[hodag]

I am trying to compute the minimum quantity of a citrate containing anti-coagulant solution needed to bind all the free calcium in a bone marrow aspirate.  I think the number should be in the range of 12ml +/- per 100ml of aspirate, but the number I come up with is about 0.5ml (versus an expected value of 12-15ml).

A standard blood collection bag designed to hold 500ml of blood comes pre-filled with 70ml of citrate solution, or 14ml of citrate per 100ml.  While there are differences between bone marrow and whole blood, the difference is not that large leading me to believe that I screwed up my calculations somewhere.  In this application, too much citrate is almost as bad as too little citrate so I need to get it right.

Can somebody please look at my calculations and tell where where I screwed up?  I have attached an MS Word file to this message with all my work laid out in MS Equations.  I suspect I dropped the ball in my grams to moles calculations.

[renge ishyo]

While there are differences between bone marrow and whole blood, the difference is not that large leading me to believe that I screwed up my calculations somewhere.

Are you sure this assumption is correct? From what I understand calcium is a chief component of bone itself as well as an intracellular signaling molecule in bone tissue, and you would expect it to be present in bone marrow in higher concentrations than in blood taken from many other places in the body where it's primarily use is that of a signaling molecule.

[hodag]

Yes, for this purpose the assumption is sufficiently correct.  Bone and the bone marrow structure does contain a lot of calcium, but bone marrow cells do not.  The purpose of this procedure is to aspirate bone marrow derived cells, after which the erythrocytes are centrifuged and discarded, leaving about 55% of the starting volume which comprised of serum and the buffy coat cells.

The goal is to chelate all the free Ca2+ in the serum as that is the bioactive species we are trying to control.  If the bone marrow did contribute substantail calcium, that would just make my answer even more wrong than it already is!!!

We want to add enough anti-coagulant to prevent clotting, but nothing extra as CaCl will be added at the end of the process to deliberately create a clot.  Under-citrating the bone marrow aspirate will cause premature clotting and over-citrating the aspirate will cause the opposite problem at the end of the process.  Getting the citrate in the range of 110% - 120% of the ideal minimum is probably ideal, but right now I am so far off I need a sanity check of my calculations.  I must have it wrong!

[JGK]

Are you are assuming that the citrate in the pre prepared bags is exactly enough to bind all of the calcium. This may not be the case, it may be in a large excess given the wide range of values seen in biological systems. The maunfacturer will produce a "one size fits all" bag rather than a number of different ones. A single bag may then be used for normal patients or patients with abnormally high or low Ca levels.

I tried to follow your calculations (only went back as far as molar levels (not atoms) and came up with a value of 1.1 to exactly bind the calcium.

[hodag]

JGK,

Thanks for taking a look.  Yes, we are planning to use standard blood bags containing citrate because that is what is commercially available.  The protocol I want to use will be to remove all the citrate from the bag, reserving some portion in a syringe, and discarding the rest.  Then, as cells are added to the bag a small quantity will be added back.

A standard blood bag is definitely overkill, hence the problem.  Indeed we do not want to inject cells directly into the bag as putting few cells into a large volume of acidic material is not good for them.  That is why I need to know what is required to bind all the free calcium, after which I will overshoot by some small percentage (say 110%) just to be sure.  The other issue is that standard blood bags are designed to store mixed cell populations for up to 35 days, and I am not sure what happens to the bound calcium during storage.  We use the cells within 2-3 hours so the requirements are different, hence I am trying to understand the requirements precisely.

How did you come up with 1.1?  I stink at chemistry and am trying to understand this so that I can develop a spreadsheet that customizes the quantity to the patient's starting calcium and hematocrit values.  Whatever detail and education you can provide is greatly appreciated.

[JGK]

By making a mistake (I reversed the chelation ratio), I now get 0.526 mL