CHAPTER ONE
1.0
INTRODUCTION
Platelets have
been documented to pay a vital role in blood coagulation (Mustard et al, 1966; Guyton and
Hall, 1996). To adequately play this role, it is important that the
quantity of these platelets in blood be maintained regularly within a narrow
range of 150-400x109 cells per liter of blood (Caucasians) (Dacie
and Lewis; 1994) and 100-400x109 cells per liter of blood
(Nigerian) (Miller and Weller; 1971). The lifespan of a platelet is
7-10days, once they are released from the bone marrow; young platelets are
trapped in the spleen for 36 hours before entering the circulation, where they
have a primary haemostatic role. (www.bioline.org.br/request). Quantitative
abnormalities of platelets have been reported to result in many disease
conditions e.g. hemorrhage, thrombosis and atherosclerosis (Bloom and Thomas 1981, Sheldon
1988).
An
anticoagulant is a substance that prevents coagulation, that is, it stops
blood. Chemical can be added to stop blood clotting. Apart from heparin, most
of these chemicals work by binding calcium ions, preventing the coagulation
proteins from using them (Buller et al; 2005). Heparin work by
binding to antithrombin (Lewis et al; 2008). The commonly
used anticoagulants are ethylemediaminetetra-acetic acid (EDTA), oxalate,
heparin, sodium citreate, and sodium fluoride/potassium oxalate (Coles
2007). Some authors also include Citrate Phosphate Dextrose and Adenine
(CPDA), fluoride oxalate, Acid Citrate Dextrose (ACD) (Chessbrough; 2006).
The
change in blood on storage are directly related to the storage time and the
type of anticoagulant used (Ochei and Kolhatka; 2008).
Haematologic sample must be analyzed as soon as possible to prevent artifacts
created by exposure to anticoagulants and cell deterioration due to storage (Willard
at al, 1999). Haematology samples should be analyzed within 3 hours or
to be refrigerated at 4oC to avoid artificial increased haematocirt,
increased mean corpuscular volume and decreased mean corpuscular haemoglobin
concentration. Platelet counts should be performed no later than 1 hour after
collection. www.vetal/tranquerancom.ar/pages/wild/smallanimal.ss.htm.)
Abnormalities blood stored in anticoagulatnt include tetany of muscles,
decreased packed cell volume (PCV), count. (Gossens et al; 1991, Date and
Lewis 1994). Erythrocyte creation, neutrophil hypersegmentation and
lymphocytic nuclear distortion may occur in aged samples. In addition, monocyte
vacuolization monocyte pseudopod formation and platelet aggregation are some of
the more common artifacts encountered in stored samples (Willard at al; 1999).
Despite all this documented abnormalities observed in blood stored in
anticoagulants, the use of anticoagulants to store blood at 4oC
still remain the most available, widely accepted and most reliable method of
blood storage in the Tropics (Nwafia et al; 2003). But the
changes in environmental factors especially the fluctuating electric power
supply and local availability of substandard blood anticoagulant bags.
There
is need, therefore to constantly monitor the procedural effect in blood
storage.
1.1
Aims and
Objectives
To determine the
effect of storage in different anticoagulants and storage
time on
platelets.
1.2
Justification of
the Study
Platelets are
vital in blood coagulation and their quantity in the body is essential. Some
disease conditions may require blood transfusion and oftentimes, it is
difficult to obtain fresh whole blood in that immediate environment, hence, the
only available choice are blood stored in anticoagulant. It then becomes very
vital that the components of these stored blood especially platelets are
maintained relatively constant to prevent post transfusion complications
especially with blood coagulation. This study/research is to determine the best
anticoagulant that can preserve blood components especially platelets in stored
blood.
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 PLATELETS
Platelets
are produced predominantly by bone marrow megakaryocytes as a result of budding
of the cytoplasmic membrane (www.bmj.com/./809). The precusor of the
megakaryocyte-the megakaryoblast arises by a process of differentiation from
the haemopoetic stem. The magakarycyte matures by edomitotic synchronic
replication, enlarging the cytoplasmic volume as the number of nuclear lobes
increase in multiples of two. Very early on invagination of plasma membrane are
seen, called the demarcation membrane, which evolves through the development of
the magakarycyte into a highly branched network. At a variable stage in
development, most commonly at the eight nucleus stage, the cytoplasm becomes
granular. Platelets form by fragmentation of magakarycyte cytoplasm,
approximately each magakarycyte giving rise to 1000-5000 platelets (Hoffbrand
et al; 2006). On average they are 1.5-3um in diameter (Lewis et al; 2008) while (Hoffbrand
et al; 2006) says they are 3.0-0.5um in diameter with a mean volume of
7-11 fl. They do not contain a nucleus and are bounded by a typical lipid
bilayer membrane. Beneath the outer band lies the marginal of microtubules,
which contains the shape of the platelet and depolymerise when aggregation
begins. The central cytoplasm is dominated by the three types of platelet
granules: the dense (δ) granules, a- Granules and lysosomal granules (Lewis
at al 2008). The more frequent specific granules contains a heparin
antagonist PF4, platelet derived growth factor (PDGF), β Thromoboglobulin,
fibrinogen, von willebrand factor and other clotting factors. Dense (δ)
granules are less common and contains adenosine diphosphate (ADP),
adenosine triphosphate (ATP) , 5-hydropxytrptamine
(5-HT), Calcium (Hoffbrand et al, 2006) while (Lewis et al; 2008) included serotonin, pyrophosphate,P5 Lectin
(CD62), transforming growth factor beta
(I), catecholamine, guanosine diphosphate/guanosine triphosphate (GDP/GTP).
Lysosomes contain hydrolytic enzymes and peroxisomes contain catalase
(hoffbrand et al; 2006).
Once
release from the bone marrow, young platelets are trapped in the spleen for 36
hours before entering the circulation, where they have a primary haemostatic role. The
platelet membrane has integral
glycoproteins essential in the site of interaction with the plasma
initial events of adhesion and aggregation leading to formation of the platelet
plug during haemostatis. (WWW.bmj.com./../809.
The platelet membrane is the
site of interaction with the plasma environment and with the
damaged vessel wall. It consists of phospholipids cholesterol, glycolipids and
at least nine glycoprotein named glycoprotein I
glycoprotein IX. After activation the
membrane also expresses binding sites
for several conagulation proteins such as factor XI and
factor VIII (Lewis et al; 2008).
2.1.1 Platelet Function
The
main function of platelet is the formation of mechanical plugs during the
normal haemostatic response to vascular injury. In the absence of platelets
spontaneous leakage of blood through small vessels may occur. The
immobilization of platelets at the sites of vascular injury requires specific platelet- vessel wall (adhension)
and platelet –platelet (aggregation interactions) (Hoffbrand et al, 2006).
Platelets have been documented to play a vital role in blood coagulation (Guyton and Hall, 1996). They
act as plugs around the opening of a
wound and release contain factors that are necessary for formation of a blood
clot to prevent loss. The platelets also maintain the integrity of the blood vessels by
plugging the gaps in the endothelial lining (Ochei and Kolhatkar, 2008)
Clotting takes place in 3 essential steps:
1.
In response to rupture of the vessel or damage
to the blood itself, a complex cascade
of chemical reactions occurs in the blood involving more than a dozen blood coagulation
factors. The net result is formation of a complex of activated substances collectively
called protheombin activator.
2.
The
prothrombin activator catalyzes conversion of prothrombin into thrombin.
3.
The
thrombin acts as an enzymes to convert fibrinogen into fibrin
fibers that enmesh platelets, blood cells and plasma to form the clot (Guyton and Hall;
1996)
2.1.2 Quantitative
and Qualitative Abnormalities of Platelets
A. congenital abnormalities which includes:
1.
Fanconi’s Anaemia: This is an autosomal recessive
preleukaemic condition that often presents as thromobocytopenia with skeletal
or genitourinary abnormalities.
2.
Giant
granular platelets in peripheral blood film are seen in Bernard –soulier
syndrome or may –Hegglin anomaly. Megakaryocytic thrombocytopenia presents with
severe neonatal thrombocytopenia (platelet count < 2 x109/L),
though this often correct itself after the first year of life.
3.
Wiskolt-aldrich
syndrome:
is an x-linked disorder with a triad of thrombocytopenia, eczema and
immunodeficiency. The platelet count is usually 20-100x109/L and the platelets
are functionally abnormal.
May
Hegglin anomaly and variants of Alport’s syndrome are both characterized by
giant platelets.
Diseases
of the platelet storage pool are deficiencies in either in the α or dense
granules causing poor secondary platelet aggregation.
1.3 Acquired Abnormalities
Decrease production of platelets due to
suppression of failure of the bone marrow is the commonest cause of the
thrombocytopenia. In aplastic anaemia, leukaemia and marrow infiltration and after
chemotherapy thrombocytopenia is usually associated with a failure of red and
white cell production but may be an isolated finding secondary to drug toxicity
(pencillamine, costrimozazole, alcohol,
malaria or viral infection (HIV, infectious mononucleosis).
Disorders with increased platelet consumption
i.
Disorders
with immune mechanism;
a.
Autoimmune-idiopathic thrombocytopenia purpura
b.
Alloimmune-post
transfusion purpura
c.
Neonatal
alloimmune-thrombocytopenia
ii.
Infection
–associated: infectious mononucleosis,
HIV, malaria
iii.
Drug
–induced, heparin, penicillin, quinine, sulphonamide rifampicin
iv.
Thrombotic
thrombocytopenic purpura/haemolytic
urmic syndrome
v.
Hypersplensim
and splenomegaly
vi.
Disseminated
intravascular coagulation
vii.
Massive
transfusion
Acquired disorders of reduced platelet
production
a.
Drug
induced e.g quinine, aspirin (Cheesbrough; 2006)
b.
Leukemia
c.
Metastatic tumor
d.
Aplastic anaemia
e.
Myelodysplasia
f.
Cytotoxic drugs
g.
Radiotherapy
h.
Infections e.g
typhoid and other septicaemias (Cheesbrough; 2006)
i.
Megalobalstic anaemia
j.
Due to bone marrow failure or replacement
Increased platelet consumption: May be due to immune
or non immune mechanisms
a.
Idiopathic thrombocytopenic purpura is a relatively
immune disorder and the most frequent
cause of isolated thrombocytopenia without anaemia or neutropenia. In this
disorder autoantibody produced is usually LgG directed against antigens on the platelet membrane. The platelet lifespan is reduced
to few hours. The platelet count may
vary from < 5 x199/L to near
normal,
b.
Spontaneous skin purpura in
severe immune theombocytopenia
c.
Bone marrow aspirate showing increased magakaryocytes in immune
thrombocytopenia
d.
Neonatal alloimmune thrombocytopenia is
similar to haemolytic disease of the new born infants except that the antigenic stimulus comes from platelet
specific antigens rather than red cell
antigens.
2.1.4 Post
–Transfusion Abnormality:
This
is an acquired abnormality. It is a rare complication of blood transfusion
presenting with severe thromobocytopenia 7-10 days after the transfusion.
Patients are usually multiparous women who are negative for the human platelet antigen
1a. Antibodies to this antigen develop that are somehow responsible for the immune destruction of the patients own platelets.
Heparin
induced thrombocytopenia occurs during heparin treatment in up to 5% patients.
It may manifest when arterial or venous thrombosis occurs during a fall in the
platelet and this is thought to be due the
formation of antibodies to heparin that are bound to platelet factor 4,
a platelet granule protein. The immune complexes activate platelets and endothelial cells, resulting
in thrombocytopenia and thrombosis co-exiting.
2.1.5 Causes
of Acquired Platelet Dysfunction;
1.
Aspirin and non-steroidal anti-
inflammatory agents
2.
Penicillins and cephalosporins
3.
Uraemia
4.
Alcohol
5.
Liver disease
6.
Myeloprolifrative disorders
7.
Myeloma
8.
Cardiopulmonary bypass
9.
Fish oils
2.1.6 Thrombocytosis;
a.
Essential (primary) thrombocytosis
b.
Reactive (secondary) thrombocytosis
c.
Infection
d.
Malignant disease
e.
Acute and chronic inflammatory diseases
f.
Pregnancy
g.
After splenectomy
h.
Iron deficiency
i.
Hemorrhage
In
essential (primary) thrombocytosis and reactive (2o) thrombocytosis,
the platelet count is raised above the upper limit of normal (www.bmj.com/..809).
Abnormalities of
blood stored in anticoagulants include tetany of muscles, decreased packed cell
volume (PCV), increased mean cell volume (MCV), artificial increase in Platelet
count (PC) and decrease white blood cell (WBC) count (Gossens et al; 1991),(Dacie and
Lewis; 1994).
2.2.2
ANTICOAGULANTS
Anticoagulants
are the chemicals which prevent clotting of blood when mixed with blood in
proper proportion (Willard et al; 1999). These chemicals, apart from hepian works
by binding calcium ions preventing coagulation proteins from using them.
Heparin interferes with the formation and/or activity of thrombin and the
activity of clothing factor 1X, X, XI, XII (Hylek et al; 2007).
Characteristics
of anticoagulants;
An
anticoagulant selected for use in hematological examination must have the
following qualities;
1.
It
must not alter the size of red cells
2.
It
must not cause haemolysis
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. (Schalm et al; 2006).
The Frequently used anticoagulants
includes;
1.
Ethylenediaminetetra acetic acid (EDTA)
2.
Oxalate
3.
Heparin
4.
Sodium citrate
5.
Sodium fluoride/potassium oxalate
6.
Citrate phosphate dextrose and adenine
(CPD-A).
7.
Fluoride oxalate (FO)
8.
Acid citrate dextrose (ACD)
Ethylenediaminetetra
acetic acid, heparin, fluoride oxalate and sodium citrate are the ones will be
used for the study and hence they are explained below;
2.2.3
Ethylenediaminetetra Acetic
Acid (EDTA)
EDTA
is a widely used acronym for the chemical compound Ethylenediamintetra acetic
acid. EDTA is a polyamino carboxylic acid with the formular (CH2N)
CH2 CO2H2)2. The colorless, water
soluble solid is widely used to dissolved scale. Its usefulness arises because
of its role as a chelating agents, that is its ability to “sequester” metal
ions such as Ca2+ Fe3+. After being bound by EDTA metal
ions remain in solution but exhibit diminished reactivity. EDTA is produced as
several salts; notably disodium EDTA and calciumdisodium.
EDTA
(Vitalongevity
2007). (Jain 2006) said that EDTA consist or comes in 2 forms;
a.
The
tripotassium salt (K3 EDTA)
b.
And
the disodium salt (Na2 EDTA). He also added that 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).
2.2.4
Synthesis of EDTA;
The compound was
first described in 1935 by Ferdinard Munz, who prepared the compound from
ethylenediamine and chloroacetic acid (Vitalongevity 2007). Today, EDTA is
mainly synthesized from ethylenediamine (1,2-diamino ethane), formaldehyde
(Methanal), and sodium salt which can be converted in a subsequent step in the
acid forms;
H2NCH2CH2NH2+4CH2O+4NaCN+4H2O®(NaO2CCH2)2NCH2
CH2N (CH2CO2Na) 2 + 4NH3
(NaO2CH2
NCH2)2 NCH2 CH2N (CH2 CO2Na)2
4HCI® (HO2CCH2)
2 NCH2 CH2N(CH2CO2H2+4NaCL.
Impurities
cogenerated by this route include glycine and niterilotriacetic acid (New
York Times; 2008).
2.2.5 Mode
of Action
It acts as a powerful calcium chelating agent. The calcium in blood is
bound in an unionzed and soluble complex with EDTA
Tests which are
performed by using EDTA blood are:
1.
Haemoglobin
2.
White
blood count
3.
Packed
cell volume determination
4.
Erythrocyte
sedimentation rate by wintrobe’s method
5.
Platelet
count
6.
Differential
white blood cell count
2.26
Advantages
1.
It
gives the best preservation of cellular morphology. Good morphology of the cell is observed even
after 2 to 3
hours of blood collection
2.
Since platelet clumping is inhibited for
platelet counts using this anticoagulant is preferred (Willard et al 1999)
2.2
Disadvantages;
1.
Excess EDTA causes shrinkage of red blood
cells and erroneous packed cell volume, mean cell volume and means corpuscular
haemoglobin (Jain 2006).
2.
Platelets
swell and disintegrate due to excess of EDTA and artificially high platelet
count may be obtained due to disintegrated platelets.
3.
EDTA
is not suitable for use in the
coagulation studies mainly in the
determination of prothrombin time
4.
EDTA 2k (potassium salt is recommended
as anticoagulant for complete blood count. (Willard et al 1999)
2.3.1 Mechanism
of Action of Anticoagulants
The thromboplastin released by damaged tissue, or platelets converted inactive
prothrombin into active thrombin in the
presence of calcium ions. Thrombin coverts soluble fibrinogen into insoluble
fibrin clot in the presence of calcium ions.
Thromboplastin
Prothrombin ----------------------------------- Thrombin
Ca++
Thrombin
Fibrinogen(soluble) ----------------------------------- Fibrin
(insoluble)
Ca++
fibrin + blood cells clot (fine threads)
Some
anticoagulants such as potassium oxalate prevent clotting of blood by
precipitating ionic calcium in plasma
while anticoagulant such as sodium citrate ions into unionized form. Since
calcium ions are not available, blood is prevented from clotting. Heparin on the
other hand acts as antithrombin and thus
prevents the formation of thrombin and blood
clotting (Willard et al; 1999)
2.4.1 A
study carried out by Mcschine RL,
sibinga S, Brozovic B
(Clin lab
Haematol, 1990) showed that EDTA amongst trisodium citrate, citrate phosphate dextrose
adenine and acid citrate dextrose is
the best anticoagulant for use in blood and blood components is required (Thompson et al, 1983) also has studied and came out with a conclusion that Na2EDTA appears to
provide the best conditions of anticoagulation for both routine clinical and research laboratory measurement of the mean platelet volume (MPV). They also
stated that this anticoagulant (Na2EDTA) inhibited platelet
activation but left the platelets in the normal discoid shape. Okada; 1999 stated that the
dipotassium salt of EDTA is the
preferred anticoagulant for platelet volume analysis in Europe and Japan, whereas in US and United Kingdom the tripotassium
salt of EDTA is more commonly used. He
also stated that if blood is collected into EDTA, platelets quickly change shape from discs with a
2 -4µm diameter and a thickness of 0.45µm to spheres covered by long
thin filopodia. Threatte et al 1984 and Bath 1993 also
added that the sphering of
platelets in EDTA is initially so volumetric but almost immediately, their apparent size changes over 1-2
until a state of semi equilibrium is
reached (Okada; 1999). This led to the suggestion that EDTA is not a
suitable anticoagulant for platelet volume analysis (Rowani, 1986) . Okada, 1999 further stated
that if blood from certain individuals is anticoagulated with EDTA, the platelets aggregate, causing an apparent
thrombocytopenia to be recorded, Jackson and Carter 1993 also added that in
some but not all, cases may result from the
presence of agglutinating antiplatelet antibodies.
Nevertheless (Macey et al, 1999) cited
that EDTA has been used more
recently in several studies of platelet status,
the national committee for clinical laboratory methods also
recommends EDTA for full blood counts
and white blood cell differential analysis
( National Committee
for clinical laboratory standards, 1996) . the international
council for standardization in Haematology currently recommends the dipotassium salt of EDTA as
the anticoagulant for full blood counts
and platelet analysis(International
council for standardization in haematology;
1993, and Lewis; 1999)
Note: Hedge et al, 1991 stated that recent
report suggested that storage of blood in EDTA gave falsely elevated results and they equally found out
in their study that little difference occurred in the values in blood
anticoagulated in EDTA at 72 hours of storage
2.51 HEPARIN
Heparin,
is a natural anticoagulant in the body found in the liver (from the Greek word”
hepar” meaning liver) and may also
be within basophils and mast cells. Heparin is also called anti
thromboplastin or antithrombin (Schalms;
2006) Heparin is isolated
from either porcine intestine or form beef
lung where it is bound to histamine and stored in the mast cell
granules, when heparin is isolated,
the purification leads to a
heterogeneous mixture of molecules it is an acidic polysaccharide with side
groups, either sulpates or N-acetyl groups
attached to individuals
sugar group. The sulfate groups
are extremely important in the anticoagulant activity by binding to
antithrombin III (at III) enhancing the rate of thrombin –Antithrombin III
complex formation by 1,000 to 10,000 fold. (platelet inhibitors, com/review
article).
Heparin
is available in a liquid or dry form as sodium, calcium, ammonium and lithium
salts, each of these will interfere with determination of their respective ions in the plasma.
2.5.2 Mode of action; it interferes
with the formation and / or activity of
thrombin and the
activity of clotting factors IX, X, XI, XII. (Schalms; 2006).
Lithium
or sodium salt of pheparin at a concentration of
10 20ul per ml of blood is a
commonly used anticoagulant for chemistry, gas analysis and emergency tests. It
does not alter the size of the red cells, and it is recommended when it is
important to reduce to a minimum the chance of
lysis occurring after blood has
been drawn. It is thus the best anticoagulant for osmotic fragility test and is
suitable for immuniphenotyping (Dacie and Lewis; 2008).
However,
heparin is not suitable for blood counts because it often induces platelet and
leucocyte clumping (Van and Simmons; 1995, Salzamin and Rosenberg; 1980, and Hirsh and
Levine; 1992).
2.5.3 Disadvantages
of Heparin Anticoagulant,
1.
It
causes clumping of leucocytes
2.
It
interferes with the staining of leucocytes
3.
It
is the most expensive of anticoagulants
4.
Blood
will clot within 8-12 hours because clotting is only delayed but
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 (Schalms;
2006).
In
a study carried out by Mahony and Fergusm; 1992 they found that heparin and
other calcium - chelating anticoagulants have differing effects on
platelets but no consensus was reached.
Platelets
tends to Clump in samples collected into
low molecular weight heparin
(LMWH) precluding its use as an anticoagulant cited from (Am
Jvet Res, 2001) . This was contrary to
what (Nwafia et al 2003)
got in
their study, they concluded from
their study that there is
minimal loss of platelet in
blood stored in heparin when compared to other anticoagulants used in their study
and said heparin have
sparing effect on platelets (Nwafia
et al, 2003).
2.6.1 SODIUM CITRATE (NAC)
Sodium
citrate is the anticoagulant of choice for studies of platelet function and
morphology. The standard concentration is 1 part 3.8% solutions to parts of
blood, (Schalms; 2006) . For coagulation studies a volume of blood is
added to 1 volume of 109mmo1/l sodium citrate solution (Ingram and Hills 1976).
For erythrocyte sedimentation rate (ESR), 4 volumes of blood are added to 1 volume of the sodium citrate solution
(109mmol/L) and immediately mixed with it (international committee for standardization
in haematology: 1977). These are some of
the uses of sodium citrate.
2.6.2 Disadvantages of Sodium Citrate;
1.
It
interferes with many chemical tests
2.
It
has a tendency to shrink cells
3.
Because
of a 10% dilution of blood,
sodium citrate is generally not used for complete blood count (CBC)
(Schalms; 2006)
For
many years, citrate was the anticoagulant preferred by most investigators
undertaking platelet studies (Zelmanovic et al, 1998), this is mainly because sodium citrate causes
less spontaneous activation of platelets
in vitro but EDTA does (Golanski
et al; 1996). a high concentration of
sodium citrate in blood samples
decreases plasma concentration of ionized calcium resulting in reduced platelet aggregation and fibrinogen binding. (Am Jvet Res; 2001).
When blood is collected into citrate,
there is initially little or no change in platelet shape and volume, however in citrate, platelets adopt a spherical shape and volume, however in citrate, platelets
adopt a spherical shape (Macey et al,
1999) and as in EDTA,
swell progressively over a period
of 1 -
2 hours (3-10% increase in volume
by impedance procedures, depending on
the concentration of sodium citrate used
(Threatte
el al; 1984; Bath; 1993 and Jackson and Carter,
1993) . citrate-base
anticoagulants have been used for their determination of platelet indices
in the ADVIA 120 (Macey et al 1999 and Zelmanovic et al 1998)
they suggested that platelet sphering
may not be essential for the analysis. As cited from (Clin
Lab, 1990 ) analysis of the mean platelet volume (MPV)
showed significant lower values in the
citrated samples as compared to the same
examples in EDTA.
2.7.1 FLOURIDE OXALATE
Flouride
is an enzyme inhibitor. It prevents the
break down of glucose to lactic acid by enzyme action (gycolysis). Blood
collected into fluoride-oxalate can also be used for measuring protein, urea
and bilirubin but not for electrolytes or enzymes (Cheesbrough; 2005).
2.7.2 Mode
of Action:
Oxalate has a mechanism similar to that of
citrate,
it combine with calcium to form insoluble calcium oxalate.
2.7.3 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.
Blood examination is preferred within
one hour of collection as some cellular distortion becomes evident thereafter (Schalms;
2006).
Not much have been documented
about the effect of fluoride oxalate anticoagulant on platelets in stored blood.
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 MATERIALS
3.1.1 Sample population and size:
The blood of
some apparently healthy volunteers from Madonna University Teaching Hospital (MUTH)
will be used for the study. The ratio of male to female would not be significant.
3.1.2.
Ethical
Clearance:
The Ethical
Committee consent will be sought and also consent from candidate will be
sought.
3.1.3.
Other materials and reagents:
Ten mls syringe,
cotton wool, tourniquet, 70% alcohol, heparin anticoagulant (3iu per bottle), ethylenediaminetetra
acetic acid (EDTA) anticoagulant (3.5mg
per bottle), sodium citrate container (6.5lmg per container), fluoride oxalate
(49mg per container), cover slips, Neubeur counting chamber, lml pipette,
automatic micropipette, plain tubes, racks and microscope.
3.2
METHODS
3.2.1
Sampling
and sample size:
The samples will
be collected by just explained to the students what their blood is going to be
used for hence questionnaires are not necessary. Eight Milliliters (8mls) of
venous blood will be collected from each subject by venous puncture using
aseptic methods.
3.2.2
Procedure:
The left median
antecubital will be used for all subjects to maintain uniformly. The blood
samples will be immediately placed in different sterile container containing
specific anticoagulants to be used for the study. Each anticoagulant container
will contain 2mls of blood, four (4) types of anticoagulants will be used for
the study. The final concentration of each of the four anticoagulants will be
2mls of blood to – dispotassium ethlenediaminetetra – acetic acid KI2EDTA
3.5mg, heparin 3iu, fluoride oxalate 49mg, and sodium citrate (NaC)
6.51mg. the anticoagulatnted blood sample will be stored at 4oC from
where samples will be taken on a daily basis for four (4) consecutive days for
platelet count (Normal range 100-400x109/Liter) Dacie and Lewis 1994).
3.2.3 Method of Platelet Count:
The
method to be adopted will be that contained in Chessobrough 2006. The platelet
count will be done using the Improved Neubeur Counting Chamber and cover slips.
The blood will be diluted with platelets diluting fluid (1% ammonium oxalate)
in the ratio of 1:20 using the improved Neubeur Counting Chamber the platelets
per liter of blood will be calculated.
3.2.4 Principle of test:
blood is diluted
I in 20 in a filtered solution of ammonium oxalate rreagent which lyzes the red
cells. This method of platelet count is that of Cheesbrough 2006.
3.2.5 How result values will be analyzed;
Three readings
will be taken in each case; the mean values and the standard deviation will be
calculated and recorded. The values obtained will be subjected to “student
test” using 150x109/L as the standard and Day as the control or base
line. P. value (degree of freedom expressed in percentage) of <0.05 will be
taken as significant statistically.