2. History
Physiologist Johannes Müller described fibrin, the
substance forming thrombus serving as the first milestone
for anti coagulants. Arthus discovered in 1890 that calcium
was essential in coagulation.
The theory that thrombin is generated by the presence of
tissue factor was consolidated by Paul Morawitz in 1905.
First anti coagulant preservative was used by Rous &
Turner in 1916 contained of citrate-glucose solution and
was used to store human blood
during first world war in 1987.
Heparin is one of the oldest drugs currently in widespread
clinical use. It was originally isolated from canine liver
cells, hence its name Heparin. Heparin's discovery can be
attributed to the research activities of two men: Jay McLean
and William Henry Howell.
1950’s Mark Rubin at Georgetown University
3. DEFINITION
A substance that prevents coagulation or clotting of
blood but doesn’t dissolve an already formed clot.
Uses
• Storage of blood for blood transfusion or
hematological testing
• Therapeutic
4.
5. Number and/or name Function
I (fibrinogen) Forms clot (fibrin)
II (prothrombin) Its active form (IIa) activates I, V, VII, VIII,
XI, XIII, protein C, platelets
Tissue factor(formerly known as
factor III)
Co-factor of VIIa
Calcium(formerly known as factor IV) Required for coagulation factors to bind
to phospholipid
V (proaccelerin, labile factor) Co-factor of X with which it forms the
prothrombinase complex
VI Unassigned – old name of Factor Va
6. VII (stable factor, proconvertin) Activates IX, X
VIII (Antihemophilic factor A) Co-factor of IX with which it
forms the tenase complex
IX (Antihemophilic factor B or
Christmas factor)
Activates X: forms tenase
complex with factor VIII
X (Stuart-Prower factor) Activates II: forms
prothrombinase complex with
factor V
XI (plasma thromboplastin
antecedent)
Activates IX
XII (Hageman factor) Activates factor XI, VII and
prekallikrein
XIII (fibrin-stabilizing factor) Crosslinks fibrin
10. ETHYLENE DIAMINE TETRA ACETIC ACID
Used for routine hematological work
MOA: EDTA has chelating effect on calcium
molecule in blood.
Three types of EDTA salt – sodium
potassium
lithium
Dipotassium salt more soluble than Disodium
salt, hence preferred
Although Dilithium salt is less soluble than
Dipotassium but the same sample of blood can
be used for chemical investigations
12. • The salt can be used in powder as well as liquid form
• Coating of inside surface of blood collection tube with
thin layer of EDTA improve speed of it’s uptake by blood
• Tripotassium salt dispensed in liquid form in blood will be
slightly diluted and produces some shrinkage in RBC
resulting in 2-3% dec. in PCV with in 4hrs of collection
followed gradual inc. in MC V
• Trisodium EDTA not recommended d/t high pH
• K2EDTA and Na2EDTA salts are commonly used in dry
form; K3EDTA is normally used in liquid. K2EDTA is
spray-dried on the wall of the tube and will not dilute the
sample and is recommended by ICSH and CLSI for
haematology testing.
14. DISADVANTAGES OF EDTA
RBC & WBC shrinkage
degenerative changes
>2mg/ml blood : dec. PCV ; inc. MCHC
PLATELETS
swell & disintegrate
artificially high platelet count
OTHERS
• Causes leucoagglutination affecting both lymphocytes &
neutrophils
also there is flowering of lymphocytes which can be
counted as neutrophil.
• Fails to demonstrate basophilic stippling of RBC in lead
poisoning.
• Monocyte activation measured by release of tissue
15. DOUBLE OXALATE
Acts by chelating calcium in blood
Also k/a Ammonium & Potassium oxalate
OR Heller and Paul double oxalate
Ammonium salt : causes swelling of RBC
Potassium salt : causes cell shrinkage
hence double oxalate retains normal shape of RBC
proportion of potassium : ammonium :: 2:3
16. USES
• ESR
• Routine Hematology (TLC, DLC, Hb)
DISADVANTAGES
Variable dilution of plasma is caused by
potassium oxalate d/t water transport from cells
to plasma.
If oxalate is added to vials and dried in an
oven, great care is to be taken to avoid
temperatures above 80ºC. As Oxalates are
converted to carbonates by prolonged exposure
to elevated temperatures.
17. TRISODIUM CITRATE
Acts by chelating calcium in
blood .
It is used in a concentration of :-
1 part 0.11 M sodium citrate to 9
parts whole blood for ESR (by
westergren)
1 part 0.11 M sodium citrate to
4parts whole blood for
coagulation profile.
Citrate is usually in blue
Vacutainer tube. It is in liquid form
in the tube and is used for
18. USES
FIBRINOGEN
PT
PTT
ESR(by westergren method)
DISADVANTAGES
Alters concentration of blood as it is always
used in solution form , hence, is not used for
routine hematology.
19. SODIUM FLOURIDE
Sodium fluoride has a double action on the blood:
It prevents clotting by chelating calcium.
It prevents all phosphatase action, inhibit glucose
oxidase activity in enzymatic glucose reaction.
It is used for determination of blood sugar. It
preserve blood for 24 hours at room temperature
and 4 to 6 day in refrigerator.
20. Common disadvantages with
calcium chelators
They inhibit various plasma enzyme activities like:
Amylase activity inhibited by oxalate and citrate
LDH and Acid Phosphatase inhibited by oxalate
Fluoride , Heparin or EDTA interfere with accurate
determination of electrolytes
21. HEPARIN
Heparin is a naturally-occurring anticoagulant
produced by basophils and mast cells. Heparin acts
as an anticoagulant, preventing the formation of clots
and extension of existing clots within the blood. While
heparin does not break down clots that have already
formed (unlike tissue plasminogen activator), it allows
the body's natural clot lysis
Mechanism of action:
Heparin binds to anti thrombin inhibiting interaction of
various clotting factors i.e. Xa, IX a, XIa, XII a & plasmin.
23. USES
ESR
Methaemoglobin
Osmotic Fragility
HLA Typing
Ammonia
Disadvantages
• Costly
• In Leishman stained peripheral blood film blue
color is imparted to the background due to
presence of plasma
25. COMPOSITION( in 1l of distilled water)
CHEMICAL ACD CPD CPDA-1
TRI
SODIUM
CITRATE
22.0 g 26.3 g 26.3 g
CITRIC
ACID
8.0 g 3.27 g 3.27 g
DEXTROSE 24.6 g 25.50 g 31.8 g
SODIUM
DIHYDROGE
N
PHOSPHATE
____ 2.28 g 2.22 g
26. 15ml of ACD and 14ml of CPD & CPDA-1 is
required for preservation of 100ml of blood
Initial pH of ACD 5.0 and of CPD & CPDA-1 is
5.6
Also storage time at 2-6ºC for ACD & CPD is 21
days and for CPDA-1 is 35days
27. FUNCTION OF VARIOUS
COMPONENTS
o CITRATE
Causes chelation of calcium
SODIUM DIPHOSPHATE
Prevents fall in pH
o DEXTROSE
Addition of glucose prolongs survival of stored RBC
as it is required for metabolism. Glucose passes from
plasma to RBC and is utilized for energy production.
2 PATHWAYS for energy production :
• 90% by Embedem Mayeroff pathway in which there
is breakdown of glucose into lactate through
anaerobic glycolysis.
28. • 10% by Pentose phosphate pathway through aerobic
glycolysis.
The various intermediaries formed are necessary for
maintaining
their ability to deliver oxygen to tissues through
generation of
2,3-DPG.
Viability correlates with the level of ATP.
o CITRIC ACID
Fairly weak tribasic hydroxyacid
Along with tri sodium citrate which is alkaline
gives an optimal pH.
Prevents carmalization of glucose in citrate
dextrose solution during autoclaving.
o ADENINE
29. ADDITIVE SYSTEM
To extend RBC storage to 42 days and to harvest
maximum amount of plasma, additive systems are
now available in which storage environment of RBC
is altered by adding certain nutrients after removal of
plasma
Its made by adding the following components to the
CPD :-
• Sodium chloride-adjusts osmotic pressure
• Adenine – maintains high level of ATP in RBC
• Dextrose
• Mannitol- prevents disintegration of RBC
31. PHYSICAL
o Shape of RBC discoid to spherical
o Loss of lipid in RBC membrane
o Increased cellular rigidity d/t decrease in deformability
o Decrease in critical hemolytic volume(CHV) in parallel
with
membrane lipid content
CHV is largest volume to which RBC swells before
haemolysis
32. o Decrease in osmotic fragility
o Blood stored for >24 hr at 2-6ºC has few viable
platelets and granulocytes
o Heat labile coagulation factors V & VIII decrease on
storage
upto 50% in first 48-72hrs
There after loss is comparatively slower
after 21 day factorV 30%
factorVIII 15%
33. BIOCHEMICAL CHANGES
Drop in pH and dextrose level causes anaerobic
glycolysis in RBC to generate ATP.
Decreased ph causes Decreased 2,3-DPG and cells
ability to release oxygen to the tissues.
Metabolic functions slow down in cold temperature,
ATP levels decreases.
ELECTROLYTE: loss of potassium from RBC to
plasma
passage of sodium from plasma to
cells
plasma ammonia levels also
increase
34. THERAPUTIC USES
Anti coagulants used for therapeutic use are :-
• Heparin and LMWH
• Oral Anticoagulants – warfarin and other Vit K
antagonists
They are used for prevention of various thrombotic
diseases
DVT
PULMONARY EMBOLISM
ACUTE MYOCARDIAL INFRACTION
