For hematology section, tests such as full blood count (FBC), erythrocyte sedimentation rate ESR), peripheral blood films (PBF), and retic count are done. there are also malaria tests and dengue panel.
For FBC. the tests are done in Celldyn Ruby analyzer which can be done under open or close module. the samples which arrive in EDTA tubes are firstly check for clots. Clotted blood are rejected and staffs will inform the staff nurse in charge. The test will also be cancelled. If the blood is not clotted, the volume of the blood that is above the cap can be done in close module while anything below the cap needs to be done under open module. The results of the tests that are within the range are aoto-validated and the samples can be filled. Some of the auto-validated samples are kept and the results are printed. the samples is then smeared, stained and observed under microscope. This is to ensure that the results between the analyzer and microscopy correlates. The results which are out of range are validated by the staffs.
Every morning and afternoon, the staffs will select one sample whicj the level of the blood is half of that of the tube. The staffs will do correlation whereby the sample is tested on the two analyzer on open module. The results are recorded to ensure that both analyzers give the same results (estimated).
Nyzah
0702888i
Saturday, September 26, 2009
Sunday, September 13, 2009
Protein precipitation
As for my MP, i have to analyze a drug in plasma sample. However, there are many impurites in plasma sample. In order to obtain as little interfering peaks when analyzing by HPLC, we have to remove the protein in the plasma.
General theory of using an organic solvent to precipitate out proteins in plasma:
- organic solvent reduces water activity
-thus water cannot dissolve the hydrophilic proteins
This is done by:
1. Adding acetonitrile to plasma sample in the ratio of 2:1
2. Shake well
3. Centrifuging at 6000rpm for 10 minutes
4. Supernatant is then spiked in with the drug for analysis
5. Vortex for 2 minutes
General theory of using an organic solvent to precipitate out proteins in plasma:
- organic solvent reduces water activity
-thus water cannot dissolve the hydrophilic proteins
This is done by:
1. Adding acetonitrile to plasma sample in the ratio of 2:1
2. Shake well
3. Centrifuging at 6000rpm for 10 minutes
4. Supernatant is then spiked in with the drug for analysis
5. Vortex for 2 minutes
_____________________EDIT________________________
Advantages of using acetonitrile for protein precipitation as compared to other method:
- it is a common solvent used in HPLC as mobile phase
- it has very low to no absorbance at wavelength more than 190nm
eriko
0700477C
eriko
0700477C
Serum protein electrophoresis
This week I am going to write about serum protein electrophoresis.
Serum protein electrophoresis is a laboratory technique for separating proteins in serum whereby the serum is placed onto an inert support medium such as agarose gel (on a plastic support sheet) and then exposed to an electrical field. Proteins in the serum will then migrate and separate based on their net surface charge and molecule size to either end of the electrodes at varying speed. This would then separate the serum protein components into five fractions, in decreasing order of migration speed - albumin, alpha-1 globulins, alpha-2 globulins, beta globulins, and gamma globulins. Once migration process has completed, the protein fractions on the gel can be fixed and stained for visual interpretation.
Scanning of gel can also be done by the densitometer for quantitation in order to provide graphical data on the total and relative amounts of the various proteins.
The factors that may affect the mobility of proteins include size of the protein, charge of the protein (negatively/positively charged), strength of the electrical field and properties of the support medium.
Serum protein electrophoresis (SPE) is used to screen the presence of protein abnormalities (especially M-band) in the serum. SPE also aids in the detection, diagnosis, and monitoring of pathophysiologic conditions associated with these abnormal proteins such as multiple myeloma.
Method:
1. 10ul of samples are pipetted into the wells of the applicator accordingly (1st well is always used for Control).
2. The applicator is then placed in wet storage chamber (teeth facing up) for 3 minutes.
3. Buffered strips are attached to the electrode.
4. The surface of SPE gel is blotted with fine filter paper to remove excess liquid.
5. 200ul of D.I water is applied onto the migration plate (approximately 1/3 from the bottom).
6. The gel is then slowly placed onto the migration plate (ensure that the water is spread across the whole width of the gel & ensure no air bubble is formed).
7. The electrode and the applicator carrier are then lowered down.
8. The applicator is retrieved from the wet storage chamber after 3 minutes.
9. The plastic protective cover of the applicator is then removed carefully.
10. The applicator is placed onto the applicator carrier at the respective position.
11. The lid of the migration module is closed and the required migration program is selected (15/30 PROTEIN [E]).
12. Electrophoresis is conducted.
13. After process is completed, the applicator & buffered strips are removed and discarded.
14. The gel is removed, placed into the gel holder and then slotted into the staining chamber (stained with amido black).
15. Once done, the dry stained gel is removed from the gel holder and scanned.
If there is presence of M-band (distinct band at the gamma region), it will be noted and quantitated. E.g.: Presence of M-band (15g/L).
If the is absence of M-band, it will be noted as well but there is no quantitation.
E.g.: M-band is absence.
If there is presence of a polyclonal protein (broad band at the gamma region), it will also be noted and quantitated.
E.g.: Presence of polyclonal protein (45g/L).
Immunofixation can be done to identify the particular subtype of immunoglobulin heavy and light chains that make up the M-band for classification purposes.
Natasha. 0703883I. :]
Serum protein electrophoresis is a laboratory technique for separating proteins in serum whereby the serum is placed onto an inert support medium such as agarose gel (on a plastic support sheet) and then exposed to an electrical field. Proteins in the serum will then migrate and separate based on their net surface charge and molecule size to either end of the electrodes at varying speed. This would then separate the serum protein components into five fractions, in decreasing order of migration speed - albumin, alpha-1 globulins, alpha-2 globulins, beta globulins, and gamma globulins. Once migration process has completed, the protein fractions on the gel can be fixed and stained for visual interpretation.
Scanning of gel can also be done by the densitometer for quantitation in order to provide graphical data on the total and relative amounts of the various proteins.
The factors that may affect the mobility of proteins include size of the protein, charge of the protein (negatively/positively charged), strength of the electrical field and properties of the support medium.
Serum protein electrophoresis (SPE) is used to screen the presence of protein abnormalities (especially M-band) in the serum. SPE also aids in the detection, diagnosis, and monitoring of pathophysiologic conditions associated with these abnormal proteins such as multiple myeloma.
Method:
1. 10ul of samples are pipetted into the wells of the applicator accordingly (1st well is always used for Control).
2. The applicator is then placed in wet storage chamber (teeth facing up) for 3 minutes.
3. Buffered strips are attached to the electrode.
4. The surface of SPE gel is blotted with fine filter paper to remove excess liquid.
5. 200ul of D.I water is applied onto the migration plate (approximately 1/3 from the bottom).
6. The gel is then slowly placed onto the migration plate (ensure that the water is spread across the whole width of the gel & ensure no air bubble is formed).
7. The electrode and the applicator carrier are then lowered down.
8. The applicator is retrieved from the wet storage chamber after 3 minutes.
9. The plastic protective cover of the applicator is then removed carefully.
10. The applicator is placed onto the applicator carrier at the respective position.
11. The lid of the migration module is closed and the required migration program is selected (15/30 PROTEIN [E]).
12. Electrophoresis is conducted.
13. After process is completed, the applicator & buffered strips are removed and discarded.
14. The gel is removed, placed into the gel holder and then slotted into the staining chamber (stained with amido black).
15. Once done, the dry stained gel is removed from the gel holder and scanned.
If there is presence of M-band (distinct band at the gamma region), it will be noted and quantitated. E.g.: Presence of M-band (15g/L).
If the is absence of M-band, it will be noted as well but there is no quantitation.
E.g.: M-band is absence.
If there is presence of a polyclonal protein (broad band at the gamma region), it will also be noted and quantitated.
E.g.: Presence of polyclonal protein (45g/L).
Immunofixation can be done to identify the particular subtype of immunoglobulin heavy and light chains that make up the M-band for classification purposes.
Natasha. 0703883I. :]
Thursday, September 3, 2009
Blood gas
Hey. Its me again. I will be talking about blood gas, the station where I was attached to. I have learnt to use the Roche cobas b 221 analyzer to test for blood gases. Actually, there are three modules in this analyzer ( ISE, Co-oximetry and BG module), but I am going to touch on the BG module only. This BG module helps to determine the pH, pO2 and pCO2.
Blood gas (BG) tests are important in evaluating the respiratory as well as the metabolic conditions of the patient. These tests help to detect whether the lungs are effective in delivering the o2 and eliminate co2 from the body. In addition, the tests help to detect abnormal blood pH that influences the metabolic conditions.
These analyzers employs the assay of potentiometry and amperometry. pH and pCO2 are measured by potentiometry whereas pO2 is measured by amperometry. These assays measure the potential generated by the electrodes so as to determine the concentration of the analytes of interest.
Specimens for BG tests are arterial blood in heparinized syringes. These syringes are transported along with ice so as to prevent clotting of blood.
To perform BG test:
1. The syringe containing blood is taken out from the plastic biohazard packet.
2. Rub the syringe with both hands so to homogenize the blood sample.
3. A clot catcher is then attached to the syringe.
4. Dispense one or two drops of blood to make sure that there is no clot in the blood.
5. The syringe is then injected into the analyzer and blood sample is aspirated.
6. Results are then obtained.
To look at how the BG analyzer looks like, you can go to this website: http://rochediagnostics.ca/lab/pct/gas/
Siti Shahimah Samat
0702717J
Blood gas (BG) tests are important in evaluating the respiratory as well as the metabolic conditions of the patient. These tests help to detect whether the lungs are effective in delivering the o2 and eliminate co2 from the body. In addition, the tests help to detect abnormal blood pH that influences the metabolic conditions.
These analyzers employs the assay of potentiometry and amperometry. pH and pCO2 are measured by potentiometry whereas pO2 is measured by amperometry. These assays measure the potential generated by the electrodes so as to determine the concentration of the analytes of interest.
Specimens for BG tests are arterial blood in heparinized syringes. These syringes are transported along with ice so as to prevent clotting of blood.
To perform BG test:
1. The syringe containing blood is taken out from the plastic biohazard packet.
2. Rub the syringe with both hands so to homogenize the blood sample.
3. A clot catcher is then attached to the syringe.
4. Dispense one or two drops of blood to make sure that there is no clot in the blood.
5. The syringe is then injected into the analyzer and blood sample is aspirated.
6. Results are then obtained.
To look at how the BG analyzer looks like, you can go to this website: http://rochediagnostics.ca/lab/pct/gas/
Siti Shahimah Samat
0702717J
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