Sample Collection Guide

BLOOD COLLECTION INSTRUCTIONS

General notes about blood collection.

For all Coagulation tests trauma associated with the venipuncture must be avoided otherwise the test results could be elevated and misleading.

Samples for coagulation studies are usually collected into 3.2% (0.105M) buffered sodium citrate (blue-topped Vacutainers®). There are some tests that do require a special tube and this is detailed in the blood collection instructions.

When there is a combination of tests to be collected that have different instructions for each, consult HRL for information on consolidating collection instructions to minimize the number of tubes collected.

Ideally all plasma samples should be platelet-free, defined as less than 5 x 10⁹/L (less than 5,000/cubic mm.) This is essential for some tests, and is achieved by double centrifugation or filtration, as outlined in Blood Collection Method 2. However, for some tests this is not as critical, and a final platelet count of less than 10 x 10⁹/L (less than 10,000/cubic mm.) is acceptable. This is achieved by single centrifugation (and careful aliquoting so as not to disturb the buffy coat), as outlined in Blood Collection Method 1.

A plasma storage temperature of -70°C is recommended, and is mandatory for samples being stored for longer than 6 months. Minus 35°C may be used up to 6 months, and for some assays      -20°C is acceptable for up to 1 week.

 A fridge-freezer, or any other -20°C freezer in which there is an automated defrost cycle, should not be used.

METHOD 1

FOR MOST COAGULATION ASSAYS BLOOD SAMPLES ARE COLLECTED INTO 3.2% (0.105M) BUFFERED SODIUM CITRATE.

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is added to the anticoagulant at a ratio of 9 parts of blood to 1 part of citrate and gently mixed.  A blue-topped 3.2% buffered sodium citrate Vacutainer® can be used.
   

2. Centrifuge the sample at 1700g for 15 minutes to obtain platelet-poor plasma. The plasma must have a final platelet count that is less than 10,000 per cubic mm (less than 10x10⁹/L).

Remove all but a few millilitres of plasma from the cells, being careful not to disturb the buffy coat.


3. Place the plasma into plastic tubes, cap and freeze at -35° C or colder (-20° C can be used for up to 1 week).
   

4. Send on dry ice using the separately listed shipping instructions.

METHOD 2

FOR TESTS REQUIRING PLATELET-FREE PLASMA BLOOD SAMPLES ARE COLLECTED INTO 3.2% (0.105M) BUFFERED SODIUM CITRATE

METHOD:

• Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is added to the anticoagulant at a ratio of 9 parts of blood to 1 part of citrate. A blue-topped 3.2% buffered sodium citrate Vacutainer® can be used.
  

• Centrifuge the sample at 1700g for 15 minutes.
  

• Transfer the supernatant plasma into a clean plastic tube and


EITHER:

re-centrifuge for an additional 5 minutes at 1700g and transfer the supernatant into plastic tubes, taking care not to disturb any cells at the bottom of the tube

OR:

filter the plasma through a 0.22 micron filter, to ensure the plasma has a final platelet count that is less than 5,000 per cubic mm (less than 5x10⁹/L).

• Cap and freeze at -35° C or colder (-20° C can be used for up to 1 week).
  




• Send on dry ice using the separately listed shipping instructions.

METHOD 3

FOR PROTHROMBIN F1.2 & THROMBIN-ANTITHROMBIN (TAT) AND FIBRIN MONOMER COAGULATION ASSAYS:

BLOOD SAMPLES ARE COLLECTED INTO 3.2% (0.105M) BUFFERED SODIUM CITRATE.

METHOD:
Trauma associated with the venipuncture must be avoided otherwise the test results could be elevated and misleading.

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The tourniquet must only be on for the minimum period possible prior to the venipuncture, must not be too tight, and must be removed at the commencement of blood flow. The blood is added to the anticoagulant at a ratio of 9 parts of blood to 1 part of citrate, and the tube is mixed gently. A blue-topped 3.2% buffered sodium citrate Vacutainer® can be used.
   

2. Centrifuge the sample within one hour of collection at 1700g for 15 minutes to obtain platelet-poor plasma. The centrifugation can be done at room temperature or at 4° C.
   

3. Remove all but a few millimetres of plasma from the cells, being careful not to disturb the buffy coat.
   

4. Divide the plasma into two equal aliquots, place into plastic tubes, cap and freeze at -35° C or colder (-20° C can be used for up to 1 week).
   

5. Send on dry ice using the separately listed shipping instructions.

METHOD 4

FOR BTG (BETA-THROMBOGLOBULIN) and PF4 (PLATELET FACTOR 4) COAGULATION ASSAYS:

BLOOD SAMPLES ARE COLLECTED INTO CTAD TUBES

METHOD:

Sample collection must be handled with extreme care to avoid any platelet activation.

1. Prepare an ice/water bath deep enough to cool the entire contents of the sample collection tube.
   

2. Ensure the refrigerated centrifuge is at operating temperature of 4 ± 2° C.
   

3. Use a CTAD tube, and break the vacuum before sample collection.
   

4. Draw blood using a syringe and 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The tourniquet must only be on for the minimum period possible prior to the venipuncture, must not be too tight, and must be removed at the commencement of blood flow. Discard the first two ml. of blood, and collect 4.5 ml. of blood per CTAD (vacuum already broken).
   

5. Immediately place the tube in the ice/water bath.
   

6. Centrifuge the sample within one hour of collection at 2,500g for 30 minutes at 4° C to obtain platelet-poor plasma. Double spinning is not necessary with the CTAD system.
   

7. As soon as centrifugation is completed, collect 1/3rd the volume of the supernatant (about 1mL) by aiming the plastic pipette tip in the middle region of the liquid portion, making sure not to aspirate the top surface of the plasma where some light platelets may be found, and not too near to the platelet layer that sits on top of the cells. The plasma must have a final platelet count that is less than 5,000 per cubic mm (less than 5x10⁹/L).
   

8. Place the plasma in plastic tubes, cap and freeze at -35° C or colder (-20° C can be used for up to 1 week).

  9.  Send on dry ice using the separately listed shipping instructions.

**CTAD tubes (previously known as Diatube-H tubes) are available on request from HRL, or from Becton-Dickinson, through VWR, catalog # 367599 (Hemoguard stopper, 4.5 mL draw.) The tubes contain buffered citrate solution, theophylline, adenosine and dipyridamole. Collection of blood into CTAD Tubes is recommended for all tests in which it is important to avoid release of platelet factors during collection and processing of blood samples. The "stabilization" of the platelets by CTAD prevents artificial entry of platelet factors into the plasma and consequently falsification of the test result.

METHOD 5

FOR FIBRINOLYTIC STUDIES:

BLOOD SAMPLES ARE COLLECTED INTO 3.2% (0.105M) BUFFERED SODIUM CITRATE.

METHOD:

1. Prepare an ice/water bath deep enough to cool the entire contents of the sample collection tube.
   

2. Ensure the refrigerated centrifuge is at operating temperature of 4 ± 2° C.
   

3. If venous occlusion studies are being done, follow the process on page 2. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. Ideally the blood should be collected without a tourniquet, but if one is used, it must only be on for the minimum period possible prior to the venipuncture, must not be too tight, and must be removed at the commencement of blood flow.
   

4. The blood is added to the anticoagulant at a ratio of 9 parts of blood to 1 part of citrate, mixed gently, and immediately placed in the ice/water bath. A blue-topped 3.2% buffered sodium citrate Vacutainer® can be used.
   

5. All samples are processed within 1 hour of collection by centrifugation at 1,700g for 15 minutes at 4° C to obtain platelet-poor plasma. The plasma must have a final platelet count that is less than 10,000 per cubic mm (less than 10x10⁹/L).
   

6. Remove all but a few millimetres of plasma from the cells, being careful not to disturb the buffy coat.
   

7. Place the plasma into plastic tubes, cap and freeze at -35° C or colder.
   

For Venous Occlusion studies:


8. The patient is placed in a supine position and encouraged to relax for 15 minutes prior to blood collection.
   

9. Take blood as above.
   

10. Label the tube as the pre-occlusion sample and immediately place in the ice/water bath.
    

11. Using the other arm, blood pressure is taken, and the pressure cuff is inflated to midway between systolic and diastolic. The cuff is maintained at that pressure for 10 minutes.
    

12. At 10 minutes, and before the pressure is released, a second blood sample is taken as above (paragraph 4). Ensure that the sample is labeled as the post-occlusion sample, mix gently and immediately place in the ice/water bath.
    

13. Process the sample as above.
    

14. Send on dry ice using the separately listed shipping instructions.
    

METHOD 6

BUFFY COAT for DNA analysis

Two alternative methods of blood processing are available:

Method A:

1. Draw 4.5 mL of blood into a 5 mL EDTA (lavender-capped), or 3.2% or 3.8% buffered sodium citrate (blue-capped) Vacutainer¨ tube.
   

2. Centrifuge the sample for 15 minutes at room temperature or 4°C. Remove and discard the upper plasma portion using a plastic transfer pipette.
   

3. Using the plastic transfer pipette, collect as much of the white cell layer (buffy coat) as possible. A few red cells are acceptable.
   

4. Place the buffy coat in plastic tubes, cap and freeze at -20°C or lower until ready to ship.
   

5. Send using the separately listed shipping instructions, which include packaging in dry ice.
    

Method B - (if being shipped to arrive within 7 days of collection)

1. Draw the blood as described in part A1 above, without subsequent centrifugation.

2. The blood should be stored at 4°C, but can be shipped at room temperature to arrive within 7 days of collection.

3. Shipping materials should conform to regulations.

METHOD 7

FOR P-SELECTIN, sCD40L & HOMOCYSTEINE ASSAYS:

BLOOD SAMPLES FOR THESE ASSAYS ARE COLLECTED INTO EDTA

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is added to the EDTA anticoagulant, using a lavender-topped EDTA Vacutainer® tube (a 5 mL tube is sufficient). Keep sCD40L and homocysteine samples on ice until separation.
   

2. Centrifuge the sample within 30 minutes of blood collection to ensure optimal recovery. Centrifuge at 1700g for 15 minutes to obtain platelet-poor plasma. The plasma must have a final platelet count that is less than 10,000 per cubic mm (less than 10x10⁹/L).
   

3. Remove the plasma from the cells, being careful not to disturb the buffy coat. A minimum of 0.4 mL of plasma is required for each assay.
   

4. Place the plasma into plastic tubes, cap and freeze at -35°C or colder. Stability at -20°C is up to 4 weeks for P-Selectin samples and indefinitely for homocysteine samples. P-Selectin samples are also stable at 2-8°C for up to 1 week, and homocysteine for 2 weeks, from collection to assay. Grossly hemolyzed samples are not acceptable.
   

5. For P-Selectin, samples can also be assayed when submitted in 3.2% (0.105M) buffered sodium citrate, heparin, or as serum or cell culture supernatant: please call for further information.
   

6. Send on dry ice using the separately listed shipping instructions.

METHOD 8

For Tests Requiring STABILYTE® Blood Collection

BLOOD SAMPLES ARE COLLECTED INTO STABILYTE® BLOOD COLLECTION TUBES (strong acidic citrate).

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is added to the anticoagulant at a ratio of 9 parts of blood to 1 part of citrate. A special STABILYTE® tube is used, which has a black top and contains a strong acidic citrate solution (American Diagnostic Product # 45TUB or Inter Medico).
   

2. The sample is placed on ice and centrifuged at 4°C within 30 minutes at 1700g for 15 minutes.
   

3. Freeze at -35°C or colder.
   

4. Send on dry ice using the separately listed shipping instructions.

METHOD 9

FOR PLASMINOGEN ACTIVATOR INHIBITOR ANTIGEN OR FUNCTIONAL ASSAY (PAI/PAI-1):

BLOOD SAMPLES ARE IDEALLY COLLECTED INTO CTAD TUBES (see footnote* below).

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is added to the anticoagulant at a ratio of 9 parts of blood to 1 part of citrate, using a DIATUBE¨-H tube (see footnote* below).
   

2. Centrifuge the sample at 1700g for 15 minutes.
   

3. Transfer the supernatant plasma into a clean plastic tube and
   EITHER:

   recentrifuge for an additional 5 minutes at 1700g and transfer the supernatant into plastic tubes, taking care not to disturb any cells at the bottom of the tube
   

   OR:
   

   filter the plasma through a 0.22 micron filter, to ensure the plasma has a final platelet count that is less than 5,000 per cubic mm (less than 5x10⁹/L).
   

4. Cap and freeze at -35°C of colder (-20°C can be used for up to 1 week).
   

5. Send on dry ice using the separately listed shipping instructions.
   

*CTAD tubes are available on request from HRL, or from Becton-Dickinson, B-D catalog # 367015 (conventional stopper), or catalog # 367599 (Hemoguard stopper.) They are 3.2% (0.105M) buffered citrated, light blue stopper, 4.5 ml draw, with special additives that prevent platelet activation. When stored they must be protected from light.

A blue-topped 3.2% (0.105M) buffered sodium citrate Vacutainer¨ can be used for sample collection provided that the sample is processed immediately and that the plasma is double centrifuged or filtered, as in 3 above, to assure platelet-free plasma (less than 5 x10⁹/L).

METHOD 10

FOR DEHYDRO THROMBOXANE B2: Urine Sample

METHOD:

1. Collect a random urine sample. Sterility is not necessary.
   

2. Aliquot 2- 1mL vials and freeze: preferably at -70°C, but -35°C is acceptable for up to 1 week.

METHOD 11

FOR E-SELECTIN ASSAYS:

BLOOD SAMPLES FOR THESE ASSAYS ARE COLLECTED INTO HEPARIN

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is added to the heparin anticoagulant, using a green-top Vacutainer® tube (a 5 mL tube is sufficient).
   

2. Centrifuge the sample within 30 minutes of blood collection to ensure optimal recovery. Centrifuge at 1700g for 15 minutes to obtain platelet-poor plasma. The plasma must have a final platelet count that is less than 10,000 per cubic mm (less than 10x10⁹/L).
   

3. Remove the plasma from the cells, being careful not to disturb the buffy coat. A minimum of 0.4 mL of plasma is required for each assay.
   

4. Place the plasma into plastic tubes, cap and freeze at -35°C or colder. Stability at –20ºC is up to 4 weeks and at 2-8°C for up to 1 week.
   

5. Lipemic, grossly hemolyzed or turbid samples are not acceptable.
   

6. Samples can also be assayed when submitted in 3.2% (0.105M) buffered sodium citrate or serum: please call for further information.
   

7. Send on dry ice using the separately listed shipping instructions.

METHOD 12

For Cytokine Assays and other assays requiring serum:

BLOOD SAMPLES FOR THESE ASSAYS ARE COLLECTED WITHOUT ANTICOAGULANT

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is drawn into a red-top Vacutainer® tube (a 5 mL tube is usually sufficient).
   

2. Allow the sample to clot at room temperature, and centrifuge the sample at 1700g for 15 minutes.
   

3. Remove the supernatant serum from the cells as soon as possible after centrifugation, being careful not to disturb the cells.
   

4. Place the serum into plastic aliquot tubes, cap and freeze at -20°C or colder; -70°C is preferred.
   

5. Send on dry ice using the separately listed shipping instructions.

METHOD 13

For Thromboxane B2 assays:

BLOOD SAMPLES ARE COLLECTED INTO CTAD TUBES (see footnote* below).

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is added to the anticoagulant at a ratio of 9 parts of blood to 1 part of citrate, using a CTAD 4.5 mL draw tube (see footnote* below).
   

2. Centrifuge the sample at 2000g for 20 minutes.
   

3. As soon as centrifugation is completed, collect 1/3rd the volume of the supernatant (about 1ml.) by aiming the plastic pipette tip in the middle region of the liquid portion, making sure not to aspirate the top surface of the plasma where some light platelets may be found, and not too near to the platelet layer that sits on top of the centrifuged cells. The plasma must have a final platelet count that is less than 5,000 per cubic mm (less than 5x10⁹/L).
   

4. Transfer the supernatant plasma into a clean plastic tube.
   

5. Cap, label and freeze at -35ºC of colder (-70°C is preferred).
   

6. Send on dry ice using the separately listed shipping instructions.
   

*CTAD tubes (previously known as Diatube-H tubes) are available on request from HRL, or from Becton-Dickinson, through VWR, catalog # 367599 (Hemoguard stopper, 4.5 mL draw.) The tubes contain buffered citrate solution, theophylline, adenosine and dipyridamole. Collection of blood into CTAD Tubes is recommended for all tests in which it is important to avoid release of platelet factors during collection and processing of blood samples. The "stabilization" of the platelets by CTAD prevents artificial entry of platelet factors into the plasma and consequently falsification of the test result.

METHOD 14

For Fibrinopeptide A (FPA) Assays:

BLOOD SAMPLES ARE COLLECTED INTO FPA ANTICOAGULANT TUBES (see footnote* below).

METHOD:

1. Blood is collected into a red-top vacutainer® tube spiked with a special FPA anticoagulant provided in the kit (see footnote* below).

2. The whole blood should be carefully mixed at a ratio of 1 part anticoagulant to 9 parts venous whole blood and centrifuged within 2 hours for 15 minutes at approximately 1700g.

3. Remove plasma supernatant and transfer to a clean plastic 12 x 75mm tube.

4. Recentrifuge for 5 minutes to ensure platelet free plasma.

5. Transfer the supernatant plasma into a clean plastic tube.

6. Cap, label and freeze within 4 hours at -35ºC of colder (-70°C is preferred).

7. Send on dry ice using the separately listed shipping instructions.
   

*FPA tubes are available on request from HRL.  The tubes contain Trisodium Citrate, Heparin, Hirudin, Aprotinin and sodium azide. Collection of blood into FPA Tubes is recommended for this test to stabilize certain fibrinogen-related plasma factors during collection and processing of blood samples which may consequently affect the test result.

METHOD 15

For Cytokine Assays:

BLOOD SAMPLES FOR THESE ASSAYS ARE COLLECTED WITHOUT ANTICOAGULANT

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is drawn into an SST Vacutainer® tube (a 5 mL tube is usually sufficient).
   

2. Allow the sample to clot at room temperature, and centrifuge the sample at 1000g for 15 minutes.
   

3. Remove the serum as soon as possible after centrifugation.
   

4. Place the serum into plastic aliquot tubes, cap and freeze at -20°C or colder; -70°C is preferred.
   

5. Send on dry ice using the separately listed shipping instructions.

METHOD 16

For Tumour Growth Factor β1 (TGF-β1)  Assays:

BLOOD SAMPLES FOR THESE ASSAYS ARE COLLECTED WITHOUT ANTICOAGULANT

METHOD:

1. Draw blood using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood is drawn into an SST Vacutainer® tube (a 5 mL tube is usually sufficient).
   

2. Allow the sample to clot at room temperature for 30 minutes.

3. For complete release of TGF-β1, incubate overnight at 2 - 8°C

4. Centrifuge the sample at 1000g for 15 minutes.
   

5. Remove the serum after centrifugation and place the serum into plastic aliquot tubes, cap and freeze at -20°C or colder; -70°C is preferred.
   

6. Send on dry ice using the separately listed shipping instructions.