Hi everyone, this is my third post. In this post, i am going to talk about processing of the gynaecology specimens in cytology lab.
Basically, the gynae specimens come in two forms, Thin Prep vials or conventional smears. The thin prep vials contain preservcyt solution which is basically a methanol-based medium. The purpose is to preserve the cells to allow proper evaluation under microscope.
Collection of specimen
1)Specimen is collected with a spatula or a cytobrush
2)It is rinsed immediately in the presercyt solution
3)The vial will be capped tightly and labeled with the patient information before being set to the lab
In the lab
1)Once specimen is received, the patient information on the form must be double check with that on the vial and thin prep number is assigned to both of them(eg.0983)
2)The vials are then taken into the processing room to be processed by the Thin Prep 2000
3)Thin prep slides are labeled with the thin prep number and the patient's ID
4)The slide is first inserted into the slide holder
5)This is then followed by putting in the fixative vial which contains 95% ethanol
6)Specimen vial is inserted next followed by slotting in the filter
7)Press program '4'to process the gynae specimens
After processing
1)Once processing is done, the slide holder will drop the slides into the fixative vial
2)The slides in the fixative vial will be left aside for around 15 minutes to adequately fix the cells before staining
3)The stain used is papanicolaou stain which is bascially carried out by the machine
4)Slides are then manually mounted using Depex
Things to take note
1)The slides must be adequately fixed before undergoing staining so as to prevent cells from dropping during that process
2)The specimen vial must always be placed into the Thinprep 2000 before the filter to prevent the filter membrane from being scratched
3)At the end of the day, make sure that grease is applied onto the black rings of the filter cap so as to reduce friction
That is the end of my post, which is generally an overview of how processing of thinprep specimens are carried out. Questions are welcome. :)
zi shuang
0703383J
Sunday, September 27, 2009
Thursday, September 17, 2009
Real Time PCR Probes
Hi there, i have talked about real time PCR in my previous post. In order to get results from RT-PCR, probes such as Taq man are used.
Taq Man is a RT Probe (the Cheaper one). Taq man is a DNA sequence with binded 2 proteins Reporter protein (at the 5' end) and quencher protein at the 3' end. The quencher protein will inhibit the reporter protein when they are both in the same DNA strand. This is due to the inhibition of proton transfer.
Basic: PCR processes are Denaturation, Annealing and Amplification.
At denaturation, DNA will become Single stranded DNA strands. At Annealing, the probes will bind to complementary base pairs along the targetted DNA strand. At Amplification, Taq Polymerase will bind to the DNA strands and produce copies of the DNA strands. The Taq polymerase will move down the targetted DNA strands to produce complementary strands. Once the Taq Polymerase will "free" the reporter protein once it hits the probe (Breaking up the probe). The released Reporter protein will fluores to give a signal. A reader in the machine will pick up the signal and the signal will be converted to picture form which is shown on the computer screen. So as the PCR cycles continues more reporter proteins will be released thus forming a graph.
Another kind of probe which is used is the molecular beacon (slightly more expensive i heard). Molecular beacons are in a hair pin structure with the 2 proteins at the ends of the DNA seqence. It will denature at the Denaturation step to form SSdna. Will bind to DNA at annealing and the reporter protein will be activated to give off the fluorescence signal.
The two probes above are the more commonly used probes due to the cost.
For pictorial form, please refer to the link below
http://www.iba-biotagnology.com/images/naps/rt_pcr.gif (Taq Man)
http://www.pentabase.com/Portals/0/EasyBeacons%20mekanisme.jpg (Molecular beacon)
Credits to
Dr. Chan for teaching me about RT-PCR on first week of SIP.
Cheers
Tiong Han
Tg01
0703762E
Taq Man is a RT Probe (the Cheaper one). Taq man is a DNA sequence with binded 2 proteins Reporter protein (at the 5' end) and quencher protein at the 3' end. The quencher protein will inhibit the reporter protein when they are both in the same DNA strand. This is due to the inhibition of proton transfer.
Basic: PCR processes are Denaturation, Annealing and Amplification.
At denaturation, DNA will become Single stranded DNA strands. At Annealing, the probes will bind to complementary base pairs along the targetted DNA strand. At Amplification, Taq Polymerase will bind to the DNA strands and produce copies of the DNA strands. The Taq polymerase will move down the targetted DNA strands to produce complementary strands. Once the Taq Polymerase will "free" the reporter protein once it hits the probe (Breaking up the probe). The released Reporter protein will fluores to give a signal. A reader in the machine will pick up the signal and the signal will be converted to picture form which is shown on the computer screen. So as the PCR cycles continues more reporter proteins will be released thus forming a graph.
Another kind of probe which is used is the molecular beacon (slightly more expensive i heard). Molecular beacons are in a hair pin structure with the 2 proteins at the ends of the DNA seqence. It will denature at the Denaturation step to form SSdna. Will bind to DNA at annealing and the reporter protein will be activated to give off the fluorescence signal.
The two probes above are the more commonly used probes due to the cost.
For pictorial form, please refer to the link below
http://www.iba-biotagnology.com/images/naps/rt_pcr.gif (Taq Man)
http://www.pentabase.com/Portals/0/EasyBeacons%20mekanisme.jpg (Molecular beacon)
Credits to
Dr. Chan for teaching me about RT-PCR on first week of SIP.
Cheers
Tiong Han
Tg01
0703762E
Saturday, September 12, 2009
cytogenetics[THERMOTRON & TECAN harvester]
Hello everybody,
in the blink of an eye, we are ending our attachment in less than 2months time. Hope everyone is still doing managing well. Anyway, back to the topic, i will be focusing about the two machines that are used in the cytogenetics lab. Cytogenetics is the study of chromosomes structures, its function and behaviour.
TECAN harvester
[The reagents]
As the name implies, it is a harvester however it is a robotic harvester that is involved in the harvesting of the cells. There are three major steps in the Harvesting procedure. The first step is Mitotic arrest,so as to arrest the cells in the metaphase stage because chromosomes are best studied at the metaphase stage as it is the clearest and at its most contracted state. The tecan harvester will add in Colcemid, a mitotic inhibitor that helps prevent spindle fiber formation, a process by which sister chromatids are pulled to opposite poles for incorporation into the 2 daughter cells. In addition,it also promotes chromosome condensation.
The second step is Hypotonic treatment whereby the cells are treated with a hypotonic saline solution to increase the cell volume so that chromosomes can spread out to allow easier identification.
The last step in harvesting is Fixation whereby the cells are fixed with addition of 3:1 methanol: glacial acetic acid so as to remove water from the cells and preserve them, by hardening the membrane and prepares chromosomes for the bending procedure.
[Drying of slides]
After which the slides are dried in the THERMOTRON before they are heated, stained and karyotyped.
hOPE this will give everybody some idea how harvesting of cells is carried out in the cytogentics lab that i am attaced to.
cheers,
Yong Herng
0702243G
in the blink of an eye, we are ending our attachment in less than 2months time. Hope everyone is still doing managing well. Anyway, back to the topic, i will be focusing about the two machines that are used in the cytogenetics lab. Cytogenetics is the study of chromosomes structures, its function and behaviour.
TECAN harvester
[The reagents]
As the name implies, it is a harvester however it is a robotic harvester that is involved in the harvesting of the cells. There are three major steps in the Harvesting procedure. The first step is Mitotic arrest,so as to arrest the cells in the metaphase stage because chromosomes are best studied at the metaphase stage as it is the clearest and at its most contracted state. The tecan harvester will add in Colcemid, a mitotic inhibitor that helps prevent spindle fiber formation, a process by which sister chromatids are pulled to opposite poles for incorporation into the 2 daughter cells. In addition,it also promotes chromosome condensation.
The second step is Hypotonic treatment whereby the cells are treated with a hypotonic saline solution to increase the cell volume so that chromosomes can spread out to allow easier identification.
The last step in harvesting is Fixation whereby the cells are fixed with addition of 3:1 methanol: glacial acetic acid so as to remove water from the cells and preserve them, by hardening the membrane and prepares chromosomes for the bending procedure.
[Drying of slides]
After which the slides are dried in the THERMOTRON before they are heated, stained and karyotyped.
hOPE this will give everybody some idea how harvesting of cells is carried out in the cytogentics lab that i am attaced to.
cheers,
Yong Herng
0702243G
Sunday, September 6, 2009
SUBJECT TITLE: Chemistry
Name of Test: G6PD Screening Test
Principle
Glucose-6-P reacts with NADP+ to produce gluconate-6-P and NADPH and H+ in the presence of G6PD. The NADPH produced in the reaction fluoresces under long-wave UV-light. If there is a marked deficiency of this enzyme, or if G-6-PD is lacking entirely, no fluorescence will be observed.
Materials
1. Timer (not necessary)
2. G6PD Reagent
3. Negative Control (Commercial Blood)
4. Pipette
5. Pipette tips
6. Filter paper (absorbent paper)
7. Gloves
8. Sample in EDTA tube
9. Hitachi Cups
10. Tube rack (to hold the sample tube & Hitachi cups)
11. Marker (for labelling purposes)
Method (using 1 patient’s sample)
1. One Hitachi cup was labelled with “Negative Control”.
2. Another Hitachi cup was labelled with the patient’s laboratory request number e.g. “08-48”.
3. “Negative Control” and “08-48” were written below respective circles on the filter paper.
4. 100ml of G6PD Reagent was pipetted into patient’s tube labelled e.g. “08-48” and “Negative Control”.
5. 5ml of patient’s EDTA Blood and Negative Control were pipetted into the labelled cups “08-48” and “Negative Control” respectively and mixed.
6. The pipette tips were remained in the cups.
7. Using the same pipette tips, 5ul of mixture from each cup was pipetted onto each circle on filter paper respectively as shown below as the 1st drop.
8. It was left at room temperature for 5 minutes.
9. After 5 minutes, the 2nd drop was pipetted and left for another 5 minutes.
10. After 5 minutes, the 3rd drop was pipetted and incubated for 10 minutes at 35°C.
11. After incubation, the filter paper was observed under long wave UV light using equipment shown below.
12. Result was recorded.
Note: EDTA tube would be passed to Haematology Section for ABO typing if the patient is an infant. Otherwise, the tube would be filed back.
Results and Interpretation
If fluorescence is seen under UV light, the result is recorded as “present” on the worksheet. For the Negative Control, there should not be any fluorescence seen. However, if fluorescence is seen using the patient’s blood, it indicates that the patient has the enzyme G6PD and therefore, it shows that the person is not suffering from G6PD Deficiency.
Before reading the results under UV light, ensure that the filter paper is dry. The sample obtained from a normal or slightly reduced G6PD activity will show a strong fluorescence. If there is absolutely no fluorescence after 10 minutes of incubation in all the 3 drops, it suggests that there is a total or significant deficiency of G6PD. If there is fluorescence in any of the 3 drops but the other 2 drops do not fluorescence, incubate for another 5 minutes and observe again.
During the test, ensure that the blood is well mixed with G6PD reagent. Otherwise, it can result in false positive result. In other words, the patient does not have G6PD Deficiency, but the result shows that he/she has.
By: Rebecca (0703363B)
Name of Test: G6PD Screening Test
Principle
Glucose-6-P reacts with NADP+ to produce gluconate-6-P and NADPH and H+ in the presence of G6PD. The NADPH produced in the reaction fluoresces under long-wave UV-light. If there is a marked deficiency of this enzyme, or if G-6-PD is lacking entirely, no fluorescence will be observed.
Materials
1. Timer (not necessary)
2. G6PD Reagent
3. Negative Control (Commercial Blood)
4. Pipette
5. Pipette tips
6. Filter paper (absorbent paper)
7. Gloves
8. Sample in EDTA tube
9. Hitachi Cups
10. Tube rack (to hold the sample tube & Hitachi cups)
11. Marker (for labelling purposes)
Method (using 1 patient’s sample)
1. One Hitachi cup was labelled with “Negative Control”.
2. Another Hitachi cup was labelled with the patient’s laboratory request number e.g. “08-48”.
3. “Negative Control” and “08-48” were written below respective circles on the filter paper.
4. 100ml of G6PD Reagent was pipetted into patient’s tube labelled e.g. “08-48” and “Negative Control”.
5. 5ml of patient’s EDTA Blood and Negative Control were pipetted into the labelled cups “08-48” and “Negative Control” respectively and mixed.
6. The pipette tips were remained in the cups.
7. Using the same pipette tips, 5ul of mixture from each cup was pipetted onto each circle on filter paper respectively as shown below as the 1st drop.
8. It was left at room temperature for 5 minutes.
9. After 5 minutes, the 2nd drop was pipetted and left for another 5 minutes.
10. After 5 minutes, the 3rd drop was pipetted and incubated for 10 minutes at 35°C.
11. After incubation, the filter paper was observed under long wave UV light using equipment shown below.
12. Result was recorded.
Note: EDTA tube would be passed to Haematology Section for ABO typing if the patient is an infant. Otherwise, the tube would be filed back.
Results and Interpretation
If fluorescence is seen under UV light, the result is recorded as “present” on the worksheet. For the Negative Control, there should not be any fluorescence seen. However, if fluorescence is seen using the patient’s blood, it indicates that the patient has the enzyme G6PD and therefore, it shows that the person is not suffering from G6PD Deficiency.
Before reading the results under UV light, ensure that the filter paper is dry. The sample obtained from a normal or slightly reduced G6PD activity will show a strong fluorescence. If there is absolutely no fluorescence after 10 minutes of incubation in all the 3 drops, it suggests that there is a total or significant deficiency of G6PD. If there is fluorescence in any of the 3 drops but the other 2 drops do not fluorescence, incubate for another 5 minutes and observe again.
During the test, ensure that the blood is well mixed with G6PD reagent. Otherwise, it can result in false positive result. In other words, the patient does not have G6PD Deficiency, but the result shows that he/she has.
By: Rebecca (0703363B)
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