Centrifugation and Aliquoting of Blood Serum and Plasma
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- Опубликовано: 1 сен 2017
- This video shows Dr. Evan Matthews explaining how to centrifuge blood samples and use transfer pipettes to remove the serum or plasma for immediate use or storage.
Link to video showing how to use transfer and precision pipettes.
• Pipetting Skills
Link to video showing how to use a microcentrifuge.
• Microcentrifuge
Link to Dr. Evan Matthews website.
sites.google.com/site/evanmat...
I attempt to make the most accurate material I can. However, mistakes happen. Below is a list of mistakes identified in this video.
Known errors in this video:
At 1:34 I state that plasma still contains platelets after centrifugation. This is generally not true. The platelets settle into the thin white "buffy coat" that also contains the white blood cells. You can alter the process to create a platelet rich plasma. I have links below on how that works.
See my error:
• Centrifugation and Ali...
How to make platelet rich plasma:
www.ncbi.nlm.nih.gov/pmc/arti...
www.hindawi.com/journals/isrn...
Discussion of the buffy coat:
onlinelibrary.wiley.com/doi/a... 
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Amazing demonstration and the principle of each and every process took part. This video serves as a great help for my project!! Thanks a lot! I was desperately searching for such videos. Keep posting various laboratory techniques that are highly utilized.
Thanks. I'm glad this helped.
Thank you. Very helpful and easy to understand.
Thank you very much for creating this video. It provides valuable information and details regarding this process. Thank you for showing how the machine works and the detailed steps necessary to accomplish this lab test. Thanks.
You are welcome!
This was *very* helpful. I breed border collies via AI and with the crazy prices of progesterone tests have decided to do my own draws and send the samples out to university vet lab. My vet tech friends showed me how to do the draw [butterflies much easier than TB tubes]. This told me how to process them before sending them out. I can do my own tests for 1/3 cost of commercial/vet labs, including shipping.
Great explanation.
I was just searching this experiment for our biology lesson and finally got it.Thanks a lot
I'm glad it helped.
I need to look at serum samples in graduate research. This was very helpful. Thank you!
I'm glad it helped!
thank you ! not even my teacher knew what to do and you saved my master degree thesis
I'm glad it helped!
(:
@@thunder440v3 :)
Great video! Super clear explanation. Thanks 🙏🏻
I'm glad it helped.
This is really good! I am a certified medical assistant and just brushing up on my skills and this video is very accurate! Thanks :)
I'm glad it helped.
incredibly helpful and right to the point. Thank you.
You are welcome.
really awesome and easy to understand thankz
Wow!!! That really helps, thank you so much❤️
I'm glad it was helpful.
Completely understood...Thank you so much!
I'm glad it helped.
Thnks sir its really helpfull video in hematology
Great video thank you for posting this!
You are welcome!
Thanks doc
Very nicely explained 🙂 thanks for sharing
You are welcome!
omg increible video ;)
كررها معي
١-استغفرالله 🌿
٢-الحمدلله🌹
٣-الله اكبر🌼
٤-سبحان الله🌻
٥-لاحول ولاقوة الا بالله 🌺
٦-لا اله الا الله🌿
٧-اللهم صًلِ علـّۓ. محمد وال محمد🍃
اجر لي ولك🥰.
thanks a lot sir
Thankyou so much it really helped
I'm happy it helped.
WELL, DETAILED VIDEO THANKS FOR THIS
You are welcome.
Hi, Great video 👍🏽 wanted to refresh on extracting prp, when using yellow top tubes with ACD solution is it best to spin for 5 minutes at 4000rpm, remove plasma and spin again for 5 min to achieve cloudy prp at the bottom or just spin for 10 minutes at 4000rpm and extract prp from bottom ? Which of the two methods would produce a more obvious cloudy separation of prp ? thank you in advance ☺️
Nice video, i am Dr vet trying to refresh after decades doing administrative, a little correction 6:50 platelets are spun down in EDTA tubes to cell portion (they lie between RBC's and the buffy coat and if spun correctly it be none in plasma, the difference in these two show on a quick assessment using field refractometar and it is about 7 percent fibrinogen of total (TP) plus 1 percent coags of total (TP) captured in coagulum so if the total blood protein (TP) human plasma is e.g 7.5 g/dl it will be 6.9 g/dl for the same blood sample using serum.
Thank you. This is a known error in this video. I attempted to provide some information on this error in the description.
@@VivoPhys ...and i made two corrections on my comment, huh now it looks much better...
Thus, in plasma, when blood is collected for anticoagulant, there will be no platelets under appropriate centrifugation conditions, but there will still be inactive coagulation factors (proteins of the blood coagulation system, including fibrinogen). When collected on a clot, not only platelets clot, but also fibrin from fibrinogen after activation of the coagulation factors, so the serum contains neither platelets nor fibrinogen.
So good explain
I'm glad it helped.
Really helpful 👍
I'm happy it helped.
Thank you ❤️
You are welcome.
Thank you!
You are welcome.
So informative 😊🙏
Thank you 😊🌄🌱
God bless you..
I'm happy you found it helpful. Please explore some of my other videos.
@@VivoPhys woah... Just now I checked out your channel (the content list!) I must say, bandwidth of content is quite extensive.. You are so knowledgeable person. it really gonna be so helpful. I am definitely going to watch them. Thanks
Stay safe 😊🙏
Sending peace n good vibes from India 🕊️🌱
Good vibes from New Jersey USA!
@@VivoPhys 😊🌱🙏
Thankyou so much sir ✨
You are welcome.
Thank you . Good video i can development my work.
You are welcome.
Thanks so much
I'm glad it helped.
figure out a way to get concentrations of the platelets for PHP. that would be very usefull
Saved my life 😌
I'm glad it helped.
Thank you for your video. Could you tell me how much time we have from blood collection to centrifugation in order to have good plasma results? I read somewhere that I shouldn't pass 4h but in my case, it is hard since the collection place is far from the research lab. Thanks
It's hard to put a firm number on it. Itty depends on the tests you plan on doing. Some things degrade faster than others.
Thanks
You are welcome.
What happens when prp injections is done without the separation of the blood?
thanks sir g verry easy ....
Thank you!
Thx sir
Thanks for the video, i want to prepare blood sample for RIA analysis of thyroid hormones.. What is the proper sample should i prepapare, serum or plasma? and what temperature should i use for storage?
You would need to check the assay kit you use for those specifics.
So Evan, after the PP plasma is extracted with the first centrifuge-spin, it can then be spun for the second time to get the PRP, right? If so, before doing that,
1. To what type of tube - or what color cap tube - should the PPP be transfer to for a second spin, and,
2. Would the same speed/time be applied to extract the PRP with the second spin? Thank you!!!
Sorry, I'm not familiar with the methods you're referring to. Hopefully someone else will reply with a better answer.
Very informative Can you recommend spin time and rpm speed protocol for Platelet Rich Fibrin (PRF) for injectable facial aesthetics.
I have no idea. Sorry.
Thank you for the great video. Out of curiosity what is the reasoning behind doing a hematocrit with the centrifuge on blood and not the mcht setting?
I'm not familiar with the setting you are referring to.
Awesome n easy to understand.I want to ask one thing.If I dont have a refrigerator to store the serum and i dont have the eliquots too,can i take the serum directly from the tube ( with serum on top and cells at bottom after centrifugation).And in how much minimum time should i process for testing with serum ,once it is seperated??
It all depends on the type of analysis. Sometimes it is ok to sample the serum directly from the tube, but you must be careful not to disrupt the cells or mix the sample. I'm guessing most analyses should be complete in an hour or less.
thanks!!
I hope it helped.
Hi I am using heparin tube for the rat blood. Since I m decapitating rat and collection blood from trunk without anesthesia. So collected blood in a falcon tube I shall darwback into vacutainer for serum and plasms both. My question will the blood be fine and won't contaminate or oxidoze by this method?
It depends on what you are trying to measure in the blood. Your best bet would be to contact the company that makes your assay kit or machine. However, if the blood is exposed to the air then it is also exposed to oxygen.
Could you explain or demonstrate what happens to the buffy coat when using a Plasma Separator Tube with gel?
Sorry I'm not sure I'm familiar with what you are talking about. Hopefully someone else can respond to help you.
Thanks for responding so quickly. I'm a Phlebotomy student and we are learning about the different tubes & additives. I need to draw a diagram of a Li Heparin Plasma Separator Tube (PST) with gel added to form a barrier after being spun down. I searched google images & think I found what I'm looking for.
Great video. Thanks. So how long typically do we have for the centrifugation step after blood collection?
I'd say 5 to 10 minutes but it depends on sample type and intended analysis.
it's a same procedure for all types of immunoglobulins
Good very good
Thank you! What kind of tube is adequate for holding the plasma? What tube does the plasma get transferred into? Any additives?
Heparin tubes are common, but you should check with whatever company performs your analysis or makes your analysis kits/machine.
Thank you for this!
I hope it helped.
Question. If you invert the tubes after the machine, can you put it again in the machine or not?
You can but you run the risk of hemolysing the blood cells.
what if the sample looks yellow on bottom ???
How do I separate plasma from frozen anticoagulated blood?
Can I use this plasma for skin Prp
is it right if i spin the sample at 5000 rpm and i want to measure antioxidant enzymes?
I don't know. I have not done that assessment before.
Thanks for the informative video!
What is the proper rpm and spin time for PRP for microneedling? I will be using tiger tops and I just need the plasma. Thanks!
Sorry but I'm not sure. It is best to consult the manual for the test you are running. Hopefully someone else will respond who is familiar with what you are doing.
Does all tubes need to be centrifuged or just the ones that have plasma & serum?
If you want the blood to separate then you must centrifuge it. If you want whole blood like when doing hemoglobin assessments then you should not centrifuge it.
When transferring serum to a transfer tube, Can I pour the serum from one tube to the other or do I need to pipette? If serum is poured, what could happen?
You need to pipette it. Otherwise you will likely mix you sample. This is especially true in a anti coagulant tube.
What do you do with the separated serum?
That what the lab will be testing
hello dear doctor.
how I can obtain buffy coat layer more specifically. then when I took the buffy coat layer, how long time I can store my separated layer. for example, can I store it for about 6 months or 12 months because I don't have a plan to perform my work directly on the sample when I collected it?
thanks for your response.
kind regard
Diyari.
Sorry. I don't have experience assessing the buffy coat. Hopefully someone else will respond.
@@VivoPhys thanks so much.
That was great
I hope it helped.
@@VivoPhys it did thank you!
Sooooo my question is can both tubes .. glass / plastic give you prp ?????
Sir,I want to extract only the pigment that is heme from blood.If you please tell me how can I do that..
I do not know. Sorry.
Is that serum Cholesterol ? Yellow
I centrifuge EDTA tube for 15mins at 1.2rcf after collection...there were small red cell particles in the plasma after centrifuging, anyone know why?
You centrifuged for two long or you didn’t centrifuge straight away
Which coloured tube must we use to collect blood samples for toxicology testing? Is it purple or red?
Sorry. I am not sure. Hopefully someone else will respond. If you find this information elsewhere please respond here so others can find it.
What gage needle is best for centrifugal blood for plasma
It's not crucial. Too small can cause hemolysis, but that can happen for other reasons too. I typically go between 18-22g depending on the size of there veins.
I'm thinking I'm more of a visual learner. and working in a pharmacy with people that take their job seriously can be hurtful. it's hard to get trained, when a starter and you don't possess enough experience. thank you sincerely
I hope this is helpful.
is red blood fluid not used for examination?, is it only plasma/serum used?
Whole blood is used for some tests like hemoglobin and hematocrit.
Can this be use to treat a herniated disc via injection
I have no idea. Sorry.
Is it ok to centrifuge the sample again when you accidentally mixed the sample after centrifuging it?
I have done it when necessary, but you run the risk of hemolysis which can alter the values. I would add a comment to the data in case the values are odd.
At 6:50, the anticoagulant tube has plasma "with unused coagulation factors" and platelets. Clot formation inside a tube is caused by fibrinogen turning into fibrin (not platelets) by action of thrombin and initially by the contact with the glass from the tube (silica).
Hi Omar Lemus. Thank you for contributing to the conversation on this. Please see the description section. This error has been discussed previously, and I list it as a known error within the video.
So the purple top gives serum and red gives plasma...where do you purchase these tubes?
They can be purchased from medical supply companies. I'm not sure if the color coding is universal across companies.
Is it safe to extract blood from the great saphenous vein?
I have no experience with that vein. So I do not know.
Just amazing sir...
😊😆
I'm glad it helped!
@@VivoPhys so nice of you sir.. Thanks for replying to me...
😊😊
FOR PRP....DOES THE TUBE HAVE TO HAVE ANTICOAGULANT AND GEL IN IT....I PREFER TO DRAW THE BLOOD INTO A STERILE CLEAR GLASS TEST TUBE WITH NO ADDITIVES...10 ML TUBES...WILL IT CLOT OR WOULD I STILL BE ABLE TO GET PRP FROM IT
Please define PRP.
PLATELET RICH PLASMA
I'm not sure. Hopefully someone else will respond. Sorry.
To get PLASMA, the tube must have an anticoagulant. If it does not have an anticoagulant, it is called SERUM.
How long can be stored the uncoagulated blood sample for serum? And doe freezing effect the blood?
Thanks
Ideally you should centrifuge the blood and remove the serum immediately. Then the isolated serum can be stored or frozen for later analysis. Keeping the blood cells in the sample you run the risk of hemolysis.
@@VivoPhys Thank you sir. How long can I hold for the blood sample to separate from the serum? Can I centrifuge the sample immediately.?
I can't say for sure but you can probably hold onto the blood for an hour maybe even 2 hours before centrifuging. If you are attempting to get serum (not plasma) then you need to wait until the blood is coagulated. That usually takes about 10-15 minutes.
Just wondering...does anyone know how to isolate a hormone from the serum/plasma?
Im sorry.. Im still im school as a medical assistant and i was wondering when im gonna separate a plasma from the blood what are the lab names i would need to know.. I dont know if i make since
There are several tests that may require it. It would be hard to give a list. Sorry.
@@VivoPhys its okay..
dumb q, but why is it soooo bad to get even the tiniest little bit of red blood in with the plasma/disturb the cells? Does it completely ruin your plasma?
Hi Kimi O. I do not know how much red blood cell in your plasma sample is "too much", but I would assume you are correct that it does not take much. The reason is the contents of the cytosol in the red blood cell is different from the contents of the plasma/serum. So if you get red blood cells into your plasma/serum sample they will likely hemolyze (burst) releasing their content. This can dramatically change the level of some things your attempting to measure like plasma/serum potassium. So you will get an inaccurate result.
@@VivoPhys thanks so much!!
Cool now I can order me a centrifuge off Ali Express and do my own prapius shots have been forking out $1500 a shot
Is it working though?
That's what I did
what tubes did the clinic use to extract your PRP? Which ones will you use?
I have a question. How and why does the silicone coated tube promote blood coagulation? is it a chemical process? Thanks doing a school report on blood centrifugation.
According to BD's website: "BD Vacutainer Plus plastic serum and SST tubes are coated with silicone and micronized silica particles to accelerate clotting. A silicone coating reduces adherence of red cells to tube walls. The silica coating can sometimes cause the inner tube wall to appear cloudy and/or filmy. This cloudy appearance does not make the tubes unacceptable for use."
www.bd.com/en-us/offerings/capabilities/specimen-collection/blood-collection/venous-collection/bd-vacutainer-blood-collection-tubes/vacutainer-blood-collection-tube-faq/general-tubes-faq
How exactly the micronized silica particles cause platelet aggregation and clotting is unclear to me but I found this: "It was reported that SiNPs penetrated the platelet plasma membrane and stimulated a rapid and prolonged NO release, IIbIIIa activation, and finally the platelet aggregation [25]."
www.hindawi.com/journals/bri/2016/2959414/
That's a really nice centrifuge! How much does it cost?
I'm not real sure. Probably a few thousand dollars.
@@VivoPhys Do you know what model it is?
I don't. Unfortunately I've been working from home due to COVID-19 so I'm unable to check to. Sorry. Any centrifuge with cooling capabilities probably works as well as this one. This was but used. So it is not a new model.
If the nurse forget to turn hiv blood sample upside down 5 times will the result come out as false?
Hi Nɪᴋɪᴛᴀ Rɪᴄᴇ. I do not know the process of testing blood for HIV. However, typically inverting the sample is just to mix the chemicals in the tube with the blood to either promote or inhibit clotting. I cannot imagine that it would cause an error in the test. At worst, it would probably just ruin the blood so it could not be tested at all.
I came here to learn about centrifusion of blood for a fanfic I'm making, and I stayed for how cool this shit is.
I hope this helped.
@@VivoPhys I'd did! :]
Thanku bro
I'm glad it helped.
I liked it video very much
Keep posting
@@Yogaabhyash Thank you. I try my best to create and upload content when my day job is not demanding. With the current COVID-19 pandemic I am much busier than normal. I will start posting again as soon as I can. Stay safe!
How does the plasma actually seperate from the blood?
I spent some time looking this up and found a lot of poor explanations out there. Hopefully someone can double check my answer to be sure it is accurate and maybe give a better one. Regardless, here is my best attempt to explain (and understand) this. The centrifuge tube holders apply a horizontal force on the tube to make it move. Because the tube is attached to a circular object that can only spin (assuming it does not break) there is a simultaneously applied force of the tube holder pushing the blood tube towards the center of the centrifuge preventing the tubes from flying away. This force is called centripetal force. Applying these two forces simultaneously causes the tubes to spin instead of moving in a straight horizontal line. Remember that inertia = mass x acceleration (in this situation the blood tube is constantly accelerating from the forces already described or it would not go in a circle). All parts of the blood have a similar acceleration because the whole tube is moving as a unit. Keep in mind the tubes of blood are spinning with the bottom of the tube facing towards the outside of the circle, and blood is liquid (plasma) with blood cells suspended throughout. The components of the blood (e.g. red blood cells) have more mass than the liquid portion (plasma). Going back to the inertia equation, the key difference between the various blood components is there mass not their acceleration. Therefore, the more mass they have the more inertia they have. So as they accelerate towards the center of the centrifuge the components of blood with more mass (more inertia) end up migrating towards the outside of the centrifuge (bottom of the tube). This is because they are more resistant to that acceleration (i.e. more inertia) than the less massive portion of the blood.
Nectar
You didn´t counterweight the load in the rotor. You´ll end up breaking it.
Counter weighting is important.
Ģrasias x Compartir
Hello what color tubes can you Not centrifuge
To my knowledge you can centrifuge any tubes. However what you can pipette out after is different depending on the type of tube you use.
You should confirm with the manufacturer.
@@VivoPhys so for example you can Centrifuge a Lavender? im asking because is whole,blood and i thought you couldn't centrifuge it 😐 i thought you can only centrifuge plasma or serum .
Centrifuging whole blood is how you separate it into serum or plasma.
@@zuley6957 we are not suppose to centrifuge whole blood.
Do doctors perform this job or nurses?
To be clear. I am not a medical doctor. I have a PhD. Drawing blood and processing it can be done without a degree.
I am in nursing school and so far what I have learned is the nurse takes the blood, sends it to the lab and the lab tech does this. hope that answers ur question! lol i'm like 4 months late responding but just came across this video!
Often, neither doctors or nurses. The person who draws the blood varies between hospitals, and may vary from time to time in the same facility. The blood may be drawn by a certified medical laboratory scientist (MLS, bachelor's degree laboratory professional), a certified medical laboratory technician (MLT, associate's degree medical professional), or a phlebotomist (may be on the job trained, or a certified graduate of a phlebotomy program), or a nurse or physician (limited training in blood collection in nursing or medical school). Centrifuging and separating the blood is done in the laboratory, and again, depending on the facility, the person who does this may be an on the job trained laboratory assistant, may be an MLS or MLT, or a combination. Who does the TESTING of that sample? That will be a college-educated laboratory professional. It may be a certified medical laboratory technician (MLT) with an associate's degree, or a certified Medical Laboratory Scientist (MLS) with a bachelor's degree.
Sir at what RPM the serum gets separated ???????
It varies depending on what you are trying to do but I believe I used around 2300 in this video.
Correction, I used about 3200.
For how much time
I believe it was 10 minutes
sir thoda hindi bhi boldiya kijiye but video is very good
mine didn't seperate it was still red
Couldn't you just put the "ruined" sample back in the centrifuge to re-separate it?
It depends on what you are trying to measure in the sample and how sensitive it is to hemolysis. Each time you spin the sample you run the risk if bursting the blood cells.
Hmm, as a home mechanic guy watching how 2 videos on YT i feel this video is the same thing for the Health workers... 🤔