Sample Handling

The sample requirements for each test offered by the Hemostasis and Thrombosis Laboratory may vary from time to time. The following notes may be helpful and should be used in conjunction with the Tests Available listing.

Tests of blood coagulation are extremely sensitive to the ways in which blood is collected, stored and transported. It is therefore very important that the specific instructions for sample handling are closely followed.

Failure to observe the sample processing procedures below may lead to erroneous results.

1	In general, 4.5 ml venous blood should be added to blue top (BT) tubes each containing 0.5 ml of 3.2% sodium citrate. Please note that stated normal ranges might not be correct if blood is collected into 3.8% sodium citrate. Short-sampled tubes give erroneous results and will not be accepted. Tubes should be kept cool (NOT frozen) and delivered to our laboratory within 2 hours. If delivery cannot be achieved in this time, perform this procedure to achieve a platelet poor plasma (<10,000/uL): Centrifuge the correctly filled 3.2% Sodium Citrate Light Blue Top Tube at 3500 RPM's for 10 minutes. Remove the supernatant plasma utilizing a plastic transfer pipette. (If processing an EDTA sample for Homocysteine, do not utilize the same plastic transfer pipette to process the citrated plasma). Transfer to a labeled plastic tube and centrifuge a second time for the same speed and time (3500 RPM for 10 minutes). Then, decant plasma into a second labeled plastic tube and freeze plasma at -20 ° C or below within 2 hours. If multiple tests have been requested on the same patient, divide the plasma into corresponding 0.5 to 1.0 mL aliquots in labeled plastic tubes (refer to stated minimum volumes for each test). Some tests may be combined together in one aliquot. See chart below for grouped tests and minimum volumes. Frozen plasma must not be allowed to thaw during transport and must be shipped on dry ice.
2	Specimens for DNA-based (PCR) assays (e.g. FV Leiden and Prothrombin Gene Mutation) may be supplied as whole blood or as the cells that remain after separation of plasma. These samples should be shipped on regular ice and must NOT be frozen. Because we reflexively test for FV Leiden in the event of an abnormal APCR test, the residual cells should accompany requests for the latter.
3	Samples for Platelet Function Analyzer (PFA)-100® test, Platelet Aggregation, Direct Platelet Antibody Test, Platelet Glycoprotein Analysis (flow cytometry), Sonoclot Analysis and Thromboelastography must be supplied as whole blood. Samples should arrive in our laboratory within 1-2 hours of collection and should be maintained at room temperature (15-25°C). Platelet aggregation testing is only performed between 9 am and 2 pm Monday - Friday. Please call the laboratory (407-303-2449) to schedule the test, before drawing the blood. Do not spin, refrigerate or freeze samples.
4	Platelet antibody direct. Due to the sample volume needed for this assay, for premature infants, neonates and children under 14 please refer to Florida Hospital Policy on maximum amounts of blood to be drawn from patients or similar reference i.e. Phlebotomy Handbook, 7th Ed., Garza, Beacon-McBride, 2005, as this test may not be appropriate in these patients. PLEASE NOTE: Number of Lt Blue Tubes (BT) stated is based on a 5.0 mL glass 3.2% sodium citrate light blue top tube (BT). EDTA lavender top is also acceptable. For all others follow per requirements listed below: For Platelet counts >150,000 per cu.mm., draw 4 Lt Blue tubes For Platelet counts 51-150,000 per cu.mm., draw 5 Lt Blue tubes For Platelet counts 21-50,000 per cu.mm., draw 7 Lt Blue tubes For Platelet counts 11-20,000 per cu.mm., draw 9 Lt Blue tubes For Platelet counts <10,000 per cu.mm., we cannot process test due to inability to concentrate sufficient amount of platelets.

Commonly Grouped tests	Plasma Volume Preferred	Minimum Volume	Comments
APCR, AT activity, Protein C, Protein S activities	2 ml	1 ml	Volumes include reflex testing and heparinase treatment if indicated
DRVVT, PTT-LA, KCT, Staclot- LA	2 ml	1 ml	Minimum Lupus Anticoagulant Panel includes DRVVT, PTT-LA and KCT with reflex testing and heparinase treatment if indicated
FVIII, FIX, FXI, FXII	2 ml	1 ml	Volume includes heparinase treatment if indicated
FII, FV, FVII, FX	2 ml	1 ml	Volume includes heparinase treatment if indicated
PT and APTT Inhibitor Screen	2 x 2 ml	1.5 ml	PT, APTT or PTT-LA with reflex immediate and incubated mix and heparinase treatment if indicated
PT, APTT, Fibrinogen, Thrombin Time	1 ml	0.5 ml	Volumes include reflex testing and heparinase treatment if indicated
Plasminogen, antiplasmin	1 ml	0.5 ml
Von Willebrand activity and antigen	1 ml	0.5 ml	Von Willebrand Multimeric analysis must be sent as a separate aliquot
Protein S free and total antigen	1 ml	0.5 ml	Volume includes heparinase treatment if indicated