sorry for the super late post!
Since the column of the HPLC is very expensive, we can have to do things to maximize the length of its life:)
Here are the things to do:
- during shutdown, always flush the column with ultrapure water to remove all the salts present from the buffer. If this is not done, salts will precipitate and cause clogging of column.
- after which, flush the column with methanol to make the environment unsuitable for microbial growth.
- the ultrapure water used should be changes every 3 days. Since when the ultrapure water is exposed to the air, the airborne bacteria/ bacterial nutrients will enter and multiply in the water. If this contaminated water is used to run experiment, the bacteria will enter and cause column clogging.
- sample injected into the column should always filter will a syringe filter to remove any contaminates.
- reagents used in HPLC should be of HPLC grade
eriko wong
0700477C
Thursday, November 12, 2009
Sunday, October 25, 2009
Human Pancreatic Polypeptide (PP)
Human pancreatic polypeptide (PP) is a polypeptide hormone secreted by the pancreatic polypeptide (PP) cells of the islets of Langerhans in the endocrine portion of the pancreas. It consists of a chain of 36 amino acids.
The function of PP is to self regulate the pancreas secretion activities (endocrine and exocrine) and it also has effects on hepatic glycogen levels and gastrointestinal secretions. It is triggered in humans by protein-rich meals, fasting, exercise, and acute hypoglycemia and is inhibited by somatostatin and intravenous glucose.
The purpose of this test is for the non-radioactive quantitation of human pancreatic polypeptide in human serum and plasma. The principle of this test is by sandwich enzyme-linked immunosorbant assay (ELISA) based sequentially on:
1. Capture of human PP molecules from samples to the wells of a microtiter plate coated by a pre-titered amount of anti-PP antibody.
2. Wash away of unbound materials from samples.
3. Binding of second biotinylated anti-PP polyclonal Ab to the captured molecules.
4. Wash away of unbound materials from samples.
5. Conjugation of horseradish peroxidase to the immobilized biotinylated Ab.
6. Wash away of free enzyme conjugates.
7. Quantification of immobilized Ab-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of substrate 3, 3’, 5, 5’-tetramethylbenzidine.
The enzyme activity is measured spectrophotometrically at 450nm with 630nm background correction. Increase in absorbency is directly proportional to the amount of captured human PP in the unknown sample.
A standard curve is generated by plotting the absorbance versus the respective reference standards of known concentrations of human PP. The concentration of human PP in the samples is then determined directly from this standard curve.
Range of this assay: 12.3pg/mL to 3000pg/mL human PP (50uL sample size)
Natasha.
0703883I
The function of PP is to self regulate the pancreas secretion activities (endocrine and exocrine) and it also has effects on hepatic glycogen levels and gastrointestinal secretions. It is triggered in humans by protein-rich meals, fasting, exercise, and acute hypoglycemia and is inhibited by somatostatin and intravenous glucose.
The purpose of this test is for the non-radioactive quantitation of human pancreatic polypeptide in human serum and plasma. The principle of this test is by sandwich enzyme-linked immunosorbant assay (ELISA) based sequentially on:
1. Capture of human PP molecules from samples to the wells of a microtiter plate coated by a pre-titered amount of anti-PP antibody.
2. Wash away of unbound materials from samples.
3. Binding of second biotinylated anti-PP polyclonal Ab to the captured molecules.
4. Wash away of unbound materials from samples.
5. Conjugation of horseradish peroxidase to the immobilized biotinylated Ab.
6. Wash away of free enzyme conjugates.
7. Quantification of immobilized Ab-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of substrate 3, 3’, 5, 5’-tetramethylbenzidine.
The enzyme activity is measured spectrophotometrically at 450nm with 630nm background correction. Increase in absorbency is directly proportional to the amount of captured human PP in the unknown sample.
A standard curve is generated by plotting the absorbance versus the respective reference standards of known concentrations of human PP. The concentration of human PP in the samples is then determined directly from this standard curve.
Range of this assay: 12.3pg/mL to 3000pg/mL human PP (50uL sample size)
Natasha.
0703883I
Friday, October 16, 2009
Immunofixation Electrophoresis
Immunofixation Electrophoresis ( IFE) is a method to detect monoclonal band (M-band). The M-band consist of monoclonal proteins (M-proteins), which are abnormal proteins produce by continous proliferation of plasma cells. The purpose of implementing IFE is to investigate the M-band, that is to know which particular immunoglobulin heavy chains, gamma(IgG), alpha(IgA) and mu(IgM) and light chains (kappa or lambda) that make up the M-proteins. In the laboratory that I am attached to, usually the test that require IFE is myeloma test. In addition, IFE also provides urine protein electrophoresis, which is useful in the study of renal excretion of proteins.
Steps involve:
1. Proteins in the samples (urine and serum) are separated by electrophoresis on agarose gel.
2. Immunofixation of electrophoresed proteins includes, the monospecific antisera diffuse into
agarose gel and bind to the antigens (proteins), if present. Thus, resulting in the precipitation
of the antigens.
3. The unprecipitated antigens are removed from the gel by blotting and washing.
4. The precipitated antigens are stained and can be evaluated visually. M-band is seen when one
of the fractions is dark in colour.
IFE only provides qualitative determination of results.
Siti Shahimah Bte Samat
0702717J
Steps involve:
1. Proteins in the samples (urine and serum) are separated by electrophoresis on agarose gel.
2. Immunofixation of electrophoresed proteins includes, the monospecific antisera diffuse into
agarose gel and bind to the antigens (proteins), if present. Thus, resulting in the precipitation
of the antigens.
3. The unprecipitated antigens are removed from the gel by blotting and washing.
4. The precipitated antigens are stained and can be evaluated visually. M-band is seen when one
of the fractions is dark in colour.
IFE only provides qualitative determination of results.
Siti Shahimah Bte Samat
0702717J
Sunday, October 11, 2009
blood collection
In this post, I am going to write about a tutorial given by one of our colleague. All of us have to attend this tutorials unless we are busy at our work stations. These tutorials are given at least once every 2 weeks.
Topic for today: blood collection
The Clinical and Laboratory Standards Institudes (previously known as NCCLS) sets guidelines of procedure to ensure that quality specimens are collected for laboratory testing. Upon collection, all plastic tubes must be gently inverted 5-10 times to provide thorough mixing of the additives. Also, it is important to note that shaking of blood tubes may cause hemolysis of the blood.
Order of draw of blood collection tubes procedure:
1st) Blood culture bottles
Culture bottles should always be drawn first. this is to reduce contamination of the blood specimen which might cause the analyzer to detect a positive bottle. thus, leading to a wrong treatment for the patient.
2nd) Citrate (light blue) tube
blood to be taken as soon as possible before formation of microclots. also, this tubes must be drawn before the collection of serum tubes (red and yellow) to prevent comtamination with clot activator which are present in them.
3rd) Serum (red/yellow) tube
4th) Heparin tube
5th) EDTA (purple) tube
they contain potassium hence, it is not advisable to collect blood into this tube before the serum tubes (chemistry tests). this is because serum tubes might be contaminated with potassium causing inaccurate results. for eg, K+ level will be falsely high and Ca+/Mg+ levels will be falsely low.
6th) Fluoride/Oxalate (grey) tube
oxalate pesent in this tube can be obstructive to cell membrane. thus, it is not advisable to collect blood into this tube before both serum tubes (chemistry tests) and EDTA tubes (haematology tests). if both tubes are contaminated with oxalate, it will also lead to inaccurate results.
wendy/0701158h
answers (there's some prob i couldnt post a comment)
1. nyzah,
heparin tube is the green top tube. most chemistry tests can be done using this tube. for eg, renal panel, lipid panel etc.
2. shemeema,
the citrated tube is used for running the coagulation panel (PTINR/aPTT). hence, the presence of clots will affect the result.
thanks,
wendy.
Topic for today: blood collection
The Clinical and Laboratory Standards Institudes (previously known as NCCLS) sets guidelines of procedure to ensure that quality specimens are collected for laboratory testing. Upon collection, all plastic tubes must be gently inverted 5-10 times to provide thorough mixing of the additives. Also, it is important to note that shaking of blood tubes may cause hemolysis of the blood.
Order of draw of blood collection tubes procedure:
1st) Blood culture bottles
Culture bottles should always be drawn first. this is to reduce contamination of the blood specimen which might cause the analyzer to detect a positive bottle. thus, leading to a wrong treatment for the patient.
2nd) Citrate (light blue) tube
blood to be taken as soon as possible before formation of microclots. also, this tubes must be drawn before the collection of serum tubes (red and yellow) to prevent comtamination with clot activator which are present in them.
3rd) Serum (red/yellow) tube
4th) Heparin tube
5th) EDTA (purple) tube
they contain potassium hence, it is not advisable to collect blood into this tube before the serum tubes (chemistry tests). this is because serum tubes might be contaminated with potassium causing inaccurate results. for eg, K+ level will be falsely high and Ca+/Mg+ levels will be falsely low.
6th) Fluoride/Oxalate (grey) tube
oxalate pesent in this tube can be obstructive to cell membrane. thus, it is not advisable to collect blood into this tube before both serum tubes (chemistry tests) and EDTA tubes (haematology tests). if both tubes are contaminated with oxalate, it will also lead to inaccurate results.
wendy/0701158h
answers (there's some prob i couldnt post a comment)
1. nyzah,
heparin tube is the green top tube. most chemistry tests can be done using this tube. for eg, renal panel, lipid panel etc.
2. shemeema,
the citrated tube is used for running the coagulation panel (PTINR/aPTT). hence, the presence of clots will affect the result.
thanks,
wendy.
Saturday, September 26, 2009
Hematology
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
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
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
Sunday, August 30, 2009
microo
hello pple.
it's my turn again.
so i'm going to write about a test done in micro lab.
Wampole™ TOX A/B QUIK CHEK®
this test is a rapid immunoassay for detecting Clostridium difficile toxins A and B.
specimen: fecal specimen
suspected disease: C. difficile disease
(before proceeding with this test, we have to bring all reagents and devices to room temperature to ensure accurate results.)
Required Reagents:
- Diluent
- Congugate
- Wash Buffer
- Substrate
ASSAY PROTOCOL
1. Dilute specimen
using a small glass test tube, add 25μL of specimen to 500μL Diluent + 1 drop of Congugate. mix well.
2. Add sample
transfer 400μL of diluted sample-conjugate mixture to sample well. incubate for 15minutes.
3. Wash Membrane Device
add 300μL Wash Buffer to Reaction window
4. Add Substrate reagent
2 drops to Reaction window. incubate and read results 10 minutes later.
5. Interpretation
read from Reaction window.
two lines -> positive
1 control line -> negative
* invalid test if there is no control line. repeat test pls.
* a positive result may be interpreted at any time during the 10minute interpretation but cannot be ruled as neg or invalid until after 10 minutes.
wendy ong/0701158h
it's my turn again.
so i'm going to write about a test done in micro lab.
Wampole™ TOX A/B QUIK CHEK®
this test is a rapid immunoassay for detecting Clostridium difficile toxins A and B.
specimen: fecal specimen
suspected disease: C. difficile disease
(before proceeding with this test, we have to bring all reagents and devices to room temperature to ensure accurate results.)
Required Reagents:
- Diluent
- Congugate
- Wash Buffer
- Substrate
ASSAY PROTOCOL
1. Dilute specimen
using a small glass test tube, add 25μL of specimen to 500μL Diluent + 1 drop of Congugate. mix well.
2. Add sample
transfer 400μL of diluted sample-conjugate mixture to sample well. incubate for 15minutes.
3. Wash Membrane Device
add 300μL Wash Buffer to Reaction window
4. Add Substrate reagent
2 drops to Reaction window. incubate and read results 10 minutes later.
5. Interpretation
read from Reaction window.
two lines -> positive
1 control line -> negative
* invalid test if there is no control line. repeat test pls.
* a positive result may be interpreted at any time during the 10minute interpretation but cannot be ruled as neg or invalid until after 10 minutes.
wendy ong/0701158h
Sunday, August 23, 2009
Week Nine
Section: Special Chemistry. (COBAS)
Hi, I shall talk about this section whereby the analyzer used is called COBAS c system. The tests usually done here are Acute Myocardial Infarction (AMI) Panel; CKMB mass and Troponin T. Other tests include hormones and HbA1c.
I shall elaborate more on HbA1c. HbA1c is one of the glycated hemoglobins. It is formed in a two steps non-enzymatic reactions of glucose with the N-terminal amino group of the -chain of the normal adult Hb (HbA). The amount of HbA converted to HbA1c is relative to that of the average concentration of glucose in the blood thus, HbA1c is suitable to monitor long term blood glucose control in individuals with diabetes mellitus.
This method uses a detergent in the hemolyzing reagent to eliminate interface from leukocytes. All glycated hemoglobins at the -chain N-terminus and which have antibody-recognizable regions identical to that of HbA1c are measured.
The sample which comes in EDTA tube is to check for clotted blood. If there is no clotted blood, the presence of bubbles is removed to avoid sampling error. The sample is the placed in a special HbA1c rack and placed into the machine. The machine will detect the barcode and do the test ordered. The sample (glycohemoglobin in HbA1c) is reacted with anti-HbA1c antibody to form antigen-antibody complexes. Then, polyhaptens are added to start the reaction by reacting with the excess anti-HbA1c antibodies to form an insoluble antibody-polyhapten complex which is the measure turbidimetrically. The final result is expressed as percent HbA1c. The results are then transmitted to the LIS system and the staffs allocated there will validate the results.
nyzah
Hi, I shall talk about this section whereby the analyzer used is called COBAS c system. The tests usually done here are Acute Myocardial Infarction (AMI) Panel; CKMB mass and Troponin T. Other tests include hormones and HbA1c.
I shall elaborate more on HbA1c. HbA1c is one of the glycated hemoglobins. It is formed in a two steps non-enzymatic reactions of glucose with the N-terminal amino group of the -chain of the normal adult Hb (HbA). The amount of HbA converted to HbA1c is relative to that of the average concentration of glucose in the blood thus, HbA1c is suitable to monitor long term blood glucose control in individuals with diabetes mellitus.
This method uses a detergent in the hemolyzing reagent to eliminate interface from leukocytes. All glycated hemoglobins at the -chain N-terminus and which have antibody-recognizable regions identical to that of HbA1c are measured.
The sample which comes in EDTA tube is to check for clotted blood. If there is no clotted blood, the presence of bubbles is removed to avoid sampling error. The sample is the placed in a special HbA1c rack and placed into the machine. The machine will detect the barcode and do the test ordered. The sample (glycohemoglobin in HbA1c) is reacted with anti-HbA1c antibody to form antigen-antibody complexes. Then, polyhaptens are added to start the reaction by reacting with the excess anti-HbA1c antibodies to form an insoluble antibody-polyhapten complex which is the measure turbidimetrically. The final result is expressed as percent HbA1c. The results are then transmitted to the LIS system and the staffs allocated there will validate the results.
nyzah
Sunday, August 16, 2009
Week 8: High Performance Liquid Chromatograph
Name: Eriko Wong (0700477C)
Section: Analytical Chemistry
Ok, so today I'm going to share with you about the components of HPLC and how does it work.
Here is a picture showing the different components of HPLC::)
Image source: http://www.protein.iastate.edu/hplc.html
1) Mobile Phase Supply System are to be filtered and degassed first before pass through the column so as to remove any small particles or dissolved gases. If this step is not done, it can cause the clogging of column.
2) Injector is where you would inject your sample in. This is done by a syringe and a needle. Sample is also filtered first before being carried to the column for analysis.
3) Packed column: is also known as the stationary phase. This is where the interactions between the sample, mobile phase and stationary phase occur. The different interaction of the different components in the sample will lead to the different time they elute out of the column. Often, a guard column is installed to trap unwanted materials. Column is usually housed in a oven, under a predetermined temperature to ensure a consistent interaction of the sample, mobile and stationary phase.
4) Detector: The one I'm using is Ultraviolet-visible spectrometry detector. At different wavelength, the peak area and height of the sample recorded will be different too.
An example of a chromatogram:: (x-axis: time, y-axis: the absorption by the detection)
Image Source: http://www.protein-ms.de/METHODS/HPLC/HPLC.htm
5) Recorder: This is to obtain a real time plot of the analysis. Once the analysis is done, the computer will print out the data, along with the chromatogram.
Hopefully you guys get the idea of how HPLC works.... :)
Saturday, August 8, 2009
Clinical Chemistry - Urine Phase Contrast
Hey, here's Natasha again:]
I’m here to talk about urine phase contrast.
The purpose of performing urine phase contrast is mainly to differentiate isomorphic red blood cells and dysmorphic red blood cells present in the urine. Apart from that, if there are presence of white blood cells, microorganisms, epithelial cells, casts or/and crystals, they will be noted as well.
Isomorphic red blood cells, can suggest urological diseases which are non-glomerular origin. They appear bright and circular in shape. Isomorphic red blood cells still retain the haemoglobin whereas dysmorphic red blood cells have lost the haemoglobin.
Dysmorphic red blood cells, however, can suggest diseases of glomerular origin such as glomerulonephritis, etc. Dysmorphic red blood cells may appear circular or slight difference in shape and also appear as dark, thick rings.
The presence of microorganisms can indicate urinary tract infection (UTI). They may appear cocci or bacilli in shape, singly, pairs or in chains.
Crystals can be found in urine specimens that are super saturated with a crystalline compound and the formation of crystals are pH dependent. Thus a change in urine pH may cause crystal precipitation. For example, calcium oxalate maybe found in deposit of both acid/alkali urine. It may be regarded as a normal constituent of urine or may indicate renal calculi, renal disease or glycol poisoning.
The type of microscope used is the urine phase contrast microscopy. As compared to normal microscopy, the optical components of this microscope are able to change the different phases of structures into differences in light intensity.
Normal microscopy is when there is no optical contrast technique is employed. It uses transmitted light to view a specimen that contains natural contrast/colour or is stained.
The type of counting chamber used is Med-Fuchs Rosenthal counting chamber. This counting chamber is a little different from the counting chamber that we get to use in school during MCT practicals. It only consists of 16 big squares with 16 smaller squares in each of the big square. Any adjacent four squares can be used to count the isomorphic and/or dysmorphic red blood cells.
For urine phase contrast, fresh first morning mid-stream urine is collected and spin down at 2000 rpm for 10mins. The supernatant is then discarded (most of the time the cell pellet is not as visible, thus usually ¾ of the urine is discarded). The remaining urine containing the cells is then mixed well and pipetted onto the counting chamber. It is then viewed under the microscope.
Drawings are not drawn to scale.
In this picture, WBCs are the larger cells with granules inside. Isomorphic RBCs are bright ones and smaller in size than WBC. Dysmorphic RBCs are the dark, ring-shaped cells.
Picture is taken from http://lab-qa.org/u_atlas/sem01_22.htm
Med-Fuchs Rosenthal Counting Chamber. Click to have a better view:] Picture is taken from http://www.hausserscientific.com/FuchsRosenthalDirect.htm
I’m here to talk about urine phase contrast.
The purpose of performing urine phase contrast is mainly to differentiate isomorphic red blood cells and dysmorphic red blood cells present in the urine. Apart from that, if there are presence of white blood cells, microorganisms, epithelial cells, casts or/and crystals, they will be noted as well.
Isomorphic red blood cells, can suggest urological diseases which are non-glomerular origin. They appear bright and circular in shape. Isomorphic red blood cells still retain the haemoglobin whereas dysmorphic red blood cells have lost the haemoglobin.
Dysmorphic red blood cells, however, can suggest diseases of glomerular origin such as glomerulonephritis, etc. Dysmorphic red blood cells may appear circular or slight difference in shape and also appear as dark, thick rings.
The presence of microorganisms can indicate urinary tract infection (UTI). They may appear cocci or bacilli in shape, singly, pairs or in chains.
Crystals can be found in urine specimens that are super saturated with a crystalline compound and the formation of crystals are pH dependent. Thus a change in urine pH may cause crystal precipitation. For example, calcium oxalate maybe found in deposit of both acid/alkali urine. It may be regarded as a normal constituent of urine or may indicate renal calculi, renal disease or glycol poisoning.
The type of microscope used is the urine phase contrast microscopy. As compared to normal microscopy, the optical components of this microscope are able to change the different phases of structures into differences in light intensity.
Normal microscopy is when there is no optical contrast technique is employed. It uses transmitted light to view a specimen that contains natural contrast/colour or is stained.
The type of counting chamber used is Med-Fuchs Rosenthal counting chamber. This counting chamber is a little different from the counting chamber that we get to use in school during MCT practicals. It only consists of 16 big squares with 16 smaller squares in each of the big square. Any adjacent four squares can be used to count the isomorphic and/or dysmorphic red blood cells.
For urine phase contrast, fresh first morning mid-stream urine is collected and spin down at 2000 rpm for 10mins. The supernatant is then discarded (most of the time the cell pellet is not as visible, thus usually ¾ of the urine is discarded). The remaining urine containing the cells is then mixed well and pipetted onto the counting chamber. It is then viewed under the microscope.
Drawings are not drawn to scale.
In this picture, WBCs are the larger cells with granules inside. Isomorphic RBCs are bright ones and smaller in size than WBC. Dysmorphic RBCs are the dark, ring-shaped cells.
Picture is taken from http://lab-qa.org/u_atlas/sem01_22.htm
Med-Fuchs Rosenthal Counting Chamber. Click to have a better view:] Picture is taken from http://www.hausserscientific.com/FuchsRosenthalDirect.htm
Tuesday, July 28, 2009
TDx/IMX
Hey. Siti Shahimah is here again. :)
Okay, so I have learnt about TDx/IMX. Basically, TDx/IMX are the name of analyzers that help to detect therapeutic dosage of drugs so as to ensure appropriate therapy for patients.
Firstly, I am going to talk about TDx analyzer. This analyzer utilizes Fluorescence Polarization Immunoassay (FPIA) technology. Briefly, this FPIA employs the antigen (patient's sample), antigen (labeled with fluorophore) and antibody reaction. When there is low concentration of antigens in patient's sample, more labeled antigens will bind to the antibodies, thus polarization increase. However, high concentration of antigens in patient's sample will cause less labeled antigens to bind to the antibodies, thus polarization decrease.
TDx analyzes drugs such as cyclosporine, methotrexate and everolimus. Cyclosporine and everolimus are immunosuppresant drugs whereas methotrexate is an antineoplastic drug.
For IMX, it utilizes Microparticles Enzyme Immunoassay (MEIA) technology. This MEIA employs the sandwich principle which involve antigens (patient's sample), antibodies and antibodies ( labeled with alkaine phosphatase). The antigens will bind to antibodies bound on microparticles, forming immune complexes. The labeled antibodies will then bind to the immune complexes and catalyzes the substrates added. The catalytic reaction will generate a fluorescent product and this product is measured.
IMX analyzes drugs such as tacrolimus, sacrolimus and also squamous cell carcinoma (SCC) antigens. Tacrolimus and sacrolimus are immunosuppresive drugs whereas SCC antigens are found on squamous cell cancer tissue on the uterine cervix.
These are the tests that I have done: Cyclosporine test, methotrexate test, tacrolimus test and everolimus test. The procedure are as follows:
Okay, so I have learnt about TDx/IMX. Basically, TDx/IMX are the name of analyzers that help to detect therapeutic dosage of drugs so as to ensure appropriate therapy for patients.
Firstly, I am going to talk about TDx analyzer. This analyzer utilizes Fluorescence Polarization Immunoassay (FPIA) technology. Briefly, this FPIA employs the antigen (patient's sample), antigen (labeled with fluorophore) and antibody reaction. When there is low concentration of antigens in patient's sample, more labeled antigens will bind to the antibodies, thus polarization increase. However, high concentration of antigens in patient's sample will cause less labeled antigens to bind to the antibodies, thus polarization decrease.
TDx analyzes drugs such as cyclosporine, methotrexate and everolimus. Cyclosporine and everolimus are immunosuppresant drugs whereas methotrexate is an antineoplastic drug.
For IMX, it utilizes Microparticles Enzyme Immunoassay (MEIA) technology. This MEIA employs the sandwich principle which involve antigens (patient's sample), antibodies and antibodies ( labeled with alkaine phosphatase). The antigens will bind to antibodies bound on microparticles, forming immune complexes. The labeled antibodies will then bind to the immune complexes and catalyzes the substrates added. The catalytic reaction will generate a fluorescent product and this product is measured.
IMX analyzes drugs such as tacrolimus, sacrolimus and also squamous cell carcinoma (SCC) antigens. Tacrolimus and sacrolimus are immunosuppresive drugs whereas SCC antigens are found on squamous cell cancer tissue on the uterine cervix.
These are the tests that I have done: Cyclosporine test, methotrexate test, tacrolimus test and everolimus test. The procedure are as follows:
- Labeled microcentrifuge tubes for each patient's sample accordingly.
- 150 uL of patient's sample is pipetted into the corresponding tubes.
- 50 uL of solubilization reagent is then added to the tubes.
- Each tubes are vortexed for 10s to ensure thorough mixing (make sure caps are tight)
- Sample tubes are centrifuged for 5min at 10800rpm so as to obtain the supernatant.
- Supernatant is decanted into corresponding sample cells on the carousel.
- Reagent pack is mixed gently.
- The reagent pack and the carousel are placed on the analyzer and samples are run.
- Results are obtained and recorded.
This is the link if you want to see how the analyzer looks like:
Saturday, July 25, 2009
urinalysis
hii it's my turn now !
so, i'll be writing about the department i was allocated last week, urinalysis. before i start on the processes of tests done by this bench, there are some preparations and QCs to mention briefly. this is also what we usually do to start off the exciting day. after rollcall, i'll be preparing the 0.1% hypochlorite. pretty easy as they're in the tablet form. only thing to note is- how many tablets to how much DI water. for QC, we have to do it for the osmometer, refractometer, ABG analyzer and the dipstix analyzer. materials used for these QCs are mainly DI water, saline and commercial solution.
okay, so now i'll begin on the tests/skills i've learnt.
1. urine FEME
this test includes both dipstix and microscopic examination (ME). we should be familiar with the procedures for this test thus i'll summarize what i've learnt. there is a correlation between the dipstix result and the ME.
if there is a presence of protein, there could be casts present under the ME. similarly, positive nitrate shows presence of microorganism, positive esterase shows presence of WBCs.
2. rapid plasma reagin (RPR) card test
to test for syphilis using the patient's serum. we use a kit, Omega Diagnostics Immutrep RPR to carry out this test with only one drop of serum. there is a positive and negative control to compare with the samples. positive results will show agglutination.
3. dengue panel
this test is also carried out by using a commercial kit, SD Dengue Duo, a one step dengue NS1 Ag and IgG/IgM test. time taken is around 15 minutes.
4. arterial blood gas (ABG)
ABG!! that's what the order entry person shouts after they have ordered this test. reason being, it must be done asap to obtain an accurate result as this test measures the pH and the partial pressure of oxygen, carbon dioxide and also, bicarbonate concentration of the blood sample. the analyzer used is called Roche OMNI C. just have to poke the needle into the syringe and the analyzer produce the results. so, it is pretty simple. however, most of the time the med tech will be doing this as their pass are needed to operate this analyzer. i've tried it couple of times tho. ;)
5. HCG test
there is a test kit for this too and patient's serum is used. altho urine can also be loaded on the kit, serum is preferred as it gives a more accurate result. it's a straightforward test with either a +/- shown after 15 minutes.
ps i have photos to upload but dontknow how :s will add in after figuring out.
enjoy weekends,
wendyong
so, i'll be writing about the department i was allocated last week, urinalysis. before i start on the processes of tests done by this bench, there are some preparations and QCs to mention briefly. this is also what we usually do to start off the exciting day. after rollcall, i'll be preparing the 0.1% hypochlorite. pretty easy as they're in the tablet form. only thing to note is- how many tablets to how much DI water. for QC, we have to do it for the osmometer, refractometer, ABG analyzer and the dipstix analyzer. materials used for these QCs are mainly DI water, saline and commercial solution.
okay, so now i'll begin on the tests/skills i've learnt.
1. urine FEME
this test includes both dipstix and microscopic examination (ME). we should be familiar with the procedures for this test thus i'll summarize what i've learnt. there is a correlation between the dipstix result and the ME.
if there is a presence of protein, there could be casts present under the ME. similarly, positive nitrate shows presence of microorganism, positive esterase shows presence of WBCs.
2. rapid plasma reagin (RPR) card test
to test for syphilis using the patient's serum. we use a kit, Omega Diagnostics Immutrep RPR to carry out this test with only one drop of serum. there is a positive and negative control to compare with the samples. positive results will show agglutination.
3. dengue panel
this test is also carried out by using a commercial kit, SD Dengue Duo, a one step dengue NS1 Ag and IgG/IgM test. time taken is around 15 minutes.
4. arterial blood gas (ABG)
ABG!! that's what the order entry person shouts after they have ordered this test. reason being, it must be done asap to obtain an accurate result as this test measures the pH and the partial pressure of oxygen, carbon dioxide and also, bicarbonate concentration of the blood sample. the analyzer used is called Roche OMNI C. just have to poke the needle into the syringe and the analyzer produce the results. so, it is pretty simple. however, most of the time the med tech will be doing this as their pass are needed to operate this analyzer. i've tried it couple of times tho. ;)
5. HCG test
there is a test kit for this too and patient's serum is used. altho urine can also be loaded on the kit, serum is preferred as it gives a more accurate result. it's a straightforward test with either a +/- shown after 15 minutes.
ps i have photos to upload but dontknow how :s will add in after figuring out.
enjoy weekends,
wendyong
Sunday, July 19, 2009
Week 4
Hello Everyone!
I’m Nyzah.
For the first two weeks of SIP, I’m attached to the central processing (CP) are whereby specimens were sorted out, processed and ordered. Specimens usually will arrive in the pneumatic tube via the pneumatic system. The pneumatic system connects all wards, specialist clinics and operating theatre. Sometimes, the healthcare workers will also dispatched the specimens to the clinical lab personally. This is done by passing it through a window. Medical technologist will then attend to the specimens.
The specimens are then processed by removing the request forms from the kangaroo pouch and stapling it to the Ziploc bag with the specimen in it. Specimens ranging from plain tube, EDTA tube, fluoride tubes, urine samples, stools and blood gas specimens are processed there. For microbiology specimens, the specimens are placed inside a red plastic box and for histopathology specimens into the green plastic box. This is because microbiology laboratory is out of bound due to the H1N1 cases and histopathology due to renovations. The other specimens are then placed on the CP workstations whereby the medical technologists or the laboratory clerk will order the tests wanted in the Laboratory Information System (LIS). Then, specimens that come in tubes (usually blood) are removed from the Ziploc bags. Barcode are pasted on it and the details of the patients are pasted on the workstation forms. For other specimens other than tubes, the barcodes are pasted on the Ziploc bags. The person pasting the barcodes have the responsibility of checking the names and the I/C number of the patient against the labeled tubes or other samples. The specimens are then dispatched to various workstation i.e. hematology, biochemistry, etc.
If the person is unsure about the tests that are need to be done, it is necessary for them to call the doctor or the staff nurse in charge so as to avoid confusion and long turnaround time.
Nurul Nyzah
0702888I
I’m Nyzah.
For the first two weeks of SIP, I’m attached to the central processing (CP) are whereby specimens were sorted out, processed and ordered. Specimens usually will arrive in the pneumatic tube via the pneumatic system. The pneumatic system connects all wards, specialist clinics and operating theatre. Sometimes, the healthcare workers will also dispatched the specimens to the clinical lab personally. This is done by passing it through a window. Medical technologist will then attend to the specimens.
The specimens are then processed by removing the request forms from the kangaroo pouch and stapling it to the Ziploc bag with the specimen in it. Specimens ranging from plain tube, EDTA tube, fluoride tubes, urine samples, stools and blood gas specimens are processed there. For microbiology specimens, the specimens are placed inside a red plastic box and for histopathology specimens into the green plastic box. This is because microbiology laboratory is out of bound due to the H1N1 cases and histopathology due to renovations. The other specimens are then placed on the CP workstations whereby the medical technologists or the laboratory clerk will order the tests wanted in the Laboratory Information System (LIS). Then, specimens that come in tubes (usually blood) are removed from the Ziploc bags. Barcode are pasted on it and the details of the patients are pasted on the workstation forms. For other specimens other than tubes, the barcodes are pasted on the Ziploc bags. The person pasting the barcodes have the responsibility of checking the names and the I/C number of the patient against the labeled tubes or other samples. The specimens are then dispatched to various workstation i.e. hematology, biochemistry, etc.
If the person is unsure about the tests that are need to be done, it is necessary for them to call the doctor or the staff nurse in charge so as to avoid confusion and long turnaround time.
Nurul Nyzah
0702888I
Monday, July 6, 2009
Third Week - Rabbit's blood collection
Done by: Eriko
Section: Haematology
YohOoo! So, last week I did blood collection from rabbits.
Firstly, to prevent the rabbit from moving as much, it is wrapped around with towel with only the head and ear exposing. Then, its ear is cleaned with alcohol swab to disinfect that area for blood collection. A needle is then injected into the central auricular artery. When this step is performing and the rabbit moves, the needle is removed immediately and the processes have to be repeated. Sometimes, we have to massage the other ear to distract the rabbits so as they don't shake/move when we are injecting with the needle.
Then, a syringe is connected to the needle to draw the blood. When the desired amount of blood is drawn, gauze is placed on top of the injected area to apply pressure so as to stop the bleeding. Blood is then injected to EDTA tube and inverted a few times to prevent blood clot.
However, during the blood collection, the blood will always clot. Thus, a pre-rinsed anti-coagulant syringe is preferred to be used in this case.
Here's a picture of blood collection from rabbit to help you picture it better:)
Retrieved on 6 July, 2009 from website media.wiley.com
That's it, guys!
Good luck and enjoy your SIP!~~~~
xo
Section: Haematology
YohOoo! So, last week I did blood collection from rabbits.
Firstly, to prevent the rabbit from moving as much, it is wrapped around with towel with only the head and ear exposing. Then, its ear is cleaned with alcohol swab to disinfect that area for blood collection. A needle is then injected into the central auricular artery. When this step is performing and the rabbit moves, the needle is removed immediately and the processes have to be repeated. Sometimes, we have to massage the other ear to distract the rabbits so as they don't shake/move when we are injecting with the needle.
Then, a syringe is connected to the needle to draw the blood. When the desired amount of blood is drawn, gauze is placed on top of the injected area to apply pressure so as to stop the bleeding. Blood is then injected to EDTA tube and inverted a few times to prevent blood clot.
However, during the blood collection, the blood will always clot. Thus, a pre-rinsed anti-coagulant syringe is preferred to be used in this case.
Here's a picture of blood collection from rabbit to help you picture it better:)
Retrieved on 6 July, 2009 from website media.wiley.comThat's it, guys!
Good luck and enjoy your SIP!~~~~
xo
Sunday, July 5, 2009
Clinical Chemistry - DP analyzer
Hey, here's Natasha:]
I am attached to a clinical chemistry lab for my SIP. There are a number of workstations consisting of different analyzers in this clinical chemistry lab. For the first week, I am assigned to the Modular Analytics Evo DP analyzer.
This DP analyzer can be divided into three sections; D module, P module and ISE module.
The D and P modules of the DP analyzer actually performs photometric assays on serum/plasma, urine and CSF samples. It measures the absorbancies of the chemical reaction in the reaction cells when the sample is mixed with the appropriate reagent. The D module performs several tests such as renal function test (urea, creatinine, etc), liver function test (AST, ALP, etc) and lipids (total cholesterol, triglycerides, etc). The P module can actually perform tests similar to the D module; however in this clinical lab, it is used to conduct the diabetic panel test (glucose) only. The only difference between the D and P modules is the method used to add the reagents into the reaction cells. The D module stands for 'dispensing' so it will dispense the reagents into the reaction cells. The P module stands for ' pipetting' so it will pipette the reagent from the reagent bottle and then release it into the reaction cells.
The ISE module, however, invloves measuring the difference in electircal potential between the sample measurement electrode and the reference electrode. The electrical poential of the KCl reference solution on the reference electrode serves as a zero baseline for the ISE calculations of the samples. The ISE module is used to determine the amount of sodium, potassium and chloride in the samples.
One of the tests that could be performed on the DP analyzer (specfically P module) is the glucose tolerance test (GTT). GTT is actually the dministration of glucose to determine how quickly glucose is cleared from the bood of the patient. The test is usually used to test for diabetes and insulin resistance in those patients.
Glucose is the major monosaccharides present in the blood and serves as an esential energy supplying substrate for cellular functions Glucose degradation occurs via glycolysis. Glucose determinations can be used in detecting hypoglycaemia and hyperglycaemia, in the diagnosis of diabetes as well as in the monitoring of glucose levels in people with diabetes.
The patient sample is taken in the form of blood in sodium fluoride/ potassium oxalate tubes (grey tubes). Sodium fluoride is an anti-glycolytic agent which aids in inhibitng glycolysis. The patient would have to fast the night before the test. For the test, the patient would have to drink a liquid containing a certain amount of glucose. Then after 2 hours, the patient's blood will betaken again. Thus there will be two grey tubes; one containing patient blood at 0 hours before the intake of glucose drink and the other containing patient blood at 2 hours after the intake of glucose drink.
In a person without diabetes, the glucose levels will rise and then fall rapidly. However in a person with diabetes, glucose levels will rise higher than normal and fail to come back down as fast.
I am attached to a clinical chemistry lab for my SIP. There are a number of workstations consisting of different analyzers in this clinical chemistry lab. For the first week, I am assigned to the Modular Analytics Evo DP analyzer.
This DP analyzer can be divided into three sections; D module, P module and ISE module.
The D and P modules of the DP analyzer actually performs photometric assays on serum/plasma, urine and CSF samples. It measures the absorbancies of the chemical reaction in the reaction cells when the sample is mixed with the appropriate reagent. The D module performs several tests such as renal function test (urea, creatinine, etc), liver function test (AST, ALP, etc) and lipids (total cholesterol, triglycerides, etc). The P module can actually perform tests similar to the D module; however in this clinical lab, it is used to conduct the diabetic panel test (glucose) only. The only difference between the D and P modules is the method used to add the reagents into the reaction cells. The D module stands for 'dispensing' so it will dispense the reagents into the reaction cells. The P module stands for ' pipetting' so it will pipette the reagent from the reagent bottle and then release it into the reaction cells.
The ISE module, however, invloves measuring the difference in electircal potential between the sample measurement electrode and the reference electrode. The electrical poential of the KCl reference solution on the reference electrode serves as a zero baseline for the ISE calculations of the samples. The ISE module is used to determine the amount of sodium, potassium and chloride in the samples.
One of the tests that could be performed on the DP analyzer (specfically P module) is the glucose tolerance test (GTT). GTT is actually the dministration of glucose to determine how quickly glucose is cleared from the bood of the patient. The test is usually used to test for diabetes and insulin resistance in those patients.
Glucose is the major monosaccharides present in the blood and serves as an esential energy supplying substrate for cellular functions Glucose degradation occurs via glycolysis. Glucose determinations can be used in detecting hypoglycaemia and hyperglycaemia, in the diagnosis of diabetes as well as in the monitoring of glucose levels in people with diabetes.
The patient sample is taken in the form of blood in sodium fluoride/ potassium oxalate tubes (grey tubes). Sodium fluoride is an anti-glycolytic agent which aids in inhibitng glycolysis. The patient would have to fast the night before the test. For the test, the patient would have to drink a liquid containing a certain amount of glucose. Then after 2 hours, the patient's blood will betaken again. Thus there will be two grey tubes; one containing patient blood at 0 hours before the intake of glucose drink and the other containing patient blood at 2 hours after the intake of glucose drink.
In a person without diabetes, the glucose levels will rise and then fall rapidly. However in a person with diabetes, glucose levels will rise higher than normal and fail to come back down as fast.
Thursday, June 25, 2009
Clinical chemistry
Hey. I am Siti Shahimah from TGO1 and I am posted in clinical chemistry lab. Firstly, I am going to talk about the urine phase contrast microscopy (UPCM). This technique helps to examine urinary constituents. These constituents are red blood cells (isomorphic and dysmorphic), white blood cells, casts, crystals, microorganism, and other (e.g. yeast cells and spermatozoa). The difference between iso/dysmorphic red cells are their appearance. Isomorphic red cells appear bright under microscope whereas dysmorphic appear dark and sometimes has a hole-like in the middle of the cell. In UPCM, Med-Fuchs Rosenthal Counting Chamber is used. This is different from the one that we used in school. This chamber consist of 9 big squares with 16 small squares in each big squares.
This is the summarised steps in performing UPCM.
1) Mix urine sample well.
2) Pour 10ml of urine sample into centrifuge tube.
3) Centrifuge the tube at 2000rpm for 10 minutes.
4) Discard supernatant.
5) Vortexed the remaining volume of urine.
6) Load the sample into Med-Fuchs Rosenthal Counting Chamber.
7) Examine under microscope (40x).
I have also performed urine and serum osmolality. In this lab, I used two osmometers and they are called Vapor Pressure osmometer. These osmometers are different from what we have in school. In school, we used the freezing point depression osmometer. Vapor Pressure osmometers help to measure osmolality by using thermocouple found in the chamber of osmometer. These osmometers are very sensitive and produce quite accurate results.There are two osmometers so that we can tally to see if the results are consistent with each other or not.
The difference in the osmometer results must not be more than 10 mOsm/kg. There was once when one of the osmometers result showed more than 10
mOsm/kg difference and the staff had to recalibrate both of the machines by using three different levels of standard. Level 1 is 290 mOsm/kg, level 2 is 1000 mOsm/kg and level 3 is the 100 mOsm/kg. After recalibration/ switch the machine off and do test for QC materials, the osmometer result is acceptable.
This is the summarised steps in performing UPCM.
1) Mix urine sample well.
2) Pour 10ml of urine sample into centrifuge tube.
3) Centrifuge the tube at 2000rpm for 10 minutes.
4) Discard supernatant.
5) Vortexed the remaining volume of urine.
6) Load the sample into Med-Fuchs Rosenthal Counting Chamber.
7) Examine under microscope (40x).
I have also performed urine and serum osmolality. In this lab, I used two osmometers and they are called Vapor Pressure osmometer. These osmometers are different from what we have in school. In school, we used the freezing point depression osmometer. Vapor Pressure osmometers help to measure osmolality by using thermocouple found in the chamber of osmometer. These osmometers are very sensitive and produce quite accurate results.There are two osmometers so that we can tally to see if the results are consistent with each other or not.
The difference in the osmometer results must not be more than 10 mOsm/kg. There was once when one of the osmometers result showed more than 10
mOsm/kg difference and the staff had to recalibrate both of the machines by using three different levels of standard. Level 1 is 290 mOsm/kg, level 2 is 1000 mOsm/kg and level 3 is the 100 mOsm/kg. After recalibration/ switch the machine off and do test for QC materials, the osmometer result is acceptable.
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