Plasma, Serum & Anti-coagulants Types of blood and their indications Plasma Overall contents of Plasma SERUM Buffy coat • This is the middle layer between the plasma and RBCs. • This will contains white cells and platelets. Difference b/w Serum and Plasma Blood Purpose of anticoagulants • To prepare the whole blood or the plasma, anticoagulants are needed. • The anticoagulants are added to the container before collecting the blood sample. • These are used to prepare whole blood or plasma during the collection of blood samples. • In routine used anticoagulants are: EDTA (Ethylenediaminetetraacetic acid) • Indications: • This is useful for the hematological examination. • It is used for cell count, hematocrit, hemoglobin estimation, and cell differential count. • EDTA is used as a disodium or dipotassium salt. • Mostly potassium EDTA is used as an anticoagulant, recommended for hematology studies. • This is more soluble. Mechanism of action: • This chelating agent binds the calcium, which is needed for coagulation. Chelation prevents coagulation. • It is effective at a final concentration of 1 to 2 mg / mL of blood. • This can be used as a powder or solution and then added to vials. Let it dry. • It is used as disodium, dipotassium, or tripotassium salt. Solution of EDTA: • EDTA solution of 0.1% can be prepared and used. Let it evaporate at room temperature. • Or 1.5 mg/mL. • More than 2 mg/mL causes shrinkage of the cells. Advantages of EDTA: • EDTA preserves the morphology of the blood cell structure. • This is the anticoagulant of choice for hematocrit, Hb, and differential count. • This is the best anticoagulant for peripheral blood smears and studies. • It has little effect on the various tests. • They produce less shrinkage of RBCs. • There is less increase in the cell volume after keeping the blood. Drawbacks of EDTA: • It inhibits alkaline phosphatase, creatine kinase, and leucine aminopeptidase activities. • EDTA is not suitable for Calcium and iron estimation. Heparin Indications: • This is used in the DVT (deep vein thrombosis) • It is used in pulmonary embolism. • This is also used in unstable angina. • This is used as a prophylactic drug in venous thrombosis. • This is the drug of choice if needed in pregnancy because it can not cross the placenta. • This is used in cardiopulmonary bypass surgery. This will maintain the patency of the blood vessels. • It can be used in DIC if there are predominantly vasoocclusive manifestations. Properties of Heparin: • This is an anticoagulant and causes the least interference with the test. • This is theoretically the best anticoagulant because it is a normal blood component and does not introduce any foreign contaminants to the blood specimen. • This is more costly than the others. • It is present in powder form but is hygroscopic and dissolves rapidly. • Mechanism of action of heparin: • The GI tract does not absorb it, so given by injection in case of therapy. • Heparin accelerates antithrombin III action, neutralizing thrombin, thus preventing fibrin formation from fibrinogen. • It forms the thrombin + antithrombin cofactor + heparin complex and prevents fibrin clot formation. • It prevents coagulation for 24 hours by neutralizing the thrombin, thus preventing fibrin clots’ formation from the fibrinogen. Solution preparation of the heparin: • Heparin is added to 0.2 mg / mL of blood in each test tube. • Or 20 units of heparin for 1 mL of blood (in another reference, 15 U/mL). • Or a drop of heparin is drawn into the syringe. • After collecting blood, invert the tubes 5 to 7 times for proper blood mixing. Advantage: • This is the best anticoagulant to dry when minimal hemolysis is desired, e.g., sodium and potassium estimation. • This is the best anticoagulant to estimate pH, blood gases, electrolytes, and ionized calcium. Drawback • It is costly. • It inhibits the acid phosphatase activity. • It gives a blue background for Wright’s stain smears, so not good for peripheral blood smear interpretation. • It interferes with the binding of calcium to EDTA. • It is not used for coagulation and hematology studies. • Ammonium heparin affects the RBC volume. Sodium Citrate • Citrate is used as trisodium citrate salt. • It is a white hygroscopic crystalline powder. Indications: • Sodium citrate is widely used for coagulation studies. • For PT and PTT. • The sample can be used for ESR by the Westergren method. Mechanism of action: • it is used in solution form. • This will chelate calcium. Inactivates Ca++ ions. Sodium citrate solution preparation and uses: • Trisodium citrate= 3.2 to 3.8 g/dL (3.2% solution). • Mix well Trisodium citrate 3.8 grams in distle water. • This can be used as 0.109 mg/mL. • In blood, its ratio is 1:9, where 9 parts are blood, and 1 part is sodium citrate. • PT and PTT= Blood: Sodium citrate = 9: 1 part (blood 9 parts: sodium citrate 1 part) • ESR = Blood: Sodium citrate = 4:1 (1.6 mL of blood: o.4 mL Sodium citrate). Drawbacks of sodium citrate • This is used in liquid form (liquid anticoagulant). • This is not a good anticoagulant for a complete blood examination. • This is not good for the estimation of calcium. • It inhibits aminotransferase and alkaline phosphatase. • It has little value in clinical chemistry. Potassium Oxalate • Mechanism: • This may be sodium, potassium, ammonium, or lithium oxalic acid salt used as an anticoagulant. • This forms an insoluble complex with calcium ions (precipitate with calcium as a salt). • This is the most popular oxalate salt used as an anticoagulant in powder form. How to prepare K-oxalate Solution: • Potassium oxalate is used at a concentration of 1 to 2 mg/mL of blood. • Bulk solution: when you mix 30 grams/dL in distal water. • Now add a few drops to the test tube side and dry it in the oven below 100 °C. • The combination of ammonium/potassium oxalate does not lead to shrinkage of the RBCs. • While other oxalates cause shrinkage. Drawbacks of potassium oxalate • If the concentration is >3 mg/mL, there are chances for hemolysis. • There is a reduction of 10% hematocrit. • Oxalates inhibit enzymes like acid phosphatase, alkaline phosphatase, amylase, and LDH. • It may cause the precipitation of calcium as oxalate salt. Sodium Fluoride • This is a weak anticoagulant but uses an antiglycolytic agent to preserve glucose. • This inhibits the system involved in glycolysis and preserves the glucose. • This can be used as a dry additive. Mechanism of action: It acts in two ways: • As an anticoagulant by binding the calcium. • As an enzyme inhibitor that prevents the glycolytic enzyme from destroying the glucose. • Sodium fluoride acts after the enolase, so it will not be effective in the first 1 to 2 hours. • Not good for clinical chemistry tests. How to prepare sodium fluoride Solution: • This is effective at 2 mg/mL of blood concentration and another anticoagulant like potassium oxalate. • When used alone, then more concentration than 2 mg/mL is needed. • This can be used in combination with oxalate as a fluoride-oxalate mixture. • Most specimens are preserved at 25 °C for 24 hours and at 4 °C for 48 hours. • Sodium fluoride is poorly soluble, so mix blood thoroughly before effective anti-glycolysis occurs. • This is mainly used for glucose estimation. Drawback • This is also an inhibitor of many enzymes. • Also, effect urease for the estimation of urea. Sodium Iodoacetate • This is an effective antiglycolytic agent and substitute for sodium fluoride. • Solution use: • It can be used at a concentration of 2 g/L and is an effective glycolytic agent. • This may be substituted for sodium fluoride. • This does not affect urease. Drawback: • It inhibits creatine kinase but does not affect other chemistry tests.
