Thursday, November 12, 2009

ways to protect the column from damaging

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

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)


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

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.


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.


Saturday, September 26, 2009


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).


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


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


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.

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. :]