Equipment to Count Stem Cells in Blood
December 17, 2010 11:28 PM Subscribe
What inexpensive equipment can be used to count stem cells in a blood sample?
I have been asked to locate used lab equipment to count stem cells from a blood sample.
I have been asked to locate used lab equipment to count stem cells from a blood sample.
Well a second-hand FACSCalibur probably wouldn't be too expensive, and a two laser system could be used to identify LSKs (Lin- Sca-1+ c-kit+). I presume it's human blood we're talking about...?
posted by kisch mokusch at 5:14 AM on December 18, 2010
posted by kisch mokusch at 5:14 AM on December 18, 2010
Cheaper is to use Ficoll or Percoll discontinuous gradient centrifugation (random cite).
I asked my dad's transplant specialist whether they were going to do any enrichment on his allo-stem cell harvest, he told me that the stem cell band is pretty distinct from the other monocytes.
If you could identify which band it is, you could just suck out that band and do a simple number count.
However, without treatment with, say, GCSF (granulocyte colony stimulating factor) the number of haematopoietic stem cells in human peripheral blood is pretty damn small and I doubt you'll get an accurate count, if any at all.
FACS is the standard way of doing this; however, the CD (center of differentiation) identity of stem cells is still kinda-sorta not agreed upon. The gold standard would be to plate the Ficoll separated stem cell band and see how many form colonies. Work back from the number of colonies to how much peripheral blood you originally started with. If you do it by FACS taking all mononuclear cells (you'll still need to do a Ficoll/Percoll separation although, iirc, there's also a salt-based lysis that you can use to get rid of the erythrocytes and leave the white blood cells unharmed, but density gradient centrifugation is better).
Ficoll/Percoll is relatively inexpensive (although it's a consumable), and when I was using it for peripheral blood mononuclear cells I was just using a centrifuge that, second hand, would only be a thousand or so. I've also done it with ancient centrifuges that you might be able to pick up as scrap metal. You'll also need some disposable plastic labware (Falcon tubes...) and a haemacytometer to count cells. Oh, butterfly needles and syringes to harvest the blood (and alcohol wipes, small bandaids, rubber tubing, a rack for the tubes that you'll transfer the blood into). A seralogical pipettor (there are cheapass bulbs and lever-type pipettors as well as electric ones) and pipettes for making the gradient and layering the blood - you'll want bleach and a plastic bottle for cleanup). A 10uL/20uL/variable pipettor and pipette tips for counting (you'll probably want some trypan blue). If you want to grow up colonies, you'll need some more disposable plastic labware (culture dishes) and solid media (you'll have to research the specific type of media for growing up stem cells - there's probably a bunch of expensive hormones and other factors involved). A laminar flow hood for working in a sterile environment. A microscope (you might be able to get by with an inverted 10/20x - which you'll also need for the haemacytometer). An autoclave. A culture incubator. Probably a variable temperature water bath for pre-warming liquid media.
I'm curious; to what ends do you want to achieve by counting human peripheral blood stem cells? It'll probably be cheaper to outsource the operation; have some research lab do it for you. It's not going to be cheap - there's time on the FACS machine, technician time, cost of antibodies... there might be regulations requiring health workers to do the phlebotomy... if you're counting stem cells in untreated peripheral blood, you're going to need a lot of peripheral blood to get an accurate count and quite a bit of FACS time.
posted by porpoise at 10:14 AM on December 18, 2010 [3 favorites]
I asked my dad's transplant specialist whether they were going to do any enrichment on his allo-stem cell harvest, he told me that the stem cell band is pretty distinct from the other monocytes.
If you could identify which band it is, you could just suck out that band and do a simple number count.
However, without treatment with, say, GCSF (granulocyte colony stimulating factor) the number of haematopoietic stem cells in human peripheral blood is pretty damn small and I doubt you'll get an accurate count, if any at all.
FACS is the standard way of doing this; however, the CD (center of differentiation) identity of stem cells is still kinda-sorta not agreed upon. The gold standard would be to plate the Ficoll separated stem cell band and see how many form colonies. Work back from the number of colonies to how much peripheral blood you originally started with. If you do it by FACS taking all mononuclear cells (you'll still need to do a Ficoll/Percoll separation although, iirc, there's also a salt-based lysis that you can use to get rid of the erythrocytes and leave the white blood cells unharmed, but density gradient centrifugation is better).
Ficoll/Percoll is relatively inexpensive (although it's a consumable), and when I was using it for peripheral blood mononuclear cells I was just using a centrifuge that, second hand, would only be a thousand or so. I've also done it with ancient centrifuges that you might be able to pick up as scrap metal. You'll also need some disposable plastic labware (Falcon tubes...) and a haemacytometer to count cells. Oh, butterfly needles and syringes to harvest the blood (and alcohol wipes, small bandaids, rubber tubing, a rack for the tubes that you'll transfer the blood into). A seralogical pipettor (there are cheapass bulbs and lever-type pipettors as well as electric ones) and pipettes for making the gradient and layering the blood - you'll want bleach and a plastic bottle for cleanup). A 10uL/20uL/variable pipettor and pipette tips for counting (you'll probably want some trypan blue). If you want to grow up colonies, you'll need some more disposable plastic labware (culture dishes) and solid media (you'll have to research the specific type of media for growing up stem cells - there's probably a bunch of expensive hormones and other factors involved). A laminar flow hood for working in a sterile environment. A microscope (you might be able to get by with an inverted 10/20x - which you'll also need for the haemacytometer). An autoclave. A culture incubator. Probably a variable temperature water bath for pre-warming liquid media.
I'm curious; to what ends do you want to achieve by counting human peripheral blood stem cells? It'll probably be cheaper to outsource the operation; have some research lab do it for you. It's not going to be cheap - there's time on the FACS machine, technician time, cost of antibodies... there might be regulations requiring health workers to do the phlebotomy... if you're counting stem cells in untreated peripheral blood, you're going to need a lot of peripheral blood to get an accurate count and quite a bit of FACS time.
posted by porpoise at 10:14 AM on December 18, 2010 [3 favorites]
Oh - for a simple simple count, my ballpark estimate is in the range of fifty to a few hundred milliliters of peripheral blood as opposed to a few drops. For FACS, I'd expect the lowest minimum to be 20 to 50mL to get a reliable count.
posted by porpoise at 10:17 AM on December 18, 2010
posted by porpoise at 10:17 AM on December 18, 2010
Uh, upon re-reading my answer I need to point out that LSKs are a population defined in the mouse (Sca-1 is a mouse antigen with no known human equivalent). Human HSCs are found within the Lin- CD34+ compartment, though I think a few other molecules are included nowadays, such as CD133.
At any rate, porpoise's answer is far better than mine. And his "random cite" raises another question for the OP - what stem cell population are you interested in? Mesenchymal stem cells or haematopoietic stem cells? I think everyone has been assuming HSCs (I certainly was), but you never actually specified...
I will add that whether you should take the FACS approach vs the CFU-assay approach will depend on what you're trying to do.
If you're planning on screening a lot of individuals, then FACS will be the cheaper option. Antibodies aren't exactly cheap, and the time spent in front of the machine isn't small (especially with older machines that can only run a couple of thousand events per second), but the CFU-assays are most definitely not cheap either.
On the other hand, CFU-assays are robust and easily interpreted. Once you know how to identify the various colony types, that is. Certainly, if you are planning on publishing any results, you will need to do CFU-assays. The reviewers wouldn't accept FACS data alone.
But FACS is quick, easy and gives you immediate results. And if, say, you were looking at whether certain treatments caused stem cell mobilisation (a la GM-CSF, as porpoise mentioned) then FACS would be a perfectly acceptable technique to screen through hundreds of samples, with the understanding that any interesting findings would be confirmed using the CFU-approach.
posted by kisch mokusch at 2:04 PM on December 18, 2010
At any rate, porpoise's answer is far better than mine. And his "random cite" raises another question for the OP - what stem cell population are you interested in? Mesenchymal stem cells or haematopoietic stem cells? I think everyone has been assuming HSCs (I certainly was), but you never actually specified...
I will add that whether you should take the FACS approach vs the CFU-assay approach will depend on what you're trying to do.
If you're planning on screening a lot of individuals, then FACS will be the cheaper option. Antibodies aren't exactly cheap, and the time spent in front of the machine isn't small (especially with older machines that can only run a couple of thousand events per second), but the CFU-assays are most definitely not cheap either.
On the other hand, CFU-assays are robust and easily interpreted. Once you know how to identify the various colony types, that is. Certainly, if you are planning on publishing any results, you will need to do CFU-assays. The reviewers wouldn't accept FACS data alone.
But FACS is quick, easy and gives you immediate results. And if, say, you were looking at whether certain treatments caused stem cell mobilisation (a la GM-CSF, as porpoise mentioned) then FACS would be a perfectly acceptable technique to screen through hundreds of samples, with the understanding that any interesting findings would be confirmed using the CFU-approach.
posted by kisch mokusch at 2:04 PM on December 18, 2010
allo = auto transplant
Again, digdan, knowing why/what your boss wants to do with human peripheral blood stem cell counts would go a lot further than saying, "oh, about a hundred grand+ upfront and ongoing consumables and technician's costs."
posted by porpoise at 7:07 PM on December 18, 2010
Again, digdan, knowing why/what your boss wants to do with human peripheral blood stem cell counts would go a lot further than saying, "oh, about a hundred grand+ upfront and ongoing consumables and technician's costs."
posted by porpoise at 7:07 PM on December 18, 2010
This thread is closed to new comments.
posted by emd3737 at 2:48 AM on December 18, 2010