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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