Electrophoresis test instructions

Electrophoresis test instructions

Target
The purpose of this guideline is to describe the process of doing the work and the quality control of the electrophoresis device.
2- Scope of application
This guideline applies to hematology.
3- Responsibility for implementation
He is responsible for implementing this implementation method with the technical staff of the hematology departments and is responsible for quality assurance.
4- Definitions
does not have
5- Description of actions
Principles:
The separation of proteins is done by this system based on the difference in their electric charge in a buffer bed.
 
Solutions Materials and equipment required for hemoflobin electrophoresis:
1. Whole blood on EDTA anticoagulants such as CBC.
2. Hemolysing solution for hemolysis.
3- Tris Glycine buffer solution.
4- Amidoblack dye solution.
5- Color solution on.
6- Dehydration solution (pure methanol).
7- Cleaning solution.
8- Cellulose acetate gel.
9- Applicator and template.
 
Preparation of solutions:
1- Preparing hemolysis: We wash the whole blood 3 times with saline physiology and in the last step, with the help of a sample of λ1000, we remove the physiology so that Packed Cell is obtained. Mix for 2-3 minutes until a clear solution is obtained. Make sure that the L20 is removed from the bottom of the tube containing the Packed Cel and the extra blood is removed after the sampler tip is removed.
2- Preparing the Tris Glycine buffer solution: Add a packet of buffer powder to 1500ml of distilled water. This solution is stable for one month at refrigerator temperature.
3- Color solution: 100ml distilled water + 100ml pure methanol + 10ml glycal acetic acid
Note that acetic acid must be freshly added to the mixture of water and methanol each time.
Other solutions can be used ready-made.
 
Cellulose acetate gel preparation:
First, remove the gel coating and cut the top right corner of the gel. This is necessary to have the order of the patients. Then, immerse the gel in a container containing Tries-Glycine buffer for 15 minutes so that the surface of the gel holding the gel is facing down. Get.
Then we take the gel out of the container and put it between two smooth papers and with the low pressure of the fingers, we get the extra moisture evenly.
Place the gel on the stencil so that the retainer is facing down.
At this stage, L10 of the hemolysis solution is placed on the stencil platform and sampled by the applicator. Sampling 2-3 times on a filter paper. Sampling should be done at a distance of 2 cm from the top edge of the gel. During the sample. The tip should touch the paper for 5 seconds.
 
Electrophoresis tank preparation:
The Electro-Forz tank is a rectangular vessel with a middle blade and is thus divided into two parts. Each part has a movable wall. First, in each part of the Trais-Glycine buffer, we place a filter paper on the movable walls. This paper should be soaked in a buffer and then placed on the walls, which are in the form of an anion bridge.
Then we put the gel on the moving walls covered with filter paper. At this stage, the gel should be turned upside down, ie the holder should be placed upwards. Then we put two glass slides on the top and bottom of the gel and close the tank lid. We close it. We connect its systems to the power supply. We set the voltage to 250 volts and turn on the device so that the electrophoresis can be done for 30 to 40 minutes. In this voltage, a current of 3-4 amps is set. After performing the electrophoresis, we turn off the device. Open the tank door and remove the gel.
 
coloring :
Dip one foot into warm paraffin for 7 minutes, pausing between layers to allow them to dry. At this stage, the holder is placed at the bottom until the end of the divorce process.
 
Color:
Remove the excess color with the help of filter paper from the edge of the gel and remove the gel from the container containing the color solution. We do coloring in 3 steps. Each step is done with a new paint solution and each step is continued for 5 minutes.
 
Dehydration or dehydration:
Dip the gel into the container containing the methanol so that it completely covers the surface of the gel. This step is 1 minute.
 
Clearing:
Put the gel in a bowl containing Clearing solution and wait for 5 minutes.
 
Transparency at 70 ف C:
Place the gel vertically on a wooden rack and place it in the oven at 70 ف C for a minimum of 10 minutes until it is completely clear. Dry.
 
Bandwidth reading by densitometer:
Refer to the attached user manual.
 
Control and maintenance:
In order to control the quality of the performed samples, Hb A2 and Hb F can be measured in the following methods and we can compare them with the results of electrophoresis.
1- Check the Hb A2 with the column method (commercial kits).
2- Hb F should also be checked by chemical method (commercial kits).
 
Service and repair:
Refer to the attached user manual.
 
Safety requirements for working with the device:
Refer to the attached user manual.
 
Differences between hemoglobin and serum electrophoresis:
Example: In electrofarm serum, we do not have the steps of hemolysis preparation and the patient's serum is used directly.
Buffer: The buffer used in serum electrophoresis is Tris Ippurate. We bring a bottle of this solution to the volume of 1000 ml.
Color: The color of the patio is S.
Color: Acetic acid is 5%.
Voltage: The voltage is 160 volts and the current will be 5-5 amps.
The rest of the steps are the same as for Electrofor Hemoglobin.