Hey all. Time sure flies and its already our last week of SIP next week! Anyway, I will continue from where i left the previous entry.
After purification is done, the sequence of the individuals DNA need to be determined by carrying out sequencing.
DNA sequencing
DNA sequencing using dideoxy-chain termination method or Sanger sequencing is a technique used to determine the exact nucleotide sequence of a gene, which includes adenine, guanine, cytosine and thymine. It uses DNA synthesis as the basis for the sequencing reaction and also incorporates dideoxynucleotide triphosphate (ddNTPs) as chain terminators. Firstly, the DNA template is denatured and primer is allowed to anneal. DNA polymerase will synthesize the complementary strand by incorporating the dNTPs, in which the strand will only terminate once a ddNTP that lacked a 3’ hydroxyl group is incorporated. After sequencing, the strands of different lengths are separated by capillary gel electrophoresis. As the fragments migrate out of the gel, they are hit by a laser beam, causing the ddNTPs to fluoresce. The data is converted to an electropherogram for visualization.
In sequencing, there are 2 major parts to it that is 1) Cycle Sequencing 2) Ethanol precipitation.
Cycle Sequencing
Cycle sequencing is carried out in three major steps, similar to PCR except one primer is used in the reaction.
1. Denaturation: The hydrogen bonding that holds the double stranded of the target DNA molecule together, are separated into single stranded DNA by heating at 940C.
2. Annealing: The two single stranded DNA are then allowed to cool, at the temperature ranging from 45oC to 60oC, where annealing of the primers to the single stranded DNA will take place. This would initiate the synthesis of the complementary sequences. However, only one primer, either forward or reverse will be used in cycle sequencing.
3. Extension: In this step, Taq polymerase will bind to the DNA, allowing the synthesis of complementary strand by incorporating dNTPs. When a dye-labelled ddNTP is incorporated, it cannot form a phosphodiester bond with a normal nucleotide, thus, terminates the synthesis of the new strand.
This reaction was carried out using the Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The components listed below will be added to the sequencing plates for sequencing. The reaction was terminated using equal volumes of stock solutions consisting of 3M sodium acetate and 125mM EDTA, after cycle sequencing.
Components of Cycle Sequencing Reaction
-ABI Dye
-Sequencing Buffer
-Primer (Forward/Reverse)
-DNA/PCR product
-Distilled Water
Ethanol Precipitation
To precipitate out the DNA, ethanol precipitation was done by adding 25µl of 100% ethanol to the PCR products in the sequencing plates. It will then be incubated for 15 minutes by wrapping the plates with aluminium foil. After which, the plate was centrifuged at 3000G, 4oC for 30 minutes to obtain the DNA pellet. To remove the 100% ethanol that is the supernatant, the plate was inverted on a blotting paper and pulsed at 200G for 20 seconds. Following this, to wash the DNA pellet, 70µl of 70% ethanol will be added and again, the plate was centrifuged at 1700G, 4oC for 15 minutes. Subsequently, it will be pulsed twice at 200G for 20 seconds to remove the 70% ethanol. To properly ensure that the pellet was completely dried, the plate was placed in the vacuum dryer for 5 minutes followed by 95oC for 5 minutes in the opened-lid thermocycler. 20µl of formamide was then added to dissolve the DNA pellet before it was placed inside the 3730 DNA Analyzer (Applied Biosystems, USA) to be sequenced.
After sequencing is done, the results will then be produced as trace files by the DNA analyzer machine and loaded into a computer program for blasting and genotyping to be done.
Blasting
Blasting is a method used to locate single nucleotide polymorphisms in the fragment. A software will then align the trace files, in which all the different DNA sequences of each individual DNA will be aligned accordingly. After which, the DNA sequences will be compared and see if there is the presence of double peaks or change in the colour of peak at the same location. This is a high indicative of a SNP. After blasting is done, and the SNPs had been located, genotyping can then be done for each individual.
Genotyping
Using Sequencing Analysis v5.2 (Applied Biosystems, USA) and SeqScape v2.5 (Applied Biosystems, USA), the resultant trace files from the DNA sequencing reactions were base called and assembled respectively. Following assembly, analysis of the DNA sequences was then done by multiple sequence alignment and comparing to the reference obtained from National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) to detect single nucleotide polymorphisms. Each individual will be either having genotypes either 1) homozygous wild type, 2)heterozygous 3)homozygous variant.
For example, for SNP A>G,
Homozygous wild type : AA
Heterozygous: AG
Homozygous variant: GG
Liyanah Zaffre
0607718D
TG02
Week 19 posting
WEEK 18
So, this week I would lke to share with you another analyzer called the Elecsys 2010 by Roche Diagnostics, USA. Elecsys 2010 is used to perform immunological tests, mainly in vitro testings for the quantitative and qualitative determination of analytes present in the human serum.
The kind of immunoassays carried out in my lab are tumor markers such as CA 125 and CA 19-9, Free T4 and Free T3, Carcinoembryonic antigen (CEA), Hep-B surface antigen type II, Hep-A virus, HIV, Thyroid Stimulating Hormone (TSH), Luteinizing Hormone (LH), Testosterone, Follicle Stimulating Hormone (FSH), Beta-HCG, Sex hormone-binding globulin (SBHG), Rubella G, Alpha Fetal Protein (AFP), Prolactin, and many more. However, for this posting, I won't go into each test individually. I will mainly focus on the principles of the analyzer, only. Elecsys 2010 works based on 3 types of principles; competitive binding, sandwich binding and bridging binding.
Elecsys 2010 by Roche Diagnostics, USA.
Retrived 24th October 2008 from http://images.google.com.sg/imgres?imgurl=http://www.rochediagnostics.pl/content/produkty_i_uslugi/diag_scentr/immunodiagnostyka/img/elecsys_2010.jpg&imgrefurl=http://www.rochediagnostics.pl/content/produkty_i_uslugi/diag_scentr/immunodiagnostyka/elecsys2010.html&h=170&w=232&sz=17&hl=en&start=7&usg=__VI1tMedzGExoyIfrzsys6Kpc8Dg=&tbnid=BQz3cQjiytYxSM:&tbnh=80&tbnw=109&prev=/images%3Fq%3Delecsys%2B2010%2Broche%26gbv%3D2%26hl%3Den
- the analyzer uses this principle for analytes with low molecular weight (eg. Free T3)
Firstly, the antigen found in the sample (human serum) is mixed with specific anti-T3 antibody labelled with ruthenium complex. After incubation, the 2 particles will bind to one another. Another reagent is added, this time containing biotinylated T3 particles and streptavidin-coated paramagnetic microparticles. The process goes through another incubation cycle, after which immune complexes are formed. The immune complexes are transported into a measuring cell where they are magnetically entrapped onto a working electrode. The unbound particles are washed away by a washing reagent called the ProCell. The ruthenium complexes are then electrically stimulated to cause a chemiluminescent reaction to produce a light signal. The analyzer then detects the amount of light produced and calculates the concentration of antigens present in the human serum. The amount of light produced is indirently proportional to the amount of antigen present. The following is an illustration done using microsoft powerpoint of how the antibodies and antigens compete with one another for binding sites.
2) Sandwich principle
- the analyzer uses this principle for analytes with higher molecular weight (eg. TSH)
The sample (human serum) containing antigens is mixed with biotinylated TSH antibodies and ruthenium-labeled TSH-specific antibodies. The mixture is then incubated for 9 minutes to allow the particles to bind to one another, after which another reagent containing streptavidin-coated paramagnetic microparticles are added and bind to the binding site of the biotinylated antibodies. The reaction goes through another incubation cycle causing the formation of immune complexes. The immune complexes are then transported into a measuring cell where they are magnetically entrapped onto a working electrode. The unbound particles are washed away by a washing reagent called the ProCell. The ruthenium complexes are then electrically stimulated to cause a chemiluminescent reaction to produce a light signal. The analyzer then detects the amount of light produced and calculates the concentration of antigens present in the human serum. The amount of light produced is dirently proportional to the amount of antigen present. The following is an illustration done using microsoft powerpoint of how the sandwich formation is formed between the antigens and antibodies. 
3) Bridging principle
- the analyzer uses this principle when the tests require detection of antibodies and not antigens in the serum.
Firstly, the machine will detect the presence of serum antibodies in the sample. The serum antibodies are then mixed with biotinylated antigen found in the test reagent and ruthenium-labeled antigens. After a period of incubation to allow the particles to bind to specific binding sites, stretavidin-coated paramagnetic particles are added and bind to the biotinylated antigen binding site, forming immune complexes. The immune complexes with the paramagnetic particles bound to them will be magnetically attracted to a working electrode when they are transported into a measuring cell. Unbound complexes are washed away by ProCell. The ruthenium antigens are then electrically stimulated to produce light signals. The light signals are detected by the analyzer which will then calculate the antibodies concentrations in the human serum. The amount of light detected is directly proportional to the amount of antibodies present. The following is an illustration done using microsoft powerpoint, of how the bindings occur during the process. 
I guess that's all for you. Hope to see all of you soon.
2 more weeks of SIP C:C:C:C:C:C:
Nur Azeimah
0607060A
TG 02
Week 17
All cells undergo the cell cycle, a process by which the cell replicates through different phases. One of which is the M phase, where an adult cell splits into two daughter cells. The M phase is further categorized into two different processes: mitosis, where cell's chromosomes are divided between the two daughter cells, and cytokinesis, where the cell's cytoplasm divides and forms distinct cells. Prometaphase, metaphase, anaphase and telophase are stages that take place within mitosis.
M Phase in Cell Cycle
(Source: http://www.infoplease.com/cig/biology/cell-cycle-interphase-mitosis-cytokinesis.html)
Metaphase is a stage in which condensed chromosomes align in the middle of the cell before being separated into each of the two daughter cells. Because the structure of chromosomes is the clearest at this stage, cells will be stimulated to stop growing at this stage for analysis. The metaphase chromosomes can be studied in spontaneously dividing cells or in cells that have been stimulated to divide in culture.
Metaphase
(Source: en.wikipedia.org/wiki/Metaphase)
3 general processes that must take place in order to study the chromosomes are:
1) Culturing – Supplying the cells with nutrients to allow to proliferation
Before obtaining the metaphases of cells, cultures may be needed. The decision of whether to culture the cells lies on the specimen type. Spontaneously dividing samples such as bone marrow and lymph node may be set up for a direct harvest. Other samples such as tissues may require culturing for several days to allow the cells to proliferate. Once the sample has been cultured, harvesting is performed.
2) Harvesting – Obtain metaphases of cells, the stage at which chromosomes can clearly be seen
When cells are growing spontaneously, they will be harvested to obtain metaphases for analysis. In harvesting, three standard protocols will be used: mitotic arrest with Colcemid, hypotonic treatment with KCl (potassium chloride) and fixation with 3:1 methanol: acetic acid.
a) Mitotic arrest with Colcemid – Cells are arrested at the metaphase stage to enable the capturing and analysis of the chromosomes. Colcemid is used to prevent spindle formation, a process by which sister chromatids are pulled to opposite poles for incorporation of into the 2 daughter cells. It also promotes chromosome condensation, a process that can be affected by increased exposure time and concentration.
Different exposure time to Colcemid can affect the quality and quantity of the chromosomes; the condensation effect is greater when exposed for a longer period of time (ie. chromosomes are smaller in size but more). However, different culture and harvest methods may react differently to the Colcemid reaction. Therefore, to obtain the desired result, cells will be exposed to Colcemid for:
2 hours (long exposure time: more but shorter chromosomes)
20 mins (short exposure time: lesser but longer chromosomes)
b) Hypotonic Treatment with KCl – After arresting the cells at metaphase stage, they need to be treated with a hypotonic saline solution to increase the cell volume so that chromosomes can spread out. However, prolonged exposure may cause weakening of the cytoplasmic membrane, thus increasing the risk of the cell bursting and chromosomes to escape.
c) Fixation (3:1 methanol: acetic acid) – The purpose of fixing the cells is to remove the water content as well as to preserve them be hardening the membranes and chromatin, and somehow prepares chromosomes for the bending procedure.
3) Karyotyping – Chromosomes will be pair up to check for abnormalities
After fixing the cells, the slide needs to be stained to be able to visualize the bands on the chromosomes. Stained slides can now be karyotyped.
Down Syndrome (extra chromosome 21)
(Source: http://www3.geneticsolutions.com/?id=1530:1873)
Klinefelter Syndrome (extra sex chromosome X)
(Source: http://www3.geneticsolutions.com/?id=1530:1873)
Answers to Week 16 posting (For Li Ping and Ernest)
I understand that the answers I provide is quite long, but it's going to give you a clearer picture in some of the applications of flow cytometer, especially regarding my MP. Please don't give up on the 'long' post and read it =)
Before I start answering questions, I would like to touch on a few terms first (you could skip this part if you wish):
1) Fluorescence-based techniques
2) What is autofluorescence?
3) What is ‘true negative’?
Answers:
1) You should know that fluorescence-based techniques use fluorochromes in many different ways; one of the ways is to utilize the ability of dye to influx in the cells and ability of cells to efflux out the dye. Examples of such dyes are: Hoechst 33342 and Rhodamine.
These fluorochromes emit a certain ‘light’ wavelength after excitation from a light source (eg. Laser beam) that is detected by a detector in the Flow cytometer. Please take note that flow cytometer is mostly used to analyze live cells.
2) Autofluorescence is ‘weak’ autofluorescence emitted by cells not stained with the fluorochromes. Therefore, they are considered to be ‘false positive’ fluorescence.
3) As you will know, before the immunofluoresence staining of a cell, the cell should not express any fluorescence, right? So, ‘true negative’ is the sum of ‘negative’ and ‘false positive’ fluorescence.
To Li Ping’s Questions
Answers:
I believe you are confuse over the word ‘true negative’, when I mention about ‘true negative’, I meant the portion of cells in a heterogeneous cell population that are not stained positive. Only 'unstained' tube consist of 'negative' results as it is not stained at all. Other 'stained' tubes consist of both stained and unstained cells, so the data from 'unstained' tube is used to identify the cells that were not stained, something like a negative control.
And yes, it is correct to say that the value obtained will be used as benchmark, however, this benchmark is to identify the amount of ‘true negative’ population in other tubes. You must know that the cell sample we used is a heterogeneous population of cells and so, not all cells will be stained with the dye we used.
Different gates are used for different experiments. For my experiment, I used that series of gating that I mentioned before:
a. Scatter gate:
used to gate cluster of cells which our cells of interest is located and exclude all other ‘rubbish’ cells.
b. Side Scatter (SSC) gate:
used together with FSC for doublets discrimination and exclude all but single cells.
c. Forward Scatter (FSC) gate:
used together with SSC for doublets discrimination and exclude all but single cells.
d. Viability gate:
identify viable, living, cells. Cells not stained by Propidium Iodide (PI), one of the viability dyes that could be used, are live cells.
e. Live gate:
exclude ‘rubbish’ cells, like red blood cells, and non-viable cells that are not successfully excluded by previous gates. Also exclude other cells that are not of interest.
f. SP gate:
compare “Hoechst 33342 only” against “Hoechst 33342 + Verapamil” tubes to identify and gate SP cells.
Note: Blockers, like verapamil, inhibit ABC transporters that give a ‘tail-like’ cell profile of SP cells that are low Hoechst blue and red, on the plot graph.
Hoechst emits a wavelength that can be detected at two different channels with two different detectors.
To Ernest Questions
1) Yes, you can interpret it as that way. ‘Unstained’ tube contains cells that are not stained. This is used to compare with other ‘stained’ tubes, which consist of both stained and unstained cells in its pool of heterogeneous cell population. It helps to identify cells that are not stained from the rest of the stained cell population. Something like a negative control.
2) ‘PI only’ stands for the tube that is only stained by propidium iodide, which is one of the viability dyes that could be used. Cells not stained by PI are live cells.
3) You can add as many dyes as you can, however, people usually 2 different dyes plus a viability dye at most. Also, it doesn’t make sense to add too many dyes as there will be complications like spectrum overlap of fluorescence that spills into other ‘light’ channels and gives false positive results.
(For example only: Imagine having PE dye fluorescent spills over into FITC dye channel and gives a false positive result in the FITC dye channel as the fluorescence detected in FITC channel is actually from the PE dye)
4) Please refer to above answers to Liping’s questions.
5) Please do not confuse my experiment to detect SP cells from an unrelated experiment of DNA analysis that also uses Hoechst 33342 dye. SP cells are known to actively efflux out drugs and dyes like Hoechst 33342, which is why Hoechst 33342 is used in our experiment design. And we compare Hoechst red to Hoechst blue channels to observe our results.
As I mentioned in answering Liping’s question:
“Blockers, like verapamil, inhibit ABC transporters that give a ‘tail-like’ cell profile of SP cells that are low Hoechst blue and red, on the plot graph.
Hoechst emits a wavelength that can be detected at two different channels with two different detectors.” (Quoted)
6) Refer to above, answer to your question 5.
I really hope both of you understand more about my SP experiment. If you have any more doubts, feel free to ask again.
Also, to understand more about my SP experiment, please read my 3rd posting of “Double posting! (2nd part: SP Profiling in Stem Cells)”, before asking me any related questions.
Many thanks
Quan Jun
Week 16: 4th Official Blog Posting
Hi guys & gals!
Woah, time sure flies, I can still remember the first day of my SIP, but it's now left with 4 more weeks of SIP! Then I can take permenant leave from my supervisor! Can't wait. Hahaz!
Hope everyone is doing well too!
This week, I would like to continue from my last blog posting. Today, I would like to explain how the SP profile is recorded to give a more complete picture to the use of flow cytometer in SP profiling.
For every experiment, the following sample tubes are needed:
1) “Unstained” tube
2) “PI only” tube
3) “Single dye only” stained tubes
4) “Combination of dyes” stained tubes (differ according to each different experiment)
Before recording the data, there are procedures to be done first.
For my experiment, I have the following sample tubes:
1) “Unstained” tube
2) “PI only” tube
3) “Hoechst 33342 only” stained tube
4) “Hoechst 33342 + Verapamil (x) + PI” stained tube
[For simple explanation sake, I will use only one tube with Verapamil as blocker at a fixed concentration (x)]
Firstly, this is what we do:
1) we will take a look at the unstained tube to view the “true negative” and auto-fluorescence stained population
2) Vary the voltages of the “light” detectors to display the desired cell cluster in all the plot graphs
3) View other tubes to take a quick look at our stained population to observe if we have any “positive” stained population
4) Steps 1 to 3 are done interchangeably to bring out our desired cell profile, once done, we proceed to the next step
5) Since I am doing on SP profiling, I will do up a series of gating in the following order:
a. Scatter gate
b. Side Scatter (SSC) gate
c. Forward Scatter (FSC) gate
d. Viability gate
e. Live gate
f. SP gate (To be done after all the cell profiles was recorded)
After we brought out our desired cell profile, we would start recording the data.
Number of events to record (for my experiment only):
1) “Unstained” tube (50K “all event” events)
2) “PI only” tube (50K “viable gate” events)
3) “Hoechst 33342 only” stained tube (200K “live gate” events)
4) “Hoechst 33342 + Verapamil (x) + PI” stained tube (200K “live gate” events)
Note: The number of events to record differs with different operators, different experiments, and ability of the flow cytometer to obtained the desired cell profile
After recording the results, the flow cytometer is prepared to shut down for the day and that’s it.
The operation of flow cytometer differs from operator to operator, however, the recording of tubes always follow a sequence:
1) “Unstained” tube
2) “PI only” tube
3) “Single dye only” stained tubes
4) “Combination of dyes” stained tubes
Please do ask question, about anything you do not understand, I would "love" to explain it to you. Haha.
.....4 more weeks to the end.....
Hope everyone take care of yourself!
Many thanks
Quan Jun
TG02
Week 14 posting
Hey all. I sincerely apologise for the late posting as I was confused over the dates and the weeks. Anyway, I’ll continue from where I left of the previous post that is gel check for primer optimization. After the gel check for primer optimization and the optimum conditions have been chosen for the specific primer, PCR can then be done for that primer.
What are the differences and similarities between primer optimization and PCR?
Differences
-Primer optimization is done using test DNA whereas PCR is done using a specific patient’s (whether healthy or diseased) DNA. This is because, when doing the primer optimization, the optimum conditions are not known, thus, it is not necessary to use patient’s DNA. (Patient’s DNA is very precious as they are retrieved only ONCE from the patient themselves while test DNA can be bought.)
-For primer optimization, a range of temperatures will be programmed at the Thermogradient, whereas, for PCR, a specific optimum temperature will be set for the Thermocycler.
Similarities
- The method for conducting both PCR and primer optimization is similar including all the reagent used.
Introduction on PCR
Polymerase Chain Reaction (PCR) is a method for the amplification of specific DNA sequences in vitro, which involves repetitive heating and cooling cycles to yield large quantities of replicated DNA. It requires two oligonucleotide primers that will flank the DNA target sequence that is to be replicated. The three major steps that are carried out in each PCR cycle are the following:
1. Denaturation: The hydrogen bonding that holds the double stranded of the target DNA molecule together, are separated into single stranded DNA by heating at 940C.
2. Annealing: The two single stranded DNA are then allowed to cool, at the temperature ranging from 45oC to 60oC, where annealing of the primers to the single stranded DNA will take place. This would initiate the synthesis of the complementary sequences.
3. Extension: When the temperature is increased to a temperature optimum for the Taq DNA polymerase, it will bind to the free 3’ end of the primers. By incorporating dNTPs, the Taq DNA polymerase synthesizes a new DNA strand in a 5’ to 3’ direction.
Method for PCR
*Please refer to previous entries on method for primer optimization as they are similar*
After which, gel check has to be done again. This time, it is to double check if the bands are present for different DNA samples. However, for the gel check of PCR, only 0.5ul of loading dye and 2ul of random PCR product are used instead of 2ul of loading dye and 10ul of primer optimization product. This is because, the remaining 8ul of the PCR product is required to be used for experiments following this step. If approximately 80% of the bands appear, purification of the PCR product can be done.
Purification
To eliminate excess PCR components after the PCR reaction, purification of the amplified PCR products was done using two hydrolytic enzymes, Exonuclease 1 (Exo 1) and Shrimp Alkaline Phosphatase (SAP). Exonuclease 1 catalyzes the removal of excess primers in the 3’ to 5’ direction while Shrimp Alkaline Phosphatase is responsible for the dephosphorylation of dNTPs to prevent it from participating in any further polymerisation steps. Both enzymes at its optimum temperature, 37oC, are involved in the digestion process. Following this, the temperature will increase to 72oC resulting in the denaturation and inactivation of the enzymes. This would prevent the enzymes from interfering in subsequent steps, for example, cycle sequencing.
Methods for purification
Following PCR and gel electrophoresis, 0.5µl of Exonuclease 1 (Exo1) and 1µl of Shrimp Alkaline Phosphatase (SAP) need to be added into each sample of the PCR products.
1. Calculations were done by multiplying the number of samples needed to be purified.
2. One 1.5ml of eppendorf tube (master mix tube) was collected and labeled ‘Exo-Sap’.
3. An ice box, filled with crushed ice, was prepared.
4. Exo 1 and SAP was retrieved from -20oC fridge and placed in the ice box.
5. The desired amount of Exo and SAP was pipetted into the master mix tube.
6. The master mix tube was then spun down using a mini centrifuge.
7. 1.5ul of the resultant mixture was pipetted into each sample in each well.
8. The samples of the PCR products were placed into the thermocycler and the program was set.
After purification is done, the next step would be sequencing. I would explain this step in further details in the next posting. Till then, I would like to wish all Muslims, Selamat Hari Raya Aidilfitri and to all non-Muslims, have a joyous holiday! J
Liyanah Zaffre
0607718D
TGO2
Answers for WEEK 13
As most of the questions asked are pretty similar and pictures are requested, this post will show some of the common casts and crystals that I've encountered in my lab. The following pictures are retrieved from websites since I wasn't allowed to snap photos in my lab. They're actually quite easy to find on the net.
1) Waxy casts
I was told that waxy casts normally appear as the end stage of casts formation. It basically means, a waxy cast appears when the patient has been suffering from renal failure for a long time. It is originally a normal cast but appears as waxy after years of suffering from renal failure. It normally appears in diseased patients. So far, I've only encountered one throughout my whole week stay under urinalysis. They are normally broad with sharp edges and their ends looked as if it has been cut or broken. They are also more distinct than hyaline casts.
Retrieved 28th September 2008 from, http://www.academic.marist.edu/~jzmz/methods/waxycast.jpg
2)Granular casts
Granular casts are quite normal and may be due to breakdown of other cellular casts such as RBC casts or leukocyte casts. Sometimes, their appearance may indicate chronic renal failure but similar to hyaline casts, they may appear due to stress or after strenuous exercise and may disappear after sometime. Notice the granules found on the cast, making it look like a big clump of granules.
Retrieved 28th September 2008 from, http://www.agora.crosemont.qc.ca/urinesediments/image/d21i003.jpg
3) Hyaline casts
Hyaline casts may be quite tricky to identify due to its faint appearance and almost blends in with the background. Therefore, as I've mentioned in my earlier post, casts are usually looked out for when protein levels are high. They appear in urine due to solidification of Tamm-Horsfall mucoprotein secreted by tubule cells. They are more common in acidic urine. Patients who have done vigorous exercises or are feeling dehydrated, may have hyaline casts in their urine. The arrows pointing in the picture are trying to show the smooth edges of the hyaline cast.
Retrieved 28th September 2008 from, http://www.med.uiuc.edu/internalMed/residency/edmod/mod1/hyaline.jpg
4) Calcium Oxalate Crystals
Calcium Oxalates are very easy to distinguish due to its "square" shape and crosses found in the middle. A senior colleague told me that sometimes, presence of calcium oxalates may be due to patients eating too much vegetables. These type of crystals are quite common. They do not necessarily indicate any diseased conditions. They may or may not develop into kidney stones depending on how hydrated the body is.
Retrieved 28th September 2008 from, http://library.med.utah.edu/WebPath/jpeg2/URIN077.jpg
5) Triple Phosphates
This is another type of common crystals found in normal patients. To me, they look like gold bars, though some people would prefer to think of them looking like coffin lids. They don't have crosses in the middle like calcium oxalates. They appear due to ammonia concentrations found in the urine making it slightly alkaline. High amounts may indicate urinary tract infection. But very small amounts may not be clinically significant and does not indicate any diseased state.
Retrieved 28th September 2008 from,
http://meded.ucsd.edu/isp/1994/im-quiz/images/struvit.jpg
6) Uric Acid
As I've mentioned, these crystals are quite normal and are usually found in urine with pH 5 or lower. The rossette form of uric acid is the more common form. It is basically the flower-shaped ones. Uric acid may accumulate and are formed due to breakdown of purines or waste products in the body. Too much uric acid may cause gout to develop. If they accumulate in the kidneys, kidneys stones may be formed.
Retrieved 28th September 2008 from, http://www.academic.marist.edu/~jzmz/methods/uacystals.jpg
These are the common crystals and casts that I've encountered so far. I'm sure there are more but are less common in patients. The results printed by the analyzer would usually show the amounts of the substances present. Usually, plus signs '+' are used to indicate the levels of the substances present. For example, 'Leukocytes ++++' means about 30-50 leukocytes are present. We would then report the results into the system as Leukocytes 4+. The system would automatically know that there are 30-50 leukocytes detected.
We usually spin the urine samples if high amounts of leukocytes are detected or when high amounts of erythrocytes are detected or both. We also spin turbid or slightly turbid samples. I was told that if the urine is not spun down, we might miss out certain substances that are present in the urine. Therefore, by spinning them down and allowing the substances to settle at the bottom and then examining the sediment, would enable us to identify almost all the substances that are present in the urine sample. If proteins appear to be 3+ or 4+, we also spin the urine sample down to observe for any casts.
The doctors who ordered for this test would usually simply indicate UAN or URINE FEME on the request form. Both would mean that urinalysis and urine microscopy is required to be done upon the urine sample.
Urine Specific Gravity is an indication of how concentrated or how diluted the urine sample is. The more concentrated the urine, the higher the specific gravity is. The values are usually fixed like 1.000, 1.010, 1.015, 1.020, 1.0250, 1.0300 and so on. Increased specific gravity may be due to dehydration or increased secretion of anti-diuretic hormone in the body. Decreased specific gravity may indicate damage to the tubule cells causing the kidneys to be unable to reabsorp water, kidneys producing too much urine or renal failure. Specific gravity of 1.002 to 1.035 is considered normal.
I hope this second posting is much more clearer to all of you and I hope I've managed to answer all the queries posted earlier. Thank you C:
Nur Azeimah
0607060A
TG 02
WEEK 13
For the past 3 weeks, I was posted to a satellite laboratory under the company I was attached to. A satellite lab basically means a smaller “branch” that carries out basic tests for walk-in patients. The satellite lab also receives samples from next-door clinics and a nearby hospital. It is more stressful in the morning as most of the samples are urgent samples, compared to the main lab.
On my last week there, I was assigned the task the carry out urine tests. Urine tests include urine pregnancy tests, urine glucose tolerance tests and urinalysis. For this entry, I shall talk about urinalysis.
The equipment used in the lab is Miditron Urine Analyzer manufactured by Boehringer Mannheim. It is actually quite an old equipment but it produces results within 5 minutes. The whole process is semi-automated. Upon receiving urine samples, the urine need to be transferred into a long conical tube up to the first marking from the top. The patient’s barcode number is registered into the machine for easy identification, results entry and results validation later on, which can only be done by the permanent staffs there. The urine is checked for its colour and clarity. The colour may be yellow, straw, reddish, or dark brown. The clarity ranges from clear to turbid. A test strip is then dipped into the urine. The test strip used is known as Combur 10 Test M manufactured by Roche Diagnostics. After dipping the test strip into the urine, the test strip is loaded into the machine. The machine then scans the test strip to read for any colour change on the test strip. After scanning the test strips, results are produced and printed about 5 minutes later.
By using the test strip, the specific gravity, pH, presence of leukocytes, nitrite, protein, glucose, ketone bodies, urobilinogen, bilirubin, and erythrocytes can be tested.
Principles:
- Specific Gravity : detection of ion concentration of the urine through a colour change from blue to blue-green or yellow when protons are released by a complexing agent.
- pH : contains pH indicators methyl red, phenolphthalein and bromothymol blue which reacts with hydrogen ions to produce a colour change.
- Leukocytes : granulocyte esterases cleave off indoxyl ester which reacts with diazonium salt to produce a violet dye.
- Nitrite : detection of organic nitrite compounds produced by nitrite-forming bacteria. Presence of nitrite will cause a colour change to pink or red.
- Protein : this component is very sensitive to albumin. Detection of albumin is based on protein error of a pH indicator. Presence of protein in high amounts may indicate presence of casts, for example hyaline casts or granular casts.
- Glucose : reaction between glucose and oxidase or glucose and peroxidase.
- Ketone bodies : detection of ketone bodies and is more sensitive to acetoacetic acid than acetone.
- Urobilinogen : reaction between diazonium salt with urobilinogen present in the urine, producing a colour change to red.
- Bilirubin : (similar to urobilinogen) reaction between diazonium salt with bilirubin to produce a pinkish colouration.
- Erythrocytes : reaction between organic hydroperoxide with haemoglobin and myoglobin to produce a peroxidase-like reaction. Positive results would show a blueish-green colouration.
After running the urinalysis using the machine, the positive urine samples are centrifuged at 2500 rpm for 5 minutes to obtain the sediment. The sediment is then observed under a microscope which is usually done by a more senior medical technologist. However, I have been given a chance to observe certain bacteria, casts and crystals that are present in abnormal urine samples. Some crystals are quite common such as Calcium Oxalates, Uric Acid, Amorphous Phosphates and Amorphous Urates. Uric acid is usually present in urine samples with pH 5. Amorphous Phosphates and Amorphous Urates look very similar. Therefore, if they are present in a urine sample, the pH of the urine is usually looked at to differentiate between the two. Amorphous Phosphates are present in urine sample with pH 7 or more, while Amorphous Urates are present in urine sample with pH lesser than 7.
Nur Azeimah
0607060A
TG 02
Week 12
Hi, this week I'll be sharing on microbiology.
In the microbiology lab, the most commonly received types of specimens would be urine, stool and swabs from any part of the body for culture. Culture basically refers to specimens being plated on specific agar/s to observe for any predominant growth of microorganisms. If culture results confirmed that the patient is infected, CDS (calibrated dichotomous sensitivity) test would be done to determine the antibiotic sensitivity of the microorganism in order for doctors to prescribe the correct type of antibiotic to the patient. 
CDS (another term for antibiotic sensitivity) Test
Because of the fact that human stool is not sterile, there is a high possibility that there would be a mixture of gram positive or negative, aerobic or anaerobic bacteria present. Therefore, differential media (eg. XLD, TCBS and Campylobacter agar) need to be used to differentiate the normal from pathogenic flora present in the stool.
Besides stool cultures, high vagina swabs (HVS) are also very commonly received in the lab. Because I work in a gynae hospital, most of the HVS received are to detect for Group B Streptococcus in pregnant women. Although group B Streptococcus can be considered as a type of normal flora in the female genital tract, the presence of this organism in pregnant women is clinically significant.
Swabs used for Culture
Retrieved from http://home.caregroup.org/departments/pathology/lab_manual/PLM_containers.htm
Group B Streptococcus can cause a relatively rare but serious infection in newborns. Approximately 10-30% of pregnant women carry this type of bacteria in the vagina or rectal area, and may pass in to their babies during labour. Some of the consequences include sepsis, pneumonia or meningitis in newborns. Some of these babies, particularly those with meningitis, will have long-term health problems such as hearing or vision loss, cerebral palsy, or developmental disabilities and about 5% would not be able to survive. Therefore, to reduce the risk of the infection in newborns, prenatal testing and treatment is often recommended.
Processing:
1. Swab would be plated on blood agar and put into TGN enrichment broth.
2. Plates and broths are then incubated for 37°C for 24 hours.
3. After incubation, sub-culturing is done by plating the TGN enrichment broth on blood plates.
4. Original blood plates incubated for 24 hours will be checked for organisms such as strep B, Candida and Listeria.
5. Subcultures will be read the following day for strep B.
Kliger Iron Agar (KIA) – Detects the ability of the microorganism to ferment glucose by observing the reaction produced be the organism in the agar. KIA also contains sodium thiosulfate to test for hydrogen sulfide. Iron salts present in the media react with the hydrogen thiosulfide to produce an insoluble black precipitate.
OF (Oxidation Fermentation) Basal Media – Used to determine oxidative and fermentative metabolism of carbohydrates by gram-negative bacteria on the basis of acid reaction in either the open or closed system. Closed system refers to the surface of the agar being covered with oil.
Motility Indole Lysine Medium – Used to demonstrate motility, indole production, lysine decarboxylase and deaminase activity and hydrogen sulfide production in microorganisms.
Ka Hang
TG02
Double posting! (1st part: Simple Introduction to Flow Cytometry)
Hi all
I had gotten some feedbacks from some of our fellow course-mate that my entries are a little too technical and very hard to understand. So I decided to skip the technical stuffs and go straight in to the topic with simple explanation.
So feel free to ask me any questions regarding any parts that you do not understand in my entry.
Simple 4 steps explanation of how an experiment with the Fluorescent Activated Cell Sorter (FACSAria), also known as a flow cytometers, works:
1) Load the samples onto the FACSAria
2) Data will be acquired
3) Set gating to select out the cells of interest
4) Record the data
Flow cytometry is simply just to analyze the data acquired from cell population that was loaded onto the FACSAria. It analyze by detecting and identifying the cell population of interest that were set aside differently from rest of the cell populations.
Some examples of how this could be done are through the uses of cell markers and certain dyes, like fluorescent-coupled antibodies or fluorescent dyes or fluorescent proteins.
Example of Fluorescent coupled antibodies:
1) Antibodies against CD44 cell marker, coupled with PE fluorochrome
Note: PE stands for phycoerythrin
Example of Fluorescent dyes:
1) Hoechst 33342
Example of Fluorescent proteins:
1) Green Fluorescent Protein (GFP) via cell transfection
Okay, that’s all to my introduction to the flow cytometers and I will stop any further complex explanation here. Hope everyone could have a rough understanding of what flow cytometry is about.
Many thanks
Quan Jun
TG02
Group 08
08 September 2008
Double posting! (2nd part: SP Profiling in Stem Cells)
Up next is my introduction to my Major Project (MP), which is related to Side Population (SP).
Short Introduction
Side population is a defined population of cells that could be distinctly identified from the rest of the heterogeneous cell population with the use of Hoechst 33342 dye in the flow cytometers. Using defined filters that allow the collection of the emission profile of the used dye does this.
The primary objective of my project is to identify the best blockers blocker and its optimal concentration in blocking ATP-binding cassette (ABC) family of transporters. The blockers are Verapamil, Fumitremorgin C and Reserpine respectively.
We will be using bone marrow cells.
ABC family of transporters is responsible for the high efflux of the Hoechst dye that contributes to the low Hoechst staining which could be identified with the use of flow cytometers. We use isolated bone marrow from murine species for SP identification.
Below is a diagram that shows how SP cells are identified and gated. .JPG)
The following is the protocol in which my laboratory follows to acquire SP profiling in flow cytometry.
(The protocol is only for understanding purposes; so certain information is left out, please understand)
DMEM = Dulbecco’s Modified Eagle’s Medium
HBSS = Hank’s buffer salt solution (Hank’s Balanced Salt Solution)
Use surgical instruments to remove the fur from the fore and hind limbs
Remove and store the fore and hind limbs in cold DMEM+(with EDTA)
Transport the specimens on ice and process in the laminar flow hood
Remove the meat from the fore and hind limbs
Cut the ends of the femur, tibia, humeral and radius
Flush the interior of the bone until it turns white
Filter the cell suspension obtained with a 100um cell strainer
Centrifuge the cell suspension
Remove all but 500ul of the supernatant
Add erythrocyte-lysing solution in a ratio of 11:1
Incubate for 5 minutes under room temperature
Centrifuge the cell suspension
Remove the supernatant and wash 2 times with cold HBSS+
After washing, centrifuge the cell suspension again
Remove the supernatant and add 2ml of cold DMEM+(without EDTA)
Count the number of cells using a hemocytometer
Calculate the total number of cells in the cell suspension
Prepare and label empty 15ml falcon tubes
Pipette in 1mL of pre-warmed DMEM+(without EDTA) in each of the tubes
Calculate the amount of cell suspension to add to achieve a final concentration of 106 cells/ml for each of the tubes
Place the tubes in the waterbath (37oC)
Add blockers to “Hoechst + blocker + PI” tubes according to the blockers to be used
Pre-incubate the cell suspension for 15mins at 37oC
After pre-incubation, add in the Hoechst 33342 dye to all the tubes and incubate for a further 90mins at 37oC
Mix the cell suspension at every 20mins interval during the incubation
After incubation, centrifuge the cell suspension
Remove the supernatant and resuspend in cold HBSS+ with PI
Store on ice and out of light before data acquisition on FACSAria
Record the SP profile acquiredWeek 10 sharings
Hi everyone!
It is time for me to introduce to you the new DropArrayTM platform that my company has developed.
DropArrayTM is a platform aimed to rival the 96-well plate. The 96-well plate is the most common platform used to run assays, especially those that require numerous washing steps. One example of such assays is Enzyme-Linked Immunosorbent Assay (ELISA).
However, running assays on a 96-well plate has been shown to consume relatively large amounts of reagents, samples, and assay time. As for the washing steps, there is the risk of cross-contamination and/or carry-over of liquids across the different wells.
This new platform has been designed and developed to overcome the problems mentioned above.
I will briefly describe the components of the DropArrayTM that I most oftenly use:
S48/S96 Plate
The plates are actually microscope glass slides patterned with Teflon coat. The coat acts as a hydrophobic surface. The S48 plate has 48 wells (12x4), and the S96, 96 wells (12x8). The diameter of the wells is 2mm.
LT100 Wash Station
This station comprises of a chamber and control buttons to set the speed, duration and volume needed for washing. There is an inlet tubing to allow wash buffers to flow into the washing chamber, and an outlet tubing to drain it out of the chamber. The plate is loaded into the chamber and the washing (or rinsing) process can then start: drain any oil > fill chamber with wash buffer > shake > drain wash buffer.
The shaking step is to remove any media or liquid from the wells.
Incubation Oil
For cell seeding. After the cells are seeded into the wells, the wells are covered with incubation oil to prevent evaporation of media during the incubation period inside the CO2 incubator.
Rinsing Oil
Rinsing oil also prevents total evaporation of liquid from the wells when running the assay. What makes the rinsing oil different from the incubation oil is that the former has lower boiling point. So, the incubation oil is more suitable for the CO2 incubator environment. The rinsing oil is also applied to prevent carry-over between wash steps.
There are few other components, such as: Plate Adapter, Dispensing Guide, Tip Cassette, and Lid. But since I seldom/do not use them, I will not explain them here. Feel free to click here if you would like to know more.
The DropArrayTM platform is also used for cell-based assays. I will describe one such assay that I had done in week 9 in the next entry. So, stay tuned!
Nor Liyana
0607927A
TG 02- Group 8
Week 9 Posting : Liyanah Zaffre :)
Hello my dearest friend, I hope all of you are well. It was great meeting up during the campus discussion. :)
Anyway, as for this week, I will continue from where I stop in the previous post.
After primer optimization is done, we then need to visualize the PCR products to determine at which temperature is the best. Thus, we need to load the product into the agarose gel, and subject it to gel electrophoresis.
How to make an agarose gel?
Depending on the fragment size, the percentage of the agarose gel differs. As for me, my fragment size is about 1.2kb and thus I use the 1.5% agarose gel.
Methods to make a 1.5% agarose gel
1. Select the suitable agarose gel mould and the combs, then place it in the fume hood.
2. Ensure that the mould is level, by using the air bubble gadget (air bubble must be at the centre of the gadget).
3. Calculate the amount of agarose, volume of the TAE buffer and ethidium bromide needed.
Calculation of the amount of agarose and volume of TAE buffer
Since the suitable mould can only hold up to 60ml, we only need 60ml of TAE buffer.
100ml -> 1.5g of agarose
60ml -> 1.5/100 x 60 = 0.9g of agarose in 60 ml of TAE buffer.
Final concentration needed for agarose gel: 0.5 ug/ml
Therefore, using M1V1=M2V2
10000ug x V1 = 0.5ug x60ml
V1= 0.5 x 60 / 10000 = 0.003ml = 3ul of ethidium bromide
4. Weigh out 0.9g of agarose powder and add it into a dry conical flask.
5. Obtain 60ml of TAE buffer using a measuring cylinder.
6. Pour the 60ml of TAE buffer into the conical flask.
7. Stuff tissue at the mouth of the conical flask and place it in the oven for 1 minute.
8. At intervals, take out the conical flask and swirl it, to check if the agarose gel has melted.
9. After which, the agarose gel solution should be clear and colourless and let the solution is cool for a few seconds.
10. Add in 3ul of ethidium bromide to the solution and swirl it again.
11. Pour the agarose gel solution to the mould and place the combs in their respective positions.
12. The gel is then left to harden and solidify for about 10-15 minutes.
Why use agarose instead of polyacrylamide?
Agarose gel is mainly used to separate smaller molecules like nucleic acids eg DNA, while polyacrylamide are capable and often used for separating larger molecules like proteins. Also, polyacrylamide gel is more expensive than that of agarose and thus, not used in this case as not necessary.
Gel electrophoresis
1. Place the agarose gel mould with the solidified agarose gel into the electrophoresis tank containing the TAE buffer. Make sure the whole gel is immersed in the buffer.
2. Add 2ul of loading dye into each of the tubes of the PCR products. Spin it down to mix it well.
3. Pipette out 10ul of the resultant mixture and load them into the respective lanes.
4. Set the electrophoresis at 120 voltage for 20 minutes, and ensure it is running.
Principle of gel electrophoresis
- Gel electrophoresis is used to separate macromolecules eg proteins or DNA that might differ in size, charge or conformation.
-When the gel is run, the molecules (eg protein in particular) will migrate towards the positive (anode) or negative (cathode) depending on their charge.
- Thus, nucleic acids foe example DNA has a negative charge due to the phosphate group attached, will migrate towards the anode.
- As noted from the methods, an electrophoresis buffer is used. Why? It is because the buffer provides ions to carry a current and also to maintain a constant pH.
Once that is done, I would then proceed on to do the gel check, to see my PCR products that will be seen as bands. For this step, a machine also known as Gel Doc is used. As this step is after primer optimization, we need to know the right temperature for the primer. The right temperature would show bright and sharp bands.
Examples
L=ladder, Temperatures: 55oC-65oC,
Best temperature: Lane 5 = 58oC
Why? Lane 1-4 shows non-specific binding. This is due to too low annealing temperatures. When that happens, the primers don’t anneal properly and thus will lead to non specific products as seen.
Lane 8-12 , although the bands are pretty bright, they are not as sharp as Lane 5.
(Sorry, pictures are not that clear so can’t really visualize the minute differences)
2nd example
As for this, there are actually 12 lanes and the ladder. However Lanes 1-6 shows smearing and non specific binding. And Lane 7 shows faint band as compared to Lane 8. Thus, it is pretty obvious in this case that Lane 8 is the best temperature. For this also, Lane 9-12 do not show any bands. This is because the temperatures are too high for the primers to anneal and thus, no products.
After which, when we have chosen the best conditions for PCR to run, we can then proceed on to do PCR for the different DNA samples at that condition. This again, will be explained in my future postings.
Alrighty, till next time then. Take care lovelies. :)
Liyanah Zaffre
0607718D
TG02
Week 8
Hey all. It's my turn to share again. For the past 2 weeks, I was attached to the Biochemistry department. Not many samples were received manually in this department as most of the samples were loaded into a sample manager and processed automatically. I believe Maya has explained what is a sample manager in one of her posts previously, so I shall not go into it. Some of the tests done in the department are urine glucose tolerance test, G6PD screening test, HbA1C test and urine drugs screening test. The rest of the tests are loaded in and processed automatically. Most of the automated tests are processed by a machine called the ADVIA 1650 by Siemens Diagnostics. However, for HbA1C test, the samples need to be ordered into the system and loaded in manually.
Retrived 16th August 2008 from, www.clpmag.com/issues/articles/2006-12_09.asp
For Drugs 5 test, one test device is used. The test device is able to test presence of all 5 drugs at one time. For Drugs 7 test, 3 test devices are used. 1 test device testing for the presence of the same 5 drugs, 1 test device testing for the presence of Barbiturates (BAR) and 1 more test device testing for the presence of Benzodiazepines (BZO).
The test is based on lateral flow chromatographic immunoassay to detect presence of the specific drugs and their metabolites. The principle of the test is competitive binding between either the drug present in the urine with their specific antibody or, the drug conjugate with the specific antibody.
3-5 drops of urine are dropped onto the each of the circular wells found at the bottom part of the test device. The urine will then move upwards by capillary action. If the drug is present below the cut-off concentration, the drug conjugate will bind to the specific antibody instead and forms a visible line across. If a visible line is observed, the result is recorded as negative. However, if the drug is present in high amounts and above the cut-off concentration, the drug will bind to the specific antibody instead of the drug conjugate. This will cause no visible line to be observed across and the result is recorded as positive. For every drug tested, there is a region for Control. If there is no visible line observed across at the Control region, the test is then considered invalid and should be tested again using a new test device.
The following figure is a visual interpretation of the results, done using Microsoft Powerpoint.
The cut-off concentrations are as follows for each type of drug:-
Amphetamine - 1000 ng/mL
Barbiturates - 300 ng/mL
Benzodiazepines - 300 ng/mL
Cocaine - 300 ng/mL
Marijuana - 50 ng/mL
Methamphetamine - 1000 ng/mL
Opiate (OPI 300) - 300 ng/mL
The antibodies present on the test devices for each drug type are mouse monoclonal antibody-coupled particles. For each drug test, drug-protein conjugates are also present specific to the drug type. For each Control region, it contains goat anti-rabbit IgG polyclonal antibodies and rabbit IgG.
This urine drugs screening test is only a qualitative test and not a quantitatinve test. Therefore, we are unable to tell the amount or drugs level present. It is also only a screening test. A gas chromatography or mass spectrometry should be carried out to confirm any positive results obtained. A negative result does not necessarily the urine is free of drugs. Other types of drugs not tested for their presence, may also be present. A positive result may also be caused by the kind of food consumed by the patient.
As most of the drugs are controlled drugs, I have never encounter any positive results ever since I have been attached to the Biochemistry department. According to one of the permanent staffs there, it is also quite rare to encounter any postive results as Singapore rules are very tight when it comes to abusing drugs.
I guess that's all for now. If you have any queries to ask, please feel free to do so. I will try to reply as soon as I can. Thank you. C:
Nur Azeimah
0607060A
TG02