Assignment 2

I have handed in these two assignments as the start of the project .

Cytometric measurement of Annexin 5 by flowcytometry
Supervised by : Dr J Vickers.
Saman Yazdani

1- Describe the hypothesis to be tested in your project
This experiment is aimed for a basic understanding of the system. It is a quantitative measurement of Annexin5 (a family of calcium-binding proteins).
There are two hypotheses on the table:
-Activation of platelets increases A5 binding to platelets.
Or
-Activation of platelets has no effect on A5binding to platelets surface. (Conference abstract)

First, the amount of A5 on the surface of platelets is measured by flow cytometry (as a base line).A5 is bound to phospholipids inside the platelets include: phosphatidyl inositol 1, phostatidyl serine and ethanol amine (conference abstract).
This measurement is kept as a base line to be compared. Then some substances include: ADP and A23187( Calcimycin, Calcium Ionophore, Antibiotic A23187 ,Calcium Ionophore A23187), (both as activator) and PGE1 (prostaglandin E1-as inhibitor)
Are separately added to the platelets (conference abstract).
If the indicated substances (ADP, A23187 and PGE1) had any effect on the presence and exhibition of those phospholipids, then it can be displayed (it is assumed). The fact is the indicated phospholipids would bind to A5 and then they expo and present Anx5 to the surface of the platelets (it is expected). In flowcytometry a material (A5 binding) would bind to A5 out side the platelets and then A5 binding is measured, it does indirectly shows the amount of A5 out side the platelets.
As stated above, the hypothesis is base on the fact that, the ADP, A23187 & PGE1 (as activator and inhibitors) could cause a change in the manifestation the Anx5 on the surface of the platelets.

2. Briefly outline the type of experimental design you intend to employ, include appropriate control strategies.

Experimental Design.
Materials & Method:
Blood samples (up to 5) .ADP and A23187 (activator) PGE1 (inhibitor), Flow cytometry machine) either 2 or 4 laser channels) and general haematology laboratory equipments.
First of all, the blood is taken from donors (not necessarily patient).It would be centrifuged and the plasma is separated and then platelet is reached from the Buffy coat layer. Then the sample will be washed to have more pure platelets.
In order to have a base line for experiment, before doing the rest of process, the level of A5 is measured by flowcytometry .In this experiment the effect of some substances is measured in order to realise the activation or inhibition of platelets.
As mentioned before this experiment is aimed to either prove or disapprove the indicated hypothesis. Further explanations are about the assumptions coming part5.However it is essential to know that why this experiment is done and what should be focused in all part of the experiment.
It is required to remember that the Anx5 cellular protein which binds with some types of phospholipids (mainly phospholipids serine) and then appears on the surface side of the platelets. (Conference abstract).
Like every other experiment, the first time is a negative control, means measuring with out adding any substance and just measuring the Anx5 as a base line.

3. What type of statistical analysis will be used to analyse your data?
Using SPSS software or simple excel spread sheets could be used to evaluate and analyse the data. The main output of this experiment is some figures by flowcytometry (and graphs) which indicate the amount of A5 in different cases include before and after adding those activator or inhibitors.
A schematic diagram like follow is expected.
Blue: A5 on the surface of platelet before adding ADP
Red: A5 on the surface of platelet before adding A23187
Yellow: A5 on the surface of platelet before adding PGE1
This could be extend with different substances and materials as well .Up to know what ever is reached is this fact that those indicated substances have effect( either increase or decrease) on the level of A5 presenting on the surface of platelets (conference abstract). Although more specific and details are always expected over any experiment.
As stated above, by changing the amount of those indicated substances their effect will be changed as well, hence in t2 the more indicated substances would be added, t3 ant t4 and t5 as well.

4- Outline the number and type of experimental observations that are required to carry out this type of analysis.
As the out put results of flow cytometry is an average of a lot of measurements of doing the test, Longobardi Givan (2001); therefore even if in each of those conditions, there are measured once, should not cause technical errors.
Up to 10 donors blood could give satisfactory data to be analysed.


5-In the majority of experimental designs it is necessary to make a number of assumptions. Outline some of the assumptions you have made when designing experiments for your project. Give reasons for these decisions

ADP-induced platelet aggregation when added in vitro and increase aggregation, Carlo patrono et al 2001, means increasing the activity of platelets. In the opposite side PGE1 and A23187 decrease the activity of platelets (conference abstract).Another fact which has already been mention is the affinity of phosphatydil serine to bind with Anx5 and presenting on the surface of the platelets. Consequently the main assumption could be finding a logical link between the activation of platelets (or inhibition) and level of presenting of Anx5 on the surface of the platelets.
One of the assumptions could be null and void, means there is not any relation ship between these parameters.
Another assumption is, the more the platelets are activated, the more Anx5 they would expose. The third assumption is vice verse the second one, the more the platelets are activated the less Anx5 they would expose.
It is assumed that the assumption would be rejected, because lots of other researchers earlier have been proved that the phosphatydil molecules inside the platelet surface would be undergone due to the activity of the platelets, E.E Nishizawa et el (2008).If so, the level of manifesting the anx5 must logically change on the surface of the platelets (either increase or decrease). Base on those three indicated assumptions the experiment and following the hypothesis would be carried on.
There are other physiological activators which have similar effect on the platelets: thrombin, collagen, thromboxane A2, adrenaline, platelet activating factor, 5-HT, vasopressin, George, JG. (2000) and Dahlbäck, B (2000). Blood coagulation. It is assumed to use other substances over the experiment as well.



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