heya.......first and foremost i would like to wish all muslim frenz SELAMAT HARI RAYA.......dun forget to collect as many green packets as possible..it might be our last chance...hehehe....aniwei this week i would post something that was shown to me on the 1st day of work......ISOLATION OF PLASMA AND WBC..yah i noe..a bit outdated....
Now2 dun get confuse...yes i am under research but my lab is also involve in clinical stuff....soo yeah it's a mixture of both...so don't get a shock if u hear people in my lab go...quick sequence this first....the person is waiting for drugs to be administered......so back to the story.....
This whole thing is called blood processing and it is done by the research officer...we attachment students are not allowed to do this because our subsequent experiments will depend on this and if we screw up the blood processing....we are soo dead....we can't possibly ask the kind subject who 'donated' his/her blood to us to 'donate' again can we......
Phlebotomist will help us to draw blood from the volunteers (healthy individuals/patients that agreed to take part in our research). Then our counterpart in the clinical trials dept will then deliver the blood to us (the blood is collected in 3 EDTA tube-purple cap). Depending on what study the blood is for....the apropriate components will be isolated.....it is usually plasma, WBC and DNA..unfortunately i'm unable to post up on DNA isolation because we are not shown on tt....perhaps this wk they'll show us........well they have been promising to show us but the time has not come yet......now..after the delivery of blood......the show begins....
A) Isolation of plasma
1. Centrifuge the 3 tubes of blood at 2000 rpm for 10 min
-this separates the plasma from the other blood components
2. Pipette the supernatant (plasma) into the 1.5ml eppendorf tubes.
-pipette as much supernatant as possible but do not disturb the pellet
3. Store the eppendorf tubes at -80oC fridge
**the plasma is used for HPLC experiment to determine the drug level before, during and after infusion to study the drug pharmacodynamics and kinetics. thus plasma is usually collected for studies that involve drugs (usually in diseased subjects/patients to see their response)
B) Isolation of WBC
1.Transfer 3ml of the pellet (from isolation of plasma) into 15 ml tubes
-the pellet is actually RBC
2. Add 9ml of RBC lysis solution to the tubes
-this forms 1:3 blood to lysis solution ratio
3. Incubate the tubes at room temperature for 10 min
4. Centrifuge the tubes at 2000 rpm at 25oC for 10 min
5. Pour away the supernatant
6. Add 1ml of RBC lysis solution and pipette up and down
7. Transfer everything from the tubes to eppendorf tubes
8.Centrifuge the tubes at 3000rpm for 3min at 4oC
9.Centrifuge at 15000rpm for 5 min at 4oC
Discard the supernatant
11. Store the pellet (WBC) at -80oC fridge
**this WBC will be used to isolate the DNA...soo die2 WBC will be collected for any studies since our lab is a very DNA lab....
tt's all for now......unfortunately this is my last post...left 3 more wks of SIP......enjoy it okies and let's fight till the end..............wishing everybody...ALL THE BEST FOR OUR SIP/MP......:D
nur zahirah tg02
Subscribe to:
Post Comments (Atom)
14 comments:
Hey
In the isolation of plasma, u stated that HPLC is used to determine the drug level before, during and after infusion to study the drug pharmacodynamics and kinetics...care to explian the principles of HPLC? Thanks
Eugene Wong
TG02
hello
what is the RBC lysis solution used ?? COz we do have a solution that causes the lysis of RBC..(sodium citrate). Wat is the rbs lysis solution u ppl use in ur lab?
Vino
TG02
Hi there!
How does RBC lysis solution targets only RBC? Whats the mechanism behind it?
Thank you
Royston
TG01
Hi!
I would also like to noe abt the HPLC that u mentioned. Is it just peaks that u will be seeing?
Thank you!
Charmaine
TG01
Hi unidentified,
Are you attached to some sort of cancer research labs? It seems that there stuff you are doing sound like you are there. hehe.
The lab I'm attached to performs blood processing too. Does your lab extract WBC using the buffy coat method too? hmm
Is the step 6 of WBC isolation to make sure that all RBC are lysed?
Why is centrifugation performed twice at 3000 and 15 000 rpm for the isolation of WBC? ( Step 8 and 9)
Loh Sharon, tg 01
hey,
you said that the blood is from known volunteers and doing research on drugs, right? or your lab uses diseased volunteers? i'm confused.
elaine
Eh sorry ah, but I suddenly noticed vino's comment. So I have to ask vino. Sodium Citrate causes Lysis of RBC?? Are you sure? ISnt sodium citrate a type of anticoagulant? Blue-cap tube?
Zahirah, slamat hari raya! hahaha.
Sharifah
Tg01
heya sories that i took quite some time to ans ur qn coz this part is not handled by me...n my research officers are in a stress situation rite now coz they haf to churn reports 4 the big boss...soo i'll try to ans which qn i can ans 1st ok....for the rest..i'll try to ask my officer if the time permits.....
zahirah
To: Eugene
HPLC involves liquid mobile and stationary phases. The mobile phase is usually a buffer to which a polarity modifier is added. The stationary phase consists of a stainless steel column of silica gel that is bonded to a nonpolar liquid. The stationary phase is less polar than the mobile phase, causing compounds which are lower in polarity to be retained longer than more polar molecules. The column is packed very tightly allow partitioning. Therefore, a pump is used to move the mobile phase through the column. The separated molecules enter an optical flowcell after eluting from the column. UV (ultra-violet) light is passed through the flowcell and a photo-multiplier tube or photodiode array detects the transmitted light. When a drug enters the flowcell it will absorb a portion of the incident UV light causing an increase in absorbance (optical density). This signal is applied to a chart recorder, which produces a peak that is proportional in height and area to the concentration of the drug. This peak will then be used to determine the level of the drug present in the plasma at varying time of the day depending on the study we are doing
To: Vino
like sharifah..me also wondering..isn't sodium citrate an anti-coagulant..it chelates the calcium ions in the blood, disrupting the blood clotting mechanism.....we use gentra puregene cell kit..from qiagen..the RNC lysis is in the kit...i'll try to find out wat the components are for u....
To: Royston
hmm...i'm also not sure how it targets only the RBC..perhaps it has something to do wif ion charge that attracts the lysis solution to RBC..and then disrupts the RBC through osmotic tension...
To: Charmaine
yupz...it's from the peaks the drug concentration will be determined..then we will plot this box looking thing (something like graph) and see their relations (drug, pk and time)....
To: Sharon
hmm..i dun think we use the buffy coat method...
yupz..step 6 is to ensure that all RBC is lysed...
1st centrifuge step is actually to allow a slow spin to make the WBC slowly settle down at the bottom of the tube and to prevent them from sudden shock...after they have sort off accustomed to spinning...then we can increase the spin to increase the rate of the separation (pellet and supernatant).
To: elaine
we work with both healthy and diseased individuals....it depends on the stage of the project...if the project is new (like wat i did for my MP)..then we need to work with healthy individuals 1st..this means that we just need their DNA to screen for any mutations that occur at our gene of interest in these healthy individuals to see their frequencies....if the project seems feasible..only do we proceed to patients...and it's from this patients will we study about how they respond to the drug that we might want to study used to target our gene....it's a step by step research and that's y our projects usually take 3-4 yrs to complete....
Post a Comment