THE PRESERVATION EFFECT OF DIFFERENT ANTICOAGULANTS ON PLATELETS IN STORED BLOOD



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.
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