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.
2.0 LITERATURE REVIEW
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)
c. Metastatic tumor
d. Aplastic anaemia
f. Cytotoxic drugs
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
5. Liver disease
6. Myeloprolifrative disorders
8. Cardiopulmonary bypass
9. Fish oils
a. Essential (primary) thrombocytosis
b. Reactive (secondary) thrombocytosis
d. Malignant disease
e. Acute and chronic inflammatory diseases
g. After splenectomy
h. Iron deficiency
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).
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)
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:
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
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)
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.
Prothrombin ----------------------------------- Thrombin
Fibrinogen(soluble) ----------------------------------- Fibrin (insoluble)
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
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.
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.
3.0 MATERIALS AND METHODS
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.
3.2.1 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.
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.