BY : Dr. Beenish Zaki, Instructor Department of Biochemistry (15 February 2012)

Learning Objectives S KILL : At the end of the practical session students will be able to identify and demonstrate: -Methods of blood sample collection. -Ideal timing for blood collection. -Type of anticoagulant used for a blood sample K NOWLEDGE : At the end of the practical session the student will be able to explain various changes involved in the metabolism of RBC in a blood sample and effects of anticoagulant of the RBC metabolism.

COLLECTION OF BLOOD SPECIMENS Sites for blood collection can be divided into: Capillary Blood Venous Blood Arterial Blood

COLLECTION OF BLOOD SPECIMENS CALILLARY BLOOD: This is usually obtained from a finger or thumb. Both heparinised and plain capillary tubes are manufactured and they can be centrifuged to obtain plasma or serum. This method is usually employed to collect volumes up to 200µl.

CAPILLARY TUBE

COLLECTION OF BLOOD SPECIMENS VENOUS BLOOD: Blood may be taken from any convenient vein usually the median cubital vein is used. Once the desired amount of blood is collected remove the needle carefully and slowly transfer the blood to an appropriate container.

Location of Median Cubital vein

COLLECTION OF BLOOD SPECIMENS ARTERIAL BLOOD: This is usually obtained by inserting a needle into the radial; brachial or femoral artery. Acid – base status can be analyzed. Done in the cases of Diabetic ketoacidosis and respiratory disorders. The composition of capillary blood is closer to that of arterial blood than to venous blood.

Arterial Blood Gas

DIFFERENCE BETWEEN PLASMA AND SERUM SERUM Serum is the supernatant fluid when coagulated blood has been centrifuged. The blood is allowed to clot at room temperature for 15 to 30 minutes. When the blood has clotted completely, it is then centrifuged for 5-10 minutes PLASMA Plasma is the supernatant fluid obtained when anti- coagulated blood has been centrifuged. The blood is mixed with an appropriate amount of anticoagulant This preparation should be mixed immediately and thoroughly to avoid clotting. The solution is then centrifuged for 5-10 minutes.

TIME OF COLLECTION OF BLOOD SPECIMENS The best time for taking blood is after fasting overnight. All the specimens should be clearly labeled with: Patient’s Details Date, The test to be done

ANTICOAGULANTS In the laboratory anticoagulants are used for preventing coagulation of blood without diluting and without causing a change in the volume of red cells.

TYPES OF ANTICOGULANT HEPARIN EDTA (Ethylene diamine tetra- acetate) Oxalates and Citrates Sodium Flouride

FUNCTION OF ANTICOAGULANT HEPARIN: A mucopolysaccharide ACTION: Acts by inhibiting the conversion of prothrombin to thrombin. Available as lithium, sodium, potassium salts. QUANTITY: 2mg/10ml of blood.The best anticoagulant except for cost CLINICAL APPLICATION: Used in the estimation for urea, creatinine, cholesterol.

FUNCTION OF ANTICOGULANT EDTA (Ethylene diamine tetra- acetate): ACTION: acts by chelating calcium ions. QUANTITY: 20mg/10ml of blood CLINICAL APPLICATION: It is a good anticoagulant used in hematology for RBC; WBC; Platelets; PCV; ESR investigations

FUNCTION OF ANTICOGULANT Citrates : ACTION: Citrates act by converting calcium into a soluble non-ionised form Quantity: 30mg/10ml. Present as Na+/K+ salts Clinical application: sodium citrate used for coagulation profile.

FUNCTION OF ANTICOGULANT Sodium Fluoride: ACTION: It acts by inhibiting red cell metabolism i.e glycolysis at enolase step. It is usually mixed with oxalates 3 parts and fluoride 1 part. Quantity: 10mg/ml large amounts are required.

STEPS OF GLYCOLYSIS

PRESERVATION OF BLOOD SAMPLE Specimen should Collected under sterile conditions. Specimen should be delivered as soon as possible to the laboratory usually with in 2 hours. If delay is inevitable, then the serum should be stored at: 4 degree for 24hours. For longer duration at -20 degrees.

AVOIDING HAEMOLYSIS Appreciable visible hemolysis makes the sample unsuitable for determinations e.g. potassium, enzymes. Hemolysis is minimized by avoiding mechanical breakdown of red cells. When taking blood: Constrict the arm minimally Draw the blood slowly and steadily into the syringe Expel it slowly and gently into the container with the syringe tip touching the side so that the blood runs slowly down.

CHANGES IN BLOOD ON STORAGE Due to hemolysis or passage through RBC membrane plasma concentration of potassium increases. Formation of ammonia from nitrogenous Conversion of pyruvate to lactate Some low molecular weight hormones like ACTH Glucagon and PTH are rapidly destroyed by enzymes present in plasma and so they should be separated quickly.