Saturday, November 8, 2008
Structural Abnormalities of Chromosomes
When chromosomes have replicated and are waiting to be separated into 2 daughter cells, chromosome breakage is frequently accompanied by exchange material from one chromatid to another. The exchange of material occurs during the pachytene period of the first meiotic division and is a mechanism built into the reproductive cycle to ensure the mixing of the gene pool. The exchange is called crossing over and it occurs when paired homologs form chiasmata. The abnormailties occur when the chromosomes break at non-homologous sites leading to unequal exchanges. 2 major classes of detectable rearrangements are rearrangements within a single chromosome and rearrangements involving more than 1 chromosome.
Examples of rearrangements within a single chromosome:
Deletion is a loss of chromosomal material. An interstitial deletion can arise from unequal crossing over at meiosis. Breaks can occur in 2 homologs at slightly different positions on the chromosomes and the pieces can be reconnected to the alternate chromosome. One chromosome could lose material while the other could gain material. This kind of mechanism is more likely to occur. Cri-du-chat syndrome is one of the examples of syndromes resulting from deletion.
Duplication occurs when a portion of one homolog may be present in duplicate. A duplicated segment may be inserted in the same order as the original segment. However, we cannot identify which piece was original. It can also become reversed. Duplication in the same order which also can be known as tandem duplication may probably results from unequal crossing over in meiosis or from a rearrangement between 2 chromatids during mitosis. Any duplication represents a trisomic state for whatever gene loci are present on the affected segment.
Inversion is a two-break structural irregularity that affects a single chromosome. The segment between the 2 breaks rotates 180 degrees and reattaches to the points of breakage. There are 2 types of inversion: Paracentric and Pericentric. In paracentric inversions, the centromere is not included in inverted segment and the arm ratio of the chromosome remains the same. In pericentric inversions, the inverted portion does include the centromere and the arm ratio may be changed.
Examples of rearrangements involving more than 1 chromosome:
Translocation is an exchange of genetic material between 2 or more non-homologous chromosomes. It is an abnormal mechanism but it can occur when 2 or more chromosomes break at the same time. Broken chromosome ends are regarded as sticky and can attract the cellular enzymatic repair service. Usually broken ends stay close to each other and are reunited. However, it is possible to mismatch the broken ends. The carrier of a balanced translocation may pass on the only part of the translocation which can result in genetically unbalanced offspring.
Dicentric chromosome possesses 2 centromeres and it is a result of the joining of homologous chromatids to form an isochromosome. An isochromosome is a chromosome in which the arms n either side of the centromere are morphologically identical ad bear the same gene loci. Dicentrics may cause problems during cell division, however, if the 2 centromeres are far apart on the derivative chromosome. If both centromeres are active, they can be drawn to opposite poles of the spindle resulting in formation of an anaphase bridge. Anaphase bridge is a chromosome that makes a bridge between 2 daughter cells at anaphase. When this happens, the dicentric can be left outside both daughter nuclei as they form or can break apart and cause loss or gain of chromosomal material.
Insertion is an addition of genetic material between 2 normally adjacent sections of a chromosome. The additional material could be a duplication of a section of the same chromosome but is more likely to come from a different chromosome. Insertions are difficult to identify unless there is a complementary deletion in the cell.
Monday, November 3, 2008
The modular design of the ARCHITECT family of analysers allow multiple processing modules which perform all sample processing activities, to be physically joined to form a single workstation or system. These ARCHITECT systems can be configured to process sample using potentiometric and photometric methoeds and/or chemiluminescent microparticle immunoassay (CMIA) methods.
The ARCHITECT ci8200 analyser is composed of two modules, which is c8000 and i2000. C8000 module is using the potentiometric and photometric methos while the i2000 module uses CMIA method.
Here are the majority tests carried out the ARCHITECT analyser and their reference range:
1. Free Thyroxine (FT4) 10.0-20.0pmol/L
2. Total Triidothyroninn (Total T3) 1.2-2.3 nmol/L
3. Anti-TPO <5.6IU/ml
4. Anti-Tg <4.11u/ml
5. Alpha-fetoprotein (AFP) 1.0-10.0ug/L
6. Carcinoembroynic Antigen (CEA) 0.0-5.0ug/L
7. Vitamin B12 (B12) 132-835 pmol/L
8. Folate (FA) 7.40nmol/L
9. Ferritin 30-323ug/L M (0-30Y) 32-294ug/L M (31Y & Above) 7.6-179ug/L F (premenopausal) 182-339ug/L F (postmenopausal)
10. Syphilis non-reactive
11. HBsAg non reactive
12. HbeAg non-reactive
13. Anti-HCV non-reactive
14. Anti-HAV IgM non-reactive
15. HAV AB non-reactive
16. Anti-HBs non-reactive
17. HIV Ag/Ab Combo non-reactive
18. Kappa light chains 1.40-3.8g/L
19. Lambda light chains 0.90-2.50g/L
20. Immunoglobulin A 0.8-4.0g/L
21. Immunoglobulin G 5.0-15.0g/L
22. Immunoglobulin M 0.3-3.0g/L
23. Rheumatoid Factor <20.0 IU/L
24. Transferrin 1.74-3.64g/L Spectrophotometric method
25. Iron F 11.27 umol M 11.29umol/L
The analyser uses these three methods to measure the concentration of analytes in sample.
1. Photometry method
Phtometry method uses
-End point assay are reactions that reach an equilibrium and at that time there is little or no further change to the absorbance readings. During this equilibrium time, the system measures the absorbance readings used for calibration and calculating results.
-Rate assay are reactions that measure the constant change in absorbance over time. The system performs readings several times during this reaction, calculates absorbance change (activity), and then uses the readings to calculate results.
2. Potentiometry (measures electrical potential in sample)
-The c8000 modules uses an ICT (integrated chip technology) modules to measure potentiometric assay (eleytrolytes). ICT methodology uses the solid state ion-selective electrodes contained in a single chip that reduces the maintenance require to perform electrolytes measurements.
3. Chemiluminescent technology
-During sample processing, microparticles (paramagnetic microparticles coated with capture molecules) are mixed with the sample binds to the corresponding capture molecules on the microparticles, forming the immune complex.
-After incubation, a magnet attracts the paramagnetic microparticles (bound to the surface analyte) to a wall of the reaction vessel. The reaction mixture is washed to remove unbound materials, further processing can now take place.
-Detection of ther eaction mixture in the reaction vessel is accomplished with a chemiluminescent acridinium-labeled conjugate. The conjugate binds to the immune complex to complete the reaction mixture.
-After incubation, the reaction micture is washed to remove unbound material. The pre-trigger solution is added and a background read is taken.
-Pre-trigger performs the following functions: - Create an acidic anvironment to prevent early release of the energy(light emission). Helps to keep microparticles from clumping. - Splits acridinium dye off the conjugate bound to the microparticle complex. This prepares the acridinium dye for the nest step.
- The system then adds Trigger solution to the reaction mixture. The acridinium alkaline solution. This causes the chemiluminescent reaction to occur. N-methylacridne is formed and releases energy (light emission) as it returns to its ground state. The CMIA optical assembly measures the chemiluminescent emissin (activated red) over a predefined time perioed to quantitate the analyte concentration or to determine qualitative interpretations for index cut-off assay.
-Human serum (including serum collected in serum separator tubes) or plasma collected in sodium heparin, lithium heparin separator tubes, or potassium EDTA anticoagulant tubes may be used. Other anticoagulants have nor been validated for use with ARCHITECT.
1. Specimen are spun via the centrifuge for 5minutes.
2. The cap of the specimen tube is removed.
3. Specimen placed on the ARCHITECT rack.
4. Rack loaded into the ARCHITECT.
5. Test are run.
So that's all for my last posting.
Thanks for reading.
Monday, October 20, 2008
Name of test: Urea Breath Test
-This test is carried out for diagnosis of Helicobacter pylori (H pylori) infection
-H pylori is a gram-negative bacillus which is highly motile due to its multiple unipolar flagella
Retrieved 21 October, 2008, from http://images.google.com.sg/imgres?imgurl=http://www.science.org.au/nobel/2005/images/invasion.jpg&imgrefurl=http://www.science.org.au/nobel/2005/05diagram4.htm&h=329&w=321&sz=30&hl=en&start=2&um=1&usg=__Xjdem48HW8H_C711OrWmzPSMn7g=&tbnid=0Tjg0gylcn5OMM:&tbnh=119&tbnw=116&prev=/images%3Fq%3DH%2Bpylori%26um%3D1%26hl%3Den%26sa%3DN
-They produce the enzyme urease which converts urea to ammonium and bicarbonate
-Some of the diseases caused by H pylori infection are:
1. Gastritis and dyspepsia
2. Stomach ulcers
3. Duodenal ulcers
4. Stomach cancer
-The UBIT Tablet taken by the patient contains 100 mg of 13 C-Urea
-If the patient has Helicobacter pylori infection, H. pylori will produce the enzyme urease. The enzyme urease will convert the 13 C-Urea to 13 CO2 and NH4+
-The 13 CO2 is then absorbed in the blood, diffused into the blood and exhaled
-In our laboratory, we use the 13 CO2 Urea Breath Analyzer UBiT-IR300 by Otsuka electronics to analyse our UBT samples
-The machine will measure the 13 CO2 in both the *pre-dose and post dose breath samples
-The difference of the 13 CO2 levels in both breath samples will also be calculated
-This measurement is done via infrared spectroscopy
2 UBT bags
1 UBIT tablet
100mL of water
Preparation for the test:
1.Fast for 8-9 hours before the test
2.Stop taking Proton Pump Inhibitors one week before the test
3.Stop taking antibiotics 4 weeks before the test
During the test:
1. The patient has to breathe into the “Pre-dose” bag without taking the UBIT tablet.
2. Within 5 sec, the patient has to swallow the UBIT tablet on an empty stomach with 100 mL of water. The tablet is not to be chewed, crushed or dissolved.
3. After taking the tablet, the patient has to lie down on his left side for 5 minutes.
4. Then, he has to remain seated for another 15 minutes.
5. After that, the patient has to breathe into the “Post-dose” bag.
6. These two bags with the request form will be sent our lab for analysis.
In the lab:
The medical technologists will:
1.Check the labels
2.Insert “Pre-dose” bag into the “Pre-dose” inlet
3.Insert “Post-dose” bag into the “Post-dose” inlet
A value of 2.5% or higher indicates that the patient is positive for H.pylori infection
*pre-dose: sample before taking tablet
*post-dose:sample after taking tablet
Sunday, October 19, 2008
i had to work from 10am till 7pm. the slot was either for Haematology section or CP.
CP stands for Corporates and Poly section.
For haematology, the workflow remain the same. i will just have to assist the medtech that is posted there and cover the section from 5pm-7pm or until the next shift staffs are in.
However i was new to CP section. So i was asked to observe the workflow first.
The specimens from the polys will arrived at the lab around 10.30am. That will be the first batch of specimens. In total, 3 batches of specimens are send to the lab within the expected timings. Upon receiving the specimens, the poly clerk will have to check the specimen sent, wether it matches will the ones indicated in the forms. Once checked, the clerk will have to 'ech' the specimen into our system. If 'ech' is not done, the specimen cannot be run as the the machines will not be able to recognise them. After which the poly medtech will take over. He/ She will then have to process the specimen according to the test requested.
Usually the bloods are sent in plain tubes and fluoride tubes.
For plain tubes, we can load it into MPA ( a machine in chemistry) directly. For fluoride tubes however, we have to spin them using centrifuge before loading them into either SWA or COBAS for processing.
The tubes are then archive into a special rack for easy indentification.
After the processing of the first batch is done, we have to check for any test that are not run. We do so by checking the incomplete log. In this system, we can check for any outstanding test that are not yet done.
If there is indeed any test that are not run, we have to find the specimen and run it.
That's for poly specimens.
For corporate specimens, the workflow remains the same. The only difference is that the specimen are not 'ech' into our system but are ordered as corporate screening specimen. What we do here is, we ordered the tests that are required like how we do order entry but the location for the test are change into their respective companies.
The workflow henceforth are similar to the polys.
After all the specimens are received and processed, we have to check the incomplte log again in case we missed out any test. If everything is smooth sailing then we can print the corporates results which are then reported back to the companies.
So that's basically the roles of a CP medtech.
Sunday, October 12, 2008
The Nexes Special Stain machine is barcode-driven and has optimizes protocols to help ameliorate productivity and consistency. The tray in the machine can hold up to 20 slides while the reagent tray can take up to 25 reagents.
1) The sections are fished from the floatation bath and onto the slide.
2) The biopsy number and the name of the special stain required is labeled on the slide.
(ie. 08/12345 GMS)
3) The slide is heated on the hot plate.
4) The barcode label is keyed in and the protocol of the test required for the slide is chosen.
5) The label is pasted on the slide.
6) The slide is sent for dewaxing.
7) The slide is placed in the slide tray in Nexes.
8) The section on the slide is hydrated with a wash solution to prevent drying up of the section and to prevent unwanted solution in the machine to dirty parts of the slide.
9) The reagents required are loaded.
10) The liquid coverslip solution is filled up if it is insufficient.
(The liquid coverslip acts as a temporary protection while the slide is still in the machine after staining)
11) The RUN button is clicked
12) The run time will be shown
13) When staining is done, the reagents are removed from the tray.
14) The slide is removed from the slide tray and sent for dehydration(70%, 80%, 95%, absolute alcohol)
15) The slide is cleared, by dipping it into xylene.
16) The slide is mounted.
So far, i have performed two stains manually, without the help of the Nexes Special Stain machine. The stains are PAS and PASD. PAS stain is used to stain carbohydrates. The difference between the two stains is, PASD uses diastase to digest glycogen. This stain is used to differentiate glycogen from other carbohydrates. Under microscopic examination, the tissues stained with PAS will consist of clusters of glycogen, stained dark pink / magenta.
1) The section of interest is fished onto the slide.
2) The slide is heated on the hotplate for 3 minutes.
3) Dewax the slide and bring the sections to water.
4) For PASD stains, treat the sections with thawed diastase for 30 minutes and subsequently, wash the slide with water.
5) For PAS stains, treat it immediately with 1% aqueous periodic acid for 5 mins.
6) Treat the slides with Schiff's reagent for 3 mins, until sections turn light pink.
6) Wash the slides in water
7) Counterstain the nuclei with alum haematoxylin for 1 min.
8) Blue the slides in water
9) Dehydrate the slides by dipping them into 70%, 80%, 95&, absolute alcohol.
10) Clear the slides by dipping them into xylene
11) Mount the slides
Monday, October 6, 2008
To all my muslim friends, Selamat Hari Raya! And to all my non-muslims friends, hang in there...5 weeks left. Sorry fot the late blog entry. My computer is not working well these days.
For the last 5 weeks, I have been attached at the processing section. Some feels that the processing section is rather simple yet boring job. But I feel that it is the MOST important section in the laboratory. Every specimen will have to undergo the processing section before the requested tests are performed. Here’s what I have learnt I have pick up along the way…
When a test is requested by physicians, specimens are collected in appropriate tubes/ containers and sent to the lab together with a request form. In the lab that I am attached to, there are four types of tests forms. Such includes the
1. External form-specimens will be sent to external laboratory upon request as test are not available in our lab.
2. Interlaboratory form- request for tests done in the clinical lab.
3. Microbiology lab form-request for test done in the micro lab.
4. Histopathology form-request for test done in histo lab.
Thus upon arrival of specimens to the processing section, it will be sort as according to the forms. Those interlaboratory tests are then further arranged. The interlaboratory test specimens usually come from wards, specialist clinics, accident and emergency section. Those ‘URGENT’ cases from any of the above department will be prioritized. All A&E tests and MICU and SICU cases are also prioritized, followed by clinics, and wards.
The laboratory admin clerk will then order tests requested in the LIS and print out the barcode for the tests requested. The barcode labels are pasted in the sample tubes. When pasting the barcode labels, one must check that the label which was already pasted by the physician tally with the name on the requested form and that the barcode labels are pasted on the right sample tubes for the right tests. E.g. Test for full blood count, specimen must be placed in the EDTA tube and not plain tube.
Medical Technologies must ensure that barcodes are labeled neatly and appropriately so that the analyzers are able to scan the barcode. The specimens are then spin (if required like plain blood tubes) and/or delivered to the respective section.
The processing section is also in charge of rejecting specimens and cancellation of tests. Here are some common reasons that specimens are rejected:
1. blood in EDTA tubes are clotted
2. insufficient specimen
3. specimen leaked
4. empty container sent
5. specimen sent in wrong tube
6. no name label on specimen
7. name/ NRIC mismatch between request form and specimen
Alrite people.Thanks for reading.See you guys soon...
Friday, September 26, 2008
Test Name: Stool for ova, amoeba & cyst test.
Hello everyone. Hope you guys are coping well. I have observed this test done at the Routine Section. The ova and parasites test is performed to identify intestinal parasites and their eggs, which are called ova, or cysts in patients with symptoms of gastrointestinal infection. Patients may have no symptoms or may not even experience diarrhea, blod in the sool, and other gastrointestinal distress. Identification of a particular parasite indicates the cause of the patient's disease and determines the medication needed to treat it.
Saline wet mount would be the best for detection of helminth eggs and larvae and it is especially good for motile protozoan cysts, which appear refractile. When cysts have been found in saline mount, they should be observed in Lugol iodine stained preparation which will reveal nuclear structure and te presence of glycogen. The nuclei stain a brownish colour and can be easily stained. For an accurate diagnosis, a concentrated method is advised so as to increase the probability of finding the parasites in faecal samples.
Para-pak spin con is a commercial kit for fecal parasite concentration of eggs, larva and protozoa. The picture below is para-pak spin con.
Picture taken from http://www.mdeur.com/products/972200.htm
Feces should be collected directly into a clean container. It must not be contaminated with urine, water, or other materials.
-10% buffered solution
-Lugol Iodine Solution
1. Stool(0.5g) was emusified with 10ml of saline in a plastic tube
2. 4 drops of CON-Trate Reagent A which helps to enhance the breakdown of fecal aggregrates and mucus therefore freeing parasites.
3. Plastic tube was capped and contents were mixed thoroughly.
4. The mixture were then filtered using the para-pak filter into the centrifuge tubes provided.
5. The filtered mixture were span at 2000rpm for 10 minutes.
6. The supernatant were removed.
7. And the sediment were resuspended.
8. Wet mount were performed on the sediment.
9. Then slide were examined microscopically for parasite.
The senior medical technologies will be observing the slide. Presence of bacteria and other microorganism in the stool would indicate patient is normal, thus no ova, amoeba or cyst seen. Presence of ova, amoeba or cyst would indicate an abnormal result. In addition is a sign of parasitic infestation.