Immunology

Experiment 8

Aim of the Experiment

Effective Separation of blood products

Principal

The decision to collect anticoagulated (plasma, buffy coat, RBC) or coagulated (serum, clot) blood samples must be made prior to collection so that proper blood draw tubes will be used. Serum (needs clot time) A serum separator tube (SST, tiger top tube). Let the blood sit for 30 minutes to one hour at room temperature to clot before spinning and separating. A delay in centrifugation may have a detrimental effect on the sample quality and may result inaccurate results. Avoid hemolysis. The decision to collect anticoagulated (plasma, buffy coat, RBC) or coagulated (serum, clot) blood samples must be made prior to collection so that proper blood draw tubes will be used.

Serum (needs clot time)

A serum separator tube (SST, tiger top tube). Let the blood sit for 30 minutes to one hour at room temperature to clot before spinning and separating. A delay in centrifugation may have a detrimental effect on the sample quality and may result inaccurate

results. Avoid hemolysis.

Separating plasma (time sensitive)

Tube with an anti-coagulant eg: Edta (lavender top)sodium heparin (green top), sodium citrate (blue top) are used for separating Plasma You need to spin and separate within one hour of receiving the specimen (time sensitive Separating plasma (time sensitive) Tube with an anti-coagulant eg: Edta (lavender top) sodium heparin (green top), sodium citrate (blue top) are used for separating Plasma You need to spin and separate within one hour of receiving the specimen (time sensitive) .

Materials required

• Collecting tubes, syringe, microscope, Haemocytometre,, cotton, isopropyl alcohol.

Procedure

Keep blood on wet ice and process within one hour of blood draw Separation of plasma

1. Blood will be collected into purple top EDTA tubes and centrifuged (2000 rpm) at 4 degrees centigrade for 20minutes.

2. After centrifugation using clean pipette technique place 1.0ml of plasma into1.5ml eppendorf tube labeled with tracking number and “plasma”

3. Freeze immediately at –80 degree freezer Separation of Serum

4. A 10 ml tube of whole blood will be collected following standard procedures using a serum separator tube (SST, tiger top tube) from each patient.

5. Allow samples to clot for one hour at room temperature

6. Centrifuge for 10 minutes at approximately 1000g

7. Using clean pipette technique Aliquot 210ul of serum into labeled cryovials.

8. Immediately freeze vials of serum at –80-degree freezer

Aliquoting whole blood

Whole blood will be aliquoted into sterile tubes upon receipt by carefully inverting the blood tube so that it is gently mixed before pipetting appropriate amounts (protocol specific) of whole blood into appropriate storage tubes using clean pipette tips between each patient.

• Gently invert the tube of blood to mix contents

• Carefully open blood tube (universal precautions; gloves, eye protection)

• With clean pipette tipp aliquot appropriate amount of whole blood into clean/labeled storage tubes.

• Freeze in –80 freezer.