Sunday, July 29, 2007
Check the fetus’ chromosomes at metaphase to see if it is normal.
A normal set of chromosome has
- a total chromosomal count of 46 consisting of a pair of each chromosome(1-22) and the sex chromosome- either XX or XY.
1) Detect abnormalities in fetus of women with advanced maternal age
With a maternal age of 35 and above, the chances of carrying a fetus with genetic abnormality increases. Common genetic abnormalities include Down’s syndrome (Trisomy 21) and Edward’s syndrome (Trisomy 18).
2) Doctors noticed abnormalities during ultrasound
During ultrasound, doctors notice something amiss about the baby’s condition and with may send AF out for test.
Why Aminotic Fluid?
AF is present all around in the placenta (which holds the baby). As the baby grows, it will shed cells in to the AF. Therefore, the AF contains baby cells that can be cultured and analysis for chromosomal abnormality.
How is it process?
Collection – Processing (Set-up) – Harvest – Drying – Baking – Staining -- Analysis
Firstly, the AF will be equally allocated into 3 tubes, one of which will be kept as a back-up. The tubes will then be centrifuged. After the supernatant is removed, culture media is added to resuspend the cells. Different media e.g. (will usually use) BioAMF and Chang’s Media will be used in the 3 tubes, this is part of quality control.
Secondly, the cell suspension will be aliquot into 4 petri dish containing cover-slips. They will then be incubated and the cells will eventually grow directly on the cover-slips.
After the cells have grown to form about 4 colonies, they will be harvested. Harvesting is done automatically using an auto-harvestor- Tecan. Tecan is able to carry out the entire harvesting process independently and during this period the technologist is able to carry out other duties.
Tecan will carry out the following processes:
-remove the culture media via a pump
-add hypotonic solution (KCl)
-timed an incubation period of 30mins in hypo
-add fixative- made up of methanol and acetic acid.
-timed an incubation of 30mins in fixative
-change another round of fixative
When cells are exposed to hypotonic solution, they will swell and this allows the chromosomes to spread out and not clump together (which will be difficult for analysis). The addition of fixative removes water and preserves cells in its swelled-up state.
After the harvesting process is done, the Petri dish containing the cover-slip will be transferred into the Thermotron for further removal of water aka Drying. Drying involves the addition and removal of fixatives up to 3 times for complete dehydration of cell and to spreading the cells out. Thermotron is a incubator that facilitates the drying process, it is able control the humidity and temperature thus promoting optimal drying. This process is important as:
- if drying is too fast(low humidity, high temp), chromosomes won't be able to spread out effectively
-if drying is too slow(high humidity, low temp), chromosomes will spread so much that it goes beyond the cover-slip and thus will be washed away.
Next, the cover-slip will be remove from the Petri dish and be mounted on a glass slide. The glass slide will then be baked in the hot plate at 90°C for 11/2 hours; this is to fix the cells on the slide to prevent it from floating away during staining. Baking also causes the A-T bonds to dissociate thus allowing the dye to be taken up by the chromosome.
After which, cells will be stain in either Wright’s or Geimsa to give a visible/prominent light and dark bands on the chromosomes for analysis. The staining process involves the trysinisation of cells. Trypsin hydrolyses protein component of the chromatin thereby allowing the dye to react(dye) with the exposed DNA.
Finally, after staining, it's ANALYSIS time. Real-experienced med techs will study and identify the chromosomes and detect any deletion, addition or presence of extra chromosome with their naked eyes. They're real quick in identifying them!
Alrights, here's the end, if there's any question, just bring it on =)
Sunday, July 22, 2007
Alright basically i've learnt tons of new stuff at microbiology but i'm going to relate to you about my experiences of working the "Affirm VPIII Microbial Identification test" by Becton Dickinson. Basically the whole process of the experiment/test, involves both manual and automated parts.d Sample preparation is done manually, while the processing is done by the machine BD MicroProbe. I will elaborate more below.
The Affirm VPIII Microbial Identification Test is a DNA probe test, used by the laboratory for the detection and identification of Candida species, Gardnerella vaginalis and Trichomonas vaginalis nucleic acid in vaginal fluid specimens from patients with symptoms of vaginitis.
Vaginitis is one of the most common problems observed in clinical medicine. There are 3 main categories of vaginitis, they are bacterial vaginosis (BV), yeast vaginitis (Candidiasis) and trichomoniasis vaginitis. BV is the most commmon form of vaginal infection, followed by vaginal candidiasis and trichomoniasis. This vaginal infections are clinically significant especially in pregnant woman, as they can lead to pre-term labor and birth and various adverse pregnancy outcomes.
Principles of the procedure
The Affirm VPIII Microbial Identification Test is based on the principles of nucleic acid hybridization. In nucleic acid hybridization tests, complementary nucleic acid strands join to form specific, double-stranded complexes called hybrids.
The test uses 2 specific single-stranded nucleic acid probes for each organism, a capture probe and a colour development probe. These 2 probes are complementary to the distinct genetic sequences of the target organism. The capture probes are immobilized on a bead embeded in a Probe Analysis Card (PAC). Each of the three organisms being tested has a separate bead on the PAC. The colour development probes are contained in a mutli-well reagent cassette.
The various components are put into the BD microprobe machine and run. At the end of the run, if there is even a slight colour change on any of the organism beads, it would indicate that that the specific organism is present in the patient's sample. Note that there are 2 beads that act as a positive and negative control. After the run, if the positive control bead does not show a change of colour or if the negative control shows a change of colour, it means that the results of the run are obsolete and that the sample must be rerun.
Procedure's after receiving patient sample
- Label a sample collection tube (SCT) with the patients identification no.
- Take the swab obtained from the patient and and cut it such that the SCT can be closed.
- Drop 12 drops of lysis solution to the tube.
- Mix the swab in the tube with the lysis solution.
- Cover the tube with a cap and place in a lysis block of 85 degrees for 10 mins.
- Remove the tube from the block and add 12 drops of buffer solution to the tube.
- This time add a filter cap to the tube
- Prepare the PAC and write down patients id no. in the space.
Prepare the reagent casette
- Add 4 drops of substrate solution to well no.7 of the reagent cassette.
- Drop the patients sample into well no.1 of the reagent cassette.
- Place the PAC into well no.1 of the reagent cassette.
- Press the run key to start the process and wait for about 32-35 minutes for the process to be done.
Reporting of results
Since we students do not have access to the hospitals system, we can only write down the test results on the test request form and wait for a medical technologist to come by verify the result and key the results in.
If the tests results show that the patient is negative for all 3 organisms, we write on the test request form "All -ve/ All Neg".
If the test results show that the patient tests results show 1 or more postive for the disease we write it on the test request form as eg. Gard +ve (Gardnerella positive)/ Cand +ve (candidiasis).
Thats all for my report! i'll get back to you all if you have any comments or queries.
Randall Chua (TG02) 0503272G
Saturday, July 14, 2007
Biosafety Laboratory level 1 and Biosafety Laboratory level 2
Biosafety Laboratory level 1
Biosafety Laboratory level 1 commmoly known as BSL 1, can be used for micro-organism or chemicals classified as level 1.Basicall there are 4 classes ranging from class 1 to class 4. Micro-organism classified under class 4 are considered very dangerous. In AS4, class 3 or 4 micro-organism are not handled. BSL 1 is suitable for work involving well- characterized agents that are not known to cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment. Work is done/ conducted on open bench top using standard microbiological practices.
The following standards, special practices, safety equipments and facilities apply to agents assigned to BSL1:
- Acces to the lab is limited when experiments with culture are in progress
- Eating, drinking, chewing gum or smoking are not permited in the lab
- Mouth pipetting is prohibited
- All procedures are performed carefully to minimize the creation of splashes &/or aerosol
- In the BSL1, there are sinks for handwashing. All personnel must wash their hands, after they handle viable materials, after ther remove their gloves, before they leave the lab
- Work surfaces are decontaminated at least once a day with 1:50 dilution of 5.25% NaOCl
- If any spillage of micro-organism or chemicals occurs, the spillage area must be soak with 1:5 dilution of 5.25% NaCl
- All cultures, stocks, contaminated needles, sharp equipments, broken glasses(such as glass pipettes), must be decontaminated before disposal, by autoclaving
- Broken glasses must not be handled directly by hand but must be remove by a brush and dustpan
- A biohazrad sign must be posted at the entrance of the lab. This only occurs if infectious agents are persent. The sign must include the name of agents i use, the name and phone no. of the investigator
- Personnel Protective Equipments (PPE) such as gloves, lab coat must be worn in the lab at all times
- Goggles should be worn, if the personnel wears contact lense or does not wear spectacles, when splashes of hazrads chemical or micro-organism is anticipated
- Bench top in BSL1 are impervision to water and are resistant to organic solvent, acids, alkalis, moderate heat.
- The bench top n BSL1 is also resistant to chemicals which are used to decontaminate the work surface and equipments
- BSL 1 is a "pressured" laboratory. That is it prevents dirty air from entering the lab when the door is open
Biosafety Laboratory 2
Biosafety Laboratory 2 is similiar to BSL 1 lab. it is suitable for work involving agents of modertate potential hazard to the personnel and the environmnet. Nevertheless there are some differences. In BSL2, lab personnel have specific training in handing pathogenic agents and are directed by a trained personnel. Acces to the lab is restricted when work is being conducted. Extreme precautions are taken with contaminated sharp items and certain procedure in which infectious aerosol or splahes may be created in the biosafety cabinets.9details are below)
The standards, special practices, safety equipments and facilities apply to agents assigned to BSL1(point 1-15) also applies to BSL2. In AS4 , instead of BSL2,AS4 has 2 BSL 2 plus lab. One for micro-organism work while another one is for mammalian cell culture work
Apart from those ponits, there are a few more points personnel must follow
- The laboratory director establishes polices and procedure whereby people who have been advised of potential hazards and meet specific entry requirements may enter the laboratory
- Biosafety procedures are incorporated in to Standard Operating Procedures (SOPs) for the lab by the lab director
- Personnel working in BSL2 are advised of specific hazards anf are required to read and follw instructions on practices and procedures
- All personnel working in BSL2 must recieve appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposure to hazardous materials and evacuation procedures
- Personnel Protective Equipments designated for BSL2 use are worn in BSL2. This protective clothing is removed and left in the lab before leaving for other lab or non-lab areas
I hope you guys had fun reading and understood everything. I hope I am able to answer all your questions. Good Luck and Good bye for now.
Eugene Wong TG02
Saturday, July 7, 2007
Quantity can be determined for Serum ferritin, folate, RBC folate, Vitamin B12, Cortisol, Urine Cortisol, PTH, testosterone, D-HEAS and BHCG using DXI. It is a little similar to the analyzer in school that we used during practical. DXI has a continuous loading capability and approximately 120 samples can be loaded at once. Racks can be loaded directly from the centrifuge and is released in less than 5 minutes for other testing. This increases productivity and efficiency. When loading into analyzer, the sample must be checked for fibrin clots. Amount of plasma must be above 1cm, if not, it must be transferred to a plastic tube. DXI uses paramagnetic particle chemiluminescent immunoassay for quantitative determination. Light generated by the reaction is measured with a luminometer. Amount of analyte in the sample is determined from a stored, multipoint calibration curve. If results are too high, dilution is required. DXI has an on board dilution for ferritin. If after dilution, it is still high, do 3X manual dilution. Folate and B12 do not require dilution. The rest is manually diluted with either saline or SO calibrator.
Calibration is done once a month, when a new lot of reagent is used, on expiry, QC is unacceptable or after major maintenance procedures or replacement of parts. Calibration determines the relation between the result and the value of the input quantity.
1. Backup System
There is an auto system backup at 5am.
The cartridge is changed daily in case of breakdown.
2. Back up daily data system
3. Check inventory
Loading bulk supplies
-Substrate- equilibrated to room temperature under reagent load door, able to remain at room temp for 14days.
-Wash buffer- used to dilute samples/reagent, wash away unbound materials in RVs, wash pipettor probes and prime system.
-Reaction Vessels- store samples on board, prepare sample dilution, incubate sample with assay specific reagent during processing.
-Liquid waste generated when- pipettors are washed between processing steps and when unreactive fluid are washed into RV.
-Solid waste- collect empty reagent packs, used RVs, and condensation during processing.
-Reagent- If test not done so often, don’t keep adding reagent – not fresh.
4. Record test count of previous day
5. Shake solid waste container – radioactive wastebag is used
6. Run daily special clean routine – not the normal cleaning because Vitamin B12 reagent is sticky. Contrad, Citranox, Methanol 70%
Quality control is important and has to be done daily to keep the analyzers in top condition and results produced are accurate and precise. Charts are compared and if controls are not within the 2SD mean, QC must be done again.
Biorad Lyphocheck Immunoassay Plus Control Levels 1- 3 and PTH Control Levels 1-3 are done daily. PTH control is run twice as it is running 24hrs. Controls are parafilmed and stored in the fridge to prevent deterioration of the constituents. Liquickek Urine Chemistry Control Levels 1- 2 for Urine free Cortisol and RBC folate Control Levels 1- 3 are done when needed. If 2 out of 3 controls are in, patient samples can be loaded.
1. Ferritin- two site immunoenzymatic “sandwich” assay. Light production is directly proportional to concentration of ferritin.
2. Vitamin B12- competitive binding immunoenymatic assay. Photon production is inversely proportional to concentration of ferritin.
3. PTH – two site immunoenzymatic “sandwich” assay. Light production is directly proportional to concentration of ferritin.
4. DHEA- competitive binding immunoenzymatic assay. Light production is inversely proportional to concentration of ferritin.
5. Testosterone - competitive binding immunoenzymatic assay. Light production is inversely proportional to concentration of ferritin.
6. Cortisol- competitive binding immunoenzymatic assay. Light production is inversely proportional to concentration of ferritin.
- Serum cortisol
- 24hr Urine free Cortisol- Two sets of controls are used in case the first set is out of range. Ethyl acetate is added which produce two distinct layers as acetate density is lighter. It is used to extract cortisol from urine. 200ul is pipetted out from the top layer and left overnight in the fumehood to dry up. SO calibrator acts as a diluent to dissolve the precipitated cortisol. Run 1 set of control first. This reduces cost as a bottle of SO calibrator is very expensive. If not within range, use second set of control. If within range, run patient sample on DXI. Procedures are done in fume hood as acetate has a strong odour and is carcinogenic. Glass tubes are used as plastic react with acetate. As glass tubes are used, centrifuge speed is 2500 rpm to prevent breakage.
7. Folate- competitive binding receptor assay. Light production is inversely proportional to concentration of ferritin.
- Serum or Plasma (heparin) Folate – do not use hemolysed sample. Folate level in red cell is much greater than serum or plasma, lead to spuriously high results.
- RBC folate – contains EDTA- prevent clotting – whole blood or plasma. Specimen sent to Haem lab for HCT. Lysing solution added to release folate in RBC from endogenous binding proteins. Placed in cupboard for 1 ½ hrs as lysing solution and the lysing reaction is light sensitive. Run controls to see if they are within range before loading patient samples.
Thats all for now.
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