Cytogenetics: chenghong

hi guys sorry for the delay in finding a good example of the abnormal karyotype.

in this case it is a patient with derivative material on the 16th chromosome, and the addtional material is a translocation from the chromosome 20 at p11.2 site to p13.3 on chromosome 16.

it is important that a med tech is able to read and write the standard nomenclature for the chromosomes to let others know what is going on.

Haematology

Kleihauer Test

It is to determine the distribution of fetal Hb in red cells and especially useful in determining the presence of fetal RBC in the maternal circulation.

Principle:
HbF will be able to resist acid elution to a greater extent than normal cells whereas HbA (adult) gets denatured in these conditions. Red cells containing HbF will be deeply stained whereas cells with HbA will give a ghost-like appearance.

Procedure:
1. Fix blood film with 80% ethanol for 5 mins

2. Rinse with distilled water and dry

3. Immerse in elution solution (haematoxylin & ferric chloride) for 20s.

4. Rinse thoroughly with distilled water

5. Counterstain with eosin for 2 mins

6. Rinse with water and dry

7. Examine under microscope

Results:
HbF level should be <0.01% for adult.
-Adult red cells appear as ghost-like pale pink.
-Fetal cells stain densely red.

QC:
A normal adult blood (negative) and cord blood (positive) should be stained together.

Clinical significance:
It is a sensitive procedure which identifies individual cells containing HbF even when few are present, and their detection in the maternal circulation has provided valuable information on the cause of hemolytic disease of the newborn.


Eunice
TG02
0503245C

Microbiology, Haematology and LMQA

Hi all, I have been posted to microbiology and now, on to haematology after serology on my first 3 weeks.
In my lab at microbiology, I was assigned for 3 quarters of my time there, to open bags at the specimen reception. So, ppl, pls spare me from questions that are too in depth. =)

It is essential to ensure that all request forms comes with the appropriate and correctly labelled specimens. There were cases when the specimen and request form does not tally. For example, the name of the patient labelled on the specimen is different from the name printed on the request form, we will write the comment that the samples were labelled as "xxxx (the name found on the sample)" and the barcoders would call the clinic and query on that. On cases where the specimen comes without a request form, we would write down the patient's details on a request form and call up the clinic to ask for the type of test. If the specimen is not properly labelled or is unlabelled, we would pass the specimen to the HOD(head of department) to deal with it.

Since Sasi has explained Urinalysis in detail, I would like to share some interesting things I had seen during my time there.


Trichomonas vaginalis

Trichomonas vaginalis is the most common parasite in urine. This organimsm is the same size and also looks like a white blood cell (wbc) especially if it is next to a wbc, it may be mistaken as an wbc. But if you look carefully, you will notice 4 flagella. Since it is mostly accompanied by wbcs and epithelial cells, its motility is the diagnostic feature. It affects females the most although males can have infection of T. vaginalis, which is rare.

For more infomation and a video of how it looks like, please follow this link: http://www.microbiologybytes.com>video>Trichomonas.html

Tyrosine crystals

Tyrosine crystals occurs in severe liver disease, tyrosinosis and Oasthhouse urine disease. They look like refractile needle-like crystals under 1000x magnification and appears black especially around the centre. They may also look yellowish due to presence of bilirubin in urine. They are found in acidic urine only.











Obtained from : http://www.medicine.uiowa.edu>cme>clia>modules.asp?testID=20

Yeast cells

Refering to pictures that Sasi posted, she showed yeast cells budding. During my experience in the lab, I managed to spot yeast cells with hyphae!








Taken from
http://www.agora.crosemont.qc.ca>
urinesediments>Imdoceng>
d05d002.htm

This picture shows the hyphae of yeast cells =)




Mucous threads

Mucous threads are present in urine in small amounts. In presence of urinary tract infection or irritation of the urinary tract, large amounts of mucous may be discharged in urine. Wide mucous threads may be confused with hyaline casts or cylindroids. So look carefully under the microscope! Cylindrical composition of casts and their rounded ends distinguishes hyline casts from mucous threads.








Mucous threads with uric acid crystals

Taken from : http://www.agora.crosemont.qc.ca
>urinesediments>Imdoceng>d05d004.htm


Stool culture
I was briefed on how to do stool culture in the microbiology lab but did not have a chance to perform. Basically, because the lab unlike hospitals, we receive samples that are relatively less pathogenic and we only indentify for samonella and shigella spps only. The media we used are specific for isolation of such organisms. They are XLD, maconkey, TCBS and selenite F broth.


LMQA of Microbiology lab

Urisys 2400: The machine will prompt around once a month for a new calibration. 2 controls will be run per day. The low/normal value and the high/abnormal value. The values are recorded into a logbook and runs are rejected if they are out of range.

Agar for cultures: Agar plates are bought commercially. When they arrive, we check macroscopically for contamination before a sample is incubated at 37 degree celcius overnight. After incubation, if there are still no growth, we conclude that it is sterile. To check for the plate's viability, we streak commercially prepared ATCC strains and incubate overnight at 37 deg celcius. The growth of the strains indicates that the plates are viable. So after the plates passed the quality check, we can then use them for testing.

This is just the general briefing I was given. Thus, for the in depth information for quality control, I am not very sure of them.


Haematology

I had so far learnt ESR and malarial parasite testing. For ESR, the principle and method is the same from what we learnt in school.
For malarial parasite, the principle is the same as what Lizzie (from 77 med tech street) described 6 weeks ago. Any slide with platelet less than 150 or more than 500 is selected for a blood smear to check for malaria parasite. An autostainer is used to stain the blood slides before microscopy.
The prevalence of the disease is rather low-according to my mentor. But I am so lucky/unlucky to spot 1 patient's slide positive for Malaria parasite (plasmodium vivax) on my 3rd day of practice while earlier that day, there was a case of plasmodium falciparium ^^



Hope that you have learnt something from my post.

Yeng Ting
TG02

Cheng Hong: Cytogenetics Q&A

Q1) Why we do not want the cells to enter into mitotic phase?
Ans: We add the colcemid(mitotic inhibitor) to prevent the cells from entering M phase so that the 2 sister chromatids will not be pulled apart so that we are able to do karyotyping.


Q2) What action can be taken to save cell cultures that have been already contaminated? OR do you just use the spares?
Ans: We will use the back up tube to harvest the cells if the back up tube is also contaminated we will add antibiotics to treat the cultures.


Q3) Does any procedures required for any types of identification of those chromosome 1-22?
Ans: There is a book on the banding for each of the chromosome so we can look out for the typical banding type to identify the respective chromosomes.

Q4) May i know how you collect "villius"??
Ans: Collection of the villius is a minor operation to be done in a operating theater. It is done with the aid of ultra sound, the villius is attached to the placenta( picture it as a tree: the roots are the villius and the soil as the placenta, the villius holds tightly to the placenta and the villius is attached to the amniotic sac which holds the fetus)

Q5) What is colcemid?
Ans: it is a mitotic inhibitor


Q6) if we forgot to subculture the cells , what are some actions you guys take?or do you guys have like a timetable thingy to remind you ouh today must subculture this 'plate'
Ans: on each tray we will stick the day/at which stage of the culture it is at, and we have to check the time table to know what to do with the cultures(subculture/harvest/feed after tiff/hold/backup)


Q7) What are some examples of mitotic inhibitors and what's the purpose of using the different media?
Ans: by using 2 different types to brand media (both media have about the same composition) is a preventive measure, if one brand of the media is contaminated/ have batch variation there is still another culture which will not be affected.


Q8) Explaining how does the humidity and temperature, height dropped at, amount of water present on the slide and concentration of cells, affect the spreading of the chromosomes, length and colour?
Ans: This really depends on the technicians, they have their preferred method. But generally if the temp is high the evaporation rate is higher we will get darker, tighter chromosome as the chromosome do not have much time to spread(thus making analyzing difficult, also affects the staining). If the temp is colder and the humidity is high, the evaporation rate is slower thus the chromosome has a lot of time to spread causing pale and long chromosomes (to counter it we use a slightly drier slide to make or we do the slide making in the thermothron, where the humidity can be controlled)


Q9)For the blood harvesting, what is the purpose of the hypotonic solution?
Ans: the hypo solution is to swell up the nucleus of the cell so that the chromosomes have space to spread making analyzing easier is there is fewer overlaps.


Q10) Pictures that show normalites & abnormalities of karyotye?
Ans: i will try to get it and post asap


Q11) You said that you have to warm up beaker containing washed glass slides, to 90C. In our lab, we dun warm up the beaker containing the slides. The slides are washed and kept in DI water in the fridge. When we wanna dropslide we fill pour away the water and fill it up with new D.I water . Why issit that u have to warm it??
Ans: the warming of the slides really depends on the preference of the technologist as in the lab is quite cold so we warm the slides to aid in the evaporation of the film of water and the spreading of the chromosome.

Cheng Hong: Cytogenetics

Basically in cytogenetics lab the lab technologist must be able to do karyotyping(sort out the chromosomes 1 to 22 and X & Y.

The lab technologist have to be able to detect any abnormalities in the fetus/ child/ adult so as to be able to help the clinicians to give accurate diagnosis and patients will be able to go for genetic counseling if needed. Some genetic diseases like trisomy 21 which gives rise to a down syndrome child/ a deletion in a certain part of the gene and might affect the heart of the fetus etc

Type of specimens:
1) Amniotic fluid culture(collected around 4months after pregnancy)
2) Chronic villi (collected 10 to 12 weeks after pregnancy so that the vilius is not too thick/ deep into the placenta)
3) Cord blood (collected around 20-22weeks after pregnancy so that the umbilical cord is think enough to be pricked)
4) Bone Marrow to detect Leukemia due to genetics
5) Product of conception(fetus/ fetal eye/fetal gonads/ fetal skin)
6) Tumor
7) Peripheral blood

The General work flow in a Cytogenetics lab:
1) Grow cells in media for harvesting (so that there is enough cells to obtain enough metaphases to study the chromosome)
2) Add mitotic inhibitor so that the cells are not able to proceed into the Mitotic phase of the cell cycle
3) Add hypotonic solution (so that the nucleus will swell up thus the chromosomes can spread out)
4) Add fixative (to “freeze the cell in the swelled up state)
5) Stain the slides (normally with Giemsa stain so that the bands in the chromosome can be seen)
6) Microscopy and Karyotyping

Cell cultures
§ The daily maintenance of the cultures is quite tedious and labour intensive, normally each specimen is cultured for 9 to 11 days. We have to check the cultures daily for any microbial contamination then action must be taken to save the culture and prevent the spreading of the contamination.
§ If the media is used up we have to change fresh media for the cells. And if the cells are very confluent, we must tiff(sub-culture) into a new culture so that the cells will not be so packed
§ Once the culture is enough for harvest(at least 4 colonies)

Amniotic fluid culture setup (to be done in the BSC)
§ The samples are sent in 25ml syringe (the doctors should collect around 20ml of fluid)
§ Prepare 2 tubes for each sample (1 conical base tube, 1 slant tube)
§ Invert the syringe to mix the cells in the fluid so that it is evenly distributed
§ Add to each of the culture tubes (10ml each)
§ Spin down at 1200rpm for 10 mins
§ Prepare culture dish(4 each for each sample in case of any contamination in 1 dish/ low growth)
§ Add a cover slip into the dish
§ Record the pellet size after spin down, colour of supernatant, with or with out feces or RBCs
§ Aspriate and discard the supernatant
§ Add Chang media to the slant tube(0.5ml)
§ Add Bio-AMF medium to the conical tube(0.5ml)
§ Resuspend pellet
§ Fill slide with the cells in the conical tube (slant tube as back up)
§ Incubate tubes in 2 different incubators at 37oC, 5%Co2, 5%O2

*to prevent any incident of unable to obtain cells due to contaminations, the lab uses to separate incubators (2 dish of the 4 dishes in each incubator), in case of event that 1 of the incubator breaks down/ contaminated the other 2 dishes will not be affected. The lab also uses media from different vendors in case the batch has any defects there will sill be back ups. The incubators are also washed and wipe down with alcohol weekly.

Blood culture setup (to be done in BSC)
§ Blood should be sent in sodium heparin tube to prevent coagulation and to preserve the cells
§ In blood culture we want to grow the White cells
§ Spin down the blood tube at 1200rpm for 10mins
§ Use a pasture pipette to aspirate up the layer of buffy coat(whitish layer where the WBC is)
§ Invert the pipette and mix the cells
§ Add into tubes containing RPMI medium and 1 M199 medium
§ Check if blood is clotted (if clotted mesh up clot and place in tube with media as the clot may trap the WBCs)
§ Add PHA to induce the white cells to enter the cell cycle as currently it is in G0 phase
§ Mix well and incubate at 37oC in a Co2 incubator (using a 45o angle rack to facilitate gaseous exchange)
§ On 2nd day mix the tubes and in the afternoon add MTX(to remove the thymidine so that the cells cant go through Sphase)
§ On 3rd day add thymidine, now the cells can proceed into S phase so that we will be able to synchronize the cells to enter metaphase together
§ Proceed to harvesting

Blood Harvesting
§ Warm up hypotonic solution to 37oC in a water bath and add 50µl of colcemid at 20mins and 2hrs interval
§ Spin down the tubes at 1200rpm for 10mins and discard supernatant
§ Dislodge cell pellet and add 8-10ml for hypotonic solution and mix well
§ Incubate for 5mins then spin at 1500rpm for 6mins and discard supernatant
§ Add 8-10ml of hypotonic solution again and incubate for 5mins and spin down again
§ Discard supernatant and add 2ml of fixative(3parts of methanol and 1 part of acetic acid)
§ Mix well and spin down at 1500rpm for 6mins and discard supernatant
§ Resuspend pellet and add 10ml of fixative and incubate for 30mins at room temp
§ Spin down at 1000rpm for 10mins is discard supernatant
§ Add 8ml of fixative and let it stand for 15mins
§ Proceed to slide making

Slide making (dropslide)
§ Warm up beaker containing washed glass slides, to 90oC
§ Depending on the humidity and temperature, height that is will be dropped at, amount of water present on the slide, concentration of cells, it will affect the spreading of the chromosomes, lenghth and colour
§ Spin down at 1000rpm for 10mins
§ Aspirate most of the fixative and transfer to an empty tube and leave some to dislodge the cells
§ Add the fixative to obtain best concentration to drop slide
§ Get slide and flick once/ twice to remove excess water
§ Hold the slide at a 45o angle and on the other hand hold the pipette and space them about 15-30cm apart and drop on the top part of the slide and allow the cells to slide down the slide
§ Wipe off excess water from the sides and let the slide dry
§ Check under microscope if the cells are too pack/ chromosomes are too short/ dark thus we have to adjust the humidity of the slide and height of dropping
§ Proceed to slide staining

Slide staining
The slides are stained with giemsa stain and wright’s stain, the combination of this 2 stains gives good contrast of the dark and light band in the chromosome, thus we will be able to do karyotyping and spot any abnormalities like translocation/ addition/ deletion/ inversion etc.

Any questions do ask thanks,

Cheng Hong TG02

Research: Lab Techniques

Hi all, week 6 is here and it is my turn to share my SIP experience. Time flies, doesn’t it?

I’ve been assigned to a research lab for my SIP, so the scope of my duties is to basically carry out my MP which revolves around mbio/mgen.

My MP in a nutshell is to study protein interactions in two different gene constructs using a yeast-two hybrid system. This is to be used as a basis for further studies by the company to determine if protein interactions are involved in the activation of nucleus translocation signal in the 2 genes I am studying (please don’t ask me how, I wish I knew how they would study it, but a student like me isn’t privy to such info).

Anyways, gene construct refers to a vector that has been ligated with an insert of interest. I need to amplify the inserts (2 types) and ligate them into a vector which confers kanamycin resistance for selection upon plating.

This week I shall focus on the first part of my MP – constructing fusion genes. For those interested, I will talk about the yeast-two-hybrid in a later posting as it is quite a bit of theory

Constructing fusion genes:

Steps:
1. Amplify gene 1 (PCR) and gene 2 (PCR)
2. Send amplified genes for sequencing (outsource to external company)
3. Digest vector, inserts – 1 and 2
4. Ligate inserts to vector
5. Transform ligated products
6. Colony PCR (screen for inserts in transformed cells)
7. Send positive clones from colony PCR for sequencing

1. Amplifying insert:
The first step is to obtain the inserts 1 and 2 by PCR (polymerase chain reaction) from a template DNA, provided in stock by the company. The underlying principle of PCR is that a single dna is all that is needed to generate many copies of replicate DNA.
There are 5 steps in total:
1) initial denaturation – 95oC, 4mins
2) denaturation - 95oC, 30s
3) Annealing - 56oC, 30s
4) Extension - 72oC, 4mins
5) Final extension - 72oC, 10mins
Step 2-4 run for 35 cycles.

The first denaturation is to separate dDNA so that the single strands can act as a template for synthesis. Annealing temperature is for the primers to anneal to the template and extension is for the polymerase to incorporate dNTPs for synthesis. I use PFU polymerase as it has proof reading ability – which is necessary to avoid errors in amplified sequence. Quality of PCR products can be affected by MgCl2 concentration, annealing temperature (too high no products, too low unspecific), genomic contaminations, amount of template DNA (I find 2ul produces good enough results for me) etc. Master mix has to be made up - not provided. Mine is as follows:

1) pfu buffer - 5ul
2) pfu polymerase - 0.5ul
3) dNTP -1ul
4) water - make up to 50ul reaction
5) Primers (forward and reverse) - 0.5uL each
6) Template (2ul)

Note: It is best to keep pfu polymerase on ice before placing to PCR to enhance its enzyme activity as it is not as heat stable as taq polymerase.

To view PCR products, need to run on agarose gel. I usually make up 1% agarose (agarose powder and TAE buffer) due to my insert size - 1=185bp, 2=1.8kbp and run for about 40mins at 100V, depending on size of the gel. Larger agarose concentrations (1.5-2%) are generally better for smaller dna chains <100bp.




















As seen in the gel picture, there are some unspecific products – probably due to low annealing temperature. I did try raising the temperature by 1oC, but it produced no bands. My PCR products are the 200bp and 2kbp bands. These bands are excised under UV light using disposable scalpels and placed into 1.5mL microcentrifuge tubes for purification to send for sequencing. As a safety precaution, it is necessary to wear protective headgear before operating the UV illuminator.


Purification/gel clean up system: Wizard SV Gel and PCR clean-up system

After obtaining the bands, need to weigh them to determine how much membrane binding solution is needed.10ul/10mg of membrane binding solution (MBS) is utilsed, and gel excised is incubated with MBS at 50-65 oC till gel is completely dissolved. Mixture is transferred to minicolumn assembly (minicolumn + collection tube) and incubated at room temperature for 1 min, before centrifuging at 16000g for 1 min. This is to get rid of waste. Flow through is discarded.
700ul of membrane washing solution is added, and tubes spun at 16000g for 1 min, flowthrough discarded. Membrane washing solution (MWS) contains ethanol to precipitate DNA, otherwise DNA will be washed away. Washing step is repeated with 500ul of MWS and spun for 5mins. Tubes are then spun for 1 min to allow for evaporation of ethanol, as ethanol contamination with DNA can cause problems in sequencing and PCR reactions.
Lastly, water is added to elucidate purified DNA in column and centrifuged down for 1min. A portion of DNA obtained will then prepared for sequencing.

2. Sequencing:
Although we send sequencing to external companies, we need to prepare and perform the sequencing reactions ourselves. We are provided with a sequencing master mix called big dye which basically contains buffer, labeled ddNTPs etc. Set up is as follows:

Big dye -8ul
Water – 9.5ul
Template (DNA to be sequenced) – 2ul
*primer – 0.5ul
Total: 20ul

PCR conditions:
1) initial denaturation – 95oC, 3mins
2) denaturation - 95oC, 30s
3) Annealing - 56oC, 30s
4) Extension - 72oC, 4mins
5) Final extension – N/A
2-4 run for 30 cycles.

*Note that forward and reverse primers are added into 2 separate tubes. For every 1 template, there are 2 tubes – 1 forward and 1 reverse. This is for counterchecking purposes in sequencing. Lets say that the forward sequence result has 1 base pair discrepancy. To check if this is a mutant or if the sequencing was read wrongly by machine (higher chances if machine reports low confidence for that nucleotide, which can be quite random due to weak signaling of labeled fluorescent), it is counterchecked against the reverse sequence (need to reverse complement it first). If the sequence is as expected, then the error was due to error in reading. If the sequence produced is identical to the forward sequence, then the gene is probably mutated – the polymerase incorporated a wrong base pair.


Below is an example of sequencing results. The circled blocks are representation of the signal strength – the stronger the signal strength, the more sure we can be that the nucleotide is read correctly by the machine. Blue is the strongest (above 50%), yellow indicates a 40% chance and anything below 30% is red. Software used is sequence scanner from applied biosystems.














To check if the insert is correctly sequenced as expected – compare actual and expected sequence using NCBI Blast program: http://www.ncbi.nlm.nih.gov/> blast>bl2seq>wblast2.cgi
If result is a 100% match, then it is considered correct. Anything lesser requires either re-sequencing or checking with reverse complement as mentioned previously (produce a forward sequence from a reverse primer). Reverse sequences can be reverse complemented manually or by using this program: bioinformatics.org>sms>rev_comp.html

3. Digestion:

Once it is confirmed that the sequences for both insert 1 and 2 are correct, they can be digested for ligation with the vector. The vector also needs to be digested.

Restriction enzymes such as EcoRI and NdeI are used. Since I do double digestion (add both enzymes at once), a compatible buffer for both must be used, such as buffer 4 from biolabs. Digested products are incubated at 37 oC (vector = 2hrs), (inserts = 4hrs).

Digested products are heated at 65 oC, 10mins to deactivate enzymes.
Digested vectors must be run on gel to obtain digested vector (non-digested vectors appear as smudge on gel. Gel picture below shows unsuccessful digestion (1) and successful digestion (2) of vector (ignore the other products).




















Vector will then be excised and purified as described earlier, then dephosphorylated to prevent re-ligation. Alkaline phosphatase only works with in alkaline conditions (hence the name), so buffer is added.

Dephosphorylation:
Alkaline phosphatase – 5ul
AP buffer – 5ul
Vector - 40ul

Incubate at 37 oC, 1 hr, heat inactivate at 65oC, 30mins.

4. Ligation:
Here comes the easiest part by far! Basically, inserts are added to vectors (ratio 3:1 for higher success of ligation), and ligase and ligase buffer (I use T4) is added to seal the “nicks”. For those who can’t recall, nicks are the missing phosphate backbone. Incubate at 16 oC overnight for best results.

----------------------------------------------------------------------------------------------------
Ligated products will then be transformed in ecoli cells via heat shock to screen for successful colonies, which is step 5 -7 of the whole procedure - I will discuss this in my next posting as it seems a bit overkill now.

Sorry for the long post! Luckily, this stuff is all sem 2 work, so won’t be tested! Feel free to ask questions.


Cheers,

Debra , TG02