Homemade Anticoagulants: Preparation guide (Trisodium Citrate and ACD)

by Lethenteron and DerMeister


If you order your blood from a butcher, there’s a very good chance that your blood has already been treated with anticoagulants, or that is has been defibrinated. Don’t hesitate to ask your provider for details. However, If you come and collect it yourself, (at a farm, at the slaughterhouse, or from your own livestock etc.), you may need to treat the blood yourself, either by vigorous stirring (traditional method) to defibrinate the blood or via the addition of anticoagulants.


Contrary to tradition, vinegar and salt are not very good options to preserve blood, and definitely not for raw consumption. It significantly alters the taste, and it is the stirring that is usually performed alongside that helps remove the fibrin and leaves the blood fluid. Keep in mind that the consumption of raw blood involves very significant risks and it’s your responsibility if you decide to engage in it. Check out this post for some information about blood pathogens and safety guidelines.

Putting defibrination and salt/vinegar aside, anticoagulants are generally a much better option, but you might not have any available or you might not know how to use them: that’s where this post comes in.

We’ll be talking about two reasonably accessible and reliable options here: trisodium citrate and ACD.

Trisodium Citrate:

Trisodium citrate has the chemical formula of Na3C6H5O7. It is sometimes referred to simply as “sodium citrate”, though sodium citrate can refer to any of the three sodium salts of citric acid. It possesses a saline, mildly tart flavor. It is mildly basic and can be used along with citric acid to make biologically compatible buffers.[n1]

Sodium citrate is chiefly used as a food additive, usually for flavouring, to add tartness to various foods, among which various club soda, sausages, wine or as a preservative. It appears as E331 on the label. It is also used to alter the texture of certain foods, like ice cream, yogurt, jams and as an emulsifier.


Another primary use of sodium citrate, and the one that concerns us here, is as an anticoagulant in blood-collection tubes and for the preservation of blood in blood banks. It is also used to treat animal blood intended for human consumption. When the blood is not simply defibrinated, sodium citrate is the additive most commonly used.[n2] [n3]
The citrate ion chelates calcium ions in the blood by forming calcium citrate complexes, disrupting the blood clotting mechanism.


Method:

To work effectively, trisodium citrate needs to be dissolved in water to form a solution. That solution is then added to the collected blood. Alternatively, the blood can be poured in a container that already contains the anticoagulant solution. In both cases, the mixture needs to be stirred gently to ensure a homogenous distribution.

You already have purchased or otherwise obtained your trisodium citrate; if not, you can find underneath a link to the product on Amazon, and, if you would prefer to make your own, the detailed How-To.

Here follows the procedure to prepare your anticoagulant solution:

      1. Dissolve 3.80g Sodium citrate in 100.0ml Distilled water. (Stir until solids are no longer observed.)
      1. Use 50ml of the solution for 450ml blood. http://himedialabs.com/TD/R014.pdf
    1. Store below 30°C in tightly closed container and away from bright light.


This is extrapolated from HiMedia‘s 3.8% sodium citrate solution. However, Meat Technology Update gives the following proportions in the context of slaughterhouse blood processing:
Sodium citrate should be added at a rate of up to 3 g/L of blood, for example, 7–8 mL of a 40% solution could be added to one litre of blood”

There’s a lot of variation in how much sodium citrate solution you should use to achieve the best results. Too little and the blood might clot, too much and the tart taste will be overwhelming. Overall, the average tends to be around 25-70 ml of a 3.8% solution for A liter of blood. As a general rule, you might want to add a little too much rather than not enough, but factors such as temperature or mixing can influence the outcome as well.

If you are not getting your blood on site, directly from the animal, you’ll want to sieve your blood through a fine mesh sieve to remove any clots that might have formed since collection before mixing it with your anticoagulant of choice.


Where to get trisodium citrate and what to get:

Buy it online:
You’ll want to make sure you get food-grade trisodium citrate. Example, on Amazon.

Making your own:
If you have some experience, you can also synthesize your own Trisodium Citrate at home with minimal effort.

Materials:
Citric Acid – C6H6O7 – 192.124 g/m
Sodium Bicarbonate (baking soda) – NaHCO3 – 84.01 g/m

Yields : Trisodium Citrate – Na3C6H5O7 – 258.06 g/m

Balanced Equation:
C6H8O7 + 3NaHCO3 = 3CO2(g) + 3H2O + Na3C6H5O7

Procedure:

      1. Dissolve 192g of citric acid in distilled water; in another container, dissolve 252g of sodium bicarbonate in distilled water.
      1. Slowly pour the sodium bicarbonate solution in to the acid solution while stirring, this must be done slowly as the reaction will liberate large quantities of CO2 gas, which can cause the solution to bubble out of its container if added too quickly. Do not do this in a confined space as the CO2 may build up and become a suffocation hazard.
      1. After the bicarbonate is all added, the solution should have a pH of 7, if the pH is not at least close to neutral, add more citric acid solution or bicarb solution until a neutral pH is reached.
    1. Next we need to remove our product from solution, there are two ways to do this:
  1. Pour the solution in to a clean glass crystallization dish (a pyrex baking dish will suffice) and gently bake in the oven at 120C (~250F) until all the water has evaporated, leaving behind a white solid (your finished trisodium citrate). This can then be collected and stored for later use.
  2. Or, place the solution containing Trisodium Citrate in a refrigerator until crystals precipitate, you can then decant the water and filter the crystals out. Allow the crystals to dry completely before storage. You may want to crush the crystals in to a powder to make it dissolve easier once you’re ready to prep your anticoagulant. Optionally, you may boil the solution gently before attempting precipitation to remove any excess water, resulting in a saturated solution which will make precipitation easier. To do so, boil gently until solids begin to appear, then remove from heat immediately and allow to cool to room temperature before placing in the fridge for crystallization.


ADC (Acid-citrate-dextrose):

ACD, or Acid-citrate-dextrose solution is any solution of citric acid, sodium citrate, and dextrose in water. It is mainly used as an anticoagulant to preserve blood specimens required for tissue typing.

It is similar to CPDA-1, one of the additives used for the preservation of whole blood and red blood cells (commonly used in blood collection bags for blood banks). But the latter allows for a much longer shelf life, up to 35 days, by extending red cell survival through the addition of adenine to the solution, needed for the maintenance of red cell ATP levels.


It is certainly possible to make your own ACD anticoagulant, the base constituents being reasonably available and safe. Somebody on TheLabRat [n4] was king enough to provide their own protocol and while I don’t have personal experience with making this myself yet, it should work properly.

      1. Dissolve 1.32g of sodium citrate in 85ml of distilled water.
      1. Dissolve 0.48g of citric acid in the solution from step 1.
    1. Dissolve 1.47g of dextrose in the solution from step 2.
      1. Add distilled water to 100ml.
      1. Filter sterilize through 0.2um filter (This step could be skipped, but it might cause some mild coagulation to occur in the treated blood).
    1. Use in proportions of about 125ml of solution for 500ml blood.


Where to get:

Dextrose can be purchased from Amazon and most specialized nutrition shops : https://www.amazon.com/NOW-Foods-6925-Dextrose-32-Ounce/dp/B008GQ2JPO?th=1

General Troubleshooting:

Time: If you satisfy all those requirements and clotting is still happening, you may be spending to much time between the blood collection and addition of the anticoagulant.

You might want to have your anticoagulant in the same container where the blood is poured so you can progressively mix the two as you collect it. Stir or inverse gently.

Storage: Buffer intended for antigen retrieval can be stored at room temperature for 3 months or at 4C for longer storage. But since this is intended for human consumption, I think it would be advisable to prepare it the same week if not the same day it will be used.[n5]


Safety:

We already have a relatively extensive article on the safety of various anticoagulants that you can view here. Trisodium citrate, for example, is discussed and shown to be reasonably safe, even in relatively high quantities.

However, pure Trisodium Citrate Dihydrate could be irritating to contact with the eyes, skin and airways. It is advised to use personal protective equipment, as gloves, safety goggles and respiratory protection when handling. Full toxicology information here.

While I touched on CPDA-1 on that post, I didn’t talk about ACD, though we can reasonably assume that they have a similar profile (the only difference being the lack of adenine in ACD).
The safety sheet for different formulations of ACD only warn about adverse risks if ingested on its own, in large quantities [n6], there is no known chronic toxicity.

That being said, we are neither doctors nor chemists. This is the result of our own experiences and/or research into the subject and while we’ve tried to provide as many sources as possible, there’s room for error. If you decide to use these guidelines to treat collected blood, we are not responsible for any potential risks taken.

If you have any expertise in the area and would like to contribute or correct any information given in this post, do not hesitate to contact us. Same-wise, if you would like to share your personal experience with these anticoagulants, let us know.


References:
[n1] Wikipedia, trisodium citrate. https://en.wikipedia.org/wiki/Trisodium_citrate

[n2]  Kostić, I. ;  Bukara, K. ;  Ilić, V. ; Bugarski, B. Effect of bovine blood storage in slaughterhouses on parameters relevant to hemoglobin isolation. Proceedings, International 57th Meat Industry Conference, Meat and Meat Products – Perspectives of Sustainable Production, Belgrade, Serbia, June 10-12, 2013 2013 pp.201-205 ref.23. https://www.cabdirect.org/cabdirect/abstract/20153427121
[n3] Meat Technology Update ; 2003 http://www.carndeporc.cat/3tres3_common/art/ca/1178/fitxers/CSIRO,2003.pdf
[n1] Weil, R. (1983). Sodium Citrate in the Transfusion of Blood. JAMA: The Journal of the American Medical Association, 250(14), 1901 : https://sci-hub.tw/10.1001/jama.1983.03340140071035

[n4] The Lab Rat, Biotech jobs and research reference material http://www.thelabrat.com/protocols/ACD.shtml

[n5] Question on ResearchGate https://www.researchgate.net/post/Sodium_citrate_buffer_vs_citrate_buffer
[n6] BD Material Safety Data Sheet for BD Vacutainer® Brand Tube with ACD Solution A or B ( https://www.rmlonline.com/images/data/attachments/0000/2069/msds-Tube_ACD_A_or_B.pdf)

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