HSBTE, 2nd Semester, DMLT Unit 1. Blood fractions, Separation of blood plasma, Separation of blood serum, Different protein precipitating reagents.

• Blood fractions

Blood present in side human body is whole blood. There are four major components in whole blood. These are Red blood cells (RBCs), White blood cells (WBCs), Platelets, and plasma. These components are easily separated by centrifugation of whole blood. They are also considered as major fraction of blood. Blood plasma consists of 90% water, 2% dissolved salts, and 8% proteins and clotting factors. Fractionation of blood plasma separates the components of blood plasma. These components are called minor fractions of blood. Albumin, immunoglobulin, and clotting factors are considered as minor fractions of blood.

“Blood fractions are constituents of blood plasma that are obtained by blood plasma fractionation technique.”

• 1. Albumin

• 2. Clotting factors: fibrinogen, prothrombin, cryoprecipitate

• 3. Colony stimulating factors

• 4. Erythropoietin

• 5. Fibrinogen

• 6. Rh factors

• 7. Prothrombin

• 8. Interferon

• 9. Interleukin

Separation of blood plasma: Whole blood remains in liquid form inside the human body. Blood clots within a few minutes outside the human body. Blood clots shrink and release pale yellow liquid. A pale yellow liquid is called serum. A blood sample containing blood clots is not suitable for hematology examination. Reason: several blood components remain inside the clot and cannot be traced during hematology examination. Thus anticoagulants are used in blood samples to prevent blood clotting. Blood plasma can be easily separated out from anticoagulated blood. Blood plasma is used to estimate blood glucose.

• Fluoride is used as an anticoagulant for blood plasma separation. Fluoride prevents glucose glycolysis during the storage period of blood plasma.

• “Blood plasma is the pale yellow liquid component of blood without blood cells but have protein components of whole blood.”

The process of separating blood plasma

Centrifugation of anticoagulated blood develops three layers in a centrifugation tube. The bottom layer is packed with red blood cells. The middle layer is a white buffy coat containing leucocytes and platelets. The upper layer is pale yellow fluid that is blood plasma. Plasma constitutes 51% to 57% of total volume.

• · Collect 5 mL blood in a centrifuge tube or bulb containing suitable anticoagulant. (Quantity of blood shall depend upon several tests to be performed). Approximately 0.5 mL plasma is separated from 2 to 3 mL anticoagulated blood.

• · Centrifuge it at 1500 RPM for 10 minutes.

• · Transfer the upper pale yellow fluid into a dry test tube using a Pasteur pipette.

• · Store it at 2 to 8o C till test is performed.

Separation of blood serum

 Blood plasma without clotting factors is called serum. Pale yellow liquids separate out from clots in blood coagulation is called serum.

• 1. Collect 5 to 7 ml of blood in a tube without anticoagulant. (Quantity of blood shall depend upon several tests to be performed). 1 mL serum is separated from 5 to 7 mL whole blood.

• 2. Keep the tube in a slanting position and set aside for 30 minutes at room temperature. The clot will loosen slowly.

• 3. Transfer the serum into a centrifuge tube by using a Pasteur pipette.

• 4. Centrifuge it at 1500 RPM for 10 minutes.

• 5. Transfer the upper pale yellow fluid into a dry test tube using a Pasteur pipette.

• 6. Store it at 2 to 7o C till the test is not performed.

Different protein-precipitating reagents

 Tungstic acid is used as a protein-precipitating reagent. The anionic part of tungstic acid combines with a cationic form of protein. It requires protein to be on the acidic side of its isoelectric point. It can be achieved by maintaining the pH of the specimen between 5 and 7.

• Tungstic acid is prepared by mixing 1 part of 1% sodium tungstate (10gm/dL) and part of 2/3N sulphuric acid and 7 parts water.

• H2SO4 + Na2WO4 = H2WO4 + Na2SO4.

• Sulphuric Sodium Tungstic Sodium

• acid Tungastate acid Sulphate

• Tungstic acid and sodium sulfate cause the precipitation of protein from the specimen.

Preparation of protein-free filtrate

 It can be studied under the following two headings

• A. Deproteinization of blood

  • Mix the following thoroughly in a 25 mL conical flask

    • · 2/3N Sulphuric acid 1 mL

    • · 10g/dL Sodium tungstate 1mL

  • · Blood 1mL

  • · Distilled water 7 mL.

Set aside at room temperature for 5 minutes. Filter it by using Whatman filter paper No44. The grade of filter paper is selected depending on the particle size of the precipitate and the method of filtration.

B. Deproteinization of Serum/Plasma/CSF/Urine:

• Mix the following thoroughly in a 25 mL conical flask

  • · 2/3N Sulphuric acid 0.5 mL

  • · 10g/dL Sodium tungstate 0.5mL

• · Blood 1mL

• · Distilled water 8 mL.

Set aside at room temperature for 5 minutes. Filter it by using Whatman filter paper No. 44. The grade of filter paper is selected depending on the particle size of the precipitate and the method of filtration

Dr Pramila Singh