Separation of Plasma and Serum from Whole Blood

WHOLE BLOOD : 

• The average person circulates about 5L of blood (1/13 of body weight), of which 3L is plasma and 2L is cells.
•   It is living tissue, composed of different types of cells suspended in fluid called plasma.
•   The cells are produced primarily by bone marrow and account for blood “solids”.
•   Plasma fluid derives from the intestines and organs and provides a vehicle for cell measurement.

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BLOOD FUNCTIONS : 

• Transportation of gases, nutrients, waste products, regulatory molecules(e.g. hormones) and metabolites.
•   Regulation of pH and osmotic pressure.
•   Maintenance of body temperature.
• Protection against infections.
•   Clot formation,

BLOOD CELLS : 

• • Blood  cells  are  classified  as
  •   Red blood cells  (erythrocytes)
  •   White blood cells (leukocytes)
  •   Platelets (thrombocytes)
  •   The size of the cells differs:
  • white cells are the largest, red cells fall into the middle, and platelets are the smallest.

RED BLOOD CELLS (RBC) :  

• Red blood cells contain hemoglobin, a complex iron-containing protein that carries oxygen throughout the body and gives blood its red color.
•   They live for approximately 120 days in the circulatory system and are eventually removed by the spleen.

WHITE BLOOD CELLS (WBC) : 

• They are responsible for protecting the body from invasion by foreign substances such as bacteria, fungi, and viruses.
•   Types of WBCs:
• Granulocytes
•   Neutrophils
•   Eosinophils
• Basophils
•   Agranulocytes
•   Monocytes
• Lymphocytes

PLATELETS : 

• They are very small cellular components of blood that help the clotting process by sticking to the lining of blood vessels.
•   They survive in the circulatory system for an average of 9-10 days before being removed from the body by the spleen.
• They help prevent massive blood loss resulting from trauma, as well as blood vessel leakage.

PLASMA : 

• Plasma is the liquid portion of the blood
•   It constitutes about 55 % of blood volume.
•   90% of plasma is water
•   It contains:
•   Albumin (the chief protein constituent),
• Fibrinogen (responsible, in part, for the clotting of blood),
•   Globulins (including antibodies).

SYNTHETIC CULTURE MEDIA

SERUM : 

• It resembles plasma in composition but lacks the coagulation factors. (Serum = Plasma – clotting factors)
•  It is obtained by
•   letting a blood specimen clot prior to centrifugation usually in a red top tube with no additives or anticoagulant.

•   Or by centrifugation of plasma to precipitate fibrinogen.
•   The serum is preferred for many tests (e.g. determination of lactate dehydrogenase) as the anticoagulants in plasma can sometimes interfere with the results.

COLLECTION OF BLOOD SPECIMENS :

• • If whole blood or plasma is desired, an anticoagulant must be added to the specimen immediately after it is drawn or placed into the tube into which the blood is collected.
  •   Types of Anticoagulants

Heparin : 

•  It is the most satisfactory anticoagulant since it does not produce a change in red cell volume or interfere with subsequent determinations.
•   It inhibits the formation of thrombin from prothrombin and thus preventing the formation of fibrin from fibrinogen.

EDTA : 

• It is a chelating agent, drives its anticoagulant activity from the fact that it binds calcium, which is essential for the clotting mechanism.

Potassium Oxalate :

• It inhibits blood coagulation by forming insoluble complexes with calcium ions, which is necessary for coagulation.

Sodium Citrate : 

•   It does not precipitate the calcium but converts it into a non-ionized form, and hence prevent clotting of blood.

Sodium Fluoride ; 

•   It acts as a weak anticoagulant.
•   It has been used chiefly as a preservative since it inhibits red cell metabolism and bacterial action. If blood is treated to prevent clotting and permitted to stand or centrifuged in a container:
•   The RBCs, which weigh more than the other components, will settle to the bottom;
•   The plasma will stay on top, and •
• The WBCs and platelets will remain suspended between the plasma and the RBCs.

CHANGES IN BLOOD ON KEEPING : 

• Loss of carbon dioxide.
•   Conversion of glucose to lactic acid (glycolysis).
•   Increase in plasma inorganic phosphate.
•   Formation of ammonia from nitrogenous substances.
•   Passage of substances through the red cell envelope.
•   Conversion of pyruvate into lactate.

MATERIALS : 

• Whole blood
•   Centrifuge (up to 5000 rpm)
•   Centrifuge tubes suitable for the rotor of the centrifuge (preferably plastic and capped).
• Disposable gloves
• Disposable Pasteur pipette.
•   Measuring cylinder 10 ml.

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METHOD :

• Into dry clean centrifuge tube, pipette 15 ml of whole blood (V1).
• Place the centrifuge tube in the centrifuge machine and run it at 3000 rpm for 10 minutes. Centrifugation of whole blood separates the solid from the supernatant plasma.
• Remove the tube, withdraw the liquid layer (plasma) by pasture pipette and measure its volume using a small measuring cylinder (V2). Determine the volume of blood cells too V3 (equal to V1 – V2).
•   Transfer the supernatant (plasma) in another centrifuge tube and make further centrifugation at 3000 rpm. This will precipitate fibrinogen and the supernatant will be SERUM. Measure its Volume (V4).

RESULT :

Record your results in the following table:

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