1. Dr Than Kyaw 27 February 2012

2. COAGULATION Complex process of blood clotting Important part of hemostasis Cessation of blood loss from damaged vessel(s) Disorders of coagulation can lead to an increased risk of bleeding (hemorrhage) or clotting (thrombosis). ( Refer to theory ) 2

3. ANTICOAGULANTS Drugs that prevent the clotting (coagulation) of blood by inhibiting the formation of one or more clotting factors The reagents employed should not bring about alteration of blood components. 3

4. USES OF ANTICOAGULANTS In vitro - experiments - blood tests In vivo - usually given as treatments - to prevent thrombus extension and embolic complications by reducing the rate of fibrin formation - Deep vein thrombosis and pulmonary embolism. - Myocardial infarction - Cerebrovascular disease - Blood transfusion 4

5. 5 Types of anticoagulants 1.Ammonium and Potassium Oxalate Ammonium oxalate – expand the RBCs Sodium oxalate – shrink the RBCs Also k/s double oxalate, balanced oxalate - Mixture of ammonium oxalate and potassium oxalate (3:2) - 1 mg of double oxalate - sufficient to prevent clotting of 1 ml of blood - action: precipitate ionic calcium and form insoluble salts. - safely used for haematocrit, Hb estimation, RBC and WBC counts, prothrombin time and bilirubin concentration etc.

6. 6 Types of anticoagulants 2. Sodium Citrate (trisodium citrate) - 3.8% aqueous solution is used. - 9 parts of blood: 1 part of sodium citrate solution - action: Chelating effect on calcium ion

7. 7 Types of anticoagulants 3. E.D.T.A (Versene or Sequestrene) - di-sodium salt of ethylene diamine tetra acetic acid (EDTA); powerful anti-coagulant - action: stable chelating effect on calcium - 1 to 2 mg per ml of blood - can be used in the blood transfusion - prevents blood platelets clumping in vitro; so can be employed for platelets count - Blood from some birds and reptiles hemolyzes when collected into EDTA

8. 8 Types of anticoagulants 4. Dicoumarol (Bishydroxycoumarin) - Obtained from spoiled sweet clover; inactive in vitro - Affect the transport of vitamin K to its site of action. - Vit K is required for normal synthesis of prothrombin and other clotting factors. - Interferes with factor VII and factor X in the liver - Reduces the Christmas factor (IX) - Reduces the adhesiveness of platelets - Increases antithrombin concentration in plasma

9. 9 Types of anticoagulants 5. Heparin - a natural anti-coagulant which aids in maintaining blood in a fluid state. - main source in the body - mast cells (liver, lungs etc) - inhibits the reaction between thrombin and fibrinogen - also interferes with the formation of thromboplastin - Heparin retards agglutination and lyses of platelets - used in the ratio of 0.1 to 0.2 mg per ml of blood - should not use for blood films; it gives faint blue back-ground on staining.

10. 10 Types of anticoagulants 6. Hirudin: - Present in the buccal glands of leech - A powerful anti-coagulant - Inhibits the conversion of prothrombin to thrombin. 7. Venoms - Venoms of certain snakes contain a powerful anticoagulant effect - Interferes with the action of thromboplastin or - Deplete the blood fibrinogen.

11. 11 8. Contact and Smoothness of Surface - The clotting of the blood can be prevented when it comes in contact with the tissue fluid or with the substance which is wet or has a smooth surface. -When the vessel in which the blood is collected is coated with thin layer of paraffin wax, blood remains in fluid state for half an hour. Types of anticoagulants

12. 12 Lab 1. (b) Erythrocyte sedimentation rate (ESR) Aim: To determine the ESR of the blood sample using Westergren/ Wintrobe tube Principle - A simple non-specific screening test - Indirectly measures the presence of inflammation in the body - A column of blood taken in a narrow tube - Keep vertically undisturbed - RBCs tend to settle down due to higher specific gravity than plasma - Tendency of RBCs to settle more rapidly in the face of some disease states - Usually due to increase in plasma fibrinogen, immunoglo-bulins, and other acute-phase reaction proteins - Changes in red cell shape or numbers may also affect the ESR.

13. Aggregate or Rouleaux formation Single rouleaux formation Medium density aggregate Small high density aggregate Small rouleaux network

14. 14 Method 1Method 2 Westergren tube -300 mm in length (graduated 0 – 200 mm) -Internal diameter 2.5 mm -Both ends open 3.8% sodium citrate solution Blood Stand (rack) Wintrobe tube -110 mm long (graduated 0 – 100 mm) -Internal diameter 3 mm -One end closed Pasteur pipette EDTA Blood Stand (rack) Requirements: ESR

15. SpeciesSite of collection and permitted conditions Dog, Cat Cephalic, saphenous, femoral and jugular veins Ruminants Jugular vein, tail vein Swine Jugular vein, anterior vena cava, ear veins Chicken Brachial wing vein, right jugular vein Blood sample collection ESR

16. 16 ESR Procedure (Westergren) Collect 2 ml of blood into a syringe by venue puncture transfer into a vial containing 0.5 ml of 3.8% sodium citrate solution (blood to citrate ratio of 4:1) Mix gently the contents of the vial by repeated inversion Suck the blood mixture carefully into the Westergren pipette up to the 0 mark without introducing any air bubbles. Fix the pipette vertically in the Westergren rack Allow it to remain for 2 hours Screw clamp at the top as well as pit at the bottom provided by rubber pads ensuring right fixing without allowing leakage. It also avoids the effect of atmospheric pressure. Observe the pipette for the column of clear plasma at the end of 1 h and the 2 nd hour. Note the readings in mm per 1 st hour and 2 nd hour.

18. ESR SpeciesWestergren tubeWintrobe tube mm at end of 1 hour mm at end of 2 hour Record your observed values after 1 hour and then 2 hour. Precautions The pipettes should be kept perfectly vertical. Blood column should be without bubles. Use the blood within 2 hours of collection (6 hours if kept 4  C). The test should be carried out at the room temperature.

19. 19 ESR Procedure (Wintrobe) - Performed similarly as Westergren method - EDTA (or double oxalate) anticoagulated blood without extra diluent is drawn into the tube. - The value is usually higher than Westergren method. - The method also is exposed to atmospheric pressure.

20. ESR Some interferences which increase ESR: increased level of fibrinogen, gamma globulins. technical factors: tilted ESR tube, high room temperature. Some interferences which decrease ESR: abnormally shaped RBC (sickle cells). technical factors: short ESR tubes, low room temperature, delay in test performance (>2 hours), clotted blood sample, excess anticoagulant, bubbles in tube. Chronic inflammatory disease (collagen and vascular diseases) increases ESR. Polycythemia decreases ESR.

21. Red blood cells have settled, leaving plasma at the top of the tube. Reading: 18 mm/hour BA A at “0” hr; B after 1 hour

22. Lab 1. (c) Hematocrit or Packed Cell Volume (PCV) Aim: to estimate the packed cell volume of the ------------------- blood Principle - PCV (hematocrit) - one of the most common blood test performed - PCV is the percentage of RBC in the blood. - Easy, inexpensive, reliable, and provides valuable information. - The term “packed cell volume” describes the principle behind the test. - Blood is drawn into a capillary tube, - Take care not to overfill the tube. - By centrifugation cellular components are ”packed” into the bottom of the capillary tube. - RBCs at the bottom of the tube, Buffy coat (leukocytes and platelets) Clear to yellowish top layer is plasma.

23. Hematocrit Procedure - Fill two hematocrit tubed ¾ full with blood containing anticoagulant (by capillary action). - Blood may be obtained directly from the patient. Use a hematocrit tube containing an anticoagulant. - Wipe the tube with a kimwipe or blotting paper - Place finger over the “non-blood” end of the tube and push the opposite end into a clay sealant 3-4 times - Place the hematocrit tube in a centrifuge with the clay end toward the periphery - Place centrifuged hematocrit tube on a reader with the top of the clay sealant at the 0% mark and the top of the plasma layer at 100% Centrifuge at 10000 rpm for 5 minutes.

24. Hematocrit Procedure (continued) Read the % of RBC which is read at the top of the RBC layer, do not include the buffy coat. Note and record the color of the plasma. (eg. Clear and transparent, white and cloudy, etc) Note an increase or decrease in the size of the buffy coat. The buffy coat is usually less than 1 mm wide. Also, note the color of the buffy coat which is normally white. Record your findings carefully and write a lab report.

25. Hematocrit

26. Format of the report DVT 1073 Fisiologi Haiwan II Lab work no. ____ Name: ____________ No. Matrik: ______________ Date: ________ Title of the laboratory work. Aim Requirements Procedures Observation and findings Discussion/Comments/conclusion

27. The End