Apologize for longness!

Hello Mazen,

with respect to the informative relies to your original post:

(also your) questions:

i) which cells from the buffy coat you will collect for extraction in the future?

ii) which method you are thinking to use for retrieval of cells and

iii) how will you store these cells then for / until future DNA extraction?

Hints (only as an electron microscopist, having done a lot of buffy coat preps myself):

i) as Jeffrey and Susan say, you need a collecting system providing anticoagulant (heparin, citrate or EDTA).

The best of these 3 IMHO has been citrate, second best EDTA coating of the inner wall.

ii) the collecting tubes should not contain small glass globes (as –at least in former times – a sort of blood collecting tubes contained these) since in the end glass beads/balls interfere with the production of an optimal buffy coat.

iii) as Susan says: normally – if collection tubes with anti-coagulants are used there should not be a problem for at least the transport and some bench-time left. Important will be: vigorously shaking is no good option, instead, when arriving in your lab, let stand for some minutes at room temperature (RT). Cooling (e.g. down to 4degrC) IMHO is not necessary (Cave: if sent via parcel service “on Ice” sometimes blood collection vessels have been frozen due to misplacing the tubes into ice rather than “on ice”).

iv) The volume of buffy coat layer of “cells” depends on the volume of blood collected. Usually for a buffy coat preparation (at least for EM, visualizing morphologically the layers of serum, platelets, white cells – lymphocytes and white cells – neutrophils, red blood cells [at the bottom]) you should get at least 2 x 4 ml of blood.

Since the interesting “cellular” layer from 1 x 4.0 ml collected blood in collecting tubes unfortunately will be reasonably thin (note: as Jeffrey pointed out: now and then there also will be patients with a low WBC-count! and therefore it will be difficult to prepare&handle the cells after the procedure of producing the buffy coat) I used to "order" 2 x 4 ml of peripheral blood and in the Lab collected those into ONE special made centrifuge glass vial (= narrower diameter than usual centrifugation vials, allowing for centrifuging at least 8 ml of blood in ONE vial.).

v) Right before centrifugation the collecting vials are gently “shaken” (= turned around), in case of the special centrifugation glass vial both collecting tubes are poured carefully into this one. Caution: always balance the weight of the blood containing vial with a second one filled with water, both vials must have exactly the same weight (2 decimals!)! Otherwise you’ll not get a perfect buffy coat anyway!

vi) centrifugation: I always used a swing-out rotor (instead using a fixed angle rotor) with my centrifuge. Independent of and (as such will be communicated even in highly scientific papers) you have to chose a speed of the centrifuge to get 900 – 1000 g (gravity force) for some 5-10 mins (since there will be heating up of the specimen vial, it is recommended to use a refrigerated centrifuge, set to RT or say ). Let the centrifuge run out without using the/a “break” function since such eventually will disturb your delicate buffy coat layers.

After centrifugation take out the centrifuge glass vial very carefully. You’ll end up (top to bottom) with SERUM (yellowish to brown) – a small whitish layer = platelets, followed by the lymphocytes’ layer and the layer of neutrophils, at the bottom RBC-layer (hopefully).

vii) If you want to collect either of the (cellular) layers you can do this by pipetting very slowly and carefully from the respective cell-layer. But in this case you won’t get the whole volume or number of cells in that layer without sucking up also cells/fluid from another layer.

[For EM-Prep. the plasma/serum layer should be removed most cautiously by pipetting, leaving a small volume upon the thrombocytes. Then the layers left guardedly will be overlayed with fresh fixative from pipetting along the inner wall of the glass. How to dislodge and take out the fixed jelly buffy coat layers is another story I can provide but not in this place.]

For your purpose (collecting cells for future DNA extraction) you will not use any fixative, nor you will then extract the fixed jelly buffy coat layers for further processing in EM (i.e. further fixation, dehydration and embedding in resin blocks),

instead, you will have to collect in a way to be able to proceed with regard to the task you intend to do: if your cell fraction has to be you should either use gradient centrifugation (Ficoll, Percoll, Lymphprep, as Jeffrey already proposed, cf. Google: search phrase ).

Best wishes and regards, and good luck Wolfgang

Mazen,

First, I assume you are working with human blood. As such, I would need to know what anti-coagulant is in the red cap vacutainers. I work with animal blood and a red top tube we use has no anti-coagulant. In human hospitals they have many types of tubes. If it is a red top tube like ours, you lose WBC in the clot. Likely not what is happening in your case. I have used heparin, EDTA and citrate to anti-coagulate whole blood for buffy coats. Depends upon what you want to look at.

Human and horse blood sediments rapidly in comparison to other species. I know in healthy horses and likely humans if you leave tube on countertop for 30 minutes or less the RBC fall out leaving plasma. I do not think that leaving for 5-10 minutes should reduce your yield. However, if you are concerned, try inverting tube several times after sitting to resuspend cells and then spin. Should provide adequate buffy coat. Some people, just like horses, cows etc may have low WBC counts so perhaps sometimes your yield will be lower. I would say on average the reduction in yield is not likely due to low WBC count in healthy subjects though.

I think BD makes a vacuatainer tube which has additive that lyses RBC immediately and you spin after lyse cycle providing simple WBC pellet. I believe this system is used primarily for lymphocyte typing, but you should check if your goal is culture or other experimental manipulations.

Alternatively, we have been using 6% dextran in PBS to keep WBC in suspension after centrifugation. For me, this is great as I want to purify neutrophils. For you likely you are looking at lymphocytes, monocytes or stem cells? Depends upon your goal. And there is always the gold standard of gradient centrifugation with percoll or lymphprep, etc.

I hope this is helpful. I am happy to provide recipes/protocols for dextran, etc. if you are interested in neutrophils. They are available on the web if you search.

Best regards,

Jeff Lakritz

Hi Jeffrey. Thanks so much for taking the time to provide me with all those details! Yes we are working with human blood. I will make sure they are using tubes with no anti-coagulant to collect a buffy coat (i have a feeling this is the problem!). Our aim is mainly to collect cells for DNA extraction in the future.

Thanks again,

Mazen

Mazen,

You definitely need to use an anti-coagulant. Otherwise blood clots and cells are trapped in clot.

Hi Mazen,

I use buffy coat preps for electron microscopy and the blood is collected in EDTA or citrate anticoagulant tubes. The blood is often shipped to us from Westmead Kids and Sydney Children's Hospital which sometimes takes a few hours. We have had no problems preparing a buffy coat even after this time period between collection and receipt. Hope this helps. Sue.

Thanks Sue, we have ordered EDTA tubes for plasma and buffy coat collection

Apologize for longness!

Hello Mazen,

with respect to the informative relies to your original post:

(also your) questions:

i) which cells from the buffy coat you will collect for extraction in the future?

ii) which method you are thinking to use for retrieval of cells and

iii) how will you store these cells then for / until future DNA extraction?

Hints (only as an electron microscopist, having done a lot of buffy coat preps myself):

i) as Jeffrey and Susan say, you need a collecting system providing anticoagulant (heparin, citrate or EDTA).

The best of these 3 IMHO has been citrate, second best EDTA coating of the inner wall.

ii) the collecting tubes should not contain small glass globes (as –at least in former times – a sort of blood collecting tubes contained these) since in the end glass beads/balls interfere with the production of an optimal buffy coat.

iii) as Susan says: normally – if collection tubes with anti-coagulants are used there should not be a problem for at least the transport and some bench-time left. Important will be: vigorously shaking is no good option, instead, when arriving in your lab, let stand for some minutes at room temperature (RT). Cooling (e.g. down to 4degrC) IMHO is not necessary (Cave: if sent via parcel service “on Ice” sometimes blood collection vessels have been frozen due to misplacing the tubes into ice rather than “on ice”).

iv) The volume of buffy coat layer of “cells” depends on the volume of blood collected. Usually for a buffy coat preparation (at least for EM, visualizing morphologically the layers of serum, platelets, white cells – lymphocytes and white cells – neutrophils, red blood cells [at the bottom]) you should get at least 2 x 4 ml of blood.

Since the interesting “cellular” layer from 1 x 4.0 ml collected blood in collecting tubes unfortunately will be reasonably thin (note: as Jeffrey pointed out: now and then there also will be patients with a low WBC-count! and therefore it will be difficult to prepare&handle the cells after the procedure of producing the buffy coat) I used to "order" 2 x 4 ml of peripheral blood and in the Lab collected those into ONE special made centrifuge glass vial (= narrower diameter than usual centrifugation vials, allowing for centrifuging at least 8 ml of blood in ONE vial.).

v) Right before centrifugation the collecting vials are gently “shaken” (= turned around), in case of the special centrifugation glass vial both collecting tubes are poured carefully into this one. Caution: always balance the weight of the blood containing vial with a second one filled with water, both vials must have exactly the same weight (2 decimals!)! Otherwise you’ll not get a perfect buffy coat anyway!

vi) centrifugation: I always used a swing-out rotor (instead using a fixed angle rotor) with my centrifuge. Independent of and (as such will be communicated even in highly scientific papers) you have to chose a speed of the centrifuge to get 900 – 1000 g (gravity force) for some 5-10 mins (since there will be heating up of the specimen vial, it is recommended to use a refrigerated centrifuge, set to RT or say ). Let the centrifuge run out without using the/a “break” function since such eventually will disturb your delicate buffy coat layers.

After centrifugation take out the centrifuge glass vial very carefully. You’ll end up (top to bottom) with SERUM (yellowish to brown) – a small whitish layer = platelets, followed by the lymphocytes’ layer and the layer of neutrophils, at the bottom RBC-layer (hopefully).

vii) If you want to collect either of the (cellular) layers you can do this by pipetting very slowly and carefully from the respective cell-layer. But in this case you won’t get the whole volume or number of cells in that layer without sucking up also cells/fluid from another layer.

[For EM-Prep. the plasma/serum layer should be removed most cautiously by pipetting, leaving a small volume upon the thrombocytes. Then the layers left guardedly will be overlayed with fresh fixative from pipetting along the inner wall of the glass. How to dislodge and take out the fixed jelly buffy coat layers is another story I can provide but not in this place.]

For your purpose (collecting cells for future DNA extraction) you will not use any fixative, nor you will then extract the fixed jelly buffy coat layers for further processing in EM (i.e. further fixation, dehydration and embedding in resin blocks),

instead, you will have to collect in a way to be able to proceed with regard to the task you intend to do: if your cell fraction has to be you should either use gradient centrifugation (Ficoll, Percoll, Lymphprep, as Jeffrey already proposed, cf. Google: search phrase ).

Best wishes and regards, and good luck Wolfgang

Hi Wolfgang,

Wow that is a long answer, much appreciated! As I can tell you have learnt a lot from first-hand experience with bloods.

Each of your points are important and will take into account. Our goals are probably not as complicated as you think (i.e. DNA extraction for future epigenetic studies) and so purity is preferred but probably not essential. We will be using EDTA+Aprotinin tubes to collect the plasma layer (for peptide analysis) and buffy coat.

These will be stored initially at -20C and transferred to -80C freezer for long-term storage. I am assuming standard DNA extraction/purification techniques will be used (commercial kits), although I don't think we have gone that far as yet.

Thanks again.

Best regards,

Mazen

Dear Mazen, you're welcome!

All the best,

regards,

Wolfgang

Hi all

I came across this post while researching pathology requirements for a clinical trial. We need to extract buffy coat. We are using a red top serum and grey top tube at most collections. Can buffy coat be collected in either of these? Unfortunately, we have very strict limitations with how much blood we can collect so if we need to use an EDTA, we will be essentially 'wasting' blood just to get buffy coat.

Another question I have is, what tube is needed for gut hormone analysis? Some people say plasma (EDTA) and some say serum (gold/red).

Any advice would be very appreciated!

Hi Mackenzie,

blood collected in Red top serum tubes will coagulate making buffy coat extraction very difficult (we had this problem initially) . I don't have experience with the grey top tubes however they have an anticoagulant and preservative which should work similarly to the EDTA tubes. 

For the gut hormones,  check out the BD P800 vacutainers which were specially developed for gut hormone analysis. Have a look at some assay kit manuals for the analytes of interest as well for blood collection recommendations,  they usually recommend keeping the tubes on ice to minimise degaradation of these sensitive proteins and peptides and quick freezing.

All the best, 

Mazen

Red top tubes coagulate blood and you would not be able to collect white cells from this tube.  EDTA (lavender top), sodium citrate (yellow top) and heparinized (green top) will prevent clotting and allow preparation of buffy coats.  I suggest you scale back volume of each collection and collect 1/3 into grey, 1/3 into red top and 1/3 in either heparin or EDTA tubes.  Conversely, if you could substitute plasma for serum, collect fluoride containing tube and either EDTA or Heparin instead of RTT. In my experience, cells collected with EDTA and Heparin will work, but yellow top tubes (citrate) provide best buffy coat for cell based assays in vitro (cell culture, flow cytometry).  I would want to know the requirements of the cell based assays as these are quite specific now.   

Hi Mackenzie,

It depends if you need your buffy coat to be viable - I can't seem to find if the fluoride in a grey top tube will affect WBC viability, but I would say there is a good chance it might as it is a glycolytic inhibitor and means that blood collected has stable glucose, lactate etc (ie. cells stop respirating). I'm not sure if this effect would be diminished upon washing. Anecdotally, when I spin these tubes 5-10 min after draw I can see a little buffy coat, so the cells are maintained for that long. If you're just hoping for buffy coat for DNA extraction I think it would be OK.

If you NEED buffy coat I'd suggest using EDTA or heparin etc instead of the grey top. Good luck with your trial.

Here's what helps us with buffy coat: We get NHP samples in blood draw tubes with lavender stoppers. These contain EDTA to prevent clotting. Vet techs drawing the blood also need to invert the tubes a few times after the draw to ensure the blood and EDTA mix. Then the tubes are kept under refrigeration: Stored by techs in small 4-degree C fridges. We pick them up in a cooler with an ice pack. They come out of the cooler for a few minutes while we log sample IDs, and then they go into a centrifuge for a spin at 4-degrees C. After the spin, they go into another 4-degree fridge until I take them into a tissue culture hood to process them. When they go into the hood, I have the tubes in rack sitting on a tub of ice. That all does help with clotting. So constant refrigeration is key. Also key is the mixing with the EDTA or some other anticoagulant. Often when we do get clots it can mean the tubes weren't inverted at the time of draw, but as I see a lot of variation in how different samples separate and such, some clotting could be do to the individual NHP we're getting samples from. Some will just be better clotters than others. (Darned, biological variation!)

Another thing I was reading about in terms of buffy coat and DNA recently was in this paper: http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0039821. I was looking at it trying to understand something about our DNA yields (we use an automated extraction system), but I found it interesting that some factors, such as age, sex, BMI and cancer, can affect the DNA yields because these things can affect the amount or composition of WBCs an individual has. So that's another thing to keep in mind and maybe look at if you do all of the other steps to protect the integrity of the buffy coats (e.g. refrigerating or keeping it on ice, processing ASAP, using anticoagulants, etc.) and are still having trouble. Is there something common to the study participants that could affect their WBCs?