Sunday, November 9, 2008
YES! Finally it’s the end of SIP. But it also means submission for reports and all are coming. Hmm.. Anyway, I was posted to blood bank for the last week. But I am going to post on another test done in the Haematology lab, which I was in for 3 weeks before blood bank lab.
In Haematology lab, it is actually one of the busiest lab. There are constantly a lot of samples sent to the lab to be tested. There are only two types of specimen sent; blood and CSF samples. I am going to share on Erythrocyte sedimentation rate test. In the lab, they make use of the method Sedi-rate P4-Micr System to test for ESR.
Principle
Erythrocyte Sedimentation Rate is the rate of sedimentation of red cells in the first 50minutes. This will happen when anticoagulated whole blood is allowed to stand for a period of time, which the red cells will settle from the plasma. The increase in ESR shows indicates some abnormalities in the body.
Sedimentation of the red cells actually occurs in 3 stages.
1) Preliminary stage: In the first few minutes, rouleaux occurs and aggregates forms.
2) The sinking period: This is where the sinking takes place at a constant speed.
3) Last stage: This is where the rate of sedimentation slows which is due to the aggregates settling at the bottom of the tube.
Materials
Ø Aquisel tube with stopper filled with 0.8ml of Trisodic Citrate. (Available commercially)
Ø Pipette (for ESR)
Ø Rack/Aquisel tube styoform holder
Ø Mechanical pipette
Ø Pipette tips
Ø Timer
Methods
1. The EDTA blood samples sent to the lab should be mix well.
2. 320µl of blood were pipetted from the EDTA sample to an Aquisel tube containing Trisodic Citrate.
3. The Aquisel tube is mixed well.
4. The pipette is introduced into the Aquisel tube. The pipette is pushed downwards until the blood fills the whole pipette. The blood level should be at “0” on the markings of the pipette.
5. The timer is set to 50minutes and time starts.
6. A small LIS generated sticker is to be stick over the Aquisel tube.
7. Another small LIS generated sticker is to be pasted on the ESR record book. (For record purposes)
8. A “miscellaneous testing” is stamped on the request form and initiated.
9. After 50minutes exactly, the values on the pipette is read and results are taken down in the ESR record book and the request form, which is also to be signed for traceability.
10. The results are to be entered and verified manually in the LIS system.
Reference value
Neonates 1 to 2 days old : 0 to 4mm/50minutes
Neonates 3 days to 1 month old : 0 to 10mm/50minutes
Paediatrics 2months to 12years old :0 to 10mm/50minutes
Adults more than 12years old :0 to 10mm/50minutes
IF the ESR values are higher than the reference value, it can means:
Ø Increase in fibrinogen and/or globulin
Ø Decrease in albumin concentration
Ø Decease in cell pack volume
Ø Tuberculosis, Rheumatic fever, kidney disorder, systemic Lupus Erythematosus (SLE)
Ø Anemia
*Things to take note
The specimen tubes should be checked, there should not be any blood clots or platelet clumps, if there are clumps present, the results would not be vaild. The test should also be done in 4 hrs after the blood collection as the red cells would become spherical after that and it affects the results. The ESR pipette should be left standing on a rack upright, if there is any tilting, it accelerates the ESR. If the blood is lysed, the results will also have error.
That's all!
Zhenling
TG02
=)
11:46 AM
Tuesday, November 4, 2008
Hey guys, this is my last posting for our SIP.
I was posted to Histopathology lab for 2 weeks and everyday was packed with things to be done due to low manpower. Anyway, in the lab everyone has to be well rounded in all aspects such as immunology, sectioning, trimming/passing and so on.
The day starts with blocking/embedding of fixed specimens into paraffin wax that were prepared overnight. Next, the lab technicians will shave the blocks and immersed them in softeners for around ten minutes to achieve a smoother and softer cut to get the ribbons for fixing onto the slides. (Note that we have learnt these during pathology so I won’t go in detail, but you guys can ask questions about it)
The most interesting task that I look forward to everyday would be the passing part in the afternoon when we will retrieve all the specimens for the day and sort them accordingly. Passing is done by lab technicians whereas trimming is done by pathologists and the dictations are assisted by the technician. The difference is that the pathologists trim/section the larger kinds of specimens such as colon, breast tissue, stomach, etc and frozen section specimens (specimens received from the operating theater) whereas the technicians will pass specimens such as breast lumps, appendix, prostatic cores, etc. In our lab, appendix, intestines and breast tissues are common and it is quite rare to receive specimens such as aborted fetus, nose (however we did received it for the first time 3 weeks ago), leg, etc. I helped out daily with the dictation part by writing the measurements of the specimens received, their description and comments given by the technicians.
The thing that I would like to further touch on would be the retrieval of prostatic cores.
It is done through needle biopsies for the diagnosis of prostate cancer by the pathologist. A minimum of 8 cores are required and it is made up of 4 from each side of the prostate gland and the 4 cores are from the apex, mid, periurethral and base. The whole procedure is done using a biopsy gun which is inserted through the wall of the rectum into the prostate gland area. I’m not clear of the whole procedure as I don’t get to see it.
Next, during passing of the prostate cores, the length are noted and inked blue before being wrapped in a filter paper and place into a cassette for fixing. Note that the cassettes are to be placed in formalin as soon as possible to prevent the cores from drying causing shrinkage leading to inaccurate diagnosis.
I was posted to Histopathology lab for 2 weeks and everyday was packed with things to be done due to low manpower. Anyway, in the lab everyone has to be well rounded in all aspects such as immunology, sectioning, trimming/passing and so on.
The day starts with blocking/embedding of fixed specimens into paraffin wax that were prepared overnight. Next, the lab technicians will shave the blocks and immersed them in softeners for around ten minutes to achieve a smoother and softer cut to get the ribbons for fixing onto the slides. (Note that we have learnt these during pathology so I won’t go in detail, but you guys can ask questions about it)
The most interesting task that I look forward to everyday would be the passing part in the afternoon when we will retrieve all the specimens for the day and sort them accordingly. Passing is done by lab technicians whereas trimming is done by pathologists and the dictations are assisted by the technician. The difference is that the pathologists trim/section the larger kinds of specimens such as colon, breast tissue, stomach, etc and frozen section specimens (specimens received from the operating theater) whereas the technicians will pass specimens such as breast lumps, appendix, prostatic cores, etc. In our lab, appendix, intestines and breast tissues are common and it is quite rare to receive specimens such as aborted fetus, nose (however we did received it for the first time 3 weeks ago), leg, etc. I helped out daily with the dictation part by writing the measurements of the specimens received, their description and comments given by the technicians.
The thing that I would like to further touch on would be the retrieval of prostatic cores.
It is done through needle biopsies for the diagnosis of prostate cancer by the pathologist. A minimum of 8 cores are required and it is made up of 4 from each side of the prostate gland and the 4 cores are from the apex, mid, periurethral and base. The whole procedure is done using a biopsy gun which is inserted through the wall of the rectum into the prostate gland area. I’m not clear of the whole procedure as I don’t get to see it.
Next, during passing of the prostate cores, the length are noted and inked blue before being wrapped in a filter paper and place into a cassette for fixing. Note that the cassettes are to be placed in formalin as soon as possible to prevent the cores from drying causing shrinkage leading to inaccurate diagnosis.
Photos retrieved from http://www.upmccancercenters.com/cancer/prostate/biopsyneedle.html
These are examples of stained prostate core slides:
Retrieved from http://www.hopkinsmedicine.org/hmn/F02/feature2.html
Yup, that is all, I’m not sure are the information sufficient cause in the histology lab, although I get to observe the procedure of the immunology staining process, but we are not allowed to do anything and there is not much learnt there. =)
Debbie
TG02
=)
3:31 PM
Sunday, November 2, 2008
This will be my last post.1 more week and internship is completed. I am still in chemistry lab (or to be more specific is chromatography lab). Last few posts have shared thin layer chromatography and column chromatography. This post will be on liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS is the last step that will be carried out for my MP. It is used for chemical analysis of samples.
Chromatography means separation of components of sample based on chemical or physical properties of sorbent. (e.g. hydrophobic interaction, hydrogen bonding etc)
(Image of LC-MS/MS: taken from www.agilent.com)What is LC-MS/MS?
LC-MS/MS instrument consisted of high performance liquid chromatography (HPLC) coupled to mass spectrometer (MS). HPLC will separate the sample and detect by UV and MS carry out mass analysis.
Since LC-MS/MS consists of 2 part, HPLC and MS, I shall explain them separately.
HPLC
HPLC has an injector that will inject the sample into the column.
A pump then force sample through column.
It is based on the same principle as column chromatography (mentioned in my second post). The difference is that in column chromatography, the sample flows through sorbent by gravity and is more manual while in HPLC, the sample is forced through the column by pressure and is more automated.
After separation of sample, the components are detected by UV detector of HPLC. Different components can be detected at different UV wavelength. Therefore, it is important to set the UV detection at a wavelength which the analyte of interest can be detected. Other than UV detection, retention time also differs for different compounds since different compounds have different affinity for the sorbent. So, different compounds remain in sorbent for different duration.
Sorbent (stationary phase) often used are reversed phase and normal phase. An e.g. of sorbent for reversed phase is C18 and e.g. of sorbent used for normal phase is silica gel. In normal phase, compounds are eluted in increasing polarity (i.e. least polar to most polar) while reversed phase is vise versa. Of course, elution of compounds depends a lot on mobile phase used. (e.g. methanol, acetonitrile water etc)
As mentioned in alex’s post (http://codec-5.blogspot.com/2008/07/attachment-experience-from-weeks-i-ii.html), parameters that affect results can include injection volume, stationary phase, mobile phase, UV wavelength, flow rate, column diameter etc.
(Example of HPLC chromatogram: Image taken fromhttp://www.ualberta.ca)
X-axis is the time and Y axis is absorbance (AU). Absorbance is proportional with the concentration. Each peak represents one fraction.
The above is just explanation on HPLC…..now is moving on to mass spectrometry instrument.
MS
Why need mass spectrometry?
UV detection can only detect compounds with double bonds or ring structure. Compounds without those properties will not be detected by UV. Thus, mass spectrometry is needed since all compounds have mass.
Why need mass spectrometry?
UV detection can only detect compounds with double bonds or ring structure. Compounds without those properties will not be detected by UV. Thus, mass spectrometry is needed since all compounds have mass.
Mass spectrometry instrument consists of a ion source, mass analyzer and mass detector.
Ionisation is required to forms ions for detection by mass spectrometry. Two main ionization modes that are often used are the electrospray ionization (ESI) and the atmospheric pressure chemical ionization (APCI).
Mass analyzers will store the ions and eject them through mass filter to be detected by mass detector. The ions are ejected by varying voltage.
(will not be elaborating more on this part since lots of physics (voltage, direct current (DC), alternating current (AC) etc) is involved and the entry will become several times longer)
A mass spectrum shows the fragmentation pattern of the analyte.i.e it shows the quantity of ions at different mass to charge (m/z) ratio. By comparing mass spectrum of sample with a database containing mass spectrums of thouasands of compounds….the identity of the compound in a sample may be figured out.
Image of a mass spectrum. Taken from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htm
X-axis is the m/z value and y-axis is the abundance.
X-axis is the m/z value and y-axis is the abundance.
Lim Xin Ni
TG02
0607325H
=)
9:56 PM