, staying away from the buffy coat layer. Spin down the plasma portion again at 1500g for 15 minutes. With another plastic Pasteur pipette, transfer the plasma to another plastic tube, staying clear of the bottom of the tube where the platelets lie. Alternatively, the plasma may be filtered using a 0.2 micron filter.
• Transfer plasma into a plastic tube using a plastic Pasteur pipette. Do not use glass tubes or glass Pasteur pipettes as glass can activate the clotting cascade.
• Label each tube “plasma.” Submit a plasma aliquot for each and every coagulation assay requested (one tube for each test). If possible, submit one additional plasma aliquot for repeat and/or test additions.