Current presentation is about the comprehensive literature of Anticoagulants, Bulbs for blood collection and their specific uses intended for Graduate and Post graduate students of Physiology and Pathology in Medical and Health Sciences.

1. ANTICOAGULANTS
Dr. Aniket A. Shilwant
BAMS, MD
1

2. DEFINITIONS
Whole Blood – A venous, arterial or capillary blood sample in which the
concentrations and properties of cellular and extra-cellular constituents remain
relatively unaltered when compared with their in-vivo state.
Anticoagulation in-vitro stabilizes the constituents in a whole blood sample for a
certain period of time.
Plasma – The virtually cell-free supernatant of blood containing anticoagulant
obtained after centrifugation.
Serum – The undiluted, extracellular portion of blood after adequate coagulation is
complete
Dr. Aniket Shilwant, GJPIASR 2

3. ANTICOAGULANTS
Additives that inhibit blood and/or plasma from clotting ensuring that the constituent
to be measured is non-significantly changed prior to the analytical process.
Anticoagulation occurs by binding calcium ions or by inhibiting thrombin activity.
Dr. Aniket Shilwant, GJPIASR 3

4. ANTICOAGULANTS
Purpose
 Coagulation studies of blood
 Blood storage and preservation
 Diagnostic point of view
Properties
 Easily soluble in blood
 Maintains the blood in fluid condition
 Does not create hemolysis in Blood cells
 Maintains the size and shape of RBCs
 Minimizes destruction of WBCs Dr. Aniket Shilwant, GJPIASR 4

5. Anticoagulants mechanism of action
Interferes plasma clotting factors
Anticoagulants
Inhibits platelet aggregation
Antiplatelets
Dissolves blood clots
Thrombolytics
Dr. Aniket Shilwant, GJPIASR 5

6. Types of Anticoagulants
IN-VITRO
 EDTA
 Tri-Sodium Citrate
 Double Oxalate mixture
 Sodium Fluoride
 Heparin
 ACD & CPD-A
IN-VIVO
 Di-coumarol & Warfarin
 Heparin
Dr. Aniket Shilwant, GJPIASR 6

7. Types of Anticoagulants
Calcium chelator
Binds with Calcium
Ex. EDTA, Tri-Sodium citrate, Double Oxalates, etc.
Non-Calcium chelator
Do not bind with Calcium
Ex. Heparin, Warfarin, etc.
Dr. Aniket Shilwant, GJPIASR 7

8. IN-VITRO ANTICOAGULANTS
1. Ethylene Diamine Tetra -acetic Acid (EDTA).
Also known as Sequestrene (Di-Potassium salt) or Versene (Di-Sodium salt)
Potassium and Sodium salts (Dipotassium, Tripotassium, Disodium)
Mostly used - dry (anhydrous) dipotassium salt of EDTA
Tri-potassium salt of EDTA causes some shrinkage of RBCs resulting 2–3% decrease
in PCV.
Mode of action
Prevents clotting by removing ionic calcium from the blood sample by chelation.
Dr. Aniket Shilwant, GJPIASR 8

9. IN-VITRO ANTICOAGULANTS
1. Ethylene Diamine Tetra -acetic Acid (EDTA).
Effective concentration
1.2 to 2.0 mg/mL blood
Note
Excess of EDTA (> 2 mg/ml blood) affects all blood cells.
RBC shrinks thus reduces PCV, WBC undergo degenerative changes
Platelets break into large enough fragments to be counted as normal platelets.
Dr. Aniket Shilwant, GJPIASR 9

10. IN-VITRO ANTICOAGULANTS
2. Trisodium Citrate (Na3 C6 H5 O2.2H2O).
Mostly used in coagulation studies
Available forms - sodium, ammonium, and potassium citrate.
Mostly preferred – Sodium citrate
Mode of action
It de-ionizes the free blood calcium which will prevent clotting.
Citrate ion combines with calcium in the blood to form an unionized calcium
compound.
Note
It does not preserve the cell morphology
Dr. Aniket Shilwant, GJPIASR 10

11. IN-VITRO ANTICOAGULANTS
2. Trisodium Citrate (Na3 C6 H5 O2.2H2O).
Effective concentration
3.8 % solution is prepared in distilled water
A citrated bulb in ratio of 1:9 (citrate : blood) is used for coagulation studies
For ESR by Westergreen method in ratio of 1:3 (citrate : blood)
Other uses
Sodium citrate used in storage of blood in blood banks
Prothrombin Time (PT) estimation used in 1:9 ratio (1Part AntiCo. : 9 Parts blood)
 ESR estimation by Westergreen method used in 1:4 ratio
Dr. Aniket Shilwant, GJPIASR 11

12. IN-VITRO ANTICOAGULANTS
3.Double Oxalate mixture
 It is a mixture of ammonium oxalate and potassium oxalate
Effective concentration
 Ammonium oxalate and potassium oxalate in ratio of 3:2
Mode of action
 Prevents clotting by forming insoluble calcium salts, thus removing ionic calcium.
Note
 Use of too little will not serve the purpose of anticoagulant
 Use of too much oxalate is hypertonic and damages all blood cells
 Avoid sodium oxalate, as it leads to Crenation of RBCs
Dr. Aniket Shilwant, GJPIASR 12

13. IN-VITRO ANTICOAGULANTS
4. Sodium Fluoride.
 Mostly used in estimation of Plasma glucose level.
 A mixture of 10 mg of sodium fluoride and 1 mg thymol is an anticoagulant as well
as a preservative.
 Mode of action
 Fluoride inhibits glycolytic enzymes, thus prevents loss of glucose.
Dr. Aniket Shilwant, GJPIASR 13

14. IN-VITRO ANTICOAGULANTS
5. Heparin (MW - Molecular weight ranging from 15000–18000)
Named so as extracted from – Liver
Naturally occurring powerful anticoagulant
A highly charged mixture of sulphated polysaccharides
Secreted by Mast cells and Basophils
Mode of action
Heparin itself has no anticoagulant activity. When it combines with anti-thrombin III, their
combined ability increases to remove thrombin as soon as it is formed
This complex of two also removes clotting factors such as – IX, X, XI, and XII.
Dr. Aniket Shilwant, GJPIASR 14

15. IN-VITRO ANTICOAGULANTS
5. Heparin (MW - Molecular weight ranging from 15000–18000)
Effective concentration
10–20 IU /ml blood
Note
Expensive
Creates black background in smear, thus is not used for smear preparation
Dr. Aniket Shilwant, GJPIASR 15

16. IN-VITRO ANTICOAGULANTS
Anticoagulants used for Blood storage
ACD – Acid Citrate Dextrose
CPD – Citrate Phosphate Dextrose
CPD-A – Citrate Phosphate Dextrose Adenine
Dr. Aniket Shilwant, GJPIASR 16

17. IN-VITRO ANTICOAGULANTS
6. ACD and CPD – A (citrate-phosphate-dextrose-adenine)
Uses
Storage of blood
Effective concentration
Donated single unit of blood (450ml) is stored under aseptic conditions into a special
plastic bag with
63 ml of CPD-A (citrate-phosphate-dextrose-adenine) mixture.
Dr. Aniket Shilwant, GJPIASR 17

18. IN-VITRO ANTICOAGULANTS
6. ACD and CPD – A (citrate-phosphate-dextrose-adenine)
Role of CPD-A
Citrate – Serves as Anticoagulant
Sodium diphosphate – Acts as Buffer, prevents fall in pH
Dextrose – Supports ATP synthesis through glycolytic pathway. Regulates Sodium
Potassium pump activity to maintain size and shape of RBCs and increases their lifespan
Adenine – Provides substrate for the synthesis of ATP, thus improving post-donation
viability of RBCs
Dr. Aniket Shilwant, GJPIASR 18

19. IN-VIVO ANTICOAGULANTS
1. Dicoumarol and warfarin
 These are Vitamin K antagonists
 These are slow acting anticoagulants as compared to Heparin.
Mode of action
 Inhibit the action of vitamin K which is essential as a cofactor for synthesis of
proteins containing glutamic acid
 Such as – clotting factors II, VII, IX, and X, protein C, and protein S.
2. Heparin
 Useful in-vivo during open-heart surgery, hemodialysis.
Dr. Aniket Shilwant, GJPIASR 19

20. OTHER ANTICOAGULANTS
Hirudin
Hirudin is an antithrombin extracted from leeches or prepared by a genetic
engineering process. Hirudin inhibits thrombin by forming a 1:1 hirudin-thrombin
complex.
Effective concentration
Hirudin is used at a concentration of 10 mg/L (40).
Dr. Aniket Shilwant, GJPIASR 20

21. Blood storage
Component Storage Temperature Expiry
Whole blood
RBC
Blood banks
Cold storages
2-6℃ ± ℃ 35 days with CPDA bags
42 days with CPD SAGM
Fresh Frozen Plasma (FFP)
Cryoprecipitate
Cold storages
Freezer
-30℃ or less 1 year
Platelet concentrate Platelet
agitator
22±2℃ 5 days
Cryo-poor plasma Freezer -30℃ or less 5 years post Plasma
fractionation
Storage conditions and expiry of different blood components
Dr. Aniket Shilwant, GJPIASR 21

22. Color codes for different anticoagulants
No Anticoagulant Serum
Sodium Fluoride Glucose estimation
EDTA Complete Hemogram (CBC), ESR
3.2% Sodium Citrate Bulb
Coagulation studies
Prothrombin Time (PT), APTT
Heparin Bulb Bone marrow studies
Citrate Blood culture
(K2) EDTA
Blood bank tests, Blood typing
Blood grouping
COLOR
CODES
OF
BULBS
FOR
ANTICOAGULANTS
Dr. Aniket Shilwant, GJPIASR 22

23. Thank You !!!
Dr. Aniket A. Shilwant
BAMS, MD
Associate Professor
Sharir Kriya Dept.
GJPIASR, CVM University
Anand, Gujarat
23