Saturday, July 28, 2007
Introduction (skip if necessary)
I'm attached to *GH Clinical Lab together with 4 others (names are omitted for confidentiality purpose). In *GH, there are 2 clinical labs. One 24hr routine lab situated in the main building for routine tests (basic chemistry, LFT, TFT, Renal function, etc), and another lab in a building that some lecturer in school has been warning us, where you gotta show your pass or risk "getting shot". Here, highly specialized tests such as specific proteins (rheumatoid factor), trace metal panel (copper, zinc) and others like G6PD, homocysteine, etc, are analyzed.
Our supervisor has kindly planned a schedule for us which indicates that for the span of 20 weeks, we will spend 10 in the 24hr routine lab, 5 purely on our MP (but still gotta report for work =/) and the last 5 at the "special" lab.
The routine lab is further divided into 5 stations, based on the type of tests performed and machines used. We will spend 2 weeks each at each station.
This week, I'm attached to a station using the Beckman Coulter Unicel DXI (below)to conduct routine tests for serum ferritin, folate (RBC and serum), Vitamin B12 (usually done with folate as both are linked by the reaction pathway for methionine synthesis), Cortisol (24h urine free cortisol and serum cortisol), PTH, testosterone, D-HEAS (precursor of sex hormones) and beta-HcG (pregnancy hormone). The other analyzer, the Johnson & Johnson's Vitros DT60 II (image further down) tests for lipase, lactate, ammonia, lithium and cholinesterase.
Beckman Coulter Unicel DXI (taken form http://www.beckmancoulter.com/products/instrument/immunoassay/UniCel_DxI_800.asp)
Johnson & Johnson's Vitros DT60 II
Assay and Analyzer
For this posting, I will elaborate on the lactate assay of the Vitros DT60 II. (I chose the DT because its small compared to other analyzers but very, very unique.)
The beauty of the DT60 lies in the fact that unlike any other analyzer, you don't have to worry about adding or changing reagents..at all. As if that isn't enough, QC is done once a day (other analyzer needs at least two QC runs a day), and calibration is only done twice a year!
Other additional but rather significant benefits will be that minimal daily maintainence is necessary (just wipe with a cotton swab) and solid waste can be dumped of really easily.
Many of you will be wondering what kind of freak analyzer is this that does not need reagent loading. Well, that is because this machine conducts tests by using a special slide containing the required reagents! So you just have to load in that slide, drop a patient fluid sample onto it, and wait for the results. The slide will then be transported into the machine to carry out the test reactions.
The below shows the various layers of this multi-layered slide (click to enlarge).
(taken from http://www.orthoclinical.com/Products/products.aspx?id=4§ion=features)
The Vitros Slide is made up of five main layers. From the top, there is an upper slide mount (for supporting purposes). The next layer is the spreading layer, which helps to distribute the patient sample evenly to the underlying layers. The third layer is the reagent layer, which contains all the reagents needed for the photometric reaction to take place. The final layers are the transparent support layer (to aid photometric absorbance reading), and the lower slide mount (similar to the upper slide, for supporting purposes).
Test principle and reactions
Principle: Colorimetric Assay
In the case of the lactate assay, the reaction at the reagent layer of the dry-slide consists of two main reactions.
First, lactate is oxidized by lactate oxidase (found in the reagent layer) to pyruvate and hydrogen peroxide.
Second, the hydrogen peroxide generated oxidizes the 4-aminoantipyrine, 1, 7-dihydroxynapthalene dye system (included in the reagent layer) in a HRP (horseradish-peroxidase)-catalyzed reaction to form a dye complex (red).
The slide is incubated and the intensity of the dye complex is measured using the in-built spectrophotometer to quantitate the lactate values using its absorbance (at 555nm).
This colorimetric assay has an approximate five minutes incubation time. When the test is complete, the results will be printed out immediately from the DT60 analyzer in the form of a narrow result slip. The results are written down on a form and filed into the LIS. The result slips are then torn off and clipped together with the result forms in a file.
Request form with attached heparinized tube in ice- Samples must be sent in ice (bag containing ice in water) to prevent anaerobic glycolysis from producing lactic acid!
Clinical Significance of Lactate test
Reference Range: 0.90 -1.70mmol/L
Elevated lactate levels indicates lactate acidosis. Lactate levels increase in many conditions and may aid the monitoring of conditions such as diabetes mellitus. In diabetes mellitus, renal failure may result, causing metabolic acidosis. Elevated lactate levels are also useful in diagnosis of tissue hypoxia, in which there is cellular glycolysis, hence causing respiratory acidosis.
Finally a raised lactate level may be used in the screening of malignancies.
Hope you've "enjoyed" reading the post. Do feel free to ask questions. Thanks!
P.S. Answer turnaround time: About 24hrs (from the next working day).
Sunday, July 22, 2007
Right, I'll be focusing on the experiment that I did for my MP rather than SIP. That's
because the SIP job scope is mainly setting and clearing up after the
As most of you know, I'm working on the same project as Joan, just only looking at different components.
After 3 continuous weeks of gavaging the mice, it was time to sacrifice them. The mice were anaesthesized with Ketamine, a dissociative anaesthesia, and Pamlin, a sedative through Intraperitoneal (IP) injection.
How much to be injected was worked out by using this formula:
Dosage to be given = (Weight of mouse(kg) X Drug Dose)/Concentration
After they were all sedated, blood was collected by Cardiac Puncture and transferred to EDTA Tubes to prevent them from clotting.
The mice were then euthanized by cervical dislocation. The mouse was placed facing down, its neck was held down in place and the tail was pulled hard swiftly. This will then break the backbone of the mouse and sever the nerves connecting the backbone and the brain. This is one of many humane methods of euthanizing.
Diagram 1 shows the position on euthanizing the mouse using cervical dislocation.
The heart was checked to see if it was still beating before continuing on with dissection.
For dissection, a dissecting tray, a pair of forceps and scissors were prepared by swapping with 70% alcohol to disinfect the equipments. The mouse was placed onto the dissecting tray with its stomach facing up, a midline incision was made by using the forceps to grab the skin and the scissors to cut. The spleen was removed and placed into a tube containing 1X Phosphate Buffer Saline (PBS). The spleen is found at the left side of the mouse, at the posterior. The liver was removed and placed into 1X PBS with 0.16mg/ml of Heparin. The liver is directly below the lungs/ribcage.
Diagram 2 shows how the dissection was done and where the organs are found.
Thereafter, each mouse was placed into the biohazard bag and placed into the freezer for the storage of carcasses.
The spleens were taken to perform the LDH Assay.
Working in a flowhood, each spleen was homogenized by scraping them against a sterile cell strainer into a suspension in 10ml of RPMI + 10%FBS medium.
Working in the dark, 5ml of Lymph M were pipetted into 12 15ml centrifuge tubes.
* Lymph M is a solution that works by density gradient. After centrifugation, different cell layers will be formed by their densities, including the lymphocyte layer.
5ml of the splenocyte suspensions were slowly aliquoted into the tubes using a glass pipette so as not to disturb the interface.
The tubes were then centrifued at 1500g, for 20 minutes at room temperature.
The yellowish lymphocyte layer was removed using a glass pipette into a new sterile 15ml centrifuge tube.
Diagram 3 shows the different layers after centrifugation.
The removing the lymphocyte layer, the lymphocytes were then washed 2 times by adding 10ml of medium and centrifuging at 800g, for 10 minutes at room temperature, decanting the medium then resuspending the cell pellet in another 10ml of medium.
After washing, 1ml of RMPI + 2%FBS medium was added into each tube and the cell pellets were resuspended.
Using a haemocytometer and tryphan blue, the lymphocytes were counted.
YAC-1 cells cultured were also counted using the above method.
Calculations were made to challenge the lymphocytes at different cell concentration, namely, 50X, 100X, and 150X.
A 96-well plate was used for the challenging of the cells.
From here onwards, RMPI + 2%FBS medium was used instead of RPMI + 10%FBS.
Blanks, High and low controls, effector controls and the test samples were done in triplicates.
The plate was then incubated in a 37oC incubator with 5% CO2 overnight.
The next day, all the suspensions were pipetted into eppendorf tubes and centrifuged at 1500g for 5 minutes at 4oC to form the cell pellets.
Cell lysing solution was made, by aliquoting 100uL of Triton X into 10ml of RPMI + 2%FBS and vortexing the tube for Triton X to dissolve.
The high control supernatants were decanted and 200uL of the lysing solution was added to lyse the cells.
2 plates of 96-wells plates were used for the assay as each tube was done again in triplicates.
A 20X dilution was made to each tube by adding 95uL of DI water to 5uL of each cell supernatants into the new 96-wells plates.
The reaction mixutre was prepared by pipetting 1ml MilliQ water to the lyophilzate catalyst for 10 minutes and vortexing it after 10 minutes.
The dye was thawed in the water bath.
250ul catalyst was pipetted to 11.25ml of dye into 15ml centrifuge tube for 100 tests and the mixture was mixed by inverting up and down.
This reaction mixture was poured into a resevoir and 100uL of it was pipetted into each well using a multi-channel pipette.
The plates were then covered with aluminium foil and incubated for 20 minutes.
The stop buffer was prepared by pipetting 80ul DI water to 20ul 5M HCl to a dilution of 1N HCl
* Always add acid to water slowly!
After incubation, 50uL of stop buffer was pipetted into each well using a multi-channel pipette to stop the reaction.
Absorbance were then read at 490nm by using a spectrophotometer.
*Any bubbles formed will affect the results, thus, all bubbles must be removed before reading the plate.
Cytotoxicity was calculated using this formula:
((Effector-Target Mixture – Effector Control) – Low Control))/(High Control – Low Control)
Diagram 4 shows YAC-1 cells at 20X
Diagram 5 shows the lymphocytes at 10X
Diagram 6 shows the lymphocytes with YAC-1 cells at 10X
All diagrams and photos taken/done by me.
Any questions, just ask me! =DD
Wednesday, July 18, 2007
I was assigned to do a project to prevent 2 proteins from interacting to prevent tumorigenesis.
Throughout my SIP, I did different things everyday unless I don’t get any expected results and need to redo. My work rotates around Mbio, CellBio, MCT and Biochemistry. I am going to focus on DNA preparation in my discussion here. I am supposed to isolate the DNA I clone from the host cells that I had transformed.
There are three types of DNA prep for plasmid isolation: maxi-, mini, midi-prep.
Below are differences between these 3 prep works:
As I only did the maxi- and miniprep, I cannot comment more on midiprep.
-large scale DNA preparation
-Quantity of DNA eluted: 500ug/ml- 850ug/ml
Steps and reagents involved:
-Uses cultured cell clones in 250ml of LB media
- Requires centrifugation at 4۫C
- Requires a big Maxi- column to bind DNA, a dish to collect flow through that pass the column for disposal and a Du-pont tube for DNA elution
- Steps are lengthy and more buffers are involved like W8 (wash buffer), E4 (elution buffer), 70% ethanol for precipitation, TE buffer for resuspension and Equilibration Buffer (EQ1) to wet the column before draining any solution
- Drain through column by gravity flow
- Principle is lysis, bind and wash DNA, precipitation and elution.Details of the protocol can be found in www.invitrogen.com under PureLink™ HiPure Plasmid Filter Purification
-Small scale DNA preparation
-Quantity of DNA: up to 40ug/ml
-Steps and reagent involved:
-Uses cultured cells in 4ml of LB media
-Only requires a normal centrifuge
- Only requires a normal spin column to bind DNA and 2ml Wash tube to elute the DNA and for flow through collection
-Involves lesser steps and buffer and column work by centrifugation to bring the solution down.
- Principle works by alkaline lysis of the host cells, bind, wash and elution purified DNA.
- Details of the protocol can be found in www.invitrogen.com under PureLink™ Quick Plasmid Miniprep Kit
-Middle scale DNA preparation
-Quantity of DNA: 100-350ug/ml
After the prep for our DNA purification, I need to do a quantitation of DNA content using a spectrophotometer to check is there sufficient DNA for the following steps of the project. Quality of DNA is also monitored. DNA with ratio measured at 1.6-2.0 are considered pure and good quality. Sometimes, ratio may not be quantitated by the spectrophotometer if the concentration is too low. The Miniprep is done before maxi prep so that we can prepare our large amount of DNA with the correct sequence based on the sequence results of the clone from miniprep. We quantitate the DNA in miniprep for sequencing reaction to monitor if the DNA cloned has the right sequence so that we can proceed on our project. 1 ug/ml is sufficient.
For maxiprep, we check the concentration of DNA to see if there are enough for in vitro translation for translating DNA to proteins in the next part of project .
Running a gel with the same aliquot of DNA can confirm the quality of DNA based on comparison of the intensity of the bands to make sure the measurement from the spectrophotometer is accurate. DNA might be contaminated with RNA, proteins and carbohydrates which can be determined by the quality ratio of the DNA and confirmed again using the gel.
I hope u all understand what I said and dont hesitate to ask. Thanks.
Saturday, July 7, 2007
In the Hematology department, for the first week, my main duty is to do ABO blood group and Rh typing using plating method. For the second week, my main duty is to use CELL-DYN 3500/3700 to run full blood count test and malaria parasite test.
ABO blood group and Rh typing
- Label the plate as below.
- Aliquot one drop of anti-A, anti-B, anti-AB and anti-D to the plate labeling A, B, AB and D respectively.
- Spin down the patient blood sample to separate the red cells and plasma.
- Aliquot one drop of plasma to the α, β, O column each.
- Dilute the red cells in saline (estimate 3-5%).
- Aliquot one drop of the diluted red cells to the A, B, AB, D column.
- Aliquot one drop of commercial A, B, O cells too the serum.
- Mix the blood with a vibrator and centrifuge it at 900rpm for 1 min 30 sec.
- Read the result manually using a stick to mix to check for agglutination.
Test Result with reference range:
- 0>no agglutination>negative
Things to take note:
- All reagent used are diluted. 1 drop of anti-A, anti-B, anti-AB, anti-D is diluted with 3 drops of saline. And one drop of commercial A, B, O cell to one drop of saline.
- If Rh negative, Du testing is needed. Add one drop of diluted patient red cells to one drop of concentrated Anti-D in a new test tube. Centrifuge. Incubate at 37°C for 15 min. Wash with saline for 3X. Add one drop of AHG and centrifuge to check for agglutination.
CELL-DYN 3500/3700 to run full blood count test and malaria parasite test
CELL-DYN3500/3700 is normally given the red cell indices, platelets, WOC (WBC optical count), WIC (WBC Impedance Count) count.
Run internal QC and control:
- Do background clean for 3-5 times to make sure the values stated on the screen is near to 0 (Stated on the PC screen connected to the machine).
- Run 3 control samples (Low, Normal, High) using open sample mode (manual). Make sure all values are within control limit.
- Run 2 Internal QC (normally is patient sample that is tested a day before and is within control limit) using closed and open mode. Make sure all values are within control limit.
- Load the sample into the machine for it to run in closed sample mode, and then wait for result.
- For sampling error, redo the test using open sample mode.
- Check whether all the values of the result are within control limit.
- Mainly is to check for WOC, Hb, MCHC and platelets values.
Test Result with reference range:
- For malaria parasite (MP), require making a smear when the platelets are <150>
- For full blood count (FBC), to do a rerun, WOC - <2.0>25, Hb - <60>180, MCHC - <300>365 and platelets - <100> 750. Require to make a blood smear when WOC - <3>15 and platelets <150>150.
- MP is test whether the patient (normally foreign worker) have malaria parasites.
- FBC is to test for any abnormal RBC, WBC and Platelets which is closely related to blood diseases such as leukemia and anemia.
- MP cells normally found in patients: Plasmodium Falciparum
Taken from http://deep6inc.com/previewher20.html
Things to take note:
- Controls and Internal QC need to be run at least 2 times a day to make sure the machines are accurate. This is to make sure that the test result for the patients sample run is reliable and accurate.
- Closed sample mode is automated method, just need to load the sample for the machine to run. Therefore, the volume of the patient blood sample must be more than 1ml.
- When using open sample mode, normally use for small amount or little sample, because is a manual method, make sure the patient sample is mix properly.
Hope you all can learn something from my post. Feel free to ask any question.
Lizzie Chew, TG01
Sunday, July 1, 2007
This is a summary of my first week attachment in school. During the 1st week, there was no SIP involved due to delay in assigning TSO to us. So we basically focus on our MP. My MP is on the effect of functional food on the murine B & T cells.
One week before SIP commence, the 20 mice models arrived. They were to be isolated in the quarantine room, away from the other animals in the animal holding unit (AHU). This is to prevent the spread of potential infectious disease to the healthy animals. Everyday, we had to go back to school to check on the mice health and update the daily environmental record. We also had to ensure that there was sufficient food and water supplied to the mice. As we were dealing with live animals, it is necessary to check on them everyday i.e Mon to Sun. Sometimes on weekends, we had to wake up early just to go school and do a 15min check on them. It was kind of tiring at first but we have 5mths to let us get used to it.
First week of SIP - We prepared the solutions for the negative/postive control group and test group. Autoclaved water was used as a negative control with basal production of lymphocytes. Vitamin C was used as a positive control which will enhanced the production of lymphocytes. The method used to adminster the article/solution was by gavaging. Gavaging refers to the force feeding of article directly into the stomach with the insertion of gavage tubes from the mouth. This process was to ensure that each mouse recieved the specific amount of article administerd to prevent any potential pre-analytical variations. It had to be done carefully to avoid inappopriate insertion into the lungs instead. One of the identification method we used was cage cards for each group. It indicates the protocol number, strain, sex, age, supplier, investigator and contact person. We had done ear punching to ease the identification of every mouse. It involve the use of a special punch to produce a small notch near the edge or in the middle of the ear.
The above content was what I'd done for this week. If u have any questions, pls feel free to ask. Take care!
Taken from: http://www.jhu.edu/animalcare/images/rat_james_mouse_identification.gif