hey guys! =) its my turn again. i'm gonna share with you one of the test that is carried out by the Architect i2000SR immunoassay.
The Rubella IgG test
WHY?
Rubella is a highly infectious virus that infects children. Primary postnatal rubella infection is a mild self-limiting disease that has the typical symptoms of maculopapular rash, fever, malaise and lymphadenopathy. However, primary prenatal infections might have fatal consequences as it might cause fatal damage to fetus especially during first month of gestation. After infection, infants might show one or more variety of defects which is otherwise recognised as the congenital rubella syndrome such as having low birth weight, cataracts, deafness, congenital heart disease and even mental retardation. There are currently rubella vaccines available to protect individual from this virus. This has proven to reduce cases of rubella virus infection.
So basically this test is actually conducted to measure the amount of rubella antibodies that is present in the human serum. This test is used to determine if the particular patient is immunised against rubella or if the patient has any past or recent rubella infection. It is also used to confirm that all pregnant ladies has enough rubella antibodies to be able to be protected against the infection.
HOW?
Partially purified rubella virus coated paramagnetic microparticles which will cause binding of rubella IgG if present in the human serum. It is then washed and anti-human IgG acridinium-labeled conjugate is added. It is then washed again with the addition of pre-trigger and trigger solution.
It is measure colourmetrically as RLUs and this RLUs detected by optical system is directly proportional to concentration of rubella Ab present in serum.
The serum left is kept for 1 year at -70 to -90 degrees celsius for traceability. =)
Done by: Joanna Yeo
Admin No. 0702054H
Wednesday, September 30, 2009
Monday, September 21, 2009
VDRL & TPHA
Done By: Janice Yeh
Department: Serology
Section: VDRL and TPHA
*Pictures: Taken with the permission from the Head of Department. No patient information or ID is revealed.
Abbreviations:
VDRL- Venereal Disease Research Laboratory (Venereal Disease refers to diseases that are transmitted by sexual intercourse)
RPR - Rapid Plasma Reagin
TPHA - Treponoma Pallidum Haemagglutination
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3 Parts to test for Syphilis:
1) RPR: a non-specific treponemal test: Screening test
2) RPR titer: to find out how infectious the disease is or to monitor progress of treatment in syphilis positive patients
3) TPHA: a specific treponemal test: Confirmatory test
The VDRL test is a screening test for syphilis. It measures antibodies, that can be produced by Treponema pallidum, the bacteria that causes syphilis.
The test is similar to the newer rapid plasma reagin (RPR) test. RPR is a screening test and if a positive result is obtained, a further confirmatory test is conducted, which is TPHA.
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(1) RAPID PLASMA REAGIN TEST
RPR is known as a non-treponemal test. It detects for antibodies in the patient that are against substances that a released from cells that are damaged by the bacterium. Unlike RPR, TPHA is more specific as it detects antibodies against the actual bacterium. Hence, we cannot based on the results of RPR alone as there could be many false positive reasons.
Procedure: Samples are collected from trays as such or from the Sample Receival (SR) department. Samples are spun down in a centrifuge to obtain seperation between the serum and the RBCs.
Procedure: An immuno RPR test card is used. The serum is dropped with the circle and smeared to fill the circle. The circle is a gauge of how much serum is needed. Then a drop of reagent is added. The reagent contains carbon particles coated with a mixture of lipid materials which will bind to the antibodies present in the patient's serum.
Purpose:
Why must we ensure that there is enough serum:
(1) As the samples will not be read immediately, we need to ensure we do not take to little serum as they may dry up after the next step, which is the mixing part.
Procedure: The card is then placed on a automated shaker for 8 minutes. The card should avoid being shifted around or touched.
Purpose:
Procedures:
1) Add one drop of cells to the wells:
Results: After 45 minutes, check the results. The results in the RED CIRCLE are positive/reactive. It means that patient is syphilis positive. It is denoted by large rins with peripheral agglutination. Negative results are denoted by concentrated buttons at the bottom of the wells.
Department: Serology
Section: VDRL and TPHA
*Pictures: Taken with the permission from the Head of Department. No patient information or ID is revealed.
Abbreviations:
VDRL- Venereal Disease Research Laboratory (Venereal Disease refers to diseases that are transmitted by sexual intercourse)
RPR - Rapid Plasma Reagin
TPHA - Treponoma Pallidum Haemagglutination
------------------------------------------------------------------------------------------------
3 Parts to test for Syphilis:
1) RPR: a non-specific treponemal test: Screening test
2) RPR titer: to find out how infectious the disease is or to monitor progress of treatment in syphilis positive patients
3) TPHA: a specific treponemal test: Confirmatory test
The VDRL test is a screening test for syphilis. It measures antibodies, that can be produced by Treponema pallidum, the bacteria that causes syphilis.
The test is similar to the newer rapid plasma reagin (RPR) test. RPR is a screening test and if a positive result is obtained, a further confirmatory test is conducted, which is TPHA.
-----------------------------------------------------------------------------------------------
(1) RAPID PLASMA REAGIN TEST
RPR is known as a non-treponemal test. It detects for antibodies in the patient that are against substances that a released from cells that are damaged by the bacterium. Unlike RPR, TPHA is more specific as it detects antibodies against the actual bacterium. Hence, we cannot based on the results of RPR alone as there could be many false positive reasons.
Procedure: Samples are collected from trays as such or from the Sample Receival (SR) department. Samples are spun down in a centrifuge to obtain seperation between the serum and the RBCs.
Procedure: Use a dropper to obtain a small amount of serum (~10microlitres) as shown in the picture. Ensure that only serum is obtained.
Purpose:
Why obtain serum only:
(1)Our main aim is to detect for antibodies present in the cell. Antibodies are only present in serum.
(2)Obtaining the serum only is important. Because if it is mixed with the blood to do the test, it will be harder to spot for agglutination (will be explained).
Procedure: An immuno RPR test card is used. The serum is dropped with the circle and smeared to fill the circle. The circle is a gauge of how much serum is needed. Then a drop of reagent is added. The reagent contains carbon particles coated with a mixture of lipid materials which will bind to the antibodies present in the patient's serum.Purpose:
Why must we ensure that there is enough serum:
(1) As the samples will not be read immediately, we need to ensure we do not take to little serum as they may dry up after the next step, which is the mixing part.
Procedure: The card is then placed on a automated shaker for 8 minutes. The card should avoid being shifted around or touched.Purpose:
Why should we avoid disturbing the card:
(1) As the samples are mixing, movement may cause cross contamination of samples as the circles are very close to each other.
.bmp)
Procedure: After 8 minutes, the card is taken off the shaker and swirled alittle to see clearly for agglutination.
Results: Figure 1 - Positive with grey agglutination
Figure 2 - Negative with a button of non-aggregated carbon particles in the centre of
the circle 
http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/images/std/vdrl.jpg
on the left: negative
on the right: positive
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(2) TITER MEASUREMENT
Titer is a measure of concentration, that employs serial dilution of the serum. Titer corresponds to the highest dilution factor that still yields a positive reading. The purpose of measuring the titer of the sample is find out how serious is disease or for doctors to monitor the progress of the treatment. 
Results: The last circle that contains agglutination is the titer of the sample. The titer from the first to the last circle is 1:1, 1:2, 1:4, 1:8, 1:16, 1:32.
Example, if there is agglutination present from the 1st circle to the 4th circle, it means that the sample has a titer of 1:8.
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(3)TREPONOMA PALLIDUM HAEMAGGLUTINATION
TPHA screening kit uses preserved avian erythrocytes coated with antigens of T.pallidum which will bind with specific antibody present in the patient's serum or plasma. The cells are suspened in a medium containing components to eliminate non-specific reactions such as reactions caused by other bacterium.
Procedures:
1st Roll: is always for the controls. Takes up 4 wells.
Sample Roll: takes up 6 wells.
1) WELL #1: for both Control and Patient Sample (PT): 190ul of diluent + 10ul of serum
2) WELL #2: No diluent + 25ul of diluted sample from well 1
3) WELL #3-4 (for control) : 25ul of diluent
WELL #3-6 (patient sample): 25ul of diluent
4) Transfer 25ul of mixed sample from well #1 to well #3. Mix well and transfer to the next well. 5) Discard the last 25ul of mixed sample.
Procedures:1) Add one drop of cells to the wells:
(a) Add Control Cells (Unsensitized) to Well#2
(b) Add Test cells (Sensitised) to Well #3-4 (for control), and Well #3-6 (Patient Sample)
2) Mix the microplate well by gently tapping on the corners of the plate. This is to ensure that sample or reagents along the side of the well will sink to the bottom of the well and be well mixed.
(b) Add Test cells (Sensitised) to Well #3-4 (for control), and Well #3-6 (Patient Sample)
2) Mix the microplate well by gently tapping on the corners of the plate. This is to ensure that sample or reagents along the side of the well will sink to the bottom of the well and be well mixed.
3) Incubate the well in room temperature for 45 minutes before reading the the results.
Results: After 45 minutes, check the results. The results in the RED CIRCLE are positive/reactive. It means that patient is syphilis positive. It is denoted by large rins with peripheral agglutination. Negative results are denoted by concentrated buttons at the bottom of the wells.That's all, I hope it's clear enough! :D
Transformation of chemically ultra-competent E.coli with plasmid DNA
Posted by Vo Thu Hong Anh [Jess] 0705364H
Purpose:
To generate a large amount of plasmid DNA containing a desired gene, with the aid of chemically ultra-competent E.coli
Principle:
Chemically ultra-competent E.coli cells have been commercially treated so that their membrane carry +ve charge, which will enhance the uptake of –ve charged DNA. On the other hand, chemically ultra-competent E.coli cells will also be heated at 42oC so that membrane molecules could move faster and more apart from each other, creating bigger ‘gaps’ for DNA to move in (DNA also move faster at this temperature).
Plasmid DNAs have been commercially cloned with a desired gene (a gene that u want to study) and a specific antibiotic resistant gene. As transformed cells contain plasmid DNA, they will survive the antibiotic in the agar. Those non-transformed cells will be killed by the antibiotic as they have no plasmid DNA, or in other words, no antibiotic resistant gene. As a result, the colonies growing on the agar surface are successfully transformed cells which will be picked and further grown in a larger scale to obtain enough plasmid DNA for further study.
Procedure:
- Place the paper disc containing plasmid DNA (commercially made and shipped) into a 1.5mL-centrifuge tube
- Add 50uL of 1xTE buffer to elute the DNA. Ensure that the paper disc is immersed in the buffer.
- Incubate for 60’ at room temperature. Flick the tube sometimes in between to enhance elution.
- In a meanwhile, place frozen chemically ultra-competent E.coli on ice to thaw
- After incubation, add 50uL E.coli and 5uL buffer containing plasmid DNA into a 2mL-centrifuge tube
- Place the tube on ice for 30’
- Heat shock at 42oC, 45s (using the thermomixer)
- Place the tube on ice for 2’
- Add 250uL of SOC medium into tube
- Incubate for 1hr, 37oC, 250 rpm
- After incubation, spread the mixture onto LB (Luria Broth) agar plate which contains the specific antibiotic which transformed cells will be resistant to (in my experiment, it is kanamycin)
- Incubate the plate upright for 10’ in 37oC incubator
- Invert the plate and incubate overnight to allow colonies to form.
Note: the volume of cell, DNA and reagents used may vary in different experiments as it depends on the type of cell and DNA used.
Thermomixer Eppendorf
Day 2:
- Add 3mL LB medium containing antibiotic (kanamycin) into a 15mL falcon tube (u can use as many as falcon tubes as u want, it depends on ur
- Use a sterile toothpick to touch a single colony and place the toothpick inside the tube.
- Incubate overnight, 37oC, 250rpm, to allow cell growth
This pix is drawn by me =D(btw, did I spell the word toothpick wrongly? o,0)
Day 3
- Transfer cell suspension into another tube and centrifuge to obtain cell pellet which will be then lyzed and subsequently, plasmid DNA will be extracted for futher experiment.
Yup, that's simple ^^ feel free to clarify with me anything yah? ^^
Sunday, September 13, 2009
Slide-making
By Rachel,
Greetings to everyone, :)
I was posted to Cytogenetics Lab. In this lab, I was given the opportunity to culture my own cells and perform karyotyping. It was very fun but quite scary in a way. The scary part was during karyotyping for my own chromosomes as I did not know what would be the result. Initially I was worried about the outcome but overall, it was not too bad. J
Contents of my post
1) Grading for the metaphase in slide-making.
What is slide-making?
Slide-making is the process of dropping cells onto the slides for observation of chromosomes under the microscope.
*Please refer to Jocelyn’s blog for the procedure for slide making
In this lab, I was taught how to make slides for blood specimens as well as to stain them. However, I need to make good slides so that the chromosome morphology can be observed after staining. The question is how am I going to know whether the slide is a good or a bad slide? This is where the grading system applies. :)
The chromosomes in a metaphase are graded based on these parameters, Mitotic Index, contrast, length of chromosome, spread and presence of cytoplasm.
1)
Mitotic Index is the number of metaphase across a microscopic field. An acceptable range would be about 4-5 metaphases in one microscopic field. This range is usually sought after so that the cytogenetic technologists have more chromosomes to read. If the number is less than 4 probably the medical techs have lesser chromosomes to read.
2)
Contrast of the chromosome is critical as this can affect chromosome morphology greatly. The ideal contrast is between 70%-80%. If the percentage is above 80%, the chromosomes might be darkly stained. If the percentage is less than 70% the chromosomes would be pale. If chromosomes are dark or pale, the bands on the chromosomes would be indistinctive and identification of these chromosomes would be a problem.
3)
Longer chromosomes are usually observed as more bands can be observed on the longer chromosome as compared to the shorter chromosome. Therefore longer chromosomes are mostly observed.
4)
Bands on the chromosomes which are spread widely can be observed clearly as compared to chromosomes which are over-lapping. If chromosomes frequently overlap, observation of the bands can be affected.
5)
Finally presence of cytoplasm is unwanted as their presence can affect staining. The stains cannot seep into the cytoplasm and chromosomes would be poorly stained. As such bands on the chromosomes would be indistinctive.
Slide-making is important because it determines whether chromosome morphology can be observed at 100x magnification after staining. If the slides are poorly made and stained, translocations, inversion or deletion would not be observable.
That would be all, feel free to ask me any questions regarding this topic. I would try to answer them as accurately as possible. :)
Greetings to everyone, :)
I was posted to Cytogenetics Lab. In this lab, I was given the opportunity to culture my own cells and perform karyotyping. It was very fun but quite scary in a way. The scary part was during karyotyping for my own chromosomes as I did not know what would be the result. Initially I was worried about the outcome but overall, it was not too bad. J
Contents of my post
1) Grading for the metaphase in slide-making.
What is slide-making?
Slide-making is the process of dropping cells onto the slides for observation of chromosomes under the microscope.
*Please refer to Jocelyn’s blog for the procedure for slide making
In this lab, I was taught how to make slides for blood specimens as well as to stain them. However, I need to make good slides so that the chromosome morphology can be observed after staining. The question is how am I going to know whether the slide is a good or a bad slide? This is where the grading system applies. :)
The chromosomes in a metaphase are graded based on these parameters, Mitotic Index, contrast, length of chromosome, spread and presence of cytoplasm.
1)
Mitotic Index is the number of metaphase across a microscopic field. An acceptable range would be about 4-5 metaphases in one microscopic field. This range is usually sought after so that the cytogenetic technologists have more chromosomes to read. If the number is less than 4 probably the medical techs have lesser chromosomes to read.
2)
Contrast of the chromosome is critical as this can affect chromosome morphology greatly. The ideal contrast is between 70%-80%. If the percentage is above 80%, the chromosomes might be darkly stained. If the percentage is less than 70% the chromosomes would be pale. If chromosomes are dark or pale, the bands on the chromosomes would be indistinctive and identification of these chromosomes would be a problem.
3)
Longer chromosomes are usually observed as more bands can be observed on the longer chromosome as compared to the shorter chromosome. Therefore longer chromosomes are mostly observed.
4)
Bands on the chromosomes which are spread widely can be observed clearly as compared to chromosomes which are over-lapping. If chromosomes frequently overlap, observation of the bands can be affected.
5)
Finally presence of cytoplasm is unwanted as their presence can affect staining. The stains cannot seep into the cytoplasm and chromosomes would be poorly stained. As such bands on the chromosomes would be indistinctive.
Slide-making is important because it determines whether chromosome morphology can be observed at 100x magnification after staining. If the slides are poorly made and stained, translocations, inversion or deletion would not be observable.
That would be all, feel free to ask me any questions regarding this topic. I would try to answer them as accurately as possible. :)
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