I am not a BB tech like most of you, so I can use your help on a few things:

1. How does the hematocrit of peripheral blood compare with the hematocrit of aspirated bone marrow?

2. Same question as above, but for Ca2+ instead of hematocrit.

3. I know that glass tubes will trigger the contact activation coagulation cascade because of the anionic surface. How about plastic? If a quantity of bone marrow is aspirated into a typical plastic syringe to be almost immediately transferred into a collection bag containing an effective quantity of an anti-coagulant, do you need to pre-coat the syringe with a bit of anti-coagulant? I say "almost" immediately because the hematologist will typically aspirate from a few different sites with the time span of a few minutes, so the first pull may be in the syringe for 3-5 minutes.

I tried to get answers for you from our Hematology department manager. She only had an answer to your third question:

I know that glass tubes will trigger the contact activation coagulation cascade because of the anionic surface. How about plastic? If a quantity of bone marrow is aspirated into a typical plastic syringe to be almost immediately transferred into a collection bag containing an effective quantity of an anti-coagulant, do you need to pre-coat the syringe with a bit of anti-coagulant? I say "almost" immediately because the hematologist will typically aspirate from a few different sites with the time span of a few minutes, so the first pull may be in the syringe for 3-5 minutes. With that length of time, clotting would definitely begin, even in a plastic syringe, and might even begin with an anticoagulant coated syringe. Typically inversion mixing is required to prevent clots.

Dear Hodag,

Are you simply doing a marrow aspirate for diagnostic purposes or, are you collecting marrow for transplant (you mentioned a bag). Usually, for a harvest, we rinse the syringes with heparin. After the physiscian has pulled several aspirates from the incision site, the marrow becomes diluted with peripheral blood. The Hct usually runs around 20-30% depending on the amount of isotonic media you have used in the collection bag.

Sarah Dickerson

Thanks for the replies.

This is for a stem cell isolate to be used eventually for transplant. Heparin is a non-starter for some very complicated molecular biology reasons that I won't detail, but CPD within certain limits may be acceptable. What is being attempted here is new technology.

I definitely understand the problem of peripheral blood aspiration by the aspirating physician. Technique is all important, which is why we never let a hematologist aspirate "unsupervised". He might have an MD, but very few of them know how to get really good stem cell aspirates. FYI, we limit them to 2.5 ml per aspiration site and require use of at least a 20 ml syringe. Anything smaller syringe does not create enough suction when the plunger is yanked and the adherent cells, well, they stay adhered to the trabecular bone!

Why not put some heparin in the syringe before the bone marrow? We mix Plasmalyte A with 50 IU/ml heparin for the marrow harvest and remove 1-2 ml of this into the syringes prior to collection.

Because heparin is just evil.

Seriously, heparin has been used for ages to process bone marrow and blood and the anticoagulant properties are well established. but the precise mechanism of action is undesirable since it is not easily reversible (we don't want to use protamine sulfate for example). Playing with citrate and CaCl concentrations gives the result we need, even if the process is a bit more complicated.