STANDARD OPERATING PROCEDURE Number 002

BUFFY COAT & PLASMA COLLECTION

FROM ANTI-COAGULATED WHOLE BLOOD:
SERUM COLLECTION FROM CLOTTED WHOLE BLOOD
______________________________________________________

I. PRINCIPLE

• Buffy coat (white blood cells) is fractionated from plasma and red blood cells from anti-coagulated
whole blood (EDTA or Heparin) by low speed centrifugation
• White blood cells (buffy coat) are nucleated, thus are suitable for DNA extraction
• DNA is not obtained from red blood cells as they have no nuclei
• Genomic DNA is not usually obtained from plasma
• Serum is obtained from plain whole blood that is left to clot
• Plasma may be retained or discarded depending on the individual study.

II. REFERENCES

1. Sambrook and Maniatis. Current Protocols in Molecular Biology (2002), Wiley, Pg. 2.1.1 - 2.1.4
2. Maniatis, T., Fritsch, E.F. and Sambrook, J. Molecular Cloning – a laboratory manual (1984). Cold Spring
Harbour Laboratory.

III. SPECIMEN

a) EDTA anticoagulated whole blood (preferred)

b) Heparin anticoagulated whole blood
c) Plain serum separator tubes (for serum and clot only)

IV. REAGENTS
None

V. STANDARDS
None

SOP 002 Buffy Coat & Plasma Collection from Anti-coagulated Whole Blood: Serum Collection from Clotted Whole Blood
©Western Australian DNA Bank Standard Operating Procedures Manual
Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood
Version 2.1
Effective date: 01/05/08
Page 1 of 8
STANDARD OPERATING PROCEDURE Number 002
BUFFY COAT& PLASMA COLLECTION FROM ANTI-COAGULATED WHOLE BLOOD:
SERUM COLLECTION FROM CLOTTED WHOLE BLOOD
______________________________________________________

VI. QUALITY CONTROL

None

VII. DEFINITIONS

RT – room temperature

VIII. EQUIPMENT

• Centrifuge (Eppendorf Model 5810R, Room Opposite F46.1)

• Aerosol resistant filter pipette tips

SOP 002 Buffy Coat & Plasma Collection from Anti-coagulated Whole Blood: Serum Collection from Clotted Whole Blood
©Western Australian DNA Bank Standard Operating Procedures Manual
Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood
Version 2.1
Effective date: 01/05/08
Page 2 of 8
STANDARD OPERATING PROCEDURE Number 002
BUFFY COAT& PLASMA COLLECTION FROM ANTI-COAGULATED WHOLE BLOOD:
SERUM COLLECTION FROM CLOTTED WHOLE BLOOD
______________________________________________________

IX. PROCEDURE

Wear gloves and laboratory safety glasses throughout procedure.

Method A:
Will receive 1x 10 ml EDTA (Purple lid) & 1x 10 ml Serum Tube (Red Lid)
(Note; 10 mls whole blood will yield approx 6 mls plasma; 6 mls whole blood will yield approx 4 mls
plasma)
• Collect serum, plasma and buffy coat
• Label 3x 2ml screw-capped tubes for serum (Sarstedt Cat. No. 72.694)
• Label 3x 2ml screw-capped tubes for plasma
• Label 2x 10ml yellow-capped tube for buffy coat (eg Sarstedt Cat. No. 62.9924.284)
• Label 2x 0.5ml screw capped tubes for DNA (Interpath Services, Cat. No. 2141-SO)
• Label with surname, Date of Birth, Study code, Study internal number and sample type

Method B:
Will receive 1x 10 ml EDTA (Purple lid) & 1x 10 ml Serum Tube (Red Lid)
• Collect serum and buffy coat NO PLASMA
• Label 3x 2ml screw-capped tubes for serum
• Label 2x 10ml yellow-capped tube for buffy coat
• Label 2x 0.5ml screw capped tubes for DNA
• Label with surname, Date of Birth, Study code, Study internal number and sample type

Method C:
Will receive 1x 10 ml EDTA (Purple lid) or 1 to 2x 5ml EDTA (Pink Lid)
• Label 2x 10ml yellow-capped tube for buffy coat
• Label 2x 0.5ml screw capped tubes for DNA
• Label with surname, Date of Birth, Study code, Study internal number and sample type

Method D:
Will receive 3x 10 ml EDTA (Purple lid)
• Label 1x 2ml screw-capped tubes for spare Buffy coat – For 1 EDTA
• Label 2x 10ml yellow-capped tube for buffy coat – For remaining 2 EDTA tubes to be
combined.
• Label 2x 2ml screw capped tubes for DNA
• Label with surname, Date of Birth, Study code, Study internal number and sample type
SOP 002 Buffy Coat & Plasma Collection from Anti-coagulated Whole Blood: Serum Collection from Clotted Whole Blood
©Western Australian DNA Bank Standard Operating Procedures Manual
Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood
Version 2.1
Effective date: 01/05/08
Page 3 of 8
STANDARD OPERATING PROCEDURE Number 002
BUFFY COAT& PLASMA COLLECTION FROM ANTI-COAGULATED WHOLE BLOOD:
SERUM COLLECTION FROM CLOTTED WHOLE BLOOD
______________________________________________________

Method E:
Will receive 1x 10 ml EDTA (Purple lid), 1x 10 ml Serum Tube (Red Lid) and 2x PAX Gene RNA
tubes (Brown Lid)
• Label 2x 10ml yellow-capped tube for buffy coat
• Label 3x 2ml screw-capped tubes for serum
• Label 2x 0.5ml screw capped tubes for DNA
• Label with surname, Date of Birth, Study code, Study internal number and sample type
• Write internal number on PAX Gene RNA tube and place in rack at room temperature in
RNA extraction Lab.

Method F:
For Saliva only (not applicable)

Sample Collection Protocol

• Samples will be sent to the PathWest Central Reception Area (CRA) and are placed in the walk-
in fridge (top shelf, left hand side, in rack labelled “Research Trials”) – check several times daily
for samples. (Note: Some study coordinators will hand deliver samples directly to the fridge in
DNA Extraction laboratory).
• Samples will arrive with a request form identifying the study type and patient details. Match the
request form with the blood tubes (EDTA, Serum Separator Tubes) and the patient (depending
on the study the samples may already be spun and separated and the plasma and serum
transferred to new tubes)
• Once samples are collected write the internal number of the form (check this in the file located in
office)
• Write the time and date of processing of plasma, serum and buffy coats onto the request form
• Keep all blood tubes at +4 ° C until ready to centrifuge
• Determine which Collection method has been used (A, B, C, D, E) (F = saliva only) and while
samples are spinning in Centrifuge, label all tubes as outlined below.

SOP 002 Buffy Coat & Plasma Collection from Anti-coagulated Whole Blood: Serum Collection from Clotted Whole Blood
©Western Australian DNA Bank Standard Operating Procedures Manual
Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood
Version 2.1
Effective date: 01/05/08
Page 4 of 8
STANDARD OPERATING PROCEDURE Number 002
BUFFY COAT& PLASMA COLLECTION FROM ANTI-COAGULATED WHOLE BLOOD:
SERUM COLLECTION FROM CLOTTED WHOLE BLOOD
______________________________________________________

PROCEDURE FOR PLASMA AND BUFFY COAT COLLECTION:

1. First check if plasma is to be retained or discarded, depending on the study protocol (method A).
2. Centrifuge EDTA/heparin anticoagulated whole blood at 2450 x g (3500 rpm) (Eppendorf Model
5810R) for 10 minutes at RT (this will separate the blood into an upper plasma layer, a lower red
blood cell (RBC) layer, and a thin interface (buffy coat) containing the white blood cells (WBC)(see
Figure 1a). Samples with high WBC counts will have a thicker buffy coat).

Figure 1(a) Figure 1(b) Figure 1(c)

Fractionation of layers Visible buffy coat at interface After buffy coat is removed
(Image LifeART) (Image Ambion®) (Image Ambion®)

3. Aspirate off the upper plasma layer (from EDTA tube (purple lid) using a wide-mouth plastic
disposable transfer pipet (1 ml graduated with bulb, Samco Scientific Cat. No 222) and if collecting
transfer to the labelled collection tubes. Be careful not to disrupt the buffy coat (see Figure 1b)
4. Expel all residual plasma from the transfer pipet before continuing (retain or discard depending on
method).
5. The EDTA tube is used for subsequent DNA extraction so do not discard.
6. Either continue on with DNA extraction or store EDTA tube - use an elastic band to bind 1 x labelled
yellow-capped 10 ml tube to the blood tube and leave in RT storage box until ready for DNA
extraction, place other 1 x labelled yellow-capped tube in DNA extraction rack (for day 2 of DNA
extraction procedure). (Note: store buffy coat at RT if extracting DNA within 14 days; store at -20°C if
extracting after more than 14 but less than 30 days or store at -40°C if DNA extraction will be
delayed beyond 30 days. If buffy coat is to be frozen do step 7 now.
7. When ready to extract DNA aspirate the buffy coat into the first labelled sterile yellow-capped 10 ml
tube (attached by elastic band). Aspirate slowly, using a circular motion, to pull all the visible buffy
coat material into the transfer pipet (see Figure 1c) (some contamination of the WBCs with the
underlying RBCs is likely but try to minimise).
SOP 002 Buffy Coat & Plasma Collection from Anti-coagulated Whole Blood: Serum Collection from Clotted Whole Blood
©Western Australian DNA Bank Standard Operating Procedures Manual
Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood
Version 2.1
Effective date: 01/05/08
Page 5 of 8
STANDARD OPERATING PROCEDURE Number 002
BUFFY COAT& PLASMA COLLECTION FROM ANTI-COAGULATED WHOLE BLOOD:
SERUM COLLECTION FROM CLOTTED WHOLE BLOOD
______________________________________________________
8. Store Plasma in appropriate study box in –20 °C freezer in DNA extraction Lab (Rm 46.1).
9. Once labeled with 2D study barcode place in study box –80 °C Molecular Biology Freezer.
10. Proceed with DNA extraction according to WADB SOP003 “DNA Extraction using Salt”.

PROCEDURE FOR SERUM COLLECTION:

A. Centrifuge Plain clotted whole blood tube or Serum Separator Tube at 2450 x g (3500 rpm) (Eppendorf
Model 5810R) for 10 minutes at RT
B. Aspirate the supernatant from the Serum Clot Tube (10ml red lid) using a wide-mouth plastic disposable
transfer pipette (1 ml graduated with bulb, Samco Scientific, USA, Cat. No 222) and aliquot into prepared
serum tubes.
C. Store in appropriate study box in -20 °C freezer in DNA extraction Lab (Rm 46.1).
D. Once labeled with 2D study barcode place in study box -80 °C Molecular Biology Freezer.

SOP 002 Buffy Coat & Plasma Collection from Anti-coagulated Whole Blood: Serum Collection from Clotted Whole Blood
©Western Australian DNA Bank Standard Operating Procedures Manual
Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood
Version 2.1
Effective date: 01/05/08
Page 6 of 8
STANDARD OPERATING PROCEDURE Number 002
BUFFY COAT& PLASMA COLLECTION FROM ANTI-COAGULATED WHOLE BLOOD:
SERUM COLLECTION FROM CLOTTED WHOLE BLOOD
______________________________________________________

X. CALCULATIONS

None

XI. REPORTING

None

XII. PRECAUTIONS AND HAZARDS

• Biological fluid – General information can be obtained in PathWest SOP PLSM001 (‘Biological
Hazards’); SOP PLSM010 (‘Waste Disposal’) and SOP PLSM017 (‘Dealing with Spills’). Waste
biological fluids (plasma, serum etc) are left in their original blood tube and placed in the Waste
Storage rack. When the rack is full, dispose all tubes into the Waste Bins in laboratory (lined with
Yellow Biohazard bag). When the Waste Bin is full, transfer the bag to the Large Yellow Biohazard
Bins on Level 2 (for incineration)
• Effervescent Haz Tabs (Na DCC - Troclosene Sodium (Chlorine) (Guest Medical, Cat. No. H8811)
– Use for disinfection of biological fluid spills, discard jars and environmental disinfection – follow
instructions on product container for preparing the relevant solutions. After Eye Contact:
Immediately flush with plenty of clean water for at least 15 minutes. If irritation persists, seek medical
attention. After Skin Contact: Promptly wash thoroughly with water for at least 15 minutes while
removing contaminated clothing. Wash any contaminated clothing well before re-use. If swallowed:
Immediately rinse mouth, then drink plenty of water or milk. Do not induce vomiting. Seek medical
attention. If Inhaled: Move to fresh air. If irritation persists, seek medical attention.

XIII. DOCUMENT HISTORY

See Master Copy

SOP 002 Buffy Coat & Plasma Collection from Anti-coagulated Whole Blood: Serum Collection from Clotted Whole Blood
©Western Australian DNA Bank Standard Operating Procedures Manual
Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood
Version 2.1
Effective date: 01/05/08
Page 7 of 8
STANDARD OPERATING PROCEDURE Number 002
BUFFY COAT& PLASMA COLLECTION FROM ANTI-COAGULATED WHOLE BLOOD:
SERUM COLLECTION FROM CLOTTED WHOLE BLOOD
______________________________________________________

BUFFY COAT & PLASMA COLLECTION FROM ANTICOAGULATED WHOLE BLOOD:

SERUM COLLECTION FROM CLOTTED WHOLE BLOOD

DOCUMENT HISTORY

Date Issue Number Author Description of Amendment Authorised by

1/11/06 1 Marion Macnish, Original document for WADB John Beilby

Simone Dowd &
Laura Greenwood

1/6/07 2 Marion Macnish, Aerosol resistant tips for all John Beilby
Simone Dowd & laboratory procedures
Laura Greenwood
1/5/08 2.1 Marion Macnish, Rearrange order of John Beilby
Simone Dowd & information flow
Laura Greenwood

Authorised by:…………………………………………..
(signature)

Date:……………………………………………………...

SOP 002 Buffy Coat & Plasma Collection from Anti-coagulated Whole Blood: Serum Collection from Clotted Whole Blood
©Western Australian DNA Bank Standard Operating Procedures Manual
Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood
Version 2.1
Effective date: 01/05/08
Page 8 of 8