MadTechs

Week 19 posting

2008-11-02T14:04:00.002+08:00

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 18

2008-10-26T11:35:00.009+08:00

Hi all. It's my last posting already. 2 more weeks and we're done with SIP. C:
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

1) Competive principle

- 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

2008-10-18T15:55:00.014+08:00

Hi, this week I shall give a brief overview on what Cytogenetics lab does. This is one of the few labs which still relies alot on manual work by medtechs.

Cytogenetics is the study if chromosome structures and their behaviour to discover any abnormalities in humans, and identify the medical condition that is caused by the abnormality in that particular chromosome. There are many different types of abnormalities and every type contributes to a different medical condition.

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.

Normal Karyotype

(Source: arnica.csustan.edu/.../cell_division.htm)

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)

Ka Hang

TG02

Answers to Week 16 posting (For Li Ping and Ernest)

2008-10-13T14:17:00.007+08:00

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

2008-10-11T09:11:00.004+08:00

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

2008-09-30T18:51:00.000+08:00

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

2008-09-28T13:52:00.005+08:00

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

2008-09-23T23:19:00.001+08:00

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

2008-09-13T22:21:00.014+08:00

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

Stool specimens are one of the most frequently received specimens in the lab. Stool cultures would be able to show the presence of certain types of abnormal microorganisms such as Samonella, Shigella, Campylobacter and Vibrio species present in the normal human body. For oncology patients, stool screening is required to detect any multi antibiotic resistant microorganisms such as ESBL producers, VRE or any unusual overgrowth of pathogenic flora such as P. aeruginosa or Candida.

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.

For all types of suspected microorganism growths on cultures, biochemical tests must be carried out to confirm their identity. Different types of mediums are used to identify the characteristics of the microorganisms based on their biochemical reactions. Below are some examples of mediums that are commonly used (brief explanation):

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.

Citrate Test (Simmon Citrate Agar) – Determines the ability of the microorganism to use sodium citrate as the sole source of carbon for metabolism and growth.

Urease Test - Urease is an enzyme that breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water.

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)

2008-09-08T15:34:00.003+08:00

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)

2008-09-08T14:54:00.013+08:00

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.

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)

Protocol (Flow chart):

Euthanize the mice/rodent

Use surgical instruments to remove the fur from the fore and hind limbsRemove 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 limbsCut 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

(The tubes were labeled “unstained”, “PI only” and “Hoechst + PI”, “Hoechst + blocker + PI”)

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

(Except for “unstained” and “PI only” tubes)
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 PIStore on ice and out of light before data acquisition on FACSAriaRecord the SP profile acquired

That's it for my entry this week.

So as to not confuse anyone, the protocol was briefly explained, so feel free to ask me questions if there is any doubts.

Many thanks

Quan Jun

TG02

Group 08

08 September 2008

Week 10 sharings

2008-09-01T17:14:00.002+08:00

Hi everyone!

It is time for me to introduce to you the new DropArray^TM platform that my company has developed.

DropArray^TM 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 DropArray^TM 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 :)

2008-08-23T01:01:00.003+08:00

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

For 1.5% (wt/vol) of agarose gel, we need 1.5g of agarose in 100ml 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.

Calculation of volume of ethidium bromide needed

Initial concentration of ethidium bromide: 10mg/ml
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

2008-08-17T16:56:00.005+08:00

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.

For now, I shall be sharing about urine drugs screening test. In the lab I am attached to, it is commonly known as Drugs 5 or Drugs 7 test. It simply means, the urine sample is being tested for the presence of either 5 drugs or 7 drugs. If Drugs 5 test is requested upon a urine sample, the 5 drugs tested for their presence are, Amphetamine (AMP), Cocaine (COC), Marijuana (THC), Methamphetamine (MET) and Opiate (OPI 300). If Drugs 7 test is requested upon a urine sample, the 7 drugs tested for their presence are Amphetamine (AMP), Cocaine (COC), Marijuana (THC), Methamphetamine (MET) and Opiate (OPI 300) with additional 2 more drugs, Barbiturates (BAR) and Benzodiazepines (BZO).

To perform these tests, special test cassettes devices manufactured by Bio-Rad Laboratories Europe Ltd. are used. It looks something like the following picture but it is not exactly the same thing. The one used in our lab tests for lesser number of drugs.

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

Replies to comments on Kahang's (Week 7) Post

2008-08-11T22:22:00.017+08:00

Hi, I shall answer all your questions here:

To Leslie, Ying Chee & Malerie
I mentioned that 3 different blood smears will be made, one using the patient’s (women’s), one using a male’s and another with cord blood. Remember this test is to test for the presence of fetal cells in the mother’s blood? So the most important factor would be the fetal cells.

Cord blood from newborns is used as a positive control as it definitely contains fetal cells. When stained, they will take up the eosin counterstain and appear as pinkish red intact cells. If red cells are not seen in the positive control, it might be an indication that the stain is not working well.

Male’s blood is used as a negative control because it is impossible that fetal cells will be present. Normal cells of adults will be dissolved after the acid elution staining and appear as ghost cells (illustrations are provided below). This is done to show that the procedures for this test are carried out correctly. If fetal cells are seen in the male’s smear, cross contamination might have occurred or there were errors in the procedures.

In fact the negative control may not necessarily be taken from a male. It can also be taken from a non-pregnant female, as long as it is from an adult. But in certain cases where women are unaware of their pregnancy, the results from the negative control may reflect as positive (presence of fetal cells). So to be more accurate, my lab uses a male’s sample as the negative control.

To Li Ping & Ying Chee
Yes it’s the HbF that affects the resistance to acid elution. The hemaglobin F does not dissolve in the presence of acid and thus takes up the eosin counterstain and appear as red. The adult hemoglobin A cells are already dissolved when acid is introduced, therefore they appear as ‘ghosts’. The reason behind this resistant is not really known. I tried consulting the medtechs in my lab, they are not sure either, just said that it’s definitely related to the HbF component.

To Malerie
The acid elution solution consists of solutions A, B and C:

Solution A – 0.75% Haematoxylin + 96% Ethanol
Solution B – 2.4g of Ferric Chloride + 0.5% Hydrochloric Acid
Solution C – 80% Ethanol

Solutions A, B and C are mixed in the ratio 2:1:1 and this working solution must be prepared fresh.

To Albert, Ying Chee & Gladys
Gladys, yes light microscope would be sufficient. Slides are examined under x40 objective. If fetal cells are observed, counting is performed under oil immersion (x100 objective).

Microscope examination of:

Negative control – Normal adult cell contents are dissolved, therefore nothing can take up the eosin stain, cells appear as ‘ghost’ (cells only have its border with no content)

Positive control (cord blood) – All cells take up the eosin stain and appear as pinkish red intact cells

Positive Result – Stained red cells denote the presence of fetal cells.

(All pictures taken from lab's SOP, with permission from medtech)

Ka Hang
TG02

Answers for 'Week 5 and Earlier Sharings' queries

2008-08-11T10:54:00.005+08:00

Hi everyone,

I hope you guys are doing well, in SIP and as well as in revising (!!) our other subjects. Hope You are in the pink of health too.. and unfortunately, I am not. haha.. it's the flu season again, i think~

I am here to answer some of the questions posed by some of you:

I will start with Sharon's -

How frequently do you clean CO2 cylinder and waterbath?

I hope you mean 'CO2 incubator', because I definitely do not clean any CO2 cylinder. >.^

- oops! I have just realised I typed 'CO2 cylinder' in my previous post~ Okay, am correcting it now -

The CO2 is stored in cylinders, and these cylinders (and the CO2) are supplied by Singapore Oxygen Air Liquide Pte Ltd. I place order for new supply/ies of CO2 every week, because the gas runs out every 7-9 days.

How frequent the cylinder has to be changed/ordered depends on 1. the amount of growing cells/microorganisms kept in the CO2 incubator, and 2. how frequent the gas leaks out of the incubator.

Say, for example, if I keep opening and closing the incubator, or open it for too long, more CO2 will be lost. So, yes, I have to be mindful when I am using the CO2 incubator.

The CO2 incubator is cleaned every month. This is to ensure no bacteria or any other microorganisms, that can contaminate the cell culture, grow in it. It will be a HUGE problem if there are contaminants! Imagine, you keep 20 flasks of rare cells in the incubator, and suddenly, they all died! And... it turns out that some fungi managed to sneak into the flasks and eat up all the nutrients in the media. nightmare!

Here is a better description of how important keeping the CO2 incubator clean is:

Cleaning and Disinfecting CO2 Incubators

A CO2 incubator is a basic piece of equipment in any laboratory involved in cultivation of in vitro cells. The culture bottles must be kept slightly opened in order to allow the penetration of CO2, and enable the formation of a bicarbonate - CO2 buffer system, which is the most common buffer system for tissue culture. Leakage of medium from the culture bottles, high humidity, suitable temperature, the water tray and the air flow, all form a fertile breeding ground for the development of contaminants in the incubator, which are liable to contaminate the tissue culture in all the semi-opened culture bottles.

As soon as contamination appears in the incubator, it is very difficult to eliminate it, and severe damage can be caused. Therefore, it is essential to systematically clean and disinfect the incubator. It is recommended to disinfect every 14 days, or once a month at the very least.

Retrieved August 11, 2008, from Biological Industries website

Firstly, I take out the tray (one at a time), place it at the sink and run DI water over it. Then, I spray 70% alcohol on the tray and wipe/dab with tissue paper. All these must be done quickly, to minimise the tray's exposure time to the surrounding air (i.e. dust may settle on the tray).

After all the trays and incubator wall (just spray with 70% alcohol) have been cleaned, the last item is the water tray, which should be cleaned and then filled with sterile water. What is the water used for? It is to maintain humidity inside the incubator.

To be safe, my lab uses Aquaguard 1 solution, a disinfectant from Biological Industries, to disinfect the water in the tray. 50mL of Aquaguard 1 to every 5L of sterile water.

Okay, as for the waterbath, the water has to be changed every 4 or 6 weeks. DI water, to be exact. We use Aquaguard 2 solution to disinfect the water. 2mL of Aquaguard 2 to every litre of water.

What is the difference between Aquaguard 1 and 2?

Simple: Aquaguard 1 is especially for CO2 incubator, while Aquaguard 2 is for waterbath. This is what stated at the supplier's website. It does not state the composition though.

Okay, hopefully this answers your question, Sharon. Do drop me a comment (or sms, even better! hehe) if you have any other doubts. See you this Friday, ya? ^_^

I-am-at-TP-library-going-home-soon,
Nor Liyana
0607927A

Week 7

2008-08-08T01:50:00.009+08:00

Hi all, for the past 3 weeks, I was attached to the haematology lab. Like other clinical labs, it operates 24 hours and is very busy almost everyday. The most common test ordered is full blood count, but since sofie has already touched on it, I shall talk about other tests.

Before that, do you know how specimens reach the lab? All along I thought they were sent in by some medical staff every now and then. But one day when I was helping out at the reception, I realised it was not. The specimens are received in the lab via a pneumatic transport system (that looks like water pipes) that is linked from every single ward or clinic to the lab. Samples from the wards to be sent to the lab are placed into a capsule, which contains a micro-chip that is recognised by the system to direct its way to its destination. In every ward or clinic, there will be this cupboard that contains the ‘pipe’. Empty capsules with reports from the lab (if available) will then be sent back to the respective wards using the same system. But because there is only one lane for sending out, the destination code must be keyed to ensure that they are sent back to the correct place. Isn’t this interesting? The WHOLE hospital is connected by all these ‘pipes’.

Capsules arriving from wards

Capsules waiting to be sent back to clinics/wards

Sending back capsules with reports through the one and only lane
(destination code keyed)

Ok, back to haematology. ESR (Erythrocyte Sedimentation Rate) is one of the most common tests performed in the haematology lab. There are many ways to test for the ESR. In my lab, the sedi-rate P4-Micro System is used. It measures the rate at which red cells fall in the first 50 mins when anti-coagulated blood is allowed to stand. Red cell sedimentation occurs in 3 stages: in the preliminary stage where aggregates form within a few minutes. This is followed by a period of time in which the sinking of the aggregates takes place at a constant speed. Finally, as the aggregated cells pack together at the bottom of the test tube, sedimentation rate slows down.

ESR is used to help diagnose conditions associated with acute and chronic inflammation. When inflammation is present in the body, certain proteins cause red blood cells to stick together and fall more quickly than normal to the bottom of the tube. These proteins may be produced when there an infection, autoimmune disease, or cancer. However, ESR is said to be nonspecific because increases do not indicate the exact site of inflammation or the causative agent. For this reason, a sedimentation rate is done in conjunction with other tests to confirm a diagnosis. Once a diagnosis has been made, a sedimentation rate can be done to help check on the disease or see how well treatment is working.

To perform ESR:

1. 320µl of blood is transferred into an aquisel tube, which contains 0.08ml of sodium citrate.
2. Then, a thin pipette is being pushed downwards into the tube, until the blood fills the whole pipette, indicated by the ‘0’ marking.
3. Next, the tube with pipette will be allowed to stand.
4. Exactly after 50 mins, the number of mm the red cells fallen would be read.
5.Results are then recorded in the ESR record book and the request form, and entered manually into the LIS.

Source taken from: http://www.globescientific.com/sedirateÂ™-autozero-westergren-esr-system-3469-pi-574.html

Another special test that I think relates to what we have learnt is the Kleihauer Betke Test (KB), which is used to detect the presence of foetal RBCs in the mother’s blood using the principle of acid elution (dissolving of cells). KB Test can be used to assess 3 conditions.

One of which is when a newborn baby is found to be anaemic. In this case, the test checks whether the baby’s blood had entered the maternal circulation. If positive, the amount of blood must be determined and from there, the doctor will be able to decide how much blood should be transfused.

The second condition is, when the mother is Rh(-ve) and baby is Rh(+). If the KB test reflects a positive result, rhogam must be administered within 72 hours to neutralize the foetal RBCs.

Also, KB test plays an important part in cytogenetic analysis. To detect genetic abnormalities such as down syndrome, blood from unborn foetus must be analysed. To facilitate the process, the foetus’s blood must be taken through the mother’s tummy. The KB test will confirm whether the taken blood belongs to that of the foetus and not the mother.

Foetal RBCs contain mainly HbF (a2g2). They resist acid elution more than that of adult RBCs, which contain mainly HbA (a2b2). With this principle, foetal cells can take up the eosin when counterstained and appear as darkly stained red cells. On the other hand, adult cells will be disintegrated by the acid and therefore will appear as ghost cells (because the cells are dissolved, there is no more cells present to take up the stain).

To perform the KB test:

1. 3 different smears (patient’s blood, blood of a male and cord blood) are first made. Cord blood smear act as the positive control, blood smear from male acts as the negative.

2. Slides are air dried.

3. Slides are fixed in 80% ethanol for 10 mins then stained using the acid elution method.

4. This is followed by counterstaining with eosin for 3 mins and then air dried.

5. Finally slides are examined under the microscope at high power.

The proportion of foetal RBCs that appear as pinkish red intact cells to that of adult RBCs (appear as ghost cells) is assessed in several fields. If foetal cells are detected, report the number of foetal cells seen in 2000 adult cells and the volume will be calculated.

Ka Hang

TG02

Reply to Han Yang question from "Basic Introduction to Flow Cytometry"

2008-08-06T13:11:00.006+08:00

Han Yang:

With regards to your questions, it would be understand best with pictures, so I decided to post a new entry for your questions, hope you don't mind.

1) 2 things:

One of the most important precautions to take note is to make sure there are as few cell clumps as possible. As this will clot your flow cytometer, where the troubleshooting will make you seriously regret not removing the cell clumps if you didn’t.

The next precaution relates more to the experiment design, if you want to isolate and sort a certain cell, you have to make sure you don’t have a lot of other unwanted cells in your sample (eg. RBCs, etc) or you are going to spend a long time sorting out the cells of interest due to very low % in your sample.

2) Lolz. I don’t mind sharing how the sample is load into the flow cytometer, you just place the sample tube on the “tube holder” on the “loading port” and load into the “sample injection chamber” of the flow cytometer by clicking on the "load" button on the "acquisition dashboard".
(Refer to figure 3 below for sample of "acquisition dashboard")

3) They are just some isotonic buffer solutions (eg. 1x PBS) that keep the cells structure intact for flow cytometry analysis. Some places even uses high grade DI water.

References:

4) Regarding the principle, I am not sure what principles are you talking about, but let me just show you the picture of how the lasers are focused onto the optical lens (also known as focusing lens):

Other than that, I think it’s best if I don’t touch on the principle too detailed as it relates to a whole new subject on physics (lasers). Let’s not make the learning confusing, okay? =)

Note: The display of some acquisition dashboard might vary a little, but they all basically have the same functions.

Hope my explanation is clear and help in your better understanding! =)

Quan Jun

6 weeks at SIP (Time sure flies)

2008-08-05T15:40:00.005+08:00

Hey guys! Time sure pass with the speed of light, it’s already the end of 6th week! And it’s my turn to present to everyone this week.

Hope everyone is still doing well with your SIP and MP! =)

Today, I am going to be presenting on what I usually do in my SIP:
1. Daily Quality Control (QC) for FACSAria
a. This has to be done everyday to verify that the FACSAria is in optimal working condition

This involve 3 main steps:
1. “On” the computer before the FACSAria
a. 1 cycle of “fluidic startup” (primes the system of bubbles)
2. “Time delay” QC calibration (optimization)
3. “Area scaling” QC adjustment

Since step 1 is self-explanatory, I will skip straight to step 2:

Time Delay

· “Time delay” is required to adjust so that the signals obtained from the firing of different lasers at different timing are recognized to be from the same cells by the computer.
(Refer to figure 1, below)
· “Time delay” QC calibration is required to be done for all lasers, except for blue laser, which acts as a reference.
(In a flow cytometer, it is usually standard to have the red and blue lasers)

Steps:
1. Load a tube of DI water into the FACSAria to clean the sample line (Flow rate: 10)
2. Prepare a tube of approximately 500uL of rainbow beads solution
a. Rainbow beads contain mixed fluorescent signals (eg. FITC, PE and etc)
3. Load the rainbow beads tube into the FACSAria (Flow rate: 1)
4. Gate the cluster of dots on the dot plot for singlets
a. Singlets are always located on the low SSC and low FSC on the graph
(Shown below in figure 2)

5. Once “gated”, change the window extension to 0
6. Start optimizing the “time delay” of the laser by getting the peak of the signal as right as possible
a. If the value of “time delay” is not optimized, the peak of the signal is always to the left. (refer to figure 5)
b. After the “time delay” is optimized, change the window extension back to its original value

Window extension:
This relates to the pulse measured, as the pulse measured from the
beads is quite narrow, window extension is set to 0 so to capture
only the data of the pulse signal from the beads.

Area Scaling

· Area and height mean value must approximately match for all parameters (eg. FSC, SSC, FITC, PE and etc)
o Producing a linear line on the FSC-Area vs FSC-Height dot plot
· This is to standardize the parameters for accurate interpretation of experiment results.
· “Area scaling” is required to be done for all lasers.

Steps:
1. Using the same tube of rainbow beads, load it into FACSAria (flow rate: 1)
2. “Gate” the cluster of dots which represents the singlets
(Shown below, figure 8)

3. Adjust the values of the area scaling to increase or decrease the mean area-value to match approximately the mean height-value of the signals acquired using the population statistics (FSC-A, SSC-A, FITC-A and etc)
(Refer to figures 9, 10 and 11)
4. After the mean area to height value has been approximately matched, record the data of 1000 events by clicking the “Record Data” button on the “Acquisition Dashboard”.
(Refer to figure 12)

=== Sorry for the late posting, need to make sure I get my supervisor permission first before posting ===

Posted by:
Low Quan Jun
0607243C
TG02
Group 08
05 August 2008

Week 5 and Earlier Sharings

2008-07-28T08:49:00.004+08:00

Good Morning, dear friends!

Okay, it's my turn to update all of you about my work at.. *ehem* somewhere in the west of Singapore. hehe..

TOPIC: Student Internship Programme

The Company

The company that I'm attached to is a bioinstrumentation start-up company. Just a few months old, it's a spin-off from one of Singapore's research institutes.

What is 'Bioinstrumentation'?

Well, from my understanding, it means instruments, or equipments, that are invented/developed to improve the works in the biology field, or more generally, in the laboratory.

So, my company has developed a technology that miniaturises cell culture and cell-based assays, both of which use smaller samples and take shorter time. *I would loVe to share with you how exactly this technology works, but I am not sure which information can be released.. and I haven't asked my supervisor. Hmm.. So, stay tuned, aite!*

I'm sure some of you know where my workplace is. And, I'd like to share with you something that I found interesting (sorry for the lack of a better word!):

Since many research institutes/pharmaceutical companies are situated here, the management has built a 'shared facilities', located in the basement. The first time I stepped into the area, the first thought that came to mind was, "Waaahh~ So clean, like hospital!" The floor's practically shiny, I tell you. Haha.. Okay, so, what are the services you can find there?

This place has many sections: supply center, media preparation (known as MediaPrep), washing and autoclaving service, confocal microscopy, flow cytometry (thanks, QJ, for enlighting us with the information *big smile!!*) and high content screening.

I like the supply center very much! How it works is that I make my order online (what kind of order, you'd ask? Let me see.. You can get all sorts of labware and reagents here from labcoat to alcohol-resistant marker to bleach to pipettes to petri dish to tube rack to polymerases to buffer solutions... and the list goes on~), and depending on the arrival time, I will go down from my laboratory to collect the items. Seriously, this integrated facility is efficient and saves everybody's time.

okay, NEXT!

My Jobscope

I do admin work as well as Research and Development (R&D).

In Admin, I make payment vouchers and purchase orders, write cheques as well as calling and emailing suppliers to ask for quotations or request for services. I'm also learning to use MYOB 'Mind Your Own Business', an accounting software.

In R&D, so far, I culture cells in flasks (plus changing medium and passaging). GoOd thing that I had learnt Mammalian Cell and Technology! However, since it had been a year ago, it took me some time (not to long, though!) to get the hang of it again.. cell culture technique, aseptic technique.. But now, yeah, I'm doing goOd =)

I also seed cells on the company's proprietary plates once the cells have reached 70%-80% confluence inside the culture flask. Haaa~ why seed cell on the plate? What's so special about this plate...? I'll tell you next time! What I can say for now is that the plate is FLAT and it's the size of the standard microscope slide. You'd think that the sample will slide off, right? right? Well, I'm going to let you imagine how cell seeding is done on a flat surface! *smile mischieviously*

Other than seeding, I also do aliquoting (of fluids, such as primary antibodies), clean CO₂ ~~cylinder~~incubator and waterbath, store the labwares in the appropriate place, and help my colleague to key in inventory.

Since my company is very new, I observe how my colleagues work hard to improve on the product. Any problem(s) and complaint(s) from client(s), they will try their best to rectify and troubleshoot it. Additionally, some marketing stuff: like, you have to find distributors to market your product, locally and internationally, or you do 'demo' of your product to potential customers at medical/research institutes, companies and universities. Needless to say, there is a lot of work to be done if you are new and have a goOd product to offer the scientific community.

Oh! I've just remembered: My laboratory is small, slightly bigger than our tutorial room, so, there aren't separate rooms or partitioned sections for washing, preparation and cell-culturing. Boxes of newly-arrived items or equipments have to be stored or put aside to minimise obstruction in the room. Fortunately, the essential equipments for cell-culturing work (laminar flow hood, inverted microscope and CO₂ cylinder) are positioned near to each other.

Okay, I guess that is all I have for now. I would have liked to post protocols and other 'scientific knowledge', but I've yet to gain one or two that's worth sharing with all of you. Still searching.. and still learning.

^_^

Till we meet again,
Nor Liyana bte Noor Mohammad
0607927A
TG02 - Group 8

Basic Introduction to Flow Cytometry

2008-07-25T11:39:00.004+08:00

Hi guys, it’s the 5th week already, everyone still going great? Hope you are =)

Anyway, it’s not my turn to post this week, but I decided to still post something this week so to help everyone understand more about the principle of Flow Cytometry which I am fortunate enough to be working with one.

Why would I want to do this, is because I am worried if everyone understand what I was posting during my 1st week where I bombarded everyone with tons of information.

First, over the weeks, I am going to introduction on the following basic principles:
1) Hydrodynamic focusing of the sample
2) Light collection & how they are measured
3) Sorting principle of Fluorescent Activated Cell Sorter (FACS)
4) Data analysis for flow cytometry

For this posting, I will only be explaining about hydrodynamic focusing of the sample:

I am sure all of you know that Flow Cytometry is used to analysis the cells profile based on varying factors (I’ll touch more about the factors under point 2 for next posting – refer to above).

However, before the data could be obtained, the cells have to first be loaded into the Flow Cytometry for sample processing. How the sample is load into the Flow Cytometry is very important, as the sample stream has to be in single cell line in order to record single cell profiles.

Figure 1: Cross-sectioned laser beam focused by laser optics lens
Retrived, July 25, from:
http://biology.berkeley.edu/crl/flow_cytometry_basic.html

The above diagram shows how the sample is being focused into a single cell stream before passing through the laser beam, which is the interrogation point where data about the cells are collected. As you can see, the flowing sheath fluid cause the focusing of the sample stream into single cell line.

Hydrodynamic focusing:
A higher pressure from the flowing sheath fluid causes the lower pressure from the sample to form a single flowing cell line.

That is why the surrounding sheath fluid has to be higher in pressure than the sample fluid, else hydrodynamic focusing could not occur.

That’s all for this posting, will post the next topic the following week.

Please do tell me if you find this useful for you in understanding, as this is something extra I plan on blogging to help everyone understand Flow Cytometry. Because I worried that my first post was too much in the content and hard for everyone to understand.

So, is it okay if I continue the postings to explain the basic principles of Flow Cytometry?

Everyone! Have a nice day =)

Posted by:
Low Quan Jun (0607243C)
TG02
Group 08
25 July 2008

Week 4 ( OMG 1 month already! ) :)

2008-07-17T22:33:00.006+08:00

Hey dearest course mates! Hope all of you are doing fine. It’s already been a month and I miss you guys and TP so much! ((:

Okay, besides those irrelevant things, I am attached to a research company together with Lyn, so our information might be similar.
My company is focusing on Pharmacogenetics. Now, you might be asking what is Pharmacogenetics? Is it the combination of Pharmacology and Molecular Biology/Genetics?

The answer is yes. Basically, pharmacogenetics is the study of genetic variations (polymorphisms) in individuals that bring about a different response to the same drug. Thus, for example, person A might take the drug and benefitted from it, whereas person B is administered with the same drug, but suffered from adverse drug reaction.

The 3 main areas that are extensively studied for their genetic variations involves gene that codes for 1) receptors, 2) transporters and 3) drug metabolising enzyme. As we have studied previously in Pharmacology, drugs need to bind to receptors, need to be transported to a specific target/ receptor and also need to be metabolized by certain enzymes. Thus, if it happens to be that the genetic variation in an individual is the one affecting the receptor, transporter or metabolism enzyme, then, the person would show a different pharmacodynamic and pharmacokinetic response.

Okay let’s recall! Pharmacodynamics is the study of what the drug does to the body while pharmacokinetics is the study of what the body does to the drug (absorption, distribution, metabolism and excretion). Thus, with that, the person would either have a lack of an efficacious response or more side effects.

Now, do you know why the same medicine might work very well for your friend (eg) and not for you, or vice versa? :)

To summarise what I am suppose to carry out for my research, my main objective is to find out the genetic polymorphism of a certain transporter gene, between 3 different ethnics group: Chinese, Malay and Indians and detect significant polymorphism of the drug that might affect its transportation factors, thus affecting the drug level in the body. The type of polymorphism that I am suppose to find out on, is SNPs, which is a single nucleotide polymorphism.

What is SNP? It is a single base (A, T, G and C) change that is found throughout the genome. For eg ATT GCC TCA to ATT GCC TAA. So, how does one nucleotide/base affect the gene? Because 3 nucleotides make up a certain codon that expresses for a certain protein, a single base change would change the codon and will result in the expression of a different protein. However, not all SNPs are significant, as it is common among individuals and does not affect the expression of proteins. Thus, for those SNPs that are significant, we can detect it by analysing the gene sequence carrying the SNPs. This will be explained in further details in later post.

As for now, I would describe the first step that is needed to be carried out, that is primer optimization using test DNA.

Primer optimization

Primer optimization is done to find the appropriate optimum annealing temperatures (temperature at which primer binds to DNA) that is needed later during the PCR (polymerase chain reaction) to make multiple copies of specific target sequence to be analysed.
As most primers’ annealing temperature is between 55-65oC and most appropriate number of cycles is 30, I can start testing the primers at that temperature and cycle number.

Methods

1. Prepare a master mix of 13 times the stated amount (need only 12 tubes but 13 times to overcome pipetting errors)

(total: 12 tubes)

Component(s): H20, 10x Buffer, MgCl2 (25mM), dNTPs (10mM), Forward Primer (10µM), Reverse Primer (10µM), Taq polymerase, Test DNA

Total Sample x 1 (µL): 6.9, 1, 0.6, 0.3, 0.2, 0.2, 0.3, 0.5 respectively. Total: 10µL

Sample x 13 (µL): 89.7, 13, 7.8, 3.9, 2.6, 2.6, 3.9, 6.5 respectively. Total: 130µL

[ Sorry I had to put it this way. Blogger doesnt allow to put table, right? I cant seem to copy and paste my table from word. :( ]

2. Prepare an ice-box.
3. Collect the different reagents and place them in the icebox. (Taq polymerase is to be collected last after adding all the other reagents as it will denature at room temperature or less than 20 degree Celsius if it is removed for too long)
4. Prepare a strip of 12 tubes and label them 1-12
5. Add in all the reagents into the master mix tube.
6. Place the remaining reagents back into the respective fridges.
7. Spin the master mix tube down for a few seconds to ensure all reagent is mixed
8. Pipette 10µL from the master mix into each of the 12 tubes.
9. Spin down the tubes again for a few seconds, to also remove all air bubbles.
10. Place the 12 tubes into the Thermogradient (thermocycler)
11. Place 4 empty tubes at each of the 4 corners to balance it.
12. Programme it at 55-65oC and carry out for 30 cycles.

Conditions of Thermogradient

Step 1: 95oC x 3min
Step 2: 94oC x 45s
Step 3: 55-65oC x 45s
Step 4: 72oC x 45oC
Step 5: Repeat steps 2-5 x 29 (total 30 cycles)
Step 6: 72oC x 7min
Step 7: 15oC x Infinity
-End-

This reaction takes about 1 and a half hours, so to not waste time, agarose gel is to be made (2nd step). I will explain about it in my next entry so my post wouldn’t be too lengthy ya, and before I bore you guys further. :)

So this is it, any queries, feel free to ask, I won’t bite. Hehe. Take good care of yourself and I’ll see you soon on the 25th!

Best regards,
Liyanah Zaffre
0607718D
TG02

Week 3 explanations...

2008-07-14T22:53:00.002+08:00

hey everybody.. i suppose it would e easier if i answer everybody's queries in a post rather than in the comments page. anyways, here goes...

to Athirah:
contamination do happen and that's why good pipetting skills is very important. i always try not to let the tip of the pipette touch the wells as it will introduce other contaminants into the wells. however, according to a colleague of mine, the test is specific for mycoplasma pneumonia antibodies and other bacteria are not easily detectable.

to Xin Yi:
interdeterminate means you can't really tell if it is positive or negative. that's why it is indicated as a plus-minus sign. however we do not report as plus-minus. we try to compare the results of one well with the result of the well next or after it. using a ruler to measure the compactness is rather ridiculous actually. so what i was taught was, to observe the tightness of the ring. in other words, which ring appears darker or bolder. the lest tighter ring will be the positive end-point titre. i hope that answers your question S:.
presence of a least compact ring should still be reported and not ignored as it could indicate presence of weaker mycoplasma antibodies. it is simply to be on the safer side lah actually.

to Liyanah:
i dont really miss you but i know you're probably getting skinnier now cos you miss me too much. right? hah!
anyways, the samples are diluted to achieve a titre. a titre is like, series of dilution. we carry out a titre to find out how weak or how strong the antibodies are. if all the dilution is the same, or if everything is done as a neat (1:1), then it would defeat the purpose. we wouldnt be able to tell how "dangerous" the antibodies are. or how wide the disease has spread.
we don't use the awesome multi channel pipettes in serology but we do use them in hematology labs to prepare the reagents in the morning but i shall not go into that for now. haha. reason being, we have to pipette different amounts of reagents or samples each time and multi channel pipette usually gives the same amount of solution each time we pipette.

to Lee Jin:
sensitised particles basically means that the particles are pre-coated with antigens. we learnt this in hematology i think. usually, they come in lyophilised form in the test kits. lyophilised means powdered form. we learnt that in LMQA if i'm not mistaken. lyophilised substances are usually not ready-to-use. therefore, we have to prepare them ourselves by adding the required amount of diluent.

to Han Yang:
- regarding your first question, i'm not very sure why. it's just the way the lab works. we receive samples from hospitals, clinics, etc. and they usually collect samples in the morning, thus we receive them in the afternoon.
- some examples of automated tests are Candida virus tests, Epstein-Barr virus tests which tests for nasopharyngeal carcinoma, Varicella-Zoster tests which is the main cause of chicken pox, and many more. there're really A LOT. we usually carry out these tests using a machine called the EVOLIS and it works using the ELISA method.
- for the third question, i can't really answer that because it is what given in the test kit. probably, rabbit's serum has the same reaction as human serum towards the specific viral antibodies.
- a positive result will show agglutination THROUGHOUT. presence of a button or a ring shows that the sensitised particles are not able to agglutinate due to absence of the specific antibodies and therefore they SETTLE at the bottom of the well. when they settle at the bottom, they form either a compact button or a ring, depending on the presence of the antibodies or the strength of the antibodies if they are present.
- yes, you may assume that the most compact ring is negative as the wells before that shows a less compact ring. however, that doesnt mean the patient is free from infection. due to the less compact ring observed in the wells before, it does indicate presence of the specific antibodies.
- do you mean aseptic environment, for example, close to a bunsen burner or something? sadly, we don't practise that in our lab. but the pipette tips and microwells used are sterile before we use them. and of course, we put on gloves too to protect ourselves and prevent introduction of our bacteria into the wells.
- the reason why some tests remain manual is, there are probably no machines invented yet to carry out such tests. however, special tests such as this MPA tests are usually carried out on certain samples as requested by the hospitals/ clinics. they're usually less than 50 per day. human error is inevitable, thus, secondary checks are very important and are usually done by someone more experienced. we usually do 4-5 samples at one time and it usually take less than half an hour. during the incubation time, of course we carry out other tests on other samples as not to waste time.
- i'm not sure if there are any confirmatory tests. what i know and was taught, the MPA test is specific to test for presence of mycoplasma pneumoniae antibodies. after carrying out the test, we record the results and the results are sent out to respective hospitals/clinics.

wow, that was long.. i hope i've answered all 8 or probably more of your questions. heh..

to Quan Jun:
haha. you're weird. nobody would mistake 1 - 9 as 1 -6 ... cos then, it would only be 5 hours of work.
i think your question is kind of similar to someone's question. well, as i said, a button means that the particles settled at the bottom of the well and not agglutinate. my supervisor told me that we should look out for agglutination THROUGHOUT the well and not settling of the particles in a button or a ring. i know it's a bit confusing. i was confused the first time too but i was explained as such so yea.. if wou try to disrupt the button, the particles would dissolve and still NOT form any agglutination.

to Farhanah:
for serology lab, the screening test that we usually do is the Venereal Disease test, followed by the VD titre. i don't think we have any other screening tests. the confirmatory test would be the TPHA (treponema pallidum haemagglutination) test. other screening or confirmatory manual tests include ATG-AMC (anti-thyroglobulin and anti-microsomal antibodies) test, ASO (anti-streptolysin O) test, IM (infectious mononucleosis) test, WWF (Widal and Weil Felix) & Brucella test.

thank you all for the great response. i'm very sorry if my explanations arent that clear or it still leaves you clueless but i've really tried my best to explain to you guys as what was taught to me during my stay in the serology lab for the first 2 weeks. if you still wanna ask me anything, go ahead. keep asking. it's good.

see you all C:

Week 3. 17 more weeks to go.

2008-07-06T09:52:00.011+08:00

Hey all. How are you guys doing?

I was attached to a private laboratory, together with Sharon, Lloyd, Maya and Ivan. It's CRAZY, I tell you. Working hours are a bit odd, but I guess we're all getting used to it already. Most of us worked from 1 pm to 9 pm daily. Each of us were posted to different departments but we still get to see a lot of one another as the departments are all located closely together. For the first 2 weeks of SIP, I was attached to the Serology department, which is a semi-automated lab where some of the tests are done manually while others are automated.

Samples are usually received at the lab around 1 pm and I usually have to wait for the specimen reception staffs to do the labelling and sorting out of the samples. My busy hours usually starts around 1.30 pm till break time which is around 5 pm or 6 pm. Samples usually come in hundreds up to thousands.

Most of the tests done in the Serology department are testing for presence of the different types of Sexually-Transmitted diseases. The most common and first test done upon receiving a sample is the Venereal disease (VD) test, which is done manually. However, I won't be talking about VD test as Farhana from another group has already talked about it in week 1. The procedures done in my department and hers are more or less the same. So, I'll explain about another test which I have carried out a few times by myself, but under the supervision of one of my Filipino colleagues.

Test:
Mycoplasma Pneumoniae Antibodies test (MPA)

Principle of test:
Agglutination of sensitised gelatin particles in the presence of Mycoplasma Pneumoniae antibodies in human serum.

Purpose of test:
An in-vitro diagnostic test to detect anti-Mycoplasma Pneumoniae antibodies in human serum. Mycoplasma Pneumoniae is the microorganism responsible for causing Mycoplasma Pneumonia, which is an infection of the lungs.

Materials:

- "U" shaped microwells plate
- 100 µl pipette
- 25 µl pipette
- 2 droppers (one for sensitised particles and the other for unsensitised particles)
- human serum
- Serodia®-Myco II test kit produced by Fujirebio Inc.

(Photo taken in lab. Permission given by supervisor.)

Contents of test kit:

- sample diluent (ready-to-use) - for diluting specimens (human serum) and reconstituting sensitised and unsensitised particles
- sensitised particles (lyophilized form) - gelatin particles coated with antigen specific to mycoplasma pneumoniae antibodies
- unsensitised particles (lyophilized form) - gelatin particles not coated with any antigen
- positive control (ready-to-use) - contains mycoplasma pneumoniae antibody positive rabbit serum diluted with sample diluent with dilution 1:10

Methods:

1. Label the sides of each row of the microplate with either 'C' for control (first row) or the last 3 digit of the patient's barcode number e.g. '319'. Every time we carry out a test, we have to do controls, thus labelling the first row of the microplate 'C'. Each sample takes up 6 wells; 1st well - neat (1:1), 2nd well - 1:20, 3rd well - 1:40, 4th well - 1:80, 5th well - 1:160, 6th well - 1:320. These are dilution factors or better known as titre.

2. Add 100 µl of sample diluent to the 1st wells of every row.

3. Add 25 µl of sample diluent from the 2nd to 6th wells of every row.

4. Add 25µl of positive control into the 1st well of the 1st row ('C'). Pipette up and down to mix the positive control and sample diluent together.

5. Obtain 25µl of solution from the 1st well and add it into the 2nd well and mix again. From the 2nd well, obtain another 25 µl of solution and add it into the 3rd well. Do this serial dilution up till the 6th well. Remember to discard the final 25 µl of solution from the last well to obtain the desired dilution.

6. Repeat steps 4 & 5 for every other patient's serum, but instead of adding positive control to the 1st well, add the respective patient's serum.

7. Add 1500 µl of sample diluent into the lyophilised sensitised particles and 500 µl of sample diluent into the lyophilised unsensitised particles. The amount of sample diluent to be added is written at the side of the bottles. This step is done to prepare the sensitised and unsensitised particles as they come in lyophilised form and are not ready to use. Shake the solutions gently to obtain homogenous solutions.

8. Add 1 drop of unsensitised particles to the 2nd wells of each row.

9. Add 1 drop of sensitised particles from the 3rd wells to the 6th wells of each row.

10. Incubate the wells for 3 hours.

Results:

The principle of the test is visible agglutination between sensitised particles, coated with antigen, in the presence of mycoplasma pneumoniae antibodies. Therefore, positive result will show agglutination while negative result will show no sign of agglutination.

A positive result will show a definite large ring with agglutinated particles spread within the ring. This type of result is read as (+). A more obvious positive result, read as (++), will show a uniform agglutination of agglutinated particles spread out evenly to cover the bottom of the well. A negative result will show a definite button at the centre of the well with a smooth round margin, and will be read as (-). Sometimes, the result is indeterminate and is read as (±). A small ring with smooth outer margin will be observed. If most of the results are indeterminate, I was taught to compare the compactness of the ring. The least compact ring will be reported as positive.

As the 2nd wells are added with unsensitised particles, a negative result is expected to be observed. Sensitised particles are added from the 3rd well to the 6th well of the positive control, 'C' row. Therefore, a positive result is expected to be observed in the 4 wells. The positive control is expected to give a 1:320 end point titre.

Positive result in patient's serum depends on the presence or absence of mycoplasma pneumoniae antibodies. The titre at which the positive results end is recorded. For example, a uniform agglutination is observed in the 4th well where the titre is 1:80 and a ring is observed in the next well, 5th well, where the titre is 1:160. The result is then recorded as '1:80'. A negative result in patient's serum will show no sign of agglutination and a compact ring or button is observed at the bottom of the well. The result is then recorded as 'NEG'.

Usually, we record down the titre at which the positive results end. After recording down the titre, we have to write our initial down so that we know who is responsible for doing and recording of the results.

(Photo taken in the lab. Permission given by supervisor in charge.)

This is a visual interpretation of some examples of how a positive or a negative result should look like. This chart is given by the supplier as a guide when reading test results. The interpretation of each well is written on the top right hand side of the well. Although the chart shows results till the last or the 12th well, we usually only do up till the 6th well in the lab. So, for example, the results for the first row shall be recorded as 1:40.

That's all I have to share with you guys this time. Hope to hear from you guys soon.
Enjoy C:

Nur Azeimah
0607060A
TG 02

Week 2 (30/6 - 4/7)

2008-07-05T02:28:00.006+08:00

Histopathology

Hi all. For the past 2 weeks, I was attached to the Histopathology lab. Basically the lab does routine work everyday: tissue processing, embedding, microtomy, etc. Of all, trimming can be considered the most interesting process, we will be able to see different tissues such as placenta and tonsils being cut. For every process, it is very crucial that the initial of the staff performing the job is indicated to facilitate traceability, as any small error may result in the wrong diagnosis.

Ok now I shall let you have an idea of how a Histopathology lab works. Following are the procedures that I observe everyday:

Receiving of Specimens – When specimens are first obtained at the lab, the information on the container/plastic bag (patient name/nature of specimen) must be checked against the requisition form. Next, the time and date is recorded and the medtech who performed the check will sign a form for the staff who sent in the specimens to prove the receipt. Labels which contain a barcode unique for every patient will then be printed and stick to the form and the specimen.

Most specimens sent in are fixed in formalin, which hardens the tissue for easy trimming. Large specimens are usually fixed overnight before trimming to ensure that formalin penetrates into all parts of the specimen. In the lab, formalin prepared would be sufficient for one week use.

Trimming and Passing – Before trimming of some large specimens such as breasts, asst pathologists will dye the specimen with different colored dyes to indicate the margins. While trimming, they will dictate the measurements and observations seen on the specimen (eg. a tumor measuring 2cm by 2cm is seen at 7 o’clock position) and these are immediately typed out by a lab assistant who sits just beside the asst pathologist. These observations would be printed out, attached with the requisition form and sent to pathologists to make the diagnosis. They will cut certain parts of the specimen where they suspect an abnormality may be present and ‘pass’ them into cassettes labeled with the patient number and year. For small samples, the number of strips of tissue will be written on the cassette to ensure that the embedder removes every single piece of tissue. Gel is also used to clump all the small strips of tissues together. Cassettes are labeled using a special marker which is resistant to formalin and other reagents. Also, different colored cassettes are used for different cases. All the cassettes are then placed into the automated tissue processor which works overnight, to perform thorough fixation as well as dehydration to remove all water from the tissue.

Embedding – First job performed every morning, after cassettes are removed from the processor. Tissues are first transferred to an embedding mould according to the size and pressed down using a weight (to ensure that the tissue will be flat), and embedded with paraffin wax. Blocks are then put onto an ice block for cooling.

Shaving, Sectioning and Fishing – After the wax is hardened, blocks are shaved at 10µm using the microtome to first expose the tissue. This is followed by sectioning, where tissues are sectioned at 3µm. Sectioned tissues are first placed into cold water and fished using pencil-labeled slides before transferring into the water bath containing hot water (43-48 deg C), which expands the tissue to remove folds. A thermometer must be placed inside the water to ensure that the water is not too hot as this may damage the tissue. The orientation of the sections is also very important in fishing. If fished too high, the tissue may not be stained by the autostainer (reagents in the autostainer are only of certain volume). Slides will then be arranged in slide holders and placed into the oven for heating for 15 mins at 85 deg C to ensure tissues are properly attached onto the slides to prevent detachment/sliding off during staining. Tissues can be fished 6, 3, 2 or 1 in a slide according to the size (smaller tissues are fished more in a slide).

Staining – Slide holders removed from the oven is then immediately placed into the auto-stainer, which performs the H & E stain. Special stains such as PAS, iron and reticulin stain are performed manually. Different programs in the autostainer perform different procedures (eg. program 8 is used for H & E staining and program 7 is used for dehydration, for special stains). This machine is connected to an autocoverslipper, which automatically mounts coverslips on slides without producing bubbles. Many racks can be loaded into the autostainer at once, thus the use of this machine had saved a lot of time compared to manual staining.

Sorting – Slides stained will then be checked against the tissue blocks (to tally the size and shape of tissue) to ensure labels are pasted correctly before they are sent out to the pathologists, who make the diagnosis. Used blocks will be kept into boxes in ascending order to ensure easy retrieval when additional special stains or re-cuts are requested by pathologists.

Other Histopatological Aspects

Frozen Sections – This is done when immediate results are required, usually when an operation is still in progress. When the doctor removes any tissue and is unsure whether the surrounding tissues are affected, they will request for frozen sections. Once the histolab receives a frozen section request from the operating theater (OT), a medtech would have to rush to the specimen room located around the OT to collect the fresh specimen. The pathologist in charge should already be on stand-by in the lab. Once the specimen reaches the lab, the pathologist will carry out the trimming personally. The trimmed tissue will then be treated with liquid nitrogen and sectioned using the cryocut. This is followed by the manually performed rapid H & E staining and microscopic analysis by the same pathologist. They will then call the OT to report the results. The whole process (from the time when the specimen reaches the lab to the delivery of results) must be completed within 20 mins, thus it can be very stressful when performing this procedure.

Immunohistochemistry Stain – Commonly used in the diagnosis of abnormal cells such as those found in cancerous tumors, this type of stain is performed when the pathologist suspects any abnormality in the morphology of the H & E stained slides and requires confirmation. The stain exploits the principle of antigen-antibody reaction; using specific antibodies to detect the antigens present in the cells. Firstly, slides with the cut sections and cover tiles (to protect the tissue from any damage during the staining process) are loaded into the machine. Then tumor markers such as CD31, estrogen and CK-7, which are contained in containers, are added. Positive reactions with these markers can indicate particular cellular events such as proliferation or apoptosis. These positive results can be visualized based on enzymatic conversion of an added substrate (DAB) into a brown colored precipitate at the sites of antigen localization (ie. if cells stained appear brown, there is a positive antibody-antigen reaction).

Special Stains – These are done also as confirmatory tests for diagnosis. When the pathologists suspect an abnormality in the H & E stain, they will order for special stains. Some examples are PAS & PASD, GMS and TB and Prussian blue stain (I will only elaborate on PAS & PASD, cos this is the most commonly used test):

PAS (Periodic Acid Schiff’s) detects the presence of glycogen, fungus and mucin. It stains these components magenta (using Schiff’s reagent) and the nucleus blue (haematoxylin as counterstain). PAS is always done with PASD, which has the same protocol as PAS; only that it has an additional step of treatment with diatase, which deploys glycogen into smaller sugar units that can be washed out after treatment. Glycogen stained magenta on the PAS stained slide and will be absent on the PASD stained slide. This absence of glycogen will clearly show the presence of fungus and mucin.

I will be changing lab next week. But I havent seen post mortem case (autopsy of babies)! Cos there is none. Not that I'm a saddist, my supervisor say must see. Nvm, this might not be a bad thing.

Anyway that's all I have for now. Have fun =)

Ka Hang
TG02

Week 1 (23/6 - 27/6)

2008-06-30T00:44:00.016+08:00

There was some problem with my documents to start work at my research laboratory, so I had to stay in school for the first week to do research on flow cytometry to prepare me for my research work which will involve dealing with flow cytometry cell sorter... It's going to be an exciting SIP for me! hahaz.

Flow Cytometry

Principle

It is a rapid technique that allows the analysis and identification of individual cells from a pool of heterogeneous sample population using multiple parameters.

Process of flow cytometry (Overview)

Make a single cell suspension of the interested cell culture
Use of antibodies bound with fluorochromes (PI) that is cell specific and incubate before washing (May involve use of multiple different specificity types of antibodies at any one time for flow cytometry)
The sample is then loaded onto the flow cytometry
Before using the flow cytometry, it is required to warm up the laser
The sample is pressurized upwards or downwards depending on the type of hydrodynamic focusing used (sheath fluid is used to propel the sample into the flow chamber where the laser is located)
The sample pass through the flow chamber where it is restricted to a single cell file flow
A laser beam (normally an argon-ion laser is used) is focused on the cell flow, in the flow chamber, where cells with bound antibodies will have the fluorochromes attached excited into giving off light (depending on the type of flurorchromes used)
The light scattered is collected by the orthogonal collection lens, which has a high numerical aperture for maximum fluorescence collection possible, to be measured by the photomultipliers where it is amplified and processed for analysis in the computer through digital conversion
If the interested cell is identified, the flow chamber is charged at the moment of cell droplet formation (eg. Negative charged)
The droplets are then pass through a pair of charged plates
The charged plates function to deflect and collect different charged cell droplets
The droplet with the interested cell is deflected towards the positive charged plate and collected into collection vessels for further analysis or use

Applications:

Immunophenotyping
Ploidy analysis
Cell counting
GFP expression analysis

Basic overview of the flow cytometry setup

The components are:

Fluidics system
Optics
Signal detection and processing
Electrostatic cell sorting (For flow cytometry equipped with cell sorting capabilities)

Fluidics system

It is a flow system that employs a concept of prssurized flowing fluid, shealth fluid, for single cell line presentation to the interrogation point and removes the waste into the waste container at the end of the flow.

Sheath fluid: usually an isotonic saline solution with the purpose of propelling the sample into the flow chamber.

The presentation of a line of single cells to the interrogation point is termed: hydrodynamic focusing

Figure 1.1: Hydrodynamic focusing of the sample core through a flow cell

Figure 1.2: Hydrodynamic focusing of the sample core through a nozzle tip
Taken from: http://www.stemcell.umn.edu/img/assets/10061/Intro_to_Flow_Cytometry_Learning_Guide.pdf
Interrogation point
The point of intersection between the laser beam and the sample delivered in the flow chamber.

Optics

The system in the flow cytometry that is concern with manipulating the properties of light for signal detection and processing.

Light source
Blue light argon-ion laser = 488nm
UV laser = 360-380nm

Three main points:

Light beam focusing
Light collection
Optical filters

Light beam focusing
Laser beam use optic lens to focus the intensity of the beam onto the sample stream where a cross-section of 50um laser beam is commonly set-up.

A flat-topped beam is commonly used for flow cytometry system in order to give a uniform lighting of the moving cells.

Figure 2: Cross-sectioned laser beam focused by laser optics lens
Taken from: http://biology.berkeley.edu/crl/flow_cytometry_basic.html

Light collection
The collection lens is required to have a high numerical aperture value for maximum collection of the low fluorescence and other light signals from cells.

Optical filters (Not important)
Not use for monochromatic light from lasers. They are used for arc lamp that requires filtration to give the right excitation light wavelength for appropriate fluorescent dyes; however, these are rarely used now.

Signal detection and processing

Signal detection

There are three types of light to be detected by a light detector:

Forward scatter light (also known as low-angle light)
Side scatter light
Fluorescent signals

Forward scatter light
The measurement of the degree of forward scatter light is approximately proportional to the cell size. Therefore, the larger the cell, the larger the degree of forward scatter light detected.

These can be used to differentiate cells with different cell sizes:

Red blood cell (6um)
Lymphocyte (8um)
Neutrophil (12um)
Monocyte (14um)

Figure 3: Picture of a forward scatter light detector with an obscuration bar in front on the right side of the picture
Taken from: http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

Side scatter light
The measurement of the degree of the side scatter light signal is approximately proportional to the granularity and structural complexity intracellularly.

These can be used to differentiate cells with varying granularity and structural complexity:

Lymphocytes (low intracellular complexity)
Monocytes (higher intracellular complexity)
Granulocytes (highest intracellular complexity)

Figure 4: Picture of a side scatter light from a cell
Taken from: http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

Fluorescent signals
These involve the use of antibodies coupled fluorescent molecules that are excited with the right laser wavelength in the flow cytometry and gives off a fluorescence signal.

Types of Fluorochromes used (excitation at 488nm):
Label Proteins:
Fluorescein Green color
R-phycoerythrin (PE) Orange color
Phycoerythrin-Texas Red conjugate (ECD) Red color
Phycoerythrin-cyanine5 conjugate (Cy-chrome) Deep red
Conjugated peridinin chlorophyll (PerCP) Deep red

Label DNA:
Propidium iodide (PI) Red color

Figure 5: Fluorescent dyes and their properties
Taken from: http://www.ab-direct.com/uploads/Flow-Cytometry.pdf

Optical filters
They are dichroic filters which are used for detection of many signals simultaneously.

Dichroic filters acts as a filter and a mirror: allows light wavelength of specific length to pass through while reflect the remaining blocked light to specifc light detectors.

There are three types of dichroic filters:

Long pass filter
Short pass filter
Band pass filter

Long pass filter
Blocks light below cut-off wavelength and transmit the light above that specific wavelength.

Short pass filter
Blocks light above cut-off wavelength and transmit the light above that specific wavelength.

Band pass filter
Block light below and above a certain specific cut-off wavelength and transmit the light from that narrow wavelength.

Figure 6: Diagram of the properties of different filters (shaded area is the light wavelength allowed to be transmitted through the filter)
Taken from: http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

Signal processing

The lights detected from the each of the light detectors are converted into voltage pulse and this data is transfer into a computer program to be analysis.
Figure 7: Diagram of the process of signal detection to processing
Taken from: http://www.ab-direct.com/uploads/Flow-Cytometry.pdf

Electronics and peripheral computer system
As the light signals detected are normally too small to be plotted on a graph, they have to be amplified first. These are done by a chain of linear or logarithmic amplifiers in a computer program before plotted on a graph, histogram.

There are three types of amplification:

Logarithmic amplification/scaling
Linear amplification/scale
Biexponential amplification/scaling

Logarithmic amplification
It amplifies weak signals and constricts strong signals so that they are evenly distributed to be plotted on a histogram. It is commonly used for fluorescence analysis.

Linear amplification
Unlike logarithmic amplification, linear amplification is used for a narrow range of signals so that the data could be seen clearly on the histogram. It is commonly used for DNA studies.

Biexponential amplification
It is a combination of logarithmic and linear scale at the upper end and lower end respectively which is also used on the graph for flow cytometry.
Figure 8: Comparing the difference in using linear and log scaling for fluorescence analysis
Taken from: http://probes.invitrogen.com/resources/education/tutorials/5Data_Analysis/player.html
Figure 9: Comparing the difference in using linear and log scaling for DNA analysis
Taken from: http://probes.invitrogen.com/resources/education/tutorials/5Data_Analysis/player.html

Electrostatic cell sorting

This is an extended function of flow cytometry with the capabilities to sort the cells according to the data inputted into the computer system on the cells of interest as the selection criteria.

This application consists of three parts:

Droplet formation
Droplet charging
Deflection plate (voltage plates)

Droplet formation
It is important to form droplets of approximately equal sizes to achieve only the formation of droplets which only contain one cell each for the purpose of accurate cell sorting. This is done through the high vibration of the nozzle part in the fluidics system where the fluid exits.

Droplet charging
When the droplet which contain the single cell of interest which fits the selection criteria. The fluid stream is charged at the precise ‘break-off point’ during the droplet formation.

Break-off point
It defines the moment where the droplet contains the cell of interest is formed and detaches from the fluid stream.

Deflection plate
After the charged droplet containing the cell of interest is release from the fluidics system and drip down towards the compartment containing the plates where it will be deflected left or right, depending on the charge. Non-charged droplets will just drip down into the waste container located right below the nozzle.

Figure 10: Diagram of the process of electrostatic cell sorting based on droplet charge
Taken from: http://www.ab-direct.com/uploads/Flow-Cytometry.pdf

(OH and ya, I absolutely hate blog posting, cause I am a blog idiot..! Posting a blog is such a frustrating task!

If only they have an attachment file tab, then I could attach microsoft word directly to each posting instead of spending my time trying to organise my documents which never fail to end up in a mess after I copy pass from my microsoft word document!!!)

Reference List:

1. Misha Rhaman. (2006). AbD SEROTEC. Introduction to Flow Cytometry. Retrieved June 29, 2008, from http://www.ab-direct.com/uploads/Flow-Cytometry.pdf

2. Nicoll, A. K. (2004). University of Dundee. SLS Flow Cytometry Core Facility: What is Fluorescent Activated Cell Sorting (FACS)? Retrieved June 29, 2008, from http://www.dundee.ac.uk/lifesciences/FACS/cell_sorting.htm

3. Invitrogen. (2008). Introduction to Flow Cytometry. Retrieved June 29, 2008, from http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html

4. Invitrogen. (2008). Data Analysis for Flow Cytometry. Retrieved June 29, 2008, from http://probes.invitrogen.com/resources/education/tutorials/5Data_Analysis/player.html

5. BD Bioscience. (2000). Introduction to Flow Cytometry: A Learning Guide. Retrieved June 29, 2008, from http://www.stemcell.umn.edu/img/assets/10061/Intro_to_Flow_Cytometry_Learning_Guide.pdf

By: Low Quan Jun 0607243C TG02 (Post: 29 June 2008 Edited: 04 July 2008)

ISO 15189

2008-06-16T01:22:00.007+08:00

ISO 15189:2003, Medical Laboratories – Particular requirements for quality and competence is an international standard specific for medical (clinical) laboratories that accredits their competence in technical operations and service and staff quality management¹. It has two categories - Management and Technical Requirements¹ – which contain 23 quality system elements² that allow the laboratories to produce credible results³ and improve their quality management systems⁴. It also enables them to ‘organize their operational procedures efficiently, meet the expectations of their clients and improve their service’¹.

Management requirements: generic requirements for quality management systems¹.
Technical requirements: specific requirements for activities carried out by clinical laboratories¹.

(100 words)

References

1 Périgo, D. M., & Rabelo, R. (2004). ISO-3: The New ISO 15189:2003 as a Quality Management Standard. Retrieved June 15, 2008 from http://www.westgard.com/iso3.htm

2 Cooper, G. (n.d.). ISO 15189 Quality System Elements. In
Preparations and Considerations When Seeking Laboratory Accreditation Under ISO 15189. Retrieved June 15, 2008 from http://www.sacb.org.sg/documents/PreparinglabforISO15189-GCooper.pdf

3 Colby Group International, Inc. (2003). Lab Accreditation Support. Retrieved June 15, 2008 from
http://www.colbygroup.com/en/iso.html

4 International Organization for Standardization. (2003). New ISO standard promotes quality and competence of medical laboratories. Retrieved June 15, 2008 from http://www.iso.org/iso/pressrelease.htm?refid=Ref857

Done By: Nor Liyana (0607927A) TG02

Management requirements

1. Organization and Management

· States identity¹ of laboratory
· Describes personnel responsibilities²
· Organization structure²
· Ensures no financial or political1 issues would affect testing

2. Quality Management System

· Implementation of quality policy¹
· Documentation and update of policies and protocols through a quality manual¹
· Educating personnel regarding quality management to meet objectives²

3. Document Control

· Approved¹ documentation
· Constant¹ updating of documents
· Reviewed often¹

4. Review of Contracts

· Regular review of personnel’s skills to meet customer’s needs²

5. Examination by Referral Laboratories

· Monitored for accreditation and through proficiency testing¹
· Evaluation of results by other labs²

6. External Services and Supply

· Evaluate quality of purchased products²

(95 words)

References

1. Cooper, G. (2004). Preparing Your Laboratory to Certify or Accredit Under ISO 15189. Retrieved June 13, 2008 from,
http://www.sacb.org.sg/documents/PreparinglabforISO15189-GCooper.pdf

2. Périgo, D. M., R.Ph. and Rabelo M.D., Ph.D. (2008). ISO-3: The New ISO 15189:2003 as a Quality Management Standard. www.westgard.com. Retrieved June 13, 2008 from,
http://www.westgard.com/iso3.htm

Done By: Nur Azeimah (0607060A) TG 02

7. Advisory services

· Advice and assistance¹ given on selection of examinations and services.²
· Meetings conducted regularly.²
· Eg. Advice on specimen collection

8. Resolutions of complaints

· Steps managing complaints and investigation records in place.¹
· Measures rectifying complaints: taken and documented.¹
· Eg. Supplier’s complaints

9. Identification and control of nonconformities

· Measures taken to rectify diversions.¹
· Diversions investigated, recorded, evaluated by personnel.²
· Eg. QC results¹

10. Corrective action

· Implemented to eliminate recurrence following investigation.¹
· Results reviewed for effectiveness.¹
· Audits done when deviations.³

11. Preventive action

· Reduce probability of non-conformance.³
· Can implement improvement³
· Eg QC data reviewed regularly4

12. Continual Improvement

· Improve process by reviewing regularly.²
· Identify opportunities for improvement.¹

(100 words)-Not inclusive of numberings and bullets

References

1. Périgo, D. M., R.Ph. and Rabelo M.D., Ph.D. (2008). ISO-3: The New ISO 15189:2003 as a Quality Management Standard. www.westgard.com. Retrieved June 13, 2008 from
http://www.westgard.com/iso3.htm

2. Charles-Diamond, Keisha (2007). Brochure ISO 15189. Trinidad and Tobagao Laboratory and Accreditation Service, Retrieved June 13, 2008, from
http://www.ttbs.org.tt/brochures/Brochure_ISO_15189.pdf

3. Chris (2005). Amendments to 15189: Annex D (Normative) – Point-of-Care Testing (POCT). Retrieved June 13, 2008, from
http://www.cdhb.govt.nz/ch_labs/POCT/ISOWD22870.pdf

4. Cooper, G. (2004). Preparing Your Laboratory to Certify or Accredit Under ISO 15189. Retrieved June 13, 2008 from,
http://www.sacb.org.sg/documents/PreparinglabforISO15189-GCooper.pdf

Done by: Nur Liyanah Bte Md Zaffre (0607718D) TG02

13. Quality and Technical Records

· Maintenance, safe disposal and retrieval of quality records¹
· States results achieved²
· Provides evidences of activities performed²

14. Internal Audits

· Checks compliance of processes to ISO 15189 and internal procedures¹
· Conducted regularly by trained laboratory staff² from other departments

15. Management Review

· Evaluates the effectiveness² of quality system and the need for resources¹
· Review occurrences and activities since last review²

Technical Requirements

1. Accommodation and Environment Conditions

· Adequate space²
· Safe environment²
· Clean workspace²
· Good structural organisation
· Controlled environmental conditions¹

2. Laboratory Equipment

· Selection and monitoring¹ of all lab equipments
· Includes monitoring, calibration and maintenance procedures¹
· Requirements for computer software and automated equipment¹

(99 words)

References

1. Cooper, G. (2008). Preparations and Considerations When Seeking Laboratory Accreditation Under ISO 15189. Retrieved June 14, 2008, from
http://www.sacb.org.sg/documents/PreparinglabforISO15189-GCooper.pdf

2. Périgo, D. M., and Rabelo, R., (2004). ISO-3: The New ISO 15189:2003 as a Quality Management Standard. Westgard QC. Retrieved June 14, 2008, from
http://www.westgard.com/iso3.htm

Done By: Liong Ka Hang (0606688J) TG02

3. Personnel

· The director of a medical laboratory must have sufficient education, experience and training. (NATA, 2005)

4. Pre-examination procedures

· Pre-analytical SOP must be clearly defined for smooth operation and validation. (NATA, 2005 and Westgard QC, 2004)

5. Examination procedures

· Involves analytical processes (validation, reference and critical intervals) control and documentation as it was implemented. (NATA, 2005 and Westgard QC, 2004)

6. Post-examination procedures

· Detailed document must specifics the storage or disposal of used samples and result evaluation before release. (Westgard QC, 2004)

7. Quality assurance of examination procedures

· Quality control implementation to provide confidence in results through documentation and traceability. (NATA, 2005 and Westgard QC, 2004)

8. Results reporting

· Evidence must be provided to support the accuracy and reliability of results for compliance to national legal requirements. (NATA, 2005 and Westgard QC, 2004)

(99 words, not counting the bullet points and in-text citations)

References:

1. University of Maryland University College (UMUC). (2007). APA Citation Examples – Information & Library Services -UMUC-. Retrieved June 15, 2008, from
http://www.umuc.edu/library/guides/apa.shtml

2. National Association of Testing Authorities (NATA), Australia. (2005). ISO 15189 – The New Standard for Medical Testing Laboratories. Retrieved June 15, 2008, from
www.nata.asn.au/index.cfm?objectid=0870E6A5-A8D1-2553-F3DBF59E30C60829

3. Westgard QC. (2004). ISO-3: The New ISO 15189:2003 as a Quality Management Standard. Retrieved June 15, 2008, from
http://www.westgard.com/iso3.htm

4. ISO 15189 Blogspot. (2006). ISO 15189: Section 5 – Technical Requirements. Retrieved June 15, 2008, from http://iso-15189.blogspot.com/2006/07/section-5-technical-requirements.html

5. Ontario Medical Association. (2004). PARTNERING TO PROMOTE AN UNDERSTANDING OF ISO 15189:2003. Retrieved June 15, 2008, from
http://wwwn.cdc.gov/dls/iqlm/2005Posters/Crawford-9-Partnering%20to%20promote%20understanding%20of%20ISO%2015189-2003.pdf

Done By: Low Quan Jun (0607243C) TG 02