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Plasma protein , Blood detection . And Haematocrit determination Experiment-1 Plasma protein , Blood detection . And Haematocrit determination

Heamatology (Hematology) Blood Color of blood ( Why ?) Plasma (function ?) Serum ( Different between plasma & Serum )

Formed Elements and Plasma Introduction Blood is suspension of cells in plasma Blood composition :- Formed Elements and Plasma

Formed Elements (45%) The formed elements are cells and cell fragments suspended in the plasma. The three classes of formed elements are:- Erythrocytes (red blood cells), Leukocytes (white blood cells), Thrombocytes (platelets).

Plasma (55%) The watery fluid portion of blood (90 % water) in which the corpuscular elements are suspended. It transports nutrients as well as wastes throughout the body. Various compounds, including proteins, electrolytes , carbohydrates, minerals, and fats, are dissolved in it

Blood Composition

Plasma protein The main plasma protein :- Albumin ( Mw 66.241) Globulins (Mw of globulins Cover a wide range Fibrinogen ( Mw 340.000) Plasma protein can be separated from each other by : Salting Out / Altracenterifuge/ Electrophoresis / chromatography

Objective To separate the principle proteins in serum and plasma To detect the presence of blood To detect haematocrit

Principle Principle of separation of plasma protein by salting out The principle of the method is to use a salt which will cause dehydration and neutralization of protein molecule leading to precipitation . The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein.

Principle Principle of identification of plasma proteins : Fibrinogen : Biuret test By appearance of clotting after adding equal volume of serum

Principle Globuloins : Biuret test By Heat Coagulation. Albumin :

Principle Biuret test Protein + Biuret reagent Blue Color Heat coagulation Protein + Dilute acid ( weak acid ) heating protein ppt ( cloudiness )

II)The detection of blood To detect the presence of small quantity of blood in urine , stomach fluid , etc Principle of benzidine test Blood in presence of peroxide (H2O2) catalyze oxidation of benzidine and guaiacum to give blue color . Benzidine Minute blood Blue color

benzidine is toxic & carcinogenic Caution : benzidine is toxic & carcinogenic

Principle Disadvantage of benzidine test Not specific Principle of Modified guaiacum test Hb in blood alkali reacidification Hematin extraction Guaiauum hematin peroxide Blue color Peroxidase present in milk , potatoes ,and pus as well as Fe+++, Cu++,K+ will give false positive results

Haematocrit (Hct) III) ( packed cell volume ) Simple screening test for anemia Hct used in conjunction with MCV and MCHC Principle of Haematocrit Blood in heparinized capillary tube centrifuge blood cell ( bottom ) Plasma ( top)

Methods Using wintrobe’s tube Using microhaematocrit capillaries

Calculation Hct =length of red blood cell column total length of blood column Normal range : Men ( 0.4 – 0.54) : Women ( 0.37 -0.47)

Interpretation Causes of reduced haematocrit – causes of anaemia Causes of raised haematocrit – causes of polycythemia. If pcv has been determined by wintrobe’ tube , one can obtain some more information .