Saturday, August 25, 2007
Laboratory Management and Quality Assurance
Hi guys...how are u guys? Anyway sorry for this late posting..just finish my AIMM exam on friday =)
Anyway here's a summary of biosafety level 2+, 3 & 4 and details about Biosafety Cabinet Class II
Biosafety Level: BSL-2+
Agents handled are associated with human disease[In the research lab I am in, we handle Stenotrophomonas maltophilia (S. maltophilia) of different strains] By the way, in the research lab I am working,it is a BSL 2+ lab
1.Only people with access can enter
2.All activities are performed in a BSC cabinet
3.Biosafety manual defining any needed waste decontaminated or medical surveillance policies are all filed into a file which is kept in the
1. Class II Biological Safety Cabinets are used
2. Personal Safety Equipments (PPE) are to be worn
3. Pipeting devices are used & pipeting is prohibited
1. A sink and disinfectant are present for hand washing
2. An autoclave machine within the BSL-2 lab is present
In addition, self closing double door access is implemented.
The air is not recirculated and Lab air pressure is lower than adjoining rooms
Biosafety Level: BSL-3
Agents which are associated with human disease and cause illness by spreading through air or aerosol
Practices which implies for BSL-2+ implies for BSL-3
In addition, there is controlled access and decontamination of lab clothing before laundering
Safety equipments which are used in BSL-2 are also used
In addition, respiratory protection is needed
Facilities present is BSL-2+ are also found in BSL-3
Other facilities includes
Physical separation from access corridors
exhaust air is not recirculated
Negative air flow into the laboratory
Biosafety Level: BSL-4
Agents which are associated with human disease and cause illness by spreading through air or aerosol or have unknown cause of transmission
Practices which implies for BSL-3 also implies for BSL-4
1. Personnel must change their clothing before entering the lab
2. There is a shower, which is found at the exit of the lab. This shower is used when personnel enters and leaves the lab
3. All materials are decontaminated immediately before disposal
4. All experiments or activities are conducted in a BSC class III in combination with full- body, air-supplied, positive- pressure personnel suit
Facilities present is BSL-3 are also found in BSL-4
BSL-4 lab is established in a separate building or isolated from other areas of the hospital or research laboratory
Dedicated supply and exhaust, vacuum and decontamination systems are also present in the BSl-4 laboratory
Biosafety Cabinet Class II
Biosafety Class II cabinet are designed to protect the laboratory environmnet, work material & even protects the suer from the exposure to infectious aerosols and splashesthat may be generated when manipulating materials conatining infectious agents. All users must attend and complete a BSC II training before they are allowed to used the equipment. Just like what we have learnt in Drug Development & Clinical Trails, training must be documeneted =). Bunsen burners cannot be used in the BSC II cabinets as heat produced will distort the air flow. As a result, this damages the HEPA filters thus affecting the shelf-life of the cabinet. Traffice behind the user should be minimised to avoid any shifts in airflow
I guess that I don't need to tell you guys about the operation of BSC II [It's almost like the laminar flow hoods we use in Mammalian Cell Technology =) ]. But if you want to tell you...just tell me
Just a point to take note is that instead of those plastic covers that are used to cover the hood when the hood is not in use, instead, a glass viewing panel, which can be pulled down vertically is used to cover the hood. Please note that the glass viewing panel must not be lifted beyond the safety level when the cabint is in use, otherwise the alarm will be sound. I will try to get a picture of the BSC II on monday.
That's all for it now...See u in the next campus discussion =)
Saturday, August 18, 2007
Name of Test: Paediatric Bilirubin Measurement
Principle of Test: Using LEICA UNISTAT Biliruninometer
This is a direct spectrophotometer that measures total bilirubin in neonatal serum samples. Light is passed through the sample cuvette. The beam is split by a dichroic mirror and directed towards the 460nm and 550nm filters. The transmittance of the light exiting of the filters is measured by the photodetectors. Total bilirubin is calculated based on the output from the photodetectors.
1. Specimen is collected in a heparinised capillary tube that is sealed at the end and firmly affixed onto a wooden spatula to prevent breakage. As it is light sensitive, it should be protected from light to avoid photolysis of the bilirubin.
2. The capillary tube is place in pre-labeled tube according to the number assigned and spun down to separate serum.
3. The serum is then expelled in the clean cuvette by safepette and measured in bilirubinometer.
4. Result is obtained directly from the display.
5. Results have to be reported immediately by calling up the wards.
- To avoid errors in readings, the outside surface of the cuvette must be free of serum, dust, dirt and smudges; clean if necessary.
- The sample seen in the clear rectangular area of the cuvette must be free of bubbles, tap the cuvette lightly to clear them.
Test results/Reference ranges:
0-1 days : 0-86 umol/L
1-2 days : 0-122 umol/L
3-5 days : 0-175 umol/L
>6 days : 3- 24 umol/L
Jaundice is a common, temporary and usually harmless condition in newborn infants. It affects both full-term and premature babies, usually appearing during the first week of the baby's life.
Jaundice occurs when there is a build-up of a naturally bilirubin in the blood. Bilirubin is an orange/red pigment which is produced by the breakdown of haemoglobin. As bilirubin begins to build up, it deposits on the fatty tissue under the skin causing the baby's skin and whites of the baby's eyes to appear yellow.
Severe hyperbilirubinemia is of great concern because it may lead to bilirubin-related brain damage (Kernicterus).
Performing bilurubin test is the first step in making sure that normal degrees of jaundice do not become more severe and can be treated early.
Bilirubin is destroyed by u.v light, phototherapy.
Loh Mun Jo-anne
Sunday, August 12, 2007
Isolation of the Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)
The diagnosis of the HSV-1 & 2 can be made from the isolation of the virus from lesions found in patient, by growing in cell culture. Swabs from the penile, vaginal or oral lesions are commonly used. The swabs are sent in Hank's transport media to the laboratory, so that the viruses remain alive, in order for us to isolate them. Different viruses grow best in different cell lines. For example, the dengue virus is sensitive to C6/36 mosquito cell line, but not in mammalian cell lines.
For HSV-1 & 2, it is most sensitive to cell lines from Mink Lung (ML) and Human Embryonic Lung (HEL). Basically, the sample will be inoculated into healthy ML and HEL cell line and incubated in 36°C and observed for cytopathic effect (CPE) daily. Healthy cell line refers to the absence of cell degeneration and cell line with around 70% confluency, when viewed under light microscope is used. This can be vague at times, but with enough experience one should be able to gauge the confluency of the cell line. Uninoculated tube of the same batch of cell line for ML and HEL will be incubated, which acts as a control. This allows us to decide whether any morphological changes in the inoculated cell are due to viral infection or degeneration of cell line. Typically, CPE for HSV infected cells occurs within 7 days after inoculation, after which the virus is identified by fluorescent-antibody staining of the infected cells. However, some slow-growing viruses, such as enterovirus and measles would require a longer time to show CPE, 21 days and 14 days respectively.
CPE is a result of virus infection of cell, which leads to many changes to host cells. Some changes include, altered shape, cell lysis, membrane fusion, and apoptosis. In the case of HSV-1 & 2 infections, a 'ballooning' effect can be seen under the light microscope. The cells will round up and look like tiny balloons. This is indicative of viral infection. Thus, a confirmatory test using direct immunofluorescence test (IF) is carried out. Since there are two types of HSV, monoclonal antibody is used to identify the type of HSV. The principle of this test is similar to the CMV IF test I posted earlier on. The only difference is that the monoclonal antibody is conjugated with Fluorescein isothiocyanate (FITC), which exhibits green fluorescence when excited by ultraviolet light, indicating the presence of HSV infected cells.
Yea, I guess that's about it.
Yong Yang (TG02)
Monday, August 6, 2007
Yo. It's just 14 weeks to go to the end of SIP. 14 weeks! I will be describing the blood ketones tests here.
Blood Ketones Test
Blood ketones are tested using the Optium Xceed handheld meter. The basis for this meter is electrochemical. This is a very easy gadget to use. First, a new test strip is inserted into the strip-port. An ‘apply blood’ (it shows a blinking drop) icon will show. Then, apply 1 drop of blood onto the other end of the test strip. Wait for about 30 seconds and the results will be shown on the screen. However, it must be ensured that the blood samples are sent in lithuim heparin tubes (green cap). The heparin acts as an anticoagulant. Blood delivered in any other types of blood tubes are rejected. The reference range for this test is 0.0 – 0.6 mmol/L. Critical values (> 1.4 mmol/L) must be reported immediately to the appropriate personnel, so that action can be taken by the doctor to treat the patient.
Calibration is done for every new box of test strips by inserting a CODE key into the strip-port to check for the correct CODE number.
Controls are done only when there are requests for blood ketones, mainly because there are very few requests for blood ketones (about 1 a day). There are 2 levels of controls; one of a low value and the other high. Only 1 level of control is done when there is a blood ketone test, and the controls are tested alternatively (i.e. one day use control of high value, the next time use the control of low value).
Blood ketone testing is usually done on patients with diabetes. In diabetes, there is not enough insulin or insulin activity to help the muscles to absorb glucose from the blood. When there is not enough glucose, the body will burn fat for energy. Breakdown products, or ketones, are formed. Ketones are toxic to the body. Accumulation of these acids in the body may lead to ketosis. A more serious disease is known as ketoacidosis or DKA.
Why use blood and not urine for measurement?
There are 3 types of ketone bodies – acetone, acetoacetate and β-hydroxybutyrate. β-hydroxybutyrate makes up 75% of all ketones in DKA. Therefore, it is more relevant to measure β-hydroxybutyrate in blood rather than urine ketone tests based on the nitroprusside reaction which detects acetoacetate instead. Urine ketone tests also give false positive results in the presence of some drugs and false negative results when the test strips are exposed to air for too long.
That's it for now.
Martin Ng (TG02)
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