RESULTS

Clotting Time (sec)

1000 800 600 400 200 0

differ between each blood type. An individuals blood contains antibodies to recognize foreign blood types, but none for the individuals own blood type. Agglutination occurs when these antibodies recognize foreign antigens on red cells of a different blood type, and bind them together, forming a clump of red blood cells (ONeil 1999).
Condition of Test Tube

Contact with a surface Coagulation time was observed to be longer in a test tube lined with paraffin wax than in an empty glass test tube. These results coincide with long established facts found in literature. The breakdown of platelets releases thromboplastin, one of the regulators of the conversion of prothrombin to thrombin, on which the rate of coagulation depends. Thromboplastin in the circulating blood is often neutralized by antithromboplastic substances in blood. The coagulability and fluidity of blood is determined by the concentrations of these substances in circulating blood (Donovan et al. 1949). Glass has been known to be an activator of blood plasma (Tocantins et al. 1950). One of the hypothesis corresponding to the result of the experiment attributes the higher rate of coagulation in a glass surface to the increased amount of thromboplastin liberated in a glass container, an ability which is significantly reduced when paraffin coats the glass container (Lozner et al. 1942). Another hypothesis considers not the lysis of the cell platelets, but the reactions undergone by the components of cell-free plasma when in a glass surface. The higher coagulability of blood was attributed to either the activation of thromboplastin

Figure 1. Clotting times of different test tubes under different conditions. DISCUSSION Blood coagulation in the body occurs when there are damaged blood vessels. The process prevents the severe physiological effects that could result as a consequence of uncontrolled blood loss from the damaged vessels. It is a cascade of reactions which occurs through either an intrinsic pathway or an extrinsic pathway, and ultimately leads to the formation of fibrin from fibrinogen. Following this is the formation aggregates of blood components and fibrin. Damages in the blood vessels causes platelets in the area to release ADP and other compounds, which makes them sticky and attracts additional blood platelets. These blood platelets have fibrin receptors to which fibrin can bind, and attracts even more platelets and fibrin to form a clot. The clot serves to plug the damaged area until repairs can be completed and the wound healed (Randall et al. 2002). Agglutination is the clustering together of red blood cells in the liquid plasma as a results of antigen-antibody interactions. Various individuals, whether within, and especially between species, have different blood types. On the surface of red blood cells are antigens which serve as the biological signature of an individuals blood type. These antigens are specific to and

already present in blood, or the inactivation of antithromboplastin (Donovan et al. 1949). This was explained by the possibility of electrostatic adsorption of the positively charged substances in cell-free plasma, or in blood, by the negatively charged glass surface. This corresponds to the results as it was also shown that the charge of a glass surface in contact with water is greater than that of a paraffin surface (Lozner et al. 1942). Results from the experiment indicate that blood clots the faster in a test tube with cotton fibers at the bottom and sides of the tube. This agrees with results obtained from a former study which used asbestos fibers instead of cotton fibers (Tocantins et al. 1951). The presence of cotton fibers in addition to some exposed glass surface further agitates and activates the coagulation of blood. Coagulation of blood when it was continuously stirred was shown to take longer than the normal clotting time of when it was left to stand in an empty test tube. Agitation of blood caused by vigorous stirring causes fibrin to adhere to the stirring apparatus, thereby preventing coagulation (Fa 2009). Anticoagulants Anticoagulants prevent coagulation or clotting of blood, but without altering the composition of the blood plasma. Sodium citrate achieves this by chelating calcium ions in the blood and rendering them inactive. This essentially removes calcium, which is required in the coagulation process, from the blood, making it unable to clot. Sodium citrate is the preferred anticoagulant due to its having the best ability in maintaining the integrity of

the cell. Specifically, it is able to preserve the components, biochemical constituents, and morphology of the cell (Pal et al. 2005). Sodium oxalate functions by binding with calcium in the blood and forming the insoluble calcium oxalate. It uses the same mechanism as sodium citrate in that calcium, which is needed in the coagulation pathway, is made inaccessible, such that the blood cannot clot. Sodium oxalate can cause the shrinkage of red cells, which alters cell morphology. To be used in coagulation studies, sodium oxalate must be combined with ammonium oxalate, which balances out the action of sodium oxalate by increasing the volume of the cell. Together, they form double oxalate, which is able to preserve cell morphology (Pal et al. 2005). The anticoagulant heparin is naturally found in the body, theoretically making it the best anticoagulant since it would not cause any unnatural changes in the cell. It prevents coagulation by inhibiting thrombin, and consequently the conversion of fibrinogen to fibrin, which is essential in making platelet clots (Pal et al. 2005). This part of the experiment allowed the experimenters to observe the effects of anticoagulants on blood. Theoretically, upon addition of the anticoagulants discussed above, no clotting of blood should be observed. The next step, which was the addition of calcium chloride, and consequently, of calcium, into the solution, resulted in blood coagulation. The calcium added served to initiate and continue the coagulation pathway which was inhibited by the anticoagulants binding with calcium in the blood. Effect of temperature on clotting time

The results from the experiment show that blood clots faster at higher than at lower temperatures. Individuals with core body temperatures below 35C are generally defined to have hypothermia. Though actual temperatures showing occurrence of hypothermia vary between individuals. The results obtained correspond to those in literature, which indicate that low temperatures have inhibitory effects on procoagulant enzymes, and cause platelet disfunction, as indicated by reduced platelet aggregation. Whether individually or collectively, these will have an impact on blood coagulation (Thorsen et al. 2011). Specifically, it was indicated that hypothermia affects the thrombin-generation and fibrinogen metabolism of the blood coagulation pathway. Particularly, the inhibition of fibrinogen synthesis, as well as the conversion of available fibrinogen to fibrin, was reported (Martini 2009). In contrast, hyperthermic temperatures, which are higher than normal, were observed to show a decrease, albeit a minimal one, in prothrombin time, which is the opposite of what was seen in hypothermic temperatures (Rohrer et al. 1992). Accordingly, results of the experiment showed a shorter coagulation time in higher temperatures as compared to lower temperatures. At very high temperatures, blood agglutinates into clumps and barely flow (Addis 1908), which might lead to grave consequences for the patient. Taking cold drinks after a tooth extraction helps ensure that clotting occurs after an optimal time. REFERENCES

Addis, T. (1908). The coagulation of blood in man. Quarterly Journal of Experimental Physiology 1: 305-334. Donovan, T.J., Zimmermann, B. (1949). The effect of artificial surfaces on blood coagulabiity, with special reference to polyethylene. Blood 4 (12): 1310-1316. Fa, J.T. (2009). Lecture 6&7: Blood Utilization. Retrieved from http://www.as.nchu.edu.tw/lab/5A/%E5%8B %95%E7%89%A9%E5%89%AF%E7%94 %A2%E7%89%A9%E5%88%A9%E7%94 %A8%E8%AC%9B%E7%BE%A9/%E5%8 B%95%E7%89%A9%E5%89%AF%E7%94 %A2%E7%89%A9%E5%88%A9%E7%94 %A8%20%20Lecture%206%207%20Blood %20%28handout%29.pdf. Lozner, E.L., Taylor, F.H.L., & MacDonald, H. (1942). The effect of foreign surfaces on blood coagulation. The Journal of Clinical Investigation 21 (2): 241-245. Martini, W.Z. (2009). Coagulopathy by hypothermia and acidosis: mechanisms of thrombin generation and fibrinogen availability. Journal of Trauma 67 (1): 202208. ONeil, D. (1999). Blood Components. Retrieved from http://anthro.palomar.edu/blood/blood_comp onents.htm. Pal G.K., & Pal, P. (2005). Textbook of Practical Physiology, Second Edition. Chennai, India: Orient Longman Private Ltd. Randall, D., Burggren, W., & French, K. (2002). Eckert Animal Physiology, 5th edition. New York: W.H. Freeman and Company.

Rohrer, M.J., & Natale, A.M. (1992). Effect of hypothermiaon the coagulation cascade. Critical Care Medicine 20 (10): 1402-1405. Tocantins, L.M., Carroll, R.T., & Holburn, R.H. (1951). The clot accelerating effect of dilution on blood and plasma. Relation to the mechanism of Coagulation of normal and hemophilic blood. Blood 6 (8): 720-739. Thorsen, K., Ringdal, K.G., Strand, K., Soreide, E., Hagemo, J., & Soreide, K. (2011). Clinical and cellular effects of hypothermia, acidosis ad coagulopathy in major injury. British Journal of Surgery 98 (7): 894-907. APPENDIX Table 1. Raw data on coagulation time. Test Tube Conditions Clotting Time (sec) Empty 126 Lined with paraffin 232 With cotton fiber 63 In an ice bath 317 In a hot water bath 80 Continuously stirred 210 With 5 drops of 0.1% 80 heparin and 5 drops of calcium chloride With a small amount of 898 sodium oxalate and 5 drops of calcium chloride With a small amount of 425 sodium citrate and 5 drops of calcium chloride