Chapter 1 ANTICOAGULANTS

 

                

1.1 Reading assignments: 

Required:

• 1. Veterinary Clinical Pathology. Coles. 4th Edition, pp 47-48.
• 2. Schalm's Veterinary Hematology. Jain. 4th Edition, pp 23-24. 

Recommended but not Required:

• 1. Outline of Veterinary Clinical Pathology. Benjamin. 3rd Edition, pp 7-10.  

1.2 Objectives and/or study questions: 

• 1. List the desirable qualities of an anticoagulant used for hematologic examination.
• 2. Be familiar with the uses of the following anticoagulants:
  • EDTA
  • Oxalate
  • Heparin
  • Sodium Citrate
  • Sodium Fluoride/Potassium Oxalate
• 3. Know the Vacutainer Evacuated Blood Collection System color code presented in Table I.1 and what is contained in each tube listed.
• 4. What is the difference between serum and plasma? What anticoagulant is used to obtain serum?
• 5. What are the advantages of using serum instead of plasma for serum chemistry evaluation?

1.3 Characteristics

An anticoagulant selected for use in hematological examination must have the following qualities:

• 1. It must not alter the size of the red cells.
• 2. It must not cause hemolysis.
• 3. It must minimize platelet aggregation.
• 4. It must minimize disruption of the staining and morphology of leukocytes.
• 5. It must be readily soluble in blood.

1.4 Specific Anticoagulants

1.4.1 EDTA (Ethylenediaminetetraacetic acid).

• a. Of the five anticoagulants used in hematological studies, EDTA best meets the above requirements, and is used most frequently.
• b. Also known as sequestrene or versenate.
• c. Two forms are used: The tripotassium salt (K3EDTA), and the disodium salt (Na2EDTA). The potassium salts (liquid or dry powder) are used in commercial tubes because they are more soluble. (Calcium EDTA is not used as an anticoagulant, but in the treatment of lead poisoning.)
• d. Mode of action: It forms insoluble calcium salts by chelation.
• e. 0.5 -2.0 mg EDTA per ml of blood will preserve blood excellently for at least 6 hours. Refrigeration will extend the preservation to 24 hours.
• f. Uses: Making a blood smear for cell morphology studies, test for microfilaria, perform cell counts, BUN, plasma protein, fibrinogen, glucose, determination of Coomb's Test. EDTA preserves the staining and morphological characteristics of leukocytes. 
• g. Disadvantages:
  • 1) Excessive concentrations of EDTA will cause shrinkage of RBC's and erroneous PCV, MCV and MCHC results. Thus, it is important to fill vacutainer tubes with the required quantity of blood. Hemoglobin concentration is not affected.
  • 2) Despite the suitability of EDTA, after several hours bovine lymphocytes may develop vacuoles and tend to resemble monocytes; also, not all the vacuoles in the monocytes may be genuine.
    • 3) EDTA interferes with blood chemistry tests as follows:
    • a) falsely decreases alkaline phosphatase by binding Mg++.
    • b) decreases the C02 combining power of blood.
    • c) interferes with Jaffe reaction for creatinine test.
    • d) alters Na+, K+ and Ca++ concentrations in plasma.

1.4.2 Oxalate

• a. Sodium oxalate is no longer in use. A mixture of dry ammonium oxalate and potassium oxalate in the ratio of 3:2 is used (Heller and Paul)
• b. Mode of action: It combines with calcium to form insoluble Ca oxalate.
• c. 2 mg of the mixture will prevent coagulation in 1 ml of blood.
• d. Each salt is an anticoagulant in itself, however potassium oxalate alone causes red cells to shrink; ammonium oxalate alone causes red cells to swell. Used together, little cellular distortion occurs in the first hour after collection.
• e. Uses: The oxalate mixture may be used for hematological sedimentation studies. Potassium oxalate alone is valid for immediate glucose determinations. Ammonium oxalate is used as a diluent in some methods for manually counting WBCs and platelets.
• f. Disadvantages:
  • 1) It does not prevent platelet aggregation in vitro as effectively as EDTA.
  • 2) It is poisonous and should not be used for blood transfusion.
  • 3) This oxalate mixture cannot be used in measurements of the levels of plasma potassium, alkaline phosphatase, amylase, LDH, uric acid, urea or nitrogen by a method which involves converting the urea to ammonia. Potassium oxalate alone, in a concentration of 2 mg/ml of blood, may be used for the estimation of plasma urea (BUN). Lithium oxalate, but not ammonium oxalate may also be used for BUN determination.
  • 4) Blood examination is preferred within one hour of collection, as some cellular distortion becomes evident thereafter.

1.4.3 Heparin

• a. It is a natural anticoagulant in the body, found in the liver (from the Greek "hepar" meaning liver), and may also be within basophils and mast cells. Heparin is also called antithromboplastin or antithrombin.
• b. It is available in a liquid or dry form as sodium, calcium, ammonium and lithium salts. Each of these will interfere with determinations of their respective ions in the plasma.
• c. Mode of action: It interferes with the formation and/or activity of thrombin and the activity of clotting factors IX, X, XI, XII.
• d. The optimum concentration is 0.1 to 0.2 mg/ml of blood.
• e. Heparin is the anticoagulant of choice for blood pH and blood gas analysis for acid-base balance. It may be used for special trace element studies and some cytology. Excessive heparin does not alter RBC volume.
• f. Disadvantages:
  • 1) It causes clumping of leukocytes.
  • 2) It interferes with the staining of leukocytes.
  • 3) It is the most expensive of the anticoagulants.
  • 4) Blood will clot in 8-12 hours because clotting is only delayed and not prevented.
  • 5) It is not suitable for agglutination tests, coagulation studies (prothrombin time tests) or plasma fibrinogen determination.
  • 6) It may interfere with some automated biochemical analyses of plasma.

1.4.4 Sodium citrate

• a. The formol-citrate solution (Dacies' solution) is used as a diluent in the counting of RBC's and platelets. 3.13 g of trisodium citrate is dissolved in 100 ml of water. One ml of formaldehyde is added to every 99 ml of the solution.
• b. Acid citrate dextrose (ACD) is prepared from disodium hydrogen citrate and is the anticoagulant of choice for blood transfusions. (Two grams of disodium hydrogen citrate and 3 g dextrose are added to 120 ml water, autoclaved for 30 min. at 20 psi, and used in the ratio of 1 part ACD to 4 parts of blood.)
• c. Mode of action: It combines with calcium to form an insoluble salt of calcium citrate.
• d. Sodium citrate is the anticoagulant of choice for studies of platelet function and morphology. The standard concentration is 1 part 3.8% solution to 9 parts of blood.
• e. Disadvantages:
  • 1) It interferes with many chemical tests.
  • 2) Used alone it preserves blood for only a few hours.
  • 3) It has a tendency to shrink cells.
  • 4) Because of a 10% dilution of blood, Na-citrate is generally not used for CBC.

1.4.5 Sodium fluoride - Potassium oxalate mixture

• a. 4 parts sodium fluoride + 5 parts potassium oxalate.
• b. Mode of action: NaF inhibits the glycolytic enzymes responsible for the breakdown of glucose in the blood. (At room temperature, about 10% of the glucose is lost per hour from an untreated sample.) The potassium oxalate is the primary anticoagulant, as NaF has a poor anticoagulant effect.
• c. Optimum concentration is 1 mg of the mixture per 1 ml of blood.
• d. Uses: Glucose determination.
• e. Disadvantages:
  • 1) It is poisonous.
  • 2) Its inhibition of urease and glycolytic enzymes may interfere with urea and glucose determinations that employ enzyme activity.
  • 3) Alkaline phosphatase, amylase and uric acid cannot be determined in blood containing sodium fluoride. 

1.5 Vacutainer Tubes

Blood obtained by venipuncture may be collected directly into tubes that have been pretreated with anticoagulants. The VACUTAINER Evacuated Blood Collection System (Becton-Dickinson Co., New Jersey) consists of the following:

• 1. An evacuated glass tube containing a premeasured vacuum to ensure that a specified volume of blood is drawn.
• 2. A sterile single-use blood collection needle suitable for drawing either single or multiple samples after venipuncture.
• 3. A specially designed holder which may be used optionally to secure the needle during venipuncture and insertion into the tube stopper.

The tubes are available in a variety of sizes and with a variety of additives. Tubes containing no anticoagulants are used for tests requiring serum. The stopper color coding for the tubes is listed in table I.1. Powdered anticoagulants are utilized in most vacutainer tubes because liquid anticoagulants may dilute the blood and affect results. 

table I.1: Vacutainer Blood Collection System

* Taken from Allied Fisher Scientific catalog 1988.

** SPS = Sodium Polyanetol Sulfate, an antibiotic neutralizer. 

1.6 The Use of Serum and Plasma for Biochemistries.

• 1. Estimates of the level of biochemical constituents in the serum or plasma are different from their levels in whole blood. This is because:
  • a. Many of the chemical substances are present in different concentrations in the plasma and the RBC's. It is the concentration in the plasma (or serum) which changes in disease and can be used as an aid to diagnosis.
  • b. If calorimetric methods of estimation are used, hemoglobin is first precipitated in order to avoid a false increase in the optical density of the blood.
• 2. Serum is preferred over plasma because:
  • a. Serum is less likely to show hemolysis.
  • b. Serum does not contain anticoagulant which may draw water out of cells and thereby dilute the sample. Because of this, plasma values tend to be a little lower than serum values
  • c. Some anticoagulants in plasma interfere with the chemical reactions used to measure levels of the blood constituents.
• 3. Separation of plasma:
  • a. The anticoagulated blood in the tube is centrifuged at 1,500 to 2,000 rpm for 10-15 minutes.
  • b. The plasma (i.e. the upper layer) is drawn off using a dropper or pipet and transferred into a clean storage tube. Care should be taken not to draw off any part of the cell layers.
• 4. Separation of serum:
  • a. The blood in the sample must be allowed to clot completely and the clot should be allowed to retract for as long as possible (about 1-2 hours) before the serum is removed. The maximum amount of serum that can be obtained is 40% of the original blood volume. Beyond 1-2 hours, red blood cells in the clot begin to hemolyze with adverse consequences. Silicone-coated walls of the tube and centrifugation (2,000-3,000 rpm for 10-15 minutes) aid in clot retraction.
  • b. The serum is drawn off as described for plasma.