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
Thursday, September 17, 2009
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)
Saturday, August 29, 2009
Cytology (Non-gynecology)
Hi everybody, This week is my turn to blog again =) I will talk about the things I have done in cytology as many people have been posted about histology stuff. For the first week in cytology lab, I had learnt to process gynecology specimens using the Thinprep 2000 processor. For the next 2 weeks, as we are not allowed to process non-gynecology specimens, so I had observed my trainers on how they process non-gynecology specimens, such as sputum, BAL, fluid, CSF and FNA. However, my supervisor allowed us to process our own urine or sputum samples so that we can learn how non-gynecology specimens are being processed. For the last week, I had learnt how to screen by screening their teaching slides.
Subject title: Lab Technique
Topic: Urine Papanicolaou Stain
Principle: The principle of this staining is to see detect whether the patient has any abnormalities in the kidney, bladder or the urethra. Such abnormalities can be infection, malignancy, reactive or carcinoma. Normal people should be negative which consists of urothelial cells. However, squamous cells might be also present in the urine as they shed from the trigone. Squamous cells consist of superficial cells, intermediate cells, parabasal cells and basal cells.
Steps involved: Mid stream urine is collected to ensure that the specimen is free from unnecessary contaminant. After the urine is collected, it is poured into a test tube and centrifuged at 2000 rpm for 10 minutes. The supernatant is poured away, leaving the cell pellet whereby it is dissolved in a few drops of Shandon fixative. The mixed solution is then put aside for 5-10 minutes to ensure that the cells are fixed. It is then cytocentrifuged where the mixture is being transferred onto the slides. The slides are then placed in 95% alcohol to fix the section so as to prevent the cells from dropping while staining. They are left aside for 15-20 minutes before putting into the staining machine for staining. The slides are mounted using Depex. They are screened to see whether there are any abnormalities such as infection or malignant.
Results
Normal urothelial and cervical cells are present. Below is a picture of normal urine papanicolaou stain.
However, if patient has any abnormalities such as infections, the papanicolaou stains will be different. Below are pictures on various types of infections.
Bacterial infection
Fungal infection
Viral infection
Patient might also be having urothelial carcinoma. Below is the picture of this example.
Things to note
Scums need to be removed every morning to ensure the quality of the slides are maintained.
If the specimen is malignant, all the staining solution must be filtered or stained to prevent contamination.
If anyone has any questions, feel free to ask me =)
Lok Pui
0704138G
References:
http://www.bostwicklaboratories.com/HOME/getdoc/f3882049-3705-4478-81b4-11112e4eb64b/Urinary-Tract-Infection.aspx
www.cytologystuff.com
Subject title: Lab Technique
Topic: Urine Papanicolaou Stain
Principle: The principle of this staining is to see detect whether the patient has any abnormalities in the kidney, bladder or the urethra. Such abnormalities can be infection, malignancy, reactive or carcinoma. Normal people should be negative which consists of urothelial cells. However, squamous cells might be also present in the urine as they shed from the trigone. Squamous cells consist of superficial cells, intermediate cells, parabasal cells and basal cells.
Steps involved: Mid stream urine is collected to ensure that the specimen is free from unnecessary contaminant. After the urine is collected, it is poured into a test tube and centrifuged at 2000 rpm for 10 minutes. The supernatant is poured away, leaving the cell pellet whereby it is dissolved in a few drops of Shandon fixative. The mixed solution is then put aside for 5-10 minutes to ensure that the cells are fixed. It is then cytocentrifuged where the mixture is being transferred onto the slides. The slides are then placed in 95% alcohol to fix the section so as to prevent the cells from dropping while staining. They are left aside for 15-20 minutes before putting into the staining machine for staining. The slides are mounted using Depex. They are screened to see whether there are any abnormalities such as infection or malignant.
Results
Normal urothelial and cervical cells are present. Below is a picture of normal urine papanicolaou stain.
However, if patient has any abnormalities such as infections, the papanicolaou stains will be different. Below are pictures on various types of infections.
Bacterial infection
Fungal infection
Viral infection
Patient might also be having urothelial carcinoma. Below is the picture of this example.
Things to note
Scums need to be removed every morning to ensure the quality of the slides are maintained.
If the specimen is malignant, all the staining solution must be filtered or stained to prevent contamination.
If anyone has any questions, feel free to ask me =)
Lok Pui
0704138G
References:
http://www.bostwicklaboratories.com/HOME/getdoc/f3882049-3705-4478-81b4-11112e4eb64b/Urinary-Tract-Infection.aspx
www.cytologystuff.com
Friday, August 21, 2009
The Staining :)
Hi everyone, it is my turn to post for this week. For this month, I have been posted to the routine staining area; thus, I have been exposed to various special stains like CAB, PAS, PASD, MT, Orcein, Fe, Ziehl Neelsen etc.
Today, I will introduce on PASD and Fe stain.
PASD (Periodic acid Schiff diastase)
Function: It helps to differentiate glycogen from other carbohydrates
Principle: Glycogen is digested by diastase where no reaction will occur with Schiff’s reaction afterwards.
Procedure:
1) Dewax and bring section to water
2) Treat section with diastase
3) Wash well with water
4) Treat with 1% aqueous periodic acid for 5 mins
5) Wash in water
6) Treat with schiff’s solution for 5 mins
7) Wash in water
8) Counter stain nuclei with haematoxylin
9) Blue in water
10)Dehydrate, clear and mount in DPX
Result:
Any presence of glycogen will be stain magenta on the PAS stained slide while for the PAS/D stained slide, there will be absence of it.
Note:
A positive control must always be used
A picture showing presence of glycogen stained magenta (PAS/D stain is used). This suggests that the patient is suffering from glycogen storage disease
Fe (Perl's reaction)
Function: Demonstration of iron and haemosiderin
Principle: Dilute hydrochloric acid solution release ferric iron that is tightly complxed to protein as hemoglobin. The ferric iron will then reacts with potassium ferrocyanide solution to produce an insoluble blue compound which is ferric ferrocyanide.
Reagent preparation:
Perl's reagent
20% hydrochloric acid -- 5 ml
20% potassium ferrocyanide --5ml
Procedure:
1) Dewax and bring section to water
2) Treat with perl's reagent for 20minutes
3) Rinse in SRW
4) Counterstain with nuclear fast red for 3 mins
5) Wash, dehydrate, clear and mount in DPX
Result:
Iron -- dark blue
Nuclei -- red
Perls Prussian blue stain showing hemachromatosis in cardiac muscle
Control: A positive control must also be used in this case
Staining is a very challenging job whereby you need to be able to multitask, however, i learn a lot of many different stains and the different things that need to be observed under the microscope.
Feel free to clarify any doubts and i will try my best to answer them :)
References:
http://images.google.com.sg/imgres?imgurl=http://img.medscape.com/pi/emed/ckb/pediatrics_genetics/941088-941089-941632-941708.jpg&imgrefurl=http://emedicine.medscape.com/article/941632-media&usg=__r1ZzgRvxBDwxsQKDdIiuE03bghw=&h=346&w=573&sz=37&hl=en&start=2&tbnid=x8c9fQVuwvM8JM:&tbnh=81&tbnw=134&prev=/images%3Fq%3Dperiodic%2Bacid%2Bschiff%2Bglycogen%26gbv%3D2%26hl%3Den
http://images.google.com.sg/imgres?imgurl=http://www.gladstone.ucsf.edu/gladstone/files/histology/Perls-Prussian-blue-stain.gif&imgrefurl=http://www.gladstone.ucsf.edu/gladstone/site/histology/section/2288&usg=__GqcH8PvwJO0Uffc2OAIeVfzDNgU=&h=331&w=504&sz=44&hl=en&start=7&tbnid=RpRio2w7otbr2M:&tbnh=85&tbnw=130&prev=/images%3Fq%3Dperls%2Breaction%26gbv%3D2%26hl%3Den%26sa%3DX
Cheers,
Zi Shuang
0703383J
Today, I will introduce on PASD and Fe stain.
PASD (Periodic acid Schiff diastase)
Function: It helps to differentiate glycogen from other carbohydrates
Principle: Glycogen is digested by diastase where no reaction will occur with Schiff’s reaction afterwards.
Procedure:
1) Dewax and bring section to water
2) Treat section with diastase
3) Wash well with water
4) Treat with 1% aqueous periodic acid for 5 mins
5) Wash in water
6) Treat with schiff’s solution for 5 mins
7) Wash in water
8) Counter stain nuclei with haematoxylin
9) Blue in water
10)Dehydrate, clear and mount in DPX
Result:
Any presence of glycogen will be stain magenta on the PAS stained slide while for the PAS/D stained slide, there will be absence of it.
Note:
A positive control must always be used
A picture showing presence of glycogen stained magenta (PAS/D stain is used). This suggests that the patient is suffering from glycogen storage disease
Fe (Perl's reaction)
Function: Demonstration of iron and haemosiderin
Principle: Dilute hydrochloric acid solution release ferric iron that is tightly complxed to protein as hemoglobin. The ferric iron will then reacts with potassium ferrocyanide solution to produce an insoluble blue compound which is ferric ferrocyanide.
Reagent preparation:
Perl's reagent
20% hydrochloric acid -- 5 ml
20% potassium ferrocyanide --5ml
Procedure:
1) Dewax and bring section to water
2) Treat with perl's reagent for 20minutes
3) Rinse in SRW
4) Counterstain with nuclear fast red for 3 mins
5) Wash, dehydrate, clear and mount in DPX
Result:
Iron -- dark blue
Nuclei -- red
Perls Prussian blue stain showing hemachromatosis in cardiac muscle
Control: A positive control must also be used in this case
Staining is a very challenging job whereby you need to be able to multitask, however, i learn a lot of many different stains and the different things that need to be observed under the microscope.
Feel free to clarify any doubts and i will try my best to answer them :)
References:
http://images.google.com.sg/imgres?imgurl=http://img.medscape.com/pi/emed/ckb/pediatrics_genetics/941088-941089-941632-941708.jpg&imgrefurl=http://emedicine.medscape.com/article/941632-media&usg=__r1ZzgRvxBDwxsQKDdIiuE03bghw=&h=346&w=573&sz=37&hl=en&start=2&tbnid=x8c9fQVuwvM8JM:&tbnh=81&tbnw=134&prev=/images%3Fq%3Dperiodic%2Bacid%2Bschiff%2Bglycogen%26gbv%3D2%26hl%3Den
http://images.google.com.sg/imgres?imgurl=http://www.gladstone.ucsf.edu/gladstone/files/histology/Perls-Prussian-blue-stain.gif&imgrefurl=http://www.gladstone.ucsf.edu/gladstone/site/histology/section/2288&usg=__GqcH8PvwJO0Uffc2OAIeVfzDNgU=&h=331&w=504&sz=44&hl=en&start=7&tbnid=RpRio2w7otbr2M:&tbnh=85&tbnw=130&prev=/images%3Fq%3Dperls%2Breaction%26gbv%3D2%26hl%3Den%26sa%3DX
Cheers,
Zi Shuang
0703383J
Saturday, August 15, 2009
Ahhh- CHOOO
I'm at a molecular lab. My current entry was very generic because i didnt mention anything about the current outbreak. Now the situation is getting better so i think it is okay to touch on the touchy subject.
In the Molecular lab, our assays are base on Polymerase Chain Reaction (PCR). There are 2 kinds of PCR, Real Time PCR (RT-PCR) and the conventional PCR. I discuss extraction of Flu sample using RT-PCRin this entry. Will talk more about the probes on the next entry and after that will be conventional method.
As all may know, at the initial stage of the outbreak, everyone is very worried. Everyone with sore throat and fever wants to know if they are infected with the famous "swine Flu". Culturing of the virus takes a long time. The faster way to get the confirmation will be detection of the virus DNA. DNA works by having complementary base pair (A--T, G---C) the number of dashes indicates the number of bonds between the different complementary base pair.
Lets start on how we (the staffs) go about testing.
Test ordered: H1N1 PCR
Specimen Type: Swab in VTM media (If specimen is for PCr and Culturing)
Dry Swab (For PCR. swab nose then throat.
Protocol: If samples is from the campus (Within the hospital), Inform the lab by calling the lab, before despatching. This is a highly recommened step to do especially if it is a VIP sample or urgent sample.
From outside campus, liase with lab incharge. SHE/he will say either we receive from you or no we are not going to do it for you.
Once we receive the sample (In the lab), information will be matched from the order from and the specimen. If it matches (matches 99.9% of the time), swabs will be sent into the Biological Safety Cabinet (BSC). Order form will be keyed in.
2 tubes will be label, 1 2ml Tube and 1 1.5ml eppendorf tube. the 2ml tube will be for preextraction and the eppendorf tube will be needed for post extraction.
500ul (Micro litres) of PBS will be dispensed into the 2ml tube and swabs will be broken in the tube and tube vortexed. Swab will then be removed (this step is to dislogde the virus into the PBS solution). If it is in a VTM media, about 1.5ml of media is dispensed into the 2ml tube.
Extraction is done by a machine. it is able to run 24 sample at 1 time. For respiratory virus, for this case it is considered, the lab uses off board lysis (lysis will be done out of the machine.)
Lysis buffer will be dispensed into a different wells. 1 well for 1 patient. samples are added and incubated for 10mins. this allows the virus to be lysed. Magnetic beads are then added to the wells. The machine does extraction by using magnetic beads. DNA is -ve charge and the machine is able to manipulate magnetivity to extract DNA.
There is about 55ul of elute/DNA buffer. so 55ul of elute is transfered to the 1.5ml tube.
For real time PCR, it enables you to amplify and get the results at the same time.
Samples wil be added to cappillary tubes which contains a DNA master mix, (F-primer, R-primer, Taq Polymerase, dNTP and some other things which is from the commerical kit (TAQ MAN PROBE) which willl flouresce when to give a reading.)
The capillary tubes are put into the lightcycler. 50 cycles of PCR will be runned. PCR- Denaturation 95ºC, annealing 56ºC, elongation 72ºC. At the end of each cycle, a sensor will read the amount of light given out by the probes to give a reading. If it is a postive case, a sigmoidal curve will appear. Then we will bless him (just kidding). Positive or negative cases, reports will be sent out. If it is a urgent sample, we will call the consultant or doctor.
ALong with this test, a FLU A RT-PCR is runned on another machine as a confirmatory test. IF Flu A Pos, h1n1 pos = Good luck home quarrentine/ observation. If Flu A pos, h1n1 neg= no h1n1. If Neg Flu A, pos h1n1 = troubleshooting to be done by lab personnel, results inconclusive.
RT-PCR will enable the lab to get results in 4-6hours, unlike culturing which may take days. Therefore, molecular techniques is still more prefered in the campus.
Disclaimer: I will not be able to find out the quantity of reagents added. Above knowledge is base on my observation in the lab and hope i dont offend anybody.
Cheers,
Yeo Tiong Han
TG01
P.s. I will get pictures from the lab. Have to ask permission first. 15/08/09
In the Molecular lab, our assays are base on Polymerase Chain Reaction (PCR). There are 2 kinds of PCR, Real Time PCR (RT-PCR) and the conventional PCR. I discuss extraction of Flu sample using RT-PCRin this entry. Will talk more about the probes on the next entry and after that will be conventional method.
As all may know, at the initial stage of the outbreak, everyone is very worried. Everyone with sore throat and fever wants to know if they are infected with the famous "swine Flu". Culturing of the virus takes a long time. The faster way to get the confirmation will be detection of the virus DNA. DNA works by having complementary base pair (A--T, G---C) the number of dashes indicates the number of bonds between the different complementary base pair.
Lets start on how we (the staffs) go about testing.
Test ordered: H1N1 PCR
Specimen Type: Swab in VTM media (If specimen is for PCr and Culturing)
Dry Swab (For PCR. swab nose then throat.
Protocol: If samples is from the campus (Within the hospital), Inform the lab by calling the lab, before despatching. This is a highly recommened step to do especially if it is a VIP sample or urgent sample.
From outside campus, liase with lab incharge. SHE/he will say either we receive from you or no we are not going to do it for you.
Once we receive the sample (In the lab), information will be matched from the order from and the specimen. If it matches (matches 99.9% of the time), swabs will be sent into the Biological Safety Cabinet (BSC). Order form will be keyed in.
2 tubes will be label, 1 2ml Tube and 1 1.5ml eppendorf tube. the 2ml tube will be for preextraction and the eppendorf tube will be needed for post extraction.
500ul (Micro litres) of PBS will be dispensed into the 2ml tube and swabs will be broken in the tube and tube vortexed. Swab will then be removed (this step is to dislogde the virus into the PBS solution). If it is in a VTM media, about 1.5ml of media is dispensed into the 2ml tube.
Extraction is done by a machine. it is able to run 24 sample at 1 time. For respiratory virus, for this case it is considered, the lab uses off board lysis (lysis will be done out of the machine.)
Lysis buffer will be dispensed into a different wells. 1 well for 1 patient. samples are added and incubated for 10mins. this allows the virus to be lysed. Magnetic beads are then added to the wells. The machine does extraction by using magnetic beads. DNA is -ve charge and the machine is able to manipulate magnetivity to extract DNA.
There is about 55ul of elute/DNA buffer. so 55ul of elute is transfered to the 1.5ml tube.
For real time PCR, it enables you to amplify and get the results at the same time.
Samples wil be added to cappillary tubes which contains a DNA master mix, (F-primer, R-primer, Taq Polymerase, dNTP and some other things which is from the commerical kit (TAQ MAN PROBE) which willl flouresce when to give a reading.)
The capillary tubes are put into the lightcycler. 50 cycles of PCR will be runned. PCR- Denaturation 95ºC, annealing 56ºC, elongation 72ºC. At the end of each cycle, a sensor will read the amount of light given out by the probes to give a reading. If it is a postive case, a sigmoidal curve will appear. Then we will bless him (just kidding). Positive or negative cases, reports will be sent out. If it is a urgent sample, we will call the consultant or doctor.
ALong with this test, a FLU A RT-PCR is runned on another machine as a confirmatory test. IF Flu A Pos, h1n1 pos = Good luck home quarrentine/ observation. If Flu A pos, h1n1 neg= no h1n1. If Neg Flu A, pos h1n1 = troubleshooting to be done by lab personnel, results inconclusive.
RT-PCR will enable the lab to get results in 4-6hours, unlike culturing which may take days. Therefore, molecular techniques is still more prefered in the campus.
Disclaimer: I will not be able to find out the quantity of reagents added. Above knowledge is base on my observation in the lab and hope i dont offend anybody.
Cheers,
Yeo Tiong Han
TG01
P.s. I will get pictures from the lab. Have to ask permission first. 15/08/09
Saturday, August 8, 2009
HISTOooing [ Reticulin stain & Frozen section]
Aloha everyone,
i was attached to HISTOPATHOLOGY on week 3 for three days. During thse 3 days, i managed to perform several technique such as tissue embedding, frozen section and special staining and observed many eye-opener stuffs. I got to personally see organs such as uterus, breast, kidney, as well as post-mortem of a stillbirth fetus(22weeks) being dissected and examined by pathologist. It was quite downhearted to see a dead feteus as from what the pathologist said, the baby has brain rupture which may have cause the stillbirth.
Anyway,I did a retic control for RETICULIN STAIN;
Reticulin is a type of fiber that provides structural support in tissues such as liver and kidney. The fibers in our normal liver are in well-defined strands however, an abnormal tissue such as a necrotic liver will have a discontinuous pattern. The positive control that was used is a normal liver, the reticular fibers should look black, when i see it under the microscope, it looks likes small little black dot, and the nuclei will be pinkish red.
Picture of reticular fiber:
http://neuromedia.neurobio.ucla.edu/campbell/connective_tissue/wp_images/34_reticular_fibers.gif
[Picture of retic control]
Next, i also managed to try out how frozen sectioning is like. Firstly frozen section is different from the paraffin sections, frozen sections comes without fixatives,or formalin. Wherever, there is frozen sections, i will follow the medical technologist to the operating theatre (OT) to obtain the fresh tissue excised during surgery.
FROZEN SECTIONING is a rapid diagnostic process whereby the pathologist will make a fast and rapid diagnosis to the surgeon whether the tissue is bengign or malignant,or to evaluate whether the tumour has been completely removed, is like the pathologist acts as a consultant to the surgeon to determine the extent of further surgery at the time of surgical procedure.
For example, if a tumour appears to have metastasized(spread),the suspected metastasis will then be sent for frozen section so that the pathologist will inform the surgeon whether or not to continue the surgery as there is no point in continuing the surgery if the tumour had metastasized.
So after the fresh tissue are obtained from OT, a small amount of OCT (optimum cooling temperature)compound is added onto the tissue and freezed immediately by liquid nitrogen. Thereafter which it is sectioned via a cryo-stat. The interesting part is the tissue section produced will be picked upon by a glass slide, is like you gently use the glas slide to touch the tissue section, the tissue will be condensed onto the slide and produce a smear. As compared to paraffin section it is different, whereby you need to fish it as we did during our histology practical. Next, the section are stained with Haemotoxylin and eosin (H&E), after which the pathologist will be called upon to examine and report the results. The whole procedure is a fast one, around 10 minutes or so, as it is an intraoperative procedure whereby the surgery is still ongoing so diagnosis has to be fast.
[Picture of OCT compound]
[cryocut]
[Picture of interior cryocut]
[Place Where staining is carried out]
[ Place where pathologist cut & examine the tissues]
Note:The above pictures taken 've been granted permission by my lab staff.=]
Signing off,
Yong Herng
0702243G
i was attached to HISTOPATHOLOGY on week 3 for three days. During thse 3 days, i managed to perform several technique such as tissue embedding, frozen section and special staining and observed many eye-opener stuffs. I got to personally see organs such as uterus, breast, kidney, as well as post-mortem of a stillbirth fetus(22weeks) being dissected and examined by pathologist. It was quite downhearted to see a dead feteus as from what the pathologist said, the baby has brain rupture which may have cause the stillbirth.
Anyway,I did a retic control for RETICULIN STAIN;
Reticulin is a type of fiber that provides structural support in tissues such as liver and kidney. The fibers in our normal liver are in well-defined strands however, an abnormal tissue such as a necrotic liver will have a discontinuous pattern. The positive control that was used is a normal liver, the reticular fibers should look black, when i see it under the microscope, it looks likes small little black dot, and the nuclei will be pinkish red.
Picture of reticular fiber:
http://neuromedia.neurobio.ucla.edu/campbell/connective_tissue/wp_images/34_reticular_fibers.gif
[Picture of retic control]
Next, i also managed to try out how frozen sectioning is like. Firstly frozen section is different from the paraffin sections, frozen sections comes without fixatives,or formalin. Wherever, there is frozen sections, i will follow the medical technologist to the operating theatre (OT) to obtain the fresh tissue excised during surgery.
FROZEN SECTIONING is a rapid diagnostic process whereby the pathologist will make a fast and rapid diagnosis to the surgeon whether the tissue is bengign or malignant,or to evaluate whether the tumour has been completely removed, is like the pathologist acts as a consultant to the surgeon to determine the extent of further surgery at the time of surgical procedure.
For example, if a tumour appears to have metastasized(spread),the suspected metastasis will then be sent for frozen section so that the pathologist will inform the surgeon whether or not to continue the surgery as there is no point in continuing the surgery if the tumour had metastasized.
So after the fresh tissue are obtained from OT, a small amount of OCT (optimum cooling temperature)compound is added onto the tissue and freezed immediately by liquid nitrogen. Thereafter which it is sectioned via a cryo-stat. The interesting part is the tissue section produced will be picked upon by a glass slide, is like you gently use the glas slide to touch the tissue section, the tissue will be condensed onto the slide and produce a smear. As compared to paraffin section it is different, whereby you need to fish it as we did during our histology practical. Next, the section are stained with Haemotoxylin and eosin (H&E), after which the pathologist will be called upon to examine and report the results. The whole procedure is a fast one, around 10 minutes or so, as it is an intraoperative procedure whereby the surgery is still ongoing so diagnosis has to be fast.
[Picture of OCT compound]
[cryocut]
[Picture of interior cryocut]
[Place Where staining is carried out]
[ Place where pathologist cut & examine the tissues]
Note:The above pictures taken 've been granted permission by my lab staff.=]
Signing off,
Yong Herng
0702243G
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