we have come to the end of SIP and this is my last post.
i am going to talk about my time in the histopathology section. in my workplace, the histo section is located right across the main lab. therefore it is separated from the main lab. there were many different sections in the histo lab. examples of the sections are the cytology section, immunochemistry section, trimming room, receiving section and sections where microtomy and embedding are done.
the trimming room is where different types organs are located at awaiting to be dissected and analysed by the pathologists. in the trimming room, there is a small section, equipped with its own bsc for passing to be done. passing is a process where the medical technologist will dissect smaller organs for example the appendix. the larger organs such as stomach, breasts, colon and other main organs will be dealt with in another bsc by the pathologists only. right before the process is done, the medical technologist will have to prepare the organ and the dissecting kits; such as scalpel, rulers and some casettes. the blade of the scalpel is disposable.the medical technologist will write down the descriptions that the pathologist will mention when dissecting a certain organ. the organs will arrive completely submerged in formalin. the formalin acts as a fixative for the organs.
when the pathologist is dissecting the organ, smaller portions will be cut out and placed in a number of casettes. these casettes will be sent for embedding and microtomy. the larger the organ, the more casettes will be needed as more portions will be cut out from different parts of the organs. in the embedding section, the tissues are completely submerged in hot wax. the metal molds will be filled with hot wax, the tissue will be placed in such a way that it will be easier to cut them into sections in the microtomy section. the mold is then placed on ice for the wax to harden. in the microtomy section, the tissue blocks will be cut into many sections. the sections will be placed in the water bath containing alcohol and then fished out onto a glass slide. the tissue on the glass slide will undergo dehydration and then staining in the automated stainer.
then the slides will be read under the microscope by the medical technologist.
Siti Nurfatin
0605853A
Saturday, November 8, 2008
Sunday, October 19, 2008
hihi everyone...3more weeks to go before the end of sip:)
anyway i was posted to the blood bank for this week... The blood bank operates 24hours everyday to provide the necessary testing so as to ensure the safe transfusion of blood and blood products. After spending the entire week in blood bank, I have a better understanding of the routine procedures that the laboratory is performing. During the week, I learnt the various types of tests that the laboratory is performing such as ABO blood grouping which consists of forward and reverse grouping, antibody screening, crossmatching and antibody titration. In addition, I also learnt how to process the blood stocks which will arrive from the CTM everyday.
now i am going to talk about something i have done everyday in the laboratory which is ABO Blood grouping and Rh (D) typing using the tube method. The ABO grouping is done to find out the blood group of the patients and to reconfirm the blood group of the donor. For ABO Blood grouping, there are forward and reverse grouping...for babies below 6months old, only forward grouping is done as babies at that age do not produce enough antibodies.
Method:
1. the patient's EDTA/clotted blood sample was centrifuged at 3000rpm for 6minutes.
2. 7 test tubes were being labeled and prepared
3. the first 4 tubes was added with one drop of each anti-sera: Anti-D, Anti-A, Anti-B and Anti-AB respectively. (forward grouping)
4. the last 3 tubes was added with one drop each of commercial A cells, B cells and O cells. (reverse grouping)
5. one drop of 3-5% saline suspension patient's red blood cells were added to each of the first 4 tubes.
6.one drop of patient's serum was added to each of the last 3 tubes.
7. the 7 tubes were mixed well and centrifuged for 15 seconds at 2000rpm.
8. after centrifuging, the tubes were gently agitated to dislodge the packed button of red cells and the tubes were examined macroscopically for any agglutination. for weak reactions, such as the big agglutinates could not be seen, they were examined under the microscope.
9. the results were recorded.
Agglutination Grading Chart
4+: A single agglutinate, no free cells can be seen
3+: A few large agglutinates
2+: Many agglutinates which consists of medium and small ones, no free cells can be seen,
1+: Prescence of many very small agglutinates, free red cells can be seen
0: An even red cell suspension with no agglutination seen.
thats about it:)feel free to ask any qns:)
Rachael
Tg01
0606168C
anyway i was posted to the blood bank for this week... The blood bank operates 24hours everyday to provide the necessary testing so as to ensure the safe transfusion of blood and blood products. After spending the entire week in blood bank, I have a better understanding of the routine procedures that the laboratory is performing. During the week, I learnt the various types of tests that the laboratory is performing such as ABO blood grouping which consists of forward and reverse grouping, antibody screening, crossmatching and antibody titration. In addition, I also learnt how to process the blood stocks which will arrive from the CTM everyday.
now i am going to talk about something i have done everyday in the laboratory which is ABO Blood grouping and Rh (D) typing using the tube method. The ABO grouping is done to find out the blood group of the patients and to reconfirm the blood group of the donor. For ABO Blood grouping, there are forward and reverse grouping...for babies below 6months old, only forward grouping is done as babies at that age do not produce enough antibodies.
Method:
1. the patient's EDTA/clotted blood sample was centrifuged at 3000rpm for 6minutes.
2. 7 test tubes were being labeled and prepared
3. the first 4 tubes was added with one drop of each anti-sera: Anti-D, Anti-A, Anti-B and Anti-AB respectively. (forward grouping)
4. the last 3 tubes was added with one drop each of commercial A cells, B cells and O cells. (reverse grouping)
5. one drop of 3-5% saline suspension patient's red blood cells were added to each of the first 4 tubes.
6.one drop of patient's serum was added to each of the last 3 tubes.
7. the 7 tubes were mixed well and centrifuged for 15 seconds at 2000rpm.
8. after centrifuging, the tubes were gently agitated to dislodge the packed button of red cells and the tubes were examined macroscopically for any agglutination. for weak reactions, such as the big agglutinates could not be seen, they were examined under the microscope.
9. the results were recorded.
Agglutination Grading Chart
4+: A single agglutinate, no free cells can be seen
3+: A few large agglutinates
2+: Many agglutinates which consists of medium and small ones, no free cells can be seen,
1+: Prescence of many very small agglutinates, free red cells can be seen
0: An even red cell suspension with no agglutination seen.
thats about it:)feel free to ask any qns:)
Rachael
Tg01
0606168C
Sunday, October 12, 2008
16th week
Welcome to the end of the 16th week of SIP/MP :)
For my final post, I would like to share my experience for the last week during TSO duty. This week, I'm tasked to calibrate the instruments in the Inorganic Chemistry Lab. The instruments present is analytical balance, pH meter and top-pan balance.
The protocols for each analytical balance differs due to their own brand which is similar to a handphone. For example; different brand of handphones has different type of functions even though the principle is similar. Hence, a protocol is set aside for each type of brand of analytical balance. Internal calibration is usually automated by the machine itself.
To calibrate the analytical balance, we must first prepare the analytical balance for calibration by cleaning it, ensure that the linearity is correct and change its setup to calibration mode. Although the setup to calibration mode differs from other brands of analytical balance, the main calibration process is similar. Weights of 100g, 200g and 500g is placed for calibration. This weights are specially made and to handle them, we must use the gloves that comes with the weights. If we handle the weights with our bare hands, moisture will be present on the weights and it might produce a wrong reading.
Once the weights are placed and the machine is calibrated, it is tested by placing a weight of 200g three times to ensure that the analytical balance is properly calibrated.
The top-pan balance is similar to the analytical balance however, instead of using weights of 100g, 200g and 500g, we use weights of 2kg and 4kg. After the top-pan balance is calibrated, weight of 4kg is placed on the top-pan balance three times to ensure that the top-pan balance is properly calibrated.
The pH meter is calibrated by using commercially-prepared buffer solution with a fix pH reading of 4.00, 7.00 and 10.00. The pH meter is prepared for calibration by deleting any previous standardization and then setting up the mode for new standardization using the buffer solution provided. The electrode, which is the tool that detects the reading of pH must be washed with DI water before being placed into the different buffer solution to prevent any mis-reading. The pH meter is then tested in the 7.00 buffer solution to ensure that it is calibrated properly.
Calibration of instruments is a very direct process and if we follow the protocol religiously, there should not be any mistakes. If under any circumstances the instruments is faulty, just inform the technician and they will repair the instruments.
Andika Putra
TG01
For my final post, I would like to share my experience for the last week during TSO duty. This week, I'm tasked to calibrate the instruments in the Inorganic Chemistry Lab. The instruments present is analytical balance, pH meter and top-pan balance.
The protocols for each analytical balance differs due to their own brand which is similar to a handphone. For example; different brand of handphones has different type of functions even though the principle is similar. Hence, a protocol is set aside for each type of brand of analytical balance. Internal calibration is usually automated by the machine itself.
To calibrate the analytical balance, we must first prepare the analytical balance for calibration by cleaning it, ensure that the linearity is correct and change its setup to calibration mode. Although the setup to calibration mode differs from other brands of analytical balance, the main calibration process is similar. Weights of 100g, 200g and 500g is placed for calibration. This weights are specially made and to handle them, we must use the gloves that comes with the weights. If we handle the weights with our bare hands, moisture will be present on the weights and it might produce a wrong reading.
Once the weights are placed and the machine is calibrated, it is tested by placing a weight of 200g three times to ensure that the analytical balance is properly calibrated.
The top-pan balance is similar to the analytical balance however, instead of using weights of 100g, 200g and 500g, we use weights of 2kg and 4kg. After the top-pan balance is calibrated, weight of 4kg is placed on the top-pan balance three times to ensure that the top-pan balance is properly calibrated.
The pH meter is calibrated by using commercially-prepared buffer solution with a fix pH reading of 4.00, 7.00 and 10.00. The pH meter is prepared for calibration by deleting any previous standardization and then setting up the mode for new standardization using the buffer solution provided. The electrode, which is the tool that detects the reading of pH must be washed with DI water before being placed into the different buffer solution to prevent any mis-reading. The pH meter is then tested in the 7.00 buffer solution to ensure that it is calibrated properly.
Calibration of instruments is a very direct process and if we follow the protocol religiously, there should not be any mistakes. If under any circumstances the instruments is faulty, just inform the technician and they will repair the instruments.
Andika Putra
TG01
Saturday, September 27, 2008
Stool Analysis
Hey guys. Previously I talked about routine section where urine is processed for different kinds of tests. For now, I want to talk about how stool is processed to test for ova or cyst. The tests are done to identify intestinal parasites and their eggs which are known as ova or cysts in patients. The patients may or may not experience any symptoms of gastrointestinal infection. One common symptom may be diarrhea. Another symptom is blood in stool. When we are able to identify the particular parasite in the patient, we may know the cause of the gastrointestinal infection and thus we can provide the appropriate medication to treat it.
The principle of the test involves the saline wet mount. It is the recommended test to detect helminth eggs and larvae. It is also good for motile protozoan cysts which appear to be refractile. Refractile means that granules in the cells that refracts light. When cysts are detected in the saline wet mount, they are observed in Lugol iodine stained preparation. This stain will reveal the nuclear structure of the cells. The nuclei stain will be stained brown and thus can be counted easily. A kit called Para-pak spin con is a commercial kit that is used to detect the faecal parasite concentration of eggs, larvae and protozoa. Saline wet mount technique involves placing the sediment of the specimen on a glass slide. It is mixed with saline and a cover slip is place over it. A drop or two of Lugol iodine is placed on the sediment. Then, the slide is directly examined under the microscope. This technique also provides information on the content of the stool such as the presence of leukocytes.
The procedure to obtain the sediment of the specimen (stool) is by emulsifying 0.5g of stool with 10ml of saline in a plastic tube. 4 drops of CON-trate Reagent A are added to enhance the breakdown of faecal aggregates and mucus, causing the parasites to be exposed. The contents are mixed thoroughly. The mixture is then filtered using the Para-pak filter into the centrifuge tubes provided. The filtered mixture is then centrifuged at 2000rpm for 10min.
10min later, the supernatant is discarded and the sediment is resuspended. This is where the wet mount saline is performed on the sediment.
The limitations of this procedure are such as false-negatives occurring due to low number of parasites in the stool specimen. The parasites may undergo intermittent shedding which contributes to the low number. Inappropriate transport or storage may also contribute to false-negatives. False positives may occur when one of the numerous structures in the stool is mistaken for a parasite. There is no reference range for this test. The presence of normal bacteria in stool is considered normal.
Siti Nurfatin
0605853a
The principle of the test involves the saline wet mount. It is the recommended test to detect helminth eggs and larvae. It is also good for motile protozoan cysts which appear to be refractile. Refractile means that granules in the cells that refracts light. When cysts are detected in the saline wet mount, they are observed in Lugol iodine stained preparation. This stain will reveal the nuclear structure of the cells. The nuclei stain will be stained brown and thus can be counted easily. A kit called Para-pak spin con is a commercial kit that is used to detect the faecal parasite concentration of eggs, larvae and protozoa. Saline wet mount technique involves placing the sediment of the specimen on a glass slide. It is mixed with saline and a cover slip is place over it. A drop or two of Lugol iodine is placed on the sediment. Then, the slide is directly examined under the microscope. This technique also provides information on the content of the stool such as the presence of leukocytes.
The procedure to obtain the sediment of the specimen (stool) is by emulsifying 0.5g of stool with 10ml of saline in a plastic tube. 4 drops of CON-trate Reagent A are added to enhance the breakdown of faecal aggregates and mucus, causing the parasites to be exposed. The contents are mixed thoroughly. The mixture is then filtered using the Para-pak filter into the centrifuge tubes provided. The filtered mixture is then centrifuged at 2000rpm for 10min.
10min later, the supernatant is discarded and the sediment is resuspended. This is where the wet mount saline is performed on the sediment.
The limitations of this procedure are such as false-negatives occurring due to low number of parasites in the stool specimen. The parasites may undergo intermittent shedding which contributes to the low number. Inappropriate transport or storage may also contribute to false-negatives. False positives may occur when one of the numerous structures in the stool is mistaken for a parasite. There is no reference range for this test. The presence of normal bacteria in stool is considered normal.
Siti Nurfatin
0605853a
Friday, September 26, 2008
Coagulation
Hey everyone. With only a few weeks down to the end of SIP, I bet everyone's been working hard on their MP. Anyway, for my turn in blogging, I'll touch on what I've learnt in the Coagulation section of the lab, which deals with blood samples that have been placed in 3.2% sodium citrate tubes. (shown above)
In this entry I'll highlight a particular test that is the most highly requested test for my section.
And that would be the test for Prothrombin Time (PT).
Prothrombin time is a test used to evaluate the extrinsic coagulation of the system. It can screen for congenital deficiencies of factors II, V, VII and X. PT can also monitor anticoagulant therapy (eg. warfarin medication), which is usually given on a long-term basis to patients who suffer from recurrent inappropriate blood clotting. The measurement of PT can aid in the control of the dosage of the drugs. PT is normally measured in seconds and the INR (Intl Normalized Ratio)
INR = (patient's results / normal patient average)
The ref range for patients on warfarin should be between 2.0-3.0. Those with high risk of clotting have a ref range of 2.5-3.5.
PT is usually evaluated with the results of Activated Partial Thromboplastin Time (aPTT) to assess the coagulation system better.
It irradiates red light at 660nm onto a mixture of blood plasma with added reagent (Dade Innovin) and it will read a change in turbidity as the fibrin clots are formed. This measures the coagulation time.
It also uses the Coagulation Point Detection Method (Percentage Detection Method) to calculate the coagulation time. This is considered as the time taken to achieve the amount of scattered light that is set for the coagulation detection point, susing the amount of scattered light that is present just after the start of detection as 0% and the amount of light scattered that is present at the completion of coagulation as 100%.
Antibiotics, aspirin, and cimetidine can increase the PT/INR. Barbiturates, oral contraceptives and hormone-replacement therapy (HRT), and vitamin K - either in a multivitamin or liquid nutrition supplement - can decrease PT. Certain foods (such as beef, green tea, broccoli, chickpeas, kale, turnip greens, and soybean products) contain large amounts of vitamin K and can alter PT results.
In this entry I'll highlight a particular test that is the most highly requested test for my section.
And that would be the test for Prothrombin Time (PT).
Prothrombin time is a test used to evaluate the extrinsic coagulation of the system. It can screen for congenital deficiencies of factors II, V, VII and X. PT can also monitor anticoagulant therapy (eg. warfarin medication), which is usually given on a long-term basis to patients who suffer from recurrent inappropriate blood clotting. The measurement of PT can aid in the control of the dosage of the drugs. PT is normally measured in seconds and the INR (Intl Normalized Ratio)
INR = (patient's results / normal patient average)
The ref range for patients on warfarin should be between 2.0-3.0. Those with high risk of clotting have a ref range of 2.5-3.5.
PT is usually evaluated with the results of Activated Partial Thromboplastin Time (aPTT) to assess the coagulation system better.
To run the test, my lab uses the Sysmex Ca-1500. We first check the tubes for clot then spin the sodium citrate tubes at 6000rpm for 3minutes. We then load the samples into racks and place them on the machine.The caps need not be removed as the machine has a probe that can pierce through the cap.
It works by using the Coagulation Reaction Detection Method (Scattered Light Detection Method)
It irradiates red light at 660nm onto a mixture of blood plasma with added reagent (Dade Innovin) and it will read a change in turbidity as the fibrin clots are formed. This measures the coagulation time.
It also uses the Coagulation Point Detection Method (Percentage Detection Method) to calculate the coagulation time. This is considered as the time taken to achieve the amount of scattered light that is set for the coagulation detection point, susing the amount of scattered light that is present just after the start of detection as 0% and the amount of light scattered that is present at the completion of coagulation as 100%.
Antibiotics, aspirin, and cimetidine can increase the PT/INR. Barbiturates, oral contraceptives and hormone-replacement therapy (HRT), and vitamin K - either in a multivitamin or liquid nutrition supplement - can decrease PT. Certain foods (such as beef, green tea, broccoli, chickpeas, kale, turnip greens, and soybean products) contain large amounts of vitamin K and can alter PT results.
hi everyone, i am here to blog again...anyway i realised that many people tend to spell my name wrongly...my name is spelt as Rachael not Racheal:) haha...anyway...in the previous post i talked about blood culturing...in this post i would like to continue from the previous post to talk about blood harvesting...
In the harvesting process, the purpose of the mitotic arrestment is to prevent spindle fibre formation so that the cell division can be blocked at metaphase stage. After which, there will be hypotonic treatment which is to increase the cell volume so that the chromosomes can be spread out and lastly, it will be the addition of fixative to remove water from the cells so as to preserve them.
Method:
1.The hypotonic solution was warmed in 37ºC water bath before the start of harvest process.
2.50µl of colcemid was added to each tube and one tube was exposed to colcemid for 20minutes while the other was exposed to colcemid for 2hours.
3.The tubes were centrifuged at 1200rpm for 10minutes and the supernatant was aspirated.
4.The pellet was loosen gently and 14ml of warmed hypotonic solution was added into each tube and the tubes were inverted 2-3 times to ensure that the suspension is well mixed.
5.The tubes were left at room temperature for 5minutes.
6.After 5minutes, the tubes were transferred into a covered centrifuge bucket and spun at 1500rpm for 6minutes.
7.The supernatant was aspirated and discarded.
8.The pellet was loosen gently and approximately 12ml of warmed hypotonic solution was added into each tube and the tubes were inverted 2-3 times to ensure that the suspension is well mixed.
9.Step 5 was repeated.
10.The required amount of fresh fix was prepared based on the number of cases to be harvested. (40ml per case at room temperature)
11.After 5minutes, 1pipetteful of fixative was added and the tubes were inverted 2-3 times to mix well.
12.Steps 6 and 7 were repeated.
13.The cell pellet was resuspended by flicking the tubes until no visible clumps can be seen.
14.Another 10-12ml of fixative was added and the tubes were inverted 2-3 times to mix well.
15.The tubes were stand at room temperature for at least 15minutes.
16.The tubes were centrifuge at 1000rmp for 10minutes and the supernatant was aspirated and discarded.
17.The pellet was resuspended in 8-10ml of fixative and step 16 was repeated. This step would be repeated if there clumpy or jelly like pellets are still present.
18.The pellet was resuspended in 5-7ml of fixative and was stored in refrigerator at 4ºC until ready to make slide.
when the slides are made, the chromosomes are ready to be analysed under the fluorescence microscope.thats about it:)
Rachael
0606168C
Tg01
In the harvesting process, the purpose of the mitotic arrestment is to prevent spindle fibre formation so that the cell division can be blocked at metaphase stage. After which, there will be hypotonic treatment which is to increase the cell volume so that the chromosomes can be spread out and lastly, it will be the addition of fixative to remove water from the cells so as to preserve them.
Method:
1.The hypotonic solution was warmed in 37ºC water bath before the start of harvest process.
2.50µl of colcemid was added to each tube and one tube was exposed to colcemid for 20minutes while the other was exposed to colcemid for 2hours.
3.The tubes were centrifuged at 1200rpm for 10minutes and the supernatant was aspirated.
4.The pellet was loosen gently and 14ml of warmed hypotonic solution was added into each tube and the tubes were inverted 2-3 times to ensure that the suspension is well mixed.
5.The tubes were left at room temperature for 5minutes.
6.After 5minutes, the tubes were transferred into a covered centrifuge bucket and spun at 1500rpm for 6minutes.
7.The supernatant was aspirated and discarded.
8.The pellet was loosen gently and approximately 12ml of warmed hypotonic solution was added into each tube and the tubes were inverted 2-3 times to ensure that the suspension is well mixed.
9.Step 5 was repeated.
10.The required amount of fresh fix was prepared based on the number of cases to be harvested. (40ml per case at room temperature)
11.After 5minutes, 1pipetteful of fixative was added and the tubes were inverted 2-3 times to mix well.
12.Steps 6 and 7 were repeated.
13.The cell pellet was resuspended by flicking the tubes until no visible clumps can be seen.
14.Another 10-12ml of fixative was added and the tubes were inverted 2-3 times to mix well.
15.The tubes were stand at room temperature for at least 15minutes.
16.The tubes were centrifuge at 1000rmp for 10minutes and the supernatant was aspirated and discarded.
17.The pellet was resuspended in 8-10ml of fixative and step 16 was repeated. This step would be repeated if there clumpy or jelly like pellets are still present.
18.The pellet was resuspended in 5-7ml of fixative and was stored in refrigerator at 4ºC until ready to make slide.
when the slides are made, the chromosomes are ready to be analysed under the fluorescence microscope.thats about it:)
Rachael
0606168C
Tg01
Thursday, September 11, 2008
Hi everyone, time flies and our sip is ending soon. Anyway, for this month, I was posted to the cytogenetics laboratory which I found it very interesting because this subject is very new to me. In this laboratory, I can analyze my own chromosomes using peripheral blood and fortunately, my chromosomes turned out to be normal. Now, I am going to talk about the procedures to culture peripheral blood for chromosomes studies.
The purpose to culture peripheral blood for chromosomes studies (karyotyping) is to diagnose genetic diseases for e.g. Down’s syndrome, determine the karyotype of families of individuals with chromosome abnormalities, determine carrier status, to confirm structure, numerical abnormalities and/or mosaicism etc. The principle of this test is that the white blood cells (T-lymphocytes) present in the peripheral blood will be exposed to a mitogen (PHA). The function of the mitogen is to stimulate cell division and the cells are arrested at metaphase by the addition of colcemid. The purpose of arresting at metaphase is because at the metaphase phase stage, the cells are at their most contracted stage and the least metabolitically active state and thus chromosomes are analyzed at this stage. Theses metaphases are harvested and spread onto a glass slide for analysis.
The reagents used are:
•Complete RPMI media which contains RPMI, fetal calf serum, penicillin and streptomycin and L-Glutamine
•Complete M199 media which contains M199, fetal calf serum, penicillin and streptomycin, L-Glutamine and sodium heparin
•Sodium heparin which contains sodium heparin and sterile water
•Phytohaemagglutinin (PHA) M Form
•Amethopterin (Methotrexate) (MTX)
•Thymidine
Specimen requirement: 3-5ml of peripheral blood in sterile tube with sodium heparin (anti-coagulant). Collection of blood is done using aseptic techniques.
Method:
•1 tube of complete RPMI medium and 1 tube of complete M199 medium were thawed in water bath at 37ºC.
•The blood specimen was centrifuge at 1200rpm for 10minutes. The volume of blood and condition of blood was recorded.
•The tubes were labeled with the patients’ accession number, patient’s name and date of set-up.
•A sterile transfer pipette was used to pick approximately 0.5-1ml of buffy coat together with some plasma from the blood sample.
•The transfer pipette was inverted and the sample was mixed thoroughly in the bulb. The sample was distributed into the culture tubes and the number of drops added into each tube was noted down.
•0.1ml of PHA was added into each tube.
•The caps were tighten and mixed well by inverting the culture tubes several times.
•The culture tubes were placed in a slant test tube rack to increase the surface area of the cultures for gaseous exchange. The rack was placed in a 37ºC incubator for 48hours.
•The tubes were inverted every morning to resuspend the pellet.
•50µl of MTX 10-5M working solution was added to each tube after 48 hours of incubation. The tubes were mixed well by inverting and were re-incubated at 37ºC for another 18hours.
•50µl of thymidine was added to each tube after 18 hours of incubation with MTX.
•The samples were mixed well and incubated at 37ºC for 4hours.
The function of MTX is to synchronize the cells by stopping the cells in the S phase of cell cycle and collect a large amount of cells which will then go into division together. Thymidine is used to release cells blocked by the MTX.
There are guidelines to determine the amount of blood needed to add to each tube. For neonates less than one year, 4-5 drops was added. For children 1-6years old, 5-6 drops was added. For children 7-13years old, 6-7 drops was added. For males older than 13years old, 7-8 drops was added and for females older than 13years old, 8-12 drops was added.
After blood culturing, the next step will blood harvesting. I will leave the harvesting procedure to the next person who will go to cytogenetics laboratory after me. :)
Rachael
Tg01
0606168C
The purpose to culture peripheral blood for chromosomes studies (karyotyping) is to diagnose genetic diseases for e.g. Down’s syndrome, determine the karyotype of families of individuals with chromosome abnormalities, determine carrier status, to confirm structure, numerical abnormalities and/or mosaicism etc. The principle of this test is that the white blood cells (T-lymphocytes) present in the peripheral blood will be exposed to a mitogen (PHA). The function of the mitogen is to stimulate cell division and the cells are arrested at metaphase by the addition of colcemid. The purpose of arresting at metaphase is because at the metaphase phase stage, the cells are at their most contracted stage and the least metabolitically active state and thus chromosomes are analyzed at this stage. Theses metaphases are harvested and spread onto a glass slide for analysis.
The reagents used are:
•Complete RPMI media which contains RPMI, fetal calf serum, penicillin and streptomycin and L-Glutamine
•Complete M199 media which contains M199, fetal calf serum, penicillin and streptomycin, L-Glutamine and sodium heparin
•Sodium heparin which contains sodium heparin and sterile water
•Phytohaemagglutinin (PHA) M Form
•Amethopterin (Methotrexate) (MTX)
•Thymidine
Specimen requirement: 3-5ml of peripheral blood in sterile tube with sodium heparin (anti-coagulant). Collection of blood is done using aseptic techniques.
Method:
•1 tube of complete RPMI medium and 1 tube of complete M199 medium were thawed in water bath at 37ºC.
•The blood specimen was centrifuge at 1200rpm for 10minutes. The volume of blood and condition of blood was recorded.
•The tubes were labeled with the patients’ accession number, patient’s name and date of set-up.
•A sterile transfer pipette was used to pick approximately 0.5-1ml of buffy coat together with some plasma from the blood sample.
•The transfer pipette was inverted and the sample was mixed thoroughly in the bulb. The sample was distributed into the culture tubes and the number of drops added into each tube was noted down.
•0.1ml of PHA was added into each tube.
•The caps were tighten and mixed well by inverting the culture tubes several times.
•The culture tubes were placed in a slant test tube rack to increase the surface area of the cultures for gaseous exchange. The rack was placed in a 37ºC incubator for 48hours.
•The tubes were inverted every morning to resuspend the pellet.
•50µl of MTX 10-5M working solution was added to each tube after 48 hours of incubation. The tubes were mixed well by inverting and were re-incubated at 37ºC for another 18hours.
•50µl of thymidine was added to each tube after 18 hours of incubation with MTX.
•The samples were mixed well and incubated at 37ºC for 4hours.
The function of MTX is to synchronize the cells by stopping the cells in the S phase of cell cycle and collect a large amount of cells which will then go into division together. Thymidine is used to release cells blocked by the MTX.
There are guidelines to determine the amount of blood needed to add to each tube. For neonates less than one year, 4-5 drops was added. For children 1-6years old, 5-6 drops was added. For children 7-13years old, 6-7 drops was added. For males older than 13years old, 7-8 drops was added and for females older than 13years old, 8-12 drops was added.
After blood culturing, the next step will blood harvesting. I will leave the harvesting procedure to the next person who will go to cytogenetics laboratory after me. :)
Rachael
Tg01
0606168C
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